Ovarian hyperstimulation syndrome: review and new classification criteria for reporting in clinical trials.

STUDY QUESTION: What is an objective approach that employs measurable and reproducible physiologic changes as the basis for the classification of ovarian hyperstimulation syndrome (OHSS) in order to facilitate more accurate reporting of incidence rates within and across clinical trials? SUMMARY ANSWER: The OHSS flow diagram is an objective approach that will facilitate consistent capture, classification and reporting of OHSS within and across clinical trials. WHAT IS KNOWN ALREADY: OHSS is a potentially life-threatening iatrogenic complication of the early luteal phase and/or early pregnancy after ovulation induction (OI) or ovarian stimulation (OS). The clinical picture of OHSS (the constellation of symptoms associated with each stage of the disease) is highly variable, hampering its appropriate classification in clinical trials. Although some degree of ovarian hyperstimulation is normal after stimulation, the point at which symptoms transition from those anticipated to those of a disease state is nebulous. STUDY DESIGN, SIZE, DURATION: An OHSS working group, comprised of subject matter experts and clinical researchers who have significantly contributed to the field of fertility, was convened in April and November 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: The OHSS working group was tasked with reaching a consensus on the definition and the classification of OHSS for reporting in clinical trials. The group engaged in targeted discussion regarding the scientific background of OHSS, the criteria proposed for the definition and the rationale for universal adoption. An agreement was reached after discussion with all members. MAIN RESULTS AND THE ROLE OF CHANCE: One of the following conditions must be met prior to making the diagnosis of OHSS in the context of a clinical trial: (i) the subject has undergone OS (either controlled OS or OI) AND has received a trigger shot for final oocyte maturation (e.g. hCG, GnRH agonist [GnRHa] or kisspeptin) followed by either fresh transfer or segmentation (cryopreservation of embryos) or (ii) the subject has undergone OS or OI AND has a positive pregnancy test. All study patients who develop symptoms of OHSS should undergo a thorough examination. An OHSS flow diagram was designed to be implemented for all subjects with pelvic or abdominal complaints, such as lower abdominal discomfort or distention, nausea, vomiting and diarrhea, and/or for subjects suspected of having OHSS. The diagnosis of OHSS should be based on the flow diagram. LIMITATIONS, REASONS FOR CAUTION: This classification system is primarily intended to address the needs of the clinical investigator undertaking clinical trials in the field of OS and may not be applicable for the use in clinical practice or with OHSS occurring under natural circumstances. WIDER IMPLICATIONS OF THE FINDINGS: The proposed OHSS classification system will enable an accurate estimate of the incidence and severity of OHSS within and across clinical trials performed in women with infertility. STUDY FUNDING/COMPETING INTERESTS: Financial support for the advisory group meetings was provided by Merck & Co., Inc., Kenilworth, NJ, USA. P.H. reports unrestricted research grants from MSD, Merck and Ferring, and honoraria for lectures from MSD, Merck and IBSA. S.M.N. reports that he has received fees and grant support from the following companies (in alphabetic order): Beckman Coulter, Besins, EMD Serono, Ferring Pharmaceuticals, Finox, MSD and Roche Diagnostics over the previous 5 years. P.D., C.C.C., J.L.F., H.M.F., and P.L. report no relationships that present a potential conflict of interest. B.C.T. REPORTS: grants and honorarium from Merck Serono; unrestricted research grants, travel grants and honorarium, and participation in a company-sponsored speaker's bureau from Merck Sharp & Dohme; grants, travel grants, honoraria and advisory board membership from IBSA; travel grants from Ferring; and advisory board membership from Ovascience. L.B.S. reports current employment with Merck & Co, Inc., Kenilworth, NJ, USA, and owns stock in the company. K.G. and B.J.S. report prior employment with Merck & Co., Inc., Kenilworth, NJ, USA, and own stock in the company. All reported that competing interests are outside the submitted work. No other relationships or activities exist that could appear to have influenced the submitted work. TRIAL REGISTRATION NUMBER: Not applicable.

Refined diagnostic criteria and classification of mast cell leukemia (MCL) and myelomastocytic leukemia (MML): a consensus proposal.

