Brasil
Ciência Rural
Sumário Anterior Atual Seguinte
Sumário
CLINIC AND SURGERY • Cienc. Rural 56 (08) • 2026 • https://doi.org/10.1590/0103-8478cr20250057 linkcopy

Open-access EDTA K2 and K3 concentrations on erythrocyte parameters in canine blood samples

Concentrações de EDTA K2 e K3 nos parâmetros eritrocitários em amostras de sangue de cães

AuthorshipSCIMAGO INSTITUTIONS RANKINGS

ABSTRACT:

Ethylenediaminetetraacetic acid (EDTA) is essential in veterinary hematology to prevent coagulation and ensure accuracy of hematological analyses. However, using outside recommended concentrations may alter hematological parameters. This study investigated the influence of different concentrations of EDTA K2 and K3 on erythrocyte parameters in canine blood. Blood samples from 20 healthy dogs were collected and allocated to groups containing varying anticoagulant concentrations. Evaluated erythrocyte parameters included red blood cell count, hemoglobin concentration, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution width (RDW). Increasing EDTA concentration resulted in reduced MCV and PCV, particularly in samples containing higher concentrations of EDTA K3. In addition, increased MCHC and decreased RDW-SD were observed at elevated EDTA concentrations. Red blood cell counts and hemoglobin concentration showed no significant changes. These findings indicated that adherence to recommended EDTA ratios is essential to avoid alterations in erythrocyte parameters, with concentrations up to twice the recommended level for EDTA K2 remaining acceptable.

Key words:
EDTA K2; EDTA K3; canine; hematological parameters; anticoagulant

RESUMO:

O anticoagulante EDTA é essencial na hematologia veterinária para evitar a coagulação e garantir a precisão dos exames hematológicos. No entanto, seu uso fora das concentrações recomendadas pode causar alterações nos parâmetros hematológicos. O objetivo deste estudo foi investigar como diferentes concentrações de EDTA K2 e K3 influenciam os parâmetros eritrocitários no sangue de cães. Para isso, foram analisadas amostras de sangue de 20 cães hígidos, distribuídos em grupos com diferentes concentrações de anticoagulante. Além disso, os parâmetros eritrocitários avaliados incluíram contagem de eritrócitos, concentração de hemoglobina, volume globular (VG), volume corpuscular médio (VCM), concentração de hemoglobina corpuscular média (CHCM) e amplitude de distribuição dos glóbulos vermelhos (RDW). Os resultados mostraram que o aumento da concentração de EDTA reduziu o VCM e o VG, especialmente nos grupos com EDTA K3 em altas concentrações. Igualmente, foi observada elevação do CHCM e uma diminuição do RDW-SD com concentrações elevadas de EDTA. A contagem de eritrócitos e a concentração de hemoglobina não apresentaram alterações significativas. Conclui-se que, para evitar alterações nos parâmetros hematológicos, deve-se respeitar a proporção recomendada de EDTA, sendo aceitável até o dobro da concentração indicada para o EDTA K2.

Palavras-chave:
EDTA-K2; EDTA-K3; cães; parâmetros hematológicos; anticoagulante

INTRODUCTION

Anticoagulants play an essential role in veterinary hematology by preventing blood clotting in vitro, which is fundamental for performing hematological analyses. Clot formation interferes with blood cell counting and may cause analyzer obstruction, thereby compromising result accuracy (BANFI et al., 2007; STOCKHAM & SCOTT, 2011; THRALL et al., 2024).

Several anticoagulants are commercially available, including heparin, sodium citrate, and ethylenediaminetetraacetic acid (EDTA). Among these, EDTA is considered the most suitable for hematological testing, particularly in mammals (DIAS et al., 2023; THRALL et al., 2024). EDTA acts by chelating calcium ions present in blood, interrupting platelet activation, and the coagulation cascade. Unlike other anticoagulants, calcium binding by EDTA is irreversible. In addition, EDTA can be used in powder form or in small volumes, minimizing the dilution effect observed with anticoagulants such as sodium citrate and contributing to its widespread use in hematological analyses (BANFI et al., 2007; STOCKHAM & SCOTT, 2011).

