Comparison of Complete Blood Count Parameters using EDTA, Sodium Citrate, and Heparin Anticoagulants

Anak Agung Made Sedana Putra¹, Yetti Hernaningsih²*

¹Student of Specialist, Clinical Pathology Study Program, Faculty of Medicine, Universitas Airlangga.

²Department of Clinical Pathology, Faculty of Medicine, Universitas Airlangga,

Dr. Soetomo General Academic Hospital, Surabaya, Indonesia.

*Corresponding Author E-mail: yetti-h@fk.unair.ac.id

ABSTRACT:

A complete blood count is a routine blood examination performed in laboratories. EDTA is the standard anticoagulant used in complete blood count examination. The use of ethylenamineintetraacetic acid (EDTA) may cause pseudo thrombocytopenia. The alternative to EDTA is sodium citrate and heparin anticoagulants. This study aimed to compare the results of complete blood count parameters using EDTA, sodium citrate, and heparin anticoagulants for the necessity of health service in the laboratory. The design of this study was a cross-sectional observational analytic done from October 2020 until November 2020. The participants of this study came from 62 adults who look healthy recruited in Medical Check-up Department. The sample was collected using K₂- EDTA, 3.2% sodium citrate, and heparin tubes with volumes of 3ml, 3ml, and 4ml respectively. The sample was then examined using Sysmex XN 1000 hematology analyzer. There were no significant differences between the result of hemoglobin (HBG), red blood cells (RBC), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cells distribution width (RDW), and white blood cells (WBC) parameters. The result of platetelet (PLT) was significantly different between the three tubes. The results of platelet examination with sodium citrate and heparin tubes were lower than those obtained with the EDTA tube. EDTA is still the best choice for a complete blood count, but sodium citrate and heparin anticoagulants can be used as alternatives for testing.

KEYWORDS: Complete blood count, EDTA, sodium citrate, heparin, health service.

INTRODUCTION:

The clinical laboratory is an inseparable part of health services because it occupies an important position in the in vitro diagnosis. This is reasonable because laboratory tests are required in screening, diagnosis, disease monitoring, and treatment monitoring. Given the importance of laboratory examinations, each laboratory is required to provide precise, fast, and accurate results for their health service.¹

Complete blood count examination is a routine test performed in laboratories. The parameter of complete blood count includes hemoglobin, red blood cell count (RBC), and erythrocyte index including mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), hematocrit (HCT), white blood cell count (WBC), platelet count (PLT), erythrocyte index (RBC).¹

The laboratory processes consist of 3 stages, namely pre-analytic, analytic and post-analytic. The pre-analytical is the initial preparation stage, where this stage greatly determines the quality of the samples that will be used and affects the next work process. The pre-analytic stage includes the specimen collection process, in which the use of the right tube containing anticoagulant for blood samples is one of the most important pre-analytic factors.²

Anticoagulants are substances that prevent blood clotting by inhibiting the coagulant factors for binding to calcium, which results in inhibiting the formation of thrombin, which is needed to convert fibrinogen to fibrin in the clotting process. If the test requires blood or plasma, the specimen should be collected in a tube containing an anticoagulant. Anticoagulant specimens should be mixed immediately after specimen collection to prevent micro clot formation. There are various types of anticoagulants used in hematological examinations, including EDTA (ethylenamineintetraacetic acid), heparin, and citrate. EDTA is used in several hematological tests because of its ability to retain the cellular components of blood cells. Di-potassium and tri-potassium (K₂ and K₃) EDTA are the standard anticoagulants recommended for routine complete blood count tests. EDTA is not recommended for the examination of electrolytes that depend on iron ions such as calcium, magnesium, iron, alkaline phosphate, creatinine kinase.³

Complete blood count with an EDTA tube can trigger EDTA-induced thrombocytopenia (pseudo thrombocytopenia). Pseudo thrombocytopenia can be found in 0.1-2% of inpatients and 15-17% of outpatients with isolated thrombocytopenia. Unrecognized pseudo thrombocytopenia can lead to unnecessary additional examinations, transfusions and prevent patients from receiving surgery. This condition can cause additional costs and inconvenience to the patient.⁴

Some cases report that EDTA-induced thrombocytopenia can be prevented by using other anticoagulants such as sodium citrate and heparin.⁴ Sodium citrate is an anticoagulant that is often used for coagulation physiological examination. Sodium citrate is used for coagulation physiological examination because it can maintain the labile factor coagulation. Coagulation with sodium citrate requires the correct ratio between blood and anticoagulants, therefore the tube should be filled properly. Sodium citrate works by binding to extracellular calcium (Ca²⁺). Calcium has an important role in the regulation of platelet function. Calcium plays a role in platelet remodeling, secretion, and aggregation. Increased calcium levels are important in platelet activation. Increased levels of calcium trigger platelet activation, such as remodelling of cytoskeleton actin which is required in platelet shape changes, and externalization of αIIbβ3 receptors which are important in platelet aggregation, therefore, in the case of calcium bound by sodium citrate, some of the roles that occur in platelet activation can be inhibited, resulting in a decrease platelet aggregation.³ Heparin, a polysulfuric mucoitin acid, is an effective anticoagulant in small amounts without significant effect in various tests. Heparin works by accelerating the action of antithrombin III, neutralizing thrombin, and preventing fibrin formation.¹

This study aimed to compare the results of complete blood counts using the anticoagulant EDTA, sodium citrate, and heparin, also determine the agreement of sodium citrate and heparin anticoagulants to EDTA for analysing complete blood count.

