フローサイトメトリー測定の問題点

―エスケープ現象―

抄録

Escapee phenomenon in flow cytometric analysis (FCM) of lymphocyte subsets is defined as a variable number of lymphocytes escaping from the conventional gate of lymphocytes to those of other hematological cells by correlated analysis of forward and side scatters. Although this phenomenon is well recognized and is an artifactual problem in FCM, little information is available about its mechanism. Recently, we examined a new mechanism of escapee phenomenon in FCM of lymphocytes, and this issue was reviewed. Using dot plots of forward scatter versus fluorescent intensity of anti-CD8 antibody labeled with FITC, CD8+ cells escaping from the conventional lymphocyte gate were separated to two populations: one was CD8+ escaping cells that were the size of granulocytes (type I escapee), and the other was CD8+ escaping cells that were the size of monocytes (type II escapee). Type I escapee also expressed CD13, and the fluorescent intensity of CD8 on these cells was equal to that of CD8 on non-escapee cells, which were within the conventional lymphocyte gate. Type I escapee appeared to contain a CD8+ lymphocyte and a granulocyte. Type II escapee expressed neither CD33 nor CD14, and the fluorescent intensity of CD8 on these cells was two times the intensity of CD8 on non-escapee cells. Type II escapee appeared to contain two CD8+ lymphocytes. Type I escapee disappeared by treatment with unlabeled anti-CD32 antibody, which can block FcRII, before staining with FITClabeled anti-CD8 antibody. Type I escapee was also reduced by treatment with anti-CD16 antibody, which can block FcRIII. Type II escapee was reduced by treatment with anti-CD32 or anti-CD16 antibodies. These results showed that escapee phenomenon of peripheral CD8+ lymphocytes in FCM occurred through Fc receptors. Thus, we propose the following model of the escapee phenomenon; peripheral CD8+ lymphocytes bind to other cells through the binding between Fc receptors on other cells and anti-CD8 antibody recognizing CD8+ lymphocytes, resulting in a shift from the conventional lymphocyte gate to the gate of granulocytes or monocytes in FCM.