Measurement of hemostatic factors in EDTA plasma.

Abstract

This study determined whether immunoassays of factors VII (FVII) and VIII (FVIII) and von Willebrand factor (vWF) in EDTA-anticoagulated plasma samples are comparable to bioassays and immunoassays of these factors in citrate-anticoagulated plasma. Blood from 40 healthy volunteers was collected in EDTA- and citrate-anticoagulant tubes and assayed using immunoassays (EDTA and citrate) and clotting assays (citrate). Linear regression analyses were performed and Pearson correlation coefficients recorded. The correlation coefficients (95% confidence intervals [CIs]) between levels in EDTA- and citrate-anticoagulated plasma samples were 0.893 (0.806-0.943) for FVII antigen (ag), 0.930 (0.870-0.962) for FVIIIag, 0.990 (0.981-0.995) for vWFag, and 0.949 (0.906-0.973) for vWF activity. Coefficients (CIs) were 0.811 (0.668-0.896) for FVII coagulant activity (c) in citrate and FVIIag in EDTA and 0.608 (0.366-0.774) for FVIIIc in citrate and FVIIIag in EDTA. Measurements of FVII, FVIII, and vWF antigens in EDTA-anticoagulated plasma samples give values comparable to similar measurements in citrate-anticoagulated samples. Clotting activity, especially of FVIII, is less well correlated. Although antigen assays using EDTA are not recommended for patients with coagulopathies, they may be suitable for population-based studies.

Full text

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Am J Clin Pathol 2008;130:811-815 811
811 DOI: 10.1309/AJCPRU5QLKLQ0OMS 811
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / Clotting FaCtors in EDta Plasma
Measurement of Hemostatic Factors in EDTA Plasma
David Green, MD, PhD,1 Brandon McMahon, MD,1 Nancy Foiles, MT,1 and Lu Tian, PhD2
Key Words: Factor VII; Factor VIII; von Willebrand factor; EDTA; Citrate
DOI: 10.1309/AJCPRU5QLKLQ0OMS
A b s t r a c t
This study determined whether immunoassays of
factors VII (FVII) and VIII (FVIII) and von Willebrand
factor (vWF) in EDTA-anticoagulated plasma samples
are comparable to bioassays and immunoassays
of these factors in citrate-anticoagulated plasma.
Blood from 40 healthy volunteers was collected
in EDTA- and citrate-anticoagulant tubes and
assayed using immunoassays (EDTA and citrate)
and clotting assays (citrate). Linear regression
analyses were performed and Pearson correlation
coefficients recorded. The correlation coefficients
(95% confidence intervals [CIs]) between levels in
EDTA- and citrate-anticoagulated plasma samples
were 0.893 (0.806-0.943) for FVII antigen (ag), 0.930
(0.870-0.962) for FVIIIag, 0.990 (0.981-0.995) for
vWFag, and 0.949 (0.906-0.973) for vWF activity.
Coefficients (CIs) were 0.811(0.668-0.896) for FVII
coagulant activity (c) in citrate and FVIIag in EDTA
and 0.608 (0.366-0.774) for FVIIIc in citrate and
FVIIIag in EDTA. Measurements of FVII, FVIII, and
vWF antigens in EDTA-anticoagulated plasma samples
give values comparable to similar measurements in
citrate-anticoagulated samples. Clotting activity,
especially of FVIII, is less well correlated. Although
antigen assays using EDTA are not recommended for
patients with coagulopathies, they may be suitable for
population-based studies.
Hemostatic factors are usually measured in citrated
plasma by bioassays. However, most other clinical laboratory
tests are performed using blood collected in EDTA. Situations
occasionally arise where a clinician requires clotting tests on a
patient who has had only EDTA-anticoagulated blood drawn,
or the blood collected in a citrate tube has clotted. If the
patient is still available and agrees, another blood sample may
be collected and placed in a citrate-containing tube, but if a
fresh sample cannot be obtained, the tests are usually omitted.
To avoid this outcome, we examined whether immunoassays
of 3 hemostatic factors (factors VII [FVII] and VIII [FVIII]
and von Willebrand factor [vWF]) using blood collected in
EDTA would provide results comparable to bioassays and
immunoassays for these factors using citrate blood.
Materials and Methods
Blood samples were obtained from 40 healthy volun-
teers, comprising an equal number of men and women aged
23 to 53 years. Excluded were persons with self-reported
anemia; people taking medications for high blood pressure,
heart disease, or diabetes; and women with self-report-
ed pregnancy or taking oral contraceptives or estrogens.
The study was approved by the Northwestern University
Institutional Review Board (Chicago, IL), and all volunteers
provided written informed consent.
For the study, 30 mL of blood was collected from each
volunteer and divided into aliquots for 2 EDTA tubes and 2
citrate Vacutainer tubes (Becton Dickinson, Franklin Lakes,
NJ). The blood was mixed with the anticoagulant and immedi-
ately centrifuged at 1,500g at room temperature for 15 minutes

