New nonimmunological method of measuring fibrinogen-fibrin degradation products

THROXBOSIS RESEARCH Printed in the united

Vol. 4, pp. 567-585, 1974 Pergamon Press, Inc.

States

NEW NONIMMUNOl0GICALMETHOD OF MEASURING FIBRINOGEN-FIBRINDEGRADATIONPRODUCTS A. E. Finkelstein,*B. Alexander, and F. Roisman Departmentof Rheumatology,FundacionCIMAE (Centro de Investigaciones Medicas Albert Einstein),Buenos Aires, Argentina, and the Coagulation Laboratory,New York Blood Center, New York, N. Y., 10021, U.S.A. + in revised form 28.2.1974. (Received 25.10.1973; Accepted by Editor S. Niewiarowski)

ABSTRACT

A new nonimmunologicalprocedure,called the Latex Fibrin Polymerization InhibitionTest, for the assay of serum fibrinogenfibrin degradationproducts (FDP) is described. This is based on the observationthat the aggregationof uncoated latex particles during the polymerizationphase of fibrin formation is inhibitedby the presence of FDP. It is sensitive,specific, and rapid. The results are expressed in PolymerizationInhibition Units (PIU). It has been applied to studies of FDP prepared in vitro and to assay of normal and pathologicalsera. The ratio between these results and those obtained with an immunologicalassay for FDP is helpful in the differentialdiagnosis of the presence of "early" versus "late" degradation products in sera of patients with elevated FDP. Introduction

The determinationof the quantity and quality of circulating fibrinogen-fibrindegradationproducts (FDP) is becoming increasinglyimportant(1,2).

*

PresentlyHead, RheumatologyResearch Department,CIMAE; Research Associate, New York Blood Center, while on leave of absence August 1, 1968July 31, 1969.

+

Supported by Grants USPRS HL-11447, USAMRDC Contract DADA 17-69-C-9022, and the Boris Garfunkel a Hijos (BGH) Fellowship,Buenos Aires, Argentina. Reprint requests: Dr A. E. Finkelstein,FundacionCIMAE, Luis Viale 2831, Buenos Aires, Argentina. 567

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Its main value is for the diagnosis and management of disseminated intravascular coagulation (DIG)*, otherwise known as the intravascularconsumption coagulopathy,described in abruptio placenta, intrauterinedeath, sepsis, blood transfusionincompatibility,pulmonary embolism,vascular thrombosis, post-partumhemorrhage,eclampsia and a host of other diseases. Plasminogen is converted by activators to plasmin, a protease which degrades fibrin or fibrinogeninto smaller fragments,which appear in the circulation. Some have a pronounced antithrombinactivity (3), and inhibit fibrin polymerization (4). This report is concerned with a new nonimmunologicprocedure for measuring FDP, based upon the observationthat uncoated latex particle aggregation, during thrombin-fibrinogeninteraction,is inhibitedby the presence of FDP in the reaction mixture (5). This new procedure is called the Latex Fibrin PolymerizationInhibitionTest (LFPIT). Sensitive and specific for quantitatingFDP, it has been applied to studies of FDP prepared in vitro, and in studies of many normal and pathologicalsera. When used side by side with the inrcunoassay procedure of Merskey, Lalezari, and Johnson (6), the LFPIT permits the differentiationbetween the "early" nonclottable fragments and the "late" components. The method is so simple as to make it valuable for screening suspicioussera, for diagnosis of defective heswstasis, and for closely monitoring thrombolytictherapy.

* Abbreviations: FDP, fibrinogen-fibrindegradationproducts; PIU, polymerization inhibitionunits; LFPIT, latex fibrin polymerizationinhibitiontest; LFPT, latex fibrin polymerizationtest; cu, caseinolyticunits; SBI, soybean trypsin inhibitor; N.I.H., National Institutesof Health; TRCRII, tanned red cell hemagglutinationinhibitionimmunoassay; SCT, staphylococcalclumping test; CA, cancer.

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Materials and Methods 1.

