Dimeski, Goce

Cameron CL

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Evidence on the cause of false positive troponin I results with the Beckman AccuTnI method

University of Queensland eSpace

Clin Chem Lab Med 2011;49(6):1079–1080  2011 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/CCLM.2011.163
2010/753
Article in press - uncorrected proof
Letter to the Editor
Evidence on the cause of false positive troponin I results
with the Beckman AccuTnI method
Goce Dimeski1,2,*
1 Department of Chemical Pathology, Pathology
Queensland, Princess Alexandra Hospital, Woolloongabba,
Queensland, Australia
2 School of Chemistry and Molecular Biosciences, The
University of Queensland, Brisbane, Australia
Keywords: false positive; insoluble fibrin; troponin I.
False positive troponin I (TnI) results with the Beckman
AccuTnI method have been reported since 2002 (1). Recent
studies from my own laboratory have demonstrated that false
positive TnI results occur with both serum and lithium hep-
arin sample types with BD tubes (2) and Greiner tubes, at a
rate of 14/1000 samples on both the Access 2 and DxI800
analysers (3). These and other publications since then have
attributed the errors to residual fibrin (1, 4, 5). The discrepant
results are rarely reproducible. The strategy employed to
minimise erroneous results from being reported has been to
utilise aliquots of serum or plasma and re-centrifuge the ali-
quot at centrifugation settings routinely used or at high
speeds (6). In our laboratory, the strategy is to utilise the
analyser’s reflex function and analyse samples in duplicate
when the result is )0.04 and -5.00 mg/L. Results outside
these limits have been very rarely observed to produce false
positive results and thus are not run in duplicate. If the dupli-
cate results exceed a difference of 20% or more, which is
considered clinically significant and needs investigation, an
aliquot is re-centrifuged and the supernatant re-analysed. The
third (and fourth replicate if produced) are reviewed in con-
junction with other pathology results and clinical information
before a result is reported. It is by no means a fool proof
process, and on very rare occasion false positive results con-
tinue to be reported at a rate of one result in approximately
4000 samples. The downside with the process, as pointed out
by Pfafflin, is that there can be delays in reporting of results
and potential delays in diagnosis of myocardial infection (6).
The delay in reporting of TnI results with this procedure is
justified by minimisation of result inaccuracies, reducing the
number of patients falsely diagnosed or being exposed to
unnecessary stress with additional testing, treatment and
*Corresponding author: Goce Dimeski, Department of Chemical
Pathology, Pathology Queensland, Princess Alexandra Hospital,
Ipswich Road, Woolloongabba, Queensland 4102, Australia
Phone: q61 7 31765 5082, Fax: q61 7 3176 7070,
E-mail: goce_dimeski@health.qld.gov.au
Previously published online March 24, 2011
admission. The cost of the reagent and other consumables
are easily accounted for by preventing the inappropriate
admission of a few patients each year, and preventing poten-
tial legal and professional implications.
The exact cause of the problem has not been identified
nor has anyone shown residual fibrin or any other analyte
being directly linked to the problem. Lithium heparin plasma
samples stored at 48C containing ‘‘insoluble fibrin’’ mesh
were used to demonstrate the impact of sample quality result-
ing in non-specific binding and the lack of method robust-
ness. Such insoluble fibrin is observed after 24 h of storage
at 2–88C in about 5% of samples in our laboratory. Insoluble
fibrin is formed from fibrinogen, and fibrinogen is present
in lithium heparin plasma. Fibrin formation and fibrin struc-
ture is stated to be influenced by a number of variables
including fibrinogen and thrombin concentrations, pH, ionic
strength, chloride ion concentration, presence of calcium
ions, and polyphosphate, a potent anticoagulant (7). Disease
state and medication can both diminish the efficacy of hep-
arin activity and lead to increased fibrin formation, and this
is enhanced when plasma is stored at low temperature (8).
In our laboratory, blood tubes (lithium heparin plasma and
serum) are centrifuged for 10 min at 208C and 3000 g (approx.
4200 rpm). Approximately 95% of samples are centrifuged
