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. INRs can be falsely elevated in patients with an lupus anticoagulants (LA). The chromogenic factor X assay has been shown to be a useful tool in the management of patients who are receiving oral anticoagulant therapy (OAT). Useful to monitor subjects converting from Agatobran and Lepirudin (direct thrombin inhibitors) to coumadin.

3. INTRODUCTION LA subjects can produce antibodies that interfere with the phospholipid-dependent clotting reactions that are part of PT assays. Antiphospholipid antibodies have been shown to artificially prolong the clotting times of PT assays, making the PT/INR unreliable for monitoring anticoagulation. When monitoring OAT subjects to prevent sub- therapeutic or supra-therapeutic dosing these patients should be individually monitored with an assay such as the chromogenic FX assay that is insensitive to the presence of an LA and does not require a phospholipid surface..

4. Diapharma Factor X Principle RVV 1. FX → FXa Ca 2+ FXa 2. FXa substrate → peptide + pNA Stage 1: Factor X is activated in the presence of calcium and the activator Russell’s Viper Venom (RVV) to FXa. Stage 2: The generated FXa hydrolyses the chromogenic substrate and liberates the chromophore, pNA. – The color is then read with a spectrophotometer at 405 nm. The intensity of the color is proportional to the FX activity in the sample. Factor X testing may be performed in a microtiter plate, or on automated analyzers.

5. 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.

6. 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.

7. 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.

8. 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.

9. Lab One RESULTS: Normal range Laboratory One NormalsN=30Correlation Range (%) Mean (%) 0.9015/0.720 Chromogenic72.0-137.6104.8 Clottable94.1-159.7126.9

10. Lab One RESULTS: OAT subjects Laboratory One OATN=30 INR=1.7-5.9 Correlation Range (%) Mean (%) 0.903/0.031 Chromogenic19.3-62.531.0 Clottable7.0-48.013.9

11. Lab One RESULTS: OATsubjects with presence of an LA Laboratory One OAT with LA N=152 INR 0.97-4.5 Correlation P=0.021 Range (%) Mean (%) 0.841 Chromogenic7.0- 122.0 33.1 Clottable2.7- 101.0 22.8

12. 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

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

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

15. 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

16. 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%)

17. SUBJECTS (N=80) NORMALS20 COUMADIN20 HEPARIN (UFH) 0.2-1.0 8 ELEVATED FIBRINOGEN (>500 mg/dl) 8 LUPUS ANTICOAGULANT 8 HEMOLYZED 4 LIPEMIC 4 RABBIT (elevated FX) 8

18. ANIARA (BIOPHEN) VS DIAPHARMA Descriptive stats DIA FX 1 DIA FX 2 BIO FX 1 BIO FX 2PTINR Mean 73.675.887.889.620.01.75 Standard Error 5.2678475.3602087.6378637.774831.0790920.118624 Standard Deviation 46.8216547.6425768.3151269.540199.651691.061008 Range 18118242544435.93.94 Minimum 19 76.50.41 Maximum 20020144445142.44.35 Sum 58155990702971681602.1140.19 Count 79 80 Confidence Level(95.0%) 10.4874810.6713515.2027815.475412.1478780.236116

19. ANIARA (BIOPHEN) VS DIAPHARMA T-TESTS DIA FX 1DIA FX 2BIO FX 1BIO FX 2 Mean73.6075975.8227887.862589.6 Variance2192.2672269.8144666.9564835.838 Observations79 80 Pearson Correlation0.9882100.988147 Hypothesized Mean Difference00 df7879 t Stat-2.69754-1.45475 P(T<=t) one-tail0.0042790.07485 t Critical one-tail1.6646251.664371 P(T<=t) two-tail0.0085570.1497 t Critical two-tail1.990847 1.99045

20. ANIARA (BIOPHEN) VS DIAPHARMA ANOVA GroupsCountSumAverageVariance DIA FX 179581573.607592192.267 DIA FX 279599075.822782269.814 BIO FX 180702987.86254666.956 BIO FX 280716889.64835.838 ANOVA Source of VariationSSdfMS FP-value F crit Between Groups15931.7435310.579 1.517660.20986 2.633364 Within Groups10987633143499.245

21. PRECISION ON QC QC STATSDIAP BIOPHEN STA-NSTA-PFX NORFX ABN Mean98.9166737.0833394.8695759.82609 Standard Error1.0319470.3556820.6233420.285708 Standard Deviation5.0554891.7424792.9894411.370208 Range206156 Minimum86348957 Maximum1064010463 Sum237489021821376 Count24 23 Confidence Level(95.0%)2.1347460.7357841.2927310.592522 CV%5.14.63.162.3

25. 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.

26. 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.