THROMBOSIS RESEARCH 65; 745-756,1992 0049-3848/92 $5.00+ .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

DETERMINATION OF SOLUBLE FIBRIN: A COMPARISONOF FOUR DIFFERENTMETHODS J.U. Wieding and C. Hosius Department of Haematology, University Hospital, 3400 Gottingen,

Germany

(Received 256.1991; accepted in revised form 20.1 .1992 by Editor F. Markwardt)

ABSTRACT

The determination of soluble fibrin (SF) in plasma was compared using four different methods. The SF-ELISA immunologically measures the concentration of desAA- and desAABB-fibrin while the SF-tPA-test is based on activation of plasminogen by tissue plasminogen activator (tPA) in the presence of fibrin; the SF-PS-turbidimetry assay relies on the protamine sulphate (PS) -induced aggregation of fibrin in plasma whereas the SF-erythrocyte-agglutination-test (SF-EAT) detects soluble fibrin by its aggregation with fibrin monomers attached to test erythrocytes. Soluble fibrin was generated in vitro by addition of thrombin or ancrod to plasma. In these experiments the soluble fibrin values of the four methods correlated well with each other and with the fibrinopeptide A release, especially in ancrod-induced fibrinogen turnover (r > 0.93). This high correlation is remarkable, considering the fact that the methods are based on different principles. Detection of thrombininduced soluble fibrin was more sensitive; differences between ancrod and thrombin action were observed as well, probably due to different forms of soluble fibrin. A delayed increase of SF-PS-turbidimetry values in particular during the thrombin action can be attributed to a lack of detectable aggregation of soluble fibrin at low concentrations due to its solubility in plasma. Subsequently, soluble fibrin was measured in samples from patients. The SF-ELISA and SF-tPA-test were highly sensitive and correlated better than the other methods with each other, but all correlations were less satisfactory compared with the in vitro These weaker correlations might be explained by the heterogeneity of studies. soluble fibrin determined by inter- and intraindividually varying concentrations of fibrinogen and its different derivatives in plasma samples from patients. All methods

provided reliable results with differences in sensitivity, specificity and practicality. The SF-tPA-test, SF-PS-turbidimetry, and SF-EAT are practical methods for routine use whereas the SF-ELISA is a highly reliable and by far the most sensitive and specific method thus offering new insights into pathogenesis of fibrinaemia and related diseases. Key words :

Soluble fibrin determination, protamine sulphate , paracoagulation , haemagglutlnation assay, tissue plasminogen activator, enzyme-linked immunosorbent assay.

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INTRODUCTION

Soluble fibrin is an early and sensitive indicator of hypercoagulative states with increased fibrinogen-fibrin turnover; it is of particular relevance in the pathogenesis and diagnosis of disseminated intravascular coagulation (DIC) (1,2,3,4,5) and thrombosis (6,7). Because of its importance many methods have been described for determination of soluble fibrin based on a variety of principles, but no single test has found widespread acceptance due to a lack of either sensitivitv, specificity or practicality. A classification of the known tests according to the underlying principle is proposed in the following scheme : - Paracoagulation tests ( non-enzymatic aggregation of soluble fibrin )

ethanol gelation test (8) tests using polyanionic reagents such as protamine sulphate ((9), many modifications) ristocetin / netropsin (10)

or

- Affinity of soluble fibrin to insolubilized fibrin(ogen) derivatives

chromatography with fibrinogen agarose (11) agglutination of fibrin monomer -coated erythrocytes (12) or latex particles (13) affinity to fibrinogen immunoprecipitates (14)

- Other fibrin-specific functional properties

activation of tissue plasminogen activator (tPA) (15) incorporation of Cl4 glycine ethyl ester (5)

- Physicochemical properties

agarose gelfiitration (16) polyacrylamide gelelektrophoresis (17) isoelectric focussing (18) determination of the N-terminal glycine (19)

