Flow cytometric analysis of platelet surface glycoproteins in the diagnosis of thirty-two Turkish patients with Glanzmann thrombasthenia: a multicenter experience

Background/aim: Glanzmann thrombasthenia (GT) is a rare autosomal recessively inherited bleeding disorder characterized by the quantitative (type 1 and type 2) or qualitative (type 3) deficiency in platelet membrane glycoprotein (GP) IIb/IIIa (CD41a/CD61) fibrinogen receptors. In type 1, 2, and 3, CD41a/CD61 expression is 5%, 5%­20% and above 20%, respectively. In this study, diagnosis of GT was confirmed and subgroups were identified in 32 Turkish patients by flow cytometry analysis. Materials and methods: CD41a/CD61 expression levels in platelet-rich plasma (PRP) obtained from peripheral venous EDTA blood samples were analyzed with a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). GT subgroup analysis was performed by counting 50,000 events in the BD FACSDiva Software v6.1.3 program of the instrument. Results: In the present study, in blood samples of 32 patients from 23 families with GT and 22 healthy controls, co-expression levels of CD41a and CD61 in PRP was analyzed. 12 out of 23 families were consistent with type 1 GT (52.2%), 4 were consistent with type 2 GT (17.4%), and 7 were consistent with type 3 GT (30.4%). Conclusion: Especially due to consanguineous marriages, GT with various glycoprotein levels may be detected. As a result of the flow cytometry analysis of the present study with the highest GT patient population in Turkey, type 1 GT patients were the most common subgroup. In the determination of the GT subgroups; especially in the detection of type 3 GT, flow cytometry is the most sensitive glycoprotein analysis method. In addition to light transmission aggregometry, CD41a/CD61 study by flow cytometer confirms diagnosis when mutation analysis cannot be performed.

Diagnostic Modalities Based on Flow Cytometry for Chronic Granulomatous Disease: A Multicenter Study in a Well-Defined Cohort.

BACKGROUND: Chronic granulomatous disease (CGD) is characterized by defective microbial killing due to mutations affecting subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Definitive genetic identification of disease subtypes may be delayed or not readily available. OBJECTIVE: Sought to investigate the role of intracellular staining of NADPH oxidase enzyme subunits in predicting the respective genetic defects in patients with CGD and carriers. METHODS: Thirty-four patients with genetically inherited CGD, including 12 patients with X-linked CGD (gp91phagocyte oxidase (phox) deficiency due to cytochrome b-245, beta polypeptide [CYBB] mutations) and 22 patients with autosomal-recessive CGD (p22phox, p47phox, and p67phox deficiency due to cytochrome b-245, alpha polypeptide [CYBA], neutrophil cytosolic factor 1 [NCF1] and NCF2 mutations, respectively) were recruited from different immunology centers and followed up prospectively. Dihydrorhodamine testing and NADPH oxidase subunit expression in white blood cells were determined by flow cytometry. RESULTS: gp91phox and p22phox defects, which result in simultaneous loss of both proteins due to their complex formation, were differentiated only by comparative analysis of patients' and mothers' intracellular staining. p47phox and p67phox protein expression was almost undetectable in patients compared with carrier mothers and healthy controls. The expression values of the respective subunits were found to be significantly higher in all controls as compared with carrier mothers, which in turn were higher than those of patients. CONCLUSIONS: Analysis of NADPH oxidase enzyme subunits by flow cytometry in patients and carriers is useful in the rapid prediction of the genetic defect of patients with CGD, thus guiding targeted sequencing and aiding in their early diagnosis.