Improvement in isolation of human peripheral blood leukocyte subpopulations: application in diagnosing human cytomegalovirus infection in bone marrow transplant patients.

OBJECTIVES High-yield isolation and purification of human leukocyte subpopulations from whole blood is fundamental to many biological and medical applications including qualitative and quantitative PCR-based techniques of determining human cytomegalovirus infection. Several procedures have been reported to purify morphologically and functionally intact human leukocyte subpopulations for diagnostic proposes. Here, we report and evaluate a technique for high-yield purification of intact and viable human leukocyte subpopulations based on modification of a previous methodology. MATERIALS AND METHODS One hundred peripheral blood samples were collected from bone marrow transplant recipients (n = 60), bone marrow donors (n = 20), and healthy blood donors (n = 20). The samples were tested in parallel using 4 different leukocyte separation methods. The methods were evaluated based on the concentration, purity, and viability of the isolated leukocyte subpopulations. RESULTS When compared with standard methods, our methods produced 99% purity for both polymorphonuclear or mononuclear leukocytes. The corresponding viability for the methods was determined to be 98%. No erythrocyte contamination was demonstrated. However, the maximum concentration for polymorphonuclear or mononuclear leukocytes obtained by standard methods was 70%. The corresponding viability for all the methods was determined to be 98%. CONCLUSIONS Our results indicate that in patients with decreased whole blood leukocyte numbers, using either a modified Ficoll NH(4)Cl or a modified dextran method would be valuable for simultaneous separation of polymorphonuclear and mononuclear leukocytes with high purity, viability, and concentration.