Journal of Clinical Investigation Vol. 42, No. 11, 1963 QUANTITATIVE STUDIES OF THE EFFECT OF RED-BLOOD-CELL SENSITIZATION ON IN VIVO HEMOLYSIS * By M. CONSTANTOULAKIS,t N. COSTEA,4 R. S. SCHWARTZ, AND W. DAMESHEK (From the Blood Research Laboratory, Pratt Clinic-New England Center Hospital, and the Department of Medicine, Tufts University School of Medicine, Boston, Mass.) (Submitted for publication January 22, 1963; accepted July 29, 1963) Although a positive antiglobulin reaction is a characteristic and distinctive feature of autoim- mune hemolytic anemia (AIHA), the precise role of the erythrocyte-sensitizing globulin in the pathogenesis of this disease is poorly understood. Earlier studies have suggested that the severity of hemolysis in AIHA is a function of the quan- tity of globulin adherent to the red blood cells (1); however, direct estimation of the amount of autoantibody was possible in only a few cases. There is, in addition, a lack of quantitative data concerning the effects of isoantibodies on erythro- cyte survival. In the experiments described in this paper, the relationships between red-cell sen- sitization, by either auto- or isoantibodies, and in vivo hemolysis were examined through the tech- niques of Cr51-labeled red-cell survival and the radioactive antiglobulin (RAG) test (2). In this test, rabbit antihuman globulin labeled with radioactive iodine is used. After appropriate in- cubation and washing, the amount of rabbit anti- human globulin reacting with the sensitized red cells may be ascertained from the specific radio- activity of these cells. The amount of human antibody sensitizing the red blood cells is calcu- lated from the combining ratio between the rab- bit antibody and the erythrocyte-bound human gamma globulin. When the human antibody was of the gamma2 globulin variety, this ratio was found to be 7 to 1, i.e., 7 molecules of rabbit anti- globulin reacted with 1 molecule of red-cell-bound antibody. The results of these experiments indi- cated that, irrespective of their serologic attri- * Aided by American Cancer Society grant T-243 and U. S. Public Health Service grant E-3091. t Present address: 2 Ersis Street, Athens 7, Greece. t Present address: West Side VA Hospital, South Damen Avenue, Chicago 12, Ill. butes, both isoantibodies and autoantibodies had qualitatively distinctive effects on red-cell survival. MATERIALS AND METHODS Patients. Seventeen patients with AIHA of the "warm" antibody variety were studied; 11 were "idio- pathic," and the rest had systemic lupus erythematosus (2), chronic lymphocytic leukemia (3), or Hodgkin's disease (1). Characteristics of the individual cases are shown in Table I. Isoantibodies. The anti-D and anti-Kell sera were of the incomplete, noncomplement-binding variety.' Eluates were prepared by Fudenberg, Barry, and Dameshek's modification (1) of the Kidd (3) method and were stored at - 200 C until used. Each of the eluates was tested serologically and physicochemically as follows. a) Serology. Eluates were titrated by the standard indirect antiglobulin technique (4), in which a constant pool of red cells 2 (Hemantigen) and commer- cial antiglobulin serum 3 were used. The tests were car- ried out at 40, 220, and 370 C. In some instances, fresh human serum was added as a source of complement. The serologic specificity of the eluates was determined by the indirect antiglobulin reaction using a panel of red cells of known antigenic compositions (Panocell). The antigens present in this panel were: C, CW, D, E, V, c, e, f, K, k, Fya, Fyb, JOa, Le', Leb, Jka, Jkb, M, N, S s, P. and Lua. Normal red cells (Hemantigen) incubated with the eluates were also tested for sensitization by the standard antiglobulin technique with specific goat anti- human 7S and antihuman 19S sera.5 b) Physicochemical studies. Electrophoresis by standard techniques on pa- per and cellulose-acetate supporting media were car- ried out on a Durrum-type cell. Protein concentrations were determined by the method of Lowry, Rosebrough, Farr, and Randall (5). A standard curve, utilizing a 1 Kindly provided by the Ortho-Research Foundation, Raritan, N. J., and the Blood Grouping Laboratory, Bos- ton, Mass. 2 Hemantigen, Knickerbocker, New York, N. Y. 3 Broad spectrum, Ortho-Research Foundation, Raritan, N. J. 4Panocell, Knickerbocker, New York, N. Y. 5 Hyland Laboratories, Los Angeles, Cal. 1790
Transcript
Journal of Clinical InvestigationVol. 42, No. 11, 1963
QUANTITATIVE STUDIES OF THE EFFECT OF RED-BLOOD-CELLSENSITIZATION ON IN VIVO HEMOLYSIS*
By M. CONSTANTOULAKIS,t N. COSTEA,4 R. S. SCHWARTZ,ANDW. DAMESHEK
(From the Blood Research Laboratory, Pratt Clinic-New England Center Hospital, and theDepartment of Medicine, Tufts University School of Medicine,
Boston, Mass.)
(Submitted for publication January 22, 1963; accepted July 29, 1963)
Although a positive antiglobulin reaction is acharacteristic and distinctive feature of autoim-mune hemolytic anemia (AIHA), the precise roleof the erythrocyte-sensitizing globulin in thepathogenesis of this disease is poorly understood.Earlier studies have suggested that the severityof hemolysis in AIHA is a function of the quan-tity of globulin adherent to the red blood cells(1); however, direct estimation of the amount ofautoantibody was possible in only a few cases.There is, in addition, a lack of quantitative dataconcerning the effects of isoantibodies on erythro-cyte survival. In the experiments described inthis paper, the relationships between red-cell sen-sitization, by either auto- or isoantibodies, and invivo hemolysis were examined through the tech-niques of Cr51-labeled red-cell survival and theradioactive antiglobulin (RAG) test (2). Inthis test, rabbit antihuman globulin labeled withradioactive iodine is used. After appropriate in-cubation and washing, the amount of rabbit anti-human globulin reacting with the sensitized redcells may be ascertained from the specific radio-activity of these cells. The amount of humanantibody sensitizing the red blood cells is calcu-lated from the combining ratio between the rab-bit antibody and the erythrocyte-bound humangammaglobulin. When the human antibody wasof the gamma2 globulin variety, this ratio wasfound to be 7 to 1, i.e., 7 molecules of rabbit anti-globulin reacted with 1 molecule of red-cell-boundantibody. The results of these experiments indi-cated that, irrespective of their serologic attri-
* Aided by American Cancer Society grant T-243 andU. S. Public Health Service grant E-3091.
