ROBERT F. SCHILLING (From the Department of Medicine, University of Wisconsin Medical School, Madison, Wisc.) cells.

Transcription

1 RADOACTVTY OVER THE SPLEEN AND LVER FOLLOWNG THE TRANSFUSON OF CHROMUM51-LABELLED ERYTH- ROCYTES N HEMOLYTC ANEMA' By LEE L. SCHLOESSER,2 DONALD R. KORST,s DALLAS V. CLATANOFF,2 AND ROBERT F. SCHLLNG (From the Deprtment of Medicine, University of Wisconsin Medicl School, Mdison, Wisc.) (Submitted for publiction April 17, 1957; ccepted June 24, 1957) The beneficil effect of splenectomy in hereditry spherocytosis is well recognized. Selective retention of spherocytes in the spleens of ptients with congenitl ws reported by Emerson, Shen, nd Cstle in 1946 (1). Subsequently, spherocyte "trpping" ws demonstrted in spleens removed from ptients without this disorder (2, 3). n ddition, these bnorml red cells in the spleen hve greter osmotic frgility thn those in the peripherl blood (1). Hm nd Cstle first postulted tht the spleen incresed the osmotic frgility of red cells by intrvsculr stsis in mnner similr to tht produced by sterile incubtion of blood (47). Recently it hs been suggested tht the spleen "conditions" spherocytes for erly osmotic hemolysis or increses their susceptibility to destruction by the mechnicl forces of the circultion (8, 9). Although exct mechnisms of spherocyte destruction remin obscure, splenectomy in congenitl llevites the process. The role of the spleen in red cell destruction in cquired is less thoroughly understood. Splenic hemosiderosis, erythrophgocytosis, nd congestion with red cells suggest the importnce of red cell sequestrtion by this orgn in the pthogenesis of mny of the cquired s. The first good response to splenectomy ws reported by Micheli in 1911 (1). This opertion hs since been performed with unpredictble results in diseses hving little in com- 1 Supported by reserch grnts from the Ntionl nstitutes of Helth; the Office of the Surgeon Generl, United Sttes Army; nd the Grdute School of the University of Wisconsin from funds supplied by the Wisconsin Alumni Reserch Foundtion. 2Trinee, Ntionl Cncer nstitute, during prt of this study. 3 Present ddress: Deprtment of Medicine, University of Michign nd Veterns Administrtion Hospitl, Ann Arbor. mon other thn. Criteri for predicting fvorble result from splenectomy in cquired, or utoimmune, hve not been provided by conventionl lbortory ids. Determintion of reltive orgn distribution of rdioctive erythrocytes is fesible by externl scintilltion counting (11). Jndl, Greenberg, Yonemoto, nd Cstle hve reported method for determining orgn sites of red cell sequestrtion using Cr5l-lbelled erythrocytes (12, 13). They reported splenectomy ws beneficil in one ptient who hd splenic ccumultion of lbelled red cells. A method of determining orgn locliztion of Cr51-lbelled red cells utilizing the rtio spleen: liver rdioctivity s estimted by externl scintilltion counting ws reported from this lbortory in 1955 (14). t ws ssumed tht high splenic uptke of rdioctivity concomitnt with n incresed rte of Crp5 red cell disppernce from the peripherl blood ws evidence for bnorml red cell ccumultion in the spleen. The present report of red cell survivls nd orgn counting dt ws derived from 27 lbelled red cell infusions. Splenic uptke of rdioctivity in ws correlted with response to splenectomy. MATERALS AND METHODS Conventionl lbortory dt n ptients suspected of, determintions of hemoglobin, reticulocytes, hemtocrit, red blood cell count, red cell frgilities (15), nd serum bilirubin (16, 17) were performed by estblished techniques. The method of Dcie ws used for the determintion of utohemolysis of red cells (18). Erythroid: myeloid rtios were computed fter counting 1, nucleted cells in the bone mrrow smer. Averge fecl urobilinogen in four-dy stool collection ws determined by the method of Wtson (19), using Colemn junior colorimeter. One stool urobilinogen determintion ws semiquntittive (2). When these dt showed evidence for 147

