Journal of Applied Hematology

: 2014  |  Volume : 5  |  Issue : 3  |  Page : 101--106

Under filled di potassium-ethylene di amine tetra acetic acid vacutainers and its effect on automated blood cell indices in healthy blood donors: Is there a need to re-investigate it as a rejection criterion?

Vibha Gupta, Vikas Shrivastav, Gita Negi, Harish Chandra, Shelly Mittal, Debasis Biswas 
 Department of Pathology, Himalayan Institute Medical Sciences, Dehradun, Uttarakhand, India

Correspondence Address:
Vibha Gupta
Department of Pathology, Himalayan Institute Medical Sciences, Swami Ram Nagar, Dehradun - 248 140, Uttarakhand


Introduction: Standard guidelines assert that any vacutainer, which is 10% less under-filled should be rejected as it may affect the results. These guidelines refer to the studies using liquid K 3 -ethylene diamine tetra acetic acid (EDTA). Although the new guidelines recommend K 2 -EDTA instead of earlier K 3 -EDTA but has not clearly commented upon whether the same holds true for the new anticoagulant K 2 -EDTA. Materials and Methods: Blood samples from 100 healthy blood donors were included. We took 7-8 ml of blood and was then transferred to three 3 ml capacity vacutainers-up to 3 ml, 2.5 ml, 1 ml (marked a, b, c respectively) containing spray-dried K 2 -EDTA as anti-coagulant. Thus, b and c vacutainers were under-filled. Hematological parameters in all the samples were analyzed immediately using automatic analyzer (Cell Dyn). Statistical analysis was done with the help of SPSS v. 17.0 (Chicago, IL, USA) using one-way ANOVA. Results: The results showed that three groups were comparable with respects to hemoglobin, hematocrit, total leukocyte count, differential leukocyte counts, total platelets, mean corpuscular volume, mean hemoglobin concentration, mean cellular hemoglobin concentration along with red cell distribution width. Conclusion: Our study suggested that under-filling till 67% (1 ml in 3 ml standard capacity) of the recommended volume in a spray-dried K 2 -EDTA vacutainers did not affect hematological parameters in healthy people. In view of these results, there is need to further investigate the same in diseased. If further studies done on under filling of the pathological samples also give similar results as in our study then only the present guidelines related to under filling could be revised when K 2 -EDTA is used as an anticoagulant.

How to cite this article:
Gupta V, Shrivastav V, Negi G, Chandra H, Mittal S, Biswas D. Under filled di potassium-ethylene di amine tetra acetic acid vacutainers and its effect on automated blood cell indices in healthy blood donors: Is there a need to re-investigate it as a rejection criterion?.J Appl Hematol 2014;5:101-106

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Gupta V, Shrivastav V, Negi G, Chandra H, Mittal S, Biswas D. Under filled di potassium-ethylene di amine tetra acetic acid vacutainers and its effect on automated blood cell indices in healthy blood donors: Is there a need to re-investigate it as a rejection criterion?. J Appl Hematol [serial online] 2014 [cited 2023 Sep 30 ];5:101-106
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Laboratory accreditation guidelines recommend rejection of blood vacutainers that contain lesser or higher than the recommended blood volume that is, the volume of blood which they are designed for. [1] This was supported by Clinical and Laboratory Standards Institute (CLSI) guidelines that the amount of anticoagulant in the vacutainers is ascertained for a given volume of blood and thus, under-filled or over-filled vacutainers can adversely affect results. [2] The other CLSI guidelines state that for all type of vacutainers, the variation in the blood volume should not be more than 10% of the recommended volume. [3]

A couple of studies suggested that under-filling of vacutainers was one of the common causes of sample rejection not only in India, but also in other parts of the world. [4],[5] Hematology laboratory in our institute receives on an average 700 blood samples each day. If we consider that the prevalence of under-filled samples in our laboratory is similar to the reported prevalence in other studies, a large number of sample should be rejected each day.

