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An investigation of the immune response of homozygotes for the Rh haplotype -D- and related haplotypes
Blood Transfusion and Immunohaematology Tome XXV. - - N ° 2. - - 1982
185
An Investigation of the Immune Response of Homozygotes for the Rh Haplotype--D and Related Haplotypes Using Cells of Rare Rh Phenotypes
by G.L. DANIELS MRC Blood Group Unit, LONDON(England).
ANY examples of h o m o z y g o t e s for the rare Rh gene complex - - D - - , in w h i c h the red cells lack any representation of either the C or E series of antigens, have been studied (RACE and SANGER [17]) since the original example was described in 1950 by RACE, SANGER and SV,LWYN [18]. Following i m m u n i z a t i o n by p r e g n a n c y or transfusion, h o m o z y g o u s - - D - - people f o r m antibody which reacts with all cells of c o m m o n Rh type [17].
M
RACE e t al. [19] f o u n d that the s e r u m of the first example of - - D - - / ~ D - - , Mrs. B., contained separable anti-e, anti-C and anti-c. They thought, however, that the m a j o r c o m p o n e n t of the antibody was anti-Cc w h i c h seemed to be directed against C and c indifferently. HACKEL [9], using the sera of Mrs. B. and a n o t h e r h o m o z y g o u s - - D - - , Mr. M.C., c o n f i r m e d these results and d e m o n s t r a t e d also
DANIELS
186
G.L.
some separable anti-E and anti-ce. He agreed t h a t m u c h of the antibody p r e s e n t was directed against an antigen p r o d u c e d b y all c o m m o n Rh gene complexes. GUNSON and DONAHUE [7] f o u n d t h a t a b s o r p t i o n s w i t h CDe/CDe, cDE/cDE, C D e / c D E or c d e / c d e r e d cells c o m p l e t e l y r e m o v e d all the antibodies p r e s e n t in the s e r u m of their CWD--/CWD- p r o p o situs; s o m e w h a t similar findings w e r e those of BUCHANAN and MCINTYRE [1] w i t h the s e r u m of their - - D - - / - - D - propositus. F r o m the results of sequential adsorption-elution of - - D - - / - - D J sera with CDe/CDe, c D E / c D E and c d e / c d e ceils in t h a t order, ROSENFIELD et al. [21] stated t h a t the i m m u n e r e s p o n s e in - - D - - / - - D - individuals is directed t o w a r d s antigenic d e t e r m i n a n t s i n d e p e n d e n t of all other k n o w n Rh specificities. This a n t i b o d y was n u m b e r e d anti-Rhl7 [21] and is the a n t i b o d y previously n a m e d anti-Hro b y WIENER [28]. The sera of s o m e of the CWD- [7] and c D - - [6, 26, 30] homozygotes, the only (C)D ~v-- h o m o z y g o t e [17, 22] and one of the two •D" h o m o z y g o t e s [3] contain an a n t i b o d y (or antibodies) like t h a t in the - - D - - / - - D - - i m m u n e sera. H o w e v e r , TIPPETT [27] s h o w e d that i m m u n e sera f r o m people w i t h " d e l e t e d " p h e n o t y p e s differ in their ability to agglutinate Rhino a cells and those of an R aa homozygote. PERRIER et al. [16] a n d HABIBI et al. [8] s h o w e d t h a t sera f r o m two - - D - - h o m o z y g o t e s contained, as well as anti-Rhl7, a second antibody to a public antigen (anti-Nou) w h i c h r e a c t e d w i t h ceils of c o m m o n and certain d e p r e s s e d Rh p h e n o t y p e s and w i t h h o m o z y g o u s (C)D ~v-- cells, b u t not w i t h - - D - - / - - D - - , c D - - / c D - - or Rhnun cells. PERRIER et al. [16] also d e m o n s t r a t e d , s e p a r a b l e anti-e in all the sera f r o m - - D - - h o m o z y g o t e s and r e l a t e d p h e n o t y p e s t h a t they tested. R e p o r t e d b e l o w is an a t t e m p t to analyze the i m m u n e of people a p p a r e n t l y h o m o z y g o u s for " p a r t i a l l y d e l e t e d " complexes (and one - - D - - / - - - - - double h e t e r o z y g o t e studying t h e i r reactions w i t h cells of r a r e Rh p h e n o t y p e s , the earlier o b s e r v a t i o n s of DANIELS et al. [5].
response Rh gene [12]) b y extending
SEROLOGICAL S T U D I E S
I m m u n e sera f r o m 16 - - D - - ~ - - - D - - , 1 - - D - - / - - - - - - , 3 cD--/ cD--, 3 CwD--/CWD- , 1 - D ' / ' D - a n d 1 (C)D~V--/(C)D I v j , all of which are c o m p a t i b l e w i t h - - D - - / - - - D - - a n d Rh~un cells, w e r e tested against the following cell s a m p l e s of r a r e Rh types: t w o e x a m p l e s of " D : / ' D (Mrs. H.D. [3] and Dor.), h o m o z y g o u s R ~ (Otto H. [23]),
I M M U N E R E S P O N S E OF H O M O Z Y G O T E S FOR T H E Rh I t A P L O T Y P E
187
a c D e N e g r o w i t h v e r y w e a k e (W.J. [2]), t h r e e e x a m p l e s o f Rhr, oa s h o w i n g v a r y i n g e x p r e s s i o n o f R h a n t i g e n s ( K u r a . [29], Hel. [25] a n d C.C. [14]) a n d a p o s s i b l e C W D ( e ) / - - D - (N.R.). A s u m m a r y of t h e R h r e a c t i o n s o f t h e s e cell s a m p l e s is g i v e n in T a b l e I.
TABLE
I
R e s u l t s of testing s o m e cells w i t h rare R h p h e n o t y p e s w i t h R h antisera. Rh
ANTISERA
CELLS D
~I.D . . . . . . . . . . . . )or ............. Otto H ........ W.J ............ Kura ........... Hel.* . . . . C.C ............ N.R ............
C
+ + + + .
.
.
. . +w +
c
--.
. . _+ --
Cw
E
+ + .
. --
.
.
. +
.
e
f
± _+
_ _+
+w +
---
G
D inc
+ +
+ +
Evans
Rh32
Rh33
+ + +
+
. --
+w
+
_+
LW
+ + + -k
+_ +
D inc = incomplete anti-D tested against saline suspensions of cells_ ± = + with some sera - - with others. +w = weaker reaction than control cells with all sera. * Other tests show Hel. is not Rh~un [4, 25]. All negative with anti-V and anti-VS.
T h e 25 s e r a w e r e t e s t e d a g a i n s t t h e r a r e cells b y t h e i n d i r e c t a n t i g l o b u l i n t e s t (IAT), u s i n g a p u r e a n t i - I g G . W h e r e n e c e s s a r y s o l u b l e A a n d B b l o o d g r o u p s u b s t a n c e s w e r e u s e d to i n h i b i t anti-A a n d a n t i - B in t h e s e r a , g r o u p A o r B Rh~un o r - - D - - / - - D - being used as a negative control. Titrations of the sera were also tested a g a i n s t C D e / c D E cells, b y IAT, i n o r d e r t o g{ve a n e s t i m a t e o f t h e relative strengths of the sera. A s u m m a r y o f t h e r e s u l t s , s h o w i n g t h e d i f f e r e n t p a t t e r n s of r e a c t i o n s of t h e s e r a , is g i v e n in T a b l e I I . I n Fig. 1 a n a t t e m p t is m a d e to r e l a t e t h e a n t i b o d y s t r e n g t h to t h e p o s i t i v e a n d n e g a t i v e r e a c t i o n s w i t h t h e r a r e cell s a m p l e s . T h e r e s u l t s i n T a b l e I I a n d Fig. 1 s h o w h e t e r o g e n e i t y o f t h e s e r a in t h e i r + a n d - - r e a c t i o n s w h i c h c a n n o t b e e n t i r e l y d u e to t h e s t r e n g t h of t h e a n t i b o d y . The positive reactions, although generally given by the higher scoring sera, show an overlap with t h e l o w e r s c o r i n g s e r a , e s p e c i a l l y w i t h O t t o H. cells. To t a k e s p e c i f i c e x a m p l e s : t h r e e s e r a s c o r i n g 91, 114 a n d 117 w i t h C D e / c D E
188
DANIELS
G.L.
