Abstract
Remarkably, classical von Willebrand disease (vWD) laboratory work-ups have been shown to require repeat laboratory testing 1 to 20 times to confirm diagnosis [1]. Furthermore, most diagnostic laboratory algorithms terminate with molecular confirmation [2, 3]. Here we present the method validation of a diagnostic laboratory approach that allows for the simultaneous functional and genetic diagnosis and subtyping von Willebrand disease (vWD) and pseudo- or platelet-type vWD within 48 hours. The clinical sensitivity of this test is approximately 85% for subtypes 2A, 2B, 2N, 2M and 3 vWD [4].
The method validation is compliant with current Clinical Laboratory Improvement Act (CLIA) and applicable current Good Laboratory Practices (cGLP, 21 CFH Part 58) guidelines and therefore is suitable for clinical utilization and clinical trial utilization. The method validation protocol included the creation of reference intervals, reportable ranges, intra-run precision, inter-run precision, accuracy, lower limit of quantification (LLQ), linearity, analytical and clinical sensitivity assessment, analytical and clinical specificity assessment, interfering substances, stability and longitudinal quality control performance [Table 1.].
Historically, no one test has been able to diagnose vWD. Classical laboratory work-ups include aPTT, factor VIII (8) activity, Ristocetin cofactor activity, vWF antigen, ratio calculations, multimeric composition and others. Each of these tests is influenced by numerous factors, such as inflammation, stress, infection, hormone replacement therapy, age, acute phase response, menstrual cycle, pregnancy exercise, ABO blood type, lupus anticoagulant and other factors, making traditional diagnosis a challenging moving target [3, 5, 6].
The methodology used in this study employed simultaneous classical vWD laboratory testing and rapid next generation sequencing (NGS) optimized for 48-hour result availability [Table 2]. Furthermore, normal healthy and vWD 2N subtype samples can clearly be distinguished in this testing approach [Table 3].
vWD occurs with bleeding symptoms at rate of 1 in 1,000 in the general population [6]. In 10% of those cases the bleeding is severe [3]. This prevalence makes vWD the most common congenital bleeding disorder known [6]. Acquire vWD (1-5% of cases) can be assessed by contrasting functional von Willebrand testing with genetic testing in a specialized coagulation, platelet and genetics laboratory. Common symptoms of vWD include menorrhagia, prolonged bleeding or bruising from minor trauma, recurrent nose bloods, excessive bleeding at childbirth and excessive bleeding associated with surgery.
In conclusion, the standard clinical approach of diagnosing vWD can take months to years and require one or more referrals to specialized clinicians to establish diagnosis and subtyping. The data presented in this study proposes that an alternative method of rapidly characterizing suspected vWD patients simultaneously with both functional and genetic tests can revolutionize speed of diagnosis and treatment.
vWD Subtypes: (each can have severe clinical presentations)
Type 1 = 60%
Type 2 = 30%
Type 2A = 15%
Type 2B = 5%
Type 2M < 5%
Type 2N <5% (clinically similar to mild hemophilia A)
Type 3 = 5-10%
Pseudo-vWD or Platelet-type vWD = rare (defect in GP1BA gene)
Acquired vWD = 1-5%
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Federici, AB. Int Jnl Lab Hem 2016; 38(Suppl. 1): 41-49.
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James PD and Lillicrap, D. Br J Haematol 2013; 161: 166-176.
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No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
