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CPT:
85250; 85335; 85730; 85732(x3)
Synonyms
- Bethesda Titer
Test Includes
Activated partial thromboplastin time (aPTT); aPTT 1:1 mix with normal plasma; aPTT 1:1 mix with normal plasma (60-minute control); aPTT 1:1 mix with saline; factor IX activity; factor IX Bethesda titer
Expected Turnaround Time
2 - 5 days
Turnaround time is defined as the usual number of days from the date of pickup of a specimen for testing to when the result is released to the ordering provider. In some cases, additional time should be allowed for additional confirmatory or additional reflex tests. Testing schedules may vary.
Related Documents
For more information, please view the literature below.
Procedures for Hemostasis and Thrombosis: A Clinical Test Compendium
Specimen Requirements
Specimen
Plasma, frozen
Volume
6 mL (2 mL in each of three tubes)
Container
Blue-top (sodium citrate) tubes
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 No. 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. Stable at room temperature for four hours.
Patient Preparation
The patient should not be anticoagulated. Do not draw from an arm with a heparin lock or heparinized catheter.
Test Details
Use
Confirmation and characterization of factor IX 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 be accurately measured in the Bethesda titer system.6
Methodology
The factor IX inhibitor (Bethesda titer) assay is performed using an activated partial thromboplastin time (aPTT)-based system.6 Serial dilutions are made of patient plasma with veronal buffered saline, then mixed with normal plasma containing close to 100% factor IX activity and are then incubated for two hours. An aPTT-based factor IX assay using factor IX-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 IX inhibitor that neutralized 0.5 IU of factor IX 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 IX inhibitors arise as alloantibodies in 3% to 5% of hemophilia B (Christmas disease) patients in response to factor IX concentrate, prothrombin complex concentrate, or fresh frozen plasma therapy.
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. 1997 Jan; 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.
LOINC® Map
| Order Code | Order Code Name | Order Loinc | Result Code | Result Code Name | UofM | Result LOINC |
|---|---|---|---|---|---|---|
| 500390 | Factor IX Inhibitor Panel | 96459-3 | 500513 | APTT | sec | 14979-9 |
| 500390 | Factor IX Inhibitor Panel | 96459-3 | 500363 | APTT 1:1 NP | sec | 5946-9 |
| 500390 | Factor IX Inhibitor Panel | 96459-3 | 500364 | APTT 1:1 Saline | Sec | 43734-3 |
| 500390 | Factor IX Inhibitor Panel | 96459-3 | 500657 | APTT 1:1 NP, 60 min Incubation | sec | 30323-0 |
| 500390 | Factor IX Inhibitor Panel | 96459-3 | 500658 | APTT 1:1NP 60min Inc. Control | sec | 44950-4 |
| 500390 | Factor IX Inhibitor Panel | 96459-3 | 500672 | Factor IX Activity | % | 3187-2 |
| 500390 | Factor IX Inhibitor Panel | 96459-3 | 500445 | Factor IX Bethesda Titer | Bethesda | 3185-6 |
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