PEDIATRIC UROLOGY Ischemia Modified Albumin and D-dimer in the Diagnosis of Testicular Torsion: An Experimental Model Fatma Sarac1*, Selman Yeniocak2, Akif Erbin3, Esma Yucetas4, Kamile Altundal5, Burak Ucpinar3, Ayse Saygili6, Macit Koldas4 Purpose: We aimed to investigate the potential early diagnostic value of ischemia modified albumin (IMA) and D-dimer in testicular torsion. Material and Methods: A total of 42 prepubertal Wistar-Hannover rats (26-30 days old, weighing 75-125 grams) were used in the study. They were randomly divided into 2 groups as torsion (21 rats) and control (21 rats). Both torsion and control groups were subdivided into three subgroups as 30th, 120th and 240th minutes. Intraperitoneal injection of 70 mg/kg ketamine (Ketalar, Pfizer, Istanbul, Turkey) plus 10 mg/kg of xylazine (Rompun, Bayer, Istanbul, Turkey) were used for general anesthesia. In the control group, scrotal incision was made and the left testis gently extracted. Then, intracardiac blood and testicular tissue were obtained at 30th, 120th and 240th minutes. In torsion group, testicular ischemia was achieved by rotating left testis 720° clockwise and maintained by fixing the testis. Blood and testicular samples were obtained at 30th, 120th and 240th minutes. All animals were sacrificed after completion of the study. Results: There was a statistically significant difference between the IMA and D-dimer levels at 30th, 120th and 240th minutes of torsion group when compared with the control group (p = .001). When compared in terms of pathological changes at 30th, 120th and 240th minutes, significant difference was found for all 3 periods (p = 0.039, p = 0.014, p = 0.03, respectively). The D-dimer and IMA estimated torsion with reasonable accuracy [Area under the curve (AUC)= 0.771 (p = 0.003, 95% confidental interval: 0.620-0.922) and AUC = 0.706 (95% confi- dental interval: 0.549-0.863, p = 0.022), respectively. Conclusion: The elevated D-dimer and IMA serum levels observed in the experimental testicular torsion model seem to have a potential role as a serum marker in the early diagnosis of testicular torsion. Keywords: D-dimer; ischemia modified albumin; testicular torsion INTRODUCTION Testicular torsion (TT) occurs due to the loss of blood flow to the testis and surrounding tissues as a result of spermatic cord rotation.(1) Testicular re- covery is likely if intervention is performed within the first 6 hours after the onset of symptoms.(2-4) Testicular torsion causes ischemic injury; detorsion causes reper- fusion damage and they both cause structural and bio- chemical changes in the testis.(5-7) In case of ischemia, cellular stress factors such as hypoxia, acidosis, free radical damage and deterioration of membrane integ- rity change the structure of the albumin molecule. At the N-terminal end of the albumin, some changes that reduce the binding capacity of transitional metals such as copper, cobalt and nickel occur. This newly-formed damaged albumin is called “ischemia modified albu- min” (IMA).(8-10) D-dimer is a degradation product of 1Department of Pediatric Surgery, Haseki Traning and Research Hospital, Istanbul, Turkey. 2Department of Emergency Medicine, Haseki Traning and Research Hospital, Istanbul, Turkey. 3Department of Urology, Haseki Traning and Research Hospital, Istanbul, Turkey. 4Department of Biochemistry, Haseki Traning and Research Hospital, Istanbul, Turkey. 5Department of Pathology, Haseki Traning and Research Hospital, Istanbul, Turkey. 6Department of Pediatrics, Arnavutkoy State Hospital, Istanbul, Turkey. *Correspondence: Department of Pediatric Surgery, Haseki Traning and Research Hospital, Istanbul, Turkey. Phone: +90 533 460 69 06. Fax: +90 212 529 4400. E-mail: fsarac75@gmail.com. Received November 2018 & Accepted April 2019 fibrin. Local fibrin formation and lysis are part of the inflammatory response and fibrin degradation products such as D-dimer, regulate the acute phase response and the production of systemic inflammatory mediators. Both markers are mainly elevated in ischemic-hypoxic and thromboembolic conditions(11-16). Examination of testicular blood flow by color doppler ultrasonography (USG) or scintigraphy are the main diagnostic methods in the diagnosis of TT. However, these methods may not be easily accessible in every case . So, there is a need for fast laboratory tests which are practical, easily accessible and have a high diagnos- tic value. Some limited animal studies have shown that IMA and D-dimer can have a significant value in the diagnosis of TT. In this experimental study, we aimed to investigate the role of serum levels of IMA and D-dimer in the early diagnosis of TT in prepubertal rats. Since TT is mostly Urology Journal/Vol 16 No. 