|
C. Kluft
*
Gaubius Laboratory, TNO-PG
P.O. Box 2215
2301 CE Leiden
The Netherlands
Phone: +31.
15181497
Fax: +31.715181904
E-Mail: kluft@euronet.nl
* on behalf of a working group within the Subcommittee on
Fibrinolysis, of the Scientific and Standardisation Committee of
the International Society on Thrombosis and Haemostasis (P.J.
Declerck, J. Jespersen, J.Gram, C.Kluft) and the active
participants, see acknowledgement.
 Download as a PDF
here
Abstract Background
Proficiency testing of enzymatic methods for plasminogen
activator inhibitor 1 (PAI-1) showed a systematic variability
indicating the aspecificity of some methods.
Methods
To define and detect specificity of enzymatic methods for PAI-1,
experts of the ISTH/SSC subcommittee defined criteria and test
samples to check the criteria. 16 samples were prepared to test (a)
specificity in depleted plasma, (b) interference by added PAI-2 or
PAI-3, (c) interference by added tissue-type plasminogen activator
(t-PA), (d) performance in dose response. To exercise the test
procedure participants were recruited via the subcommittee,
literature and company data. Coded samples were distributed to
participants. The NIBSC standard for PAI-1 activity was included
for the normalisation of results. Adherence to the predetermined
criteria was judged blind in the ISTH/SSC subcommittee meeting.
Results
In total 17 laboratories with 15 different assay methods
participated in the study. Methods were based on four detection
principles. 8 methods failed to detect sufficiently low activity in
depleted plasma and were sensitive to added PAI-2. 10 methods were
sensitive to interference by endogenous t-PA as revealed by
additions of t-PA. Not all methods could adequately measure in
acidified plasma. Two methods were fulfilling all the criteria
Conclusions
Most methods were not specific for PAI-1 (enz. procedure) in
acidified plasma. Definition of criteria and test methods by
experts of the ISTH/SSC proved a valuable concept, according to the
exercise undertaken.
Introduction
Proficiency testing of assays for the fibrinolytic variable
Plasminogen activator inhibitor 1 (PAI-1, imm; procedure, enz;
procedure) has been executed by collaborative studies within the
framework of the SSC (Scientific and Standardisation Committee) of
the ISTH (International Society on Thrombosis and Haemostasis) and
showed large interlaboratory and intermethod variation (1,2).
In particular the results of studies on the activity methods for
PAI-1 showed a systematic variation which could not expected to be
solved by harmonising with a suitable standard. It was decided that
an effort was required to define criteria for specificity of
enzymatic PAI-1 methods.
The situation was more complex since many methods were in use both
as commercial methods and in-house methods and documentation was
insufficient for decisions on specificity. It was decided that in
addition to defining criteria an exercise would be executed by
providing a set of samples to test the methods for the specificity
claimed or theoretically expected.
The present report, in addition to defining criteria and test
samples describes an exercise of experimental testing and decisions
based on the data and criteria. The data have thus far only been
presented without the identification of the methods involved.
Materials and
Methods
Samples:
Sample A was a PAI-1-depleted plasma sample prepared by
immunoadsorption of plasma as previously described (3); samples B,
C and D were prepared by adding a defined amount of purified
reactivated recombinant PAI-1 to depleted plasma (4).
Samples E and F were prepared by adding to immunodepleted plasma
a defined amount of recombinant PAI-2 (Delta Biotechnology Ltd,
Nottingham, UK; batch 25/7; purity >95%) (plasma dilution <
1%), or PAI-3 / Protein C inhibitor (Technoclone GMBH, Vienna,
Austria) (plasma dilution 30%).
Platelet-poor plasma for sample G (see table I) was prepared as a
pooled plasma from blood collected from 15 volunteers in 0.129
mol/l of sodium citrate (1:10), kept on ice and centrifuged within
30 minutes at 2000 g for 20 minutes at 4_C. PAI-1 was inactivated
by incubation of the pooled plasma for 24 hours at 37_C according
to (5).
