Chronic Hemolytic Anemia due to Cold Agglutinins

II. THEROLEOFC' IN REDCELLDESTRUCTION

ROBERTS. EVANS, ELIZABETH TuiNER, MARGARETBINGHAM, andRICHAIRD WOODS

From the Medical Service of the Veterans Administration Hospital and theDepartment of Medicine of the University of Washington School ofMedicine, Seattle, Washington 98108

AB S T RA C T The sera of four patients withchronic hemolytic anemia due to cold agglutininsdeposited C' globulins on normal red cells at 370C.The circulating cells of the patients were heavilycoated with C' complex and were relatively re-sistant to C' hemolysis by cold agglutinin. Suchred cells were removed from the patients' circu-lation at an exponential rate with 51Cr tj thatvaried from 7 to 19 days. Normal red cells wereremoved rapidly by hepatic sequestration duringthe first hours in the patients' circulation. There-after, a slower rate of abnormal destruction oc-curred which was associated with the accumula-tion of C' complexes on the red cell and thedevelopment of resistance to C' hemolysis by coldagglutinin. Normal red cells coated with sufficientC' complex by action of cold agglutinins in vitroto produce resistance to C' hemolysis by coldagglutinins demonstrated varying degrees of im-proved survival during the first hours in thecirculation of three of the patients.

The levels of serum C' were reduced in all fourpatients with chronic hemolytic anemia due tocold agglutinins. Transfusion of large volumes ofnormal red cells into two patients further reducedserum C'. 51Cr-labeled normal red cells survivedlonger after red cell transfusions than before,because of less rapid destrction during the firsthours in the circulation. The reduction in serumC' levels appeared responsible for the improvedsurvival.

Received for publication 13 March 1967 and in re-vised form 4 December 1967.

In subjects without cold agglutinins, the pres-ence of the spleen decreased the survival of redcells from a patient who had previously undergonesplenectomy. Splenic removal also predominatedin the reduced survival of autologous red cells inone patient. Neither hepatic nor splenic mecha-nisms predominated in removing autologousC'-coated cells in the other two patients.

INTRODUCTION

The hemolytic anemia associated with high titersof anti-I cold agglutinins is sometimes inter-mittent, with acute episodes of hemolysis afterexposure to low temperatures. In the majority ofour patients abnormal blood destruction has beenchronic and independent of reduction in body tem-perature. It is probable that the episodes of hemo-globinuria that follow chilling in some patientsare due to complement (C') hemolysis by thesemacroglobulin antibodies which have hemolytic aswell as agglutinating properties (1). The patientswith the chronic form of hemolytic anemia whichwe have studied have not manifested hemoglo-binuria when exposed to low temperatures, despitethe occurrence of acrocyanosis secondary to intra-vascular agglutination. There are two features ofthe disease in such patients which appear to pre-vent acute hemolysis. Serum C' values are usuallylow and the circulating red cells are abnormallyresistant to C' hemolysis in vitro by anti-I coldagglutinins.'

1 The traditional term "cold agglutinins" will be re-tained in denoting these red cell antibodies in this report.

The Journal of Clinical Investigation Volume 47 1968 691

In a preceding report we have presented evi-dence that accumulation of C' globulins by thered cell resulting from repeated transient reac-tions with cold agglutinin at body temperature isthe principal cause of the resistance of the pa-tients' red cells to C' hemolysis in the chronicform of the disease (2). Data presented here indi-cate that incomplete C' complexes, which accumu-late on the circulating red cells at normal bodytemperatures, protect red cells from rapid destruc-tion by C' and cold agglutinin, but lead to a moregradual removal in which splenic sequestrationmay predominate.

METHODS

Patient material

Studies of four patients with chronic hemolytic ane-mia associated with cold agglutinins with anti-I speci-ficity are reported. The basic hematologic and serologicdata appear in Table I.

Case 1. E. M., a 60-yr-old male with high titers ofcold agglutinin, has been reported on previously (3).Splenectomy in August 1962 was followed by improve-ment in hematocrit for 18 months. The patient's red cells,which had exhibited relatively normal morphology andhypotonic fragility before splenectomy, developed sphero-cytosis and a marked increase in hypotonic fragility.After this period of improvement a gradual fall in he-matocrit began. 1 yr later, after prolonged exposure tocold, he was readmitted with gangrene of both lower ex-tremities. Studies reported here were carried out afterthe onset of relapse, before and after recovery from bi-lateral below-the-knee amputations.

Case 2. A. J., an 81 yr old woman, had noted acro-cyanosis for 3 yr but never observed change in color ofthe urine after chilling. Her symptoms remained much

the same during the 3 yr before the study but her coldagglutinin titer had increased from 1: 32,000 to 1: 200,000.Physical examination was without significant abnormali-ties.

