1. A Multi-Laboratory Evaluation of a Chromogenic Factor X Assay for Monitoring Oral Anticoagulation Therapy DL McGlasson 1*; PN Shaklee 2; 1 59 th Clinical Research Squadron, Wilford Hall Medical Center, Lackland AFB, TX; 2 Research Laboratory, BioCascade Incorporated, Arlington, WI This information is for education only and is not a product endorsement.

2. INTRODUCTION Monitoring subjects with the presence of a lupus anticoagulant (LA) on oral anticoagulant therapy (OAT) with a prolonged clottable PT/INR assay may be difficult. The chromogenic factor X assay has been shown to be a useful tool in the management of patients with lupus anticoagulants (inhibitors) who are receiving oral anticoagulant therapy (OAT). Useful to monitor subjects converting from Agatobran and Lepirudin (direct thrombin inhibitors) to coumadin.

3. INTRODUCTION: CONTINUED A multi-site and multi-instrument validation of the chromogenic DiaPharma Factor X Assay kit (DFX) was undertaken in order to evaluate the utility of the assay for measuring FX in subjects receiving OAT. The chromogenic FX assay has been suggested as an alternative approach to monitor patients on OAT who have the presence of an LA.

4. MATERIALS AND METHODS The DFX micro titer method was compared to a clottable FX (CFX) method in Laboratory 1. All clottable assays were performed on the Diagnostica Stago Inc., STA automated coagulation analyzer using Neoplastine CI+ with a low ISI and other Stago reagents. All testing was performed on citrated platelet-poor plasma with platelet counts <10 9 /L A normal range was established with 30 normal subjects with no known clinical abnormalities.

5. MATERIALS AND METHODS: 2 Clinical sensitivity was tested using 30 specimens subjects on OAT. The specimens were assayed for FX levels by DFX and CFX and PT/INR tests were performed. Thirty-one specimens positive for the presence of either hemolysis (n=9), icteric color (n=2), lipemia (n=5), heparin (n=10) or LAs (n=5) were analyzed by DFX and CFX to check for the influence of interfering substances. Nineteen subjects with the presence of an LA on OAT and an unstable INR with specimens taken at 8 time points were evaluated by both methods.

6. MATERIALS AND METHODS: 3 Laboratory 2 used an STA compact and reagents from Diagnostica Stago, Inc., to evaluate both the CFX and DFX methods. A normal range was established using 25 normal subjects on both methods. Fifty-five subjects on OAT were evaluated by both the CFX and DFX methods. Precision and accuracy testing using different levels of FX were analyzed by all methods at both institutions.

7. Lab One RESULTS: Normal range Laboratory One NormalsN=30Correlation Range (%) Mean (%) 0.720 Chromogenic72.0-137.6104.8 Clottable94.1-159.7126.9

8. Lab One RESULTS: OAT subjects Laboratory One OATN=30 INR=1.7-5.9 Correlation Range (%) Mean (%) 0.903 Chromogenic19.3-62.531.0 Clottable7.0-48.013.9

9. Lab One RESULTS: OATsubjects with presence of an LA Laboratory One OAT with LA N=152Correlation Range (%) Mean (%) 0.841 Chromogenic7.0- 122.0 33.1 Clottable2.7- 101.0 22.8

10. Lab One RESULTS: Interfering substances Laboratory One Interfering substances N=31 INR= 1.0-1.2 T- test/Correlation Range (%) Mean (%) P=0.59/0.906 Chromogenic101.2-126.4113.8 Clottable97.4-120.7109.5

11. Lab Two RESULTS: Normal range Laboratory Two NormalsN=25Correlation Range (%) Mean (%) 0.871 Chromogenic83.0-147.0120.4 Clottable69.0-139.0105.7

12. Lab Two RESULTS: OAT Laboratory Two OATN=55Correlation Range (%) Mean (%) 0.948 Chromogenic17.0-65.032.5 Clottable2.0-41.010.4

13. RESULTS: Precision and Accuracy Testing Lab One and Two Precision Data CV (%) Laboratory One Laboratory Two Chromogenic1.9-10.42.5-5.1 Clottable<5.0%4.6-9.2

14. RESULTS: Precision and Accuracy Testing Lab One and Two Precision Data: Laboratory 1 performed precision testing using times 10 replicates on 6 specimens, run on the DFX in the range of 10- 120% activity. CV ranged from 1.9-10.4%. Using 5 known standards for the DFX, assay accuracy ranged from 99.2-100.8% recovery. Laboratory 2 performed precision testing on 3 levels of FX (n=12) for DFX (CV=2.5-5.1%), CFX (CV=4.6-9.2%)

15. CONCLUSIONS The present studies of the DFX kit demonstrated the assays robustness, precision, accuracy and utility for monitoring patients on OAT with and without interfering substances, the presence of an LA or unstable INR. This assay should offer health care providers an option for monitoring patients receiving OAT, especially those where INR values may not be reliable when an LA is present, and when bridging Agatroban® subjects to warfarin.

16. REFERENCES Moll S and Ortel. Monitoring Warfarin Therapy in patients with Lupus Anticoagulants; Annals of Internal Medicine. August 1, 1997, 127(3). Thom J, Ivey L, Gilmore G, Eikelboom JW. Evaluation of the phospholipid-rich dilute Russell’s Viper Venom assay to monitor oral anticoagulation in patients with lupus anticoagulant. Blood Coagulation and Fibrinolysis 2004,, 15:353-357. Sanfelippo MJ, Sennet J, McMahon EJ. Falsely Elevated INRs in Warfarin-Treated Patients with the Lupus Anticoagulant. Wisconsin Medical Journal, June 2000:62-64, 43.