Evaluation of a new single-parameter volumetric flow cytometer (CyFlow(green)) for enumeration of absolute CD4+ T lymphocytes in human immunodeficiency virus type 1-infected Thai patients

Abstract

Use of the standard dual-platform flow cytometric method for determination of CD4(+) T-lymphocyte counts, which needs both a flow cytometer (FCM) and hematological analyzer, would inevitably lead to increased variability. The development of new single-platform (SP) FCMs that provide direct CD4(+) T-lymphocyte counts for improved assay precision and accuracy have recently attracted attention. This study evaluated one of those systems, CyFlow(green) (Partec), a single-parameter SP volumetric FCM. The performance of CyFlow(green) was compared with those of two reference standard SP microbead-based technologies of the three-color TruCOUNT tube with the FACScan FCM and a two-color FACSCount system (Becton Dickinson Biosciences). Absolute CD4(+) and CD8(+) T-lymphocyte counts in 200 human immunodeficiency virus type 1-seropositive blood specimens were determined. Statistical analysis for correlation and agreement were performed. A high correlation of absolute CD4 counts was shown when those obtained with CyFlow(green) were compared with those obtained with the bead-based three-color TruCOUNT system (R(2)=0.96; mean bias, -69.1 cells/microl; 95% confidence interval [CI], -225.7 to+87.5 cells/microl) and the FACSCount system (R(2)=0.97; mean bias, -40.0 cells/microl; 95% CI, -165.1 to+85.1 cells/microl). The correlation of the CD4(+) T-lymphocyte counts obtained by the two bead-based systems was high (R(2)=0.98). Interestingly, CyFlow(green) yielded CD4(+) T-lymphocyte counts that were 21.8 and 7.2 cells/microl lower than those obtained with the TruCOUNT and the FACSCount systems, respectively, when CD4(+) T-lymphocyte counts were <250 CD4(+) T-lymphocyte counts/microl range or 17.3 and 5.8 cells/microl less, respectively, when CD4(+) T-lymphocyte counts were <200 cells/microl. The single-parameter CyFlow(green) volumetric technology performed well in comparison with the performance of the standard SP bead-based FCM system. However, a multicenter comparative study is needed before this FCM machine is implemented in resource-limited settings.

FIG. 1.

Representative flow cytometric histograms illustrating the software algorithm of the three-color TruCOUNT MultiSET system (A to C), the two-color FACSCount system (D to F), and the single-parameter CyFlow^green FlowMax system (G and H).

FIG. 2.

Linear regression analysis of absolute CD4⁺ and CD8⁺ T-lymphocyte counts between the volumetric CyFlow^green flow cytometric method and the two standard bead-based flow cytometric methods. Correlation plots for the CyFlow^green system versus the three-color TruCOUNT system with FACScan (A and D), the CyFlow^green system versus the FACSCount system (B and E), and the FACSCount system versus the TruCOUNT system (C and F) are shown.

FIG. 3.

Bland-Altman (2) bias plots of absolute CD4⁺ and CD8⁺ T-lymphocyte counts between the volumetric CyFlow^green flow cytometric method and the two standard bead-based flow cytometric methods. The differences between the CyFlow^green system and the three-color TruCOUNT with FACScan system (A and D), the CyFlow^green system and the FACSCount system (B and E), and the FACSCount system and the TruCOUNT system (C and F) are shown. The horizontal line in the center indicates the mean bias of the two methods. The lower and upper limits of agreement are indicated by the lower and the upper horizontal lines, respectively.

FIG. 4.

Bland-Altman (2) bias plots of absolute CD4⁺ T-lymphocyte counts of <250 cells/μl between the volumetric CyFlow^green flow cytometric method and the two standard bead-based flow cytometric methods. The differences between the CyFlow^green system and the three-color TruCOUNT with FACScan system (A), the CyFlow^green system and the FACSCount system (B), and the FACSCount system and the TruCOUNT system (C) are shown. The horizontal line in the center indicates mean bias of the two methods. The lower and upper limits of agreement are indicated by lower and upper horizontal lines, respectively.