t Present address: 2 Ersis Street, Athens 7, Greece.t Present address: West Side VA Hospital, South
Damen Avenue, Chicago 12, Ill.
butes, both isoantibodies and autoantibodies hadqualitatively distinctive effects on red-cell survival.
MATERIALS AND METHODS
Patients. Seventeen patients with AIHA of the"warm" antibody variety were studied; 11 were "idio-pathic," and the rest had systemic lupus erythematosus(2), chronic lymphocytic leukemia (3), or Hodgkin'sdisease (1). Characteristics of the individual cases areshown in Table I.
Isoantibodies. The anti-D and anti-Kell sera were ofthe incomplete, noncomplement-binding variety.'
Eluates were prepared by Fudenberg, Barry, andDameshek's modification (1) of the Kidd (3) methodand were stored at - 200 C until used. Each of theeluates was tested serologically and physicochemicallyas follows. a) Serology. Eluates were titrated by thestandard indirect antiglobulin technique (4), in which aconstant pool of red cells 2 (Hemantigen) and commer-cial antiglobulin serum 3 were used. The tests were car-ried out at 40, 220, and 370 C. In some instances, freshhuman serum was added as a source of complement.The serologic specificity of the eluates was determinedby the indirect antiglobulin reaction using a panel of redcells of known antigenic compositions (Panocell). Theantigens present in this panel were: C, CW, D, E, V, c, e,f, K, k, Fya, Fyb, JOa, Le', Leb, Jka, Jkb, M, N, S s, P.and Lua. Normal red cells (Hemantigen) incubatedwith the eluates were also tested for sensitization by thestandard antiglobulin technique with specific goat anti-human 7S and antihuman 19S sera.5 b) Physicochemicalstudies. Electrophoresis by standard techniques on pa-per and cellulose-acetate supporting media were car-ried out on a Durrum-type cell. Protein concentrationswere determined by the method of Lowry, Rosebrough,Farr, and Randall (5). A standard curve, utilizing a
1 Kindly provided by the Ortho-Research Foundation,Raritan, N. J., and the Blood Grouping Laboratory, Bos-ton, Mass.
2 Hemantigen, Knickerbocker, New York, N. Y.3 Broad spectrum, Ortho-Research Foundation, Raritan,
N. J.4Panocell, Knickerbocker, New York, N. Y.5 Hyland Laboratories, Los Angeles, Cal.
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RED-CELL SENSITIZATION AND IN VIVO HEMOLYSIS
TABLE I
Some clinical and laboratory features in 17 patients with AIHA *
¶ A = antimetabolites.** SLE = systemic lupus erythematosus.
solution in which the protein-nitrogen concentrationwas determined by the micro-Kjeldahl technique, wasprepared for each new batch of Lowry reagents. Im-munodiffusion in agar plates was performed with amodification of the Ouchterlony method (6). Specificantihuman 7S and antihuman 19S globulins sera of goatorigin were employed. Since some eluates were slightlycontaminated with free hemoglobin, each gel-diffusiontest was controlled for the possible precipitation ofmethemalbumin in the agar medium by allowing theeluate to react against normal human serum in a sepa-rate agar plate. In no instance was a precipitin lineobserved in these control tests. Eluates were next in-cubated with equal volumes of 0.2 M mercaptoethanol(7, 8) at room temperature for 48 hours in tightlysealed tubes, then dialyzed in the cold against 20 L ofphosphate buffer (pH 7.2) containing 0.02 M iodoacetate.Controls in which saline was substituted for 2-mercap-toethanol were run simultaneously. As an additionalcontrol, a serum containing a high titer of cold ag-glutinin was treated in a similar manner and then testedfor cold agglutinin activity. Indirect antiglobulin testswere carried out, as described above, on the eluatestreated in this manner.
RAG tests (2) were performed on red cells from pa-tients with AIHA immediately before the red-cell sur-vivals were started and on the artificially sensitized nor-mal erythrocytes just before the Cr" labeling procedure.
(0) to severe illness (+ + + +) .
Determiniation of the combbining ratio of RAG withthe red-cell antibodies. The globulin fractions of po-tent anti-D and anti-K sera obtained from individualdonors were dissolved in borate buffer, pH 8. Immuno-electrophoretic analysis of these fractions showed onlyone component, gamma2 globulin. These preparationswere labeled with I"'5 by the method of Helmkamp andhis associates (9). In some experiments, the anti-D serawere treated by absorption on thoroughly washed Rh-positive red cells and subsequent elution by Fudenbergand his co-workers' (1) modification of the Kidd (3)technique. Other experiments showed that this pre-liminary isolation of the antibody did not affect the re-sults, and later studies dealing with the combining ratiowere performed directly with the labeled globulin frac-tions. The radioactive antibody solutions containing 6%bovine or human serum albumin, added to minimize utakeof nonspecific proteins (2, 10), were incubated with 0.1-ml portions of packed red cells. After 15 minutes ofincubation at 370 C, the cells were washed in chilled 0.15MA saline until the radioactivity of the red-cell buttonremained constant. All washing procedures were car-ried out in a refrigerated centrifuge. The amount ofantibody protein attached to the red-cell surface wasthen determined from the SA of the antibody solution.After this, the cells were incubated with RAG labeledwith I131 for 30 minutes at 37° C. The cells were nextwashed in cold saline until less than 5% of the radio-
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CONSTANTOULAKIS,COSTEA, SCHWARTZ,AND DAMESHEK
activity was recovered in the supernatant fluid of twosuccessive washings. After each washing, the amountsof 112' and I" were determined in a single-channel spec-
trometer. Interference o-f the J131 radiation spectrumwith that of Ib", which was less than 10%, was correctedby appropriate calculations. Radioactivity was con-
verted to micrograms of protein nitrogen according topreviously described methods (2).
