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Correspondence to: Anthony J. Janckila, Veterans Affairs Medical Center, 800 Zorn Avenue, Louisville, KY 40206. E-mail: anthony.Janckila@med.va.gov
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Summary |
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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 51:11311137, 2003)
Key Words: tartrate-resistant acid, phosphatase, cytochemistry, flow cytometry, cytofluorography, dendritic cell
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Introduction |
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TARTRATE-RESISTANT ACID PHOSPHATASE (TRACP) is a well-accepted specific cytochemical marker for leukemic cells in hairy cell leukemia (
We have used cytochemistry on cell smears and polyacrylamide gels to study TRACP expression in dendritic cells (DCs) derived from human blood monocytes (
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Materials and Methods |
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Cell Sources
Human monocyte-derived phagocytic dendritic cells (DCs) were prepared as previously described (
Cytochemistry of TRACP on Cell Smears
Cytocentrifuged smears were prepared from day 6 DCs and stained for TRACP activity using published methods (
Cell Extraction and Immunoassay
In four experiments, DCs were harvested for immunoassay of intracellular TRACP activity and protein. Washed DCs were counted, pelleted, and resuspended to 107 cells /ml in lysis buffer (10 mM Tris/300 mM NaCl/0.5% NP-40/1 mM EGTA, pH 7.5) containing 1 mM phenyl methylsulfonyl fluoride and 1 µg/ml each pepstatin and leupeptin. Lysates were placed on ice for 30 min, then frozen at -70C until analyzed. Lysates were thawed and centrifuged to remove insoluble material and the supernatant assayed for TRACP activity and protein by published methods (
Cytochemical TRACP Reaction for Cytofluorometry
Cryopreserved cells were rapidly thawed and washed twice in PBS. Afterward, the viability was always greater than 85%. Viable cells were placed in microcentrifuge tubes and pelleted. All subsequent incubations were carried out on ice. Cells were fixed for 1 min in a solution of 4% paraformaldehyde in PBS and then permeabilized by adding Triton X-100 to a final concentration of 0.02% and incubating for another 2 min. Fixed cells were washed by centrifugation once in PBS and once in TRACP stain buffer (50 mM MES, pH 6.3, containing 50 mM Na tartrate) and adjusted to 2 x 105 to 4 x 106 cells/ml. Aliquots of 50 µL cells in TRACP stain buffer were then prewarmed to 25C. A 2 x staining solution was prepared immediately before use by adding 80 µl stock naphthol-ASBI phosphate (12.5 mg/ml in dimethyl formamide) and 20 µl stock fast red violet (10 mg/ml in DMF) to 900 µl prewarmed MES/tartrate buffer. Both ASBI-P and FRV solutions were stored in single-use aliquots. To initiate the enzymatic reaction, 50 µl of prewarmed 2 x staining solution was added to the warmed cells and incubated for precisely 9 min. The reaction was stopped by adding 900 µl ice-cold PBS. To control for background fluorescence of cytochemical reaction product, ASBI-P substrate was omitted while fast red violet salt remained in the solution. For some experiments to optimize the method, the reaction time was varied from 3 to 15 min and the cell number varied from 2 x 105 to 2 x 107 cells/ml.
Simultaneous Staining of Surface Antigens and TRACP Activity
Anti-CD86fluorescein isothiocyanate (FITC) and anti-CD11cphycoerythrin (PE) were purchased from BD Biosciences (Singapore). To demonstrate surface membrane antigens together with cytoplasmic TRACP activity, surface antigens were labeled first, followed by TRACP staining. Fixed and permeabilized cells were suspended in 50 µl PBS with pre-tested optimal amounts of commercial fluorochrome-labeled monoclonal antibodies and incubated on ice for 30 min. After washing twice in PBS containing 2% BSA, the cells were subjected to TRACP cytochemical staining as described above. The triple-labeled cells were used immediately for cytofluorometric analysis.
