Enumeration of CD4(+) T-cells in the peripheral blood of HIV-infected patients: An interlaboratory study of the FACSCount system

The aim of the present study was to assess the interlaboratory reproducibil ity of the FACSCount system for the enumeration of peripheral blood (PB) CD 4(+) T-cells. In each of the seven participating centers, both previously s tained and unstained PB samples (n = 49) were received and either analyzed or stained and then analyzed. Interlaboratory reproducibility was checked i n two different groups of centers (n = 3 and n = 4) where the study was per formed in parallel. In addition, both the intralaboratory precision and acc uracy of this system were analyzed in comparison with results obtained with conventional flow cytometry. Accordingly, upon comparing both methods, a h igh degree of correlation was observed in the total number of CD3(+) T-cell s (coefficient of correlation of 0.9750 +/- 0.0184, slope of the best linea r fit: 0.9214 +/- 0.0311, y-intercept of 12 +/- 47) as well as in the numbe r of CD3(+)/CD4(+) (coefficient of correlation of 0.9794 +/- 0.1457, slope of the best linear fit: 0.9463 +/- 0.0753, y-intercept of -11 +/- 36) and C D3(+)/CD8(+) (coefficient of correlation of 0.9728 +/- 0.0192, slope of the best linear fit: 0.9682 +/- 0.0735, y-intercept of 7 +/- 95) major subsets . In addition, tow coefficients of variation (CV) were obtained for replica tes, indicating the method's high degree of accuracy. The present study sho ws that with respect to the interlaboratory reproducibility reported for mo st techniques used for the enumeration of PB CD4(+) T-cells, the FACSCount system results in data with much lower coefficients of variance (CVs) (mean CV of less than 10%), Upon measuring the impact on results of different va riables associated with either sample preparation or data acquisition and a nalysis, our study clearly shows that data acquisition and analysis does no t influence the results by increasing variability since the coefficients of variation obtained for samples prepared in the same laboratory under the s ame conditions and read in different laboratories with different instrument s were identical to those obtained far the replicates of the same samples r ead in each individual center. In contrast, interlaboratory variability, al though low, significantly increased when sample preparation was carried out in different laboratories, suggesting that pipetting still represents the major source of variability in the FACSCount system. (C) 1999 Wiley-Liss, I nc.