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Tartrate-resistant acid phosphatase (TRAP) isoform 5b is a potential serum marker for osteoclastic activity. Biochemical assays for serum TRAP activity with para-nitrophenylphosphate (pNPP) have low specificity for bone because of hydrolysis by unrelated nontype 5 TRAPs of blood cells and by related isoform 5a. Our purpose was to increase the specificity of TRAP assay for osteoclastic activity by using naphthol-ASBI phosphate (N-ASBI-P) as a substrate for serum type 5 TRAP activity and heparin as an inhibitor of isoform 5a. TRAP activity in individual and pooled sera of normal subjects and patients with endstage renal disease (ESRD) and rheumatologic diseases was quantitated using pNPP and N-ASBI-P as substrate at pH 5.5 and 6.1. For some experiments, heparin (23U/ml) was added as a specific inhibitor of isoform 5a activity. Isoforms 5a and 5b were separated from serum pools by cation exchange chromatography and identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). N-ASBI-P was selectively hydrolyzed by TRAP isoform 5b. TRAP assays with pNPP and N-ASBI-P correlated only in ESRD sera, which contained primarily isoform 5b. The two assays did not correlate in normal or rheumatic sera with significant amounts of 5a. Heparin inhibited isoform 5a activity approximately 50% but had little effect on isoform 5b activity. Biochemical assay of serum TRAP activity can be made specific for isoform 5b by using N-ASBI-P and heparin. This method can be adapted to simple microplate biochemical or immunochemical assays. This simplified method for assessment of osteoclastic TRAP 5b activity warrants a detailed investigation in diseases of bone metabolism.

Background: Tartrate-resistant acid phosphatase (AcP) 5b is a marker of osteoclastic activity and bone resorption. Immunoassays for serum TRAcP may lack sensitivity and specificity because of the presence of non-bone isoform 5a. The purpose of this study was to isolate the serum isoforms, quantify their disease-related expressions, and test an improved immunoassay for TRAcP 5b., Methods: We separated TRAcP isoforms chromatographically from pooled sera of healthy, rheumatoid arthritis (RA) and endstage renal disease (ESRD) subjects. TRAcP isoforms were identified by electrophoresis and quantified by biochemical and immunochemical assays. Serum TRAcP activity in healthy, RA, and ESRD cohorts was assessed at pH 5.5 and 6.1, and compared with bone alkaline phosphatase (BAP) and N-telopeptides of type I collagen (NTx)., Results: TRAcP isoforms 5a and 5b were present in all sera; 5b was identical to osteoclastic TRAcP. In serum from healthy subjects, 5a accounted for 87% of the enzyme protein but only 55% of the activity. In RA, both isoforms were increased two- to threefold in protein, but their specific activities were subnormal. In ESRD, only 5b was abnormal, being increased fivefold in protein and threefold in activity. In RA sera, TRAcP activity did not correlate with either BAP or NTx. In ESRD sera, TRAcP activity correlated with BAP and NTx only when measured at pH 6.1., Conclusions: All sera contained both TRAcP isoforms 5a and 5b, but only 5b was present in bone. TRAcP isoform expression was variable in different diseases. Measurement of TRAcP activity at pH 6.1 improves the specificity of immunoassay for isoform 5b.

