fibrinogen degradation products in plasma using a monoclonal antibody

THROMBOSIS RESEARCH 44; 715-728, 1986 0049-3848/86 $3.00 + .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

A LATEX

IMMUNOASSAY OF FIBRIN/FIBRINOGEN IN PLASMA USING A MONOCLONAL

DEGRADATION ANTIBODY

PRODUCTS

Massoud

Mirshahi, Jeannette Soria, Claudine Soria, Jean-Yves Perrot and Claude Boucheix Department of Internal Medicine, Hotel-Dieu, Place du Parvis Notre-Dame, 75101 PARIS CEDEX DC. Laboratories of Haematology and Biochemistry, Hotel-Dieu and H&pita1 Lariboisiire, PARIS. U.268, INSERM, Hbpital Paul Brousse, 94800 VILLEJUIF, FRANCE. (Received 6.5.1986; Accepted in revised form 6.8.1986 by Editor C.L. Arocha-PiEango)

ABSTRACT We have developed a latex immunoassay using an anti D antibody recognizes an neo monoclonal (F2C5) which epitope present in fragment D but which is hidden in intact fibrinogen and in early fibrinogen degradation products. This technique was applied directly to plasma of both healthy donors and patients, and was very shown to be convenient for clinical investigation, especially in emergency cases for diagnosis of intravascular coagulation. use of plasma samples The serum offers several advantages: it is not instead of since time consuming blood clotting is not required; overestimation cannot be it avoids when fibrinogen underestimation due to the totally clotted, and binding of nonclottable fibrin degradation products to during the clot clotting in vitro. This monoclonal antibody, which more with FbDP (expressed in reacts than fragment D, does not allow fragment D) with fibrin and fibrinogen degradation products to be this discrimination does not differentiated. However, for its clinical use since the level of seem critical fragment D neo remained within the normal antigen 12 cases of spontaneous or venous occlusionrange in induced hyperfibrinolysis. ____________________~~~~~~~~~~~~-~ Key words: Intravascular coagulation; Latex assay. 715

Monoclonal

antibodies;

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The presence of fibrin degradation products (FbDP) in blood samples is a good indication of disseminated intravascular coagulation (DICI Reliable (1). techniques using polyclonal antisera such as the latex particle agglutination assay (2) and the tanned red cell hemagglutination inhibition immunoassay (31, are available to measure the total nonclottable fibrinlogen) derivatives in serum. Notwithstanding their usefulness and general acceptance, the simple immunoassay of FOP (fibrin(ogenI degradation products) using antisera directed against fibrinogen or its degradation products, presents several basic defects, notably the inability to distinguish between fibrinogen (Fg) and FDP. When the fibri_ nogen from a patient is not totally clottable (due to dysfibrinogenemia, the presence of heparin or other inhibitors of fibrin formation), fibrinogen interferes with FDP determination in serum, leading to an overestimation. Furthermore, as reported by Whitaker et of fibrin derivatives may al (41, the concentration be lower in serum than in the corresponding plasma, because nonclottable fibrin derivatives bind to the fibrin clot when may plasma is allowed to clot in vitro. We have developed a practi_ cal and highly specific assay for FDP determination using latex particles coated with an anti fragment D neo antibody, in order to avoid the influence of fibrinogen. This antibody interacts with an epitope accessible in fragment D and to a larger extent with the D dimer from stabilized fibrin but does not interact with undsgraded fibrinogen fibrin monomers and early (Fg). fibrinogen degradation products. This assay sensitive enough for biolgical investigation of disseminated intravascular coagu_ lation, can be carried out directly on plasma. Consequently, this test offers several advantages over the usual test : it is suitable for cases since it is time consuming to emergency obtain serum devoided in fibrinogen, especially for completely patients under heparin therapy. Furthermore, using this test, the underevaluation of FDP levels due to occlusion of FDP into fibrin is eliminated. In this report, we present the technical details of the procedure and assess its sensitivity, specificity and its application to plasma and serum samples from selected clinical cases.

