A NEW METHOD FOR THE DETERMINATION OF THE FIBRIN PERCENTAGE IN BLOOD AND PLASMA.

BY H. C. GRAM.

(From the Medical Clinic of the University of Copenhagen, Copenhagen, Denmark.)

(Received for publication, April 1, 1921.)

The variations in the fibrin (fibrinogen) content of the blood have interested several observers, though the technique for these determinations has never been very good or convenient.

The spontaneously coagulable matter of the blood may be deter- mined in two ways, by precipitating either the fibrinogen as such or as fibrin by the natural process of clotting. The last named method is the oldest and has generally been carried out so that the fibrin has been whipped out of a measured or weighed amount of blood (Hoppe-Seyler, 1; Samuely, 2), or the blood has been shaken with small rods and the fibrin adhering to these removed (Dastre, 3). A still older method used by Andral (4) and others was to leave a measured quantity of blood to clot, then whipping out and washing the fibrin (“fib&e du cadlot”). This procedure is described by Doyon, Morel, and Kareff (5).

All these methods require a rather large quantity of blood (20 to 100 cc.), and they are not very accurate since the fibrin obtained is not pure and small amounts may be lost during the whipping and washing. A better way of determining the fibrin has been chosen by the authors, using a stable plasma for analysis because this may be freed of leucocytes. Often, however, this was not done. Pfeiffer (6) determined the fibrin by recalcifying oxalated plasma and calculating the fibrin percentage from the difference in the nitrogen content of plasma and serum. As it is necessary to knom the cell volume for these calculations, it was either arbitrarily fixed as 40 per cent or was estimated by the method of Bleibtreu.

279

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280 Fibrin Percentage in Blood

Cullen and Van Slyke (7) have proposed to isolate the clot from recalcified oxalate plasma and to determine its nitrogen content; the fibrin percentage is then calculated by multiplying by 6.25. It must also be mentioned that Bang (8) has proposed to determine the fibrin in blood by weighing 1 drop absorbed on filter paper and making a micro-analysis of its nitrogen content after extracting with a highly diluted alkaline solution.

The possibilities of fibrinolysis (Green, 9; Goodpasture, lo), which in my opinion are only to be feared when one does not use a cell-free plasma, have caused a great number of authors to pre- fer to determine the fibrinogen. For this purpose a stable plasma of some sort must be used.

Reyhe (11) used 12 cc. of fluoride plasma in which the fibrinogen was precipitated by adding 30 cc. of distilled water and 16 C.C. of an ammomium sulfate solution having a specific gravity of 1.245. The precipitated fibrinogen was washed with an identical dilu- tion of this salt and then with hot water, alcohol, and ether, and is weighed after drying. Doyen, Morel, and PBju (12) recom- mended the use of 1 cc. of 10 per cent acetic acidto 12 cc. of plasma for the same purpose.

The specific coagulation temperature of the fibrinogen has been used by Frederick (13) and later by Whipple (14), who heated 20 cc. of clear, oxalated plasma to 56-60°C. for 10 minutes, and washed and weighed the precipitate.

With such methods the different authors have found very vary- ing results (Table I), which may partly be explained by the physio- logical variations in the fibrin content; the number of observations in each series has often been very small.

A practically useful method for determining the fibrin (fibrin- ogen) percentage must fulfill the following conditions : (1) Quan- tities of blood must be used which may be easily obtained and several times without inconvenience to the patients. (2) A deter- mination of the percentage both in plasma and in blood must be allowed. (3) The complete precipitation of the fibrin (fibrinogen) must be controlled. (4) Fibrinolysis must be excluded. (5) The fibrin and fibrinogen must be pure and cellular elements, which may either increase the weight or cause fibrinolysis (Rulot), should not be included.

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TABL

E I.

Fibr

in

and

Fibr

inog

en

Perc

enta

ges

in

Bloo

d an

d Pl

asm

a of

Nor

mal

M

en

and

Anim

als.

Autho

r. Te

chniq

ue.

Anim

al.

Rey

he

(11)

....

......

......

......

. M

tiller

(1

5).

......

......

......

....

Fred

erick

(1

3) .

......

......

......

. W

hipp

le

(14)

. ...

......

......

.....

Whi

pple

, M

ason

, an

d Pe

ight

al

(16)

. Pf

eiffe

r (6

). ...

......

......

......

. Bu

nge

(17)

. ...

......

......

......

. Ri

chet

(1

8).

