Journal of Clinical InvestigationVol. 41, No. 3, 1962

REACTIVITY OF RED CELL ELUATESAND SERUMSIN PATIENTS WITHACQUIREDHEMOLYTICANEMIA AND CHRONIC

LYMPHOCYTICLEUKEMIA*

By JEROMEI. BRODYWITH THE TECHNICAL ASSISTANCE OF DOROTHYA. CHERTOK

(From the Department of Medicine, Veterans Administration Hospital, and the University ofMiami School of Medicine, Coral Gables, Fla.)

(Submitted for publication June 15, 1961; accepted October 27, 1961)

The purpose of this study was to define furtherthe immunologic characteristics of the Coombs-positive red cell eluates and serums of patientswith acquired hemolytic anemia and chroniclymphocytic leukemia by determining whetherthey are reactive with and related to autologousneoplastic lymphocytes. These erythrocyte-coat-ing globulins have provoked a great deal of investi-gative and clinical interest and as a result certainof their properties are well recognized. It is gen-erally agreed that they are heterogeneous globu-lins (1) which sensitize human erythrocytes bothin vitro and in vivo without regard to cell type(2) and that they may stimulate antiglobulinswhen injected into an appropriate recipient (3).Nevertheless, the immunologic origin of theseproteins has not been adequately delineated.

The frequency with which Coombs-positive ac-quired hemolytic anemia occurs in chronic lympho-cytic leukemia as opposed to other leukemias andlymphomas (4-7) and the improvement whichmay follow the administration of lympholyticdrugs, such as the adrenocortical steroids (8),suggested that the autologous, circulating, neo-plastic lymphocyte might function as an antigenicchallenge in these patients. This concept has beendiscussed on prior occasions (9, 10), but experi-mental evidence to sustain this supposition hasbeen inconclusive.

The technique of immune adherence (11), inwhich an antigen either macroscopically or mi-croscopically adheres to a primate red cell or non-primate platelet indicator, only in the presence ofcomplement and specific antibody, was employedin this investigation because its suitability for thedetection of antigen-antibody reactions has beenwell demonstrated (12-14).

* Presented in part to the National Meeting of theAmerican Federation for Clinical Research (Section I,Hematology), Atlantic City, N. J., April 30, 1961.

METHODSAND MATERIALS

A. Subjects1. Patients with chronic lymphocytic leukemia. A to-

tal of 9 male patients, varying in age from 56 to 72 years,was employed in this study. Despite the fact that theperipheral blood lymphocyte morphology was variableand not all patients exhibited the small, characteristic,dark-staining lymphocyte as the predominant cell typeat all times, a diagnosis of chronic lymphocytic leukemiawas made in all patients because of their age and pro-longed clinical course. Except for the patient who diedas the result of an acute blastic crisis 9 months afterdiagnosis of his blood disorder, the patients were knownto have had leukemia for a minimum of 12 months. Twopatients were, for all intents and purposes, clinically welland had as significant abnormalities only an elevated totalleukocyte count with an absolute lymphocytosis, andminimal or no splenomegaly or lymphadenopathy.There was marked splenomegaly and peripheral lymph-adenopathy in four patients with lymphoblasts, immaturelymphoid elements resembling reticular lymphocytes, andprolymphocytes in their peripheral blood. In two pa-tients there was moderate lymphadenopathy and spleno-megaly, moderate anemia, and mature-type "cleft" leuke-mic lymphocytes in the peripheral blood. The presenceof marked splenomegaly and nucleolated lymphoid cellsin the peripheral blood of one patient without lymphade-nopathy suggested that his disease began as a primarysplenic lymphocytic lymphoma with eventual dischargeof these neoplastic lymphocytes into the peripheral blood.Six patients did not receive antileukemic therapy duringthe study, but serum and red cell eluate reactivity wereevaluated in one patient during prednisone therapy, inone patient before and after the administration of predni-sone and chlorambucil, and in one patient before and afterhe was treated with chlorambucil alone.