Survival of artificially sensitized, normal erythrocytes.Three to five ml of blood from normal donors was takenin acid-citrate dextrose,6 washed twice, and incubatedwith a solution of the desired isoantibody for 15 minutesat 370 C. After the cells were washed twice, a smallsample was removed for RAGdetermination. The re-
maining cells were labeled with Cr`1 (11). The sensi-tized, labeled erythrocytes were injected into the donor,and samples were taken at different time intervals, de-pending upon the rapidity of disappearance of the la-beled cells from the circulation. A simultaneous de-termination of blood volume was carried out with eachred-cell survival study; this value was then comparedwith the expected blood volume (12) to calculate thefraction of red cells destroyed within the first 10 min-utes of their injection.
Survival studies with erythrocytes from patients withAIHA. Ten to fifteen ml of the patient's blood was
labeled with 30 to 60 Ac of Cr51. One sample was in-jected into the patient (autosurvival), and another was
administered to a normal recipient of the same ABOand Rh groups as the patient (isosurvival). A simul-taneous blood-volume determination was carried out ineach recipient. Samples were taken at various time in-tervals, depending upon the rapidity of destruction ofthe injected cells. Patients with findings in any way in-dicative of past or present hepatitis were excluded fromthis part of the study, as were normal recipients whogave histories of previous blood transfusions.
Determination of radioactivity. Radioactivity of thespecimens was counted in a well-type, iodide-crystal
6 Abbott Laboratories, North Chicago, Ill.
scintillation counter,7 and the time of counting was setto maintain the counting error within 5%o. Referencestandards were prepared for purposes of decay correction.
RESULTS
Characterization of eluates in AIHA (Tab.'eII). All eluates reacted in the indirect antiglobu-lin test, the highest titer being 128, the lowest, 4.During these studies, we found that reprocessing
of samples of erythrocyte stroma through addi-tional cycles of the elution procedure, in some in-stances as many as five cycles, yielded serologicallyactive eluates. Apparently, a single elution of thered-cell stroma brought only partial recovery ofantibody. Wetherefore made no further attemptsto quantitate erythrocyte-coating globulins by an
elution technique. No serologic activity was de-tected in the eluates prepared from the red cells offour normal individuals. Test red cells were in-cubated with the eluates at 40, 220, and 370 C, andin no instance were significant deviations in speci-ficity or antiglobulin titer noted as the result ofthese temperature differences. The protein con-
tent of the eluates ranged from 41.3 to 101.3 jugN (nitrogen) per ml. No serologic specificitycould be demonstrated in seventeen of the twentyeluates studied. Of the remaining three, one be-haved like anti-e (the patient's red cells were
e-positive), and two showed some specificity thatcould not be completely characterized.
Although the red-cell sensitizing protein was
obviously a gammaglobulin, since it reacted with7 Scintillation detector 810B, Baird Atomic, Cambridge,
Mass.
TABLE II
Eluate characteristics in ten patients with A IHA
Indirect Coombs Agar-gel diffusionTiter
Titer after Anti- Anti- Anti- Anti-Patient Protein N 370 40 2-ME* 7S 19S 7S 19S Specificity
rabbit aDtihuman gamma globulin in the indirectCoombs test, we tried to characterize this materialfurther. On paper electrophoresis, the eluatesmigrated in the gammnia-globulin region, and insome instances, they appeared as a diffuse bandextending from the gamma region to the cathodaledge of the beta position. Migration into theleading edge of the beta position was observedwx-hen cellulose acetate was the supporting medium.
Four of eleven eluates tested reacted in the(louble-diffusion gel system with specific goat anti-human 7S serum (Figure 1). These reactionswere visible as well-defined arcs whose convexityfaced the antigen well. This type of precipitinarc indicates that the molecular weight of the anti-gen (eluate) is equal to or less than the molecularweight of the antiserum (hyperimmune goat se-
rum) and is compatible with the reaction of gamma
globulin of the 7S variety (13). None of theeluates reacted in this system with specific goatantihuman 19S serum. The failure of seven elu-ates to show precipitin bands in the agar may havebeen due either to insufficient quantities of antigenor to inadequate amounts of antibody in the goatserum. When the latter antiserum was used as the"developing" reagent in the indirect antiglobulin
FIG. 1. TVWO-DIMIENSIONAL AGARDIFFUSION OF RED-CELL
ELUATE FROM PATIENTS WITH AUTOIMMUNEHEMOLYTIC
ANEMIA (AIHA). The center well contains goat anti-human 7S antibody; wells 1, 2, and 4 were charged witheluates from AIHA red cells, whereas well 3 contains an
eluate from normal erythrocytes.
TABLE III
The combining ratio of RAGwith erythrocyte antibodies*
* Three typical experiments, in which three differentisoantibodies were tested, are shown. In the range ofantibody excess, the ratio of RAGto sensitizing antibodyis approximately 7.0.
t Antibody nitrogen/0.1 ml packed red cells.
tests of the eluates, all reactions were negative.On the other hand, a rabbit serum reacting spe-cifically with human gamma2 globulin gave strongagglutination patterns (Table II). In everyinstance, incubation of the eluates in 0.1 MI 2-mer-captoethanol failed to reduce the titer signifi-cantly (Table II), although this procedure re-duced the cold agglutinin titer of the positive con-trol serum.