6/ November-December2019/ pp. 567-571. [DOI: 10.22037/uj.v0i0.4974] seen in pediatric age group and young adults, we pre- ferred to use prepubertal rats. MATERIAL AND METHODS Study design The present animal stud y was approved by Bezmialem Vakif University (BVU) Local Ethics Committee of the Animal Experiments (IRB number: 2018/18) and was carried out in the BVU Experimental Animal Research Laboratory. Animals used in the experiment were kept in steel cages at a room temperature of 22°C and were fed with normal water and standard food until the day of the study. Water-only diet was provided for the last 12 hours before the induction of the study. A total of 42 experimentally naïve and drug-naïve male prepuber- tal Wistar-Hannover rats were used in the study. They were randomly divided into 2 groups as torsion (21 rats) and control (21 rats) group. Both torsion and control groups were subdivided into three groups as 30th, 120th and 240th minutes. Intraperitoneal injection of 70 mg/ kg ketamine (Ketalar, Pfizer, Istanbul, Turkey) plus 10 mg/kg of xylazine (Rompun, Bayer, Istanbul, Turkey) were used for general anesthesia. In control group, scro- tal incision was made and the left testis gently extract- ed. Then intracardiac blood and testicular tissue were obtained at 30th, 120th and 240th minutes. In torsion group, testicular ischemia was achieved by rotating left testis 720° clockwise and maintained by fixing the tes- tis. Blood and testicular samples were obtained at 30th, 120th and 240th minutes. All animals were sacrificed after completion of the study. Biochemical investigations To measure serum IMA and D-dimer levels, rat IMA ELISA kit (Catalog No. CK-E91024, Eastbiopharm., Hangzhou Eastbiopharm Co. Ltd.) and rat D-dimer (D2D) ELISA kit (Catalog No. CK-E91432, Eastbio- pharm., Hangzhou Eastbiopharm Co. Ltd.) were used, respectively. Specimen absorbances were determined on a Biotek ELX800 (Biotek, Winooski, VT, USA) mi- croplate reader at a wavelength of 450 nm. The IMA results were expressed in IU/mL and the minimum detectable level was 1 IU/L. The d-dimer results were expressed in ng/mL and the minimum detectable level was 5 ng/L. Histopathological examinations Testicular tissues were fixed in 10% formaldehyde solution and they were embedded into paraffin for fol- low-up procedures. Standard sections of four microns were prepared and they were stained with hematoxylin and eosin (H&E). The slides were evaluated by using a light microscope and classified according to the clas- sification system which was designed by Cosentino et al.17: Stage 1: Normal testicular tissue (Figure 1.A) Stage 2: Less regular germ cells, irregular convergent seminiferous tubules (Figure 1.B) Stage 3: Irregular germ cells, diminished pycnotic nu- clei and destructed bounded seminiferous tubules (Fig- ure1.C) Stage 4: Seminiferous tubules filled with irregular germ Table1. Summary of population characteristics of the rats Torsion group Control group n 21 21 Mean age days, (range) 27.9 (26-30) 28.1 (27-29) Mean weight days, (range) 101.2 (75-125) 103,4 (75-125) D-Dimer IMA Control group Median Torsion group Median Pa Control group Median Torsion groupc Median Pa (IQR) (min-max) (IQR) (min-max) (IQR) (min-max) (IQR) (min-max) 30 min 118.2 (34.1) 127.7 (23.7) .001 32.0 (17.5) 36.3 (6.0) .001 (105.5-171.6) (113.1-160.6) (24.7-48.0) (32.1-41.6)d, e 120 min 110.1 (14.4) 142.6 (25.7) .001 35.6 (12.3) 37.2 (10.3) .001 (94.0-234.3) (126.4-208.4) (25.5-46.4) 9(30.3-54.5)f 240 min 116.0 (18.3) 149.3 (54.7) .001 35.5 (16.4) 61.9 (22.4) .001 (86.5-187.9) (119.6-258.5) (22.5-47.3) (43.4-82.9) Pb .428 .967 .174 .02 Abbreviations: IMA: Ischemia modified albümin; IQR: Interquartile range; min-max: minimum-maximum a Wilcoxon test b Kruskal-Wallis test cMann-Whitney U test was performed to test the significance of pairwise differences using Bonferroni correction (p = .05/3=.017) to adjust for multible comparisons; dp = .565 (comparison of 30 min and 120 min); e p = .002 (comparison of 30 min and 240 min); fp = .006 (comparison of 120 min and 240 min). Table 2. Comparison of serum IMA and D-dimer levels at 30, 120 and 240 minutes between torsion and control groups Figure 1. Histopathological findings of each stage A. normal tes- ticular tissue (stage 1) B. less regular germ cells, irregular conver- gent seminiferous tubules (stage 2) C. irregular germ cells, dimin- ished pycnotic nuclei and destructed bounded seminiferous tubules (stage 3) D. seminiferous tubules filled with irregular germ cells which have coagulation necrosis (stage 4) Early diagnosis of testicular torsion-Sarac et al. Pediatric Urology 568 cells which have coagulation necrosis (Figure 1.D) Statistical analysis Data were analyzed by using