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1949 4: 1196-1213 ROBERT S. EVANS and ROSE T. DUANE SIGNIFICANCE OF THROMBOCYTOPENIA AND LEUKOPENIA ANTIBODY PRODUCTION TO ACTIVITY OF THE DISEASE. II. THE ACQUIRED HEMOLYTIC ANEMIA : I. THE RELATION OF ERYTHROCYTE http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information 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 For personal use only. by guest on May 10, 2013. bloodjournal.hematologylibrary.org From

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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

    http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

    http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

    http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlInformation 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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  • ACQUIRED HEMOLYTIC ANEMIA

    I. THE RELATION OF ERYTHROCYTE ANTIBODY PRODUCTION TO ACTIVITY OF THEDISEASE. II. THE SIGNIFICANCE OF THROMBOCYTOPENIA AND LEUKOPENIA

    By ROBERT S. EVANS, M.D., and ROSE T. DUANE, A.B.

    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.

    1196

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  • ROBERT S. EVANS AND ROSE T. DUANE 1197

    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.

    METHODS

    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

    agglutination.9

    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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  • I 198 ACQUIRED HEMOLYTIC ANEMIA

    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 adsorbed rabbit serum may be determined

    by precipitin titers. However, the authors found that the most practical method of assaying the rabbit

    serum svas to determine the highest dilution at which cells sensitized in a uniform manner with Rh0

    blocking antibody were agglutinated. This was done by exposing Rh1 cells of one individual to a

    high concentration of Rh0 blocking antibodies for one-half hour at 37 C. The Rh0 blocking serum used

    showed an agglutinin titer of i-aaa in beef albumin and was used in a dilution of ,-a to sensitize

    the Rh, cells. The sensitized cells were washed three times in normal saline and added to the saline

    dilutions of the rabbit serum and incubated for one-half hour at 37 degrees before brief centrifugation

    and microscopic observation of the end point of agglutination. The two sera used have shown a con-

    sistent ability to agglutinate Rh, cells so sensitized in dilutions of I-3aa plus or minus one dilution.

    Red cells from patients with acquired hemolytic anemia tested for sensitization were collected from

    venous or capillary blood and diluted directly in normal saline. The cells were washed three times with

    normal saline and a final 2. per cent suspension was approximated. A drop of the suspension was then

    added to a drop of rabbit serum and left for one-half hour at room temperature and subjected to brief

    centrifugation before observation of agglutination. Cells from patients svith active acquired hemolytic

    anemia showed a nearly complete agglutination in a to io dilution of the anti-globulin rabbit serum,

    indicating that most if not all the cells were sensitized. Suitable control suspensions showed no agglutina-

    tion. It was noted that cells from patients with acquired hemolytic anemia were agglutinated by varying

    dilutions of the rabbit serum, so observations were made to determine if the amount of antibody on the

    cell surface had an inverse relationship to the concentration of rabbit serum required to produce agglu-

    tination. That such an inverse relationship exists is shown by the analogy with cells sensitized by varying

    concentrations of Rh0 blocking antibody. Rh, cells were incubated one hour at 37 C. in serial dilutions

    of an anti-Rho blocking serum, washed, and exposed to dilutions of antiglobulin serum and the end

    point of agglutination observed microscopically. The results are shown in table

    It is evident from the above that cells exposed to decreasing amounts of sensitizing antibody, below

    a concentration which saturates (1-la to I-32.a), require increasingly high concentrations of antiglo-

    bulin serum to produce agglutination. From this it may be inferred that avidity of patients cells to

    agglutinate in the dilutions of the rabbit sera is proportional to the amount of antibody on the cell

    surface.

    In the observations reported here we have used two preparations of antihuman globulin serum.

    Both show comparable activity for agglutination of Rh0 cells that have been sensitized by blocking

    antibody. However, we have observed certain variations in the ability of these sera to agglutinate cells

    from patients with active disease. The cells of some patients show a consistently greater susceptibility

    to agglutination in one serum than the other. These observations indicate the importance of using several

    rabbit sera simultaneously to detect sensitization of erythrocytes in acquired hemolytic anemia.

    DIIsRIPTI0N OF PATIENTS

    Eleven patients with acquired hemolytic anemia are included in this series.

    One of the patients has been described in detail in a previous report.8 The diagnosis

    of hemolytic anemia in each patient was based on the presence of a chronic anemia,

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  • ROBERT S. EVANS AND ROSE T. DUANE 1199

    reticulocytosis, an increase in serum bilirubin and, in most patients, the demon-

    stration of an increased fecal urobilinogen. All patients showed erythroid hyper-

    plasia of the bone marrow. The patients in the series were grouped together under

    the heading of acquired hemolytic anemia because of an absence of a family history

    of anemia or jaundice and a lack of any personal history suggestive of hemolytic

    anemia prior to the onset of the present illness in adult life. In most instances,

    patients did not exhibit well-marked spherocytosis and increased osmotic fragility.

