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Background:  With the recent dramatic increase in access to ART, simpler and less costly methods to monitor treatment—including absolute CD4⁺ T-cell enumeration—are urgently needed. Accordingly, the Guava Technologies, Inc. microcapillary flow cytometric EasyCD4⁺ assay was evaluated vs the predicate method used in 5 highly experienced flow cytometry laboratories to compare the inter-laboratory variability  and to examine the difference between the CD4⁺ T cell counts obtained by the EasyCD4 assay and the laboratories’ usual method.

Methods:  Whole blood specimens (n = 66) were collected by the NIAID Immunology Quality Assurance Laboratory (IQA) and shipped overnight to each of the other 4 participating laboratories. Specimens were stratified by CD4⁺ T-cell count (determined by the usual IQA method), > 300 cells/µL (n = 23) and ≤ 300 to 600 cells/µL (n = 43). Each site tested each specimen in triplicate by the EasyCD4 assay; a tube of matched blood was shipped for analysis by the usual (predicate) method in each site’s licensed clinical laboratory.

Results:  To estimate inter- and intra-site variability, percentage of coefficient of variation were calculated using each site’s predicate result and a randomly chosen value from the 3 EasyCD4 replicates at each site. This analysis reveals:  the median inter-site percentage of coefficient of variation for CD4⁺ T-cell counts >300 cells/µL were 11.3 and 16.2 (p = 0.0006) for Guava EasyCD4 and site predicate methods, respectively; the median inter-site percentage of coefficients of variation for CD4⁺ T cell counts ≤ 300 cells/µL were 9.5 and 10.0 (p = 0.7627) for Guava EasyCD4 and site predicate methods, respectively; the median intra-site percentage of coefficients of variation for CD4⁺ T-cell counts < 300 cells/µL were 5.0, 4.7, 6.1, 6.9, and 6.2 (p = 0.3875); d) the median intra-site percentage of coefficients of variation for CD4⁺ T-cell counts ≤ 300 cells/µL were 3.2, 4.4, 4.6, 5.2, and 4.5 (p = 0.0459). The Wilcoxon signed rank statistical test was used for inter-site comparisons. To estimate intra-site variability, percentage of coefficients of variation were calculated using the 3 EasyCD4 replicates and the Friedman statistical test.

Conclusions:  The EasyCD4 assay inter- and intra-site variability and correlation with predicate methods were excellent, at both CD4⁺ T-cell ranges (> 300 and ≤ 300 cells/µL) analyzed. The EasyCD4 assay represents a simple, low-cost alternative to traditional methods for CD4⁺ T-cell enumeration.

Keywords: CD4+ T cell enumeration; multi-site study; ARV monitoring