SPECIFICITY OF AUTO-ANTIBODIES IN HEMOLYTIC ANEMIA
I S R A E L D A V I D S O H N , M . D . , AND ABE OYAMADA, M . D .
Departments
of Pathology, the Chicago Medical School and the Mount Sinai Medical
Foundation and Hospital, Chicago,
Illinois
Research
The facts that an individual may produce antibodies against his own red blood
cells and that such hemolyzing and agglutinating antibodies may be responsible
for hemolytic anemia have been known since the beginning of the century.
Donath and Landsteiner described in 1904 hemolysins for the patient's own red
cells in paroxysmal cold hemoglobinuria.9 Soon afterward, Widal, Abrami and
Brul6,16' " Chauffard and Troisier,2 and Chauffard and Vincent3 incriminated
so-called auto-antibodies for some hemolytic anemias. A review of the literature
during 30 years following the significant observations of the French investigators
reveals only occasional reports on the role of auto-antibodies in hemolytic
anemia. The difficulty in demonstrating these antibodies seems to have been
the reason.
The subject remained dormant until Dameshek and Schwartz 7,8 revived it
by their ingenious experimental studies. They produced hemolytic anemia in
guinea pigs with a rabbit immune serum containing hemolytic antibodies against
guinea-pig red cells. From these results and from clinical observations, Dameshek and Schwartz postulated the responsibility of an antigen-antibody reaction
for certain forms of hemolytic anemia in man.
The next important event was the discovery in the middle forties by workers
in the Rh field of so-called incomplete antibodies capable of reacting demonstrably only in a favorable environment, provided by a suitable diluent and temperature of incubation. Clumping of red cells becomes manifest in the presence
of large molecular diluents (albumin, serum, certain synthetic compounds such
as dextran and polyvinylpyrrolidone), but not when one employs physiologic
solution of sodium chloride, the time-honored reagent for dilution of antibody
containing serum and for suspension of red blood cells.
Most significant for the study of auto-antibodies in hemolytic anemia was the
introduction by Coombs4 of the antiglobulin test, the principle of which was
discovered by Friedberger and Moreschi. 10 ' 13
Another step forward was the demonstration of increased sensitiveness of test
red blood cells treated with proteolytic enzymes (trypsin, papain, etc.).14 The
various methods for detection of auto-antibodies are listed in Table 1.
Received for publication September 15, 1952.
This investigation was supported in p a r t by a research grant (A-77) from the N a t i o n a l
I n s t i t u t e of Arthritis and Metabolic Diseases, of t h e National I n s t i t u t e s of H e a l t h , U. S.
Public Health Service, and bj' a research grant from the Hematology Research Foundation,
Chicago, Illinois.
Presented in p a r t a t t h e T h i r t i e t h Annual Meeting of t h e American Society of Clinical
Pathologists, Chicago, Illinois, October 17, 1951.
Dr. Davidsohn is Professor of Pathology and Chairman of D e p a r t m e n t a t the Chicago
Medical School; Dr. Oyamada is a R u t h Berger Reader Fellow of the Hematology Research
Foundation, Chicago.
101
102
DAVIDSOHN AND OYAMADA
Application of the new technics to the study of serum of patients with acquired hemolytic anemia has shown that the antibodies are of the incomplete
type and that they are present in a large proportion of cases of the disease.
These developments explain the failure of so many workers to demonstrate autoantibodies as long as they used only saline as diluent in their tests.
The purpose of this paper is to show that a further increase in detection of
warm auto-antibodies in the serum can be achieved by using the patient's own
red cells in the tests for auto-antibodies, and that certain characteristic reaction
patterns can be observed depending on the type of cells employed.
It would seem unnecessary to emphasize the use of an individual's own cells
in view of the fact that the term "auto-antibodies" implies the use of the tested
person's own red cells. However, a review of papers on the subject, which mention
TABLE 1
METHODS FOR DEMONSTRATING
A. A U T O - A N T I B O D I E S ATTACHED TO P A T I E N T ' S RED CELLS
1. Direct antiglobulin test
a. Qualitative
b. Quantitative
2. Technic employing enzyme-treated red
cells
3. 1 + 2.
Direct antiglobulin test, using enzymetreated red cells
4. Elution of antibody from red blood cells
AUTO-ANTIBODIES
B. F R E E AUTO-ANTIBODIES I N P A T I E N T ' S SERUM. A G GLUTINATION OF P A T I E N T ' S OWN RED CELLS AND F R E QUENTLY ALSO RED CELLS OF OTHER P E R S O N S ,
INCLUDING T H O S E OF GROUP O
1. Indirect antiglobulin test
a. Qualitative
b. Quantitative
2. Enzyme-treated red cells
3. 1 + 2.
Indirect antiglobulin test, using enzymetreated red cells
4. Suitable medium (human serum, bovine
albumin, etc.) used as diluent for patient's serum and for suspension of
red cells
5. Patient's red cells, washed and unwashed,
may clump in a suitable medium (human serum, bovine albumin, etc.)
specifically the type of red cells used in tests for auto-antibodies in the patient's
serum, showed relatively many that stated that only normal cells of group 0
were employed. Few authors are specific on this point. Dacie 6 in his recent monograph recommends the use of "normal group 0 corpuscles." The impression
seems to prevail that it matters little what type of red cells is used. This attitude
was expressed clearly by Wiener in a recent paper:18 "Auto-antibodies have the
ability to react not only with an individual's own red cells but also with the red
cells of all other human subjects. Therefore, auto-antibodies are specific for antigens present in every human blood, in contrast to antigens such as A, B, M, N
and Rh, which are type-specific." On the other hand, Dameshek 6 found "that
often the best way to demonstrate an antibody is to test the patient's serum
against his own red blood cells." Table 2 in Dameshek's paper.illustrates convincingly the advantage of using the patient's own cells.
