Immunostaining of whole agar cultures by APAAP

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.