Mast cell leukemia (MCL), the leukemic manifestation of systemic mastocytosis (SM), is characterized by leukemic expansion of immature mast cells (MCs) in the bone marrow (BM) and other internal organs; and a poor prognosis. In a subset of patients, circulating MCs are detectable. A major differential diagnosis to MCL is myelomastocytic leukemia (MML). Although criteria for both MCL and MML have been published, several questions remain concerning terminologies and subvariants. To discuss open issues, the EU/US-consensus group and the European Competence Network on Mastocytosis (ECNM) launched a series of meetings and workshops in 2011-2013. Resulting discussions and outcomes are provided in this article. The group recommends that MML be recognized as a distinct condition defined by mastocytic differentiation in advanced myeloid neoplasms without evidence of SM. The group also proposes that MCL be divided into acute MCL and chronic MCL, based on the presence or absence of C-Findings. In addition, a primary (de novo) form of MCL should be separated from secondary MCL that typically develops in the presence of a known antecedent MC neoplasm, usually aggressive SM (ASM) or MC sarcoma. For MCL, an imminent prephase is also proposed. This prephase represents ASM with rapid progression and 5%-19% MCs in BM smears, which is generally accepted to be of prognostic significance. We recommend that this condition be termed ASM in transformation to MCL (ASM-t). The refined classification of MCL fits within and extends the current WHO classification; and should improve prognostication and patient selection in practice as well as in clinical trials.

Proposed diagnostic algorithm for patients with suspected mastocytosis: a proposal of the European Competence Network on Mastocytosis.

Mastocytosis is an emerging differential diagnosis in patients with more or less specific mediator-related symptoms. In some of these patients, typical skin lesions are found and the diagnosis of mastocytosis can be established. In other cases, however, skin lesions are absent, which represents a diagnostic challenge. In the light of this unmet need, we developed a diagnostic algorithm for patients with suspected mastocytosis. In adult patients with typical lesions of mastocytosis in the skin, a bone marrow (BM) biopsy should be considered, regardless of the basal serum tryptase concentration. In adults without skin lesions who suffer from mediator-related or other typical symptoms, the basal tryptase level is an important parameter. In those with a slightly increased tryptase level, additional investigations, including a sensitive KIT mutation analysis of blood leucocytes or measurement of urinary histamine metabolites, may be helpful. In adult patients in whom (i) KIT D816V is detected and/or (ii) the basal serum tryptase level is clearly increased (>25-30 ng/ml) and/or (iii) other clinical or laboratory features suggest the presence of 'occult' mastocytosis or another haematologic neoplasm, a BM investigation is recommended. In the absence of KIT D816V and other signs or symptoms of mastocytosis or another haematopoietic disease, no BM investigation is required, but the clinical course and tryptase levels are monitored in the follow-up. In paediatric patients, a BM investigation is usually not required, even if the tryptase level is increased. Although validation is required, it can be expected that the algorithm proposed herein will facilitate the management of patients with suspected mastocytosis and help avoid unnecessary referrals and investigations.

Increased serum baseline tryptase levels and extensive skin involvement are predictors for the severity of mast cell activation episodes in children with mastocytosis.

BACKGROUND: Despite the good prognosis of pediatric mastocytosis, some patients suffer from severe mast cell (MC) mediator-associated symptoms. The aim of this study was to identify predictors for severe MC mediator release symptoms in children with mastocytosis in the skin (MIS). METHODS: Serum baseline total tryptase (sbT) levels in 111 children with MIS - 80 maculopapular cutaneous mastocytosis/plaque mastocytosis, 22 nodular mastocytosis, and nine diffuse cutaneous mastocytosis - were investigated as a predictive biomarker for the occurrence of MC mediator-related signs and symptoms within the first 18 months after disease onset. RESULTS: Twelve children (11%) who showed extensive cutaneous disease involving >90% of body surface area (BSA) suffered from severe symptoms requiring hospitalization, with (n = 5) or without (n = 6) management in the intensive care unit (ICU) owing to life-threatening complications. The median sbT was significantly (P < 0.001) higher in patients with extensive cutaneous disease vs those with <90% of BSA involved (45.5 vs 5.2 µg/l, respectively), as well as in children with grade 4 (severe mastocytosis-related symptoms requiring emergency therapy and hospitalization) vs those with grade <4 (46.2 vs 5.2 µg/l, respectively). Receiver operating characteristics curve analyses showed that the optimal cutoff s for sbT to predict the need for daily antimediator therapy, hospitalization, and the management in an ICU were 6.6, 15.5, and 30.8 µg/l, respectively (sensitivity and specificity of 77% and 79%, 100% and 95%, and 100% and 96%, respectively). CONCLUSIONS: Increased sbT in association with extensive cutaneous involvement identifies patients at risk for severe MC activation events in pediatric mastocytosis.