Stabilyte plasma for samples H,I,J,K was prepared from blood of
volunteers collected in Stabilyte�tubes (6)(Biopool AB Umea,
Sweden) and handled as above. PAI-activity was initially assessed
using method 4, and retrospectively assigned using method 14 and
expressed in U/ml (NIBSC 87/512). Two samples with 7 U/ml and 15
U/ml were used. Samples I and K were spiked with a specified amount
of concentrated two-chain melanoma tissue-type plasminogen
activator (t-PA) (7) (plasma dilution < 1%) with a value
assigned according to t-PA activity assay using method 4.
Samples L and M were NIBSC 87/512 and 92/654 from the National
Institute for Biological Standards and Control, Potters Bar, UK and
were provided as lyophilised samples. NIBSC 87/512 had a certified
value of 25 IU/ml; 92/654 was certified later with 27,5 IU/ml
(8).
Methods:
Assay 1 is an assay based on the measurement of t-PA/PAI-1
complexes present in the sample before addition and after addition
of an excess of t-PA (9). The difference between the two values
reflects the amount of active PAI-1. Assay was performed by P.J.
Declerck and I. Knockaert using lot MA-15H12/MA-62E8-HRP, detection
limit 1 U/ml.
Assay 2 is an assay based on the measurement of t-PA/PAI-1
complexes present in the sample before addition and after addition
of an excess of t-PA (10). The difference between the two values
reflects the amount of active PAI-1. The study was performed by Dr
W. Nieuwenhuizen and R. Laterveer using a prefinal version without
addition of PPACK and not yet adapted for use on acidic plasma as
in the reference (10); detection limit 0.4 ng/ml.
Assay 3 measures active PAI-1 by addition of excess single chain
t-PA and measurement of t-PA-PAI-1 complex in both the original and
treated samples. Active PAI-1 is determined by subtraction and
expressed as ng/ml (11). N. Booth and A.M. Croll performed the
study.
Assay 4 is a titration assay of PAI-1 in the sample with a
series of two-chain melanoma t-PA concentrations according to
Verheijen et al (12) using CNBr fragments to stimulate t-PA
activity and measurement of plasmin/chromogenic substrate activity.
Results are expressed in t-PA international units calibrated
against NIBSC 83/517. C.Kluft and P. Meijer performed the
study.
Assay 5 is Coatest� PAI from Chromogenix AB, Molndal, Sweden and
measures residual single chain t-PA after incubation with plasma,
by means of chromogenic substrate activity of plasmin formed in 50
minutes in the presence of a fibrin-derived stimulator (13). S.
Rosen and L. Wejkum performed the study on lot X0074 employing the
manual version in tubes. Results are expressed in AU/ml; 1 AU /ml
PAI activity being defined as the amount required to inhibit 1
IU/ml t-PA.
Assay 6 is Spectrolyse� /pl Biopool AB, Umea, Sweden; a
two-stage indirect enzymatic assay assessing residual melanoma
single chain t-PA after incubation with plasma (14-16). t-PA is
measured after an acidification step to destroy plasma plasmin
inhibitors and by its ability to generate plasmin activity on a
chromogenic substrate in the presence of the stimulator
poly-lysine. Results are expressed in units defined as the amount
of PAI that inhibits 1 IU of human sct-PA calibrated against NIBSC
83/517. A Takada and T Urano performed the study using v1-1, lot
1102025.
Assay 7 is also Spectrolyse� /pl (see assay 6) and performed by
I. Juhan-Vague and J. Ansaldi using v1-1, lot 1102025.
Assay 8 is a Bioimmunoassay employing anti-PAI-1 IgG coated on
wells, incubation with the sample, incubation after washing with
two-chain t-PA and a subsequent assay of residual t-PA activity by
its ability to convert Glu-plasminogen assessed by the activity of
plasmin formed on H-D-Val-Leu-Lys-pNA. One unit is defined as the
amount inhibiting 1 unit of two-chain t-PA. K. Okada and O. Matsuo
performed the study.
Assay 9 is the simplified version of the SOFIA-PAI assay (17)
measuring residual melanoma sct-PA after incubation with plasma.
Residual t-PA is bound to solid-phase fibrin. t-PA activity is
assessed by plasmin activity measured on a chromogenic substrate
after addition of Glu-plasminogen. PAI activity is expressed in
units defined relative to single-chain t-PA calibrated against
NIBCS 83/517. The assay is not designed for acidified plasma. E.
