Thalassemia carrier screening in pregnant women based on targeted quantification of hemoglobin subunits by LC-MS/MS

<title>Abstract</title> <p> Thalassemia complicates prenatal carrier screening because pregnancy alters red-cell-based indices. We developed a pregnancy-adapted LC–MS/MS hemoglobin subunit-based screening strategy. We prospectively enrolled 1,726 reproductive-age women (1,052 pregnant; 674 non-pregnant) and performed hematological testing, hemoglobin electrophoresis, globin gene sequencing, and targeted LC–MS/MS quantification of hemoglobin subunits. Machine-learning models evaluated transferability and built pregnancy-specific classifiers. A subset of 287 samples (189 pregnant, 98 non-pregnant) underwent DIA proteomics to explore pregnancy-associated proteomic features. Among 1,726 participants, 145 carriers were identified (99 α-thalassemia, 46 β-thalassemia). Pregnancy significantly altered hematological indices and hemoglobin subunit concentrations, causing systematic misclassification of normal pregnant women as -α ^3.7 /αα carriers when using models trained in non-pregnant cohorts. A pregnancy-specific model achieved an ROC-AUC of approximately 0.98 and reduced false-positive predictions compared to RBC-index-only models. DIA proteomics revealed that pregnancy remodels the blood proteome in β ⁰ /β ^N carriers, involving erythropoiesis-related proteins such as AHSP and indicating that pregnancy amplifies β-thalassemia-associated proteomic disturbances. Pregnancy modifies hematological, hemoglobin-subunit, and proteomic features, limiting non-pregnant screening models. LC-MS/MS with gestation-specific modeling improves carrier screening, while DBS-DIA proteomics supports pregnancy-aware biomarker development for β-thalassemia screening and classification. </p>