Introduction

Anticoagulants are used as additives to blood specimens so the samples being measured are unlikely to be significantly changed before analyzing. There are many types of anticoagulants available, depending on the sample and testing required. Anticoagulation occurs by binding calcium ions (ethylenediaminetetraacetic acid [EDTA], citrate) or by inhibiting thrombin activity (heparin).¹ Citrate is used when evaluating coagulation disorders whereas heparin is used for chemistry panels.¹ EDTA is used to prevent platelet aggregation and is the preferred anticoagulant for hematology; it allows for preservation of cellular components and morphology of cells.^2–4 EDTA chelates calcium ions and prevents enzymatic reactions that lead to coagulation, maintaining blood in its liquid state.³ However, increased concentrations of K₃ EDTA in relation to the quantity of blood increases the ionic concentration leading to red cell shrinkage as water moves out of the red blood cells (RBCs) to equalize the osmotic pressure in the surrounding plasma.^3–5 Most EDTA tubes use spray-dried EDTA rather than liquid, decreasing the osmotic effect on blood cells.^2,3 The reported optimal concentration is 1.6 mg of K₃ EDTA per milliliter of blood.⁶ In 2004, the Clinical and Laboratory Standards Institute recommended discarding samples if the proportion of blood to EDTA is inappropriate because of alteration in measured hematology variables.^7–10

Most animal research regarding the effects of excess EDTA on hematology results focuses on calculated hematocrit (HCT) using a machine rather than measured packed cell volume (PCV). The research described in this statement focuses on centrifuged PCV rather than results obtained through cell analyzers. PCV is a bedside blood test used to measure the amount of red cells in whole blood. The PCV is directly quantified as the height of the red cell column in a micro-HCT tube after centrifugation measured on a designated card.⁶ The remainder of this paper will consider PCV as a measured, centrifuged sample, not obtained using a cell analyzer. Studies have shown that PCV gives values ∼1.5–3% higher compared with HCT because of plasma trapped in the RBC layer.¹¹ Benefits to using PCV are a small sample volume is needed and results are obtained quickly. Micro-HCT tubes to determine PCV are generally more precise than obtaining RBC count manually, and visual examination of plasma in the tube can provide assessment for icterus, hemolysis, or lipemia.⁵ According to Willard and Tvedten, micro-HCT tubes should be centrifuged for 5 min.⁵ The amount of time a blood sample sits before being run in a cell analyzer can also impact results. According to Wu et al., HCT and mean cell volume (MCV) were stable at room temperature for up to 8 hr if an appropriate concentration of EDTA/blood was submitted.¹² Willard and Tvedten state that red cell indices should be analyzed within 2–3 hr or the sample should be refrigerated at 4°C. RBC swelling can occur after 6–24 hr when stored at room temperature, raising PCV and MCV and lowering mean corpuscular hemoglobin concentration.⁵ These findings were primarily associated with blood samples in EDTA tubes and how long a sample sits before obtaining complete blood count (CBC) results; no research could be found on the impact on PCV from micro-HCT tubes.

This study used nonheparinized micro-HCT tubes as the control. The World Health Organization recommends no anticoagulant in micro-HCT tubes if the blood sample used is previously anticoagulated. However, no research could be found on the impact of time to processing on PCV from whole blood placed in nonheparinized micro-HCT tubes.¹³

Since 1935, studies have been performed in humans and many mammalian species to determine the extent to which hematology results are impacted by EDTA.^12,14–20 In 1970, Penny et al. observed the effect of concentration of K₂ EDTA per milliliter of blood on PCV in dogs, cats, horses, pigs, and sheep. Excess concentration of EDTA/blood (underfilled tubes) can lead to shrinkage of the red cells, altering hematology results.⁵ Research in 2005 by Nemec and Drobnič-Košsorok assessed the impact of K₃ EDTA on red cell indices in healthy beagle dogs. This study found that underfilling tubes resulted in a higher concentration of EDTA, decreasing HCT and MCV. This study only used two concentrations of EDTA/blood (an ideally filled tube with 3 mL of blood versus an underfilled tube with 1 mL of blood).¹⁷ There are no veterinary studies that have evaluated additional serial dilutions of EDTA/blood. There are also no studies that have included samples directly placed into micro-HCT tubes without anticoagulant. This research is relevant to the veterinary field because it is common to have difficulties collecting adequate volumes of blood samples because of patient behavior or size leading to underfilling of EDTA tubes, which may then produce erroneous results and affect clinical decision making.² This study includes a larger sample size and with more EDTA/blood dilutions compared with previous studies in dogs. Using PCV instead of calculated HCT from an automated analyzer will allow for the use of a bedside, cost-effective, reliable test, which can be integrated into clinical practice.