Angles-Cano and S. Loyau performed the study.
Assay 10 is TC� Actibind-PAI-1 from Technoclone GMBH, Vienna,
Austria and is based on the immobilisation of functionally active
t-PA to plates by means of a monoclonal antibody. PAI-1 in the test
sample binds to t-PA and is then quantified using labelled
monoclonal anti-PAI-1 antibody. Results are expressed in U/ml of a
PAI standard; detection limit 4.0 U/ml. B.R. Binder and R. Beckmann
performed the study with lot 593 of TC 16070.
Assay 11 is TC� R Actibind-PAI-1 CL , which is a prototype kit not
further developed, based on method 10, however, with t-PA
cross-linked to the monoclonal antibody, using a PAI-1 standard
calibrated according to NIBSC 87/512.
Assay 12 is TC� PAI activity kit from Technoclone GMBH, Vienna,
Austria. And is based on the inhibition of rt-PA added to plasma.
Remaining t-PA activity is determined by its Glu-plasminogen
activating properties stimulated by CNBr-fragments of fibrinogen
and a synthetic plasmin substrate. Plasmin inhibitor is quenched by
a special reagent.(18).
Results are expressed in U/ml of a PAI standard. B.R. Binder and V.
Carroll performed the study with lot 593 of TC 15070. The method
was not suitable for Stabilyte plasma and results on samples H-K
should be interpreted accordingly. The high recording in
PAI-depleted samples in this study was questioned in view of in
house results and previous results (1). The mixture of single and
two-chain rt-PA was later replaced by a single chain preparation to
reduce sensitivity for PAI-2 (19).
Assay 13 is a two stage method with incubation of plasma with an
excess of single-chain t-PA followed by assay of residual t-PA
enzymatic activity with a plasminogen/chromogenic plasmin substrate
assay utilizing poly-D-lysine as a t-PA stimulator (16). Values are
expressed in IU/ml relative to t-PA. The study was performed by E.
Eriksson, P.Falk and A. Hansevi.
Assay 14 is an u-PA-PAI-1 complex ELISA (20), that assesses the
u-PA complex formed after incubation with added u-PA. Assay with
lot 2F5013 was performed by M.Philips and B.Lillethorup and results
were reported in pmol/l active PAI-1; the experience is that when
analyzing NIBSC 83/517 29 fmol equals 1 IU.
Assay 15 is similar to assay 15 with reagents obtained from Novo
Nordisk, Baegsvaerd, Denmark and performed by E. Eriksson, P.Falk
and A. Hansevi with lot 774-776.
Assay 16 is Stachrom� PAI , from Diagnostica Stago,
Asni�res-sur-Seine, France. After incubation of the plasma with
u-PA, the excess u-PA is measured by its capability to generate
plasmin in an environment with inhibitors for plasmin inhibitor and
alpha-2-macroglobulin (21).The assay was run on a semi-automated
device ST 888, by F. Nicham and N. Barat with lot 922190.
Assay 17 is Berichrom� R PAI from Behringwerke AG, Marburg,
Germany, an assay which employs u-PA as the target enzyme for PAI
in plasma and measurement of uninhibited u-PA via
plasminogen/plasmin conversion and subsequent cleavage of a
chromogenic plasmin substrate in an environment with oxidative
inactivation of plasmin inhibitor (22,23). The study was performed
by H. Keuper and P. Lenz with a two-point method ; lot 25258 (test
version). Results are expressed in u-PA IU/ml which according to
the experience is 1/5 to 1/6 of the t-PA inhibiting units.
Results
Criteria for specificity of methods for active PAI-1 in plasma
In plasma we can expect different molecular forms of PAI-1, such
as inactivated PAI-1 (originating from platelets, or generated
spontaneously by time-dependent thermal inactivation of active
PAI-1), active PAI-1 and PAI-1 in complex with proteases, notably
t-PA (plasminogen activator, tissue-type) or u-PA (urokinase-type
PA). Besides this complexity we can expect that other inhibitors in
plasma compete for the target protease (t-PA or u-PA) used in a
test principle. Another complexity was the co-occurrence of active
t-PA besides PAI-1 in plasma. This t-PA could compete with the
assay of activity or continue to react with PAI-1 after blood
sampling. To avoid the latter, a method was introduced around
1988-1990 to acidify blood as soon as possible after its collection
or to use acid anticoagulants to achieve a pH of around 6 (6,24).
