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Lymphocyte subset analysis on frozen whole blood.


3rd Conf Retro and Opportun Infect. 1996 Jan 28-Feb 1;:155. Unique

Background: Lymphocyte subset analysis by flow cytometry is generally performed on fresh blood samples, which is not cost-efficient for small batches and precludes analysis of serial samples in controlled runs. We report an alternative method utilizing frozen whole blood. Methods: Aliquots of 20 EDTA-anticoagulated whole blood samples (11 HIV+, 9 HIV-) were tested with anti-CD3/4/45 and anti-CD3/8/45 fresh (less than 18 hrs. post draw), and after freezing for 7-180 days at -80C or 160C (LN2), with and without 10% DMSO. Analysis utilized gating on bright CD45 cells with low side 70 scatter. Lymphocyte counts were obtained on fresh samples with an automated analyzer. Results: Percentage of CD3 + , CD4 + , CD8 + lymphocytes correlated closely in fresh and frozen aliquots. + Figure shows linear regression for % CD3+4+ cells, fresh vs. frozen at -80C (slope = 1.015x, y-intercept = -0.35). Net mean fluorescence intensity was slightly lower in frozen cells compared to fresh (-10% loss for CD4+ without DMSO; -5% with DMSO). Conclusions: Ability to accurately enumerate lymphocyte subsets on frozen whole blood by combining fresh cell counts with immuno-phenotyping on frozen samples should facilitate: 1) cost-effective batch testing, 2) analysis of frozen repository samples, and 3) testing of serial samples in the same run, thereby diminishing run-to-run variability and allowing detection of small changes in subset markers for monitoring disease progression and effects of therapy. See original to view figure.

Antigens, CD3/ANALYSIS Antigens, CD4/ANALYSIS Antigens, CD8/ANALYSIS Cell Separation Flow Cytometry Human *Lymphocyte Subsets Lymphocytes/IMMUNOLOGY Regression Analysis ABSTRACT


Information in this article was accurate in November 30, 1996. The state of the art may have changed since the publication date. This material is designed to support, not replace, the relationship that exists between you and your doctor. Always discuss treatment options with a doctor who specializes in treating HIV.