J. clin. Palh. (1953), 6, 15.

THE MEASUREMENT OF PROTHROMBIN IN PLASMAA CASE OF PROTHROMBIN DEFICIENCY

BY

ROSEMARY BIGGS AND A. S. DOUGLASFrom the Department of Pathology, Radcliffe Infirmary, Oxford

(RECEIVED FOR PUBLICATION OCTOBER 7, 1952)

Quick's (1935) one-stage prothrombin test isinvaluable as a routine method, and it has beencarried out on the plasma of patients with manytypes of clotting defect. When the one-stageclotting time is lengthened the patient is often saidto have "hypoprothrombinaemia." This is mis-leading, because the test cannot be relied upon tomeasure prothrombin. Patients with a markedreduction in prothrombin may show little changeof clotting time by Quick's method, and patientswith no significant reduction in prothrombin mayhave a grossly lengthened one-stage clotting time.Quick's test is influenced by accelerators of bloodcoagulation, by the concentration of fibrinogen,and by the presence of inhibitors, such as heparin,in the blood. At present the one-stage test cannotbe used as a measure of any specific substance.The important accelerators which influence the

one-stage test are factors V (Owren, 1947) .andVII (Koller, Loeliger, and Duckert, 1951). FactorV is a protein in the globulin fraction of plasmawhich is not adsorbed by inorganic precipitatessuch as Ca3(PO4)2, BaSO4, and AI(OH)3. Thissubstance has been given various names, accelera-tor globulin (Ware and Seegers, 1948), prothrom-bin accelerator (Fantl and Nance, 1948), and thelabile factor (Quick, 1943). Factor V may bedeficient from birth or childhood (Owren, 1947;Frank, Bilhan, and Ekren, 1950; Brink andKingsley, 1952), or may become deficient as aresult of acute infection (Koller, Gasser, Krusi,and de Muralt, 1950), or liver disease (Owren,1949). Factor VII (Koller et al., 1951) is a proteinin the globulin fraction of serum which is adsorbedby BaSO4 and Al(OH)3. The activity of factorVII may be identical with the phenomena attri-buted to prothrombin conversion factor (Owenand Bollman, 1948), serum prothrombin conver-sion accelerator (de Vries, Alexander, and Gold-stein 1949), proconvertin and convertin (Owren,1951a, 1951b), and co-thromboplastin (Mann andHurn, 1951). The term factor VII is preferredbecause Koller et al. made the most illuminating

study of its properties. Factor VII is importantbecause the plasma of patients under treatmentwith any of the dicoumarin group of drugs lacksthis factor.A rough, quantitative distinction between the

effects of factors V, VII, and prothrombin can beachieved by the use of plasma adsorbed withAl(OH)3, which contains factor V but not pro-thrombin or factor VII, and normal serum, whichcontains little factor V and prothrombin but muchfactor VII. The one-stage clotting time of a plasmadeficient in factor V will be shortened by the addi-tion of Al(OH)3-treated plasma. The one-stageclotting time of a plasma deficient in factor VIIwill be shortened by the addition of serum but notby the addition of Al(OH)3-treated plasma. Theone-stage clotting time of a plasma deficient inprothrombin will not be shortened by the additionof AI(OH)3-treated plasma or serum.

Although a number of patients with so-calledidiopathic hypoprothrombinaemia have been de-scribed, in none can the defect be attributed withcertainty to a lack of prothrombin alone. In fiveinstances the defect was probably a reduction offactor V (Owren, 1947; Frank et al., 1950;de Vries, Matoth, and Shamir, 1951; Stohlman,Harrington, and Moloney, 1951; Brink andKingsley, 1952). In four instances factor VII mayhave been deficient (Giordano, 1943; Crockett,Shotton, Craddock, and Leavell, 1949; Landwehr,Lang, and Alexander, 1950; Alexander, Goldstein,Landwehr, and Cook, 1951). In nine instances thedefect was not clearly defined (Rhoads and Fitz-Hugh, 1941; Plum, 1943; Murphy and Clark,1944; de Marval and Bomchil, 1944; Hauser,1945; Quick. 1947; Hagen and Watson, 1948;Covey, Cohen, and Papps, 1950; Ferguson, 1950).The patient to be described had uncomplicated

prothrombin deficiency which is, apparently, avery rare condition. The plasma of this patientwas useful for the study of coagulation factors.From the two-stage method carried out on thepatient's plasma a method for the measurement of

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ROSEMARY BIGGS and A. S. DOUGLAS

prothrombin was devised. This method will bedescribed in this paper. The patient's plasma wasalso useful for the study of plasma thromboplastin,and this work is described elsewhere (Biggs, 1952;Biggs, Douglas, and Macfarlane, 1953).