2. Controls. This group consisted of 8 subjects: 2healthy laboratory technicians; and 6 patients, one eachwith sickle cell trait, diabetes mellitus adequately con-trolled, chronic glomerulonephritis with azotemia, rheu-matoid arthritis with mild anemia, post-hepatitic cirrho-sis, and peptic ulcer.

B. Tests for erythrocyte sensitization

Qualitative direct and indirect Coombs test (15) wereperformed on leukemic and control patients with a po-

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tent, broad-spectrum antihuman globulin serum.' Thistest was performed in duplicate, both with and withoutfresh human serum as a complement source, because ofprior observations which suggested that complement in-creases the sensitivity of the Coombs reaction (16, 17).Human serum was employed, as opposed to guinea pigserum, in a deliberate attempt to avoid the introductionof a heterologous agent that might interfere with theevaluation of the test.

Quantitative direct Coombs tests were determined bythe ability of serially diluted red cell eluates to resensi-tize normal erythrocytes. The technique consisted of in-cubating 0.1 ml of a 2 per cent suspension of normal, typeO Rh negative erythrocytes, 0.3-ml aliquots of the redcell eluate (obtained as outlined below) serially dilutedwith normal saline, 0.25 ml of human complement di-luted 1: 10 with gelatin-veronal buffer (GVB), and 0.25ml GVB in a 370 C water bath for 1 hour. The mixturewas then centrifuged and washed with saline four times,the supernate removed, and the Coombs test performed.

Quantitative indirect Coombs tests were similarly per-formed by incubating 0.25 ml of a 2 per cent suspensionof type 0, Rh negative erythrocytes, 0.75-ml aliquotsof patient serum serially diluted with normal saline, and0.25 ml of fresh human complement diluted 1: 10 withGVB. After incubation in a 370 C water bath for 1hour, the reactants were centrifuged, washed, and theCoombs test performed. Serum and eluate dilutionsvaried from full strength to 1: 72 and were used in theseconcentrations because they afforded optimum reactivityin the test. The Coombs antiglobulin serum was usedundiluted, at full strength, in all determinations. In allinstances the Coombs reaction was considered positivewhen either macroscopic or microscopic red cell agglu-tination was observed.

C. Preparation of reactants for immune adherence (IA)assay

1. Complement. Normal, human, type AB, Rh nega-tive serum was used as complement source. It was keptat - 70° C in 1-ml aliquot portions in screw-top vials.After quick thawing it was used in a constant dilution of1: 40 with GVB.

2. Lymphocytes. To prepare leukemic lymphocytes,10 ml of blood was aspirated into a syringe containing 0.5mg heparin, expressed into a clean, dry 15 X 150 mmscrew-top test tube, and centrifuged at room temperaturein a PR-2 International centrifuge (head no. 269) at 2,000rpm (900 G) for 20 minutes. The supernatant plasmaand 3/4 of the red cells deposited in the lower portion ofthe tube were transferred to a similar test tube with acapillary pipet. The mixture was agitated gently to in-sure adequate mixing and incubated in a 37° C waterbath for 45 minutes. The supernatant plasma was cen-trifuged at room temperature at 600 rpm (80 G) for 7minutes. The supernatant plasma was discarded and theresultant leukocyte mixture resuspended in 5 ml of cold

1 Ortho Pharmaceutical Corp., Raritan, N. J.

physiologic saline. This suspension was permitted toprecipitate at room temperature for approximately 30minutes or until the major portion of the leukocytes settledout. The supernatent was then discarded and the leuko-cyte sediment resuspended in 5 to 10 ml of normal saline.

In order to obtain a lymphocyte suspension as freeas possible of contaminating polymorphonuclear leuko-cytes, a modification of the glass-wool column leukocyteseparation technique (18) was employed. This methodremoved approximately only 50 per cent of the con-taminating polymorphonuclear leukocytes but is, never-theless, one of the more practical methods available forthis type of separation. The lymphocyte suspension, af-ter filtration through the glass-wool column, was washedthree times with GVB. These procedures did not alterthe morphologic integrity of the lymphocytes. A 2 percent lymphocyte suspension (0.2 ml packed lymphocytessuspended in 9.8 ml GVB) was considered to be fullstrength, and serial dilutions up to 1: 16 were made witbGVB to be used in the immune adherence assay.