The combining ratio of RAGwith red-cell iso-antibodies. In the experiments shown in Figure2, an(l Table III, Rh-positive red cells were sen-sitized with a standard amount of I'25-labeledanti-D; 0.1-ml samples of these cells were thendistributed into seven tubes. Different amounts ofRAGcontained in a constant volume were addedto each of them, and the adsorptions of 1125_anti-D and I131-RAG were determined. Afterreaching a maximal value, the amount of anti-human globulin adsorbed by sensitized erythro-cytes showed no further increase. In the zoneof extreme antibody excess, the ratio betweenI131-RAG and I125-anti-D was found to be 7 to 1,assuming a mol wt of 160,000 for both the rabbitantihuman globulin and the human anti-D globu-
1793
CONSTANTOULAKIS,COSTEA, SCHWARTZ,AND DAMESHEK
lins. The same result was obtained with a sec-
ond sample of anti-D and a sample of anti-K.The ratio of 7 to 1 was constant regardless ofthe amount of antibody adherent to the red cell,and in one experiment this ratio remained con-
stant, i.e., 7 to 1, during the entire washing pro-
cedure, since the human antibody dissociatedfrom the red cell in combination with 7 moleculesof rabbit antibody.
Isoantibodies. a) Effect of varying amountsof a given isoantibody (anti-D) on red-cell sur-
vival. Serum no. 60-665, obtained from an in-
z4
z
zw
C-zJ4
0
49
.0
0
z
0 5 10 Is
SUg RAG N
20 25
FIG. 2. THE COMBINING RATIO OF RADIOACTIVE ANTI-
GLOBULIN (RAG) WITH ANTI-D AT INCREASING CONCEN-
TRATIONS.
dividual donor and containing a powerful in-
complete anti-D antibody, was used to sensitizeRh-positive red cells and obtain final concentra-tions of antibody bound to red cells of 0.21, 0.54,0.97, 1.11, 1.37, 1.53, and 3.29 ,ug N per ml redcells. These antibody concentrations were cal-culated from the RAG absorption, assuming a
combining ratio of 7 to 1 (RAG to anti-D).The survival of these variously sensitized cells isshown in Figure 3, which demonstrates the re-
lationship between amount of red-cell-bound anti-
body and red-cell survival (ti). Several char-
acteristics of this relationship bear comment.
20-
10 -
5-
4
a
Nel
1.
0.5
0.1 I II0 1 2 3
ANTI - D, jyg N/ml RBC
FIG. 3. RELATION BETWEEN AMOUNT
BOUNDANTI-D AND RED-CELL SURVIVAL.
from one sensitized person was used.
OF RED-CELL-
Serum 60-665
Low levels of sensitization (0.21 and 0.54 fg anti-body N per ml RBC) did not appreciably affectthe red-cell survival, even though the antiglobulintest of the injected cells was positive (0.54 ,uganti-D N per ml RBC gave a 4 + antiglobulinreaction). Above a certain level of sensitization,small increments in absorbed antibody producedmarked changes in red-cell survival. When anti-body concentration was less than one-half themaximal binding capacity of the red-cell popula-tion (i.e., 3 pg anti-D N per ml RBC), red-cellsurvival was extremely short (ti, 35 minutes)
FIG. 4. HEMOLYTIC EFFECTIVENESS OF ERYTHROCYTEISOANTIBODIES. The amount of red-cell-bound antibodywas virtually identical in each experiment. Note, how-ever, the marked variations in red-cell survival times.
and approached the minimal survival of red cellssensitized with noncomplement dependent uni-valent antibody (14).
b) Effect of antibodies with identical serologicspecificities on red-cell survival. Three sera con-taining high-titer incomplete anti-D were ob-
100
Splenectomnized
o 50 i0
o | \ a_ Non-Splenectomized
- 20
I
0 5 10 15 20 25 30DAYS
FIG. 5. EFFECT OF THE SPLEEN ON SURVIVAL OF SENSI-TIZED RED BLOOD CELLS. The degree of red-cell-boundantibody was identical in each experiment (1.11 jug anti-Dantibody N per ml RBC).
U)
-IV
0 2 3 4 5
yg ANTIBODY N/ml RBC
6 7
FIG. 6. RELATION BETWEENAMOUNTOF AUTOANTIBODYAND SURVIVAL TIME OF AUTOLOGOUSRED CELLS IN THE
PATIENT (open circles). Closed circles represent resultsshown in Figure 3, superimposed for comparison.
40-
20-
10-
5.Un
0
-1-
1.
.5.
0.10 I 2 3 4 5 6 7
Jyg ANTIBODY N/mi RBC
FIG. 7. RELATION BETWEENAMOUNTOF AUTOANTIBODY
AND SURVIVAL TIME OF THE PATIENT'S RED CELLS IN A
NORMALRECIPIENT (open circles). Closed circles repre-sent results shown in Figure 3, superimposed for com-
parison.
1795
00 0 0 000
0 0
0
0
00
,.*ANTI-D SERUM*60-665
CONSTANTOULAKIS,COSTEA, SCHWARTZ,AND DAMESHEK
100,002.24 Days 72 Days T/2. 7 .4 Days
~~~~~~~~~~~~~~~~~~~~~~38%*lDay
50 62%.I1.4 Days
20
100,
. * YN. , B. , M,T~2 4 3Days T~'2.8.3 Days *T/2. 4 5Days
50
20 1
000injectd in2t7DaysTa. 7.2 Days Tpe 4.5 Days
ae~ ~ ~ o69 0.04 Days
50- ~~~~~~~~~~~~~~~~~~~~~~~~~~317.13.5 Days
20-
16t 25 0 5 10 15 20 25 0 5 10 1520 25
DAYS DAYS DAYS
FIG. 8. PATTERNS OF RED-CELL DESTRUCTION IN AUTOIMMUNEHEMOLYTIC ANEMIA.