Case 3. 0. A., a 76 yr old man, noted cyanosis andpain in his fingers and toes during exposure to cold in1955. These symptoms increased gradually until, in 1964,the hematocrit was 34%, reticulocytes were 4%o, the se-rum bilirubin was elevated, and cold agglutinins werepresent in high titer. The patient then received 10 mg ofChlorambucil daily for 4 months. This was discontinued1 month before our observations. The liver was not en-larged and the spleen was not palpable.

Case 4. B. C., a 73 yr old man who had been losingweight for 1 yr, developed symptoms of gall bladder dis-ease and underwent cholecystectomy. At that time he wasanemic and was found to have cold agglutinins. Therewas no adenopathy and liver and spleen were not palpable.

The first three patients had high titers of cold agglu-tinins associated with acrocyanosis. The fourth had lowerlevels of cold agglutinins and had not noted acrocyanosis.The cold agglutinins of all four patients produced C'hemolysis of normal cells at 30°C; the cold agglutininsof two (E. M. and A. J.) were hemolytic at 37'C withina pH range of 7.2-7.4. The sera of all four deposited C'globulins on normal red cells at 37'C even though C' he-molysis was not produced at this temperature by the seraof two (0. A. and B. C.).

The red cells of all four patients washed at 37'C wereagglutinated by antiglobulin serum with anti-p13 (C'3)and anti-,8de (C'4) activity (2, 4, 5). Agglutination wasnot produced by antisera with anti-yG and anti--yM ac-tivity. The adsorption of "MI-labeled anti-f3-globulin se-rum was always three to four times that adsorbed by nor-mal red cells and within the same range as normal redcells that had been reacted with cold agglutinin and C' soas to produce accumulation of C' globulins (2). The pa-tients' red cells were resistant to C' hemolysis by coldagglutinins at 25'C within the pH range of 7.2-7.4, andalthough slight C' hemolysis occurred at pH 6. 8, the

ABLE I

Summary Data from Patients with Chronic Hemolytic Anemia and Cold Agglutinins

C' hemolysisof pts' cells

tj sur- by cold ag- Uptake 181I-vival of glutinin of labeled anti-#-

Titer cold patients C' E. M.* at globulinPatient Hct. Retic. agglutinin 5"Cr cells levels pH 7.2, 25'C serum:

% C'H5O U§ %Case 1 E. M. 27 3 1:500,000 20 35 0 4.0 XCase 2 A. J. 26 3 1:200,000 16 40 0 4.0 XCase 3 O. A. 37 4 1:32,000 11 70 0 3.6 XCase 4 B. C. 24 5 1:512 7 34 0 4.0 X

* Normal cells have shown 9-52% C' hemolysis under the same conditions.4 Recorded as 1311-labeled anti-,8-globulin activity adsorbed by patient's cells compared to the activity adsorbed bynormal cells.§ Normal levels, 85-125 C'H50 units.

692 R. S. Evans, E. Turner, M. Bingham, and R. Woods

percentage hemolysis was always less than the limits ofvariation shown by normal cells.

The subjects who volunteered for the studies of sur-vival of 'Cr-labeled cells were normal individuals or pa-tients with any one of a variety of disorders. None hadevidence of blood loss or hemolytic activity. Most asplenicsubjects had received previous transfusions. Isoantibodiesfor red cells could not be demonstrated by in vitro tests.Subjects with normal splenic circulation had not beentransfused previously.

Experimental methods

The techniques employed in the isolation and study ofradioiodinated cold agglutinin have been described else-where (1).

Coating of normal cells with C' globulins. The methodsused to accumulate C' complex on normal cells by actionof cold agglutinin are described in a report on the invitro studies of the resistance of such cells to C' he-molysis by cold agglutinins (2). The same technique hasbeen employed under sterile conditions to coat normalcells with C' complex for in vivo survival studies. In laterexperiments, it was demonstrated that the plasma of E. M.collected in acid citrate dextrose (ACD) solution in theproportion of 10 paris whole blood to 1 part ACDcouldbe used as the source of cold agglutinin with productionof only 1-2%o C' hemolysis during the coating of normalcells. The plasma was mixed with 3 volumes of normalserum, and 1 volume of normal cells was suspended inan equal volume of the serum plasma mixture. The rea-gents were mixed at 370C and then cooled to 250C. Thetemperature was again raised to 370C. Alternation of tem-perature was accomplished five times in 30 min. At theend of 30 min, the cells were sedimented at 370C and themixture of plasma and normal serum was replaced. Thisprocedure was repeated for 3 hr. The degree of hemolysiswas measured by comparing the amount of hemoglobinin the supernatant fluid with 100% hemolysis of an equalvolume of red cells. The coated red cells, normal redcells, and the red cells of the patients were tested forsusceptibility to C' hemolysis by cold agglutinin as de-scribed below. Red cells coated with C' complex in vitroby action of cold agglutinin and normal serum will bereferred to as C'1423 cells though it is recognized thatother fractions of C' globulins may also be present (2).

