Effects of Pre-analytical Variables Effects of Pre-analytical Variables on the on the anti-Xa anti-Xa Chromogenic Assay when Chromogenic Assay when Monitoring Unfractionated Heparin and Low Monitoring Unfractionated Heparin and Low Molecular Weight Heparin Anticoagulation Molecular Weight Heparin Anticoagulation David L. McGlasson, MS, CLS/NCA David L. McGlasson, MS, CLS/NCA United States Air Force United States Air Force This information is for education only and is not a product endorsement. This information is for education only and is not a product endorsement.
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Effects of Pre-analytical Variables on the anti-Xa Chromogenic Assay when Monitoring Unfractionated Heparin and Low Molecular Weight Heparin Anticoagulation.
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Effects of Pre-analytical Variables on the Effects of Pre-analytical Variables on the
anti-Xaanti-Xa Chromogenic Assay when Monitoring Chromogenic Assay when Monitoring Unfractionated Heparin and Low Molecular Weight Unfractionated Heparin and Low Molecular Weight
Heparin AnticoagulationHeparin Anticoagulation
David L. McGlasson, MS, CLS/NCADavid L. McGlasson, MS, CLS/NCAUnited States Air ForceUnited States Air Force
This information is for education only and is not a product endorsement.This information is for education only and is not a product endorsement.
INTRODUCTION: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).
INTRODUCTIONINTRODUCTION 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.
MATERIALS AND METHODSMATERIALS 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.
MATERIALS AND METHODSMATERIALS 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.
MATERIALS AND METHODSMATERIALS 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.
MATERIALS AND METHODSMATERIALS 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.
MATERIALS AND METHODSMATERIALS 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.
Universal Calibrator/Calibration CurveUniversal Calibrator/Calibration Curve Recoveries of Different Commercial Heparins in Plasma SamplesRecoveries of Different Commercial Heparins in Plasma Samples
Target Values (IU/ml)
Cal Curve 0.40 0.80 1.20 1.60 2.00
LMW Cal 0.42 0.77 1.12 1.49 1.80
Fragmin UFH Cal 0.44 0.80 1.16 1.53 1.83
Univ. Cal 0.42 0.78 1.14 1.54 1.83
LMW Cal 0.44 0.81 1.16 1.49 1.83
Innohep UFH Cal 0.46 0.83 1.19 1.54 1.87
Univ. Cal 0.44 0.81 1.17 1.53 1.87
LMW Cal 0.39 0.83 1.18 1.57 1.92
Lovenox UFH Cal 0.42 0.86 1.21 1.61 1.97
Univ. Cal 0.39 0.84 1.20 1.60 1.97
LMW Cal 0.40 0.84 1.19 1.58 1.93
LMWH UFH Cal 0.43 0.87 1.22 1.62 1.97
Univ. Cal 0.40 0.85 1.21 1.61 1.97
LMW Cal 0.42 0.77 1.14 1.62 1.93
UFH UFH Cal 0.44 0.80 1.18 1.66 1.97
Univ. Cal 0.42 0.77 1.16 1.66 1.97
CONCLUSIONS: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.
REFERENCESREFERENCESMcGlasson 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.