Recombinant human stem cell factor (kit ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo.

Stem cell factor (SCF), also known as mast cell growth factor, kit ligand, and steel factor, is the ligand for the tyrosine kinase receptor (SCFR) that is encoded by the c-kit proto-oncogene. We analyzed the effects of recombinant human SCF (r-hSCF, 5-50 micrograms/kg/day, injected subcutaneously) on mast cells and melanocytes in a phase I study of 10 patients with advanced breast carcinoma. A wheal and flare reaction developed at each r-hSCF injection site; by electron microscopy, most dermal mast cells at these sites exhibited extensive, anaphylactic-type degranulation. A 14-d course of r-hSCF significantly increased dermal mast cell density at sites distant to those injected with the cytokine and also increased both urinary levels of the major histamine metabolite, methyl-histamine, and serum levels of mast cell alpha-tryptase. Five subjects developed areas of persistent hyperpigmentation at r-hSCF injection sites; by light microscopy, these sites exhibited markedly increased epidermal melanization and increased numbers of melanocytes. The demonstration that r-hSCF can promote both the hyperplasia and the functional activation of human mast cells and melanocytes in vivo has implications for our understanding of the role of endogenous SCF in health and disease. These findings also indicate that the interaction between SCF and its receptor represents a potential therapeutic target for regulating the numbers and functional activity of both mast cells and cutaneous melanocytes.

Serum total tryptase levels are increased in patients with active chronic urticaria.

BACKGROUND: We have demonstrated previously mast cell histamine release upon incubation with chronic urticaria (CU) sera, presumably by degranulation. OBJECTIVE: To explore total and mature tryptase in order to assess whether any increase in total tryptase levels is due in part to mast cell degranulation or to mast cell burden. We also wanted to explore differences between the autoimmune groups called idiopathic (serum unable to activate basophils), and to correlate total and mature tryptase levels with different urticaria features. METHODS: We measured total and mature tryptase serum levels in 81 CU patients, 16 atopic donors and 21 healthy control sera. We assessed autoimmunity by measuring the CD63 expression in normal basophil donors upon incubation with CU sera. RESULTS: We found significantly higher levels of total tryptase in the sera of CU patients (6.6 ±4.1 µg/L) than in sera from healthy non-atopic subjects (4.4 ±2.8 µg/L) and from atopic subjects (4.5 ±1.7 µg/L). Mature tryptase levels were undetectable (<1 ng/mL). Total tryptase levels in the autoimmune urticaria group were significantly higher (9.8 ±5.4 µg/L) than the idiopathic urticaria group (4.4 ±2.2 µg/L). A significant difference in total tryptase was found between symptomatic patients (7.3 ±4.1 µg/L) compared with asymptomatic ones (5.7 ±4.1 µg/L) at the time of venesection. No difference was found in mature tryptase levels either. CONCLUSION: Total elevated tryptase levels are not accompanied by an elevated mature tryptase levels, as might be expected if the serum levels reflected mast cell degranulation.

Practical allergy (PRACTALL) report: risk assessment in anaphylaxis.

Effector mechanisms in anaphylaxis were reviewed. Current approaches to confirmation of the clinical diagnosis were discussed. Improved methods for distinguishing between allergen sensitization (which is common in the general population) and clinical risk of anaphylaxis (which is uncommon) were deliberated. Innovative techniques that will improve risk assessment in anaphylaxis in the future were described.

Exercise-induced hypersensitivity syndromes in recreational and competitive athletes: a PRACTALL consensus report (what the general practitioner should know about sports and allergy).

Exercise-induced (EI) hypersensitivity disorders are significant problems for both recreational and competitive athletes. These include EI-asthma, EI-bronchoconstriction, EI-rhinitis, EI-anaphylaxis and EI-urticaria. A group of experts from the European Academy of Allergology and Clinical Immunology and the American Academy of Allergy Asthma and Immunology met to discuss the pathogenesis of these disorders and how to diagnose and treat them, and then to develop a consensus report. Key words (exercise with asthma, bronchoconstriction, rhinitis, urticaria or anaphylaxis) were used to search Medline, the Cochrane database and related websites through February 2008 to obtain pertinent information which, along with personal reference databases and institutional experience with these disorders, were used to develop this report. The goal is to provide physicians with guidance in the diagnosis, understanding and management of EI-hypersensitivity disorders to enable their patients to safely return to exercise-related activities.