cells f a i l e d to r e a c t w i t h t h e cells of H.D. a n d O t t o H. w h e r e a s a n o t h e r s c o r i n g 87 r e a c t e d w i t h b o t h s a m p l e s a n d t h r e e o t h e r s s c o r i n g 50, 64 a n d 68 r e a c t e d w i t h O t t o H. cells. T h e a b i l i t y to 120
110 100 9O
80
7o 8 •
>
60
o~ 50 4o
fl 30 E-~ 2O
10
+
H.D. •D"
-
~
Dor, "D"
+
~
Otto H. Rh33
+
-
Kura. P~mod
+
-
Hel. Rhmod
Redcells
FI6. 1 . - Diagram relating the antibody strength of sera from people with "deleted" phenotypes (Table II) to their + a n d - reactions with cells of rare Rh phenotypes. Dots on the left hand line of each pair of lines indicate titration scores against CDe/cDE cells of sera which reacted with I-I.D., Dor., Otto H., Kura. and Hel. ceils, whereas the dots on the right hand lines indicate scores against CDe/cDE cells of sera that failed to react x~'ith H.D., Dor., Otto H., Kura. and Hel. cells. r e a c t w i t h Rhmod cells is c l e a r l y m o r e d e p e n d e n t o n t h e s t r e n g t h of t h e a n t i b o d y a l t h o u g h t h e r e a r e e x c e p t i o n s ; f o r e x a m p l e o n e s e r u m w i t h a s c o r e a g a i n s t C D e / c D E ceils of 57 r e a c t e d w i t h K u r a . a n d Hel. cells w h e r e a s a n o t h e r , w i t h a s c o r e of 83, d i d n o t . T h e t w o h o m o z y g o u s "D" s a m p l e s a r e d i s t i n g u i s h e d f r o m e a c h o t h e r b y o n l y o n e s e r u m ; t h a t is, o n l y t h r e e of t h e f o u r s e r a t h a t r e a c t e d w i t h H.D. cells r e a c t e d w i t h Dor. cells. L i k e w i s e , t w o of t h e RhmoaS ( K u r a . , Hel.) a r e d i s t i n g u i s h e d b y o n l y o n e s e r u m ( T a b l e II).
189
I M M U N E RESPONSE OF HOMOZYGOTES FOR THE Rh HAPLOTYPE
TABLE II Results of indirect antiglobulin tests (IAT) using sera f r o m people w i t h " d e l e t e d " p h e n o t y p e s against cells of rare R h phenotypes.
Cells
SERA
Phenotypes
No,
-DH.D.
7
--D
2
cD--
3
--D
1 1
CwD(C)Dr v -
3
2
--D
1
CwD--
1
--D
3
1 --D--/-- ---- + 1 cD--
9
5
c(D)(e) Dor. Otto H.
m
C.C.
N.R.
--
+
+
+
+
+
+
+
+
+
+
+
-b
+
+
+
+
+
--
+
q-
--
+
+
b
m
m
+
I m
---D-CwDm
m
+ +
+ ÷
-b
u
--D--
-D-
cD(e) Rhraod Hel. W.3. Kura.
+
m
+
+
m
All sera compatible with - - D - - / - - D - - and Rhnull cells. None of the reactions due to anti-A or anti-B
T a b l e I I also s h o w s t h a t t h e a n t i b o d y c o n t e n t of t h e six t y p e s of " d e l e t e d " g e n o t y p e s ( h o m o z y g o u s - - D - - , c D - - , CWD- , "D' a n d (C)D ~v-- a n d - - D - - / - - - - - - ) as j u d g e d b y t h e s e , r e a c t i o n s w i t h r a r e p h e n o t y p e s does n o t d i s t i n g u i s h o n e f r o m a n o t h e r . T h e o n l y except i o n is t h e s i n g l e e x a m p l e of s e r u m f r o m "D'/-D- (H.D.), t h e o n l y o n e t h a t f a i l e d to r e a c t w i t h W.J. cells. T i t r a t i o n s of m o s t of t h e s e r a w h i c h gave p o s i t i v e r e a c t i o n s w i t h t h e cells of r a r e p h e n o t y p e s w e r e t e s t e d i n p a r a l l e l w i t h t h o s e cells a n d cells of c o m m o n R h t y p e a n d t h e r e s u l t s a r e s h o w n i n T a b l e I I I . T h e s c o r e s w e r e a l w a y s less f o r t h e r a r e cells t h a n f o r c o n t r o l s as m i g h t b e e x p e c t e d f r o m t h e p a t t e r n of r e a c t i o n s i n T a b l e I I : t h e d i f f e r e n c e i n s c o r e s was, i n g e n e r a l , m o s t m a r k e d for ' D ' / ' D " cells (H.D. a n d Dor.), less so f o r Otto H. a n d Rhmoa ( K u r a . ) , still less f o r N.R. a n d Rhmod C.C. a n d l e a s t f o r W.J.. H o w e v e r v a r i a t i o n i n d e g r e e of r e d u c t i o n of s c o r e does exist b e t w e e n d i f f e r e n t s e r a (e.g. s e r a of Mrs. B. a n d Hal. a g a i n s t N.R. a n d C.C. cells).
DAN1ELS G.L.
190
The sera of M.C. ( - - D - - / - - D - - ) a n d of Mrs. H. ( c D - - / c D - - ) and an eluate of M.C. s e r u m a d s o r b e d on C D e / c D E cells, all of w h i c h r e a c t e d w i t h " D ' / ' D ' and Rhmoa cells w e r e f u r t h e r investigated to see if the a n t i b o d y for these r a r e cells was s e p a r a b l e f r o m t h a t for c o m m o n Rh p h e n o t y p e s . All eluates w e r e m a d e b y the digitoninacid-stromal m e t h o d [11, 13]. TABLE I I I
Titration scores (IAT) [or sera tested against cell samples o[ rare R h phenotypes and controls.
Sera Name Mrs. B. M.C. Ca. Cus. M.I. D.O.
Ota. Pep. V,M. Mrs. H. Joh.
Cells
Phenotype
H.D.
Dor.
Otto H.
W.J.
--D-"' ~ ~ '~ '~ ~
0
0 85 0 0 0 0 0 0 23 29 0 0 0 0 0 0
0 86 0
103
') --D--/--
* Control
--
cD-"
Po.
Coo. Hal. MacD. Nou.
0 0 0 0 0 0
~'
CwD-~'
~ (C)DIV-= mean
--
32 0 0 19 0 0 0
score of titrations
Kura.
C.C.
N.R.
0
34 90 12 38 19 19 42 76
67 99 15 41 16 32 40 82
101 43 25 69 93 51 87
106 6O 22 53 90 42 76
0 46
0 0 0
58 0 0 0 7 0
0 0 0
103
56 97
93 0 0
tested against cells of common
Control* 109 112 24 66 28 56 81 94 91 108 71 44 7t 93 69 102
Rh phenotypes
Fig 2 shows t h a t a b s o r p t i o n w i t h C D e / c D E cells r e m o v e d all a n t i b o d y for ' D ' / ' D " cells a n d for cells of c o m m o n p h e n o t y p e s . A b s o r p t i o n w i t h " D ' / : D ' (H.D.) ceils, on the o t h e r hand, r e m o v e d all a n t i b o d y for "D'/'D" ceils but, even a f t e r continued a b s o r p t i o n s left a strong a n t i b o d y for cells of c o m m o n p h e n o t y p e . The separable a n t i b o d y in these sera w h i c h r e a c t e d w i t h " D ' / ' D cells is not, however, anti-Evans. An eluate f r o m M.C. s e r u m a d s o r b e d on "D'/'D: cells r e a c t e d equally strongly w i t h "D'/'D" and cells of c o m m o n Rh p h e n o t y p e giving the following t i t r a t i o n scores: "D'/'D" cells 33; CDe/CDe 41; c D E / c D E 32; c d e / c d e 42; CDe/-D" 39; cde/'D" 41. Dot. "D'/'D" cells w e r e also u s e d in a b s o r p t i o n / e l u t i o n tests w i t h Mrs. H. s e r u m ( t i t r a t i o n s not done) a n d gave similar results to those o b t a i n e d w i t h H.D. cells thus s u p p o r t i n g the conclusions d r a w n f r o m using H.D. cells.