- Immunological properties

- anti fbnl7 for detection of desk&fibrin

(20)

For more than 20 years, paracoagulation tests have proved to be practical in patients’ diagnostics (8,s); especially ethanol or protamine sulphate paracoagulation tests are often employed in rOUtit% clinical use and investigation of DIC (3,2). Protamine sulphate (PS) causes an aggregation of fibrin in plasma and the arising turbidity can be estimated (9). However, none of the numerous modifications were successful due to either a lack of sensitivity or of specificity especially to fibrinogen. The new SF-PS-turbidimetry overcomes the problems of earlier versions by providing a sensitive measurement of the protamine sulphate -induced aggregation of soluble fibrin in a turbidimeter (21,4). A major advance was achieved by an approach based on the discrimination between fibrinogen and fibrin by means of the monoclonal antibody anti-fbn17 (20). In order to detect the antigen structure determining de&A-fibrin the synthetic hexapeptide Gly-Pro-Arg-Val-Val-Glu, representing the amino terminus of the alpha chain of human fibrin after cleavage of fibrinopeptide A, was used as immunogen. This antibody is employed in an enzyme linked immunological assay system (=SF-ELISA) to quantify fibrin in plasma. The newly developed SF-tPA-test is based on the activation of plasminogen by tissue plasminogen activator (tPA) in the presence of fibrin (22); the subsequent turnover of a chromogenic substrate through the resulting plasmin activity is dependent upon the plasma fibrin concentration (15). A very convenient method for detection of soluble fibrin especially in single specimens is the erythrocyte agglutination test (SF-EAT) : The affinity of the sample’s fibrin to fibrin monomer coated test erythrocytes leads to their visible agglutination (12). The aim of the present study was to compare these four methods using plasma samples with soluble fibrin generated in vitro by thrombin or ancrod addition; subsequently, plasma samples were examined from patients.

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MATERIALS

AND

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METHODS

Citrate plasma was obtained from 1: 10 v:v blood anticoagulated with sodium-citrate (0.015M) after centrifugation at 2000 g for 10 minutes. Plasma, for in vitro experiments, was obtained from one healthy volunteer with a fibrin concentration of 1.95 g/l and without any history or sign of hypercoagulability, as well as normal results in routine blood coagulation tests, determination of antithrombin III and fibrin degradation products. Plasma samples from patients in intensive care or with DIC were collected on three days for routine diagnostics. The 92 samples were stored for a maximum of 8 hours (appr. 2 to 3 hours at 20°C then at 1OC). Soluble fibrin was determined (n=92) by the SF-ELISA, SF-tPA-test, and additionally by the SF-PS-turbidimetry and SF-EAT in 72 of the 92 samples. In examining different storage conditions fibrin was measured in 36 plasma samples from patients before and after storage periods of 1 or 4 day(s), at +22, + 1, -20 and -75 OC, with and without addition of aprotinin. The antifibrholylic agent aprotinin (Antagosar? from Behring, FRG) was added to all plasma samples to a final concentration of 5 KIE/ml plasma unless otherwise stated. Standards for soluble fibrin are included in the kits for SF-ELISA as well as the SF-tPA-test. Furthermore desAA- and desAABB-fibrin, kindly provided by Dr.Lill (Boehringer Mannheim GmbH, FRG), and desAA-fibrin from Biopool (S) were used for analysis in the SF-PSturbidimetry; serial dilutions of these standards were performed in 1 M NaBr to give final concentrations of 5 to 400 ug/ml after adding 100 ul to 900 ul plasma. Plasma samples with in vitro induced soluble fibrin were obtained by incubation of normal citrate plasma with thrombin (n= 40) or ancrod (n= 45) : The action of thornbin (purchased from Hoffmann-LaRoche (CH), final concentration 0.04 NIH/ml plasma) at 25OC was stopped by addition of hirudin (from Pentapharm (CH), final concentration 100 IE per ml) to eight aliquots taken at time intervals of 14, 15, 16, 18 or 20 seconds, respectively, after addition of thrombin to the plasma sample. Analogously, the action of ancrod (Arwi# donated from Knoll AG (FRG), final concentration 0.2 IE/ml) at 25OC was stopped by addition of the specific antiserum (granted from Knoll AG, 0.30 IE anti-Arwin to 1 ml plasma) to nine aliquofs taken at time intervals of 12, 13, 14, 15 or 16 seconds, respectively, starting with the beginning of ancrod action. After inhibition of either thrombin or ancrod the sample mixture was further incubated for seven hours at 25 or 37OC, respectively. In those plasma samples the fibrinopeptide A release was measured by the FPA-ELISA and the generation of soluble fibrin was determined by the SFELISA, SF-tPA-test, SF-PS-turbidimetry and the SF-EAT. FibrinopeptideA (FPA) determination is based upon a modification of Nossel’s procedure (23) : FPA was measured enzyme-immunologically in citrate plasma after precipitation of fibrinogen with bentonite employing the FPA-ELISA kit from Boehringer Mannheim, FRG. Soluble fibrin (SF) was determined by four different methods : The SF-ELBA (20) was performed as recommended by the manufacturer Boehringer Mannheim, who provided the required reagents solely for research purposes (reagents are not commercially available). Polystyrene microtiter plates were coated with the monoclonal antibody specifically directed against fibrin. The plates were subsequently incubated with plasma samples diluted 1:lOO in phosphate buffered solution, then with a peroxidase iabelled polyspecific antibody against fibrinogen, and washed after each step; bound enzyme activity was measured with the chromogenic substrate ABTS. The fibrin concentration of a given sample was calculated from a standard containing a definite amount of desAA-fibrin.

