Hereditary alpha tryptasemia is not associated with specific clinical phenotypes.

BACKGROUND: Hereditary alpha tryptasemia (HαT) is found in approximately 7% of the population. Associations with a variety of clinical symptoms including gastric reflux, joint hypermobility, dysautonomia, flushing and pruritus, and hymenoptera allergy have variably been described in prior reports. However, our understanding of this genetic trait is limited by a paucity of published studies, referral bias, and conflicting findings at clinical presentation. OBJECTIVE: The purpose of this study was to assess the clinical phenotype of HαT in a random biorepository population and in patients with and without mastocytosis referred to the allergy clinic. METHODS: Tryptase copy number allele was assessed using digital droplet PCR. Participants with or without HαT were interviewed and examined by a clinician and surveyed regarding their medical history and symptomology. RESULTS: HαT was identified in 7.5% of the random biorepository samples and in 18% of patients with mastocytosis. There was no difference in the clinical symptomology or medical history of individuals with HαT compared to controls. Average baseline serum tryptase was higher in individuals with HαT compared to controls, but there was no difference in urinary mast cell activation products. CONCLUSIONS: Elevated baseline serum tryptase was the only consistent phenotypic marker for HαT in this study. There was a higher frequency of HαT in patients with mastocytosis than in the general population.

Genetic Regulation of Tryptase Production and Clinical Impact: Hereditary Alpha Tryptasemia, Mastocytosis and Beyond.

Tryptase is a serine protease that is predominantly produced by tissue mast cells (MCs) and stored in secretory granules together with other pre-formed mediators. MC activation, degranulation and mediator release contribute to various immunological processes, but also to several specific diseases, such as IgE-dependent allergies and clonal MC disorders. Biologically active tryptase tetramers primarily derive from the two genes TPSB2 (encoding ß-tryptase) and TPSAB1 (encoding either α- or ß-tryptase). Based on the most common gene copy numbers, three genotypes, 0α:4ß, 1α:3ß and 2α:2ß, were defined as "canonical". About 4-6% of the general population carry germline TPSAB1-α copy number gains (2α:3ß, 3α:2ß or more α-extra-copies), resulting in elevated basal serum tryptase levels. This condition has recently been termed hereditary alpha tryptasemia (HαT). Although many carriers of HαT appear to be asymptomatic, a number of more or less specific symptoms have been associated with HαT. Recent studies have revealed a significantly higher HαT prevalence in patients with systemic mastocytosis (SM) and an association with concomitant severe Hymenoptera venom-induced anaphylaxis. Moreover, HαT seems to be more common in idiopathic anaphylaxis and MC activation syndromes (MCAS). Therefore, TPSAB1 genotyping should be included in the diagnostic algorithm in patients with symptomatic SM, severe anaphylaxis or MCAS.

Sensitive KIT D816V mutation analysis of blood as a diagnostic test in mastocytosis.

The recent progress in sensitive KIT D816V mutation analysis suggests that mutation analysis of peripheral blood (PB) represents a promising diagnostic test in mastocytosis. However, there is a need for systematic assessment of the analytical sensitivity and specificity of the approach in order to establish its value in clinical use. We therefore evaluated sensitive KIT D816V mutation analysis of PB as a diagnostic test in an entire case-series of adults with mastocytosis. We demonstrate for the first time that by using a sufficiently sensitive KIT D816V mutation analysis, it is possible to detect the mutation in PB in nearly all adult mastocytosis patients. The mutation was detected in PB in 78 of 83 systemic mastocytosis (94%) and 3 of 4 cutaneous mastocytosis patients (75%). The test was 100% specific as determined by analysis of clinically relevant control patients who all tested negative. Mutation analysis of PB was significantly more sensitive than serum tryptase >20 ng/mL. Of 27 patients with low tryptase, 26 tested mutation positive (96%). The test is furthermore readily available and we consider the results to serve as a foundation of experimental evidence to support the inclusion of the test in diagnostic algorithms and clinical practice in mastocytosis.

Impact of age and heterophilic interference on the basal serum tryptase, a risk indication for anaphylaxis, in 1,092 dermatology patients.

