Test Catalog

Test Id : F8NGS

Hemophilia A, F8 Gene, Next-Generation Sequencing, Varies

Useful For
Suggests clinical disorders or settings where the test may be helpful

Molecular confirmation of a clinical diagnosis of hemophilia A in affected male patients

 

Identification of the causative alteration in the F8 gene for prognostic and genetic counseling purposes

 

Helping determine hemophilia A carrier status for female patients with a family history of hemophilia A

 

Molecular prenatal confirmation of hemophilia A

Genetics Test Information
Provides information that may help with selection of the correct genetic test or proper submission of the test request

This test detects pathogenic alterations within the F8 gene to delineate the underlying molecular defect in a patient with a laboratory diagnosis of hemophilia A and for determining carrier status for female patients with a family history of hemophilia A. This test may also be used to prenatally identify and confirm hemophilia A in a male fetus at risk for inheriting the disease. Diagnostic prenatal testing for female fetuses is typically medically unnecessary and not performed except in very rare cases where the fetus is known to be at risk of inheriting a pathogenic F8 alteration from both parents.

 

The gene target for this test is:

Gene name (transcript): F8 (GRCh37 [hg19] NM_000132)

Chromosomal location: Xq28

Highlights

-This test identifies pathogenic alterations in the F8 gene associated with hemophilia A.

-It is recommended that the F8 alteration be confirmed in the affected male or obligate carrier female prior to testing at-risk individuals.

-This test allows for the prenatal identification of pathogenic alterations in the F8 gene associated with hemophilia A. Prenatal genetic testing is not routinely performed without the prior identification of a familial hemophilia alteration.

Reflex Tests
Lists tests that may or may not be performed, at an additional charge, depending on the result and interpretation of the initial tests.

Test Id Reporting Name Available Separately Always Performed
CULAF Amniotic Fluid Culture/Genetic Test Yes No
CULFB Fibroblast Culture for Genetic Test Yes No
MATCC Maternal Cell Contamination, B Yes No
_STR1 Comp Analysis using STR (Bill only) No, (Bill only) No
_STR2 Add'l comp analysis w/STR (Bill Only) No, (Bill only) No

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

Prenatal genetic testing is not routinely performed without the prior identification of a familial hemophilia alteration in an affected male relative or a female relative who is a confirmed carrier of the alteration. Requests for this prenatal testing without a known familial alteration are performed at the discretion of a Molecular Hematopathology Laboratory Director.

 

For any cord blood or prenatal specimen that is received, maternal cell contamination studies will be added. A maternal whole blood sample is required to perform this test.

 

If amniotic fluid is received, amniotic fluid culture for genetic testing will be added and charged separately. If chorionic villus specimen is received, fibroblast culture for genetic testing will be added and charged separately.

 

The following algorithms are available in Special Instructions:

-Hemophilia Testing Algorithm (for testing affected male patients)

-Hemophilia Carrier Testing Algorithm (for female patients with a family history of hemophilia)

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Method Name
A short description of the method used to perform the test

Custom Sequence Capture and Targeted Next-Generation Sequencing (NGS) followed by Polymerase Chain Reaction (PCR) and Sanger Sequencing when appropriate.

NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.

Yes

Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test

F8 Gene, Full Gene NGS

Aliases
Lists additional common names for a test, as an aid in searching

F8

Factor VIII gene

HA

Hemophilia A

Factor VIII deficiency

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

Prenatal genetic testing is not routinely performed without the prior identification of a familial hemophilia alteration in an affected male relative or a female relative who is a confirmed carrier of the alteration. Requests for this prenatal testing without a known familial alteration are performed at the discretion of a Molecular Hematopathology Laboratory Director.

 

For any cord blood or prenatal specimen that is received, maternal cell contamination studies will be added. A maternal whole blood sample is required to perform this test.

 

If amniotic fluid is received, amniotic fluid culture for genetic testing will be added and charged separately. If chorionic villus specimen is received, fibroblast culture for genetic testing will be added and charged separately.

