David L. McGlasson, MS, MLS(ASCP)
Posted: August 20, 2019
The laboratory work up of von Willebrand Disease (vWD) involves determining the level of VWF by both functional and antigenic methods. Per recommendations from the National Heart Lung and Blood Institute (NHLBI) that were developed by a working group in 2008, initial testing for VWD should include a: 1) VWF Ristocetin Cofactor assay VWF:RCo (or equivalent), 2) VWF antigen assay (VWF:Ag), and 3) Coagulation FVIII activity assay (FVIII). Second tier specialized testing should include multimeric analysis, the von Willebrand Factor collagen binding assay (VWF:CBA), ristocetin induced platelet aggregation (RIPA), platelet VWF assessments, genetic sequencing, FVIII binding assessment and the VWF propetide assay (VWF:pp).
Despite these testing guidelines, a number of variables can affect the accuracy of VWF testing in general and this in turn may impact result interpretation and VWD diagnosis.
One such variable is that the results of screening tests (bleeding time, PFA-100®, and APTT) can be normal in patients with type 1 VWD 1,2,3. Typically, these patients have VWF:Ag levels of approximately 45–55%, usually producing normal screening test results. Therefore, these screening tests should not be relied upon for patients with significant bleeding histories. In these cases specific assays (e.g. von Willebrand Factor activity (VWF:A) and VWF:Ag)) are warranted.
Another variable is the fact that due to stress an endogenous release of VWF stored in the Weible-Palade bodies of the endothelium can result in a transient increase in circulating VWF antigens. Thus patients with mild type 1VWF can appear to have falsely elevated antigen levels particularly if they, for example, have undergone a difficult blood draw at sample collection. In fact, the stress of a difficult blood draw, especially in anxious young children, can double or triple the VWF level, making it impossible to rule out VWD without repeated testing.4 For optimal sample collection, the patient should avoid exercise, stress, caffeine, and be as calm as possible before the blood is collected..
The third variable is a technical one involving the processing of the citrated sample. Samples that are not centrifuged properly at 1500 g for 5–10 minutes can have residual platelets in the plasma as high as 30,000 to 40,000/µL. Freezing this plasma releases platelet proteases, altering VWF multimeric structure and resulting in an apparent increase in the VWF antigen level and decrease in the VWF activity.59 Only citrated PPP samples with platelet counts less than 10,000/µL should be used for von Willebrand testing.
The fourth variable is the reference standard used in the clinical laboratory, which can affect the various assays for VWF. Clinicians often use the ratio of VWF:Ag to VWF:A (or vice versa) to subclassify type 2 variants. Therefore, it is imperative that both of the assays be calibrated using a reference plasma traceable to the WHO standard or equivalent.
The fifth variable relates to known associations that result in higher VWF antigen levels. One such association is the influence of blood type on VWF plasma level. Individuals with blood group AB have a 60–70% higher level of VWF than those with blood group O. As a result, some laboratories interpret VWF levels using specific reference intervals for blood typesi 2.
A variety of clinical disorders, including pregnancy, inflammatory disease, and use of oral contraceptives, also can cause increased levels of VWF.3,4 Transient elevations in VWF levels could mask actual low or borderline levels.
The treatment for type 1 VWD would include VWF replacement therapy, but alternatively desmopressin (desamino-D-arginine vasopression [DDAVP]) or Stimate may be used, which causes a transient two- to fivefold increase in VWF plasma level in most patients with type 1 disease. 3,4 A DDAVP or Stimate challenge is performed for VWD:1 patients prior to assessing their response. Baseline citrated plasma samples, 30 minutes and 4 hours post-treatment (DDAVP is infused, whereas Stimate is sprayed into nasal passages). The samples are assayed for FVIII:C, VWF:Ag, and VWF:A. PFA can be used to measure the efficacy of DDAVP or Stimate treatment, but has variable response to specific factor replacement therapy.5,6 Care must be taken in handling, centrifuging, and labeling these timed samples. Patients with no apparent increase in VWF after challenge or have increased VWF clearance may not be suitable candidates for this therapy. Patients with rapid VWF clearance should be assessed for VWF-pp to rule out Vicenza type VWD7,8,9.
