Factor VIII Inhibitor Profile, Comprehensive

CPT: 85240; 85730; 85732(x3)
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Test Details

Synonyms

  • Bethesda Titer

Test Includes

Activated partial thromboplastin time (aPTT); aPTT 1:1 mix with normal plasma; aPTT 1:1 incubated mix with normal plasma; aPTT 1:1 mix with saline; factor VIII activity; factor VIII Bethesda titer

Use

Confirmation and characterization of factor VIII inhibitor

Limitations

If there is residual coagulation factor activity, it could falsely lower the Bethesda titer result. Lupus anticoagulant activity and antithrombotic agents that function as inhibitors should be ruled out prior to assaying for factor inhibitors in order to avoid factitiously positive results. Some inhibitors may fully neutralize factors in less than two hours. Autoimmune inhibitors that demonstrate second order kinetics cannot always be accurately measured in the Bethesda titer system.6

Methodology

The factor VIII inhibitor (Bethesda titer) assay is performed using an activated partial thromboplastin time (aPTT)-based test system.6 Serial dilutions are made of patient plasma with veronal buffered saline, then mixed with normal plasma containing close to 100% factor VIII activity and are then incubated for one hour. An aPTT-based factor VIII assay using factor VIII-depleted plasma substrate is then performed on these incubated mixtures. Results are compared to those of incubated normal plasma. One Bethesda unit is defined as the amount of factor VIII inhibitor that neutralized 0.5 IU of factor VIII in this system. The number of serial dilutions tested is based on the anticipated level of the inhibitor.

Additional Information

Factor inhibitors (also called circulating anticoagulants or inactivators) are endogenously produced antibodies that interfere with coagulation in vivo or in vitro.6 These inhibitors are frequently specific for their respective coagulation factors. Specific factor inhibitors can be classified as neutralizing or non-neutralizing. Neutralizing inhibitors interact with the functional component of the coagulant protein while non-neutralizing antibodies react with the protein somewhere other than the functional epitopes. Factor inhibitors are not normally present in plasma. When present, factor inhibitors are measured by Bethesda titer units (BU). Titers <5 BUs are classified as low responders, titers >10 BUs as high responders. Factor VIII inhibitors may arise as IgG4 (noncomplement fixing) alloantibodies or autoantibodies. Factor VIII alloimmunization occurs in 15% to 20% of hemophilia, when the patient's plasma factor VIII concentration is <1%. Alloimmunization is idiosyncratic, affecting some, but not all, severe hemophilia A patients. Autoimmune factor VIII inhibitors occur in 1 of 1,000,000 individuals, primarily in pregnancy and the postpartum period, in patients older than 55 years of age, and in people with primary autoimmune disorders.

Porcine factor VIII inhibitors may arise in hemophilia A patients treated with porcine factor VIII (HyateC®) therapy. HyateC® is no longer available from the manufacturer. Alloimmune factor VIII inhibitors are suspected when hemophilia A patients' symptoms increase in severity, or bleeding continues despite infusion of ever-increasing dosages of factor concentrates. The normal plasma half-life of factor VIII, is 12 hours; however, hemophilia A patients with inhibitors may have undetectable factor VIII levels a few minutes after factor VIII-concentrate infusion and may require specialized therapy to halt acute bleeding. Neutralizing factor inhibitors form covalent complexes with their target coagulant factor molecules, rendering the factors nonfunctional. For alloimmune factor VIII inhibitor, this is a first order reaction with complete in vitro inactivation of available factor VIII. Autoimmune factor VIII inhibitors generally exhibit in vitro second order kinetics, achieving equilibrium with factor VIII leaving some residual factor VIII unbound in the plasma. In vivo, however, autoimmune factor VIII inhibitors cause “acquired hemophilia” with symptoms as severe as the more common congenital severe hemophilia. The mortality rate for acquired hemophilia is up to 22%.

Specimen Requirements

Specimen

Plasma, frozen

Volume

6 mL

Container

Blue-top (sodium citrate) tube

Patient Preparation

The patient should not be anticoagulated. Do not draw from an arm with a heparin lock or heparinized catheter.

