QUANTIFICATION OF THE VARIATION DUE TO LYSING TECHNIQUE IN IMMUNOPHENOTYPING OF HEALTHY AND HIV-INFECTED INDIVIDUALS

Objective: 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-st ain method using hypotonic NH4Cl. The major difference between these m ethods is that only in the latter the aliquots of sample to be distrib uted into diverse tubes for the various antibody combinations were obt ained from a lysis step performed in the same tube. Design and Methods : 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 us e of the minimal acceptable antibody combinations, i.e., CD45/CD14, CD 3/CD4 and CD3/CD8, according to CDC recommendations. All samples were processed in triplicate to assess tube-to-tube variability. Results: I n healthy subjects, erythrocytes pre-lysing provided the highest purit y and recovery in the lymphocyte gate, and allowed the best identifica tion of CD4(+) lymphocytes. Most remarkably, erythrocytes pre-lysing s ignificantly outdid all other methods in reducing tube-to-tube variabi lity. This allowed the attainment of highest correlation between CD3() cells identified by CD3/CD4 and CD3/CD8 antibody combinations and th e minimum variability between the sum of the %CD3(+)CD4(+) and %CD3(+) CD8(+), and the total %CD3(+). This higher reliability of the pre-lysi s method was particularly evident with HIV+ patients in which the lymp hocyte gate was often and unpredictably contaminated by debris and oth er cell types. Conclusions: The present study demonstrates that lysing erythrocytes in a single tube and distributing aliquots of lysed bloo d into different tubes for the various antibody combinations provides superior results for routine immunophenotyping. Copyright (C) 1998 The Canadian Society of Clinical Chemists.