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Moniek P.M.
de Maat
Department of Thrombosis Research, University of Southern Denmark
and department of Clinical Biochemistry, Ribe County Hospital,
Esbjerg, Denmark and Gaubius Laboratory TNO-PG, Leiden, the
Netherlands
Jorgen Gram
Department of Thrombosis Research, University of Southern Denmark
and department of Clinical Biochemistry, Ribe County Hospital,
Esbjerg, Denmark
Jorgen Jespersen
Department of Thrombosis Research, University of Southern Denmark
and department of Clinical Biochemistry, Ribe County Hospital,
Esbjerg, Denmark
Cornelis Kluft
Gaubius Laboratory TNO-PG, Leiden, the Netherlands
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Background.There is no common standardisation of
different commercially available kits for both t-PA and PAI-1
antigen.Aim.The aim of this project was to study whether the
exchange of the kit calibrator with the common calibration
materials of the WHO would harmonise the results produced by five
different commercially available t-PA and PAI-1 antigen kits when
analysing the SSC secondary standard.Methods.WHO international
standards were used as calibrator and the SSC secondary standard
and a commercially available plasma standard were used as test
plasma in 5 commercially available kits measuring total t-PA and
PAI-1 antigen. For t-PA only, the SSC secondary standard was spiked
with purified t-PA and recovery was studied.Results.There was a
large variation in the concentrations of t-PA antigen (ranging from
<0.5 to 6.6 ng/ml for the SSC secondary standard and from 3.3 to
10.9 ng/ml for the commercial plasma standard, respectively)
produced by the different kits. Also, PAI-1 antigen results of the
different kits showed a large variation (ranging from 20.3 to 51.2
ng/ml for the SSC secondary standard and from 41.8 to 89.7 ng/ml
for the commercial plasma standard, respectively). Results of the
two test samples and spiking with t-PA were not in agreement in all
methods, indicating differences in specificity of tests. Data point
to a specific effect of the matrix of standards.Conclusions.The use
of a common calibration material does only marginally harmonise
data for t-PA and PAI-1 antigen assays. There is a need for
improvement of methods to cope with standards and
standardisation.
Correspondence to: Moniek P.M. de Maat,
PhD
Dept. Clinical Biochemistry, Ribe County
Hospital, Ostergade 80 DK 6700 Esbjerg, Denmark Tel. +45 79 182423,
Fax +45 79 182430, E-mail Mpm.demaat@pg.tno.nl# within the frame work of the ISTH/SSC
subcommittee on Fibrinolysis
Increased protein concentrations of tissue-type
plasminogen activator (t-PA) and increased activity and protein
concentration of plasminogen activator inhibitor-1 (PAI-1) in
plasma have been associated with the evolution of ischemic heart
disease (IHD)(1-5). Therefore measurement of t-PA and PAI-1 antigen
can be useful as a prognostic indicator of the risk of IHD.
Kits for total t-PA and PAI-1 antigen are
commercially available, and we have previously demonstrated that
the major problem with the measurement of e.g. PAI-1 antigen and
activity is the large variation in the results obtained by the
different commercially available kits (6,7). In these studies the
calibrators provided with each kit were used, but even after
harmonisation with the use of the NIBSC reference material a
considerable variation in the results existed. Obviously such a
situation is inconvenient when t-PA and PAI-1 are used in the
clinical laboratory, because it makes it very difficult, if not
impossible, to transfer data from scientific studies to daily
clinical practice and also to compare results from one laboratory
to another. In addition, the development of a secondary plasma
standard by the SSC required decisions about the method to be used
in conjunction with the WHO standards.
A logical step to try to reduce inter-assay
variation and standardise measurement results would be to use the
same calibrator in the different kits. Therefore, we studied within
the framework of the Subcommittee of Fibrinolysis of the Scientific
and Standardisation Committee (SSC) whether substitution of the kit
calibrator by the NIBSC reference plasma would reduce or eliminate
inter-kit variation.
We studied five different commercially available
t-PA and PAI-1 antigen kits reported to measure total
concentrations of each analyte, and in order to eliminate
inter-laboratory variation we produced all the results at the same
laboratory. The first batch of the SSC secondary standard and a
commercial plasma standard were used as test plasmas.
