W. Nieuwenhuizen, Ph.D.,
TNO-PG Gaubius Laboratory, P.O. Box 2215, 2301 CE
Leiden, The Netherlands
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Information about the status of the haemostatic balance can be derived from the end products of coagulation and fibrinolysis, i.e. soluble fibrin and fibrin degradation products (FbDP), such as D-dimer, respectively. Assays for FbDP have been available for more than 15 years. Examples are semi-quantitative latex agglutination assays, and quantitative enzyme immunoassays for D-dimer. It is known that the use of serum can lead to erroneous and even false-positive or false-negative results. Little is known about the use of the assays in the Netherlands (type of test; serum or plasma as sample; requested by which specialism; for which indications; cito assay or not; numbers of assays). To collect such data we sent out questionnaires to 116 clinical centres. On the basis of the responses received from 82 centres we can conclude that the vast majority of the centres (76) use semi-quantitative latex tests. Of these 76 centres 59 used plasma samples (28000 tests/year); and 17 used serum (4800 test/year). The assays are done at the request of gynaecologists, internists, intensive care units and cardiologists for a variety of indications such as DIC, pregnancy complications, DVT and PE. In most cases cito assays were involved.
The D-dimer assays are discussed with special reference to standardisation, (biochemical) specificity, reproducibility, and the reasons why serum can cause erroneous results.
Under normal conditions there is equilibrium between on the one hand the activity of the coagulation system, and on the other hand the activity of the fibrinolytic system. Disturbance of this equilibrium, designated as the haemostatic balance, can cause bleedings when the fibrinolytic system is relatively more active than the coagulation system, and can cause thrombotic phenomena when coagulation is more active than fibrinolysis.
An activated coagulation system leads to thrombin formation. The thrombin formed converts fibrinogen to fibrin, and activates factor XIII to factor XIIIa, which cross-links the fibrin formed. Crosslinked fibrin is insoluble.
An activated fibrinolytic system (often as a reaction to the activation of coagulation) yields active plasmin. Plasmin degrades the insoluble, cross-linked fibrin to soluble (still cross-linked) degradation products (FbDP) such as �D-dimer�.
In its most elementary form the haemostatic balance can thus be described as the equilibrium between fibrin formation and fibrin degradation. As a result measurements of derivatives of fibrinogen, such as D-dimer can help in the diagnosis of disturbances in the haemostatic balance.
Fibrin(ogen) derivatives have been measured for many years by using a variety of methods (1). Elevated concentrations of FbDPs have been found in serum of patients with venous thrombo-embolism and disseminated intravascular coagulation (DIC), but also in the case of trauma, surgical procedures, infections, malignancies, and sickle-cell anaemia (2-12). It was postulated that increased FbDP concentrations confirm the diagnosis of diseases which are characterized by an activated coagulation system, and that normal FbDP concentrations exclude such diseases (8,12).
The observation that FbDP concentrations rise during thrombolytic therapy of acute myocardial infarction patients, and subsequently decrease again, suggested that the measurement of FbDPs could be used to monitor the effect of the therapy (13,14).
In general a test for a disease, which is characterized by an activated coagulation, is clinically useful when that test excludes the patients without the disease (specificity) and confirms the diagnosis in patients with the disease (sensitivity).
However, the lack of specificity and accuracy of serum FbDP assays has caused a lot of confusion with regard to the value of FbDPs as markers for haemostatic disturbances. Most FbDP assays were traditionally performed with serum samples, since the assays were usually based on polyclonal antibodies which, in plasma, would cross-react with the huge excess of fibrinogen. As will be elaborated in the discussion section the use of serum samples is a source of artifacts. For almost twenty years now, however, FbDP assays have been available, which are based on monoclonal antibodies, which do not cross-react with fibrinogen; and those assays can thus be carried out on plasma samples without those risks of artifacts. But the modern assays for D-dimer also have their limitations, which will be discussed below.
Several types of assay are available e.g. semi-quantitative latex agglutination assays, and quantitative enzyme immunoassays. Relatively little is known about their use in the Netherlands.
One purpose of the present study was to investigate which types of assay are used, and how frequently; by which specialisms and for which indications; and whether or not the assays are carried out as cito assays. The impression existed that serum samples are still being used on a quite considerable scale, notwithstanding the fact that it has been known for many years (15) that serum can be a source of erroneous results in assays for fibrin degradation products (16). A second purpose of this study was to investigate whether this notion has led to the abandonment of the use of serum samples.
Design of the study
A questionnaire was sent to 123 addresses in 116 different clinical centres (some centres have more than one laboratory) throughout the Netherlands.
The following questions were asked:
Do you use as a sample serum, plasma or blood?
What is the principle of the assay you use: ELISA, latex agglutination or an other principle?
Is the assay used quantitative or semi-quantitative?
Requested by which specialism?
For which indication/question is the assay used ?
Is the assay used in cito situations or not?
What is the approximate number of assays which you performed in the past month?
The centres were asked to return the completed forms within two weeks. After some weeks those who had not responded were contacted by phone.
Of the 123 laboratories approached (in the 116 centres) a total of 94 responded either via the completed questionnaire or by phone. Of those who reacted only by phone ten indicated that they did not, not anymore, or not yet use the assays; five indicated that they did not have time to respond; the remaining were repeatedly unreachable by phone. A total of 82 (71% of all centres) completed forms were received.
