Background:

North American and European guidelines for dual-platform (DP) flow cytometry recommendabsolute CD4 T-cell counts to be calculated from two parameters: the absolute lymphocyte counts obtainedon a hematology analyzer and the percentages of CD4 cells among lymphocytes (CD4%/lympho) obtainedby flow cytometry. Nevertheless, the identification of lymphocytes is error-prone: a poor match betweenthese common denominators in the two systems is the main source of inaccuracy. In contrast, total leucocytecounts (white cell counts [WCC]) and CD4% among the gated CD45 leucocytes (CD4%/leuco) can bedetermined with greater accuracy.

Methods:

We introduced “PanLeucogating,” i.e., we used total leucocytesas the common denominator for improving the precision of DP absolute CD4 counting. Correlations andBland-Altman tests were used for statistical analysis.

Results:

First, 22 stabilized blood product sampleswere provided by U.K. National External Quality Assessment Scheme (NEQAS) and a higher accuracy andprecision of CD4 counts were documented using PanLeucogating compared with lymphocyte gating. Next,183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and single-platform (SP) methodology, including both volumetric and bead-based techniques. A particularly highcorrelation and comparable precision of absolute CD4 counts were observed between the SP volumetricmethod and DP PanLeucogating (R2 0.990; bias 6 SD 17%). The SP volumetric method showed lowerlevels of agreement with the DP lymphocyte gating (R2 0.758; bias 14 SD 51%) and with the SPbead-based method (R2 0.923; bias 4 SD 31%).

Conclusions:

These observations show that DPleucocyte counts (WCC) should replace lymphocyte counts as the “common denominator” although CD4%/lymphovalues can, as an extra step, be also provided readily if requested. When coupled with quality controlfor WCC on hematology analyzers, the DP method with CD45 PanLeucogating represents a robust CD4 T-cellassay that is as accurate as the SP volumetric technique. This DP method uses only two, CD45 and CD4,antibody reagents and can be run on any pair of hematological analyzer plus flow cytometer.

Background:

North American and European guidelines for dual-platform (DP) flow cytometry recommendabsolute CD4 T-cell counts to be calculated from two parameters: the absolute lymphocyte counts obtainedon a hematology analyzer and the percentages of CD4 cells among lymphocytes (CD4%/lympho) obtainedby flow cytometry. Nevertheless, the identification of lymphocytes is error-prone: a poor match betweenthese common denominators in the two systems is the main source of inaccuracy. In contrast, total leucocytecounts (white cell counts [WCC]) and CD4% among the gated CD45 leucocytes (CD4%/leuco) can bedetermined with greater accuracy.

Methods:

We introduced “PanLeucogating,” i.e., we used total leucocytesas the common denominator for improving the precision of DP absolute CD4 counting. Correlations andBland-Altman tests were used for statistical analysis.

Results:

First, 22 stabilized blood product sampleswere provided by U.K. National External Quality Assessment Scheme (NEQAS) and a higher accuracy andprecision of CD4 counts were documented using PanLeucogating compared with lymphocyte gating. Next,183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and single-platform (SP) methodology, including both volumetric and bead-based techniques. A particularly highcorrelation and comparable precision of absolute CD4 counts were observed between the SP volumetricmethod and DP PanLeucogating (R2 0.990; bias 6 SD 17%). The SP volumetric method showed lowerlevels of agreement with the DP lymphocyte gating (R2 0.758; bias 14 SD 51%) and with the SPbead-based method (R2 0.923; bias 4 SD 31%).

Conclusions:

These observations show that DPleucocyte counts (WCC) should replace lymphocyte counts as the “common denominator” although CD4%/lymphovalues can, as an extra step, be also provided readily if requested. When coupled with quality controlfor WCC on hematology analyzers, the DP method with CD45 PanLeucogating represents a robust CD4 T-cellassay that is as accurate as the SP volumetric technique. This DP method uses only two, CD45 and CD4,antibody reagents and can be run on any pair of hematological analyzer plus flow cytometer.