Archives of Pathology and Laboratory Medicine: Vol. 127, No. 6, pp. 752–754.

Pathologic Quiz Case: A 2-Year-Old Boy With a Hereditary Bleeding Disorder

Malcolm Schinstine, PhD, MD,a and Donna Robert, MT(ASCP)SHa

aFrom the Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH

Accepted September 17, 2002

A 2-year-old boy was brought to a tertiary care hospital by his mother and grandmother for evaluation of a bleeding disorder. He had no other significant medical history. The patient's mother and maternal grandmother shared a bleeding disorder and were concerned that the boy had inherited the same disease. The mother had previously used Humate P (40 IU/kg q8–12h) as needed for bleeding episodes. The patient's laboratory studies demonstrated a prothrombin time of 13.1 seconds (normal range, 11.9–14.1 seconds), an international normalized ratio of 1.0 (normal range, 0.9–1.2), a platelet count of 206 × 103/L (normal range, 160–450 × 103/L), and an activated partial prothrombin time of 34 seconds (normal range, 24–33 seconds). Additional tests showed a factor VIII activity level of 44% (normal range, 50%–150%), a von Willebrand antigen level of 29% (normal range, 50%–150%), and a von Willebrand activity of 19% (normal range, 50%–150%). A bleeding time test was not performed. Platelet aggregation studies demonstrated marked aggregation of the patient's platelets in the presence of 1.0% and 0.5% ristocetin, channels 1 (blue) and 2 (olive), respectively (Figure 1 ; channel 3 (red)—5 g/mL collagen, channel 4 (green)—8 M epinephrine). Platelet aggregation was also seen in the presence of 0.2% to 0.4% ristocetin (data not shown). The reaction of normal control platelets to 1.0% (Figure 2 ; channel 1, blue) and 0.5% (Figure 2 ; channel 2, olive) ristocetin is also shown.

What is your diagnosis?

Pathologic Diagnosis: von Willebrand Disease, Subtype 2B Return to TOC

von Willebrand disease is the most common inherited bleeding disorder and is usually inherited in an autosomal-dominant fashion. The dysfunction is due to a quantitative or qualitative defect in von Willebrand factor, leading to the disruption of primary hemostasis. Normally, von Willebrand factor mediates the adhesion and aggregation of platelets to the subendothelium in areas of high shear force (as found in arteries). In addition, von Willebrand factor protects factor VIIIc from inactivation by forming a complex with it.

Inherited von Willebrand disease is divided into 3 main categories depending on whether the dysfunction is due to a quantitative (types 1 and 3) or a qualitative (type 2) deficiency in the factor. Type 1 is by far the most common, accounting for the majority of cases, and is the mildest form of the disease.1 In contrast, in type 3 von Willebrand disease, there is an almost total absence of von Willebrand factor. This form is very rare and the most severe.

Type 2 von Willebrand disease can be further subclassified as type 2A, 2B, 2M, or 2N. Type 2A is the most common of this group and refers to qualitative variants of von Willebrand factor that express decreased platelet-dependent function associated with the absence of high-molecular-weight multimers. Type 2M (M for multimers) may involve a mutation that effectively inactivates binding sites for ligands on platelets and collagen. Thus, the multimeric distribution of von Willebrand factor is normal in patients with type 2M disease, but the binding to platelets is impaired. Type 2N (N for Normandy) resembles hemophilia A in that plasma levels of factor VIII are decreased. In contrast to hemophilia A, the low circulating levels of factor VIII in type 2N von Willebrand disease are caused by the decreased half-life of factor VIII due to its inability to bind the abnormal von Willebrand factor expressed in these patients.

The patient described in this report had type 2B von Willebrand disease. Type 2 variants account for 20% to 30% of all cases of von Willebrand disease.2 As with most forms of von Willebrand disease, inheritance of type 2B von Willebrand disease is predominantly autosomal dominant; however, cases with apparent recessive inheritance patterns have been described. This subtype of von Willebrand disease is characterized by von Willebrand factor molecules that express an increased affinity for the platelet membrane glycoprotein GPIb. The multimeric structure of von Willebrand factor found in platelets and cultured endothelial cells from patients with the disorder appears normal.3 To date, at least 21 missense mutations and 1 small insertion have been identified in type 2B von Willebrand disease.2 These mutations appear to be restricted to an area containing the GPIb-binding site, the von Willebrand factor A1 domain. As can be surmised, there is a high degree of heterogeneity in type 2B disease. Patients expressing different mutations can exhibit increased platelet aggregation, thrombocytopenia, and some loss of multimers.

The different subtypes of von Willebrand disease can be delineated by a good clinical and family history in conjunction with laboratory studies.4 General screening tests include a bleeding time, a platelet count, and an activated partial prothrombin time. The bleeding time is usually prolonged in von Willebrand disease. This test is not specific but will detect quantitative or qualitative changes in von Willebrand factor or platelets. Impairments in the function of the vessel wall can also be shown with a bleeding time. The value of the activated partial prothrombin time is minimal in most cases of von Willebrand disease. Where this test may be of import is for patients with low levels of factor VIIIc and for patients with type 2N disease. The total platelet count is important to obtain because it allows the exclusion of bleeding disorders related to thrombocytopenia not related to von Willebrand disease and because it can indicate the presence of a qualitative defect in von Willebrand factor, particularly in patients with type 2B disease. Other screening tests that can be used are the filter method with high shear stress and adhesion and retention tests that attempt to simulate the high shear stress present in arterioles.