To obtain lymphocytes from the control patients, 40ml of heparinized blood was aspirated, divided into 10-mlaliquots, and treated in a manner similar to that for leuke-mic lymphocytes. The combined lymphocyte yields of 4tubes were pooled and, because technically it was notpossible to obtain as many lymphocytes from control asfrom leukemic patients, the lymphocyte suspension wasadjusted to 20 per cent transmittance on a Beckman DUspectrophotometer at a wave length of 400 mu. Thisserved as the full-strength concentration in the controlassays, and subsequent dilutions up to 1: 16 were madewith GVB.

3. Materials to be used as antibody. In order to evalu-ate the lymphocyte-antigen hypothesis, heterologouslymphocyte antibody was made by injecting human leu-kemic lymphocytes into rabbits. A 3-ml suspension oflymphocytes, containing a total of 7.5 X 106 cells, was in-jected twice weekly for 3 weeks into the marginal earvein of albino rabbits weighing approximately 2 kg.The animals were rested for 7 days; 10 ml of blood wasthen harvested from the ear vein. The blood was al-lowed to clot and the serum was removed and stored in2-ml aliquots at - 350 C. Prior to use in the assay theserum, containing the heterologous neoplastic antibody,was heated to 560 C for 30 minutes to inactivate com-plement. It was then subsequently absorbed, in succes-sion, with erythrocytes from the lymphocyte donor andwith the IA indicator erythrocytes (2 parts red cells to1 part serum) at 00 C for 45 minutes to remove anyheterologous erythrocyte antibody. The absorption wasrepeated until full-strength rabbit antiserum no longercaused microscopic red cell agglutination. Heterologouslymphocyte antibody in immune adherence was used indilutions from full strength to 1: 2,560, made with GVB.

Red cell eluates were prepared by acid elution (2)after washing the erythrocytes six times with normalsaline at room temperature. Reactivity of the washingswas not evaluated by conventional Coombs or IA tests.Eluate reactivity from unwashed cells or from red cells

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REACTIVITY OF RED CELL ELUATESAND SERUMS

washed fewer than six times was not determined. Thismethod, after the final step in the procedure, resulted inapproximately 20 to 30 ml of a brownish-tinged fluidwhich was dialyzed overnight in cellophane casing (d2 inchdiameter)2 against a 15 per cent solution of Plasdone-C(polyvinylpyrrolidone).3 The residuum, amounting to

about 0.5 ml of concentrated eluate, was diluted to a

standard volume of 3 ml with GVBand was considered to

be "full-strength eluate." Eluates in immune adherencewere used in dilutions from full strength to 1: 16,384,made with GVB.

Serums to be used as antibody were prepared as fol-lows: 15 ml of blood was placed in a 15 X 150 mmdry,clean test tube and allowed to clot at room temperaturefor 1 hour; the clotted blood was refrigerated at 40 Cfor 4 hours. The blood was then centrifuged at room

temperature and the serum removed with a capillary pipetand stored in 2-ml aliquots at - 350 C until used. Se-rum dilutions in immune adherence ranged from fullstrength to 1: 640, made with GVB. All antibody dilu-tions were devised to give optimum reactivity in theassays.

4. Indicators. Indicator red cells were collected, stored,and employed as previously described in detail (14). Thefinal constant red cell concentration was adjusted to an

optical density of 0.390 at a wave length of 541 mu witha slit opening of 0.02 on a Beckman DU spectrophotom-eter. Guinea pig platelets were collected and preparedas previously described (19). They were standardized to20 per cent transmittance at a wave length of 400 m~uon

a Beckman DU spectrophotometer and used in this con-

stant concentration.