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3 1472 L. L. SCHLOESSER, D. R. KOST, D. V. CLATANOFF, AND R. F. SCHLLNG, Cr" red cell survivl studies with orgn counting were performed. Technique of Cr" red cell survivl studies A. Lbelling nd dministrtion of erythrocytes. Sterile technique ws used throughout. Seventy-five to 9 ml. of venous blood were collected by grvity flow into 1-ml. siliconized bottle contining 1 ml. cid-citrtedextrose4 solution. Seventy-five to 1 microcuries Cr" (specific ctivity rnge,.7 to 26. mc. per mg.) s sodium chromte diluted in.85 per cent NCl were dded t once. The mixture ws incubted t 37 C. for 45 minutes with frequent gentle gittion. Following incubtion, 2 mg. scorbic cid were dded to prevent 4Abbott Lbortories, North Chicgo, llinois. The ACD solution hd the following composition per 1 ml.: Dextrose, USP 132 mg.; sodium citrte, USP 25 mg.; citric cid, USP 8 mg. further uptke of Cr" by red cells (21). A single wshing procedure, using cold sterile sline, ws done s follows: The mixture ws centrifuged nd the superntnt plsm ws removed. The red cells were wshed once with sline nd finlly diluted with sline to volume pproximting tht of the whole blood withdrwn. These infusions were designted "wshed." The wshing procedure removed Cr" which ws not bound to erythrocytes so tht more thn 97 per cent of Cr" infused ws red-cell bound. n some instnces the blood ws not wshed nd such infusions were designted "non-wshed." Cr"-lbelled red cells were given intrvenously through n 18-guge needle by grvity drip from the collecting bottle or in 5-ml. volumes by clibrted syringe. B. Determintion of Cr" red cell surivl. Al smples of peripherl blood were collected in blnced oxlte (15) in pproximtely 6-ml. quntities. Pcked cell volumes were determined. Exctly 4 ml. were pipetted into counting tubes clibrted t 4 ml. Plsm ws re- LE Donors nd recipients of Cru-kbelled red ceus Recipient of Cr red cells Spleen size Cr" Averge Donor of Cru red cells (cm. below red-celi rtio Study costl hlf-life spleen Cry: No. Subject Dignosis Subject Digs mrgin) (dys) liver Cr 1 D. D. Norml D. D. Norml D. D. Norml J. H. Norml T. M. Norml T. M. Norml M. M. Norml M. M. Norml D. H. Norml D. H. Norml D. H. Norml L. R. Congenitl J.K. Polycythemi J.K. Polycythemi L.K. Myelofibrosis L.K. Myelofibrosis L. R. Congenitl D. H. Norml L. R. Congenitl L. R. Congenitl Norml E. Z. Congenitl nemi 12 E. Z. Congenitl E. Z. Congenitl V. H. Congenitl V. H. Congenitl V. H. Congenitl V. H. Congenitl 29 (post splenec tomy) 15 N. H. Congenitl N. H. Congenitl

4 SPLENC TRAPPNG OF RED CELLS N HEMOLYTC STATES TABLE -Continued Recipient of Cru red cells Spleen size Cr Donor of Cru red cells (cm. below red-cell Axer rtio Study costl hlf-life spleen Cru: No. Subject Di s Subject Dignosis mrgin) (dys) liver Cr" 16 Norml A. K. Proxysml nocturnl hemoglobinuri 17 A. K. Proxysml A. K. Proxysml nocturnl nocturnl hemoglobinuri hemoglobinuri 18 L. H. diopthic L. H. diopthic Norml P. P. diopthic Norml N. A. Myelofibrosis Norml W. M. Chronic lymphocytic leukemi 22 Norml L. W. L.E.D.* J. T. diopthic J. T. diopthic G. S. Myelofibrosis G. S. Myelofibrosis Norml J. L. diopthic 6-lit J. L. diopthic J. L. diopthic Norml L. Y. Chronic 1 <1 6.4 lymphocytic leukemi * L.E.D. = Disseminted lupus erythemtosus. t The spleen enlrged by clinicl estimte from 6 cm. below costl mrgin to 11 cm. below costl mrgin during the study. moved fter centrifugtion nd the red cells were hemolysed by the ddition of distilled wter to the 4-ml. mrk. Rdioctivity ws mesured in well-type scintilltion counter. Blood smples were drwn in most instnces t frequent intervls for 24 hours following infusion. The smple showing the most rdioctivity during this period ws chosen the "1 per cent smple of Red Cell Cr"." n some, single 2- or 24-hour smple served s the "1 per cent smple." -Subsequent smples were collected t intervls of 1 to 14 dys depending upon the rte of disppernce of rdioctivity from the peripherl blood. Corrections for Cr" decy were obvited by counting both the "1 per cent smple" nd the intervl smples on the sme dy. Corrections for vrition in plsm volumes bsed on heitocrit detmintions were not used, nd no corrections were mde for the elution 1473 of Cr" from red cells. The per cent survivl of Cr" red cells t ny time fter infusion ws given by: CPM per 4-ml. intervl smple CPM per 4-ml. "1 per cent smple" X 1 where CPM = net rdioctivity in counts per minute. Studies were usully crried to 1 per cent survivl of red cell Cr" except when splenectomy terminted the study sooner. Externl orgn counting Orgn counting ws done with n uncollimted scintilltion probe t the time of smple collection for Cr" red cell survivl. With the ptient supine, the pproximte centers of both nterior nd lterl skdn projections of the liver nd lterl projection only of the spleen were estimted by physicl emntion. The detector ws