I M M U N E R E S P O N S E OF' H O M O Z Y G O T E S FOR T H E Rh I-1APLOTYPE
191
A Absorbed with CDe/cDE cells
Absorbed with H,D. cells
80
6O
o~ 40~
~ ~0"~,, .~
01 "-~ .... • 1
2
4
i
2
4
Number of absorptions
.B Absorbed with H.D. cells
Absorbed with CDe/c~E cells 1 20
100
m
8O LI
o o
60
o~ 4= q~
4o 20
.\
~k I
2
4
o\
2
4
~umbe= of absorptions
FIG, 2. - - Absorption graphs. A.
M . C . (--D--/--D--) eluate (from CDe/cDE) cells against H.D. ( ' D / ' D ' ) and control ceils. B. Mrs. H. (cD---/cD--) serum against H.D. and control cells. --x-- Titration scores against H.D. cells. - - e - - Average titration scores against CDe/CDe, cDE/cDE and cde/cde cells.
A b s o r p t i o n a n d e l u t i o n t e s t s w i t h M.C. s e r u m (Fig. 3) u s i n g Rhmoa ( K u r a . ) a n d C D e / c D E cells gave a d i f f e r e n t p i c t u r e a n d s u g g e s t t h a t a s i n g l e a n t i b o d y is r e s p o n s i b l e f o r t h e r e a c t i o n s w i t h b o t h Rhmod a n d cells of c o m m o n R h p h e n o t y p e s . This a n t i b o d y w a s r e m o v e d less e f f i c i e n t l y b y Rhmoa t h a n b y C D e / c D E cells; t h e e l u a t e s f r o m
192
DAN1ELS G.L.
b o t h p h e n o t y p e s b e h a v e d a l i k e i n r e a c t i n g less s t r o n g l y w i t h Rhmod t h a n w i t h C D e / c D E cells. Some time after the above work was carried out homozygous (C)D ~ v - (Nou.) cells b e c a m e a v a i l a b l e f o r t e s t i n g ( k i n d l y s u p p l i e d b y Drs. Ph. ROUGER a n d B. HABIBI). T h e s e cells r e a c t e d w i t h M.C. Absorbed with CDe/cDE cells
Absorbed with Ku~a. cells
8O
vl
•
~60~
X
~40 4~
.~ 20 0
"~ ...............
1
2
""
4
0
1
2
4
Number of absorptions
FIG. 3. - - Absorption graphs. M.C. (--D--/---D--) serum against Kura. (Rhmod) and CDe/cDE
cells. --x-- Titration scores against Kura. cells. - - e - - Titration scores against CDe/cDE cells. M.C. serum diluted 1:10 in A and B solutions. Eluates from M.C. serum adsorbed on CDe/cDE cells gave titration scores of 25 with Kura. cells and 47 with CDe/cDE cells. Eluates from M.C. serum adsorbed on Kura. cells gave scores of 27 with Kura. cells and 42 with CDe/cDE cells. a n d Mrs. H. s e r a a n d t h e p a t t e r n of r e a c t i o n w i t h t h e s e r a i n T a b l e I I r e s e m b l e d t h a t f o r O t t o H. m o r e c l o s e l y t h a n t h a t f o r H.D. o r Dor., a l t h o u g h s o m e of t h e s e r a w e r e n o l o n g e r a v a i l a b l e f o r t e s t i n g . N o u . cells r e a c t e d s t r o n g l y w i t h M.C. s e r u m a b s o r b e d f o u r t i m e s w i t h H.D. o r w i t h D o t . cells b u t d i d n o t r e a c t w i t h M.C. s e r u m a b s o r b e d f o u r t i m e s w i t h K u r a . cells o r w i t h C D e / c D E cells. N o n e of t h e s e f o u r a b s o r b e d s e r a r e a c t e d w i t h H.D. o r Dor. ceils a n d all b u t t h e l a s t r e a c t e d w i t h C D e / C D e cells. Nou. cells also r e a c t e d w i t h a n e l u a t e p r e p a r e d f r o m Dor. ceils s e n s i t i z e d w i t h M.C. s e r u m . T h i s e l u a t e also r e a c t e d w i t h H.D., Dot. a n d C D e / e D e cells.
DISCUSSION
Homozygotes for the " d e l e t e d " Rh gene complexes ( - - D - - , cD--, CwD- , (C)D w - a n d "D') w h e n i m m u n i z e d b y p r e g n a n c y o r t r a n s f u -
I M M U N E R E S P O N S E OF H O M O Z Y G O T E S F O R T H E R h H A P L O T Y P E
193
sion f o r m antibody which reacts with cells of all c o m m o n Rh phenotypes but, with the exception on occasion of ' D ' / ' D ' [3] and (C)D~V--/ (C)D ~ v - [8, 16] cells, are all m u t u a l l y compatible. It has been suggested that these i m m u n e sera contain a mixture of antibodies resembling those m a d e by people of c o m m o n p h e n o t y p e together with a c o m p o n e n t directed at the Cc and Ee antigen indifferently [9, 17, 19] or, alternatively, that one antibody called anti-Hro [28] or anti-Rhl7 [21] is responsible. The results discussed here suggest that neither of the above is the whole answer. The heterogeneity of these i m m u n e sera was d e m o n s t r a t e d by investigations with cells of rare Rh p h e n o t y p e (Tables I I and I I I ) . Results with "D'/'D" and R~a/R 3a (Otto H.) ceils could be explained by a qualitative as well a quantitative difference in the antibody c o n t e n t of the sera (Tables I I and I I I , Fig. 1). The titration scores for these cells were lower than those for cells of c o m m o n phenotypes (Table I I I ) : some sera, n o t a b l y Hal., failed to react with ' D ' / ' D " and Rh33, yet s h o w e d not reduction in scores for other rare phenotypes (C.C., N.R.) suggesting that m o r e than one antigen is involved. Absorption and elution studies with D ' / D cells suggest that M.C. ( - - D - - / - - D - - ) and Mrs H. ( c D - - / c D - - ) sera have at least two antibodies: one which reacts with cells of c o m m o n phenot~pes but not with ' D ' / ' D ' (anti-Rhl7) and one which reacts with ceils of all c o m m o n p h e n o t y p e s and "D'/'D" (Fig. 2). There was no evidence of separable anti-Evans, specific for 'D" cells, in these sera. Since D / ' D ' cells are negative with the s e r u m of the original hr ~ negative, Mrs. Sh. [4], this second a n t i b o d y c a n n o t be the same as anti-Hr (anti-Rhl 8). The different p a t t e r n of reactions of " d e l e t e d " sera against Otto H. (Rh33) and W.J. cells (Table II) is evidence against the sera containing a mixture of antibodies against C, c, E and e antigens. These cell samples are alike in their C, c, E and e reactions and differ only in their D, G and Rh33 antigens; b o t h D and G are present on cells of all of the " d e l e t e d " pbenotypes. However, Otto H. shows that some Rh antigens, e.g. c, can be expressed normally in the absence of n o r m a l expression of Rhl7. The tests with Rhmoa cells, three unrelated examples, suggest that their relatively weak reactions (Tables I I and I I I , Fig. 1) can be a t t r i b u t e d to a quantitative r a t h e r t h a n qualitative difference. C.C., who has the strongest Rh antigens, gives the strongest reactions failing to react with only one of the sera (a weak antibody). McGuIREMALLORY et al. [14] state that C.C. cells show no depression at all of Rh17 antigen as d e t e r m i n e d by an a u t o m a t e d qualitative blood typing technique using an AutoAnalyser. U n f o r t u n a t e l y they do not say how
194
DAN1ELS
