REPOSITORY BNL Keeond's
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MARCH 25, 1961
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A. C. ALLISON
B. S. BLUMBERG
M.D. Columbia, D.Phil. Oxon.
OF THE NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND
DtrinxG the past decade it has become apparent that
human beingsdiffer in their serum-proteins as well as in
Genetically controlled variations
have been described in haptoglobins and transferrins
(Smithies and Connell 1959), y-globulins (Grubb 1957,
Ropartz 1960), and in the Ge system definable by immunoelectrophoresis (Hirschfeld and Beckman 1960). Though
the chemical differences between the alternative. proteins
are not yet known, they are generally assumed to be small
—analogous to the single aminoacid substitutions that distinguish the different human hemoglobin half-molecules
(Hunt and Ingram 1959) and the small sugar substitutions
that distinguish the blood-group substances (Morgan
1959). Nevertheless, the possibility remains that these or
other serum-proteins are sufficiently different in structure
to induce serum-protein isoimmunisation when blood is
transfused.
Such a reaction is already known in rabbits. Oudin
(1956), Dray and Young (1959), and others have shown
that rabbits can be immunised with serum from other
rabbits of different y-globulin type. In the original
experiments the foreign plasma was introduced with
adjuvants, but recent observations (S. Dray, personal
communication) have shown that adjuvants are unneces-
sary for immunisation, Three or four intravenous injec-
tions of serum from rabbits of another y-globulin type led
to the production in recipients of well-defined precipitating
antibodies.
‘The experiments described here were undertaken to
find out whether any similar reaction can be shown in man.
Sera were obtained from subjects who had received multiple transfusions and were tested for precipitating
antibodies against a panel of sera chosen to representall
the available genetically controlled serum-protein types.
A serum has been found which gives strong precipitation
with some, bur not ali, sera; and this reaction defines a
polymorphism involving the «,-globulins. Besides being
of genetic interest, this finding raises the possibility that
serum-protein isOimmunisation takes place in man and
represents a clinical problem in persons receiving bloodtransfusions.
Materials and Methods
Post-transfusion sera.—These were obtained from patients at
the Clinical Center of the National Institutes of Health,
The
indications for transfusion included open-heart surgery,
hemolytic and aplastic anemias, pelvic tumours, and leuk-
gmias. Wherever possible, blood-samples were drawn between
10 and 30 days after the last transfusion. Sera were stored at
—20°C and were tested within 5 days of collection. Many of
the subjects had received 30 or more transfusions.
Panel sera.—These were selected from the collection of the
Section on Geographic Medicine and Genetics of the National
Institute of Arthritis and Metabolic Diseases. The 24 sera
included haptoglobin types 1-1, 2-1, 2-2, 2-1M, and O;
transferrin types CC, CD, and BC; and y-globulin types Gm
a+, Gma—, Gm b—-, Gm b—, Gm x-++, and Gm x—. In
addition, 3 sera from patients with definite rheumatoid arthritis
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L. «actor,
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ton, .actor
and 1 serum fran
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titres ofofHRIOR
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25, 1961
7
for 2 mont
wetued
waa} tran fusions ths
ow au.
Because of th
aumerous megakar
od, z
st rer heral blo
did not alter the reaction to be described. The sera Were fgg
OF THE NATIONAL INSTITUTE FOR MEDICAL RESEARCH, LONDON, N.W.7
Bethesda, who had received 5 or more transfusions.
g
Brookhaven National Labora
ignt with well-developed lupus erythematosus (Lz)
.
positive L.£. histological preparations and high titres fet
nuclear antibodies, were used. The sera had been
20°C for periods varying from 2 days to 2 years, S,.”
-
ALA., B.AML., D.Phil. Oxon.
their red blood-cells.