Tests that can confirm the diagnosis of von Willebrand disease include assays for factor VIII activity and plasma levels of von Willebrand factor antigen. In most patients with von Willebrand disease, factor VIII activity and plasma levels of von Willebrand factor are decreased. A caveat in evaluating plasma levels of von Willebrand factor is that persons with type O blood normally express lower levels of von Willebrand factor than persons with other blood types.

Ristocetin cofactor activity (VWF/Rco) is a confirmatory assay that reflects the functional property of von Willebrand factor by mimicking the platelet interaction with the GPIb/IX complex. Ristocetin, a small glycopeptide antibiotic, binds to both von Willebrand factor and GPIb, causing a von Willebrand factor–dependent platelet agglutination. This test has the same diagnostic power as von Willebrand factor antigen levels and is required to discern between patients with type 1 and 2M von Willebrand disease. Unfortunately, VWF/Rco demonstrates considerable interassay and interlaboratory variability. This has led to the use of alternative assays in an attempt to increase reproducibility. Thus, many laboratories have begun to use the von Willebrand factor/collagen-binding activity assay. This assay is capable of detecting the absence of high- and intermediate-molecular-weight von Willebrand factor. Moreover, the normalization of the von Willebrand factor/collagen-binding activity assay to the von Willebrand factor antigen demonstrates a greater difference (ie, greater sensitivity) in distinguishing between type 2A and 2B von Willebrand disease than does the normalization of VWF/Rco to the von Willebrand factor antigen. In combination with ristocetin-induced platelet aggregation and VWF/Rco, the von Willebrand factor/collagen-binding activity assay may help delineate between type 2A, 2B, and 2M as well as type 1 von Willebrand disease.

Ristocetin-induced platelet aggregation is used to show a higher than normal affinity of von Willebrand factor for the platelet GPIb/IX complex. This test is particularly useful for evaluating patients with type 2B von Willebrand disease or those with platelet type von Willebrand disease (mutations in GPIb, a very rare occurrence). In this assay (as in Figures 1 and 2 ), varying concentrations of ristocetin (0.5, 1.0, and 1.5 mg/mL) and platelet-rich plasma are mixed. The minimal concentration of ristocetin able to cause 30% aggregation is recorded. Patients that demonstrate aggregation in 0.5 mg/mL of ristocetin usually suffer from type 2B von Willebrand disease.

As mentioned, the von Willebrand factor gene is highly polymorphic. The traditional method used to analyze polymorphisms is restriction fragment length polymorphisms. These arise when a single nucleotide difference exists between genomic DNA sequences. These differences result in either the creation or destruction of restriction enzymatic sites. Southern blot tests can subsequently be used to analyze digestion differences. Polymorphisms may also be analyzed by polymerase chain reaction amplification of the region of interest, followed by restriction enzyme digestion and electrophoresis. More recent developments that may be useful in detecting mutations and polymorphisms are conformation sensitive gel electrophoresis, and denaturing high-performance liquid chromatography. In conformation sensitive gel electrophoresis, DNA heteroduplexes are detected by their migration pattern on a mildly denaturing gel. Similarly, denaturing high-performance liquid chromatography relies on the separation of heteroduplexes formed by the mixing, denaturing, and reannealing of 2 or more chromosomes using an alkylated, nonporous poly(styrene-divinylbenzene) stationary phase. The molecular methods used to diagnose bleeding disorders have been reviewed elsewhere.5

There are multiple accepted therapies for the treatment of von Willebrand disease. The efficacy and appropriateness of the treatment depend on the type of disease that is present; however, the general goal of treatment is to correct coagulopathies related to low factor VIII levels and to correct prolonged bleeding times. For example, desmopressin is the therapy of choice for mild forms of von Willebrand disease. Desmopressin is counterindicated in the treatment of type 2B von Willebrand disease because of transient thrombocytopenia.6 For those resistant to desmopressin, factor VIII/von Willebrand factor concentrates is the recommended therapy. Other therapies that have been used to treat von Willebrand disease are antifibrinolytic amino acids (eg, epsilon aminocaproic acid and tranexamic acid) and estrogens.

References Return to TOC

1. Van Cott, EM, and M. Laposata. Coagulation, fibrinolysis, and hypercoagulation. In: Henry JB, ed. Clinical Diagnosis and Management by Laboratory Methods. 20th ed. Philadelphia, Pa: WB Saunders Co; 2001:642–659.

2. Fressinaud, E, C Mazurier, and D. Meyer. Molecular genetics of type 2 von Willebrand disease. Int J Hematol 2002;75:9–18.

3. De Groot, PG, AB Federici, and HC De Boer. et al. VWF synthesized by endothelial cells from a patient with type II B supports platelet adhesion normally but has an increased affinity for platelets. Proc Natl Acad Sci U S A 1989;86:3793–3797.

4. Budde, U, E Drewke, and K Mainusch. et al. Laboratory diagnosis of congenital von Willebrand disease. Semin Thromb Hemost 2002;28:173–189.

5. Goodeve, AC. Laboratory methods for the genetic diagnosis of bleeding disorders. Clin Lab Haematol 1998;20:3–19.

6. Federici, AB, and PM. Mannucci. Advances in the genetics and treatment of von Willebrand disease. Opin Pediatr 2002;14:23–33.