From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., “Coagulation.” In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.
Related Information
Synonyms Multimer Assay; Ristocetin Cofactor; Ristocetin-Induced Platelet Aggregation Assay; von Willebrand Factor Antigen; von Willebrand Factor Assay; von Willebrand Factor Collagen-Binding Assay; von Willebrand Factor Multimer Assay
Applies toTest Includes Assays for von Willebrand factor (vWF) activity (ristocetin cofactor), vWF antigen, and factor VIII should be ordered. If indicated by these results, a vWF multimer analysis and/or low-dose ristocetin aggregation assay can be ordered.
Abstract von Willebrand factor (vWF) mediates platelet adhesion to injured endothelium, the first step in hemostasis. It also helps maintain factor VIII levels. When vWF is deficient, patients have a bleeding disorder called von Willebrand disease (vWD). vWD is the most common hereditary bleeding disorder, of which several subtypes are recognized (see below).
Specimen Plasma
Container Three blue top (sodium citrate) tubes
Collection Routine venipuncture. Deliver tubes to laboratory immediately, otherwise falsely low factor VIII values may occur (factor VIII is labile). If multiple tests are being drawn, draw blue top tubes after any red top tubes but before any lavender top (EDTA), green top (heparin), or gray top (oxalate/fluoride) tubes. Immediately invert tubes gently at least 4 times to mix. Tubes must be appropriately filled.
Storage Instructions Separate plasma from cells as soon as possible. Plasma can be stored for 2 hours on ice, otherwise store frozen. For vWF antigen only, plasma can be stored for 8 hours at room temperature or 24 hours on ice, otherwise store frozen.
Causes for Rejection Specimen received more than 4 hours after collection, tubes not filled, clotted specimens
Turnaround Time Several days (longer if follow-up testing is needed, such as multimer analysis)
Reference Interval Varies with blood type through an unknown mechanism. Results are reported as a percent of the amount expected in normal plasma. By definition, the mean value in pooled normal plasma is 100%. In a large study of normal persons, the mean vWF level was 75% in blood type O, 106% in type A, 117% in type B, and 123% in type AB individuals. The overall mean vWF level was 100%.1 Newborns have higher vWF levels than do adults. Values for vWF gradually decrease into the adult normal range by age 6 months.2
Use Determine if a patient with a personal or family history of bleeding has von Willebrand disease (vWD); assist in determining hemophilia A carrier status in females
Methodology
Initial tests:
The ristocetin cofactor assay assesses vWF function by measuring ristocetin-mediated binding of vWF to platelet GPIb, which leads to platelet agglutination.3 The ristocetin cofactor assay is performed by mixing patient plasma with ristocetin and formalin-fixed normal platelets and measuring the amount of platelet agglutination in an aggregometer. Note: The term “agglutination” is often used to describe ristocetin-induced platelet aggregation, because true platelet aggregation links platelets through fibrinogen and GPIIb/IIIa, whereas ristocetin links platelets through von Willebrand factor and GPIb. The von Willebrand factor antigen assay is an enzyme-linked immunosorbent assay (ELISA), which measures the quantity of vWF, independent of vWF function. An alternative automated assay involves latex particles coated with antibodies directed against vWF. In the presence of vWF, the latex particles form aggregates that absorb light passing through the specimen. The amount of light absorbance is directly related to the amount of vWF in the specimen. Rocket immunoelectrophoresis is an older antigen assay that is still in use in some laboratories. The factor VIII assay is a PTT-based clotting assay which measures factor VIII activity.
More recently, alternative immunoassays have been designed to assess vWF function.4 The collagen-binding assay is an ELISA in which collagen is the antigen. If vWF is functional, it binds to collagen and is subsequently detected. Another vWF “functional” ELISA uses monoclonal antibodies that recognize a functional epitope on vWF, but there is conflicting evidence whether this test correlates better with ristocetin cofactor or with vWF antigen. These newer functional assays are not yet as established as the ristocetin cofactor assay.