......

......

......

....

Erbe

n (1

9).

......

......

......

...

Arth

us

(29)

. ...

......

......

......

Br

rone

t (2

1)

......

......

......

...

Schn

eide

r (2

2).

......

......

......

Bi

erna

cki

.(23)

....

......

......

.. Be

cque

rel

and

Rodi

er

(24)

. ...

...

Berg

grtin

(2

5)

......

......

......

. Kr

uger

(2

6).

......

......

......

...

Mee

k (2

7).

......

......

......

.....

Andr

al (4

). ...

......

......

......

.

See

text

. R

eyhe

’s.

Heat

co

agul

atio

n.

‘I I‘

“ “

See

text

. W

hipp

ing.

“ I‘ I‘ ‘I “ “ “ “ ‘I I‘

Fibr

ine

du

caillo

t.

Cat

tle.

Rabb

its.

Hors

es.

Men

. I‘ “ 6‘

“ “ “ “ “ ‘I “

Child

ren.

Ne

wAbo

rn.

Dogs

. M

en.

.

Fibr

inog

en.

“ I‘ “ “

Fibr

in. “ “ “ “ <‘

“ “ “ “ ‘<

“ ‘I

- --

-

0.41

0.

47

0.29

-0.4

3 Ab

out

0.40

. 0.

30-0

.40

<0.3

1

Perce

ntage

in

blood

.

0.10

-0.4

0 0.

19-0

.28

<0.3

0 0.

160.

20

Abou

t 0.

20.

“ 0.

20.

“ 0.

19.

“ 0.

22.

0.29

-0.4

2 0.

12

0.17

X1.2

2 0.

25-9

.35

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282 Fibrin Percentage in Blood

I shall now describe the technique for determining the fibrin content followed by me and afterwards the experiments, which have proved its reliability.

About 4.5 cc. of venous blood are run into a graduated 5 cc. centrifuge tube (Oluf Thomsen) divided into one-tenth of a cc. and containing 0.5 cc. of 3 per cent sodium citrate. The titrated blood is shaken and the blood adhering to the cork and upper part of the glass is wiped off.

After the specimen has stood for some time the corpuscles have sedi- mented sufficiently to draw off 0.025 cc. of plasma for the platelet count (Thomsen, 28; Gram, 29, 30) and 0.4 cc. for the determination of the coagulation time (Gram, 31). The velocity of this sedimentation depends upon the cell volume percentage and the fibrinogen percentage in the plasma (Gram, 32).

The specimen is then centrifuged for 90 minutes at 3,000 revolutions per minute and stopping very slowly. The tubes must be securely fixed in the corresponding holes of the centrifuge.

The amount of titrated blood and precipitate is noted, the cell volume being calculated by the equation

P.100 Volume percentage = B

P = Precipitate in cc. B = Blood in cc.

The clear cell-free titrated plasma is drawn off with a pipette, and 2 cc. are transferred into a 50 mm. wide cylindrical vessel, whose bottom is slight~ly rounded off against the sides. 9 cc. of 0.9 per cent sodium chloride and 2 cc. of 1 per cent CaCh * 6HzO solution are then added and t.he vessel is placed in the thermostat at 35” C. for 13 hours. When the glass is taken out the contents will always be found clotted, except possibly in very severe cases of hemophilia or melena neonatorum.

By inclining and turning the glass around the clot will always loosen completely, not even leaving traces on the vessel.

It is thrown out on several layers of filter paper on which it forms a jelly-like cake. The water is absorbed very quickly by the paper, leaving a round shining membrane, which may easily be detached with a glass rod when the top layer of paper is thrown into a jar of water.

The det,ached membrane is kept in distilled water for 15 minutes and is then transferred into absolute alcohol for 5 minutes and finally into ether for another 5 minutes to dehydrate and extract lipoids.

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H. C. Gram

The hardened, dehydrated fibrin which resembles a piece of paper is gripped by small wire pincers of known weight and hung in the thermostat for some hours or in an oven for a shorter period.

When the weight is constant the fibrin is weighed either on the analytical balance or on a fine torsion balance. The balance generally used only gave an accuracy of $ mg.

In the cylindrical glass vessel there is always left a small amount of liquid (diluted serum), which may be used as a control, since clotting either spontaneously or by addition of a little serum shows that the precipitation of the fibrinogen has not been complete. This control test is very fine, giving positive results with quantities of fibrin too small to influence the weight; i. e., less than $ mg.

