FROM T H E BACTERIOLOGICAL LABORATORY OF T H E N O R W E G I A N A R M Y

CHIEF: P R O F E S S O R T H . THJBTTA, M . D . A N D FROM T H E N O R W E G I A N R E D CROSS IILOOU

TRANSFUSION SERVICE I N OSLO. CHIEF: O T T O H A R T M A N N .

SUBGROUP AZB WITH WEAK A-RECEPTOR AND IRREGULAR AGGLUTININ ai (LANDSTEINER)

AND ITS RELATION TO BLOOD- TRANSFUSION. By Otto Ha?trnurin,.

(Received for Publication November 28th, 1938).

According to Landsteiner, Moss and Jansky, four types of the human blood is to be distinguished. These types are named according to their contents of agglutinogen in the red globules, with 0, A, B and AB. Each type is further characterised by the contents of agglutinins in the serum, type 0 having the agglutinins anti-A and anti-B, type A having the agglutinin anti-B, type B having the agglutinin anti-A and type AB hav- ing no agglutinins in the serum. The agglutinin therefore cannot be present together with the corresponding aggluti- nogen (receptor). According to A . Lauer and F. Bernstein’s theories, all individuals produce both agglutinins (anti-A and anti-B), but i f a receptor is present for one or both agglutinins, the agglutinin is fixed in the moment of nascency and is con- sequently not present in the serum. The properties of the bloodtypes are inheritable. According to F . Bernstein’s hypo- thesis there are three allelomorphe genes, A, B and R ; of these A and B are dominating and R is recessive; only two of these genes are present in one individual.

E . von Dungern and L. Hirzfeldl) have pointed out that

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there seems to be two different forms within the A-type. This was shown by an absorption-experiment. The fact was first fully cleared by Thomsen and his collaborators’ researches 1928-31. Two subgroups within the A-type are now to be distinguished, A, and A,; and in the same way we have two subgroups within the AB-type, A,B and A,B. 0. Thomsen, V. Friedenreich and E . Worsaae2) completed Bernsteins theory of heredity by the supposition of two different genes A, and A,, both dominating R, and A, dominating A,. While these properties from a hereditary point of view differ in quality, they differ only appearently in quantity when examined sero- logically in iso-sera, as both are agglutinated in anti-A serum to a different extent, A, stronger, A, weaker. This mere quan- titative difference in serological respect has long time ago been pointed out by Thomsen3). Further Thomsen4) has shown that the A-property can be so much the less prominent when combined with B, that it may appear as i f B to a cer- tain extent dominates A. This is most marked in the case of A,, i. e. in the A,B-group. The A-property of this group may therefore, even in adults, be hidden and found only with great difficulty by agglutination in iso-sera. Every one per- forming clinical determination of bloodgroups with iso-sera may thus be inclined to take A,B for B. Two such cases with non-demonstrable A-receptor in the AB-group in adults have been described by Thomsen6). Similar cases have been men- tioned in the literature several times, but often discoveries of this kind have been explained in a wrong way, or not comp- letely understood.

The sheepblood immune-sera of rabbits may be of great help in demonstrating the A-receptor in cases of this kind. F. Schiff and L. Adelsberger6) have shown that human A-corpuscles and sheep-corpuscles have a common antigen, namely a heterophilic antigen, and serum from rabbits im- munised with sheep-corpuscles consequently agglutinate human A-corpuscles. Not all sheepblood immune-sera are of equal fitness, but we can produce sera that distinctly agglutin- ates A-corpuscles in cases where no agglutination is produced

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even by the strongest iso-sera. Dolter?) and Sachs8, 9, suggest that these sera ought to be used for blood-grouping instead of iso-serum anti-A. When a sheepblood immune-serum is used, this is only as a control on group 0 and group B after the usual typing in iso-sera. Biirkle- de la CarnpJO) recom- mends a similar method by determination of blood groups before blood-transfusion. A positive result ought to be verified by a subsequent demonstration of the agglutinins in the serum of the individual examined.

The contents of agglutinogen in the red corpuscles is veri- fied by the common clinical blood-grouping method. By closer examinations the contents of agglutinin in the serum is also tested, as the agglutinating power of serum is also charac- teristic of each of the four blood-types. In this way double security would be gained. But, as emphasized by Raestrupl l ) , false results by the blood-type AB may slip in, even by using this course, which we are going to describe in the following case.