5 1474 L. L. SCHLOESSER, D. R. KORST, D. V. CLATANOFF, AND R. F. SCHLLNG plced on the skin t these sites so the fce of the crystl ws pproximtely prllel to the body surfce. When multiple determintions of rdioctivity were mde from single orgn t the sme time, the results were verged. A minimum of 5, counts over ech orgn site ws obtined. Rdioctivity vlues over either orgn less thn 1.5 times bckground were not used. The rtio net spleen counts: net liver counts ws determined. Selection of subjects for Cr' studies Twenty-one subjects were studied by Cr'-lbelled red cell survivl nd orgn counting techniques. Five were helthy young men without splenomegly. Studies were mde on two ptients who hd splenomegly without. Rdioctivity studies were mde in 14 ptients who hd clssicl evidence of. With the exception of Ptient N. A., ll subjects with cquired were given drenl steroids prior to splenectomy without significnt benefit. We hve hesitted to dvise splenectomy for cquired when the process ws controlled by steroids. RESULTS nitil dt on ptients with Pertinent clinicl fetures nd lbortory dt on the 14 ptients showing evidence for re presented in Tble. The finl dignosis for ech subject is entered in Tble for completion, but ws not mde in ll ptients prior to lbelled erythrocyte studies. All subjects but N. H. nd L. H. hd incresed (> 28 mg. per dy) fecl urobilinogen. The osmotic frgility of red cells fter incubtion ws bnormlly incresed in ptients with hereditry spherocytosis. ncreses in men frgility upon incubtion were not seen consistently in ptients with cquired. Autohemolysis fter 48 hours of sterile incubtion ws strikingly incresed in hereditry spherocytosis nd in the one subject (A. K.) with proxysml nocturnl hemoglobinuri. n cquired the degree of utohemolysis vried within wide limits from norml to bnorml. Mechnicl frgility ws regulrly incresed only in the hereditry spherocytosis group. Cr5l red cell survivl nd orgn counting There were 26 simultneous determintions of Cr5l red cell survivl nd spleen Cr51: liver Cr5' rtios in the 21 subjects. Five of these subjects received lbelled red cells from suitble donor (homologous), nd t nother time n infusion of their own lbelled red cells (utologous). An dditionl survivl study ws done in one subject (V. H.) fter splenectomy. Pertinent dt on donor red cells nd recipients re shown in Tble. n Figures 1 to 9 dt on Cr5' red cell survivl nd orgn counting for ech recipient re plotted 1 j 8 o 6 cc - z 3 4 X 2 1 Cr5 LABELLED (WASHED AUTOLOGOUS NORMAL RED CELLS 5 % SURVVAL' 42 8 zj L i 5G Cr LABELLED (WASHED) AUTOLOGOUS NORMAL RED CELLS 5 % SURVVAL 3 8 h 6-3. P 2. 9 SPLEEN' NOT PALPABLE AVERAGE RATO SrEN Or"' S t 51'2 '4& ' 36 ' 4:8 ' ' 1'7'2 RECPENT T. M. NORMAL i r-2 SPLEEN' NOT PALPABLE AVERAGE RATO SLVER Cr5 ' 1*5 '2 24' 36 4*8 6' RECPENT M. M. NORMAL FG. 1. NoRMAL RD CULLs NFUSED rnto NORMAL RECPENTS Recipients T. M. nd M. M. (Studies No. 3 nd 4-Tble ). 72

6 o 6- t- zw SPLENC TRAPPNOG OF RM CELLS N HEMOLYTC STATES Cr LABEL :ED (WASHED) HOMOLOGOJJS SPHEROCYTES (FROH L R.) 5% SURVVAL' 6 1~ loo. 4-~ 1 _2o elm LABELLED (NON-WASHED) AUTOLOGOUS NORMAL RED CELLS 5% SURVVAL, _._._._*_ *C6 3. F AVERAGE RATO WO ' 3.4 t 6 SPLEEN' NOT PALPOGLE 5 : ~~~~~O72 12' RECPENT. H. SPLEEN' NOT PALPABLE o SPLEEN AVERAGE RATO LVER Wr * 12 ' & ' S ' 48 ' 6 DLAYS 4' NORMAL. 72 loot 88 hi D 6- e 5f 4-4 A 2. 1 Oro LAELA.ED (WASNECO AUTLOGOU5 ~8 8 rpnerocytes i 5% SURVVALs ~~~~~ o Aus 2C 6- To Crc LABELLED (NON-WASHEW NOlOOKLUS NORMAL RED CELLS (FROM D. N) 5% SURVVAL' 3 i,4- F 2- SPLEEN' 9en BELOW COSTAL MARGN AVERAGE RATO SPL'EEN%-$ LVER Cr RECPENT L. R.. 6. G. J ml '- SPLEEN S9 BELOW COSTAL MARGN AVERAG RATO SPLEEN C s, '376W ' CONGENTAL HEMOLYTC ANEMA 6 72 FG. 2. A MODEL OF RED CELL SEQUESTRATON Recipients D. H. nd L. R. (Studies No. 5, 6, 9, nd 1-Tble ). Ptient L. R. served s spherocyte donor in the upper nd lower left studies which were crried out simultneously. There ws rpid ccumultion of splenic rdioctivity s evidenced by rising spleen Cr': liver Cr' rtios concomitnt with grossly reduced red cell survivl in both subjects. On the right re presented the dt on the' infusion of Cr' red cells from norml Subject D. H. into ech subject simultneously. n contrst, the red cell survivl times were in the norml rnge nd the spleen Cr': liver Cr' rtios did not rise pprecibly reltive to chnges which occurred fter infusion of spherocytes. on rithmeticl coordintes. Figures 1 to 7 hve A. Norml studies. ndividul studies in northe sme scle to fcilitte comprison of studies. ml subjects re depicted in Figures 1 nd 2. n The dt in two subjects with severe hemolysis the five studies of norml donors nd recipients, re shown on expnded scles in Figres 8 nd 9. the Cr51 red cell hlf-life vried from 3 to 42 dys,