Collection

Citrated plasma samples should be collected by double centrifugation. Blood should be collected in a blue-top tube containing 3.2% buffered sodium citrate.1 Evacuated collection tubes must be filled to completion to ensure a proper blood to anticoagulant ratio.2,3 The sample should be mixed immediately by gentle inversion at least six times to ensure adequate mixing of the anticoagulant with the blood. A discard tube is not required prior to collection of coagulation samples, except when using a winged blood collection device (ie, "butterfly"), in which case a discard tube should be used.4,5 When noncitrate tubes are collected for other tests, collect sterile and nonadditive (red-top) tubes prior to citrate (blue-top) tubes. Any tube containing an alternate anticoagulant should be collected after the blue-top tube. Gel-barrier tubes and serum tubes with clot initiators should also be collected after the citrate tubes. Centrifuge for 10 minutes and carefully remove 2/3 of the plasma using a plastic transfer pipette, being careful not to disturb the cells. Deliver to a plastic transport tube, cap, and recentrifuge for 10 minutes. Use a second plastic pipette to remove the plasma, staying clear of the platelets at the bottom of the tube. Transfer the plasma into a LabCorp PP transpak frozen purple tube with screw cap (LabCorp N° 49482). Freeze immediately and maintain frozen until tested. To avoid delays in turnaround time when requesting multiple tests on frozen samples, please submit separate frozen specimens for each test requested.

Please print and use the Volume Guide for Coagulation Testing to ensure proper draw volume.

Storage Instructions

Freeze

Clinical Information

Footnotes

1. Adcock DM, Kressin DC, Marlar RA. Effect of 3.2% vs 3.8% sodium citrate concentration on routine coagulation testing. Am J Clin Pathol. 1997Jan; 107(1):105-110. 8980376
2. Reneke J, Etzell J, Leslie S, Ng VL, Gottfried EL. Prolonged prothrombin time and activated partial thromboplastin time due to underfilled specimen tubes with 109 mmol/L (3.2%) citrate anticoagulant. Am J Clin Pathol. 1998 Jun; 109(6):754-757. 9620035
3. National Committee for Clinical Laboratory Standardization. Collection, Transport, and Processing of Blood Specimens for Coagulation Testing and General Performance of Coagulation Assays; Approved Guideline. 5th ed. Villanova, Pa: NCCLS; 2008. Document H21-A5:28(5).
4. Gottfried EL, Adachi MM. Prothrombin time and activated partial thromboplastin time can be performed on the first tube. Am J Clin Pathol. 1997 Jun; 107(6):681-683. 9169665
5. McGlasson DL, More L, Best HA, Norris WL, Doe RH, Ray H. Drawing specimens for coagulation testing: Is a second tube necessary? Clin Lab Sci. 1999 May-Jun; 12(3):137-139. 10539100
6. Adcock DM, Bethel MA, Macy PA. Coagulation Handbook. Aurora, Colo: Esoterix−Colorado Coagulation; 2006.

References

Goodnight SH, Hathaway WE. Disorders of Hemostasis and Thrombosis: A Clinical Guide. 2nd ed. New York, NY: McGraw-Hill; 2001.
Green D, Lechner K. A Survey of 215 nonhemophiliac patients with inhibitors to factor VIII. Thromb Haemost. 1981 Jun 30; 45(3):200-203. 6792737
Hay CR, Ludlam CA, Colvin BT, et al. Factor VIII inhibitors in mild and moderate severity haemophilia. Thromb Haemost. 1998 Apr; 79(4):762-766. 9569189
Kasper CK, Aledort L, Aronson D, et al. Proceedings: A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. 1975 Nov 15; 34(2):612. 1198543
Kasper CK. Laboratory tests for factor VIII inhibitors, Their variation, significance, and interpretation. Blood Coagul Fibrinolysis. 1991; 51(suppl 1):7.
Triplett DA. Use of the dilute Russel viper venom time (dRVVT): Its importance and pitfalls. J Autoimmune. 2000 Sep; 15(2):173-178. 10968905

LOINC® Map

Order Code Order Code Name Order Loinc Result Code Result Code Name UofM Result LOINC
117157 F-VIII INH, Comprehensive 117158 aPTT sec 14979-9
117157 F-VIII INH, Comprehensive 117159 aPTT 1:1 Normal Plasma sec 5946-9
117157 F-VIII INH, Comprehensive 117160 aPTT 1:1 Mix Saline sec 43734-3
117157 F-VIII INH, Comprehensive 117163 aPTT 1:1 NP Mix, 60 Min,Incub. sec 30323-0
117157 F-VIII INH, Comprehensive 086264 Factor VIII Activity % 3209-4
Reflex Table for Factor VIII Activity
Order Code Order Name Result Code Result Name UofM Result LOINC
Reflex 1 117164 Factor VIII Bethesda Titer 117164 Factor VIII Bethesda Titer Bethesda 3204-5

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