THROMBOSIS RESEARCH Printed in the United States

Vol. 91.~~. 1-8, 1976 Pergamon Press, Inc.

METHODS FOR CORRECTING INHIBITORY EFFECTS OF FIBRINOGEN DEGRADATION PRODUCTS IN FIBRINOGEN DETERMINATIONS

Barbara M. Alving and William R. Bell Division of Hematology, Department of Medicine

The Johns Hopkins Medical Institutions Baltimore, Maryland, U.S.A.

(Received 23.4.1976; in revised form 3.5.1976. Accepted by Editor M.I. Barnhart)

ABSTRACT To assess the influence of fibrinogen degradation pro- ducts (FDP) on fibrinogen determinations, Clauss and Ratnoff-Menzie assays were performed with different con- centrations of human plasma and FDP. When varied con- centrations of plasma were mixed with an equal volume of FDP at a final titer of 1:512 (tanned red cell assay), fibrinogen levels were decreased by approximately 30% in the Ratnoff assay. Inhibition was overcome by clotting the fibrinogen in a smaller volume of saline. Inhibition was also corrected by clotting more concentrated mix- tures of plasma and FDP with thrombin. The influence of FDP in fibrinogen assays is significant when low levels of fibrinogen are measured.

INTRODUCTION The Clauss and Ratnoff-Menzie assays are widely used for

fibrinogen determinations in clinical coagulation laboratories. In the Clauss method, the fibrinogen concentration is estimated by determining the rate at which clotting occurs after the addi- tion of a standard solution of thrombin (1). In the Ratnoff- Menzie technique, which is not rate-dependent, fibrinogen is clotted by an excess of thrombin; the fibrin concentration is measured by a protein assay (2).

Fibrinogen degradation products (FDP) cause falsely low fibrinogen levels in rate-dependent assays because of their anti- thrombin and anti-polymerization activities (3,4,5,6). Fibrino- gen cannot be measured accurately by the Clauss method in plasma

1

2 FIBRINOGEN ASSAYS/FDP CORRECTION Vol.9,No.l

of patients with disseminated intravascular coagulation (DIG) be- cause FDP may be present in high titers (7). Accurate assays for fibrinogen measurement are essential in experimental and clinical studies of the effect of FDP on fibrinogen metabolism.

The effect of FDP in the Ratnoff-Menzie assay has not been described. The purpose of this study was to determine the in- fluence of FDP in both techniques and to explore ways of over- coming any observed inhibition. Ancrod, a purified coagulant enzyme from the crude venom of the Malayan pit viper, was com- pared to thrombin as a clotting agent. Ancrod cleaves only fibrinopeptide A from fibrinogen and does not activate Factor XIII (fibrin stabilizing factor) or plasminogen (8). The mechan- ism by which ancrod clots fibrinogen is different from that of thrombin. Therefore, the possibility that clotting of fibrinogen by ancrod might not be inhibited by FDP was explored.

MATERIALS AND METHODS ACD plasma. Nine volumes of human blood were collected into class tubes containinu one volume of acid citrate dextrose (0.73 gm citric acid, 2.2 gm sodium citrate, and 2.45 gm dextrose in each 100 ml. Travenol Laboratories, Inc., Morton Grove, Illi- nois). Plasma was obtained after the blood was centrifuged for 10 minutes at 1,100 g at room temperature and was used the same day or stored overnight at 4O C. Thrombin. Topical thrombin of bovine origin (Parke, Davis and Company, Detroit, Michigan) was diluted with barbital buffer to a concentration of 100 units/ml. Small aliquots were then stored at -20° C. Streptokinase. Streptokinase (Streptase, Hoechst Pharmaceutical Companv, Somerville, New Jersey) was obtained in vials of loo;oo~-I.u. The enzyme was diluted to a concentration of 20,000 units/ml in sterile H20. Barbital buffer. 2.05 gm sodium diethylbarbiturate, 2.75 gm diethylbarbituric acid, and 7.3 gm sodium chloride were dissolv- ed in one liter of distilled water and heated gently. The pH was 7.5 f 0.1. EACA-CaC12 thrombin. A solution containing equal volumes of 0.3 PT epsilon-aminocaproic acid (EACA), 0.075 M calcium chloride (CaC121, and thrombin at a concentration of 10 units per ml of phosphate citrate buffer was used to clot plasma prior to FDP determinations. The phosphate citrate buffer (pH 6.4) contained one volume of 0.15 M primary sodium phosphate and one volume of 0.1 M sodium citrate. Measurement of FDP. Fibrinogen degradation products were measur- ed by the tanned red cell hemagglutination inhibition immuno- assay (TRcHII), as described by Merskey, et al. (9). Normal values in this laboratory are titers of 1:4 or less. Ancrod. Ancrod was obtained as Venacil (Abbott Laboratories, North Chicago, Illinois) in concentrations of 100 units/ml. One unit of ancrod is that amount of material that will clot a stan- dard solution of fibrinogen at 37O C in the same time as one NIH unit of thrombin.