AUTO-ANTIBODIES IN HEMOLYTIC ANEMIA
103
Moolten and Clark12 found that a patient's own red cells were more readily
clumped in viral agglutination tests than were normal 0 cells exposed to the
patient's serum.
It may well be that the reason why the original investigators of auto-antibodies in hemolytic anemia succeeded where the later ones failed was that Widal,
Abrami and Brule16' " state specifically that they mixed 10 drops of the patient's
serum with 1 drop of his own red cells. Thus they created conditions favorable
for demonstrating incomplete antibodies and in addition used particularly
sensitive test cells.
We have investigated the antiglobulin (Coombs) test and warm and cold
agglutinins in the serum of 88 patients with various forms of hemolytic anemia.
Our observations, extending over several years, are in accord with Dameshek's
report 6 of greater agglutinability of the patient's own red blood cells, as compared with red cells of other persons.
MATERIALS
Test tubes, 75 by 10 mm.
Glass droppers were prepared to deliver 20 drops per ml. Droppers of identical
size were used in each experiment.
Patient's serum. Blood was permitted to clot at room temperature. The clotted
blood specimen was kept overnight in the refrigerator at from 2 to 5 C. The serum
was separated and stored in the refrigerator. Some specimens of serum were
stored frozen at — 20 C.
lied cells. Four varieties of red cells were used: the patient's own cells, cells of
the same group and Rh type as the patient's, and cells of group O of the same
Rh type as the patient's; in addition, in the case of Rh-negative patients, O
Rh-positive cells were also used, and in the case of Rh-positive patients, O Rhnegative cells were used frequently. The cells were obtained from clotted blood,
occasionally from citrated blood. Red cells of different types were as a rule of the
same age. Occasionally the patient's own red cells were of necessity somewhat
older, especially when so-called comparative titrations with stored serums were
done. In view of the fact that aging decreases the agglutinability of red cells,
the higher agglutinability of the patient's own red cells could not be attributed
to the occasional older age of these cells. The cells were washed 3 times with
large volumes of saline, and 2 per cent suspensions were prepared from packed
cells.
METHODS
Reading of agglutination. The test tubes were shaken gently and clumping was
noted. In tubes without grossly visible clumping, presence of agglutination was
determined by placing the test tube horizontally on the stage and viewing it
with the low-power objective (25- or 35-mm. scanning lens) of the microscope.
In case of doubt the suspension, after shaking, was poured on a slide and read
with the low-power (16-mm.) objective. The reciprocal of the highest serum dilution giving microscopically detectable agglutination was recorded as the titer.
104
DAVIDSOHN AND OYAMADA
1. Direct Antiglobulin Test
a. Qualitative test. Red blood cells were obtained from the clot and washed
3 times with large volumes of saline. T o 1 drop of 2 per cent suspension of red
cells in a 75 by 10 mm. test tube 2 drops of a standardized antihuman-globulin
rabbit immune serum were added. The result was read with the naked eye after
the mixture was incubated 30 minutes in a water bath at 37 C. In the absence
of agglutination, the tube was centrifuged for 2 minutes at 1000 r.p.m. The term
"positive Coombs test" when used alone means the qualitative direct antiglobulin test.
6. Quantitative test. The red cell suspension was prepared as under a. Progressive dilutions of the antihuman globulin serum were prepared and to 2 drops of
each dilution a drop of the 2 per cent suspension of red cells was added. The
incubation and the recording of results were done as outlined.
2. Indirect Antiglobulin Test
a. Qualitative test. Two drops of the patient's serum were mixed with 1 drop
of a 2 per cent saline suspension of washed and packed red cells, of the 4 varieties
listed under "Red Cells."
Incubation at 37 C. for 30 minutes. The results were read with the naked eye
and microscopically as described previously. If negative, the cells were washed
4 times with saline. After the fourth centrifugation the supernatant saline was
discarded as completely as possible, and 1 drop of antiglobulin serum was added.
After incubation for 30 minutes at 37 C , results were read as described
previously.
b. Quantitative test. Two drops of progressive dilutions of the patient's serum
were placed in a row of 75 by 10 mm. test tubes. Otherwise the technic was the
same as outlined in 2a.
8. Test for Warm Agglutinins
a. Saline diluent. To 2 drops of progressive dilutions of the serum with saline,
1 drop of a 2 per cent saline suspension of red cells of the varieties mentioned
was added. For each variety of red cells a separate row of tubes was set up.
After incubation in a water bath at 37 C. for 2 hours, results were recorded as
described previously.
Tests giving entirely negative results and higher dilutions of tests beyond
those in which positive results were obtained, were retested using the indirect
antiglobulin technic as indicated under 2.
b. Bovine albumin diluent. The procedure was identical with a except that 20
per cent bovine albumin was used as diluent for the serum and for the red cell
suspension.
J).. Test with Enzyme-Treated Red Cells
Reagents. Papain, 1 per cent stock solution, was prepared by suspending 1 Gm.
of papain in 100 ml. of 0.85 per cent solution of sodium chloride. This keeps well
in the refrigerator.
