Tartrate-resistant acid phosphatase isoform 5a as an inflammation marker in end-stage renal disease.

AIM: End-stage renal disease (ESRD) is often complicated by chronic inflammation and malnutrition. We tested whether serum tartrate-resistant acid phosphatase (TRACP) isoform 5a relates to other markers of inflammation in ESRD. MATERIAL: Predialysis serum was collected from 99 ESRD patients (51 male, 48 female) aged 55 +/- 15 years and a control group of 36 healthy subjects (8 male, 28 female) aged 43.2 +/- 10.5 years. METHODS: Serum TRACP 5a activity and protein, TRACP 5b activity and C-reactive protein (CRP) were estimated by in-house immunoassays. Commercial kits were used for serum bone-specific alkaline phosphatase, Ntelopeptides of Type I collagen, interleukin-6 (IL-6) and fetuin-A. Intact parathyroid hormone was determined by chemiluminescent assay. Albumin, cholesterol, triglycerides, ferritin and hemoglobin were compared to the hospital reference ranges. Bone mineral density (BMD) was measured at the heel in 69 patients and all control subjects and expressed as g/cm2 and age-corrected T-score. RESULTS: Mean (median) levels of all serum markers were significantly elevated in ESRD except fetuin-A, which was significantly reduced. Mean BMD (g/cm2) was not different than control, but mean T-score was significantly reduced. TRACP 5a protein correlated with CRP, triglycerides and ferritin, but not with IL-6 or any other nutritional or bone markers or BMD. TRACP 5b activity correlated with all bone markers and BMD, but not with inflammation or nutritional markers. CONCLUSION: Our findings suggest that TRACP 5a may be a useful marker to estimate the degree of inflammation in ESRD patients on chronic hemodialysis.

Cytochemistry of tartrate-resistant acid phosphatase: 15 years' experience.

Tartrate-resistant acid phosphatase (T-AcP) has been used in the past 15 years as a specific test for the diagnosis of hairy cell leukemia (HCL). However, enzyme activity has been reported to be absent from the hairy cells of rare cases of HCL and to be present in the neoplastic cells of diseases other than HCL. In order to fully utilize T-AcP for the diagnosis of HCL, it is necessary to maximize the sensitivity and specificity of the staining method, to have adequate quality control to ensure technical and interpretative accuracy, and to prepare optimal cytologic and histologic materials for study. In blood, only the presence of cells with intense T-AcP activity is diagnostic of HCL (positive T-AcP test). In tissues other than blood, the presence of cells with intense enzyme activity may not be diagnostic for HCL; assessment of cell morphology and appreciation of the pattern of enzyme localization are also important. In studying the blood of over 1,000 patients, we have found a negative T-AcP test in two of 200 cases of HCL and a positive T-AcP test in three of 800 patients with diseases other than HCL. We believe that the T-AcP test, when performed and interpreted properly, is a useful diagnostic test for HCL.

Protein-tyrosine phosphatase activity of hairy cell tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAcP) is a reliable cytochemical marker for the diagnosis of hairy cell leukemia (HCL). The enzyme has been the subject of much biochemical investigation yet its function in the hairy cells (HC) is still unknown. Two TRAcPs have been purified from HCL spleen tissues by a series of chromatographic separations. The two enzymes, provisionally called peak 1 and peak 2, had specific activities of greater than 600 U/mg and 800 U/mg respectively when p-nitrophenyl phosphate (p-NPP) was used as substrate and had Km values in the range of 1 to 5 mM p-NPP. The two TRAcPs had the same substrate specificities and inhibitor sensitivities, therefore could be isoforms of the same enzyme. Their pH optima were between 5 and 6 for all substrates tested including the phosphotyrosine-containing peptide, Raytide, which was still hydrolyzed efficiently at neutral pH. Neither phosphoserine nor phosphoserine-containing casein were hydrolyzed by either enzyme. The TRAcPs of HC may thus be capable of functioning as protein-tyrosine phosphatases (PTP). High activity of a PTP could regulate the activities of protein-tyrosine kinases and thereby influence the growth and differentiation of the hairy cells.

Bone marrow necrosis. Diagnosis and assessment of extent of involvement by radioisotope studies.

In two patients with extensive marrow necrosis, the diagnosis of marrow necrosis was established by morphologic and radioisotopic studies, and the extent of involvement was accurately assessed by marrow scanning with technetium Tc 99m sulfur colloid while the patients were still alive. The literature on marrow necrosis was briefly reviewed and the clinical features of this condition were characterized. It was found that patients with this condition often have malignancies, underlying marrow disorders, sepsis, bone pain, and pancytopenia. Their marrow is often difficult to aspirate and they may require frequent transfusions to maintain a stable hemoglobin level. Radioisotopic studies are useful in the diagnosis and assessment of extent of involvement of this condition. They should be used in patients with clinical findings suggestive of marrow necrosis.

