Inversion of Factor VIII Intron 22 Test at Chughtai Lab

Inversion of Factor VIII Intron 22 (Form NIBD) at Chughtai Lab

The Inversion of Factor VIII Intron 22 (Form NIBD) test is a highly specialized molecular genetic assay designed to detect the most common genetic mutation responsible for severe Hemophilia A. Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency or dysfunction of coagulation Factor VIII (F8), a critical protein in the blood clotting cascade. Genetic studies indicate that approximately 40% to 50% of all cases of severe Hemophilia A are caused by a specific structural rearrangement: the inversion of intron 22 within the F8 gene. Chughtai Lab, a premier diagnostic network in Pakistan, offers this advanced molecular test to provide definitive genetic confirmation for patients, carriers, and families affected by this debilitating bleeding disorder.

The F8 gene, located on the long arm of the X chromosome (Xq28), is highly complex. The intron 22 inversion occurs due to an intrachromosomal homologous recombination event. Within intron 22 of the F8 gene lies a sequence called int22h-1. Two additional, highly homologous copies of this sequence, known as int22h-2 and int22h-3, are located telomeric to the F8 gene. During meiosis, misalignment and subsequent crossing over between the intragenic int22h-1 and one of the extragenic copies (either int22h-2 or int22h-3) results in a complete inversion of the intervening DNA segment. This structural alteration completely disrupts the reading frame of the F8 gene, splitting it into two separate parts and preventing the synthesis of functional Factor VIII protein. The resulting clinical phenotype is severe Hemophilia A, characterized by spontaneous joint and muscle bleeds, prolonged bleeding after trauma, and a high risk of life-threatening hemorrhages.

By utilizing state-of-the-art molecular diagnostic technologies, Chughtai Lab performs this analysis with exceptional precision. The test is invaluable for confirming a clinical diagnosis in affected males, identifying heterozygous female carriers who may pass the mutation to their offspring, and facilitating prenatal diagnosis in high-risk pregnancies. Understanding the specific genetic mutation also plays a vital role in clinical management, particularly in predicting the risk of developing neutralizing antibodies (inhibitors) against therapeutic Factor VIII concentrates, which is a major complication in the treatment of Hemophilia A.

Clinical Procedure: What to Expect

Patient Preparation

Proper preparation is essential to ensure sample integrity and accurate molecular analysis. Patients undergoing the Inversion of Factor VIII Intron 22 test at Chughtai Lab should observe the following guidelines:

• No Fasting Required: There is no requirement for dietary restriction or fasting before this test. The patient may eat and drink normally.
• Clinical Documentation: It is highly recommended to provide a detailed clinical history, including previous Factor VIII activity levels, coagulation profile results (such as aPTT), and a comprehensive family pedigree chart if available.
• Informed Consent: Because this is a genetic test, informed consent must be signed by the patient or a legal guardian prior to sample collection.
• Medication History: Inform the laboratory staff of any recent blood transfusions or factor replacement therapies, although these do not typically interfere with DNA-based molecular testing.
• Form NIBD Requirements: Ensure that the specific referral form (Form NIBD) is fully completed by the referring hematologist or geneticist, as it contains essential clinical parameters required for accurate reporting.

During the Procedure

The collection and processing of the sample follow strict molecular biology protocols to prevent contamination and preserve genomic DNA:

• Sample Collection: A trained phlebotomist at Chughtai Lab will perform a standard venipuncture to collect a peripheral blood sample.
• Tube Type: The blood is drawn into an EDTA (ethylenediaminetetraacetic acid) tube (purple top), which prevents coagulation and preserves cellular integrity for DNA extraction.
• Volume: Approximately 3 to 5 mL of whole blood is required for the analysis.
• Patient Experience: The procedure is quick, typically taking less than five minutes, and involves minimal discomfort associated with a standard needle prick.
• DNA Extraction: Once received at the molecular diagnostics division of Chughtai Lab, genomic DNA is extracted from the white blood cells using high-purity extraction kits.
• Molecular Analysis: The extracted DNA undergoes specialized amplification techniques, such as Inverse Polymerase Chain Reaction (I-PCR) or Long-Range PCR, designed specifically to identify the structural rearrangement of intron 22.
• Quality Control: The assay includes internal controls to verify DNA quality and amplification efficiency, ensuring the highest level of diagnostic accuracy.