AUTO-ANTIBODIES IN HEMOLYTIC ANEMIA
105
Papain, 0.1 per cent test solution, was prepared by diluting 1 volume of stock
solution with 9 volumes of buffered saline.
Buffered saline was prepared by diluting 1 part of one-fifteenth molar Sorenson phosphate buffer (pH 7.3) with 9 parts of saline. This keeps well in the
refrigerator.
Procedure. Cells to be used are washed 3 times with large amounts of saline.
To 1 volume of washed red cells are added 2 volumes of 0.1 per cent papain
solution. The mixture is incubated 30 minutes in the water bath at 37 C , shaking
frequently. After centrifugation, the supernatant is decanted and the red cell
sediment is washed with large volumes of saline.
A 2 per cent suspension in saline is used.
TABLE 2
A U T O - A N T I B O D I E S IN H E M O L Y T I C A N E M I A
NO. OF
PATIENTS
Acquired
Idiopathic
Symptomatic
Hereditary spherocytosis
Congenital nonspherocytic
anemia
Hemolytic transfusion reactions
Total
33
28
14
POSITIVE ANTIGLOBULIN TEST
WARM AUTOAGOLUTININS PRESENT
COLD AGGLUTININS
PRESENT*
No.
%
No.
%
No.
%
32
14
3
97
50
21
20
7
1
61
25
7
11
6
0
33
21
0
4
Of
0
0
0
0
0
9
7
78
3
33
2
22
i
88
* Cold agglutinins are listed as present if the titer was 1:20 or higher,
f 0 indicates no agglutinins.
Serum or serum dilutions prepared as described under 3a are incubated with
the papain-treated red cells. After incubation in the water bath at 37 C. for 30
minutes, the mixture is centrifuged and the results are read.
5. Comparative Tests
Hemagglutination tests done with the same serum but with different red cells
frequently show considerable variations in titers. This source of error can be
reduced by testing serums obtained on several occasions with the same lot of
red cells. The serums remaining after tests were kept in the refrigerator and then
retested along with a fresh serum". The results of our tests have been regularly
checked and rechecked in this fashion.
RESULTS
Table 2 records the incidence of positive antiglobulin tests, of warm autoagglutinins and of cold agglutinins, in 88 patients with various forms of hemolytic anemia. The positive warm auto-agglutinins include tests done with bovine
106
DAVIDSOHN AND
OYAMADA
albumin and with enzyme-treated cells. Tests for auto-agglutinins with saline
diluent were uniformly negative. Cold agglutinins were recorded as present
if obtained in a titer of 1:20 or higher. Free auto-antibodies were present in
only 1 patient diagnosed as having hereditary spherocytosis. This patient presented special features and will be discussed later. The scarcity of warm autoagglutinins in the serum of patients with hereditary spherocytosis is of interest.
In the early reports on the antiglobulin test in hemolytic anemia hope was held
out that the test may permit differentiation of the acquired form of the disease
from the hereditary, 1 ' u but this has not been verified by subsequent reports.
Several papers with positive antiglobulin tests in hereditary spherocytosis have
been published; 16 ' 19 hence the possible significance of negative results in tests
for warm auto-antibodies. The incidence of cold agglutinins in titers regarded
as being above normal was not strikingly different in the 2 forms of acquired
hemolytic anemia.
Serologic Reaction Patterns
In the course of our studies on auto-agglutinins we became aware as early as
1947 that several patterns of reactions can be observed if, in addition to the
patient's own red cells, other cells are used in the agglutination tests. Under
such circumstances we have observed consistent reaction patterns.
The studies included tests with 4 varieties of red cells, each with 2 diluents,
saline and 20 per cent bovine albumin, the third with papain-treated red cells,
and finally, each set-up at 37 C. and at from 2 to 5 C. Frequently these tests
were repeated several times, testing stored specimens of different dates with
the same batch of red cells, in order to reduce to a minimum the influence of
technical factors, especially those caused by variations in agglutinability of red
cells. Relatively large amounts of serum were needed for such studies. The blood
of 26 patients, 18 with idiopathic acquired anemia, and 8 with symptomatic
anemia, were studied in this manner. In Table 3 the essential serologic, hematologic and clinical data have been summarized.
The first 11 cases show no significant differences in the reaction with the various red cells, in tests employing bovine-albumin diluent. This type of reaction,
in which the titer for the patient's own cells and the other cells tested is similar,
has been designated as reaction pattern + + .
The serum of the next 4 patients (Nos. 12-15) agglutinated their own red
cells in a considerably higher titer than the other cells. Twice the difference was
fourfold, once eightfold and once a thousandfold. This pattern has been labeled
as 4 + + . No differences were noted in this and in the preceding group between
the titer for red cells of the same group and type as the patient's, and cells of
group 0 .
The last 11 cases show a still further pronounced difference between the reactivity of the patient's own cells and cells of other persons. The latter did not
react at all, whereas the former showed titers of varying height. The symbol
H— is used to express the difference in the reactivity.
