IMMUNOSTAINING OF WHOLE AGAR CULTURES BY APAAP

We have adapted the alkaline phosphatase-anti alkaline phosphatase (AP AAP) technique to demonstrate cell antigen distributions in intact aga r culture. The method facilitates batch processing and is no less conv enient to perform than standard APAAP procedures. Myeloid and lymphoid antigens generally demonstrated strong staining intensity. However, s taining at day 0 consistently produced no antigen expression for two m onoclonals (CD11c and CD34) in contrast to positivity in parallel cyto spins. CD11c showed rapidly increasing antigen expression over subsequ ent days of culture whereas the expression of CD34 could not be shown in conventional agar culture at any time from day 0 to day 14. Positiv ity was only restored in CD34-positive leukaemic cells using a modifie d culture technique in which cells were cultured as pre-formed small a ggregates. Assessment of these aggregates extended to cell cycle analy sis using anti-bromodeoxyuridine. CD71 positivity in normal culture sa mples correlated with colony configuration (whether clones were 'sprea d' or 'tight' in appearance). CD38 staining of normal bone marrow cult ure at day 7 showed asymmetrical staining of cells in a small number o f micro-groups. The clonal detection of aberrant antigens (CD7, CD2) f or assessment of minimal residual disease in AML was a disappointment due to the relative frequency of positive clones in normal culture.