We developed this study to assess the effect of various concentrations of EDTA/blood in tubes on PCV. We hypothesized that there will be a progressive decrease in PCV with progressive underfilling of EDTA tubes, compared with directly filled nonanticoagulated micro-HCT tubes.

Materials and Methods

Results

A total of 94 blood samples were used for this research from healthy dogs. The results of the analyses revealed that there is a statistically significant difference between the mean PCV of all groups (control, 0.25 mL, 0.5 mL, 1 mL, 1.3 mL, and 1.5 mL) such that progressive underfilling of the EDTA tubes resulted in a decreased PCV compared with the control. The mean PCV of the control group was 53.6, whereas the mean PCV for the overfilled group was 51.17. The range of PCVs in the control was 39–69; 1.5 mL: 41–68; 1.3 mL: 41–66; 0.75 mL: 39–64; 0.5 mL: 39–62; and 0.25 mL: 38–62. The closest difference was between 1.5 and 1.3 mL (P = .0138), where the means were 51.17 and 50.63, respectively. All other pairwise comparisons were P < .0001. The mean PCV for 0.75 mL was 49.06, 0.5 mL was 47.64, and 0.25 mL was 46.44. The control group revealed a higher mean PCV than the appropriately filled 1.3 mL of K₃ EDTA; the overfilled 1.5 mL group also had a lower mean PCV than the control. The results of PCV with different concentrations of K₃ EDTA/blood are shown in Table 1 and Figure 1.

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TABLE 1 Influence of K₃ EDTA on PCV Based on Increasing Concentration of EDTA/Blood in 94 Healthy Dogs

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Discussion

We developed this study to interpret the effect on PCV of overfilling, accurately filling, and progressively underfilling K₃ EDTA tubes. We hypothesized that a progressive decrease in PCV based on the amount a tube was underfilled would occur. This study found a statistically significant decrease in the mean PCVs between overfilled, appropriately filled, and progressively underfilled EDTA tubes compared with control samples. To elaborate, the mean PCVs between the EDTA/blood groups (including the control) revealed enough of a difference to consider that the concentration of EDTA/blood can impact PCV. The less blood within the EDTA tube, the lower the PCV compared with the control group.

Even though the 1.3 mL samples were considered appropriately filled based on Sarstedt’s product specification recommendations, the PCVs were statistically lower than the control groups (no K₃ EDTA).²¹ We attribute the difference in PCV to shrinking of erythrocytes because K₃ EDTA is a hypertonic medium. All salts of EDTA are hyperosmolar, causing water to leave the erythrocytes, leading to cell shrinkage.^5,6 This impacts the PCV results, which should be considered in light of our findings. The higher the concentration of EDTA/blood, the greater the osmotic withdrawal of water from the cells decreasing PCV.¹⁷ Based on our results, even the overfilled EDTA tubes had lower PCVs than the control group. We can consider this to be the effect EDTA salts have on blood samples. However, overfilling tubes can dilute the anticoagulant, predisposing the sample to clotting, which we cannot entirely rule out in our study.⁵ Grossly clotted blood was not administered into the micro-HCT tubes, but we cannot rule out clots not visible to the naked eye. If a sample becomes clotted, the blood that is obtained and submitted in the micro-HCT tubes are not parallel to the entire blood sample, possibly allowing for abnormal results. Research did not reveal whether clotted blood in HCT tubes would cause the PCV to be higher or lower than the control. Overall, there was a progressive decline in mean PCV results compared with the control.