    TABLE i-Relation of Amount of Antibody on the Cell Surface to Agglutinab:lity of Sens:t:eed Cells in

    Dilutions of Anti-globulin Serum

    Dilutions of anti-Rh serumDilutions of anti-

    globulin serum -1-20 1-320 1-640 1-1200 1-2400 1-501)0 1-10,00))

    _5 ++++ ++++ +++ +++ ++ - ++ +I-Ia ++++ ++++ +++ +++ ++ + ai-ia ++++ ++++ +++ ++ ++ a a

    1-40 ++++ ++++, ++ ++ + a ai-8o - +++ +++ ++ + a a a

    ,-i6a - ++ ++ + a a a a1-32.0 + + a a a a a1-640 a a a a a a a

    TABLE i-Basic Data of ii Patients with Acquired Hemolytic Anemia

    Pt., Sex, Age Known factor associated with onsetHen1at-

    Hemo- RBC . Fecal0bi7 Million Itejus linogen

    100 cc. cmm g/day

    7.7 ii 8 30 2,080

    6.o i.6 9 509.5 1.46 iS 2.0 450+

    4.1 1.1 2.0 15

    9.8 1.75 13 40 I 950

    9.5 1.5 2.2. 10 530

    7.5 1.5 12. 10 1360

    10.2. 3.3 13 2.0 ,8ao

    i.6 9 Ia 42,5-i-

    9.2. 1.9 8. i i5oo

    7.8 1.3 2.0 30

    C. S., F, 2.2- 0 2.1

    C. A., F, i4 Pneumonitis i8.W. G., M, i Hepatitis, ? Sulfonamides i6

    J.F.,M,34 0

    H. S., F, - Gold therapy for arthritis 2.7

    F. R., F, 42. Pregnancy, thrombopenic pur- 2.8pura

    0. \V., F, 47 0 2.3A. D., M, 50 Injury 34

    D. B., F, a

    B.T., F,64 a i8

    E. S., M, 78 Chronic lymphatic leukemia i6

    In further distinction to congenital hemolytic jaundice, the rapid destruction of

    normal transfused cells was evident when measured in seven of the patients.

    Other varieties of hemolytic anemia were excluded by appropriate tests.

    In of the ii patients there was nothing to suggest a precipitating cause for

    the onset of hemolysis. In 6 of the patients there were a variety of conditions as-

    sociated with onset of the disease which have been recorded in table 2., along with

    a summary of the basic data during the first few days of observation. The disease

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  • 12.00 ACQUIRED HEMOLYTIC ANEMIA

    varied in severity from the mild to the very acute form, and in all patients the

    course was prolonged over a period of weeks, months and even years, so there was

    ample opportunity to make serial observations to confirm the initial data recorded

    in the table.

    RESULTS

    I . Activity of the Hemolytic Process in Relation to the Amount of Antibody on the Cell

    Surface as Measured by Agglutinabilit,y of the Erythrocytes in the Anti-,lobulin Serum

    For this purpose the patients may be divided into three groups as follows:

    I . Four patients were studied with the Coombs reagent in both the active phase

    and during a remission. In 2. of the cases (Nos. i and 7) the remission followed a

    splenectomy, and in the other 2. (Nos. 3 and 8) the remission occurred spon-

    taneously after weeks of observation of the active state.

    2.. Two patients with persistently active disease did not have splenectomy.

    One of these (No. i) died without benefit of splenectomy after several weeks ofobservation. The second patient is an elderly man (No. ii) who has hemolytic

    anemia in association with chronic lymphatic leukemia. Results of x-ray therapy

    will be discussed below.

    3 . Five patients have been studied with the antiglobulin serum technic follow-

    ing splenectomy. Two of these patients (Nos. 4 and ) have active disease withanemia and rapid blood destruction, although there was evidence of some im-

    provement following splenectomy. Three patients (Nos. 6, and io) were studied

    at periods of one year to eighteen months after continued remissions induced by

    splenectomy. In all examinations the erythrocytes showed agglutination in the

    antiglobulin serum.

    In general, good correlation was found between activity of the disease and the

    amount of antibody on the surface of the red cell as measured by the technic de-

    scribed above. As shown in table 3, erythrocytes of patients with active disease

    were agglutinated by dilutions of antiglobulin serum which ranged from i-8o

    tO 1-12.80. On the other hand, the erythrocytes of patients in whom the disease

    process had subsided so that the rate of hemolysis approached normal were ag-

    glutinated in ranges of i to 2. dilution up to i-8o.

    There seemed to be some variation between individual patients as to the amount

    of antibody on the surface of the red cells in the active phase of the disease as

    compared to the amount present during a complete or partial remission. While

    most patients with active disease showed agglutination of cells by i-i6o to 1-310

    dilution of the anti-globulin serum during the active phase of the disease, one

    patient (E. S., No. ii) had erythrocytes which were frequently agglutinated by

    dilutions of 1-12.80 or I-2.5oo. On the other hand, the red cells of A. D., No. 8,

    who had a somewhat milder degree of anemia, were never agglutinated by dilu-

    tions greater than i to 8o. However, when this patient entered a remission, which

    for a time appeared nearly complete, agglutination of erythrocytes was not present

    in dilutions greater than i-s.

    An exception to the generalization concerning agglutinability of the cells and

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  • Patient State of Disease

    i. C. S. Active

    Quiescent post-splenectomy

    C. A. Active

    21

    4

    . W. G. Active

    Spontaneous remission

    8.a 30 io8o I--p.o

    1.2. 5 12.5 12.0

    2.8

    4. J. F.

    2.1.0 iao 1-640

    Active disease after splenectomy - 17 70.0 40

    i6 iS.o io 450* i-i6o

    36 ti.a io i-to

    . 0. W.

    8. A. D.

    Active 2-3

    Quiescent 3 months after splenectomy 33

    i-i6o

    2.0.0 3a 700 1-32.0

    1.0 10 1-40

    12.0 10 1360 1-32.0

    i.6 S iia i-i

    . D. B. Quiescent iS months post-splenectomy 42. 0.5 1-5

    ,o. B. T. Quiescent IL months post-splenectomy 4?. 4.0 306

    ii. E. S. Active 2.8 2.2. 30 1-640

    ROBERT S. EVANS AND ROSE T. DUANE 12.01

    activity of the disease was observed in a patient (H. S., No. ) in whom thedisease seemed to be persistently active but whose erythrocytes on several oc-

    casions showed diminished susceptibility to agglutination as shown in table 4.

