, Cornelis Kluft [1,2], J�rgen Jespersen 
- Department of Thrombosis Research, University of Southern
Denmark, and Department of Clinical Biochemistry, Ribe County
Hospital in Esbjerg,
- Gaubius Laboratory TNO-PG, Leiden, The Netherlands
Dr. J�rgen Gram, Department of Clinical Biochemistry, Ribe
Hospital in Esbjerg, �stergade 80, DK-6700 Esbjerg, Denmark. Fax
no.: + 45 79 18 24 11,
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There is compelling evidence that measurement of analytes of the
fibrinolytic system may be helpful for the clinician in a number of
situations. Except for measurement of plasma fibrin d-dimer such
quantities have not been widespread introduced in the clinical
laboratory. One main reason for this is a documented large method
and interlaboratory variation for specific analytes.
This manuscript describes historical aspects and the current
status of standardization with respect to measurement of
fibrinolytic quantities. Also, it describes recent initiatives
taken within the framework of International Society of Thrombosis
and Haemostasis (ISTH) and the International Federation of Clinical
Chemistry (IFCC) to promote standardization within the field of
fibrinolysis (and coagulation).
There has been a great increase in the number of publications
dealing with the role of the fibrinolytic system, since
thrombolytic treatment of myocardial infarction was introduced in
the mid 1980's. Many of such publications have indicated an
important role of fibrinolysis in health and disease. Consequently,
it has been suggested that measurement of analytes of the
fibrinolytic system may be helpful for the clinician in a number of
situations (Table 1).
Table 1. Clinical use
of analytes of the fibrinolytic system
|Identification of factor deficiencies
|Monitoring critically ill patients
Despite the potential importance of the use of fibrinolytic
analytes in a number of patient situations, measurements have only
to a limited extent been introduced in the clinical laboratory. One
requirement, which must be met, if such analytes should be more
widely introduced, is that the results produced in one laboratory
are very similar to the results produced in another laboratory. At
present, this is hardly the case, and proficiency testing reports
have documented a large interlaboratory method-dependent variation
in some key variables of the fibrinolytic system (1,2). This
significant problem is well-known from other fields within clinical
biochemistry. It is the experience that one way to improve the
situation is to introduce a reference measurement system (3).
However, such a work is complex and comprehensive and it demands
cooperation between national and international scientific societies
of different disciplines, industry, and official institutions
Here, we briefly summarize the first steps taken in order to try
to introduce a standardization system within the field of
Four decades ago Astrup proposed that the major role of the
fibrinolytic system is to regulate the amount of fibrin deposition
in tissue and blood vessels following tissue injury (5,6). Since
then comprehensive biochemical studies have elucidated the system
in detail. The conversion of M=92,000 g/mol plasminogen to M=70,000
g/mol plasmin is one central step in fibrinolysis. This so-called
activation of plasminogen is related to three distinct activator
pathways (Fig. 1).
It is believed that the most important activator in blood is
plasminogen activator, tissue type, which is a M=60,000 g/mol
protein, probably produced and released to blood from the
endothelial cells. Plasminogen activator, urokinase type is another
M=55,000 g/mol activator, which can probably be produced and
released to blood from endothelial cells in a one-chain proenzyme
(single chain urokinase plasminogen activator) form and converted
to a two-chain form during fibrinolysis. The third pathway is
related to contact activation and is yet less
Two serpin class inhibitors contribute significantly to the
regulation of activation of blood fibrinolysis. One is plasminogen
activator inhibitor 1, which is a 52,000 g/mol plasma protein
produced by hepatocytes and endothelial cells. This inhibitor has
affinity to both plasminogen activator, tissue type and urokinase
type. The primary inhibitor of plasmin is plasmin inhibitor, - a
M=70,000 g/mol protein produced by hepatocytes.
Fibrin affinity of fibrinolytic components and the presence of
inhibitors secure that the action of plasmin is confined to fibrin.
The fibrin affinity is partially regulated by thrombin activable
fibrinolysis inhibitor (TAFI), which is a carboxypeptidase that
removes COOH-terminal lysine and arginine residues from fibrin (7).
The plasmin-mediated degradation of fibrin results in a number of
When coagulation factor XIII cross-links the formed fibrin, the
degradation products contain fibrin d-dimer, which is a
heterogenous class of degradation products with widely variable
molecular weights (average M=182,600 g/mol).