MethodsCollection of Plasma-Venous blood is collected

and mixed with 3.8% sodium citrate in the propor-tion of 1 part of citrate to 9 parts of blood. Theplasma is separated after centrifuging for five minutesat 2,000 r.p.m.The one-stage method is carried out essentially as

described by Quick (1942). A full account of themethod, including the preparation of brain emulsionand M-40 calcium chloride, is given by Biggs (1951).

Two-stage Method.-Fibrinogen is prepared by thephosphate-buffer method of Jaques (1943) describedby Biggs (1951).Test.-For the test 0.4 ml. of undiluted citrated

plasma is mixed with 0.4 ml. of brain emulsionwarmed to 37' C. in a water-bath and 0.4 ml. of M-40CaCl2 added. As the CaCl2 is added a stop-watch isstarted, and at 15, 30, and 45 seconds, 1 minute, andthereafter at minute intervals 0.1 -ml. samples areremoved from this incubation mixture which is form-ing thrombin and added to 0.4-ml. amounts offibrinogen warmed to 37' C. in small tubes. Theclotting times of the fibrinogen samples are recordedand the test continued until these exceed three min-utes. When the two-stage method is carried out onundiluted plasma thrombin formation and its neutrali-zation by antithrombin are both very rapid. Usuallyin normal plasma no significant amount of thrombincan be detected after four minutes' incubation. Atechnical difficulty results from clotting in the incuba-tion mixture. When this occurs the clot must beremoved. It has been found that the removal of theclot can be achieved quite easily by winding it on toa wooden swab stick, the end of which has been splitand the halves slightly separated. Inexperiencedworkers may find that this test is best carried out bytwo operators. One worker removes the 0.1-ml.samples from the incubation mixture and the otherrecords the clotting times of the fibrinogen samples.

Thrombin-Fibrinogen Dilution Curve.-The pre-paration of this curve is described by Biggs (1951).The clotting times are -read from the- thrombin-fibrinogen curve in terms of thrombin units. A curveof thrombin generation and disappearance can thenbe drawn and the area enclosed by this curve iscomputed either with a planimeter or by the morelaborious method of counting the squares on thegraph paper. The procedure is carried out onnormal and abnormal plasma samples and the areaobtained from the abnormal is expressed as a percen-tage of the normal. As an example the figuresobtained as an average of 15 normal plasma samplesare given in Table I. These can be converted to

RESULTS OF ATABLE I

TWO-STAGE PROTHROMBIN TEST ONNORMAL PLASMA

Incubation Time in Minutes

1 2 3 4 5

Clotting time(seconds) .. 20 17 18 3 1 77 170 180

Thrombin units 6-2 7 6-8 3 5 1-2 0-2 0

thrombin units from the thrombin-fibrinogen dilu-tion curve (Table I). From these figures the curveof thrombin generation and its disappearance canthen be drawn (Fig. 1).Thrombi nUnits

FIG. 1.-The curve represents the resultsof the two-stage test carried out onnormal plasma. The amount ofthrombin formed after various timeintervals is recorded.

31

21

Time in minutes6

A ntithrombin.-It is an essential prerequisite ofthis test that the antithrombin content of the normaland test plasmas should be similar. The anti-thrombin content of plasma can be tested by themethod of Astrup and Darling (1942). To ensurethat the antithrombin is sufficiently normal for thetwo-stage test a simpler technique may be employed.Sufficient thrombin to clot 1 ml. of plasma in threeto four seconds is added to 1 ml. of plasma. At half-minute intervals thereafter 0.1 ml. of the thrombin-plasma mixture is added to 0.4 ml. of fibrinogen andthe clotting times are recorded. The lengthening ofthe clotting time with the passage of time should besimilar in the normal and patient's plasma. " Throm-bin topical" prepared by Roche may be used for thistest.