Statistical Package for the Social Sciences software package version 16 (SPSS Inc., Chicago, IL, USA). Descriptive analyses were presented using median, interquartile range (IQR), minimum and maximum for non-normally distributed variables. The Wilcoxon test was used to compare tor- sion group with it’s control group. More than two group comparisons were made by Kruskal Wallis test; if there was a significiant difference, Mann-Whitney U test was performed to test the significance of pairwise differ- ences using Bonferroni correction (p = .05/30 = .017) to adjust for multiple comparisons. The comparison of torsion group with its control group in terms of patho- logical staging was performed with the chi-square test. Cut-off point value was determined by ROC analysis. Statistical significance was accepted as p <.05. RESULTS The characteristic of the rats is shown in Table1. Also, serum IMA and D-dimer levels of torsion and control groups are summarized in Table 2. There was a sta- tistically significant difference between the IMA and D-dimer levels at 30th, 120th and 240th minutesin the torsion group when compared tothe control group (p = .001). There was a significant difference in terms of IMA levels between subgroups of the torsion group (30th vs 240th minutes and 120th vs 240th minutes, p = .002 and p = .006, respectively). However, no signifi- cant difference was detected in terms of D-dimer values (p = .174). When torsion and control groups were compared in terms of pathological changes at 30, 120 and 240 min- ute according to the Cosentino classification, signifi- cant difference was found for all 3 periods (p = .039, p = .014, p = .03, respectively). In the torsion group, the mean Cosentino stage was 2.6, 3.3 and 3.4 at 30, 120 and 240 minute, respectively. However, these values were between 1.1 and 2.1 in the control group. The receiver operating characteristics (ROC) curves of both markers are shown in Figure 2. The D-dimer and IMA estimated torsion with reasonable accuracy [Area under the curve (AUC) = .771 (p = .003, 95% confiden- tal interval: 0.620-0.922) and AUC = 0.706 (p = .022, 95% confidental interval: 0.549-0.863), respectively. Sensitivity, specificity and predictive values of D-dim- er and IMA are shown in detail in Table 3 and Table 4. At a cut-off point of 118.9 mg/dL, the D-dimer has a sensitivity of 90.5%, specificity of 61.9%, PPV of 70.4% and NPV of 86.7%. The IMA was 81% sensitive and 52.4% specific in the diagnosis of TT at a cut-off point of 35.5 mg/dL. DISCUSSION Viability and preservation of testis in TT is dependent on the degree and the duration oftorsion. It has been shown that 360 degrees of TT does not have an effect on fertiliy, whereas, 720 degrees and above has nega- tive impacts on fertiliy. It has been stated that chances of testicular preservation in 6, 12 and 24 hours of TT is 90%, 50% and 10%, respectively.(6) Therefore, immedi- ate diagnosis and treatment of TT is required in order to preserve testis and fertility. Sensitive and specific laboratory parameters which may aid in the early diagnosis of TT are limited. Suspicion of TT generally ends up with surgical exploration of the testis. Sensitive, fast and practical biochemical mark- ers are of importance as they would serve as adjunct to diagnosis and increase efficiency of TT management. The D-dimer and IMA assays are fast and practical lab- oratory tests that are routinely available in an outpatient setting via quantitative assays. Therefore, in the pres- ent animal model, we studied the D-dimer and IMA markers. Both markers are mainly elevated in ischem- ic-hypoxic and thromboembolic conditions. Because of fact that torsion is an ischemic condition and it creates thrombotic formations in arterial and venous vascula- ture, it is expected that the D-dimer and IMA levels in- crease in ovarian and testicular torsion. IMA measurement has recently been proposed as a sen- sitive marker for the diagnosis of myocardial ischae- mia. Clinical usage of IMA in pathological conditions has grown in number, with additional application in deep venous thrombosis, pulmonary thromboembo- lism, lower limb ischemia, cerebrovascular events and Pediatric Urology 569 Early diagnosis of testicular torsion-Sarac et al. Table 3. The predictive characteristics of D-dimer at different cut-off values D-Dimer (mg/dL) Sensitivity Specifity PPV NPV 118.9 90.5% 61.9% 70.4% 86.7% 123.3 81% 66.7% 70.8% 77.8% 131.4 66.7% 81% 77.8% 70.8% Abbreviations: PPV: positive predictive value; NPV: negative predictive value Table 4. The predictive characteristics of IMA at different cut-off values IMA (mg/dL) Sensitivity Specifity PPV NPV 35.5 81% 52.4% 63% 73.3% 35.63 76.2% 57.1% 64% 70.6% 36.84 66.7% 66.7% 66.75% 66.7% Figure 2. The receiver operating characteristics (ROC) curves of D-dimer and IMA disseminated intravascular coagulation. Also, IMA is regarded as a marker of oxidative stress related to is- chaemia reperfusion in any organ, because it is found elevated in various clinical entities associated with oxi- dative stress such as systemic sclerosis, type-2 diabetes and polycystic ovary syndrome.