Quantitative Immunology of Immune Hemolytic Anemia

Transcription

1 Quntittive Immunology of Immune Hemolytic Anemi II. THE RELATIONSHIP OF CELL-BOUND ANTIBODY TO HEMOLYSIS AND THE EFFECT OF TREATMENT WENDELL F. ROSSE From the Duke University Medicl Center, Durhm, rth Crolin 2776 A B S T R A C T The concentrtion of cell-bound nd serum ntibody ws determined in series of ptients with wrm ntibody immune hemolytic nemi by determining the mount of Cl fixed to the cells by nti-igg. This ws compred to the rte of hemolysis s determined by hemoglobin concentrtion nd reticulocyte count, or the endogenous production of crbon monoxide. The rte of hemolysis ws, in generl, proportionl to the concentrtion of cell-bound ntibody. In splenectomized ptients, the rte of hemolysis ws very much less thn in unsplenectomized ptients for given concentrtion of cellbound ntibody. When prednisone ws given, three effects were noted: () t high doses of drug, the concentrtion of cell-bound ntibody decresed rpidly nd the concentrtion of serum ntibody incresed, suggesting tht the ffinity of ntibody for ntigen hd been ltered; (b) in ptients chieving remission, the concentrtion of serum ntibody fell to low levels but rose gin if the dose of prednisone ws insufficient; (c) in one ptient, prednisone ppered to inhibit sequestrtion of highly sensitized cells. INTRODUCTION The commonest form of utoimmune hemolytic nemi is chrcterized by the presence of ntibody of the IgG immunoglobulin clss on the red cell (1). These ntibodies chrcteristiclly rect with red cell ntigens t body temperture, nd they fix complement reltively inefficiently since two ntibody molecules in juxtposition re required to initite complement sequence (2). The exct mechnism by which the red cells re destroyed in this syndrome is not completely understood. Recently it hs been demonstrted tht the presence of the IgG ntibody on the surfce of the red cell my cuse it to dhere to monocytes nd splenic mcrophges (3, ). In this rection, portion of the red cell mem- Received for publiction 2 July 197 nd in revised form 7 December 197. brne my be removed, resulting in spherocytosis. Sequestrtion of the cells by elements of the reticuloendothelil system, especilly the spleen, is probbly responsible for the ultimte destruction of the cells. When complement is fixed, direct cytolysis my ccount for smll prt of the cellulr destruction. The rte of hemolysis in ptients with hemolytic nemi due to wrm-recting ntibody my vry gretly, nd reltionship between the degree of nemi or the rte of hemolysis nd the mount of ntibody on the red cell surfce hs been suspected but hs not been estblished (5, 6). The investigtion of this reltionship hs been hmpered by the difficulty in determining the mount of ntibody on the cells nd in the serum of ptients with this disese. The mount of ntibody on the cell hs been estimted by elution (7), with rdiolbeled ntiglobulin (6) nd, more recently, by n indirect estimte of the mount of nti-igg which dheres to cells coted with IgG (8). In the present studies, we hve undertken to estimte the mount of ntibody present on the red cell surfce by mesuring the fixtion of the first component of complement, C1. Since IgG utoimmune ntibodies fix complement only very poorly, if t ll, we hve rected IgGcoted cells with rbbit nti-igg. The combintion of two nti-globulin molecules brings bout the fixtion of C1 molecule (9). The concentrtion of nti-globulin cn be djusted so tht the direct proportion exists between the number of Cl molecules fixed nd the number of IgG utoimmune ntibody molecules present on the red cell. By this method, we re ble to derive minimum but proportionl estimte of the number of ntibody molecules present on the surfce of the red cell. The rte of hemolysis in these ptients ws mesured by modifiction' of the endogenous crbon monoxide 1Logue, G. L., W. F. Rosse, W. T. Smith, H. A. Slzmn, nd L. A. Guttermn Endogenous crbon monoxide production mesured by gs phse nlysis: n estimtion of heme ctbolic rte. Submitted for publiction. 73 The Journl of Clinicl Investigtion Volume