The haemostatic balance describes the dynamic balance between
activation of coagulation and fibrinolysis, which in turn
determines the amount of fibrin present following tissue injury.
This concept can favourably be used in a clinical setting as
depicted in table 2. Table 2. Clinical expression of deviations in
activity of coagulation and fibrinolysis
Table 2: Clinical
expression Activity of coagulation Activity of fibrinolysis
Activity of coagulation
Activity of fibrinolysis
Many reports have demonstrated that a defective fibrinolysis
characterized by low levels of plasminogen activator, tissue type
(enz; procedure), high levels of plasminogen activator, tissue type
(imm; procedure), and high levels of plasminogen activator
inhibitor 1 (imm; procedure, enz; procedure) is associated with an
enhanced risk for evolution of cardiovascular disease. Similarly,
it has been reported that determination of plasma fibrin d-dimer is
helpful in the diagnosis of exclusion of venous thromboembolic
disease. Deficiency of serpin inhibitors (plasminogen activator
inhibitor 1, plasmin inhibitor) are associated with an enhanced
risk of bleeding (8,9).
Despite the fact that individual analytes of the fibrinolytic
system have a prognostic power that equals cholesterol with respect
to evolution of future cardiovascular disease, these analytes have
not widely been used in daily clinical risk stratification.
Current status of
Most efforts within standardization in fibrinolysis have been
associated with determination of the potency of pharmaceutical
materials such as recombinant proteins/enzymes used in fibrinolytic
treatment. Much less has been done with respect to international
harmonization of measurements of fibrinolytic analytes in
biological fluids. However, it has been appreciated by different
scientific societies that there is a need for increasing the level
of standardization, and new activities are now emerging.
According to ISO a reference material is a material or substance
comprising preparations, whose properties are sufficiently
homogenous and well-established to be used for the calibration of
an apparatus, the assessment of a measurement method, or for
assigning values to materials (10). A number of such reference
materials exists for measurement of quantities of the fibrinolytic
system (11). The values of most materials are assigned in arbitrary
units, i.e. international units (Table 3).
Table 3. Available
reference materials within the field of fibrinolysis
||66/46 urokinase type
||87/594 urokinase type (high molecular weight)
||86/670 tissue type
|Plasminogen activator inhibitor 1
IRP - International Reference Preparation: IS - International
Standard :BS - British Standard :NIBSC - National Institute of
Biological Standards and Control
These standards have primarily been created by a painstaking
work done by dr. P. Gaffney, The National Institute for Biological
Standards and Control, United Kingdom. However, it is apparent from
the table that the values of the standards are in arbitrary units
(IU). This may be a problem, because the results of a given assay
can exclusively be traced back to IU and not to SI units. It is
obvious that in complex measurement systems such as e.g. plasma
coagulum lysis; time (formerly euglobulin clot lysis time) it would
be appropriate to express results in arbitrary units, but with
respect to well-defined analytes it would be possible to have
reference materials with traceability to SI units. Another
potential problem with the reference materials listed in table 3 is
that the values are usually assigned by collaborative studies,
frequently with the use of methods available at time - a procedure
which makes it difficult to transfer values from an old to a new
In 1991-1992 meetings between representatives from the
Scientific and Standardization Committee (SSC) of the International
Society of Thrombosis and Haemostasis (ISTH), the
Committee/Commission on Quantities and Units of the International
Federation of Clinical Chemistry (IFCC), and the International
Union of Pure and Applied Chemistry (IUPAC) resulted in
recommendations regarding the use of nomenclature and units within
the whole field of haemostasis (12,13). In the foreword of one of
the documents it is stated: "The technology used by one laboratory
speciality may vary even within the speciality, and may be
incomprehensible to another speciality. This is a minor
inconvenience to the laboratory specialities, each one essentially
operating within its own area of activity. However, for the user
this is highly unsatisfactory and also it may hinder the treatment
of the patient" (13). Thus, the organisations involved in these
standardization activities clearly stress the importance of
introducing harmonization in nomenclature and units. Despite this,
the recommended nomenclature has so far not been used
systematically, neither in daily practice nor in scientific
publications. As a first step towards a wide-spread use of a
harmonized nomenclature, it may be advised that scientific journals
more strictly observe the recommendations.