An adult individual X. is typed as B by the slide aggluti- nation method. By the examination of the serum against fresh test-corpuscles from type A and B, the A-corpuscles are agglu- tinated. This fact verifies type B, as B-individuals' serum con- tains the agglutinin anti-A.

When, however, titration of the agglutinin was made, a titre of only 8 at room-temperature was found. This is a lower titre than that generally found by titration of iso-agglutinins. The corpuscles of X. were therefore on a later occasion exa- mined anew by means of a sheepblood immune-serum. Strong agglutination immediately occured. By a new attempt with iso-sera on a slide no agglutination was seen in a serum with a titre of 256 against most A-corpuscles, but, on the other hand, distinct agglutination occured in a serum with a titre of 1024.

Next titration by the test-tube method was made with the corpuscles, compared with two different A,-corpuscles. The titration was made both with iso-serum at Oo, 20° and 3 7 O C, and with sheepblood immune-serum at 3 7 O C . The concen- tration of the corpuscles was 112 per cent.

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Anti-A iso-serum Titre Corpuscles of type

00 c 1200 (: 1370 c Sheepblood immune- serum of rabbit. Titre

A1 I1

I 2048 I 1024 ~ 512 I 2560

2560

APB (Corpuscles of X) I 64 1 16 1 8 I 640

It appears from this experiment that, while there is a large difference between the A,-receptor and A,-receptor when tested in iso-sera, this difference is by far less prominent in sheepblood immune-serum.

According to these experiments, there is no doubt that the individual X. belongs to the group AB. This group is not, according to the general rule, likely to have agglutinins in the serum. In this individual, however, there has been found an agglutinin that acts on A-corpuscles. It does not, of course, act on the indicidual’s own corpuscles and is therefore not the regular agglutinin anti-A, but the ,irregular<< agglutinin a, (Landsteiner)12s 13, 14), that acts only on A,- and not on A,- corpuscles.

In order to understand the existence of this agglutinin, we must try to find out in which relation A, and A, stands to each other when we use an anti-A iso-serum.

Friedenrei~hl~) has shown that i f an anti-A serum is ab- sorbed with a given quantity of A,-corpuscles, the absorbed serum only agglutinates A,- and not A,-corpuscles; the iden- tical result can be obtained by using a smaller quantity of A,-corpuscles in the absorption-test instead of using a greater quantity of A,-corpuscles. There consequently seems to be only a quantitative difference in the power of fixing agglu- tinin by A, and A,; A, fixes more agglutinin than A,. Lattes and Cavazzutil6) and Mino17) have previously shown that i f a sufficient quantity of A,-corpuscles is used, all the agglu- tinin anti-A can be resorbed.

Friedenreichls) has further shown that the regular iso-

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agglutinin is no unity, but composed of fractions with various thermal amplitudes. As an example he uses an anti-A serum which is absorbed with 1/64 volum of corpuscles A, at 37" C:

37oc 20oc oo c Titre before the absorption 16 64 64-128 Titre after the absorption 1 16 64

SO the absorption at 37" C has evidently removed the agglu- tinin-fractions that act at this temperature; to a lesser degree the fractions that act best at 20" C and least those with it

maximal action at 0" C. If the serum is absorbed anew at 21" C, the remaining agglutinin will show a strong conformity with the agglutinin GI,. Such an absorbed serum represents, according to FriedenreichlO) , an artificial a,.

As an further example that the difference between agglu- tinogen A, and A, only appears as a quantitative difference, we can here mention that A, in the group A,B, where the property of A to a certain extent can be suppressed by B, not always is sharply agglutinated by such an absorbed serum or by the agglutinin al. In this case A, acts in just the same way as A, because the power of fixing the agglutinin has been lowered.

According to these facts we can explain the presence of the agglutinin a, in the serum of some A,B-individuals. The A,-receptor is in this case weakly developed and only fixes the agglutinin anti-A slightly. A,B-individuals produce, like all the rest, both anti-A and anti-B agglutinins. The latter is easily fixed to the B-receptors of the organism, while A,- receptors are not able to fix all the anti-A that are produced, but only the greater part, and especially the fractions acting at body-temperature.

