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Background Tartrate-resistant acid phosphatase (TRACP) is an enzyme common to cells of the mononuclear phagocyte system and a clinically relevant biomarker for osteoclasts and inflammatory macrophages. The purpose was to assess applications and performance of six anti-TRACP monoclonal antibodies. Methods Mab9 C5, 14 G6, 162, 203, 220, and 89 were used as capture and detection antibodies in quantitative immunoassay, and for western blot (WB), immunoprecipitation, and immunohistochemistry of paraffin sections containing chronic inflammatory infiltrates. The clinical performance of mab14 G6 for immunoassay of serum TRACP5b activity was compared to two commercial kit methods. Results Mab9 C5 is useful for WB and immunohistochemistry methods only. Mab14G6, 162, and 203 are useful for quantitative immunoassay and immunoprecipitation, however, mab203 causes inactivation of enzymatic activity. Mab220 and 89 are specific for TRACP5a and useful in all applications. Mab14G6 has similar clinical sensitivity and specificity as two commercial methods. Conclusions TRACP is an important marker in osteoimmunology. Specific antibodies with unique specificity for TRACP isoforms and defined applications will be valuable for clinical evaluation of bone metabolic, inflammatory and autoimmune diseases and will aid in basic research of TRACP biochemistry and biology.

Human serum contains two related isoforms of TRACP: TRACP 5a and TRACP 5b. Serum TRACP 5a protein is increased in about one third of rheumatoid arthritis (RA) sera. This study was undertaken to examine the significance of serum TRACP isoforms 5a and 5b as disease markers of inflammation and bone destruction in RA. One hundred eighteen patients were recruited including 50 with RA (25 with nodules), 26 with osteoarthritis (OA), and 42 with other rheumatic diseases. Twenty-six healthy adults served as controls. Serum TRACP 5a activity, TRACP 5a protein, and TRACP 5b activity were determined by in-house immunoassays. C-reactive protein (CRP) was determined by in-house immunoassay using commercial antibodies and CRP. Other commercial markers included bone-specific alkaline phosphatase (BALP), C-telopeptides of type-I collagen (ICTP), cartilage glycoprotein-39 (YKL-40), and IgM rheumatoid factors (IgM-RF). Mean TRACP 5a protein was significantly elevated only in RA compared with healthy controls and other disease groups. TRACP 5a protein correlated significantly only with IgM-RF in RA. Among RA patients, mean TRACP 5a protein and IgM RF were significantly higher in nodule formers. In contrast, TRACP 5b activity was slightly elevated in RA and correlated with BALP, ICTP, and YKL-40 but not with IgM-RF or CRP. Mean TRACP 5b activity was no different in RA patients with or without nodules. TRACP isoforms could be useful disease markers in RA; TRACP 5a protein may be a measure of systemic inflammatory macrophage burden and disease severity. TRACP 5b activity is a marker for osteoclast number and perhaps local or systemic bone destruction.

Background/Aims: Secondary hyperparathyroidism (SHP) is characterized by high bone turnover and elevated serum bone remodeling markers. Elevation of serum interleukin-6 (IL-6) levels is also characteristic of end-stage renal disease. This study investigates the effects of intravenous calcitriol on serum bone resorptive markers, namely, type 5b tartrate-resistant acid phosphatase (TRACP5b) and IL-6 in patients with SHP. Methods: Intravenous calcitriol therapy was given for 16 weeks to 24 patients on maintenance hemodialysis with plasma intact parathyroid hormone (iPTH) levels >300 pg/ml. Blood was drawn at baseline and every 4 weeks for 16 weeks for determination of the levels of biochemical parameters, iPTH, IL-6 and bone remodeling markers, including bone-specific alkaline phosphatase (bAP) and TRACP5b. Results: Only 21 patients responded to the calcitriol therapy, with significant decrements in serum iPTH after 4 weeks of therapy and thereafter. After 16 weeks of calcitriol therapy, 21 patients had significant decrements in serum iPTH (707.9 ± 317.8 vs. 205.0 ± 63.1 pg/ml, p < 0.01). Prior to treatment, a significant correlation was found between increased levels of serum iPTH and IL-6 levels (r = 0.45, p < 0.05). After treatment, there was also a significant and parallel lowering of levels of serum iPTH, IL-6 (8.52 ± 3.59 vs. 7.24 ± 2.81 pg/ml, p < 0.01), bAP (54.68 ± 36.17 vs. 24.55 ± 13.84 U/l, p < 0.01) and TRACP5b (3.41 ± 1.89 vs. 1.80 ± 0.55 U/l, p < 0.01). Our results additionally showed significant positive correlationsbetween baseline levels of serum IL-6 and those of iPTH, bAP and TRACP5b. After 16 weeks of calcitriol treatment, the correlation between IL-6 and iPTH levels lost significance but levels of serum IL-6, bAP and TRACP5b remained significantly correlated. Conclusions: Elevated levels of serum IL-6 and bone remodeling markers, namely, bAP and TRACP5b which are common features of SHP, are effectively suppressed by calcitriol therapy. This indicates that hyperparathyroidism not only accelerates bone remodeling but may also aggravate inflammation in patients on maintenance hemodialysis.