In order to secure the highest possible level of accurate
results in the clinical laboratory there is a requirement to adhere
to the hierarchical structure of a comprehensive coherent
measurement system (3,4,14) in which there is a coupling of the
analytical method (definitive method, reference method) with
reference material (primary reference material, secondary reference
material, standards). For quantities for the coagulation system
there exists one method adhering to a WHO calibration scheme
(plasma coagulation, tissue factor induced (15)), and a number of
written national reference measurement procedures (reviewed 16). In
contrast, there exist no reference measurement procedures as
written standards within the system of fibrinolysis. However, the
need for standardization, or at least harmonization, has been
appreciated. It should be noted that the European Concerted Action
on Thrombosis and Disabilities Programme, initiated under the
Commission of the European Union, disclosed a great need on
harmonization also of quantities of the fibrinolytic system. This
has resulted in the publication of a comprehensive manual on
Laboratory Techniques in Thrombosis, in which leading scientists
describe in detail "state of the art" methods (17).
Despite these efforts it is obvious that the coherent reference
measurement system within fibrinolysis is incomplete (18). There is
a need for internationally accepted referent measurement
procedures. A sufficient level of standardization cannot be reached
solely with the use of reference materials/standards with assigned
Within the Scientific and Standardization Committee
(Subcommittee of Fibrinolysis) of ISTH surveys have been carried
out on comparability of methods for analytes of the fibrinolytic
system. These surveys have shown great variation in results - up to
a factor five difference despite the use of common calibrators and
harmonization procedures. This is the main background that the
Subcommittee of Fibrinolysis at the annual meeting in New York,
1993, decided to be more deeply involved in method standardization
and to study the possibilities to adhere to the principles of a
coherent reference measurement system. As a consequence a Project
Group of Materials and Methods (PMG) was formed within ISTH
focussing on the specificity of methods in relation to available
calibrators, and criteria and test procedures for specificity of
methods were prepared for the following analytes: Plasminogen
activator inhibitor 1 (imm. procedure; enz. procedure); plasminogen
activator, tissue type (imm. procedure); plasmin inhibitor (enz.
procedure; plasminogen (enz. procedure).
It was soon recognized that a proper standardization requires a
coherent reference measurement system, well-known from clinical
biochemistry. Therefore, contact was taken to IFCC to join forces
(19,20). As a result a joint IFCC/ISTH Committee on the
Standardization of Coagulation Tests (C-SCT) was formed at the
annual ISTH Scientific and Standardization Committee meeting in
Florence, 1997. The C-SCT has replaced PGM, and it is the intention
that this new Committee promotes standardization of analytes of the
fibrinolytic system as well as the coagulation system. Thus, with
the establishment of C-SCT new powerful perspectives have evolved
with respect to standardization of laboratory procedures of
fibrinolytic (and coagulation) quantities.
In this issue of JIFCC a summary of the work of PGM is presented
with PGM method guidelines and method reports on the following
analytes: Plasminogen activator; tissue type (imm; procedure);
plasmin inhibitor (enz; procedure); plasminogen (enz; procedure).
In addition, problems and progress regarding fibrin d-dimer and
plasminogen activation inhibitor 1 (imm; procedure) standardization
are presented in separate reports.
1. Gram J, Declerck PJ, Sidelmann J, Jespersen J, Kluft C.
Multicentre evaluation of commercial kit methods: Plasminogen
activator inhibitor activity. Thromb Haemost 1993; 70: 852-7.
2. Declerck PJ, Moreau H, Jespersen J, Gram J, Kluft C.
Multicentre evaluation of commercially available methods for the
immunological determination of plasminogen activator inhibitor-1
(PAI-1). Thromb Haemost 1993; 70: 858-63.
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clinical chemistry. Clin Chem 1979; 25: 833-9.
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reference measurement system in laboratory medicine - a major
international meeting organized by the International Federation of
Clinical Chemistry (IFCC), Geneva, October 5-7, 1994. Eur J Clin
Chem Clin Biochem 1996; 33: 977-9.
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6. Astrup T. The biological significance of fibrinolysis. Lancet
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the mechanism of inhibitor of fibrinolysis by activated
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