Hereditary Alpha Tryptasemia: Validation of a Single-Well Multiplex Digital Droplet PCR Assay in a Cohort of Symptomatic Patients.

BACKGROUND: Hereditary alpha tryptasemia (HαT) has significant prevalence and potential morbidity in the general population. However, it remains largely undiagnosed in routine clinical diagnostics due to low availability of efficient assessment methods. To address this issue, we developed a reliable and efficient single-well multiplex digital droplet PCR assay. METHODS: The assay was based on the reconstruction of the TPSAB1 gene through quantification of the ratio of α- and ß-tryptase copy number variants (CNV) in a single-well measurement. We performed analytical validation by determining CNV measurement clustering around the expected copy numbers in 281 cases and determined the diagnostic accuracy of basal serum tryptase (BST) to predict HαT and HαT subtypes in 141 symptomatic patients. RESULTS: The assay determined α- and ß-tryptase CNVs with an overall accuracy, expressed as a 99% prediction interval, of 0.03 ± 0.27 copy numbers. The optimal BST cutoff level to predict HαT in symptomatic patients, who had no other explanation for relatively high tryptase levels (i.e., no diagnosis of systemic mastocytosis, myeloid neoplasm, or end-stage renal failure), was 9.2 ng/mL (sensitivity: 98.1%; specificity: 96.6%). HαT showed a linear gene-dose effect, with an average gene-dose increase of 7.5 ng/mL per extra α-tryptase gene. CONCLUSION: Our single-well multiplex digital droplet PCR assay accurately determined HαT and could be implemented as a state-of-the-art routine diagnostic test. The assay demonstrated a strong correlation with BST and the optimal threshold for identifying HαT in symptomatic patients with unexplained high tryptase concentrations was at a BST level of 9.2 ng/mL.

Effectiveness of a vitamin D regimen in deficient multiple myeloma patients and its effect on peripheral neuropathy.

PURPOSE: Peripheral neuropathy (PN) is common in multiple myeloma (MM) patients. More insight has been gained concerning the role of vitamin D in preventing PN. However, studies evaluating the effects of vitamin D3 supplementation on PN are lacking. The aims of this study are to (1) evaluate the effectiveness of a vitamin D3 regimen on achieving adequate vitamin D levels in deficient MM patients and to (2) exploratively evaluate the effect of vitamin D3 supplementation on PN. METHODS: Thirty-nine MM patients with inadequate (< 75 nmol/L [= 30 ng/mL]) 25-hydroxyvitamin D (25(OH)D) levels were included in this multicenter, prospective, single-arm study, of whom 35 patients completed the study. They received oral vitamin D3 for 6 months according to a dose escalation regimen that consisted of one or two loading doses of 200,000 international units (IU), and maintenance doses of 800, 1600, or 3200 IU/day depending on the 25(OH)D level. A validated questionnaire was used to measure PN. RESULTS: Median 25(OH)D increased from 38 (IQR 32-52) nmol/L at baseline to 77 (IQR 72-87) nmol/L after 6 months (P < 0.001). Adequate 25(OH)D levels were achieved by 66% of the subjects, and 34% were within the range of 50-75 nmol/L. Furthermore, in 37% of the participants, PN severity decreased (P = 0.007). CONCLUSION: The use of substantially higher vitamin D3 doses than recommended in current guidelines resulted in a significant increase in vitamin D levels in MM patients. Furthermore, evaluation of PN showed a significant decrease in PN grading. However, this exploratory evaluation needs further confirmatory research.

Detection of clonal mast cell disease in wasp venom allergic patients with normal tryptase.

Background: Clonal mast cell disease (CMD) is an underlying aggravating condition in wasp venom allergy (WVA) which requires a different treatment strategy. CMD is increasingly recognized in patients with normal basal serum tryptase (bsT). However, methods to identify at risk patients have not yet been assessed in large cohorts of WVA patients with normal bsT. Methods: This retrospective study evaluated the reliability of the REMA score in detecting CMD in a cohort of grade IV WVA patients with normal bsT and assessed the added value of other clinical parameters, KIT D816V mutation analysis in peripheral blood (PB) and the diagnosis of hereditary alpha tryptasemia (HAT). All patients had a conclusive bone marrow evaluation that demonstrated or excluded underlying CMD. Results: In total 35 CMD and 96 non-CMD patients were included. REMA score had a sensitivity of 72% (95% CI 56%-88%) and specificity of 79% (95% CI 70%-87%) in this cohort. Loss of consciousness during systemic reaction and bsT between 6.3 and 11.4 ng/ml were additional parameters independently associated with CMD. Sensitivity of KIT in PB was relatively low, 56% (95% CI 36%-75%), but had added value as screening method in patients with a low REMA score due to 100% specificity. Conclusion: The REMA score is a relatively reliable method to detect patients at risk of CMD among WVA patients with normal bsT. KIT mutation analysis in PB could serve as additional screening method in patients with low REMA scores.