7 1476 -J w L. L. SCHLORSSER, D. R. KORST, D. V. CLATANOFF, AND R. P. SCHLLNG, 8o 61 - z 4. - i* 2 w : 12 1 z or.,-_> , 4, 3. 2.,1 1 -J 8 hi O hi h F , 4. 3'i 2..1 Cr' LABELLED (WASHED HOMOLOGOUS NORMAL RED CELLS 5SUOWAs22 SPLEEN 18 cm BELOW COSTAL MARGN AVERAGE RATO C * 6 * 72 * 1'2 ' Ce 1 LABELLED (NON -WASHED) AUTOLOGOUS SPHEROCYTES 5 % SURVVAL. SPLEEN ANOT PALPABLE 1.j it 2 hi h E: 4c SPLEEN- 18 cm BELOW RECPENT E. Z. CONGENTAL HEMOLYTC ANEMA s Joi 4-1' 1 -i 8 - X 6 L SPLEEN SCp 14 5 AVERAGE RATO ru '6 ' 48 ' d ' 7'2' &. J i~ 3. RECiENT V.H CrO LABELLED (WASHED) AUTOLOGOUS SPHEROCYTES 5 % SURVVAL: COSTAL MARGN AVERAGE RATO LVEEN Gr 3,3?Y%~~~VRC 12 2w4 * 36 * \Cr' LABELLED (NON -WASHED) AUTOLOGOUS SPHEROCYTES 5% SURVVAL, 29 POST- SPLENECTOMY T2 CONGENTAL HEMOLYTC ANEMA FG. 3. CONGENTAL HEMOLYC ANinu Recipient E. Z. (Studies No. 11 nd 12-Tble ). Homologous norml nd utologous (spherocyte) studies re shown in the upper plots. The homologous norml red cell dt is shown on the left. The ptient suffered n infection between the 18th nd 25th dys of this study coincident with fll in hemtocrit. The drop in the red cell survivl curve t this time produced red cell hlf-life slightly less thn norml (22 dys). After recovery the curve becme norml. On the right is shown the utologous (spherocyte) plot. The red cell hlflife ws grossly reduced concomitnt with rpid ccumultion of splenic rdioctivity. This study ws terminted by splenectomy on the 12th dy fter infusion. Recipient V. H. (Studies No. 13 nd- 14-Tble ). Autologous studies before nd fter splenectomy re shown in the lower plots. The pre-splenectomy study plotted on the left demonstrtes only slight erly rise in the spleen: liver rdioctivity rtio. The verge rtio ws not high. The post-splenectomy survivl of red cells (spherocytes) shown on the right ws norml. The spleen weighed 2 Gm. k) 1.

8 SPLENC TRAPPNG OF RED CELLS N HEMOLYTC STATES nd verge spleen: liver rdioctivity rtios from.9 to 1.8 (Tble -Studies 1 to 5). B. Splenomegly without. These studies re illustrted in Figure 4. n recipient J. K. (polycythemi ver in remission), the Crl red cell hlf-life ws 27 dys, nd the verge spleen: liver rdioctivity rtio ws 2.8. Ptient L. K. hd myelofibrosis without. The Crl1 red cell hlf-life ws 27 dys, nd the verge spleen: liver rdioctivity rtio ws 1.8. A very slight increse in rtios occurred concomitnt with the disppernce of lbelled red cells from the peripherl blood in both of these subjects. C. Congenitl. For the observtions shown in Figure 2, Subjects L. R. (congenitl ) nd D. H. (norml) served dul role, i.e., s utologous nd reciprocl homologous recipients of Cr"l red cells (Tble -Studies 5, 6, 9, 1). Lbelled spherocytes infused into norml person or bck into the donor were rpidly eliminted from the peripherl circultion, nd the spleen Crl1: liver Crl rtios incresed to bnormlly high levels. n contrst were the norml survivl of norml red cells in the sme subjects. Three other subjects with congenitl were studied (E. Z., V. H., N. H.-Tbles nd, Figures 3 nd 3). D. Proxysml nocturnl hemoglobinuri. One ptient with proxysml nocturnl hemoglobinuri ws studied. nitil dt (Subject A. K., Tble ) indicted modertely severe. There ws mild thrombopeni nd leukopeni. There hs been no gross hemoglobinuri. Studies with rdioctive red cells re shown in Figure 5. The dignosis of proxysml nocturnl hemoglobinuri ws not considered until norml red cell survivl ws obtined fter the infusion of lbelled cells from norml donor. Survivl of the ptient's own lbelled red cells ws mrkedly reduced suggesting n intrcorpusculr defect. Previous dt did not suggest hereditry spherocytosis (Tble ). The spleen Cr'l: liver Cr'1 rtios did not indicte selective splenic sequestrtion of red cells in either study. The cid hemolysis test of Hm (15) ws positive. E. Acquired. Nine ptients with cquired were studied with rdioctive red cell techniques (Tble -Stud- J O 6. w - z 4. C or Z C z hi w A > 1O ro U 'U. nru 6 '5, 14' i3. 2 'Cr51 LABELLED (WASHED) AUTOLOGOUS SPHEROCY1 5% SURVVAL17 DAY' TES s AVERAGE RATO SPLEEN Cr 51 AVERAGE RATO LVER Cr5' ' 2.4 SPLEEN : NOT PALPABLE ' 2 2'4D' 3 S6 ' 4' ' 7@2 RECPENT N. H. CONGENTAL HEMOLYTC ANEMA FG. 3. CONGENTAL HEMOLYTC ANEMA Recipient N. H. (Study No. 15-Tble ). Ptient N. H. ws the son of V. H. He ws symptomtic with initil evidence for miniml. The utologous red cell survivl ws modertely reduced nd there ws prominent rise in spleen Cr": liver Cr" rtios. ies 18 to 27). The dt re shown in Figures 6 to 9. Ptient L. Y., with chronic lymphocytic leukemi, hd the most severe (Tble ). Dily blood trnsfusions were necessry to mintin stble hemtocrit. Lbelled red cell studies re shown in Figure 9. The scle used in this grph is enlrged so tht the rpid chnges my be clerly seen. The 5 per cent survivl of Crl-lbelled red cells from norml donor ws less thn one dy. With the rpid decline in the peripherl blood rdioctivity there ws n pproximtely reciprocl rise in the spleen Cr1: liver Cr"5 rtio. The verge rtio of spleen to liver rdioctivity ws 6.4. Splenectomy The effect of splenectomy upon the process in eight ptients is given in Tble. The two ptients with congenitl, V. H. nd E. Z., hd the nticipted good response.