Anti-,8-globulin serum. The production and testingof antisera for human C' globulins, as well as the radio-iodination of the antisera and its use in measuring C'globulins on normal cells, have been described elsewhere(2). The adsorption of radioiodinated anti-8-globulinserum by red cells of patients is compared with the ad-sorption of the antiserum by normal cells and expressedas the ratio of radioactivity of patients' cells to normalcells. Red cells from patients with hemolytic disorders notassociated with immunological abnormalities adsorbedthe same amount of 'I activity as normal red cells.

C' hemolysis by cold agglutinin. 0.1 ml of cells wassuspended in 1 ml of a mixture of 1 part patient's serumcontaining cold agglutinins and 3 parts normal serum.

The sera had been adjusted to pH 7.2 or 6.8 by additionof mineral acid. The cell serum suspension was mixedat 37°C and then incubated at 250C, or at a higher tem-perature if so designated. After 30 min the hemoglobinconcentration was compared to that produced by 100%osmotic hemolysis of an aliquot of the same suspension.

When 'Cr-labeled cells were present, the percentagehemolysis of these cells was determined by comparingthe 51Cr counts in the supernatant hemolysate with thetotal 5'Cr counts of the specimen.

Titration of serum complement. The titration of humancomplement was performed by the method of Ecker, Pil-lemer, and Seifter (6).

51Cr labeling of normal and C'-coated red cells. 10 mlof whole blood was collected with 2.0 ml of ACD solu-tion which served as anticoagulant and a medium to hastenthe labeling of red cells by 51Cr (7). 100 ,uc of 5'Cr as

Na2CrO was added to the blood with constant agitation.The final concentration of the chromium salt was lessthan 2.0 ,ug/ml of blood (8). The suspension was main-tained at 37°C for 30 min before the red cells were

sedimented and the supernatant removed. The cells were

then washed three times at 37°C in sterile isotonic salineand resuspended to the original volume of whole blood.Measurement of cells for injection was done with a cali-brated syringe. The first venous sample was removedfrom the opposite arm 5 min after completion of injectionand at designated intervals thereafter. Dried heparin or

EDTA was used as an anticoagulant for the specimens.Duplicate 2-ml samples were pipetted into disposableplastic tubes and the radioactivity was determined in a

Packard well-type gamma counter. The radioactivity ofthe sample removed at 5 min was used as the referencepoint and the percentage values of subsequent specimenswere plotted against time on semilogarithmic scale.Plasma concentrations of 'Cr were determined after in-jection of some cell suspensions.

Body scanning was performed with a directional scin-tillation counter (Packard auto-gamma spectrometer)with the patient supine. The counts per minute over theliver, spleen, and precordium were expressed as ratiosof liver to precordium counts and spleen to precordiumcounts according to the method of Jandl, Greenberg,Yonemoto, and Castle (9).

RESULTS

Survival of the patients' red cells in the autolo-gous circulation and in the circulation of normaland asplenic subjects. The red cells of E. M.(case 1) labeled with 51Cr and washed at 37°Cwere observed for survival in vivo in four sepa-rate experiments. Survival of aliquots of labeledcells in the patient and other subjects withoutspleens was compared with survival in normalsubjects. The results of these studies are sum-

marized in Table II. The tj of survival was alwaysshorter in the normal subject than in the patient

Chronic Hemolytic Anemia due to Cold Agglutinins 693

TABLE I Itj of Survival of 5'Cr-Labeled Cells of E. M. in the Autologous

Circulation and in the Circulation of Subjects withand without Normal Splenic Circulation

at of survival

Patientwith

Expt. spleen Asplenic NormalNo. absent subjects subjects

days1 20 13 52 17 33 19 - 74* 19 26 13

* This survival study was started after the patient re-ceived 2 U of red cells.

or asplenic subject. Surface counting in the fournormal subjects demonstrated that the spleen wasthe principal site of sequestration of the 51Cr-labeled cells in the two with the shortest survival.Splenic localization was not demonstrated in theother two. It should be noted that the red cellsof E. M. were spherocytic and susceptible tohemolysis in hypotonic solution. This change inshape had appeared during the partial remissionafter splenectomy.