This "new" anticoagulation procedure complicates the situation as
well, since not all assay methods were designed for or adapted to
the use of acidified plasma.
In figure 1 the situation in plasma is summarised
and in particular it should be noted that we aim to measure active
PAI-1 and not the residual activity of PAI-1.
There were four test principles involved in the methods
evaluated (see table II) and criteria focus on these principles.
The criteria were established during the Meeting of the ISTH/SSC
subcommittee on Fibrinolysis in 1992.
Criterium 1a and b:
General : No interference by other plasma (inhibitory)
components
Specific 1a: Immunodepleted and thermally inactivated plasma should
have an activity < 5% of that of NIBSC standard 87/512 (grading
A = both < 5%; grading B = only one < 5%; grading C = both
> 5%)
Specific 1b: Added PAI 2 or PAI 3 added to immunodepleted plasma
should have an activity < 5% of that of NIBSC standard 87/512
(grading A = both < 5%; grading C = one or two < 5%)
Criterium 2:
General: No interference by endogenous t PA
Specific: Added t PA to two (acid) plasmas with different PAI-1
activity level should not reduce recorded PAI-1 activity with an
amount > 20% of the initial activity (Grading A = mean reduction
< 20%; grading C = mean reduction > 20%).
Criterium 3: Precision / performance
Specific: Dose-response of added PAI-1 to plasma (0; 4.6; 17; 55.8
U/ml) should be of high quality (grading A = r > 0.999; grading
B = r > 0.9; grading C = r < 0.9)
The criteria (1a, 1b, 2, 3) were to be independently applied and
any C should result in exclusion and A and B both in inclusion. The
criterium on performance was limited to criteria for the
dose-response curve and not intended at this stage to evaluate
whether the coefficient of variation was half of that of the total,
including biological variation, which should also be evaluated
(25).
Samples to evaluate
adherence to the criteria
The following samples were prepared and included in the set that
was distributed:
Table I
r-PAI-1 = recombinant PAI-1; r-PAI-2 = recombinant PAI-2; tc-t-PA = two chain melanoma t-PA.
Samples were coded and frozen and distributed on dry ice except
for the NIBSC standards which were freeze-dried. The original set
was larger including additions of r-PAI-1 to buffer, but these were
later excluded.
Procedure of the
evaluation and follow-up.
An invitation to participate voluntarily (by direct mail) to
volunteers responding at the subcommittee meeting, to scientists of
original publications of method development, method evaluation
and/or modification (in total 33) and company scientists
responsible for commercial methods (in total 14) resulted in 18
responses concerning 16 different methods. The general requirement
and criteria were stated in a letter in advance.
Thus evaluation was confined to expert laboratories or laboratories
that had designed a test and were informed about the aims and
procedures.
It had been decided to perform the analysis blind in two ways
(a) the test samples were coded for participants (b) the methods
were coded for discussion in the SSC subcommittee meetings.
The results of the evaluation were presented coded at the SSC
subcommittee on fibrinolysis meeting in 1993, New York and on the
basis of the criteria and predetermined judgement, two methods only
fulfilled all criteria (see below).
Subsequently, individual results were distributed to
participants for comment, and to acknowledge that the single
evaluation of each assay method (except in two cases) might have
been subject to incidental error (misfortune) or that the
engineered samples might have created unexpected problems. For
instance the use of acidified plasma (pH 6) was quite recent.
Additional data were welcomed. The reactions have been taken into
account. Notably, the evaluation with added recombinant PAI-2 was
at variance with the experience of some investigators: this issue
was not resolved.
After the formal presentation of coded results and the
evaluation of comments by the organisers, the results were not
decoded on any occasion and were only subject to consideration by
the participants for their own method / information. The selection
of a potential reference method and potential recommendation within
the framework of the SSC was not considered appropriate. The
present manuscript is the first decoded account of the results.