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The SF-ffA-test was done using the COA-set Fibrin Monomer (kindly provided from Kabi, Sweden) which contains the chromogenic substrate S2390 to determine the tissue plasminogen activator (tPA) mediated plasmin activity (15). In a micro-titer plate 100 ul of the diluted specimen (plasma + Tris buffer with the ratio 1 t40, I t80 or 1 t200) were incubated with 100 ul plasminogen and 50 ul tPA/S2390 dilution at 20°C, the increase in optical density during 20 minutes of incubation was measured at 405 nm. The fibrin concentration of a sample was calculated from the desAA-fibrin standard. The inhibitory effect of aprotinin on the SF-tPA-test was readjusted in consideration of the 14.6% decrease in values due to 5 KIE/ml aprotinin in analyzed samples. This calculation was made from prior experiments, using aprotinin with concentrations up to 10 KIE per ml plasma, in which a linear dependence was demonstrated (r= 0.98, n= 20). The SF-PS-turbidirnefry assay measures the aggregability of soluble fibrin in plasma after addition of protamine sulphate (PS) with a turbidimeter (21): 40 ul citrate plasma and 185 ul 0.1 M Tris-NaCI-buffer (pH 6.5) were pre-incubated 156 seconds at 25’C; 25 ul of a 0.2% PS-dilution (containing 1 mmol/l ZnCI,) were added and after 12 seconds the increase in turbidity was measured at 334nm over a timespan of 7 seconds. Turbidity was measured in the photometer ‘EPOS-Analyzer 5060’ from Eppendorf (FRG), equipped for programmable automatic pipetting. The results were calculated from AE/At by AE/min = (Elssec - E,_c) . 60/7 with Eosec= addition of PS; analyses were performed in triplicate and the median taken as the result. The SF-erythrocyte-agglutinationtest (SF-EAT) according to Largo (12) was performed with the FM-Test kit from Boehringer M. : 0.1 ml of the plasma sample or the enclosed control plasma were incubated for 10 minutes at 37OC with 0.05 ml solution of the fibrin monomer coated erythrocytes. Then the mixture was transferred to a slide and gently swung for 6 minutes. If an agglutination of the erythrocytes was seen due to soluble fibrin, the analyzed sample was diluted 1:2 with sodium citrate in order to yield semiquantitative titer results.