* Relationship between Blood Groups and von Willebrand Factor Level
|Blood Groups||von Willebrand factor level (%)|
von Willebrand Factor Activity (VWF:A)/Ristocetin cofactor (VWF:RCo) assay
Ristocetin was used as an antibiotic until the early 1970s when it was recognized to cause thrombocytopenia in normal individuals though not in certain individuals with bleeding problems. It is now used in the VWF:RCo assay however since ristocetin is derived from a fungus it is important to know that this material is highly variable and responses between different lots of reagent are not always reproducible. Traditional aggregometers or automated coagulation analyzers capable of measuring light scattering changes are usually the instruments of choice for measuring VWF:RCo. The test principle is based on the fact that in the presence of ristocetin and normal exogenous formalin-fixed platelets, VWF from patient plasma induces agglutination of the platelets. The slope of the agglutination is plotted versus the percent VWF activity, and results are extrapolated from a calibration curve determined from a reference (standard) curve. Other modifications of the assay use the time elapsed expressed in millimeters on the graph instead of slope of agglutination. The control and each patient sample are tested in duplicate and at two separate dilutions (aggregometry method), with the resulting slopes or rates calculated from the reference curve; the results are averaged and reported as a percentage of activity of VWF:RCo. Note that the VWF:RCo assay is very time consuming, labor intensive, and has a high coefficient of variation (CV), though all of this has been improved with the use of automated analyzers performing the assay. 10,11
VWF:Activity using ristocetin independent methods
Newer assays that measure von Willebrand activity independent of ristocetin include latex particle-enhanced immunoturbidometric assay (LIA) and chemiluminescent methods using automated instruments that reduce the variability associated with ristocetin and manual testing. For LIA based methods, latex particles contain either a specific anti-VWF monoclonal antibody directed against the functional GPIb binding site on VWF or mutated recombinant GPIb fragments12,13 which react with the VWF in patient plasma. The degree of agglutination is directly proportional to the activity of VWF in the sample and it is determined by measuring the decrease of transmitted light caused by the aggregates. These ristocetin independent methods also may not be affected by single nucleotide polymorphisms (SNPs) that are common in people of African descent who tend to present as having VWD when relying on VWF:RCo assays13 .
The general reference interval for VWF:A is 60–150%. VWF:A is usually decreased in plasma from patients with all three types of VWD and the platelet-type pseudo-VWD type 3 has undetectable levels of VWF and FVIII.
Normal fluctuations occur with the diagnostic tests just discussed. Large variations occur in test results in normal individuals as well, emphasizing the need for commercial reference plasmas to be standardized against international standards when preparing the standard curve for all VWF testing.
The laboratory professional should understand the test results regarding hemophilia versus VWD and be able to evaluate each patient’s test results, repeat them, and perform verifications as needed.
von Willebrand Factor Antigen Assay (VWF:Ag)
As with clinical testing for most bleeding disorders, the antigenic measurement for VWF protein was available before functional assays. Zimmerman first used the “rocket” immunoelectrophoresis technique of Laurell14,15,16 to measure VWF:Ag. This assay was time consuming while measuring and calculating decreased levels of vWF was difficult.
Solid phase methods currently used for measuring VWF:Ag include ELISA assays or automated, immunoturbidimetric assays. The latex immunoassay (LIA) test procedure coats microlatex particles with rabbit antihuman VWF antibodies, producing agglutination when mixed with plasma containing VWF. The extent of agglutination (turbidity) correlates to the level of VWF:Ag present in the citrated plasma sample. The methodology is reliable, easy to automate, and produces results in a timely manner. However, it has a limited lower limit of quantitation and may not be able to distinguish severe forms of type I from type 3 VWD.
von Willebrand Factor (VWF) Multimer Analysis
The availability of multimeric analysis has improved diagnosis and treatment of VWD patients. VWF in normal plasma exists as multimers ranging in size from dimers of approximately 600,000 daltons (Da) to very large multimers of up to 20 million Da. Plasma electrophoresis using low concentrations of agarose (0.65%) or SDS-PAGE and staining with radiolabeled antibody against VWF allows the full range of multimers to be visualized. 15,16
Because these multimeric analyses are difficult to perform and interpret and require a very pure and specific antibody, specialized reference laboratories or coagulation centers usually perform VWF multimeric analyses.
