A comparison of erythrocyte sedimentation rate between blood anticoagulated with sodium citrate and EDTA among Tuberculosis suspected patients at University of Gondar Hospital, North West Ethiopia

Objective: The main aim of this study was comparison of erythrocyte sedimentation rate results of sodium citrate and ethylene diamine tetra-acetic acid anticoagulant. A comparative cross-sectional study was conducted at University of Gondar Referral Hospital, Northwest, Ethiopia. A total of 70 TB suspected study participants were recruited. From each study participants, 3ml blood was collected in to EDTA tube and 1.6ml blood was collected in to a test tube containing 0.4ml Sodium Citrate anticoagulant. Results: The mean ± SD values for ESR were 57.9 ± 41.45 in EDTA and 50.99 ± 43.5 in citrate anticoagulated blood. The mean difference of ESR values between EDTA and citrated blood was 6.91 ± 13.66 mm/hr with a significant difference. The mean ± SD of ESR for EDTA and citrate blood was 59.57 ± 42.31 and 54.71 ± 40.44 mm/hr for males and 53.57 ± 44.61 and 46.04 ± 41.82 mm/hr for females, respectively. This study indicated that there was a significant difference between ESR values with EDTA and tri-sodium citrate anticoagulants.


Introduction
Erythrocyte sedimentation rate (ESR) is a common inexpensive and non-specific hematology test frequently ordered in clinical medicine. Still, it remains the most widely used laboratory test for monitoring the course of infections, inflammation, some types of cancer. In addition, it serves as a general sickness index when it is used in conjunction with the patient's clinical history and physical examination (1)(2)(3).
Increased amount of the plasma proteins like fibrinogen are the major factors increasing the ESR test results by reducing the negative electrostatic force between red blood cells (RBCs) leading to an increased rate of rouleaux formation and the RBCs easily falling down within the plasma (4,5). In case of inflammatory and infectious processes, the fibrinogen concentration in the blood increases the rouleaux formation and the RBCs settle faster than normal (6,7).
The ESR test is commonly increased in some chronic infectious conditions like active tuberculosis, infective endocarditic, systemic infection, bone infections, rheumatic fever and severe skin infections.
It is also important for the assessment of severity of inflammatory bowel disease in children (8)(9)(10)(11). It also has been used as a marker of response to treatment in tuberculosis. Moreover, increased ESR can be an early predictive marker of HIV seropositive progression towards AIDS. Additionally, increased ESR can be used as an inexpensive "sickness index" in the elderly (12).
In general, the result of ESR is raised in a wide range of infectious, inflammatory, generative, malignant conditions and associated factors including anemia, pregnancy, hemoglobinopathies, hemoconcentration, and the treatment of anti-inflammatory drugs which changes the plasma proteins that increases in fibrinogen, immunoglobulin, and C-reactive protein (13 -14).
The principle of the ESR determination is based on the measurements of the sedimentations rate of aggregated erythrocytes in plasma. The International Council for Standardization in Hematology (ICSH) recommended the Westergren method as the method of choice for ESR determination. When anticoagulated blood is placed in Westergren tube in vertical column, the erythrocytes normally settle quite slowly by the influence of gravity and the distance of erythrocytes falling down in a vertical column from the plasma in within one hour (15)(16)(17).
Undiluted specimens anticoagulated with EDTA reduce the risk of pre-analytic mistakes due to a partially coagulated specimen or to small clots, an altered blood/sodium citrate (SC) ratio, and problems linked to the final volume, inherent mainly in techniques using special tubes for both specimen collection and ESR measurement (18,19). Using undiluted blood with K3EDTA is a recommended specimen for ESR determination by ICSH because it gives a more reliable result than the traditional SC (20,21). However, according to the 2011 ICSH recommendations, the reference method for the measurement of the ESR should be based on the Westergren method with modifications that use either whole blood anticoagulated with EDTA and later diluted with SC or saline (4:1) or whole blood anticoagulated with SC (4:1) in Westergren pipettes (22).
Even though various researches were conducted in relation to ESR value among different disease, no studies have conducted on comparison of ESR value between blood mixed with SC and EDTA in the study area. Even though ESR test is one of the commonest investigations carried out in the clinical hematology laboratory, there is no recognized standard control sample available for monitoring the test. The reliability and reproducibility of the results depends on the use of correct methodology. So, this study was aimed to assess the variability of the two commonly used anticoagulants.

Methods And Materials Study setting and population
A comparative cross-sectional study was conducted on a total of 70 study subjects who come to the TB clinic at University of Gondar Comprehensive Specialized Hospital (UOGCSH) from March to May 2018. UOGCSH is found in Gondar town, Amhara regional state, Northwest Ethiopia. Gondar town is located at 727 km far from Addis Ababa and 185 km far from Bahir Dar town to the northwest part of the country. The town is situated at a latitude and longitude of 12°36′N 37°28′E with an elevation of 2133 meters above sea level.
All patients who come to the TB clinic and willing to participate during the study period and whose ages 15 and above were included in the study. Whereas, patients who did not give enough amount of blood sample, and who are not willing to participate in the study, were excluded from the study.

Sample size determination
A double population mean formula was used to calculate the required sample size using mean of citrate and EDTA with a null hypothesis of, Ho: μ1 = μ2 or (μ1-μ2) = 0 and an alternative hypothesis of, HA: μ1 ≠ μ2 or (μ1-μ2) ≠ 0.
[Please see supplementary files for formula] So, this comparative study was conducted on 70 individuals.