107
AUTO-ANTIBODIES IN HEMOLYTIC ANEMIA
TABLE 3
SBBOLOGrc REACTION P A T T E R N S IN AUTO-AGGLUTININS IN ACQUIRED H E M O L Y T I C A N E M I A
WARM AGGLUTININS PRESENT FOR
SEROLOGIC REACTION PATTERN; PATIENT NO., SEX,
AGE; GROUP AND TYPE
PATIENT'S OWN CELLS
OTHER CELLS I N CLUDING THOSE OF
SAME GROUP AND
R h TYPE AS PATIENT'S AND O CELLS
ANTIGLOBULIN
TEST. DIRECT
COLD AGGLUTININS FOR
O CELLS OF
SAME Rh
TYPE
(Diluent:
saline)
(Diluent: bovine albumin 20%)
I. Pattern + +
A. Idiopathic
1. F , 25. A, R h 2 (cDE)
2. F , 58. A R h 0 ( D ) positivc
3. F , 57. 0 rh (cde)
(a) 9/1/51
1:2
1:2
1:4
1:4
+*
+
1:20
1:20
1:20
(1:20 papain)§
1:20
(— papain)
+
(1:1 papain)
1:2
1:32
(1:128 papain)
(1:1 papain)
1:2
1:32
(l:64 papain)
1:12S{
1:128
1:10
(1:10,240 papain)
1:10
(1:80 papain)
(1:2048 papain)
(1:12S papain)
-t
(b) 6/21/52
4. F , 53. 0 R h , (CDe)
5. F , 66. 0
Rhaihs
(CDE)
6. F , 28. 0 R h , (CDe)
(a) 4/9/52
(b) 5/18/52
B . Symptomatic
7. M, 79. A R h 0 ( D ) positive
S. M, 59. B R h , (CDe)
9. M , 65. A 2 R h , (CDe)
10. F , 58. A R h , R h 2
(CDE)
C. Transfusion reaction
11. F , 72. 0 R h 2 (cDE)
I I . P a t t e r n 4.4- +
A. Idiopathic
12. M , 19. B R h , (CDe)
13. M , 1. A, rh (cde)
... ' (a) 8/21/51
(b) 9/10/51
(c) 7/23/52
13. Symptomatic
14. M, 38. 0 rh (cde)
15. F , 34. AB R h , (CDe)
1:32
—
1:8192
—
1:512
1:1
1:1
+
-
1:1
1:4
(1:128 papain)
1:2
(1:2 papain)
+
'+
—
—
1:2
1:4
+
-
1:16
1:2
+
—
1:2
1:2
(1:32 papain)
1:2
(1:8 papain)
1:512
+
—
1:512
—
1:1
1:2
+
+
1:1
1:8
(1:256 papain)
1:4
(1:2 papain)
1:8
1:2048
(1:2048 papain)
1:16
(1:128 papain)
1:4
1:8
—
1:512
108
DAVIDSOHN AND OYAMADA
T A B L E 3—Continued
WARM AGGLUTININS PRESENT FOR
SEROLOGIC REACTION PATTERN; PATIENT NO., SEX,
AGE; GROUP AND TYPE
PATIENT'S OWN CELLS
OTHER CELLS I N CLUDING THOSE OF
SAME GROUP AND
R h TYPE AS PATIENT'S AND O CELLS
ANTIGLOBULIN
TEST, DIRECT
COLD AGGLUTININS FOR
O CELLS OF
SAME Rh
TYPE
(Diluent:
saline)
(Diluent; bovine albumin 20%)
III. Pattern + A. Idiopathic
16. F , 10. A Rh„ (D)positive
(a) 9/3/47
(b) 1/15/48
17. F , 56. B Rh„ (D)positive
18. M , 8. A,B R h 0 (D)positive
19. F , 23. A RlhRh..,
(CDE)
20. F , 47. B R h 0 ( D ) positive
21. M , \y2. A 2 B R h j l h o
(CDE)
(a) 7/13/50
(b) 11/7/50
22. F , 24. O Rh» (cDE)
(a) 5/19/50
(b) 6/23/50
23. F , 87. A R h . R h ,
(CDE)
24. F , 4 . A R h , R h , ( C D E )
(a) 7/31/51
(b) 5/12/52
25. F , 39. O RhiRh*
(CDE)
B . Symptomatic
26.' M, 46. A R h 0 (cDe)
1:1
1:4
1:32
-
+
+
+
1:32
1:32
-
+
—
1:32
—
+
—
1:1024
-
+
-
1:8
1:32
-
1:640
1:640
-
1:2
1:1
1:4
—
1:80
1:40
1:2048
1:8192
+
—
1:32
(1:8 papain)
1:4
(1:512 papain)
1:1
(1:2 papain)
1:2
1:8
(1:2 papain)
—
-
"
+
1:128
+
—
* Qualitative direct antiglobulin test is read as positive ( + ) or negative (—).
f Negative; in t h e case of cold agglutinins this means a titer of 1:16 or less.
§ Titers in parentheses were obtained with t h e papain-cell technic.
| Q u a n t i t a t i v e direct antiglobulin t e s t is expressed in dilutions of t h e antiglobulin
serum.
CLINICAL D A T A AND F O L L O W - U P
Patient
No.
1. 41-47. Severely ill. H b . 4.0 Gm. Jaundice; elevated urinary urobilinogen. M a n y
transfusions. Splenectomy day after test for auto-agglutinins. Antibodies of similar
low titers persisted for at least 3J^ m o n t h s ; 3 years later clinically well, H b . 14.7
Gm., no antibodies.
2. 72-48. Splenectomy 1 year previously. Severe relapse. H b . 5.2 G m . Platelets 25,000.
P u r p u r a . Moderately improved with transfusions. J a n u a r y 1949; H b . 9.5 Gm., platelets 57,000.