Follow-up tests (performed if indicated, see table):
Multimer analysis is performed when type 2 vWD is suspected.5 A plasma sample is electrophoresed on a gel to separate the multimers by size. The multimers are then visualized using 125I-labeled anti-vWF antibody or other techniques. Low-dose ristocetin platelet aggregation assay is performed when type 2B vWD is suspected.6 This test is similar to the ristocetin cofactor assay, except that the patient’s own platelets are used instead of normal platelets, and lower doses of ristocetin are used. The patient’s own platelets and plasma are mixed with ristocetin, and platelet aggregation is measured in an aggregometer. This assay is less sensitive than the ristocetin cofactor assay for diagnosing vWD, but it is useful for confirming a diagnosis of type 2B vWD. Type 2B patients’ platelets become abnormally coated with vWF in vivo, due to increased affinity of the mutant vWF for platelet GPIb. As a result, the patient’s platelets show increased aggregation in this assay. Platelet-type vWD also shows increased aggregation in this assay, due to a mutation on platelet GPIb which increases its affinity for vWF. In contrast, other types of vWD may show decreased ristocetin-induced platelet aggregation due to decreased vWF quantity and/or function. Further specialized coagulation testing can be performed to distinguish type 2B from platelet-type vWD.
Additional Information von Willebrand disease (vWD) is the most common hereditary bleeding disorder, occurring in up to 1% of the general population.7,8 Many cases remain undiagnosed because of the mild nature of bleeding in many patients and the fact that acute phase reactions can mask the diagnosis. vWF is a polypeptide synthesized in endothelial cells and megakaryocytes, which polymerizes to form multimers containing up to 100 subunits. Bleeding symptoms resemble those of a platelet function defect, since platelet adhesion is impaired. Thus, the most common symptoms are epistaxis, easy bruising, bleeding with dental extractions, and menorrhagia.
Laboratory testing for vWD is summarized in the table. Repeat testing is often required, because both vWF and factor VIII become elevated above baseline during acute phase reactions (including even minor illnesses, injury, or stress), pregnancy, estrogen use, or in newborns. An elevation of a low or borderline value for vWF into the normal range during any of these conditions often masks the diagnosis of vWD. Measurement of an acute phase reactant such as fibrinogen is helpful in assessing the likelihood that a patient is in an acute phase reaction at the time of testing.
vWF serves as the carrier protein for factor VIII, and levels of factor VIII are often decreased when vWF is decreased. When vWF is markedly decreased, the factor VIII level can also become very low, which prolongs the PTT. In most vWD patients, the disease is mild or moderate and the PTT is therefore normal.
Many variants of vWD have been described, but the classification scheme has recently been simplified into three types (see table).9 Type 1 is by far the most common form, accounting for the majority of cases. Type 1 vWD is characterized by a partial quantitative deficiency of vWF. Although the quantity of vWF is reduced, the function of the individual vWF molecules which are synthesized is normal.
Interpretation of von Willebrand Factor Assays
RCoF + vWF Ag + FVIII + Fibrinogen
(or other acute phase reaction marker):
* Normal:* vWD unlikely if no acute phase reaction, pregnancy, estrogen use, newborn
* Normal* but fibrinogen (or factor VIII) elevated: repeat vWF assays when fibrinogen and factor VIII are normal
* RCoF, vWF Ag, FVIII reduced to a similar extent: type 1 vWD likely
* RCoF, vWF Ag, FVIII severely reduced (<10%) or undetectable: type 3 vWD likely * RCoF reduced more severely than vWF Ag and FVIII:** consider type 2 vWD (2A, 2B, or 2M); perform multimer analysis and low-dose ristocetin cofactor to determine subtype: - Multimer analysis normal: type 2M likely (subtle abnormalities in some variants) - Multimer analysis missing high molecular weight multimers: type 2A likely - Multimer analysis missing high and intermediate molecular weight multimers: type 2B or platelet type likely - Increased low-dose ristocetin aggregation: type 2B or platelet type*** - Normal or decreased low-dose ristocetin aggregation: not type 2B or platelet type * FVIII reduced (5% to 40%), RCoF and vWF Ag normal:** consider type 2N vWD; or in males, mild hemophilia A. In female hemophilia A carriers, factor VIII is approximately 50% with large variability. Consider also factor VIII degradation if