1. Effects of Pre-analytical Variables on the anti-Xa Chromogenic Assay when Monitoring Unfractionated Heparin and Low Molecular Weight Heparin Anticoagulation David L. McGlasson, MS, CLS/NCA United States Air Force This information is for education only and is not a product endorsement.

2. INTRODUCTION: The purpose of this study was to determine if the chromogenic anti-Factor Xa (anti-Xa) assay is less affected by pre- analytical variables in monitoring patients on unfractionated heparin (UFH) and low molecular weight heparin (LMWH) than the activated partial thromboplastin time (APTT).

3. INTRODUCTION APTT most commonly used assay to monitor UFH therapy. APTT usually cannot be reliably used to monitor LMWH therapy. Anti-Xa assay can be used to monitor LMWH and other heparin analogues and UFH. Previous studies have cited the interference of anticoagulants, factor deficiencies, interfering substances, specimen collection, APTT reagent sensitivity, and instrumentation on the APTT. Our study used different concentrations of anticoagulants, collection tubes and blood to anticoagulant ratio to see if the variables affected both the APTT and anti-Xa assay on the UFH and LMWH dosing regimens.

4. MATERIALS AND METHODS Forty-six subjects receiving either enoxaparin (LMWH) or UFH were randomly selected and consented for this study. Protocol was approved through the local IRB under the tenets of the Helsinki protocol for human subjects experimentation and monitored through the US Air Force Surgeon Generals Office. Twenty-six subjects were receiving LMWH and twenty individuals were receiving UFH.

5. MATERIALS AND METHODS: UFH subjects: 10 males and 10 females, 20-85 y/o. Range assayed: 0.05-2.0 IU/ml. LMWH subjects: 13 males and 13 females, 26- 91 y/o. Range assayed: 0.06-1.22 IU/ml. Pre-existing conditions included but not limited to coronary disease, antiphospholipid antibody syndrome, deep vein thrombosis, recurrent spontaneous abortion, pulmonary embolism. Some of the subjects were also on oral anticoagulant therapy.

6. MATERIALS AND METHODS: Each subject had 6 vacutainer collection tubes obtained in a single atraumatic venipuncture. Specimens were split into 3 groups: 3.8% sodium citrate, 3.2% sodium citrate and a CTAD tube. Each tube had a normal draw of 9:1 blood to anticoagulant ratio and a short draw of 6:1. Data was analyzed using descriptive statistics, t-test or ANOVA, and linear regression.

7. MATERIALS AND METHODS: Platelet-poor plasma was obtained by centrifuging the whole blood at 2500g for 15 minutes guaranteeing a platelet count of <10,000/uL and stored at –70°C until ready for testing. Specimens were thawed for 5 minutes at 37°C just prior to testing. All specimens had an APTT and a chromogenic anti- Xa assay performed on each specimen regardless of the type of heparin being given. Each specimen had an anti-Xa assay result for the UFH, LMWH and a HYBRID calibration curve run.

8. MATERIALS AND METHODS: APTT reagent used was the PTT-A® from Diagnostic-Stago, Inc. STA-Rotachrom® Heparin Assay, Diagnostic- Stago, Inc. Analayzer was an STA-R® automated coagulation analyzer. All collection tubes were non-wettable, siliconized glass obtained from BD Vacutainer Systems.

9. APTT RESULTS UFH SUBJECTS: ANOVA GROUPSAve: seconds P-value/F APTT 3.8 ND105.40.9481/0.9 APTT 3.8 SD127.2 APTT 3.2 ND107.7 APTT 3.2 SD105.5 APTT CTAD ND104.3 APTT CTAD SD100.1

10. ANTI-FXa RESULTS ON UFH SUBJECTS: ANOVA GROUPSAve: IU/ml P-value/F UFH 3.8 ND0.360.9878/0.12 UFH 3.8 SD0.32 UFH 3.2 ND0.37 UFH 3.2 SD0.33 UFH CTAD ND 0.37 UFH CTAD SD 0.36

11. anti-Xa ANOVA RESULTS ON UFH WITH THE HYBRID CURVE

12. Table 5: Examples of comparisons of UFH APTT results with anti-FXa heparin results. SUBJECTS UFH 3.8 ND APTT 3.8 SD APTT 3.2 ND APTT 3.2 SD APTT CTAD ND APTT CTAD SD APTT APTT MEAN (SEC.) ANTI- Xa MEA N IU/ml 628436.948.938.441.443.347.842.80.41 0297114.5109.9116.2107.1112.8110.9111.90.35 579369.164.966 69.667.567.20.29 113068.390.863.174.364.478.273.20.45 0807111.2133.6118.6117110.6128.6119.90.45 044736.463.335.843.634.351.144.10.46 031670.775.678.569.573.866.172.40.57 6296114.7234.291.6114.8124.3139.7136.60.05