Statistical analysis

was performed by calculation of correlation coefficients according to Pearson as well as Spearman. Pearson’s coefficients are given for a better differentiation of the inter-method correlation in soluble fibrin determination. In the in vitro experiments soluble fibrin results were compared with the fibrinopeptide A release (table 1, figure 1 shows mean values as well as computer-fitted curves).

RESULTS After addition of thrombin or ancrod to normal citrate plasma the release of fibrinopeptide A (FPA) directly depended on the time of proteolysis (figure not shown) which was stopped by addition of hirudin (n=40) or specific antiserum (n= 45 samples). As seen in other investigations (24) clotting phenomena were noticed after the release of relatively less FPA when induced by thrombin instead of ancrod; thus the generation of soluble fibrin by thrombin was observed in a smaller range (figure 1). In thrombin or ancrod-induced fibrinogen-fibrin turnover the results from SF-ELISA, SFtPAtest, SF-EAT, SF-PS-turbidimetry and FPA-ELISA correlated well with each other and with the FPA-release (table 1 : 0.88 < r < 0.99). All correlations were better in experiments with ancrod (r 2 0.93) than with thrombin. Results of the SF-ELISA showed a nearly linear dependence on the FPA-release bin as well as ancrod action on fibrinogen (r= 0.98), more so than either the especially the SF-PS-turbidimetry. Thrombin-induced fibrin could be measured greater sensitivity than when induced by ancrod. From the SF-ELISA results,

during thromSF-tPA-test or with 1.25-fold an average of

DETERMINATION

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500

OF SOLUBLE

749

FIBRIN

35 35_ 30

400

0A

30_ 25 25_ 20_ :20_ G E -15 % L G .g10_ E L) ;

5_

::

15_

;;lOG .=

5_

5

I bl o_ % 0, b 4

I

600

600

400

Fibrinopeptide

I

I

I

I

200

0

A

(nmol/l)

35. 35_ 30. 30_

0

25.

B

25_

I

0

Figure 1 :

I

200

I

I

600 400 Fibrinopeptide

I

600 A (nmol/l)

I

1000

I

1200

I

1400

Soluble fibrin (SF) detectable by SF-EUSA ( ?? ), SF-tPA-test (o), SF-EAT (v) and SF-PS-turbiiimetry (A) in dependence on the FPA - release induced by time-limited action of thrombin ( diagram A ) and ancrod ( diagram B ); shown are mean values from data of 2 series performed on different days with equal enzyme action times.

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one fibrin(monomer) molecule (mol.wt. 1530) was detectable after the release of 2.3 molecules fibrinopeptide A by thrombin or 2.9 molecules FPA by ancrod. As compared to the FPArelease, the results of the SF-tPA-test, SF-EAT and SF-PS-turbidimetry depended more on the different manner of action of the two enzymes than did the SF-ELISA. The results obtained with the SF-PS-furbidimetrycorrelated well with the release of fibrinopeptide A only when induced by ancrod, but showed a steep increase during thrombin action, causing FPA levels exceeding 450 nmol/l in the investigated plasma with 1.95 g/l fibrinogen. This increase and the final decline in sensitivity for high concentrations of soluble fibrin led to a sigmoid curve (figure 1 A). In the range of greatest sensitivity (release of 400 to 650 nmol/l FPA) the SF-PS-turbidimetry correlated with the FPA-release with r= 0.99. After release of 600 nmol/l fibrinopeptide A the SF-PS-turbidimetry values were approximately 5fold higher when induced by thrombin instead of ancrod. Up to a release of 400 nmol/l FPA only ancrodinduced fibrin could be detected but not the thrombin-induced fibrin. A moderate increase in sensitivity could also be seen during the ancrod action: After release of more than 400 nmol/ml FPA the soluble fibrin was measured with approximately twofold sensitivity.

