33 shaking red-cap blood collection tube without additive substances is recommended to accelerate the blood clotting process gilang nugraha1, rohayati2 1department of medical laboratory technology, faculty of health, universitas nahdlatul ulama surabaya, surabaya, indonesia 2department of medical laboratory technology, poltekkes kemenkes bandung, bandung, indonesia correspondence: gilang nugraha, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code : 60237 email: gilang@unusa.ac.id received: february 26, 2019 revised: march 21, 2019 accepted: march 26, 2019 abstract many blood collection tube manufacturers do not recommend shaking the red tube. shaking the red tube to avoid the intensity of the changed of blood that interacts with the glass surface of the tube will trigger the contact path in the coagulation cascade. generally, the blood takes 30-60 minutes to clots at room temperature without shaking. the aim of this study was to determine the effectiveness of shaking the red-cap blood collection tube in producing serum volume. the method of this study was experimental as much as 5 ml of blood was taken and put into 3 tubes with a volume of 1ml each tube. the first and the second tube were shaken 8 times. the first tube was incubated for 10 minutes while the second tube was incubated for 25 minutes. meanwhile, the third tube (as a control) was not shaken but was incubated for 40 minutes. the tube were centrifuged at 3000 g for 10 minutes. the serum volume was measured using micropipette and collected into eppendorf tube. the results showed that there were a difference in the number of serums formed after tube shaking by time variation (p = 0.002), the results of the post hoc test using bonferroni test while showed that the second tube did not have a difference in serum volume with control (p = 0.751). it can be concluded that the red-cap blood collection tube, which was shaken 8 times and incubated for 25 minutes long could accelerate the coagulation process. keywords shaking, blood collection tube, blood clotting ina j med lab sci tech 2019; 1(1): 33-37 gilang nugraha, rohayati 3 4 introduction many blood collection tube manufacturers do not recommend shaking the red-cap blood collection tubes (not containing additives). the purpose of shaking blood collection tubes is to mix additives with blood such as anticoagulants (1). red-cap collecting tubes are generally used for clinical chemistry and immunoserology to obtain serum specimens and to incubate the specimen allowed to clot in this tube (2). shaking the red-cap blood collection tube increase the interaction of blood to the glass surface of tube, then it can trigger the contact path which causes activation of factor xii (fxiia) and leads to the blood clotting cascade (3–5). activating the blood-clotting cascade through contact factors may accelerate the blood clotting process in the red-cap blood collection tube, then the specimen can be performed immediately in the pre-analytic stage. generally, the unshaken red-cap blood collection tube takes 30-60 minutes to clot the blood at room temperature in order to obtain serum with a good quality (6). therefore, the aim of this study was to determine the effectiveness of shaking the red-cap blood collection tube in producing serum volume. this research was carried out experimentally on 30 volunteers. materials and methods design, samples, and techniques the research was conducted experimentally on august 2018. as much as 5 ml blood samples were collected from 30 students of medical laboratory technology study program, faculty of health, nahdlatul ulama university of surabaya. the research was conducted in b tower of laboratory of nahdlatul ulama university of surabaya. the blood samples were drawn about 5 ml using syringe, then put into 3 blood collection tubes without additives 1 ml for each tube (red lid), tubes 1 (first tube) and 2 (second tube) are used as test tubes and tube 3 (third tube) as a control. the test tubes were immediately shaken 8 times, except the control tube. incubation was performed to give the chance for blood to clot, the first tube was incubated for 10 minutes, the second tube was incubated for 25 minutes and third tube was incubated for 40 minutes. incubated blood samples were immediately centrifuged with thermo scientific sl 16 r r (thermo fisher scientific inc, langenselbold, germany) at 3000 g for 10 minutes. the serum volume was measured using micropipette and collected into eppendorf tube. ethical approval ethics commission of nahdlatul ulama university of surabaya, east java, indonesia, approved this research. informed consents ina j med lab sci tech 2019; 1(1): 33-37 gilang nugraha, rohayati 3 5 were obtained from all patients before drawing blood specimens. statistical analysis significant test was used in this study; anova test was used to determine the effect of shaking and incubation time in formation of serum volume compared to the control. post hoc analysis was used to determine the same treatment with the control and determine the optimum treatment that can be used. p value <0.05 was stated to be statistically significant. results characteristics of respondents as much as 30 respondents were enrolled in this study after obtaining individual approval and institutional informed consent. respondents were active students in the medical laboratory technology study program, faculty of health, nahdlatul ulama university of surabaya. the range of age was 20 to 23 years old with an average of 20.7 years old. the ratio of gender between men and women was 1:27. serum volume the obtained blood samples were treated based on research methods. the first treatment was given by shaking the tube 8 times and by incubating for 10 minutes. the second treatment was shaken 8 times and incubated for 25 minutes. furthermore, the control was not carried out by shaking. however it was incubated for 40 minutes. the characteristics of formed serum volume presented in table 1. table 1. characteristics of serum volume in various treatments parameter mean (µl) sd 1st treatment 308 103.54 2nd treatment 368 77.07 control 390 88.49 the effect of shaking on serum volume the anova test result revealed that the probability/sig results was 0.002. since the probability the probability value/sig was <0.05, it can be concluded that there was a difference in the amount of formed serum after shaking the tube by time variation. post hoc test was used to these three homogeneous group variants (p = 0.243) then using bonferroni test to determine serum volume compared to the control. significant value of shaken blood tube incubated for 10 minutes was 0.002. it means that there was a difference in serum volume with control. meanwhile, significant value of another shaken blood tube, which incubated for 25 minutes, was 0.751, which means that there was no difference in serum volume compared with control. discussion vacuum tube or commonly known as vacutainer is vacuum containers used for blood collection (7). there are two types of ina j med lab sci tech 2019; 1(1): 33-37 gilang nugraha, rohayati 3 6 the red-cap vacuum tubes, which are made from glass and plastic. red plastic tube contains the blood clotting activator while red glass tube does not contain any additives. both tubes are used to obtain serum for clinical chemistry or immunoserology tests. most vacuum tube manufacturers suggest to not shaking red glass tube due to excluded from additives (2,7). the surface of glass on collecting tube is negative charge, and then it easily triggers the contact path in coagulation cascade during the blood clotting process. zymogen factor xii (fxii) is activated into fxiia after contact with the glass tube surface and activated multistep cascades, while thrombin (fiia) is formed as an activator forming fibrin threads (5). it means that the shaking of the red-cap blood collection tube is recommended to increase contact of blood with the tube surface and to activate the blood clotting process. hence, blood-clotting process is formed faster. shaking test of two blood collection tubes incubated in 10 and 25 minutes produced significant different in serum volume (p=0.002). the serum volume formed in the first treatment was significantly different compared to the control (p = 0.002) and serum volume was less than that in the control treatment. the differences in serum volume formed could be due to incomplete blood hemostasis, because blood clot retraction process was not optimal when serum was trapped by imperfect blood clot, thus producing less serum volume (8). the serum volume formed in the second treatment was not significantly different compared to the control (p=0.751). this means that the shaking of the red-cap tube incubated for 25 minutes had the same volume obtained from the commercial technique (control), while in this study the control was incubated for 40 minutes, thereby saving 15 minutes. conclusions the red-cap blood collection tube without additives is recommended with 8 times shaking and 25 minutes incubation. the limitations of this study were measuring serum volume without considering debris that might be carried in serum. conflict of interest there are no conflicts of interest. ina j med lab sci tech 2019; 1(1): 33-37 gilang nugraha, rohayati 3 7 references 1. clsi. procedures for the collection of diagnostic blood specimens by venipuncture ; approved standard-fifth edition. vol. 23, clinical and laboratory standards institute. 2003. 1-52 p. 2. bowen rar, sattayapiwat a, gounden v, remaley at. blood collection tube-related alteration in analyte concentrations in quality control material and serum specimens. clinical biochemistry. 2015;47(3):150-157. 3. simão f, feener ep. the effects of the contact activation system on hemorrhage. front med [internet]. 2017;4(july):1–10. available from: http://journal.frontiersin.org/article/10.3389/fme d.2017.00121/full 4. wu y. contact pathway of coagulation and inflammation. thromb j. 2015;13(1). 5. zhu s, diamond sl. contact activation of blood coagulation on a defined kaolin/ collagen surface in a microfluidic assay shu. trombos res. 2014;134(6):1335–1343. 6. yin p, lehmann r, xu g. effects of preanalytical processes on blood samples used in metabolomics studies. anal bioanal chem. 2015;407(17):4879-4892. 7. nugraha g. panduan pemeriksaan laboratorium hematologi dasar. ke-2. jakarta: trans info media; 2017. 8. li z, li x, mccracken b, shao y, ward k, fu j. a miniaturized hemoretractometer for blood clot retraction testing. small. 2016;12(29):3926–34. 74 study of hemoglobin levels on hemolysis sample noor febryani1, ikke nanda amalia2, intan dwi anggraeni3, gilang nugraha3 1science of biological resources, the united graduate school of agriculture science, gifu university, japan 2department of basic medical science, faculty of medicine, airlangga university, indonesia 3department of medical laboratory technology, faculty of health, nahdlatul ulama university of surabaya, indonesia correspondence: gilang nugraha, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code : 60237 email: gilang@unusa.ac.id received: september 11, 2019 revised: october 4, 2019 accepted: october 18, 2019 abstract hemolysis can significantly affect the reliability of test results and occur in the pre-analytical phase. the aim of this study is to reveals the correlation of hemoglobin levels on hemolysis sample. this experimental study was carried out using samples from thirty students of medical laboratory technology study program of the nahdlatul ulama university of surabaya. blood samples were treated by hard shaken in 30 times in order to damage the middle part of the blood sample. data on hemoglobin levels were collected and analyzed using the pearson correlation test before and after treatment with significant value p < 0.05 indicating that there was a significant correlation. hemoglobin concentrations were strongly positive for the hemolysis of the sample (p = 0.000). the conclusion is that sample hemolysis has a potency to be the confounding factor on the hemoglobin test. keywords hemoglobin, sample hemolysis, pre-analytical phase introduction hematology routine examination is a general hematological examination which are frequently requested in laboratory because it is usually used as an early detection in providing a diagnosis of disease or monitoring condition of patients. hemoglobin examination is one of hematological routine examinations which is often in laboratory. there are four principal techniques for measuring hemoglobin, which are tallquist, sulfate of copper, sahli and ina j med lab sci tech 2019; 1(2): 74-79 noor febryani, et al. 7 5 cyanmethemoglobin (1). the cyanmethemoglobin technique uses a spectrophotometer or photometer to measure colorimetrically. cyanmethemoglobin technique has been recommended in hemoglobin examination because that is a reference method, all of types of the hemoglobin can be measured except sulfhemoglobin (8). this technique has been recommended in hemoglobin examination since the error only reaches around 2% (1). although the cyanmethemoglobin technique is a reference method, errors that might occur in this examination can be caused by preanalytical, analytical and post analytical phase. error which is occur in the preanalytical phase gives the contribution of 61% of the total laboratory error (9). the errors in the pre-analytical phase may interfere with the reliability of laboratory resultan (2), this happens when especially taking blood samples are often occur hemolysis. hemolysis is the most common cause of pre analytical error. prevention of medical errors is a goal of health care system. the laboratory errors are due to pre analytical variables that has received a great deal of attention. it has been analyzed that hemolysis of patient specimen may interfere with accurate measurement of analytes. basically, hemolysis occurs when red blood cells become damaged or destroyed. red blood cells contain hemoglobin molecules which are released during hemolysis. once a whole blood specimen is hemolyzed, the hemoglobin molecules within the red blood cells are released causing the serum or plasma to have a pink to red color (10). one of the causes of hemolysis is in vitro hemolysis (3). in vitro hemolysis is a major instance of error in laboratory testing as it is mainly caused by improper collection and handling of specimens, such as extended use of venous stasis, blood differentiation by means of intravenous catheters and blood collection (4). furthermore, in vitro hemolysis also affects the quality of analyte and can lead to incorrect interpretation of the obtained resultan (5). when hemolysis occurs in blood cells due to in vitro hemolysis, the erythrocyte cell membrane collapses so that the hemoglobin release will broken off the cell membran (6). the examination in the laboratory indicated that the increasing of hemoglobin values are due to the examination result test which was inaccurate. this study of the hemoglobin examination revealed that the range of value was not far apart or still in normal order. therefore, the purpose of this study reported here was to more thoroughly investigate the correlation of hemolysis in hemoglobin concentration. materials and methods this study was experimental research which are true experimental with measured ina j med lab sci tech 2019; 1(2): 74-79 noor febryani, et al. 7 6 pretest-post test control group design. sample was measured hemoglobin before and after being treated. the blood examination were performed in laboratory nahdlatul ulama university of surabaya, faculty of health, medical laboratory technologist study program. a total of thirty samples were collected from the students of medical laboratory technology study program, faculty of health, nahdlatul ulama university in february – april 2019. ethical clearance was held by ethics commission of nahdlatul ulama university of surabaya which was approved this research. informed consent was given to patient before taking blood samples followed by anonymity and confidentiality data of patient would be guaranteed by researchers. samples of blood were taken as much as 3 ml and divided into two sections of the tube. in the first tube, the hemolysis sample was measured directly as a control. while in the second tube, the hemolysis sample was treated by hard shaking of the center portion of the sample vigorously in thirty times and wounded the middle part of the blood sample, then the sample was centrifuged (write down the merek/type of machine) at 4000 rpm for 10 minutes. after the plasma was lysis visible then the hemoglobin levels and percentage of hemolysis were measured. hemoglobin levels were measured using spectrophotometer genesys 10s uv-vis with 540nm wavelength, while on the percentage of hemolysis was calculated as two measurements. the first measurement of hemolysis in another tube was measured percentage of hemolysis by nacl 0.1% solution (a1) and the second measurement of hemolysis was measured percentage of hemolysis by nacl 0.9% solution (a2) then both were measured of the absorbance with spectrophotometer genesys 10s uv-vis in 540 nm wavelength. the percentage of hemolysis was calculated by using: %hemolysis = abs. nacl 0,9% solution abs. nacl 0,1% solution × 100% *abs: absorbance the data of hemoglobin levels before and after treatment were collected and analyzed using pearson correlation test with spss version 16.0 to determine the correlation of hemoglobin levels on hemolysis sample with the significance value p < 0.05 showed in statistically, there was a significant correlation. results percentage of sample hemolysis the research were conducted from 30 respondent which were active students of medical laboratory technologist study program, faculty of health, nahdlatul ulama university of surabaya. there were consist of men and women student which especially first was measured the percentage of hemolysis, obtained data as follows: ina j med lab sci tech 2019; 1(2): 74-79 noor febryani, et al. 7 7 table 1. percentage of hemolysis hemolysis (%) total 0.7 1 0.8 3 0.9 7 1 1 1.1 2 1.2 6 1.3 4 1.9 1 2 1 2.3 1 2.9 1 characteristics of hemoglobin levels in various treatments the sample of blood was taken as much as 3 ml, subsequently divided into two parts. in the first tube, the hemolysis sample was measured directly as a control. whereas the hemolysis sample in the second tube was treated by hard shaking the center portion of the sample vigorously in thirty times and wounded the middle part of the blood sample, then the sample was centrifuged at 4000 rpm for 10 minutes in room temperature (2025℃). after the plasma were lysis visible then the hemoglobin levels were measured. this treated sample is called by hemolysis sample. characteristics of the samples are summarized in table 2. table 2. characteristics of hemoglobin level parameter mean sd range control 13.1 1.7 9.7-16.4 hemolysis sample 13.8 1.8 10.4-17.2 correlation of sample hemolysis on hemoglobin levels based on the statistical analysis of the paired samples t-test, the results of the significance value were 0.000. it is indicated that the levels of hemoglobin in the hemolysis sample were different. correlation of hemoglobin levels on hemolysis sample using the pearson correlation test determined the strength of the correlation of hemoglobin levels on sample hemolysis11. it showed that the significance value was 0.000 and it could be concluded that there was a strong positive correlation of hemoglobin levels on hemolysis sample. these results are shown in the following scatter diagram. fig 1. the scatter diagram of hemoglobin levels y = 1,0434x + 0,0955 r² = 0,9832 9 11 13 15 17 19 9 11 13 15 17 h e m o ly si s s a m p le ( g /d l ) non-hemolysis sampel (gr/dl) ina j med lab sci tech 2019; 1(2): 74-79 noor febryani, et al. 7 8 discussion hemolysis is caused by the release of hemoglobin and internal components of erythrocyte membranes into the plasma. generally, hemolysis is caused by biochemical, physical and immunologic mechanisms (2). in vitro hemolysis is usually happened due to errors in collecting and handling of specimen2. in the laboratory experiment, the prevalence of hemolysis to reject samples in the hemoglobin test is not known yet. however, the clsi recommended that the lyzed samples were not be tested (2). the result of this study revealed that a comparison of normal sample and hemolysis sample had significant differences from a blood sample of healthy volunteers who were mechanically induced hemolysis performed in vitro. hemoglobin examination with cyanmethomoglobin technique is used the drabkins solution with the composition of potassium ferricyanide which binding heme (ferro) become (ferri) methemoglobin. cyanide ion were changed the methemoglobin become cyanmethemoglobin, kh2po4 are set the ph of solution (7.0-7.4) and non ionic detergent is functioning to accelerate the lysis of erythrocyte. turbidity in blood sample is consequence of leukositosis, hyperlipidemia and globulin can cause absorbance measurements to false increase (8). the hemoglobin levels have been shown to increase in hemoglobin tests with the hemolysis sample in this study. this is not same as the fact that when erythrocyte cells are lysis, it can lead to the disappearance of fluidity or fragility the membranous of the erythrocyte which is easily broken that cause the reduction in hemoglobin level (7). however, statistical analysis on pearson correlation test indicated that was a strong positive correlation of hemoglobin levels on hemolysis sample. the result of this study revealed that hemolysis sample has a potency to be the confounding factor on hemoglobin test. therefore, further research should be carried out to affect hemoglobin levels on hemolysis sample. conclusions based on the result of this study, it can be concluded that there was a strong positive correlation of hemoglobin levels on hemolysis sample. the hemolysis sample has a potency to be the confounding factor on hemoglobin test. this is caused by the turbidity of hemolysis sample so that the hemoglobin levels is false increase. acknowladgements we would like to thank the laboratory technicians at the laboratory of nahdlatul ulama university of surabaya for technical support. ina j med lab sci tech 2019; 1(2): 74-79 noor febryani, et al. 7 9 conflict of interest there are no conflicts of interest. references 1. nugraha g. laboratory practice guide on basic hematology. trans info media; 2017. 2. hernaningsih y, akualing js. the effect of hemolysis on plasma prothrombin time and activated partial thromboplastin time test using photo-optical method. medicine. 2017; 96 (38): 15. 3. michael p, phelan, edmund z, reineks, jesse, schold, frederic, chamberlin j, gary, procop. pre analytic factors associated with hemolysis in emergency department blood samples. arch pathol lab med. 2018; 142: 229-235. 4. adiga us. hemolytic index-a tool to measure hemolysis in vitro. iosr journal of biotechnology and biochemistry. 2016;2(2): 4952. 5. multinovic d, andrijevic i, licina m. & andrijevic l. confidence level in venipuncture and knowledge on causes of in vitro hemolysis among healthcare professionals. biochemia medica. 2015: 25(3): 401-409. 6. kazmierczak sc. accurate results in the clinical laboratory:a guide to error detection and correction. amsterdam (netherlands): elsevier academic press; 2019. chapter 5, interferences of hemolysis, lipemia and high bilirubin on laboratory tests; 57-65. 7. saputro da, junaidi s. giving the vitamin c on physical practice maximum and change the hemoglobin levels and erythrocyte count. 2015; 4(3): 32-40. 8. norsiah w. the difference of hemoglobin levels use sianmethemoglobin method with and without centrifugation on leucositosis. 2015; 1(2): 72-83. 9. praptomo, aj. quality control of medical laboratory. deepublish; 2018. 10. colvin, h. the breakdown on hemolyzed specimens. 2018; 14(1): 1-2. 58 identification of ascorbic acid content in carica papaya l. using iodimetry and uv-vis spectrophotometry nosy maria riscahyani1, evy ratnasari ekawati1, khoirul ngibad1 1department of medical laboratory technology, faculty of health science, maarif hasyim latif university, indonesia correspondence: khoirul ngibad, jl. raya ngelom megare no. 30, ngelom, taman, sidoarjo, east java, indonesia zip code : 61257 email: khoirul_ngibad@dosen.umaha.ac.id received: july 10, 2019 revised: julay 30, 2019 accepted: october 15, 2019 abstract ascorbic acid mostly comes from vegetables and fruits, especially fresh fruits. vitamin c is a vitamin that can be formed by several types of plants. one of them is papaya which has various contents including vitamin c that can increase endurance, help skin rejuvenation and repair body tissues. the purpose of this study was to determine the levels of vitamin c contained in papaya using iodimetry and uv-vis spectrophotometry. the sample used in this study was papaya fruit. determination of vitamin c levels in papaya samples using the titration method bas done by adding sample filtrate with starch indicator then titrated with titrant i2 until the endpoint color of blue titration is formed. other hands, the determination of vitamin c levels in papaya samples using the uv-vis spectrophotometry method bas conducted by making an ascorbic acid calibration curve then the filtrated sample was added with h2so4 5% and ammonium molybdate reagent, then the absorbance of the sample was measured at 494 nm wavelength. the results of the determination of vitamin c levels using the iodimetry was 0.0147% and the results of the determination of vitamin c levels using the uv-vis spectrophotometry method was 0.1313%. in conclusion, vitamin c levels analyzed by using uv-vis spectrophotometry methods were greater than vitamin c levels analyzed by using the iodimetry method. keywords ascorbic acid, papaya, iodimetry, uv-vis spectrophotometry introduction papaya is a fruit that has good nutritional value. it can be used in the form of fresh fruit and processed products. papaya contains 1.0-1.5% of protein, 1.0-1.5% of vitamin a, and 69-71 mg (100 g)-1 of vitamin c. minerals that are contained in papaya including 11-31 mg (100 g)-1 of calcium, and 39-337 mg (100 g)-1 of potassium. the other vitamin content in papaya fruit is fat which is 0.1%, 7-13% of carbohydrate. meanhwile, the enrgy content in papaya is 200 kj and 85 ina j med lab sci tech 2019; 1(1): 58-64 nosy maria riscahyani, et al. 5 9 90% of water. the vitamin c can increase the body resistance and help in body skin and tissue rejuvenation (1). deficiency of vitamin c can can affect bone strength, chronic energy deficiency, weakness, depression and increase risk of various disorders (2). vitamin c is often present together with substances or other vitamins in food. foodstuffs contained vitamin c are mainly fruits and vegetables (3). one of the fruit that contains of many antioxidant compounds such as carotenoids, is papaya fruit. ripe papaya has betacarotene which is 276 micrograms per 100 gram, 761 micrograms per 100 gram of betacryptoxanthin, and 75 micrograms per 100 g lutein and zeaxanthin. the betacarotene is provitamin a and also act as an effective antioxidant to ward off free radical attacks (4). there are several methods developed for the determination of vitamin c level including the uv-vis spectrophotometry method (5) and the iodimetry method (6). the spectrophotometric method can be used to determine the mixture content with an overlapping spectrum without prior separation, because the software is easy to use for analysis and is microcomputer instrumentation. spectrophotometry is widely used in various fields of chemical analysis, especially pharmaceuticals. while, the iodimetry method was a simple method, which is easy to be applied in a research and can give good result in the measurement of analyte content (7). the reaction of the iodimetry method between vitamin c and amilum was the double bond that is added by iodine that will break into a single bond. if all of the vitamin c has been added to iodine, the iodine which drops during titration will react with the starch indicator solution to form blue iod-amilum. the formation of blue color indicated that the titration process has been completed, because all of vitamin c has been added to iodine so that the volume of iodine needed during titration was equivalent to the amount of vitamin c. the titration treatment must be done quickly because there are many factors that cause oxidation of vitamin c, for example, it occurs when sample preparation. this is because vitamin c easily reacts with o2 in the air to become dehydroascorbic acid (8). materials and methods material the materials included california papaya, aquadest, starch solution, iodine 0.01 n solution, ki solution, h2so4 solution, sodium thiosulfate, ascorbic acid, oxalic acid, and ammonium molybdate. equipment the equipments included glass equipments, erlenmeyer flask, volumetric ina j med lab sci tech 2019; 1(2): 58-64 nosy maria riscahyani, et al. 6 0 flask, burette, statif, clamp, analytic balance and uv-vis spectofotometry. titrimetric method standardization of 0.01 n sodium thiosulfate as much as 10 ml of kio3 was taken with volume pipette, then it was poured into 250 ml erlenmeyer. then, it was added with 10 ml of 10% ki and 2 ml of 2 n h2so4 . then, it was titrated with sodium thiosulfate solution until the solution was formed, indicated by light yellow. then it was added with 1 ml of starch until the right blue color disappears. standardization of 0.01 n i2 as much as 10 ml of sodium thiosulfate was taken. then, it was added with 1 ml of starch. then, it was titrated with i2 solution until blue color. determination of vitamin c levels as much as 25 grams of sample was weighed and dissolved with distilled water. then, it was put into 100 ml volumetric flask and was added with distilled water to mark boundaries and was homogenized. as much of 10 ml of filtrate sample was taken and put it into erlenmeyer. then, the solution was added with 1 ml of starch and 10 ml of distilled water. the titration process using 0.01 n 12 standard solutions until blue color endpoint was formed. uv-vis spectrophotometry method preparation of 1000 ppm ascorbic acid stock solution as much as 25 mg of standard ascorbic acid was weighed carefully and bas put into 25 ml volumetric flask and was diluted with 0.4% oxalic acid until 25 ml. determination of maximum wavelength as much as 1 ml of 10 ppm ascorbic acid solution was pipetted and bas put into 25 ml volumetric flask. then, the solution was added with 10 ml of h4so4 and 5% ammonium molybdate until the mark limit and then it was homogenized. preparation of standard curve as much as 100 ppm of ascorbic acid solution was pipetted for 7 times, 0.5; 0.75; 1; 1.25; 1,5; 1.75 and 2 ml. each of the solution was put into 25 ml volumetric flask and then it was added with 5 ml of 10% h2so4 and the volume was sufficient with 5% ammonium molybdate until the mark limit and then it was homogenized. from this procedure, concentration of 0.2 will be obtained; 0.3; 0.4; 0.5; 0.6; 0.7 and 0.8 ppm. then, incubation process for 30 minutes was done. the absorption was measured by uv-visible spectrophotometry at wavelength of 494 nm and linear regression equation was determined. determination of sample content using uv-vis spectrophotometry method papaya fruit was peeled and the seed was removed and it was cut into small pieces and then it was crushed in blender until resemble ina j med lab sci tech 2019; 1(1): 58-64 nosy maria riscahyani, et al. 6 1 slurry (juice). samples that have been blended was weighed as much as 10 gram and was put into a100 ml beaker glass. then, it was added with distilled water and filtered by using filter cloth to separate residue and filtrate. the filtrate was added to 100 ml volumetric flask and was added with distilled water to the mark mark. the filtrate obtained was ready to be used as samples in this research. then, it was pipetted 2,5 ml of filtrate and was put into 25 ml volumetric flask. the solution was added with 4 ml of 5% h2so4 and the volume was sufficient with 5% ammonium molybdate to the limit and then incubated for 30 minutes and measured using spectrophotometry at wavelength of 494 nm (9). results the results of determination of vitamin c levels in carica papaya l. samples using titrimetric method can be seen in table 1. table 1. result of determination of vitamin c levels using titrimetric method description: n1 = 1 st test, n2 = 2 nd test, n3 = 3 rd test the calibration curve of vitamin c standard solutions of various concentrations in determination of vitamin c levels in carica papaya l. samples using uv-vis spectrophotometry method can be shown in figure 1. fig 1. the calibration curve of vitamin c standard solutions of various concentrations sample volume (ml) volume average (ml) vitamin c levels (%) sd %rsd n1 0.6 0.6 0.0147 0 0 n2 0.6 n3 0.6 ina j med lab sci tech 2019; 1(2): 58-64 nosy maria riscahyani, et al. 6 2 table 2 show the result of determination of ascorbic acid level in carica papaya l. samples using the uv-vis spectrophotometry method. table 2. results of ascorbic acid using the uv-vis spectrophotometry method description: n1 = 1 st test, n2 = 2 nd test, n3 = 3 rd test discussion in previous research, determination of vitamin c levels was done by using iodimetry method with amilum as an indicator and iodine as standard solutions (6). the results can be seen in table 1. the principle of this measurement is vitamin c (ascorbic acid) as a strong reducing agent and can simply be titrated with iodine standard solution. in the results of the study using the iodimetry method, iodine standardization was replicated three times. when standardization process of iodine, there was an error during the making of the solution so that the remodeling of the iodine solution was made with the results obtained as desired by 0.0071 n with a change in yellow and will turn to blue color immediately so that it can be used for titration. in the study, vitamin c that analyzed using the iodimetry method produced vitamin c levels of 0.0147 % with addition of sulfuric acid as an acidic giver. this titration treatment must be done immediately because many factors will cause oxidation of vitamin c, for example oxidation by air, especially when exposed to heat. the longer the fruit was stored in the open place (outdoor), the higher the chace of deacreasing in the vitamin c levels. vitamin c is easily reacts with o2 in the air to become dehydroascorbic acid (10) so that the vitamin c level will be lower. furthermore, the analysis of vitamin c on papaya was done by using uv-vis spectrophotometry. this method was used to test the amount of light absorbed at wavelength in the ultraviolet region so that the light of ray will be splited directly through transparent cells containing solvents, then the light will pass through compounds in the analyte to be absorbed and recorded as absorbance. analysis of vitamin c levels using uvvis spectrophotometry method begins by determining the maximum wavelength and was obtained in the wavelength of 494 nm. when the sample preparation was done, oxalic acid was added to keep vitamin c in the sample to remain stable and as an inhibitor of the oxidation process. addition sample absorbance (a) vitamin c levels in 10 g of sampel (%) average (%) sd %rsd n1 0.062 0.130 0.1313 0.001 0.0088 n2 0.062 0.132 n3 0.062 0.132 ina j med lab sci tech 2019; 1(1): 58-64 nosy maria riscahyani, et al. 6 3 of ammonium molybdate reagents to form color so that the shifting wavelengths that occur will be easily observed at visible wavelengths. based on data (figure 1) from the measurement using uv-vis spectrophotometry method of a standard solution of vitamin c, it can be made the curve of relationship between absorbance and concentration vitamin c with linearity equation of y = 0.015x + 0.0133. based on table 2, it can be seen that the results of the analysis of vitamin c content in california papaya fruit using uv-vis spectrophotometry method was 0.1313% in 10 grams of fruit. incubation samples were analyzed for optimum reaction between vitamin c and ammonium molybdate to form a stable blue complex. based on this research, there are differences between the results of the determination of vitamin c using the iodimetry method and the uv-vis spectrophotometry method. the vitamin c content in papaya which measured using the iodimetry method was 0.0147% while the vitamin c content in papaya which measured using the uv-vis spectrophotometry method was 0.1313%. the result of uv-vis spectrophotometry method was higher than the result of uv-vis spectrophotometry method. based on the result, there are significant differences between the vitamin c level contained in papaya using the iodimetry method and uv-vis spectrophotometry method. the result was in line with the result of identification of vitamin c content in kiwifruit (actinidia deliciousa) by using iodimetry and uv-vis spectrophotometry method (11). it was because the uv-vis spectrophotometry method provided the simple way to measured very small quantity of substances and the number which read directly was recorded by the detector (6). conclusions based on the results obtained, it can be concluded that the vitamin c levels using the spectrophotometric method was 0.1313% in 10 gram samples which is nine times greater than the results of the determination of vitamin c using the iodimetry method (0.0147%). conflict of interest there are no conflicts of interest. references 1. k. suketi, r. poerwanto, s. sujiprihati, and w. d. widodo, “karakter fisik dan kimia buah pepaya pada stadia kematangan berbeda,” j. agron. indones. (indonesian j. agron., vol. 38, no. 1, 2010. 2. l. gillberg, a. d. ørskov, m. liu, l. b. s. harsløf, p. a. jones, and k. grønbæk, “vitamin c–a new player in regulation of the cancer epigenome,” in seminars in cancer biology, 2018, vol. 51, pp. 59–67. 3. p. aghajanian, s. hall, m. d. wongworawat, and s. mohan, “the roles and mechanisms of actions of vitamin c in bone: new developments,” j. bone miner. res., vol. 30, no. 11, pp. 1945–1955, 2015. 4. m. adib, “ekstrak buah pepaya (carica papaya l.) meningkatkan kadar catalase dan glutathione hati tikus yang terpaparlead acetate,” j. sainhealth, vol. 2, no. 1, pp. 8–12, 2018. ina j med lab sci tech 2019; 1(2): 58-64 nosy maria riscahyani, et al. 6 4 5. k. ngibad and d. herawati, “perbandingan pengukuran kadar vitamin c menggunakan spektrofotometri uv-vis pada panjang gelombang uv dan visible,” borneo j. med. lab. technol., vol. 1, no. 2, pp. 77–81, 2019. 6. k. ngibad and m. s. pradana, “effect of starch and sulfuric acid on determination of vitamin c in papaya fruit using iodimetri,” indones. j. med. lab. sci. technol., vol. 1, no. 1, pp. 15–21, 2019. 7. j. w. munson, analisis farmasi metode modern. parwa b. diterjemahkan oleh harjana. surabaya: airlangga university press, 1991. 8. n. rahman, m. ofika, and i. said, “analisis kadar vitamin c mangga gadung (mangifera sp) dan mangga golek (mangifera indica l) berdasarkan tingkat kematangan dengan menggunakan metode iodimetri,” j. akad. kim., vol. 4, no. 1, pp. 33–37, 2015. 9. m. tahir, a. t. kusuma, and e. ekawati, “analysis of lycopene and vitamin c levels of pomelo citrus fruit (citrus maxima (burm) merr) red n white varieties from south sulawesi,” jcps (journal curr. pharm. sci., vol. 2, no. 1, pp. 125–130, 2018. 10. f. g. winarno, food chemistry and nutrition. jakarta: gramedia, 1997. 11. e. mulyani, “perbandingan hasil penetapan kadar vitamin c pada buah kiwi (actinidia deliciousa) dengan menggunakan metode iodimetri dan spektrofotometri uv-vis,” pharmauho, vol. 3, no. 2, 2018. 52 evaluation of erythrocyte sedimentation rate (esr) of motorcycle workshop worker exposed to benzene moch. sahri1, maharani pertiwi koentjoro2, shamsul bahari shamsudin3 1department of occupational health and safety, faculty of health, nahdlatul ulama university of surabaya, indonesia 2department of medical laboratory technology, faculty of health, nahdlatul ulama university of surabaya, indonesia 3department of community & family medicine, faculty of medicine & health sciences, universiti malaysia sabah, malaysia correspondence: maharani pertiwi koentjoro, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code : 60237 email: maharani@unusa.ac.id received: september 9, 2019 revised: october 7, 2019 accepted: october 12, 2019 abstract occupational exposure to benzene from fuel oil in workshop motorcycle workshop causes several health effects, depending upon the level and duration of exposure. analysis of blood is important to access the status of worker’ health. the study aimed to assess the gather basic information required for protecting workers’ health and improve working conditions in the works sites by investigating the esr levels. a comparative cross-sectional study conducted in surabaya city, east java-indonesia, which involved 100 workers. the occupational data collected using a structured questionnaire while blood analysis parameters measured with an automated hematology analyzer. the result showed that leukocytes, erythrocytes, hemoglobin (hgb), hematocrit values (hct), platelet, mcv, rdw, eosinophil, basophils, neutrophil rods, neutrophil segment, lymphocyte, monocytes were in normal value. whereas, mch, mchc, esr has shown in abnormal value as average 24.5 pg, 29.18 g/dl and 8.21 mm/h, respectively. those value, especially in esr value indicated the increasing concentration plasma viscosity. this is may cause by inflammation in the workers’ body. to prevent the hazardous effect of benzene exposure, occupational health should be implemented for workers in order to protect them from exposure to benzene. keywords motorcycle workshop workers, esr, surabaya city, benzene introduction the level of benzene exposure in the workshop motorcycle workshop during operations may occur. mechanical workshop sites process various chemical products based on highly volatile and flammable liquid hydrocarbons (1, 2), namely premium, pertamax, solar which are the most widely ina j med lab sci tech 2019; 1(2): 52-57 moch. sahri, et al. 5 3 used types of fuel oil. it presents many dangers associated with toxic chemical materials, including carcinogens such as benzene. however, recent reports have indicated a link between occupational diseases such as cancer and benzene exposure in workshop site (2). workers in sites exposure to benzene are more risk in occupational health (1, 3). erythrocyte sedimentation rate (esr) is a blood test that measures a “sed rate”. this rate is depends on various factors, namely hemoglobin concentration, the ratio of plasma proteins, serum lipid concentration, and plasma ph. it has been a widely used marker for inflammation and indicates disease (5). in the occupational health of fuel oil, esr determination is an important part of critical and emergent diagnoses duc as acute and chronic lymphocytic leukemia; nonhodgkin’s lymphoma, multiple myeloma, anemia due to hematopoietic organs dysfunction, malignancy, temporal arteritis, renal disease, collagen vascular disease (6) (7). furthermore, the role of esr in the clinical management of various disease (6). routine tasks in the workshop motorcycle workshop include such as replacing spark plugs or changing the oil, repair engines and transmissions, removing dents from fenders. thus, evaluation of the effect of benzene exposure is one of occupational health requirement at workshop motorcycle workshop (8) . this study aims to gather basic information required for protecting workers’ health and improve working conditions during works sites by investigating the hematological parameters, especially in the esr levels. materials and methods study design a comparative cross-sectional study of workers conducted at a workshop motorcycle workshop. the study subject involved 100 occupationally workshop motorcycle workers exposed to fuel oil. all worker require hematological parameters evaluation, especially in esr levels. all workers were asked to give informed consent. data collection demographic and occupational data were collected using a structured questionnaire, and the subjects ade interviewed by the researcher. before the blood collection, resting blood pressure was measured by using sphygmomanometer. blood samples from worker respondents were taken at the end of the work shift. 4 ml of blood samples were collected using venipuncture techniques into a vacutainer tube containing potassium edta from the antecubital area of participants. the tubes were then placed in a holder or rack and placed/stored undisturbedly inin cold box. all samples were analyzed using an automated hematology analyzer horiba type abx micros 60 (kx-21n; sysmex, kobe, japan). ina j med lab sci tech 2019; 1(2): 52-57 moch. sahri, et al. 5 4 data processing and analysis all the data were cleaned, double entered into microsoft excel spreadsheets and analyzed using stata software version 13 (statacorp, college station, tx, usa). ethical considerations the proposal in this study was approved by the surabaya occupational safety and health technical implementation unit. all respondents in this study gave written agreements to participate in this study. all respondents were given an explanation of the purpose and results of this study. laboratory test results were given to respondents in this study. results general characteristics personal characteristics of the workshop motorcycle workers are presented in table 1 they were comparable with regard to age and sex. 91% of workers were males. the average age of the workers was 28 years. protective measures were poorly followed during work in workshop. table 1. personal charateristics of workshop motorcycle workers age group range percentage (%) age-group (years) 18-27 55 28-37 33 38-53 12 sex female 9 male 91 table 2. major activities of the workshop motorcycle workers job task mechanical engineer 1. dismantle engines and repair or replace defective parts, such as magnetos, carburetors, and generators. 2. remove cylinder heads, grind valves, and scrape off carbon, and replace defective valves, pistons, cylinders and rings, using hand tools and power tools. 3. hammer out dents and bends in frames, weld tears and breaks; then reassemble frames and reinstall engines. 4. repair or replace other parts, such as headlights, horns, handlebar controls, gasoline and oil tanks, starters, and mufflers. 5. repair and adjust motorcycle subassemblies such as forks, transmissions, brakes, and drive chains, according to specifications. ina j med lab sci tech 2019; 1(2): 52-57 moch. sahri, et al. 5 5 fig 1. partial view of working practice in of the workshop motorcycle. workers are engaged in routine activities without proper personal protective equipment (mask and gloves) based on this study, the possible routes of chemical entry into the body trough inhalation. it has shown in figure 1 that the workers did not wear a mask during workpractice table 3. blood analysis and hematological parameters of workshop motorcycle workers parameters average value normal (%) abnormal (%) leukocytes 8.53 x103/mm3 80 20 erythrocytes 5.07 x 106/mm3 93 7 hemoglobin (hgb) 12.51 g/dl 96 4 hematocrit values (hct) 43.26 % 98 2 platelet 298.96 91 9 mcv 84.36 µm³ 86 14 mch 24.5 pg 11 89 mchc 29.18 g/dl 7 93 rdw 13.62 % 96 4 eosinofil 1 % 100 0 basophils 0 % 100 0 natrofil rod 0.65 % 100 0 neutrophil segment 63.94 % 95 5 lymphocyte 30.99 % 98 2 monocytes 3.44 % 35 65 esr 8.21 mm/h 8 92 mcv (mean corpuscular volume); mch (mean corpuscular hemoglobin); mchc (mean corpuscular hemoglobin concentration); rdw (rbc distribution width); esr (erythrocyte sedimentation rate). discussion the results presented here are based on the study in workshop motorcycle worker to evaluate the esr value during benzene exposure in their worksites. workshop motorcycle workers are exposed to different chemicals of fuel oil in their workshop. studies have found that they can be exposed to numerous types of heavy metals duc as lead, chromium, and cadmium, including carcinogens such as benzena (1, 9). benzene was induced poisoning occurs almost entirely by inhalation of benzene vapors in the air. workshop motorcycle workers in our study exposed to benzene showed mean rbcs (average value is 5.07 106/mm3) and ina j med lab sci tech 2019; 1(2): 52-57 moch. sahri, et al. 5 6 hemoglobin (12.5 g/dl) were in the normal range. rbc indicates mcv, mch and mchc which are calculated from hemoglobin, hematocrit, and erythrocytes count. normal mcv indicates normocytic (normal average rbc size). mean cell hemoglobin concentration (mchc) refers to the average concentration of hemoglobin in the rbcs contained within the sample. abnormally low mchc in workshop motorcycle workers is called hypochromic. mean cell hemoglobin (mch) refers to the average weight of hemoglobin in the rbcs in the sample (units are picograms (pg/cell). among workshop motorcycle worker, the esr abnormal rate is as much as 92%. it is suggested that some inflammation reaction in the body (9). the esr value indicated the increasing concentration of fibrinogen, the main clotting protein and alpha globulin (10). it leads to plasma viscosity increased and red blood cell start to aggregate (10, 11). we assumed that raised esr is observed in workshop motorcycle workers caused by benzene in fuel oil exposure. it is well known that benzene causes haematological toxins, such as aplastic anemia and leukemia. recent studies demonstrated that low-level of benzene exposure (<1 ppm) has potential in hepatotoxicity (12). benzene is slightly soluble in water. hence, this is easy to circulating blood enters the tissue and fat tissue. the primary route of benzene exposure is entering the body from inhalation (13). this is caused by benzena which is very easy to evaporate into the air very quickly. benzene will enter into the bloodstream, and travel into the entire organ. in temporally, benzene can be stored in the bone marrow and fat. the metabolism benzene includes in oxidations of the benzene rings by cytochrome p450 monooxygenase. this enzyme is primarily produced in liver cells. therefore, accumulation of benzene in long periodic could affect liver function. normally, benzene is degraded into phenol (14). further, phenol is absorbed into the blood and distributed to other tissue. short term exposure to fuel oil, especially in benzene results in neurotoxic, irritatory, reproductive and developmental effects (15). long-term exposure to benzene may cause a hazardous effect on health, such as hematopoietic cancer. evaluation of hematological parameters, including esr is useful for monitoring the exposure of workshop motorcycle workers. this exposure carries a serious risk. therefore, it is important to keel fuel oil securely from motorcycle workshop workers. this study has some limitations. we evaluated the hematological parameters, namely esr in workshop motorcycle workers exposed to fuel oil. we did not measure the specific chemicals to which the workshop motorcycle workers were exposed. it would be better to explore the effect of types of occupational chemical exposure on ina j med lab sci tech 2019; 1(2): 52-57 moch. sahri, et al. 5 7 health in future studies. conclusions benzene contamination in the air may cause reduced mch and mchc level and esr. it was possible that the workers’ exposure to benzene in fuel oil during work. the advice that can be given in this research is to reduce benzene exposure should install exhaust ventilation and workers using personal protective equipment such as gas masks and gloves. acknowladgements we would like to acknowledge all the workshop motorcycle workers who were willing to be respondents in this study. conflict of interest there are no conflicts of interest. references 1. chung e-k, shin j-a, lee b-k, kwon j, lee n, chung k-j, et al. characteristics of occupational exposure to benzene during turnaround in the petrochemical industries. saf health work. 2010 sep;1(1):51–60. 2. kamal a, rashid a. benzene exposure among auto-repair workers from workplace ambience: a pioneer study from pakistan. int j occup med environ health. 2014 oct;27(5):830–9. 3. chaiklieng s, suggaravetsiri p, autrup h. risk assessment on benzene exposure among gasoline station workers. int j environ res public health. 2019 jul 16;16(14). 4. hashemi r, majidi a, motamed h, amini a, najari f, tabatabaey a. erythrocyte sedimentation rate measurement using as a rapid alternative to the westergren method. :5. 5. siemons l, ten klooster pm, vonkeman he, van riel pl, glas ca, van de laar ma. how age and sex affect the erythrocyte sedimentation rate and c-reactive protein in early rheumatoid arthritis. bmc musculoskelet disord. 2014 dec;15(1):368. 6. bray c, bell ln, liang h, haykal r, kaiksow f, mazza jj, et al. erythrocyte sedimentation rate and c-reactive protein measurements and their relevance in clinical medicine. 2016;115(6):6. 7. salehiforouz b, vahdati a, malekirad aa, edalatmanesh ma. evaluaton of hematological indices of workers exposed to benzene. 2017;15:41–9. 8. tualeka ar, salesman f, jalaluddin j, wahyu a. safe concentration of benzene exposure in work environment at motor workshop. glob j health sci. 2018 oct 25;10(11):p188. 9. salemcity a. toxicological assessment of consumption of benzene-polluted water on the heart and blood of albino rats. [cited 2019 aug 28]; available from: https://www.academia.edu/6439376/toxicologi cal_assessment_of_consumption_of_benzene -polluted_water_on_the_heart_and_blood_ of_albino_rats 10. harrison m. erythrocyte sedimentation rate and c-reactive protein. aust prescr. 2015 jun;38(3):93–4. 11. yin w, xu z, sheng j, xie x, zhang c. erythrocyte sedimentation rate and fibrinogen concentration of whole blood influences the cellular composition of platelet-rich plasma obtained from centrifugation methods. exp ther med. 2017 sep;14(3):1909–18. 12. koh d-h, jeon h-k, lee s-g, ryu h-w. the relationship between low-level benzene exposure and blood cell counts in korean workers. occup environ med. 2015 jun;72(6):421–7. 13. brekalo-lazarevic s, begic a, ademovic z, horozic e. determination of benzene metabolite phenol in the urine and analysis of blood parameters of workers exposed to benzene. :6. 14. brekalo-lazarevic s, begic a, ademovic z, horozic e. determination of benzene metabolite phenol in the urine and analysis of blood parameters of workers exposed to benzene. :6. 15. tuomi t, veijalainen h, santonen t. managing exposure to benzene and total petroleum hydrocarbons at two oil refineries 1977-2014. int j environ res public health. 2018 24;15(2). 15 effect of starch and sulfuric acid on determination of vitamin c in papaya fruit using iodimetri khoirul ngibad1, m. sungging pradana1, ingrid retno y. 1 1department of medical laboratory technology, faculty of health science, universitas maarif hasyim latif, sidoarjo, indonesia correspondence: khoirul ngibad, jl. raya ngelom megare no. 30, ngelom, taman, sidoarjo, east java, indonesia zip code : 61257 email: khoirul_ngibad@dosen.umaha.ac.id received: february 11, 2019 revised: march 16, 2019 accepted: march 26, 2019 abstract vitamin c is an antioxidant that can be used to inactivate oxidation reactions and prevent the formation of free radicals. sources of vitamin c are fruits, such as papaya fruit. the purpose of this study was to determine the effect of the indicator volume of 1% starch and 2 n sulfuric acid on the determination of vitamin c in papaya fruit samples. this study used the iodimetri method with a standard iodine solution, starch indicator and the addition of sulfuric acid. the variations of starch indicator volume include: 0.25; 0.5, 1, 2 and 3 ml and the variations of sulfuric acid volume include: 0, 2, 4, 6, and 8 ml. the results showed that the optimum 1% starch indicator volume was 1 ml and the optimum volume of 2 n sulfuric acid was 2 ml. keywords vitamin c, sulfuric acid, starch, iodimetri. introduction one of the vitamins needed by humans is vitamin c, also known as l–ascorbic acid. vitamin c is a chemical compound with formula c6h8o6. vitamin c is an antioxidant, which is a compound that can act to inactivate oxidation reactions and prevent the formation of free radicals. on the other hand, vitamin c also can act as a calogen–forming compound (1). consumption of vitamin c that is not sufficient enough can cause the vitamin c deficiency. in contrast, excess of vitamin c intake in adolescents will also cause problems in the gastrointestinal system (2). recommended daily allowance (rda) is ina j med lab sci tech 2019; 1(1): 15-21 khoirul ngibad, et al. 1 6 the basis for adjusting the amount of adequate vitamins per day. the intake of at least 10 mg of vitamin c per day can prevent the occurrence of scurvy deficiency disease. according to the regulation of the minister of health of the republic of indonesia no. 75 in 2013 concerning the recommended nutritional adequacy rate for the indonesian nation reveals that vitamin c is needed every day at least 40 to 50 mg (infants under 1 year), 40 mg (ages 1 – 3 years), 45 mg (ages 4 – 6 years), 45 to 50 mg (ages 7 –12 years), 100 mg (pregnant women) and 150 mg (breastfeeding mothers) (3). sources of vitamin c are derived from fruits such as guava fruit, raisins, kiwi, lychees, oranges, persimmon, lime, papaya, strawberries, lemons, pineapples, melons, mangoes, star fruit, grapes, passion fruit, breadfruit, durian, avocado, jackfruit, pomegranate, banana, and watermelon. determination of vitamin c in food and beverage packaging samples can be determined using the titrimetric method (4– 7) and spectrophotometry (8–10). among the two methods, the titrimetric method has the advantage of not requiring standard analyte solutions, simple equipment, easy to do and its accuracy is high. determination of vitamin c used the titrimetric method based on iodimetri, namely vitamin c will be oxidized by iodine standard solution and the endpoint of titration was indicated by the formation of blue color with the addition of starch indicators. the accuracy in determination of vitamin c was influenced by several measurement parameters, such as: volume of starch indicator and sulfuric acid used for the titration process. some studies used variations of starch volume indicators: 2 drops (2), 3 drops (11), 2 ml (4), a few drop (3,12), and 3 ml (1). on the other hand, there are several studies that used the addition of sulfuric acid in the titration process (1,3,12). the uncertainty in the use of volumes of starch and sulfuric acid is feared to affect the accuracy in determining vitamin c levels in food and beverage ingredients. thus, this study will know the effect of volume of the 1% starch indicator and 2 n sulfuric acid on the determination of vitamin c in samples of papaya fruit using iodimetri. materials and methods material the materials used include carica papaya l. (papaya fruit), 1% starch indicator, 0.01 n iodine standard solution, distilled water, filter paper, label paper, 0.01 n na2s2o3, 0.01 n kio3, 10% ki, and h2so4. equipment the equipments used include beaker glass, erlenmeyer flask, volumetric flask, burette, statif, clamp, volume pipette, measuring pipette, scissors, knife, blender, spatula, and analytic balance. ina j med lab sci tech 2019; 1(1): 15-7 21 khoirul ngibad, et al. 1 7 procedure na2s2o3 standard as much as 10 ml of 0.1 n kio3 solution was taken and then was put into the erlenmeyer flask. then, as much 5 ml of 10% ki solution and 2 ml of h2so4 was added into the flask. then, solution was titrated using the na2s2o3 solution until a light yellow color was formed. next, mixtured was added with 0.5 ml of 1% starch indicator and was titrated with na2s2o3 solution until a blue color disappears (12). i2 standard as much as 10 ml of i2 solution was taken into the erlenmeyer flask. furthermore, solutions was titrated using na2s2o3 solution to form a light yellow color. next, mixtured was added with 0.5 ml of 1% starch indicator. then, mixtures was titrated with a na2s2o3 solution until a blue color disappears (12). preparation of sample papaya fruit was shelled and the seed was removed. then, it was cut into small pieces and was weighed as much as 100 grams. furthermore, was blended until it resembles a slurry (juice). then, mixtures was filtered with a filter cloth to separate between residue and filtrate. the residue was removed and the filtrate was put into 1000 ml volumetric flask and added with distilled water to the boundary mark. the filtrate obtained is ready to be measured using the iodimetri method. effect of 1% starch indicator volume on the determination of vitamin c in papaya fruit as much as 25 ml of papaya sample solution was taken and put into erlenmeyer flask. then, solution was added by indicator solution of 1% starch with volume variation of 0.25; 0.5, 1, 2 and 3 ml. then, mixtures was titrated with i2 standard solution until a blue color was formed. from this treatment, the optimum volume of starch indicator will be obtained. effect of addition of 2 n sulfuric acid on determination of vitamin c in papaya fruit as much as 25 ml of papaya sample solution were taken and put into erlenmeyer flask. then, as much as 1 ml of 1% starch indicator solution was added. then, as much as 2 n h2so4 solution were added with volume variations include 0, 2, 4, 6, and 8 ml. then, mixtures were titrated with i2 solution standard until a blue color was formed. from this treatment, the optimum volume of sulfuric acid will be obtained. results effect of 1% starch vitamin c levels in papaya fruit are tabulated in table 1 and fig.2. the obtained results revealed that papaya fruit contain maximum amount of vitamin c that is 12.91 mg/ml. ina j med lab sci tech 2019; 1(1): 15-21 khoirul ngibad, et al. 1 8 table 1. effect of 1% starch indicator volume on determination the vitamin c levels in papaya fruit indicator volume (ml) titrant volume (ml) vitamin c levels (mg) n1 n2 n3 average standard deviation n1 n2 n3 average standard deviation 0,25 1.1 0.9 1.1 1.03 0.12 9.68 7.92 9.68 9.09 1.02 0,5 0.6 1.1 0.9 0.87 0.25 5.28 9.68 7.92 7.63 2.21 1 1.4 1.5 1.5 1.47 0.06 12.32 13.2 13.2 12.91 0.51 2 0.9 1 1 0.97 0.06 7.92 8.8 8.8 8.51 0.51 3 1.4 1 0.5 0.97 0.45 12.32 8.8 4.4 8.51 3.97 description: n1 = test 1, n2 = test 2, n3 = test 3 fig 1. effect of 1% starch indicator volume on determination the vitamin c levels in papaya in order to established methods for determination of vitamin c in papaya fruit, sulfuric acid was used in treatment (fig.2 and table 2). after incubation with sulfuric acid, a colored product is formed which is in a blue color. fig 2. effect of sulfuric acid addition on the determination of vitamin c levels in papaya 0 2 4 6 8 10 12 14 16 0 0,5 1 1,5 2 2,5 3 3,5 v it a m in c l e v e ls ( m g ) 1% starch indicator volume (ml) v it a m in c l e v e ls ( m g ) sulfuric acid volume 2n (ml) ina j med lab sci tech 2019; 1(1): 15-7 21 khoirul ngibad, et al. 1 9 table 2. effect of volume of 2 n sulfuric acid on determination of vitamin c levels in papaya volume of sulfuric acid (ml) titrant volume (ml) levels of vitamin c (mg) n1 n2 n3 ave rage standard deviation n1 n2 n3 ave rage standard deviation 0 0.8 1 1 0.93 0.12 7.04 8.8 8.8 8.21 1.02 2 0.8 0.9 0.9 0.87 0.06 7.04 7.92 7.92 7.63 0.51 4 0.8 0.6 0.8 0.73 0.12 7.04 5.28 7.04 6.45 1.02 6 0.8 0.8 0.8 0.8 0 7.04 7.04 7.04 7.04 0 8 0.8 0.8 0.8 0.8 0 7.04 7.04 7.04 7.04 0 description: n1 = test 1, n2 = test 2, n3 = test 3 discussion the principle of determination the vitamin c contents using the iodimetri method is based on the reaction between vitamin c which is a strong reducing agent and standard solution of iodine which is an oxidizer with starch indicator. in this study, vitamin c in papaya fruit samples react with iodine standard solution so that an addition reaction occurs between iodine and double bonds of vitamin c on number 2 and 3 of carbon atoms become a single bond. if the whole vitamin c has been supplemented by standard iodine solution, then a reaction occurs between the droplets of the next iodine solution and the starch indicator solution so that iod–amilum was formed. the end point of the titration was indicated by the presence of blue color indicating that the titration has been completed because all vitamin c has been supplemented by iodine (5). reactions that occur can be seen in figure 3. fig 3. the reaction between vitamin c in papaya fruit with iodine produced the dehydroascorbic acid effect of 1% starch indicator volume on determination of vitamin c in papaya in this study, the effect of 1% starch indicator volume on the determination of vitamin c levels in papaya fruit was studied using iodimetric titration. in the process of titration, the addition of an indicator serves to show that the endpoint of the titration has occurred so that the titration process must be stopped immediately. the addition of incorrect volume indicators is feared to affect the accuracy of the measurement of vitamin c levels in a sample. ina j med lab sci tech 2019; 1(1): 15-21 khoirul ngibad, et al. 2 0 table 1 and figure 1 showed that the addition of the indicator volume by 1 ml resulted the optimum vitamin c levels of 12.91 mg with the smallest standard deviation of 0.51. on the other hand, the addition of volume indicators of 1% starch as much as 2 and 3 ml decreased the vitamin c levels and increased the standard deviation value for addition the volume indicator of 1% starch by 3 ml. this is probably caused by the excess volume of 1% starch indicator, the less the volume of iodine standard solution needed so that the vitamin c levels in the sample reduced. thus, the optimum volume of 1% starch indicator in determination the vitamin c in papaya fruit was 1 ml. the effect of addition of 2 n sulfuric acid on determination the vitamin c levels in papaya this study also was studied the effect of addition of 2 n sulfuric acid on determination of vitamin c levels in fruit papaya using iodimetri. in the process of titration, the addition of sulfuric acid give an acidic condition in the solution of vitamin c when the titration process occurs with iodine standard solution. the effect of 2 n sulfuric acid on determination of vitamin c in papaya fruit was studied with 2 n sulfuric acid volume variations of 0, 2, 4, 6 and 8 ml. based on table 2 and figure 3, we can calculated that the addition of 2 ml of sulfuric acid volume produced the optimum vitamin c levels of 7.63 mg with standard deviation of 0.51. on the other hand, the more volume of 2 n sulfuric acid added caused slight decrease in fluctuating of vitamin c levels. in general, the standard deviation value in the study of the effect of sulfuric acid volume on determination of vitamin c levels in papaya fruit was smaller than the study of the effect of 1% starch indicator volume on the determination the vitamin c levels in papaya. it was also supported by visual observations in the laboratory proving that the color yield of the titration end point of all sulfuric acid addition treatments produced more stable color. this shows that at low ph vitamin c is more stable than high ph. thus, the optimum volume of 2 n sulfuric acid on determination of vitamin c levels in papaya fruit was 2 ml. conclusions the addition of 1% starch indicator and 2 n sulfuric acid volume had an effect on the determination of vitamin c levels in papaya fruit. the optimum 1% starch indicator volume obtained was 1 ml and the optimum volume of sulfuric acid 2 n was 2 ml. conflict of interest no potential conflict of interest. ina j med lab sci tech 2019; 1(1): 15-7 21 khoirul ngibad, et al. 2 1 references 1. mulyani e. perbandingan hasil penetapan kadar vitamin c pada buah kiwi (actinidia deliciousa) dengan menggunakan metode iodimetri dan spektrofotometri uv–vis. pharmauho, vol. 3, no. 2, 2018. 2. siti n, agustina a, and nurhaini r. penetapan kadar vitamin c pada jerami nangka (artocarpus heterpophyllus l.). j. farm. sains dan prakt., vol. 2, no. 1, pp. 1–5, 2016. 3. damayanti et and kurniawati p. perbandingan metode penentuan vitamin c pada minuman kemasan menggunakan metode spektrofotometer uv–vis dan iodimetri. 2017. 4. rahmawati f and hana c. penetapan kadar vitamin c pada bawang putih (allium sativum, l) dengan metode iodimetri,” cerata j. ilmu farm. (journal pharm. sci., vol. 4, no. 1, 2016. 5. rahman n, ofika m, and said i. analisis kadar vitamin c mangga gadung (mangifera sp) dan mangga golek (mangifera indica l) berdasarkan tingkat kematangan dengan menggunakan metode iodimetri. j. akad. kim., vol. 4, no. 1, pp. 33–37, 2015. 6. najwa fr and azrina a. comparison of vitamin c content in citrus fruits by titration and high performance liquid chromatography (hplc) methods. int. food res. j., vol. 24, no. 2, p. 726, 2017. 7. suntornsuk l, gritsanapun w, nilkamhank s, and paochom a. quantitation of vitamin c content in herbal juice using direct titration. j. pharm. biomed. anal., vol. 28, no. 5, pp. 849–855, 2002. 8. buhari i. analisis kadar vitamin c dalam produk olahan buah salak (salacca zalacca) secara spektrofotometri uv–vis. universitas islam negeri alauddin makassar, 2010. 9. raghu v, platel k, and srinivasan k. comparison of ascorbic acid content of emblica officinalis fruits determined by different analytical methods. j. food compos. anal., vol. 20, no. 6, pp. 529– 533, 2007. 10. khan mmr, rahman mm, islam ms, and begum sa. a simple uv–spectrophotometric method for the determination of vitamin c content in various fruits and vegetables at sylhet area in bangladesh. j. biol. sci., vol. 6, no. 2, pp. 388– 392, 2006. 11. rahim a and alimuddin a. analisis kandungan asam askorbat dalam buah naga merah (hylocereus polyrhizus) dengan iodimetri. j. kim. mulawarman, vol. 14, no. 1, 2016. 12. karinda m, fatimawali f, and citraningtyas g. perbandingan hasil penetapan kadar vitamin c mangga dodol dengan menggunakan metode spektrofotometri uv–vis dan iodometri. pharmacon, vol. 2, no. 1, pp. 86–89, 2013 38 the effect of test tube sterilization from serum lipemic against level of triglyceride gpo-pap method fitri fadhilah1, ana bina sari1, astika apriliani1 1department of medical laboratory technology, sekolah tinggi analis bakti asih, bandung, indonesia correspondence: fitri fadhilah, jl. padasuka atas no.233, padasuka, cimenyan, city of bandung, west java, indonesia zip code : 40192 email: fitrifadhilahssimkes@gmail.com received: february 8, 2019 revised: march 25, 2019 accepted: march 26, 2019 abstract in terms of analytic factors, it is important to define acceptable levels of common interferences, such as lipemia or hemolysis. for triglyceride, the laboratory technician must define whether samples with excess lipemia will be included in the study; this depends, in part, on whether the interferences affect the methods. in most laboratories, glass or plastic that is in direct contact associated with bio hazardous material is usually disposable. if not, it must be decontaminated according to appropriate protocols. immediately rinsing glass or plastic supplies after use, followed by washing with a powder or liquid detergent designed for cleaning laboratory supplies and several distilled water rinses, may be insufficient. to ensure that all remaining fat from lipemic serum that attached to the tube wall has been removed, then the sterilization process is carried out so that a sterile tube is obtained. the purpose of this study is to determine the effect of test tube sterilization from serum lipemic against levels of triglyceride gpopap (glycerol-3-phosphate oxidase-p-aminophenazone) examination. this research method was a laboratory experiment. we used 8 times repetition with tubes used first are given liquid fat and cleaned by sterilization, washed with surfactant and washed with water only. by using statistical tests anova obtained of this study showed results p>0.05 which is mean the treatment that used did not show a significant difference in the treatment of ordinary water-washed tubes with sterilized tubes and surfactant washed tubes. the conclusion of this study is cleaning of the test tube with the sterilization method is recommended because to avoiding the fear of remaining pollutants that can affect the results, it can also minimize the life of bacteria and viruses from the sample to be examined. however, if the sterilization method is difficult to do because of limited equipment and so on, the use of surfactants and the correct method of cleaning the tube is enough to remove impurities such as fat. keywords tube sterilization, serum lipemics, triglyceride ina j med lab sci tech 2019; 1(1): 38-43 fitri fadhilah, et al. 3 9 introduction pre-analytic refers to all steps that must be taken before the sample is analyzed. over the years, a series of studies have shown that 61% of all testing errors occur in the pre-analytic phase. meanwhile, along with technological advances and procedures for quality assurance, it has significantly reduced the number of analytical errors (1,2). potential sources of errors or failures in the pre-analytic process in the laboratory include the type of test requested, identification of samples, improper time, improper fasting, incorrect type and comparison of anticoagulation with blood, improper mixing, appropriate equipment, and hemolysis or lipemic specimens (2). lipemic serums provide additional challenges in laboratory analysis. the lipemic serum is a cloudy or milky serum (cloudy white). this condition is mainly due to increased levels of fat in the blood. some chemical tests can be carried out on lipemic specimens due to turbidity and disrupting testing procedures (3). other pre-analytic that must be considered is the cleanliness of the tools used. the cleanliness of the tool can affect the results of the examination. if the tool to be used is not clean, things will not be desired. for example, if there are chemicals, fats, or impurities remaining on these devices, then the substance can react with the substances we use afterwards and can result in failures in the examination (3). equipment in laboratory examinations in general must be clean, dry conditions do not contain ingredients that change substances in the sample, and easily washed from former specimens. in some clinical laboratories glass test tubes was used for clinical examination, which will be read on a spectrophotometer, which is a component that will be passed by light. in laboratory test tubes glass is used repeatedly, but sometimes the procedure for treating glass test tubes is not done properly and correctly (4,5). sterilization is a process in which this activity aims to free tools or materials from various types of microorganisms. a material should be steriled if it is free from living microorganisms that are pathogenic or not, both in vegetative form and nonvegetative forms (spores) (6). materials and methods experimental research methods have various types of designs, the experimental method in this study uses the design type one group pretest-post test design. we used as 8 times repetition with tubes used first are given liquid fat and cleaned by sterilization, washed with surfactant and washed with water only, which is a ina j med lab sci tech 2019; 1(1): 38-43 fitri fadhilah, et al. 4 0 laboratory tests to determine the effect of sterilization of reaction tubes from lipemic serums on triglyceride levels in the gpopap method. the sample in this study were the serum of the officers of the citama bojong gede hospital laboratory, bogor. the research was conducted at the laboratory of citama bojong gede hospital, bogor. the time of the study conducted in august until september 2018. results the results of triglyceride levels were examined in serum and worked using a test tube that had been cleaned by sterilization, washed with surfactant, and washed with water only were shown at figure 1. triglyceride examination was carried out with 3 treatments. each uses 8 different samples, namely using a sterile tube obtained an average yield of 127, a minimum result of 95 and a maximum yield of 183, a tube washed with surfactant obtained an average yield of 149.75, a minimum yield of 109 and a maximum yield of 202, the tube is washed with water just got an average yield of 198.13, a minimum yield of 153 and a maximum yield of 254. in shapiro-wilk test, it was obtained significant results of 0.359 on examination with sterile tubes, 0.813 on inspection with surfactant washing tubes, and 0.811 on inspection with ordinary water wash tubes. fig 1. graph of triglyceride level results 0 50 100 150 200 250 300 1 2 3 4 5 6 7 8 t ri g ly c e ri d e c o n c e n tr a ti o n ( m g /d l ) repetition triglyceride level sterilization surfactant water ina j med lab sci tech 2019; 1(1): 38-43 fitri fadhilah, et al. 4 1 because of the three significance results were >0.05 (95% confidence level where 5% or 0.05 is the error limit that is still accepted) then the data was declaring to be normally distributed. in this homogeneity test, it was obtained the results of 0.150 with a significance of 0.862, so that the data can be declared homogeneous because the significance results of 0.862>0.05. because of the results of the data were normal and homogeneous followed by further statistical tests. in anova test, it was obtained significant results of 0.000. in addition, a confidence level of 95% the results of the significance of 0.000<0.05, so that the conclusions obtained there were significant differences. because the results show that there was a significant difference, further testing followed it, namely the post hoc test. the results of the post hoc test on the treatment of sterile tubes with washing tubes plus surfactants were obtained at an average difference of -22.750, and a significant result of 0.461. from these data the significance of 0.461>0.05, the results did not show a significant difference. the results of the post hoc test on the treatment of sterile tubes with normal water wash tubes obtained results of an average difference of -71.125 and a significance result of 0.000. from these data the significance of 0.000<0.05, the results show there were a significant differences. the results of the post hoc tests on the treatment of washing tubes plus surfactants with sterile tubes obtained results of an average difference of 22.750 and a significant result of 0.461. from these data the significance of 0.461> 0.05, the results showed no significant differences. the post hoc test results on the treatment of washing tubes plus surfactants with ordinary water wash tubes obtained results of an average difference of -48.375 and a significant result of 0.015. from these data the significance of 0.015 <0.05, the results showed a significant difference. the results of the post hoc test on the treatment of ordinary water wash tubes with sterile tubes obtained the results of an average difference of 71.125 and a significance result of 0.000. from these data the significance of 0.000<0.05, the results showed a significant difference. the results of the post hoc test on the treatment of ordinary water wash tubes with washing tubes plus surfactants obtained results of an average difference of 48.375 and a significant result of 0.015. from these data the significance of 0.015<0.05, the results showed a significant difference. discussion after the study examined triglycerides using a tube that had been given liquid fat and then washed and handling with three methods the first sterilized tube, washed with surfactants, and the last washed using water ina j med lab sci tech 2019; 1(1): 38-43 fitri fadhilah, et al. 4 2 only. triglyceride examination was performed on a spectrophotometer using 8 samples. in this study on sterilized tubes and tubes equipped with surfactants the results were not much different, whereas in tubes equipped with air the results were far different from those of the sterilized tubes. this shows the cleanliness of the tool that can produce the results of the inspection. if the tool to be used is not clean, things might not be desired. for example, if there are chemicals, fats, or impurities remaining on these tools, then these substances can be considered with substances that we can use and can be returned to the lab (7,8). by performing statistical data, the test results should the average number using sterile tubes obtained an average yield of 127.00, the tubes connecting with surfactants obtain an average yield of 149.75, the tubes carry with air only obtained an average yield of 198.13. then proceed with the normality test, using the shapiro-wilk test because the sample that passes is less than 50. the data obtained from the shapiro wilk test results in a significance of 0.359 on examination with a sterile tube, 0.813 on examination with a surfactant washing tube, and 0.811 when receiving a washing tube ordinary water. because the results of the three significance >0.05 (95% confidence degree while 5% or 0.05 is the error limit that is still accepted) then the approved data is normally distributed. furthermore, the homogeneity test could be declared homogeneous because the significance results are 0.862>0.05 (9). since the results of normal and homogeneous data are carried out by the next statistical test which is using anova test. in this anova test obtained significant results of 0,000. so with a confidence level of 95% the results of the significance of 0.000<0.05, so that the conclusions obtained are significant differences (9). because the results showed a significant difference then continued with the further test namely the post hoc test. in the post hoc test the results obtained did not show a significant difference in the sterilized tubes with tubes equipped with surfactants, then the packaging tubes were added with a sterile surfactant. while for the results that show a significant difference from the results of a sterile tube with an air wash tube, then on the treatment tube plus surfactant with a normal air wash tube, and on a normal tube air wash tube with a sterile tube, also a normal air washing tube with a washing tube plus surfactant (10). this shows that the release of the test tube with the sterilization method is highly recommended because it could be avoided that the remaining impurities that are feared to affect the results can also minimize viruses and bacteria from the samples to be discussed. however, if the sterilization method is difficult because of the limitations of the tools and so on, the use of surfactants ina j med lab sci tech 2019; 1(1): 38-43 fitri fadhilah, et al. 4 3 and how to store the tubes is really enough to remove impurities such as fat (11). conclusions the cleaning of the test tube with the sterilization method is recommended to avoiding the fear of remaining pollutants that can affect the resultan. it can also minimize the life of bacteria and viruses from the sample to be examined. however, if the sterilization method is difficult to do because of limited equipment and so on, the use of surfactants and the correct method of cleaning the tube is enough to remove impurities such as fat. conflict of interest there are no conflicts of interest. references 1. plebani m. diagnostic errors and laboratory medicine – causes and strategies. j int int fed clin fed chem clin lab med chem lab med. 2015;26(1):7–14. 2. conte r. management of errors in a clinical laboratory. off j inst sci technol [internet]. 2018;spring 201:22–3. available from: https://istonline.org.uk 3. sampath s, bhatia k, batra hs. analysis of various errors in receipt of samples in the biochemistry laboratory of a tertiary care hospital. 2016;(september):280–4. 4. guder wg. history of the preanalytical phase: a personal view. biochemia medica. 2014. 5. cornes mp, atherton j, pourmahram g, borthwick h, kyle b, west j, et al. monitoring and reporting of preanalytical errors in laboratory medicine: the uk situation. ann clin biochem. 2016;53(2):279–84. 6. bearss jj, honnold sp, picado es, davis nm, lackemeyer jr. validation and verification of steam sterilization procedures for the decontamination of biological waste in a biocontainment laboratory. appl biosaf. 2017;22(1):33–7. 7. farrell cjl, carter ac. serum indices: managing assay interference. ann clin biochem. 2016;53(5):527–38. 8. arifuddin n, saad ma, lakshmi vu. jermb . com jermb . com case report. :21–2. 9. kim tk. understanding one-way anova using conceptual figures. korean j anesthesiol. 201. 10. shingala mc, rajyaguru a. comparison of post hoc tests for unequal variance. int j new technol sci eng. 2015. 11. rutala wa, weber dj. disinfection and sterilization in healthcare facilities. in: practical healthcare epidemiology. 2018. 19 comparation between mac conkey and coconut water medium as a growth medium for escherichia coli endah prayekti1, suliati2, dwi agustin wulandari1 1department of medical laboratory technology, faculty of health, universitas nahdlatul ulama surabaya, surabaya, indonesia 2department of medical laboratory technology, poltekkes surabaya, surabaya, indonesia correspondence: endah prayekti, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code: 60237 email: endahphe@unusa.ac.id received: january 28th, 2021 revised: march 15th, 2021 accepted: march 15th, 2021 published: april 28th, 2021 doi: 10.33086/ijmlst.v3i1.1906 abstract escherichia coli is the bacteria that can cause diarrhea in humans and often used as a parameter of stool environmental pollution. culture of e. coli from the sample often requires mac conkey as commercial media which is able to distinguish it from other bacteria in the enterobacteriaceae group. commercial media such as mac conkey certainly has a price that is quite expensive because of its ability as a growth medium for enterobacteriaceae. therefore, in the study tested natural ingredients that can be used for growth media, such as coconut water. the purpose of this study was to compare the ability of mac conkey media and coconut water to support the growth of e. coli. this research is an experimental study with a completely randomized design. the concentration of coconut water tested was 0%, 20%, 40%, 60%, 80%, and 100%. the results showed that at the concentration of coconut water 20% to 60% the number of e. coli colonies on coconut water media was slightly below the mac conkey agar media, while in coconut water a concentration of 80% showed a greater number of colonies than mac conkey. the mann whitney test showed a significant difference between the number of colonies on 80% coconut water media and mac conkey agar, which was equal to 0.004 (p < 0.05). based on these results, coconut water has the potential to be used as a growth medium for e. coli. keywords coconut water, escherichia coli, mac conkey. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. mailto:endahphe@unusa.ac.id https://journal2.unusa.ac.id/index.php/ijmlst/article/view/1906/version/2390 ina. j. med. lab. sci. tech. 2021; 3(1): 19-25 endah prayekti, et al. 2 0 introduction indonesia is an archipelago which produces coconut fruit in most of provinces (1). indonesia is one of country in the world which produces a high number of coconut fruit per year. coconut were being consumed for its water and flesh, and or its product as kopra and coconut oil (1). in coconut water contain carbohydrate, protein, fat, vitamin, and minerals (2,3). coconut water can be used for a body electrolytes subtitution, antidotes, antioxidants, to antibacterial beside for being consumed (4,5,6). coconut water can also be used as a medium for making food products such as nata de coco (7, 8). some traditional markets in indonesia sell coconut meat and often not use the water. abundance and ease of the access to obtain coconuts can be used as opportunities for wider use of coconut water. utilization of coconut water for bacterial growth media in the laboratory is one of the ways to access it. the examination parameter which often carried out in the laboratory is the examination of escherichia coli. e. coli is the bacteria that can cause diarrhea in humans (9, 10, 11) and often used as a parameter of stool environmental pollution (12, 13, 14). culture of e. coli from the sample often requires mac conkey as commercial media which is able to distinguish it from other bacteria in the enterobacteriaceae group. commercial media such as mac conkey certainly has a quite expensive price because of its ability as a growth medium for enterobacteriaceae. therefore, in this study were tried to compare viable count of e. coli recovered from coconut water medium and mac conkey as gold standard for growing e. coli. materials and methods this study was an experimental study using complete randomized design. variable of this study were coconut water concentration (0%, 20%, 40%, 60%, 80%, 100%) and numbers of e. coli colony. this study conducted in microbiology laboratorium at universitas nahdlatul ulama surabaya, indonesia. this study used e. coli atcc 25922 that were obtained from bblk (balai laboratorium kesehatan) surabaya. e. coli need to subculture in nutrient agar slant before used. twenty-four bacterial culture in nutrient agar slant then suspend in sterilize 0.85% natrium chloride until it reaches mac farland 0.5 turbidity. coconut water between 6 and 8 months of age were obtained from traditional market. coconut water was extracted by breaking coconut endocarp. coconut water obtained then analyse for chemical characters, namely carbohydrate, protein and fat component in balai riset dan standarisasi industri surabaya (baristand industri surabaya) indonesia. composition of the coconut water media per 100ml were ina. j. med. lab. sci. tech. 2021; 3(1): 19-25 endah prayekti, et al. 2 1 lactose 10 g; agar 1.5 g; neutral red 2% 2 ml; aquadest (varying concentration of the coconut water); coconut water (0%, 20%, 40%, 60%, 80%, 100%). each of media component were mixed and boiled to allow completely dissolve. media was sterilized using autoclave for 15 minutes at 121oc, 1 atm. for pure e. coli, a uniform cell suspension was pepared by dispersing the overnight colony growth from nutrient agar media. the turbidity of the bacterial suspension used was according to the mc farland 0.5 standard (0.05 ml of 1.175% barium chloride dihydrate (bacl2.2h2o), with 9.95 ml of 1% sulfuric acid (h2so4)). bacterial suspension was inoculated using pour plate method with a proper dilution. bacterial suspension diluted in series of 0.85% natrium chloride. each of serial dilution bacterial suspension were taken as much as 1 ml and placed it in sterile petridish. media were poured to sterile petri dish and left to solid. media were in icubated at 37oc for 24 hours in incubators. the replication used in the study was 5 times for each treatment. the result of this work was observation of the the number of colonies‐forming units and its macroscopic colonies of e. coli. numbers of e. coli (cfu) obtained from medium after incubation then analyze statisticly using mann whitney. colony morphology was illustrated in diagram. results this study used young coconut water for growth medium of e. coli and comparing it with mac conkey as commercial medium. base component of coconut water used were illustrated in table 1. the bacterial colony recovered in each group has vary in its colony size. e. coli growth in mac conkey show medium category in colony size while in coconut water medium show vary colony size from small pin point category until small colony category (figure 1). bacterial colony number which recovered in each group of coconut water medium treatment shows slightly increasing in colony number while number of e. coli colony at 80% group were exceed mac conkey group (figure 2). colony colour in coconut water group were white while colony in mac conkey were red (data not shown). table 1. composition per 600 ml of young coconut water componet percentage protein 0.453 carbohydrate 4.488 fat 0.257 discussion coconut water contain numerous nutrition components for bacterial growth, including macro and micro component (15, 16). there are various types of coconuts, with different mineral content in coconut water. chuku & calagbour (16) compared various coconut species for its coconut water content ina. j. med. lab. sci. tech. 2021; 3(1): 19-25 endah prayekti, et al. 2 2 which are mineral and protein content. in general, the largest to lowest mineral content in a row were cl k na ca s fe cu. the mineral components in coconut water are needed for bacterial growth. in addition to minerals, coconut water also contains sucrose, glucose, and fructose (15). the presence of these sugar in coconut water createthe colony of e. coli bacteria can not express pink color at 24-hour incubation, which indicates the absence of lactose fermentation in coconut water media. e. coli tends to use the simplest sugar available in the media first before complex ones (17). e. coli colony shows discoloration change to pink after one week of incubation (data not shown). in this study, the 80% e. coli treatment group had a large count of colony growth but has smaller colony size compared to growth using mac conkey media. this condition is possible because in medium coconut water has a low source of nitrogen and phosphorus. limitation of phosphorus in the media will slowing the translation process because of the low certain ribosomes and the rna is very much so that it requires a lot of phosphorus. meanwhile, nitrogen limitation cause a slow elongation process of the translation process (18). figure 1. escherichia coli colony size scoring in each of group treatment. treatment group: control using mac conkey agar and coconut water media agar by adding coconut water in media approximately 20%, 40%, 60%, 80% and 100%. growth of e. coli in each treatment then scored based on size, score 3 for medium size; 2 for small size; 1.5 for pin point size; 1 for small pin point size; 0 for no growth. 0 0,5 1 1,5 2 2,5 3 3,5 control (mac) 20% 40% 60% 80% 100% group treatment b a c te ri a l c o lo n y s iz e s c o ri n g ina. j. med. lab. sci. tech. 2021; 3(1): 19-25 endah prayekti, et al. 2 3 the differences between both testing media were absence of nitrogen source, bile salt, and crystal violet in the coconut water medium. one of mac conkey media component are peptone, in which can promote enrichment for bacterial colony. peptone is nitrogen source for bacteria, and oftenly used as enrichment purpose (19). the phytochemical testing of several types of coconut water qualitatively shows the presence of tannin content in coconut water. tanin is one of phytochemical which can inhibit bacterial growth (20). it explains no e. coli growth in 100% group. figure 2. numbers of escherichia coli colony recovered in mac conkey and coconut water media. treatment group: control using mac conkey agar and coconut water media agar by adding coconut water in media approximately 20%, 40%, 60%, 80% and 100%. conclusions coconut water ia able to use as component medium for the e. coli cultivation. the coconut water has a good source for carbohydrate. further evaluation needs to be carried out to explore the coconut water physiological influenced bacterial growth. author contributions endah prayekti: conceptualization, methodology, writing-original draft, visualization, supervision, funding ina. j. med. lab. sci. tech. 2021; 3(1): 19-25 endah prayekti, et al. 2 4 acquisition. suliati: methodology, supervison. dwi agustin wulandari: formal analysis, investigation, resources. acknowladgements researchers are grateful for the funding provided by the research institute and community service at universitas nahdlatul ulama surabaya. conflict of interest there are no conflicts of interest. references 1. directorate general of estate crops, tree crop estate stattistic of indonesia 2015-2017. jakarta: secretariate of directorate general of estate crops, directorate general of estate crops, ministry of agriculture, 2016. 2. yong jwh, ge l, ng yf, tan sn. the chemical composition and biological properties of coconut (cocos nucifera l.) water. molecules. 2009;14(12):5144–5164. doi: 10.3390/molecules14125144. 3. appaiah p, sunil l, kumar pkp, krishna agg. physico-chemical characteristics and stability aspects of coconut water and kernel at different stages of maturity. j food sci technol. 2015;52(8):5196–5203 doi: 10.1007/s13197-0141559-4. 4. debmandal m, mandal s. coconut (cocos nucifera l.: arecaceae): in health promotion and disease prevention. asian pac. j trop med. 2011;4(3): 241–247. doi: 10.1016/s19957645(11)60078-3. 5. zulaikhah s. health benefits of tender coconut water (tcw). int j pharm sci res. 2019; 10(2).474-8 doi: 10.13040/ijpsr.09758232.10(2).474-8. 6. halim hh, dee ew, dek msp, hamid aa, ngalim a, saari n, jaafar ah. ergogenic attributes of young and mature coconut (cocos nucifera l.) water based on physical properties, sugars and electrolytes contents. int j food prop. 2018;21(1):2378–2389. doi: 10.1080/10942912.2018.1522329. 7. gayathry g. production of nata de coco a natural dietary fibre product from mature coconut water using gluconacetobacter xylinum (sju-1). int j food ferment technol. 2015;5:231 doi: 10.5958/2277-9396.2016.00006.4. 8. thankappan g, anitha p. effect of sources of coconut water and acidulants on physico chemical properties of nata-de-coco. j trop agric. 2018;56(2):206–209. 9. yang sc, lin ch, aljuffali ia, fang jy. current pathogenic escherichia coli foodborne outbreak cases and therapy development. arch microbiol. 2017;199(6):811-825. doi: 10.1007/s00203-0171393-y. 10. zhou y, zhu x, hongyan h, lu y, lu j, mau l, mau l, sun z. characteristics of diarrheagenic escherichia coli among children under 5 years of age with acute diarrhea: a hospital based study. bmc infect dis. 2018;18(63):1-10. doi: 10.1186/s12879-017-2936-1. 11. jiang zd, dupont hl. etiology of travellers’ diarrhea. int soc travel med. 2017;24(1):13-16. doi: 10.1093/jtm/tax003. 12. sasakova n, gregova g, takacova d, mojzisova j, papajova i, venglovsky j, szaboova t, kovacova s. pollution of surface and ground water by sources related to agricultural activities. front sustain food syst. 2018;2:42. doi: 10.3389/fsufs.2018.00042. 13. jang j, hur hg, sadowsky mj, byappanahalli mn, yan t, ishii s. environmental escherichia coli: ecology and public health implications—a review. j appl microbiol. 2017;123(3):570–581. doi: 10.1111/jam.13468. 14. ercumen a, pickering aj, kwong lh, arnold bf, parvez sm, alam m, sen d, islam s, kullmann c, chase c, ahmed r, unicomb l, luby sp, jr jmc. animal feces contribute to domestic fecal contamination: evidence from e. coli measured in water, hands, food, flies, and soil in bangladesh. 2017. doi: 10.1021/acs.est.7b01710. 15. prades a, dornier m, diop n, pain jp. coconut water uses, composition and properties: a review. fruits. 2012;67(2):87-107. doi: 10.1051/fruits/2012002. 16. chuku lc, kalagbor gi. protein and mineral element content of coconut (cocos nucifera) water from different species. am j adv drug deliv. 2014;2(4):451-457. [online]. available: ina. j. med. lab. sci. tech. 2021; 3(1): 19-25 endah prayekti, et al. 2 5 www.ajadd.co.uk. 17. bren a, park jo, towbin bd, dekel e, rabinowitz jd, alon u. glucose becomes one of the worst carbon sources for e. coli on poor nitrogen sources due to suboptimal levels of camp. sci rep. 2016:2-11. doi: 10.1038/srep24834. 18. li shj, li z, park jo, king cg, rabinowtiz jd, wingreen ns, gitai z. carbon, nitrogen and phosphorus limitation conditions. nat microbiol. 2018;3(8):939-947. doi: 10.1038/s41564-0180199-2.escherichia. 19. husin n, kamal smm, chuan lt, muhammad nf, jusoh n. comparison of microbial growth on fish waste peptones from different hydrolysis methods, 2015 5th int conf biomed eng technol (icbet 2015). 2015;81(1):54-57. doi: 10.7763/ipcbee. 20. mulyanto a, mujahid i, khasanah tu. kemampuan air kelapa muda sebagai antimikroba terhadap bakteri escherichia coli penyebab diare [the ability of young coconut water as an antimicrobial against the bacteria escherichia coli that causes diarrhea]. j bio-site. 2018;(4)1:18. 8 correlation of malnutrition, worm infection, parents, income and knowledge on anemia prevalence among 6–9 year old students of liliba inpres elementary school marni tangkelangi1 1department of health analyst, poltekkes kemenkes kupang, kupang, indonesia correspondence: marni tangkelangi, jl. farmasi, liliba, kupang, east nusa tengga, indonesia zip code : 85111 email: mari.tangkelangi@gmail.com received: february 26, 2019 revised: february 20, 2019 accepted: april 4, 2019 abstract anemia in school–age children will not only cause harm to health but also will have impact on students learning achievement. thus, anemic children will indirectly affect the national development. the aim of this study is to observe the correlation between malnutrition, worm infection, parents income and knowledge on anemia prevalence among 6–9 years old children. the method of this research was a cross sectional study with a simple random sampling technique, consisted of 222 participants from liliba inpres elementary school. this study was done by measuring children body mass indeks (bmi)–for–age to measure the z score and compare to who children growth standard, by using microscope examination with direct method for identifying helminthiasis, by using questionaries to measure the parent knowledge and parent income and also by measuring haemoglobin values using poct device. the results showed that there are correlations between malnutrition and worm infection on anemia prevalence (p value 0.000). however, there are no correlations between parent’s knowledge (p value 0.469) and parent’s income on anemia prevalence among 6–9 years old children on liliba inpres elementary school (p value 0.606). in conclusion, these findings confirm that malnutrition and worm infection was correlated with anemia prevalence on liliba inpres elementary school students so that they are advised to manage their nutritional intake and to practice personal hygiene. keywords anemia, malnutrition, worm infection, knowledge, income ina j med lab sci tech 2019; 1(1): 8-14 marni tangkelangi 9 introduction children are one of the vulnerable groups affected by anemia beside pregnant and breastfeeding group and the elderly (1). the prevalence of anemia on school–aged children in developing countries and developed counties was estimated about 42% and 17%, respectively. anemia prevalence in indonesian children at age 5–14 years is 42.8 % for boys and 49.2% for girls (2). anemia is a condition where the haemoglobin (hb) levels fall below the normal value (11 g/dl) (3). anemia can happen as a consequence of extrinsic and intrinsic factors. the intrinsic factors are blood disorders, low iron intake, and worm infection. meanwhile, the extrinsic factors are poor knowledge about nutrition, level of parent education, economy status, and lifestyle (4). anemia in children can disrupt the growing process, decrease learning concentration, and are vulnerable to have other diseases. research proved that there was a correlation between hemoglobin level (anemia status) on children learning ability. iron deficiency anemia could decrease the child concentration which will further affect their study achievement (5). liliba inpres elementary school is a school that has the most student compared to all elementary school in kupang city, as many as 1.047 students (6). this school is not only has the most students, but also has very diverse population variation. the students come from different background races, economic status, parent’s education background and parent’s occupation. six until nine years old female students were selected as the inclusion criteria because the majority of female students in this range of age has not experience menstruation. moreover, the female students are less likely manage their personal hygiene, are vulnerable to have infection, and are in a transisional age from childhood to adolescent. therefore, the research aim to observe a correlation between malaria and malnutrition, worm infection, parent’s knowledge and income on anemia in liliba inpres elementary school students aged six until nine years old. materials and methods this research was an analytical study with a cross sectional study design. the research setting was at liliba inpres elementary school. the research held on october until november 2016. the population were students aged 6–9 years old who attended liliba inpres elementary school in oebobo, kupang. the total amount of students was 338 children. the sample on this research were 222 students chosen by simple random sampling technique. the tools used in this research were hemoglobinmeter (easy touch®), hemoglobin test strips, body scales and height gauge. ina j med lab sci tech 2019; 1(1): 8-14 marni tangkelangi 1 0 nutritional status determined by the z score based on children bmi–for–ages compared to who children growth standard. the analysis of worm infection were done directly by microscope observation for worms’ infection. the data of parent’s knowledge and income was conducted by interview using a questionnaire. haemoglobin (hb) examination was done by taking blood capillary then was measured with haemoglobinmeter and was compared with anemia at child age school based–who standards. the research using chi–square test for knowing the correlation between variables. results and discussion the research subject were 222 student of liliba inpres elementary school aged 6–9 years who met the inclusion criteria and received written approval from their parents. the subject consisted of 106 boys (47.7%) and 116 girls (52.3%). the majority of the subjects research were 6–7 years old (66.2%). the body weight of the subject was mostly in the range of 17–21.9 kg as many as 130 people (58.6%). the highest body height of the subjects were in the range of 1.11–1.20 meters as many as 111 people (50%). the occupation of most fathers was private employee as many as 86 people (38.7%) and mother’s occupation as many as 91.9% were housewives. table 1 shows the number of subjects who had anemia (hb <11 g / dl), as many as 118 people (53.2%). the percentage of students who classified as malnutrition (bmi–for–age z score <–2 sd) were 47.3%. the amount of students who had positive worm infection were 60 people (27%). meanwhile, 70.7% of parent knowledge was good (the questionnaire score ≥ 24) and 67.1% parent’s income level (income > 386,139 rupiah /person/ month) fall in the low category. table 1. research subject categories no variable category n % min max mean 1. anemia condition anemia 118 53.2 6.9 g/dl 14.4 g/dl 10.7 g/dl normal 104 46.8 2. malnutrition yes 105 47.3 bmi 8.5 bmi 30.2 bmi 13.2 not 117 52.7 3. worms infections positive 60 27 _ _ _ negative 162 73 4. parents knowledge low 65 29.3 score of 13 score of 38 score of 26 well 157 70.7 5. parents income level low 149 67.1 83,333 idr/ capita/month 1,500,000 idr/capita/month 379,500 idr/capita/month ina j med lab sci tech 2019; 1(1): 8-14 marni tangkelangi 1 1 correlation between malnutrition and anemia table 2 implicated that there was a significant relationship between malnutrition and anemia prevalence. the description of malnutrition in children is obtained by the z score (bmi–for–ages). table 4. shows that there were 105 children (47.3%) who fall in the category of malnutrition. this is consistent with the study observed by rosanti (7) which stated that there was a strong relationship between nutritional status (malnutrition) and the incidence of anemia in children in the benan ngisor area. the national heart, lung and blood institute states that one of the main risk factors of anemia is consumption of low iron foods, vitamins and minerals. nutrients in foods such as protein, folic acid, vitamin b 12 and iron are the main raw materials for the formation of erythrocytes (8). malnutrition is a condition where a person does not get enough nutrients that will decrease the erythrocyte synthesis so that it will cause anemia in children. the six until nine years of age is a crucial moment where a child's body needs nutrients in sufficient quantities in order to grow according to the standards of growth. however, lack of consumption of nutritious food can also be caused by the parent’s income. they cannot provide nutritious food for their children if the income is lower than the average. based on the observations, some children with malnutrition showed the signs of anemia such as weakness, lethargy, pallor, dry lip angle and pale eye conjunctiva. if the condition of anemia that occurs in children is still at a mild level (hb 9.4–10.9 g / dl), it can be managed by increasing the intake of nutrients, especially protein and minerals. the iron (fe) tablets can also increase the hb levels in malnourished children (9). table 2. correlation of malnutrition with the incidence of anemia malnutrition anemia condition total sig. (p) anemia normal n % n % n % yes 76 34.2 29 13.1 105 47.3 0,000 not 42 18.9 75 33.8 117 52.7 total 118 53.1 104 46.9 222 100 correlation between infection and anemia indriati (10) wrote about the incidence of anemia in school children due to lack of iron and worms infection. in line with that, the research conducted by supriadi (11) concluded that there was a significant relationship between helminthiasis and anemia in students of dembol in karanganyar sub district. ina j med lab sci tech 2019; 1(1): 8-14 marni tangkelangi 1 2 chi–square test analysis results (table 3) concluded that there was a significant relationship between the incidences of anemia with helminthiasis from a total of 222 stool samples examined microscopically. there were 60 samples that were positive with the type of infecting worm. worms are often found in school–age children, especially in children who live in the environment with poor sanitation, unavailability of latrines, lack of clean water supplies, and lack of personal hygiene. those are the underlying cause of the occurrence of helminthiasis in children (12). this study involved six until nine years old children. at this range of age, most children spend their time by playing with sand because the location of the elementary school is mostly a land field. therefore, the chance of worm infection is higher when children did not wear a footwear when playing on the ground. worms that enter the human body will live and develop in the large intestine. the worm will suck blood ("seize" nutrients) needed by the body. in the case of severe parasitemia, worms can cause severe anemia in children (<7 g / dl). in addition to sucking blood on the surface of the intestine where the mouth of the worm attaches, blood leakage can also occur. this causes the body to lose blood and leads to a decrease in hemoglobin levels resulting in an anemia condition (8). table 3. worm relationship with the incidence of anemia worms anemia degree total sig. (p) anemia normal n % n % n % positive 46 20.7 14 6.3 60 27 0,000 negative 72 32.4 90 40.6 162 73 total 118 53.1 104 46.9 222 100 correlation between parent’s knowledge and anemia table 4 shows that 70.7% parents have good knowledge about anemia based on questionnaire filling scores. based on the statistical analysis of anemia variables with parental knowledge, it was concluded that there was no correlation between the incidence of anemia and knowledge of parents. this is contrary to the research conducted by syafri, et al. (13) which concluded that there was a correlation between family income and the incidence of anemia in children in cilallang inpres elementary school, makassar. the results obtained in this study are in line with the research by ikhmawati (14) which concluded there was no relationship between knowledge and hb levels. in addition, wetipulinge (15) stated there was no association between anemia knowledge and the incidence of anemia. based on the results of interviews to the mothers of the students, some children who have low hb ina j med lab sci tech 2019; 1(1): 8-14 marni tangkelangi 1 3 levels often have problems with eating patterns. even though their parents have knowledge about anemia and its effects, they tend to let their children to eat snack instead of eating main meal so it decrease the children’s appetite. table 4. worm relationship with the incidence of anemia parent’s knowledge anemia degree total sig. (p) anemia normal n % n % n % less 37 16.7 28 12.6 65 29.3 0.469 well 81 36.5 76 34.2 157 70.7 correlation between parent’s knowledge and anemia the parent’s income of liliba inpres elementary school as much as 67.1% is in the low category, with the average income was 379,500 idr/capita/month (table 1). it still below the standard of gkm according to bps, which is 386,139 idr/capita/ month. most income comes only from the fathers’ income because 91.9% of mothers are housewives. statistical analysis (table 5) showed that there was no relationship between the incidence of anemia and the income of parents. this result is in line with the research by hendro (16) which states that there was no relationship between family income and anemia status. different opinions expressed by liow et al. (17) which stated that there was a significant relationship between income and the incidence of anemia. syafri et al. (13) stated that there was a relationship between family income and the incidence of anemia. during the observation, children with sufficient parent’s income have low hb levels (<11 g/dl), whereas students with low parent’s income have normal hb levels. this condition could happen because even their parents has low income, they could provide nutritious food such vegetable which rich in fe minerals that could prevented them from being anemic. family income will influence the ability to buy nutritious food. however, parent’s income did not always guarantee that children cannot be anemia, especially if parents obey unhealthy eating patterns of their children. in other words, less income does not mean that children will suffer from anemia due to malnutrition. nevertheless, having high income tends to provide pocket money for their children so that the children consume snacks more often and consume less food provided at home. ina j med lab sci tech 2019; 1(1): 8-14 marni tangkelangi 1 4 table 5. correlation between parental income and anemia income anemia degree total sig. (p) anemia normal n % n % n % weak 81 36.5 68 30.6 149 67.1 0.606 intermediate 37 16.7 36 16.2 73 32.9 total 118 53.2 104 46.8 222 100 conclusions anemia prevalence in liliba inpres elementary school students was 52.3%. this research also find out that there were students suffer from malnutrition and worm infection. besides, most of student parents have low income although they have good knowledge. anemia prevalence in students of inpres liliba elementary school was mostly caused by the intrinsic factor (malnutrition and worms infection) compared to the extrinsic ones (parent’s knowledge and income). parental control on children eating pattern and personal hygiene was recommended to decrease the anemia prevalence in children. conflict of interest there are no conflicts of interest. references 1. indonesian ministry of health. 2001. nutrition anemia prevention program on woman age fertile (wus). jakarta: directorate nutrition society directorate general bina health society. 2. who. 2004. iron deficiency anemia and prevalence anemia. http://www.who//html (access: 18 march 2016). 3. who. 2001. iron deficiency anemia: assessment, prevention and control. geneva: world health organization. 4. siswono. 2004. time best consuming vitamins. http://www.gizi.net/ (access: march 8, 2016). 5. sediaoetama, achmad d. 2006. science nutrition for college student and professional vol. jakarta: dian rakyat. 6. indonesian ministry of health. 2001. nutrition anemia prevention program on woman age fertile (wus). jakarta: directorate nutrition society directorate general bina health society. 7. rosanti, ika. 2009. relationshipbetweennutrition al status with event anemia on child toddlers i n bendan region visiting semarang city. thesis. semarang: university semarang state 8. masrizal. 2007. study anemia deficiency literat ure iron. 2nd edition: journal health society. 9. indriati, ira. 2006. influence supplementation iron and vitamin a against hemoglobin levels and achievent learn child school basic negeri 3 neglasari banjar city. semarang: university diponegoro. 10. indriawati, r. 2002. study to examination hemoglobin (hb) method sahli and talquist. jakarta: mutiara medika. 11. supariasa, et la. 2002. assessment of nutritional status. jakarta: egc. 12. fitri, j., et la. 2012. analysis factors risk infection worms student school elementary in district ang kola est disctrits sourth tapanuli in 2012. journal science enviroment. riau: university of riau. 13. syafri, m., et la. 2013. relations factor family and child with genesis anemia in child school basic presidential instuction cilallang makassar city. makassar: hasanuddin univeritiy. 14. ikhmawati , y., et la. 2013. relations between knowledge about anemia and habits eat against hemoglobin level st teenagers daughter ini surakarta high school mta dormitory. surakarta: muhammaadiyah university of surakarta. 15. wetipulinge, s. 2006. knowledge of anemia and habits eat against hb on teenagers daughter of yogyakarta muhammadiyah ii high school. yogyakarta: ugm. 16. sumito, w. 2004. fortification substance iron on food ai companion. jakarta: center studies policy food and nutrition. 17. liow, f., et la. 2012. relationship between social status economy with anemia on mother pregnant in the village say hello sub-district tenga disctricts south minahasa. manado: sam ratulangi university. ina j med lab sci tech 2019; 1(1): 8-14 marni tangkelangi 1 5 42 the effect of environmental factor and use of personal protective equipment on the symptoms of acute respiratory tract infections in furniture industry workers merry sunaryo1 1department of safety and occupational health, faculty of health, universitas nahdlatul ulama surabaya, surabaya, indonesia correspondence: merry sunaryo, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code : 60237 email: merry@unusa.ac.id received: september 9, 2019 revised: december 18, 2019 accepted: april 11, 2020 abstract dust is one type of potential hazardzs in the workplace that can affect the health of the workers. the occupation that are always exposed to dust is furniture industry workers so that they have higher risk of getting acute respiratory tract infection (ari) disorder which can interfere with breathing. the wood dust is formed from some of the sawn wood and sanding that will lead to an acute respiratory tract infection. the study aimed to determine the effect of environmental factor and the use of personal protective equipment (ppe) against the symptoms of acute respiratory infections in the furniture industry workers. the research method used was quantitative method with observational and cross-sectional research types and it was analysed by using logistic regression test. the population in this study was the workers of the furniture industry at semarang street, surabaya city, with a total of 57 people, of which 37 furniture workers as a sample. the results show that most of the workers has symptoms of acute respiratory tract infection. it could be influenced by the environmental factor such as dust exposure that produced wood dust in each manufacturing processes. additionally, the use of ppe also affected the occurrence of acute respiratory tract infections symptoms in the workers. in conslusion, many factors can influence the occurrence of acute respiratory tract infection symptoms in the furniture industry workers. therefore, it is necessary to minimize the dust exposure in workers by wearing ppe such as respirators. keywords environmental factor, personal protective equipment, dust, acute respiratory tract infection symptoms introduction occupational illness is any disease contracted primarily as a result of an exposure to risk factors arising from work activity, such us illnesses and medical conditions can be caused by unsafe actions and conditions. one of the unsafe conditions in the environment is the presence of dust hazards in the workplace. the majority of work-related death based on the international ina j med lab sci tech 2020; 2(1): 42-49 merry sunaryo 4 3 labor organization (ilo) data is cancer (34%), followed by occupational accidents (25%), respiratory diseases (21%), cardiovascular disease (15%), and others (5%) (1). meanwhile, an acute respiratory tract infection (ari) is a major health problem in indonesia, with the prevalence of such disease is as much as 25.5%, where 16 provinces of which have a prevalence above the national rate. acute respiratory tract infections rank first out of the 10 most diseases suffered by people in indonesia. the results of riskesdas of indonesia (basic health research) in 2013 showed that the prevalence of acute respiratory tract infections was 24%. in addition, pneumonia also becomes the health problem in indonesia which is as much as 2.1%. acute respiratory tract infections (ari) are acute inflammatory events in the acute or lower respiratory tract caused by microbial or bacterial, viral, or rickets infections, without or accompanied by inflammation of the lung parenchyma (2). the factors that affect a person's risk of acute respiratory tract infections exposure are environmental factors, individual characteristics and employee behavior. one of the environmental factors is air pollution in the workplace while the individual factors includ age, gender and education level which can affect the risk of susceptibility to acute respiratory tract infections. meanwhile, the worker behavior includes the use of personal protective equipment (3). dust is a solid chemical, caused by natural or mechanical forces such as processing, pulverizing, softening, fast packing, blasting from objects, both organic and inorganic, which have a diameter between 0.1 µm to 500 µm (4). there are several types of dust namely coal dust, cement, cotton, asbestos, wood dust, dust during the rice milling process or organic dust and others. exposure to dust in the workplace can cause acute or chronic respiratory distress in the workers. various factors affect the onset of such disease. those factors include dust particles size and shape, dust concentration, dust chemical properties and duration of dust exposure. individual factors also affect the onset of the disease such as lung defense mechanisms, anatomy and physiology of the respiratory tract (5). the wood furniture industry is one of the industries that has a very rapid development. the physical process of the raw materials of the furniture manufacture tends to produce pollution such as wood dust particles. approximately, 10 to 13% of the sawn wood and sanding will produce wood dust. this wood dust can generate particle pollution in the air and environment. in addition, the wood furniture industry workers can be exposed to dust (6). dust level that exceed the threshold and limit value for key air pollutans can cause health problems such as respiratory ina j med lab sci tech 2020; 2(1):42-49 merry sunaryo 4 4 problems which can later become acute respiratory infections. high concentration of pollutants in the environment can damage the lung defense mechanism. however, dust exposure can be a cause of ari even though the level of wood dust is below the not available (nav) (7). therefore, this study was analyzed the influence of environmental factor which in this case is the wood dust and the use of personal protective equipment against the symptoms of acute respiratory tract infections in the furniture industry workers in semarang street, surabaya city, indonesia. materials and methods this research method was a quantitative research using observational research types and cross-sectional approach. this research was carried out in semarang street, surabaya city, east java. the population in this study were the workers in the furniture industry in semarang street, surabaya city, indonesia, with a total of 57 people, of which 37 furniture workers were as the sample. the sampling technique in this study was done by simple random sampling technique. the variables in this study consisted of independent variables and dependent variable. the independent variables were environmental factor (dust) and the use of personal protective equipment. meanwhile, the dependent variable was acute respiratory tract infections. data collection techniques was done by using interview and observation with a questionnaire. data were calculated by logistic regression analysis. results distribution of acute respiratory tract infection symptoms based on the results regarding the distribution of workers who experienced ari symptoms are shown in table 1. table 1. distribution of acute respiratory tract infection symptoms in personal characteristics of the respondents variable acute respiratory tract infection symptoms description number of participants % no 13 35 yes 24 65 total 37 100 table 1 shows that the number of workers who experienced symptoms of acute respiratory tract infections was as many as 24 people out of 37 respondents (65%). based on the data above, it can be concluded that the majority of workers in the semarang road furniture industry has symptoms of acute respiratory tract infections. ina j med lab sci tech 2020; 2(1): 42-49 merry sunaryo 4 5 analysis of environmental factors on symptoms of acute respiratory infection the analysis of environmental factors (wood dust) that affects the symptoms of acute respiratory tract infections in the respondents using bivariate logistic regression tests is presented in tabel 2. table 2. environmental factor analysis of symptoms of acute respiratory tract infections variable b wald df sig exp(b) ci 95% cox & snell r square environmental factor (dust exposure) 1.805 5.640 1 0.018 6.080 0.152 *logistic regression analysis table 2 elaborates the results of an analysis of environmental factors (wood or environmental dust) with criteria above the threshold value and below the threshold value. these results indicated that dust above the threshold value has a significant effect on symptoms of acute respiratory tract infections with p-value 0.018 < 0.05. the level of risk of dust on the occurrence of acute respiratory tract infections symptoms in the furniture workers based on the value of exp (b) is 6.080. it can be interpreted that the workers exposed to dust above the threshold value have 6 times more risk of experiencing acute respiratory tract infections symptoms compared to the workers who were exposed to dust below the threshold value. analysis of the use of personal protective equipment against acute respiratory tract infections symptoms the results of the analysis on the effect of the use of personal protective equipment against symptoms of acute respiratory tract infections in respondents by using a bivariate logistic regression test showed in table 3. table 3. results of analysis of ppe on symptoms of acute respiratory tract infections variable b wald df sig exp(b) ci 95% cox & snell r square ppe (mask) 1.569 4.496 1 0.034 4.800 0.121 *logistic regression analysis based on the results of the logistic regression test in table 3, the use of mask has a significant effect on the symptoms of acute respiratory tract infections p-value 0.034 < 0.05. these results also show level of risk of the use of personal protective equipment against the occurrence of acute respiratory tract infections symptoms in the furniture ina j med lab sci tech 2020; 2(1):42-49 merry sunaryo 4 6 workers based on an exp (b) value of 4.800. this can be interpreted that the workers who worked without using personal protective equipment have a 4.8 times higher risk of experiencing acute respiratory tract infections symptoms compared to those who use personal protective equipment. discussion symptoms of acute respiratory infection ari is an acute inflammation of the above and below respiratory tract due to microbial or bacterial, viral, and rickets infection, without or accompanied by inflammation of the lung parenchyma (2). ari (acute or lower respiratory tract disease), according to who, can usually be contagious which can then cause a variety of diseases, from asymptomatic or mild infections to severe deadly diseases (8). ari is an infectious disease that attacks one part and or more of the airways, starting from the nose (acute channel) to the alveoli (lower channel) including related tissues, such as sinus, middle ear cavity and pleura (9). there are several factors that can influence a person's risk of getting ari, such as environmental factors, individual characteristics and employee behavior. environmental factors include air pollution that can be generated from industrial product, dust, cigarette smoke, and others. meanwhile, individual factors affecting ari are age, sex and education level. lastly, the worker behaviors such as smoking and the use of masks or other personal protective equipments also influence the risk of getting ari (3). exposure to dust can cause either acute or chronic respiratory health problems. dust particles that can cause acute respiratory problems are industrial products that can pollute the air such as wood dust, coal, cement, asbestos, chemicals, and the others. there are several factors that influence the emergence of diseases or disorders of the respiratory tract due to dust. these factors are dust particles, dust shape, concentration of dust, dust solubility and dust chemical properties as well as duration of dust exposure. whereas, the individual factors include defense mechanisms or lung strength, anatomy and physiology of the respiratory tract (5). based on the results of this research, the number of workers who experienced symptoms of acute respiratory tract infections was 24 out of 37 correspondent. it represents 65% of the workers. it can be concluded that the majority of workers have symptoms of ari. acute respiratory infections have several symptoms, for example cough, flu, nasal congestion, sore throat, shortness of breath, fever, headache to muscle aches. however, many symptoms of acute respiratory tract infections were not examined properly. many cases of ari can cause complications, access to timely health ina j med lab sci tech 2020; 2(1): 42-49 merry sunaryo 4 7 services will reduce the risk of disease severity and even death. therefore, the need for supervision of symptoms of acute respiratory tract infections in workers. effect of environmental factors on symptoms of acute respiratory tract infection the result of this research indicated that dust exposure above the threshold limit value has a significant effect on symptoms of acute respiratory tract infections p-value <0.05. the risk level of dust on the occurrence of symptoms of acute respiratory tract infections in the furniture workers based on the value of exp (b) was 6.080. it can be interpreted that the workers who was exposed to dust above the threshold limit value have 6 times more risk of experiencing symptoms of acute respiratory tract infections compare to those who were exposed to dust below the threshold limit value. dust is one material that is often referred to as particles floating in the air with a size of 1-500 µm. dust is often used as an indicator of pollution both inside and outside the building. it is also used to indicate the level of danger to the environment and to the occupational health and safety. high dust concentrations can be related to ari events. pm10 (particulate matter below 10 µm) or dust is found in the furniture manufacturing process or it can also be found in household fuel use. pm10 particles consist of complex particles of 0.1-10 µm, covering all sizes of viruses (0.1-1 µm) and bacteria (0.5-5 µm). the pathogen is free and it can move in the air (10). recent findings suggest that shortterm dust exposure is associated with health effects even in low concentrations ≤100 µg/m3. in this study, it was found that an average pm10 concentration of 70.60 µg/m3 resulted in ari in the furniture industry workers. pm10 is a pollutant oxidant that can be inhaled by the respiratory tract. oxidants are electrophilic chemicals that can move the electrons from various molecules which will lead to oxidation from these molecules. oxidants can damage body cells through lung parenchyma cells, both alveolar cells and its matrix (11). the threshold limit value for dust is a criterion in the workplace as a preventive guideline in order that the workers are still able to work without causing adverse health effect for no more than 8 hours a day or 40 hours a week (minister of manpower and transmigration regulation, 2011). the threshold limit value for dust as stated in minister of manpower and transmigration regulation (no. per.13/men/x/2011 about the threshold limit values of physical and chemical substances in the workplace air) must not exceed 3.0 mg/m3. if the workplace containts dust exceeding the threshold limit value, it is very risky to cause health problems, one of which is an ari. ina j med lab sci tech 2020; 2(1):42-49 merry sunaryo 4 8 effect of use of personal protective equipment on symptoms of acute respiratory tract infections the results of the analysis in this study showed that the use of personal protective equipment (masks) has a significant effect on symptoms of acute respiratory tract infections. these results can be known from the significance value or p-value 0.034 < 0.05. the risk level of the use of personal protective equipment against the occurrence of acute respiratory tract infections symptoms in the furniture workers based on an exp (b) value was 4.800. it can be interpreted that the workers who were not wearing protective mask have 4.8 times higher risk of experiencing acute respiratory tract infections symptoms compared to those who did. the results of this study are in line with the previous research (12) about pm10 on ari in the furniture industry workers. it was elaborated that there was a significant relationship between the use of personal protective (masks) on the occurrence of ari (p-value 0.001; ci (95%) 2.39 – 148.010) of the 43 furniture workers who have ari, almost all workers do not use ppe, such as masks or other nose caps. the results of this research are also consistent with another study. rizki (14) analyzed the risk factors of ari events on the workers in the block rubber production section of pt. sri trang, indonesia. it is stated that there was a significant relationship between the use of ppe (masks) and the incidence of ari in those workers with a p-value of 0.010 ci (95%) 1.375-4.253 (13). personal protective equipments can prevent harmful particles such as gas, steam, and dust that can enter the respiratory system. therefore, masks or respirators can be used as personal protective equipments to prevent the workers from inhaling dangerous particulates. conclusions based on the results of the study, the furniture industry workers have a risk of experiencing acute respiratory tract infections symptoms. the workers who were exposed to dust above the threshold limit value have 6 times more risk of experiencing acute respiratory tract infections symptoms compared to those who were exposed to dust below the threshold limit value. in addition, the workers who were not using personal protective equipment (mask) have 4.8 times more risk of experiencing acute respiratory tract infections symptoms compared to those who used personal protective equipment while working. the management of the furniture industry at semarang street, surabaya, should technically improve a routine supervision of the dust exposure control and their employee’ working performance, especially the use of personal protective equipments. the workers ina j med lab sci tech 2020; 2(1): 42-49 merry sunaryo 4 9 must obey the rule of using masks to protect themselves from ari. moreover, the employee’ health status should be checked regularly. lastly, a healthy lifestyle recommendation should also be implemented by the workers. conflict of interest there are no conflicts of interest. references 1. ap h. the relationship between work period and the use of personal protective equipment with vital lung capacity in the spinning unit workers 1 part of the ring frame pisma putra pekalongan textile. unnes j public heal. 2013;2(3):1-9. 2. trisnawati yt, juwarni. the relationship of smoking behavior of parents with ari in toddlers in the working area of the rembang health center in purbalingga district. scientific journal of public health. 2013;6(1):35-42. 3. sormin kr. the relationship between the characteristics and behavior of workers exposed to cotton dust and ari events at pt. unitex. depok; 2012. 4. rohilla a. sharma v, kumar s, sonu. upper respiratory tract infections: an overview. int j curr pharm res. 2013;5(3):1–3. 5. cahyana a, djajakusli r rm. factors associated with the occurrence of lung function disorders in coal miners pt. indominco mandiri east kalimantan in 2012. makassar; 2012. 6. halim f. the relationship between physical work environment factors and the occurrence of acute respiratory infection in tukrejo hamlet furniture industry workers, bondo village, bangsri district, jepara regency, central java province in 2012. jakarta; 2012. 7. ministry of health republic of indonesia. guidelines for control of acute respiratory infection. 2012. 8. who. the who air quality guideline. 2010. 9. aditama t. guidelines for controlling acute respiratory infections. bandung: cv dipenogoro; 2012. 10. wijayanto a. pm10 exposure and ari symptoms in batako manufacturing plant workers in banyuasin district. jakarta; 2008. 11. osman e, pala k. occupational exposure to wood dust and health effects on the respiratory system in a minor industrial estate in bursa/ turkey. int j occup med environ heal. 2009;22(1):43-50. 12. yusnabeti, wulandari aw, luciana r. pm10 and acute respiratory infection in furniture industry workers. makara journal of health reseach. 2010;4(1):25-30. 13. sudarmaji, anindya m. relationship between characteristics of workers and total dust levels with respiratory complaints in wood x industry workers in lumajang regency. journal of environmental health perspective. 2015;1:1-12. 14. rizki sr. analyzed the risk factors of ari events on the workers in the block rubber production section of pt. sri trang, indonesia. universitas sriwijaya. 2014. 64 the potential use of edta as an alternative to defibrination in preparing blood agar plates with human ab blood type on staphylococcus aureus culture dora dayu rahma turista1,2, eka puspitasari2, fanny kurnanda2 1biology education departement, faculty of teacher training and education, mulawarman university, samarinda, east borneo, indonesia 2departement of medical laboratory technology, stikes hutama abdi husada, tulungagung, east java, indonesia correspondence: dora dayu rahma turista, jl. kuaro, gunung kelua, samarinda ulu, paser, east borneo, indonesia zip code: 75119 email: doraturistaofficial@gmail.com received: february 6th, 2021 revised: march 8th, 2021 accepted: march 31th, 2021 published: april 28th, 2021 doi: 10.33086/ijmlst.v3i1.1923 abstract blood agar plates (bap) are composed of blood as one of the compositions. sheep’s blood is usually used, but since it is difficult to be obtained, human ab blood type was used as an alternative. in preparing bap, blood is defibrinated to lyse the blood clotting factors. blood clots can also be prevented by adding anticoagulants, such as ethylenediaminetetraacetic acid (edta). this study aims to investigate the potential use of edta as a substitute for defibrination in preparing bap with human ab blood type. this study employed a completely randomized design with true experimental method using staphylococcus aureus as the sample. the parameters were the number of colonies, types of hemolysis, and hemolysis zone. the results showed that the s. aureus grown on bap with edta-human ab blood type was 64 colonies (mean), produced β-hemolytic pattern, and 6 mm hemolytic zone. in contrast, the s. aureus grown on bap with defibrinated human ab blood type showed 82 colonies (mean), β-hemolytic pattern, and 5 mm hemolytic zone. there were significant differences in the number of colonies (0.000 < α) and hemolytic zones (0.02 < α). however, there was no difference in the hemolysis type (both treatments produced β-hemolysis). edta was possible to be used as a substitute for defibrination in preparing bap to assess the hemolysis type of s. aureus, but it might not be able to be used as a benchmark for counting the number of colonies and determining the hemolysis zone of s. aureus. keywords ab blood type, blood agar plates, defibrination, edta, staphylococcus aureus this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. mailto:doraturistaofficial@gmail.com https://journal2.unusa.ac.id/index.php/ijmlst/article/view/1923/version/2407 ina. j. med. lab. sci. tech. 2019; 1(1): 64–71 dora dayu rahma turista, et al. 6 5 introduction blood agar plates (bap) are universal culture media for growing various bacteria and also can be used as a differential medium to differentiate hemolytic bacteria from nonhemolytic bacteria. in general, bap contains defibrinated mammalian blood (1). defibrination refers to the process of removing clotting factors from blood to allow the perfect mixture of blood and medium. blood clots formation can also be prevented by adding anticoagulants, such as ethylenediaminetetraacetic acid (edta) (2). edta is frequently used since it is unable to distort blood cells (3). bap are also used to differentiate pathogenic bacteria based on the hemolytic patterns (4). standard bap are commonly prepared using sheep’s blood. however, since ensuring the sterility of sheep venous blood after the collection process is challenging, human a, b, o, or ab blood types can be used as alternatives (1). bap with human ab blood type offers easier identification of staphylococcus aureus due to its wider hemolytic zone when compared to bap with other blood types (1). s. aureus can induce red blood cells (rbc) lysis by four hemolysis types (α, β, γ, and δ). hemolysis is observed on the appearance of a clear zone around the bacterial colony on the bap. hemolysis is caused by a toxin called hemolysin. it is one of the virulence factor of s. aureus. it also determines the virulence factor of coagulase positive staphylococci (cps) and coagulase negative staphylococci (cns) (5). it plays a role in bacterial invasion and also helps in disengaging from immune response (6). edta has long served as an anticoagulant with competitive advantages as opposed to other anticoagulants. its major advantage is that it does not promote blood cells distortion and hence an excellent option for most hematological tests (3). to the best of our knowledge, the use of edta as a substitute for defibrination in preparing bap with human ab blood type as culture media for the growth of s. aureus has never been conducted. based on this basis, this study aims to investigate the potential use of edta for such usage. materials and methods the materials were blood agar base (merck) media, human ab blood type, edta vacutainer tube, pure culture of s. aureus, nacl 0.9%, and a 0.5 mcfarland solution. a completely randomized design was employed in this study. the s. aureus pure culture was obtained from the microbiology laboratory collection (stikes hutama abdi husada tulungagung, indonesia). human ab blood type was collected from sterilized venous blood. for the control group, the blood was defibrinated manually using marbles, while for the experimental group, it was directly collected ina. j. med. lab. sci. tech. 2021; 3(1): 64–71 dora dayu rahma turista, et al. 6 6 using an edta vacutainer tube (1.8 mg/ml blood). after the collection, the blood was prepared for making bap. s. aureus was inoculated on nacl 0.9% and the turbidity was matched to a 0.5 mcfarland solution for density comparison of a bacterial suspension with 1.5 x 108 cfu/ml. t streak method was used to inoculate s. aureus from nacl 0.9% on bap. s. aureus was incubated for 48 hours, then the type of hemolysis was observed by measuring the hemolytic zone. the s. aureus colonies were also counted. the results were analyzed using manova with 0.05 significance level. results the colonies of s. aureus were round, small, smooth, convex, and white after 48 hours of incubation on the bap with edtahuman ab blood type. in contrast, when grown on bap with defibrinated human ab blood type, s. aureus colonies were medium, smooth, convex, and white. the growth of s. aureus on the bap, both with edta-human ab blood type and defibrinated human ab blood type, was observed based on three parameters: a) the number of colonies; b) zones of hemolysis; and c) types of hemolysis (table 1). analysis using manova for number of colonies and hemolytic zones showed that the p-values were 0.000 and 0.02, respectively. table 1. the growth of s. aureus on the bap with edta-human ab blood type and defibrinated human ab blood type. blood type number of colonies hemolytic zones (mm) hemolysis types (α, β, γ, or δ) 1 2 3 1 2 3 1 2 3 edta-human ab blood type 62 65 65 6 6 6 β β β defibrinated human ab blood type 82 84 80 5 5 5 β β β discussion carbohydrates in blood are the main sources of nutrition to support bacterial growth. blood can boost the growth of certain bacteria (7). defibrination is only intended to remove blood clotting factors in erythrocytes rather than to eliminate the nutritional content (4). the mean number of s. aureus colonies grown on bap both with edta-human ab blood type and defibrinated human ab blood type is depicted in figure 1. ina. j. med. lab. sci. tech. 2019; 1(1): 64–71 dora dayu rahma turista, et al. 6 7 figure 1. the mean number of s. aureus colonies grown on bap both with edta-human ab blood type and defibrinated human ab blood type. the p-value of number of colonies was 0.000, indicating that there was a significant difference between the number of s. aureus colonies that grow on bap with edtahuman ab blood type and bap with defibrinated human ab blood type. the number of s. aureus colonies grown on bap with defibrinated human ab blood type was higher than grown on bap with edtahuman ab blood type. this occurs since edta has inhibitory power, although it is very low. in a previous study, the addition of edta to egg white lysozyme extract reduced the number of s. aureus and salmonella typhosa colonies (8). na-edta combined with nisin at certain concentrations and ph also inhibited the growth of gram-positive and gram-negative bacteria (9). combination of edta/tromethamine and oxytetracycline did not significantly affect staphylococcus hominis, whereas edta/tromethamine with several other antibiotics had low inhibitory effects on other antibiotic-resistant bacteria (10). edta is able to inhibit the growth of s. aureus because it can damage the membrane of bacterial cells. bacterial cell membranes consist of various types of lipids. the types of lipid in each bacteria (even in one species) are different. they differ due to environmental conditions (11). edta is believed to have a detergent-like mechanism to the chelating mechanism because the solution of the tetrasodium salt in edta can dissolve grease (12,13,14). this alkalinity, however, does not occur in low concentrations (15). this study underlines the abundance of s. aureus colonies which were able to grow on bap containing edta since there was only a small amount of edta in the blood (1.8 mg/ml blood). the concentration used in this study is extensevily used in clinical laboratories for chemical blood assay. the mean of s. aureus hemolytic zones grown on bap both with edta-human ab blood type and defibrinated human ab blood type is depicted in figure 2. 64 82 0 20 40 60 80 100 edta ab defibrinated ab n u m b e r c o lo n ie s in t h e p la te blood type ina. j. med. lab. sci. tech. 2021; 3(1): 64–71 dora dayu rahma turista, et al. 6 8 figure 2. the mean of s. aureus hemolytic zones grown on bap both with edta-human ab blood type and defibrinated human ab blood type. s. aureus colonies grown on bap with edta-human ab blood type showed wider hemolytic zones compared to those grown on bap with defibrinated human ab blood type. the complete hemolytic zones could only be observed after 48 hours of incubation. a 24-hours incubation did not display any hemolytic zones on all plates. bacterial colony characteristics on culture media after 48 hours of incubation are easier to distinguish (16). hemolytic zones are the clear areas around the bacterial colonies that grow on bap. they are determined by lifting the agar plates to a light source coming from behind (transmitted light), and then the diameter is measured by a ruler in millimeters (17). the p-value of hemolytic zones was 0.02, indicating that there was a significant difference between s. aureus colonies grown on bap with edta-human ab blood type and those grown on bap with defibrinated human ab blood type. s. aureus colonies on bap with defibrinated human ab blood type grew more than those on bap with edtahuman ab blood type. s. aureus was able to grow and produce hemolytic zones in both bap with edta-human ab blood type and bap with defibrinated human ab blood type. previous study showed that s. aureus can grow well in bap with all of the human blood types (a, b, ab, and o) (1). this is because the morphology and nutrient content in the blood are consistent (18). hemolysis type of s. aureus grown in bap both with edta-human ab blood type and defibrinated human ab blood type are presented in figure 3. both treatments showed the same type of hemolysis which was β-hemolysis. in a previous study, s. aureus was able to produce β-hemolysis when grown on bap with the four human blood types (a, b, ab, and o) (1). 6 5 4,5 5 5,5 6 6,5 edta ab defibrinated ab t h e h e m o ly si s z o n e a v e ra g e ( m m ) blood type ina. j. med. lab. sci. tech. 2019; 1(1): 64–71 dora dayu rahma turista, et al. 6 9 figure 3. hemolysis type of s. aureus on (a) bap with edta-human ab blood type and (b) with defibrinated human ab blood type. s. aureus can produce the four types of hemolysis: α, β, γ, and δ. certain s. aureus strains produce β toxin, which is a neutral sphingomyelinase (19). this toxin is produced in large quantities by some strains of s. aureus and secreted into culture media as an exotoxin with a molecular weights of 35,000 (20). β-hemolysis is also referred to as complete hemolysis. β-hemolysin is highly influential on human immunity. apart from hemolysin, there are other toxins that affect the virulence of s. aureus in humans. one of them is panton-valentine leucocidin (pvl), which is a cytotoxin that is highly toxic to human neutrophils (21). certain strains of s. aureus also produce the toxic shock syndrome toxin-1 (tsst-1) which triggers toxic shock syndrome (tss) (22). other strains produce additional exoproteins, including tsst-1, staphylococcal enterotoxins (sea, seb, secn, sed, see, seg, seh, and sei), exfoliative toxins (eta and etb ), and leucocidin (20). defibrination requires expertise and timeliness. an error may cause the blood to clot, leading to the imperfection of the media mixture and nutrients damage. edta could be used as a substitute for blood defibrination to grow s. aureus on bap since it was able to have s. aureus produce the same type of hemolysis in defibrinated blood bap, but with wider hemolytic zone and less colony growth. furthermore, edta can simplify the process of preparing bap as defibrination step was eliminated, thereby reducing the error factor in making bap. conclusions s. aureus could grow on bap with edta-human ab blood type and defibrinated human ab blood type. edta could be used as a substitute for defibrinated blood in the process of making bap to observe the hemolytic patterns of s. aureus, but could not be used as a benchmark for counting the bacterial colonies and measuring the hemolytic zones of s. aureus. a b ina. j. med. lab. sci. tech. 2021; 3(1): 64–71 dora dayu rahma turista, et al. 7 0 author contributions dora dayu rahma turista: conceptualization, methodology, analyzing, writing-reviewing and editing. eka puspitasari: data curation, supervision, validation. fanny kurnanda: visualization, investigation, validation. acknowledgment gratitude was expressed to the departement of medical laboratory (stikes hutama abdi husada tulungagung, indonesia) for the support in completing this study. conflict of interest no conflict. references 1. turista ddr., puspitasari e. the growth of staphylococcus aureus in the blood agar plate media of sheep blood and human blood groups a, b, ab, and o. j teknol lab. 2019; 8(1): 1-7. 2. russell fm, biribo ssn, selvaraj g, oppedisano f, warren s, seduadua a, et al. as a bacterial culture medium, citrated sheep blood agar is a practical alternative to citrated human blood agar in laboratories of developing countries. j clin microbiol. 2006; 44(9): 3346-51. 3. shabnam i, chuphal ds, joshi bc. ethylenediaminetetraacetic acid (edta) dependent pseudothrombocytopenia: a case report. j clin diagnostic res. 2014; 8(10): 4. yeh e, pinsky ba, banaei n, baron ej. hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests. plos one. 2009; 4(7). 5. moraveji z, tabatabaei m, shirzad aski h, khoshbakht r. characterization of hemolysins of staphylococcus strains isolated from human and bovine, southern iran. iran j vet res. 2014; 15(4): 326-30. 6. silva er da, boechat jud, martins jcd, ferreira wpb, siqueira ap, silva n da. hemolysin production by staphylococcus aureus species isolated from mastitic goat milk in brazilian dairy herds. small rumin res [internet]. 2005; 56(1-3): 271-5. 7. bonnet m, lagier jc, raoult d, khelaifia s. bacterial culture through selective and nonselective conditions: the evolution of culture media in clinical microbiology. new microbes new infect. 2020; 34. 8. melani d, radiati le, thohari i. the addition of edta (ethylenediaminetetraacetic acid) with egg white lysozyme extracts as the antimicrobial activity on salmonella sp and staphylococcus. 2013; 1-8. 9. khan a, vu kd, riedl b, lacroix m. optimization of the antimicrobial activity of nisin, na-edta and ph against gram-negative and gram-positive bacteria. lwt food sci technol [internet]. 2015; 61(1): 124-9. 10. farca am, nebbia p, re g. potentiation of antibiotic activity by edta-tromethamine against three clinically isolated gram-positive resistant bacteria. anin vitro investigation. vet res commun [internet]. 1994; 18(1): 1-6. 11. sohlenkamp c, geiger o. bacterial membrane lipids: diversity in structures and pathways. narberhaus f, editor. fems microbiol rev [internet]. 2016; 40(1): 133-59. 12. colobert l. [inactivation by lysozyme of the somatic antigen of heated pathogenic salmonella]. c r hebd seances acad sci. 1957;244(23): 2863-5. 13. noller ec, hartsell se. bacteriolysis of enterobacteriaceae. i. lysis by four lytic systems utilizing lysozyme. j bacteriol [internet]. 1961; 81(3): 482-91. 14. noller ec, hartsell se. bacteriolysis of enterobacteriaceae. ii. preand co-lytic treatments potentiating the action of lysozyme. j bacteriol [internet]. 1961; 81(3): 492-9. 15. gray gw, wilkinson sg. the effect of ethylenediaminetetra-acetic acid on the cell walls of some gram-negative bacteria. j gen microbiol [internet]. 1965; 39(3): 385-99. 16. oktari a, vanawati n, kurniawati ui. the optimization of human blood agar (hba) for streptococcus pneumonia growth. j phys conf ser. 2019; 1280(2): 3-9. ina. j. med. lab. sci. tech. 2019; 1(1): 64–71 dora dayu rahma turista, et al. 7 1 17. buxton r. blood agar plates and hemolysis protocols. in: american society for microbiology. 2016. p. 1-9. 18. makhro a, huisjes r, verhagen lp, mañúpereira m del m, llaudet-planas e, petkovakirova p, et al. red cell properties after different modes of blood transportation. front physiol. 2016; 7(jul): 1-21. 19. huseby m, shi k, kent bc, digre j, mengistu f, keun ss, et al. structure and biological activities of beta toxin from staphylococcus aureus. j bacteriol. 2007; 189(23): 8719-26. 20. dinges mm, orwin pm, schlievert pm. exotoxins of staphylococcus aureus. clin microbiol rev. 2000; 13(1): 16-34. 21. löffler b, hussain m, grundmeier m, brück m, holzinger d, varga g, et al. staphylococcus aureus panton-valentine leukocidin is a very potent cytotoxic factor for human neutrophils. plos pathog. 2010; 6(1): 1-12. 22. laham n al, mediavilla jr, chen l, abdelateef n, elamreen fa, ginocchio cc, et al. mrsa clonal complex 22 strains harboring toxic shock syndrome toxin (tsst-1) are endemic in the primary hospital in gaza, palestine. plos one. 2015; 10(3): 1-17. 1 analysis of apo–b serum level in balb/c mice hypercholesterolemic against temulawak extract (curcuma xanthoriza roxb) ryadatus sholihah1, m. shofwan haris1, yogi khoirul abror1 1department of medical laboratory technology, sekolah tinggi ilmu kesehatan ngudia husada madura, madura, indonesia correspondence: ryadatus sholihah, jl. r. e. martadinata, no. 45 mlajah, bangkalan, east java, indonesia zip code : 69112 email: ryads.lpm@gmail.com received: january 23, 2019 revised: march 24, 2019 accepted: march 26, 2019 abstract apo–b serum levels is the most predictive value for the incidence of atherosclerosis and coronary heart disease. curcuma xanthorrhiza roxb contains curcumin, which can be used as an antioxidant, anti–inflammatory and antihypercholesterol. the mechanism of curcumin contained in ginger to reduce cholesterol is due to its function as a cholagoga or bile stimulant. this study aims to determine the effect of temulawak extract on the levels of apo–b serum in hypercholesterolemia mice. this research were a true experimental study with a post–test only control group design carried out in february 2018. the extraction as much as 25 mice were divided into 5 groups where are group consisted of 5 mice. positive control group (k+) were treated with high cholesterol feed and water, negative control group (k–) were given standard feed and water, treatment group 1 (p1) were given high cholesterol food and 25mg/kg bw of curcuma extract for 14 days, treatment group 2 (p2) were treated with foods high in cholesterol and 50mg/kg bw of curcuma extract for 14 days and treatment group 3 (p3) treated with high cholesterol and ginger extract 75mg/kg bw for 14 days. examination of apo–b levels were measured using the spectrophotometric method. data were analyzed using one–way anova. the results showed that the average of apo–b level at (k+) was 209.7 ± 1.02 mg / dl, at (k–) 115.3 ± 1.04 mg / dl, at (p1) 180.4 ± 1.07 mg / dl, at (p2) 147.6 ± 1.12 mg / dl, at (p3) 119.1 ± 1.10 mg / dl. based on the results of statistical test it was found that there was a significant decrease in apo–b levels with p–value=0.001 at alpha 0.05 (p<α). keywords temulawak extract, apo–b serum levels, hypercolesterolemic. introduction coronary heart disease is still one of the diseases feared by most people because the mortality rate of coronary heart disease is still high, in both developed countries and developing countries. according to the data from the world health organization (who), ina j med lab sci tech 2019; 1(1): 1-7 ryadatus sholihsh, et al. 2 in 2012 around 17.5 million people, deaths in the world were caused by heart and blood vessel disease. it represented 31% of the causes of death worldwide. one of the laboratory markers that can be assessed the risk of cardiovascular disease is by checking cholesterol levels. laboratory tests that can be carried out to include blood total cholesterol levels, levels of low density lipoportein (ldl), high density lipoprotein (hdl), and apolipoprotein b (apo–b). apolipoprotein b is an amphipatic protein and the only protein known to require lipids for its secretions(1). in humans there are two apo– b isoforms, namely apo b–100 containing 4536 amino acids and apo b–48 containing 2152 amino acids. both types of apo–b have the function of binding to ldl receptors and also play a role in the cholesterol (2). the current dyslipidemia drug is nicotinic acid, fibric acid, hmg coa reductase inhibitor (statin), and bile acid binder. the use of statins in maximum doses such as atorvastatin (80 mg/day) has been shown to reduce ldl to 58% in hypercholesterolemic patients. the use of niacin at a dose of 3 g/day up to 4.5 gr/day can reduce ldl up to 25%. however, the use of statins and niacin can distrub side effects, such as hepatotoxic, myopathic and teratogenic effects on growing cells. peptic gastritis or ulcer is a common reason for not using niacin. hepatysis occurs in up to 3% in individuals treated with niacin (3). development of new drugs is needed which is safer, cheaper and more effective in improving lipid profiles. one of the natural ingredients that is widely used in society empirically is temulawak. curcuma xanthorrhiza is a native plant that is growing in indonesia and its resources are very abundant in madura, especially in bangkalan. bangkalan is a district that is well known for its richness of plants that can function as an alternative treatment. the part of this plant that is often used is the rhizome. this plant can grow well and adapt in the open or under the shade of trees to a shade level of 40%. several studies have shown that ginger can be used as a drug, antimicrobial, antibacterial and antioxidant (4). the chemical content of the temulawak could be distinguished from the starch fraction, which is the largest fraction, in the form of yellowish–white powder; the curcuminoid fraction, these oil yellow to reddish–giving component in the ginger rhizome and essential oil fraction, which is a component consisting of monoterpenes and sesquiterpenes derived compounds. curcumin is a fraction of curcuminoids, which have a broad–spectrum biological activity. curcumin in temulawak can work as an antioxidant, anti–inflammatory, and antihypercholesterol. the mechanism of ina j med lab sci tech 2019; 1(1): 1-7 ryadatus sholihsh, et al. 3 curcumin in ginger to reduce cholesterol is due to its function as a cholagoga or bile stimulant. the activity of the ginger rhizome is characterized by increasing production and secretion of bile; by increasing excretion of bile it will reduce high cholesterol levels (4). based on the background described above, the researcher intends to conduct a research on the effect of temulawak extract on apo– b levels in hypercholesterolemic mice materials and methods this type of research is true experimental, using quantitative analysis methods. the population of this study was male mice (mus musculus) of balb/c strain with a body weight of 20–30 grams, 2–3 months of age obtained from the faculty of veterinary medicine, airlangga university, surabaya. the reseach was conducted at january 2018. samples from this study were mice, male strain balb/c taken from the population randomly as many as 42 tails then grouped into 3 groups. material curcuma rhizome, quail egg yolk, 96% ethanol, 0.5% na cmc (natrium carboxymethyle cellulose), standard feed, drinking water, apo–b reagent kits from human companies. tool blades, blenders, analytic balance, waterbath shakers, buchner, rotary evaporator, glassware, 30x20 cm mouse cages, sonde, 1 ml syringe, centrifuge, bottles, surgical instruments, and photometers, sample cups, micropipets, tubes. procedure method of making ethanol extract of curcuma rhizome extraction was carried out by maceration method, by means of 75 grams of dried ginger powder put into maserator. then, as much as 96% ethanol solvent 400 ml were added and shaked for 1 hour to achieve a homogeneous condition in the waterbath shaker at a speed of 120 rpm for 1 hour. the solution was macerated for 24 hours at room temperature. after 24 hours, the solution was filtered or separated using a buchner filter. the residue from the filtering process was winded and remaserated again for 24 hours. the maceration was repeated up to 3 times. then, the mixed solution filtering results 1 to 3 with the rotary vacuum evaporator at 50 until a concentrated extract with a concentration of 100% was obtained. then, extract made a dose of 25 mg/kg bb, 50 mg/kg bb and 75 mg/kg bb using na cmc 0.5% solvent. animal preparation a total of 25 mice were weighed and recorded their weight, they are divided into 5 treatment groups. positive control group (k+) were treated with high cholesterol and drinking water, negative control group (k–) were treated with standard feed and drinking ina j med lab sci tech 2019; 1(1): 1-7 ryadatus sholihsh, et al. 4 water, treatment group 1 (p1) were given high cholesterol food and 25mg/kg bw of curcuma extract for 14 days, treatment group 2 (p2) were given high cholesterol food and 50 mg/kg bb of curcuma extract for 14 days and treatment group 3 (p3) were given high cholesterol and ginger extract 75 mg/kg bb for 14 days. each group was placed in a cage measuring 30x20 cm cage. each cage contained 5 mice and was adapted to be given a standard cp 511 feed and drinking water in ad libitum for 7 days as an acclimati zation period. after 7 days of acclimati zation, the mice were weighed again and the object of the study were mice weighing approximately 25 grams, mice that fit the criteria in groups k(+), p1, p2, and p3 were given quail egg yolks using a sonde to the stomach 1 time/day for 7 days. on the 8th day, one mouse was taken from each groups to be checked its total cholesterol levels. mice were fasted for 10 hours before being examined for total cholesterol levels. the blood of mice was taken using a 1ml syringe through the heart, then a total cholesterol level was examined using a poct cholesterol device. mice were included in the category where are hypercholesterolemia (cholesterol level above 82 mg/dl) from the examination of total cholesterol levels, then given the following treatment: a. positive control (k+) group was given high cholesterol and drinking water in ad libitum. b. the negative control group (k–) was only given standard feed and drinking water in ad libitum c. the first treatment group (p1) was given temulawak extract using a sonde to the stomach at a dose of 25 mg/kg bb for 14 days, giving standard feed and drinking water in ad libitum. d. the second treatment group (p2) was given temulawak extract using a sonde to the stomach at a dose of 50 mg/kg bb for 14 days, standard feed and drinking water in ad libitum. e. the third treatment group (p3) was given temulawak extract using a sonde to the stomach at a dose of 75 mg/kg bb for 14 days, standard feed and drinking water in ad libitum. the treatment for giving curcumin extract is given for 14 days. mice were fasted on the 15th day for 10 hours but the mice were still given a drink. mice were taken in 5 treatment groups or a total of 25 mice in the three treatment groups to be examined for apo–b levels. mice anesthetized using chlorophome. after losing consciousness, mice are dissected using a scalpel and take the blood. blood was taken using 1 ml syringe through the heart as much as 1 ml from each mouse. mice that have been taken for blood were not used anymore because they have been sacrificed (mice destroyed). ina j med lab sci tech 2019; 1(1): 1-7 ryadatus sholihsh, et al. 5 examination of apo–b levels the blood that has been taken from mice then centrifuged to take the serum, then the serum was inserted into prestige 24 i. analysis techniques analysis of data used in this study was to compare differences in apo–b serum levels in the three treatment groups using spss. if the data is normally distributed then it will use the parametric statistical test one way anova test, but if the data is not normally distributed, it using the kruskall–wallis non–parametric statistical test. results the results of the average, total cholesterol level in mice after giving a sonde of quail egg yolk for 7 days was 128 ± 1.02 lmg/dl, where the total cholesterol level was normal in mice ranging from 26–82 mg/dl. based on the average, it can be seen that mice have been hypercholesteloremic. after being given treatment in accordance with the treatment group for 14 days, the average examination results of apo–b levels were 209.7±1.02 mg/dl in the positive control group (k+); 115.3±1.04 mg/dl in the negative control group (k–); 180.4±1.07 mg/dl in treatment group 1 (p1); 147.6 ± 1.12 mg/dl in treatment group 2 (p2) and 119.1±1.10 mg/dl in treatment group 3 (p3). the lowest average was found in the treatment group 3 given temulawak extract at a dose of 75 mg/kg bb. fig 1. the effect of temulawak extract to apo–b serum levels discussion effect of high fat feeding on total cholesterol levels in the positive control group (k+) was 128±1.02 mg/dl. the results of this study are in line with the research conducted by santiago (5) which proved that each mouse given a high–fat feed diet had dyslipidemia or increased levels of lipid profiles. high fat feed is given for 2 weeks, this 0 50 100 150 200 250 k(+) k(-) p1 p2 p3 a p o -b s e ru m l e v e ls groups the effect of temulawak extract to apo-b serum levels ina j med lab sci tech 2019; 1(1): 1-7 ryadatus sholihsh, et al. 6 means that the mice remain in a state of dyslipidemia. the results of this study showed a higher lipid profile in the positive control group (k+) compared to the treatment group. this shows a high–fat diet has an positives effect on lipid profiles. fats contained in food will be broken down into cholesterol, triglycerides, phospholipids and free fatty acids when digested in the intestine. these four fat elements will be absorbed from the intestine and enter into the blood. the increase in absorption of fat will cause the condition of hypercholesterolemia. the higher the level of fat in the food, the higher the level of lipid profiles total cholesterol and apo b in the body (6). effect of temulawak extract on apo–b levels the results of apo b levels in the positive control group (k+) tended to be higher than in the treatment group (p1, p2 and p3), there were significant differences in apo b levels in each groups, where the higher dose was given, the value apo b levels will decrease. the temulawak extract dose given is 25 mg/kg bb, 50 mg/kg bb and 75 mg/kg bb respectively. this is in line with the statement that the content of flavonoids in ginger in the form of hesperidin can inhibit the secretion of apo b–100 by the liver, thus affecting the level of apo b in the blood. hesperidin is the main flavonoid in temulwak has been shown to be hypocholesterolemic. hesperidin works through a mechanism of inhibition of the enzyme activity hmgcoa reductase and acat and inhibits the secretion of apolipoprotein b by the liver (7). curcumin also contains an active ingredient, namely curcumin, curcumin plays a role in stimulating cholesterol–7a– hydroxylase hepatic enzyme activity. this enzyme contained in liver cells catalyzes the change in cholesterol into bile salts. the increase activity of this enzyme shows an increase in cholesterol catabolism. the reaction of 7a–hydroxylase in cholesterol biosynthesis is the first step that must be present in bile acid biosynthesis. as a result of stimulation of this enzyme by curcumin, the change in hepatic cholesterol into bile salts increases, resulting in reduced cholesterol levels in the liver. in order to meet the cholesterol needs, the number of ldl receptors in the liver will be increased, so that there is an increase in ldl uptake. the decreased cholesterol levels, ldl and increased levels of hdl plasma cholesterol (7). in a previous study conducted by (4), the study concluded that ginger extract at a dose of 100 mg/kg bb and 400 mg/kg bb could reduce total cholesterol levels by >20% in rats given high fat feed, where total cholesterol consisted of ldl, hdl and triglycerides. ina j med lab sci tech 2019; 1(1): 1-7 ryadatus sholihsh, et al. 7 conclusions based on the results of research that has been conducted on the effect temulawak extract on apo–b levels in mice, it can be concluded that there was a significant difference in the decrease in apo–b levels : in the positive control group (k+) tended to be higher than in the treatment group (p1, p2 and p3), there were significant differences in apo b levels in each groups, where the higher dose was given, the value apo b levels will decrease. where is the lowest decrease occurred in the treated group 3 with an extract dose of 75 mg/kg. acknowladgements acknowledgments are given to several parties who have helped and been involved, including: 1. dr. mustofa haris as chairman of the ngudia husada madura foundation which has provided research funding. 