Laboratory Issues in the Identification and Diagnosis of von Willebrand Disease On the 80th Anniversary of Erik von Willebrand's Original Publication

Eighty years ago, Erik von Willebrand published his original description of what is now called von Willebrand disease (vWD). It appears timely that Seminars in Thrombosis and Hemostasis dedicates two issues to this disorder. The present issue focuses on laboratory aspect of the identification of vWD, and the following one on clinical and management issues. While considerable progress has been made in the understanding of this disease, there still remain many uncertainties that will likely be clarified in the next 80 years. A brief historical perspective is offered by Koutts, who emphasizes the development of the test procedures presently in use. When Eric von Willebrand first described this disorder, he termed it “pseudohemophilia” to illustrate its clinical resemblance to hemophilia (at that time hemophilia B had not yet been described), but recognizing the differences from hemophilia with regard to pattern of heredity, prolonged bleeding times and mucocutaneous hemorrhages. Then Rudolf Jürgens, a German clinician living in Switzerland, suggested that the disorder might be a platelet defect because of the prolonged bleeding times. He persuaded von Willebrand to adopt the term “hereditary constitutional thrombocytopathy.” In Europe the disorder then became known as the von Willebrand-Jürgens syndrome. The abnormal bleeding times but normal clot retraction (the only functional platelet test known at that time) led Angloamerican clinicians to propose the term “angiohemophilia” or “vascular hemophilia.” The situation became more complex when, with the introduction of the so-called thromboplastin generation test, low factor (F) VIII levels were found in these patients. Now there were two disorders with low FVIII levels-one autosomal dominant, the other one recessive sex linked. Both presented with different bleeding patterns and with some differences in laboratory tests. The breakthrough came with the development of new test procedures that allowed the measurement of not only clotting factors in plasma, but also some platelet functions. These new tests ultimately allowed the recognition of von Willebrand disease (vWD), distinctly different from hemophilia A. Dr. Koutts expertly traces these developments.

Favaloro next reviews the various test procedures presently in use for diagnosing vWD and its various forms that are different from a molecular perspective. The difficulties encountered with a diagnostic work-up, the limitations of certain tests, and the complexities with which von Willebrand factor (vWF) exerts its various functions are delineated in this article. An entire battery of tests needs to be employed to derive to the proper classification of vWD. However, even with all tests applied, there still remain difficulties and problems. This article very clearly describes the presently available options for a laboratory to attempt to diagnose vWD.

Adcock and coworkers provide a retrospective analysis of the diagnosis of vWD made in 497 patients. Comparisons are reported between test procedures and how they correlate, and which tests are advisable to avoid unnecessary testing on one hand, but also to avoid over- and underdiagnosis and misdiagnosis on the other. The article contains a wealth of useful information.

Kouides discusses the difficulties and pitfalls of diagnosing vWD in women. Levels of vWF vary considerably in women related to menses, pregnancy, oral contraceptive use, and postmenopausal hormonal replacement therapy. Many of these variables are not well understood so that it is difficult at this time to make recommendations as to when the laboratory tests should be performed. In addition, other variables that impact the diagnosis are reviewed.

The next four articles describe efforts from various countries and continents to standardize laboratory tests for the diagnosis of vWD. The first assessment, reported by Meijer and Haverkate, describes the experiences of the European Concerted Action on Thrombosis and Disabilities Foundation. This Foundation has provided external quality control samples since 2003, and 180 laboratories participate in this program. vWF activity and antigen tests are performed. Antigen assays generally provide better agreement between laboratories than activity tests. Although normal samples were correctly identified by most participating laboratories, type 1 vWD samples were incorrectly interpreted by 20 to 40% of the laboratories. Clearly, further improvements are needed.

Similar findings are reported by Kitchen and coworkers from the United Kingdom, where ~200 laboratories participate in the U.K. National External Quality Assessment Program. Again, reasonable agreements were reported for vWF antigen assays, but activity measurements had a coefficient of variation (CV) between 40 and 50%. These authors also find that assays for vWD still are problematic.

Hayes and colleagues describe the results of proficiency testing by the U.S. College of American Pathologists. Also in this program vWF antigen assays performed fairly well, whereas ristocetin cofactor activity assays showed greater variability (CVs between 23 and 30%). The need for more reliable test procedures is stressed.

Favaloro and associates report on the outcomes of the Royal College of Pathologists of Australasia Quality Assurance Program that encompasses laboratories from Australia, New Zealand, and some Asian countries. The experience is similar to that reported for the other programs, but errors can apparently be reduced substantially when test panels are employed, and even better results are seen when vWF:collagen binding activity assays are included. However, major problems are noted with the subclassification of type 2 vWD. These studies clearly indicate that major efforts must be made to standardize tests so that errors in the diagnosis of vWD are reduced.

Budde and coworkers review the issue of multimer analysis of vWF in the classification of the various qualitative and quantitative forms of vWD. The methods used are labor intensive, technically difficult to perform, and subject to reader variability. Yet, experienced readers can analyze > 90% genotype-phenotype relations when a sensitive multimer analysis technique is used. The authors describe the multimeric patterns of all type 2 subclasses. They emphasize, however, that all other test panels should be performed before multimeric analysis is requested.

Hubbard reports on the progress that is being made in establishing a laboratory standard for the diagnosis of vWD and for control of potencies of vWF concentrates. Due to the wide range of normal values for vWF in plasma, such standards should clearly improve the diagnostic and treatment modalities. A World Health Organization International Standard has been developed to assess vWF activity and antigen levels in plasma, but calibration of therapeutic vWF concentrates is much more difficult. It is hoped that future attempts at standardization will yield results that are similar to those available for FVIII concentrates.

Mazurier discusses the vWF concentrates that are presently available for the treatment of vWD. The products vary with regard to protein composition, purity, and amounts of vWF and FVIII, and are not standardized. There appears to be no agreement on what the composition should be. The author offers certain suggestions as to what standard products should be able to accomplish therapeutically.

Favaloro examines the usefulness of the PFA-100 (platelet function analyzer) in the identification of vWD. This system was basically designed to mimic primary hemostasis in vitro. It is not specific for any individual platelet contribution to primary hemostasis, but is a useful screening test for platelet dysfunction, congenital or acquired. Given that lack of platelet adhesion is one of the major defects in patients with vWD, the PFA-100 results will be abnormal in the majority of patients with vWD with a sensitivity of 85 to 90%. A normal PFA-100 result rules out vWD with great likelihood, but an abnormal result is not specific for vWD because many variables, especially the presence of antiaggregating medications (aspirin), influence the test results. Overall however, the system is a useful tool to assess platelet function and it is thus a useful assay as part of a von Willebrand work-up.

In the last article James and Lillicrap describe the role of genetic testing in the diagnosis of vWD. The determination of the genomic DNA not only assists in making the correct diagnosis in type 1 and 3 cases, but is also useful in classifying type 2 forms of vWD. This investigation is in its early stages of development and is part of the Canadian national network of inherited bleeding disorders. The technology appears to be a useful tool to complement the more routine diagnostic modalities for vWD.

I would like to thank all contributors for their excellent articles that make this issue of Seminars in Thrombosis and Hemostasis a state-of-the-art work on the laboratory diagnosis of vWD. My special thanks are offered to Drs. Favaloro and Michiels for compiling this important information.