Results of the
exercise
a) General aspects
From_the 18 applicants, 17 continued co-operation and received 16
coded samples. From these sixteen samples, thirteen (see table I)
were used for evaluation. On retrospect, the samples with PAI-1
added to buffer were excluded from evaluation due to poor and
erratic recovery of added PAI-1 evident from all data.
As two independent participants participated per two methods,
the whole procedure could be evaluated. For method 6 and 7 and for
14 and 15 (see table II) the data showed excellent agreement as
expressed by the mutual correlation of all data r>0.97.
b) Method principles
The methods evaluated in the present study had different
principles for detecting the active PAI-1.
The principles involved either addition of t-PA to the samples (13
cases) or addition of u-PA (4 cases): The t-PA preparation could be
a one- or two-chain molecule.
The detection principle involved the measurement of the complex of
PAI-1 formed with the added enzyme (7 cases). In view of the
normal, endogenous presence of t-PA*PAI-1 complex the increase in
the amount of complex was evaluated. In the case of u-PA addition
it was assumed that normally no u-PA*PAI-1 complex was circulating,
restricting the use to normal situations where this applies.
The detection principle involved in 10 cases the detection of the
excess of added enzyme, either t-PA (8 times) or u-PA (2 times)
In Table II, the methods are arranged and numbered according to
the principles of assay as mentioned above. As can be observed from
the table as well, the units in which the results are provided
differ, despite the availability of standard NIBSC 87/512 (assigned
value 25 U/ml). For comparison, the results of the participants for
the standard in their own units is given in the table. It
illustrates the variability existing at that time in reporting data
on PAI-1 activity.
Table II
Assays are identified by number and described in materials and
methods. AU = arbitrary units.
c) Normalization for evaluation.
For evaluation all results were normalised relative to the
included sample of NIBSC 87/512 and presented in percentages of
this standard throughout the manuscript, unless specified
otherwise. For validation of this procedure, for all criteria,
normalisation was also exercised with the other standard NIBSC
92/654 and with one of the frozen samples ( Stabilyte sample H, see
table I) and similar results and conclusions were obtained in all
cases. For nine methods with an excellent analytical performance
(dose-response curve with r> 0.999) the two NIBSC standards were
compared showing close agreement on an activity of NIBSC 92/654 of
86 % of that of NIBSC 87/512 (interquartile range: 81-96, standard
deviation 18%).
d) Criterium 1: absence of interference by other plasma
(inhibitory) components
The evaluation on criterium I could not use the samples with
buffer matrix due to poor recovery/stability of such samples as
concluded retrospectively, preventing direct evaluation of matrix
interferences
Interference by plasma components and other inhibitors was
evaluated by
1) the results in immunodepleted plasma and a pooled citrated
plasma incubated overnight at 37_C to inactivate PAI-1
(activity-depleted)
2) the results of assay in immunodepleted plasma supplemented with
PAI-2 or PAI-3
Table III
Results are summarised in table III in three categories I -
III.
Category I concerns methods with a combination of a low recording
in depleted plasmas (criteria A = both < 5%) and low
interference by PAI-2 (criterium A = below 5%).
Category II does not fulfil criteria A and/or B but is less clearly
aberrant compared to category III
Category III is clearly not adequate due to criterium C applying to
depleted plasma or the addition of PAI-2.
There was no sensitivity to PAI-3; only occasional values did not
fulfil criterium A.
e) Criterium 2: Absence of interference by endogenous free
t-PA
To test absence of interference by endogenous active t-PA, the
procedure was chosen to add to Stabilyte plasma (where added t-PA
is preserved and does not react with endogenous PAI-1 in the
sample) an additional amount of t-PA. This extra amount should not
interfere with the assay of the PAI-1 activity in the sample. The
addition to plasma H of 4 IU/ml t-PA and to plasma J of 7 IU/ml
t-PA would be comparable to about 50% potential neutralisation of
the endogenous PAI-1 amount.
As shown in Table IV, most methods show significant reduction in
the recording of PAI-1 activity following the addition of t-PA.
According to the criteria of change <20% only 4 methods
fulfilled the criterium, and out of these only two methods from
category I and II from table III.