Table 1 Inter-method correlation coefficients of linear regression

(A )

Fibrinopeptid A (FPA) and soluble fibrin (SF) generated in vitro SF-ELISA SF-tPA-test SF - EAT

by - Ancrod action

- Thrombin action

( B)

FPA SF-ELISA : SF-tPA-test : SF-EAT :

0.978

FPA SF-ELISA SF-tPA-test SF-EAT

0.985

: :

0.926

0.975

0.945

0.971 0.931

0.957 0.928

0.972 0.982

: :

0.982

SF-PS-test 0.988 0.981 0.948 0.977 0.929 0.884 0.982 0.939

Soluble fibrin in plasma samples from patients SF-tPA-test SF - EAT

SF-PS-test

- n=8samples collected on 1 day from 1 patient

SF-ELISA : SF-tPA-test : SF-EAT :

0.984

< 0.3 < 0.3

0.941 0.887 < 0.3

- n = 44 samples collected on 1 day from 5 patients

SF-ELISA : SF-tPA-test : SF-EAT :

0.704

< 0.3 < 0.3

< 0.3 0.430 < 0.3

- 72 samples from 2 days

SF-tPA-test :

- 92 samples from 3 days

SF-ELISA

:

0.771 0.923

( A ) Correlations of results from SF-ELISA, SF-tPA-test, SF-EAT, SF-PS-turbidimetry (= SF-PS-test) and FPA-ELISA in fibrinogen-fibrin turnover induced in vitro by thrombin and ancrod; a representative correlation from data obtained with one calibration comprising 2 test series each with thrombin (16 samples) and ancrod (18 samples) Is shown. ( B ) Correlations of results from SF-ELISA, SF-tPA-test and SF-PS-turbidlmetry In samples from patients in intensive care.

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751

The SF-tfA-test was approximately 2.5fold more sensitive for soluble fibrin generated by thrombin instead of ancrod. One fibrin(monomer) molecule (on average) was detectable after the release of 2.0 molecules of fibrino-peptide A by thrombin or 4.7 molecules FPA by ancrod. Figure 1 A illustrating the correlation between thrombin-induced FPA-release and measured soluble fibrin, shows a slight lack in the sensitivity of the SF-tPA-test between releases of approximately 150 and 300 nmol/l FPA, followed by a recovery in sensitivity. Results of the SF-EAT (mean values of titers) demonstrated a nearly linear correlation with the FPA-release by the action of both enzymes, and were almost comparable to results from the SF-ELISA (table 1 ); however, thrombin-induced fibrin was detectable with a 3.5fold higher sensitivity than with ancrod action. Altogether, when compared with the FPA-release the four methods were more sensitive in detecting fibrin generated by thrombin than by ancrod, if SF-PS-turbidimetry values in the initial lag phase were not considered (figure 1 ). Method evaluation Precision controls of the SF-ELISA and the SF-PS-turbidimetry were performed by lo-fold analysis of 6 different plasma samples. The intra-test variation coefficients of SF-ELISA results were calculated in relation to the mean value as 16.6ug/ml+2.8%, 13.2ug/ml+2.8%, 8.9ug/ml ~7.6%, 2.2ug/ml+6.1%, 1.9ug/ml+4.2%, 1.5ug/ml_+4.0%. In SF-PS-turbidimetry the withinrun coefficients of variation ranged from 1.7 to 7.4 inversely proportional to the mean value (21). A further assessment of the SF-PS-furbidimetry was performed with standardized fibrin (desAAand desAABB-fibrin dissolved in NaBr) added to normal plasma: 100 ug/ml desAA-fibrin could be measured with the same sensitivity as approximately 40 ug/ml desAABB-fibrin. DesAAfibrin added to plasma was detected down to a level of 12 ug/ml, which resulted in an increase in values from 2.5 to 5 mE/min, for example. Fifteen to 45 minutes after adding 200 ug desAAfibrin to 1 ml plasma with 2.0 g/l fibrinogen (soluble fibrin < 10 us/ml), the SF-PS-turbidimetry values continuously rose 1.5-fold but values decreased when a plasma sample containing 5 g/l fibrinogen was used. Whereas the sensitivity of SF-PS-turbidimetry and SF-EAT was found to be limited to about 10 to 15 ug/ml fibrin in plasma, the sensitivity of the SF-ELISA and the SF-tPA-test depends on the predilution of samples, dilutions of 1: 100 and 1:40, respectively, are recommended. Determination

