Impact of Common Anticoagulants on Complete Blood Count Parameters Among Humans

Abstract Introduction Among the most frequently used anticoagulants in hematological testing are tetra-acetic acid (EDTA), sodium citrate, and sodium heparin. However, there is a noticeable gap in literature concerning the effects of these anticoagulants on hematological parameters specifically in humans. This study aims to assess the effectiveness of EDTA, sodium citrate, and sodium heparin for conducting complete blood count (CBC). Methods This cross-sectional study conducted at Smart Health Tower from January to April 2024 involved 250 participants who underwent CBC using K2EDTA, sodium citrate, and sodium heparin. The acquired data were analyzed using SPSS, with a significance level of p < 0.05, employing Intra-class correlation coefficient and one-way ANOVA to assess consistency and agreement among anticoagulants. Results A total of 250 participants, with 138(55.2%) males and 112(44.8%) females, underwent CBC testing with di potassium EDTA(K2EDTA), sodium citrate, and sodium heparin. Comparing K2EDTA with sodium heparin showed comparable values in 14 out of 23(60.87%) CBC parameters. Using K2EDTA as the standard, citrate showed perfect or substantial agreement in assessing 8 out of 23 CBC parameters (34.78%). Regarding the comparison of anticoagulants to K2EDTA to determine their agreement levels while sodium heparin was accurate and precise in 13(56.52%) parameters. Conclusion Citrate was found to be a less reliable anticoagulant for CBC estimation compared to K2EDTA, potentially leading to inaccurate readings. On the other hand, sodium heparin showed comparable performance to K2EDTA, making it a suitable alternative under specific conditions. Introduction The Complete Blood Count (CBC) is a widely requested blood test by clinicians, assessing the total quantities and characteristics of cellular constituents within the bloodstream. The CBC parameters include red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs). This comprehensive assessment includes determining the total and differential count of WBCs, also measuring RBC count, hemoglobin (HGB) levels, and hematocrit (HCT), as well as their indices such as mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW). Additionally, CBC evaluates platelet (PLT) count indices [1,2]. A CBC serves as an important diagnostic tool for assessing human health, detecting congenital abnormalities, and identifying functional changes due to various pathological factors [3]. Its findings can reveal various conditions such as infections with elevated WBC counts, leukemia with abnormal WBC counts, anemia with low HGB levels, and liver cirrhosis with reduced PLT counts. Recent studies suggest that specific combinations of CBC components, along with derived secondary results, can predict risks of different diseases like cardiovascular disease, cancer, type 2 diabetes, and metabolic syndrome [1,2]. It's widely recognized that collection and sampling of blood, laboratory techniques and storage conditions, and the choice of anticoagulant can substantially impact the outcomes derived from hematological analysis, especially CBC results [4]. Among the various anticoagulants used for both sample collection and routine laboratory analysis, the most commonly utilized ones in hematology