    There seemed to be no relationship between the severity of the anemia and the

    reticulocytosis to the amount of immune body on the surface of the red cell during

    the periods of observation. There are several possible explanations of this observa-

    TABLE 3.-Typical Hematologic Data in Relationship to Agglutinability of the Erythrocytes in Dilutions

    of the Anti-globulin Serum

    . H. S. Active disease after splenectomy

    6. F. R. Quiescent after splenectomy 4a

    Greatest- (-. dilution of

    Hemat- - Reticu- Icterus Fecal Urobi- antiglobulinocrit bytes Index linogen serum show-

    ing agelutination

    Active 34 13 2-0 iSoo i-So

    Spontaneous remission ,% 10 -

    tion. The method is rough at best, and perhaps variations in technic of prepara-

    tion of the cells accounted for loss of some immune body in those observations

    when the cells were not agglutinated by higher dilutions of the rabbit serum.

    Also, there may be fairly rapid variations in the amount of antibody present

    which are not reflected in the degree of anemia or reticulocytosis.

    A second exception to the generalization that activity of the disease is related

    closely to the amount of antibody on the cell was observed in W. G., who entered

    a phase of spontaneous remission after six weeks of active disease with a well-

    marked drop in agglutinahility of the erythrocytes and then showed a return of

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  • 12.02. ACQUIRED HEMOLYTIC ANEMIA

    the susceptibility of his cells to agglutination in higher dilutions (i-i6o) of the

    rabbit serum without immediate return of active anemia as shown in table .

    Of particular interest are the two patients (Nos. i and 7) who were studied

    before and after splenectomy, since they provide data as to the mechanism of the

    response to splenectomy. In both patients there was a decrease in the aggluti-

    nability of the red cells in antiglobulin serum and a cessation of abnormal hemolysis

    during the week following splenectomy as shown in table 6.

    TABLE 4.-Serial Observations on a Patient with Active Hemolytic Anemia whose Cells Occasionally Seemed

    to Show Diminished Amount of Antibody without Varying the Activity of the Disease

    Greatest dilution of

    Date Hematocrit % Reticubocytes antigbobulin serum showingagglutination

    9/2-2-/47 2.4 2.2. 1-32-a

    9/2-9/47 2.7 2.0 1_ia

    10/13/47 2.5 2-a ,-,6o

    10/2-1/47 2.4 39 1-32-a

    11/13/47 2.3 2-4 1-40

    1/13/48 2.5 2. 1-40

    1/2-9/48 2.3 1-2-0

    3/ 8/48 2-4 2.2. 1-32.0

    3/2-2-/45 2-2- 2.1 1-32-0

    4/ 6/48 2.4 2.1 i-i6o

    TABLE 5.-Spontaneous Remission Associated with or Follouing Diminished Agglutinability of the Red Cells.

    So far there has been no evidence of recurrence after the cells again became susceptible to agglutination in

    dilutions of i-i6o

    Hematocrit Reticubocytes Antiglobulin serum dilution

    717 17 9.5 -- 1-32-0

    7-2-S 3, 7.7 i-i6o

    S-i 31 11.5 i-i6o

    8-13 32- 11.5 I-Ia

    S-ia 36 3 I-

    9-Ia 36 ii i-So

    9-2-7 45 3.0 i-i6o

    10-30 49 0.9 i-i6a

    One of the patients (C. S.) showed a fall in the hematocrit and a rise in icterus

    index to i six weeks after splenectomy, accompanied by evidence of an increase

    in the amount of erythrocyte antibody on the cells, but no further evidence of

    relapse occurred. She was well and free of signs and symptoms of hemolytic disease

    six months later. The second patient (0. W.) had a relapse and died in a distant

    part of the state about three months after our last observations showed the process

    to be quiescent.

    Serial observations have also been made of one patient (E. S.) during the course

    of x-ray therapy of chronic lymphatic leukemia. The essential data are shown in

    table 7.

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  • ROBERT S. EVANS AND ROSE T. DUANE 12.03

    TABLE 6.-Data Showing the Effect of Splenectomy in Two Patients. There was rapid bematologic improve-

    ment and a drop in the concentration of antibody on the cell surface following splenectomy

    Patient Hematocrit.

    1 e icu-ocy es

    rusIfl CX

    Fecal Urobi-linogen

    Mg/day

    Greatestdilution of

    the anti-globulin

    serum pro-ducing ag-glutination

    i. C. S., F, 2.2.Before splenectomy 19

    wk. after splenectomy - 43

    6 wk. after splenectomy 35

    6 mo. after splenectomy 41

    a. O.W.,F,47

    Before splenectomy 2.3

    i wk. after splenectomy 36.

    3 mo. after splenectomy 33

    (.,

    7.0

    I .0

    ,.6

    i. i

    12..0

    i .

    i .6

    aa

    5

    15

    5

    10

    8

    8

    aa8a

    2.10

    12.5

    1360

    2.2.0

    i-p.o

    I-I

    i-,6o

    I-la

    1-32.0

    i-ia

    I-I

    TABLE 7.-Serial Observations in Patient with Active Acquired Hemolytic Anemia and Chronic lymphatic

    Leukemia in Relation to X-ray Therapy. There appeared to be some lessening of the hemolytic process, improve-

    ment in the anemia and possible reduction in the amount of antibody on the cell following the second course of

    x-ray therapy, which depressed the lymphocyte count in the peripheral blood

    Hematocrit ReticulocytesLeukocytesper cu.mm.

