Scand. I . Haemat. (1973) 11,204-209

Quantitation of Plasma Fibrinogen in the Presence of Fibrinogen Degradation Products

HARALD ARNESEN, M.D.

Haematological Research Laboratory (Chief, H . C. Godal), Department IX (Chief, K . Aas), Ullevdl Hospital, University Clinic, Oslo, Norway

Three methods for fibrinogen determination in plasma were compared as to their reliability in the presence of fibrinogen degradation products (FDP). The method of Clauss gave erroneously low values, the semiquantitative method of Schneider was less influenced by FDP, whereas the method of Jacobsson gave fairly accurate results. Additional experiments suggested that dilution of the test plasma was responsible for the very low values obtained with the method of Clauss, whereas the final pH of 6.4 in the method of Jacobsson was found to be critical for the limited effect of FDP in this assay.

In the presence of fibrinogen degradation products (FDP), the thrombin clotting time is prolonged, and the polymerization of fibrin is impaired (Alkjaersig et al 1962, Amesen & Godal 1973, Bang et al 1962, Godal & Helle 1963, Hirsh et al 1965). Fibrinogen assays based on clot formation, therefore, may give erroneous results in .the presence of FDP. To evaluate a fibrinolytic state correctly, genuine fibrinogen and FDP, should be assayed separately. Consequently, fibrinogen assay methods, which are not af- fected by the presence of FDP should be aimed at. In the present investigation, the influence

of FDP on the results obtained with three different methods, widely used for routine purposes (Schneider 1952, Jacobsson 1955, Clauss 1957), was studied. As considerable discrepancies between the methods were observed, additional experiments were per-

formed to elucidate the basis for these variations.

MATERIALS AND METHODS

Patients on urokinase treatment. 29 consecutive urokinase-treated patients were part of the European urokinase trial in myocardial infarc- tion (1972). Samples were taken at regular inter- vals 4 times during the 18 h treatment in all patients.

Citrated plasma. 9 vol of blood were collected into precooled (+ 4 O C) plastic tubes containing 1 vol of 0.1 M sodium citrate. Plasma was pro- cessed by centrifugating 2000 g for 20 min at + 4 O C and was used fresh or after stored deep- frozen at -200 C for less than 3 months.

Fibrinogen. Purified human fibrinogen, more than 90 % clottable (Jacobsson 1955), and con- taminated with plasminogen, was obtained from Kabi, Stockholm, Sweden. Solutions containing 12 mg/ml were dialysed against 200 vol of (9 parts of 0.3 M NaCl and 1 part of barbiturate buffer pH 7.4) and stored at -20° C. Before use, the solutions were thawed and diluted to desired

FDP AND FIBRINOGEN DETERMINATION 205

concentrations with distilled water and barbiturate buffer, ionic strength and pH being adjusted to 0.15 and 7.4 respectively.

Urokinase. Kindly supplied by Hoffmann-La Roche, Basle, Switzerland. Vials containing 20,000 CTA-U were dissolved in phosphate buffer pH 7.6 to a concentration of 2000 CTA-U/ml, and stored at -2OOC.

Thrombin. Bovine thrombin, Topostasine, Hoff- mann-La Roche, Basle, Switzerland, was dissolved in 0.15 M NaCl to a final concentration of 30 NIH-U/ml, and stored in lusteroid tubes at -2OO C. Further dilutions were made with 0.15 M NaCl and kept at +4O C during the experi- ments.

Soybean trypsin inhibitor (STI), type I-S from Sigma, St. Louis, Mo., USA, was dissolved in phosphate buffer pH 8.0 to a concentration of 10 mg/ml, and stored at -20° C. Final concentration in all experiment: was 0.4 mg/ml.

Immunoelectrophoresis was performed accord- ing to NilChn (1967), using rabbit antihuman fibrinogen antiserum (1 %) in the agarose gel.

Rabbit antihuman antiserum against fibrinogen was obtained from Behringwerke, MarburgLahn, Western Germany.

Fibrinogen determinations were performed ac- cording to Schneider (1952), Clauss (1957) and Jacobsson (1955), the latter as modified by Blom- back (1958) and Godal (1961).

Thrombin clotting times were performed using 0.2 ml of preheated (37O C) test sample or control plasma and 0.1 ml of thrombin (3 NIH-U/ml) at 370 c.

Barbiturate buffer p@ 7.4 containing 0.74 % NaCl, ionic strength 0.15 (Owren 1947), was used.

Phosphate buffer pH 7.6. 4.00 g NaCl, 7.15 g NazHP04, 1.30 g KHzPO4 dissolved in 900 ml distilled water. pH was adjusted to 7.6 with 1 N NaOH and the vol to 1000 ml with distilled water.

Phosphate buffer pH 6.1. 4.30 g NaCI, 6.00 g NaHzP04, 1.90 g NaaP0.1 dissolved in 1000 ml distilled water.

