Von Willebrand Disease Type 2N DNA, Select Exons (Sendout)

Test Information: Von Willebrand Disease (VWD) is one of the most common inherited bleeding disorders. This disorder is divided into three types, Type 1, Type 2 and Type 3. All types of this condition cause prolonged bleeding episodes of varying severity; Type 1 is defined by a partial quantitative VWF deficiency, while Type 3 is defined by the absence of detectable VWF. Type 1C is defined by low VWF levels and rapid VWF clearance. Type 2 is defined by a functional (qualitative) VWF defect and is further divided into four subtypes. Type 2A VWD is the most common form of Type 2, accounting for 70% of the Type 2 diagnosis.

Type 2N VWD is defined by low coagulation factor VIII (FVIII) levels due to a defect in the ability of VWF to bind FVIII. Nearly all variants that cause Type 2N VWD are located in the exons that encode VWF D’-D3 region. Some type 2N variants affect the VWF propeptide.

Variants in the von Willebrand Factor (VWF) gene cause VWD. This disease can have different inheritance patterns. Type 1, Type 1C, and Type 2 (2A, 2B, and 2M) are generally inherited in an autosomal dominant pattern, which means one copy of the altered gene in each cell is sufficient to cause the disorder. Type 3 and Type 2N are inherited in an autosomal recessive pattern, which means both copies of the gene in each cell have variants.

Additional Test Information: This Von Willebrand Factor Type 2N VWD Screen sequences exons 4, 9, 18-21, 24, 25, and 27. Confirmation is performed by sequencing the exon(s) in which the variant(s) is/are located.

If a causative pathogenic variant is not identified, further screening can be performed by Von Willebrand Disease DNA Evaluation (Sendout) [RVWDEV]. Request "reflex to DNA von Willebrand Disease Evaluation (Test 3250-17)" on the Bloodworks requisition or via Epic order question.

Overview of Type 2N von Willebrand Disease (VWD): Type 2N von Willebrand disease (VWD) is characterized by decreased von Willebrand factor (VWF) affinity for factor VIII. This condition can clinically resemble hemophilia A. Typically, concerning laboratory values for Type 2N VWD include:

• Factor VIII activity < 50%
• Factor VIII activity/VWF antigen ratio < 0.5

These values indicate that the factor VIII is disproportionately lower than VWF. VWF binds to factor VIII in the blood and stabilizes it, preventing its degradation by proteolytic enzymes. Therefore, when both the factor VIII activity and the Factor VIII activity/VWF antigen ratio are disproportionately low, Type 2N VWD or mild hemophilia may be considered.

Diagnostic Flow Chart: An algorithm for the diagnosis of type 2N VWD²

Recommended Testing Steps:

• Begin with Von Willebrand Disease Type 2N Genotyping (this test) to determine the status of relatives.
• If there is a VUS (Variant of Uncertain Significance), or if no variant is found: Perform the Von Willebrand Disease Type 2N Binding (Sendout) to validate or complete the evaluation for Type 2N VWD.
• If Type 2N VWD is not identified, but clinical concern is high: Testing for Hemophilia A DNA Evaluation (Sendout) [RDHEMA] may be warranted to rule out alternative diagnoses.

Ordering Requirements:

• Orders from Washington Center for Bleeding Disorders (SEAWBD) and FHCC Hematology are pre-approved. Orders from other locations require Laboratory Medicine Resident approval.
• For orders not submitted via Epic or ISEAWBD interface, providers must complete a Bloodworks NW Eastlake Genomics Laboratory Requisition.

Whole Blood: 10 mL LAVENDER TOP EDTA tube

Cultured Amniocytes: Two T-25 flasks of cells cultured to confluency.

• Amniotic fluid is not accepted.
• An EDTA whole blood sample from the mother of the fetus must accompany cultured amniocyte orders.