    Highest dilution

    X-ray of antiglobulinserum producing

    agglutination

    %

    1-2.2. 2.8 2.2. 90,000 1-640

    1-2.9 2.8 15 i6,ooo 8o r total body radiation i-i6o

    2.-17. 2.8 15 2.4,000 1-32.0

    a-ao a6 17 a4,000 1-32.0

    3-15 17 17 2.1,000 1-640

    4-12. 2.3 ,8 2.8,000 I z-n.8o

    5-3 2.0 2.9 37,500 1-2.500

    5-13 2.3 19 40,400 6oor 1-640

    5-17 i6 i6 36,000 Total to i-i6o

    5-2.0 i6 41,000 Spleen 1-310

    5-a4 13 8 11,500 5-13 to 1-640

    5-2.7 14 41.8 17,640 6-i in i-ia8o

    6-i 2.1 2.1 5,2.00 6 doses I-3ao

    6-4 17

    6-7 i6.I

    7-2. 2.7

    8-ao 32.

    98 30

    9-15 30.5

    19

    32.

    7.6

    8.i

    10.0

    5,400

    7,000

    5,000

    1,400

    8,oo

    7,500

    1-2.500

    1-12.80

    i-i6o

    i-8o

    1-32.0

    i-i6o

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  • AI-

    20 40 80

    12.04 ACQUIRED HEMOLYTIC ANEMIA

    It is of interest that there was an improvement in the anemia following a reduc-

    tion in the lymphocyte count in the response to x-ray therapy. At the same time

    the amount of antibody on the cells appeared to reach lowest concentration of any

    time during his course. These variations may be chance variations in the disease

    and bear no relationship to x-ray therapy. However, exposure to x-ray has been

    shown to diminish antibody response in animals, and we have previously observed

    improvement in a patient with acquired hemolytic anemia#{176}given x-ray therapy.

    With a method of assay available, further data may be obtained on the effect of

    x-ray on the amount of antibody produced in acquired hemolytic anemia.

    -J

    >

    >a:

    (I)I-

    zLUU

    lxLUa:

    60DAYS

    FIG. i-Survival time of transfused cells in patients with acquired hemolytic anemia and in patients

    with other types of hemolytic disease. Patients S., E. S., J., B., T., G., all showed sensitization of theerythrocytes. Patients C., P. and A. had congenital hemolytic jaundice, Mediterranean anemia and

    paroxysmal nocturnal hemoglobinuria.

    tOO 120

    II. The Longevity of Transfused Cells

    Observations of the survival of normal transfused cells was made by the technic

    of differential agglutination in 6 of the ii patients. The results of these observa-

    tions are shown in figure i. In all 6 cases the destruction of the transfused cells was

    several times the rate of destruction of transfused cells in normal individuals and

    in patients with other types of hemolytic disease. Observations in figure i include

    congenital hemolytic jaundice, Mediterranean anemia, and paroxysmal nocturnal

    hemoglobinuria.

    In three additional patients in this series (Nos. 2., 4, ), repeated transfusionswere necessary, and it soon became apparent from simple calculation that the

    transfused red cells were being destroyed rapidly because of the transitory effect

    on the severity of the anemia. In each case enough red cells were given during

    the space of a few days to replace entirely the patients cells and to produce a

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  • ICC-

    60 I

    60

    ROBERT S. EVANS AND ROSE T. DUANE 12.05

    normal or greater than normal circulating red cell volume. Since the anemia

    quickly developed again in the absence of blood loss, it must be assumed that the

    transfused, as well as the patients own red cells were rapidly destroyed. In one

    of these patients (No. ), exhibiting very marked spherocytosis, it was shownproviously9 that transfused cells showed a tendency toward spherocytosis and an

    increase in hypotonic fragility within forty-eight hours after injection.

    In the remaining 2. patients, no data were obtained as to the longevity of trans-

    fused cells.

    I

    U

    Uw0.

    21

    00 90 70 60PER CENT IS0T0NICIT

    Fio. 2--Curves of hypotonic fragility in ii patients with acquired hemolytic anemia. The central

    dotted line is the average of 30 control determinations, while the lateral lines represent the extremes.

    H. S., H. J. and J. F. were patients who continued to have active disease after splenectomy. There isevidence that the curve of hypotonic fragility was close to normal prior to splenectomy. C. A. showed

    the most severe disease prior to splenectomy. One, H. S., showed a normal fragility curve, even though

    the disease was very active.

    III. Susceptibility to Hemolysis in Hypotonic Solution

    Representative samples of the quantitative curves of hypotonic fragility are

    shown in figure 2.. An average control curve is shown and the limits of variation of

    some 30 control curves are also represented. It can be seen that most of our patients

    with acquired hemolytic anemia had curves of hypotonic fragility which were

    increased above average but were close to or within the widest limits of normal

    variation by the method employed. In one patient (C. S.) with active hemolytic

    anemia and an output of 2.080 mg. of fecal urobilinogen per day, the curve of

    hypotonic fragility was identical with a normal curve done at the same time.

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  • iio6 ACQUIRED HEMOLYTIC ANEMIA

    On the other hand, 3 patients in this series (H. S., J. F., C. A.,) and one otherpatient (H. J.), reported elsewhere,9 showed curves of hypotonic fragility whichwere considerably increased above the normal range. Three of these 4 continued

    to have chronic hemolytic anemia following splenectomy. The fourth patient

    (C. A.) showing greatly increased hypotonic fragility of the red cells had by

    some criteria the most severe hemolytic anemia in the series and died without

    splenectomy.