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812 Am J Clin Pathol 2008;130:811-815
812 DOI: 10.1309/AJCPRU5QLKLQ0OMS
© American Society for Clinical Pathology
Green et al / Clotting FaCtors in EDta Plasma
and then recentrifuged for 10 minutes to prepare platelet-poor
plasma. The plasma was rapidly frozen and stored in aliquots at
–70°C until assayed. Frozen samples were thawed at 37°C and
tested in batches of 20. All samples were assayed in duplicate.
FVII coagulant activity (FVIIc) was assayed in citrate
plasma using a 1-stage method based on the prothrombin time.
The assay was performed on the STA Compact (Diagnostica
Stago, Asnieres, France) using Neoplastine CL Prothrombin
Time reagent and calibrator plasma and system controls from
Diagnostica Stago, Parsippany, NJ. FVIIc-deficient plasma
was obtained from George King Biomedical, Overland Park,
KS. The coefficient of variation (CV) of the assay was 6.9%.
FVIII coagulant activity (FVIIIc) was assayed in citrate
plasma using a 2-stage method based on a modified throm-
boplastin generation test.1 The assay was calibrated using the
Unicalibrator from Diagnostica Stago and standardized using
World Health Organization standards, and all samples were
tested at 2 dilutions. The CV of the assay was 5.2%.
FVII antigen (FVIIag) was measured in EDTA-
anticoagulated plasma using the Asserachrom VII-Ag
reagents and enzyme-linked immunosorbent assay (ELISA;
Diagnostica Stago). The CV of the assay was 4.6%. FVIII anti-
gen (FVIIIag) in EDTA-anticoagulated plasma was assayed
with the VisuLize FVIII Antigen Kit (Affinity Biologicals,
Ancaster, Canada), which uses a double-antibody ELISA. The
CV was 4.7%.
vWF was measured in citrate- and EDTA-anticoagulated
plasma using 2 assay methods. One is an immunoassay that
recognizes an epitope on the vWF factor involved in its
interaction with GP1b-α2 (Imubind vWF Activity ELISA,
American Diagnostica, Stamford, CT). The CV of the assay
was 6.5%, and we have used the designation vWFac to differ-
entiate this activity antigen from the antigen measured by the
immunoturbidimetric method (vWFag, Liatest vWF Antigen
Kit, Diagnostica Stago). The CV of that assay was 3.3%.
Statistical Methods
Based on data in the literature,3,4 it was estimated that
measurements of hemostatic factors in 40 subjects would
provide a better than 97% chance of correctly concluding that
the correlation coefficient for clotting and immunologic assays
using citrate or EDTA anticoagulants would be greater than
0.85. The correlation coefficients and the corresponding 95%
confidence intervals (CIs) between various measurements in
citrate and EDTA were determined for the 4 antigen measure-
ments: FVIIag, FVIIIag, vWFag, and vWFac. In addition, the
correlation coefficients between FVIIc in citrate and FVIIag
in EDTA and between FVIIIc in citrate and FVIIIag in EDTA
were calculated. Simple linear regression analyses were used
to examine the relationships between the various measure-
ments of the hemostatic factors in citrate and EDTA.
Results
The mean and SD of the various measurements in the 40
participants are summarized in zTable 1z. The mean values
for the antigens measured when EDTA was the anticoagulant
were generally higher than when citrate was the anticoagulant,
probably reflecting a small degree of dilution owing to the
liquid citrate (EDTA is a powder). The mean and range of
values for FVII clotting activity measured in citrate (0.985
[0.66-1.46]) were very close to the antigen values, whether
measured in citrate (0.995 [0.63-1.72]) or in EDTA (0.997
[0.60-1.39]). The mean and range of values for FVIII clotting
activity measured in citrate (1.19 [0.68-2.10]) were slightly
higher than the antigen values measured in citrate (1.109
[0.75-2.20]) or in EDTA (1.147 [0.75-1.89]).
zFigure 1z includes 4 scatter plots for each antigen
(FVIIag, FVIIIag, vWFag, and vWFac) in citrate vs their
counterparts in EDTA. The fitted regression lines were plotted
to describe the relationship between the antigen measurements
in citrate vs those in EDTA. The Pearson correlation coeffi-
cients (95% CIs) were 0.893 (0.806-0.943) for FVIIag, 0.930
(0.870-0.962) for FVIIIag, 0.990 (0.981-0.995) for vWFag,
and 0.949 (0.906-0.973) for vWFac. Least square analysis
yielded the fitted lines EDTA = 0.518 + 1.177 × citrate for
vWFag, EDTA = 12.98 + 0.969 × citrate for vWFac, EDTA
zTable 1z
Measurements of FVIIag, FVIIc, FVIIIag, FVIIIc, vWFag, and vWFac*
Analyte
Mean (SD) EDTA
EDTA (Range)
Mean (SD) Citrate
Citrate (Range)
FVIIag
FVIIc
FVIIIag
FVIIIc
vWFag
vWFac
0.997 (0.199)
—
1.147 (0.319)
—
1.441 (0.396)
1.225 (0.318)
0.60-1.39
—
0.75-1.89
—
0.92-2.28
0.63-1.95
0.995 (0.232)
0.985 (0.261)
1.109 (0.342)
1.196 (0.393)
1.221 (0.333)
1.131 (0.311)
0.63-1.72
0.66-1.46
0.75-2.20
0.68-2.10
0.75-2.17
0.61-1.80
FVIIag, factor VII antigen; FVIIc, factor VII coagulant activity; FVIIIag, factor VIII antigen; FVIIIc, factor VIII coagulant activity; vWFac, von Willebrand factor activity
antigen; vWFag, von Willebrand factor antigen.
* Values are given in IU/mL.