EDTA is available in different formulations, including prefilled collection tubes and powder or liquid preparations that require prior handling. The salt associated with the EDTA molecule varies, with disodium EDTA, dipotassium EDTA (EDTA K2), and tripotassium EDTA (EDTA K3) representing the most used forms in hematology (BANFI et al., 2007; THRALL et al., 2024). Disodium EDTA is less frequently used because of its lower solubility, although it remains effective as an anticoagulant (HADLEY & WEISS, 1955). EDTA K2 and EDTA K3 are widely employed in blood collection tubes, typically identified by a lavender or purple cap. EDTA K2 is generally present as a dry powder applied to plastic tubes, whereas EDTA K3 is commonly used in liquid form in glass tubes (BANFI et al., 2007).

Several consensus statements and clinical pathology guidelines in both human and veterinary medicine recommend preferential use of EDTA K2 at the appropriate blood-to-anticoagulant ratio, advising a maximum of twice the manufacturer’s recommended concentration. This recommendation aims to prevent alterations in cell morphology and ensure accurate determination of hematological parameters, particularly erythrocyte indices, which may be affected by the hypertonicity of EDTA salts (SBPC, 2018; DIAS et al., 2023; THRALL et al., 2024). However, in veterinary practice, blood collection does not always proceed as planned because of factors such as patient size, stress, or limited cooperation. In addition, economic constraints or limited awareness during procurement may result in acquisition of tubes containing EDTA K3, making their use unavoidable in routine laboratory settings. Therefore, this study evaluated the effects of varying concentrations of EDTA K2 and EDTA K3 on erythrocyte parameters in canine blood samples.

MATERIALS AND METHODS

Blood samples from 20 clinically healthy dogs, with no breed or sex predilection, were used in this study. To be included, animals were required to weigh more than 5 kg, be at least 1 year of age, and present no clinical abnormalities. Sample collection was performed after owner authorization through written informed consent, either at the animals’ residences or at the Veterinary Clinic of UNIGRAN.

Following local antisepsis with cotton soaked in 70% alcohol, blood samples were collected by jugular venipuncture using 5 mL syringes and needles measuring 25 × 0.7 mm or 25 × 0.8 mm. Each sample was divided into six aliquots: three placed into disposable BD Vacutainer® tubes containing 3.6 mg of EDTA K2 (TK2 group) and three into disposable Vacuplast® tubes containing 3.6 mg of EDTA K3 (TK3 group). According to manufacturer instructions, both tube types were designed for the addition of 2 mL of blood.

Based on anticoagulant type, samples were further subdivided according to the anticoagulant-to-blood ratio. Groups were identified with the suffix 1 (TK2.1 and TK3.1) for the recommended anticoagulant-to-blood ratio (1.8 mg/mL), as indicated by the manufacturer; suffix 2 (TK2.2 and TK3.2) for a twofold higher ratio (3.6 mg/mL); and suffix 3 (TK2.3 and TK3.3) for a fourfold higher ratio (7.2 mg/mL). These different anticoagulant concentrations were obtained by adding varying blood volumes to the tubes to achieve the desired ratios: 2.0 mL for TK2.1 and TK3.1, 1.0 mL for TK2.2 and TK3.2, and 0.5 mL for TK2.3 and TK3.3.

After group identification, samples were transported to the UNIGRAN Clinical Pathology Laboratory and processed according to the recommendations of STOCKHAM & SCOTT (2011) using an automated hematology analyzer (Sysmex® PocH-100iV Diff®).

Erythrocyte parameters evaluated included red blood cell count (RBC), hemoglobin concentration (Hb), packed cell volume (PCV) or hematocrit, and erythrocyte indices, namely mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution width expressed as standard deviation and coefficient of variation (RDW-SD and RDW-CV, respectively). These variables were analyzed to determine the influence of anticoagulant type and anticoagulant-to-blood ratio on erythrocyte parameters.

