Effects of Pre-analytical Variables on the

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 91 blood to
  anticoagulant ratio and a short draw of 61.
• 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 lt10,000/uL and
  stored at 70C until ready for testing.
• Specimens were thawed for 5 minutes at 37C 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
10
ANTI-FXa RESULTS ON UFH SUBJECTS ANOVA
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.  
13
COMPARISON OF UFH VS HYBRID CURVE
14
(No Transcript)
15
COMPARISON OF HYBRID CURVE S-HYBRID CURVE AND
UFH
16
APTT RESULTS OF LMWH
17
anti-Xa RESULTS ON LMWH SUBJECTS
18
anti-Xa RESULTS OF LMWH WITH THE HYBRID CURVE
19
COMPARISON OF LMWH RESULTS VS HYBRID CURVE
20
(No Transcript)
21
COMPARISON OF HYBRID CURVE S-HYBRID CURVE AND
LMWH
22
COMPARISON OF ANIARA anti-FXa TO UFH STAGO HYBRID
CURVE
23
(No Transcript)
24
COMPARISON OF ANIARA anti-FXa TO LMWH STAGO
HYBRID CURVE
25
(No Transcript)
26
Calibration with Different LMWHs
27
Universal Calibrator/Calibration Curve
HemosIL Heparin Xa/ACL TOP (IU/mL
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
Pending FDA Clearance, Not available in US
28
Universal Calibrator/Calibration Curve
Arixtra Calibration Curve (ACL TOP)
Orgaran Calibration Curve (ACL 10000)
Recovery of Arixtra and Orgaran Samples
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.
  200536(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. 200516(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 199892Suppl 1123b.
• Adcock DM et al Minimum volume requirements for
  routine coagulation testing Dependence on
  citrate concentrations. AJCP 1998109595-599.
• 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.
•