prolonged specimen transportation. *Consider blood type when determining if values are normal. **Mean RCoF:vWF Ag ratio is 0.3 for type 2A, 0.6 for type 2B, and uncertain (<1) for type 2M. Mean FVIII:vWF Ag ratio is 0.28 for type 2N (see Footnote 13). ***Thrombocytopenia may occur with type 2B or platelet-type (and rare type 2A variants). RCoF = ristocetin cofactor assay; vWF Ag = von Willebrand factor antigen assay; FVIII = factor VIII assay Type 2 vWD is characterized by qualitative (functional) deficiencies of vWF. Often the quantity of vWF is also reduced. Type 2 vWD is further subdivided into four categories (see table). Type 2A and type 2B are characterized by a loss of high molecular weight multimers of vWF. The highest molecular weight multimers have more hemostatic function than the lower molecular weight multimers. Therefore, in these disorders, the overall function relative to the quantity of vWF molecules is reduced. Thus, the functional assay (ristocetin cofactor) result is reduced more than the quantitative assay (von Willebrand factor antigen). In type 2A, the loss of high molecular weight multimers is due to defective multimer assembly and secretion or increased proteolysis of multimers.10,11 Type 2B vWD mutations lead to increased binding of vWF to GPIb, the platelet vWF receptor.6 Platelets coated with vWF are cleared from the bloodstream at an increased rate, leading to loss of high molecular weight multimers as well as thrombocytopenia. Platelet-type or pseudo-vWD is a rare disorder in which a mutation in the platelet GPIb gene (not the vWF gene) leads to increased binding of vWF to GPIb, resulting in the same findings described above for type 2B vWD.12 Types 2M and 2N vWD are rare subtypes of type 2 vWD. Type 2M vWF mutations cause decreased function despite the presence of normal-sized multimers, often because the mutation impairs the ability of vWF to bind to platelet GPIb.9,13 In type 2N (Normandy) vWD, the factor VIII-binding ability of vWF is impaired, and the half-life of factor VIII is consequently shortened. Thus, vWF is normal in quantity (normal antigen assay) and has normal platelet-adhesion function (normal ristocetin cofactor assay), but factor VIII levels are decreased. As a result, type 2N patients are frequently misdiagnosed as having hemophilia A.14 The family history may be useful in distinguishing type 2N vWD from hemophilia A. Type 2N vWD is inherited autosomally (males and females are affected), whereas hemophilia A is an X-linked recessive disorder (males are affected and females are carriers). An assay which measures the ability of vWF to bind factor VIII is available in a limited number of specialized laboratories. Type 2N patients show decreased binding of factor VIII in this assay. Type 3 vWD is a rare, severe bleeding disorder characterized by a severe quantitative deficiency of vWF such that vWF is typically undetectable. The bleeding time is often prolonged in vWD. However, it is neither a necessary nor a reliable test for diagnosis. In hemophilia A carriers (who are females only), the factor VIII:vWF ratio is ~0.5, instead of the normal ratio of 1. Definitive determination of carrier status may require DNA-based testing for mutations that cause hemophilia A. Bleeding episodes, in most patients, can be treated with DDAVP (desmopressin) if needed, as DDAVP temporarily increases the levels of vWF and factor VIII two- to threefold. As a small percentage of patients do not respond to DDAVP, patients are usually given a trial dose of DDAVP while asymptomatic, with measurement of their vWF level before and after DDAVP, to ensure that their vWF levels do increase with DDAVP. Bleeding patients who do not respond to DDAVP or patients with severe vWD can be treated with vWF-containing factor VIII concentrates (eg, Humate-P®, Alphanate®, Koate®). Some consider DDAVP contraindicated in type 2B because it can cause thrombocytopenia. However, others report DDAVP is a beneficial treatment for type 2B patients. Acquired vWD is a rare condition that can occur spontaneously or in association with a variety of underlying disorders, such as hematologic neoplasms or autoimmune diseases. Thrombotic thrombocytopenic purpura (TTP) is due to a deficiency of a vWF-cleaving protease, usually due to an autoantibody against the protease.15,16 This could account for the microvascular platelet-rich thrombi and thrombocytopenia that are characteristic of TTP. Unusually large vWF multimers may also be seen in TTP. Footnotes
1. Gill JC, Endres-Brooks J, Bauer PJ, et al, “The Effect of ABO Blood Group on the Diagnosis of von Willebrand’s Disease,”Blood, 1987, 69(6):1691-5.