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m a n y sera were used to d e t e r m i n e this. Table I I I shows that, by m a n u a l titration, the sera f r o m " d e l e t e d " p h e n o t y p e s give varying results with C.C. cells. To take extreme examples f r o m Table I I I ; if Mrs. H. or Hal. sera were used no depression could be seen, b u t if the s e r u m f r o m the original - - D - - / - - D - - , Mrs. B., was used a large depression could be seen c o m p a r e d with C D e / e D e cells, C.C. cells scoring only 30% of that of the c o n t r o l cells. The suggestion of a quantitative r a t h e r than a qualitative antigenic difference in Rhmoa is s u p p o r t e d b y a b s o r p t i o n and elution tests using Kura. cells (Fig. 3). The results p r e s e n t e d above, together with previously r e p o r t e d results [1, 4, 7, 8, 15, 16, 21], suggest that these i m m u n e sera do not contain a m i x t u r e of antibodies directed against C, c, E and e antigens, although separable anti-e m a y sometimes be present. By a b s o r p t i o n tests using CDe/CDe, c D E / c D E and c d e / c d e cells PERRIER et al. [16] f o u n d anti-e to be p r e s e n t in all seven samples tested (4 f r o m - - D - - / - - D - - , 2 f r o m c D - - / c D - - and one f r o m (C)D~V---/ (C)D Iv--- people). Daniels [4] tested sera in a similar way and f o u n d separable anti-e in b o t h sera f r o m - - D - - h o m o z y g o t e s tested b u t could not separate anti-e f r o m the s e r u m of a C~D - homozygote, although an a n t i b o d y was p r e s e n t w h i c h b e h a v e d like anti-e h u t w h i c h was a b s o r b e d by c D E / c D E cells: he f o u n d no trace of anti-e in the s e r u m of tbe "D" h o m o z y g o t e H.D. However, m o s t of the reactivity is due to one antibody, anti-R17, in w h i c h case the antigen R h l 7 must, like D and e, be a mosaic, or there is a mixture of antibodies to high f r e q u e n c y antigens present in all c o m m o n phenotypes. Some sera f r o m people with " d e l e t e d " p h e n o t y p e s contain anti-Rh18 (anti-Hr or anti-Hr9 as well as anti-Rhl7 (anti-Hro) [10, 24] and PERRIER et al. [16] and HABIBI et al. [8] described a n o t h e r antibody to a high f r e q u e n c y antigen (anti-Nou) in some such sera. ISSITT [10] suggested that some sera m a y also contain anti-Rh34 [20]. Absorption-elution tests using "D:/'D" ceils (Fig. 2) show the presence of a n o t h e r a n t i b o d y to a high f r e q u e n c y antigen, different f r o m anti-Rhl7 and anti-Rhl8. It is suggested that this specificity be referred to as anti-Day. The reaction of Nou. cells with M.C. s e r u m a b s o r b e d f o u r times with -D'/-D- cells, and consequently no longer reactive with these cells, shows that anti-Day is not the same as anti-Nou t h o u g h anti-Nou m a y contain anti-Dav. Anti-Dav has not been s h o w n to differ f r o m anti-Rh34. SUMMARY I m m u n e sera f r o m people h o m o z y g o u s for the " d e l e t e d " gene complexes - - D - - , cD--, CwD- , "D" and (C)D ~ v - as well as hetero-
I M M U N E R E S P O N S E OF H O M O Z Y G O T E S F O R T H E Rh H A P L O T Y P E
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zygous - - D - - / - - - - - were studied by their reactions, and in some cases by absorption-elution tests, with cells of the following rare Rh types: two unrelated examples of h o m o z y g o u s "D'; three unrelated examples of Rhmod; h o m o z y g o u s Raa; cDe with a very weak e; a p p a r e n t CWD(e)/--D - . The results showed that these i m m u n e sera are clearly heterogeneous: this heterogeneity was not entirely due to the strength of the a n t i b o d y (as d e t e r m i n e d by titration against CDe/cDE cells) or to the p h e n o t y p e of the a n t i b o d y maker. A b s o r p t i o n and elution tests using two of the i m m u n e sera and •D~/'D" cells d e m o n s t r a t e d a " n e w " antibody, anti-Dav, for a high f r e q u e n c y Rh antigen in addition to anti-Rhl7. RESUME Les s6rums i m m u n s d'individus p r 6 s e n t a n t des ph6notypes Rh partiellement s i l e n c i e u x , - - D - - , cD--, CWD- , "D" et (C)D ~v---, ainsi q u ' u n h6t6rozygote - - D - - / - - - - - ont 6t6 6tudi6s par leurs r6actions et e n certains cas p a r d e s tests d ' a h s o r p t i o n et d'6lution h l'aide de globules rouges h ph6notypes Rh e x c e p t i o n n e l s suivants: deux exempies n o n apparent6s de " D ' / ' D ' ; trois exemples non apparent6s de Rh~oa; et un exemple de RaaR a3, de cDe avec un e tr~s faible, et de CWD(e)/--D - probable. Les rdsultats ont m o n t r 6 que les r6actions de ces s6rums i m m u n s sont tr6s h6t6rog~nes: cette h6t6rog6n6it6 n'6tait pas enti6rement lide h la puissance de l'anticorps (d6termin6 p a r le titrage c o n t r e les globules rouges CDe/cDE) ni a u ' p h d n o t y p e de l'individu ayant ddvelopp6 l'anticorps. Les tests d ' a b s o r p t i o n et d'61ution, utilisant deux de ces sdrums immuns, avec les globules rouges "D-/-D-, ont ddmontr6 un nouvel anticorps, anti-Day, c o r r e s p o n d a n t ~ un antig6ne Rh de haute fr6quence ainsi q u ' u n anti-Rh 17. ACKNOWLEDGEMENTS. -The author thanks the many colleagues who sent samples of immune sera and cells of rare phenotype. All are acknowledged individually in reference 4.
Request reprints f r o m : Dr. G.L. DANIELS MRC Blood Group Unit Wolfson House University College London 4 Stephensen Way LONDONNWl 2HE England.
DANIELS G.L.
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REFERENCES [1] BUCHANAND.I. and MCINTYRE J. - - Consanguinity and two r a r e matings: - - D - - / - - D - - × C D e / - - D - - and C D e / - - D - - × c D e / - - D - - . Brit. J. Haemat., 1, 304-307, 1955. [2] CHAMPNEY A., WILSON B. and TIPPETT P.A. - - An u n u su al Rh p h e n o t y p e in a black A m e r i c a n family. Lab. Med., 11, 277-278, 1980. [3] CONTRERAS M., ARMITAGE S., DANIELS G.L. and TIPPETT P. - - Homozygous .D-. Vox Sang., 36, 81-84, 1979. [4] DANIELS G.L. - - Blood group antigens of high frequency: a serological and genetical study. PhD thesis, University of London, 1980. [5] DANIELS G.L., GAVIN J. and TIPPETT P. - - H e t e r o g e n e i t y of i m m u n e response of - - D - - / - - D - - , c D - - / c D - - and CwD--/CwD- people. Abstract. 17th Congr. int. Soc. Hemat., 15th Congr. int. Soc. Blood Transf., Paris, p. 439, 1978. [6] DELMAS-MARSALET Y., GOUDEMAND M. and TIPPETT P. - - Un nouvel exemple de g6notype rh6sus c D - - / c D - - . Rev. ft. Transfi, 12, 233-238, 1969. [7] GUNSON H.H. and DONOl~IUE W.L. - - Multiple examples of the blood genotype CwD--/CwD- in a Canadian family. Vox Sang., 2, 320-331, 1957. [8] HABIBI B-, PERRIER P, and SALMON Ch. - - Antigen Nou. A n ew high f r eq u en cy Rh antigen. Blood Transl. Immunohaemat., 24, 117-120, 1981. [9] HACKEL E. - - Rh antibodies in the s e r u m of two - - - D - - / - - D - - people_ Vox Sang., 2, 331-341, 1957. [10] ISSlTT P.D. - - Serology and genetics of the Rhesus b l o o d group system. M o n t g o m e r y Scientific Publications, Cincinnati, 1979. [11] JENKINS D.E. J r and MOOREW.H. - - A r a p i d m e t h o d for the p r e p a r a t i o n of high p o t e n c y auto and alloantibody eluates. Translusion, Philad., 17, 110-114, 1977. [12] KENDRICK L., DUNSTAN-ADAMS C., HUMPHREYS J., PLANTOS M., MOORE B.P.L., LOMAS C. and DANIELS G.L. - - The r a r e Rh haplotypes - - D - and - - - - - - in a family w i t h a - - D - - / - - - - - propositus. J. I m m u n o genet., 8, 243-247, 1981. [13] KOCHWA S. and ROSENFIELO R.E. - - I m m u n o c h e m i c a l studies of the Rh system. I. I s o la ti o n and c h a r a c t e r i z a t i o n of antibodies. J. Immunol., 92, 682-692, 1964. [14] McGuIRE-MALLORY D., ROSENFIELD R.E. WONG K.Y., HELLER C., ROnlNSTEIN P., ALLEN F.H. fir., "WALKERM.E. and LEWIS M. - - Rhmoa, a second kindred (Craig). Vox Sang., 30, 430-440, 1976. [15] MOORE B.P.L., CORNWALLS., CROCKFORDJ. and Vos G_H. - - The i m m u n e response in D - - - - , DC w---- and D c - - homozygotes. Abstract. 14th Congr. Int. Soc. Blood Transl., Helsinki, 1975. [16] PERRIER P., HABIBI B. and SALMON Ch. - - Allo antibodies in p ar t i al l y silent Rh phenotypes. E v i d e n c e for a possibly n e w high f r e q u e n c y Rh antigen. Blood Transl. Immunohaemat., 23, 327-339, 1981. [17] RACE R.R. and SANGER R. - - Blood Groups in Man, 6th ed., Blackwell, Oxford, 1975.