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Am J Clin Pathol 2008;130:811-815 813
813 DOI: 10.1309/AJCPRU5QLKLQ0OMS 813
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / original artiClE
= 23.32 + 0.768 × citrate for FVIIag, and EDTA = 18.37 +
0.868 × citrate for FVIIIag. zFigure 2z shows 2 scatter plots:
FVIIc in citrate vs FVIIag in EDTA and FVIIIc in citrate vs
FVIIIag in EDTA. The Pearson correlation coefficients (95%
CIs) were 0.811 (0.668-0.896) between FVIIc in citrate and
FVIIag in EDTA and 0.608 (0.366-0.774) between FVIIIc in
citrate and FVIIIag in EDTA. The lines do not pass through
the origin because clotting activity was proportionately higher
than antigen concentration.
Discussion
The anticoagulant action of citrate solutions was dis-
covered more than a century ago and the anticlotting effects
of EDTA in 1944.5,6 Citrate forms un-ionized calcium salts;
adding calcium reverses the anticoagulation. On the other
hand, EDTA irreversibly chelates calcium and permanently
prevents progression of the coagulation cascade. Therefore,
citrate has been the anticoagulant of choice for bioassay of
coagulation factors. However, citrate may fail to prevent acti-
vation of coagulation if technical difficulties permit thrombin
generation. For example, a difficult venipuncture may cause
hemolysis and expose blood to tissue fluids, delays in trans-
ferring the blood from the collection device to the citrate
tube may allow time for thrombin formation, and improper
or inadequate mixing of the sample with the citrate may per-
mit activation of coagulation by calcium ions. Under these
circumstances, citrate plasma is unsuitable for measurement
of clotting factor concentrations. We performed this study
to answer the question of whether immunoassay of clotting
factors performed using EDTA-anticoagulated plasma could
substitute for assays using citrate-anticoagulated plasma.
The mean values for FVIIag in citrate and EDTA were
identical and for FVIIIag differed by only 0.04 IU/mL.
Scatter plots for the 2 factors in citrate and EDTA showed
correlation coefficients of 0.89 and 0.93, respectively, indi-
cating that there is no loss of antigen when EDTA is used as
the anticoagulant. This is in marked contrast with the major
0.6
0.8
1.0
1.2
0.60.8 1.01.2
Factor VIIag in EDTA (IU/mL)
Factor VIIag in Citrate (IU/mL)
1.41.6
1.4
0.8
1.2
1.6
2.0
0.8 0.6 1.01.2
vWFac in EDTA (IU/mL)
vWFac in Citrate (IU/mL)
1.4 1.61.8
1.0
1.5
2.0
0.81.0 1.2
vWFag in EDTA (IU/mL)
vWFag in Citrate (IU/mL)
1.41.61.8
0.8
1.2
1.01.5
Factor VIIIag in EDTA (IU/mL)
Factor VIIIag in Citrate (IU/mL)
2.0
1.6
AB
CD
zFigure 1z Scatter plots showing the measured concentrations of factor VII antigen (A; FVIIag), factor VIII antigen (B; FVIIIag),
von Willebrand factor antigen (C; vWFag), and von Willebrand factor activity (D; vWFac) for plasma samples collected in EDTA
or citrate anticoagulant. A, r = 0.89. B, r = 0.93. C, r = 0.99. D, r = 0.95.