2 method of Coburn, Willims, nd Khn (1). This method ws chosen since frequent seril estimtes of the red cell survivl time were needed in order to follow the effect of therpy. Using these methods, we hve investigted the effect of ntibody coting of the red cells, the effect of splenectomy in these ptients, nd the effects of drenocorticl steroid therpy. METHODS Ptients. 3 ptients with utoimmune hemolytic nemi, wrm ntibody type, in whom the direct nti-globulin test (Coombs test) ws positive using nti-igg ntiserum, nd in whom the mount of IgG on the cells could be quntitted by the methods described below, were studied. A summry of the clinicl course nd lbortory dt of these ptients mentioned specificlly in the text re given in Tble I. The ntibodies present in serum or elutes did not hve specificity for known red cell ntigens when tested on pnel of cells or when bsorbed with cells of known Rh genotype. Blood smples. Whole blood or wshed red cells obtined fter defibrintion were mixed with one-qurter volume of Alsever's solution (11). Since preliminry tests indicted tht the mount of ntibody on the red cell surfce decresed during storge, smples were nlyzed no lter thn 2 hr fter being drwn. Serum ws obtined either by removing the serum fter defibrintion or by llowing the clot to retrct t room temperture for 1-2 hr. The serum ws heted t 56C for 3 min nd stored t - 2'C until use Ḃuffer. Veronl-buffered sline (VBS), ph 7., ws mde ccording to the method of Myer (11). When buffers of reduced ionic strength were required, VB S ws mixed with isotonic sucrose buffer, ccording to the directions given in reference 12. Antiserum. Rbbit ntiserum to humn immunoglobulin G (IgG) ws obtined by repeted injection of rbbits with purified IgG from ptients with multiple myelom. In some instnces, nti-whole serum obtined from Ortho Phrmceuticl Corp., Rritn, N. J. ws used. The nti-igg molecules were IgG immunoglobulins. The C1 fixtion nd trnsfer test. The Cl fixtion nd trnsfer test ws performed ccording to the methods given in references 2 nd 9. In order to test the mount of cellbound ntibody, the cells of the ptient were wshed three times in VBS..25 ml of pcked cells ws suspended in 9. ml of 6%o VBS-sucrose nd the suspension ws djusted so tht 1: 25 dilution hd n opticl density of.21 t 51 mi. This stndrd suspension contins pproximtely 2.2 X 18 red blood cells per ml..1 ml of this suspension ws mixed with.2 ml of n pproprite dilution of rbbit nti-igg nd incubted for 3 min t 3'C. The dilution used ws such tht slope of the plot of the logrithm of mount of primry ntibody ginst the logrithm of the mount of Cl fixed ws equl to 1 (see references 9 nd 13). The cell suspension ws wshed twice with 8 ml of 6o VBS-sucrose t bout 5 g. After the pellet hd been thoroughly dispersed with vortex mixer,.3 ml of Ci, which hd been dsorbed twice with * volume of pcked humn red cells, ws dded nd the mixture ws incubted t 3C for 3 min. After incubtion, the cells were wshed, trnsferred to new set of tubes, nd wshed three further times with 8 ml of 6%o VBS-sucrose t ech wshing. After dilution in VBS, the mount of CY fixed ws determined s outlined. In order to determine the concentrtion of ntibody in serum or elute solutions, norml cells from single O+ donor were wshed nd stndrd suspension ws mde..1 ml of cells ws mixed with.2 ml of serum nd the mixture ws incubted for 3 min t 3 C. The cells were wshed twice with 6% VBS-sucrose nd.2 ml in pproprite dilution (see bove) of rbbit nti-igg ws dded. After incubtion for 3 min t 3 C,.3 ml of Cl ws dded nd the cells were further treted s in the quntittive direct Coombs test. The results were expressed s