Cytofluorometric Analysis
Cytofluorometry was performed with a BectonDickinson FACSCalibur system equipped with a 488-nm blue laser and a 635 red diode laser for multicolor fluorescence, in addition to forward scattering and side scattering measurements. FITC-labeled antibodies and PE-labeled antibodies were measured using FL1 at 530 nm and FL2 at 585 nm, respectively. TRACP activity revealed by naphthol-ASBI/FRV-LB reaction was detected in FL3 at 670 nm. All cytofluorometric data were subsequently analyzed and graphically displayed using B-D Cell Quest software.
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Results |
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Cytofluorometry of TRACP Activity by Cytochemical Staining
Using standardized final concentrations of substrate (1 mM) and coupler (0.25 mM) for cytochemical staining of intracellular TRACP in fixed cells, we optimized the number of cells per 100 µl test and the reaction time to achieve a mean fluorescence index (MFI) of the DC population in the linear portion of the kinetic curve. This way, strong positive and weak positive cells could be differentiated. The reaction time to maximal staining intensity was approximately 15 min. The kinetics of fluorochrome production were linear from 3 to 9 min using 105 cells per test or less (Fig 1). When the cell number exceeded 5 x 105 per test the reaction kinetics deviated from linearity resulting in substantially lower fluorescence at 9 min due to substrate depletion. We also observed that the MFI for TRACP cytochemistry increased in a time-dependent manner when cells were stored at 4C in the fixed state before staining and analysis (data not shown). This was probably due to mild reduction of the di-iron center of TRACP or to proteolytic activation of TRACP by intracellular proteases. Proteolytic activation of TRACP has been reported previously for purified TRACP stored at 4C (
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TRACP was expressed in greater than 90% of DCs from all eight cases tested. Although the intensity of TRACP expression varied from donor to donor, there was a high degree of reproducibility when the enzyme reaction was performed on freshly thawed cells and analyzed immediately (Table 1). We also observed that subpopulations of smaller DCs often expressed more TRACP activity than larger cells, as defined by forward scatter (data not shown). The case-by-case variation in TRACP expression was validated in DCs from four donors by light microscopy of stained DCs (Fig 2A) and by immunoassay of TRACP in cell extracts (Fig 2B). These findings suggest that flow cytofluorometry can be used for quantitative assessment of intracellular TRACP expression.
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Co-expression of Cytoplasmic TRACP Activity and Surface Markers of Dendritic Cells
To use flow cytofluorometry of TRACP most effectively, it would be advantageous to simultaneously reveal surface markers with specific fluorescent antibodies. In Fig 3 are data from a representative experiment showing the results of this method. The gated population of DCs (Fig 3A) co-expressed CD86 and CD11c (Fig 3B), which are commonly used as markers for DCs. Background fluorescence caused by FRV coupler alone in control TRACP activity incubations is nil (Fig 3C and Fig 3D) and did not affect detection of FITC-labeled CD86 or PE-labeled CD11c (Fig 3E and Fig 3F). On staining TRACP activity with ASBI-P substrate, greater than 90% of DC were positive (Fig 3G and Fig 3H), and the numbers of CD86-positive cells (Fig 3I) and CD11c-positive cells (Fig 3J) remained mostly unchanged. There were apparent correlations among CD86, CD11c, and TRACP expression by DCs from this donor. However, these relationships were not evident in DCs from all donors.
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Sometimes we observed that the MFI of some surface marker stains was diminished after the TRACP cytochemical reaction compared to cells that were not stained for TRACP. Experiments (not shown) using antibodies from different sources and adjustments of TRACP reaction buffer concentration revealed that this was probably due to elution of lower-affinity antibodies during the TRACP reaction. The effect could be minimized, but not completely eliminated, by keeping the ionic strength of the TRACP reaction at approximately 50 mM. Therefore, to compare surface antigen staining before and after TRACP staining, the threshold settings were readjusted for analysis of triple-stained cells to partially correct for the elution effect. Table 2 summarizes quantitative data from cytofluorometric analysis of cell surface CD11c and CD86 expression before and after TRACP staining in preparations from five normal donors repeated on two to five separate occasions. CD11c and CD86, like TRACP, varied from case to case, were slightly reduced after TRACP cytochemical staining, but were reproducible. Table 3 summarizes data showing that surface labeling of different fluorescent antibody pairs had no effect on the intensity of detectable TRACP expression.