The closed circles are the survival curves of autologous red cells in the patient (auto-
survival), and the open circles are the results obtained when the patient's cells were
inj ected into a normal recipient (isosurvival). Various patterns are represented.
Left: short autosurvival time and normal isosurvival; Middle: short autosurvival andshort isosurvival; Right: short autosurvival and short, two-component isosurvival.
tained from three separate persons (no. 60-665,San, and no. 3077). Normal Rh-positive red cellswere treated with these antibodies as describedabove. Cells incubated with serum San were sen-
sitized with 0.91 pg antibody N per ml RBC;those sensitized with serum 3077 absorbed 3.5 ug
antibody N per ml RBC. Two samples of cellssensitized with serum 60-665 absorbed 0.97 and3.29 ug antibody N per ml RBC. The survivaltimes (t1) of these cells were 14, 1.0, 264, and0.5 hours, respectively. Two sera containing high
titers of incomplete noncomplement dependentanti-K obtained from two sensitized persons (no.CK-11 and 60-1214) were treated in a similarfashion. Cells sensitized with serum CK-11 ab-sorbed 0.91 ug antibody N per ml RBC, and thosesensitized with serum 60-1214 absorbed 0.50 Mug.The red-cell survivals (tj) were 45 minutes and10 days, respectively (Table IV, Figure 4).
c) Survival of sensitized red cells in splenecto-mized recipients. Normal Rh-positive red cellswere sensitized with 1.11 Mig anti-D antibody N
1796
RED-CELL SENSITIZATION AND IN VIVO HEMOLYSIS
per ml RB(C and labeled with Cr51; half of thesecells was injected into a normal person, who alsoserved as the RBCdonor, and the other half wasinjectel into an otherwise normal recipient whosespleen had been removed several years previouslybecause of traumatic rupture. The red-cell sur-vival in the first recipient had a tj of 3.8 days,whereas the to in the splenectomized recipientwas 30 days (Figure 5). A second experimentinvolving two other recipients, one normal andone splenectomized, was carried out with cellssensitized with 3.47 /g anti-D antibody N per mlRBC. The survival times (to) of these cells were60 minutes and 16.4 days, respectively.
Autoantibodies. a) Autosutrvivals. Cr51-la-beled red-cell survival studies with autologouscells were done in 17 patients with AIHA. Inthree patients, two survival studies were done.The relationship between red-cell survival andamount of sensitizing autoantibody bound to thered cells is shown in Figure 6. There was nocorrelation between these two parameters.
b) Isosurvivals. \With red cells from the pa-tients with AIHA, Cr51-labeled red-cell survival
In 2c
0
r)RI-4
8 12 16
T/2 (AUTO) DAYS
FIG. 9. CORRELATIONBETWEENAUTOSURVIVAL AND ISO-SURVIVAL IN AUTOIMMUNEHE'MOLYTIC ANEMIA. In ten
patients, there was correlation between the two determi-nations (open circles connected by solid line), while infive, autosurvival was short, and isosurvival was normal(open circles within the dashed box).
studies were performed in 15 normal recipients.These determinations were made simultaneouslywith the autologous red-cell survival studies de-scribed above. The results of these experimentsare shown in Figures 7 and 8. Three patternsof isologous red-cell survival were found: normalsurvival (6), shortened survival (4), and a two-phase curve with shortened survival (4). Therewas no correlation between the survival of thesecells and the amount of bound antibody. Inmost patients, the tj of the isosurvival was similarto that of the autosurvival (Figure 9). In fivepatients, however, there was actually an inversecorrelation: normal isosurvival and short autosur-vival. In three instances, the isosurvival wasnormal for 10 to 16 days, after which there wasan abrupt disappearance of the remaining cells.This type of "collapse" curve was regarded as anindication of isosensitization of the recipient andwas not taken into account in calculating the to ofthe red-cell survival; the latter was obtained byprojection of the slope of the original regressioncurve to 50% of the radioactivity contained in thefirst blood sample.
DISCUSSION
Isoantibodies. During the past decade, it hasbecame clear that human red cells differ consider-ably in their isoantigenicity. For example, theantigen D is a powerful immunizing substance,although Jkb and s are only weakly antigenic (14).Similarly, the antibodies provoked by these anti-gens vary considerably in their in vivo hemolyticactivity; anti-D almost always produces severehemolysis, whereas anti-Lua, although present inappreciable titer, may be without apparent effecton red-cell survival (14). Isoantibodies maythus be said to manifest at least two kinds ofbehavior: serologic and biologic.
Our experiments demonstrated that differencesin biologic activity occurred, not only among anti-bodies of different serologic specificities, but alsoamong antibodies with identical serologic charac-teristics. When red cells were sensitized with0.97 ug of anti-D nitrogen per ml RBC, their tAwas 11 days, but red cells sensitized with 0.91Kg anti-K nitrogen per ml RBC had a tj ofonly 45 minutes. The marked variations notedwhen antibodies of identical serologic specificitywere tested are illustrated by a specimen of
1797
CONSTANTOULAKIS,COSTEA, SCHWARTZ,AND DAMESHEK
anti-i) that sensitized cells with 0.97 ug N perml RBC, with a resulting survival time of11 days (ti), whereas another specimen of anti-D,which sensitized cells with 0.91 jug N per mlRCB, caused rapid destruction of red cells witha tj of less than 1 day. These differences wereapparently not due to the action of complement,since each of the antibodies studied was ofthe incomplete, noncomplement-fixing variety.Probably, these differences represent an intrinsiccharacteristic of a particular red-cell antibodythat we shall refer to as "hemolytic effectiveness."