Therapeutic leukapheresis in hairy cell leukemia.

Therapeutic leukapheresis was performed on three patients, and plasmapheresis on two patients with far-advanced hairy cell leukemia. Two of the three patients who were treated with leukapheresis had many hairy cells in their peripheral blood, while the other had relatively few. In each patient, dramatic clinical and hematologic improvements were observed that have sustained for more than 23, 10, and 26 months, respectively. Plasmapheresis of similar intensity failed to show any appreciable therapeutic effects on two other patients with similar clinical and hematologic findings. We believe that the favorable therapeutic effects of leukapheresis are due to the removal of factors capable of inhibiting normal hematopoiesis. This factor(s) is present in the cells that were removed by leukapheresis. The exact nature of this factor(s) or the cells that produce this factor(s) remains to be identified.

Naphthol-ASBI phosphate as a preferred substrate for tartrate-resistant acid phosphatase isoform 5b.

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.

Systemic mast cell disease. Analysis of 58 cases and literature review.

Based on study of 58 histologically proved cases of SMCD, we believe that the prognosis of most SMCD patients can be anticipated at the time of initial diagnosis by using 5 independent significant predictors developed in a multivariate model. Our study confirms the significance of several previously reported poor prognostic factors: absence of skin involvement and the presence of hepatosplenomegaly, cytologic atypia, and a hypercellular bone marrow. However, in contrast to previous reports we did not find a uniform correlation between the presence or absence of skin involvement and prognosis. The observation that anemia was strongly related to so many prognostic variables may be due to the number of patients in our study with associated hematologic disorders. Alternatively, this evidence of ineffective erythropoiesis may support the concept that SMCD is a myeloid stem cell disorder and frequently affects other hematopoietic cell lines. The observation that death occurs within the first 3 years in most fatal cases of SMCD suggests that these patients should be followed carefully for this interval after initial diagnosis, especially if poor prognostic features are present. Currently there is no curative therapy for SMCD.

Hepatic involvement in systemic mast cell disease.

Thirteen patients with systemic mast cell disease were studied in order to define the hepatic changes in this disease and to correlate the histologic lesions in the liver with the clinical findings. These patients often presented with multisystem disorders and 10 had hepatomegaly. Microscopically, the liver tissues in all patients showed fibrosis and chronic inflammatory cellular infiltration with plasma cells, lymphocytes, eosinophils, and mononuclear fibroblast-like cells in the portal area. The hepatic sinusoids were not significantly involved. A histologic diagnosis of systemic mast cell disease is seldom entertained in liver biopsy specimens embedded in paraffin and stained with hematoxylineosin, but can be facilitated in biopsy specimens embedded in plastic such as methacrylate. Tissue mast cells in the cellular infiltrate can be demonstrated best by special staining techniques with Giemsa, toluidine blue, and chloroacetate esterase. The severity of the histologic changes in the liver does not correlate well with the size of the liver or biochemical changes in the blood. Abnormal serum biochemical values were noted primarily in those with dehydration caused by diarrhea and vomiting, and in those with malnutrition. Hepatic function test results were usually normal, except for alkaline phosphatase level, which was elevated in all 13 patients. Although the clinical significance of hepatic involvement in systemic mast cell disease cannot be established with certainty in this study, it is believed that the prognosis of systemic mast cell disease is most intricately related to the systemic effects of mast cell involvement in many other organs, and not to hepatic involvement per se.

Occupational carcinogenesis: the Louisville experience with vinyl chloride-associated hepatic angiosarcoma.

Hepatic angiosarcoma in man was first associated with exposure to vinyl chloride in Louisville, Kentucky, where it was identified in 10 persons from a single vinyl chloride polymerization plant; clinical manifestations are summarized herein. Following prolonged exposure to vinyl chloride, the onset of this disease is insidious and the clinical picture is that of nonspecific hepatic injury with mildly abnormal biochemical liver test results. Carcinoembryonic antigen and alpha fetoprotein are undetectable. Radionuclide and angiographic studies of liver show characteristic but nondiagnostic abnormalities. A definite diagnosis is usually made only by open liver biopsy. Treatment is unsatisfactory but chemotherapy seems to prolong survival. Average survival from diagnosis is about 12 months. Overt liver failure usually occurs only as a preterminal event and was the major cause of death in all of our patients. Preventive measures are now in effect in the plant. This experience illustrates the importance of the clinician in occupationally-related cancer.

