Hemophilia A DNA Evaluation (Sendout)

General Information

Lab Name

Hemophilia A DNA Evaluation (Sendout)

Lab Code

RDHEMA

External Test Id

3250-02

Description

Test Information: Patients with severe hemophilia A that are negative for the DNA Factor VIII inversion or patients that have moderate or mild hemophilia can be evaluated for variants by Sanger sequencing of the 26 exons of the F8 gene and the 5’untranslated and 3’ untranslated regions. Through current screening methods, a variant is found ~96% of the time.

If the specific variant cannot be identified, intragenic linkage is often useful to trace the affected allele through the family tree. Linkage studies also help identify the de novo origin of mutations in families with an isolated occurrence (eg: maternal grandparents). Linkage studies may be informative in families who have hemophilia A where no variant is able to be identified in the proband Intragenic linkage may be useful in 80-90% of families where key family members (patient and both parents of potential carrier) are available.

    Additional Test Information: For approximately half of the severe hemophilia A patients (and some moderate cases with FVIII levels of ~1%), the DNA Factor VIII Inversion testing will be negative. For these patients, along with the moderate and mild patients, the Hemophilia A Mutation Evaluation will be performed to identify the causative variant. Screening of the F8 gene will be performed by Sanger sequencing. If no causative variant is identified through Sanger sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA) is performed. Confirmation is performed by sequencing the exon in which the variant is located or by MLPA if necessary.

      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.

      References

      Synonyms

      3250-02, F8 Genotyping, HemA Genotyping, Hemophilia A

      Components

      Code Name
      RHEMAS Hemophilia A DNA Evaluation Specimen Type
      RHEMAR Hemophilia A DNA Evaluation Result
      RHEMAL Hemophilia A DNA Evaluation Performing Lab

      Interpretation

      Method

      Polymerase Chain Reaction (PCR) Based

      Reference Range

      See individual components

      Ref. Range Notes

      See report.

      Ordering & Collection

      Specimen Type

      Blood, Extracted DNA, Cultured Amniocytes

      Collection

      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.

          Forms & Requisitions

          Approval Required

          Pre-approved for Washington Center for Bleeding Disorders (SEAWBD) and FHCC Hematology. Orders from other locations require LMR approval.

          Handling Instructions

          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.

          Quantity

          requested: 10 mL EDTA whole blood
          minimum: 3 mL EDTA whole blood

          Processing

          Result-at-entry:

          • RHEMAS (Hemophilia A DNA Evaluation Specimen Type): <Specimen type will file automatically for orders placed in Epic. For others, enter the appropriate specimen type code.>
          ETC Translation
          WB Whole Blood
          CULAMN Cultured Amniocytes
          DNAEXT Extracted DNA

          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: 3250-02
          • 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).

          Performance

          LIS Dept Code

          Sendouts (SO)

          Performing Location(s)

          Sendout Bloodworks Northwest Eastlake Genomics Laboratory
          206-568-2184

          1551 Eastlake Ave E
          Suite 100
          Seattle, WA 98102

          Also known as: Bloodworks Northwest Bleeding Disorders Laboratory

          Frequency

          Turnaround Time: 2-4 weeks from sample receipt at Bloodworks NW. If Multiplex Ligation-dependent Probe Amplification (MLPA) testing is necessary, results may take up to a couple of months.

          Available STAT?

          No

          Billing & Coding

          CPT codes

          81407

          LOINC

          Interfaced Order Code

          UOW6143