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Date Posted: 18:09:04 07/03/02 Wed
Author: jalada
Subject: Re: Anti-Globulin Test
In reply to: Rita 's message, "Anti-Globulin Test" on 20:15:07 05/14/02 Tue

>Anti-Globulin Test
>Sir,i am miss jalada patel from Gujrat, india. i want some information about anti human globulin reagent- like it's preparaion technique, anti C3d or C3c concentraion in it, titre of anti-C3d, quality assurance techniques for Monospecific AHG and polyspecific AHG with anti C3d.
i will be thankful to u if u will give me some idea about this,
yours faithfully
jalada
> 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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