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The Sensitivity and Specificity of a Red Blood Cell Agglutination D-Dimer Assay for Venous Thromboembolism When Performed on Venous Blood
Sanjeev D. Chunilal, MB, ChB;
Patrick A. Brill-Edwards, MD;
Pamela B. Stevens, RN;
Jody P. Joval, RN;
Joanne A. McGinnis, RN;
Mala Rupwate, ART;
Jeffrey S. Ginsberg, MD, FRCPC
Arch Intern Med. 2002;162:217-220.
ABSTRACT
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Background Studies evaluating the accuracy of the SimpliRED D-dimer assay for venous
thromboembolism (VTE) have used a capillary fingerstick blood sample, which
requires the test to be performed immediately at the bedside. Initial studies
showed a sensitivity for VTE of 90% to 95% when the assay was performed by
a finite number of experienced health care workers. However, because of the
test's subjectivity, misinterpretation of the result is possible when performed
by inexperienced staff. Recent reports by other investigators indicated a
low sensitivity of this assay for VTE and noted a reduction in sensitivity
(84%) for pulmonary embolism.
Objective To determine the sensitivity and specificity of the D-dimer test performed
in the laboratory by experienced technologists on venous whole-blood samples
in routine collection tubes. If D-dimer testing results accurately detect
VTE when performed in this manner, concerns about the sensitivity of this
assay would be solved.
Methods One hundred forty-eight consecutive patients with suspected VTE underwent
D-dimer testing at the bedside using a fingerstick sample and venous blood
collected into a plain tube. Venous blood was also collected into tubes containing
tri-potassium EDTA, sodium citrate, or a combination of lithium and heparin
for D-dimer testing in the laboratory. In addition, the EDTA tube was refrigerated
overnight at 4°C for retesting at approximately 24 hours. The presence
or absence of VTE was determined by means of objective results of testing
and a 3-month follow-up.
Results Thirty-four subjects (23%) had confirmed VTE (25 with deep vein thrombosis;
9 with pulmonary embolism). All laboratory venous blood D-dimer results showed
sensitivities of 97%, specificities of 61% to 64%, and negative predictive
values of 99%, compared with 88%, 71%, and 95%, respectively, when the results
were obtained by means of fingerstick at the bedside.
Conclusions The SimpliRED D-dimer assay performed in the laboratory on venous blood,
collected into any of 3 routine laboratory tubes, is sensitive and moderately
specific for VTE. Based on this study, immediate bedside testing (particularly
by inexperienced personnel) under suboptimal conditions is unnecessary. Furthermore,
the high sensitivity of refrigerated EDTA samples allows specimens to be stored
or transported (on ice at 4°C) for testing for 24 hours after collection.
INTRODUCTION
D DIMER is a fibrin degradation product formed by the enzymatic activity
of plasmin on cross-linked fibrin polymers. Plasma levels can be measured
and are useful in patients who present with symptoms of pulmonary embolism
(PE) or deep vein thrombosis (DVT),1-2
because negative test results (below a predefined cut point) rule out the
likelihood of these diseases.
The SimpliRED D-dimer assay (Agen, Inc, Brisbane, Australia) is a unique
whole-blood assay that uses a bispecific antibody to human red blood cells
and D dimer. In a whole-blood sample, in the presence of elevated levels of
D dimer (>0.2 g/mL), this antibody will cause visible agglutination of red
blood cells. When performed by experienced personnel, this assay has a high
sensitivity (89%-94%), moderate specificity (66%-77%), and a high negative
predictive value (96%-98%) for DVT and PE.1-5
A high sensitivity of a D dimer for DVT and PE is critical, because the consequences
of missing the diagnosis can be fatal or nonfatal recurrence. However, as
most patients (approximately 80%) with suspected DVT or PE do not have the
disease, the specificity of a D-dimer assay is also important to ensure that
a sufficient number of these patients have a negative result to make the test
useful. In our institution, a limited number of nurses and technologists perform
the assay, and the result is a high sensitivity and moderate specificity.