The NIBSC plasma t-PA (94/730), pure t-PA (86/670),
and plasma PAI-1 (92/654)(Gaffney 1996) were used as calibrators
(NIBSC, ). The plasma t-PA standard (94/730) contains 25 ng t-PA
per ampoule (by assignment). The purified second international
standard for t-PA (86/670) is highly purified, 98% single chain
t-PA from a Bowes melanoma cell line (8), and contains 2 �g/ml t-PA
antigen (assigned by amino acid analysis). The PAI-1 plasma
standard (92/654) is enriched with added reactivated recombinant
PAI-1, lyophilised and assigned 185 ng/ml PAI-1 antigen (9). The
calibration curves were constructed as dose-response curves by
dilutions of the NIBSC plasma standards of t-PA and PAI-1, as
described by the manufacturers; i.e. dilution buffer or plasma
depleted of t-PA or PAI-1, respectively. The SSC secondary standard
( Lot # 1)(Immuno, Vienna, Austria ) and the Biopool reference
plasma (Biopool, Umea,Sweden ) were used as test materials.
An EIA measuring total t-PA, i.e. free and
complexed t-PA, single-chain and double-chain t-PA. Two monoclonal
antibodies, directed against different epitopes of t-PA, are used
for coating and a third monoclonal antibody for tagging.
(Chromogenix, Molndal, Sweden) (10)
Two different monoclonal antibodies are used as
capture and tagging antibody, directed against different epitopes
of t-PA (Technoclone, Vienna, Austria)
A goat anti-human t-PA is used as coating antibody
and anti-t-PA Fab fragments are used as tagging antibody measuring
free and complex forms of t-PA (Tintelize t-PA, Biopool, Umea,
Sweden)(11).
A one-step EIA measuring total, bound and free
forms of molecules (12)(Organon Teknika, Turnhout, Belgium)
An EIA measuring total circulating t-PA, i.e. free
and complexed t-PA, single-chain and double-chain t-PA. The EIA
uses two different mouse monoclonal antibody against human t-PA for
coating and tagging (Asserachrom � t-PA, Stago,
Asnieres-sur-Seine, France)
An EIA with equal sensitivity for PAI-1/recombinant
t-PA complex, PAI-1/melanoma t-PA complex, PAI-1/u-PA complex,
inactive PAI-1 and active PAI-1 (Chromogenix, Molndal,Sweden)
(13,14)
Two different monoclonal antibodies, directed
against different epitopes of PAI-1, are used as capture and
tagging antibody. The EIA measures free and complexed t-PA
(Technoclone, Vienna, Austria)
An EIA using a combination of monoclonal and
polyclonal antibodies that measures human PAI-1, endothelial type.
It detects active and inactive (latent) forms of PAI-1, as well as
that complexed as t-PA/PAI-1 and u-PA/PAI-1 (Tintelize PAI-1,
Biopool, Umea, Sweden)(14,15).
A one-step EIA for PAI-1 measuring total, bound and
free forms of molecules (16) (Organon Teknika, Turnhout,
Belgium).
An EIA measuring total circulating PAI-1, i.e. free
and complexed with t-PA, bound or unbound to vitronectin, active or
inactive forms, and platelet PAI-1. The EIA uses two different
mouse monoclonal antibody against human PAI-1 for coating and
tagging (Asserachrom � PAI-1, Stago, Asnieres-sur-Seine,
France)
In the first experiment (both t-PA and PAI-1) the
calibration curve was constructed by means of the NIBSC plasma
(matrix) standards, diluted according to the instructions given by
the manufacturers (figure 1). The SSC secondary standard and the
Biopool reference plasma were used as samples and the
concentrations were determined using the NIBSC calibration curve.
The experiment was performed eight times on two different
days.
In the second experiment (for t-PA only) the SSC
secondary standard was spiked in a dose-dependent manner with
increasing amounts of NIBSC purified t-PA (figure 2). A
dose-response curve was constructed and the absorbance at the
cut-off point with the Y-axis was detected. The cut-off point
represents the amount of t-PA in the SSC secondary standard and the
absorbance was converted to concentration units with the use of the
calibration curve, which goes through the point of origin and is
parallel to the original dose-response curve. The experiment was
performed six times on two different days.