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814 Am J Clin Pathol 2008;130:811-815
814 DOI: 10.1309/AJCPRU5QLKLQ0OMS
© American Society for Clinical Pathology
Green et al / Clotting FaCtors in EDta Plasma
loss of FVIII clotting activity that is observed with EDTA-
anticoagulated plasma. Many years ago, EDTA was shown
to inactivate FVIII, probably by disrupting calcium ion–de-
pendent noncovalent interactions among the FVIII domains.7
This is not surprising because calcium ions are integral to the
structural integrity of the FVIII molecule. Recent studies have
shown that 2 calcium ions are located within the molecule.8
One is in the A1 domain, tightly coordinated by a single glu-
tamate and 3 aspartate residues. It may function to maintain
a stable metal ion–dependent linkage of the heavy chain (the
A1, A2, and B domains) and the light chain (the A3, C1, and
C2 domains).9 The second is in the A2 domain coordinated
by 2 aspartate residues; it functions to weakly associate the
A1 domain with the A2 domain through electrostatic interac-
tions.10 EDTA likely disrupts these critical interactions and
accounts for the loss of procoagulant activity.
The mean values for FVIIc and FVIIag were close to
1 IU/mL, and the correlation coefficient between the 2 was
0.81. This result likely reflects the different standards used
for each method. Kleinveld et al11 suggest that the differences
among the various assays are probably due to intercompany
variations in standardization rather than to differences attrib-
utable to the anticoagulant. On the other hand, FVIIIc in
citrate plasma was higher than FVIIIag measured in citrate or
EDTA plasma, and the correlation coefficient was only 0.61
between the clotting assay and the antigen assay. FVIII levels
more than 1.0 U/dL in the FVIII clotting assay were found to
correlate imperfectly with levels measured by a chromogenic
substrate assay for FVIII.11 Although it is possible that some
activation of FVIII occurs during sample collection, storage,
or assay, a more likely explanation is that the higher values
again reflect a problem with assay standardization.
In this study, we examined the feasibility of assay-
ing FVII, FVIII, and vWF in EDTA-anticoagulated blood.
It has previously been shown that fibrinogen, assayed in
EDTA-anticoagulated plasma, provides results comparable
to clotting assays using citrate-anticoagulated plasma.4
Using clot-rate assays, the investigators reported a correla-
tion coefficient of 0.972.
A limitation of our study is that we measured hemostatic
factors in healthy volunteers, and the results may not be gen-
eralizable to patients with high or low clotting factor levels.
However, as shown in Table 1, the range of values in these
volunteers was quite broad and included values more than
1.50, which implies that the assays would be suitable for epi-
demiologic studies of thrombotic risk factors.
Measurements of FVII, FVIII, and vWF antigens in
EDTA-anticoagulated plasma from healthy volunteers gave
values comparable to similar measurements in citrate-antico-
agulated plasma. Although antigen assays using EDTA are not
recommended for patients who have coagulopathies, they may
be suitable for population-based studies, especially when there
is concern about the quality of citrate-anticoagulated samples.
From the 1Division of Hematology/Oncology, Department of
Medicine, and the 2Department of Preventive Medicine, Feinberg
School of Medicine, Northwestern University, Chicago, IL.
0.6
0.8
1.0
1.2
1.4
0.80.61.01.2
Factor VIIag in EDTA (IU/mL)
Factor VIIc in Citrate (IU/mL)
1.41.6
0.8
1.0
1.2
1.4
1.6
1.8
1.01.5
Factor VIIIag in EDTA (IU/mL)
Factor VIIIc in Citrate (IU/mL)
2.0
AB
zFigure 2z Scatter plots showing the measured concentrations of factor VII coagulant activity (FVIIc) in citrate plasma and factor
VII antigen (FVIIag) in EDTA plasma (A) and factor VIII coagulant activity (FVIIIc) in citrate plasma and factor VIII antigen (FVIIIag)
in EDTA plasma (B). A, r = 0.81. B, r = 0.61.

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Am J Clin Pathol 2008;130:811-815 815
815 DOI: 10.1309/AJCPRU5QLKLQ0OMS 815
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / original artiClE
Supported by grant AG032136 from the National Institute
on Aging, National Institutes of Health, Bethesda, MD. The
funding agency had no role in the design and conduct of the study;
collection, management, analysis, and interpretation of the data;
and preparation, review, or approval of the manuscript. The
authors had full access to all of the data in the study and take
responsibility for the integrity of the data and the accuracy of the
data analysis.
Address reprint requests to Dr Green: 676 N St Clair St,
Suite 850, Chicago, IL 60611.
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