This D-dimer assay is one of a few that has undergone extensive clinical
testing; it has been evaluated in published studies enrolling more than 1500
patients with suspected venous thromboembolism (VTE).1-4
However, concerns have been raised that the subjectivity of the assay reduces
its accuracy. In the initial studies when the assay was performed by selected
personnel, it showed a sensitivity of 90% to 95% for VTE.2-3
In a subsequent, larger multicenter study of patients with suspected PE that
used multiple personnel to perform and interpret the assay, we observed a
sensitivity of only 84%.4 Moreover, in the
hands of other investigators, the sensitivity for VTE has been reported to
be as low as 65%.6 Based on our clinical experience,
we reasoned that these reports of the low sensitivities were probably the
result of inexperienced, busy health care workers who performed the assay
under suboptimal conditions.
To overcome these problems, 2 options are available. First, performance
and interpretation of the assay should be limited to a finite number of experienced
personnel working under optimum conditions. Second, an objective technique
should be incorporated to read the assay; this technique is currently not
available.
To explore the first solution, we tested the hypothesis that this D-dimer
assay has as high a sensitivity for VTE when performed by a relatively small
group of experienced, well-trained laboratory technologists as it has when
performed by a small group of well-trained research personnel. To facilitate
laboratory testing, we collected samples of venous whole blood into 3 routine
blood collection tubes (containing sodium citrate, EDTA, or a combination
of lithium and heparin). We also wanted to estimate the accuracy of the assay
when the blood specimen was stored overnight, to explore whether laboratories
could send the blood sample within 24 hours to another laboratory that has
the assay.
PATIENTS AND METHODS
Consecutive patients with suspected DVT or PE who were referred to the
thromboembolism service at the McMaster Division of the Hamilton Health Sciences
Corporation, Hamilton, Ontario, were enrolled from June 1, 1997, through November
30, 1998, and the last 3-month follow-up occurred in February 1999. Patients
were excluded from the study if (1) they were receiving ongoing warfarin sodium
therapy or they had received more than 24 hours of unfractionated or low-molecular-weight
heparin therapy; (2) they were pregnant; or (3) they had undergone major surgery
in the past 3 days. Consent was obtained from all patients after the study
was explained, and all patients underwent assessment by a physician specializing
in thromboembolism. The study was approved by the institutional review board
of the hospital.
Patients with suspected DVT received a diagnosis of DVT on the basis
of a positive finding on compression ultrasonography (CUS) or venography.
No DVT was indicated if findings on serial CUS, impedance plethysmography
(IPG),7 or venography were negative and if
the patients remained free of VTE within the 3 months of follow-up.
Patients with suspected PE underwent ventilation perfusion (VQ) lung
scanning, and further testing was performed according to the scan results.8 Patients received a diagnosis of PE on the basis of
a positive finding on a pulmonary angiogram, a high-probability lung scan,
or a nondiagnostic VQ lung scan and a positive test result for DVT. No PE
was indicated if they had a normal finding on a pulmonary angiogram or VQ
lung scan or nondiagnostic VQ lung scan and negative results of serial CUS,
serial IPG, or venography and if they remained free of VTE within 3 months
of follow-up.
The IPG and CUS were performed and interpreted as previously described9-10; CUS was reported as diagnostic of
DVT if 2 contiguous proximal deep venous segments were noncompressible. Venography
was performed using the technique of Rabinov and Paulin,11
and findings were interpreted as positive if an intraluminal filling defect
was visible in at least 2 views. Lack of filling of vessels was considered
to be nondiagnostic. The VQ lung scans and pulmonary angiography were performed
as previously described.12 The results of the
lung scan were classified as normal, high probability (segmental or greater,
or a perfusion defect with normal ventilation and visible on 2 views), or
nondiagnostic (segmental defects with matched ventilation defects, or subsegmental
perfusion defects with or without matched ventilation defects). Diagnostic
criteria for positive angiographic findings consisted of a constant intraluminal
filling defect seen in multiple views or a sharp cutoff in a vessel greater
than 2.5 mm in diameter.