When the values for t-PA were determined in the SSC
secondary standard, using the NIBSC t-PA reference plasma as a
calibrator, the levels obtained with the different kits varied from
<0.5 ng/ml to 5.2 ng/ml (Fig 3,4). Also in the second
experiment, where the SSC secondary standard was spiked with NIBSC
purified t-PA, the t-PA antigen levels measured for the SSC
secondary standard varied widely from 1.6 ng/ml to 7.3 ng/ml. The
tests that measured the highest and lowest levels in experiment 1
were similarly ranked in the second experiment (Fig 5).
When the values for t-PA were determined in the
Biopool reference plasma, again using the NIBSC t-PA reference
plasma as a calibrator, the levels obtained with the different kits
also greatly varied, and the kits that measured relatively low
levels in the SSC secondary standard were not the same as those
that measured relatively low levels in the Biopool reference plasma
(Fig 6).
There was no association between the variation in
the levels measured in the SSC secondary standard with the
different kits and the kind of reference material (buffer or
plasma) originally provided in the kits. For all kits the slopes of
the calibration curve in experiment 1 and the slope of the curve of
the SSC secondary plasma standard spiked with purified t-PA in
experiment 2 were different (results not shown).
When the values for PAI-1 were determined in the
SSC secondary standard, using the NIBSC PAI-1 reference plasma as a
calibrator, the concentrations obtained with the different kits
varied from 22.0 ng/ml to 44.8 ng/ml (Fig 7). There was a weak
relationship between concentrations measured in the SSC secondary
plasma standard and the Biopool reference plasma (Fig 8).
There was no association between the variation in
the levels measured in the SSC secondary plasma standard with the
different kits and the kind of reference material originally
provided with the kits.
We observed that the introduction of a common
calibrator for determination of total t-PA and PAI-1 antigen
concentrations did not harmonise results which was produced by the
different available kits. To a great extent the problem is related
to t-PA determination, while the variation of PAI-1 determination
is fairly stable, at least when we include only kits 1, 2, 4 and
5.
The NIBSC matrix standards for t-PA and PAI-1 were
used as calibrator materials for each of the kits, and all the
measurements were performed in the same laboratory and by the same
technician. In order to have optimal compliance to kit procedures
we used for each kit its own dilution reagent to dilute the NIBSC
standard plasmas and the NIBSC purified t-PA. We used this
experimental set-up in order to try to harmonise the calibration
process of the different kits, but we cannot exclude that the
matrix of the NIBSC plasma standards influenced the measurement
results. It should be noted that some differences were observed for
t-PA in the series of experiments in which the SSC secondary plasma
standard was spiked with purified t-PA using a constant matrix
back-ground. In figure 5, the kits 4 and 5 show a systematic
deviation either due to differences in specificity for added t-PA
and plasma t-PA or sensitivity for matrix effects.
Various monoclonal and polyclonal antibodies have
been developed and used in the development of commercially
available immunological enzyme-immuno-assays (EIA). Because the
antibodies are directed against different epitopes and t-PA and
PAI-1 can be found in various forms, the different EIA will have
different specificities for the various forms. For each kit, it was
stated by the companies that the total t-PA or PAI-1 antigen was
measured. Data regarding sensitivity towards the different forms of
t-PA and PAI-1 was only in the kits from Chromogenix, Biopool and
Organon. Since no information was available for the other kits, it
was not possible to evaluate whether the specificity of the
antibodies explains the differences in values found with the
different kits. It cannot be excluded that the additives of the
calibrator and test plasmas or the lyophilisation method may affect
the assays differently. We studied this by calculating for each kit
the ratio between results of the SSC secondary standard and the
Biopool reference plasma. This ratio would be expected to be equal
for all kits, i.e. one kit would be expected to measure relatively
high levels in both plasmas while another kit would be expected to
measure relatively low levels in the two plasmas. However, we
observed an inter-kit variation, in particular with respect to the
t-PA measurements (Fig 6). These data indeed suggest that other
factors (such as additives and lyophilisation) affect the
measurement procedures - an observation which is also of importance
for the preparation of future reference materials.