Background: Tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) is a product of osteoclasts and a biochemical marker of bone resorption rate. However, erythrocytes and platelets contribute to total TRAP activity in serum, reducing the specificity of direct biochemical assays in serum. Osteoclast TRAP is also known as type-5 TRAP and is antigenically unique. Immunoassays are sought to improve the specificity and sensitivity of TRAP as a bone marker., Methods: We developed two colorimetric microplate assays for type-5 TRAP: an enzyme capture immunoassay to measure antibody-bound enzymatic activity, and a two-site immunoassay to measure bound enzyme protein. Both use the same monoclonal antibody (14G6) to capture type-5 TRAP, which permits determination of specific activity of serum TRAP in health and disease., Results: Both TRAP assays were linear from one-tenth to fivefold the mean value in 18 healthy subjects. In these subjects, the mean (SD) TRAP activity was 3.2 (0.54) U/L for the enzyme capture assay and 37 (13) microg/L for the two-site assay. Mean TRAP activity was not significantly increased in 64 patients with endstage renal disease requiring hemodialysis (HD) or 99 unselected patients with rheumatic diseases. By contrast, TRAP protein was increased in both the HD and rheumatic disease groups. The specific activity of TRAP in the 17 of 64 HD sera that had increased TRAP activity (0.088 U/microg) was similar to that in healthy subjects (0.091 U/microg). By contrast, the specific activity of TRAP in the 31 of 99 rheumatic sera with increased TRAP protein (0.035 U/microg) was significantly decreased., Conclusions: Wide sample distributions for TRAP activity in HD patients and TRAP protein in rheumatic disease patients suggest the presence of subpopulations of HD patients with increased TRAP activity and of rheumatic patients with increased TRAP protein. Each assay for TRAP activity and protein may have its own biological significance and clinical applications in specific groups of patients.

The demonstration of tartrate-resistant acid phosphatase (TRAP) activity has long been a cornerstone in the diagnosis of hairy cell leukemia (HCL). Recently a monoclonal antibody to this enzyme has been developed that can be used in an immunoperoxidase method on paraffin-embedded tissues. By using a peroxidase-labeled streptavidin biotin method, paraffin sections of B5 and formalin-fixed tissue from 86 cases of HCL (41 bone marrow, 36 spleen, 9 liver) were stained with the antibody to TRAP and compared against staining for CD20 (L26) and DBA.44 (DAKO, Carpinteria, Calif). In addition, 193 specimens (127 bone marrow, 42 lymph node, 19 spleen, 5 other) from a variety of neoplastic and nonneoplastic hematologic conditions were stained using the monoclonal antibody to TRAP. For comparison, these cases were also stained with DBA.44. In the cases of HCL, 80 of 86 specimens were immunoreactive for TRAP. While the antibody to TRAP generally stained less than 50% of the hairy cells, CD20 and DBA.44 stained 90% and 50% to 60% of hairy cells, respectively. Two of three cases of marginal zone lymphoma showed weak immunoreactivity to the TRAP antibody. Two specimens from a patient with Gaucher's disease and 8 of 13 cases of mastocytosis also showed positivity to the TRAP antibody in the macrophages and mast cells, respectively. In contrast, staining for DBA.44 was positive in 3 of 9 cases of B-cell large cell lymphoma, 1 of 4 cases of mantle cell lymphoma, and in the paraimmunoblasts of 1 of 7 cases of small lymphocytic lymphoma. Only HCL was TRAP and DBA.44 positive. This antibody to TRAP is a useful addition to the diagnosis of HCL but should be used in conjunction with CD20 and DBA.44. The use of this antibody to determine minimal residual disease after chemotherapy was not addressed.

Immunohistochemical studies were done on formalin-fixed, paraffin-embedded tissues to evaluate the specificity of a newly developed monoclonal antibody (9C5) against tartrate-resistant acid phosphatase. Sections from 195 specimens were examined, which included 33 types of tissues/organs. These tissues included normal, inflammatory, and neoplastic processes. Neoplastic tissues from 14 patients with hairy cell leukemia served as positive controls. Epitope enhancement was accomplished either by microwave irradiation in citrate buffer or by boiling in water followed by trypsin digestion. Tissues were reacted with monoclonal antibody 9C5 and stained with either the avidin-biotin peroxidase method or the alkaline phosphatase anti-alkaline phosphatase method. The hairy cells of all cases of hairy cell leukemia reacted positively with 9C5. Other positively stained cells included osteoclasts, activated macrophages and giant cells. Immunohistochemical studies with 9C5, when interpreted within the context of the specificity of this antibody, are useful for the diagnosis and assessment of treatment results for hairy cell leukemia. Monoclonal antibody 9C5 also may be useful as a marker for osteoclasts and the activated macrophages and for the diagnosis of disorders involved by these cells.

