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 



 

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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 pre-

analytical, analytical and post analytical 

phase. Error which is occur in the pre-

analytical 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 



 

 

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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: 

  



 

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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 (20-

25℃). 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)



 

 

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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. 

 



 

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CONFLICT OF INTEREST 

There are no conflicts of interest. 

 

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