Subtyping lymphocytes in peripheral blood by direct immunoalkaline phosphatase labeling and light scatter/absorption flow cytometric analysis.

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.

journal_name

Am J Clin Pathol

authors

Kim YR,Paseltiner L,Kling G,Yeh CK

doi

10.1093/ajcp/97.3.331

subject

Has Abstract

pub_date

1992-03-01 00:00:00

pages

331-7

issue

3

eissn

0002-9173

issn

1943-7722

journal_volume

97

pub_type

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