Table IV
f) Criterium 3: Dose-response curve quality
As shown in Table IV, 7 assays showed an excellent dose-response
(r>0.999) while 4 performed poorly (r< 0.9). The amount of
sample provided did not allow for dilution experiments, causing in
some cases a poor performance for the higher value of PAI-1 in
sample D.
g) Overall scoring
When using either exclusion or inclusion as an approach only
methods 10 and 14 were not excluded. Grading C on criterium C did
not contribute independently to any exclusion.
Discussion
The study presented focused on the specificity of methods for
active PAI-1 in acidified plasma.
For the evaluated methods for PAI-1 activity we showed that the
majority was not specific according to the tests and criteria
applied. Only 2 out of 15 methods were potentially specific.
The three major problems causing these results were (a) the
sensitivity of about half of the methods to interference by another
inhibitory factor in plasma as evident from the results in PAI-1
depleted plasma, (b) the fact that some methods were not adapted to
the use of acid or Stabilyte plasma , and (c) the sensitivity of
many methods to endogenous t-PA as revealed by the addition of
extra t-PA. In addition, most methods aimed to assess elevated
PAI-1 and performed suboptimally at very low levels (cf 1).
The presence of an interfering plasma factor had been described
previously (5,26); the identity of this interfering factor is still
unknown. The sensitivity to this interference coincided with
sensitivity to the addition of PAI-2. The effect of PAI-2 was
debated in subsequent correspondence with some participants and at
variance with their experience. This should be noted, but was not
further evaluated.
The sensitivity to added t-PA was previously not considered a
major aspect to circumvent since various methods aimed at studying
elevated PAI-1 where endogenous t-PA is a minor component. Also the
level of endogenous active t-PA was underestimated before the
introduction of acidification of blood to inhibit in vitro
interaction of t-PA and PAI-1. At present it is state-of-the art to
use acidified plasma to avoid in vitro interactions and to attempt
to measure active PAI-1 (see figure 1). This allows the use of methodology for
all purposes independent of the t-PA status. It can be observed
that methods using u-PA as target enzyme for PAI-1 were relatively
insensitive to endogenous t-PA.
The two methods that fulfilled the criteria provided specificity
for PAI-1 by the use of specific antibodies either to immobilise
t-PA and detect only newly formed PAI-1-t-PA or to detect
specifically the formation of the u-PA*PAI-1 complex. In method
with u-PA the excess of u-PA used was apparently sufficient to
avoid interference by endogenous t-PA. In later development also
method 2 solved the problem of endogenous t-PA by the addition of
PPACK (10). Later assay formats with the principle of methods 8 and
10 maintained a pH 6 during binding of the PAI-1, using acidified
plasma.
The results of the exercise demonstrate the value of the
criteria and test samples for the methods evaluated. It is possible
that new criteria and test samples are necessary when other assay
principles or standards are introduced. It should be noted that the
exercise was not a complete evaluation; for instance matrix effects
and analytical variability relative to the total variability were
not assessed.
The exercise was limited to the evaluation of a limited number of
samples for each aspect of specificity and to one laboratory, for
most methods and results should be confirmed in subsequent more
elaborate and detailed experiments for definite conclusions.
The process in the subcommittee of fibrinolysis of the ISTH/SSC
resulted in an increased awareness of the importance of specificity
of methods and the experimental documentation, especially for
standardisation and when a reference method is required. In
addition it seemed adequately possible in all methods to use the
NIBSC standards, though, this issue has not been formally
investigated. It should be recognised that the evaluation did not
focus on possible matrix interference. An inventory on this aspect
in the present exercise was not possible because of the problems
with the analyte added to buffer: the samples were excluded from
evaluation. In future evaluations of these aspects require
additional attention.
The ISTH/SSC can be considered a suitable expert forum for the
definition of criteria and testing principles in haemostasis. The
testing as executed for the method for PAI-1 activity was seen as a
single exercise and in future the responsibility of parties who
develop the methods and standards.
Acknowledgements
Dr. P.J. Declerck (Immunodepleted plasma, rPAI-1), Dr. B.R.
