The effect of erythrocyte lysing reagents on enumeration of leukocyte subpopulations compared with a no-lyse-no-wash protocol.

INTRODUCTION

Standard protocols in flow cytometry (FCM) require lysis of erythrocytes, which may induce an unwanted loss of leukocytes as bystander effect.

METHODS

In the present study, we investigated the influence of 6 laboratory protocols using 4 different lysing reagents, FACS Lysing Solution (FacsL), QUICKLYSIS (QuickL), IOTest 3 Lysing Solution (NH4Cl), VersaLyse (VersaL), and VersaLyse with added fixative (VersaFix) on the relative quantity of leukocyte subsets identified by CD3, CD4, CD8, CD19, CD14, CD16, CD56, and CD45, applying a no-lyse-no-wash (NoL) protocol as reference. In addition, we compared the efficiency of red blood cell (RBC) lysis.

RESULTS

Peripheral blood samples from 52 individuals were analyzed. NoL was suitable as reference method, but led to less clear-cut gating of lymphocyte and monocyte populations due to a wider distribution of light scatter. Best completeness of RBC lysis with remaining erythrocytes below 10% was achieved using NH4Cl and VersaL. We observed a loss of 11% of monocytes after QuickL. Lymphocyte counts were 19% lower after FacsL. Cell subsets within the lymphocyte compartment were rather similar between the different methods with the exception of lower B-cell counts (-8%) and higher NK-cell counts (+11%) after FacsL. NH4Cl and VersaL were in good accordance with the NoL method and also with the mean values of all methods.

CONCLUSION

Our data show that the lysing reagents tested lead to specific deviations in the quantitation of leukocyte subsets and show different efficiency of erythrocyte lysis.