9 1478 L. L. SCHLOESSER, D. R. KORST, D. V. CLATANOFF, AND R. F. SCHLLNG OU 8 :sow o 6 z w 4 w 2 Cr5' LABELLED (WASHED) AUTOLOGOUS RED CELLS 5 % SURVVAL : O 4-2. Cr LABFLLED (NON -WASHED) AUTOLOGOUS RED CELLS 5% SURVVAL, g 3 21 : SPLEEN 8 cm BELOW COSTAL MARGN SPLEEN Cr AVERAGE RATO 7*gEE' St 2.8 LVER Cr RECPENT J. K. SPLENOMEGALY; NO ANEMA ' 6 5-1: z w 4- i. 3. F 2 4 i SPLEEN, 17 cm. BELOW COSTAL MARGN AVERAGE RATO SLVEERN Cri 1.8 -~~~~~LVRC ' 36 ' 48 '6 7 RECPENT L. K. SPLENOMEGALY; NO ANEMA FG. 4. SPLENOMEGALC SUBJECTS WTHOUT HEMOLYTC ANEMA: AUToLxoUs CRe RED CEL NFUSONS Recipient J. K. nd L. K (Studies No. 7 nd 8-Tble ). These studies serve to illustrte the effect of splenomegly without on the spleen Cr': liver Cr' rtios. Subject J. K. hd polycythemi ver in remission, nd L. K. hd myelofibrosis. 1. so. Cr,Le LD (WASHED HoMLGOU NORMAL RED CELLS SO SURVVAL. 3 1 q 8: Ael (WASHED) AUTNLȦ.No RED 51 SURVVAL 6 CGLLS 6. 4 bi 4. 2 & V 8 6 l u 4 SPLEEN. 2 ct BELOW COSTAL MARG5 AVERAGE RATO LER Goin LVER Cr SPLEEN2 n BELOW COSTAL MARG AVERAGE RATO SLEER Go", 1.5 UVER Cr S6 48 ' * 72 RECPENT A, K. PAROXYSMAL NOCTURNAL HEMOGLOBNURA ' FG. 5. PAROXYSMAL NocTURNAL HEMOGLOBNURA: HOMOLOGOUS NORMAL AND AUTOLOGOUS CR' RED CELL NFUSONS Recipient A. K. (Studies No. 16 nd 17-Tble ). The homologous infusion dt re shown on the left, nd the survivl of these cells ws norml. On the right, the utologous infusion dt re given. Note the rpid rte of destruction of the ptient's own lbelled cells suggesting n intrcorpusculr defect. There ws no selective splenic uptke of Cr' in either study s determined by spleen Cr': liver Cr' rtios.