A U T O - A N T I B O D I E S I N HEMOLYTIC
ANEMIA
109
T a b l e 3—Concluded
3. 152-50. (a) Chronic anemia; A C T H and numerous transfusions given. H b . 9.1 G m .
Splenectomy 11/28/51; 9 months later, no improvement.
(b) Tests repeated 13 times. Last positive direct antiglobulin test 9/1/51, negative
10/1/51. Auto-antibodies with bovine albumin persisted until 1/8/52; negative
4/25/52.
4. 27-51. Chronic anemia and jaundice. Active hemolysis. H b . 5.8 Gm. Reticulocytes
24 per cent. Elevated fecal urobilinogen. Splenectomy 5/19/51. Cortisone, temporary improvement. H b . 10.3 Gm. M a n y transfusions.
5. 67-51. In relapse. Moderately active hemolysis. H b . 7.8 Gm. Reticulocytes 12 per
cent. Transfusions; A C T H . Follow-up 6 m o n t h s ; immunohematologic studies repeated 5 times; results similar.
6. 18-52. (a) Severely ill; H b . 3 Gm. Reticulocytes 47 per cent.
(b) Improved with transfusions and A C T H . H b . 8.7 Gm. Relapse after 2 months.
7. 73-48. Acute leukemia. R B C , 0.85 million. Transfused many times. Died.
8. 124-49. Chronic lymphocytic leukemia. R B C , 1.73 million.
9. 63-51. Chronic lymphocytic leukemia with anemia. On A C T H and Cortisone therapy for some time.
10. 4-52. Hodgkin's disease. H b . 7.0 Gm. Transfused m a n y times. Died.
11. 116-49. Aplastic anemia t r e a t e d with transfusions. R B C , 1.5 million.
12. 57-49. Splenectomy 3 months previously. Mild relapse. H b . 12.5 Gm.
13. 50-51. (a, b) Seriously ill. Despite numerous transfusions and 8 days of A C T H , H b .
9.1 Gm., reticulocytes 18 per cent. Splenectomy 7 months later. Improved. H b .
14.2 Gm., then relapse.
(c) Follow-up 7/23/52. H b . 9.5 Gm., 8 antibody studies, all of same p a t t e r n .
14. 153-48. Lymphoblastoma. Two-year history of anemia and irradiation to spleen.
H b . 2.6 Gm. Reticulocytes 12 per cent. Followed for 1 year. Antibodies absent about
9 months later, b u t antiglobulin test remained positive.
15. 6-51 Postnecrotic cirrhosis. H b . 7.1 G m . Died 1 year later.
16. 51-47. Splenectomy 2 years previously; accessory spleen removed 1 year ago. Clinically improved for 1 year. In clinical relapse a t the first examination. H b . 5 Gm.
Erythroblastemia. P u r p u r a .
17. 19-48. Severely ill. H b . 5.1 G m . Reticulocytes 60 per cent. Jaundiced for 10 m o n t h s .
Splenectomy on same day as blood tested.
18. 164-48. Splenectomy 2 years previously. R e t u r n e d in relapse. Transfused many
times. Also received irradiation and P32. Moderately anemic. Follow-up after 5
y e a r s : well.
19. 119-49. Severely ill. H b . 4.3 Gm. Reticulocytes 44 per cent. Splenectomy 7 days
previously; 100 blood transfusions in 5 m o n t h s . Died.
20. 22-50. Severely ill. R B C , 1.5 million. Reticulocytes 45 per cent. Splenectomy 4/21/50;
rapid recovery.
21. 27-50. (a) Severely ill. H b . 5.6 G m . Reticulocytes 25 per cent.
(b) Transfused man}' times; 20 antibody studies, p a t t e r n unchanged. H b . remains
low in spite of numerous transfusions. Died.
22. 92-50. Severe anemia, 0.7 million R B C . After numerous blood transfusions R B C
4.08 million. Urine urobilinogen 4 + , bile present. Splenectomy same d a t e as first
blood stud}'. Improved. Alive August 1952.
23. 65-51. Severely ill. H b . 5.1 Gm. Reticulocytes 9 per cent. A C T H . Died.
24. 66-51. (a) Severely ill. Strong urobilinogenuria. H b . 3.8 Gm. Reticulocytes 50 per
cent. Transfused 3 times. Splenectomy 8/24/51. A C T H and m a n y transfusions.
(b) Improved, H b . normal; 8 antibody studies in 10 months, all of same p a t t e r n .
25. 41-52. Anemia unsuccessfully treated 2 yr. H b . 7.5 Gm. Reticulocytes 2.6 per cent.
Transfused several times. Improved. H b . 9.1 Gm.
26. 168-49. Gastric resection 13 years previously for lymphosarcoma of stomach. Anemia ( l i b . 11.8 Gm.) and jaundice for 3 weeks. Elevated urine urobilinogen. Reticulocytes 16 per cent.
Serologic p a t tern
T i t e r of
Own cells
Other cells
Serologic p a t tern
T i t e r of
Own cells
Other cells
13. 50-51*
13. 50-51J
9/10/51
4+ +
1:2048
1:32
1:32
1:32
++
8/28/51
+-
9/22/51
1:32
0
Clinically improved
+-
1/20/48
1:8
0
Clinically improved
after transfusions
and A C T H
Seven a n t i b o d y studies in one m o n t h :
4 + + , p a t t e r n persisting
1:2048
1:2
Splenectomy 3
m o n t h s before
9/10/51
4+ +
1:80
1:5
1 year after splenect o m y ; relapse, H b .