(17) In an animal torsion model study by Mentese et al., detorsion was performed 4 hours later and testicular tis- sues were histopathologically examined 2 hours and 2 weeks after. IMA values were found to be elevated in early and late stages. The authors stated that IMA val- ues were valuable in evaluation of acute and long-term testicular injury and evaluation of fertility capacity. (11) In contrary to our study, the samples were obtained af- ter TT, thus, the effect of reperfusion on histopatholog- ical results wasinevitable. In our study, we have inves- tigated markers which can aid in early diagnosis of TT. Ischemia was performed but detorsion was not applied and effects of reperfusion was not investigated. In an experimental ovarian ischemia/reperfusion (I/R) model, IMA values were found to be higher when compared with the control group and also, positive correlation between IMA values and histopathologic results were detected in the I/R group.(12) In an experimental testicular torsion, it was shown that an increase of D-dimer level could be detected in the blood of rats within 4 hours.(13) Other experimental studies showed that D-dimer started to increase in min- utes after the onset of ischemia and reached its highest value in 6-12 hours.(14-15) All these results suggest that D-dimer can be a potential valuable marker in the early diagnosis of TT. In the present study, the predictive characteristics of the both markers (D-dimer and IMA) were satisfactory (AUC = 0.771 and AUC = 0.706, re- spectively; these results can be interpreted as reasona- ble accuracy). In the patients who had ovarian torsion, D-dimer sensitivity was detected to be 71.4% and speci- fity was detected to be 85% in ROC curve analysis. (14) Other than IMA and D-Dimer, some new biomarkers have been proposed in early diagnosis of TT. In a ran- domized, controlled, experimental study, Turedi et al studied plasma SCUBE1 (a novel marker of platelet ac- tivation) protein and they proposed that its measurement may have diagnostic, therapeutic or prognostic value in TT. (7) In a clinical study, Gunes et al investigated some hematological parameters (neutrophil / lympho- cyte ratio; NLR, platelet/lymphocyte ratio (PLR), mean platelet volume (MPV), and platelet) and they claimed that NLR may be used as a predictive factor for testicu- lar viability following TT. (3) Peretti et al. proposed that lower MPV value in "early-presenting" patients with TT playsa role in predicting testis viability.(18) Gul et al. reported that caspase-3 immunoreactivity increases in the torsion group and that melatonin and melatonin plus pulsed magnetic field (PMF) treatment reduces the rate of immuno-reactivity.(19) Despite these promising results, there is a need for further studies to routinely use these markers in clinical practice. Conducted studies have generally focused on the is- chemia/reperfusion injury and approaches to treatment. (20) Postpubertal rats were used in almost all of them. We investigated the ischemia markers on pre-puber- tal rats. However, our study hadsome limitations. The major limitation was the relatively small sample size; thus, large-scale randomized experimental and clinical trials are encouraged to be designed, so that the above conclusions can be verified with an increased statistical power. Other biochemical markers were not studied in our study and this can be cited as another limitation. CONCLUSIONS On the basis of the findings ofthis experimental study, serum D-dimer and IMA levels are significantly high- er in rats with TT compared to the control group. The elevated serum D-dimer and IMA levels seem to have a potential role as a serum marker in the early diagno- sis of TT. Future investigations about biomarkers for the early diagnosis of TT should be the focus of further clinical studies. ACKNOWLEDGEMENT We are thankful to the Academic Studies Commitee of Haseki Training and Research Hospital for providing financial support for the expenses (including IMA ELI- SA kit and D-Dimer ELISA kit) associated with this study. CONFLICTS OF INTEREST The authors report no conflict of interest. REFERENCES 1. Da Justa DG, Granberg CF, Villanueva C, Baker LA. Contemporary Review of Testicular Torsion: New Concepts, Emerging Technologies and Potential Therapeutics. 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