D. Assay

Heterologous antibody reactivity was determined inKontes hemagglutination tubes as previously described(14). In a checkerboard titration pattern, 0.25-ml ali-quots of serially diluted heterologous lymphocyte anti-body were added slowly to 0.25-ml portions of seriallydiluted leukemic lymphocytes. Complement, 0.5 ml, di-luted 1: 40 with GVB, was added to each tube and, after

2 Arthur H. Thomas Co., Philadelphia, Pa.3 Antra Chemical Div. General Aniline and Film Corp.,

Charlotte, N. C.

a 15-minute incubation period in a 37° C water bath,0.1 ml of the standard red cell indicator suspension was

added to each tube with gentle mixing. After another45-minute incubation, IA patterns (0 to 4+) were de-termined macroscopically and confirmed by wet coverslippreparations under dark-field microscopy. An example ofthis titration is illustrated in Table I.

Red cell eluate and serum reactivity were determinedas previously described in detail (19). With separatecheckerboard titrations for each antigen, serial dilutionsof red cell eluates and serums were added in 0.25-mlaliquots to 0.25-ml portions of serially diluted erythrocyteand lymphocyte antigen in 2-ml screw-top vials. Thevials were rotated at 6 rpm in a 37° C incubator for 10minutes. They were removed from the incubator, 0.5 mlcomplement (1: 40) was added to each vial, and thenwere reincubated on the rotator. Then 0.1 ml of plateletindicator suspension was added to each vial and the in-cubation repeated for 40 minutes. Wet coverslip prepa-rations were examined microscopically for immune ad-herence. The reaction was read as negative when no

antigen adhered to the platelets, as a trace when lessthan 10 per cent adhered, + 1 for IA of 10 to 25 per cent,+ 2 for IA of 25 to 50 per cent, + 3 for IA of 50 to 75per cent, and + 4 with IA of more than 75 per cent of theantigen.

E. Tests for lymphocyte sensitization

During the course of the study, when initial resultssuggested that the Coombs-positive globulins might berelated to neoplastic lymphocytes, qualitative Coombstests, as described previously, were performed on bothleukemic and control lymphocytes.

RESULTS

A. Tests for erythrocyte sensitization. Of thenine patients with leukemia, five had a positive di-rect Coombs test, two had a positive indirect test,one was positive both direct and indirect, and one

patient was completely negative. In all instancesfresh serum either increased the positivity of a

reaction or converted a negative test to a positive

JE I

Immune adherence assay of heterologous lymphocyte antibody

Antigen* Antibody dilutiont

Conc. Dilution Undil. 1:5 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1,280 1:2,560

2 Undiluted +4 +4 +4 +4 +4 +3 +3 +3 +2 +2- +22 1:2 +3 +3 +4 +4 +3 +3 +2 +2 +2 +2 f+22 1:4 0 +1 +2 +3 +3 +2 +2 +2 +1 +1 +12 1:8 0 0 +11 +1 +2 +2 +2 +1 +1 0 02 1:16 0 0 0 +1 +1 +1 +1 +1 0 0 0

* Human leukemic lymphocytes.f Heterologous rabbit antiserum.

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JEROMEI. BRODY

TABLE II

Qualitative and quantitative Coombstests in patients with leukemia

Qualitative QuantitativeCoombs test Coombs test

Patient Direct* Indirect Directt Indirect:

1 0 02 dt + 0 1:43 bt + 0 1:32

at + + 1:4 1:44 bt O + 1:8

at + + 1:16 1:45 + 0 1:486 + 0 Undil.

only7 + 0 1:48 0 + 1:249 + 0 1:2

* During treatment, dt; before treatment, bt; aftertreatment, at.

t Dilution of red cell eluate reacting with undilutedCoombs antiserum after resensitization of normal, type 0Rh negative erythrocytes.

t Dilution of serum reacting with undiluted Coombsantiserum after resensitization of normal, type 0, Rhnegative erythrocytes.

result and therefore only this test is recorded.Quantitative Coombs tests performed with redcell eluates and serums varied in titer from fullstrength to a 1: 48 dilution. These results aresummarized in Table II.