Plasma and serum donors_ : obtained Plasma was after 10 min centrifugation (3,000 g) of blood collected on 0.13 M sodium citrate (1 vol for 9 vol of blood). Serum was obtained by allowing 2ml of blood to clot with thrombin in the presence of calcium and amino caproic acid. After 1 hour incubation at 37-C. serum was collected and allowed to stand for an additional hour at 37’C. Plasma and serum samples were obtained from - 20 healthy volunteers (controls) - 29 patients presenting DIC : The tests used for routine investigation of DIC have been described by Caen et al (5) except for fibrin monomer complexes which were detected by the hemagglutination assay according to Largo et al (6) using

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Lab,FranceI. For many of these patients, FS test (Stag0 was clearly well compensated. The consumption coagulopathy FOP serum performed using latex particles coated assay was products anti fibrinogen degradation with polyclonal Ig (Thrombowellcotest). - 7 patients with hyperfibrinogenemia. - 1 patient with hyperfibrinolysis. The only anomaly detected suffering from an haemorrhagic tendency, was in this patient, fibrinolytic activity as shown by a shortening an increased of the euglobulin lyris time (30 to CO min) in the absence of occlusion and an increased lysis area on fibrin any venous hyperfibrinolysis was due to an increase in the plates. This biological activity of tissue plasminogen activator (tpAI (control : 0.26 from 0.1 to 0.6 ngfml and ngiml, ranging patient : 1.65 ngfml). TpA determination was performed actor_ ding to Ranby (7). -1 patient under thrombolytic therapy with Streptokinase (SKI(Hoechst lab.) for pulmonary embolism. Blood was collec_ ted during SK therapy, 12 hours after the beginning of treatment. At the time of examination, the fibrinogen level was 35 mg/lOO ml. -In 11 patients plasma was obtained from blood collected after venous occlusion (V.0.) For the V.O. test, a blood pressure cuff was inflated during 10 min. at the mean value of maximal and minimal blood pressure. After V.O., euglobulin lysis time was reduced to 30 min or less. FOP levels were determined on fresh, post V.O. plasma and also after 6 hours incubation at room temperature. - 5 patients presenting with severe rheumatoid arthritis and having a positive Waaler Rose test. Re: The following substances were used : purified fibrinogen (grade and plasmin (Kabi, Stockholm), Streptokinase L), plasminogen (Behring), tween 20, bovine albumin, orthophenylene diamine (OPD) (Sigma chemical Co St Louis). Peroxfdase-labelled goat immunoglobulin anti-mouse immunoglobulin was obtained from E.Y. Lab. (San Mateo). 96 Well polystyrene microtiterplates for ELISA (Dynatech). Latex particles u diameter) (Rhone-Poulenc). (0.3 Early and late cross-linked fibrin degradation products (FbDP) were prepared according to Gaffney et al (6,9) and their strut_ ture checked by SDS agarose electrophoresis according to Conna_ ghan et al The early degradation products were shown to (10). consist of a serie of high molecular weight (HMWI derivatives and of 0x0, XY, X0. DY and DO. The late degradation products contained a larger quantity of DO fragment and a smaller quan_ tity of HMW derivatives and fragment DY than the early degrada_ tion products -fragment D at a low concentration was also present in these products. Their concentrations are expressed in fragment 0 equivalent, taking into account the initial concen_ tration of fibrinogen. X and Y fibrinogen degradation products (FgDP) were a kind gift of Or Nieuwenhuizen (Gaubius Institute, Leiden). Their purity was checked by SDS polyacrylamide gel electrophoresis. D and E FgDP were obtained by plasmin digestion of Fg and separation by DEAE cellulose chromatography according to Nilhen (11).