A tartrate-resistant acid phosphatase was isolated from a huma spleen infiltrated with reticulum cells of leukemic reticuloendotheliosis. The purified enzyme was used to establish the optimal conditions for quantitative analysis of this enzyme by electrophoresis on acrylamide gel. As little as 0.1 unit of enzyme activity (or 1 ng of the purified enzyme protein) could be quantitatively detected when it was mixed with 4 mg of albumin before electrophoresis.

Tartrate-resistant acid phosphatase activity determined by enzyme immunoassay was higher in the serum of cancer patients than that in normal blood donors. The highest activity was found among patients having malignancy metastatic to bone. The classic colorimetric method showed a broad range of values among normal blood donors, and the contrast between normal and cancer patients was less obvious. Most of the cancer patients had normal to low alkaline phosphatase activities.

The authors have developed an immunoalkaline phosphatase method and have applied it with success to the study of blood cells. They have now observed that macrophages in tissues and in serous effusions may be nonspecifically stained when immunoalkaline phosphatase methods are used. A systematic study of this endogenous macrophage phosphatase activity has shown it to have a pH optimum of 5.0-6.0 (acid phosphatase), but it remains weakly active in the mildly alkaline conditions used in the immunoalkaline phosphatase procedure. At its pH optimum, this macrophage phosphatase is mostly tartrate resistant, however, when 50 mM tartrate is added to a staining medium of pH 7.6-8.0, the residual endogenous phosphatase activity effectively is inhibited. When immunochemical studies are conducted by immunoalkaline phosphatase methods, the authors recommend addition of 50 mM tartrate to a buffer of pH 7.6-8.0. This modification does not significantly decrease the sensitivity of the specific staining of surface antigens.

A tartrate-resistant acid phosphatase was isolated from a human leukemic spleen by freeze-thawing in saline and purified by repeated chromatography on carboxymethyl-cellulose. The purified enzyme has a molecular weight of 64 000. It catalyzes the hydrolysis of inorganic and organic pyrophosphate as well as the phenolic ester of monoorthophosphate, with optimal activity between pH 5 and 6. However, there is no activity toward mono-orthophosphate esters of aliphatic alcohols. The present data have identified its catalytic function as a pyrophosphatase. However, it has properties different from the pyrophosphatase previously observed in normal animal tissues.

Radiological studies were done on 23 patients with systemic mast-cell disease (SMCD). Significant changes occur most often in bones and less commonly in the gastrointestinal tract and other visceral organs. These changes may be related either to tissue infiltration by mast cells, or to the effect exerted on tissues by chemical mediators of the mast cells, although in some instances findings may be coincidental. Because the radiological changes are not unique to SMCD, their main value, in association with the clinical information, is in directing further studies for diagnostic confirmation and in estimating the extent of systemic involvement.

Histochemical markers were used to identify the various cellular and structural components of the human spleen, and to investigate the histogenesis of the splenic lesions of Hodgkin's disease. The early lesions appear in areas near the central artery (periarterial lymphatic sheath) in the white pulp. The white pulp becomes hypertrophic. The lesions enlarge, extend into the red pulp, and compress the sinuses and the cords of Billroth. The derivations of various "histiocytes" contained with the lesions are differentiated by using cytochemical stains for lysosomal enzymes and for granulocytes. The epithelioid cells in the granulomas are rich in those lysosomal enzymes typically seen in phagocytic histiocytes, suggesting that they are indeed true histiocytes. The malignant "histiocytes," including the mononuclear Hodgkin cells, the binucleated Sternberg-Reed cells, and the multinucleated giant cells, do not contain significant amounts of lysosomal enzymes and more closely resemble stimulated lymphocytes. The splenic lesions in Hodkin's disease may be the result of a lymphocytic and histiocytic cellular response to an unknown agent, which reaches the spleen through the central artery in the white pulp.