 

The following algorithms are available in Special Instructions:

-Hemophilia Testing Algorithm (for testing affected male patients)

-Hemophilia Carrier Testing Algorithm (for female patients with a family history of hemophilia)

Specimen Type
Describes the specimen type validated for testing

Varies

Ordering Guidance

Genetic testing for hemophilia A should only be considered in males if clinical and family history, initial coagulation screens, and FVIII activity (F8A / Coagulation Factor VIII Activity Assay, Plasma) indicate a diagnosis of hemophilia A. Causes of acquired (non-genetic) hemophilia A should be excluded prior to genetic testing.

 

Most patients with less than 1% factor VIII activity and severe hemophilia A have large rearrangements in the F8 gene called inversions that are not detectable by this testing method. Full gene sequencing for a patient with severe hemophilia A should be performed only if genetic testing for these inversions is negative. See F8INV / Hemophilia A F8 Gene, Intron 1 and 22 Inversion Mutation Analysis, Whole Blood.

 

Genetic testing for hemophilia A in females should only be considered if a first-degree male relative has been diagnosed with hemophilia A, if there is a maternal family history of hemophilia A and her mother has not been excluded as a carrier, or if the patient has abnormally low FVIII activity (F8A / Coagulation Factor VIII Activity Assay, Plasma).

 

For females with bleeding symptoms and no known personal or family history of hemophilia A, consider BDIAL / Bleeding Diathesis Profile, Limited  or the specific factor assays.

 

Prenatal genetic testing should NOT be performed without the prior identification of a familial hemophilia alteration because diagnostic prenatal testing requires an invasive procedure that carries a small but real risk of inducing spontaneous abortion.

Additional Testing Requirements

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

Shipping Instructions

Prenatal Specimens: Advise Express Mail or equivalent if not on courier service. Prenatal specimens can be sent Monday through Thursday and must be received by 3 p.m. CST on Friday in order to be processed appropriately.

Blood: Ambient and refrigerate specimens must arrive within 7 days  and frozen specimens must arrive within 14 days.

Collect and package specimen as close to shipping time as possible.

Necessary Information

Hemophilia A Patient Information (T712) is required, see Special Instructions. 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
Defines the optimal specimen required to perform the test and the preferred volume to complete testing

Submit only 1 of the following specimens:

 

Specimen Type: Peripheral blood or cord blood

Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD) or light-blue top (sodium citrate)

Specimen Volume: 3 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send specimen in original tube.

Specimen Stability Information: Ambient (preferred) 7 days/Refrigerated 7 days/Frozen 14 days

 

Due to the complexity of prenatal testing, consultation with the laboratory is required for all prenatal testing.

 

Specimen Type: Amniotic fluid

Supplies: Refrigerate/Ambient Shipping Box, 5 lb (T329)

Container/Tube: Amniotic fluid container

Specimen Volume: 10-20 mL

Collection Instructions:

1. Optimal timing for specimen collection is during 14 to 18 weeks of gestation, but specimens collected at other weeks of gestation are also accepted.

2. Discard the first 2 mL of amniotic fluid.

3. Place the tubes in a Styrofoam container.

4. Fill remaining space with packing material.

5. Unavoidably, about 1% to 2% of mailed-in specimens are not viable.

6. Bloody specimens are undesirable.

7. If the specimen does not grow in culture, you will be notified within 7 days of receipt.

Additional Information:

A separate culture charge will be assessed under CULAF / Culture for Genetic Testing, Amniotic Fluid

Specimen Stability Information: Refrigerated (preferred) <24 hours/Ambient <24 hours

 

Specimen Type: Chorionic villi

Supplies: CVS Media (RPMI) and Small Dish (T095)

Container/Tube: 15-mL tube containing 15 mL of transport media

Specimen Volume: 20-30 mg

Collection Instructions:

1. Collect specimen by the transabdominal or transcervical method.

2. Transfer the chorionic villi specimen to a Petri dish containing transport medium.

3. Using a stereomicroscope and sterile forceps, assess the quality and quantity of the villi and remove any blood clots and maternal decidua.