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Fibrin monomers were obtained, according to Belitser et al (12) 1 ml of lOmg/ml fibrinogen with 2 units of thrombin by clotting presence of 1 mM EDTA. The clot was squeezed out, washed in the M NaCl and then dissolved in 4M urea (pll 7.5). 4 times with 0.15 Fibrin monomer concentration was determined by measuring the 0.0. at 282 nm. Production

and selection of monoclonal antibodies (McAb): Six week old BALBIC mice were immunized with 100 ug early FbDP dissolved in 0.3 ml 0.15 M NaCl and mixed with an equal volume l/3 of the suspension was injec_ Freund's adjuvant. of complete ted intraperitoneally and subcutaneously. An intravenous 2/3 of 100 ug FbDP was given on day 31. Four days booster injection sacrificed for the prepafation of spleen later, the mice were between 3x10 mouse immune Hybridization cell suspensions. spleen cells and 5x10 myeloma NSl cells were performed actor_ of Kohler and Milstein (13) modified by ding to the technique Each fusion product and Scheidegger (14). Fazekas de St Groth (Limbro Division, Flow Lab: Ayr_ was seeded in 5 microplates grown in selective medium. Hybridoma supernatants shire), and were tested between day 11 and day 13 by the immunoenzymological described previously (15) by using Fg. screening procedure as fragment E) and FbDP coated plates. In FgDP (fragment D and reactivity of the culture fluid with fibrin mono_ parallel, the fibrin monomers immobilized on poly_ mers was assessed using Stanislawski et al Supernatant styrene according to (16). culture fluids reacting with fragment 0, but not with fibrinogen the same procedure on fragment X and fragment Y were tested by Cells of the selected wells were transferred to coated plates. limiting dilution frozen. The macroplates, cloned and by selected clones were also injected intraperitoneally into mice were given an fluid: female mice to produce ascitic Balb/c intraperitoneal injection of 0.5 ml Pristane (Algrich .Lab, prior to the injection of 2.10 hybridoma Gillingham) 14 days cells.

.

.

.

. . McAbina assay : The capacity of fg, fragment D and FbDP in solution to inhibit McAb to fragment D coated plates was also mea_ the binding of assay was performed by a 2 step procedure sured. The competition as previously described (15). etitive

lnhibltlon

of

latex oarticle aaalutination assav : Latex particles were coated with selected Ig according to Singer and Plotz (17) by incubating latex particles with IgG in 0.15 M NaCl 0.1 M glycine, pH 8.35, for 4 hours at 37'C. The latex par_ then centrifuged the pellet kept in 0.15 M titles were and saline buffer containing O.ltl glycine, 10 mg/ml bovine albumin and lmglml sodium azide 8.35). Purified Ig were obtained (PH from ascitic fluid according to Boschetti et al (18). 50 ul of plasma or serum used at progressive dilutions performed in the same buffer as that used for latex particles (the starting dilu_ tion had to be l/5 in order to be in good conditions of pH and ionic strength for agglutination) were transferred onto a glass ul of a previously shaken latex suspension were added plate: 20 and the mixture stirred with a glass rod, thus forming a ring of

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Presence or absence of macroscopic agglutinain diameter. 2 cm tion was noted after gently rocking the plate for 6 min.. and the sensitivity of the reaction were deter_ The specificity mined using serial dilutions of fibrinogen, fragment X. fragment D and early or late FbDP solutions. Y, fragment

loISelection of the D neo antibodv used for the coatina of the latex parti(lg. 20. 21) : only C presented the characteantibodies tested, Of the 216 D neo antibody. These antibodies bound only to ristics of anti fragment D coated plates and not to fragment E, nor fibrinogen, activity was nor fibrin monomer coated wells. Immune peroxidase detected only on immobilized fragment D (0.0. more than 1.5 on D less than 0.05 on fibrinogen, fragment E or coated Plates and the antibody selected fibrin monomer coated wells). Furthermore, in this test, called F2C5, did not react with either fragment X or with fragment Y. The competitive inhibition binding assay was performed in order of reactivity between the D neo antibody to check the absence the steric conformation of and fibrinogen in solution, since from that of fibrinogen in may be different bound fibrinogen solution (15). Both fragment D and FbDP prevented the binding of plates. F2C5 to D-coated The inhibition curves generated with in fig 1. either fragment D or FbDP are shown O.D. 1.5 Fibrinogen