    Sweeping conclusions cannot be drawn from these observations, but it is evident

    that the susceptibility of circulating erythrocytes to hemolysis in hypotonic solu-

    tion may be quite normal in the presence of active disease even with sensitive

    methods of measurement. The increase in hypotonic fragility when the disease

    persists following splenectomy suggests that the spleen in situ may remove the

    spherocytic cells from the circulation and keep the curve of hypotonic fragility in

    TABLE 8.-Platelet and Leukocyte Counts before and after Splenectomy in Patient. tiho Shoued Thromboiytopenia

    or Leukopenia along with Ative Hemolytic Anemia

    Platelets LeukocytesPatient ______________________ ________________ _____________________________________________

    Before splenectomy Following splenectomy Before splenectomy Following splenectomy

    C. S. 70,000 2.00,000 1,000 7,000

    .1-F. 6o,aaa 70,000 13,000 iS,aoaF. R. ia,aoa 45,000 9,500 6,700

    0. \V. ii,aaa 675,000 1,700 8,ooo

    190,000

    D. B. 35,000 Normal 5,000 Normal

    ia6,aoo

    the peripheral blood close to normal range unless the disease becomes very active,

    as was the case in the patient (C. A.) who died without splenectomy.

    IV. Thrombocytopenia and Leukopenia

    Five patients in this series had persistently low platelet counts prior to splenec-

    tomy, and 2. of the had a marked degree of leukopenia. These findings are sum-

    marized in table 8, which shows representative platelet and leukocyte counts

    before and after splenectomy.

    Only one of the patients with thrombocytopenia had clinical manifestations of

    purpura. This patient (F. R.) was hospitalized because of purpura, and the hemo-lytic anemia was not suspected at first. The remaining 4 patients did not exhibit

    purpuric manifestations, although the platelet count was below 6o,ooo per cu.

    mm. in several determinations. Following splenectomy, the platelet counts of

    three of the five patients rose to a normal level or above, concomitant with a

    subsidence of the hemolytic process. The patient with thrombocytopenic purpura

    and hemolytic anemia continued to have few platelets in the peripheral blood with

    platelet counts of 10,000 or less for the remaining two months of her pregnancy

    and during the postpartum period. In spite of the failure of her platelets to rise,

    there appeared to be a definite improvement in the purpura after operation, and

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  • ROBERT S. EVANS AND ROSE T. DUANE 12.07

    the bleeding time fell to a normal range. The hemolytic anemia improved slowly,

    beginning about one month following splenectomy, but was still active at the

    time of delivery. She was delivered without incident with one transfusion given

    at the time of delivery. The platelet count one year later was still only 5o,ooo

    per cu. mm., and there was evidence of some sensitization of the erythrocytes,

    though the hematocrit was 40 and the reticulocytes were o. per cent.

    A second patient (J. F.) continued to exhibit a persistent thrombocytopeniafollowing splenectomy. The chronic hemolytic anemia also persisted without

    real remission, although there was evidence of some decrease in severity following

    operation.

    Both patients with leukopenia showed a prompt and consistent elevation of the

    leukocyte count to normal range following splenectomy, along with a response

    of the other blood elements.

    DISCUSSION

    The demonstration of the direct relationship of the amount of adsorbed anti-

    body to the rate of destruction of red cells is another step in our understanding of

    the pathogenesis of acquired hemolytic anemia. Activity of the disease is as-

    sociated with evidence of maximal adsorption of the immune body, whereas

    remission in the hemolytic activity is, in the main, associated with distinctly less

    adsorbed immune substance. So far we have not observed the complete disap-

    pearance of the abnormality, even in patients who have been in remission for a

    year or more, which explains perhaps why relapse of this disease occurs so readily.

    The immediate effect of splenectomy when it is successful in producing a remission

    appears to be brought about by a sharp reduction of the amount of adsorbed

    sensitizing agent on the cell. This suggests that the spleen is the principal site of

    production of the sensitizing agent. Wagley and co-workers#{176} have recently been

    able to demonstrate the persistence of the sensitizing agent in the washed pulp

    of spleens from patients with acquired hemolytic anemia. The failure of splenec-

    tomy to produce a remission in some patients is evidently due to the production

    of sufficient hemolysin in other lymphatic or reticulo-endothelial tissues to keep

    the disease active. Even when the hemolytic process continues there is usually

    evidence that splenectomy has diminished its severity. This observation implies

    that some proportion of the total quantity of hemolysin is always produced in the

    spleen.

    The exact nature of the hemolysin is still not entirely clear. The chief question

    seems to be whether or not it is a true immune body or some entirely different, as

    yet unknown, type of variant of normal plasma protein. Evidence is needed to

    show that the hemolytic agent is a specific immune response to an antigen common

    to erythrocytes. Perhaps a complex of some component of the red cell and a foreign

    substance such as a virus or medication provides the necessary antigenic stimulus.

    This explanation would be more clear-cut if it were shown that hemolytic anemias

    definitely associated with sulfonamide medication exhibited the same evidence of

    sensitization of the erythrocytes. In our series one patient received gold therapy

    prior to and during the onset of her disease, but this cannot be regarded as more

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  • 12.08 ACQUIRED HEMOLYTIC ANEMIA

    than suggestive evidence that erythrocyte antibody production may be stimulated

    by a medication, since hemolytic disease is not generally reported as a complica-

    tion of gold therapy.