ICLES
sed.
obama Was: rai
ed.
sas Deen obtain
different population groups, including American hing
(sixteen), American Negroes (three), Micronesians (threes ~
Alaskan Eskimo, and a Viet-Namese. “*
En
Gel-diffusion precipitation technique-—The Ouchterlony yg.
cedure was carried out in 5 cm. petri dishes containing Buty
Without transfusic#
ry
vo. and in Februa
a
vote? of the Nation
0-9% ‘Oxoid Ionagar” (w/v) in 0:07 M sodium p
buffer, pH 7-0, with 0-001 Af sodium ethylenediaming,
tetra-acetate and 0-001 .VM sodium azide. With a die, Six wel
6 mm.in diameter were cut round a centre well of the same
The circumferences of the centre well and peripheraj wh
were 4 mm. apart. After removal of the agar cores from mw,
wells, the bases of the latter were sealed with small volumey
molten agar. The post-transfusion sera were placed in yy
centre wells and panel sera in the peripheral wells. The
were stored at room temperature and observed at 18 hours (hg
which time clear-cut precipitation was usually visible), 48
;
and 4 days. The precipitation is best seen by oblique illum,
tion from below against a dark background.
‘ea
Immunoelectrophorests,—This was done by the mi
sf
of Grabar and Williams (1955) and Scheidegger (1955) unig
standard lantern-slide plates coated with agar made oy
barbitone buffer 0-01 M. pH 8-4. Antibody (or antigen) ag.
placed in the side well and examined after reacting for 18 boy
by which time weli-defined precipitates were visible. ~Thy- J
reaction was continued to 36 hours, after which theplates weg.
washed in saline and stained.
Se
Starch-gel electrophoresisStarch gel (Connaught Lab
tories, Toronto) was made up in ¢ris-(hydroxymeth]-ameap:
methane buffer (Poulik 1957) and poured into 6 x 80 x 150 ma.§
trays. Samples were inserted as a starch paste (Smuchies 192%
into a slot 3 mm. wide. Electrophoresis was carried outS.
5 hours at 6V per cm. The position of the component.eej
identified in a smail strip of gel; the various components wee:
eluted by the method of Gordon (1960) and were concennzat
by ultrafiltration.
Eee |
Preparation of y-globulin components.—Separation of 7Sist:
19S y-globulins was achieved by sucrose density-gredies.
centrifugation (Fudenberg and Kunkel 1957) using 40, 30.2%.
and 10% solutions of sucrose.
z
Bentonite-flocculation test-—The method of Bozicevichcoal
(1958) was used.
. ESS
Latex-fixation test.—This was carried out as described Wf
Singer and Plotz (1956), using the Hyland ‘ RA-test Cet
(Hyland Laboratories, Los Angeles, California).
oy
aS
Observations
Fig. ?
svg. 1--The precipitiz
plate, between the -
seme, Dut not all,
persons. Unstained
Fig. 2—The precipitin
tse centre well agai
wr.
The uo degrees of
ee 2+ reactors and r.
s~pital the patient
sacs. The leucocyt
fe phitelet count ¥
7 ‘Se differential cou
2 relative increase oO
‘Ss scwrmalities.
Ref
Lignosed, The pa:
wah raised his hi
Lwharged,
When the patien
soy were well tole
suts of blood. Th
there has never bee
anor bloods. In t
transfusion reaction
‘ransfusions there
+22 F} accompar
‘ler symptoms. 7
A precipitin was found in the serum ofa patient we.
had received manytransfusions. He had a long and varet
history of illness.
ee
The patient is a 64-year-old white American retired execua
of Hungarian birth. At the age of 16 he had albuminuria
Y
lasted for 4 years. In 1934,while still living in Budapest,bebad?
an episode of sudden onset of parazsthesize and paralysis afmer
legs. This resolved spontaneously in 2 months. A
=
episode, involving complete paralysis up to the waist, wens
of the arms, and loss of sensation and reflexes in the legs,
O™. §
place in the United States in 1958. Cerebrospinal fuid cm. |
tained 73 mg. protein and 150 lymphocytes per ml. FOR:;
neuritis of unknown cause was diagnosed, and again rempisroe
ibe
was spontancous.
;
n
dant
*
The patient also had symptomsof peptic ulcerato
from 1921, with gastrointestinal hemorrhage in 1938 and 1*
In 1941 a gastroenterostomy was performed, but sy@ ek
and hemorrhage have recurred at intervals. The patieot 8%.
again admitted to hospital in 1958, at which time he was repo
to have low red-cell counts, Jow hemoglobin levels, 28
i
white-cell and platelet counts. X-ray examination suger:
an ulcer crater in the proximal duodenum. The blee
ss
Prize. 4—Agar-g
el i
The serum of a
“et by ultrafitera:
2€ the patient
was
“™st:cn of the pre
Stlobulin, Aboy