FROM T H E S T A T E I N S T I T U T E OF I’UHLIC H E A L T H , S B R O D I d C N O S T I C D E P A R T M E N T , OSLO, N O R W A Y (CHI15’F: O T T O HARTMA”, M.D. )

ANTI-A AGGLUTININS IN SERA FROM A 8 INDIVIDUALS

h l

ERIK JUEL Ilecrived 3.xii.58

In 1926 Landsteiner & Witf (23 ) deliionstrated anti-A agglutinin in some AB sera, an announcement which was surprising since i t con- flicted with “Landsteiner’s Law”. Later Lantisteiner & Leuine ( 2 4 ) showed that these anti-A sera were specifically directed against the subgroup A ] , that they gave the same reactions as an anti-At (alphal) produced ad modum u. Dungern & Hirszfeld ( 4 ) by absorption of B- sera, and that these naturally occurring anti-A1 sera were only found in individuals with blood group AzR, or occasionally in A 2 bloods.

Investigations are later carried out to reveal the incidence of anti-At in Ad3 and ‘42 individuals. By using the normal slide or tuhe technique and performing the reactions at + 5 ” C the majority of workers have found that anti-AI is present in 25-35 per cent of all ArR sera and in 2-5 per cent of A:! sera (2 , 19, 29, 3 0 ) .

Friedenreich & W a d e r ( 1 1 ) maintain that there is a connection between the strength of the 4 receptor and the anti-A1 content of the serum, i.e. that the strength of the A receptor is conversely proportionate to the quantity of the “irregular” anti-A1.Gammelgaard (12) found no anti-A1 in 50 A3 and 3 .43B bloods, and Friedenreich ( 9 ) investigated sera from 46 A 3 and 3 A:rB individuals without being able to demonstrate the “irregular” agglutinin reacting at room temperature. The weaker A4 groups, howerer, A4 and A: ( 5 , 6 , 12, 13 .26 ) show a regular anti-AI, and in some cases a n anti-A which agglutinates A2 cells also. Gammel- gaard ( 1 2 ) found anti-Al in 5 out of 10 samples of A I blood and in 13 of 15 A5 bloods a t room temperature. The cases reported by Fischer & Hahn (8 ) and Hartmann ( 1 8 ) where the serum contained anti-A1 were probably of group A+ or A:. The A \ blood reported by Gammelgnard ( 1 2 ) , and the A,, (151 , 4, ( 3 2 ) or C (31) mentioned by others writers only exceptionally contain anti-A agglutinin, and then only reacting a t low temperatures.

Treatment of red cells with the proteolytic enzymes trypsin ( 2 8 ) , papain ( 2 2 ) and ficin ( 27, 33) has provided an important means of assistance in the diagnosis of hlood group immune antibodies. A tech-

91

nique using ficin-treated cells is often more sensitive than hemaggluti- nation in albumin and than an indirect Coombs' technique (16) . It is generally recognized that the enzyme technique is far less effective in systems normally reacting best in saline, for example the ARO system (22, 23, 14) even though Makinotlnn et nl. (27 ) by using ficin-technique showed that the sensitivity of A and B cells were increased by 2 4 titer steps.

The observations (21 ) that anti-A1 sera agglutinated enzyme treated At cells stronger than untreated, and that 42 cells treated with ficin, trypsin or papain also were agglutinated by anti-AI sera has led to this investigation into the frequency of the so-called "irregular" agglutinin anti-A1 in A& blood.

a1 E T H 0 D S

Sera f rom 40 healthy A# hlood donors were collected anti stored deep-frozen. All the bloods were typed with two different anti-AI sera,- -the one ahsairhed ad modum v. Dungern and Hirszfeltl and the o ther a naturally occurring serum f rom a n AxI% in d ividua 1.

Hrnningscn (20) has shown tha t P-neg. sera in most cases contain anti-P, and Crawford, Cutbush & Mollison (3) anti Bird (1) that a large proportion ( i f the in- complete "non-specific" cold antihodies are in fact either anti-H or a mixture ( i f

anti-H and other specific blood group antibodies with low thermal range. For this reason o u r specimens were first ahsorhed with an equal volume (if OP+ blood at +4" C fo r two hours to remcive a s much as possihle of the ccild-reacting iso- agglutinins.

