In recent years, HIV lymphocyte immunophenotyping has evolved significantly. New fluorochromes and multicolor reagents, enhanced instruments, and the capacity to provide absolute cell counts using single-platform techniques have all contributed to the reliability of T-cell subset measurements. Previous studies in aging specimens have shown that the choice of gating strategy had a dramatic impact on immunophenotyping results in specimens that were analyzed beyond 48 hours [10
]. Specifically, the largest deviations from baseline values occurred at 96 hours and gating protocols that included dual light scatter gates provided the greatest shift of T-cell subset values over time. In contrast, gating protocols that were based exclusively on cell lineage-specific gates gave the most robust T-cell values up to 96 hours, thus demonstrating that by selecting the most appropriate gating protocol, the temporal integrity of specimens can be extended up to 4 days [10
]. Because there is only limited literature data for time and temperature requirements of dual-platform methods, we performed an internal study regarding the effect of specimen age and refrigeration on T-cell subsets. The impetus for performing this analysis stemmed from the fact that, while hematology analyzers were available around the clock, no immunophenotyping was available during off shifts. Thus, peripheral blood specimens would potentially have to be analyzed for T-cell subsets beyond the recommended 48-hour window, even though complete blood count data could have been performed within 24 hours from the draw.
When comparing absolute CD4 and CD8 counts obtained from aging specimens store at RT, the CD4 subset showed at positive bias at both 72 and 96 hours, while the CD8 subset demonstrated a similar trend only at 96 hours. Of note, reported acceptable intralaboratory absolute count ranges are within 75 cell/μL for CD4 and within 150 cells/μL for CD8 [11
], which are similar to the differences seen in our study at RT. Aging specimens preserved at 4°C showed a negative bias and less variation in the CD4 absolute counts, as compared to specimens held at RT. CD8 absolute counts demonstrated a negative trend at 72 hours of refrigeration, as well. Of note, the proportions of outliers were higher for the cold-stored specimens; however, the outliers did not occur preferentially at either high or low absolute T-cell counts. When comparing mean % CV for subset determinations at different time points, the values were similar to values reported for aging specimens on single-platform methods [10
]. Once again, measurements performed on refrigerated specimens showed a lower % CV compared to those performed on samples stored at RT. One limitation of the study is that the number of cold-stored specimens with CD4 absolute counts <300 cells/μL was relatively small (n=12).
Our findings support the robustness of T-cell subset enumeration by a dual-platform methodology in aging specimens, up to 96 hours at RT, and up to 72 hours after refrigeration at 4°C. Similar to results reported in other, single-platform studies, our gating strategy takes advantage of the fact that lymphocytes maintain a uniform, high-density expression of CD45 at least up to 96 hours. This is unlike traditional, dual light scatter gates, in which there is significant drift of lymphocytes outside the initial gate when the specimen is older than 24 hours. Also, while initial studies have reported that single-platform methods resulted in lower interlaboratory variations of absolute T-cell counts (as compared to dual-platform methods) [12
], a recent report actually did not show statistical differences between the two methods [11
]. At least in this context, dual-platform methods show similar performance characteristics to single-platform techniques, thus suggesting the successful employment of either methodology in achieving accurate results.
From a logistical standpoint, our results suggest that T-cell subset immunophenotyping can be performed on aging specimens stored at RT or at 4°C, with results generated within published acceptable ranges. This allows off shift processing of specimens, which in our laboratory account for <10% of the total subset immunophenotyping volume.