10 SPLENC TRAPPNG OF RED CELLS N HEMOLYTC STATES 1479 TABLE Efect of splenectomy in eight ptients with CrO Averge Cru-lbelled red-cell rtio Spleen red-cell hlf-life spleen CrO: wt. Ptient Dignosis infusion (dys) liver Cru (Gm.) Result V. H. Congenitl Autologous Good E. Z.* Congenitl Autologous , Good L. H.* diopthic Autologous Good J. L.* diopthic Autologous ,38 Fir J. T. diopthic Autologous Poor N. A. Myelofibrosis Homologous Good norml W. M. Chronic lymphocytic Homologous ,86 Good leukemi norml L. Y. Chronic lymphocytic Homologous < ,45 Good leukemi norml * Crl-lbelled red cell survivl with orgn counting ws interrupted by splenectomy t 4 to 5 per cent survivl of lbelled erythrocytes. Ptient V. H. (Tble -Study 14, nd Figure 3) hd norml utologous Cr51 red cell survivl fter splenectomy. Ech of the five ptients with cquired benefitting from splenectomy hd n verge spleen: liver rdioctivity rtio of 3. or higher following the infusion of Cr51-lbelled red cells. Probbly more importnt thn the verge rtio is evidence for rising rtio of spleen: liver rdioctivity, suggesting selective splenic trpping of red cells. Dt from Ptients N. A. (Figure 6), J. L. (Figure 8), nd L. Y. (Figure 9) show striking evidence of incresing splenic rdioctivity s compred to the liver during the period of rpid removl of lbelled red cells from the circultion. Studies on W. M. (Figure 6) show high initil spleen: liver rdioctivity rtio, but this point ws obtined 12 hours fter the infusion of cells nd my not be representtive of erlier splenic rdioctivity. The dt from Ptient L. H. (Figure 6) re suggestive of splenic ccumultion of trpped red cells, but we lck dt for the first severl dys of the study. n contrst, Ptient J. T. (Figure 7) hd n verge rtio of 1.9 nd ws not improved by splenectomy. There ws no evidence of rising spleen: liver rdioctivity rtio in this ptient. The results given in Tble represent the effect of splenectomy upon the nd not the primry disese. Following splenectomy, Ptients L. H. nd W. M. hve hd sustined remissions for 2 nd 3 months, respectively. Ptient J. L., who in the six months prior to splenectomy required t lest 19 blood trnsfusions, did not require trnsfusions for three months post-opertively. He expired t home four nd one-hlf months following splenectomy. A primry dignosis ws not estblished. Ptient N. A., who hd developed myelofibrosis nd following polycythemi ver, received drmtic relief of for t lest 12 months post-splenectomy. Fourteen months fter splenectomy this ptient hd developed leukemi without evidence of. He died two months lter in the hospitl with severe nd blsts of undetermined type in the peripherl blood nd bone mrrow. Ptient L. Y. (chronic lymphtic leukemi), whose hemolysis ws so severe s to require dily blood trnsfusions, ws drmticlly relieved by splenectomy. There ws no evidence for when this ptient ws seen in the hospitl few dys prior to his deth three nd one-hlf months postopertively. DSCUSSON The technique of red cell lbelling nd estimting erythrocyte survivl with rdioctive chromium used in this study is similr to tht employed by Ebugh, Emerson nd Ross (22). Red cell survivl dt reported here re bsed upon the rdioctivity in red cells per unit volume of whole blood

11 148 L. L. SCHLOESSER, D. R. KORST, D. V. CLATANOFF, AND R. F. SCHLLNG 1 :1 8- Cro LABELLED (WASHEC AUTO US RED CELLS 5% SURVAL: 8 Crt LABELLED (WASHED) NORMAL RED CELLS 5% SURVVAL i p.. SPLEEN S L BELOW COSTAL MARGN AVERAGE RATO UVER -LCt 3. L : *472_ '6 4T8'6o RECPENT L. H. DOPATHC HEMOLYTC ANEMA 2 SPLEEN. A em BELLOW AVERAGE COSTAL E AS RATOLECrs WW LVER Cr' '?P RECPENT P. P. DOPATHC HEMOLYTC ANEMA w1 6 Cr' LABELLED (NON -WASHED HOMOLOGOUS- NORMAL RED CELLS 5% SURVVAL m 6 S Cr LABELLED (* HOMOLOGOUS NORMAL NASHEM RED CELLS 5 % SURVVAL.6 z. 4 F21 4 1! 2. i4>> l SPLEENs 7 el BELOW COSTAL MARN SPLEEN. 14 c BELOW COSTA. marg - AVERAGE RATO PLEEN 54 UVER Cr AVERAGE RATO UVER Cr *, TLVlt olk :.o 112 * 24 * 36 * "CS * CO * 7f2 ' -n? ' t _' ' = 48 ' * _ ---- REPENT NA. W. M. ACQURED HEMOLYTC ANEhUA REGPMENT ACQURED HEMOLYTC ANEMA FG. 6. ACQURED HEMOLYTC ANEMA Recipient L. H. (Study No. 18-Tble ). Ptient L. H. hd splenomegly, thrombopenic purpur nd mild of unknown cuse. Autologous infusion dt re given in the upper left grph. The ptient's own lbelled cells were eliminted t modertely incresed rte (5 per cent survivl in 16 dys). The verge spleen Cr': liver Cr' rtio ws 3.. This study ws terminted by splenectomy on the 25th dy fter infusion. There ws good response to splenectomy. Recipient P. P. (Study No. 19-Tble ). On the upper right homologous infusion dt for Ptient P. P. re shown. The ptient hd miniml. The survivl of homologous Cr'-lbelled red cells substntited moderte process (5 per cent survivl in 17 dys). The rise in spleen Cr': liver Cr' rtios, however, is striking nd pproximtes in reciprocl mnner the fll in peripherl blood red cell Cr'. The verge spleen Cr': liver Cr' rtio ws 4.2. Splenectomy ws not performed. Recipient N. A. (Study No. 2-Tble ). This ptient hd myelofibrosis nd cquired following polycythemi ver. He ws given Cr"-lbelled red cells from fi