0.19 0.38 0.75

1.5

3

Antigen

6

12

in solution

25

50

-

(ug:ml)

log.scale FIG. 1 Inhibition of binding of the McAb F2C5 on immobilized t,agment by fibrinogen, fragment 0 and FbOP at different concentrations. In solution, observed with

D

fragment D had a lower inhibitory effect than that : 50 % inhibition was observed with 0 ug/ml FbOP

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of fragment D and 0.6 ug/ml of FbDP (corresponding to 0.6 ug/ml of fragment D). Fibrinogen, even at high concentrations is unable to inhibit the binding of F2C5 to fragment D coated wells. 2') FDP determination usinq F2C5 coated latex particles_ : Agglutination of F2C5 coated latex particles was observed with 1 whether this was early or uglml FbDP (fragment D equivalent), late FbDP and with 7 ug/ml fragment D. For FbDP determination in biological samples, we thus considered the sensitivity of F2C5 latex coated particles to be 1 ug/ml. and fragment Y, whatever Fibrinogen, fragment X the concentrainduce tion used, not the agglutination of these latex did particles. subjects, - In the 20 normal the fragment D neoantigen level determined in the plasma was less than 5 ug/ml, expressed in has to fragment D. It be noted that 5 ugfml of FbDP was the limit of the sensitivity of our test according to the fact that the first dilution of plasma used was 1/S. - In the seven hyperfibrinogenemic patients (fibrinogen concen_ tration above 700 mg/lOO ml, three being under heparin therapy). found to the FOP level was be less than 5 ug/ml, using F2C5 anti coated latex particles. On the other hand, using polyclonal D + E-coated particles, the serum FDP level of these patients normal (from was higher than 30 to 160 ug/mll but dropped to normal when the sera were incubated in the presence of 5U/ml thrombin for an additional hour at 37'C. - Twenty-nine patients with DIC were tested. The results of the for these patients are summarized in biological investigation were determined both in plasma using F2C5 table 1. FDP levels coated latex particles and in serum using anti D + E polyclonal plasma, the level of FOP was [g-coated latex particles. In always increased, as compared to normal plasma, and ranged from 0.92) was found between CO to 600 ug/ml. A good correlation (r the FDP levels in plasma using F2C5 and that obtained in serum. stressed that for Z patients in this It should, however, be of FDP had been previously noted in the group. an overestimation anti D serum, using polyclonal + E latex particles, and this when overestimation disappeared the serum was incubated for an additional hour in the presence of excess thrombin. The results expressed in table 1 are those obtained after the second incuba_ tion period. correlation Despite the good between the plasma level (latexF2C5) and the serum level (latex-polyclonal), in five patients (case n'3, 7, 15, 19. 211, the FDP level was in the upper limit of the normal in serum using latex particles sensitized range with polyclonal Ig whereas it was two to three (16-32ug/ml), times higher in the corresponding plasma. Differences observed between plasma and serum FDP levels may not appear very but significant one has to take into account that the range of normal values was much higher in serum (16-32ug/ml) than in plasma allowing the clear affirmation that (<5pg/ml) plasma FDP levels were elevated in these patients. In order to ??

1.

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150 130 160 50

170 220 200 130 90 200 130

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with

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Fg = Fibrinogen, S.C. = Soluble of chronic myeloid leukemia

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Thrombin Fg Cephalin clotting clotting (mg/.ll) time (set) time (set) Patient/Control

of

_________________________~~__~~~~~~

data

_______________________________________________-________~~_~~~~_~~~~~~___~~~_~~~~~~~~~~~~~~~~~~~~~~~~-~~~~~-~

12. 13. 14. 15. 16. 17. 16. 19. 20. 21. 22. 23. 24. 25. 26. 27. 26. 29.

