Immunostaining of whole agar cultures by APAAP

Summary We have adapted the alkaline phosphatase-anti alkaline phosphatase (APAAP) technique to demonstrate cell antigen distributions in intact agar culture. The method facilitates batch processing and is no less convenient to perform than standard APAAP procedures. Myeloid and lymphoid antigens generally demonstrated strong staining intensity. However, staining at day 0 consistently produced no antigen expression for two monoclonals (CD11c and CD34) in contrast to positivity in parallel cytospins. CD11c showed rapidly increasing antigen expression over subsequent 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. Positivity was only restored in CD34-positive leukaemic cells using a modified culture technique in which cells were cultured as pre-formed small aggregates. Assessment of these aggregates extended to cell cycle analysis using anti-bromodeoxyuridine. CD71 positivity in normal culture samples correlated with colony configuration (whether clones were ‘spread’ or ‘tight’ in appearance). CD38 staining of normal bone marrow culture at day 7 showed asymmetrical staining of cells in a small number of micro-groups. The clonal detection of aberrant antigens (CD7, CD2) for assessment of minimal residual disease in AML was a disappointment due to the relative frequency of positive clones in normal culture.