6.8 G m .
. 7/23/47
4+ +
* Tests done with bovine albumin diluent,
f Tests done with p a p a i n - t r e a t e d red cells.
Serologic p a t tern
T i t e r of
Own cells
Other cells
18. 164-48*
Case No.
Two a n t i b o d y s t u d ies in 11 m o n t h s :
H— p a t t e r n
( N O T R E L A T E D TO A P P A R E N T T E C H N I C A L F A C T O R S )
TABLE 4
C H A N G E S IN SEROLOGIC R E A C T I O N P A T T E R N
1:16
1:2
Clinically
proved
7/23/52
4+ +
im-
1:8
0
Well, M a y 1951
+-
10/4/49
3
s
>
>
o
o
o
W
3
AUTO-ANTIBODIES I N HEMOLYTIC
111
ANEMIA
The pattern was consistent in most cases on repeated testing and through
many months of observation. For example, in Case 13 there was no change
during an observation period of 11 months, and in Case 24 during 10 months.
The behavior was similar in most other patients in whom repeated tests could
be done. In a few isolated instances the pattern did change. They will be discussed later.
Changes in reaction patterns caused by increased sensitiveness of red cells. It is
well known that enzymes (trypsin or papain) increase the agglutinability of
red cells. The titers in parentheses in Table 3 have been obtained with papaintreated red cells. In 5 of 8 cases tested, considerable increase in titer was noted.
In Case, 3 no auto-agglutinins were detectable with 20 per cent bovine albumin
and the antiglobulin test was negative 10 months after splenectomy, but lowtitered auto-agglutinins were still detectable with the papain technic. A similar
phenomenon was observed in Case G, except that the titer with the papain technic was high and the antiglobulin test was positive. The tendency to maintain
TABLE 5
SUMMARY OF SEROLOGIC R E A C T I O N P A T T E R N S OF W A R M A G G L U T I N I N S IN ACQUIRED
HEMOLYTIC A N E M I A
P A T I E N T ' S OWN CELLS
+
4+
CELLS OF SAME GROUP AND R h
TYPE AS PATIENT'S GROUP AND 0 CELLS
+
+
+
Total
NO. OF CASES
PER CEXT OF CASES
11
4
11
42.5
15
42.5
26
the pattern is apparent. A change in pattern was noted in 2 instances: from + +
to 4 + + in Case 6, and from +— to + + in Case 25.
These results indicate that the reaction pattern is influenced by the sensitiveness of the technic employed, but even the most sensitive method available has
modified the reaction pattern in only 2 of 8 cases.
Auto-antibodies in hereditary spherocytosis. Free auto-antibodies were found
in only 1 of 14 patients diagnosed as having hereditary spherocytosis. Three of
them, including the one discussed here, had positive direct antiglobulin tests.
The diagnosis in the patient with free auto-antibodies has been questioned for
several reasons. Splenectomy had little if any beneficial effect, and the osmotic
fragility of red cells was only slightly increased. It is interesting that the free
antibodies reacted only with the patient's own enzyme-treated red cells and only
in undiluted serum. The pattern was H—.
Changes in reaction patterns not related to technic. In addition to changes in the
serologic reaction patterns brought about by increased sensitivity of red cells,
changes have been observed in tests done with the identical technic. Such
changes are shown in Table 4. It may be that one or the other form of therapy
employed (blood transfusion, ACTH, splenectomy) is responsible for the alteration, but no convincing relation could be established.
112
DAVIDSOHN AND OYAMADA
The distribution of the 3 main serologic patterns is summarized in Table 5.
Reaction pattern + -\—. In 3 cases of idiopathic hemolytic anemia, a pattern
different from the 3 previously described was noted. In addition to the patient's
own red cells, cells also of the same blood group and Rh type were agglutinated,
but not cells of group 0 . This pattern was designated as + H — . The first plus
sign refers to the reaction with the patient's own cells, the second to the reaction
with cells of the same group and Rh type as the patient's, but not his own, and
the third sign to cells of group 0 . In 2 of 3 instances this pattern was transient, <
and was preceded and replaced by the + + pattern in 1 patient and by the -\—
n the other; in the third patient, only 1 specimen of blood could be examined.
DISCUSSION
One is tempted to speculate on the possible significance of these various reaction patterns. No relation could be established between the patterns and the
severity of the anemia. On the other hand, a comparison of the titers of the
patient's own red cells in Table 3 shows a tendency toward higher values in
TABLE 6
D I S T R I B U T I O N O F SEROLOGIC R E A C T I O N P A T T E R N S IN ACQUIRED H E M O L Y T I C A N E M I A
SEROLOGIC PATTERNS
,•++
++
No.
Per cent
No.
+-
Per cent
No.
Total
Per cent
No.
Per cent
Idiopathic
Symptomatic
6
5
55
45
2
2
50
50
10
1
91
9
18
8
69
31
Totals
11
100
4
100
11
100
26
100
reaction pattern -\—. In patients with the -f- + pattern, the highest titer was
1:32 and this occurred only once. In the same number of patients with the -\—
pattern, this titer was present 5 times.
The distribution of the 3 patterns in the 2 forms of anemia (Table 6) shows a
higher incidence of the -\— pattern in idiopathic anemia. The small number of
cases in this series precludes generalizations regarding any relationship between
the type of anemia and the serologic reaction pattern.