MATERIALS AND METHODS:

Sample Collection:

Participants were recruited by consecutive sampling between October-November 2020. The participants were taken from adults look healthy, aged >18 years who underwent a medical check-up examination. All participants have understood and signed the informed consent. This research has obtained ethical clearance from the Health Research Ethics Committee, Dr. Soetomo Hospital with number 0083/KEPK/X/2020. Samples were collected in three tubes with a volume of 3mL for K₂ EDTA tubes, 3mL for 3.2% sodium citrate tubes, and 4mL for heparin tubes.

Sample Processing:

Complete blood count was performed using Sysmex XN 1000 hematology analyzer. The complete blood count parameters were 18, including hemoglobin (HGB), hematocrit (HCT), red blood cells (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), red cell distribution width (RDW), white blood cells (WBC), neutrophils (%), lymphocytes (%), monocytes (%), eosinophils (%), basophils (%), platelets (PLT), platelet distribution width (PDW), mean platelet volume (MPV), platelet large cell ratio (P-LCR) and plateletcrit (PCT). Especially sample with sodium citrate anticoagulant will be corrected by multiplying with constanta 1.1 to overcome the dilution effect which is resulted by sodium citrate itself.

Statistical Analysis:

Statistical analysis using SPSS version 23.0. A normality test was performed using Shapiro Wilk. The comparative test was carried out with the Kruskal Wallis test for data that has abnormal distribution and the Annova test for data that has normal distribution. The agreement test used the Bland Altman plot for the data which was statistically different. The percentage of bias between the two tubes is calculated according to the following formula:

%mean differences: [(Mean EDTA-Mean sodium citrate or heparin/Mean EDTA) X100%]. The results of calculating the percentage mean differences of all the parameters studied were compared with the percentage desirable bias that had been determined based on biological variation⁵, except for PDW, P-LCR, and PCT because the percentage desirable bias for these two parameters had not been determined

RESULTS AND DISCUSSION:

The total participants obtained in this study were 62 adults who look healthy who visited the Medical Check-up Department. The proportion of the male was 21 and women were 41 participants with an age range of 18-58 years old as in the following table (Table 1):

Comparison of the results of statistical analysis between the three anticoagulants were shown in Table 2. From the results, there were no statistical differences between EDTA, sodium citrate, and heparin for the parameters of hemoglobin, hematocrit, RBC, MCV, MCH, RDW, WBC, eosinophils, basophils, neutrophils, lymphocytes, and monocytes. Statistical differences were obtained on examinations for parameters PLT, PDW, MPV, P-LCR, and PCT.

Table 1. Characteristics of Participants

Characteristics	Result
Sex - n (%)
Male	21 (33,9%)
Female	41 (66,1%)
Age – years old
Minimum-maximum	21 – 57
Median	35
Mean ± SD	35.93 ± 9.21

Table 2. Complete blood count using EDTA, sodium citrate, and heparin anticoagulants.

	Mean (SD)			Mean % Differences (p-value)		Desirable Bias%
Parameter	EDTA	Sodium Citrate	Heparin	EDTA-Sodium Citrate	EDTA-Heparin	Desirable Bias%
Haemoglobin (g/dL)	13.5 (1,6)	13.3 (1,5)	13.5 (1,5)	1.2 (0.529)	0.07 (0.977)	1.8
Hematocrit (%)	40.24 (3.88)	40.10 (3.78)	40.17 (3.79)	-0.32 (0.842)	0.14 (0.925)	1.7
RBC (x10⁶/µL)	4.95 (0.40)	4.87 (0.42)	4.95 (0.42)	1.4 (0.356)	0.00 (0.998)	1.7
MCV (fL)	81.37 (4.81)	82.29 (4.96)	81.24 (4.80)	-1.13 (0.290)	0.16 (0.881)	1.2
MCH (pg)	27.28 (2.22)	27.46 (2.30)	27.30 (2.20)	-0.64 (0.660)	-0.04 (0.227)	1.4
MCHC (g/dL)	33.50 (1.15)	33.15 (1.16)	33.57 (1.16)	1.01 (0.096)	-0.20 (0.734)	0.8
RDW-CV (%)	13.30 (1.11)	13.39 (1.09)	13.32 (1.13)	0,60 (0.669)	-0,07 (0.955)	1.7
WBC (x10³/µL)	8.05 (1.73)	8.06 (1.76)	8.10 (1.78)	-0.12 (0.963)	-0.62 (0.883)	5.6
Eosinophil%	2.26 (1.9)	2.26 (1.87)	2.53 (2.67)	0.00 (1.00)	-12.3 (0.474)	19.8
Basophil%	0.50 (0.24)	0.51 (0.23)	0.66 (1.19)	-2 (0.920)	-32 (0.206)	15.4
Neutrophil%	59.7 (8.5)	59.93 (8.37)	59.44 (8.94)	-0,23 (0.926)	0,56 (0.822)	9.1
Limphocyte%	30.15 (7.27)	29.92 (7.31)	30.41 (7.54)	0.76 (0.861)	-0.86 (0.751)	7.4
Monocyte%	7.3 (1.95)	7.34 (1.77)	7.10 (1.82)	-1.09 (0.801)	2.8 (0.844)	13.2
Platelet (x10³/µL)	323.95 (68.21)	274.04 (56.17)	245.54 (64.85)	15.4 (0,00)*	24.2 (0.00)*	5.9
PDW	11.23 (1.73)	9.56 (1,19)	10.56 (1.49)	14.9 (0.00)*	0.67 (0.013)*	NA
MPV (fL)	10.10 (0.82)	9.30 (0,70)	9.92 (0.71)	7.9 (0.00)*	5.96 (0.17)	2.3
P-LCR	25.25 (6.77)	19.37 (5.34)	23.98 (5.48)	23.28 (0.00)*	5.02 (0.23)	NA
PCT (%)	0.32 (0.06)	0.23 (0.05)	0.24 (0.06)	28.12 (0.00)*	25 (0.00)*	NA