IMMUNE RESPONSE OF HOMOZYGOTES FOR THE Rh HAPLOTYPE
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[18] RACE R.R., SANGER R. and SELWYN J.G. - - A p r o b a b l e deletion in a h u m a n Rh c h r o m o s o m e . Nature, Lond., 166, 520, 1950. [19] RACE R.R., SANDER R. and SELWYN J.G. - - A possible deletion in a h u m a n Rh c h r o m o s o m e : a serological and genetical study. Brit. J. Exp. Path., 32, 124-135, 1951. [20] ROSENFIELD R.E., ALLEN F.H. Jr. and RUBINSTEIN P. - - Genetic m o d el for the Rh blood-group system. Proc. Natl. Acad. Sci_, USA, 70, 13031307, 1973. [21] ROSENFIELD R.E., ALLEN F.H. Jr., SWISHER S.N. and K0CHWA S. - - A r e v i e w of Rh serology and p r e s e n t a t i o n of a n ew terminology. TransIusion, Philad., 2, 287-312, 1962. [22] SALMON C., GERBAL A., LIBERGE G., SY B., TIPPETT 1~. and SANGER R. - Le c o m p l e x e g6nique DIv(C)--. Rev. ft. Transl., 12, 239-247, 1969. [23] SCHNEIDER W. and TIPPETT P. - - Rh33, a n o t h e r possible homozygote. Abstract. 30th Ann. Mtg. AABB, Atlanta, 1977. [24] SHAPIRO M. - - Serology and genetics of a new blood factor: h r s. J. forens. Med., 7, 96-105, 1960. [25] STEVENSON M.M., ANIDO V., TANNER A.M. and SWOYER J. - - Rh " n u l l " is not always null. Brit. Med. J., 1, 417, 1973. [26] TATE H., CUNNINGHAMC., MCDADE M.G., TIPPETT P.A. and SANDER R. - An Rh gene c o m p le x Dc--. Vox Sang., 5, 398-402, 1960. [27] TIPPETT P. - - Depressed Rh phenotypes. Rev. ft. Transl. I m m u n o Hdmat., 21, 135-150, 1978. [28] WIENER A.S. - - Blood-group n o m e n c l a t u r e . Science, 128, 849-852, 1958. [29] YAMAGUCHI H., OKUBO Y., TANAKA M., TOKUNAGA E. and AMARI S. - R a r e blood type Rhmoo o c c u r r i n g in two J a p a n e s e families. Proc. Jap. Acad., 51, 763-766, 1975. [30] YAMAGUCHIH., OKUBOY., TOMITA T., YAMANOH. and TANAKA*M.- - A case of R h gene c o m p l e x c D - - / c D - - f o u n d in a Japanese. Proc. Jap. Acad., 45, 618-620, 1969.
185
An Investigation of the Immune Response of Homozygotes for the Rh Haplotype--D and Related Haplotypes Using Cells of Rare Rh Phenotypes
by G.L. DANIELS MRC Blood Group Unit, LONDON(England).
ANY examples of h o m o z y g o t e s for the rare Rh gene complex - - D - - , in w h i c h the red cells lack any representation of either the C or E series of antigens, have been studied (RACE and SANGER [17]) since the original example was described in 1950 by RACE, SANGER and SV,LWYN [18]. Following i m m u n i z a t i o n by p r e g n a n c y or transfusion, h o m o z y g o u s - - D - - people f o r m antibody which reacts with all cells of c o m m o n Rh type [17].
M
RACE e t al. [19] f o u n d that the s e r u m of the first example of - - D - - / ~ D - - , Mrs. B., contained separable anti-e, anti-C and anti-c. They thought, however, that the m a j o r c o m p o n e n t of the antibody was anti-Cc w h i c h seemed to be directed against C and c indifferently. HACKEL [9], using the sera of Mrs. B. and a n o t h e r h o m o z y g o u s - - D - - , Mr. M.C., c o n f i r m e d these results and d e m o n s t r a t e d also
DANIELS
186
G.L.
some separable anti-E and anti-ce. He agreed t h a t m u c h of the antibody p r e s e n t was directed against an antigen p r o d u c e d b y all c o m m o n Rh gene complexes. GUNSON and DONAHUE [7] f o u n d t h a t a b s o r p t i o n s w i t h CDe/CDe, cDE/cDE, C D e / c D E or c d e / c d e r e d cells c o m p l e t e l y r e m o v e d all the antibodies p r e s e n t in the s e r u m of their CWD--/CWD- p r o p o situs; s o m e w h a t similar findings w e r e those of BUCHANAN and MCINTYRE [1] w i t h the s e r u m of their - - D - - / - - D - propositus. F r o m the results of sequential adsorption-elution of - - D - - / - - D J sera with CDe/CDe, c D E / c D E and c d e / c d e ceils in t h a t order, ROSENFIELD et al. [21] stated t h a t the i m m u n e r e s p o n s e in - - D - - / - - D - individuals is directed t o w a r d s antigenic d e t e r m i n a n t s i n d e p e n d e n t of all other k n o w n Rh specificities. This a n t i b o d y was n u m b e r e d anti-Rhl7 [21] and is the a n t i b o d y previously n a m e d anti-Hro b y WIENER [28]. The sera of s o m e of the CWD- [7] and c D - - [6, 26, 30] homozygotes, the only (C)D ~v-- h o m o z y g o t e [17, 22] and one of the two •D" h o m o z y g o t e s [3] contain an a n t i b o d y (or antibodies) like t h a t in the - - D - - / - - D - - i m m u n e sera. H o w e v e r , TIPPETT [27] s h o w e d that i m m u n e sera f r o m people w i t h " d e l e t e d " p h e n o t y p e s differ in their ability to agglutinate Rhino a cells and those of an R aa homozygote. PERRIER et al. [16] a n d HABIBI et al. [8] s h o w e d t h a t sera f r o m two - - D - - h o m o z y g o t e s contained, as well as anti-Rhl7, a second antibody to a public antigen (anti-Nou) w h i c h r e a c t e d w i t h ceils of c o m m o n and certain d e p r e s s e d Rh p h e n o t y p e s and w i t h h o m o z y g o u s (C)D ~v-- cells, b u t not w i t h - - D - - / - - D - - , c D - - / c D - - or Rhnun cells. PERRIER et al. [16] also d e m o n s t r a t e d , s e p a r a b l e anti-e in all the sera f r o m - - D - - h o m o z y g o t e s and r e l a t e d p h e n o t y p e s t h a t they tested. R e p o r t e d b e l o w is an a t t e m p t to analyze the i m m u n e of people a p p a r e n t l y h o m o z y g o u s for " p a r t i a l l y d e l e t e d " complexes (and one - - D - - / - - - - - double h e t e r o z y g o t e studying t h e i r reactions w i t h cells of r a r e Rh p h e n o t y p e s , the earlier o b s e r v a t i o n s of DANIELS et al. [5].
response Rh gene [12]) b y extending
SEROLOGICAL S T U D I E S
I m m u n e sera f r o m 16 - - D - - ~ - - - D - - , 1 - - D - - / - - - - - - , 3 cD--/ cD--, 3 CwD--/CWD- , 1 - D ' / ' D - a n d 1 (C)D~V--/(C)D I v j , all of which are c o m p a t i b l e w i t h - - D - - / - - - D - - a n d Rh~un cells, w e r e tested against the following cell s a m p l e s of r a r e Rh types: t w o e x a m p l e s of " D : / ' D (Mrs. H.D. [3] and Dor.), h o m o z y g o u s R ~ (Otto H. [23]),
I M M U N E R E S P O N S E OF H O M O Z Y G O T E S FOR T H E Rh I t A P L O T Y P E
187
a c D e N e g r o w i t h v e r y w e a k e (W.J. [2]), t h r e e e x a m p l e s o f Rhr, oa s h o w i n g v a r y i n g e x p r e s s i o n o f R h a n t i g e n s ( K u r a . [29], Hel. [25] a n d C.C. [14]) a n d a p o s s i b l e C W D ( e ) / - - D - (N.R.). A s u m m a r y of t h e R h r e a c t i o n s o f t h e s e cell s a m p l e s is g i v e n in T a b l e I.
