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

www.aegis.org