The red cells of A. J. (case 2) and B. C. (case4) exhibited normal survival in both normal andasplenic subjects. The tj survival of autologouscells was 16 days for A. J. and 7 days for B. C.There was slight parallel increase in both spleen-precordium and liver-precordium ratios in A. J.,whereas the spleen-precordium ratios increasedsignificantly in B. C. Despite the relatively lowtiter of cold agglutinins in the latter patient, thetj of 7 days exceeded the rate of removal ofautologous cells of any other patient in the series.

Survival in patients of normal red cells and nor-mal red cells coated with C' complex in vitro.When 51Cr-labeled group 0 I-positive cells from anormal donor were given to E. M. (case 1) therate of removal was rapid. About 50% of the redcells remaining in the circulation 5 min after in-jection was removed at the end of 1 hr. The rateof disappearance then diminished and the remain-ing cells were removed at an exponential ratewith a tj of 12-14 days (Fig. 1). A week later,cells from this donor were coated with C' globu-lins by incubation with cold agglutinin and whole

serum. Approximately 7% hemolysis had oc-curred during the 3 hr of incubation. The residualcells were resistant to C' hemolysis by cold agglu-tinins and showed a slight increase in hypotonicfragility. The survival of the C'1423 cells is com-pared with the survival of the normal cells andsurvival of autologous cells in Fig. 1.

After injection of the normal red cells 10% ofthe 51Cr counts present in whole blood at 5 mmwas in the plasma as opposed to 1.5%o with C'1423cells. Blood volume determinations based on the5-min 51Cr counts of whole blood were 3200 and3500 ml for the normal and C'1423 cells respec-tively. The higher plasma counts after the injec-tion of normal red cells indicate that appreciableintravascular hemolysis occurred without clearanceof the 51Cr, though some of the plasma counts mayhave been contributed by C' hemolysis duringcollection before mixing with EDTA.

This experiment was repeated 7 months laterwhen the hematological status was unchanged.C'1423-coated cells from the donor used in theprevious experiment were administered before thenormal cells. The survival curves of each wereapproximately the same as those shown in Fig. 1,indicating that the order of administering thenormal and C'1423-coated red cells did not influ-ence their survival.

Similar observations were made with normal

10090

70 e.

*2 50

WW 40

@ 30

........... Pts. Cells20

Normal Cells

C' 1423 Cells

10 * . I * * I * I2 4 6 8 10 12 14 16 18 20

Days

FIGURE 1 Survival of normal group 0 I-positive cellsin E. M. and survival after the cells were coated withG'1423 complex through the action of cold agglutinincompared with survival of autologous cells.

694 R. S. Evans, E. Turner, M. Bingham, and R. Woods

Normal Cells

--- C' 1423-Coated Colls

I I I I I I I I a I I I

1 2 3 4 5 6 7 8 9 10 11 12

Days

FIGURE 2 Survival of normal group 0 I-positive cells in A. J. and survival, of cells fromthe same donor after coating with C'1423 complex.

group 0 I-positive cells in patient A. J. (case 2).More than 50% of the normal cells was removedin 40 min. Thereafter, the rate of removal was

more gradual with a tj of about 5 days. 6 wk afterthis survival curve, cells from the same donorwere coated with C' globulins by incubation withACDplasma of E. M. and normal human serum.

There was minimal hemolysis during preparationand the cells were demonstrated to be resistant to

C' hemolysis by cold agglutinin at pH 7.2. TheseC'1423 cells did not undergo the initial period ofrapid destruction since 100% of the counts pres-

ent at 5 min was present at 3 hr (Fig. 2). Therate of destruction then corresponded to the sec-

ond, slower phase of destruction of the normal redcells with a t4 survival of approximately 5 days.6 wk later, when the hematologic status of the pa-

tient was unchanged, 51Cr-labeled cells from the

same donor were again injected without priortreatment with C'. The rate of destruction dupli-cated the first survival of the normal 5lCr-labeledcells. An increase in the liver-precordium ratiooccurred during the rapid removal of normal cells,but did not occur during removal of the C'1423cells.

When the normal red cells were given, approxi-mately 10%o of the 51Cr counts present in wholeblood was in the plasma at 5 min as opposed to0.5%o with the C'1423 cells. Blood volumes calcu-lated from the 5-min samples varied from 2400 to

2800 ml for this 40 kg woman. Measurements ob-tained with autologous red cells were within thisrange.