TABLE
I
R e s u l t s of testing s o m e cells w i t h rare R h p h e n o t y p e s w i t h R h antisera. Rh
ANTISERA
CELLS D
~I.D . . . . . . . . . . . . )or ............. Otto H ........ W.J ............ Kura ........... Hel.* . . . . C.C ............ N.R ............
C
+ + + + .
.
.
. . +w +
c
--.
. . _+ --
Cw
E
+ + .
. --
.
.
. +
.
e
f
± _+
_ _+
+w +
---
G
D inc
+ +
+ +
Evans
Rh32
Rh33
+ + +
+
. --
+w
+
_+
LW
+ + + -k
+_ +
D inc = incomplete anti-D tested against saline suspensions of cells_ ± = + with some sera - - with others. +w = weaker reaction than control cells with all sera. * Other tests show Hel. is not Rh~un [4, 25]. All negative with anti-V and anti-VS.
T h e 25 s e r a w e r e t e s t e d a g a i n s t t h e r a r e cells b y t h e i n d i r e c t a n t i g l o b u l i n t e s t (IAT), u s i n g a p u r e a n t i - I g G . W h e r e n e c e s s a r y s o l u b l e A a n d B b l o o d g r o u p s u b s t a n c e s w e r e u s e d to i n h i b i t anti-A a n d a n t i - B in t h e s e r a , g r o u p A o r B Rh~un o r - - D - - / - - D - being used as a negative control. Titrations of the sera were also tested a g a i n s t C D e / c D E cells, b y IAT, i n o r d e r t o g{ve a n e s t i m a t e o f t h e relative strengths of the sera. A s u m m a r y o f t h e r e s u l t s , s h o w i n g t h e d i f f e r e n t p a t t e r n s of r e a c t i o n s of t h e s e r a , is g i v e n in T a b l e I I . I n Fig. 1 a n a t t e m p t is m a d e to r e l a t e t h e a n t i b o d y s t r e n g t h to t h e p o s i t i v e a n d n e g a t i v e r e a c t i o n s w i t h t h e r a r e cell s a m p l e s . T h e r e s u l t s i n T a b l e I I a n d Fig. 1 s h o w h e t e r o g e n e i t y o f t h e s e r a in t h e i r + a n d - - r e a c t i o n s w h i c h c a n n o t b e e n t i r e l y d u e to t h e s t r e n g t h of t h e a n t i b o d y . The positive reactions, although generally given by the higher scoring sera, show an overlap with t h e l o w e r s c o r i n g s e r a , e s p e c i a l l y w i t h O t t o H. cells. To t a k e s p e c i f i c e x a m p l e s : t h r e e s e r a s c o r i n g 91, 114 a n d 117 w i t h C D e / c D E
188
DANIELS
G.L.
cells f a i l e d to r e a c t w i t h t h e cells of H.D. a n d O t t o H. w h e r e a s a n o t h e r s c o r i n g 87 r e a c t e d w i t h b o t h s a m p l e s a n d t h r e e o t h e r s s c o r i n g 50, 64 a n d 68 r e a c t e d w i t h O t t o H. cells. T h e a b i l i t y to 120
110 100 9O
80
7o 8 •
>
60
o~ 50 4o
fl 30 E-~ 2O
10
+
H.D. •D"
-
~
Dor, "D"
+
~
Otto H. Rh33
+
-
Kura. P~mod
+
-
Hel. Rhmod
Redcells
FI6. 1 . - Diagram relating the antibody strength of sera from people with "deleted" phenotypes (Table II) to their + a n d - reactions with cells of rare Rh phenotypes. Dots on the left hand line of each pair of lines indicate titration scores against CDe/cDE cells of sera which reacted with I-I.D., Dor., Otto H., Kura. and Hel. ceils, whereas the dots on the right hand lines indicate scores against CDe/cDE cells of sera that failed to react x~'ith H.D., Dor., Otto H., Kura. and Hel. cells. r e a c t w i t h Rhmod cells is c l e a r l y m o r e d e p e n d e n t o n t h e s t r e n g t h of t h e a n t i b o d y a l t h o u g h t h e r e a r e e x c e p t i o n s ; f o r e x a m p l e o n e s e r u m w i t h a s c o r e a g a i n s t C D e / c D E ceils of 57 r e a c t e d w i t h K u r a . a n d Hel. cells w h e r e a s a n o t h e r , w i t h a s c o r e of 83, d i d n o t . T h e t w o h o m o z y g o u s "D" s a m p l e s a r e d i s t i n g u i s h e d f r o m e a c h o t h e r b y o n l y o n e s e r u m ; t h a t is, o n l y t h r e e of t h e f o u r s e r a t h a t r e a c t e d w i t h H.D. cells r e a c t e d w i t h Dor. cells. L i k e w i s e , t w o of t h e RhmoaS ( K u r a . , Hel.) a r e d i s t i n g u i s h e d b y o n l y o n e s e r u m ( T a b l e II).
189
I M M U N E RESPONSE OF HOMOZYGOTES FOR THE Rh HAPLOTYPE
TABLE II Results of indirect antiglobulin tests (IAT) using sera f r o m people w i t h " d e l e t e d " p h e n o t y p e s against cells of rare R h phenotypes.
Cells
SERA
Phenotypes
No,
-DH.D.
7
--D
2
cD--
3
--D
1 1
CwD(C)Dr v -
3
2
--D
1
CwD--
1
--D
3
1 --D--/-- ---- + 1 cD--
9
5
c(D)(e) Dor. Otto H.
m
C.C.
N.R.
--
+
+
+
+
+
+
+
+
+
+
+
-b
+
+
+
+
+
--
+
q-
--
+
+
b
m
m
+
I m
---D-CwDm
m
+ +
+ ÷
-b
u
--D--
-D-
cD(e) Rhraod Hel. W.3. Kura.
+
m
+
+
m
All sera compatible with - - D - - / - - D - - and Rhnull cells. None of the reactions due to anti-A or anti-B
T a b l e I I also s h o w s t h a t t h e a n t i b o d y c o n t e n t of t h e six t y p e s of " d e l e t e d " g e n o t y p e s ( h o m o z y g o u s - - D - - , c D - - , CWD- , "D' a n d (C)D ~v-- a n d - - D - - / - - - - - - ) as j u d g e d b y t h e s e , r e a c t i o n s w i t h r a r e p h e n o t y p e s does n o t d i s t i n g u i s h o n e f r o m a n o t h e r . T h e o n l y except i o n is t h e s i n g l e e x a m p l e of s e r u m f r o m "D'/-D- (H.D.), t h e o n l y o n e t h a t f a i l e d to r e a c t w i t h W.J. cells. T i t r a t i o n s of m o s t of t h e s e r a w h i c h gave p o s i t i v e r e a c t i o n s w i t h t h e cells of r a r e p h e n o t y p e s w e r e t e s t e d i n p a r a l l e l w i t h t h o s e cells a n d cells of c o m m o n R h t y p e a n d t h e r e s u l t s a r e s h o w n i n T a b l e I I I . T h e s c o r e s w e r e a l w a y s less f o r t h e r a r e cells t h a n f o r c o n t r o l s as m i g h t b e e x p e c t e d f r o m t h e p a t t e r n of r e a c t i o n s i n T a b l e I I : t h e d i f f e r e n c e i n s c o r e s was, i n g e n e r a l , m o s t m a r k e d for ' D ' / ' D " cells (H.D. a n d Dor.), less so f o r Otto H. a n d Rhmoa ( K u r a . ) , still less f o r N.R. a n d Rhmod C.C. a n d l e a s t f o r W.J.. H o w e v e r v a r i a t i o n i n d e g r e e of r e d u c t i o n of s c o r e does exist b e t w e e n d i f f e r e n t s e r a (e.g. s e r a of Mrs. B. a n d Hal. a g a i n s t N.R. a n d C.C. cells).
DAN1ELS G.L.