11.

2. 3. 4. 5. 6. 7. 6. 9. 10.

Patients

Table 1: Underlying disease and main biological __________________________________-___________-__________________________

El

2 U

m

E

”z

.

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44,

No.

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analyse if the differences observed in these 5 patients were due to the samples tested or to the reagent used, serum FOP levels latex particles. The were also determined using F2C5 coated in table 1 show that the discrepancy between results presented the level of FOP in plasma and that of the corresponding serum was also observed using latex particles sensitized with F2C5.

DIC

Heparin

therapy or

FDP

SK

therapy

hyperfibrinogenomia

300

200

0

100

0

0

0 0

?? ?? ??

0 ??

0

A

“6 PATIENTS

FIG. Discrepancies and

serum

between FOP using

2

plasma FOP using the polyclonal

the latex

1 1 or n' 3, 7.

McAb latex assay assay before (0

OIC cases are patients after (A 1 addition of thrombin. 15, 19, 21 (see the text for comments of the figure).

(0

activity were hyper-fibrinolytic - Thirteen patients with These patients were divided into 3 groups: - In the patient suffering from a hemorrhagic tendency due to a hyper-fibrinolytic activity, the FOP levels both in plasma and in serum were below 5 pg/ml. the patient under SK therapy, the level of FOP in plasma, - In measured by anti 0 latex particles, was 12 pg/ml, neo McAb whereas the fibrinogen level was 35 mg/lOO ml. On the contrary, the level of FOP using polyclonal Ig latex coated particles, in the serum (315 )Ig/ml). Immunoelectrophoresis was very high

tested.

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performed on plasma (data not shown) detected only early fibri_ nogen degradation products. - In the 11 patients under venous occlusion, even when euglobu_ time had dropped to 39 min or even 5 min, FOP levels lin lysis fresh and in plasma after 8 hours determined both in plasma both cases below room temperature, were in incubation at 5ug/ml. Fig. 2 summarizes some discrepancies observed between plasma and serum FOP. - In the 4 patients presenting severe rheumatoid arthritis, an FOP found in plasma, since in the overestimation of may be any coagulation disorder, the FOP level was found to absence of be increased in 3 cases (respectively 30, 8 and 5ug/ml). DISCUSSION assay of The immunological fibrin (ogen) degradation products prominent in the laboratory diagnosis of OIC. Simple has become antifibrinogen or anti 0 + E FOP assays, using either polyclonal not allow distinction between fibrinogen and FOP. It is sera do thus impossible, practically, to measure FOP in plasma, and it is necessary to measure the FOP in serum. a cumbersome approach since one has to be sure that all This is the fibrinogen has been clotted and thus does not interfere with the FOP assay. However as reported here, the opportunity to perform the FOP on plasma exists - providing that a monoclonal 0 assay directly neo antibody is available. neoantibodies Polyclonal anti 0 have already been prepared to fibrinogen degradation products and unde_ differentiate between Theoretically, graded fibrinogen such antisera, after (22). fibrinogen adsorption to remove panspecific antibodies, could be applied to the measurement of fibrinogen degradation products in the presence of fibrinogen. In practice, these polyclonal anti_ useful because of the low titer of antibody which sera are not remains after fibrinogen adsorption. pro_ With this aim, McAb were raised against fibrin degradation selected by screening the cell culture ducts, and the hybridomas using a fluid supernatants solid phase enzyme immunoassay. 4 antibodies were anti 0 neo detected among the panel of McAb. These anti 0 neo McAb recognized an determinant antigenic expressed on fragment 0 or FbOP, but not in intact fibrinogen 21). F2C5 McAb was selected in an immunoenzymological (19, 20, competitive binding assay because it was shown to interact pre_ rather than with the fragment 0 obtained ferentially with FbOP from plasmin digestion of fibrinogen, similarly to the antisera Budzynski et reported by al (23) and by Ciernewski et al (24) because it did not react with early FgOP. As observed and also immunoenzymological in the assay, using latex agglutination, F2C5 reacted more strongly with FbOP than with fragment 0 and did not react with fragments X and Y. Enzymoimmunological assays are becoming popular but these tech_ niques are not convenient for emergency cases. The latex proce_ dure using the McAb F2C5 developed here, should become an useful the common FOP assay, since our test is sensi_ alternative for