    Another group of anemias which require further study with the special im-

    munologic technic are the so-called symptomatic hemolytic anemias. This term

    is used to describe hemolytic disease associated with a large variety of disease

    states including lymphomas, leukemias and cirrhosis. So far we have studied only

    one such patient, but the mechanism of hemolysis seems to be the same as in other

    patients with acquired disease. It is of interest that diseases of lymphocytic tissue,

    which is known to be active in the production of antibodies, are associated with

    hemolytic anemia and that treatment of the underlying disease by x-ray is said to

    be helpful in controlling the hemolytic process. It is probably significant that

    an improvement in the anemia and transient decrease in the amount of adsorbed

    antibody followed x-ray therapy of the lymphatic leukemia in our patient.

    In the study of patients with atypical or acquired hemolytic anemia several

    technics should be employed to determine the presence or absence of adsorbed

    antibody. Our experience, with two separately prepared anti-human serum rabbit

    sera, indicates that specificity may vary and that two or more sera should be used

    to demonstrate the presence of adsorbed antibody. Less specific but equally sensi-

    tive methods should also be used in conjunction with the Coombs test. Washed

    erythrocytes should be suspended in human serum and in 30 per cent beef albumin,

    incubated, subjected to centrifugation and inspected for agglutination before the

    possibility of sensitization is discarded. The same technics should be employed in

    an attempt to demonstrate free antibody in the serum. Normal cells will adsorb

    the free immune body if present in the patients serum and become agglutinable

    in the Coombs reagent or in beef albumin. However, our efforts to demonstrate

    free antibody in the patients serum have been inconstant as opposed to the con-

    sistency with which it has been demonstrated to be adsorbed on circulating cells.

    The exact mechanism of cell destruction brought about by the antibody is not

    entirely clear. We have, as yet, no evidence that hemolysis occurs as a result of

    lysis with the fixation of complement. There is, on the other hand, evidence to

    show that destruction of sensitized cells is relatively slow. Observations with

    antiglobulin serum indicate that the great majority of cells in the peripheral blood

    are sensitized, since nearly all are involved in the agglutination. However, studies

    of pigment excretion and observations of longevity of transfused cells indicate a

    survival time of several days for the average cell. It is evident that sensitization

    does not bring about immediate destruction.

    There is evidence from transfusion experiments to show that sensitization is

    reversible, since cells from patients with acquired hemolytic anemia may have a

    normal survival time when transfused into a normal individual.t It has also been

    demonstrated that A cells sensitized by B agglutinin are not irreversibly damaged

    and exhibit a normal survival time after being used in a transfusion.2 We have ob-

    served that transfused cells which have been in the patients circulation for several

    days prior to splenectomy and demonstrated to be involved in the hemolytic

    process show a normal rate of disappearance when splenectomy has produced a

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  • ROBERT S. EVANS AND ROSE T. DUANE 12.09

    remission. We may conclude that sensitization brings about cell destruction slowly

    over the course of days and that it does not immediately damage the red cell

    irreversibly.

    If the sensitized cells are susceptible to agglutination in vivo we have an cx-

    planation of cell destruction, since it has been shown that the injection of a simple

    agglutinin, such as Concanavalin A, will produce a hemolytic anemia in animals.3

    We have not observed agglutination of red cells from patients with acquired

    hemolytic anemia in vivo, but agglutination does occur under optimum conditions

    in vitro. When the washed cells are suspended in whole human serum and sub-

    jected to light centrifugation, agglutination is usually observed. The reaction is

    qualitative, but we have the impression that the intensity of the agglutination is

    proportional to the degree of sensitization as measured by the anti-human-serum

    serum technic as described above. In several instances, cells from patients in mild

    or inactive phases of the disease failed to agglutinate when centrifuged in whole

    serum, probably because of a lack of sufficient concentration of antibody on the

    cell surface.

    It seems probable that agglutination of sensitized cells in vivo is the most im-

    portant mechanism in cell destruction. Agglutination produces stasis ofcells which

    leads to increased osmotic and mechanical fragility and probably susceptibility to

    phagocytosis. If a critical concentration of immune globulin on the cell surface is

    necessary to produce agglutination in vivo we have an explanation for the cessation

    of hemolysis in the quiescent state since the amount of antibody on the cell appears

    to be considerably reduced. The suggestion that a certain concentration of immune

    body on the cell surface is necessary for cell destruction in vivo may explain the

    absence of hemolytic disease in the newborn even when maternal sensitization has

    occurred and there is maternal-fetal incompatibility. In these instances it is possible

    that the concentration of immune substance on the babys cells may not be great

    enough to produce agglutination and hemolysis.

    The implications of the association of thrombocytopenia and leukopenia with

    acquired hemolytic anemia are clear. It strongly suggests that the leukopenia and

    thrombopenia in these patients is due to the presence of an immune body with a

    broader range of activity than the red cells or to a separate immune substance or

    substances more specific for platelet and white cell tissue. The latter explanation is

    more likely since there is no correlation between severity of the hemolytic process

    and the degree of thrombocytopenia or leukopenia. It is possible that a similar

    mechanism will be found for thrombocytopenic purpura and splenic neutropenia,

    which occur as single disease states unaccompanied by hemolytic anemia. Previous

    observations of the occurrence of leukopenia, thrombocytopenia and hemolytic

    anemia in the same patient were made by Wiseman and Doan4 in their original

    report of primary splenic neutropenia, and by Dameshek and Estren.24 The latter

    authors refer to these cases as hyperspienic hemolytic anemia, and account for

    the leukopenia and thrombocytopenia on the basis of an unusual degree of splenic

    inhibition upon the bone marrow. Such cases often show a remarkable response to

    splenectomy. It is probable that the hemolytic anemia in these patients was of the

    acquired variety, especially since a family history of hemolytic jaundice was lack-

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  • 12.10 ACQUIRED HEMOLYTIC ANEMIA

    ing. A patient with splenic neutropenia described by Rogers and HalP5 showed

    thrombocytopenia and a mild anemia with slight polychromatophilia, normoblasts

    in the peripheral blood and elevation of the indirect reacting serum bilirubin.

