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Journal of Histochemistry and Cytochemistry, Vol. 51, 1131-1135, September 2003, Copyright © 2003, The Histochemical Society, Inc.

ARTICLE

Flow Cytoenzymology of Intracellular Tartrate-resistant Acid Phosphatase

Anthony J. Janckilaa,c, Wen-Kuang Yangd, Ruey-Jen Lind, Chia-Jen Tsengd, Hsin-Yu Changd, Jia-Ming Changd, and Lung T. Yama,b
a Special Hematology Laboratory, Veterans Affairs Medical Center, Louisville, Kentucky
b Departments of Medicine, University of Louisville, School of Medicine, Louisville, Kentucky
c Microbiology and Immunology, University of Louisville, School of Medicine, Louisville, Kentucky
d Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC

Correspondence to: Anthony J. Janckila, Veterans Affairs Medical Center, 800 Zorn Avenue, Louisville, KY 40206. E-mail: anthony.Janckila@med.va.gov

Tartrate-resistant acid phosphatase (TRACP) is a cytochemical marker for hairy cell leukemia, macrophages, dendritic cells, and osteoclasts. Our purpose was to develop multicolor cytofluorometric methods to evaluate intracellular TRACP enzymic activity using a fluorogenic cytochemical reaction in combination with immunochemical stains for distinct surface membrane antigens. Monocyte-derived dendritic cells (DCs) were the model TRACP-expressing cells studied. Intracellular TRACP activity was disclosed using naphthol-ASBI phosphate as substrate with fast red-violet LB salt as coupler for the reaction product. Before the TRACP enzymic reaction, surface antigens, CD86 and CD11c of DCs, were bound with specific fluorescent antibodies to test compatibility of surface labeling and intracellular staining. TRACP activity varied in DCs from donor to donor but was reproducible on repeated examinations of each sample. Samples could be stained for simultaneous analysis of surface antigens and intracellular TRACP activity, provided certain technical details were observed. The TRACP reaction time should not exceed 9 min and the cell number should not exceed 2 x 105/100 µl test. Fluorescent surface labels did not affect the intensity of the TRACP stain, but the intensity of some surface labels may be diminished by elution of low-affinity antibodies during the TRACP reaction. Readjustment of the threshold settings in triple-labeled cells is needed to compensate for this phenomenon. Intracellular TRACP activity can be quantitated in subpopulations of cells within mixed cell populations by flow cytofluorometry using simple cytochemical methods in combination with fluorescent antibodies to cell-surface and other differentiation antigens. The cytochemical method should be useful for basic investigations of differentiation, maturation, and function of macrophages, DCs, and osteoclasts, and for diagnosis and management of hairy cell leukemia. (J Histochem Cytochem 51:1131–1137, 2003)

Key Words: tartrate-resistant acid, phosphatase, cytochemistry, flow cytometry, cytofluorography, dendritic cell


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