Hemolytic effectiveness seems to be a charac-teristic of red-cell antibodies that is independentof their serologic properties. The nature of thisquality is unknown, but it may be the result ofelution rate, electrostatic charge, steric configura-tion, and mode of union with antigen on the red-cell surface. Although the effects of some ofthese factors are obscure, elution of antibodiesfrom the red-cell membranes has been well-demon-strated in vitro (10, 15-19) and in vivo (20). Agiven antibody solution is composed of a heteroge-nous population of molecules with varying disso-ciation constants. The dissociation constants mayhave a broad range; hence elution of some anti-bodies will occur spontaneously (19), but othersadhere to the red-cell surface despite repeatedwashings. Hughes-Jones and his associates (10)have suggested that "each successive dissociationof antibody from red cells should contain antibodywith a progressively higher range of intrinsicbinding constants." In our experiments, anti-bodies resisting elution despite serial washingswere studied for their effects on red-cell survival.Thus the rapid in vivo hemolysis of the sensitizedred cells was presumably due to antibody withrelatively low dissociation constants. Neverthe-less, since survival studies of the sensitized cellsshowed two-component curves, additional elutionof antibody in vivo (20) probably contributed tothe improved viability of a portion of the in-jected red cells. We found a linear relationshipbetween the amount of red-cell-bound antibody(anti-D), as determined by the RAG technique,and the red-cell survival time (Figure 3). Sincethe points on this curve were derived from sur-vival-time determinations in seven different nor-mal recipients with their own erythrocytes, theprincipal cause of the shortened red-cell survival
time was evidently the action of the antibody.Such factors as zygosity of the red cells, distri-bution of the antigen on the red-cell surface, vari-abilities in elution rates either because of anti-genic factors or the number of antibody mole-cules on the red-cell surface, if important, wouldhave disturbed the linearity of this curve.
The shape of the curve shown in Figure 3 il-lustrates several additional features of the inter-action between isoantibody and red cells. a) Sen-sitization of erythrocytes does not necessarily leadto their premature destruction, as Mollison andPaterson (21) have already pointed out. In oneexperiment, the amount of anti-D absorbed by redcells was 0.54 fug N per ml RBC. This amountwas enough to give a 4 + direct antiglobulin test;nevertheless, survival of these cells was withinnormal limits. The limitations of red-cell-survi-val techniques with the Cr5' label are such, how-ever, that appreciable diminution in the mean celllife may go undetected. "Normal" survivals ofCr5'-labeled, sensitized red cells should, therefore,be cautiously interpreted (22, 23). b) The steepslope of the curve indicates that, above a certainlevel of sensitization (approximately 0.90 MAg N perml RBC), minute increases of red-cell-boundantibody induce pronounced effects on red-cellsurvival. Since antibody molecules possess elec-trostatic charges, minute increments in these coat-ing proteins may conceivably result in membranechanges favoring phagocytosis of the sensitizedcell by the spleen or liver. c) Complete satura-tion by antibody of the antigenic sites of the redcell is not necessary to cause their destruction ata maximal rate. When red cells absorbed ap-proximately 30% of their anti-D combining ca-pacity, their survival was measured in minutes.Thus, the zygosity of the sensitized red cells wouldnot be a factor in their survival under theseconditions.
That antibodies of the incomplete noncomple-ment-dependent type are not the sole factors lead-ing to premature destruction of red cells was dem-onstrated by the striking differences between thedisposal of equally sensitized red cells in normaland in splenectomized recipients. Although thesurvival of anti-D-sensitized cells in splenecto-mized recipient E.B. was shortened (tj, 16.4days), she had mild autoimmune hemolytic ane-mia, which may account for this finding. These
1798
RED-CELL SENSITIZATION AND IN VIVO HEMOLYSIS
results confirm those of Jandl, Jones, and Castle(24) and of Hughes-Jones, Mollison, and Veall(25), who demonstrated by a surface countingtechnique that the principal site of destruction ofred cells sensitized by anti-D is the spleen. Ear-lier studies had shown only hours difference be-tween the survival of anti-D-sensitized red cellsin splenectomized and in normal recipients. Sincequantitative estimation of red-cell sensitizationwas not performed, however, comparing these re-sults with ours is difficult. One problem, for ex-ample, is that in the absence of the spleen, cellssensitized with more than 3.5 ug of anti-D N perml RBCmay be removed from the circulation bythe liver.
Autoantibodies. Because the RAG techniquedetermines only the amount of rabbit antihumanglobulin absorbed by sensitized red cells, the com-bining ratio of sensitizing antibody to rabbit anti-human globulin must be determined to calculatethe amount of red-cell-bound human antibody.Utilizing two isotopes of iodine, we found thisratio for incomplete anti-D and anti-K antibodiesof the 7S variety. In the zone of extreme anti-body excess, it was 7 to 1, a value previously ob-tained immunochemically with soluble gamma,globulin (26, 27).
The derivation of combining ratios in agglu-tinating systems would depend not only on thequantitative relationship between antigen and anti-body, but also upon the availability of the cellularantigen and steric hindrances intrinsic to the sys-tem. These complications in applying the pre-cipitin reaction to cell-bound antibody are illus-trated by Rappaport's finding that even in largeantibody excess, all antigenic groups of the to-bacco mosaic virus failed to combine with antibody(28-29). In calculating the combining ratio bythe RAG technique, however, determination ofthe intrinsic red-cell antigens is not employed, butrather the amount of antibody adherent to themembrane. This antibody actually functions inthe RAGtest as the antigen. Since the same num-ber of antigenic (D) sites on the red-cell surfacewas found by the RAGtechnique as by the use ofonly I131-labeled anti-D, antigen-antibody reactionson the red cell surface must be equally availableto both the red cell antibody and the RAGreagent.This evidence implies that unavailability of mem-brane antigens (either D or anti-D) is not a fac-
tor in these reactions and accounts for the coml)in-ing ratio's being the same as for soluble systems.
