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.

APTT RESULTS UFH SUBJECTS: ANOVAAPTT RESULTS UFH SUBJECTS: ANOVA

GROUPS Ave: seconds P-value/FAPTT 3.8 ND 105.4 0.9481/0.9

APTT 3.8 SD 127.2

APTT 3.2 ND 107.7

APTT 3.2 SD 105.5

APTT CTAD ND 104.3

APTT CTAD SD 100.1

ANTI-FXa RESULTS ON UFH SUBJECTS: ANTI-FXa RESULTS ON UFH SUBJECTS: ANOVAANOVA

GROUPS Ave: IU/ml P-value/FUFH 3.8 ND 0.36 0.9878/0.12

UFH 3.8 SD 0.32

UFH 3.2 ND 0.37

UFH 3.2 SD 0.33

UFH CTAD ND

0.37

UFH CTAD SD

0.36

anti-Xa ANOVA RESULTS ON UFH WITH THE anti-Xa ANOVA RESULTS ON UFH WITH THE HYBRID CURVEHYBRID CURVE

Groups Count Sum Average VarianceHYBRID 3.8 ND 20 7.41 0.3705 0.107931HYBRID 3.8 SD 20 6.84 0.342 0.107196HYBRID 3.2 ND 20 7.4 0.37 0.102168HYBRID 3.2 SD 20 6.81 0.3405 0.084426HYBRID CTAD ND 20 7.86 0.393 0.177475HYBRID CTAD SD 20 7.61 0.3805 0.140058

ANOVASource of Variation SS df MS F P-value F critBetween Groups 0.044034 5 0.008807 0.073466 0.996077 2.293909Within Groups 13.66583 114 0.119876

Total 13.70986 119

Table 5: Examples of comparisons of UFH APTT results with anti-FXa heparin results. 

SUBJECTS UFH

3.8 NDAPTT

3.8 SD APTT

3.2 ND APTT

3.2 SDAPTT

CTAD ND

APTT

CTAD SD

APTT

APTT MEAN(SEC.)

ANTI-Xa

MEAN

IU/ml

6284 36.9 48.9 38.4 41.4 43.3 47.8 42.8 0.41

0297 114.5 109.9 116.2 107.1 112.8 110.9 111.9 0.35

5793 69.1 64.9 66 66 69.6 67.5 67.2 0.29

1130 68.3 90.8 63.1 74.3 64.4 78.2 73.2 0.45

0807 111.2 133.6 118.6 117 110.6 128.6 119.9 0.45

0447 36.4 63.3 35.8 43.6 34.3 51.1 44.1 0.46

0316 70.7 75.6 78.5 69.5 73.8 66.1 72.4 0.57

6296 114.7 234.2 91.6 114.8 124.3 139.7 136.6 0.05

COMPARISON OF UFH VS HYBRID CURVECOMPARISON OF UFH VS HYBRID CURVE

Anova: Single FactorUFH VS HYBRID CURVESSUMMARY

Groups Count Sum AverageUFH 3.8 ND 20 7.24 0.362UFH 3.8 SD 20 6.45 0.3225UFH 3.2 ND 20 7.43 0.3715UFH 3.2 SD 20 6.54 0.327UFH CTAD ND 20 7.49 0.3745UFH CTAD SD 20 7.19 0.3595HYBRID 3.8 ND 20 7.41 0.3705HYBRID 3.8 SD 20 6.84 0.342HYBRID 3.2 ND 20 7.4 0.37HYBRID 3.2 SD 20 6.81 0.3405HYBRID CTAD ND 20 7.86 0.393HYBRID CTAD SD 20 7.61 0.3805

ANOVASource of Variation SS df MS F P-valueBetween Groups 0.105245 11 0.009568 0.093564 0.999936Within Groups 23.31499 228 0.102259

Total 23.42023 239

Rotachrom Heparin: UFH Samples 3.2% ND and SD

y = 1.0927x - 0.0046

R2 = 0.9586

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 0.2 0.4 0.6 0.8 1 1.2 1.4

UFH Calibration Curve: IU/mL

Hyb

rid

Cal

ibra

tio

n C

urv

e: I

U/m

L

COMPARISON OF HYBRID CURVE; COMPARISON OF HYBRID CURVE; S-HYBRID CURVE AND UFHS-HYBRID CURVE AND UFH

Groups Count Sum Average Variance

UFH 50 25.11 0.5022 0.067618

HYBRID 50 23.62 0.4724 0.097276

S-HYBRID 50 24.23 0.4846 0.098389

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 0.022444 2 0.011222 0.127871 0.880065 3.057621

Within Groups 12.90081 147 0.087761

Total 12.92326 149        

APTT RESULTS OF LMWHAPTT RESULTS OF LMWH

GROUPS Ave: seconds P-value/F

APTT 3.8 ND 37.9 0.4665/0.9

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

anti-Xa RESULTS ON LMWH SUBJECTSanti-Xa RESULTS ON LMWH SUBJECTS

GROUPS Ave: IU/ml P-value/FLMWH 3.8 ND 0.42 0.9059/0.26

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

anti-Xa RESULTS OF LMWH WITH THE anti-Xa RESULTS OF LMWH WITH THE HYBRID CURVEHYBRID CURVE

Groups Count Sum Average VarianceHYB 3.8 ND 26 11.92 0.45846 0.08717HYB 3.8 SD 26 11.1 0.42692 0.07445HYB 3.2 ND 26 12.23 0.47038 0.07908HYB 3.2 SD 26 10.9 0.41923 0.0682HYB CTAD ND 26 12.84 0.49385 0.08948HYB CTAD SD 26 12.07 0.46423 0.08403