A novel heparin-dependent processing pathway for human tryptase. Autocatalysis followed by activation with dipeptidyl peptidase I.

Tryptase is the major protein constituent of human mast cells, where it is stored within the secretory granules as a fully active tetramer. Two tryptase genes (alpha and beta) are expressed by human mast cells at the level of mRNA and protein, each with a 30 amino acid leader sequence. Recombinant precursor forms of human alpha- and beta-tryptase were produced in a baculovirus system, purified, and used to study their processing. Monomeric beta-protryptase first is shown to be intermolecularly autoprocessed to monomeric beta-pro'tryptase at acid pH in the presence of heparin by cleavage between Arg-3 and Val-2 in the leader peptide. The precursor of alpha-tryptase has an Arg-3 to Gln-3 mutation that precludes autoprocessing. this may explain why alpha-tryptase is not stored in secretory granules, but instead is constitutively secreted by mast cells and is the predominant form of tryptase found in blood in both healthy subjects and those with systemic mastocytosis under nonacute conditions. Second, the NH2-terminal activation dipeptide on beta-pro'tryptase is removed by dipeptidyl peptidase I at acid pH in the absence of heparin to yield an inactive monomeric form of tryptase. Conversion of the catalytic portion of beta-tryptase to the active homotetramer at acid pH requires heparin. Thus, beta-tryptase homotetramers probably account for active enzyme detected in vivo. Also, processing of tryptase to an active form should occur optimally only in cells that coexpress heparin proteoglycan, restricting this pathway to a mast cell lineage.

Cloning and characterization of a second complementary DNA for human tryptase.

A second cDNA for human tryptase, called beta-tryptase, was cloned from a mast cell cDNA library in lambda ZAP. Its nucleotide sequence and corresponding amino acid sequence were determined and compared with those of a previously cloned tryptase cDNA, now called alpha-tryptase. The 1,142-base sequence of beta-tryptase encodes a 30-amino acid leader sequence of 3,089 D and a 245-amino acid catalytic region of 27,458 D. The amino acid sequence of beta-tryptase is 90% identical with that of alpha-tryptase, the first 20 amino acids of the catalytic portions being 100% identical. This identity, together with recognition of each recombinant protein by monoclonal antibodies directed against purified tryptase validate the tryptase identity of both alpha-tryptase and beta-tryptase cDNA molecules. Modest differences between the nucleic acid sequences of alpha- and beta-tryptase occurred throughout the cDNA molecules except in the 3' noncoding regions, which were identical. Although most highly conserved regions of amino acid sequence in the trypsin superfamily are conserved in both tryptase molecules, beta-tryptase has one carbohydrate binding site compared to two in alpha-tryptase, and one additional amino acid in the catalytic sequence. Regions of the substrate binding pocket in beta-tryptase (DSCQ, residues 218-221; SWG, residues 243-245) differ slightly from those in alpha-tryptase (DSCK, residues 217-220; SWD, residues 242-244). The presence of both alpha- and beta-tryptase sequences in each haploid genome was indicated by finding alpha- and beta-tryptase specific fragments after amplification by PCR of genomic DNA in 10 unrelated individuals. Localization of both alpha- and beta-tryptase sequences to human chromosome 16 was then performed by analysis of DNA preparations from 25 human/hamster somatic hybrids by PCR. It is now possible to assess the expression of each tryptase cDNA by mast cells and the relationship of each gene product to the active enzyme.

Cloning and characterization of complementary DNA for human tryptase.