Purified human fibrinogen (95% clottable). Grade L, A.E. Kabi, Stockholm, Sweden, and Cutter Lab, Berkeley, California.

2.

Human Plasmin, A.B. Kabi, Grade A, containing 3 caseinolyticunits (cu)/ mg protein. The product was dissolved in 50% glycerol to contain 5 cuiml.

3.

Soybean trypsin inhibitor (STI), WorthingtonBiochemicalCorp., Freehold, N.J.; an aqueous solutionwas prepared to contain 20 mg/ml.

4.

Two forms of thrombin were used: a) Thrombin (Parke, Davis Co., Detroit, Mich., lyophilized),dissolved in 50% glycerol saline to contain 100 N.I.H. units/ml, and stored in aliquots at 4'C; b) Thrombin Reagent (ClinicalScience, Inc., Whippany,N.J., lyophilised),dissolved in distilled water to contain 1 or 10 N.I.H. units/ml, and used promptly.

5.

Fibrinogen for FDP Test (ClinicalSciences, Inc., Whippany, N.J.)

6.

Latex reagent, 0.8n (ClinicalSciences, Inc., Whippany,N.J.)

7.

Aminocaproicacid (Afibrin,250 mg/ml), (Lederle,Pearl River, N.Y.)

8.

Glycerine-salinebuffer (GSB), 0.1 ml glycine, 0.17 M NaCl, 0.0025 M NaOH, pH 8.2.

9.

Sera for FDP determinationswere obtained from 7-10 ml of venous blood drawn with a plastic syringe and disposableneedle, placed into tubes containing0.1 ml of a 10 N.I.H. units/ml of thrombin-calcium(0.025M CaC12) solution and 0.1 ml of Afibrin. The tubes were kept at room temperature for two hours, the evolved clot was gently freed with a wooden applicator,the tubes were spun at 1,000 xg for 10 min, and the sera removed. Some were tested promptly, others were kept overnight at 4OC. These were again centrifuged,and the supernatenttested. Other biological fluids (ascitic, synovial and pleural) were handled exactly as blood and the material obtained after clotting was kept overnight at 4'C, centrifugedand used.

-

-

570

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The tanned red cell hemagglutinationinhibition immunoassaymethod (TRCHII)was performed according to Merskey, Lalezari, and ;Tohnson(6), with rabbit antihuman fibrinogengenerously suppliedby Clinical Sciences, Inc., Whippany, N.J.

11.

The staphylococcalclumping test (SCT) was performed according to Iiawiger et. al. (7). The staphylococcalpowder was kindly provided by Dr. Hawiger.

12.

Immunoelectrophoresis was performed according to Scheidegger (8).

Preparation-of Fibrin DegradationProducts To 10 ml aliquots of 1.0 ml of a solution of fibrinogen (5 mg/ml in 0.5 M Tris buffer (pli7.4) kept at 4oC, 0.1 ml of cold (0-4OC) plasmin (5 cu/ml in 50% glycerol) were added, mixed, and put on ice. Iusnediately, 0.1 ml of thrombin (10 N.I.H. units/ml) was added and the mixture kept on ice until clotted. When clotted, the samples were warmed to 37'C, and to the tubes were added 0.1 ml of ST1 (20 mg/ml) at the following intervals:3', S', lo', 15', 18', 20', 30', 60', 120' and 24 hours. After 10' time interval,clot lysis was completed in all the tubes. Clots evident in the first two tubes (3' and 5') were removed, and protein content of the supernatantsof all the tubes were determined and diluted into aliquots to contain 1 mg/ml of protein each. The same procedure was followed to prepare FDP with decreasingconcentrationof plasmin: 2.5, 1.2, 0.6, 0.1 cu/ml.

