Comparison of immunophenotyping by slide-based cytometry and by flow cytometry

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Flow cytometry (FCM) is the standard for peripheral blood leukocyte (PBL) immunophenotyping.
• Slide-based cytometry (SBC) offers additional capabilities like repeated staining and morphological analysis.
• Clinical utility of SBC requires direct comparison with established FCM methods.

Purpose of the Study:

• To systematically compare immunophenotyping results from FCM and SBC (LSC).
• To evaluate the correlation between PBL immunophenotyping data generated by both methods.

Main Methods:

• Peripheral blood leukocytes (PBLs) were stained using directly labeled monoclonal antibodies and the whole blood method.
• Specimens were analyzed in parallel using a FACScan (FCM) and a laser scanning cytometer (LSC) with various triggers (forward scatter, CD45 FITC, 7-AAD).
• Acetone fixation was used for 7-AAD measurements with LSC.

Main Results:

• High correlation (regression coefficients near 1.0) was observed between LSC and FCM for major PBL populations and lymphocyte subsets.
• The 7-AAD trigger demonstrated the best performance for LSC analysis.
• LSC effectively quantifies major leukocyte populations and lymphocyte subsets.

Conclusions:

• LSC is a viable method for PBL immunophenotyping, particularly when sample volume is limited or morphological assessment is needed.
• LSC's ability to detect rare leukocytes or weak antigens is currently limited and may necessitate amplification strategies.
• The study recommends LSC for specific immunophenotyping applications based on its strong correlation with FCM.