We used ion-exchange column chromatography and electrophoresis on polyacrylamide gel to compare the acid phosphatase isoenzymes of prostate and leukocytes. The major isoenzyme of the prostate is band 2A; only a trace of band 2B was observed. However, the major isoenzymes of leukocytes are band 4 and band 2B, and only a small amount of band 2A was observed. The three isoenzymes isolated from leukocytes or prostate gland react to the antiserum prepared against the aicd phosphatase isoenzyme of seminal fluid. Acid phosphatases of leukocytes other than the three isoenzymes mentioned above did not interact with the antiserum.

Spectrophotometry of total acid phosphatase activity in children's sera showed an average value of 22.4 +/- 2.9 and 7.4 +/- 0.8 U/liter, for the hydrolysis of p-nitrophenyl phosphate and alpha-naphthyl phosphate, respectively. Analyses of "band 5b", after electrophoresis on acrylamide gel, gave even higher values. The values for children's sera were much higher than those for sera from adults. The multiplicity of acid phosphatases in sera of children and adults was studied by electrophoresis on acrylamide gel and by chromatography on CM-Sepharose. Both methods showed the major acid phosphatase in children's sera to be an acid pyrophosphatase, band 5b. Its catalytic properties are indistinguishable from the enzyme previously isolated from the spleen of leukemic reticuloendotheliosis.

Hairy cell populations of greater than 90 purity were prepared from six samples obtained from four patients with leukemic reticuloendotheliosis (LRE). These then were analyzed for surface immunoglobulin (SIg) and antigens specified by a panel of monoclonal antibodies. The hairy cells from all patients displayed SIg and antigens reactive with OKIa-1 and OKM-1 antibodies; these markers often were expressed simultaneously. T-cell-specific antigens were not displayed on the surface of hairy cells. The simultaneous expression of SIg and OKM-1 which ordinarily are unique for B cells or myeloid cells, respectively, suggests that hairy cells may represent an aberrant form of either cell type with defective regulation of antigen expression. It alternatively suggests the possibility that B cells and myeloid elements develop along a common pathway and that the hairy cell is a component of such a pathway.

A practical immunocytochemical method of demonstrating surface antigens of human blood cells on air-dried smears or other cytologic preparations has been developed. This method uses monoclonal antibodies as the primary antibodies and calf intestinal alkaline phosphatase as the enzymatic indicator. Combined staining with cytochemical stains for myeloperoxidase or nonspecific esterase on the same slide is also possible when needed. These methods are very useful for accurate identification of human blood cells on the commonly available clinical specimens and are very helpful in the diagnosis and classification of various hematologic neoplasms, including chronic lymphocytic leukemias, acute leukemias, and related diseases.

Immunocytochemical methods were evaluated in order to find a practical one for cell identification on cytologic preparation of body fluids. The effects of fixatives and Triton X-100 treatment on the preservation of cell-type-specific antigens and cell morphologic characteristics were also examined. The method using the alkaline phosphatase-antialkaline phosphatase (APAAP) complex as the indicator is recommended because of its high specificity and sensitivity. With this method, currently available and potentially useful monoclonal antibodies were examined, and the antibodies that were useful for the identification of normal and neoplastic cells commonly present in body fluids were selected for practical applications.

An immunochemical method for quantitative analysis of the tartrate-resistant acid phosphatase (EC 3.1.3.2), band 5, is presented. This method involves precipitation of the enzyme from the serum by the antibody specific to band 5 and by sheep anti-rabbit immunoglobulin, followed by analysis of the enzyme activity in the precipitate. The precipitation procedure eliminates the interferences of the tartrate-sensitive phosphatase of all tissues, of the tartrate-resistant phosphatase of erythrocytes, and of unknown substances that interfere with the colorimetric method. We compare the present method with previously described colorimetric and electrophoretic methods.