A fingerstick D-dimer assay was performed (by a research assistant)
at the bedside using the method described by John et al.13
Subsequently, with the use of a sterile technique, venous blood was obtained
in 4 tubes containing the following additives: none (plain), 0.105M buffered
3.2% sodium citrate (blue top), a combination of lithium and heparin (green
top), and tri-potassium EDTA (purple top) (Becton Dickinson, Mississauga,
Ontario). The D-dimer assay was repeated by a second research nurse or assistant
at the bedside in the plain tube. The remaining 3 tubes were sent to the laboratory
for testing. A laboratory technologist performed the D-dimer test within 4
hours of collection on all 3 remaining tubes using whole blood (samples kept
at room temperature before testing). In addition, the EDTA tube was placed
in a refrigerator (at 40°C) overnight and retested the following morning;
this tube is referred to as the 24-hour EDTA sample.
The D-dimer results from the EDTA tubes tested within 4 hours are referred
to as the 4-hour EDTA samples. All 4 laboratory personnel
(including M.R.) performing D-dimer assays were unaware of the results of
objective tests for VTE and the capillary fingerstick and plain-tube D-dimer
test results already performed at the bedside.
We decided a priori to perform the study in 2 parts. In the first part,
50 patients would be enrolled, and if the results were poor, we planned to
terminate the study, whereas if the results looked promising, we would continue
enrollment. Of the first 50 patients, 10 had confirmed VTE, and the 24-hour
EDTA sample showed a sensitivity of 100% for VTE compared with a sensitivity
of 70% for bedside fingerstick testing. Based on these results and on our
objective demonstration that laboratory testing had a high sensitivity, we
estimated that an additional 100 patients would need to be enrolled. By enrolling
150 patients, and with an expected prevalence of VTE of 20% (30 patients)
if the observed sensitivity of the 24-EDTA samples (and other samples) was
93% (28 of 30 patients), the sensitivity of laboratory testing would be sufficiently
high and the 95% confidence interval (CI) would be sufficiently narrow that
we would satisfy our objective.
We calculated descriptive statistics with means and SDs using SPSS software
(Version 8.0; SPSS Inc, Chicago, Ill). Sensitivities, specificities, and negative
predictive values were calculated for each collection method. We compared
the capillary fingerstick sensitivity and the most sensitive laboratory test
result using the Fisher exact test. The 95% CIs of proportions were calculated
using CIA software (Version 1.1; ISO Data Centre, Saclay, France).
RESULTS
One hundred fifty-three patients were enrolled. For technical reasons,
D-dimer assay results were unavailable for 2 patients, and only the results
of bedside testing were available for 3 patients; results of these latter
3 patients are included in the final analysis. Three patients died before
their 3-month follow-up and were excluded from the final analysis. For the
148 patients whose results were included in all analyses, mean (SD) age was
58 (16) years. Ninety-four patients (64%) were female. Of the 30 patients
presenting with suspected PE, 9 had diagnositic findings positive for the
disease. Of 118 with suspected DVT, 25 had positive diagnostic findings.
Of the 3 patients who died, none were considered to have VTE. One was
an 86-year-old woman who had negative findings of initial objective tests
for DVT and negative results of the D-dimer assay. Four weeks later, she died
of a massive brainstem hemorrhage proven by findings on computed tomographic
scanning. A second woman of the same age died at her nursing home after a
clinically diagnosed stroke; results of her D-dimer assay by means of capillary
fingerstick sampling were negative, but results of laboratory tests were weakly
positive. The third patient died 5 days after enrollment. At initial assessment,
he had negative findings on CUS, and all results of D-dimer assay were positive.
Three days later, he was admitted to another hospital with hematuria, urinary
retention, and obstructive renal failure. Ventricular fibrillation developed
shortly after cystoscopy, and he could not be resuscitated. An autopsy was
not performed.
Table 1 summarizes the sensitivities,
specificities, and negative predictive values and the corresponding 95% CI
for all test formats. The median time for testing of the 24-hour EDTA tube
after initial bedside testing was 22.6 hours (SD, ± 8.5 hours). The
sensitivity of the capillary fingerstick D-dimer assay was 88% (95% CI, 73%-97%);
specificity, 71% (95% CI, 63%-79%). Although the point estimates of the sensitivities
all favored laboratory testing compared with bedside testing, the differences
between the sensitivities of the capillary fingerstick bedside method and
of the other D-dimer assay methods are not statistically significant (P = .36).
