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Immunostaining of whole agar cultures by APAAP
1University Department of Clinical and Laboratory Haematology, Manchester Royal Infirmary, UK.
Clinical and Laboratory Haematology
|March 1, 1995
Summary
The alkaline phosphatase-anti alkaline phosphatase (APAAP) technique effectively stains cell antigens in agar cultures. However, CD34 antigen expression requires modified culture methods for accurate detection in myeloid and lymphoid cells.
Area of Science:
- Immunohistochemistry
- Cell Biology
- Hematology
Background:
- The alkaline phosphatase-anti alkaline phosphatase (APAAP) technique is a standard method for detecting cell antigens.
- Intact agar culture is a valuable model for studying cell antigen expression over time.
- Standard APAAP protocols may face challenges in demonstrating certain antigens in specific culture conditions.
Purpose of the Study:
- To adapt and evaluate the APAAP technique for cell antigen demonstration in intact agar cultures.
- To investigate the expression patterns of myeloid and lymphoid antigens (CD11c, CD34, CD71, CD38) during agar culture.
- To assess the feasibility of detecting aberrant antigens for minimal residual disease monitoring in acute myeloid leukemia (AML).
Main Methods:
- Adaptation of the APAAP technique for intact agar cultures, allowing for batch processing.
- Parallel analysis of cytospins and agar cultures to compare antigen expression.
- Culturing cells in conventional agar and modified aggregate cultures.
- Cell cycle analysis using anti-bromodeoxyuridine.
- Immunophenotyping using monoclonal antibodies against CD11c, CD34, CD71, CD38, CD7, and CD2.
Main Results:
- The adapted APAAP technique demonstrated strong staining intensity for most myeloid and lymphoid antigens.
- CD11c antigen expression increased over culture days, while CD34 expression was undetectable in conventional agar cultures.
- A modified culture technique using pre-formed aggregates restored CD34 positivity in leukaemic cells.
- CD71 positivity correlated with colony morphology, and CD38 showed asymmetrical staining in bone marrow cultures.
- Detection of aberrant antigens (CD7, CD2) for AML minimal residual disease was limited by positive clones in normal cultures.
Conclusions:
- The adapted APAAP technique is a convenient and effective method for analyzing cell antigen distribution in agar cultures.
- Specific antigen expression, like CD34, may be culture-dependent and require optimized techniques for accurate assessment.
- The utility of detecting aberrant antigens for minimal residual disease in AML using this method is currently limited due to cross-reactivity in normal cells.