Vol.9,No.l FIBRINOGEN ASSAYS/FDP CORRECTION 3

FDP. FDP were made by incubating fresh human ACD plasma with streptokinase (5,000 units/ml plasma) at 37O C for 90-120 minutes. The digest was mixed with an equal volume of thrombin EACA-CaC12 solution for 2 hours at O" C to remove any remaining early clot- table products and to prevent continued digestion. The solution was then centrifuged for 10 minutes at 1,100 g; the supernatant was removed and measured for FDP concentration by the TRCHII. Titers between 1:1,024 and 1:2,048 were obtained consistently.

Clauss technique. 0.2 ml of plasma was mixed with equal volumes or FDP in increasing concentrations in a series of test tubes; a 0.1 ml sample from each tube was then diluted 1:5 to 1:lO with barbital buffer. An 0.1 ml aliquot of the dilution was then mixed with 10 units of thrombin (10 units/O.1 ml) or 4.5 units of ancrod at 37O C. A platinum wire hook was used to detect the exact time of the first fibrin strand formation. The seconds required for clotting were converted to fibrinogen concentration according to a standardized curve.

Ratnoff-Menzie technique. Aliquots of fresh human plasma, un- diluted or diluted 1 2 : or 1:4 with barbital buffer were mixed with an equal volume of FDP. The final titer of FDP in this mixture was 1:512. One ml of each aliquot was clotted according to the Ratnoff-Menzie method, using 100 units of thrombin or 10 units of ancrod as the clotting agent. The sample was twirled for 3 minutes in a centrifuge tube containing 10 ml normal sal- ine (NS), one ml glass beads, and thrombin or ancrod. The tubes remained undisturbed for 10 minutes and were then centrifuged at 700 g for 5 minutes. The supernatant was removed and the sam- ples were washed twice more by addition of normal saline and centrifugation.

After the washing process, the fibrin was heated in 10% NaOH solution. A protein assay was then done, using tyrosine as the standard and Folin-Ciocalteu phenol reagent (2).

RESULTS

Effect of FDP on fibrinogen measurement in the Ratnoff- Menzie technique:

Figure 1 shows the effect of FDP on measurements of various fibrinogen concentrations.

In this experiment, FDP were constant at a final titer of 1:512. When fibrinogen was 116 mg/lOO ml of plasma in the absence of degradation products, the level apparently decreased to 90 mg/lOO ml of plasma in the presence of FDP, representing a 22% error (p c.10). At a control fibrinogen of 54 mg/lOO ml of plasma, the apparent level decreased to 34 mg/lOO ml of plasma in the presence of FDP. This 37% error was significant (p c.0025). A 42% error occurred when control fibrinogen was 31 mg/lOO ml of plasma.

Effect of initial clotting volume in the Ratnoff-Menzie procedure:

When control plasma was mixed with FDP at a final titer of

FIBRINOGEN ASSAYS/FDP CORRECTION Vol.9,No.l

Influence Of FDP On Varied Fibrinogen Concentrations FDP= I:512

120-

100 -

Dilutions Of Plasma

FIG. 1: Influence of FDP in the Ratnoff-Menzie procedure. The solid line shows fibrinogen levels in the absence of FDP. The interrupted line shows the apparent decrease of fibrinogen in the presence of FDP at a final titer of 1:512. The numbers in parentheses indicate the num- ber of separate determinations performed.