In our study the companies had the opportunity to
comment on our results and conclusions and in general they were in
agreement with our results. The companies also stressed that
difference in the matrix of the calibration material, the
lyophilisation and the specificity of the antibodies could be the
factors causing the discrepancies. The only significant
disagreement was that for kit number 3 the company reported that
they had measured a higher t-PA concentration in the first
experiment. This supports the assumption that matrix effects
contribute to the observed variation in measured levels. For kit
number 3 the company also reported interference in the assay by the
buffer HEPES. Enquiry about the SSC secondary standard revealed
that this standard contained HEPES (H. Lang, personal
communication)
We have previously demonstrated a large inter-assay
variation of PAI-1 measurements when the kit calibrators provided
were used (6,7). The present study shows a similar result when a
common reference material with an approved value is used and
standard plasmas are measured Despite the fact that the information
with all kits claimed to measure total protein concentration of
t-PA and PAI-1, we observed a highly significant variance in
measurement results of both t-PA and PAI-1 antigen in the standards
in this study (Fig 3,7). We realise that we have increased the
complexity by using lyophilised, stabilised standards, but only
when kits also can cope with such standards, harmonisation becomes
possible. It was observed that information on the additives to
standards is meagre. It can be suggested that such information be
improved to allow a better interplay in development between
standards and methods.
We have now definitively demonstrated that the use
of calibration materials with certified values is not sufficient to
secure a low inter-kit variation of measurements. Standardisation
efforts to improve the situation should, in addition to the
improvement of reference materials, certainly focus on the
evaluation of the specificity of kits. Work on the establishment of
such criteria is ongoing within a working group of the SSC.
Furthermore, we must focus on the development of reference methods.
Such a strategy would be a first step in the development of a
coherent measurement system (17) in which there is a close
association between reference materials (primary, secondary etc.)
and the analytical methods (definitive, reference and field
methods). Such a procedure would also be of great help to
manufacturers since so far there has been no general calibration
system within fibrinolysis.
Design of the first experiment where a calibration
curve was made using the NIBSC matrix standards. The SSC secondary
standard or the Biopool reference plasma were then used as sample
and the concentration was determined using the NIBSC calibration
curve from the particular kit.
Design of the second experiment (for t-PA only)
where the SSC secondary standard was spiked in a dose-dependent
manner with increasing amounts of purified t-PA. A dose-response
curve was then constructed and the absorbance at the cut-off point
with the Y-axis was detected. The cut-off point represents the
amount of t-PA in the SSC secondary standard.
t-PA antigen concentrations measured in the SSC
secondary standard using five different kits that measure total
t-PA protein concentrations (first experiment with direct
measurement of the SSC secondary standard)(mean and 1 SEM).
t-PA antigen concentrations measured in the SSC
secondary standard (estimated as the cut-off point on the
ordinate)(second experiment) using five different kits that measure
total t-PA protein concentrations (mean and 1 SEM)
t-PA antigen concentrations measured in the SSC
secondary standard in the first experiment (abscissa) and the
second experiment (ordinate) using five different kits that measure
total t-PA protein concentrations.
t-PA antigen concentrations in SSC secondary
standard and Biopool reference plasma measured by the use of the
different kits and the NIBSC t-PA plasma standard as calibrator
(first experiment).
PAI-1 antigen concentrations measured in the SSC
secondary plasma standard (first experiment) using five different
kits that measure total PAI-1 protein concentrations(mean and 1
SEM)
PAI-1 antigen concentrations measured using the
different kits in SSC secondary plasma standard and Biopool
reference plasma and using the NIBSC PAI-1 plasma standard as
calibrator.
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We wish to thank Linda Huisman, Katrine Overgaard,
Mette Toft for excellent technical assistance, Chromogenix,
Technoclone, Organon Teknika, Stago and Biopool for giving us kits
to perform the studies and for their participation in interpreting
the data, Immuno for giving us the SSC secondary standard and Drs.
Patrick J Gaffney and Tracy Edgell from the NIBSC for providing the
NIBSC reference material
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