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Evaluation of a semi‐automated von Willebrand factor multimer assay, the Hydragel 5 von Willebrand multimer, by two European Centers

Abstract Background The phenotypic diagnosis of von Willebrand disease (VWD) is a multistep process with classification dependent on the quantification of von Willebrand factor (VWF) multimeric structure. VWF multimer analysis is a technically challenging, lengthy and non‐standardised assay, usually performed in specialist laboratories. Recently, a new semi‐automated multimer assay, the Hydragel 5 von Willebrand multimers (H5VWM) has become available. Objectives This study, performed in two European centres, compared existing in‐house multimer assays to the H5VWM in individuals with and without VWD. Results Overall agreement of 91.1% was observed in 74 individuals with normal VWF levels, 57 patients grouped as type 1 VWD, 33 type 2A, 16 type 2B, 28 type 2M, 11 type 2N. Patients tested following Desmopressin or VWF concentrate, with thrombotic thrombocytopenic purpura and acquired von Willebrand syndrome were also evaluated. Many of the discrepancies between methods were in patients with genetic mutations linked to more than one type of VWD including p.R1374C/H and p.R1315C. Quantifiable multimer results were available within one working day. Densitometry improved the interpretation of the multimers with slight structural variations that were not apparent by visual inspection of the in‐house method. Conclusions 5VWM was a rapid, sensitive, standardised assay which used existing technology and could be included as an initial screen of VWF multimers in a VWD diagnostic algorithm in conjunction with traditional multimer analysis.


| INTRODUCTION
Von Willebrand disease (VWD) is a hemorrhagic disorder that is caused by a reduction, dysfunction, or absence of von Willebrand factor (VWF). 1 VWF is a large multimeric glycoprotein that binds platelets to the damaged subendothelium and also circulates in complex with factor VIII (FVIII). VWF comprises subunits of variable size from the low molecular weight multimers (LMWM) to subunits of more than 50 in size, the high molecular weight multimers (HMWM). VWD is sub-typed by the ratio of the activity (VWF Ac) to the concentration of protein (VWF antigen, VWF:Ag). 2 A VWF Ac/VWF:Ag ratio of >0.6 indicates a quantitative type 1 disorder. A ratio of <0.6 is indicative of qualitative type 2 disorders, which generally have a disproportionate reduction of VWF activity compared to VWF:Ag. 3 Type 2 VWD is further divided into four subtypes; type 2A which is generally ineffective multimer assembly or secretion, type 2M which is linked to reduced binding to platelet GPIb, type 2B which is an increased binding to GPIb, and type 2N which is a decrease or absence in binding to FVIII with preservation of the ability of VWF to bind platelets. 4,5 VWF is absent in type 3.
The diagnosis of type 2 VWD requires a number of specialized assays including analysis of multimer distribution. 3 Multimer analysis is not widely undertaken by routine laboratories 6 due to lack of method standardization, length of test, requirement of specialist equipment, and often variable and subjective results. In-house VWF multimers (IHVWM) may be performed using sodium dodecyl sulphate (SDS) agarose gel electrophoresis, often followed by Western blotting, then visualization by methods including via radioactive Iodine (I 125 ) autoradiography, chemiluminescence, 7 infrared fluorescent imaging, 8 or Alkaline Phosphatase (AP). 9 Densitometry of the agarose gels may be performed to aid interpretation of the multimer pattern. 10 Interpretation of densitometry is also not standardized between laboratories; some report peaks 1-3 to represent the LMWM, 4-5 the intermediate molecular weight multimers (IMWM), and peaks 6 and above represent the HMWM, 11 while others report peaks 1-5 as LMWM, 6-10 as IMWM, and peaks 10 and above as HMWM. 9 A new semi-automated VWF multimer assay kit has recently been launched. The Hydragel 5 von Willebrand multimers test (H5VWM) is performed with existing Hydrasys 2 Technology (Sebia, France) and the kit includes ready to use buffers, agarose gels, and antibodies.
In this study, two centers compared H5VWM with an existing inhouse SDS agarose gel electrophoresis method in healthy donors and patients with different types of VWD or acquired von Willebrand syndrome (AVWS). Subgroups of patients were also tested following treatment with endogenous or exogenous VWF or in the presence of interferring substances. Possible interference in the H5VWM method by high rheumatoid factor was assessed in eight samples provided by Sebia. Rheumatoid factor levels were determined by both rate Nephelometry (Beckman Coulter, Villepinte, France) with levels ranging from 1000 to 8750 IU/mL (reference interval <20 IU/mL) and by immunoturbidimetry (Randox, Crumlin, UK) with levels ranging from 932 to 4834 IU/mL (reference interval <12.5 IU/mL). The influence of anticoagulant was assessed with three patients whose blood was taken into both citrate and EDTA.