So there evidently is a connection between the A,-receptor and the agglutinin a, in the way that the weaker the receptor, the stronger the agglutinin. Friedenreich and Waaler2") hold that they have found such a connection. It must, however,

be taken for granted that more or less anti-A is produced in the various A,B-individuals as well as in B- and O-indivi- duals. Even i f therefore the power of fixing agglutinin by the A,-receptor is the same in several individuals of this group, the production can vary, and the resultant, a,, be of different force.

Landsteiner's irregular agglutinin a , is consequently a fraction of the regular agglutinin anti-A. It hardly acts at body-temperature, but can act even at a high room-tempe- rature; it acts best at a low temperature. It has a thermal amplitude that stands nearer the cold-agglutinins than the type-specific iso-agglutinins.

The knowledge of the cold-agglutinins we essentially owe researches by Kette121). They are unspecific, acting on the corpuscles whatever the type might be, altough to a various extent; they act upon own corpuscles too. They rarely act at a usual room-temperature and never at a higher room-tem- perature. They are more thermo-labile than the specific agglu- tinins. Heating to 56O C for 60 minutes does not destroy them, but at 60° C they are distinctly weakened in 5 minutes and destroyed in 30-60 minutes. When the cold-agglutinins are kept at room-temperature, they will, as a rule, be destroyed in a week or earlier, while they can keep unchanged for a couple of months in a refrigerator. If the serum is separated from the coagulum at O0 C, the cold-agglutinin will be fixed by own corpuscles. If there are only a small amount of cold- agglutinins in the serum, the greater part may be removed in this way, but i f the amount is large, only the smaller part can be removed.

The cold-agglutinins are often abundantly present together with the agglutinin a,, and if the cold-agglutinins are not removed, a , will be able to show its specific reaction only within a very small space of temperature, for instance be- tween 1 5 O C and 1 8 O C, as the cold-agglutinins will enter in action below this temperature.

In the case here mentioned, there was in the serum, be- sides the agglutinin a,, an abundance of cold-agglutinins,

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which acted strongly on own corpuscles. Various experiments were done to separate these agglutinins from each other. The table below shows the titre of the cold-agglutinins an the agglutinin a , against corpuscles of different blood-groups at various temperatures, and the titre against the same cor- puscles after various attempts to differentiate the agglutinins.

Corpuscles from type :

A1 1 A1 11 A2B (own) B I R I1 0 1 0 I1

Serum from individual A2B

Titre: I Titre: I Titre: I I Titre:

Kept hy 37O (: for 5 days

Titre :

Both heating to 570 C for 2 hours and 5 days’ keeping at 37O C destroyed all cold-agglutinins, but a great part of the agglutinin a , too. The absorbtion at Oo C with 8/1 volum own corpuscles (own corpuscles were used in order to avoid misinterpretings as far as possible) removed in this case all cold-agglutinins, while a , was very little influenced. The ag- glutinin a , had so, even at Oo C, so little avidity to A, occurring in the same individual that only a very little part of the agglu- tinin is absorbed even with this large excess of corpuscles.

The table shows that a, is quite different from the un- specific cold-agglutinin that occurs in the same serum, even if the thermal amplitude approaches that of the cold-agglu- tinin. Schematically one can compare the amplitude of the

13 Acta path. Vol. XVI. 2

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ordinary iso-agglutinin anti-A, the fraction a,, and the cold- agglutinin in the following way:

The agglutinin a, does not act upon A-corpuscles a t body- temperature. On the other hand, in this case lysins could be demonstrated against A,-corpuscles at 37O C in the active se- rum. By inactivating the serum at 56O C for a quarter of an hour, the lytic effect was lost, but could be reproduced when new complement in form of small quantities of fresh guinea- pig-serum was added.