Of the eight control subjects six had negativedirect and indirect Coombs reactions. However,the patients with peptic ulcer and post-hepatitic

FIG. 1. UNDERDARK-FIELD MICROSCOPY,THE LEUKEMICLYMPHOCYTESIN THE CENTEROF THE FIELD ARE ADHERENT

TO THE MORELUMINESCENT, PERIPHERAL INDICATOR RED

CELLS (X450).

FIG. 2. IMMUNE ADHERENCE OF THE AUTOLOGOUS,LEUKEMIC LYMPHOCYTESTO THE STELLATE, INDICATORGUINEA PIG PLATELETS UNDERDARK FIELD (X450).

cirrhosis both had positive direct Coombs tests.Neither patient, however, was anemic and neitherpatient had an elevation of the reticulocyte count.The red cell eluate from the patient with cirrhosisresensitized normal erythrocytes only when un-diluted, and the patient with post-hepatitic cirrhosishad a resensitization titer of 1: 8.

B. Immune adherence assays. Heterologousleukemic lymphocyte antibody was reactive (+ 2)in a serum dilution up to 1: 2,560 and indicates thatIA is a valid method for detecting lymphocyte anti-body (Figure 1). The + 2 reaction in this dilu-tion is evidence that the antibody most probablywould have retained its reactivity for at least an-other tube dilution.

TABLE III

Leukemic red cell eluate and serum reactivityin immune adherence (IA)

IA re- Coombs-positive Coombs-positiveactivity* eluatest serumst

no. no.1:4 1 01:8 1 01:32 1 01:48 1 01:72 1 11:128 1 01:192 0 21:384 0 11:16,384 2 0Total 8 4

* Dilution of eluate or serum reactive in IA with autol-ogous leukemic lymphocytes.

t Represents 2 patients studied before and after anti-leukemic therapy.

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REACTIVITY OF RED CELL ELUATESAND SERUMS

TABLE IV

Clinical variables and immune adherence reactivity

IA reactivity tPatient Therapy Predominating cell type* Anemia Eluate Serum

1 0 MTL 0 0 02t Prednisone MTL 0 1:72 03§ Chlorambucil and bt: lymphoblasts, prolymphocytes Severe 1:16,384 0

prednisoneat: ML, occ. prolymphocyte Mild 1:8 1:192

4 Chlorambucil bt: nucleolated retic. lymphocytes Severe 0 1:1925 0 at: nochange Severe 1:48 1:725 0 ML, prolymphocytes, occ. lymphoblast Severe: patient died 1: 16,384 06 0 MTL 0 1:4 07 0 MTL Severe: patient died 1:128 08 0 MTL Moderate 0 1:3849 0 MTL Mild 1:32 0

* MTL, mature-type lymphocyte; ML, mature lymphocytes.t Dilution of eluate or serum reactive with autologous, neoplastic lymphocytes.t This patient was admitted with icterus, severe anemia, and septicemia. He showed an excellent therapeutic re-

sponse. It is felt that the titer of 1: 72, obtained after his discharge, would have been much higher if it had been oppor-tune to study him during the acute phase of his illness.

§ This patient, subsequent to completion of this investigation, developed a fulminating lymphoblastic leukemia andexpired.

Immune adherence was present when Coombs-positive red cell eluates and serums were reactedwith autologous, leukemic lymphocytes, indicatingthat an antigen-antibody reaction had taken place(Figure 2). With both the eluates and serumsthe highest dilution giving a + 1 reaction is re-ported as the end point of the assay. Reactivity ofeluates varied from 1: 8 to 1: 16,384 and that ofserums from 1: 72 to 1: 384. These findings aresummarized in Table III. Eluate and serum reac-tivity were influenced by the clinical variables ofmorphologic maturity of the lymphocyte, the se-verity of the leukemic process, and whether thepatient had received antileukemic therapy. Ingeneral, but not without exception, antibody reac-

TABLE V

Qualitative immune adherence reactivity *

Antigen Antibody Indicator IA

Human leukemic Heterol. (rabbit) Human red cells +lymphocyte lymphocyte antiserum

