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Date Posted: 20:15:07 05/14/02 Tue
Author: Rita
Subject: Anti-Globulin Test

Anti-Globulin Test

When the Michigan healthcare society removed blood tests
standards in the 1970's, I believe that the fact should have been placed on
the calendar. It could be an anniversary date that would be celebrated each
year, instead of many blood tests arguing subjects.

The first special antibody listed on the 1983 Blood Type
Identification Card was +DAT. The American Red Cross furnished me with
Professor Coombs' address. Professor Coombs was the man that the +DAT test
was named after.

Professor Coombs sent the following:

The Discovery of the Anti-Globulin Test
written by A. E. Mourant
pages 180 to 183
Vox Sang. 45: 180-83 (1983)

Vox Sanguinis
Editor-in-Chief: C. P. Engelfriet, Amsterdam
Reprint
Publisher: S. Karger AG, Basel
Printed in Switzerland

Milestones in Blood Transfusion and Immunohaematology

When the Second World War broke out in 1939, the British Government's
medical advisers realized that large numbers of blood transfusions were
likely to be needed for military and civilian casualties, and that for this
purpose great quantities of blood-group testing serum would be necessary.
The only blood group system of clinical importance then known was the ABO
system.

A few years before the war a group of workers, supported by the national
Medical Research Council, and including Dr. G. L. Taylor as Director and Dr.
R. R. Race, had begun work on the genetics of the blood groups. They were
housed in Professor R. A. Fisher's Galton Laboratory at University College,
London. At the outbreak of war they were instructed to move to the
Department of Pathology of Cambridge University, where they were to select
human donors whose serum was suitable for use as anti-A and anti-B grouping
sera, and to process and distribute such sera. They became known as the
Galton Laboratory Serum Unit.

When, in 1940, the rhesus blood groups were discovered by Landsteiner and
Wiener, and shown to be of clinical importance, the unit, and Dr. Race in
particular, began to carry out fundamental work on the new system. This
work [Race and Taylor, 1943; Race et al., 1943, 1944] became the basis of
Fisher's CDE hypothesis and notation of the
immunology and genetics of the system. The first publication of these was
by Race [1944] who, in the same paper, also showed that, in addition to the
supposedly normal form of anti-Rh (henceforth to be known as anti-D)
antibody, which agglutinated D-positive red cells directly, when they were
suspended in a saline medium, there existed a variant, known as 'incomplete'
antibody. This could at first be detected only by the 'blocking test' - the
blocking of Rh-positive cells, which had been suspended in the
incomplete serum, so that they became inagglutinable by the normal antibody.

The existence of this type of antibody, and its blocking properties, were
also discovered independently by Diamond [1944] and Wiener [1944]. Soon
after this, Wiener [1945] described the 'conglutination' test for incomplete
antibodies, the forerunner of the bovine albumin suspension test.

At this time (1944-45) there was, working in the Department of
Pathology, a veterinarian immunologist, R. R. A. Coombs, who became
interested in the possible nature of incomplete antibody. He tried
observing under the microscope the electrophoresis, or migration in an
electric field, of red cells suspended in a saline medium, and coated
respectively with 'complete' and 'incomplete' antibody, with uncoated cells
as a control. It was found that the cells coated with incomplete antibody
migrated at a similar speed to those with complete antibody, and very
differently from the uncoated cells.

It was at this stage, in July 1945, following the death of Dr. Taylor,
and the appointment of Dr. Race as Director, that I joined the Unit in a
junior capacity.

Almost simultaneously with my arrival, R. Coombs, by a brilliant feat of
intuition, had conceived the principle of the anti-globulin test. He states
that he was traveling on an ill-lit wartime train from London to Cambridge,
trying to read some papers by Ehrlich on the side-chain theory, and
speculating idly (like Kekule on the tetravalent carbon
atom) on the behaviour of red cells and antibodies, when he visualized the
cells, already coated with molecules of incomplete antibody, which was of
course a globulin, but still floating free, becoming linked together by
molecules of another antibody, an antiglobulin antibody!