of soluble fibrin in patient’s plasma

Examining plasma samples (n=92) from patients in intensive care or with suspected DIC the results of SF-PS-turbidimetry (n=72) correlated moderately well with those obtained with the SF-ELISA (r= 0.70) or with the SF-tPA-test (r= 0.77). Values from SF-ELISA and SF-tPA-test correlated better (r= 0.92, n = 92); the plot (figure 2) shows that the SF-tPA-test is less sensitive in determination of low fibrin concentrations than the SF-ELISA; however, the linear regression including all values resulted in a line with a slope of 1.05. The correlation of the SF-EAT with the three other methods ranged below r= 0.2; no visible agglutination occurred in over 80% of the plasma samples in which the presence of fibrin was indicated by more than one other method although previous studies have demonstrated a high validity of SF-EAT for fibrinaemia in pre-thrombotic state (7) and DIC (3). Plasma samples were analyzed as fresh as possible, since the study of different storage conditions could not lead to clear recommendations. After addition of 5 KIE aprotinin per ml plasma and storage over 36 hours at + l°C or over 4 days at -75’C the values of SF-ELISA and SF-PS-turbidimetry increased by an average of 2.1 and 2.4 %, respectively, but 6 of 36 samples differed by more than ~10% in comparison to the initial value.

752

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DETERMINATION OF SOLUBLE FIBRIN

1000

SF-ELBA (nmol/l) !z

100 E

10 5

I

E

.

. “.

.

.

OS1E

O,OIL’ 1

.

’ “““’

.

i

(n= 92, r,in= 0.924)

,

I

I1111111

IO SF-tPA-test

100

I111111,

1000

(nmol/l)

Figure 2 : Correlation of SF-ELBA and SF-tPA-test in determining soluble fibrin in 92 samples from ps-

tients; curvilinear form of the line from linear regression (r=0.92) due to the logarithmic scales

DISCUSSION

The determination of soluble fibrin (SF) by four methods with different basic principles was compared (12,20,21,15). All methods provided reliable results with differences in sensitivity, specificity and practicality. In vitro thrombin- or ancrod-induced fibrinogen-fibrin turnover was measured equally well by the release of fibrinopeptide A as well as by the formation of soluble fibrin detected by the FPA-ELISA as well as the SF-ELISA, SF-tPA-test and SF-EAT but marginally less satisfactory by SF-PS-turbidimetry (table, figure 1). All methods correlated well with each other and with the fibrinopeptide A release, especially in ancrod experiments. This high correlation is particularly striking, considering the fact that the underlying principles of each method are very different. However, differences were observed in the detection of soluble fibrin induced in vitro by either ancrod or thrombin, and in samples from patients. Whereas thrombin initially induces a fibrinogen-fibrin turnover by splitting the two fibrinopeptides A and later the fibrinopeptides B from a fibrinogen molecule (25,26,27), ancrod’s action is restricted to cleavage of both fibrinopeptides A (28), similar to reptilase or batroxobin (29). Therefore ancrod action only generates desAA-fibrin whereas thrombin produces both de&IA- and desAABB-fibrin. The fibrin(monomer)s aggregate and form complexes with other fibrin structures, fibrinogen and/or fibrin(ogen) degradation products (30,31,32); these fibrin complexes have various sizes and are heterogeneous in nature (25,29). In plasma they are held in solution mainly by fibrinogen (31,33,34), consequently the term ‘soluble fibrin’ has been Depending on the concentration, size and composition of the circulating established. complexes, fibrin may become insoluble and precipitate (25). The relevance of fibrinogen in solubilizing fibrin could be demonstrated by SF-PS-turbidimetry which reflects the aggregability of fibrin: This was greater, for example, when plasma with low instead of high fibrinogen levels were used to dissolve desAA-fibrin.