    Fisher#{176} recently commented on the presence of leukopenia and thrombocytopenia

    in one of a series of patients with acquired hemolytic anemia.

    The association of thrombocytopenia with acquired hemolytic anemia seems to

    be more common than the occurrence of a panhematopenia. In 1941 we observed

    severe thrombocytopenia with fatal termination in a 3 i year old man who had had

    splenectomy three years previously for hemolytic anemia that was evidently of

    the acquired variety since there was no past or family history of hemolytic dis-

    order. A mild thrombocytopenia was present with the severe anemia prior to

    surgery. There was response of both anemia and thrombopenia following operation,

    and he was well until the development of purpura four years later in which blood

    platelets were close to the zero level. He died of a cerebral hemorrhage and autopsy

    showed no evidence of an accessory spleen. In 1942. one of us studied a patient7

    with acquired hemolytic anemia who exhibited severe thrombocytopenia during

    most of seventeen weeks of hospitalization. Platelet counts became normal for a

    short interval following splenectomy, but the thrombocytopenia recurred along

    with continuing hemolysis.

    The patient in the present series who showed clinical purpura during pregnancy

    seems to represent a transition between acquired hemolytic anemia and classical

    thrombocytopenic purpura in that the bleeding tendency and anemia were both

    important clinical features. To complete the transition from one disease to the other

    we have recently studied a young woman with idiopathic thrombocytopenia who

    showed a relatively mild anemia which may have been caused by the persistent

    vaginal bleeding. However, her red cells gave a positive test with the anti-globulin

    serum in a dilution of 1-40 on several occasions. Evidence of red cell sensitization

    ceased abruptly and completely following splenectomy, although the thrombo-

    cytopenia continued with some improvement over the course of months.

    The various explanations of the cause of primary thrombocytopenic purpura

    resolve into two principal points of view. The first holds that thrombocytopenia

    occurs because of deficient formation of platelets in the bone marrow. Dameshek

    and Miller (i8) found an increased number of megakaryocytes in the marrow

    which were qualitatively abnormal in that they did not seem to be producing

    platelets. They postulated a hormonal influence of the spleen in depressing platelet

    formation. Excessive destruction of platelets, particularly in the spleen, is the

    alternative explanation for deficiency of platelets in the peripheral blood. Principal

    exponents of this view are Doan and his co-workers, who have observed excessive

    phagocytosis of platelets in supravital preparations of splenic tissue.9 Excessive

    phagocytosis is also advanced as the explanation of primary splenic panhemato-

    penia with hemolytic anemia, thrombocytopenia and leukopenia.

    Abnormal phagocytosis of damaged blood elements probably does occur, but

    we doubt if the macrophages of the intact spleen have the capacity to ingest and

    digest the amount of cellular elements necessary to produce a severe panhemato-

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  • ROBERT S. EVANS AND ROSE T. DUANE 12.1 I

    penia in view of the functional capacity of the marrow. The suggestion that an

    accessory spleen consisting of a few grams of tissue is capable of doing the same

    thing seems less plausible.

    If thrombocytopenic purpura is due to the formation of an antibody-like sub-

    stance similar to that found in acquired hemolytic anemia both deficient formation

    and excessive destruction may be important in producing the extreme degrees of

    thrombocytopenia sometimes observed. Sensitized platelets may be susceptible to

    agglutination, and phagocytosis and the presence of an anti-platelet antibody in

    the circulation may damage the cytoplasm of the megakaryocyte so as to inhibit

    the formation of platelets.

    In view of the possibility of a common etiologic mechanism for acquired hemo-

    lytic anemia and thrombocytopenic purpura, it is worth noting that the available

    data as to the effect of splenectomy in the two diseases shows a similarity. In both

    conditions the effect of splenectomy is uncertain. It is usually beneficial to some

    degree, but in only one half to two thirds of the patients is remission complete.2#{176}22

    Relapse after a remission has been observed in both groups of patients.

    SUMMARY AND CONCLUSIONS

    I . Observations of i i patients with acquired hemolytic anemia are reported.

    2.. In contrast to patients with congenital hemolytic jaundice, all patients in

    this group exhibited evidence of sensitization of their erythrocytes by an antibody-

    like agent. In all patients studied there was abnormal destruction of transfused

    cells in vivo.

    . The sensitizing agent was found to be adsorbed on the erythrocytes when it

    could not be demonstrated in the serum. A rough method of assay of the amount

    adsorbed was devised by making serial dilutions of the anti-globulin serum. With

    this technic a fairly consistent correlation was found between the amount of anti-

    body on the cell and activity of the disease.

    4. Splenectomy when successful appears to exert a curative effect by sharply

    reducing the amount of antibody substance on the cell. Patients who had not

    responded to splenectomy in the past showed evidence of saturation of their cells

    with adsorbed antibody. The erythrocytes of patients who had responded to

    splenectomy and were in remission when studied showed distinctly less antibody

    on the cell by the same technique.

    . Two patients were observed to enter spontaneous remission after a long period

    of activity. The onset of remission in both was associated with a decrease in the

    amount of adsorbed immune body. However, one patient has shown evidence of

    return of antibody production without immediate recurrence of the hemolytic

    anemia. This inconsistency is not explained.

