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Validation of improved performance characteristics for the automated von Willebrand factor ristocetin cofactor activity assay

2010; Elsevier BV; Volume: 8; Issue: 12 Linguagem: Inglês

10.1111/j.1538-7836.2010.04123.x

1538-7933

Emmanuel J. Favaloro, Soma Mohammed, Jane McDonald,

Heparin-Induced Thrombocytopenia and Thrombosis

We were very interested to read reports in this journal of a modified (m) protocol for performance of an automated von Willebrand factor (VWF) ristocetin cofactor (VWF:RCo) assay, first in abstract form [1Stadler M. Hillarp A. Haderer C. Zapfl C. Pock K. Weinberger J. Roemisch J. Improved automated VWF:RCo assay for VWF‐FVIII concentrates and plasma samples by adjustment of assay reagents, composition and performance parameters.J Thromb Haemost. 2009; 7Google Scholar] (as also presented by poster at the XXII ISTH Congress in 2009), and subsequently as a full paper [2Hillarp A. Stadler M. Haderer C. Weinberger J. Kessler C.M. Römisch J. Improved performance characteristics of the von Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol.J Thromb Haemost. 2010; 8: 2216-23Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. These authors reported improved sensitivity of this modified assay [VWF:RCo(m)] to low levels of VWF, as well as improved repeatability and reproducibility, primarily in terms of VWF concentrate assessment, but also as feasibly applied to von Willebrand disease (VWD) testing. The theoretical utility of the VWF:RCo assay in VWD testing stems from its representation as a surrogate activity assay for VWF‐platelet interaction as well as displaying some selective sensitivity to high molecular weight (HMW) forms of VWF [3Favaloro E.J. An update on the von Willebrand factor collagen binding (VWF:CB) assay: 21 years of age and beyond adolescence, but not yet a mature adult.Semin Thromb Hemost. 2007; 33: 727-44Crossref PubMed Scopus (84) Google Scholar]. Thus, when used in combination with VWF antigen (VWF:Ag) testing, a low VWF:RCo/VWF:Ag (RCo/Ag) ratio will suggest either a defect in VWF‐platelet adhesion (i.e. type 2M VWD) or a loss of HMW VWF (i.e. as feasibly present in either type 2A or 2B VWD). Two major problems with VWF:RCo testing, however, compromise utility for detection and discrimination of severe type 1 vs. type 3 as well type 1 vs. type 2 VWD, and namely poor sensitivity to low levels of VWF (lower limit of sensitivity or limit of detection [LOD] typically around 10–15 U dL−1 [4Favaloro E.J. Bonar R. Marsden K. on behalf of the RCPA QAP Haemostasis CommitteeLower limit of assay sensitivity: an under‐recognised and significant problem in von Willebrand disease identification and classification.Clin Lab Sci. 2008; 21: 178-85PubMed Google Scholar]), and poor reproducibility (i.e. high assay variability, often yielding cross laboratory coefficients of variations [CVs] of over 40% [5Favaloro E.J. Bonar R. Kershaw G. Sioufi J. Baker R. Hertzberg M. Street A. Marsden K. on behalf of the RCPA QAP in HaematologyReducing errors in identification of von Willebrand disease: the experience of the royal college of Pathologists of Australasia Quality Assurance Program.Semin Thromb Hemost. 2006; 32: 505-13Crossref PubMed Scopus (64) Google Scholar]). Although automated VWF:RCo assays tend to be more ‘reproducible’ (lower CVs) than those performed on aggregometers, they may conversely suffer inferior performance with regards to LOD [4Favaloro E.J. Bonar R. Marsden K. on behalf of the RCPA QAP Haemostasis CommitteeLower limit of assay sensitivity: an under‐recognised and significant problem in von Willebrand disease identification and classification.Clin Lab Sci. 2008; 21: 178-85PubMed Google Scholar]. The standard automated VWF:RCo assay [VWF:RCo(s)] utilizes a commercially available reagent (Siemens BC VWF reagent; Catalogue no. 10446425; Siemens Healthcare, Marburg, Germany) containing premixed lyophilized platelets and ristocetin that is reconstituted using 4 mL of water. The reported [1Stadler M. Hillarp A. Haderer C. Zapfl C. Pock K. Weinberger J. Roemisch J. Improved automated VWF:RCo assay for VWF‐FVIII concentrates and plasma samples by adjustment of assay reagents, composition and performance parameters.J Thromb Haemost. 2009; 7Google Scholar, 2Hillarp A. Stadler M. Haderer C. Weinberger J. Kessler C.M. Römisch J. Improved performance characteristics of the von Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol.J Thromb Haemost. 