Comparative assessment of von Willebrand factor multimers vs activity for von Willebrand disease using modern contemporary methodologies

Abstract

Introduction Diagnosis of von Willebrand disease (VWD) is challenging due to heterogeneity of VWD and test limitations. Many von Willebrand factor (VWF) assays are utilized, including antigen (Ag), activity and multimer analysis. Activity assays include ristocetin cofactor using platelets (VWF:RCo) or other particles incorporating recombinant glycoprotein I (‘VWF:GPIbR’), or other GPI binding assays using gain‐of‐function mutations (‘VWF:GPIbM’), or collagen binding (VWF:CB). Aim To comparatively evaluate modern contemporary VWF activity assays vs VWF multimer analysis using modern contemporary methods. Materials and methods Several VWF activity assays (VWF:RCo, VWF:GPIbR, VWF:GPIbM, VWF:CB) assessed (typically as a ratio against VWF:Ag) against a new semi‐automated procedure for different types of VWD (1, 3, 2A, 2B, 2M), plus control material (n = 580). The evaluation also focussed on relative loss of high and very high molecular weight multimers (HMWM and VHMWM) by densitometric scanning. Results All evaluated VWF activity/Ag ratios showed high correlation to the presence/absence of HMWM and VHMWM, although VWF:CB/Ag and VWF:GPIbR/Ag ratios using an automated chemiluminescence method yielded highest correlation coefficients (r = .909 and .874, respectively, for HMWM). Use of the investigative procedure (VHMWM) identified fewer false positives for ‘loss’ in type 1 VWD. Conclusions This comparative investigation identified that new automated chemiluminescence VWF activity assays best identified relative loss or presence of HMWM and VHMWM according to activity to Ag ratios and an alternative investigative method for identifying VHMWM in multimer testing for a new commercial multimer method may lead to fewer false identifications of HMW loss in type 1 VWD.

Improving diagnosis of von Willebrand disease: Reference ranges for von Willebrand factor multimer distribution

“…Favaloro et al 20 and Oliver et al 21 have also reported the presence of all HMWM in their type 2M patients, but their studies do not provide any information about its quantification and the diagnosis of type 2M. On the other hand, Bowyer et al 17 and a recent report by Favaloro et al 39 published quantitative HMWM loss in some of the type 2M patients. However, Crist et al 18 and Pikta et al 19 do not mention type 2M VWD in their studies about H5/11VWM.…”

Navigating the Myriad of von Willebrand Factor Assays

“…22,23 A commercial semiautomated method is now available which offers better standardization and faster turnaroundtime, but which may not be able to evaluate fine details around VWF multimer structures. 24,25 Finally, there are other assays used to further interrogate VWF function, for example (1) the VWF propeptide assay (VWF:pp), which mostly helps to identify VWD types sitting in type 1 with faster VWF clearance, 26 and (2) the VWF-FVIII binding (VWF:FVIIIB). 27 Neither of these assays is widely employed, and instead tends to be only offered by specialist laboratories.…”

“…7,[15][16][17]30 This is "analogous" to adding platelets from a patient with PT-VWD to plasma, and assessing how well plasma VWF binds. There is only one commercial offering for this assay by LIA (Siemens Innovance VWF Ac assay) and this can be performed on Siemens analyzers, 7,16,24,28 and in theory can also be adapted to non-Siemens analyzers. The other main assay type is ELISA, 17,30 but this reflects different methodologies and reagents and is not yet commercially available to this author's knowledge.…”

“…An alternative commercial method is now available that can also in general distinguish 2A and 2M VWD. 24,25 Recognize, however, that VWD represents a continuous variable, with hundreds of separate VWF mutations identified, 39 and thus some patients will still remain in an equivocal zone with exhaustive testing, even including VWF multimers and genetic testing. Be also aware that the commercial VWF multimer method cannot distinguish multimer changes in a triplet structure, as sometimes used to identify rarer forms of type 2 VWD.…”

Classification of von Willebrand disease in the context of modern contemporary von Willebrand factor testing methodologies

“…The main strengths of the new methodology include high reproducibility, ability for same‐day test results, automation of many test steps, and ability to “accurately” identify loss or retention of HMW VWF by both visual and quantitative methods (densitometry). Nevertheless, there are also some limitations, as summarized in another recent publication 18 . One main limitation is the current single agarose gel concentration, limiting identification of VWF structural changes, including an absence of any triplet banding.…”