Autologous leu- CP red cell eluate GPP +kemic lymphocyte

Autologous leu- CP serum GPP +kemic lymphocyte

Autologous CP red cell eluate GPP 0erythrocyte

Autologous CP serum GPP 0erythrocyteAutologous non- CP nonleuk. red cell GPP 0leuk. lymphocyte eluate

* CP, Coombs-positive; GPP, guinea pig platelets.

tivity was more marked when the lymphocyteswere immature and when the clinical state of thepatient was poor. Reactivity appeared to diminishduring or after treatment. However, in Patient 3a marked drop in eluate reactivity was accom-panied by a moderate rise in serum reactivity, andin Patient 4 the reverse occurred, although thischange was not so pronounced. These observa-tions are summarized in Table IV. In both in-stances the alterations were reflected in similarchanges in the direct and indirect Coombs reac-tions (Table II). In addition, two Coombs-posi-tive red cell eluates and serums (Patients 3 and 4)were reactive in a 1: 2 dilution in IA with non-leukemic, control lymphocytes. Reactivity withthese cells was less than that obtained with autolo-gous, neoplastic lymphocytes.

Immune adherence was not observed, indicatingthe absence of an antigen-antibody reaction, whenCoombs-positive red cell eluates and serums werereacted with autologous erythrocytes, whenCoombs-negative eluates and serums were reactedwith autologous erythrocytes and lymphocytesand, particularly, when nonleukemic, control,Coombs-positive red cell eluates were reacted withautologous lymphocytes. The results of the sig-nificant qualitative eluate and serum IA assays aresummarized in Table V.

C. Tests for lymphocyte sensitization. Lympho-cytes from leukemic patients with positive Coombs

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JEROMEI. BRODY

tests were agglutinated by the Coombs antiserumin a manner similar to that for erythrocytes. Allcontrol lymphocytes gave a negative reaction withthe antihuman globulin.

DISCUSSION

The most significant observation of this studywas the occurrence of immune adherence whenCoombs-positive leukemic red cell eluates and se-rums were reacted with autologous, neoplasticlymphocytes. This is evidence of an antigen-anti-body reaction and suggests an immunologic re-lationship between these substances as demon-strated by the IA technique.

In view of the fact that the lymphocyte is acomplex mosaic of proteins, lipids, and carbo-hydrates and because various agents such as cold,heat, X-ray, and microorganisms have been postu-lated as irritants for conversion of normal tissueconstituents into antigens (20), it is conceivablethat this cell, after being taken up by the reticu-loendothelial system, might function as an anti-gen and provoke an autologous immunologic re-sponse in a previously unresponsive organism.In this way the abnormal lymphocyte may be re-sponsible, in part, for the stimulation of these ab-normal globulins. It is of particular importance,therefore, that immune adherence did not occurwhen Coombs-positive red cell eluates from thecontrol patients with peptic ulcer and cirrhosiswere reacted with their own lymphocytes. It wasof related interest, however, that two Coombs-positive red cell eluates and serums were reactivewith control lymphocytes. These latter two ob-servations suggest that nonleukemic lymphocytesare not autoantigenic under non-neoplastic con-ditions but that normal and leukemic lymphocytesmay contain cross-reacting antigens.

Although an immunologic relationship has beendemonstrated by the immune adherence methodbetween these Coombs-positive globulins and theintact neoplastic lymphocyte, it is not apparentwhether these globulins are directed against thewhole autologous lymphocyte, per se, or againstits individual integral components. The possibilityexists, therefore, that these globulins are whollyor in part stimulated by an externally introducedantigen that is retained and transported by theleukemic lymphocyte which then becomes a con-jugated antigen (21). The process of conjuga-

tion, during which the original antigen or struc-ture undergoes some alteration, creates new cate-gories of antigen specificity to which patients withleukemia may so respond as to produce abnormalantibody globulins. For example, a virus (22) orvirus-like agent (23), containing or composedof foreign protein, may combine with the autolo-gous, leukemic lymphocyte and contribute to oraccelerate its malignant transformation. As aparallel example, it has been suggested that theautoantibodies and autoimmunity of ulcerativecolitis, demonstrated as tissue precipitins, may re-sult from antigenic stimuli provided by the greatnumber of microorganisms known to invade thetissues in this disease (24).