are ethylene diamine tetra acetic acid (EDTA), citric acid salts, sodium and lithium oxalates, and heparin [5,6]. The National Committee for Clinical Laboratory Standards has suggested using EDTA for CBC due to its ability to preserve cell structure [7]. However, limited evidence exists on the effects of other anticoagulants on CBC parameters among animal species. Among humans, heparin is typically avoided for blood smears and WBC counts due to staining and clotting issues, respectively. Conversely, EDTA is considered unsuitable for erythrocyte osmotic fragility assessment and may cause cell damage if overused [8]. The majority of studies documented in existing literature have focused on evaluating the impacts of different anticoagulants on CBC results or its specific components across diverse animal species. The current study aims to estimate variations in CBC parameters using different anticoagulants, employing dipotassium EDTA(K2EDTA), sodium citrate, and sodium heparin, among humans to evaluate their effectiveness. Methods Study Design, population, and criteria This cross-sectional laboratory-based study was conducted at Smart Health Tower from January to April 2024. Prior to participation, all individuals were thoroughly briefed about the study and required to provide informed written consent. The study included a total of 250 participants, all of whom underwent complete blood count tests utilizing K2EDTA, sodium citrate, and sodium heparin as anticoagulants. The study population consisted of patients attending Smart Health Tower, representing both genders without any gender bias. Inclusion was restricted to those who had visited the facility, while individuals or their guardians ( in case of minors) who declined to provide consent were excluded from the study. Determination of the sample size The effective sample size was determined using G*Power statistic 3.1.9.7, employing linear multiple regression as the statistical test with a two-tailed approach. With an effective sample size of 0.35, an α error probability of 0.01, and a statistical power of 0.99, along with a predictor value of 1, the minimum required sample size was 158. Therefore, a sample size of 250 was utilized for the comparison in CBC parameters between these three different anticoagulants. Sample collection and statistical analysis Trained health workers collected blood samples from participants using sterile syringes and needles, drawing 5 mL from either the median cubital or prominent forearm vein. The samples were distributed as follows: 1.8 mL into sodium citrate tubes and 1.6 mL into K2EDTA and sodium heparin tubes. After gentle mixing, complete blood counts (CBC) were analyzed with the Medonic M51 automated hematology analyzer within 3 to 6 hours post-collection. Tube characteristics are detailed in Table 1. Various hematological parameters were assessed, including WBC, percentages of neutrophils, lymphocytes, monocytes, eosinophils, basophils, as well as RBC, HCT, HGB, MCV, MCHC, RDW-SD, PLT, MPV, PDW, PCT, and PLCR. Participant demographics, such as age and gender, were also recorded. Data were initially processed in Microsoft Excel 2019 for accuracy and completeness before being transferred to SPSS version 25.0 and MedCalc version 20 for statistical analysis. Intra-class correlation coefficient (ICC) analysis was