Analysis of discrepancies between one-stage clotting assay and chromogenic substrate assay for coagulation factor Ⅷ activity and their underlying mechanisms in patients with mild hemophilia A

doi:10.16150/j.1671-2870.2026.02.011

Abstract

Abstract:

Objective The one-stage clotting assay (OSA) for measuring coagulation factor Ⅷ activity (FⅧ:C) is currently the most commonly used method in clinical practice. However, in patients with hemophilia A carrying specific missense mutations, the FⅧ:C level measured by OSA may be higher than that indicated by the clinical bleeding phenotype, leading to underestimation of disease severity or even missed diagnosis of hemophilia. The chromogenic substrate assay (CSA) is often used as a supplement to OSA. This study analyzes the genotypes and laboratory phenotypes of patients with mild hemophilia A (MHA) and explores the diagnostic value of OSA, CSA, and thrombin generation test (TGT) in MHA. Methods From June 2019 to September 2025, five male patients suspected of having MHA, whose FⅧ:C levels measured by OSA were inconsistent with their actual clinical bleeding phenotype, were retrospectively and consecutively enrolled at our center. The patients' ages ranged from 24 to 75 years. Clinical data were collected from all patients. FⅧ:C levels were measured using both OSA and CSA, and the differences between the two assays were compared. TGT was performed to evaluate the patients' global coagulation potential. The F8 genotype was determined using next-generation sequencing combined with Sanger sequencing. Structural visualization of FⅧ domains and key amino acid residues was performed using PyMOL software to explore the effects of mutations on the spatial structure of FⅧ. Results OSA indicated that among the five patients (A-E), three patients (A, C, and E) had FⅧ:C levels <40%, while the other two patients (B and D) had FⅧ:C levels of 55.3% and 63.7%, respectively. CSA results showed that the FⅧ:C levels of the five patients ranged from 7.8% to 25.7%, and all five patients were diagnosed with MHA based on CSA results. The ratio of OSA to CSA results ranged from 1.3 to 5.5, and CSA results were more consistent with the clinical bleeding phenotype in patients'. TGT was performed in four of the five patients. Compared with normal pooled plasma, the patients showed reduced endogenous thrombin potential, decreased peak thrombin, and lower thrombin generation velocity, along with prolonged time to peak. Four previously reported F8 missense mutations were identified among the five patients: p.V285M (patient A), p.A303P (patient C), p.S308L (patients B and D), and p.R546W (patient E). PyMOL structural analysis indicated that the first three mutations were located at the interaction interface of the A1, A3, and A2 domains of FⅧ, potentially affecting the stability of activated FⅧ (FⅧa) and causing inconsistency between OSA results and clinical bleeding phenotype. The p.R546W mutation was located near this interaction interface and had a relatively smaller effect on FⅧa stability. Conclusions FⅧ:C levels measured by OSA may be inconsistent with the clinical bleeding phenotype in some patients with MHA. The combined use of OSA and CSA can help improve diagnostic accuracy. TGT can be used to assist in evaluating patients' global coagulation function and provide a reference for precise diagnosis.

Key words: Mild hemophilia A, Laboratory diagnosis, Assay discrepancy, Thrombin generation test

CLC Number:

R446
R554.1

CHEN Guangming, LIN Liya, WU Haoyang, WU Wenman, DING Qiulan, WANG Xuefeng, DAI Jing. Analysis of discrepancies between one-stage clotting assay and chromogenic substrate assay for coagulation factor Ⅷ activity and their underlying mechanisms in patients with mild hemophilia A[J]. Journal of Diagnostics Concepts & Practice, 2026, 25(02): 200-208.

Figures/Tables 6

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References 24

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Patients	APTT (s)	FⅧ:C (OSA, %)	FⅧ:C (CSA, %)	Ratio (OSA/CSA)	Variant
Patients	APTT (s)	FⅧ:C (OSA, %)	FⅧ:C (CSA, %)	Ratio (OSA/CSA)	(HGVS)
Patient A	29.1	38.5	12.3	3.1	c.853G>A, p.V285M
Patient B	26.8	55.3	13.7	4.0	c.923C>T, p.S308L
Patient C	33.7	15.3	7.8	2.0	c.907G>C, p.A303P
Patient D	27.2	63.7	11.6	5.5	c.923C>T, p.S308L
Patient E	28.4	34.4	25.7	1.3	c.1636C>T, p.R546W

Patients	ETP(nmol·L^-1·min^-1)	Peak (nmol/L)	TTP (min)	Velocity (nmol·L^-1·min^-1)	LT (min)
NPP	1250.75	153.06	9.00	32.80	4.33
Patient A	780.68	40.83	13.67	4.00	3.50
Patient B	291.87	21.50	10.67	2.93	3.33
Patient C	527.89	30.91	13.33	3.31	4.00
Patient D	554.47	34.58	11.33	4.43	3.50