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The threshold of FPA-release necessary for fibrin detection by the SF-PS-furbidimetryresulted in a delayed increase of SF-PS-turbidimetry values during the in vitro thrombin action (sigmoid curve in figure 1 A) . This finding can be attributed to a lack of detectable aggregation at low plasma fibrin concentrations. Brass (34) described a comparable phenomenon with two stages of fibrin clot formation : initially no fibrin polymerization occurs and then particle size rapidly increases. If fibrin in plasma is kept stable in solution no spontaneous aggregation occurs. By neutralizing fibrin’s negative charges protamine sulphate augments the ability of soluble fibrin to aggregate thus leading to its precipitation (35,9,21). At very low plasma fibrin concentrations protamine sulphate is obviously not capable of inducing a measurable aggregation, an effect which is strongly dependent also upon the protamine concentration : Either no substantial aggregation occurs or, more probably, the formed fibrin complexes are so small that they can be further held in solution in plasma. Interestingly, this lack of fibrin aggregability at the initially low concentrations was more obvious in thrombin than ancrodinduced fibrinogen-fibrin turnover. When compared with the FPA-release, beyond the initial lag phase thrombin-generated soluble fibrin could be measured by the SF-PS-turbidimetry with a distinctly greater sensitivity than fibrin induced by ancrod. This may be explained by the finding that in comparison to ancrodinduced desAA-fibrin the desAABB-fibrin is able to form stronger and thicker aggregates through both longitudinal as welf as lateral polymerization (24,29). This stronger aggregation can also explain the 3.5-fold increased sensitivity of the SF-EAT when using thrombin instead of ancrod or of SP-PS-turbidimetry in detection of de&A- and desAABB-fibrin added to plasma. different types of aggregation and subsequent forms of resulting fibrin Analogously, complexes may contribute to differences in sensitivity of the SF-ELISA as well as SF-tPA-test for measurement of thrombin and ancrod action, respectively. For example, the ELISA detects only fibrin with desAA-fibrin specific antigen which is freely accessible for the antibody and not hidden in the structure of the fibrin complex. The SF-tPA-test depends on the trimolecular interaction of fibrin, tPA, and plasminogen (15), which may also be influenced by the structure of fibrin complexes. The slight lack of sensitivity of the SF-tPA-test during an intermediate stage of thrombin-induced fibrinogen-fibrin turnover (figure 1) might be due to steric problems during the formation of the cyclic ternary complex and subsequent activation of plasmin (22). In this context Halverson et al. assumed that fibrinogen interferes with the stimulating effect of fibrin on the tPA-catalyzed activation of plasminogen (36). With this goal in mind, they carried out a similar in vitro study : They correlated the SF-tPA-test, SF-EAT and the ethanol gelation test (8) with the FPA-release. Depending on the fibrinogen levels and the inhibition of thrombin action, molar levels of FPA were 2 to 25 times higher than that of soluble fibrin (SF-tPA-test). Discrepancies with the present results may well be due to different ways in preparing soluble fibrin (36). In ana/yzing patients’plasma the inter-method correlation could not approximate the excellent results of the in vitro experiments. Heterogeneity of soluble fibrin in patients’ plasma might be the most important reason. Variations in formation of fibrin complexes from fibrin monomers, -dimers or oligomers with fibrinogen and fibrin(ogen)olytic products cause intra- and interindividual differences in size and composition (17,25,29,31). For this reason, in the in vitro experiments concentrations of fibrinogen and fibrin(ogen) degradation products were kept quite constant by using the same sample with an antifibrinolytic additive; in contrast, the concentrations of fibrinogen and its derivatives in patient plasma are always subject to great variations.