.41 A2 and OP+ cells were treated with ficin according to the technique descrihcd hy Makinodan 6.t a l . (27), and to two drops of serum were added two drops ?h per cent suspension of untreated and ficin-treated A1 A:! and 01'+ cells. The reactions were carried out in tubes and incuhated a t +4" (1 for one hour. After reading at this temperature the tubes were shaken well, and af te r a fur ther two hours at room tcmperature (ca 18" (1) a new reading was made.

I1 E s u I. 'I' s Twelve of the 40 A2B sera, i .e. 3 0 per cent agglutinated untreated ;\I

red cells a t 5" C. Nine of these 12 sera agglutinated A I cells a t rooin temperature, i.e. 22 per cent of total (Table 1 ) .

None of the sera agglutinated untreated A:! or OP+ cells :it 5" or 18" C.

Alle of the 40 sera agglutinated ficin-treated A1 cells a t 5" and 36 of them at 18" C.

26 sera agglutinated ficin-treated A2 cells a t 5 " . and 13 of them also a t 18" C. The reactions were noticeably weaker than with ficin-treated A I cells.

Six sera gave weak agglutination with enzymetreated OP + cells a t 5" C but none at 18" C.

TABLE 1

10368 10372 10377 10389 19321 10462 10525 10539 19320 10640 10664 10740

11129 11195

11256 11292 11446 10414

13814 13543 12976 12780 12703 12668

13838

11248

13876

12.568 12548 12345 12312 12308 12129 12111 12108 12088 11938 11573 14418 14203

++ ( + I + ++ +

+(+) ++ ( $ 1 + ( + I +

+ ( + I + ( + I + + +

+ (+ ) +(+) + ( + I + +

( + I ( + I +

+ ( + I ++ + ( + I

( + I ++ ++ + (+ ) + + + +

+(+)

W

w

Reactions o f sera from A2B individuals, ahsorhed wit equal volumes OP+ cells, with untreated and firin-treated A1 A:! and O P + cells at 5" C and 18" (1.

D I S C IT S S I 0 N

In the present work it is shown that each of the 40 investigated A2B sera contain agglutinin with A specificity, mainly of the cold reacting type. As the agglutinins also agglutinate A2 cells with great regularity, it can not he classified as alphal, hut has to he a common alpha or a mixture of alpha and alphal.

Friedenreich (10) has shown that the so-called "irregular" agglutinin

anti-Al is identical with that part of the alphal--fraction, normally occurring in all 0 and B sera, which has the lowest thermal amplitude, and that the alpha-fraction of an anti-A serum is that with the highest temperature optimum. Erwin &: Young ( 7 ) , Young ( 3 4 ) and Hartmnnn ( 1 7 ) have reported cases where A. and A z H recipients after transfusion with 0 blood partially neutralized the anti-A agglutinin leaving a speci- fically reacting anti-Al. It seems in these cases likely that the common alpha and those parts of the alpha1 with the highest temperature ampli- tude are first neutralised hy the antigens of the A:! individuals, leaving alpha1 of the cold variety. This alphal, according to Frietlenreich iden- tical with the "irregular" anti-Al, continues to circulate for a consider- able time because of its slight avidity a t hody temperature.

The finding of hoth alphat and common alpha, especially of the cold reacting variety in sera froin normal 4 . H individuals seeins to be in agreement with the above mentioned investigations. A. receptors react, even if weakly, with anti-A1 (25) and then usually a t low temperatures. A s the in vivo interaction between the A-receptors and agglutinins takes place prefereably a t 3 7 " C, the anti-A with the highest temperature amplitude will thus he neutralised and the cold reacting fractions con- tinue to circulate freely, even though they have A-specificity.

In connection with the present investigation it is of practical iin- portance to mention that false positive reactions may occur when cross- matching is carried out with ficin-treated cells, either in saline or by an indirect Coombs' technique, if the recipient is o f blood group Ad3, even if the reactions with A:! cells a t 18" C or higher temperatures, a s a rule, are weak.

s t T X I X I A H Y

Sera from 40 4.B hloods have been investigated to find the frequency of the naturally occurring, so-called "irregular" anti-Ar. Twelve (30 per cent) of these sera contained anti-Al reacting a t 5" C. Nine (22 per cent) also agglutinated untreated A I cells a t 18" C.

