SOURCES OF THE ANTIBODIES DEVELOPING AFTER
BY OSWALD H. ROBERTSON, M.D., AND PEYTON ROUS, M.D.
(From the Laboratories of The Rockefeller Institute for Medical Research.)
(Received for publication, October 1, 1921.)
The recent wide utilization of transfusion as a therapeutic measure
has brought to light many facts of theoretical as well as practical
significance. Perhaps most interesting from both points of view is
the gradual decrease in beneficial effect, and the appearance sometimes
of positive injury from the frequently repeated injection of alien
blood. Were it not for this complication one might reasonably ex-
pect to maintain cases of pernicious anemia in good blood condition
for an indefinite period of time. Needless to say, the sources
of the failure have been the subject of much discussion and of
some research. One fact of great weight has been clearly shown.
Blood derived from a donor originally compatible, as proven both by
in vitro tests and by the clinical result, may not only cease to be use-
ful when too frequently injected into the same individual but may
give rise to serious reactions. A change has occurred, not in the donor
but in the recipient, such that the alien blood is no longer tolerated in
Boycott and Douglas 1 have noted that blood is destroyed more
rapidly after repeated transfusions in normal animals than it is at
first, as attested by an increase in the rate at which plethora disappears.
One of us, with Oliver, 2 has utilized the phenomenon to induce in
rabbits a hemosiderosis closely resembling that of hemochromatosis
in man. No doubt a part of the pigmentation observed at autopsy
in pernicious anemia patients who have been repeatedly transfused
is due to a like destruction of alien blood. But how is this blood
destroyed? By circulating antibodies or within special organs?
t Boycott, A. E., and Douglas, C. G., J. Path. and Bact., 1909, xlii, 414.
Rous, P., and Oliver, 7., J. Exp. ]fled., 1918, xxviii, 629.
ANTIBODIES DEVELOPING AFTER TRANSFUSION
Recent observations indicate that circulating antibodies are to a
considerable degree involved, s It has been found that repeated
transfusions of compatible blood in rabbits are followed often by the
appearance in the recipients' plasma of hemagglutinins so strong
that the red cells come together into a firm mass practically as soon
as the blood has been shed, while, furthermore, a fulminant destruc-
tion of corpuscles may take place in vivo with result in anemia. Rob-
ertson 4 has presented evidence that the elements destroyed are the
alien cells which, little by little, under the circumstances of plethora
and diminished bone marrow activity consequent thereon have taken
the place of cells proper to the host.
The hemagglutinins just mentioned are in the immediate sense
autoantibodies. They clump practically all of the circulating eryth-
rocytes, are especially effective at low temperatures, and persist
in high titer for months after the transfusions have been discontinued
and after recovery from the severe intercurrent anemia which may
develop soon after their appearance.
engaged in eliminating unusually large amounts of blood will elaborate
antibodies directed against its own cells? This is a point of major
interest in any study of the source of the hemagglutinins, and one
not without a practical bearing. In human beings true autohemag-
glutinins have repeatedly been observed 5 in association with anemia
of obscure origin; while autohemolysins are known to bear an impor-
tant relation to paroxysmal hemoglobinuria.
Is it possible that an organism
Three to six compatible donors were selected for each recipient by the examina-
tion of mixtures of the citrated bloods, e The recipients received, 6 days in every
7, 10 cc. of blood taken by cardiac aspiration into 1 to 2 cc. of 0.9 per cent salt
solution containing 1 per cent of sodium citrate. The donors were employed in
rotation. The small amount of citrate mentioned was sufficient to prevent
clotting during the short period required to introduce the blood into the recipient's
ear vein. About half of the transfused animals failed to develop autoagglutinins
at any time, even when the injections were continued for many weeks. In the
8 Rous, P., and Robertson, O.H., J. Exp. Med., 1918, xxvii, 509,
4 Robertson, O. H., J. Exp. Med., 1917, xxvi, 221.
5 Clough, M. C., and Richter, I. M., Bull. Johns Hopkins Hosp., 1918, xxix, 86.
6 Rous, P., and Turner, J. R., J. Am. Meal. Assn., 1915, lxiv, 1980.
OSWALD H. ROBERTSONAND PEYTON ROD'S
other individuals they appeared early, as a rule after only five to ten transfusions;
and when three or four more had been given, that is to say, after some 10 days
to 3 weeks in all, they were well marked. The shed blood of the rabbits, when
examined at room temperature, now showed prior to clotting a massive clumping
of the red cells due, as our previous work has shown, to the presence in the
plasma of true hemagglutinins, a
The Mixed Content of the Blood.
The transfused rabbits became, as it were, mixing vessels for
several alien corpuscles and plasmas, perhaps indeed concentrators of
immune principles through the elimination of the fluid wherewith
the latter were introduced. The clumping of the cells of the shed
blood might conceivably have been the result of:
1. The injected sodium citrate---a possibility ruled out by nega-
tive findings in control animals given citrate alone.
2. Antibodies introduced with the donor's plasma and (a)active
against the recipient's cells. These might either have developed
during the period of experiment, or have been so weak as to escape
detection in vitro. No tests had been made to rule out (b) interag-
glutination among the donor bloods.