2010; 8: 2216-23Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar] modifications for VWF:RCo(m) comprise: (i) addition of extra ristocetin and saline to this commercial reagent post its normal reconstitution, (ii) use of VWF‐deficient plasma (VWFDP) as test diluent (instead of saline), and (iii) some adjustment of sample and reagent volumes in the assay procedure. Given that use of excessive ristocetin is reported [6Scott J.P. Montgomery R.R. Retzinger G.S. Dimeric ristocetin flocculates proteins, binds to platelets, and mediates von Willebrand factor‐dependent agglutination of platelets.J Biol Chem. 1991; 266: 8149-55Abstract Full Text PDF PubMed Google Scholar] to precipitate proteins and agglutinate platelets in a VWF‐independent, or HMW‐VWF less‐dependent, manner, we were somewhat skeptical that the improved sensitivity reported for VWF:RCo(m) was not at the cost of assay ‘specificity’. That is, we were concerned that improved detection and discrimination of severe type 1 vs. type 3 (i.e. improved LOD performance) would come at the cost of poorer discrimination of type 1 vs. type 2 VWD. Indeed, no evidence was provided against this concern in the original poster report [1Stadler M. Hillarp A. Haderer C. Zapfl C. Pock K. Weinberger J. Roemisch J. Improved automated VWF:RCo assay for VWF‐FVIII concentrates and plasma samples by adjustment of assay reagents, composition and performance parameters.J Thromb Haemost. 2009; 7Google Scholar], although some limited VWD case data (7× type 2 and 7× type 3) were subsequently provided in the full report [2Hillarp A. Stadler M. Haderer C. Weinberger J. Kessler C.M. Römisch J. Improved performance characteristics of the von Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol.J Thromb Haemost. 2010; 8: 2216-23Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. Accordingly, we have evaluated the relative performance of the modified [VWF:RCo(m)] vs. standard automated assay [VWF:RCo(s)] using a BCS analyzer (Siemens, Sydney, NSW, Australia) and a range of samples from individuals with VWD. In brief, the two methodologies were compared using samples from (i) type 3 VWD [n = 4, plus a commercial VWFDP (Affinity Biologicals, Ancaster, ON, Canada)], (ii) type 1 VWD [n = 17, comprising eight ‘severe’ type 1 (VWF levels < 16 U dL−1) and nine ‘moderate/mild’ type 1 (VWF levels 16–35 U dL−1)], (iii) ‘possible’ or ‘borderline’ type 1 VWD (positive clinical bleeding history but VWF levels 36–65 U dL−1; n = 11), and (iv) type 2A VWD (n = 15), and (v) type 2B VWD (n = 4). The present study was performed as a diagnostic testing quality assurance project, utilizing either stored samples frozen from previous clinical investigations (‘retrospective study’), or samples recently referred to us for testing (‘prospective study’). VWD diagnosis and typing was performed according to current guidelines [7Sadler J.E. Budde U. Eikenboom J.C.J. Favaloro E.J. Hill F.G.H. Holmberg L. Ingerslev J. Lee C.A. Lillicrap D. Mannucci P.M. Mazurier C. Meyer D. Nichols W.L. Nishino M. Peake I.R. Rodeghiero F. Schneppenheim R. Ruggeri Z.M. Srivastava A. Montgomery R.R. et al.Working Party on von Willebrand Disease ClassificationUpdate on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor.J Thromb Haemost. 2006; 4: 2103-14Crossref PubMed Scopus (921) Google Scholar] and as previously extensively outlined [8Favaloro E.J. Thom J. Patterson D. Just S. Dixon T. Koutts J. Baccala M. Rowell J. Baker R. Desmopressin therapy to assist the functional identification and characterisation of von Willebrand disease: differential utility from combining two (VWF:CB and VWF:RCo) von Willebrand factor activity assays?.Thromb Res. 2009; 123: 862-8Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar]. VWF:Ag and VWF collagen binding (VWF:CB) activity testing was also performed on the same samples using our standard assay protocols [8Favaloro E.J. Thom J. Patterson D. Just S. Dixon T. Koutts J. Baccala M. Rowell J. Baker R. Desmopressin therapy to assist the functional identification and characterisation of von Willebrand disease: differential utility from combining two (VWF:CB and VWF:RCo) von Willebrand factor activity assays?.Thromb Res. 2009; 123: 862-8Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 9Favaloro E.J. Evaluation of commercial von Willebrand factor collagen binding assays to assist the discrimination of types 1 and 2 von Willebrand disease.Thromb Haemost. 2010; 104: 1009Crossref PubMed Scopus (53) Google Scholar] on the same day as VWF:RCo testing. Our VWF:CB assay has been extensively validated for its sensitivity to HMW VWF, as previously reviewed [3Favaloro E.J. An update on the von Willebrand factor collagen binding (VWF:CB) assay: 21 years of age and beyond adolescence, but not yet a mature adult.Semin Thromb Hemost. 2007; 33: 727-44Crossref PubMed Scopus (84) Google Scholar] and most recently reported [9Favaloro E.J. Evaluation of commercial von Willebrand factor collagen binding assays to assist the discrimination of types 1 and 2 von Willebrand disease.Thromb Haemost. 2010; 104: 1009Crossref PubMed Scopus (53) Google Scholar]. VWF:RCo(m) was performed as per the previous reports [1Stadler M. Hillarp A. Haderer C. Zapfl C. Pock K. Weinberger J. Roemisch J. Improved automated VWF:RCo assay for VWF‐FVIII concentrates and plasma samples by adjustment of assay reagents, composition and performance parameters.J Thromb Haemost. 