Which blood collection tubes are appropriate for peripheral blood flow cytometry?

For flow cytometry on peripheral blood, the acceptable anticoagulants are ethylenediaminetetraacetic acid (EDTA; lavender top), sodium heparin (green top), and acid citrate dextrose (ACD; yellow top).

EDTA and ACD act similarly by binding calcium in the blood, which inhibits the clotting process. EDTA, however, binds calcium more strongly and irreversibly while preserving cellular components and morphology of blood cells. EDTA is the anticoagulant of choice if a complete blood count and white blood cell differential are performed from the same sample used for flow cytometry. Since ACD is in liquid form, it is not acceptable for single platform quantitative flow cytometric assays such as lymphocyte subset absolute count.

Heparin binds to the enzyme inhibitor antithrombin III. This complex accelerates the inactivation of thrombin and factor X to prevent the formation of fibrin from fibrinogen. Heparin tends to cause white blood cell clumping and faint blue background in Wright-Giemsa stained smears. It is, therefore, not the ideal anticoagulant if both hematology and flow cytometry studies are to be performed on the same specimen. However, it is preferred for bone marrow aspirates.

All three anticoagulants have different stability. Sodium heparin samples are reportedly stable for 48-72 hours, EDTA samples for 48 hours, and ACD samples for 72 hours. However, flow cytometry laboratories should do their own validation to determine stability time for each anticoagulant.

Reference:
Davis BH, Dasgupta A, Kussick S, Han J-Y, Estrellado A; on behalf of ICSH/ICCS working group. Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS- Part II- Preanalytical issues. Cytometry Part B 2013; 84B: 286-290.