Test for Factor V Deficiency.-The preparation ofAI(OH)3a is by the method of Bertho and Grassmann(1938). Al(OH)3-adsorbed plasma was prepared anddescribed by Biggs (1951).

Test.-To 0.9 ml. of the plasma to be tested isadded 0.1 ml. of Al(OH)s-treated normal plasma.The mixture is tested by the one-stage prothrombintest. Al(OH)3-treated plasma is rich in factor V butpoor in prothrombin and factor VII. If the clotting

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THE MEASUREMENT OF PROTHROMBIN IN PLASMA

time of the mixture is appreciably shorter than thatof the patient's plasma factor V is deficient.

Test for Factor VII Deficiency.-Normal serum isprepared by collecting 3 ml. of normal blood into aglass tube containing three glass beads. The tube isshaken during clotting to ensure that most of theprothrombin is converted to thrombin. After onehour or more at 37° C. the serum can be separatedby centrifuging.Test.-To 0.9 ml. of the plasma to be tested is

added 0.1 ml. of serum. The mixture is tested bythe one-stage method. Serum is rich in factor VII,but contains little factor V or prothrombin. Amarked reduction in clotting time indicates factor VIIdeficiency.

Case ReportG. W. was a man aged 26.The patient had been well until 15 months before

investigation. He then developed haematuria, investi-gation of the renal tract revealing no cause. Ninemonths later he developed large spontaneous bruiseson the right calf, on both arms, and in the neck.There was a history of epistaxis and bleeding fromthe gums, and also of transient pains in the knees. Asa child the patient underwent tonsillectomy, and twoyears before the onset of his present illness a toothwas removed with no excessive bleeding. There wasno history of abnormal bleeding in his relatives. Atthe time of onset of the bleeding the patient wasworking in a brewery. Soon afterwards he went toa rubber factory, where he was employed in cuttingout from sheets of rubber.The abnormality in this patient was acquired in

adult life and could not easily be attributed to hisoccupation; nor did he admit to taking any form ofmedicine.

Clinical examination was negative.Laboratory Investigations. - Haemoglobin was

13.7 g.%, the white-cell count 5,100 per c.mm. Theblood film appeared normal.Plasma proteins were 6.6 g./100 ml. (albumin, 3.8 g.;

globulin, 2.4 g.; fibrinogen, 360 mg.). The thymolturbidity test was negative, as also was the colloidalgold test. The sucrose tolerance curve was normal.The bilirubin level was 0.3 mg.%.

Fat excretion was normal, with an intake of fat of280 g. per day and an excretion of fat of 7.2 g. (2.6%).The bleeding time (Ivy's method) was 5 minutes

(normal 2{-7 minutes). A tourniquest test was nega-tive. A platelet count gave 645,000-1,121,000/c.mm.

Investigation of Coagulation Defect.-The whole-blood clotting time, determined by the modifiedmethod of Lee and White (1913), was 13{-20 minutes(normal 5-10 minutes); the one-stage prothrombintime was 18-22 seconds (normal 14 seconds). Theantithrombin measured by Astrup and Darling's (1942)method was 179 units (normal plasma 193 units). Thereaction of plasma to thrombin was normal.

Test for Factor V.-The one-stage clotting time ofthe patient's plasma was not shortened by the addi-

B

tion of normal plasma treated with Al(OH)3. Sincethe Al(OH)3-treated plasma contains an excess offactor V the patient's plasma did not appear to bedeficient in factor V.

Tests for Factor VII.-The one-stage clotting timeof the patient's plasma was not shortened by theaddition of normal serum, which contains an excessof factor VII. Additions of the patient's plasmashortened the clotting time of plasma from a patientunder treatment with " tromexan " (which lacks factorVII) as well as did similar additions of normalplasma.

It appears that the patient's plasma lacked neitherof the known accelerators of blood coagulation.

Two-stage Prothrombin Test.-The curves illus-trating thrombin formation in normal and in thepatient's plasma, using the two-stage method, areshown in Fig. 2. The interpretation of this test isconsidered in detail in a later part of this paper.Thrombin

Units

FIG. 2.-The curves represent theresults of the two-stage testcarried out on normal plasma(e *) and on theprothrombin-deficient patient'splasma(x x).