The following 3 observations are significant: (1) The patient's own red cells
were always agglutinated whenever free auto-agglutinins were present in the
serum; (2) the serum of some patients clumped their own red cells in a considerably higher dilution than the red cells of other persons; (3) the serum of other
patients clumped only their own red cells.
These findings indicate that warm auto-agglutinins manifest varying degrees
of specificity. It seems permissible to speak of specific auto-agglutinins when
the -\— serologic reaction pattern is present, in order to emphasize the specific
reaction exclusively with the patient's own red cells; to speak of specific and
nonspecific auto-agglutinins when the 4 + + reaction pattern is seen, in order
AUTO-ANTIBODIES IN HEMOLYTIC ANEMIA
113
to express the dominance of the reaction with the patient's own cells; of
nonspecific auto-agglutinins in relation to the + + pattern, to express the lack
of selectivity.
According to Dameshek, 6 the stronger reaction with the patient's own cells
may be explained by the previous coating of such cells by antibodies in the
circulation. The antibody in the serum used in the in vitro tests could be conTABLE 7
RELATION OK THE QUANTITATIVE DIRECT ANTIGLOBULIN TEST TO WARM AUTO-AGGLUTININS
AND TO COLD AGGLUTININS
WARM AGGLUTININS FOR
TYPE OF ANEMIA, CASE NO.
DIRECT
ANTIGLOBULIN TEST
PATIENT'S
OWN CELLS
OTHER CELLS INCLUDING
THOSE OF SAME GROUP
AND R h TYPE AS P A T I E N T ' S AND O C E L L S
COLD
AGGLUTININS
(Diluent: saline)
(Diluent bovine albumin)
Idiopathic hemolytic
anemia
1. 126-48
2. 27-50
3. 33-50
4. 92-50
5. 152-50
6. 200-50
7. 27-51
8. 34-51
9. 50-51
10. 53-51
11. 65-51
12. 67-51
13. 18-52
1:2
1:640
1:32
1:80
1:16
1:64
1:128
1:1
1:512
1:64
1:2048
1:128
1:32
0
1:32
0
1:2
1:2
0
1:2
0
1:8
1:4096
1:4
1:32
1:10
0
0
0
0
1:1
0
1:2
0
1:2
1:32
0
1:32
1:10
N o t elevated
N o t elevated
1:32
N o t elevated
N o t elevated
N o t elevated
N o t elevated
N o t elevated
1:32
1:4096
1:S192
1:8192
N o t elevated
Symptomatic
anemia
1. 153-4S
2. 39-50
3. 56-50
4. 82-50
5. 139-50
6. 158-50
7. 63-51
1:1
1:1
1:1
1:40
1:4
1:4
1:4
1:4
0
0
0
0
0
1:8
0
0
0
0
0
0
1:4
Not
Not
Not
Not
Not
Not
Not
hemolytic
elevated
elevated
elevated
elevated
elevated
elevated
elevated
ceived as exerting an additive effect. This concept is difficult to reconcile with
the fact that in our experience the patient's own cells were unable to reduce the
antibody titer in absorption experiments. Why should such cells give a higher
titer with antibodies which they are unable to absorb in vitro? Furthermore,
it is in serums with the pattern -\— that the agglutination titer for the patient's
own cells was particularly high (see Table 3). Finally, it would be difficult to
explain why some serums are capable of clumping cells of group O and others
114
DAVIDSOHN A N D OYAMADA
fail to do it even with the sensitive technic employing papain-treated red cells.
One is forced to conclude that previous sensitization of the patient's own red
cells is not sufficient to explain the differences in the specificity of auto-antibodies.
Relation between Antiglobulin Test and Auto-agglutinins
The quantitative direct antiglobulin test was done in 13 cases of idiopathic
and in 7 of symptomatic anemia (Table 7). Eight of the 13 patients with idiopathic anemia had titers of 1:64 or higher, whereas 6 of the 7 patients with
symptomatic anemia had titers of 1:4 or lower. Hence, if we assume that free
warm auto-agglutinins are an indication of a higher degree of sensitization, the
figures in Table 7 would support such an assumption. They were present in 9
of the 13 cases of the idiopathic and only in 2 of the symptomatic group. Significantly high titers of cold agglutinins were also present in 5 cases of the idiopathic group and in none of the symptomatic.
The prevalent opinion is that there is no direct relationship between the titer
of the direct quantitative antiglobulin test and the severity of the hemolytic
process. On the other hand, presence of auto-agglutinins in the serum may be
interpreted as indicative of an overflow of antibodies remaining after the red
cells have been maximally sensitized. This concept is supported by our experiments in which it was not possible to reduce the titer of auto-agglutinins by
absorbing serums containing such antibodies with the patient's own cells.
The existence of auto-antibodies capable of reacting only with the individual's
own red cells is an example of an exceptionally high degree of specificity. This
interesting phenomenon is certainly deserving of further study. One possible
practical application may be implied: On the face of it, it should be easier to
find suitable blood donors for such patients than for patients with agglutinins
capable of clumping red cells of the same blood group and Rh type as their own
and with cells of group 0 . Our own observations have thus far not been sufficient to permit such conclusions.
SUMMARY
The antiglobulin test was positive in 32 of 33 patients (97 per cent) with
acquired idiopathic hemolytic anemia, in 14 of 28 patients (50 per cent) with symptomatic hemolytic anemia and in 3 of 14 patients (21 per cent) with hereditary
spherocytosis. The incidence of warm auto-agglutinins in these patients was 61,
25 and 1 per cent, respectively. The rarity of warm auto-agglutinins in the
serum of patients with hereditary spherocytosis may be significant.