Extracted DNA: >10 ug extracted DNA at a concentration of >25ng/ul

Collection Timing:

• Collect any time Monday through Thursday.
• Samples collected on a Friday must be timed appropriately to ensure delivery at Bloodworks Eastlake Lab by the 1 p.m. cutoff.

Samples sent on Fridays must be received at Bloodworks Northwest Genomics Lab by 1 p.m.

Outside Laboratories:

• Store and transport whole blood at ambient temperature.
• Expedite transport; samples must be received by Bloodworks NW within 72 hours of collection and before 1 p.m. on Fridays. Allow for one day of transport from UW-MT to Bloodworks Northwest.
  • Unless the cells are cultured at UW-MT Cytogenetics and Genomics Lab, clients should arrange to send cultured amniocyte samples directly to Bloodworks NW due to the short stability window.
• Submit a completed Bloodworks Northwest Eastlake Genomics Requisition along with the DLMP Requisition.* Leave the billing section blank to be completed by Sendouts staff.
  • *Use client-specific requisition when available. To obtain a client-specific requisition, contact Client Support Services via email at: commserv@uw.edu.
• WA Center for Bleeding Disorders only: A separate Bloodworks NW requisition is not required for orders submitted via interface.

Stability (Whole Blood): Ambient (preferred): 72 hours; Refrigerated: 72 hours; Frozen: -20°C or -70/80°C. Avoid thawing/re-freezing.

• Frozen EDTA tube(s) are accepted if samples are frozen the day of the blood draw, never thawed, and are shipped and received on dry ice. Contact Eastlake Bleeding Disorders at (206) 568-2184 with questions about sample acceptability.

Result-at-entry:

• RVWDNS (VWD Type 2N DNA Specimen Type): <Specimen type will file automatically for orders placed in Epic. For others, enter the appropriate specimen type code.>

Specimen Type Codes:

Processing:

• Maintain whole blood and cultured amniocyte samples at ambient temperature.
• Whole blood received frozen should be stored frozen.
• If sample is collected on a Friday, notify Sendouts immediately upon sample receipt to ensure delivery to Bloodworks NW by the 1 p.m. cutoff.

Sendouts (UW-MT/HMC) and FHCC SPS:

• Bloodworks NW Test: 3250-09
• Submit a copy of the ISEAWBD eREQ or Epic requisition with the sample (ensure that all pages are printed to accommodate the order questions). For orders not placed in Epic or via the ISEAWBD interface, a separate Bloodworks Northwest Eastlake Genomics requisition is required.
• Package sample(s) with the associated requisition in a biohazard bag (one patient per bag). Orders received with multiple
  patient samples/requisitions in the same biozard bag will be rejected by Bloodworks NW.
• Blood:
  • Send blood orders as received with the UW DLMP or Delivery Express courier directly to the Bloodworks NW Eastlake location.
  • Transport whole blood at ambient temperature. Whole blood received frozen should be stored frozen and transported on dry ice.
  • Samples may be sent Monday through Friday only. If sent on a Friday, samples must be in lab at the Eastlake location by 1 p.m.
  • Ambient or refrigerated whole blood samples must arrive at Bloodworks within 72 hours of collection.
• Cultured amniocytes:
  • Send cultured amniocytes same-day to the Bloodworks Central Genomics Lab (921 Terry Ave, Seattle WA 98104).
  • Cultured amniocytes must arrive at Central Genomics within 48 hours of collection and by 1 p.m. on Fridays.

Stability (Whole Blood): Ambient (preferred): 72 hours; Refrigerated: 72 hours; Frozen: -20°C or -70/80°C. Avoid thawing/re-freezing.

• Frozen EDTA tube(s) are accepted if samples are frozen the day of the blood draw, never thawed, and are shipped and received on dry ice. Contact Eastlake Bleeding Disorders at (206) 568-2184 with questions about sample acceptability.

Stability (Cultured Amniocytes): Samples must arrive within 48 hours to the Central Genomics location (amniocytes should not be sent to the Eastlake laboratory).