Human serum tartrate-resistant acid phosphatase exists as two enzyme isoforms (TRACP 5a and 5b), derived by differential, post-translational processing of a common gene product. Serum TRACP 5b is from bone-resorbing osteoclasts (OC) and becomes elevated in diseases of increased bone resorption. TRACP 5a is secreted by macrophages (MΦ) and dendritic cells (DC) and is increased in many patients with rheumatoid arthritis. Our purpose was to fully characterize the properties of human TRACP isoforms and to produce an antibody specific to TRACP 5a for use as a biomarker in chronic inflammatory diseases. Partially purified, natural serum TRACP isoforms and recombinant TRACP 5a (rTRACP 5a) were compared with respect to specific activity and subunit structure and presence of sialic acid. Mice were immunized with rTRACP 5a, and resulting hybridomas were screened for monoclonal antibody to serum TRACP 5a. One antibody, 220, was tested for its epitope specificity and use in various immunological techniques. rTRACP 5a had properties identical to serum TRACP 5a. Antibody 220 was specific for the trypsin-sensitive epitope in the loop peptide, present only in TRACP 5a. Antibody 220 was effective for specific immunoprecipitation, immunoassay, and immunoblot of TRACP 5a. Intact TRACP was present in MΦ, DC, and OC. TRACP 5a was the predominant isoform secreted by MΦ and DC, whereas TRACP 5b was the predominant isoform secreted by OC. TRACP isoforms 5a and 5b may have different functions inside and outside of monocyte-derived cells. Antibody 220 is an important resource for studies of the biosynthetic relationship among TRACP isoforms and of the significance of serum TRACP 5a as a marker in diseases of bone metabolism and inflammation.

Objective: Our purpose was to develop a specific immunoassay for tartrate-resistant acid phosphatase (TRACP) 5b using naphthol ASBI phosphate (N-ASBI-P) as a selective substrate for isoform 5b and heparin as a selective inhibitor of isoform 5a. Methods: Serum TRACP 5a and 5b and recombinant TRACP 5a were used to optimize and standardize the immunoassay for specificity, linearity, analytical recovery, sensitivity and reproducibility. Serum N-telopeptide cross-links (NTX) and bone alkaline phosphatase (bone ALP) were also measured. The clinical sensitivity and specificity were assessed in healthy control subjects and patients with osteoarthritis (OA), rheumatoid arthritis (RA) and endstage renal disease (ESRD). Results: TRACP 5b specificity was achieved at pH 6.3 with 2.5 mmol/l substrate and 25 U/ml heparin. Isoform 5b specificity was increased over our original immunoassay using 4-nitrophenyl phosphate (4-NPP) without heparin. The alternative immunoassay was linear with 110% analytical recovery and no serum matrix effects. The average intra-assay error was 10.65%; the average inter-assay error was 10.11% for values of 1–3 U/l and 6.5% for values of 7–11 U/l. Mean serum TRACP 5b in OA and RA were not significantly different from control using either immunoassay. Mean serum TRACP 5b was significantly increased in ESRD with both immunoassays. Serum TRACP 5b levels correlated significantly with NTX and bone ALP in the disease groups, but not always in the control group. Conclusion: This alternative immunoassay for TRACP 5b activity is highly specific. It should have applications in evaluating patients with bone disease and will improve our understanding of the biological significance of TRACP 5b expression in health and disease.