An alternative hypothesis may be that the redcell, which itself may be altered in leukemia, anti-genically stimulates these abnormal lymphocytesto produce red cell antibodies. There are severalobservations that do not support such an ap-proach, however, but do not rule it out completely.First, it postulates that the lymphocyte synthesizesantibody. While this may be true, in part, thereis no complete agreement at present as to whichcells are responsible for this function (25). Theplasma cell (26), reticuloendothelial cell (27),and mononuclear cell of lymphoid origin (28)have all been implicated in this activity. Fur-thermore, it presupposes that these cells are im-munologically competent, a thesis not in accordwith the increased homograft tolerance of pa-tients with lymphoma and related disorders (29),their diminished de novo antibody synthesis (30),

land their cutaneous anergy (31).The observation that Coombs-positive red cell

eluates did not react with autologous erythrocytesindicates the failure of an antigen-antibody union,as determined by the IA technique, and suggeststhe absence of an immunologic relationship be-tween the red cell and the globulin. This, ofcourse, still does not rule out erythrocyte antibod-ies or antibody-like substances which escape de-tection by this particular serologic assay. How-ever, it should be pointed out that lack of reactivityof the autologous Coombs-positive erythrocyte isnot necessarily due to the fact that its surface isglobulin coated, because similar red cells have beenemployed successfully as indicators in consistentlyreproducible starch antigen-antibody IA titrations(32).

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REACTIVITY OF RED CELL ELUATESAND SERUMS

The variability of the antibody reactivity asnoted in Table IV is consistent with the observa-tion that, during the evolution of malignant cells,associated histochemical differences may effectcellular antigenicity. Furthermore, the breadth ofleukoagglutinin activity stimulated by leukocytesobtained from a single donor (33) favors the con-cept that leukocytes have a complex antigenicstructure. In addition, the patient with the ma-ture-type leukemic lymphocytes had a negativeCoombs test and his eluate and serum were notreactive in immune adherence, suggesting that thepotential autoantigenicity of the leukemic lympho-cyte is partially dependent on its increasing im-maturity. In Patient 4, whose disease may haveoriginated as a splenic lymphocytic lymphoma, thedisparity between antibody reactivity and the pro-nounced morphologic cellular immaturity in theperipheral blood may be associated with the de-layed discharge of neoplastic cells into the periph-eral blood.

The Coombs-positive eluates and serums weremore reactive in patients with severe, progressive,and terminal disease than in less seriously ill pa-tients. This implies that during the uncontrolledor resistant phase of leukemia more of these abnor-mal proteins may be produced, perhaps because theimmune mechanism of such patients is capable ofrecognizing, and therefore regarding as foreign, adegree of change in an autologous cell that is ig-nored by the immune mechanism of healthy indi-viduals.

Diminution of eluate and serum reactivity wasnoted in patients treated with adrenocortical ste-roids and chlirambucil. The precise method bywhich the adrenocortical steroids ameliorateCoombs-positive acquired hemolytic anemia is notknown, but several explanations may be attempted.The steroid hormones perhaps exert their influ-ence by accelerating the rate of removal of lymph-oid cells or by inhibiting their production (34)and therefore decreasing the amount of antigenicmaterial to which the immune mechanism is ex-posed. Another factor may be that the adreno-cortical steroids influence the antibody-producingmechanism itself and prevent its responsivenessto antigenic stimuli (35). Finally, the efficacy ofsteroids may depend on augmenting effective bonemarrow erythropoiesis (36). The reactivity-re-ducing mechanism of chlorambucil, a polyfunc-

tional alkylating compound, may be intimately re-lated to its ability to denature or inactivate cellu-lar nucleoprotein (37). The drug may reduce thetotal amount of potentially antigenic material oralter the biologic properties of the neoplastic cellso that its antigenicity is decreased.