By using ficin-treated A1 and A:! cells it is shown that all the 40 sera contained anti-A reacting a t 5" C and 36 of them a t 18" C. 26 sera con- tained an agglutinin reacting with ficin-treated A:! cells at 5' C, and 13 at 18" C.

I, I T E II A rr 11 II E

1. Bird, G . W . G . : Nature, 172: 734, 1953. 2. Hiickrlar, R . : Hlut, 1 : 129, 1955. 3. Crawford, H . , Cutbush, M . &Moll ison, P.L.: I.ancet, 268: 566. 1953. 4. I ) . DungPm, E . & Hirszfi>lrl, I..: Ztschr. Immunitatsf., 8 : 526, 1911. 5. I)unsford, I . : Hull. Centr. I d ) . of Netherlands Hed Cross 'I'ransf. Serv., 2 : 209.

6. Ilunsfortl, I . R: ;Ispintrll, P.: Ann. Eugen., 1 7 : 30, 1952. 7. Erwin, I ) . M. & Young, L. E. : Hlood, 5: 61, 1950. 8. Fisrhrr. W. & Ilnhn, F.: Ztschr. Immunitiitsf., 8 4 : 177, 1935.

1952.

95

9. Frietlenreich, V.: Ztschr. Immunitatsf., 8 9 : 409, 1936. 10. Fricdenreich, V.: Ztschr. Immunitiitsf., 71 : 283, 1931. 11. Frietlenreich, V. & W a d e r , G.: C. R. Sec. d . Riol., 106: 773, 1931. 12. Cammelgaartl,.4.: Om Sjzeldne Svage A-Receptorer (As A4 A5 og A,) hos hfen-

nesket, Thesis, Nyt Nordisk Forlag, Copenhagen 1942. 13 . Gammelgaard, A. C Marcossrn, P. V.: Ztschr. Immunitltsf., 98: 411,1940. 14. Goldsmith, K.: Lancet, 268: 76, 1955. 15. Groi)e-Rasmussen, M., Soutter, L. & I,eoine, Ph.: J . Clin Path., 22: 209, 1952. 16. Haber, G. & Rosenfield, R. E.: Papers in dedication of the 60th birthday of dr.

17. Hartmann, 0.: Papers in dedication of the 60th birthday of dr. P. H. Andresen,

18. Hurtmann, O., Hatlland, K . Jt Luntlermll, J . IT.: N o r d . Med., 10:1136, 1941. 19. nan tier Heitle, H. M . & unn Loghem, J . J . j r . : Ned. Tschr. Geneesk., 40: 2812, 1947. 20. Hrnningsen, K.: Om Blodtypesystemet P, Thesis, Dansk Videnskabs Fortag, Co-

21. Juel, E.: In preparation. 22. Kuhns, W . J . & Bailey, A . : Am. J. Clin. Path., 20: 1067, 1950. 23. Landsteiner, K. & Wit t , D. H.: J . Immunol. 1 1 : 221, 1926. 24. Landsteiner, K. & Lcoinr, I’h.: J . Immunol., 12: 441, 1926. 25. Lattes, L. & Cauarutti, 4 . : J. Immunol., 9 : 407, 1924. 26. uan Loghrrn, J . J . j r . & uan cler Hart, M.: Vox Sanguinis, 4 : 69,1954. 27. Makinodan, T. C Macris, N. T.: J. Immunol., 75: 192, 1955. 28. Morton, J. A. & Pickles, M. M.: Nature, 159: 779,1947. 29. Taylor, G. L . , Race, R. R., Prior, ‘4. M . & Ikin, E.: J . Path. Hact., 54: 514, 1942 30. Wiener, A . S.: J . Immunol., 41 : 181, 1941. 31. Wiener, .4. S.: Ann. Eugen., 18: 1, 1953. 32. Wiener, A. S. & Cordon, E. C.: Brit. J. Haemat., 2: 305, 1956. 33. Wiener, A . S . : & K n t z , L.: J. Immunol., 66: 51, 1951. 34. Young, L. E.: J . Imrnunol., 5 1 : 101,1945.

P. H. Andresen, Munksgaard, Copenhagen 1957, p. 45.

Munksgaard, Copenhagen 1957, p. 76.

penhagen, 1952.