The association between elevated serum type 5 TRACP activity and metabolic bone diseases has been recognized for many years. However, serum type 5 TRACP exists as two related isoforms: 5a and 5b. Only isoform 5b is osteoclast-derived; the origin and significance of isoform 5a has hardly been explored. We have used simultaneous immunoassays for type-5 TRACP activity and total type-5 TRACP protein in conjunction with non-denaturing gel electrophoresis and column chromatography to investigate the nature and significance of TRACP isoforms 5a and 5b in end-stage renal disease (ESRD) and rheumatoid arthritis (RA). Our studies have shown that TRACP activity and protein are elevated in approximately 50% of sera from ESRD patients, which is caused by osteoclastic isoform 5b. We have also shown that total TRACP protein, but not TRACP activity, is elevated in approximately 30% of sera from RA patients, which is caused by non-osteoclastic isoform 5a. When macrophages or dendritic cells (DC) were cultured in vitro, abundant TRACP 5a was secreted into the culture medium, whereas TRACP 5b was retained intracellularly by both cell types. This implicates macrophages and DC as potential sources of elevated TRACP 5a in RA. Because TRACP isoform expression may be disease-specific, it is important to be able to distinguish TRACP 5a from 5b. There are four criteria by which to do so: (1) TRACP 5a bears sialic acid residues while TRACP 5b does not; (2) the pH optimum for TRACP 5a is 5.2 while that for TRACP 5b is 5.8; (3) the specific activity of TRACP 5a is significantly lower than that of TRACP 5b; and (4) TRACP 5a is as an uncleaved polypeptide, whereas TRACP 5b is a proteolytically nicked disulfide-linked "heterodimer." The differences in biochemical properties and disease-specific expression of TRACP isoforms 5a and 5b suggest that they are regulated differently and perform separate functions in a tissue-specific manner.

Our objective was to evaluate the significance and source of serum tartrate-resistant acid phosphatase (TRACP) in patients with rheumatoid arthritis (RA).Thirty-five RA, 32 osteoarthritis (OA) and 16 control subjects were studied. Serum TRACP-5b activity and total TRACP protein were determined by immunoassay. TRACP isoforms were analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Serum bone alkaline phosphatase (BAP), cross-linked N-terminal telopeptides (NTx), and C-terminal telopeptides (ICTP) of type I collagen were estimated as markers of bone turnover. C-reactive protein (CRP) was measured as a marker of chronic inflammation. Macrophages and dendritic cells (DC) were developed from peripheral blood monocytes. Cell lysates and culture supernatants were analyzed for TRACP isoforms by immunoassay and PAGE.In RA, mean TRACP-5b activity was normal, but median total TRACP protein was increased twofold (p0.001). In OA, TRACP-5b activity and protein were normal. In RA, TRACP-5b activity correlated weakly with ICTP (r=0.56) while TRACP protein levels correlated weakly with NTx (r=0.43). Additionally, TRACP protein, but not TRACP-5b activity correlated significantly with CRP (r=0.42). Macrophage and DC lysates contained TRACP-5b, while tissue culture supernatants contained TRACP-5a.Increased total TRACP protein in RA sera was probably due to TRACP-5a and not derived from osteoclasts. Rather, it could be a secreted product of inflammatory macrophages and DC.

Langerhans cell histiocytosis (LCH) is an intriguing disorder characterized by the accumulation of specialized dendritic cells called Langerhans cells in several diverse tissues and body sites. It has been cited in numerous case reports to be associated with a wide variety of malignant neoplasms. Although many hypotheses have been suggested, the basis for such associations remains essentially unknown. We describe another association here that to our knowledge has not been reported thus far: a solitary plasmacytoma occurring at a site of previous involvement by LCH. This constitutes a new addition to the now fairly lengthy list of malignant neoplasms that have been reported to occur in the setting of LCH. The possible reasons for such an association are discussed along with a brief review of LCH.

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) μg/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/μg) was similar to that in healthy subjects (0.091 U/μg). By contrast, the specific activity of TRAP in the 31 of 99 rheumatic sera with increased TRAP protein (0.035 U/μg) 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.