The amino acid sequence of human mast cell tryptase was determined from corresponding cDNA cloned from a lambda ZAP library made with mRNA derived from a human mast cell preparation. Tryptase is the major neutral protease present in human mast cells and serves as a specific marker of mast cells by immunohistologic techniques and as a specific indicator of mast cell activation when detected in biologic fluids. Based on nucleic acid sequence, human tryptase consists of a 244-amino acid catalytic portion of 27,423 D with two putative N-linked carbohydrate binding sites and a 30-amino acid leader sequence of 3,048 D. A His74, Asp120, Ser223 catalytic triad and four cystine groups were identified by analogy to other serine proteases. Regions of amino acid sequence that are highly conserved in serine proteases, in general, were conserved in tryptase. The catalytic portion of human tryptase had an 84% amino acid sequence similarity with that of dog tryptase; their leader sequences had a 67% similarity. Asp217 in the substrate binding pocket of human tryptase is consistent with a specificity for Arg and Lys residues at the site of cleavage (P1), whereas Glu245 is consistent with the known preference of human tryptase for substrates with Arg or Lys also at P3, analogous residues also being present in dog tryptase. Asp244, which is substituted for the Gly found in dog tryptase and in most serine proteases, is present in the putative substrate binding pocket and may confer additional substrate specificity on human tryptase for basic residues. Further studies now can be designed to elucidate these structure-function relationships.

Time course of appearance and disappearance of human mast cell tryptase in the circulation after anaphylaxis.

Tryptase, a neutral protease of human mast cells, is a potentially important indicator of mast cell involvement in various clinical conditions. The current study examined the time course of appearance and disappearance of tryptase in the circulation after an anaphylactic event and the stability of both endogenous and exogenous tryptase in terms of freeze-thawing and temperature. The peak level of tryptase after an experimentally induced systemic anaphylactic reaction occurred 1-2 h after the initiating bee sting in each of three subjects, in contrast to histamine levels which peaked at 5-10 min. In some cases elevated levels of tryptase may not be detected during the initial 15-30 min. Tryptase levels then declined under apparent first order kinetics with a t1/2 of approximately 2h. Similar disappearance kinetics were observed for two subjects presenting in the emergency room with immediate type reactions, one with severe asthma after indomethacin ingestion, the other with systemic anaphylaxis after a bee sting. Histamine levels declined rapidly and were back to baseline by 15-60 min. Measured levels of tryptase in serum or plasma were not diminished by up to four freeze-thaw cycles. Incubation of serum samples taken from subjects with elevated levels of tryptase at 22 and 37 degrees C indicated that greater than 50% of endogenous tryptase was still detected after 4 d. Purified tryptase added to serum or plasma and incubated as above was less stable: approximately 50% of exogenous tryptase in serum and approximately 15% in plasma was detected after 2d of incubation. Therefore, optimally samples should be stored frozen, but even those stored at room temperature for up to 4 d should be satisfactory. The best time to obtain samples for tryptase determinations is 1-2 h after the precipitating event, but depending on the magnitude of the initial response elevated levels of tryptase may be present in the circulation for several hours.

A common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects.

Many patients with asthma have increased wheezing with colds. We hypothesized that rhinovirus colds might increase asthma by augmenting airway allergic responses (histamine release and eosinophil influx) after antigen challenge. Seven allergic rhinitis patients and five normal volunteers were infected with rhinovirus type 16 (RV16) and evaluated by segmental bronchoprovocation and bronchoalveolar lavage. Segmental challenge with saline and antigen was performed 1 mo before infection, during the acute infection, and 1 mo after infection. Lavage was performed immediately and 48 h after antigen challenge. Data were analyzed by two-way analysis of variance, and a P value of < or = 0.05 was considered to be significant. All volunteers inoculated with RV16 developed an acute respiratory infection. BAL fluid obtained from allergic rhinitis subjects during the acute viral infection, and 1 mo after infection, showed the following significant RV16-associated changes after antigen challenge: (a) an enhanced release of histamine immediately after local antigen challenge; (b) persistent histamine leak 48 h afterwards; and (c) a greater recruitment of eosinophils to the airway 48 h after challenge. These changes were not seen in non-allergic volunteers infected with RV16 and challenged with antigen, nor in allergic volunteers repetitively challenged with antigen but not infected with RV16, nor in RV16 infected allergic volunteers sham challenged with saline. We conclude that rhinovirus upper respiratory infection significantly augments immediate and late allergic responses in the airways of allergic individuals after local antigen challenge. These data suggest that one mechanism of increased asthma during a cold is an accentuation of allergic responses in the airway which may then contribute to bronchial inflammation.

Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation.

