Rapid diagnosis of leukaemia: a three-stage immunophenotyping technique

We have developed a three-stage avidin-biotin alkaline phosphatase system (ABAP) for immunophenotyping acute and chronic leukaemias. Air-dried blood and bone marrow films or cytospins were fixed in acetone, methanol-acetone, or methanol-acetone-formalin. A primary monoclonal antibody layer was employed with a corresponding biotinylated anti-species second layer. The third stage was avidin-conjugated alkaline phosphatase and visualisation was achieved with Fast Red TR as diazo salt. This was compared using a limited range of primary antibodies with the alkaline phosphatase anti-alkaline phosphatase (APAAP) method, routinely performed as a five-stage technique. The ABAP technique provided a 2.5 h, reliable, three-stage immunophenotyping procedure without the need for amplification steps. A wider range of first layer monoclonals of any species could be utilised by using the appropriate secondary antibody. Preservation of cell morphology was very good. Smaller amounts of expensive monoclonal antibodies could be used compared to the quantity required with other slide techniques. This approach to immunophenotyping can consequently be employed by any general haematology laboratory to provide a sensitive and inexpensive service. The technique has been in routine diagnostic use for the last 2 years, and consistently good results have so far been achieved in a national external immunophenotyping quality assurance scheme.