Tartrate-resistant acid phosphatase (TRAP) is expressed abundantly by osteoclasts and is required for bone resorption. This enzyme is emerging as an important biomarker in bone pathology, both for histochemical identification of osteoclasts and as a serum marker of osteoclast activity and increased bone turnover. Rat and mouse models are becoming popular systems for studying osteoclast development, bone physiology and morphogenesis, and bone diseases such as osteoporosis. We have developed two unique antibodies to human TRAP purified from hairy cell leukemia spleen. Both antibodies (9C5 and 14G6) are suitable for immunohistochemistry of osteoclasts and macrophages. Only one (14G6) is capable of immunoprecipitating active TRAP from human cell lysates. Antibody 9C5 reacts with a denatured epitope of TRAP while antibody 14G6 probably reacts with a native, conformational determinant. The high degree of homology among TRAPs of various species predicts that these antibodies should be suitable for work in experimental animals as well as humans. Immunohistochemical staining, electrophoretic analyses, immunoprecipitation and immunoblotting assays of human rat and mouse TRAP were carried out to test the validity of these antibodies as cell markers in rodents. Both antibodies were suitable for immunohistochemistry in all species. Antibody 9C5 was suitable for immunoblotting of denatured TRAP of all species tested. Antibody 14G6 reacted with the native TRAP of humans only and failed to immunoprecipitate mouse or rat TRAP activity. Although TRAP is a phylogenetically conserved protein, subtle, species-specific determinants exist. Care should be exercised when anti-TRAP antibodies are used for immunoassay in experimental animals.

Tartrate-resistant acid phosphatase (TRAP) is an important cell marker in both hematology and bone pathology. Cytochemical stains for TRAP activity are used to identify leukemic cells of hairy cell leukemia, inflammatory or activated macrophages, and bone resorbing osteoclasts. A monoclonal antibody to TRAP (9C5) was developed for immunohistochemical identification of these and other TRAP-expressing cells in paraffin sections. In our continuing efforts to maximize the sensitivity and specificity of 9C5 for diagnostic and investigational purposes, we conducted studies to compare steam to microwave irradiation for heat induced epitope retrieval (HIER) and to compare formalin fixation to B-5 fixation. We found that a simple and inexpensive household vegetable steamer provided equivalent or superior HIER for demonstration of TRAP with 9C5 in a variety of tissues. We also found no significant differences in immunoreactivity or nuclear detail between formalin and B-5 fixed bone marrow biopsy sections. ...

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.

Tartrate-resistant acid phosphatase (TRAcP) has been an indispensible marker for hairy cell leukemia (HCL) for over two decades. However, the traditional TRAcP cytochemical stain cannot be performed effectively on sections of paraffin-embedded tissues that are important resources for histopathologic evaluation in diagnosis and treatment of HCL. Wide variation in expression of TRAcP activity by hairy cells (HCs) within and among patients is an interesting biologic phenomenon that has not been explained and can cause some diagnostic uncertainty as well. To solve the problem of staining TRAcP in paraffin sections and to begin to address the questions of variable TRAcP expression in HCL, we developed a monoclonal antibody to TRAcP, 9C5, for immunohistochemical identification of HCs. In smears of blood and bone marrow, immunocytochemistry of TRAcP using 9C5 was as specific but slightly less sensitive than direct cytochemical staining of enzymatic activity. In paraffin sections of spleen and bone marrow from HCL patients, immunohistochemistry with 9C5 stained the HCs with high sensitivity and specificity and clearly showed the characteristic diffuse infiltration by HCs. Other cells noted to stain strongly with 9C5 were occasional macrophages in bone marrow smears and osteoclasts and occasional tissue macrophages in paraffin sections. These are cells known to express abundant TRAcP activity. Immunohistochemistry with anti-TRAcP monoclonal antibody 9C5 may have utility as an added option in the diagnosis of HCL, as a means to evaluate residual disease in HCL patients undergoing new treatments, and as a way to address questions regarding variable expression of TRAcP activity by HCs within and among patients with HCL. Also, 9C5 has potential as a reagent for the immunoassay of bone-derived serum TRAcP in patients with certain bone diseases and cancers with bone metastasis.

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.