2009; 7Google Scholar, 2Hillarp A. Stadler M. Haderer C. Weinberger J. Kessler C.M. Römisch J. Improved performance characteristics of the von Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol.J Thromb Haemost. 2010; 8: 2216-23Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar], using an equivalent final ristocetin concentration, except that we utilized Helena ristocetin (Catalogue no. 5370; Helena Laboratories, Melbourne, FL, Australia). We used Standard Human Plasma (Siemens) as a calibrator for all assays, and ran an additional low‐level type 1 VWD‐like control [Coagulation Control A (COAGA); Technoclone, Vienna, Austria] with all assays. Summary data are shown in Fig. 1. As expected, in linear regression analysis our VWF:CB assay showed only limited correlation with VWF:Ag (Fig. 1A), driven mostly by the type 1 VWD samples, with divergence driven by type 2 VWD samples. There was better correlation of VWF:CB with VWF:RCo(s), which improved further with VWF:RCo(m). In confirmation of the previous reports [1Stadler M. Hillarp A. Haderer C. Zapfl C. Pock K. Weinberger J. Roemisch J. Improved automated VWF:RCo assay for VWF‐FVIII concentrates and plasma samples by adjustment of assay reagents, composition and performance parameters.J Thromb Haemost. 2009; 7Google Scholar, 2Hillarp A. Stadler M. Haderer C. Weinberger J. Kessler C.M. Römisch J. Improved performance characteristics of the von Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol.J Thromb Haemost. 2010; 8: 2216-23Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar], VWF:RCo(m) showed superior VWF LOD sensitivity compared with VWF:RCo(s) (see VWFDP, type 3 VWD, severe type 1 VWD and type 2A & 2B VWD data; Fig. 1B,C). This facilitated better discrimination of type 3 from severe type 1 VWD, as VWF:RCo(s) yielded similar values for both VWD types, with VWF LOD around 10–15 U dL−1, whereas VWF:RCo(m) showed better VWF LOD at around 3 U dL−1. The VWF LOD for our VWF:Ag and VWF:CB assays is < 2 U dL−1 (Fig. 1B,C). VWF:RCo(m) also permitted better discrimination of type 1 from types 2A and 2B VWD (Fig. 1C,D), with greater and more consistent VWF functional discordance, and lower RCo/Ag ratios, evident in type 2 VWD. Our typical cut‐off VWF:CB/VWF:Ag (CB/Ag) ratio value for discriminating type 1 vs. HMW VWF deficient types 2A/2B VWD is around 0.6–0.7 [3Favaloro E.J. An update on the von Willebrand factor collagen binding (VWF:CB) assay: 21 years of age and beyond adolescence, but not yet a mature adult.Semin Thromb Hemost. 2007; 33: 727-44Crossref PubMed Scopus (84) Google Scholar, 8Favaloro E.J. Thom J. Patterson D. Just S. Dixon T. Koutts J. Baccala M. Rowell J. Baker R. Desmopressin therapy to assist the functional identification and characterisation of von Willebrand disease: differential utility from combining two (VWF:CB and VWF:RCo) von Willebrand factor activity assays?.Thromb Res. 2009; 123: 862-8Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 9Favaloro E.J. Evaluation of commercial von Willebrand factor collagen binding assays to assist the discrimination of types 1 and 2 von Willebrand disease.Thromb Haemost. 2010; 104: 1009Crossref PubMed Scopus (53) Google Scholar], and this cut‐off permitted complete discrimination for the data set evaluated in the present study (Fig. 1D). However, although RCo(s)/Ag ratios with the same sample data set showed some overlap between types 1 and 2A/2B VWD (mostly as a result of LOD sensitivity issues highlighted previously), RCo(m)/Ag ratios permitted complete discrimination, similar to CB/Ag test data (Fig. 1D). In conclusion, we report additional validation of the recently reported modified VWF:RCo(m) assay [1Stadler M. Hillarp A. Haderer C. Zapfl C. Pock K. Weinberger J. Roemisch J. Improved automated VWF:RCo assay for VWF‐FVIII concentrates and plasma samples by adjustment of assay reagents, composition and performance parameters.J Thromb Haemost. 2009; 7Google Scholar, 2Hillarp A. Stadler M. Haderer C. Weinberger J. Kessler C.M. Römisch J. Improved performance characteristics of the von Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol.J Thromb Haemost. 2010; 8: 2216-23Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. We have now incorporated this modified assay into our diagnostic practice, performing the standard VWF:RCo(s) assay initially, and then repeating all samples yielding values below 30% with the modified VWF:RCo(m) assay. In practice, this is like performing a high‐ and low‐factor assay curve, with the high curve suitable for the bulk of testing, and the low curve applied selectively. Notably, the VWF:RCo(m) assay is only applied to some 10% of all VWF:RCo tests, as the bulk of routinely tested samples yield normal VWF test results in our diagnostic practice. We will continue to monitor the assay behaviour prospectively, and we encourage other laboratories to also investigate its suitability within their own diagnostic practice. The authors state that they have no conflict of interest.