conducted to evaluate consistency among the three anticoagulants, with interpretations as follows: <0.50 for poor consistency, 0.50-0.75 for moderate, 0.75-0.90 for good, and >0.90 for excellent consistency. A p-value of <0.05 was considered significant. One-way ANOVA assessed variations in CBC parameters among samples collected in K2EDTA, sodium citrate, and sodium heparin tubes. Additionally, the concordance correlation coefficient (CCC) was used to evaluate agreement, with K2EDTA as the standard, and interpreted as follows: ≥0.99 for almost perfect agreement, 0.95-0.99 for significant agreement, 0.90-0.95 for moderate agreement, and <0.90 for poor agreement [9]. Table 1. Properties of Laboratory Test Tubes Utilized for CBC Evaluation. Tube details EDTA Heparin Citrate Type of tube K2EDTA Sodium (vacuum blood collection tube) PT Tube (Sodium citrate) Dimension 13 x 75 mm 13 x 75 mm 13 x 75 mm Storage 5- 25°C 5-25°C 5-25°C Expiration date 31-3-2025 24-11-2027 19-12-2025 Tube capacity (volume) 5 ml 5 ml 5ml Required volume 1.5-2 ml 1.5-2ml 1.8ml Tube material Plastic glass glass Manufacturer Vacutest kima sri MR+ MR+ Origin/country Italy China China Anticoagulant concentration 5.4 mg 18iu 3.2% Results Among the 250 participants involved, 138 (55.2%) were male, and 112 (44.8%) were female. The participants had an average age of 41.20 ± 16.51 years (5-91). Consistency in CBC results using sodium heparin, K2EDTA, and sodium citrate indicated excellent consistency in the determination of WBC, %Neu, %Lymph, Neu, Lymph, RBC, HGB, HCT, MCV, MCH, MCHC, RDW-SD, MPV, PDW, and PLCR among these anticoagulants with ICC >0.90 (Table 2).  Table 2. CBC results consistency using K2EDTA, Sodium heparin, Sodium citrate. CBC parameters Intra-class correlation coefficient Confidence interval 95% Lower Upper WBC 0.991 0.972 0.996 %Neu 0.961 0.880 0.981 %Lymph 0.987 0.984 0.990 %Mon 0.494 0.038 0.717 %Eos 0.869 0.838 0.895 %Bas 0.733 0.636 0.801 Neu 0.988 0.961 0.994 Lymph 0.987 0.973 0.993 Mon 0.612 0.120 0.802 Eos 0.182 0.038 0.367 Bas 0.803 0.719 0.858 RBC 0.922 0.328 0.976 HGB 0.923 0.321 0.976 HCT 0.902 0.449 0.964 MCV 0.998 0.994 0.999 MCH 0.996 0.992 0.998 MCHC 0.963 0.921 0.979 RDW-SD 0.924 0.901 0.941 PLT 0.536 0.276 0.689 MPV 0.915 0.843 0.948 PDW 0.921 0.880 0.945 PCT 0.563 0.104 0.763 PLCR 0.930 0.856 0.960 Regarding variation in CBC parameters using K2EDTA, sodium citrate, and sodium heparin, no statistically significant variation was found in the median %Lymph, Eos, MCV, and MCH among these three different anticoagulants (Table 3).   Table 3. Variations among CBC parameters using different anticoagulants. CBC parameters Sodium Heparin Median (Min-Max) CitrateMedian (Min-Max) K2EDTAMedian (Min-Max) P-value WBC 7.54(2.52-26.26) 7.21(2.26-23.85) 7.51(2.54-26.10) 0.046 %Neu 61.15(37.2-92.6) 56.70(37.60-90.90) 56.30(30.70-91.80) <0.001 %Lymph 33.2(3.8-50.30) 33.55(4-50.50) 33.45(3.90-52.60) 0.718 %Mon 1.9(0.0-12.20) 6(0.20-13) 6.45(0.80-14.10) <0.001 %Eos 2.5(0.10-22.50) 2.50(0.20-24.30) 6.45(0.80-14.10) <0.001 %Bas 0.65(0.20-3.10) 0.50(0.10-2.20) 0.50(0.10-1.50) <0.001 Neu 4.92(0.94-24.31) 4.40(0.99-21.68) 4.65(0.78-23.96) 0.015 Lymph 2.41(0.39-5.76) 2.31(0.37-5.40) 2.49(0.45-5.99) 0.049 Mon 0.15(0.00-0.93) 0.43(0.01-1.06) 0.49(0.05-1.17) <0.001 Eos 0.20(0.01-2.08) 0.18(0.01-2.19) 0.16(0.01-2.55) 0.730 Bas 