Diagnostic Value of Bone Marrow Tryptase in Systemic Mastocytosis
Diagnostic Value of Bone Marrow Tryptase in Systemic Mastocytosis
The hypothesis of the study is that Bone Marrow Tryptase (MT) level is a diagnostic marker of
 Systemic Mastocytosis (SM). Determination of the bone marrow tryptase in Bone Marrow Aspirate
 (BMA) could be a new diagnostic criteria for systemic mastocytosis with sensitivity close to
 100% and a low false negative rate. This new test could be useful to improve the ability to
 diagnose accurately systemic mastocytosis (in particular the indolent forms). Because of its
 limited invasiveness compared to bone marrow biopsy, it could also be considered as a test
 performed before bone marrow biopsy. Only patients with high bone marrow tryptase would then
 undergo bone marrow biopsy.

 In the future and if validated by this study, bone marrow tryptase could be a useful marker
 of mast cell load and help to monitor the efficacy of treatment in systemic mastocytosis.
Mastocytosis are a group of disorders characterized by clonal accumulation of mast cells in
 various organs. Diagnosis of mastocytosis is challenging and patients remain undiagnosed for
 years. Using the diagnostic criteria defined by the World Health Organization (WHO)
 mastocytosis can be divided between Cutaneous Mastocytosis (CM) and Systemic Mastocytosis
 (SM).

 Study suggested that measurement of tryptase in plasma from a Bone Marrow Aspirate (from BMA)
 could be useful for the diagnosis of SM. Results from a preliminary study at Toulouse
 University Hospital suggested that a Bone Marrow Tryptase Level (MT) of 50 µg/L or more may
 be a more sensitive diagnostic criterion (sensitivity of 94.7%) than the histological result
 of BMB (the major diagnostic criterion for SM with a sensitivity of 68 to 80% in expert
 centers) for diagnosis of SM in patients with mastocytosis in skin.

 Because this preliminary study was done on a limited number of patients and in a single
 center, the diagnostic value of MT for the diagnosis of SM, has to be confirmed on a larger
 and more representative population.

 In addition, the investigators propose to evaluate:

 -  The diagnostic accuracy of MT for diagnosis of SM in patients without mastocytosis in
 skin;

 -  The diagnostic accuracy of absolute and corrected MT for diagnosis of SM with and
 without mastocytosis in skin;

 -  The diagnostic accuracy of MT/ST ratio for diagnosis of SM with and without mastocytosis
 in skin;

 -  The diagnostic accuracy of flow cytometry performed on cells collected by BMA and
 maintained in a preservative solution for diagnosis of SM with and without mastocytosis
 in skin;

 -  The diagnostic accuracy of quantification of the fraction of KIT mutation-positive cells
 in peripheral blood for diagnosis of SM with and without mastocytosis in skin;

 -  Correlation the results of quantification of the fraction of KIT mutation-positive cells
 in peripheral blood with results of MT level, results of MT/ST ratio and results of
 absolute and corrected MT;

 -  Correlation the results of the CF performed both on the EDTA/TransFix® tubes and on
 sodium heparin tubes and validate the transport conditions (20-25°C within 24 to 96H)
 for the CF; these analyzes will be performed on the first 50 samples of BMA.

 The samples for these diagnosis tests will be made on the day of inclusion. For each enrolled
 patient, one 5 mL sample of peripheral blood (EDTA) and 1 mL samples of BM aspirate (0,5 mL
 in a EDTA/TransFix® tube et 0,5 mL in a sodium heparinate tube) will be collected the same
 day, and shipped in less than 24 to 96H (+20-25°C) to a central laboratory.

 For each patient, genomic DNA from total leucocytes will be purified from the peripheral
 blood EDTA samples, and used later for the quantification of the kit mutations by real-time
 qPCR. The plasma tryptase level will be measured from the peripheral blood EDTA sample after
 an aliquot has been taken for DNA extraction. The bone marrow aspirate sample will be used
 first for immunophenotyping of BM mastocytes by flow cytometry (FC), and then, after
 centrifugation, for measurement of BM tryptase level. The degree of dilution of the BM
 aspirate by peripheral blood will be estimated by comparing the percentage of CD16bright vs.
 CD16low granulocytes in BM samples, and used to adjust the BM tryptase. Tryptase measurements
 will be made from EDTA or Na, heparinate plasma, using a standardized fluoro enzyme
 immunoassay on an automated analyzer. Immunophenotyping of BM mastocytes will be performed on
 BM aspiration stabilized by TransFix® (a Cellular Antigen Stabilisation Reagent) (and for
 firsts 50 samples on BM aspiration in Na, heparinate) using a pre-defined mixture of
 anti-CD45/CD117/CD34/CD2/CD25/CD16 antibodies, coupled to appropriate fluorochromes. Genomic
 DNA from BM leucocytes will be extracted from 300-500µL of peripheral blood (both anti
 coagulated with EDTA) using a Promega DNA automated extractor, and subsequently stored at
 +4°C. Quantitative PCR will be performed using a pre-validated qPCR assay (Life Technologies,
 Foster City, CA) with a 7900HT Fast Real-Time PCR System (Life Technologies).

 The end of study visit is performed the same day that the patient is seen to be made aware of
 the results of the clinical and laboratory investigations made. This is also the day when the
 planned medical management of mastocytosis is discussed.
Inclusion Criteria:

 -  Suspicion of systemic mastocytosis, for whom bone marrow sampling (bone marrow biopsy
 and/or bone marrow aspiration) is carried out as part of normal workup of the disease
 in the center of reference and centers of competence in mastocytosis.

 -  Patient whose written informed consent has been obtained.

 Exclusion Criteria:

 -  Patients with a contra-indication to marrow sampling (anticoagulant and/or
 anti-clotting treatments,

 -  Thrombocytopenia < 50 000/mm2) for whom it is impossible to formally conclude the
 final type of mast cell disease: SM, CM, mast cell activation syndrome.