Preoarationof FibrinogenDegradation Products To 10 aliquots of 1 ml of a solution of fibrinogen (5 s&ml), 0.1 ml of plasmin (5 cu/ml) were added and the mixture incubatedat 37'C. The digestion was stopped at selected intervalsby adding to the tubes 0.1 ml of ST1 (20 mg/ ml) at the following time intervals:O', 3', S', lo', 15', 18', 20', 30', 60', 1201, and 24 hours. The 0' time control containing fibrinogenin the LFPIT contrary to the TRCHII gives a_negative result because it is clotted during the procedure (see below). In order to separate nondigestedremaining fibrino-

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gen, to aliquots of each tube 0.1 ml of thrombin (10 N.I.H. units/ml) were added; the clots removed by centrifugation,and the protein content of the supernatantswere determinedand diluted to a concentrationof 1 mglml. To obtain many intermediatepoints between 0 and 20' of digestion,another series of fibrinogen-plasmin digestingmixtures were similarlyprocessed at 2-3 min. intervals. Latex Fibrin PolymerizationInhibition Test (LFPIT) -Procedure: 1.

Set up two series of 9 tubes each. The last two, labelled C1 and C2, serve as controls.

2.

Add 0.5 ml of the test serum (previouslyheated at 45OC for 15' to inactivate latex stabilizer),to the first tube of one row of tubes, and 0.5 ml of normal control serum (heated as the test serum) to the first tube of the second row; 0.25ml of glycerine buffer is added to the rest of the tubes.

3.

Make serial dilutionsup to tube No. 7.

4.

Add to each tube, except C2, 0.05 ml of plasma diluted with saline to contain 25 to S$,g/ml of fibrinogen.

5.

Then add to each tube 0.05 ml thrombin reagent containing 1.0 N.I.H. unit/ ml and iunaediately add 0.15 ml of latex reagent.

6.

Shake each tube to make sure the particles are evenly suspended,and incubate at 37'C for one hour. C1 and C2 are controls: C1 for the thrombinfibronogeninteraction(latex aggregationor clot), and C2 for the latex stability (even latex suspension).

7.

After one hour, compare each dilution of the test serum with the corresponding dilutionsof the control serum. The amount of FDP present in the test sample is reflected by the ultimate dilution of the serum sample at which fibrin polymerizationis inhibited,as evidencedby comparing the degree of latex aggregationwith the same dilution of the control sample.

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The

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degree of aggregationis expressed as C

2+, l+ and 2.

WITH LATEX

(clot),

F

-Jo1.4,No.4

(floccule),4+, 3+,

The degree of inhibition is expressed as Polymerization

InhibitionUnits (PIU), obtained by multiplying the highest dilution of the test senun sample at which inhibitiontakes place by 2.5 pg*, which is the fibrinogenconcentrationof the reaction mixture. If the last tube which inhibits aggregationcorrespondsto a dilution 1:16 of the test serum, the sample contains 16 x 2.5 = PIU/ml. If the next tube to the one which shows total inhibitionis partially inhibited as compared with the same dilution of the control serum, an average dilution value is obtained. For instance, if the last tube which presents complete inhibitioncorresponds to a dilution of 1:32, and the next 1:64 dilution gives a reading of 2+ as compared with a C value of the same dilution of the control, the following formula is applied: 32+64 2

~48;

48 x 2.5 - 120 PIU/ml

Results As already reported (S), uncoated latex particles aggregate during fibrinogen conversion to fibrin. This was called the Latex Fibrin Polymerization Test (LFPT). The degree is quantitativelyrelated to the amount of fibrinogen converted. The addition of the LFPT system of FDP, some of which inhibit polymerization,could inhibit aggregation in direct proportion,not only to their concentration,but also to their quality. The degree of inhibition would be evidenced by the quantity of nonaggregatedparticles present in the reaction muxture.

Thus, no, or relatively little, inhibitionwould be indicated

by no, or relative little change in optical density. The inhibitoryeffects of "late" FDP upon the LFPT are shown by optical density changes, as measured

* This concentrationwas found-opt_imal (see below in Discussion).

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by a PMQ II Zeiss spectrophotometer,and naked eye readings (Table I). As little as 0.025 ug/ml of FDP can still be detected in this system (Table I). Table I Latex Fibrin PolymerizationInhibition (As evidencedby optical density readings) Tube No.

