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


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.
Clinical Virology Section, Organ Transplant Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran. Behbahani_2000@yahoo.com
A
A
Behzad-Behbahani
R
R
Yaghobi
F
F
Sabahi
M H
MH
Rostaei
A
A
Alborzi
D016026:Bone Marrow Transplantation; D002469:Cell Separation / Q000379:methods; D003586:Cytomegalovirus Infections / Q000175:diagnosis*; D007962:Leukocytes / Q000166:cytology*; D007963:Leukocytes, Mononuclear / Q000166:cytology; D009504:Neutrophils / Q000166:cytology; D011183:Postoperative Complications; D014019:Tissue Donors