When is an Inversion of Factor VIII Intron 22 Test Performed?

Diagnosis of Severe Hemophilia A

Physicians request this test for male patients presenting with clinical signs of severe bleeding and laboratory findings showing Factor VIII activity levels below 1%. Confirming the intron 22 inversion provides a definitive genetic diagnosis, distinguishing Hemophilia A from other bleeding disorders with similar clinical presentations, such as von Willebrand disease or Factor XI deficiency.

Carrier Testing in Female Relatives

Since Hemophilia A is an X-linked recessive disorder, female relatives of an affected male (including mothers, sisters, and daughters) are at risk of being carriers. Because carriers typically have normal or near-normal Factor VIII activity levels, phenotypic coagulation tests are unreliable for carrier determination. This molecular test definitively identifies whether a female carries the intron 22 inversion, allowing for informed reproductive choices.

Prenatal Diagnosis for High-Risk Pregnancies

In families with a documented history of the F8 intron 22 inversion, pregnant carriers can opt for prenatal diagnosis. The test can be performed on fetal DNA obtained via chorionic villus sampling (CVS) at 10 to 12 weeks of gestation or amniocentesis at 15 to 18 weeks. This helps expectant parents and healthcare providers prepare for the birth and immediate management of an affected infant.

Predicting Inhibitor Development Risk

One of the most challenging complications in managing severe Hemophilia A is the development of neutralizing IgG antibodies (inhibitors) against infused Factor VIII. Patients with large structural gene disruptions, such as the intron 22 inversion, have a significantly higher risk (approximately 20% to 30%) of developing inhibitors compared to those with missense mutations. Identifying this mutation helps hematologists design proactive treatment strategies and monitor the patient closely.

Unexplained Prolonged Activated Partial Thromboplastin Time (aPTT)

When routine coagulation screening reveals an isolated, unexplained prolongation of the activated partial thromboplastin time (aPTT) in a male infant or child with a history of easy bruising or bleeding, this genetic test is indicated as part of the diagnostic workup. It provides a conclusive answer when baseline coagulation factor assays are ambiguous or difficult to perform due to age or prior factor administration.

What Does an Inversion of Factor VIII Intron 22 Test Detect?

The molecular genetic analysis is highly specific and is designed to detect, characterize, or rule out several genetic and clinical parameters:

• Presence of the F8 Intron 22 Type I inversion (resulting from recombination with the distal extragenic homolog int22h-3).
• Presence of the F8 Intron 22 Type II inversion (resulting from recombination with the proximal extragenic homolog int22h-2).
• Absence of the intron 22 inversion, ruling it out as the cause of Hemophilia A.
• Hemizygous affected status in male patients, confirming severe Hemophilia A.
• Heterozygous carrier status in female individuals, indicating one normal and one mutated F8 allele.
• Homozygous normal status in female individuals, ruling out carrier status for this specific mutation.
• Adequacy of genomic DNA yield and purity extracted from the blood sample.
• Successful PCR amplification of target genetic regions.
• Presence of atypical structural rearrangements within the F8 locus.
• Genetic confirmation of clinical severe phenotype.
• Correlation with low baseline Factor VIII coagulant activity.
• High-risk genetic profile for the development of Factor VIII inhibitors.
• Familial mutation linkage for cascade testing of at-risk relatives.
• Feasibility of pre-implantation genetic diagnosis (PGD) for future pregnancies.
• Clarification of sporadic versus inherited cases of severe Hemophilia A.
• Exclusion of other large deletions or duplications within the F8 gene.
• Genetic data necessary for enrollment in clinical trials for novel therapies, including gene therapy.
• Validation of prenatal diagnostic samples (CVS or amniotic fluid).
• Distinction between Hemophilia A and other inherited bleeding diatheses.
• Confirmation of genetic lineage in complex family pedigrees.

