Bleeding disorder of unknown cause: Is there a role for expanded platelet testing?

Abstract Introduction: Only 30% of patients (pts) presenting with abnormal bleeding symptoms receive a clear diagnosis, with many classified as having bleeding disorder of unknown cause, or BDUC (Baker et al. 2021). Currently, the recommended diagnostic workup for BDUC by the International Society on Thrombosis and Haemostasis (ISTH) includes plasmatic assays for coagulation factors, von Willebrand factor (vWF) antigen/activity, and platelet function testing (PFT), specifically platelet function analyzer-100 (PFA-100) and light transmission aggregometry (LTA). However, PFTs are not widely available and cannot be mailed to reference laboratories. We studied the diagnostic yield of an expanded testing panel which includes quantitative platelet glycoprotein flow cytometry (GpFL) and platelet electron microscopy (PTEM), which we have optimized for specimen mailing. Methods: We conducted a minimal risk, retrospective study by reviewing the electronic health records of pts (18 years and older) who were seen in the Coagulation Clinic of a tertiary academic medical center for evaluation of a bleeding disorder between October 2017 and June 2025. Testing included plasma-based assays including PT, APTT, vWF assay, FVIII, FIX, FXIII screen, and thrombin time (and additional reflex assays as needed). At the discretion of the treating clinician, PFTs (PFA-100 and LTA), GpFL, PTEM, and fibrinolysis assays (plasminogen activator inhibitor-1 antigen [PAI-1 Ag] and alpha-2 plasmin inhibitor) were included. We abstracted clinical data on pt demographics, ISTH bleeding scores, and the results of coagulation testing. Pts with an established diagnosis of a bleeding disorder, underlying malignancy, and those who did not have plasma-based coagulation testing were excluded from this study. Results: 109 pts met study criteria, and 82.6% were females (n = 90) with a median age of 45 years (range = 18-88 years). The median ISTH-BAT score was 7 (range: 2-21). About half of the pts, 49.5% (n = 54), had normal coagulation profiles and PFTs. All 54 had PTEM performed, of which 14.8% (n = 8) were abnormal (alpha and/or dense granule deficiencies). 85.2% (n = 46) had GpFL testing performed, with one showing reduced Gp2b/CD42 and Gp3a/CD61 expression, four showing reduced Gp1a/CD49b expression, and three showing reduced GpVI expression. Half of the pts, 50.5% (n = 55) had an abnormal coagulation profile from plasma and/or PFTs, of which 20% (n = 11) had a clinically significant coagulation factor deficiency (FV, FVII, FX). Roughly 9.1% (n = 5) had type 1 vWD of which 1 also had PAI-1 deficiency. One pt had isolated PAI-1 deficiency. Abnormal coagulation profiles were noted in 53 pts who had PFA-100 and LTA done, and both tests were normal in 22.6% (n = 12). Within the abnormal coagulation profile group, 10.9% (n = 6) had ultrastructural abnormalities on PTEM (dense and alpha granule abnormalities). Majority of the pts (n = 46) had GpFL done, and one had abnormal reductions in Gp1b-aCD42b, three in Gp1aCD49b, and seven in GPVI. Comparison of the ISTH BAT score between the normal and abnormal coagulation profiles/PFTs groups by non-parametric (Mann-Whitney U test) and parametric (Welch's t-test) showed no significant difference (p = 0.56 and p = 0.87) respectively. Conclusion: In this cohort of pts with abnormal bleeding, standard recommended testing resulted in a diagnosis in 66.4% pts. The remainder would have been classified as BDUC. The addition of GpFL and PTEM resulted in establishing a diagnosis that explains the bleeding phenotype in 14.8% of pts with an otherwise normal coagulation profile and PFT. Although abnormal PFTs indicate a platelet function defect, GpFL and PTEM may further define the abnormalities particularly differentiating syndromic from non-syndromic inherited platelet disorders. Additional studies are needed to confirm our findings and explore the possibility of addition of GpFL and PTEM for patients with BDUC.