A raised baseline serum tryptase is a risk indicator for anaphylactic reactions, especially in patients with hymenoptera venom allergy. Borderline elevations (> 11.4 µg/l) occur frequently and may necessitate invasive diagnostic procedures to rule out systemic mastocytosis. We retrospectively analysed 1,092 non-mastocytotic patients from our general dermatology clinic with respect to age- and gender-associated effects and investigated the impact of heterophilic antibody interference on the tryptase assay. The results were stratified by gender and five age classes. Sera with raised tryptase (n = 106) were re-tested after pre-incubation with Heterophilic Blocking Tubes (HBT(®), Scantibodies Laboratory; Santee, CA, USA). A significant increase in baseline tryptase was observed with increasing age. Incubation with HBT(®) caused a decline of more than 50% in only one case. In conclusion, older patients showed significantly higher serum tryptase levels and heterophilic interference was of subordinate relevance.

Clinical and laboratory parameters of mast cell activation as basis for the formulation of diagnostic criteria.

Mast cell (MC) activation occurs in a number of different pathologic conditions. Acute activation is commonly seen in patients with allergic reactions, with consecutive massive release of vasoactive and proinflammatory mediator substances from MCs, leading to the clinical signs and symptoms of anaphylaxis. In these patients, serum tryptase concentrations usually increase subtantially above baseline levels. Chronic MC activation is more difficult to diagnose, especially when symptoms are mild or atypical, and no underlying disease is found. In these patients, serum tryptase levels usually are normal. In a smaller group of patients, tryptase levels are constantly elevated and may point to an occult form of mastocytosis. These patients have to be examined for MC monoclonality, other criteria of a primary MC disease, non-MC hematopoietic neoplasms, and reactive disorders producing chronic MC activation or MC accumulation. In most patients in whom MC activation is found, histamine-induced symptoms can be documented and usually respond to treatment with histamine receptor antagonists or MC stabilizers. If this is not the case, alternative explanations for symptoms and differential diagnoses have to be considered.

Alpha 2-macroglobulin capture allows detection of mast cell chymase in serum and creates a reservoir of angiotensin II-generating activity.

Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by alpha(2)-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that alpha(2)-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used alpha(2)-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of approximately 1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. alpha(2)-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to alpha(2)-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that alpha(2)-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.

Expulsion of secondary Trichinella spiralis infection in rats occurs independently of mucosal mast cell release of mast cell protease II.

Our aim was to elucidate the contribution of mucosal mast cells to the effector phase of a secondary immune response to Trichinella spiralis. During secondary infection, rats expel 90-99% of T. spiralis first-stage larvae from the intestine in a matter of hours. This phenomenon appears to be unique to rats and has been called rapid expulsion. Primary intestinal infection by T. spiralis induces mastocytosis, and mast cell degranulation occurs when challenged rats exhibit rapid expulsion. These observations have engendered the view that mast cells mediate rapid expulsion. In this study, we report that immunization of adult Albino Oxford rats by an infection limited to the muscle phase did not induce intestinal mastocytosis, yet such rats exhibited rapid expulsion when challenged orally. Although mastocytosis was absent, the protease unique to mucosal mast cells, rat mast cell protease II (RMCPII), was detected in sera at the time of expulsion. We further evaluated mast cell activity in neonatal rats that display rapid expulsion. Pups born to infected dams displayed rapid expulsion, and RMCPII was detected in their sera. By feeding pups parasite-specific mAbs or polyclonal Abs before challenge infection, it was possible to dissociate mast cell degranulation from parasite expulsion. These results indicate that rapid expulsion can occur in the absence of either intestinal mastocytosis or RMCPII release. Furthermore, release of RMCPII is not sufficient to cause expulsion. The data argue against a role for mast cells in the mechanism underlying the effector phase of protective immunity against T. spiralis in rats.

Fate of two mast cell tryptases in V3 mastocytosis and normal BALB/c mice undergoing passive systemic anaphylaxis: prolonged retention of exocytosed mMCP-6 in connective tissues, and rapid accumulation of enzymatically active mMCP-7 in the blood.

