Test Id : 8INHE

Testing begins with coagulation factor VIII activity assay with dilutions to evaluate assay inhibition; if the factor VIII activity assay is normal or increased, a technical interpretation will be provided.

If the factor VIII activity assay is decreased, an inhibitor screen will be performed at an additional charge to look for specific factor VIII inhibition and a professional interpretation will be provided. If specific inhibition is apparent, the titer of the inhibitor will be determined.

This test is for factor VIII inhibitors only. If the patient is known to have hemophilia A, this is the correct test to order. If the presence or type of inhibitor is unknown, first order APROL / Prolonged Clot Time Profile, Plasma. When screening studies are needed for patients with known hemophilia B, order 9INHE / Factor IX Inhibitor Evaluation, Plasma.

Send all vials in the same shipping container.

Specimen Type: Platelet-poor plasma

Patient Preparation:

1. Patient must not be receiving Coumadin (warfarin) or heparin therapy

2. Fasting preferred

Collection Container/Tube: Light-blue top (3.2% sodium citrate)

Submission Container/Tube: Plastic vials

Specimen Volume: 3 mL in 3 plastic vials, each containing 1 mL

Collection Instructions:

1. Specimen must be collected prior to factor replacement therapy.

2. For complete instructions, see Coagulation Guidelines for Specimen Handling and Processing.

3. Centrifuge, transfer all plasma into a plastic vial, and centrifuge plasma again.

4. Aliquot plasma (1-2 mL per aliquot) into 3 separate plastic vials, leaving 0.25 mL in the bottom of centrifuged vial.

5. Freeze plasma immediately (no longer than 4 hours after collection) at -20 degrees C or, ideally, at -40 degrees C or below.

Additional Information:

1. Double-centrifuged specimen is critical for accurate results as platelet contamination may cause spurious results.

2. Each coagulation assay requested should have its own vial.

• Coagulation Guidelines for Specimen Handling and Processing

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

2 Plastic vials, each containing 1 mL

Detecting the presence and titer of a specific factor inhibitor directed against coagulation factor VIII

This test is not useful for the detection of a lupus-like circulating anticoagulant inhibitor, a nonspecific circulating anticoagulant, or other inhibitors that are not specific for coagulation factors.

Testing begins with coagulation factor VIII activity assay with dilutions to evaluate assay inhibition; if the factor VIII activity assay is normal or increased, a technical interpretation will be provided.

If the factor VIII activity assay is decreased, an inhibitor screen will be performed at an additional charge to look for specific factor VIII inhibition and a professional interpretation will be provided. If specific inhibition is apparent, the titer of the inhibitor will be determined.

Factor VIII (FVIII) inhibitors are IgG antibodies directed against coagulation FVIII that typically result in development of potentially life-threatening hemorrhage. These antibodies may develop in 1 of 4 different patient populations:

-Patients with congenital FVIII deficiency (hemophilia A) in response to therapeutic infusions of factor VIII concentrate

-Older nonhemophiliac adult patients (not previously factor VIII deficient)

-Women in postpartum period

-Patients with other autoimmune illnesses

FACTOR VIII ACTIVITY ASSAY

Adults: 55-200%

Normal, full-term newborn infants or healthy premature infants typically have levels greater or equal to 40%.*

*See Pediatric Hemostasis References in Coagulation Guidelines for Specimen Handling and Processing.

FACTOR VIII INHIBITOR SCREEN:

Negative

GENERAL FACTOR BETHESDA UNITS:
< or =0.5 Bethesda Units

Normally, there is no inhibitor (ie, negative result).

If the screening assays indicate the presence of an inhibitor, it will be quantitated and reported in Bethesda (or equivalent) units.

Occasionally, a potent lupus-like anticoagulant may cause false-positive results for a specific factor inhibitor (eg, factor VIII or IX).

1. Hoffman R, Benz Jr EJ, Silberstein LE, et al, eds. Hematology: Basic Principles and Practice. 7th ed. Elsevier; 2018

2. Kasper CK. Treatment of factor VIII inhibitors. Prog Hemost Thromb. 1989;9:57-86

3. Peerschke EI, Castellone DD, Ledford-Kraemer M, et al. Laboratory assessment of FVIII inhibitor titer. Am J Clin Pathol. 2009;131(4):552-558. doi:10.1309/AJCPMKP94CODILWS

4. Pruthi RK, Nichols WL. Autoimmune factor VIII inhibitors. Curr Opin Hematol. 1999;6(5):314-322. doi:10.1097/00062752

5. Kottke-Marchant. K, ed. Laboratory Hematology Practice. Wiley Blackwell Publishing; 2012

The factor VIII assay is performed on the Instrumentation Laboratory ACL TOP using the activated partial thromboplastin time (aPTT) method and a factor-deficient substrate. Patient plasma is combined and incubated with a factor VIII-deficient substrate (normal plasma depleted of factor VIII by immunoadsorption) and an aPTT reagent. After a specified incubation time, calcium is added to trigger the coagulation process in the mixture. Then the time to clot formation is measured optically using a wavelength of 671 nm.(Owen CA Jr, Bowie EJW, Thompson JH Jr. Diagnosis of Bleeding Disorders. 2nd ed. Little, Brown and Company; 1975; Cielsa B. Defects of plasma clotting factors. In: Hematology in Practice. 3rd ed. FA Davis; 2019:chap 17)

The factor VIII inhibitor screen consists of measuring the difference in factor VIII activity (partial thromboplastin time-based assay) before and after incubation of a mixture of normal plasma and patient's plasma for 1 hour at 37 degrees C. For optimal sensitivity, the factor VIII value of the normal plasma is adjusted to approximately 20%, because the factor VIII assay is more sensitive in this area of the curve. In addition, an excess of patient's plasma will make the test more sensitive to small amounts of inhibitors.(Owen CA Jr, Bowie EJW, Thompson JH Jr. The Diagnosis of Bleeding Disorders. 2nd ed. Little, Brown, and Company; 1975:143-145; Cielsa B. Defects of plasma clotting factors. In: Hematology in Practice. 3rd ed. FA Davis; 2019:chap 17)

If the inhibitor screen is positive for an inhibitor of factor VIII, the inhibitor will be quantitated by the Bethesda assay. In the Bethesda procedure, inhibitors are quantified by mixing equal volumes of serially diluted plasma with normal plasma. This mixture is incubated 2 hours at 37 degrees C, and its factor VIII activity is measured and compared to a control run at the same time. The difference between the factor VIII activity of the patient's incubation mixture and that of the control is used to calculate the titer. The residual factor VIII activity is converted to Bethesda units: 50% residual factor VIII is equal to 1 Bethesda unit. Assays using the same basic principle as the Bethesda assay are used to quantitate the inhibitors of the other coagulation factors. (Kasper CK, Aldedort LM, Counts RB, et al. A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. 1975;34:869-872; Cielsa B. Defects of plasma clotting factors. In: Hematology in Practice. 3rd ed. FA Davis; 2019:chap 17)

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This test has been modified from the manufacturer's instructions. Its performance characteristics were 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.

85390-Factor VIII Tech Interp

85240-Factor VIII activity assay

85335-Bethesda titer (if appropriate)

85335-Factor VIII inhibitor screen (if appropriate)

85390-Factor VIII Professional Interp (if appropriate)