When the recalcified plasma was kept in the thermostat for the usual length of- time the control never was found positive even in hemophilia.

In order to calculate the fibrin percentage in plasma (FP) and blood (Fb) a knowledge of the following values is necessary:

Wfi = Weight in gm. of fibrin in 2 cc. of titrated plasma. Cb = Citrated blood (0.5 cc.). c = Citrate (0.5 cc.). P = Cell precipitate in cc.

The formula for calculating the percentage in pure plasma is:

F P

= (Cb - P) *:W.fz. 100 (Cb-C--P)*2

The corresponding formula for the percentage in pure blood is:

Fb = (Cb - P) * W’fi * 100 (Cb - C) -2

The technique in the first instance has been based upon a study of the coagulation time of titrated plasma on recalcificationwhich has been published elsewhere (Gram, 31).

The exactitude of the cell volume determinations has been dealt with in another publication (Norgaard and Gram, 33).

In this paper we shall, therefore, only present the experiments concerning: (1) the proper recalcification of the titrated plasma; (2) the possibilities of fibrinolysis and fibrinogenolysis; (3) the accuracy of the method and the mean error on it; and (4) com-

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284 Fibrin Percentage in Blood

parisons between my technique and the results gained by simple. defibrination of blood and by determination of the fibrinogen after Whipple’s method of heat coagulation.

Proper Recalcijication.-1 drop of 1 per cent CaC12-6H20, solution to 0.1 cc. of plasma equals 1 cc. of the solution to 2 cc. of plasma, since the pipette used in the coagulation experiments. gave 20 drops to the cc.

It was found that the optimal.recalcification of titrated plasma from a mixture of 0.5 cc. of 3 per cent citrate + 4.5 cc. of blood was

TABLE II.

Efects of Various RecalciJication of Plasma Kept in the Thermostat for 13. Hours After RecalciJication.

Speci- mens.

Recalci- fication

peLi lx. citmted plasms.

cc.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

-

1

0

P _

-

I -

Fibrin per-

entage in

‘laBma

0.33 0.33 0.33 0.33 0.32 0.33 0.32 0.33

-

%:

1

C’

P _

-

II -

Fibrin per-

entage in

desma _

0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32

-

TX

- - - - - - - - - -

T _ -

c

F _-

-

III

Fibrin per-

entage in

hsma

0.31 0.34 0.34 0.34 0.33 0.34 0.34 0.34

“ix

+ - - - - - - -

- I IV

Fibrin per-

en* in

0.51 0.51 0.49 0.49 0.51 0.51 0.49 0.51

1 to 4 cc. of 1 per cent CaClz * 6Hz0 per 2 cc. varying recalcification are found in Table II. -

- I

Eli:

- - - - - - - -

V

Fibrin per-

:entagc in

,lfMlX3

0.39 0.37 0.39 0.39 0.37 0.39 0.37 0.33

The effects of a.

In two cases the coagulation is not complete after 14 hours, the recalcification in these specimens being respectively 0.5 and 4 cc. per 2 cc. of titrated plasma. With recalciflcations between 1 and 3.5 cc. of 1 per cent CaClz - 6Hz0 the result is always the same except for the experimental error.

A recalcification of 2 cc. of 1 per cent CaC12 - 6Hz0 per 2 cc. of titrated plasma must be considered safe, which also has been proved in practice.

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H. C. Gram 285

TABLE III.

Fibrin Percentages in Equ@ly Recalcijied Specimens from the Same Individual Left for Various Periods in the Thermostat.

Time after recalcification (2ac. of 1 per cent C&l% 6HzO)

min.

5 10 15 20 25 30 35 60

Fibrin percentage in plasma.

No coagulation. “ ‘<

0.09 0.32 0.32 0.33 0.32 0.32

TABLE IV.

Control.

++

++

++

+

-

Double Specimens, of Which No. I Is Left for I$. Hours, and No. II’for 2.J Hours in the Thermostat after Recalcijication.

Speoi- men NO.

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Diagnosis.

Convalescence after pneumonia. ..... Rheumatic fever. .................... Carcinoma of the liver. ............... Normal .............................. Lymphatic leucemia ................. Polycythemia .......................

“ ........................ “ ........................

Febricula ............................. ‘I Stenosis pylori ..............