molecules Cl fixed per red cell. The constrints upon this method of determining ntibody in solution re given in references 2 nd 9. When ppinized cells were required for the quntittive indirect Coombs test, they were prepred by incubting equl volumes of stndrd suspension of cells with 1%yo cysteine-ctivted ppin, t ph 7. for 2 min. After incubtion, the cells were wshed twice nd resuspended in the originl volume of 6% VBS-sucrose buffer. Red cell survivl time. Red cell survivl time ws mesured by the endogenous production of crbon monoxide, by use of gs phse nlytic system described by Logue et l. The red cell life spn estimted from crbon monoxide ws clibrted with the red cell life spn determined by the diisopropylflourophosphte (DFP3) method of Cline nd Berlin (1). The vlues expressed re for the corrected men red cell life spn; norml is 1-13 dys. RESULTS Reltionship between hemolytic rte nd cell-bound ntibody. The mount of cell-bound ntibody ws determined in 25 ptients with immune hemolytic nemi, wrm ntibody type, before tretment with steroids, splenectomy, or immunosuppressive gents. The reltionol x , Reticulocyte Count * 15.-D *>. Spenectomized & O. n._ J 15 Hemoglobin, g/ioomi FIGURE 1 The reltionship between the hemoglobin concentrtion in peripherl blood nd the mount of ntibody on the circulting red blood cells s mesured by Cl fixtion in the presence of nti-igg ntibody. ne of the ptients were receiving drenocorticosteroid or immunosuppressive therpy t the time of the test. The results from ptients who hd undergone splenectomy re shown in the boxed re. Reltionship of Cell-Bound Antibody to Immune Hemolysis In Vivo 735

3 ) u Go ~ ) L, - et..e._' Cd -n.q.(n: ) Aw.U :9 ) o 8 d : u ) o g 2) L US o._ id 93 oo.2.~~~~~~~~~~ X X z o - 3 co XX U) p X p p) :z; o P S go '.I )) E c o c e cc *-F. e ) u,) X.2.2 U..5) ) U +.u U v I I I + ' Q - *. e es U U US. U. co i. 8i o o ' NNCC t'- WI) i)u - - -s N N oo ') r N O ' eu (U O O r N o N N - N.o US.t. l * 1._ ) oo e ' co in) N N 1 U U ii -. W NO ') W) In NO (d *to ) CD..1 fi - U UA \ ) ~~du; N) ~ U) c.s~ '. o X o. ' o) o m NCc NO ' m ' N 'I I Ns N N N~ r _ o m N N 'o ') U) r N U) u) ' N (n ~T.~-. " ~T(z (z T ir~ ~ (Z N i m Xm Y pi; ui V5, 3 b i 736 W. F. Rosse

4 IQ 12 _ g _ u-6. 3 r I - OgO Red Cell Men Life Spn, Dys FIGURE 2 The reltionship between men red cell life spn, mesured by the endogenous production of crbon monoxide, nd the mount of ntibody on the circulting red blood cells, s mesured by fixtion of C1 in the presence of nti- IgG ntibody. The results for splenectomized ptients re shown in the boxed re. ship of the mount of ntibody on the cell to the hemolytic rte, s judged by either the decrese in hemoglobin nd increse in reticulocyte count or by the production of crbon monoxide is shown in Figs. 1 nd 2. Although smll mounts of cell-bound ntibody, in some instnces not redily detectble by this ssy, pper to reduce the red cell life spn s mesured by endogenous - 6 x 1l C._ 6 t '-._ 2 C-) -2 Pt.: C.H. Unsplenectomized Reticulocyte Count - * 15- % * >%. _ * Hemoglobin, g/looml FIGURE 3 The reltionship between hemoglobin concentrtion in the peripherl blood nd concentrtion of cell-bound ntibody in ptient C. H. during drenocorticosteroid therpy. crbon monoxide production, mrked reduction in the life spn nd consequent nemi occurs when lrge mounts of ntibody re present on the cell. There is considerble vrition from ptient to ptient in the mount of cell-bound ntibody which effects given mount of hemolysis or dimunition in hemoglobin concentrtion. In individul ptients, the reltionship between the mount of cell-bound ntibody nd the rte of hemolysis or reduction in hemoglobin is more clerly shown (Fig. 3). In eight ptients, the mount of cell-bound ntibody ws mesured before nd fter the onset of hemolytic excerbtion (Tble II). These dt indicte tht the min determinnt for the rte of destruction of red cells in wrm ntibody hemolytic nemi is the mount of cellbound ntibody. * *. *. TABLE I I Chnges in Concentrtion of Cell-Bound Antibody during Excerbtion of Immune Hemolytic Anemi Therpy before Reticulocyte Cell-bound Serum Ptient excerbtion Dte Hemoglobin count ntibody ntibody g/1 ml % M. P. Steroids* G. R. Steroids Chlormbucil B. B C. F R. F. Steroids Splenectomy A. G. Steroids Splenectomy * Usully prednisone. %. Reltionship of Cell-Bound Antibody to Immune Hemolysis In Vivo 737