2. dr. m. hasinuddin, s.kep., ns, m.kep as chair of the school of health ngudia husada madura which has given us space to be able to conduct research. 3. the second and third colleagues who have helped in the completion of this article. conflict of interest there are no conflicts of interest. references 1. setiawan hg, kaligis ahm, assa ya. description of serum apolipoprotein b (apo-b) levels in lacto-ovo vegetarians. ebiomedik. 2017;5(1):2–5. 2. cesar tb, aptekmann np, araujo mp, vinagre cc, maranhão rc. orange juice decreases lowdensity lipoprotein cholesterol in hypercholesterolemic subjects and improves lipid transfer to high-density lipoprotein in normal and hypercholesterolemic subjects. nutr res [internet]. elsevier inc.; 2010;30(10):689–94. available from: http://dx.doi.org/10.1016/j.nutres.2010.09.006. 3. sargowo d. lipid , dyslipidemia and cv impact : the urgency to treat. 2012;0–18. 4. anggraini s. pengaruh ekstrak etanol rimpang temulawak (curcuma xanthorrhiza roxb.) terhadap kadar kolesterol total pada tikus putih hiperlipidemia. universitas muhammadiyah surakarta. 2012;9. 5. santiago vaj, jayachitra j, shenbagam m. & nalini n. d-limonene attenuates bloodpresssure and impoves the lipid and antioxidant status in high fat diet and l-name treated rats.j. pham. 11,pp.752-758.2010. 6. hermawati, manalu w, suprayogi a, astuti da. dietary fiber supplements in the diet to improve the blood lipid parameters of hypercholestrolemia mice. makara seri kesehatan. 2013;17(1):1-9. 7. murfian aa, aznam n. the effect of giving instant ginger on the total cholesterol level of male white rats wistar hypercholestrolemia strain. jurnal kimia dasar. 2017;6(4):159-466. 60 collagen-vi specific primer design identification in rats (rattus norvegicus) pancreas endin nokik stujanna1, sri suci ningsih1, rizkyana avissa1, novi putri ayu1, zahra nurussofa1, dewi jantika djuarna1, rini latifah1, wawang setiawan sukarya1 1faculty of medicine, muhammadiyah prof dr hamka university, tangerang, banten, indonesia correspondence: endin nokik stujanna, faculty of medicine, muhammadiyah prof dr hamka university, raden patah, parung serab, ciledug, tangerang, banten, indonesia zip code: 13460 email: endin_stujanna@uhamka.ac.id received: 27 november, 2021 revised: 15 january, 2022 accepted: 22 february, 2022 published: april 28, 2022 doi: 10.33086/ijmlst.v4i1.2515 abstract type 2 diabetes mellitus, the most common diabetes type characterized by hyperglycemia, is caused by abnormal secretion and activity of pancreatic insulin enzymes. the extracellular matrix (ecm) plays a vital role in keeping β pancreatic cells intact and undissociated. the ecm in the pancreas can play a role in influencing insulin function and production. the most abundant ecm in the pancreas is collagen type vi. collagen type vi has an essential role in the survival of pancreatic islet cells, including pancreatic β cells. nowadays, polymerase chain reaction (pcr) technology is widely utilized for molecular biology analysis. one of the most critical factors for successful polymerase chain reaction (pc)r is designing the correct specific pcr primers. the objective of this study was to design a specific primer for collagen vi in the pancreas of rattus norvegicus. the primer was designed and analyzed using mega.11, primer three-plus, and primer-blast. five primer pairs were analyzed based on the characteristics of primer length, product amplicon length, tm value, gc percentage, and secondary structure. primer pair 3 (f:5’tgtttggctttgtcgcgggc-3’ and r:5’ttgttgctgccgacactggc-3’); col6a2 (f:5’tgtggtcaacaggctgggcg-3’ and r:5’tctggcgccggctctctttg-3’) were considered as the best primer for the collagen vi expression detection from the pancreas of rattus norvegicus, which produce amplicon about 250pb and 245pb, respectively. keywords diabetes mellitus, primer, collagen vi, rattus norvegicus. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 6 1 introduction type 2 diabetes mellitus (t2dm), a global major health problem, ranks first as a non-communicable disease that causes the highest death in indonesia (1). dmt2 is closely related to the insulin hormone. this hormone is produced by pancreatic β cells, whose primary function for maintaining glucose homeostasis. deficiency of insulin secretion by pancreatic β cells is one of the characteristics of the emergence of t2dm, which results in high blood glucose levels (2). the glucose stimulates cellular signaling pathways to synthesize and translocate insulin granules. this series of processes is supported by the pancreatic cell’s ability to produce an oscillatory effect that triggers insulin secretion. these oscillatory effects play a role in synchronizing secretory activity between pancreatic cells (3). the molecular and cellular mechanisms can be studied further by using an in vitro model using 3d spheroidal cultures of the igl cell line. igl is a cell derived from a subculture of pancreatic β cells of rats capable of expressing insulin-glase in response to high environmental glucose levels (4). research by suzuki et al. (4) has proved that igl cell (insulin-glase) spheroids exhibit an oscillatory effect of insulin secretion in response to high environmental glucose levels. furthermore, high glucose stimulation in the 3d culture of igl cells showed a similar oscillatory effect of insulin secretion as the pancreatic islet of langerhans in vivo. the 3d culture model was considered more representative of natural conditions in vivo (5). the synchronization between cells in the pancreatic β islet will be weak if the cells dissociate. the extracellular matrix (ecm) plays a vital role in keeping pancreatic β cells intact and undissociated. ecm in the pancreas can play a role in protecting cells, influencing insulin function and production, and influencing the susceptibility of pancreatic islets toward cytokines. ecm can be found in large quantities in the pancreas, including laminin, collagen type iv, and collagen type vi (6). the component of type vi collagen was higher than collagen types 1 and 4 in adult human pancreas research (7). another research stated that the administration of type vi collagen in the pancreatic islets treatment could increase the viability of the pancreatic islet cells when treated in vitro (8). it indicates that type vi collagen has an essential role in the survival of pancreatic islet cells, including pancreatic cells. however, how type vi collagen functions on pancreatic islet function and insulin expression is still unknown. the role of type vi collagen was explored by looking at the expression pattern of type vi collagen at the gene and protein levels. expression of type vi collagen at the gene level can be detected by the polymerase chain reaction (pcr) method. dna primer ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 6 2 is dna sequences complementary to the sequence to be amplified. therefore, the primer used in the pcr process serves as a barrier to the target dna fragment to be amplified. a pair of primers consists of a forward primer and a reverse primer (9). the success of dna amplification depends on the accuracy of the primer. the primer parameters include melting temperature (tm), percentage of g and c (% gc), 3′dimer, stability, repeats, and hairpins. the primer designed must meet the criteria used in the pcr process and produce products according to the desired regional range (10). this research was aimed to design the primer with good specificity to detect the expression of the cx36 gene (type vi collagen gene) in the pancreas of rattus norvegicus by pcr technique. materials and methods this research used bioinformatics software online instruments for designing primer, namely primer3plus (website: https://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi), integrated dna technologies (idt), blast (website: https://blast.ncbi.nlm.nih.gov/blast.cgi) at the national center for biotechnology information (ncbi), and molecular evolutionary genetic analysis (mega). the material used in this research was dna sequence data of collagen vi r. norvegicus downloaded in fasta format from ncbi database. the method consists of searching gene sequences on the ncbi site in the gene sub-search, selecting the “refseq” menu, and selecting “rattus norvegicus” in the organism option (figure 1). figure 1. collagen vi gene search result in ncbi ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 6 3 the gene sequences obtained may have several isoforms. mega.11 application was used to carry out alignment to obtain conserved sequences from these isoforms. then the conserved sequences were used for primer design with primer3plus (figure 2). figure 2. primer2plus front page display ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 6 4 figure 2 shows primer2plus setting. some settings on the “general and advance settings” menu for the desired primer criteria. for example, it was related to the primer and amplicon size, tm, gc composition, etc. then select “pick primers” to get the primer selection. the results displayed were several choices of primer pairs (forward and reverse) accompanied by data on primer length, product or amplicon length, tm, %gc (11). in order to designing primers for pcr, two types of secondary structures should be analyzed, namely dimers and hairpins. the primers obtained were further analyzed with idt to determine how big the primers were to form dimer and hairpin primer. furthermore, the primers obtained were further analyzed with primerblast to determine the specificity of the primer to the target gene. figure 3. display of primer-blast. results results the collagen vi gene (col6a1: id 294337 and col6a2: id 361821) in rattus norvegicus is located on chromosome number 20 on nc_051355.1. this gene, which has two isoforms, is aligned using the mega application. the primer design result was carried out by using primer3plus and further analyzed with idt and primerblast, which are summarised in the following tabel 1. discussion the pcr method has been widely used in the biomolecular and medical world. good primer design is an important factor for a successful pcr process. several essential characteristics that need to be considered in designing pcr were primer length, gc composition, melting temperature (tm), dimer primer, and hairpin primer (12). the recommended primer length for optimum pcr application is 18-30 base length (bp). if ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 6 5 the primer is too short, it will cause nonspecific amplification, while too long primer tends to form secondary structures such as hairpin loops (13). all primers designed for collagen vi had a length of 20 bp, which was categorized as a good primer. the gc composition in the primer was also essential to note. the gc proportion was the percentage that described the ratio of g and c nucleotides presented in the primer sequence. the primer gc proportion was 3080%. however, the recommended optimal gc proportion was at a value of 40-60% (12). the primer design results obtained had a value of 60-65%. although this design value was slightly higher, the higher gc composition was considered less adverse on the pcr results (10). the nucleotide composition was also closely related to the melting temperature (tm) value. the reaction specificity of the pcr was highly dependent on the primer tm value. the recommended optimal tm value was in the range of 50-60°c. the two primers should have the same tm value or only had a difference of about 2-3°c. different tm can cause a decrease in the primer annealing efficiency (13). all primer pairs obtained from the collagen vi primer design had a tm value difference of not more than 3°c between the forward and reversed primer. in addition, almost all primer pairs had tm values within the recommended range of 58.8-59.6°c. only col6a1 1 (forward, reverse), 2 (reverse), 3 (reverse), and 4 (forward) primer pairs have a tm value slightly higher than the recommended value of 60.2°c, and only col6a2 primer pairs 2 (reverse), which had a tm value slightly higher than the recommended value of 60.0°c. an important factor to be considered in designing a good primer was the possibility of primer forming a secondary structure. the secondary structure can be formed from one primer sequence itself that forms a hairpin loop structure or between pairs of primer that form dimer primer or heterodimer (12,13). this secondary structure should be avoided because it can reduce the specificity of the primer. all primers could form self-dimer or heterodimer structures based on the primer design results. the primer pair with the lowest selfdimer, heterodimer, and hairpin numbers was the number four primer pair with selfdimer and hairpin values 1-2 and heterodimer 15. the five pairs of primer obtained were further analyzed for specificity using primerblast (11). all tested primer pairs showed specific results with the col6a1 gene in rattus norvegicus (>xm_215375.8 rattus norvegicus collagen type vi alpha 1 chain (col6a1), mrna) with a product amplicon length of 247pb. ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 6 6 table 1. collagen vi primer design results gene name gene id primer sequences primary length (bp) product amplico n length (bp) tm (°c) gc (%) selfdimer hetero dimer hairpin blast result col6a 1 >lcl|nc_05 1355.1_cds _xp_2153 75.5_1 f-ttggctttgtcgcgggctcc r-ttgtggctgccgacactggc 247 f-20 r-20 f-60,2 r-60,2 f-65 r-65 f-10 r-12 13 f-4 r-3 >xm_215375.8 predicte d: rattus norvegicus collagen type vi alpha 1 chain (col6a1), mrna. 247bp. f-tgaccggctgagcaaggacg r-atgtgccgccagccatccac 179 f-20 r-20 f-59,1 r-60,0 f-65 r-65 f-8 r-12 17 f-4 r-1 >xm_215375.8 predicte d: rattus norvegicus collagen type vi alpha 1 chain (col6a1), mrna. 179bp. ftgtttggctttgtcgcgggc rttgtggctgccgacactggc 250 f-20 r-20 f-59,0 r-60,2 f-65 r-65 f-8 r-9 13 f-5 r-3 >xm_215375.8 predicte d: rattus norvegicus collagen type vi alpha 1 chain (col6a1), mrna. 250bp. ftggctggcggcacattcacc racgttgagctggtcggagcc 223 f-20 r-20 f-60,2 r-59,1 f-60 r-65 f-13 r-10 15 f-1 r-2 >xm_215375.8 predicte d: rattus norvegicus collagen type vi alpha 1 chain (col6a1), mrna. 223bp. ftgaccggctgagcaaggacg rtccggtgaatgtgccgccag 187 f-20 r-20 f-59,1 r-59,7 f-65 r-65 f-8 r-10 16 f-4 r-2 >xm_215375.8 predicte d: rattus norvegicus collagen type vi alpha 1 chain (col6a1), mrna. 187bp. col6a 2 >lcl|nc_05 1355.1_cds _np_0010 94211.1_1 f-tgagctggcgctatggtggc r-acgtgctgccggatctgctg 172 f-20 r-20 f-59,5 r-59,8 f-65 r-65 f-12 r-10 15 f-2 r-4 >xm_006256300.4 predi cted: rattus norvegicus collagen type vi alpha 2 chain (col6a2), transcript variant x1, mrna. 172bp. https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291 https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291 https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291 https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291 https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958758291 https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483 ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 6 7 gene name gene id primer sequences primary length (bp) product amplico n length (bp) tm (°c) gc (%) selfdimer hetero dimer hairpin blast result >lcl|nc_05 1355.1_cds _xp_0062 56362.1_2 f-ttccgcaggggcaccttcac r-acggcaaagagccggatgcc 188 f-20 r-20 f-59,3 r-60,0 f-65 r-65 f-9 r-9 19 f-4 r-1 >xm_006256300.4 predi cted: rattus norvegicus collagen type vi alpha 2 chain (col6a2), transcript variant x1, mrna. 188bp. ftgtggtcaacaggctgggcg rtctggcgccggctctctttg 245 f-20 r-20 f-59,6 r-59,4 f-65 r-65 f-11 r-7 13 f-5 r-1 >xm_006256300.4 predi cted: rattus norvegicus collagen type vi alpha 2 chain (col6a2), transcript variant x1, mrna. 245bp. fttccgcaggggcaccttcac rttgggggccacggcaaagag 197 f-20 r-20 f-59,3 r-59,5 f-65 r-65 f-9 r-9 16 f-4 r-1 >xm_006256300.4 predi cted: rattus norvegicus collagen type vi alpha 2 chain (col6a2), transcript variant x1, mrna. 197bp. fatgcccagcagcaggaagcc rtaggccaccatagcgccagc 248 f-20 r-20 f-59,7 r-58,9 f-65 r-65 f-14 r-15 18 f-5 r-1 >xm_006256300.4 predi cted: rattus norvegicus collagen type vi alpha 2 chain (col6a2), transcript variant x1, mrna. 248bp. https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483 https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483 https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483 https://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=1958757483 ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 6 8 figure 4. position of primers in the gene col6a2 thus, primer pair 3 was considered the closest to a good primer category for detecting collagen vi expression from the pancreas of rattus norvegicus at the gene level by pcr method with amplicon length. 250pb product. as for the col6a2 gene in rattus norvegicus (>xm_006256300.4 rattus norvegicus collagen type vi alpha 2 chain (col6a2), mrna) with a product amplicon length of 245pb. besides all the considerations above, the selection of designed primer outcome must be considered after performing experimental tests in the laboratory. researchers can redesign the primer if the pcr results are not good by changing the primary parameter criteria settings. for example, change the gc proportion value, shift the tm number, increase the resulting amplicon length to 150-259pb [12], or use other primary design applications available online for free. ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 6 9 conclusions the applications mega.11, primer3plus, and primer-blast can facilitate designing the specific collagen vi primers. col6a1 gene primer pairs 3 (f:5’tgtttggctttgtcgcgggc-3’ and r:5’-ttgttgctgccgacactggc-3’); col6a2 (f:5’tgtggtcaacaggctgggcg-3’ and r:5’-tctggcgccggctctctttg-3’) are the best primer in this study for collagen vi expression detection in rattus norvegicus pancreas and produce amplicon around 250pb and 245pb, respectively. future research is required to analyze the specificity of the primer design. author contributions endin nokik stujanna: substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data, drafting the article or revising it critically for important intellectual content. sri suciati ningsih: substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data. rizkyana avissa: substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data. novi putri ayu: substantial contributions to acquisition of data. zahra nurussofa: substantial contributions to conception and design dewi jantika djuarna: substantial contributions to conception and design rini latifah: substantial contributions to conception and design. wawang setiawan sukarya: final approval of the version to be published acknowledgments the authors would like to thank all supporting staff at the faculty of medicine and thank lemlit uhamka. conflict of interest the authors declare that there is no conflict of interest. references 1. soelistijo sa, lindarto d, decroli, permana h, sucipto kw, kusnadi y, budiman, ikhsan r. pedoman pengelolaan dan pencegahan diabetes melitus tipe 2 dewasa di indonesia 2019 guidelines for the management and prevention of adult type 2 diabetes mellitus in indonesia 2019. perkumpulan endokrinologi indonesia. 2019:1–117. 2. rachdaoui n. insulin: the friend and the foe in the development of type 2 diabetes mellitus. international journal of molecular sciences. 2020:21(5). https://doi.org/10.3390/ijms21051770. 3. tengholm a, gylfe e. oscillatory control of insulin secretion. molecular and cellular endocrinology. 2009:297(1–2), 58–72. https://doi.org/10.1016/j.mce.2008.07.009. 4. suzuki t, kanamori t, inouye s. quantitative visualization of synchronized insulin secretion from 3d-cultured cells. biochemical and biophysical research communications. 2017: 486(4); 886–892. https://doi.org/10.1016/j.bbrc.2017.03.105. ina. j. med. lab. sci. tech. 2022; 4(1): 60–70 endin nokik stujanna, et al. 7 0 5. suzuki t, kanamori t, inouye s. novel technology for studying insulin secretion: imaging and quantitative analysis by a bioluminescence method. yakugaku zasshi. 2020:140(8); 969–977. https://doi.org/10.1248/yakushi.20-00012-2. 6. llacua la, faas mm, de vos p. (2018). extracellular matrix molecules and their potential contribution to the function of transplanted pancreatic islets. diabetologia. 2018: 61(6); 1261–1272. https://doi.org/10.1007/s00125-0174524-8. 7. hughes sj, clark a, mcshane p, contractor hh, gray dwr, johnson prv. (2006). characterization of collagen vi within the isletexocrine interface of the human pancreas: implications for clinical islet isolation? transplantation. 2006: 81(3); 423–426. https://doi.org/10.1097/01.tp.0000197482.91227. df. 8. llacua la, hoek a, de haan bj, de vos p. collagen type vi interaction improves human islet survival in immunoisolating microcapsules for treatment of diabetes. islets. 2018; 10(2), 60– 68. https://doi.org/10.1080/19382014.2017.1420449. 9. chuang ly, cheng yh, yang ch. specific primer design for the polymerase chain reaction. biotechnol lett. 2013;35(10):1541–9. 10. álvarez-fernández r. explanatory chapter: pcr primer design. methods enzymol. 2013;529:1– 21. 11. ye j, coulouris g, zaretskaya i, cutcutache i, rozen s, madden tl. primer-blast: a tool to design target-specific primers for a polymerase chain reaction. bmc bioinformatics. 2012;13:134 12. basu c. preface. pcr primer design. methods mol biol. 2015;1275:vii 13. apte a, daniel s. pcr primer design. cold spring harb protoc. 2009;4(3):1–10. 38 liver histopathological change and malondialdehyde level of rattus norvegicus on administration of curcuma zedoaria and paracetamol toxic dose putu oky ari tania1, puja ayu misuari2, satya yudhayana2, ketut ayesha edelwise prayoga2 1biomedical department and biomolecular research, faculty of medicine wijaya kusuma surabaya university, indonesia 2faculty of medicine wijaya kusuma surabaya university, indonesia correspondence: putu oky ari tania, jl. dukuh kupang xxv no. 54, kecamatan dukuh pakis, surabaya, east java, indonesia zip code: 60225 email: putuoky@uwks.ac.id received: february 8th, 2021 revised: march 28th, 2021 accepted: march 29th, 2021 published: april 28th, 2021 doi: 10.33086/ijmlst.v3i1.1927 abstract high doses of paracetamol create necrosis in the liver and produce free radicals. when liver function decreased in a long time, it will lead to severe liver damage and it will be irreversible. rhizome of curcuma zedoaria has the potential effect as an antioxidant. it is assumed that its properties inhibit the formation of free radicals which formed from toxic doses of paracetamol. the aim of this study was to examine the histological structure of the liver and to determine malondialdehyde (mda) levels in the administration of c. zedoaria toxic dose and paracetamol on the rats (rattus norvegicus). the study was used twenty-four rats divided into four groups (positive control: carboxy methyl cellular (cmc) 0.5%; negative control: paracetamol 1.35g/kg body weight; treatment group 1 (t1): c. zedoaria 105mg/200g and paracetamol 1.35g/kg body weight 2 hours later, and treatment group 2 (t2): paracetamol 1.35g/ kg body weight and c. zedoaria 105 mg/200g 2 hours later). the kruskall-wallis test results showed mda level did not significantly different between groups (p = 0.087). hepatocellular changes were observed descriptively with hematoxylin-eosin staining. positive control showed greater hepatocellular changes rather than other groups, hepatocyte cells were enlarged with cytoplasm showing eosinophilic granules infiltrates, enlarged irregular nuclei, nucleolus prominent, there are many necrosis cells. keywords antioxidant, curcuma zedoaria, hepatocyte, malondialdehyde, paracetamol. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. https://journal2.unusa.ac.id/index.php/ijmlst/article/view/1927/version/2411 ina. j. med. lab. sci. tech. 2021; 3(1): 38–46 putu oky ari tania, et al. 3 9 introduction white turmeric (curcuma zedoaria) are found in tropical climates, which are often used as traditional medicine (1). rhizome of c. zedoaria is widely used for the treatment of cancer, indigestion, chronic pelvic inflammation, blood clots, coronary heart disease and anemia (2). c. zedoaria has bioactive components of flavonoids, curcuminoid polyphenols, terpenoids and essential oils. the polyphenols are thought to be a potential antioxidants of this plant (3). the phenolic or polyphenols were include to flavonoids, which are known to be modifiers biological response, functions as metal chelators and eradicating free radicals (4). the ability of a component to capture free radicals and thus inhibit the oxidation process is called antioxidants (5). free radicals including various other oxidants such as reactive oxygen species (ros) have detrimental effects and induce oxidative stress (6). according to ji et al (2), oxidative stress occurs when there is an imbalance between the formation and elimination of ros. free radicals are highly reactive and damage important molecules such as nucleic acids, proteins and lipids (2). lipids will produce peroxidation when a free radical attack and break down the chain hydrogen from methylene, and form lipid radicals. when oxygen binds to lipid radicals form peroxyl lipid radicals which eventually become malondialdehyde (mda) (6). inappropriate use of paracetamol or acetaminophen will induce necrosis and death in liver cells (7). toxic doses of paracetamol leads to the production of the reactive metabolite n-acetyle-pbenzoquinone imine (napqi) (8). napqi escalation and degradation of the amount of natural antioxidant hepatic gluthathione create hepatotoxicity (7). the hepatoprotector effect of c. zedoaria to liver damage by paracetamol has been published yet, but the effect of administration of c. zedoaria and paracetamol toxic doses simultaneously has not been widely observed. the aims of this study was to examine the histological structure of the liver in the administration of c. zedoaria and paracetamol, conversely paracetamol and c. zedoaria, as well as the mda level of rattus norvegicus. materials and methods plants the preparation of the extract from c. zedoaria and the experiment conducted in january, 2020. the c. zedoaria rhizome washed and cut into smaller pieces, then dried in a cold place. the pieces are mashed, filtered, until it obtains to a yellowish powder. the c. zedoaria powder then can be macerated using ethanol for 72 hours to extract all the compound in c. zedoaria that ina. j. med. lab. sci. tech. 2021; 3(1): 38–46 putu oky ari tania, et al. 4 0 dissolved with polar solvent (ethanol absolute). futhermore, the extract was obtained by evaporation with a vacuum evaporator, extraction with evaporator temperature 60o c and 100 rpm (9). dose used in this study were 105 mg/ 200 g body weight (10). animals the total of 24 adult male rats (rattus norvegicus) were divided into 4 groups, namely 2 control groups (positive control and negative control), and 2 treatment groups (treatment 1 and treatment 2). the treatment was carried out for 7 days in the animal experimental laboratory of the faculty of medicine, wijaya kusuma university, surabaya. measurement of mda levels was carried out at the biochemistry laboratory of the faculty of medicine, wijaya kusuma surabaya university. histopathological analysis was carried out at laboratory of pathology anatomy, airlangga university. this study received ethical approval by komisi etik penelitian kesehatan faculty of medicine, wijaya kusuma surabaya university (reg. no. 06/sle/fk/uwks/2020. experimental design hepatotoxicity effect in this study was performed by paracetamol at dose 1.35g/kg body weight (11). oral route was used to paracetamol and c. zedoaria administration, because the treatment was done repeatedly for 7 days and consider this route was safer, it took slower onset of action which is important in drug effect experiment. used of paracetamol single doses was performed in positive control. in negative control was indicated as normal indicator was administered by carboxyl methyl cellulose (cms) 0.5%. the treatment groups divided into 2 groups, which represent the effect of administration c. zedoaria extract at dose 105mg/ 200 g body weight and paracetamol at dose 1.35g/ kg body weight. the treatment for group 1 (t1) was administered by paracetamol and 2 hours later was given c. zedoaria extract. the treatment for group 2 (t2) was given c. zedoaria extract and administered by paracetamol 2 hours later. this study was conducted in 7 days. sample analysis/ sampling analysis on the day 8th after treatment, liver tissues were collected. liver tissue were homogenized and measure of mda levels by spectrophotometer. spectrophotometric determination of liver malondialdehyde (mda), according to the thiobarbituric acid (tba). histopathological analysis the livers were collected in formaldehyde solution (10%). these samples were paraffined embedded, sectioned of 5 µm, and stained by hematoxylin-eosin staining. histological changes were observed by light microscope descriptively. ina. j. med. lab. sci. tech. 2021; 3(1): 38–46 putu oky ari tania, et al. 4 1 statistical analysis/research approach the data were analyzed using the nonparametric kruskal-wallis test to analyze liver mda levels, while descriptive analysis was used to observe changes in the structure of cells and tissues in the liver. results the differences in cell structure and tissue in the liver in each group are observed and analyzed descriptively. the results of this observations are presented in figure 1. histopathological changes observed by paraffin embedding methods use hematoxylin-eosin staining. nucleus were stained by hematoxylin colored blue, and cytoplasm were red stained by eosin. figure 1. hepar histopathology in (a) negative control, (b) positive control, (c) treatment 1/ t1, (d) and treatment 2/ t2: normal cell (thick red arrow) and nucleus (thick black arrow). positive control: observed enlarged hepatocytes with nucleus (thick red arrow), eosinophils granules were found within cytoplasm (thin black arrow). cell death and necrosis were found (thin red arrow) with inflammatory cells (thick black arrow). irregular shaped of nucleus (thick yellow arrow). t1: showed enlarged nucleus of hepatocytes (thick black arrow), necrosis hepatocytes with pale and fragmented nucleus (thick red arrow) and granule eosinophils within cytoplasm (thin red arrow). t2: showed enlarged nucleus of hepatocytes (thick black arrow), more death or necrosis cell than t1 (thick red arrow) and prominent of nucleolus (thin red arrow), found hepatic vacuolization (thin yellow arrow). a b c d ina. j. med. lab. sci. tech. 2021; 3(1): 38–46 putu oky ari tania, et al. 4 2 histological changes at the negative control group, the liver is in normal conditions, the normal hepatocyte cells were seen. the positive control group was induced by toxic doses of paracetamol show enlarged hepatocyte cells with cytoplasm showing eosinophilic granules, enlarged nucleus and irregular shape of cells. the nucleolus was prominent, there are many cells in the mitosis phase. the liver histological in positive control are also appear in the necrosis of cell. in addition, around the central vein appear inflammatory cells such as lymphocytes, histiocytes and polymorphonuclear (pmn) cells. the hepatocytes also experienced necrosis characterized by pale colored and fragmented nucleus, or even the cells loss of their nucleus. meanwhile, t2 group show the cell histology that was not slightly different from the t1 group, but there were more necrotic of hepatocyte. liver oxidative stress represented by malondialdehyde level in liver. the mean of mda levels for each group are presented in figure 2. figure 2. the mean of mda level on negative control, positive control, t1 and t2. the highest mean level of mda from the table above described in the t2 group, which is received c. zedoaria extract before giving paracetamol (8.18 nmol/ml). whereas administered of paracetamol before c. zedoaria extract (t1) show the lowest average of mda level, which was 7.22 nmol/ml. giving c. zedoaria after paracetamol (t1) compared to give c. zedoaria before paracetamol (t2) show that mda levels were significantly different. the statistical analysis using spss 16.0 software with the non-parametric kruskal-wallis test. the results of statistical data analysis obtain p-value of 0.087 (α > 0.005). the difference in liver mda levels between groups is not significantly different. ina. j. med. lab. sci. tech. 2021; 3(1): 38–46 putu oky ari tania, et al. 4 3 discussion the toxic doses of paracetamol used are known create a hepatic damage and hepatoxic. in accordance with the research of rivera et al (12) it is known that the acute and repeated administration of paracetamol for 4 days found severe histological changes the sinusoidal dilatation, ballooning hepatocytes, lymphocyte infiltration (inflammation), hemorrhage and extensive pericentral hepatic necrosis. acute and repeated administration of paracetamol lower the expression of peroxisome proliferator-activated receptor alpha (pparα). pparα is a receptor that plays a role in controlling metabolic homeostasis, inflammation and fibrogenesis. the expression of hepatic pparα receptors will decrease when there is an advanced inflammation and the fibrosis condition of non-alcoholic steatohepatitis (nash) (13). this also illustrates that a decrease in pparα indicates a lack of anti-inflammatory agents and an increase in ros (12). the results show an inconsistency, especially in the positive control group where the level of mda in the liver show a lower value than the t2 group. paracetamol administration in positive control showed lower mda when compared to t2 group. it is presumed because the levels of mda show greater changes in the kidneys rather than the liver, as demonstrated in the study of hamza and al-harbi (14) giving paracetamol dose of 2g/kg for 30 consecutive days can increase mda levels in the liver and kidneys. mda level in the liver were 12.35 nmol/g and mda level in the kidney were of 38.68 nmol/g. mda level of the liver in control group was 1.74 and 8.67 nmol/g in the kidney. paracetamol will show higher levels of mda in the kidneys. the used of paracetamol dose to induce liver damage for seven days and demonstrate toxicity were insufficient. in the study of bharatham et al (15), the toxic dose of paracetamol used are 3000mg/kg or 600mg/200g per day given for 7 days, whereas in this study are 1.35g/ kg body weight per day. the effect of c. zedoaria which is given 2 hours before paracetamol administration is not effective because of the short time of c. zedoaria activity to neutralize the oxidant from paracetamol. to analyze the effect of curcuma as hepatoprotector and reduce liver enzymes, when antioxidants properties of curcuma are given for seven days, the paracetamol is given on day eight of a single dose (16). the research model or design used to describe the hepatoprotector activity of curcuma should be given antioxidants and paracetamol together several days in one group, followed by a single dose of paracetamol in the next day. research by dogaru et al (17) was given paracetamol along with sylimarin antioxidant properties for five days and after that, the next day paracetamol was given. in group t2 of this ina. j. med. lab. sci. tech. 2021; 3(1): 38–46 putu oky ari tania, et al. 4 4 study, c. zedoaria as an antioxidant was given two hours before paracetamol without administered of paracetamol again in the next day. it is the possibility that antioxidant effect of c. zedoaria is not visible. repetitive paracetamol administration for 7 days could be resistance to hepatic damage. hepatotoxic due to upregulation of the enzymes glucose-6-phospatease dehydrogenase (g6pd) and glutathione reductase activity as an adaptive and protective response due to oxidative stress and lack of glutathione (18). repetitive dose of paracetamol in positive control did not shows statistically differences of mda level to t2. the effect of paracetamol and curcuma towards mda level slightly different with the histopathological changes. administration of paracetamol show many changes in the hepatocellular. the effect of paracetamol at dose 1.35g/ kg body weight on liver histopathology is observed in the positive control group. in this group, liver tissue damage was seen with hepatocellular necrosis, which is indicated by enlargement of the hepatocytes with granulated cytoplasm or vacuolation. it is related with several studies which illustrate that paracetamol overdose will produce centrilobular hepatic necrosis which can be fatal (19). enlarged of nucleus and hepatocytes due to cell were prepared to undergo proliferation and mitosis. liver is an organ has capability to regenerate after damaged by paracetamol. exposure of paracetamol induce cell death and necrosis that can be recover by proliferating hepatocytes. according to thonda and shivalinge (20), paracetamol administration causes necrosis in centrilobular hepatic necrosis which is characterized by nuclear picnosis and eosinophilic cytoplasm followed by excessive hepatic lesions. the covalent bond of n-acetyl-p-benzoquinone imine (napqi) which is an oxidative product of paracetamol, with sulfidrile group proteins, results in lipid peroxidation, degradation of gluthathione levels and produces necrotic cells in the liver. histopathological changes in the t1 and t2 groups show several enlarged hepatocyte cells and nuclei with irregular shapes. administration of c. zedoaria before paracetamol (t2) show more necrotic cells and inflammatory cells rather than in t1. however, when those are compared with positive control given toxic doses of paracetamol, it shows improvement in morphology and cell necrosis, although not all of the hepatocytesbecomes normal. inflammatory cell appearance in positive control and t2 indicate that necrotic hepatocytes produces by paracetamol are phagocyted by lymphocytes and pmn as inflammatory cells. previous study reported that administration of paracetamol dose 1,000 mg/kg show the increase number of inflammatory cells i.e. neutrophil (21). ina. j. med. lab. sci. tech. 2021; 3(1): 38–46 putu oky ari tania, et al. 4 5 between all groups, it shows that t1 is the group that point out the hepatocellular recover from paracetamol toxic dose. it is consistent with the mda level, in t1 is the lowest level of mda as well. the role of curcumin as an antioxidant agent is the scavenging activity against ros. the antioxidant capacity of curcumin is also related to the inhibition of lipid peroxidation and induction of translocation of nuclear factor e2 related factor 2 (nrf2) to the nucleus. curcumin administration can reduce serum transminase, mda and inflammatory cytokines in experimental animals given apap (22), which is indicated by the absence of inflammatory cells such as pmn or lymphocytes. in this study represented by t1 where mda showed lowest level than in other groups. the limitation of this study is the inaccurate research design, then the exact biomarker testing that should be tested is metalloproteinases (mmps) as a marker of liver damage. the antioxidant and antiinflammatory activities were more precisely observed with the ability to overcome ros and improve cell viability in reducing cell death. conclusions the administration of c. zedoaria and paracetamol on mda levels has no effect. there were no differences in mda levels between treatments. the most histopathological changes are shown in paracetamol alone within 7 days of treatment. author contributions putu oky ari tania: conceptualization, writing-reviewing, validation. puja ayu misuari: data analysis, methodology. satya yudhayana: sampling and data collection. k. ayesha eldelwise prayoga: writing-original draft preparation. acknowladgements thank you to the lembaga penelitian dan pengabdian masyarakat (lppm), wijaya kusuma surabaya university as a funder, dr. heriyawati, sp.pa from pathology anatomy department, airlangga university. conflict of interest the authors declare no conflicts of interest regarding of this research. references 1. rahman a, afroz m, islam r, islam kd, amzad hossain m, na m. in vitro antioxidant potential of the essential oil and leaf extracts of curcuma zedoaria rosc. j appl pharm sci. 2014;4(2):107– 11. 2. ji s, fattahi a, raffel n, hoffmann i, beckmann mw, dittrich r, et al. antioxidant effect of aqueous extract of four plants with therapeutic potential on gynecological diseases; 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4(2): 168–176 dian anggraini, et al 1 6 9 introduction kidney failure or end stage renal disease (esrd) is caused by a gradual decline in kidney function. esrd requires therapy for renal replacement with interventional kidney transplantation or dialysis. dialysis can be done with hemodialysis (hd) and capd (continuous ambulatory peritoneal dialysis) (1). the number of active patients having kidney replacement therapy worldwide in 2018 was 132,142, or 499 per million people, according to data from the indonesian renal registry (irr), with an increase in patients undergoing renal replacement therapy of 66,433, or 251 per million people. this number has gone up. in comparison to the prior year, it nearly doubled (2). meanwhile, in 2015 east java ranked 4th after central java for cases of kidney failure requiring dialysis therapy (3). this shows that every year there is an increase in patients with kidney failure on dialysis. kidneys have an essential function in homeostasis, namely removing metabolic waste, maintaining fluid and electrolyte balance, and producing hormones that can affect other organs; one example is blood pressure control (4). peritonitis in capd more often comes from micro-contamination of organisms on the skin during fluid replacement dialysate, contamination during catheter change, bacterial colonization at exit sites and tunnel infections. capd (continuous ambulatory peritoneal dialysis is a dialysis technique performed with the peritoneal membrane (abdominal cavity), which functions as a filter, namely the peritoneal membrane (abdominal cavity membrane). therefore, capd is often called "dialysis" through the stomach as a dialysis membrane that separates the dialysate fluid and blood plasma in the peritoneal cavity from the peritoneal blood vessels during capd (5). blood is transferred using a machine and tubing. the water and garbage are subsequently removed using a filter. along with fresh fluid, the blood is reintroduced to the body. in general, this treatment is carried out from 12 to 24 hours a day. in addition, it qualifies as ambulatory since the patient still removes urea and extra fluid during dialysis and maintains the electrolyte balance that ckd patients experience (6). in the capd process, the dialysate fluid can be changed up to 3-4 times a day. the composition of this dialysate fluid is differentiated according to the type of osmotic, buffer, and electrolyte (2). treatment of kidney failure with capd can be done every day (7). serum albumin concentrations strongly predict an increased risk of morbidity and death in peritoneal dialysis (capd) patients. the amount of albumin in the body and serum albumin concentration is determined by several factors, including liver albumin ina. j. med. lab. sci. tech. 2022; 4(2): 168–176 dian anggraini, et al 1 7 0 synthesis (which is influenced by diet), protein intake), protein catabolism, and extravascular distribution of albumin, in addition to these factors. because the protein leaks into the peritoneal effluent in capd patients, the serum albumin concentrations are vulnerable to adverse effects. patients with hypokalemia had lower serum albumin concentrations than those without hypokalemia, and serum albumin concentrations correlated significantly with serum potassium concentrations (8). according to research by riyadi et al., (9), the body's electrolyte alterations in chronic kidney failure are associated to hypertension. the levels of sodium and potassium can have an impact on the patient's blood pressure. inhibiting renin secretion prevents angiotensinogen from being converted into angiotensin i, which is one of multiple ways that higher potassium concentrations might lower blood pressure. second, decreased aldosterone secretion due to elevated potassium concentrations can result in a reduction in intravascular fluid due to sodium release. a higher risk of morbidity and mortality in peritoneal dialysis (capd) patients is substantially predicted by serum albumin concentrations. the production of albumin by the liver (which is controlled by diet and protein intake), protein catabolism, and extravascular distribution of albumin are a few of the factors that affect serum albumin concentration and albumin levels in the body. capd patients are susceptible to side effects on serum albumin concentrations because the protein is lost into the peritoneal effluent. patients with hypokalemia had lower serum hypoalbuminemia in patients with kidney failure is closely related to poor nutritional intake. the membrane used in the process of renal replacement therapy/dialysis can cause inflammation by activating white blood cells and increasing inflammatory mediators (10). albumin is very important in these conditions. at the same time, the decrease in albumin concentrations in capd therapy patients can be caused by an increase in higher capd fluid concentrations so that excess water that will be removed with dialysate fluid is wasted and albumin concentrations in the blood are wasted, which can cause albumin to fall (11). there is a previous study on the description of albumin concentrations in capd patients, which stated that 80% of capd patients had albumin concentrations lower than the usual standard or hypoalbuminemia (12). laboratory examinations carried out at the phc surabaya hospital on patients with chronic kidney failure with capd therapy included bun/ureum, and creatinine. whereas in some cases on capd therapy, albumin and electrolyte concentrations were found the low. examination of albumin and electrolytes is a prognostic factor that is also important to know in patients with renal ina. j. med. lab. sci. tech. 2022; 4(2): 168–176 dian anggraini, et al 1 7 1 failure with hypertension. this study aims to determine the relationship between albumin concentrations and sodium and potassium concentrations in capd patients. the provisional hypothesis in this study is that there is a relationship between albumin concentrations and sodium-potassium in capd patients. materials and methods study design and participants the types of research in this study are analytical and cross-sectional. the population in this study was patients with kidney failure with capd therapy at the phc surabaya hospital. the sample for this study was 60 samples that were determined with purpose sampling using the slovin formula (13). the inclusion criteria for samples are the 40-50 age group, the 5160 age group and the 61-70 are group in cpad patients. this research was carried out at phc surabaya’s laboratory of the clinical pathology in october 2021 may 2022. data collection and measurements the sample used was fresh serum obtained from total blood without anticoagulants and centrifuged at 3000 rpm for ± 15 minutes at room temperature (14). the serum was measured using an architect c4000 clinical chemistry analyzer using the bromocresol green (bcg) method (albbcg). the intensity of this green colour indicates the concentrations of albumin in the serum. the intensity of the colour produced is proportional to the concentration of albumin in the sample measured at a wavelength of 620 nm. examination of serum sodium and potassium concentrations obtained from serum that has been filtered and measured using a caretium medical instruments with an ion selective electrode (ise) method. the principle of measurement is to calculate the ion content of the sample by comparing the unknown ion value with the known ion concentrations value (21) in this study, primary data were collected by sampling patients receiving capd medication and by analyzing serum albumin, sodium, and potassium levels. measurements of the data continued with albumin examination with the architect c4000 clinical chemistry analyzer and examination of sodium and potassium with the caretium ise method. additionally, analytical processing will be done on the collected data. this research has received ethical approval from the hospital ethics committee phc surabaya with the number: 001/kepk/rsps-2022. statistical analysis the data obtained from this study was analyzed using spss (statistical product and service solutions) 26 program. the data obtained were processed using the spearmen correlation statistical test to determine whether there was a relationship between ina. j. med. lab. sci. tech. 2022; 4(2): 168–176 dian anggraini, et al 1 7 2 albumin concentrations with sodium and potassium in capd patients based on normal values/threshold values for each examination. the inspection data obtained were then analyzed using the spearman correlation test. the spearman correlation test is a statistical test aimed at knowing the relationship between two or more ordinary scale variables. since the data are interval data, they are converted into the original data by categorizing according to the threshold for each variable. including low, average, and three high categories (15). results the results of the analysis of the albumin, sodium, and potassium concentrations in capd patients between the ages of 40 and 50 who had examinations at the phc surabaya hospital's clinical pathology laboratory are presented in table 1. it shows that the average albumin concentration of capd patients aged 40 to 50 is 3.24 g/dl, while the sodium and potassium contents are 136.91 mmol/l and 3.72 mmol/l, respectively. according to the age distribution of patients with capd had average albumin values of 3.24 g/dl, 3.0 g/dl, and 2.86 g/dl between the ages of 61 and 70. the average sodium concentration in capd patients aged 41 to 50 was 136.91 mmol/l, compared to 134.21 mmol/l for patients aged 51 to 60 and 134.19 mmol/l for those aged 61 to 70. in capd patients aged 41 to 50, the average potassium level was 3.72 meq/l, 3.70 meq/l in those aged 51 to 60, and 3.68 meq/l in those aged 61 to 70. table 1. results of analysis of albumin, sodium and potassium in patients with capd aged 40-50 years mean min max std. deviation albumin (g/dl) 3.24 4.80 2.00 0.65 sodium (mmol/l) 136.9 142.7 130.9 2.90 potassium (mmol/l) 3.72 4.53 3.01 0.45 according to table 1, capd patients between the ages of 40 and 50 tend to have low albumin concentrations as well as normal sodium and potassium concentrations. capd patients between the ages of 51 and 60 were tested for albumin, sodium, and potassium concentrations. according to the findings, the average sodium concentration was 134.2 mmol/l, the average potassium concentration was 3.70 mmol/l, and the average albumin concentration was 3.00 g/dl. this data indicated that patient is in normal condition. normal sodium levels are usually between 136 145 mmol/l. normal blood potassium levels were considered to be between 3.5 and 5.5 mmol/l. ina. j. med. lab. sci. tech. 2022; 4(2): 168–176 dian anggraini, et al 1 7 3 table 2. results of examination of albumin, sodium and potassium in patients with capd aged 51-60 years mean min max std. deviation albumin (g/dl) 3.00 3.90 1.80 0.59 sodium (mmol/l) 134.2 139.6 130.2 2.78 potassium (mmol/l) 3.70 6.00 2.62 0.82 table 2 reported that potassium and sodium concentrations are within normal ranges. table 2 demonstrates that the average albumin concentrations in capd patients aged 51 to 60 years tend to be low. the average albumin, sodium, and potassium values in capd patients between the ages of 51 and 60 were lower than those between the ages of 40 and 50. according to a descriptive examination of capd patients aged 61 to 70, average albumin concentrations were 2.82 g/dl, sodium concentrations were 134.19 mmol/l, and potassium concentrations were 3.68 mmol/l. according to table 3, the findings of testing capd patients between the ages of 61 and 70 years old revealed that, on average, albumin and salt concentrations tended to be lower than normal, but potassium concentrations were within normal ranges. in comparison to capd patients aged 40–50 years and 51–60 years, capd patients aged 61–70 years had lower average findings for albumin, sodium, and potassium concentrations. the spss program's spearman correlation test was then used to analyze the inspection data. the received data is transformed into ordinal data by categorizing in accordance with the threshold of each variable because the data are in the form of interval data. table 4 demonstrates result from spss with details on the characteristic sample (gender, bmi, etc.). table 3. results of examination of albumin, sodium and potassium in patients with capd aged 61 – 70 years mean min max std. deviation albumin (g/dl) 2.82 4.10 1.90 0.65 sodium (mmol/l) 134.19 144.9 116.4 6.16 potassium (mmol/l) 3.68 4.81 6.16 0.55 table 4. results of data analysis from spss group variable a variable b sig r 40-50 years old albumin sodium 0.006 0.548 potassium 0.042 0.418 51-60 years old albumin sodium 0.045 0.478 potassium 0.034 0.502 61-70 years old albumin sodium 0.031 0.509 potassium 0.045 0.478 ina. j. med. lab. sci. tech. 2022; 4(2): 168–176 dian anggraini, et al 1 7 4 sig.(2-tailed) was used to determine significance. if the value of sig.(2-tailed) <0.05 then the relationship contained in r is considered significant. it means that there is a relationship. both variables are moderate/moderate and unidirectional between albumin concentrations and sodium and potassium concentrations. in the age group of 40-50 years, there is a strong and unidirectional relationship between albumin concentrations and sodium concentrations where the sig value < 0.05 in the r correlation. in the age group 51-60 years, there was a moderate and direct relationship between albumin concentrations and sodium concentrations where the sig value <0.05 in the r correlation. in the 61-70-year group there is a strong and direct relationship between albumin concentrations and sodium concentrations where the sig value <0.05 in the r correlation. discussion renal failure patients with capd therapy showed most capd patients had low albumin concentrations. this study's results align with purwanto et al., (16) which examined the albumin profile of capd patients at rsup. dr. sardjito yogyakarta stated that most capd patients were more in hypoalbuminemia conditions, as much as 80.7%. decreased concentrations of albumin in the blood causing edema or disfigurement in the patient's body (10). the results of the data analysis found a strong and unidirectional relationship between albumin concentrations and sodium concentrations in capd patients. the results of this study are in line with chang et al., (8), which examined hyponatremia as a predictor of mortality in peritoneal dialysis (capd) patients. in the study, one of the factors was that serum sodium concentrations were positively related to serum albumin in capd patients. the relationship in this study is related to the age of capd patients and the complications of peritonitis that cause hypoalbuminemia to cause overhydration, causing hyponatremia in capd patients who were examined at the clinical pathology laboratory of phc hospital surabaya. the glomerular filtration rate decreases as you age because the kidneys' renal mass decreases as a result of losing numerous nephrons. after reaching adulthood, body tissues' cells start to deteriorate. in the elderly, there is a decrease in the number of nephrons by 5-7% every decade starting at the age of 25 (17). patients receiving peritoneal dialysis (capd) are at an elevated risk of morbidity and death, which is highly predicted by serum albumin concentrations. the amount of albumin in the body and serum albumin concentration is determined by several factors, including liver albumin synthesis (which is influenced by diet), protein intake, protein catabolism, and ina. j. med. lab. sci. tech. 2022; 4(2): 168–176 dian anggraini, et al 1 7 5 extravascular distribution of albumin. in addition to these factors (18). because the protein is lost into the peritoneal effluent in individuals with capd, the serum albumin concentrations are sensitive to adverse effects. serum albumin concentrations were significantly associated with serum potassium concentrations, and patients with hypokalemia had lower serum albumin concentrations than patients without hypokalemia (19). the concentration of potassium is approximately 30 times higher in the intracellular space than it is in the extracellular space. numerous factors affect how potassium is distributed between the extracellular and intracellular fluid compartments. the na-k atpase transporter's active transport in the opposite direction causes the potassium to go through the cellular membrane following passive diffusion from the intracellular to the extracellular compartment (na–k pump). according to our examination, albumin concentration and serum potassium concentration in people with peritonitis were statistically correlated. even though capd is simple to implement, it can result in a number of problems that, if unchecked, can be fatal. peritonitis is a common side effect. inflammation (peritonitis) and hypoalbuminemia are linked to quantifiable excess body water (overhydration) in capd patients (20). conclusions a relationship between albumin concentrations and sodium and potassium concentrations in capd patients in the three age-difference groups, which give values below the average, can be inferred from the research findings and the spearmen correlation test. according to our research, people with hypokalemia have lower serum albumin concentrations than those without the condition. author contributions dian anggraini: conceptualization, methodology, investigation, writingreviewing, editing, software, data curation, and writing-editing, methodology. edy haryanto: validation. syamsul arifin: validation. ayu puspitasari: editing – reviewing. conflict of interest there are no conflicts of interest. references 1. liu x, dai c. advances in understanding and management of residual renal function in patients with chronic kidney disease. kidney dis. 2016. 2:187–196. https://www.karger.com/article/fulltext/doi:44 9029. 2. lydia a. peran continous ambulatory peritoneal dialysis dalam pemerataan layanan pengganti ina. j. med. lab. sci. tech. 2022; 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[the effect of health education on knowledge and attitudes in the prevention of hypoglycemia in patients with diabetes mellitus at the andalas padang public health center in 2018]. fakultas kedokteran universitas andalas. j fak kedokt univ andalas. 2018;1:6–9. http://scholar.unand.ac.id/doi:61716/2/2. bab 1 (pendahuluan).pdf 11. yaswir r, ferawati i. fisiologi dan gangguan keseimbangan natrium, kalium dan klorida serta pemeriksaan laboratorium. [physiology and disorders of sodium, potassium and chloride balance and laboratory examination]. j kesehat andalas.2012;1(2):80-85 https://doi.org/10.25077/jka.v1i2.48 12. alharbi ma. is low serum albumin a predictor sign of the incidence of peritoneal dialysisassociated peritonitis? a quasi-systematic review. saudi j kidney dis transplant.2020; 31:320–334. doi: 10.4103/1319-2442.284006 13. nalendra ara, rosalinah y, priadi a, et al. (2021). statistika seri dasar dengan spss. [basic series statistics with spss]. bandung: penerbit media sains indonesia 14. verdiansyah. pemeriksaan fungsi ginjal. [kidney function check]. 2016; 43(2):148-154. doi: http://dx.doi.org/10.55175/cdk.v43i2.25 15. suyanto, amal ai, noor a, astutik it. analisis data penelitian petunjuk praktis bagi mahasiswa kesehatan menggunakan spss. [analysis of research data practical instructions for health students using spss]. 2018. semarang: unisula press. 16. purwanto b, yuniasih d, puspitasari m. albumin description of continuous ambulatory peritoneal dialysis (capd) patients in sardjito hospital yogyakarta. dahlan med j. 2021; 2:10–18. doi: 10.1016/j.nut.2021.110818. 17. sandala ga, mongan ae. gambaran kadar kalium serum pada pasien penyakit ginjal kronik stadium 5 non dialisis di manado kandidat. [overview of serum potassium levels in patients with chronic kidney disease stage 5 non-dialysis in manado candidate]. j e-biomedik. 2016; 4:4– 9 18. fan x, huang r, wang j, et al. risk factors for the first episode of peritonitis in southern chinese continuous ambulatory peritoneal dialysis patients. plos one. 2014; 9:1–7. https://dx.plos.org/doi:10.1371/journal.pone.010 7485 19. davies sj, zhao j, morgenstern h, et al. low serum potassium concentrations and clinical outcomes in peritoneal dialysis—international results from pdopps. kidney int reports. 2021; 6:313–324. https://doi.org/10.1016/j.ekir.2020.11.021 20. dao bqq, pham nht, nguyen dl, pham vm, nguyen hd, nguyen dt, tran vt, do q, le vvt. overhydration and low serum prealbumin predict peritoneal dialysis-related peritonitis in continuous ambulatory peritoneal dialysis patients. bmc nephrol. 2020; 21:1–8. https://bmcnephrol.biomedcentral.com/articles/d oi:10.1186/s12882-020-02178-w 21. statlyte. electrolyte analyzer ( k / na / cl / ca / ph ) service manual. available at https://sunrisesurgicalhouse.com/brochure?broch ure=products/wltzykqvabxmd432lxasbbw kkpfz2n7vrxpn3hg5.pdf 22 microbial pattern of diabetic foot ulcer patient in jemursari islamic hospital surabaya period 2012–2016 adyan donastin1, aisyah1 1department of medicine, faculty of medicine, nahdlatul ulama university of surabaya, surabaya, indonesia correspondence: aisyah, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code : 60237 email: aisyahdr@unusa.ac.id received: february 26, 2019 revised: march 26, 2019 accepted: april 1, 2019 abstract diabetic foot ulcers (dfu) are complications in people with diabetes mellitus (dm) in the form of wounds or tissue damage resulting in vascular insufficiency and or neuropathy that can develop into an infection. early detection of germs of diabetic foot ulcers may be used as a recommendation of empirical therapy before the definitive treatment based on culture results and appropriate antibiotics treatment, which may reduce hospitalization time and amputation events. according to riskesdas in 2013, state that the number of antibiotic used without prescriptions in indonesia about 86.1%. the study aims to retrospectively analyze the bacterial culture and drug susceptibility test results for patients with diabetic foot ulcers (dfu) in jemursari islamic hospital surabaya during 2012–2016 to help clinicians choose a more appropriate empirical antibiotic treatment for dfu. this study used cross– sectional designed with retrospective approaches, which analyzed descriptively and samples were taken by the total sampling of 11 samples. this research was conducted at islamic hospital of jemursari surabaya in may–september 2017 by using medical record data which are outpatient and inpatients who treatment at jemursari islamic hospital. the result was found 6 types of bacteria consisting of staphylococcus aureus (18%), staphylococcus non– haemolytic (18%), klebsiella pneumonia (27%), enterobacter aerogenes (18%), burkholderia cepacia (9%), escheria coli (9%). the most sensitive antibiotics in the gram–positive bacteria in this study are amikacin, teicoplanin and oxacillin and the most resistant to amoxicillin and ampicillin whereas the most sensitive antibiotics in the gram–negative bacteria in this study were meropenem and the most resistant to ciprofloxacin and trimethroprim–sulfamethoxazole. keywords microbial pattern, antibiotics sensitivity pattern, diabetic, foot ulcer, diabetes mellitus ina j med lab sci tech 2019; 1(1): 22-32 adyan donastin, aisyah 2 3 introduction diabetes mellitus (dm) is one of the main problems in public health system that has increased dramatically over the past 2 decades and continues to increase (1–3). based on research by the world health organization (who) in developing countries showing the highest increase in dm patients in southeast asia including indonesia and it is estimated that in the next 1 or 2 decades the frequency of dm in indonesia will increase dramatically to rank number 5 in the world (4). diabetes mellitus that is not treated properly will cause complications, as is the most common and often occurs is diabetic foot ulcer (dfu). damage will arise if in the long term there is a decrease in blood flow accompanied by nerve damage (neuropathy) in the legs, thereby increasing the likelihood of dfu. diabetic foot ulcer is a wound that occurs in the legs of people with type 1 diabetes and 2, then infection and or tissue damage resulting from neuropathy (nerve disorders), angiopathy (impaired blood flow in the legs) or both that often become the place of entry of bacteria into the legs (5–6). gardner (7) states that around 15% of patients suffering from dm will develop into dfu during their lifetime (7). further infections without good treatment and adequate can be the most common cause of amputation, and based on non–traumatic events with the risk of amputation 10–20 times more often in patients with dm compared with non–dm (8), and about 85% amputation in dm patients associated with dfu (9), can even end in disability or death (10). the current dfu prevalence in indonesia is 12%, while the prevalence of dfu risk factors in indonesia is 55.4% (11). several studies show that there are variations in the types of germs that cause dfu, both aerobic and anaerobic germs. akbar et al. (29) in arifin achmad hospital for 23 samples received a. baumanii (34.8%), k. pneumoniae (26.2%), e. coli (17.4%), e. cloacae (8.7%), p. stuartii (4.3%), r. ornithinolytica (4.3%), and p. aeruginosa (4.3%). research by akhi et al. (2015) of 60 samples obtained s. aureus (28%), enterobacteriaceae (24%), e. coli (15%), citrobacter spp. (4%), enterobacter spp. (4%), and staphylococcus spp. negative coagulase (17%), enterococcus spp. (15%), p. aeruginosa (7%), acinetobacter spp. (4%), and bacteroides fragilis (4%). data from previous studies show that early detection of germs in dfu can be used as a recommendation for empirical therapy before definitive therapy based on the results of culture and appropriate antibiotics, so as to reduce the time of hospitalization and the incidence of amputation. the results of riskesdas in 2013 also stated that the use of prescription antibiotics in indonesia was 86.1%. this study aims to retrospectively analyze the bacterial culture and drug ina j med lab sci tech 2019; 1(1): 22-32 adyan donastin, aisyah 2 4 susceptibility test results for patients with diabetic foot ulcers (dfu) in jemursari islamic hospital surabaya during 2012–2016 to help clinicians choose a more appropriate empirical antibiotic treatment for dfu. materials and methods the data collected are secondary data based on research variables taken from the clinical pathology laboratory installation of jemursari surabaya islamic hospital for the 2012–2016 periods. the population in this study was medical records of patients with diabetes mellitus with complications of diabetic foot ulcers at jemursari islamic hospital surabaya in january 2012– december 2016. the samples in this study were medical records of patients with diabetes mellitus with complications of diabetic foot ulcers in january 2012– december 2016, which fulfills the inclusion and exclusion criteria, and samples are taken in total sampling. the inclusion criteria in this study were medical records of dm patients with dfu who are hospitalized at jemursari hospital with complete identity, results of pus culture test, and antibiotic sensitivity test and exclusion criteria were grade 0 and grade 1 ulcers. this research was carried out descriptively because the observations were carried out according to the conditions as they were without any direct treatment from the researchers on the test subjects and using a cross–sectional design with a retrospective approach. evaluate the results of medical records regarding germs that cause dfu and the rational use of antibiotics in these cases. results dfu patients who were hospitalized at jemursari hospital from 2012 to 2016 totaled 291 patients. data on dfu patients were then compared with data on patients who underwent pus culture at jemursari hospital. the data of the patients taken were 57 data on dfu patients with a history of undergoing a specimens culture of pus at jemursari hospital. the inclusion criteria in this study were medical records of dm patients with dfu who were hospitalized at jemursari hospital with a complete identity, results of pus culture test, and antibiotic sensitivity test. based on the inclusion criteria of this study, 46 patient data were excluded from the study because there were no forms of pus culture test results and antibiotic sensitivity tests from the laboratory, so that the samples used in this study were 11 patients. distribution of dfu patients according age and sex table 1 showed that the results are differentiated by age group (16), with the age group 1–12 years and 12–18 years there are no dfu patients, in the age group 18–60 years, 195 patients (67.01%), and at age> 60 years there were 96 patients (32.99%), with an average age of 55.55 years. in this study, ina j med lab sci tech 2019; 1(1): 22-32 adyan donastin, aisyah 2 5 there were 152 male patients (52.23%), while 139 female patients (47.77%). table 1. distibution of dfu patients according age and sex patient freq. percentage (%) age 1–12 years old 0 0 > 12–18 years old 0 0 > 18–60 years old 195 67.01 >60 years old 96 32.99 sex man 152 52.23 woman 139 47.77 distribution of pathogenic germs to pus specimens the results of germ culture from pus specimens in dfu patients at jemursari hospital showed in figure 1. it showed that the 11 germ samples obtained two types of germs, namely gram–positive and gram– negative germs. the gram–positive germs found in this study were staphylococcus nonhaemolyticus and staphylococcus aureus, while the gram–negative germs found in this study were escherichia coli, enterobacter aerogenes, burkholderia cepacia, and klebsiella pneumonia. fig 1. distribution of pathogenic germs to pus specimens the sensitivity pattern of gram–positive germs to some antibiotics the most sensitive antibiotics used in gram–positive germs in this study were amikacin, teicoplanin, and oxacilin, while the antibiotics most resistant to gram– positive germs were amoxycilin and ampicillin. ina j med lab sci tech 2019; 1(1): 22-32 adyan donastin, aisyah 2 6 fig 2. the sensitivity pattern of gram–positive germs to some antibiosis fig 3. the sensitivity pattern of non haemolytic staphylococcus antibiotics the sensitivity pattern of gram–negative germs to some antibiotics the most sensitive antibiotic used in gram–negative bacteria in this study is meropenem, while the antibiotic most resistant to gram–negative germs is ciprofloxacin and trimethroprim– sulfamethoxazole. p e rc e n ta g e o f se n ti v it y p e rc e n ta g e o f se n ti v it y ina j med lab sci tech 2019; 1(1): 22-32 adyan donastin, aisyah 2 7 fig 4. the sensitivity pattern of the antibiotic klebsiella pneumonia fig 5. the sensitivity pattern of the antibiotic enterobacter aerogenes p e rc e n ta g e o f se n ti v it y p e rc e n ta g e o f se n ti v it y ina j med lab sci tech 2019; 1(1): 22-32 adyan donastin, aisyah 2 8 fig 6. the sensitivity pattern of escherichia coli antibiotics fig 7. the pattern of antibiotic sensitivity of burkholderia cepacia discussion distribution of dfu patients according age and sex the distribution of the age groups of patients with the most dfu in this study was 18–60 years as many as 195 (67.01%) with an average age of 55.55 years. old age is one of the factors that influence dm, which can cause neuropathy complications in patients with dfu (12). the results of this study are in accordance with decroli’s research in rsup dr. m djamil padang in 2007, which obtained the highest age group, was 40–59 years as many 65.8% (13–14). the age of patients suffering from diabetes and age at complications (one of them is diabetic ulcers) is related, this is in accordance with tarigan's study at herna hospital in medan in 2009– 2010 where the highest dm patients in the age group >40 years as dm age groups the most is 128 (95.5%) (15). p e rc e n ta g e o f se n ti v it y p e rc e n ta g e o f se n ti v it y ina j med lab sci tech 2019; 1(1): 22-32 adyan donastin, aisyah 2 9 based on these studies it can be concluded that the age group with the most dfu is in the productive age group to old age. this could be attributed to people's lifestyles and eating patterns, especially those that are not good (16). based on the results of the basic health research (riskesdas) in 2007, it was found that the proportion of deaths due to diabetes mellitus in the 45–54 year age group in urban areas was ranked second, namely 14.7% and national prevalence of dm based on examination of population gaet >15 years in urban areas is 5.7%. this illustrates that dm disease, especially in urban areas are serious and impactful problem productive age group productivity (17). diabetic ulcer often occurs at the age of >50 years due to decreased physiological body functions such as decreased insulin secretion or resistance, so that the ability of the body to function on high blood glucose control is less optimal. uncontrolled blood sugar levels will result in long chronic complications, both macro– and micro vascular, one of which is diabetic ulcer (18). factors that influence dm complications and are related to dfu other than age are gender. in this study, it was found that dfu patients in jemursari hospital were more prevalent in men (52.23%) than women (47.77%). this study is in line with the research of decroli (2008) who obtained a sex distribution that was more dominant in men (71%) in rsup dr. m. djamil padang, and in accordance with the gaol study (19), which gets the sex distribution more dominant in men (54%) in dr. rsup m. djamil padang in 2011–2013 (19). this study was also in accordance with commons research at the royal darwin hospital in 2015 with data on 177 patients found to be predominantly male (60%) (20). the research conducted by danmusa (21) explained that the incidence of dfu was more prevalent in men (67.2%) compared to women (32.8%). this study also according to chomi et al. (22) diabetic ulcer distribution in men can be caused by men compared with women who consult doctors less often, and information given to doctors tends to be less (22). the research conducted by danmusa (21) explained that the incidence of dfu was more prevalent in men (67.2%) compared to women (32.8%). jobs for men spend more time outdoors and do more work severe, making it easier for dfu to occur and increasing the risk of amputation. amputation in male dm patients has twice the risk (22–23). this research is not in line with fahmi's (24) research in cengkareng regional general hospital in 2013–2014 where women were dominant (57.6%), and witanto's research at immanuel hospital in bandung, which had more dominant female distribution (63%) (24–25). ina j med lab sci tech 2019; 1(1): 22-32 adyan donastin, aisyah 3 0 the difference in research between the two groups can be overcome if this research is conducted with more and more representative sample sizes. distribution of gram–positive and gram– negative germs based on this study and other studies it can be seen that germs found in dfu patients at different places and at different times not exactly the same, but some of the same germs like s. aureus, k. pneumoniae, e. coli and enterobacter are obtained. the gram– positive germs that were present in the dfu in this study were s. aureus (18%) and non haemolytic staphylococcus (18%). gram– negative germs present in dfu in this study were k. pneumoniae (27%), e. aerogenes (18%), b. cepacia (9%), e. coli (9%). in this study, it was found that infection in dfu was still caused by a polymicrobial infection so that the pattern of diabetic foot infection specifically could not be ascertained. each study has different characteristics of germ patterns depending on the patient's condition and environment so different empirical antibiotic therapies are needed as well (26) the sensitivity pattern of gram–positive germs to some antibiotics s. aureus was found to be 100% sensitive to antibiotics amikacin, ceftriaxone, teicoplanin, clindamycin, azithromycin, linezolid, oxacilin, and chloramphenicol, 50% sensitive to amoxicilin–clavulanic acid, piperacillin–tazobactam, meropenem, cefepime, cefoxitin, levofloxacin, trimethroprim–sulfamethoxazole, and erythromycin. s. aureus was found to be 100% resistant to ofloxacin, vancomycin, amoxycilin, and ampicillin, 50% resistant to amoxicilin–clavulanic acid, piperacillin– tazobactam, meropenem, cefepime, cefoxitin, levofloxacin, and trimethroprim–sulfamethoxazole. infection patients who are given penicillin as the first therapy and their incomplete administration can cause resistance since the emergence of methicillin–resistant s. aureus, vancomycin glycopeptide is the only uniformly effective treatment for staphylococcus infection. resistance of vancomycin glycopeptide to s. aureus (27). this study is in accordance with the research of chaudhry et al. (28) who received staphylococcus aureus were resistant to penicillin (100%), vancomycin (80%), and ampicillin (30%) (28). the sensitivity pattern of gram–negative germs to some antibiotics k. pneumoniae was found to be most sensitive to amikacin, gentamycin, amoxicilin–clavulanic acid, cefoperacilin sulbactam, piperacilin–tazobactam, meropenem, and ceftrizoxime. k. pneumonia was found to be 100% resistant to the antibiotic moxifloxacin fluoroquinolone, resistant 66.67% of ciprofloxacin, levofloxacin, ofloxacin, trimethroprim– sulfamethoxazole, and chloramphenicol, ina j med lab sci tech 2019; 1(1): 22-32 adyan donastin, aisyah 3 1 while 50% were resistant to cefepime, cefotaxime, ceftazidime, ceftriaxone, and aztreonam. akbar et al. (29) obtained k. pneumonia sensitive to amikacin antibiotics (100%) (29). this study is in accordance with the research of chaudhry et al. (28) who received k. pneumonia resistant to ceftazidime (100%), ceftriaxone (100%), cefepime (100%), cefotaxime (80%), aztreonam (60%), and chloramphenicol (20%) (28). conclusions in this research, it can be concluded that the age group of patients with the most dfu is the age group 18–60 years as many as 195 (67.01%) patients, with an average age of 55.55 years. based on gender, the majority of dfu patients were men (152.23%). the germs found in this study were s. aureus (18%), non–haemolytic staphylococcus (18%), k. pneumoniae (27%), e. aerogenes (18%), b. cepacia (9%), e. coli (9%) the dominant germ found in diabetic foot ulcer patients at jemursari hospitali period 2012– 2015 is a gram–negative germ, namely k. pneumonia (27%). the sensitivity of antibiotics to germs consist of the most sensitive antibiotics in gram–positive germs in this study were amikacin (100%), teicoplanin (100%), and oxacillin (100%), and the most resistant to amoxicillin (0%) and ampicillin (0%). the most sensitive antibiotic used in gram–negative germs in this study is meropenem (100%), while the antibiotic most resistant to gram–negative germs is ciprofloxacin (0%) and trimethroprim–sulfamethoxazole (0%). conflict of interest there are no conflicts of interest. references 1. shahbazian, h. yazdanpanah,l. latifi, s. 2013. risk assessment of patients with diabetes for foot ulcers according to risk classification consensus of international working group on diabetic foot (iwgdf). pak j med sci, 29. pp.730-734. 2. whiting, d. guariguata, l, weil,c, shaw, j. 2011. idf diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. diabetes res clin pract, 94. pp. 311-321. 3. shaw,j. sicree, r. zimmet, p. 2010. global estimates of the prevalence of diabeters for 2010 and 2030. diabetes res clin pract, (87), pp.4-14. 4. suyono, s. 2014. diabetes melitus di indonesia. in alwi, a.s.b.m.s.s.s.buku ajar ilmu penyakit dalam, 6th ed. jakarta:interna publishing. 5. aragon-sanchex, 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staphylococcus aureus. new england journal of medicine, (340),pp.493-501. 28. chaudry, w. badar, r. jamal, m. jeong, j. 2015. clinico-microbiological study and antibiotic resistance profile of merca and esbl gene prevalence in patients with diabetic foot infections. experimental anda therapeutic medicine, 11. pp.1031-38. 29. akbar, g.t., karimi, j. anggraini, d. 2014. pola bakteri dan resistensi antibiotik pada ulkus kaki diabetik grade dua di rsud arifin achmad periode 2012. jom, 1(2). 90 electrolytes levels (na, k, cl) in serum stored at 4°c temperature amalia nurul fauziah1, m. atik martsiningsih2, budi setiawan3,4 1pratama hospital yogyakarta, yogyakarta city, yogyakarta, indonesia 2d-iii of medical laboratory technology, poltekkes kemenkes yogyakarta, yogyakarta, indonesia 3bachelor’s degrees in medical laboratory technology, poltekkes kemenkes yogyakarta, yogyakarta, indonesia 4pusat unggulan iptek inovasi teknologi bidang kesehatan masyarakat (pui-novakesmas), poltekkes kemenkes yogyakarta, yogyakarta, indonesia correspondence: amalia nurul fauziah, jl srandakan km 01, pandak, bantul, yogyakarta, indonesia zip code: 55761 email: amalianurul112@gmail.com received: january 2, 2021 revised: march 5, 2021 accepted: june 6, 2021 published: october 30, 2021 doi: 10.33086/ijmlst.v3i2.1870 abstract the samples used for serum electrolyte measurement should be analyzed immediately after being received in the laboratory within 1-2 hours to avoid an increase in the error of the results. serum should be stored at 4°c for a period to prevent damage. the analyst should consider maximum delay time in the examination to maintain the serum's quality. this study compared the 2-hour and 3-hour delays in sodium (na), potassium (k), and chlorine (cl) tests. the method used in this study is an observational analysis with a cross-sectional study design. the samples in this study used 35 patient serum residues. the study was conducted in november 2020 with a continuous sampling technique. electrolyte levels in the sample were measured by avl 9180 electrolyte analyzer using ion-selective electrode (ise) method. the differences in electrolyte (na, k, cl) levels were analyzed by the kruskal-wallis statistical test at a 95% confidence level. the results showed that the content of sodium, potassium, and chlorine were 0.719; 0.976; and 0.772. this study showed that there was no significant difference in the electrolyte content of sodium (na), potassium (k), and chlorine (cl) in the serum directly detected from the serum stored at 4°c for 2 hours and 3 hours. in conclusion, it is acceptable to postpone the serum test for 3 hours with various considerations. keywords chloride, delay time, potassium, serum, sodium. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2021; 3(2): 90–98 9 1 amalia nurul fauziah, et al. introduction fluid and electrolyte balance disorders are common in clinical practice, especially in hospitals (1). the fluid amount and electrolyte concentration changes can cause a variety of problems if not appropriately managed. the electrolyte tests are usually performed as regular check-ups for specific medical conditions, such as decreased renal function, heart failure, and diarrhea (2–4). sodium (na+), potassium (k+), and chloride (cl-) are the primary electrolytes (ions) in the body. changes in electrolyte concentration and or ratio of anions and cations will cause changes in cell activity that can endanger life (5). na+ is the primary electrolyte of extracellular fluid, which in the case of hyponatremia or hypernatrium, the na+ concentration is regulated by the kidneys. k+ is the central intracellular cation and plays a vital role in cell metabolism. changes in plasma potassium levels (hyperkalemia or hypokalemia) can affect neuromuscular and heart function (6). clinical features of potassium disorder can be the most life-threatening disorder compared to others (7). besides, chloride is the primary extracellular anion in humans. it is essential to maintain serum neutrality, acid-base balance, homeostasis of body fluids, osmotic pressure, production of hydrochloric acid (hcl) in the gastrointestinal tract, kidney function, and electrical activity in muscular activity (8). someone should give immediate treatment when their body electrolytes are imbalanced (9). if not managed properly, changes in fluid and electrolyte concentration can cause serious problems. laboratory error leads to unnecessary delays and additional costs by obligatory repeated samples and causes unnecessary suffering to patients. currently, preanalytical errors account for up to 70% of all errors in laboratory diagnosis, most of which arise from problems in patient preparation, sample collection, transportation, and preparation for analysis and storage (10,11). the samples used to measure serum electrolytes should be analyzed as soon as possible, preferably within 1-2 hours after being received in the laboratory. the sodium, potassium, and chlorine stability will change after a few hours of centrifugation (12). proper specimen storage is an option to avoid repeated sampling in patients. if the serum should be stored for a moment, it should be closed and stored in the refrigerator before analyzing the serum at room temperature (13). the limitation of sample analysis in some laboratories is the delay in sample analysis after the centrifugation process. this condition is due to follow-up requests from clinicians, large sample loads, tool damage, and unavailability of backup equipment in the laboratory. according to preliminary research, the average time required to refer a serum sample to the laboratory is 2 to 3 hours. ina. j. med. lab. sci. tech. 2021; 3(2): 90–98 9 2 amalia nurul fauziah, et al. the study by baruah et al. (12) also showed that the validity of the sodium and chlorine results was affected 3 hours after centrifugation. in contrast, the validity of the potassium results was affected 1 hour after centrifugation. therefore, it is essential to find out the differences in the electrolyte levels of sodium (na), potassium (k), and chloride (cl) in the serum after being stored for 2 hours and 3 hours at 4oc. materials and methods this study used an analytical observational study with a cross-sectional study design. the study was conducted in november 2020. a total of 35 serum samples residue from patients who performed chemical examinations in the laboratory of one hospital in yogyakarta city, indonesia was collected by consecutive sampling. inclusion criteria in this study were the remaining serum from patients examined in the laboratory with a volume of ≥1 ml. the samples used were visually clear, not hemolysis, icteric, and lipemic. the research procedure was carried out by inserting a blood sample into the vacutainer tube of the clot separator gel. the blood was allowed to clot at room temperature. then the sample serum is grouped into two treatments. we directly examined the first sample group, and the second sample group was stored in a refrigerator. the serum was stored in a fridge at 4°c ± 2°c for 2 hours and 3 hours. the serum was allowed to stand for 30 minutes at room temperature before the test (13). the samples obtained were centrifuged at 3,000 rpm for 10 minutes. the serum was separated into eppendorf tubes. thirty-five serum samples were analyzed for na+, k+, and cllevels were measured by avl 9180 electrolyte analyzer using the ionselective electrode (ise) method. the differences in sodium, potassium and chloride levels in samples between direct-used and cold-stored (2 hours and 3 hours at 4oc) samples were analyzed by a statistical test. the data obtained were primary data and scaled ratio data. the statistical analysis was performed by spss 24.0 for windows. the data was analyzed with a one-sample kolmogorov-smirnov test. abnormal result resulting caused by data dissemination. therefore, we were analyzed further by statistical tests, namely non-parametric statistical tests used kindependent samples (kruskal-wallis h). the data is presented as tables and statistical tests of non-parametric kruskal-wallis with a confidence level of 95% using statistical programs. the ethics commission has approved this research for health research of politeknik kesehatan kementrian kesehatan (poltekkes kemenkes yogyakarta) under the ethics feasibility letter no. ekepk/polkesyo /0629/x/2020 dated october 20, 2020. ina. j. med. lab. sci. tech. 2021; 3(2): 90–98 9 3 amalia nurul fauziah, et al. results the average time required to send a serum sample to the laboratory was 2-3 hours (after time for collection). the results of serum electrolyte testing for several treatments (direct-tested and stored for 2 hours and 3 hours at 4°c) are shown in table 1. table 1. serum electrolyte level for several treatments (direct-tested and stored for 2 hours and 3 hours at 4° c) electrolyte test n mean (mmol/l) min (mmol/l) max (mmol/l) sd p-value na directly tested 35 138.7 133 144 2.64 0.719 2 h 35 139.2 134 146 2.70 3 h 35 139.0 134 145 2.82 k directly tested 35 4.31 3.2 6.1 0.65 0.976 2 h 35 4.32 3.3 6.0 0.63 3 h 35 4.31 3.3 6.0 0.63 cl directly tested 35 103 96 110 3.38 0.772 2 h 35 104 97 110 3.24 3 h 35 104 97 110 3.21 source: primary data, 2020 table 1 present the sodium levels of 2 hours-stored-serum (139.2 mmol/l) is 0.5 mmol/l higher than directly-tested-serum (138.7 mmol/l). while 3 hour-stored-serum sodium (139.0 mmol/l) is 0.3 mmol/l higher than directly-tested-serum (table 1). potassium levels of serum stored for 2 hours (4.32 mmol/l) is 0.01 mmol/l higher than 3hours-stored and direct-tested one (4.31 mmol/l) (table 1). chloride levels of both serums stored for 2 hours and 3 hours (104 mmol/l) is 1 mmol/l higher than directtested one (103 mmol/l) (table 1). in this statitiscally analysis, a p-value higher than 0.05 (> 0.05). it is means that no diffreny was observed. discussion fluid and electrolyte balance is crucial for understanding the maintenance of homeostasis and the successful treatment of many metabolic disorders. there are many regulatory mechanisms for electrolyte balance in organisms. disturbances of these mechanisms lead to electrolyte imbalance, which may be a life-threatening clinical condition. electrolyte imbalance is a common manifestation of many diseases. all electrolyte imbalances should be comprehensively considered. the examination is essential to clarify the clinical scenario for effective and successful treatment. most electrolyte imbalances are ina. j. med. lab. sci. tech. 2021; 3(2): 90–98 9 4 amalia nurul fauziah, et al. low and high sodium, potassium, calcium, and magnesium (14). potassium maintains the cardiac rhythm and contributes to neuromuscular conduction. k level imbalance, indicated by hyperkalemia or hypokalemia, will cause cardiac arrhythmias and neuromuscular weakness. chloride (cl) helps to maintain electrical neutrality with na. cl also maintains acid-base balance by buffering h+ equal hydrogen changes and cl changes to maintain electrical neutrality through the movement of bicarbonate ions (hco3-). kidney and endocrine disorders are usually characterized by plasma electrolyte imbalance. changes in electrolyte levels are associated with pathological consequences and increased mortality (15). analysts use laboratory diagnostic for diagnosis, monitoring, and prognosis in patients. laboratory medicine has the potential to improve patient safety since it crosses many pathways and organizational boundaries. clinicians can implement proactive interventions to highlight high-risk situations, such as pre-assessment and drug therapy monitoring. laboratories have a considerable role to play in diagnostic and therapeutic decision-making and monitoring safety. there are many such examples highlighted in this paper, but increasing the knowledge of the use and abuse of testing, and studying the outcomes, should be part of the value-added function of laboratory services. this condition will improve the quality of care and affect the results (16). distinguishing between the normal and abnormal fluid balance in the patient can be challenging. the diagnosis of fluid balance abnormalities requires the informed and reasonable interpretation of clinical and laboratory data (17). several pre-analytical variables affect electrolyte results, including the type of anticoagulant, storage conditions, and hemolysis. hemolysis of blood causes a false increase in plasma k results by releasing intracellular k. however, grossly hemolyzed specimens will affect the analyses of na and cl levels due to a dilutional effect. the presence of excess anticoagulants when small volumes of blood are collected will similarly cause a dilutional effect and falsely decreased plasma levels of na and cl. refrigeration of unseparated whole blood may enhance the intracellular release of k from erythrocytes (15). various studies on the stability of chemical metabolites, especially sodium, potassium, and chloride in serums, give mixed results. hedayati et al. (18) revealed that after the first cycle (24 h) at 2 to 8°c, changes in all the analytes (sodium, potassium, and chloride) were less than 10%. after serum stored in refrigeration in three cycles (72 h). at this moment, serum chloride was changes as 0.4% and statistically significant at the 5% level. donnelly et ina. j. med. lab. sci. tech. 2021; 3(2): 90–98 9 5 amalia nurul fauziah, et al. al. (19) compared sodium, potassium, and chloride levels in serums stored at room temperature (4oc) and -20oc for 48 hours, 14 days, and four months. while o'keane et al. (20) showed that all metabolites, including sodium, potassium, and chloride, have the stability of up to 48 hours if stored at a temperature of 4oc. some researchers discover that sodium and chloride levels in the serum can be stable for up to 12 hours with serum separation. after rapid centrifugation and storage at 4°c, the electrolytes can be stable for 48 hours (21). these data indicate that changes in these analytes are not consistent across all cycles and storage temperatures tested (18). in the cooling process, glycolysis is inhibited. thereby na-k atpase-depending power cannot maintain its gradient. consequently, intracellular potassium will exit the erythrocytes, leading to an increase in potassium levels in plasma (22). furthermore, storing of erythrocytes showed increased potassium leakage, and all these effects increased with increasing storage time (23). since sodium density is lower than the chloride (24), there is only one-tenth of sodium in erythrocytes, so serum testing delays do not cause sodium leakage into the serum (13). various electrolyte results depended on the temperature conditions of each country. research conducted in tropical countries will provide higher temperatures so that the stability of sodium, potassium, and chloride can change after a few hours of centrifugation. na and cl results are affected at 3 hours, but k results are affected at 1 hour. climatic conditions and uncovered sample cups left under the fan for a few hours are responsible for this evaporation and falsely high serum electrolyte values (12). research conducted in tropical indonesia gave similar results. the effect of long delays in serum examination for 0, 3, 5, and 7 hours was observed by other researchers that performed a serum examination of sodium and chloride levels (25). other researchers discovered that delayed sample-processing of over 2 (two) hours did not affect the serum sodium and chloride level. on the other hand, delayed sample-processing of more than 2 (two) hours can affect potassium levels (13). according to an et al. (26), na, k, and cl examination samples stored at room temperature will consistently increase over time, whereas low-temperature storage will prevent such changes. the evaporation of samples can cause an increase in such metabolites. therefore, cold sample conditions and closed containers or tubes are highly recommended for long-term storage. this research reveals that the average na, k, and cl levels in all treatments (directly examined and stored for 2 hours and 3 hours at a temperature of 4oc) are relatively comparable. there was no significant difference in sodium (p = 0.719), potassium ina. j. med. lab. sci. tech. 2021; 3(2): 90–98 9 6 amalia nurul fauziah, et al. (p = 0.976), or chloride (p = 0.772) levels in serums of all treatments, according to statistical tests. the findings are comparable to those of donnelly et al. (19) and o'keane et al. (20), who discovered that serum can be stored at 4 degrees celsius for up to 48 hours. this discovery indicates that the storage of serum specimens for examination of sodium, potassium, and chloride should pay attention to the container, conditions, and storage temperature, making the serum's stability can last longer (27). serum storage in clinical laboratories is still widely used in indonesia. as a result, each laboratory's guidelines should state the stability of each analyte's storage. the laboratory must follow standards of operational procedure for storage optimization. the researcher should consider the type and stability of specimen, anticoagulants, preservatives, and containers when storing samples for electrolyte analysis. serum samples for the study of na, k and cl can be stored for 14 days at a temperature of 2025°c. alternatively, if stored at 4°c, it can last for 14 days (27). this study shows that the storage of serum for sodium, potassium, and chloride analysis can be done at a maximum of 3 hours at a temperature of 4oc. the limitation of this study is the determination of the storage length of serum samples, which is limited to only 2 hours and 3 hours. as a result, it is unclear how long the maximum storage time will affect the serum sample's stability. in previous studies by trisna et al. (13), sampleprocessing delays of over 2 hours do not affect the results of sodium and chloride examinations, while sample-processing delays of more than 2 hours can affect potassium results. conclusions this study found no significant difference in sodium (na+), potassium (k+), and chloride (cl-) levels of the serum with all treatments (directly examined or stored for 2 hours and 3 hours at 4oc), indicating that postponing the serum examination for 3 hours with various considerations is still permissible. according to these findings, the analyst should keep serum specimens for electrolyte analysis (na+, k+ and cl-) at 4°c for a maximum of 3 hours. in previous studies, sodium and chloride were affected 3 hours after centrifugation at room temperature, while potassium results were affected 1 hour after centrifugation at room temperature. further research is needed to determine the maximum storage time at 4oc, which can affect the stability of serum electrolytes. author contributions amalia nurul fauziah: term, conceptualization, methodology, formal analysis, investigation, resources, data ina. j. med. lab. sci. tech. 2021; 3(2): 90–98 9 7 amalia nurul fauziah, et al. curation, writing-original draft, visualization, supervision, project administration, funding acquisition. m. atik martsiningsih: conceptualization, methodology, validation, writing review & editing, visualization, editing or revision of the manuscript. budi setiawan: conceptualization, methodology, formal analysis, data curation, writing review & editing, visualization, editing, or manuscript revision. acknowladgements appreciation goes to the management of laboratory unit, pratama hospital yogyakarta city, which commits to carry out this research successfully. conflict of interest the authors declare no conflict of interest. references 1. capasso g, unwin r. electrolytes and acid–base: common fluid and electrolyte disorders. medicine (baltimore) [internet]. 2011 jun;39(6):317–24. available from: https://linkinghub.elsevier.com/retrieve/pii/s135 7303911000636 2. rahmawati f. aspect of chronic kidney disease. j ilm kedokt wijaya kusuma. 2018; 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3(2): 90–98 9 8 amalia nurul fauziah, et al. pediatr pathol lab med. 1995; 20. o’keane mp, cunningham sk. evaluation of three different specimen types (serum, plasma lithium heparin and serum gel separator) for analysis of certain analytes: clinical significance of differences in results and efficiency in use. clin chem lab med [internet]. 2006 jan 1;44(5). available from: https://www.degruyter.com/view/j/cclm.2006.44. issue-5/cclm.2006.099/cclm.2006.099.xml 21. dupuy am, cristol jp, vincent b, bargnoux as, mendes m, philibert p, et al. stability of routine biochemical analytes in whole blood and plasma/serum: focus on potassium stability from lithium heparin. clin chem lab med [internet]. 2018 feb 23;56(3):413–21. available from: https://www.degruyter.com/doi/10.1515/cclm2017-0292 22. scott mg, legrys va, hood jl. electrolytes and blood gases. in: tietz textbook of clinical chemistry and molecular diagnostics. 2012. 23. burger p, kostova e, bloem e, hilarius-stokman p, meijer ab, van den berg tk, et al. potassium leakage primes stored erythrocytes for phosphatidylserine exposure and shedding of procoagulant vesicles. br j haematol. 2013; 24. apriliani i, santosa b, sukeksi a. difference in electrolyte levels (na, k, cl) in samples immediately and delayed by 150 minutes. repository.unimus.ac.id. 2018. 25. azizah n, aliviameita a. effect of delay of serum examination on electrolyte levels of sodium and chloride. j med lab sci technol. 2019; 26. an b, park c-e. evaluation of stability of serum on different storage temperatures for routine chemistry analytes. korean j clin lab sci. 2014; 27. departemen kesehatan ri. pedoman praktek laboratorium yang benar (good laboratory practice). jakarta; 2008. 81 the relationship between leukocytes numbers and consciousness level of craniotomy patients at the jemursari islamic hospital surabaya in 2018-2019 ainul rofiq1, utami ambarsari2, hafizh auliyan sodali3, misbakhul munir3 1departement of anesthesiology and reanimation, faculty of medicine, universitas nahdlatul ulama surabaya, jawa timur, indonesia 2departement of radiology, faculty of medicine, universitas nahdlatul ulama surabaya, jawa timur, indonesia 3faculty of medicine, universitas nahdlatul ulama surabaya, jawa timur, indonesia correspondence: misbakhul munir, jl. jemursari no. 51-57, surabaya, east jawa, indonesia zip code: 60237 email: munirdr04@gmail.com received: january 15, 2021 revised: april 3, 2021 accepted: july 31, 2021 published: october 30, 2021 doi: 10.33086/ijmlst.v3i2.1889 abstract this study investigates the correlation between leukocyte number and the patient's awareness level after craniotomy surgery, both high (leukocytosis) and low (leukopenia). several studies reveal that an increase in leukocytes affects mortality rates due to the high level of leukocytes affecting our body's functionalities, including awareness. previous studies revealed that a leukocyte count exceeding 17.5x106/l was associated with a lower gcs score, a longer hospital stay, and worsening ct scan results, regardless of the type of focal lesion that occurred. this study uses observational methods in the form of retrospective case studies. the data was collected from the medical records of the operation room at jemursari islamic hospital in 20182019 with a sample size of 89. the research was conducted at jemursari islamic hospital surabaya from may 2018 to august 2019. data were analyzed using the spearman correlation test with p <0.05. results showed no significant relationship between the leukocyte count and the consciousness level of post-craniotomy patients. future research could estimate the specific effects on the morphology of specific leukocyte cells that are elevated in post-craniotomy patients. this research is expected to provide insight into the effect of leukocytes on consciousness to reduce patient mortality after craniotomy. keywords craniotomy, leucocytes, patient this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2021; 3(2): 81–89 8 2 ainul rofiq, et al. introduction the craniotomy is a surgical procedure that opens the cranium to access the brain. craniotomy means making a hole (s) in the skull (cranium). traumatic brain injury is the most common reason for craniotomy (1). in many countries, traumatic brain injury is the leading cause of death and disability in the early decades of life. an estimated 1.4 million people experience a traumatic brain injury each year in the united states, and more than 5 million people experienced disability from traumatic brain injury (2). the operation is performed in a hospital with an adequate neurosurgical department and intensive care unit (icu) (3). craniotomy procedure, like any other operative procedure, carries a death risk. the research conducted at dr kariadi semarang from february 2010 to february 2012 reveals that 51 of 103 dead patients underwent craniotomy surgery treated at the high care unit (hcu) and icu (4). in comparison, white blood cells or leucocytes, other cells found in the blood, has a different function from erythrocytes. white blood cells or leukocytes play a role in defending the body against foreign objects that possible harm the survival of the individual (5). leucocytes type consisting of granular (include basophils, eosinophils, rod neutrophils, neutrophils) and agranular (includes lymphocytes, monocytes, and plasma cells) (6). as an immune system component, white blood cells flow in the human blood and kill germs or diseases. the normal white blood cell count (wbc) in peripheral blood is in the reference range from 3.2 x 103 to 10 x 103 wbc/µl (7). leukocytes are the main cell of the body defence system. leukocytes serve for protection, the body's defences against infection, and kill mutant cells (8). monocytes, the largest blood cells, are the second layer of body defence that can phagocytose and belong to macrophages (9). an increase in the percentage of monocytes in the leukocyte count indicates inflammation (10). some research shows that leukocytosis without infection is associated with increased hospital mortality, but this finding is not supported by complete data. al-gahtany (6) in saudi arabia found a significant association between leukocyte count and gcs (glasgow coma scale) score. leukocyte count exceeding 18 x 106/l has a predictive value for low gcs scores (6). likewise, gurkanlar et al. (11) in turkey show that leukocyte counts have a significant relationship to gcs scores, length of stay in the hospital, worsening head ct scan results, glasgow outcome scale (gos), and leukocyte counts exceeding 17.5 x 106/l. in addition, patients with traumatic subarachnoid hematoma had a higher mean leukocyte rate than a normal one. therefore, this study would assess a relationship ina. j. med. lab. sci. tech. 2021; 3(2): 81–89 8 3 ainul rofiq, et al. between the blood leukocyte count and the gcs score in craniotomy surgery patients in the icu room at jemursari islamic hospital. al-gahtany (6) showed a significant relationship between leukocyte count and gcs score, where a high leukocyte total (exceeding 14.18x106/l) is associated with the low gcs scores (6). rovlias & kotsou (12), from a prospective analysis of 125 patients, find that severe head injuries had a significantly higher leukocyte count than moderate and mild head injuries. the study of gurkanlar et al. (11) on 59 head injury patients found that a leukocyte count exceeding 17.5x106/l was a predictive factor for poor gcs score, prolonged hospital stay, and worsening ct scan results, regardless of the type of focal lesion that occurred. syed shahzad hussain et al. (13) in pakistan show that patients with severe head injuries experienced increased total leukocytes. some literature proposes that catecholamines and corticosteroids have a significant role in increased leukocytes in the injured patient. corticosteroids increase the leukocyte count by releasing leukocyte cells from their storage sites in the bone marrow into the bloodstream (14). in the brain, the microglia cell bodies become hypertrophied by a long process after trauma. then microglia cell bodies branch in the first sixty minutes after trauma (15). microglial cells express mhc class i and ii antigens. these antigens are presented to lymphocytes in regional lymph nodes and activate lymphocytes circulating in the central nervous system (12). furthermore, new leukocytes are increased in severe head injuries. leukocyte cells are less elastic than erythrocytes, requiring a higher pressure to push them into small diameter capillaries. in a state of decreased perfusion pressure, the capillaries can act as a net and trap leukocyte cells to increase the leukocyte count. after this process, the leukocyte cells stick to the endothelium and cannot be released even though the perfusion pressure has returned to normal (15). the purpose of this study was to determine the relationship between the leukocytes number and the consciousness level of craniotomy patients at the jemursari islamic hospital surabaya in 2018-2019. in addition, this research is expected to provide scientific information about the relationship between the number of leukocytes and the level of awareness in craniotomy surgery patients at the icu jemursari islamic hospital. the research results on post craniotomy patients at the jemursari islamic hospital in surabaya during 2018-2019 can give essential information for surgery doctors. materials and methods we performed an analytical observational study without direct treatment ina. j. med. lab. sci. tech. 2021; 3(2): 81–89 8 4 ainul rofiq, et al. to subjects and used a case study design with a retrospective approach. the health research ethics committee has approved this research at the jemursari islamic hospital surabaya by letter number 0102/kepk-rsi js/ix/2019. the data collected is secondary data collected from the operation room of the jemursari islamic hospital in surabaya for the period 2018-2019. the population in this study was the medical records of post craniotomy patients at the jemursari islamic hospital in surabaya from may 2018 to august 2019. the sample in this study was the medical records of post craniotomy patients at the jemursari islamic hospital in surabaya in may 2018 august 2019 grouped into the inclusion or exclusion criteria. sampling was done by purposive sampling. inclusion criteria for craniotomy patients are post-craniotomy gcs data and post-craniotomy complete blood (leukocyte) lab results. exclusion criteria are incomplete medical records such as missing gcs and leukocyte laboratory data. the collected data were analyzed and grouped based on the inclusion and exclusion criteria. thus, these data are considered valid and reliable for our study. the collected data were entered into a computer system for tabulation and statistical analysis using spss software (version 17.0). baseline data were analyzed using quantitative techniques where variables are expressed as frequencies and percentages or as mean with standard deviation. both the independent and dependent variables use a nominal measurement scale. the correlation between the leukocyte count and the patient's level of consciousness was analyzed using the spearman correlation test. statistical significance was defined if p <0.05. we declare that there is no intervention on the research subjects. patient data confidentiality was protected by not including the patient's name and identity to comply with research ethics. data processing will be performed in the following ways: • collecting medical records of craniotomy patients in 2018 • collecting data per month proportionally until the samples number reaches the target • entering the required data per medical record into the recapitulation table • entering data including patient's initials, age, gender, level of consciousness (gcs number), and the leukocyte count in the table. results we found that ninety samples of patients selected by the purposive sampling technique had met the inclusion criteria. the sample characteristics were described based on the leukocytes number and the consciousness level of the patient. ina. j. med. lab. sci. tech. 2021; 3(2): 81–89 8 5 ainul rofiq, et al. table 1. data on age range and gender of post-craniotomy patients at the jemursari islamic hospital, surabaya, 2018 patient freq (n=90) percentage (%) age (years) 0-20 2 2.2 21-40 16 17.8 41-60 68 75.6 > 60 4 4.4 gender male 20 22.2 women 70 77.8 we found that the highest population age group is 41-60 years of 68 (75.6%), and the lowest population age group is 020 years of 2 (2.2%) (table 1). eighty-three research subjects were male (20 people or 22.2%) and female subjects (70 people or 77.8%). table 2. data on the number of leukocytes in post craniotomy patients at jemursari islamic hospital surabaya in 2018 number of leukocytes freq (n=90) percentage (%) high 73 81.1% normal 17 18.9% total 90 100.0% we found that 73 patients (81.1%) have an increase in leukocyte numbers, and 17 patients (18.9%) have normal leukocytes numbers (table 2). table 3. data on awareness level of post craniotomy patients at jemursari islamic hospital surabaya in 2018 level of consciousness freq (n=90) percentage (%) nomal 84 93.3 decreased 6 6.7 the patients with normal consciousness levels were 84 (93.3%), and those with decreased consciousness levels were 6 (6.7%) (table 3). the spearman's correlation statistical test of leukocyte count and awareness level shows a not significance value of 0.026 (p > 0.05), which means that there was no significant relationship between the number of leukocytes and the level of consciousness in post craniotomy patients. the correlation of the two variables between the consciousness level and the leukocytes number is very weak by the correlation coefficient of -0.126. discussion the highest craniotomy patient age group is 41-60 years (75.6%) (table 1), indicating that most of the craniotomy patients at the jemursari islamic hospital surabaya are older people or one with degenerative diseases that require craniotomy surgery. these results are in line with (16), in which the incidence of brain tumors increased rapidly at the age of 40-70 years in 2014-2016 and then increased sharply in its development. on the other hand, male craniotomy patients were 83 (22.2%) and 77.8% female. this result shows that most of the craniotomy patients at the jemursari islamic hospital are women. this data aligns with the research conducted at rsud dr. hasan sadikin bandung in 2010– 2013 that most brain tumor sufferers were women (17). ina. j. med. lab. sci. tech. 2021; 3(2): 81–89 8 6 ainul rofiq, et al. several reports propose the role of catecholamines and corticosteroids in the presence of trauma due to craniotomy (table 2). corticosteroids increase the leukocyte number by releasing them from their storage sites in the bone marrow into the bloodstream (14). in the brain, the microglia cell bodies become hypertrophied by a long process after trauma. then microglia cell bodies branch in the first sixty minutes after trauma (15). microglial cells express mhc class i and ii antigens. these antigens are presented to lymphocytes in regional lymph nodes and activate lymphocytes circulating in the central nervous system (12). because leukocyte cells are less elastic than erythrocytes, they require more force to be pushed into small capillaries. when the perfusion pressure is low, the capillaries act as a net, trapping leukocyte cells and increasing the leukocyte count. even though the perfusion pressure has returned to normal, the leukocyte cells have adhered to the endothelium and cannot be released (15). there were 84 (93.3%) patients with normal consciousness levels and six patients with decreased consciousness levels (6.7 percent) (table 3). the number of craniotomy patients who experience an increase in leukocytes is above 50%. this result does not fit the findings of guranlar et al. that higher leukocyte counts (more than 17.5 x 106/l) are correlated to poor gcs scores, length of hospital stay, and worsening ct scan results in 59 head injury patients (11). the spearman's correlation statistical test of leukocyte count and awareness level gave a nonsignificant value of 0.026 (p > 0.05), indicating no significant relationship between the number of leukocytes and the level of consciousness in post-cranial surgery patients. the relationship between the two variables is not unidirectional. the higher awareness level is not correlated to the higher leukocyte number. this result is not in line with the research by al-gahtany (6) in saudi arabia, which showed a significant relationship between leukocyte count and gcs score (leukocyte count exceeding 14.18 x 106/l is correlated to low gcs scores) (6). the increase in leukocytes itself can occur due to the inflammatory response in the body, stimulated by both microbes and nonmicrobes. the case related to this study is an increase in leukocytes due to tissue and muscle damage, where the response produced by one person is different from another (18). decreased level of consciousness is caused by the failure of the projection and reception of afferent impulses by the reticular activating system (ras) and the brain's two hemispheres so that alertness and awareness are not achieved (19). this condition can be caused by various factors, for example, lesion destruction caused by infection and trauma. it is often characterized by an increase in leukocytes as an indication ina. j. med. lab. sci. tech. 2021; 3(2): 81–89 8 7 ainul rofiq, et al. of inflammation (18). these results indicate that the leukocyte number alone cannot indicate infection or trauma, especially trauma to the center of consciousness. subsequent investigations need to be performed to assess other factors, including the presence of infection or trauma associated with loss of consciousness. these factors can be combined with the leukocyte count, although the leukocyte count is not an ideal biomarker. deepak (20) shows that the leukocyte count can be used to evaluate and measure brain injury after intracranial surgery and can have enormous utility in routine neurosurgical treatment. in the subsequent research, we need to collect a larger population to get more accurate and significant results. further studies need to analyze the impact of craniotomy surgery on the morphology of specific leukocytes so that the specific effects of these cells can be estimated. improvements to research data can be made by recording medical records of research locations and classifying research data. we found that 73 patients (81.1%) have an increase in leukocyte numbers, and 17 patients (18.9%) have normal leukocytes numbers (table 2). this mechanism occurs in the presence of trauma due to craniotomy. the patients with normal consciousness levels were 84 (93.3%), and those with decreased consciousness levels were 6 (6.7%) (table 3). this result is in accordance with the fact that the percentage of craniotomy patients with increased leukocytes is more than 50%. the spearman's correlation statistical test of leukocyte count and awareness level gave a non-significant value of 0.026 (p> 0.05), indicating no significant relationship between the number of leukocytes and the level of consciousness in post-craniotomy patients. this result is different from algahtany's research (6) in saudi arabia which showed a significant relationship between leukocyte count and gcs scores. an increase in leukocytes number can occur due to an inflammatory response in the body, stimulated by both microbes and nonmicrobial, such as lesions damaged by infection and trauma (18). these findings suggest that the number of leukocytes alone cannot indicate infection or trauma, particularly trauma to the center of consciousness. other factors, such as the presence of infection or trauma associated with loss of consciousness, must be investigated further. conclusions in conclusion, most of the craniotomy patients at jemursari islamic hospital surabaya are elderly and female patients, where elderly patients often have degenerative diseases that require craniotomy surgery. ina. j. med. lab. sci. tech. 2021; 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(maria ham meilania saraswati, ed.). elsevier saunders; 2015. 19. aninditha t, prawiroharjo p. buku ajar neurologi. departemen neurologi fkui-rscm; 2017. 20. deepak a, nilesh k, bhawani, ss. leukocytosis after routine cranial surgery: a potential marker for brain damage in intracranial surgery. asian j neurosurg. 2016;2:109-113. 1 analysis of aptt based clot waveform parameters in various clinical conditions – a study at a tertiary care center rachana lakhe1, amit nisal1, preeti doshi1, ravindra nimbargi1 1department of pathology, bharati vidyapeeth deemed to be university and medical college, pune, india correspondence: doshi preeti, department of pathology, bharati vidyapeeth deemed to be university and medical college, pune, india zip code: 411043 email: prdoshi22@gmail.com received: june 8, 2022 revised: january 17, 2023 accepted: february 10, 2023 published: april 29, 2023 doi: 10.33086/ijmlst.v5i1.3064 abstract various coagulation tests like prothrombin time (pt) and activated partial thromboplastin time (aptt) are estimated by automated coagulation analyzers. the newer fully automated analyzers generate clot wave forms apttcwa for these parameters are derived. in this study, the objective was to analyze clot wave form characteristics morphology and its first and second derivative values in cases with abnormal aptt. acl top 300 generated curves for aptt in a total 125 patients with 20 normal controls are included. first derivative, second derivative, morphology of curve: sigmoid, biphasic, prolonged pre-coagulation phase, second derivative morphology like early and late shoulder, biphasic peak, delayed deceleration were the analyzed parameters. wave clot forms of 125 patients were included in this study. patients (m:f 2.2:1, mean age: 46.9 ± 20 years). a spectrum of clinical conditions was covid (20%), liver disease (23%), polytrauma (10.4%), cardiac diseases (8.8%), sepsis/dic (7.2%), thromboembolism (7.2%), renal diseases (6.4%), bacterial infections (4%), dengue (4%), snake bite (1.6%) and factor deficiency (1.6%). liver and heart disease showed a significant difference in acceleration and deceleration peaks followed by sepsis, dengue, polytrauma and sepsis/dic. deceleration peak was prolonged in patients of covid (p<0.05). sepsis and liver diseases showed prolonged first derivative peak (p<0.05). cwa is very easily available on all automated coagulation analyzers. it is inexpensive with fast turn round time. both quantitative as well as qualitative informations such as velocity, acceleration of clot formation and wave pattern details were recorded. our study highlights importance of quantitative and qualitative cwa parameters acquired by performing aptt test for the automated analyzers. keywords aptt, clot waveform, velocity acceleration. citation: lakhe r, nisal a, doshi p, nimbargi r. analysis of aptt based clot waveform parameters in various clinical conditions – a study at a tertiary care center. indones j med lab sci technol. 2023;5(1):1–9. doi: 10.33086/ijmlst.v5i1.3064 this is an open access article distributed under the creative commons attribution-sharealike 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2023 by author. mailto:prdoshi22@gmail.com mailto:https://doi.org/10.33086/ijmlst.v5i1.3064 https://creativecommons.org/licenses/by-sa/4.0/ ina. j. med. lab. sci. tech. 2023; 5(1): 1–9 rachana lakhe, et al. 2 introduction clot waveform analysis (cwa) is an extended interrogation of the curve generated by an optical detection system during the measurement of coagulation assays such as prothrombin time (pt) and activated partial thromboplastin time (aptt). it detects light transmittance basd on its absorbance. this is a global hemostatic assay, that reflects the overall hemostatic factor (1). the automated photo-optical coagulation analyzers used for the estimation of pt and aptt display the clot reaction curves along with the first and second derivative curves (first and second dcs) (2). the height of the first dc in the apttcwa is used to reflect to the “thrombin burst” as a hemostatic ability. the low height of the first dc in aptt-cwa suggests a risk of bleeding. the height of the second dc in aptt-cwa is useful for detecting coagulation factor deficiency (3). thromboelastography (teg) also shows a different pattern of information, but is slightly expensive and time-consuming (4). the coagulation system has a specific mechanism that includes the cascade system, thrombin burst and enhancement of clotting activation by phospholipids (pls) (5). the various available assays which evaluate the coagulation system these days are activated partial thromboplastin time (aptt), pt, thromboelastography (teg), and thrombin generation test (tgt) (6). cwa is a global coagulation assay that evaluates the kinetics of fibrin formation during testing of aptt or pt. clot waveforms provide information on light transmittance during clot formation (7). automatic optical end-point coagulation analyzers have the ability to show the clot reaction curve of the pt and aptt and reflect the “thrombin burst” with “enhancement of clotting activation by pls (8). the plot waveform analyses the slope generated by optical detection during routine coagulation tests, such as aptt or pt. the optical detection system generates a clot formation process with respect to the change in transmittance and absorbance of the light beam through the sample (9). continuous measurement of the change in light transmission or absorbance during the pt and aptt assays is performed and the data are given in the form of a wave. this generated clot wave has three phases: 1) precoagulation, 2) coagulation: either decreased light transmittance or increased absorbance along with formation of fibrin is seen in the clotting process which is seen as slope on waveform, 3) post coagulation: towards the end of coagulation, the light transmittance or absorbance stabilizes, which is seen as a linear segment on the waveform (10). tgt and cwa provide similar information, as both correlate with the rate ina. j. med. lab. sci. tech. 2023; 5(1): 1–9 rachana lakhe, et al. 3 and velocity of thrombin formation and reflect the entire process of thrombin generation. clinical conditions such as dic/sepsis and prediction of bleeding risk in dic (3), factor viii deficiency (3), bacterial infections (11), covid 19 (12), patients on anticoagulant therapy exhibit different clot waveform morphology. the aim of the present study was to analyze and compare clot waveform characteristics, such as morphology and firstand second-derivative values, in cases with abnormal aptt. materials and methods it is a prospective cross-sectional study with a duration of 6 months. a total of 125 patients and 20 age matched controls were included in the study. all the abnormal aptt samples were thoroughly selected through the analyzer. patients receiving unfractionated heparin (ufh) or lowmolecular-weight heparin (lwmh) were excluded from the study. blood samples were collected in anticoagulant tubes containing 1:9 volumes of 3.2% trisodium citrate. for obtaining platelet poor plasma: platelet-poor plasma was obtained by centrifugation of the blood samples at 3000 rpm for 15 min. the plasma is analyzed for platelet count on a cell counter, which should be less than 10000/ per microliter. pt/aptt was performed using an acl top 300 cts coagulation analyzer. the morphology of clot waveform in all the conditions with abnormal aptt was studied. the parameters studied were the first derivative (maximum velocity of clot formation), second derivative (maximum and minimum acceleration and deceleration during clot wave formation). statistical analysis was performed using sps15 software. the mean and standard deviation were calculated for the aptt, first and second derivatives. mann whitney u test was performed and p value was determined. statisctical significance was set at p<0.05. results a total of 145 patients were included in the study, with 20 control samples. the mean age group in the present study was 46.9 ± 20 years with male to female ratio of 2.2:1. the ranges of aptt, first, and second derivatives in controls and cases were determined (table 1). the morphology of the clot waveforms was studied in all 125 cases. the various clot wave patterns studied were sigmoid, biphasic, prolonged pre-coagulation phase, slow or steep slope, second-derivative morphology, biphasic peak, and delayed deceleration. the liver disease and corona virus disease 2019 (covid-19) were the two most prevalent conditions among the 125 cases in the current investigation. (figure 1). the most common pattern was a sigmoid pattern and prolonged pre-coagulation phase. various morphologies of the clot waveforms in different clinical conditions are shown in ina. j. med. lab. sci. tech. 2023; 5(1): 1–9 rachana lakhe, et al. 4 figure 2. the mean values of aptt and the first and second derivatives in each clinical condition were calculated and compared with those of the controls (table 2). a significant association was found between cases of liver disease and sepsis with respect to the first derivative (p<0.05). a significant association was found in cases of covid-19, sepsis, heart disease, and liver disease with respect to the second derivative (p<0.05) (table 3). table 1. range of aptt, first and second derivatives in cases and controls parameters controls cases aptt 29.7 to 36.5 seconds first derivative 98.08 354.87 tu/l 11.43 344.81 tu/l second derivative (acceleration) 376.1 1212.81 tu/l 21.29 865.02 tu/l second derivative (deceleration) 225.52 563.867 tu/l 8.95 270.29 tu/l figure 1. spectrum of clinical cases and various morphology patterns of clot wave 5 7 25 5 2 11 29 13 8 9 2 9 0 5 10 15 20 25 30 35 spectrum of clinical condition 97 28 40 85 97 28 98 27 morphology of clot wave formation ina. j. med. lab. sci. tech. 2023; 5(1): 1–9 rachana lakhe, et al. 5 figure 2. waveforms of normal and biphasic aptt clots. on the mda system, photo-optical monitoring of clot formation yields a sigmoid pattern that looks like a wave. table 2. mean values of aptt, first and second derivative in various clinical condition mean aptt mean velocity (first derivative) mean acceleration (second derivative) mean deceleration (second derivative) controls 33.3 217.95 742.05 362.38 covid-19 57.3 257.78 624.72 253.05 heart disease 45.3 200.34 513.29 229.30 chronic diseases 49.9 209.07 569.16 264.75 bacterial infections 47.5 344.81 865.02 270.29 liver diseases 42.6 166.62 430.30 202.71 dengue 42.5 151.39 385.33 146.68 factor deficiency 52.6 256.20 667.98 232.26 renal diseases 62.5 283.29 640.51 263.04 sepsis/dic 73.6 144.24 295.31 121.39 snake bite 117.4 11.43 21.29 8.95 venous thromboembolism 55.3 254.34 452.47 199.88 ina. j. med. lab. sci. tech. 2023; 5(1): 1–9 rachana lakhe, et al. 6 table 3. association of aptt, first and second derivatives in various clinical conditions clinical condition aptt first derivative second derivative (+) second derivative (-) covid mann whitney u test 14.000 207.000 186.000 128.000 p value 0.000* 0.575 0.284 0.013* sepsis mann whitney u test 0.000 41.000 16.000 12.000 p value 0.000* 0.047* 0.001* 0.001* heart disease mann whitney u test 0.000 88.000 58.000 53.000 p value 0.000* 0.364 0.032* 0.019* liver disease mann whitney u test 6.000 185.000 113.000 90.000 p value 0.000* 0.033* 0.000* 0.000* *p value < 0.05 there were two cases of snakebite with abnormally raised aptt and abnormal morphology of the clot waveform. the precoagulation phase was entirely distorted and the first and second-derivatives were suggestive of consumptive coagulopathy (dic). in addition, two cases of factor deficiency (factor v and factor viii) showed corresponding changes in the clot wave pattern with early and late shoulders in the second derivative curves. discussion multiple factors, such as the blood vessel wall, plasma proteins, platelets and coagulation factors are involved in the coagulation cascade. pt and aptt are the routinely performed tests that give information regarding hemostasis. coagulation assays are of utmost importance in this new era. (13). cwa is based on aptt and pt tests, which are global coagulation tests and is studied on the principle of optical detection system through an automated coagulation analyzer. various acl series are widely available and use an automated coagulation analyzer that works on the light absorbance principle and helps in studying the entire process of hemostasis depicted in the form of waves as described above (14). in the present study, clot waveforms detected hemostatic alterations and abnormal patterns in various clinical cases like covid19, bacterial infection, liver diseases, dic/sepsis, hemophilia, venous thromboembolism. a rise in the incidence of thrombotic events, such as pulmonary embolism, has been observed in critical ill cases of covid-19 with severe hypercoagulability is been seen in various studies (15). ina. j. med. lab. sci. tech. 2023; 5(1): 1–9 rachana lakhe, et al. 7 m.f rubereto et al., (16) studied cwa in 191 patients with liver cirrhosis and found values of maximum acceleration and deceleration were lower in the cirrhotic patients as compared to the control groups which correlate with our study. takuya et al., (12) studied the clot wave forms of aptt in 26 patients with covid-19 and the results showed abnormal patterns of second derivative morphology (early shoulder type and late shoulder type), which were similar to those found in our study. in a study by tan et al., (1) of 101 patients, it was concluded that patients with bacterial infections showed significantly higher cwa parameters than controls. in contrast, patients with dengue infection had significantly lower cwa parameters. this similar observation was also seen in our study. kei et al., (3) studied the clot wave form in 211 patients of sepsis and showed first and second derivatives curves were useful in diagnosis and prediction of bleeding risks. there was a significant association between the second derivative and the disease condition in this study. a study by dave et al., (17) showed that the risk of severe bleeding in patients with hemophilia a invariably accompanied by an aberrant clot waveform and thrombin generation test, while the factor viii level did not always reflect the actual bleeding severity. similarly, we had two cases of factor deficiency in our study with abnormal clot wave formation, thus providing new insight into the potential utility of cwa in detecting hypercoagulability or risk of bleeding in various clinical conditions (17). a study by kanouchi et al., (18) showed significant atypical peak and deceleration/acceleration ratio extension using clotting waveforms, specifically in patients with la-positive aps. oka et al., (19) studied the cwa for the assessment of doac effects and provided valuable insights into the relevance of anticoagulation to therapeutic efficacy and bleeding risk from the perspective of fibrinolysis. cwa is an extended study of the routine aptt test that utilizes pre-existing test protocol and equipment assay. many automated analyzers use changes in light transmittance or absorbance to measure clotting times, and these optical changes over time as clot forms are captured and presented as a clot waveform curve in the software of the analyzers (20). as more studies showing the correlation between the clot wave patterns have surfaced, the importance of full utilization of the data provided by the automation machines can be interpreted without any additional cost and turn over time (14). clot waveform analysis provides similar information to tgt, as it correlates with the rate and velocity of thrombin formation, and reflects the whole process of thrombin ina. j. med. lab. sci. tech. 2023; 5(1): 1–9 rachana lakhe, et al. 8 generation. this contrasts with routine coagulation assays, in which clotting time only reflects coagulation initiation (14, 16). conclusions cwa is readily available in newer coagulation analyzers. in addition to routine coagulation tests, cwa, which is readily available without any extra cost, helps study the process of hemostasis in normal as well as abnormal patients without any additional turnaround time. quantitative as well as qualitative information was obtained from the clot waveform analysis, which can be used for clinical decisions. our study highlights the importance of quantitative and qualitative cwa parameters obtained using a simple aptt test. author contributions rachana lakhe and preeti doshi: design, acquisition and analysis of data and drafting of manuscript, statistical analysis with critical revision. amit nisal and ravindra nimbargi: the project was supervised. acknowledgements the author thanks the reviewers for their constructive reviews to the paper. conflict of interest there is no financial relationship between them. the authors declare that there are no conflicts of interest. references 1. tan cw, wong wh, cheen mh, chu ym, lim ss, ng lc et al. assessment of aptt-based clot waveform analysis for the detection of haemostatic changes in different types of infections. scientific reports. 2020; 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27(1):337-42. doi: 10.1080/16078454.2022.2043573 9 chemometric analysis of serum magnesium calculations using mg-xylidyl blue-i method based on molar absorptivity ally kafesa1, nadira nur hajah lutfi1, cep wahyu1 1medical laboratory of technology study program, rajawali institute of health, bandung, indonesia correspondence: ally kafesa, rajawali barat no.38, kota bandung, west java, indonesia zip code: 40184 email: kafeally@gmail.com received: january 7th, 2021 revised: march 27th, 2021 accepted: april 2nd, 2021 published: april 28th, 2021 doi: 10.33086/ijmlst.v3i1.1876 abstract the concentration of magnesium is determined based on the absorbance of the mg-xylydil blue-i complex solution use spectrophotometer. based on the lambert-beer rule, the calculation of sample concentration is based on the formula a = ԑ. b. c. generally, the thickness of the cuvette (b) and the molar absorptivity (ԑ) factor will be ignored because it is considered to have a fixed value, therefore the sample concentration is measured based on the ratio of the absorbance of the sample against the standard solution. however, the standard solution contains pure magnesium and has a different matrix than the sample matrix, so this condition can give analytical errors and lead to misinterpretation of the results. the purpose of this study was to determine the accuracy and the precision of serum magnesium calculation by the principle of the mgxylydil blue-i complex reaction based on molar absorptivity compared to the general method. this research uses comparative study design methods. the serum sample used was the patient's serum specimen who has a normal magnesium level. the results showed that the significance value of the paired t-test statistical was 0.000 (p < 0.05). the accuracy value (d%) of the calculation formula uses ɛ is 0.00 and the precision value (cv%) is 0.53. while the accuracy value (d%) of the calculation formula without ɛ is 0.00 and the precision value (cv%) is 0.38. calculations based on molar absorptivity (ɛ) can measure more significant serum magnesium than those calculated based on standard magnesium solutions. keywords epsilon, magnesium, mg-xylydil blue-i complex reaction, serum, uv-vis spectrophotometry. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. mailto:kafeally@gmail.com https://journal2.unusa.ac.id/index.php/ijmlst/article/view/1876/version/2360 ina. j. med. lab. sci. tech. 2021; 3(1): 9–18 ally kafesa, et al. 1 0 introduction long-term deficiency of magnesium (mg) can cause hypomagnesemia with clinical manifestations including numbness, tingling, muscle cramps, seizures, personality changes, and abnormal heart rhythm. on the other hand, excessive consumption of magnesium from drugs containing mg (laxatives or antacids) led to hypermagnesemia, hyperthyroidism, kidney failure, and liver failure. therefore, it is important to maintain the stability of the balanced micronutrient magnesium content in the body (1,2). magnesium is an essential element that composes the coenzyme for signal transfer in neurons and enzymes for cardiac contraction. in assition, magnesium is required for the metabolism of carbohydrates, fats, and amino acids as micronutrients. magnesium has medicinal value as a general laxative, antacid (e.g. milk of magnesia), and to stabilize abnormal nerve excitation or spasm of blood vessels in conditions such as eclampsia (2). an analytical method (qualitative and quantitative analysis), e.g. chemometric analysis method, is needed to determine the content of magnesium. chemometrics work by combining statistical values with chemistry, especially analytical chemistry. the chemometric analysis uses statistical principles to design; select an optimal analytical procedure and experiment, and provide maximum and relevant chemical information through chemical data analysis (4,5). the spectrophotometer is widely used in quantitative measurements of magnesium because the amount of light absorbed by the particles in the solution depends on the type and number of particles (6,7). the photometer spectrum is based on the lambert-beer law. lambert-beer law states that the concentration of the standard solution is directly proportional to the value of light absorption (absorbance) (8). this law applies to monochromatic rays e.g. light with a single wavelength or that has an adjacent wavelength band. to calculate the absorbance of a sample, it is necessary to know the molar absorption denoted by ɛ (epsilon), which is a molecule or ion that absorbs a solvent with a certain wavelength but does not depend on a particular concentration and wavelength or through radiation (9). the ɛ (epsilon) cannot be removed because it is affected by solvents. the solvent determines the addition of the electron transition energy in the compound, thus the wavelength that becomes the energy will be absorbed in a certain color. if the solvent has many mixtures, it will require a large amount of energy due to the interaction of the solvent (energy π-π* can be smaller or greater). therefore, mathematically, the ɛ factor in the calculation equation cannot be ignored. ina. j. med. lab. sci. tech. 2021; 3(1): 9–18 ally kafesa, et al. 1 1 the use of different measuring formulas of magnesium content may provide different interpretations of the results. comparative analysis of results is needed to compare the accuracy, precision, and statistical difference between the measurement results of the two formulas. materials and methods this research used comparative study design methods. the sample used in this study was serum of healthy patients with normal magnesium levels in june 2020. the equipment used was a spectrophotometer (genesystm 10s). reagents used in this study are magnesium xl fs from diasys diagnostic systems (germany) contains ethanolamine ph 11.0 750 mmol/l, glycoletherdiamine-tetraacetic acid (gedta) 60 µmol/l, xylydil blue-i (248266 sigma aldrich, cas number 14936-97-1) 110 µmol/l and 2 mg/dl standard. the sample was measured by a photometer at a wavelength of 520 nm (11). samples that met the inclusion criteria were 3 samples. the inclusion criteria in this study were patients who were willing to become respondents by filling out an informed consent sheet and the sample volume had to reach 3 ml. the sampling technique used was random sampling with the criteria of non-lysed, non-icteric, and non-lipemic blood sample examination (11). the samples obtained were centrifuged at 3.000 rpm for 15 minutes. total samples were analyzed for magnesium levels with the photometric test method using xylydil bluei. the magnesium ion forms a purple complex with xylydil blue-i in an alkaline condition. in the presence of the calcium ion complex gedta, the reaction is specific. the intensity of the purple color is proportional to the concentration of magnesium (11). after the data on serum magnesium absorbance are collected, the magnesium content was calculated based on two formulas: formula type a and formula type b. calculation a cspl = aspl x cstd astd ........................................ 1 cspl: sample concentration (mg/dl) aspl: sample absorbance cstd: standard concentration (mg/dl) astd: standard absorbance calculation b cspl = aspl x mw ɛ .1𝑐𝑚 1% ....................................... 2 cspl: sample concentration (mg/dl) aspl: sample absorbance mw: molecular weigth of xylydil bluei (513,5 g/mol) ɛ .1𝑐𝑚 1% : molar absorptivity of xylydil blue-i (49.000 l m-1cm-1) the calculation results of the two formulas were analyzed using the paired t ina. j. med. lab. sci. tech. 2021; 3(1): 9–18 ally kafesa, et al. 1 2 test. the accuracy and precision analysis are calculated based on the deviation from repeated measurements of 3 times. results this research begins by creating a calibration curve to get the liner value. standard curve determination was performed with several concentrations (2, 4, 6, 8 mg/dl). figure 1. curve calibration of magnesium standard solution the calibration data shows that r = 0.9278, which means that the data shows a linear correlation between the concentration of magnesium and absorbance value. the data collected was analyzed by statistical tests following certain conditions. data on 20 healthy patients were obtained with a mean serum magnesium level of 3.077 mg/dl, the lowest value was 1.387 mg/dl, and the highest value was 5.581 mg/dl. all patients had normal serum magnesium levels (one day before sampling) and did fasting for 10 – 12 hours to minimize the influence of food and activity. the accuracy and precision of the magnesium level of patients was determined by statistical analysis (table 1). table 1. quality test data no parameter quality 1 mean 3.077 2 standard deviation (sd) 1.712 3 coefficient of variation (cv) 0.556 4 accuracy (d%) 0.057 5 total error (te) 1.17 % 6 total error allowable (tea) 4% *based on magnesium test normal data distribution is obligatory before paired t-test conducted. the data normality test results show in table 2. result shows that all variable is normality distributed (p-value < 0.05). y = 0,0174x + 0,6435 r² = 0,9278 0,66 0,68 0,7 0,72 0,74 0,76 0,78 0,8 0 1 2 3 4 5 6 7 8 9 a b so rb a n c e a t 5 2 0 n m concentration (mg/dl) abs linear (abs) ina. j. med. lab. sci. tech. 2021; 3(1): 9–18 ally kafesa, et al. 1 3 figure 2. serum magnesium level curve with 2 formulas: non (ԑ) and (ԑ) table 2. variable test of normality test results for each sample kolmogorov-smirnov shapiro-wilk statistic df sig. statistic df sig. kit_insert 0.198 16 0.093 0.898 16 0.076 epsilon 0.116 16 0.200 0.925 16 0.205 the sample size is lower than 50, therefore the shapiro-wilk normality test was used. a significance value lower than 0.05 means the data is normally distributed. the significance value of epsilon is 0.205 (p < 0.05) and the significance value of the insert kit is 0.076 (p < 0.05) so that the next test uses the paired t-test. table 3. results of t-test statistics – paired one-sample test test value = 0 t df sig. (2-tailed) mean difference 95% confidence interval of the difference lower upper kit_insert 10.40 15 0.000 2.42 1.92 2.91 epsilon 7.52 15 0.000 2.88 2.06 3.69 statistical test (table 3) shows that the significance value is 0.000 (p < 0.05) for both the formula using the kit-insert and epsilon, therefore there is a significant difference between the calculation formula for the results of the chemometric analysis with ɛ and analysis without ɛ. 0 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 m g s e ru m c o n c e n tr a ti o n m g /d l sample no. non (ԑ) (ԑ) ina. j. med. lab. sci. tech. 2021; 3(1): 9–18 ally kafesa, et al. 1 4 discussion spectrophotometric analysis work by white light or radiation passed through a colored solution, then radiation with a certain wavelength will be absorbed and other will be transmitted. the absorbance value depends on the content of the substances, the more molecules absorb light, the greater the absorption value. therefore, the absorbance value will be directly proportional to the concentration of the substance contained in a sample (9). curve calibration of magnesium standard solution have slope 0.0174 and intercept 0.6435 with equation y = 0.0174x + 0.6435. the correlation between analyte level (x) and instrument response (y) is expressed as the correlation coefficient (r = 0.9278) (figure 1). ideally, the intercept is zero. it is expected that no instrument response will occur when analyte free water or blanks are measured. but in this research, we find instrument response occurs due to small interaction, interference, noise, contamination or other sources of bias. therefore, the intercept (a) in this calibration curve can be considered as the signal from the blank. while the slope (b) is a measure of the sensitivity of a test method. we have greater the value of b, so this method provides a higher sensitivity or the instrument's response is strong enough to change the magnesium existing levels. based on the correlation coefficient obtained, it shows a linear relationship between magnesium concentration and absorbance. the linear relationship that occurs is positive and strong. in this study, curves of serum magnesium levels in 20 serum samples with 2 formulas (non (ԑ) and (ԑ)) showed an average difference in yield of 15.27%, where serum magnesium levels calculated using ԑ had higher levels (figure 2). at magnesium levels <2.5 mg/dl, the difference in calculations is not too far away, but at magnesium levels >2.5 mg/dl, there is a very large difference. this shows that the calculation of serum magnesium using the molar absorption of xylydil blue-i can bind magnesium ions more than calculated compared to the absorbance of standard magnesium solutions. the molecule that receives visible light at the appropriate frequency will experience a transfer of energy to a higher level (transfer of electrons from the ground state to the excited state). this electron transition (µ-µ*) absorbs specific energy and can be detected at certain wavelengths. the specificity and quantity of absorbed light energy are determined based on solubility. the more dissolved a compound, the more energy it will absorb. conversely, the harder it is to dissolve, the lighter energy is transmitted and this will give false results of the measured number of molecules (8). the calculation formula used to measure magnesium in serum is as follows formula 3: cspl = aspl x cstd astd ....................................... 3 ina. j. med. lab. sci. tech. 2021; 3(1): 9–18 ally kafesa, et al. 1 5 this calculation formula comes from: 1. 𝐴𝑠𝑝𝑙1 = 𝑏1 𝑥 ԑ1𝑥 𝐶𝑠𝑝𝑙1 2. 𝐴𝑠𝑝𝑙2 = 𝑏2 𝑥 ԑ2 𝑥 𝐶𝑠𝑝𝑙2 if there are two similar solutions measured, the fixed factor in the formula can be removed and combine: 1. 𝐴𝑠𝑝𝑙1 = 𝑏 𝑥 ԑ 𝑥 𝐶𝑠𝑝𝑙1 2. 𝐴𝑠𝑝𝑙2 = 𝑏 𝑥 ԑ 𝑥 𝐶𝑠𝑝𝑙2 cspl1 = aspl x cspl2 aspl2 cspl1: sample concentration aspl1: sample absorbance (sample 1) cspl2: standard concentration aspl2: standard absorbance (sample 2) in this formula, the concentration of the magnesium in the standard solution (pure solvent) is used as a reference for calculating the level of magnesium in serum. the serum is a matrix containing solutes (enzyme, protein, clotting agents, immune system); body essentials such as vitamins and hormone; and dispersed cell components with a ph between 7.35 – 7.45. the solubility of magnesium ions in the serum matrix differs from the solubility of magnesium in pure solvents which do not contain other solutes. a standard solution of magnesium dissolved in water at a ph of 9.0. the difference in ph of this solution reduces the reaction of magnesium with xylydil blue-i the magnesium ion forms a purple complex with xylydil blue-i in an alkaline condition. in the presence of the calcium ion complex gedta, the reaction is specific. the intensity of the purple color is proportional to the magnesium concentration (8). figure 3. chemical structure of xylidyl blue-i (c25h20n3nao6s) (7). figure 3 show the structure of xylydil blue-i. iupac name of xylidyl blue-i is sodium; 3-[[3-[(2,4-dimethylphenyl) carbamoyl] -2-hydroxynaphthalen-1-yl] diazenyl]-4-hydroxy benzene sulfonate. xylydil blue-i is a synthetic colorimetric reagent for mg detection with a molecular weight of 513.5. the aqueous solution of xb1 is red and turns reddish-violet in the presence of mg at ph 9 (maximum wavelength: 510 nm, molar absorptivity: 49,000, detection range 0.02-0.4 ppm. xylydil blue-i can react specifically with magnesium ions in the presence of a glycoletherdiamine tetraacetic acid ina. j. med. lab. sci. tech. 2021; 3(1): 9–18 ally kafesa, et al. 1 6 (gedta). gedta is a substance to bind and control metal ions because it can remove water hardness (chelating agent). that is the greater affinity of chelating ligands for a magnesium ion than that of similar nonchelating (monodentate) ligands for the same metal (6). xylydil blue-i has a very good molar absorption (ɛ = 49,000) value in polar solvents therefore it can be used for the detection of compounds in polar matrices. when xylydil blue-i reacts with magnesium ions at ph < 9, the stoichiometry of the reaction will shift to the left and the complex mg-xylydil blue-i products formed are getting less. polar sulfonyl groups (so3 -) can interact favorably with similar water molecules. therefore, the short-chain hydroxyl-benzene is soluble in water. however, since the organic portion (more c atoms) gets bigger (the longer chain in this case), this interaction is less effective and water solubility decreases (8,6). serum ph does not support the mg xylydil blue-i reaction because the electrons in the sulfonyl group do not have enough energy to bind protons to the magnesium ion at ph 7. on the other hand, the standard solution of magnesium has a ph of 9. if this difference is calculated using formula type a, the magnesium test results will be inconsistent, thus the calculation of serum magnesium levels must be divided by the molar absorptivity of xylidyl blue-i. cspl = aspl x mw ɛ .1𝑐𝑚 1% ....................................... 4 type b formula (lambert-beer) involves the molecular weight and molar absorptivity of xylydil blue-i. consequently, the measured mg-xylydil blue-i complex reaction corresponds to the actual reaction product. the ph changes and the number of magnesium ions in the serum matrix will not affect the detected reaction product suitability. calculation of serum magnesium using this formula has units of mole/liter, therefore it must be converted to mg/dl. this research was done in the patient who had normal magnesium levels. more comprehensive studies must be conducted in patients with pathological conditions that allow the maximum amount of magnesium ions that can form bonds with xylydil bluei. conclusions the serum magnesium level formula that include molar absorptivity factor have a significant difference result (α<0.05) compared to formula without molar absorptivity factor. there is a significant difference between serum magnesium levels using the absorbance calculation formula with formula a (without ɛ factor) and formula b (with ɛ factor). calculations based on molar absorptivity can measure more significant serum magnesium than those ina. j. med. lab. sci. tech. 2021; 3(1): 9–18 ally kafesa, et al. 1 7 calculated based on standard magnesium solutions. author contributions ally kafesa: conceptualization, methodology, writing-original draft, visualization, supervision, funding acquisition. cep wahyu: methodology, supervison. nadira nur hajah lutfi: formal analysis, investigation, resources. acknowladgements the authors express their sincere gratitude to the rajawali institute of health, bandung, indonesia. conflict of interest we declare that we do not have any commercial or associative 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2015; 3(2). 90 in vitro anticoagulant activity of crude protease of bacillus tequilensis hsfi-5 stalis norma ethica1, tri joko raharjo2, dewi seswita zilda3, nur hidayati4 1master program of clinical laboratory science, universitas muhammadiyah semarang, semarang, indonesia 2department of chemistry, universitas gadjah mada, yogyakarta, indonesia 3research center for deep sea, earth sciences and maritime research organization, national research and innovation agency, jakarta, indonesia 4klinik pratama subekti medical, pemalang indonesia correspondence: stalis norma ethica, perumahan btn rejomulyo ii/44, kediri, indonesia zip code: 64129 email: norma@unimus.ac.id received: january 1, 2023 revised: april 10, 2023 accepted: may 15, 2023 published: may 22, 2023 doi: 10.33086/ijmlst.v5i2.3791 abstract bacillus tequilensis hsfi-5 is a food-grade bacterial isolate obtained from the fermented intestine of holothuria scabra (sand sea cucumber). strain hsfi-5 had been reported to be able to produce proteases, which had shown several characteristics of an antithrombotic agent, i.e., fibrinolytic and clot-lysis activities. however, its anticoagulation activity test is yest to be done. this study aimed to determine the anticoagulant activity of the crude protease hsfi-5 in vitro. the study design was a completely randomized design with a sample size of 90 calculated using the federer formula. the material used was crude protease from b. tequilensis in skim milk broth. prothrombin time (pt), activated partial thromboplastin time (aptt), and plasma recalcification time (prt) were carried out to test the anticoagulant activity. citrated platelet poor plasma samples were divided into positive control, normal control, direct examination with crude enzyme in volumes of 50 and 100 µl and pre-incubation at 37ºc for 5, 10, and 15 min with crude enzyme volumes of 50 and 100 µl. the data normality was tested with the kolmogorov-smirnov test and the different tests were analyzed by one-way anova with the post hoc lsd test. the results of one-way anova both on pt, aptt, and prt examinations showed that there was a significant difference between the treatment groups (p<0.05). the longest results of pt, aptt, and prt are positive controls, and the shortest results are normal controls for pt, and 15’ 50 group for aptt and prt. it is clear that crude protease b. tequilensis hsfi-5 exhibits anticoagulant as well as thrombolytic action, raising the possibility that it could function as an antithrombotic drug. keywords crude protease, bacillus tequilensis hsfi-5, anticoagulant. citation: ethica sn, raharjo tj, zilda ds, hidayati n. in vitro anticoagulant activity of crude protease of bacillus tequilensis hsfi-5. indones j med lab sci technol. 