Time in minutes

From these curves it was calculated that the patient'splasma contained about 11% of the normal amountof prothrombin.The Effect of Vitamin K.-Vitamin K was given

to the patient in three different ways with an intervalof three days between each trial: 100 mg. of a water-soluble analogue, " synkavit," was given intra-venously; 1,000 mg. of vitamin K, was given orally;and 500 mg. of vitamin Ki was given intravenouslyby the method of Davidson and MacDonald (1943).In none of these trials did the vitamin K cause a risein prothrombin tested by the two-stage method.

Effect of Intravenous Administration of NormalPlasma.-Fresh citrated plasma, 860 ml., was givento the patient. The plasma was given within twohours of its collection from three donors. On asecond occasion 500 ml. of stored citrated plasmawas given to the patient. On each occasion therise in prothrombin as tested by the two-stage methodwas slight and the effect transient (Fig. 3). Theineffectiveness of transfusion suggests either that thenormal rate of prothrombin utilization was very highor that it was unusually fast in this patient.

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ROSEMARY BIGGS and A. S. DOUGLAS

Prothrombin

TransfusionI Stored Plasma

TransfusionI Fresh Plasma

'i

Tume in hours1 IG. 3.-The curves show the level of prothrombin (measured by the

two-stage test) in the patient's plasma before and after plasmatransfusions.

This patient is apparently the first to be describedin whom a coagulation defect can be attributed to anuncomplicated deficiency of prothrombin. The un-usual features of the case are the relatively slightalteration in the one-stage " prothrombin " time andthe lengthened whole-blood clotting time. The slightlengthening of the one-stage clotting time suggeststhat the one-stage test may be relatively more sensi-tive to changes in the accelerators of blood coagula-tion than it is to prothrombin. The lengthened whole-blood clotting time may be related to a very low levelof thrombin generated by the plasma thromboplastinsystem when prothrombin is grossly deficient.

The Measurement of ProthrombinThe two-stage prothrombin test is usually con-

sidered to give the most reliable measure of pro-thrombin, but it is to be doubted whether thistest, as usually interpreted, often gives a truemeasure of prothrombin in plasma. In experi-ments in which prothrombin is separated fromantithrombin the test can be used to give ameasure of prothrombin (Biggs, 1951 ; Biggs andMacfarlane, 1953). In these artificial conditionsprothrombin is quantitatively converted to throm-bin, and the amount of thrombin formed givesa measure of prothrombin. In plasma thrombinis neutralized by antithrombin as it is formed, andthe amount of thrombin detected in a plasmamixture forming thrombin is not the total amountof thrombin formed, but the amount from whichthe amount neutralized has been substracted.The two-stage test is therefore usually carried

out on high dilutions of plasma samples to avoidthe effects of antithrombin. But it can be shown(Biggs, 1951 ; Biggs and Macfarlane, 1953) thatdilution does not remove the effects of anti-thrombin. Moreover, plasma samples in whichprothrombin is reduced cannot be greatly dilutedbecause the amount of thrombin to be detected istoo low in these diluted samples." If the effedts

of antithrombin cannot be eliminated by dilution,it is obviously necessary either to remove anti-thrombin by some method other than dilution orto devise a test in which the effect of antithrombinis allowed for.

In Fig. 4 are shown the results of carrying outthe two-stage test as described in this communica-tion on an undiluted normal plasma sample, on theplasma of the patient with prothrombin deficiency,and on the plasma of a patient treated with" tromexan." In the prothrombin-deficient patient'splasma thrombin formation is complete intwo minutes and the level of 2.2 units is main-tained only for a few seconds, whereas in the" tromexan" plasma thrombin can be detected upto eight minutes, and a level of between 1 and

ThrombinUnits7

6

5

4

3

2

FIG. 4.-The curves 1of the two-stagenormal plasmaplasma of a pi" tromexant"

\ ~~~the prothrombil0 ~~~~plasma ( x

represent the results:test carried out ont( *), theoatient treated with(o-o), andin-deficient patient's-x ).