within 1 h from the time the blood is collected. The total
turn-around time (TAT) for TnI results from collection to
result validation is -70 min for approximately 90% of sam-
ples, and the remaining samples have longer TAT mainly
because they are requested at a later time. The sample mix for
routine TnI analysis is 60% serum and 40% lithium heparin
plasma. Routinely, three levels of control material are run and
the mean CV values at the time of the study were 13.5%
(0.058 mg/L), 9.6% (0.532 mg/L) and 8.3% (6.87 mg/L).
To determine if insoluble fibrin can lead to false positive
TnI results, two experiments were performed. In the first
experiment, a sample with TnI above the cut-off threshold
and containing insoluble fibrin was analysed 12 times; 10
times with fibrin mesh intact and two times following re-
centrifugation or using clean aliquot of the sample. Both
clean sample TnI concentrations were 0.077 and 0.077 mg/L.
The 10 replicates produced results ranging from 0.09 to
0.162 mg/L.
For the second experiment, lithium heparin plasma from
two samples containing insoluble fibrin were pooled. The
sample was centrifuged at 100 rpm for 1 min to concentrate
the fibrin, but not destroy the fibrin mesh. The clear super-
natant (plasma) was removed and replaced with an equal
volume of normal saline. The sample was gently inverted
and re-centrifuged at 100 rpm for 1 min. The saline wash
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1080 Dimeski: Cause of false positive TnI results with Beckman AccuTnI method
Article in press - uncorrected proof
Table 1 Effects of insoluble fibrin from lithium heparin plasma
on the Beckman AccuTnI assay.
Fibrin sample cTnI, mg/L Actual time
of analysis
Fibrin aliquot 1 0.061 17:45
Fibrin aliquot 1 (reflex) 0.016 17:59
Fibrin aliquot 2 0.050 17:46
Fibrin aliquot 2 (reflex) 0.023 18:00
Fibrin aliquot 3 0.038 17:47
Fibrin aliquot 4 0.035 17:47
Fibrin aliquot 5 0.034 17:48
Centrifuged aliquot 0.015 18:00
Centrifuged aliquot 0.013 18:14
step was repeated two more times and the sample tube filled
with saline to the original plasma level in the tube to resus-
pend the fibrin. An aliquot was also re-centrifuged and the
clear supernatant analysed in duplicate. Five aliquots of the
sample containing fibrin were analysed for TnI. The results
are summarised in Table 1.
The results indicate fibrin is likely to have crossed linked
the assay antibodies to produce false positive TnI results. The
results also show that as fibrin settles out in the saline, inter-
ference was reduced as indicated by the reflex results. Addi-
tionally, the false positive TnI results are higher when the
fibrin concentration is higher in the sample, before it settles.
In plasma, insoluble fibrin does not settle to the same extent
and stays dispersed throughout the sample either as result of
networks with other proteins or binding/anchoring to the tube
wall.
The mechanism suggested by Er et al. is that the antibody
either binds non-specifically to fibrin or the indicator enzyme
may be physically trapped by fibrin in the separation matrix
(4). This is unlikely to cause false positive TnI results. For
the false positive to occur, both antibodies (capture and sig-
nal) have to be bound by the fibrin and be retained in the
cup following the wash to cause the error. To overcome this
problem, method redesign is essential. This may involve a
combination of factors, such as modification of reagent pH
to minimise non-specific binding, introducing an additional
wash step after the sample and capture antibody-paramag-
netic particle have been incubated and bound, and the second
wash step occuring after the signal antibody has been incu-
bated to form the sandwich. Additionally, increasing the
wash time and rigor needs to also be considered. The data
represents direct evidence of non-specific binding leading to
false positive TnI results, and confirms that insoluble fibrin,
if present in the aliquot aspirated for analysis, is capable of
causing false positive results.
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors stated that
there are no conflicts of interest regarding the publication of this
article.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
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Evidence on the cause of false positive troponin I results with the Beckman AccuTnI method

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