At a later date, the observations of red cell sur-

vival were repeated except that red cells coatedwith C' globulins by the action of cold agglutininsof A. J. were given first. The tj surival of theC'1423 cells was 4 days, whereas that of the nor-

mal red cells was between 1 and 2 days.Additional observations of red cell survival in

these patients were of interest. Red cells coatedwith C' complexes by the action of cold agglutininand zymosan-adsorbed serum (R3) were removedfrom the circulation of E. M. as rapidly as normalred cells even though they were resistant to C'hemolysis in vitro. Red cells coated with C' globu-lins by incubation with cold agglutinin and one-

third the amount of normal serum usually em-

ployed did acquire a coating of 8-globulins butwere not significantly more resistant than un-

coated cells to C' hemolysis in vitro. These red cellswere removed from the circulation of A. J. almostas rapidly as normal red cells with 32% removedin 1 hr and a t1 of survival of 2 days. These ex-

periments indicate that insufficient coating of C'globulins, whether through removal of some frac-tions of the third component or reduction in totalC' available, failed to improve red cell survival.

The survival of group A I-positive normal cellslabeled with 51Cr was also observed in patient

Chronic Hemolytic Anemia due to Cold Agglutinins 695

so706050

40

30

20

a

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0Sb

10 -.&^ I

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0. A. (case 3). 30%o of the counts present at 5min was removed at the end of 2 hr. Thereafter,the rate of removal was relatively slow but con-tinued to be abnormal with a t4 of 10 days. At alater date, 51Cr-labeled C'1423-coated cells fromthe same donor were injected. 20%o of C'1423cells was removed at the end of the first 2 hr.Thereafter, the rate of removal approximated theremoval of the normal cells. The tj survival wasalso 10 days. The ratios of liver-precordium andspleen-precordium counts during the two observa-tions of red cell survival in case 3 are shown inFig. 3. During the initial period of removal of thenormal cells the liver-precordium ratio increasedrapidly. WhenC'1423 cells were injected the liver-precordium ratio did not increase, a finding whichsuggests a different mechanism of removal.

Survival of 51Cr-labeled cells before and after ad-ministration of red cells from 2 U of blood. Pre-vious observations had indicated discrepancies inthe rate of destruction of transfused cells in case 1.51Cr-labeled normal cells had been removed rap-idly with a half-life of less than 24 hr; yet red celltransfusions were given without reactions and thehalf-life of these cells was measured on one occa-

0I=a

DC

aI.'-I

1.5 I

1.0

0.5

0

Normal Group A Cells

sion by the Ashby technique to be 19 days orroughly one-third normal. This finding suggestedthat the mechanism for removal of normal cellsby cold agglutinin was limited and that the rapidremoval of 51Cr-labeled cells might not occur afterthe administration of 500 ml of red cells.

5 ml of red cells was removed from each of 2pints of group 0 I-positive blood within 4 hr ofwithdrawal from the donors. The cells were labeledwith 51Cr and injected into the patient. 48%o of thecounts present at 5 min was removed from the cir-culation at 30 min and 52% was removed in 16hr. During the next 6 hr, a small but significantrise in counts occurred, indicating that some se-questered cells had been returned to the circula-tion. An additional 5 ml of cells was removed fromeach of the 2 pints of blood for 51Cr labeling. Theremaining cells, washed to remove C', were ad-ministered without incident. After completion ofthe transfusion, the second suspension of 51Cr-labeled cells was injected. If one assumes that the51Cr-labeled cells already present in the circula-tion were now being removed at the constantrate observed after 24 hr in previous studies, onemay calculate the survival of this second suspen-

a

)o

Liver-Precordium Ratio- - - -Spleen-Precordium Ratio

C' 1423-Coated Group A Cellsa

U.

.e

r - - I I I I I I

1 2 3 4 5 6 7 8 9 10 11

Days

FIGURE 3 Liver-precordium and spleen-precordium ratios during the survivals of '1Cr-labeled normal and C'1423-coated I-positive red cells in the circulation of 0. A.

696 R. S. Evans, E. Turner, M. Bingham, and R. Woods

10090so70

a

0

a.

40

30

20

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0._

aI-S

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a

0

.z

._.

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u110mgS

I I a a a I aI i

2 4 6 8 10 2 4 6 8 10

DaysFIGURE 4 Survival of 'Cr-labeled group 0 I-positive cells in E. M. before and after administration ofthe washed red cells of 2 pints of blood from the same donors. The projected survival of the first pretrans-fusion infusion of GCr-labeled cells after 24 hr is shown by the dotted line and is based on data from pre-vious survival curves.

The observation was repeated with normal cells from other donors except that the red cell transfusionswere given 72 hr after starting the pretransfusion survival study.

sion of 51Cr-labeled cells given after the transfu-sion. 13%o of the counts present at 5 min was re-moved at 30 min and 23%7o was removed at 16 hr.Thereafter, the rate of removal was more gradualwith a tj of 10 days (Fig. 4).