Whenever we reject any sample, a corrective action is to be taken. This action results in the series of activities. First, in the case of under-filled vacutainers, a fresh sample is expected to be drawn as corrective action, making sure that adequate amount of blood is drawn. WHO guidelines on phlebotomy describe that the phlebotomy is not a risk-free procedure and hematoma formation, vasovagal reaction, arterial puncture or nerve damage may occur with each attempt of phlebotomy. [6] The risk is not limited to the patient, but also the health care workers are at a higher risk to catch the blood borne infections from contaminated surfaces, blood splashes and accidental needle pricks during phlebotomy. [6]

Besides the risks mentioned so far, repeat phlebotomy is associated with multiple other problems. One of them is the patient's dis-satisfaction. According to a study done, 35% subjects undergoing phlebotomy experienced more than expected discomfort and thus the dis-satisfaction with the procedure may appear. [7] Second, the time spent in re-drawing the blood for under-filled vacutainers leads to a delay in reporting critical results that can have an impact on the clinical care. [8] Third, the hospital has to bear economic burden not only related to the cost of vacutainers, but also related to the manpower engaged in repeat phlebotomy. [9],[10]

Looking at the consequences of the rejection of under-filled vacutainers, we should ask ourselves does under-filling has any clinically significant effect on the blood parameters? In a routine, ethylene di amine tetra acetic acid (EDTA) is recommended as the anticoagulant of choice in hematology (prevents clotting by chelating calcium, which is an important cofactor in coagulation reactions) when compared to other anticoagulants as Heparin. [11] EDTA on one hand cause minimal alteration of red blood and leucocytes cells size or number, on the other hand, it reduces platelet activation by protecting the platelets clumps formation that may occur if calcium is not chelated. [12] Although both dipotassium (K 2 -EDTA, K 3 -EDTA) and disodium salts (Na 2 EDTA) are powerful anticoagulants, yet Na 2 EDTA is less soluble with blood. Hence, potassium salts are preferred (K 2 -EDTA and K 3 -EDTA). [12] Due to slight dilution caused by K 3 -EDTA, the preparation of choice is K 2 -EDTA. [13] K 2 -EDTA in concentration of 1.5-2.0 mg/ml shows maximum chelating effect on calcium. [14] According to the available literature, high concentration of liquid K 3 -EDTA (which is inevitable in under-filled vacutainers) increases the ionic concentration (and thus makes the plasma hypertonic), which in turn, may lead to shrinkage of red cells and their morphology may change. [15],[16],[17] These results have been supported by a recent study as well. [18] The effects of excess of K 3 -EDTA are not limited to the erythrocytes, but it can also affect the leucocytes and cause membrane damage. [16] Thus, an under-filled vacutainer containing K 3 -EDTA significantly affects the blood parameters.

However, contradictory results have been reported when spray-dried K 2 -EDTA containing under filled vacutainers were used instead of liquid K 3 -EDTA. To the best of our knowledge, only one study has been done on under-filled vacutainers containing spray dried K 2 -EDTA, and it reported that under-filling up to 75% of the standard volume did not lead to any significant change in hematological parameters. [19] Thus, underfilled K 2 -EDTA vacutainers may be accepted, however, this type of study needs to be replicated in other laboratories.

Further problem arises when we follow the CLSI guidelines. [2],[3] On one hand, these guidelines recommend that K 2 -EDTA vacutainers should be used in hematology. On the other hand, they also mention that inadequate filling of EDTA vacutainers leads to changes in blood parameters and they should be rejected. [2],[3] However, these guidelines are based on the reference studies that were conducted using liquid K 3 -EDTA as an anticoagulent, [2] If we use the vacutainers containing K 2 -EDTA, probably we may reach a different conclusion.

Hence considering the proportion of under-filled vacutainers that are rejected, problems associated with repeat phlebotomy and dearth of literature regarding the effect of under-filling in spray-dried K 2 -EDTA motivated us to conduct this study. In short, this study was aimed to find out the effect of underfilling of K 2 -EDTA vacutainers on hematological parameters in a healthy population.


This experimental study was carried out after obtaining approval from the institutional ethics committee. A total of 100 healthy blood donors who were thoroughly screened and physically examined and found fit to donate blood were included in this study. Before, including in the study, they were explained the purpose of this research and an informed consent was taken. Before starting we did an initial experiment. We took expired blood and with the help of calibrated pipette we filled two empty K 2 -EDTA vacutainers up to 2.5 ml and 1 ml respectively. The level of filling was marked externally on the tubes on the labels. Considering that each lot of vacutainers have the labels at same position on each vacutainers, we took all the 300 vacutainers of the same lot. Out of these 300 vacutainers 100 vacutainers were marked at volume of 1 ml on the label, in the same way, 100 vacutainers from the same lot were marked at volume of 2.5 ml according to the marking Done during the initial experiment. Thus for every case we labeled them as "a," "b" and "c" as 3 ml, 2.5 ml, 1 ml respectively (3 ml as 100% filling, 2.5 ml as 16.27% under filled; close to the volume recommended by CLSI guidelines as ± 10% criteria of acceptance) and only 1 ml (66.7% under-filled). From each donor, approximately 7-8 ml of venous blood from ante-cubital vein was drawn after tying tourniquet, using aseptic precautions with needle and syringe. After collecting, the needle was detached in each case, the stopper of each vacutainers was removed. With the manual pressure on the piston of the syringe, blood was dispensed in standard 3 ml along with marked vacutainers with 2.5 ml, 1 ml (K 2 -EDTA). Stopper was replaced, and each vacutainers was then rotated to 180° for 5-6 times for proper mixing anticoagulant and blood. The same procedure was followed with each of the subjects included in this study. These samples were run on a five part differential (Cell Dyn 3700). This analyzer used four systems to analyze the blood samples.

