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Gram, Cornelis Kluft [1,2], Trevor Barrowcliffe , Paul
Declerck , Charles W. Francis , Patrick Gaffney , J�rgen
- Department of Clinical Biochemistry, Ribe County Hospital and
Institute for Thrombosis Research, University of Southern Denmark,
- Gaubius Laboratory TNO-PG, Leiden, the Netherlands
- National Institute for Biological Standards and Control,
Hertfordshire, United Kingdom;
- Laboratory of Pharmaceutical Biology, Katholieke University,
- Hematology Unit, University of Rochester Medical Centre,
- St. Thomas Hospital, Department of Surgery, London, United
Format of the document
Principle of the assay procedure
Criteria for specificity
Test methods for criteria
Standardisation and quality assurance
Preparation of the patient
Preparation of specimen
Description of materials
In 1990 the Subcommittee of Fibrinolysis within the framework of
the Scientific and Standardization Committee of the ISTH decided to
point out a working group, which should study whether different
commercial methods for measurement of plasminogen activator
inhibitor activity and plasminogen activator inhibitor type 1
antigen produced results transferable from one laboratory to
another (1,2). The study documented that there was not consensus of
the results produced by the different methods in different
laboratories, and furthermore that some of the commercial methods
might be characterized by a high imprecision and a poor accuracy
(1,2). Subsequently, the Subcommittee of Fibrinolysis (Amsterdam,
1991) decided a further strategy to improve this situation, i.e.
the introduction of reference measurement procedures as the most
reliable approach to obtain accurate results in clinical
laboratories for measurement of quantities in the fibrinolytic
system. Such an approach makes use of the experience from clinical
To achieve this goal was more complex than originally
anticipated and the involvement of various organizations and
players in the field had to be defined and organized. However, in
September 1995 a comprehensive procedure proposed by the above-
mentioned working group was accepted by the executive director of
the SSC and started as a pilot project within the subcommittee of
The pilot project was run by a project group on methods (PGM)
including Drs. T. Barrowcliffe, P. Declerck, C.W. Francis, P.
Gaffney, J. Gram, J. Jespersen, C. Kluft (Chairperson).
Briefly, the procedure defines the role of the SSC and its
subcommittees as experts on methods and materials in haemostasis;
the NIBSC as the body produce and characterize standard materials,
and the IFCC as the body to select and execute reference method
procedures. The aim is to develop methods and materials in
combination and coordinated in effort and time.
It was recognized that in order to provide a forum for selection
of a reference method, the SSC should contribute with expertise by
providing a platform of defining criteria for specificity and
testing these criteria for methods of quantities in the haemostatic
Scientists and companies can document their methods according to
the expert reports on criteria and test methods in order to obtain
methods for determination of quantities in the fibrinolytic system
with ensured day-to-day, intra- and interlaboratory consistency in
clinical laboratories as well as research laboratories. The aim is
to improve transferability of accurate data from one laboratory to
another, from publications to daily laboratory work, and to obtain
more clearly defined reference ranges and, if possible, decision
limits for results of quantities.
A method fulfilling the said criteria should be characterized by
high quality measurement, which depends on detailed information on
principle of reaction, method of measurement, preanalytical and
analytical steps, data reduction, reliability characteristics,
reference limits, and quality assurance.
The present guidance may not be applicable for description of
all methods of fibrinolytic quantities. The structure of the
present guidance is therefore optional, but the clauses included
should be considered.
3. Written information
3.1. Format of the document
3.1.1. Title page
The title should be short and concise. Included in the title
should be the name of the quantity being the objective of the
measurement procedure. The name of the quantity should
preferentially follow the latest edition from the International
Union of Pure and Applied Chemistry (IUPAC) or otherwise a name
officially accepted by ISTH (4).
As a footnote to Title and Authors should be mentioned: This
work was carried out by the authors as a working group within the
frame-work of the Subcommittee on Fibrinolysis of the SSC of the
ISTH. The report was approved by the Project group on methods and
materials (Drs. T. Barrowcliffe, P. Declerck, C.W. Francis, P.
Gaffney, J. Gram, J. Jespersen, C. Kluft (Chairperson) and a
plenary session of the subcom-mittee.
The document should include a short introduction which describes
the use of the quantity in health care, general problems with
measurement of the quantity in clinical laboratories, and the place
of the recommended measurement procedure in a binary analyte
reference system based on true values (when such exist) (5).
Molecular forms of the component which can occur in several body
fluids, can occur in pathological conditions and during medical
interventions, including quantitative information should be briefly
Explanation on which situation the selected analytical method
can be applied and which molecular forms are addressed. (Note: it
is important to balance between completeness and practicability and
it is possible to state that a limitation applies for example that
total protein assay in blood plasma is aimed at and special
molecular forms in extreme conditions such as polytrauma, lethal
sepsis, and in rarely studied body fluids like liquor are not
Note: In principle, particular attention should be paid to
blood, but study of other biological fluids can be included when
information is adequate to decide so.
