Test Id : F81B

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, Blood is required to perform this test. See Additional Testing Requirements.

The following algorithms are available:

-Hemophilia Carrier Testing Algorithm

-Hemophilia Testing Algorithm

If a familial variant has not been identified in a severely affected hemophilia A patient, order F8INV / Hemophilia A F8 Gene, Intron 1 and 22 Inversion Mutation Analysis, Whole Blood.

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.

Due to the complexity of testing nonperipheral 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, Blood on the maternal specimen.

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.

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.

• Informed Consent for Genetic Testing
• Hemophilia Carrier Testing Algorithm
• Hemophilia Testing Algorithm
• Hemophilia A Patient Information
• Informed Consent for Genetic Testing (Spanish)

1. Hemophilia A Patient Information (T712) is required.

2. New York Clients-Informed consent is required. Document on the request form or electronic order that a copy is on file. The following documents are available:

-Informed Consent for Genetic Testing (T576)

-Informed Consent for Genetic Testing-Spanish (T826)

3. If not ordering electronically, complete, print, and send a Coagulation Test Request (T753) with the specimen.

1 mL

First-tier molecular testing for male patients affected with severe hemophilia A, when a familial intron 1 inversion has been previously identified

Determining hemophilia A carrier status for at-risk female patients, ie, individuals with a family history of severe hemophilia A due to F8 intron 1 inversion

This test detects the intron 1 inversion in the F8 gene. The intron 1 inversion variant accounts for approximately 5% of the variants associated with severe hemophilia A.

Intron 1 inversion known variant analysis can only be performed for individuals when an intron 1 inversion has already been identified in the family. For testing options, see Ordering Guidance.

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, Blood is required to perform this test. See Additional Testing Requirements.

The following algorithms are available:

-Hemophilia Carrier Testing Algorithm

-Hemophilia Testing Algorithm

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 inversion variant accounts for approximately 5% of variants associated with severe HA. These inversions are typically not identified in patients with mild or moderate HA.

Intron 1 inversion known variant analysis is only recommended for individuals when an intron 1 inversion has already been identified in the family.

If a familial mutation has not been identified in a severely affected HA patient, the F8 gene intron 1 and 22 inversion analysis (F8INV / Hemophilia A F8 Gene, Intron 1 and 22 Inversion Mutation Analysis, Whole Blood) should be ordered.

If the intron 1 inversion analysis is negative, the tested individual has not inherited the familial variant.

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.

An interpretive report will be provided.

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

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.

Intron 1 inversion known variant analysis is only recommended for individuals when an intron 1 inversion has already been identified in the family.

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

The intron 1 inversion variant targeted by this assay is found in approximately 5% of individuals with severe hemophilia A; if an intron 1 inversion has not been already identified in the family, the assay may be uninformative.

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.

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

Genomic DNA from whole blood or cord blood is amplified by polymerase chain reaction with primers specific for the F8 intron 1 inversion variant.(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 Jan;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)

Weekly

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• Prospective clients should contact their account representative. For assistance, contact Customer Service.

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.

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