The 82 forms showed:
76 centres use a semi-quantitative latex agglutination test
2 centres use a quantitative ELISA
3 centres use a quantitative or semi-quantitative version of a filtration-type assay
1 centre does not use fibrin degradation products assays
4 centres use more than one type of assay
Of the 76 centres which perform semi-quantitative latex agglutination assays, 59 indicated that they use plasma samples; the remaining 17 use serum. Extrapolation of the number of assays performed by each centre during the last month indicates that 28000, and 4800 plasma and serum assays, respectively, are performed per year by using a latex agglutination assay.
The majority (49 of the returned forms) of the centres indicated the use of cito determinations.
As can be concluded from the above the use of other types of assays (ELISA, filtration, turbidimetric) is relatively small.
The aforementioned data plus the requesting specialisms and the indications for which the assays are used are summarized in the table.
It is obvious that by far most assays are requested by gynaecologists, internists, intensive care units (ICU) and cardiologists; and that DIC, pregnancy complications, DVT and pulmonary embolism are the most important indications.
It can be derived from the table that 39000 assays are performed on a yearly basis. When all types of assays are taken together, this figure is rather modest. A possible reason for this may be derived from remarks made on the forms by several of the centres. One important reason mentioned was the lack of standardization. Results obtained for the same sample by using different assay methods may vary by a factor of ten in absolute value. Several reasons can be mentioned for these differences:
the terms fibrin degradation products, and even more so D-dimer suggest a molecular uniform identity of the fibrinolysis products measured. This is misleading. Degradation products are highly heterogeneous, and this heterogeneity varies from patient to patient. The enormously long fibrin stands in a blood clot consist of a very large number of fibrin monomer subunits, which are kept together by among others crosslinks (iso-peptide bonds). Each subunit consists of a central E-domain, flanked by two D-domains. The crosslinks are situated between the D-domains of two neighboring subunits. Hence a pattern is performed which can be represented as: (= DED = DED = DED =)n (in which = stands for the crosslinks).
The plasmin digestion (fibrinolysis) of the fibrin strands is a random process which occurs via proteolytic cleavage of the subunits. This leads to the formation of fragments of those strands. These fragments are soluble, and are composed of a very variable (even large) number of subunits, which contain the motive designated as D-dimer, but are not D-dimer. Examples are ED = DED = DED and ED = DED = DED = DED = D, whereas the term D-dimer is limited to D = D.
The various assays used are based on different (mostly monoclonal) antibodies. These antibodies have different reactivities with the various fibrin degradation products comprising D-dimers. Each manufacturer uses his own calibrator, and assigns a concentration to it.
On the basis of the above considerations it will be clear that standardisation of FbDP assays is not simple. Yet, the situation is not hopeless. This author has (in his capacity as chairman of the Fibrinogen Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society of Thrombosis and Haemostasis (ISTH)) made a successful attempt at generating a reference material. Although this is not an international standard, it enables manufacturers and users to compare the results obtained with the various assays.
Some users have the impression that the lower the numerical results obtained with a particular assay for normal plasma samples, the more specific the particular assay. From what is said above about calibration, it will be clear that this is a mistake: the numerical values found are the direct result of the values assigned by the manufacturers to the calibrators.
It has become clear that the diagnosis of DVT or pulmonary embolism cannot be made on the basis of a positive D-dimer assay. This is not surprising considering the fact that fibrin degradation products can occur in all cases of (even local) fibrin formation. It has been shown in a number of studies that a negative result can be used as an exclusion criteria for the occurrence of DVT (16). However, negative results of latex agglutination assays appear less suitable as exclusion criteria than normal results of quantitative ELISAs. These conclusions are mostly based on studies on reasonably large numbers of clinical samples. A possible reason for the greater value of ELISAs could be the lower lowest detection limit of the ELISAs. Recently we came across another possible reason for the lesser value of latex agglutination assays for the exclusion of thrombosis. We found (in at least one latex agglutination test kit) that the batch-to-batch variation of the detection limit is at least a factor of 2. This variation is not indicated by the manufacturer in the insert. The consequences of this for a particular study are obvious, when different batches of such test kits are used, and it is assumed that the FbDP concentrations at which they become positive, are identical.
Although in many cases the assays for degradation products have undoubtedly added to the diagnostic potential, it is quite likely that the aforementioned points have not helped to implement assays for fibrin degradation products in the clinical routine.
Serum samples should not be used. As early as 1985 Gaffney (15) emphasised that the use of serum causes erroneous results and artifacts (16). Too high or even false-positive results can be found, when anti-coagulant degradation products are present (17-19); in the case of an abnormal fibrinogen with a reduced coagulability ; or when a patient is anticoagulated with heparin. Another possible cause is the partial lysis of the clot during serum preparation, not only in hyperfibrinolytic patients, but even in normals (20).
Also too low or false-negative results are possible. Some degradation products coagulate (18,21) or adsorb to the clot (15,20,22).
In the coming years existing assays will be improved and new improved assays will become available. It is our intention to repeat this study after a couple of years to monitor whether the clinical acceptance of fibrin degradation products assays has increased.
The author acknowledges Mrs Jongsma for collecting the data, and Prof.Dr. P. Brakman, Dr. F. Haverkate and Dr. E. Brommer for critically reading the manuscript, and their constructive comments.
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Correspondence should be directed to:
W. Nieuwenhuizen, Ph.D.,
TNO-PG Gaubius Laboratory,
P.O. Box 2215, 2301
CE Leiden, The Netherlands
Copyright © 2001 International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). All rights reserved.