12 L 2 SPLENC TRAPPNG OF RED CELLS N HEMOLYTC STATES Cro LABELLED (WASpE HOMOLOGOUS ORMAL RED CELLS 5 % SURV' S - - zi 1 so Cro LABELLED (NON -WASHED) AUTOLOGOUS RED CELLS 5 % SURVVAL: i i l SPLEENS3mm LOW COSTAL MARG AVERAGE RATO ER Cry 2. * > *2t RECPENT L W. ACQURED HEMOLYTC ANEMA 4. *i 3. F 2 2 SPLEEN' 2 m BELOW COSTAL MARGN RTOLVER Cr AVERAE AVERGE RATO SPLEEN Or L9L e ' 72 *2 * R4 ' 36 * 48 '6 RECPENT J. T. DOPATHC HEMOLYTC ANEMA FG. 7. ACQURED HEMOLYTC ANEMA Recipient L. W. (Study No. 22-Tble ). Ptient L. W. hd positive "LE test." The lbelled red cell dt in this subject re shown on the left. The survivl of norml donor's cells ws modertely reduced (hlf-life, 19 dys) nd there ws no evidence for selective sequestrtion of lbelled erythrocytes in the spleen by externl counting (verge spleen Cr': liver Cr" rtio, 2.). Splenectomy ws not done. Recipient J. T. (Study No. 23-Tble ). Ptient J. T. received his own lbelled red cells nd is illustrted on the right. The red cell survivl ws reduced (hlf-life, 15 dys), nd there ws no indiction from spleen Cry: liver Cr" rtios for splenic sequestrtion of lbelled erythrocytes. This ptient did not benefit from splenectomy. which ssumes constnt whole blood volume. t is believed this method gives better estimtion of Cr51 red cell survivl thn techniques bsed on mesurements of rdioctivity per unit volume of pcked cells. The ltter method ssumes constnt red cell volume, n ssumption which my not be correct in sttes (23). There re mny errors involved in the estimtion of reltive orgn rdioctivity by externl monitoring of gmm emissions. Vritions in counter positioning, counter-to-source distnce, orgn size, nd rdioctivity in djcent viscer ll ffect the results. We hve estimted the ntomic center of the surfce projection of the liver nd spleen by percussion, nd, by using externl lndmrks, repositioned the probe t the sme re in subsequent observtions. Prticulr cre must be employed in positioning the detector over the smller orgn (e.g., smll spleen). f the pproximte center is not locted or there re moderte vritions in dy-to-dy positioning, reltively lrge errors my be mde in evluting orgn rdioctivity. There re geometric limits of rdioctivity ccepted by the scintilltor t close rnge. Consequently, less rdioctivity is detectble over the lrge orgn s compred to the smll one, even though both contin equl quntities of isotope. This problem cn theoreticlly be solved by norml donor. The dt re shown in the lower left grph. There ws rpidly rising spleen Cry: liver Cr" rtio concomitnt with rpid rte of Cr" red cell removl from the peripherl blood. This ptient responded well to splenectomy but 14 months lter the clinicl fetures of cute leukemi led to his demise. Recipient W. M. (Study No. 21-Tble ). This ptient hd chronic lymphtic leukemi with n cquired. The survivl of Cr"-lbelled red cells from norml donor ws grossly reduced (hlf-life, 6 dys). There ws n ssocited rise in spleen Cr": liver Cr" rtio (verge rtio, 3.5). This ptient hd n excellent response to splenectomy. He hs hd no tretment or during the two nd one-hlf yers since.

13 1482 L. L. SCELOESSER, D. R. KORST, D. V. CLATANOF, AND R. P. SCHLLNG incresing the counter to source distnce; however, without elborte shielding pprtus, specificity of orgn rdioctivity is lost. Orgn res hve been counted with n unshielded probe touching the skin, ssuming this procedure would give vlid dt. There is evidence tht not ll of the Cr5' found in the spleen fter the infusion of lbelled red cells is hemoglobin bound (24). t is considered unlikely, however, tht such rdioctivity is derived from sources other thn Cr5l-lbelled red cells destroyed in the spleen. Cr5lCl3 (13, 25) nd Crl-lbelled hemoglobin infusions (13) showing low flt curves of splenic rdioctivity support this conjecture. n Figure 1, verge spleen to liver rdioctivity rtios in cquired ptients were grouped ccording to splenectomy response g Cr1 LABEL.LED (WASHED) HOMOLOGOUS NORMAL RED CELLS 5 % SURVVAL, 3 nd compred with the rtios obtined in norml nd splenomeglic subjects not demonstrting mechnisms. The ltter group noted by n sterisk in Figure 1 includes verge orgn rdioctivity rtios from Studies 6 to 8, 11 nd 25, Tble. Averge rtios of spleen: liver rdioctivity in the five norml subjects receiving lbelled norml red cells rnged from.9 to 1.8. The higher rtios of spleen: liver rdioctivity in splenomeglic subjects without re presumptive evidence for greter lbelled red cell volume "seen" by the detector over the lrger orgn. These studies provided control observtions. From these dt it ppers tht ptients with reduced Cr5' red cell survivl nd rising or high spleen: liver rdioctivity rtio will hve fvorble response to splenectomy. Obviously mny more ptients will hve to be studied by 1] bso 6 4 Crm LABELLED (WASHED AUTOLOGOUS RED CELLS 5 % SURVVAL: i l SPLEEN, 6 cm BELOW COSTAL MARGN AVERAGE RATO SPLVEERN Cr ' 2.4 -~~~~~VRC e , 2. l SPLEEN. t cm BELOW COSTAL MARGN AVERAGE RATO EELN Cr. 6 t RECPENT J. L. DOPATHC HEMOLYTC ANEMA FG. 8. ACQuED HEMOLYTC ANEMA (Note chnge in scle of bsciss.) Recipient J. L. (Studies No. 25 nd 26-Tble ). Ptient J. L. hd severe disese. The survivl of comptible lbelled donor red cells ("homologous"), given on the left, ws norml. During this study the spleen incresed in size from 6 cm. below costl mrgin to 11 cm. below costl mrgin. The verge spleen Cr": liver Cr' rtio ws 2.4 prior to interruption of the study t 3 dys for blood trnsfusions. The utologous study given on the right showed grossly reduced red cell hlf-life of the ptient's cells, nd concomitntly rpid selective ccumultion of rdioctivity in the spleen. The spleen Cr': liver Cr" rtios verged 3.4 when the study ws terminted by splenectomy on the ninth dy. Subsequent detiled study of the Rh genotype of the ptient nd comptible donor reveled difference. The ptient ws found to be CDE/CDe nd the donor CDe/Cde. These findings re comptible with, though not dignostic of, n nti-rh type specific. The cid hemolysis test of Hm ws negtive.

