Acquired Hemolytic Anemia

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  • 1949 4: 1196-1213

    ROBERT S. EVANS and ROSE T. DUANE SIGNIFICANCE OF THROMBOCYTOPENIA AND LEUKOPENIAANTIBODY PRODUCTION TO ACTIVITY OF THE DISEASE. II. THE ACQUIRED HEMOLYTIC ANEMIA : I. THE RELATION OF ERYTHROCYTE about reproducing this article in parts or in its entirety may be found online at: about ordering reprints may be found online at: about subscriptions and ASH membership may be found online at:

    Copyright 2011 by The American Society of Hematology; all rights reserved.20036.the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by

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    I T IS NOW evident that the syndrome of acquired hemolytic anemia represents adistinct entity which is separate in pathogenesis and course from the commonly

    described familial hemolytic jaundice. This distinction, which was recognized

    by the writers of the early part of the century, was lost sight of by many more

    recent observers, who suggested that acquired hemolytic anemia was simply a

    sudden outcropping of a latent inborn defect. Since spherocytosis of the red cells

    is always present in congenital hemolytic jaundice and is sometimes observed in

    acquired hemolytic anemia, the confusion was natural, particularly when a sharp

    distinction could not always be made on clinical grounds. Beginning with the red

    cell survival experiments of Dacie and Mollison, it has become increasingly

    evident that acquired hemolytic anemia is caused by a hemolysin active for

    all erythrocytes, while congenital hemolytic jaundice is due to a defect in red cell

    structure. During the last few years it has been possible to demonstrate sensitiza-

    tion of erythrocytes from patients with acquired hemolytic anemia with im-

    munologic technics developed in the field of Rh sensitization.37 We have

    some evidence, then, by analogy, that the hemolytic agent in acquired hemolytic

    anemia is an immune body similar to the univalent or hyperimmune Rh antibody

    and may be a response to antigenic stimulus. The ready demonstration of the

    abnormal immune mechanism in acquired hemolytic anemia elevates this rather

    rare disease from the position of an obscure hematologic phenomenon of uncertain

    etiology to the general field of abnormal immunology. Because of the unique

    properties of erythrocytes, the affected tissue can be isolated and subjected to

    close observations so that variations in the rate of production of the hemolysin

    can be measured in relation to severity of the disease and to any type of thera-

    peutic procedure.

    It is worthwhile at this point to summarize our knowledge of the antibody-

    like agent which appears to he responsible for the destruction of red cells in ac-

    quired hemolytic anemia.

    i. The destructive agent appears to be a fraction of plasma protein, probably

    a globulin, since erythrocytes from persons with acquired hemolytic anemia are

    agglutinated with the anti-human serum rabbit serum of Coombs, Mourant and

    Race,3 as are cells sensitized by Rh hyperimmune antibody. Red cells from normal

    individuals and from patients with other types of disease are not agglutinated by

    this reagent.

    From the Department of Medicine, Stanford University School of Medicine, San Francisco, Calif.

    * The svord hemolysin is used as an all-inclusive turn for agents known to bring about destruction of

    red blood cells.


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    2.. The hemolytic agent is also similar to the univalent or hyperimmune Rh

    antibody in that sensitized cells do not usually agglutinate in saline but require a

    more complex colloidal medium. Whole serum, 30 per cent beef albumin and 2.per cent acacia in i . per cent calcium chloride have been found to provide the

    necessary factors to allow agglutination to take place.

    3 . Since accelerated hemolysis proceeds at a fairly constant rate in acquired

    hemolytic anemia, it appears that the agent does not require special conditions of

    temperature or pH for activity as is the case in some hemolytic syndromes. In

    keeping with this property of constant activity of the hemolysin it has been our

    experience that the immune body could be found on the surface of the cell when it

    could not be demonstrated in the serum.

    4. We have been able to remove the sensitizing agent from the surface of the

    red cell by heating a suspension of the sensitized cells to 6 C. in normal saline.

    The immune body appeared to remain active to some degree in the saline eluate

    since normal cells exposed to it became agglutinable in the Coombs reagent. This

    observation is considered further evidence that the hemolytic agent is an immune

    body. So far this observation that the lytic agent can be transferred to normal

    cells has not been confirmed by others, and we have been successful in further

    attempts in only one of three patients.

    . The agent is active for all erythrocytes since transfused cells appear to be

    destroyed at a rate which approximates the rate of destruction of the native cells.

    Although the etiologic significance of the antibody-like abnormality found in

    acquired hemolytic anemia appears to be established, the study of patients who

    appeared to have recovered completely following splenectomy showed a persistence

    of the abnormality. The erythrocytes from patients in remission were found to be

    agglutinable in the anti-human globulin serum even though all evidence of ac-

    celerated hemolysis had subsided. This observation appeared to throw some doubt

    on the significance of sensitization as a primary cause of the disease. It seemed

    possible that the agglutinability of the erythrocytes in the various media could be

    the result rather than the cause of the disease, and that splenectomy produced a

    remission by removal of the principal site of destruction of abnormal cells. On

    the other hand, it appeared more likely that a quantitative relationship between

    the amount of immune body present and the rate of blood destruction might

    explain the apparent paradox. We attempted, therefore, to devise a method of

    quantitating the amount of antibody on the red cell so that measurements could

    be made during active and quiescent phases of the disease. In this report we are

    presenting evidence of a direct relationship between the amount of antibody on

    the cell and the activity of the hemolytic process.


    The general methods used in the study of hemolytic disease have been outlined previously.8 A modi-

    fication of the Ashby technic was employed in following the longevity of transfused cells by differential


    The anti-human serum rabbit serum* for performance of the Coombs test was produced as follows,

    * This reagent will be referred to as the anti-human globulin serum or antiglobulin serum.

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    Ten rabbits were injected subcutaneously at weekly intervals with a. cc. of fresh human serum for

    six weeks. Following an interval of one month, a second course of injections was given before the ani-

    mals were sacrificed and the sera collected. The red cell agglutinins in the rabbit serum were adsorbed

    by mixing the sera in cc. amounts with equal quantities of a a per cent suspension in normal saline

    of pooled Group A, B and 0 cells that had been washed repeatedly to remove serum protein. The mix-

    ture was then incubated at 37 C. for one hour with frequent agitation before centrifugation and removal

    of the supernatant fluid. Two such absorption procedures sufficed to remove all of the cell agglutinins.

    It was found important to use 0 cells as well as those of Group A and B, since agglutinins specific for0 cells appeared in low titer if 0 cells were not included in the adsorption process.

    Once the rabbit serum was completely adsorbed, it did not agglutinate washed human cells of any

    group or type in any concentration. It was then filtered, placed in small vials and kept in the frozen

    state, where it maintained its original activity.

    The amount of antibody for human serum protein in the