2 3 4 S 6 7 8Time in minutes

2.5 units of thrombin is maintained for fiveminutes. The prothrombin-deficient patient'splasma has a rapid prothrombin conversion. Inthe "tromexan " plasma thrombin formation isdelayed. These two curves suggest that muchmore thrombin is formed in the " tromexan "-

treated patient's plasma than in that of the pro-thrombin-deficient patient. It would seem that,in assessing the amount of prothrombin present,the duration of thrombin formation must be asimportant as the level reached. The duration ofthrombin formation and the level of thrombinattained can, both be taken into account by com-puting the areas enclosed by the curves as ameasure of prothrombin. When this is done thepatient's plasma would be said to contain 11%of prothrombin and the " tromexan " plasma 68 %.This assessment would appear to be more reason-able than the comparison of the levels of thrombinformed. Common sense suggests that this is areasonable method of measuring prothrombin, butis it really more rational than the previousmethods?

--f

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THE MEASUREMENT OF PROTHROMBIN IN PLASMA

The use of the two-stage test on undilutedplasma involves an understanding of the effectsof accelerating substances and antithrombin on thelevels of thrombin detected when carrying out thetest. To achieve this understanding it is con-venient to consider a simplified theoretical modelof the blood coagulation system. Suppose that asubstance A is converted to another substance Bby a first order reaction, and substance B is simul-taneously converted to C by a first order reaction.

ThrombinUnits

10

8]

3'

2 4Tin

1 2 3 4 5 6 7 8 1 2Time in minute. Ti

(c)

FIG. 5.-Theoretical curves to show the progress of two consecuti. A-B .B- --+C. (a) These curves show the disappearaiance of B and C in such a reaction. In the blood coagulation saprothrombin, B thrombin, and C neutralized thrombin. In tmeasured. (b) These curves represent the progress of form.different initial concentrations of A are present. The speedits conversion to C are constant. (c) These curves represent theB when the initial amount of A is constant and the speed of nchanged but the speed of formation of B is varied. (d) These cgress of formation of B when the initial amount of A and thB are constant but the speed of neutralization of B is varied.

Then the progress of the reaction may be repre-sented in Fig. Sa. The concentration of A fallsas A is converted to B. The concentration of Brises until the conversion of B to C balances theconversion of A to B. Then when most of A isconverted to B the concentration of B falls andthat of C rises. In blood coagulation A wouldrepresent prothrombin, B thrombin, and C aneutralized thrombin-antithrombin association. Inthe two-stage method it is B which is measured.

The speed of formation of Bwill depend on the presenceof activating factors and thespeed of disappearance of Bon the concentration of neu-tralizing factors.When the amount of A is

varied, but the speeds of for-mation and neutralization ofB are unchanged, the curvesshown in Fig. 5b are obtained.In these curves the peak levelsof B and the areas enclosedby the curves are both pro-portional to the amount of Ainitially present.When the amount of A is

constant and the speed ofneutralization of B is un-

. changed, variation in the6 8 10 12 speed of formation of B gives

ne in minutes(b) curves such as those in

Fig. 5c. The peak levels ofthrombin reached are now noindication of the amount of Ainitially present, but the areasenclosed by the curves areproportional to the amount ofA present at the beginning.From Fig. 5c it is clear thatthe constancy of the areas isdue to the slower disappear-ance of B which leads to acrossing over of the curvesduring the decline of B. Inthe blood coagulation system

3 4 5 6 this crossing over of theme in minutes curves is often easier to detect

(d)ive first order reactions: than a change in the speed ofnce of A and the appear- appearance of thrombin.vstem A would represent[he two-stage test B is When the amount of A isation of B when threeI of formation of B and constant and the speed of for-progress of formation of mation of B is unchanged, butkeutralization of B is un-,urves represent the pro- the speed of neutralization ofe speed of formation of B is varied, then the curves of

ThrombinUnits

1011

19

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ROSEMARY BIGGS and A. S. DOUGLAS

Fig. 5d are obtained. In these conditions neitherthe peak levels of B nor the areas enclosed by thecurves are proportional to the amount of A initi-ally present.

If this theoretical model can be followed it isclear that the measurement of the area enclosedby the two-stage curve will give the most generallyreliable proportional measure of prothrombin, butthat prothrombin cannot be measured unless thelevel of antithrombin is normal.

It is obviously important to know how closelythe blood coagulation reactions approach thetheoretical model. When the average of 15 curves

ThrombinUntls

I

I %

I

IIIIIIIIII

FIG. 6.-The curves represent progress ofthrombin formation in the two-stagetest (e .) comparedwithatheoretical model of the reaction,assuming that the reaction can beinterpreted as two consecutive firstorder reactions A (prothrombin)

* B (thrombin) - r C(neutralized thrombin). The theo-retical curve is shown as a discon-tinuous line.