This machine measured white blood cell (WBC) white optical count, and the WBC Differential counts with the help of optical flow channel. The WBC impedance count was calculated in one of the electrical impedance channels. Another electrical impedance channel was used to calculate the total red blood cell (TRBC) and total platelet counts (TPLT). This machine also contained a spectrophotometric channel that measured the hemoglobin (HGB).

Hematocrit (Hct) mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration (MCHC) were calculated from above values. Mean corpuscular volume (MCV) was derived from RBC size distribution data. The laboratory was accredited under NABL. The analyzer had its regular calibration done with last calibration was done 2 months ago. There was regular quality control checks 3 times a day using "BIO RAD" quality controls. All the samples were analyzed and included in the study only when the quality controls were within acceptable limits.

Statistical analysis

Statistical analysis was performed with the help of SPSS v. 17.0 (Chicago, IL, USA). Descriptive statistics was calculated. The data from the under filled vacutainers (i.e. b and c) were compared with the results obtained from the standard collections (i.e. vacutainers "a"). One-way ANOVA was run to compare the difference of hematological parameters among standard volume (3 ml) and under filled vacutainers (2.5 ml and 1 ml).


The results showed that absolute neutrophils counts were comparable between three samples (4.22 ± 1.34 in 1 ml; 4.14 ± 1.31 in 2.5 ml and 4.16 ± 1.31 in 3 ml) in all the 100 cases. Similarly, absolute lymphocyte count was also comparable (1.90 ± 0.82 in 1 ml; 2.06 ± 0.83 in 2.5 ml and 2.17 ± 1.7 in 3 ml). Same held true for absolute eosinophil count (0.41 ± 0.39 in 1 ml; 0.40 ± 0.39 in 2.5 ml and 0.40 ± 0.38 in 3 ml) and absolute basophil count (0.80 ± 0.03 in 1 ml; 0.07 ± 0.03 in 2.5 ml and 0.08 ± 0.08 in 3 ml). The results from important parameters in hematology are shown in [Table 1]. The results of this study showed that none of the CBC parameters differ until the standard vacutainers contained upto 66.67% lesser volume in healthy donors.{Table 1}


In short our study depicted that up to 67% under-filling of the vacutainers containing spray-dried K 2 -EDTA did not affect any of the hematological parameters including Hct, MCV and MCHC in all healthy donors. Considering the problems associated with the phlebotomy procedure itself and economical losses associated with rejection of under-filled K 2 -EDTA vacutainers, the outcome of our study appears noteworthy.

To the best of our knowledge, so far, only few studies have addressed this issue using under-filled vacutainers containing K 2 -EDTA and the results of our study confirm the findings of one of the very few studies done earlier. [19] According to this study the under filled spray-dried K 2 -EDTA tubes show comparable results with regards to complete blood count, WBC, differential WBC count and reticulocyte count even when the blood volume as low as 1 ml was filled in vacutainers of 4 ml. (75% lesser filled compared to that of recommended volume. [19]

On the contrary, results with under filled vacutainers containing liquid K 3 -EDTA showed a pattern, which was different from underfilling of K 2 -EDTA. [18],[20] The 30% under filling in K 3 -EDTA containing vials did not affect the hematological parameters, (this is well above the extent of acceptable under-filling (i.e. upto 10%) as suggested by the standard guidelines) (CLSI). [3] When the under filling was increased to 60% (i.e. 2 ml blood in a vacutainer designed for 5 ml) a significant change in some of the hematological parameters was observed. [18] The MCV was decreased which in turn lowered Hct and MCHC values. [18] Similar observations were reported by Nemec et al. [20] at 67% under-filling in liquid K 3 -EDTA vials. These observations confirmed the results of earlier studies that have found the shrinkage of RBC during under-filling. [3],[15],[16],[17] Thus, it must also be noted that K 2 -EDTA and K 3 -EDTA containing vacutainers differ with regards to the magnitude of under-filling before a change in hematological parameters becomes apparent (more than 30% under-filling in K 3 -EDTA vacutainers had adverse effects on hematological parameters, while these effects did not appear in K 2 -EDTA vacutainers even at 75% under-filling).