190
The sera of M.C. ( - - D - - / - - D - - ) a n d of Mrs. H. ( c D - - / c D - - ) and an eluate of M.C. s e r u m a d s o r b e d on C D e / c D E cells, all of w h i c h r e a c t e d w i t h " D ' / ' D ' and Rhmoa cells w e r e f u r t h e r investigated to see if the a n t i b o d y for these r a r e cells was s e p a r a b l e f r o m t h a t for c o m m o n Rh p h e n o t y p e s . All eluates w e r e m a d e b y the digitoninacid-stromal m e t h o d [11, 13]. TABLE I I I
Titration scores (IAT) [or sera tested against cell samples o[ rare R h phenotypes and controls.
Sera Name Mrs. B. M.C. Ca. Cus. M.I. D.O.
Ota. Pep. V,M. Mrs. H. Joh.
Cells
Phenotype
H.D.
Dor.
Otto H.
W.J.
--D-"' ~ ~ '~ '~ ~
0
0 85 0 0 0 0 0 0 23 29 0 0 0 0 0 0
0 86 0
103
') --D--/--
* Control
--
cD-"
Po.
Coo. Hal. MacD. Nou.
0 0 0 0 0 0
~'
CwD-~'
~ (C)DIV-= mean
--
32 0 0 19 0 0 0
score of titrations
Kura.
C.C.
N.R.
0
34 90 12 38 19 19 42 76
67 99 15 41 16 32 40 82
101 43 25 69 93 51 87
106 6O 22 53 90 42 76
0 46
0 0 0
58 0 0 0 7 0
0 0 0
103
56 97
93 0 0
tested against cells of common
Control* 109 112 24 66 28 56 81 94 91 108 71 44 7t 93 69 102
Rh phenotypes
Fig 2 shows t h a t a b s o r p t i o n w i t h C D e / c D E cells r e m o v e d all a n t i b o d y for ' D ' / ' D " cells a n d for cells of c o m m o n p h e n o t y p e s . A b s o r p t i o n w i t h " D ' / : D ' (H.D.) ceils, on the o t h e r hand, r e m o v e d all a n t i b o d y for "D'/'D" ceils but, even a f t e r continued a b s o r p t i o n s left a strong a n t i b o d y for cells of c o m m o n p h e n o t y p e . The separable a n t i b o d y in these sera w h i c h r e a c t e d w i t h " D ' / ' D cells is not, however, anti-Evans. An eluate f r o m M.C. s e r u m a d s o r b e d on "D'/'D: cells r e a c t e d equally strongly w i t h "D'/'D" and cells of c o m m o n Rh p h e n o t y p e giving the following t i t r a t i o n scores: "D'/'D" cells 33; CDe/CDe 41; c D E / c D E 32; c d e / c d e 42; CDe/-D" 39; cde/'D" 41. Dot. "D'/'D" cells w e r e also u s e d in a b s o r p t i o n / e l u t i o n tests w i t h Mrs. H. s e r u m ( t i t r a t i o n s not done) a n d gave similar results to those o b t a i n e d w i t h H.D. cells thus s u p p o r t i n g the conclusions d r a w n f r o m using H.D. cells.
I M M U N E R E S P O N S E OF' H O M O Z Y G O T E S FOR T H E Rh I-1APLOTYPE
191
A Absorbed with CDe/cDE cells
Absorbed with H,D. cells
80
6O
o~ 40~
~ ~0"~,, .~
01 "-~ .... • 1
2
4
i
2
4
Number of absorptions
.B Absorbed with H.D. cells
Absorbed with CDe/c~E cells 1 20
100
m
8O LI
o o
60
o~ 4= q~
4o 20
.\
~k I
2
4
o\
2
4
~umbe= of absorptions
FIG, 2. - - Absorption graphs. A.
M . C . (--D--/--D--) eluate (from CDe/cDE) cells against H.D. ( ' D / ' D ' ) and control ceils. B. Mrs. H. (cD---/cD--) serum against H.D. and control cells. --x-- Titration scores against H.D. cells. - - e - - Average titration scores against CDe/CDe, cDE/cDE and cde/cde cells.
A b s o r p t i o n a n d e l u t i o n t e s t s w i t h M.C. s e r u m (Fig. 3) u s i n g Rhmoa ( K u r a . ) a n d C D e / c D E cells gave a d i f f e r e n t p i c t u r e a n d s u g g e s t t h a t a s i n g l e a n t i b o d y is r e s p o n s i b l e f o r t h e r e a c t i o n s w i t h b o t h Rhmod a n d cells of c o m m o n R h p h e n o t y p e s . This a n t i b o d y w a s r e m o v e d less e f f i c i e n t l y b y Rhmoa t h a n b y C D e / c D E cells; t h e e l u a t e s f r o m
192
DAN1ELS G.L.
b o t h p h e n o t y p e s b e h a v e d a l i k e i n r e a c t i n g less s t r o n g l y w i t h Rhmod t h a n w i t h C D e / c D E cells. Some time after the above work was carried out homozygous (C)D ~ v - (Nou.) cells b e c a m e a v a i l a b l e f o r t e s t i n g ( k i n d l y s u p p l i e d b y Drs. Ph. ROUGER a n d B. HABIBI). T h e s e cells r e a c t e d w i t h M.C. Absorbed with CDe/cDE cells
Absorbed with Ku~a. cells
8O
vl
•
~60~
X
~40 4~
.~ 20 0
"~ ...............
1
2
""
4
0
1
2
4
Number of absorptions
FIG. 3. - - Absorption graphs. M.C. (--D--/---D--) serum against Kura. (Rhmod) and CDe/cDE
cells. --x-- Titration scores against Kura. cells. - - e - - Titration scores against CDe/cDE cells. M.C. serum diluted 1:10 in A and B solutions. Eluates from M.C. serum adsorbed on CDe/cDE cells gave titration scores of 25 with Kura. cells and 47 with CDe/cDE cells. Eluates from M.C. serum adsorbed on Kura. cells gave scores of 27 with Kura. cells and 42 with CDe/cDE cells. a n d Mrs. H. s e r a a n d t h e p a t t e r n of r e a c t i o n w i t h t h e s e r a i n T a b l e I I r e s e m b l e d t h a t f o r O t t o H. m o r e c l o s e l y t h a n t h a t f o r H.D. o r Dor., a l t h o u g h s o m e of t h e s e r a w e r e n o l o n g e r a v a i l a b l e f o r t e s t i n g . N o u . cells r e a c t e d s t r o n g l y w i t h M.C. s e r u m a b s o r b e d f o u r t i m e s w i t h H.D. o r w i t h D o t . cells b u t d i d n o t r e a c t w i t h M.C. s e r u m a b s o r b e d f o u r t i m e s w i t h K u r a . cells o r w i t h C D e / c D E cells. N o n e of t h e s e f o u r a b s o r b e d s e r a r e a c t e d w i t h H.D. o r Dor. ceils a n d all b u t t h e l a s t r e a c t e d w i t h C D e / C D e cells. Nou. cells also r e a c t e d w i t h a n e l u a t e p r e p a r e d f r o m Dor. ceils s e n s i t i z e d w i t h M.C. s e r u m . T h i s e l u a t e also r e a c t e d w i t h H.D., Dot. a n d C D e / e D e cells.