Binder (PAI-3), Dr D.J. Ballance (rPAI-2) and Dr P.J. Gaffney
(NIBSC standards) are gratefully acknowledged for supply of
materials and samples. Ing. P. Meijer is gratefully acknowledged
for sample preparation and distribution
The following groups are acknowledged for their voluntary
participation in the analysis:
Dr S. Thorsen, M. Philips and B. Lillethorup, Rigshospitalet,
Section for Hemostasis & Thrombosis, Dept of Clinical
Biochemistry, Copenhagen, Denmark
Dr. P.J. Declerck and I. Knockaert, Laboratory of pharmaceutical
biology and phytopharmacology, Institute for Pharmaceutical
Sciences, Leuven, Belgium
Dr. W. Nieuwenhuizen and R. Laterveer, Dept of Lipids and
Endothelium, Gaubius Laboratory, IVVO-TNO, Leiden, The
Netherlands
Dr. N. Booth and A.M. Croll. Dept of Molecular and Cell biology,
University of Aberdeen, Marischal College, Aberdeen, Scotland
Dr C.Kluft and P. Meijer. Gaubius Laboratory, IVVO-TNO, Leiden, the
Netherlands
Dr S. Rosen, E. Ersdal-Badju and L Wejkum. Chromogenix AB, Molndal,
Sweden
Dr A. Takada and T. Urano.Dept of physiology, Hamamatsu University
School of Medicine, Hamamatsu, Shizuoka, Japan.
Dr I. Juhan-Vague and J. Ansaldi. Laboratoire d'hematologie,
H�pital d'adultes Timone et Universitaire de Marseille, Marseille,
France.
Dr K. Okada and O. Matsuo. Dept of Physiology, Kinki University
School of Medicine, Osakasayama City, Japan.
Dr. E. Angles-Cano and S. Loyau. INSERM U.143, Institut de
pathologie cellulaire, H�pital de Bicetre, Paris, France.
Dr. B.R. Binder, R. Beckmann and V. Carroll. Dept of Medical
Physiology, Lab. for Clinical Experimental physiology, University
of Vienna, Vienna, Austria.
Dr E. Eriksson, P. Falk and A. Hansevi. Fibrinolyslab, Dept of
Surgery , East Hospital, Gothenburg, Sweden.
Dr G. Contant, F. Nicham and N. Barat. Serbio Laboratory,
Gennevilliers, France.
Dr H. Keuper and P. Lenz. Dept of Coagulation and Fibrinolysis
diagnostics, Behringwerke AG, Marburg, W-Germany.
References
1) Gram J, Declerck PJ, Sidelmann J, Jespersen J, Kluft C.
Multicentre evaluation of commercial kit methods: plasminogen
activator inhibitor activity. Thromb Haemostas 1993;70:852?7.
2) Declerck PJ, Moreau H, Jespersen J, Gram J, Kluft C.
Multicenter evaluation of commercially available methods for the
immunological determination of plasminogen activator inhibitor?1
(PAI?1). Thromb Haemostas 1993;70:858?63.
3) Declerck PJ, De Mol M, Alessi M-C, Baudner S, Paques GP,
Preissner KT, Muller-Berghaus G. Collen D. Purification and
characterization of a plasminogen activator inhibitor 1 binding
protein from human plasma. J Biol Chem 1988;263:15454-61.
4) Alessi M-C, Declerck PJ, De Mol M, Nelles L, Collen D.
Purification and characterization of natural and recombinant
plasminogen activator inhibitor 1(PAI-1). Eur J Biochem
1988;175:531-40.
5) Kluft C, Jie AF, Sprengers ED, Verheijen JH. Identification
of a reversible inhibitor of plasminogen activators in blood
plasma. FEBS Lett. 1985;190:315?8.
6) Ranby M, Sundell IB, Nilsson TK. Blood collection in strong
acidic citrate anticoagulant used in a study of dietary influence
on basal tPA activity. Thromb Haemost 1989;62:917?22.
7) Kluft C, van Wezel AL, van der Velden CAM, Emeis JJ,
Verheijen JH, Wijngaards G. Large scale production of extrinsic
(tissue-type) plasminogen activator from human melanoma cells. In:
Mizrahe A, van Wezel AL, eds. Advances in biotechnological
processes, 2nd ed. New York: AR Liss, 1983:97-110.