Collagen-Binding Assays for von Willebrand Disease (VWD)
Collagen-binding assays (VWF:CB) are specialized VWF activity assays that are used to differentiate VWD type 2A and 2B from type 2M. Performing both VWF:CB and RCoF assays increases the ability to differentiate VWD type 2 variants (discrimination of VWD types 2A or 2B from type 2M). Specifically, all three subtypes (2A, 2B, 2M) give abnormal RCoF results, but only types 2A and 2B give abnormal VWF:CB results. Type 2M patients have normal VWF:CB. 17,18,19
Note that while the VWF:CB assays were initially thought to be replacements for the VWF:RCoF assay –due to their low variability and because both tests are sensitive to a reduction in high-molecular-weight multimers of VWF –they are best used in conjunction with the VWF:RCoF assay.
Von Willebrand Factor ProPeptide (VWFpp)
The increased clearance of VWF from plasma can be responsible for low VWF levels (i.e. type 1 Vicenza VWD). VWF and its propeptide (VWFpp) are released into plasma in equimolar amounts but have very different half-lives (8 – 12 hours and 2-3 hours, respectively). An increased VWFpp to VWF:Ag ratio (VWFpp/VWF:Ag, > 3)has been described in both VWD and in acquired von Willebrand syndrome (AVWS). This finding indicates that measuring VWFpp can be useful in assessing VWF synthesis, secretion, and clearance and it may even be necessary when information on family history of the disorder is not available. Reduced VWFpp levels indicate reduced VWF synthesis or secretion. An increased VWFpp/VWF:Ag ratio is expected when VWF is cleared rapidly from plasma (type 1 Vicenza), while decreased VWF secretion/intracellular retention results in a normal ratio. Therefore using a VWFpp ELISA combined with the VWF:Ag can help in distinguishing between patients with an elevated increase in VWF clearance (as in AVWS or VWD type 1 Vicenza) from VWD patients with a moderate increased clearance that is seen in the majority of VWD subjects20,21.
VWD is a hugely complex disorder that requires complex testing. Some of the methods are not available in the United States and are only performed in a few reference laboratories. This presents a problem for the laboratories that deal in regular or special coagulation complex testing. Expense and expertise have to be taken into consideration when performing these assays. Interpretation of these assays also requires a lot of knowledge into the diagnosis of these disorders.
Studies are still ongoing into these issues.
REFERENCES FOR VON WILLEBRANDS DISEASE FOR CLOT CLUB
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2 Schlammadinger A., Kerenyi A., Muszbek .L., & Boda Z. (2000). Comparison of the O’Brien filter test and the PFA-100 platelet analyzer in the laboratory diagnosis of von Willebrand’s disease. Thrombosis and Haemostasis.84,88–92.
3 Cariappa R., Wilhite T.R., Parvin C.A., & Luchtman-Jones L. (2003). Comparison of PFA-100 and bleeding time testing in pediatric patients with suspected hemorrhagic problems. Journal of Pediatric Hematology and Oncology. 25, 474–479.
4 Abildgaard C.F., Suzuki Z., Harrison J., Jefcoat K., & Zimmerman T.S. (1980). Serial studies in von Willebrand’s disease: variability versus “variants.” Blood. 56, 712–16.
5 Sousa N.C., Anicchino-Bizzacci J.M., Locatelli M.F.., Castro V. &, Barjas-Castro M.L. (2007). The relationship between ABO groups and subgroups factor VIII and von Willebrand factor. Haematologica. 92, 236-39.
6 Montgomery R.R., Gill J.C., Scott J.P. (2003). Hemophilia and von Willebrand disease. In: Nathan DG, Ginsburg D, Look AT et al., eds. Nathan and Oski’s Hematology of Infancy and Childhood, 6th ed. Philadelphia: W. B. Saunders.
7 Fressinaud E., Veyradier A., Sigaud M., Boyer-Neumann C., Le Boterff C., & Meyer D. (1999). Therapeutic monitoring of von Willebrand disease: interest and limits of a platelet function analyser at high shear rates. British Journal of Haematology. 106, 777-783.