2023;5(1):90–9. doi: 10.33086/ijmlst.v5i2.3791 this is an open access article distributed under the creative commons attribution-sharealike 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2023 by author. mailto:norma@unimus.ac.id https://creativecommons.org/licenses/by-sa/4.0/ ina. j. med. lab. sci. tech. 2023; 5(2): 90–99 stalis norma ethica, et al. 9 1 introduction coagulation is a process involving several components, such as blood vessels, platelet cells, and blood coagulation factors. normal coagulation pathways are a collaboration between clot formation and clot decay processes. an imbalance of the coagulation system can occur in an illness that causes bleeding or thrombosis (1,2). one of the diseases causing blood coagulation disorders is coronary vascular disease (cvds). data from the world health organization (who) states that 17.9 million people (32%) in the world die every year due to cvds. as many as 85% of people with cvd die from heart attacks and strokes. the risk of venous thromboembolism (vte) increases in the first 3 months after the occurrence of an ischemic stroke (3,4). ischemic stroke can occur due to a sudden reduction in blood flow to the brain, leading to reduced neurologic function. blood flow is reduced as a result of thromboembolic blockage. after 4 to 14 days from the time of commencement, antithrombotic therapy may be given (5,6). the antithrombotic potential of bacterial proteases can be explored in order to minimize costs (7). antithrombotic activities of bacterial proteases encompass mainly three characteristics: fibrinolysis, clot lysis and anticoagulant. the anticoagulant activity of bacterial proteases has been reported in several studies (7,8). proteases with fibrinolytic and thrombolytic activities had also been reported, mostly from bacillus subtilis (9,10). bacillus tequilensis hsfi-5 is a bacterial isolate obtained from the fermented intestine of the sand sea cucumber, holothuria scabra. the sea cucumber was isolated from sand sea cucumber from kodek gulf village, lombok, west nusa tenggara. strain hsfi-5 was found to have the ability to produce fibrinolytic protease, which is an important characteristic of an antithrombotic agent (11,12). another important characteristic of the protease of b. tequilensis that underlines its potential as an antithrombotic agent as clot lysis activity. this study represents novelty as the anticoagulant activity of the protease from b. tequilensis hsfi-5 has not yet been tested. such studies are important as a basis for further exploring bacterial protease activity through in vivo antithrombotic and toxicity assays. the commonly used antithrombotic agents work through the tissue plasminogen activator (tpa) mechanism. trypsin-like serine protease has been reported to be an anticoagulant agent because it contains sulfated polysaccharides that play a role in anticoagulant, antiangiogenic, and immunostimulating processes (7,13). the presence of thrombolytic activity is one of the potential anticoagulant (11). pt, aptt, and prt were used to test anticoagulant activity of crude protease. the pt, aptt, and prt ina. j. med. lab. sci. tech. 2023; 5(2): 90–99 stalis norma ethica, et al. 9 2 assays are the most commonly used tests for the examination of blood coagulation factors (14–16). discovery of alternative anticoagulants is needed to minimize the risks and costs of drug use. the purpose of this study was to measure the anticoagulant activity of the crude protease hsfi-5 in vitro. materials and methods a completely randomized design was used in this research. the study used three examination parameters, namely pt, aptt, and prt. the study was conducted at the laboratory of hematology of universitas muhammadiyah semarang in october 2022. the controlled variables of this study included a varied volume of crude protease from b. tequilensis hsfi-5 (of known concentration) and length of incubation time. the number of samples used in the study was calculated using the federer’s formula. the total number of treatment in this study was 10 group, namely (1) positive control; (2) normal control; (3) direct incubation with addition of 50 µl crude protease (d 50); (4) direct incubation with addition of 100 µl crude protease (d 100); (5) 5 mins pre incubation with addition of 50 µl crude protease (5’ 50); (6) 5 mins pre incubation with addition of 100 µl crude protease (5’ 100); (7) 10 mins pre incubation with addition of 50 µl crude protease (10’ 50); (8) 10 mins pre incubation with addition of 100 µl crude protease (10’ 100); (9) 15 mins pre incubation with addition of 50 µl crude protease (15’ 50); (10) 15 mins pre incubation with addition of 100 µl crude protease (15’ 100). each experiment had three replicate plants per treatment. the total study sample for each study parameter was 30. the total number of study samples was 90 samples. the blood samples used for the study were taken from a healthy volunteer who was willing to be a respondent. the research was conducted with permission from the ethics committee of the faculty of public health, university of muhammadiyah semarang with number 377/kepk-fkm/unimus/2020. isolation of crude protease enzyme b. tequilensis hsfi-5 b. tequilensis hsfi-5 isolates were subcultured on skim milk broth (smb) medium and incubated at 37ºc for 72 hours. bacterial cultures were centrifuged at 3000 rpm for 15 min at 4º c. the supernatant (crude protease) was harvested from the centrifugation results and could be used for in vitro anticoagulation assay (17). in vitro assays pt, aptt, and prt were performed to examine the anticoagulant activity of crude protease b. tequilensis hsfi-5. ina. j. med. lab. sci. tech. 2023; 5(2): 90–99 stalis norma ethica, et al. 9 3 anticoagulant heparin was used as a positive control. b. tequilensis hsfi-5 crude protease treatment normal and positive controls were treated as standard procedures, while direct assay treatments with crude protease b. tequilensis hsfi-5 50 µl and 100 µl was carried out by means of 50 µl ppp with 50 µl/100 µl crude protease, which was analyzed for clotting that occurred as standard procedure. test samples were prepared at 50 μl and 100 μl raw protease volumes with pre-incubation times of 5, 10, and 15 minutes. prothrombin time (pt) the pt examination used a sample of citrated platelet poor plasma (ppp). as much as 50 µl of ppp was added with 50 µl of pt reagent (teclot pt-s, germany) and incubated at 37ºc for 2 min before reading photometrically on a coagulometer (coatron m1, germany). pt results were expressed in seconds. the resulting clotting was analyzed using a coatron m1 coagulometer according to its manufacturing method (teclot pt-s, germany). activated partial thromboplastin time (aptt) coagulation analysis with aptt was performed photometrically with the teclot aptt-s reagent and read with a coatron m1 device. as much as 50 µl of ppp was reacted with 50 µl of aptt reagent and then incubated for 2 min at 37º c. the preparation of the test sample was carried out like the sample treatment for the pt test, and then the clotting time was expressed in seconds (s) following the teclot (germany) manufacturing procedure. plasma recalcification time (prt) prt examination was carried out manually with the addition of nacl and cacl2 0.025 m at 37ºc. the preparation of examination samples was carried out as in the treatment for pt and aptt examinations. as much as 100 µl ppp was added to 100 µl physiological nacl and incubated for 1 min. the mixture was then added to 100 µl cacl2 0.025 m and incubated for 90 s. the clot that appeared every 30 seconds was observed. prt results expressed in seconds were recorded (18). statistical analysis data were analyzed using spss 25 software (ibm, usa). kolmogorov-smirnov test for analyzing normality of data. normal data were followed by the one-way anova difference test. a post hoc lsd test was performed to determine the differences between the treatment groups in the study. a significant value of less than 0.05 was expressed as the result with a significant difference. ina. j. med. lab. sci. tech. 2023; 5(2): 90–99 stalis norma ethica, et al. 9 4 results the bacterial isolate of b. tequilensis hsfi-5 used in this study was from a previous study originally deposited in a microbank of the microbiology laboratory of universitas muhammadiyah semarang (11). after subculturing in 250-ml of smb medium, culturing and centrifugation, 35 ml of crude protease was obtained as a supernatant. this sample was then subjected to pt, aptt, prt and hematology assays. initial analysis of the protein content of the crude protease yielded a nano drop value of 7,7 mg/ml. hematology results showed that the highest mean pt examination was in the positive control group, and the lowest mean was in the normal control group. the highest and lowest aptt test result were determined in the positive control and 15’ 50 groups, respectively. the highest and lowest mean prt values in the positive control and 15’ 50 groups, respectively. baseline values for the pt assay were 10-14 seconds, aptt 22-30 seconds, and prt 90-250 seconds. the kolmogorov-smirnov normality test was normal for all groups, so the one-way anova test was used and performed. the p-value was 0.000 (p<0.05). an lsd post hoc test was used to determine differences in each group. the results of the post hoc lsd test are shown in figure 1. ina. j. med. lab. sci. tech. 2023; 5(2): 90–99 stalis norma ethica, et al. 9 5 figure 1. post hoc lsd test results of (a) pt, (b) aptt, (c) prt. *significant. discussion the crude protease b. tequilensis hsfi-5 was recovered from the bacterial culture supernatant of skim milk broth medium and previous studies based on fibrin plates and gravimetric analysis reported promising fibrinolytic and clot-lytic activities respectively. crude bacterial proteases are also expected to have anticoagulant activity, supporting their potential development as antithrombotic agents (11). in this study, we analyzed ina. j. med. lab. sci. tech. 2023; 5(2): 90–99 stalis norma ethica, et al. 9 6 the anticoagulant activity of bacterial proteases by performing pt, aptt and prt. the existence of side effects of available anticoagulants has led researchers to investigate the anticoagulant activity of the crude protease b. tequilensis hsfi-5 (19). anticoagulant activity of crude protease b. techylensis was analyzed by direct examination of pt, aptt, and prt (no delay), and by varying incubation times and crude protease volumes. the results of the descriptive analysis of the pt studies showed a trend for the studied proteases to prolong the pt time, consistent with the increased volume of hsfi-5 crude protease. the highest yield was obtained in the group with 100 µl of crude protease b. tequilensis hsfi-5 added and ppp incubated for 15 min. research data show that the longer the pre-incubation period and the more crude protease added, the longer the pt time. the most significant result of the pt examination was a 57.45% elongation compared to the normal controls. the difference in pt assay results appeared to greater with the addition of 100 µl of crude protease, suggesting that the addition of 100 µl gave optimal pt result at 37º c and 15 pre-incubation time. these results are in line with studies suggesting that fibrinolytic proteases can cause prolongation of pt (17). the pt test is a coagulation test to assess the potency of clotting factors in the extrinsic and common pathways. the presence of elongations in pt indicates that the crude protease b. tequilensis hsfi-5 can inhibit the coagulation processes in the extrinsic and common pathways (7). aptt testing is commonly used to detect coagulation factor deficiencies in intrinsic signaling pathways. the results showed that the addition of 100 µl of crude protease significantly prolonged the aptt results for the same pre-incubation time. however, the addition of 50 µl compared to 100 µl of crude protease reduced the sample. this was probably due to the low protease concentration, which required a larger amount and longer time to inhibit clotting. prolongation of the aptt results compared to the normal controls resulted in up to 2.75-fold time prolongation. this indicates that the crude protease b. tequilensis hsfi-5 is able to inhibit the coagulation process. all groups had extended prt test times, but a pattern was observed in which the addition of 100 µl of crude protease significantly increased the number of samples. a 3.87-fold extension was observed in the 15 min pre-incubation group compared to controls. these results demonstrate that the optimal anticoagulant activity of crude protease is preincubated for 15 minutes in a volume of 100 µl. ina. j. med. lab. sci. tech. 2023; 5(2): 90–99 stalis norma ethica, et al. 9 7 extending the prt results, we showed that the crude protease hsfi-5 can inhibit coagulation via an intrinsic pathway. longer incubation times and higher volumes increase anticoagulant activity because the low concentration of crude protease requires longer volumes and times to work optimally. a lower concentration of crude protease increases the amount and time to activate the anticoagulant mechanism through the serpin mechanism. the anticoagulant mechanism of the crude protease hsfi-5 is believed to be based on the serpine mechanism. coagulation and fibrinolysis are primarly controlled by the protease inhibitor serpine superfamily. antithrombin (at) is a physiological anticoagulant that targets procoagulant enzymes, particularly factor xa and thrombin (20–23). serpine participate in the anticoagulant mechanism by carrying out the proteolytic activity of clotting proteases in intrinsic and extrinsic pathways (24–26). antithrombin inhibits the activity of factor xa and thrombin (27–29). serpines have been identified in bacteria, archaea, eukaryotes, and viruses. several types of serpines include subtilisin, papain, and caspase families. the pci/at inhibited interaction between thrombin and thrombomodulin causes diffusion of the serpine-protease complex (30). chieving higher anticoagulant activity, smaller volumes, and minimal pre-incubation times requires the use of concentrated proteases. as a result, it can be used more effectively in the treatment of thrombosis (24,29,31). conclusions the crude protease b. tequilensis hsfi-5 exhibits anticoagulant activity based on pt, aptt, and prt vacuum and may be an alternative anticoagulant in the future. the longest results for pt, aptt, and prt represent the positive controls, and the shortest results were obtained from the normal control for pt, and 15-min incubation with 50 µl protease for aptt and prt. however, in vivo protease anticoagulant studie are required to confirm the results of these in vitro anticoagulation studies. author contributions stalis norma ethica: were involved in planning and designing the work, dewi seswita zilda: performed the enzyme preparation, tri joko raharjo and stalis norma ethica performed the statistical analysis. nur hidayati: assisted in interpreting the results and worked on the manuscript. acknowledgements this study was funded by national competitive applied research grant ina. j. med. lab. sci. tech. 2023; 5(2): 90–99 stalis norma ethica, et al. 9 8 (penelitian terapan kompetitif nasional, ptkn) 2022 from indonesian ministry of education, culture, research technology and higher education (kemendikbud ristek). conflict of interest authors declare that no conflict of interest in this study. references 1. putri a. diathesis hemorrhagic, coagulation and fibrinolytic system. biomol heal sci j. 2022;5(1):54–61. doi: 10.20473/bhsj.v5i1.35280. 2. palta s, saroa r, palta a. overview of the coagulation system. indian j anaesth. 2014;58(5):515–23. doi: 10.4103/00195049.144643. 3. who. cardiovascular diseases. 2021. available from https://www.who.int/healthtopics/cardiovascular-diseases#tab=tab_1. 4. virani ss, alonso a, aparicio hj, benjamin ej, bittencourt ms, callaway cw, et al. heart disease and 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https://www.ncbi.nlm.nih.gov/pmc/articles/pmc 3624763/pdf/nihms412728.pdf. 80 lycopene improves the metformin effects on blood glucose and neutrophil counts in type 2 diabetic rats medina sianturi1, neni susilaningsih2, heri nugroho3,4, maria suryani1 1department of nursing, stikes elisabeth, semarang, central java, indonesia 2department of histology, faculty of medicine, diponegoro university, semarang, central java, indonesia 3department of internal medicine, faculty of medicine, diponegoro university, semarang, central java, indonesia 4department of interbnal medicine, kariadi teaching hospital, semarang, central java, indonesia correspondence: medina sianturi, stikes elisabeth, kawi street 11, semarang, indonesia zip code: 50252 email: challenia@gmail.com received: february 5, 2023 revised: march 6, 2023 accepted: april 20, 2023 published: april 29, 2023 doi: 10.33086/ijmlst.v5i1.3865 abstract in patients with type 2 diabetes mellitus (t2dm), both innate and acquired immunity are weakened by hyperglycemia. lycopene is one of the hydrocarbon carotenoids that has been widely studied for its powerful antioxidant and anti-inflammatory properties, furthermore act as hypoglycemic and immunomodulator. herein, we investigated the effect of lycopene and metformin combination on fasting blood glucose (fbg) and neutrophil counts. the rats were divided randomly into six groups, each containing five rats. group 1 consisted of normal rats (n) and group 2, t2dm (dm) rats, which were administered 0.5 ml of coconut oil; group 3 t2dm rats were administered 250 mg/kg of metformin in 0.5 ml of coconut oil; groups 4,5 and 6 rats were administered a combination of metformin 250 mg/kg with 10 mg/kg (dml-10), 20 mg/kg (dml-20) and 40 mg/kg (dml-40) of lycopene in 0.5 ml of coconut oil, respectively. treatment was administered every day for 28 days. a model of t2dm rats was induced by a high-fat diet for two weeks combined with streptozotocin–nicotinamide. data were analyzed with a one-way anova test followed by the least significant difference (lsd) test. there were significant differences in fbg levels and the number of neutrophils in all groups. lycopene combined with metformin had lower fbg concentrations and higher neutrophil counts compared to metformin monotherapy (p<0.001), and these observations were dose-dependent. lycopene combined with metformin can improve blood glucose and neutrophil counts in rats with diabetes. the highest effect was observed in combination with lycopene at a dose of 40 mg/kg and metformin at a dose of 250 mg/kg. keywords blood glucose, lycopene, metformin, neutrophil, t2dm. citation: sianturi m, susilaningsih n, nugroho h, suryani m. lycopene improves the metformin effects on blood glucose and neutrophil counts in type 2 diabetic rats. indones j med lab sci technol. 2023;5(1):80–9. doi: 10.33086/ijmlst.v5i1.3865 this is an open access article distributed under the creative commons attribution-sharealike 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2023 by author. mailto:challenia@gmail.com https://doi.org/10.33086/ijmlst.v5i1.3865 https://doi.org/10.33086/ijmlst.v5i1.3865 https://creativecommons.org/licenses/by-sa/4.0/ ina. j. med. lab. sci. tech. 2023; 5(1): 80–89 medina sianturi, et al. 8 1 introduction patients with type 2 diabetes mellitus (t2dm) experience disorders of blood glucose metabolism that affect immunity. it has been found that diabetes mellitus (dm) patients experience disturbances in the number and function of neutrophils (1–4). research on neutrophil counts in dm patients is still controversial. a previous study showed an increase in the number of neutrophils in t2dm patients. it was found that the number of neutrophils with dm was 1.25 times higher than that of those without dm (5,6). the number of neutrophils was found to be higher in females (7), which differed from the study by eze et al. (1), which reported a decrease in neutrophil counts in dm (1). no correlation was found between fasting blood glucose and neutrophil counts (6). neutrophils are cells of the innate immune system that are the first to arrive at the site of infection and primarily kill microorganisms (5). neutrophil count is impaired due to decreased neutrophil production and migration (8), which increases the risk of infection and thus the morbidity and mortality of dm patients (9). the results of studies that have been conducted so far suggest that the use of lycopene as a single therapy can improve glycemic status by reducing fasting blood glucose by 30 40% (10,11) and hba1c by 41% (12), improving insulin resistance levels, and increasing insulin sensitivity (11). lycopene was also able to reduce the neutrophil counts, although this was not significant, and the neutrophil-lymphocyte ratio (1). lycopene is a carotenoid (c40h56) found in red fruits and vegetables, including tomatoes, papaya, red peppers and watermelon, that has been made into an extract. the role of lycopene as a nutraceutical has been widely studied. lycopene treatment is given either in single doses or in combination with other therapies such as metformin and statins. the results showed that lycopene is very beneficial for diabetic patients who are intolerant to statins (13). the combination of metformin with low doses of lycopene can reduce fasting blood glucose levels after two weeks of intervention (14), while high doses of 45 mg/kg can reduce blood glucose by 50%, and the results are the same as with single or combined doses of metformin and lycopene (15). treatment with lycopene can reduce neutrophil counts, but it is no different from treatment with a different dose of lycopene (1). this is the first study to evaluate the effects of combination therapy with lycopene and metformin on neutrophil counts in rats with t2dm at different doses of lycopene. the aim of this study was to evaluate the synergy of lycopene with metformin and its effects on blood glucose levels and rat immunity against type 2 dm induced by a combination of a high-fat diet, streptozotocin, and nicotinamide. ina. j. med. lab. sci. tech. 2023; 5(1): 80–89 medina sianturi, et al. 8 2 materials and methods materials thirty male albinos wistar rats 6 weeks old and weighing approximately 160–200 g was purchased from the laboratory animals of the center for food and nutrition studies (cnfs), gadjah mada university in yogyakarta, indonesia. they were housed in an animal room with a 12-hour light–dark cycle, a temperature of 24⁰c ± 2⁰c, and a relative humidity of 50%–60%. the rats were housed in individual stainless-steel cages and had ad libitum access to water and food, in accordance with the animal laboratory guidelines of the cnfs (center for natural sciences and fisheries) at gadjah mada university. metformin in the form of metformin hydrochloride (99.6%) (phapros tbk, indonesia) and tomato extract powder containing 98% lycopene from sigmaaldrich (st. louis, mo, usa) were used. all groups of rats with and without diabetes were treated with a single dose by gavage daily for 28 days. doses of lycopene and metformin were chosen according to eze et al. (1) and figueiredo et al. (15), respectively. the research was carried out in august– september 2022 in the laboratory animals of cnfs, gadjah mada university. the experimental procedures approved by the local ethics committee of the medical faculty of diponegoro university (approval no. 28/ec/h/fk-undip/iv/2022) and were performed in accordance with the principles expressed in the declaration of helsinki. all subjects were cared for according to the animal laboratory guidelines of the cnfs, gadjah mada university. experimental design this study used a completely randomized experimental design. wistar rats were randomly divided into a normal control group (n group, n = 5) and a type 2 diabetic rats’ group (n = 25), which were fed a high-fat diet (comfeed par-s 60%, flour 27.8%, cholesterol 2%, folic acid 0.2%, and lard 10%). after two weeks, the type 2 diabetic rats’ group were injected with streptozotocin–nicotinamide (nacalai tesque, japan) at 45 mg/kg/bw and 110 mg/kg, respectively, in citrate buffer (ph 4.6) intraperitoneally. after 72 h, fasting blood glucose ≥ 200 mg/dl was determined in type 2 diabetic rats (16). in total, thirty wistar rats were divided into six groups for treatment with a combination of metformin and lycopene. the first and second groups were normal (n) and type 2 diabetic rats (dm) given 1 ml of coconut oil (cnfs). the third group was type 2 diabetic rats treated with a combination of metformin 250 mg/kg in coconut oil (dmet). the four groups were type 2 diabetic rats treated with a combination of metformin and lycopene with a dose of 10 mg/kg lycopene in 1 ml of coconut oil (dml-10). the fifth group were ina. j. med. lab. sci. tech. 2023; 5(1): 80–89 medina sianturi, et al. 8 3 type 2 diabetic rats treated with a combination of metformin and lycopene with a dose of 20 mg/kg lycopene in 1 ml of coconut oil (dml-20). the sixth group were type 2 diabetic rats treated with a combination of metformin and lycopene at a dose of 40 mg/kg lycopene in 1 ml of coconut oil (dml-40). all treatments were administered every day for 28 days through an oral feeding tube. coconut oil can increase the bioavailability of lycopene but does not influence blood glucose concentration (17). biochemical assays after 28 days of final intervention and overnight fasting, all experimental animals were euthanized under ketamine anesthesia. blood samples were collected from the retroorbital flexure using a glass capillary, placed in a tube containing edta for neutrophil count, and allowed to clot. serum was separated by centrifugation at 3,500 rpm (2,000 g) for 10 minutes for the blood glucose exam. blood glucose concentration was determined by the glucose oxidase method using a dyasis reagent kit (holzheim, germany) following the manufacturer’s instructions (18). neutrophil counts of the rats were determined using an automated analyzer, following the manufacturer’s manual for hematology analyzer sysmex xp-100 (19). statistical analysis results were expressed as the mean ± standard deviation. differences between groups were analyzed using one-way analysis of variance followed by the least significant difference (lsd) test. statistical analyzes were performed with spss version 23. significant differences between mean values were considered at p < 0.05. results fasting blood glucose figure 1a shows the levels of fasting blood glucose concentrations in the experimental groups after 28 days of treatment. there was a significant difference (p<0.001) in fbg in all groups. there was a less significant effect of the combination of metformin with lycopene activity at doses of 20 and 40 mg/kg compared to metformin monotherapy. the mean differences were 16.63 and 23.45 mg/dl, respectively. fbg levels in the combination of metformin and lycopene dosed at 10 mg/kg were lower than those in the metformin monotherapy group, with a mean difference of -16.25 (figure 1b). there was a strong negative correlation between fbg concentration and neutrophil counts (figure 2). ina. j. med. lab. sci. tech. 2023; 5(1): 80–89 medina sianturi, et al. 8 4 figure 1. fbg concentration and neutrophil counts in rats with t2dm. values are expressed in terms of the mean ± standard error of the mean. differences between groups were analyzed using a one-way analysis of variance followed by the least significant difference (lsd) test, *p<0,001, **p<0,05 compared to an group; compared to bdm group: compared to cdmet group; compared to ddml-10 group; compared to ddml-20 group; and compared to ddml-40 group. ina. j. med. lab. sci. tech. 2023; 5(1): 80–89 medina sianturi, et al. 8 5 neutrophil counts the number of neutrophils in the experimental groups after 28 days of treatment is shown in figure 1. there was a significant difference in the number of neutrophils between all groups (p< 0.001). the combination of metformin and lycopene activity had a greater significant effect at doses of 20 and 40 mg/kg compared to metformin monotherapy. the mean differences were 0.19 and -0.34 x 106/µl, respectively. neutrophil count levels in the combination of metformin and lycopene dosed at 10 mg/kg were higher than those in the metformin monotherapy group, with a mean difference of 0.19 x 106/µl (fiure 1b). there was a significant and strong negative relationship between fbg concentration and neutrophil count (figure 2). figure 2. correlation of fbg and neutrophil counts in rats with t2dm discussion the findings indicate that the induction of t2dm in rats leads to disruptions in glucose metabolism and immune function, as evidenced by increased blood glucose and decreased neutrophil levels. however, administering metformin monotherapy or in combination with lycopene resulted in a decrease in blood glucose and an increase in neutrophil counts, indicating that these treatments effectively improve glucose control and immune function in t2dm rats. metformin is the first-line therapy as an oral hypoglycemic in t2dm (20,21). treatment with metformin monotherapy has been able to reduce blood glucose levels. metformin lowers blood glucose by ina. j. med. lab. sci. tech. 2023; 5(1): 80–89 medina sianturi, et al. 8 6 inhibiting glycogenesis in the liver, which results in the reduction of hepatic glucose and an increase in the hepatic cytosolic redox state (22). administration of metformin and lycopene combination therapy with different doses resulted in a better lowering of blood glucose when compared to metformin monotherapy. these results are in accordance with the research of haribabau et al., (10); figueiredo, et al., (11); yin et al., (14); and zheng, et al., (15); which stated that lycopene, as an antioxidant, can act as an anti-diabetic. lycopene, as an antidiabetic, can regulate glycolipid metabolism, preventing insulin resistance, inflammation, and fat accumulation (11). it increases insulin sensitivity through lycopene, and increases insulin sensitivity through upregulation of insulin receptors and igf-1 receptors, pi3k, and the expression of akt proteins phosphorylated in the hippocampus and cerebral cortex of insulin-resistant rats. treatment with a combination of lycopene and metformin resulted in a decrease in fasting blood glucose that was 1327% higher than a single dose of lycopene (10,11). the addition of lycopene at doses of 20 mg/kg and 40 mg/kg in metformin therapy provided better blood glucose reduction results than the figueiredo (15) study, where the addition of 45 mg/kg lycopene had the same blood glucose reduction results as metformin monotherapy. this is the first study to evaluate the effects of combination therapy with lycopene and metformin on neutrophil counts in rats with t2dm. this study showed that treatment with a combination of lycopene and metformin increased neutrophil counts by 53 – 67,6%. this result is different from the studies of bathia at al., (23) and maldovan et al.,(24) which found a decrease in neutrophil count by administering a single dose of lycopene to patients with hepatocellular carcinoma. these results are consistent with research by eze et al., (1) which showed that giving lycopene monotherapy to diabetic rats could increase the number of neutrophils, although it was not significantly higher when compared to this study. the results of this study prove that the combination of lycopene and metformin is effective in increasing neutrophil counts in t2dm rats. the mechanism of action of lycopene can improve the immune status of patients with dm, because lycopene is immunomodulatory and increases the number of neutrophils (1,25). neutrophils can improve immunity by reducing infections, which are common in patients with t2dm. the combination of metformin and lycopene can improve the mechanism of neutrophil production and recruitment, causing the number of neutrophils to be higher than with metformin treatment monotherapy. this improvement occurs ina. j. med. lab. sci. tech. 2023; 5(1): 80–89 medina sianturi, et al. 8 7 through the mechanism of action of lycopene through non-oxidative pathways that can improve immune status (26). in contrast to the study by huang et al., (6) reducing fgd increased the number of neutrophils, proving a relationship between blood glucose levels and the immunity of t2dm patients. this suggests that with a decrease in fasting blood glucose concentration, there is an increase in neutrophil counts. the increasing doses of lycopene that improved blood glucose and neutrophil count more effectively than metformin monotherapy were 20 and 40 mg/kg. this indicates that the best doses to improve blood sugar and immunity start at 20 mg/kg, which is consistent with the study by imran et al. (27), that the most effective and recommended dose is 20 mg/kg. the results of this observation prove that lycopene synergizes with metformin in decreasing blood glucose and improving the immune system by increasing neutrophil counts in dm rats, and is expected to help type 2 dm patients (20). the results of this study can be used to support the effectiveness of antioxidants, especially lycopene, in patients with type 2 diabetes and be considered when providing antioxidant based nutritional supplements. conclusions in a type 2 diabetes mellitus rat model, the combination of lycopene and metformin was founding to lower blood glucose concentrations and increase neutrophil counts. additionally, lycopene was founding to improve the performance of metformin in lowering blood glucose concentrations and increasing neutrophil counts. the dosedependent effect of the lycopene and metformin combination was observed. the combination of lycopene at a dose of 40 mg/kg and metformin at a dose of 250 mg/kg has the highest effect. author contributions medina sianturi: conceptualization, methodology, validation, formal analysis, investigation, data curation, writing original draft, writing review and editing, and project administration. neni susilaningsih: conceptualization, methodology, validation, formal analysis, review, and editing. heri nugroho: conceptualization, methodology, validation, review, and editing. maria suryani: conceptualization, review, and editing. acknowledgements we would like to thank the ministry of education, culture, research, and technology for providing funding for this study. conflict of interest the author declars there is no conflict of interest. ina. j. med. lab. sci. tech. 2023; 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p=0.001). this study concluded that there was a significant difference between urinary pdx in dm with and without diabetic nephropathy, there was a significant difference between urine acr levels in dm with and without diabetic nephropathy, and there was a relationship between urinary pdx levels and urine acr in dm subjects with diabetic nephropathy. keywords albumin-creatinine ratio, diabetic nephropathy, urine podocalyxin. citation: pelu je, kurniawan lb, widaningsih y, zainuddin a, umar h, nurahmi n, et al. analysis of urine podocalyxin in type 2 diabetes mellitus subjects with and without diabetic nephropathy. indones j med lab sci technol. 2023;5(1):20–28. doi: 10.33086/ijmlst.v5i1.3933 this is an open access article distributed under the creative commons attribution-sharealike 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2023 by author. mailto:https://doi.org/10.33086/ijmlst.v5i1.3933 https://creativecommons.org/licenses/by-sa/4.0/ ina. j. med. lab. sci. tech. 2023; 5(1): 20–28 jusni ekasari pelu, et al. 2 1 introduction type 2 diabetes mellitus (t2dm) is a growing global health problem related to the obesity epidemic. individuals with type 2 dm are at risk of experiencing microvascular complications such as retinopathy, nephropathy and neuropathy and macrovascular complications like cardiovascular comorbidities due to hyperglycemia and the individual components of the (metabolic) insulin resistance syndrome (1). diabetic nephropathy (dn) caused by diabetes mellitus is one of the major causes of end-stage kidney failure worldwide (2). clinically, it is characterized by the development of proteinuria with decreased glomerular filtration rate, which lasts for a long time, often more than 10-20 years. if it is untreated, the resulting uremia is morbidity (3). diabetic nephropathy is a complication of diabetes mellitus in the kidneys which can end up as kidney failure. nephropathy is the leading cause of death and disability in people with dm (3,4). podocalyxin (pdx) is an anionic transmembrane sialoglycoprotein from podocytes, a member of the cd34 family (cluster of differentiation 34). podocytes are visceral epithelial cells that play a role in the glomerular filtration barrier (gfb) formation. the destruction of podocytes and their release in the urine results in the detection of pdx in urine (u-pdx), making it a marker that may be useful in the early diagnosis of diabetic nephropathy (5). podocyte injury plays an essential role in the pathological mechanisms of dn. typical signs of podositopathy are podocyte hypertrophy, foot process effect, epithelial-mesenchymal transdifferentiation (emt), release from the glomerular basement membrane and apoptosis (6,7).the early stages of dn are characterized by a progressive decrease in the number of podocytes, loss of their foot processes, release of podocytes through urine, and damage of the diaphragmatic filtration slits, which causes proteinuria (8). the existence of podocytes and their specific proteins in the urine can be considered as potential markers in the early detection of dn. currently new markers for early detection of dn are being evaluated when most of the research focus on podocyte-specific proteins such as podocalyxin, nephrin, synaptopodin, podocin, mindin, etc (9). the research results of sun, d., zhao, x., & meng, l (10) defined a new role for podocalyxin in constructing cell morphology. when ectopicly expressed in kidney epithelial cells, podocalyxin dramatically enhances microvillus formation. in addition, podocalyxin is essential for foot processes (fp) elongation in kidney podocytes. podocalyxin was originally identified in the glomeruli of the kidney, where it is not only abundant but also essential for kidney development (10). this study aims to ina. j. med. lab. sci. tech. 2023; 5(1): 20–28 jusni ekasari pelu, et al. 2 2 determine and compare urine podocalyxin levels in subjects with type 2 diabetes mellitus with and without diabetic nephropathy. materials and methods study design and patients this research was an observational study with a cross-sectional research design. this research received ethical approval from the health research ethics commission (kepk) hasanuddin university faculty of medical-unhas (rsptn uh) with ethical number 596/un4.6.4.5.31/pp36/2022. collection of clinical data sampling locations were carried out at the endocrine polyclinic at hasanuddin university hospital, the clinical pathology laboratory at hospital of hasanuddin and the clinical pathology laboratory at rsup. wahidin sudirohusodo makassar. furthermore, the research carried out at the hasanuddin university medical research center (humrc) laboratory, hasanuddin university makassar state university hospital and the parahita makassar clinical laboratory. a total of 50 participants were grouped into the 25 participants of t2dm with dn and 25 participants of t2dm without dn using a non-probability sampling technique. pathological data the inclusion criteria in this study were participants who were willing to take part in a series of studies, participants t2dm with or without dn, and excluded pregnant women, preeclampsia, lupus nephritis, cancer, hypertension, and coronary heart disease. the diagnosis of t2dm participants was based on the results of laboratory tests, namely fasting blood glucose levels of 126 mg/dl or hba1c values of 6.5%. participants diagnosis with and without dn by examining the urine albumin creatinine ratio (acr) with an interpretation of the results of <30 mg/g means not diagnosed with diabetic nephropathy and if  30 mg/g means diagnosed with diabetic nephropathy. measurement of urine podocalyxin the measurement of urine podocalyxin levels used the enzyme-linked immunosorbent assay (elisa) method with assay pharmagenie® (sbrs1004) at a wavelength of 450 nm and reported in ng/ml. urine albumin and creatinine levels were examined by the cobas 311 instrument with the immunoturbidimetric method and stated in mg/g. statistical analysis data processing was performed using the windows computer program spss (statistical product and science service) version 23. the data analysis used was bivariate analysis using the mann-whitney test to compare urine podocalyxin levels in ina. j. med. lab. sci. tech. 2023; 5(1): 20–28 jusni ekasari pelu, et al. 2 3 subjects of t2dm with and without dn, compared acr in t2dm subjects with and without dn, followed by a correlation test using the spearman test. the test results are significant if p*<0.05. results the research results are presented in a table accompanied by an explanation. participant characteristics (table 1) such as gender, age, diagnosis of t2dm participant based on measurement results of hba1c and diagnosis of t2dm with dn based on acr ≥ 30 mg/g. table 2 and table 3 present a comparison test of urine podocalyxin levels and acr levels in t2dm participant with and without dn, then table 4 presents a correlation test of urine podocalyxin levels with acr in t2dm with dn. table 1. participant characteristics characteristics n % sex male female 24 31 43.6 56.4 ages (years old) 30 – 40 41 – 50 51 – 60 61 – 70 > 70 3 8 23 18 3 5.5 14.5 41.8 32.7 5.5 hba1c <6.5 % ≥6.5 % 5 50 9.1 90.9 urine albumin/creatinine ratio < 30 mg/g (t2dm with dn) ≥ 30 mg/g (t2dm without dn) 27 28 49.1 50.9 table 1 shows the characteristics of research participant based on gender showing female participant (56.4%) were dominant compared to male participant (43.6%). based on age category, most of the participant was aged between 51–60 years (41.8%). hba1c level as an indicator of glycemic control in the category ≥6.5% for 50 participants (90.9%) and <6.5% for 5 participants (9.1%). acr as a biomarker in the ≥30 mg/g category were 28 paticipants (50.9%), the average urine podocalyxin value in nephropathy participant was 1.160 ng/ml, while the average urine podocalyxin in participant without nephropathy including, diabetics was 0.167 ng/ml. ina. j. med. lab. sci. tech. 2023; 5(1): 20–28 jusni ekasari pelu, et al. 2 4 table 2. comparison test of urine podocalyxin levels in participant with diabetic nephropathy and without diabetic nephropathy parameter characteristics mean sd p* urine podocalyxin (ng/ml) t2dm with dn 1.160 2.066 <0.001 t2dm without dn 0.167 0.047 *mann whitney test table 2 shows that the average urine podocalyxin value in diabetic nephropathy participants is 1.160 ng/ml with a standard deviation of 2.066 ng/ml, while the average urine podocalyxin value in participants without diabetic nephropathy is 0.167 ng/ml with a standard deviation of 0.047 ng/ml. this result indicates that the average urinary podocalyxin in diabetic nephropathy is bigger than participant without diabetic nephropathy (p <0.05). this result showed a significant difference between urine podocalyxin levels in diabetic nephropathy participants and participants without diabetic nephropathy. table 3. comparison test of albumin/creatinine ratio levels in participants with diabetic nephropathy and without diabetic nephropathy parameter characteristics mean sd p* albumin/creatinine ratio levels (mg/g) t2dm with dn 633.74 1546.11 <0.001 t2dm without dn 10.071 7.883 *mann-whitney test the average albumin creatinine ratio in diabetic nephropathy subjects were 633.74 mg/g with a standard deviation of 1546.11 mg/g, while the average value of albumin creatinine ratio in conditions without diabetic nephropathy was 10.071 with a standard deviation of 7.883 (table 3). this result showed that the average albumin creatinine ratio in diabetic nephropathy is higher than the albumin creatinine ratio in conditions without nephropathy (p <0.05). this showed a significant difference between the albumin creatinine ratio in participants with diabetic nephropathy and those without diabetic nephropathy. table 4. correlation test of urine podocalyxin levels with albumin/creatinine ratio in diabetic nephropathy participant albumin/creatinine ratio urine podocalyxin r 0.510 p < 0.001 n 55 *spearman-rho test, r=correlation, p=level of significance, n=number of samples ina. j. med. lab. sci. tech. 2023; 5(1): 20–28 jusni ekasari pelu, et al. 2 5 table 4 showed the correlation test results between urine podocalyxin and albumin creatinine ratio in participant with diabetic nephropathy. there is a correlation between urinary podocalyxin and albumin creatinine ratio in diabetic nephropathy participants (p<0.001). discussion this study determined and compared urine podocalyxin levels in dm participants with and without diabetic nephropathy (11). podocalyxin is a main component of the charge barrier of the glomerular basement membrane (gbm) and plays an important role in regulating the permeability of the glomerular filtration barrier (12). based on table 2, the urine podocalyxin comparison test in nephropathy and nonnephropathy participants showed a significant difference between urine podocalyxin in participants with nephropathy and urine podocalyxin without nephropathy. kostovska i, et al (13) reported an increase in urine podocalyxin levels in 48.2% of patients with normoalbuminuria, 64% of patients with microalbuminuria and 100% of patients with macroalbuminuria (13). similar result research by hara m, et al. (2012) demonstrated that podocalyxin is found in urine in the early stages of diabetic nephropathy, before the apperance of microalbuminuria (14). research by rongzhen wang, et al (15) reported that pcx excretion through urine increased along with decreased pcx expression in the kidneys, which would be consistent with the release of pcx protein from the kidney into the urine (15). previous investigations determined that elevated urinary levels of pcx could be used as a biomarker to predict early kidney damage and the development and progression of complications in patients with early-stage diabetic nephropathy, anaphylactic purpuric nephritis, lupus nephritis, or iga nephropathy (16,17). moreover, measuring pcx in urine is a non-invasive method that can be applied soon in clinical settings. although previous studies focused on patients with early-stage diabetic nephropathy, this study included patients with diabetic nephropathy and clinical albuminuria. the present study's finding that urinary pcx levels are increased in nephropathy patients and are significantly associated with reduced kidney function suggests the potential clinical utility of this parameter as a biomarker in patients with early-stage dn (17). based on table 2, the results of comparison test of albumin creatinine ratio for dm with and without nephropathy showed a significant difference between the albumin creatinine ratio in nephropathy participant and participant without nephropathy. shoji m, et al (18) in their research reported a significant positive ina. j. med. lab. sci. tech. 2023; 5(1): 20–28 jusni ekasari pelu, et al. 2 6 correlation between the urinary microalbumin creatinine ratio and podocalyxin (18). this finding is different from that obtained from the results of a study by kostovska i, et al (13) which detected a weak positive correlation between urine podocalyxin levels and albumin creatinine ratio. furthermore, a previous study conducted by kostovska i, et al (13) reported that urine podocalyxin levels were higher in type 2 diabetes mellitus patients compared to control subjects with a p-value <0.05 (13). the urinary podocalyxin level increased gradually with the degree of diabetic nephropathy (p<0.029). urinary podocalyxin levels positively correlated with urinary microalbumin/creatinine ratio (um/cr) (r=0.227; p=0.002). measurement of podocyte molecules in urine samples can serve as a specific marker of kidney podocyte cell damage (19). further research is needed on urinary podocyte cytology as a noninvasive test to assess the level of glomerular damage (20). research conducted by asao r, et al (20) revealed a significant correlation between urinary podocalyxin levels and the degree of acute extra capillary damage (r=0.72; p<0.001), but the level of protein excretion did not correlate with acute glomerular disorders (20). hypertension in t2dm patients is one of the factors that affect podocalyxin levels because it is directly related to kidney function. research conducted by abe, h, et al (21) explains that exposure to ages can interfere with the rii-smad3 signal, giving rise to the elf3 protein, which indicates irreversible podocyte injury (r=0.73; p <0.05) (21). akankwasa, g, et al (22) in his research on the comparison of podocalyxin and nephrin as a marker of impaired kidney function (22) which was updated through the research of watanabe, k, et al (23), which explained that increased urine albumin leakage is closely related to glycocalyx injury in glomeruli and renal podocytes (23). a cross-sectional study conducted by ikuma, d, et al (24) concerning the correlation of urine podocyte count and urinary podocalyxin levels to lupus nephritis histology showed significant results (r=0.50; p=0.0012) (24). apart from pathological factors, several technical factors play a role in measuring podocalyxin levels. research conducted by kimura, m, et al (25) regarding the detection of podocyte cells in urine samples of dn patients using the cytocentrifugal method can detect positive wt-1 cells in dn (25). this study may have limitations such as the research method used cross-sectional which only looks at one event at a time, in addition to the small sample size of the study. we recommend further prospective studies with a larger sample size to compare of urine podocalyxin levels and microalbuminuria (mau) as a marker for early detection of dn. ina. j. med. lab. sci. tech. 2023; 5(1): 20–28 jusni ekasari pelu, et al. 2 7 conclusions the study concluded there was a differences in urine podocalyxin levels in t2dm with and without dn, found differences of acr in t2dm with and without dn, and a significant correlation between urine podocalyxin and acr in t2dm with dn. author contributions jusni ekasari pelu: conceptualization, methodology, writing-original draft, formal analysis, and funding acquisition. liong boy kurniawan & yuyun widaningsih: supervision, data curation, writing-original draft preparation. alfian zainuddin, husaini umar & nurahmi: writing-original draft, writing-reviewing. theosobia grace orno: project administration, writingoriginal draft, visualization. acknowledgements thanks to all contributors to this research to be done. conflict of interest there is no conflict interest. references 1. durruty p, & sanzana, sanhueza m, sanhueza l. pathogenesis of type 2 diabetes mellitus. intechopen. 2019. doi: 10.5772/intechopen.83692 2. davies mj, aroda vr, collins bs, gabbay ra, green j, maruthur nm, rosas se, del prato s, mathieu c, mingrone g, rossing p, tankova t, tsapas a, buse jb. management of hyperglycemia in type 2 diabetes, 2022. a consensus report by the american diabetes association (ada) and the european association for the study of diabetes (easd). diabetes care. 2022; 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5(1): 10–19 paul c. inyang-etoh, et al. 1 1 introduction diabetes is a group of frequently occurring metabolic disorders which has similar physical properties with hyperglycemia. it is associated with decreased insulin synthesis and utilization, which impairs the body’s ability to ineffectively use nutrients (1). urinary tract infections which are usually classified as symptomatic and asymptomatic are reported to be common infections among diabetic patients (2). diabetics are more likely to get urinary tract infections because their immune systems are weaker and they often have problems with their bladder control, which makes them more likely to let bacteria and uropathogens get into their genitals. the high level of glucose in their urine makes it perfect for these organisms to grow (3,4). there is a significant increase in urinary tract infections in people with diabetes, which is likely caused by the presence of other infections (uropathogens in the kidneys) (5). most pathogens that can cause urinary tract infections in people with diabetes are very likely to change their antibiotic resistance patterns over time. this means that many of these pathogens are less likely to respond to antibiotics (6-8). o'neil et al., (9) has reported that a rise in antibiotics resistance could result to global mortality of ten million annually. due to the health importance of the pathogens which cause urinary tract infections, it is germane to adequately identify the causative agents and the effective antimicrobial agents against them for successful treatment of the infections (10). despite the fact that there have been several researches on antibiotic resistance, the situation still exists and requires pragmatic investigations that will provide up-to-date information on antimicrobial resistance. there is also paucity of data on the asymptomatic bacteriuria in patients with diabetes in calabar. this study was to identify the pathogens associated with asymptomatic bacteriuria among patients with diabetes and the antibiogram of those isolates in calabar. this will provide information on the asymptomatic bacteriuria in diabetic patients and provide guide for treatments. materials and methods study area the area of study is calabar, the capital of cross river state, which includes the calabar municipal and calabar south local government areas in the south-south region of nigeria. study design this was a cross-sectional study of 200 healthy asymptomatic diabetic patients in calabar who volunteered to take part in the study. personal information was obtained from each of the study participants. the inclusion criterion was participants from the ina. j. med. lab. sci. tech. 2023; 5(1): 10–19 paul c. inyang-etoh, et al. 1 2 ages of 21 years with history of diabetes while the exclusion were participants with participants with pregnancy, signs and symptoms of urinary tract infections, antibiotic usage within one week preceding the study and history of underlying illness and less than 21 years. specimen collection and processing early morning midstream urine and blood specimens were aseptically obtained from each diabetic patient, labeled and transported to the laboratory for processing. the glucometer described by pickering & marsde (11) has been used to measure the level of fasting blood sugar in the blood. macroscopy of the urine specimens was carried out in the laboratory to examine for the physical properties of the urine samples. the samples were analyzed microscopically by centrifuging 10 ml of each sample in a test tube at 3,000 revolution per minute (rpm) for 5 minutes. the supernatant was poured off while the sediment was well mixed for a wet preparation and a drop of the sediment was placed on a clean, grease-free glass slide and covered with cover glass. the preparation microscopically examined using the 10x and 40x objectives with the condenser iris closed sufficiently to provide good contrast of white blood cells (leukocytes), red blood cells (erythrocytes), bacterial debris and casts (12). the sample was inoculated with 1µl of a standard quantitative loop to a cysteine lactose electrolyte deficient (cled) agar, macconkey, and blood agar plates (oxoid, ltd., basingstoke, hampshire, england). the plates were incubated aerobically at 37oc for 24 hours. the result was reported as significant/non-significant growth, or contaminated (discarded). urine culture plates showing ≥105 colony-forming units (cfu)/ml of single bacterial species were considered as significant bacteriuria (13). the presumptive identification criteria of the organisms were gram-stain reaction of the organisms, microscopic appearance and colony characteristics. indole production, citrate utilization, h 2 s production, gas production, urea hydrolysis, lysine decarboxylation, lactose fermentation and motility were used for further identification of gram-negative bacteria. coagulase, catalase, and mannitol fermentation assays were used to further identify gram-positive bacteria (12). antibiogram (antiobiotic testing) test was performed on all positive isolates using the standardized kirby bauer disc diffusion technique according to the criteria of the clinical and laboratory standards institute (14). antibiotic-impregnated discs containing amoxicillin-clavulanic acid (amc, 30μg), pefloxacin (def, 30µg), gentamycin (gn, 10μg), chloramphenicol (ch, 10µg), ciprofloxacin (cip, 5μg), sulfamethoxazole/trimethoprim (sxt, 30µg), augmentin (au, 30µg) and septrin ina. j. med. lab. sci. tech. 2023; 5(1): 10–19 paul c. inyang-etoh, et al. 1 3 (sp, 30μg) were placed onto the surface of mueller-hinton agar. statistical analysis data obtained were analyzed using the statistical package for social science (spss) version 20 manufactured by international business machines (ibm corp, armonk, new york). proportions were used for categorical variables. differences in infection rates among participants were determined by chi-square and p-value <0.05 was considered significant. ethical approval this was sought for and obtained from the cross-river state health research ethics committee of the cross-river state ministry of health research ethics committee (crs-hrec) with approval number: crs/mh/hrec/020/vol.v1/255 and a written consent form was also duly signed by the participants before taking part in the study. results the mean fasting blood sugar levels of the study cohort by age is shown in table 1. the highest mean fasting blood sugar level was among those between the ages of 41-50 years (12.3+4.38) while the least mean fasting blood level was among those between the ages of 21-30 years (8.9+2.84). table 1. mean fasting blood sugar of the study population by age age (years) no. examined mean fasting blood sugar (fbs) level (mmol/l) 21-30 12 8.9+2.84 31-40 48 11.2+4.65 41-50 48 12.3+4.38 51-60 58 10.6+3.59 61-70 34 11.0+3.77 total 200 11.08+4.07 the mean fasting blood sugar of the study population by gender as presented in table 2 shows that the fasting blood level of the males (11.3+4.46) was higher than that of the females (10.9+3.70). table 2. mean fasting blood sugar of the study population by gender gender no. examined mean fbs level male 98 11.3±4.46 female 102 10.9±3.70 total 200 11.08±4.07 the prevalence of bacterial pathogens in the study is 26% based on the incidence of infections among participants by age (table 3). the highest rate of infection was found in those between the ages of 41 and 50 (37.5%) while the lowest rate was found in people between the ages of 61 and 70 (11.8%). the difference between the rate of infection and age was not statistically significant (p>0.05). prevalence of bacterial pathogen = total number of infected participants total number of the study population × 100 ina. j. med. lab. sci. tech. 2023; 5(1): 10–19 paul c. inyang-etoh, et al. 1 4 table 3. occurrence of infections of the study population by age age (years) no. examined no. (%) infected (n=52) statistics 21-30 12 2 (16.7) χ2=37.5245, p=0.1106 at df=4 31-40 48 12 (25.0) 41-50 48 18 (37.5) 51-60 58 16 (27.6) 61-70 34 4 (11.8) total 200 52 (26.0) according to table 4, females had a greater infection rate of 29.4% (30/102) than males did (22.4%, 22/98). gender differences in infection rates were not statistically significant (p>0.05). table 5 lists the biochemical processes used to determine the presence of bacterial pathogens. table 4. occurrence of infections in the study population by gender age (years) no. examined no. (%) infected (n=52) statistics male 98 22 (22.4) χ2=0.1380, p=0.7102 at df=1 female 102 30 (29.4) total 200 52 (26.0) table 5. biochemical reactions of pathogenic bacteria in urine samples biochemical reaction bacterial pathogen e. coli klebsiella staphylococcus aureus proteus gram reaction -ve -ve +ve -ve microscopic appearance rod rod spherical rod colony on cled agar opaque yellow yellow mucoid golden yellow translucent blue colony on macconkey agar pink pink red-pink colourless colony on blood agar yellow grey-white mucoid yellow grey-white swarm indole +ve -ve -ve -ve citrate -ve +ve +ve +ve urease -ve +ve +ve +ve lysine +ve -ve +ve -ve lactose +ve +ve +ve -ve h2s -ve -ve -ve +ve gas production +ve +ve -ve +ve motility test +ve -ve -ve +ve coagulase +ve catalase +ve mannitol +ve key: +ve = positive, -ve = negative ina. j. med. lab. sci. tech. 2023; 5(1): 10–19 paul c. inyang-etoh, et al. 1 5 bacterial pathogens in the study population showed that escherichia coli had the highest prevalence at 46.2% (24/52), as show in table 6. conversly, proteus spp. had the lowest distribution of 11.5% (6/52). table 6. occurrence of bacterial pathogens in the study population (n=52) bacteria frequency of occurrence (%) escherichia coli 24 (46.2) klebsiella spp. 14 (26.9) staphylococcus aureus 8 (15.4) proteus spp 6 (11.5) total 52 (26.0) table 7 shows the rate of growth inhibition to bacterial pathogens by antibiotics. ciprofloxacin was fully susceptible (100%) to all the bacterial pathogens. gentamicin is primarily sensitive to e. coli (87.5%), pefloxacin was mostly sensitive to klebsiella (71.4%), septrin was mostly sensitive to klebsiella and proteus (50%), augmentin was mostly sensitive to staphylococcus aureus (50%), chloramphenicol and amoxillin were mostly sensitive to e. coli (45.8% and 41.7% respectively). ciprofloxacin (cpx) was the most sensitivity (100%) on the isolates while amoxicillin (am) was the least sensitivity (46.2%) as presented in figure 1. discussion urinary tract infections are more frequent with more severity among those with diabetes which are most times caused drug resistant microorganisms (15). in this study, people with diabetes had a significant prevalence (26.0%) of urinary tract infections. this was lower than the prevalence of urinary tract infections among the same subjects as was previously reported by shah et al., (16) in malaysia, dave et al., (17) at ahmedabad in india, 92.0% and higher than 10.6% by worku et al., (18) at debre tabor and 10.7% by mohammed et al., (19) at hawassa both in ethiopia. these disparities in prevalence might have been as a result of the variations in sample size, geographical location, personal hygiene, and the screening tests used. the highest risk of infection was found in people between the ages of 41 and 50. this was consistent with the findings of other studies that majority of asymptomatic bacteriuria occurred among adults over the age of 40 (17,20). this may be due to physiologic changes related to aging and comorbid illnesses in elderly adults. females were mostly infected in this study. similar reports have been made by nabaigwa et al., ina. j. med. lab. sci. tech. 2023; 5(1): 10–19 paul c. inyang-etoh, et al. 1 6 (21) kumar et al., (22) and nadia et al., (23) who noted that females were more higher infections in than males. females are susceptible to bacteriuria due to the shorter distance between the female urethra and the anus compared to the male urethra. table 7. inhibition rate of to pathogenic bacteria by antibiotics bacterial pathogen sensitivity to antibiotics (%) cipro gen pef sep aug chlo amo e. coli (n=24) 24 (100) 21 (87.5) 17 (70.8) 10 (41.6) 9 (37.5) 11 (45.8) 10 (41.7) klebsiella spp (n=14) 14 (100) 12 (85.7) 10 (71.4) 7 (50.0) 5 (35.7) 6 (42.9) 5 (35.7) s. aureus (n=8) 8 (100) 5 (62.5) 5 (62.5) 3 (37.5) 4 (50.0) 3 (37.5) 3 (37.5) proteus spp (n=6) 6 (100) 4 (66.7) 3 (50.0) 3 (50.0) 2 (33.3) 1 (16.7) 2 (33.3) key: cipro=ciprofloxacin, gen=gentamicin, pef=pefloxacin, sep=septrin, aug=augmentin, chlo=chloramphenicol, amo=amoxicillin figure 1. percentage of sensitivity of antibiotics escherichia coli were the most observed organisms (46.2%) in this study followed by klebsiella spp. (26.9%). the findings of akram et al., (24), kalaichelvi & daranendaranchellapa (25), durmaz et al., (26) and bhagat & sahu (27), who observed that escherichia coli were the most uropathogens concurred with this. this might have happened as a result of escherichia a typical human intestine bacterium that can quickly turn opportunistic in the urinary tract. most of the isolates were resistant to the tested antibiotics in this study. this high antibiotic resistance is a global 100 80.8 67.3 44.2 40.3 40.3 38.5 0 20 40 60 80 100 120 % o f se n si ti v it y antibiotics ina. j. med. lab. sci. tech. 2023; 5(1): 10–19 paul c. inyang-etoh, et al. 1 7 issue which is commonly due to abuse of antibiotics (28). the study area reported that antibiotics were severly misused and significantly associated with antibiotic resistance (29). conclusions the prevalence of asymptomatic bacteriuria among diabetic patients in calabar was 26.0%. the most commonly observed organisms were escherichia coli. the study found that widespread antibiotic resistance existed and that ciprofloxacin was the most sensitive medication. in order to prevent complications, it is advised that diabetes individuals regularly get checked for urinary tract infections. a study with larger sample size and power should be conducted to evaluate the distribution of uropathogens among diabetic patients. patients should be educated about the appropriate antibiotic use based on culture results. implementation of management program to explain the usage of antibiotic is needed. author contributions etefia u. etefia and sonia o. ejiofor: designed the study. etefia u. etefia: analyzed the study data. paul c. inyang-etoh: approved the final version for submission. all authors critically reviewed the manuscript. acknowledgements we appreciate all the participants who voluntarily took part in the study. conflict of interest all the authors declare that there is no conflict of interest. references 1. londhe a, naik m, shinde v, patel p, wyavahare s. study of diastolic dysfunction in asymptomatic type 2 diabetes mellitus. int j curr med appl sci 2016; 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approach and treatment. arc journal of diabetes and endocrinology. 2018; 4(2):31-3. doi: 10.20431/2455-5983.0402005 27. bhagat zp, sahu mc. pervasiveness of urinary tract infection in diabetic patients and their causative organisms with antibiotic sensitivity ina. j. med. lab. sci. tech. 2023; 5(1): 10–19 paul c. inyang-etoh, et al. 1 9 pattern. apollo med. 2020; 17:26-30. doi: 10.4103/am.am_2_20 28. craig j, simpson j, williams g, lowe a, reynolds g, mctaggart s, et al. antibiotic prophylaxis and recurrent urinary tract infection in children. n engl j med. 2009; 361:1748-59. doi: 10.1056/nejmoa0902295 29. asuquo ae, epoke j, asuquo ee. antibiotic misuse among high school students in calabar, nigeria. mary slessor j med. 2023; 3(1): 72-4. doi: 10.4314/msjm.v3i1.11001 128 validation of rapid antibody (igm – igg) test kit for sars-cov-2 infection in surabaya, indonesia museyaroh1, puspa wardani2, aryati2, evy diah woelansari1 1health polytechnic ministry of health surabaya, surabaya, east jawa, indonesia 2department of clinical pathology, faculty of medicine, universitas airlangga/dr. soetomo general academic hospital, surabaya, indonesia correspondence: museyaroh, jl. karangmenjangan 18 a, surabaya, east java, indonesia zip code: 60286 email: museyaroh21@gmail.com received: may 31, 2022 revised: september 18, 2022 accepted: october 6, 2022 published: october 30, 2022 doi: 10.33086/ijmlst.v4i2.3027 abstract at the beginning of the corona virus disease 2019 (covid-19) pandemic, rapid test examinations were widely used as a screening for severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infection. the purpose of this examination was to detect sars-cov-2 igm/igg antibodies in the patient's body. one of these tests uses the immunochromatographic method. this study aims to determine the validity of immunochromatography. the study was conducted from august to september 2020. the sample used in this study was 100 patients. the research was conducted at husada utama hospital surabaya, indonesia. according to the study's findings, the zybio brand reagent kit has an accuracy of 85%, a sensitivity of 82%, a specificity of 88%, a positive predictive value of 87%, and a negative predictive value of 83%. in the group of patients who experienced clinical symptoms, < 7 had a sensitivity of 50%, specificity of 88%, positive predictive value of 60%, negative predictive value of 83%, and accuracy of 77.94% while the group of patients experiencing clinical symptoms > 7 days, had a sensitivity value of 100 %, specificity of 88%, positive predictive value of 84%, negative predictive value of 100%, and accuracy of 92.68%. based on these results, the conclusion is that the zybio brand reagent kit has a relatively high sensitivity, specificity, positive predictive value, negative value, and sample accuracy. in the group with clinical sensitivity < 7 days, the positive predictive value and accuracy are lower than the sample group with clinical symptoms > 7 days but have the same specificity. keywords igg, igm, immunochromatography, sars-cov-2, validation. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2022; 4(2): 128–138 museyaroh, et al 1 2 9 introduction in december 2019 the world was shocked by the outbreak of an acute respiratory disease that occurred in wuhan, china (1). this outbreak was initially thought to be transmission from animals to humans (zoonosis), but recently it was discovered that transmission of this virus occurs between humans through droplets (2). according to data as of april 13, 2022, in indonesia the number of confirmed cases of corona virus disease 2019 (covid-19) was 6,036,909 with recovery cases of 5,814,688 (96.3%) and the death rate of 155,746 people (2.6%) (3). the clinical symptoms of covid-19 are very wide ranging, from asymptomatic, fever, dry cough, anosmia, sore throat, fatigue, conjunctivitis, nausea, vomiting, diarrhea, shortness of breath, and sepsis (4). clinical manifestations of covid-19 can progress to pneumonia, respiratory failure and even death (5). about 80% of cases were classified as mild or moderate and 13.8% were serious illness, and 6.1% of patients fell into a critical condition (6). deterioration and death generally occur in older people with congenital disease (50-75%) (5). diagnostic speed and accuracy are very important for the diagnosis and control of the covid-19 outbreak. the examination recommended by world health organization (who) is molecular examination using nucleic acid amplification or real-time polymerase chain reaction (rt-pcr) because pcr has high sensitivity and specificity, but has drawbacks. one of the drawbacks of the pcr method is that this examination requires a relatively long time, the process is quite complicated, expensive, and requires experts. therefore, this examination cannot be carried out in all health care facilities, especially in first-level health services such as health centers and hospitals in remote areas. currently, many antibody-based examinations are used to detect the presence of immunoglobulin m (igm) and immunoglobulin g (igg) antibodies against the sars-cov-2 (severe acute respiratory syndrome coronavirus-2) virus. it is widely known that igm was the body's first line of defense during viral infections before the appearance of igg (7). according to li z et al., (8) igm can be detected in the blood of a person infected with the sars-cov2 virus for 3-6 days after the onset of clinical symptoms and igg can be detected 8-14 days after infection with the sars-cov-2 virus. many methods are currently widely used to detect anti-sars-cov-2 igm and igg antibodies. one of them is the immunochromatography method. this method is relatively fast, low cost, and does not require experts (8). there are many brands of rapid diagnostic tests for igm and igg sars-cov-2 antibodies available. one of the rapid test brands that are widely used in health ina. j. med. lab. sci. tech. 2022; 4(2): 128–138 museyaroh, et al 1 3 0 care centers, both hospitals and clinical laboratories, is the zybio brand rapid test. this rapid test has a limit of detection (lod) igm: 0.25 g/ml and igg: 0.23 g/ml. the purpose of this study was to determine the validity of the zybio brand rapid test including sensitivity, specificity, positive predictive value, and negative predictive value. materials and methods study setting the study is of the descriptive diagnostic test kind and has a prospective crosssectional study design. study population the population used in this study were inpatients or outpatients with symptoms of covid-19 or without symptoms of covid19. pcr is the method recommended by who to detect the sars-cov-2 virus, which is based on the principle of nucleic acid amplification. positive samples, if at least two genomic targets are detected, namely n (nucleocapsid), e (envelope), s (spike), or rdrp (rna-dependent rna polymerase) gene specific to the sarscov2 virus (3). pcr test and samples were taken from inpatients or outpatients who had performed a positive or negative pcr swab examination at husada utama hospital surabaya, east java-indonesia. sample size and sampling technique sampling in the study was conducted from august 2020 to october 2020 at husada utama hospital surabaya. there are 100 samples total, 50 of which are covid-19 positive patients who were verified by a positive pcr test and 50 of which are noncovid-19 positive patients who were verified by a negative pcr test. consecutive sampling was used to collect the samples. data management and analysis the analysis of the data was descriptive. analysis of the diagnostic value of commercial sars-cov-2 rapid immunochromatographic test kits was done by immunochromatography principle. when a patient's sample contains anti-sars-cov2 igm/igg antibody, it will bind to an antigen labeled colloidal gold (sars-cov-2 recombinant antigen). a color band will be formed. the performance of rapid diagnostic test (rdt) by zybio brand was expressed by determining diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value, diagnostic efficiency. ethical considerations the study obtained ethical approval from the health research ethics committee, faculty of medicine universitas indonesia with no 273/ec/kepk/fkua/2020. ina. j. med. lab. sci. tech. 2022; 4(2): 128–138 museyaroh, et al 1 3 1 results characteristics of covid-19 and noncovid-19 patients gender distribution in patients with confirmed covid-19, male patients amounted to 30 patients (60%) and female patients amounted to 20 patients (40%) while in healthy patients or non-covid-29 patients, gender patients 23 patients (46%) male and 27 female patients (56%). table 1. reactivity of anti-sars-cov-2 igm or igg antibodies anti-sars-cov-2 igm and igg antibody reactivity antibody reactivity covid-19 patient non covid-19 patient igm/igg and igm+igg reactive 41 6 igm/igg and igm+igg non-reactive 9 44 number of samples 50 50 table 2. analysis of anti-sars-cov-2 igm and igg antibody diagnostic tests anti-sars-cov-2 igm and igm antibody diagnostic test diagnostic value igm/igg and igm + igg igm igg diagnostic sensitivity 82% 62% 72% diagnostic specificity 88% 94% 88% positive predictive value 87% 91.1 % 85.7 % negative predictive value 83% 71.2 % 75.8 % accuracy 85% 78% 80% based on table 2, the results of the analysis of the diagnostic test for anti-sarscov-2 igm and igg antibodies are: the sensitivity of igm/igg and igm + igg is greater than the sensitivity of igm or igg alone, the specificity of igm/igg and igm + igg is equal to specificity of igg alone and smaller than igm alone, positive predictive value of igm/igg and igm + igg is greater than igg but lower than igm, negative predictive value of igm/igg and igm + igg is greater than igm or igg, accuracy of igm /igg and igm + igg is greater than igm or igg. table 3. analysis of anti-sars-cov-2 igm and igg antibody diagnostic tests based on the patient's day of illness analysis of anti-sars-c0v-2 igm and igg antibody diagnostic tests diagnostic value < days after the onset of symptoms > days after the onset of symptoms > days after the onset of symptoms > days after the onset of symptoms diagnostic sensitivity 50 % 100% diagnostic specificity 88% 88% ina. j. med. lab. sci. tech. 2022; 4(2): 128–138 museyaroh, et al 1 3 2 diagnostic value < days after the onset of symptoms > days after the onset of symptoms > days after the onset of symptoms > days after the onset of symptoms positive predictive value 60% 84 % negative predictive value 83 % 100% accuracy 77.94% 92.68 % based on table 3 the results in the sample group < days after the onset of symptoms: rapid diagnostic tests for antisars-cov-2 igm and igg antibodies have lower sensitivity, positive values, negative predictive values, and accuracy than the sample group > days after the onset of symptoms but have the same specificity. table 4. cross-reaction of igm and igg sars-cov-2 with non-sars-cov-2 antibody antibodies non covid -19 patient other antibody tests other antibody test results sars-cov-2 antibody results number igm salmonella positive negative 10 igg dengue positive negative 7 igm dan igg dengue positive negative 1 anti hcv positive negative 2 total 20 of the 50 samples of non-covid-19 patients known through negative pcr examination results, there were 20 samples that were positive for antibodies other than anti-sars-cov-2 igm and igg antibodies, namely 10 samples of positive igm salmonella patients through tubex-tf and ict salmonella examinations, 7 positive dengue igg patients through the dengue virus ict examination, and 1 positive patient for dengue igm and igg through the dengue virus ict examination, and 2 positive patients with anti-hepatitis c virus (hcv) antibodies through the anti-hcv ict examination. discussion characteristics of covid-19 patients and non-covid-19 patients the results of the analysis based on the sex distribution of confirmed covid-19 patients showed that there were more males than females, namely 30 male patients (60%) and 20 female patients (40%). according to hidayati (9) which states that men dominate the population of confirmed positive for ina. j. med. lab. sci. tech. 2022; 4(2): 128–138 museyaroh, et al 1 3 3 covid-19 in indonesia, the positive male population accounts for more than half of the total confirmed covid-19 patients. clinical manifestations in male patients are much worse than in female patients. the percentage of men who die is much higher than that of women. this may be related to the habit of men who smoke more often, therefore respiratory tract diseases in men are often worse than women (9). reactivity of sars-cov-2 igm and igg antibodies in covid-19 patients based on the of the anti-sars-cov-2 antibody reactivity analysis, the results were obtained, namely in covid-19 patients, igm/igg or igm+igg reactive were 41 patients, and igm/igg or non-reactive igm+igg were 9 patients. in healthy or noncovid-19 patients, the results were igm/igg or igm+igg reactive as many as 6 patients and igm/igg or non-reactive igm+igg as many as 44 patients. the results of the analysis of anti-sarscov-2 antibody reactivity based on the type of antibody, namely in covid-19 patients, reactive igm in 31 patients, non-reactive igm in 19 patients, and reactive igg in 36 patients, non-reactive igg in 14 patients while in healthy patients, or non-covid-19 patients, 3 patients has reactive igm, 47 patients with non-reactive igg and 6 reactive igg, 44 patients with non-reactive igg. according to the indonesian association of clinical pathology doctors, negative results on rapid diagnostic tests of antisars-cov-2 igm and igg antibodies (rapid tests) occur: (a) when someone is not infected with the sars-cov-2 virus, (b) in the window period where the patient infected with the sars-cov-2 virus but antibodies have not been formed so that the antibody is not detected by the device, (c) in immunocompromised patients whose immune function is impaired so that they cannot produce enough antibodies to be detected by the device or because the patient's antibody levels are below the device's detection level, while positive results on rapid diagnostic tests of anti-sars-cov-2 igm and igg antibodies can be caused by (a) exposure/infection with the sars-cov-2 virus, (b) cross-reaction with other coronavirus antibodies or other viruses that resemble the sars-cov-2 virus and rheumatic factors (10). according to spicuzza et al., (11) in their research, two cases of confirmed covid-19 patients had negative igm/igg antibody test results. this could be due to seroconversion from covid-19 patients and related to inappropriate timing of sampling, such as in the first week of infection when the body has not yet formed antibodies so that the results of the igm and igg examine analysis of igm and igg antibody diagnostic tests -sarscov-2. based on the analysis of the anti-sarscov-2 antibody validation test, this reagent ina. j. med. lab. sci. tech. 2022; 4(2): 128–138 museyaroh, et al 1 3 4 kit has a sensitivity of 82%, specificity of 88%, positive predictive value of 87%, negative predictive value of 83% and accuracy of 85%. in the analysis of the anti-sars-cov-2 antibody diagnostic test, based on the type of antibody, the results obtained are igm has a sensitivity of 62%, specificity of 94%, positive predictive value of 91.1%, negative predictive value of 71.2% and diagnostic accuracy of 78%, while igg antibody has a sensitivity of 72%, specificity of 88%, positive predictive value of 85.7%, negative predictive value of 75.8%, and accuracy of 80%. the above results are slightly different from the research of liu et al., (12) which states that the igm/igg rdt has a sensitivity of 85.6%, specificity 91%, positive predictive value 95.1%, negative predictive value 82.7%, and accuracy of 88.3%. the reasons underlying the discrepancy in the results are still not known with certainty but may be due to the analytical differences between the tests. in addition, the difference in the results may also be caused by differences in immune responses between patients in population groups and differences in sampling time. the exact timing for detecting igm and igg responses after infection with sars-cov-2 is so far unclear as few studies are available with differing results nations become negative. if this result is confirmed in a larger sample, the test may be considered a potential tool for measuring population immunization. according to barbosa et al., (13) the rate of increase in sars-cov-2 antibodies is different for each individual. in patients with mild clinical symptoms, specific antibodies appear earlier, usually on day 7 when igm is lower and igg continues to increase. in patients with severe clinical symptoms of sars-cov-2, antibody seroconversion appeared longer, usually on day 12 and igm continues to increase. diagnostic sensitivity is the ability to diagnose a patient with a disease that is correctly identified as a positive result through screening tests. in this study, the sensitivity value of this reagent kit was 82%. these results indicate that the sensitivity of this reagent kit is relatively low, so it is hoped that manufacturers can increase the sensitivity of the detection of this sarscov-2 igm and igg test reagent kit, because the lower the sensitivity, the falser negative cases, and this will result in increased transmission of the sars-cov-2 virus in people who are in close contact with patients infected with the sars-cov-2 virus. for the specificity of this reagent kit, it has a relatively high value of 88%. this result shows that the reagent kit is likely to correctly identify people who are not sick with screening tests / screening for 88%. with regard to the positive predictive value of 87%, this result shows that the proportion of ina. j. med. lab. sci. tech. 2022; 4(2): 128–138 museyaroh, et al 1 3 5 people with positive test results who actually have the disease is 87%. with regard to the negative predictive value of 83%, this result shows that the proportion of patients with negative results who do not suffer from covid-19 is 83%. with regard to the diagnostic accuracy of 85%, this result shows that the proportion of true test results (true value) among all those examined is 85%. however, according to several studies, pcr as a reference examination in research has a sensitivity of around 75%. therefore, this rapid diagnostic test still has the possibility of having higher sensitivity, specificity, positive predictive value, negative predictive value, and accuracy. reactivity of igm and igg antibodies based on the day of onset of symptoms based on the results of this study, the sars-cov-2 igm and igg reagent kit obtained results in the group of patients who experienced clinical symptoms < 7 days having a sensitivity of 50%, specificity of 88%, positive predictive value of 60%, negative predictive value of 83% and an accuracy of 77.94% based on the type of antibody, namely igm antibody has a sensitivity of 50%, specificity of 94%, positive predictive value of 75%, negative predictive value of 83.9% and accuracy of 82%, while for igg antibody it has a sensitivity of 27.7%, 88% specificity, 45.45% positive predictive value, 77% negative predictive value and 59.75% accuracy. patients in this group may be in the early stages of infection or the window period or because the antibody concentration is too low so that the antibody cannot be detected. (12). the results above are different from the results in the group of patients who have clinical symptoms > 7 days, where this reagent kit has higher sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, namely sensitivity of 100%, specificity of 88%, positive predictive value of 84%, a negative predictive value of 100%, and an accuracy of 92.68%. based on the type of antibody, igm antibody has a sensitivity of 68%, specificity of 94%, positive predictive value of 88%, negative predictive value of 82.45%, and accuracy of 84%, while antibody igg has a sensitivity of 96.87%, specificity of 88%, a positive predictive value of 83.78%, a negative predictive value of 97.7%, and an accuracy of 91.46%. these results are in line with the research of liu et al., (12) which is in 16 confirmed covid-19 patients with symptoms of 0-7 days the sars-cov-2 igg/igm reagent kit only had a sensitivity of 18.8%, while in group 8 15 days the igm/igg has a sensitivity of 50%, and in the group of patients with clinical symptoms > 16 days the igm/igg sensitivity is 100%. according to andrey et al., (14) most of the false negatives in the sars-cov-2 igm/igg antibody test were in the subgroup ina. j. med. lab. sci. tech. 2022; 4(2): 128–138 museyaroh, et al 1 3 6 of samples taken 0-6 days after the onset of clinical symptoms. this may be related to seroconversion from covid-19 patients, but until now seroconversion in covid-19 patients is still not clearly known, because there are only a few studies and there are differences in the results of some of these studies. according to hoffman et al., (15) seroconversion in covid-19 patients occurs between 7-12 days after the onset of symptoms., generally, igm is produced first and igg is produced later. the presence of igg lasts for a long time in the body. hsueh et al., (16) added that igg seroconversion occurs on average 10 days after the onset of clinical symptoms in covid-19 patients and the peak of this antibody seroconversion is at 15 days. according to döhla et al., (17) seroconversion occurs sequentially for igm and then igg with a median time of 11 and 14 days, respectively. therefore, if the sample is taken less than that time, it is likely that antibodies have not been formed and the test results will be false negative. according to research by long et al., (18) mentioning seroconversion in 26 patients who were initially seronegative during the observation period, the results obtained were 3 types of seroconversion, namely synchronous seroconversion of igg and igm, igm seroconversion earlier than igg, and igm seroconversion slower than ig. igm can be detected in the blood of a person infected with the sars-cov2 virus for 3-6 days after the onset of clinical symptoms and igg can be detected 8-13 days after infection with the sars-cov-2 virus (19,20). reactivity of sars-cov-2 igm and igg with other antibodies based on the results of this study, from 20 samples of healthy or non-covid-19 patients with negative pcr results and patients experiencing clinical symptoms resembling covid-19 such as fever and diarrhea, there were 10 positive patients with igm salmonella, 1 patient positive for igm and igg dengue virus, 7 patients positive for dengue virus igg and 2 positive patients with anti-hepatitis c (hcv) antibodies, and the results of the sars-cov-2 igm and igg examinations were negative. therefore, from these results the researchers concluded that this reagent kit did not cross-react with salmonella bacteria antibodies, viral antibodies dengue, and hepatitis c virus (hcv) antibodies. these results can be used as additional information, because the reagent insert kit only states that these reagents do not cross-react against antibodies to parainfluenza virus, chlamydia pneumonia, mycoplasma pneumonia, and adenovirus. who does not recommend the sarscov-2 igm/igg rapid test as a tool for the diagnosis of covid-19 because this test is not specific (does not detect the presence of the virus directly) so that if the rapid test results show non-reactivity, it does not rule ina. j. med. lab. sci. tech. 2022; 4(2): 128–138 museyaroh, et al 1 3 7 out the possibility of not being infected with the sars-cov-2 virus because the performance of this test is influenced by many factors such as the time of onset of illness, the patient's immune response, the time of sampling and inter-analytical testing (13). however, this assay can be a reliable option in controlling the prevalence of covid-19, especially in situations where rt-pcr and elisa assays are not available, or cannot be used reliably. according to liu et al., (12) the rapid diagnostic test of igg/igm sars-cov-2 is reliable if it is not performed < 6 days after the onset of clinical symptoms. the advantage of the rapid test for antisars-cov-2 igm and igg antibodies is that this test provides a fast response that only takes a few minutes. in patients who come with a discrepancy between the clinical/radiological picture and molecular tests, the detection of these antibodies can be used as an additional element that helps the doctor to make the correct diagnosis. the limitations of this study were that the researcher could not determine the seroconversion of the anti-sars-cov-2 igm and igg antibodies, the sampling was limited to < 7 days after the onset of clinical symptoms and > 7 days after the onset of clinical symptoms, so that the researchers could not determine the seroconversion and the best timing in sampling igm and igg antibody tests for sars-cov-2. in the negative pcr samples, the researchers were unable to determine other diseases other than covid-19. conclusions in patients with corona virus disease-19 (covid-19) with clinical symptoms > 7 days, rapid diagnostic tests for anti-sarscov-2 igm and igg antibodies have greater sensitivity, specificity, positive predictive value, negative predictive value, and accuracy than rapid diagnostic tests in patients with corona virus disease-19 (covid-19) with clinical symptoms < 7 days. author contributions muza: conceptualization, methodology, investigation, writing-reviewing editing, software, data curation, and writing-editing, methodology. puspa wardani: validation, aryati: validation. evy diah woelansari: editing – reviewing. acknowledgements husada utama hospital surabaya 2020 gave this work its support. clinical pathology laboratory was where this work was done. conflict of interest there is no conflict of interest. ina. j. med. lab. sci. tech. 2022; 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3(2): 135–145 ririh jatmi wikandari, et al. 1 3 6 introduction worms infection caused by worm parasites, with some species of worm known as soil transmitted helminth (sth) (1). sth is a worm that in its life cycle requires suitable soil to develop into an infective form. common sth species are roundworm (ascaris lumbricoides), whipworm (trichuris trichiura) and hookworm (ancylostoma duodenale, necator americanus) (2). sth infections occur worldwide. this infection is widespread in tropical and subtropical regions, where about one-third of the world's population are infected by at least one sth species. estimation globally globally, it is estimated that ascaris lumbricoides infect 820 million people, trichuris trichiura infect 460 million people, and hookworms infect 440 million people (1). the highest prevalence occurs in asia, sub-saharan africa and latin america (3). worms infection occurs in southeast asia, including indonesia. indonesia as a tropical country with a warm and humid climate provides an ideal environment for the survival of the eggs or larvae of the sth parasite. the prevalence of worms in indonesia varies from 2.5% to 62%. worms infection enable attack all ages from children to adults (2). the prevalence of worms was related to the low socialeconomic conditions of the community, poor environmental sanitation and poor personal hygiene. sth infection rarely causes death but causes diarrhea, abdominal pain and a dropin hemoglobin level. as a result, the longterm effects of this infection lead to decreased cognitive, intellectual abilities and low work productivity (4). because the symptoms are generally not specific, sth infection is considered a normal condition by the affected individual, or as a symptom of another disease that occurs more often in certain conditions so that this disease is considered not a dangerous disease (4,5). worms transmission occurs due to soil contamination by sth’s species eggs or larvae. in warm tropical environments, parasite eggs are excreted in the feces of infected individuals and contaminate the soil. humans become infected through ingestion of eggs or larvae that are excreted in the feces of an infected person. in addition, hookworm eggs hatch in the soil, releasing larvae that mature into a form that can actively penetrate the skin (1). community behavior contributes to the incidence of worms, for example, lack of personal hygiene and poor environmental sanitation. work related to or using the land has a high risk of contracting worms, one of the jobs is farmers (6). based on the research of ali (7), as many as 70% of farmers in maharatu village, pekanbaru suffer from sth infection. vegetable farmers have 9 times the risk of getting a worm infection. the risk of sth infection in farmers is caused by poor sanitation hygiene, and not ina. j. med. lab. sci. tech. 2021; 3(2): 135–145 ririh jatmi wikandari, et al. 1 3 7 using personal protective equipment (ppe) (7). studies by adeola (8) showed that the habit of not wearing shoes and not washing hands before eating was found to be associated with an increased likelihood of sth infection. based on preliminary study farmers in batur wetan getasan hamlet, work with out used proper personal protective equipment, for example using shoes or footwear, wearing gloves, or even not using both. the floor of the houses in batur wetan hamlet, mostly are still on land. this situation enables cause the farmers by the worms that are transmitted through the soil. this study aims to determine the factors associated with worm infection in vegetable farmers in batur wetan hamlet, getasan regency, semarang-indonesia. materials and methods study area the type of study is observational (nonexperimental), with cross-sectional. this study was conducted in june until august 2019. dusun batur wetan, one of part of batur village is located at an altitude of 1,200 m above sea level with a slope-shaped topography and an average temperature of 30°c (figure 1). the soil conditions are quite fertile so it is very suitable for agriculture, especially horticulture with an average rainfall of 2,500 mm/year. irrigation and watering system using soil water. the sensus done at 55 vegetable farmers population, in batur wetan getasan hamlet, central java province. figure 1. location of batur wetan getasan hamlet inclusion and exclusion criteria inclusion criteria for vegetable farmers were aged 17-55 years with minimum 1 year working period. exclusion criteria for vegetable growers who were sick. data collection used a structured questionnaire to ina. j. med. lab. sci. tech. 2021; 3(2): 135–145 ririh jatmi wikandari, et al. 1 3 8 collect the sociodemographic data (including gender, age). the given questioner consisted of the using of gloves when working, the latrine outside the house, washing hands with water and soap after defecating, washing hands with soap and water before eating, consumption of uncooked, cut nails once a week, using toilet facilities in place work, drinking water from wells. after the interview, respondents collect feces in a stool container. this study did not use a control group, because the respondents did not receive any intervention or treatment. ethics statement this study was approved by the health research ethics committee of ministry of health polytechnic semarang with no. 168/ea/kepk/2019. data collection each respondent was given a plastic dry screw-top container, applicator wand, toilet paper, and given appropriate instructions on how to obtain and carry a fresh stool sample. stool samples were collected, labeled, and transported to the health analyst parasitology laboratory, campus 3 of the health polytechnic of the ministry of health, semarang. microscopic examination (olympus) of infection with worm eggs sth in fecal specimens using the flotation method. briefly, emulsify 1 g of feces with 3-4 ml of saturated salt solution in a test tube, then stir until homogeneous, add the saturated salt solution to the brim. place the coverslip on it, leave it for 10-60 minutes, then remove the coverslip, observed for the presence of eggs/larvae (9). statistical analysis the data obtained from the study were analyzed by spss statistics for windows version 20, both univariate and bivariate analyzes. univariate analysis was conducted to obtain a description of each variable were calculated using descriptive statistics, while bivariate analysis to determine the relationship between independent variables to the dependent variable, using chi square analysis with a 95% confidence degree. if result shows p-value <0.05, it means that statistically or indicate has a significantly relationship between variables, and vice versa. results based on field observations, irrigation and watering systems use ground water, fertilizer comes from animal waste, and there are no toilets available in the workplace. the sth infection of respondents by gender and age, showed in table 1. table 1. sth infection of the respondent characteristic sth infection total yes no n % n % n % gender male 2 3,6 29 51,4 31 55 female 1 2,3 23 42,7 24 45 age (years) 20-39 2 3,6 17 30,9 19 34,5 40-59 1 2,5 35 63 36 65,5 ina. j. med. lab. sci. tech. 2021; 3(2): 135–145 ririh jatmi wikandari, et al. 1 3 9 the respondent’s population were 55 people, consist of 31 (56%) men and 24 (44%) women. the infected with worms of respondent population consist of 2 women and a man. the incidence of sth infection is most prevalent in the age of 20-39 years (3.6%) and age 40-59 years (2.5%). table 2. frequency distribution of stool examination result sth f % infected 3 5,1 no infected 52 94,9 total 55 100 tabel 2 showed there are 3 (5.1%) vegetable farmers infected with sth. based of the identification of sth positive stool samples found a. lumbricoides eggs (figure 2) and t. trichiura eggs (figure 3). figure 2. ascaris lumbricoides eggs stool examination magnification 40x (black arrow) there were 3 respondents who were infected with sth (table 3). the relationship between using of gloves while working with the incidence of helminth infections is not significant (p = 0.214). the relationship between the location of the latrine outside the house and the incidence of worms is not significant (p = 0.347). the washing hands with soap and water after defecating habits was not significant (p = 0.233). figure 3. trichuris trichiura eggs stool examination magnification 40x (black arrow) regarding the relationship between the consumption of uncooked food and cutting nails once a week with the incidence of worms is significant (p = 0.911) (0.711). however, the relationship between the washing hands with water and soap before eating habits with helminth infectios showed a significant relationship (p = 0.000). all of 55 respondents has been used toilet facilities at work, source of clean water from wells, uses footwear when working. discussion worms are found in areas with high humidity, moist soil conditions. the nature of clay and high humidity and tropical climate is very suitable for the growth of the eggs of ascaris lumbricoides and trichuris trichiura (10,11). in warm tropical environments ina. j. med. lab. sci. tech. 2021; 3(2): 135–145 ririh jatmi wikandari, et al. 1 4 0 where tsh is widespread, parasite eggs are excreted in the feces of infected individuals and contaminate the soil (12). worm infections mostly attack workers whose activities are more related to soil. vegetable farmers are a group of workers who are at risk of infection with helminthiasis because their activities are directly related to the soil. sth infection is higher in male farmers than female farmers. it is not in line with statement of ross (13), that women are more at risk of sth infection than men, but it’s in line to aribodor (14) statement, that there are varying levels of infectivity associated sexes differ according to socioeconomic and cultural factors. the incidence of sth infection can affect all gender, ages, from children to adults, depending on the good and bad of personal hygiene and environmental sanitation (15). table 3. statistic analysis variable sth infection total p-value yes no n (%) wearing gloves when working yes 38 38 (69) 0,214 no 3 14 17 (31) location of latrine outside the house yes 43 43 (78) 0,347 no 3 9 12 (22) washing hands with water and soap after defecating yes 3 35 38 (69) 0,233 no 17 17 (31) washing hand with water and soap before eating yes 2 52 54 (98) 0,00 no 1 1 (2) comsumption of uncooked food yes 2 52 54 (98) 0,911 no 1 1 (2) cutting nails once a week yes 1 23 24 (44) 0,711 no 2 29 31 (56) use the toilet facilities at the workplace yes 3 52 55 (100) no source of clean water from wells yes 3 52 55 (100) a no wearing footwear for work yes 3 52 55 (100) a no a = no data computed because result constan this study used flotation method, because this method produces a clean preparation for microscopic examination need with minimal residual dirt that interfere. this method increases the likelihood of detecting parasitic organisms when the amount is small, fairly easy to do and inexpensive (16). the result showed 2 types of worm eggs, namely ascaris lumbricoides and trichuris trichiura. these eggs are a types of worms eggs that are commonly found in indonesia. this result is in line with apsari (17), which found eggs of a. lumbricoides and t. trichiura. a. ina. j. med. lab. sci. tech. 2021; 3(2): 135–145 ririh jatmi wikandari, et al. 1 4 1 lumbricoides and of t. trichiura. boko, taiwo and aribodor (12,14,18), reported that a. lumbricoides and t. trichiura were the most dominant and important among sth, as well as intestinal parasites. the relationship of using gloves when working with the incidence of worm infections, is not significant. this result is in line with research imansyah (19) showing that stated no significant relationship between the use of ppe (gloves or shoes) and sth infection. this indicates that the sth infection does not originate in the workplace but may originate elsewhere. however, these findings is not in line with by baidowi (20) which showed workers who did not wear gloves were 8.8 times more likely to be infected with sth than workers who wore gloves while working. gloves are one of the personal protective equipment. personal protective equipment aims to protect all parts of the hand, prevent direct contact to the soil, cut the chain of transmission of sth infection, and prevent entering eggs of a. lumbricoides and t. trichiura from nails or sticking to the hands (20,21). a latrine facility is very important for life. the latrine is used as a place to defecate and urinate. the use of latrines must meet health requirements, toilets must be clean, clean water and soap are provided for washing hands (2,22). the use of clean latrines is one of the ways to live clean and healthy. an unsanitary latrine can cause the spread of disease due to human waste (23). hands are the main transmission body organ of germs and diseases. hand hygiene is important to avoid transmission of germs, dangerous diseases and prevent infection (24). efforts to control the risk factors for worms can be done by washing hands before eating or after defecating with water and soap (25). the results of washing hands with soap are in line with ali's research (7) on the habit of washing hands with soap in vegetable farmers in maharatu village, marpoyan damai district, pekanbaru city, which is related to the incidence of worms. the same results were also shown by research alamsyah on vegetable farmers in lingga village, sungai ambawang district, kubu raya regency, which showed that the habit of washing hands with soap was related to the incidence of worms (26). the habit of washing hands with water is not enough to reduce the number of disease-causing microorganisms that stick to the hands. the indonesian ministry of health stated that washing hands properly is using soap and water (27). washing hands with water and soap are more effective at removing dirt and dust and reducing the number of diseasecausing microorganisms such as viruses, bacteria, other parasites that stick to the surface of the skin, nails and fingers on both hands such as worm eggs (25). it can be stated that it is very possible that the ina. j. med. lab. sci. tech. 2021; 3(2): 135–145 ririh jatmi wikandari, et al. 1 4 2 handwashing behavior of vegetable farmers in batur wetan hamlet is carried out inappropriately and correctly so that it is one of the factors causing worm infection. the soil condition of batur wetan hamlet is quite fertile so it is suitable for agriculture, especially horticulture (28). vegetable planting land can be a source of transmission of sth worms because moist soil is a good growth medium for the development of worms (29,30). the moist soil is very suitable for fertile eggs of a. lumbricoides and t. trichiura worms to develops into infective stage (31). the optimum growth temperature for a. lumbricoides eggs is approximately 250c, and for t. trichiura eggs at 300 c (2). farmers' habit of using manure also has the potential to contaminate sth to vegetables. animal waste posibly transmits these worms (29). in addition to local suitable soil and environmental conditions, is also influenced by the number of infective eggs and intering the host. the more eggs found in the source of contamination (soil, dust, vegetables, etc.), the higher the endemicity in an area (2). the result of consumption of uncooked food in this research, this is different from the results of yavari (32) which states that there is a relationship between raw food consumption and the incidence of worm infection. consumption of fresh vegetables plays an important role in the transmission of parasites in humans, if vegetables are not washed properly (33). parasites that have been linked to foodborne infections include worm eggs (34). when vegetables contaminated with worm eggs are eaten by humans, the person will become infected with worms. personal hygiene is a person's efforts to maintain health, one of which can be done by maintaining nail hygiene. transmission of worm infection to humans can occur in several ways, namely direct transmission through worm eggs attached to nails that have been contaminated by soil contaminated with sth. to prevent the transmission of intestinal worms, nails should always be cut short, and clean, uneven nail surface (there are wounds on the nails), color is not clear, the skin under the nails the length of the nails exceeds the fingertips (2). efforts to control helminthic risk factors can be carried out through personal hygiene efforts or environmental hygiene. personal hygiene is carried out, including the use of clean water for bathing purposes, consumption and washing hands with soap using clean water (2). availability of latrines is needed on agricultural land where work is carried out as a means of standardised disposing of feces for disease prevention and control. for disease prevention, workers must use personal protective equipment such as gloves and footwear (35). footwear can protect feet from the entering of worm larvae into the skin (12). footwear that is used for ina. j. med. lab. sci. tech. 2021; 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[guidelines for guidance on clean and healthy living behavior]. kementerian kesehatan ri. jakarta; 2011. 1–97 p. 42 a comparative study of mean platelet volume in diabetic population with and without vascular complication ajantha swamy vasudhevan1, dhivya mohan sumathi2, ashwath kumar chinnaraju selvakumar3, rajabalaji3 1department of physiology, saveetha medical college and hospital, chennai, tamil nadu, india 2department of anatomy, arunai medical college, and hospital, tiruvannamalai, tamil nādu, india 3department of orthopaedics, saveetha medical college and hospital, chennai, tamil nadu, india correspondence: dhivya mohan sumathi, no. 61 krishnaswamy nagar, koothappakkam, cuddalore, tamilnadu, india zip code: 607002 email: dhivyambbsms@gmail.com received: september 12, 2022 revised: march 26, 2023 accepted: april 3, 2023 published: april 29, 2023 doi: 10.33086/ijmlst.v5i1.3465 abstract diabetes mellitus leads to long-term complications involving multiple organs and systems of the human body. among the list of complications, a relatively vascular complication increases the morbidity of the condition. patients with diabetes mellitus are influenced by various factors like hyperglycaemic state, insulin resistance, oxidative stress, and metabolic condition like obesity, and all the mentioned conditions also present with platelet hyperactivity. mean platelet volume (mpv) can be used as one of the laboratory parameters to know the function and activation of the platelets, which reflects the vascular profile of the patient. so, the present study compares the values of mean platelet volume among the diabetic groups to determine the relation between the vascular complication and the mean platelet volume. this study was conducted with 90 participants, who were divided into three groups. group a is non-diabetics, group b is type 2 diabetics, and group c is type 2 diabetics with vascular complications and mpv. on analyzing the statistical mean value of mean platelet volume, group b's (type 2 diabetics) value was higher than group a's (non-diabetics) and statistically significant with a p – value of 0.001. similarly, the mean value of group c (type 2 diabetes with complications) was higher than group b (type 2 diabetics) and statistically significant with a p – value of 0.049 in the diabetics with and without vascular complications. on comparing the mpv of different study groups, the mpv is higher in the diabetic group with complicatiosn compared with the diabetics without complications. keywords mean platelet volume, type ii diabetes mellitus, vascular complication. citation: vasudhevan as, sumathi dm, selvakumar akc, rajabalaji r. a comparative study of mean platelet volume in diabetic population with and without vascular complication. indones j med lab sci technol. 2023;5(1):42–52. doi: 10.33086/ijmlst.v5i1.3465 this is an open access article distributed under the creative commons attribution-sharealike 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2023 by author. mailto:dhivyambbsms@gmail.com mailto:https://doi.org/10.33086/ijmlst.v5i1.3465 mailto:https://doi.org/10.33086/ijmlst.v5i1.3465 https://creativecommons.org/licenses/by-sa/4.0/ ina. j. med. lab. sci. tech. 2023; 5(1): 42–52 ajantha swamy vasudhevan, et al. 4 3 introduction diabetes is a comprehensive health burden which is worldwide. microand microvascular complications are common in long term diabetic cases and these complications can involve other systems of the human body (1,2). diabetes mellitus is one of the most common conditions that is prevalent but undetected in many cases. most of the cases are incidental findings during the routine investigation of other conditions. certain cases can present with macrovascular changes like atherosclerotic disease or microvascular changes like retinopathy, neuropathy, and nephropathy even during the time of detection of a new diabetic case (3). vascular complications from diabetes mellitus are caused by numerous factors, including transformation in the growth factors involving the endothelium of the blood vessels, advanced glycation products, chronic inflammation, altered fibrinolytic ability, and increased platelet aggregation and activation (4). endothelial dysfunctions and vascular lesions are prominent features of the complications of sustained hyperglycemia. the state of hyperglycemia causes microvascular changes and macro vasculopathy in diabetic patients. platelets is a type of blood cell that has a major role in homeostatis, i.e. the spontaneous arrest of bleeding. platelets take part in temporary homeostatic plug formation in response to stimuli from the site of the endothelial defect by changing their shape, adhering to the sub endothelial surface, and aggregating and releasing their intracellular contents to form a platelet plug. platelets also augment the process of permanent clot formation by coordinating with activated clotting factors (5). the normal vasomotion of the blood and its components in the blood vessels determine the normal blood flow and the thromboresistance. there is always an interaction between the blood cells and the endothelial lining of the blood vessels. these interactions initiate the atheromatous plaque formation and also the response from the blood components to the rupture of the plaque. the interaction of the platelet with the plaque rupture site results in the coagulation cascade, which eventually results in vascular events. in a chronic, ongoing inflammatory state, the release of proinflammatory cytokines like interleukin 6 is associated with thrombopoietin generation. interleukin released produces effects on platelet precursor megakaryocyte like increase in megakaryocytic cytoplasmic volume and ploidy of nuclei, which in turn result in the a in large number of platelets (6). the pathophysiological state of diabetes mellitus with vascular disease can be analyzed by various factors, like the state of hyperglycemia in the patient, oxidative stress, the relation of insulin resistance with atherosclerosis, microrna, and thrombosis. ina. j. med. lab. sci. tech. 2023; 5(1): 42–52 ajantha swamy vasudhevan, et al. 4 4 in all the above-mentioned states, platelet hyperactivity is of major relevance (3). hyperglycemia leads to a pro-thrombotic state in diabetic patients. the platelet adhesion is increased by the glycation of the surface proteins of the platelet. both chronic and acute states of hyperglycemia trigger platelet activation. insulin resistance is another prime factor in the development of type 2 diabetes mellitus. pancreatic beta cells are responsible for the production of compensatory insulin in pre-diabetic cases, eventually leads to a decrease in the pancreatic beta cell mass due to its apoptosis, resulting in insulin deficiency. insulin controls platelet action by binding to the insulin receptor on the surface of the platelets. the interaction of insulin and platelets also affects plasminogen activator secretion, insulin receptor substrate 1, and insulin-like growth factor -1 (igf-1) (7). increased oxidative stress has been reported in type 2 diabetes mellitus. in these cases, platelet activation and platelet aggregation are due to increased platelet calcium concentration. oxidative stress causes platelet hyperactivity are due to increased production of f2 isoprostane, signaling platelet reception, and decreased activity of e-nos (8). obesity is one of the major metabolic conditions that exacerbates insulin resistance. obesity presents with an increased platelet count, increased platelet volume and also affects other platelet indices indices, indicating increased platelet adhesion and activation (9). activation of the metabolic changes in diabetic patients further leads to vasoconstriction and leads to thrombus formation involving the platelets. all vascular complications are initiated by the inflammatory and thrombotic changes that occur in the endothelium of the vascular structure. when the vascular complication begins, the equilibrium of the vasoconstriction and vasodilation, along with the anti coagulating system is disturbed. the impaired endogenous inhibition of the platelets increases the susceptibility to the platelet activation, and so the altered endothelium becomes a medium for adhesion molecules and platelet agonists. longstanding cases of diabetes show changes in the morphology and adhering quality of the platelets. increased platelet activity can be witnessed in acute hyperglycemic states even in a normal individual without diabetes mellitus. all these factors make it necessary to understand the mechanism of endothelium and platelet dysfunction, which are involved in the pathophysiology of diabetes mellitus with vascular complications (10). mean platelet volume is a laboratory parameter that quantifies the volume of the platelet and infers its function and activation (11). morphological variation in the platelet size is associated with differences in the amount of the production of serotonin, ina. j. med. lab. sci. tech. 2023; 5(1): 42–52 ajantha swamy vasudhevan, et al. 4 5 β-thromboglobulin, and procoagulants. thus, mean platelet volume could be an economical laboratory parameter that reflects the state of thrombogenesis in diabetic patients. evidence from previous literature has shown that the range of mpv is higher in the diabetic population compared to the nondiabetic population (12). including mean platelet volume and other platelet activity in the routine investigations can assess the burden of the complication to diabetics. since many methods are costly, require specialized equipment, and are time-consuming, these investigations are given less importance in routine assessment methods. potentially, there are lacunae in the scientific data on the cut-off value, methodology, and procedure, making more interpretation of the results and clinical utility of results even the uncertain. mpv is also found to be higher in various other conditions such as hypertension, cardiovascular diseases, hypercholesterolemia, obesity, and smoking, suggesting a common mechanism and possible comorbid conditions along with diabetes mellitus (13). the aim of the study is to compare the values of mean platelet volume among the diabetic groups and to determine the relationship between vascular complications and mean platelet volume, with the following objectives: to determine the difference in the mean platelet volume between diabetics and non-diabetics, and to review the difference in the mpv between diabetics with and without complications (established vascular disease). materials and methods this cross-sectional analytical study was conducted after the approval from the institutional ethical committee. with consent, the study was conducted among the patients who attended master health check-ups in a medical college for one month. patients aged above and 18 were included in the study, except for those with conditions involving blood, liver, bone marrow, chronic systemic inflammatory disorders, any infectious diseases, cancer chemotherapy, patients on antiplatelet therapy, smoking, alcoholism, and pregnancy. a detailed history was elicited from the patients regarding their diabetic profile and the associated complications. all the participants were examined clinically to screen for the presence of any macro or microvascular complications. venous blood samples were collected in the hemogram tubes containing sodium fluoride and dipotassium edta. samples were transported to the lab within one hour of collection to minimize variations due to sample aging. the blood samples were used to assess the mean platelet volume (mpv), hba1c and plasma glucose. mpv was measured by an automatic complete blood count. hba1c and plasma glucose were ina. j. med. lab. sci. tech. 2023; 5(1): 42–52 ajantha swamy vasudhevan, et al. 4 6 performed to know the diabetic state of the patient. plasma glucose was measured by the glucose oxidase method in an autoanalyzer. hba1c was measured by an automated ionexchange high-performance liquid chromatograph (in a laboratory performed using an ngsp-certified method and dcct assay standardized) (11). following the screening of 150 participants for inclusion and exclusion criteria, a total of 90 participants were included in the study and divided into three groups based on the following criteria: group a: non-diabetic controls. healthy non-diabetic controls based on their fasting and postprandial blood glucose levels according to the american diabetic association criteria. group b: type 2 diabetes mellitus without any diabetes-related complications. patients diagnosed with type 2 diabetes mellitus according to american diabetic association criteria (14) on oral antihyperglycemic drug therapy and without complications related to diabetes. group c: type 2 diabetes mellitus with complication patients diagnosed with type 2 diabetes mellitus based on american diabetic association criteria (14) on oral antihyperglycemic drug therapy and with established complications were analyzed. statistical analysis statistical analysis was carried out by using statistical package for social science (spss) 22 with the student’s independent sample two-tailed t-test. values were considered statistically significant when the p value was less than 0.05. results among the ninety participants of the study group, 58% were male and 42% were female. statistically, the mean value was calculated between the three groups of study and they were compared. the major parameter compared in the study was the mean platelet volume and the hba1c among the control and diabetic patients with and without complications. mean platelet volume in comparing the mean platelet volume between the diabetic without complication (group b) and the non-diabetic group (group a), the mpv value was higher in the diabetic (group b) and statistically significant (table 1). on comparing the mean platelet volume of diabetics with complications (group c) and diabetics without complications (group b), the mpv value was higher in diabetics with complications (group c) and statistically significant (table 1 and figure 1). ina. j. med. lab. sci. tech. 2023; 5(1): 42–52 ajantha swamy vasudhevan, et al. 4 7 table 1. comparison of mpv between diabetics and non-diabetics group mean std. deviation p-value mpv (fl) non-diabetic 7.5267 0.91196 0.001* diabetic 8.5033 1.20444 mpv (fl) diabetic without complication 8.5033 1.20444 0.049* diabetic with complication 9.1970 1.45780 figure 1. comparison of mpv among diabetics with and without complication and non-diabetics discussion diabetes is a major health burden for the world. it is a metabolic disorder that leads to various long-term vascular complications, such as retinopathy, nephropathy, neuropathy, and cardiovascular and cerebrovascular diseases (15). various factors, such as the state of hyperglycemia, insulin resistance, oxidative stress, and metabolic conditions like obesity are responsible for the pathophysiological state of the condition and have relevance with platelet hyperactivity. complications are higher among diabetics with poor glycemic control, longer duration of diabetes, and associated comorbidities such as obesity and hypertension, which increase the risk of mortality in diabetics (16). platelets plays an important physiological role in primary hemostasis (17). they are the main component of the blood, which maintains the integrity of the blood's homeostasis. the size of the platelet determines their thrombogenic nature, in which the large platelets have an increased number of dense granules when compared with the smaller platelets. in the 0 1 2 3 4 5 6 7 8 9 10 non-diabetic diabetic without complication diabetic with complication m p v ( fl ) ina. j. med. lab. sci. tech. 2023; 5(1): 42–52 ajantha swamy vasudhevan, et al. 4 8 diabetic population, increased platelets aggregation can cause both micro-and macrovascular complications. along with diabetes mellitus, other illnesses like hypertension, cardiovascular diseases, hypercholesterolemia, obesity, and smoking are also associated wuth higher mpc levels, pointing to a potential shared cause. the mpv can be used to measure how large highly reactive and pleasant pletelets are. diabetes results in the deregulation of signaling pathways that can lead to platelet hyperactivity and thrombosis, which can cause vascular lesions to develop and progress (18). in this study, we compared the platelet activity in diabetics and nondiabetics using the mean platelet volume. our present study has shown an increased value of mean platelet volume in the diabetic group compared to the nondiabetic group, and similar results have been established in the previous studies, which also show significance in the value of mpv and platelet distribution width in a similar comparison (19,20). a study was conducted in the north indian population, in which the case of diabetes and the control group were categorized with the fasting and based on the american diabetes association. the diabetic patients were also examined for vascular complications and categorized based on them. similar to our present study, the mean platelet volume and other platelet indices were higher in the diabetics with or without complications. however, upon analyzing and comparing the platelet indices between the diabetics with and without complications,the values of platelet distribution width showed significant variability, but the mpv was normal (19). contrary to the aforementioned results, another study done to compare the platelet indices in diabetic patients with and without vascular complication showed complications an increased mpv value in diabetics with vascular complications, similar to our study results. in the same study, the other platelet indices values of the diabetic group were higher than those of the control group (20). few studies have shown a decrease in the platelet count among diabetics when compared to non-diabetics, suggesting that factors such as short survival times, impaired production, and increased turnover of platelest in diabetic patients may be at play. however, the mpv appears to be higher in their studies as well (21). in another study done to assess the correlation between the mean platelet volume and diabetes, the case and the control groups were divided based on the value of hba1c. cases with thrombotic and hematological diseases those and with low hemoglobin counts were excluded. the statistical correlation of the study showed that the mean platelet count in the diabetes was significantly lower than the control group. however, a significant correlation was ina. j. med. lab. sci. tech. 2023; 5(1): 42–52 ajantha swamy vasudhevan, et al. 4 9 established between the mean platelet volume values in both groups. in another study, the prognosis of diabetes mellitus was assessed by the effectiveness of the platelet parameter analysis. even though the values of platelet indices were higher in diabetic patients with complications, they were not statistically significant (22). most of the studies are done in cases of diabetics with vascular complications, since this leads to increased morbidity and mortality in these cases. few studies have also concentrated on the involvement in the initiation of the atherosclerotic process, as it is of rising interest. therefore, the status of platelets was studied in both type 1 and type 2 diabetics based on arachidonic acid metabolism. in the aforementioned study, increased platelet aggregation was detected even in a diabetic patient who did not suffer from vascular complications and presented with increased basal arachidonic acid metabolism. this can later stages contribute to increasing the risk of occurrence of vascular complications, both at the micro and the macro level, in diabetic patients (23). similar to the previous studies which compared the mean platelet volume between diabetics and the diabetics with retinopathy (a vascular complication). our present study has also shown an increased mean platelet volume in the diabetic with complication group compared to the diabetic group. this suggests a role for platelets in the vasculopathy of diabetics. the statistical correlation between the hba1c and the mpv has also been studied previously, and a similar positive correlation has been observed (15,24). a study was conducted to investigate the relationship between glycaemic control and platelet activity in type 2 diabetes mellitus. in the study, two groups were assessed based on hba1c. cases with > 7% hba1c were put under the glycaemic control measures for 3 months and weekly assessments were done for those cases. at the end of 3 months, 80 % of the cases showed good glycaemic control and also a significant decrease in the level of the mean platelet volume, which supports the result that increased hba1c concentration increases the mean platelet volume (15). a cohort study was conducted to estimate the correlation between platelet indices and type 1 and type 2 diabetes mellitus. a large sample group with both types of diabetes was taken and the platelet indices were studied under normal and glucose-controlled conditions. in this study, the platelet mass was higher and the mpv value was lower in both type – 1 and type – 2 diabetes when compared with the control group. the assessment of short-term glucose control did not have any effect on the values of the platelet indices (25). a similar study demonstrated the correlation between the platelet indices and ina. j. med. lab. sci. tech. 2023; 5(1): 42–52 ajantha swamy vasudhevan, et al. 5 0 diabetes with and without complications. they also studied the demographic details of the study group and the statistical influence on the different cases with various complications. 40% of the cases presented with cardiovascular complications, 30% with diabetic foot, and 14% had multiple complications, including retinopathy, neuropathy, and nephropathy. cases with complications showed increased values of platelet indices in diabetics with complications similar to our study (26). a similar study was conducted, excluding patients with all possible vascular co-morbid conditions. in the department of ophthalmology, fundoscopic examination was done and the degree of retinopathy was assessed. mpv values were studied for both the case and the control groups. the results projected that the patients with diabetic retinopathy at a proliferative state showed significantly higher values of mpv than normal, healthy individuals like our findings (24). studies conclude that glycemic control decreases the hyperactivity of the platelet function, thus decreasing diabetic vascular complications. the results regarding the duration of diabetes and mpv are similar to a few studies, but are contradictory to others. it is necessary to rule out qualitative platelet disorder to confirm the findings, which is the limitation of the present study. conclusions our work leads us to the conclusion that diabetics with vascular problems have greater mean platelet volumes. comparing the mean platelet volumes of the various study groups, the diabetes group with complications has a higher mean platelet volume than the diabetics without complications. our findings suggest that an increase in platelet size may be one of the triggers for atherosclerosis linked to diabetes macrovascular and microvascular problems. these findings require confirmation by larger-scale research. as a result, mean platelet volume can be utilized to predict the development of vascular problems in people with diabetes.in this study, we compared the platelet activity in diabetics and nondiabetics using the mean platelet volume. author contributions ajantha swamy vasudhevan: conceptualization, methodology and investigation. dhivya mohan sumathi: original draft writing and reviewing and data curation, ashwath kumar & rajabalaji: investigation and data collection. conflict of interest the author declars there is no conflict of interest. ina. j. med. lab. sci. tech. 2023; 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decreased antioxidant status. diabetes. 2004 apr 1;53(4):1046-51. doi: 10.2337/diabetes.53.4.1046 24. ji s, zhang j, fan x, wang x, ning x, zhang b, yan h. the relationship between mean platelet volume and diabetic retinopathy: a systematic review and meta-analysis. diabetology & metabolic syndrome. 2019. 11, 1-8. doi: 10.1186/s13098-019-0420-3 25. goyal p, mahajan s, parmar c. evaluation of platelet function in subjects with type-2 diabetes mellitus: a case control study. national journal of physiology, pharmacy and pharmacology. 2020. 10(8), 667-667. doi: 10.5455/njppp.2020.10.03057202014052020 26. gohil a, dhotre s, faldu b, goswami h. study of platelet indices in diabetes mellitus patient with respect to hba1c and its correlation with diabetic complications. diabetic foot. 2021. 30, 30-61. doi: 10.33545/pathol.2021.v4.i3b.391 https://doi.org/10.1111/jdi.13198 99 assessment of platelet indices profile of pregnant women attending university of abuja teaching hospital, nigeria amos dangana1, anthony uchenna emeribe2, hezekiah alkali isah3, sanusi musa4, joel monday abu5, solomon oloche onoja6, nkechi onukegbe7, idris nasir abdullahi4 1department of medical laboratory services, university of abuja teaching hospital, pmb 228 gwagwalada, abuja nigeria 2department of medical laboratory science, faculty of allied medical sciences, university of calabar, pmb 1115 calabar, nigeria 3department of haematology, university of abuja teaching hospital, pmb 228 gwagwalada, abuja nigeria 4department of medical laboratory science, faculty of allied health sciences, ahmadu bello university, pmb 05 zaria, nigeria 5department of family medicine, university of abuja teaching hospital, pmb 228 gwagwalada, abuja nigeria 6department of medical laboratory science, university of nigeria, enugu, nigeria 7department of strategic information and research, institute of human virology correspondence: idris nasir abdullahi, department of medical laboratory science, faculty of allied health sciences, ahmadu bello university, zaria, pmb 05 samaru road, zaria, nigeria email: eedris888@yahoo.com, inabdullahi@abu.edu.ng received: may 19, 2021 revised: july 26, 2021 accepted: august1, 2019 published: october 30, 2021 doi: 10.33086/ijmlst.v3i2.2110 abstract platelets initiate hemostasis by aggregating at the site of injury and participate in ensuring endothelial integrity. a defect in this process could lead to intravascular blood loss. this case-control study sought to determine the platelet counts and indices among pregnant women in the university of abuja teaching hospital gwagwalada, abuja, nigeria. a total of 120 pregnant women as case and 60 non-pregnant women as control were enrolled for this study. blood samples were collected in edta tubes, and complete platelet count and indices were carried out using an automated five-part haematology analyzer. the mean ± standard deviation of the platelets count among the pregnant women, 226.54 ± 69.76 109 cells/l was not significantly different from that of the non-pregnant women, 214.95 ± 52.22x 109 cells/l (p=0.295). there was a significant differences in mean platelets volume (mpv) of the case and control groups (p=0.036). after post-hoc test, the significant difference was between the pregnant women in 3rd trimester and the control group (p=0.014). however, there was no diffences in the mena platelets larger cell ratio and platelet distribution width in the case and control groups. fifteen (11.0%) and 7 (12.1%) of the case and control control groups, respectively had mild thrombocytopenia. however, there was no significant association between pregnant status and thrombocytopenia (p=0.836). based on these findngs, it can be infered that platelet count and mpv decreases while pdw increase with the progression of gestation age compared to the non-pregnant women. keywords platelet indices, haemostasis, coagulopathy, nigeria. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. mailto:eedris888@yahoo.com mailto:inabdullahi@abu.edu.ng ina. j. med. lab. sci. tech. 2021; 3(2): 99–108 1 0 0 amos dangana, et al. introduction pregnancy is a physiological condition and usually does not affect the general health of the pregnant woman. however, pregnancy results in hormonal, hemodynamic and haematological changes (1). these physiological changes need to be considered normal adaptations. increased total blood volume and hemostatic changes help to combat possible consequences of haemorrhage at delivery (2). it is often argued that altered hemostatic status is required, as the maternal coagulation system prepares for the challenges of parturition, and aims to minimize intrapartum blood loss. however, the alterations in the hemostatic system begin as early as the first trimester, suggesting a requirement for such changes in the proper progression of the early stages of pregnancy, in addition to their role in the regulation of post-partum bleeding (3). for instance, alterations in hemostasis enable the necessary changes in the uteroplacental vasculature to support the establishment of the trophoblast invasion of the spiral arteries of the uterus early in gestations (4). the altered hemostatic status during normal pregnancy presents several physiological challenges in the vasculature and results in an increased risk of excessive thrombosis, especially within the uteroplacental circulation (5). this enhanced pregnancy-associated thrombotic risk may provide the mechanistic basis for many of the major pregnancy complications, such as preeclampsia, hellp syndrome (hemolysis, elevated liver enzymes and low platelet count) and intrauterine growth retardation (iugr) (6). assessing platelet count in pregnancy is of clinical importance. it is useful in making a diagnosis of hellp syndrome (7). the hellp syndrome can often be a complication of pre-eclampsia and eclampsia, determining the severity of these clinical disorders and assessing response level to treatment in hellp syndrome (8). pregnancy is associated with endothelial stress and increased platelet aggregation in the uteroplacental circulation resulting in a progressive decline in platelet count with increasing gestational age (9). furthermore, the increase in plasma volume associated with pregnancy results in dilutional thrombocytopenia (10). thus, platelet counts are generally lower in pregnancy compared to non-pregnant women, and thrombocytopenia could occur in about 10% of pregnancies (11). rarely, this may be severe enough to cause maternal and neonatal morbidity and mortality. the elevated platelet aggregation in pregnancy has been attributed to increased formation of thromboxane a2, more intracellular calcium mobilization and reduced synthesis of cyclic adenosine monophosphate (cyclic amp) (12). with this cognizance, the current prospective study aims to determine the values of platelet ina. j. med. lab. sci. tech. 2021; 3(2): 99–108 1 0 1 amos dangana, et al. counts and indices among pregnant women attending the university of abuja teaching hospital gwagwalada abuja, nigeria. data from these could assist to also evaluate pregnant women with potential haemostatic defect and preeclampsia (mpv is useful in this regard). materials and methods study area this study was conducted at the university of abuja teaching hospital (uath), gwagwalada, federal capital territory (fct) abuja. the laboratory investigations were carried out at the haematology laboratory of uath. study design this was a case-control study on pregnant women who consented to be part of the study at the antenatal clinic at the university of abuja teaching hospital. controls were enrolled from the female medical students undergoing clinical postings and rotations in the hospital. this study took place from 20th april to 30th december 2018. selection criteria for the case group pregnant women who gave written informed consent, and healthy with no history of diabetes mellitus, coagulationrelated diseases (such as hemophilia, thalasemia), malaria, hiv/aids, hepatitis-b and c viruses were enrolled as the case subjects. their biodata alongside parity and gestational ages were extracted from their hospital folders through the assistance of the attending physicians and nurses. ethics approval ethical approval was obtained from the ethical committee of the university of abuja teaching hospital (approval number: uath / hrec / pr / 2018 / 004.018) gwagwalada, abuja, nigeria. consent to participate informed written consent was obtained from all participating subjects before recruiting into the study, following the standards of human experimentation and with the helsinki declaration of 1975. selection criteria for the control group healthy females (>18 years) who were not pregnant in the last 12 months, with no history of diabetes mellitus, coagulationrelated diseases (such as hemophilia and thalasemia), malaria, hiv/aids, hepatitis-b and c viruses were enrolled as the control subjects. they provided their biodata through a short questiionnare administered to them by nurses. analytical laboratory methods sample collection about 3 millileter (ml) of whole blood samples were collected from all the subjects and dispensed into edta (ethylenediaminen,n,n,n-tetra acetate) tube. blood samples were analysed in batches within 2 hours of collection until the required number (n=160) was attained. ina. j. med. lab. sci. tech. 2021; 3(2): 99–108 1 0 2 amos dangana, et al. platelet count and indices the full blood count was carried out using the genesis ha6000 automated hematology analyzer (calatog number: 9027809900) (perlong medical equipment company, china). among other parameters, the analyzer determined platelet count and its various platelet indices (absolute platelet count, mean platelet volume, platelet distribution width and platelet larger cell ratio). daily and per-run quality control for all the procedures for automated platelet indices analyses were ensured. statistical analysis data were presented as mean and standard deviation on the statistical package for social science (spss version 26) and analyzed using student’s t-test and anova for a significant difference in platelet indices between groups. also, two tailed chi squared test was used to determine association between 2 catigorical variables. probability (p<0.05) was used to determine the level of significance for all the statistical analyses. results in this study, one hundred and eighty (180) participants who met the inclusion criteria were successfully enrolled. among which 120 were pregnant women (case group) and 60 non-pregnant women (control group). the demographic data obtained for the subjects showed that the most of the control group aged within 21 to 25 years (41.4%) while the majority of the pregnant women (43.2%) were within 26-30 years. majority of the control group (58.6%) had never been pregnant, whereas (28.0%) of the case group had been pregnant at least once (table 1). there was a statistically significant difference in age and parity, between the cases and the control groups (p<0.0001) (table 1). although, there was an increase in mean platelet count (plt) in the pregnant women when compared with the non-pregant group (226.54± 69.76 x109 cell/l) versus 214.95±52.22x 109 cell/l), the mpv, pdw, and platelet larger cell ratio were slightly lower in pregnant women than the nonpregnant group. these changes were not statistically different between the 2 study groups (p˃0.05) (table 2). from our findings, the mean plt count in the first, second and third trimester of pregnancy (228.64± 45.25 x 109 cell/l, 237.38± 82.39 x 109 cell/l and 212.48± 61.08 x 109 cell/l) did not differ significantly from the mean plt count of the nonpregannt women (214.95± 52.22 x 109 cell/l) (p=0.184) (table 3). moreover, the mean mpv of pregnant women were lowest in 3rd trimester and significantly differ from pregnant women in their 1st, 2nd trimester and the control group (p=0.036) (table 3). the mean mean platelet volume (mpv) in the first, second and third trimester of pregnancy (10.41± 0.83 fl, 10.33± 0.92fl ina. j. med. lab. sci. tech. 2021; 3(2): 99–108 1 0 3 amos dangana, et al. and 8.73± 5.70fl) differed significantly from the mean mpv of the non-pregannt women (10.27± 1.81fl) (p=0.036) (table 3). after post-hoc test, the significant difference was between the pregnant women in 3rd trimester and the control group (p=0.014) (table 4). however, there was no significant differences in the platelet distribution width and platelet larger cell ratios among pregnant women of all the three trimesters and control groups (p>0.05) (table 3). out of the the 180 subjects in this study, 105 (89.0%) and 53 (87.9%) of the case and control groups had normal platelet counts (150-400 x 109 cells/l), respectively. moreover, 15 (11.0%) and 7 (12.1%) of the case and control groups had mild thrombocytopenia (100-150 x 109 cells/l), respectively. none of the 2 study groups had moderate and severe thrombocytopenia. also, there was no significant association between pregnancy status and thrombocytopenia (p=0.836) (table 5). table 1. association between age, parity and pregnancy status of study participants variables pregnancy status x 2 p-value pregnant, n (%) non-pregnant, n (%) age (years) 15-20 0 (0.0%) 13 (19.0%) 52.125 <0.0001* 21-25 16 (11.9%) 24 (41.4%) 26-30 51 (43.2%) 14 (24.1%) 31-35 39 (33.1%) 5 (8.6%) 36-40 14 (11.9%) 4 (6.9%) parity 0 0 (0.0%) 34 (58.6%) 52.125 <0.0001* 1 33 (28.0%) 14 (24.1%) 2 30 (25.4%) 6 (10.3%) 3 27 (22.9%) 1 (1.7%) ≥4 28 (23.7%) 3 (5.2%) table 2. comparison of platelet indices between non-pregnant and pregnant women platelet indices non-pregnant women (n=60) pregnant women (n=120) mean difference t value p-value platelet count (x 109 cells/l) 214.95 ± 52.22 226.54 ± 69.76 -11.59 ±17.54 -1.119 0.295 mean platelet volume (fl) 10.27 ± 1.81 9.74 ± 3.63 0.53 ±1.82 1.046 0.297 platelet distribution width 13.60 ± 2.07 13.44 ± 3.60 0.16 ±1.53 0.320 0.749 platelet larger cell ratio 29.61 ± 7.65 28.38 ± 8.33 1.23 ±0.68 0.972 0.333 ina. j. med. lab. sci. tech. 2021; 3(2): 99–108 1 0 4 amos dangana, et al. table 3. comparison of platelet indices between non-pregnant and the various trimesters of pregnant women platelet indices non-pregnant state (n=60) first trimester (n=24) second trimester (n=52) third trimester (n=44) f value p-value platelet count (x 109 cells/l) 214.95± 52.22 228.64± 45.25 237.38± 82.39 212.48± 61.08 1.632 0.184 mean platelet volume (fl) 10.27± 1.81 10.41± 0.83 10.33± 0.92 8.73± 5.70 2.913 0.036* platelet distribution width 13.60± 2.07 13.28± 2.17 13.48± 3.29 13.45± 4.45 0.056 0.983 platelet larger cell ratio 29.61± 7.65 28.29± 7.74 27.90± 6.89 29.00± 10.12 0.440 0.725 table 4. comparison of mean platelet volume between non-pregnant and the various trimesters of pregnant women using the least significant difference (lsd) post hoc parameter groups mean difference p-value non-pregnant state first trimester mean platelet volume (fl) 10.27± 1.81 10.41± 0.83 -0.14± 0.79 0.861 non-pregnant state second trimester mean platelet volume (fl) 10.27± 1.81 10.33± 0.92 -0.06± 0.59 0.917 non-pregnant state third trimester mean platelet volume (fl) 10.27± 1.81 8.73± 5.70 1.55± 0.62 0.014* table 5. incidence and severity of thrombocytopenia in non-pregnant and pregnant women variables pregnancy status x2 p-value pregnant, n (%) non-pregnant, n (%) platelet count normal (150-400 x 109 cells/l) 105 (89.0%) 53 (87.9%) 0.043 0.836 mild (100-<150 x 109 cells/l) 15 (11.0%) 7 (12.1%) moderate (50-<100 x109cells/l) 0 (0.0%) 0 (0.0%) severe (<50 x 109 cells/l) 0 (0.0%) 0 (0.0%) discussion platelet indices studies in pregnancy have become of great interest owing to the recurrent hypercoagulability crisis accompanied by pregnancy. this study showed that there are changes in the various platelet indices in pregnancy and the mean values of these parameters differ from the control group (non-pregnant women). however, the absolute platelet count (plt) did not significantly change in pregnant women compared to the contril subjects. this is not in agreement with an earlier study by boehlen et al. (13) where significant platelet counts decrease was reported among pregnant women. physiological changes in haematological parameters during pregnancy was reported to have significantly decreased the platelet count in pregnant women (13). our findings were also not similar to those of fahmi et al. (14) where they noted that thrombocytopenia was caused by either increased platelet destruction or decreased platelet production. in pregnancy, increased platelet destruction may be mediated by ina. j. med. lab. sci. tech. 2021; 3(2): 99–108 1 0 5 amos dangana, et al. immunological mechanisms, abnormal platelet activation, or platelet consumption. increased destruction or utilization of platelets during pregnancy occurs in microangiopathies (affecting small blood vessels) such as thrombotic thrombocytopenic purpura, haemolytic uraemic syndrome, haemolysis, elevated liver enzymes, low platelet (hellp) syndrome, and pre-eclampsia (14). another study agrees with the physiologic findings in pregnancy where platelet counts decrease possibly due to haemodilution, which majorly occurs in the third trimester (15). however, the difference in platelet count reported in our study compared to others could be attributed to the nature of sampling techniques or variation in laboratory analytical protocols, as these might have impacted on the results (16). indeed, there are various hematology analyzers with different technologies and principles of operations (such as the impedance, optical methods and immunofluorescence) current used to count the platelets. besides, platelet counts are seldom subjected to variations due to artifacts that needed to be ruled out before validation of protocol (16). however, the 5-part automated hematology analyser used in our study is insignifcanly affected by these factors as it uses the latest hematology analysis technology with the best performance caharcteristics (17). these data emphasize the need for additional studies to accurately describe the course of platelet counts throughout normal pregnancies, to document the period platelet counts begin to decrease, and to determine the range of platelet counts among normal women during pregnancy. findings from this study showed nonsignificant decreased levels of mpv, pct and pdw in pregnant women compared to the control subjects (p˃0.05). these changes might be related to the blood volume expansion and hemodilution that occurs during pregnancy. during pregnancy, platelet count decreases gradually from the 1st till the 3rd trimester. in addition, hemodilution has been shown to accelerate platelet consumption which may contribute to a decline in platelet count throughout gestation (9). interestingly, platelet activation can occur several weeks prior to the clinical onset of pre-eclampsia and increased mean platelet volume (mpv) in the late first trimester of gestation are suggestive of iugr and pe (9). our findings was similar to an earlier study by babah et al. (18), where decreased mpv and pct in pregnancy was highlighted (18). findings from our study are also in consonance with that of nooh and abdeldayem (19) where they also reported a decrease in the level of mpv, pct and pdw during pregnancy. findings from this revealed an increase in the platelet count in the first and second ina. j. med. lab. sci. tech. 2021; 3(2): 99–108 1 0 6 amos dangana, et al. trimester of pregnancy. however, there was a decrease in platelet count among subject in their 3rd trimester when compared with the control group. these are similar to the findings by babah et al. (18) where they reported a statistically non-significant increase in platelet count in the first to the third trimester of pregnancy. these observations could be attributed to the feeding habit of the subject (7). it is known that supplements or diet rich in vitamins, iron and folate could enhance erythropoietic activities in the bone marrow, which could assist in boosting platelet counts during pregnancy (20). in most lowand middleincome settings, pregnant women consistent consumption of iron/folate tablets reduces with gestation age (poor adherence) (21, 22). nevertheless, the progressive fall in platelet count with an increase in gestation did not cause thrombocytopenia in all trimesters of pregnancy as the values were within the normal range (150–450 × 109/l). there was a significant increase in mean platelet volume among subjects in the first and second trimester when compared with the control group. but a significant decrease was observed in the third trimester when compared with the control group. mean platelet volume (mpv) and platelet distribution width (pdw) were reported to increase during platelet activation (23). essentially, the decline in the third trimester could be attributed to the physiologic haemodilution. in normal pregnancy, there is often an increase in platelet aggregation and a decrease in the number of circulating platelets with gestation (24). as the platelet lifespan decreases, the mpv increases minimally during pregnancy (24). thus, the mpv is an accurate measure of platelet size and its considered a biomarker of platelet function (25). hence, larger platelets with higher mpv counts are reactive and raise higher amounts of the prothrombotic factor thromboxane a2, increasing the tendency to thrombosis (25). the pdw values appeared to have reduced in the three groups of pregnant women, which were statistically nonsignificant. on the contrary, an increase in pdw was reported by omorogiuwa and aigborhuan (26), which was attributed to physiologic compensation for the decreasing platelet count and volume with progression in gestation age (26). platelets having denser granules are bigger and metabolically active (27), hence pdw is more specific than mpv in the identification of platelet activity. it is a simple and specific marker for enhanced coagulation (23). there was a statistically non-significant increase in the mean of mean± sd of mpv in the first and second trimester among the case group when compared with the mean± sd value of the control group. however, in the ina. j. med. lab. sci. tech. 2021; 3(2): 99–108 1 0 7 amos dangana, et al. third trimester, there was a significant decrease in mpv among pregnant women. on the contrary, a longitudinal study by giles (28) showed that mpv increased with gestational age in a statistically nonsignificant manner. the decline in mpv in the third trimester could be attributed to platelet consumption and hemodilution, conditions common in the third trimester. findings from this study showed mild thrombocytopenia among the pregnant women when compared with the control group. this explains the normal physiologic changes in pregnancy where hemodilution effects overwhelm the erythropoietic activities of the bone marrow (30). however, pregnant women with thrombocytopenia may be at a higher risk of coagulopathy. conclusion this study is not without limitation. the heterogenous nature of the biodata (especially age) of the case and control groups might signify a bias in subjects selection which could influence the statistics of some analytes. also, the concurrent analyses of certain platelet factors (such as fibrinogen and von willebrand) would have provided more information about the coagulation profile of the pregnant women with low platelet counts and mpv. it is worthy to follow up the pregnant women and neonates with higer risk of developing coagulation complication so that approparite therapeutic interventions could be made available to arrest possible complications. author contributions amos dangana: conceptualization, formal analysis, investigation, supervision, writing original draft, writing review & editing. anthony uchenna emeribe: formal analysis, methodology, writing original draft, writing review & editing. hezekiah alkali isah: writing original draft, writing review & editing. sanusi musa: writing original draft, writing review & editing. joel monday abu: writing original draft, writing review & editing. solomon oloche onoja: writing original draft, writing review & editing. nkechi onukegbe: writing original draft, writing review & editing. idris nasir abdullahi: conceptualization, formal analysis, investigation, methodology, supervision, writing original draft, writing review & editing. conflict of interest none declared by authors. references 1. capasso g, unwin r. electrolytes and acid–base: common fluid and electrolyte disorders. medicine (baltimore) [internet]. 2011 jun;39(6):317–24. available from: 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santosa b, sukeksi a. difference in electrolyte levels (na, k, cl) in samples immediately and delayed by 150 minutes. repository.unimus.ac.id. 2018. 25. azizah n, aliviameita a. effect of delay of serum examination on electrolyte levels of sodium and chloride. j med lab sci technol. 2019; 26. an b, park c-e. evaluation of stability of serum on different storage temperatures for routine chemistry analytes. korean j clin lab sci. 2014; 27. departemen kesehatan ri. pedoman praktek laboratorium yang benar (good laboratory practice). jakarta; 2008. 72 retraction notice to ‘‘n-terminal pro-brain natriuretic peptide (nt-probnp) in stage 1 and stage 2 hypertension patients” [ina. j. med. lab. sci. tech. 2019;1(2): 64-67] supriati wila djami1 1department of health analyst, health ministry polytechnic of kupang, indonesia correspondence: supriati wila djami, jl.farmasi, liliba, kupang, east nusa tenggara, indonesia zip code: 85111 email: djamiwila@gmail.com doi: 10.33086/ijmlst.v3i1.2061 this article has been retracted at the request of the editor in chief of the indonesian journal of medical laboratoy science and technology (ijmlst) volume 1 number 2 of 2020 pages 65-67 with doi: 10.33086/ijmlst.v1i2.1159 because it has been published in the journal of health information volume 17 number 1 year 2019 pages 64-74 with doi: 10.31965/infokes.vol17.iss1.232. papers submitted to ijmlst should neither published previously nor be under consideration for publication another journal. therefore, the author should make a statement upon submitting. reusing data in any form must follow good citation principles, therefore this paper has ethical violations in the scientific publishing system. https://journal2.unusa.ac.id/index.php/ijmlst/article/view/2061/version/2545 https://journal2.unusa.ac.id/index.php/ijmlst/article/view/1159 http://jurnal.poltekeskupang.ac.id/index.php/infokes/article/view/232 45 spermatozoa morphology examination using lenshooketm sqa x1 pro compared with manual method adhipireno purwanto1, purwanto edy2,3, suyono seso sulijaya4, ismawatie emma1,5, santoso budi6, limijadi edward kurnia setiawan3 1clinical pathology laboratory department of central general hospital dr. kariadi semarang, semarang, central jawa, indonesia 2master program of medical laboratory science/clinical, faculty of nursing and health sciences, university of muhammadiyah semarang, semarang, central jawa, indonesia 3islamic hospital fatimah cilacap, central jawa, indonesia 4gladiool ivf centre, magelang city, central jawa, indonesia 5clinical pathology laboratory department of mother and child hospital restu ibu sragen, central jawa, indonesia 6muhammadiyah semarang university, central jawa, indonesia correspondence: purwanto edy, jl. kedungmundu no.18, kedungmundu, kec. tembalang, kota semarang, semarang, central java zip code: 50273 email: ichtiar9@yahoo.co.id received: april 17, 2021 revised: januari 1, 2022 accepted: february 22, 2022 published: april 28, 2022 doi: 10.33086/ijmlst.v4i1.2059 abstract according to world health organization data, 30-40% of infertility is caused by male factors. the morphology of normal spermatozoa is an indicator of male fertility, and it is known by manual or automatic sperm analysis. lenshooketm sqa x1 pro automatic equipment comes along with the development of laboratory equipment automation technology. the working principle of this tool is by shining light on the object of examination, then the camera with high resolution, with the facility of an optical lens will take a picture of the object. the database recorded by the camera is analyzed by the algorithm. the research objective was to test the suitability of the lenshooketm sqa x1 pro automatic tool with manual method as the gold standard. subjects in this study were patients who carried out semen analysis tests at the clinical pathology laboratory of rsia "restu ibu" sragen from june to august 2020. the examination method used an automatic method with the lenshooketm sqa x1 pro tool and a manual method with papanicolaou staining. the results of the study, conformity test with who 2010 normal standards, automatic methods reached 94.4% compared to manual methods. the next statistical test was with standard mean, normal sperm morphology data had a significance of 0.001, abnormal sperm head data had a significance value of 0.956 and abnormal sperm tail data had a significance value of 0.339. the lenshooketm sqa xi pro device based on automatic technology can be used in laboratory services for sperm analysis in addition to manual methods. suggestions for using the lenshooketm xi pro automatic tools are still accompanied by the manual method. keywords automatic, microscope, morphology, spermatozoa. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. 59 adhipireno purwanto, et all. introduction the morphology of spermatozoa refers to the whole form of spermatozoa cells consisting of the head, middle and tail (1). spermatozoa cells form in the seminal tubules that are in the testes. this tubule contains a complex series of cells, namely the development or division of cells from the germinal to the formation of spermatozoa or male gametes (2). about 300 million spermatozoa carry out the process of spermatogenesis mean in one day there are approximately 300 million spermatozoa newly manufactured (3). according to data from the world health organization (who), 30 – 40% of infertility is caused by male factors, hence it is important to evaluate fertility of men as part of routine check. the basic test for infertility by performing semen analysis is the most commonly used diagnostic option. the result of semen analysis of 25% of infertile men was asthenozoospermia (abnormality of spermatozoa movement). the rest are disturbances in number (oligozoospermia) and morphology (teratozoospermia) or a combination of the three (4). semen analysis includes macroscopic and microscopic examinations. microscopic analysis of normal semen results from parameters of concentration, motility and morphology to determine fertility in men. normal spermatozoa cell morphology as a clinical tool for male fertility (5). morphological abnormalities can make it difficult for spermatozoa to fertilize an egg (6). spermatozoa show tremendous variability in size and shape. there is a correlation between the morphology of spermatozoa and reaching the female reproductive tract during the fertilization process (7). analysis of semen in manual method is the gold standard (who), which is widely used in laboratory of clinical pathology. this method has the advantage and disadvantage. some of the findings in the field for the shortcomings of the manual method of semen analysis include that there are still differences in the interpretation of the results of inter laboratory examinations. the analysis process takes a relatively long time between 30 to 60 minutes, and the equipment used does not have the same standard (4). the morphological examination of spermatozoa using an automatic method is present amidst the need for semen analysis. the development of accessible, fast and standard methods for semen analysis is urgently needed. the automated method provides a solution for semen analysis checks with fast results and good quality control standards (4). semen analysis using automated with computer-based method to cover the shortage of existing shortcomings in the semen analysis manual methods (8). the basis for the authors’ consideration as medical laboratory technologist experts ina. j. med. lab. sci. tech. 2022; 4(1): 45–59 adhipireno purwanto, et all. 4 7 in conducting research entitled spermatozoa morphological examination using the lenshooketm sqa x1 pro tool compared to the manual method is because the authors still find that there are differences in the interpretation of semen analysis results between one laboratory and another. some literature also states that manual semen analysis is a simple and inexpensive test, but has high variability and is very subjective (9). automatic method selections for spemartozoa analysis may use the lenshooketm sqa x1 pro. this tool consists of software and hardware and the technical analysis works automatically (10). this is very practical and simple tool, which has four key parameters for evaluating male fertility, namely concentration, morphological, motility and ph. lenshooketm sqa x1 pro yields very fast examination results which only take 3 to 5 minutes to get all the test results. good quality control would be giving results in a accurate and reliable (11). as a comparison, the researchers conducted a manual morphological examination of spermatozoa using papanicolaou stain because this is one of the who gold standards for spermatozoa morphological staining (12). the aim of this study was to evaluate the spermatozoa morphology using lenshooketm sqa x1 pro compared with manual method in order to provides reliable results. materials and methods morphology of spermatozoa were grouped into normal and abnormal morphology (5). sampling was performed by using a consecutive sampling, that is sampling technique to assign subjects that met the study criteria. samples were taken from patients who visited the clinical pathology laboratory of restu ibu hospital, sragen, indonesia from june 2020 to august 2020. the number of samples in this study was 48 patients, which are consist of the 36 patients who met the criteria for the study. the inclusion criteria in this study were men in reproductive age between 20 to 45 years old and with spermatozoa concentration in excess of 2 million/ml. exclusion criteria were semen samples with blood mixture, abstinence more than 7 days, liquefaction more than 60 minutes, increasing number of leukocyte in semen, and the number of immature spermatozoa cells. sperm fluid release the release of sperm fluid for good results is by masturbating without using tools, such as gels, detergents and others. sampling through sexual intercourse is not recommended. if circumstances compel sampling by sexual intercourse then use condoms and lubricants that are non-toxic and fertility-friendly if necessary. collect complete sperm fluid, especially the first fraction rich in sperm. 59 adhipireno purwanto, et all. sample handling prepare two sample pots, a and b, each with a patient identification label. the sample is accommodated in a clean, dry and widemouthed sample pot. the research sample for one identity is divided into two in the sample pot a and the sample pot b. sample pot a for inspection of the lenshoke sqa x1 pro automatic method tool. sample pot b for manual method examination with papanicolaou stain. each sample pot is done at the same time using two different inspection methods. to maintain the quality of the sample in the specimen pot, the temperature is kept around 20 – 37°c. manual method in the manual method, papanicolau dye was used and considered that this dye is one of the dyes recommended by who. staining papanicolaou gives the results of the examination both for the morphology of spermatozoa and other cells. papanicolaou staining has been proven and recommended by the who (12). polychromatic staining is considered a very reliable staining technique. factors that affect the coloring in addition to the use of dye solution, the time of painting, the duration of immersion, rinsing and immersion currents follow the standards that have been set. figure 1 shows slides stained using the papanicolaou procedure. this stain can be permanently installed and stored for use as internal quality control (13). the principle of the staining of papanicolau the harirs's haematokxylin dye stains the cell nucleus blue, orange g and ea 50 alcohol-based green coloring will work to color the cytoplasma. ethanol 50% 80% 95% 100% for fixation and make cells become dehydrated and ethanol acid removes dyes undesirable but still attached especially to the cytoplasmic area. water rehydrates cells (13). procedure for manual method shows in figure 1. figure 1. preparation; 5-20 µl of semen is dripped on the object glass. (a) move the slide to another object to make an erase. (b) dry in the air 5-15 minutes (who, 2010). papanicolaou coloring soak the dried slides sequentially on ethanol 80% for 30 seconds, continue to ethanol 50% 30 seconds and soak in pure water for 30 second. put into harris hematoxylin stain for 4 minutes, then into pure water 30 seconds, put into ethanol acid for 8 seconds, flux with cold tap water for 5 minutes, and then into alcohol 50% for seconds, and 80% for 30 seconds, then dip (a) (b) ina. j. med. lab. sci. tech. 2022; 4(1): 45–59 adhipireno purwanto, et all. 4 9 into ethanol 95% for 15 minutes. stain with g-6 orange dye for 1 minute, and dip repeatedly into ethanol 95% for three times with 30 seconds each. stain with ea-50 green dye for 1 minute, and then rinse with ethanol 95% two times with 30 second each. final clearing rinse with ethanol 100% two times for 15 second each. figure 2. morphology of spermatozoa with papanicolau dye. (a) showing spermatozoa with amorphous head with thickened midpiece. (b) round head (14). result reading morphology reading is the one semen analysis parameter in medical labotatory.the slides then read on a microscope with at least two technical officers. using 1,000 times magnification assisted with immersion oil, observe the morphology of normal and abnormal spermatozoa. report the percentage of observations of spermatozoa morphology as a result of the study (figure 2). the morphology of spermatozoa includes the assessment of the head, neck, middle and tail. the normal form of spermatozoa is a tadpole which consists of a blunt head in which there is a nucleus, and has a tail that contains an apparatus for moving. morphologically abnormal spermatozoa are categorized into subgroups according to defects in the head, neck, midsection and tail (2). several recent studies have demonstrated the importance of assessing the morphology of abnormal spermatozoa more carefully to establish the diagnosis of infertility (13). head normal the head of the sperm cell is oval with a size of 3 – 5 µm, there is a cell nucleus (nucleus) containing genetic information in the form of dna in it. this genetic information will meet the genetic information from the egg and will determine whether the fetus is male or female. in the head of the spermatozoa, there are also enzymes, such as the hyaluronidase enzyme, which functions to penetrate the corona layer above the ovum, and protease enzymes (18). a b 59 adhipireno purwanto, et all. neck normal neck is the area just behind the head that contains the centrioles. the middle part contains mitochondria arranged in a spiral, which contains energy (atp) as an energy source for spermatozoa, for locomotion to the site of fertilization and for spermatozoa metabolism (3). tail the tail of the spermatozoa is long with a size of 50 µm divided into the neck, the main/middle and the end (19). the main part is the longest part of the tail, and the end is the pointed end of the tail. the tail of the spermatozoa is in the form of flagella as a means of locomotion in the form of a long cytoskeleton that functions to propel the spermatozoa forward, at a speed of 30 inches/hour (18). abnormal abnormal is an abnormal form of spermatozoa. morphologically abnormal spermatozoa are categorized into subgroups according to defects in the head, neck, midsection and tail (1). several recent studies have demonstrated the importance of assessing the morphology of abnormal spermatozoa more carefully to establish the diagnosis of infertility (13). term the results of semen analysis used to describe the morphological abnormalities of spermatozoa shows in figure 3.. figure 3. abnormal spermatozoa morphology (5). head large or small, tapering, bulb-shaped / pyriform, amorphous, hollow >20% of the head area is occupied by a cavity that is not stained, the head is broad and a combination of the above. midpiece midpiece defects includes tailless spermatozoa that appear as free heads, or loose heads, uninserted tails or swollen/irregular central bent tails. abnormally thin midsection e.g. no ina. j. med. lab. sci. tech. 2022; 4(1): 45–59 adhipireno purwanto, et all. 5 1 mitochondrial sheath or various combinations of these abnormalities. tail short tails, double tails, shaped like hairpins, broken, curled tails with drips at the ends, or a combination of these abnormalities. automatic method the tool used is the lenshooketm sqa x1 pro, a product from bonraybio, a device that works automatically for human semen analysis by integrating mechanical, optical, electronic and algorithmic technologies. with the use of this semen quality analyzer, it will facilitate and improve performance in the laboratory so that work is more efficient. semen analysis using equipment equipped with a computer is an automated method that can provide objective and precise information about the characteristics of semen samples, such as morphology, concentration, and motility (16). quality control of the cement quality analyzer can be standardized for each tool, so as to minimize differences in the results of cement analysis between laboratories. semen analysis in combination with computer technology has evolved over the past 40 years, through advances in devices for capturing images from microscopes, massive increases in computing power along with tremendous reductions in computer size, new computer languages, and updated software algorithms (17). in this study, the automatic tool used is the lenshooketm sqa x1 pro, a product from bonraybio, a device that works automatically for human semen analysis by integrating mechanical, optical, electronic and algorithmic technologies. the working principle of the lenshooketm sqa x1 pro tool is with a beam of light on the object of the examination, and then a high-resolution camera, with an optical lens facility, will take pictures of the object. furthermore, the clinical database that has been recorded by the camera is analyzed for calculations with the algorithm (15). the initial rare work procedure prepares the sample by putting this sperm collection device for the semen quality analyzer using a special consumable cup test, and then wait 30 to 60 minutes for sperm to thaw. homogenized the sample in the cup by turning it back and forth 8 – 10 times, check the color and volume of the sperm sample (figure 4-5). figure 4. how to operate the lenshooketm sqa x1 pro appliance (11). 