1:512, the fibrinogen level apparently decreased from 48 mg/lOO ml of plasma to 33 mg/lOO ml of plasma when 10 ml NS was the initial clotting volume. A similar error was found when ancrod was substituted for thrombin as the clotting agent, as seen in Figure 2.

Effect Of Volume In The Ratnoff Technique

60

Iml NS Control f

)ml NS 4ml NS 1P(l:512) FDP(k512)

cl Thrombin

LB Ancrod

FDP=I:512

FIG. 2: Effect of initial clotting volume in the Ratnoff-Menzie technique. The clear bars represent determinations made with thrombin. tions with ancrod.

The lined bars represent determina- Each value is the average for two

separate assays. The range is indicated by brackets. The diluent is normal saline (NS).

Vol.9,No.l FIBRINOGEN ASSAYS/FDP CORRECTION

The inhibitory effects of FDP in the presence of thrombin or ancrod were completely overcome when the initial clotting volume was 4 ml NS.

Effect of allowing prolonged clotting in the Ratnoff-Menzie procedure in the presence of FDP:

Plasma was allowed to clot with thrombin or ancrod in the presence of FDP 12-14 hours at 4O C prior to the first wash. The control fibrinogen was 62 mg/lOO ml of plasma in the stan- dard Ratnoff-Menzie technique. All samples clotted with ancrod for 12-14 hours were the same as control values, regardless of initial clotting volume or the presence of FDP, as shown in Figure 3.

Prolonged Clotting In The Rotnoff Technique

T 60

50

5 z? 40

.c

$ 30

s g 20

IO

0 _L

I( Iml NS IOml NS 4mlNS Control FDP(k512) FDP(l:512)

cl Thrombin

Ea Ancrod

FDP = I:512

FIG. 3: Effect of prolonged clotting in the Ratnoff-Menzie tech- nique. Clear bars show determinations made with ancrod. Each value is the average of two separate assays; the range is indicated by brackets. Control fibrinogen by the standard assay was 62 mg/lOO ml of plasma.

Thrombin-clotted samples were erroneously low, even in the absence of added FDP, with an average of 44 mg/lOO ml of plasma or 30% error. Regardless of clotting volume, the error remained essentially unchanged in the presence of FDP.

Effect of FDP on the fibrinogen measurement by the Clauss technique:

When samples of plasma containing fibrinogen at 90 mg/lOO ml of plasma and various concentrations of FDP were assayed, the fibrinogen levels appeared to decrease in the presence of in- creasing concentrations of FDP, as seen in Figure 4.

Inhibition could be overcome by clotting more concentrated solutions of plasma containing FDP. This was achieved by using a lower dilution with barbital buffer prior to the addition of thrombin. Ancrod was unsatisfactory as a thrombin substitute in this rate-dependent technique because the end point was not sharp, and the rate of clotting was prolonged.

FIBRINOGEN ASSAYS/FDP CORRECTION Vol.9,No.l

Clauss Technique

I 1 I I I I

VI6 i/32 l/64 VI28 l/256 l/512

Titer - FDP

FIG. 4: Apparent decrease in fibrinogen levels measured by the Clauss technique in the presence of increasing concen- trations of FDP. For each point the control fibrinogen was 90 mg/lOO ml of plasma. The dilution with barbital buffer was 1:5.

DISCUSSION

The present study shows that erroneously low levels of fibrinogen are measured by the Ratnoff-Menzie and Clauss techni- ques when FDP are present and fibrinogen is less than 100 mg/ 100 ml of plasma. Inhibition in both assays was overcome by clotting more concentrated plasma. This was achieved in the Ratnoff-Menzie assay by using an initial clotting volume of 4 ml NS instead of 10 ml.