The control was considered the undiluted PCV in nonanticoagulated micro-HCT tubes. According to Hughes et al., no significant difference in the spun micro-HCT among 30 samples of whole blood in K₃ EDTA existed between heparinized and nonheparinized capillary tubes.²² Bamberg et al. agreed that their results indicated that the additional dilution produced in a heparinized capillary tube, when being filled with an EDTA-anticoagulated sample, is not sufficient to produce clinically different micro-HCT results.²³ Research shows there is an insignificant difference on PCV whether micro-HCT tubes use anticoagulants or not.^22,23 Micro-HCT tubes (whether anticoagulated or not) are easily accessible, user friendly, and an appropriate means to obtain consecutive and rapid results. Ideally, comparing PCV to a calculated HCT using blood analyzers to evaluate CBC findings would allow for further comparisons. This was not an option at this time because of limited funding and is considered a limitation in this study.

We chose to use micro-HCT tubes without anticoagulant to determine PCV as the control in this study because this method is a rapid, cost-effective, repeatable test that can be performed in general practice, emergency, and specialty hospital settings. It can also be used for serial evaluation of RBC count and levels of anemia during hospitalization, and we wanted a test that could be clinically applicable to daily practice. Despite recommendations for appropriately filled EDTA tubes, underfilling is still a concern for when phlebotomy may be difficult in veterinary patients (hypoperfusion, coagulopathy, patient temperament, size, etc.). The results of this study showed that there is a statistically significant difference (and a progressive decrease) in PCV with underfilling of EDTA tubes as expected, but there was also a difference in the ideally filled tube and directly filled micro-HCT tubes. The primary focus of this research was to assess the effect of sample handling of PCV in a population of healthy patients without underlying abnormalities in PCV. Clinically, this information is most applicable to patients with anemia in which serial measurement of PCV is performed to monitor red cell levels and assess the need for interventions (such as blood transfusions). Further studies would be needed to confirm that the findings of this study are similar in a population of anemic patients as well.

Based on the results of this study, it is recommended that a standard method of determination of PCV is performed on patients with manual measurements of PCV to avoid errors in interpretation, especially if serial measurements are taken during a period of hospitalization. This study underscores the need for consistency in practice when assessing and monitoring anemia. The results of this study illustrate the importance of a consistent means of measurement of RBC count to allow for appropriate interpretation of blood results.

This study used a much larger sample size compared with previous studies supporting the irregularities with underfilled EDTA tubes. A control was also used in this study to allow for comparison to no anticoagulant as well as considering what happens if tubes were overfilled. This was the first time, based on our literary research, that these two aspects were evaluated.

There were severe limitations to this study. There are different types of EDTA, K₂ and K₃ EDTA. The majority of the United States and United Kingdom uses K₂ EDTA; however, K₃ EDTA is more commonly used in Europe and Japan. K₂ EDTA has less pronounced osmotic effect on blood cells than K₃ EDTA.^3,17 The International Council for Standardization of Hematology recommends K₂ EDTA for hematological testing; however, our hospital did not use these sampling tubes, and K₃ EDTA was the medium available. Comparing the different osmotic properties and impact they have on PCV can allow for further results potentially allowing for improved accuracy of blood results. The laboratory technicians were aware of potential decrease in PCV based on concentration of EDTA/blood. This allows for reader bias possibly causing skewed results. Not all micro-HCT tubes have no additives. Frequently they contain heparin, which could be another variable to consider in future studies assessing EDTA/blood in micro-HCT tubes with and without anticoagulant. Current literature does not reveal statistical differences between heparinized and nonheparinized micro-HCT tubes; however, the effect of different concentrations of EDTA/blood or the use of whole blood was not a factor.²² The centrifuge at the facility was not standardized to the recommendation in the literature; however, all samples were spun for the same length of time at the same force so as not to cause intersample variability.⁵ The amount of time the EDTA tubes with blood sat before obtaining results was unlikely to cause intersample differences based on research. Filled EDTA tubes need to sit for at least 2–6 hr to have skewed results.^5,12 Further research can also compare different concentrations of EDTA/blood on CBC red cell analysis and the correlation of these results to the PCVs. Lastly, all the subjects were healthy, which does not translate into the population of clinically unwell hospitalized patients. More clinical studies would need to be performed to determine clinical applicability for patients with disease processes causing hematologic abnormalities.

Conclusion

This study revealed that underfilled K₃ EDTA tubes result in lower PCVs when compared with the control. This study also revealed that overfilled K₃ EDTA tubes have lower PCVs than the control group, which is a new finding in the literature. In the future, studies can assess PCV compared with calculated HCT and research PCV in diseased patients. Clinicians performing serial assessments of canine PCV should consider using the same sample volume for the most consistent and accurate results.