Sephadex G-200, from Pharmacia, Uppsala, Sweden, was used. Gel filtration was performed at room temperature in a 5 x 55 cm column with 1.0 M NaCl, 0.05 M Tris-hydrochloric acid buffer at pH 8.3. The flow rate was 30 mlh, sample size 20 ml (about 10 mg/ml), and eluate aliquots about 5 ml. STI was added to the elution solution to prevent further proteolysis.

Purified FDP. Fibrinogen was incubated with urokinase at 37O C. Purified early FDP were ob- tained from early stage I1 FDP (according to Marder et a1 1969), giving maximal prolongation of a standard thrombin clotting time, and purified late FDP (D+E) from stage 111 FDP. Purification was performed on Sephadex G-200. The peak frac- tions were subjected to concentration and dialysis, assuring pH 7.4 and ionic strength 0.15. Protein concentration was calculated from optical density at 280 nm using extinction coefficient 16 as for fibrinogen.

Inclusion into clots of FDP, and their influence on fibrinogen determination according to Jacobs- son, at various pH. 1 vol of citrated bovine plas- ma (fibrinogen conc. 4.5 mg/ml), 1 vol of purified early FDP, (D+E) or normal saline, and 4 vol of phosphate buffer pH 6.1 or 7.6, were clotted with 2 vol of thrombin (30 NIH-U/ml) for 2 h at room temperature. The clots were thoroughly syneresed on a wooden rod and dissolved in 10 ml of alkaline urea for spectrophotometric deter- mination at 280 nm.

Inclusion of FDP into clots was calculated from immunoelectrophoretic quantitation of anti- human fibrinogen reacting antigens in the clot liquors, using standards from purified early FDP or (D +E) respectively.

Bovine plasma gave no precipitation with the antihuman fibrinogen antiserum used.

RESULTS

Fibrimgen determination in patients on uro- kinme treatment. 28 of 116 samples in 15 of 29 patients gave unmeasurably low values with the method of Clauss. According to the method of Jacobsson, the mean value in these samples was 97 mg/100 ml (42-154), and the thrombin clotting time was pro- longed from 15 to 32.5 s (23-48) as a mean. None of the patients showed clinically im- portant bleeding tendency.

Fibrinogen determinations in plasma added various amounts of purified early or late FDP. As shown in Figure 1, the Clauss method gave decreasing values with in-

206 HARALD ARNESEN

creasing amounts of early FDP. When the concentration of FDP was increased to about 2/3 of that of fikinogen, unmeasurably low estimates were obtained. The semiquan- titative method of Schneider gave decreased yield with increasing amounts of FDP. The method of Jacobsson gave fairly accurate results.

In a similar experiment with products (D+E) (Figure 2), the same tendency, al- though less striking, was encountered. When the concentration of (D+E) was about twice that of fibrinogen, giving a prolongation of the thrombin clotting time from 21 to 59 s, the Clauss method gave only about !A of the original value, while the Schneider and Jacobsson methods remained unaffected.

The influence of plasma dilution on the thrombin clotting time in the presence of FDP. As shown in Table I, prolongation of the thrombin clotting time during serial dilu- tion of plasma was much more pronounced in the presence of FDP.

The influence of FDP on fibrinogen deter- mination according to Jacobsson, at various pH. The use of bovine plasma, not reacting

t* Schneider titre

-A Clauss method Jacobsson method

F in solution ...... I 34 x----.x Thrombin time 2c

I I 25 5'0 7b 160

Early FDP(fina1 conc)(mg/lOOmi)

Figure 1. Fibrinogen determinations and thrombin clotting time in plasma (2 vol) added various amounts of purified early FDP (1 vol).

TABLE I The effect of dilution on the thrombin clotting

time of plasmas containing fibrinogen (F) or fibrinogen/FDP respectively

Thrombin concentrations were selected to give the same clotting time in the two plasmas

prior to dilution Serial dilution with barbiturate buffer pH 7.4

135 13 136 92 13 68 20 68 46 24 34 30 34 23 60 17 49 17 12 >I80 8 >180

with antihuman fibrinogen antiserum, and FDP from human fibrinogen, permitted the estimation of non-included FDP in the clot liquors by immunoelectrophcwesis. As shown in Figure 3, the inclusion of early FDP was markedly higher at pH 7.65 than at pH 6.4 ( 4 0 4 5 % and 20-30 % respectively). The inclusion of products (D+E) was very small at both pH tested, but regularly higher at pH 7.65 (Figure 4).

At pH 6.4, the estimated fibrinogen was practically unaffected by FDP (Figures 3 and 4). At pH 7.65, low concentrations of early FDP gave slightly increased values,

*-* Schneider titre C-. Jacobrson method

I I 1 5b 100 150 260 250 360

Roductr, (D*E)(final c o w ) hg/lOOmi)

Figure 2. Fibrinogen determinations and thrombin clotting time in plasma (2 vol) added various amounts of products (D+E) (1 vol).

