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 ROUS
The fact that repeated subcutaneous or intraperltoneal injections
with a compatible blood will cause the development of antibodies
effective against such blood has been generally accepted since Ehrlich's
study of induced isolysins in goats; but it has failed of recognition
in its important practical bearing on the outcome of repeated trans-
fusions in man. Our experience with rabbits repeatedly injected
from compatible donors, especially such animals as showed a sudden
and great blood destruction, serves to illustrate in little what must
not infrequently occur in human beings. It follows that the most
careful blood tests are called for with patients repeatedly retrans-
fused at short intervals. In special, a thick spread of the patient's
blood should be examined for autoagglutinatlon which is often, as
we have shown, an evidence that newly developed isoagglut~n~n.~
are in circulation. Throughout the tests the temperature factor
should be carefully controlled. Serum separated from the clot at
37°C. will often yield antibodies not demonstrable in that taken in
the cold, and conversely agglutination mixtures examined at room
temperature are far more likely to yield positive findings than those
kept at blood heat.
Whether massive transfusions are preferable in pernicious anemia
to repeated small ones remains uncertain. Sudden large increments
of blood tend to lessen the reparative activity of the bone marrow. 4
But, on the other hand, they may also act to prevent fulminant de-
struction of the introduced blood by the recipient's serum antibodies.
In the work already mentioned on experimental hemosiderosis in
rabbits ~ some very large transfusions were given to animals that had
developed strong serum antibodies, in the hope that abundant and
continued blood destruction would ensue.
• The amount of antigen introduced so greatly exceeded that of the
circulating antibodies that these latter failed to be destructive and
plethora was maintained. It is a curious fact that in animals trans-
fused 6 days out of every 7 during a period of 6 months the agglu-
tinins gradually and completely disappeared, although following the
first few injections they were often strong. A similar disappearance
of precipitins upon long immunization has been recorded by Tschisto-
witsch and Nuttall. n Yet the transfused blood is somehow destroyed
with great rapidity in animals frequently injected.
This was never the case.
12 Nuttall, G.H.F., Bloodimmunityand blood relationship, Cambridge, 1904,127.
ANTIBODIES DEVELOPING A~TER TRANSFUSION
The massive agglutination observable in the shed blood of trans-
fused rabbits, and associated not infrequently with sudden marked
blood destruction, has a practical significance in connection with the
untoward results of repeated transfusion from donors originally
compatible; and it has special theoretical interest because the clumping
of the cells is apparently all autoagglutination. To determine the
actual source of the antibodies has been the object of the present work.
The agglutination ill its most marked form has been traced to
isoantibodies elicited by the presencein the body of corpuscles originally
found compatible; and the frequently associated, rapid blood destruc-
tion is doubtless of similar origin. Occasionally antibodies develop
in the donor bloods during the period of transfusion, but they are so
weak as to be negligible. There remain instances of what would
seem to be true autoagglutination due to serum bodies induced by the
transfusions as a by-product, so to speak, in the manufacture of iso-
agglutinlns. The antigenic relationship between the red cells of
different rabbits is so close that normal isoagglutinins became fixed ill
the cold upon their elaborator's own corpuscles.
Agglutinins exist within the red cells of rabbits--as has been claimed
by Klein. They are readily demonstrable ill watery extracts of the
dried corpuscles. Whether similar agglutinins ever exist within
human cells remains to be determined. We have not found them
in the normal corpuscles.