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Discussion |
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Cytofluorometry is a powerful technology that allows precise quantitative description of complex cell mixtures. With it, it is now possible to define the type and differentiation stage of neoplastic cells and offer more accurate diagnoses of leukemias, lymphomas, and other cancers (
In these studies we established a cytochemical method for quantitation of intracellular TRACP activity in cell suspensions via flow cytoenzymology. The method can be performed alone or in combination with immunolabeling of cell surface antigens to demonstrate TRACP expression in phenotypically defined cell populations. The availability of these novel methods to demonstrate TRACP activity would be of considerable practical and scientific value.
In practice, TRACP enzyme cytofluorometry is easy to perform. However, certain technical details require careful attention. The reaction is rapid with linear kinetics, reaching maximal intensity in approximately 15 min at pH 6.3 and 25C. By incubating for 9 min (within the linear range), the method can discriminate cells with high activity from those with low activity. Because the reaction is so rapid and sensitive, careful control of the incubation time and temperature is essential for reproducible results. The number of cells per assay should be kept at or below 2 x 105/100 µl sample. Beyond this, the reaction becomes non-linear, with diminishing fluorescence due to substrate depletion. Therefore, for consistent results, the concentration of cells in the test sample must be determined beforehand and adjusted to within a defined range. Finally, samples should be stained and analyzed on the same day they are prepared. TRACP activity in DCs increased significantly on refrigerated storage in the fixed state. Pure recombinant TRACP has been observed to undergo limited proteolytic cleavage of the "loop" peptide on storage to cause a marked rise in specific activity (
Using this cytochemical technique under well-controlled conditions, the TRACP activity within individual DCs still varied considerably among different normal donors, although the percentage of positive cells was consistently greater than 90%. Most DCs (8595%) from five donors tested strongly expressed CD11c. CD86 was variably expressed to a lesser degree (6685%). We observed that some antibodies were significantly desorbed from cells after subsequent cytochemical staining for TRACP. We could minimize this effect by maintaining the ionic strength of the TRACP reaction buffer at approximately 50 mM. Even with careful readjustment of the threshold settings in the triple-labeled preparation, there was a measurable decline in surface marker fluorescence intensity in some instances. On the other hand, TRACP enzymatic activity was unaffected by prior labeling of cell surface antigens with fluorescent antibodies. To provide consistency throughout this study, all experiments were performed on cells that were fixed and permeabilized before any labeling reactions. This eliminated the problem of differential light scattering by fixed and unfixed cells. We realize that fixation may crosslink or denature some important epitopes on some target antigens recognized by monoclonal antibodies. It must be determined empirically, by each laboratory, which surface antigens are stable to fixation and which are not. For this method to work with labile antigens, the surface antigens must be labeled first, followed by fixation, permeabilization, and TRACP staining.
From a scientific point of view, cytofluorometry of intracellular TRACP will allow study of the temporal expression and possible functional relationships among TRACP and other defining markers of the various macrophage lineages as the cells grow, differentiate, and mature. The present method is a relatively simple one that allows the measurement of TRACP enzymatic activity at a defined endpoint, together with other cell surface antigenic markers. Therefore, it cannot make kinetic measurements of enzymatic activity in individual cells by repeated or continuous monitoring of the reaction, as can laser scanning cytometry (
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Acknowledgments |
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Supported by a grant from the Research Service of the US Department of Veterans Affairs (AJJ) and a grant (DOH91-TD.1001) from the Department of Health, Taiwan, ROC (WKY).
We thank Ms Luann Jaggers for her technical assistance. We gratefully acknowledge Dr Jussi Halleen for his generous gift of anti-TRACP J1B antibody.
Received for publication July 30, 2002; accepted April 16, 2003.
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