The above observation was repeated at a laterdate except that the initial survival curve was ob-served for 3 days to record the beginning of theperiod of more gradual destruction after the first24 hr (Fig. 4). The initial rate of removal ofthe pretransfusion 51Cr-labeled cells was againrapid, with 46%o removal in 1 hr followed by a re-turn of some cells to the circulation at the end of 24hr. The gradual slope of red cell removal then be-gan. After transfusion of approximately 400 mlof red cells, the second aliquot of 51Cr-labeled cellswas given. Initial destruction was more gradualin that 2%o of the counts was removed at 40 minand 12%o was removed at 3 hr.

Similar studies were carried out with case 2.After completion of a survival study of group 0I-positive cells in the patient, she received group

A I-positive cells from 2 pints of blood. A secondsurvival study of the normal group 0 I-positivecells was begun promptly thereafter. As shownin Fig. 5, there was only a slight decrease in therate of removal of group 0 cells. There was noevidence of isoimmunization to the red cells ofthis group 0 donor and when cells from this donorwere coated with C' complex before injection theyshowed the improved survival depicted in Fig. 2.

A second study was carried out with group AI-positive red cells. The red cells of this donorwere found in many in vitro observations to beless susceptible to C' hemolysis by cold agglutininthan the group 0 cells used in the previous ob-servation. The rate of removal of the group A cellswas less rapid (Fig. 5). After transfusion of redcells from 2 pints of blood the survival of cellsfrom the group A I-positive donor was repeated.The initial period of rapid destruction did not oc-cur, and the tj survival of 16 days was equal tothat of the autologous cells.

Measurements of serum C' before and after red

Chronic Hemolytic Anemia due to Cold Agglutinins 697

%%**4b"I'Ab

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-

-

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60

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C

30

A.

20 _ Group 0 Cells

10

Group A Cells

- Before Transfusion--- After Transfusion

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10

Days

FIGURE 5 Survival of group 0 I-positive cells in A. J. (group A) before and after adminis-tration of 400 ml of washed group A red cells and survival of group A I-positive cells beforeand after subsequent transfusion of group A cells.

cell transfusions. Serum C' levels of the patientswere measured before and after the transfusionsof cells from 2 U of blood. There was significantreduction in the depressed levels of serum C', asshown in Tables III and IV. This reduction can beattributed to the adsorption of C' globulins to thered cells by action of the cold agglutinin. Before

TABLE I I IEffect of Transfusing 400 ml of Washed Red Cells

on Serum C' Levels in E. M.*

Patient ControlTime

C'H100 C'H50 C'H1OO C'H50

hrPretiansfusion

24 20 8949 139

0 19 82

Posttransfusion0.1 0 600.5 0 50 42 127

24 0 78

* Recorded in 100 and 50% hemolytic units.

transfusion, all of the circulating cells were ag-glutinated in anti-p8-globulin serum. After trans-fusion, a high proportion of cells was not aggluti-nated; this indicated a lack of 8-globulin (C')coating. 24 hr later the agglutination of the circu-

TABLE IVEffect of Transfusing 400 ml of Washed Red Cells

on Serum C' Levels in A. J.

Patient Control

Time C'H100 C'H50 C'H0oo C'HSO

hrPretransfusion 15 33 61 124Posttransfusion

0.5 0 0 61 12496 0 10

288 8 25 62 123

Pretransfusion 15 24 58 109Posttransfusion

0.1 0 0 68 11024 0 0.6

268 0 5 60 114480 11 33 58 119

698 R. S. Evans, E. Turner, M. Bingham, and R. Woods

TABLE VSusceptibility to Complement Hemolysis by Cold Agglutinin

of Normal Group A I-Positive Cells Labeled with 5"Crduring Survival in the Circulation of A. J.

Hemolysisof 51Cr-

Hemolysis labeledUlCr- of whole cells in

labeled cell samples sample atcell at 25IC, 25-C.

Time survival pH 6.8 pH 6.8

hr % % %0.1 100 5 530.5 90 5 491 88 5 453 84 5 43

24 72 3 1772 61 3 12

144 54 3 6

lating cells in anti-fl-globulin serum was againcomplete.

Levels of serum C' were measured before andafter transfusions of red cells in 10 patients with avariety of hematologic disorders. Change in C'H50units varied between + 3 and -14% with anaverage change of - 3%.

Development of resistance to heMolysis of 5'Cr-labeled cells in vivo. The susceptibility of 51Cr-labeled group A I-positive red cells to complementhemolysis after their introduction into the circu-lation of case 2 was studied by determining thepercentage hemolysis of the 51Cr-labeled cellscompared with the hemolysis of samples of thetotal cell population.