Tests for warm auto-agglutinins revealed 3 consistent serologic reaction patterns, depending on variations in agglutination of the patient's own cells, other
cells of the same group and Rh type as the patient's, and 0 cells. Changes iii
the reaction pattern were seen rarely, except when the use of enzyme-treated
red cells increased their agglutinability.
The pattern designated as -\—, and characterized by failure of the patient's
serum to clump cells other than his own, was particularly frequent in idiopathic
acquired hemolytic anemia.
AUTO-ANTIBODIES IN HEMOLYTIC ANEMIA
115
A tendency toward higher titers for one's own cells was observed in patients
with the H— pattern.
The quantitative direct antiglobulin test showed significantly higher titers
in idiopathic hemolytic anemia than in symptomatic hemolytic anemia.
CONCLUSION
The study of warm auto-aggiutinins in the serum of patients with acquired
hemolytic anemia may furnish information not supplied by the antiglobulin
test alone.
REFERENCES
1. BOORMAN, K . E . , D O D D , B. E . , AND L O U T I T , J. F . : Hemolytic icterus (acholuric j a u n dice); congenital and acquired. Lancet, 1: 812-814, 1946.
2. CHAUFFARD, M . A., AND T R O I S I E R , J . : Anemie grave avec hemolysine dans le s6rum;ictere h6molysinique. Semaine m6d., 28: 904, 1908.
3. CHAUFFARD, M. A., AND VINCENT, C.: Hfimoglobinurie hemolvsinique avec ictere polvcholique aigu. Semaine med., 29: 601-604, 1909.
4. COOMBS, R. R. A., M O U R A N T , A. E . , AND R A C E , R. R . : A new t e s t for t h e detection of
weak and " i n c o m p l e t e " R h agglutinins. Brit. J . Exper. P a t h . , 26: 255-266, .1945.
5. D A C I E , J. V.: Practical Haematologv. Brooklyn, New Y o r k : Chemical Publishing Co.,
Inc., 1951, 172 pp.
6. D A M E S H E K , W . : Acquired hemolytic anemia. Physiopathology with particular reference
to auto-immunization and therapy. Proc. Third International Congress of the International Society of Hematology, Cambridge, England (1950). New York: Grime &
S t r a t t o n , I n c . , 1951, p p . 120-133.
7. DAMESHEK, W., a n d SCHWARTZ, S. O.: Hemolysins as the cause of clinical and experimental hemolytic anemias. Am. J . M . S c , 196: 769-792, 1938.
S. DAMESHEK, W., AND SCHWARTZ, S. O . : Acute hemolytic anemia (acquired hemolytic
icterus, acute t y p e ) . Medicine, 19: 231-327, 1940.
9. D O N A T H , J . , AND LANDSTEINER, K . : Ueber paroxysmale Haemoglobinurie. Miinchen.
med. Wchnschr., 51: 1590-1593, 1904.
10. F R I E D B E R O E R , E . , AND M O R E S C H I , C : Ueber Haemolyse beschleunigende I m m u n s u b stanzen. Centralbl. f. B a k t . , 45: 346-352, 1907.
11. LOUTIT, J . F . , AND MOLLISON, P . L . : Haemolytic icterus (acholuric jaundice), congenital
and acquired. J . P a t h . & Bact., 58: 711-728, 1946.
12. MOOLTEN, S. E . , AND CLARK, E . : The red blood cell as a vehicle of virus t r a n s p o r t : I .
Isolation and identification of blood-borne viruses. I I . Role of blood-borne viruses in
autohemagglutination and in hemolytic anemia. T r a n s . New York Acad. S c , 14:
232-238 (April) 1952.
13. MORESCHI, C . : Besclileunigung u n d Verstiirkung der Bakterienagglutination durch
Antieiweissera. Centralbl. f. Bakt., 46: Orig., 456-460, 190S.
14. MORTON, J . A., AND P I C K L E S , M. M . : Use of trypsin in t h e detection of incomplete a n t i R h antibodies. N a t u r e (London), 159: 779-780, 1947.
15. SINGER, K., AND MOTULSKY, A. G.: The developing (Coombs) test in spherocytic hemolytic anemias, its significance for t h e pathophysiology of spherocytosis and splenic
hemolysis. J . Lab. & Clin. Med., 34: 768-783, 1949.
16. W I D A L , F . , ABRAMI, P . , AND B R U L E , M . : Les icteres d'origine hemofytique. Arch. mal.
coeur, 1: 193-231, 1908.
17. WIDAL, F . , ABRAMI, P . , and B R U L E , M . : Auto-agglutination des hematies dans l'ictere
heinolytique acquise. Compt. rend. Soc. biol., 64: 655, 190S.
IS. W I E N E R ' , A. S., SAMWICK, A. A., M O R R I S O N , M . , AND L O E W E , L . : Acquired h e m o l y t i c
anemia. Am. J . Clin. Path., 22: 301-312, 1952.
19. W R I G H T , C-S., D O D D , M . C , BOURONCLE, B . A., D O A N , C. A., AND Z O L L I N G E R , R . M . :
Studies of hemagglutinins in hereditary spherocytosis and in acquired hemolytic
anemia; their relationship to the hypersplenic mechanism. J. L a b . & Clin. Med., 37:
165-181, 1951.