RBC = red blood cells; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; RDW-CV = red cells distribution width; PDW = platelet distribution width; MPV = mean platelet value; P-LCR = platelet large cells ratio; PCT = plateletcrit; NA : not available; * = statistically significant, p value <0.05. All parameter were analyzed using Anova test.

The anticoagulants of choice for complete blood counts are dipotassium EDTA and tripotassium EDTA.⁶ The International Council For Standardization In Hematology (ICSH) recommends the use of EDTA as the anticoagulant of choice for hematological examinations.⁷

The results of complete blood count for the parameters of HGB, RBC, HCT, MCV, MCH, WBC, eosinophils, basophils, neutrophils, lymphocytes, and monocytes with sodium citrate and heparin anticoagulants were not significantly different from the results with EDTA. These results are consistent with studies conducted by Perrotta et al. (1998) who compared complete blood counts with EDTA anticoagulants and sodium citrate and Sakoguchi et al. who compared the results of EDTA complete blood counts with heparin from blood gas samples.^8,9

The results of complete blood count for the parameters of PLT, PDW, MPV, P-LCR, and PCT with sodium citrate and heparin anticoagulants were significantly different from the results of tests with EDTA. The results of PLT on sodium citrate and heparin anticoagulants were significantly lower than the results of PLT with EDTA. This can be due to platelet aggregation and adhesion that is not visible to the appliance. McShine et al. (1990) found that aggregate formation on sodium citrate is evidence of platelet activation which in turn depends on several factors including the calcium-binding efficiency of the anticoagulant. EDTA is a more efficient Ca²⁺ chelating agent than citrate; in terms of molarity, approximately five times as much citrate is required to prevent coagulation. Although theoretically more citrate than is required in citrate blood to prevent coagulation activation, McShine et al. suspected that a small population of platelets may be activated to form microaggregates. Researchers suspected that the formation of small amounts of platelet micro-aggregates is an inevitable event that begins with blood collection; This phenomenon occurs more frequently in citrate than in EDTA, most likely due to the lower affinity for the lower divalent cations.¹⁰^,11

The results of the suitability test using the Bland Altman plot for the parameters of PLT, PDW, MPV, P-LCR, and PCT are shown in Figure 1.

Figure 1. The Bland Altman plot for EDTA-sodium citrate and EDTA-heparin for statistically different parameters with a limit of agreement (LoA) of 95%

McShine et al also found lower MPV citrate values than EDTA. These results illustrate the ability of the anticoagulant to bind calcium upon initial contact of a blood sample with a foreign surface.¹⁰^{,11,12,13,14,15,16,17}

Lower PLT results on heparin than EDTA were also found in the study by Shojania and Turnbull (1987). There are several reasons for the low yield of invitro PLT. In vitro heparin aggregates or potentiates the aggregation activity of other substances. Heparin potentiates platelet aggregation by adenosine diphosphate (ADP). Heparin can also increase the sensitivity of platelets to ADP and epinephrine in heparin plasma.¹¹^,18,19,20,21

CONCLUSION:

In summary, sodium citrate and heparin anticoagulants can be used as alternatives for health service, however, EDTA is still the best choice for complete blood counts, because because of its superiority in maintaining platelet. Especially in cases with pseudo thrombocytopenia due to EDTA, the anticoagulants of sodium citrate and heparin could replace the EDTA even though resulted lower count than that of EDTA. It is necessary to carry out further studies with larger sample size and confirmed by a blood smear.

ACKNOWLEDGEMENT:

We thank Arif Nur Muhammad Ansori for editing the manuscript.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 21.09.2021 Modified on 30.12.2021

Research J. Pharm. and Tech 2022; 15(10):4687-4691.

DOI: 10.52711/0974-360X.2022.00786