Clinical validation of CD16b as a standardized biomarker for inherited GPI deficiencies

OBJECTIVES: Inherited glycosylphosphatidylinositol (GPI) deficiencies (IGDs) impair the expression of GPI-anchored proteins and produce diverse clinical phenotypes that complicate diagnosis. Since we had previously identified CD16b as a useful diagnostic biomarker, we aimed to establish a standardized flow cytometric assay suitable for routine clinical application. METHODS: We analyzed granulocyte expression of CD16b in 29 IGD patients and 101 controls (21 non-IGD and 80 healthy adults). Granulocyte CD16b expression was quantified as geometric mean fluorescence intensity (gMFI) by flow cytometry in our laboratory and a commercial testing company. Receiver operating characteristic (ROC) analysis was used to define diagnostic cut-offs. RESULTS: CD16b on granulocytes showed the largest decrease (approximately 85%) among GPI-anchored proteins in IGD patients. Mean gMFI was 41,660 ± 36,286 in IGD versus 88,370 ± 24,856 in healthy controls (p < 0.001). ROC analysis yielded an optimal cut-off of 53,431 (sensitivity 0.73, specificity 0.96, area under the curve (AUC) 0.84, Youden index 0.69). We defined 40,000 as the definitive diagnostic threshold and 60,000 as the borderline value. These cut-offs were validated with the commercial assay, which provides results within 2-3 days and is reimbursed by Japan's national health insurance. CONCLUSIONS: Measurement of granulocyte CD16b provides a reliable and rapid screening method for IGD that complements genetic testing. Implementation of this assay by a commercial provider, together with validated cutoffs, enables nationwide, insurance-covered diagnostic testing and functional confirmation of GPI pathway gene variants, representing a significant advance in IGD diagnosis.