Immunohistochemical detection of monocytes by the antiserum specific to monocytic esterase.

An antiserum specific to esterase Ib was produced in a rabbit. The antigen-antibody reaction was visualized by the strong esterase activity in the precipitin band in Ouchterlony double diffusion and immunoelectrophoresis. Immunohistochemical procedure demonstrated strong staining in the monocyte-infiltrated splenic sections and tissue sections of true histiocytic lymphoma. Negative results were observed in T- and B-cell lymphomas, granulocytic sarcoma, and chronic granulocytic leukemia. This antibody may be useful for the identification of monocytes and histocytes in paraffin-embedded tissue sections.

Assessment of a method for immunochemical detection of antigen on nitrocellulose membranes.

Immunoblotting techniques are widely used for detection of antigen immobilized on nitrocellulose membranes. There are many immunolabeling methods and staining methods available to disclose the presence of antigen in such techniques. Five common staining methods each for alkaline phosphatase and horseradish peroxidase were examined. The staining methods with the highest sensitivity and the lowest background were selected for studies comparing five immunological labeling methods using human IgG as a model antigen. Results were evaluated on the basis of the least amount of detectable antigen and background staining. The most sensitive dot-blot method was then tested for its applicability to Western blots. For both dot-blots and Western blots, the immunoalkaline phosphatase methods are more sensitive than the corresponding immunoperoxidase methods. The use of biotinylated secondary antibodies and an avidin-enzyme conjugate is recommended. Disclosure of alkaline phosphate is best achieved with naphthol AS phosphate as substrate and fast blue BB as chromogen. Peroxidase is best stained using H2O2 and diaminobenzidine (DAB). Potential endogenous enzyme activities are demonstrable by blotting methods but can be inhibited by including levamisole in the disclosure reaction medium for calf intestinal alkaline phosphatase indicators, or by incubation of blots with sodium azide and hydrogen peroxide before immunolabeling when using horseradish peroxidase indicators.

Epitope enhancement for immunohistochemical demonstration of tartrate-resistant acid phosphatase.

We have developed a monoclonal antibody (9C5) for immunohistochemical localization of tartrate-resistant acid phosphatase (TRAcP). This antibody reacts with a denatured epitope of TRAcP and requires enhancement methods to promote antigenicity in paraffin-embedded tissues. We used this antibody to systematically examine proteolytic digestion and heat denaturation conditions for epitope enhancement in both paraffin sections and fixed smears. The goal was to increase the sensitivity of the immunohistochemical stain for TRAcP. Optimal conditions for proteolytic digestion were established. Denaturation in a conventional boiling water bath was compared to microwave irradiation in several commonly used solutions. Immunohistochemistry was compared directly to TRAcP cytochemistry in fixed smears from hairy cell leukemia specimens to gauge the level of sensitivity of our improved method. Attempts were made to "retrieve" the 9C5 epitope from overfixed tissues and aged smears. Maximal immunoreactivity of TRAcP was achieved by microwave irradiation in a citrate or Tris buffer of pH 6.0-8.0 without the need for a subsequent protease digestion step. With this method of epitope enhancement, immunohistochemistry with antibody 9C5 was as sensitive as direct cytochemical staining of TRAcP activity. However, once a tissue specimen had been overfixed or a smear stored for a year or more, the 9C5 epitope was no longer retrievable. The key element in epitope enhancement for 9C5 immunohistochemistry is heat denaturation of the target epitope. Immunohistochemistry of TRAcP in paraffin sections would be a great asset to the study of specialized forms of the monocyte/macrophage lineage and to the process of macrophage activation. It would also provide another means for more precise evaluation of residual disease in bone marrow of patients treated for hairy cell leukemia.

Cytochemical characterization of leukemic cells with numerous cytoplasmic granules.

Ten cases in which leukemic cells contained numerous cytoplasmic granules were examined by using a panel of cytochemical reactions. The diagnoses in the 10 cases were mast cell leukemia, chronic basophilic leukemia, and acute myeloid leukemia with basophilic differentiation in one case each, acute promyelocytic leukemia in two cases, acute megakaryoblastic leukemia in two cases, and blastic hairy cell leukemia in three cases. The cytochemical panel consisted of peroxidase, toluidine blue, chloroacetate esterase, aminocaproate esterase, tartrate-resistant acid phosphatase, and immunoalkaline phosphatase for platelet/megakaryocyte-specific antigen. The unusual cytologic features of leukemic cells in cases similar to our 10 cases have caused considerable diagnostic difficulties. In our 10 cases, however, the effective use of cytochemical studies helped to achieve accurate identification of the various types of leukemic cells. We conclude that the intelligent application of cytochemical techniques continues to be useful for the accurate cytodiagnosis of hematopoietic neoplasms.

