| 1.1 Reasons for the necessity of proper data analysis Even today, many laboratories still continue the less than desirable practice of reporting antigenic expression as the percentage of positive cells. In this approach to FCM analysis, (1) the cell population is gated first by light scatter, then the antibody fluorescence analyzed on single parameter histograms or dual parameter plots; (2) for each marker, a cursor is moved and set to measure the fraction of cells with fluorescence greater than that of the control sample (in which cells are exposed to an irrelevant immunoglobulin); 3) the results are then reported as percent positive per antibody tested. （上述分析方式都是免疫组化时代遗留下来的） The origins of this approach to data reporting can be traced back to the microscopic evaluation of immunostaining performed on glass slides (smears, cytospin preparations) and the reporting techniques used for lymphocyte subset analysis (e.g., CD4 counts in human immunodeficiency virus [HIV]-infected patients). In many laboratories, the larger share of the FCM workload is composed of T- (or other) cell subset determinations on peripheral blood samples that do not harbor malignant cells. In this setting, a change in the number of cells in each subset is clinically important. Furthermore, the cells analyzed are discrete subpopulations of normal lymphoid cells with relatively bright fluorescence. Therefore, it is appropriate to report each subset as a percent positive for each antibody and, where applicable, to calculate the CD4 : CD8 ratio. The numerical values thus generated are reminiscent of those obtained for chemistry tests, in which the abnormalities consist of altered levels of the normal components in the blood. 但在血液肿瘤分析时，更注重的不是表达百分比，而是其表达模式（见下段文字） In samples suspected of harboring a hematopoietic malignancy, however, determining the exact number of neoplastic cells is less important than determining whether or not neoplastic cells are present and, if present, the type of hematopoietic neoplasm they represent. Unfortunately, this information is not always apparent from the “percent-positive” data format. The percent-positive format assumes, incorrectly, that within a leukemia or lymphoma, all of the tumor cells uniformly either lack or exhibit the same degree of clear-cut expression for a given antigen. However, in contrast with benign lymphocytes, neoplastic hematopoietic cells of the same clone often do not express the same amount of a given antigen on their cell surface and, therefore, display variability in the fluorescence intensity for that marker. The degree of variability depends on the particular surface antigen. For instance, reporting a case of leukemia as being 40% CD20 positive is ambiguous. This number could represent either (1) a case in which 40% of the cells formed a distinct population with a fluorescence intensity well above the negative control or (2) a single population in which 100% of the cells displayed a shifted fluorescence intensity, but only 40% of the cells were brighter than the background. The latter occurrence is frequently observed when the tumor cell expression for a surface antigen is weak. |
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