Additional Information:

A separate culture charge will be assessed under CULFB / Fibroblast Culture for Genetic Testing

Specimen Stability Information: Refrigerated (preferred) <24 hours/Ambient <24 hours

 

Specimen Type: Confluent cultured cells

Container/Tube: T-25 flask

Specimen Volume: 2 Flasks approximately 90% confluent

Collection Instructions: Submit confluent cultured cells from another laboratory

Additional Information: There will be no culture charge.

Specimen Stability Information: Ambient (preferred) <24 hours/Refrigerated <24 hours

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Forms

1. Hemophilia A Patient Information (T712) is required, see Special Instructions.

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 in Special Instructions:

-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.

Specimen Minimum Volume
Defines the amount of sample necessary to provide a clinically relevant result as determined by the Testing Laboratory

Blood: 1 mL

Amniotic fluid: 10 mL

Chorionic villi: 20 mg

Confluent cultured cells: 2 full flasks

Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected

All specimens will be evaluated at Mayo Clinic Laboratories for test suitability.

Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included

Specimen Type Temperature Time Special Container
Varies Varies (preferred)

Useful For
Suggests clinical disorders or settings where the test may be helpful

Molecular confirmation of a clinical diagnosis of hemophilia A in affected male patients

 

Identification of the causative alteration in the F8 gene for prognostic and genetic counseling purposes

 

Helping determine hemophilia A carrier status for female patients with a family history of hemophilia A

 

Molecular prenatal confirmation of hemophilia A

Genetics Test Information
Provides information that may help with selection of the correct genetic test or proper submission of the test request

This test detects pathogenic alterations within the F8 gene to delineate the underlying molecular defect in a patient with a laboratory diagnosis of hemophilia A and for determining carrier status for female patients with a family history of hemophilia A. This test may also be used to prenatally identify and confirm hemophilia A in a male fetus at risk for inheriting the disease. Diagnostic prenatal testing for female fetuses is typically medically unnecessary and not performed except in very rare cases where the fetus is known to be at risk of inheriting a pathogenic F8 alteration from both parents.

 

The gene target for this test is:

Gene name (transcript): F8 (GRCh37 [hg19] NM_000132)

Chromosomal location: Xq28

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

Prenatal genetic testing is not routinely performed without the prior identification of a familial hemophilia alteration in an affected male relative or a female relative who is a confirmed carrier of the alteration. Requests for this prenatal testing without a known familial alteration are performed at the discretion of a Molecular Hematopathology Laboratory Director.

 

For any cord blood or prenatal specimen that is received, maternal cell contamination studies will be added. A maternal whole blood sample is required to perform this test.

 

If amniotic fluid is received, amniotic fluid culture for genetic testing will be added and charged separately. If chorionic villus specimen is received, fibroblast culture for genetic testing will be added and charged separately.

 

The following algorithms are available in Special Instructions:

-Hemophilia Testing Algorithm (for testing affected male patients)

-Hemophilia Carrier Testing Algorithm (for female patients with a family history of hemophilia)

Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

Hemophilia A (HA) is a bleeding diathesis that most commonly affects male individuals. Symptoms include soft tissue bleeding and articular hemorrhage, such as joint bleeds, deep muscle hematomas, intracranial bleeding, prolonged oozing after surgery, and unexplained gastrointestinal (GI) bleeding. In male patients with severe deficiency, spontaneous bleeding may occur. In individuals with mild HA, bleeding may occur only after surgery or trauma. In severe hemophilia, affected male patients typically present with these symptoms within the first 2 years of life. In moderate hemophilia, affected male patients will usually present in their toddler years. Mild hemophiliacs are typically diagnosed later in life and sometimes well into adulthood.

