Highly Reproducible Flow Cytometric Analyses of T-Cell Subsets Following Prolonged Storage of Blood Samples in Liquid Nitrogen 

            Experimental protocols for the treatment of HIV-1 infection, conducted by the AIDS Clinical Trials Group or by various pharmaceutical companies, frequently mandate same-day shipment of blood samples to certified laboratories for determination of CDC-recommended T-cell subsets.  For geographically distant sites, such as Hawaii, this requirement poses a recurrent logistical challenge and contributes to additional expense.  To investigate if blood samples for flow cytometric analysis can be stored for later batch shipment and analysis, we compared CD3, CD4, CD8, CD19 and CD56 counts in blood samples from 52 HIV-1-infected individuals and 21 uninfected healthy controls, analyzed at the time of collection and after storage in vapor-phase liquid nitrogen for 6 weeks.  Blood samples were processed for flow cytometry within 6 hrs of collection; an aliquot was analyzed on the day of collection and an aliquot was stored in liquid nitrogen for analysis 6 weeks after collection, using a Coulter XL-MCL flow cytometer. Statistical comparison between flow cytometric analyses of T- and B-cells and monocytes in fresh and frozen samples revealed low degrees of variance and high correlation coefficients (r ≥ 0.95, P < 0.01), even in samples from HIV-1-infected individuals with relatively low CD4 counts (<100/mm3; r = 0.98, P < 0.01).  These data indicate that blood samples, collected from HIV-1-infected and uninfected individuals at distant sites, can be routinely processed then rapidly frozen in liquid nitrogen for later analysis of T- and B-cells. This knowledge will make flow cytometric studies of remote populations technically more feasible and logistically less daunting. These data also suggest that samples for studies requiring flow cytometric analyses could initially be processed, then frozen for batch shipment, leading to substantial savings in transportation costs.

Nerurkar, V.R., Kimura, L.H, Dashwood, W-M., Johnson, A.C. III, Hill, R., and Yanagihara, R.:  Highly reproducible flow cytometric analyses of T-cell subsets following prolonged storage of blood samples in liquid nitrogen.  Journal of Clinical Microbiology  (in preparation).  

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