Because the amount of sensitizing antibodyelutable from red cells is relatively minute inAIHA, determining the combining ratio directlyis impossible. Attempts were therefore made toobtain additional information concerning thephysicochemical properties of these autoanti-bodies. As Table III shows, there are a numberof indications that these proteins are 7S gammaglobulins, and in no instance were reactions com-patible with a macroglobulin type of antibodyobtained. These findings agree with those ofFudenberg and Kunkel (30), who found that thecirculating antibodies in AIHA of the warm va-riety were 7S gamma2 globulins. To simplify thecomparisons between the autoantibodies of AIHAand the isoantibodies under investigation, we as-sumed that their combining ratio was also 7 to 1.
Of the 17 patients with autoimmune hemolyticdisease in whom autosurvival studies were done,15 showed varying degrees of shortened erythro-cyte life span, but the two patients who were inremission showed no abnormal hemolysis. Nocorrelation was found between the values for red-cell survival (t4) and the amount of autoantibodyon the erythrocytes. Thus, patients with less than1 pg antibody N per ml RBC were found tohave highly abnormal degrees of hemolysis; pa-tients with 5 or more pug antibody N per ml RBChad only mild hemolysis. Dacie (31) has dis-cussed the possibility that the autoantibodies ofAIHA are less effective in causing red-cell dam-age than are isoantibodies. The data of the pres-ent experiments indicate, however, that smallamounts of some autoantibodies may be associ-ated with severe hemolysis (e.g., patient H.C.with 0.4 ,ug antibody N per ml RBCand a red-cellsurvival t, of 3.5 days), and some isoantibod-ies may have only moderate effects on red-cell life span (e.g., red cells sensitized with 0.97,ug anti-D serum 6055 N per ml had a t4 of 11.1days). Yet red-cell survival times of less than 1day are never seen in AIHA (such a conditionwould be rapidly fatal), whereas isoantibodiesmay readily induce this degree of hemolysis, evenwhen they sensitize erythrocytes to the same ex-tent as autoantibodies.
The belief, widely held, that acquired hemo-lytic anemia can be differentiated from hereditary
1799
CONSTANTOULAKIS,COSTEA, SCHWARTZ,AND DAMESHEK
spherocytosis by determining the survival of anAIHA patient's red cells in a normal recipient,is not supported by the results of our experi-ments. The survival in normal recipients of redcells from patients with AIHA has been reportedpreviously; in most of these studies, the Ashbymethod was used. Loutit and Mollison (32) andLUdin (33) reported that such red cells sur-vived as usual in normal recipients. Selwyn andHackett (34), Owren (35), and Strumia and hisassociates (36) found an initial rapid disappear-ance of approximately 50% of the transfusedred cells, but normal survival of the remainingcells. Of the 15 isosurvivals with the red cells ofour patients, the red cells of 7 had normal iso-survivals, those of 4 showed a two-component be-havior, and the remaining 4 had shortened iso-survivals. Moreover, the cells that showed atwo-component curve had abnormally reduced sur-vivals even in the "long" components of the curve.In contrast to Mollison's experience (37), ourwork showed no correlation between the severityof the hemolytic disease and the results of theisosurvivals. There is no reason to believe thatred cells from patients with AIHA should havenormal isosurvivals, since the rate of destructionof these cells in both the donor and the recipientis a function of the hemolytic effectiveness of theantibody, its rate of in vivo elution, and the ac-tion of complement. Although factors such asphagocytic activity of the spleen, fever, and in-fection undoubtedly further influence red-cellautosurvival in AIHA, in 10 of 15 patients therewas a close correlation between auto- and isosur-vival (Figure 9). This finding indicates that inthese patients, autoantibody was the predominantfactor influencing red-cell survival. In the re-maining patients, such a correlation was not found;we therefore assume that factors other than auto-antibody contributed to the hemolytic process.
SUMMARY
The quantitative relationships between eryth-rocyte sensitization and survival were studied bythe techniques of Cr51-labeled red-cell survival andthe radioactive antiglobulin (RAG) test. Thetwo major types of red-cell antibodies, isoanti-bodies and autoantibodies, have two types of be-havior, serologic and biologic. Regardless of theirserologic attributes, these antibodies had dis-
tinctive effects on erythrocyte survival. Evenantibodies of the same serologic specificity haddifferent effects on in vivo hemolysis. This bio-logic attribute was termed "hemolytic effective-ness."
When either isoantibodies or autoantibodiesfrom different individuals were studied, there wasno correlation between the amount of antibodyabsorbed by erythrocytes and their survival time.In 10 of 15 cases of autoimmune hemolytic ane-mia, however, there was a positive correlation be-tween the survival time of the patient's red cellsin himself and their survival time in a normal per-son. This finding we interpreted as indicatingthat, in these cases, the major cause of the abnor-mal hemolysis was the autoantibody.
ACKNOWLEDGMENT
The expert technical assistance of Miss Jane Reins-borough is gratefully acknowledged.
REFERENCES
1. Fudenberg, H., I. Barry, and W. Dameshek. Theerythrocyte-coating substance in autoimmune he-molytic disease: its nature and significance. Blood1958, 13, 201.
2. Costea, N., R. Schwartz, M. Constantoulakis, andWA. Dameshek. The use of radioactive antiglobulinfor the detection of erythrocyte sensitization. Blood1962, 20, 214.
3. Kidd, P. Elution of an incomplete type of antibodyfrom the erythrocytes in acquired hemolytic anaemia.J. clin. Path. 1949, 2, 103.
4. Dunsford, I., and J. Grant. The Antiglobulin(Coombs) Test in Laboratory Practice. Edin-burgh, Oliver and Boyd, 1949.
5. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, andR. J. Randall. Protein measurement with theFolin phenol reagent. J. biol. Chem. 1951, 193,265.
6. Ouchterlony, 0. Diffusion-in-gel methods for im-munologic analysis in Progress in Allergy. Ba-sel, S. Karger, 1958, vol. 5, p. 1.
7. Deutsch, H. F., and J. I. Morton. Dissociation ofhuman serum macroglobulins. Science 1957, 125,600.
8. Grubb, R., and B. Swahn. Destruction of some ag-glutinins but not of others by-two sulfhydryl com-pounds. Acta path. microbiol. scand. 1948, 43,305.