Type 5 tartrate-resistant acid phosphatase (TRAP) has been a clinically relevant biomarker for about 50 years. It has always been a reliable and specific cytochemical marker for hairy cell leukemia and for differentiated cells of monocytic lineage. Only recently has the test for serum TRAP activity been accepted as sensitive and specific enough for clinical use as a marker of osteoclasts and bone resorption. This has come about through steady advances in knowledge about TRAP enzymology, structure, function, and molecular regulation and a consequent appreciation that TRAP isoforms 5a and 5b have very different clinical significance. As a measure of osteoclast number and bone resorption, TRAP 5b has diagnostic and prognostic applications in osteoporosis, cancers with bone metastasis, chronic renal failure, and perhaps other metabolic and pathologic bone diseases. Serum TRAP 5a, on the other hand, has no relationship to bone metabolism but seems instead to be a measure of activated macrophages and chronic inflammation. Exploration of the real clinical usefulness of serum TRAP 5a for diagnosis and disease management in a wide variety of chronic inflammatory diseases is only now beginning. This perspective traces the important basic scientific developments that have led up to the refinement of serum TRAP isoform immunoassays and their validation as biomarkers of disease. Many unanswered questions remain, providing a wealth of opportunity for continued research of this multifaceted enzyme.

Human serum contains 2 isoforms of type-5 tartrate-resistant acid phosphatase (TRACP): 5a and 5b. TRACP-5b is osteoclastic. Our goal was to determine if serum TRACP-5a could originate from inflammatory macrophages (MPhi). We stained 246 paraffin-embedded tissue samples for TRACP using monoclonal antibody 9C5 (mab9C5) to isoforms 5a and 5b and a novel mab220 specific to isoform 5a. CD68 and lysozyme were also stained. MPhi of chronic and granulomatous inflammation and in tissues that undergo strong antigenic stimulation were strongly positive for TRACP, more so with mab220 than with mab9C5. Noninflammatory MPhi in lymph node sinuses or germinal centers and red pulp MPhi of spleen were weak or negative for TRACP. Marginal zone lymphocytes and sebaceous glands of skin were weakly positive for TRACP. Tissue mast cells displayed strong TRACP staining. Neuroendocrine cells of gastrointestinal tissues were strongly immunoreactive with mab9C5 but negative with mab220. Restricted expression of TRACP primarily in inflammatory MPhi supports our hypothesis that circulating TRACP-5a could be a biomarker of chronic inflammatory disease activity.

Diagnosis and follow-up of bone metastases in breast cancer patients usually rely on symptoms and imaging studies. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is a specific marker of osteoclasts and is herein proposed as a marker of bone metastasis in breast cancer patients. An immunoassay using a monoclonal antibody, 14G6, was used to measure the activity of serum TRACP 5b at pH 6.1 in 30 early breast cancer patients without bone metastasis and in 30 aged-matched breast cancer patients with bone metastasis. Another 60 normal volunteers were recruited as controls. Bone alkaline phosphatase (BAP), a traditional marker of bone turnover, was also measured in selected cases. The overall mean TRACP 5b activity in normal women was 2.83 +/- 1.1 U/l, and it increased with age. The mean TRACP 5b activity in early breast cancer patients did not differ from that of the normal group (2.93 +/- 0.64 vs. 2.83 +/- 1.1 U/l; p = 0.66), whereas it was significantly higher in breast cancer patients with bone metastasis (5.42 +/- 2.5 vs. 2.83 +/- 1.1 U/l; p0.0001). BAP activity was significantly higher in breast cancer patients with bone metastasis than in early breast cancer patients (p = 0.004). Serum TRACP 5b activity correlated well with BAP activity in breast cancer patients with bone metastasis (p0.0001), but not in normal individuals or in patients without bone metastasis. TRACP 5b activity can be considered a surrogate indicator of bone metastasis in breast cancer patients.

SUMMARY 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 � 10 5 /100 � l 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)

Objective: In human serum, type-5 tartrate-resistant acid phosphatase (TRACP) exists as two closely related isoforms: 5a and 5b. Serum isoform 5b is an osteoclast product that reflects bone resorption rate and is frequently increased in diseases of increased bone turnover. Isoform 5a protein is often increased in rheumatoid arthritis (RA) sera and may be a product of inflammatory macrophages. Our objective was to compare the biochemical characteristics of TRACP 5a and 5b. Methods: We transfected the human ACP 5 gene into CHO cells and cloned a stable cell line (CHO/TRACP 8F5) that expresses high levels of TRACP activity both intracellularly and as a secreted product. Both enzyme preparations were purified on an anti-TRACP antibody column. Their biochemical properties were compared to the natural serum isoforms using colorimetric assays for activity and total protein. Their structural properties were compared to natural serum isoforms using denaturing and nondenaturing polyacrylamide gel electrophoresis. Results: Both enzyme preparations were heterogeneous. The combined secreted recombinant TRACPs (rTRACPex) had all the characteristics of natural serum TRACP 5a. There were seven uncleaved glycoproteins with a pH optimum of 5.2, relatively low specific activity (278 U/mg) and differentially sialylated. The combined intracellular TRACPs (rTRACPin) had all the characteristics of natural serum TRACP 5b. They consisted of two proteins, one of which was a processed heterodimer, with a pH optimum of 5.8, a relatively high specific activity (887 U/mg) and lacked sialic acid. Conclusion: This cell line provides an avenue for the simultaneous study of the regulation, function and intracellular trafficking of separate TRACP isoforms and the identification of their physiologic substrates in a single uniform cell source.