    6. The tendency toward spherocytosis as measured by increased osmotic fragility

    may or may not be present in acquired hemolytic anemia. Prior to splenectomy the

    most marked increase in hypotonic fragility was observed in the patient with the

    most active disease. Continued activity of the disease following splenectomy was

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  • 12.12. ACQUIRED HEMOLYTIC ANEMIA

    productive of the most extreme increases in spherocytosis. This suggests that the

    spherocytic cells are removed from the circulation by the spleen - -

    7. Agglutination of red cells when the amount of adsorbed antibody reaches a

    critical level, together with such other phenomenon as stasis, spherocytosis,

    increased mechanical fragility and possibly phagocytosis probably explain the

    increased cell destruction.

    8. The occurrence of definite and sustained leukopenia with neutropenia and

    thrombocytopenia in several patients with hemolytic disease due to an immune

    body agent raises questions as to the etiology of classic thrombocytopenic purpura

    and of splenic neutropenia. Patients have been observed who seem to represent

    transition forms between acquired hemolytic anemia and thrombocytopenic pur-

    pura. Abnormal immune mechanisms could account for both excessive destruction

    of platelets and deficient formation.

    REFERENCES

    I DAdE, J. V., AND M0LLIs0N, P. L. : Survival of normal erythrocytes after transfusion to patientswith familial haemolytic anemia. Lancet i. 1943.

    2 EMERSON, C. P. , JR. , SHEN, S. C., HAM, T. H. , AND CASTLE, W. B. : The mechanism of blood destruc-

    tion in congenital hemolytic jaundice. J. Clin. Investigation 26: uSa, 1947.

    3 COOMBS, R. R. A. , MOURANT, A. E. , AND REECE, R. R. : A new test for the detection of weak and

    incomplete Rh agglutinins. Brit. J. Exper. Path. 26.- 2-55-2.66, 1945.

    4 BOORMAN, K. E. , DODD, B. E. , AND Lou-ri-c, J. F. : Haemolytic icterus (acholuric jaundice); Con-

    genital and acquired. Lancet I: 512-, 1946.

    5 EVANS, R. S. , DUANE, R. T. , AND BEHRENDT, F. : Demonstration of antibodies in acquired hemolytic

    anemia with anti-human globulin rabbit serum. Proc. Soc. Exper. Biol. & Med. 64: 372., 1947.

    6 NEBER, J., AND DAMESHEK, W. : The improved demonstration of circulating antibodies in hemolyticanemia by the use of a bovine albumin medium. Blood 2. 371, 1947.

    7 STURGEON, P. :A new antibody in serum of patients with acquired hemolytic anemia. Science io6:2-93, 1947.

    8 EVANS, R. S.: Chronic hemolytic anemia. Observations of the effect of fat content of the diet and

    multiple red cell transfusions. Arch. mt. Med. 77? 544, 1946.DUANE, R. T.: Observations on the effect of irradiation in chronic acquired hemolytic anemia

    exhibiting hemolytic activity for transfused erythrocytes. Blood 2? 72--54, 1947.

    is WAGLEY, P. F., SHEN, S. C., GARDNER, F. H., AND CASTLE, W. B.: Studies on the destruction of redblood cells. VI. The spleen as a source of a substance causing agglutination of the red blood cells of

    certain patients with acquired hemolytic jaundice by an anti-human serum rabbit serum (Coombs

    serum). J. Lab. & Clin. Med. 33. Ii97, 1948.

    STATS, D., ROSENTHAL, N., AND WASSERMAN, L. R.: Hemolytic anemia associated with malignantdiseases. Am. J. Clin. Path. 57. August, 1947.

    12 LOUTIT, J. F., AND MOLLISON, P. L.: Haemolytic icterus (acholuric jaundice), congenital and ac-quired. J. Path. & Bact. j8: 711, 1946.

    i3 HAM, T. H., AND CASTLE, W. B.: Relation of increased hypotonic fragility and of erythrostasis to the

    mechanism of hemolysis in certain anemias. Tr. A. Am. Physicians 55? 12-7, 1940.

    14 \VI5EMAN, B. K., AND DOAN, C. A.: Primary splenic neutropenia; A newly recognized syndrome

    closely related to congenital hemolytic icterus and essential thrombocytopenic purpura. Ann.

    mt. Med. i6: 1097, 1942-. ROGERS, M. H., AND HALL, B. E.: Primary splenic neutropenia. Arch. Int. Med. 75: 191, 1945.16 FISHER, J. A.: The cryptogenic acquired hemolytic anemias. Quart. J. Med. i6. 2-45, 1947.

    EVANS, R. S.: Acute hemolytic anemia with autoagglutination: A case report. Stanford M. Bull. I..

    August. 1943.

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  • ROBERT S. EVANS AND ROSE T. DUANE 12.13

    DAMESHEK, W., AND MILLER, E. B.: The megakaryocytes in idiopathic thrombocytopenic purpura,

    a form of hyperspienism. Blood i. 2.7, 1946.19 DOAN, C. A., AND WRIGHT, C. S.: Primary congenital and secondary acquired splenic panhematopenia.

    Blood I. 10, 1946.20 WINTROBE, M. M., HANRAHAN, E. M., JR., AND THOMAS, C. B.: Purpura hacmorrhagica, with special

    reference to course and treatment. J.A.M.A. 109. 1170, 1937. WISEMAN, B. K., DOAN, C. A., AND WILSON, S. J.: Present status of thrombocytopenic purpura, with

    special reference to diagnosis and treatment. J.A.M.A. ix;. 8, 1940.22 VAUGHAN, J. M.: Treatment of thrombocytopenic purpura. Brit. M. J. 2? 841-845, 1937.

    23 RATHER, L. J.: Arch. Int. Med. In Press.24 DAMESHEK, WILLIAM, AND ESTREN, S.: Spleen and Hypersplenism. New York, Grunc & Stratton, 1948.

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