Quantification of the variation due to lysing technique in immunophenotyping of healthy and HIV-infected individuals

We performed a side-by-side comparison between three stain-then-lyse commercially available methods (Ortho-mune Lysing solution, FACS lysing solution and ImmunoPrep reagent system) and a lyse-then-stain method using hypotonic NH4Cl. The major difference between these methods is that only in the latter the aliquots of sample to be distributed into diverse tubes for the various antibody combinations were obtained from a lysis step performed in the same tube.

Lymphocytes from 20 healthy and 20 HIV+ subjects were phenotyped by dual color flow cytometry using a standard procedure that included the establishing of a lymphocyte gate on light scatter bit map and the use of the minimal acceptable antibody combinations, i.e., CD45/CD14, CD3/CD4 and CD3/CD8, according to CDC recommendations. All samples were processed in triplicate to assess tube-to-tube variability.

In healthy subjects, erythrocytes pre-lysing provided the highest purity and recovery in the lymphocyte gate, and allowed the best identification of CD4+ lymphocytes. Most remarkably, erythrocytes pre-lysing significantly outdid all other methods in reducing tube-to-tube variability. This allowed the attainment of highest correlation between CD3+ cells identified by CD3/CD4 and CD3/CD8 antibody combinations and the minimum variability between the sum of the %CD3+CD4+ and %CD3+CD8+, and the total %CD3+. This higher reliability of the pre-lysis method was particularly evident with HIV+ patients in which the lymphocyte gate was often and unpredictably contaminated by debris and other cell types.

The present study demonstrates that lysing erythrocytes in a single tube and distributing aliquots of lysed blood into different tubes for the various antibody combinations provides superior results for routine immunophenotyping.