Chronic polyarthritis ................. Cirrhosis of the liver. ............... Pernicious anemia .................... Normal ............................. Fatty degeneration of the liver. ..... Polycythemia ....................... Chronic constipation. ................ Neurasthenia ........................ Chronic nephritis ..................... Splenectomy (postoperative). ......... Stenosis pylori ...................... Tertiary syphilis ............ .? ........ Fatty degeneration of the liver. ..... Pseudoleucemia ..................... Carcinoma of the liver. .............. Normal .............................

-

-

Fibrin ercentage in p P amna.

I

0.39 0.55 0.72 0.32 0.32 0.32 0.33 0.25 0.55 0.82 0.65 0.18 0.18 0.25 0.08 0.30 0.29 0.28 0.49 0.43 0.37 0.66 0.12 0.39 0.07 0.27

-

.-

-

II

0.39 0.55 0.72 0.32 0.32 0.32 0.33 0.25 0.55 0.82 0.65 0.18 0.18 0.25 0.08 0.30 0.31 0.27 0.47 0.44 0.39 0.63 0.13 0.41 0.07 0.26

i- Differ- ence.

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0

+:.02 -0.01 -0.02 +0.01 $0.02 -0.03 +0.01 +0.02

0 -0.01

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286 Fibrin Percentage in Blood

If we consider how coagulation progresses in a series of identical specimens of titrated plasma recalcified in the same way, we find the results shown in Table III.

The coagulation is complete after 25 minutes, and the control is fine enough to show a residue of fibrin, which is too small to influence the weight of the precipitated fibrin.

Possibilities of Fibrinolysis and Fibrinogenolysis by an Analysis of Double Specimens from the Same Person.-The first question

TABLE V.

Analysis of Double Specimens of Which No. I Is Centrifuged at Once and No. II after Standing 24 Hours at Room Temp&c&e.

Speci- men NO.

6 7 8 9

10 11 12 13 14 15

Diagnosis.

Normal ...................... I‘ ..........................

Febricula (cancer?). ........... “ ..................... .....

Convalescense after rheumatic fever .........................

Pernicious anemia. ........... Nephritis; anemia. ............ Enteritis ...................... Pernicious anemia. ............ Enteritis ...................... Normal. ..................... Abdominal carcinoma. ........ Renal tumor. ................ Constipation ................. Normal ......................

-

-

P

Fibrin percentage in

plaSma.

I II I

er em

37 41 40 43

v em

39 41 40 42

0.27 0.27 0.23 0.23 0.54 0.54 0.62 0.62

38 38 0.32 29 29 0.22 23 23 0.66 40 41 0.28 27 27 0.24 43 44 0.33 44 46 x).35 44 45 0.52 26 26 0.62 39 39 0.28 42 42 0.28

-

I

‘II

0.32 0.22 0.66 0.28 0.24 0.33 0.36 0.54 0.60 0.29 0.30

-

0 0 0 0

0 0 0 0 0 0

kO.01 kO.02 -0.02 to.01 to.02

was settled by letting Specimen I stand for 18 hours and No. II for 24 hours in the thermostat after recalcification (Table IV).

This shows that an appreciable fibrinolysis does not take place in cell-free plasma within 24 hours. Specimens 12, 13, 15, 23, and 25 show that the low fibrin percentage found in some diseases of the liver is not due to fibrinolysis.

In the same way a series of double specimens (Table V) showed that a dissolution of the fibrinogen did not take place, when a specimen of titrated blood was left at room temperature for 24 hours before being centrifuged.

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H. C. Gram 287

Error of the Method.-First, a series of double specimens of titrated blood were analyzed and showed very concordant results (Table VI).

TABLE VI.

Analysis of 29 Simple Double Specimens.

Speci- men No.

1

2

3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

25

26 27 28 29

Diagnosis.

Normal ....................... Enterocolitis .................. Polycythemia ................. Diabetes ...................... Ulcus ventriculi .............. Normal ....................... Ischias ........................ Bronchitis. ..................... Nephritis ...................... Febricula ...................... Nephritis ...................... Pregnancy ..................... Normal ....................... Nephritis ......................

“ ...................... Polycythemia ................. Chronic polyarthritis .......... Cirrhosis of the liver. .......... Pleurisy ....................... Normal ....................... Influenza1 pneumonia. ......... Nephritis (1) ................... Rheumatic fever. .............. Convalescence after rheumatic

fever. ....................... Influenza1 pneumonia; fatty de.

generation of the liver. ...... Pleurisy ....................... Influenza ....................... Rheumatic fever. .............. Normal .......................