1 2 3

Time in minutes4 5

for normal plasma is compared with a theoreticalmodel it is found that there is a surprisingly closeagreement (Fig. 6).The amount of prothrombin may be varied by

adding prothrombin to the plasma of the pro-thrombin-deficient patient. When this was doneit was found that the experimental curves agreedreasonably well with those of the theoretical model(Fig. 7).

In the plasma of patients treated with " tro-mexan" thrombin formation is delayed, and thedelay can be reduced by the addition of normalserum. The effect of accelerators on the two-stage test can therefore be studied by comparingthrombin formation in "tromexan " plasma withthat in " tromexan " plasma to which serum hasbeen added and with that in normal plasma. FromFig. 8 it is clear that the experimental curves are

of the same general pattern as those of thetheoretical model.

ThrombinUnits

12T

10.

8-

6~

4'

2

FIG. 7.-The curv

of the two-st;the plasmadeficient 'paplasma to Mprothrombincurves shouldin Fig. 5 (b).

2 4 6Time in minutes

ves represent the resultstage test carried out onof the prothrombin-

atient and the samewhich two amounts ofwere added. These

I becompared with those

i 1'o

The speed of neutralization of thrombin can bevaried by adding various amounts of heparin tonormal plasma. When the two-stage test is carriedout on samples containing various amounts ofheparin the curves resemble those of the theoreticalmodel (Fig. 9).The close agreement between the experimental

results and the theoretical model does not implythat the reactions of blood coagulation can beexplained in terms of this simple model. Otherexplanations may also be adequate. Slight alter-ations in the conditions of carrying out the testsmay affect the results. From this close agreementall that can be deduced is that, provided the con-ditions for making the test are not altered, the areamethod of measuring prothrombin is likely to givemore generally reliable results than other methods.The calculations for deriving the theoretical curvesare described by Daniels (1948).Thrombin

3,

FIG. 8.-The curves represent the results

of the two-stage test carried out on

the plasma of a patient treated

with " tromexan " ( x -..x ),

the same plasma to which 10% of

normal serum was added(o-o), and normal plasma

*). These curves shouldbe compared with those in Fig. 5 (c).

7

6.

4o

3-

W w * v s

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THE MEASUREMENT OF PROTHROMBIN IN PLASMA

6I AFIG. 9.-The curves represent the6 I \ results of the two-stage test

carried out on normal plasmaO and the same plasma to which

5. B \was added i, i, and i a unit ofheparin. These curves shouldbe compared with those in

4 / 1/8 Fig. 5 (d).

3.

1142-

11/2

2 4 6 8 10Time in minutes

There are two other methods by which thereliability of this method of measuring pro-thrombin can be assessed. The amount of pro-thrombin in a normal and prothrombin-deficientplasma sample can be measured. The two samplescan then be mixed in known proportions and theamount of prothrombin in the mixtures can bemeasured and the measured results compared withthose calculated from the original samples. Whenthis is done there is a reasonable correlation be-tween the observed and expected figures (Fig. 10).The prothrombin content of a plasma sample

can also be measured in the globulin fraction con-

CakubedArea

1600j

1400t

200'

000

800;

600

400

200

S*eA* 0

S00

0

00

I.S

260 400 600 800 1000 1200 1600Obsemed Ara

FIG. 10.-This diagram represents the results of carrying out thetwo-stage test on normal and prothrombin-deficient samples andon mixtures of the two in known proportions. In the mixturesthe calculated and observed values for the areas enclosed bythe curves are compared.

taining prothrombin separated from antithrombin(Douglas, 1953). When this is done a con-stant level of thrombin is reached in the two-stage test. This constant level of thrombin isproportional to the amount of prothrombinpresent. The globulin fraction of 10 samples ofplasma from patients treated with "tromexan "were prepared. The amounts of prothrombinfound by two-stage tests on the original plasmasamples agreed very well with the results of thetwo-stage tests on the globulin fractions.From these results it appears that a simple two-

stage procedure on unmodified plasma can be usedto give a reasonably reliable measure of pro-thrombin.

