Six and more color immunophenotyping on the slide by laser scanning cytometry (LSC)

The request for a more profound immunophenotyping and sometimes the lack of material demands more measurable fluorescence colors to increase the number of detectable antigens per specimen. Six different fluorescences are distinguishable in the Laser Scanning Cytometer (LSC). In the present study we wanted to increase this number to eight colors per measurement. Combined with an earlier study it is likely possible to measure n fluorescences i.e. n leukocyte subsets by a series of measurements followed by subsequent restraining steps. The new method is realized by s-ing the combination of filter change and a subsequent re-measurement for the distinction between the fluorescent dyes Cy5 and Cy5.5. The optical filters are replaced after the first measurement and the same specimen is remeasured without removing it from the microscope. For the second measurement a filter is inserted that detects Cy5.5 but not Cy5 (710/10nm). After the second measurement of the same specimen both data files are combined. With the aid of this feature it is possible to line out the differences between both measurements. If the data from the second measuring (Cy5.5 only) is subtracted from the first, Cy5 data is the result. After the first two measurements when eight different fluorescences (i.e. antigens or leukocyte subsets) were analyzed, the same cells are restained and a new measurement is performed. In theory, one can perform n re-measurements with eight fluorescences respectively. The information gained per specimen is only limited by the number of available antibodies and b sterical hindrance.