 

HA is an X-linked recessive disorder that affects approximately 1 in 10,000 live male births, among all ethnic populations. Female carriers of HA alterations have a 50% chance of passing on the alteration to each child they have. If the child is male, there is a 50% chance of him inheriting the alteration and being affected by HA. If the child is female, there is a 50% chance of her inheriting the alteration and being a carrier of HA. While female carriers generally do not have bleeding symptoms, they are at risk of having affected sons. However, not all women with an affected son are germline carriers of a F8 alteration as de novo alterations in F8 occur. Overall, there is a 2% to 20% chance of her not being a carrier of an alteration associated with HA, depending on the type of alteration in the son. Importantly, there is a small risk for recurrence even when the familial F8 alteration is not identified in the mother of the affected patient due to the possibility of germline mosaicism. All of the daughters of a man with HA will inherit the disease-causing alteration. None of his sons will inherit the alteration or be affected by HA. Daughters of a man with hemophilia are considered obligate carriers because it is a virtual certainty that they carry the alteration by virtue of their biological relationship with their father. If a woman has a brother or maternal nephew who is affected with hemophilia and then has a son with hemophilia, she too is considered an obligate carrier.

 

HA is caused by a deficiency of clotting factor VIII (FVIII), an essential blood coagulation protein. Factor VIII increases the catalytic activity of factor IXa to convert factor X to Xa by more than 100,000-fold, propelling further steps in the coagulation cascade. FVIII is encoded by the factor VIII (F8) gene. Approximately 98% of patients with a diagnosis of HA are found to have an alteration in F8.

 

Hemophilia is classified according to bleeding severity, which correlates with FVIII activity levels. Severe HA is associated with FVIII activity levels of less than 1% in a male. Moderate HA is associated with 1% to 5% activity. Mild hemophilia is associated with 5% to 40% of factor VIII activity.

 

Affected male patients are diagnosed with hemophilia A on the basis of their FVIII activity (F8A / Coagulation Factor VIII Activity Assay, Plasma) and clinical evaluation, while obligate carrier female patients are identified by family history assessment. For affected male patients, genetic testing to identify the causative alteration is indicated if factor studies confirm an abnormally low FVIII clotting activity (less than 40%) and von Willebrand factor antigen testing is normal. In affected male patients, there is good correlation between genotype, FVIII plasma levels, and hemorrhagic risk.

 

Genetic testing for HA in women should only be considered if she has a first-degree male relative diagnosed with HA, if there is a maternal family history of hemophilia A and her mother has not been excluded as a carrier, or if she has abnormally low FVIII activity (F8A / Coagulation Factor VIII Activity Assay, Plasma). Carrier status in females is not excluded if the female patient has normal FVIII activity. In females, the wide range of normal factor FVIII activity in women precludes an accurate assessment of carrier status, thus making molecular testing essential in assessment of carrier status in women maternally related to males affected by HA. Carrier testing is made much easier and conclusive when the specific familial alteration has been identified in an affected male relative of obligate carrier.

 

For prenatal testing, a specific familial alteration should be known in order to perform prenatal testing on any male fetus at risk of inheriting a genetic alteration causing hemophilia from his mother. This is because diagnostic prenatal testing requires an invasive procedure (ie, amniocentesis or chorionic villi sampling) that carries a small but real risk of inducing spontaneous abortion. Thus, prior to any prenatal genetic testing, every effort should be made to 1) identify the familial alteration in an affected male relative or in an obligate carrier and 2) confirm the mother carries the alteration. This ensures an invasive procedure is not performed unnecessarily on a pregnancy that is not at risk for hemophilia and that the test results are informative and conclusive.

 

Causes of acquired (non-genetic) HA that should be excluded prior to genetic testing include heparin use, disorders associated with antibodies to clotting factors such as systemic lupus erythematosus or antiphospholipid syndrome, pregnancy or the postpartum period, rheumatic disease, tumors or hematologic malignancies, use of certain drugs (eg, penicillin, sulfamides, phenytoin, interferon, fludarabine).

 

Obtaining a medical genetics or hematology (coagulation) consultation prior to ordering is advised.

Reference Values
Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

An interpretive report will be provided

Interpretation
Provides information to assist in interpretation of the test results

An interpretive report will be provided.

 

Evaluation and categorization of variants is performed using the most recent published American College of Medical Genetics and Genomics (ACMG) recommendations as a guideline. Variants are classified based on known, predicted, or possible pathogenicity and reported with interpretive comments detailing their potential or known significance.

 

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.

