A Multi-Laboratory Evaluation of a A Multi-Laboratory Evaluation of a Chromogenic Factor X Assay for Chromogenic Factor X Assay for Monitoring Oral Anticoagulation Monitoring Oral Anticoagulation

TherapyTherapy

DL McGlasson1*; PN Shaklee2; 159th 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.

INTRODUCTIONINTRODUCTION

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.

INTRODUCTIONINTRODUCTION 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..

Diapharma Factor X PrincipleDiapharma Factor X Principle

RVV

1. FX → FXa Ca2+

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.

INTRODUCTION: CONTINUEDINTRODUCTION: 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.

MATERIALS AND METHODSMATERIALS 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 <109/L

A normal range was established with 30 normal subjects with no known clinical abnormalities.

MATERIALS AND METHODS: 2MATERIALS 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.

MATERIALS AND METHODS: 3MATERIALS 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.

Lab One RESULTS: Normal rangeLab One RESULTS: Normal range

Laboratory One

Normals N=30 Correlation

Range

(%)

Mean

(%)

0.9015/0.720

Chromogenic 72.0-137.6 104.8

Clottable 94.1-159.7 126.9

Lab One RESULTS: OAT subjectsLab One RESULTS: OAT subjects

Laboratory One

OAT N=30 INR=1.7-5.9

Correlation

Range

(%)

Mean

(%)

0.903/0.031

Chromogenic 19.3-62.5 31.0

Clottable 7.0-48.0 13.9

Lab One RESULTS: OATsubjects with Lab One RESULTS: OATsubjects with presence of an LApresence of an LA

Laboratory One

OAT with LA

N=152

INR 0.97-4.5

Correlation

P=0.021

Range

(%)

Mean

(%)

0.841

Chromogenic 7.0-122.0

33.1

Clottable 2.7-101.0

22.8

Lab One RESULTS: Interfering Lab One RESULTS: Interfering substancessubstances

Laboratory One

Interfering substances

N=31 INR=

1.0-1.2

T-test/Correlation

Range

(%)

Mean

(%)

P=0.59/0.906

Chromogenic 101.2-126.4 113.8

Clottable 97.4-120.7 109.5

Lab Two RESULTS: Normal rangeLab Two RESULTS: Normal range

Laboratory Two

Normals N=25 Correlation

Range

(%)

Mean

(%)

0.871

Chromogenic 83.0-147.0 120.4

Clottable 69.0-139.0 105.7

Lab Two RESULTS: OATLab Two RESULTS: OAT

Laboratory Two

OAT N=55 Correlation

Range

(%)

Mean

(%)

0.948

Chromogenic 17.0-65.0 32.5

Clottable 2.0-41.0 10.4

RESULTS: Precision and Accuracy RESULTS: Precision and Accuracy Testing Lab One and TwoTesting Lab One and Two

Precision Data

CV (%) CV (%)

Laboratory One

Laboratory Two

Chromogenic 1.9-10.4 2.5-5.1

Clottable <5.0% 4.6-9.2

RESULTS: Precision and Accuracy RESULTS: Precision and Accuracy Testing Lab One and TwoTesting 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%)

SUBJECTS (N=80)SUBJECTS (N=80)

NORMALS 20

COUMADIN 20

HEPARIN (UFH) 0.2-1.0 8

ELEVATED FIBRINOGEN (>500 mg/dl)

8

LUPUS ANTICOAGULANT

8

HEMOLYZED 4

LIPEMIC 4

RABBIT (elevated FX) 8

ANIARA (BIOPHEN) VS DIAPHARMA ANIARA (BIOPHEN) VS DIAPHARMA Descriptive statsDescriptive stats

DIA FX 1

DIA FX 2

BIO FX 1

BIO FX 2 PT INR

Mean 73.6 75.8 87.8 89.6 20.0 1.75

Standard Error 5.267847 5.360208 7.637863 7.77483 1.079092 0.118624

Standard Deviation 46.82165 47.64257 68.31512 69.54019 9.65169 1.061008

Range 181 182 425 444 35.9 3.94

Minimum 19 19 19 7 6.5 0.41

Maximum 200 201 444 451 42.4 4.35

Sum 5815 5990 7029 7168 1602.1 140.19

Count 79 79 80 80 80 80

Confidence Level(95.0%) 10.48748 10.67135 15.20278 15.47541 2.147878 0.236116

ANIARA (BIOPHEN) VS DIAPHARMAANIARA (BIOPHEN) VS DIAPHARMAT-TESTST-TESTS

  DIA FX 1 DIA FX 2 BIO FX 1 BIO FX 2

Mean 73.60759 75.82278 87.8625 89.6

Variance 2192.267 2269.814 4666.956 4835.838

Observations 79 79 80 80

Pearson Correlation 0.988210 0.988147

Hypothesized Mean Difference 0 0

df 78 79

t Stat -2.69754 -1.45475

P(T<=t) one-tail 0.004279 0.07485

t Critical one-tail 1.664625 1.664371

P(T<=t) two-tail 0.008557 0.1497

t Critical two-tail 1.990847   1.99045  

ANIARA (BIOPHEN) VS DIAPHARMAANIARA (BIOPHEN) VS DIAPHARMAANOVAANOVA

Groups Count Sum AverageVarianc

e

DIA FX 1 79 5815 73.60759 2192.267

DIA FX 2 79 5990 75.82278 2269.814

BIO FX 1 80 7029 87.8625 4666.956

BIO FX 2 80 7168 89.6 4835.838

ANOVA

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

Between Groups 15931.74 3 5310.579 1.51766 0.20986 2.633364

Within Groups 1098763 314 3499.245

     

PRECISION ON QCPRECISION ON QC

QC STATS DIAP DIAP BIOPHEN BIOPHEN

STA-N STA-P FX NOR FX ABN

Mean 98.91667 37.08333 94.86957 59.82609

Standard Error 1.031947 0.355682 0.623342 0.285708

Standard Deviation 5.055489 1.742479 2.989441 1.370208

Range 20 6 15 6

Minimum 86 34 89 57

Maximum 106 40 104 63

Sum 2374 890 2182 1376

Count 24 24 23 23

Confidence Level(95.0%) 2.134746 0.735784 1.292731 0.592522

CV% 5.1 4.6 3.16 2.3

Bland Altman of DIA FX and BIO FX

mean 81.1

mean-1.96sd -29.6

mean+1.96sd 191.9

-300

-200

-100

0

100

200

300

0 50 100 150 200 250 300 350

Average of DIA FX and BIO FX

Diff

eren

ce b

etw

een

DIA

FX

and

BIO

FX

CONCLUSIONSCONCLUSIONS

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.

REFERENCESREFERENCES 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.