DISCUSSION
Homozygotes for the " d e l e t e d " Rh gene complexes ( - - D - - , cD--, CwD- , (C)D w - a n d "D') w h e n i m m u n i z e d b y p r e g n a n c y o r t r a n s f u -
I M M U N E R E S P O N S E OF H O M O Z Y G O T E S F O R T H E R h H A P L O T Y P E
193
sion f o r m antibody which reacts with cells of all c o m m o n Rh phenotypes but, with the exception on occasion of ' D ' / ' D ' [3] and (C)D~V--/ (C)D ~ v - [8, 16] cells, are all m u t u a l l y compatible. It has been suggested that these i m m u n e sera contain a mixture of antibodies resembling those m a d e by people of c o m m o n p h e n o t y p e together with a c o m p o n e n t directed at the Cc and Ee antigen indifferently [9, 17, 19] or, alternatively, that one antibody called anti-Hro [28] or anti-Rhl7 [21] is responsible. The results discussed here suggest that neither of the above is the whole answer. The heterogeneity of these i m m u n e sera was d e m o n s t r a t e d by investigations with cells of rare Rh p h e n o t y p e (Tables I I and I I I ) . Results with "D'/'D" and R~a/R 3a (Otto H.) ceils could be explained by a qualitative as well a quantitative difference in the antibody c o n t e n t of the sera (Tables I I and I I I , Fig. 1). The titration scores for these cells were lower than those for cells of c o m m o n phenotypes (Table I I I ) : some sera, n o t a b l y Hal., failed to react with ' D ' / ' D " and Rh33, yet s h o w e d not reduction in scores for other rare phenotypes (C.C., N.R.) suggesting that m o r e than one antigen is involved. Absorption and elution studies with D ' / D cells suggest that M.C. ( - - D - - / - - D - - ) and Mrs H. ( c D - - / c D - - ) sera have at least two antibodies: one which reacts with cells of c o m m o n phenot~pes but not with ' D ' / ' D ' (anti-Rhl7) and one which reacts with ceils of all c o m m o n p h e n o t y p e s and "D'/'D" (Fig. 2). There was no evidence of separable anti-Evans, specific for 'D" cells, in these sera. Since D / ' D ' cells are negative with the s e r u m of the original hr ~ negative, Mrs. Sh. [4], this second a n t i b o d y c a n n o t be the same as anti-Hr (anti-Rhl 8). The different p a t t e r n of reactions of " d e l e t e d " sera against Otto H. (Rh33) and W.J. cells (Table II) is evidence against the sera containing a mixture of antibodies against C, c, E and e antigens. These cell samples are alike in their C, c, E and e reactions and differ only in their D, G and Rh33 antigens; b o t h D and G are present on cells of all of the " d e l e t e d " pbenotypes. However, Otto H. shows that some Rh antigens, e.g. c, can be expressed normally in the absence of n o r m a l expression of Rhl7. The tests with Rhmoa cells, three unrelated examples, suggest that their relatively weak reactions (Tables I I and I I I , Fig. 1) can be a t t r i b u t e d to a quantitative r a t h e r t h a n qualitative difference. C.C., who has the strongest Rh antigens, gives the strongest reactions failing to react with only one of the sera (a weak antibody). McGuIREMALLORY et al. [14] state that C.C. cells show no depression at all of Rh17 antigen as d e t e r m i n e d by an a u t o m a t e d qualitative blood typing technique using an AutoAnalyser. U n f o r t u n a t e l y they do not say how
194
DAN1ELS
G.L.
m a n y sera were used to d e t e r m i n e this. Table I I I shows that, by m a n u a l titration, the sera f r o m " d e l e t e d " p h e n o t y p e s give varying results with C.C. cells. To take extreme examples f r o m Table I I I ; if Mrs. H. or Hal. sera were used no depression could be seen, b u t if the s e r u m f r o m the original - - D - - / - - D - - , Mrs. B., was used a large depression could be seen c o m p a r e d with C D e / e D e cells, C.C. cells scoring only 30% of that of the c o n t r o l cells. The suggestion of a quantitative r a t h e r than a qualitative antigenic difference in Rhmoa is s u p p o r t e d b y a b s o r p t i o n and elution tests using Kura. cells (Fig. 3). The results p r e s e n t e d above, together with previously r e p o r t e d results [1, 4, 7, 8, 15, 16, 21], suggest that these i m m u n e sera do not contain a m i x t u r e of antibodies directed against C, c, E and e antigens, although separable anti-e m a y sometimes be present. By a b s o r p t i o n tests using CDe/CDe, c D E / c D E and c d e / c d e cells PERRIER et al. [16] f o u n d anti-e to be p r e s e n t in all seven samples tested (4 f r o m - - D - - / - - D - - , 2 f r o m c D - - / c D - - and one f r o m (C)D~V---/ (C)D Iv--- people). Daniels [4] tested sera in a similar way and f o u n d separable anti-e in b o t h sera f r o m - - D - - h o m o z y g o t e s tested b u t could not separate anti-e f r o m the s e r u m of a C~D - homozygote, although an a n t i b o d y was p r e s e n t w h i c h b e h a v e d like anti-e h u t w h i c h was a b s o r b e d by c D E / c D E cells: he f o u n d no trace of anti-e in the s e r u m of tbe "D" h o m o z y g o t e H.D. However, m o s t of the reactivity is due to one antibody, anti-R17, in w h i c h case the antigen R h l 7 must, like D and e, be a mosaic, or there is a mixture of antibodies to high f r e q u e n c y antigens present in all c o m m o n phenotypes. Some sera f r o m people with " d e l e t e d " p h e n o t y p e s contain anti-Rh18 (anti-Hr or anti-Hr9 as well as anti-Rhl7 (anti-Hro) [10, 24] and PERRIER et al. [16] and HABIBI et al. [8] described a n o t h e r antibody to a high f r e q u e n c y antigen (anti-Nou) in some such sera. ISSITT [10] suggested that some sera m a y also contain anti-Rh34 [20]. Absorption-elution tests using "D:/'D" ceils (Fig. 2) show the presence of a n o t h e r a n t i b o d y to a high f r e q u e n c y antigen, different f r o m anti-Rhl7 and anti-Rhl8. It is suggested that this specificity be referred to as anti-Day. The reaction of Nou. cells with M.C. s e r u m a b s o r b e d f o u r times with -D'/-D- cells, and consequently no longer reactive with these cells, shows that anti-Day is not the same as anti-Nou t h o u g h anti-Nou m a y contain anti-Dav. Anti-Dav has not been s h o w n to differ f r o m anti-Rh34. SUMMARY I m m u n e sera f r o m people h o m o z y g o u s for the " d e l e t e d " gene complexes - - D - - , cD--, CwD- , "D" and (C)D ~ v - as well as hetero-
I M M U N E R E S P O N S E OF H O M O Z Y G O T E S F O R T H E Rh H A P L O T Y P E
195
zygous - - D - - / - - - - - were studied by their reactions, and in some cases by absorption-elution tests, with cells of the following rare Rh types: two unrelated examples of h o m o z y g o u s "D'; three unrelated examples of Rhmod; h o m o z y g o u s Raa; cDe with a very weak e; a p p a r e n t CWD(e)/--D - . The results showed that these i m m u n e sera are clearly heterogeneous: this heterogeneity was not entirely due to the strength of the a n t i b o d y (as d e t e r m i n e d by titration against CDe/cDE cells) or to the p h e n o t y p e of the a n t i b o d y maker. A b s o r p t i o n and elution tests using two of the i m m u n e sera and •D~/'D" cells d e m o n s t r a t e d a " n e w " antibody, anti-Dav, for a high f r e q u e n c y Rh antigen in addition to anti-Rhl7. RESUME Les s6rums i m m u n s d'individus p r 6 s e n t a n t des ph6notypes Rh partiellement s i l e n c i e u x , - - D - - , cD--, CWD- , "D" et (C)D ~v---, ainsi q u ' u n h6t6rozygote - - D - - / - - - - - ont 6t6 6tudi6s par leurs r6actions et e n certains cas p a r d e s tests d ' a h s o r p t i o n et d'6lution h l'aide de globules rouges h ph6notypes Rh e x c e p t i o n n e l s suivants: deux exempies n o n apparent6s de " D ' / ' D ' ; trois exemples non apparent6s de Rh~oa; et un exemple de RaaR a3, de cDe avec un e tr~s faible, et de CWD(e)/--D - probable. Les rdsultats ont m o n t r 6 que les r6actions de ces s6rums i m m u n s sont tr6s h6t6rog~nes: cette h6t6rog6n6it6 n'6tait pas enti6rement lide h la puissance de l'anticorps (d6termin6 p a r le titrage c o n t r e les globules rouges CDe/cDE) ni a u ' p h d n o t y p e de l'individu ayant ddvelopp6 l'anticorps. Les tests d ' a b s o r p t i o n et d'61ution, utilisant deux de ces sdrums immuns, avec les globules rouges "D-/-D-, ont ddmontr6 un nouvel anticorps, anti-Day, c o r r e s p o n d a n t ~ un antig6ne Rh de haute fr6quence ainsi q u ' u n anti-Rh 17. ACKNOWLEDGEMENTS. -The author thanks the many colleagues who sent samples of immune sera and cells of rare phenotype. All are acknowledged individually in reference 4.
Request reprints f r o m : Dr. G.L. DANIELS MRC Blood Group Unit Wolfson House University College London 4 Stephensen Way LONDONNWl 2HE England.
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