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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.

The objective of this study was to identify the isoform, type-5a or type-5b, responsible for increased tartrate-resistant acid phosphatase (TRAP) activity in endstage renal disease (ESRD) and TRAP protein in rheumatoid arthritis (RA). We studied 24 sera each from healthy, ESRD and RA subjects. Type-5 TRAP activity and protein were quantitated by immunoassays. Isoform expression was determined by computerized imaging of non-denaturing polyacrylamide gels (PAGE) stained for TRAP activity. Other biochemical markers included: intact parathyroid hormone (iPTH), total and bone-specific alkaline phosphatase (TAP, BAP), N-telopeptides of type-I collagen (NTx), and free pyridinoline (Pyd). Isoform 5a was normal in both ESRD and RA. Isoform 5b was elevated in ESRD only. Serum TRAP activity correlated with both isoforms 5a and 5b in RA, but only with 5b in ESRD. TRAP protein assays did not correlate with PAGE assays for 5a or 5b. TRAP activity, but not protein, correlated with BAP and NTx in RA sera. Both TRAP activity and protein correlated with iPTH, TAP and Pyd in ESRD sera. Increased TRAP activity in ESRD was due to increased osteoclastic isoform 5b and related to bone turnover. Increased TRAP protein in RA was suspected, but not proven, to be isoform 5a and not related to bone turnover. Heterogeneity of serum TRAP and preferential expression of isoforms has clinical significance in different diseases including ESRD and RA.

Magnetization fluctuations of uniformly magnetized media have been measured in sputtered CoP thin films as a function of Cr underlayer thickness. Increasing underlayer thickness from 20 to 400 nm in nine steps yields decreasing magnetization fluctuation noise from 210 nm to about 25 nm, similar to the known decrease of transition noise. For virtually every disk, the noise power dependance on magnetization is well fit by the square of the magnetization versus current loop derivative, (dM/dI)2. Thus, the noise mechanism appears to be a modulation process over the entire series. Analysis of measured magnetization noise spectra yields approximate correlation lengths which decrease with increasing Cr underlayer thickness.

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.

A major product of osteoclasts, tartrate-resistant acid phosphatase (TRAP) is an essential but insufficient enzyme for bone resorption. TRAP is an excellent cell marker for osteoclasts and macrophages and is being investigated as a serum marker for osteoclast activity in diseases of bone destruction. For decades, TRAP has also been used as a marker for hairy cell leukemia. Immunoassays for TRAP are sought to increase the sensitivity and specificity of the TRAP test for bone and hairy cells. Our laboratory recently developed a monoclonal antibody to TRAP (9C5) useful for immunohistochemical identification of TRAP-positive cells in paraffin sections. Herein, we characterize 9C5 in greater detail and report production of another anti-TRAP monoclonal antibody antibody (14G6) reactive with native, active enzyme antigen. Enzyme immunoassay, immunoprecipitation, western blot, and immunohistochemical analyses revealed the contrasting properties of 9C5 and 14G6. Antibody 9C5 reacts with a heat-denatured epitope and is suitable for denaturing western blot analysis and for immunohistochemistry. Antibody 14G6 reacts with a conformational determinant destroyed by heat and is suitable for immunoprecipitation of active TRAP, although 20% to 30% of activity is inhibited in the immune complexes. Having characterized several properties of these anti-TRAP antibodies, 9C5 and 14G6 may be useful for development of TRAP-specific immunoassays in bone pathology and hematology. They will certainly be of use for the study of biosynthesis, regulation, expression, and function of TRAP.

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.

Tartrate-resistant acid phosphatase is an inducible marker of cell differentiation and activation expressed by specialized cells of macrophage lineage and some activated lymphocytes. Clinically, this phosphatase is a diagnostic marker for hairy cell leukaemia and osteoclast activity. The cDNA for this enzyme has been cloned from a placental expression library, yet the cell(s) expressing the enzyme protein has not been determined with certainty. Our laboratories have developed a monoclonal antibody, 9C5, suitable for immunohistochemical localization of tartrate-resistant acid phosphatase in paraffin sections. The purpose of this study was to use antibody 9C5 to identify cells expressing tartrate-resistant acid phosphatase in sections of paraffin-embedded, normal, full-term placenta and to determine if those cells expressed other macrophage markers including CD68 (PG-M1 antibody), LN5, lysozyme, alpha 1-antitrypsin and alpha 1-antichymotrypsin. Histochemical localization of activity in frozen sections was compared with immunohistochemical localization in paraffin sections of the same tissue specimens. The activity and antigenicity of this enzyme were detected in decidual cells, syncytiotrophoblast, and some macrophages distributed throughout maternal and embryonic tissues, but not in neutrophils. Unlike other tissues previously examined, placenta contains significant numbers of the phosphate-positive cells that are not of macrophage origin.

Leukopenia, thrombocytopenia, and anemia are important features of hairy cell leukemia (HCL). They are generally considered to be due to hypersplenism and to inadequate production by bone marrow which is heavily infiltrated by the neoplastic hairy cells (HC). However, the cytopenias may also be caused by hemopoietic inhibition by cytokines derived from the mononuclear cells (MNC) of HCL. We studied the MNC of HCL with an in vitro assay for granulocyte-macrophage progenitors (CFU-GM) to search for this hemopoietic inhibitor(s) and to determine the cell source and mechanism of its production/release. We found that MNC conditioned media from 7 of 9 HCL cases exerted substantial inhibitory effect (23% to 66%) on normal marrow cells. Peak inhibitory activity was obtained in media conditioned with 10(6) MNC/ml for 90 minutes to 24 hours. Both HC and lymphocytes could release inhibitor(s) through mutually synergistic cell interactions. HC alone were inactive and lymphocytes alone were only weakly active. Mixtures of conditioned media of HC and of lymphocytes were not synergistic. The lymphocytes responsible for the inhibition were present in preparations depleted of cells bearing the cluster designation 4 antigen (CD4+) and B cells and were most likely the CD8+ T-cells. In one patient so examined, a partial reversal of inhibition was achieved by treating MNC-CM with antibodies to tumor necrosis factor (TNF)-alpha suggesting that TNF-alpha was at least partly involved in the inhibition of CFU-GM. This mechanism of cytokine release may be operative in vivo to account for the cytopenias in HCL and, if so, could alter the concept of hypersplenism in this disease.

The coexistence of two relatively rare disorders in 1 patient often induces speculation about possible cause and effect of these disorders. The suitable therapeutic regimen for both diseases and roentgenologic manifestations are described.

Acid phosphatase (E.C.3.1.3.2) in a giant cell bone tumor and a spleen infiltrated with hairy cells was extracted by citrate buffer and then by 0.3 mol/L NaCl. The cationic acid phosphatase in the crude extract was isolated by CM-cellulose chromatography, and further separated by high pressure liquid chromatography. The majority of the tartrate resistant acid phosphatase in the hairy cell spleen was unabsorbed on CM-cellulose and was insensitive to iron. A much larger portion of the acid phosphatase in the bone tumor, than in the spleen, was cationic and was eluted from the column by 0.8 mol/L NaCl. The cationic acid phosphatase was further separated into consecutive peaks of acid phosphatases with different sensitivity to iron. A major portion of acid phosphatase in the giant cell bone tumor was enhanced by iron, while the amounts of iron-enhanced and iron-insensitive acid phosphatase were about the same in the spleen. The differences of the phosphatases in these two types of pathologic specimens indicate the occurrence of two types of enzymes with different biological significance.