59 adhipireno purwanto, et all. figure 5. display results on the lenshooketm sqa x1 pro tool showed ph of the semen, total spermatozoa in million per mililiters semen, total moved of the spermatozoa, and amount of the total normal morphology of the spermatozoa (11). spermatozoa morphology parameters that emerged from readings using the lenshoketm sqa x1 pro device were normal morphology with units of percent. abnormalities in sperm size and morphology assessment includes the percentage of head length, head width, head circumference, head area and tail length (15). results conformity test the suitability of the results of the automatic method of spermatozoa morphology examination with manual method of spermatozoa morphology using the 2 x 2 contingency table and the chi square test. the characteristic and examination result are shown in table 1 and 2. referring to who 2010 standard value of normal spermatozoa morphology 4%. the conformity test used a 2 x 2 contingency table from normal morphological data (table 3). the results of this study could not be analyzed. because of the 36 samples tested by the manual method, all showed normal results. this results in abnormal morphological data of 0. the agreement that can be made with the 2010 who standard normal value ≥ 4% is by looking at the percentage of automatic normal results compared to manual reaching 94.4% and high and low yields from both methods illustrate that the average the automatic method shows that the normal results are lower than the manual method shown as 83.3%. clinical based suitability according to the 2010 revision of who guidelines, men with normal sperm cell ina. j. med. lab. sci. tech. 2022; 4(1): 45–59 adhipireno purwanto, et all. 5 3 morphology is ≥4%. this is observed by namely by macroscopic and microscopic assessment of the semen (table 4-5). the sperm concentrationof normal results automatically compared to manual, reaching 94.4% and the high and low results of the two methods illustrating that the average automatic method results show more normal results lower than the method. manual, namely 83.3%, as shown in table 6. table 1. subject characteristics age (year) ʃ sampling technique ʃ abstinence (day) ʃ 21 30 23 masturbation 48 <2 0 3140 20 coitus interruptus 0 2 – 7 46 > 40 5 special condoms 0 >7 2 amount 48 48 table 2. exclusion research samples description ʃ abstinence for more than 7 days 2 the sample is red / mixed with blood 1 liquifaction more than 60 minutes 2 the concentration of spermatozoa is less than 2 million 6 reagent cassette damaged reading part is subject to hand grease 1 amount 12 statistical based conformity conformity standard mean between automatic normal morphology and manual normal morphology table 3. contingency 2x2 mean normal spermatozoa morphology automatic normal mean * manual normal mean mean normal manual total positive negative automatic normal mean positive count 10 1 11 % of total 27.8% 2.8% 30.6% negative count 7 18 25 % of total 19.4% 50.0% 69.4% total count 17 19 36 % of total 47.2% 52.8% 100.0% testing the results of research based on statistics using the standard mean value of the data; normal morphology automatic method and normal morphology manual method. abnormal head area automatic method and abnormal head area manual method abnormal tail length automatic method with abnormal tail length manual method. 59 adhipireno purwanto, et all. a. sensitivity = 10/17 = 0.588% b. specificity = 18/19 = 0.947% c. positive prediction value = 10/11 = 0.909% d. negative prediction value = 18/25 = 0.720% table 4. suitability test for the mean morphology of normal spermatozoa value df asymp. sig. (2-sided) exact sig. (2-sided) exact sig. (1-sided) pearson chi-square 12,130 a 1 0.000 continuity correction 9,737 1 0.002 likelihood ratio 13,446 1 0.000 fisher's exact test 0.001 0.001 linear-by-linear association 11,793 1 0.001 n of valid cases 36 the suitability test is based on the calculation of the mean morphological value of spermatozoa with significance 0.001 <0.05. it can be concluded that h0 is rejected and h1 is accepted, which means that by means of statistical analysis, there is a difference between automatic spermatozoa morphology and manual spermatozoa morphology (table 7). standard conformance mean between automatic abnormal head area and manual abnormal head spermatozoa with slightly abnormal 'borderline' heads are classified as abnormal. the suitability test was based on the calculation of the mean morphology of abnormal spermatozoa head with a significance of 0.956 > 0.05 (p < 0.05; r = 0.10). then by means of statistical analysis, we can observe that h0 is accepted and h1 is rejected, which means there is no difference between the calculation of abnormal morphology of spermatozoa head automatic method with abnormal sperm morphology head manual methods. table 5. contingency 2 x 2 mean abnormal head morphology mean head area * mean head manual mean head manual total positive negative mean head area positive count 7 10 17 % of total 19.4% 27.8% 47.2% negative count 8 11 19 % of total 22.2% 30.6% 52.8% total count 15 21 % of total 41.7% 58.3% ina. j. med. lab. sci. tech. 2022; 4(1): 45–59 adhipireno purwanto, et all. 5 5 a. sensitivity = 7/15 = 0.467% b. specificity = 11/21 = 0.524% c. positive predicted value = 7/17 = 0.412% d. prediction value negative = 11 / 19 = 0,579% table 6. suitability test for mean morphology of normal spermatozoa value df asymp. sig. (2-sided) exact sig (2-sided) exact sig. (1-sided) pearson chi-square 0.003 1 0.955 continuity correction 0.000 1 1,000 likelihood ratio 0.003 1 0.955 fisher's exact test 1,000 0.611 linear-by-linear association 0.003 1 0.956 n of valid cases 36 the suitability test was based on the calculation of the mean morphological value of abnormal spermatozoa tail with a significance value of 0.339 > 0.05. hence, by means of statistical analysis, it can be concluded that h0 is accepted and h1 is rejected, which means that there is no difference between the calculations of the abnormal spermatozoa tail morphology of the automatic method. standard conformance mean between automatic abnormal tail and manual abnormal tail table 7. contingency 2 x 2 mean abnormal morphology of tail automatic tail mean * manual tail mean manual mean equipment total positive negative automatic tail means positive count 8 9 17 % of total 22.2% 25.0% 47.2% negative count 12 7 19 % of total 33.3% 19.4% 52.8% total count 20 16 36 % of total 55.6% 44.4% 100.0% a. sensitivity = 8/20 = 0.400% b. specificity = 7/16 = 0.438% c. positive prediction value = 0.471% d. negative prediction value = 7/19 = 0.368% 59 adhipireno purwanto, et all. table 8. contingency 2 x 2 mean abnormal morphology of tail value df asymp. sig. (2-sided) exact sig. (2-sided) exact sig. (1-sided) pearson chi-square 0.942 1 0.332 continuity correction 0.403 1 0.526 likelihood ratio 0.945 1 0.331 fisher's exact test 0.503 0.263 linear-by-linear association 0.916 1 0.339 n of valid cases 36 discussion this research was conducted at the clinical pathology laboratory of mother and child hospital "restu ibu" sragen from june 2020 to august 2020. researchers carry out the rules of practice in the laboratory examination stages as should the preanalytic, analytic and post-analytic. overall in this study, researchers conducted 48 patient samples, of which 12 patient samples were included in the exclusion criteria. 36 patient samples entered the inclusion criteria according to the needs of the sample in this study. the sample characteristics of the 36 samples studied showed as many as 20. at the stage of carrying out the analysis using the leenshoke sqa x1pro automatic tool, there are several things that need to be considered to get the results according to the patient's clinical condition, namely: measure the use of electricity used for automatic devices is stable, perfect sample homogeneity, the cleanliness of the cassette in the reading lens area must be completely clean, free of grease and dirt, cross checking reads using the manual method on samples with low spermatozoa concentrations. because at concentrations below 2 million spermatozoa readings on the automatic instrument will be read with a result <1 for concentration, morphology and motility, and then immediately remove the cassette from the instrument after the reading is complete. in the suitability test of normal morphological results with automatic methods compared to manual methods as gold standard, using 2x2 contingency tables to see sensitivity, specificity, normal predictive values and abnormal predictive values. with the normal standards set by who 2010, cannot be tested with 2x2 contingency tables. because the abnormal value in the manual method is 0. all results from the 36 samples from the manual method fall within the normal value range of ≥4 million/ml. automatic morphological comparisons that can be done clinically with who 2010 standards by looking at the number of normal results achieved by the automatic method of the 36 samples studied. the achievement was 34 samples entered the normal range. this means that this automatic achievement ina. j. med. lab. sci. tech. 2022; 4(1): 45–59 adhipireno purwanto, et all. 5 7 compared to the manual method reaches 94.4%. the normal morphological average yield of the automatic method is lower than the manual method. data shows that of the 36 samples, 83,3% lower than the manual method. because the standard normal value set by who 2010 cannot be clinically tested using a 2 x 2 contingency table, the automatic method suitability test is performed statistically by determining the standard of the mean value. the statistical test consisted of data on normal morphology, abnormal morphology of the head and abnormal morphology of the tail. after testing the three data with the standard mean as standard, the following results are obtained: a. normal morphological data shows a significance value of 0.001 < 0.005, it can be concluded that h0 is rejected and h1 is accepted, which means that there is a difference between statistical calculations for spermatozoa morphology with automatic method and manual method of spermatozoa morphology. b. data on abnormal morphology of the head had a significance value of 0.371 > 0.05. it can be concluded that h0 is accepted and h1 is rejected, which means that statistically there is no difference between the calculation of abnormal morphology of spermatozoa head automatic method with abnormal sperm morphology manual methods. c. data on abnormal morphology of tails with a significance value of 0.339> 0.05. it can be concluded that h0 is accepted and h1 is rejected, which means that statistically there is no difference between the calculation of abnormal spermatozoa tail morphology automated method with abnormal sperm morphology manual methods. the presence of an automatic tool lenshooketm sqa xi pro, has its advantages such as simple physical tools, easy operation with touch screen technology, quick reading of results that only takes 3 to 5 minutes, visualization of spermatozoa in the form of a video, focus setting and hdmi connection to the monitor can display ph, motility, concentration and morphology. disadvantages of this device are in conditions of low spermatozoa concentration, the device cannot read spermatozoa completely, and the investment costs for equipment and operational costs for reagents are quite high compared to manual ones. in the manual method with papanicolaou dye, the results of the head, tail and cytoplasmic morphology of spermatozoa were more clearly read than other dyes. tool investment costs and operating costs are lower than automated methods. the data from the research were carried out by descriptive tests with the results of the standard deviation of the morphology of normal or abnormal spermatozoa, the manual method showed the 59 adhipireno purwanto, et all. results with the same value, namely 2.253 and the standard deviation value of the morphology of normal / abnormal spermatozoa, the automatic method also showed the same results, namely 2.247. in the suitability test for normal morphology results, the automatic method is compared to the manual method as the gold standard, using a 2x2 contingency table to see the sensitivity, specificity, normal predictive value and abnormal predictive value, with the normal standard set by who 2010, cannot be tested with a 2x2 contingency table because the abnormal value in the manual method was 0. all the results from 36 samples of the manual method were within the normal value range of 4 million/ml. the automatic morphological comparison that can be done clinically with the 2010 who standard is by looking at the number of normal results achieved by the automated method. of the 36 samples studied, the achievement is that as many as 34 samples are in the normal range. this means that if the automatic achievement is compared to the manual method, it reaches 94.4%. the average yield of the normal morphology of the automatic method is lower than that of the manual method. the data shows that of the 36 samples, 83.3% lower than the manual method. conclusion spermatozoa morphology examination is important in terms of determining a man’s overall fertility potential. morphology examination take into observation multiple aspects, namely a sperm’s shape such as its head, midpiece, tail, and the presence of cytoplasmic droplets. the most common spermatozoa morphology examination in medical laboratory is microscopic observation by manual and eutomatic machine. in general, the presence of the lenshooketm sqa xipro device which is based on automatic technology is compatible with the manual method. hence, this automated tool can be used in laboratory services complementing the manual method as the gold standard. author contributions all author has equal contribution for the study conception and design, data collection, analysis and interpretation of results, and manuscript preparation. acknowledgement the authors would like to thank our colleagues of at the clinical pathology laboratory of mother and child hospital "restu ibu” sragen, their help is most grateful. we also like to thank the patients who participated in this study. conflict of interest the authors declare that there is no conflict of interest. ina. j. med. lab. sci. tech. 2022; 4(1): 45–59 adhipireno purwanto, et all. 5 9 references 1. lasiene k. assessment of human sperm cells morphological parameters. spermatozoa facts and perspectives. 2018 ; 51-62. 2. susilowati t. buku spermatology. 1st edition . malang. universitas brawijaya press. 2011. 3-7p. 3. tortora gj, derrickson b. principles of anatomy & physiology .14 ed. america . kaye pace . 2014. 1078p. 4. agarwal a, henkel 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infertilitas laki-laki spermatozoa dna fragmentation detection and detection in male infertility. e-journal kedokteran indonesia. 2015;3(2):1–2. 10. engel km, grunewald s, schiller j, paasch u. automated semen analysis by sqa vision ® versus the manual approach—a prospective double-blind study. andrologia. 2019 ; 51(1): 110. 11. bonraybio. lenshooke xi pro semen quality analyzer. 2019; 1-4. http://bonraybio.com/en/product.php?act=view& id=8. 12. who. laboratory manual for the examination and rrocessing of human semen. 5 ed. who library. 2010. 13. auger j. spermatozoa and sperm structure. second edi. vol. 1, encyclopedia of reproduction. elsevier; 2018. 62-67 p. 14. aksoy e, aktan tm, duman s, cuce g. assessment of spermatozoa morphology under light microscopy with different histologic stains and comparison of morphometric measurements. international journal of morphology. 2012 ; 30 (4):1544-550. 15. narulita p. the effectiveness of lenshooketm semen quality analyzer x1 pro for human semen analysis. fac med univ airlangga, surabaya. 2020;1(1). 16. sugiyono. metode penelitian kuantitatif kualitatif dan r&d quantitative qualitative and r&d. graha ilmu. edisi1.bandung, alfabeta.2019. 286 292p. 17. amann rp, waberski d. computer-assisted sperm analysis (casa): capabilities and potential developments. theriogenology.2014 ; 81(1)5-17. 18. apriora vd, amir a, khairsyaf o. gambaran morfologi spermatozoa pada perokok sedang di lingkungan pe group yang datang ke bagian biologi fakultas kedokteran universitas andalas description of spermatozoa morphology in medium smokers in the pe group who came to the biology section of the faculty of medicine, andalas university. j kesehat andalas. 2015;4(2):425–9. 19. marzano g, chiriacò ms, primiceri e, dell’aquila me, ramalho-santos j, zara v, et al. sperm selection in assisted reproduction: a review of established methods and cutting-edge possibilities. biotechnology advances. 2019 ; ( 19 ) : 3. 68 incidence of hepatitis b surface antigen (hbsag) infection and its relationship with risk factors at janitors at regional general hospital dr. m. m. dunda limboto gorontalo indra elisabet lalangpuling1, zumrotul abidah dakio1, nikma2, dwi setiyo prihandono3, lendawati4 1department of medical technology laboratory, politeknik kesehatan kementerian kesehatan manado, manado, indonesia 2department of medical technology laboratory, politeknik kesehatan kementerian kesehatan ternate, ternate, indonesia 3department of medical technology laboratory, politeknik kesehatan kementerian kesehatan kalimantan timur, samarinda, indonesia 4department of medical technology laboratory, politeknik kesehatan kementerian kesehatan tanjung karang, bandar lampung, indonesia correspondence: indra elisabet lalangpuling, politeknik kesehatan kemenkes manado, jalan r.w. monginsidi malalayang ii manado, indonesia zip code: 95263 email: indra_elisabet@yahoo.com received: november 24, 2022 revised: march 30, 2023 accepted: april 17, 2023 published: april 29, 2023 doi: 10.33086/ijmlst.v5i1.3623 abstract hepatitis b is an inflammatory liver disease caused by the hepatitis b virus, which can be acute or chronic. the outer membrane protein of hepatitis b virus (hbv) is known as hepatitis b surface antigen (hbsag). janitors are employees who are tasked with cleaning the hospital environment to keep it clean, because of the the dangers that exist in hospitals, such as disease transmission, can occur if the hospital environment is not kept clean. the purpose of this study was to describe the results and their relationship to risk factors of the hepatitis b examination on janitors at the regional general hospital (rsud) dr. mansyoer mohammad dunda limboto gorontalo. this study used a descriptive method with an accidental sampling technique. the sample in this study amounted to 33 respondents who met the inclusion criteria. specimen were examined using the rapid test method. the data were presented descriptively and statistically to see the relationship between work and the incidence of hepatitis b infection. the results obtained were as many as 33 respondents by conducting an hbsag examination, with the results obtained being 2 reactive people and 31 non-reactive people. the results of statistical tests on the relationship between infection status and age showed a p-value of 0.019 and the relationship between infection status and symptoms showed a p-value of 0.002. the study’s findings revealed that there was a substansial correlation between the respondents’ reported symptoms and their infection status (6% of respondents teste for hepatitis b). keywords hbsag, janitors, rapid test method, regional general hospital dr. m. m. dunda limboto. citation: lalangpuling ie, dakio za, nikma n, prihandono ds, lendawati l. incidence of hepatitis b surface antigen (hbsag) infection and its relationship with risk factors at janitors at regional general hospital dr. m. m. dunda limboto gorontalo. indones j med lab sci technol. 2023;5(1):68–78. doi: 10.33086/ijmlst.v5i1.3623 this is an open access article distributed under the creative commons attribution-sharealike 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2023 by author. mailto:indra_elisabet@yahoo.com https://doi.org/10.33086/ijmlst.v5i1.3623 https://doi.org/10.33086/ijmlst.v5i1.3623 https://creativecommons.org/licenses/by-sa/4.0/ ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 6 9 introduction the liver is the center of the body's metabolism, which serves a variety of purposes and is critical to sustaining life. the reserve capacity is very large, with only 10-20% of liver tissue that is still functioning, it is enough to maintain the body of the owner. the ability of the liver to replace dead tissue with new tissues (regenerate) is also quite large. because of this, the removal of some diseased or damaged liver tissue is swiftly followed by the growth of new tissue. there are four different types of liver functions; the formation and excretion of bile, metabolism of substances important for the body, body defense, and vascular function (1). hepatitis b is an inflammatory liver disease caused by the hepatitis b virus (hbv), which can be acute or chronic. active chronic forms can lead to cirrhosis, liver cancer, to death. hepatitis b is difficult to recognize because the symptoms are not immediately felt and some may not appear at all. for this reason, many people do not realize that they have been infected. this virus usually takes 1-5 months from exposure to the virus until the appearance of the first symptoms appear. hepatitis b virus surface antigen (hbsag) is the outer envelope protein of hbv, and is a sign that the individual has been infected with hbv. hbsag positive can be found in healthy people (healthy carrier), acute hepatitis b, chronic hepatitis b, liver cirrhosis and primary liver cancer (2). based on the type, the causes of hepatitis are divided into two categories: infectious and non-infectious hepatitis. in noninfectious hepatitis, inflammation of the liver occurs due to non-infectious causes, such as chemicals, alcohol, and drug abuse. noninfectious hepatitis, including drug-induced hepatitis, is not classified, as an infectious disease, because the cause of hepatitis is not caused by infectious agents such as fungi, bacteria, microorganisms and viruses (3). hbv transmission can also occur through blood transfusions contaminated with hbv and those who frequently receive hemodialysis. in addition, hbv can enter the body through cuts or abrasions to the skin and mucous membranes, for such as needle sticks or sharp object wounds, ear piercings, tattoos, needle stick treatment (acupuncture), selfinjection habits, and the use of unsterile needles. the use of medical equipment and dental care equipment that is not properly sterilized can transmit hbv (4). hbv that enters the bloodstream through a portal of entry, such as lymph vessels enters the circulation. after reaching the blood circulation, the virus will spread throughout the body and finally reach the target organ, namely liver cells. the entry of the hbv particle occurs through a receptor-mediated process. virions enter the cytoplasm, open the nucleocapsid, and then enter the ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 7 0 hepatocyte nucleus. the incomplete doublestranded viral dna sequence becomes covalently intact dna with the help of host cell enzyme activity, which not only "perfects" the viral dna but also releases dna polymerase virus. this dna then becomes the template for forming rna polymerase ii, which produce rna for the translation of proteins important to viruses. these proteins are then used for the encapsulation process and the formation of new viruses (5). based on the 2013 world health organization (who) report, two billion people in the world suffer from hepatitis, 240 million of them suffer from chronic hepatitis b and 1.46 million of them die. deaths from this disease are comparable to hiv deaths (1.3 million), tb deaths (1.2 million), malaria deaths (0.5 million). however, hepatitis has not received the same level of serious attention as the three diseases. data from the indonesian liver research association (pphi) at the national consensus for the management of hepatitis b in indonesia shows that the prevalence rate of hepatitis b in indonesia is between 4.020.3%. based on data from the ministry of health in 2013, nationally there were 2,981,075 (1.2%) of the population in indonesia suffering from hepatitis. this condition had doubled from 2007 (6). indonesia is the thirdmost country hepatitis b in with the world sufferers, after china and india with thirteen million sufferers, while in jakarta it is estimated that one in twenty people suffer from hepatitis b. most of the population is infected with the hbv since childhood. a number of countries in asia, 810% of the population suffer from chronic hepatitis b (7). indonesia is a country with endemic hepatitis b in 2007, it was 0.6% of hepatitis cases and increased to in 2013 by 1.2% of hepatitis cases (8). according to riskesdas data in 2018, districts / cities in gorontalo province, had a prevalence of hepatitis cases of 10,997, with a percentage of 0.55% (9). data from regional public hospital (rumah sakit umum daaerah, rsud) of dr. m.m. dunda limboto showed the prevalence of reactive hepatitis cases in 2019 was 2 reactive cases, 11 reactive cases in 2020, and 19 reactive cases in 2021. hospitals are places that have complex potential hazards for workers. they produce large amounts of waste, some of which is hazardous to health in their environment. hospital activities will produce waste, both solid, liquid, and gaseous waste containing pathogenic germs, chemical substances and medical devices, which are generally dangerous to the environment (1). hospital are one of the places of employment that are at danger since there is a chance that patients, staff members, and even visitors could contract infectious diseases there. examples of infectious diseases that can occur in hospitals includes ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 7 1 tuberculosis (tb), hepatitis b, hepatitis c, and hiv/aids. in addition to infectious diseases, hospitals also have other risks or hazards, such as explosion, fires, accidents caused by electrical installations, radiation exposure, toxic and hazardous chemicals, anesthetic gases, psychological and ergonomic related disorders. all of the potential hazards mentioned above can clearly interfere and cause a feeling of insecurity and discomfort for workers, patients, and visitors in the hospital environment (10). one type of work that is prone to accidents and occupational health problems is janitors work. janitors are at high risk of experiencing health problems because they are directly exposed to garbage disposal, thus putting them at risk of being exposed to the hbv (11). janitors are employees who are tasked with cleaning the hospital environment to keep it clean and minimize disease transmission. the job of cleaning the hospital environment makes the janitors vulnerable to exposure to hazards that can interfere with their health. according to rita (12) it is explained that, when hbsag was examined on 30 samples in kendari city (indonesia), 6.66% of the samples were reactive. this proves that janitors in hospitals are very much at risk of being infected with diseases due to things that are not taken into consideration when working, one of which is about the use of personal protective equipment when beginning work. some of the factors that cause workers to be reluctant to use personal protective equipment include difficulty to use, discomfort, lack of understanding of the importance of safety equipment, and indiscipline in use. the aim of this research is to describe the results and their relationship to risk factors of the hepatitis b examination on janitors at the regional general hospital (rsud). dr m. m. dunda limboto gorontalo. materials and methods study design and area the type of research used is descriptiveanalytical. the study was conducted from february to may 2022. sampling and examination were carried out in the laboratory of the regional general hospital dr. m. m. dunda limboto gorontalo. respondents in this study were janitors. approval for this study was obtained from the health research ethics committee of manado health polytechnic ministry of health no. kepk.01/05/080/2022. sample size determination and sampling techniques the sample used was the total population that met the inclusion criteria; there were as many as 33 respondents, selected using the accidental sampling technique. primary data was obtained from interviews with respondents and the results of laboratory examinations of hbsag samples and ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 7 2 secondary data was obtained from journals and literature related to this study. data collection tool and methods the tools and materials used in this study were immunology tubes, tube racks, tourniquets, centrifuges, 1 ml mini pipettes, timers, masks, 3 ml disposable syringes, alcohol swabs, dry cotton, hbsag strips and gloves. the materials used in this study were serum samples obtained using the immunochromatography test or rapid test method. sampling was carried out in the morning after filling out the questionnaire. specimen collection and processing whole blood was collected by venipuncture using vacutainer tubes, with (bd vacutainer® containing the anticoagulant edta). serum samples were taken after kept at 4º for 30 minutes and the examined in the laboratory of the regional general hospital dr. m. m. dunda limboto. serological test the examination method used was the immunochromatography test or rapid test method, where the serum sample was observed for the results through the presence or absence of a red line. the results were then interpreted as reactive interpretation if two red lines were formed in the control (c) and test (t) areas; and negative if a red line was formed in the control area (c). if no line was formed on the strip, it was declared invalid and the test had to be repeated. data analysis the data were collected, processed, analyzed descriptively, and stasistically using the spss program to examine the relationship between variables then presented in tabular form, narrated, and concluded. results the hbsag examination in this study was carried out on 33 respondents who were taken from the janitors of rsud. dr. m. m. dunda limboto. the results of the laboratory examination of the respondents showed a reactive result of 6% (table 1). table 1. frequency of hbsag test result result frequency (n) frequency (%) occurence reactive 2 6 non-reactive 31 94 total 33 100 according to research findings based on gender, respondents were predominantly female, numbering up to 21 (64%) and men, numbering up to 12 (36%). see table 2 for more information. table 2. distribution of respondents according to gender gender frequency (n) percentage (%) man 12 36 woman 21 64 total 33 100 ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 7 3 based on table 3, the majority of respondents were in the age range of 21-30 and 31-40 with a total of 61% each. table 3. distribution of respondets by age age group frequensy (n) pencentage (%) 21 30 7 21 31 40 20 61 41 50 6 18 total 33 100 table. 4 showed no significant relationship between infection status and the respondent's age. according to table 5 regarding the symptoms of hepatitis, it showed that the majority of respondents (90%) did not feel any symptoms of hepatitis. table 4. relationship between status infection with age result age (year) total (%) p-value 21-30 (%) 31-40 (%) 41-50 (%) positive 2 (6) 0 0 (0) 2 (6) 0.019 negative 5 (15) 20 (61) 6 (18) 31 (94) total 7 (21) 20 (61) 6 (18) 33 (100) table 5. distribution respondents based on disease symptoms symptomps of hepatitis frequency (n) percentage (%) feel symptomps 2 6 no feel symptomps 31 94 total 33 100 table 6 shows the infection status along with symptoms. the statistical test results showed that there was a relationship between infection status and the symptoms experienced. table 6. relationship between infection status with symptom result symptom total (%) p-value yes (%) no (%) positive 2 (6) 0 (0) 2 (6) 0.002* negative 0 (0) 31 (94) 31 (94) total 2 (6) 31 (94) 33 (100) *signifikan if p-value <0.005 discussion regional general hospital (rsud). dr. m.m dunda limboto, originally named limboto general hospital, is a hospital owned by the gorontalo district government located in the administrative area of ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 7 4 gorontalo regency, founded on november 25, 1963, now accredited b. this study began with filling out informed consent. in this study, the sample used was a venous blood sample, which was taken by performing a phlebotomy with an open system. three milliliters of blood were transferred to an immunology tube and then centrifuged for 10-15 minutes. the method used was the immunochromatography method by looking at the red lines in the control area (c) and the test area (t). janitors are at risk of being infected with infectious diseases due to small factors that are not taken into consideration when working, such as the use of personal protective equipment when starting work and whether the janitors has previously suffered from hepatitis. the results of this study are contradicted research conducted by rita (12) where the majority of respondents were male, at 60%. this research is in line with the proposed by bertelli (12) that the female sex is more susceptible to the effects of infection due to the low level of cell-mediated immunity in women and their physical weakeness compared to men. research conducted on work accidents caused by biological materials shows that they occur mostly in female workers who have low education and employed in the health sector (13). research conducted by rita (12) showed that the highest cleaning service at from the age of 26 30. research conducted by indarti (14) showed that the majority of respondents were between the ages of 21-30 years, indicating that most of the workers were dominated by young people (15). human age can be classified into several groups, each of which describes a stage of human growth. one of the age group divisions or age categories issued by the ministry of health of the republic of indonesia on its official website, namely depkes.go.id as follows: infancy, childhood, early adolescence, late adolescence, early adulthood, late adulthood, early elderly, late old age and old age. at the age of 20 years and over, the maximum oxygen capacity in the body will gradually decrease. at the age of around 50-60 years, the ability of muscle strength will decrease, resulting in a decrease in the body's physical ability to do work (15). research conducted in china showed that 90% of cases of hbv were experienced by the population over the age of 20 years (17). all age groups can be at risk of experiencing hepatitis b infection, as transmission of the virus can occur through actions, behaviors, or habits. table. 4 shows no significant relationship between infection status and the respondent's age. research conducted by amtarina et al (18) showed that respondents who had hepatitis b infection were typically 20 years of age or older.(18). hepatitis b in children can occur acutely or chronically, and transmission can occur ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 7 5 vertically through childbirth and intrauterine. however, the incidence of acute hepatitis b infection in children and adolescents is low due to vaccine administration in childhood (19). based on the results of interviews with respondents, it was shown that none of the respondents had received the hepatitis b vaccine. similarly, research conducted by samiun (20) found that most respondents had not received the hepatitis b vaccine compared to those who had received the vaccine. according to these findings, people who have never received a hepatitis b vaccination have a higher risk of contracting the disease. this is in line with the theory that the administration of the hepatitis b vaccine can prevent both hepatitis b and illnesses like liver cancer and cirrhosis that are brought on by hepatitis b. one method of preventing the spread of hepatitis b is vaccination. currently, there are two forms of immunization available; active immunization and passive immunization. active immunization is achieved by administering the hepatitis b vaccine, which contains purified hbsag. this hbsag is taken from purified serum of hepatitis b patients or from the recombination of yeast cell dna. hepatitis b vaccination can provide protection against hepatitis b infection for more than 20 years. the success of the vaccination is assessed by the detecting completing the hepatitis b immunization (3-4 times) (19). the hepatitis b vaccine (recombivax hb, comvax, and engerix-b), which is a vaccine made from an inactivated virus, can be given three or four times within six months (21). vaccines are one way to prevent infections by germs such as bacteria and viruses from entering our bodies (21). since 1997, the government has supported the national immunization program as a menas of preventing the spread of hepatitis b. workers in the health industry, including cleaning officers, should have received the. hepatitis b vaccine is given to prevent the occurrence of hepatitis b (23). not everyone who is infected with hbv develops symptoms of hepatitis. between 30 and 40 percent of people infected with this virus do not experience any symptoms. if there are symptoms. they usually appear within four to six weeks after infection and can last from several weeks to several months. some people who experience symptoms of acute hepatitis b feel so sick and tired that they cannot do anything for weeks or months. if the immune system is unable to control hbv infection within six months, symptoms of chronic hepatitis b may appear. not everyone with chronic hepatitis b experiences symptoms. some people may occasionally experience symptoms that go away after a while, while others may experience persistent symptoms. symptoms of chronic hepatitis b can be similar to those experienced with acute ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 7 6 hepatitis b. these symptoms tend to be mild to moderate and are usually temporary. additional symptoms may occur, especially in people who have had chronic hepatitis b for a long time. these symptoms include rashes, hives (an allergic reaction characterized by itching, red spots and swelling), arthritis (inflammation of the joints), and polyneuropathy (tingling or burning sensations in the arms and legs) (22). the statistical test results showed that there was a relationship between infection status and the symptoms experienced. histopathologically, hepatitis b is classified into persistent chronic hepatitis b, chronic lobular hepatitis b, and active chronic hepatitis b. all three can develop into cirrhosis and primary liver carcinoma. the clinical symptoms of hepatitis b depend on the severity of the infection. the course of hepatitis b infection consists of several phases, namely the incubation phase, the acute phase, the convalescent window phase and the healing phase. hbv is transmitted through percutaneous and mucous membranes infected with body fluids containing the virus. the virus can survive for more than one week on dry surfaces, thereby increasing the risk of infection (23). personal protective equipment (ppe) is a set of tools used by workers to protect part or all of their body from potential hazards or work accidents. ppe is equipment used to protect healthcare workers from microorganisms that cause infection. the use of ppe is very influential in the transmission of disease. the risk of nurses contracting diseases such as hepatitis hiv/aids increases if the use of ppe is neglected, resulting in a risk of infection. hepatitis and hiv/aids can attack nurses if they do not use ppe due to exposure to body fluids or puncture needle (25). the greatest risk of transmission through needle sticks is experienced by healthcare workers and cleaning staff in health facillities (25). according to research, the causes of workers not using personal protective equipment (ppe) are weak management and supervision, sanctions, lack of facilities and infrastructure, carelessness or negligence of humans, and inconvenience (21). some of the factors that influence workers' reluctance to use personal protective equipment include difficulty, discomfort, disturbance when using it; low understanding of the importance of safety equipment and indiscipline in its use (1). to improve the health of employees and their families by performing routine checks, administering vaccinations, wearing ppe at work and properly disposing of used needles. employees is also washing hands before and after working to prevent the spread of disease in the workplace. there are several methods of examining the hbv, and the rapid diagnostic test (rdt) method is one that can be used with ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 7 7 good sensitivity and specificity results. the rdt method is cost-effective (27). it can be used for examinations aimed at treatment but is not recommended for screening tests for potential donors (28). conclusions examination of hepatitis b surface antigen (hbsag) using the immunochromatography 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[tinjauan pustaka virus hepatitis b ditinjau dari aspek laboratorium]. j kesehat andalas. 2019;8(4):247–54. 25. muljono dh, wijayadi t, sjahril r. hepatitis b virus infection among health care workers in indonesia. euroasian j hepato-gastroenterology. 2018;8(1):88–92. doi: 10.5005/jp-journals10018-1269 https://doi.org/10.1016/s2468-1253(19)30042-1 https://doi.org/10.1016/s2468-1253(19)30042-1 ina. j. med. lab. sci. tech. 2023; 5(1): 68–79 indra elisabet lalangpuling, et al. 7 9 26. savoia e, argentini g, gori d, neri e, piltchloeb r, fantini mp. factors associated with access and use of ppe during covid-19: a crosssectional study of italian physicians. plos one. 2020; 15(10), e0239024. doi: 10.1371/journal.pone.0239024 27. okawa s, komada k, ichimura y, sugiyama m. comparison between a rapid diagnostic test and dried blood spot-based immunoassay for hepatitis b surface antigen testing : performance and cost implications in a population-based serosurvey in vietnam. int j infect dis [internet]. 2022;125:51– 7. doi: 10.1016/j.ijid.2022.10.011 28. magnus s, orlien s, abdosh t, yusuf n, berhe n, kran ab, et al. field performance of hbsag rapid diagnostic tests in rural ethiopia. j virol methods [internet]. 2021;289 (december 2020):114061. doi: 10.1016/j.jviromet.2020.114061 122 differences of spermatozoa concentration analysis between manual and automatic methods emma ismawatie1,2, purwanto adhipireno3, seso sulijaya suyono4, edy purwanto5, budi santoso6, edward kurnia setiawan limijadi7 1master program of medical laboratory science / clinical, faculty of nursing and health sciences, university of muhammadiyah semarang, central java, indonesia 2clinical pathology laboratory department, mother and child hospital restu ibu sragen, central java, indonesia 3clinical pathology laboratory department of central general hospital dr. kariadi semarang, central java, indonesia 4gladiool ivf centre, magelang city, central java, indonesia 5islamic hospital fatimah cilacap, central java, indonesia 6faculty of nursing and health, university of muhammadiyah semarang, central jawa, indonesia 7clinical pathology laboratory department of central general hospital dr. kariadi semarang, central java, indonesia correspondence: emma ismawatie, wantilan rt 10 rw 05, jelobo, wonosari, klaten, central jawa, indonesia zip code: 57473 email: emma11ismawatie@gmail.com received: february 26, 2021 revised: august 12, 2021 accepted: september 16, 2021 published: october 30, 2021 doi: 10.33086/ijmlst.v3i2.1961 abstract the examination of sperm concentration in the laboratory is the calculation of the number and motility using a microscope or using a device. there are still some clinicians who doubt the accuracy of the sperm count results using a semen analyzer rather than using the manual method. this study aims is to determine the differences of the sperm concentration examination between the manual method and the automatic method. subjects in this study were patients who carried out semen analysis tests at the clinical pathology laboratory of rsia "restu ibu" sragen from june to august 2020. the object of this research is the examination of sperm concentration, using a manual method using a hemocytometer and an automatic method using the lenshooke ™ sqa x1 pro. the results of statistical tests using the mann whitney methods show that the significance value (p) was 0.960, which means that there was no difference in the results of the sperm concentration examination between the manual method and the automatic method. result of this research shows that there is no weakness or significant difference if compared between manual and automatic methods. keywords spermatozoa, semen analyzer, manual spermatozoa, lenshooke sqa x1 pro, hemocytometer. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 2 3 introduction the infertility rate for married couples worldwide according to the world health organization (who), it is estimated that 1015% of couples who experience infertility problems during the reproductive period. male factors alone account for about 20% of infertility cases and a combination of male and female factors accounts for about 5060% of all infertility cases (1). semen analysis is one of the initial tests performed on infertility cases to evaluate the male side. the purpose of semen analysis is to determine the condition of sperm, the results can determine whether the man is fertile or infertile (2). routine evaluation of male infertility cases currently uses semen analysis as a standard covering concentration, motility and morphology (3). the concentration of spermatozoa in the future decreases, therefore examination of the concentration of spermatozoa is very important to establish cases of infertility. manual method of spermatozoa concentration examination using this simple tool is an examination of spermatozoa concentration that has been recommended by the who as the gold standard (2). the semen analysis examination includes macroscopic examination and microscopic examination. the macroscopic parameters of the semen that were examined included volume, appearance, color, coagulation, liquefaction and viscosity. meanwhile, the microscopic parameters examined from the semen sample include the concentration, motility and morphology of spermatozoa and other cell components (4). in calculating the concentration of the manual method using a hemacytometer counting chamber, it has the advantages of being easy to use, low cost of equipment and operational investment. while the drawbacks require high accuracy, the time required is relatively long between 30 and 60 minutes, is subjective, there are intra and inter-laboratory differences, not all laboratories have other trained verification personnel (5). along with technological developments in the laboratory field, there is now an examination method for automatic semen analysis. which combines computer technology and has been developing for approximately 40 years. through the advancement of devices for capturing images from the microscope, a large increase in computing power along with a reduction in computer size, and updated software algorithms are urgently needed (3). in an automatic method based on the development of digital imaging technology to obtain fast sperm concentration results, the reading process only takes 3-5 minutes. the level of accuracy, which has a good correlation and agreement of results overcomes the subjectivity problems of the assessment and is able to improve and standardize the concentration parameters. in its use, the semen quality analyzer tool is still found to ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 2 4 be deficient, including the ability of the tool to not be able to read sperm concentration if the number of spermatozoa is less than 16 per field of view, the investment costs for the equipment and operation of the equipment are quite high (6,7). there are still some clinicians who doubt that semen analysis uses an automatic method, this further strengthens researchers to carry out and test the quality of this automatic tool with spermatozoa concentration parameters (6,8). examination of spermatozoa using the automatic method of semen quality analyzer can improve accuracy, one of which is about the results. reading of spermatozoa concentration (9). in the research conducted by dearing et al. (10) the criteria examined were comparisons with the improved neubauer and leja 20-μm spaces, within and between field accuracy, linearity of sperm concentrations from diluted stocks in semen and media, accuracy against internal and external quality materials, assessment of uneven flow effects and receiver. analysis of surgical characteristics for predicting fertility was compared with the neubauer method. this work demonstrates that casa's sca technology is not a standalone 'black box', but rather a tool for trained staff that enables rapid and multiple sperm counts, providing identified and corrected errors. this system will produce accurate, linear, and precise results, with less analytic variance than the manual method which correlates well to the improved neubauer space. in the one of study comparing manual and automatic using the sqa-icp type, the sample used in the study was 50 samples. the difference in research on the use of the leenshoke x1 pro automatic tool and the number of samples analyzed was 40 samples (9). the differences in the above research have sparked the interest of researchers to test different spermatozoa concentrations, using two different methods, namely, the manual method with a computation chamber and an automatic method with a semen quality analyzer (11). the purpose of this study was to compared the examination of sperm concentration, between manual methods using a hemocytometer and an automatic method using the lenshooke ™ sqa x1 pro. result of the comparison calculation of sperm concentration between the manual method and the automatic method showed with the mann whitney method, the sig value is 0.960. because the value of sig> 0.05, it can be concluded that h0 is accepted and h1 is rejected. materials and methods spermatozoa analysis includes macroscopic examination by direct observation, which consists of volume, liquefaction (liquefaction), appearance, odor, consistency, viscosity, and ph. while the microscopic examination using a ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 2 5 microscope, which consists of assessment of concentration and motility, agglutination, morphology and vitality of spermatozoa (12,13). microscopic sperm analysis refers to three parameters, namely sperm concentration or number, sperm motility or movement speed, and sperm shape or morphology. the method of calculating the concentration of spermatozoa starts from the preparation of the patient where special instructions are needed to the patient before releasing the sperm, then the method of collecting sperm which includes a special room for sperm production, containers, labeling, storage, delivery, and examination forms before proceeds with processing and examination specimen (2,14). hemacytometer chamber to count the concentration of spermatozoa are has 25 large boxes, each large box is divided into 16 small boxes consisting of an upper and a lower side each side. semen analysis macroscopic observations without using a microscope but with a visual, examination includes : liquefaction at room temperature with normal semen thaw within 60 minutes, normal semen appereance is gray white or pearl white, characteristic odor or smell like a flamboyant flower. consistency and viscosity are liquefied, will drip small and slowly, if they are in the form of tendrils more than 2 cm or do not want to break are hyperviscosity, and if it looks like water and clear it is hypoviscosity. normal ph value of spermatozoa are 7.2 – 7.8 and the normal volume is 1.5 – 5 ml (15,16). microscopic semen analysis filter test is the examination of semen fluid using a microscope. microscopic observations include assess the concentration and motility of spermatozoa, do homogeneity manually, drop 10 microliters on a slide, cover with a cover glass, allow 1 minute to stabilize, check at room temperature and magnification 400x. observe the entire rectangular area of the cover slip directed left-right-up-down, if the distribution of spermatozoa is uneven, a new preparation is made. agglutination, performed when assessing the movement of spermatozoa, observe 10 fields of view randomly, the assessment is: head-head, head-neck, head-tail, tail-tail or mixed, average agglutination is estimated at 5%. vitality of spermatozoa, the assessment is carried out if the number of moving sperm is more than 30% and to determine the sperm that are actually alive at the time of expulsion (15). counting the concentration of macroscopic spermatozoa. this research uses observational analytic laboratory, where to find the differences sperm concentration using two methods: the manual method and automated method hemocytometer semen quality analyzer. the subjects in this study were patients who carried out semen analysis tests at the clinical pathology laboratory of rsia ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 2 6 "restu ibu" sragen from june to august 2020 with legal letter number 079-17/b/rsia ri/iii/2020. determination of the sample size of the population used the formula isaac and michael (14) according to sugiono (16). s = 𝜆2. 𝑁. 𝑃. 𝑄 𝑑2. (𝑁 − 1) + 𝜆2. 𝑃. 𝑄 s = 3.481 × 40 × 0,5 × 0,5 0.052(40 − 1) + 3,841 × 0,5 × 0,5 s = 35.9700 s = 36 the number of samples obtained by the researchers was 36 samples. sampling was carried out on respondents who came to be tested for semen analysis who had been educated and promised to come for the examination in sequence. inclusion criteria in this research were patients aged 20 to 45 years or reproductive age and patients with spermatozoa yields of more than 20 spermatozoa cells per field of view. exclusion criteria were hemospermic semen samples or blood-stained semen samples, semen sample with leukospermia, semen sample with many immature sperm cells and semen sample less than 1.5 ml. the independent variables are the manual method of the hemacytometer and the automatic method of the semen quality analyzer. dependent variable is the result of examination of spermatozoa concentration. the object of this research is the examination of sperm concentration, using a manual method using a hemocytometer and an automatic method using the lenshooke ™ sqa x1 pro. the results obtained was calculated statistically with spss 20 to determine the difference in the results of the examination between the two methods. this research get approval from the ethics commision for health research of muhammadiyah semarang university. manual method the reading of spermatozoa concentration, manual method using a counting chamber (improved neubauer) consists of an upper and a lower side, each side has 25 large boxes, each large box is divided into 16 small boxes. the calculation is a large box, starting with the middle box (2,17,18). the steps for calculating the concentration by dilution are as follows: a diluent solution is prepared: dissolve 50 grams of sodium bicarbonate (nahco3) and 10 ml of 35% formalin in 1,000 ml of aquabidest. stir the sample until homogeneous. determine the dilution to be used in the sample by observing the wet preparation: drop 10 μl of homogeneous semen on the object glass, cover with a cover glass (size 22 mm x 22 mm), observe with a 400x magnification light microscope. after determining the dilution to be used, make a tube containing 50 μl of the homogeneous ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 2 7 sample plus a diluent solution. a computation chamber with a cover glass (thickness size 4; 0.44 mm) was prepared. as much as 10 μl sample mixture were taken and a homogeneous diluent solution, then fill it in one of the counting rooms for the improved neubauer haemocytometer. let stand for 4 minutes, then count the number of spermatozoa in 10-15 minutes to avoid evaporation. the spermatozoa concentration is counted, until at least 200 spermatozoa have been observed and the rows of the five large squares are fully examined. record the number of rows when counting reaches 200 spermatozoa. calculations were carried out in other counting rooms as many as the rows obtained during the first calculation. if the difference between the number of spermatozoa in the two counting chambers is high, the calculation is repeated. if the difference does not differ greatly, the spermatozoa concentration is calculated (1921). in the examination of sperm concentration using the manual method, it begins with the preparation of a sample that meets the criteria. the sample is left to stand for a maximum of 60 minutes until it melts (liquefaction), then dilution is carried out and analyzed by dropping the semen liquid into the improved neubower counting chamber, the calculation is carried out in 1 large box in the middle. in this study, following standard criteria, the counted cell is the cell in the middle and touches the left and top lines (figure 1). the cells that touch the right and bottom lines are completely uncountable. the drawback in this manual method is that the sperm cells that are constantly moving make it difficult for visual capture, so it is possible that the cells that should be counted are not counted and vice versa. table 1 shows table of correction factors calculation of spermatozoa concentration with "improved neubauer" counting chamber. figure 1. spermatozoa in the middle of chamber field (red box) determine the required dilution of the initial semen sample and make a wet preparation, to estimate the number of spermatozoa per large field of view (x 200 or x 400) (2). ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 2 8 table 1. table of correction factors calculation of spermatozoa concentration with "improved neubauer" counting chamber spermatozoa per x400 field spermatozoa per x200 field dilution required semen (µl) fixative (µl) chamber area to be assessed >101 >404 120(1+19) 50 950 improved neubauer grids 5,4,6 16-100 64-400 1:5(1+4) 50 200 improved neubauer grids 5,4,6 2-15 8-60 1:2(1+1) 50 50 improved neubauer grids 5,4,6 <2 <8 1:2(1+1) 50 50 improved neubauer or large-volume all 9 grids or entire slide source : laboratory manual for the examination and processing of human semen 5 ed who library (2010) automatic method this automatic semen analysis was performed using the lenshooketm x1 pro (bonraybio co., cn) semen quality analyzer for sperm concentration, world health organization (who). lenshooketm x1 pro technology is based on a high-resolution optical lens and built-in autofocus combined with an artificial intelligence autoculation system, the rationale for using autofocus optical lenses to replace laboratory microscopes is based on the concept of using an automated optical inspection system (5). specifications of the lenshooke™ sqa x1 pro semen quality analyzer, measuring 140mm wide, 70mm thick, 71.2mm high, 3.5” resistive touch panel control panel, usb 2.0 input and hdmi output, resolution 320 (h) x 480 (v) color dot matrix, ac100240v/50-60hz power supply; dc output 5v/2a rechargeable li-polymer battery (6). the accuracy of the sca semen quality analyzer is generally very good with optimal accuracy at 200-600 spermatozoa per field, this is directly from the casa (computerassisted sperm analysis) screen sca developed for single and multiple plane precision tests. the semen quality analyzer is accurate with its concentration of latex beads. in the semen quality analyzer, quality control is carried out using special reagents, if ten thousand samples of semen analysis are carried out on the automatic semen quality analyzer, or it can be done every six months (12). the semen quality analyzer provides increased precision over manual methods and can now be applied to routine spermatozoa analysis provided adequate quality control procedures and high measurement standards are followed. spermatozoa concentration and motility data on semen quality analyzers have been shown to be associated with various fertility measures, although over-calculation of specific semen quality analyzer methods because spermatozoa measurements have been well documented, several systems have been shown to accurately calculate ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 2 9 spermatozoa concentrations (13). the sperm collection device for the semen quality analyzer uses a special cup test consumable. wait 30 to 60 minutes for sperm to thaw and then homogenized the sample in the cup by turning it back and forth 8-10 times. sperm collection device for the semen quality analyzer uses a special consumable test cup. wait for 30 to 60 minutes for sperm thawing, then the sample is homogenized in the cup by inverting it 8-10 times. check the color and volume of the sperm sample (figure 2). figure 2. procedure for a semen cassette quality analyzer. the first to know is to not touching the double drip test zone inside and outside (under the area), touching the test zone with hands or gloves could contaminate the detection window and will lead to inaccurate results. the correct semen test cassette will be displayed on the monitor, if the cassette is not properly inserted there will be sound. the results will appear on the screen (13). the technology used artificial intelligence or light to capture in the tool system, apply the sample to the cassette, use the thumb to open the cassette. the concave design of the cassette opening allows analyzing the concentration, motility and morphology of the semen and detecting, analyzing the ph of the semen. operational way with automatic tool cassette are checked for unexpired test cassette that has not changed color (no green). do not touching the double-drip test zone inside and outside (underneath the area) touching the test zone with your hands or gloves can contaminate the detection window, and will lead to inaccurate results. applying the first ph drop then drop it to the “concentration”. avoid bubbles, don't drip the sample on the top cover of the cassette, then pressing the yellow area to close the cassette. the correct semen test cassette will be displayed on the monitor. all patients who have come are asked to fill out an informed consent to perform a semen analysis examination and will be educated about preparation, from abstinence to how to remove the sample and collect it. ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 3 0 the way to store spermatozoa so that the results are better is by masturbating without using tools, such as gel, detergent and other aids, may be accompanied by his wife in a special room provided by the hospital, because it will affect the spermatozoa samples that are accommodated and will be analyzed semen. spermatozoa samples will be divided into two pots to be carried out by two methods simultaneously. results the results of the study from the target population of all patients who carried out semen analysis tests at the clinical pathology laboratory of rsia "restu ibu" sragen period from june 2020 to august 2020 obtained a total sample size of 36. the number of samples is in accordance with the calculation of the sample size needed by the researcher to obtain the appropriate data. sampling is carried out on all semen samples who come to be examined for spermatozoa analysis, which have been educated and promised to come for the examination in sequence. table 2. subject characteristic based on age age category age range (year) amount sampling methods late adolescence 17-25 7 masturbating early adulthood 26-35 21 masturbating late adulthood 36 -45 8 masturbating total 36 table 2 show characteristics of patients who are willing to be research subjects are in the age range of late adolescence to early old age. the majority of respondents are in the age range of 26-35 years or in this phase referred to as early adulthood as many as 21 people. then at the age range of 17 to 25 years or called late adolescence as many as 7 people. medium in the age range of 36-45 years or the phase of late adulthood as many as 8 people (table 3-4). table 3. sample characteristic based on volume and color semen volume semen color (ml) amount criteria amount < 1,5 2 white gray 17 1,5 – 5 30 pale yellow 10 > 5 4 yellow 9 total 36 36 table 4. sample characteristic based on liquefaction time liquefaction time abstinence time (minutes) amount (days) amount < 30 13 2 – 3 14 30 – 60 23 4 – 5 14 > 60 0 6 – 7 8 total 36 36 the inclusion criteria table shows samples that meet the requirements to be included in the study including the volume of semen, the number of samples is 36 which will then be calculated by the researcher using manual methods and automatic methods. the aim of the researcher to get a normal sample is to make it easier to read the sperm concentration, so that the effective ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 3 1 time for examining each sample can be achieved. table 5. count result of spermatozoa subject characteristic manual methods automatic methods minimum value (million cell/mm3) 5.3 4.0 maximum value (million cell/mm3) 240.0 243.8 mean (million cell/mm3) 86.731 87.347 standart deviation 76.0004 76.4370 > mean 14 (38.9 %) 22 (61.1 %) < mean 14 (38.9 %) 22 (61.1 %) table 5 contain simple descriptive test to determine the lowest and highest values, the mean value, and the standard deviation of each examination. in the manual method, the lowest examination score was 5.3 million/mm3, the highest value was 240 million/mm3, the average examination result was 86.731 million/mm3 with a standard deviation of 76.0004 million/mm3. whereas in the automatic method, the lowest examination value was 4 million/mm3, the highest value was 243.8 million/mm3, the average examination result was 87.347 million/mm3 with a standard deviation of 76.4370 million/mm3. this research is a type of quantitative research, the data that has been collected is analyzed statistically using the statistical package for the social science version 20 (spss). before analyzing the data, a normality test was conducted to determine whether the data was normal or not. normality test using kolmogorow smirnow. the data is classified as normally distributed if the p value > 0.05. furthermore, if the data is normally distributed, then the data is tindependent test, but if the data distribution is not normal p value < 0.05 the data is analyzed by the mann whitney test. the normality test of the data obtained is used to determine the distribution of the data in order to determine the statistical test used. with the mann whitney method, it can be seen the difference between the calculation of the sperm concentration between the manual method and the automatic method. the table 5 above also shows the comparison of the results against the mean on the examination of sperm concentration using the manual method. the results of the examination were below the mean value of 22 samples or as much as 61.1%, above the mean value of 14 samples or as much as 38.9%. in the examination of sperm concentration using an automatic method. the results of the examination were below the mean value of 22 samples or as much as 61.1%, above the mean value of 14 samples or as much as 38.9%. this study shows the p value (significance) of the manual method with the kolmogorov-smirnov test of 0.002 and the shapiro-wilk test of 0.000. in the automatic method, the kolmogorov-smirnov test is 0.001 while the shapiro-wilk test is 0.001. both data groups show a significance ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 3 2 value below 0.05, which means that both data have an abnormal distribution value. because the two groups of data were not normally distributed, the next hypothesis test was to use the mann whitney method. the comparison of the calculation of sperm concentration between the manual method and the automatic method show with the mann whitney method, the sig value is 0.960. because the value of sig> 0.05, it can be concluded that h0 is accepted and h1 is rejected, which means that there is no difference between the limitation of this study of sperm concentration between the manual method and the automatic method. discussion data were obtained from patients who came to the clinical pathology laboratory of rsia “restu ibu” sragen for the period june to august 2020. the sample was a population that met the criteria for semen analysis examination. the number of samples in this study were all semen samples in the clinical pathology laboratory of rsia "restu ibu" taken sequentially according to the hour and day the semen sample was received by the laboratory. determine the sample size of the population using the formula isaac & michael according to (15) and as many as 36 samples. examination of sperm concentration using the manual method in this study showed results above the average by 14 and below the average by 22 samples. the data obtained from this study have been tested for normality using the kolmogorov smirnov and shapiro wilk methods. the normality test used in this study was shapiro wilk because the amount of data used was less than 100. the expected p value (significance) was 0.05. the p-value on the examination of sperm concentration with the manual method was 0.000 and the automatic method was 0.001. because both groups of data have a significance value lower than the same value of 0.05, it can be said that the data distribution is not normal. the statistical test used next is the mann-whitney test. the results of statistical tests using the mann whitney method obtained a significance value (p) of 0.960. in this study, h0 is accepted if the result is p > 0.05. because the results of the study showed a p value > 0.05, h0 was accepted and h1 was rejected, meaning that there was no difference in the results of the sperm concentration examination between the manual method and the automatic method. basically, there are two categories of automatic sperm analyzers on the market which can be characterized by their detection technology. sqa-v gold is a fully automated system, which is based on the detection of electro-optical signals generated by moving sperm and interpreted by a special algorithm. this signal processing for sperm motility is combined with ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 3 3 spectrophotometric technology to determine sperm concentration. the computer assisted sperm analysis (casa) is based on another principle: microscopic image capture and image processing to detect both motile and immotile spermatozoa via rapid and sequential frame acquisition (18). this study controls for factors ranging from pre-analytic, analytic and post-analytic. in the pre-analytic factors, starting with an explanation of the criteria for the accepted sample, including abstinence, methods of dispensing and holding, and sending samples to the laboratory. in terms of analytic factors, sample processing is also carried out carefully from the stages of observing sample quality, liquefaction, and sample processing. at the post analytic stage, the researcher controls the results and records the data. lenshooketm sqa x1 pro provides higher normal morphological values and average concentrations compared to the gold standard. this result occurs because lenshooketm sqa x1 pro uses different examination criteria, directly without staining on morphology. although direct concentration tests were carried out in the selection of spermatozoa for intracytoplasmic sperm injection (icsi), until now the spermatozoa morphological criteria have not been applied in semen analysis (6). the results of the examination of sperm concentration in this study showed that there were no significant differences in sperm concentration between the manual method and the automatic method. this shows that the gold standard used is still used as a reference for assessing sperm concentration. and the use of automatic tools can also be considered in terms of speed, which will make it easier to work if the sample inspection is carried out in large quantities. conclusions the results of the sperm concentration examination in this study showed that there was no significant difference in sperm concentration between the manual method and the automatic method. acknowledgements research included in this review was partly completed at the clinical pathology laboratory of rsia “restu ibu” under the supervision of purwanto adhipireno and seso sulijaya suyono. author contributions emma ismawatie: conceptualization, formal analysis, investigation, writing original draft, writing review & editing. purwanto adhipireno: supervision, conceptualization, methodology, writing original draft. seso sulijaya suyono: validation, formal analysis. edy purwanto: validation, reviewing. santoso budi: validation, reviewing. edward kurnia ina. j. med. lab. sci. tech. 2021; 3(2): 122–134 emma ismawatie, et al. 1 3 4 setiawan limijadi: writing review & editing, investigation. conflict of interest there are no conflict of interest to report. references 1. ring jd, lwin aa, köhler ts. current medical management of endocrine-related male infertility. asian j androl. 2016;18(3):357–63. 2. who. laboratory manual for the examination and processing of human semen. 5 ed. who library. 2010. 3. amann rp, waberski d. computer-assisted sperm analysis (casa): capabilities and potential developments. theriogenology.2014 ; 81(1)5-17. 4. dohle g, arver s, bettocchi c, jones t., kliesch s, punab m. guidelines on male hypogonadism. eur assoc urol. 2011;1–24. 5. agarwal a, henkel r, huang cc, lee ms. automation of human semen analysis using a novel artificial intelligence optical microscopic technology. andrologia.2019; 2. 1-9. 6. bonraybo. lenshooke xi pro semen quality analyzer. 2019; 1-4. http://bonraybio.com/en/product.php?act=view& id=8. 7. maggavi rr, pujari sa, vijaykumar cn. motility analysis with morphology: study related to human sperm. procedia computer science. 2019 ; 152. 179-85. 8. narulita p. the effectiveness of lenshooke tm semen quality analyzer x1 pro for human semen analysis. fac med univ airlangga, surabaya. 2020;1(1). 9. chaurasia a, sinha a, upahdyay p. comparison of semen analysis by manual and automated method. j pathol nepal. 2016;6(12):990–3. 10. dearing c, jayasena c, lindsay k. can the cperm class analyser (sca) casa-mot system for human sperm motility analysis reduce imprecision and operator subjectivity and improve semen analysis?. human fertility. 2014; 17(1): 37–44. 11. baig a, shoebuddin m, ahmed m. comparison of manual sperm analysis with computer-assisted sperm analysis: a comparative cross-sectional study.national journal of physiology, pharmacy and pharmacology. 2019 ; 9.1-3. 12. syauqy a. evaluasi kromatin sperma sebagai indikator kualitas sperma. 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[qualitative research methods and r&d]. graha ilmu. edisi1. bandung, alfabeta. 2019. 286 292p. 16. singh s, sharma s, jain m, chauhan r. importance of papanicolaou staining for sperm morphologic analysis: comparison with an automated sperm quality analyzer. american journal of clinical pathology. 2011;136 (2) : 24751. 17. shahnaz, ayesha. infertility: a review on causes, treatment and management. womens health & gynecol. 2016;2(6): 35-40. 18. park ys, park s, ko ds, park dw, seo jt, yang km. observation of sperm-head vacuoles and sperm morphology under light microscope. clinical and experimental reproductive medicine, 2014;41(3), 132–136. https://doi.org/10.5653/cerm.2014.41.3.132 19. ariagno ji, mendeluk gr, furlan mj, sardi m, chenlo p, curi sm, pugliese mn, repetto he, cohen m. computer-aided sperm analysis: a useful tool to evaluate patient's response to varicocelectomy. asian journal of andrology, 2017;19(4), 449–452. https://doi.org/10.4103/1008-682x.173441 20. franken dr, oehninger s. semen analysis and sperm function testing. asian journal of andrology, 2012;14(1), 6–13. https://doi.org/10.1038/aja.2011.58 21. kevin, kl, raymond hwl, ernest hyn, pak ch, william sby. semen analysis – what a clinician should know. continuing medical education. 2012. avalaible online at https://www.mims-cpd.co.id/ 53 stability of lyophilized homemade control serum after reconstitution on sgot and sgpt levels stored in freezer at temperature (-2° to -4°c) and -20°c for 8 weeks kadek profit hartani1, anik handayati1 1department of medical laboratory technology, poltekkes kemenkes surabaya, east jawa, indonesia correspondence: anik handayati, jl. karang menjangan no.18a, airlangga, kec. gubeng, surabaya, east jawa, indonesia zip code: 60286 email: anik_handayati@yahoo.co.id received: july 28, 2022 revised: february 15, 2023 accepted: april 16, 2023 published: april 29, 2023 doi: 10.33086/ijmlst.v5i1.3278 abstract improving health laboratory services is closely related to strengthening the quality of health laboratories. serum control is part of the quality assurance of health laboratories. lyophilized homemade serum is a type of homemade control serum. this research aims to determine the stability of lyophilized homemade control serum against serum glutamic oxaloacetic transaminase (sgot) and serum glutamic pyruvic transaminase (sgpt) levels. this research used an experimental method with pooled sera as research material stored at -2°c to -4°c and -20°c for 8 weeks. pooled sera were obtained from respondents who had no history of disease, were free from hiv, aids, and hbsag. the research was conducted at the reference laboratory and clinical chemistry laboratory politeknik kesehatan (poltekkes) kementerian kesehatan, surabaya. the results of the study were data analysis using linear regression test. the sgot parameter at freezer temperature (-2 to -4°c) showed that the storage time had an effect of 90.97% with an error component of 9.03%. at freezer temperature (-20°c), the result of storage time has an effect of 78.71% with an error component of 21.29%. in the sgpt parameter with freezer temperature (-2 to -4°c), the result of storage time has an effect of 96.71% with an error component of 3.29%. at freezer temperature (-20°c), the result of storage time has an effect of 91.47% with an error component of 8.53%. the results of the sgot and sgpt examinations did not exceed the limits of ±2sd and ±3sd the cv on the sgot and sgpt examinations also did not exceed the ccv limits, which is to be stable. keywords lyophilized homemade control serum, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase. citation: hartani kp, handayati a. stability of lyophilized homemade control serum after reconstitution on sgot and sgpt levels stored in freezer at temperature (-2° to -4°c) and -20°c for 8 weeks. indones j med lab sci technol. 2023;5(1):53–67. doi: 10.33086/ijmlst.v5i1.3278 this is an open access article distributed under the creative commons attribution-sharealike 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2023 by author. mailto:anik_handayati@yahoo.co.id https://doi.org/10.33086/ijmlst.v5i1.3278 https://doi.org/10.33086/ijmlst.v5i1.3278 https://creativecommons.org/licenses/by-sa/4.0/ ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 5 4 introduction clinical laboratory services are an integral part of health services needed to make a diagnosis, by determining the cause of the disease. clinical laboratories need to be organized in a quality manner to support efforts to improve the quality of public health. given the importance of the function of laboratory examination results, the quality of laboratory results must always be guaranteed. in accordance with government regulations, clinical laboratories are required to carry out laboratory quality assurance (1). laboratory quality assurance is the entire process or action taken to ensure the accuracy and accuracy of examination results. there are two types of quality assurance in laboratories, namely internal quality assurance and external quality assurance. internal quality assurance is a prevention and control activity carried out by each laboratory on an ongoing basis to prevent and reduce the occurrence of errors or inconsistency examination results. external quality assurance is an activity held regularly by other parties outside the laboratory concerned to monitor and evaluate the performance of a laboratory in a particular field of examination (2,3). measuring the accuracy of laboratory examination results can be carried out using the control materials (4,5). control materials used in laboratories to monitor the accuracy of examination results or the quality of clinical examination results (6). control materials can be obtained from commercial or prepared from pond materials (secondary control materials). secondary control material is made from the remaining serum of patients with normal levels and collected (pooled sera) as a substitute for commercial control materials. external quality assessment (eqa) requires stable quality control (qc) materials (7). nowadays, the control serum used in the laboratory in the commercial ones. this control serum is taken from animals which may not be the same as human serum and quite expensive. meanwhile, in terms of efficiency, the homemade lyophilized human serum does not require costs to make. besides that, to utilize the samples used in clinical chemistry examinations, there are usually only a few, while the remaining will be discarded (8). for small laboratories, health centers and educational laboratories that use the homemade lyophilized human serum as a control serum, it is hoped that it can be an alternative to control materials (9). material quality control (qc) is critical to internal quality control (iqc) and external quality assessment (eqas) schemes. however, many developing countries disadvantaged by unavailability and high cost of commercial control agents. therefore, preparing homemade lyophilized human sera would be cost-effective as a quality control agent. the lyophilized serum is widely used in clinical laboratories because it is stable for ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 5 5 1 – 2 years when stored at 2-8°c and several years at -20°c or lower (10). pooled serum qc is also stable and low cost compared to commercial qc. another use of pooled serum is to monitor a continuous flow analysis system throughout a long estimation chain, to ensure that qc is maintained. serum pools are valuable in detecting and correcting aberrations, especially for automated analyzer techniques. the stability of the serum pool is known to have a shorter duration than the lyophilized control sera. moreover, the serum pool only checks for precision at one level (11,12). examination of liver function is one of the clinical chemistry examinations that is often requested by doctors. it is because the role of the liver as an essential body organ and the central organ of metabolism. two types of enzymes that are often associated with liver cell damage included in the aminotransferase group. in liver disease, the levels of sgot (serum glutamic oxaloacetic transaminase) and sgpt (serum glutamic pyruvic transaminase) tend to change in parallel. if liver cells are damaged, these enzymes, which are normally present in the cells, enter the bloodstream. the more liver cells that are damaged, the higher the levels of sgot or sgpt measured in the blood (13). previous studies demonstrated that at -20°c the maximum change was shown by sgot and sgpt (decreased by 17% and 6%, respectively) at 6 months for the serum pool (14). alt activity decreased after 3 days of storage at room temperature (22°c) and decreased after 7 days at 4°c (15). the purpose of this study was to determine the stability of post-reconstitution homemade lyophilizate control serum stored in the freezer at -2°c to -4°c and -20°c against sgot and sgpt parameters for 8 weeks. materials and methods this research is an experiment study with a time series research design. the material used in this study was homemade lyophilized serum after reconstitution stored at -2°c to 4°c and -20°c for 8 weeks. the experiment was performed from november 2021 to may 2022. the selected respondents were female students of the surabaya ministry of health politeknik kesehatan (poltekkes) aged 18-21 years, had no history of disease, were free of hiv and hbsag. this study used purposive sampling as sampling technique. respondents previously asked to fill out informed consent form, for respondent who agreed with the consent. the ethics commission has approved this research under the ethics eligibility letter no. ea/824/kepk-poltekkes_sby/v/2022. collection of blood the research procedure started with injecting the respondent's blood into a red cap vacutainer tube. the blood obtained was ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 5 6 allowed to stand at room temperature. the vacutainer tube was centrifuge at 3000 rpm for 15 minutes. the sample criteria were not hemolyzed, not lipemic, and not icteric. the anti-hiv and hbsag tests were performed to confirm serum sample was not infectious. the pooled serum was then homogenized using a vortex mixer and transferred into vials, each 3 ml using a volume pipette. furthermore, the serum sample was lyophilized. homemade lyophilized control serum preparation freeze dryer used to make lyophilized serum. firstly, serum stored in the freezer at -80°c until frozen. then, the inside of the freeze dryer cleaned. the tool cover and tube supports were smeared with vaseline gel. the power button on the back of the vacuum pump and the front of the freeze dryer were turned on. the display on the device would showed a temperature of -50°c. the sample tube is connected with rubber on the tube support also closed using aluminum foil. the valve connecting the sample tube to the instrument is turned. the freezer drying process can be started and considered complete when all the serum frozen into powder. serum in the form of lyophilizate is dissolved by tapping on the bottle so that the powder falls to the bottom of bottle. then, the powder was dissolved in 3 ml of distilled water using a volume pipette. the dissolved powder was stand 30 minutes and homogenized back and forth. after homogenized, the liquid was divided into microtube packages, 500 microliters of serum each, then stored at the appropriate temperature and storage time. 60 microtubes were obtained from 10 vials of homemade lyophilizate serum. the microtubes divided into three groups. the first group, as many as 10 micro tubes was performed duplicate examination at 5 reference laboratories to test homogeneity and determine the initial value or base line. the second group, as many as 24 microtubes stored at -2°c to -4°c, and the third group of 24 microtubes stored at -20°c. samples in microtubes were used to test the stability of sgot and sgpt parameters, examined by replication 3 times, once a week for 8 weeks. determination of sgot and sgpt level the enzymatic kinetic method performed to determine of sgot and sgpt levels. the principle of determine sgot is that sgot/ast in the sample catalyzes the transfer of the amino group from l-aspartate to 2-oxoglutarate to form oxaloacetate and lglutamate. oxaloacetate in the presence of nadh and malate dehydrogenase (mdh) reduced to l-malate. in this reaction nadh is oxidized to nad. the principle for determining sgpt is that alanine aminotransferase (alt) catalyzes the transaminase of l-alanine and 2-oxoglutarate to form l-glutamate and pyruvate. the pyruvate formed is reduced to lactate by the ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 5 7 lactate dehydrogenase (ldh) enzyme and nicotinamide adenine dinucleotide (nadh) is oxidized to nad. the amount of nadh oxidized is directly proportional to alt activity. the examination begins with removing the microtubes of homemade lyophilized serum samples, which have been dissolved and stored in the freezer at -2°c to -4°c and -20°c, then allowed to stand until room temperature. the sample pipette on the microtube is transferred to the sample cup and placed on the sample tray in the automatic clinical chemistry determination device. check the selected sgot and sgpt then click start/run to start the determination. statistical analysis the data analysis technique used in this research is the examination results of the sgot and sgpt levels. the test is performed with a simple linear regression analysis test with spss, which is tabulated (presenting data in tabular form). from the data obtained, then data analysis was carried out by calculating the average of the results of the examination (mean), standard deviation (sd), and coefficient of variation (cv). the data obtained were then performed with a linear regression test to determine the effect of storage time on homemade lyophilized serum after reconstitution on the stability of sgot and sgpt levels. results this study was initiated by conducting anti-hiv and hbsag screening tests. the aim was to detect the presence or absence of hiv and hepatitis b viruses in each respondent used the rapid test immunochromatography method. the results of the anti-hiv and hbsag screening tests were declared non-reactive, meaning that the respondent's serum was negative for hiv and hepatitis b viruses. after the serum resulted infection-free, it was followed by the initial examination as a control test. this check aims to get a reference value or baseline value. the data obtained in this examination is used as a reference value for further research. in this examination, the sample used was homemade lyophilizate serum after being dissolved with sgot and sgpt parameters. the results of the initial test showed in table 1. table 1. results of initial examination and homogeneity test of homemade lyophilized control serum sgot and sgpt parameters examination results (u/l) sgot sgpt mean 19.75 5.35 sd 2.20 0.85 cv (%) 11.16 15.86 table 1 showed the average value of sgot, which is 19.75; sd 2.20; and cv 11.16%, while for the average value of sgpt 5.35; sd 0.85; and cv 15.86%. the cv value on examination of sgot and sgpt ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 5 8 levels at -2℃ to -4℃ storage from week 1 to week 8, does not exceed the limit of cv (ccv). value chosen coefficient of variation (ccv) for sgot 12.5% and sgpt 17.3%, meaning that the levels of sgot and sgpt serum lyophilizate homemade are homogeneous to variations in vials. figure 1. levey-jennings graph based on initial value of sgot levels homemade lyophilized control serum based on figure 1, it shows that the warning limit (±2sd) of the control serum lyophilized homemade for sgot examination. the upper warning limit (+2sd) is 24.16 u/l and the lower warning limit (-2sd) is 15.34 u/l. while, the control limit (±3sd) is the upper control limit is 26.36 u/l and the lower control limit is 13.13 u/l with an average value of 19.75 u/l. figure 2. levey-jennings graph based on initial value of sgpt. levels homemade lyophilized control serum +3sd; 26.36 +2sd; 24.16 mean; 19.75 -2sd; 15.34 -3sd; 13.13 10 12 14 16 18 20 22 24 26 28 s g o t l e v e l (u /l ) grafik levey-jennings based on initial value of sgot +3sd; 7.89 +2sd; 7.05 mean; 5.35 -2sd; 3.65 -3sd; 2.81 2 3 4 5 6 7 8 9 s g p t l e v e l (u /l ) grafik levey-jennings berdasarkan nilai awal sgpt week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 5 9 based on figure 2, it figured out that the warning limit (±2sd) of the control serum lyophilized homemade for sgpt examination, namely the upper warning limit (+2sd) is 7.05 u/l and the lower warning limit (-2sd) is 3.65 u/l. while the control limit (±3sd), the upper control limit is 7.89 u/l and the lower control limit is 2.81 u/l with an average value of 5.35 u/l table 2. results of homemade lyophilized control serum stability examination sgot parameter at temperature -2°c to -4°c week 1 2 3 4 5 6 7 8 amount 102 102 102 95 94 93 92 89 mean 20.4 20.4 20.4 19 18.8 18.6 18.4 17.8 sd 1.67 1.67 1.52 1 1.48 1.14 1.14 0.84 cv (%) 8.20 8.20 7.43 5.26 7.89 6.13 6.20 4.70 table 2 showed the average data for weekly examination results, standard deviation, and cv stability of lyophilized homemade control serum sgot parameters at storage temperature -2°c to -4°c for 8 weeks of storage. table 3. results of homemade lyophilized control serum stability examination sgot parameter at temperature (-20°c) week 1 2 3 4 5 6 7 8 mean 19.8 20.2 19.6 20 19.6 18.8 18.6 18.2 sd 1.30 1.64 1.95 1.41 0.89 0.84 0.89 0.45 cv (%) 6.60 8.13 9.94 7.07 4.56 4.45 4.81 2.46 table 3 showed the average data of weekly examination results, standard deviation, and cv stability of lyophilized homemade control serum sgot parameters at storage temperature (-20 °c) for 8 weeks of storage. table 4. results of homemade lyophilized control serum stability examination sgpt parameters at temperature -2°c to -4°c week 1 2 3 4 5 6 7 8 mean 4.62 4.5 4.4 4.32 4.18 4 3.98 3.96 sd 0.27 0.38 0.37 0.39 0.22 0.49 0.59 0.66 cv (%) 6.0 8.46 8.50 9.02 5.18 12.37 15.01 16.71 table 4 showed the average data of weekly examination results, standard deviation, and cv stability of lyophilized homemade control serum sgpt parameters at storage temperature -2°c to -4°c for 8 weeks of storage. ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 6 0 table 5. results of homemade lyophilized control serum stability examination sgpt parameters at temperature (-20°c) week 1 2 3 4 5 6 7 8 mean 4.68 4.6 4.62 4.56 4.3 4.28 4.2 4.18 sd 0.29 0.33 0.33 0.38 0.37 0.27 0.47 0.61 cv (%) 6.30 7.21 7.24 8.29 8.54 6.48 11.16 14.68 table 5 showed the average data of weekly examination results, standard deviation, and cv stability of lyophilized homemade sgpt control serum parameters at storage temperature (-20°c) for 8 weeks of storage. in tables 2 and 3, it presented that the mean data, standard deviation, and cv stability of lyophilized homemade sgot control serum parameters during 8 weeks of storage at temperature -2°c to -4°c and (20°c) which can be displayed in graphical in figure 3. in tables 4 and 5, it presented that the mean data, standard deviation, and cv stability of lyophilized homemade sgpt control serum parameters during 8 weeks of storage at temperature -2°c to -4°c and 20°c, which can be displayed in graphical in figure 4. figure 3. graph of weekly average results sgot levels of homemade lyophilized control serum figure 4. graph of weekly average results sgpt levels of homemade lyophilized control serum 17 18 19 20 21 s g o t l e v e l (u /l ) sgot levels (-2°c) – (-4°c) (-20°c) s g p t l e v e l (u /l ) sgpt level (-2°c) – (-4°c) (-20°c) week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 5 4.5 4 3.5 ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 6 1 figure 5. results of examination of homemade lyophilized serum parameters sgot 8 weeks of storage on the levey-jennings chart based on figure 5, it displayed that the results of the examination of the stability of the homemade lyophilized control serum on the levels of sgot stored in the freezer at a temperature of -2°c to -4°c and temperature (-20°c) with a storage time of 8 weeks were stable because they did not exceed the warning limit (±2sd) and control limit (±3sd). figure 6. results of examination of homemade lyophilized serum parameters of sgpt 8 weeks of storage on the levey-jennings chart based on figure 6, it displayed that the results of the examination of the stability of the homemade lyophilized control serum on the levels of sgpt stored in the freezer at a temperature of -2°c to -4°c and temperature (-20°c) with a storage time of 8 weeks were stable because they did not exceed the warning limit (±2sd) and control limit (±3sd). 10 12 14 16 18 20 22 24 26 28 s g o t l e v e l (u /l ) stability of sgot level 2 3 4 5 6 7 8 9 s g p t l e v e l (u /l ) stability of sgpt levels +3sd ; 26.36 +2sd ; 24.16 mean ; 19.75 -2sd ; 15.34 -3sd ; 13.13 week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 +3sd ; 26.36 +2sd ; 24.16 mean ; 19.75 -2sd ; 15.34 -3sd ; 13.13 ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 6 2 figure 7. graph of sgot linear regression test at temperature (-2) to (-4) °c based on figure 7, it presented that the sgot level of homemade lyophilized control serum stored in a freezer temperature -2°c to -4°c for 8 weeks obtained the linear equation y = -0.4024x + 21.036 with r2 = 0.9097. value analysis r2 (r square) or the coefficient of determination is 0.9097, which means that the storage time has an effect of 90.97% on the stability of sgot stored at -2°c to -4°c. while the rest (100% 90.97% = 9.03%) influenced by other variables outside this regression equation or variables that are not examined (error component). figure 8. graph of sgot linear regression test at temperature (-20°c) based on figure 8, it displayed that the sgot level of homemade lyophilized control serum stored in a freezer temperature (-20°c) for 8 weeks obtained a linear equation y = -0.2619x + 20.529 with r2 = 0.7871. value analysis r2 (r square) or the coefficient of determination is 0.7871, which means that the storage time has an effect of 78.71% on the stability of sgot stored at a temperature of (-20°c). while, the rest (100% 78.71% = 21.29%) influenced by other variables outside this regression equation or variables that are not examined (error component). y = -0.4024x + 21.036 r² = 0.9097 17 18 19 20 21 0 1 2 3 4 5 6 7 8 9 s g o t l e v e l (u /l ) storage time (week) kadar sgot pada suhu (-2°c) – (-4°c) y = -0.2619x + 20.529 r² = 0.7871 18 19 20 21 0 1 2 3 4 5 6 7 8 9 s g o t l e v e l (u /l ) storage time (week) sgot level at temperature (-20°c) ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 6 3 figure 9. graph of sgpt linear regression test at temperature -2°c to -4°c based on figure 9, it presented that the sgpt level of homemade lyophilized control serum stored in a freezer temperature (-2) to (-4) °c for 8 weeks obtained a linear equation y = -0.1019x + 4.7036 with r2 = 0.9671. value analysis r2 (r square) or the coefficient of determination is 0.9671, which means that the storage time take effect 96.71% of the stability of sgpt stored at temperature -2°c to -4°c. while, the rest (100% 96.71% = 3.29%) influenced by other variables outside this regression equation or variables that are not examined (error component). figure 10. graph of sgpt linear regression test at temperature -20°c based on figure 10, it displayed that the sgpt level of homemade lyophilized control serum stored in a freezer temperature (-20°c) for 8 weeks obtained a linear equation y = -0.0807x + 4.7907 with r2 = 0.9147. value analysis r2 or the coefficient of determination is 0.9147, which means that the storage time has an effect of 91.47% on the stability of sgpt stored at a temperature of -20 °c. while, the rest (100% 91.47% = 8.53%) is influenced by other variables outside this regression equation or variables that are not examined (error component). y = -0.1019x + 4.7036 r² = 0.9671 0 1 2 3 4 5 6 7 8 9 s g p t l e v e l (u /l ) storage time (week) sgpt levels at temperature (-2°c) – (-4°c) y = -0.0807x + 4.7907 r² = 0.9147 0 1 2 3 4 5 6 7 8 9 s g o t l e v e l (u /l ) storage time (week) sgpt level at temperature (-20°c) 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.8 4.6 4.4 4.2 4.0 3.8 ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 6 4 discussion in the calculation of the average level of sgot stored at a temperature of -2°c to -4°c and temperature (-20°c) entered on the levey-jennings’ chart, there was no mean result from week 1 to week 8 that exceeds the warning limit (±2sd) and control limit (±3sd). at the level of sgpt stored at a temperature of -2°c to -4°c and temperature (-20°c) result average calculation included in the levey-jennings’ chart, there was no mean result from week 1 to week 8 that crossed the warning limit (±2sd) and control limit (±3sd). these results indicate that homemade lyophilized control serum stored at (-2) to (-4) °c and (-20°c) for 8 weeks were stable for sgot and sgpt parameters. the results of cv calculations obtained on sgot examination stored for 8 weeks at freezer temperature, namely (-2) to (-4) °c and temperature (-20°c), which showed different results (table 4 and 5). the results of the cv calculation do not exceed the ccv limit, it is stable and the inspection method used is compatible for the sgot parameter. cv calculation results on sgpt inspection at freezer temperature -2°c to -4°c and temperature (-20°c), which showed different results (table 4 and 5). the results of the cv calculation on the sgpt examination did not exceed the ccv limit, so it was stable for the sgpt parameters and the examination method used was compatible. the linear regression test showed the value of the coefficient of determination (r square), which serves to find out what percentage of the influence is given by the independent variables (temperature and storage time) simultaneously on the dependent variable (sgot and sgpt levels). r2 has a value between 0 – 1 with the provision that the closer to the number one, the better. if the r square is 0.6, it means 60% of the distribution of the dependent variable is explained by the independent variable. the remaining 40% is explained by variables outside the independent variable (error component). if the value of the r square is small, it means that the error component is high. previous research stated there was no significant difference between sgot and sgpt examination stored at -20°c. it concludes that prepared homemade lyophilized human serum can be used up to 9 months if stored at -20°c and 7 months at 28°c (2). the sgot and sgpt levels decreased consistently in the time interval of sample storage, but the rate of change of the decrease in levels was considered more influential than storage temperature (16). the previous results studies in the collected serum examined enzyme parameters, including sgot and sgpt, which were stable for up to 30 days at a temperature of -20°c. the variation was not clinically significant. decreased sgpt levels ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 6 5 can cause by loss of enzyme activity on long storage and interference by lactate dehydrogenase (ldh) in alt (17). all common clinical chemistry analytes examined, aside from serum amylase, showed adequate stability following up to 30 days of storage at -20°c. these results indicate that deep freezing at -20°c could serve as an effective tool for additional analyses at later time points along with research purposes, which require that samples be stored for longer until batch analysis can be conducted (18). in this stability test, the factors that can affect the stability of sgot and sgpt are storage temperature and storage time. the enzymatic reactions in sgot and sgpt influenced by several factors, one of which is the influence of temperature, both the specimen storage temperature and the working temperature. at high temperatures, the protein portion of the enzyme begins to break down (denature) by that inhibited the reaction (18). storage temperature is an essential factor that causes the stability of the control material. the control serum stability temperature when it is stored at -20℃ is relatively better than storage in the refrigerator (2). the working reagent factor and the storage time of the reagents can also affect the results of the examination, due to how the working reagents are used when mixing, and the working reagents that have been mixed and stored, even though the expiration date on the reagents have not exceeded the time limit stated in the reagent kit or guidelines work (19). another factor that can affect the measurement of the level of examination parameters is turbidity because turbidity in the serum can affect the measurement absorbance. the process of making homemade lyophilized control serum can also affect the results of the examination. incomplete centrifugation and contamination are unavoidable but can be reduced to a minimum (20). the type of freezer used in the laboratory to determine the level of stability of pooled sera. most small laboratories use household refrigerators whose freezer temperature is higher than the freezer for serum storage (21). each laboratory has standard procedures and different quality reagents and instruments, so further research is needed if you want to use the homemade lyophilized serum as a commercial control serum with different types of parameters and for a longer time. many things can affect the results of control serum or lyophilized homemade at the pre-analytical stage, which consists of patient preparation, specimen identification, specimen collection and storage, specimen handling, specimen ina. j. med. lab. sci. tech. 2023; 5(1): 53–67 kadek profit hartani & anik handayati 6 6 delivery, specimen processing and preparation, and analysis consisting of specimen examination, equipment maintenance and calibration, reagent quality test, accuracy test, and post-analytic, which consists of writing, interpreting, and reporting results (3). the advantage of using homemade lyophilized serum is that it is easy to obtain and relatively inexpensive, while commercial control serum is expensive. however, the stability of commercial control serum is longer than the homemade lyophilized serum, especially in certain analytes. with proper storage and handling homemade lyophilized serum can be used instead of commercial control serum for internal and external quality assurance (20). conclusions there is an effect of storage time on the stability of sgot and sgpt levels stored in the freezer at -2°c to -4°c and -20°c for 8 weeks, but the results of sgot and sgpt stored for 8 weeks still meet the requirements levey-jennings control chart. author contributions kadek profit: original draft writing and reviewing and data curation, investigation and data collection. anik handayati: conceptualization, methodology and investigation. conflict of interest there are no conflicts of interest. references 1. hedayati m, razavi sa, boroomand s, kheradmand kia s. the impact of pre-analytical variations on biochemical analytes stability: a systematic review. journal of clinical laboratory analysis. john wiley and sons inc; 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60(7): 1003-1010. doi: 10.1515/cclm-2022-0063 18. dupuy am, cristol jp, vincent b, bargnoux as, mendes m, philibert p, badiou s. stability of routine biochemical analytes in whole blood and plasma/serum: focus on potassium stability from lithium heparin. clinical chemistry and laboratory medicine (cclm). 2018; 56(3): 413421. doi: 10.1515/cclm-2017-0292 19. warsyidah aa. stability test of working reagent storage after mixing at room temperature against blood urea examination. media laboran. 2016;6:1–4. https://uit.ejournal.id/medlab/article/view/299 20. pawlik-sobecka l, sołkiewicz k, kokot i, kiraga a, płaczkowska s, schlichtinger am, kratz em. the influence of serum sample storage conditions on selected laboratory parameters related to oxidative stress: a preliminary study. diagnostics. 2020; 10(1): 51. doi: 10.3390/diagnostics10010051 21. maulidiyanti ets the effect of storage time on pooled sera in freezers on the quality of clinical chemical examination. j med lab sci. 2021;4(2):78–82. doi: 10.21070/medicra.v4i2.1613 https://doi.org/10.1515/cclm-2019-0732 https://doi.org/10.33846/hn61203 https://doi.org/10.1373/clinchem.2017.277186 https://doi.org/10.52403/ijrr https://doi.org/10.1515/cclm-2019-0586 https://doi.org/10.1515/cclm-2022-0063 https://doi.org/10.1515/cclm-2017-0292 https://doi.org/10.3390/diagnostics10010051 68 comparison of leaves and bark mangkokan (nothopanax scutellarium) extracts against the death of house flies (musca domestica) darmadi1, harni sepriyani1 1department of medical laboratory technology, faculty of health, universitas abdurrab, riau, indonesia correspondence: darmadi, jl. riau ujung no. 73, tampan, air hitam, payung sekaki, air hitam, payung sekaki, pekanbaru city, riau, indonesia zip code : 28291 email: darmadi@univrab.ac.id received: june 29, 2020 revised: july 8, 2020 accepted: august 20, 2020 abstract flies (musca domestica) are vectors of disease that can transmit to humans. generally, these flies controlled by using chemical insecticides. excessive use of insecticide causes resistance and environmental pollution. the alternative way in fly control is to use natural ingredients from the extract of mangkokan stem bark and leaves (nothopanax scutellaria). this study aims to determine the ratio of fly mortality after administration of ethanol extract of bark and methanol extract of mangkokan leaves (n. scutellarium). the method used in this study is a post-test only control group design. the average mortality rate of house flies using ethanol extract of mangkokan stem bark (n. scutellarium) was 5 for 55 minutes while methanol extract of mangkokan leaves (n. scutellarium) was 5 for 135 minutes with a p-value of 0.374 (p>0.05). it concludes that there is no significant difference in fly mortality with the ethanol extract of stem bark and methanol extract of mangkokan leaves (n. scutellarium). result of this study shows that the ethanol extract of stem bark and mangkokan leaf extract (n. scutellarium) are equally potential natural insecticides. keywords nothopanax scutellaria, leaves ethanol extract, barks ethanol extract, musca domestica introduction flies (musca domestica) are one of the common insects that are easy to found in a clean or dirty environment. flies easily to found in homes, markets, livestock, landfills, and waste. flies also interfere in terms of calm and cleanliness (1). in general, flies are a vector of disease that could transmit to humans. transmission occurred when flies carried diseases from dirty sources, for examples are garbage, mailto:darmadi@univrab.ac.id ina j med lab sci tech 2020; 2(2): 68-75 darmadi, et al. 6 9 faeces, household waste and animal waste. m. domestica as a vector meaning that flies carry and transfer disease from one place to another place. flies transmitted disease from the mouth or other body parts such as contaminated feet and alight on human food or animal food (2). flies controlled by chemical insecticides. this method of control would cause environmental pollution. therefore, an alternative way to fly control is to use natural ingredients. natural insecticides toxicity is low, so it did not have a negative effect and safe (3). some plants could use as a natural insecticide, one of them is leaves and bark ethanol extract of mangkokan (nothopanax scutellarium). mangkokan leaves extract used as larvacide, which given mortality effect on culex sp, reported by ahdiah and purwani (4). marina and astuti (5), have been added that mangkokan and pandan leaves extract as a repellent in aedes albopictus was able to reject it. the utilization leaves and barks mangkokan extract used as a natural insecticide on several insects. the compound of leaves and barks mangkokan extract were tannins, saponins, and flavonoids. research carried out by darmadi and anita (6), lancium domesticum corr methanol extract containing were flavonoids, saponins, and triterpenoids at 5, 10, 15% concentration as a natural insecticide for flies. based on the background above, the researcher was interested in the comparison between leaves and barks ethanol mangkokan extract to m. domestica mortality. fig 1. bark mangkokan (nothopanax scutellarium) fig 2. leaves mangkokan (nothopanax scutellarium) materials and methods the research was conducted at the researcher's home because the covid (corona virus disease) pandemic of research could not carry out in the micropar ina j med lab sci tech 2020; 2(2): 68-75 darmadi, et al. 7 0 laboratory of abdurrab university for one month. research is an experiment in laboratory and post test only control group design. the population for this research was m. domestica from a farm field in pekanbaru, indonesia. samples were 75 m. domestica, five flies for each extract and three times for each extract. the equipment was analytical scales, knives, measuring flasks, measuring cups, stopwatches, erlenmeyer, spray bottles, breeding boxes, research boxes, blenders, evaporators and dark bottle. while, the phytochemical test equipment is dropper pipette, drip plate, spatula, test tube, test tube rack, volume pipette, suction ball, filter paper, cotton. the ingredients were leaves and barks mangkokan, magnesium, hydrochloric acid, fecl3 1 b/v%, chloroform, sulfuric acid, major reagents, lieberman burchard reagents. musca domestica preparation m. domestica was taken from the farm field. the characteristics of the flies caught were observed to ensure that they were m. domestica to be put in the box. leaves and barks mangkokan extract preparation leaves and barks mangkokan washed and dried by aerated, then chopped into small pieces and blended. the 250 grams for each sample was extracted by maceration procedures in ethanol for 3 x 24 hours. the extract was filtered and separated between residue and filtrate. the filtrate put into the evaporator to separated between extract and solvent. furthermore, viscous extract put in erlenmeyer flask. the result of thick extract diluted to be 10, 20, and 30% extract concentration (7). formula of extract (%) = 𝑔𝑟𝑎𝑚 𝑉𝑜𝑙𝑢𝑚𝑒 x 100% the preparation of various concentration of extract has conducted by adding 0.5 g; 1 g; and 1.5 g of extract then diluted in 5 ml of aquadest to produce a concentration 10%; 20% and 30% of the extract, respectively. transfultrin 0.1% (v/v) used as a positive control. meanwhile, aquadest applied as negative control. extract test samples extract and control put in spray bottles. as much as five test boxes were prepared and in each box were added by five flies. in addition, extract was sprayed as much as 8 – 10 spray with a distance of approximately 10 cm for each concentration. observation every box for 30 minutes and calculated died flies and mortality time. ina j med lab sci tech 2020; 2(2): 68-75 darmadi, et al. 7 1 results musca domestica mortality for barks extract (nothopanax scutellarium) fig 3. positive control musca domestica died fig 4. negative control of musca domestica is still alive fig 5. musca domestica die at a concentration of 10% fig 6. musca domestica die at a concentration of 20% fig 7. musca domestica die at a concentration of 30% ina j med lab sci tech 2020; 2(2): 68-75 darmadi, et al. 7 2 musca domestica mortality for leaves extract (nothopanax scutellarium) fig 8. positive control musca domestica died fig 9. negative control of musca domestica is still alive fig 10. musca domestica die at a concentration of 10% fig 11. musca domestica die at a concentration of 20% fig 7. musca domestica die at a concentration of 30% ina j med lab sci tech 2020; 2(2): 68-75 darmadi, et al. 7 3 observation result based on observation of barks extract on m. domestica mortality, the result of research presented on table 1. based on table 1, m. domestica mortality for barks ethanol extract at a concentration of 30% with 5 flies’ deaths for 30 minutes, at 20% concentration of dead 5 flies for 45 minutes and at 10% concentration for 5 flies for 1 hour. for negative control (–) there were no flies that died. tabel 1. musca domestica mortality for barks extract based on research of leaves extract on m. domestica mortality, the result of research presented on table 2. based on table 2, m. domestica mortality for leaves ethanol extract at a concentration of 30% with 5 flies’ deaths for 10 minutes, at 20% concentration of dead 5 flies for 20 minutes and at 10% concentration for 5 flies for 25 hours. for negative control (–) there were no flies that died. tabel 2. musca domestica mortality for leaves extract no concentration musca domestica mortality average death time i ii iii 1 10% 5 5 5 5 25 minutes 2 20% 5 5 5 5 20 minutes 3 30% 5 5 5 5 10 minutes 4 control (+) 5 5 5 5 2 minutes 5 control (–) 0 0 0 0 0 minutes discussion based on this research, it showed that there was five (5) death m. domestica at a concentration of 10%, 20% and 30% with a total duration of 135 minutes for barks extract and 55 minutes for leaves extract. based on data indicated that the death of m. domestica using ethanol extract from mangkokan barks and leaves have the same effect as using insecticide, even though the death time varies within 1 hour. death of flies caused by the presence of secondary no concentration musca domestica mortality average death time i ii iii 1 10% 5 5 5 5 1 hour 2 20% 5 5 5 5 45 minutes 3 30% 5 5 5 5 30 minutes 4 control (+) 5 5 5 5 1 minutes 5 control (–) 0 0 0 0 0 minutes ina j med lab sci tech 2020; 2(2): 68-75 darmadi, et al. 7 4 metabolite compounds found in the ethanol mangkokan extract. based on research conducted by eden, et al. (5) on the phytochemical test of leaves methanol mangkokan extract contain secondary metabolites of flavonoids and saponins. while the leaves ethanol mangkokan extract contains flavonoid compounds, saponins, tannins and alkaloids (8). the two extract types have differences content of secondary metabolites. leaves ethanol mangkokan extract contains more secondary metabolites when compared with bark extract. based on those contents, it concluded that ethanol extract from mangkokan leaves has the highest death rate of m. domestica rather than the bark extract. the total duration of death time is 55 minutes for ethanol mangkokan leaves extract while the bark extract has a period of death time of 135 minutes. the mechanism of flavonoid compounds action is by attacking the respiratory tract and metabolic system in insects. respiratory organs that are attacked in the form of spiracles on the surface of the body and will cause wilting of the nerves, and damage to spiracles so that the insects cannot breathe and eventually die (3). also, flavonoid compounds will damage cell wall permeability and inhibit the work of enzymes that affect the metabolic system that interferes with eating activity and can act as a stomach poison in insects (9). in addition to flavonoid compounds, saponin is one of the secondary metabolites. saponins are compounds that are toxic to insects because they can enter the body of insects which can be toxic to the stomach, which causes decrease absorption of digestive enzyme activity and can interfere with the body's metabolic processes (9). saponin compounds also interfere with the physical exterior (cuticles) that are similar to detergent properties. this content can disrupt the process of changing the skin (moulting) and can reduce the surface tension of the skin so it damages the surface of the skin and can cause death in insects (10). tannin is the highest content after alkaloids. tannins are polyphenol compounds that can form complex compounds with tannin proteins which cannot be digested by the stomach and have a binding ability to protein, carbohydrates, vitamins, and minerals. tannins can interfere with insects in digesting food because tannin will bind to proteins in the digestive system that are needed by insects for growth, it predicted that the digestion process of flies (m. domestica) becomes disrupted due to these tannins (4). conclusions the conclusion of this research is there was five (5) death m. domestica at a concentration of 10%, 20% and 30% with a total duration of 135 minutes for barks extract ina j med lab sci tech 2020; 2(2): 68-75 darmadi, et al. 7 5 and 55 minutes for leaves extract. there is no significant difference in the death of m. domestica by added barks and leaves ethanol mangkokan extract. given those points, the extract of mangkokan leaves and barks are both capable as natural insecticides. conflict of interest there are no conflicts of interest. references 1. ahdiah, i., and purwani, i. k. 2015. effect of mangkokan leaf extract (nothopanax scutellarium) as mosquito larvasida culex sp. journal of science and art in its, 3(1) : 32-36. 2. aseptianova., fitri., w. t. and nurina., n. 2017. efectiveness of utilization plant conservation as electrical insectiside for dbd. bioexperiment: journal of biological research, 3(2) : 10-19. 3. azizah., adnan, m. r., and su'udi, m. 2018. potential of sengor wood sawdust as a botanical insecticide. journal of bioscience, 4(1) : 45–54. 4. darmadi., and anita, d. 2018. mortality test for house flies (musca domestica) after giving duku skin ekstract. clinical journal science, 6(1) : 18-23. 5. eden, w. t. et al. 2016. antioxidant activity of mangkokan leaf methanol extract (polyscias scutellaria (burn. f.) fosberg). indonesia pharmaceutical media, 11(2) : 1126–1135. 6. marina, r., and astuti, e. p. 2012. potential of pandan leaves (pandanus amaryllifolius) and mangkokan (nothopanax scutellarium) as repellent of aedes mosquitoes albopictus. aspirator, 4(2) : 85–91. 7. mirnawaty., supriadi., and jaya, b. 2012. test the efectiveness of lansat skin extract (lansium domesticum) as an electrict anti mosquito against aedes aegyipti. academic journal. 1(4) : 147–152. 8. poedji, h., and enggar, f. l. 2007. potential of musca domestica linn. as a vector some disease. briwijaya malang health journal, 23(3). 9. sadiah, s., herlina, n. and indriati, d. 2015. the effectiviness of a ethanol extract emulsion 70 percent of transplant leaves (nothopanax scutellarium (burm.f) merr) as hair growth stimulants, the journal fitophyhence, 4(1). 10. sembel, d. t. 2009. entomology of medicine. cv andi offset. yogyakarta. 11. yuliana, v., yamtama., and kadaruso, a. h. 2016. the inflamed applications of cambodian leaves (plumeria acuminata) againts the death of house flies (musca domestica). ward health journal, 13(1) : 300-306. 34 laboratory trial of protein determination in urine using different ph values of acetic acid and acetate buffer method dinar rahayu1, tuti rustiana1 1department of medical laboratory technology, sekolah tinggi analis bakti asih, bandung, indonesia correspondence: dinar rahayu, jl. padasuka atas no.233, padasuka, cimenyan, city of bandung, west java, indonesia zip code : 40192 email: dinarrahayu91071@gmail.com received: february 6, 2020 revised: march 17, 2020 accepted: april 10, 2020 abstract the determination of protein in urine is important in clinical examination along with other parameters in urine. the presence of protein in urine can be interpreted that there is a disorder in kidney. acid and heat coagulations method is still widely used in many areas to determine protein in urine. in this method, the characteristic of protein that will precipitate in the presence of acid or if exposed to heat is deployed to gain information about the amount of protein. the geater amount of protein, the more prominence is the coagulation. urine ph also varies according to the condition, classic acidosis will give an acidic urine and the presence of ammonium producing bacteria can cause basic urine. in this research acetic acid method with 6% of ch3cooh and ph value of 2.9 and buffer acetic with ph 4.5 are used to determine the certain amount of protein (+3 value, corresponds with 2-4 mg/dl protein in urine) in varied ph values of urine samples. in order to compare the results, first in control urine with ph 6.8 the results of both methods is compared with mann-whitney test and shows no significant different, then the kruskall-wallis test is used to compare the results in other ph values to control and the test is shown also there are no significant difference. this shows that either acetic acid at ph 2.9 or acetic buffer at ph 4.5 can be used to determine protein amount in urine. keywords proteinuria, acetic acid, acetate buffer, urine ph, buffer introduction under normal physiological state, urine is expected to be protein free. high molecular weight proteins in plasma (e.g., albumin and globulin) could not pass through the filtration membrane due to the effects of the size barrier and charge barrier of the glomerular capillary filtration membrane. low molecular weight proteins (e.g., β2microglobulin (β2-m), α1-microglobulin (α1-m), and lysozyme), however, can freely pass through the filtration membrane, although the filtration amount is low and 95% of these proteins are reabsorbed when entering the proximal convoluted tubule (1,2). the final urine protein content is ina j med lab sci tech 2020; 2(1): 34-41 dinar rahayu, et al. 3 5 therefore low (only 30-130 mg/24h) and consists primarily of plasma albumin (40%), immunoglobulin fragments (15%), other plasma proteins (5%), and urinary systemoriginating tissue proteins (40%). the protein concentration in a random urine sample is 080 mg/l, and the results of qualitative tests for urokinase protein are typically negative. when the urine protein exceeds 150 mg/24h or the concentration is above 100 mg/l, the result for the qualitative protein test becomes positive. this is known as proteinuria (3). proteinuria is common finding in chronic renal failure patients and current evidence indicates that the presence of proteinuria is an early marker of an increased risk of progessive kidney disease, poor cardiovascular and death (2). as the measurement and sampling procedures for proteinuria assessment have not been standardized yet, it is of clinical importance to take into account different types of urinary proteins, albumins, laboratory techniques, and urine sampling methods in order to have the best approach for an individual patient (4). total urinary protein can be assessed using dipstick, precipitation, and electrophoresis methods. urine specimen for proteinuria assessment can be obtained either from a timed collection or a spot urine sample. nevertheless, currently spot urine proteinor albumin-to-creatinine ratios are preferred to a 24-hour urine sample in routine practice (4). the proteinuria is commonly assessed by the heat and acetic acid test. now dipstick test is replacing the old methods (5). heat coagulation test may be used in under-resourced settings as an alternative to dipstick testing or other methods that are unavailable or too costly. a test tube is filled to two-thirds with urine. a few drops of dilute acetic acid are added to make the urine sample acidic. the upper part of the test tube containing urine is heated (but not boiled) over a burner. acidic environment is to ensure that the coagulation formed is protein because on heating, phosphates also coagulate but dissolves in acidic environment (6). the result is considered to be negative (no protein presents in sample) if there is no cloud in solution after the test, a +1 (0.1 g/dl) result if there is a definite cloudiness, if held against a typed papers, letters typed can be seen through the cloud in upper part of the test tube, +2 (<0.3 g/dl) if there is definite cloudiness, if held against the typed paper, letters typed are seen as faint lines in the backgound at top of the test tube, +3 (0.3-1.0 g/dl) if there is a definite cloudiness, if held against the typed paper, letters typed are seen as faint lines in the backgound at top of the test tube, +4 (>1.0 g/dl) if there is a thick solid precipitation, clot present at top of the test tube (6,7). the newer method after heat and acid coagulation is dipstick method to detect ina j med lab sci tech 2020; 2(1): 34-41 dinar rahayu, et al. 3 6 proteinuria. the reaction is based on the phenomenon known as the ‘protein error’ of ph indicators (a dye) where an indicator that is highly buffered will change color in the presence of proteins (anions) as the indicator releases hydrogen ions to the protein. the dye is acid-base indicators such as tetra bromophenol blue. when the dye is buffered at ph 3, it is yellow; the addition of increasing concentration of protein changes the color to geen then to blue. the developed color is compared with a color chart which allows protein concentration to be gaded from trace to +4, corresponding to concentrations from 1 to 10 mg/dl to geater than 500 mg/dl (8). at a constant ph, the development of any geen color is due to the presence of protein. colors range from yellow to yellow-geen for negative results and geen to geen-blue for positive results. but the clinical judgement is required to evaluate the significance of trace results. a color to be defined as proteinuria differ from each manufacturer, but usually geater than 30 mg/dl indicates significant proteinuria (8). urine protein mainly consists of albumin. this protein can reversibly and drastically change its conformation when exposed to changes in solution ph (transitions occurring at ph 2.7, 4.3, 8, and 10) (9). as for urine, the normal ph range is 5 to 7, with low urine ph can be caused by high protein diet because the increased endogenous acid production from sulfurcontaining amino acids or metabolic acidosis (e.g., chronic diarrhea) while high urine ph (usually >7) is caused by metabolic alkalosis (e.g., vomiting), distal renal tubular acidosis, urea-splitting organisms (e.g., proteus), urine that is infected will become alkaline over time due to formation of ammonia (nh3) from bacterial urease, urine that is exposed to air for a long time can also have elevated ph due to loss of co2 from urine (8). so the aims of this study was to examine the results from acetic acid test with using 6% of acetic acid (ph 2.9) and acetate buffer (ph 4.5) of urine in varied ph values or urine samples with addition of known amount of protein to mimic proteinuria conditions. materials and methods apparatus used in this research are 100 ml beaker glasses, dirui dr-7000e photometer, stirrer, micropipette, spiritus burner, test tubes and test tube-rack, manti lab mt-103 ph meter, fanmed 80-i centrifuge, and its centrifuge tubes. materials used are biuret reagent. biuret reagent is prepared by dissolved 1.5 g of copper sulfate pentahydrate (cuso4.5h2o) and 6 g of sodium potassium tartrate (knac4h4o6.4h2o) in 500 ml of water, and add 300 ml of 10% (w/v) of naoh, and add 1 g of potassium iodide and make a 1l solution (10). ina j med lab sci tech 2020; 2(1): 34-41 dinar rahayu, et al. 3 7 acetic acid (ch3cooh) 6%, sodium acetate (ch3coona), acetate buffer (ph 4.5) is made with 1.544 g of sodium acetate anhydrous and 0.076 g of acetic acid to make 200ml of solution. ph is checked with manti lab mt-103 ph-meter and adjusted with hcl or naoh to achieve ph of 4.5. serum and urine were taken from four healthy volunteers from students of sekolah tinggi analis bakti asih wtih normal plasma protein level and negative test of protein in urine. to make serum, 3 ml of blood is drawn from vein and put in centrifuge tube for 10 minutes before being centrifuged at 1500 rpm for 15 minutes to separate serum from blod clot. carefully serum is pipetted and tested for protein with biuret method. twenty microliters of serum is added to 1ml of biuret reagent. the mixture is incubated for 5 minutes at room temperature (25oc) then put in photometer. the reading of absorbance at 546 nm is compared to absorbance of protein standard solution and protein concentration in serum is calculated. as much as 5 g/dl of protein in serum is obtained. urine sample that is used is a random time and collected in a clean and dry plastic container with a lid. the urine specimen is tested with two methods to check its protein contents then proceed to the addition of serum to make simulated urine that contains protein at +3 (4 mg/dl). for each of ph group, a 40 ml portion of urine is needed. simulated urine with protein that can be detected by heat and acid coagulation is achieved by adding serum with the known concentration of protein to urine, the result is urine sample with protein concentration is 4 mg/dl (+3). urine batch is divided to make urine samples with different ph by adding ammonium hydroxide or citric acid (ph range is 6, 6.5, 6.8, 7, and 7.5). we made four replications for each method in each ph value. simulated urine with ph 6.8 is chosen as normal ph. to compare the median value between acetic acid results goup and buffer acetate results goup at the normal condition (control at ph 6.8), the mann-whitney test is used, then kruskal-wallis test is used for statistical treatment of these data (among three groups, i.e.; acetic acid results, acetate buffer result at each ph , and control) because it analyzes whether there is a difference in the median values of three or more independent samples (11). all experiment is conducted in chemistry laboratory of sekolah tinggi analis bakti asih bandung, west javaindonesia during march until may 2019 and this study was approved by the ethics committee of sekolah tinggi analis bakti asih bandung. ina j med lab sci tech 2020; 2(1): 34-41 dinar rahayu, et al. 3 8 results the results of this study can be seen in the following table 1: table 1. results of experiments treatment test results replicate 1 replicate 2 replicate 3 replicate 4 blank acetic acid ( ) ( ) ( ) ( ) acetic buffer ( ) ( ) ( ) ( ) ph 6.0 acetic acid ( +++ ) ( +++ ) ( +++ ) ( +++ ) acetic buffer ( +++ ) ( +++) ( +++ ) ( +++ ) ph 6.5 acetic acid ( +++ ) ( ++) ( +++ ) ( +++ ) acetic buffer ( +++ ) ( +++ ) ( +++ ) ( +++ ) ph 6.8 acetic acid ( ++ ) ( ++ ) ( ++ ) ( +++ ) acetic buffer ( ++ ) ( +++ ) ( +++ ) ( +++ ) ph 7.0 acetic acid ( ++ ) ( ++ ) ( ++ ) ( +++ ) acetic buffer ( ++ ) ( ++ ) ( ++ ) ( +++ ) ph 7.5 acetic acid ( ++ ) ( ++ ) ( ++ ) ( +++ ) acetic buffer ( ++ ) ( ++ ) ( ++ ) ( ++ ) a = (+++) b = (+++) a = (+++) b = (+++) a = (+++) b = (+++) a = (+++) b = (+++) a; left test tube is simulated urine sample with acetic acid. b; right test tube is simulated urine sample with acetic buffer fig 1. results for simulated urine samples at ph 6.0. white arrows indicated the cloud/precipitation of protein. a = (+++) b = (+++) a = (++) b = (+++) a = (+++) b = (+++) a = (+++) b = (+++) a; left test tube is simulated urine sample with acetic acid. b; right test tube is simulated urine sample with acetic buffer fig 2. results for simulated urine samples at ph 6.5. white arrows indicated the cloud/precipitation of protein. ina j med lab sci tech 2020; 2(1): 34-41 dinar rahayu, et al. 3 9 a = (+++) b = (++) a = (+++) b = (++) a = (++) b = (++) a = (++) b = (+++) a; left test tube is simulated urine sample with acetic acid. b; right test tube is simulated urine sample with acetic buffer fig 3. results for simulated urine samples at ph 6.8. white arrows indicated the cloud/precipitation of protein. a = (++) b = (++) a = (++) b = (++) a = (++) b = (++) a = (+++) b = (+++) a; left test tube is simulated urine sample with acetic acid. b; right test tube is simulated urine sample with acetic buffer fig 4. results for simulated urine samples at ph 7.0. white arrows indicated the cloud/precipitation of protein. a = (+++) b = (++) a = (++) b = (++) a = (++) b = (++) a = (+++) b = (++) a; left test tube is simulated urine sample with acetic acid. b; right test tube is simulated urine sample with acetic buffer fig 5. results for simulated urine samples at ph 7.5. white arrows indicated the cloud/precipitation of protein ina j med lab sci tech 2020; 2(1): 34-41 dinar rahayu, et al. 4 0 the mann-whitney test between acetic acid result and acetate buffer result at control ph (6.8) shows no significant difference,that means we can say both group gave the same result at normal ph. then we continued to the kruskall-wallis test. it was used to compare the median values among groups in each ph compared to control. the test also shows there is no significant differences. discussion the determination of urinary protein is important for its significance use in clinical diagnosis (12). the rapid test can be performed by dipstick but the heat and acid coagulation test are still widely used. in determining proteinuria itself, classically this is done by a 24-hour collection, but as creatinine is excreted at a constant rate, a ratio of urine albumin to creatinine or protein to creatinine is sufficient in most patients (13). nevertheless in urinalysis if the protein persists and the amount is significant one can suspect the patients has proteinuria symptoms. in this experiments it has been shown that heat and acid coagulation test can be used in wide range of ph (figure 1-5). since there can be variation in urine ph from time to time because of many reasons among other the bacteria that produces ammonium hydroxide that can cause basic urine, or acidosis case which can make urine acidic. this implies to the environment of protein in urine. since each protein has a definite isoelectric point and albumin which is the dominant protein in urine has isoelectric point of 4.5 then the change of ph nearing to that point will cause albumin to coagulate which we can correspond to the amount of protein presents in urine (14). with +3 protein (0.2-0.4 g/dl) in urine samples, it has been shown in this experiment that acetic acid test is in accordance with acetic buffer test which used the prominent properties of protein that will coagulate in the presence of heat and/or acid. this process if known as denaturation of protein. that means the change of protein environment will cause several damages in protein structures. the clear sign of denaturation is precipitation in which soluble protein has lost several bonds in its structure and protein precipitates. as for these two methods, it has been shown that they can be used in many ph values of the urine samples. a method to be a useful method must endure and still gives a reliable result in several clinical conditions. conclusions according to the results of experiments and kruskall-wallis test, the determination of protein in urine at ph 6.5 to 7.5 can be done with acetic acid test with 6% (ph 2.9) or acetate buffer (ph 4.5). both of method were give +3 positive value which correspondent of 0.2-0.4 g/dl of protein. ina j med lab sci tech 2020; 2(1): 34-41 dinar rahayu, et al. 4 1 conflict of interest there are no conflicts of interest. references 1. stankov s v. definition of inflammation, 1. zhang a, huang s. progress in pathogenesis of proteinuria. int j nephrol. 2012;2012. 2. prakash m, phani nm, kavya r, supriya m. urinary peptide levels in patients with chronic renal failure. online j heal allied sci. 2010;9(3):1–3. 3. hong dsc, oh ih, park js, lee ch, kang cm, kim gh. evaluation of urinary indices for albuminuria and proteinuria in patients with chronic kidney disease. kidney blood press res. 2016;41(3):258–66. 4. toblli je, bevione p, di gennaro f, madalena l, cao g, angerosa m. understanding the mechanisms of proteinuria: therapeutic implications. int j nephrol. 2012;2012. 5. vaidyanathan k. textbook of biochemistry for medical students. textb biochem med students. 2016;(august). 6. beaufils ma an. mmm von d. the figo textbook of pregnancy hypertension. vol. 6, nephrologie et therapeutique. 2016. 200–214 p. 7. mauliddina j. detecting proteinuria: a comparison of diagnostic tests. paediatr indones. 2011;51(4):207–12. 8. kwong t, robinson c, spencer d, wiseman oj, karet frankl fe. accuracy of urine ph testing in a regional metabolic renal clinic: is the dipstick accurate enough? urol res. 2013;41(2):129–32. 9. pergande mr, cologna sm. isoelectric point separations of peptides and proteins. proteomes. 2017;5(1). 10. janairo, gerardo, linley sy, marianne, yap, leonisa, llanos-lazaro, nancy, robles j. determination of the sensitivity range of biuret test for undergraduate bio...: discovery service for universiti tunku abdul rahman. ejournal sci technol. 2011;6(5):p77-83. 11. nahm fs. nonparametric statistical tests for the continuous data: the basic concept and the practical use. korean j anesthesiol. 2016;69(1):8–14. 12. matica j. disease : national recommendations. 2017;27(1):153–76. 13. chen yt, hsu hj, hsu ck, lee cc, hsu kh, sun cy, et al. correlation between spot and 24h proteinuria: derivation and validation of equation to estimate daily proteinuria. plos one. 2019;14(4):1–12. 14. audain e, ramos y, hermjakob h, flower dr, perez-riverol y. accurate estimation of isoelectric point of protein and peptide based on amino acid sequences. bioinformatics. 2016;32(6):821–7. 24 severe anemia and six-month all-cause mortality in chronic kidney disease patients undergoing hemodialysis langgeng perdhana1, shofa chasani1,2 1hemodialysis unit, roemani muhammadiyah hospital semarang, indonesia 2department of internal medicine, roemani muhammadiyah hospital, semarang, indonesia correspondence: langgeng perdhana, jl. wonodri baru raya no. 22, semarang, central java, indonesia zip code: 50242 email: langgeng.p@gmail.com received: february 27, 2021 revised: july 1, 2021 accepted: february 9, 2022 published: april 28, 2022 doi: 10.33086/ijmlst.v4i1.1963 abstract anemia is often found in patients with chronic kidney diseases (ckd). anemia can affect poor outcomes in hemodialysis patients. however, studies examining role of severe anemia as a predictor factor in six-month all-cause mortality in hemodialysis patients in semarang are limited. therefore, further study was needed to answer these questions. we therefore designed this study to determine the role of severe anemia and six-month all-cause mortality in hemodialysis patients. the cohort design study was carried out from october 2019 to march 2020 at the hemodialysis unit of roemani muhammadiyah hospital semarang. the dependent variable was severe anemia, defined as hemoglobin levels <8.0 g/dl. the mortality rate was investigated over six months after the baseline measurements. among 85 respondents, 35 (41.2%) respondents were categorized into severe anemia, and 50 (58.8%) respondents were categorized into non-severe anemia groups. the respondents consisted of 54 (63.5%) males and 31 (36.5%) females. the hemoglobin level in the severe and non-severe anemia groups was 7.2 ± 0.6 and 9.5 ± 1.2 (mean ± standard deviation). the kaplan meier survival curve showed that the non-severe anemia group had a higher survival rate than the severe anemia group (98.0% vs. 82.9%, p = 0.011). the cox regression analysis found a significant relationship between anemia severity and 6month all-cause mortality in hemodialysis patients (p = 0.027; hazard ratio: 9.3). severe anemia plays a role as a predictor factor in six-month all-cause mortality among hemodialysis patients. keywords anemia, dialysis, end-stage kidney disease, fatality, hemoglobin. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 2 5 introduction anemia is often found in patients with chronic kidney disease (ckd). in ckd, hemoglobin (hb) levels begin to decrease in ckd stages 2 in male patients and stage 3 in female patients. however, anemia is more common in ckd stage 4 and the end-stage kidney disease (eskd) population; anemia is present in 90% of patients (1). anemia defined as decreasing of hb levels <13.0 g/dl in male or <12.0 g/dl in female. anemia is classified into non-anemia, mild anemia, moderate anemia, and severe anemia. the intervals differ between men and women. in men, anemia is classified into non-anemia if hb levels ≥13.0 g/dl, mild anemia if hb levels 11.0 to 12.9 g/dl, moderate anemia if hb levels 8.0 to 10.9 g/dl, and severe anemia if hb level <8.0 g/dl. in women, anemia is classified into non-anemia if hb levels ≥12.0 g/dl, mild anemia if hb levels 11.0 to 11.9 g/dl, moderate anemia if hb levels 8.0 to 10.9 g/dl, and severe anemia if hb levels <8.0 g/dl. moderate and severe anemia have the same definition in men or women (2). guidance for the management of anemia in dialysis patients in indonesia uses a combination of intravenous iron supplementation, erythropoietin therapy, and nutritional therapy. the targeted therapy was hb levels ≥10.0 g/dl. blood transfusion is the last therapeutic option for increasing hb levels in chronic dialysis patients. too many risks may occur during blood transfusions, such as transfusion reaction or blood borne virus (bbv) transmission related to blood transfusions, such as hepatitis b, hepatitis c or human immunodeficiency virus (hiv) (3). world health organisation (who) makes some recommendations about blood transfusion indication in chronic anemia is given if hb level <7.0 g/dl in adults (4). in indonesia, as many as 68,153 (78%) hemodialysis patients had hb levels <10.0 gr/dl, and 19,557 (22%) hemodialysis patients had hb levels ≥10.0 gr/dl (5). anemia in ckd is caused by various factors, including erythropoietin deficiency, uremicinduced erythropoiesis inhibition, decreased red blood cell survival, nutritional (folate, iron, and vitamin b12) deficiency, anorexia, and loss of dialysate (6). anemia can affect poor outcomes in hemodialysis patients. anemia in hemodialysis patients decreased the quality of life, decreased the systemic hemodynamic capacity and cardiac function, increased the incidence of left ventricular enlargement of the heart, and decreased cognitive and sexual abilities of patients. anemia also increase morbidity and hospitalization in the hemodialysis population. meanwhile, studies investigating the association between anemia with mortality in hemodialysis patients are still limited. a long-term study is needed to determine its relationship (3). ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 2 6 a study published by robinson et al., (7) showed that patients with hb levels ≥11.0 g/dl have a longer survival among maintenance hemodialysis patients but show no additional survival advantages for patients with hb levels ≥12.0 g/dl. kuo et al., (8) carried out similar study and showed similar results. patients with hb levels <9.0 g/dl, 9.0 to 9.9 g/dl had a higher mortality than patients with hb levels 10.0 to 10.9 g/dl. nevertheless, patients with hb levels 11.0 to 11.9 g/dl and ≥12.0 g/dl had lower mortality than patients with hb levels 10.0 to 10.9 g/dl. these include the studies conducted by guinn et al., (9) which are showed that severe anemia is associated with increased myocardial ischemia and mortality in patients declining transfusion. based on the description above, no publication examined the effect of severe anemia as a predictor factor on six months – all-cause mortality in hemodialysis patients. so that further study is needed relating to this matter to answer these problems. this study aimed to determine the effects of severe anemia as a predictor factor on all-cause mortality within six months in ckd patients undergoing hemodialysis. materials and methods patients starting regular hemodialysis at the roemani muhammadiyah hospital semarang, indonesia from october 2019 to march 2020 were enrolled. the inclusion criteria were patients who underwent hemodialysis ≥3 months, frequency of hemodialysis twice a week, and were willing to participate in the study. patients with incomplete data moved to other hemodialysis units and experienced changes in the frequency of hemodialysis during the study period were excluded from this study. respondents included in this study had given permission and signed for informed consent. this research also has obtained authorization from roemani muhammadiyah hospital. it has passed the ethical approval from the health research ethics committee of roemani muhammadiyah hospital semarang by number ea-012/kepkrsr/iii/2021. the independent variable was severe anemia, categorized into severe anemia and non-severe anemia. severe anemia defined as hb level <8.0 g/dl and non-severe anemia group if defined as hb level ≥8.0 g/dl. three milliliters (ml) pre-dialytic blood sample was taken and checked for hb levels using the spectophotometry method in the laboratory unit of roemani muhammadiyah hospital in october 2019. all-cause mortality was observed from october 2019 until march 2020. the collected data was then analyzed using kaplan meier and cox regression analysis using spss 18.0 program. the kaplan meier was used to approximates the survival function using at most one predictor. ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 2 7 for purpose of this research, approximately 115 respondents underwent hemodialysis in roemani muhammadiyah hospital semarang. seven-teen respondents were excluded from the study because did not meet inclusion criteria. it was considered since six respondents underwent hemodialysis less than twice a week, and 11 respondents underwent hemodialysis <3 months. ninety eight respondents were met inclusion criteria, threeten respondents were excluded from the study because seven respondents found incomplete data. as much as two respondents moved to another hemodialysis unit during the study period, and four respondents experienced changes in the frequency of hemodialysis during the study period. after going through inclusion and exclusion, there were 85 respondents whose data had been proceed and analyzed. as shown in figure 1, details the numbers excluded and reasons for their exclusion in this study. figure 1. sample selection flow chart results a fotal of 85 patients were enrolled in the study. as much as 33 (38.8%) respondents were diabetic, and 52 (61.2%) respondents were non-diabetic. of them, 54 (63.5%) respondents were male and 31 (36.5%) respondents were female. the mean age of respondents is 50.9 ± 10.6 years. the 115 respondents underwent hemodialysis did not meet inclusion : • 6 respondents underwent hemodialysis less than twice a week • 11 respondents underwent hemodialysis less than 3 months 98 respondents met inclusion respondents were excluded form this study, beacause : 7 respondents found incomplete data 2 respondents moved to other hemodialysis unit during study period 4 respondents experienced changes in frequency of hemodialysis during study period 85 respondents followed the study to the end ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 2 8 duration of hemodialysis was 24 ± 19.5 months. from the clinical examination, we got the mean systolic blood pressure (sbp), diastolic blood pressure (dbp), and mean arterial pressure (map) was 161.2 ± 21 mmhg, 86.1 ± 15.7 mmhg, and 111.1 ± 15.2 mmhg, respectively. the mean hb level of all in these studies was 8.5 ± 1.5 g/dl. result from chemical laboratory parameters test were the mean ureum and creatinin levels were 133.1 ± 38 mg/dl and 13.1 ± 4.5 mg/dl. based on the calculation, we found that the mean estimated glomerulus filtration rate (egfr) was 5.9 ± 2 ml/min/1.73m2. in addition, dialysis-related factor parameters were showed that the mean adequacy of dialysis (kt/v) was 1.1 ± 0.2. the mean ultrafiltration (uf) rate and quick of blood (qb) were 2,610 ± 969 ml and 195.5 ± 21.1 ml/min. table 1 shows sociodemographic data and clinical examination of the respondents. table 1. sociodemographic data and clinical examination of the respondents variables n (%) diabetes mellitus (dm) • yes • no 33 (38.8) 52 (61.2) gender • male • female 54 (63.5) 31 (36.5) anemia levels • severe anemia (hb <8 mg/dl) • non severe anemia (hb ≥8 mg/dl) 35 (41.2) 50 (58.8) variables min max mean ± sd ages (years) 27 79 50.9 ± 10.6 duration of hd (months) 3 109 24 ± 19.5 egfr (ml/min/1.73m2) 3.2 13.1 5.9 ± 2 pre dialysis sbp (mmhg) 108 224 161.2 ± 21 pre dialysis dbp (mmhg) 52 147 86.1 ± 15.7 pre dialysis map (mmhg) 79 167 111.1 ± 15.2 hb (g/dl) 6 12.9 8.5 ± 1.5 ureum (mg/dl) 53 219 133.1 ± 38 creatinin (mg/dl) 5.2 24.8 13.1 ± 4.5 kt/v 0.7 1.8 1.1 ± 0.2 uf rate (ml) 500 4,500 2,610 ± 969 qb (ml/min) 180 300 195.5 ± 21.1 n: number of cases in the severe anemia group, 20 (57.1%) respondents were male, and 15 (42.9%) respondents were females. while in the nonsevere anemia group, 34 (68%) respondents were male, and 16 (32%) respondents were female. in the severe anemia group, 15 ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 2 9 (42.9%) respondents had diabetes, and 20 (57.1%) respondents were non-diabetic. while in the non-severe anemia group, 18 (36%) respondents had diabetes, and 32 (64%) respondents were non-diabetic. the mean hb level in the severe and non-severe anemia groups was (mean ± standard error; 7.2 ± 0.6 vs 9.5 ± 1.2 g/dl). the mean age of respondents in the severe anemia and non-severe anemia group was 50.4 ± 8.8 vs 51.2 ± 11.7 years, and the mean duration of hemodialysis in the severe anemia and non-severe anemia group was 23.5 ± 16.9 vs 24.3 ± 21.4 months. in table 2, we measured and compare sbp, dbp, and map in severe anemia and non-severe anemia group were 164.2 ± 20.1 vs 159.2 ± 21.5 mmhg; 83.8 ± 14.6 vs 87.6 ± 16.4 mmhg; and 110.6 ± 14.5 vs 111.5 ± 15.8 mmhg). the mean egfr in the severe anemia group was 5.8 ± 1.7 ml/min/1.73m2, and in the non-severe anemia group was 6.0 ± 2.2 ml/min/1.73m2. other parameters such as mean of ureum and creatinin levels in severe anemia and non severe anemia group were 138.6 ± 37.5 vs 129.2 ± 38.2 mg/dl; and 13.0 ± 4.1 vs 13.2 ± 4.9 mg/dl. dialysis parameters were mean of kt/v, uf rate, and qb namely 1.1 ± 0.2 vs 1.1 ± 0.2; 2,900 ± 900 ml vs 2,400 ± 100 ml; 195.7 ± 20.4 vs 195.3 ± 21.7 ml/min. the overall survival in kaplan-meier analysis for respondents in the non-severe anemia group was higher than respondents in the severe anemia group (figure 2). however, the overall survival rates in the two groups were significantly different (98.0% vs 82.9%; p-value = 0.011). table 2. characteristic data on each group variables severe anemia group non-severe anemia group male n (%) female n (%) male n (%) female n (%) gender 20 (57.1) 15 (42.9) 34 (68) 16 (32) yes n (%) no n (%) yes n (%) no n (%) diabetes melitus 15 (42.9) 20 (57.1) 18 (36) 32 (64) variables sa group (mean ± sd) non sa group (mean ± sd) p value 95% ci ages (years) 50.4 ± 8.8 51.2 ± 11.7 0.101 5.408 – 3.905 duration of hd (months) 23.5 ± 16.9 24.3 ± 21.4 0.157 7.897 – 9.601 egfr (ml/min/1,73m2) 5.8 ± 1.7 6 ± 2.2 0.437 1.060 – 0.683 pre dialysis sbp (mmhg) 164.2 ± 20.1 159.2 ± 21.5 0.751 4.156 – 14.196 pre dialysis dbp (mmhg) 83.8 ± 14.6 87.6 ± 16.4 0.845 10.681 – 3.058 pre dialysis map (mmhg) 110.6 ± 14.5 111.5 ± 15.8 0.830 7.569 – 5.826 hb (g/dl) 7.2 ± 0.6 9.5 ± 1.2 0.000 2.760 – 1.877 ureum (mg/dl) 138.6 ± 37.5 129.2 ± 38.2 0.750 7.179 – 26.059 creatinin (mg/dl) 13 ± 4.1 13.2 ± 4.9 0.355 2.153 – 1.855 kt/v 1.1 ± 0.2 1.1 ± 0.2 0.594 0.075 – 0.099 uf rate (liters) 2,900 ± 900 2,400 ± 100 0.279 0.045 – 0.876 qb (ml/min) 195.7 ± 20.4 195.3 ± 21.7 0.390 8.869 – 9.698 n: number of cases ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 0 figure 2. kaplan meier overall survival curve. the survival in nsa (non-severe anemia) group was higher than in sa (severe anemia) group (98.0% vs 82.9%; p-value = 0.011). table 3 shows he output of this multiple cox regression analysis. the results show that severe anemia is a predictive factor of six months of all-cause mortality among hemodialysis patients (p = 0.027, hazard ratio: 9.3). we find that the patient with severe anemia (hb level less than 8 mg/dl) had a 9.3 times higher mortality rate than patients without severe anemia (hb level more than equal to 8 mg/dl). on the other hand, other variables like gender, ages, duration of hemodialysis, ureum levels, creatinin levels, egfr, dialysis adequacy, and diabetes mellitus did not play a role in predicting six months of all-cause mortality in hemodialysis patients (p-value ≥ 0.05). table 3. cox regression analysis for mortality parameters p-value hazard ratio (95% ci) gender 0.663 1.4 (0.312 – 6.236) age 0.614 1.0 (0.950 – 1.090) duration of hd 0.608 1.0 (0.944 – 1.035) severe anemia 0.027 9.3 (1.118 – 77.194) ureum 0.356 1.0 (0.990 – 1.290) creatinine 0.570 1.1 (0.788 – 1.541) egfr 0.441 0.9 (0.021 – 39.9) kt/v 0.926 0.9 (0.021 – 39.9) diabetes mellitus 0.809 0.8 (0.186 – 3.714) ci, confidence interval the mortality rate in this study was 8.2%. during six months, seven respondents died, including six respondents (three males and three females) in severe anemia groups and one respondent (a male) in non-severe anemia groups. the limitation of this study is that there is no data related to the cause of death. in comparison, based on indonesian ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 1 renal registry (irr) data in 2018, the most common cause of death among hemodialysis patients in indonesia was cardiovascular events that around 42%, then followed by sepsis around 10%, cerebrovascular events 8%, gastrointestinal bleeding 3%, other cause 6% and no data 31% (5). the overall mortality in cox regression analysis for respondents in the non-severe anemia group was lower than respondents in the severe anemia group as shows in figure 3. however, the overall mortality rates in the two groups were significantly different (2.0% vs 17.1%; p-value = 0.027). figure 3. cox regression overall mortality curve discussion anemia in ckd and its management the prevalence of severe anemia in this study was 41.2%. if we compare with the general population in the united states (us), the prevalence of severe anemia was 1.5% (10). compare with previous studies, severe anemia prevalence among hospitalized patients was 14.7% (11). no other reference describes the prevalence of severe anemia among hemodialysis populations. this difference is due to differences in the people in this study were only hemodialysis patients. anemia in ckd is caused by various factors, including erythropoietin deficiency, circulating uremic-induced inhibitors of erythropoiesis, shortened red blood cell survival, nutritional deficiencies such as folate, iron, and vitamin b12, anorexia, and dialysate losses (6). renal anemia is anemia in ckd mainly due to decreased erythropoietin production capacity. other factors contributing to renal anemia are iron deficiency, the short lifespan of erythrocytes, secondary hyperparathyroidism, infection inflammation, hemoglobinopathy, hypothyroidism, and folic acid deficiency. ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 2 based on the indonesian society of nephrology (inasn) recommendation, iron status should be checked first before giving erythropoietin stimulating agents (esa). for the optimal erythropoiesis response, iron status must be sufficient. the iron status that should be checked includes serum iron, total iron binding capacity (tibc), transferrin saturation, and ferritin serum. if we find a condition of iron deficiency anemia, it should be corrected first. iron therapy indicated in absolute iron deficiency anemia, functional iron deficiency anemia, and the maintenance stage of iron status. among hemodialysis patients, it is categorized to be absolute iron deficiency anemia if transferrin saturation <20%, and ferritin serum <200 ng/ml, functional iron deficiency anemia if transferrin saturation <20% and ferritin serum ≥200 ng/ml, and enough iron if transferrin saturation ≥20% and ferritin serum ≥200 ng/ml. parenteral iron therapy is divided into the correction phase and maintenance phase. in absolute and functional iron deficiency anemia, the correction phase is done by giving 100 mg iron sucrose or iron dextran twice a week for five weeks. the targeted therapy reached if transferrin saturation >20% and ferritin serum >200 ng/ml. on the other hand, the maintenance phase ensures that erythropoiesis has sufficient iron during esa therapy. the targeted therapy reached if transferrin saturation ranged between 20 – 50% and ferritin serum ranged between 200 – 500 ng/ml (3). the use of parenteral iron supplementation among hemodialysis patients has increased from year to year. in 2017, there were 27.2% increases in the use of parenteral iron supplementation to 34,430 ampoules per year. data from irr, only 6% of patients with serum fe levels ≥150 mg/dl and 52% patients with transferrin saturation ≥20 (5). the limitation of this study is that the respondents did not check for serum iron, tibc, transferrin saturation, and ferritin serum. furthermore, it is unknown whether the patient’s iron status is sufficient, absolute iron deficiency anemia, or functional iron deficiency anemia. the use of esa therapy is determined based on some factors, such as clinical evidence of the benefit of esa therapy, costeffectiveness, and the negative impact that may be found in the administration of esa therapy. esa therapy can be given in patients with hb levels <10 g/dl, and other causes of anemia have been ruled out. esa therapy can improve hb levels in renal anemia patients with no absolute iron deficiency anemia and no severe infection. the targeted therapy of hb in esa therapy is 10 12 g/dl. esa therapy is divided into correction and maintenance phases based on the inasn recommendation of anemia therapy in ckd. the correction phase has been done if target therapy of hb has not been reached by giving esa 2,000 – 5,000 iu twice a week or 80 ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 3 120 unit/kg/weeks. in contrast, the maintenance phase is done if target therapy of hb has been reached by giving esa 2,000 – 5,000 iu per week. if the patient’s hb level is >13 g/dl or esa hypersensitivity reaction occurs, esa therapy should not be given. some side effects that may be occurred in esa therapy are hypertension and thrombosis. however, these recommendations in indonesia have not been implemented optimally. it can be caused by many factors, including differences in regulations in each hemodialysis unit or hospital differences in hemodialysis financing claims between hospitals depending on the level of the hospital (3). esa therapy use among hemodialysis patients also increased from year to year. in 2017, there were 36.6% increases in the use of esa therapy to 652,708 injections per year (5). nutritional therapy plays an important role in increasing hb levels in renal anemia patients. there are various benefits of nutritional therapy in hemodialysis patients, including fixing and maintaining optimal nutritional status, preventing excess metabolic waste, regulating water and electrolyte balance, and controlling ckdrelated conditions such as anemia, hypertension, bone disorder, and cardiovascular disease. energy intake in ckd patient is recommended at 35 calories/kg/day in non geriatric (age <60 years) patients, and 30 – 35 calories/kg/day in geriatric (age ≥60 years) patient. protein intake is also important. the recommendation is 1.2 g/kg/day in hemodialysis patients. the protein given contains at least 50% with high biological content (animal protein). the recommendation of lipid intake in hemodialysis patients ranged around 25 30% total daily calories with restriction of saturated fat 10% (12). blood transfusion becomes the last choice for hb correction in renal anemia. blood transfusion can be given in under the conditions: patient with hb level <7 g/dl with or without anemia symptoms, hb level <8 g/dl with cardiovascular disorder, acute bleeding with hemodynamic disturbance symptoms, and pre-surgical procedure. target hb in transfusion is different than in esa therapy. the target is ranged between 7 – 9 g/dl. blood transfusion is a relatively safe procedure, but it does not mean that there are no side effects. some side effects that may be occurred during blood transfusion are circulation overload, allergic or anaphylactic reaction, iron overload, hemolytic reaction, febrile non-hemolytic reaction, blood-borne infection such as hepatitis b, hepatitis c, and hiv (3). the use of blood transfusion among hemodialysis patients has increased from year to year. in 2017, there were 22.6% increases in the use of blood transfusion to 46,362 bags per year (5). ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 4 factors related to mortality prediction the patient’s age does not play a role as a prediction factor in six months of all-cause mortality in hemodialysis patients (p-value = 0.614). it is the opposite of msaad et al. (13) study, that described the surviving patients were younger than the deceased (43.07 ± 13.52 years vs 53.09 ± 13.56 years). the survival of non-geriatric patients is 82% and 53% in non-geriatric patients after four years of follow-up (13). gender does not play a role as a predictive factor of all-cause mortality in the next six months in hemodialysis patients (pvalue = 0.663). it is similar to vongsanim et al., (14) study that described gender does not play a role as prediction mortality in hemodialysis patients. women have a higher survival rate than men in the general population, but it is not similar in hemodialysis populations (14). urea blood level does not play as a predictive factor of all-cause mortality in the next six months in hemodialysis populations (p-value = 0.356). it is similar to stosovic et al., (15) study that described the lowest mortality observed in urea levels 28 – 31 mmol/l at the baseline data and 25 – 27 mmol/l in the whole observation period. in conclusion, urea level was not a predictor of mortality in the whole cohort. however, low urea and high urea were independent predictors of mortality in the corresponding models using cox regression (15). in this study, creatinine level does not play as a predictive factor of all-cause mortality in hemodialysis patients within the next six months (p-value = 0.570). it contradicts with ajiro et al., (16) studies that explained a significant relationship between serum level creatinine with mortality after ten years undergoing hemodialysis. in the population who had undergone hemodialysis >10 years, pre-dialysis serum creatinines plays a role as a predictor of mortality risk than in the population who had undergone hemodialysis ≤ 10 years. we found that low serum creatinine (<11.0 mg/dl) was associated with a high death risk. patients with high serum creatinine may be better nourished with a greater somatic protein mass (16). in this study, egfr does not play a role as a predictive factor of mortality in the next six months among hemodialysis populations (p-value = 0.444). no study correlated egfr and mortality among hemodialysis patients. haas et al., (17) showed reduced egfr at the time of admission is a strong and dependent predictor for 30 days mortality among populations of patients admitted to medical emergency departments. the 30 days mortality risk was 1.8%, 3.5%, 6.9%, 11.1%, 13.6%, and 14.2% in patients with egfr of ≥90, 60 89, 45 59, 30 44, 15 29, and <15 ml/min/1.73m2. the egfr was also significantly associated with in-hospital mortality, the percentage of icu admission, ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 5 and a longer hospital stay. lin et al., (18) showed that early dialysis initiation for eskd patients is associated with increased mortality risk among east asian populations. however, cardiocerebrovascular mortality between the early and late dialysis showed no survival differences. the limitation of this study is that there are no data about egfr at dialysis initiation among respondents. in this study, adequacy dialysis does not play as a predictor factor for six months of all-cause mortality among hemodialysis patients (p-value = 0.926). the mean kt/v in this study is 1.1 ± 0.2. this value is considerably lower than the kt/v target for the population undergoing hemodialysis twice a week (kt/v target = 1.8). it contradicts with hong et al., study (19) that showed an association between kt/v and mortality in hemodialysis patients might be modified by body mass index (bmi). a higher kt/v and bmi were independently associated with lower mortality risk for allcause mortality in hemodialysis patients. among patients with low bmi (<20 kg/m2) and normal bmi (20 to <23 kg/m2), higher kt/v was associated with lower all-cause mortality compared to the reference group (kt/v 1.2 to 1.4). on the other hand, among patients with high bmi (>23 kg/m2), the association between higher kt/v and lower all-cause mortality was attenuated. on the other hand, compared to patients with normal bmi and kt/v within the target range, those with low bmi had a higher risk for all-cause mortality. however, increasing kt/v values was associated with narrowing the mortality risk gap. compared to patients with normal bmi and kt/v within the target range, those with high bmi and kt/v <1.2 did not have an increased risk for all-cause mortality despite low dialysis adequacy. moreover, high bmi patients with kt/v >1.2 were at lower risk for all-cause mortality than those with normal bmi and kt/v within the target range. the differences between the low and high bmi groups may be explained in several ways: malnutrition inflammation complex syndrome (mics) and protein energy wasting (pew) likely account for the more significant mortality among hemodialysis patients. then, the improvement of dialysis adequacy has been associated with better nutritional status as assessed by serum albumin, which is linked to more remarkable survival in hemodialysis patients. on the other hand, high bmi also indicates good nutritional conditions. al sahow et al. study (20) showed that low kt/v was strongly associated with higher mortality in women but not in men. women hemodialysis patients with lower kt/v had a 1.91 higher mortality risk than in higher kt/v women hemodialysis patients. since we know that dialysis dose as measured by kt/v can be influenced by so many factors, such as time of dialysis, qb, dialysate flow, interruption session (hypotension or clotting), access ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 6 functionality (stenosis and recirculation), needle size, and placement, dialyzer characteristics, and proper blood sampling. in order to achieve higher survival among the hemodialysis population, qb must be increased to >350 ml/min. the dialysis time must be increased to ≥4 hours thrice weekly, which will reduce low kt/v prevalence and improve survival in hemodialysis patients. in this study, diabetes does not correlate with a predictor factor in all-cause mortality among hemodialysis patients (p-value = 0.809). it is similar to ajiro et al., (16) study that explained that patients with diabetes had a higher mortality rate than patients without diabetes in the group with ≤10 years of hemodialysis. however, they were not at a greater risk for death in the group with >10 years of hemodialysis (16). racki et al., (21) reported a similar result that diabetic patients had significantly lower survival than nondiabetic patients, and cardiovascular disease remained the primary cause of death in both groups. this study found that severe anemia plays a role as a prediction factor in six months of all-cause mortality in hemodialysis patients (p-value = 0.027, hazard ratio: 9.3). hemodialysis patients with severe anemia have a 9.3 times higher mortality risk in the next six months than in hemodialysis patients without severe anemia. no previous study examined the effect of severe anemia and six months of all-cause mortality in hemodialysis patients. robinson et al., (7) and kuo et al., (8) were showed that hemodialysis patients with hb level ≥11 g/dl had high survival. the difference between these studies and robinson et al., with kuo et al., (8) are the respondents grouped into severe anemia with hb level <8 g/dl and non-severe anemia, which hb level ≥ 8 g/dl. while in robinson et al., (7) and kuo et al., (8) have found that the respondents were grouped into patients with hb level ≥11 g/dl and patients with hb <11 g/dl. anemia as predictor factor for mortality there are so many different definitions of terms for malnutrition in the dialysis populations, such as protein energy wasting (pew), protein energy malnutrition (pem), mics, malnutrition inflammation atherosclerosis (mia), and uremic wasting syndrome depending on the involvement of inflammation, hypercatabolism, and increased in uremia levels. however, there are no uniform diagnostic criteria for malnutrition. comorbidities such as heart failure and ckd-mineral bone disorder have a bidirectional correlation with nutritional status. the pathomechanism relates to malabsorption due to gut edema, poor appetite due to cytokine production, and difficulty in oral intake and food preparation arising from fatigue and breathing difficulty. contributing factors related to the development of malnutrition are categorized as iatrogenic and non-iatrogenic origin. ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 7 iatrogenic or physician-induced malnutrition develops from a medical procedure, pharmacological treatment, prolonged hospitalization, nosocomial infection, or delayed wound healing. on the other hand, iatrogenic malnutrition is an adverse dialysis event in eskd patients. the factors related include dialysis-induced nutrient losses, multiple dialyzers use dialysis-induced inflammation, the efficacy of uremia corrections, dialysis adequacy, dialysis frequency, dialysis duration, and efficacy of metabolic acidosis correction (22). a major goal of dialysis therapy is to remove uremic waste products through partial removal of uremic solutes. in uremia conditions, transamination activities inhibited and impacted protein and amino acid metabolism. the removal of uremic solutes by the hemodialysis process depends on many factors, such as duration of the dialysis session, dialysis adequacy, dialyzer membrane permeability, and dialysis frequency (22). the dialysis process impacts chronic nutrient losses. around 6 – 12 g of amino acids and 7 – 8 g protein losses during each dialysis session. it contributes to hypoalbumin conditions that play a strong predictor of malnutrition and mortality among hemodialysis patients. nutrient losses depend on the dialyzer / hollow fiber (hf) membrane and the mechanism of solute removal. increasing the pore size of the hf membrane allows the release of a larger intermediate molecule and increases the involuntary loss of albumin by about 2–14 g. it depends on the degree of membrane permeability condition such as membrane bio-incompatibility, high flux membrane, hemofiltration technique, hemodiafiltration technique (hdf), and the practice of multiple dialyzers that induce greater membrane permeability and facilitate the greater loss of amino acids into the dialysate (22). in some countries, including indonesia, dialyzer reuse is common. inasn recommends that the hemodialysis unit reuse the dialyzer seven times during the hemodialysis process (23). however, dialyzer reuse may contribute to adverse effects such as infection risk, biochemical and immunologic reactions, improper sterilization, increased membrane permeability, and performance loss, leading to inadequate dialysis (22). many factors lead to inflammation in hemodialysis patients, such as biocompatibility of the dialyzer membrane, infection related to dialysis access, and impure dialysate containing endotoxins. generally, the high flux dialyzer membrane and hdf technique are associated with lower inflammation grades in hemodialysis patients when compared to the low flux dialyzer membrane. the differences are attributed to processing technology for structuring and composing the membrane, dialyzer ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 8 bioincompatibility, water permeability, clearance, and appropriate sieving coefficients. in some studies, hemodialysis patients with catheter access had a significantly higher malnutrition inflammation score and lower serum albumin. inflammation also occurs with dialysate contamination by a microorganism that produces endotoxin that passes through the dialyzer membrane and enters into blood circulation, affecting the production of proinflammatory cytokines such as interleukin (il)-1, il-6, and tumor necrosis factor (tnf-a) that are not effectively removed by dialysis treatment using low flux dialyzer. overall, dialysis patients are vulnerable to oxidative stress with increased reactive oxygen species (ros) production and antioxidant depletion. ros induced activation of nuclear factor kappa b (nfkb), which translocated the cell nucleus stimulating cytokine production that causes inflammation. another impact of hemodialysis treatment is the activation of polymorphonuclear leucocytes that trigger ros production. on the other hand, low antioxidant levels in hemodialysis patients may also occur from limited consumption of vegetables and fruits to prevent hyperkalemia. when malnutrition coexists with inflammation, the combination is mics, reduces albumin production, and fosters poor appetite (22). metabolic acidosis develops in the early stages of ckd from the kidney’s inabilities to excrete non-volatile acids and synthesize bicarbonate to maintain acid-base imbalance. metabolic acidosis contributes to malnutrition by reducing protein synthesis and increasing muscle degradation. the malnutrition pathway in hemodialysis patients involves protein catabolism, secondary insulin resistance, inflammation, and increased serum leptin level. in metabolic acidosis, muscle degradation occurs due to two mechanisms. this mechanism increased branched chain keto acid dehydrogenase (bckad) and the atp-dependent ubiquitin proteosome system (ups) pathway. in acidosis conditions, there is an increase in gene transcription and activity of bckad enzyme to degrade the branched chain amino acid (bcaa) that play a role as precursors for protein synthesis and is mainly metabolized in the muscle. differently, metabolic acidosis activates ups by increasing gene transcription of the proteasome and atpdependent ubiquitin, leading to increased caspase three activity, which promotes cleaving of muscle fibers, resulting in poor muscle mass. additionally, the acidic environment affects insulin binding to receptors, reducing tissue sensitivity to insulin and affecting glucose uptake, and inhibiting insulin’s anabolic effect, causing muscle depletion (22). ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 3 9 while non-iatrogenic factors develop spontaneously from contributive factors accompanying ckd progression, they are not related to the primary treatment. noniatrogenic factors may contribute to malnutrition conditions in hemodialysis patients. the factors related include suboptimal dietary intake, taste alteration, poor appetite, insulin resistance, and psychosocial factors, such as depression, lack of social support, financial constraints, and decreased physical functioning (22). suboptimal dietary intake is a primary contributing factor to malnutrition and mortality among hemodialysis patients. adult recommendation for dietary energy intake (dei) and dietary protein intake (dpi) to achieve nutrient adequacy within 25 – 35 kcal/kg ideal body weight (ibw)/day and 1.0 – 1.2 g protein/kg ibw/day. however, achieving dei and dpi goals is still challenging among hemodialysis patients. the inadequate diet may be attributed to monotonous dietary patterns, poor diet quality, anorexia, alterations in taste, and poor appetite. taste alterations develop food aversion learning, which impacts appetite and reduces overall dietary quality, contributing to malnutrition. the reduction in taste perception itself may be related to zinc deficiency. on the other hand, the pathomechanism of poor appetite is explained by changes in appetite hormones. ghrelin, an orexigenic hormone mainly secreted by the stomach, regulates appetite by stimulating spontaneous food intake (22). insulin at physiological levels bears catabolic and anabolic effects on skeletal muscle by promoting bcaa transport, regulating protein synthesis in the muscle, and facilitating glucose transport and uptake by muscle tissues. insulin resistance is associated with peripheral resistance of glucose uptake at the skeletal muscle site and manifests as impaired insulin signaling through the phosphorylation of insulin receptor substrate-1. in another pathway, high retinol binding protein 4 (rbp4) plays a role in glucose metabolism by inducing gluconeogenesis and inhibiting glucose uptake in the muscle. educes insulin activity affects bcaa transport and blunts insulin’s anabolic effects for decreasing skeletal muscle breakdown (22). psychological factors such as depression, lack of social support, financial constraints, and decreased physical functioning may negatively impact nutritional status in hemodialysis patients. hemodialysis patients lacking social support have a higher prevalence of diminished appetite, depression, reduced physical functioning, and poor adherence to hemodialysis treatment that may be associated with malnutrition conditions. lack of income is common among hemodialysis patients, and it is linked to poor dietary adherence because ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 4 0 patients’ access to healthy food options is limited by cost (22). in guinn et al., study (9), it was shown that severe anemia is associated with increased myocardial ischemia and mortality in patients declining transfusion. the allcause mortality rate within 30 days was 19.8%, and the risk of death increases 55% per 1g/dl decrease in nadir hb. the result is similar to this study: severe anemia is associated with six-month all-cause mortality in hemodialysis patients. the difference between guinn et al., (9) study and this study is in its population. the population in this study is only ckd patients who are undergoing hemodialysis. so, the population sample is more homogenous than in guinn et al., (9) study. the population of guinn et al., (9) study is all patients with blood transfusion refusal, which has many different underlying causes of anemia. on the other hand, the duration of observation on mortality patients is also different. guinn et al., (19) study observed all-cause mortality within 30 days, but in this study, we observed the all-cause mortality within six months. in park et al., study (24), it was shown that a significant correlation between low hb and the risk of left ventricular hypertrophy (lvh). adaptive changes in cardiac geometry are common in chronic anemia patients, including those with ckd. low hb patients’ echocardiograms reveal an abnormal pattern of cardiac remodeling. hemodynamic compensation for anemia includes reducing afterload due to decreased systemic vascular resistance, increased preload due to increased venous return, and increased sympathetic activity and inotropic factors. these adaptive physiological changes may lead to cardiac volume overload, resulting in eccentric lvh. in eskd, these geometric changes are accompanied by arterial stiffening that can worsen lvh and abnormal coronary perfusion, increasing mortality due to cardiac events. unfortunately, there were no data regarding the result of the patient’s echocardiography in this study. perdhana et al., (25) studies have found that a significant correlation between anemia and intradialytic hypertension (idh). patients with hb level <10 g/dl had 5.9 more significant risks of experiencing idh than patients with hb level ≥10 g/dl. idh is one of several complications that may be occurred during hemodialysis. idh increases blood pressure from the beginning to the end of the hemodialysis process. the patient’s blood pressure may be normal when hemodialysis started but then increased to hypertensive during and at the end of hemodialysis (26). there are several definitions of idh. in some studies, idh was defined as increasing mean arterial pressure (map) >15 mmhg within or immediately post-dialysis. in others defined as increasing >10 mmhg in systolic ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 4 1 blood pressure (sbp), and in some others defined as blood pressure rising to any degree during the second or third intradialytic hours. however, there are no accurate definitions considered as criteria for adjusting the diagnosis of intradialytic hypertension (27). in several studies, idh has been associated with poor outcomes in hemodialysis patients, including increased hospitalizations, higher blood pressure, cardiovascular morbidity, and all-cause mortality (28). in addition, previous studies have found that idh had a significant relationship as a predictor factor of six-month all-cause mortality among hemodialysis patients (29). hemodialysis patients with idh complications had 7.6 times higher risk of all-cause mortality within six months than hemodialysis patients without idh. shamir et al., (28) study showed an association of idh with an increase in the left ventricular mass index (lvmi). lvmi is a strong and independent predictor of survival and cardiovascular events in hemodialysis patients. idh and left ventricular hypertrophy (lvh) may share common underlying pathomechanisms like extracellular volume overload and increased peripheral vascular resistance due to endothelial dysfunction. on the other hand, idh has also been associated with increased ambulatory blood pressure in hemodialysis patients, increasing cardiovascular events and mortality (28). iron deficiency is one of the causes of anemia in ckd patients (6). in ckd, imbalance in iron homeostasis results from increased iron losses, reduced iron absorption, and iron storage and mobilization disruption. so, intravenous iron supplementation plays a vital role in treating anemia in eskd (30). on the other hand, a high iron level could increase the risk of infection through impairment of neutrophil and t cell function and promotion of microbial growth (31). even though hemodialysis patients also have a high risk of infection due to immunity dysfunction (32). any such increase in risk may be substantial because the infection is a significant cause of mortality in dialysis patients (33). unfortunately, there were no data regarding the results of the patient’s iron levels in this study. the hemopoietic tissue has a high nutritional requirement. malnourished people are more likely to develop anemia and leucopenia. the lack of protein reserves and other nutrients to support the hemopoiesis process is the cause of this condition. 34 it means that lower hemoglobin is a marker of low protein intake and malnutrition. malnutrition and anemia are reported as specific cardiovascular risk factors for dialysis patients, and each of the mia syndrome components worsens the survival of these patients. malnutrition is related to metabolic acidosis due to increased protein ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 4 2 catabolism, decreased protein synthesis, endocrine abnormalities, and inflammation among dialysis patients. metabolic acidosis resulting in inflammatory stimulation, lipids oxidation, and oxidative stress can increase the risk of atherosclerosis and cardiovascular events (35). any other limitation of this study was that it did not perform regular evaluations of clinical and laboratory parameters during this study period. on the other hand, there is no clear information about the cause of death in the recruited respondents. conclusions we showed that severe anemia is a predictor factor in all-cause mortality among hemodialysis patients in the next six months. anemia affects the incidence of cardiomegaly, which increases the risk of cardiovascular death. in addition, anemia decreases immunity in patients and increases the risk of infection. many factors cause anemia among hemodialysis patients, such as malnutrition, inadequate hemodialysis, iron deficiency, and erythropoietin deficiency. if anemia can be appropriately treated, the survival of hemodialysis patients will also improve. author contributions langgeng perdhana: conceived and designed the analysis, collected the data, wrote the paper; shofa chasani: contributed data or analysis tools, wrote the paper. acknowledgment we acknowledge the staff and patients at the hemodialysis unit of roemani muhammadiyah hospital for their support, help and cooperation in this study. conflict of interest there are no conflict of interest declared in this study. references 1. cases a, isabel e, salvador t, vicente p, raquel o, jose lg, jose mp. anemia of chronic kidney disease; protocol of study, management and referral to nephrology, 2018; vol. 38; issue 1; pages 1 108. doi : 10.1016/j.nefroe.2018.01.007 2. world health organization. hemoglobin concentration for the diagnosis of anaemia and assessment of severity, department of nutrition for health and development, world health organization, geneva, 2011. 3. suhardjono, lubis hari, lydia a, widodo, bakri s, widiana igr, nassution sr, effendi i martakusumah ah, djarwoto, b, mohani ci, azmi s, syukri m, partiningrum dl, rasyid h, palar s, nugroho p, hustrini nm. konsensus manajemen anemia pada penyakit ginjal kronik consensus on the management of anemia in chronic kidney disease, perhimpunan nefrologi indonesia (pernefri), jakarta, 2011. 4. world health organization, clinical transfusion practice: guidelines for medical interns, world health organization, geneva. 5. perhimpunan nefrologi indonesia. 11th report of indonesian renal registry. jakarta: perhimpunan nefrologi indonesia, 2019. ina. j. med. lab. sci. tech. 2022; 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doi: 10.3390/nu12103147 23. dharmeizar, 2016, surat pernefri no. 310 / pb pernefri / x / 2016 tentang pemakaian dialiser ulang, pengurus besar perhimpunan nefrologi indonesia re-use of dialyzers, executive board of the indonesian nephrology association, jakarta. 24. park s, jung jy, kang jg, hong hp, oh cm,. association of left ventricular hypertrophy with hemoglobin levels in nonanemic and anemic populations, cardiology 2020; 145; 485 491. https://doi.org/10.1159/000508034 25. perdhana l, shofa c. faktor – faktor yang mempengaruhi kejadian hipertensi intradialisis pada pasien penyakit ginjal kronis yang menjalani hemodialisis di rumah sakit roemani muhammadiyah semarang dalam proceeding book : pertemuan ilmiah tahunan konferensi kerja perhimpunan nefrologi indonesia 2019 https://doi.org/10.1111/sdi.12817 https://dpoi.org/10.1186/s12882-019-1570-0 https://doi.org/10.1093/ckj/sfz195 https://doi.org/10.1016/j.diabres.2006.05.015 ina. j. med. lab. sci. tech. 2022; 4(1): 24–44 langgeng perdhana & shofa chasani 4 4 (pit – konker pernefri 2019) : the practice in kidney disease and hypertension care in indonesia, padang : perhimpunan nefrologi indonesia, 2019. 26. kandarini y, raka w, ketut s. association between ultrafiltration volume and intradialytic hypertension in maintenance hemodialysis, medicina 2017, volume 48, number 2: 152 – 156. 27. georgianos pi, pantelis as, carmine z. intradialysis hypertension in end-stage renal disease patients: clinical epidemiology, pathogenesis, and treatment, hypertension 2015. ; 66: 456 463. https://doi.org/10.1161/hypertensionaha. 115.05858 28. shamir ra, ameet k, jonathan y, yi y, dana m, jennifer g, ploth d, negrea l, paine s, rahman m, kwong ry, zager p, manisha j. association of intradialytic hypertension with left ventricular mass in hypertensive hemodialysis patients enrolled in the blood pressure in dialysis (bid) study; kidney blood pres 2018; 43: 882 892. doi: 10.1159/000490336 29. perdhana l, shofa c. intradialytic hypertension and six month all cause mortality in hemodialysis patients. j hypertension: may 2021 volume 39 issue p e11 doi: 10.1097/01.hjh.0000752516.20104.12 30. metivier f, sylvain jm, alain pg, bruno p, gerard ml. pathophysiology of anemia: focus on the heart and blood vessel nephrol dial ,transplant 2000; 15 (suppl 3): 14 18. 31. thomas g, george ra, carlo ajm, gallard lt, goodnough ic, macdougall gm, mayer g, porto g, winkelmayer wc, jay bw. iron administration, infection, and anemia management in ckd: untangling the effects of intravenous iron therapy on immunity and infection risk, kidney medicine 2020; vol.2; issue 3; pg: 341 353. https://doi.org/10.1016/j.xkme.2020.01.006 32. julie hi, kristen lj. iron and infection in hemodialysis patients, semin dial 2015; 27 (10); 26 36. doi: 10.1111/sdi.12168 33. ahmed ms, mohanram n. immune dysfunction and risk of infection in chronic kidney disease; ackd 2019; vol. 26; issue 1; p8 15. https://doi.org/10.1053/j.ackd.2019.01.004 34. borelli p, solange lb, marcelo mr, ricardo af, haematological alterations in protein malnutrition, rev. bras. hematol. hemoter. vol. 26 no. 1, sao jose do rio petro. https://doi.org/10.1590/s1516848420040001000010 35. raikou vd, despina k. the association between intradialytic hypertension and metabolic disorders in end-stage renal disease, hindawi international j hypertension 2018. https://doi.org/10.1155/2018/1681056. https://doi.org/10.1590/s1516-848420040001000010 https://doi.org/10.1590/s1516-848420040001000010 1 a powerful elisa technique to test the potential of extra virgin olive oil in reducing tnf-α level and edema volume in male rattus norvegicus exposed to carrageenan andina putri aulia1, suprapto maat2, aryati3 1department of clinical pathology, faculty of medicine, universitas islam sultan agung, semarang, indonesia 2department of medical laboratory technology, faculty of health, universitas nahdlatul ulama surabaya, surabaya, indonesia 3department of clinical pathology, faculty of medicine, universitas airlangga, surabaya, indonesia correspondence: andina putri aulia, jl. kaligawe raya km.4 semarang, central java, indonesia zip code : 50112 email: auliaputri.dr@unissula.ac.id received: november 30, 2020 revised: march 3, 2020 accepted: march 31, 2020 abstract extra virgin olive oil is extracted from fruit that can be used as anti-inflammatory agent. this research aimed to test the potential of extra virgin olive oil in reducing edema volume and tnf-α plasma in carrageenan-induced rats. this research was purely experimental research with the post test control group design. a total of twenty eight wistar rats were divided randomly into four treatment groups. group i was a control negative group, while the group ii, iii, and iv were orally administered with extra virgin olive oil at the dose of 0.9 ; 1.8 ; 2.7 ml/day, respectively. paw edema was measured one hour before the rats was induced to carrageenan and every hour until four hours after it was induced to carrageenan. tnf-α plasma was measured at four hours. analysis of the data was done by calculating the presentation of edema inhibition in every group, then the data was statistically analyzed by anova, repeated anova, lsd and kruskal wallis test with 95% confidence interval. the result showed that extra virgin olive oil has an antiinflammatory effect. the highest decrease in edema volume percentage was in group iii (14.21%). there was a significant difference in the edema volume of all treatment groups at each time of the experiment with tnf-α value (p < 0.05). in conclusion, the administration of extra virgin olive oil can lower the volume of carrageenan-induced edema in rats depend on the dose. also, the administration of extra virgin olive oil can be dose-dependent in reducing the levels of tnf-α in carrageenan-induced edema in rats. keywords anti inflammation, extra virgin olive oil, edema volume, tnf-α, carrageenan introduction inflammation is a physiological response to various stimuli such as infection, irritation or tissue injury. inflammation is also known as a type of non-specific immune response, and it involves many mediators (1,2). inflammation is a beneficial response but it can be detrimental to the host because it contributes to numerous pathogenesis of diseases including allergic, autoimmune, ina j med lab sci tech 2020; 2(1): 1-10 andina putri aulia, et al. 2 infectious, heart disease, arthritis, osteoporosis, diabetes, myopathy and cancer (3,4). during the inflammatory process, proinflammatory and cytokines are secreted. the vascular reactions cause fluids, blood elements, leukocytes, mediators and proinflammatory cytokines to accumulate at the site of injury to remove harmful agents and to repair damaged tissue. the cytokines are immune system proteins regulating interactions between cells and stimulating immune reactivity, either in a specific or nonspecific immunity (2,5). inflammatory cytokine is a small peptide secreted primarily by macrophages and lymphocytes that will activated the tissue in response to trauma stimuli, such as endotoxin, immune complexes, physical and chemical trauma (3,6). the pro-inflammatory mediators respond to various stimuli, including bacterial lipopolysaccharide (lps), cytokines, and uv radiation that will further induces the activation of nuclear factorkappa b (nf-kb) and activator protein-1 (ap-1). the nf-kb activates a number of molecules involved in the inflammatory response (proinflammatory cytokines), including inos, cox-2, tnf-α, il-1β, and il-6 (7,8). meanwhile, tumor necrosis factor-α (tnf-α) is a cytokine that has a different reaction in different cells. it involved in all process of inflammations and it can be used as an indicator of oxidative stress, apoptosis or necrosis that occur in the cells. this cytokine induces acute phase reaction and activates the vascular endothelium, leukocytes, platelets and fibroblasts, so the cascade of inflammatory process is initiated by vascular, cellular and humoral immune system (6,9). the vascular changes due to pro inflammatory cytokines induction that will cause the movement of fluid to interstitial tissue called edema, one of the cardinal signs of inflammation (3). the prevalence of inflammation is associated with place, race and disease. nonsteroidal anti-inflammatory drugs (nsaids) is one of the most commonly prescribed for the treatment of inflammation (10). thirty million tablets of non-steroidal antiinflammatory drugs are sold in the united states annually. this number reflects the dependency on anti-inflammatory drugs (4). additionally, the incidence of inflammatory disease such as osteoarthritis and gout disease has increased. more than fifty percent of nsaids are administrated to patients over 60 years old, so it leads to the increase in the side effect of nsaids. there are several ways to prevent or slow down the progress of inflammation, either by using drugs or medicinal plants. up to now, it is estimated that the indonesian people still use a variety of plants for an alternative therapy. the use of plants as drugs are expected to have relatively low side effects compared to anti-inflammatory drugs. the ina j med lab sci tech 2020; 2(1): 1-10 andina putri aulia, et al. 3 long term use of nsaids can cause erosion and bleeding in the lower gastrointestinal tract. it was reported that nsaids cause injury and affect the integrity of the mucous membrane of the gastrointestinal tract (10). one of the plants widely used as a drug is olive’s fruit. olive can be processed into olive oil, virgin olive oil and extra virgin olive oil. extra virgin olive oil has different characteristic from other types of olive oil because of its refining process and its composition (11). olive oil is used as a dietary component by the mediterranean community to reduce the risk of illness and death. the mediterranean people highly value the high oleic acid in olive oil as well as its minor components; the phenolic compounds (12). phenolic compounds in extra virgin olive oil have been shown to have anti-inflammatory and anti-oxidant properties, as well as anti-microbial activity. various phenolic compounds in extra virgin olive oil play an anti-inflammatory role in decarboxy methyl ligstroside aglycone (oleocanthal), hydroxytyrosol, flavonoid, and oleoropein (13). the phenol compound of extra virgin olive oil has been shown to decrease the concentration of interleukin-6 (il-6), a pro-inflammatory cytokine secreted by response to trauma. other studies have shown that phenolic compounds in olive oil can inhibit the cyclooxygenase-2 (cox-2) activity. impellizzer et al. also reported that oleuropein could reduce tnf-α, il-1β and no in carrageenan-induced pleura in rats (8,14). based on the afore mentioned reasons, the researcher wanted to analyzed the effects of extra virgin olive oil on carrageenaninduced paw edema volume and levels of tnf-α in rats. materials and methods this research method is an experimental with post-test only design only randomized control group design. the population in this study were 28 male wistar rats (rattus norvegistus) aged 2 3 months, weighing about 200 grams. animals were acclimatized for 7 days and were divided into 4 groups. group i was the control group. group ii, iii and iv were orally administrated with a single dose of extra virgin olive oil at 0.9 ml/day, 1.8 ml/day, and 2.7 ml/day, respectively. all groups were given 2% of carrageenan injection (0.1 ml). one hour before the injection of carrageenan, all labolatory animals were subjected to paw volume evaluation. the extra virgin olive oil was produced in italy with the brand bertolli. it was administrated 30 minutes after carrageenan injection, and was followed by edema paw volume measurement at h1 (after 1 hour), h2 (after 2 hour), h3 (after 3 hour) and h4 (after 4 hour) after injection. in h4, all groups were sacrificed under ether anesthesia. blood samples were taken through the heart after surgery. the blood samples were centrifuged ina j med lab sci tech 2020; 2(1): 1-10 andina putri aulia, et al. 4 at 1500 rpm for 30 minutes to obtain serum for tnf-α levels analysis. this research has been approved by the research ethics committee of the faculty of veterinary medicine, airlangga animal care and use committee (acuc) with the ethical clearance number 576-ke. all data was analyzed using saphiro wilk normality test (p > 0.05) and homogeneity of variance levene's (p > 0.05). the differences between unpaired groups were analyzed using anova, repeated anova and lsd test for rat paw edema volume, and oneway anova (p < 0.05) and lsd (p < 0.05) test for the tnf-α. all statistical tests were performed with spss program. results the increased volume of edema (table 1) showed that the control group experienced an increase in edema volume during the first, second and third hours after induction of carrageenan. afterwards, there was a decrease in edema volume at the h4. in group ii and iii, edema volume increased up to 1 hour after injection of carrageenan, and it began to decrease in the second hour after the injection of carrageenan (figure 1). the groups were sacrificed by anesthetizing using ether, and the blood samples were taken through the heart after surgery. blood samples were centrifuged at 1500 rpm for 30 min to obtain serum for tnf-α levels analysis. in group iv, there was an increase in edema volume occurred shortly after the injection of carrageenan (t0) and there was a decrease in edema volume at 1 hour after injection of carrageenan (t1). table 1 shows the percentage of a decline in edema volume in each group. the percentage of reduction in edema volume in the group iv was 14.21%. table 1. mean and standard deviation of rat’s paw edema volume (ml) group h-1 h0 h1 h2 h3 h4 δvolume (%) k* 2.63 ± 0.40 3.34 ± 0.46 3.42 ± 0.45 3.48 ± 0.44 3.6 ± 0.40 3.29 ± 0.54 10,25 ± 5,6 p1* 2.53 ± 0.38 3.14 ± 0.29 3.17 ± 0.36 2.99 ± 0.33 2.88 ± 0.44 2.87 ± 0.37 11,8 ± 11,6 p2* 2.47 ± 0.22 3.13 ± 0.23 3.22 ± 0.27 3.06 ± 0.23 2.94 ± 0.2 2.8 ± 0.25 13,28 ± 6,0 p3* 2.59 ± 0.31 3.23 ± 0.30 3.16 ± 0.33 3.16 ± 0.29 3.08 ± 0.33 2.85 ± 0.28 14,21 ± 4,5 *anova test : p < 0.05 fig 1. mean increase in edem volume (ml) 2,00 2,50 3,00 3,50 4,00 t before t0 t1 t2 t3 t4 control kp1 kp2 kp3 interval time of measurement (hour) m e a n e d e m a v o lu m e (m l ) ina j med lab sci tech 2020; 2(1): 1-10 andina putri aulia, et al. 5 the anova test showed a significant difference in all groups. thus, there was a significant difference among the time of administration in each group. lsd test of edema volume variables showed that there was much difference in edema volume in the time between h-1 and h3 (p = 0.001). in the group i, the most significant difference in edema volume was found between h-1 and h3 (p = 0.001). meanwhile, in the treatment group iii, the most different in edema volume was the time between h-1 and h1 (p = 0.001), and time between h-1 and h1 (p = 0.001). furthermore, in the treatment group iv, the most different in edema volume was the time between h-1 and h0 (p = 0.001). a repeated anova test was used to determine whether there were mean differences in the repeated measurements. the results showed an overall significant difference between the time of measurement (p = 0.001) and a significant difference in the mean of edema volume between the groups based on the time of measurement (p = 0.013). the lsd test based on the time of measurement that showed a significant difference were: the time group between h-1 and h0 (p = 0.001), h-1 and h1 (p = 0.001), h1 and h2 (p = 0.001), h-1 and h3 = 0.001), h1 and h4 (p = 0.001), h0 and h4 (p = 0.001), h1 and h3 (p = 0.019), h1 and h4 (p = 0.001), h2 and h4 (p = 0.001) and h2 and h4 (p = 0.001). there was a significant difference between h-1 and h0 (p = 0.001). therefore, it can be concluded that all groups has edema in the rat paw after injection of carrageenan. the lsd results also showed that there were significant differences in edema volume based on the time of repeated measurement which was found between group i and ii (p = 0.037), and group i and iii (p = 0.042). the percentage of reduction in edema volume has a normal distribution yet the variance was not homogeneous, so that the different test used was kruskal wallis test. the different test results of the percentage of reduction in edema volume after administration of extra virgin olive oil showed that there was no significant difference (p = 0.268) among the groups (table 1). thus, there was no difference in the percentage of reduction in edema volume in all of groups. in the variable of tnf-α level (figure 2), the highest mean was found in the control group (2,736.6 ± 1,535.2 pg/ml) while the lowest one was found in the group p3 (380.64 ± 90.0 pg/ml) anova test result p = 0.001). it indicated that there were significant differences between groups. the result of lsd test showed that there was a significant difference between group i and group iii (p = 0.003), group i and group iv (p = 0.001), group ii and group iii (p = 0.033), group ii and group iv (p = 0.001), as well as group iii and group iv (p = 0.012). the most ina j med lab sci tech 2020; 2(1): 1-10 andina putri aulia, et al. 6 significant difference was between group i and group iv, along with group ii and iv. fig 2. difference in the mean of tnf-α discussion anti-inflammation test of olive oil to reduce edema volume based on the table 1, we can analyse the change of rat paw volume in each treatment group. in h0 (approximately 15 minutes after injection of 2% of carrageenan), there was an inflammation triggered by 2% of carrageenan as shown by the increasing volume of rat paw in all of groups. the control group showed a difference in the increase of edema volume of 0.71 ml compared to the previous leg volume at the time h-1. group ii, group iii and group iv showed a difference in the increase of edema volume of 0.61 ml, 0.66 ml, and 0.63 ml, respectively. this research is in accordance with the research conducted by hidayati et al. (5), showing that there was an increase in edema volume at 15 minutes after the injection of carrageenan (5). table 1 also showed that the largest dose of 2.7 ml of extra virgin olive oil administered to the group iv has the most rapid anti-inflammatory effect compared to that of the other treatment groups and has the smallest percentage in the increase of edema volume. the highest decrease in edema volume was the treatment in the group iv (14.21%). based on result in table 1, there was a significant difference in edema volume between the measurement time in all of groups. this research is supported by fezai et al (14), which stating that there was a significant effect on the volume of rat paw edema injected with extra virgin olive oil due to the olive oil phenolic compounds that can lower the prostaglandin level by inhibiting the cyclooxygenase. the inhibition of edema volume by extra virgin olive oil was due to the inhibition of 0 500 1000 1500 2000 2500 3000 k p1 p2 p3 t n f -α (p g /m l ) group tnf-α ina j med lab sci tech 2020; 2(1): 1-10 andina putri aulia, et al. 7 cox-2 which play an important role in the conversion of arachidonic acid to prostaglandin formation, so that the inflammation will not occur (15). during the inflammation, various inflammatory mediators including 5-hydrocytriptamine (5ht), chemotactic factors, bradykinin, leukotrien and prostaglandin are released. prostaglandins and prostacyclin cause erythema and vasodilatation as well as increase the local blood flow in vitro. histamine and bradykinin also play a role in the increase of vascular permeability yet the vasodilation effect is not as much as that in the prostaglandins. there is an inflammation in the initial phase due to the release of histamine, serotonin and other similar substances. then, in the next phase, there is an activation of prostaglandins, protease, lysosomes and other quinine substances (16). this research is also supported by hidayati (5) study showing that flavonoids can decrease the volume of inflammatory edemainduced rat. another study conducted by favacho et al. (17) also said that oleic acid in euterpe oleracea martmay’s fruit has decrease the volume of rat paw edema injected with carrageenan (17). the repeated anova test result shows overall significant differences between the measurement time (p = 0.001). there were significant differences in mean between repeated measurement edema volume period (p = 0.013) among the groups. the lsd test results showed that there was a significant difference (p = 0.001) between the time group h-1 the time group h0 (shortly after the injection of carrageenan), indicating that there was a significant increase in rat edema volume (edema) after carrageenan injection. the result of lsd test among all treatment groups based on repetition of measurement showed that there was a significant difference between group i and group ii treated with olive oil at the dose of 0.9 ml. furthermore, a significant difference was also obtained between group i and group iii treated with olive oil at the dose of 1.8 ml while those in groups i and group iv treated with olive oil at 2.7 ml showed no significant difference. this result is consistent with the previous research elaborated by fezai et al., (14) showing that the largest dose of extra virgin oil has no statistically significant difference. it is possible that the relation between the volume of carrageenan injected was slightly different in each animal so that the volume of edema was not similar. the results of different tests indicated the percentage of reduction in edema volume p = 0.480 (p > 0.05), meaning that there was no significant difference in the effect of extra virgin olive oil on the percentage of reduction of edema volume among the groups. however, the clinical percentage of reduction in edema volume was greater along with the increasing dose. it is likely due to the absorption, distribution, metabolism and ina j med lab sci tech 2020; 2(1): 1-10 andina putri aulia, et al. 8 excretion of the active compounds in extra virgin olive oil will vary depending on the body. therefore, the dose of olive oil in this study can not be used as a reference of effective dose of olive oil as an antiinflammatory agent. anti-inflammation test of olive oil on tnf-α the carrageenan injected in rat's paw involves several mediators. in the early phases of inflammation, the first detected mediators are histamine, serotonin and bradykinin. in the next phase, the detected mediator is prostaglandin, a mediator that causes an increase in the vascular permeability. additionally, local or systemic inflammation due to carrageenan was associated with the increase in proinflammatory cytokines such as tnf-α, il-1 and il-6 (18). the administration of extra virgin olive oil containing phenolic compounds such as hydroxytyrosol, oleochantal and flavonoids can lower tnf-α levels through the blocking of ikk phosphorylation resulting in inhibition of the degradation of ikb proteosomal, thus preventing the activation of nf-kb (19). the level of tnf-α increased four hour after injection, it is based on the research conducted by ogata et al. (20) showing that elevated levels of the highest plasma tnf-α present at the fourth hour in rats injected by carrageenan intraperitonially. furthermore, the results showed that the highest level of tnf-α was in the control group, and there was a decrease along with an increasing dose. this is in line with the research conducted by amijaya et al. (21) which suggested that flavonoids may lower the levels of tnf-α in inflammatory-induced mice. the research conducted by hardyanto et al. (22) also showed that flavonoids can decrease tnf-α in rats induced by urolithiasis. the result of lsd test showed that there was a significant difference between control group and p2 group administrated with olive oil at the dose of 1.8 ml. additionally, there was a significant difference between control group and p3 group treated with 2.7 ml olive oil. this statistic difference was not similar from clinically decreased levels of tnf-α. the highest dose of olive oil has the highest anti-inflammatory effect in lowering plasma tnf-α levels. it is possible that the absorption, distribution, metabolism and excretion of active compounds of olive oil in the body vary depending on the microenvironment. in a study conducted by nugraheni (11), the percentage of oleic acid in olive oil was 77.478%. oleic acid contained in olive oil acts as an anti-inflammatory agent by inhibiting cox-2 regulation. at 30-50 g of olive oil is consumed, with a concentration of 180 mg/kg of phenol compounds, it is estimated that 8 mg of phenolic compounds ina j med lab sci tech 2020; 2(1): 1-10 andina putri aulia, et al. 9 such as hydroxytyrosol, tyrosol, oleocanthal and flavonoids are absorbed (23). the molecular mechanism of phenolic compounds as anti-inflammatory agents includes enzyme inhibition of proinflammatory, such as cyclooxygenase (cox-2) and lipoxygenase (lox) and inducible nitric oxide synthase (inos). it involves the activation of the peroxisome proliferator activated receptor gamma (ppar γ) and inhibition of nuclear factor-kappa b (nf-kb) (24). the result of volume edema showed significant differences between group i and group ii, group i and group iii. also, tnf-α levels showed significant differences between group i and group iii as well as group i and group iv. this is presumably related to the capture point difference of each active compounds of extra virgin olive oil, causing differences in the effectiveness of extra virgin olive oil in various dosage levels of the edema volume and serum tnf-α. therefore, this study could not show the effective dose of anti inflammatory response of extra virgin olive oil. conclusions this study concludes that extra virgin olive oil can reduce the volume edema in rats according to the dose and it can be dosedependent in reducing the levels of tnf-α in carrageenan-induced edema in rats. further studies are needed to determine the efectiveness and toxicity of the extra virgin olive oil dose that has an anti inflammatory activity. in addition, the purification, identification, and quantification of the active subtance in the extra virgin oil that has an anti inflammatory activity is also necessary to be analysed. tnf-α tissue examination and immunohistochemistry examination of rat’s paw expression are also needed. conflict of interest there are no conflicts of interest. references 1. stankov sv. definition of inflammation, causes of inflammation and possible antiinflammatory strategies. open inflamm j. 2012 jul;5(1):1–9. 2. baratawidjaja kg, rengganis i. basic immunology. xi. edition xi. jakarta, indonesia: university of indonesia faculty of medicine publisher agency; 2014. 3. chen l, deng h, cui h, fang j, zuo z, deng j, et al. inflammatory responses and inflammationassociated diseases in organs. oncotarget. 2018;9(6):7204–18. 4. beyaert r, loo g van. inflammation. in: lang f, editor. encyclopedia of molecular mechanisms of disease. volume 2. belgium: springer vdrlag berlin heidelberg; 2009. 1052–3. 5. hidayati nura, listyawati s, setyawan adwi. chemical content and antiinflammatory test of ethanol extract of lantana camara l. in white rats (rattus norvegicus l.) male. 2008;5(1):10–7. 6. huang w, glass ck. nuclear receptors and inflammation control: molecular mechanisms and pathophysiological relevance. arterioscler thromb vasc biol. 2010;30(8):1542–9. 7. khan s, shehzad o, cheng m, li r, kim ys. pharmacological mechanism underlying antiinflammatory properties of two structurally divergent coumarins through the inhibition of ina j med lab sci tech 2020; 2(1): 1-10 andina putri aulia, et al. 1 0 pro-inflammatory enzymes and cytokines. j inflamm. 2015;1–11. 8. khalatbary ar, zarrinjoei g. antiinflammatory effect of oleuropein in experimental rat spinal cord trauma. 2012;14(4):229–34. 9. cruvinel wm, júnior dm, araújo jap, catelan ttt, de souza aws, da silva np, et al. immune system part i fundamentals of innate immunity with emphasis on molecular and cellular mechanisms of inflammatory response. rev bras reumatol. 2010;50(4):443–61. 10. kurniawan b, n carolia, a sukohar, thamrin a. antiinflammatory effectiveness of binahong leaves extracts (anredera cordifolia (ten.) steenis) in male sprague dawley rats induced by carrageenan. 2014;4(8):10–7. 11. nugraheni b. decreased blood sugar levels due to giving extra virgin olive oil (study on sprague dawley mouse mice induced by high fat feed). 2012;35(0215):116–21. 12. cicerale s, lucas l, keast r. biological activities of phenolic compounds present in virgin olive oil. 2010;458–79. 13. parkinson l, keast r. oleocanthal , a phenolic derived from virgin olive oil : a review of the beneficial effects on inflammatory disease. 2014;12323–34. 14. fezai m, senovilla l, jemaà m, ben-attia m. analgesic , anti-inflammatory and anticancer activities of extra virgin olive oil. 2013;2013. 15. kumar, abbas, fausto, levy mm. robbins and cotran pathologic basis of disease 9th ed. 2013. 16. bhagat r, misar a v, ambavade sd, kulkarni dk. hptlc analysis and anti-inflammatory activity of jatropha gossypifolia l . root in mice and wistar rats. 2013;3(024929):4–8. 17. favacho hs, oliveira br, santos kc, medeiros bjl, sousa pjc, perazzo ff, et al. antiinflammatory and antinociceptive activities of euterpe oleracea mart., arecaceae, oil. rev bras farmacogn. 2011 feb;21(1):105–14. 18. necas j, bartosikova l. carrageenan : a review. 2013;2013(4):187–205. 19. yang jh, kim sc, shin by, jin sh, jo mj, jegal kh, et al. o-methylated flavonol isorhamnetin prevents acute inflammation through blocking of nf-κb activation. food chem toxicol. 2013 sep;59:362–72. 20. ogata m, matsui t, kita t. carrageenan primes leukocytes to enhance lipopolysaccharideinduced tumor necrosis factor alpha production. 1999;67(7):3284–9. 21. amijaya app, murwani s, wardhana aw. effect of moringa oleifera leaf water extract on tumor necrosis factor alpha levels and histopathological features of coronary artery endothelial cells in white rats (rattus norvegicus) fed atherogenic diet. univ brawijaya; 2013. 22. hardyanto j, trisunuwati p, winarso d. effects of leaf extract and water clover stem (marsilea crenata) on decreased levels of alpha necrosis and interleukin 1 beta tumors in white rats (rattus norvegicus) urolithiasis. univ brawijaya; 2013. 23. romero m gomez, villaba rga, pancorbo alegria c, gutierrez alberto f. metabolism and bioavailability of olive oil polyphenols. in: boskou dd, editor. olive oil constituens, quality, health properties and bioconversions. in tech; 2012. 333–56. 24. chen l, teng h, jia z, battino m, miron a, yu z, et al. intracellular signaling pathways of inflammation modulated by dietary flavonoids: the most recent evidence. crit rev food sci nutr. 2018;58(17):2908–24. 76 purple sweet potato (ipomoea batatas l.) peels extract as an alternative dye for bacteria gram staining nastasya nunki1, diah titik mutiarawati2, endah prayekti1 1department of medical laboratory technology, faculty of health, universitas nahdlatul ulama surabaya, surabaya, indonesia 2department of medical laboratory technology, poltekkes surabaya, surabaya, indonesia correspondence: nastasya nunki, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code : 60237 email: nastasya.nk14@student.unusa.ac.id received: july 12, 2020 revised: july 22, 2020 accepted: august 25, 2020 abstract crystal violet and safranin are dyes in gram staining, which are carcinogenic. alternative safe materials are needed to minimize the use of carcinogenic properties. purple sweet potato (ipomoea batatas l.) peels were the candidate of the alternative dye source because of its high anthocyanin pigment. the purpose of this study was to determine purple sweet potato (ipomoea batatas l.) peels extract as an alternative to gentian violet in gram staining of bacteria. extracts obtained from purple sweet potato peels studied with varying concentrations of 50%, 60%, and 75% for 1, 3, and 5 min as a substitute for gentian violet on bacillus sp. the parameters observed from this study based on visual field clarity, glass slide cleanliness, contrast, bacterial shape, bacterial colour. each extract concentration compared with a control group using gentian violet. the results showed that optimum staining in 50% concentration for 5 min, 60% concentration for 5 min, 75% concentration for 3 min, and 5 min respectively. the present study exhibited the potency of ipomoea batatas l. peels extract as an alternative staining agent. keywords ipomoea batatas l., gentian violet, bacteria gram taining, bacillus sp. introduction the microorganism is a living organism that is very small in size, so microscopic observation aids are needed. microorganisms studied in the laboratory for various purposes. the study of these microorganisms intends to facilitate and assist in the examination service in the laboratory (1). bacteria have a strong cell wall as an outer part and maintain the shape of bacteria and protect against osmotic pressure. in staining, there are variations in the structure of cell walls. where the complex structure, semi-rigid with bacterial cell wall thickness ina j med lab sci tech 2020; 2(2): 76-84 nastasya nunki, et al. 7 7 ranges from 10 to 35nm and surrounds the cytoplasmic membrane, serves to give shape to the cell and protect the contents of the cell from outside cell influences (1). bacterial cell walls are essentials for growth and division. based on the chemical composition of the cell wall, which causes cell wall rigidity is peptidoglycan. the mechanism of gram staining based on the structure and composition of the cell wall (2). in the laboratory world, especially in the field of staining, microbiology is one of the keys to helping to provide information about the diagnosis of a disease. the existence of the development of staining procedures to assist in roughly observing the morphology of microorganisms helps in identifying parts of the cell structure of microorganisms and helps differentiate similar microorganisms (1). the importance of observing the morphology of microorganisms, where the appearance of microorganisms in living conditions is quite complex, not only because of their size but also because they are transparent and colourless when suspended in a liquid medium. to study the properties and divide microorganisms into specific groups for diagnostic purposes, the biological dyes and staining procedures with a light microscope have become the main tools in microbiology (3). the most basic and primary staining methods used are gram staining. the gram reaction is related to morphological characteristics in phylogenetic-related forms (4). synthetic dyes are including crystal violet, safranin, carbol fuchsin, are dyed commonly used in gram staining. crystal violet and safranin are dyes that often used in gram staining. crystal violet is a human carcinogen (5). to minimize the use of carcinogenic properties for gram staining, the research to find alternative materials are needed. to get natural dyes that can be used as alternative dyes, extraction methods are needed to get the best pigment (anthocyanin) from the colour of these natural ingredients, thereby maximizing the quality and quantity. purple sweet potato peel has an average anthocyanin level of 521.84–729.74 mg / 100g (6). thus, in this research conducted further testing, using peel extracts of purple sweet potatoes (ipomoea batatas l.). in the previous study (7), purple sweet potato extract can be obtained manually and can produce reddish-purple colour. for this reason, it needs to develop research to maximize the content of dyes (anthocyanin) in the purple sweet potatoes peels using extraction techniques mae (microwaveassisted extraction) method. the previous study only examined the coccus grampositive group without being compared with other groups. in this study, gram staining of bacillus sp tested and also the optimum concentration ina j med lab sci tech 2020; 2(2): 76-84 nastasya nunki, et al. 7 8 and time of gram staining were studied. the purpose of this study was to determine purple sweet potato (ipomoea batatas l.) peels extract as an alternative to gentian violet in gram staining of bacteria. materials and methods the materials used in this study were the pure culture of bacillus sp. obtained from central health laboratory (balai besar laboratorium kesehatan/bblk) in surabaya, nacl 0.85% (0,85 gr nacl [sap chemicals]/100 ml distilled water), gram stain hucker method, immersion oil (olympus), staining from ipomoea batatas l. peel extract, ethanol 96% (sap chemicals), hcl 1n (8.3 ml hcl 37% (sap chemicals)/100 ml distilled water), hcl 2n (16.6 ml hcl 37% (sap chemicals)/100 ml, nh4oh 1n (16 ml hcl 37% (merck)/100 ml distilled water), nh4oh 2n (32 ml hcl 37% (merck) 100 ml distilled water). the sample used was the peels of purple sweet potato (ipomoea batatas l.) not other types of sweet potato. this study consisted of several stages. the first stage was the process of sample preparation using mae (microwave-assisted extraction) method extraction (8), determination of the concentration formula, the staining process, observation under a microscope, and lastly data analysis. extraction techniques mae (microwave-assisted extraction) method was selected to maximize the content of dyes (anthocyanin) in the purple sweet potatoes peels. in sample preparation, the sample extracted using the mae. the extraction process carried out with a concentration variation of 50%, 60%, 75%, which approved with a combination of staining time of 1 minute, 3 minutes, and 5 minutes. at each concentration of ipomoea batatas l. peel extract, a formulation was performed to obtain the appropriate color. 50% concentration obtained by adding (extract 0.5 gr/10 ml ethanol 96%, 4ml hcl 2n, 3ml nh4oh 2 n), 60% concentration obtained by adding (extract 0.6 gr/10 ml ethanol 96%, 5 ml hcl 2n, 3.5 ml nh4oh 2 n), a concentration of 75% was obtained by adding (extract 0.75 gr/10 ml ethanol 96%, 6 ml hcl 2n, 4.2 ml nh4oh 2n). the smear prepared from culture bacillus sp. on a clean glass slide allowed it to air-dried and fixed it by flaming. for the experimental group, at first, the smear on a glass slide was added with ipomoea batatas l. extracts at each concentration for 1 minute, 3 minutes and 5 minutes. the ipomoea batatas l. extract was poured off and applied lugol for 1 minute and washed with water, the smear decolourized with an organic solvent-alcohol for 30 seconds, or until the colour was oozed from the slide and washed with water. ina j med lab sci tech 2020; 2(2): 76-84 nastasya nunki, et al. 7 9 the safranin applied for 1-minute washed with water and blot dried. the slide was observed under magnification 100x after putting a drop of immersion oil then the results were compared with staining using gentian violet for 1 minute. for the control group, the first step did not use extracts but gentian violet and the next steps as in the experimental group until the observation was processed. the results were compared directly between the gram staining using ipomoea batatas l. peel extract tested on grampositive bacteria as an experimental group. furthermore, in gram staining using gentian violet as a control group. the sampling technique in this study was a simple random sampling. the parameters observed in this study included visual field clarity, glass slide cleanliness, contrast, bacterial shape, and bacterial colour. in the data analysis, the observation was results of each extract concentration of 50%, 60%, and 75% with a staining time of 1 minute, 3 minutes, and 5 minutes compared to the control, coding was carried out based on 5 observation parameters to obtain quantitative data. then, the calculation carried out with the formula for the total number of coding values with 3 times repetition / 15 (5 parameters repeated 3 times) x 100%. results ipomoea batatas l. peel extract concentration of 50%, 60%, 75% based on observations obtained by the results of colouring where the extraction concentration of 50% showed the best results when staining for 5 minutes, the extract was inadequate to penetrate bacteria, and some bacteria were not completely stained. the extraction concentration of 60% showed the best results when staining for 5 minutes (figure 1). the dye appears to penetrate the bacterial cell wall, but some bacteria were not completely stained. the extraction concentration of 75% showed the best results when staining for 3 minutes and 5 minutes, although some bacteria were not stained (figure 2). in the gentian violet stain, visible dyes penetrate the bacterial cell wall and also the spores of bacillus sp. (figure 3). at the percentage of the staining results, at the extraction of 50% concentrations obtained staining approaching control with the results of the staining showed the optimum results when staining for 5 minutes. for extraction of a concentration of 60%, it obtained staining that approached the control with the results of the staining showed the optimum results when staining for 5 minutes. the extraction concentration of 75% attained a colour approaching the control with the results of the staining showed the optimum results when staining for 3 minutes and 5 minutes (figure 4). ina j med lab sci tech 2020; 2(2): 76-84 nastasya nunki, et al. 8 0 fig 1. microscopic observation results of bacillus sp. 100x magnification with staining of ipomoea batatas l. for 5 minutes. (a) extract concentration of 50%, (b) extract concentration of 60% fig 2. microscopic observation results of bacillus sp. 100x magnification with staining of ipomoea batatas l. extract concentration of 75%. (a) stainding for 3 minutes, (b) staining for 5 minutes fig 3. microscopic observation results of bacillus sp. 100x magnification with gentian violet for 1 minute a b a b ina j med lab sci tech 2020; 2(2): 76-84 nastasya nunki, et al. 8 1 fig 4. comparison percentage of staining results of ipomoea batatas l. extract concentration and time of staining of gentian violet the results analyzed statistically using the one-way anova test with spss instrument version 16.0. where the concentration of 50% obtained α value of 0.06, a concentration of 60% obtained α value of 0.227, a concentration of 75% obtained α value of 0.297, α value of >0.05. there was no significant difference between the results of staining using ipomoea batatas l. extracts and control (gentian violet). the determination of colour tendency was using the colour matching with the rgb table, then compared with the control (table 1). 60% 68% 68% 100% 68% 68% 80%80% 80% 86% 50 60 75 control t im e o f s ta in in g extract concentration time of staining 1 minute time of staining 3 minute time of staining 5 minute table 1. rgb code for bacterial gram staining (rgb color chart.cdr aws) bacteria time of staining (minute) rgb code for bacterial gram staining color name concentration of staining control 50% 60% 75% bacillus sp. 1 rgb: 180 4 133 rgb: 173 14 145 rgb: 180 4 133 – 153 0 122 rgb: 70 20 111 purple 3 rgb: 173 14 145 rgb: 152 4 110 rgb: 153 0 122 5 rgb: 152 4 110 rgb: 180 4 133 – 153 0 122 rgb: 141 4 123 ina j med lab sci tech 2020; 2(2): 76-84 nastasya nunki, et al. 8 2 discussion the extraction concentration of 50% showed the best results when staining for 5 minutes. the extract was inadequate to penetrate bacteria, and some bacteria were not completely stained. the extraction concentration of 60% showed the best results when staining for 5 minutes. the dye appears to penetrate the bacterial cell wall, but some bacteria were not completely stained. the extraction concentration of 75% showed the best results when staining for 3 minutes and 5 minutes, although some bacteria were not stained. the colour produced by the ipomoea batatas l. peel is purple. the colour produced by ipomoea batatas l. peel is more stable under acidic conditions and the increase in ph affects the resulting colour. besides the use of polar solvents makes it easier to dissolve the peel content of ipomoea batatas l. (6). based on the study results of yuniarti and misbach (7) that ipomoea batatas l. can give colour to bacteria. on microscopic observations, staphylococcus aureus shaped coccus with reddish-purple. in this study, the formulation was needed as an anthocyanin proof test that was appropriate in processing ipomoea batatas l., so that the colour produced under the microscope can resemble the purple colour produced by gentian violet. phenolic compounds of ipomoea batatas l. peel which included in the flavonoid group and the content of phenol compounds which is ipomoea batatas l. peel is the same as gentian vielot content. gentian violet is a triaryl methane dye with three phenol groups arranged by methane groups. the composition contained in gentian violet is (2 gr crystal violet, 20 ml ethanol 95%, 0.8 gr ammonium oxalate, 100 ml distilled water). based on a study conducted virgianti and lucyana (9), kmno4 used as an oxidizer when added to the solution of angkak and teak leaves as a cover dye in gram staining. the alleged positive charge of potassium and dye complexes forming dissociation, so that it can be bound to bacterial cell components which have a negative charge. for this reason, the process of extraction results requires the addition of ethanol to match the composition of gentian violet. besides, the addition of acid-base was useful to get the appropriate violet colour. violet colour obtained when it was at anthocyanin equilibrium, which was a quinoidal base for the addition of nh4oh and hcl serves as a counterweight when trial and error. bacillus sp. bind dyes from ipomoea batatas l., after the formulation and was obtained purple colour, which comes from the reaction between hcl and nh4oh, forming compounds nh4cl. ina j med lab sci tech 2020; 2(2): 76-84 nastasya nunki, et al. 8 3 in this study, the dyes formed tend to be alkaline dyes, to maintain the purple colour of the extraction of the peel of ipomoea batatas l. alkaline dyes are substances that produce cations. the ionization of the chromogen component shows a positive charge that can bind to the cell component, which is a negative charge. the nucleic acid is a negatively charged cell component (9, it also can accept positively charged dyes such as the gentian violet, or in this study, the dyes derived from the peel extract of ipomoea batatas l. assumes that the positive charge of nh4 ions dissociates with anthocyanin dyes in the form of cyanide which has a negatively charged oh group from the results of ipomoea batatas l. peel extracts. positive charge ions resulting from dissociation with anthocyanin dyes bind with protoplasm ions of bacterial cells that have negatively charged nucleic acids in the results of the ipomoea batatas l. peel extract form of phosphate groups (9). in this study, there was a control group and an experimental group. the control group bacillus sp. were stained with gentian violet, lugol, alcohol, and safranin. this study has limitations. the further research is needed to use ipomoea batatas l. peel extract as a substitute for gentian violet in gram stain. the author assumes that the dye produced from the peel extract of ipomoea batatas l. has not maximally penetrated the bacterial wall so that the colour of bacteria did not entirely derive from the extract. the author assumes this was coming from the residual colouring of safranin. to minimize information bias, the authors recommend that further study on this topic, especially regarding the mechanism of alternative dyes in penetrating bacterial cell walls, so that we can find out more about the ability of alternative dyes to stain bacterial cell walls. conclusions the present study demonstrated that ipomoea batatas l. peel extract can be used as an alternative staining agent, but to substitute gentian violet in gram staining of bacteria, need conducting further studies on this topic and observe to several aspects. besides, the matter that needs to be considered is the storage method and storage time for the extraction of ipomoea batatas l. peel. the author recommended conducting further studies use the extract 100% concentration and the staining time used for 5 minutes. conflict of interest there are no conflicts of interest. acknowledgment(s) the author would like to thank the laboratory assistants and several related parties who have provided direction and ina j med lab sci tech 2020; 2(2): 76-84 nastasya nunki, et al. 8 4 support in this study. the author also would like to thank bblk surabaya, microbiology laboratory faculty of health at the universitas nahdlatul ulama surabaya, chemistry laboratory at the universitas hang tuah surabaya and department of medical laboratory technology faculty of health at the universitas nahdlatul ulama surabaya. references 1. pelczar, m j, chan e c s. fundamentals of microbiology volume 1. ui press; 1988. 2. sri harti a. health microbiology. andi offset; 2015. 3. cappuccino j g, sherman n. microbiology laboratory manual edition 8. egc; 2014. 4. geo f b, karen c c, janet s b, stephen a m, timothy a m. jawetz, melnick, adelberg. medical microbiology edition 25. egc; 2012 5. sun m, ricker k, osborne g, marder m e, schmitz r. evidence on the arcinogenicity of gentian violet. oehha; 2018. 6. andarwulan, nuri, faradilla r h f. natural dyes for food. seafast center ipb; 2012. 7. yuniarty t, misbach s r. utilization of purple sweet potato extract (ipomoea batatas l.) as a staining substance in staphylococcus aureus staining. teknolab. 2016; 5(2): 59-63. 8. agung l, yunianta. anthocyanin extraction from purple sweet potato skin waste (ipomoea batatas l.) microwave-assisted extraction method (study of extraction time and solvent). artikel ilmiah malang: universitas brawijaya. 9. virgianti d p, lucyana c. use of combination of angkak extract and teak leaves as closing dyes in gram staining. jurnal kesehatan bakti tunas husada. 2017;1 7(1): 66-72. 10. subandi. microbiology. development of studies and observations in islamic perspectives: remaja rosdakarya; 2010. 11. aballe, l m, questo i a r, dimacuta s s. basella alba (alugbati) berries and hibiscus rosa sinensis (gumamela) flower crude extracts as alternative dyes for gram staining. medical laboratory science of san pedro college; 2016. 12. aishah b, nursabrina m, noriham a, norzizah a r, shahrimi m. anthocyanins from hibiscus sabdariffa, melastoma malabathricum, and ipomoea batatas and its color properties. international food research journal. 2013; 20(2): 827-834. 13. jiwintarum y, rohmi, prayuda i d p m. dragon fruit (hylocereus polyrhiuz) as a natural color for bacterial staining. jurnal kesehatan prima. 2016; 10 (2): 1726-1734. 14. kamel f h, najmaddin c. use of some plants color as alternative stain in staining of bacteria. kirkuk university journal scientific studies (kuiss). 2016; 11(3): 248-253. 15. azmi, a n, yunianta. extraction of anthocyanin from mulberry fruit (morus alba l.) method of microwave-assisted extraction (study of extraction time and the ratio of ingredients: solvent). jurnal pangan dan agroindustri. 2015; 3 (3): 835-846. 16. martha e, kresno s. qualitative research methodology for health. raja grafindo persada; 2016. 17. notoatmodjo. health research methodology. rineka cipta; 2012. 18. misnadiarly, djajaningrat h. microbiology for clinic and health. rineka cipta; 2014. 19. radji m. microbiology textbook for pharmacy and medical students. egc; 2011. 1 ability of ethanol extract from ajwa and sukkari dates (phoenix dactylifera l.) in inhibiting the growth of methicillin-resistant staphylococcus aureus (mrsa) putra rahmadea utami1, sri indrayati1, nur hayatang1 1department of medical laboratory technology, faculty of health science, universitas perintis indonesia, padang, indonesia correspondence: putra rahmadea utami, jl. adinegoro km 17, simpang kalumpang, lubuk buaya, padang, west sumatera, indonesia zip code: 25586 email: putrarahmadeautami123@gmail.com received: december 8th, 2020 revised: march 12th, 2021 accepted: march 15th, 2021 published: april 28th, 2021 doi: 10.33086/ijmlst.v3i1.1848 abstract staphylococcus aureus is a pathogenic bacterium that spread throughout the world and still a problem that continues to increase both in hospitals and the community. infections due to s. aureus usually treated with antibiotics, but in some cases, several strains of s. aureus found to be resistant to antibiotics, such as methicillin-resistant staphylococcus aureus (mrsa). based on the previous research, the ethanol extract from ajwa and sukkari dates formed an inhibitory zone against the mrsa bacteria growth. this study aims to determine the inhibition of the ethanol extract from ajwa and sukkari variety of dates (phoenix dactylifera l.) on the s. aureus growth. the ethanol extract from ajwa and sukkari dates with a concentration of 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml resulted in the same inhibition zone with a diameter of ≤ 6 mm which categorized as weak (resistant), whereas the positive control ciprofloxacin had a resistance zone with a diameter of 9 mm. this study results concluded that the ethanol extract of ajwa and sukkari dates only has a maximum concentration of 20 mg/ml, which is still classified as a low concentration and has not been able to inhibit mrsa bacteria growth. keywords dates ajwa, dates sukkari, extract ethanol, mrsa. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. introduction staphylococcus aureus is treatable with antibiotics, but several strains of s. aureus found to be resistant to antibiotics, such as methicillin-resistant staphylococcus aureus (mrsa). the spread of mrsa has been the subject of several studies because the danger of antibiotic resistance is a serious world health problem in both developed and developing countries. in 2010, the prevalence mailto:putrarahmadeautami123@gmail.com https://journal2.unusa.ac.id/index.php/ijmlst/article/view/1848/version/2332 ina. j. med. lab. sci. tech. 2021; 3(1): 1–8 putra rahmadea utami, et al. 2 of mrsa is 28% (hong kong and indonesia) and 70% (korea) among all clinical isolates of s. aureus, while the s. aureus infections found in related communities in asian countries varied widely, from 5–35% (1). the presence of mrsa found in the hospital environment. the study of vysakh & jeya (2) obtained 450 s. aureus isolates collected from patients from several hospitals in india, which 121 positives for mrsa (27%) and 329 as msa (73%). the university of alexandria, which collected 50 isolates of s. aureus strains from several hospitals, found 40% strains resistant to the antibiotics oxacillin and cefoxitin, which indicates the presence of mrsa in these isolates (2). however, research conducted in indonesia found that the prevalence of mrsa in rsud dr. sutomo surabaya, indonesia was 8.2%. despite the results, the study showed that with the low prevalence of mrsa in indonesia, a coping strategy still needed to prevent further infection (1,3). dates are one of the plants in their role as medicine and food. dates are a food ingredient that is rich in vitamins, minerals, fiber and sugar. in some varieties, the sugar content of dates can reach up to 88%. many researchers researched twelve dates varieties to find out their chemical content, such as flavonoid compound (1,2). aldaihan and bhat examined the benefits of dates (phoenix dactylifera l) in vitro and found that one of its benefits is that they have antibacterial properties. this research showed that flavonoids are the active substances in dates. flavonoids can be used as immunomodulators to increase the work of macrophages on pathogenic microbial phagocytes that attack the body (4,5). based on the previous research, the ethanol extract from ajwa and sukkari dates formed an inhibitory zone against the mrsa bacteria growth (2). extraction is a process of separating a substance based on differences in its solubility in two different insoluble liquids, usually water and other organic solvents such as nutritious substances or active substances from a part of medicinal plants, animals, and several types of fish, including marine biota (3). due to the different thickness of plant and animal cells, an extraction method with a particular solvent needed. this extraction method is based on the principle of mass transfer agent component in a solvent, wherein the displacement began in the interface layer and then diffuses into the solvent (6). this study aims to determine the inhibition of the ethanol extract from ajwa and sukkari variety of dates (phoenix dactylifera l.) on the s. aureus growth. materials and methods this research used an experimental research design and conducted in julyaugust 2020 at the microbiology laboratory of andalas university, padang, indonesia. ina. j. med. lab. sci. tech. 2021; 3(1): 1–8 putra rahmadea utami, et al. 3 this study performed to determine the inhibition zone of the ethanol extract from ajwa and sukkari dates against the mrsa. this study used the ethanol extract from ajwa and sukkari dates. extraction was carried out using 96% ethanol solvent for 3x24 hours and evaporated using a rotary evaporator to obtain a thick extract of ± 150 g. the extract concentrations used were 5 mg/ml, 10 mg/ml, 15 mg/ml and 20 mg/ml. the study used mrsa bacteria, namely s. aureus. bacterial isolates obtained from rsup dr. m. djamil padang, indonesia. bacteria isolates were resistant to penicillin-derived antibiotics. the positive control used ciprofloxacin, while the negative control used sterile aquadest for the treatment. results characteristics of ethanol extract of sukkari and ajwa dates the pulp of ajwa and sukkari dates each ±500 g separated from the seeds. furthermore, from the ethanol extract, each obtained ±150 g of thick extract. the thick extracts from ajwa dates and sukkari dates shown in figure 1. the color of sukkari dates is darker than ajwa dates. characteristics of methicillin-resistant staphylococcus aureus (mrsa) the bacteria used were mrsa bacteria obtained from the microbiology laboratory of andalas university, padang. the mrsa bacterial suspension is already present in the media (figure 2). inhibition test of ethanol extract of ajwa dates and sukkari on the growth of methicillin-resistant staphylococcus aureus the test results of the inhibition of the ethanol extract of ajwa and sukkari dates with various concentrations of 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml can be seen in figure 3. this study result showed that the ethanol extract compounds from sukkari and ajwa dates could not inhibit mrsa bacteria growth. after that, it observed that there was no clear zone or zone of inhibition around the disc. in this study, the positive control were used ciprofloxacin 50 mg, while the negative control were used sterile distilled water. table 1 showed that the extract from ajwa and sukkari dates could not inhibit mrsa bacteria growth. a seen in figure 2, the zone of clearance appears from disk diameter paper discs. repetition performed three times to show the inhibition zone diameter ≤ 6 mm (diameter paper discs). ina. j. med. lab. sci. tech. 2021; 3(1): 1–8 putra rahmadea utami, et al. 4 figure 1. dates extract using ethanol solvent. (a) sukkari, (b) ajwa. figure 2. characteristics of mrsa. (a) mrsa turbidity compared to mcfarland standard of 0.5 (left) and bacterial suspense (right), (b) the arrow in figure b shows the mrsa colony of bacteria, (c) the arrow in figure c shows cefoxitin resistance. . figure 3. inhibition test. (a) sukkari dates ethanol extract concentrations of 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml, (b) ajwa date ethanol extract concentrations of 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml, (c) and ciprofloxacin. a b a b c a b c ina. j. med. lab. sci. tech. 2021; 3(1): 1–8 putra rahmadea utami, et al. 5 table 1. the results of the inhibition test of mrsa ethanol extract dates concentration (mg/ml) extract repetition (mm) x 1 2 3 ajwa dates 5 ≤ 6 ≤ 6 ≤ 6 ≤ 6 10 ≤ 6 ≤ 6 ≤ 6 ≤ 6 15 ≤ 6 ≤ 6 ≤ 6 ≤ 6 20 ≤ 6 ≤ 6 ≤ 6 ≤ 6 sukkari dates 5 ≤ 6 ≤ 6 ≤ 6 ≤ 6 10 ≤ 6 ≤ 6 ≤ 6 ≤ 6 15 ≤ 6 ≤ 6 ≤ 6 ≤ 6 20 ≤ 6 ≤ 6 ≤ 6 ≤ 6 control positive ciprofloxacin 50 mg 9 negative control aquadest ≤ 6 note: description of disc paper diameter = 6 mm discussion the study results showed no inhibition zone formed from the ethanol extract of ajwa and sukkari dates against the growth of mrsa bacteria. ajwa and sukkari dates ethanol extract with concentrations of 5 mg/ml, 10 mg/ml, 15 mg/ml and 20 mg/ml formed weak (resistant) inhibition zone diameter of ≤6 mm. meanwhile, the positive control ciprofloxacin formed an inhibition zone with a diameter of 9 mm (7). this condition happened because the bacteria used for this study was mrsa, where these bacteria are a group of bacteria that are already resistant to antibiotics. mrsa is a s. aureus that immune to methicillin-type antibiotics. mrsa experiences resistance due to genetic changes caused by irrational exposure to antibiotic therapy (8). besides, the research results also prove that mrsa is also resistant to other antibiotics. this methicillin resistance will be followed by the simultaneous emergence of resistance to a large number of other antibiotic classes through the acquisition of additional receptor determinants, or as a result of mutations. all of which will cause the receptors at the target site to become resistant (9,10). one of the factors of differences in sensitivity patterns or determinants of bacterial resistance to antimicrobials carries by genetic information outside the chromosomes, namely plasmid (11). s. aureus is a bacterium that has small plasmids and large plasmids that have more than one resistance gene. based on the study result, it can be assumed that there are large plasmids that carry more than one antibiotic-resistant gene in mrsa. additionally, the methicillin ina. j. med. lab. sci. tech. 2021; 3(1): 1–8 putra rahmadea utami, et al. 6 resistant gene may be linked to other antibiotic-resistant genes. mrsa bacteria, which are gram-positive bacteria, have a peptidoglycan layer that is 20–80 nm thick. gram-positive bacteria also have a cell wall containing teichoic acid, which is a watersoluble polymer that functions as a transport for positive ions in and out (11,12,13). other studies conducted on the use of plant extracts to inhibit mrsa growth. one of the studies showed that the clove flower extract has an antimicrobial effect, which containing eugenol, flavonoids, tannins, saponins, alkaloids and phenols that can damage the structure of bacterial cells. the results showed that the minimum bactericidal concentration (mbc) was 0.39%. one way anova test showed a significant difference (p < 0.05) between clove flower extract concentration and the number of mrsa colonies (1). clove flower extract can kill mrsa by damaging the structure of bacterial cells. another study was to test the inhibition of the ethanol extract of bitter melon (momordica charantia) against the growth of mrsa. the ethanol extract of bitter melon fruit used as a test solution with a concentration (w/v) of 20%, 40%, 60%, and 80%. the results showed that the average diameter of the inhibition zone of the ethanol extract of bitter melon against the growth of mrsa with concentrations of 20%, 40%, 60%, and 80% was 6.16 mm, 9.5 mm, 10.83 mm, 12.3 mm respectively. the higher the ethanol extract concentration of bitter melon fruit, the higher the inhibition power of the growth of mrsa (14,15,16). the plant extracts used also contain content that is almost the same as other plants, namely eugenol, flavonoids, tannins, saponins, alkaloids and phenols, which can damage the structure of bacterial cells. however, in this study, the ethanol extract of dates did not have a significant ability to inhibit the growth of mrsa (2). whereas in research, a combination of extracts with different plants can inhibit the growth of other bacteria. in the research of utami et al. (13), the results of testing the combination of chinese petai extract and aloe vera showed significant differences (p < 0.05) at the concentrations of 25 g, 50 g, 75 g, and 100 g. the ethanol extract of chinese petai and aloe vera can inhibit the growth of e. coli. the results showed the most effective concentration of 100 g/ml resulted in an interaction between the ethanol extract of aloe vera and china petai in inhibiting the growth of e. coli (12,17). in contrast, this ajwa and sukkari date research, only have 20 mg/ml as the maximum concentration, which still classified as a low concentration. as a result, the active compound content is the only low level and unable to inhibit the growth of mrsa bacteria (4,18,19). ina. j. med. lab. sci. tech. 2021; 3(1): 1–8 putra rahmadea utami, et al. 7 conclusions the ethanol extract of ajwa and sukkari dates with a concentration of 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml formed the same inhibition zone with a diameter of ≤6 mm. it conclusion, this study indicate that the ethanol extract of ajwa and sukkari dates only has a maximum concentration of 20 mg/ml, which is still classified as a low concentration and has not been able to inhibit mrsa bacteria growth by damaging the bacterial cell structure. author contributions putra rahmadea utami: conceptualization, methodology, writingreviewing and editing. sri indrayati: data curation, writing-original draft preparation, supervision. nur hayatang: visualization, investigation. acknowladgements this study was supported by the universitas perintis indonesia. conflict of interest there are no conflicts of interest. references 1. setiawan hg, kaligis ahm, assa ya. description of serum apolipoprotein b (apo-b) levels in lacto-ovo vegetarians. ebiomedik. 2017;5(1):2–5. 2. kuntaman k, hadi u, setiawan f, koendori eb, rusli m, santosaningsih d, et al. prevalence of methicillin resistant staphylococcus aureus from nose and throat of patients on admission to medical wards of dr soetomo hospital, surabaya, indonesia. southeast asian j trop med public health. 2016;47(1):66–70. 3. kader oa, el-batouti ga, ghazal aa, baraka km. hospital-acquired methicillin resistant staphylococcus aureus: analysis of mec a gene and taphylococcal cassette chromosome. int j curr microbiol appl sci. 2015;4(9):805–15. 4. nismawati, rizalinda s, agus r. deteksi methicillin resistant staphylococcus aureus (mrsa) pada pasien rumah sakit universitas hasanuddin dengan metode kultur detection of methicillin resistant staphylococcus aureus (mrsa) in hasanuddin university hospital patients by culture method. pros semin nas biol. 2018;4(1):978–602. 5. hamad i, abdelgawad h, al jaouni s, zinta g, asard h, hassan s, et al. metabolic analysis of various date palm fruit (phoenix dactylifera l.) cultivars from saudi arabia to assess their nutritional quality. molecules. 2015;20(8):13620–41. 6. hariadi b, widodo a. the effect of dates (phoenix dactylifera l.) extract of ajwa varieties on no levels in balb/c mice infected with salmonella typhimurium. diponegoro med j (jurnal kedokteran diponegoro). 2018;7(2):751– 61. 7. syarif ra, sari f, ahmad ar, indonesia um, urip j, km s. rimpang kecombrang (etlingera elator jack.) as a phenolic source. j feto ina. 2000;2(2):102–6. 8. fiţ ni, chirilă f, nadăş g, pall e, mureşan r. comparative testing of antimicrobial activity of aqueous extracts of aloe vera and lycium barbarium. bull univ agric sci vet med clujnapoca vet med. 2013;70(1):72–6. 9. hu w, li c, dai j, cui h, lin l. antibacterial activity and mechanism of litsea cubeba essential oil against methicillin-resistant staphylococcus aureus (mrsa). ind crops prod. 2019;130(november 2018):34–41. 10. bussmann rw, glenn a, sharon d. antibacterial activity of medicinal plants of northern peru can traditional applications provide leads for modern science?. indian j tradit knowl. 2010;9(4):742– 53. 11. craft km, nguyen jm, berg lj, townsend sd. methicillin-resistant: staphylococcus aureus (mrsa): antibiotic-resistance and the biofilm ina. j. med. lab. sci. tech. 2021; 3(1): 1–8 putra rahmadea utami, et al. 8 phenotype. medchemcomm. 2019;10(8):1231– 41. 12. suryati n, bahar e, ilmiawati i. antibacterial effectiveness test of aloe vera extract against escherichia coli growth in vitro. j kesehat andalas. 2018;6(3):518. 13. rahmadea up, chairani, yudha h. combination test of chinese leaf extract (leucaena leucocephala folium) and aloe vera inhibiting growth escherichia coli. indones j med lab sci technol. 2020;2(2):60–7. 14. utami pr, chairani c, ilhamdi i. interaksi ekstrak etanol daun petai cina (leucaena leucocephala folium) dan lidah buaya (aloe vera l.) menghambat pertumbuhan staphylococus aureus secara invitro [the interaction of ethanol extract of chinese petai leaves (leucaena leucocephala folium) and aloe vera (aloe vera l.) inhibiting the growth of staphylococus aureus by invitro]. j kesehat perintis (perintis’s heal journal). 2019;6(2):186–92. 15. azizah a, suswati i, agustin sm. anti-microbial effect of clove flower extract (syzygium aromaticum) against methicillin-resistant staphylococcus aureus (mrsa) in vitro. saintika med. 2018;13(1):31. 16. suhaimi s, indrawati t, kumala s. combination test of crude fruit extracts (aloe vera. (l) brum. f.) and blue leaves (piper crocatum ruiz & pav) extracts for purple leaf (piper crocatum ruiz & pav) antibacteries for acne. jiffk j ilmu farm dan farm klin. 2018;15(01):12. 17. cuny c, friedrich a, kozytska s, layer f, nübel u, ohlsen k, et al. emergence of methicillinresistant staphylococcus aureus (mrsa) in different animal species. int j med microbiol. 2010;300(2–3):109–17. 18. stefani s, chung dr, lindsay ja, friedrich aw, kearns am, westh h, et al. meticillin-resistant staphylococcus aureus (mrsa): global epidemiology and harmonisation of typing methods. int j antimicrob agents. 2012;39(4):273–82. 19. pratama p, purwanta m, qurnianingsih e. the effectiveness of ethanol extract of egyptian date palm seeds (phoenix dactylifera l.) as an antibacterial agent against streptococcus pyogenes in vitro. 2019;19(3):135–40. 20. vysakh pr, jeya m. a comparative analysis of community acquired and hospital acquired methicillin resistant staphylococcus aureus. j clin diagnostic res. 2013;7(7):1339–42. 56 the antibacterial activity of thermoactinomyces sp. (h24) extract against escherichia coli and staphylococcus aureus julia nanda puspita1, rikhsan kurniatuhadi1, rahmawati 1 1department of biology, faculty of mathematics and natural sciences, tanjungpura university, west kalimantan, lndonesia correspondence: rahmawati, jl. prof. dr. h. hadari nawawi, pontianak, west kalimantan, indonesia zip code: 78124 email: rahmawati@fmipa.untan.ac.id received: august 15th, 2020 revised: february 13th, 2021 accepted: march 31th, 2021 published: april 28th, 2021 doi: 10.33086/ijmlst.v3i1.1700 abstract bacteria of the genus thermoactinomyces have the ability to produce antibacterial bioactive compounds. this bioactive compound can be used for combating diarrheal agents such as escherichia coli and staphylococcus aureus. this study aims to determine the antibacterial activity of the metabolite extract from thermoactinomyces sp. (h24) against e. coli and s. aureus. methanol was used as a solvent for the extraction of bacterial bioactive compounds. antibacterial activity was analyzed by the diffusion method with several extract concentrations (0.75 ml, 1.5 ml, 2.25 ml, and 3 ml), 10% dmso (dimethyl sulfoxide) as the negative control, and ciprofloxacin as the positive control. our result shows that termoactinomyces sp. (h24) extract has an inhibitory effect on the growth of e. coli and s. aureus with an effective concentration of 2.25 ml (inhibition strength: very strong). keywords antibacterial, escherichia coli, staphylococcus aureus, thermoactinomyces sp. (h24). this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. introduction eschericia coli and staphylococcus aureus are two common causative agents of serious infections in humans (1,2). e. coli can infect the respiratory and digestive tracts and cause several diseases like pneumonia, endocarditis, wound infection, meningitis (3), and diarrhea by producing enterotoxins (4). s. aureus can cause several diseases such as pneumonia, osteomyelitis, arthritis, and inflammation of the brain. s. aureus cause hemolysis of blood, and have the ability to rahmawati@fmipa.untan.ac.id https://journal2.unusa.ac.id/index.php/ijmlst/article/view/1700/version/2183 ina. j. med. lab. sci. tech. 2021; 3(1): 56–63 julia nanda puspita, et al. 5 7 produce enterotoxins that led to diarrhea, seizures, and fever (5). the emergence of various kinds of diseases caused by pathogenic bacteria encourages the researcher to find alternative antibacterial compounds. thermoactinomyces sp. (h24) is one of the bacteria with the ability to produce the antibacterial compound (6) . jawetz et al. (7) find that thermoactinomyces sp. (h24) have antifungal activity. thermoactinomyces sp., in addition to antifungal compound, also produce antibacterial aromatic compound namely cyclic hexapeptides thermoactinoamide a-f with ability to inhibit lipophilic bacteria (8). currently, there is no reported research on the antibacterial analysis of thermoactinomyces sp. (h24) for inhibiting the growth of several pathogenic bacteria such as e.coli and s. aureus. therefore, this research was conducted to analyze the antibacterial activity of thermoactinomyces sp. (h24) for inhibiting the growth of s. aureus and e. coli. materials and methods preparation and sterilization the media used were nutrient agar (na), mueller hinton agar (mha), and nutrient broth (nb) dissolved in 1,000 ml of distilled water. all media, petri dishes, and test tubes were sterilized using an autoclave at 121°c, 2 atm for 15 minutes (9). extraction of termoactinomyces sp. (h24) metabolites thermoactinomyces sp. (h24) was cultivated by the streak plate method on nutrient agar, then incubated at 50°c for 24 – 72 hours. a single colony of thermoactinomyces sp. (h24) was inoculated on 50 ml of nutrient broth, then incubated in an incubator shaker at 110 rpm, 50°c for 84 hours until the culture reached a stationary phase. thermoactinomyces sp. (h24) metabolite was extracted by centrifuging the culture at 3,000 rpm for 15 minutes to obtain supernatant. the supernatant obtained was a source of secondary metabolites (9). the supernatant was filtered using whatman paper no. 1. the filtrate was immersed in 100% methanol for 24 hours in different bottles with a ratio of v/v (1:1). the solvent was removed using a rotary evaporator and then allow to stand for 24 hours (9). qualitative test of thermoactinomyces sp. (h24) extract a qualitative test was done to analyze the presence of phenols, alkaloids, flavonoids, saponins, tannins, steroids, and terpenoids in thermoactinomyces sp. (h24) extract. preparation of escherichia coli and staphylococcus aureus culture the colony of e. coli and s. aureus was transferred into 9 ml of sterile distilled water until the optical density (od) value similar to 0.5 mc farland standard. mcfarland ina. j. med. lab. sci. tech. 2021; 3(1): 56–63 julia nanda puspita, et al. 5 8 standard was prepared by mixing h2so4 with 0.05 ml of bacl 1% in a test tube (10). antibacterial activity test of thermoactinomyces sp. (h24) extract various concentration of thermoactinomyces sp. (h24) extract (0.75 ml, 1.5 ml, 2.25 ml, and 3 ml) was transferred into 3 ml sterile distilled water. antibacterial activity test was performed by well diffusion method. e. coli and s. aureus culture were swabbed evenly on the all surface of the mha medium using a sterile cotton swab. the well was made on a medium with a diameter of 6 mm, then filled with 25 µl of each extract with several concentrations, dmso 10% as a negative control, and ciprofloxacin 0.25 µg/ml as a positive control. the medium was incubated at 37° c for 24 – 48 hours (11). a clear zone formed after incubation was measured using a caliper. the clear zone categorized into very strong (diameters >20 mm); strong (diameter 11 – 20 mm); moderate (diameter 6 – 10 mm); and weak (<5 mm) (12). results qualitative test of thermoactinomyces sp. (h24) extract the qualitative test was revealed the presence or absence of secondary metabolites from thermoactinomyces sp. (h24) extract (table 1). table 1. qualitative test results of thermoactinomyces sp. (h24) extract no. secondary metabolites reactor change result 1. alkaloids wagner brown deposits are formed + 2. flavonoids magnesium (mg)andamylalkohol no yellow discoloration occurs 3. saponins hcl no foam 4. tannins fecl3 no dark blue discoloration 5. phenol etanoland fecl3 no change into green color 6. steroidsandterpenoids ch3cooh glacial, h2so4 no change into red or green note: + : contains secondary metabolites; – : not contains secondary metabolites ina. j. med. lab. sci. tech. 2021; 3(1): 56–63 julia nanda puspita, et al. 5 9 a qualitative test of the thermoactinomyces sp. (h24) extract showed the presence of the alkaloid compounds in the extract, indicated by the formation of brown deposits when wagner's reagent was added to the extract. antibacterial activity of thermoactinomyces sp. (h24) the formation of the clear zone indicated the ability of thermoactinomyces sp. (h24) extract to inhibit the growth of e. coli and s. aureus. the diameter of the clear zone showed that the extract had various bacterial growth inhibition level against e. coli from moderate to very strong depending on extract concentration (table 2). statistics analysis showed that the different concentrations give significantly different results. table 2. average diameter of clear zone against escherichia coli concentration of extract (ml) diameter (mm) category 24 hrs 48 hrs dmso 10% 0.00 0.00 0.75 9.44 9.62 moderate 1.5 16.18 16.71 strong 2.25 21.78 28.49 very strong 3 26.85 32.94 very strong ciprofloaxcin 37.57 41.20 very strong figure 1 showed the clear zone formed after incubation 24 hours and 48 hours, indicated the ability of thermoactinomyces sp. (h24) extract against e. coli. figure 2 showed the clear zone formed after incubation 24 hours and 48 hours, indicated the ability of thermoactinomyces sp. (h24) extract against s. aureus. figure 1. the clear zone of thermoactinomyces sp. (h24) extract against escherichia coli: (a) 24 hours, (b) 48 hours of incubation a b ina. j. med. lab. sci. tech. 2021; 3(1): 56–63 julia nanda puspita, et al. 6 0 table 3. average diameter of clear zone against staphylococcus aureus concentrationof extract (ml) diameter (mm) category 24 hrs 48 hrs dmso 10% 0.00 0.00 0.75 7.41 7.43 moderate 1.5 13.52 13.71 strong 2.25 22.31 22.68 very strong 3 26.12 36.48 very strong ciprofloaxcin 36.16 36.48 very strong figure 2. the clear zone of thermoactinomyces sp. (h24) extract against staphylococcus aureus: (a) 24 hours, (b) 48 hours of incubation discussion thermoactinomyces sp. (h24) extract has the ability to inhibit the growth of e. coli and s. aureus, indicated by the formation of a clear zone around the well. the effective concentration of thermoactinomyces sp. (h24) extract against e. coli and s. aureus was 0.75 ml, 1.5 ml, 2.25 ml, and 3 ml with inhibition strength from moderate to very strong. thermoactinomyces sp. (h24) extract concentration of 0.75 ml could inhibit e. coli and s. aureus at a moderate level (table 2 and table 3). teta et al. (8) was reported that thermoactinomyces vulgaris isolated from a hydrothermal vent on the coast of iceland possessed moderate level antibacterial activity with a concentration of at least 0.35 ml against s. aureus and 0.14 ml against escherichia coli. bratchkova et al. (13) showed that thermoactinomyces sp. isolated from penguin droppings on livingston island, antarctica had a strong level of antibacterial activity at the volume of 0.10 ml against s. aureus and e. coli. this high antibacterial activity at a low concentration from thermoactinomyces sp. extract reported by teta et al. (8) and bratchkova et al. (13) because of the different extraction solvents a b ina. j. med. lab. sci. tech. 2021; 3(1): 56–63 julia nanda puspita, et al. 6 1 and the use of chromatography result in the more pure extract. the concentration of extract positively correlated with the diameter of the clear zone. the clear zone formed is produced from the antibacterial active substance in the extract. the most effective concentration of extract for inhibiting the growth of e. coli and s. aureus is 2.25 ml. e. coli cell walls have three layers (multilayer) with high-fat content (11 – 22%) and peptidoglycan layer inside stiff layer (10% dry weight) that cause e. coli less absorb antibacterial compound (14). the effective concentration of antibacterial extract produces a biological response. the effective concentration is defined as the lowest concentration that provides a significant response (15). the diameter of the inhibition zone at the 48-hours incubation time was greater than 24-hours incubation, indicated that the antibacterial activity is bactericidal or has the ability to kill bacteria (16). the average diameter of the inhibition zone resulted from the thermoactinomyces sp. (h24) extract against e. coli was greater than s. aureus. this difference indicates that gram-negative and gram-positive bacteria have different responses to antibacterial compounds. e. coli, a gram-negative bacteria, is more sensitive to non-polar (hydrophobic) antibacterial compounds such as alkaloid compounds than gram-positive bacteria such as s. aureus (20). the difference in response between grampositive and gram-negative bacteria to alkaloid compounds is due to the cell walls of gram-negative bacteria contain 11–22% lipids which will more easily react with nonpolar alkaloid compounds, resulting in cell lysis. in contrast, gram-positive bacteria, such as s. aureus, contains 4% lipids which make it difficult to react with alkaloid compounds. bratchkova et al. (13) reported that e. coli (gram-negative) was more sensitive than s. aureus (gram-positive) bacteria in response to thermoactinomyces sp. impact-14 extract. the antibacterial activity of thermoactinomyces sp. (h24) extract against e. coli and s. aureus is due to the content of metabolite compounds produced by thermoactinomyces sp. (h24). the qualitative test showed the presence of alkaloid compounds in thermoactinomyces sp. (h24) extract (table 1). several studies also show that many thermoactinomyces strains extract contained alkaloid compounds as antibacterial agents. bratchkova et al. (13) explained that thermoactinomyces sp. impact-14 isolated from penguin droppings on livingston island, antarctica contains βcarboline alkaloid compounds which have antibacterial activity against escherichia coli, staphylococcus aureus, bacillus subtilis, bacillus mycoides, streptomyces viridoch, proteus vulgaris, candida ina. j. med. lab. sci. tech. 2021; 3(1): 56–63 julia nanda puspita, et al. 6 2 tropicalis, candida albicans, mucor miehei, and penicillium notatum. alkaloid compounds found in the thermoactinomyces sp. (h24) extract play a role in inhibiting the growth of e. coli and s. aureus. the nitrogen group in the alkaloid will react with the amino acids that cause the change in the structure of the amino acid in the bacterial cell wall. changes in the structure of amino acids trigger lysis in bacterial cells led to bacterial death (17). conclusions thermoactinomyces sp. (h24) extract contained an alkaloid compound and has antibacterial activity against e. coli and s. aureus. strong inhibition activity against e. coli and s. aureus was achieved by the concentration of 50% thermoactinomyces sp. (h24) extract. author contributions julia nanda puspita: conceptualization, methodology, formal analysis, investigation, data curation. rahmawati: validation, writing-review & editing, project administration, funding acquisition. rikhsan kurniatuhadi: validation, writing-original draft, project administration. author contributions julia nanda puspita: conceptualization, methodology, formal analysis, investigation, data curation. rahmawati: validation, writing-review & editing, project administration, funding acquisition. rikhsan kurniatuhadi: validation, writing-original draft, project administration. acknowladgements the autors are grateful to jerliman et al. which has permitted to using thermoactinomyces sp. (h24) and microbiology labotarory, faculty of mathematics and natural science, tanjungpura university for the facilities and assistance in completing the research work. conflict of interest there are no conflicts of interest. references 1. arenz s, wilson dn. blast from the past: reassessing forgotten translation inhibitors, antibiotic selectivity, and resistance mechanisms to aid drug development. mol cell. 2016;61(1):112. 2. amalia s, wahdaningsih s, untari ek. activity testdragon fruit skin n-hexane fraction antibacterial red (hylocereus polyrhizus britton & rose) against staphylococcusaureus atcc 25923. pontianak: faculty tanjungpura university medicine. j phy ina. 2016; 61-64. 3. entjang i. microbiology and parasitology for the nursing academy and health power school of the equals. pt. citra aditya bakti; 2018: 56-58 4. brooks gf, karen cc, butel js, stephen am. jawetz, melnick & adelberg’s, book 1 medical microbiology. jakarta: egc; 2015. 5. plata fx, ebergeny x, resendiz jl, villarreal o, barcena r, viccon ja, mendoza gd. palatability and chemical composition of feeds ingested in captivity by yucatan white-tailed deer (odocoileus virginianus yucatanensis). arc med vet. 2019; 123-129. ina. j. med. lab. sci. tech. 2021; 3(1): 56–63 julia nanda puspita, et al. 6 3 6. manalu j, rahmawati, hidayat n. antifungal activity of actinomycetes isolates from hot springs ai sipatn lotup sanggau against isolate hortaea werneckii (t1). protobiont. 2019; 69-77. 7. jawetz e, melnick jl, adelberg ea. medical microbiology. translated by mudihardi e, kuntaman, wasito eb, mertaniasih nm, harsono s, alimsardjono l, edition xxii, 327-335, 362363, publisher salemba medika, jakarta; 2015. 8. teta r, marteinsson vt, longeon a, klonowski am, groben r, bourguet km, costantinov, mangonia. thermoactinoamide a, an antibiotic lipophilic cyclopeptide from the icelandic thermophilic bacterium thermoactinomyces vulgaris. j nat prod. 2017; 2530-2535. 9. mulyadi, sulistyani n. activity of 12 actinomycetes isolates against bacteria resistance: public health. 2013;7(2): 55-112. 10. zheng l, chen h, han x, yan x. antimicrobial screening and active compound isolation from marine bacterium nj6-3-1 associated with the sponge hymeniacidon parleve. world j microb biotech. 2015; 201-206. 11. moningka ks, kojong ns, sudewi s. antibacterial activity test of leaf extract of cats (acalypha hispida burm. f.) against staphylococcus aureus and escherichia coli in vitro. j pharm sci. 2015; 2302-2493. 12. warbung yy, wowor vns, posangi j. inhibition of sea sponge extract callyspongia sp. against the growth of staphylococcus aureus bacteria. egigi journal (eg). 2013; 1 (2): 2. 13. bratchkova a, ivanova v, gousterova a, laatsch l. β-carboline alkaloid constituents from a thermoactinomyces sp. strain isolated from livingston island. biotechnology & biotechnological equipment: antarctica. 2012: 3005-3009. 14. todar, k. pathogenic e. coli, todar’s online textbook and bacteriology, http://textbookofbacteriology.net/e.coli.html., 2018. 15. liu j. minimum effective dose. encyclopedia of biopharmaceutical statistics. taylor & francis. p. 1493. doi:10.1081/e-ebs3-130001128. 2019. isbn 978-1-4398-2246-3. 16. limaye l, patil r, ranadive p, kamath g. application of potent actinomycete strains for bio-degradation of domestic agro-waste by composting and treatment of pulp-paper mill effluent. adv micro. 2017; 94-108. 17. gunawan i. preliminary extraction of bioactive compounds as antibacterials as well as toxicity test and minimum inhibitor concentration (mic) test of soft rocks of origin perairan pulau pulau, kepulauan seribu. description: faculty of fisheries and marine science, bogor. 2019. 18. bagg j, mcfarlane, wallace t, poxton, ian r, smith, andrew j. essentials of microbiology for dental students, 2nd edition. oxford university, 2017. 19. chaudhary hs, yadav j, shrivastava ar, singh s, singh ka, gopalan n. antibacterial activity of actinomycetes isolated from different soil samples of sheopur. j adv pharm technol. 2017; 118–123. 20. levinson w. review of medical microbiology, the mcgraw-hill companies, 25-26, 78-79, 2018. 21. sharma sk, gupta vk. in vitro antioxidant studies of ficus racemosa linn root, pharmacognosy magazine, india. 2008. 73 modified spin column-based rna extraction methods of staphylococcus aureus using purelink® rna mini kit and basic laboratory instrument muhammad taufiq hidayat1, endry nugroho prasetyo1 1department of biology, faculty of science and data analytics, institut teknologi sepuluh nopember, east java, indonesia correspondence: muhammad taufiq hidayat, laboratory of microbiology and biotechnology, department of biology, institut teknologi sepuluh nopember, sukolilo, surabaya zip code: 60111 email: muhammadtaufiqhidayat7@gmail.com received: december 22, 2020 revised: may 31, 2021 accepted: juni 1, 2021 published: october 30, 2021 doi: 10.33086/ijmlst.v3i2.1863 abstract rna extraction is an important process before gene expression assessment at the transcriptomic level. rna is a sensitive material to environmental factors such as temperature and contaminants, so the rna extraction process generally requires sophisticated and expensive laboratory instruments. in this study, we extract rna from staphylococcus aureus bacteria using the purelink® rna mini kit. the instruments used in this study are basic instruments such as a hand homogenizer and non-thermal centrifuge. the results of rna extraction were visualized using agarose gel electrophoresis. these results indicate that bacterial rna extraction can be performed using the purelink® rna mini kit even with inexpensive basic laboratory instruments. keywords basic laboratory instrument, purelink® rna mini kit, rna extraction, staphylococcus aureus. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2021; 3(2): 73–80 muhammad taufiq hidayat & endry nugroho prasetyo 7 4 introduction total rna extraction from bacterial cells is used to analyze gene expression at the transcriptional level. high-quality total rna is a prerequisite for a variety of downstream applications (1). rna quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other rna-based downstream applications (2,3). the purity and integrity of rna can impact the accuracy of techniques such as real-time quantitative polymerase chain reaction (4). spin column-based is one of the rna extraction methods that comprise of four stages namely lysis of cells, binding of nucleic acid to silica gel membrane, washing the nucleic acid bound to the silica gel membrane, and elution of the nucleic acid (5). first, the cell membrane was broken by a lysis solution in order to free nucleic acid from cell. the binding solution that contains buffer solution and ethanol is added to a spin column, and the column is put in a centrifuge. samples are lysed and homogenized in the presence of guanidinium isothiocyanate, a chaotropic salt capable of protecting the rna from endogenous rnases (6). the centrifuge forces the binding solution through a silica gel membrane inside the spin column. the nucleic acid will bind to the silica gel membrane as the solution passes through if the ph and salt concentration of the binding solution is optimal. the column is put in a centrifuge again, forcing the wash buffer through the membrane to removes any remaining contaminant from the membrane, leaving only the nucleic acid bound to the silica gel. lastly, the column is put in a centrifuge again, forcing the elution buffer through the membrane. the elution buffer removes the nucleic acid from the membrane and the nucleic acid is collected from the bottom of the column. staphylococcus aureus is an important nosocomial gram-positive pathogen that causes various infectious diseases in humans, ranging from harmless localized skin lesions to systemic infections, such as endocarditis, pneumonia, and other life-threatening diseases (7). gram-positive bacteria have a thick peptidoglycan layer and no outer lipid membrane (8). rna extraction is an important step to assess genetic expression, for example, to determine the effect of the antibiotic compound on the genetic expression of s. aureus. generally, rna extraction uses commercial kits that permit for fast purification of high-quality nucleic acids. the success of commercial kits largely relies on the spin columns assembled with solid-phase nucleic acid binding material which allows easy binding, washing, and elution of nucleic acids in the purification process (9). the extraction method should be subject to various considerations such as budget, organism, and in particular, the aim of the ina. j. med. lab. sci. tech. 2021; 3(2): 73–80 muhammad taufiq hidayat & endry nugroho prasetyo 7 5 experiment (10). rna extraction process generally requires sophisticated and expensive laboratory instruments. with limited laboratory instruments, we evaluate rna extraction using purelink® rna mini kit and basic instruments such as a hand homogenizer and non-thermal centrifuge. materials and methods materials used in this study are purelink® rna mini kit (thermo fisher scientific, united states) (comprises of lysis buffer, wash buffer i, wash buffer ii, rnase-free water, spin cartridges, collection tubes), 2-mercaptoethanol, s. aureus culture, and luria bertani broth. the instrument used are hand homogenizer (mini hand homogenizer mt-13k) and nonthermal centrifuge (br technologies). s. aureus cultivation and cell harvesting bacterial cultivation and cell harvesting was done based on (11). a oneloop single colony of s. aureus from mannitol salt agar and luria bertani agar was transferred to luria bertani broth. cultured was incubated for 24 hours at 37oc. culture in luria bertani broth was harvested by centrifuging at 4,000 rpm for 5 minutes and removes supernatant. rna extraction rna extraction was done based on the manufacturer’s guide protocol (purelink® rna mini kit) (5) with modification. lysis and homogenization cell pellet was transferred to rnase-free tube. 0.6 ml lysis buffer prepared with 2mercaptoethanol was added to sample using rnase-free pipette tips. sample was vortexed at high speed until the cell pellet is completely dispersed and the cells appear lysed. sample was homogenized with hand homogenizer (mini hand homogenizer mt13k) for 2 minutes. binding, washing, and elution one volume 70% ethanol was added to each volume of cell homogenate. sample was vortexed to mix thoroughly and to disperse any visible precipitate that may form after adding ethanol. sample up to 700 µl (including any remaining precipitate) was transferred to the spin cartridge (with the collection tube). sample was centrifuged at 8,000 rpm for 15 seconds at room temperature. the flow-through was discarded and the spin cartridge was reinserted into the same collection tube. steps (transfer to spin cartridge and centrifuge) was repeated until the entire sample is processed. seven hundred µl wash buffer i was added to the spin cartridge. sample was centrifuged at 8,000 rpm for 15 seconds at room temperature. the flow-through and the collection tube was discarded. the spin cartridge was placed into a new collection tube. five hundred μl wash buffer iiwith ethanol was added to the spin cartridge. sample was centrifuged again at ina. j. med. lab. sci. tech. 2021; 3(2): 73–80 muhammad taufiq hidayat & endry nugroho prasetyo 7 6 8,000 rpm for 15 seconds at room temperature. the flow-through was discarded and the spin cartridge was reinserted into the same collection tube. steps wash buffer ii was repeated once. the spin cartridge was centrifuged at 8,000 rpm for 1-2 minutes to dry the membrane with attached the rna. the collection tube was discarded and the spin cartridge was inserted into a recovery tube. 50 μl rnase–free water was added to the center of the spin cartridge. sample was incubated at room temperature for 1 minute. the spin cartridge was centrifuged for 2 minutes at 8,000 rpm at room temperature to elute the rna from the membrane into the recovery tube. agarose gel electrophoresis the integrity of total rna isolated and the extent of genomic dna contamination were analyzed through agarose gel electrophoresis (12). the integrity and size of rna can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining. unlike dna, rna molecules quickly form secondary and tertiary structures (13). 5 μl sample was mixed with 5 μl nuclease-free water and 2 μl loading dye, then transferred to 1.2% agarose gel well. electrophoresis was run at 100v for 1 hour. agarose gel was dipped in ethidium bromide for 30 minutes and then visualized on high performance ultraviolet transilluminator (thermo fisher scientific, united states). results the visualization of agarose gel under ultraviolet illumination showed rna indicated by the formation of the single bands pointed by arrow (figure 1). figure 1. visualization of agarose gel under uv after 3 days stored at -40oc. visualization is done in duplicate (shown in wells 1 and 2). the bands formed are shown in a and b. this result indicated that purelink® rna mini kit with basic laboratory instruments such as a hand homogenizer and non-thermal centrifuge can be used to extract total rna from s. aureus culture. the result of rna isolation was stored at -40oc for 3 days then was visualized again under uv transilluminator to determine whether the extracted rna still survived or degraded after the storage period. the results of the second electrophoresis are shown in figure 2. ina. j. med. lab. sci. tech. 2021; 3(2): 73–80 muhammad taufiq hidayat & endry nugroho prasetyo 7 7 figure 2. visualization of agarose gel under uv after 3 days stored at -40oc. visualization is done in duplicate (shown in wells 1 and 2). the bands formed are shown in a and b. discussion the success of rna isolation is influenced by several factors, including the choice of method and the availability of instruments. rodriguez et al. (10) stated that the extraction method should be subject to various considerations such as budget, organism, and the aim of the experiment. purelink® rna mini kit is designed for fast purification of high-quality nucleic acids based on the spin column-based method. the purelink™ rna mini kit provides a simple method for isolating high-quality rna. bacterial cells are lysed and homogenized in the presence of guanidine isothiocyanate, and ethanol is added to the sample (1). guanidine isothiocyanate is a chaotropic salt capable of protecting the rna from endogenous rnases (6). the sample is then processed through a spin cartridge containing a clear silica-based membrane to which rna binds. the binding mechanism of nucleic acid adsorption on the silica surface mainly includes four factors: hydrogen bond, salt bridge and electrostatic force formed between the nucleic acid and the silica surface, and the solubility of nucleic acid (14). any impurities are effectively removed by subsequent washing. the purified rna is then eluted in rnase-free water (1). yang et al. (15) also reported that the rna isolated using e silica spin columnbased was relatively less contaminated by protein, polysaccharide, and phenolic compounds or other reagents. the application of high concentration guscn (w/v 50%) in subsequent binding and washing buffers also enhanced removing denatured proteins and other inhibitors. rna extraction process generally requires sophisticated and expensive laboratory instruments. with limited laboratory instruments, we evaluate rna extraction using purelink® rna mini kit and basic instruments such as a hand homogenizer and non-thermal centrifuge. in the manufacturer's protocol (5), it is stated that homogenization can be done with 3 alternative tools, namely the homogenizer, syringe and needle, and rotor-stator. the homogenizer provides highly consistent results and is more convenient than other homogenization methods (5). xu et al. (16) ina. j. med. lab. sci. tech. 2021; 3(2): 73–80 muhammad taufiq hidayat & endry nugroho prasetyo 7 8 stated that the rna extraction results increased from 35.4–37.6 (ct values) without homogenization to 37.8–40.2 (ct values) with homogenization. the lysis step is combined with a homogenization step that includes denaturation with a guanidinethiocyanate containing buffer to inactivate the rnases released from the cell (17). wever et al. (18) indicate that the important factor that determines the results of rna extraction is the buffer volume added during homogenization according to the recommendation of the homogenization protocols. the results of this study prove that hand homogenizer can be used for the sample homogenization process in the cell lysis process. another factor that affects the rna extraction results is temperature. a centrifuge is a vital tool in the rna extraction process. the centrifuge forces the binding solution through a silica gel membrane inside the spin column, forces wash buffer through the membrane to removes any remaining contaminant from the membrane, and forces elution buffer through the membrane (5). in this study, we used a non-thermal centrifuge for the rna extraction process. rna extraction in the field reported by breitler et al. (19) can be carried out at high temperatures (25–38°c) using a trizol reagent. abdallah et al. (20) also reported that the stability of mers-cov rna on spin columns of rna extraction kit at room temperature. the results of this study indicate that the use of a non-thermal centrifuge can be an effective alternative for rna extraction if a thermal centrifuge is not available in the laboratory. novelty this study reports a new rna isolation strategy using simple laboratory instruments. according to the purelink® rna mini kit guide, homogenization can use one of 3 alternatives, namely 1. homogenizer; 2. syringe and needle; 3. rotor-stator. another important process in rna isolation is centrifugation. in the manufacturer’s guide, it is recommended to use a temperaturecontrolled centrifuge because rna is sensitive to high temperatures. however, in some laboratories, these sophisticated instruments are not available, so in this study, alternative instruments that are widely available in public laboratories are used, namely non-thermal centrifuge and hand homogenizer. the results of this study indicate that this simple instrument can be used to isolate s. aureus rna. even after being stored for 3 days at -40oc, the results of electrophoresis still showed that the isolated rna was not degraded. these results are expected to be an alternative for laboratories with limited instruments in order to practice rna isolation. ina. j. med. lab. sci. tech. 2021; 3(2): 73–80 muhammad taufiq hidayat & endry nugroho prasetyo 7 9 conclusions based on the results of visualization of agarose gel on the uv transilluminator, we conclude that rna isolation with the purelink® rna mini kit can be carried out in a laboratory with basic instruments such as a hand homogenizer and centrifuge without temperature control. author contributions muhammad taufiq hidayat: conceptualization, methodology, formal analysis, investigation, writing original draft, writing review & editing, visualization. endry nugroho prasetyo: conceptualization, resources, supervision, project administration, funding acquisition. acknowladgements this study was supported by the ministry of research, and technology / national research and innovation agency (ristekbrin), republic of indonesia, and institut teknologi sepuluh nopember (its), surabaya, indonesia under a grant number of 3/amd/e1/kp.ptnbh/2020 and 1177/pks/its/2019. conflict of interest there are no conflicts of interest. references 1. wang h, han y. chapter 4 extraction of total rna from yersinia pestis. yersinia pestis protoc., singapore: springer; 2018, p. 29–34. 2. rodrıguez a, duyvejonck h, belleghem jd van, gryp t, simaey l van, vermeulen s, et al. comparison of procedures for rna-extraction from peripheral blood mononuclear cells. plosone 2020;15:1–17. https://doi.org/10.1371/journal.pone.0229423. 3. ahmad j, baig ma, ali aa, al-huqail a, ibrahim mm, qureshi mi. comparative assessment of four rna extraction methods and modification to obtain high-quality rna from parthenium hysterophorus leaf. 3 biotech 2017;7:373. https://doi.org/10.1007/s13205-0171003-3. 4. bezerra d da s, neves bg, guedes sf de f, regis wfm, stipp rn, rodrigues lka. extraction and purification of rna from human carious dentine : an approach to enable bacterial gene expression studies. j heal biol sci 2019;7:145–51. https://doi.org/10.12662/23173076jhbs.v7i2.2562.p145-151.2019. 5. ambion. purelink ® rna mini kit. carlsbad: life technologies corporation; 2012. 6. zavala-alvarado, crispin, benaroudj n. the single-step method of rna purification applied to leptospira. leptospira spp, new york: humana; 2020, p. 41–51. 7. kitichalermkiat a, katsuki m, sato j, sonoda t, masuda y, honjoh k ichi, et al. effect of epigallocatechin gallate on gene expression of staphylococcus aureus. j glob antimicrob resist 2020;22:854–9. https://doi.org/10.1016/j.jgar.2020.06.006. 8. liu y, breukink e. the membrane steps of bacterial cell wall synthesis as antibiotic targets. antibiotics 2016;5:1–22. https://doi.org/10.3390/antibiotics5030028. 9. shi r, lewis rs, panthee dr. filter paper-based spin column method for cost-efficient dna or rna purification. plosone 2018;13:1–14. 10. rodríguez ml, rosa ac, jewtuchowicz v. rna extraction from the yeast candida parapsilosis sensu stricto using two commercially available silica column-based purification methods. j microbiol mod tech 2018;3:201. 11. kher mn, sheth nr, bhatt vd. in vitro antibacterial evaluation of terminalia chebula as an alternative of antibiotics against bovine subclinical mastitis. anim biotechnol 2019;30:151–8. https://doi.org/10.1080/10495398.2018.1451752. ina. j. med. lab. sci. tech. 2021; 3(2): 73–80 muhammad taufiq hidayat & endry nugroho prasetyo 8 0 12. nadiya f, anjali n, gangaprasad a, krishnannair k. high-quality rna extraction from small cardamom tissues rich in polysaccharides and polyphenols. anal biochem 2015;485:25–7. https://doi.org/10.1016/j.ab.2015.05.017. 13. bhat ai, rao gp. agarose gel electrophoresis for nucleic acids. charact. plant viruses. springer protoc. handbooks, new york: humana; 2020. 14. hu wp. improve sample preparation process for mirna isolation from the culture cells by using silica fiber membrane. sci rep 2020;10:1–9. https://doi.org/10.1038/s41598-020-78202-8. 15. yang f, wang g, xu w, hong n. a rapid silica spin column-based method of rna extraction from fruit trees for rt-pcr detection of viruses. j virol methods 2017;247:61–7. https://doi.org/10.1016/j.jviromet.2017.05.020. 16. xu r, shieh yc, stewart ds. comparison of rna extraction kits for the purification and detection of an enteric virus surrogate on green onions via rt-pcr. j virol methods 2017;239:61–8. https://doi.org/10.1016/j.jviromet.2016.10.016. 17. hay m, li ym, ma y. rna extraction of escherichia coli grown in lysogeny broth for use in rt-qpcr. jemi methods 2017;1:1–6. 18. wever j de, tulkens d, verwaeren j, everaert h, rottiers h, dewettinck k, et al. a combined rna preservation and extraction protocol for gene expression studies in cacao beans. front plant sci 2020;11:992. https://doi.org/10.3389/fpls.2020.00992. 19. breitler j, campa c, georget f, bertrand b, etienne h. a single-step method for rna isolation from tropical crops in the field. nat publ gr 2016;6:1–6. https://doi.org/10.1038/srep38368. 20. abdallah nma, zaki am, abdel-salam sa. stability of mers-cov rna on spin columns of rna extraction kit at room temperature. diagnostic microbiol infect dis 2020;98:115182. https://doi.org/10.1016/j.diagmicrobio.2020.1151 82. 44 comparison of glucose reduction in urine using benedict method heated by methylated flame with 100°c waterbath fitri fadhilah1, noviana vanawati1 1departement of medical laboratory technology, school of analyst bakti asih, indonesia correspondence: fitri fadhilah, jl. padasuka atas no.233, padasuka, cimenyan, city of bandung, west java, indonesia zip code : 40192 email: fitrifadhilahssimkes@gmail.com received: april 24, 2019 revised: may 5, 2019 accepted: august 19, 2019 abstract the high prevalence of diabetes melitus (dm) is a global problem that must be solved by health workers around the world. this study aims to determine the differences in the results of urine reduction examination using benedict method heated by spirtus flame and waterbath 100°c. this research method is a laboratory experiment. the results of this study indicated that the urine reduction examination by heating methylated flame and waterbath 100oc shows the same results from negative (-) until positive (+4). examination of urine reduction by heating the methylated flame and waterbath 100oc did not affect the results. however, there are a difference in the process of urine reduction examination by heating flame which was need a longer time up to 3-5 minutes. additionally, the solution in the tube could be exploded. it was also time consuming which could only carry out one by one sample. meanwhile, the heating of urine by using 100oc waterbath is relatively faster, which only took 2 minutes. the urine was not also exploded when it was boiled and the heating process could perform 6-8 samples at the same time (depending on the tube rack). in conclusion, the heating method of urine by using waterbath was better than spirtus (methanol) flame since it could carry out large sample in one time and it was safer for the laboratory personnel. keywords urine reduction, glucose, benedict, methylated flame, waterbath introduction diabetes mellitus is a group of metabolic disorders of carbohy-drate metabolism in which glucose is underutilized, producing hyperglycemia. some individuals may experience acute life-threatening hyperglycemic episodes, such as ketoacidosis or hyperosmolar coma. as the disease progresses, individuals are at increased risk for the development of specific complications, including retinopathy (which may lead to blindness), renal failure, neuropathy (nerve damage), and atherosclerosis. the last condition may result ina j med lab sci tech 2019; 1(2): 44-51 fitri fadhilah, et al. 4 5 in stroke, gangrene, or coronary artery disease (1–3). diabetes was initially diagnosed by the oral glucose tolerance test (ogtt). in 1979 a work group of the national diabetes data group proposed modified criteria for diagnosis. this classification scheme recognized two major forms of type i diabetes (insulin-dependent) diabetes mellitus (iddm) and type i1 (non-insulindependent) diabetes mellitus (niddm). to base the classification on etiology rather than treatment, in 1995 the american diabetes association (ada) established a work group to reexamine the classification and diagnosis of diabetes mellitus. the revised classification, published in 1997, eliminates the terms insulin dependent diabetes mellitus and non insulin dependent diabetes mellitus, which now are termed type 1 diabetes and type 2 diabetes, respectively. another significant change is the elimination of the categories of previous abnormality of glucose tolerance and potential abnormality of glucose tolerance (1, 2, 4). various sugars may be found in the urine under certain circumstances, both pathologic and physiologic condition. these include glucose, fructose, galactose, lactose, maltose, pentose and sucrose (5). the presence of detectable amounts of glucose in urine is termed glycosuria; this condition occurs whenever the glucose level in the blood surpasses the renal tubule capacity for reabsorption. glucose may appear in the urine at different blood glucose levels. however, it is not usually indicated as hyperglycemia. glomerular blood flow, tubular reabsorption rate, and urine flow will also influence its appearance. when hyperglycemia is present, however, glycosuria usually occurs when the blood level is greater than 180–200 mg/dl (1,6). although hyperglycemia per se is not necessarily indicative of diabetes mellitus, the appearance of glucose in the urine necessitates further workup. when glycosuria is present, it is typically accompanied by polyuria and thirst. inadequate carbohydrate utilization in these patients results in elevated ketone levels in the blood and urine due to increased in fat metabolisme (1). the advantage of a urine method over a blood test for glucose is that it is painless and inexpensive for diabetic individual. urine glucose measurements are most useful for well-controlled diabetic individuals who do not have to make frequent adjustments in their insulin/hypoglycemic agents. in insulindependent diabetes, a negative urine measurement could correspond to a wide range of serum glucose levels; this is attributed to the great variation in renal threshold for glucose in diabetic patients. therefore, urine measurements may be misleading, and home blood glucose monitoring is more preferable (7). ina j med lab sci tech 2019; 1(2): 44-51 fitri fadhilah, et al. 4 6 copper reduction tests (bennedict test) as a screening test, the glucose oxidase method will not detect increased levels of galactose or other sugars in urine. it is therefore important that a copper reduction method should be used, especially for young pediatric patients. of the copper reduction methods used for screening purposes, the qualitative benedict method is more sensitive in reducing substances in urine than is the single-tablet (clinitest) copper reduction method (5,8). urinary glucose examination using benedict method utilizes the glucose as a reducing agent. the principle of examining benedict is that glucose in the urine will reduce cuprisulfate to cuprosulfat, which can be seen by changing the color of the benedict solution. positive results are indicated by turbidity and color changes from blue to yellowish green to brick red. examination of the benedict method can be done by using heating with methylated flame to boil. the weakness of this method is time consuming which can only perform one sampel per process. there is also a risk of accidents in the laboratory (1,5). waterbath laboratory equipment can also be used for the diagnosis of disease. for the diagnosis of samples from a patient, temperature stability is needed, so that the diagnostic results are truly appropriate. the principle of waterbath is that when cold sterilizes the plug is turned on, the temperature is desired and set. arrangements must be made according to the readings of the thermostat or according to a temperature monitoring system. the advantages of waterbath are faster to get the results, 8-10 samples can be done at a once time, and the risk of accidents in the laboratory is relatively poor (9,10). materials and methods benedict qualitative reagent contains cupric ion complexed to citrate in alkaline snlution. reducing substances convert cupric to cuprous ions, forming yellow cuprous hydroxide or red cuprous oxide. the, tablets contain anhydrous cupric sulfate, naoh, citric acid, and sodium bicarbonate (nahco). five drops (0.25 ml) of urine are mixed with 10 drops of water in a test tube. it was then mixed and immediately observed the color. a chart orovided by the manufacturer is used to the result. heat was generated by contacting naoh and water. the initial reaction between citric acid and nahco caused the release of carbon dioxide, which blankets the mixture and reduces contact with oxygen from the air to prevent reoxidation of cuprous ions. if large quantities (>2 g/dl) of sugar are present in the urine, the solution goes through the range of colors and returns to greenish-brown. (8) material that used in this experiment was methylated falme, waterbath, tubes, benedict reagent and timer. the experimental method ina j med lab sci tech 2019; 1(2): 44-51 fitri fadhilah, et al. 4 7 in this study used the design type one group pretest-postes design, we used 3 times repetition with tubes used methylated fire and waterbath 100oc to determine benedict test result by using a sampel that were known for the glucose level. the population in this study were laboratory workers who worked in the pelopor ii regiment clinical laboratory, north bogor. the sample in this study was fresh urine of pelopor ii troop regiment clinic laboratory officers, north bogor. the study had begun in august 2018 until september 2018. the research was conducted at pelopor ii regiment clinical laboratory, north bogor. results the results of the comparison study of glucose reduction in urine using heating method of methylated flame and 100oc waterbath can be seen in the following tabel_1. table 1. results of benedict tests heating method result repetition 1 repetition 2 repetition 3 methylated flame negative (-) negative (-) negative (-) positive (+1) positive (+1) positive (+1) positive (+2) positive (+1) positive (+1) positive (+3) positive (+3) positive (+3) positive (+4) positive (+4) positive (+4) waterbath 100oc negative (-) negative (-) negative (-) positive (+1) positive (+1) positive (+1) positive (+2) positive (+1) positive (+1) positive (+3) positve (+3) positive (+3) positive (+4) positive (+4) positive (+4) fig 1. glucose reduction in urine negative results methylated flame waterbath ina j med lab sci tech 2019; 1(2): 44-51 fitri fadhilah, et al. 4 8 fig 2. glucose reduction in urine positive (+1) results fig 3. glucose reduction in urine positive (+2) results fig 4. glucose reduction in urine positive (+3) results fig 5. glucose reduction in urine positive (+4) results from the picture above, the examination of glucose by benedict test heated by methylited flame and waterbath 100oc showed exactly same result. the number of samples used in the examination of glucose reduction in urine done by the benedict test heated by methylated flame and 100oc waterbath were 15 samples each. the total number of samples were 30 samples with 3 repetitions. the results were obtained by using urine samples that have been given additional glucose with various treatments in accordance with predetermined values (5,11). there result is that normal urine samples without addition of glucose showed a negative result with blue color ( there was no change in color). meanwhile, urine samples with the addition of 0.05gr of glucose dissolved in 10ml of urine showed a positive result (+1) with green / yellowish green color. besides, the urine samples with the addition of 0.15gr glucose dissolved in 10 ml of urine showed a positive result (+2) with cloudy yellow color. similarly, urine samples with methylated flame methylated flame methylated flame methylated flame waterbath waterbath waterbath waterbath ina j med lab sci tech 2019; 1(2): 44-51 fitri fadhilah, et al. 4 9 the addition of 0.30g of glucose dissolved in 10 ml of urine showed a positive result (+3) with cloudy orange color. lastly, the sample with the addition of 0.50gr of glucose dissolved in 10 ml of urine showed a positive result (+4) with red brick color (1,8,10). discussion sugars may be called as reducing or nonreducing based on their ability to reduce copper during the benedict’s test. the reducing property of sugar is based on the presence of free aldehyde or ketone group in them. most of monosaccharides and disaccharides are reducing sugars, while sucrose is nonreducing sugar. reducing sugars are capable of reducing cu2+ (cupric ions) to cu+ (cuprous ions) in alkaline medium which produces red precipitate of cuprous oxide or yellow precipitate of cuprous hydroxide. the urine of normal individuals contains small amount of reducing substances which are not sufficient to give positive test with benedict’s test or fehling’s test. various reducing sugars present in the urine are glucose, galactose, fructose, and lactose. examination of urine for glucose is rapid, inexpensive, and noninvasive and is used to screen large numbers of samples. the monitoring of urine glucose lacks sensitivity and specifity and provides no information about blood glucose concentrations below the renal threshold (usually 180 mg/dl). the older screening tests detect all sugars that reduce copper and also react with reducing substances other than sugars. specific tests for measurement of glucose that are quantitative or semiquantitative are widely available and have essentially replaced the nonspecific tests in adults. the copper reduction test is used to screen neonates and infants for inborn errors of metabolism that may result in the appearance of reducing sugars other than glucose (e.g., galactose or fructose) in the urine (12,13). heating is part of the urine glucose examination with benedict method and heating only serves to help the occurrence of the reduction process so that the temperature used is boiling either with methylated fire or waterbath will not affect the results of urine reduction (1,6,14). both methylated or waterbath are common tools in laboratories, both of these tools have the same function which is to heat. the optimum temperature that can be achieved by both is 100oc so that basically these two tools have the same in heating, although methylated has an affordable price and easy to obtain but thre is weaknesses also accompany its use, for example the perform can only be done one by one so that it slows down the performance in the laboratory. waterbath is the preferred source of heat for several things. waterbath is popularly used mainly to heat flammable chemicals so ina j med lab sci tech 2019; 1(2): 44-51 fitri fadhilah, et al. 5 0 there is no need to use open fires and can prevent fires. waterbaths are made from containers filled with hot water. all watebaths in the laboratory are equipped with interfaces, these interfaces can be either digital or analog, this allows the user to set the desired temperature. waterbath can also be used as a heating reagent. in addition, this tool can also be used to activate certain chemical reactions (such as the urine reduction test reaction bennedict method) that can occur at high temperatures. heating is part of the urine glucose examination with benedict method and heating only serves to help the occurrence of the reduction process so that the temperature used is boiling either with methylated fire or waterbath will not affect the results of urine reduction (1,6,14). conclusions examination of urine reduction by heating the methylated flame and waterbath 100oc does not affect the results. but there is a difference in the process with heating flame need a longer time is 3-5 minutes, when boiling the solution in the tube could be explodes and can only work one by one sample, while the heating of 100oc waterbath the time required is relatively faster, 2 minutes, not explode when boiling and can work 6-8 samples at the same time. acknowladgements the source of funding for this research came from researchers' personal funds conflict of interest there are no conflicts of interest. references 1. bourey re, kaw mk, lester sg, ghanem ss, najjar sm. diabetes. in: diet, exercise, and chronic disease: the biological basis of prevention. 2014. 2. of d, mellitus d. diagnosis and classification of diabetes mellitus. diabetes care. 2014. 3. forouhi ng, wareham nj. epidemiology of diabetes. medicine (united kingdom). 2019. 4. egan am, dinneen sf. what is diabetes? medicine (united kingdom). 2019. 5. lankelma j, nie z, carrilho e, whitesides gm. analysis of glucose in urine. anal chem. 2012. 6. andrade jam, kang hc, greffin s, garcia rosa ml, lugon jr. serum uric acid and disorders of glucose metabolism: the role of glycosuria. brazilian j med biol res. 2014. 7. olagbuji bn, atiba as, olofinbiyi ba, akintayo aa, awoleke jo, ade-ojo ip, et al. prevalence of and risk factors for gestational diabetes using 1999, 2013 who and iadpsg criteria upon implementation of a universal one-step screening and diagnostic strategy in a sub-saharan african population. eur j obstet gynecol reprod biol. 2015. 8. ricchiuti v. tietz textbook of clinical chemistry. am j clin pathol. 2016. 9. james ca, richardson aj, watt pw, maxwell ns. reliability and validity of skin temperature measurement by telemetry thermistors and a thermal camera during exercise in the heat. j therm biol. 2014. 10. bishop m, fody e, schoeff l. clinical chemistry techniques, principles, correlations [internet]. techniques, principles, correlations. 2010. 1-788 p. available from: papers3://publication/uuid/ac48e5e0-82784589-88e8-ef544b4da818 11. ottosson-laakso e, tuomi t, forsén b, gullström m, groop ph, groop l, et al. influence of familial renal glycosuria due to mutations in the slc5a2 gene on changes in glucose tolerance over time. plos one. 2016. ina j med lab sci tech 2019; 1(2): 44-51 fitri fadhilah, et al. 5 1 12. gupta y, kalra s. screening for diabetes. j pak med assoc. 2015. 13. pippitt k, li m, gurgle he. diabetes mellitus: screening and diagnosis. am fam physician. 2016. 14. clarke sf, foster jr. a history of blood glucose meters and their role in self-monitoring of diabetes mellitus. british journal of biomedical science. 2012. 47 relation of parasites in soil with the existence of parasites on farmer's nails edza aria wikurendra1, merry crismiati2, globila nurika3 1doctoral school of management and organizational science, faculty of economic science, hungarian university of agriculture and life science, hungaria 2environmental health study program, widyagama husada college of health, malang, indonesia 3public health study program, faculty of public health, university of jember, jember, indonesia correspondence: edza aria wikurendra, 7400, kaposvar, guba sandor utca 40 email: edza.wikurendra@phd.uniszie.hu received: december 13th, 2020 revised: march 11th, 2021 accepted: march 15th, 2021 published: april 28th, 2021 doi: 10.33086/ijmlst.v3i1.1850 abstract worms is an infectious disease caused by parasitic worms that can endanger health. worms that often infect and have a very detrimental impact are soil-borne worm infections or soil-transmitted helminths. soil-transmitted helminths still considered insignificant because it is considered not to cause harm or cause death. this study aims to determine the relationship of parasites in the soil with the presence of parasites on the nails of farmers sumber urip 1 village wonorejo, east java, indonesia. the research method was used observational analytic with a cross-sectional study design which involved 18 sumber urip 1 farmers in wonorejo village. the sampling technique used was total sampling. the bivariate analysis uses pearson correlation with decision making using significant <0.01. the identification of parasites using the floating method in 18 soil samples contained 12 flattering pieces of hookworm larvae and roundworm eggs. while the results of parasite identification with sedimentation method in 18 nail samples of farmers, there are 11 positive hookworm larvae samples, i.e. ancylostoma duodenale. the correlation test result showed a relationship between parasites in the soil and nails of sumber urip 1 farmers in wonorejo village (significant as p < 0.01). the use of gloves and footwear (shoes) when working on agricultural land, wash hands with soap and brush nails so that dirt is lost, and consume worm medicine can prevent worms infection. keywords infection, farmers' nails, parasites, soil-transmitted helminths. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. https://journal2.unusa.ac.id/index.php/ijmlst/article/view/1850/version/2334 ina. j. med. lab. sci. tech. 2021; 3(1): 47–55 edza aria wikurendra, et al. 4 8 introduction worms is an endemic and chronic disease caused by parasitic worms with a high prevalence and is not lethal but affects the human body's health, resulting in decreased nutritional and public health conditions. the worm species enterobius vermicularis, taenia saginata, ascaris lumbricoides, and ancylostoma duodenale often infect and have devastating effects on soil-borne or soil-transmitted helminths. because it is not considered dangerous or causes death, soil-transmitted helminths is not a problem or a severe threat to society. however, the impact of soil-transmitted helminths infection can cause decreased health and even death (1). soil-transmitted helminth is a term that refers to a group of parasitic diseases caused by nematode worms that are transmitted to humans through fecal contaminated soil. types of worms transmitted by significant soil concern to humans are ascaris lumbricoides, trichuris trichiura, necator americanus, and ancylostoma duodenale. the highest prevalence occurs in areas with inadequate sanitation and unsafe water (2). more than 1.5 billion people, or about 24% of the world's population, have soiltransmitted helminths infection in 2015 (3). world health organization data in 2015 stated that more than 270 million preschoolaged children and more than 600 million school-age children suffering from soiltransmitted helminths infection require intensive care. meanwhile, in indonesia, worms are a common folk disease, and the condition can coincide with several types of worms at once (3). the worm disease survey results stated that 31.8% of elementary school students had worms (4). in the 2017 worm disease data at poncokusumo health center, malang regency, indonesia, there were 11 worm disease cases. in areas with a prevalence of around ≥20% – <50%, drug administration is carried out en masse to all elementary school children in a district/city once a year. if the prevalence is ≥50%, deworming is administered to all elementary school children in a district/city two times a year (5). selective treatment is given when the majority is <20%. poncokusumo puskesmas in april 2018, 689 people in wonorejo village, indonesia, needed worm medicine. soil-transmitted helminths worm infection can occur in related jobs or using moist soil as its primary raw material because it is the right place for ascaris lumbricoides, trichiuris trichiura, necator americanus, ancylostoma duodenale, and strongyloides stercoralis. often groups of agricultural workers (farmers) who are in direct contact with the ground get this worm infection (2). vegetable farmers who are at risk of being infected with worms are the sumber urip 1 farmer group in wonorejo village, where the farmers only use gloves ina. j. med. lab. sci. tech. 2021; 3(1): 47–55 edza aria wikurendra, et al. 4 9 made of cloth, and footwear in the form of boots is rarely use. therefore, this study analyzes the relationship between parasites in the soil and parasites in sumber urip 1 farmers' nails in wonorejo village, poncokusumo district, malang regency. materials and methods the study was an analytic observational study with a cross-sectional study design conducted from june to august 2018. the study's population were farmers of sumber urip 1 in wonorejo village with a sample size of 18 people, and the sampling conduct by comprehensive selection. in this study, the samples were soil samples and nail samples from the farmer members of sumber urip 1, wonorejo village, poncokusumo district, malang regency. this research has passed the ethical test with letter number 684/un.8/kepk/dl/2018. the instrument in this study used a secondary data search guide covering the respondent's characteristics and primary data using laboratory tests. the laboratory examination in this study aims to identify parasites in sumber urip 1 farmland in wonorejo village using the floating method. this method uses a saturated salt solution or saturated sugar to float the eggs. the floating method generally used for checking feces that contain a small number of worm eggs. this method works based on the lighter specific gravity. the solution used has a heavier density compared to worm eggs so that the eggs and the large particles in the feces will float on the surface (6). the sedimentation method is a suitable method for examining old stool samples, thus the method used to identify parasites on the nails of sumber urip 1 farmers in wonorejo village. the principle of the sedimentation method is that the use of centrifugal force can separate the suspension and supernatant so that the worm eggs can settle (7). there are two data processing steps in this research, namely manual data processing and computer data processing. for the statistical analysis, the univariate and bivariate analysis used the pearson correlation test at spss 16.0 for windows. results the respondent characteristics grouped into four categories based on gender, age, education, and work period in years. the results of observations made on 18 farmer members of sumber urip 1, wonorejo village, poncokusumo district, malang regency were 100% male. at the beginning of the formation of the sumber urip 1 farmer group, women farmers did not join so that currently, the members of the sumber urip 1 farmer group are male. the identification of parasites in the sample of farmers' agricultural land in sumber urip 1, wonorejo village, poncokusumo district, malang regency ina. j. med. lab. sci. tech. 2021; 3(1): 47–55 edza aria wikurendra, et al. 5 0 used the floating method. the result of the floating method showed in table 1. based on table 1, it is known that the results of the examination of parasites on the soil with a sample size of 18, there were positive results for parasites in 12 samples with a percentage of 66.7% and negative consequences for parasites in 6 samples with a ratio of 33.9%. the results of identifying positive soil samples for parasites found hookworm larvae (figure 2) and roundworm eggs (figure 1) with the following picture. table 1. frequency distribution of parasite identification results in soil using floating method number result frequency percentage (%) 1 positive 12 66.7 % 2 negative 6 33.9 % total 18 100 % figure 1. roundworm eggs with a size of 60–45 microns (black arrow). the results of the identification of parasites in the nail samples of sumber urip 1 farmers, wonorejo village, poncokusumo district, malang regency using the sedimentation method with observations of the results using a microscope are shown in table 2. 20 µm ina. j. med. lab. sci. tech. 2021; 3(1): 47–55 edza aria wikurendra, et al. 5 1 figure 2. hookworm larvae with a size of 600 x 25 microns (black arrow). table 2. frequency distribution of identification results of parasites in nails using the sedimentation method number result frequency percentage (%) 1 positive 11 61.6 % 2 negative 7 38.9 % total 18 100 % figure 3. hookworm larvae with a size of 600 x 25 microns (black arrow). 150 µm 150 µm ina. j. med. lab. sci. tech. 2021; 3(1): 47–55 edza aria wikurendra, et al. 5 2 based on table 2, it is known that the results of parasite examination on the nails of sumber urip 1 farmers with a total sample of 18 are as much as sample of 11 samples of positive parasites with a percentage of 61.1%, and a negative result of parasites in 7 samples with a ratio of 38.9%. the results of identifying positive samples of parasites on farmers' nails found in the following picture of hookworm larva (figure 3). the results of the correlation test carried out on the identification results of soil samples and farmer nail samples are shown in table 3. table 3. the results of the relationship between parasites in the soil and parasites on the farmers' nails variable significance correlation soil samples 0.004* nail samples *correlation is significant (p < 0.01) based on table 3, it is known that the results of the pearson correlation test on soil samples and nail samples, amounting to 18 samples each, were significant at 0.004. the significance result of 0.004 is less than 0.01, so the soil sample and nail sample are correlated, or there is a significant relationship. discussion the respondents' age mostly is around 45 – 50 years. that age is likely because people who work as farmers are not in the adolescent or old age group, although a small proportion is productive (8). the education level of farmer members is mainly elementary school education. it showed that the community does not consider education as essential and does not have the awareness to complete at least nine years of education regardless of the type of work. all farmer members' working period is more than ten years. sumber urip 1 members' working period is more than ten years because sumber urip 1 farmer members are members from the beginning of the group's formation until now, and no new members added. hookworm is a type of soil-transmitted helminths that can easily infect its host because, in addition to eggs, infective larval forms can actively enter the host's body. plantation lands, schoolyards, and settlements are places where human activities often occur; soil contamination by hookworm eggs and larvae coupled with a lack of public awareness to maintain personal hygiene while doing activities outside the home will further increase the risk of hookworm transmission (9). ina. j. med. lab. sci. tech. 2021; 3(1): 47–55 edza aria wikurendra, et al. 5 3 roundworms are generally distributed throughout the world, air humidity is high, especially in tropical and subtropical areas. these roundworm eggs can survive up to one year in soil (10). from the results of parasite research on the ground at the research location, there were hookworm larvae and roundworm eggs because the wonorejo village location was a highland area with relatively cold temperatures and was close to mountains so that the humidity of the soil was high. the form of agricultural land in wonorejo village is loose soil, so it is suitable for agriculture and is a breeding ground for hookworms and roundworms. the presence of hookworm larvae can cause sumber urip 1 farmers have poorly maintained nails, and agricultural land processing does not use gloves. the identification results obtained hookworm larvae because they are thin shape, 600 microns elongate, white to gray, and the oral cavity does not look perfect. the spread of hookworm is widespread throughout the world, especially in tropical and subtropical areas which have high temperatures and humidity (10). hookworm is a type of soil-transmitted helminths that can easily infect its host because, in addition to eggs, infective larvae can actively enter the host's body (9). eggs or worm larvae are often stuck on dirty nails. this condition usually occurs in children who often play in the ground and adults who work in the garden or the fields. transmission of worm infection can be through long fingernails where there is a possibility that worm eggs tucked in so that the worm eggs swallowed when eating (11). the relationship between parasites in the soil and the farmer's nails is probably due to the habit of farmers who do not use gloves when processing agricultural land, lack of personal hygiene, lack of knowledge about a parasitic infection; or the use of fertilizer from livestock manure as a soil fertilizer. soil pollution is the cause of the transmission of worm eggs from the soil to humans through hands or nails that contain worm eggs, which then enter the mouth with food. the existence of agricultural/plantation land, resident habits, and occupation of the population can be risk factors for hookworm infection incidence in humans. hookworm larvae can grow and develop very well in loose soil because, the larvae can freely take oxygen than if they are in clay. the use of manure containing worm eggs on plantation land is likely to cause contamination of plantation soil by hookworms and other types of worms (9). larvae or adult worms can cause symptoms of hookworm infection. according to the place of preference, adults hookworms can live in the small intestinal cavity of the host animal. adult hookworms cause slow blood loss because adult worms can suck blood from 0.2 to 0.3 ml every day. ina. j. med. lab. sci. tech. 2021; 3(1): 47–55 edza aria wikurendra, et al. 5 4 chronic infection can cause progressive anemia, hypochromic microsites, iron, and hb deficiency can decrease to 2 g/dl. if infection situation continues to worsens, it can cause shortness of breath, fatigue, and dizziness to heart weakness. hookworm larvae can penetrate to skin and can cause itching (ground itch). hookworm larvae can also migrate to the lungs and cause pneumonitis. ancylostoma duodenale infection is more severe than disease caused by necator americanus this is in line with research that there is a significant relationship between cutting nails once a week using footwear, washing hands before eating, and washing the feet with the incidence of worms (13). in other studies, it has shown there was a relationship between the habit of washing hands with antiseptic soap and the incidence of worms (14). in line with this, this study showed an association between handwashing habits and worm infection (15). worm infection can be affected by personal hygiene, such as hand and nail hygiene. worm infection mainly transmitted through dirty hands and feet because worm eggs are usually hiding in long and dirty fingernails (16). lack of personal hygiene, especially defecation and sanitation habits, low socioeconomic status, lack of education levels are risk factors for hookworm infection. improved sanitation, hygiene, and chemotherapy have made hookworm infestations rare in developed countries but are still endemic in the world (17). hookworm infection can prevent by giving worm medicine to sufferers, and it is better if mass treatment also carried out for all residents in endemic areas. health education is given to residents to make adequate latrines to prevent soil contamination. when walking on the ground, they always use footwear to avoid skin infection by hookworm filariform larvae (10). conclusions this study found there was a significant relationship between parasites in the soil and the nails of farmers (p-value < 0.01). suggestions for sumber urip 1 farmers in wonorejo village for use gloves and footwear in the form of shoes when working on agricultural land, wash hands with soap and brush nails so that dirt is lost, and regularly consume worm medicine to prevent the worm infection. author contributions edza aria wikurendra: conceptualization, writing-reviewing, validation, data analysis, methodology. merry chrismiati: sampling and data collection. globila nurika: writing-original draft preparation. ina. j. med. lab. sci. tech. 2021; 3(1): 47–55 edza aria wikurendra, et al. 5 5 acknowladgements our thanks go to the researchers and all parties involved, as well as universitas nahdlatul ulama surabaya, widyagama husada college of health and university of jember. conflict of interest there is no conflict of interest in this study. references 1. ministry of health of the republic of indonesia. indonesia health profile 2019 [internet]. 2019. available from: https://www.kemkes.go.id/download.php?file=d ownload/pusdatin/profil-kesehatanindonesia/profil-kesehatan-indonesia-2019.pdf 2. wijaya nh, anies, suhartono, hadisaputro s, seyawan h. risk factors for hookworm infection in albasia breeding farmers in kemiri district, purworejo regency. j epidemiol kesehat komunitas. 2016;1(1):15–24. 3. world health organization. taenia solium: who endemicity map update [internet]. world health organization; 2016. available from: https://apps.who.int/iris/bitstream/handle/10665/ 251908/wer9149_50.pdf;jsessionid=ecde8bc 2f24ebf63788372248a50e3f9?sequence=1 4. ministry of health of the republic of indonesia. regulation of the minister of health of the republic of indonesia number 15 year 2017 concerning worms control [internet]. jakarta; 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2011. 1–379 p. 11. sofiana l, kelen msj. factors related to soil transmitted helminth infection on primary school children. unnes j public heal. 2018;7(1):55–61. 12. noviastuti ar. soil transmitted helminths infection. med j lampung univ. 2015;4(8):107– 16. 13. baidowi ii, armiyanti y, febianti z, nurdian y, hermansyah b. the correlation between the use of personal protective equipment (ppe) and soiltransmitted helminths infection in the workers of kaliputih plantation jember regency. j agromedicine med sci. 2019;5(2):61–8. 14. putra tri, loesnihari r, panggabean m. soiltransmitted helminth infection and eosinophil levels among waste collectors in banda aceh. indones j trop infect dis. 2018;7(2):27–34. 15. mahmudah u. relationship of home environmental sanitation to the incidence of worm infection in elementary school children. j kesehat. 2017;10(1):32–9. 16. nugraha ti, semiarty r, irawati n. the relationship between environmental sanitation and personal hygiene with soil transmitted helminths (sth) infection in school-age children in koto tangah district, padang city. j kesehat andalas. 2019;8(3):590–8. 17. rahmawati y, mustika s, ahmad h. case report: diagnosis of loeffler’s syndrome and duodenal nekatoriasis by endoscopy. j kedokt brawijaya. 2014;28(1):58–61. 