The diagnosis of systemic mastocytosis depends on the proper recognition of extensive mast cells infiltration in tissues. Accurate identification of the mast cells in tissues may be difficult when there is no clinical suspicion to initiate a special search for these cells. Four patients with atypical clinical features and eosinophilia in tissues and three with eosinophilia in blood had hematologic or histologic findings suggestive of myelofibrosis with myeloid metaplasia, and malignant lymphoma. It was the presence of blood and tissue eosinophilia in these patients that led to further investigation and recognition of the diagnosis of systemic mastocytosis. Methods for identification of the mast cells are examined. The combined use of Wright-Giemsa stain, toluidine blue, and aminocaproate esterase is preferred. The eosinophils are intimately related to the mast cells. In cases of eosinophilia of unknown etiology, systemic mastocytosis should be considered one of the possible etiologic factors.

Cytochemical demonstration of tartrate-resistant acid phosphatase activity is essential for the diagnosis of leukemic reticuloendotheliosis. In order to perform this test correctly and to interpret the results propertly, it is necessary to understand the technical details of the cytochemical methods thoroughly. The method using naphthol--ASBI phosphoric acid--fast garnet GBC is recommended for this purpose, and factors crucial to the cytochemical study, such as fixation, substrate, coupler, pH and temperature of incubation buffer, counterstains, and mounting media are examined and discussed. Conventional methods for acid phosphatase in the presence and absence of L(+) tartaric acid are also critically examined. The naphthol--ASBI phosphoric acid--fast garnet GBC method is sensitive, technically simple and easily reproducible. Its reaction product is highly chromogenic and is most suitable for cytochemical demonstration of acid phosphatase and tartrate-resistant acid phosphatase activity in cytologic preparations. The naphthol--ASBI phosphoric acid--pararosaniline method is highly specific and is best for histochemical demonstration of acid phosphatase and tartrate-resistant acid phosphatase in tissue sections.

The authors have used two immunoalkaline phosphatase methods to study nonhematopoietic tumor tissues of four patients, one each with alveolar cell carcinoma of the lung, renal cell carcinoma, gastric adenocarcinoma, and colon carcinoma. They found, regardless of specific antibodies used, definite enzyme activity in the tumor cells of these four patients. Although it was possible to determine that the tumor cells were epithelial in origin because of their intense staining with antibodies to epithelial cell antigens, control slides labeled with nonimmune mouse ascites also contained cells with definite enzyme activity. In two of these cases, unlabeled smears were stained for alkaline phosphatase and showed that the tumor cells contained endogenous levamisole-resistant enzyme activity. This endogenous enzyme activity is not demonstrable in either the benign cells of these cases or the benign or malignant cells of other control cases. The findings suggest that the immunoalkaline phosphatase methods also have their inherent endogenous enzymic problems. They also suggest that cytochemical demonstration of levamisole-resistant alkaline phosphatase may be a useful cell marker for the identification of tumor cells in serous effusions.

A carboxylic-ester hydrolase was isolated from the leukocytes of a patient with myelomonocytic leukemia. Its relative molecular mass as estimated by sucrose density-gradient sedimentation is about 70 000. The purified enzyme is specific for acetyl esters of aromatic alcohols. It is inhibited by fluoride, but insensitive to eserine or p-chloromercuriphenylsulfonate. Hydrolysis of 1-naphthyl acetate was optimal above pH 6.0; of o-nitrophenyl acetate, above 8.0. The common catalytic site for the two types of substrates on the enzyme was confirmed by competitive inhibition data.

We purified acid phosphatase isoenzyme 5b from a human spleen affected by leukemic reticuloendotheliosis and used it to produce a specific antiserum. The antiserum was used to show complete immunological identity among isoenzymes 5a and 5b in human serum, and 5b isolated from a giant-cell bone tumor and from the spleen of a case of Hodgkin's disease. Acid phosphatase 5b in a giant-cell bone tumor was isolated for biochemical characterization. Its pH optimum and substrate specificity were very similar to those of isoenzyme 5b from human spleen.