0.05(0.01-0.23) 0.04(0.01-0.17) 0.04(0.01-0.13) <0.001 RBC 5.12(2.47-7.65) 4.62(2.21-6.35) 5.13(2.48-7.05) <0.001 HGB 14.2(6.90-21.30) 12.6(6.20-16.9) 14.10(6.90-18.20) <0.001 HCT 43.15(21.4-64.8) 38.90(19-51.20) 43.45(3.72-54.60) <0.001 MCV 85.45(56.90-108.5) 85.15(56.8-108.4) 85.9(57.3-108.6) 0.534 MCH 28.4(18.10-35.70) 28.10(18-37.5) 28.25(18.10-36.40) 0.425 MCHC 33(30.50-37.60) 32.70(30.4-39.10) 32.60(30-38.40) <0.001 RDW-SD 43.5(34.70-63.10) 43.40(34.60-64.00) 44.10(35.30-82.20) 0.016 PLT 159(32-424) 176(21-1584) 250(86-482) <0.001 MPV 9.30(6.90-12.10) 8.80(4.40-11.80) 9.10(7.10-13.00) <0.001 PDW 11.85(7.30-21.10) 11.10(2.60-20.00) 11.60(8.10-23.60) <0.001 PCT 0.15(0.03-0.34) 0.15(0.02-0.70) 0.22(0.09-0.37) <0.001 PLCR 31.75(15.30-51.10) 28.15(5.20-48.80) 30.40(15.40-57.90) <0.001 Regarding variation in estimation of CBC parameters using the results of two anticoagulated blood such as K2EDTA-sodium citrate, K2EDTA-sodium heparin, sodium citrate-sodium heparin, the results of the comparison of K2EDTA-sodium citrate indicated comparable results in median %Neu, %Lymph, %Eos, Neu, Bas, MCV, MCH, and MCHC with a p-value of ≥0.05. Comparison of K2EDTA-sodium heparin results indicated comparable results in median WBC, %Lymph, %Eos, Neu, Lymph, RBC, HGB, HCT, MCV, MCH, RDW-SD, MPV, PDW, and PLCR with a p-value of ≥0.05. In comparing the results of CBC between sodium citrate-sodium heparin, the result indicated a nonsignificant difference in median WBC, %Lymph, %Eos, Lymph, MCV, MCH, RDW-SD, and PCT (Table 4). Table 4. Variation in estimation of CBC using two anticoagulants’ results. CBC parameters Sodium CitrateMedian (Min-Max) Sodium HeparinMedian (Min-Max) P-value K2EDTAMedian (Min-Max) Sodium HeparinMedian (Min-Max) P-value K2EDTAMedian (Min-Max) Sodium CitrateMedian (Min-Max) P-value WBC 7.21(2.26-23.85) 7.54(2.52-26.26) 0.121 7.51(2.54-26.10) 7.54(2.52-26.26) 0.941 7.51(2.54-26.10) 7.21(2.26-23.85 0.05 %Neu 56.70(37.60-90.90) 61.15(37.2-92.6) <0.001 56.30(30.70-91.80) 61.15(37.2-92.6) <0.001 56.30(30.70-91.80) 56.70(37.60-90.90) 0.788 %Lymph 33.55(4-50.50) 33.2(3.8-50.30) 0.922 33.45(3.90-52.60) 33.2(3.8-50.30) 0.695 33.45(3.90-52.60) 33.55(4-50.50) 0.903 %Mon 6(0.20-13) 1.9(0.0-12.20) <0.001 6.45(0.80-14.10) 1.9(0.0-12.20) <0.001 6.45(0.80-14.10) 6(0.20-13) 0.003 %Eos 2.50(0.20-24.30) 2.5(0.10-22.50) 0.801 6.45(0.80-14.10) 2.5(0.10-22.50) 0.994 6.45(0.80-14.10) 2.50(0.20-24.30) 0.740 %Bas 0.50(0.10-2.20) 0.65(0.20-3.10) <0.001 0.50(0.10-1.50) 0.65(0.20-3.10) <0.001 0.50(0.10-1.50) 0.50(0.10-2.20) 0.07 Neu 4.92(0.94-24.31) 4.92(0.94-24.31) 0.011 4.65(0.78-23.96) 4.92(0.94-24.31) 0.288 4.65(0.78-23.96) 4.92(0.94-24.31) 0.345 Lymph 2.41(0.39-5.76) 2.41(0.39-5.76) 0.282 2.49(0.45-5.99) 2.41(0.39-5.76) 0.633 2.49(0.45-5.99) 2.41(0.39-5.76) 0.040 Mon 0.15(0.00-0.93) 0.15(0.00-0.93) <0.001 0.49(0.05-1.17) 0.15(0.00-0.93) <0.001 0.49(0.05-1.17) 0.15(0.00-0.93) <0.001 Eos 0.20(0.01-2.08) 0.20(0.01-2.08) <0.001 0.16(0.01-2.55) 0.20(0.01-2.08) <0.001 0.16(0.01-2.55) 0.20(0.01-2.08) <0.001 Bas 0.05(0.01-0.23) 0.05(0.01-0.23) <0.001 0.04(0.01-0.13) 0.05(0.01-0.23) <0.001 0.04(0.01-0.13) 0.05(0.01-0.23) 0.547 RBC 5.12(2.47-7.65) 5.12(2.47-7.65) <0.001 5.13(2.48-7.05) 5.12(2.47-7.65) 0.993 5.13(2.48-7.05) 5.12(2.47-7.65) <0.001 HGB 14.2(6.90-21.30) 14.2(6.90-21.30) <0.001 14.10(6.90-18.20) 14.2(6.90-21.30) 