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Subtyping lymphocytes in peripheral blood by direct immunoalkaline phosphatase labeling and light scatter/absorption
Y R Kim1, L Paseltiner, G Kling
1Research and Development Division, Technicon Instruments Corporation, Tarrytown, New York 10591.
Insights
A new rapid method uses alkaline phosphatase-conjugated antibodies for immunophenotyping on standard flow cytometers. This technique offers speed, sensitivity, and accuracy for clinical blood cell analysis.
Area of Science:
- Clinical immunology
- Flow cytometry
- Biochemical analysis
Background:
- Immunophenotyping is crucial for assessing immunologic status.
- Wider clinical laboratory use of flow cytometry requires efficient methods.
- Existing methods may lack speed or accessibility for routine diagnostics.
Purpose of the Study:
- To develop a rapid method for detecting monoclonal antibody-labeled cells.
- To adapt immunophenotyping for clinical flow cytometers (Technicon H*1/H*2).
- To enable direct immunoenzymatic labeling for cell analysis.
Main Methods:
- Conjugating calf-intestinal alkaline phosphatase to monoclonal antibodies (anti-CD2, CD3, CD4, CD8, CD19).
- Utilizing a specific buffer/substrate (5-bromo-4-chloro-3-indolyl phosphate/nitroblue-tetrazolium salt) for intense precipitate formation.
- Employing mild cell fixation and incubation at 38°C for accelerated reactions.
Main Results:
- Successful direct immunoenzymatic labeling of antibody-labeled cells.
- Generation of stable, intense purplish-blue precipitates for detection.
- Demonstrated competitive performance with immunofluorescence methods in speed, sensitivity, and accuracy.
Conclusions:
- The developed method enables rapid immunophenotyping on absorption-based clinical flow cytometers.
- This technique enhances the clinical utility of flow cytometry in laboratories.
- The immunoenzymatic approach provides a viable alternative to fluorescence-based methods.
Abstract:
Evaluation of blood cells to determine immunologic status is becoming an important clinical application of flow cytometric analysis. For a wider use of immunophenotyping technology in clinical laboratories, the authors developed a rapid method to detect monoclonal antibody-labeled cells using forward light scatter/absorption clinical flow cytometers such as the Technicon H*1 and Technicon H*2 differential complete blood count analyzers. Calf-intestinal alkaline phosphatase was conjugated to mouse monoclonal antibodies (anti-CD2, CD3, CD4, CD8, CD19) for direct immunoenzymatic labeling. The combination of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue-tetrazolium salt in diethanolamine buffer at pH 9.6 was selected as buffer/substrate to yield stable, insoluble, and very intense purplish-blue precipitates on the surface of the cells labeled with monoclonal antibody-alkaline phosphatase conjugates. Endogenous alkaline phosphatase in granulocytes was inhibited with levamisole. Early mild fixation of the white cells permitted incubation at 38 +/- 1 degrees C, which accelerated each step of the reaction without disrupting the cells throughout the procedure. The method is competitive with the direct immunofluorescence whole-blood method used on fluorescence flow cytometers in speed, sensitivity, and accuracy, as demonstrated with alkaline phosphatase-conjugated anti-CD2, CD3, CD4, CD8, CD19 monoclonal antibodies.