4 ml of red cells of a normal group A donorwas labeled with 51Cr and transfused into the pa-tient. The time of withdrawal of the sample, the percent hemolysis of the cell suspension, and the percent hemolysis of the 51Cr-labeled cells are pre-sented in Table V. The 51Cr-labeled cells weresusceptible to C' hemolysis by cold agglutinin afterinjection, but acquired resistance during -the firstfew hours. After 24 hr the hemolysis of the trans-fused group A I-positive cells had decreased from53 to 17%, even though 72% of the labeled redcells remained in the circulation. The pH of 6.8and temperature of 250C were selected in orderto produce maximum hemolysis of the patient'scells as well as of the injected 51Cr-labeled cells.These observations were repeated at a later datewith similar results.

DISCUSSION

Consistent with observations of others, the redcells of the four patients with chronic hemolyticanemia due to cold agglutinins were agglutinatedby antisera containing anti-,l81- or anti-Pfle-activity(4, 5). There is evidence that the intensity of ag-glutination in anti-fl-globulin serum and the ad-sorption of '31I-labeled anti-fl-globulin serum wasrelated to the amount of C' globulin accumulatedon the red cell surface (2). Normal I-positive redcells, incubated with cold agglutinin and C' forseveral hours, adsorbed 131I-labeled anti-fl-globu-lin serum with the same avidity as the circulatingcells of the patients. The resistance to C' hemoly-sis increased as C' complex accumulated.

The accumulation of C' globulins by the patient'scells was consistent with the demonstration thatthe cold agglutinins of all four patients had a ther-mal range of activity sufficient to cause attach-ment of C' globulins to red cells during incubationat 370C. Several other observations indicated thatC' fixation by cold agglutinins occurred in the cir-culation at normal body temperature. An abruptdrop in serum C' level invariably followed the in-fusion of large volumes of red cells into two of thepatients. In addition, there was evidence that thereaction with C' continued for some time aftertransfusion since it was several hours before allof the circulating red cells were again agglutinatedby anti-fl-globulin serum. Further evidence forgradual accumulation of C' globulins in vivo wasdemonstrated in the development of resistance bythe transfused red cells to C' hemolysis by coldagglutinin (Table V). The normal 58Cr-labeledcells acquired considerable resistance to C' hemoly-sis during the first 24 hr and continued to do sofor several days.

Several lines of evidence indicate that the ac-quisition of C' coating affected the rate and routeof destruction of red cells in the patients' circula-tion. The red cells of the four patients that werelabeled with 51Cr and returned to the circulationwere removed at an exponential rate. The red cellsof two patients, A. J. and B. C., were observed tohave a normal survival time in the circulation ofnormal and asplenic subjects. It is evident that thered cells of these patients, although coated withC' globulins, were not irreversibly damaged andrequired continuing action by cold agglutinin andC' to produce accelerated destruction. Only the

Chronic Hemolytic Anemia due to Cold Agglutinins 699

red cells of E. M. were removed from the circu-lation of normal subjects at an accelerated rate.His red cells had developed a marked spherocyto-sis after splenectomy, suggesting that red cellswhich would have been removed by the spleenwere allowed to remain in the circulation. Thesplenic filter mechanism has been shown to beparticularly effective in removing spherocytic cellsfrom the circulation in a variety of hemolyticstates (10-12).

The injection of normal red cells into the cir-culation of the three patients with chronic hemo-lytic anemia resulted in an initial phase of rapidremoval followed by a second phase of more grad-ual destruction. The slope of the survival curveduring the second phase was exponential and ap-proximated the rate of removal of autologous redcells. The slower second phase of abnormal de-struction was associated with the accumulation ofC' complexes and the acquisition of resistance toC' hemolysis by the surviving red cells. Coatingnormal red cells from the same donor with C'complex by interaction with cold agglutinin and C'in vitro before injection produced slight to markedimprovement in the survival during the first fewhours. The disappearance was then exponentialand also resembled the survival of autologous redcells. It appears from these data that red cells canbe partially protected from the initial rapid phaseof abnormal destruction in vivo by a coating ofC' complex. Since all three patients had been trans-fused before our studies, it is possible that isoanti-bodies for red cells influenced the survival curves.It has been demonstrated that small quantities of51Cr-labeled cells may be eliminated rapidly byisoantibodies that cannot be detected in vitro byavailable serological techniques (13). In some in-stances of suspected isoimmunization a secondinjection of red cells from the same donor resultedin a normal survival; therefore, the improved sur-vival of the C'1423-coated cells after the injectionof normal cells does not entirely eliminate the pos-sibility of isoimmunization (14). However, it wasdemonstrated that a repeat injection of the normalred cells into A. J. after the improved survival ofthe C'1423-coated cells resulted in a typical bi-phasic curve of disappearance identical to the firstsurvival curve. Furthermore, in separate obser-vations with E. M. and A. J. administration ofC'1423-coated cells before administration of nor-mal red cells also resulted in improved survival

