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Mayo Test ID F8INV Hemophilia A F8 Gene, Intron 1 and 22 Inversion Mutation Analysis, Whole Blood


Ordering Guidance


For evaluation of a patient with bleeding symptoms and no known personal history of a bleeding disorder, consider ALBLD / Bleeding Diathesis Profile, Limited, Plasma or the specific factor assays.



Additional Testing Requirements


Due to the complexity of testing non-peripheral blood, consultation with the laboratory is required for all cord blood samples. Order this test on the cord blood specimen (only 1 specimen tube required) and order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.



Necessary Information


Hemophilia A Patient Information(T712) is required. Testing may proceed without the patient information, however, the information aids in providing a more thorough interpretation. Ordering providers are strongly encouraged to fill out the form and send with the specimen.



Specimen Required


Patient Preparation: A previous bone marrow transplant from an allogenic donor will interfere with testing. Call 800-533-1710 for instructions for testing patients who have received a bone marrow transplant.

Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD) or blue top (3.2% sodium citrate)

Specimen Volume: 4 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send whole blood specimen in original tube. Do not aliquot.


Useful For

First-tier molecular testing for male patients affected with severe hemophilia A when a variant has not been identified in the family

 

Determining hemophilia A carrier status for at-risk female patients, ie, individuals with a family history of severe hemophilia A

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
MATCC Maternal Cell Contamination, B Yes No

Testing Algorithm

Maternal cell contamination testing will be performed for all cord blood specimens. A maternal whole blood sample with an order for MATCC / Maternal Cell Contamination, Molecular Analysis, Varies is also required to perform this test. See Additional Testing Requirements.

 

The following algorithms are available:

-Hemophilia Carrier Testing Algorithm

-Hemophilia Testing Algorithm

Method Name

Polymerase Chain Reaction (PCR) or Inverse Shifting-Polymerase Chain Reaction (IS-PCR)

Reporting Name

HA F8 Intron 1/22 Inversion, B

Specimen Type

Whole blood

Specimen Minimum Volume

1 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Whole blood Ambient (preferred) 7 days
  Frozen  7 days
  Refrigerated  7 days

Reject Due To

Gross hemolysis OK
Gross lipemia OK

Clinical Information

Hemophilia A (HA) is caused by a deficiency of clotting factor VIII (FVIII). HA is an X-linked recessive bleeding disorder that affects approximately 1 in 5000 male individuals. Male patients are typically affected with bleeding symptoms, whereas female carriers generally do not have bleeding symptoms but are at risk of having affected sons. Rarely, approximately 10% of female carriers have FVIII activity levels below 35% and are at risk for bleeding.

 

Bleeding, the most common clinical symptom in individuals with HA, correlates with FVIII activity levels. FVIII activity levels below 1% are associated with severe disease, 1% to 5% activity with moderate disease, and 5% to 40% with mild disease. In male patients with severe deficiency, spontaneous bleeding may occur. In individuals with mild HA, bleeding may occur only after surgery or trauma.

 

FVIII is encoded by the factor VIII (F8) gene. Approximately 98% of patients with a diagnosis of HA are found to have a variant in F8 (ie, intron 1 and 22 inversions, point mutations, insertions, and deletions). The intron 1 and 22 inversion variants account for approximately 50% of variants associated with severe HA. These inversions are typically not identified in patients with mild or moderate HA.

 

It is recommended that the F8 variant be confirmed in the affected male patient or obligate female carrier prior to testing at-risk individuals. Affected male patients are identified by FVIII activity (F8A / Coagulation Factor VIII Activity Assay, Plasma) and clinical evaluation, while obligate female carriers are identified by family history assessment. If the intron inversion assays do not detect an inversion in these individuals, additional analysis (ie, F8 sequencing) may be able to identify the familial variant. Of note, not all women with an affected son are germline carriers of a F8 variant, as de novo variants in F8 do occur. Approximately 20% of mothers of isolated cases do not have an identifiable germline F8 variant. Importantly, there is a small risk for recurrence even when the familial F8 variant is not identified in the mother of the affected patient due to the possibility of germline mosaicism.

Reference Values

An interpretive report will be provided.

Interpretation

The interpretive report will include assay information, background information, and conclusions based on the test results.

Cautions

Obtaining a medical genetics or hematology (coagulation) consultation prior to ordering is advisable. Consultations with the Mayo Clinic Special Coagulation Clinic, Molecular Hematopathology Laboratory, or Thrombophilia Center are available for DNA diagnosis cases. This may be especially helpful in complex cases or in situations where the diagnosis is atypical or uncertain.

 

This assay detects only F8 intron 1 and 22 inversion variants. Thus, a negative result does not exclude the presence of other variants in F8.

 

The intron 1 and 22 inversion variants targeted by this assay are found in approximately 50% of individuals with severe hemophilia A; the assay may be uninformative for a number of families.

 

Test results should be interpreted in the context of clinical findings, family history, and other laboratory data. Errors in the interpretation of results may occur if the information provided is inaccurate or incomplete.

Clinical Reference

1. Antonarakis SE, Rossiter JP, Young M, et al: Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood 1995 Sep;86(6):2206-2212

2. Rossiter JP, Young M, Kimberland ML, et al: Factor VIII gene inversions causing severe hemophilia A originate almost exclusively in male germ cells. Hum Mol Genet 1994 Jul;3(7):1035-1039

3. Castaldo G, D'Argenio V, Nardiello P, et al: Haemophilia A: molecular insights. Clin Chem Lab Med 2007;45(4):450-461

4. Johnsen JM, Fletcher SN, Huston H, et al: Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative. Blood Adv 2017 May;1(13):824-834. doi: 10.1182/bloodadvances.2016002923

5. Pruthi RK: Hemophilia: A practical approach to genetic testing. Mayo Clin Proc 2005 Nov;80(11):1485-1499

Method Description

Genomic DNA from whole blood or cord blood is digested with restriction enzyme, ligated with T4 DNA ligase, and amplified by polymerase chain reaction (PCR) with primers specific for the F8 intron 22 inversion variants.(Rosetti LC, Radic CP, Larripa IB, De Brasi CD: Developing a new generation of tests for genotyping hemophilia-causative rearrangements involving int22h and int1h hotspots in the factor VIII gene. J Thromb Haemost. 2008 May;6(5):830-836)

 

Genomic DNA from whole blood or cord blood is amplified by PCR with primers specific for the F8 intron 1 inversion mutation.(Bagnall RD, Waseem N, Green PM, Giannelli F: Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood 2002;99(1):168-174; Meijer P, Verbruggen, Spannagi M: Clotting factors and inhibitors: Assays and Interpretation. In: Kottke-Marchant K, ed. Laboratory Hematology Practice. Wiley Blackwell Publishing. 2012:435-446)

Day(s) Performed

Weekly

Report Available

14 to 21 days

Specimen Retention Time

Whole blood: 2 weeks; DNA: Indefinitely; from New York State: 90 days

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information

81403

LOINC Code Information

Test ID Test Order Name Order LOINC Value
F8INV HA F8 Intron 1/22 Inversion, B 81761-9

 

Result ID Test Result Name Result LOINC Value
35760 HA F8 Int 1/22 Reason for Referral 42349-1
35761 HA F8 Intron 1/22 Inversion, B 81761-9
35762 F8INV Interpretation 69047-9
35763 HA F8 Intron 1/22 Reviewed By 18771-6