3. Antibodies elicited in the recipients and directed against (a) the
strange corpuscles and (b) against the animal's own corpuscles.
4. Changes in the circulating cells resulting in a greater
5. Antibodies within the injected corpuscles, liberated by their
More remote possibilities could be invoked, as for example, a non-
specific agglutination brought about through physical changes in
the plasmas pooled in vivo, but, as will be shown, those above listed
suffice to account for the findings.
Tests with Preserved Cells.
In an initial series of experiments for the analysis of the conditions,
blood specimens were taken aseptically from the individuals of sev-
eral donor-recipient groups prior to any transfusions, and set aside
in a citrate-glucose mixture in which rabbit corpuscles remain viable
ANTIBODIES DEVELOPING APTER TRANSFUSION
for several weeksJ Later when the transfusions had elicited the
clumping phenomenon, these preserved corpuscles were several times
washed, made to a 5 per cent suspension with salt solution, and used
in agglutination tests paralleling others carried out at the same time
with freshly taken cells of the same animals.
For the purpose, equal parts of cell suspension and serum were mixed, allowed
to stand for 15 to 30 minutes at room temperature, and examined microscopically.
Room temperature is far more favorable to hemagglutination than blood heat.
Clumping of the cells is ordinarily completed in about 10 minutes.
The tests yielded clear-cut results. Prior to transfusion no agglu-
tination reaction was ever observed between the bloods of the pro-
spective donors and recipients, when examined either according to
the method just described or by that with whole citrated bloods
previously referred to. Weak interagglutinations were sometimes
noted between the bloods of certain donor groups but they were
entirely absent from others. The fresh and preserved cells of the
same individual were regularly found to behave almost identically
on test in vitro. No evidence was obtained for possibility No. 4
of those above listed, namely an increase in agglutinability of cells
circulating in an alien organism; but several other causes for the
clumping were readily demonstrated. Most important, and most
frequent, were isoagglutinins, often of high titer, developing in
the recipient and effective against the cells of one or more donors
(possibility 3, a). Such antibodies commonly appeared, and were
readily demonstrable in serum separated from the clot at room
temperature. Their association with the most marked clumping
noted makes it highly probable that the sudden anemia supervening
on plethora which occurs only in association with such marked clump-
ing is due to isoantibodies.
Agglutinins interactive between the donor bloods (possibility 2,
b) sometimes developed, but they were never strong. Occasionally
agglut~n~ns for the recipients' cells appeared ill the donors' plasma dur-
ing the period of transfusion (possibility 2, a). These were always
so weak as to be negligible in vivo.
There remained a series of instances in which, with all such anti-
bodies ruled out, there were yet hemagglutinins in the recipient that
7 Rous, P., and Turner, J. R., J. Exp. Med., 1915, xxiii, 219.
OSWALD H. ROBERTSON AND PEYTON ROUS
led to a marked clumping not only of the mixture of cells present after
transfusion but of the recipient's cells taken prior to transfusion and
kept in vitro.
Such antibodies, like true autoagglutinins, s were read-
fly bound to, and completely freed from, the cells by cooling and wann-
ing respectively. Tests on their origin were now begun; and to rule
out wholly the possibility that undetected antibodies had been in-
jected with the donors' bloods, the transfusions were carried out with
Transfusions with Washed Cells.
Five donor-recipient groups were chosen in the usual way and the donors bled
in rotation. 10 cc. of whole blood was taken each time into about 20 cc. of a
sterile isotonic solution of sodium citrate (3.8 per cent) in water and allowed to
stand in the ice box for 2 days, during which period practically complete sedimen-
tation occurred. The supernatant fluid was now pipetted off and the cells were
suspended in about 20 cc. of Ringer's solution, to which had been added 0.125
per cent of gelatin and 0.2 per cent of sodium citrate. The gelatin prevented
mechanical injury of the cells during the manipulations, and the citrate did away
with the slight tendency to clotting. After a further 2 days of sedimentation in
the cold the corpuscles were again suspended, but this time in gelatin-Ringer's
solution lacking citrate, and immediately thrown down with the centrifuge; made
to a total bulk of 10 cc. with an ordinary Ringer's mixture containing only 0.76
per cent NaC1; and injected. During all the handling no hemolysis occurred
except occasionally to a negligible degree in the case of a donor with unusually
Prior to the first transfusion, blood specimens from the prospective recipients
were taken for preservation as already described.
In three of five rabbits repeatedly injected with the washed cells
a well defined clumping in the shed blood was soon noted.
two better marked cases the serum of the recipient, separated from
the cells at 37°C., caused at room temperature outspoken agglutina-
tion of the preserved ceils of the same individual.
which caused the red ceils to come together remained fixed upon them
in the cold, and the cell masses could be repeatedly washed in salt
solution without its liberation. But when the fluid was wanned to
body heat the agglutinins passed into it and the cell mass separated
into its components. By such means the antibody proved readily
obtainable in salt solution, as is the case with autoagglutinins. 8
s Landsteiner, K., M iind,, reed. Woch., 1903, 1, 1812.