Michela Rondoni

A rare case of Hemoglobin Leiden interfering with the DIFF channel of Sysmex XE-2100

SIR: The Hub Laboratory of The Greater Romagna Area is a ‘ Shared Resource Laboratory ’ , opened in 2009, that provides Laboratory Medicine service for more than one million inhabitants living in an area of about 5000 square km in North Italy. In the Laboratory more than 3000 Complete Blood Cell Counts (CBCs) are daily processed with a good chance of fi nding really rare cases. Recently we investigated a 37-year-old Italian woman who attended one of the 94 blood drawing centers served by our laboratory with a request of CBC in August 2014. All the hematological parameters including Red Blood Cells (RBC), White Blood Cells (WBC) and Platelets (PLT) were within reference limits but the hematology analyzer (Sysmex XE-2100) measured a very low fl uorescence signal in DIFF scattergram (Figure 1), failed to measure the differential WBC and gave the warning fl ag ‘ abnormal scattergram ’ . The surfactant applied in DIFF channel induces complete lysis and shrinkage of the RBC membrane and increases the permeability of WBC, thus permitting a polymethine fl uorescent dye to combine with nucleic acids of permeabilized cells. The intensity of fl uorescence detected by analyzer refl ects the nucleic acid content of the WBC [1]. A technical artifact appears implausible because the DIFF defect was confi rmed using the other fi ve XE-2100 analyzers equipping our laboratory, no other samples measured in that day showed the same DIFF channel defect and Quality Control yielded satisfactory results. Since the same fl uorescence DIFF defect was persistently present in three samples collected and analyzed in June 2012, April 2013 and August 2014, we suspected a stable cause of the phenomenon. We hypothesize that the RBC lysis could interfere with the WBC staining. A blood smear was observed and a normal WBC differential count with several irregularly contracted RBC were detected (Figure 2). Oxidative damage, particularly in patients with G6PDH defi ciency and some hemoglobinopathies including unstable hemoglobins, are characteristic causes of irregularly contracted RBC morphology [2]. Scandinavian Journal of Clinical & Laboratory Investigation, 2015; 75: 436–437

SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis

The molecular basis of advanced systemic mastocytosis (SM) is not fully understood and despite novel therapies the prognosis remains dismal. Exome sequencing of an index-patient with mast cell leukemia (MCL) uncovered biallelic loss-of-function mutations in the SETD2 histone methyltransferase gene. Copy-neutral loss-of-heterozygosity at 3p21.3 (where SETD2 maps) was subsequently found in SM patients and prompted us to undertake an in-depth analysis of SETD2 copy number, mutation status, transcript expression and methylation levels, as well as functional studies in the HMC-1 cell line and in a validation cohort of 57 additional cases with SM, including MCL, aggressive SM and indolent SM. Reduced or no SETD2 protein expression—and consequently, H3K36 trimethylation—was found in all cases and inversely correlated with disease aggressiveness. Proteasome inhibition rescued SETD2 expression and H3K36 trimethylation and resulted in marked accumulation of ubiquitinated SETD2 in SETD2-deficient patients but not in patients with near-normal SETD2 expression. Bortezomib and, to a lesser extent, AZD1775 alone or in combination with midostaurin induced apoptosis and reduced clonogenic growth of HMC-1 cells and of neoplastic mast cells from advanced SM patients. Our findings may have implications for prognostication of SM patients and for the development of improved treatment approaches in advanced SM.

Serum total tryptase level confirms itself as a more reliable marker of mast cells burden in mast cell leukaemia (aleukaemic variant).

Mast cell leukemia (MCL) is a very rare form of systemic mastocytosis (SM) with a short median survival of 6 months. We describe a case of a 65-year-old woman with aleukaemic variant of MCL with a very high serum total tryptase level of 2255u2009μg/L at diagnosis, which occurred following an episode of hypotensive shock. She fulfilled the diagnostic criteria of SM, with a bone marrow smear infiltration of 50–60% of atypical mast cells (MCs). She tested negative for the KIT D816V mutation, without any sign of organ damage (no B- or C-findings) and only few mediator-related symptoms. She was treated with antihistamine alone and then with imatinib for the appearance of anemia. She maintained stable tryptase level and a very indolent clinical course for twenty-two months; then, she suddenly progressed to acute MCL with a serum tryptase level up to 12960u2009μg/L. The patient died due to haemorrhagic diathesis twenty-four months after diagnosis. This clinical case maybe represents an example of the chronic form of mast cell leukemia, described as unpredictable disease, in which the serum total tryptase level has confirmed itself as a reliable marker of mast cells burden regardless of the presence of other signs or symptoms.