8 Patzke J., Budde U., Huber A., Méndez A., Muth H., Obser T, . . . Schneppenheim R. (2014). Performance evaluation and multicentre study of a von Willebrand factor activity assay based on GPIb binding in the absence of ristocetin. Blood Coagulation and Fibrinolysis. 25, 860-870.
9 Flood V.H., Gill J.C., Morateck P.A, Christopherson P.A., Friedman K.D., HaberichterS.L., Branchford BR, . . . Montgomery R.R. (2010). Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor. Blood. 116, 280-286
10 Mannucci P.M., Franchini M., Castaman G., & Federici A.G. (2009).. Italian Association of Hemophilia Centers. Evidence-based recommendations on the treatment of von Willebrand disease in Italy. Blood Transfusion. 7, 117–126.
11 Ewenstein B.M. (2001). Use of ristocetin cofactor activity in the management of von Willebrand disease. Haemophilia. 7(suppl 1), 10–15.
12 Patzke J., Budde U., Huber A., Méndez A., Muth H., Obser T., . . . Schneppenheim R. (2014). Performance evaluation and multicentre study of a von Willebrand factor activity assay based on GPIb binding in the absence of ristocetin. Blood Coagulation and Fibrinolysis. 25, 860-70.
13 Flood V.H., Gill J.C., Morateck P.A.. Christopherson P.A., Friedman K.D., Haberichter S.L., . . Montgomery R.R. (2010). Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor. Blood. 116,:280-286.
14 Favoloro E.J., Bonar R., Kershaw G., Hertzberg M., & Koutts J. (2004). Laboratory diagnosis of von Willebrand’s disorder: quality and diagnostic improvements driven by peer review in a multi-laboratory test process. Haemophilia 10, 232-242.
15 Favaloro E.J., Thom J., Baker R., (2000). Assessment of current diagnostic practice and efficacy in testing for von Willebrand’s disorder: results from the second Australiasian multi-laboratory survey. Blood Coagulation and Fibrinolysis 2000.;11(8):729-37.
16 Laurell C.B. (1966). Quantitative estimation of proteins by electrophoresis on agar gel containing antibodies. Analytical Biochemistry. 14, 45–52.
17 Riddell, A. F., Jenkins, P. V., Nitu-Whalley, I., McCraw A.H., Lee C.A., & Brown S.A. (2002) Use of the collagen-binding assay for von Willebrand factor in the analysis of type 2M von Willebrand disease: a comparison with the ristocetin cofactor assay. British Journal of Haematology. 116, 187–92
18 Hayes T.E., Brandt J.T., Chandler W.L, Eby C.S., Kottke-Marchant K., Krishnan J., . . Cunningham M.T. (2006). External peer review quality assurance testing in von Willebrand disease: the recent experience of the United States College of American Pathologists proficiency testing program. Seminars in Thrombosis and Hemostasis. 32, 499–504.
19 Kitchen S., Jennings I., Woods T.A., Kitchen D.P., Walker I.D., & Preston F.E. (2006). Laboratory tests for measurement of von Willebrand factor show poor agreement among different centers: results from the United Kingdom National External Quality Assessment Scheme for Blood Coagulation. Seminars in Thrombosis and Hemostasis. 32, 492–498.
20 Riddell, A. F., Jenkins, P. V., Nitu-Whalley, I. C., McCraw, A. H., Lee, C. A. & Brown, S. A. (2002), Use of the collagen-binding assay for von Willebrand factor in the analysis of type 2M von Willebrand disease: a comparison with the ristocetin cofactor assay. British Journal of Haematology, 116: 187–92
21 Haberbrichter SL et al: Critical Importance of VWF Propeptide (VWFpp) In The Diagnosis Of Type 1 Von Willebrand Disease (VWD). Blood 2013 122:331.
23 Stufano F et al: Evaluation of the Utility of vonWillebrand and Factor Propeptide in the Differential Diagnosis of von Willebrand disease and Acquired von Willebrand Syndrome.
Parts of this communication have been taken from the upcoming book chapter from McGlasson DL, Gosselin RC. Hemostasis: Laboratory Testing and Instrumentation. Clinical Laboratory Hematology, Pearson 4th ed. Boston, MA. Hemostasis Laboratory Testing and Instrumentation. 2019 in press.