0.816 14.10(6.90-18.20) 14.2(6.90-21.30) <0.001 HCT 43.15(21.4-64.8) 43.15(21.4-64.8) <0.001 43.45(3.72-54.60) 43.15(21.4-64.8) 0.999 43.45(3.72-54.60) 43.15(21.4-64.8) <0.001 MCV 85.45(56.90-108.5) 85.45(56.90-108.5) 0.874 85.9(57.3-108.6) 85.45(56.90-108.5) 0.808 85.9(57.3-108.6) 85.45(56.90-108.5) 0.503 MCH 28.4(18.10-35.70) 28.4(18.10-35.70) 0.391 28.25(18.10-36.40) 28.4(18.10-35.70) 0.773 28.25(18.10-36.40) 28.4(18.10-35.70) 0.807 MCHC 33(30.50-37.60) 33(30.50-37.60) 0.009 32.60(30-38.40) 33(30.50-37.60) <0.001 32.60(30-38.40) 33(30.50-37.60) 0.502 RDW-SD 43.5(34.70-63.10) 43.5(34.70-63.10) 0.709 44.10(35.30-82.20) 43.5(34.70-63.10) 0.110 44.10(35.30-82.20) 43.5(34.70-63.10) 0.014 PLT 159(32-424) 159(32-424) 0.001 250(86-482) 159(32-424) <0.001 250(86-482) 159(32-424) <0.001 MPV 9.30(6.90-12.10) 9.30(6.90-12.10) <0.001 9.10(7.10-13.00) 9.30(6.90-12.10) 0.211 9.10(7.10-13.00) 9.30(6.90-12.10) <0.001 PDW 11.85(7.30-21.10) 11.85(7.30-21.10) <0.001 11.60(8.10-23.60) 11.85(7.30-21.10) 0.207 11.60(8.10-23.60) 11.85(7.30-21.10) 0.019 PCT 0.15(0.03-0.34) 0.15(0.03-0.34) 0.022 0.22(0.09-0.37) 0.15(0.03-0.34) <0.001 0.22(0.09-0.37) 0.15(0.03-0.34) <0.001 PLCR 31.75(15.30-51.10) 31.75(15.30-51.10) <0.001 30.40(15.40-57.90) 31.75(15.30-51.10) 0.143 30.40(15.40-57.90) 31.75(15.30-51.10) 0.001 The agreement levels between different anticoagulants, using K2EDTA as the standard, were evaluated. Sodium citrate showed perfect agreement in assessing MCV and MCH (CCC = 0.990) but displayed significant agreement in determining WBC, %Neu, %Lymph, Neu, Lymph, and Eos (CCC between 0.95 and 0.99). Moderate agreement was observed in assessing MCHC (CCC = 0.929), while poor agreement was found in all other parameters with CCC<0.90. Similarly, sodium heparin demonstrated perfect agreement in determining MCV (CCC=0.994) and MCH (CCC=0.990), with substantial agreement in other parameters such as WBC, %Lymph, Neu, Lymph, RBC, and HGB (CCC between 0.95 and 0.99), but poor agreement in parameters with CCC<0.90. Regarding the comparison of K2EDTA and sodium citrate, citrate was highly precise and accurate in the estimation of WBC, %Neu, %Lymph, Neu, Lymph, Eos, MCV, MCH, and MCHC. While comparing sodium heparin to K2EDTA, it was highly precise in the estimation of WBC, %Neu, %Lymph, Neu, Lymph, Eos, RBC, HGB, HCT, MCV, MCH, MCHC, and PLCR (Table 5). Table 5. Concordance correlation coefficient (CCC) for the estimation of the level of agreement between K2EDTA with sodium citrate and sodium heparin. CBC parameters K2EDTA-Citrate Pearson ρ (precision) Accuracy K2EDTA-Sodium heparin Pearson ρ (precision) Accuracy WBC 0.97(0.9571 -0.9718) 0.988 0.977 0.985(0.981- 0.989) 0.986 0.999 %Neu 0.972(0.964-0.978) 0.974 0.998 0.847(0.814-0.875) 0.925 0.916 %Lymph 0.984(0.980- 0.988) 0.985 0.999 0.951(0.937- 0.961) 0.954 0.997 %Mon 0.663(0.593 - 0.723) 0.705   0.941 0.157(0.111 -0.201) 0.440 0.355 %Eos 0.636(0.567 - 0.697) 0.688 0.926 0.594(0.525 - 0.656) 0.675 0.881 %Bas 0.460(0.361 -0.548) 0.480 0.958 0.305(0.209 - 0.396) 0.371 0.822 Neu 0.980(0.975 - 0.984) 0.990 0.990 0.972(0.964 - 0.978) 0.980 0.991 Lymph 0.956(0.949 - 0.966) 0.984 0.974 0.969(0.961 - 0.976) 0.973 0.997 Mon 0.733(0.675 - 0.782) 0.795 0.922 0.209(0.157 - 0.261) 0.500 0.419 Eos 0.968(0.960 - 0.975) 0.973 0.995 0.927(0.909 - 0.941) 0.942 0.983 Bas 0.565(0.477 - 0.642) 