Turnaround Time and Report Access at Chughtai Lab

Due to the complex nature of molecular genetic testing, which involves DNA extraction, PCR amplification, and fragment analysis, the turnaround time for the Inversion of Factor VIII Intron 22 test is typically longer than routine blood tests. Chughtai Lab is committed to delivering accurate results within a clinically appropriate timeframe, usually within 10 to 14 working days.

Once the analysis is complete, the report undergoes a rigorous multi-step verification process by qualified molecular pathologists and genetic specialists. Patients and referring physicians can access the reports conveniently through multiple channels. Reports can be downloaded directly from the official Chughtai Lab website or accessed via the My Chughtai mobile application. Additionally, physical copies can be collected from any of the numerous Chughtai Lab collection centers located across Pakistan, or delivered directly to the patient’s registered address upon request.

Inversion of Factor VIII Intron 22 Findings Overview

The following table outlines the clinical interpretation of various findings obtained from the Inversion of Factor VIII Intron 22 molecular test:

Structure / Parameter Evaluated	Normal Findings	Possible Abnormal Findings
F8 Intron 22 Status (Male)	Inversion Not Detected (Normal F8 gene)	Inversion Detected (Type I or Type II); confirms severe Hemophilia A
F8 Intron 22 Status (Female)	Inversion Not Detected (Homozygous normal)	Inversion Detected (Heterozygous); confirms carrier status
DNA Quality & Concentration	Adequate DNA yield and high purity	Degraded DNA or low concentration (requires recollection)
PCR Amplification Control	Successful amplification of internal controls	Failed amplification (indicates PCR inhibition or technical error)
Factor VIII Activity Correlation	Normal activity levels (50% to 150%)	Extremely low activity levels (<1%) in affected males
Inhibitor Development Risk	Baseline risk associated with general population	Significantly elevated risk of developing neutralizing antibodies
Clinical Bleeding Phenotype	Normal hemostasis; no abnormal bleeding	Severe bleeding diathesis, frequent hemarthrosis, hematomas
Pedigree Risk Assessment	No genetic risk identified for offspring	50% risk of transmission to offspring from carrier females

Note: Diagnostic findings should always be interpreted by a qualified healthcare professional together with the patient’s symptoms, medical history, physical examination, laboratory investigations, previous imaging studies, and other relevant clinical information. Additional investigations or specialist consultation may be recommended depending on the findings.

Why Choose Chughtai Lab for Inversion of Factor VIII Intron 22 Testing?

• Experienced Healthcare Professionals: Our molecular diagnostics division is led by highly qualified pathologists, geneticists, and technologists specializing in inherited bleeding disorders.
• Patient-Focused Care: We prioritize patient comfort, confidentiality, and clear communication throughout the testing process.
• Quality Diagnostic Services: Chughtai Lab maintains strict quality control measures and participates in international external quality assurance programs.
• Professional Reporting: Reports are detailed, clinically structured, and verified by senior specialists to ensure absolute diagnostic accuracy.
• Modern Diagnostic Approach: We utilize state-of-the-art thermocyclers and genetic analysis platforms to perform high-resolution molecular testing.
• Comfortable Environment: Our nationwide network of diagnostic centers offers a clean, welcoming, and professional environment for sample collection.
• Convenient Location: With hundreds of collection centers across Pakistan, patients can easily access our services close to home.
• Commitment to Accurate Diagnosis: We are dedicated to providing reliable genetic insights that empower clinicians to make informed treatment decisions.