9. Helmkamp, R. W., R. L. Goodland, W. F. Bale, I. L.Spar, and L. E. Mutschler. High specific activityiodination of -y-globulin with iodine-131 mono-chloride. Cancer Res. 1960, 20, 1495.
1800
RED-CELL SENSITIZATION AND IN VIVO HEMOLYSIS
10. Hughes-Jones, N. C., B. Gardner, and R. Telford.The kinetics of the reaction between the blood-group antibody anti-c and erythrocytes. Bio-chem. J. 1962, 85, 466.
I1. Read, R. C., G. W. Wilson, and F. H. Gardner. Theuse of radioactive sodium chromate to evaluate thelife span of the red blood cell in health and cer-
tain hematologic disorders. Amer. J. med. Sci.1954, 228, 40.
12. Wennesland, R., E. Brown, J. Hopper, Jr., J. L.Hodges, Jr., 0. E. Guttentag, K. G. Scott, I. N.Tucker, and B. Bradley. Red cell, plasma andblood volume in healthy men measured by radio-chromium (Cr51) cell tagging and hematocrit: in-fluence of age, somatotype and habits of physicalactivity on the variance after regression of volumesto height and weight combined. J. clin. Invest.1959, 38, 1065.
13. Korngold, L., and G. van Leeuwen. The effect ofthe antigen's molecular weight on the curvature ofthe precipitin line in the Ouchterlony technic. J.Immunol. 1957, 78, 172.
14. Greendyke, R. M., and F. W. Chorpening. Normalsurvival of incompatible red cells in the presence
of anti-Lua. Transfusion 1962, 2, 52.15. Jandl, J. H., and M. E. Kaplan. The destruction of
red cells by antibodies in man. III. Quantitativefactors influencing the patterns of hemolysis inviZtvo. J. clin. Invest. 1960, 39, 1145.
16. Bowman, W. N., M. M. Mayer, and H. J. Rapp.Kinetic studies of red cell antibody combination andthe resultant transfer of antibody from cell to cellduring hemolysis. J. exp. Med. 1951, 94, 87.
17. Talmage, D. W. The primary equilibrium betweenantigen and antibody. Ann. N. Y. Acad. Sci. 1957,70, 82.
18. Goodman, H. S., and L. Masaitis. The dissociationof hemolytic antibody from sensitized cells as
measured by cell-to-cell transfer. J. Immunol.1960, 85, 391.
19. Evans, R. S., M. Bingham, and P. Boehni. Auto-immune hemolytic disease. Antibody dissociationand activity. Arch. intern. Med. 1961, 108, 338.
20. Costea, N. V., M. Constantoulakis, and R. Schwartz.The in vivo behavior of specific erythrocyte anti-bodies in man (abstract). Blood 1962, 20, 792.
21. Mollison, P. L., and Paterson, J. C. S. Survivalafter transfusion of Rh-positive erythrocytes prev-
iously incubated with Rh antibody. J. clin. Path.1949, 2, 190.
22. Berlin, N. I., T. A. Waldmann, and S. M. Weissman.Life span of red blood cell. Physiol. Rev. 1959,39, 577.
23. Stohlman, F., Jr. The use of Fe59 and Cr51 for esti-mating red cell production and destruction: an
interpretive review. Blood 1961, 18, 236.24. Jandl, J. H., A. R. Jones, and W. B. Castle. The
destruction of red cells by antibodies in man. I.
Observations on the sequestration and lysis of redcells altered by immune mechanisms. J. clin. In-vest. 1957, 36, 1428.
25. Hughes-Jones, N. C., P. L. Mollison, and N. Veall.Removal of incompatible red cells by the spleen.Brit. J. Haemat. 1957, 3, 125 .
26. Jager, B. V., E. L. Smith, M. Nickerson, and D. M.
Brown. Immunological and electrophoretic stud-ies on human y-globulins. J. biol. Chem. 1948,176, 1177.
27. Kabat, E. A., and J. P. Murray. Comparison ofhuman -y-globulins in their reactivity with rabbitanti-'y-globulin by the quantitative precipitinmethod. J. biol. Chem. 1950, 182, 251.
28. Rappaport, I. The antibody-antigen reaction. Anhypothesis to account for the presence of uncom-
bined antigenic sites in the presence of excess
antibody. J. Immunol. 1957, 78, 246.29. Rappaport, I. The reversibility of the reaction be-
30. Fudenberg, H. H., and H. G. Kunkel. Physicalproperties of the red cell agglutinins in acquiredhemolytic anemia. J. exp. Med. 1957, 106, 689.
31. Dacie, J. V. The haemolytic anaemias, congenitaland acquired. Part II. in The AutoimmuneH-aemolytic Anaemias, 2nd ed. New York, Grune
& Stratton, 1962.32. Loutit, J. F., and P. D. Mollison. Hxmolytic ic-
terus (acholuric jaundice), congenital and ac-
quired. J. Path. Bact. 1946, 58, 711.33 Liidin, H. Zur Differential diagnose himolytischer
Anamien. Acta haemat. (Basel) 1948, 1, 28.34. Selwyn, J. G., and W. E. R. Hackett. Acquired
haemolytic anaemia: survival of transfused erythro-cytes in patients and normal recipients. J. clin.Path. 1949, 2, 114.
35. Owren, P. A. Acquired hemolytic jaundice. Scand.
*J. clin. Lab. Invest. 1961, 1, 313.
36. Strumia, M. M., L. Taylor, A. B. Sample, L. S.Colwell, and A. Dugan. Uses and limitations ofsurvival studies of erythrocytes tagged with Cr51.Blood 1955, 10, 429.
37. Mollison, P. L. Blood Transfusion in Clinical Medi-cine, 3rd ed. London, Blackwell Scientific Publi-