5 ~~~~r X. >16 2.-S.C x > * C (-) *~~~~~+>. 12 The effect of splenectomy. The reltions the mount of cell-bound ntibody nd the hiemolytic rte in the serum to decrese. When the dose of prednisone s judged by endogenous crbon ws monoxide p roduction or gin rised to 6 mg/dy on dy 25, the cell-bound reduction in hemoglobin ws determined in six ptients ntibody gin decresed (see Fig. 7). who hd been splenectomized (Figs. 1 nd 2). These In five ptients, the rpid removl of cell-bound ntiptients ppered to hve considerbly less hiemolysis *e i for body ws not ccompnied by n increse in the ntibody given concentrtion of cell-bound ntiboody thn p- cell-bound in the serum (Fig. 8). In three ptients, the mount of cell-bound ntibody diminished only slowly over period of 1-2 dys. In one ptient, prednisone Pt. -M. P Reticulocyte Count ppered to be virtully without effect on the concentrtion of cell-bound ntibody. This ptient did not hve -E16 _ I o <2. I _x remission in hemolytic rte s judged by decresed hemtocrit nd continued trnsfusion requirement. * >. l 12 U- ~1Z -8 Count tients who hd not been splenectomized. However, hemolysis occurred when he mount of cell-bound ntibody becme excessive (Fig., Tble II, ptients A. G. nd R. F.). One ptient, M. P., ws extensively studied be- D fore nd fter splenectomy (Fig. 5). The incresed tolernce for cell-bound ntibody fter splenectomy is clerly shown. In five ptients, the mount of ntibody ttched to 51 red cells extruded from the spleen t opertion ws compred to the mount present in the peripherl blood on IL. the sme dy (Tble III). In ech cse, the mount of oontibody on the red cells from splenic blood exceeded tht Rnge Unsplenectomize present on the red cells in the peripherl blood. TePtients The effect of predisone therpy. 15 ptients were given 6 mg of prednisone per dy t the beginning of 2 therpy. In 1 ptients, the mount of ntibody present on the cells fell rpidly In eight of these ptients, this II rpid dimunition of the cellbound ntibody ws ccompnied by n increse in the mount of ntibody present Heh m og-nbin(g/idnd l) FIGURE The reltionship hemoglob between in concentr- in the serum. When tested, the men red cell life spn tion in the peripherl blood nd the concentr~ tion of cell- ws seen to increse s the mount of ntibody decresed bound ntibody in ptient R. F. who hd underrgone splenec- (see tomy. The rnge for unsplenectomized Figs. 6 nd 7). This effect ppers to be function ptients Fig. 1) is shown in the shded re. The ptieint ws tking of the high dose of prednisone given since, in ptient -1 mg of prednisone t the time of the det( =rmintions. R. A. t dy 2, when the dosge of prednisone ws reduced from 6 mg/dy, the mount of ntibody present on ship between the cell begn to increse nd the mount of ntibody Postsplenectomy TABLE I I I Cell-Bound Antibody on Peripherl Red Cells nd on Red Cells Removed from the Spleen fter Splenectomy C.),6~ N Hemoglobin, g /1 ml FIGURE 5 The reltionship between the concentrtion of hemoglobin in peripherl blood nd the concentrtion of cell-bound ntibody in ptient M. P. before nd fter splenectomy. 738 W. F. Rosse Presplenectomy * + o I. 1*.o+;. ;- Molecules of CT fixed per red cell Men red cell life spn Peripherl Splenic Before After Ptient blood blood splenectomy splenectomy dys D. S P. P M. P M.S M. G M. W.* * Ptient with hereditry spherocytosis.