There are four other observations regardingthese globulins which require comment. Patient4, prior to therapy, and Patient 8 had only indirectpositive Coombs tests. It may be suggested thatthis is unusual and represents an acquired isohe-magglutinin as the result of blood transfusions.However, it is more likely that this is a type ofcold antibody, often found in reticuloendothelialmalignancies, which did not bind to the red cellat body temperature or is a globulin similar to themacromolecular cold antibody (38). The fact thatit resensitized type 0, Rh negative erythrocytesmakes it less probable that this is a type-specificantibody stimulated by isosensitization. The as-sociated change in the location of the globulins inPatients 3 and 4 after therapy indicates not onlythat their reactivity was modified but also sug-gests that the red cell membrane or the structuresupon which globulin binding is dependent mayhave been altered by the adrenocortical steroidsand the chlorambucil. The agglutination of leu-kemic lymphocytes from patients with Coombs-positive acquired hemolytic anemia by the Coombsantiserum suggests that these cells, as well aserythrocytes, are coated with abnormal proteins.Finally, the observation of the marked reactivityof two of the eluates (dilution to 1: 16,384) doesnot necessarily imply that this is characteristicof the eluates, per se, or that the eluate globulinsdiffer in potency from the serum globulins whichartificially resensitize erythrocytes in vitro. Itmore likely suggests that for reasons as yet un-disclosed, under certain circumstances, more glob-ulin coats the erythrocyte than remains floatingfree in the serum. This is supported by instances,as noted in Table IV, in which the serum reac-tivity was obviously higher than that of theeluates.

These observations do not completely explainthe genesis of the Coombs-positive globulins inthis type of acquired hemolytic anemia. It is pos-sible that the globulins will cross react with anti-gens that are as yet unknown, aside from the in-tact leukemic lymphocyte. In addition, they may

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react more strongly with some fractions of thelymphocyte and be weakly or nonreactive withothers. These experiments, however, representanother approach to an unsolved problem and mayhelp to explain the immunologic alterations inthis syndrome.

SUMMARY

1. The most important observation of this studywas that the Coombs-positive red cell eluates andserums of patients with acquired hemolytic ane-mia and chronic lymphocytic leukemia reacted inimmune adherence with autologous leukemiclymphocytes. This is evidence of an antigen-anti-body reaction and suggests an immunologic rela-tionship between these substances, as demonstratedby the immune adherence method.

2. On the other hand, red cell eluates and serumsdid not react with autologous erythrocytes nor didcontrol, nonleukemic red cell eluates react withcontrol, nonleukemic lymphocytes, suggesting theabsence of a similar relationship between theseelements.

3. Mild reactivity of two Coombs-positive redcell eluates and serums with control lymphocytessuggests that normal and leukemic lymphocytesmay contain cross-reacting antigens.

4. It is suggested that the autologous leukemiclymphocyte may be one of the factors that stimu-late the development of the Coombs-positive glob-ulins.

REFERENCES

1. Fudenberg, H. H., and Kunkel, H. G. Physicalproperties of the red cell agglutinins in acquiredhemolytic anemia. J. exp. Med. 1957, 106, 689.

2. Kidd, P. Elution of an incomplete type of antibodyfrom the erythrocytes in acquired haemolyticanaemia. J. clin. Path. 1949, 2, 103.

3. Chaplin, H., Jr., and Cassell, M. Studies on anti-eluate sera. I. The production of antiglobulin(Coombs) sera in rabbits by the use of antibodieseluted from sensitized red blood cells. Vox Sang.(Basel) 1960, 5, 32.

4. Davis, L. J. Symptomatic haemolytic anaemia; re-port of 4 cases. Edinb. med. J. 1944, 51, 70.

5. Stats, D., Rosenthal, N., and Wasserman, L. R.Hemolytic anemia associated with malignant dis-eases. Amer. J. clin. Path. 1947, 17, 585.

6. Jonsson, U., Hansen-Pruss, 0. C., and Rundles,R. W. Hemolytic anemia in myelogenous leu-kemia with splenectomy. Blood 1950, 5, 920.

7. Kyle, R. A., Kiely, J. M., and Stickney, J. M. Ac-quired hemolytic anemia in chronic lymphocytic

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