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Semiautomatic densitometric analysis of von Willebrand factor (VWF) multimers appears to have excellent accuracy in clinical practice, according to the findings of a study presented by Ferdows Atiq, MD, of the University Medical Center Rotterdam, in the Netherlands, at the virtual 62nd American Society of Hematology (ASH) Annual Meeting and Exposition.
“[T]here are some limitations of [standard VWF multimer classification] technique,” said Dr Atiq. “Subtle changes can be missed because it’s based on visual examination. [It] is also time consuming, and the amount of high-molecular-weight [multimers] cannot be quantified.”
Therefore, Dr Atiq and colleagues sought to validate the accuracy of VWF multimer semiautomatic densitometric analysis in clinical practice and to identify patient characteristics associated with VWF multimer densitometry outcomes in von Willebrand disease (VWD).
Patients from the Willebrand in the Netherlands (WiN) study were included based upon personal hemorrhagic diathesis or family history of VWD and VWF antigen (VWF:Ag), VWF activity (VWF:Ab), VWF collagen binding (VWF:CB), and FVIII activity (FVIII:C) levels. Blood samples were collected from each patient upon enrollment in the WiN study, and all patients completed a self-administered questionnaire to determine Tosetto bleeding score.
Multimers from patient samples were analyzed by Western blotting, and ImageJ was used for densitometric analysis. The visualized bands were classified as small, medium, or large multimers. A medium-large VWF index was calculated based on the intensity of medium and large multimer bands to all multimer bands in a patient sample and then divided by the results for a normal control (or assigned 0 if no patient band was present).
A total of 561 patients with VWD were included in the analysis (62.7% women; type 1, n=328; type 2, n=211; type 3, n=21). Median age was 44 years (interquartile range [IQR], 29-58). From the densitometric analysis, the median medium-large VWF multimer index was 1.06 (IQR, 0.99-1.12) in type 1 VWD, 0.53 (IQR, 0.29-0.89) in type 2 VWD, and 0.00 (IQR, 0.00-0.00) in type 3 VWD.
When the bands were classified visually for each patient as normal, reduced, or absent, the corresponding median VWF medium-large multimer indices were 1.07 (IQR, 1.02-1.12), 0.84 (IQR, 0.71-0.91), and 0.31 (IQR, 0.20-0.44; P <.001).
Using gold standard visual classification results for comparison, the medium-large VWF multimer index accurately distinguished normal from reduced high-molecular-weight VWF multimers (area under the curve [AUC], 0.96; P <.001), accurately distinguished reduced high-molecular-weight VWF multimers from absence of high-molecular-weight VWF (AUC, 0.95; P <.001), and distinguished type 2A and 2B from type 2M and 2N (AUC, 0.96; P <.001).
Medium-large VWF multimer index correlated most strongly with (VWF:CB; ρ=0.79; P <.001) and the VWF:CB/VWF:Ag ratio (ρ=0.80; P <.001). Additionally, after adjustment for age, sex, blood group, and type of VWD, higher medium-large VWF multimer index was associated with a lower bleeding score (β=-4.6; P =.001).
“[W]e found in this study that VWF multimer densitometric analysis has an excellent accuracy in comparison with visual examination,” concluded Dr Atiq.
Disclosure: Some authors have declared affiliations with or received funding from the pharmaceutical industry. Please refer to the original study for a full list of disclosures.
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Atiq F, Boender J, Cnossen MH, et al. Semiautomatic VWF multimer densitometric analysis: validation of the clinical accuracy and clinical implications in von Willebrand disease. Presented at: American Society of Hematology (ASH) 62nd Annual Meeting and Exposition; December 5-8, 2020. Abstract 571.