1 mir-141-3p relative expression level from ffpe samples as biomarker of prostate adenocarcinoma carcinogenesis in yogyakarta, indonesia sari eka pratiwi1, sri nuryani wahyuningrum2, rachma greta perdana putri3, danarto4, didik setyo heriyanto5, nur arfian6, sofia mubarika haryana7, indwiani astuti8 1department of biology and pathobiology, faculty of medicine, universitas tanjungpura, indonesia 2magelang unit of health research and development, ministry of health, indonesia 3department of histology, faculty of medicine, universitas ahmad dahlan, yogyakarta, indonesia 4department of urology, sardjito hospital, yogyakarta, indonesia 5department of anatomy pathology, faculty of medicine, public health and nursing, universitas gadjah mada, yogyakarta, indonesia 6department of anatomy, faculty of medicine, public health and nursing, universitas gadjah mada, yogyakarta, indonesia 7department of histology and cell biology, faculty of medicine, public health and nursing, universitas gadjah mada, yogyakarta, indonesia 8department of pharmacology, faculty of medicine, public health and nursing, universitas gadjah mada, yogyakarta, indonesia correspondence: sari eka pratiwi, jl. prof. dr. hadari nawawi, pontianak, west kalimantan, indonesia zip code: 78124 email: sariekapratiwi@medical.untan.ac.id received: september 7, 2021 revised: october 1, 2021 accepted: february 4, 2022 published: april 28, 2022 doi: 10.33086/ijmlst.v4i1.2355 abstract globally, prostate cancer (pca) is the second leading cause of male cancer-associated mortality. micro-rnas (mirnas) are small non-coding rnas considered promising biomarkers for diagnosis, prognosis, and treatment options. a mir-141 expression is frequently dysregulated and influences the development and progression of pca. this study aimed to identify mir-141 expression level as a marker to differentiate pca from another prostate anomaly, especially in yogyakartaindonesia. formalin-fixed paraffin-embedded (ffpe) tissues for each three groups: benign prostatic hyperplasia/bph, high-grade prostatic intraepithelial neoplasia/hgpin, and pca (n=7/group) were stored in a commercial clinical laboratory in yogyakarta. the total rna was extracted from ffpe sections using mirneasy ffpe kit, followed by the quantification of mir-141-3p expression level by rt-pcr. the result showed that mir141 relative expression level on pca was higher than other groups and significantly different (p<0.05, kruskal wallis test). the mean of the mir-141 relative expression level of bph, hgpin, and pca were 1.04±0.87, 6.44±7.8, and 7.06±8.83, respectively. the relative expression level of mir-141 can potentially be a prognostic biomarker in pca and could differentiate aggressiveness in prostate anomaly, especially bph, hgpin, and pca. keywords bph, hgpin, mir-141, pca, prostate anomaly markers. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech2022; 4(1): 1–9 sari eka pratiwi, et al. 2 introduction prostate cancer (pca) is the most common malignancy in men and the second leading cause of male cancer-associated mortality around the world (1,2). the early pca stage is localized and can be treated with a range of treatments, including chemotherapy, radiation therapy, radical prostatectomy, cryotherapy, and others. unfortunately, following initial treatment, around 23-40% of these patients will acquire metastatic cancers. prostate tumors frequently spread to the bone and other organs, resulting in death (2). although pca is ordinary, there are no diagnostic or prognostic biomarkers that can be used to determine its aggressiveness. the prostate-specific antigen (psa) test, utilized in the clinic as a standard assay, is not specific for pca. infections, inflammation, hyperplasia, and other things can affect psa levels to vary. these limitations of psa level usually lead to overdiagnosis and overtreatment (1,2). recent research has demonstrated the capability of nucleic acids as a disease marker. microrna (mirna) is a type of rna that has been demonstrated to be a disease marker (1–3). micro-rnas (mirnas) are small non-coding rnas and one of the regulatory rnas arranged by 18-25 nucleotides. mirnas play a role in the posttranscriptional regulation of gene expression by targeting almost 30% of all messenger rnas (mrnas). it silences the mrnas by degrading or inhibiting the mrna translation and expression. the dysregulation of mirnas can lead to some diseases such as cancer, indicating their essential role in carcinogenesis (4–7). today, mirna is considered as promising biomarker for diagnosis, prognosis, and treatment options in several solid cancers, including pca (8). mirnas tend to be oncomir or tumor suppressor mirnas. as a tumor suppressor, mirna targets the oncogenic mrna (6,7). mir-141 is one member of the mir-200 family (mir200) that plays a role in proliferation, epithelial-mesenchymal transition (emt), migration, invasion, and drug resistance (8,9). in malignant tumors, mir-141 expression is frequently dysregulated and influences the tumor's development and progression. dysregulation of mir-141 (microrna141) in many types of cancer also modulates cellular motility and regulates stemness. mir-141 is commonly known to have a dual role in tumor development, as a tumor suppressor gene or an oncomir depends on the type and severity of cancer. this phenomenon provides more benefit of mir-141 utilization for therapeutic targeting agents, diagnostic or prognostic biomarkers (10). mir-141-3p (microrna-141-3p) directly inhibits mrna zeb1 (zinc finger e ina. j. med. lab. sci. tech. 2022; 4(1): 1–9 sari eka pratiwi, et al. 3 box binding homeobox 1) and zeb2 (zinc finger e-box binding homeobox 2), a gene that plays an essential role in the emt process of invasive tumors (11). conversely, one study in pancreatic, colorectal, and breast cancer cell lines showed that zeb1 triggers a mirna-mediated feed-forward loop by directly inhibiting mir-141 transcription, which stabilizes emt and promotes invasion of cancer cells (12). mir-141 also repressed stat4 (signal transducer and activator of transcription 4) and targeting transcriptional co-activator with pdz-binding motif that regulates cancer invasion and proliferation in gastric cancer (13). meanwhile, as an oncomir, mir-141 was found up-regulates in many prostate cancer studies compared to healthy controls (14). in patients with bone-metastatic prostate cancer, the mir-141 expression level was elevated and positively correlated with the bone lesion (15). dysregulation of mir-141 contributes to a deeper understanding of tumorigenesis and becomes a potential diagnostic and prognostic biomarker in the development of prostate cancer. studies of mir-141 in prostate cancer and another prostate anomaly were rarely done in indonesia, especially in special region of yogyakarta, in the form of ffpe sample. therefore, in this study, we aimed to identified mir-141 expression level as a marker to differentiate prostate cancer (pca) to high-grade prostatic intraepithelial neoplasia (hgpin) and benign prostatic hyperplasia (bph), based on formalin-fixed paraffin-embedded (ffpe) tissue sample of patients from yogyakarta, indonesia. materials and methods sample collection the study was approved by the medical and health ethics committee, faculty of medicine, universitas gadjah mada, yogyakarta, with approval number of ke/fk/0888/ec/2018. samples were collected from formalin-fixed paraffinembedded (ffpe) blocks stored in a commercial clinical laboratory in yogyakarta from 2017 until 2018. as many as seven ffpe blocks from each bph, hgpin, and pca group were sliced into five sections with a 10-µm thickness each and prepared for rna extraction. total rna extraction and reverse transcriptase-pcr (rt-pcr) total rna extracted from ffpe sections with mirneasy ffpe kit (qiagen, germany, cat no. 217504). after ffpe blocks were sliced, the sections were placed in safe lock tubes (1.5 ml), and 160 µl xylene was added to each tube for the deparaffinization process. the rnas were eluted using 30 µl rnase-free water. cdna synthesis was performed with mircury lna rt kit (qiagen, germany, cat. no 339340). for the synthesis of cdna from mirna, the rna template was diluted by adding nuclease-free ina. j. med. lab. sci. tech2022; 4(1): 1–9 sari eka pratiwi, et al. 4 water until the concentration achieved 5 ng/ µl, then placed 2 µl into 0.2 ml tube. quantitative real time pcr for mir141-3p quantification of mir-141-3p (3' gguagaaauggucu--gucacaau 5') in ffpe section used mircury lna mirna pcr assay kit (qiagen, germany, cat no. 339306) in biorad cfx96 real-time machine. the preparation step was started by diluting 1 µl cdna template with 59 µl nuclease-free water (1:60), then placed 4 µl diluted cdna template into a white qpcr strip tube. the initial pcr condition was 950c for 10 minutes, followed by 40 cycles of denaturation step at 950c for 10 seconds, with annealing and extension steps at 580c for 1 minute. this study used u6 for internal control and normalized mirna, h2o for negative control, and all the samples were performed in duplicate. mir-141-3p expression was analyzed based on the value of δcq of mir-141-3p, which was normalized with δcq of u6. then, the expression was calculated using the livak method. the microrna amplification curve was read as cycle quantification (cq). the cq of mir-141-3p value was then normalized with the cq of u6 value, and the value of δcq was conducted. then the δδcq quantification with the comparison was the average value of the bph group’s δcq (as a comparative group). after the δδcq results were obtained, the next step was to analyze the target microrna expression by the livak method with the formula was 2–(δδcq). the type of quantification in this study used relative quantification by comparing the groups of hgpin and pca to bph. the mann-whitney test and kruskal-wallis test were used to analyze the differences of mir141 expression levels in the tumor tissue according to the clinicopathological characteristics (p value significant < 0.05). results subject characteristics this study involved 21 subjects that consisted of 7 bph patients, 7 hgpin patients, and 7 pca patients. patients were dominated by the elderly, with an average of age was 68 years old. gleason score for pca patients showed 2 people having mediumgrade cancer (score 7) and 5 people having high-grade cancer (score 8 and 9). detail for subject characteristics is shown in table 1. mean difference of mir-141-3p relative expression mir-141 relative expression level of three groups of subjects was significantly different (p<0.05, kruskal wallis test). the difference showed that the mir-141 level in pca samples was higher than hgpin and bph samples. the mean difference of mir141 in the three groups is described in figure 1. ina. j. med. lab. sci. tech. 2022; 4(1): 1–9 sari eka pratiwi, et al. 5 table 1. subject characteristics of samples characteristics of subject age (years), mean ± sd (min – max) 67.71±11.48 (44 83) bph 67.71±11.64 (55 83) hgpin 63.29±12.34 (44 80) pca 72.14±10.27 (55 82) prostate anomaly status (n, %) bph 7 (33.3) hgpin 7 (33.3) pca 7 (33.3) gleason score for pca samples (n) score 7 2 score 8 2 score 9 3 grade of pca samples (n) grade 3 2 grade 4 2 grade 5 3 relative expression mean of mir-141-3p bph 1.04±0.87 (0.07-2.17) hgpin 6.44±7.8 (0.59-22.15) pca 7.06±8.83 (1.04-26.44) bph: benign prostatic hyperplasia, hgpin: high-grade prostatic intraepithelial neoplasia, pca: prostate cancer figure 1. clustered coloumn of mir-141 mean relative expression in benign prostatic hyperplasia (n=7, bph), prostate cancer (n=7, pca), and high-grade prostatic intraepithelial neoplasia (n=7, hgpin). *p=0.023,three-way kruskal-wallis test; **p=0.009 bph versus pca, **p=0.048 bph versus hgpin, p=565 pca versus hgpin, mann-whitney test 1,04 7,06 6,44 0 1 2 3 4 5 6 7 8 bph pca hgpin m e a n o f m ir -1 4 1 r e a lt iv e e x p r e ss io n l e v e l (f o ld -c h a n g e ) p=0.009** p=0.048** p=0.023* ina. j. med. lab. sci. tech2022; 4(1): 1–9 sari eka pratiwi, et al. 6 discussion regulation of mir-141 in prostate cancer development involves complex mechanisms between the target gene and other interfering molecules. considering that regulation of mir-141 was cancer type-dependent, the expression level of mir-141 could be different based on cancer severity. our finding shows that the mir-141 expression levels of prostate cancer patients was significantly higher than hgpin and bph patients. sample of prostate cancer tissue with upregulating mir-141 expression was known as a prostate cancer patient in a condition of grades 3, 4, and 5. in this term, we could report that mir-141 in the high stadium of prostate cancer, in this case, grades 3-5, played a role as oncomir. oncomirs or oncogenic mirnas are mirna which are associated with cancer. various mirnas act as emt repressors, particularly inhibiting the zeb1 expression, which involves mir-200s families (mir200a, mir-200b, mir-200c, mir-141, and mir-429). the function of the mir-200s families is to promote met and inhibit emt by targeting zeb1. in other ways, zeb1 represses the gene transcription of mir-200s by directly binding on the gene's promoter region (16,17). mir-141-3p is involved in targeting mrna zeb1 and regulates the androgen receptor (ar) mrna expression by targeting the ar’s open reading frame (orf), particularly at early pca but not in the advanced stages (18). the results of this study are in line with studies conducted by cheng et al., (19) which showed an increment of mir-141-3p expression in the serum of prostate adenocarcinoma patients who have undergone metastasis compared to the serum of healthy patients. cancer metastases involve angiogenesis, a formation of new blood vessels, which is required for tumor expansion, and sufficient tumor nutrition. a previous study showed that overexpression of mir-141 correlated with higher blood vessel formation in mouse models and inducing secretion of vegfa (vascular endothelial growth factor-a), one of the potent blood vessel growth factor (20,21). another mechanism that relates mir-141 in worsening tumor conditions is inducing oxidative stress response. a prior study reported that mir-141 could modulate higher reactive oxygen species (ros) in oxidative stress conditions, including tumor microenvironment, by directly targeting the p38a gene that blocks proliferation and promotes apoptosis. this condition implies disruption of cellular components, induces cell proliferation, and stimulates tumor growth (22). fluctuation of mir-141 expression level in any cancer was capable of distinguishing malignant or non-malignant tissue and used ina. j. med. lab. sci. tech. 2022; 4(1): 1–9 sari eka pratiwi, et al. 7 to differentiate cancer staging (10). in a prostate cancer, overexpression of mir-141 is the most potent biomarker of metastatic phase. it can differentiate prostate cancer patients with local advanced from those with metastasis (14). in line with our results, a study reported that a higher level of mir-141 was related to more aggressive and advanced disease of prostate cancer (high gleason score, large tumor size, and lymph node metastases). upregulation of mir-141 also associated with increased risk of biochemical recurrence (23). other studies confirm that overexpression of serum mir-141 could be used to differentiate non-metastatic from metastatic group patients with 69% sensitivity and 94% specificity (24). otherwise, a recent study revealed that higher expression of mir-141 enforces a strong epithelial phenotype and minimizes partial loss of mesenchymal phenotype by directly binding to the zeb1 gene, an emt transcription factor. this mechanism correlated with mir-141 down-regulation in metastatic prostate cancer patients (25). our study, to our knowledge, is the first study to look for the possible role of mir-141 on prostate cancer compared to high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia in indonesian people using ffpe samples. our result suggests that elevating mir-141 expression shows a higher stadium of prostate cancer. this study had limitations in representing the small number of the patients. a large number of prostate cancer patient subjects is required to validate diagnostic and prognostic relevance of mir141 to represent prostate cancer progression. conclusions in conclusion, our research has shown that mir-141 level in a higher grade of pca plays a role as oncomir and can be a prognostic biomarker. the mir-141 level also could differentiate aggressiveness in bph, hgpin, and pca. author contributions sari eka pratiwi: conceptualization, methodology, investigation, writingreviewing and editing. sri nuryani wahyuningrum: software, data curation, and writing-editing. rachma greta perdana putri: investigation. danarto: conseptualization: didik setyo heriyanto: supervision. nur arfian: methodology, supervision, and validation. sofia mubarika haryana: supervision and validation. indwiani astuti: methodology, supervision, writing reviewing and editing. acknowledgements this work was supported by a research grant from faculty of medicine, public health, and nursing 2018. this work was conducted at the laboratory of anatomy, molecular biology laboratory, and integrated laboratory of faculty of medicine, ina. j. med. lab. sci. tech2022; 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4(1): 1–9 sari eka pratiwi, et al. 9 21. tejero r, navarro a, campayo m, viñolas n, marrades rm, cordeiro a, et al. mir-141 and mir-200c as markers of overall survival in early stage non-small cell lung cancer adenocarcinoma. plos one. 2014;9(7):1–9. 22. hui l, bakiri l, mairhorfer a, schweifer n, haslinger c, kenner l, et al. p38α suppresses normal and cancer cell proliferation by antagonizing the jnk-c-jun pathway. nat genet. 2007;39(6):741–9. 23. richardsen e, andersen s, melbø-jørgensen c, rakaee m, ness n, al-saad s, et al. microrna 141 is associated to outcome and aggressive tumor characteristics in prostate cancer. sci rep. 2019;9(1):1–9. 24. ali r, el tabbakh s, el delgawy w, kotb a, desouky mn. microrna-141 as a diagnostic and prognostic biomarker for prostate cancer in egyptian population: pilot study. african j urol. 2018;24(4):347–52. 25. liu c, liu r, zhang d, deng q, liu b, chao hp, et al. microrna-141 suppresses prostate cancer stem cells and metastasis by targeting a cohort of pro-metastasis genes. nat commun. 2017;8. 21 cytotoxicity assay using brine shrimp lethality test on collagenchitosan wond dressing sterilized by ultraviolet light ary andini1, endah prayekti1, devyana dyah wulandari1, ersalina nidianti1 1department of medical laboratory technology, faculty of health, universitas nahdlatul ulama surabaya, surabaya, indonesia correspondence: ary andini, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code : 60237 email: aryandini@unusa.ac.id received: february 14, 2020 revised: february 17, 2020 accepted: march 23, 2020 abstract collagen gives a moist state on the wound area to accelerate the wound healing process. chitosan is a polymer known as non-toxic, antibacterial, antifungal, biodegradable, and biocompatible material. combination of collagen and chitosan is expected to be the best biomaterials as a wound dressing for the healing process. the study aimed to determine the cytotoxicity assay on collagen-chitosan wound dressing sterilized by ultraviolet (uv) light using brine shrimp lethality test (bslt) method. the test groups were divided into k0, k1, k2, and k3 groups. k0 contained pure chitosan as a control group, k1 contained collagen 25%-chitosan 75%, k2 contained collagen 50%chitosan 50%, k3 contained collagen 75%-chitosan 25%. collagen was extracted from the skin and scalp of snakehead fish (channa striata), then it was mixed with chitosan until collagen-chitosan wound dressing formed. this study used brine shrimp level test (bslt) method with solution concentration: 10, 50, 100, 250, 500, 750 and 1000 ppm. based on the results, it showed that k0, k1, k2, and k3 group had lc50 > 1000, proved that collagen-chitosan wound dressing was non-toxic material. the conclusion of the study explain that composite wound dressing based on collagen-chitosan in all groups that was sterilized under uvlight for 15 minutes was not toxic. also, it showed lc50 > 1000 based on brine shrimp lethality test. keywords collagen, chitosan, bslt, wound dressing, ultraviolet introduction there are two kinds of wound dressing, such as traditional wound dressing including gauze, plasters, lint, bandages, cotton wool, and modern wound dressing including films, hydrogel, hydrocolloids, foam and composites (1,2). wound dressing aplication has main roles in healing the wound and preventing infection on wound area. best wound dressing with good quality could accelerate the wound healing, prevent the wound from infection of bacteria and fungal, and reduce pains (2). the particular wound dressing can promote a newly epithelium ina j med lab sci tech 2020; 2(1): 21-26 ary andini, et al. 2 2 layer that may not be damaged during the dressing removal around the wound (3). the composite dressing is made from a combination of any kind of materials for dressing. it is suitable to burn wounds, surgical wounds, infectious wound and refractory chronic wound (2). collagen and chitosan are known as biomaterials that have bioproperties including biocompatible and biodegradable (4). therefore, it can be developed into composite dressing for wound healing. however, its application is limited due to clinical test before using for human. few steps were needed before clinical test of a wound dressing which are toxicity (5), quality (6), in vitro and in vivo evaluation test (7). based on ariyadi and dewi (8) study, it showed that dry sterilization using ultraviolet (uv) irradiation for 10 and 15 minutes would not be contaminated by bacillus subtilis colony (8). therefore, this research used composite collagen-chitosan extracted from channa striata collagen that was sterilized under ultraviolet (uv) light for 15 minutes to determine lc50 using brine shrimp lethally test (bslt). previously, the brine shrimp was utilized in various bioassay such as analysis of pesticide residues, stream pollutant, anesthetics, dinoflagellate toxins, mycotoxins, cocarcinogenecity of phorbol esters, and toxicants in marine (9). materials and methods skin and scales of sneakhead fish were collected from srikandi fishmonger at banjar asri village, tanggulangin sub-district, sidoarjo, east java province, indonesia. the 100 mesh chitosan powder (food and medical grade) was made from black tiger shrimp shells and it was obtained from monodon (marine natural product). the cytotoxicity assay was conducted by using artemia salina (golden west artemia, supreme plus). the collagen was extracted from the skin and scales of sneakhead fish by macerating it in hcl 2% for 48 hours and it was neutralized with naoh 1m. chitosan powder was dissolved in ch3cooh 1% (4). collagenchitosan composite was synthesized by mixing collagen 25% : chitosan 75% as k1 group; collagen 50% : chitosan 50% as k2 group; collagen 75% and chitosan 25% as k3 group; and pure chitosan as a control group (k0). the cytotoxicity assay was carried out with the brine shrimp lethally test (bslt) by using a. salina. each collagen-chitosan composite of k0, k1, k2 and k3 was dissolved with dmso 1 % then was divided into 10 ppm, 50 ppm, 100 ppm, 250 ppm, 500 ppm, 750 ppm and 1000 ppm. as much as 5 ml of each concentration was added into petri dish then it was added with 5 ml of seawater contained in ten a. salina. hereafter, it was incubated for 24 hours (10). ina j med lab sci tech 2020; 2(1): 21-26 ary andini, et al. 2 3 % mortality = total larvae mortality total larvae x 100 % lc50 assessment was analyzed by microsoft excel with probit analysis. afterward, the toxicity categories of each sample could be determined based on the table 1. table 1. lc50 categories (9) categories lc50 (ppm) very toxic <30 toxic 30-1000 non-toxic > 1000 (source: meyer et al, 1982) results brine shrimp lethally test (bslt) was conducted using artemia saline larvae to study the toxicity of collagen-chitosan composite. this study was a preliminary test before the composite applied in vivo. the result of probit analysis of larvae mortality percentage for each group sample can be seen in table 2, 3, 4, and 5. table 2. percentage of larvae mortality in k0 group concentration (ppm) log 10 mortality total larvae %mortality probit 10 1,00 0 10 0% 50 1,70 3 10 30% 4,48 100 2,00 0 10 0% 250 2,40 0 10 0% 500 2,70 1 10 10% 3,72 750 2.88 0 10 0% 1000 3,00 0 10 0% table 3. percentage of larvae mortality in k1 group concentration (ppm) log 10 mortality total larvae %mortality probit 10 1,00 0 10 0% 50 1,70 0 10 0% 100 2,00 1 10 10% 3,72 250 2,40 2 10 20% 4,16 500 2,70 1 10 10% 3,72 750 2.88 0 10 0% 1000 3,00 2 10 20% 4,16 ina j med lab sci tech 2020; 2(1): 21-26 ary andini, et al. 2 4 table 4. percentage of larvae mortality in k2 group concentration (ppm) log 10 mortality total larvae %mortality probit 10 1,00 3 10 30% 4,48 50 1,70 1 10 10% 3,72 100 2,00 0 10 0% 250 2,40 2 10 20% 4,16 500 2,70 2 10 20% 4,16 750 2.88 2 10 20% 4,16 1000 3,00 0 10 0% table 5. percentage of larvae mortality in k3 group concentration (ppm) log 10 mortality total larvae %mortality probit 10 1,00 2 10 20% 4,16 50 1,70 0 10 0% 100 2,00 0 10 0% 250 2,40 0 10 0% 500 2,70 1 10 10% 3,72 750 2.88 3 10 30% 4,48 1000 3,00 1 10 10% 3,72 tabel 6. lc50 assesment no. group lc50 toxicity 1 k0 > 1000 non-toxic 2 k1 > 1000 non-toxic 3 k2 > 1000 non-toxic 4 k3 > 1000 non-toxic (source: meyer et al, 1982) based on the results, it showed that collagen-chitosan composite sterilized by uv light was not toxic. this is based on the interpretation of the data, according to meyer et al, (9). discussion the composit dressing is used for wound treatment to reduce the contamination risk. the research about wound dressing had developed rapidly due to the demand for wound treatment which increase every year. chitosan and collagen are popular biomaterials for wound dressing because of its bioproperties. sterilization of materials before in vivo assay must be done to prevent microbial and fungal contamination. the uv light exposure on materials could mantain sterility of materials. however, not every material suitable for this methods. therefore, cytotoxicity assay has to be conducted to analyse its bioproperties before it used for humans. brine shrimp lethally test was a ina j med lab sci tech 2020; 2(1): 21-26 ary andini, et al. 2 5 simple cytotoxicity assay by using a. salina to obtain lc50. if lc50 value is more than 1000, it means that the materials tested were not toxic (9). based on the results of the study, it showed that wound dressing for skin wound healing that sterilized by uv light at the whole group was not toxic because lc50 > 1000. chitosan was also known as antibacterial and anti-fungal properties (4). exposure of uv sterilization used was 254 nm to inactivate the microbe. absorption of uv light on microbe could damage nucleic acids and their constituents that caused mutagenetic effect and cell retardation (11). ultra-violet (uv) radiation on the material causes chemical modification of nucleoprotein compounds and occurs the cross-linked of timin molecules that could generate false genetic code, promoting mutation that will cause the damage and weaken the vital functions of the organism until dead (8). based on the results of ariyadi and dewi (8) study, it showed that the best uv method as anti-bacterial treatment was obtained in 10 and 15 minutes. therefore, this study was using 15 minute for uv exposure as a sterilization method on the wound dressing. collagen could heal chronics wound by stimulating fibroblast and promoting endogenous collagen synthesis on wound area (12). based on lei et al. (12) research, it showed that collagen hydrogel dressing could promote the rate and quality of diabetic fullthickness wound healing on rats. moreover, collagen from snakehead fish contains glycine, glutamine and arginine. glycine has a role in collagen synthesis around the wound area, while glutamine has a role as an energy source during the inflammatory phase and proliferation phase of wound healing. arginine has a role in immunity system and stimulates endothelial cell function. a chitosan was known as a biomaterial for drug release on wound area due to its hydrogels properties (13). therefore, the combination of collagen and chitosan can enhance the bioproperties of collagen-chitosan composite dressing. conclusions collagen-chitosan based composite wound dressing in all groups (k0, k1, k2 and k3) that was sterilized under uv-light for 15 minutes were not toxic based on brine shrimp lethally test which showed lc50 > 1000. acknowladgements the authors gratefully acknowledge the financial supports of this research from the institute of research and community development of universitas nahdlatul ulama. conflict of interest there are no conflicts of interest. ina j med lab sci tech 2020; 2(1): 21-26 ary andini, et al. 2 6 references 1. dhivya s, vijaya v, santhini e. review article wound dressings – a review. biomedicine. 2015;5(4):24–8. 2. lei j, sun l, li p, zhu c, lin z, mackey v, et al. the wound dressings and their applications in wound healing and management. heal sci j. 2019;13:1–8. 3. shabunin as, yudin ve, dobrovolskaya ip, zinovyev e v, zubov v, ivan em, et al. chitin / chitosan nanofibers : processing and biomedical applications. cosmetics. 2019;6:1–10. 4. andini a, prayekti e. chitosan as antifungal in channa striata collagen-chitosan for wound healing. med heal sci j. 2019;3(2):8– 11. 5. leliani hn, seniwati ds. collagen extraction from bone of lutjanus sp. and toxicity assay. indones chim acta. 2019;12(1):67–72. 6. mennini n. quality of wound dressings: a first step in establishing shared criteria and objective procedures to evaluate their performance. j wound care. 2016;25(october 2017). 7. kumar pts, lakshmanan v, anilkumar t v, ramya c, reshmi p, unnikrishnan ag, et al. retraction of “flexible and microporous chitosan hydrogel/nano zno composite bandages for wound dressing: in vitro and in vivo evaluation.” acs appl mater interfaces. 2019;11:28596. 8. ariyadi t, dewi ss. pengaruh sinar ultra violet terhadap pertumbuhan bakteri bacillus sp. sebagai bakteri kontaminan (effect of ultra violet rays on the growth of bacillus sp. as contaminant bacteria). j kesehat. 2009;2:20–5. 9. b.n meyer, n.r. ferrigni, j.e putnam, l.b jacobsen, d.e nichols j. m. brine shrimp : a convenient general bioassay for active plant constituents. j med plant res. 1982;45:31–4. 10. muaja ad, koleangan hsj, runtuwene mrj. uji toksisitas dengan metode bslt dan analisis kandungan fitokimia ekstrak daun soyogik ( saurauia bracteosa dc ) dengan metode soxhletasi (toxicity assay using bslt method and phytochemical content analysis of soyogik leaf extract (saurauia bracteosa dc) using soxhletation method). j mipa unsrat online 2. 2013;2(2):115–8. 11. antonio c, silva ds, andrade nj de, fátima n de, soares f, ferreira so. evaluation of ultraviolet radiation to control microorganisms adhering to low-density polyethylene films. brazilian j microbiol. 2003;34(1517–8382):175–8. 12. lei j, chen p, li y, wang x, tang s. collagen hydrogel dressing for wound healing and angiogenesis in diabetic rat models. int j clin exp med. 2017;10(12):16319–27. 13. ou a, louis h, pi a, oc a, ei n, ef o. biochemistry & pharmacology : open access the chemistry of chitin and chitosan justifying their nanomedical utilities. biochem pharmacol (los angel). 2018;7(1):1– 6. 27 in vitro citotoxicity assays of seagrass (enhalus acoroides) methanol extract from soropia coastal waters in southeast sulawesi province theosobia grace orno1, agnes rantesalu2 1department of health analyst, poltekkes kemenkes kendari, kendari, indonesia 2department of health analyst, poltekkes kemenkes kupang, kupang, indonesia correspondence: theosobia grace orno, poltekkes kemenkes kendari, jl. jend a.h. nasution no. g14 anduonohu, kendari, southeast sulawesi, indonesia zip code : 93231 email: theosobiagraceorno@gmail.com received: february 11, 2020 revised: march 13, 2020 accepted: april 13, 2020 abstract the studies analysing the use of natural ingredients as an alternative treatment in the field of pharmacology are developing very rapidly. one of researches that is quite promising in the pharmaceutical industry is the application of marine materials. marine materials that are frequently used consist of shellfish, algae, sponges and seagrass. several studies on toxicity tests have shown that the methanol extract of seagrass (from species enhalus acoroides) is more toxic than the other seagrass family. this study aims to test the toxicity level of seagrass (e. acoroides) extract from soropia coastal waters. the research method in this study was an experimental laboratory using e. acoroides seagrass as a sample that was obtained from soropia coast, konawe regency, southeast sulawesi province. the sample was extracted using methanol as a solvent by macerating it and was tested for its toxicity using the brine shrimp lethality test (bslt) method. toxicity test results showed that the samples with a concentration of 10 ppm, 100 ppm and 1000 ppm in leaves extracts produced an lc50 value of 404.88 ppm, while the stem and root extracts has a value of lc50 > 1000 ppm. the test was continued with higher concentration of leaves extracts consisted of 250 ppm, 500 ppm, and 1000 ppm. the toxicity test showed an lc50 value of 0.7309; which means that it was very toxic. the methanol extract of seagrass (e. acoroides) is potential to be used for further analysis and anticancer formulations. keywords cytotoxicity, enhalus acoroides, soropia coastal waters, southeast regency introduction the research and development on new compounds with unique structures and interesting pharmacological activities have been isolated from marine biota. in general, these compounds are beneficial for humans as it containts high-value bioactive compounds that can be used in both the research and industrial setting. a very complex and dynamic interaction of marine ina j med lab sci tech 2020; 2(1): 27-33 theosobia grace orno, et al. 2 8 ecosystems trigger marine biota to produce secondary metabolites that has a function to defend their lives against competitors, predators and parasite. since the last ten years, the study of marine natural products has began to be carried out in indonesia (1). corals, sponges, algae and seagrasses are marine organisms that are often used as research materials to find new raw material for medicines. however, among these organisms, seagrass is still relatively new in the development of raw materials of new drugs. seagrass is a group of closed seeded plants (angiosperms) and single piece plants (monocots) that are able to live permanently below the sea level. because seagrasses live permanently below the sea level, seagrass is classified as benthic organisms. these organisms produce secondary metabolites to maintain their survival from external disturbances both physicochemically and biologically (2). several studies have shown that seagrass contains compounds that are potential as an anticancer (3), antioxidants (4), and antibacterial agents (5). the research on the potential of some seagrasses as anticancer have been carried out in severeal palces in indonesia including the pramuka island waters, lampung waters, morotai and spermonde waters. the results declared that it has acute cytotoxic potential which can be applied as a new raw material for anticancer drug (6). the toxicity tests intend either to evaluate the safety of a compound or to detect the anticancer activity of a compound. in the soropia waters of konawe regency, southeast sulawesi province, the data on the distribution of seagrass has been extensively studied, but the research on the level of toxicity of active compounds contained in seagrass is still lacking. the soropia sub-district is a coastal area where natural products in the form of marine materials can be studied and utilized for various sectors including the health sector. the reason of choosing enhalus acoroides as a research sample is based on aprilyani et al's (15) research which analysed the seagrass distribution in southeast sulawesi waters, where e. acoroides ranks first in the seagrass distribution in all stations (7). this study tests the cytotoxicity of seagrass in soropia waters by using the brine shrimp lethality test (bslt). this test is usually used in screening natural marine bioactive compounds because it shows a correlation within in vitro cytotoxic methods and a specific anticancer test (8). some methods can also be employed in this test, such as lemna assay, potato disc as well as sell culture methos (microculture tetrazolium salt/mtt). among the four methods, bslt is highly recommended by anderson in the toxicity test because it has a correlation of up to 95% confidence level in specific anticancer tests. although mtt also gets the same results as bslt, bslt is ina j med lab sci tech 2020; 2(1): 27-33 theosobia grace orno, et al. 2 9 easier, faster, cheaper and practical method (9). this study used methanol as an extraction solvent based on the results of previous studies by aulia fajarullah on the extraction of secondary metabolites. seagrass extraction process uses several types of solvent, where methanol solvent yields the highest extract (10.09%) and secondary metabolite compounds namely tannins, saponins, triterpenoids and steroids (10). materials and methods this research was conducted at the biochemistry laboratory of hasanuddin university for approximately one month. this research was an analytical study with a laboratory observation approach. chemical and reagents methanol, e. acoroides, artemia salina larvae were purchased in laboratory of poltekkes kemenkes kendari, indonesia. fresh leaves, root and rhizome of e. acoroides were used for experiments. it was collected from soropia district, south sulawesi province, indonesia. sample preparations the wet weight of seagrass samples was collected and was immediately placed in plastic bags containing sea water in order to prevent evaporation. then, it was transported to the laboratory under cool condition. the seagrass samples of e. acoroides was washed thoroughly in deionized water to removed the biota that attached to it. in addition, the samples were separated into the roots, stems and leaves and each parts was arranged in aluminum trays. the e. acoroides samples were dried using oven dryer with a temperature of 50°c for 4 days. drying process was conducted until the samples reached a moisture content below 10%. furthermore, the samples were removed from the oven and were stored in a jar then was put in a dry place (total solid). the dried samples were milled by using grinder machines and the powder was then sieved manually by using mesh filter with size 30 mm. the fresh samples used in this experiment were 2 kg and it yielded 800 g powder after the drying and grinding process. weight shrinkage in the seagrass samples were 60%. the powdered samples were stored in a refrigerator for further use. e. acoroides extract extraction were conducted by maceration method, namely by immersing the sample in a solvent within a few days. the maceration method was chosen for extraction because it is easy to do and only uses tools that are simple and easy to obtain (10,11). the dried seagrass sample was weighed as much as 50 g and was soaked in a 250 ml methanol solvent in a glass bottle and was macerated for 24 hours. the sample solutions were then filtered using filter paper through glass funnel and whatman no. 42. the filtrate was concentrated to dryness by ina j med lab sci tech 2020; 2(1): 27-33 theosobia grace orno, et al. 3 0 rotation using evaporator under reduced pressure at temperature of 4°c. finally, the crude extract from the seagrass sample was obtained in the form of a paste. then, it was weighed to get the percentage of yield, and it was kept at -20°c prior to further analysis. preparation of the artemia salina larvae a. salina eggs was prepared by soaking it in sea water for 10-15 minutes. good egges would be settle while bad eggs would be float. as much as 50 mg of a. salina eggs were hatching in a container filled with sea water for 10-15 minutes. then, the eggs at the bottom of the container were taken and were hatched in a container that also contained sea water under a 25-watt lamp and was equipped with an aerator. a. salina eggs hatched and became larvae after 24 hours. a. salina larvae that were good for the bslt test were those that have a lifetime of 48 hours. if it died more than 48 hours, it was feared that a. salina's death was not caused by extract toxicity but rather by limited food supply (12). toxicity analysis the toxicity of e. acoroides crude extract was analyzed by using the brine shrimp letality test (bslt). this test were applied on a. salina l. this test aims to determine the level of toxicity of a natural material (13). bslt test was carried out by describing a total of 10-15 shrimp larvae in each test bottle which was then added with a crude extract of seagrass from each tests. test solutions with concentration of 250 ppm, 500 ppm and 1000 ppm were inserted into the bottle. incubation was carried out for 48 hours and it was aerated. this test was carried out 3 times in each concentration series. the observation was run for 24 hours. the number of shrimp larvae death to determine the lethal concentration 50 (lc50) value was counted. lc50 is an assessment of the level of toxicity of a substance against 50% of larval deaths. shrimp larvae defined have died while they were motionless for 10 seconds. the data were analyzed using probit analysis to obtain the value lc50 by microsoft excel (2009) for windows. analysis were conducted by comparing the lc50, if the value of lc50 was higher than 1000 ppm, it would be categorized as toxic (13). results extract of e. acoroides were found to inhibit the growth of a. salina on blst test (table 1). based on the results of the toxicity test of seagrass extract (e. acoroides) which was divided into leaf extract, stem extract and root extract, leaf extract gave an lc50 value of 404.88 ppm (low toxic) while stem and root extract gave an lc50 value of >1000 which means it's not toxic. ina j med lab sci tech 2020; 2(1): 27-33 theosobia grace orno, et al. 3 1 table 1. toxicity test results of seagrass (e. acoroides) extract with bslt method sample conc (μg/ml) log conc replication 1 (larvae) replication 2 (larvae) replication 3 (larvae) % dead % corrected probit lc50 (ppm) dead life dead life dead life e. acoroides (leaf) 100 3 5 5 7 3 5 5 56.67 56.67 5.17 404.88 10 2 4 6 4 6 4 6 40 40.00 4.75 1 1 2 8 2 8 2 8 20 20.00 4.16 e. acoroides (stem) 100 3 2 8 2 8 3 7 23.33 23.33 4.27 >1000 10 2 2 8 1 9 3 7 20 20.00 4.16 1 1 0 10 0 10 3 7 10 10.00 3.72 e. acoroides (root) 100 3 2 8 2 8 4 6 26.67 26.67 4.38 >1000 10 2 2 8 1 9 0 10 10 10.00 3.72 1 1 1 9 0 10 1 9 6.67 6.67 3.50 note: lc50 <1 (very toxic), >1 and <100 (moderate toxic), >100 (low toxic) (16). the toxicity test on leaf extracts with several levels of concentration including 250 ppm, 500 ppm and 1000 ppm represents in table 2. table 2. toxicity test results of seagrass (e. acoroides) extract with bslt method no. concentration (µg/ ml) total larvae dead precentation of larvae dead (%) lc50 (µg/ ml) 1 250 11 37 0,7309 2 500 21 70 3 1000 20 100 note: lc50 <1 (very toxic), >1 and <100 (moderate toxic), >100 (low toxic) (16). discussion in this study, researchers chose seagrass species e. acoroides as research samples. the selection of this sample type was guided by aprilyani et al's (15) research on mapping of seagrass distribution in southeast sulawesi waters, where e. acoroides ranks first in seagrass distribution in all stations (7). e. acoroides seagrass was then extracted using methanol as a solvent. among the various types of solvents used in screening the secondary metabolites of the seagrass e. acoroides, methanol has been shown to have very high rendering activity and has the highest secondary metabolite content of four compounds namely tannins, saponins, triterpenoids and steroids (10). methanol is a universal solvent that has polar (-oh) and nonpolar deciduous (-ch3) groups so that it can attract polar and nonpolar compounds (14). bioactive compounds are usually having toxic at high doses. therefore, in this study, the acute toxicity test used bslt. the mechanism of death of a. salina was thought to be related to the function of compounds dissolved in seagrass extracts which can inhibit the feeding power of larvae (antifeedants/food dodgers). therefore, toxicity analysis was performed to know the toxicity level of the e. acoroides extracts in order to determine their toxicity level. the way these compounds work is by acting as stomach poisoning (stomach poisoning). ina j med lab sci tech 2020; 2(1): 27-33 theosobia grace orno, et al. 3 2 thus, if these compounds enter the larva body, the digestive apparatus will be disrupted. in addition, these compounds can inhibit the taste receptors in the mouth area of the larvae. this results in larvae failing to get a taste stimulus so they are unable to recognize their food until finally the larvae starve and die (13). the results of bslt on leaf extract samples (e. acoroides) with dilution of 1 ppm, 10 ppm and 100 ppm gave lc50 values of 404.88 ppm with low toxicity interpretation, while stem extracts and root extracts gave lc50 values of >1000 ppm with non-toxic interpretation. because only leaf extract that gave an lc50 value of 404.88 ppm, we conducted further tests on leaf extract with dilution variations of 250 ppm, 500 ppm and 1000 ppm. the calculation results show lc50 of 0.7309 ppm with a very toxic interpretation. tannin is one of the secondary metabolites contained in the methanol extract of e. acoroides that can be used as an anticancer compound. anti-cancer tannin activity occurs through the mechanism of inhibiting the work of enzymes, prevention of mutagenesis of cells that can cause cancer, and activation of cancer macrophage cells. the mechanism of action of this tannin uses histone deacetylase inhibitors (hdac) (15). the role of saponin as an anticancer has been known to inhibit the formation of bcl-2 which is expressed too high, induce caspase-3 protein that is expressed too low, and can trigger g1 cell cycle arrest (16). whereas, triterpenoids and steroids act as anti-cancer by activating apoptosis as well as making anti-proliferation (17). conclusions the finding of this study revealed that e. acoroides methanol extract had bioactivity potentials. the extracts with a concentration of 250 ppm, 500 ppm and 1000 ppm has an acute toxic potential againts a. salina larvae, which is indicated by lc50 values. lc values of e. acoroides methanol extract is 0.7309 ppm, thus proving the existence of bslt method. it is necessary to do further study to identify and isolate the active compounds that serve as anti-cancer agents to maximize the benefits of e. acoroides to human medicine. conflict of interest there are no conflicts of interest. references 1. permana ah, husni a, budhiyanti sa. antioxidant activity and toxicity of seagrass cymodocea sp. journal of agricultural technology. 2016 apr;17(1):37-46. 2. jaafarzadeh n, hashempour y, angali ka. acte toxicity test using cyanide on daphina magna by flow-through system. journal of water chemistry and technology. 2013;35(6):281-286. 3. satria d, silalahi j, haro g, ilyas g, hsb paz. antioxidant and antiproliferative activities of an ethylacetate fraction of picria fel-terrae lour. herbs. asian pacific journal of cancer prevention. 2017;18(2):399-403. available from: https://www.ncbi.nlm.nih.gov/pmc/ articles/ pmc5454734/ ina j med lab sci tech 2020; 2(1): 27-33 theosobia grace orno, et al. 3 3 4. ahmad a, usman h, natsir h, karim a. isolation and characterization of bioactive protein from green algae halimeda macrobola acting as antioxidant and anticancer agent. 2014;2(5):134-140. 5. fajarullah a, irawan h, pratomo a. ekstraksi senyawa metabolit sekunder lamun 4. thalassodendron ciliatum pada pelarut berbeda. repository umrah. 2014. 6. puspitasari r. evaluation of the use of seagrass extract as active antifouling material for aquatic producers. jurnal segara. 2016 mar 5;12(1). 7. amudha p, jayalaksmi m, pushpabharathi n, vanitha v. identification of bioactive components in enhalus acoroides seagrass extract by gas chromatography–mass spectrometry. asian journal of pharmaceutical and clinical research. 2018;11(10):131-137. 8. widiastuti e, arfiani r, kairani ia, christyanto y, ara nf, maharani hw. antioxidant effect of clerodendrum sp and acanthus illicifolius methanol extraction on blood profile of male mice induced by benzo(α)pyrene. the 4th international conference on biological science and biotechnology. 2019 july:1-9 9. permana ah, husni a, budhiyanti sa. antioxidant activity and toxicity of seagrass cymodocea sp. extracts. journal of agricultural technology. 2016 apr 23;17(1):37-46. 10. kurnia y, widiastuti el, nurcahyani e. effect of antioxidant taurine, seagrass (enhalus acoroides l.), and red algae (eucheuma cottonii l.) on oxidative stress in some organ homeostasis in which infected by glifosat. proceedings of the national seminar on indonesian medicinal plants 55th. 2019;1(1):296-303. 11. baehaki a, widiastuti i, herpandi h. antioxidant activity of extracts of halodulepinifoliaseagrass from solvents with different polarities. oriental journal of chemistry. 2017;33(1):181-185. 12. wardani g, farida n, andayani r, kuntoro m, sudjarwo sa. the potency of red seaweed (eucheuma cottonii) extracts as hepatoprotector on lead acetate-induced hepatotoxicity in mice. pharmacognosy research .2017;9(3):282-286. 13. rengasamy krr, arumugam r, thangaradjou t, anantharaan p. phytochemical constituents, antioxidant properties and p-coumaric acid analysis in some seagrasses. food research international. 2013;54(1):1229-1236. 14. wang xb, sun zh, fan lx, liu yy, feng j, ma gx, chen dl. two novel diterpenes from the stems and leaves of tropical seagrass enhalus acoroides in the south china sea. natural product research. 2019 aug 19:1-9. 15. aprilyani v, hamdi a, arami h. types of diversity and seagrass distribution patterns in the waters of holimombo village, buton regency. journal of water resource management. 2018;3(4):309-317. 16. wagner jg. pharmacokinetics for the pharmaceutical scientist. technomic pub., lancarter-basel. 1993. 85 ureum and creatinine health study in patients diabetes mellitus muhammad rizki kurniawan1, eni kusrini1 1departement medical laboratory technology, faculty of science and technology, universitas binawan, jakarta, indonesia correspondence: muhammad rizki kurniawan, jl. kalibata raya-dewi sartika no. 25-30 east jakarta, indonesia zip code : 13630 email: kurniawan@binawan.ac.id received: june 1, 2020 revised: juni 4, 2020 accepted: august 21, 2020 abstract diabetes mellitus is a metabolic disease that is marked by an increase in blood glucose levels exceeds normal limits. one of the effects of diabetes mellitus is kidney function disorder. many researches about diabetes mellitus found that patients have uncontrolled levels of urea and creatinine. the purpose of this study was to determine urea and creatinine levels in patients with diabetes mellitus. the research method uses a descriptive approach to determine urea and creatinine levels among diabetes mellitus patients. the results of this study based on the participant characteristics were 47.3% male respondents and 52.7% female respondents, then based on diabetic aged >45 years was 90.95% and <45 years 9.1%. based on disease duration, illness for five years was 65.5%, 6-10 years 16.3% and >10 years was 18.2%. the overall creatinine and urea levels were normal. the conclusion of this study is a general description of urea and creatinine levels in the study sample within normal limits. however, the age factor of patients less than 45 years and the duration of illness from 6 to 10 years has a risk of increased levels of urea and creatinine in the blood. keywords diabetes mellitus, urea, creatinine, age introduction metabolic diseases are medical disorders that affect the production of energy in the human body cells. generally, genetic disorders result in disruption of metabolism, so the enzymes that play a role in cell metabolism processes are lost or damaged. a degenerative disease that needs to be our concern recently is diabetes mellitus because it is one of a severe health problem worldwide. after all, there tends to be an increase case number in future. diabetes mellitus is a metabolic disease characterized by an increase in blood glucose levels exceeding the normal. the cause is impaired insulin secretion or insulin action; therefore, it happens to result in metabolic disorders of fats, and protein failure of various microvascular organs, especially that eyes, ina j med lab sci tech 2020; 2(2): 85-92 muhammad rizki kurniawan, eni kusrini 8 6 kidneys, nerves, and complications including heart disease and stroke (1,2). data from the world health organization (who) stated that from various types of diabetes mellitus. diabetes mellitus type 2 (dmt2) is a type that has the highest percentage (90–95%). 80% of dmt2 patients are in low-income countries or medium with an age range of 40–59 (3). the prevalence of dmt2 in several countries developing due to increased prosperity in the country. according to the data released there are known to be 415 million people in the world suffering from diabetes mellitus (4). based on the 2019 international diabetes federation (idf), 10,7 million people are living with diabetes mellitus in indonesia. henceforth, the predicted prevalence of diabetes mellitus in indonesia during the year 2030 and 2045 will reach 13.7 and 16,6 million (4). changes in lifestyle that hit most of indonesian resulted in an increase of some diseases such as obesity, dyslipidemia and diabetes mellitus. according to perkeni (endocrinology society of indonesia), a person with diabetes if the blood sugar levels when ≥200 mg/dl, fasting blood sugar levels ≥126 mg/dl and blood sugar levels postprandial which is a continuation of the fasting blood sugar test ≥ 200 mg/dl (5). diabetic nephropathy (microvascular complications) is a disorder that occurs in the kidney caused by diabetes mellitus (6). this kidney function disorder characterized by decreased glomerolus filtrate rate (gfr) followed with an increase in urea and creatinine. diabetic nephropathy is a condition kidney that not only experiences disposal failure but lost large amounts of protein, specifically albumin. albumin is the resulting metabolism of proteins stored in the blood. laboratory tests related to diabetes mellitus as a diagnostic examination is when blood sugar, fasting blood sugar, blood sugar postprandial, blood sugar tolerance test and hba1c. fasting blood sugar, i.e. examination of sugar levels when the patient is fasting 12 hours before examination (7,8). hba1c is the specific glycated haemoglobin as a result addition of glucose to the nterminal valine in the beta chain haemoglobin (b-n [1-deoxy] fructosyl-hb). the concentration of hba1c depends on blood glucose concentrations and erythrocyte life span. hba1c describes the average patient’s long-term blood glucose for 2–3 months with good control value (hba1c level <6,5%), moderate control (hba1c level 6,5%–8%), and poor control (hba1c level >8%). the most hba1c examination widely used and is a gold standard for glycemic monitoring as well as inhibits inflammation that often occurs (9). hba1c is checking with using blood ingredients, to obtain information on blood sugar levels in ina j med lab sci tech 2020; 2(2): 85-92 muhammad rizki kurniawan, eni kusrini 8 7 fact, because the patient cannot control the test results, in a period time 2–3 months. this test is useful for measuring the level of sugar bonds in haemoglobin a (a1c) throughout the life of red blood cells (120 days) (9). the laboratory test to know and prevent complications in the kidneys is urea and creatinine. urea is the result of protein metabolism. it came from the amino acids that the ammonia has transferred in the liver and reach the kidneys, and excreted an average of 30 grams a day. normal blood urea level is 20–40 mg per 100 ml of blood, but this depends on the amount of normal protein that is eaten and liver function in urea formation. creatinine is a waste product from the creatine phosphate repairment that occurs in muscle, excreted through the kidneys. (10,11,12). according to medical record data of outpatients, 9600 patients are with diabetes mellitus. outpatients are mostly patients from the social security administrator for health (badan penyelenggaraan jaminan sosial kesehatan/bpjs kesehatan) and they do not do kidney examination since the beginning because of the limited examination fees provided by the bpjs kesehatan. based on the background above, the researcher is interested in determining the description of ureum and creatinine in patients with diabetes mellitus. materials and methods the tools used in this study include equipment blood sampling (syringes, nonanticoagulant vacuum tubes, alcohol swabs, plasters, tourniquet, label), chemistry analyzer (pentra 400), centrifuge and micropipette. the ingredients used in this study include serum blood; ureum abx reagents; abx creatinine reagents; multical and normal controls calibrator. this research was conducted in the laboratory of the qadr hospital and used descriptive research type. the primary purpose of this research was giving a description or description of urea and creatinine in patients with diabetes mellitus. the source of data used in this study is primary data. the populations in this study were diabetics who carried out blood sugar control to the laboratory. the sampling technique in this study was simple random sampling, which is a simple sampling, where each element of the population has an equal chance of being selected as a subject. the sample in this study was diabetic patients as many as 55 respondents. this study approved by qadr hospital ethical commission with the letter of ethical approval number 016/rsq/e.p/i/2018. microsoft excel used for data collection and the data analysis used ibm spss 25. data analysis included test the frequency data for respondent characteristics based on sex, age, creatinine level, and urea level. the ina j med lab sci tech 2020; 2(2): 85-92 muhammad rizki kurniawan, eni kusrini 8 8 description testing for urea levels and creatinine levels of respondents used the bar graphs. results the results of research conducted at the laboratory of qadr hospital obtained from 52.7% of female patients and 47.3% male patients. based on the age characteristic, patients aged ≥45 years was 90.9%. the respondent's characteristics; previous urea and creatinine levels showed in table 1. table 1. characteristics of diabetes mellitus patients no parameter % n (number) 1 gender male female 47.3 52.7 26 29 2 age < 45 years ≥ 45 years 9.1 90.9 5 50 3 long been sick ≤5 years 6–10 years >10 years 65.5 16.3 18.2 36 9 10 4 urea levels (mg/dl) ≤40 > 40 80 20 44 11 5 creatinine levels (mg/dl) ≤ 1.5 > 1.5 83.6 16.4 46 9 table 2. creatinine quality control table 3. ureum quality control name unit name unit tv 40.0 tv 40.0 average 39.9 average 39.9 sd pabrik 2 sd pabrik 2 sd 1.84 sd 1.84 cv % 4.61 cv % 4.61 d% -0.32 d% -0.32 min 37 min 37 max 43 max 43 normal value of male 8–24 mg/dl normal value of male 8–24 mg/dl normal value of female 6–21 mg/dl normal value of female 6–21 mg/dl ina j med lab sci tech 2020; 2(2): 85-92 muhammad rizki kurniawan, eni kusrini 8 9 table 2 and table 3 described the quality control of creatinine and urea. the objective of the quality control is that the inspection quality complies with westgard standards and the results are valid. fig 1. creatinine level fig 2. urea level based on the figure 1, the urea level <40 mg/dl was 80% (44 people) and> 40 mg / dl was 20% (11 people). in addition, creatinine levels <1.5 mg / dl was 83.6% (46 people) and >1.5 mg/dl which was 16.4% (9 people) (figure 2). based on data, urea and creatinine levels are still within normal limits for patients with diabetes mellitus discussion one of the effects of diabetes mellitus is impaired kidney function, where urea and creatinine levels rise above the normal (11). in this study, patients with diabetes mellitus in patients at qadr hospital were determined based on the doctor's diagnosis and recorded on the patient's medical record status. the examination of urea and creatinine levels was usual examination requested by doctors at qadr hospital to see impaired kidney function. not all diabetics who control blood sugar also do urea and creatinine checks, so people with diabetes do not know the state of their kidney function at that time. based on table 2 and 3, the mean value of creatinine and urea level is 39.9%, the standard deviation is 1.84, variation coefficient 2.1%, limit d% 3 and limit cv 5. valid accuracy of conclusion, accurate results of precision and also the precise distribution of numbers generated nothing exceeds the rating limit westgard rules. the results of research on quality control are the same, the value of the limit d% 5 and the cv 3 limit according to the westgard method. precision and accuracy are both as a result of the examination (13). ina j med lab sci tech 2020; 2(2): 85-92 muhammad rizki kurniawan, eni kusrini 9 0 the description obtained by researchers on the sex characteristics in table 1 showed that female is suffering from diabetes mellitus more than male. this result is in line with the results of the basic health research (riset kesehatan dasar/riskesdas). riskesdas result in 2013 showed that there is an increase in women by 7.7% and men by 5.6% in the number of patients with diabetes mellitus. diabetes mellitus in women cause by less physical activity than men moreover women's habits consume foods containing chocolate, sugar and fast food snacks (14). this little physical activity makes the body do not use a lot of carbohydrates or glucose and trigger the occurrence of metabolic diseases such as diabetes. the results of research in patients with diabetes mellitus aged ≥45 years is more than 90%. this evidence is in line with the data obtained from idf that most diabetics have an average age of 40-59 years. in this age range, the ability of pancreatic β-cells to produce insulin begins to decline according to the ageing process in humans. but that cannot be cured, only reduce the severity of diabetes mellitus (2). the results of the research based on the duration of diabetes mellitus (table 1), showed more than 65% of respondents have diabetes mellitus for more than five (5) years. the period of diabetes mellitus sufferers up to 10 years shows the success of patients with good control of blood sugar levels. it is possible because of the influence of various factors, namely adherence of patients taking medication, adherence to checking their blood sugar levels to the laboratory or compliance of patients with maintaining their lifestyle and diet. in theory, diabetes mellitus can cause kidney dysfunction (8). based on the study result in table 1, showed that more than 80% urea levels of diabetic respondents were at a normal level (≤40 g/dl). the average urea level was 34.54 mg/dl in the age group ≥45 years or still within normal limits. this result showed that people with diabetes mellitus still have normal kidney function therefore it is possible that people with diabetes mellitus success in controlling blood sugar levels. the parameters of kidney function are creatinine levels. the result of this research obtained that 83.6% of creatinine levels of patients were within the normal range (≤ 1.5 mg/dl). the highest creatinine level (2.48 mg/dl) was in the age group of more than 45 years. this evidence shows that the kidney function of patients with diabetes mellitus is still normal. however, the level of creatinine significantly increased in patients aged <45 years. as long as the length of patients has diabetes mellitus, the level of urea and creatinine will increase in the range of 6 to 10 years. the average condition of kidney function in patients with diabetes mellitus is in good condition. however, there is a tendency for ina j med lab sci tech 2020; 2(2): 85-92 muhammad rizki kurniawan, eni kusrini 9 1 an increase in urea and creatinine levels the age of <45 years or the range of illness in 610 years. that evidence is almost in line with the results of a study of 35–45% of people with diabetes mellitus experiencing complications of diabetic nephropathy, which results in kidney failure. although the risk factors that influence are not examined (15) conclusions this study concludes that urea and creatinine levels in the study sample were within normal limits. however, the age factor of patients less than 45 years and the duration of illness from 6 to 10 years has a risk of increased levels of urea and creatinine in the blood. further research needs to be done with a focus on type-2 diabetes mellitus using a different research design, especially at the age of <45 years with a length of 6–10 years. conflict of interest there is no conflict of interest in this study. acknowledgment(s) thank you to qadrs hospital as a place of research. references 1. ranjani g. asymptomatic bacteriuria in type 2 d iabetic women patients who are attending medic ine opd of government dharmapuri medical c ollege, dharmapuri.iam journal. 2017;4(9): 3642. 2. hayashi j, hasegawa a, hayashi k, suzuki t, i shii m, otsuka h, yatabe k, goto s, tatsumi j, and shin k. effects of periodontal treatment on t he medical status of patients with type 2 diabete s mellitus: a pilot study.bmc oral health.2017; 17:77.doi 10.1186/s12903-017-0369-2. 3. international diabetes federation (idf). idf di abetes atlas nine edition, international diabete s federation (idf). 2019. 4. saeedi p, petersohn i, salpea p, malanda b, kar uranga s, unwin n, colagiuri s,guariguata l, motala a a, ogurtsova k, shaw e j, bright d, williams r. global and regional diabetes preval ence estimates for 2019 and projections for 2030 and 2045: results from the international diabet es federation diabetes atlas, 9th edition. diabet es research and clinical practice. 2019;157;107 843.https://doi.org/10.1016/j.diabres.2019.1078 43. 5. indonesian association of endocrinologists (pe rkeni). consensus on the use of insulin. jakart a.2015 6. abu m a. clinical and biochemical asspciation s with dabetic retinopathy in male patients in t he gaza strip. in endrocinology. 2017.doi: 10.3 389/fendo.2017.00302. 7. sharma s, sharma k, singh ka, kumar m, trip athi n. biochemical profile in type 2 diabetes mellitus with special referenceto dyslipidemia: a retrospective study. ijmscr.2018;1:27-34. 8. chutani a, pande s. correlation of serum creati nine and urea with glycemic indexand duration o f diabetes in type 1 and type 2 diabetes mellitu s: a comparative study. national journal of phy siology. 2017;7:914-919. 9. ahmed sh, abd-ali e, m. r. abdullah. bioche mical study on diabetic nephropathy patients.i hjpas. 2010;23(3). 10. puspitasari and aliviameita a. relationship bet ween renal function test serum and lipid prof ile in patients with diabetes mellitus.journal of physcis. 2018;012011. 11. renda r. can salivary creatinine and urea levels be used to diagnose chronickidney disease in ch ildren as accurately as serum creatinine and urea levels? a case–control study. renal failure. 20 17;39:1:1525-6049.https://doi.org/10.1080/0886 022x.2017.1308256. 12. aminu kb, johnson dw, feehally j, haarris d, ina j med lab sci tech 2020; 2(2): 85-92 muhammad rizki kurniawan, eni kusrini 9 2 jindal k, lunney m, okpechi ig, salako bl, w iebe n, ya f, tonelli m, levin a. global kidne y health atlas (gkha): design and methods. ki dney international suplemments. 2017; 7:145-1 53. 13. jemani, kurniawan mr. hematology quality c ontrol analysis in an-nisa hospitals laborator y, tangerang. binawan student journal. 2019; 1; 82-85. 14. sutawardana jh, yulia, waluyo a. phenomenol ogy study of experiences of people with diabet es mellitus who have had hypoglycemia episod es. web. 2017. 15. rindiastuti y. 2008. nefropati diabetik. web. 20 17. 50 association between triglyceride serum levels and glomerular filtration rate (egfr) in patients with chronic renal failure at jemursari islamic hospital surabaya, indonesia uswatun hasanah1, suhariyadi2, andreas putro ragil santoso1 1department of medical laboratory technology, faculty of health, universitas nahdlatul ulama surabaya, surabaya indonesia 2departement of medical laboratory technology, poltekkes kemenkes surabaya, surabaya, indonesia correspondence: uswatun hasanah, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code : 60237 email: uswatun.ankes15@gmail.com received: july 17, 2020 revised: august 23, 2020 accepted: august 25, 2020 abstract chronic renal failure (crf) is a progressive and irreversible decrease in kidney function. one risk factor that affects the progression of crf is dyslipidemia due to abnormalities of lipid metabolisms. dyslipidemia is characterized by the increase level of total cholesterol, ldl cholesterol, triglycerides, and a decrease in hdl cholesterol level. egfr is a parameter for assessing the excretion function, by calculating the amount of filtrate produced by the renal glomerulus. this study aims to determine the relationship between serum triglyceride levels with egfr in patient suffering from crf at jemursari islamic hospital surabaya. this type of research is descriptive experimental with cross–sectional design. the study population was the patients with crf. the primary data in this study was the results of examination of serum triglyceride levels in patients with crf. the secondary data (serum creatinine levels, body weight, age, and sex) was obtained from medical records of crf patients in february 2019. as much as 12% of patients were in the 40–50 years old category, 36% of patients were 51–60 years old, 44% of patients were 61–70 years old, and 8% of patients were 71–80 years old. 72% of patients were male. the mean triglyceride level was 146.68 mg/dl and the average egfr was 19.86 ml/min/1.73m2. the relationships between triglyceride levels and egfr was carried out by the spearman–rho test where r value was –0.442 and 𝜌 value was 0.027. it was concluded that there was a significant negative correlation between serum triglyceride levels and egfr in patients with crf. keywords chronic renal failure, triglycerides, egfr, gpo–pap introduction renal failure is a condition in which the kidney decrease the function, so the kidneys are unable to maintain body homeostasis. in general, there are 2 types of kidney failure, namely chronic renal failure and acute renal failure. acute renal failure is a condition in which the kidneys suddenly stop functioning ina j med lab sci tech 2020; 2(2): 50-59 uswatun hasanah, et al. 5 1 entirely or almost entirely but may still be able to return to normal kidney function in a short time. meanwhile, chronic renal failure is a condition in which there is a progressive damage to the function of many nephrons that have an effect on reducing the kidney function. both of those categories produce specific renal failure which results in renal blood vessels, glomerulus, tubules, interstitial kidneys, and parts of the urinary tract outside the kidney (1). renal failure is defined as chronic if it is chronic, permanent, and progressive, so that the glomerular filtration rate (gfr) will progressively decrease and will eventually reach terminal renal failure. patients with chronic renal failure (crf) are often asymptomatic until there is an increase in kidney damage (2). based on data in various centers of nephrology in indonesia, the prevalence of chronic renal failure increases with age. it sharply increases in the age group of 35–44 years old (0.3%), followed by 45–54 years of age (0.4%) and 55–74 years of age (0.5%). the highest prevalence of chronic renal failure are ≥75 years of age (0.6%). in addition, the prevalence of crf in men is 0.3% which is higher than that of women (0.2%) (3). triglycerides (also called triacylglycerol) are one of the fats in the blood formed by esterification of glycerol and three fatty acids carried by lipoproteins in plasma (4). the digestive process of triglycerides begins with absorption in the intestine and it is circulated in the blood to the liver in the form of chylomicrons (exogenous). the people who consume foods which high in lipids will cause the appearance of cloudy serum such as milk or cream (lipemic). the liver has a role in the treatment of triglycerides. most triglycerides are stored as fat in adipose tissue. the function of triglycerides is to provide energy to the heart muscle and skeletal muscle as well as to reserve of energy that can produce a lot of atp. triglycerides are a major cause of arterial disease and are often compared to cholesterol through lipoprotein electrophoresis test. the increase in triglyceride concentration will cause hyperlipoproteinemia (4). according to a study conducted by bhagaskara, et.al (5), the mean triglyceride levels in patients with chronic renal failure was 163.26 mg/dl (it belongs to fairly high category). the results of this study are in accordance with raju’s research, et.al (5), which stated that chronic renal failure patients with dyslipidemia showed the characteristics such as elevated triglyceride levels and ldl cholesterol levels (5). hyperlipidemia or an increase in lipid profile contributes not only to heart disease, but also contributes to the progression of renal failure. glomerular filtration rate (gfr) is one of the physiological examinations of the kidneys in assessing excretory function by ina j med lab sci tech 2020; 2(2): 50-59 uswatun hasanah, et al. 5 2 calculating the filtrate released by the glomerular kidney. patients with gfr 60–89 ml/min/1.73m2 showed an increase in lipid profile. according to sengsuk(6) in the diabetes and obesity international journal, the determination of gfr estimation was based on the cockroft–gault formula which uses 3 variables (age, weight, and sex) with the normal value of egfr is 90 ml/min/1.73m2. circulating lipoproteins play a direct role in the pathogenesis of glomerulosclerosis and tubulointerstitial changes (6). based on the explanation above, the authors are interested in examining the correlation of triglyceride serum levels to estimated glomerular filtration rate (egfr) in patients with chronic renal failure at jemursari islamic hospital surabaya. materials and methods the research method used was descriptive experimental research with a cross–sectional study design. the equipment’s that used in this research were tms 24i premium automatic device (tokyo boeki medisys, japan), 10–1000 µl micropipette, blue micropipette tip, and eppendorf tube. the primary data in this study was the result of examination of serum triglyceride levels in patients with chronic renal failure. secondary data (serum creatinine levels, body weight, age, and sex) was obtained from medical records of patients with crf at jemursari hospital surabaya in february 2019. the study population was patients with chronic renal failure at the jemursari islamic hospital surabaya in the period of february 2019 who had fulfilled the retention criteria to be taken as research subjects. the sample used was fresh serum (fasting 12 hours), which was obtained from whole blood without anticoagulants that was centrifuged at 3.000 rpm for ±15 minutes at room temperature (12). the sample size was calculated using slovin formula and was resulted as many as 25 serum (8). the research sample was taken using purposive sampling technique which was based on certain considerations that had met the inclusion criteria set by the researcher. the sample inclusion criteria used in this study were: patients with chronic renal failure, men and women aged ≥40 years old, patients with chronic renal failure who were either have or have not undertaken hemodialysis, patients with chronic renal failure who perform creatinine examination and measurement of body weight in the same time, and patients with chronic renal failure who were undergoing hospitalization or outpatient care. the exclusion criteria for the study sample included: patients aged <40 years, patients with a diagnosis of crf+hypertension, crf+diabetes mellitus, crf+renal cysts, and crf+urosepsis. ina j med lab sci tech 2020; 2(2): 50-59 uswatun hasanah, et al. 5 3 the samples obtained were centrifuged at 3.000 rpm for ±15 minutes. the serum was separated into eppendorf tubes. a total of 25 fresh serum samples were analyzed for triglyceride levels using the gpo–pap enzymatic colorimetric method using the automatic tms 24i premium (tokyo boeki medisys, japan). determination of egfr value was calculated manually using the cockcroft–gault (c–g) formula: 𝐺𝐹𝑅 = (140 − 𝑎𝑔𝑒) × 𝑤𝑒𝑖𝑔ℎ𝑡(𝑘𝑔) × 0,85(𝑓𝑒𝑚𝑎𝑙𝑒) 72 × 𝑆𝑐𝑟 ( 𝑚𝑔 𝑑𝐿 ) scr : serum creatinine the results of the analysis of triglyceride levels were carried out to invistigate the correlation analysis of the egfr value. the variables in this study were age, gender, triglyceride level, and egfr of crf patients. the data were analyzed by univariate and bivariate. bivariate analysis (egfr and triglycerides) used the spearman correlation test with the data normality test. data is presented in textular, graphical and tabular forms. the statistical program used is ibm spss statistics 23.0. ethical clearance this research was approved by the research ethics committee of jemursari islamic hospital surabaya (005/kepk–rsi js/i/2019). involvement of respondents was based on a written agreement by filling a consent form. respondents may withdraw at any time if they do not agree or are not satisfied with any study procedures. results table 1 shows the distribution of samples by age and gender. the age of the men and women respondents was ≥40 years. about 75% of respondents were outpatient care and 25% of respondents were undergoing hospitalization. most of crf patients were 61–70 years old (44%), followed by 12% of patients belongs to 40–50–years age group, 36% of patients falls in 51–60 years age group and 8% of patients were categorized in 71–80 years age group. table 2 shows the distribution of samples according to laboratory examination results. the egfr results was calculated manually using the cockcroft–gault formula. the variable serum creatinine levels, body weight, age, and sex data were obtained from medical records of patients in february 2019. based on the table 2, there were 0% of crf patients with egfr values between 60–89 and ≥90 ml/min/1.73m2. a total of 5 stage iii patients (20%) have egfr values 30–59 ml/min/1.73m2. ten stage iv patients (40%) have egfr values 15–29 ml/min/1.73m2 and 10 stage v patients (40%) have egfr values <15 ml/min/1.73m2. the mean egfr value was 19.87 with standard deviation 11.41. all of respondents suffered from dyslipidemia. there were 64% of ina j med lab sci tech 2020; 2(2): 50-59 uswatun hasanah, et al. 5 4 respondents have triglyceride level <150 mg/dl. four patients (16%) have triglyceride level of 150–199 mg/dl and 5 patients (20%) have tg level 200–499 mg/dl. the mean triglyceride level was 146.68 with standard deviation 86.95. table 1. sample distribution by gender and age gender aged additional inspection hemodialysis (hd) % weight (kg) scr (mg/dl) male 40–50 53 5.54 non–hd 4% 51–60 61–78 1.49–18.28 non–hd 28% 61–70 61–78 1.76–9.93 non–hd 32% 71–80 68–74 1.79–2.48 non–hd 8% female 40–50 48–51 1.79–6.01 non–hd 8% 51–60 49–51 1.45–7.12 non–hd 8% 61–70 50–53 3.70–9.74 non–hd 12% 71–80 0 0 non–hd 0% table 2. sample distribution according to laboratory examination results laboratory test range mean std. dev. % egfr (ml/min/1.73m2) ≥90 19.87 11.41 0% 60–89 0% 30–59 20% 15–29 40% ≤15 40% triglyceride (mg/dl) <150 146.68 86.95 64% 150–199 16% 200–499 20% ≥500 0% figure 1 shows the data distributions of triglyceride levels in patient with crf. kolmogrov–smirnov normality test shows that the distribution was normal (𝜌<0.05). figure 2 shows the data distributions of egfr, which were normal (𝜌<0.05). table 3 shows the statistical distribution of triglyceride and egfr. it shows the results of examination of triglyceride levels (a minimum value was 33 mg/dl and a maximum value was 357 mg/dl. while, the results of the egfr value were a minimum value 4.75 ml/min/1.73m2 and a maximum value of 47,76 ml/min/1.73m2. the statistical correlations between serum triglyceride levels and egfr can be seen in table 4. the spearman–rho correlation test results obtained significance value 0.027 < α (0.05). ina j med lab sci tech 2020; 2(2): 50-59 uswatun hasanah, et al. 5 5 fig 1. distributions of triglyceride levels (ml/min/1.73m2) fig 2. distributions of egfr (mg/dl) table 2. min–max and median values of tg and egfr laboratory test value’s range min–max median tg 33–357 mg/dl 123 mg/dl egfr 4.75–47.76 ml/min/1.73m2 15.59 ml/min/1.73m2 table 3. statistical correlations egfr triglyceride levels correlation coefficient –.442* sig. (2–tailed) .027 n 25 0 1 2 3 4 5 6 0 100 200 300 400 0 1 2 3 4 5 .00 10.00 20.00 30.00 40.00 50.00 ina j med lab sci tech 2020; 2(2): 50-59 uswatun hasanah, et al. 5 6 discussion this study aims to determine the correlation between serum triglyceride levels and egfr values in subjects with chronic renal failure. the population of this study were all patients with chronic renal failure at jemursari islamic hospital in surabaya during february 2019. the number of patients with crf was 93 patients, but only 25 patients met the inclusion criteria. a total of 8 excluded patients were under 40 years old. moreover, 60 excluded patients had a diagnosis of crf with complications. the results showed that 40% of respondents was stage iv crf patients, 40% of respondents was stage v patients, followed by 20% of stage iii patients, 0% of respondents was stage i and ii patients. the degree of severity is obtained through the calculation of the egfr value with cockcroft–gault (c–g) formula in ml/min/1.73m2. gfr is parameter for assessing excretion function, by measuring the amount of filtrate produced by the kidney glomerulus (2). the lower value of gfr indicates the more severity of the kidney damage. table 1 shows that the most respondents (44%) with crf occur at 61–70 years of age. 12% of respondents was in the 40–50–year age group, 36% of respondents was in the 51– 60–year age group and 8% of them was in the 71–80–year age group. a decline in kidney function is a normal process as the age increase. the increasing age shows a progressive decrease in gfr and renal blood flow (rbf). the decrease occurs around 8 ml/min/1.73m2 per decade since the 40 years of age (9). of 25 samples, 18 patients (72%) were male, while 7 patients (28%) were. this is in accordance with the indonesian renal registry (9) data, which shown hemodialysis patients throughout indonesia were dominated by men in 2007–2012 (10). these results might be related to the incidence of crf, such as kidney stones, which also occur mostly in male. other studies show that the prevalence of kidney stones in men was 10,6% and those in women was 7,1%. based on table 2, that was 0% of crf patients who has egfr values between 60– 89 and ≥90 ml/min/1.73m2. a total of 5 stage iii patients (20%) has egfr values 30–59 ml/min/1.73m2. ten stage iv patients (40%) has egfr values of 15–29 ml/min/1.73m2 and 10 stage v patients (40%) has egfr values <15 ml/min/1.73m2. a decrease in gfr value can be affected by the increasing age and the cause of kidney damage itself. based on table 2, the results of examination of triglyceride levels were a minimum value of 33 mg/dl, a maximum value of 357 mg/dl, and a mean of 146,68 mg/dl. while the results of the egfr value obtained a minimum value 4,75 ml/min/1.73m2, a maximum value of 47,76 ml/min/1.73m2 with an average 19,86 ml/min/1.73m2. the results of the study at ina j med lab sci tech 2020; 2(2): 50-59 uswatun hasanah, et al. 5 7 the laboratory unit at jemursari islamic hospital surabaya stated that there was a relationship between triglyceride levels and egfr values in crf patients. the spearman–rho correlation test results has a significance value (0.027) < α (0.05) then h1 was accepted, which means that there was a statistically significant correlation between serum triglyceride levels and egfr values. the number of correlation coefficients is negative, which is –0.444 *. a negative sign indicates that the correlation of the two variables is not in the same direction (the type of relationship is not unidirectional). while the asterisk (*) shows the level of strength (closeness) of the relationship between the two variables (table 4). thus, it can be concluded that there was a strong and unidirectional significant correlation between the variables of triglyceride levels and egfr values. the higher the serum triglyceride level, the lower the egfr value. furthermore, 11 subjects had high triglyceride levels. this is in accordance with the research conducted by anggun (16) in kariadi hospital semarang, where 73 patients with chronic renal disease were accompanied by hypertriglyceride (52.9%). whereas, senge, et al. (17) in the kidney– hypertension polyclinic of rsup dr. r. d. kandou manado, found that there was a positive relationship between triglyceride levels and egfr in ckd patients (ρ = 0.030), meaning that the higher triglyceride levels, the higher the egfr value will be. this mismatch may be caused by several factors, such as a high creatinine diet, malnutrition, ketoacidosis and drugs (cimetidine, sulfa, trimethoprim) which results in decreased creatinine secretion which indicates as one of the determinants of the glomerular filtration rate (12). indonesian renal regulations (irr) data stated that in 2007 there were 6,862 people who suffered from chronic kidney failure and experienced an increase in 2012, amount to 28,782 people. chronic kidney failure is kidney damage that occurs for 3 months, based on pathological abnormalities or markers of kidney damage such as proteinuria. if there is no sign of kidney damage, the diagnosis of crf can be made if the glomerular filtration rate is less than 60 ml/min/1.73m2 (10). decreasing the gfr value is related to the severity. the national kidney foundation recommends that the egfr can be calculated according to serum creatinine. calculation of gfr based on serum creatinine, age, body size, gender, and race without the need for urinary creatinine levels using the cockcroft and gault equation (13). the classification of crf stage based on gfr values is as follows: stage i with a gfr value of 90 ml/min/1.73m2, stage ii with a gfr value of 60–89 ml/min /1,73m2, stage iii with a gfr value amounting to 30–59 ml/min/1.73m2, stage iv with a gfr value of 15–29 ina j med lab sci tech 2020; 2(2): 50-59 uswatun hasanah, et al. 5 8 ml/min/1.73m2, and stage v with a gfr of 15 ml/min/1,73 m2 (14). one risk factor that affects the progression of chronic renal failure is dyslipidemia. people with crf are at an increased risk of cardiovascular disease and have a higher prevalence of hyperlipidemia (or dyslipidemia) than the general population. dyslipidemia occurs due to abnormalities of lipid metabolism in patients with crf. most of patients (47%) with chronic kidney failure died from cardiovascular disease as the main cause (10). in fact, mild renal insufficiency has been shown to be associated with an increased rate of cardiovascular events (15). dyslipidemia characterized by increased triglyceride levels, total cholesterol and ldl cholesterol and decreased hdl cholesterol levels are often associated with crf and contribute to an increased risk of cardiovascular disease. various experimental studies have shown that lipid abnormalities can worsen the progression of kidney damage (17). the process of forming triglycerides is derived from food. the fatty foods we eat consist of triglycerides and cholesterol. in addition to cholesterol derived from food, in the intestine, there is also cholesterol from the liver which is excreted with bile into the small intestine. triglycerides and cholesterol in the small intestine will be absorbed into the intestinal mucosal enterocytes. triglycerides will be absorbed as free fatty acids while cholesterol remains cholesterol (4). inside the small intestine, free fatty acids will be converted into triglycerides, while cholesterol will be esterified to cholesterol esters and both together with phospholipid and apolipoproteins will form lipoprotein, known as chylomicron. this chylomicron will enter the lymph channels and eventually through the thoracic duct will enter the bloodstream. triglycerides in chylomicron will undergo hydrolysis by the lipoprotein lipase enzyme derived from endothelium into free fatty acids. free fatty acids can be stored as triglycerides again in fat tissue, but if they are present in large amounts, some of them will be taken by the liver to become a material for the formation of triglycerides in the liver (17). high triglyceride levels contribute to the process of atherosclerosis. poor circulation to most organs causes hypoxia and tissue injury, and stimulates inflammatory reactions in the walls of blood vessels. if atherosclerosis occurs, the blood supply to the kidneys will decrease and can cause gfr abnormalities and decreased kidney function (17). conclusions in conclusion, there was a significant negative correlation between serum triglyceride levels and the estimated value of the glomerular filtration rate (egfr) in patients with chronic renal failure at ina j med lab sci tech 2020; 2(2): 50-59 uswatun hasanah, et al. 5 9 jemursari islamic hospital, surabaya. the higher the triglyceride level, indicate the lower the egfr value. further research is needed to be done by considering the etiology and clinical picture of crf patients which are confounding factors in this study. conflict of interest the authors declare no conflict of interest. acknowledgment(s) appreciation goes to all respondents and management of laboratory unit, jemursari islamic hospital surabaya who gave the commitment that this study can be implemented successfully. references 1. rahardjo m, koendhori eb, setiawati y. guyton h. textbook of medical physiology. 11st ed. pennsylvania: elsevier saunders; 2006. 2. barret kim e, barman sm, boitano s, brooks hl. textbook of ganong medical phisiology. 24th ed. jakarta: egc; 2015. 3. ministry of health. riset kesehatan dasar (riskesdas 2013). jakarta:, badan penelitian dan pengembangan kesehatan; 2013. 4. nugraha g. lipid : classification, metabolism, atherosclerosis, and laboratory analysis. jakarta: trans info media; 2017. 5. bhagaskara, liana p, santoso b. correlation of lipid levels with urea&creatinine levels in patient with chronic renal failure in dr. mohammad hoesin palembang general hospital from january 1 to december 31, 2013. medical and health journal. 2015;2(2):223– 230. 6. bulum t, kolaric b, prkacin i, duvnjak l. total and ldl cholesterol are associated with glomerular filtration rate in normoalbuminuric type 1 diabetic patients. coll antropol. 2013;37(3):771–776. 7. sevilla c. research methods quezon city: rex printing company; 2007. 8. weinstein jr, anderson s. the aging kidney: physiologycal changes. adv chronic kidney dis. 2010;17(4):302–307. 9. indonesian nephrology association. 5th report of indonesian renal registry [update 2012; cited 2019 march 14]. available from: https://www.indonesianrenalregistry.org/ 10. scales c d, smith, a c, hanley j m, saigal, c.s. prevalence of kidney stone in the united states. neurology. 2012;62(1):160–165. 11. effendi i, markum h. supporting examination in kidney disease. jakarta: interna publishing; 2006. 12. verdiansah. examination of kidney function. ckd journal. 2016; 43(2): p. 148–154. 13. toussaint n. screening for early chronic kidney disease australia: saunder; 2012:30-55 14. chan cm. hyperlipidemia in chronic kidney disease. annals academy med. 2005;34(1):31– 35. 15. agarwal r. effects on statins on renal function. in mayo clinic proceedings. 2007;82(11):1381–1390. 16. wulandari ad, chasani s, ismail a. association of dyslipidemia with urea and blood creatinine levels in patient with diabetic nephropathy [skripsi]. semarang: universitas diponegoro; 2012. 17. senge ce, moeis es, sugeng. correlation of serum lipid levels with estimated values of glomerular filtration rate in chronic kidney disease. eclinic journal. 2017;5(1):44–50. 139 neutrophil lymphocyte ratio (nlr) in covid-19 patients receiving convalescent plasma therapy desyani ariza1, andi maya kesrianti1, tazya anggraini ruslan1 1d-iv of medical laboratory technology, megarezky university, makassar, indonesia correspondence: desyani ariza, jl. antang raya no.43, makassar, indonesia zip code: 90234 email: desyaniariza@yahoo.co.id received: october 30, 2021 revised: july 12, 2022 accepted: september 14, 2022 published: october 30, 2022 doi: 10.33086/ijmlst.v4i2.2460 abstract recently, a new rna virus from the coronaviridae family was discovered, known as sars-cov-2. this virus causes pneumonia and inflammation in the body. one of the laboratory tests used to see inflammation in the body is the neutrophil lymphocyte ratio, or often abbreviated as nlr. nlr is one of the markers of inflammation that can be used simply, efficiently, and reliably because of its high stability and sensitivity. higher nlr values tend to lead to a severe and poor prognosis, so this test can be done by monitoring patients with confirmed corona virus disease 2019 (covid-19). the purpose of this study was to determine the neutrophil lymphocyte’s ratio in covid-19 patients receiving convalescent plasma therapy. this research method is a quantitative observation using a descriptive approach. quantitative observational research is used to analyze data in the form of numbers from the results of laboratory tests. the results obtained from 17 research subjects, namely the nlr values before convalescent plasma therapy were obtained in as many as 6 patients (35%) with normal nlr values below 3.13 and nlr values are increasing above 3.13 in as many as 11 patients (65%). meanwhile, after convalescent plasma therapy, there were 8 patients (47%) with normal nlr below 3.13 and nlr values increasing above 3.13 as many as 9 patients (53%). this research concerns about changes in nlr values before and after convalescent plasma therapy, which before convalescent plasma therapy there were 6 patients with normal nlr values and after convalescent plasma therapy increased to 8 patients with normal nlr values which indicate a better good prognosis. keywords convalescent plasma therapy, corona virus disease 2019, neutrophil lymphocyte ratio. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2022; 4(2): 139–147 1 4 0 desyani ariza, et al introduction at the end of 2019, the whole world was shocked by the news of the spread of disease outbreaks such as pneumonia, the cause of which was not yet known. this disease was discovered for first time in china, specifically in the city of wuhan. patients infected with this pneumonia-like disease are mainly animal dealers at the huanan market located in the city of wuhan. on january 7, 2020, several researchers successfully identified the cause of the spreading pneumonia epidemic, the new type of novel coronavirus. over 800 confirmed cases, including health workers infected by covid-19 have been identified in wuhan. a number of other confirmed cases have been discovered in other provinces in china, south korea, thailand, japan and the united states (1). the world health organization (who) has officially named the disease covid -19 (2019 coronavirus disease) and the virus called sars-cov-2 (severe acute respiratory syndrome coronavirus-2) (2). initially the symptoms of covid-19 were not so severe and only occurred with fever and cough symptoms which then spontaneously recovered. in some individuals, it doesn't even produce symptoms (3). however, these symptoms can develop shortness of breath, dyspnea, pneumonia, which can cause acute respiratory distress syndrome (ards), kidney failure, coagulation dysfunction, multiple organ failure and can even cause death (4). the cytopathic effect of the virus and its ability to control the immune response determines how severe the infection is. a diminished and weak immune response can lead to rapid reproduction of the virus, resulting in tissue damage (5). a number of other treatments have been offered to treat patients with covid-19. one of them is a convalescent plasma therapy (tpk). convalescent plasma therapy is a therapy or treatment that uses plasma from donors who have rescued patients with covid-19 patients to covid-19 patients who are still suffering from the disease. previously, convalescent plasma therapy had been applied to treat diseases ebola-related illnesses and was a recommended by the who in 2014 (6). the characteristics of recipients of convalescent plasma therapy are covid-19 patients who have been confirmed by throat swabs using reverse transcription polymerase chain reaction (rt-pcr) examinations, show signs and symptoms towards the progression of illness, and have critical conditions. those who are entitled or able to donate or as donors are patients who have recovered from covid-19 infection, which has been proven by conducting a negative swab examination with rt-pcr. in addition, the donor has no signs and symptoms in the last 10 days. the donor also tests seronegative is for hepatitis b virus ina. j. med. lab. sci. tech. 2022; 4(2): 139–147 1 4 1 desyani ariza, et al (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv), and syphilis and has high polyclonal antibody titers (igm and igg), and neutralizing antibody titers >40 (7). in addition to treatment and therapy for patients with covid -19, several medical measures to detect sars-cov-2 have been widely extensively implemented. many initial tests are carried out so that people infected with this virus are immediately treated and isolated so that this virus does not spread to a large extent. abnormal laboratory results play an important role in classifying and assessing a patient’s prognosis so that early treatment is given, which is should lead to better outcomes. laboratory tests may describe inflammation and immune status, which may be useful as a possible predictors of the prognosis for patients with covid -19 (8). one of the parameters that are simple, rapid, and widely available is the hematological parameter that can determine the number and ratio of inflammatory cells (9). neutrophils are the most common type of leukocyte, the greatest number, and play a significant role in the body's response to inflammation (10). neutrophils have an affinity for immune complexes, as well as phagocytosis capacity (11). one of the inflammatory markers which may be used is the neutrophil lymphocyte ratio (nlr) (12). according to fuad et al., (13), an increase in the nlr was found in patients with confirmed covid-19. nlr provide an illustration of the balance between the innate immune response (neutrophils) and adaptive immunity (lymphocytes). the nlr value is calculated by dividing the absolute number of neutrophils cells by the absolute number of lymphocytes. the nlr value is also calculated from the percentage of neutrophils divided by the percentage of lymphocytes. the nlr calculation can be used as an important point in covid-19 patients care at intake. higher levels of nlr tend to have a severe and poor prognosis, requiring particular care and treatment (13). in the study above, we have not examined the value in covid-19 patients undergoing treatment or therapy, whether or not there are changes. therefore, in this study, we are examining the specificities of the neutrophil lymphocyte ratio value in patients with covid-19 undergoing convalescent plasma therapy. materials and methods the method used in this study is a quantitative observation using a descriptive approach to determine the value of neutrophil lymphocyte ratio (nlr) in patients with covid-19 undergoing convalescent plasma therapy. quantitative observational research aims to analyses and process data in the form of figures derived from the laboratory test results. the nlr value is calculated by dividing the neutrophils percentage by the ina. j. med. lab. sci. tech. 2022; 4(2): 139–147 1 4 2 desyani ariza, et al lymphocytes percentage (17) and then record the results of the data obtained. data obtained in the raw data via laboratory examinations, then processed and presented in tables and descriptions. tools and materials used in this study included total blood samples, edta vacuum tubes, spout, alcohol and cotton, and the use of the sysmex xn-10™ automated hematology analyzer as an examination tool. the method of the tool is flowcytometry. in this study, blood was collected twice, namely blood collection before therapy and blood collection after therapy. the collection of covid-19 healing plasma is based on routine plasma collection by plasmapheresis. plasma products are processed in the form of as fresh frozen plasma. the convalescent covid-19 plasma transfusion dose is approximately 4 -13 ml/kg of the recipient's body weight. the abo type of the transfused convalescent plasma is consistent with the patient's abo type. in addition, the convalescent plasma is intersected with the patient's red blood cells to ensure compatibility. a conventional plasma transfusion is given at approximately 10 ml during the first 15 minutes, which is then increased to approximately 100 ml per hour with careful monitoring (14). the study population in this study were covid-19 patients confirmed by a physician. patients were subjected to routine blood sampling prior to and following convalescent plasma therapy to see the value of the neutrophil-lymphocyte ratio. the interval between blood collection before and after treatment was 1-2 days, but there was no measurement of the amount of treatment given to patients who received convalescent plasma donors. this research was completed in 2021 in the clinical laboratory of rsup. dr. wahidin sudirohusodo, makassar, indonesia. this research was approved by the ethics committee of the faculty of medicine, hasanuddin university and bears the letter number 685/un4.6.4.5.31/pp36l 2021. results clinical laboratory tests are conducted to confirm the diagnosis of patients with confirmed covid-19. among these are performed by out by various examination methods, namely rapid diagnostic test (rdt) antigen, rdt antibodies, polymerase chain reaction (pcr) and culture. in the diagnosis covid-19, the rt-pcr exam is the gold standard recommended due to its high sensitivity and specificity (15). the subjects of this study were mainly patients with covid-19 with moderate to severe symptoms. these patients were treated at the rsup dr. wahidin sudirohusodo. 14 of the 17 patients who had convalescent plasma therapy made a full recovery and were permitted to continue receiving ambulatory care (26). ina. j. med. lab. sci. tech. 2022; 4(2): 139–147 1 4 3 desyani ariza, et al according to the study results, 17 subjects obtained samples of covid-19 patients that met the inclusion criteria. table 1 presents the general characteristic of the subjects after data processing. table 1. patient characteristics (n=17) characteristics criteria n % age (years old) 12-25 26-45 46-65 >65 1 2 8 6 5.9 11.76 47.05 35.29 gender man woman 11 6 64.70 35.30 total 17 100% table 1 shows the age characteristics and obtained by the age of 12-25 years there is one patient (5.9%). subjects at the age of 2645 years there are two patients (11.76%), at the age of 46-65 years there are eight patients (47.05%) and aged over 65 years there are 6 patients (35.29%). meanwhile, gender characteristics revealed that among males there were 11 patients (64.70%) and among females there were 6 patients (35.30%). table 2 shows the normal pre-treatment nlr values lower than 3.13 in 6 patients (35%) and increasing above 3.13 before therapy in 11 patients (65%). whereas the nlr value after therapy was obtained that was normal below 3.13 on 8 patients (47%) and increased by 9 patients (53%). table 2. overview of nlr values before and after convalescent plasma therapy check indicator before therapy after therapy n % n % nlr ≤ 3,13 6 35% 8 47% ≥ 3,13 11 65% 9 53% total 17 100% 17 100% discussion an investigation was conducted at rsup dr. wahidin sudirohusodo on 17 investigate subjects. these subjects have undergone an nlr examination to see a description of the nlr value in patients with covid-19 receiving convalescent plasma therapy using the sysmex xn-10™ automated hematology analyzer. the most amount of subject is between age of 46-65 years with up to eight patients (47.05%). as a result, we can infer that growing older also contributes to an increase in the number of covid-19 instances that have been confirmed. this study is in line with research undertaken by liu et al., (18), where the age of people symptomatic covid-19 is older than 40 years old (84%). this is due to the relationship between age and immunity level, where older people are at greater risk of infection than body immunity decreases (18). with regard of gender criteria, 11 subjects (64.70%) had more covid-19 infection than women. this study is ina. j. med. lab. sci. tech. 2022; 4(2): 139–147 1 4 4 desyani ariza, et al consistent with research of liu et al., (18) which lead us to believe that man with covid-19 are more infected than women (18). this is due to the fact that men are known to have superior ace2 expression. it is related to sex hormones that cause men to be more likely to be infected more easily. furthermore, men's daily activities are more predominant when they work outside for long hours. however, in fact, both of men and women have a high risk of contracting covid-19 when performing day to day activities without implementing the health protocols established by the government. in table 2, pre-treatment nlr results for 6 patients with nlr values are normal or below 3.13. as many as 11 patients indicated that nlr values increased above 3.13 posttherapy. currently, there is a change for 8 patients with normal nlr values and 9 patients with higher nlr values. a high nlr value describes the prognosis of the disease in the direction of severe and poor behavior in order for it to require more intensive treatment. additionally, the value of the nlr can serve as a simple test to help determine the severity of the illness and predict the outcome of covid-19. the results of table 2 demonstrate that the normal nlr values are higher therapy, which means that there is a change. this study is in line with the research of maulana (19) which indicates that convalescent plasma therapy has potential to reduce mortality in covid19 patients and help reduce hospitalizations. it also indicates that there is a change or a better prognosis after plasma therapy. however, this requires even more research with larger research subjects to see changes that occur in the nlr value or other examination parameters performed in patients with covid-19 receiving convalescent plasma therapy. convalescent plasma therapy has previously been applied to treat diseases caused by virus, such as 1918 influenza, avian influenza a (h5n1), ebola, and other viral infection. this therapy has also been used in hong kong for the outbreak of sars-cov-1 in 2003, h1n1 in 2009-2010 and mers-cov in 2012. convalescent plasma therapy was also performed in wuhan, china and in new york, united states (us). the us food and drug administration (fda) has issued a decision allowing convalescent plasma therapy to for patients with covid-19 (20). although it still requires further research with larger research subjects, the results of the study indicated that there was a shift in the nlr value to a better prognosis. what is known if the normal nlr value means describe the successful completion of therapy. because the nlr value describes how severe the inflammation is in the body. in severe infections or systemic inflammation, the neutrophil/lymphocyte ratio will increase so that it may be used for ina. j. med. lab. sci. tech. 2022; 4(2): 139–147 1 4 5 desyani ariza, et al clinical assessment of patients with systemic inflammation. this description shows that in severe systemic inflammation there is a greater increase in neutrophil lymphocytes ratio compared with mild systemic inflammation conditions (21). the sample of subjects in this study were covid-19 patients with severe symptoms and hospitalized. this is indicated that the nlr value was increasing. from this finding, it may be indicated that the body's immune system reacts by increasing the neutrophillymphocyte ratio. this ratio is higher for severe inflammation than for mild systemic inflammation. it's just that there aren’t any other studies that compare the nlr value in patients with covid-19 with various categories of symptom. an increased number of neutrophils compared with lymphocytes ratio is one of the immune system’s physiological responses to systemic inflammation (22). this is due to changes in the dynamics and regulation of apoptosis in the systemic inflammatory versus non-inflammatory condition (21). the delay in the mechanism of neutrophil apoptosis will have an impact on the prolonging the function of neutrophil in the inflammatory process. in addition, it also delays the production of toxic metabolites. on the other hand, increased lymphocyte apoptosis may have an impact on reducing inflammatory effectors and causing immunosuppression (23,24). activated neutrophils and inflammatory cytokines will damage the function of tissues and organs as a result of the effects of the toxic metabolites produced. in addition, the process of lymphocyte apoptosis can lead to immunosuppression of the adaptive immune system, making it more susceptible to infection which will trigger a further systemic inflammatory response (25) . finally, the occurrence of high neutrophil counts and decreased lymphocyte counts will increase the absolute ratio of neutrophils to lymphocytes relative to patients without a systemic inflammatory response. an rise in the neutrophil-lymphocyte ratio will raise the patient's risk of morbidity and mortality, which are characterized by organ damage and dysfunction (21). this study is strengthening the theoretical basis and other studies that the ratio of neutrophil to lymphocyte or nlr changes when appropriate therapy or treatment is carried out. it also indicates a change or improved prognosis after convalescent plasma therapy. however, this requires even more research with larger research subjects to see changes that occur in the nlr value or other examination parameters carried out in covid-19 patients receiving convalescent plasma therapy. for information other than the laboratory tests results from 17 patients undergoing convalescent plasma therapy, 14 patients ina. j. med. lab. sci. tech. 2022; 4(2): 139–147 1 4 6 desyani ariza, et al were able to recover and were allowed to be outpatients (26). conclusions this study emphasizes how secure nlr treatment is. there is a change to the nlr values before and after convalescent plasma therapy. in pre-treatment, up to 6 patients with normal nlr values. afterwards, after therapy grew to 8 patients with normal nlr values. this suggests a positive prognosis. author contributions desyani ariza: processing data, conceptualizing, writing and submitting articles, supervision, administration. andi maya kesrianti: sample and data collection, data verification and validation. tazya anggraini: article revision, field review, reviewing and editing articles. acknowledgments sincere gratitude is expressed to all patients and donors who participated in this study. conflict of interest according to the author there is no conflict. references 1. huang c, wang y, li x, ren l, zhao j, hu y, zhang l, fan g, xu j, gu x. articles clinical features of patients infected with 2019 novel coronavirus in wuhan, china. 2020;497–506. https://doi.org/10.1016/s0140-6736(20)30183-5 2. levani y, prasetya ad, ramadhani sm. coronavirus disease 2019 (covid-19): pathogenesis, clinical manifestations and treatment options. j med heal [internet]. 2021;17(1):44–57. https://doi.org/10.26714/magnamed.6.1.2019.3137. 3. mardewi iga, yustiani nt. gambaran hasil laboratorium pasien covid-19 di rsud bali mandara: sebuah studi pendahuluan [overview of laboratory results of covid-19 patients at bali mandara hospital: a preliminary study]. intisari sains medis. 2021;12:374–8. https://doi.org/10.15562/ism.v12i2.1000 4. harahap rj. clinical characteristics of coronavirus disease 2019. j prof nurse res. 2020;2(3):317– 24.https://doi.org/10.37287/jppp.v2i3.145. 5. susilo a, rumende cm, pitoyo cw, santoso wd, yulianti m, sinto r, singh g, nainggolan l, nelwan ej, khie l, widhani a, wijaya e, wicaksana b, maksum m, annisa f, jasirwan om, yunihastuti e, penanganan t, new i, cipto r. coronavirus disease 2019 : tinjauan literatur terkini coronavirus disease 2019 [review of current literatures] . 2020;7(1):45–67. https://doi.org/10.7454/jpdi.v7i1.415. 6. monica th, triyono t, harly pr. management of convalescent plasma therapy for covid-19 patients third edition book. surabaya: media partners persada; 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pmid: 25214990. 24. fabiano ps vn. cell death during sepsis : integration of disintegration in the inflammatory response to overwhelming infection. apoptosis. 2009;14:509–21. https://doi.org/10.1007/s10495009-0320-3. 25. jacqueline pbc. an overview of the immune system. lancet. 2001;357:1777–90. https://doi.org/10.1016/s0140-6736(00)04904-7. 26. ariza, desyani, ferdhyanti au. routine hematological overview of covid-19 infected patients before and after convalescent plasma therapy. j med lab sci technol [internet]. 2021;4(2):93–8. https://doi.org/10.21070/medicra.v4i2.1615. 80 the effect of beetroot (beta vulgaris l.) juice on cholinesterase activity in farmers exposed to organophosphate pesticides devyana dyah wulandari1, andreas putro ragil santoso1, devyani diah wulansari 2 1department of medical laboratory technology, faculty of health, nahdlatul ulama university of surabaya, indonesia 2department of pharmacy, faculty of pharmacy, university of surabaya, indonesia correspondence: devyana dyah wulandari, jl. jemursari no. 51-57, surabaya, east java, indonesia zip code : 60237 email: devyanadyah@unusa.ac.id received: june 20, 2019 revised: june 27, 2019 accepted: october 6, 2019 abstract a decrease in cholinesterase (che) enzyme activity is an indicator of pesticide poisoning, especially organophosphate pesticides. the che activity reduction will result in an increase in the amount of acetylcholine and will bound to muscarinic and nicotinic receptors in the central and peripheral nervous system which can lead to the onset of alzheimer's disease. however, antioxidant products can slow the progression of alzheimer's disease by protecting neurons from oxidative stress. red beetroot (beta vulgaris l.) contains betalains and phenolic compounds which act as an antioxidants that are capable of preventing such illness. this study aims to prove the concept that antioxidant compounds in red beetroot juice can increase the che activity in subjects exposed to organophosphate pesticides. this research is an observational study with an experimental approach. this research used 25 farmers who were exposed to organophosphate pesticides as respondents. they were given 500 ml of beetroot juice twice a day for 2 consecutive months. cholinesterase levels were measured before and after consuming red beet juice. measurement of cholinesterase levels was carried out using a kinetic photometric test. this method was developed based on recommendations from the german society of clinical chemistry (dgkc). the results showed that the average of che (u/l) level before treatment was 8.102 and 8.380 after treatment with a p value 0,62. it can be concluded that there was an increase in the level of cholinesterase but it was not statistically significant. this may be caused by a different response to activity of cholinesterase after consuming beetroot juice in each subjects, age difference, smoking habits, and personal protective equipment. keywords beetroot, cholinesterase, organophophate, pesticide ina j med lab sci tech 2019; 1(2): 80-87 devyana dyah wulandari, et al. 8 1 introduction pesticides are poisons intentionally made by humans to kill plant-disturbing organisms and disease-spreading insects (1). the high usage of pesticides are detrimental to health, particularly for the workers who contact with pesticides and the impacted population. the health risk of pesticides exposure in the workers may due to improper handling of that poisoning substance. besides, the population are also vulnerable to pesticides poisoning due to the contamination of such substance that enter the food chain, either because of ingestion and inhalation of pesticides residue or direct contact of pesticides through the skin. every day, a thousands of farmers working on agriculture land are poisoned by pesticides. it is estimated that millions of people involved in agriculture sector suffer from pesticide poisoning every year. based on data from the world health organization (who) and the united nations environment program (unep), 1-5 million cases of poisoning occur in people working in the agricultural sector. in addition, the population around the agricultural sites are very at risk of being exposed to pesticides either through air, soil or contaminated water. moreover, organophosphate pesticides are the most toxic insecticides that is usually used in the consumer goods so that the population will also be impacted. pesticides exposure in a small amount can cause death, but it takes more than a few mg to cause death in adults. organophosphate will inhibit the action of pseudokholinesterase in plasma and cholinesterase in red blood cells and in synapses. based on previous research, exposure to pesticides can cause cancer, alzheimer's disease and even birth defects. pesticides also have the potential to damage the nervous system, reproductive system, and endocrine system (2). in a nutrients journal have reported that betalains has high antioxidant and anti-inflammatory abilities in vitro and in vivo tests. this has sparked interest in researchers to examine the role of beets in the field of clinical pathology in several diseases characterized by oxidative stress and chronic inflammation such as liver disease, arthritis and even cancer (3). red beetroot (beta vulgaris l.) is preferred as a rich source of betacyanin, including a group of fruits that have reddish to purple betalains pigments consisting mainly of betanin and isobetanins. betalains and phenolic compounds present in red beetroot have been reported to increase resistance in low density lipoprotein (ldl) to oxidize and to prevent cancer and cardiovascular disease by reducing the oxidative effects of free radicals on lipids (4). free radicals or reactive oxygen species (ros) play many important roles in physiology and pathological processes. oxidative stress is a biological phenomenon that results from a biochemical imbalance ina j med lab sci tech 2019; 1(2): 80-87 devyana dyah wulandari, et al. 8 2 between the formation and cleansing / buffering of free radicals (5). workers in the agrochemicals and biological fertilizers company are the most often exposed to pesticides, especially organophosphate pesticides. based on the results of the situation analysis, many complaints arose from the operators who indicated that there were symptoms of poisoning. while the temporary solution that has been done is to move positions in other parts for a while and there is no way to minimize the symptoms of poisoning that arise. whereas, from the results of previous studies, (5) explained that natural antioxidant products can slow the progression of alzheimer's disease by protecting neurons from oxidative stress. over the past few years, oxidative stress has been described as a co-lethal factor in cases of poisoning caused by organophosphate pesticides. many studies emphasize the role oxidative stress in cases of organophosphate poisoning. previous studies have shown that the administration of date palm extract is able to restore atrazine poisoning, a herbicides that cause oxidative damage to sperm parameters, reduce testicular antioxidant enzymes, gsh, sod, cat, and gpx levels in mice (6). another studies found the presence of oxidative stress and inhibition of acetylcholinesterase on farmers exposed to organophosphate pesticides (7). antioxidants have been suggested as organophosphate antidotes. meanwhile, nurulain et al. did not find that giving glutathione (gsh) and nac do not provide a survival effect on acute exposure induced paraoxon (a very dangerous type of op pesticide) in wistar mice. this shows that antioxidants have no role in the toxicity of acute phase ii pesticides (8). several studies have shown that the administration of antioxidant activity compounds can minimize pesticide poisoning. thus the researchers want to examine whether red beetroot juice (beta vulgaris l.) that is proven to have antioxidant activity is able to minimize pesticide poisoning through increased cholinesterase activity. materials and methods the equipments that used in this research were terumo volume 3 ml syringe, edta vacutainer tube, torniquet, cotton, centrifuge brand: tomy, type: mx-105, genesys ™ 10s uv-vis spectrophotometer thermo fisher scientific, thermometer, dropper, dropper measure, ice box, ice pack. meanwhile, the materials used were 96% alcohol, distilled water, deionized aquades, phosphate buffer ph 7.4, thiol 5.5'-dithio-bis (2-nitro benzoic acid) {dtnb} 0.33 mm, acetylcholine 5 mm. red beetroot juice was made by mixing 150 mg of red beetroot in 1 l of water. afterward, it was blended using a blender (servings per person), divided into 2 servings ina j med lab sci tech 2019; 1(2): 80-87 devyana dyah wulandari, et al. 8 3 to drink twice a day (500 ml/serving), at the morning and at the evening (9). they consume this juice every day for 2 months. blood sampling was done by damming with tourniquet. alcoholic cotton was applied in a circular manner as an antiseptic and was allowed to dry. the syringe needle was inserted in positions 30-45°c until a sign of blood comes out of the needle then the syringe was drawn to the limit of 2 with a blood volume of 2 ml. the needle was taken from the vein and the blood was inserted into the vacuum tube containing the edta and was homogenized (10). separation of erythrocytes and plasma was done by inserting the blood that has been taken into the vacuum container and it was centrifuged using centrifuge brand: tomy, type: mx-105 for 10 minutes at 4000 rpm. erythrocytes that have been separated from plasma were taken and placed in test tubes (11). measurement of che was done by taking erythrocytes that have been separated from plasma and it was then added with deionized water to the same as the initial volume of blood. afterwards, it was diluted 60 times using a buffer (phosphate buffer 0.1 m, ph = 7.4), and it was frozen for hemolysis. the mixture was thawed again and diluted with buffer 9 times. after that, it was added with thiol reagent 5.5'-dithio-bis (2-nitro benzoic acid) {dtnb} (dtnb concentration 0.33 mm) and was allowed to stand for 10 minutes. then the mixture was added to the atch substrate (atch concentration of 1.0 mm) and was read with absorbance of 412 nm using a uv-vis genesys ™ 10s spectrophotometer thermo fisher scientific by using a blank containing erythrocyte hemolysis in a buffer (12). this research is an observational study with an experimental approach since the data was taken through laboratory tests. additionally, the participant was interviewed by using a questionnaire which ask data on age, length of work and symptoms felt. the population and sample in this study were male farmers in the mojokerto region who were exposed to organophosphate pesticides. the inclusion criteria were 30 years old and above, and has been working for at least 1 year. the data was analyzed by a paired t-test using spss 16 software. statistical tests were performed at 95% confidence level and a significant difference if p <0.05. the participants were given an informed consent and the number of ethical clearance certificate is 091/ke/vii/2019. results the study population (respondents) were farmers exposed to organophosphate pesticides. all of the respondents were male as many as 25 people with different working periods as shown in table 1. respondents were exposed to organophosphate pesticides ina j med lab sci tech 2019; 1(2): 80-87 devyana dyah wulandari, et al. 8 4 daily because they work as farmers on an agricultural land. the parameters of the examination carried out were the levels of cholinesterase. cholinesterase levels were measured before and after administration of red beet juice. the results of the examination of cholinesterase levels can be seen in table 1. the normal range of cholinesterase levels ranged from 4,620 11,500 u / l. the higher levels of cholinesterase the better because cholinesterase has a function to convert acetylcholine into acetate and choline that can prevent the accumulation of acetylcholine. conversely, if the level of cholinesterase is lower than 4,620 u / l, it will show the symptoms of poisoning due to the accumulation of acetylcholine. table 1. results of cholinesterase level before and after treatment patient code age (years old) che (u/l) level before treatment che (u/l) level after treatment normal value of che (u/l) p value 1 50 11.340 9.405 4.620 – 11.500 0.620 2 46 3.967 10.479 3 62 5.670 6.436 4 54 5.666 6.830 5 54 9.371 8.276 6 50 8.759 7.759 7 53 7.302 9.915 8 57 9.775 5.770 9 63 8.718 4.547 10 50 8.441 7.357 11 60 7.446 9.047 12 41 6.655 9.342 13 38 4.351 7.380 14 63 11.284 9.685 15 60 9.927 9.611 16 60 7.133 9.954 17 64 6.673 8.013 18 32 8.198 7.995 19 64 11.086 6.007 20 52 6.840 10.999 21 65 9.410 10.035 22 35 10.583 7.428 23 60 6.542 8.723 24 61 6.121 7.989 25 50 11.287 10.516 average 8.102 8.380 *u/l = unit per liters ina j med lab sci tech 2019; 1(2): 80-87 devyana dyah wulandari, et al. 8 5 the statistical analysis of the effect of red beet juice consumption on cholinesterase levels can be seen in table 1. the results show that consumption of red beet juice on cholinesterase levels was not statistically significant with a p value 0.620. it means that consumption beetroot juice was not able to increase cholinesterase levels significantly. discussion farmers are one of the subjects who are vulnerable to pesticides. in a plantation area in mojokerto, east java, the farmers use organophosphate pesticide. organophosphate pesticides are the most toxic pesticides than the other groups. this pesticide is able to inhibit the performance of the cholinesterase enzyme. the enzyme normally hydrolyzes acetylcholine into acetate and cholin. when the enzyme is inhibited, it causes the amount of acetylcholine to increase and binds to the muscarinic and nicotinic receptors of the central and peripheral nervous system. therefore, this causes symptoms of poisoning that affect all parts of the body. there are some people who have shown the indications of poisoning symptoms marked by a decrease level of the enzyme cholinesterase, characterized by complaints such as dizziness and nausea. it was also found in the farmers working on agricultural land in mojokerto. however, symptoms of organophosphate poisoning vary greatly. each symptom is very dependent on the presence of persistent acetylcholine or depression stimulation followed by central or peripheral nerve stimulation. early symptoms such as salivation, lacrimation, urination and diarrhea (slud) occur in acute organophosphate poisoning due to stimulation of muscarinic receptors so that the acetyl kholin content in the blood increases in the eyes and smooth muscle (13). organophosphate is a type of pesticide that is most widely used in agriculture. cholinesterase levels are known as biomarkers in patients exposed to organophosphate pesticides. in the results of the study in table 1 showed that some cholinesterase levels are still in the normal range, but there are some others that have che levels close to the lower limit (symptoms of poisoning), as in patient coded by number 2 and number 13. based on the results of statistical calculations obtained, the p value was 0.62 which indicates that consumption of red beet juice did not significantly influence the levels of cholinesterase. it may be caused by a different habits and personal protective equipment. it can be seen in patient coded by number 2, he was experienced a very significant increase in cholinesterase levels with an increase of 6512 che (u/l). furthermore, the patient coded by number 11 increase in cholinesterase levels by 2613 che (u/l), the patient coded by the number 12 ina j med lab sci tech 2019; 1(2): 80-87 devyana dyah wulandari, et al. 8 6 increase in cholinesterase levels by 2687 che (u/l), the patient coded by the number 13 with an increase 3029 che (u/l). lastly, the patient coded by the number 20 increase in cholinesterase levels by 4159 che (u/l). from this data, there was an increase in the level of cholinesterase but it was not statistically significant. one study suggested that the nitrate content was not the only compound contained in red beetroot that were proposed to have beneficial effects on health and disease. beetroot is a rich source of phytochemical compounds including ascorbic acid, carotenoids, phenolic acids and flavonoids. beets are also one of the few vegetables that contains a group of highly bioactive pigments known as betalains. members of the betalains family are categorized as red-purple betacyanin pigments or yellow-orange betaxanthin pigments. based on other studies, it mentioned that phenolic compounds can react with residues of active amino acids found on the active side of che through hydrogen bonds, hydrophobic interactions and π-π interactions. the results show that hydroxyl groups in the structure of phenolic compounds are believed to increase cholinesterase activity (14). this is the underlying mechanism why antioxidant in beetroot can increase cholinesterase level. this supports the results of this study on several patient codes that indicate an increase in cholinesterase activity. however, there are several other factors that can influence the level of cholinesterase, including nutritional status, health conditions, age, sex, ambient temperature, smoking habits, and habits of using personal protective equipment (ppe) (15). in the results of this study, it was note that each respondent has a different response after consuming red beet juice on cholinesterase activity. this can be due to the influence of age, smoking habits and habits of using ppe that are different from each respondent. unfortunately, this research did not examine the effect of age, smoking habits and habits on cholinesterase levels, but it could be that these factors are the cause of not being statistically significant results. this research is preliminary data that can be used as a basis for further research to determine the efficacy of red beet juice in increasing levels of cholinesterase by involving age and habits. conclusions based on the results of the study concluded that there was an increase in the level of cholinesterase but it was not statistically significant. the increasing of cholinesterase levels can be caused by antioxidant properties of red beetroot juice (beta vulgaris l.) so it can be minimize the pesticide poisoning through increased cholinesterase activity. in further studies it is recommended to increase the dose, duration ina j med lab sci tech 2019; 1(2): 80-87 devyana dyah wulandari, et al. 8 7 of treatment and involve factors of age and habits. acknowladgements thank you to the ministry of higher education research for providing funding for this research, thank you to the nahdlatul ulama surabaya university for providing easy access to research facilities and thanks to all respondents who have helped this research. conflict of interest there are no conflicts of interest. references 1. soemirat juli, 2003. environmental toxicology, gadjah mada university press, bandung. 2. sarwar, muhammad. the dangers of pesticides associated with public health and preventing of the risks. international journal of bioinformatics and biomedical engineering. 2015.vol. 1, no. 2, pp. 130-136. 3. clifford, tom. glyn howatson, daniel j. west and emma j. stevenson. the potential benefits of red beetroot supplementation in health and disease. nutrients 7.2015, 2801-2822 4. guldiken burcu, gamze toydemir, kubra nur memis, sena okur, dilek boyacioglu and esra capanoglu. home-processed red beetroot ( beta vulgaris l.) products: changes in antioxidant properties and bioaccessibility. int. j. mol. sci. 2016, 17, 858. 5. pervin, mehnaz., md. abul hasnat, yoon mi lee, da hye kim, jeong eun jo and beong ou lim. antioxidant activity and acetylcholinesterase inhibition. of grape skin anthocyanin (gsa). molecules. 2014, 19, 9403-9418 6. ganga, u.k, hemalatha c., kishori b. protective role of date fruit extract against chlorpyrifosinduced reproductive toxicity in albino male rats. international journal of green pharmacy. 2018 ,12 (1). 7. bayrami m, hashemi t, malekirad aa, ashayeri h, faraji f, abdollahi m. electroencephalogram, cognitive state, psychological disorders, clinical symptom, and oxidative stress in horticulture farmers exposed to organophosphate pesticides. toxicol indl health 2012;28:90-6 8. nurulain, s.m, szegi, p., tekes k., and syed nh naqvi. antioxidants in organophosphorus compounds poisoning. arh hig rada toksikol 2013;64:169-177 9. maria, rita a. porto, vivian s. okina, tatiana c. pimentel and sandra helena prudencio. physicochemical stability, antioxidant activity, and acceptance of beet and orange mixed juice during refrigerated storage. beverages,2017,3, 36 10. eflm (european federation of clinical chemistry and laboratory medicine) recommendation for venous blood sampling, 2017 11. standard operating procedure for collecting, proccessing, and storage of blood samples, 2017 12. jamshhidzade, akram, hossein nicknahad, mohammadi-bardbori a, talati m.. comparative measurement of serum acetyl cholinesterase enzyme using three different methods. iranian journal of toxicology, 2009. volume 2 , no.4 13. aroonvilairat, soraya., wannapa kespichayawattana, thiwaree sornprachum, papada chaisuriya, taweeratana siwadune and kavi ratanabanangkoon.. effect of pesticide exposure on immunological, hematological and biochemical parameters in thai orchid farmersa cross-sectional study. int. j. environ. res. public health.2015, 12, 5846-5861 14. szwajgier. d. anticholinesterase activity of selected phenolic acids and flavonoids – interaction testing in model solutions. annals of agricultural and environmental medicine 2015, vol 22, no 4, 690–694. 15. purba, ig. analysis of factors related to cholesteresterase levels in fertile age women in agricultural areas. universitas diponegoro; 2009. tesis 65 n-terminal pro-brain natriuretic peptide (nt-probnp) in stage 1 and stage 2 hypertension patients supriati wila djami1 1department of health analyst, health ministry polytechnic of kupang, indonesia correspondence: supriati wila djami, jl.farmasi, liliba, kupang, east nusa tenggara, indonesia zip code : 85111 email: djamiwila@gmail.com received: july 10, 2019 revised: july 30, 2019 accepted: october 7, 2019 abstract the increased levels of nt-probnp in the blood occur when heart function, especially the left ventricular muscle chambers of the heart increases. therefore, nt-probnp is used as a biomarker to detect heart failure.the level of n terminal – pro brain natriuetic peptide was independently associated with an increased risk of hypertension. this study aimed to determine the difference of nt-probnp serum levels and the correlation between the levels of nt-probnp in patients with stage 1 and stage 2 hypertension. this research was conducted at rsup dr. wahidin sudirohusodo in august september 2018. the study used a crosssectional design with the total of 72 hypertensive patients, who had met the inclusive criteria. nt-probnp levels were measured using the elisa (enzyme linked immunosorbent assay) method. the collected data was processed using mann whitney different test and spearman's rho correlation test. the study results indicated that the level of nt-probnp in the hypertensive patients with stage 2 was higher and significantly different (p = <0.001) compared to stage 1 hypertensive patients. ntprobnp levels were higher in the hypertensive group of >6 years than in the hypertensive group <6 years. there were significant differences between the two groups statistically (p=0.010). it can be conclude that there is a significant difference in the levels of nt-probnp with a degree of hypertension where nt-probnp levels were higher in patients with stage 2 hypertension compared to stage 1 hypertension, although there was not statistically significant correlation between levels of nt-probnp with hypertension degree. further research was needed to determine the relationship of nt-probnp levels with the degree of hypertension, which can confirm the diagnosis, especially in patients with hypertension. also, it is suggested to consider the accuracy of the data length of a patient suffering from undiagnosed hypertension. keywords nt-probnp, hypertension, degree of hypertension, duration of hypertension ina j med lab sci tech 2019; 1(2): 65-73 supriati wila djami, et al. 6 6 introduction hypertension or high blood pressure is a global health problem, including in indonesia because of the high prevalence, although different in various state. hypertension does not give complaints and typical symptoms so many people do not realize it since it was dubbed as the silent killer (1). hypertension is defined as someone who had a systolic blood pressure ≥ 140 mmhg or diastolic blood pressure ≥ 90 mmhg, on repeated examinations (2). hypertension is one of the most common diseases found in primary medical practice which is also a risk factor of myocardial infarction, stroke, acute kidney failure, and death (3). according to nhlbi (national heart, lung, and blood institute), 1 in 3 (patients) suffers from hypertension. in a metaanalysis study that included 61 prospective international studies in 1 million patients, which was equivalent to 12.7 million personyears, it was found that a decrease in mean systolic blood pressure of 2 mmhg could reduce the risk of mortality from ischemic heart disease by 7% and reduce the risk of stroke mortality by 10%. achieving the target of reducing blood pressure is very important to reduce cardiovascular events in hypertensive patients (4). the health profile of south sulawesi province in 2016 showed the prevalence of hypertension in south sulawesi province obtained through population blood pressure measurements at the age of > 18 years with a total of 142,571 cases (5). based on data from the makassar city health profile in 2016, hypertension was included in the top 10 cases of the highest cause of death in makassar (6). primary hypertension is hypertension that does not have a known cause, faced by the majority (90%) of high blood pressure patients who come to the practice. primary (essential) hypertension has several factors at play, namely hormonal factors in the renin angiotensin aldosterone system, autonomic nervous system, peripheral resistance, salt intake (naci), and others. secondary hypertension can be determined as the cause which is generally experienced by a small percentage of patients (10%) with high blood pressure. t he most common cause of secondary hypertension is chronic kidney disease. other causes are obstructive sleep apnea, primary aldosteronism, renal artery stenosis, cushing's syndrome, pheochromocytoma, hyperparathyroidism, coarctation of aorta, hypo and hyperthyroidism and drugs (1). complications of hypertension may affect various organs such as the heart (ischemic heart disease, left ventricular hypertrophy, heart failure), brain (stroke), kidney (renal failure), the eyes (retinopathy) also peripheral arteries (intermittent claudication) . the damage of these organs depend in high blood pressure patients and the duration of the high blood pressure is not ina j med lab sci tech 2019; 1(2): 65-73 supriati wila djami, et al. 6 7 controlled and untreated (4). the division of the severity of hypertension in a person is one of the basis for determining the treatment of hypertension according to the seventh report of the joint national committee on prevention, detection, evaluation and treatment of high blood pressure. the classification of hypertension in adults divided into groups of normal, prehypertension, stage i hypertension and stage ii hypertension. systolic blood pressure is the main measurement that is the basis for determining the diagnosis of hypertension (2). nt-probnp test can be used as a new parameter to identify at an early stage and monitor the progress of side effects of chemotherapy on the heart, in addition to the measurement of left ventricular ejection fraction. aside from being used as a biomarker for acute and chronic heart failure, nt-probnp can also be used to protect against the decrease in left ventricular function of asymptomatic patients with risk factors for cardiovascular disease. hypertension is one of the main risk factors for cardiovascular disease, such as heart failure, acute myocardial infarction and even sudden death. patients with hypertension can control the abnormalities and heart functions such as left ventricular hypertrophy / lvh) and left ventricular systolic dysfunction (lvsd), and the effect of their use by the left ventricular hypertrophic response associated with hypertension. detection of this condition is very important in the management of hypertension (1). diagnosis of hypertension with the most accurate physical examination is by using mercury sphygmomanometer. you should do more than one measurement in a sitting position with your elbows bent on the table with your palms facing up and your arms should be at heart level. measurements are made in a calm state. patients are expected to not consume food and drinks that can affect blood pressure such as coffee, soda, foods high in cholesterol, alcohol and so on (7). biomarkers such as nt-probnp has been investigated as a test that can help the diagnosis and management of heart failure at the same prognosis. steffanus (8), also declared that there is a relationship between elevated levels of nt-probnp with cirrhosis of the liver disease. reseach conducted by khairunnisa (9), stated that there is a relationship between increased levels of ntprobnp and the impairment of left ventricular ejection fraction-probnp. ntprobnp different on various diseases, because it needs to do further research to determine differences in the levels of the various diseases that cause heart failure. however, other studies have suggested that the increased nt-probnp was independently associated with an increased risk of hypertension (10).this makes the researcher interested to know the different levels of nt ina j med lab sci tech 2019; 1(2): 65-73 supriati wila djami, et al. 6 8 probnp and nt-probnp levels relationship with the degree of hypertension. the purpose of this study was to determine the differences in the levels of nt-probnp in stage 1 hypertensive patients and stage 2 hypertension patients. also, the researcher intended to know the relationship between the levels of nt-probnp in stage 1 hypertension and stage 2 hypertension. materials and methods this research was conducted in the medical record room and outpatient installation of dr. wahidin sudirohusodo general hospital makassar, august september 2018. 72 sampel collected in the analysis in research unit at university rsptn hasanuddin. this research was a cross-sectional study. the population in this study were all patients with hypertension who underwent outpatient rsws who aged > 30 years and suffering from hypertension stage i and stage ii and has a systolic blood pressure > 140 mmhg and diastolic blood pressure > 90 mmhg. data from medical records collected by age, sex, duration of hypertension and blood pressure are presented in tabular form to explain the characteristics of the study sample. the tools used in this study were elisa (enzyme-linked immunosorbent assays) reader organon model 680 (biorad) and microwell shaker tool (incubator 1000 heidolph). nt-probnp levels in patients with stage i and stage ii hypertension previously done kolgomorov-smirnov test to determine whether the data have normal distribution. the mean of the two groups were then tested for statistical significance by mann whitney test and spearman's rho. results revealed significant when p < 0.05. results this study was conducted on 72 patients with hypertension based on blood pressure, hypertension degree and medical record data in the outpatient installation of rsup dr. wahidin sudiro husodo. the research subjects were divided into 2 groups consisting of 36 patients per group with systolic blood pressure 140-159 mmhg; diastolic 90-99 mmhg (hypertension stage 1) and systolic blood pressure ≥160 mmhg; diastolic ≥100 mmhg (stage 2 hypertension). the characteristics of sex of the subjects was illustrated on table 1. the subject consisted of a total of 32 male patients and female 40 patients. based on the characteristics of the age, there was a total of 11 patients whose age below 45 years old (15.3%) and age greater than or equal to 45 years old amounted to 61 people (84.7%). meanwhile, the total patients based on the characteristics of the length of hypertensive which less than 6 years was 56 patients (77.8%) and the number of hypertension patients which the length of hypertensive ina j med lab sci tech 2019; 1(2): 65-73 supriati wila djami, et al. 6 9 greater than or equal to 6 years was 16 patients (22.2%). lastly, the number of patients based on blood pressure characteristics was 36 patients (50%) with first degree hypertension and 36 samples (50%) with second degree hypertension. table 1. characteristics of research subjects characteristics n percentage (%) min max mean gender male female 32 40 44.4 55.6 age (year) <45 11 15.3 32 80 56.17 ≥ 45 61 84.7 older hypertension (years) <6 56 77.8 0 11 2.64 ≥ 6 16 22.2 blood pressure (mmhg) hypertension stage 1 36 50 140/90 220/121 161.01/98.15 hypertension stage 2 36 50 levels of nt-probnp analysis based research subject characteristics the results of statistical analysis of the study subjects showed significant differences in the levels of nt-probnp based on old patient suffering from hypertension (p=0.010) and degree of hypertension (p=<0.001). the results of statistical analysis in table 2 showed that the average age of the subjects in this study was 56.17 ± 10.35 years old with the largest age was > 45 years old amounted to 84.7%, performed different tests based on the characteristics of the age of the patients (p=0,863). the test results showed statistically significant difference between the levels of nt-probnp in patients with stage 1 and stage 2 hypertension (p=<0.001). table 2. analysis of differences between nt-probnp levels with characteristics of the research subjects characteristics nt-probnp levels (ng / l) p-value * min max mean sd gender male 5.47 250.22 51.49 48.51 0.803 female 3.46 1252.65 145.86 305.55 age (year) <45 10.60 128.55 46.77 35.10 0.863 ≥ 45 3.46 1252.65 114.22 252.24 older hypertension (year) <6 3.46 1252.65 62.79 164.66 0.010 ≥ 6 16.65 1150.68 247.83 361.01 blood pressure (mmhg) stage 1 3.46 128.55 34.98 28.99 <0.001 stage 2 11.06 1252.65 172.85 316.26 * mann-whitney (p=<0.05) ina j med lab sci tech 2019; 1(2): 65-73 supriati wila djami, et al. 7 0 the results of statistical analysis in table 3 is based on the characteristics of the study subjects which showed no correlation with the levels of nt-probnp after spearman's rho test (p<0.05) so that it can be concluded that there was no relationship between the levels of nt-probnp with the degree of hypertension. table 3. correlation analysis levels of nt-probnp with characteristics of research subjects characteristics nt-probnp levels (ng / l) p-value* median min max sd gender male 31.93 5.47 250.22 48.51 0.762 female 46.14 3.46 1252.65 305.55 age (years) <45 50.17 10.60 128.55 35.10 0.979 ≥ 45 36.20 3.46 1252.65 252.24 older hypertension (years) <6 32.32 3.46 1252.65 164.66 0.183 ≥ 6 78.17 16.65 1150.68 361.01 blood pressure (mmhg) stage 1 24.33 3.46 128.55 28.99 0,862 stage 2 53.27 11.06 1252.65 316.26 *correlation spearman's rho discussion this research shows that there were more patients with hypetension in women than men and those whose age> 45 years old than those whose age <45 years old. age is an importan factor of hypertension. when someone get older, the risk of hypertension is also higher. an increase in hypertension cases will develop in their fifties and sixties. increase in blood pressure with the increase in age is a normal condition. however, if the change of blood pressure is too striking and accompanied by other factors, it triggers hypertension with its complications (11). according to azhar (12), it was stated that hypertension is more common in women than in men, this happens because premenopausal women are protected by the hormone estrogen which can increase the concentration of hdl and decrease ldl concentrations. however, when women experience menopause, estrogen will decrease. mainly experienced by women who are elderly, so that the blood pressure in elderly women tend to be high (12). as a biochemical marker that gives new hope to the cardiovascular field, the normal value of nt-probnp still cannot be fully determined because it depends on the examination method and the time of sampling. however, the concentration mentioned can be influenced by age and sex, which tends to increase in older age and female sex. some clinical conditions such as ina j med lab sci tech 2019; 1(2): 65-73 supriati wila djami, et al. 7 1 acute coronary syndrome, kidney failure and diabetes mellitus can also increase the concentration of cardiac natriuretic peptides including nt-probnp. this study concluded that there was no difference in the levels of nt-probnp in both men and women. these results are consistent with renardi (2009) showed no significant difference between the groups in the study of sex where nt-probnp levels were higher in men than in women. meanwhile, in this study, nt-probnp levels were higher in women than in male. this contradiction can be understood because it uses a different sample size and uneven distribution in the two groups of hypertension. the results of another study showed that women and men have nt-probnp concentrations were relatively the same. ntprobnp concentration varies, depending on gender and age. in males, the nt-probnp concentration increases with age as well as women's nt-probnp plasma concentration relatively increases with age (9). this study also shows that there is no significant difference between age and levels of ntprobnp. in contrast to these results, sarzani et al (13), which an average age of study subjects is 88.1 ± 5.1 years concluded that there were significant differences between the age factor with nt-probnp in patients with heart failure. this contradiction can be understood as research conducted on different age groups and different groups of cases. the average levels of nt-probnp is based on the longtime characteristics of patients suffering from hypertension that concluded that there were differences in levels of nt-probnp which was significantly longer based on the characteristics of hypertension. the results of statistical tests performed concluded that there was a significant difference between the levels of nt-probnp in patients with stage 1 and stage 2 hypertension (p<0.001). there was no significantly (relationship) between nt-probnp and sex of the subject. these results are consistent with the research conducted by rosello et al (2012) which showed no significant relationship based on the characteristics of sex in hypertensive patients. there was no significantly between age and levels of nt-probnp. furthermore, there was no correlation between nt-probnp levels were significantly associated with hypertension old. the results of this study for statistically consistent with research done by munir & sargowo (14), showed no significant correlation between levels of nt-probnp with duration of hypertension (old hypertension) in patients with hypertension. statistical test results concluded that there was no significant correlation between levels of nt-probnp in degrees 1 and degrees 2 hypertension patients. levels of ina j med lab sci tech 2019; 1(2): 65-73 supriati wila djami, et al. 7 2 nt-probnp is based on the characteristics of the systolic blood pressure 140-159 mmhg; diastolic 90-99 mmhg (hypertension grade 1) and systolic blood pressure ≥160 mm hg; ≥100 mmhg diastolic (hypertension grade 2), after statistical correlation test value of p = 0.862 (p = <0.05). this is consistent with the observation asterina et al (15) also concludes that there was a significant relationship between levels of nt-probnp in patients with hypertensive heart disease. this research agreement can be concluded there was no significant correlation between levels of nt-probnp with the degree of hypertension. the limitations of this study include inaccurate data and information regarding the length of time patients suffer from hypertension because some patients do not remember clearly, other patients are late to check up on health facilities, so it is difficult to determine the duration of suffering from true hypertension. conclusions it can be concluded that there was a significant difference in the levels of ntprobnp with a degree of hypertension, found nt-probnp levels were higher in patients with grade 2 hypertension compared to group 1 degree hypertension, although it was not statistically significant correlation between levels of nt-probnp with hypertension degree. further research are needed to determine the relationship of nt-probnp levels with the degree of hypertension, which can confirm the diagnosis, especially in patients with hypertension and to consider the accuracy of the data length of a patient suffering/undiagnosed hypertension. acknowladgements acknowledgements are given to several parties who have helped and been involved, including : ppsdm agency of the ministry of health of the republic of indonesia which has provided research funding. prof. dr. dr. haerani rasyid, m.kes.sp.pd-kgh,sp.gk who have helped in the completion of this research. prof. dr. mansyur arif, ph.d., sp.pk(k) who have helped in the completion of this research. uleng bahrun, dr., ph.d., sp.pk(k) who have helped in the completion of this research. dr. yuyun widaningsih, dr. m.kes., sp.pk who have helped in the completion of this research. dr. ilhamjaya pattelongi, dr., m.kes who have helped in the completion of this research. conflict of interest there are no conflicts of interest. ina j med lab sci tech 2019; 1(2): 65-73 supriati wila djami, et al. 7 3 references 1. rilantono li. cardiovascular disease. jakarta: faculty of medicine, university of indonesia; 2016. 2. cardiovascular specialist doctors association of indonesia (csdai). hypertension management guidelines on cardiovascular disease. issue i. jakarta: cardiovascular specialist doctors association of indonesia; 2015. 3. joint national committee (jnc) 8. the eighth joint national committee, hypertension: the silent killer: update jnc-8 guideline; 2015. 4. muhadi. evidence-based guideline. handling hypertension adult patients, (online). 2016;43(1).(www.cdkjournal.com/index.php/cd k/article/, accessed january 14, 2018). 5. south sulawesi provincial health office. health profile of south sulawesi province in 2015. makassar: south sulawesi provincial health office; 2015. 6. makassar city health office. 2015. 2015 makassar city health profile makassar: makassar city health office. 7. hulaima, i.s. 2017. factors associated with control of blood pressure in hypertensive patients at the kedaton city bandar lampung health center. bandar lampung: faculty of medicine, university of lampung. 8. steffanus m, wibawa idn, nadha ikb. positive correlation between degree of liver cirrhosis and n terminal-pro brain natriuretic peptide (nt-probnp). 9. khairunnisa. correlation levels of nt probnp with left ventricular ejection fraction function in children heart failure. padang: graduate program in biomedical sciences medical specialist education program unand rs. m. djamil; 2017. 10. bower et al., .n-terminal pro-brain natriuretic peptide (nt-probnp) and risk of hypertension in the atherosclerosis risk in communities (aric) study.american journal of hypertension. 2015;28(10):1262-1266. 11. hikmah, n. relationship between smoking time and hypertension in rannaloe village, bungaya district, gowa regency. makassar. faculty of medicine and health uin alauddin; 2017. 12. azhar i. hypertension patient characteristics picture in sleman gamping puskesmas yogyakarta. yogyakarta: college of health sciences of the university general achmad yani yogyakarta; 2017. 13. sarzani r. et al . nt-probnp and its correlation hospital mortality in the very elderly without and admission diagnosis of heart failue.plos journals.2016;11(4): e0153759. 14. munir a,sargowo n. n terminal pro brain natriuretic peptide as indicator of left ventricle hypertropy in hypertensive patients compared with left ventricle mass index in echocardiography. jurnal kardiologi indonesia. 2010;31(2):87-98. 15. asterina f, nasution b, akbar n. the level of n terminal-pro brain natriuretic peptide in hypertensive heart disease patients. indonesian journal of clinical pathology and medical laboratory. 2007;14(1):42–46. 93 utilization of 1% of methylene blue in staining histopathological preparations at anatomic pathology laboratory tri rahmawati1, yadi apriyadi2, mamay1 1department of medical laboratory technology, stikes karsa husada, garut, indonesia 2departement pathology of anatomi, regional public hospital dr. slamet, garut, indonesia correspondence: mamay, jl. nusa indah no.1, jayaraga, kec. tarogong kidul, kabupaten garut zip code : 44151 email: mamay.1745@gmail.com received: may 31, 2020 revised: june 19, 2020 accepted: august 25, 2020 abstract tissue staining using hematoxylin-eosin (he) is a standard method of histopathological staining. the tissue staining is hampered when there is no hematoxylin reagent in laboratory. therefore, other reagents are needed that can replace the use of hematoxylin. methylene blue is a basic dyes that interact with cell nuclei which has a negative ionic charge of the tissue. it can be used as an alternative nuclei staining. this study aims to evaluate the use of 1% of methylene blue in cell nuclei staining in histopathological preparations. the research sample were 15 pathology preparations which were randomly selected including breast cancer, cervical cancer and ovarian cancer in the bank of sampel at anatomical pathology laboratory of rsud dr. slamet garut, indonesia. the experiment showed that the methylene blue dyes yielded “worth” result (40%) and “poorly” result (60%). further research can be carried out by modifying the ph of 1% of methylene blue reagent so that it can maximize the staining preparations results as good as those using hematoxylin. keywords cancer, hematoxylin, histopathology, methylene blue introduction cancer prevalence is increasing in the world every year. according to the international agency for research on cancer (iarc), the number of cancer patients worldwide increases since 2012. more than14 million new cases of cancer, of which 8 million or more cases, died from cancer. based on the ministry of health of the republic of indonesia data in 2018, the prevalence of tumors or cancer in indonesia shows an increase from 1.4 per thousand populations in 2013 to 1.7 per thousand populations in 2018 (1). in garut regency, especially in the anatomic pathology laboratory of rsud dr. slamet garut, there were 189 cancer patients (44.9% of breast cancer patients, 10.5% of cervical cancer, 8.9% of lymph node cancer, 7.4% of colon cancer, 5.8% of ovarian cancer, 4.7% of ina j med lab sci tech 2020; 2(1): 93-100 tri rahmawati, et al. 9 4 thyroid cancer, 3.7% of bone cancer, 2.1% of prostate cancer, 1.05% of parotid cancer (salivary glands), 1.05% of molar pregnancy, 1.05% of cancer of body fluids, and other types of cancers which count up to 9.5%). precancerous and cancer is characterized by excessive cell proliferation due to the increase in mitotic cells (2). the increased and abnormal mitosis indicates a damaged tissue of an important feature in precancerous and cancer tests. the identification and quantification of mitotic cells is used in histological examination to support the diagnosis (3). histological staining has been widely used in pathology (4). examination of the development of cancer can be conducted microscopically through histopathology examination. important stages of histopathological examination starts from fixation, processing, embedding, cutting and staining (5). tissue staining using the hematoxylin-eosin (he) is a standard method of histopathological staining. hematoxylin is a basic dye that is commonly used for staining cell nuclei and provides a bluish tint while eosin yields a pink dye (2). the principle of such coloration is chemical interactions binding between tissue and dyes (4). the tissues with negative or anionic charges are more easily stained with a blackish-blue color with an alkaline staining called basophilic using hematoxylin dyes. meanwhile, tissue components with a positive or cationic charge (cytoplasm) are more easily colored into pink with an acidic staining called acidophilic using a substance eosin color (7). the services in the anatomic pathology laboratory are often hampered when the hematoxylin reagent runs out and it takes a long time for the reagent to arrive since the availability of hematoxylin is quite rare. thus, the process of hematoxylin maturation requires a long time (8). nevertheless, the examination service in the anatomic pathology laboratory must be done continuously. therefore, other reagents are needed that can replace the use of hematoxylin in histopathological preparations staining. the other reagents that can be applied for staining are crystals violet (2, 3, 9), toluidine blue, neutral red, methylene green, and methylene blue (4, 10, 11). methylene blue is a cationic dye that has a positive ionic charge so that it will interact with cell nuclei that have a negative ionic charge of the tissue (4) löffler's alkaline of methylene blue is used in histopathologically conjunctival epithelial cells and blue-colored neutrophils and more intense colored nuclei (12). previous stain with 1% of methylene blue detect oral dysplasia and carcinoma resulted in good validity (11). however, the utilization of 1% of methylene blue for histopathological staining cancer mitosis has not been reported. therefore, the author aims to apply methylene blue compound in the process of ina j med lab sci tech 2020; 2(1): 93-100 tri rahmawati, et al. 9 5 histopathological preparations as a substitute for hematoxylin in the hematoxylin-eosin staining. materials and methods the tools used in this study were microtome knife, pencil, camera, tissue, label paper, oven, cover glass, object-glass, beaker glass, microscope, microtome, water bath, cassette, tweezers, mold, refrigerator, pipette, and staining jar. the materials used include tissue cuts, 10% of buffered formalin, absolute alcohol, 96% of alcohol, 80% of alcohol, 70% of alcohol, xylol, paraffin, lithium carbonate, mayer hematoxylin, eosin, blue methylene, tap water, distilled water, and entelan. this research was conducted in the sitohistotechnology laboratory from july to august 2019. sample were obtained from 15 pathology preparations and were randomly selected, including breast cancer, cervical cancer and ovarian cancer in the bank of sampel at anatomical pathology laboratory of rsud dr. slamet garut, indonesia. the quality assessment of the preparation is carried out by an anatomist pathologist. the sample was fixed in the second stage with a 10% of formalin buffer solution for 24 hours. the tissue preparation consisted of several stages, including dehydration, clearing, impregnation, embedding and tissue cutting. the dehydration process used alcohol in an oven at a temperature of 65oc– 70oc for 45 minutes, starting from 70%, 80% and 95%. the clearing step used xylol i and xylol ii, respectively, for 45 minutes in the oven with a temperature of 65oc–70oc. the impregnation step used liquid paraffin for 45 minutes in an oven with a temperature of 65oc–70oc. furthermore, the embedding step was done to plant the tissue into paraffin molds then it was stored in the refrigerator for 2 hours until it was solidified completely. lastly, a gross cut with a thickness of 4 microns was done to obtain a network band. the tissue tape was affixed to the glass object and was followed by mounting in a water bath with a temperature of 40oc to obtain histopathological preparations that were ready to be coloured. histopathological preparations for the control group used the mayer hematoxylineosin staining method while the experimental group used methylene blue-eosin staining. the blue methylene used 1% of distilled water. the results of staining was analysed under a microscope with a magnification of 400x. an optimal-quality staining as nuclei exhibiting ‘blue hematoxylin’ with chromatin patterns, permit the differentiation of distinct cell type (13). a sub-optimal staining, the nuclei were weakly stained and appear pale (14). the data were categorized based on the quality of the stained nuclei (chapman category modified), it is classified as “good” if the nucleus was clearly stained and the chromatin was clearly visible; ina j med lab sci tech 2020; 2(1): 93-100 tri rahmawati, et al. 9 6 “worth” if the nucleus was stained but less clear and the chromatin was lacking clearly visible, and it was classified as “poorly” if the cell nucleus is not colored. results the quality of staining cell nuclei using hematoxylin-eosin in five breast cancer preparations has good quality (the cell nuclei were visible with coarse chromatin). the results of the staining showed the presence of mitotic cell nuclei (observed cell nuclei that were three lobes in one cell), as shown in figure 1 (a). the results of staining the cell nucleus with methylene blue-eosin in a breast cancer preparation showed poor staining quality that was characterized by colorless cell nuclei, mitotic cell nuclei and chromatin were not observed, as shown in figure 1 (b). meanwhile, the four breast cancer preparations have a pretty good staining quality, the cell nucleus was colored but it was not seen and the chromatin was less clear, as shown in figure 1 (c). fig 1. staining cell nuclei in breast cancer preparations with 400x magnification. (a) hematoxylin, (b–c) methylene blue the quality of staining of cell nuclei with hematoxylin-eosin in four cervical cancer preparations showed good staining quality with clear observations of cell nuclei. the appearance of mitotic cell nuclei is shown in figure 2 (a). the results of cell nucleus staining with methylene blue-eosin in a cervical cancer preparation showed a quite good quality. the cell nucleus was stained but it was not seen and chromatin was less clear so that there should be a cell nucleus, as in figure 2 (b). meanwhile, the four cervical cancer preparations showed poor quality colour which means that the cell nucleus was not stained and chromatin was not observed, as shown in figure 2 (c). a b c ina j med lab sci tech 2020; 2(1): 93-100 tri rahmawati, et al. 9 7 fig 2. staining cell nuclei in cervical cancer preparations with 400x magnification. (a) hematoxylin, (b–c) methylene blue the results of cell nucleus staining with hematoxylin-eosin in five ovarian cancer preparations showed good staining quality, dye the cell nucleus was stained and observed with coarse chromatin as shown in figure 3 (a). the results of nucleus cell staining with methylene blue-eosin in four ovarian cancer preparations showed poor quality meaning that the cell nucleus was not stained with chromatin which was not observed, as shown in figure 3 (b). meanwhile, the four ovarian cancer preparations showed quite good quality of staining. it means that the cell nucleus was colored but it was not seen, it only looked like a small dot from the cytoplasmic outline so that it could not be seen, as well as the cell nucleus and chromatin nuclei were also not observed, as shown in figure 3 (c). the results of the breast cancer, cervical and ovarian cancer staining preparations using hematoxylin-eosin method and methylene blue-eosin in the control group can be seen in table 2. fig 3. staining cell nuclei in ovarian cancer preparations with 400x magnification. (a) hematoxylin, (b-c) methylene blue a b c a b c ina j med lab sci tech 2020; 2(1): 93-100 tri rahmawati, et al. 9 8 table 2. results of staining cell nuclei of breast, cervical and ovarian cancer dye cancer preparation quantity (percentage%) good worth poorly hematoxyilin breast 5 (100%) 0 0 servical 4 (80%) 1 (20%) 0 ovarian 5 (20%) 0 0 total 14 (93%) 1(7%) 0 methylen blue breast 0 4 (80%) 1 (20%) servical 0 1 (20%) 4 (80%) ovarian 0 1 (20%) 4 (80%) total 0 6 (40%) 9 (60%) discussion methylene blue is a cationic dye which is also known as basic dyes. it has a positive ionic charge so it will interact with cell nuclei that have a negative ionic charge of the tissue (4). this dye can react with anionic groups as phosphate groups of nucleic acids (dna and rna) which is commonly used as nuclear stains. it is a basophilic dye which will bind to acidic tissue. this relates to the concept of tissue staining, where acidic tissue is more easily stained with base or basic dyes (7). bonding staining to tissue is not different from chemical bonds and the mechanism is similar in other organic bonding components. the bonding of tissue coloring involves ion interactions. it is a combined form and it can bind to tissues and dyes as long as two different ion poles interact properly (4). 