The next question is: What could be the probable reason for the different behaviors of K 2 -EDTA and K 3 -EDTA during under-filling? The possibility is that they affect the cells differently. This was answered by Van Cott et al. [21] who compared the effect of liquid K 3 -EDTA and spray-dried K 2 -EDTA on the hematological parameters in standard volumes. In this study, they found that K 3 -EDTA affected many of the hematological parameters, e.g. total leukocyte count, TRBC, TPLT, reticulocyte count, HGB, Hct, MCV, MCHC, RDW and reduced their values. The results of above study matches with the earlier study done by Brunson et al. [22] in which it was proposed that dilutional effect of K 3 -EDTA could be responsible for these results. [21],[22] These findings were then accepted by the International Council for Standardization in Hematology and National Committee of Clinical Laboratory Standards (NCCLS). [23],[24] Thus, it appears that the same effect was either carried or might even amplify when the ratio of K 3 -EDTA to blood was reduced due to under-filling in earlier studies. EDTA is a recommended anticoagulant for hematological parameters as it best preserves cellular morphology of blood cell. [25] Since, EDTA is common in both the compounds (K 3 -EDTA and K 2 -EDTA), the observed difference could also be related to the K 2 and K 3 parts of the compounds.

Last question arises about the effect of the storage of under filled K 2 -EDTA vacutainers. It is stated that film should be made as early as possible as significant changes occur after 3 h of collection. [14] It is known that changes occur with time in EDTA anticoagulated blood. These are significant and early to develop if EDTA anticoagulated blood is kept at room temperature. Although TRBCs, WBCs and platelets, as well as red blood cell indices, are usually stable for 8 h after collection. Blood kept at 4° in refrigerator show statistically insignificant changes till 24 h. [14]

Although, we did not analyze the effect of under filling on storage related changes in our study, this has been answered in another study using the pathological samples by Giron et al. [26] who found that K 2 -EDTA did not affect most of the hematological parameters in under-filled vacutainers (27.5%) even after 24 h. In the same study, total WBC count, monocytes count and MPV showed a small significant difference, which was although statistically significant yet it was too small to be clinically significant. [26] On the other hand, study done K 3 -EDTA showed an increase in cell volume on standing (1.6% increase after 4 h). [27] Hence, under filled K 2 -EDTA provides better stability as compared to K 3 -EDTA during storage. The results of the present study must be seen in the light of few facts. First, we have included the blood samples from healthy donors. Pathological samples that include leukemic patients also may not behave in a similar manner and thus further studies are required. Second, neonates were not included deliberately as neonate samples were routinely collected already in pediatric vacutainers (500 μl). This volume of blood is minimal volume enough for the analyzer. Hence the experiment of under filling was not possible. Thirdly, we have analyzed the results up to 67% under-filling. Whether further under-filling affects, the results is yet to be seen. Finally, effect of under-filling on stored samples is yet to be analyzed as previous studies have assessed under-filling to a limited extent.

In conclusion, the present study suggests that even 67% under-filling blood from healthy donors in a K 2 -EDTA vial does not affect hematological parameters, The results of present study, shows the direction to conduct more and more studies of similar type in healthy as well as various pathological conditions. If further studies done on under filling of the pathological samples also give similar results as in our study then then only the present guidelines related to under filling could be revised when K 2 -EDTA is used as an anticoagulant. This will help the institutions not only to prevent phlebotomy related accidents but also, reduce the economic burden (cost imparted towards vacutainers used for repeat sample, towards the reagents and running cost of machines, toward manpower e.g. phlebotomist, technician etc.) and available resources can be utilized somewhere else. Last and most importantly, this will help to reduce the delay in reporting the results caused due to rejections related to under-filling K 2 -EDTA without affecting the results. This will add another milestone towards patient safety and care.


We are thankful to the Himalayan Institute of Medical Sciences, Dehradun (India) for providing us the financial support for this study vide project no PATH/2012/01.


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