Transcript Multi-Laboratory Evaluation of a Chromogenic Factor X

A Multi-Laboratory Evaluation of a
Chromogenic Factor X Assay for
Monitoring Oral Anticoagulation
Therapy
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.
INTRODUCTION

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.
INTRODUCTION



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 subtherapeutic 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 Principle
RVV
FX
1.
→
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: 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 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: 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: 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 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 subjects
Laboratory
One
OAT
Range
(%)
N=30
Correlation
INR=1.7-5.9
Mean
(%)
Chromogenic 19.3-62.5
31.0
Clottable
13.9
7.0-48.0
0.903/0.031
Lab One RESULTS: OATsubjects with
presence of an LA
Laboratory
One
OAT
with LA
Range
(%)
N=152 Correlation
INR
P=0.021
0.97-4.5
Mean 0.841
(%)
Chromogenic
7.0122.0
33.1
Clottable
2.7101.0
22.8
Lab One RESULTS: Interfering
substances
Laboratory
One
Interfering
substances
Range
(%)
N=31
INR=
1.0-1.2
Mean
(%)
Chromogenic 101.2-126.4
113.8
Clottable
109.5
97.4-120.7
Ttest/Correlation
P=0.59/0.906
Lab Two RESULTS: Normal range
Laboratory
Two
Normals
Range
(%)
N=25 Correlation
Mean 0.871
(%)
Chromogenic 83.0-147.0 120.4
Clottable
69.0-139.0 105.7
Lab Two RESULTS: OAT
Laboratory
Two
OAT
N=55
Correlation
Range
(%)
Mean
(%)
0.948
Chromogenic 17.0-65.0
32.5
Clottable
10.4
2.0-41.0
RESULTS: Precision and Accuracy
Testing Lab One and Two
Precision Data
CV (%)
CV (%)
Laboratory
One
Chromogenic
1.9-10.4
Laboratory
Two
2.5-5.1
Clottable
<5.0%
4.6-9.2
RESULTS: Precision and Accuracy
Testing 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 10120% 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)
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
Descriptive stats
DIA FX
1
Mean
DIA FX
2
BIO FX
1
BIO FX
2
INR
20.0
1.75
73.6
75.8
87.8
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
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
5815
5990
7029
7168
1602.1
140.19
79
79
80
80
80
80
10.48748
10.67135
15.20278
15.47541
2.147878
0.236116
Range
Sum
Count
Confidence
Level(95.0%)
89.6
PT
ANIARA (BIOPHEN) VS DIAPHARMA
T-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
79
79
80
80
Observations
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 DIAPHARMA
ANOVA
Groups
Count
Sum
Average
Variance
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
Between Groups
15931.74
3
5310.579
Within Groups
1098763
314
3499.245
F
P-value
1.51766 0.20986
F crit
2.633364
PRECISION 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
Difference between DIA FX and BIO FX
300
mean+
1.96sd 191.9
200
100
mean 81.1
0
mean1.96sd -29.6
-100
-200
-300
0
50
100
150
200
250
Average of DIA FX and BIO FX
300
350
CONCLUSIONS


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.
REFERENCES



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.