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Comparison between coagulation factor VIII quantified with one-stage activity assay and with mass spectrometry in haemophilia A patients: Proof of principle.

ABSTRACT: INTRODUCTION:Haemophilia A is a hereditary bleeding disorder caused by a factor VIII (FVIII) deficiency. As biomarker, FVIII activity is used to classify disease severity and to monitor treatment. The one-stage clotting assay (OSA) is performed to measure FVIII activity, but OSA's limitations may result in misclassification of disease severity or suboptimal monitoring of treatment. Measurement of FVIII plasma concentration with liquid chromatography-tandem mass spectrometry (LC-MS/MS) might overcome these challenges. The objective is to investigate the correlation between FVIII activity and concentration, and determinants for differences between the two methods. METHODS:In this cross-sectional study, all haemophilia A patients receiving standard-of-care were eligible for inclusion. Within the activity categories of <1 IU/dL, 1-5 IU/dL, >5-40 IU/dL, >40-150 IU/dL and >150-600 IU/dL, we randomly selected 15-20 plasma samples and compared FVIII concentration (LC-MS/MS) to FVIII activity (OSA) with linear regression and Bland-Altman analysis. Potential determinants for differences were analysed with linear regression. RESULTS:Inclusion was 87 samples. Bland-Altman analysis demonstrated an overall mean difference of -1% with an SD of 64% between the two methods. Large differences were correlated with the presence of anti-FVIII antibodies (133% [95% CI: 81, 185] n = 5) and use of exogenous FVIII products (-37% [95% CI: -65,-9] n = 58), for example plasma-derived and B-domain-modified FVIII products. CONCLUSIONS:Despite good overall correlation between the two methods, relative differences were large, especially for samples with anti-FVIII antibodies or exogenous FVIII products. These differences may have clinical impact. More research is needed to determine the value of FVIII plasma concentration in comparison with FVIII activity.

SUBMITTER: Donners AAMT

PROVIDER: S-EPMC7754357 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Comparison between coagulation factor VIII quantified with one-stage activity assay and with mass spectrometry in haemophilia A patients: Proof of principle.

Donners Anouk A M T AAMT van Maarseveen Erik M EM Weetink Yrea R J YRJ El Amrani Mohsin M Fischer Kathelijn K Rademaker Carin M A CMA Egberts Toine C G TCG Huisman Albert A Musson Ruben E A REA

International journal of laboratory hematology 20200706 6

<h4>Introduction</h4>Haemophilia A is a hereditary bleeding disorder caused by a factor VIII (FVIII) deficiency. As biomarker, FVIII activity is used to classify disease severity and to monitor treatment. The one-stage clotting assay (OSA) is performed to measure FVIII activity, but OSA's limitations may result in misclassification of disease severity or suboptimal monitoring of treatment. Measurement of FVIII plasma concentration with liquid chromatography-tandem mass spectrometry (LC-MS/MS) mi ...[more]

PMID: 32633067

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Project description:IntroductionDiagnosis, treatment monitoring and assessment of desmopressin effect in haemophilia A patients are performed by measurement of factor VIII activity (FVIII). The two assays commonly applied are the one-stage assay and the chromogenic assay. Especially in non-severe haemophilia A, discrepancies between these assays are common. It is still unestablished which assay corresponds best with bleeding phenotype and desmopressin effect.AimTo correlate FVIII levels measured by the one-stage assay and by the chromogenic assay with bleeding phenotype and, additionally, to compare FVIII assay discrepancies before and after desmopressin administration.MethodFactor VIII was measured in 130 non-severe haemophilia A patients during routine visits to the outpatient clinic and/or during desmopressin testing. FVIII was measured by both the one-stage assay and the chromogenic assay. Discrepancies between assays were defined as at least a twofold difference of FVIII or an absolute FVIII difference between measurements of ≥0.10 IU/mL. Bleeding phenotype was defined as annual number of treated bleedings (adjusted ABR).ResultsHundred and thirty non-severe haemophilia A patients were included. In 31/130 patients, assay results were discrepant. However, FVIII measurements with both assays correlated adequately with adjusted ABR. In addition, in 27/130 patients FVIII measurements at baseline and after desmopressin administration were analysed. In 13/27 patients, all measurements were either equivalent or discrepant when results were compared. In 14/27 patients, this was not the case as both equivalent measurements and discrepant measurements at different time points within one patient were observed.ConclusionNeither the one-stage assay nor the chromogenic assay is superior in predicting bleeding phenotype. In addition, equivalent or discrepant FVIII results measured before desmopressin do not always predict FVIII assay results after desmopressin administration.

Project description:In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methylcytosine to detect the presence of 5 methylcytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates of these experiments. The methylated genes show a uniform distribution over all the chromosomes. The gene ontology of these also indicates no functional correlation between the genes that are methylated. We detect the presence of methylation in IGF2, an imprinted gene and thus known to harbour DNA methylation. The method is extremely specific and offers a quick and efficient way to analyse the methylation landscape on a high throughput scale. This method uses existing technology to assess methylation and thus can integrate very efficiently into any platform used. A method that detects DNA methylation that is not restricted to specific sequences would provide a useful platform to analyse methylation distribution among the genes in any given phenotypic condition.Availability of additional approaches to examine DNA methylation would lead to increase in our understanding of the methylome and would help define the intrinsic and extrinsic factors which govern it. Microarray based high throughput methods have been extensively used for gene expression analysis. The method described uses a microarray to analyse DNA methylation levels in a gene/cDNA/oligo specific manner. Here, genomic DNA is fragmented and used for hybridization to microarray slides. The presence of DNA cytosine methylation in the hybridized DNA is detected by employing Cy3 labelled anti 5mC( 5-methyl cytidine) antibody (monoclonal). The resolution provided by this method is dependent upon the microarray platform used. This method can be adapted to any platform thus enabling seamless integration with existing technology of gene expression analysis

Dataset Information

Comparison between coagulation factor VIII quantified with one-stage activity assay and with mass spectrometry in haemophilia A patients: Proof of principle.

Publications

Comparison between coagulation factor VIII quantified with one-stage activity assay and with mass spectrometry in haemophilia A patients: Proof of principle.

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