Data collection and ESR determination
Questionnaire and check lists prepared in English language and then translated to the local Language which is Amharic were used to collect the socio-demographic and clinical data of the study participants. Patient blood was collected both in the K3EDTA tubes and the citrated tubes. The ESR of blood samples in both tubes was determined by Westergren method. For each participant, two tests of ESR determination were performed. First 5 ml of fresh venous blood was collected from each study participant and expressed into appropriately labeled EDTA and SC tubes and mixed properly. The ESR of blood samples in both tubes was measured within 2 hours in accordance with the recommendation of ICSH. We filled the Westergren tube exactly to "0"mark and place the tube in the rack for 1 hour and the ESR results were recorded in mm/h. Then results from the K3EDTA tubes were compared to those of citrated tubes.

Quality assurance
Standard operating procedures (SOPs) and manufacturers' instruction were strictly followed for all laboratory activities as much as possible. All blood collection test tubes were checked for their expiry date and laboratory results were recorded on standard report formats using participants' identification number. The data of each patient were reviewed for the presence of mislabeling and completeness.

Data analysis
EpiInfo version 3.5.4 was used for data entry and SPSS version 20 for analysis. The statistical analysis was performed using the Pearson correlation, linear regression, and paired and independent t-test to establish the relationship between the 2 methods. Paired and independent t-test at 95% confidence level were used to compare the two methods and to determine the factors affecting the agreement of the two methods, respectively. The ESR values were expressed as means ± SD and in all cases Pvalue <0.05 was considered as statistically significant.

Socio-demographic information
A total of 70 TB suspected patients were included in the study. The study participants age ranged from 15 to 82 years but the majority were in the age group of 36-50 years, 21 (30%). From the total study participants, majority 46 (65.7%) were males and 21 (30%) were aged between 36-50 years.

Mean and standard deviations for EDTA and citrated blood
The ESR value obtained from the study was higher in EDTA anticoagulant compared to citrate anticoagulant for all independent variables. The mean ± SD values for ESR were 57.9 ± 41.45 in EDTA and 50.99 ± 43.5 in citrate anticoagulated blood. The mean difference of ESR values between EDTA and citrated blood was 6.91±13.66 mm/hr with a significant difference. The mean ± SD of ESR for EDTA and citrate blood was 59.57±42.31 and 54.71±40.44 mm/hr for males and 53.57±44.61 and 46.04 ± 41.82 mm/hr for females, respectively (Table2).
The paired t-test analysis showed that there was a statistically significant difference between the mean ± SD ESR values of the two anticoagulated blood. The mean difference of ESR values between EDTA and citrated blood was 6.91 ± 13.66 mm/hr with a significant difference (t: 4.24; P <0.001; 95%CI: 3.66-10.17).

Discussion
The ESR value obtained from this comparison study showed that the mean value of EDTA anticoagulated blood (57.90 mm/hr) was greater than that of citrated blood (50.99 mm/hr) by 6.91 mm/hr. It may however be that EDTA increases rouleaux formation than sodium citrate leading to increase ESR in EDTA blood or it might be a difference in viscosity, where citrated blood may be more viscous than EDTA blood resulting to lower values of ESR in citrated blood. This observation coincides with the result reported from Tehran University, Iran, which stated that there was significant difference between the results of ESR test by using two anticoagulants (24).
Our study was also similar with that of a study conducted in India at Yenepoya University hospital patients. They found that the values of ESR using EDTA showed 4-6 mm less than that of sodium citrate. Finally they conclude and recommended that ESR can be performed using EDTA blood instead of sodium citrate since this utilizes only limited amount of blood for the procedure (25).
The result of our study was also concurrent when compared to the study conducted in Nigeria, and its final finding was stated that mean ± SD ESR values of the blood by EDTA anticoagulant is higher than mean ± SD ESR values of the blood by citrate. When compared to our finding, its result was within the normal range of ESR, the reason for this difference were the study participants as it was conducted from healthy individuals and our study was conducted on TB suspected patients which is one consequence for the increment of ESR value. In addition to this, the mean ± SD ESR values of males were less than that of females which contradicts this finding. This may due to the fact that most of our male study participants were old aged and had a history of tuberculosis (23). However, the result of our study was found to be in contrary with an earlier report conducted in India, which stated that the ESR measurement values of Citrated blood was greater by 4-6 mm/hr than EDTA (7).
A study carried out in Philippines got a mean ESR value of 36.7 and 43.09 for EDTA and sodium citrate respectively, which is not coincided with our finding, i.e., 57.90 and 50.99 for EDTA and Citrate respectively, this is may be due to some physiological and genetic differences between white and black men (26).

Conclusion
In this study, the mean ESR value is higher in EDTA anticoagulated blood than mean ESR value of trisodium citrate anticoagulated blood. Measured value of ESR for male is greater than female. Patients of age >50 years have larger mean ESR value than other age groups. Patients having a previous history of TB and those who had confirmed TB shows greater mean ESR value than the counter parts.
Therefore, it is better to use only one of the two anticoagulants consistently as there is a difference between them.

Limitations
The study was conducted at TB suspected and patients only and the study area was also limited in one district and therefore the result of the research may not be representative for the whole population. Ethical Review Committee. Permission letter was obtained from the hospital director to conduct the study. The objectives of the research were explained to the study participants and written informed consent was taken. To ensure confidentiality of data, participants were identified using codes.

Consent for publication
Not applicable.

Availability of data and materials
All the data on which the conclusions of this manuscript are drawn are available in the corresponding author. So that any who needs the data can get it upon reasonable request.

Supplementary Files
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