0.576 0.990 0.458(0.371 -0.537) 0.543 0.848 RBC 0.723(0.691 - 0.764) 0.983 0.742 0.973(0.966-0.979) 0.974 0.999 HGB 0.742(0.705 - 0.775) 0.988 0.752 0.977(0.971 - 0.982) 0.979 0.998 HCT 0.670(0.620 - 0.714) 0.911 0.735 0.907(0.882 - 0.926) 0.909 0.998 MCV 0.990(0.987 - 0.992) 0.995 0.995 0.994(0.992 - 0.995) 0.995 0.998 MCH 0.990(0.987 - 0.992) 0.991 0.998 0.990(0.987 - 0.992) 0.992 0.998 MCHC 0.929(0.910 - 0.944) 0.933 0.995 0.874(0.844 - 0.898) 0.932 0.937 RDW-SD 0.721(0.661 - 0.772) 0.762 0.946 0.738(0.681- 0.786) 0.771 0.958 PLT 0.313(0.236 - 0.386) 0.470 0.668 0.290(0.235 - 0.343) 0.648 0.448 MPV 0.784(0.734 - 0.826) 0.830 0.945 0.873(0.841 - 0.899) 0.887 0.985 PDW 0.790(0.740 - 0.832) 0.815 0.970 0.819(0.774 - 0.856) 0.832 0.983 PCT 0.319(0.256 - 0.380) 0.598 0.534 0.238(0.187- 0.289) 0.585 0.408 PLCR 0.833(0.794- 0.866) 0.879 0.948 0.876(0.847- 0.901) 0.901 0.973 Discussion The choice of anticoagulants and storage time significantly affect blood sample analysis [10]. In a study by Akorsu et al. involving 55 healthy individuals, consistency in blood parameters across three anticoagulants was observed: K3EDTA, sodium citrate, and lithium heparin [3]. Similarly, a current study utilized K2EDTA, sodium citrate, and sodium heparin, finding excellent consistency in various blood parameters, with ICC values exceeding 0.90. Regarding variation in CBC parameters using different anticoagulants, in a study which is conducted on 30 clinically healthy dogs from different breeds, no significant variation between sodium citrate and K3EDTA was found in 4 out of 8 CBC parameters (50%) including HGB, HCT, PLT, and PCT [11]. Similarly, in a study conducted on humans, in which variation in the estimation of CBC parameters was evaluated using three different anticoagulants, namely, K3EDTA, sodium citrate, and lithium heparin, no statistically significant difference was observed in 5 out of 14 CBC parameters (35.7%) including MCV, MCH, MCHC, %Lymph, and %Neu among the three anticoagulants examined [3]. In the present study, regarding variation in CBC parameters using K2EDTA, sodium citrate, and sodium heparin, no statistically significant variation was found in 4 out of 23 CBC parameters (17.40%) including %Lymph, Eos, MCV, and MCH among these three different anticoagulants. The significant variations observed in other CBC parameters underscore the need for careful consideration when selecting anticoagulants, particularly in clinical settings where precise and consistent CBC measurements are crucial for accurate diagnosis and monitoring of conditions [12]. In a study of 50 healthy dogs comparing EDTA and sodium citrate, no comparable results were found among 9 CBC parameters, suggesting citrate may lead to inaccurate results compared to EDTA [13]. Another study of 55 healthy individuals comparing heparin and citrate revealed significant differences in 5 out of 14 CBC parameters (35.71%), with the remaining parameters showing variations. Similar patterns were observed when comparing citrate to EDTA. Comparing heparin to K3EDTA showed significant variations in three parameters (21.43%) [3]. In the current study, comparing K2EDTA to sodium citrate showed similar results in 8 out of 23 CBC parameters (34.78%), while comparing K2EDTA to sodium heparin showed comparable values in 14 out of 23 CBC parameters (60.87%). Comparing PLT results between K2EDTA and sodium citrate with sodium heparin, significantly lower PLT counts were found in the latter two in the current study, contradicting findings in existing genuine literature [14-16]. One study suggested that citrate's strong platelet activation in sick animals may lead to decreased PLT counts due to platelet clumping [17]. Additionally, lower HGB and HCT values were observed in citrated blood samples compared to EDTA, consistent with previous studies [3,11]. This discrepancy may be attributed to citrate's interference with HGB oxidation, resulting in higher HGB levels in EDTA samples. The CBC is commonly conducted on venous blood specimens anticoagulated with EDTA. Among various EDTA subtypes, the dipotassium salt form, K2EDTA, is endorsed by the International Council for Standardization in Hematology as the preferred anticoagulant for blood cell enumeration and sizing [7]. The study evaluated agreement levels between different anticoagulants, using K2EDTA as the standard. Sodium citrate showed substantial agreement in 8 out of 23 CBC parameters (34.78%), including MCV, MCH, WBC, %Neu, %Lymph, Neu, Lymph, and Eos. Similarly, sodium heparin demonstrated substantial agreement in determining MCV, MCH, WBC, %Lymph, Neu, Lymph, RBC, and HGB. These findings align with previous literature, which indicated substantial agreement with heparin in assessing 4 out of 14 CBC parameters (28.57%), including RBC, HGB, HCT, and MCH [3]. Conclusion Citrate was found to be a less reliable anticoagulant for CBC estimation compared to K2EDTA, potentially leading to inaccurate readings. On the other hand, sodium heparin showed comparable performance to K2EDTA, making it a suitable alternative under specific conditions. Declarations Conflicts of interest: The authors have no conflicts of interest to disclose. Ethical approval: The study was approved by the Institutional Ethics Committee and utilized data obtained from hospital archives Ethical Approval for this study was obtained from the Ksciens ethical committee (Approval Number 43. 2025). Patient consent (participation and publication): Written informed consent was obtained from all patients (or their legal guardians, where applicable) for participation in the study and for the publication of all associated clinical information and images. Source of Funding: Star Lab Company. Role of Funder: The funder remained independent, refraining from   involvement   in   data   collection, analysis, or   result formulation, ensuring unbiased research free from external influence. Acknowledgements: Not applicable. Authors' contributions: RQS and SQO were major contributors to the conception of the study, as well as to the literature search for related studies. DAH and AMM were involved in the literature review and the writing of the manuscript. SLE, HAY, HSA and MTT were involved in the literature review, the design of the study, the critical revision of the manuscript, and the processing of the tables.  QOS and AMM confirm the authenticity of all the raw data. All authors have read and approved the final manuscript. Use of AI: AI was not used in the drafting of the manuscript, the production of graphical elements, or the collection and analysis of data. Data availability statement: The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.