He realized that this idea could be the basis of a practical test, and
he formulated in some detail its possible applications -- essentially as the
direct and the indirect anti-globulin tests. The general principle had
indeed already been proposed and confirmed in practice by Moreschi [1908a,
b] but his work was very little known and his method had not got through
into the general corpus of immunological techniques. We rediscovered his
papers after completing most of our own work, and a note on them was added
in proof to the paper by Coombs et al. [1946]. Moreschi had died some years
previously. Coombs [1954] subsequently discussed them in some detail,
generously acknowledging his full priority.

Coombs theoretical work was followed by many weeks of intensive activity
by Coombs, Race, myself, and our technicians. First we had to secure (or
prepare) an anti-serum against human globulin. We were fortunate to find
that Dr. Muriel Adair, in the neighbouring Physiological
Laboratory, did have a stock of sera from rabbits immunized respectively
against human globulin, against whole human serum, and against human
pseudoglobulin. She gave us a generous supply of these sera, which we
proceeded to absorb with human group AB Rh-positive (R1 R2) red cells
uncoated with blood group antibodies, until such cells ceased to be
agglutinated by the absorbed serum. To the best of my remembrance the
original critical experiments, and the majority of the others, were carried
out with the anti-human-globulin serum.

Rh-positive red cells (probable genotype cDE/cde) were sensitized with
the incomplete form of anti-D, washed three times with physiological saline
and then exposed to suitably absorbed and diluted anti-globulin serum.

Sensitized and washed cells suspended in saline gave no agglutination,
while the same treated cells, when exposed to the various anti-globulin
sera, were agglutinated to a titre of 64 or more. This was confirmed by
tests using numerous different incomplete anti-D sera. Homologous cells
treated with an incomplete anti-c serum were also shown to be agglutinated
by rabbit anti-human globulin serum.

D-positive cells only weakly agglutinated by certain anti-D sera were
found to be strongly agglutinated after washing and exposing to a rabbit
anti-globulin serum. Similar results were obtained using e-positive cells
and the then newly discovered but very weak anti-e serum. The results of
this work were published immediately in a short paper [Coombs et al., 1945a]
and subsequently in greater detail [Coombs et al., 1945b].

A wide range of positive results, and of negative control tests, had now
established the effectiveness and specificity of the anti-globulin test for
detecting incomplete and weakly reacting anti-Rh sera, by sensitizing
homologous cells artificially with such sera. The next step was to use the
test for detecting natural in-vivo sensitization of the cells of infants
suffering from haemolytic disease of the newborn. It was already known that
though there was marked haemolysis of the cells of such infants,
spontaneous agglutination was very rare.

Cord blood specimens were now obtained from numerous infants diagnosed
clinically and by routine Rh testing as suffering from haemolytic disease of
the newborn, and from normal healthy babies. The red cells from each were
washed three times in physiological saline, and then exposed to the
anti-human globulin reagent. All the specimens from healthy infants
remained unagglutinated while nearly all those from affected infants were
agglutinated. Of three babies with somewhat anomalous symptoms, and
negative anti-globulin test results, two were finally diagnosed as suffering
from jaundice of a non-immunological variety, and one case was unsolved
(but the baby survived).

One infant without clinical symptoms but giving a positive result was of
probable Rh genotype CDe/cde, as were both its parents. The mother's
serum yielded a positive indirect anti-globulin result with the father's red
cells. The non-Rh antigen involved in this case was that subsequently known
as Kell. Thus at the very outset the test had detected a previously unknown
blood group system which has since proved to be of some clinical importance.

The work on the direct anti-globulin test on cord bloods was published
by Coombs et al. [1946]. Subsequent work [Coombs and Mourant, 1947],
using various human protein fractions to see whether they inhibited the
anti-globulin test, showed that the protein fraction responsible for
sensitization was gamma-globulin. The 'incomplete' antibody mainly
responsible for positive anti-globulin test results is now known as IgG
while the 'complete' antibody is IgM.

This was the last paper in which I collaborated directly with Coombs,
though I subsequently become both a manufacturer and a user of
anti-human-globulin sera. Work on the anti-globulin reaction and its
various modifications still goes on, and the history of some of its
developments has been described by Coombs [1970, 1981].