A,B-individuals of the type described here, can be used as blood-donors for all AB-individuals, both A,B and A,B. In

occur that acts upon A,-corpuscles and O-corpuscles, but it cannot gain any influence upon the weak A-receptor of this A,B-group. In the recipient A,B, a, can act on A, of the A,B- group and it is therefore not advisable to use a donor of this type. If it is impossible to obtain a donor of the same type A,B, which, of course, in most cases cannot be done, a donor of the A,B-type shall not be used, but a donor of the B-type. The corpuscles of this type cannot be influenced by the re-

A,R-individuals an agglutinin a, (Landsteiner) 13, l4 ) may

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cipient's serum, and the B-donors agglutinin anti-A will not act upon the corpuscles of the A,B-recipient because of the weakly developed A-receptor; all the same, the proper thing to do is to use a B-donor with known, low titre of anti-A in his serum.

Individuals of the type mentioned here are therefore to be considered as blood-donors of type AB, but as recipients of type B.

Summary . In the corpuscles of an adult individual of group AB the

A-receptor could be demonstrated only by using an exception- ally strong anti-A iso-serum; but readily with a potent anti- sheepcorpuscle rabbit immune-serum.

The serum from this individual agglutinated A-corpuscles. Agglutination was caused by the agglutinin a, (Landsteiner) . The origin of this agglutinin is discussed together with the possible relationship between this agglutinin and a weak A-receptor. Agglutinin al is a fraction of the regular iso-agglu- tinin anti-A, with a thermal amplitude between that of the typical iso-agglutinin and that of the non-specific cold-agglu- tinin, being situated nearer to the latter than to the former. The irregular agglutinin a, is quite different, however, from the ordinary cold-agglutinin. This fact is shown in our case by absorbing all cold-agglutinins from the serum, and also by destroying the cold-agglutinins, leaving agglutinin a, in pure state in the serum.

In the active serum from this case, lysins against A-cor- puscles could be demonstrated at 3 7 O C.

In relation 'to blood-transfusion, individuals of this type belong to type AB as donors; but they belong to type B as recipients, in the cases where a donor of the identical type A,B cannot be obtained.

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REFERENCES.

1. Dungern, E. von , und Hirschfeld, L.: Zeitschr. f. Immunitatsf. 8:526, 1911.

2. T h o m s e n , O., Friedenreich, V., og Worsaae , E.: Acta pat. et micr. scand. 7:157, 1930.

3. T h o m s e n , 0.: Zeitschr. f. Immunitatsf. 57:3-4.:301, 1928. 4. T h o m s e n , 0.: Zeitschr. f . Rassenphys. 1:3-4:198, 1929. 5. T h o m s e n , 0.: Ugeskr. Lager 91:46:1017, 1929. 6. S c h i f f , F., und Adelsberger, L.: Zeitschr. f . Immunitatsf. 40:4-

5:335, 1924. 7. Dolter, W. : Zeitschr. f . Immunitatsf. 43:l-2:95, 1925. 8. Sachs , H., und Klopstock, A.: Methoden der Hamolyseforschung.

I Abderhalden: Handbuch der biol. Arbeitsmeth. Abt. XIII, Teil 2, Heft 6 9 7 , Berlin 1927.

9. Sachs , H.: Klinische Wochenschr. 6:51:2422, 1927. 10. Biirkle- de l a C a m p , H.: I Steffan: Handb. der Blutgruppenk.

p. 270, Munchen 1932. 11. Raes t rup , G.: I Steffan: Handbuch der Blutgruppenkunde, p. 337. 12. Landsteiner , K., and Witt, D . H.: Journal of Immunology 11:3:221,

1926. 13. Landsteiner , K . and Levine, Ph.: Journal of Immunol. 12:6:441,

1926. 14. Landsteiner , K., and Lewine, Ph.: Journal of Immunol. 17:1:28,

1929. 15. Friedenreich, V., et Worsaae , E.: Compt. rend. SOC. biol. 102:884,

1929. 16. Lattes , L., et Cavazzut i , A.: Journal of Immunol. 9:5:407, 1924. 17. Mino, P.: Munch. med. Wochenschr. 71:1129, 1924. 18. Friedenreich, V.: Zeitschr. f. Immunitatsf. 71:3-4:283, 1931. 19. Friedenreich, V.: Compt. rendu SOC. biol. 106:770, 1931. 20. Friedenreich, V., et W a d e r , G.: Compt. rend. SOC. biol. 106:773,

1931. 21. Kettel , K.: Undersegelser over Kuldehemaggl. Kebenhavn 1930.