Prolongation of time allowed for clotting in the Ratnoff- Menzie test did not overcome inhibition in the presence of FDP. In fact, fibrinogen levels were lowered by prolonged clotting, even in the absence of FDP. This may have been due to proteo- lytic properties of thrombin manifested over a period of hours or to contamination of commercial thrombin by plasminogen (10).

Ancrod clotted fibrinogen completely in the presence of FDP when small initial clotting volumes were used and when clotting was prolonged. Fibrinogen levels may not have decreased with prolonged clotting because the proteolytic activity of ancrod is less than that of thrombin (8). Ancrod was ineffective in the Clauss test in the presence of FDP; this may have been due to the short time period allowed for clotting.

Arnesen (3,4) has evaluated the influence of purified early and late human FDP in the Clauss and Jacobsson techniques. He found a greater inhibition of the Clauss by early FDP than by late FDP; the inhibition was overcome by clotting more concen- trated plasma.

Vol.9,No.l FIBRINOGEN ASSAYS/FDP CORRECTION 7

The Jacobsson technique allows clotting of fibrinogen to occur for one hour in a solution of phosphate buffer and throm- bin. Arnesen found inhibition by early FDP in this assay only when the buffer pH was 7.65 or greater (3). When the pH was 6.4, fibrinogen values were unaffected by the presence of early FDP. However, 20-30% of early FDP was incorporated into the clot, implying that equal amounts of fibrin remained soluble.

In the present study, the Ratnoff procedure was performed at pH 7.2-7.4. When clottable FDP were mixed with plasma, no decrease in fibrinogen level was found. This may well have been due to incorporation of FDP into the clot, as shown by Arnesen.

In clinical DIC and in exoerimental work concerninc fibrino- gen metabolism in the presence-of FDP, significant errors occur in fibrinogen measurement. Simple modifications in the Clauss and Ratnoff-Menzie assays can be done in order decrease the probability of falsely low fibrinogen values presence of high titers of FDP.

may both to in the

ACKNOWLEDGMENTS

Supported in part by research grant number HL-01601 from the National Heart and Lung Institute.

Dr. William R. Bell is a Hubert E. and Anne E. Rogers Scholar in Academic Medicine.

Ancrod was generously supplied as Venacil by Dr. Joseph F. Donahoe of Abbott Laboratories, North Chicago, Illinois, U.S.A.

REFERENCES

1.

2.

3.

4.

5.

6.

CLAUSS,A.: Gerrinnungsphysiologische schnellmethode zur bestimmug des fibrinogens. Acta Haematol. 17, 237, 1957.

RATNOFF,O.D. and MENZIE,C.: A new method for the determina- tion of fibrinogen in small samples of plasma. J. Lab. Clin. Med. 37, 316, 1951.

ARNESEN,H.: Quantitation of plasma fibrinogen in the pre- sence of fibrinogen degradation products. Stand. J. Haematol. 11, 204, 1973.

ARNESEN,H. and GODAL,H.C.: Studies on the thrombin clotting time. Stand. J. Haematol. 10, 232, 1973.

KOWALSKI, E.: Fibrinogen derivatives and their biologic activities. Semin. Hematol. 5, 45, 1968.

MARDER,V.S. and SHULMAN,N.R.: High molecular weight deriva- tives of human fibrinogen produced by plasmin. J. Biol. Chem. 244, 2120, 1969.

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7. COLMAN,R.W., ROBBOY,S.J., and MINNA,J.D.: Disseminated intravascular coagulation (DIG): an approach. Am. J. Med. 52, 679, 1972.

8. PIZZO,S.V., SCHWARTZ,M.L., HILL,R.L., et al.: Mechanisms of ancrod anticoagulation. J. Clin. Invest. 51, 2841, 1972.

9. MERSKEY,C., LALEZARI,P., and JOHNSON,A.J.: A rapid simple, sensitive method for measuring fibrinolytic split products in human serum. Proc. Sot. Exp. Biol. Med. 131, 871, 1969.

10. MILLS,P.A. and KARPATKIN,S.: The molecular components and fibrinolytic activity of human thrombin. Haemostasis 2, 181, 1973.