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tive enough (lug/ml in fragment D equivalent) for clinical investigation in an emergency. It should be noted that since the McAb F2C5 recognizes a single epitope on monomeric fragment D, agglutination of F2C5-coated latex particles by fragment D can only be induced by a modification of the charge of latex parti_ cles due to the specific binding of the fragment 0, and not to a clustering of latex particles which would require the presence of several epitopes on fragment D (25). study, it appears From our that the application of a D neo McAb latex assay to plasma is of great interest : the applica_ tion of this method using F2C5 for the study of patients presen_ ting consumption coagulopathy is illustrated by the analysis of our 29 patients (table 1) who had circulating soluble complexes action of thrombin. indicating the Some of these patients pre_ sented a well compensated DIC syndrom since they had a platelet count and/or a fibrinogen level normal or moderately decreased because of an inflammatory process and since factor V was not always decreased due to the fact that this process had been eve_ luting for a long time. can be concluded that this technique results. it From these 1 - the test is very convenient in emer_ offers many advantages: it is time saving (blood clotting is not required); gency since the underestimation of FDP as observed in 5 pa_ 2It avoids tients (cases 3, 7, 15, 19, 21). In these cases FDP levels were in the upper limit of the normal range in serum using polyclonal they were increased in the plasma using McAb sensitized Ig. but As FDP serum levels were identical using either latex particles. the monoclonal latex assay, it was concluded the polyclonal or these patients probably contained non-clottable that plasma of fibrin fragments which remained occluded in the clot or bound to fibrin when blood was allowed to clot in-vitro, as already shown due to a by Whitaker et al (4); 3 -it also avoids overestimation advantage was demons_ coagulation. This defective or delayed trated in 4 of the 29 DIC cases studied and in the 7 seven cases we have demonstrated these cases, of hyperfibrinogenemia. In second incubation of serum in the presence of additional that a Latex assay, in thrombin was required, when using the polyclonal order to avoid this overestimation. discrimination between fragment D and FbDP does not Furthermore, since even in severe hyperfibrinolysis as be important, seem to patients with hyperfibrinolytic activity (1 pa_ observed in 12 and severe hyperfibrinolysis and 11 pa_ spontaneous tient with activity related to a release of hyperfibrinolytic tients with venous occlusion), no fragment D had been detected since tpA by plasma FDP levels were normal when using F2C5. This indicates an fibrinogen degradation or a non-extensive degrada_ absence of F2C5 does not react with early FgDP. Despite hyper_ tion, since FDP in plasma is the value of fibrinolytic activity, normal that fibrinogen is not degraded (or not the fact explained by because on one hand tpA. the most extensively degraded), the activator, low affinity for important plasminogen has a very absence of fibrin plasminogen in the (26). and on the other 2 anti plasmin is a very potent and immediate inhi_ hand, alpha plasmin in plasma and thus prevents extensive bitor of (27) extensive absence of fibrinogen degradation in plasma. The