Cell volume. Fibrin

percentage in plrtsma.

I II I II

er em er cem

40 40 39 41 67 67 40 40 42 43 43 43 40 41 44 44 46 45 39 40 36 36 32 31 46 48 46 46 49 50 72 72 41 41 37 37 37 39 47 49 42 43 50 50 34 34

0.22 0.22 0.32 0.32 0.32 0.31 0.38 0.38 0.32 0.32 0.32 0.32 0.32 0.32 0.46 0.44 0.56 0.59 0.82 0.82 0.28 0.28 0.49 0.47 0.31 0.32 0.58 0.56 0,59 0.61 0.37 0.37 0.38 0.37 0.25 0.25 0.66 0.68 0.30 0.32 0.48 0.49 0.90 0.90 0.94 0.93

41 44

40 44

0.35 0.39

0.35 0.40

42 42 0.50 0.49 48 49 0.33 0.32 42 40 0.56 0.57 48 47 0.35 0.34:

-

I

-

,

,

,

I

,

I

,

/

,

i -

Xffer- ence.

0 0

0.01 0 0 0 0

0.02 0.03 0 0

0.02 0.01 0.02 0.02 0

0.01 0

0.02 0.02 0.01 0

0.01

0 0.01

0.01 0.01 0.01 0.005

Then in three cases the mean error was determined by analysis of respectively 9, 9, and 10 specimens (Table VII-A, B, and C).

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288 Fibrin Percentage in Blood

Specimen No.

TABLE VII-A.

Convalescence after Angina.

Cell volume.

per csnt

50 49 49 49 49 50 50 49 49

-

_ -

-

Fibrin percentage in plasma.

Fibrii n&age k .

0.3745 0.1865 0.3707 0.1833 0.3543 0.1806 0.3548 0.1798 0.3712 0.1892 0.3575 0.1780 0.3756 0.1875 0.3712 0.1892 0.3875 0.1949

The mean error is calculated by the formula:

E=mean error d=variations of each determination from the average n=number of determinations

The mean error on the fibrin percentage in plasma was f 0.011, in blood f 0.0055.

Specimen No.

TABLE VII-B.

Graves’ Disease (Severe Case).

Cell volume. 1 Fib~p~~~~ Fibrii em&age fl .

per cent 44 44 44 44 44 44 44 45 44

-I 0.4075 0.2276 0.4075 0.2276 0.3912 0.2185 0.4075 0.2276 0.4081 0.2294 0.4075 0.2276 0.4075 0.2276 0.4112 0.2256 0.3912 0.2185

According to the above equation the mean error on the fibrin percentage in plasma was f 0.0075, in blood f 0.0041.

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H. C. Gram 289

TABLE VII-C.

Normal Individual.

Specimen No.

1 2 3 4 5 6 7 8 9

10

Cell volume.

per cent

39 40 40 41 40 40 41 40 40 39

Fibrin percentage in plasma.

0.2840 0.2683 0.2667 0.2691 0.2675 0.2667 0.2815 0.2807 0.2675 0.2807

0.1725 0.1622 0.1600 0.1595 0.1611 0.1600 0.1652 0.1678 0.1611 0.1715

Accord&g to the above equation the mean error on the fibrin percentage in plasma was f 0.0073, in blood f 0.0048.

My Technique for the Fib& Determination Has Been Compared with a Determination of the Fibrin Percentage by Whipping Out the .Fibrin (Table VIII).-The following technique was used: Fy venous puncture a specimen of 4.5 cc. was taken into citrate and treated in the ordinary way. Then 50 cc. of blood were placed in a graduated glass. This quantity is whipped for 10 minutes with a glass rod whose end is slightly widened.

TABLE VIII.

Fibrin Determinations by My Technique (I) Compared with Fibrin Determin- ations by Simple Whipping (II).

Speci- men NO.

Cdl Diagnosis. volume.

I

per csnt

1 Polycythemia.. . . 71 2 Nephritis.. . . . . . . 47 3 ‘< . . . . . . . 36 4 “ . . . , . . . 49 5 Polycythemia.. . . 73 6 Neurasthenia. . . . 44 7 Observation.. . . . 49 8 Normal.. . . . . . 40 9

‘I 48

-

I i

--

-

Fibrin ,ercentage n pllwms.

I

- I 7

I II

%k”,” fibrin

~epentape in blood.

(II-I)

0.25 0.07 0.09 +0.02 0.59 0.31 0.34 +0.03 0.28 0.18 0.16 -0.02 0.59 0.31 0.34 +0.03 0.36 0.10 0.12 +0.02 0.33 0.18 0.18 0 0.37 0.19 0.19 0 0.25 0.15 0.14 -0.01 0.30 0.16 0.16 0

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290 Fibrin Percentage in Blood

One then adds 5 cc. of fresh serum and whips for another 5 minutes. The defibrinated blood after standing for an hour is diluted with water and examined for clots, which may have been detached during the whipping or have been formed later.

The fibrin has collected itself as a reddish tube around the rod and may easily be slid over the top of it. It is washed in physio- logical saline solution till it is nearly colorless and then successively in water, hot distilled water, hot alcohol, and ether.

After drying to constant weight, the weight of fibrin in grams multiplied by 2 gives the fibrin percentage in blood.

TABLE IX.

A Comparison between Fibrin Determinations by My Method (I) and Fibrin- ogen Determinations by the Method of Whipple (II).

NO. Diagnosis.

I I ___-

per cent Nephritis.. . . . . . . . . . . . . . . . . . 41 0.53

“ . . . . . . . . . . . . . . . . . . . 49 0.59 Febricula . . . . . . . . . . . . . . . , . . 52 0.45 Diabetes. . . . . . . . . . . . . . . . . . . 41 0.33 Febricula; acromegalia . . . . . . 36 0.39

“ . . . . . . . . . . . . . . . . . . . 46 0.46 Pernicious anemia.. . . . . . . . . 27 0.23 Normal.. . . . . . . . . . . . . . . . . . . . . 44 0.25

“ . . . . . . . . . . . . . . . . . . . . . 43 0.31

7

Fib& mgen per. :entrtge ir

plasma.

II

-

: ;fi

f

. -

0.56 $0.03 0.60 +0.01 0.50 f0.05 0.35 +0.02 0.42 $0.03 0.52 +0.06 0.24 +0.01 0.29 $0.04 0.36 +0.05

Several specimens had to be rejected either because particles of the clot were detached or because the clot would not lose its color and so might be supposed to contain other elements than pure fibrin.

The determinations shown in Table VIII gave very concordant results. It must, however, be noticed that the values compared are the smaller fibrin percentages in blood, so that the differences would be larger if the percentages in plasma could be compared. A comparison between my technique for the fibrin determination and a determination of fibrinogen by the method of Whippk gave the results shown in Table IX.

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EC. C. Gram

Instead of oxalated plasma I used 20 cc. of titrated plasma, which were heated to 56-60” C. for 10 minutes. The precipitated fibrinogen was washed, dried, and weighed and the calculation of the percentage was made in the same way as by my own method.

From the lowest to the highest fibrin (fibrinogen) contents there was a very good concordance between the results of the two methods, though the fibrinogen percentages on the average were somewhat higher.

The results in pathological cases1 have been published in brief elsewhere (Gram, 32), so I shall give only the results of a series of observations o normal men and women.

TABLE X.

65 Normal Men.

Fibrin percent- Fibrin percent- age in plasma. age in blood.

Cell volume

per cent

Highest . . . . . . . . . . . . . . . . . . . . . . . . 0.36 0.19 51 Lowest . . . . . . . . . . . . . . . . . . . . . 0.20 0.11 43 Average . . . . . . . . . . . . . . . . . 0.27 0.14 48

TABLE XI.

86 Normal Women.

Fibrin percent- Fibrin ercent- age in plssma. age in \ load.

Cell volume.

per cent Highest . . . . . . . . . . . . . . . . . . . . . . . . 0.38 0.21 45 Lowest . . . . . . . . . . . . . . . . . . . . . . . 0.21 0.12 37 Average . . . _ . . . . . . . . . _ . . . . . . . . 0.29 0.17 41

The fibrin percentage in plasma is slightly lower in men than in women. The difference between the percentage per 100 cc. of blood is even larger, owing to the larger average cell volume in men’s blood.

The examination of the fibrin content in cases of anemia and polycythemia has shown that the percentage in plasma is the value kept constant during variations in the cell volume, so that the percentage per 100 cc. of blood is abnormally high in anemia and abnormally low in polycythemia.

1 Hyperinosis in infectious diseases, cancer, pregnancy, nephritis, chronic polyarthritis, etc. Hypinosis only in severe degeneration of the liver. The results will appear in full in the Acta med. Scandinav.

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NO. 1 2 3 4 5 6 7 8

- Re

peat

ed

Anal

ysis

fo

r ‘F

ibrin

in

8

Norm

al

Pers

ons

(Nos

. d

and

8 Ar

e M

en,

the

Res

t Ar

e W

omen

).

I II

III

IV

V

Jan.

22

Ja

n.

2!

Feb.

5

Feb.

12

In

terv

al

of 1

yr.

“ 24

“

31

“ 20

Fe

b.

1

“ 20

“

1

‘l 20

Ja

n.

2;

“ 20

“

2;

“ 21

“

2f

” 24

“

31

“ 6

“ 8

“ 4

“ 3

“ 3

‘( 3

I‘ 7

“ 12

“ 16

-J

“

‘I 11

“

“ 10

“

“ 10

“ 10

‘I 12

“

“ 1

“

“ 1

“

‘I 1

“

“ 1

“

-

D&3.

Fibrin

pe

rcenta

ge

in pla

sma.

Fibrin

pe

rcenta

ge

in blo

od.

II III

- IV

V

- I I

0.38

0.

38

0.35

0.

341

0.21

0.

22

0.19

0.

20

0.35

0.

33

0.35

0.

33

0.18

0.

17

0.18

0.

17

0.33

0.

33

0.30

0.

29

0.19

0.

19

0.16

0.

17

0.32

0.

34

0.33

0.

30

0.20

0.

21

0.20

0.

18

0.27

0.

24

0.27

0.

16!

0.15

0.

16

*

0.23

0.

26

0.27

0.

24

0.14

0.

15

0.15

0.

14

0.22

0.

13

0.20

0.

11

0.25

0.

24

0.24

0.

15

0.15

0.

14

0.22

0.

23

0.20

0.

21

0.11

0.

12

0.11

0.

12

-

0.37

0.

21

0.31

0.

18

0.31

0.

19

0.29

0.

18

Gzt

t- va

ria-

tion

from

av

er-

age.

0.02

0.01

0.02

0.02

0.03

0.02

0.02

0.02

- I

-

- I - 44

- II - 43

- III

- 45

- -

IV

V -

-

43

42

48

47

49

48

41

37

38

40

40

44

-

43

44

42

42

38

40

39

38

39

40

38

42

43

42

41

42

40

46

49

46

- -

-

Cell

volum

e.

37

43

-

*At

the

four

th

exam

inat

ion

No.

5

suffe

red

from

a

cold

. Th

e sp

ecim

en

neve

rthel

ess

was

anal

yzed

an

d sh

owed

a

sligh

t, no

t ab

solu

te

incr

ease

; fib

rin

per

cent

in

pla

sma

0.35

, in

blo

od

0.22

. Th

is re

sult,

of

cou

rse,

ca

nnot

be

use

d as

nor

mal

, sin

ce

all

infe

ctio

ns

will

prod

uce

a m

ore

or l

ess

pron

ounc

ed

hype

rinos

is.

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H. C. Gram 293

It has also to be settled whether the large variations in the normal fibrin percentage occur in the same person or whether each normal person has a certain level of fibrin content, which is kept fairly constant.

The last seems to be the case according to the results given in Table XII.

The fibrin was determined on six women (Table XIII) at differ- ent times of the day in order to see whether any variations could be traced or whether the digestive leucocytosis did influence the fibrin content.

This does not seem to be the case, the variations being always very small and independent of the digestive leucocytosis, which is very pronounced in the cases of Nos. 3, 5, and 6.

TABLE! XIII.

Fibrin Percentage in Plasma, Cell Volume Percentage, and Number of Leuco- cytes at Different Times of the Day.

NO.

1 0.33 2 0.32 3 0.33 4 0.29 5 0.35 6 0.21

. : _

-

!p.m. E

0.33 0.32 0.33 0.31 0.36 0.22

-

- I-

ipI& 1 - 1

0.37 0.21

-

Cell volume.

10a.m. 2p.m. l3p.m. --- oer cmt per cent per wnc

39 38

42 41 38 40 42 42 44 43 43 42 40 41

STJMMARY.

-

-

I _ - t

-

Leucocytes per cmm.

10% m. 2 p. tn. 6 p.m. ---

7,500 7,100 5,900 7,700 6,500 11,700 8,200 9,900 6,500 10,900 ~11,800 5,400 5,700 9,100

1. A technique is described by which the fibrin percentage in blood and plasma may be determined by recalciflcation of 2 cc. of clear cell-free plasma obtained from 5 cc. of titrated blood (0.5 cc. of 3 per cent citrate + 4.5 cc. of blood). This includes a determi- nation of the cell volume.

2. On the same specimen one may determine the platelet count by Thomsen’s method and the coagulation time by a method indicated by the author.

3. The technique described is shown to fulfill the conditions exacted for a trustworthy method.

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Fibrin Percentage in Blood

4. The results by this technique in the blood of normal men and women are put forward. When the stress is laid upon the fibrin percentage per 100 cc. of plasma, this is caused by the results found in diseases of the blood which together with other pathologi- cal results have been published in brief elsewhere.

BIBLIOGRAPHY.

1. Hoppe-Seyler, F., Handbuch der physiologisch- und pathologisch- chemischen Analyse fur Aertze und Studirende, Berlin, 8th edition, 1909.

2. Samuely, in Abderhalden, E., Handbuch der biochemischen Arbeits- methoden, Berlin and Vienna, 1910, ii, 375.

3. Dastre, quoted by Richet, C., Dictionnaire de physiologie, Paris, 1904, vi, 411.

4. Andral, Essai d’hematologie pathologique, Paris,’ 1845. 5. Doyon, M., Morel, A., and Kareff, N., Compt. rend. Sot. biol., 1906, lviii,

681. 6. Pfeiffer, I., 2. l&n. Med., 1897, xxxiii, 215. 7. Cullen, G. E., and Van Slyke, D. D., Proc. Sot. Ezp. Biol. and Med.,

1915-16, xiii, 197. 8. Bang, I., Mikromethoden, Wiesbaden, 1920. 9. Green, J. R., J. physiol., 1887, viii, 373.

10. Goodpasture, E. W., Bull. Johns Hopkins Hosp., 1914, xxv, 330. 11. Reyhe, Nachweis und Bestimmung des Fibrinogens, Inaugural disser-

tation, Strassburg, 1898. 12. Doyon, M., Morel, A., and Peju, G., Compt. rend. Sot. biol., 1905, lvii,

657. 13. Frederick, Recherches sur la coagulation du sang, Gand, Paris, and

Leipsic, 1878. 14. Whipple, G. H., Am. J. Physiol., 1914, xxxiii, 50. 15. Miiller, P. T., Beitr. them. Physiol. u. Path., 1905, vi, 454. 16. Whipple, G. H., Mason,V. R., andPeighta1, T. C., Bull. Johns Hopkins

Hosp., 1913, xxiv, 207. 17. Bunge, quoted by Sahli, H., Z. klin. Med., 1905, lvi, 264. 18. Richet, C., Dictionnaire de physiologie. Fibrine, Paris, 1904, vi, 410. 19. Erben, F., Z. klin. Med., 1902, xlvii, 302. 20. Arthus, Precis de chimie physiologique, Paris, 1909. 21. Arronet, Quantitative Analyse des Menschenblutes, etc., Dissertation,

Dorpat, 1887, 65. 22. Schneider, Die Zusammensetzung des Blutes, etc., Dissertation, Dor-

pat, 1891, 22. 23. Biernacki, E., Z. klin. Med., 1897, xxxi, 279. 24. Becquerel and Rodier, Recherches sur la composition du sang, Paris,

1844. 25. Berggriin, Arch. Kiderh., 1895, xviii, 178.

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H. C. Gram 295

26. Kruger, ober das Verhalten des foetalen Blutes, etc., Dissertation, Dorpat, 1886,37.

27. Meek, W. J., Am. J. Physiol., 1912, xxx, 161. 28. Thomsen, O., Acta med. Scundinav., 1920, liii, 507. 29. Gram, H. C., Arch. Int. Med., 1920, 325. xxv, 30. Gram, H. C., Acta med. Scandinav., 1920-21, liv, 1. 31. Gram, H. C., Bull. Johns Hopkins Hosp., 1920, xxxi, 364 (see corrections

in following issues). 32. Gram, H. C., Arch. Int. Med., 1921, xxviii, 312. 33. Norgaard, A., and Gram, H. C., J. Biol. Chem., 1921, xlix, 263.

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H. C. GramPLASMA

PERCENTAGE IN BLOOD ANDDETERMINATION OF THE FIBRIN

A NEW METHOD FOR THE

1921, 49:279-295.J. Biol. Chem. 

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