Acknowledgement

I am indebted to Prof. R. R. A. Coombs for reading a draft of this paper
and suggesting a number of factual corrections. He is, of course, not
responsible for the ultimate wording.

References

Coombs, R. R. A.: Moreschi e alcuni recenti sviluppi nello studio dell'
aggluntinazione. Informaz. Med. 9: 126 (1954).
Coombs, R. R. A.: History and evolution of the anti-globulin reaction and
its application in clinical and experimental medicine. Am. J. clin.
Path. 53: 131-135 (1970).
Coombs, R. R. A.: Assays utilizing red cells as markers; in Collor,
Immunoassays for the 80's, pp. 17-34 (MTP Press, Lancaster 1981).
Coombs, R. R. A.: Mourant, A. E.: On certain properties of antiserum
prepared against human serum and its various protein fractions: their use
in the detection of sensitization of human red cells with 'incomplete' Rh
antibodies, and on the nature of this antibody. J. Path. Bact. 59: 105-111
(1947).
Coombs, R. R. A.: Mourant, A. E.; Race, R. R.: Detection of weak and
'incomplete' Rh agglutinins: a new test. Lancet ii: 15 (1945a).
Coombs, R. R. A.: Mourant, A. E.; Race, R. R.: A new test for the
detection of weak and 'incomplete' Rh agglutinins. Br. J. exp. Path. 26:
255-266 (1945b).
Coombs, R. R. A.: Mourant, A. E.; Race, R. R.: In vivo isosensitization of
red cells in babies with haemolytic disease. Lancet i: 264 - 266 (1946).
Diamond, L. K.: Progress report to committee on Medical Research of the
Office of Scientific Research and Development. OEM. cmr. 384 (1944).
Landsteiner, K.; Wiener, A. S.: An agglutinable factor in human blood
recognised by immune sera for rhesus blood. Proc. Soc. exp. Biol. Med.
43: 223 (1940).
Moreschi, C.: Neue Tatsachen uber die Blutkorperchen- agglutination.
Aentbl. Bakt. 46: 49 - 51 (1908a).
Moreschi, C.: Beschleunigung and Verstarkung der Bakterienagglutination
durch Eiweisssera. Zentbl. Bakt. 456 (1908b).
Race, R. R.; Taylor, G. L.: A serum that discloses the genotype of some
Rh positive people. Nature, Lond. 152: 300 (1943).
Race, R. R.; Taylor, G. L.; Boorman, K. E.; Dodd, B.E.:
Recognition of Rh genotypes in man. Nature, Lond. 152: 563 (1943).
Race, R. R.; Taylor, G. L.; Cappell, D. F.; McFarlane, M.:
Recognition of a further common Rh genotype in man.
Nature, Lond. 153: 52 - 53 (1944).
Race, R. R.: An 'incomplete' antibody in human serum.
Nature, Lond. 153: 771 (1944).
Wiener, A. S.: A new test (blocking test) for Rh sensiti-
zation. Proc. Soc. exp. Biol. Med. 56: 173-176 (1944).
Wiener, A. S.: Conglutination test for Rh sensitization.
J. Lab. clin. Med. 30: 622 (1945).

Dr. A. E. Mourant,
The Dower House,
Maison de Haut,

End of the article sent by Professor Coombs.

After reading the anti-globulin test article, I looked up the immunoglobins
in the list of blood tests. They were the following:

IMMUNOGLOBINS

Test Lab figures My personal figure

IgG (723.0-1685.0 dl) 1200
IgA (60-333) 195
IgM (45-145 dl) 95
IgD (0.5-3.0 dl) 1.5
IgE (0-380 ml) 150


Then I looked at the 1983 Immunoglobin Assay, which was the following:

The Patient's IGG figure was 5080 MG/DL.
The Patient's IGA figure was 964 MG/DL
The Patient's IGM figure was 812 MG/DL

The three listed patient figures were each flagged as high to signal cell
abnormalities, yet, the hematologist would not prescribe any
antitumor/antiviral antibiotics to treat what was causing the clearly
visible neck tumor.

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