Cautions
Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

Clinical:

Some individuals may have a mutation that is not identified by the methods performed. The absence of a variant, therefore, does not eliminate the possibility of hemophilia A (HA). This assay does not distinguish between germline and somatic alterations, particularly with variant allele frequencies (VAF) significantly lower than 50%. Test results should be interpreted in context of clinical findings, family history, and other laboratory data. Misinterpretation of results may occur if the information provided is inaccurate or incomplete.

 

Technical Limitations:

Next-generation sequencing (NGS) may not detect all types of genetic variants. Additionally, rare alterations may be present that could lead to false negative or positive results. Therefore, test results should be interpreted in the context of activity and antigen measurements, clinical findings, family history, and other laboratory data. If results do not match clinical findings, consider alternative methods for analyzing these genes, such as Sanger sequencing or large deletion/duplication analysis. Misinterpretation of results may occur if the information provided is inaccurate or incomplete.

 

If multiple alterations are identified, NGS is not able to distinguish between alterations that are found in the same ("in cis") and alterations found on different alleles ("in trans"). This limitation may complicate diagnosis or classification and has implications for inheritance and genetic counseling. To resolve these cases, molecular results must be correlated with clinical history, activity and antigen measurements, and family studies.

 

Unless reported or predicted to cause disease, alterations found deep in the intron or alterations that do not result in an amino acid substitution are not reported. These and common polymorphisms identified for this patient are available upon request.

 

Reclassification of Variants Policy:

At this time, it is not standard practice for the laboratory to systematically review likely pathogenic variants or variants of uncertain significance that are detected and reported. The laboratory encourages health care providers to contact the laboratory at any time to learn how the status of a particular variant may have changed over time.

Clinical Reference
Recommendations for in-depth reading of a clinical nature

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;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;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. Oldenburg J, Rost S, El-Maarri O, et al: De novo factor VIII gene intron 22 inversion in a female carrier presents as a somatic mosaicism. Blood 2000;96(8):2905-2906

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

6. Konkle BA, Huston H, Fletcher SN: Hemophilia A. In GeneReviews. Edited by MP Adam, HH Ardinger, RA Pagon et al. University of Washington, Seattle. Updated 2017 June 17. Accessed March 2020. Available at ncbi.nlm.nih.gov/books/NBK1404/

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Method Description
Describes how the test is performed and provides a method-specific reference

Next-generation sequencing and/or PCR and Sanger sequencing are performed.

 

Regions of homology, high guanine-cytosine (GC)-rich content, and repetitive sequences may not provide accurate sequence. Therefore, all reported alterations detected by next-generation sequencing in these regions are confirmed by an independent reference method. However, this does not rule out the possibility of a false-negative result in these regions.

 

Sanger sequencing is used to confirm alterations detected by next-generation sequencing when appropriate.(Unpublished Mayo method)

PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information

No

Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.

Monday through Friday

Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

21 to 28 days

Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

DNA: Indefinitely

Performing Laboratory Location
Indicates the location of the laboratory that performs the test

Rochester

Fees
Several factors determine the fee charged to perform a test. Contact your U.S. or International Regional Manager for information about establishing a fee schedule or to learn more about resources to optimize test selection.

  • Authorized users can sign in to Test Prices for detailed fee information.
  • Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
  • Prospective clients should contact their Regional Manager. For assistance, contact Customer Service.

Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.

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

CPT Code Information
Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.

CPT codes are provided by the performing laboratory.

81407

LOINC® Information
Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the order and results codes of this test. LOINC values are provided by the performing laboratory.

Test Id Test Order Name Order LOINC Value
F8NGS F8 Gene, Full Gene NGS 94234-2
Result Id Test Result Name Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
113044 F8NGS Result 50397-9
113038 Alterations Detected 82939-0
113036 Interpretation 69047-9
113039 Additional Information 48767-8
113040 Method 85069-3
113041 Disclaimer 62364-5
113042 Panel Gene List 21673-9
113043 Reviewed By 18771-6

Test Setup Resources

Setup Files
Test setup information contains test file definition details to support order and result interfacing between Mayo Clinic Laboratories and your Laboratory Information System.

Excel | Pdf

Sample Reports
Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports
International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports