EFFECT OF HaMORRHAGE ON THE HXMO- LYTIC TITRE OF THE SERUM OF RABBITS.

JOHN MILLS, M.B., Ch.B. (Grocers’ Scholar). From the Department of Pathology, University of Cambridge.

THIS s tudy is concerned wi th the effects of hzmorrhage on the antibody titre of the serum of rabbits immunised against aheep erythrocytes. A considerable number of experiments bearing on this problem have already been carried out bu t the results obtained are by no means concordant.

Historical.-Most of the work so far carried out has been concerned with the production of diphtheria antitoxin or typhoid agglutinins. Salomonsen and Madsen (1898), in experiments on horses and goats immunised against diphtheria toxin, found that after a large bleeding there was a fall in the antibody content of the serum proportional to the volume of blood removed. Following this fall there was a rise, which in one case was sufficient to restore the titre of the serum to the level at which it stood previous to the bleeding. This experiment was carried out during the sharp rise in the antibody curve which occurs a few days after an injection of antigen. In their other experiments also the fall was followed by a rise, but in none of them did the titre reach the level a t which it had stood previous to the hEmorrhage. The same authors showed that the antibody content of the milk followed a similar course to that of the serum. Nicolle (1904) studied the effects of small bleedings (10 c.c.) on rabbits immunised against B. typhosus. He found that the effect of bleeding during the period of fall in the agglutinins was either to arrest the fall or convert it into a rise. He also observed an increase in the number of white cells which he considered might be a source of agglutinins. Friedberger and Dorner (1905) bled rabbits before and after injection of antigen (goat blood). They concluded, from comparison of the titres of bled animals with unbled controls, that small bleedings before and during immunisation increase the ability of animals to produce hamolysin but that large bleedings have the opposite effect. Rothberger (1906) also studied the effects of bleeding rabbits immunised against B. typhosus. He found that a fall occurred immediately after the bleeding to be followed later by a rise if the bleeding had been carried out within fourteen days of the last injection of antigen. He considered that any rise that was observed was due rather to the last injection of antigen than to the bleeding. He gives a review of the literature and criticises Friedberger’s results on the grounds that individual variations iu ability to produce antibody render valueless comparisons between different animals. Whitehead (1911) showed that in typhoid patients a marked increase in the agglutinating power of the serum is to be expected after intestinal hsmorrhage and considered that this increase was due to the bleeding. His work was based on the findings of Schroeder whose work he quotes at great length. The latter worker concluded, from experiments on rabbits, that it was possible to convert a falling agglutinin curve into a rising curve by means of bleeding. When the titre was falling steadily he was

579

able to produce, by repeated small bleedings, a rise to a level above that at which it stood previous to the hcemorrhage. O’Brien (1914) concluded that bleeding had only a limited adverse influence on the antibody titre of a horse.

Methods.-Rabbits were treated a t three or four-day intervals by intravenous or subcutaneous injections of a 50 per cent. suspension of sheep erythrocytes. The cells were washed five times in twenty times their bulk of 0’85 per cent. sodium chloride solution. Four or five injections were given, the dose varying between 1 C.C. and 5 C.C. according to the size of the animal and the stage of treatment a t which the injection was given. After the last injection the rabbit was bled 5 C.C. from the ear vein on several successive days. The blood was collected in sterile test tubes and the serum drawn off twenty- four hours later and heated for half an hour a t 56°C. on two successive days to inactivate and ensure sterility. When a number of daily samples had been obtained, the rabbit was subjected to a bleeding large enough to reduce the haemoglobin percentage of the blood to 50 per cent. or less. During this large bleeding the blood was collected in 5 C.C. amounts and treated as described above. I n this way several samples of serum were obtained and the course of any change that might occur in the antibody content of the serum during the bleeding could be followed more closely than had hitherto been done. During the period of recovery from the ansmia induced by the haemorrhage a blood sample was taken each day.

The hzemoglobin estimations were made on the Kober colorimeter. A series of test tubes was prepared each tube containing 10 c.c of distilled water to which a trace of ammonia had been added. For each estimation 40 c.mm. of blood was added to one of these tubes and coal gas bubbled through until the hamoglobin was saturated. Before the bleeding was commenced four such samples were taken, pooled and used throughout the estimations as a 100 per cent. standard. All readings were made against this standard, or against a 7 5 per cent, standard made by diluting it. I n several of the experiments these estimations were compared with readings on the Kaldane hamoglobinometer. The two sets of readings were found to correspond to within 5 per cent., usually the difference was 2 or 3 per cent. During the bleeding a t least one such estimation wits made for each 5 C.C. of blood taken. A reading was also taken each day during the period of recovery.

The titrations of the hsmolytic power of the serum were carried out in 3”xV test tubes, each tube receiving 1 C.C. of a dilution of the rabbit serum, 1 C.C. of a 1/20 dilution of fresh guinea-pig serum and 0% C.C. of an erythrocyte suspension standardised by counting to contain approximately 700,000 cells per c.mm. The unit of hzmolysin was fixed as the smallest amount of serum which, in the presence of an excess of complement, caused complete haemolysis of 0’5 C.C. of the standard cell suspension. The tubes were incubated a t 40°C. for two hours, during which time they were shaken frequently, then removed to the ice chest and left overnight to settle, the reading being taken the following morning. The titre of the first sample of serum obtained during the large bleeding was taken, for convenience, to represent 100 per cent.

Results.

A total of nine rabbits were bled, of these seven were immunised by intravenous injection, two by subcutaneous injection. Five of them were bled 18 days or more, one 11 days and three 4 days after the last injection of antigen. All, except one tha t died during the

HKMOL YSIN AFTER NEMORRh-AGE 561

165 165 125 100

100 83 71 71 71 62 62

50 41 41 48 48 62 62 62

bleeding, were watched during the period of recovery. Rabbit 548 is selected as a typical experiment and the results are given in detail in table I. and charts I. and 11. I n the other experiments identical results were obtained with the exception of experiments 549 and 731 which are referred to again below.

...

...

...

... 100

94 76 70 68 63 63

53 46 47 56 56 71 71 93

TABLE I.-Xabbit 548.

Weight 2850 grms. Last injection of antigen 9.11.23. On 4.12.23 was bled 80 C.C. from ear vein. After 14.12.23 the serum titre fell steadily.

6.12.23 7.12.23 8.12.23

10.12.23 12.12.23

Date.

3 1.

5 39

5 ,, 5 ,9

5 ,9

5 1,

30.11.23 1.12.23 2.12.23 3.13.23

4.12.23

Amount of bleeding.

5 C.C. 5 ,, 5 9 9

5 1,

5 ,9

20 ,, 20 ,, 10 ,, 10 ,, 10 ..

5.12.23 1 5 ,,

14.12.23 1 5 ,,

Time since beginnlng of bleeding.

Hamolysin Hsmoglobin per cent. per cent.

kaDbit 548 shows a marked fall in the lytic power of the serum as a result of the bleeding, and this fall runs parallel with the fall in the concentration of the hsmoglobin of the blood. This was observed in every experiment. One must conclude either that the extravascular j u i d s of the body, which are called upon to restore the blood volume to i ts normal level during and after the hmnorrhage, contain no hmmolysin or, if they do contain uny hcemolysin, that it i s unable to penetrate the endothelium along with the other constituents of the j u i d s .

During the period of recovery, when the hsmoglobin percentage of the blood is steadily increasing, there is an increase in the con- centration of the hsmolysin of the serum. This rise in the antibody curve was observed in all but two of the experiments, 549 and 731 showed a continued fall after the bleeding.

Rabbit 549, immunised by intravenous injection, WRS the second experiment of the series. I n the titrations of the Rerum samples the dilutions were so arranged that each tube contained 2 5 per cent. less serum than the preceding one. In the later experiments it was realised that this difference was too

58 2

1 io

160

150

140 .

1%

120

110

ml%. g o .

8 0 .

70

6 0 .

50

40

J. MlLLS

.

,

.

.

.

.

.

.

Chart I.

Rabbit 548

@--9 60 .

50

40

30

20

LO

Raemoglobin 0- - -e- - -e

Xaemolysin -

4 Bled 75 c.c..-}

1 2 3 4 3 0 1 Hours since beginning to bleed-

Chart I1 Rabbit 545

I 22 23 24 25 26 27 28 29 30 31 32 33 34 55 Days since last injection of antigen.

HAZMOL YSIN AFTER HATMORRHAGE 583

large and the titrations were arranged to show much smaller variations in the haemolytic power. It is very probable that a rise in the hsmolysin curve would have been observed i n this experiment, if the titrations had been carried out as in the later experiments.

Rabbit 531 (chart III.), immunised by subcutaneous injection, also showed a continuous fall in titre after hsmorrhage. This is the only experiment in which the bleeding coincided with the summit of the curve of hsemolysin production and an explanation of the continued fall is suggested later. That the route of immunisation is not sufficient explanation is shown by rabbit 841 (chart IV.) which also received subcutaneous injections and shows a curve similar to rabbit 548.

I n no case did the increase in titre observed after the bleeding exceed 25 per cent. of the titre of the first sample taken a t the commencement of the bleeding. The hzmolysin curve follows approximately that of the hsmoglobin until the top of the rise is reached ; afterwards however the hsmoglobin continues to rise while the hemolysin falls.

Boycott and Douglas (1909) showed that after a large bleeding overdilution of the blood may occur to such an extent that the volume exceeds the normal by as much as 30 per cent. The question arises whether the rise in the hsmolysin curve is a true one, or merely an apparent one dependent on the return of the blood volume to. normal after overdilution. Before the haemorrhage rabbit 548 had a calculated blood volume of 135 c.c., assuming 48 C.C. of blood per kilo of body weight (Boycott 1912). The bleeding amounted to 80 c.c., ie. 59 per cent. of the original volume. A period of two hours elapsed between obtaining the first and last samples of blood, and during this time there was a considerable amount of dilution as is evidenced by the fall in the hzernoglobin percentage of the blood. As the 80 C.C. of blood removed from the vessels is in part represented by the extravascular fluids which had entered the circu- lation whilst the bleeding was being carried out, considerably less than 60 per cent. of the rabbit’s original blood was removed by the hsmorrhage.

It is indeed possible to calculate the proportion of original blood to tissue fluid in the 80 C.C. removed: thus 5 C.C. removed when hsmoglobin is 100 per cent.=5 C.C. blood and 5 C.C. removed when hemoglobin is 96 per cent.=4*8 C.C. blood. By correcting for the dilution it is found that only 65 C.C. or 48 per cent. of the rabbit’s original blood was taken out by the bleeding. The lowest point reached by the ha?moglobin was 46 per cent. i.e. 6 per cent. below what was to be expected. Overdilution to 150 C.C. is sufficient to. account for this difference. A return to normal volume would cause an increase in serum titre up to 45 per cent., whilst the increase observed was to 62 per cent., and overdilution to a volume of 205 C.C. would have to occur if this is the sole explanation of the phenomenon.

584 j. MILLS

100

90

80

70

60

50

40

50

20

10

1W

120

110

LOO

90

Bo

10

60

50

40

jo

20

10

Chart 111. Rabbit 731

Haemoglobin + - -+ -

0 I

I Haemolyein I I

I

9 10 11 12 1J 14 15 16 17 18 19 Day8 e ince last Lnjection of antigen-

Haemoglobin . .+ - -0

Haemolysin

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Day0 since last injection of antigen

HAMOL YSlN AFTER H&-MORRHAGE 586

Weight

sj&.

I n table 11. the above details are given for seven animals, and it is seen that in experiments 548, 550 and 841 the calculated amount of overdilution falls a long way short of that necessary to account for the rise in serum titre.

Original blood Estimated removed.

blood amouIlt ___

c.c. Amount volume. of c,c. bleeding.

C.C.

TABLE IT.

zowest. Per

cent.

-

No.

__

548 549 550 607 608 731 841 -

Highest Per cen6

~- 80 55 50

105 G O 50 90

I ---- i

65 45 41 76 47

' 40 68

1780 1650 3000 2235 1800 2200

80 75

145 100

80 100

__

Per cent.

50 52 27 43 34 45

48 56 55 52 47 50 66 -

50 61 41 52 34 66

Lowest hatno- globin. %er cent.

46 45 46 33 43 43 42

Overdilution.

Cal- ulated C.C.

-

150 none none 210 123

93 none __

rhat necessary to account for rise in

liremol ysin. C.C.

205

88 220 120

147

...

...

A consideration of these figures renders it extremely doubtful that overdilution is the sole explanation of the phenomenon. It was thought that there might be an increased production of antibody along with the increased activity of the blood forming organs, because the hemolysin curve follows the hsmoglobin for some days after the hsmorrhage. If this is the explanation it ought to be possible to diminish or abolish the rise in the hsemolysin curve by replacing the blood removed during the bleeding with an equal or greater amount of blood from normal rabbits. The transfused blood would be retained within the circulation of the recipient and there would be no stimulus to the blood forming organs. That blood transfused to make good a hemorrhage is retained has been shown by various workers (Boycott and Douglas 1910, Ashby 1919). The former found this to be the case even though the animal had been previously trained in getting rid of foreign blood cells.

Transfusion experiments. The transfusions were carried out by means of the tubes described by

Kimpton and Brown (1913) by which method it was possible to measure the amount of blood transfused. An immunised rabbit was bled 50 C.C. from the carotid artery. The first 10 C.C. of blood were collected in a sterile tube and named first bleeding. Immediately afterwards approximately the same volume of blood from a normal donor was injected into the jugular vein. This procedure was repeated as many times as possible, the first 10 C.C. of each bleeding being set aside and numbered. After the last transfusion a final blood sample was taken and named last bleeding. Usually between 30 C.C. and 50 C.C. of blood could be transfused a t each step without the use of any anti-coagulant, and it was possible to replace about 80 per cent. of the rabbit's blood with blood from normal animals. Al l rabbits were examined for the presence of normal hamolysin before being used as donors. I n most cases the serum of the donors contained not more than ten units normal hemolysin per c.c., two showed twenty units per C.C. : the immunised rabbits both gave a titre of one

586 J MILLS

thousand units. At the second transfusion rabbit 836 had a titre of five hundred units per C.C. All operations were carried out under general anssthesiaand with aseptic precautions. Ether was the anaesthetic except when otherwise stated.

Rabbit 836.--23.9.24 to 3.10.24 received four intravenous injections of sheep cells.

11.10.24 was bled 55 C.C. from the carotid artery and transfused 45 C.C. into the jugular vein. Normal rabbit 844, whose serum contained twenty units normal h~molys in per c.c., was used as donor. The rabbit recovered rapidly from the effects of the operation and three hours later was taking food.

14.10.24 there was a slight swelling at upper end of wound. 15.10.24. swelling increased, two sutures removed and about 2 C.C. of pus

17.10.24 still suppurating, animal killed. evacuated.

TABLE 111.-Rabbit 835.

I I Titre.

I Date. I Units.

1 9.10.24 . . . . I 1000 11.10.24-

First bleeding , Second bleeding . .

12.10.24 . . . . 444 13.10.24 . . . 500 14.10.24 . . . * . I 500 15.10.24 . . . . 570 17.10.24 - . . . 1 666

Chart V

Rabbit 855

Percentage.

125

100 42 55 62 62 71 84

7 8 9 10 11 12 1> 14

Days since laat Injection of antigen.

In table 111. and chart V. are shown the variations in titre of the serum of rabbit 835.

HAMOL YSIN AFTER HAMORRHAGE 587

15.10.24- First bleeding . Second bleeding . Third bleeding . Last bleeding .

Rabbit 8S6.-Immunised at the same time and in the sa,me way as 835. On 15.10.24 was bled 150 C.C. and transfused 165 C.C. From 16.10.24 to

8.11.24 frequent samples of serum were obtained. On 8.11.24 a second bleeding and transfusion was carried out, using the carotid and jugular of the opposite side. The steps of the operations are given below. The rabbit stood both operations very well.

TABLE IV.-Rabbit 836.

8.11.24- 1000 First bIeeding .

625 Second bleeding . 415 Third bleeding , 310 Fourth bleeding .

Last bleeding .

Time.

1

Amount of bleeding.

9.11.24 10.11.24 11.11.24 12.11.24 13.11.24 20.11.24

15.10.24- 10.30 to 10.66 . 11.12 ,, 11.16 . 11.26 ,, 11.36 . 11.45 . . . 11.50 . . . 11.30 to 11.40 . 11.42 ,, 11.54 . 12.11 ,, 12.15 .

8.11.24--

12.1 ,, 12.10 .

55 C.C.

46 ,. 42 ,, ...

8 $9

55 C.C. 35 I ,

50 ,, 50 ,.

Amount of transfusion.

Unit8 of normal lysin per C.C. of donor's 8erum.

Rabbit 845, less than 10

,, 847, ,, 10 ,, 854, ,, 10

1 9 846, 9, 10

...... Rabbit 864, 10 units.

,, 866, less than 10 ,, 868, ,. 10 ,, 867, 20 units.

The above table shows the steps of the operations on rabbit 836. In the second operation the artery was let go whilst carrying out the last bleeding and a volume of blood estimated a t 10 C.C. was lost. The carotid artery was tied and a last transfusion carried out. A tinal blood sample was taken from the ear vein two hours later, when the rabbit had recovered from the anaesthetic. Ether was used as the anaesthetic for the recipient. The donors each received a subcutaneous injection of 10 C.C. of a 25 per cent. solution of urethane.

The results of the titrations of the serum samples of this rabbit are shown in table V. and chart TI.

TABLE V.-Rabbit 836.

1 1 Date. Units of lysin per C.C. serum. Date.

11.10.24 . ... ... 12.10.24 . : : I :% 1 1

16.10.24 . . . 17.10.24 . . . 18.10.24 . . . 19.10.24 . . . 20.10.24 . . . 22.10.24 . . . 25.10.24 . . . 27.10.24 . . . 30.10.24 . . .

355 415 500 625 830 830 625 585 585

...

... ...

Units of lysin per C.C. serum.

...

...

500 250 200 200 142

200 220 290 350 445 445 ... ... ...

In both the transfusion experiments the rise in the hamolysin It would curve is far more marked than in the bleeding experiments.

588 J. MILLS

seem therefore that the rise cannot be explained by an increased activity of the blood forming organs leading to a renewed production of antibody. It is suggested that the two following factors are concerned in the production of this rise. First, a t any given point in the antibody curve, formation and destruction of haemolysin are so balanced as to maintain the serum titre a t a certain definite level. If any cause reduces the serum titre the tendency will be for it to swing back to the point normal for the period of immunity. Second, after any considerable reduction in serum titre, one would expect that a redistribution of antibodies occurs. Normally these are present in the 150

120

110

100

90

30

:i,

60

50

40

30

20

10

lymph and presumably also in the tissue fluids (Greer and

. 12 16 li, 2o 22 24 26 20 X, 32 54 36 38 40 42 44 ~ a p since l a a t inject ion of antigen.

Eecht 1910). After bleeding the blood volume is maintained at the normal level by transferring fluid from the alimentary tract and from the tissues to the vessels. The former fluid contains no anti- bodies, and the antibodies in the latter are probably unable to penetrate the enclothelium along with the other constituents. If this is the case the concentration of antibodies in the tissue fluids after bleeding will be greatly in excess of the normal relation to their concentration in the serum, and the antibodies will then be transferred from the former to the latter until the normal relation is re-established. If the bleeding is performed a t the maximum of the curve, a very large volume of blood will have to be removed in order to cause a fall in serum titre which will be greater than will normally occur in both the serum and tissue fluids. Experiment 731 was carried out on the eleventh day after the last injection of antigen, which is about the time that one

N E M O L YSIN AFTER HEMORRHAGE 589

would expect the maximum of the antibody curve. Therefore, even if the rabbit had not been bled, a very marked fall in the curve was to be expected.

If this hypothesis is correct, a rise should always occur subsequent t o haemorrhage, except when the maximum falls a t or near the time of bleeding. If the bleeding is effect,ed previous to the maximum the rise may be to a point beyond that a t which it stood immediately before the bleeding. After the maximum the rise could never be continued beyond this point.

My thanks are due to Dr R. A. Webb and Dr H. Florey for considerable assistance in the operative procedures.

ASHBY, W. . . . . . . . BOYCOTT, A. E., AND DOUGLAS,

BOYCOTT, A. E., AND DOUGLAS,

BOYCOTT, A. E. . . . . . . FRIEDBERGER AND DORNER . GREER, J. R., AND BECHT, F. C . KIMPTON, A. R., AND BROWN.,

NICOLLE, C. . . . . . . . O’BRIEN, R. A. . . . . . . ROTHBERGER, C. J. . . . . SALOMONSEN, C. J., A N D

WHITEHEAD, H. . . . , . .

C. G.

C. G.

J. H.

MADSEN, T.

REFERENCES.

Journ. Exp. Xed., 1919, xxix. 267. This Journu,Z, 1909, xiii. 256.

This Journal, 1910, xiv. 294.

This Journal, 1912, xvi. 485. Centralbl. f. Baht., Orig., 1905, xxxviii. 544. Amer. Journ. Phys., 1910, xxv. 292. Journ. Anzer. Med. ASSOC., 1913, lxi. 117.

Ann. de l’Inst. Past., 1904, xviii. 209. This Journal, 1914, xviii. 69. Centralbl. f. Bakt., Orig., 1906, xli. 562. Ann. de Z’hst. Past., 1898, xii. 763.

h n c e t , 1911, ii. 1067.

JOURN. OF PATA.-VOL. XXVIII. 2 P