6 A second effect of prednisone is dimunition in the mount of ntibody present in the serum (Tble IV). This effect ws seen in ll ptients undergoing clinicl remission with prednisone tretment. The time required for the concentrtion in serum ntibody to rech low levels rnged from to 87 dys. This time period ppered to be longer for ptients with chronic lymphocytic leukemi thn for ptients not hving this disese. Once the mount of ntibody present in the serum hd fllen, prednisone dosge could be sfely reduced to levels of less thn 1 mg/1 ml in ll ptients chieving this response. Three ptients did not hve mrked dimunition in the mount of ntibody present in the serum. In these ptients, the mount of ntibody on the cells, the mount of ntibody in the serum, nd the hemolytic rte remined high. A third effect of prednisone ws deduced from nlysis of the results of ptient R. A. (see Fig. 6). In this ptient, the mount of cell-bound ntibody present on dy 2 ws pproximtely tht present before the onset of prednisone therpy. However, the men red cell life spn ws pproximtely four times the initil vlue. This suggests tht prednisone my interfere with the sequestrtion of the ntibody-coted red cells by the reticuloendothelil system. DISCUSSION The red cells of ptients with utoimmune hemolytic nemi of the wrm ntibody -type re rndomly destroyed s the result of the immunologicl rections which tke plce on their surfce. In some instnces, complement is fixed. However, in mny, if not most instnces complement is not fixed efficiently since two molecules of the IgG ntibody re needed for the fixtion of single molecule of the first component of complement. Therefore, the gretest proportion of the hemolysis which occurs in these ptients is probbly the result of the presence of ntibody on the cell surfce. The quntittive reltionship between the mount of ntibody present on the cell surfce nd the rte of destruction of the cells hs been somewht obscure in the pst. Jndl nd Kpln showed D-positive red cells sensitized with nti-d nd injected into norml recipients were rpidly destroyed (15). The rte of destruction ws roughly prllel to the mount of ntibody used in sensitiztion. These findings were confirmed nd expnded by the studies of Mollison, Crome, Hughes-Jones, nd Rochn (5). These investigtors used much higher concentrtions of ntibody nd observed much more rpid rtes of red cell destruction. C-) _c -% C ō! M I C w c VCuIJ Cl o E - _=. I I I I c FIGURE 6 The chnges in cell-bound nd serum ntibody concentrtion, hemoglobin concentrtion in the peripherl blood, nd men red cell life spn fter prednisone therpy in ptient B. C. Reltionship of Cell-Bound Antibody to Immune Hemolysis In Vivo 739

7 Dys FIGURE 7 The chnges in cell-bound nd serum ntibody concentrtion, hemtocrit, nd men red cell life spn fter prednisone therpy in ptient R. A. Constntoulkis, Coste, Schwrtz, nd Dmeshek mesured the mount of ntibody on the red cells of ptients with utoimmune hemolytic nemi using n nti- IgG lbeled with 'I nd sought to relte the level of ntibody to the red cell life spn s mesured by 51Cr (6). Although they could demonstrte with single exmple of nti-d tht the mount of ntibody on cells sensitized in vitro ws relted to their life spn, they found no reltionship between the mount of ntibody coting nd destruction rte in ptients with immune hemolytic nemi. They ttributed this lck of correltion to vribility in the vidity of ntibody for the red cell or to fctors other thn concentrtion of ntibody. The present studies suggest tht the concentrtion of ntibody bound to the red cell is, in fct, prime determinnt of the red cell survivl time in these ptients. Although there ws considerble vrition mong the ptients studied, those who were most nemic t presenttion nd/or who hd the gretest production of endogenous crbon monoxide s mesure of heme destruction were those whose