1

2

3

4

5

6

7

Fibrinogen irgfml

0.4

0.4

0.4

0.4

0.4

0.4

-

FDP ;rg/ml

0.4

0.2

0.1

0.05

0.025

OD 600 rqr

0.78

0.72

0.62

0.56

0.55

0.44

1+

2+

2+

2+

3+

Naked Eye Reading *

-

0.1 0.80 -

* Aggregationof latex particles after 0.07 NIH units of thrombin added to each tube.

In order to study the effect upon the LFPIT of progressivefibrin and fibrinogendigestion by plasmin, aliquots of such digests (see procedures) were studied simultaneouslyby inmumoelectrophoresis, LFPIT, TRCHII and the of plasmin digests of fibrinogen. SCT. Fig. 1 shows the imrmnoelectrophoresis The difference in electrophoreticmobilitiesof the "late" digests as compared to the “early" products can be seen.

Fig.

1

Innnunoelectrophoresis of plasmin digests of fibrinogen. Incubation time noted in minutes and ON (overnight); cathode at top, anode at bottom of each slide. F= fibrinogencontrol. Migration time: 180 min at 3 ma/slide; buffer: RCl-veronal,pH 8.2; 50 ~1 of rabbit anti-human fibrinogenanti-serumadded to troughs.

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Fig. 2 shows the PIU values/ml obtained in relation to different digestion times of fibrinogenand fibrin, respectively.

PIU ’ ml

7000 6000 -

I III lo 20 30

0

40

II 50

60

1 70

’

60

“1

90

Digestion

100 HO 12Omin

24hs

time

PIU ml 7000

I

1000

-

=

11”““““’ 0

IO 20

It---@ I

30

40

50

60

70

60

90

Digestion

IO0 II0

120min

24hs

time

Fig. 2

Curves of the PolymerizationInhibitionUnits (PIU) per ml obtained by LFPIT in relation to plasmin digestion times of fibrinogen (above) and fibrin (below),respectively. Each value is the average of five determinations. _

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Fig. 2 shows that the 15' digests have the highest PIU/ml activity, which was further confirmed with intermediatepoints of digestionmixtures processed at 2'-3' intervals,between 0 and 20 minutes. This would indicate "early" (fragmantY) degradationproducts (9). With prolonged enzymatic digestion, PIU activity decreases until a constant value is obtained with "late" fibrin(ogen)degradationproducts. With decreasingconcentrationof plasmin in the incubationmixtures (2.5, 1.2, 0.6 cufml), prolonged incubationwas required to obtain the highest PIU activity (Fig. 3) expressed as a percentageof the highest titers obtained by the LFPIT.

Per cent of the highest titer t

151s

30’

dasmin

60' 1--_-I

1

CU/ml

-1

0.5

CU/ml

Izl

025 CU/ml

I-_-I

0.12 CU/ml

30-

0

10

20 30 40 50 60 70 80 90 min

Digestion

time

Fig. 3 Progressivelydecreasingamounts of plasmin required prolonged incubationperiods for same PIU activity.

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In Fig. 4 the values are expressed as a percentageof the initial titers obtained by the TRCHII and SCT, in assays of fibrinogendigests and fibrin digests at progressivedigestion times. As can be seen, the highest activity is also present in the "early" material. Percent of the initd A titer ‘00 w. 90 -

‘:. \

80 70 -

'4. .**....* TRCHII *..... *..* X.??..........._........................... *.,

,

60 50 40

-

30

-

20

-

0

SCT

K)

20

‘....

x. , .. I : k.,.

30

40

60

60

70

60

90

IO0 II0

120mm

*... “,

24hs

Digestion time

percent of the initiol titer 100 -a.. 90 -

l.

8070 60

0

-

SCT

10

20

*... .... '.... ..*._ TRCHII *... .-...... *.....

--,.

30

40

50

60

70

60

90

*... *...

‘...

“‘-y

. . . . . ..i ! . . . . .. . .. 4

100 110 120nnn

24hs

Digestion time

Fig. 4 Tanned Red Cell HexagglutinationInhibitionImnmoassay (TRCHII) and StaphylococcalClumping Test (SCT) titers obtained with digests of fibrinogen (above)and fibrin (below) by plasmin. Each value representsthe average.of-fivedeterminations.