The mouse mast cell protease granule tryptases designated mMCP-6 and mMCP-7 are encoded by highly homologous genes that reside on chromosome 17. Because these proteases are released when mast cells are activated, we sought a basis for distinctive functions by examining their fates in mice undergoing passive systemic anaphylaxis. 10 min-1 h after antigen (Ag) was administered to immunoglobulin (Ig)E-sensitized mice, numerous protease/proteoglycan macromolecular complexes appeared in the extracellular matrix adjacent to most tongue and heart mast cells of normal BALB/c mice and most spleen and liver mast cells of V3 mastocytosis mice. These complexes could be intensively stained by anti-mMCP-6 Ig but not by anti-mMCP-7 Ig. Shortly after Ag challenge of V3 mastocytosis mice, large amounts of properly folded, enzymatically active mMCP-7 were detected in the plasma. This plasma-localized tryptase was approximately 150 kD in its multimeric state and approximately 32 kD in its monomeric state, possessed an NH2 terminus identical to that of mature mMCP-7, and was not covalently bound to any protease inhibitor. Comparative protein modeling and electrostatic calculations disclosed that mMCP-6 contains a prominent Lys/Arg-rich domain on its surface, distant from the active site. The absence of this domain in mMCP-7 provides an explanation for its selective dissociation from the exocytosed macromolecular complex. The retention of exocytosed mMCP-6 in the extracellular matrix around activated tissue mast cells suggests a local action. In contrast, the rapid dissipation of mMCP-7 from granule cores and its inability to be inactivated by circulating protease inhibitors suggests that this tryptase cleaves proteins located at more distal sites.

Clonal mast cell disorders in patients with systemic reactions to Hymenoptera stings and increased serum tryptase levels.

BACKGROUND: Anaphylaxis after Hymenoptera stings has been reported in subjects with mastocytosis, but few data exist regarding disease prevalence in populations allergic to these insects. OBJECTIVE: The incidence of clonal mast cell (MC) disorders in subjects with both systemic reactions to Hymenoptera stings and increased serum baseline tryptase (sBT) levels was assessed by using bone marrow (BM) aspirates and biopsy specimens. METHODS: Subjects with a history of a systemic reaction caused by a Hymenoptera sting underwent the standard diagnostic work-up for Hymenoptera allergy, and sBT levels were measured. Subjects with an increased sBT level had BM evaluation that included histology/cytology, flow cytometry, and detection of KIT mutations. RESULTS: Forty-four (11.6%) of 379 subjects with systemic reactions had increased sBT levels (>11.4 ng/mL), and 31 (70.5%) of these had a history of anaphylaxis. Thirty-four subjects with increased sBT levels underwent a BM analysis. Histology detected diagnostic or subdiagnostic MC infiltrates in 22 (65%) of 34 patients. Abnormal MCs were identified by means of flow cytometry and cytology in 26 (78.8%) of 33 and 20 (58.8%) of 34 subjects, respectively. A KIT mutation was detected in 17 (54.8%) of 31 subjects. The diagnosis was indolent systemic mastocytosis in 21 (61.7%) of 34 subjects and monoclonal MC activation syndrome in 9 (26.5%) of 34 subjects. All subjects with anaphylaxis had one of those 2 disorders. CONCLUSION: The concomitant presence of systemic reactions (especially anaphylaxis) after Hymenoptera stings and increased sBT levels strongly suggests that a BM examination is indicated for the diagnosis of clonal MC disease.

Altered Metabolism of Phospholipases, Diacylglycerols, Endocannabinoids, and N-Acylethanolamines in Patients with Mastocytosis.