red cells were most hevily coted with ntibody. This ppers to be so whether or not the ntibody ws cpble of fixing complement. In individul ptients studied serilly, the red cell life spn ws, in generl, inversely relted to the mount of ntibody present on the red cell. When hemtologic relpse occurred, the concentrtion of ntibody on the cells ws invribly incresed bove levels present during remission, nd when remission occurred, the mount of ntibody ws lower in nerly ll instnces thn during the hemolytic episode. The mechnism by which the presence of ntibody on the cell surfce brings bout the destruction of the cell is not t ll certin. Archer (3) nd Lo Buglio, Cotrn, nd Jndl () found tht ntibody-coted cells were dherent to the monocytes or splenic mcrophges. This ppered to result in engulfment or loss on the prt of the membrne resulting in spherocytes. This dherence phenomenon ppers to be function of the IgG molecule since none of the other immunoglobulins or complement pper to be ble to effect this rection. This phenomenon ppers to be different from erythrophgocytosis since polymorphonucler leukocytes cnnot engulf IgG ntibody-coted cells in the bsence of complement (16). From the present studies, it ppers tht the greter the number of IgG molecules coting the cell, the greter 7 W. F. Rosse

8 likelihood tht the cells will dhere to mcrophges of the reticuloendothelil system. The dt on cells extruded from the spleen might indicte tht more hevily sensitized cells were sequestered preferentilly by the spleen. However, the increse in cell-bound ntibody on these cells might lso be due to locl production of ntibody in splenic cells. The spleen is of prmount importnce in the process of destruction of the red cells in ptients with IgG wrm gglutinin disese. Removl of the spleen reduces the rte of hemolysis in mny ptients. The dt from the ptients studied here suggests tht when the spleen is removed, up to 1 times s much ntibody must be present on the red cell to effect the sme degree of lysis. This would suggest tht the other sites of ttchment of IgG-coted cells within the reticuloendothelil system were less efficient thn splenic sites. When, however, the concentrtion of ntibody on the cell reches sufficient concentrtion, sequestrtion does occur nd the hemolysis results, despite the bsence of the spleen. These findings corroborte single studies by Consttoulkis et l. (6) nd by Jndl nd Kpln (15). The former group found tht cells from ptient with immune hemolytic nemi survived longer in splenectomized ptient thn in n unsplenectomized ptient. Jndl nd Kpln found tht more ntibody hd to be dded to cells in vitro in order to obtin the sme rte of red cell destruction in splenectomized ptient compred to n unsplenectomized ptient. Dys FIGURE 8 The chnges in cell-bound nd serum ntibody concentrtion, hemoglobin concentrtion, nd reticulocyte count fter prednisone therpy in ptient B. B. The drenocorticl steroids hve been known for t lest 2 yr to induce remission in ptients with utoimmune hemolytic nemi of the wrm ntibody type (17). Three mechnisms were proposed for the effect: () dimunition of ntibody production (18), (b) dimunition of sequestrtion (19), nd (c) ltertion of the red cell- Ptient R. F. G. R.* L. R. M. W. C. F. M. P. B. C.* R. A.* S. M. B. B. E. B. J. B.* E. K.* C. H. J. T. TABLE IV Chnges in Cell-Bound nd Serum Antibody fter High Dose Prednisone Tretment (6 mg/dy) in Ptients with Immune Hemolytic Anemi Cell-bound ntibody, molecules of CY/cell of dys until cell-bound ntibody 1 molecules CT or less 16 S > >213t > 92t Incresed ntibody in serum Initil serum ntibody, molecules Cl/cell * Ptients with prior chronic lymphocytic leukemi. t Remission s defined by these prmeters ws not chieved during the lifetime of the ptient of dys to 1 molecules of CT or less > > >213t > 92T Reltionship of Cell-Bound Antibody to Immune Hemolysis In Vivo 71