8) Gaffney PJ, Edgell TA. The international standard for
plasminogen activator inhibitor?1 (PAI?1) activity. Thromb Haemost
1996;76:80?3.
9) Declerck PJ, Verstreken M, Collen D. An immunofunctional
assay for active plasminogen activator inhibitor-1 (PAI-1).
Fibrinolysis 1988;2 suppl. 2:77-8.
10) Nieuwenhuizen W, Laterveer R, Hoegee B. An enzyme
immunoassay for the simultaneous determination of active type-1
plasminogen activator inhibitor (PAI-1), and t-PA/PAI-1 complexes.
Blood Coagul Fibrinolysis 1995;6:520-6.
11) Booth NA, Croll A, Bennett B. The activity of plasminogen
activator inhibitor-1 (PAI-1) of human platelet. Fibrinolysis
1990;4 supp 2:138-40.
12) Verheijen JH, Chang GT, Kluft C. Evidence for the occurrence
of a fast?acting inhibitor for tissue?type plasminogen activator in
human plasma. Thromb Haemost. 1984;51:392?5.
13) Wejkum L, Chmielewska J. A new adaptation of Coatest PAI for
measurement of low inhibitor concentrations in plasma. Fibrinolysis
1990;4:130.
14) Chmielewska J, Ranby M, Wiman B. Evidence for a rapid
inhibitor to tissue plasminogen activator in plasma. Thromb Res
1983;31:427-36.
15) Chmielewska J, Wiman B. Determination of tissue plasminogen
activator and its fast inhibitor in plasma. Clin Chem
1986;32:482-5.
16) Eriksson E, Ranby M, Gyzander E, Risberg B. Determination of
plasminogen activator inhibitor in plasma using t-PA and a
chromogenic single-point poly-D-lysine stimulated assay. Thromb Res
1998;50:910101.
17) Angles-Cano E, Masson-Lunven C, Gaussem P. Development of an
internal standard for plasminogen activator inhibitor-1 PAI-1 and
its use in a simplified assay for measuring PAI-1 activity in human
plasma. Fibrinolysis 1990;4 supp 2: 127-9.
18) Korninger C, Wagner O, Binder BR. Tissue plasminogen
activator inhibitor in human plasma: development of a functional
assay system and demonstration of a correlating Mr=50.000
antiactivator. J Lab Clin Med 1985;105:718-24.
19) Thorsen S, Philips M, Selmer J, Lecander I, Astedt B. Kinetics
of inhibition of tissue-type and urokinase-type plasminogen
activator by plasminogen-activator inhibitor type 1 and type 2. Eur
J Biochem 1988;175:33-9.
20) Philips M, Juul A-G, Selmer J, Lind B, Thorsen S. A specific
immunological assay for functional plasminogen activator inhibitor
1 in plasma. Standardized measurements of the inhibitor and related
parameters in patients with venous thromboembolic disease. Thromb
Haemost 1992;68:486-94.
21) Contant G, Nicham F, Martinoli JL. Determination of
plasminogen activator inhibitor (PAI) by a new venom based assay.
Fibrinolysis 1992; 6 supp 3:85-6.
22) Stief TW, Lenz P, Becker U, Heimburger N. Determination of
plasminogen activator inhibitor (PAI) capacity of human plasma in
presence of oxidants: a novel principle. Thromb Res
1988;50:559-73.
23) Stief TW, Lenz P, Becker U, Heimburger N. Functional
determination of plasminogen activator inhibitor (PAI) based on
oxidative inactivation of alpha-2-antiplasmin: no influence of
sample heparin and fibrinogen split products (FSP). Fibrinolysis
1988;2 supp 2:73-4.
24) Kluft C, Verheijen JH. Fibrinolysis Working Party: Blood
collection and handling procedures for assessment of tissue-type
plasminogen activator (t-PA) and plasminogen activator inhibitor
1(PAI-1). Fibrinolysis 1990;4 supp 2:155-61.
25) Fraser CG, Harris EK. Generation and application of data on
biological variation in clinical chemistry. Crit Rev Clin Lab Sci
1989;27:409-37.
26) Sprengers ED, Kluft C. Plasminogen activator inhibitors.
Blood 1987;69:381?7.
|