BACKGROUND: Mastocytosis is a condition characterized by the expansion and accumulation of mast cells (MCs) in various organs. The symptoms are related to the increased release of MC-derived mediators that exert local and distant effects. MCs are a source and target of phospholipase enzymes (PLs), which catalyze the cleavage of membrane phospholipids releasing lipid mediators (e.g., diacylglycerols (DAGs) and the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG)). To date, there are no data on the role of these lipid mediators in mastocytosis. Here, we analyzed plasma levels of PLA2, PLC, DAG, ECs, and EC-related N-acylethanolamines in patients with mastocytosis. METHODS: In 23 patients with mastocytosis and 23 healthy individuals, we measured plasma PLA2 and PLC activities, DAG, 2-AG, anandamide (AEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA). RESULTS: Plasma PLA2 and PLC activities were increased in mastocytosis patients compared to controls. Concentrations of DAG (18:1 20:4 and 18:0 20:4), two second messengers produced by PLC, were higher in mastocytosis compared to controls, whereas the concentrations of their metabolite, 2-AG, were not altered. AEA was decreased in mastocytosis patients compared to controls; by contrast, AEA congener, PEA, was increased. PLA2 and PLC activities were increased only in patients with mediator-related symptoms. Moreover, PLC activity was positively correlated with disease severity and tryptase concentrations. By contrast, AEA was negatively correlated with tryptase concentrations. CONCLUSIONS: PLs and some lipid mediators are altered in patients with mastocytosis. Our results may pave the way for investigating the functions of these mediators in the pathophysiology of mastocytosis and provide new potential biomarkers and therapeutic targets.

KIT and mastocytosis.

KIT is a receptor tyrosine kinase that is functionally relevant for hematopoiesis, mast cell development and function, gametogenesis and melanogenesis. Normal KIT signaling requires binding to stem cell factor, and PI3K-Akt is one of the putative effector pathways. In humans, germline loss-of-function KIT mutations have been associated with piebaldism - an autosomal dominant condition characterized by depigmented patches of skin and hair. Gain-of-function KIT mutations are usually acquired and have been associated with myeloid malignancies including core binding factor acute myeloid leukemia and systemic mastocytosis (SM), germ cell tumors, gastrointestinal stromal tumors and sinonasal T cell lymphomas. KITD816V is the most prevalent KIT mutation in mast cell disease and occurs in more than 90% of the cases that fulfill the World Health Organization diagnostic criteria for SM. However, its precise pathogenetic contribution is not well understood. In clinical practice, SM is considered either indolent or aggressive depending on the respective absence or presence of symptomatic target organ dysfunction aside from skin disease. In general, conventional therapy for SM is suboptimal, and efforts are under way to develop and employ small molecule drugs that target mutant KIT.

Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.

Mast cells are widely distributed throughout the body and express effector functions in allergic reactions, inflammatory diseases, and host defense. Activation of mast cells results in exocytosis of preformed chemical mediators and leads to novel synthesis and secretion of lipid mediators and cytokines. Here, we show that human mast cells also express and release the cytotoxic lymphocyte-associated protease, granzyme B. Granzyme B was active and localized in cytoplasmic granules, morphologically resembling those present in cytotoxic lymphocytes. Expression and release of granzyme B by mast cell-lines HMC-1 and LAD 2 and by cord blood- and mature skin-derived human mast cells depended on the mode of activation of these cells. In mast cell lines and cord blood-derived mast cells, granzyme B expression was mainly induced by non-physiological stimuli (A23187/PMA, Compound 48/80) and substance P. In contrast, mature skin-derived mast cells only produced granzyme B upon IgE-dependent stimulation. We conclude that granzyme B is expressed and released by human mast cells upon physiologic stimulation. This suggests a role for granzyme B as a novel mediator in mast cell biology.

Distribution of mast cell subtypes in interstitial cystitis: implications for novel diagnostic and therapeutic strategies?