DiscussionVery numerous methods for measuring pro-

thrombin are now in use, but none of these can berelied upon to measure prothrombin in all plasmasamples. Two-stage tests on diluted plasma towhich various accelerator substances have beenadded (Ware and Seegers, 1949) are very com-plicated and depend on the assumption that anexcess of activators is present. It is probable thatin some plasma samples a very large excess ofactivators may be necessary and this large excesscannot be achieved easily.

One-stage tests in which a large excess of acti-vators is added have also been devised (Owrenand Aas, 1951 ; Koller et al., 1951). Thesemethods do not always give the same results as thetwo-stage test described in this communication(Douglas, 1953). The reason for the discrepancyis as yet unknown. The modified one-stage test isbased on the assumption that its results mustmeasure prothrombin if an excess of activators ispresent. If the excess of activator required is verylarge this assumption may not be justified. More-over, the modified one-stage tests require reagentssuch as Seitz-filtered ox plasma and normal serum,which may vary in constituents from one sample toanother, and the test is difficult to standardize.The two-stage test described above requires no

special reagents, and possible variations in thepotency of brain thromboplastin do not affect theresults. In addition there are methods of testingthe validity of the two-stage test. The results ofthis test must at present be considered to give amore probable measure of prothrombin than modi-fications of the one-stage method. The only recog-nized limiting factor in the application of this testis variability in the antithrombin content of thecontrol and test samples. For this reason themethod cannot be used to compare the pro-

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ROSEMARY BIGGS atnd A. S. DOUGLAS

thrombin content of plasma and serum becauseplasma contains more antithrombin than serum.The two-stage test is useful in distinguishing

between the coagulation defects caused by reduc-tion in factor V, factor VII, and prothrombin.The test is also useful for determining the extentof true prothrombin deficiency. It is perhaps tooobvious to emphasize that the test will not beuseful for measuring the extent of a coagulationdefect that is not primarily one of prothrombin.In " tromexan " therapy, for example, the mainabnormality is a reduction in factor VII which isreliably measured by the one-stage test. The pro-thrombin test would not be a good method forthe control of anticoagulant therapy with dicou-marol or "tromexan." In this form of therapythe one-stage test should be used.

Finally, it must be emphasized that an isolateddeficiency in prothrombin is an extremely rare con-dition. This fact may be difficult to appreciatebecause the term "hypoprothrombinaemia " hasbeen used to describe many different coagulationdefects and because the results of the one-stagetest are so often erroneously reported in terms of" percentage of prothrombin." The one-stage testalone is a useful empirical test. When the coagu-lation defect has been defined in terms of factorV. factor VII, or prothrombin deficiency it maybe possible to use the one-stage method as a quan-titative measure of some specific substance. Forexample, in " tromexan " therapy the one-stagetest probably gives a measure of factor VIIdeficiency. In the patient with 10% of pro-thrombin recorded in this communication the one-stage prothrombin time was little lengthened. Inthe plasma of other cases we have never found lessthan 25%-,, of prothrombin by the two-stage test.Thus it is probable that the classical "prothrorn-bin" test seldom gives a measure of prothrombindeficiency because prothrombin is not usuallysufficiently reduced to influence the results of thetest.

SummaryA case of prothrombin deficiency is described.

In this patient prothrombin was reduced in amount,but factors V and VII were normal. It appearsthat isolated prothrombin deficiency is a very rarecondition.From a study of this patient a method for the

measurement of prothrombin was devised. Thistest is simple, requires no special reagents, and,as far as can be determined, it gives a reliablemeasure of prothrombin.

This two-stage method is useful in distinguish-ing between deficiencies of prothrombin and

factors V and VIl, reduction in any of which mavlengthen the clotting time by the one-stage test.The test is naturally not useful for following the

course of " tromexan "

or dicoumarol therapy be-cause in this form of anticoagulant treatment themain abnormality is a reduction of factor VII andnot of prothrombin.We should like to thank Dr. R. G. Macfarlane for

his advice and interest in this work and Dr. A. H. 1.Robb-Smith, who kindly read the manuscript; alsoProfessor L. J. Witts for permission to publish detailsof his case. One of us (A. S. D.) wishes to thank theMedical Research Council for a fellowship in clinicalresearch enabling him to carry ouit this work.

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