9 ntibody reltionship (2). Without the quntittive methods used in these studies, it hs been very difficult to test these hypotheses in ptients. Certinly, in experimentl nimls, it is possible to demonstrte dimunition of ntibody production fter tretment with steroids, especilly IgG ntibody of the secondry immune rection (18, 21, 22). Using dilution techniques, Evns, Binghm, nd Boehni, mong others, hve found tht the mount of serum ntibody decresed during prednisone therpy in ptients with utoimmune hemolytic nemi (18). The present studies demonstrte tht the totl mount of ntibody (cell bound + serum) is usully decresed fter prednisone therpy. The time intervl between the initition of prednisone therpy nd the reduction in serum ntibody vries from to 9 dys. This intervl tended to be longer in ptients with chronic lymphocytic leukemi nd reduction in serum ntibody sometimes did not occur until the leukemic process ws brought under control by other chemotherpeutic gents. If the reduction in serum ntibody did not occur, remission ws difficult to obtin nd mintin. This reduction in serum ntibody, when it did occur, could usully be mintined by reltively smll doses of prednisone (1 mg or less per dy). If the dose ws reduced too fr, the mount of ntibody in the serum rose s did the mount of cell-bound ntibody nd incresed hemolysis occurred (see Fig. 7). Clerly the dimunition in totl concentrtion (serum + cell bound) is probbly the most importnt effect of prednisone; how it is brought bout is unknown. An effect of prednisone in decresing sequestrtion my lso be inferred from these dt. In ptient R. A., the men red cell life spn ws longer for the sme or greter doses of cell-bound ntibody when prednisone ws given thn before the initition of tht therpy. This effect of prednisone hs been postulted by Kpln nd Jndl in studies on rts injected with ntibodycoted cells (19). They found tht prednisone decresed the sequestrtion of cells in the reticuloendothelil system other thn the spleen. The present dt do not llow determintion of the importnce of this effect of steroids nor locliztion of the effect to ny prt of the reticuloendothelil system. Prednisone my lter the ffinity of ntibody for the red cell. In more thn hlf the ptients studied, the mount of ntibody on the cells fell rpidly nd the mount in the serum rose upon the initition of high doses of the drug. Where mesured, the red cell survivl incresed in prllel s the mount of cell-bound ntibody decresed. The rise in serum ntibody might likewise be explined by sudden relese of stored ntibody from lymphocytes or other cells. This effect of prednisone ppers to be seen only with high doses of the drug nd probbly ccounts for the rpid, erly meliortion of hemolysis fter the institution of therpy. The bsis of this effect is the subject for further investigtion. ACKNOWLEDGMENTS This work ws supported in prt by NIH Grnt 5R1-CA Dr. Rosse is the recipient of Reserch Creer Development Awrd 5 K CA Hospitliztion of ptients ws crried out on the Clinicl Reserch Unit, Duke University Medicl Center under Grnt M1-FR3. REFERENCES 1. Dcie, J. V The congenitl nemis. In The Hemolytic Anemis. Grune & Strtton, Inc., New York. Prt I. 2. Rosse, W. F Fixtion of the first component of complement (C'l) by humn ntibodies. J. Clin. Invest. 7: Archer, G. T Phgocytosis by humn monocytes of red cells coted with Rh ntibodies. Vox Sng. 1: 59.. Lo Buglio, A. F., R. S. Cotrn, nd J. H. Jndl Red cells coted with immunoglobulin G: binding nd sphering by mononucler cells in mn. Science (Wshington). 158: Mollison, P. L., P. Crome, N. C. Hughes-Jones, nd E. Rochn Rte of removl from the circultion of red cells sensitized with different mounts of ntibody. Brit. J. Hemtol. 11: Constntoulkis, M., N. Coste, R. S. Schwrtz, nd W. 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