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577.

When the results obtained on the same samples by the LFPIT were compared with those obtained by the TRCHII, a highly significantcorrelationwas obtained, dependingupon whether the digests were "early" or "late". As can by observed in Table II, the average ratio of PIU/TRCHII of "early" (< 18') FDP material is approximately3, whereas the "late" (60' - 24 hrs) digests the ratio approaches 1.

Table II PIU/TRCHIIRatios of "Early" and "Late" FDP of Fibrinogenand Fibrin Digests Fibrinogen Time of Digestion (Minutes) LFPIT (PIU/ml) TRCHII (&ml) PIU/TRCHIIRatios

3

5

10

15

18

20

30

60

2600 3000 4000 5180 3920 2520 1400 770 1400 1400 1400 1400 1400 1400 1000 700 1.8 2.1 2.8 3.7 2.8 1.8 1.4 1.1

120 - 24 hours 770 330 700 400 1.1 1.1

Fibrin LFPIT (PIU/ml) TRCHII (tie/ml) PIU/TRCHIIRatios

2000 3500 4000 6000 4625 3250 3000 2000 1250 1250 1250 1250 1250 1250 1250 950 1.6 2.8 3.2 4.8 3.7 2.6 2.4 1.6

600 500 1.2

600 500 1.2

More precisely, 1 bg/ml of "early" FDP is approximatelyequivalentto 3 PIU/ml for 10' to 18' fibrinogendigests, and for 5' to 20' fibrin digests (Table II). The range of PIU, as determined in the serum of 150 random healthy blood donors measured by the LFPIT, indicatedthat 91.4% contained less than 5 PIU; 5.2% contained 5 PIU; and 2.5% had 10 PIU/ml. To explore whether the "in vitro" ratios 3 PIU/l TRCHII and 1 PIU/l TRCHII for the "early" and "late" products respectivelyalso exist in vivo, the LFPIT was applied concomitantlywith the TRCHII to the study of sera from different diseases. As can be seen in Table III, patients with myocardial infarctionexhibit a ratio of 3, whereas other disorders significantlyshow l&er,or indeed higher,ratios. Nearly all

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five patients with malignant diseases showed a ratio of 1, suggestingdegradation products of "late" nature, (Table IV).

Table III

Incidence of PIU/TRCRIIRatios of Sample

LPPIT

D.I.C. (Acute Meningitis) Acute Myocardial Infarc. 1,

,I

PDP Present in Different Diseases

8,

Post-AbortionHemorrage

TRCHII

PIU/TRCHII

(PIU/ml)

(ug/ml)

750

240

3.1

25

8

3.1

50

16

3.1

60

20

3.1

30

10

3.0

75

24

3.1

50

16

3.1

120

48

3.5

Brucellosis

20

Incompat. Blood Transfusion

80

80

1

Thrombophlebitis

60

65

0.9

Septic IntrauterineDeath (J. F.)

800

486

1.6

Septic IntrauterineDeath (M. V.)

400

250

1.6

4.8

4.0

Intermediateratios (between 0.4 and 3.5) are seen in studies performed with biological fluids (Table V).

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Table IV LFPIT and TRCHII Assays in Neoplastic Diseases LFPIT

Sample

(PIU/ml)

TRCHII

PIU/TRCHII

(pg/ml)

B.L. Metastatic Breast CA (8/12)

100

96

1.04

B.L. Metastatic Breast CA (8/16)

50

24

2.08

V.F. Acute MyelogenousLeukemia

200

240

0.83

C.L. Acute ErythroblasticLeuk.

200

240

3.83

J.R. CA of Prostate (10/25)

400

300

1.33

J.R. CA of Prostate (10/28)

600

600

1.00

20

20

1.00

CA of Lung

Table V FDP in Ascitic, Synovial and Pleural Effusions as Assayed by the LFPIT and TRCHII LFPIT (PIU/ml)

Sample

L.C. Ascitic Fluid (Liver Cirrhosis)

TRCHII

PIU/TRCRII

Gglml)

480

158

3.5

A.R.