AIMS: To identify the presence and geographical distribution of mast cell (MC) subtypes: MCT (tryptase positive-chymase negative) and MCTC (tryptase positive-chymase positive) in bladder tissue. METHODS: Bladder tissue was obtained from patients with painful bladder syndrome/interstitial cystitis (n=14) and normal histology from University Hospital Southampton tissue bank. Sequential tissue slices were immunohistochemically stained for MC subtypes using anti-MC tryptase (for MCT and MCTC) and anti-MC chymase (for MCTC). Stained sections were photographed, and positively stained MCs were quantified using ImageJ. Data were analysed using descriptive statistics and individual paired t-tests. RESULTS: There was a significant difference in the density of MCs between each layer of the disease bladder, with the greatest accumulation within the detrusor (p<0.001). There was a significant increase in MCTC subtype in the lamina (p=0.009) in painful bladder syndrome/interstitial cystitis. CONCLUSIONS: Our results suggest that mastocytosis is present within all layers of disease bladder, especially the muscle layer. The varying increase in MC subtypes in the lamina and mucosa may explain the variability in painful bladder syndrome/interstitial cystitis symptoms. A high influx of MCTC in the mucosa of individuals who also had ulceration noted within their diagnostic notes may be of the Hunner's ulcer subclassification. These findings suggest a relationship between the pathogenesis of MC subtypes and the clinical presentation of painful bladder syndrome/interstitial cystitis. A cohort study would further elucidate the diagnostic and/or therapeutic potential of MCs in patients with painful bladder syndrome/interstitial cystitis.

The alpha form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis.

Tryptase, a protease produced by all mast cells, was evaluated as a clinical marker of systemic mastocytosis. Two sandwich immunoassays were evaluated, one which used the mAb G5 for capture, the other which used B12 for capture. The B12 capture assay measured both recombinant alpha- and beta-tryptase, whereas the G5 capture assay measured primarily recombinant beta-tryptase. G5 binds with low affinity to both recombinant alpha-tryptase and tryptase in blood from normal and nonacute mastocytosis subjects, and binds with high affinity to recombinant beta-tryptase, tryptase in serum during anaphylaxis, and tryptase stored in mast cell secretory granules. B12 recognizes all of these forms of tryptase with high affinity. As reported previously, during systemic anaphylaxis in patients without known mastocytosis, the ratio of B12- to G5-measured tryptase was always < 5 and approached unity (Schwartz L.B., T.R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J.K. Van der Zwan and P.-W.G. Van der Linden, J. Clin. Immunol. 14:190-204). In this report, most mastocytosis patients with systemic disease have B12-measured tryptase levels that are elevated (> 20 ng/ml) and are at least 10-fold greater than the corresponding G5-measured tryptase level. Most of those subjects with B12-measured tryptase levels of < 20 ng/ml had only cutaneous manifestations. The B12 assay for alpha-tryptase and beta-tryptase, particularly when performed in conjunction with the G5 assay for beta-tryptase, provides a more precise measure of mast cell involvement than currently available assessments, a promising potential screening test for systemic mastocytosis and may provide an improved means to follow disease progression and response to therapy.

Matriptase expression in the normal and neoplastic mast cells.

Matriptase, a type II transmembrane serine protease, is distributed in almost all normal human epithelium. Several studies have demonstrated that matriptase expression is correlated with tumor progression in epithelium-derived cancer cells. Mast cells, which originate from pluripotent hematopoietic cells in the bone marrow, can produce and store almost cellular-specific neutral serine proteases, such as tryptase and chymase, and are functionally involved in both the immediate hypersensitivity response and anaphylactic shock. Mast cells are significantly increased in several neoplasms, indicating that they most likely play a role in degrading the tissue matrix. Recently, trypsin has been revealed to activate the latent matriptase on the surface of several human cancer cell lines, suggesting that matriptase and trypsin cooperatively function in extracellular proteolysis. In our study, almost all mast cells in tissues throughout the body stained positive for matripase. Matripase was also found in neoplastic mast cells. To our knowledge, this is the first time that matriptase has been shown to be expressed by mast cells. Therefore, we suggest that this expression of matriptase may not only be useful as an additional marker for mast cells but also be involved in their physiopathological function.

Kit receptor tyrosine kinase dysregulations in feline splenic mast cell tumours.