1% of methylene blue dye in previous research can detect the dysplasia and oral carcinoma. the validity of methylene blue to detect the dysplasia and carcinoma was increase (11). in this study, 1% of methylene blue dyes in coloring the cell nucleus from histopathological preparations resulted in poor quality cell nuclei with as much as 60% of the cell nucleus was not successfully stained. while, the other experiment has a pretty good staining quality (40%) where the nucleus cells were colored but the mitosis could not be seen from the cell nucleus itself. meanwhile, by using hematoxylin-eosin staining, 93% of the preparations were well colored and 7% were quite well colored. the nucleus staining in the control group underwent mitosis with three lobes, whereas in the cell nucleus experiment, the shape was visible from the methylene blue staining. it was also visible from outside the cytoplasm so that there should be a cell nucleus division with three lobes yet it was only visible in one lobe. the extracellular ph environment has more acidic ph than normal cells (the ph of normal cells was 7.4 and ph of of tumor cells was 6.5) (15). the charge variation of the breast cancer cells and fibroblasts was good at ph of 2.5–9 (16). therefore, the interaction between methylene blue dye is greater with breast cancer tissue, so that the quality of the ina j med lab sci tech 2020; 2(1): 93-100 tri rahmawati, et al. 9 9 staining is better compared to cervical cancer and ovarian cancer. the lack of staining quality by using methylene blue is affected by the composition of the dye and the acidity or ph which is different from hematoxylin. hematoxylin is mixed with potassium or ammonium alum, sodium iodate, and chloral hydrate (8). meanwhile, methylene blue was not added with other strong alkaline materials to increase the ph. in acidic condition, the ph of methylene blue will produce a different color that will color the cell nuclei but the protein remains colorless. cell nuclei that are not successfully stained by methylene blue are also caused by changing in ph because the ionic bonds in dyes are very sensitive to ph. to be conclude, there was no interaction between tissue ions and dye ions. based on previous research, maximum coloration obtained by methylene blue with loffer formula produced best result, which used potassium hydroxide to increase the ph to color all protein components and nucleic acids. ultimately, the tissue will appear blue (12). conclusions staining preparation with 1% of methylene blue can color the cell nucleus quite good. from the results of this study, it is necessary to modify the ph of the 1% of methylene blue reagent in order to maximize the staining preparations to get good results as well as those using hematoxylin. acknowladgements the source of funding for this research came from researchers' personal funds. conflict of interest there are no conflicts of interest. references 1. ministry of health republic of indonesia. cancer situation. data and information center. 2018 2. ankle mr, kale ad, charantimath s, charantimath s. comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma. indian j dent res. 2007;18:101–105. 3. jadhav kb, ahmed mujib b r, gupta n. crystal violet stain as a selective stain for the assessment of mitotic figures in oral epithelial dysplasia and oral squamous cell carcinoma. indian j pathol microbiol. 2012;55:283-287. 4. veuthey, t., georgina herrera, g, dodero, v.i. dyes and stains: from molecular structure to histological application. front in biosci. 2014;19: 91–112. 5. titford m, bowman b. 2012. what may the future hold for histotechnologists? labmedicine. 2012;43;5–10. 6. alturkistani ha, tashkand fm, saleh zmm. histological stains: a literature review and case study. glob j health sci. 2016;8;72–79. 7. mescher al. histologi dasar junqueira : teks & atlas. ed.12. jakarta : egc. 2011. 8. suvarna k, christopher l, bancroft jd, theory and practice of histological techniques, 7th edition. philadhelphia : elsivier. 2013. 9. kadoo p, dandekar r, kulkarni m, mahajan a, kumawat r, parate n. correlation of mitosis obtainned by using 1% crystal violet stain with ki67li in histological grades of oral squamous cell carcinoma. j oral biol craniofacial res. 2017;8;234–240. 10. tandon a, singh nn. brave, vr sreedhar g . image analysis assisted study of mitotic figures in oral epithelial dysplasia and squamous cell ina j med lab sci tech 2020; 2(1): 93-100 tri rahmawati, et al. 1 0 0 carcinoma using differential stains. j oral biol craniofacial res. 2016;6;18–23. 11. soman c, lingappa a, mujib, a. topical methylene blue in-vivo staining as a predictive diagnostic and screening tool for oral dysplastic changes – a randomised case control study. journal of dental sccience. 2016;4;118–123. 12. kiuchi k. rapid alkaline methylene blue supravital staining for assessment of anterior segment infections. clin ophthalmol. 2016;10;1971–1975. 13. bendzinski ecm, chu k, johnson ji, brous m, copeland k, bolon b. optical density-based image analysis method for the evaluation of hematoxylin and eosin staining precision. j histotechnology. 2020;43;29–37. 14. chapman cm. troubleshooting in the histology laboratory. j histotechnology. 2019;42;137–149. 15. jafari m, hasanzadeh m. cell-specific frequency as a new hallmark to early detection of cancer and efficient therapy: recording of cancer voice as a new horizon. biomedicine & pharmacoherapy. 2020;122;109770 16. dobrzyńska i, skrzydlewska e, and figaszewski z a. changes in electric properties of human breast cancer cells. j membrane biol. 2013;246; 161–166. https://www.sciencedirect.com/science/article/pii/s0753332219353922#! https://www.sciencedirect.com/science/article/pii/s0753332219353922#! 109 pigeonpea (cajanus cajan l.) and cowpea (vigna unguiculata l.) water as alternative growth medium for escherichia coli and staphylococcus aureus in laboratory with minimum infrastructure norma tiku kambuno1, ninda p. y. amtaran1, sherly dewu1, kuntum e. nurdin1, ni made susilawati1, yoan novicadlitha1 1department of medical laboratory technology, poltekkes kemenkes kupang, east nusa tenggara, indonesia correspondence: norma tiku kambuno, jl jalan piet a. tallo, kupang, east nusa tenggara, indonesia email: norma.kambuno@gmail.com received: may 3, 2021 revised: august 8, 2021 accepted: september 8, 2021 published: october 30, 2021 doi: 10.33086/ijmlst.v3i2.2076 abstract the availability of non-synthetic media from natural ingredients is needed to answer the needs in laboratories where the price of nutrient media is quite expensive and there are limited supplies of material ware houses. cowpea (vigna unguiculata l.) and pigeonpea (cajanus cajan l.) are the local foods of people of ntt (east nusa tenggara) which have a high enough nutritional content which has the potential to be developed into cheap, easy and simple nonsynthetic media in making. the purpose of this study was to determine whether the agar media contained nutrient from cowpea and pigeonpea water can be used as a alternative for nutrient agar for the growth of escherchia coli and staphylococcus aureus bacteria. this research is a true experiment with posttest-only control design. the growth rate of s. aureus bacteria on pigeonpea medium, cowpea medium, nutrient agar medium, were 164 cfu/ml (sd=3,13), 161 cfu/ml (sd=3,02) and 164 cfu/ml (sd=3,21), respectively. the average growth of e. coli on cowpea medium, pigeonpea medium, and nutrient agar control medium were 163 cfu/ml (sd=2,79), 167 cfu/ml (sd-2,63) and 164 cfu/ml (sd=2,75) respectively. test results anova between pigeonpea medium, cowpea medium and nutrient medium in order to obtain p value = 0.145 (p> 0.05) for the growth of e. coli bacteria and p value = 0.393 (p> 0.05) for growth s. aureus. it was concluded that there was no difference between the number of bacterial colonies of e. coli and s. aureus on three medium. the pigeonpea medium and cowpea can be used to grow and alternative nutrient agar in order to grow bacteria e. coli and s. aureus. keywords cowpea water, pigeonpea water, alternative medium, e. coli, s. aureus. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 0 norma tiku kambuno, et al. introduction cultivation medium is a nutrient medium that is prepared to grow bacteria on a laboratory scale (1,2). most of the bacteria can grow well on any media, while others require special media (3). the cultivation medium must be able to provide the energy needed for bacterial growth (4). bacteria that are inoculated on a cultivation medium are called inoculum and bacteria that have grown and reproduced are called bacterial grow (5). microbiology laboratories really need bacterial growth media for growing, isolating, counting the number, and testing the physical properties of bacteria so that a bacterium can be identified (6,7). the nutrients needed by microorganisms for their growth include carbon, nitrogen, non-metal elements such as phosphorus, metal elements such as ca, na, fe, vitamins, water, and energi (8,9). synthetic media commonly used to grow microorganisms in laboratories such as bacteria is a nutrient agar medium (10,11). some conditions that must be requirements by the bacterial growth medium are that it must contain the right nutrients for the specific bacteria to be grown, the humidity must be sufficient, the ph is appropriate, and the oxygen content is good enough, the growth media must be sterile and does not contain other microorganisms, the media is incubated at room temperature certain (9,12,13). synthetic media contain high nutrition, consisting of meat extract, yeast, plant extracts, proteins, vitamins, minerals, other organic materials. anaerobic media is a media used to grow anaerobic bacteria, containing sodium thioglycolate (14,15). selective and differential media were used to detect the presence of specific bacteria, suppress the growth of unwanted bacteria, for example mac conkey. macconkey agar media is a selective medium and a differential medium used to isolate gram-negative bacteria based on the ability of bacteria to ferment lactose or not. macconkey agar media is used primarily for the enterobacteriaceae family such as e. coli (15,16). contains crystal violet that can selectively inhibit gram-positive bacteria (8,17). the availability of synthetic media requires high costs, time to order to the industry, limited stock in the material warehouse, thus limiting repeated use, especially for research purposes (17). this condition requires non-synthetic media which is considered practical, cheap, easy to obtain and can be produced by the laboratory itself. non-synthetic media is an alternative media that uses ingredients found in nature (10). the chemical content of these ingredients is not known in detail but can be used because they are abundant in nature, easy to prepare and cheap (18). deivanayaki et al. (19) conducted research on bacterial growth media from vegetables such as carrots, tomatoes, ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 1 norma tiku kambuno, et al. cabbage, and pumpkin. these vegetables showed good results for bacterial growth in both liquid and solid medium (19). some fruits are also used as a medium for bacterial growth, such as avocados, beets, and cucumbers and orange peels (20). a part from grains, vegetables, and fruit, bacterial growth media can also be made from various types of tubers that are rich in carbohydrates (21). some researchers have succeeded in showing the growth of good bacteria in growth media from various carbohydrate sources such as cassava (manihot utilissima) (18,22), potatoes (solanum tuberosum), palmirah (borassus) and sagu (metroxylon sagu) (17,23). utilization of legumes, local names is red beans (phaseolus vulgaris l), green beans (phaseolus radiatus l.) and black soy beans (glycine max l. merr) has also been reported (3,10). alternative media from natural ingredients such as cassava starch, tunggak beans (cajanus cajan), green beans (24), black soy beans and soy bean, ganyong (canna discolor), gembili (dioscorea esculenta), garut (maranta arundinacea), have been reported to be used for the growth of aspergillus niger and fusarium oxysporum (25,26). timor island is one of the areas in east nusa tenggara (ntt), indonesia, which is rich in local nuts (27). it is recorded that there are about 29 types of local beans that are there. several types of local nuts that are commonly used in managing agriculture in timor, especially as food, are pigeonpea (cajanus cajan or pigeon pea) and cowpea (vigna unguiculata l.) both types of beans are used as local food or daily food ingredients by the local community. both of been is available in large quantities, cheap and easy to find both in traditional markets and in the home page of residents (27). the absence of research on the use of local ntt legumes in the manufacture of bacterial growth media has attracted researchers to make bacterial growth media using local legumes from timor island, ntt, namely cowpeas which in everyday life the people of timor island call them cowpea and pigeonpea. the medical laboratory technology study program is one of the study programs under the poltekkes of the ministry of health kupang, which has a microbiology, mycology and parasitology laboratory. these three laboratories require a large number of bacterial growth media for student practicum and research by lecturers and students. the availability of synthetic media such as nutrients is limited because the delivery process from outside the province often experiences obstacles causing vacancies in the laboratory. the study were aimed to find out whether or not cowpea medium and pigeonpea medium can be used as substitution media of nutrient agar for e.coli and s. aureus bacteria’s growth expecially in the laboratory with minimum infrastructure. ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 2 norma tiku kambuno, et al. materials and methods the research method used was true experiment with a posttest-only control design in which the control group and sample group were randomly selected and the effect of the treatment was analyzed using a different test, namely independent test sample t-test and one-way anova test. this research was carried out in february march 2020 in the bacterial laboratory, majoring in medical laboratory technology, poltekkes of the ministry of health kupang, indonesia. the variables in this study were the bacterial growth media and the number of e. coli (atcc 25922 and s. aureus (atcc 25953) colonies growing on the growth media. s. aureus bacteria used in this study were taken from pus samples patients at prof. dr. w. z. johannes kupang general hospital. s. aureus was maintenance in mannitol salt agar media. process of making cowpea medium and pigeonpea medium according to khaerunisa (22) and it show in figure 1; figure 1. procedure of making pigeonpea and cowpea medium ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 3 norma tiku kambuno, et al. suspensions of s. aureus and e. coli were diluted with a concentration equivalent to 0.5 mc farland. immediately after being suspended inoculated as much as 0.1 ml on each medium for cowpea, for pigeonpea medium and nutrient agar medium. we did six iterations in each test. incubated in a temperature incubator 370c for 24-48 hours then the size of the colony was measured and followed by the calculation of the number of colonies using the colony counter. nutrient agar to be used as a control medium. nutrient agar is a general purpose medium supporting growth of a wide range of nonfastidious organisms. it typically contains (mass/volume): 0.5% peptone (this provides organic nitrogen), 0.3% beef extract/yeast extract (the water-soluble content of these contribute vitamins carbohydrates, nitrogen, and salts), 1.5% agar (this gives the mixture solidity), 0.5% sodium chloride (this gives the mixture proportions similar to those found in the cytoplasm of most organisms), distilled water (water serves as a transport medium for the agar's various substances), ph adjusted to neutral (6.8) at 25°c (77°f) (28). primary data obtained from examination of total plate numbers from alternative media for pigeonpea and cowpea as well as nutrient agar media, then the data were displayed in tabular form. in addition, to obtain differences in the number of bacteria, the data obtained were processed using statistical tests, namely the independent test sample t test and anova to see the differences in bacterial growth in each medium. results general description of e. coli and s. aureus growth based on the number of bacterial colonies on cowpea medium, pigeonpea medium and nutrient agar medium can be seen in table 1. table 1 shows that the average growth rate of s. aureus bacteria on pigeonpea medium was 164 cfu/ml, on cowpea medium was 161 cfu/ml and on nutrient agar as much as 164 cfu/ml on media. the same table shows the average growth of e. coli bacteria on cowpea medium as much as 163 cfu/ml, pigeonpea medium as much as 167 cfu/ml and nutrient agar medium as much as 164 cfu/ml. table 1. growth results of e. coli and s. aureus bacteria on cowpea medium and pigeonpea medium bacteria media type repetition min cfu/ml max cfu/ml mean sd e. coli nutrient agar 6 160 168 164 2,75 cowpea 6 160 168 163 2,63 pigeonpea 6 163 170 167 2,79 s. aureus nutrient agar 6 160 170 164 3,21 cowpea 6 158 168 161 3,02 pigeonpea 6 157 166 164 3,13 https://en.wikipedia.org/wiki/growth_medium https://en.wikipedia.org/wiki/fastidious_organism https://en.wikipedia.org/wiki/mass_concentration_(chemistry) https://en.wikipedia.org/wiki/peptone https://en.wikipedia.org/wiki/beef_extract https://en.wikipedia.org/wiki/yeast_extract https://en.wikipedia.org/wiki/yeast_extract https://en.wikipedia.org/wiki/agar https://en.wikipedia.org/wiki/sodium_chloride https://en.wikipedia.org/wiki/cytoplasm https://en.wikipedia.org/wiki/distilled_water ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 4 norma tiku kambuno, et al. based on the number of e. coli bacterial colonies growing on these three media, it can be seen that the average number of e. coli bacteria colonies grows more on pigeonpea medium, namely as much as 170 cfu/ml while s. aureus bacteria grow more on nutrient control medium so that is as much as 170 cfu/ml. based on the homogeneous test, it shows a significant value> 0.05 so that it means that the data is homogeneous and can be continued with the anova test. in addition to growing pure cultures of e. coli and s. aureus, we also carry out tests on clinical specimen from pus samples from patients. it was found that the growth of e. coli could be identified properly. our findings indicate that pigeonpea medium and cowpea medium can be used as alternative media to grow e. coli. the form of bacterial growth on both media is shown in the image below: figure 2. growth of e. coli bacteria from pus samples. (a) on cowpea and (b) on pigeonpea medium figure 3. growth of pure e. coli bacteria (a) on cowpea media and (b) on pigeonpea media ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 5 norma tiku kambuno, et al. figure 2 above shows that the e. coli bacteria from pus clinic samples can grow well, the form of colonies that grow is different and the specific growth is found in cowpea medium, namely large, slimy and wetter colonies while in pigeonpea medium, the form of colonies is dry, small and spread. figure 3 show pure cultures of e. coli bacteria can grow well and show specific growth, in the medium of cowpea the form of large colonies, spreading and wetter, while in the medium of pigeonpeas the form of small colonies and drier. in pigeonpea medium, the size of the s. aureus bacteria colony was smaller than the colonies that grew on the control media. the colony size of e. coli on cowpea medium was the same as the colony size of e. coli in the control medium, while the colony size of e. coli on pigeonpea medium was smaller than the colony size of e. coli that grew on the control medium. the differences in the size of bacterial colonies are caused by several factors including nutritional factors, temperature, and osmotic pressure (23). our literature study shows that the nutrients that can be used by bacteria for growth are sources of energy, carbon, nitrogen, minerals, sulfur, and vitamins (22). the difference in nutrient content in the three media was one of the causes of the difference in colony size(20). in addition to nutritional factors and incubation temperature, another influential factor is the adaptation phase. the adaptation phase is a situation when bacteria are transferred to a new environment, the bacteria will undergo an adaptation process including the synthesis of new enzymes from the previous growing medium (21). in addition, figure 5 shows the growth of e. coli and s. aureus on nutrient agar as a control medium. figure 4. growth of pure culture of s. aureus bacteria (a) on cowpea and (b) on pigeonpea media. ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 6 norma tiku kambuno, et al. figure 5. growth of pure culture of s. aureus bacteria (a) on cowpea and (b) on nutrient agar media. the analysis was continued with the anova test and obtained p value = 0.145 (>0.05) for the number of e. coli bacteria and p value = 0.393 (>0.05) for the number of s. aureus bacteria so that there was no significant difference number of bacteria in the cowpea, pigieonpea and nutrient agar media. it was found that the significant value of cowpea medium and pigeonpea medium could substitute the use of nutrient agar medium. discussion factors that can affect bacterial growth include nutritional factors, temperature, ph and osmotic pressure (6,10). the nutrients contained in cowpea are incomplete compared to the nutrients contained in pigeonpea and in nutrient medium so that some of the nutrients needed for bakery growth cannot be fulfilled, as a result, it is difficult for bacteria to grow on cowpea medium. based on our literature study, we found that pigeonpea contain nitrogen, vitamin b12, iron/fe, phosphorus and fiber which are not found in cowpea. the nutrients needed by microorganisms for their growth include carbon, nitrogen, non-metal elements such as sulfur and phosphorus, metal elements such as ca, zn, na, k, cu, mn, mg, and fe, vitamins, water, and energi (19,21). our literature study also shows that pigeonpea beans and cowpea beans contain nutrients including, water, calories, protein, fat, carbohydrates and calcium which are needed for bacterial growth (19). in addition to nutritional factors, these bacteria are in an adaptation phase, which is when the bacteria is transferred to a new environment, it will undergo an adaptation process including the synthesis of new enzymes that are different from the previous ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 7 norma tiku kambuno, et al. growth media and recovery of toxic metabolics such as acids, alcohols and alkalis (8,10). the adaptation response can be due to nutrient deficiencies in cowpea medium and pigeonpea medium, indicated by the small size of the bacteria (18,19). the protein hydrolysis process is carried out because the protein molecules are too large to be able to enter through the bacterial cell membrane, so the bacteria excrete the protease enzyme which hydrolyzes the protein into simpler peptides (22). then the peptides formed with the help of peptidases are converted into amino acids, so that the amino acids formed can enter bacterial cells (20). in bacterial cells the amino acids that are formed are catalyzed by the enzyme lactic acid dehydrogenase and reduced by nadh to produce energy, so that it can be used for growth in colony size (1,29). research by rizki & syahnitya (24) which utilizes jicama and bean sprouts as growth media for s. aureus and e. coli bacteria shows that the growth of s. aureus and e. coli bacteria is more in jicama media, this is because jicama has a higher starch content than the starch sprouts contained in jicama is composed of 2 kinds of carbohydrates, namely amylose and amylopectin (22,24). the same thing was conveyed by khaerunisa (5) which used boiled water for yellow tubers and purple tubers as an alternative medium for the growth of s. aureus and e. coli bacteria, the results showed that the number of s. aureus and e. coli bacteria grew more on media made using yellow tuber boiled water, because the carbohydrate content of yellow tuber boiled water is higher than purple tuber boiled water, where the carbohydrate contained in yellow tubers is raffinose. raffinose is a trisaccharide consisting of the monomers fructose, glucose and galactose which are used as an energy source to increase the number of bacterial growth (22). widya's research (23) states that bacterial growth in alternative media is influenced by several factors, namely nutrient content, extract-making process, fiber content, storage and the effect of changes in ph after autoclaving sterilization (23). purwati (21) reports that the use of different natural sources of carbohydrates can be used to substitute nutrient agar (na) media for bacterial growth. suweg tuber (amorphophallus paeoniifolius), taro tuber (cyrtosperma merkusii), and kimpul tuber (xanthosoma sagittifolium) can be used as nutrient substitution media for the growth of gram-positive and gram-negative bacteria however, the best medium is the medium from the suweg tuber (amorphophallus paeoniifolius) (21). test results of anova between pigeonpea media and nutrient media in order to obtain p value = 0.145 (p > 0.05) for the growth of e. coli bacteria and p value = 0.393 (p > 0.05) for the growth of s. aureus bacteria. these results indicate that there is ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 8 norma tiku kambuno, et al. no significant effect between the number of bacterial colonies e. coli and s. aureus grown on pigeonpea media, cowpea and nutrient agar as control media. the statistical test shows that non-synthetic media from cowpea and pigeonpea can replace the use of nutrient media for daily use. the results of this study, we recommend the use of natural media for the needs of student practicum and lecturer / student research. in the initial isolation stage that requires large amounts of media, we recommend the use of these two media. we hope that the use of these two media will help with laboratory activities and make it easier to prepare and use them. several previous studies have reported the use of natural materials as a substitute for nutrient agar to grow s. aureus and e. coli (12,15,16,22). anisa (12) has also developed several different carbohydrate sources including canna tubers (canna discolor), gembili tubers (dioscorea esculenta), and garut tubers (maranta arundinacea) as an alternative medium for bacterial growth. jannah (16) has also conducted research and showed that sweet potato (ipomoea batatas (l). lam) cilembu as a substitute for carbohydrates in potato dextrose agar (pda) media for the growth of the fungus trichophyton rubrum. indrayana (15) also proved that sweet corn cobs waste flour (zea mays) can be used for the growth of e. coli and s. aureus bacteria. safitri et al. (13) have also developed a tofu whey substrate as a growth medium for pediococcus pentosaceus lactic acid bacteria. the development of specific media that grows e. coli bacteria using pure head water was developed by wulandari, it was found that there were differences in the number of colonies at each concentration, e. coli growth was found in young coconut water media with concentrations of 20%, 40%, 60%, 80%, while there was no growth at concentrations of 0% and 100%. the potential of red beans and green beans to grow lactobacillus acidophilus bacteria was developed by kurniasih et al. (5), the results showed that mung bean juice was more effective as a growth medium for lactobacillus acidophilus than red bean juice (5). other natural ingredients that have also been reported to have potential as sources of nutrition, carbohydrates, protein, vitamins and minerals for the growth of several types of bacteria include tofu industrial wastewater (4) for bacillus sp. bacteria, soybeans for pseudomonas aeruginosa (3), industrial liquid waste tapioca for lactic acid bacteria pediococcus pentosaceus (2), cassava, white sweet potato, yellow sweet potato for e. coli and bacillus sp. (23), bacteria, tofu pulp flour as a growth medium for serratia marcescens (17), bacteria, jicama for the growth of e. coli and s. aureus (24), suwes tubers, taro tubers, and kimpul tubers as a substitute for nutrient agar media (21), arum manis mango seeds (mangifera indica l.) for the growth of candida albicans and aspergillus sp. (20). ina. j. med. lab. sci. tech. 2021; 3(2): 109–121 1 1 9 norma tiku kambuno, et al. the results of our study provide recommendations to the microbiology laboratory which is difficult to obtain nutrient agar media from the industry due to budget constraints, the use of natural ingredients such as pigeonpeas and cowpeais one solution. especially for student practicum needs that require bacterial cultivation, as well as for research purposes for students and lecturers who need it. conclusions there was no difference between the number of bacterial colonies of e. coli and s. aureus on pigeonpea, cowpea and nutrient agar medium. agar medium contained nutrient from cowpea water and pigeonpea water can be used as a alternative medium to substitute nutrient agar in order to grow bacteria e. coli and s. aureus. author contributions norma tiku kambuno: conceptualized the study, developed the proposal, and performed sample collection, data management, analysis, and manuscript writing. ninda p. y. amtaran: participated in laboratory work and performed sample collection. sherly dewu: participated in laboratory work and performed sample collection. kuntum e. nurdin: participated in result interpretation, data analysis, data management and manuscript writing. ni made susilawati: participated in result interpretation, study supervision, and manuscript writing and editing. yoan novicadlitha: participated in result interpretation, data analysis, data management and manuscript writing. acknowladgements we thank dr. rh kristina skm, m.kes, director of poltekkes of the ministry of health. thus, we would like to thank the head of department of medical laboratory technology, agustina w djuma, spd, msc. we also express our deepest gratitude to all technicians department of medical laboratory technology, for their participation in this study. we are also very grateful to irwan budiana for the fruitful discussions to complete this manuscript. conflict of interest the authors declare no conflict of interests. references 1. fachraniah, fardiaz d, idiyanti t. peptone production from soybean press cake and yeast by papain enzyme for the bacterial growth media. j teknol dan ind pangan. 2002;xiii(3):260–6. 2. wulan r, meryandini a, sunarti tc. the potential of tapioca industrial liquid waste as a growth media for starter lactic acid bacteria pediococcus pentosaceus e.1222. j sumberd hayati. 2017;3(1):27–33. ina. j. med. lab. sci. tech. 2021; 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3(2): 109–121 1 2 1 norma tiku kambuno, et al. types of legumes growing on the island of timorntt. semin nas tek kim kejuangan. 2017;0(0):05. 28. american public health association, american chemical society a of oac (u. s. standard methods for the examination of water and sewage. in: association of official agricultural chemists (us). 1920. 29. martyniuk s, oroń j. use of potato extract broth for culturing root-nodule bacteria. polish j microbiol. 2011;60(4):323–7. 26 in silico analysis of antiviral activity and pharmacokinetic prediction of brazilein sappan wood (caesalpinia sappan l.) against sars-cov-2 spike glycoproteins dwi krihariyani1, edy haryanto1, retno sasongkowati1 1department of medical laboratory technology, poltekkes kemenkes surabaya, indonesia correspondence: dwi krihariyani, jl. wiguna tengah xix no.19, surabaya, east java, indonesia zip code: 60294 email: dwikrihariyani@gmail.com received: december 18th, 2020 revised: march 20th, 2021 accepted: march 22th, 2021 published: april 28th, 2021 doi: 10.33086/ijmlst.v3i1.1854 abstract brazilein is one of the secondary sappan wood metabolites which can be used empirically as an antivirus. the sarscov-2 spike (s) glycoproteins play significant roles in attaching and entering the virus into the host cell. this study aims to predict the antiviral activity and pharmacokinetic properties of brazilein of the sappan wood against the in silico sars-cov-2 s glycoproteins with vitamin c as the reference compound. molegro virtual docker 5.5 was used to predict antiviral activity by docking process. sars-cov2 s glycoprotein with nag ligand available in protein data bank (pdb) (pdb id: 7c01) was the receptor used. the pkcsm online tool was used to predict the pharmacokinetic properties and toxicity of brazilein. data were analyzed on the target receptors by comparing the docking bond energies between nag, brazilein, and vitamin c. the smaller the ligands’ bond energy to the target receptor, the more stable the bonds are. the bond energy of nag, brazilein, and vitamin c was –59.2864 kcal/mol, –65.8911 kcal/mol, and –53.9093 kcal/mol, respectively. these results suggested that brazilein has a greater capacity as an antivirus compared to nag and vitamin c. in silico test using the pkcsm online tool demonstrated that brazilein had strong pharmacokinetic properties and relatively low toxicity. keywords adme, brazilein, nag, toxicology, vitamin c. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. introduction in december 2019, mysterious cases of pneumonia were first recorded in wuhan, hubei province, china (1,2). this disease was initially referred to as the 2019 novel coronavirus (2019-ncov). on february 11, 2020, world health organization (who) declared a new name which was coronavirus disease 2019 (covid-19). the disease was caused by severe acute respiratory syndrome file:///d:/users/maharanipertiwikoentjoro/downloads/dwikrihariyani@gmail.com https://journal2.unusa.ac.id/index.php/ijmlst/article/view/1854/version/2338 ina. j. med. lab. sci. tech. 2021; 3(1): 26–37 dwi krihariyani, et al. 2 7 coronavirus 2 (sars-cov-2) (3). to date, covid-19 has become a pandemic. coronavirus is one of the significant pathogens that can infect the human respiratory system and cause mild to severe symptoms. seven coronaviruses are known that can infect humans. four of them (hcovnl63, hcov-229e, hcov-oc43, and hcov-hku1) can infect immunocompetent patients with only moderate symptoms. in contrast, middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sarscov), and sars-cov-2 cause severe symptoms in patients (4,5). the pathogenesis of sars-cov-2 is still unknown, although it is believed not too different from that of sars-cov. sarscov-2 mainly infects alveoli-lining airway cells (6,7). host tropism is the key for virus infection. if sars-cov-2 has identified the host cell according to its viral tropism, it will bind to the host cell via spike (s) glycoprotein (8,9). the s glycoprotein binds to the host cell receptor which is the angiotensinconverting enzyme 2 (ace2). ace2 is also present in other organs, such as the oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, large intestine, skin, thymus, bone marrow, spleen, liver, kidney, brain, pulmonary alveolar epithelial cells, enterocyte cells of the small intestine, endothelial cells of the venous artery, and smooth muscle cells (9,10). therefore, sars-cov-2 does not only infect the human respiratory system, but also other organs. the host cell receptor for sars-cov-2 is the same as sars-cov which is ace2. the receptor-binding domain (rbd) sequences include the sars-cov-2 receptor-binding motif (rbm) that specifically contacts the ace2. sars-cov2 rbm interacts with ace2 in humans with residual effects (gln493), thereby making sars-cov-2 capable of infecting human cells (10,11,12). covid-19 pandemic is a major health concern which requires immediate treatment. one of the efforts that can be made to combat this disease includes enhancing the humans’ immune systems. flavonoids, curcumin, limonoids, vitamin c, vitamin e (tocopherol), and catechins are good candidates which can strengthen the immune system (13,14). sappan wood (caesalpinia sappan l.) consists of five flavonoid compounds, namely brazilin, brazilein, 3'-omethylbrazilin, sappanin, chalcone, and sappancalchone. brazilein is the major constituent in sappan wood that is used empirically as an antivirus. the five compounds can be used as anti-stressants, growth promoters, appetite stimulants, antiviral drugs, aphrodisiacs, and antimicrobial agents (13,15). in silico test to determine the potential use of brazilein as an antivirus is an attractive ina. j. med. lab. sci. tech. 2021; 3(1): 26–37 dwi krihariyani, et al. 2 8 method due to several reasons. this test is safe, free of chemical waste, simple, costeffective, and can shorten research time (16,17). this test is an approach to the prediction of chemical properties of molecular physics, pharmacokinetic properties (absorption, distribution, metabolism, and excretion (adme)), the interaction of compounds with receptors, mechanisms of action, compound selectivity, and compound toxicity. vitamin c, which has been demonstrated to have a role in the treatment of covid-19, was used as a reference in this study (18). the protein data bank (pdb) 7c01 (molecular basis for a potent human neutralizing antibody targeting sars-cov-2 rbd) was used for ligands that have demonstrated good biological activities and are able to bind to the desired biological target (receptor) of the docking. here, we present a study on the prediction of the antiviral activity and pharmacokinetic properties of brazilein of the sappan wood against the in silico sarscov-2 s glycoprotein with vitamin c as the reference compound. materials and methods the materials included the 3d structures of 7c01 which were downloaded from rcsb pdb. the 3d structures of 2-acetamido-2deoxy-beta-d-glucopyranose (nag), brazilein, and vitamin c were downloaded from pubchem®. the tool used in this study was a computer with processor specifications: windows 8 64-bit. the software used was chemdraw professional 16.0, chem3d 16.0, and molegro virtual docker 5.5. prediction of operation (molecular docking) chemdraw professional 16.0 was used to draw the 2d structures of the nag, brazilein, and vitamin c ligands. chem3d 16.0 was then used to convert them to 3d structures. this software was also used to find the most stable conformation. after the minimum energy of nag, brazilein, and vitamin c ligands were calculated, {sybyl2 (*. mol2)} was stored in the format of mol2. the results were in the form of rerank score (rs), which was the energy needed in the process of ligand-receptor interaction. from this score, the antiviral activity of brazilein can be predicted. physicochemical, pharmacokinetic, and toxicity prediction of compounds (pkcsm) the pkcsm online method has been used to predict physicochemical properties, such as the molecular weight (mw), octanol/water partition coefficient logarithm (log p), the number of bonds between atoms that can rotate (torsion), hydrogen bond acceptors (hba), hydrogen bond donors (hbd), and polar surface area (psa). in this study, the pkcsm online tool was used to predict the ina. j. med. lab. sci. tech. 2021; 3(1): 26–37 dwi krihariyani, et al. 2 9 pharmacokinetic properties and toxicity of nag, brazilein and vitamin c. firstly, the 2d molecular structures of nag, brazilein, and vitamin c were drawn with the chemdraw professional 16.0 program. secondly, they were copied to the chem3d 16.0 program to be converted into 3d structures (saved as *.sdf files). thirdly, online smiles translator was used to transform the structures of nag, brazilein, and vitamin c into smiles format. compounds are subsequently processed in smiles format using the pkcsm online tool to predict the adme and toxicity. protox online tool (http://tox.charite.de/tox/) was used to predict the oral toxicity (ld50) in the globally harmonized system (gsh) (17,19). results prediction of operation (molecular docking) the 2d structures of nag, brazilein, and vitamin c are depicted in figure 1, whereas the 3d structures of nag, brazilein, and vitamin c are depicted in figure 2. (a) (b) (c) figure 1. 2d structures. (a) nag, (b) brazilein, (c) and vitamin c (chemdraw professional 16.0) (a) (b) (c) figure 2. 3d structures. (a) nag, (b) brazilein, (c) and vitamin c (chem3d 16.0) http://tox.charite.de/tox/ ina. j. med. lab. sci. tech. 2021; 3(1): 26–37 dwi krihariyani, et al. 3 0 prediction of docking and amino acid the protein structures of pdb 7c01 are depicted in figure 3, whilst the interaction between the ligands (nag, brazilein, and vitamin c) and receptors on the 7c01 protein is depicted in figure 4. figure 5 and table 1 indicate the amino acids involved in the interactions of nag, brazilein, and vitamin c compounds with the 7c01 protein receptor. table 2 displays the redocking effects of nag, brazilein, and vitamin c with 7c01 protein receptor. figure 3. protein structures (pdb 7c01) (molegro virtual docker 5.5) figure 4. receptor interactions between the ligands of (a) nag, (b) brazilein, (c) and vitamin c (molegro virtual docker 5.5) figure 5. amino acids involved in the interactions of (a) nag, (b) brazilein, (c) and vitamin c (c) with 7c01 protein receptor (h-bond, electrostatic, and steric) (molegro virtual docker 5.5) a b c a b c ina. j. med. lab. sci. tech. 2021; 3(1): 26–37 dwi krihariyani, et al. 3 1 table 1. amino acids involved in the interactions of nag (a), brazilein (b), and vitamin c (c) with 7c01 protein receptor (h-bond, electrostatic, and steric). ligands hydrogen bonds and amino acid residues electrostatic interactions and amino acid residues steric interactions and amino acid residues nag 0 – 0 – 1 asn 343(a) brazilein 1 asn 343(a) 0 – 2 asn 343(a) leu 368(a) vitamin c 1 asn 343(a) 0 – 1 asn 343(a) table 2. redocking results using the molegro virtual docker 5.5. redocking/ligand nag brazilein vitamin c i –51.5568 –58.0565 –45.9314 ii –59.2864 –65.8911 –53.9093 iii –51.2831 –55.7692 –46.8643 physicochemical, pharmacokinetic, and toxicity prediction of compounds (pkcsm) the results of the in silico predictions for physicochemical parameters of nag, brazilein, and vitamin c are presented in table 3. the in silico predictions of pharmacokinetic properties and toxicity of nag, brazilein, and vitamin c are presented in table 4. table 3. in silico predictions for physicochemical parameters of nag, brazilein, and vitamin c. struktur smiles nag brazilein vitamin c mw 221.209 284.267 176.124 log p –3.0776 1.624 –1.4074 torsion 2 0 2 hba 6 5 6 hbd 5 3 4 psa (a2) 86.290 119.652 67.321 mw = molecular weight; log p = logarithm of the octanol/water partition coefficient; torsion = bonds between atoms that can rotate; hba = hydrogen bond acceptors; hbd = hydrogen bond donors; psa = polar surface area. ina. j. med. lab. sci. tech. 2021; 3(1): 26–37 dwi krihariyani, et al. 3 2 table 4. in silico predictions of pharmacokinetic properties and toxicity of nag, brazilein, and vitamin c. pharmacokinetic properties and toxicity nag brazilein vitamin c intestinal absorption (human) (%) 19.518 90.021 39.716 skin permeability (log kp) –3.264 –3.498 –3.198 vss (human) (log l/kg) –0.101 0.194 –0.156 bbb permeability (log bb) –1.023 –0.662 –1.031 cyp2d6 substrate (yes/no) no no no cyp2d6 inhibitior (yes/no) no no no total clearance (log ml/min/kg) 0.718 0.264 0.631 oct2 substrate (yes/no) no no no ames toxicity (yes/no) no yes no ld50 (mol/kg) 1.714 2.246 1.271 vss: steady-state volume of distribution; bbb: blood-brain barrier; cyp2d6: cytochrome p2d6; oct2: organic cation transporter 2. discussion activity prediction (molecular docking) in this study, sars-cov-2 s glycoprotein with nag_601[a] ligand (pdb 7c01) was the target molecular receptor. pdb 7c01 was selected since it is homologous with sars-cov-2 s glycoprotein. the host cell receptor for sars-cov-2 is the same as sars-cov which is ace2. the rbd sequence involves sars-cov-2 rbm in direct contact with the ace2 (20). nag_601[a] ligand was selected since it demonstrated good biological activity and can bind to the desired biological target during the docking process to determine the molecules’ physical and chemical properties. the functional groups of nag, brazilein, and vitamin c responsible for pharmacophores which can reduce activity, as well as the lipophilic, electrostatic, and steric/geometric properties of the functional groups, were searched using molegro virtual docker 5.5. this was conducted in order to design the minimum structural characteristics needed for further medicine production development (21). physical and chemical properties (e.g. lipophilic and electrostatic properties) of the drugs play an essential role in transporting the drugs to reach the virus (i.e. absorption and delivery). only drugs that have a high specificity structure can interact and cause activity with the biological receptors. furthermore, the drugs’ electrostatic and steric properties play a role in promoting the precise orientation of the receptor surface molecule (21). ina. j. med. lab. sci. tech. 2021; 3(1): 26–37 dwi krihariyani, et al. 3 3 docking and amino acid analysis activity prediction the protein receptor (pdb 7c01) that was downloaded and imported into the molegro virtual docker 5.5 is presented in figure 3. figure 4 depicts the detection results of the interaction between the three ligands and s glycoprotein. cavity (volume 2587.14) with active nag_601[a] ligand was used because there is a region where the nag ligand interacts with the s glycoprotein. there was a ligand interaction with multiple amino acid residues from the 7c01 protein receptor in the interaction between the ligands and receptors. figure 5 and table 1 indicate the amino acids involved in the nag, brazilein, and vitamin c interaction pathway with the 7c01 protein receptor. the lipophilic/hydrophobic bond, electronic, and steric interactions of the amino acid residues of the protein receptor with these compounds take place. there were variations in the interactions with the s glycoprotein receptor between each of the nag, brazilein, and vitamin c compounds (figure 5 and table 1) since there were differences in the spatial arrangement of the three compound structures. the effects of redocking with the 7c01 protein receptor for nag, brazilein, and vitamin c are displayed in table 2. the binding energy of brazilein with the 7c01 protein receptor was lower than nag and vitamin c ligands. brazil had a rs of –65,8911 kcal/mol, whereas nag and vitamin c had rss of –59,2864 kcal/mol and –53,9093 kcal/mol, respectively. it indicated that brazilein supplied lower energy than nag and vitamin c, hence binding to the receptor would be more stable compared to nag and vitamin c. physicochemical, pharmacokinetic, and toxicity prediction of compounds (pkcsm) the results of the in silico prediction of nag, brazilein, and vitamin c physicochemical parameter values are displayed in table 3. lipinski et al. (1997) analyzed 2,245 drugs from the baseline world drugs index and concluded that if the molecular weight is greater than 500 da, the compounds would be difficult to be absorbed, have low permeability, have a log value of +5 octanol/water (log p) partition coefficient, have hbd expressed by the number of groups o-h and n-h greater than 5, and have an h-bond. since all values are the multiplication of five, this analysis is known as lipinski's rule of five (20). it can be analyzed from table 3 that nag, brazilein, and vitamin c had molecular weight of less than 500 da, logp values of less than 5, acceptor and donor values of less than 10, hence it can be inferred that it is easy to absorb these three compounds. ina. j. med. lab. sci. tech. 2021; 3(1): 26–37 dwi krihariyani, et al. 3 4 the in silico pharmacokinetic properties and toxicity predictions of nag, brazilein, and vitamin c are presented in table 4. according to chander et al. (21), a compound is categorized as having strong absorption capacity if the absorption value is > 80%, whereas it has poor absorption capacity if the absorption value is < 30%. the primary site for absorption of oral medications is the intestine (22). table 4 shows that nag’s human intestinal absorption value was less than 30%, whereas brazilein’s and vitamin c’s were more than 80%. these results suggested that brazilein and vitamin c had better absorption capacity than nag. pires et al. (20) stated that a compound is described to have relatively low skin permeability if its value is log kp > –2.5 (23). table 4 displays that the skin permeability (log kp) of nag, brazilein, and vitamin c was lower than –2.5, hence these three compounds were supposed to have strong skin permeability. the volume of distribution (vd) refers to the theoretical volume that is required to distribute the total dose of the medicine equally in order to give the same concentration as in the blood plasma. the higher the vd value, the more drugs than blood plasma are distributed to the body’s tissues. pires et al. (20) stated that a compound is described to have high vd if the value of log vd is > 0.45, but low vd if the value of log vd is < –0.15 (19). the steadystate volume of distribution (vss) values for nag, brazilein, and vitamin c were –0.101, 0.194, and –0.156 (table 4), thus it could be estimated that all the derivatives of these compounds can be uniformly distributed to have the same concentration as in blood plasma. another significant parameter to consider is the ability of drugs to cross the blood-brain barrier (bbb) to help decrease side effects and toxicity, or to improve the effectiveness of drugs whose pharmacological activity is present in the brain. bbb permeability is calculated as log bb (the logarithmic ratio of brain-to-plasma concentrations) in vivo in an animal model. pires et al. (20) stated that compounds are believed to be able to pass the bbb effectively if their log bb values are > 0.3, and cannot be adequately distributed if their log bb values are < –1 (23). the log bb values of nag, brazilein, and vitamin c were –1.023, –0.662, and –1.031, respectively (table 4). the log bb value of brazilein was higher than –1, while nag’s and vitamin c’s were lower than –1, hence it was expected that brazilein was capable of penetrating the bbb moderately, while nag and vitamin c compounds were less capable. most of the metabolic reactions require oxidation process. cytochrome p450 is an essential detoxification enzyme in the body, and it is primarily found in the liver. cytochrome p450 acts by oxidizing and ina. j. med. lab. sci. tech. 2021; 3(1): 26–37 dwi krihariyani, et al. 3 5 promoting the excretion of unidentified organic compounds, including narcotics. enzyme inhibitors, such as grapefruit juice, are contraindicated against cytochrome p450 enzymes since it can affect drug metabolism. it is therefore important to assess the ability of compounds to inhibit cytochrome p450, which is represented as the cytochrome p2d6 (cyp2d6) isoform in this study. table 4 displays that the cyp2d6 enzyme is not impaired or inhibited by nag, brazilein, and vitamin c, thus it could be expected that these derivatives appear to be metabolized by the p450 enzyme (19). the compound excretion process can be carried out by calculating the total clearance (cltot) and renal organic cation transporter 2 (oct2) substrate constants. cltot is a mixture of liver clearance (liver and bile metabolism) and renal clearance (excretion through the kidneys). this is related to bioavailability. to achieve steady-state concentrations, it is necessary to determine the dosage level (19). cltot values of nag, brazilein, and vitamin c were 0.718, 0.264, and 0.631, respectively (table 4). therefore, the rate of compound excretion could be estimated from these values. oct2 is a kidney-based transporter that plays a significant role in drug and endogenous compound disposal and clearance. when given along with oct2 inhibitors, oct2 substrates also have the potential to cause side interactions. the three compounds did not affect the oct2 substrates (table 4), thus it could be interpreted that the nag, brazilein, and vitamin c derivatives were not substrates of oct2. to assess the toxicity of the compounds, the ames toxicity test was carried out. the ames toxicity test is a commonly used method to determine the mutagenic ability of bacteria-based compounds. a positive test result indicates the mutagenicity of the compound and may thus serve as a carcinogen (19). brazilein might cause mutagenic effects, while nag and vitamin c were not expected to cause mutagenic effects (table 4). conclusions the bond energy of brazilein was lower than nag and vitamin c. the comparison of the bond energy values showed that brazilein had higher antiviral capacity than nag and vitamin c in silico using the molecular docking process. the physicochemical, pharmacokinetic, and toxicity properties of brazilein showed that it was projected to have strong skin permeability, be distributed uniformly to provide the same concentration as in the blood plasma, be penetrating the bbb moderately, be very well absorbed in the intestine, be metabolized by the p450 enzyme, and have relatively low toxicity. ina. j. med. lab. sci. tech. 2021; 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1: 3-25. 25. pires dev, blundell tl, ascher db. pkcsm: predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures. j med chem. 2015; 58: 4066-4072. 11 blood lead concentrations and the neuropsychology scores of pregnant women in klang valley, malaysia shamsul bahari shamsudin1, jamal hisham hashim2, nik nasri nik ismail3, a. jamal a. rahman3, maharani pertiwi koentjoro4 1department of community and family medicine, faculty of medicine & health sciences, universiti malaysia sabah, sabah, malaysia 2united nations university – international institute for global health (unu–iigh) building, ukm medical centre, kuala lumpur, malasyia 3universiti kebangsaan malaysia medical centre, kuala lumpur, wilayah persekutuan, malasyia 4department of medical laboratory technology, faculty of health, universitas nahdlatul ulama surabaya, surabaya, indonesia correspondence: shamsul bahari shamsudin, faculty of medical and health sciences, universiti malaysia sabah, kota kinabalu, sabah. malasyia zip code : 88400 email: shamsul@ums.edu.my received: march 13, 2020 revised: march 16, 2020 accepted: april 4, 2020 abstract pregnant women with high blood lead posed high risk to their fetus as placental transfer can occurs to the fetus. the objective of this study was to identify the relationship between blood lead and the neuropsychological score of women who were in their 3rd trimester of pregnancy. these respondents were undergoing a routine antenatal checkup at a teaching hospital located in klang valley areas. blood lead concentrations were analyzed using graphite furnace atomic absorption spectrophotometer (aas). the neuropsychological scores were measured with who neurobehavioral core test battery (nctb). the test consists of 7 items, which made up of the digit symbol, trail making, digit span, benton visual retention test, pursuit aiming, santa ana manual dexterity, reaction time and movement time tests. the mean blood lead was 7.78±4.77 µg/dl. the mean score for the total nctb test was 50.00±5.24. statistical analysis showed blood lead concentrations were inversely correlated with the total nctb score (r= –0.462, p≤0.01). the correlation was about 21.3%. the general linear model (glm) showed that age (β= –0.15, p = 0.017), weight (β = 2.67, p = 0.05) and height (β= –1.97, p = 0.05) also influence the total neuropsychological scores. in conclusion, blood lead reduces the total neuropsychological scores. the scores for each of the 7 items were inversely and significantly correlated with blood lead concentrations except for the trail making and santa ana manual dexterity tests. keywords neuropsychological scores, blood lead concentrations, pregnant mothers ina j med lab sci tech 2020; 2(1): 11-20 shamsul bahari shamsudin, et al. 1 2 introduction lead could be produced by human activities such as industrial, mining and agricultural activities that can spread by air, water and soil (1). lead route of entry to human body through the inhalation, ingestion and skin contact. lead has no function in human body. previous study suggested lead has a negative effect on human health even though at very low level. lead is a neurotoxin and its toxicity affect the function of the central nervous system and peripheral nerve. for adults, the main effects are peripheral neuropathy whereby the impulse conduction in the nerve is slowed down. peripheral neuropathies influence the motor and sensory nerve. the effects are the wrist and the ankle drop that are the result of the defect in the radial and peroneal nerves (1). pregnant woman is at very high risk to the lead toxicity because pregnant mothers need high level of nutrient such as calcium as well as iron to support the growth and the development of the fetus. lead could replace the function of calcium and iron. tests on the biochemical and physiological process found that the concentration of calcium and iron in the blood are inversely proportional to the lead concentrations (2). study shows blood lead levels (bbls) in pregnant women at 20.8 μg/dl can cause maternal disorders, when blls reaches 2.57 μg/dl, it can cause stress and fatigue levels in pregnant women (4). blls <5 μg/dl may cause pregnant women to have pre– eclampsia and endanger the mother’s kidney, while blls <10 μg/dl can increase blood pressure or hypertension (4). another research done by bayat et al, 2016 showed there was an increase in systolic blood pressure (0.014 mmhg) and diastolic (0.013 mmhg) following the increase of 1 μg/dl of blls (p = 0.04) (5). this study aims to investigate the association of blls with neuropsychology of pregnant women in klang valley, malaysia. klang valley area was an ex-mining erea in peninsular malaysia. active mining activity was in 1950-1970. the residue of lead and others heavy metal was still high in air, water and soil. the neurobehavioral core test battery (nctb) can detect early signs of disturbance or failure on the functions of nervous system. who was introduced nctb identifying the nervous dysfunction among workers who are working with the neurotoxin chemicals (3). materials and methods the study population is women in their 3rd trimester of pregnancy who reside in the klang valley areas. these pregnant women were attending their routine antenatal checkup at a hospital in kuala lumpur. total 202 respondents were selected through purposive sampling based on specific criteria, that she must be malaysian citizen, in the 3rd trimester pregnancy and have ina j med lab sci tech 2020; 2(1): 11-20 shamsul bahari shamsudin, et al. 1 3 signed consent to participate. their pregnancies were categorized as “high–risk” because they were referral cases from private or government clinics within the areas. these women were either in their 1st or 5th pregnancies, had hypertension, diabetes, anemia or a history of previous delivery complication. questionnaire interviews were carried out to collect information on the demographic as well as socioeconomic background and their health status. the questionnaire consists of the questions on their age, ethnicity, number of children, educational level, occupation and areas of residential. while the questions on health status include of number of deliveries, medical history, smoking habit, alcohol intake and medication during pregnancy. blood sampling and analysis venous blood was sampled during or after the 28 weeks of pregnancy. five (5) ml of the blood samples were put into a special vacuum container tube, which contained heparin (anticoagulant). all sample of blood lead were analyzed using graphite furnace atomic absorption spectrophotometer model hitachi z5700 with polarized zeeman. blood sample were diluted with matrix modifier solution in a ratio of 1:5. the modified matrix solution also acts as antifreeze agent during storage. the modified matrix solution consists of 10 ml triton–x, 10% 0.3 g ethylene diamine tetra acetic acid (edta) and 5 g ammonium dehydrogenate phosphate in 1–liter deionizer distilled water. lypocheck sample were used as a reference sample for quality control procedure during the analysis. neuropsychological assessment a neuropsychological test conducted in this study were neuropsychological core test battery (nctb) and it is commonly used by who (3). it was an instrument used to detect early nervous systems failure because of the exposure of neurotoxin agents. this instrument is very sensitive and can detect early sign of nerve disturbance at a very low level of exposure. it consists of the benton visual retention, digit span, digit symbol, pursuit aiming, reaction time, santa ana manual dexterity and profile of mood states (poms) tests. however, the poms test was not be conducted in this study, instead it was replaced by two more tests, movement time and trail making tests. the description and functional domain of the tests are shown in table 1. standard score calculation the raw score obtained from the neurobehavioral tests, was transformed into standard scores. from these transformed data or standard scores, the mean value is 50 and the standard deviation is 10. the calculation for the standard score is shown: standard score = [(raw data – mean) / standard deviation] x 10 + 50. ina j med lab sci tech 2020; 2(1): 11-20 shamsul bahari shamsudin, et al. 1 4 table 1. descriptions of neuropsychological (nctb) tests test functional domain tested description reaction/movement time attention/response & movement speed it measures how fast a person reacts/moves. it requires sustained attention by the subject. digit span auditory memory it is a test of immediate (short–term) auditory memory that requires focused attention. santa ana manual dexterity manual dexterity it requires rapid eye–hand coordination movements. digit symbol perceptual–motor speed it is a test of perceptual motor speed that also requires learning of associations. benton visual retention visual perception/memory it is a short–term visual memory that requires focused attention. it also measures the ability to organize geometrical patterns in space and memorize them. pursuit aiming motor steadiness it measures the ability to make quick and accurate movements with the hand. trail making motor tracking it measures visual motor tracking and visual scanning. it requires an attention. source: who. 1983. operational guide for the who neurobehavioral core test battery. world health organization, geneva. 15 februari (14). ethical clearance this research was approved by the research ethics committee of hospital universiti kebangsaan malaysia (ukm/ec711002105465-1). involvement of respondents is based on a written agreement with filling a consent form. respondents may withdraw at any time if they do not agree or are not satisfied with any study procedures. results table 2 shows the demography and socioeconomic background information of the respondents. the mean age for mothers was 29.6 years old and their mean gestation period was 31.6 weeks. the ethnic and religious distributions of the respondents are shown in table 2. the malays made up 58.4 % of the respondents. about 35.6% respondents are full time housewives; while others work as general worker, operate a business, self–employed or part–time wage earner. the average household income is rm 2884.7 (table 2). the mean blood lead level was 7.78 with standard deviation of 4.77 µg/dl. figure 1 shows the data distribution of blood lead for the respondents. kolmogorov– smirnov normality test shows that the distribution was normal (p > 0.05). ina j med lab sci tech 2020; 2(1): 11-20 shamsul bahari shamsudin, et al. 1 5 venous blood lead conc. (ug/dl) 24.0 22.0 20.0 18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0 f re q u e n c y 30 20 10 0 fig 1. distributions of blood lead concentrations (µg/dl) table 2. respondents’ demographic and socioeconomic background variable range mean std. dev. mother age (year) 19 – 44 29.6 5.57 mother education (year) 6 – 19 12.1 2.08 father’s education (year) 6 – 22 12.8 2.64 total household income (rm) 750 – 8500 2884.7 1383.98 mother’s height (m) 1.43 – 1.72 1.57 7.12 mother’s weight (kg) 50.0 – 92.1 68.3 7.68 pregnancy duration (weeks) 28 – 36 31.6 2.13 number of pregnancies 1 – 6 1.7 1.07 number of children 1 – 6 1.6 1.04 n = 202 table 3 shows the scores for neuropsychological test (nctb) that was carried out on the respondents. it took about 30–45 minutes to complete all the 7 types of nctb test. standard score index values were calculated from the raw scores and used for all statistical analysis. figure 2 shows the standardized nctb score test distributions, which were normal (p > 0.05). ina j med lab sci tech 2020; 2(1): 11-20 shamsul bahari shamsudin, et al. 1 6 nctb average score 68.0 66.0 64.0 62.0 60.0 58.0 56.0 54.0 52.0 50.0 48.0 46.0 44.0 42.0 40.0 38.0 36.0 f re q u e n c y 40 30 20 10 0 fig 2. distributions of neuropsychological scores table 3. neuropsychological scores (nctb) of pregnant mothers neuropsychological scores (nctb) range mean std. dev. digit symbol 29.0 – 78.3 50.0 10.00 pursuit aiming 24.5 – 80.0 50.0 10.00 trail making 26.4 – 73.1 50.0 10.00 digit span 35.8 – 85.2 50.0 10.00 benton visual retention test 24.8 – 67.3 50.0 10.00 santa ana manual dexterity 23.1 – 70.8 50.0 10.00 reaction time 23.5 – 72.0 50.0 10.00 total score total score 36.3 – 67.6 50.0 5.54 n = 202 table 4 shows the correlation between blood lead and each nctb test. the total nctb scores also showed inversely significant correlation with the respondents’ blood lead except for the pursuit aiming and santa ana manual dexterity tests. the linear model shows that about 21.3% of the variations in blood lead contributed to the neuropsychological scores. table 5 shows that the total nctb scores were influenced by the respondents’ blood lead, age, weight and height after all the confounding factors were adjusted. this model shows that these factors contribute 27% of the variations in the nctb scores. ina j med lab sci tech 2020; 2(1): 11-20 shamsul bahari shamsudin, et al. 1 7 venous blood lead conc. (ug/dl) 3020100-10 n c t b a v e ra g e s c o re 70 60 50 40 30 rsq = 0.2131 fig 3. correlation between blood lead concentrations and neuropsychological scores table 4. correlation blood lead and neuropsychological scores neuropsychological test (nctb) blood lead (µg /dl) r p value digit symbol –0.347 < 0.001** pursuit aiming – 0.021 0.762 trail making – 0.254 < 0.001** digit span – 0.447 < 0.001** benton visual retention test – 0.256 < 0.001** santa ana manual dexterity – 0.016 0.817 reaction time – 0.450 < 0.001** total score –0.462 < 0.001** n = 202 ** significant at p < 0.01 table 5. correlations of blood lead on the neuropsychological scores after adjustment of confounders dependant variable (mean neuropsychological score) regression coefficient β statistict p value (constant) c 61.74 7.42 < 0.001** blood lead (µg /dl) – 0.47 – 7.51 < 0.001** age (years) – 0.15 – 2.40 0.017* weight (kg) 0.12 2.67 0.008** height (m) – 0.10 – 1.61 0.110 educational level (years) 0.08 1.18 0.239 household income (rm) – 0.13 – 1.79 0.075 n = 202 ** significant at p ≤ 0.01 f value = 12.787, p < 0.001 * significant at p ≤ 0.05 r = 0.531, r2 = 0.282 ina j med lab sci tech 2020; 2(1): 11-20 shamsul bahari shamsudin, et al. 1 8 discussion the mean blood lead of the respondents was 7.78 µg/dl (figure 2), which is more than the blood lead of electronic industries soldering workers (6.10 µg/l) (4). pregnant women need an optimum nutrient such as ca2+ and fe2+ or fe3+, for the growth of the babies (5,6). the body tends to absorb lead if the calcium and iron intake were insufficient in the diet. lead in the mothers’ blood systems has a potential of being transferred to the fetus through the placenta and this process began 2–3 months at an early stage of pregnancies. blood lead concentrations for pregnant mothers should not exceed 10 µg/dl (7). however, almost 27% of the respondents have blood lead concentrations of more than 10 µg/dl. studies (8) in african reported that mean blood lead for pregnant mothers who live in the city and the rural ranged from 0.83 to 99 µg/dl. the difference of those data was significantly difference. this proves that environment can influence lead concentrations in the blood. there was no significant relationship between blood lead with age and the gestation period. however, the mean blood lead in previous study was higher than this study. another study (9), on pregnant women reported that the mean blood lead concentration was 8.59 ± 4.45 µg/dl and about 27.8% respondents had blood lead concentrations higher than 10 µg/dl. the study found that housewives had higher blood lead (9.55±5.5 µg/dl) than those working in offices (7.44±2.77 µg/dl), factories (8.61±3.39 µg/dl) and shops (7.01±3.13 µg/dl). however, the difference was not statistically significant. study by (10) reported that the mean blood lead for women in the avon are of the uk was range 0.41– 19.14 µg/dl. studies at port pirie, australia (11) reported that 646 women who are 30 to 36 weeks pregnant had an almost similar mean blood lead level of 7.2 µg/dl and 28% had blood lead of more than 10 µg/dl. in general, neuropsychological scores were calculated from the 7 tests in the nctb. correlation tests indicated that blood lead for these pregnant mothers had an inverse significant correlation with digit symbol, digit span, trail making, benton visual retention test and reaction time (table 4). only pursuit aiming and santa ana manual dexterity scores were not significantly correlated with blood lead (table 4). the nctb test result shows that pregnant mother with high blood lead have difficulties in concentrating and have short– term hearing and visual abilities (digit span test, benton visual retention test). they have slow motor speed through vision (digit symbol test) slow reaction toward visual stimulation (reaction time test) and low attention ability, visual scanning and visual motor trailing (trail making test). in figure 3 shown ultiple regression test result demonstrated that mean ina j med lab sci tech 2020; 2(1): 11-20 shamsul bahari shamsudin, et al. 1 9 neuropsychological score for pregnant mothers were influenced by blood lead (β = –0.56, p < 0.001), age (β = 0.12, p < 0.017), weight (β = 0.12, p = 0.08) and height (β = – 0.01, p = 0.050). the model was significant (f = 18.23, p < 0.001) with the r2 value, which shows that 27% of the variations in the nctb scores were influenced by the variables shown below: nctb mean score = 65.71 – 0.56 (blood lead) – 0.15 (age) – 0.10 (height) + 0.12 (weight) previous studies showed that blood lead among female workers had the abilities to lower the nctb score (12,13). studies (12) showed that 140 female factory operators had a mean blood lead concentration of 30.77 µg/dl and it was inversely correlated only with the reaction time test (r = –0.201, p = 0.017). meanwhile (4) found that mean blood lead for female soldering workers at an electronic factory was 6.1 µg/dl, and the comparative group was 4.6 µg/dl. the respondents' blood lead had significant relationships with digit span test (p = 0.003), santa ana manual dexterity test (p = 0.007) and total nctb scores (p = 0.001). the exposed workers had significantly lower mean score compared to unexposed workers for digit symbol test, trail making test and pursuit aiming test. pregnant mothers with high blood lead had problems with the ability to concentrate and have poor short–term auditory and visual memory and have the inability to organize geometry patterns in space and memorizing them. they have poor perceptual motor speed and fail to match between symbol and digits. they also had slow reaction to stimulation due to poor ability to pay attention and low attention ability, which cause poor visual motor tracking as well as poor visual scanning. blood lead concentrations, age, weight and height influenced 27% of the variations in neuropsychological score after adjusting for confounders. about 1 µg/dl increment in blood lead will reduce 0.4 of the neuropsychological mean score of these pregnant mothers. conclusions in conclusion, even though the blood lead concentration is quite low, it affects the neuropsychological ability of the respondents. digit symbol, digit span, trail making, benton visual retention and reaction time test scores had significant inverse correlations with the blood lead. acknowladgements appreciation goes to all respondents and management of environmental health laboratory, hospital universiti kebangsaan ina j med lab sci tech 2020; 2(1): 11-20 shamsul bahari shamsudin, et al. 2 0 malaysia who gave the commitment that this study can be implemented successfully. conflict of interest there are no conflicts of interest. references 1. rubens o, logina i, kravale i, eglite m, donaghy m. peripheral neuropathy in chronic occupational inorganic lead exposure: a clinical and electrophysiological study. j neurol neurosurg psychiatry. 2009; 71: 200–204. 2. mahaffey kr. (1995). nutrition and lead: strategies for public health. env health perspect. 1995; 103(6):191–196. 3. anger wk. reconsideration of the who nctb strategy and test selection. 2015; 0: 224–231. 4. forsyth je, islam ms, parvez sm, raqib r, rahman ms, muehe em, fendorf s, luby sp. prevalence of elevated blood lead levels among pregnant women and sources of lead exposure in rural bangladesh: a case control study. env res. 2018; 166:1–9. 5. marangoni f, cetin i, verduci e, canzone g, giovannini m, scollo p, corsello g, poli a. maternal diet and nutrient requirements in pregnancy and breastfeeding. an italian consensus documents. nutrients. 2016; 8(629): doi:10.3390/nu8100629. 6. saunders av, craig wj, baines sk, posen js. iron and vegetarian diets. 2012: 2: 11–16. 7. león ol, pacheco jms, martínez se, rodríguez ee, juárez fxc, carrilo as, quiñones, alanís fv, vargas gg, hernández mm, sustaita jd. the relationship between blood lead levels and occupational exposure in a pregnant population. bmc pub health. 2016; 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33(1):83–100. 13. shamsul bs, jamal hh, nasri nin, rahman ajb. the effects of blood lead on the neuropsychology score of 3rd trimester pregnant women in the klang valley, malaysia. j dis glo health. 2016; 11(3): 134–141. 60 combination test chinese leaves extract (leucaena leucocephala folium) and aloe vera inhibiting growth escherichia coli putra rahmadea utami1, chairani1, hendra yudha1 1department of medical laboratory technology, fakultas of health science, universitas perintis indonesia, padang, indonesia correspondence: putra rahmadea utami, universitas perintis indonesia, jl. adinegoro km 17, simpang kalumpang, lubuk buaya, padang, west sumatera, indonesia zip code : 25586 email: putrarahmadeautami123@gmail.com received: june 10, 2020 revised: juni 29, 2020 accepted: august 20, 2020 abstract chinese petai (leucaena leucocephala folium) and aloe vera (aloe vera l.) have medicinal properties among the plants. the objective of this study was to determine the number of inhibitory zones produced by the ethanol extract of chinese petai and aloe vera on the growth of escherichia coli. the research method was in-vitro experimental laboratory research design with the kirby bauer method. the samples used was chinese petai and aloe vera with pure strains of e. coli. one way anova was used to compare the differences in inhibition of chinese petai and aloe vera on the growth of e. coli. the results of a combination of chinese petai and aloe vera extract test showed that there were significant differences in the concentration of 25 g, 50 g, 75 g, and 100 g (p <0.05). the ethanol extract of chinese petai and aloe vera can inhibit the growth of e. coli. from the results of this study found that there was an interaction on the combination of ethanol extract of chinese petai and aloe vera inhibiting the growth of e. coli with the most effective concentration being 100 g/ml. this study can find out the benefits of petai cina and aloe vera also the public will know the benefits and efficacy of chinese petai and aloe vera leaves in medicine. keywords leucaena leucocephala folium, aloe vera l., escherichia coli, inhibition, combination introduction chinese petai leaf (leucaena leucocephala folium) and aloe vera (aloe vera) are one of the traditional medicines that have the potential to be developed and utilized by the people of indonesia (1). chinese petai contains alkaloids, saponins, lectins, tannins, mimosin, leucanin, protein, calcium, phosphorus, iron, fatty acids, fibre, vitamin a and other vitamins. besides that, chinese petai leaves also contain various flavonoids that become anti-inflammatory and antioxidants. lectins-contained to stimulate skin cell growth while the alkaloids function as antimicrobials. aloe vera beneficial properties, including tannin, amino ina j med lab sci tech 2020; 2(2): 60-67 putra rahmadea utami, et al. 6 1 acids, anthraquinone which is a phenolic compound that acts as a laxative, antimicrobial agent and has a strong analgesic effect. it also has campesterol, sitosterol and lupeol compounds. these compounds act as an anti-inflammatory and antibacterial agent (1,2). escherichia coli is the bacteria that most often cause urinary tract infections and nosocomial infections. e. coli can cause primary intestinal infection and also used to assess whether a water supply for household use is good or not. e. coli is a bacteria commensal which can pathogenic, acting as a primary cause of morbidity and mortality throughout the world (1,3,4). chinese lamtoro and petai plants come from tropical america that has a deep root system and resistant to dry conditions. chinese petai contains alkaloids, saponins, flavonoids, lectins, calcium, phosphorus, iron, tannins, mimosin, leukanin, proteins vitamin a and vitamin b (6). the leaves contain 22% protein (4). seeds, leaves and all parts of the plant can use to treat several diseases, such as diabetes, fracture, intestinal worms, ulcer, late menstruation, inflammation of the kidneys (nephritis) and insomnia. aloe vera is a traditional medicinal plant that has many benefits and benefits. it is a cactus-like plant originating from africa, which belongs to the liliaceae family and closed seed plants, can grow in dry areas and also in cold climates (7,8). e. coli belongs to the family enterobacteriaceae. this bacterium is a gram-negative bacterium, in the form of a short stem, has a flagellum, measuring 0.4 to 0.7 μm x 1.4 μm. e. coli grows well in almost all breeding media can ferment lactose and is micro-aerophilic. these bacteria become pathogens when outside intestinal tissue. clinically frequently infected sites are the urinary tract, bile duct, and other places in the abdominal cavity and any anatomic site (e.g. prostate gland, lung, bone) can be the site of disease (9). e. coli is a gut flora in the digestive tract that can turn into pathogenic opportunists and cause diseases such as diarrhoea. this bacterium has various pathogenic strains, including enteropathogenic escherichia coli (epec), enterotoxigenic escherichia coli (etec), enteroinvansive escherichia coli (eiec) and enterohemorrhagic escherichia coli (ehec) (10,11). combination therapy uses to broaden the spectrum of antimicrobials, minimize toxicity, slow down the process of antibiotic resistance and reduce the dose of the drug and has considerable antimicrobial activity. recently, there has been no information about the effective concentration of the combination of extracts of chinese petai leaves and aloe inhibiting the growth of the bacteria e. coli. the purpose of this study ina j med lab sci tech 2020; 2(2): 60-67 putra rahmadea utami, et al. 6 2 was to analyze the interaction effects of ethanol extracts of chinese petai leaves and aloe vera leaves in inhibiting the growth of e. coli (4,5). materials and methods this research conducted using the completely randomized experimental laboratory method. the research method involves making ethanol extract from simplicia (crude drugs) by maceration using 96% ethanol solvent. it also examines the interaction between ethanol extracts of chinese petai leaves and aloe vera stem on the inhibitory power of e. coli and in vitro by measuring the inhibitory by diffusion method according to kirby bauer. the principle of the kirby bauer method is the inhibition of the growth of microorganisms, the diameter of the zone of inhibition of bacterial growth shows the sensitivity of the bacteria, the wider the zone diameter formed by bacteria, the better it is to inhibit bacterial growth. the tools used in this study were petri dishes, tissue cotton, test tubes, wire mesh, syringes, matches, bunsen burner analytical scales, slide callipers, microscopes, sterile stick cotton, erlenmeyer, beaker glass, parchment paper, tweezers, ovens, incubators, label paper, measuring pipettes, water baths, paper cutters, rotary evaporators. the materials used in this study are pure strains of e. coli ethanol extracts of chinese petai leaves and aloe vera, sterile physiological nacl (in packaging), steril empty disks, aquades as negative controls and antibiotic as the positive control. while, the culture media used were mha (muller hilton agar), nb (nutrient broth), endo agar, nutrient agar, and mc farland solution. the extraction method used to extract chinese petai plants and aloe vera is a cold method extraction method, namely maceration. chinese petai leaves that turned into simplicia powder are added with 70% ethanol solvent until submerged. maceration poured and squeezed. the pulp is macerated again with a new solution until submerged. the maceration process carried out three times then it was evaporated on a rotary evaporator until a thick extract of chinese petai leaf obtained then lastly weighed to determine its weight. extraction of chinese petai leaves and aloe vera aloe vera leaves peeled first to get as much as 1000 grams of aloe vera gel mashed with a blender then soaked with 96% ethanol solvent until aloe vera is submerged completely, after that it is allowed to stand for 2-3 days in a closed jar. then, the liquid extract was filtered with gauze filter and collected the extract in a bottle. steering of all filtrates obtained from the immersion. concentrated with a rotary evaporator until thick extract is obtained (1). ina j med lab sci tech 2020; 2(2): 60-67 putra rahmadea utami, et al. 6 3 extract determination the extract yield calculated by comparing the weight of the ethanol extract obtained with the initial sample weight. the yield obtains through this formula: (extract weight obtained/initial sample weight) x 100%. results inhibition test of the ethanol extract of chinese petai leaves and aloe vera with the disc diffusion method that carried out with various concentrations of the extract. table 1. results of inhibitory tests of chinese petai leaf extract against escherichia coli extract concentration (mg/ml) extract repetition (mm) x̄ sd p. sig 1 2 3 25 0 0 0 0 ± 0. 0.00 50 0 0 0 0 ± 0. 75 6 6 7 6.3 ± 0.58 100 7 8 8 7.7 ± 0.58 control (+) chloramphenicol 30 29 30 29.7 ± 0.58 table 2. results of inhibitory aloe vera leaf extract against escherichia coli bacteria (mm) extract concentration (mg/ml) extract repetition (mm) x̄ sd p. sig 1 2 3 25 8 10 9 9 1 0.00 50 10 11 10 10.3 0.58 75 12 12 11 11.7 0.58 100 14 15 14 14.3 0.58 control (+) chloramphenicol 30 28.5 30 29.5 0.87 table 3. test results of combination inhibition of chinese petai leaf extract and aloe vera on escherichia coli (mm) extract concentration (mg / ml) extract repetition (mm) x̄ sd p. sig 1 2 3 25 6 7 6 6.3 0.58 0.00 50 8 9 8 8.3 0.58 75 10 11 10 10.3 0.58 100 12 12 11 11.7 0.58 control (+) chloramphenicol 27 28.5 26 27.2 1.26 ina j med lab sci tech 2020; 2(2): 60-67 putra rahmadea utami, et al. 6 4 fig 1. the results of the test for the inhibition of aloe vera against escherichia coli with a concentration of 4 samples. each sample size was carried out 3 times. in the middle is the (+) chloramphenicol control based on table 1, the inhibitory test results of chinese petai leaf extract against e. coli (mm) bacteria with a concentration of 75 mg/ml formed inhibition zone 6.3 ± 0.58 mm. moreover, the amount of 100 mg/ml extract created an inhibition zone of 7.7 ± 0.58 mm. a positive control using the chloramphenicol antibiotic obtained the broadest inhibition zone (29.7 ± 0.58 mm) compared to other concentrations. based on table 2, the inhibitory test results of aloe vera extract against e. coli (mm) bacteria with a concentration of 25 mg/ml formed inhibition zones 9± 1 mm and it increases following the increase of the extract concentrations. a positive control using the chloramphenicol antibiotic obtained the broadest inhibition zone (29.5 ± 0.87 mm) compared to others. based on table 3, the extract concentration of 25 mg/ml formed an inhibition zone 6.3 ± 0.58 mm, its increases following the increase of the extract concentrations. a positive control using the chloramphenicol antibiotic obtained the broadest inhibition zone (27.2 ± 1.26 mm) compared to others. discussion interaction of the combination of ethanol extract of chinese petai leaves and aloe vera against e. coli bacteria showed in the test of direct mixing of the two antibacterial agents. it formed the inhibitory zone when placed close together. the test to identify the synergistic effect of antibacterial combinations, discs used and treated with a single antimicrobial agent each, then both are placed at a distance equal to the average number of the radius of the zone of inhibition ina j med lab sci tech 2020; 2(2): 60-67 putra rahmadea utami, et al. 6 5 of the antimicrobial agent when tested separately (12,4). the interaction showed the formation of a bridge at or near the intersection of two inhibitory zones or no longitudinal inhibitory zones formed. the combination is said to be synergistic if it created like a bridge at or near the intersection point of two inhibitory zones, or an obstacle to growth which is the combined effect of the two antimicrobial agents (13). based on observations of the results of a combination of inhibitory test of chinese petai leaf extract and aloe vera on e. coli (mm) bacteria, the concentration was 25 mg/ml producing a zone of inhibition 6.3 ± 0.58 mm, a concentration of 50 mg/ml resulted in an inhibition zone of 8, 3 ± 0.58 mm, concentration of 75 mg/ml produces inhibition zone 10.3 ± 0.58 mm, concentration of 100 mg/ml with inhibition zone 11.7 ± 0.58 mm. positive control using the antibiotic chloramphenicol obtained the biggest inhibition zone compared to other concentrations of 27.2 ± 1.26 mm. according to davis stout in a'lana (14), the strength criteria for the combination of chinese petai leaves and aloe vera concentrations of 25 mg/ml and 50 gr/ml categorized as moderate, at a concentration of 75 mg/ml and 100 mg/ml classified as strong criteria. petai china has saponins to inhibit the growth of bacteria e. coli by damaging the cytoplasmic membrane in bacteria that cause leakage of metabolites that inactivate the bacterial enzyme systems. damaged cytoplasmic membranes can prevent the entry of nutrients needed by the nutrients the bacteria produce energy. it causes bacteria to experience growth retardation and even cause bacterial death (6). aloe vera contains active substances such as anthraquinone, tannins and flavonoids. anthraquinone binds with nucleic acids and forms a complex that can damage the acid (dna and rna) of inhibited bacteria. tannin as antibacterial works by inactivating adhesin so that bacteria cannot stick to host epithelial cells. aloe vera also contains flavonoids, which will cause lysis and inhibit the formation of cell walls. this mechanism causes aloe vera to kill or inhibit the formation of bacteria (15,16,17). flavonoids are efficient to inhibit the growth of gram-positive bacteria. flavonoids are polar compounds which are easy to penetrate the peptidoglycan layer. furthermore, the gram-positive cell wall contains polysaccharides (acidic polysaccharides) is a water-soluble polymer. this cell wall functions as the media of positive ions transport to get in and out. this solubility indicates that the gram-positive cell wall is polar. flavonoids disrupt cell wall function. this results in cell lysis (3,20,21). the mechanism of bacterial inhibition of tannin compounds is by protein reproduction, ina j med lab sci tech 2020; 2(2): 60-67 putra rahmadea utami, et al. 6 6 ie through reaction with cell membranes, enzyme inactivation and inactivation of genetic material functions, in addition to inhibiting the reverse transcriptase enzyme and dna topoisomerase so that bacterial cells cannot be formed. saponins which can provide antibacterial activity are avenacin. saponin compounds will damage the cytoplasmic membrane and kill cells (20,22,23). conclusions the conclusion of this research is there is an interaction in the combination of ethanol extracts of chinese petai leaves with aloe vera inhibited the growth of e. coli. the most efficient extract concentration was 100 mg/ml. the combination of extracts can provide effective results in inhibiting the growth of bacteria, especially e. coli bacteria. conflict of interest there are no conflicts of interest. acknowledgment(s) this study is supported by ministry of research, technology and higher education of indonesia through the research funding for beginner lecturers in 2019. references 1. utami pr, chairani c, ilhamdi i. interaction of ethanol extract of chinese petai leaves (leucaena leucocephala folium) and aloe vera (aloe vera l.) inhibits the growth of staphylococus aureus by invitro. j kesehat perintis (heal journal). 2019;6(2):186–92. 2. rahardjo m, koendhori eb, setiawati y. antibacterial activity test of aloe vera (aloe vera) ethanol extract against staphylococcus aureus bacteria. j kedokt syiah kuala. 2017;17(2):65–70. 3. ramasubramaniam ts, sivakumar v, arasu t. antimicrobial activity of aloe vera (l.) burm. f. against pathogenic microorganisms. j biosci res. 2010;1(4):251–8. 4. suryati n, bahar e, ilmiawati i. antibacterial effectiveness test of aloe vera extract against escherichia coli growth in vitro. j kesehat andalas. 2018;6(3):518. 5. suhaimi s, indrawati t, kumala s. combination test of crude fruit extracts (aloe vera. 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250, 500 and 1000 mg/kg/d respectively) and diabetic treated with metformin (km; 500 mg/kg/d). this was carried out for 28 days. the inhibition mode of r. damascene extract was examined by measuring hba1c levels and expression of nfκb by immunohistochemistry. the results showed p values > 0.05 for hba1c and p < 0.05 for nfκb. from immunohistochemical staining, it seen the expression of nfκb was low in treated group (p1, p2, p3 and km) compared with kp. thus, oral administration of r. damascene extract for 28 days could not reduce hba1c levels, but can supress nfκb expression. keywords diabetes mellitus, rosa damascene, hba1c level, nfκb expression. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 4 7 choirotussanijjah, et al. introduction diabetes mellitus or dm is a chronic hyperglycemic that can cause complications in several organs including coronary artery disease, cataracts, renal failure and stroke (1). globally, an estimated 422 million adults had diabetes in 2014, compared to 180 million in 1980. this escalation was associated with an increase in risk factors including overweight or obese. in indonesia, enhancement diabetes mellitus started around 4% in 1980 and has been increased to 7.5% in 2014 (2). compared to 2013, there was an increase diabetes mellitus prevalence of 2% in population ≥ 15 years in 2018 (3). glycosylated hemoglobin or hba1c examination is one of the criteria for diagnosing diabetes mellitus. diabetes mellitus is established if hba1c level ≥ 6.5% by the method standardized by the national glycohaemoglobin standardization program (ngsp) (4). rate glycosylated hemoglobin reflects the blood sugar level during the previous 2-3 months. diabetes mellitus could lead glycosylated hemoglobin or hba1c formation which has ability undergo further changes into advanced glycation end products (ages) (5). the binding of ages or other ligands to rage (receptor for advanced glycation end product) can stimulate various signal transduction cascades leading to formation of ros and activation of transcription factors such as nuclear factor kappa b (nf-κb). increased production of ros has been linked to impaired endothelial function and structural damage in several macromolecules including carbohydrates, protein, lipid and dna (6). nuclear factor kappa b or nf-κb plays an important role in the pathogenesis of complications of diabetes mellitus. activation of nf-b will trigger the emergence of various inflammatory mediators such as cytokines, chemokines, and cell adhesion molecules. overexpression of nf-κb tnf-α, interleukins, tgf-β, bcl2 and other proinflammatory proteins and proapoptotic genes is the key to the development of vascular complications in diabetes. in addition, the high expression of nf-b will cause vascular calcification (7). rosa damascene is one of the ornamental plants and is cultivated extensively around the world, including indonesia. badan pusat statistik (bps) stated that the harvested area of rosa damascena ranks second after chrysanthemum was 3,723,228 m2. while the province of east java ranks first for the production of roses (8). in addition to various commercial products, rosa damascena has other benefits as anti-cancer, antimicrobials, relaxants and anti-depressants, antioxidant, analgesic, and anti-inflammatory. it is the most important plant from the rosacea family and has received less attention to therapeutic application (9). furthermore will be discussed regarding the effect of r. ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 4 8 choirotussanijjah, et al. damascene to improve one of the chronic diseases, diabetes. saptarini et al. (10) compared total content between r. damascene and china red rose which extracted by maceration procedure. it was found that total anthocyanin content of r. damascene and china red rose was 0.459 ± 0.003 mg/l and 0.186 ± 0.006 mg/l, respectively. anthocyanins are a group of water-soluble pigments and widely distributed in colorful fruits and vegetables such as berries, cherries, hawthorns, peaches, grapes, apples, red onions, red radishes, black beans, eggplants, red cabbage, and purple sweet potatoes (11). the antioxidant activity of anthocyanins is influenced by the structure of the anthocyanins, the number and arrangement of groups aromatic hydroxyl, presence of electron-donating properties and electron withdrawal in the ring structure. this causes anthocyanins to be highly effective hydrogen donor for reactive free radicals thereby preventing the formation of further radicals (12). previous study stated that anthocyanins have antioxidant properties and antiinflammatory activity by inhibiting the expression of nfκb pathway induced by palmitic acid in dose dependent way (13). administration of cyanidin-3-glucoside (20 mg/kg/day for 12 weeks) in db/db mice, proved can reduce body weight, glucose levels, hba1c, c-peptide, serum insulin, blood pressure, and several parameters related to kidney damage (serum creatinine, urinary albumin, bun, albumin/creatinine ratio, fibronectin, collagen iv, tgf 1, mmp9, tnf-α, monocyte chemotactic protein-1, il-1 and α-smooth muscle actin expression) (14). in another study in vitro on visceral adipose tissue, administration of protocateccuic acid (pca) can reduce the activity of protein-tyrosine phosphatase 1b, phospho-p65 nf-κb and il-6 (15). we thus investigated whether supplementation of r. damascene extract inhibits the hba1c formation and nf-κb activation in diabetic rats. materials and methods plant r. damascene were obtained from bumi aji sub-district, batu city. r. damascene extract was done by maceration. it washed and dried at a temperature of 32-350c (avoid direct sunlight). furthermore, the dried petals were blended and macerated with 96% ethanol food grade for 24 hours. the mixture was evaporated to obtain extract. for the dosages, researcher followed gholamhoseinian et al. (16) which using red rose extract at a dose of 100 mg/kgbw/d, 250 mg/kgbw/d, 500 mg/kgbw/d and 1000 mg/kgbw/d. it had been reported that r. damascena in methanol extract was able to reduce sugar levels blood significantly compared to controls. therefore, in this ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 4 9 choirotussanijjah, et al. research, researcher will using a dose of rosa damascena extract at a dose of 250 mg/kgbw/d, 500 mg/kgbw/d and 1,000 mg/kgbw/d. animals twenty-four male rattus norvegicus (810 weeks; weighing 150-200 gram) have been used for experiment. they were caged and kept under normal laboratory condition. rats were classified into 6 groups, within 4 rats in each group. group kn was a normal rat, group kp was a diabetic rat, animal from group p1, p2 and p3 received doses 250, 500 and 1,000 mg/kg/d of r. damascene extract, and group km was treated with metformin (500 mg/kg/d) by gavage. metformin is a first-line drug for diabetes and effective as monotherapy or in combination with other antidiabetics (17). r. damascene extract and metformin were dissolved into distillated water and administered orally by intra-gastric route (in a final volume 1 ml). all studies procedure and method have been approved by health reasearch ethical clereance commission of universitas airlangga faculty of dental medicine with a number 080/hrecc.fodm/ii/2021. location and time rats were treated in the pharmacology laboratory of the faculty of medicine, airlangga university surabaya. examination of hba1c level were carried out at the central health laboratory surabaya. meanwhile, immunohistochemical examination was carried out in the laboratory biochemistry universitas brawijaya malang. this research was conducted for 3 months. experimental design rats (n = 24) devided into 6 groups (kn, kp, p1, p2, p3 and km) and have been acclimatizated for 7 days. they were placed in cages with well ventilation and cared under standardized eviromental circumstances (2228⁰c, 12 h dark light cycle and free access to food and water). for induction of diabetes, 50 mg/kg streptozotocin (stz) was injected in rats (all groups, except kn), intraperitoneally. five days after streptozotocin administration, rats blood glucose levels were checked in the tail vein with a glucometer. sugar levels above 200 mg/dl were declared as hyperglycemia and included in inclusion criteria of study. kn and kp were received normal feed and water ad libitum, while p1, p2, and p3 were treated with r. damascene extract with doses of 250 mg/kgbw/d, 500 mg/kgbw/d, and 1,000 mg/kgbw/d, respectively. then, group km was administrated metformin 500 mg/kgbw/d. administration of r. damascena extract and metformin was fixed for 28 days. after the period was over, the rats were weighed and blood sugar levels were checked with glucometer. rats were euthanized and blood samples were collected for hba1c levels and aorta was excised for nf-κb expression by immunohistochemistry. ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 5 0 choirotussanijjah, et al. hba1c levels levels of hba1c were measured using glycohemoglobin a1c assay kit (classification no:30168000, the pharmaceuticals, medical evices and other therapeutic product act of japan). samples were sentrifuged (800 g for 5 minutes), then collect 25 μl from the bloodcell layer and add it into pretreatment solution, and mix. measure this mixture with an automated analyzer and read the absorbance. the measurement need to be converted into ngsp values (%). immunohistochemical analysis of nf-κb expression paraffin block were cut into 3-5 μm thick and attached to a glass slide was fixed with acetone for 2-3 minutes and stored at 4˚c. fixing the slide with methanol and allowed to stand for 5 minutes, then dried, aerated and washed with phosphate buffer saline (pbs) ph 7.4 for 5 minutes with three repetitions. added h2o2 for 10 minutes and washed with pbs for 5 minutes with three repetitions. fbs (fetal bovine serum) containing 0.25% triton x-100 was added and incubated for 1 hour at room temperature for blocking process. furthermore, slide washed with pbs for 5 minutes with three repetitions. primary antibody (monoclonal anti p65) was added and slide was incubated for 24 hours and washed with pbs for 5 minutes with three repetitions. then secondary antibody was dripped and incubated for one hour and washed with pbs for 5 minutes with three repetitions. the next step was addition of sahrp for 40 minutes (washed with pbs for 5 minutes with three repetitions), and dripped with dab for 5-30 minutes. then counterstain with mayer's hematoxylin for 10 minutes, then rinsed and washed with distilled water for 10 minutes, dried and covered with cover glass (18). slides were examined under microscope and cells expressing nf-κb (there was a color change to brown) was calculated with a magnification of 100 x. this calculation is carried out in 20 fields of view and divides the number obtained by 20 to get the average. statistical analysis saphiro-wilk and levene test were performed for normality and homogeneity of data (p>0.05). for the data normally distributed and homogenous could be followed with one way anova test (p<0.05) and lsd test was used to compare between groups. if the data did not confirm homogenity of variances, games-howell tests can be performed to see differences between groups. if the data does not meet one of the requirements for parametric analysis, non-parametric statistical tests can be performed using the kruskal-wallis test, then continued with the mann-whittney with confidence level 95% (α = 0.05) to see the difference between treatment group. ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 5 1 choirotussanijjah, et al. results effect of rosa damascene on hba1c level the average kp had a lower mean hba1c value than the treatment rat group. based on the results of the normality test, it can be seen that the p value > 0.05. data analysis was continued by looking for homogenity. the lavene test showed p value < 0.05, which means data was not homogeneous. furthemore, brown-forshyte test was conducted and p < 0.05 was obtained. thus, h0 was rejected and h1 is accepted or there was a difference between the control and treatment groups. in this study, the games-howell post hoc test was carried out because the variation in the study sample population was not homogeneous. there was a significant difference between kn and treatment groups (p2, p3 and km (p < 0.05)). whereas, no significant difference was found between kp and other groups kn, p1, p2, p3 and km (p > 0.05) (figure 1). figure 1. comparison of mean of hba1c. values marked with # showed no difference (p >0.05) compared to positive controls (kp) effect of rosa damascene on nfκb level the shapiro-wilk test showed p > 0.05 and lavene test of the nfκb data was p = 0.062 (p > 0.05). furthermore, one way anova test was performed, the p value was 0.00 (p < 0.05), therefore, tukey-hsd method was used to see differences between groups. based on the tukey-hsd test, p value < 0.05 was found between kn and kp; p1. in addition, there were also significant differences between kp and kn; p2; p3; km (figure 2). differences in nfκb expression between groups by immunohistochemistry were shown in figure 3. # # # # # ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 5 2 choirotussanijjah, et al. figure 2. mean comparison of the nfκb expressions. values marked with *were significantly different (p<0.05) compared to kp. while # was not significantly (p>0.05) different to kp figure 3. immunohistochemical comparison between kn, kp, p1, p2, p3 and km groups. in the kn group, it was seen few expressions of nfκb compared to kp (black arrow). while in the treatment group there were decreasion nfκb expression in p3 and km. (400x magnification) # * * * * kn kp p1 ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 5 3 choirotussanijjah, et al. discussion saptarini et al. (9), compared total content between r. damascene and china red rose which extracted by maceration procedure. it was found that total anthocyanin content of r. damascene and china red rose was 0.459 ± 0.003 mg/l and 0.186 ± 0.006 mg/l, respectively. anthocyanin have several benefits for diabetic patients, including reducing fasting blood sugar, serum insulin, triglyceride levels, cholesterol, hba1c, increasing insulin secretion and insulin sensitivity (19). anthocyanins influence expression gen of glut4 and its translocation, enhance pparγ activation in skeletal muscle and adipose tissue, enhance amp-activated protein kinase activation, incerase adiponectin and leptin, and, anthocyanins also inhibit secretion of α-glucosidase from intestine and α-amylase from pancreas (20). in this study, average hba1c in kn group was low at 3.25%, while in the kp was 6.6% (data not shown). furthermore, km group obtained a value of 7.4%. furthermore, p1, p2, and p3, hba1c values was 7.7%, 7.6%, and 7.4%, respectively. after statistical tests were carried out, data showed p > 0.05. so that it did not show any effect of r. damascene on hba1c levels in diabetic rats. streptozotocin has been known as an agent that causes an increase free radicals and damage to pancreatic beta cells to cause hyperglycemia. furthermore, this condition is an important key for diabetes mellitus complications. the role of antioxidants is very important to break the chain of radical reactions. antioxidant enzymes that functions to oxidize oxidant molecules is superoxide dismutase (sod) (converting superoxide radicals to hydrogen peroxide), gsh-peroxidase (gpx) and catalase (cat) which convert hydrogen peroxide into water and oxygen. some previous studies reported inconsistent results on sod and catalase activity in various tissues. it could influence by differentiation in expression of antioxidant enzymes as well as techniques for producing rat models of diabetes mellitus (21). this mechanism may underlie the low hba1c in kp and p1. pelargonidin administration in rats induced by streptozotocin for 5 weeks showed a decrease in hba1c levels (22). qin et al. (14) also reported that a decrease in hba1c was observed in db/db mice given c3g for 12 weeks of therapy. falah et al. (23) concluded that the consumption of anthocyanins will provide benefits on fasting blood sugar levels and 2 hours pp, hba1c and homa-ir with a duration of > 8 weeks and with a dose of > 300 mg/day. contrary to this, in this study, the average increase in hba1c was 6.6% in positive control group (kp) or diabetes group. the average value is lower than treatment groups (p1, p2, and p3), which ranged from 7.4 to 7.7%. it can be ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 5 4 choirotussanijjah, et al. concluded that statistically administration of r. damascene to rats model diabetes mellitus cannot reduce hba1c levels or not showed significant differences between groups. previous study by yang et al. (20) showed there was an escalation in the number of sod and decrease in the enzyme catalase in brain tissue at the 8th week post-induction with stz. meanwhile, at 16th week after stz induction, there were reduction of sod and catalase activity in the cortex, hippocampus and blood vessels. another study conducted for 2 weeks it was mentioned that there was an insignificant expression of sod in rats control and diabetic mice. in addition, ho-1 mrna expression was increased in group of diabetic rats compared with a group of healthy rats (24). several previous studies stated that experimental research for diabetes mellitus showed inconsistent results in sod and catalase activity in various tissues. some of the factors that causes of this include differences in the expression of antioxidant enzymes as well as technique to generate a rat model of diabetes mellitus (20). these mechanisms may underlie low level of hba1c on kp and p1. meanwhile, studies conducted on humans did not show consistent results. administration of capsules containing concentrated powder of juice cranberries for 12 weeks did not show significant changes in levels of fasting blood sugar, hba1c, fructosamine, triglycerides and hdl or ldl levels between the treatment and placebo groups (25). another study reported that sour cherry juice given to female patients with type 2 diabetes can reduce body weight, blood pressure and hba1c after 6 weeks (26). based on the explanation above, giving r. damascene did not have an effect on hba1c levels and probably caused by duration of study was relatively short. besides, it needs others investigation for endogenous antioxidants level from rats thought to play role in low levels of hba1c in the positive control group compared with treatment group. nucleus factor kappa b or nfκb is protein transcription factors involved in cellular responses to various stimuli, including stress, cytokines, free radicals, bacterial and viral antigens. this factor is present in all types of tissue cells and is involved in various cellular responses (26). nfκb activation can lead to increased expression of pro-inflammatory and proapoptotic genes as well as associated with an increased incidence of complications in diabetes mellitus (27). nfκb contain homo or heterodimer protein (p65 or rela, p50/p105, c-rel, p52/p100 and rel b). nfκb forms a complex with inhibitory protein iκb. degradation of iκb inhibitor protein lead nfκb translocate to the nucleus and bind to dna and induce ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 5 5 choirotussanijjah, et al. transcription of proinflammatory genes. several studies have shown that the anthocyanidin delfinidine can inhibit the degradation of the inhibitory iκb protein and the translocation of p65 into the nucleus. in addition, the anthocyanin is also the most potent cox-2 inhibitor, both at the mrna and protein levels, suppressing lps which stimulates the activation of other transcription factors, namely c/ebp, ap-1, however, not creb (11). paixao et al. (28) reported that malvidin3-glucoside can suppress nfκb inhibition on arterial endothelial cells from bovine. in addition, malvidin can inhibit p65 which is one of the subunits of nfκb. in vitro studies on visceral adipose tissue (vat) with protocatechuic acid could reduce the activity of ptp1b, phosphor-p65, nfκb and il-6 in vat from obese subjects (15). in addition, in vitro study used hk-2 cells treated with pure c3g, showed the presence of increased cholesterol efflux, abca1 expression, pparα, lxrα, decreased proinflammatory molecules such as icam1, mcp1, tgfβ1, and nfκb (29). furthermore, administration of medox capsules containing pure anthocyanins isolated from bilberry and blackcurrent in human monocyte cultured cells and healthy human subjects (for 3 weeks) showed direct inhibition of nfκb transactivation and decreased concentrations of proinflammatory, chemokines and nfκbregulated immunoregulatory cytokines (30) in addition, hypercholesterolemic subjects who were given mixed anthocyanin purified (320 mg/day) for 24 weeks showed a decrease in nfκb-associated inflammatory response (31). this study showed that administration of r. damascene could reduce nfκb expression in the rat aorta (p < 0.05). it can be seen from the immunohistochemical results, the cell count expressing nfκb in the rat aorta decreased with the administration of r. damascene extract. this is in accordance with several previous studies. conclusions we observed that extract r. damascene has potentially effect to improve nfκb in diabetic rats, but need further some investigation about endogen antioxidant and duration of study to prove effect of r. damascene to hba1c level. author contributions choirotussanijjah: conceptualization, methodology, writing – original. harianto notopuro: supervision, conceptualization, validation. ema qurnianingsih: supervision, reviewing and validation. acknowladgements the authors are grateful to ministry of research, technology, and higher education, directorate general of learning ina. j. med. lab. sci. tech. 2021; 3(2): 146–157 1 5 6 choirotussanijjah, et al. and students of indonesia for sponsoring this study. conflict of interest the authors declare no conflict of interest. references 1. who. diabetes. https://www.who.int/healthtopics/diabetes#tab=tab_1. 2021. 2. who. global report on diabetes. isbn 5. 2016. doi:isbn 978 92 4 156525 7. 3. kemenkes. hasil utama riskesdas tentang prevalensi diabetes mellitus di indonesia 2018. 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3(2): 146–157 1 5 7 choirotussanijjah, et al. 22. roy m, sen s, chakraborti as. action of pelargonidin on hyperglycemia and oxidative damage in diabetic rats : implication for glycation-induced hemoglobin modi fi cation. 2008;82, 1102–1110. 23. fallah aa, sarmast e, jafari t. effect of dietary anthocyanins on biomarkers of glycemic control and glucose metabolism: a systematic review and meta-analysis of randomized clinical trials. food res. int. 2020;137, 109379. 24. koya d, hayashi k, kitada m, kashiwagi a, kikkawa r, haneda m. effects of antioxidants in diabetes-induced oxidative stress in the glomeruli of diabetic rats. j. am. soc. nephrol. 2003;14, 250–253. 25. balasubramanian k, dabadghao p, bhatia v, colman pg, gellert sa, bharadwaj u, agrawal s, shah n, bhatia e. high frequency of type 1b (idiopathic) diabetes in north indian children with recent-onset diabetes. diabetes care. 2003;26, 2697. 26. ataie-jafari a, hosseini ., karimi f, pajouhi m. effects of sour cherry juice on blood glucose and some cardiovascular risk factors improvements in diabetic women a pilot study. nutr. food sci. 2008;38, 355–360. 27. meyerovich k, ortis f, cardozo ak. the noncanonical nf-κb pathway and its contribution to β-cell failure in diabetes. j. mol. endocrinol. 2016.61, f1–f6. 28. paixão j, dinis tcp, almeida lm. malvidin-3glucoside protects endothelial cells up-regulating endothelial no synthase and inhibiting peroxynitrite-induced nf-kb activation. chem. biol. interact. 2012.199, 192–200. 29. du c, shi y, ren y, wu h, yao f, wei j, wu m, hou y, duan h. anthocyanins inhibit highglucose-induced cholesterol accumulation and inflammation by activating lxrα pathway in hk-2 cells. drug des. devel. ther. 2015;9, 5099–5113. 30. karlsen a, retterstøl l, laake p, paur i, bøhn sk, sandvik l, blomhoff r. anthocyanins inhibit nuclear factork b activation in monocytes and reduce plasma concentrations of proinflammatory mediators in healthy adults 2007;1(3). 1951–1954. 31. zhu y, ling w, guo h, song f, ya q, zou t, li d, zhang y, li g, xiao y, liu f, li z, shi z, yang y. anti-inflammatory effect of purified dietary anthocyanin in adults with hypercholesterolemia: a randomized controlled trial. nutr. metab. cardiovasc. dis. 2013;23, 843–849. 120 differences in diameter of the growth inhibition zone of klebsiella pneumonia bacteria after incubation at 37°c and 25°c juwy trianes1, bastian1, dewi hartati1 1medical laboratory technology div study program, institut ilmu kesehatan dan teknologi muhammadiyah palembang, palembang, south sumatra, indonesia correspondence: bastian, institut ilmu kesehatan dan teknologi muhammadiyah palembang, jl. jenderal ahmad yani, 13 ulu, kec. seberang ulu ii, kota palembang, south sumatra, indonesia zip code: 30262 email: bastiandarwin51@yahoo.com received: april 24, 2022 revised: agustus 25, 2022 accepted: september 22, 2022 published: october 2022, 2022 doi: 0.33086/ijmlst.v4i2.2919 abstract pneumonia is an infection that causes the largest death in children worldwide (south sumatra province), which occupies the 6th position. one of the laboratory reidentification of klebsiella pneumonia is a multi-antibiotic testing. the high sensitivity antibiotic that is still used is amikacin. the uniqueness of the diameter of the inhibition zone using the antibiotic amikacin is that it is active against most gram-negative bacilli. but one of the several factors that affect the diameter of the inhibition zone is the incubation temperature. the optimum temperature for pathogenic bacteria is 37oc by using an incubator; however, several factors in the use of instruments such as frequent instability and disruption of installation lead to a need of incubation at 25oc. the study aimed to determine the difference in the diameter of the growth inhibition zone of k. pneumoniae after incubation at 37oc and 25oc. this research is an experimental research conducted at the microbiology laboratory of ikest muhammadiyah palembang. the sample is k. pneumoniae which will be subjected to gram staining, biochemical tests, followed by a sensitivity test on mueller hinton media which is given an amikacin antibiotic disk and incubated at 370c and 250c in order to calculate the diameter of the zone of inhibition for the growth of k. pneumoniae bacteria. the data was analyzed using the alternative wilcoxon test which obtained a p value of 0.014. the results of this investigation showed that k. pneumoniae incubated at 37°c and 25°c had a significantly different diameter of the growth inhibition zone. keywords growth inhibition zone, inhibitory zone diameter, klebsiella pneumonia. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2022; 4(2): 120–127 bastian, et al 1 2 1 introduction indonesia is a developing country that has a tropical climate where microbes can thrive. infectious diseases are the main health problem in developing countries, one of which is indonesia. according to riskesdas (indonesia basic health research) 2018, one of the main causes of infectious disease in indonesia was transmission through the air, as 44.0% was transmitted by respiration (1). especially in south sumatra province in 2018, it was found that the number of cases of pneumonia was in the 6th position with 12.707 cases or 39.24%. the highest percentage coverage of pneumonia sufferers based on the existing target of discovery is achieved by muara enim regency with 1.997 cases (88.97%) (2). pneumonia is an infectious disease that causes the largest death in children worldwide. one of the bacteria that cause pneumonia is klebsiella pneumonia (3). k. pneumoniae is a type of klebsiella group of bacteria that infects humans a lot. these bacteria are short gram-negative (-) rods, which has a size of 0.5-0.5 x 1.2μm, has a capsule, but does not form spores. the species k. pneumoniae showed mucoid growth, large and non-motile colonies (4). k. pneumoniae laboratory diagnostics include bacterial isolation, gram staining, biochemical assays, and sensitivity tests to antibiotics. antibiotic that is still widely used in the treatment of respiratory tract infections that have high sensitivity is amikacin (6,7). antibiotic susceptibility test could be carried out using the kirby bauer diffusion method by attaching the antibiotic to mueller hinton (mha) media using tweezers and then incubating it at 37°c for 24 hours. during incubation, antimicrobial compounds will diffuse into the agar medium. the results were observed by measuring the drag diameter (mm) and compared it with kirby bauer's clsi standard (8). one of the factors that affects the sensitivity test of bacterial growth was incubation temperature. temperature is the most important environmental factor that can affect microorganisms. therefore, the temperature can affect the growth of bacteria (9). the optimum temperature for pathogenic bacteria is 37°c using an incubator, but there are constraint factors in the use of the tool, such as frequent instability and disturbances in electrical installations and lack of an incubator in a laboratory at a certain temperature, namely an ambient temperature that can grow bacteria, namely at 25°c (10). according to research conducted by saïd1 et al., (11) the effect of temperature on the sensitivity test of escherichia coli ho25 antibiotics with the diffusion method in order to state that at temperatures ranging between 25°c and 44°c can be used with the same results as the standard. based on the description above, the researchers found several problematic factors in the use of an incubator at a temperature of 37°c, one of ina. j. med. lab. sci. tech. 2022; 4(2): 120–127 bastian, et al. 1 2 2 which is a laboratory test that requires an incubator for bacterial growth at a temperature of 37°c. therefore, in this work, we compared the diameter of the growth inhibitory zone of k. pneumoniae bacteria at incubation temperatures of 37°c and 25°c. materials and methods this study used true experimental design. it was conducted on december, 2021 at the microbiology laboratory of institut ilmu kesehatan dan teknologi muhammadiyah palembang, indonesia. the sample used was the bacterium k. pneumoniae atcc 10031 using posttest-only control. this study begins with the process of testing a pure strain of bacteria k. pneumoniae atcc 10031 with gram staining for biochemical test imvic (indole, methyl red, vogesproskauer, and citrate), triple sugar iron agar (tsia), glucose, lactose, maltose, mannitol, sucrose, and decarboxylases test), macroscopic colony readings followed by instrument sterilization, working procedures for glassware sterilization processes, followed by manufacturing media by weighing 38g of mueller hinton agar and putting it in an erlenmeyer 1000 ml, then distilled water was added and then heated on a hotplate. furthermore, the media was sterilized using an autoclave at 121oc for 15 minutes and poured into a petri dish (12). then, the manufacture of a bacterial suspension; one cycle of bacterial culture from the pure culture of the test bacteria was suspended in 0.9% of nacl solution in a sterile test tube. moreover, the bacterial suspension was vortexed until it became homogeneous and the turbidity was obtained according to the standards of mc farland 0.5 (13). the sensitivity test for the growth of k. pneumoniae bacteria was carried out using kirby bauer method by taking the bacterial suspension and then inoculating into the mueller hinton media using a sterile swab, waiting a few minutes for the inoculated media to allow the bacterial suspension to seep into the media. then, an antibiotic disk amikacin was prepared, after that each antibiotic was attached to the mueller hinton media using tweezers and incubated at 37°c and ambient temperature at 25°c for 24 hours. the diameter measurement of the inhibition zone used a vernier caliper (11). the study was conducted to determine the difference in the diameter of the growth inhibition zone of k. pneumoniae bacteria after incubation at 37°c and 25°c. based on the federer formula, there were 16 repetitions of each solvent on mueller hinton agar (mha) media which was incubated at 37°c and 25°c, and the difference in the diameter of the inhibition zone was identified after the media was incubated at 37°c and 25°c for 24 hours. the diameter of the inhibition zone (mm) and the data obtained from laboratory ina. j. med. lab. sci. tech. 2022; 4(2): 120–127 bastian, et al 1 2 3 examinations are processed using the spss program. due to the small amount of data (less than 50), the shapiro-wilk test was utilized for the normality test. the results obtained were seen from the sig value; if the value of sig was > 0.05 then the data was declared normally distributed, while if sig < 0.05 then the data was declared not normally distributed. if the results were normally distributed then it would be proceeded with a paired t-test (14). if the results obtained were not normally distributed, the data was transformed, the analysis carried out depends on the distribution and variance of the transformation results. if the distribution was not normal, the analysis was continued with the wilcoxon test (15). results the identification test of k. pneumoniae bacteria was carried out before the research in order to ensure the purity of the strain to be used. the result of the macroscopic test of k. pneumoniae bacteria was large colonies (about 4 to 6 mm), gray, opaque, and slightly mucoid. these results can be seen after incubation at 37°c for 24 hours. the identification of k. pneumoniae in the imvic test (indole, methyl red, vogesproskauer, and simmons citrate) obtained negative results on the indole and methyl red tests, then positive results on the voges proskauer and simmons citrate tests. in the tsia test, the results were a/a, h2s (-), gas (+). the urease test was positive, then the sugars (glucose, lactose, maltose, mannitol and sucrose) were positive. the decarboxylases tests (arginine, lysine, and ornithine) were negative and the phenyl alanine test was negative. figure 1 shows the results of the average value of measurement of the inhibitory zone diameter in k. pneumonia bacteria on the media mueller hinton incubated at a temperature of 37°c, which is 21 mm and figure 2 shows the measurement of the inhibitory zone diameter of k. pneumonia bacteria on the media mueller hinton incubated at a temperature of 25°c, which is an average yield of 20 mm. figure 1. diameter of the inhibition zone of k. pneumoniae bacteria growing at incubation temperature. (a) at 37°c, (b) at 25°c. (a) (b) ina. j. med. lab. sci. tech. 2022; 4(2): 120–127 bastian, et al. 1 2 4 figure 2. diameter of the inhibitory zone of bacteria k. pneumonia after incubating at the 37°c and 25°c temperatures. the study has found that k. pneumoniae bacteria incubated at 370c and 250c had different diameters of inhibition zones (mm). the results of the analysis of the shapiro wilk normality test showed that the diameter of the inhibition zone of k. pneumoniae bacteria incubated at 37°c was sig 0.014, while the diameter of the inhibition zone of k. pneumoniae bacteria incubated at 25°c was 0.046. because the value obtained was sig < 0.05 based on these results, the normality of the data was not normally distributed, followed by data transformation. data transformation test was carried out to convert the original data into another form. the results of the analysis showed that the results of the data transformation test on the diameter of the inhibition zone at 37oc and 25°c were 0.014 (table 1). therefore, it can be inferred that the data are not normally distributed from the results of the statistical data transformation test of the diameter of the growth inhibition zone of amikacin antibiotics of k. pneumoniae bacteria after incubation at 37°c and 25°c. as a result, the nonparametric wilcoxon test was conducted. (16). table 1. result of spss statistical program p normality p transformation p wilcoxon 37°c temperature 0.014 0.014 0.014 25°c temperature 0.046 0.049 0.014 p value (sig 2 tailed) = 0.05 ina. j. med. lab. sci. tech. 2022; 4(2): 120–127 bastian, et al 1 2 5 wilcoxon test is an alternative test and the wilcoxon test showed p value (sig 2 tailed) = 0.014, p < therefore a significant hi value was obtained, namely there is a difference in the diameter of the inhibition zone at a temperature of 37°c and 25°c. discussion incubation at a temperature of 25°c has a diameter of growth inhibition zone using amikacin antibiotic that is significantly different from incubation at an optimum temperature of 37°c. the findings of this study diverge from those of studies by smith et al., (17), which used precision disc diffusion antimicrobial sensitivity test data carried out at 35°c, 28°c, 22°c. as a result, incubation at constant room temperature was to blame for the diameter of the inhibitory zone in the 2018 investigation in the alzair region. the temperature instability in the microbiology laboratory for the incubation of k. pneumoniae is likely one of the reasons for the disparity in results between our study and smith et al., (17). condition at 25°c produced by excessive ventilation and variable weather that significantly alters the diameter of the inhibitory zone. the ambient temperature will rise during the day, and at night it will drop once more. the measurement of the diameter of the inhibition zone shows that the test conditions have a significant influence on the accuracy of the data obtained. the accuracy of the inhibition zone diameter data decreased as the temperature decreased and the incubation time increased so that the inhibition zone diameter measurement was disrupted. temperature greatly affects enzyme activity when catalyzing a reaction; the higher the temperature the more active the enzyme activity. enzyme activity increases at the rate of reaching the optimum. an increase in temperature exceeding the optimum temperature causes weak bonds in the enzyme (17,18). according to the research conducted by smith et al., (17) temperature conditions have a significant effect on the accuracy of the data set obtained for the precision of measuring the diameter of the inhibition zone. our research is in line with saïd1 et al., (11), which states that temperature greatly affects the antibiotic sensitivity test with the agar diffusion method because room temperature shows the most significant effect compared to other temperatures, namely at 37°c and 30°c, while in our study there was a significant difference in the diameter of the inhibition zone incubated at 37°c and 25°c. temperature is one of the environmental factors that affect microbial growth. each microbe has a certain optimum temperature range for its growth. microorganisms will develop less rapidly at temperatures that are below or beyond their optimal range. the optimum temperature for pathogenic bacteria was 35±2°c. pathogenic bacteria that grow ina. j. med. lab. sci. tech. 2022; 4(2): 120–127 bastian, et al. 1 2 6 best in the middle of this range are referred to as mesophilic, which include all human and opportunistic pathogens (19). the results of the study are the same as previous studies by saïd1 et al., (11). this can occur because temperature can affect the growth of the diameter of the inhibition zone. temperatures with optimum conditions are needed for the speed of cell growth. there are several obstacles faced during the research, namely the occurrence of room temperature instability due to laboratory conditions that are often open. this greatly affects the measurement of the diameter of the inhibition zone of the bacteria on muller hinton (mh) media. each bacterium has an optimal temperature where they can grow very fast and has a temperature range in which they can grow (20). conclusions based on the research, with regard to the average diameter of the amikacin antibiotic inhibition zone on k. pneumoniae bacteria, there was a significant difference after incubation at a temperature of 37°c and 25°c, and laboratory personnel can use incubation at a temperature of 25°c as an alternative temperature by adjusting the temperature in a certain area. author contributions bastian: conceptualization, methodology, drafting, monitoring and editing. dewi hartati: revision, data curation, and validation. juwy trianes: data processing and data retrieval. conflict of interest there is no conflict of interest in the process of carrying out the research until completion. acknowledgment(s) this work was supported by private funds and this work was carried out at the microbiology laboratory of the muhammadiyah palembang institute of health sciences and technology. references 1. sari dp, rahmawati r, elvi rpw. detection and identification of coliform bacteria genera isolated from aloe drink. j labora med. 2019;3(1):29–35. https://doi.org/10.26714/jlabmed.3.1.2019.29 35 2. provincial health office. deconcentralized annual performance plan of the health office; 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(1):1–5. https://doi.org/10.35472/indojam.v2i1 17. smith p, finnegan w, ngo t, kronvall g. influence of incubation temperature and time on the precision of mic and disc diffusion antimicrobial susceptibility test data. aquaculture. 2018;490:19–24. 10. doi: 1016/j.aquaculture.2018.02.020 18. rahman iw, prihartini a. test of antibiotic sensitivity to growth of klebseilla pneumonia from sputum of patients with lower respiratory tract infections. 2021,3:81–87. https://doi.org/10.36339/jhest.v3i2.53 19. cahyati wh, sari mp. trends in toddler pneumonia in semarang city 2012-2018. higeia j public heal. 2019;3(3):408. https://doi.org/10.15294 /higeia/v3i3/30266 20. halla s, rohmi r. agrijanti, a. the effectiveness of portable incubators as an innovation tool to support microbiology laboratories. jambs. 2019;6(1):66.https://doi.org/10.32807/jambs.v6i 1.126. https://orcid.org/0000-0002-5085-3584 https://orcid.org/0000-0002-5085-3584 https://doi.org/10.25077/jka.v8i2s.955 https://doi.org/10.35472/indojam.v2i1 https://doi.org/10.36339/jhest.v3i2.53 https://doi.org/10.15294%20/higeia/v3i3/30266 111 potential of garlic filtrate as an alternative anticoagulant for whole blood samples ari nuswantoro1, jessica ningtyas berlianti1 1department of medical laboratory technology, politeknik kesehatan kemenkes pontianak, pontianak, west kalimantan, indonesia correspondence: ari nuswantoro, jln. danau sentarum komplek sentarum mandiri no. b16, pontianak, west kalimantan, indonesia zip code: 78116 email: arinuswantoro82@gmail.com received: january 18, 2022 revised: may 25, 2022 accepted: september 13, 2022 published: october 30, 2022 doi: 10.33086/ijmlst.v4i2.2683 abstract synthetic anticoagulants such as heparin, citric, and ethylenediaminetetraacetic acid (edta) are commonly used to prevent blood clots. in contrast, its widespread use in clinical laboratories is still constrained by price, its toxic nature, and its short shelf life. therefore, an alternative material that is relatively cheap, non-toxic, and easy to obtain and process in a ready-to-use form is needed. garlic contains allicin and ajoene, which are anti-platelet and antithrombogenic. this study’s aim is to explore the potential of garlic filtrate as an alternative anticoagulant. blood from 16 individuals was used and separated into four groups: nonanticoagulant, 50 μl/ml garlic filtrate, 100 μl/ml garlic filtrate, and 150 μl/ml heparin, for a total of 64 treatments. the lee and white method showed that non-anticoagulated blood had normal clotting times (mean 8 minutes and 56 seconds), whereas heparin plasma and garlic filtrate plasma had longer clotting times (more than 20 minutes); and this is statistically different based on the friedman test with a significance value (p) of 0.000 < 0.05. on spectrophotometric measurements, the levels of calcium ions in heparinized plasma and serum were 8.66 mg/dl and 8.52 mg/dl, respectively, while in garlic plasma filtrate of 50 μl/ml and 100 μl/ml were 4.13 mg/dl and 3.58 mg/dl, respectively; this is also statistically different based on the anova test with a significance value of 0.000 < 0.05. the differences indicate that garlic filtrate can extend clotting time and reduce calcium ions therefore it is worth reviewing as an alternative anticoagulant. keywords anticoagulant, calcium ion levels, clotting time, garlic filtrate. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2022; 4(2): 111–119 ari nuswantoro & jessica ningtyas berlianti 1 1 2 introduction the hematological examination includes extensive laboratory measures and is essentials for diagnosing disease. although modern technology has influenced many aspects of laboratory work, and examination procedures have become increasingly diverse, the basic principles of sample preparation remain the same. knowledge of sample preparation relates to the technique or parameter to be performed, attempts to increase the validity of the results and, ultimately, to establish the diagnosis of the disease. although almost anybody fluid can be a representative sample and provide information about a person's biological status, blood is the most widely used sample because it is easy to obtain also the analytes in it tend to be stable (1). there are several causes of blood samples for hematological examination to be unusable, including hemolysis, insufficient volume, wrong container and undue clotting (2). the presence of clots, even if they are small, can affect the examination then the results will be inaccurate (3). when platelets, proteins, and blood cells adhere to one another, a blood clot form. blood needs to be anticoagulated also known as blood thinners, to stop clots from forming. several anticoagulants are used in the laboratory to prevent clotting or to obtain plasma. the three anticoagulants that are most often are heparin, ethylenediaminetetraacetic acid (edta), and citrate. although novel anticoagulants like hirudin are available, they are not often utilized (4). however, the use of anticoagulants does not always perform smoothly. some laboratories that could not perform hematological examinations properly because they had run out of stock of anticoagulants. this condition worsens by its high price, dependence on distribution, short expiry date and its toxic or irritating nature. for this reason, the need to find alternative natural anticoagulants that address the challenges mentioned above, especially those from nature. natural products are usually made of several constituents, which have several targets. therefore, providing a pleiotropic and synergistic effect to promote positive results. in addition, the constituents of natural products usually have minimal side effects, especially on the digestive system (5). garlic (allium sativum) is known to have various biological activities (6), including in-vitro anticoagulant effects (7). garlic has been used in many regions of the world since the sumerians (2600–2100 bc); it is taken as food and medicine, particularly in the tuber; it contains antibacterial, antiviral, antifungal, and antiprotozoal properties; and it has favorable effects on the cardiovascular and immunological systems (8). garlic's chemical composition changes depending on ina. j. med. lab. sci. tech. 2022; 4(2): 111–119 ari nuswantoro & jessica ningtyas berlianti 1 1 3 the circumstances. fresh and entire garlic bulbs contain γ-glutamyl cysteine, which can be oxidized and hydrolyzed to alliin when kept at low temperatures. when garlic is sliced, crushed, chewed, or dried, a vacuole enzyme called alliinase is released, causing alliin to be converted to allicin. diallyl sulfide (das), diallyl disulfide (dads), diallyl trisulfide (dats), dithiins, and ajoene are some of the chemicals that allicin decomposes into (9). garlic has the potential to be employed as an alternative source of anticoagulant replacements since allicin and ajoene exhibit antithrombin and antiplatelet action (10,11) in addition to antibacterial (12), antifungal (13), and anticancer (14–16) properties. although there is a known relationship between garlic products with platelet aggregation (17) and calcium levels (18), the active element in garlic's capacity to inhibit blood coagulation in comparison to synthetic anticoagulants has not been thoroughly explored. this study investigated the potential of garlic filtrate to be an alternative anticoagulant for hematology laboratory tests by analyzing blood clotting time and plasma calcium ion levels. the findings of this study are expected to pave the way for the use of garlic filtrate to replace synthetic anticoagulants like heparin and edta in emergencies, to avoid chemical side effects, or for efficiency reasons, particularly in laboratories with distance and operational cost issues. materials and methods the health research ethics committee of the health polytechnic of pontianak, ministry of health (177/kepkpk.pkp/vii/2020, july 8, 2020) approved this study, which was conducted in july– august 2020 at the hematology laboratory of the department of medical laboratory technology, health polytechnic of pontianak, ministry of health. the blood of 16 volunteers who have stated their readiness and do not have blood coagulation issues were used in the study. this study used garlic filtrate because it is more accessible to collect than garlic extract. the research design was quasi-experimental, with each blood sample divided into four groups: without the addition of garlic filtrate (group a), with the addition of 50 μl/ml garlic filtrate (group b), with the addition of 100 μl/ml garlic filtrate (group c), and with the addition of 150 μl/ml heparin (group d) (19), for a total of 64 samples. the clotting time (11) and calcium level of each sample tested used spectrophotometry with the reagent kit from biosystems (barcelona, spain). the following methods were used to create garlic filtrate: (a) garlic bulbs were peeled, rinsed, and drained; (b) shredded; and (c) filtered through a funnel using whatman paper and collected in an erlenmeyer. the ina. j. med. lab. sci. tech. 2022; 4(2): 111–119 ari nuswantoro & jessica ningtyas berlianti 1 1 4 garlic filtrate is the filtered and collected solution in the erlenmeyer, while the residue is the solid left on the filter paper. measurement of clotting time is performed in the following ways: (a) the time measured with a stopwatch when blood enters the syringe; (b) 5 ml of blood collected and placed into the test tube according to the treatment group; (c) left the tube at room temperature (25°c); (d) all tubes tilted at a 45° angle after 5 minutes, and repeated every minute; and (e) the time recorded until the blood clots completely. if the blood did not clot for more than 20 minutes, the observation was over. clotting periods are measured in minutes and categorized as follows: less than 5 minutes, 5–15 minutes, 15–20 minutes, and more than 20 minutes (20). for examination of calcium ion levels, samples were obtained from venous blood. fresh venous blood was centrifuged for 10 minutes at 1500 g, then left at room temperature to 2 hours; two layers formed and the top layer (supernatant) was serum. on the other hand, fresh blood was inserted into an anticoagulated tube and centrifuged for 15 minutes at 2000–3000 g to extract plasma (21). heparin was employed as an anticoagulant, while 50 μl/ml and 100 μl/ml garlic filtrate were used as candidate anticoagulants in this investigation. according to biosystems (22), calcium measurement is as follows: (a) 1000 μl of reagent a and 1000 μl of reagent b were inserted into each blank, standard, and sample tube; (b) 20 μl of calcium standard solution was inserted into a standard tube; (c) 20 μl of serum/plasma was inserted into the sample tube; (d) homogenized and then incubated for 2 minutes at room temperature and (e) read the absorbance of the standard and sample against the blank at a wavelength of 610 nm and not more than 60 minutes. statistical studies of the variations in clotting time and calcium ion levels between the four treatment groups were carried out using the friedman test and the anova test, respectively, using spss (version 26). results this study compared the impact of adding garlic filtrate as an alternative to anticoagulants in extending clotting time in groups that received anticoagulants and those that received heparin. in non-anticoagulated samples, blood clots occurred between 5 and 15 minutes (mean time was 8 minutes 56 seconds), whereas in samples given 50 μl/ml garlic filtrate, 100 μl/ml garlic filtrate or heparin, no blood clots occurred for up to 20 minutes (figure 1). the friedman test revealed a significant difference in clotting time between non-anticoagulated samples and those given 50 μl/ml garlic filtrate, 100 μl/ml garlic filtrate, and heparin with a significance value of 0.000 < 0.05. ina. j. med. lab. sci. tech. 2022; 4(2): 111–119 ari nuswantoro & jessica ningtyas berlianti 1 1 5 figure 1. the four treatment groups' clotting times the calcium levels in heparinized plasma and serum were almost identical, at 8.66 mg/dl and 8.52 mg/dl, respectively, and did not differ statistically (significance value (p) 0.438). however, the calcium level in 50 μl/ml garlic filtrate plasma dropped dramatically to 4.13 mg/dl and was even lower in 100 μl/ml garlic filtrate plasma, at 3.58 mg/dl (figure 2). there was a significant difference in serum calcium levels between 50 μl/ml garlic filtrate plasma and 100 μl/ml garlic filtrate plasma (p 0.000). a difference in calcium levels exists between plasma of 50 μl/ml garlic filtrate and heparinized plasma, as well as between plasma of 100 μl/ml garlic filtrate and heparinized plasma (p 0.000 and 0.004 respectively). figure 2. the four treatment groups' calcium levels ina. j. med. lab. sci. tech. 2022; 4(2): 111–119 ari nuswantoro & jessica ningtyas berlianti 1 1 6 discussion platelets get activated after vascular damage and produce thromboxane a2, adenosine diphosphate (adp), serotonin, pselectin, calcium ion, and fibrinogen, among other prothrombotic mediators. platelets will stick to the subendothelial matrix receptors and produce aggregates (18). the calcium ion (ca2+) is a coagulation factor iv that is essential for blood coagulation and homeostasis. after platelet activation, calcium binds to phospholipids, providing a surface for assembly with different coagulation components. calcium ions and factor viia, in particular, stimulate alterations in the conversion of factor x to xa (23,24). heparin binds to various proteins after administration, but it is binding to antithrombin, which is significant because it induces a surface change and inactivates thrombin. antithrombin binds to clotting factors, but two are particularly important: thrombin (factor iia) and factor xa. it inhibits the development of clots and prolongs blood clotting time by inactivating thrombin and blocking the conversion of fibrinogen to fibrin (25). allicin (diallythiosulfinate) and propyl propane-thiosulfinate are two onion thiosulfinates that suppress human platelet aggregation by up to 90% (26). allicin may easily pass across cellular membranes and bind to sulfhydryl (sh)-containing proteins, preventing them from forming free sulfhydryl residues. garlic extract's antiplatelet action is attributable, at least in part, to the suppression of collagen binding to particular platelet receptors. platelet adhesion and activation might be hindered as a result (27). ajoene, another active chemical, inhibits the binding of fibrinogen and von willebrand factor (vwf) to the gpiib/iiia receptor (28). because of its interaction with fibrinogen receptors, ajoene has anti-aggregatory and antithrombotic properties, and it also blocks arachidonic acid-stimulated platelet aggregation irreversibly (29). in this study, similar calcium levels in serum and plasma heparin confirmed that heparin did not inhibit calcium ions. however, the decrease in calcium levels in the plasma of garlic filtrate gave evidence that the active ingredients in it bind calcium ions, prolonging clotting time, as shown by heparinized blood. it showed that garlic filtrate has the possibility as an anticoagulant replacement, especially under the conditions outlined above. garlic filtrate is considered to be superior to garlic extract. apart from making the procedure accessible, the combination of components in garlic filtrate has an anticoagulant effect balanced by synthetic anticoagulants such as heparin. because just one set of active chemicals is acquired when the extract is used, the anticoagulant's ina. j. med. lab. sci. tech. 2022; 4(2): 111–119 ari nuswantoro & jessica ningtyas berlianti 1 1 7 potential is thought to be diminished. if the goal is efficiency, a dose of 50 μl/ml garlic filtrate is preferable to 100 μl/ml garlic filtrate due to their shared impact of lengthening clotting time. this study, however, was unable to determine how long garlic filtrate may be stored before use, what parameters are safe or influenced when using garlic filtrate as an anticoagulant, or how long the effect lasts after being combined with blood. conclusions non-anticoagulated blood had an average clotting time of 8 minutes and 56 seconds, but heparinized and garlic filtrate plasma had a longer clotting time. this result might be attributed to the active components' performance, allicin and ajoene, which was statistically supported by the friedman test findings, which revealed a significant difference between the four groups. similarly, calcium ion levels in serum and heparinized plasma were somewhat similar, at 8.52 mg/dl and 8.66 mg/dl, respectively, but calcium ions were low in both treatments of garlic filtrate plasma (4.13 mg/dl and 3.58 mg/dl, respectively). this result suggests that garlic filtrate might be used as an anticoagulant alternative. author contributions ari nuswantoro: concept and design, writing original and revising manuscript, analysis and interpretation of data, supervision and final approval of the version to be published. jessica ningtyas berlianti: concept and design, methodology, laboratory analysis, administration, and research permission. acknowledgement the authors wish to acknowledge the contributions of participating volunteers on this study. conflict of interest the authors declare that there is no conflict of interest. references 1. giavarina d, lippi g. blood venous sample collection: recommendations overview and a checklist to improve quality. clin biochem. 2017;50(10–11):568–73. doi: 10.1016/j.clinbiochem.2017.02.021 2. lippi g, von meyer a, cadamuro j, simundic am. blood sample quality. diagnosis. 2019 ;6(1):25–31. https://pubmed.ncbi.nlm.nih.gov/29794250/. doi: 10.1515/dx-2018-0018 3. lippi g, favaloro ej. laboratory hemostasis: from biology to the bench. clin chem lab med. 2018;56(7):1035–45. https://www.degruyter.com/document/doi/10.151 5/cclm-2017-1205/html?lang=de. doi: 10.1515/cclm-2017-1205 4. cedrone e, neun bw, rodriguez j, vermilya a, clogston jd, mcneil se, et al. anticoagulants influence the performance of in vitro assays intended for characterization of nanotechnologybased formulations. mol a j synth chem nat ina. j. med. lab. sci. tech. 2022; 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4(2): 99–110 durrotul hasanah, et al. 1 0 0 introduction convention on international trade in endangered species of wild fauna and flora (cities) stated that the use of medical materials uses approximately 60,000 plant species worldwide for both traditional and modern medicines (1). mistletoes tea (escurrula atropurpurea) and mango mistletoes (dendrophthoe pentandra l.) miq is a plant that has benefits for maintaining health and fitness (2,3). tea mistletoes have been shown to reduce contractility of the tail arteries of hypertensive rats in vitro. while in vivo, tea mistletoes reduce blood pressure by improving oxidative stress and endothelial dysfunction, lowering mda (malondialdehyde) levels, and increasing no (nitrix oxygen) levels (2,4,5,6). in addition, the flavonoid and tannin compounds in the dendrophthoe pentandra l. act as antihypertensives. hypertension, known as the silent killer, is a condition of increased systolic of more than 140 mmhg and diastolic blood pressure of more than 90 mmhg (7,8). in the incidence of cardiac and blood vessels (vascular system) diseases, hypertension is one of the most influential risk factors (9,10,11,12,13). people with hypertension have a narrow blood vessel, known as vasoconstriction, which increases the cardiac work to pump blood throughout the body. this condition can weaken the cardiac muscle and cause hypertrophy. traditional medications can be an alternative to prevent and treat hypertension. therefore, on this occasion, the authors examine the histopathological profile of the cardiac in male wistar rats with a preventive model of hypertension after administration of a methanolic extract combination of tea and mango mistletoes. materials and methods this study used a completely randomized experimental design with 25 wistar rats in total for five treatments of administration of the extract combination methanolic tea leaf mistletoe and mango mistletoe, namely negative control (-), positive control (+), p1 with a dose of 50 mg/kg bw, p2 with a dose of 100 mg/kg bw and p3 with a dose of 200 mg/kg bw. a methanolic extract combination of tea and mango mistletoes was exposed to rattus novergicus (male wistar rats) for 28 days and followed by the administration of deoxycorticosterone acetate (doca)salt in the last 14 days as a preventive model. the research was carried out in march – may 2021 in the animal house faculty of dentistry, laboratory of anatomical histopathology, faculty of medicine universitas brawijaya malang, laboratory of the faculty of medicine ina. j. med. lab. sci. tech. 2022; 4(2): 99–110 durrotul hasanah, et al. 1 0 1 islamic university of malang, and laboratory of balai materia medica batu, east java. all experiments were performed with ethical approval from the islamic university malang faculty of medicine health research ethics commission (no. 006/le.001/iv/03/2020). preparation methanolic extract combination of mistletoe tea leaves and mistletoe of mango mistletoe the methanolic extract combination of tea and mango mistletoes is produced using simplicia derived from mashed dried mistletoe leaves. the maceration process includes immersing the material with a solvent that is by following per under the active compound to be extracted (14). the powder or simplicia leaves of tea parasite and mango parasite was measured at 100 grams were put into five 1.5 liter plastic bottles. then, 1 litre of 90% methanol solution was put into each bottle and shaken until the solution was homogeneous for 1 hour. after 1 hour, the homogeneous solution was allowed to stand and precipitated for 24 hours. this process was stopped when equilibrium was reached (14). the result of the deposition process for 24 hours will form a bottom layer in the form of a natant, while the top layer is called a supernatant. part supernatant will be accommodated and proceed to the extraction stage using a rotary evaporator to produce leaf extract of tea mistletoe and mango mistletoe in the form of a paste. test animal acclimatization process wistar rats (rattus norvegicus) 2-3 months old with a bodyweight of 150250 were acclimatized at the animal house, faculty of dentistry, universitas brawijaya malang for five days at room temperature ± 24oc with humidity of approximately 50-60% protected from the smoke of industry, motor vehicle fumes, and other pollutants as well as being fed and drinking. test animal care the test animals were weighed before and after the acclimatization (pretreatment) once a week for 28 days of treatment. weekly monitoring of body weight is required to determine the dose amount. wistar rats use a cage with a cover in the form of woven wire and wood shavings given as a base for mice. the husks are regularly replaced once every two days. feeding was performed every morning and evening with a feed weight of 7 grams per rat. treatment methanolic extract combination tea and mango mistletoes this study used a dose of methanolic extract combination of tea and mango mistletoes in a ratio of 3:1 with the first ina. j. med. lab. sci. tech. 2022; 4(2): 99–110 durrotul hasanah, et al. 1 0 2 dose of 50 mg/kg bw, the second dose of 100 mg/kg bw, and the third dose of 200 mg/kg bw. the methanolic extract combination of tea and mango mistletoes was administered for four weeks (5 times/week). provision doca-salt as a preventive hypertension model doca is a precursor of the adrenal cortex mineralocorticoid hormone, which has an analogous effect to the mineralocorticoid aldosterone, which causes retention of na+ and h2o in the distal tubule. doca-salt is one model of secondary hypertension due to endocrine (hormonal) influences. doca was administered twice a week during the last two weeks of treatment which was also at the same time as the administration of a methanolic extract combination of tea and mango mistletoes. doca was administered by subcutaneous injection in the positive control (+), the first treatment (p1), (p2), and (p3). blood pressure readings were taken in rats before and after doca treatment. doca given to rats was first homogenized with sesame oil and according to the dose. surgery surgery on the test animals was performed after 28 days of treatment by taking organs for histopathological examination. the organ observed is cardiac. after 3 hours of fasting, the rats were given either intramuscular ketamine injections of anesthesia. then dissection was performed after the rat was unconscious. then the cardiac organ was taken and put in an organ tube with a 10% formalin solution. the organ taken was used as a microscopic preparation and then processed following the standard histological method with hematoxylin and eosin (he) staining. histopathological examination histopathological observations of cardiac organs were performed after staining using hematoxylin and eosin by taking 400x and 200x magnification photos with five fields of view under a trinocular microscope olympus utv0.5xc-3, t7 tokyo, japan. the photos with 400x magnification were followed by observations with the imagej application to measure the diameter of cardiac muscle cells, while the photos with 200x magnification were used as visualizations. data analysis the data obtained were statistically tested using the jamovi application version 1.1.9. one-way analysis of variance (anova). a post-hoc test was conducted after additional tests. we employed anova with post hoc testing to examine differences between means while reducing the family error rate. ina. j. med. lab. sci. tech. 2022; 4(2): 99–110 durrotul hasanah, et al. 1 0 3 results the measure of cardiac muscle cell diameter microscopic measurement of the diameter of the cells in the cardiac muscle was performed using the imagej application. previously, previously, cardiac histopathology was observed under a trinocular microscope with an objective magnification of 400x in 5 fields of view. histopathological features of the cardiac in male wistar rats were observed after administration of a methanolic extract combination of tea and mango mistletoes for 28 days and followed by injection of doca-salt as a model of hypertension in rats in the last two weeks. the results were obtained based on the treatment in each group (table 1). table 1. histopathological observations on the mean cell diameter in cardiac muscle (µm) male wistar rats after treatment 28 day. treatment mean cell diameter in cardiac muscle (µm) negative control 15.5 ± 0.918a positive control (doca-salt) 26.2 ± 2.489b p1 (dose 50 mg/kgbb + doca-salt) 15.6 ± 0.982a p2 (dose 100 mg/kgbb + doca-salt) 15.1 ± 0.691a p3 (dose 200 mg/kgbb + doca-salt) 14.4 ± 0.357a the different letters in the same column indicate α significant difference at p<0.05. data are presented as mean ± sd based on table 1, the positive control (k+) group had the widest cell diameter. the result also showed there was a significant difference between treatment groups. observations were done by measuring the diameter of cardiac muscle cells in the myocardium layer. the normality test showed that the data were normally distributed (p<0.05). the results of the one way anova test obtained a p-value of <0.001, in which there was a significant difference statistically. the post hoc test was done by comparing the positive control (k+) induced by doca-salt, the negative control (k-) without doca-salt induction, p1, p2 and p3. the result showed a statistically significant difference between positive control (k+) induced by doca-salt and the negative control (k-) without doca-salt induction. in contrast, there was no significant difference in the p1, p2, and p3 (treatment group). histopathological observation observations obtained photos of cardiac muscle cells both at 200x and 400x magnification (figure 1-4). from this microscopic observation, the positive control mice were significantly different from the negative control mice (-) and the treated mice (p1, p2, and p3). on the contrary, there was no significant ina. j. med. lab. sci. tech. 2022; 4(2): 99–110 durrotul hasanah, et al. 1 0 4 difference between the negative control and treatment groups (p1, p2, and p3). figure 1. microscopic observations (cross section: 200x) myocardial tissue. (a) control group (-), (b) control group (+), (c) group p1 (50 mg / kgbw), (d) group p3 (200 mg / kgbw). description: 1,2,3,4 : cell nucleus figure 2. microscopic observations (vertical section: 200x) myocardial tissue. (a) control group (-), (b) control group (+), (c) group p1 (50 mg / kgbw), (d) group p2 (100 mg / kgbw), (e) group p3 (200 mg / kgbw). description: 1,2,3,4,5 : cell nucleus e a 1 2 3 4 1 2 3 4 5 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm ina. j. med. lab. sci. tech. 2022; 4(2): 99–110 durrotul hasanah, et al. 1 0 5 figure 3. microscopic observations (cross section: 400x) myocardial tissue. after 28 (a) control group (-), (b) control group (+), (c) group p1 (50 mg / kgbw), (d) group p2 (100 mg / kgbw), (e) group p3 (200 mg / kgbw). description: 1,2,3,4,5: cell nucleus. figure 4. microscopic observations (ventricular section: 400x) myocardial tissue. (a) control group (-), (b) control group (+), (c) group p1 (50 mg / kgbw), (d) group p2 (100 mg / kgbw), (e) group p3 (200 mg / kgbw). description: →1,2,3,4,5 : cell nucleus, →1 : hypertrophy, → 1 : intercalated disc discussion tea and mango mistletoes are parasitic plants but have also proven to be efficacious as herbs. these two plants contain flavonoids, compounds in herbal medicines or alternative phytopharmacology preparations. the research conducted by atiroh et al., (2,3) found that mistletoe tea reduced the contractility of the artery blood vessels on e 1 2 3 4 5 1 2 1 3 5 4 1 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm ina. j. med. lab. sci. tech. 2022; 4(2): 99–110 durrotul hasanah, et al. 1 0 6 the rat tail and blood pressure by improving oxidative stress and endothelial dysfunction. the parameters observed in this study were cardiac histopathology, precisely by measuring the diameter of cardiac muscle cells. the cardiac diameter muscle cell chose because of the cardiac function as a blood pump throughout the body (15). if cardiac function decreases because of cardiac muscle infection, this can affect body oxygen (o2) and nutrient demand (16). in the same way as the adaptation mechanism of hypertension, cell hypertrophy will occur in the cardiac muscles that receive an additional workload. cardio output increases because it experiences vasoconstriction then the cardiac is forced to work extra to meet the blood supply for the body. as a result, the cardiac muscle is forced to work hard while the cardiac muscle is not strong enough then the cells develop hypertrophy. the administration of the methanolic extract of the combination of tea mistletoe and mango mistletoe showed a significant difference with a p-value <0.001 (p<0.05) between all treatment groups. there are differences in each treatment group. the longest mean cell diameter of cardiac muscle was found in the positive control group (k+), which was 26.2 µm. this positive control group is a group of hypertensive rats that received doca-salt treatment. meanwhile, treatment groups 1, 2 and 3 showed the results of the p-value calculation obtained from the three treatments >0.05 to the negative control group (k-). this means that exposure to the methanolic extract combination of tea and mango mistletoe can prevent the increase in the diameter of cardiac muscle cells. the average of the three treatment groups (p1, p2, p3) has a value close to the control group negative (k-). this indicates that the administration of dose variations in each treatment group has been able to prevent the increase in the diameter of cardiac muscle cells. the p1 group (50 mg/kg bw) had an average of 15.6 µm and was able to reduce hypertrophy. tea mistletoe and mango mistletoe have antioxidants and can protect the body against damage caused by ros (reactive oxygen species) (4). from various studies, tea and mango mistletoes contain flavonoid compounds. flavonoids can act as natural antioxidants that protect biological systems and inhibit cell oxidation by reducing and capturing active oxygen and free radicals, especially superoxide. one of the efficacious flavonoids is quercetin. quercetin is found in abundance both in tea mistletoe and in mango mistletoe. the activity of this compound as an antioxidant is by releasing ina. j. med. lab. sci. tech. 2022; 4(2): 99–110 durrotul hasanah, et al. 1 0 7 or donating hydrogen ions to peroxyl free radicals to become stable. in addition, flavonoid compounds also have antithrombosis and anti-inflammatory effects that can reduce the risk of cardiac disease (17). from the anova test, the standard deviation (sd) value was also obtained, namely in the negative control group (k-) of 0.918, positive control (k+) of 2.488, p1 of 0.982, p2 of 0.691, and p3 of 0.357. the results of the analysis data obtained show that the standard deviation value in all groups has a value smaller than the average value. the data obtained in this study shows that the overall average of the data obtained is representative. the cell data obtain from cells that are not tangential or cells that have a ratio. histopathological examination results showed that the p1, p2, and p3 groups improved precisely in the diameter of cardiac muscle cells. those groups had a wider diameter size of the cardiac muscle or myocardium than the average size (10µm-20µm). meanwhile, in the group with doca-salt injection or the group with hypertension, the diameter of the cardiac muscle cells exceeded the normal size (hypertrophy). hypertrophy is a progressive disorder of an increase in the volume of cells in a tissue or organ. the increase in cardiac muscle cell diameter is an adaptation response to the increased workload of the cardiac due to hypertension (18). hypertension occurs with an increase in intracellular pressure which then causes high blood pressure experiencing shear stress on endothelial cells. the endothelial cells which are found in the tunica intima blood vessel will cause shear stress leading to endothelial dysfunction when exposed to high blood pressure (19,20). endothelial dysfunction causes vasoconstriction, a decrease in o2 and an upgrade growth factor. as a result of endothelial dysfunction and increased growth factor, the arteries of people with hypertension usually lose their elasticity. therefore, the cardio output cannot be the same as in people without hypertension. the cardiac of hypertensive people will increase its work to pump blood to meet the amount of co. on the contrary, the cardiac muscle is not strong, and the cells swell because they are forced to work, which causes hypertrophy of the cardiac muscle cells (21). one of the flavonoid contents in tea mistletoe and mango mistletoe is quercetin. in this case, quercetin can act on the smooth muscle of the arteries by stimulating or activating endothelium derived relaxing factor (edrf) that can cause vasodilation. quercetin in endothelial cells has the potential to increase the production of no. quercetin ina. j. med. lab. sci. tech. 2022; 4(2): 99–110 durrotul hasanah, et al. 1 0 8 can diffuse directly and synthesize no in the endothelium and smooth muscle, which stimulates guanylate cyclase to form cgmp resulting in vasodilation (4). the occurrence of vasodilation can prevent hypertrophy because the cardiac will not work harder, which causes cardiac muscle cells to widen. conclusions a methanolic extract combination tea leaves and mango mistletoes on male wistar rats in a preventive model of hypertension could significantly prevent the enlargement/widening of auto cells cardiac. the three variations of the dose showed that the result data did not show a significant difference. this effect was controlled by a methanolic extract combination tea and mango mistletoes at a dose of 50 mg/kgbw which was the optimum dose in reducing the diameter of cardiac muscle cells in wistar rats. the methanolic extract of the combination of tea mistletoe leaves and mango mistletoe can be used as herbal ingredients to increase preparations in the field of phytopharmacology. author contributions durrotul hasanah: conceptualization, methodology, formal analysis, investigation, writing-original draft. nour athiroh as: supervision, project administration, funding acquisition. nurul jadid mubarokati: data curation, writingoriginal draft preparation, visualization, and writing-reviewing. acknowledgement i am grateful to ministry of education, culture, research and technology of the republic of indonesia, number: 549/g164/u.lppm/k/b.07/viii/2021 which has supported grant funds for research, university leading applied research (ptupt) with the title “combination herbal mistletoe as a phytopharmaceutical product preparation an alternative candidate for indonesian traditional natural antihypertensive drugs”. conflict of interest the author declares that there is no conflict of interest. references 1. annisatul h, athiroh n, mubarakati nj. histopathological profile of therapeutic doses of mango mistletoe methanolic. jsmartech. 