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Performance of calibration standards for antigen quantitation with flow cytometry
R Lenkei1, J W Gratama, G Rothe
1Flow Cytometry Laboratory, S:t. Görans Hospital, Stockholm, Sweden.
Cytometry
|October 17, 1998
Summary
Quantitative flow cytometry (QFCM) requires specific calibration systems for consistent antibody-binding capacity (ABC) values between labs. Standardized microbead analysis helps achieve reliable results for antigen quantitation.
Area of Science:
- Immunology
- Biotechnology
- Analytical Chemistry
Background:
- Quantitative flow cytometry (QFCM) is crucial for accurate cell analysis.
- Standardization of microbead standards is essential for reliable QFCM results.
- Evaluating monoclonal antibody performance requires consistent calibration methods.
Purpose of the Study:
- To evaluate microbead standards for quantitative flow cytometry.
- To assess the performance of monoclonal antibodies (mAbs) for CD3, CD4, and CD8.
- To compare different calibration systems for antigen quantitation.
Main Methods:
- Established a unified window of analysis (UWA) on three flow cytometers.
- Tested monoclonal antibodies from three manufacturers using QC3 microbeads.
- Compared QIFI, QuantiBRITE, and relative fluorescence intensity (RFI) methods.
Main Results:
- Significant correlations were observed in antibody-binding capacity (ABC) values across different calibrators and antibodies.
- ABC values varied by 20-40% when determined using different fluorochrome conjugates and calibrators.
- Consistent ABC values are achievable with specific calibration systems, reagents, and protocols.
Conclusions:
- Standardized calibration systems are vital for inter-laboratory consistency in QFCM.
- Further refinement of QFCM protocols is needed for precise antigen quantitation.
- The study highlights the importance of specific calibrators and reagents for reliable QFCM.