This study investigated Kit receptor dysregulations (cytoplasmic immunohistochemical expression and/or c-KIT mutations) in cats affected with splenic mast cell tumours. Twenty-two cats were included. Median survival time was 780 days (range: 1-1219). An exclusive splenic involvement was significantly (P = 0.042) associated with longer survival (807 versus 120 days). Eighteen tumours (85.7%) showed Kit cytoplasmic expression (Kit pattern 2, 3). Mutation analysis was successful in 20 cases. Fourteen missense mutations were detected in 13 out of 20 tumours (65%). Eleven (78.6%) were located in exon 8, and three (21.6%) in exon 9. No mutations were detected in exons 11 and 17. Seven mutations corresponded to the same internal tandem duplication in exon 8 (c.1245_1256dup). Although the association between Kit cytoplasmic expression and mutations was significant, immunohistochemistry cannot be considered a surrogate marker for mutation analysis. No correlation was observed between c-Kit mutations and tumour differentiation, mitotic activity or survival.

Diagnostic value of tryptase in anaphylaxis and mastocytosis.

Serum (or plasma) levels of total and mature tryptase measurements are recommended in the diagnostic evaluation of systemic anaphylaxis and systemic mastocytosis, but their interpretation must be considered in the context of a complete workup of each patient. Total tryptase levels generally reflect the increased burden of mast cells in patients with all forms of systemic mastocytosis (indolent systemic mastocytosis, smoldering systemic mastocytosis, systemic mastocytosis associated with a hematologic clonal non-mast cell disorder, aggressive systemic mastocytosis, and mast cell leukemia) and the decreased burden of mast cells associated with cytoreductive therapies in these disorders. Causes of an elevated total tryptase level other than systemic mastocytosis must be considered, however, and include systemic anaphylaxis, acute myelocytic leukemia, various myelodysplastic syndromes, hypereosinophilic syndrome associated with the FLP1L1-PDGFRA mutation, end-stage renal failure, and treatment of onchocerciasis. Mature (beta) tryptase levels generally reflect the magnitude of mast cell activation and are elevated during most cases of systemic anaphylaxis, particularly with parenteral exposure to the inciting agent.

Purification of tryptase from a human mast cell line.

The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCl, HgCl2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenyl-alanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.

Chymase expressing bone marrow mast cells in mastocytosis and myelodysplastic syndromes: an immunohistochemical and morphometric study.

BACKGROUND: Two cell specific neutral proteases, tryptase and chymase, are produced by human mast cells (MC). Tryptase is constitutively expressed by all MC, whereas chymase is found only in an MC subset. Very little is known about chymase expression in MC proliferative disorders (mastocytosis). AIMS AND METHODS: Routinely processed, formalin fixed, and paraffin wax embedded bone marrow trephine biopsy specimens obtained from patients with various subtypes of mastocytosis (n = 47) and myelodysplastic syndromes (MDS; n = 28) were immunostained with antibodies against chymase and tryptase. Normal/reactive bone marrow specimens with intact haemopoiesis (n = 31) served as controls. The numbers of chymase expressing (C+) and of tryptase expressing (T+) MC were assessed morphometrically using a computer assisted video camera system. RESULTS: In normal/reactive bone marrow, the numbers of C+ MC (median, 8/mm(2); maximum, 159/mm(2)) were in the same range as those of T+ MC (median, 4/mm(2); maximum, 167/mm(2)). Because normal MC express both chymase and tryptase, these findings indicate that the common phenotype of bone marrow MC in normal/reactive states is MC(TC) (MC expressing both tryptase and chymase). In contrast, in MDS and mastocytosis, the bone marrow exhibited far more T+ MC than C+ MC in almost all cases. CONCLUSIONS: According to these findings, the predominant MC type in the bone marrow in neoplastic states such as MDS and mastocytosis is MC(T) (MC expressing only tryptase). Although the pathophysiological basis of this apparent lack of chymase expression in most neoplastic MC in mastocytosis and MC involved in MDS remains unknown, this study has produced further evidence of the superior value of antitryptase antibodies in the diagnosis of mastocytosis.

Serum tryptase and the laboratory diagnosis of systemic mastocytosis.

Total tryptase levels of 20 ng/mL or higher in a baseline serum sample when the ratio of total to beta-tryptase is 20 or greater strongly suggest underlying systemic mastocytosis. Whether these criteria prove to be more sensitive than a bone marrow biopsy will require further study. Although the absolute level of total tryptase does not predict disease severity, it may provide a practical method for assessing the efficacy of therapeutic interventions designed to reduce the mast cell burden.