13. COMPARISON OF UFH VS HYBRID CURVE

15. COMPARISON OF HYBRID CURVE; S-HYBRID CURVE AND UFH GroupsCountSumAverageVariance UFH5025.110.50220.067618 HYBRID5023.620.47240.097276 S-HYBRID5024.230.48460.098389 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups0.02244420.0112220.1278710.8800653.057621 Within Groups12.900811470.087761 Total12.92326149

16. APTT RESULTS OF LMWH GROUPSAve: seconds P-value/F APTT 3.8 ND 37.90.4665/0.9 APTT 3.8 SD 41.2 APTT 3.2 ND 37.1 APTT 3.2 SD 39.6 APTT CTAD ND 37.7 APTT CTAD SD 41.2

17. anti-Xa RESULTS ON LMWH SUBJECTS GROUPSAve: IU/mlP-value/F LMWH 3.8 ND 0.420.9059/0.26 LMWH 3.8 SD 0.37 LMWH 3.2 ND 0.43 LMWH 3.2 SD 0.38 LMWH CTAD ND 0.46 LMWH CTAD SD 0.43

18. anti-Xa RESULTS OF LMWH WITH THE HYBRID CURVE

19. COMPARISON OF LMWH RESULTS VS HYBRID CURVE

21. COMPARISON OF HYBRID CURVE; S-HYBRID CURVE AND LMWH GroupsCountSumAverageVariance LMWH5022.670.45340.070892 HYBRID5025.10.5020.062645 S-HYBRID5023.850.4770.059707 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups0.05906520.0295330.4584770.6331473.057621 Within Groups9.4689721470.064415 Total9.528037149

22. COMPARISON OF ANIARA anti-FXa TO UFH STAGO HYBRID CURVE GroupsCountSumAverageVariance Aniara 14025.780.64450.19041 Aniara 24025.810.645250.19459 Stago4023.070.576750.17191 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups0.12377220.0618860.3333710.717183.073763 Within Groups21.719471170.185636 Total21.84324119

24. COMPARISON OF ANIARA anti-FXa TO LMWH STAGO HYBRID CURVE GroupsCountSumAverageVariance Aniara 14021.920.5480.097652 Aniara 24021.860.54650.097105 Stago4022.190.554750.092303 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups0.00154520.0007720.0080730.991963.073763 Within Groups11.19534751170.095687 Total11.1968925119

26. Error bars represent 2SD limits of calibration Calibration with Different LMWH’s

27. Universal Calibrator/Calibration Curve * Pending FDA Clearance, Not available in US Gabbeta J, Krougliak V, Quiazon E, Rawal D, Kung C, Triscott M. “Liquid Heparin Assay: Rapid Monitoring of Clinically Used Heparins” Abstract # P-S-671. XXIst ISTH Congress, Geneva, Switzerland. July 2007 HemosIL Heparin Xa*/ACL TOP (IU/mL

28. Recovery of Arixtra and Orgaran Samples Arixtra Calibration Curve (ACL TOP) Orgaran Calibration Curve (ACL 10000) Universal Calibrator/Calibration Curve

29. Universal Calibrator/Calibration Curve Recoveries of Different Commercial Heparins in Plasma Samples

30. CONCLUSIONS: The anti-Xa heparin results were not statistically affected by any of the collection tubes or blood to anticoagulant ratio. Individual APTT results and the anti-Xa assay showed a high degree of discordance. This could lead to inappropriate heparin management. The HYBRID curve could be used to perform the anti- Xa testing on the UFH and LMWH anticoagulants tested.

31. REFERENCES McGlasson DL. Monitoring Unfractionated Heparin and Low Molecular Weight Heparin Anticoagulation with an anti-Xa Chromogenic Assay using a Single Calibration Curve. Lab Medicine. 2005;36(5):297-299. McGlasson DL et al: Effects of Pre-analytical Variables on the anti-Fxa Chromogenic Assay when Monitoring Unfractionated Heparin and Molecular Weight Heparin Anticoagulation. Blood Coagulation and Fibrinolysis. 2005;16(3):173-176. Meyers BL, Plumhoff EA, Gastineau DA, et al. A heparin assay system capable of measuring unfractionated Heparin and LMWH using the same standard curve. Blood 1998;92:Suppl 1:123b. Adcock DM et al: Minimum volume requirements for routine coagulation testing: Dependence on citrate concentrations. AJCP 1998;109:595-599. Gilbert M et al: Validity of the new STA-Calibrator LMWH for Assaying most Commonly used LMWH Preparations with STA-Rotachrom Heparin. Abs CD 3224. Supplement to J Throm Haemos, July 2007. McGlasson DL, Fritsma G. Abs Comparison of two Anti-Xa assays using a single calibration curve for monitoring heparin anticoagulation. Supplement to J throm Haemos, July 2007. Chan AKC et al: Do we need different standard curves for measuring diggerent LMWH? Supplement J Thromb Haemost 2007.