2021;02(02):48–54. doi: https://doi.org/10.21776/ub.jsmartech.2021.002. 02.48 2. athiroh n, purnomo y, mubarakati nj. sub chronic diagnosis of administration with scurrula atropurpurea to blood biochemichal analysis. iop conf ser mater sci eng. 2020;846(1). doi: 10.1088/1757-899x/846/1/012002 ina. j. med. lab. sci. tech. 2022; 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[relationship between duration of hypertension http://dx.doi.org/10.33474/j.sa.v3i1.6621 ina. j. med. lab. sci. tech. 2022; 4(2): 99–110 durrotul hasanah, et al. 1 1 0 and features. electrocardiogram of left ventricular hypertrophy and old myocardial infarction]. jurnal kedokteran diponegoro. 2018;7(2):1251– 65. doi: https://doi.org/10.14710/dmj.v7i2.21198 71 the correlation results of examination of hemoglobin and the erythrocyte index in patients with suspected covid-19 in the hospital of kendari city sri aprilianti idris1, firdayanti1, susanti1, muh. azdar setiawan2 1study program medical laboratory technology, politeknik bina husada kendari, kendari, southeast sulawesi, indonesia 2study program pharmacy, politeknik bina husada kendari, kendari, southeast sulawesi, indonesia correspondence: sri aprilianti idris, politeknik bina husada kendari jl. sorumba, anaiwoi, kec. kadia, kota kendari, southeast sulawesi, indonesia zip code: 93117 email: sriaprilianti.aakkdi@gmail.com received: mei 17, 2021 revised: januari 10, 2022 accepted: maret 8, 2022 published: april 28, 2022 doi: 10.33086/ijmlst.v4i1.2105 abstract coronavirus disease 2019 has become a global problem causing hundreds of thousands of deaths worldwide. haemoglobin is more susceptible to covid-19 virus attacks. haemoglobin functions as a carrier of oxygen to organs in the body. when the concentration of haemoglobin in the blood circulation is low, the transport of oxygen to several organs in the body can be disrupted. sars-cov-2 interacts with haemoglobin in red blood cells. this interaction causes the virus to break the haemoglobin chain and cause hemolysis. the erythrocyte index which consists of mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch), and mean corpuscular hemoglobin concentration (mchc) values are used to indicate the occurrence of anemia in covid-19 patients. this research method used descriptive analytic with a cross sectional study design. the population of this study was suspected covid-19 patients with a sample of 27 people. sampling was carried out using purposive sampling. this study aims to determine the relationship between haemoglobin examination results and erythrocyte index in suspected covid-19 patients.the instrument in this study used secondary data which included the results of examination of haemoglobin values and erythrocyte index. the results of the test using a parametric statistical approach with the correlation analysis method showed that there was a correlation between the results of the haemoglobin and mcv test (p-value 0.057 > 0.05), while the haemoglobin and mch test had no correlation (p-value 0.777 > 0.05), and there is no correlation between haemoglobin and mchc examination (p-value 0.372 > 0.05). keywords anemia, erythrocyte indeks, haemoglobin. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2022; 4(1): 71–80 sri aprilianti idris, et al. 7 2 introduction coronavirus disease 2019 (covid-19) has become a global problem causing hundreds of thousands of deaths worldwide since december 2019. the main challenge in handling this new outbreak is the limited data regarding variations in clinical and epidemiological findings (1). in december 2019, in wuhan, hubei province, china, 44 cases of pneumonia were reported due to infection with a new type of coronavirus that had not been identified. the virus was later known as sars-cov-2 because it has 82% similarity in its genome sequence to severe acute respiratory syndrome-corona virus (sars-cov) which became an outbreak of the disease in guangzhou, china in 2003. the disease caused by infection of sarscov-2 is then referred to as coronavirus disease 2019 (covid-19). since march 11, 2020, world health organization (who) has designated covid-19 as a global pandemic (1,2). according to who, data as of march 30, 2020, there were 693,224 cases and 33,106 deaths worldwide. europe and north america have become the epicenter of the covid-19 pandemic, with cases and deaths surpassing china. the united states ranks first with the most covid-19 cases with the addition of 19,332 new cases on march 30, 2020, followed by spain with 6,549 new cases. italy has the highest mortality rate in the world at 11.3% (3). the first covid-19 was reported in indonesia on march 2, 2020 (4). by april 9, 2020 the pandemic had spread to all provinces in indonesia after gorontalo confirmed its first case, with jakarta, west java, and east java being the worst-affected provinces. so far, indonesia has recorded 496 deaths, more than any other southeast asian country. the fatality rate is also one of the highest in the world. the researchers suggest the main reason for this high number might be a lack of testing, hence many cases went undetected (5). in southeast sulawesi, 332 covid-19 cases were confirmed as positive cases, with 113 patients undergoing treatment, 214 declared cured and 5 people died. while 535 cases were asymptomatic (otg), 15 cases were patients under surveillance (pdp), and 39 were declared as people under monitoring (odp). meanwhile, in kendari city the number of positive cases was 66 cases, with 57 cases of patients were declared cured, and 3 cases of patients dead (6). coronavirus is a positive single-strain rna virus, encapsulated and unsegmented. coronavirus belongs to the order nidovirales of the coronaviridae family. the structure of the coronavirus forms a cube-like structure with the s protein located on the surface of the virus. s protein or spike protein is one of the main viral antigen proteins and is the main structure for gene writing. this s protein plays a role in the attachment and ina. j. med. lab. sci. tech. 2022; 4(1): 71–80 sri aprilianti idris, et al. 7 3 entry of the virus into host cells (interaction of protein s with its receptors on the host cell). coronavirus is sensitive to heat and can be effectively inactivated by disinfectants containing chlorine, lipid solvents at 56oc for 30 minutes, ether, alcohol, peroxyacetic acid, non-ionic detergents, formalin, oxidizing agents, and chloroform (7). haemoglobin is more susceptible to covid-19 virus attacks. in patients with covid-19 cases, most of them come with complaints of shortness of breath, causing less and less haemoglobin that can carry oxygen and carbon dioxide to be spread throughout the body, so that it can interfere with the respiratory tract. the attack of the covid-19 virus can damage many organs and cause inflammation that occurs in the human lungs. materials and methods this research method used descriptive analytic with a cross-sectional study design that was carried out from june 2020 to july 2020. the population of this study was suspected covid-19 patients at the kendari city general hospital with a sample of 27 people, and sampling was carried out using purposive sampling with sample criteria. inclusion criteria patients with suspected covid-19 with symptoms (fever and at least one sign/symptom of respiratory illness, such as cough, shortness of breath); history of travel or living in a community area of covid-19; age 16 53 years; gender male or female; and patients with haemoglobin and erythrocyte index examinations. exclusion criteria patients with suspected covid-19 who do not perform haemoglobin and erythrocyte index examinations (8). to address the research objectives, this research used secondary sources, which included the results of the examination of haemoglobin values and erythrocyte index. the erythrocyte index consists of the value of mean corpuscular haemoglobin (mch), mean corpuscular volume (mcv), mean corpuscular haemoglobin concentrate (mchc). haemoglobin values and erythrocyte index were obtained from the results of routine blood examinations at the laboratory of suspected covid-19 patients . statistical analysis in this research, all data analyses were performed by using spss for windows version 20.0 (spss inc., chicago) and microsoft excel. data consist of the haemoglobin and erythrocyte index examinations. these data were tested for correlation, including to determine the relationship between haemoglobin, erythrocyte index, and suspected covid-19 patients. ina. j. med. lab. sci. tech. 2022; 4(1): 71–80 sri aprilianti idris, et al. 7 4 results this research was conducted at the kendari city general hospital with a total of 27 patients. the patients were suspected covid-19 patients who came to the hospital with covid-19 symptoms. table 1-7 shows classification of patients with suspected covid-19 based on haemoglobin examination results. the type of data used in this study is quantitative data (medical records of covid-19 patients at the kendari city general hospital) in the form of haemoglobin and erythrocyte index examination results {mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch), mean corpuscular hemoglobin concentration (mchc)}. these data were evaluated by using a parametric statistical, since data does follow normal distribution. table 1. classification of patients with suspected covid-19 based on haemoglobin examination results results of haemoglobin (g/dl) frequency (n) (%) normal 26 96.29 low 1 3.70 amount 27 100 table 1 shows laboratory result of patient enrolled in this study. based on table 1, patients with suspected covid-19 with normal haemoglobin examination results were 26 patients (96.29%), while patients with low examination results were 1 patient (3.70%). table 2. classification of patients with suspected covid-19 based on mcv results result of mcv (fl) frequency (n) (%) normal 25 92.6 low 2 7.40 amount 27 100 based on table 2, the results of the examination of mcv values with normal values were 25 people (92.6%) while patients with low examination results were 2 patients (7.40%). table 3. classification of patients with suspected covid-19 based on mch results result of mch (pg) frequency (n) (%) normal 10 37.4 low 17 62.7 amount 27 100 based on table 3, the results of the examination of mch values with normal examination results were 10 patients (37.4%), while patients with low examination results were 17 patients (62.7%). table 4. classification of patients with suspected covid-19 based on mchc result of mchc (g/dl) frequency (n) (%) normal 10 37.4 low 17 62.7 amount 27 100 ina. j. med. lab. sci. tech. 2022; 4(1): 71–80 sri aprilianti idris, et al. 7 5 based on table 4, the results of the examination of mchc values with normal examination results were 10 patients (37.4%), while patients with low examination results were 17 patients (62.7%). table 5. correlation test for suspected covid-19 patients based on the results of the haemoglobin and erythrocyte index examination correlation test category sig. low normal haemoglobin 1 26 0.057 mean corpuscular volume 2 25 *statistically significant (p<0.05) based on table 5, the results of the correlation analysis obtained a probability value of 0.057>0.05, which means that there is no relationship between the results of the haemoglobin examination and the mcv value. a p-value less than 0.05 (typically ≤ 0.05) is statistically significant table 6. correlation test for suspected covid-19 patients based on the results of the haemoglobin and erythrocyte index examination correlation test category sig. low normal haemoglobin 1 26 0.777 mean corpuscular haemoglobin 17 10 *statistically significant (p<0.05) based on table 6, the probability value is above 0.05 (0.777) which indicates that there is no relationship between the results of haemoglobin examination and the mch value. based on table 7, the significant value is above 0.05 (0.372), which means that there is no relationship between the results of the haemoglobin examination and the mchc value. table 7. correlation test for suspected covid-19 patients based on the results of the haemoglobin and erythrocyte index examination correlation test category sig. low normal haemoglobin 1 26 0.372 mean corpuscular haemoglobin concentration 17 10 *statistically significant (p<0.05) discussion there were 26 patients with suspected covid-19 with normal haemoglobin examination results (96.29%), while 1 patient with low test result. in this case, the results of haemoglobin examination found more normal results, this study is in line with previous research mahrania et al., (1), which found normal haemoglobin examination results in covid-19 patients. the results of the examination of mcv values with normal values were 25 people (92.6%) while patients with low examination results were 2 patients (7.40%). the results of this examination are following previous research conducted by mahrania et al., (1), many normal results were also found in the mcv value. a low mcv value indicates that the red blood cell volume is below normal, a ina. j. med. lab. sci. tech. 2022; 4(1): 71–80 sri aprilianti idris, et al. 7 6 condition also known as microcytosis (small cells). erythrocytes that are too small mean that they are only able to carry oxygen in small amounts, causing the body to become weak or tired easily (9). the results of the examination of more mch values obtained low examination results as many as 17 patients (62.7%). low mch levels are usually affected by various types of anemia, such as microcytic anemia, which occurs when red blood cells are too small to contain a certain amount of haemoglobin. the main cause is a lack of nutrients or nutrients from food, especially iron. in addition, other conditions cause low mch, namely iron deficiency anemia, celiac disease, gastric surgery, excessive menstruation, and vitamin b deficiency (10). the results of the examination of mchc value found that the results of the examination were low in 17 patients (62.7%). in this case, if the mchc value is low, it means that the haemoglobin level in each red blood cell is lower which indicates the cells are hypochromic. where the causes of low mchc values are usually caused by hypochromic microcytic anemia, iron deficiency, premature destruction of red blood cells, lead poisoning, cancer, and parasitic infections (11). based on the results of the correlation test analysis of the haemoglobin and erythrocyte index results, it was found that there was no correlation between haemoglobin and mcv, as well as haemoglobin and mch, and there was no correlation between haemoglobin and mchc. these results indicate that haemoglobin levels do not affect the erythrocyte index value in suspected covid-19 patients. although normal haemoglobin levels are not followed by erythrocyte index values. at the beginning of the covid-19 infection, especially in suspected patients, they showed normal hematological examinations including haemoglobin and erythrocyte index examinations. however, in covid-19 positive patients, the virus can attack the haemoglobin in the red blood cells through a series of cellular actions, which ultimately makes the red blood cells unable to carry oxygen, resulting in a decrease in haemoglobin levels (12). several research literatures reported that the results of blood examinations from covid-19 patients showed abnormal results, namely a decrease in the patient's haemoglobin and neutrophil values, while the opposite results were shown in the serum ferritin index, erythrocyte sedimentation rate, c-reactive protein, albumin, and lactate dehydrogenase in many patients, was significantly elevated (12). a decrease in the value of haemoglobin implies that the body will accumulate a lot of harmful iron ions, which will form inflammation in the body and an increase in ina. j. med. lab. sci. tech. 2022; 4(1): 71–80 sri aprilianti idris, et al. 7 7 c-reactive protein and albumin, causing cells to experience oxidative stress and will react, causing inflammation (13). the effect of sars-cov-2 other than on red blood cells, namely on white blood cells (leukocytes) (13). in covid-19 positive patients, a decrease in the number of white blood cells, and lymphocytes and an increase in neutrophils was found. the substantial decrease in the total number of lymphocytes indicates that the coronavirus affects many immune cells and inhibits the function of the cellular immune system. an increase in the number of neutrophils and a decrease in the number of lymphocytes was found in covid 19 patients. an increase in the number of neutrophils indicates the intensity of the inflammatory response, while a decrease in the number of lymphocytes indicates a damaged immune system (14). sars-cov-2, the pathogen responsible for covid-19, has caused morbidity and mortality at an unprecedented scale globally. scientific and clinical evidence is evolving on the subacute and long-term effects of covid-19, which can affect multiple organ systems. early reports suggest residual effects of sars-cov-2 infection, such as fatigue, dyspnea, chest pain, cognitive disturbances, arthralgia and decline in quality of life. cellular damage, a robust innate immune response (14). infants and young children are usually at high risk for admission to hospital for respiratory tract infections with viruses such as respiratory syncytial virus and influenza virus. in contrast, in children infected with covid-19, patients have relatively mild symptoms in general compared to adults. the reason for this discrepancy is still not clearly understood, as recent reports have shown a correlation between the severity of covid19 and the amount of viral load (virus shedding) that infects children less than adults (15). the following is a summary of post-acute covid-19 by organ system: 1. respiratory disorders complications that most often occur due to corona virus infection are disorders of the respiratory tract, such as respiratory failure or ards (acute respiratory distress syndrome) and pneumonia. this condition occurs when the lung tissue becomes inflamed and filled with fluid, thus interfering with the breathing process. when experiencing complications, patients with corona virus infection can experience a lack of oxygen. this developes and increase many covid-19 patients requires respiratory assistance, such as installing a ventilator and giving oxygen. 2. cardiovascular corona virus infection can make the heart work harder, making it dangerous for people who have a history of heart problems, such as heart disease and heart failure. several studies have also shown that the risk ina. j. med. lab. sci. tech. 2022; 4(1): 71–80 sri aprilianti idris, et al. 7 8 of dying from covid-19 is much higher in people with a history of heart disease than in previously healthy people (14). 3. kidney and liver disorders several case reports related to corona virus infection stated that some patients with severe symptoms may experience impaired kidney and liver function. until now, the cause of these complications is not known. however, the body's immune reaction to the corona virus is thought to be one of the causes (14) 4. gastrointestinal and hepatobiliary gastrointestinal disturbances can occur in patients with covid-19 even after a negative nasopharyngeal swab result. covid-19 has the potential to alter the gut microbiome, including enrichment of opportunistic organisms and depletion of beneficial commensals (14). 5. dermatologic hair loss (baldness) is the predominant symptom and has been reported in approximately 20% of covid-19 survivors (15). in addition to some of the complications above, patients with corona virus infection are also at risk of developing sepsis. this condition is more likely to occur in many patients with severe covid-19 showed general signs of shock, weak and have been hospitalized for a long time (14). a strong immune system is able to fight the corona virus well so that the symptoms of covid-19 that appear are mild and this disease can heal by itself. on the other hand, if the immune system is unable to fight the corona virus, severe covid-19 symptoms can appear and there is a risk of complications (14). bergamaschi et al., (16) reported that patients who were heavily infected with covid-19 produced low haemoglobin values. patients with anemia will have low haemoglobin levels. in the circulation system, haemoglobin serves as a carrier for oxygen to target organs in the body. when the concentration of the haemoglobin in the circulation is low, the transport of oxygen to several organs in the body will be disrupted, therefore causing hypoxia that will eventually result in multiple organ dysfunction, especially respiratory organ dysfunction. multiple organ dysfunction will contribute to the development of severe outcomes in covid-19 infection. moreover, in covid-19 infections, the state of anemia in the patients could be worsened (17-21). there are some limitations in our study, namely the number of samples (patient) and multiple blood samples were not taken at different time points of the disease course to see the status of rbc profiles. further, the comprehensive analysis of the rbc parameters (size, shape, and quality of rbc) with many samples would be helpful for early identification and better management of covid-19 disease. ina. j. med. lab. sci. tech. 2022; 4(1): 71–80 sri aprilianti idris, et al. 7 9 conclusions to conclude, this study highlighted and compared haemoglobin and mcv, mch and mchc examinations of covid-19 patient cases in kendari city hospital. our study results indicate that there is no correlation between the results of haemoglobin and mcv, mch and mchc examinations. author contributions sri aprilianti dan firdayanti: conceptualization, writing-reviewing, validation. muh. azdar setiawan: data analysis, methodology. susanti: data collection. acknowledgements we would like to thank the kendari city regional general hospital and the bina husada kendari politeknik. conflict of interest there are no conflicts of interest references 1. mahrania, hana kpf, paramita k, iffa m, fitriana nr, sarah sm, gadistya na, uti ns, pukovisa p. mild covid-19 cases in medical personnel: evaluation of clinical findings and transmission risk. j indon med assoc. 2020,70(4): 80-84. 2. wu z, mcgoogan jm. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention. jama. 2020;323(13):1239-1242. doi:10.1001/jama.2020.2648 3. world health organization. situation report–10 [internet]. 2020 [updated 2020 january 30; 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4(1): 71–80 sri aprilianti idris, et al. 8 0 14. ani n , kartik s , aakriti g,  elaine yw. postacute covid-19 syndrome. nature medicine. volume 27. april 2021(601–615). doi: https://doi.org/10.1038/s41591-021-01283-z 15. koichi y, miho f, sophia k. covid-19 pathophysiology: a review. clinical immunology. elsevier. clinical immunology 215 (2020) 10842. https://doi.org/10.1016/j.clim.2020.108427. 16. bergamaschi g, andreis fb, aronico n, lenti mv, barteselli c, merli s, pellegrino i, coppola l, cremonte em, croce g, mordà f, lapia f, ferrari s, ballesio a, parodi a, calabretta f, ferrari mg, fumoso f, gentile a, melazzini f, sabatino ad. anemia in patients with covid-19: pathogenesis and clinical significance. clin exp med. volume 21. january 2021 (239-246). doi: https://dx.doi.org/10.1007%2fs10238-02000679-4 17. ratcliffe r. first coronavirus cases confirmed in indonesia amid fears nation is ill prepared for outbreak. retreived juni, 2020, march, from guardian news & media limited: https://www.theguardian.com/world/2020/mar/0 2/first-coronavirus-cases-confirmed-inindonesia-amid-fears-nation-is-ill-prepared-foroutbreak. 18. susilo a. coronavirus disease 2019: a literature review. j penyakit dalam indonesia. 2020, 7(1): 45-54. 19. widmann fk. clinical review of laboratory examination results. egc. 2005. jakarta. 20. wenzhong l, hualan l. covid-19: attacks the 1-beta chain of hemoglobin and captures the porphyrin to inhibit human heme metabolism. chemrxiv. preprint. 2020. https://doi.org/10.26434/chemrxiv.11938173.v9 21. gaetano b, federica ba. internal medicine covid-19 collaborators. anemia in patients with covid-19: pathogenesis and clinical signifcance. springer. clinical and experimental medicine. 2021. volume 21, 239–246. https://doi.org/10.1007/s10238-020-00679-4. 10 neutrophil-lymphocyte ratio (nlr) and lymphocyte-monocyte ratio (lmr) as covid-19 screening parameters heny syahrini1, trinugroho heri fadjari2, nadjwa zamalek dalimoenthe3 1haematology and medical oncology division, internal medicine departement, universitas sumatera utara, medan, indonesia 2haematology and medical oncology division, internal medicine departement, hasan sadikin hospital, bandung, indonesia 3clinical pathology departement, hasan sadikin hospital, bandung, indonesia correspondence: heny syahrini, jln. bunga lau no.17 medan north sumatra, indonesia zip code: 20136 email: henylubis01@gmail.com received: august 21, 2021 revised: october 23, 2021 accepted: january 1, 2022 published: april 28, 2022 doi: 10.33086/ijmlst.v4i1.2281 abstract coronavirus disease 2019 (covid-19) diagnosis generally uses rt-pcr as the gold standard to detect coronavirus-2 (sars-cov-2); however, this method requires advanced laboratory equipment. alternatively, neutrophillymphocyte ratio (nlr) and lymphocyte-monocyte ratio (lmr) can be used to identify viral infection. the study aimed: (1) to compare each nlr and lmr ratio in patients with and without covid-19 and (2) to test the effectiveness of these ratios in identifying covid-19. the study was conducted at the haji adam malik central general hospital, medan, indonesia by acquiring 87 medical records data. the complete hematologic profile was analyzed from patients with and without covid-19. the nlr and lmr ratio accuracy were analyzed as a screening tool for covid-19. the auc of nlr was 0.638, with cut-off ≤ 2.49, 47.6% sensitivity, and 80% specificity; therefore, the nlr accuracy as a screening for covid-19 was defined as not good (just sufficient) because of auc <0,7. the auc of lmr was 0.661, with cut-off ≥ 3.23, 45.2% sensitivity, and 82.2% specificity; therefore, the lmr accuracy as a screening parameter for covid-19 is defined as not good (just sufficient) because of auc <0,7. there were significant differences in hematologic profile in neutrophil, lymphocyte, nlr, lmr between the patients in the covid-19 group and non-covid-19 group. nlr and lmr cannot be used as a screening tool because the area under curve (auc) is not good enough (just sufficient) in detecting covid-19. keywords covid-19, neutrophil-lymphocyte ratio, lymphocytemonocyte ratio. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 1 1 introduction the world went into chaos around december 2019 when a new virus outbreak emerged in wuhan, china. this virus spread into every corner of the world; therefore world health organization (who) stated coronavirus diseases 2019 (covid-19) as a pandemic that is caused by a virus entity named severe acute respiratory syndrome corona virus-2 (sars-cov-2) (1,2). the clinical manifestations of covid19 consisted of mild symptoms (e.g., fever, cough, myalgia, fatigue, and diarrhea) and severe symptoms (e.g., life-threatening dyspnea, respiratory failure, coagulation disturbance, headache, loss of consciousness, hemoptysis, multiorgan failures, and even death) (3,4). several parameters that describe severe clinical manifestations are respiratory rate >30 times/minute, oxygen saturation below 93%, the ratio of oxygen partial gas pressure and oxygen inspiration fraction <300 mmhg (pao2/fio2 <300), and infiltrates on lung x-ray increasing more than 50% in 24 to 48 hours (5). early detection and screening should be conducted for every patient that needs medical attention at every hospital or medical center. a patient classified as a suspect or probable covid-19 must undergo gold standard real-time polymerase chain reaction (rt-pcr) to detect sars-cov-2 virus nucleic acid in the sputum, an oropharyngeal and nasal swab of the patient (6,7). however, rt-pcr often shows late results, as it needs complex procedures, many samples, and a highly advanced laboratory workplace. rt-pcr is less efficient to use as a screening method by several considerations; however, this method remains the gold standard in diagnosing covid if the equipment is adequate (6). neutrophil-lymphocyte ratio (nlr) is a ratio of absolute neutrophil count to absolute lymphocyte count. this value can be easily calculated according to findings from routine hematology profiles. it is reported that this ratio has an impactful effect on inflammation in the patient. peripherally acquired samples of blood also can be used to calculate this ratio. this ratio is also beneficial in determining the cause of the infection, such as described in a retrospective study where a high ratio value indicates a bacterial infection in a fever of unknown origin (8,9). lymphocyte-monocyte ratio (lmr) can also be used to identify viral infection in a human. a ratio < 2 according to one study can be applied to diagnosing influenza infection instead of the standard rapid test (10). these ratios are even more convenient to use in covid cases as the percentage of leukocytes, lymphocytes, and neutrophils are greatly affected by the sars-cov-2 virus. compared to an unaffected person, a person ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 1 2 with covid-19 has much lower leukocyte and lymphocyte counts and much higher neutrophil counts. one study shows that a covid-19 patient may have leucopenia with 2,91 x 109/l, with 70% neutrophils (11). another study shows that the nlr ratio is significantly lower in covid-19 patients. the absolute leukocyte, lymphocyte, and monocyte counts are much lower than noncovid-19 patients (12). the severity of covid-19 is also distinguished according to the ratio where the nlr ratio is higher in severe covid-19, while the lmr ratio is lower in less severe covid-19 infection (12,13). although promising, these ratios have not been fully utilized yet as more precise data are needed to further evaluate the effectiveness of these ratios as sensitive and specific screening tools for covid-19. the study aims of this study are to compare each nlr and lmr ratio in patients with covid-19 and without covid-19 in severe and less severe covid-19 infection and (2) to test these ratios’ effectiveness in identifying covid-19. materials and methods study population the study was conducted at the haji adam malik central general hospital, medan cityt approved by the research ethics committee, faculty of medicine, universitas sumatera utara (no. 252/tgl/kepk fk usu-rsup ham/2020). the population of this study is patients categorized as probable of covid-19. this study was conducted from april 2020 until june 2020. the subjects of this study are patients categorized as definitive covid-19 with the positive result of rt-pcr sars-cov-2. we gathered secondary medical records and collected the hematologic profile of patients categorized as probable covid-19. the patients older than 18 and confirmed covid-19 by rt-pcr were included. patients younger than 18 years old and still categorized as probable covid-19 were excluded. medical record data this study used secondary data (the medical record of the patients) with parameters including the number of medical records, age, sex, rt-pcr result, severity, primary diagnosis, additional diagnosis, the endpoint of the patients, full hematologic profile (hemoglobin, leukocyte, thrombocyte, total leukocyte count: eosinophil, basophil, band neutrophil, segmented neutrophil, lymphocyte, monocyte), procalcitonin, and chief complaint. operational definition probable covid-19 is defined as subjects seeking medical attention with symptoms related to covid-19. covid-19 confirmed defined as patients with the positive result of rt-pcr. severe covid ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 1 3 19 is defined as patients confirmed with covid-19 who had one of these criteria: respiratory rate more than 30 times/minute; oxygen saturation < 93%, pao2/fio2 < 300 mmhg. less severe covid-19 is defined as confirmed covid-19 without severe covid-19 criteria. neutrophil and lymphocyte ratio is the ratio of absolute neutrophil count to absolute lymphocyte count, acquired by analyzing peripheral blood smear prepared on the day of the admission into the hospital. lymphocyte and monocyte ratio is defined as the ratio of absolute lymphocyte count to absolute monocyte count, acquired by analyzing peripheral blood smear, prepared on the day of the admission into the hospital. statistical analyses all data were analyzed using spss version 11.0 statistics for windows software. the baseline characteristics of the study population were presented in descriptive distribution tables. data are presented as mean ± standard deviation (sd) or 𝑛 (%). data analysis was conducted by unpaired t-test if the data had a normal distribution. if the data were unevenly distributed, data analysis was conducted by the mannwhitney test. to evaluate nlr and lmr ratios as a screening method for covid-19, we used parameters sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), and area under the curve (auc) from the receiver operating curve (roc). auc value 0.9-1 categorized as exceedingly accurate, 0.8-0.9 categorized as highly accurate, 0.7-0.8 categorized as well accurate, 0.6-0.7 categorized as fairly accurate, and < 0.5 categorized as useful. results there were 87 data of medical records from patients (18 – 71 years old) included in this study. there were 52 male patients (59.8%) and 35 female patients (40.2%), 21 female and 21 male in the covid-19 group, and 31 male and 14 female in the noncovid-19 group. there were no statistically significant differences in the hemoglobin, leukocyte, thrombocyte, eosinophil, basophil, neutrophil, and monocyte amount in covid-19 and non-covid-19. however, the median of the lymphocyte in the covid19 group (1.4%-57.7%, average: 19.8%) was higher than lymphocyte in the non-covid19 group (1.8%-46.4%, average: 15.7%), and this was statistically significant with a pvalue of 0.016 (p<0.05). the median of the absolute neutrophil count in the covid-19 group (2,250.00–14,297.22, average: 5,471.77) was lower than in the noncovid-19 group (1,170.78–47,329.25, average: 7756,56), and this was statistically significant, with a p-value of 0.0007 (p<0.05). there were no significant differences in severity, endpoint, comorbidity, and duration ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 1 4 of complaint in the covid-19 group compared to the non-covid-19 group. the symptoms of fever and cough were more prevalent in the covid-19 group than the non-covid-19 group, and this was statistically significant, p-value 0.004 for fever and 0.008 for cough. there were no significant differences in comorbidity between the covid-19 group and the noncovid-19 group. table 1. baseline characteristic variable total (n=87) median (range) covid-19 (n=42) median (range) not covid-19 (n=45) median (range) p-value* age (year) 44 (18-71) 43 (18-71) 48 (19-71) 0.637a sex male, n (%) 52 (59.8) 21 (50.0) 31 (68.9) 0.073b female, n (%) 35 (40.2) 21 (50.0) 14 (31.1) hematologic parameters hemoglobin 12.9 (5.8-17.4) 13.2 (5.8-17.1) 12.7 (6.4-17.4) 0.997a leukocyte 9,130 (2,370-49,250) 8,475 (4,010-16,200) 10,060 (2,370-49,250) 0.070a thrombocyte (x103) 282 (32-644) 274 (46-600) 283 (32-644) 0.728a eosinophil (%) 0.8 (0.0-8.2) 0.8 (0.0-6.0) 0.8 (0.0-9.2) 0.547a basophil (%) 0.3 (0.0-9.2) 0.3 (0.0-1.1) 0.3 (0.0-9.2) 0.188a neutrophil (%) 73.7 (33.9-96.1) 68.7 (33.9-95.8) 75.6 (40.7-96.1) 0.058a limphocyte (%) 17.0 (1.4-57.5) 19.8 (1.4-57.7) 15.7 (1.8-46.4) 0.016 a* monocyte (%) 7.0 (0.7-18.0) 7.0 (2.7-15.4) 7.5 (0.7-18.0) 0.792a absolute neutrophil 6,232.54 (1,170.78-47,329.25) 5,47177 (2,250-14,297.22) 7,756.56 (1,170.78-47,329.25) 0.007a* absolute limphocyte 1,693.2 (201.32-5,158.38) 1,843.02 (421.80-5,158.38) 1,377.88 (201.32-4,538.4) 0.159 absolute monocyte 690.2 (170.8-3,145.5) 572.77 (312.9-1,328.1) 701.1 (170.8-3,145.5) 0.114 severity severy 18 (20.7) 10 (23.8) 8 (17.8) 0.488b less severe 69 (79.3) 32 (76.2) 37 (82.2) endpoint death 22 (25.3) 11 (26.2) 11 (24.4) 0.851b cured 65 (74.7) 31 (73.8) 34 (75.6) comorbidity, n (%) none 64 (73.6) 27 (64.3) 37 (82.2) 0.356b dm 13 (14.9) 9 (21.4) 4 (8.9) esrd 4 (4.6) 3 (7.1) 1 (2.2) sepsis 4 (4.6) 2 (4.8) 2 (4.4) hiv 2 (2.3) 1 (2.4) 1 (2.2) duration of complaint ≤14 days 59 (84.3) 29 (82.9) 30 (85.7) 0.743b >14 days 11 (15.7) 6 (17.1) 5 (14.3) symptoms fever, n (%) 46 (52.9) 29 (69.0) 17 (37.8) 0.004 b* cough, n (%) 37 (42.5) 24 (57.1) 13 (28.9) 0.008 b * dyspneu, n (%) 33 (37.9) 13 (31.0) 20 (44.4) 0.195b dysphagia, n (%) 6 (6.9) 3 (7.1) 3 (6.7) 1.000c others, n (%) 9 (10.3) 4 (9.5) 5 (11.1) 1.000c p-value measured using a mann whitney test, b chi-square test, c fisher exact test; * significant if p <0.05 there were 42 patients in the covid-19 group, about ten patients categorized as severe covid-19 group, and 32 patients categorized as less severe covid-19 group ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 1 5 (table 1). the median age in the covid-19 group was 43 years (range: 18 – 71), and the median age in the severe group was 54 (range 28 – 69) and 36.5 (range 18-71) in the less severe group. this was statistically significant with p-value of 0.026 (p < 0.05). the severe covid-19 group consisted of older patients. the hematologic parameters, like hemoglobin, leukocyte, monocyte, had no significant differences in covid-19 and non-covid-19 groups. however, for other parameters, there are some statistically significant differences. the median of thrombocytes (x103) was 223.5 in the severe covid-19 group and 295.5 in the less severe group. this study showed that patients with severe covid-19 had a lower median of thrombocyte than less severe covid-19 (p-value 0.045) (table 2).. the median of eosinophil was 0.75 (0.0-6.0) in overall covid-19 patients, 0 (0-2.9) in the severe covid-19 group and 1.2 (0-6) in less severe covid-19 group. this study showed that patients with severe covid-19 had a lower median of eosinophil than less severe covid-19, p-value 0.001. table 2. the hematologic profile of severe covid-19 group and not severe covid-19 group total (n=42) median (range) severity of covid-19 variable severe (n=10) median (range) not severe (n=32) median (range) p-value age (years) 43 (18-71) 54 (28-69) 36.5 (18-71) 0.026a* sex male, n (%) 21 (50) 6 (60) 15 (46.9) 0.174 b female, n (%) 21 (50) 4 (40) 17 (53.1) hematologic parameters hemoglobin 13.2 (5.8-17.1) 11.95 (6.0-15.5) 13.4 (5.8-17.1) 0.231 a leukocyte 8,475 (4,010-16,200) 10,265 (4,010-16,200) 8,370 (5,000-16,000) 0.535a thrombocyte (x1000) 274.0 (46.0-600.0) 223.5 (76.5-318.0) 295.5 (46-600) 0.045a* eosinophil (%) 0.75 (0.0-6.0) 0 (0-2.9) 1.2 (0-6) 0.001 a* basophil (%) 0.25 (0.0-1.1) 0.1 (0-0.6) 0.4 (0-1.1) 0.003 a* neutrophil (%) 68.7 (33.9-95.8) 79.35 (74.9-92.1) 60.1 (33.9-95.8) 0.001 a* limphocyte (%) 19.8 (1.4-57.7) 12.6 (4.8-17.4) 29.4 (1.4-57.7) 0.001 a* monocyte (%) 6.9 (2.7-15.4) 6.95 (3.1-11) 7 (2.7-15.4) 0.636 a absolute neutrophil 5,471.77 (2,250-14,297.22) 6,489.76 (3,845.4-13,867.2) 5,224.92 (2,250-14,297.22) 0.111 absolute lymphocyte 1,843.02 (421.8-5,158.38) 1,218.19 (421.8-2,001.6) 2,219.78 (529.32-5,158.38) 0.012* absolute monocyte 572.77 (312.9-1,328.1) 523.47 (356.81-1,159.26) 596.41 (312.9-1,328.1) 0.859 p-value measured using a mann whitney test, b chi-square test, c fisher exact test * significant if p <0.05 the median of basophil was 0.25 (0.01.1) in overall covid-19 patients, 0.1 (00.6) in the severe covid-19 group and 0.4 (0-1.1) in less severe covid-19 group. this ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 1 6 study showed that patients with severe covid-19 had a lower median of basophil than less severe covid-19, p-value 0.003. the median of neutrophils was 68.7 (33.995.8) in overall covid-19 patients, 79.35 (74.9-92.1) in the severe covid-19 group, and 60.1 (33.9-95.8) in the less severe covid-19 group. this study showed that patients with severe covid-19 had a higher median of neutrophil than less severe covid-19, p-value 0.001. the median of neutrophils was 68.7 (33.9-95.8) in overall covid-19 patients, 79.35 (74.9-92.1) in the severe covid-19 group 60.1 (33.9-95.8) in the less severe covid-19 group. this study showed that patients with severe covid-19 had a lower median of neutrophil than less severe covid-19, p-value 0.001. the median of absolute lymphocyte count was 1,843.02 (421.8-5,158.38) in overall covid-19 patients, 1,218.19 (421.8-2,001.6) in the severe covid-19 group and 2,219.78 (529.32-5,158.38) in less severe covid-19 group. this study showed that patients with severe covid-19 had a lower median absolute lymphocyte count than less severe covid-19, p-value 0.012. the median of nlr in the covid-19 group (0.59-68.43, average: 3.55) was lower than the non covid-19 group (0.97-53.39, average: 4.75), and this was stastitically significant with p-value 0.027 (p < 0.05) (table 3).. the median lmr ratio in th covid-19 group (0.52-16.49, average: 28.5) was higher than non covid-19 group (0.369,87, average: 2.01). this was statistically significant with p-value 0.010 (p < 0.05). table 3. nlr and lmr value in covid-19 and non covid-19 variable total (n=90) median (range) covid-19 (n=43) median (range) non covid-19 (n=47) median (range) p-value nlr 4.35 (0.59-68.43) 3.55 (0.59-68.43) 4.75 (0.97-53.39) 0.027* lmr 2.26 (0.36-16.49) 2.85 (0.52-16.49) 2.01 (0.36-9.87) 0.010* p value calculated using mann-whitney test*significant if p-value < 0.05 the median nlr ratio in the covid-19 group with severe category (4.30-19.19, average: 6.20) was lower than in the noncovid-19 group (5.27-53.39, average: 13.59). however, this result was statistically insignificant because of the p-value of 0.110 (p>0.05) (table 4). the median nlr in the non-severe covid-19 group was 2.00 (0.5968.53). this value was lower than in the noncovid-19 group 4.26 (0.97-19.74), with a pvalue of 0.01 (p < 0.05). p value of less than 0.05 can be interpreted statistically significant. ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 1 7 the median lmr ratio in the severe covid-19 group (1.04-3.36, average: 1.77) was higher than the non-covid-19 group (0.73-2.52, 1.19). however, this result is defined ad statistically insignificant as the pvalue is 0.131 (p > 0.05). the median lmr ratio was 3.47 (0.52-16.49). this value was lower when compared to the non-covid-19 group compared with control 2.11 (0.369.87). this result was statistically significant with a p-value of 0.008 (p<0.05). table 4. the nlr and lmr ratio in covid-19 patients and non covid-19 patients based on the severity severe cases not severe cases variable covid-19 (n=10) median (range) not covid-19 (n=8) median (range) covid-19 (n=32) median (range) not covid-19 (n=37) median (range) p-value p-value nlr 6.20 (4.30-19.19) 13.59 (5.27-53.39) 0.110 2.00 (0.59-68.43) 4.26 (0.97-19.74) 0.010* lmr 1.77 (1.04-3.36) 1.19 (0.73-2.52) 0.131 3.47 (0.52-16.49) 2.11 (0.36-9.87) 0.008* p-value calculated using mann-whitney test*significant if p-value < 0.05 the median nlr of the severe covid19 group was 6.20 (4.30-19.19), higher than the less severe covid-19 group 2.00 (0.5968.43). with p-value 0.001 (p < 0.05), this result was statistically significant. the median lmr ratio of the severe covid-19 group was 1.77 (1.04-3.36), lower than the less severe covid-19 group 3.47 (0.5215.49). with p-value 0.003 (p < 0.05), this result was statistically significant (table 5). table 5. the nlr and lmr ratio in severe covid-19 patients and less severe covid-19 patients severity variable severe (n=10) median (range) less severe (n=32) median (range) p value nlr 6.20 (4.30-19.19) 2.00 (0.59-68.43) 0.001* lmr 1.77 (1.04-3.36) 3.47 (0.52-16.49) 0.003* p-value calculated using mann-whitney test *significant if p-value < 0.05 the accuracy of the nlr parameter was defined as not good (just sufficient) because of auc < 0.7 (cut-off value ≤ 2.49, auc 0.638, sensitivity 47.6%, and specificity 80%) (table 6). the lmr value, with a cutoff lmr ≥ 3.23, auc 0.661, sensitivity: 45.2%, specificity: 82.2%, the accuracy of the lmr parameter as a screening for covid-19 cases also defined as not good (only sufficient) because of auc < 0.7. discussion ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 1 8 the number of leukocytes in the covid19 group was lower than in the non-covid19 group, based on the baseline characteristics of the study subjects. however, in this study, the difference was not significant (8,475 [4,01016,200] vs 10,060 [2,3704,9250], p = 0.070). this study is in line with previous studies by mardani et al. (11) and song cy et al. (13). however, in both of these studies, the difference in leukocytes was indicated statistically significant differences. tabel 6. cut-off of auc nlr dan lmr ratio as a screening tool for covid-19 variable auc 95% ci p value cut-off nlr 0.638 0.528-0.738 0.022 ≤2.49 sensitivity: 47.6% specificity: 80.0% ppv: 69.0% npv: 62.1% lmr 0.661 0.552 – 0.759 0.006 >3.23 sensitivity: 45.2% specificity: 82.2% ppv: 70.4% npv: 61.7% figure 1. roc curve (a) nlr on covid-19 (b) lmr on covid-19 this study also found that approximately 75% of covid-19 cases were not severe, with relatively younger age in less severe cases than severe cases (median age 36.5 versus 54 years). the following baseline characteristic is the discovery of lymphopenia in both groups, both in covid-19 and non-covid-19 groups. previous studies stated that lymphopenia is often found in covid-19 patients. lymphopenic conditions have been discovered to be common, especially in severe covid-19 patients, due to a variety of mechanisms, including direct lymphocyte (a) (b) ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 1 9 inhibition, lymph node destruction, inflammatory cytokines, the emergence of lactic acidosis suppression lymphocytes, and attachment of coronavirus to angiotensinconverting enzyme 2 (ace) 2 receptors on lymphocytes (14). the difference in lymphocyte percentages between the covid-19 and noncovid-19 patient groups is quite significant, with the median lymphocyte value in the covid-19 group significantly higher than the non-covid-19 group (median values 19.8 [1.4-57.7] vs. 15.7 [1.846.4], p 0.016). although the absolute lymphocyte count in the covid-19 group was higher than the non-covid-19 group (1843.02 [421.8-5158.38] vs. 1377.88 [201.32-4538.4], the difference was not statistically significant. in addition, the cycle threshold from the positive rt-pcr sars cov-2 results might further explain why the covid-19 group has the absolute median value of lymphocytes that are still normal (1843.02 [421.8-5158.38]), with no lymphopenia. the sars cov-2 positive rtpcr results with high cycle threshold results indicate low viral loads. the rt-pcr cycle threshold ≤ 29 indicates the amount of viral nucleic acid tested is enormous and vice versa if the cycle threshold value ≥ 30 indicates the minimum to moderate amount of viral nucleic acid tested (16). viral load measurements from samples tissue indicate an active viral replication. they can be routinely monitored for severe viral respiratory tract infections, including clinical progression, response to treatment, healing, and recurrence (17). increasing severity is also associated with the presence of lymphopenia (15). thus, if the lymphocytes count has a normal range, the condition is probably associated with less viral load and a higher cycle threshold value (≥30). unfortunately, this study’s sars-cov-2 positive rt-pcr examination did not include the cycle threshold value. in addition, in this study, it was found that the rt-pcr group of sars-cov-2 negative experienced lymphopenia. this condition, characterized by a decrease in lymphocyte value, can be explained by comorbidities such as diabetes mellitus, sepsis, human immunodeficiency virus, and end-stage renal disease (18,19). the data on baseline characteristics in this study also showed that the percentage of neutrophils, absolute neutrophils, monocytes, and the absolute monocytes in the covid-19 group was lower than that in the noncovid-19 group, with only the absolute neutrophils statistically significant (5471.77 [2250-14297.22] vs. 7756.56 [1170.7847329.25], p = 0.007). in another study, mardani et al. (11) presented different results, that the percentage of neutrophils in the covid-19 group was significantly higher than in the non-covid-19 group. phagocytic cells such as dendritic cells, macrophages/monocytes, ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 2 0 and neutrophils play an essential role in the presence of sars-cov virus infection. in addition to airway epithelial cells and nk cells, it is also said that sars-cov-2 can infect monocyte immune cells and circulating t lymphocytes in the early stages of the disease. the infection rate in lymphocytes was 51.9% and 27.9% in monocytes. monocytes and t cells are involved in the innate and adaptive immune systems. the destruction of these cells can result in a compromised immune response (20). the decrease in monocyte value is associated with the destruction of these monocyte cells. the characteristic data also found that the types of complaints often found in covid19 cases were fever (69%) and cough (57.1%), and these complaints were significantly different from non-covid-19 cases. in this study, between the covid-19 group with severe and less severe symptoms, the percentage of neutrophils and the absolute value of neutrophils in the severe group was higher than in the less severe group. however, statistically, the significant value was only in the percentage of neutrophils (median value of 79.35 [74.9-92.1] vs 60.1 [33.9-95.8], p = 0.001). the percentage of monocytes in the study was also not significantly different between the severe and non-severe covid-19 groups (median value of 6.95 [3.1-11] vs. 7 [2.715.4], p = 0.636). for the absolute value of monocytes in this study, the severe covid19 group had a lower absolute monocyte value than the non-severe group. however, this difference was not significant (523.47 [356.81-1159.26] vs 596.41 [312.9-1328.1], p = 0.859). the nlr result in the covid-19 group was lower than the non-covid-19 group (median value 3.55 [0.59-68.43] vs 4.75 [0.97-53.39], p = 0.027). in the severe symptom group, the nlr value was lower in covid-19 compared to non-covid-19, but this difference was not significant (median 6.20 [4.30-19.19] vs. 13.59 [5.27-53.39], p = 0.110). in the moderate symptom group, the nlr value was significantly lower between covid-19 and non-covid-19 (median 2.00 [0.59-68.43] vs 4.26 [0.97-19.74] , p = 0.01). this result is inversely proportional to the previous study conducted by mardani et al. (11), which showed a higher percentage of neutrophils and a lower percentage of lymphocytes in the covid-19 group than in the non-covid-19 group so that in the end, the nlr value was higher in the covid-19 group than in the non-covid-19 group. the nlr results were different in severe and nonsevere covid-19. nlr in severe covid19 group was significantly higher than those in the moderate-grade group (6.20 [4.3019.19] vs 2.00 [0.59-68.43], p = 0.001). this result is in line with the research of yang ap et al. (2020) and song cy et al. (2020), who stated that the nlr in severe covid-19 was ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 2 1 significantly higher than those that were not severe. the lmr value in the covid-19 group was higher than the non-covid-19 group. this study also compared lmr based on the degree of disease severity between covid19 and non-covid-19. in the severely ill group, there was no significant difference in lmr between covid-19 and non-covid19 (median 1.77 [1.04-3.36] vs 1.19 [0.732.52], p = 0.131). when the lmr ratio of the less severe covid-19 group was compared to the non-covid 19 groups, the lmr ratio was higher in the severe covid-19 group, median 3.47 (0.52-16.49) vs. 2.11 (0.369.87), p = 0.008. there was no previous research comparing the lmr value between the covid-19 and non-covid-19 groups that has been published, so the results of this study provide new information for covid19 cases. the results of the lmr in this study for severe degrees of covid-19 were significantly lower than the non-severe group (1.77 [1.04-3.36] vs. 3.47 [0.52-16.49], p = 0.003). the nlr and lmr values were studied to find their accuracy as screening parameters for covid-19 cases. nlr auc was 0.638, with a cut-off of ≤2.49, 47.6% sensitivity, and 80% specificity; therefore, the accuracy of the nlr parameter as a screening for covid-19 cases is defined as not good enough (only sufficient) because of auc <0.7. in a previous study, song cy et al. (2020) used a cut-off >5.8 as one of the assessment parameters to determine cases of covid-19 along with other parameters made in the form of a covid-19 early detection score. a cut-off value of ≥3.3 indicates a covid-19 patient presenting with less severe symptoms will develop into a severe condition as much as 46.1% within 6.3 days. conversely, the cut-off of ≤3.3 indicates that covid-19 patients with non-severe symptoms will experience improvement and be outpatient within 13.5 days (13). lmr auc was 0.661, with cut-off ≥3.23, 45.2% sensitivity, 82.2% specificity; therefore, the accuracy of the lmr parameter as a screening for covid-19 cases is defined as not good enough (just sufficient) because of auc <0.7. previous research regarding lmr parameters as filter parameters for covid19 is not available. previous studies related to lmr parameters in covid-19 were prognostic studies to assess the clinical outcome of covid-19 patients. yang ap et al. (2020) explained that the lmr could not be used as a good prognostic parameter in the case of covid-19 because the auc value obtained is around 0.265 (auc <0.5). several comorbidities were not excluded from this study’s samples, affecting the ina. j. med. lab. sci. tech. 2022; 4(1): 10–23 heny syahrini, et al. 2 2 results. the research data obtained only from one of the hospitals is one of the limitations of this study. we recommended that future studies focus on searching for better and newer covid-19 screening tools with high sensitivity and specificity, cheap, easy, and non-invasive. conclusions there were significant differences in hematologic profile in neutrophil, lymphocyte, nlr, and lmr between the covid-19 and non-covid-19 groups. nevertheless, nlr and lmr cannot be used as screening tools because the area under curve (auc) is not good enough in detecting covid-19. author contributions hs had full access to all the data and took responsibility for the integrity of the data and the accuracy of the data analysis. hs and thf contributed to the concept and design of the study hs and nzd contributed to the critical revision of the manuscript for important intellectual content and statistical analysis all authors contributed to data acquisition, analysis, or interpretation and reviewed the final manuscript. acknowledgments the authors thank colleagues from universitas padjajaran (division of haematology and medical oncology, faculty of medicine), hasan sadikin general hospital, universitas sumatera utara (faculty of medicine), and haji adam malik general hospital for providing insight, assistance, and expertise for authors. we thank dr. rico andryan simatupang, dr. hafiz syaifullah siregar, dr. giovani christin purba for assistance during data collection and data entry, typing, drafting, and writing the manuscript. we also thank mr. evan susandi for his assistance in analyzing the data. conflict of interest there are no conflicts of interest references 1. xu p, zhou q, xu j. mechanism of thrombocytopenia in covid-19 patients. annals of hematology. 2020;99:1205–1208 2. world health organization. who director general’s remarks at the media briefing on 2019 ncov on 11 february 2020. 3. huang c, wang y, li x, ren l, zhao j, zang li, fan g, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. the lancet. 2020;395: 497-506. 4. liu j, liu y, xiang p, pu l, xiong h, li c, et al. neutrophil-to lymphocyte ratio predicts severe illness patients with 2019 novel coronavirus in the early stage. medrxiv. available from https://doi.org/10.1101/2020.02.10.20021584 5. he f, deng y, li w. coronavirus disease 2019 (covid-19): what we know?. j med virol [internet]. 2020 mar 14; 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4(2): 157–167 fitriani kahar, et al 1 5 8 introduction cigarettes are one of the products of tobacco processing, using or without additives. cigarettes contain additives and their use can pose health risks to individuals and society (1). cigarettes are one of the tobacco products that are burned, smoked and/or inhaled, including kretek cigarettes, white cigarettes, cigars or other forms produced from the plants nicotiana tabacum, nicotiana rustica, and other types or synthetic products whose smoke contains nicotine and tar with or without additives (2, 9). smoking is practiced repeatedly and on a continuous basis. smoking activity occurs in all environments, children, teens, adults, and the elderly. that is because the transaction process to purchase and sell cigarettes is easy and affordable for youth and students (2). smoking provides pleasure and fulfillment for smokers. apart from that, several factors behind someone to smoke include the desire to try the taste (menthol, cappuccino, black tea, etc.), the price is cheap and easy to obtain. it is believed that tobacco use relieves stress; relieves boredom, loneliness and confusion (3). smoking habits have become part of people's lifestyle. smoking is considered the leading preventable cause of death worldwide and around 6 million people die each year from causes related to exposure to secondhand smoke (4). according to the riskesdas ((baseline health research from agency of health research and development) survey (2018) the percentage of smoking in the population aged 18 years at the national level is increased. the survey indicated that from 2016 to 2018, tobacco use increased from 3.39% in 2016, 3.9% in 2017 and 9.65% in 2018 (5). smoked cigarettes have a negative impact on the human body, especially on the respiratory organs. various lung diseases that arise due to smoking are lung cancer and chronic obstructive pulmonary disease (1). cigarettes also greatly affect the hemoglobin in the body. smoking is also known to have a significant relationship with a person's immune system, because it can weaken the immune system, which affects the innate and adaptive immune systems and plays a dual role in regulating immunity by exacerbating the body's immune response (6). according to paskaria et al., (7) study findings from 2018, there was a substantial difference in co levels between students who smoked (12.21 ppm) and nonsmokers (5.11 ppm). the consensus recommendations of european respiratory society (ers) suggest that the expiratory concentration of co level in non-smokers is < 4 ppm. additionally, waseem and alvi (8) noted that hb levels rise as smoking intensity rises. smoking can affect blood components such as platelets, hemoglobin, erythrocyte sedimentation rate (21, 14). hemoglobin is a ina. j. med. lab. sci. tech. 2022; 4(2): 157–167 fitriani kahar, et al 1 5 9 protein pigment that makes red blood cells red. each hemoglobin is made up of a protein called hemoglobin. the hemoglobin in the blood, which is a protein in red blood cells that contains iron atoms and binds oxygen in the lungs and carries it to the tissues (9). cigarette smoke contains more than 4000 toxic compounds, including free radicals, nicotine, and carbon monoxide that have a negative impact on human health. nicotine induces clotting of the coronary arteries, reduces vascular activity, and increases endothelial dysfunction (11, 9). co (carbon monoxide) gas inhaled during smoking cigarette smoke, concentrations ranging from 400-5000 ppm (parts per million) (10). continued exposure to carbon monoxide gas induces the formation of carboxyhemoglobin. it is an inactive form of hb and does not carry oxygen. carboxyhemoglobin causes the dissociation curve hb to move to the left. thus, reducing hb’s capacity to supply oxygen to tissues (11, 9). the results of the study from malenica et al., (11) showed that continuous smoking had a severe adverse effect on hematological parameters (e.g., hemoglobin, white blood cell count, mean cell volume, mean corpuscular hemoglobin concentration, red blood cell count, hematocrit). results from the qadir (9) study also stated that smoking had an effect on hemoglobin concentration and pcv. based on this description, the researcher wanted to know whether there was an effect of smoking duration on blood hemoglobin levels. the objective of this study was to determine the impact of smoking duration on hemoglobin levels in smokers. materials and methods the type of research was experimental laboratory research with a descriptive approach. the hemoglobin level measured with cyanmethemoglobin method. five ml of venous blood was collected per patient using a venous puncture technique. then, 5 ml of the drabkin's reagent was put into a test tube and 20 µl of blood was mixed and then allowed to stand for 5 minutes at room temperature. the absorbance of the solution was then measured in a photometer at a wavelength of 540 nm (12). the absorbance of pure parental hb standard was also recorded at each run using a 5010 photometer. the population in this study was all students at the university of east indonesia. a total 20 subjects who were smokers were included in this study. male chronic smokers with smoking duration of 3,4, and 5 years. the sampling technique used a purposive sampling method. the exclusion criteria for this study were (1) patients with severe hypertension, endocrine disorders, liver disease, heart and respiratory illness; (2) patients taking medication; and (3) patients with habits such as alcohol and tobacco. the results obtained were subsequently analyzed in a descriptive ina. j. med. lab. sci. tech. 2022; 4(2): 157–167 fitriani kahar, et al 1 6 0 manner. further analysis is a two-variable statistic statistical using the anova with a 95% degree of confidence. therefore, if the p-value <0.05, it means that statistically there is a significant effect between the variables and vice versa. results table 1 results of hemoglobin examination using the cyanmethemoglobin method in students who smoked with the lowest hb level of 14.10 g/dl and the highest level of 18.48 g/dl. the results of the study revealed an effect between 3, 4 and 5 years of smoking duration. on the examination of hb levels, and 5 years of smokers gave the greatest effect or increase on the results of the examination of hemoglobin levels using the cyanmethemoglobin method (table 1). table 1. results of hemoglobin examination using cyanmethemoglobin method in students who smoke sample code smoking duration number of cigarettes smoked / days hemoglobin (gr/dl) a 4 years 10 15.40 b 5 years 10 18.34 c 5 years 12 15.55 d 5 years 10 14.32 e 5 years 10 18.48 f 3 years 8 14.95 g 5 years 12 15.06 h 5 years 12 15.22 i 4 years 8 15.02 j 3 years 8 15.49 k 3 years 12 14.40 l 3 years 11 14.48 m 4 years 8 14.12 n 5 years 10 16.45 o 4 years 10 14.12 p 3 years 9 14.12 q 5 years 10 14.12 r 4 years 12 14.45 s 4 years 8 14.10 t 3 years 10 15.49 ina. j. med. lab. sci. tech. 2022; 4(2): 157–167 fitriani kahar, et al 1 6 1 the results of the study, the duration of smoking with hemoglobin levels, the results of the examination of hb levels are presented in the table 2. table 2. duration of smoking with hb levels no duration of smoking total mean 1 3 years 6 14.8217 2 4 years 6 14.5350 3 5 years 8 15.9425 total 20 15.1840 table 2 shows the findings of a descriptive analysis of smokers, namely 6 smokers with a duration of smoking of 3 years with an average value of 14.8217, hb levels of 6 smokers who have smoked for 4 years with an average value of 14.5350, and 8 smokers with a duration of smoking of 5 years with an average value 15.942. table 3. anova table (one way) total variation dk sum of squares average squared f intergroup in group 2 17 7.917 23.074 3.959 1.357 2.917 total 19 30.992 table 3 shows the results of anova test to determine the effects of smoking on hemoglobin levels with dk = 19 and f = 2.917 and f. the table obtained is 2.05 (f count > f table), indicating that smoking duration has an impact on the outcomes of hemoglobin examination. figure 1. result of anova according to means plots on smokers figure 1 shows that the means plots from the anova analysis (versus smokers) are not parallel. it may be inferred that there is an interaction of effect between groups. table 2 displays the results for smokers who have been smoking for three years or less. the average score for these smokers was 14.8217, for smokers who have been smoking for four years or more, it was of 14.5350, and for smokers who have been smoking for 5-year ina. j. med. lab. sci. tech. 2022; 4(2): 157–167 fitriani kahar, et al 1 6 2 as many as 8 people with an average score of 15.1840. table 3 shows the results of the anova test to determine the effect of smoking duration on hemoglobin levels with dk = 19 and f = 2.917, and the f table obtained is 2.05 (f count> f table) means that there is an effect of smoking duration on the results of hemoglobin testing. in the subsequent test, namely the post hoc test, the highest effect was seen on smokers who were 5 years old. figure 1 depicts means plots. the influence in each group is depicted in the image of the smokers anova analysis (one way). discussion in the cyanmethemoglobin method, a certain volume of blood is diluted with a reagent and the hemoglobin concentration is calculated using a precise and well-calibrated photometer after a specific amount of time. cyanmethemoglobin determination is a reference test method for quantitatively measuring hemoglobin and is used to compare and standardize other methods. hb is converted into methemoglobin, which is converted into cyanmethemoglobin (hicn) by potassium cyanide. the absorbance of the solution is then measured in a spectrophotometer at a wavelength of 540 nm (12, 14). hb has the main physiological function of transporting oxygen to tissues and plays a role in the carrying of carbon dioxide. according to the analysis of 20 samples, it was discovered that there was an increase in hb levels in smokers. this results in an increase in hemoglobin levels is influenced by smoking habits. furthermore, co levels in the blood increase which greatly results in the formation of carboxyhemoglobin. the blood's level of hemoglobin will rise as a result of the synthesis of co. sulfohemoglobin, which is a component of carboxyhemoglobin and can impact high levels of hb in male smokers, cannot be measured by the cyanmethemoglobin method, which is utilized in this study (7). in addition, high levels of hb in the blood in male smokers are influenced by co2 in large quantities entering the body. as a result, it can stimulate the production of the hormone erythropoietin in the kidneys and the synthesis of hb in the blood can be further increased. this is because the hormone erythropoietin stimulates the coenzyme pyridoxal phosphate (vitamin b) in the process of hb synthesis in the bone marrow. heme synthesis occurs in the mitochondria through a series of biochemical reactions starting with the condensation of glycine and succinic coenzyme pyridoxal phosphate (vitamin b6) is the coenzyme for this process, which is limited by the activity of the important delta-aminolevulinic acid (al) enzyme, which is increased by erythropoietin and inhibited by hem. protoporphyrin ix and ferrous iron unite to produce heme, each of ina. j. med. lab. sci. tech. 2022; 4(2): 157–167 fitriani kahar, et al 1 6 3 which molecules is connected to a chain of globin comprised of polyribosomes. the hb molecule is then held together by the four globin chains, each of which has a separate haem group in a "pocket." its primary effect are on the sympathetic nervous system and the carbon monoxide-induced desaturation of hemoglobin (hb). cigarettes greatly affect hemoglobin (hb) in the body. carbon monoxide (co) is a colorless, odorless, flammable and highly toxic gas. the effect of carbon monoxide gas is harmful to the body because the binding capacity of carbon monoxide gas to hemoglobin is 240 times that of oxygen (o2) to hemoglobin. if carbon monoxide is inhaled by humans, the molecule will enter the respiratory tract and then enter the lungs and then attach to blood hemoglobin to form carboxy hemoglobin (15). mariani and kartini (16) reported that there was a significant relationship between the degree of smoking and hemoglobin levels. the process of synthesis of hb in the body begins with the process of co₂ from the tissues to the lungs. to carry out this gas exchange, blood cells contain the special protein hemoglobin. each red blood cell's main function is to transport o₂ to the tissues and restore it. red blood cells contain approximately 640 million hemoglobin molecules and each normal adult hemoglobin (hba) molecule consists of 4 a2b2 polypeptide chains, each with its own hb group. the molecular weight of hb a is 68,000. normal adult blood also contains small amounts of 2 other hemoglobin and hb a₂ which also contains a chain but y chain and s chain respectively instead of b 65% of hemoglobin b is synthesized in erythroblasts and 35% of hemoglobin is synthesized in the erythroblast stage reticulosis. most of the synthesis occurs in the mitochondria by a series of biochemical reactions initiated by the condensation of glycine succinyl coenzyme a under the action of the key al enzyme. finally, protoporphyrin combines with fe to form heme, each molecule of which is joined by a globin chain made up of polyribosomes, then a 4 meter of globin chain with each of its own heme groups is formed in the hemoglobin molecule (17). table 3 displays the findings of the anova test to determine the impact of smoking duration on smokers' hemoglobin levels. with dk = 19 and f = 2,917, and f table obtaining a value of 2.05 (f count > f table), it is clear that smoking duration has an impact on the test's outcomes hemoglobin. this result is supported by putri (18) with the wilcoxon test, there is a relationship between the number of cigarettes smoked and co with a p value of 0.000. hemoglobin at concentrations as small as 0.1%, carbon monoxide will bind to half of the total hemoglobin in the blood and reduce the oxygen carrying capacity of the blood by 50%. in an effort to boost the amount of ina. j. med. lab. sci. tech. 2022; 4(2): 157–167 fitriani kahar, et al 1 6 4 oxygen delivered to the tissues, the body will launch a compensation mechanism if this persists, which will result in an increase in the erythropoiesis process. hemoglobin levels will rise and surpass those in normal circumstances as a result. smoking is one of the factors that contribute to hypoxia brought on by high amounts of carbon monoxide. the body's levels of monoxide (co) are extremely harmful. one of the health issues brought on by excessive levels of carbon monoxide (co) in the body is an interruption in the heart's rhythm and blood circulation. (18). table 1 demonstrates a trend for smokers who have been smoking for of 3, 4 and 5 years to have higher hemoglobin levels. according to riskesdas (baseline health research from agency of health research and development) in 2018, smokers were categorized into three groups according to cigarette smoking intensity, namely light, moderate and heavy smokers. light smokers are individuals who smoke 1 to 10 cigarettes a day, while smokers in the moderate category consume 11 to 20 cigarettes a day. individuals who smoke more than 20 cigarettes a day are categorized as heavy smokers. numerous factors, including smoking behaviors, affect normal hematological results. smoking 10 cigarettes or more in a day will cause an increase in hemoglobin and hematocrit (packed cell volume) (5). in addition to nicotine, tar, 3,4benozopyrene, carbon monoxide, carbon dioxide, nitrogen oxides, ammonia, and sulfur, cigarette smoke also contains over 4000 other substances. about 210–300 times more so than its affinity for oxygen, carbon monoxide has a strong affinity for hemoglobin (19). the content of substances in cigarettes, especially nicotine, also affects psychological conditions, the nervous system, as well as brain activity and function, both in active and passive smokers. given the many dangers of nicotine in cigarettes, the organs in the body do not operate properly, especially in the main organs in the body, one of which is the heart pumping blood that will run abnormally because of disturbances in the heart work system due to nicotine and carbon monoxide. smoking habits affect cardiovascular endurance because carbon monoxide is released by smoke by 4% and binds hb levels faster than oxygen (20). hemoglobin is a tetrameric protein in erythrocytes that binds to oxygen and is responsible for releasing oxygen into tissues. carbon dioxide will bind to hemoglobin and be transported back to the lungs. carbon monoxide contained in cigarettes has a high affinity for hemoglobin, making it easier for the two to bind to each other to form carboxyhemoglobin, an inactive form of hemoglobin. due to hemoglobin's inability to bind oxygen, oxygen is released to different ina. j. med. lab. sci. tech. 2022; 4(2): 157–167 fitriani kahar, et al 1 6 5 tissues, leading to tissue hypoxia. by raising hemoglobin levels, the body will attempt to make up for the drop in oxygen levels (21). the results of this study are consistent with studies done by makawekas et al., (1), namely a significant difference between hemoglobin levels in active smokers and passive smokers in seventh semester students of the faculty of medicine, sam ratulangi university, manado. according to this study, active smokers had hemoglobin levels that were higher than passive smokers. this is because the carbon monoxide content in cigarettes has a stronger affinity for hemoglobin than oxygen does. the findings of a study done in makassar also indicated a connection between active smokers' hemoglobin and hematocrit levels. hematocrit and hemoglobin have a positive relationship (1). different results were found by katari et al., (15) which showed that there was no significant effect between the length of exposure to cigarette smoke on hemoglobin (hb) levels in the wistar strain of white rats. this can occur due to several factors, namely the time and dose of exposure to cigarette smoke, the type of cigarette used, the laboratory environment and the food consumed (15). the findings of katari et al., (15) are confirmed by ramadhanti et al., (22), who report that the hemoglobin levels in active smokers in 31 respondents who were active smokers an still within normal limits. because the body's response to obtaining oxygen and maintaining homeostasis and metabolism is still functioning normally. however, if this condition persists for a long time, the body will lose its ability to maintain homeostasis, which will allow smokingrelated illnesses like lung cancer to develop. additionally, study from medan revealed that smokers' hemoglobin levels were both normal and abnormal, though not by a great margin (15). conclusions based on the study's findings, it was discovered that the length of smoking 3, 4, and 5 years had an impact on the results of the cyanmethemoglobin method used to measure hemoglobin levels. smokers who had smoked for 5 years or longer had the biggest impact. your hb level will increase the longer you smoke. due to hb's affinity for the carbon monoxide in cigarettes, this is one example of how the body compensates for a lack of oxygen. therefore, it is advised that smokers cut back on their cigarette intake and try to quit because doing so will prevent health issues including a rise in hemoglobin levels that have an effect on lung conditions like. therefore, it is advised that smokers cut back on their cigarette consumption and try to quit because smoking has a negative impact on their health by raising their hemoglobin levels, which can lead to lung ina. j. med. lab. sci. tech. 2022; 4(2): 157–167 fitriani kahar, et al 1 6 6 conditions like pulmonary fibrosis, congenital heart disease, cor pulmonale, and polycythemia. by using masks, engaging in sufficient exercise, and establishing a healthy lifestyle, passive smokers can avoid being directly exposed to cigarette smoke. the findings of this study are anticipated to serve as a guide for future investigations into the many factors that can impact adult hemoglobin levels. to verify the findings of this study, additional research utilizing sizable samples from a range of age groups is required. author contributions all authors have contributed equally to a study. acknowladgements a big thank you to the students who have become respondents, and the university of east indonesia for facilitating the research activities. conflict of interest this research has no conflict of interest. references 1. makawekes mt, kalangi sjr, pasiak tf. comparison of blood hemoglobin levels in men smokers and non-smokers. j e-biomedik. 2016;4(1):1–2. https://doi.org/10.35790/ebm.4.1.2016.11250 2. kemenkes ri. peraturan menteri kesehatan republik indonesia nomor 56 tahun 2017 tentang perubahan atas peraturan mentri kesehatan nomor 28 tahun 2013 tentang pencantuman peringatan kesehatan dan informasi kesehatan pada kemasan produk tembakau (regulation of the minister of health of the republic of indonesia number 56 of 2017 concerning amendments to regulation of the minister of health number 28 of 2013 concerning the inclusion of health warnings and health information on tobacco product packaging ) [internet]. peraturan menteri kesehatan republik indonesia nomor 56 tahun 2017 tentang perubahan atas peraturan mentri kesehatan nomor 28 tahun 2013 tentang pencantuman peringatan kesehatan dan informasi kesehatan pada kemasan produk tembakau 2017 p. 1–37. available from: https://peraturan.bpk.go.id/home/details/112234 /permenkes-no-56-tahun-2017 3. kemenkes ri. hidup sehat tanpa rokok (healthy life without smoking) [internet]. kementrian kesehatan indonesia. 2017. p. 06–7. available from: http://p2ptm.kemkes.go.id/dokumenptm/hidup-sehat-tanpa-rokok 4. world health organization. raising tax on tobacco [internet]. 2014 p. 1–16. available from: http://apps.who.int/iris/bitstream/handle/10665/1 12841/who_nmh_pnd_14.2_eng.pdf;jsession id=72e4c2b9973787d46f81f04d5618dd1f?s equence=1 5. riskesdas k. hasil utama riset kesehatan dasar (baseline health research from agency of health research and development) (riskesdas) [internet]. vol. 44, kemenkes ri. 2018. available from: https://www.litbang.kemkes.go.id/laporanriset-kesehatan-dasar-riskesdas/ 6. rona s. the relationship between smoking and hemoglobin on endurance. compet j pendidik kepelatihan olahraga. 2020;12(1):41. https://doi.org/10.26858/com.v12i1.9133 7. paskaria c, fransisca, kurnia j, gunawan z, gunawan d. profile of smoking behavior and analysis of carbon monoxide levels on students in sukatani village, purwakarta. j respirologi indones. 2018;39(4):199–202. https://doi.org/10.36497/jri.v38i4.21 8. waseem s, alvi a. correlation between anemia and smoking: study of patients visiting different outpatient departments of integral institute of medical science and research, lucknow. natl j physiol pharm pharmacol. 2019;10(2):149–54. https://doi.org/10.5455/njppp.2019.9.041280512 2019 9. qadir ht. the effect of smoking on hemoglobin concentration and packed cell volume and comparing them between smokers and non ina. j. med. lab. sci. tech. 2022; 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2011. 468 p. 18. putri me. correlation of number of cigarettes with co levels in teenage smokers with vocational schools in jambi city. j akad baiturrahim jambi. 2018;7(2):123. https://doi.org/10.36565/jab.v7i2.76 19. amelia r, nasrul e, basyar m. the relationship between smoking degrees based on brinkman index and hemoglobin levels. j kesehat andalas. 2016;5(3):619–24. https://doi.org/10.25077/jka.v5i3.587 20. cintya cvd, sri widati. the influence of attitude factors, subjective norms, behavioral control on smoking habits in athletes at badminton ukm x surabaya. j biometrika dan kependud. 2017;6(1):26–34. https://doi.org/10.20473/jbk.v6i1.2017.26-34 21. wibowo dv, pangemanan dhc, polii h. the relationship between smoking and hemoglobin and platelet levels in adult smokers. j e-biomedik. 2017;5(2). https://doi.org/10.35790/ebm.5.2.2017.18510 22. ramadhanti m, amelia r, luhulima d. an overview of hemoglobin levels in active smokers at the kayuringin terminal, bekasi city. j mitra kesehat. 2019;2(1):49–52. https://doi.org/10.47522/jmk.v2i1.30 29 analysis of exopolysaccharides in lactobacillus casei group probiotics from human breast milk nur kusmiyati1, yuni puspitasari2, ulfah utami3, anggeria oktavisa denta4 1agricultural product technology department, faculty of agricultural technology, universitas brawijaya, malang, east java, indonesia 2biology study program, faculty of science and technology, universitas islam negeri maulana malik ibrahim, malang, east java, indonesia 3biology study program, faculty of science and technology, universitas islam negeri maulana malik ibrahim, malang, east java, indonesia 4nursing study program, politeknik negeri madura, sampang, east java, indonesia correspondence: nur kusmiati, jl. veteran, lowokwaru, malang, east java, indonesia zip code: 65145 email: kusmiy4tinur@gmail.com received: april 16, 2022 revised: january 6, 2023 accepted: march 27, 2023 published: april 29, 2023 doi: 10.33086/ijmlst.v5i1.2872 abstract exopolysaccharides get a lot of attention because they can improve the host immune system. exopolysaccharide is a polysaccharide that is produced and secreted from microbes outside the cell, usually found on the outside of the bacterial structure. the lactobacillus casei group from human breast milk is thought to have the ability to produce exopolysaccharides. the purpose of the study was to examine the exopolysaccharide of the l. casei group that was isolated from breast milk. the methods used include the gravimetric, the phenol-sulfuric acid and the fourier transform infra-red (ftir). the results showed that the l. casei group could produce exopolysaccharides, and had high exopolysaccharide total sugar content. lactobacillus paracasei had the highest exopolysaccharide and total sugar content of 3660 mg/l and 80.6%, respectively. the ftir results of the l. casei group exopolysaccharides showed the presence of hydroxyl functional groups o-h (3425.763295.98 cm-1), methyl c-h (2930.86-2856.70 cm-1), carbonyl c=o (1660.11-1647.27 cm-1), c-h (1456.161373.44 cm-1) and c-o-c ether (1071.08-1056.82 cm-1) which are specific characters of exopolysaccharides. since the ftir profile demonstrates that the l. casei group can produce exopolysaccharides, it has greater potential as a a probiotic. keywords exopolysaccharide, functional ggroup, lactobacillus casei. citation: kusmiyati n, puspitasari y, utami u, denta ao. analysis of exopolysaccharides in lactobacillus casei group probiotics from human breast milk. indones j med lab sci technol. 2023;5(1):29–41. doi: 10.33086/ijmlst.v5i1.2872 this is an open access article distributed under the creative commons attribution-sharealike 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2023 by author. introduction changes in lifestyle and poor diet in the community can disrupt the balance of the digestive tract microbiota, resulting in a decrease in the body's immune system (1). one of the prevention efforts against this is by restoring the balance of the immune system, like improving the health of the mailto:https://doi.org/10.33086/ijmlst.v5i1.2872 mailto:https://doi.org/10.33086/ijmlst.v5i1.2872 https://creativecommons.org/licenses/by-sa/4.0/ ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 0 digestive tract. according to ahamad et al. (2), the digestive tract is a reflection of the immune system because almost 80% of the components of the immune system are found in the digestive tract. improving the health of the digestive tract can be done by consuming probiotics. lactobacillus is one of the largest genera of lactic acid bacteria (lab) and is often used as a probiotic. research related to lactobacillus probiotics is limited to the requirements that must be met as a probiotic, namely acid and bile salt resistance, can adhere to intestinal cells and survive in the intestinal tract, able to produce antimicrobial compounds, antagonists against pathogens, safe in food and clinically, proven to affect health, and comes from humans (1). the advantage of the lactobacillus genus is that it has been declared a safe microorganism when added to food because it does not produce toxins, otherwise known as generally recognized as safe (gras) microorganisms (3). according to teame et al. (4), several microorganisms are probiotic which can produce exopolysaccharides. exopolysaccharides (eps) are sugar polymers or polysaccharides secreted by microorganisms out of cells. eps has a health effect because it can improve the immune system, so it is essential to analyze probiotics in producing eps. according to ahamad et al. (2), the lactobacillus casei group derived from human breast milk is eligible as a probiotic. l. casei group consisting of lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus can be used as candidates to produce eps. eps analysis can be done through eps production and total sugar analysis (5). in addition to these two analyzes, to ensure the compound is an eps is also needed. one method that can be used is the characterization of the functional group of a compound using fourier transform infrared (ftir) spectrophotometry (6). eps functional group analysis using ftir can provide complete information about the functional groups contained in the structure of an eps compound. the exploration of probiotic microorganisms that produce eps is increasing because the ability of these microorganisms to synthesize eps is considered very important for health. this study aimed to analyze the exopolysaccharide profile of the l. casei group derived from human breast milk based on parameters of crude extract content, total sugar, and identification of exopolysaccharide functional groups using ftir. materials and methods materials used in this study are l. casei group (l. casei, l. paracasei, l. rhamnosus) isolated from human breast milk (7), deman rogosa and sharpe broth (mrsb) ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 1 (himedia), deman rogosa and sharpe agar (mrsa) (merck), sucrose, ethanol, trichloroacetic acid 10% (tca), 70% alcohol (onemed), spirits, distilled water, phenol 5%, concentrated h2so4. bacterial cultivation this study used the l. casei group isolated from human breast milk in the study of kusmiyati et al. (7). the cultivation of the l. casei group was carried out through culture. one ose of pure culture was taken and grown on 5 ml of mrsa-tilted media at 37 ºc for 24 hours. the rejuvenated species were used for inoculum stock preparation and exopolysaccharide (eps) production analysis (8). exopolysaccharide production analysis one ose of the isolate was inoculated into 20 ml of mrsb, which 5% sucrose had been added and incubated at 37 ºc for 24 hours. inoculum was added to 20 ml of 10% tca and homogenized with a 90 rpm shaker incubator for 30 minutes. then it was centrifuged for 30 minutes at 4 ºc at 4500 rpm. the supernatant was taken and added to 96% cold ethanol twice. the sample was allowed to stand at 4 ºc for 24 hours, then centrifuged at 4 ºc at 4500 rpm for 30 minutes. the pellets were dried in an oven at 60 ºc and the eps dry weight was weighed. eps levels were calculated based on the method of nurhasanah et al. (5) and are presented in equation 1. eps content ( mg l ) = eps dry weight (mg) media volume (l) analysis of total sugar content the total sugar content of eps was determined by the phenol sulfuric acid method using glucose as the standard curve. the standard curve was made by means of 1 ml of a standard glucose solution containing 10, 20, 30, 40, 50 and 60 ppm. glucose was put into a test tube and 1 ml of 5% phenol solution and 5 ml of concentrated sulfuric acid were added. the solution was heated in a water bath at 40 ºc for 15 minutes and the absorbance was measured at 490 nm (9). exopolysaccharide: as much as 2 mg of crude eps was dissolved in 50 ml of distilled water. a total of 1 ml of eps solution was quickly added to 1 ml of 5% phenol and 5 ml of concentrated sulfuric acid and left for 10 minutes. next, the solution was heated in a water bath at 40 ºc for 15 minutes. the absorbance was measured at 490 nm (9). identification of exopolysaccharide compound profile with fourier transform infra-red (ftir) the fourier transform infra-red provides complete information about the functional groups contained in the structure of a compound. thus, in this analysis, it can be ascertained that the isolate group can produce exopolysaccharides (10). 2 mg of dry exopolysaccharide was put into an agate mortar and mixed with 200 mg of potassium bromide (kbr). after that, the material is ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 2 ground until homogeneous. next, the mixture is pressed and formed into pellets. the pellet is placed in the cell holder of the ftir instrument. dry exopolysaccharides and kbr were suppressed with a hydraulic suppressor with an ftir spectrum at a wave number of 500-4500 cm-1 (11). data analysis data analysis was carried out descriptively, quantitatively and qualitatively. quantitative data analysis used the product and total sugar of exopolysaccharides, while qualitative data analysis used the results of the ftir profile of exopolysaccharide compounds. product data and total sugar exopolysaccharide were tested for normality using shapiro-wilk and homogeneity tested using levene test. if the data is normal and homogeneous, then it is analyzed using the analysis of variance (anova). if the variance in the data gives a significant difference, then the duncan multiple range test (dmrt) is carried out with a level of = 5%. results exopolysaccharide production analysis exopolysaccharide production analysis showed that the l. casei group could produce exopolysaccharides (figure 1). the calculation results show that l. paracasei can produce the highest exopolysaccharide, which is 3660 mg/l. based on statistical analysis, the results were not different with l. casei and different (p<0.05) with l. rhamnosus. figure 1. exopolysaccharide content of the l. casei group ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 3 exopolysaccharide total sugar analysis the total sugar content of the exopolysaccharide group of lactobacillus casei showed different results (figure 2). the calculation results showed that the total sugar content of the highest exopolysaccharide in l. paracasei was 80.6%. this value was not significantly different in l. casei by 72.6%, and significantly different from l. rhamnosus. the research of xu et al. (12) showed that the total sugar content of l. casei exopolysaccharide isolated from sauerkraut, northeastern china, by the phenol-sulfuric acid method was 88%. identification of exopolysaccharide functional groups using a fourier transform infra-red (ftir) spectrophotometer the analysis of exopolysaccharide spectra produced by the l. casei group showed different wave numbers (figure 3). all species have functional groups indicating the presence of an exopolysaccharide in general, namely o-h (hydroxyl), c=o (carbonyl), c-h (methyl), and c-o-c (ether). figure 2. total sugar content of the l. casei group of exopolysaccharides the results of the exopolysaccharide spectra produced in the l. casei group of bacteria, namely the spectra at a wave number of 3295.98 cm-1, indicated the presence of a hydroxyl group (o-h) with the type of stretching molecular vibration. the absorption band in the wave number region of 2926.59 and 2856.70 cm-1 represents the methyl group (c-h) with the type of stretching molecular vibration. the absorption band in the 1660.11 cm-1 region represents the stretching c=o vibration of the carboxyl group. spectra at wave numbers 1453.31 and 1373.44 cm-1 indicate the presence of a group (c-h) with a bending molecular vibration type. the sharp absorption band at the wave number of 1071.08 cm-1 indicates the stretching ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 4 vibration of c-o-c of the ether functional group. the results of the ftir spectra showed the presence of exopolysaccharides in the l. casei group. an analysis of the results of the ftir spectra of exopolysaccharides from the l. casei group regarding the similarity of the wave numbers obtained for each functional group is presented in table 1. the l. casei group has eleven to twelve functional groups. l. casei had more functional groups than l. paracasei and l. rhamnosus. figure 3. ftir spectra of l. casei group exopolysaccharide; (a) l. casei, (b) l. paracasei, (c) l. rhamnosus. table 1. ftir spectra of exopolysaccharides from the l. casei group functional groups vibration wave number (cm-1) wave number (cm-1) (13) l. casei l. paracasei l. rhamnosus (o-h) hydroxyl stretching 3295.9 3425.7 3412.9 3650-3200 (c-h) methyl stretching 2926.5 and 2856.7 2929.4 2930.8 3000-2850 (c=o) carbonyl stretching 1660.1 1650.1 1647.2 1850-1630 (c-h) bending 1453.3 and 1373.4 1454.7 and 1414.8 1456.1 and 1414.8 1465-1370 (c-o-c) ether stretching 1071,08 1058.2 1056.8 1090-1000 ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 5 exopolysaccharide ftir results in l. casei have similarities to previous studies. differences in previous studies often occur in shifting wavenumbers but still show the similarity of functional groups. the l. casei group has all the functional groups required for a polysaccharide compound. according to zhang et al. (14) functional groups that indicate the presence of an exopolysaccharide are o-h (hydroxyl), c=o (carbonyl), c-h (methyl), and c-o-c (ether) groups. this is following the research of chambi et al. (15), who stated that ftir analysis of exopolysaccharides had several carbohydrate-related peaks, including hydroxyl, methyl, carbonyl, and ether groups. discussion exopolysaccharide production analysis genetics and individual phenotypes influence differences in the production of exopolysaccharides in lab (16). genetic traits are inherited traits of each bacterial species that are influenced by gene composition, while phenotypic traits tend to be influenced by environmental factors. in addition, the origin and species differences of lab strains will be able to contribute to the richness and diversity of glucosyltransferase (gtf) genes, which will affect the diversity of enzymes in exopolysaccharide synthesis so that variations in exopolysaccharide production occur. anindita (16) reproted that four strains of weisella confusa derived from breast milk produced different exopolysaccharides, namely w. confusa as3 (1883 mg/l), w. confusa as14 (1570 mg/l), w. confusa as18 (1369 mg/l) and w. confusa as21 (1480 mg/l). these results indicate that the ability to produce exopolysaccharides is influenced by differences in strains. the same species have varying capabilities in the production of exopolysaccharides. fatih (17) added that the differences in the production of exopolysaccharides were caused by the different enzyme activities of each species. the exopolysaccharide content of l. casei isolated from palm sap was lower (106.33 mg/l) (10) compared to the fermented colostrum kefir (1340 mg/l). in another study, l. paracasei exopolysaccharide levels were lower after 48 hours of fermentation (376.4 mg/l) (18) compared to fermented yoghurt (932.0 mg/l) (19). this study showed that l. paracasei produced an exopolysaccharide of 3660 mg/l, which is a fairly high value compared to existing studies. the production of exopolysaccharides in the l. casei group resulted in a high value because the mrsb medium was added with 5% sucrose. according to malick et al. (20), bacterial culture conditions such as medium composition, ph, temperature, and incubation period have been shown to ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 6 significantly affect exopolysaccharide production. the exopolysaccharide content of l. rhamnosus with the addition of hydrogen peroxide (h2o2) supplementation after 24 hours of incubation was 567 mg/l (21). the same study showed an increase in exopolysaccharides when given the addition of calcium chloride (cacl2) and h2o2 after 12 hours, namely 2498 mg/l. research by rajoka et al. (22) showed that l. rhamnosus could produce 1808 mg/l exopolysaccharides, while the study of bertsch et al. (23) l. rhamnosus could produce 1095 mg/l exopolysaccharides without the addition of media supplementation. another factor that affects the production of exopolysaccharides is incubation time. in this study, the incubation time used was 24 hours. incubation time must pay attention to the adequacy of nutrients in the media to ensure that bacteria can still grow by producing exopolysaccharides and do not reach a static or death phase when harvested. according to angelin & kavitha (24), exopolysaccharides are synthesized in the logarithmic or final logarithmic, and the stationary phase. however, maximum production occurs only in the late logarithmic phase rather than the stationary phase. according to fan et al. (25), the production of exopolysaccharides is often associated with the growth phase of bacteria. some exopolysaccharides are only synthesized in the late logarithmic or stationary growth phase, while others are synthesized during the microbial growth process. the quantity of exopolysaccharide produced varies between strains, media composition, and culture conditions such as ph, temperature and carbon ratio. according to al-manhel (26), the concentration of carbon sources is one of the most important factors in achieving high levels of exopolysaccharide production. in this study, the 5% sucrose addition increased the production of exopolysaccharides more than previous studies, which were without the addition of sucrose. according to bibi et al. (27) weisella cibaria c43-11 grown in liquid media and enriched with 10% sucrose showed high exopolysaccharide production. the addition of excessive sugar in the culture medium also increased the production of lab exopolysaccharides. the increase in sucrose concentration in mrs media was suitable for exopolysaccharides production in lactobacillus confusus tistr 1498. the exopolysaccharide produced by the l. casei group in this study showed that the l. casei group had the potential to be developed other than as a probiotic. exopolysaccharide total sugar analysis the total sugar content of exopolysaccharides is influenced by the amount of sugar contained in the production medium and the activity of the invertase ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 7 enzyme, which plays a role in converting the substrate into its constituent sugar monomers (28). according to imran et al. (29), the total sugar content in the exopolysaccharide l. plantarum ntmi05 was 95.45% and l. plantarum ntmi20 was 92.35%. this indicates the presence of total sugar in the exopolysaccharide produced by each different strain. in the same study, the total sugar content of l. helveticus mb2-1 was obtained at 95.45%. in another study, the total sugar content of l. pentosus 14fe was 81.38%, l. plantarum 47fe was 83,28%, l. pentosus 68f was obtained 85.19%, indicating that all exopolysaccharides consist of carbohydrates (30). the total sugar content of bacillus subtilis exopolysaccharide in the research of razack et al. (31) with media without supplementation showed a yield of 53%, while media supplemented with 2% sucrose showed a yield of 76%. this indicates that the medium has an affect on the total sugar content of the exopolysaccharide. exopolysaccharide production and exopolysaccharide sugar content obtained from the three bacteria in the l. casei group showed varying results due to different species or strains. according to nurhasanah et al. (5) differences in yield and total sugar content of exopolysaccharides due to differences in strains between species. differences in bacterial strains cause the number of metabolites produced to also be different. fatih (17) added that the number of exopolysaccharides produced by different lab was caused by inherited traits or genetic traits. the analysis of the total sugar exopolysaccharide of the l. casei group in this study showed a relationship between the total sugar content of the exopolysaccharide and the crude production of exopolysaccharides. this analysis supports the presence of polysaccharides produced by the l. casei group and improves the quality of the probiotics of the l. casei group. identification of exopolysaccharide functional groups using a fourier transform infra-red (ftir) spectrophotometer the ftir results of this exopolysaccharide can provide information that the l. casei group is capable of producing exopolysaccharides. exopolysaccharides can be used as a form of self-defence by the l. casei group. in addition, exopolysaccharides in lactic acid bacteria can also be used to improve the host immune system. according to jurášková et al. (32), exopolysaccharides produced by lactic acid bacteria can increase macrophage activity and cytokine production and stimulate the formation of immunoglobulin-a (iga). according to mundiri et al. (33), exopolysaccharides are known to have the potential to act as immunomodulators that play a role in the innate immune system in ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 8 digestion. exopolysaccharides of lactic acid bacteria can enhance the innate immune system through the role of gut-associated lymphoid tissue (galt). the use of exopolysaccharides as immunomodulators has been widely carried out and has been proven in-vitro and in-vivo to increase macrophage activity, cytokine production and ability to stimulate iga formation. in the research of inturri et al. (34), exopolysaccharides from lactic acid bacteria, bifidobacterium longum can increase the production of ifn-γ , il-1β , and il-6. according to domingos-lopes et al. (35), exopolysaccharides from the lactic acid bacteria leuconostoc citreum l3c1e7 can suppress the synthesis of allergen-specific ige and exopolysaccharides from lactic acid bacteria have been shown to have immunomodulatory activity with non-toxic effects. according to rajoka et al. (36), the exopolysaccharides of lactobacillus contain various functional groups (such as hydroxyl groups, phosphate groups, and carbonyl groups), which help to exert their immunomodulatory, antimicrobial, antioxidant, and anticancer activities. exopolysaccharides from lactobacillus can be used as nutritional and therapeutic agents to regulate the immune system, which can help fight various diseases such as cancer, diabetes, and hypertension. therefore, the addition of lactobacillus as exopolysaccharide agents immunomodulatory in functional foods has potential in the future. conclusions the l. casei group was able to produce exopolysaccharides (eps). l. paracasei was able to produce the highest eps (3660 mg/l) and total sugar (80.6%). confirmation by ftir showed that the l. casei group could produce eps characterized by hydroxyl, methyl, carbonyl and ether functional groups. this study shows that the l. casei group has more value than just being a probiotic. author contributions nur kusmiyati: substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data, drafting the article or revising it critically for important intellectual content, final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. yuni puspitasari: rafting the article or revising it critically for important intellectual content. ulfah utami and anggeria oktavisa denta: agreement to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ina. j. med. lab. sci. tech. 2023; 5(1): 29–41 nur kusmiyati, et al. 3 9 acknowledgements this research was funded by lembaga penelitian dan pengabdian masyarakat (lp2m) uin maulana malik ibrahim malang through bantuan operasional perguruan tinggi negeri (boptn) litapdimas kementerian agama indonesia. conflict of interest the authors declare no conflict of interest. references 1. othman nz, mohd din arj, mohammad z, rosli ma, sarmidi mr. statistical optimization of medium compositions for high cell mass and exopolysaccharide production by lactobacillus plantarum atcc 8014. appl food biotechnol. 2018;5(2):87–96. doi: 10.22037/afb.v5i2.19299 2. ahamad n, kar a, mehta s, dewani m, ravichandran v, bhardwaj p, et al. immunomodulatory nanosystems for treating inflammatory diseases. biomaterials. 2021;274(april):120875. doi: 10.1016/j.biomaterials.2021.120875 3. sasikumar k, kozhummal vaikkath d, devendra l, nampoothiri km. an exopolysaccharide (eps) from a lactobacillus plantarum br2 with potential benefits for making functional foods. bioresour technol. 2017;241:1152–6. doi: 10.1016/j.biortech.2017.05.075 4. teame t, wang a, xie m, zhang z, yang y, ding q, et al. paraprobiotics and postbiotics of probiotic lactobacilli, their positive effects on the host and action mechanisms: a review. front nutr. 2020;7(october). doi: 10.3389/fnut.2020.570344 5. nurhasanah, fu’adah it, satria h, yuwono sd. analisis eksopolisakarida dari bakteri asam laktat hasil fermentasi kefir kolostrum. 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4(1): 81–90 8 2 azzaubadilluah & andreas putroragil santoso introduction puberty is a condition marked by the functioning of reproductive organs such as menstruation that arises due to physiological changes. menstruation releases blood from the uterine wall gradually to the vagina. the menstrual cycle generally ranges from 21 to 35 days, while the length of menstruation ranges from 3 to 7 days (1). in chronic blood loss, women often have difficulty absorbing large amounts of iron from the small intestine to form hemoglobin proportional to the blood loss. the new created red blood cells contain only a small amount of hemoglobin (2). most menstrual blood flow occurs on the second and third day of menstruation based on research on daily menstrual blood loss and quality of life in women with heavy menstrual bleeding (3). the highest mbl (menstrual blood loss) day occurs on the second and third day of the menstrual cycle, causing limited physical activity. each menstrual phase, the quantity of blood that comes out is an average of 50 ml; when the blood loss is more than 80 ml, it is suspected to be pathological. iron in the blood will experience a reduction of 12.5-15 mg/month or approximately 0.4-0.5 mg/day. the condition of reduced iron will trigger a decrease in hemoglobin levels which appears as a form of iron deficiency anemia (4). hemoglobin is an essential protein in carrying oxygen from the lungs to the peripheral tissues and carrying carbon dioxide from the peripheral tissues to the lungs. hemoglobin is part of erythrocytes, a biomolecule that seeks to bind oxygen (5). erythrocytes are biconcave, do not have a nucleus, do not move, have red color because they contain hemoglobin. erythrocytes are 7 to 8 in diameter and 2.0 thick (6). the importance of hemoglobin causes hemoglobin examination to have an essential role in diagnosing anemia and is one of the routine examinations in the laboratory. the prevalence of anemia globally, according to world health organization (who), is estimated at around 40-88%. the majority of anemia cases in women ranged from 13.4 to 87.5% (7). those numbers show the relatively high prevalence of anemia in adolescent women (8). when the blood hemoglobin level is low, it does not necessarily mean that a person is anemic. the emergence of iron deficiency anemia or anemia due to morphological causes (normochromic normocytic anemia, hypochromic microcytic anemia, and hyperchromic macrocytic anemia) will provide an overview of the erythrocyte morphological changes. so, that other investigations can be added, such as observing the morphology of erythrocytes on a peripheral blood smear to confirm the presence of erythrocyte abnormalities. the results of the previous study showed that 60% of menstruating women had hemoglobin levels of less than 12.0 g/dl ina. j. med. lab. sci. tech. 2022; 4(1): 81–90 8 3 azzaubadilluah & andreas putroragil santoso which was below the normal limit and 40% of menstruating women had normal hemoglobin levels. based on 20 research subjects, it was found that there were deformities of erythrocytes, ovalocytes, target cells, elliptocytes, spherocytes, lacrimacytes, centrocytes, burr cells, sickle cells, helmets, stomatocytes, and crenated cells. the color of the erythrocytes obtained was hypochromic and normochromic. the size of erythrocytes obtained anisocytosis and normocytic results (9). the condition of female students in the health faculty who have a lot of student activities plus menstruation can cause iron loss, decreased hemoglobin levels and anemia. if anaemia treatment is not carried out properly, it will result in fatigue that interferes with productivity and cannot perform activities perfectly. anaemia can be overcome by taking iron supplements, vitamin b12, folic acid, and iron-rich foods, such as brown rice, green vegetables, and beans that help produce haemoglobin formation. this reason triggered researchers to find out whether there is a relationship between haemoglobin levels and erythrocyte morphology on the third day of menstruation. materials and methods this type of research is an analytical observational study with a cross-sectional approach to determine the relationship between hemoglobin levels and erythrocyte morphology on the third day of menstruation. the population in this study were female students of the health faculty at universitas nahdlatul ulama surabaya. the sample in this study were 30 female students who were having their third day of menstruation. the inclusion criteria in this study were female students of the faculty of health who were willing to be respondents, aged 15-24 years, had normal menstrual cycles and were menstruating on the third day. the respondent's blood sample was taken intravenously as much as three ccs, then blood was stored in an edta vacuum blood collection tube and at a temperature of 4-8°c. determination of respondents was carried out by purposive sampling and based on the permission of the ethics committee at universitas nahdlatul ulama surabaya. purposive sampling technique is the selection of respondents independently by researchers who will use research-based on subjective and practitioner considerations (10). the hemoglobin level was tested using the impedance sysmex xp-300™ automated hematology analyzer method, while the erythrocyte morphology examination was carried out by making a peripheral blood smear using giemsa 1:19 dye for 20-30 minutes. the normal value of haemoglobin is 12-15 g/dl for adult women (11). the reference value for erythrocyte morphology is considered normal, as long as the peripheral blood smear does not change the ina. j. med. lab. sci. tech. 2022; 4(1): 81–90 8 4 azzaubadilluah & andreas putroragil santoso morphology of the erythrocytes in terms of shape, colour, or size. data analysis in this study used the chisquare test performed with ibm spss version 21.0. the chi-square test is a nonparametric test statistical analysis. therefore, the application of the chi-square test was used for categorical data. the chi-square test can be used in research to determine the relationship between variables located in rows and columns. results based on the research, there were 30 respondents presented by the number of respondents who have standard, high, and low hemoglobin levels (figure 1). figure 1. graph showing student’s hemoglobin level based on figure 1, the lowest hemoglobin level of the respondent is 8.9 g/dl, the highest hemoglobin level is 15.2 g/dl with an average hemoglobin level of 12–15 g/dl. table 1. distribution of the frequency of inspection results hemoglobin levels reference value frequency (f) percentage (%) <12 g/dl 10 33% 12 – 15 g/dl 18 60% >15 g/dl 2 7% total 30 100% observations of erythrocyte morphology under a microscope were reported in table 2. those results obtained based on normocytic erythrocyte size and anisopoikilocytosis (figure 2). several erythrocyte deformities found, including ovalocytes, target cells, schistocytes, stomatocytes, sickle cells, cigar cells, spherocytes, lacrimacyte, and acantocyte. based on the color, normochromic and hypochromic erythrocyte colors were found. ina. j. med. lab. sci. tech. 2022; 4(1): 81–90 8 5 azzaubadilluah & andreas putroragil santoso table 2. observation results of erythrocyte morphology sample code observation results of erythrocyte morphology size form color 01 anisopoikilocytosis ovalocytes, cigar cell, tear drop, target cell, scistocyt hypochrome 02 anisopoikilocytosis acantocyte, tear drop, ovalocytes, target cell, scistocyt normochrome 03 anisopoikilocytosis ovalocytes, tear drop, acantocyte, scistocyt normochrome 04 anisopoikilocytosis ovalocytes, scistocyt, tear drop normochrome 05 anisopoikilocytosis ovalocytes, tear drop, acantocyte, scistocyt, targett cell normochrome 06 anisopoikilocytosis acantocyte, tear drop normochrome 07 anisopoikilocytosis tear drop, scistocyt, sferosit, ovalocytes normochrome 08 anisopoikilocytosis ovalocytes, cigar cell, stomatocyte normochrome 09 anisopoikilocytosis acantocyte, scistocyt, ovalocytes, target cell normochrome 10 anisopoikilocytosis ovalocytes, scistocyt, tear drop, acantocyte normochrome 11 anisopoikilocytosis ovalocytes, acantocyte, sickle cell normochrome 12 normocytic normocytic normochrome 13 anisopoikilocytosis tear drop, ovalocytes, sickle cell hypochrome 14 anisopoikilocytosis tear drop, ovalocytes, scistocyt, stomatocyte normochrome 15 anisopoikilocytosis tear drop, ovalocytes, scistocyt, sickle cell normochrome 16 anisopoikilocytosis tear drop, ovalocytes, scistocyt normochrome 17 anisopoikilocytosis ovalocytes, tear drop, stomatocyte, scistocyt hypochrome 18 normocytic normocytic normochrome 19 anisopoikilocytosis target cell, stomatocyte, ovalocytes, sferosit cigar cell hypochrome 20 anisopoikilocytosis ovalocytes, tear drop, stomatocyte normochrome 21 anisopoikilocytosis stomatocyte, ovalocytes, scistocyt, cigar cell normochrome 22 anisopoikilocytosis tear drop, ovalocytes normochrome 23 anisopoikilocytosis ovalocytes, sickle cell, stomatocyte, tear drop normochrome 24 anisopoikilocytosis ovalocytes, sickle cell, stomatocyte, sferosit, target cell, tear drop hypochrome 25 anisopoikilocytosis ovalocytes, target cell normochrome 26 anisopoikilocytosis ovalocytes, stomatocyte, scistocyt normochrome 27 anisopoikilocytosis ovalocytes, scistocyt, sickle cell, stomatocyte, target cell, cigar cell normochrome 28 anisopoikilocytosis ovalocytes, stomatocyte, tear drop, cigar cell hypochrome 29 anisopoikilocytosis ovalocytes, tear drop, scistocyt normochrome 30 anisopoikilocytosis scistocyt, stomatocyte normochrome figure 2. microscopic observation results oferythrocyte morphology. (a) anisopoikilocytosis, (b) hypochrome a b ina. j. med. lab. sci. tech. 2022; 4(1): 81–90 8 6 azzaubadilluah & andreas putroragil santoso based on the data analysis using the chisquare test is known that the p-value of hemoglobin levels in erythrocyte size is 0.836, where p-value > 0.05, which indicates that there is no relationship between hemoglobin level and erythrocyte size (table 3). the p-value of hemoglobin levels in the form of erythrocytes is 0.419, where the pvalue >0.05 indicates that there is no relationship between hemoglobin levels and the condition of erythrocytes. the p-value of hemoglobin level in erythrocyte color is 0.001, where the p-value <0.05 indicates an association between hemoglobin level and erythrocyte color. tabel 3. chi-square test results category pearson chi-square p-value* hemoglobin level with erythrocyte size 0.836 hemoglobin level with erythrocyte form 0.419 hemoglobin level with erythrocyte color 0.001 *significance p-value <0,05 discussion in this study, the research subjects used were adolescents aged 15-24 years. these age ranges are at risk of developing anaemia. one of the risk factors for anaemia is bleeding or blood loss during menstruation, so the blood will lose iron. reduced iron will have an impact on hemoglobin levels. anaemia interferes with the development of women in the decreased ability to concentrate on learning, decreased physical ability, and pale face (12). all respondents have regular menstrual cycles, as well as the body mass index (bmi) values obtained tend to be in line with expectations. bmi values can affect the menstrual cycle, which can be seen through the role of the hormone estrogen. with the increase in calories in the body, therefore an increase in body weight contributes to an increased level of estrogen in the blood. this condition occurs when the patient experiences gained body fat, androgens in the body also elevate. androgens convert into estrogens through the aromatization process in granulosa cells and fat tissue. increased estrogen in the blood will impair gnrh secretion (13). in this study, a sample of 18 respondents (60%) showed normal hemoglobin levels. this result is in line with kirana's research (2011), which stated that a woman who experiences menstruation is not necessarily anemic. it means that the respondent's body has received nutritional intake from food sources that contain a lot of iron (fe) so that it can increase the production of hemoglobin in the blood such as meat, fish, seafood, fruit, vegetable and fruit juice drinks containing vitamin c (ascorbic acid) (14). iron is an essential micronutrient for the production of haemoglobin, which functions to synthesize iron-containing enzymes to use oxygen during the cellular energy production ina. j. med. lab. sci. tech. 2022; 4(1): 81–90 8 7 azzaubadilluah & andreas putroragil santoso process (15). adequate iron in the body is the minimal amount of iron from food to avoid iron deficiency anemia (16). based on this study result, ten respondents (33%) had haemoglobin levels <12 g/dl. a study by irianti (2014) showed that the more blood loss during menstruation, the higher risk of developing anaemia. one of the triggering factors for anaemia is a lack of nutrients that play a role in the formation of haemoglobin due to impaired absorption. nutrients that catalyze heme in the haemoglobin molecule include iron, protein, and pyridoxine (vitamin b6) (17). the first three days of menstruation are days that tend to bleed a lot. decreased hemoglobin levels can make the body's metabolic disorder and nerve cells do not process optimally, resulting in an acceleration of reduced nerve impulses and disrupting the work of dopamine receptors (2). if the haemoglobin level is too low, it can also affect blood viscosity. this process will disrupt the body because it has low oxygen levels (hypoxia) and reduced oxygen supply to the organs, especially vital organs such as the brain and heart (18). the results of the observation of erythrocyte morphology in this study are in line with research conducted in mataram stated that respondents experience bleeding due to menstruation, it will trigger anemia to provide an overview of erythrocyte morphology changes that occur in abnormalities in erythrocyte size, erythrocyte shape, and erythrocyte color (4). the occurrence of pale erythrocyte coloration is due to the inhibition of the hemoglobin formation process due to interference with iron-binding. erythrocytes formed with small cytoplasm indicate that the cell is immature and does not contain hemoglobin. low iron content causes failure to form nucleated erythrocyte cytoplasm (19). the color of the erythrocytes is uneven, but the center is paler because it is thinner than the less colored periphery, which is called hypochromic. under normal circumstances, the center of the size does not exceed 1/3 of its diameter, the cell is called normochromic. the results of this study for the erythrocyte size obtained were abnormal. it means that on the third day of menstruation, a person experiences blood loss resulting in anemia due to decreased production of iron (fe) in the blood so that erythrocyte synthesis continues and produces smaller cells (microcytic). an insufficient amount of iron causes a decrease in hemoglobin in each cell, resulting in hypothermia. erythrocytes that have a larger size (macrocytic) will resemble reticulocyte cells due to the effectiveness of erythropoiesis by the bone marrow.. the standard erythrocyte shape on a peripheral blood smear is a biconcave disc with a red border compared to the centre part. this study found an abnormality in changes in the shape of erythrocytes. the occurrence ina. j. med. lab. sci. tech. 2022; 4(1): 81–90 8 8 azzaubadilluah & andreas putroragil santoso of an abnormal shape of erythrocytes describes a chemical or physical change in the cell membrane or cytoplasm. recent research in cell biology has contributed to increasing knowledge of these mechanisms. this condition occurs due to specific hematological or non-hematological abnormalities whenever an increase in the number of poikilocytosis is found (20). based on the results of data analysis, this study showed there was no relationship between hemoglobin levels and erythrocyte size on the third day of menstruation (p-value = 0.836). it was also showed that there was no relationship between hemoglobin levels and the shape of erythrocytes on the third day of menstruation (p-value = 0.419). in contrast, this study found a relationship between hemoglobin levels and erythrocyte colour on the third day of menstruation (p < 0.05), the direction of this relationship can indicate that if the subject has a more normal hemoglobin level, the result of erythrocyte staining is also more normochromic and vice versa. overall, it can be concluded that there is no relationship between hemoglobin levels and erythrocyte morphology on the third day of menstruation. based on research conducted by zhong et al., (2021) hemoglobin concentration can affect changes in erythrocyte morphology. the activity of the na-k pump will be disrupted if there is insufficient atp in the red blood cell membrane during storage, resulting in abnormal erythrocytes (21). according to research conducted by hadijah et al., (9) it was found that there was an effect of the menstrual period on hemoglobin levels and morphology of erythrocytes. this study aims to determine hemoglobin levels and morphology of erythrocytes in menstruating women. the difference from the research of hadijah et al., (9) the sample studied was the sample of the sixth day of menstruation. bleeding during menstruation will cause iron levels to decrease. the longer the menstrual period, the more loss of iron deposits and a decrease in hemoglobin levels. the longer the menstruation duration, the more influential on anemia. during menstruating, it is necessary to maintain a balanced diet so the nutritional intake is adequate and hemoglobin levels remain normal and the morphology of erythrocytes does not experience many abnormalities (9). conclusions based on the study results, it can be concluded that during menstruation on the third day of menstruation, the average hemoglobin level is 12.3 g/dl. menstruation is a condition that is quite vulnerable because it can cause iron loss, decrease hemoglobin levels and cause anemia. so that the emergence of risk factors for experiencing fatigue and disrupting productivity. on the third day of menstruation, women experience changes in erythrocyte morphology, with ina. j. med. lab. sci. tech. 2022; 4(1): 81–90 8 9 azzaubadilluah & andreas putroragil santoso abnormalities in erythrocyte size (anisocytosis), erythrocyte shape (poikilocytosis), and erythrocyte colour (normochromic, hypochromic). there was no relationship between hemoglobin level and erythrocyte morphology on the third day of menstruation, such as erythrocyte size and shape. however, there is a relationship between hemoglobin levels and erythrocyte colour on the third day of menstruation. author contributions azzaubadilluah: conceptualization, formal analysis, investigation, writing original draft, writing review & editing. andreas putro ragil santoso: supervision, conceptualization, methodology, writing original draf, formal analysis, validation, reviewing. acknowladgements the author would like to thank the laboratory assistants and several related parties who have provided direction and support in this study. the author also would like to thank the hematology laboratory faculty of health at the universitas nahdlatul ulama surabaya. conflict of interest there are no conflicts of interest. references 1. aryan r. research procedures a practical approach. in the ; 2010; jakarta: selemba medika. 2. suhanda p, suyatini. the relationship of menstruation length with hemoglobin levels. medical journal. 2016; 3(2): p. 143-148. 3. lukes a, baker j, eder s, adomako tl. daily menstrual blood loss and quality of. women's health. 2012; 8(5): p. 503-511. 4. asfaraini r, zaetun,. differences in hemoglobin levels and erythrocyte morphology before menstruation and after menstruation in young women. journal of health. 2017; 11(2). 5. maylina la. the relationship between consumption of food sources of protein, iron, and vitamin c with the incidence of anemia in elementary school students. essay. jember:; 2010. 6. nugraha g. east jakarta hematology laboratory examination guide: cv trans info media; 2017. 7. world health organization. adolescent health information system. in ; 2013: geneva http://www.who.int/adolescenthealth [diakses 31 mei 2021]. 8. wahyuningsih a, astuti sp. the relationship between hemoglobin levels and menstrual cycle regularity in d iii midwifery study program students level iii stikes muhammadiyah klaten. midwifery involution. 2012; 2(3): p. 34-35. 9. hadijah, h, hafid mp. effect of menstruation period on hemoglobin levels. journal of health analyst media. 2019; 1(1): p. 12-20. 10. satroasmoro s. fundamentals of clinical research methodology. in; 2014; jakarta: sagung seto. 11. world health organization. haemoglobin concentrations for the diagnosis of severity who 2011. 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[cited 2021 november 28. available from: https://www.who.int/vmnis/indicators/haemoglo bin.pdf. 12. sholicha ca, muniroh l. relationship of intake of iron, protein, vitamin c and menstrual pattern with hemoglobin levels in adolescent girls at sman 1 manyar gresik. indonesian nutrition media. 2019; 14(2): p. 147-153. 13. simbolon p, sukohar a, ariwibowo c, susianti. relationship between body mass index and menstrual cycle length in class 2016 students of the faculty of medicine, university of lampung. 2018; 7(2): p. 164-170. ina. j. med. lab. sci. tech. 2022; 4(1): 81–90 9 0 azzaubadilluah & andreas putroragil santoso 14. kirana dp. relationship between nutrient intake and menstrual pattern with anemia incidence in adolescent girls at sma negeri 2 semarang. 2011. 15. rachmaningrum ca, candra a. effect of zinc (zn) and iron (fe) supplementation on hemoglobin levels of toddlers 3-5 years old. journal of nutrition college. 2016; 5(4): p. 328333. 16. almatsier s, soetardjo s, soekarti m. balanced nutrition in the life cycle jakarta: pt. gramedia main library; 2011. 17. irianti b. relationship of blood volume during menstruation with the incidence of anemia in pekanbaru international midwifery academy students 2014. encyclopedia journal. 2019; 1(2): p. 257-261. 18. proverawati a. anemia and anemia of pregnancy yogyakarta: nuha medika; 2011. 19. almatsier s. basic principles of nutrition science 2011: pt. gramedia main library; 2011. 20. kiswari r. hematology and transfusion jakarta: erlangga; 2014. 21. zhong r, han d, wu x, wang h, li w, he z, et al. an evaluation of morphological changes and deformability of suspended red blood cells prepared using whole blood with different hemoglobin levels of tibetans. transfusion medicine and hemotherapy. 2021 march 18;: p. 1-10. 148 effect of bay leaf (syzygium polyanthum) extract on antioxidant activity, mda levels, and liver histopathology feature of ethambutol induced wistar rats bambang edi suwito1, lea maera shanty2, retna gumilang3, handayani4, renata alya ulhaq5 1department of anatomy histology, universitas nahdlatul ulama, surabaya, indonesia 2department of internal medicine, rsi jemursari surabaya, indonesia 3department of parasitology, universitas nahdlatul ulama, surabaya, indonesia 4department of medical pharmacology, universitas nahdlatul ulama, surabaya, indonesia 5faculty of medicine, universitas airlangga, surabaya, indonesia correspondence: bambang edi suwito, manyar adi 1 no. 31 surabaya, east java, indonesia zip code: 60282 email: bamsedi@unusa.acid received: november, 2021 revised: september 26, 2022 accepted: october 13, 2022 published: october 30, 2022 doi: 10.33086/ijmlst.v4i2.2471 abstract bay leaf extract (syzygiun polianthum) is one herbal element that may be used to lessen liver function issues, lessen symptoms of nausea, vomiting, discomfort and improve adherence and the effectiveness of tuberculosis treatment. the objective of this study was to determine the effect of bay leaf extract on antioxidant activity, malondialdehyde (mda) levels, and liver histopathology of ethambutol-induced wistar rats. this research is a laboratory experiment. antioxidant activity of bay leaf extract was evaluated by comparing vitamin c with spectrophotometry methods. white wistar rats were separated into 6 groups and used to test the label of mda and livers histopathology. group 1 serves as the control group and received dmso (placebo); group 2 was received ethambutol 50mg/kg bw; group 3 was received ethambutol and silymarin with dose 50mh/kg bw. group 4-6 had been given ethambutol and extra ethanol extract of bay leaves of 75,150, and 300mg/kg bw, respectively. the results of the spectrophotometry showed that the bay leaf extract had antioxidant activity comparable to that of vitamin c, with an ic50 of 11.4 g ± (4.4%). there was a significance difference in each group’s mda levels (p=0.002). although there was no significant difference in the liver histopathology of treated rats (p>0.05). while bay leaf extract significantly lowers mda levels in ethambutol-induced wistar rats, it has no discernible impact on the liver histopathology of ethambutol-induced wistar rats. bay leaf extract possesses antioxidant activity comparable to vitamin c. keywords antioxidant, histopathology, malondialdehyde, sygyzium polyanthum. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech. 2022; 4(2): 148–156 bambang edi suwito, et al 1 4 9 introduction tuberculosis (tb) is a serious infectious disease in the world. according to a 2015 report by the world health organization (who), tuberculosis is still the 10th leading cause of death globally. in indonesia, the prevalence of tb in 2014 was 297 per 100,000 population. in 2014, 9.7 million new cases of tb were discovered, with approximately 480,000 people progressing to multi-drug resistant (mdr) tb (1). it is reported that only 161,365 people (82.8%) that experienced to cure rates for lung disease tuberculosis patients from 194.853 pulmonary tuberculosis patients in indonesia and only 14,964 cases (7.7%) received complete treatment (2). isoniazid (h), rifampicin (r), pyrazinamide (z), ethambutol (e) are the first-line standard therapy for tb. long-term use of antituberculosis therapy drugs, especially isoniazid, rifampicin and ethambutol, tends to have side effects of hepatotoxicity, gastrointestinal disorders, neurological disorders, and hypersensitivity reactions. impair liver function (hepatotoxicity) is one of the most serious and common side effects of anti-tuberculosis (3,4). based on previous study, states that bay leaf (syzygium polyanthum) was utilized to treat of gout, gastritis, diarrhea, high cholesterol, type 2 diabetes mellitus and hypertension. saponins, triterpenoids, flavonoids, polyphenols, alkaloids, tannins, and essential oils made up of sesquiterpenes, lactones, and phenol are among the chemical compounds found in this leaf (5). plants produce flavonoids, which are polyphenol secondary metabolites, in a variety of plant components, including fruit, seeds, leaves, stems, and flowers. flavonoids are classified into eight classes as flavones, anthocyanidins, flava, flavanones, flavonolignans, isoflavones, isoflavones, and chalcones (6). according to another study, the active ingredients in propolis from south sulawesi are phenolic compounds like flavonoids which have been demonstrated to have hepatoprotective effects in mice. the results showed that the total polyphenol and flavonoid test of propolis contained 1.1% and 2.7%, respectively. in addition, the antioxidant activity test showed an ic50 (the half maximal inhibitory concentration) value of 9849 ppm and liquid chromatography with tandem mass spectrometry (lc-ms-ms) analysis supported phenolic compounds in propolis from south sulawesi (7). according to a prior study, the phenolic extract ingredient in bay leaves has an antioxidant activity (8). we hypothesis that bay leaf extract significantly affects malondialdehyde (mda), antioxidant and histological characteristics of the liver. this study aimed to determine the effect of ethanol extract from bay leaf on antioxidant activity, mda levels, and liver histopathology of ethambutolinduced wistar rats. ina. j. med. lab. sci. tech. 2022; 4(2): 148–156 bambang edi suwito, et al 1 5 0 materials and methods preparation of 70% ethanol extract of bay leaves the bay leaves dried for 7 days at room temperature, sorted, and mechanically ground into a coarse powder of s. polyanthum. a total of 1000 g of s polyanthum powder were macerated in 7000 ml of ethanol 70% for 4 hours, then the supernatant was progressively filtered through filter cloth, cotton and whatman paper no 1. a vacuum rotary evaporator was then used to evaporate the solution at a regulated temperature of 40°c while operating at a reduced pressure of 204 mbar. the extraction procedure was repeated three times after the remaining material was recovered. determination of dosage previous research has used mice at doses of 1250mg/kg bw, 2500mg/kg bw, and 5000mg/kgbw to investigate the acute toxicity of bay leaf extract. no experimental animals perished from either the control group or the treatment group, according to the findings of observations made over the course of fourteen days of treatment. this suggests that test animals can still receive a single oral dose of an ethanolic extract of bay leaves up to the maximal dose (5000mg/kg bw), or nearly 200 times the usual human dose, without the animals dying (9). as a result, the maximum dose in this investigation was set at 300 mg/kg bw, or 10% of the acute toxicity limit. in this study, bay leaf ethanol extract was administered at doses of 75 mg/kg bw, 150 mg/kg bw, and 300 mg/kg bw. treatment the implementation of this study has been approved and issued a certificate of ethics from the universitas nahdlatul ulama surabaya ethics committee with the number 242/uc/kepk/unusa/2020. wistar rats weighing about 200 gr were obtained from faculty of medicine, universitas nahdlatul ulama surabaya. rats were adapted for one week and fed regular animal pellets with water ad libitum. the animals were grouped into six groups. each group consisting of 6 male rats. group i (normal control): rats were merely given food, water, and a placebo in place of any form of treatment for each mouse. group ii (positive control): ethambutol was given to this group in combination with a dose of 50 mg/kg bw of rats. group iii: ethambutol and antihepatotoxic medicines (silymarin) were given to this group in doses of 50 mg per kg bw of rats. group iv: this group received 75 mg/kg of a 70% ethanol extract of bay leaf together with 75 mg/kg of ethambutol. group v: 150 mg/kg of the 70% ethanol extract of s polyanthum (eesp) and ethambutol were given to this group. group vi: 300 mg/kg of the 70% ethanol extract of s polyanthum (eesp) and ethambutol were given to this group. ina. j. med. lab. sci. tech. 2022; 4(2): 148–156 bambang edi suwito, et al 1 5 1 determination malondialdehyde (mda) plasma mda levels were measured according to the wills method (10). the 200 µl plasma sample was added 1 ml 20% trichloro acetate (tca) and 2 ml 0.67% thiobarbituric acid (tba). the solution was well mixed until homogeneous and heated on a water bath for 10 minutes. after cooling the solution, centrifuge it at 3000 rpm (1 rpm = 1/60 hz) for 10 minutes. the pink colored filtrate was measured at a wavelength of 532 nm using a uv-vis spectrophotometer. mda levels calculated using standard mda curves with concentrations 0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 nmol/ml, respectively (11). liver histopathology wistar rats’ liver tissue were dissected from the body and fixed in 10% formalin solutions, then dehydrated it in gradually ethanol solution in stages (50-100%). tissue were cleaned with xylene and embedded in paraffin. using a rotary microtome, sections were cut at a nominal thickness of 5–6 m and stained with hematoxylin and eosin to observe microscopic histopathological alterations and their severity. the microscopic observation was observed by a single observer, performed by an anatomy and histology lecturer from the medical faculty. the parameters of liver damage observed are cell degeneration consisting of hydropic generation, fat degeneration, necrosis, and congestion. they were scored as follows: 1) if no damage was visible in the field of view magnification at 100x and 400x; 2) if the percentage of damage was visible at or below 10% in the field of view at 100x and 400x; and 3) if the percentage of visible damage was between 10% and 30% in the field of view at 100x and 400x (12). results this research is an experimental study which uses experimental animals that are treated with ethambutol as an analogue of tuberculosis patients receiving ethambutol of the antioxidant activity of bay leaf extract (s polianthum) using the dpph (2,2 dipheniyl1-picyrylhydrazyl). the scavenging assay method was carried out at the testing service unit of the faculty of pharmacy, airlangga university. the antioxidant activity test of the bay leaf extract was carried out with a comparison of vitamin c. the reading of the results using the spectrophotometric method showed that the bay leaf extract (s polyanthum) had antioxidant activity comparable to vitamin c, with an ic50 of 11,4 µg± (4,4%). the lower the ic50 value were indicated the greater the antioxidant activity in reducing free radicals. these findings suggest that bay leaf extract has a powerful, comparable to vitamin c ability to decrease free radicals. the antioxidant activity of polyphenolic compounds can be formed in the neutralization reaction of free radicals or ina. j. med. lab. sci. tech. 2022; 4(2): 148–156 bambang edi suwito, et al 1 5 2 the termination of the chain reaction that takes place because flavonoids, which are polyphenolic compounds, have the capacity to donate hydrogen atoms to free radical molecules. malondialdehyde (mda) test ibm spss statistics 25 are used to analyze data in this study. the one-way anova test was carried out and achieved a significance value (sig) of 0,002 (table 2). the difference between the average mda levels in each group is indicated by the significance value of 0.05. table 1-4 showed the result for the data analyzed. according to the results of these experiments, giving the bay leaf extract to wistar rats after they had been given ethambutol to induce mda levels had a substantial impact. table 1. test of normality of malindialdehyde (mda) shapiro-wilk mda levels sample group statistic df sig. group 1: normal control 0.935 4 0.625 group 2: positive control 0.963 6 0.840 group 3: ethambutol + silymarin 0.947 6 0.713 group 4: eesp 75 mg/kg 0.910 6 0.343 group 5: eesp 150 mg/kg 0.980 5 0.932 group 6: eesp 300 mg/kg 0.801 4 0.105 table 2. one-way anova test of malindialdehyde (mda) sum of squares df mean square f sig. between groups 1047221.735 5 209444.347 5.446 0.002 within groups 961546.200 25 38461.848 total 2008767.935 30 histopathological test of wistar rats histopathological feature showed no liver organ changes from all the groups. no abnormalities between hepatocyte among groups was observed. statistical test was performed using the kruskal wallis method because the data were not normally distributed, then the asymptotic significance 2-tailed (asymp. sig value) was 0.106. the asymp. sig value >0,05 indicates that there is no significant difference in the liver histopathology of treated rats. this result indicated that the administration of the bay leaf extract did not have a significant effect on the liver histopathology of wistar rats which are induced by ethambutol. there is no statistically detected difference between the groups in our study ina. j. med. lab. sci. tech. 2022; 4(2): 148–156 bambang edi suwito, et al 1 5 3 figure 1. liver histopathology of wistar rats, hepatocytes are showed representatively by arrows in each figure; b1 (normal control), b2 (positive control), b3 (ethambutol + silymarin), b4 (eesp 75mg/kg), b5 (eesp 150mg), b6 (eesp 300mg). table 3. tests of normality liver histopathology of wistar rats shapiro-wilk histopatological score sample group statistic df sig. group 1: normal control 0.630 4 0.001 group 2: positive control 0.822 6 0.091 group 3: ethambutol + silymarin 0.853 6 0.167 group 4: eesp 75 mg/kg . 6 . group 5: eesp 150 mg/kg 0.881 5 0.314 group 6: eesp 300 mg/kg 0.630 4 0.001 table 4. kruskal-wallis test of wistar rats induced ethambutol and ethanol extract of bay leaf histopathological score kruskal-wallis h 9.085 df 5 asymp. sig 0.106 discussion antioxidant properties of bay leaf (s polianthum) indonesia is renowned for having high level of biodiversity, including a variety of medicinal herbal plants. one of the plants that are often used in herbal medicine is bay leaf. one of the many functions of the active ingredients in bay leaves is as an antioxidant. earlier studies utilizing the dpph technique ina. j. med. lab. sci. tech. 2022; 4(2): 148–156 bambang edi suwito, et al 1 5 4 demonstrated that the antioxidant activity of bay leaf extract was 68.55%. (13). the antioxidant activity of bay leaf extract demonstrates the variety of chemicals that contribute to the antioxidant process in preventing the formation of free radical (14). in this case, the form of chemicals 1,1diphenyl-2-picrylhydrazyl (dpph) as free radical compounds. the findings of this study suggest that bay leaf extract has a robust, comparable to vitamin c ability to decrease free radicals. this is in consistent with the study conducted by widyawaty et al., (15), which demonstrated that the antioxidant activity of the ethanolic extract of bay leaf had values of 12,65 ppm and 48,98 ppm by the dpph and abts methods, respectively, and flavonoids and phenol totals of 15,666 qe/g and 206 gae/g, respectively. with an ic50 value under 50 ppm, it can be said that the ethanolic extract of s. polyanthum belongs to the very powerful antioxidant group. malondialdehyde levels research on oxidative stress and redox signaling have long used the measurement of mda content as a lipid peroxidation marker, notably in those studies focusing on plant responses to abiotic and biotic challenges (16). the present study suggests that elevated serum levels of mda and cortisol are strongly associated with major depressive disorder. the elevations of mda and cortisol in serum level arise independently in the blood and they may acs as as biomarkers for major depressive disorder (17). the significant value of 0,05 in this study demonstrates that there are differences in the average mda levels between the groups. based on the results of these experiments, it can be deduced that giving bay leaf extract to wistar rats after they had been given ethambutol to generate mda had a considerable impact on their levels of mda. this finding was related to the possible antioxidant activity of bay leaf. liver histopathology the first-line adjunctive antituberculosis drug ethambutol is only used in conjunction with drugs like isoniazid and rifampicin. ethambutol is nearly always combined with isoniazid, rifampicin, or other anti-tuberculosis medications, so it is impossible to estimate how frequently ethambutol alone will produce serum aminotransferase increases. the addition of ethambutol to isoniazid, rifampicin or pyrazinamide does not appear to increase the rate of transient alt elevations during therapy. in addition, ethambutol is a rare cause of acute, symptomatic liver damage as well (4). it has been demonstrated that the flavonoid components in bay leaves can inhibit the rise in igg. the highest igg inhibition was observed at 100 ppm, reaching 1.01 g/ml as opposed to the 57.19 g/ml of normal controls. along with its flavonoid components, the bay leaf also contains a ina. j. med. lab. sci. tech. 2022; 4(2): 148–156 bambang edi suwito, et al 1 5 5 chemical called eugenol, which has a good affinity of -6.3 kcal/mol for inhibiting covid-19 proteases (18). additionally, high-nitric oxide macrophage production from bay leaf extract can boost the immune system (19,20). the liver histology of the treated rats in this investigation did not significantly differ from that of the control rats, indicating that the administration of bay leaf extract did not significantly affect the liver histopathology of wistar rats induced by ethambutol as show in figure 1. this outcome could be attributed to ethambutol's hepatotoxicity, which is not significant when administered without the addition of isoniazid or other anti-tuberculosis medications. conclusions the bay leaf extract has antioxidant activity comparable to vitamin c, has a significant effect on mda levels in ethambutol-induced wistar rats, but does not have a significant effect on liver histopathology of ethambutol-induced wistar rats. we suggest for the future study to add another anti-tuberculosis drugs that primarily caused hepatotoxic. author contributions all authors contributed equally to this work (conceived and designed the analysis; collected the data; contributed data or analysis tools; performed the analysis; wrote the paper). acknowladgements this study was supported by the universitas nahdlatul ulama surabaya. conflict of interest there is no conflict of interest. references 1. macneil a, glaziou p, sismanidis c, date a, maloney s, floyd k. global epidemiology of tuberculosis and progress toward meeting global targets worldwide, 2018. mmwr morb mortal wkly rep. 2020;69(11):281–5. https://doi.org/10.15585/mmwr.mm6911a2 2. lei s, gu r, ma x. clinical perspectives of isoniazid-induced liver injury. vol. 5, liver research. 2021;45–52. https://doi.org/10.1016/j.livres.2021.02.00 3. pennington km, kennedy cc, chandra s, lauzardo m, brito mo, griffith de, et al. management and diagnosis of tuberculosis in solid organ transplant candidates and recipients: expert survey and updated review. j clin tuberc other mycobact dis. 2018;11:37–46. https://doi.org/10.1016/j.jctube.2018.04.001 4. cao j, mi y, shi c, bian y, huang c, ye z, et al. first-line anti-tuberculosis drugs induce hepatotoxicity: a novel mechanism based on a urinary metabolomics platform. biochem biophys res commun. 2018;4:497(2):485–91. https://doi.org/10.1016/j.bbrc.2018.02.030 5. dewijanti i, mangunwardoyo w, astari d, hanafi m, artanti n. short communication: effects of the various source areas of indonesian bay leaves (syzygium polyanthum) on chemical content and antidiabetic activity. biodiversitas j biol divers. 2020;21(3):1190–5. https://doi.org/10.13057/biodiv/d210345 6. tungmunnithum d, thongboonyou a, pholboon a, yangsabai a. flavonoids and other phenolic compounds from medicinal plants for ina. j. med. lab. sci. tech. 2022; 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of extracts from syzygium polyanthum leaves. am j drug discov dev. 2014;4(2):90–101. https://dx.doi.org/10.3923/ajdd.2014.90.101 20. hasan r, lindarto d, siregar ga, mukhtar z. the effect of bay leaf extract syzygium polyanthum (wight) walp. on c-reactive protein (crp) and myeloperoxidase (mpo) level in the heart of rat model of myocardial infarction. medicinski glasnik: official publication of the medical association of zenica-doboj canton, bosnia and herzegovina. 2020;17(1): 41–45. https://doi.org/10.17392/1068-20 91 analysis of d-dimer level and prothombin time (pt) activated prothombin thromboplastin (aptt) on heparin administration to covid-19 patients dela yorike1, muhammad rizki kurniawan1, mohamad syafaat1 1departement medical laboratory technology, faculty science and technology, universitas binawan, jakarta correspondence: dela yorike, gg. sadar 1 no. 95 bekasi, west java, indonesia zip code: 17431 email: delayorike02@gmail.com received: september 18, 2021 revised: february 22, 2022 accepted: march 14, 2022 published: april 28, 2022 doi: 10.33086/ijmlst.v4i1.2487 abstract coronavirus disease 2019 (covid-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2). coronavirus is a dangerous pathogen that affects both humans and animals. symptoms and indicators of covid-19 infection include fever, cough, and shortness of breath, which are common in respiratory illnesses. covid-19 has spread globally with high mortality and morbidity rates. prothrombin time (pt), activated prothrombin thromboplastin (aptt), and ddimer parameters are blood tests to evaluate the coagulation status of covid-19 patients, all these parameters increased in covid-19 patients. this study aims at determininig the levels of d-dimer, pt, and aptt in covid-19 patients. clinical and laboratory records were retrospectively reviewed from 100 cases of covid-19 admitted to hospitals. this study used a a cross-sectional, descriptive, secondary data analysis design. the sample included patients from the bhayangkara tk. i r.said sukanto hospital between april-july 2021. the sample is a covid19 patient who was treated in the intensive care unit (icu), worsened, and given heparin. an independent variable is the administration of intravenous heparin at prophylactic doses. dependent variables are d-dimer, pt, and aptt values. the collected data is processed using microsoft office excel and then being analyzed. d-dimer, pt, and aptt levels in covid-19 patients were initially high or increased after heparin administration decreased or became normal. in conclusion, there was a decrease in d-dimer levels, pt values, and aptt after administration of intravenous heparin at prophylactic doses. keywords activated prothrombin thromboplastin, covid-19, ddimer, heparin, prothrombin time. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. ina. j. med. lab. sci. tech2022; 4(1): 91–98 dela yorike, et al. 9 2 introduction coronavirus is a dangerous pathogen that attacks humans and animals. coronavirus comes from the subfamily orthocoronavirinae in the family coronaviridae and the order nidovirales. this virus attacks the respiratory system in humans and causes several diseases such as severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and coronavirus disease 2019 (covid-19) (3). symptoms and signs of covid-19 infection include respiratory symptoms such as fever, cough, and shortness of breath (3). people who have a history of co-morbidities and are elderly are at high risk of contracting covid-19 (4). coronavirus disease 2019 (covid-19), the clinical syndrome associated with infection with severe acute respiratory syndrome coronavirus-2 (sars-cov-2), has been able to affect various worlds and scientific investigations have not occurred, previously there was a shortage of pharmacotherapy to fight the disease. the lack of treatment strategies with a variety of practice patterns already exists. hypercoagulability in covid-19 patients has sparked debate in clinical care regarding the use of heparin therapy (7). the rapid spread of covid-19 cases in various countries has put health status in a dangerous condition. the latest data from the world health organization reports that confirmed covid-19 sufferers in three days on 26 april 2020 in europe (1,341,851 patients), asia (43,846 patients) and africa (20,316 patients) with a global death rate of 193,710, while in indonesia, based on data from the national disaster management agency on 26 april, 2020, covid-19 sufferers numbered 8,882 and 743 people died. the most covid-19 sufferers are in dki jakarta (3,798 patients), west java (912 patients), east java (785 patients), central java (649 people) and south sulawesi (440 patients) (4). laboratory parameters to identify the blood coagulation disorders occurrence in covid-19 patients are d-dimer, fibrin/fibrinogen, prothrombin time (pt/time required for blood to form clots or blood platelets) (5). d-dimer is a fibrin degeneration product that is useful for detecting abnormal clot formation or thrombotic events and assessing the presence of a clot breaking or fibrinolytic process. quantification of d-dimer levels plays an important role in guiding therapy. d-dimer level is usually elevated in patients with covid-19 (1). prothrombin time (pt) is the test of time length in blood clotting based on the extrinsic and common pathways. this test is performed to determine the presence of bleeding disorders and assess the treatment taken to prevent bleeding (1). activated prothrombin thromboplastin time (aptt) is the test of time length in blood clotting ina. j. med. lab. sci. tech. 2022; 4(1): 91–98 dela yorike, et al. 9 3 based on the basic pathway (intrinsic pathway). the aptt test is usually paneled with the pt test to determine the presence of a bleeding disorder and the possibility of profuse bleeding during surgery (2). covid-19 patients have not been supported by sufficient research until now, so the treatment has not been established significantly. heparin is a drug to treat and prevent blood clots caused by certain medical conditions or procedures. heparin works by inhibiting the work of proteins that play a role in the blood clotting process, so the blood clots formation can be prevented. however, for the time being, the use of heparin and low molecular weight heparin (lmwh), can be used. the use of low-dose heparin was associated with a significant reduction in mortality within 28 days in patient with sepsis. other studies have shown a reduction in the risk of death at days 7 to 28 and a significant increase in the pao2/fio2 ratio in acute respiratory distress syndrome (ards) patients receiving high-dose lmwh therapy (6). in a previous retrospective study, 449 patients with severe covid-19 in china, 99 of whom received heparin (mainly lmwh) for at least 7 days, found that prothrombin time was positively correlated with 28 days. mortality and platelet count were negatively correlated. in general, no significant difference was found in mortality between heparin users and nonusers at 28 days (30.3% vs 29.7%, p = 0.910), but the mortality of heparin users at 28 days was lower than that of nonusers in patients with d-dimer levels were more than six times the upper limit of normal (32.8% vs 52.4%, p = 0.017) (18). the primary purpose of this study was to give an analysis of d-dimer, pt, and aptt in covid-19 patients who were used heparin. the source of data used in this study is secondary data. the data that has been collected is processed using microsoft office excel and made into a graph. data analysis included testing the frequency data for respondent characteristics based on sex, age, d-dimer level, pt level and aptt level. materials and methods source of patients diagnosis criteria this study was conducted in the laboratory of the bhayangkara t.k.i raden said sukanto hospital, jakarta-indonesia used a descriptive research type and has been approved by bhayangkara t.k.i raden said sukanto hospital ethical commission with the letter of ethical approval number b/2245/vii/2021. the populations in this study were all patients who confirmed positive for covid-19 in the icu/hcu bhayangkara t.k.i raden said sukanto hospital between april-july 2021. a total of 100 patients, all diagnosed with covid-19 which had confirmed a positive result of the nucleic acid test of sars-cov-2 by realtime fluorescence rt-pcr. ina. j. med. lab. sci. tech2022; 4(1): 91–98 dela yorike, et al. 9 4 diagnosis criteria the sampling technique in this study was purposive sampling, which uses the criteria that have been selected by the researcher in selecting the sample. the sample selection criteria are divided into inclusion and exclusion criteria. the inclusion criteria were above 30 years old, have comorbidities such as liver disease, kidney, heart, lung, digestive tract, urinary tract, brain, and others, and covid-19 patients who deteriorated and need icu. meanwhile, the exclusion criteria are abortus, hypertension, and thrombocytopenia. the tools used in this study include equipment blood sampling (syringes, citrate vacuum tubes, alcohol swabs, plasters, tourniquet, label), coagulation analyzer (sysmex® ca-600), centrifuge, and micropipette. the ingredients used in this study include plasma citrate blood, dade innovin reagents, pathrombin sl reagents, cacl2 reagents, ovb reagents, and normal controls. the measurement method was used cross-sectional by looking at the decrease in the value of d-dimer, pt, and aptt levels in the administration of heparin to covid-19 patients. results the distribution of respondent characteristics based on age when giving heparin to covid-19 patients at bhayangkara t.k.i raden said sukanto hospital obtained as shown in the figure 1. figure 1. frequency distribution of respondent’s age based on figure 1, most of the respondents have an age category of 46-60 years (63.75%), followed by 31-45 years (21.25%), and > 60 years (15%). moreover, the distribution of respondent characteristics based on gender when giving heparin to covid-19 patients at bhayangkara t.k.i raden said sukanto hospital is shown in figure 2. figure 2. frequency distribution of respondent’s sex based on figure 2, most respondents were male (53.75%). parameters of pt, aptt, and d-dimer levels in the administration of heparin to patients with ina. j. med. lab. sci. tech. 2022; 4(1): 91–98 dela yorike, et al. 9 5 coronavirus disease 2019 (covid-19) at bhayangkara t.k.i raden said sukanto hospital showed in figure 3. figure 3 parameters of pt, aptt and ddimer levels on heparin based on figure 3, a decrease in pt and aptt levels as well as in d-dimer levels showed after heparin administration in covid-19 patients. most respondents with covid-19 had a high pt value (63.75%) before heparin administration. meanwhile, after heparin administration, there were 53 respondents with normal pt values (66.26%) and 27 respondents with high pt values (33.75%). before heparin administration, most respondents with covid-19 had high aptt (long) values (53.75%). meanwhile, after heparin administration, there were 55 respondents with normal appt values (68.76%) and 25 respondents with high appt values (31.25%). before heparin administration, most respondents with covid-19 had high (increase) d-dimer levels (66.25%). after heparin administration, there were 47 respondents with normal appt values (58.75%) and 33 respondents with high appt values (41.25%). discussion parameters pt, aptt, and d-dimer are blood tests to evaluate the patient's coagulation status. pt examination is to evaluate extrinsic coagulation factors, while ptt can detect the function of intrinsic coagulation factors and coagulation components. both tests can help explain the cause of a bleeding or blood clotting disorder. aptt is part of ptt. the aptt panel is the endpoint of blood coagulation testing time that serves to assist in the diagnosis of coagulation factor deficiency in the intrinsic pathway. d-dimer is a protein residue formed by the breakdown of blood clots. ddimer is a fibrin degradation product formed during the degradation of blood clots by fibrinolysis. elevated d-dimer in the blood is a marker of suspected thrombosis found in deep vein thrombosis, pulmonary embolism, arterial thrombosis, pregnancy, inflammation, chronic liver disease, cancer, surgery, vasculitis, and disseminated intravascular coagulation (dic) (19). prolonged pt, aptt, and increased ddimer values are often found in severe covid-19 patients and are predictors acute respiratory distress syndrome (ards) occurrence, the need for care in the intensive care unit (icu), and death. prolongation of ina. j. med. lab. sci. tech2022; 4(1): 91–98 dela yorike, et al. 9 6 pt > 3 seconds or aptt > 5 seconds is a marker of coagulopathy (blood clotting disorder resulting in excessive bleeding) and a predictor of thrombotic complications in covid-19 patients. covid-19 patients with an increase in d-dimer (3-4 times) need to be hospitalized even if there are no severe symptoms because it indicates there is an increase in thrombin generation and risk for thrombotic events. efforts to prevent the occurrence of coagulopathy in these covid19 patients, it is necessary to provide prophylactic anticoagulants, namely low molecular weight heparin (lmwh) or unfractionated heparin (ufh) (20). the results of this study are in line with coagulation parameter studies, namely prothrombin time (pt), activated partial thromboplastin time (aptt) and d-dimer, which used a sample of 183 patients with 162 patients who survived. the study showed the average level of d-dimer was 0.61 g/ml (normal: <0.5 g/ml), pt with a time of 13.6 seconds (normal: 11.5-14.5 seconds), and aptt with a time of 41.2 seconds (normal: 29-42 seconds). this study's results indicate an increase in coagulation levels among covid-19 patients (21). the results of this study are in line with study by adie & farina (21), that there is an increase in coagulation levels in covid-19 patients, where the average d-dimer level is 0.61 g/ml (normal: <0.5 g/ml), pt with a time of 13.6 seconds (normal: 11, 5-14.5 seconds), and aptt with a time of 41.2 seconds (normal: 29-42 seconds) (21). the results of this study are in line with the study of rusdiana & akbar (22), which showed that the levels of d-dimer in the five patients had varied patterns. in case 1, there was an increase in d-dimer levels >2 mg/l in the middle of treatment, while cases 2 and 3 were stable with normal d-dimer levels <2 mg/l, but at the time of the last examination, all patients' d-dimer levels were normal < 2 mg/l after heparin administration and on discharge. in the group of patients who died, the d-dimer levels were both > 7 mg/l. ddimer is derived from the lysis of crosslinked fibrin, where an increase in its level may indicate activation of coagulation and fibrinolysis (22). similar studies have also shown that an increase in d-dimer >1.0 l/ml is the strongest predictor of mortality in covid-19 patients (23). d-dimer >1.5 l/ml was a predictor of venous thromboembolism in covid-19 patients with a sensitivity of 85% and specificity of 88.5% (24). the available evidence suggests that severe covid-19 disease can lead to coagulopathy complications in the form of disseminated intravascular coagulation (dic), which is prothrombotic with a high risk of venous thromboembolism, where pt, aptt, and d-dimer markers can be used as a parameter for blood coagulation disorders (24). based on this, the experts recommend ina. j. med. lab. sci. tech. 2022; 4(1): 91–98 dela yorike, et al. 9 7 anticoagulants, reflecting the recognition of clotting dysregulation in this situation (24) because of the effectiveness of heparin therapy (especially low molecular weight heparin / lmwh) for covid-19 treatment therapy. before heparin administration, most respondents had high or increased levels of d-dimer, pt values, and aptt values. therefore, after heparin administration, ddimer levels, pt values, and aptt values were decreased or became normal (25). the limitations of this research were the lack of monitoring of d-dimer, pt, and aptt levels among covid-19 patients with heparin administration. the recommendation for future research is to add parameters for evaluating the coagulation status of covid19 patients, such as c-reactive protein (crp), ferritin, and lactate dehydrogenase (ldh) levels. conclusions heparin are widely used anticoagulants by inhibitit coagulation. in covid-19 patients, d-dimer, pt, and aptt values before heparin administration were high or increased. meanwhile, after heparin administration, d-dimer, pt, and aptt values levels were decreased or became normal. 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