ANOVA

Source of Variation SS df MS F P-value F critBetween Groups 0.10164 5 0.02033 0.25283 0.93785 2.27449Within Groups 12.06 150 0.0804

Total 12.1617 155

COMPARISON OF LMWH RESULTS VS HYBRID COMPARISON OF LMWH RESULTS VS HYBRID CURVECURVE

Groups Count Sum AverageHYB 3.8 ND 26 11.92 0.4585HYB 3.8 SD 26 11.1 0.4269HYB 3.2 ND 26 12.23 0.4704HYB 3.2 SD 26 10.9 0.4192HYB CTAD ND 26 12.84 0.4938HYB CTAD SD 26 12.07 0.4642LMWH 3.8 ND 26 10.84 0.4169LMWH 3.8 SD 26 9.71 0.3735LMWH 3.2 ND 26 11.1 0.4269LMWH 3.2 SD 26 9.86 0.3792LMWH CTAD ND 26 11.88 0.4569LMWH CTAD SD 26 11.09 0.4265

ANOVASource of Variation SS df MS F P-value F critBetween Groups 0.3711 11 0.0337 0.4101 0.9512 1.8206Within Groups 24.679 300 0.0823

Total 25.05 311

Rotachrom LMWH 3.2% ND and SD

y = 0.979x + 0.0501

R2 = 0.9884

0

0.2

0.4

0.6

0.8

1

1.2

0 0.2 0.4 0.6 0.8 1 1.2

LMWH Cal Curve (anti-Xa IU/mL)

Hyb

rid

Cal

Cur

ve (a

nti-X

a IU

/mL)

COMPARISON OF HYBRID CURVE; COMPARISON OF HYBRID CURVE; S-HYBRID CURVE AND LMWHS-HYBRID CURVE AND LMWH

Groups Count Sum Average Variance

LMWH 50 22.67 0.4534 0.070892

HYBRID 50 25.1 0.502 0.062645

S-HYBRID 50 23.85 0.477 0.059707

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 0.059065 2 0.029533 0.458477 0.633147 3.057621

Within Groups 9.468972 147 0.064415

Total 9.528037 149        

COMPARISON OF ANIARA anti-FXa TO COMPARISON OF ANIARA anti-FXa TO UFH STAGO HYBRID CURVEUFH STAGO HYBRID CURVE

Groups Count Sum Average Variance

Aniara 1 40 25.78 0.6445 0.19041

Aniara 2 40 25.81 0.64525 0.19459

Stago 40 23.07 0.57675 0.17191

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 0.123772 2 0.061886 0.333371 0.71718 3.073763

Within Groups 21.71947 117 0.185636

Total 21.84324 119        

COMPARISON OF ANIARA anti-FXa TO COMPARISON OF ANIARA anti-FXa TO LMWH STAGO HYBRID CURVELMWH STAGO HYBRID CURVE

Groups Count Sum Average Variance

Aniara 1 40 21.92 0.548 0.097652

Aniara 2 40 21.86 0.5465 0.097105

Stago 40 22.19 0.55475 0.092303

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 0.001545 2 0.000772 0.008073 0.99196 3.073763

Within Groups 11.1953475 117 0.095687

Total 11.1968925 119        

Anti-Xa IU/mL

0.00 1.00 2.00

0.200

0.300

0.400

0.500

0.600

0.700

0.8000.900

0.100

1.000

OD

/min

STA-Calibrator LMWH

Nadroparin

Enoxaparin

Tinzaparin

Dalteparin

Error bars represent 2SD limits of calibration

Calibration with Different LMWH’s

100

300

500

700

900

1100

0.0 0.4 0.8 1.2 1.6 2.0Heparin (IU/mL)

mA

bs/m

in

InnohepFragminLovenoxLMWHUFHUniversal

Universal Calibrator/Calibration CurveUniversal Calibrator/Calibration Curve

Cal point mAbs (U/mL) %CV

2.0 3.390.8 1.610.0 1.17

* 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

100

300

500

700

900

1100

0 0.4 0.8 1.2 1.6 2

Heparin (IU/mL)

ΔO

D (m

Abs

/min

)

ARXLMWHFRGUFH

0

200

400

600

800

1000

0 0.4 0.8 1.2 1.6 2

Heparin (IU/mL)ΔO

D (m

Abs

/min

)Orgaran

Fr

UFH

Recovery of Arixtra and Orgaran Samples

Arixtra Calibration Curve (ACL TOP)

Orgaran Calibration Curve (ACL 10000)

Universal Calibrator/Calibration CurveUniversal Calibrator/Calibration Curve

Cal Curve

0.40 0.80 1.20 1.60 2.00 0.32 0.64 0.96 1.28 1.60

LMW Cal 0.48 0.84 1.20 1.58 1.91 0.38 0.67 0.96 1.26 1.53UFH Cal 0.44 0.83 1.22 1.61 1.97 0.35 0.66 0.97 1.29 1.58Orgaran Cal 0.44 0.87 1.30 1.69 2.06 0.35 0.70 1.04 1.35 1.65

Cal Curve

0.40 0.80 1.20 1.60 2.00 0.36 0.71 1.07 1.42 1.78LMW Cal 0.41 0.81 1.23 1.58 1.96 0.36 0.72 1.09 1.40 1.74

Arixtra Cal 0.42 0.82 1.23 1.57 1.95 0.37 0.73 1.09 1.40 1.74

Orgaran Target Values

Arixtra Target Value

(IU/ml) (μg/ml)

(IU/ml) (Orgaran units/ml)

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.