Fibrinogen
July 18, 2002
13:00 to 17:00
Whittier Hill Room
Boston Park Plaza Hotel
Chairman: S. Lord, USA
Co-chairs: J. Koopman, The Netherlands; R. McIntosh, UK; N.
Weinstock , Germany
Dr. Nicodemo Weinstock presented the summary of the evaluation procedure
for a high fibrinogen standard. The project was started at the XVIIth ISTH
Congress in Washington (1999), where there was considerable support for the
type of standard presented. The main problem is that fibrinogen is a risk
factor for CHD, but studies show large discrepancies in fibrinogen levels
measured.
The results of a large European study were presented and discussed at the
SSC Annual Meeting in Maastricht (2000), where those attending the Subcommittee
meeting voted for establishment of a high fibrinogen standard.
The complete approved report of the international study (19 laboratories
in 10 countries) was presented at the XVIIIth ISTH Congress in Paris (2001),
where the participants voted for the introduction of the presented standard.
In addition, the results were evaluated by scientists and clinicians not
involved in the study, reaching general agreement. Stability of the plasma
preparation was extensively evaluated. Comparison with 1st International
fibrinogen standard (WHO) showed acceptable agreement. The audience agreed
to present the data to the SSC Business Meeting and to recommend it as the
first International High Fibrinogen Reference Plasma with a potency of 5
g/l.
Dr. Colin Longstaff reported on a collaborative study of thrombin
standards in fibrin sealants. Two thrombin preparations were prepared, 10000
Ampoules each, approximately 100 units per ampoule (similar to current 1st
international standard).
The goal was to replace the currently used standards that are running low
and to re-unite IU and NIH-U to a single unit. Preliminary results from 25
participating laboratories were presented.
Each lab received four ampoules of A (current international standard), two
ampoules of B (US standard), and four ampoules each of the two new preparations,
C and D.
Overall variability was 8.8 % in clotting and 6.5 % in chromogenic assays.
There were no differences between human and bovine fibrinogen as substrate
concerning activity and the value for plasma as substrate lower than with
fibrinogen was not significant. Chromogenic assays displayed a trend towards
higher levels of thrombin activity. Candidate C seemed to be quite similar
to B, whereas D was close to A concerning amount of thrombin activity in
clotting and chromogenic assays.
Sample D was selected as best candidate for determination of final potency.
Geometric mean is 110 units per ampoule using A and B as standards and human
and bovine fibrinogen as well as plasma.
There are no stability data yet, but since there are only very small quantities
of preparations A and B left, replacement is urgent. Dr. Barrowcliffe commented
that differences between IU and NIH U are up to 15 % in earlier studies,
therefore, a common standard is a great achievement.
Carl-Erik Dempfle proposed a collaborative study on measurement of
fibrinogen in animal plasma. Results of different studies on fibrinogen in
various animal models showed a high degree of fibrinogen levels reported.
A specific problem is imposed by the fact that current fibrinogen assays are
optimized for use with human plasma, not with animal fibrinogen, and human
plasma/fibrinogen is used as calibrator. Reactivity with enzyme (bovine thrombin,...)
may be different from human fibrinogen, and the fibrin polymerization rate
may be different from human fibrin. Presence of fibrin(ogen) degradation products
influences clotting rate (underestimation of fibrinogen concentration). In
optical methods, turbidity of fibrin clots may be different from the human
system. Clot turbidity is also influenced by presence of fibrin(ogen) degradation
products (overestimation in PT-derived fibrinogen). For PT-derived fibrinogen:
Differences in PT influence rate of thrombin formation, and fibrin structure
(overestimation of fibrinogen concentration in prolonged PT / high INR).It
is suggested to study mouse, rat, rabbit and pig, and dog fibrinogen.
Study proposal consists of Phase 1: collection of plasma samples using
defined protocol for blood collection, plasma preparation and storage. Fibrinogen
concentration is measured by clot recovery method. Assays are screened. In
Phase 2, aliquots of 3 plasma samples per species are given to participating
laboratories for analysis. Phase 3 includes normal range studies for various
animal species and subspecies, using the pooled plasma material of same species
for calibration. Audience and readers are asked for support with collection
of animal plasma samples, and analysis of samples. For help and support: E-mail:
carl-erik.dempfle@med.ma.uni-heidelberg.de
Kunihiko Nakahara from Iatron Inc. presented data on a new soluble
fibrin assay. The assay is based on MAb IF-43. IF-43 does not react
with fibrinogen, but reacts with FM and fragments E of fibrin in immunoblot.
In native material, the epitope alpha 52-78 is exposed only when FM binds
to fibrinogen (or fragment D), resulting in a trimolecular complex with an
apparent MW of 1 000 kD, equaling trimer of 2 x fibrinogen and 1 x FM. Using
IF-43, a latex enhanced photometric immunoassay (LPIA) was developed. The
assay uses serial dilutions of desAABB-FM in normal plasma for calibration.
Human plasma may be replaced by bovine plasma for preparation of the calibrators.
There was no correlation with D-dimer assay (Iatron) in clinical plasma samples.
The time course in clinical samples (before/after surgery) differs both from
TAT and D-dimer, with highest levels observed on days 2 and 3 after surgery.
Elevated levels of soluble fibrin were found in patients with disseminated
intravascular coagulation.
Gordon Lowe gave an update on fibrinogen and cardiovascular disease
risk from the Fibrinogen Studies Collaboration (FSC), a collaborative meta-analysis
of prospective studies of fibrinogen and risk of CHD and stroke. Since the
collaboration started in 1999, individual data has been collected from 38
cohorts (total subjects included 137,000). After an additional 20 % of data,
10,000 coronary events and 2000 stroke events will be included.
At the next meeting of the ESC in 9/2002 in Berlin, preliminary analyses
of CVD endpoints will be presented.
A sub-analysis on type of fibrinogen assay used and standardization issues
will be discussed. Input from the Fibrinogen Subcommittee was invited. Discussions
included recalibration with the high fibrinogen standard.
Next steps of Fibrinogen Subcommittee:
- Gordon Lowe: fibrinogen as a risk factor; standardization and re-evaluation
of epidemiological studies.
- Nicodemo Weinstock: Establishment of generally accepted reference
values and risk percentiles by re-measuring available samples of risk factor
studies at a central laboratory using the newly established high fibrinogen
standard. Correlation of high fibrinogen values from different fibrinogen
assays.
- Carl-Erik Dempfle: Initiation of study on standardization of fibrinogen
measurement in animal models, in collaboration with Susan Lord.
- Colin Longstaff: Stability studies on thrombin standards.
N. Weinstock
C.E. Dempfle
G.D.O. Lowe