| Description |
Immunophenotyping of helper lymphocytes, known as CD4+ T-cells, is important to assess the functional immune system of HIV+ patients and determine the appropriate strategy for further therapy. The increasing demand for absolute CD4 counts indicates a need for evaluation of different parameters that may affect the CD4 count by flow cytometry. Major areas of concern are factors (type of anticoagulant, room temperature, time) and can affect CD4 results and sensitivity, specificity, accuracy, and precision of different analytical methods to count CD4+ T-cells. To evaluate the effect of different anticoagulants, room temperature and time (up to 72 h after the draw) on absolute CD4 count and hematology analysis (WBC, lymphocyte%) blood specimens were collected in different anticoagulants (heparin, EDTA, and ACD). A standard dual color staining with the CDC recommended panel (CD3/CD4+, CD3/CD8, CD3/CD19, CD14/CD45, and CD3/CD56 or 16) was used for flow cytometry analysis. Statistical analysis shows that absolute CD4+ T-cell results are dependent on the type of method used to obtain the lymphocyte%. Heparin can be used up to 72 h to obtain manual lymphocyte%. ACD is anticoagulant of choice for CD4% by conventional flow cytometry. EDTA showed significant differences for CD4% at all times. By using the manual lymphocyte% up to 72 h after blood collection, ACD and heparin can safely be used as anticoagulants. By using the automated lymphocyte%, heparin was the only anticoagulant that showed no significant difference for absolute CD4 counts over 72 h incubation at room temperature. EDTA can be used for hematology analysis (WBC, lymphocyte%) up to 48 h after blood collection. The WBC results of 48- and 72-h EDTA specimens showed no significant difference from the 0-6 h values. The type of method to count absolute CD4 is also an important issue. In the conventional method (using CDC recommended panel) to a different flow cytometry method that uses fluorescent beads as an internal standard, and manual method in which the rosette formation of CD4+ T-cells with anti-CD4 coated spheres are visualized and counted under microscope. As the statistical analyses of these methods indicate both flow cytometry methods provide fairly close sensitivity, specificity, accuracy, and precision results to each other. The manual method results were not as close but showed enough sensitivity to be used as an alternative to flow cytometry where flow is not available. |
