of 19 /19
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

Acquired Hemolytic Anemia

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Page 1: Acquired Hemolytic Anemia

1949 4: 1196-1213   


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:

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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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Page 2: Acquired Hemolytic Anemia





I T IS NOW evident that the syndrome of acquired hemolytic anemia represents a

distinct 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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Page 3: Acquired Hemolytic Anemia


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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Page 4: 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 for

0 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 patient’s cells to

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


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.


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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Page 5: Acquired Hemolytic Anemia


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:e�ed 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 ++++ ++++ +++ +++ ++ + a

i-ia ++++ ++++ +++ ++ ++ a a

1-40 ++++ ++++, ++ ++ + a a

i-8o -� +++ +++ ++ + a a a

,-i6a - ++ ++ + a a a a

1-32.0 + + a a a a a

1-640 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 . Fecal�0bi7 Million � I�tejus 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 2.3

A. 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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Page 6: 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.


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 of

observation. 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 with

anemia 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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Page 7: Acquired Hemolytic Anemia

Patient State of Disease

i. C. S. Active

Quiescent post-splenectomy

C. A. Active



�. W. G. Active

Spontaneous remission

8.a 30 io8o I--p.o

1.2. 5 12.5 12.0


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


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


activity of the disease was observed in a patient (H. S., No. �) in whom the

disease 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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Page 8: 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 Follou’ing 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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Page 9: Acquired Hemolytic Anemia


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


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



I .0


i. i


i .

i .6




















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



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


















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Page 10: Acquired Hemolytic Anemia


20 40 80


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.








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 the

erythrocytes. 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


In three additional patients in this series (Nos. 2., 4, �), repeated transfusions

were 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 patient’s cells and to produce a

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Page 11: Acquired Hemolytic Anemia


60 I



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 patient’s own red cells were rapidly destroyed. In one

of these patients (No. �), exhibiting very marked spherocytosis, it was shown

proviously9 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.





00 90 70 60


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 is

evidence 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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Page 12: 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 which

were 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


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 Shou’ed Thromboiytopenia

or Leukopenia along with A�tive Hemolytic Anemia

Platelets � Leukocytes

Patient ______________________ ________________ _____________________________________________

� 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,aoa

F. 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


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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Page 13: Acquired Hemolytic Anemia


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 thrombocytopenia

following splenectomy. The chronic hemolytic anemia also persisted without

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


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.


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


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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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 patient’s serum and become agglutinable

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

free antibody in the patient’s 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 patient’s 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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Page 15: Acquired Hemolytic Anemia


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


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 baby’s 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 Doan’4 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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Page 16: 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 patient’7

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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Page 17: Acquired Hemolytic Anemia


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.


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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Page 18: 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.


I DAdE, J. V., AND M0LLIs0N, P. L. : Survival of normal erythrocytes after transfusion to patients

with 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 hemolytic

anemia 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 red

blood 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 malignant

diseases. 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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‘� 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.

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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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