FDP AND FIBRINOGEN DETERMINATION 207

whereas with higher concentrations the val- ues dropped markedly (Figure 3). In the presence of products (D+E), the yield was practically unchanged, except with very high concentrations (Figure 4).

DISCUSSION

In the method of Clauss, the fibrinogen con- tent is reflected by the thrombin clotting time of diluted plasma. Using high concen- trations of thrombin, the method is un- dected by the presence of thrombin in- hibitors, but is sensitive to factors which influence fibrin polymerization. As the anti- coagulant effect of FDP is largely due to polymerization inhibition (Alkjaersig et a1 1962, Arnesen & Godal 1973), their pres- ence should give erroneously low fibrinogen values with this method. The present find-

I la75 3% 75 150 360

Purified Early FDP (mg1100ml)

Figure 3. Inclusion into plasma clots (from bovine plasma) of purified early FDP, and their influence on the determination of clottable proteins at various pH. Inclusion of FDP calculated by im- munoelectrophoretic quantitation of anti-human fibrinogen reacting antigens in the clot liquors, using standards of purified early FDP.

log scale

ings are in agreement with this concept. It has been suggested that the influence

of FDP on the method of Clauss may be useful in the evaluation of the actual haemo- static defect in fibrinolytic states (Donati et a1 1970). As shown above, about half of the patients treated with urokinase were found to have unmeasurable low fibrinogen values with the method of Clauss, but no clinically important bleeding tendency. Thus, the im- portance of FDP for the actual haemostatic defect as judged by the Clauss method is probably overestimated. The present find- ing that dilution of plasma increases the influence of FDP on thrombin clotting times, may contribute to explain the very low values obtained by the Clauss method in fibrinolytic states.

The semiquantitative method of Schneider

pH 6.4

1 37.5 75 150 360 660

Products(D+E)(mg~OOml) log scale

Figure 4. Inclusion into plasma clots (from bovine plasma) of products (D+E), and their influence on the determination of clottable proteins at various pH. Inclusion of FDP calculated by im- munoelectrophoretic quantitation of anti-human fibrinogen reacting antigens in the clot liquors, using standards of products (D+E).

208 HARALD ARNESEN

is also based on plasma dilutions, but an impaired polymerization is counteracted by prolonged incubation of the clotting mix- ture. Further, coagulation is carried out at room temperature, where polymerization in- hibitors are probably less effective than at higher temperatures (Godal 1960). Accord- ingly, this method gives measurable results in the presence of even high concentrations of FDP (Figure 1).

In the method of Jacobsson, fibrinogen is determined spectrophotometrically as fibrin, after moderate dilution of plasma with phos- phate buffer, giving a final pH of 6.4, and coagulation with thrombin at room temper- ature. In the presence of FDP this assay might theoretically be influenced by reduced fibrinogen clottability or by incorporation of FDP into the clot. Nilkhn (1967) found the incorporation of early FDP into plasma clots (pH about 7.8) to be approximately 50 %, whereas a much smaller degree of incorpo- ration (about 10 %) was obtained with late FDP. In the present study (Figure 3), 20- 30 % of early FDP was included into the clots at pH 6.4, but the fibrinogen values were virtually un,afEected, implying that equal amounts of fibrin must have been. kept soluble. By increasing the final pH to 7.65, reduced fibrinogen clottability gave ’m- accurate estimates, more pronounced with increasing amounts of FDP. What is then probably of main importance for the fairly exact results obtained with the method of Jacobsson in the presence of FDP, is the final pH of 6.4 in this assay.

The present model for calculation of the degree of incorporation of FDP into clots, using an immunoelectrophoretic procedure, necessitates the use of a species different fibrinogen as substrate. Reservations must thus be taken for species differences regard- ing fibrinogen polymerization.

For routine purposes a rapid and reli- able assay should be preferred. The Clauss method is rapid, but unreliable in the pres- ence of FDP. The method of Schneider is semiquantitative, but quickly done and only moderately influenced by the presence of FDP. The method of Jacobsson is more time consuming, but is accurate and virtually un- influenced by the presence of FDP within a range of concentrations.

In the author’s opinion, the haemostatic defect caused by FDP is probably best evaluated with the thrombin clotting time of undiluted plasma. When accurate es- timates of fibrinogen are required, the method of Jacobsson is recommended.

ACKNOWLEDGEMENTS

The skilled technical assistance of Mrs. Lise-Mette Aamodt and Mrs. Renate Ruyter is gratefully ac- knowledged.

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FDP AND FIBRINOGEN DETERMINATION 209

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Accepted for publication April 26, 1973.

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(Stuttg.) 18, 3-14.

Correspondence to

Dr. Harald Arnesen Haematological Research Laboratory Department IX, Ullevll Hospital Oslo, Norway

Scand. J . Haemat. (1973) 11 14