Diagnostic and clinical evaluation of in hibitors in a cohort of haemophilia patients in a quaternary care centre
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Date
2016-10-12
Authors
Wan, Yuen On
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Abstract
Background: Haemophilia A is a congenital bleeding disorder due to deficiency of clotting Factor VIII (FVIII) and is treated by replacement therapy using FVIII clotting factor concentrates. The most serious complication is the development of a neutralizing inhibitor to exogenous FVIII requiring treatment with a bypassing agent. Routine measurement of inhibitors is important in the diagnosis, therapeutic monitoring and surveillance of inhibitor patients. The testing of inhibitor is historically performed with the Bethesda assay (BA) which lacks specificity and reliability when it comes to the low-titre inhibitor ranges. The Nijmegen modification of the Bethesda assay (NA) was developed and proven superior to the BA. With routine inhibitor monitoring, the phenomenon of transient low-titre inhibitor is noted, they disappear spontaneously and patients do not require a change in treatment. The aim of this study was to validate NA in the routine National Health Laboratory Service (NHLS) haematology laboratory at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) and the haemophilia clinic at CMJAH using different buffered reagents and to determine the significance of the transient inhibitors using a cohort of inhibitor patients attending our haemophilia clinic. Methods: A total of 100 samples from both low- and high-titre inhibitor patients were collected in our centre over the period of 2007 to 2013 when patients had their routine clinic visits. Validation of NA, including the set-up and optimization of the NA, was done as described by Verbruggen (2014). The normal pooled plasma (NPP) and Dade® Citrol 1® Coagulation Control Level 1 (Citrol 1) were buffered with imidazole. Using either the NPP or Citrol 1, the NA was performed on 3 control samples (provided by External quality Control of diagnostic Assays and Tests (ECAT) foundation) and on all 100 patient samples. Precision
analysis was performed on a control sample to determine standard deviation (SD) and coefficient of variation (CV). Accuracy analysis was performed on patient- samples against BA and results expressed as bias and 95% confidence interval. Linearity analysis was performed in the low-titre range between 0 to 5 BU/mL. Clinical data of the corresponding patients were collected and analyzed to determine the clinical significance of transient inhibitor. Results: Of the 100 patient-samples,100 were analyzed with NPP and 43 with Citrol 1. In the NA using NPP, the control plasma CV was 8.44% (95%CI of 0.77 ± 0.05) with a SD of 0.06. In the NA using Citrol 1, the control plasma CV was 10% (95%CI of 0.93 ± 0.02) and SD was 0.09. In the Bland-Altman plot of NA against BA using the NPP, the bias was 0.49 (95%CI of -8.1 to 9.1). The bias between NA and BA using Citrol 1 was 0.8 (95%CI of -6.8 to 8.5). The correlation coefficient of NA vs BA using the NPP was 0.93 and that of the NA vs BA analysis using the Citrol 1 was 0.76. All control plasma analyses using NPP buffered by 4M imidazole solution were within reference values whilst only 2 of 3 values were within the assigned values using Citrol 1. Only 3 of 20 high-titre inhibitor patients have a history of transient inhibitor. The inhibitors in the residual 44 patients with low titre inhibitors were of transient nature and these patients have not developed clinically significant inhibitors to date. Conclusion: In this analysis of patient and control samples with inhibitors, the analytical performance of the NA was comparable to BA. The performance of the Nijmegen assay with buffered pool plasma was better than that of the same assay using buffered Citrol 1. In this study, the presence of transient inhibitor did not seem to predict future inhibitor development in our haemophilia cohort.
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A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in Haematology
Johannesburg, 2016