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Interestingly, SF-ELISA and SF-tPA-test correlated in patients’ samples nearly as well as in the in vitro experiments even though the SF-tPA-test may be influenced by plasminogen activator inhibitor and tPA in the given sample and therefore by the technique of blood withdrawal. In contrast to high inter-method correlations of SF-PS-turbidimetry values in the in vitro experiments only moderate correlations were found with the SF-ELISA and SF-tPA-test in clinical studies. Apart from the heterogeneity of soluble fibrin, the influences of other plasma proteins upon the protamine-induced paracoagulation and the fibrin network structure (37) must also be taken into consideration. In contrast to the other methods, the SF-PS-furbidimetry measures the aggregability of fibrin in plasma rather than an exact concentration of a defined antigen (SF-ELISA) or the fibrin aggregability with foreign substances (SF-EAT) or other functional properties ( SF-tPA-test). Thus, the SF-PS-turbidimetry provides information about the patients risk of intravascular fibrin aggregation and precipitation with its known complications, which is a major clinical concern for example in DIC (1,4). In comparison, the SF-ELISA is more sensitive in the detection of the generation of fibrin which, however, does not necessarily have to aggregate. Due to its sensitivity, specificity and reliability this method is predestined to offer new insights into the process of fibrin generation as well as fibrinaemia in DIC and thromboembolic diseases.

ACKNOWLEDGEMENT

The authors are indebted to Dr. U. Scheefers-Borchel

(Prof. G. Miiller-Berghaus’ Department) for her support in the initiation of this project by providing first SF-ELISA results in the preceeding test series as well as Prof. A. Henschen for encouragement and stimulating discussions in fibrinogen -related issues.

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Miiller-Berghaus, G. and Hasegawa, H. Pathophysiology Semin. ThrombHemost. 3,209-246, 1977

of generalized intravascular coagulation.

2.

Bowie, E.J. and Owen, C.A. The clinical pathology of intravascular coagulation. Bib/. Haematol. 49, 217-224, 1983

3.

Oehler, G., Klaus, H., Stiitzer, K.E. and Spanuth, E. Detection of soluble fibrin monomer complexes. Comparison of a haemagglutination assay with the ethanol gelation test. Folia HaematoLLeipz. 115, 278-283, 1988

4.

Wieding, J.U., Eisinger, G. and Kiistering, H. Diagnostik der disseminierten intravasalen Gerinnung: Aussagekraft von ldslichem Fibrin, D-Dimeren und Fibrin(ogen)-Spaltprodukten. K/in. Wochenschr. 67, 764-773, 1989

5.

Kisker, CT. and Rush, R. Detection of intravascular coagulation.

6.

Halvorsen, S., Skjonsberg, O.H. and Godal, H.C. Comparison of methods for detecting soluble fibrin in plasma from patients with venous thromboembolism. Thromb.Res. 67,341-346, 1991

7.

Vogel, G. and Spanuth, E. Predictive value of fibrin monomers in postoperative deep vein thrombosis. K/in. Wochenschr. 68, 1020-l 026, 1990

8.

Godal, H.C. and Abildgaard, U. Gelation of soluble fibrin in plasma by ethanol. Stand. J.Haematol. 3,343-350,1966

9.

Lipinski, B. and Worowski, K. Detection of soluble fibrin monomer complexes in blood by means of protamine sulphate test. Thromb.Diath.Haemorrhag. 20,44-49, 1968

10.

Watanabe, K. and Tullis,J.L. Ristocetin precipitation test. A new simple test for detection of fibrin monomer and fibrin degradation products. Am.J.C/in.Patho/. 70,691-696, 1978

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