Suitability of Lower K2 EDTA Sample Volumes for Absolute CD4 Count Enumeration by Flow Cytometric Technique

Abstract

Background: Incorrect blood sample volume-anticoagulant ratio has been the cause of both haematological and immunological errors especially when K3 EDTA-containing blood collection tubes were used. Lower whole blood sample volumes collected into 4.0 millilitres spray-dried K2 EDTA has been shown to overcome incorrect haematology results when analysed on automated haematology analyzers but there is no experimental evidence for the same in the CD4 count enumeration by flow cytometric technique. Materials and Methods: 9.0 ml of whole blood was collected from each of fifteen retroviral and ten normal volunteers and aliquot into five different 4.0 ml plastic spray-driedK2 EDTA blood collection tubes containing 4.0, 2.0, 1.5, 1.0 and 0.5 ml respectively. Each well-mixed sample was analysed on Partec Cyflow counter within 4 hours of collection for absolute CD4+T lymphocyte count. Results:  Both the reference sample volume 4.0 ml  and experimental lower sample volumes (2.0, 1.5, 1.0 and 0.5 mls) of retroviral volunteers in 4.0 ml plastic spray-driedK2 EDTA blood collection tubes gave comparable CD4 count results with percentage mean difference of 1.82%, -1.48%, 2.25% and 0% for 2.0 ml, 1.5 ml 1.0 ml and 0.5 ml respectively. Irrespective of sample volumes, the normal volunteers had higher CD4 count results. There was no statistically and clinically significant difference in the CD4 counts and the percentage mean difference were 0.4%, 0.17%, 1.00% and 0.23% for 2.0 ml, 1.5 ml, 1.0 ml and 0.5 ml respectively. The correlation (slope)and  modest logistic regression coefficient (R2) of experimental lower sample volumes of both retroviral and normal volunteers were between 0.9500 and 1.0000 showing excellent agreement in the CD4 counts of both reference and experimental sample volumes(p<0.01). Conclusion: Quality CD4 count results can be obtained with a minimum sample volume of 0.5 ml in 4.0 ml spray-dried K2 EDTA vacutainer blood collection tubes both in HIV and healthy individuals with intact immune function. Funding: This research did not receive any grant from any funding agency in the public, commercial or non-profit sector.