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Summary of D-Dimer Assay Accuracy Indices of Each Tube*
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COMMENT
The results of this study confirm that the SimpliRED D-dimer assay performed
in the laboratory on venous blood samples collected in laboratory tubes containing
one of the 3 anticoagulants has a high sensitivity and negative predictive
value for VTE. These results have 2 important practical implications. First,
samples can be tested in the laboratory by experienced technologists under
optimal conditions without compromising sensitivity. Second, if the assay
is not available in one institution, samples can be transported within 24
hours (refrigerated or on ice) to another site that has the test, if an EDTA-containing
tube is used.
The observed sensitivity for the laboratory tubes of 97% (95% CI, 85%-99%)
is consistent with the 94% sensitivity seen in the initial studies2-3 with D-dimer testing performed at the
bedside by selected, trained personnel. Furthermore, for each of the laboratory
tubes, the lower limit of the 95% CI is 85%, which is higher than the observed
sensitivity for this assay (performed at the bedside) in a recent large multicenter
trial (84%).4 This finding is consistent with
our hypothesis that the fall in sensitivity is due to performance of the assay
by less experienced personnel, perhaps under suboptimal conditions.
Several factors could influence the accuracy of the laboratory D-dimer
results, including the expertise of the observer and, for laboratory testing,
the additives in the tubes. In almost all medical centers, laboratory technologists
have more experience at reading red blood cell agglutination (also an end
point when crossmatching of red blood cells is performed) than nonlaboratory
staff, which will likely maintain the accuracy when the test is performed
on blood samples taken from laboratory tubes. Although we were initially concerned
that dilution of blood by citrate (and to a lesser extent, heparin) would
dilute the D dimer and decrease the sensitivity of the assay, we did not find
this.
To minimize bias in D-dimer interpretation, laboratory staff were unaware
of the results of objective tests for VTE and the capillary fingerstick and
plain-tube D-dimer assay results. Laboratory samples were all batch tested,
often by one technician, before storage of the EDTA tube overnight. We did
not specify an order of tube testing, and it was not practical to blind the
technologist to the SimpliRED D-dimer assay results for each of the individual
laboratory tubes. The results of the first laboratory tube could have biased
interpretation of agglutination for the other tubes. In some instances, laboratory
staff who tested the 24-hour EDTA sample tested the 4-hour EDTA sample from
the same patient. Therefore, we cannot discount the possibility of biased
assessment of the 24-hour EDTA results.
Although the point estimates of sensitivities favor laboratory testing,
this study was not designed to, and does not have sufficient power to, establish
superiority of laboratory testing compared with fingerstick testing at the
bedside.
CONCLUSIONS
This study shows that the SimpliRED D-dimer assay can be performed on
venous blood from routine laboratory samples without compromising accuracy.
This finding is important because it means that the assay should be performed
on venous whole-blood samples by laboratory staff, in centers where trained
personnel are not available to perform bedside fingerstick testing. In addition,
the accuracy of the 24-hour EDTA results means that centers that do not have
access to D-dimer testing can refrigerate and transport samples, within 24
hours, to a laboratory that has this D-dimer assay.
AUTHOR INFORMATION
Accepted for publication November 16, 2001.
Dr Ginsberg is the recipient of a Career Investigator Award from the
Heart and Stroke Foundation of Ontario, Toronto. Dr Chunilal is a recipient
of the Noonan Fellowship, McMaster University, Hamilton.
Presented in abstract form as a poster at the International Society
of Thrombosis and Haemostasis, Washington, DC, August 14-21, 1999.
Corresponding author and reprints: Jeffrey S. Ginsberg, MD, FRCPC,
McMaster University Medical Center, 1200 Main St, West HSC 3X28, Hamilton,
Ontario, Canada L8N 3Z5 (e-mail: ginsbrgj{at}mcmaster.ca).
From the Department of Medicine, McMaster University (Drs Chunilal,
Brill-Edwards, and Ginsberg and Mss Stevens, Joval, and McGinnis), and the
Department of Laboratory Medicine, McMaster Division of the Hamilton Health
Sciences Corporation (Ms Rupwate), Hamilton, Ontario.
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