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.

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.

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.

An immunohistochemical method for detecting prostatic acid phosphatase is described for the diagnosis of metastatic prostatic carcinoma. The specific antiserum against prostatic acid phosphatase was prepared from rabbit by injection of acid phosphatase purified from seminal fluid. This method gives a selective staining of the cytoplasm of the glandular epithelial cells of prostatic tissue specimens on paraffin section. Most of the non-prostatic tissues were negative except for occasional weak staining in granulocytes, islet cells of pancreas, parietal cells of stomach, tubular epithelial cells of kidney, and liver cells. Also examined were 50 consecutive cases of metastatic tumor involving the bone marrow and 5 cases of metastatic prostatic carcinoma involving the lymph node or lung. All 20 cases with prostatic primary lesion showed positive staining. All other cases were negative, except 5 of the 14 cases of metastatic breast carcinoma in women showing weakly positive results. The method is fairly specific for identification of metastatic prostatic carcinoma. Occasional positive staining in breast tumor needs further study to establish whether the staining is due to the same isoenzyme or to certain cross immunoreactivity.

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.

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.

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.

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.

Cytochemistry of acid phosphatases has recently enjoyed an increase in clinical application. Better known among these methods is the tartrate-resistant acid phosphatase stain for the hairy cells of hairy-cell leukemia. The demonstration of prostatic acid phosphatase in tumor cells outside the prostate may further aid in the detection of metastatic prostate cancer. Aside from these methods for cytologic and histologic materials, it is now possible to cytochemically demonstrate acid phosphatase activity in subcellular organelles, extracts of whole tissue and cellular preparations, and in biological fluids and secretions. The methods for optimal staining of acid phosphatase activity in these various materials are necessarily different in one or more respects. The methods for cytochemical demonstration of acid phosphatase are reviewed in the context of the applications for which they may be used. Emphasis is placed on those methods for cytologic and histologic preparations. Applications where cytochem...

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.

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.

Human prostatic acid phosphatase isoenzyme 2 (HPAcP-2) was isolated from semen. This purified enzyme was immunized to rabbit to produce polyclonal antibodies. The specificity of the antibodies was tested by Western blot transfer method. Rabbit IgG-peroxidase conjugate was prepared from the antiserum and used to localize HPAcP-2 in prostatic carcinoma. It was found that in the tumor glandular acinus the normal basal cells were replaced by tumor cells containing reaction product. In the tumor cells, the reaction product was seen in the cisternae of rough endoplasmic reticulum (ER) and Golgi apparatus. The secretory vesicles which contained reaction product-stained granules and some amorphous material were seen to fuse with the apical plasma membrane and discharged their content into the glandular lumen. On the other hand, some secretory vesicles in the tumor cells facing to the basement membrane also discharged their similar content into the extracellular spaces. Reaction product-stained granules were found in the interstitial spaces surrounding the tumor cells. These findings suggest that HPAcP-2 is synthesized on the bound ribosomes and discharged into the cisternae of rough ER. The molecules are transported to the Golgi cisternae. After concentration and packaging, HPAcP-2 molecules are then transferred to the secretory vesicles, and discharged into the glandular lumen and to the extracellular spaces. The isoenzyme released in the extracellular space may reach the blood stream through the interstitial spaces or the lymphatic system, resulting in the elevation of serum HPAcPase level in some prostatic cancer patients.