A simple immunocytochemical method using calf intestinal alkaline phosphatase as the enzymatic indicator to demonstrate surface antigens on human blood cells has been developed. The blood cells were labeled with cell specific monoclonal antibodies followed by linkage with an antiimmunoglobulin alkaline phosphatase conjugate. Cytochemical demonstration of alkaline phosphatase activity on the blood cells reflects the presence of surface antigens on these cells. The effects on the cytochemical reaction of fixation, substrates, couplers, activators and inhibitors, and storage of cytologic materials have been examined systematically. The best staining conditions are to incubate labeled smears in a 0.04 M barbital buffer at pH 7.6 containing 30 mg% naphthol AS-TR phosphate, 40 mg% fast red ITR, and 1 mM levamisole. This method is both sensitive and specific and appears most practical for objective identification of the human blood cells.

Serum acid phosphatase activity in normal children (newborn to 18 years) is several fold that in normal adults. Activity is age-related but not sex-related. The isoenzyme pattern in children is similar to that in adults and contains no prostatic fraction. Quantitatively, most of the enzyme activity in the serum of children is tartrate-resistant and correlates well with heat-labile fractions of alkaline phosphatase activity in serum, suggesting that the source of the higher acid phosphatase activity in children is bone. Significant tartrate-resistant acid phosphatase activity was demonstrated in the giant cells in three patients with giant-cell tumors, but not in the "osteoblasts" in six patients with osteogenic sarcomas and many other normal or abnormal tissues. This work suggests that the higher enzyme activity in the serum of children represents a normal physiological phenomenon resulting from their greater osteoclastic activity.

Differential characterization of the "reticulum cell" in lymphoreticular neoplasms. Am J Clin Pathol. 64: 171-179, 1975. The term "reticulum cell" is confusing, having been applied to the cells involved in many hematopoietic neoplasms, such as reticulum-cell sarcoma, histiocytic medullary reticulosis, leukemic reticuloendotheliosis, and monocytic or histiocytic leukemias. In histologic sections, even the cells from poorly differentiated extramedullary lesions of chloroma or myeloblastic leukemia have been called "reticulum cells."A combined morphologic and cytochemical approach has been used to study "reticulum cells"in smears and tissue sections of neoplasms involving "histiocytes" or "reticulum cells."The cytochemical markers are: chloracetate esterase for neutrophilic granulocytes; nonspecific esterase and fluoride-resistant esterase for monocytes and histiocytes (phagocytes); tartrate-resistant acid phosphatase for the reticulum cells of leukemic reticuloendotheliosis; pyronin for the lymphatic reticulum cells (germinal center cells). The morphology of these cells is very well appreciated in smears, and the locations of these marked cells in tissue sections are easily recognized. The use of cytochemical and immunochemical methods and functional studies, in addition to simple morphology, may be useful in subclassification of lymphoreticular neoplasms.

Two patients with systemic angioendotheliomatosis had prominent constitutional symptoms such as fever, loss of weight, and general weakness, and had multiple organ dysfunctions, including bizarre neurologic findings and dementia. Severe anemia that required frequent blood transfusions also was present. One patient developed severe hemolysis and hypersplenism that required splenectomy for relief; the other patient had intravascular hemolysis and autoimmune hemolytic anemia, which were treated unsuccessfully with conservative measures. In both cases, postmortem examination showed many large, noncohesive malignant cells within the lumen of the blood vessels in many of the organs. There was no infiltration or replacement of the normal tissues by the tumor cells. Histochemical studies showed that the tumor cells were pyroninophilic but did not have cytoplasmic immunoglobulins or activity of chloroacetate esterase and muramidase. The cells showed factor VIII antigen in their cytoplasm. Systemic angioendotheliomatosis may represent a true neoplastic process of the vascular endothelial cells.