For statistical analysis, data were first assessed for normality using the D’Agostino-Pearson test. Subsequently, hematological parameters were compared using the Friedman test, adopting a significance level of 5%.

RESULTS

Of the dogs included in the study, 13 were male and 7 were female, with a median age of 5 years. Table 1 presents the medians and ranges of values obtained for the TK2 and TK3 groups at different anticoagulant-to-blood ratios (1.8 mg/mL, 3.6 mg/mL, and 7.2 mg/mL).

Thumbnail
Table 1
Median and minimum-maximum values of erythrocyte parameters in dogs (n = 20) at varied EDTA K2 and EDTA K3 concentrations.

DISCUSSION

The effects of EDTA K2 and EDTA K3 on blood cells have been investigated in both human and animal blood samples under different conditions; however, results remain inconsistent across studies (GOOSSENS et al., 1991; BANFI et al., 2007; LUDTKE et al., 2013; MEHMOOD et al., 2018; ROY et al., 2024). Understanding how anticoagulants influence hematological values is essential in veterinary medicine to prevent misinterpretation and misdiagnosis, particularly when alterations are related to the blood-to-anticoagulant ratio. This consideration is especially relevant when factors such as patient cooperation, health status, or body size hinder collection of the appropriate blood volume, thereby compromising the anticoagulant concentration recommended by the tube manufacturer (NEMEC et al., 2005).

In this context, mean corpuscular volume (MCV) represents a key parameter for interpreting erythrocyte alterations associated with excess EDTA. MCV reflects erythrocyte size distribution, and reduced values are commonly associated with pathological conditions such as chronic anemia, iron deficiency, and hypernatremia (THRALL et al., 2024). However, under in vitro conditions, MCV reduction may result from addition of osmotic substances, including anticoagulants. Excess EDTA increases plasma osmolarity, promoting water efflux from erythrocytes, cellular dehydration, and consequent volume reduction (BANFI et al., 2007).

In the present study, MCV decreased in tubes TK2.3, TK3.2, and TK3.3 compared with TK2.1. Similar findings have previously been reported in human and dog samples collected with EDTA K3 (GOOSSENS et al., 1991; NEMEC et al., 2005). In contrast, LUDTKE et al. (2013) did not observe statistically significant MCV changes in human blood samples containing EDTA K3 at concentrations four times higher than recommended; although, a numerical decrease in MCV was noted with increasing anticoagulant concentration.

GOOSSENS et al. (1991) also evaluated the effects of elevated EDTA K2 concentrations in human blood and reported variable results depending on the method used for MCV determination. Notably, no significant MCV changes were observed when electrical impedance methodology was applied, which is consistent with the analytical approach used in the present study.

The reduction in MCV observed in tubes TK2.3, TK3.2, and TK3.3 is most likely attributable to excess EDTA in the samples. Increased anticoagulant concentration elevates plasma osmolarity and promotes erythrocyte dehydration, resulting in decreased cell volume. EDTA K3 contains one additional potassium ion compared with EDTA K2, which is released upon chelation, contributing to a hyperosmotic environment at lower mass concentrations when compared with EDTA K2 (3.6 mg/dL for EDTA K3 versus 7.2 mg/dL for EDTA K2) (GOOSSENS et al., 1991; NEMEC et al., 2005; BANFI et al., 2007).

A decrease in packed cell volume (PCV) was observed exclusively in tube TK3.3 and reflects the reduction in MCV. In automated impedance-based analyzers, such as the instrument used in this study, PCV is calculated from erythrocyte count and MCV, with the latter being directly proportional to PCV. Consequently, a reduction in MCV results in a proportional decrease in PCV. Similar reductions in PCV have been reported in canine blood samples containing two- and threefold higher concentrations of EDTA K3 (ROSS et al., 2021; NEMEC et al., 2005), supporting the findings of the present study.