"

"

II

1,

1200

400

3.0

A.F.

"

,I

11

II

100

91

1.1

R.L.

"

"

II

,I

200

182

1.1

N.B.

"

"

I*

It

37

29

1.3

37

30

1.2

R.T. Synovial Fluid (RheumatoidArth.) D.C.

"

1,

,,

8,

100

60

1.7

D.G.

"

l,

‘,

8,

50

60

0.8

A.M. Pleural Fluid (CA of Lung)

37

77

0.5

L.G.

80

32

2.5

I,

I,

II

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Discussion One of the features characteristicof D.I.C. (1) or consumptioncoagulopathy (2) is the appearance in the blood of FDP derived from fibrinogenand fibrin digestion. several laboratoriesare presently engaged in the evaluation of simple, specific, sensitive,and rapid quantitativetests to measure FDP. Following the report of Nussenzweigand Seligman (10) that fibrinogen and fibrin digestions by plasmin releases fragmentswhich retain antigenic determinantsof the parent molecule, Ferreira and Murat (11) first applied inrsunological procedures for their detection. Merskey, Lalezari and Johnson

(6) described a more sensitive quantitativeimmunoassaymethod which is more widely used. Most of the tests for FDP

are based on immunologicalreactivity

and therefore must be considered in the light of the antigenicityof FDP vis-a-vis that of the parent material and other importantvariables as discussed in detail by Merskey,Lalezariand Johnson (6). Hawiger et al (7) published a very highly sensitiveand rapid serum test involved staphylococcalclumping. All these methods for measuring serum FDP are quantitativebut the procedures fail to characterizethem further. Recently methods for determininglate FDP by means of anti-fragmentE antibody were developed (12, 13). Finkelsteinet al(14) used uncoated particles to detect antigamma globulin factors that could be eluted from red cells. During studies performedby the same investigatorwith high dilutions of thrombin-defibrinatedred cell washings, it was observed that when uncoated latex particles are present at a given interval of time after fibrinogen-thrombininteraction,they become aggregated and incorporatedinto the evolved fibrin, otherwise invisiblewith the naked eye (5). The Latex Fibrin PolymerizationInhibitionTest is a system in which uncoated particles are used to detect and measure the presence of FDP through the specific inhibitoryactivify of the latter upon fibrin polymerizationCon-

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sequent to the action of thrombin. c FDP inhibit this aggregation. The data herein presented indicate tha, The degree of inhibitionis directly related to the amount and quality of FDP present in the test sample.

The highest dilution of the sample which

inhibits aggregation,multipliedby the fibrinogenconcentrationof the reaction mixture, measures the amount of FDP. This is expressed as Polymerization inhibitionUnits (PiU/ml). These reflect the amount of anti-coagulantaction of the FDP in the test material. Tests based on the inhibitionof the aggregationof latex particles coated with polymerizingfibrin are likely to be sensitive to trace amounts of other anticoagulantslike heparin and anti-thrombinV.

liowever, we have found

experimentallythat the addition of heparin to o given serum, in a dilution comparableto the dilution obtained "in vivo" after the administrationof the relativelyhigher doses of heparin used clinically,does not interferewith the LFPIT. With higher concentrationof heparin added "in vitrotrit is necessary to perfortn the test in the presence of protamine sulfate diluted in the glycinebuffer to a final concentrationof 3.75 mg. % The LFPIT using purified fibrinogenhas proven valuable for experimental studies of FDP produced in a'controlledand purified system. For practical clinicalpurposes, however, using oxalated plasma to provide fibrinogenwas found preferablebecause of its stabilityand ready availability. The fibrinogen concentrationis, however, critical and should be such that its conversionto fibrin by thrombin in the presence of the latex suspensionwill take from 45' to 1 hour + 15'.

