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

Abstract

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.

• Authors: A Behzad-Behbahani, R Yaghobi, F Sabahi, M H Rostaei, A Alborzi
• Journal: Experimental and clinical transplantation : official journal of the Middle East Society for Organ Transplantation (Exp Clin Transplant) Vol. 3 Issue 1 Pg. 316-9 (Jun 2005) ISSN: 1304-0855 [Print] Turkey
• PMID: 15989676 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Topics

• Bone Marrow Transplantation
• Cell Separation (methods)
• Cytomegalovirus Infections (diagnosis)
• Leukocytes (cytology)
• Leukocytes, Mononuclear (cytology)
• Neutrophils (cytology)
• Postoperative Complications
• Tissue Donors