A true increase in mean corpuscular hemoglobin concentration (MCHC) is uncommon (SCOTT & STOCKHAM, 2011; THRALL et al., 2024). When observed, it is usually attributable to analytical or preanalytical factors. Because MCHC is calculated by dividing hemoglobin concentration by PCV, a decrease in the divisor leads to an apparent increase in MCHC. This phenomenon was evident in tubes TK2.3 and TK3.3 in the present study (Table 1). Elevated MCHC values may also result from analyzer calibration errors or hemolysis (SCOTT & STOCKHAM, 2011; THRALL et al., 2024); however, neither calibration issues nor hemolysis were detected in this study.

In vivo, RDW-SD reflects the dispersion of erythrocyte size; greater anisocytosis corresponds to higher RDW-SD values, as observed in regenerative anemias. However, an isolated decrease in RDW-SD has no clinical significance (THRALL et al., 2024). In the present study, RDW-SD decreased in tubes TK2.3 and TK3.3, in which EDTA concentration was four times higher than recommended. This reduction may be attributed to erythrocyte dehydration caused by the hypertonic environment, which reduced anisocytosis by causing erythrocytes to reach more uniform volumes.

In contrast, GOOSSENS et al. (1991) reported increased RDW values in samples with elevated EDTA concentrations, with a more pronounced effect in EDTA K2 tubes than in EDTA K3 tubes. The authors suggested that EDTA K2 may alter erythrocyte membrane integrity in a subset of cells, allowing water influx and subsequent cell swelling, which would increase size variability. This phenomenon was not observed in the present study. Such discrepancies may be related to species differences, as canine erythrocyte membranes may exhibit greater resistance to anticoagulant-induced alterations than human erythrocytes.

Erythrocyte count, hemoglobin concentration, and RDW-CV remained unchanged across all EDTA concentrations evaluated. Although erythrocyte volume decreased, cell number was preserved, indicating absence of hemolysis at any anticoagulant concentration tested. Because hemoglobin is contained within erythrocytes, stable cell counts are consistent with unchanged hemoglobin concentrations. RDW-CV represents a coefficient of variation calculated automatically from the erythrocyte volume histogram width divided by MCV. When both parameters decrease proportionally, RDW-CV remains unchanged (SCOTT & STOCKHAM, 2011; THRALL et al., 2024).

CONCLUSION

Therefore, both EDTA K2 and EDTA K3 are suitable for routine hematological testing in dogs when the manufacturer-recommended anticoagulant-to-blood ratio is respected, with EDTA K2 remaining acceptable at up to twice the recommended concentration.

ACKNOWLEDGMENTS

This study was supported by Centro Universitário da Grande Dourados (UNIGRAN); Universidade Federal de Mato Grosso do Sul (UFMS/MEC), Brazil; and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil (Funding Code 001 ).

REFERENCES

  • CR-2025-0057.R2
  • BIOETHICS AND BIOSECURITY COMMITTEE APPROVAL
    This study was approved by the Ethics Committee on the Use of Animals of Centro Universitário da Grande Dourados (CEUA/UNIGRAN) under protocol number 112/19.
  • DATA AVAILABILITY STATEMENT
    The raw data supporting the conclusions of this study are available from the corresponding author upon reasonable request.
  • DECLARATION OF USE OF ARTIFICIAL INTELLIGENCE
    Artificial intelligence was used to improve the language and readability of the manuscript. All revisions were carefully reviewed and approved by the authors to ensure the accuracy and integrity of the scientific content.

Edited by

Data availability

The raw data supporting the conclusions of this study are available from the corresponding author upon reasonable request.

Publication Dates

  • Publication in this collection
    31 July 2026
  • Date of issue
    2026

History

  • Received
    04 Feb 2025
  • Accepted
    30 Jan 2026
  • Reviewed
    14 Apr 2026
Creative Common - by 4.0
This is an open-access article distributed under the terms of the Creative Commons Attribution License
location_on
Universidade Federal de Santa Maria Universidade Federal de Santa Maria, Centro de Ciências Rurais , 97105-900 Santa Maria RS Brazil , Tel.: +55 55 3220-8698 , Fax: +55 55 3220-8695 - Santa Maria - RS - Brazil
E-mail: cienciarural@mail.ufsm.br
rss_feed Acompanhe os números deste periódico no seu leitor de RSS
Reportar erro