of the C'1423 cells. The removal of normal 51Cr-labeled cells from different donors all followed abiphasic pattern which has been observed withsome isoantibodies, most of which utilize C' (10).Differences in rate of removal could be correlatedwith the amount of I antigen (Fig. 5), and thesecond phase of slower removal of normal red cellswhich had become coated with C' approximatedthat of the patient's cells, suggesting that the samemechanism of removal was responsible.

It is of interest that red cells coated with C'1423complex without acquiring resistance to C' he-molysis in vitro because of the reduced amounts ofC' used in preparation were removed from thecirculation almost as rapidly as normal red cells.It is not clear why coating with C' globulins by R3serum failed to produce significant protection invivo even though C' hemolysis in vitro was in-hibited.

C' coating by cold agglutinin also influences thesite of removal of red cells. In all three patientswho received normal cells, counts accumulated inthe liver during the initial rapid phase of removalof cells from the circulation. This finding is con-sistent with the concept that the normal red cellswere susceptible to acute C' damage in vivo. Ob-servations by Jandl and Kaplan (12) and by Cut-bush and Mollison (10) have previously shownthat cells subjected to sufficient damage by otherC'-fixing red cell antibodies were removed at arapid rate and principally by the liver.

Normal red cells that survived the acute phaseof rapid destruction were then removed at aslower rate without predominance of either theliver or the spleen. The autologous red cells ofcases 2 and 3 also failed to accumulate predomi-nantly in either the liver or the spleen, which is inkeeping with the observation of Lewis, Szur, andDacie (15). Only in the fourth patient, B. C., wasthere unequivocal evidence of splenic accumulationof autologous cells. It may be significant that thered cells of this patient had the shortest survivaltime with a tj of 7 days. Failure of organ accumu-lation of radioactivity in the other two patients mayhave been due to the less rapid rate of abnormaldestruction so that accumulation of radioactivityfrom red cell destruction could have been balancedby loss of 51Cr by the organs (9). /

The survival studies of -normal red cells beforeand after transfusions in E. M. and A. J. indicatethat the mechanism that removed cells rapidly had

700 R. S. Evans, E. Turner, M. Bingham, and R. Woods

limitations and could be partially overwhelmed bylarge volumes of red cells. The fall in serum C'levels which followed the introduction of large vol-umes of red cells is the most obvious explanationof this partial suppression of the removal mecha-nism, since there was no reduction in cold agglu-tinin titer. Adsorption of an isoantibody by thelarge amount of transfused cells may be postulatedto be a mechanism of improved survival of thesecond injection of 51Cr-labeled cells. Such a mech-anism should have allowed a normal survival ini-tially followed by rapid destruction as antibodyreappeared, which is the opposite of what was ob-served. Temporary saturation of the reticuloendo-thelial tissue might have been a factor in the im-proved survival of labeled cells after transfusion.If this were the major factor, however, destructionof the damaged cells should have continued asreticuloendothelial tissue recovered. The survivalcurves in case 2 (Fig. 5), particularly those withgroup A cells, suggested that no such suppressionhad occurred.

The destruction of red cells by cold agglutininsappears to be dependent on several variables, in-cluding the thermal range of the cold agglutinin, itsC' fixing activity, the susceptibility of the redcells to C' damage, and the levels of serum C'.The syndrome presented by the four patients de-scribed in this report can occur if the cold agglu-tinins react with red cells at normal body tempera-ture. The circulating red cells become coated withC' globulins and though still susceptible to agglu-tination by cold agglutinins are relatively resistantto acute damage by C'. Such patients have acro-cyanosis from intravascular agglutination whenchilled but may escape acute exacerbations of he-molysis. This mechanism appears insufficient toprevent intravascular hemolysis in all patientsafter they are chilled, since episodes of hemoglobi-nuria have been reported to occur in some (16,17).

ACKNOWLEDGMENTThis study was supported by a grant-in-aid from theU. S. Public Health Service, Project No. A-548, C16.

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The mechanism of resistance of red cells to C'hemolysis by cold agglutinins. J. Clin. Invest. 46: 1461.

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9. Jandl, J. H., M. S. Greenberg, R. H. Yonemoto, andW. B. Castle. 1956. Clinical determination of the sitesof red cell sequestration in haemolytic anemias. J.Clin. Invest. 35: 842.

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