Karin Plank1, Carla Dorn2, Stefan W Krause2. 1. Universität Erlangen-Nürnberg, Erlangen, Germany. 2. Medizinische Klinik 5, Universitätsklinikum Erlangen, Erlangen, Germany.
Abstract
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.
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.