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fibrinogen after an important release of tpA was degradation of also noted by testing plasmas of good responders obtained before venous occlusion by a very sensitive Elisa test (cap_ and after immobilized F2C5 McA6, and then FDP on polystyrene ture of FDP using polyclonal anti D Ig labelled with pero_ detection of lysis 12 cases tested, even if the euglobulins In t,he xidase). 5 min. after venous occlusion, plasma FDP were time dropped to identical whether the plasma came from blood collected before or occlusion (results not shown). The increase in FDP after venous was only detected in serum when blood was allowed to clot in the aminocaproic acid and aprotinin (results not shown). absence of that the differentiation between fragment D rela_ This suggests ted to fibrinogenolysis and fragment DD related to fibrin degra_ dation is not very important for clinical investigation since it appeared that degradation of fibrinogen is never so extensive and never reaches the stage of fragment D formation. This diffe_ rentiation between fragment D and fragment DD might be perhaps of importance for the monitoring of thrombolytic therapy. With this aim, Whitaker et al (41, Rylatt et al (28) and Perry and Gaffney (29) pointed out the interest of using McAb specific for D dimer and cross-linked fibrin derivatives. Using latex parti_ cles coated with F2C5, in one case of thrombolytic therapy by SK, despite the severe degradation of fibrinogen (fibrinogen level in plasma dropped from 360 mg to 35 mg/lOO ml in 8 hours), the level of FDP in plasma using F2C5 coated latex particles was only moderately increased and this increase might be due to the thrombus lysis. reactivity The low of FgDP of this patient's plasma with F2C5 was due to the fact that only early FgDP, which do not react with F2C5, were found in patients under SK therapy as shown by immunoelectrophoresis. However, low quantities of fragment D related to fibrinolysis are not totally excluded, since immunoelectrophoresis is not very sensitive. Also Conna_ ghan et al technique for identifi(10). using a new sensitive cation of fibrinfogen) derivatives consisting of electrophoresis in the presence of sodium dodecyl sulfate followed by immunolo_ gical identification of the separated derivatives by autoradioto demonstrate presence of fragment D in grahy, failed any patients under urokinase therapy 2 hours after the beginning of but detected a small amount of fragment D after 24 the treatment hours.

It has

to be noted that our assay is affected by plasma with rheumatoid factor, as is the case using poly_ clonal Ig. It might be possible to avoid this overestimation by the use of Fab'2 instead of the whole F2C5 Ig. In conclusion, the use of the D neo McAb latex coated beads assay for the direct determination of FDP in plasma in cases of haemorragic disorders should prove to be a striking progress due to its rapidity and its reliability. Furthermore, using a D neo McAb which does not react with frag_ ment X and Y, this procedure .4llows the proc*sse:; of early fibrinogenolysis (as fibrinogen is undegraded or poorly degra_ ded) and fibrinolysis to be distinguished. This method may have obvious applications in the diagnosis and management of a varie_ ty of intravascular coagulation, above all in emergency cases. from

also

patients

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GFFRENCFS 1. MERSKEY C., JOHNSON fibrinogen-fibrin related &&LQQ$t 13, 313-315, 1971.

A.J.: The clinical antigen in serum.

significance of Stand. J Haemat.

ALLINGTON M.J.: Detection of fibrintogen) 2. degradation products by a latex clumping method. Stand. J. Haemat. u. IlS119, 1971. MERSKEY C., LALEZARI P., JOHNSON A._).: A rapid simple 3. sensitive method for measuring fibrinolytic split products. Proc. Sot. EXD. BioI.. Med. N.Y. u, 871-875, 1969. WHITAKER A.N., ELMS M.J., MASCI P.P., 8UNDESEN P.G., RYLATT 4. D.B, WEBER A.J., BUNCE I.H.: Measurement of cross-linked fibrin derivatives in plasma an immunoassay using monoclonal 882-887, 1984. antibodies. ,J. Clin. Path. z, 5. CAEN J., d'exbloration Paris, 1975.

SAMAMA LARRIEU M.J., et diaqnostic oratiaue.

M. L'himostase. Methodes L'expansion scientifique,

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