This optimal "latex thrombin time" requires

a final fibrinogenconcentrationof 2.5 - 5 pg/ml. The best correlationwith the TRCHII was found under these conditions. Above 5 pgfml, readings were obtained faster (within 12 min.); below 2.5 ug/ml, more time was required. and the results were not reproducible.

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It has been shown by simultaneoususe of TRCHII and SCT that during progressive fibrinogendigestion the sensitivityof both proceduresdecreased (15). This is in accord with our observations (Figs.2 and 4).

With pro-

longed digestion the sensitivitydecreases also with the LFPIT. The results obtained in the sera of 150 random presumablyhealthy donors indicated that 91.4% contained less than 5 PIU/ml, which correlateswith the immunoassayprocedures. The elevated FDP in sera of patients with acute myocardial infarction (Table III) is not in accord with the report of others (16), a discrepancyrequiring further studies of patients with myocardial infarction, with special focus on the stage of the disease. Studies by the LFPIT, to be reported in a later conuaunication, on the sera of 106 patients with rheumatoid arthritis revealed elevated PIU in about 60%. *This was paralleled by an increase of FDP, as measured by the TRCHII. There was no evidence of elevated antithrombinV,

as described in

one unusual case (17). Furthermore,the PIU/TRCHII ratio in all the rheumatoid arthritis patients with elevated serum FDP levels, was 1.2, pointing out the "late" nature of the FDP present in this disease. Validation of the specificityof the LFPIT as a measure of FDP is supported by the observed significantcorrelationwith the TRCHII values, and by the immunoelectrophoreticprofile of the digests used in the present study (Fig. 1). Under certain experimentalconditions and in certain clinical disorders this correlation is approximately3 to 1 or 1 to 1. Although under the experimentalconditions of Table II at 60' the fibrin PIU/TRCHIIratio is similar to the 3' fibrin PIU/TRCHII ratio, when sera with a PIU/TRCHIIratio of FDP equal to 1 were tested simultaneouslyby immunoelectrophoresis, they all appeared to contain "late" products. It seems likely that the low PIU/TRCHII ratios in pathologicalsera are due to the late stage of digestion. Further studies continue in comparing the sensitivityof the LFPIT with

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the conventionalplasma thrombinclotting time. Since FDP have little or no anticoagulantactivity in the orthodox plasma clotting system, and the LFPIT measures FDP, there is a possibilitythat the simultaneoususe of the LFPIT and the thrombin time determinationcould indeed differentiatedegradation products derived from fibrinogenand fibrin. Whereas concurrentuse of LFPIT and TRCHII is necessary to characterize the u

of FDP, the LFPIT by itself will quickly indicate the presence of

abnormal FDP serum levels. Besides its simplicityit has special significance in that it mirrors in biologicalterms what occurs in vivo, and when expressed in PIU, gives meaningful informationof the pathologicalprocess. When the LFPIT's value as a diagnostictool for measurementof serum FDP levels is added to the value of existing tests, it opens a new avenue to explore further the physiopathologicmeaning of qualitativedifferencesof circulating fibrinogen-fibrinderivativesin different disease conditions.

Acknowledgements The able assistanceof Dolores Lawlor, formerly of the New York Blood Center, N.Y., and Marta Volosin, Silvia Bather, and Esther Vila, of Fundacion, CIM&lI,Buenos Aires, is gratefullyacknowledged. We express our deep gratitude to Dr. Clarence Merskey for his continuous stimulationand many constructivesuggestions. We are indebted to Dr. R. Smud, Hospital Fernandez, Buenos Aires, for the sera from patientswith incompatibleblood transfusion,post-abortion hemorrhage,septic intrauterinedeath ,and the several effusions; to Dr. Harry Spiera, Mt. Sinai Hospital, New York, N.Y. for the sera from patients with rheumatoid arthritis; and to Dr.

Ii.Al-Mondhiry,Memorial Hospital, New York,

for the sera from patients with various malignancies.

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References

1.

MERSKEY, C., JOHNSON, A.J., KLEINER, J.G., and WOHL, H. The defibrination syndrome: clinical features and laboratory diagnosis. Brit. J. Haemat. 13, 528, 1967.

2.

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