| VWF multimer analysis
IHVWM at site 1 was by discontinuous SDS agarose gel electrophoresis, a modification of two methods by omission of acrylamide. 14,15 This was routinely performed at 1.6% agarose (SeaKem Multimer analysis from site 2 was performed by the laboratory of Professor Budde in Hamburg using a previously described method. 16 The Hydragel 5 VWF multimers kit (H5VWM) was used with the Hydrasys 2 Scan instrumentation to perform agarose gel electrophoresis using preformed 2% agarose gels, direct immunofixation, visualization with peroxidase-labelled antibody, and densitometry. has since revised the methodology to dilute samples greater than

| RESULTS
Inter-assay coefficient of variation (CV), calculated using the  Figure 1A-D).  and 29.1%, respectively, had no genetic analysis performed (see Figure 3A). P119, an 80-year-old male, had 21.9% HWMW and subsequent mutation analysis demonstrated a p.P1337L change in exon 28 linked to 2B VWD (see Figure 3B and  Table 2). P213 is the daughter of P119 who was grouped as normal VWF.
A reduction of HMWM by both methods was observed in 16 individuals known to have VWD type 2B. Six to 10 triplet bands were visible by IHVWM whilst H5VWM HMWM ranged from 6.5% to 32.5%.
Twenty-eight individuals classified as type 2M had normal IHVWM on visual inspection. 6 (P121, P124, P125, P214, P216, and P251) had reduction to HMWM (20.2%-33.4%) with H5VWM densitometry exhibiting a flattened appearance of the HMWM peak (see Figure 3D and Table 2). An increase in LMWM peak (see Figure 3E) was observed in the four individuals with TTP. The ULMWM using the IHVWM were less obvious, particularly at higher concentrations of ADAMTS13. Agreement in multimer results was observed in eight patients with elevated rheumatoid factor and three patients whose blood was drawn in citrate or EDTA. between multimer methods was observed in 77% of patients. The reasons for the differences are unknown but may be linked to the underlying mechanism of the disorder; further investigation of these patients is therefore warranted.

| DISCUSSION
Plasma from 55 normal donors was initially included to establish adult reference ranges. Inter-quartile ranges (IQR) were calculated; however, four donors, one female and three males, had LMWM (28.5%-33.5%) which exceeded the upper quartile +1.5 IQR value of 26.2%. These donors were considered statistical outliers and excluded from further reference range calculations. The results of limited groups of patients suggested that H5VWM was not affected by the presence of rheumatoid factor or use of EDTA in place of sodium citrate as anticoagulant. CV of less than 10% were observed for IMWM and HMWM with the normal QC sample, but LWMW was unexpectedly slightly more variable than the other parameters.
Ideally, a pathological QC sample with reduced HMWM would also be included on each gel. This would not be practical with the five-well H5VWM gel used for this present study, however, an eleven-well gel (Hydragel 11 VW) is available which offers more scope for the inclusion of two levels of QC plasma.
The sensitivity of VWF activity assays to different VWF mutations clearly impacts on the diagnosis and classification of VWD.
Innovance VWF Ac, performed at site 1, is a VWF:GP1bM type assay 2 that utilizes two gain of function mutations to glycoprotein 1b (GP1b) in place of Ristocetin and platelets. The VWF:Ag and VWF Ac of multimer discrepant patients were confirmed on repeat. It is possible that the VWF Ac assay was less sensitive to certain mutations than a traditional VWF:RCo. A previous study reported some slight differences in particular in type 2A (IIE), Vicenza VWD, and several type 1 VWD patients. 26 Laboratories which use one of the new generation of VWF activity assays should contemplate including a second activity method during the initial diagnosis of VWD. We propose an algorithm for the inclusion of H5VWM as a screening tool in the diagnosis of VWD in the laboratory (see Figure 4). This algorithm uses a VWF Ac/VWF:Ag ratio cut-off of 0.8 to determine whether VWF format, using a single agarose concentration and without the sensitivity to visualize the multimer triplet structure, H5VWM is unlikely to completely replace in-house multimer analysis but as demonstrated in our proposed algorithm, it would be a useful addition to VWD diagnosis in the routine laboratory. Multimer abnormalities could then be further explored in more detail using in-house methods with larger gels and at different agarose concentrations as required.

ACKNOWLEDGMENTS
We are grateful to Barbara Sampson for assistance with visualization of the in-house multimer gels and the staff in Coagulation at Leeds St James Hospital for the provision of additional plasma and test results for their patients. We would like to thank Ghislaine Beaulieu, Hector Bautista, and Georges Nouadje from Sebia, France for provision of the Hydrasys analyzer, H5VWM kits, rheumatoid factor samples, training, and advice throughout this study.

RELATIONSHIP DISCLOSURE
AB has received investigator, speaker fees, and travel funding from Sebia, France.

AUTHOR CONTRIBUTIONS
A. Bowyer