Evaluation of Different Mixing Procedures for K2 EDTA Primary Samples on Hematological Testing

Giuseppe Lippi, MD; 1 Gian Luca Salvagno, MD; 1 Martina Montagnana, MD; 1 Giuseppe Banfi, MD; 2 Gian Cesare Guidi, MD1


Lab Med. 2007;38(12):723-725. 

In This Article


An accurate test result begins with a high-quality specimen. Therefore, the awareness of the main sources of preanalytical variability and their effects on hematology testing is crucial to ensure reliable and accurate test results. There is a variety of preanalytical variables that can seriously affect the reliability of laboratory testing, some of which are barely preventable and identifiable by the laboratory staff. These include patient-related physical variables (physical exercise, diet, stress, positional effects), incorrect sample identification, and inappropriate procedures for collection and handling of the specimens, such as the use of improper collection tools, prolonged stasis during venipuncture, and unsuitable storage.[6,7,8] The governance of each of these preanalytical variables optimizes patient care, reduces laboratory costs, and enhances the physician-laboratory confidence.

The EDTA salts are widely used as anticoagulants in blood collection tubes. EDTA anticoagulates blood by chelating calcium, which is necessary for blood clotting since its removal inhibits a wide series of enzymatic reactions of the coagulation pathway. The International Council for Standardization in Hematology (ICSH) currently recommends K2 EDTA as the anticoagulant of choice for hematological testing.[2] This indication has been widely acknowledged in Europe and Japan, whereas K3 EDTA is still frequently used in the United States and the United Kingdom.[9] K2 EDTA is available in a spray-dried form, which does not introduce dilutional effects on small sample volumes. It is associated with a less pronounced osmotic effect on blood cells than K3 EDTA,[2] but it appears to be associated with more complaints regarding blood clotting.[9] In fact, as K2 EDTA is dispensed as a powder on the inside walls of the vial, primary tubes containing this additive may need to be properly mixed to allow a complete miscibility between the blood and the anticoagulant.

The present investigation confirms that results of hematological testing on unmixed primary K2 EDTA samples may suffer from a slight but significant preanalytical bias, which is, however, clinically acceptable according to the current analytical quality specifications for desirable bias. The analytical modules of most automated cell counters are currently provided with autosamplers that invert the primary tubes several times before they are analyzed. This procedure is necessary to enable the resuspension of the blood prior to analysis, but it has little influence on the appropriate blood-to-anticoagulant mixing that occurs immediately after venipuncture. Our results are consistent with the hypothesis that K2 EDTA coating on the walls of the tube, and natural inversion when drawing from a primary tube may have permitted sufficient mixing/contact with anticoagulant to prevent excess clotting. Accordingly, the blood-to-K2 EDTA mixture that occurs during the blood collection procedure may still be acceptable to achieve results within acceptable limits of bias, provided the tubes are adequately filled to their nominal volume. We are aware of some limitations in this study. First, we analyzed blood samples collected from healthy volunteers, and we could not confirm these results in patient's samples with abnormal counts and where these effects might be greater and of clinical relevance. Although these conclusions mainly apply to our experimental conditions, however, we hypothesize that the large majority of clotted specimens received in clinical laboratories may be due to different causes than inappropriate mixing.[9]


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