MALDI–TOF–MS for Rapid Screening and Typing of β-Globin Variant and β-Thalassemia through Direct Measurements of Intact Globin Chains

Abstract

Background Traditional phenotype-based screening for β-globin variant and β-thalassemia using hematological parameters is time-consuming with low-resolution detection. Development of a MALDI–TOF–MS assay using alternative markers is needed. Methods We constructed a MALDI–TOF–MS-based approach for identifying various β-globin disorders and classifying thalassemia major (TM) and thalassemia intermedia (TI) patients using 901 training samples with known HBB/HBA genotypes. We then validated the accuracy of population screening and clinical classification in 2 separate cohorts consisting of 16 172 participants and 201 β-thalassemia patients. Traditional methods were used as controls. Genetic tests were considered the gold standard for testing positive specimens. Results We established a prediction model for identifying different forms of β-globin disorders in a single MALDI–TOF–MS test based on δ- to β-globin, γ- to α-globin, γ- to β-globin ratios, and/or the abnormal globin-chain patterns. Our validation study yielded comparable results of clinical specificity (99.89% vs 99.71%), and accuracy (99.78% vs 99.16%) between the new assay and traditional methods but higher clinical sensitivity for the new method (97.52% vs 88.01%). The new assay identified 22 additional abnormal hemoglobins in 69 individuals including 9 novel ones, and accurately screened for 9 carriers of deletional hereditary persistence of fetal hemoglobin or δβ-thalassemia. TM and TI were well classified in 178 samples out of 201 β-thalassemia patients. Conclusions MALDI–TOF–MS is a highly accurate, predictive tool that could be suitable for large-scale screening and clinical classification of β-globin disorders.

Electrospray triple quadrupole mass spectrometry guides pathologists to suggest appropriate molecular testing in the identification of rare hemoglobin variants

“…To resolve overlapping peaks and improve accuracy, high resolution MS (HRMS) can be employed for specific cases [18] , [19] . The quality of spectral data and the selection of appropriate regions for deconvolution contribute to the precision of mass measurements.…”

“…In recent years, mass spectrometry (MS), a powerful analytical technique, has been used in research laboratories to identify hemoglobin variants based on the intact mass information of the variant globin chain [11] , [12] , [13] . However, the use of MS in clinical diagnostic laboratories has been mainly limited to low-resolution MS for quantification of small molecules [14] , [15] and peptides [16] , [17] , while high-resolution MS and Matrix Assisted Laser Desorption Ionization - Time-of-Flight (MALDI-ToF) MS have been explored for analyzing intact protein diagnostic markers [18] , [19] .…”

A high level of Hb F unmasks a new case of Hb Wanjiang (β (F3‐F4) Ala87_Thr88delinsSer_Gln (HBB:c.255_264 delinsTTTTTCTCAG)) in a pregnant woman of African ancestry

“…Before our study, two new variants were found which appears to have the same molecular trace than as our variant: Hb Dongguan-Dongcheng found in healthy individuals during population screening for Hb Pathies in China with a MALDi-ToF method 5 and, Hb Wanjiang, discovered studying an asymptomatic patient. 6 Hb Wanjiang is supposed to arises from a double recombination event as Hb Palencia (γ-δ-β fusion gene). 7 Therefore, Hb Yogoupon appears to be a new case of the same mutant but in an African background without Chinese admixture.…”