3.1.4. Principle of the assay
The principle of the measurement procedure should be
It should be summarized which mechanisms are involved in the
biological function of the component, and its appearance in blood,
and on which information the method focuses. In case of an
immunological method the fraction addressed or the total protein
addressed should be explicitly defined.
In case of an activity assay the question should be addressed
whether the method approaches closely the biological function
and/or what has been excluded due to the principle of the assay
method. The PGM favours for activity method procedures as complete
as possible representing biological function, such as the clot
lysis assay for plasminogen activators. This should minimize the
risk that purified material is not representative and cannot be
quantified at a higher metrological level by protein assay
procedures such as amino acid analysis.
In relation to the assay principle interfering factors should be
identified and listed for consideration.
The working group can identify presently available manuals
containing a prescription of the same method and comment upon the
suitability or need for revision (ECAT book, Bergmeyer series,
Methods in Enzymology, etc.).
3.1.6. Criteria for specificity
Based on the above considerations the working group defines a
concise set of criteria.
3.1.7. Test methods for criteria
Based on the criteria set the working group advises about
techniques of testing a method for adherence to the criteria. This
can be in the form of a set of clinical and manipulated samples
that should be tested and have specific results (example depleted
plasma should be zero; plasma spiked with excess of interfering
factors should give the same answer as non-spiked plasma).
and quality assurance
The working group should identify which available reference
materials and standards (6) from NIBSC, WHO, or other sources are
suitable or potentially suitable and define the status of the SSC
secondary matrix sample. In addition the present activities of EQAS
which include the analyte should be listed (7).
This section can contain remarks on various aspects and include
the following one when relevant.
126.96.36.199. Preanalytical factors
The document could describe potential preinstrumental sources of
variation for the quantity to be measured.
188.8.131.52. Preparation of the patient
This sub-clause could contain brief information (preferably
referring to appropriate source summary documents) on essential
requirements for fasting, posture of the patient before sampling,
which time of the day should the specimen be collected, time of
rest before collection of samples, which specimens can be used,
venipuncture techniques (e.g. two-tube techniques), and optionally,
the physiologic effect of drugs (e.g. steroids, fish oil).
184.108.40.206. Preparation of specimen
This sub-clause could include information on site of blood
drawing, which type of collection tubes and anticoagulants should
be used, the addition to the sample of preservatives or
stabilizers, storage conditions of the sample, storage conditions
of the specimen, specifications of centrifugation procedures. It
could be observed whether bilirubin, haemoglobin or lactescence of
the samples interfere with the measurement procedure.
220.127.116.11. Description of materials
The degree of the necessary purity or source (recombinant?) of
the reagents used should be commented on when necessary. Trade
names, systemic name or formula can be given. Similarly, the origin
of calibration materials with information on purity, the procedure
for preparation of calibrators with different concentrations should
It could be identified if the measurement procedure is dependent
on specific equipment or alternatively, whether it can be applied
with the use of equipment with similar measurement principles.
18.104.22.168. Genetic analysis
It could be identified whether presently genetic analysis of
polymorphisms in the gene for the analyte is relevant to be
included in view of composition of the analyte or in relation to
22.214.171.124. Reference ranges
The reference ranges of the method should be reported according
to accepted guidelines (8) and should be based on a sufficiently
large study population (9). Possible sex and age differences or
reference values should be described.
The references should be listed consecutively as listed in the
text and follow the Vancouver declaration.
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). Thromb
Haemost 1993; 70: 858-63.
3. B�ttner J. Reference methods as a basis for accurate measuring
systems. Eur J Clin Chem Clin Biochem 1991; 29: 223-35.
4. Nomenclature, IUPAC.
5. Bowes GN. Clinical chemistry analyte reference systems based on
true value. Clin Chem 1991; 37: 1665-6.
6. Uldall A. Quality assurance in clinical laboratories. An updated
supplement to a bibliography. Eur J Haematol 1990; 45/Suppl 53:
7. Dybk�r R. Reference materials - a main element in a coherent
reference measurement system. Eur J Clin Chem Clin Biochem 1991;
8. Dybk�r R, Soberg HE. Approved recommendation (1987) on the
theory of reference values. Part 6. Presentation of observed values
related to reference values. J Clin Chem Clin Biochem 1987; 25:
9. Henderson AR. Chemistry with confidence: Should clinical
chemistry require confidence intervals for analytical and other
data. Clin Chem 1993; 39: 929-35.