Myelodysplastic syndrome: diagnostic implications of cytochemical and immunocytochemical studies.

Cytochemical and immunocytochemical studies were performed on bone marrow aspirates from 96 cases of primary myelodysplastic syndrome (MDS), 11 cases of secondary MDS, 22 cases of non-MDS hematologic disorders, and 10 cases of nondiagnostic, apparently normal marrow specimens to determine the practicality and utility of these stains for diagnosing MDS. Cytochemical studies included iron stain, periodic acid-Schiff (PAS), peroxidase, butyrate esterase, chloroacetate esterase, and double esterase stains. Immunocytochemical staining was done with monoclonal antibody HP1-1D, which recognizes the glycoprotein IIb/IIIa complex in megakaryocytes. The iron stain remained most helpful in identifying abnormal ringed sideroblasts, a feature of dyserythropoiesis, and thus in supporting the diagnosis of MDS. The PAS stain was helpful, if positive, in identifying patients with MDS; however, when it was negative, this stain did not help distinguish MDS from non-MDS hematologic disorders. The immunocytochemical stain with HP1-1D monoclonal antibody was also helpful in identifying atypical micromegakaryocytes, indicative of dysmegakaryopoiesis. Other cytochemical abnormalities were infrequently observed and were less specific for the diagnosis of MDS. The combination of two stains--for example, PAS and iron stain or PAS and double esterase--was helpful, however, in excluding MDS, inasmuch as neither the miscellaneous nor the control group stained positively with these combinations.

Acute myelomonocytic leukemia: an unusual variant with both granulocytic and monocytic esterases in the leukemic cells.

By using the combination of alpha-naphthyl butyrate esterase and chloroacetate esterase for cytochemical detection of monocytes and granulocytes, respectively, we examined and identified five adult patients with acute myeloid leukemia whose leukemic cells often (20 to 30%) had the characteristics of both monocytes and granulocytes. All five patients were men, 23 to 82 years of age. Two patients had manifestations of preleukemia. One of these two patients had received treatment for lymphoma for 12 months before acute myelomonocytic leukemia was diagnosed. One patient had gum hypertrophy, and two had leukemia cutis. At the time of initial examination, four patients had blood leukocyte counts higher than 80,000/mm3 and one had leukopenia. Four patients received chemotherapy; three responded temporarily but died within 1 year after the myelomonocytic leukemia had been diagnosed. The patient with leukopenia has remained in complete remission for 2 years. With more frequent use of double esterase stains for classification of acute myeloid leukemias, this variant of acute myelomonocytic leukemia should be detected more often and its clinical behavior should be better understood.

T-cell chronic lymphocytic leukemia with morphologic and immunologic characteristics of cytotoxic/suppressor phenotype.

The human cytotoxic/suppressor T-cell subpopulation has characteristic cytoplasmic granules and unique surface antigens that are recognized by OKT8 monoclonal antibody. Using immunocytochemical techniques, we identified three patients with an indolent T-cell lymphoproliferative disorder having morphologic and immunocytochemical characteristics of a cytotoxic/suppressor T-cell phenotype. Review of the literature revealed 22 similar cases, which were frequently associated with neutropenia and anemia. These cases may represent a distinct clinicopathologic entity with an apparently indolent clinical course that differs from other T-cell lymphoproliferative disorders, which are generally considered to be more aggressive diseases.

Orbital metastasis from prostatic carcinoma. Identification by an immunoperoxidase technique.

A patient was seen initially with an orbital tumor causing blindness of the left eye. Although disseminated prostatic carcinoma was found, the origin of the orbital tumor was not established until prostate antigen was demonstrated in biopsy specimens by an immunoperoxidase technique. Orbital metastasis from prostatic carcinoma has been reported in 16 cases. Confirmation of the diagnosis may be difficult and, in the past, has largely been indirect as judged by clinical trial. The immunoperoxidase technique for detecting prostate antigen in tissue sections is both sensitive and specific. Application of this technique resulted in the diagnosis of this technique resulted in the diagnosis of this unusual manifestation of metastatic prostate cancer. The possibility of additional clinical applications of this immunohistochemical technique within the field of ophthalmology is raised.

Histogenesis of splenic lesions in Hodgkin's disease.

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.

Differential characterization of the "reticulum cell" in lymphoreticular neoplasms.

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.

Immunoalkaline phosphatase cytochemistry. Technical considerations of endogenous phosphatase activity.

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.