Nineteen patients with hairy cell leukemia were studied in order to define the hepatic changes in this disease and to correlate the morphologic changes in the liver with the clinical and biochemical findings. Although only eight of the patients had hepatomegaly, all 19 had microscopic mononuclear cell infiltration in the sinusoids or the portal areas or both. The severity of mononuclear infiltration in the liver correlated poorly with the size of the liver or spleen, the biochemical changes, or number of hairy cells in the blood. Abnormal serum biochemical values were present occasionally and were usually due to associated diseases or to complications of this disease. Elevated serum alkaline phosphatase activity was noted in four patients; three of them bad granulomatous lesions in the liver. Unless the characteristic “clear cell” pattern is seen, the hepatic mononuclear cell infiltration may not be diagnostic of hairy cell leukemia and, in many instances, may not even be suggestive of neoplasia. A new technique for demonstrating tartrate-resistant acid phosphatase activity in methacrylate-embedded sections was developed, which allowed identification of hairy cells in the liver biopsy specimens of all five patients so studied. The authors concluded that liver involvement is common in this disease.

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 p H 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.

Immunoelectron microscopic studies were done on prostatic tissues obtained from patients with benign hyperplasia. Rabbit IgG-peroxidase conjugate against purified human prostatic acid phosphatase band 2 (HPAP-2) was used for studies. Under the light microscope, the columnar secretory epithelia of prostatic glands showed different intensity and distribution of immunostaining whereas the basal cells were unstained. Under the electron microscope, the secretory epithelial cells often showed electron-dense reaction product in the Golgi apparatus and secretory vesicles and vacuoles, and only sparingly in the cisternae of nuclear envelope and rough ER. Sometimes, fusion of secretory vacuolar membrane and plasma membrane and discharge of the vacuolar contents into the extracellular space were noted. The surfaces of microvilli at the apical portion of the columnar epithelia and the lumen of the glandular acini always showed reaction product. These findings suggest that HPAP-2 may be synthesized in the rough ER and transported to the Golgi apparatus where it is concentrated and transferred to the secretory vesicles and vacuoles. HPAP-2 is finally discharged into the extracellular spaces through exocytosis, a secretory mechanism similar to that of other secretory proteins.

Several histochemical methods were applied to identify the various cellular and structural elements of the human spleen. These included naphthol AS acetate esterase and reticulin stain for the sinuses, fluoride-resistant esterase for macrophages, alkaline phosphatase for vascular structures, chloroacetate esterase for neutrophils, cyanide-resistant peroxidase and chlorazol fast pink for eosinophils, toluidine blue for basophils and pseudoperoxidase for erythrocytes and erythroblasts. The methods are simple, sensitive and readily reproducible and are best used in combination, so that several types of cells can be identified simultaneously. The location of these cells in the spleen and the interaction between these cells are readily appreciated. These methods can be used to study the morphologic changes of spleen in various hematologic disorders.

Seven acid phosphatase isoenzymes, namely 0, 1, 2, 3, 3b, 4 and 5, in human leukocytes have been separated both by acrylamide gel electrophoresis and by ion exchange chromatography. The study of these isoenzymes in preparations of various cell types indicates that at least some of the isoenzymes are cell type-specific, i.e., isoenzyme o in Gaucher cells, 1, 2 and 4 in neutrophils, 1 and 4 in monocytes, 3 in lymphocytes and platelets, 3b in primitive blasts and 5 in reticulum cells of leukemic reticuloendotheliosis. Isoenzyme 5 is different from others in its resistance to l(+)-tartaric acid treatment. The persistent presence of abnormally high isoenzyme 5 in the reticulum cells of leukemic reticuloendotheliosis may be important for the differential diagnosis of this disease. Possible relationships between these isoenzymes and subcellular organelles are discussed.

Chromosome studies were performed in four cases of erythroleukemia, in one case of acute erythremic myelosis and in one case of chronic erythremic myelosis. Abnormalities were encountered in all of these cases. No consistent patterns were found although clonal lines could be detected in three cases. A common feature in five of the cases was a variable degree of involvement of the chromosomes of the G group (presence of G monosomy in two cases, Ph1-like chromosome in two cases, and a slight reduction in size of a small acrocentric chromosome in 40 per cent of the metaphases in the case of chronic erythremic myelosis). C trisomy was present in one case. These findings indicate that the involvement of groups G and C may represent an important feature of the Di Guglielmo syndrome and suggest a close relationship of this disorder with other myeloproliferative diseases.

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.

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.

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.

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.