2. Andrew M, Paes B, and Johnston M, “Development of the Hemostatic System in the Neonate and Young Infant,”Am J Pediatr Hematol Oncol, 1990,12(1):95-104.
3. Weiss HJ, Hoyer LW, Rickles FR, et al, “Quantitative Assay of a Plasma Factor Deficient in von Willebrand’s Disease That Is Necessary for Platelet Aggregation. Relationship to Factor VIII Procoagulant Activity and Antigen Content,”J Clin Invest, 1973, 52:2708-16.
4. Favaloro EJ, “Collagen Binding Assay for von Willebrand Factor (VWF:CBA): Detection of von Willebrand’s Disease (VWD), and Discrimination of VWD Subtypes, Depends on Collagen Source,”Thromb Haemost, 2000, 83(1):127-35.
5. Ruggeri ZM and Zimmerman TS, “Variant von Willebrand’s Disease: Characterization of Two Subtypes by Analysis of Multimeric Composition of Factor VIII/von Willebrand Factor in Plasma and Platelets,”Blood, 1980, 65(6):1318-25.
6. Ruggeri ZM, Pareti FI, Mannucci PM, et al, “Heightened Interaction Between Platelets and Factor VIII/von Willebrand Factor in a New Subtype of von Willebrand’s Disease,”N Engl J Med, 1980, 302:1047-51.
7. Rodeghiero F, Castaman G, and Dini E, “Epidemiological Investigation of The Prevalence of von Willebrand’s Disease,”Blood, 1987, 69(2):454-9.
8. Werner EJ, Broxson EH, Tucker EL, et al, “Prevalence of von Willebrand Disease in Children: A Multiethnic Study,”J Pediatr, 1993, 123:893-8.
9. Sadler JE, “A Revised Classification of von Willebrand Diseases,”Thromb Haemost, 1994, 71:520-5.
10. Lyons SE, Bruck ME, Bowie EJ, et al, “Impaired Intracellular Transport Produced by a Subset of Type IIA von Willebrand Disease Mutations,”J Biol Chem, 1992, 267:4424-30.
11. Nichols WC, Seligsohn U, Zivelin A, et al, “Mutations in the ER-Golgi Intermediate Compartment Protein ERGIC-53 Cause Combined Deficiency of Coagulation Factors V and VIII,”Cell, 1998, 93(1):61-70.
12. Miller JL, “Platelet-Type von Willebrand’s Disease,”Thromb Haemost, 1996, 75(6):865-9.
13. Meyer D, Fressinaud E, Gaucher C, et al, “Gene Defects in 150 Unrelated French Cases With Type 2 von Willebrand’s Disease: From the Patient to the Gene,”Thromb Haemost, 1997, 78(1):451-6.
14. Mazurier C, “von Willebrand’s Disease Masquerading as Haemophilia A,”Thromb Haemost, 1992, 67:391-6.
15. Furlan M, Robles R, Galbusera M, et al, “von Willebrand Factor-Cleaving Protease in Thrombotic Thrombocytopenic Purpura and the Hemolytic-Uremia Syndrome,”N Engl J Med, 1998, 339(22):1578-84.
16. Tsai HM and Lian EC, “Antibodies to von Willebrand Factor-Cleaving Protease in Acute Thrombotic Thrombocytopenic Purpura,”N Engl J Med, 1998, 339(22):1585-94.
References
Ewenstein BM, “von Willebrand’s Disease,”Annu Rev Med, 1997, 48:525-42.
Ginsberg D and Sadler JE, “von Willebrand’s Disease: A Database of Point Mutations, Insertions and Deletions,”Thromb Haemost, 1993, 69:177-84.