Mast cells have been implicated in the pathogenesis of the matrix degradation observed in the cartilaginous and osseous structures of the rheumatoid joint. We previously reported that human mast cell tryptase, a 134-kD granule-associated neutral protease, is present in rheumatoid synovium and can activate collagenase in crude culture medium in vitro. the present study attempts to depict the precise mechanism of this activation. To express full activation of latent collagenase, matrix metalloproteinase 3 (MMP-3) or stromelysin, can be activated by tryptase in a time and dose-dependent manner. Tryptase was not capable of generating active collagenase in the crude media from cultured rheumatoid synoviocytes depleted of proMMP-3 by immunoadsorption. In addition, the function of the tissue inhibitor of metalloproteinases (TIMP) was not altered by tryptase, and SDS-PAGE analysis revealed no degradation of TIMP by tryptase. The tryptase dependent activation of synoviocyte procollagenase thereby appears to be entirely dependent upon its ability to activate proMMP-3.

Human mast cells stimulate vascular tube formation. Tryptase is a novel, potent angiogenic factor.

The presence of mast cells near capillary sprouting sites suggests an association between mast cells and angiogenesis. However, the role of mast cells in blood vessel development remains to be defined. In an attempt to elucidate this relationship, we investigated the effect of human mast cells (HMC-1) and their products on human dermal microvascular endothelial cell (HDMEC) tube formation. Coculture of HMC-1 with HDMEC led to a dose-response increase in the network area of vascular tube growth. Moreover, the extent of neovascularization was enhanced greatly when HMC-1 were degranulated in the presence of HDMEC. Further examination using antagonists to various mast cell products revealed a blunted response (73-88% decrease) in the area of vascular tube formation if specific inhibitors of tryptase were present. Tryptase (3 microg/ml) directly added to HDMEC caused a significant augmentation of capillary growth, which was suppressed by specific tryptase inhibitors. Tryptase also directly induced cell proliferation of HDMEC in a dose-dependent fashion (2 pM-2 nM). Our results suggest that mast cells act at sites of new vessel formation by secreting tryptase, which then functions as a potent and previously unrecognized angiogenic factor.

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.

Mast cells: function and contents.

Mast cells, when activated, release a spectrum of mediators that initiate and modulate immediate-type hypersensitivity reactions. Recently is has been reported that expression of mast cell neutral proteases, specific markers of mast cells, is regulated by various growth factors. Human mast cells, like those in rodents, are now known to produce cytokines that modulate IgE production and inflammation.

Human mast cell proteases and mast cell heterogeneity.

Mast cell neutral proteases are distinctive markers of the MC(T) and MC(TC) cells in humans. Measurements of tryptase levels in vivo serve as an overall indicator of mast cell activity. Further research is needed to evaluate the functional role of these proteases as well as each mast cell type in situations related to both health and disease.

The tryptase positive compact round cell infiltrate of the bone marrow (TROCI-BM): a novel histopathological finding requiring the application of lineage specific markers.

AIMS: Compact tryptase-positive round cell infiltrates of the bone marrow (TROCI-BM) are very rare histopathological findings and may pose challenging problems with regard to the cell type involved (either mast cells or basophilic granulocytes) and the exact diagnosis. METHODS: A selected panel of immunohistochemical markers against mast cell and basophil related antigens, including CD25, CD34, CD117/Kit, and the 2D7 antigen (which is found only in basophilic granulocytes) on a total of 410 routinely processed bone marrow biopsy specimens (including 88 cases of systemic mastocytosis (SM), 20 cases of chronic myeloid leukaemia (CML), 92 cases of myeloid neoplasms other than CML, and 210 controls with normal/reactive bone marrows). RESULTS: In total, 17 cases with TROCI-BM could be identified: 11 SM (including two cases of well-differentiated SM and two mast cell leukaemias; MCL), 2 myelomastocytic leukaemia (MML), 2 CML with excess of basophils (secondary basophilic leukaemia (CMLba)), and 2 tryptase positive acute myeloid leukaemia (AML). Regarding the cell types involved, TROCI-BM cells were found to express CD117/Kit in all cases of SM and MCL. In MML and tryptase postitive AML, TROCI-BM cells were found to coexpress CD34 and Kit. The basophil specific antigen 2D7 was only detected in CD34/Kit negative TROCI-BM cells in two patients with CMLba. The activating point mutation D816V was detected in 8/11 patients with SM but not in any of the other haematological malignancies. CONCLUSIONS: In summary, a total of six rare myeloid neoplasms may present with a novel immunohistochemical phenomenon tentatively termed TROCI-BM.

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7.

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD. METHODS: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+) bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.