14 1O o 8 -J -i 6 w z in 7- O 6- J 4 3 2A = r SPLENC TRAPPNG OF RED CELLS N HEMOLYTC STATES Cr5 LABELLED (WASHED HOMOLOGOUS NORMAL RED CELLS 5 % SURVVAL:.< DAY SPLEEN; 1 cm. BELOW COSTAL MARGN AVERAGE RATO SPLEEN Cr, 6.4 LVER Crtm RECPENT L. Y. AGQURED HEMOLYTC ANEMA FG. 9. ACQURED HEMOLYTC ANEMA (Note chnge in scle of bsciss.) Recipient L. Y. (Study No. 27-Tble ). Ptient L. Y. hd chronic lymphtic leukemi with severe. The ccumultion of rdioctivity in the spleen is striking nd the plot of splee Crc: liver Cr' rtios is pproximtely reciprocl to the plot of disppernce of Cr'1-lbelled erythrocytes from the peripherl blood. The verge spleen Cry: liver Cr' rtio ws 6.4. This ptient hd good respnse to splenectomy. these techniques before one cn sy tht they provide vlid criteri for preopertive prediction of the results of splenectomy. n correltion of splenic sequestrtion with results of splenectomy the following combintions re possible: 1) 2) 3) 4) Evidence for sequestrtion Benefit from splenectomy Our dt from ptients with cquired show five exmples of the first combintion nd one exmple of the fourth. We hve not observed No. 2 or 3. Thus it cnnot be climed tht the dt provide evidence enbling one to 1483 predict filure to benefit from removl of the spleen. The dt re considered evidence in fvor of the theory tht splenectomy will be beneficil in those ptients demonstrting selective splenic sequestrtion of erythrocytes. We hve no dt from ptients with cquired without detectble splenomegly (Tble ), nd hve studied only the one ptient (J. T.-Tble ) with miniml splenomegly who hd splenectomy. However, it is likely tht ptients with cquired, red cell sequestrtion in the spleen nd norml spleen size (should these fetures co-exist) would show spleen: liver rdioctivity rtios similr to tht in subjects without splenomegly infused with lbelled spherocytes (Figures 2 nd 3). We re unble to explin the filure to demonstrte Cr11- lblled spherocyte ccumultion in the spleen of the ptient with without splenomegly (V. H.-Figure 3). Cr5l-lbelled erythrocyte studies, in ddition to the estimtion of orgn ccumultion of red cells, my occsionlly suggest mechnisms of disese previously unsuspected. Clinicl evlution of Ptient A. K. hd indicted brisk, mild thrombopeni nd leukopeni. Splenectomy might hve been dvised -for "hypersplenism" hd not erythrocyte survivl studies indicted n intrcorpusculr defect (Figure 5). There ws no evidence for congenitl (Tble ), nd the cid hemolysis test ws unequivoclly positive estblishing the dignosis of proxysml nocturnl hemoglobinuri. Orgn rdioctivity rtios in this ptient did not suggest splenic ccumultion of utologous or homologous erythrocytes. These studies re in ccord with the usul observtion tht ptients with this disorder re not helped by splenectomy (18). On the other hnd, Jndl nd his collegues hve reported tht splenectomy ws beneficil in ptient with this disorder in whom splenic sequestrtion of utologous Cr51-lbelled red cells hd been demonstrted (13). Studies with rdioctive red cells in Ptient J. L. were of prticulr interest. This ptient hd severe of unknown type. The survivl of "homologous" norml Cr51-lbelled red cells ws norml. The reltively mrked rise of the spleen: liver rdioctivity rtio during this study (Figure 8) ws considered secondry to n

15 1484 L. L. SCHLOESSER, D. R. KORST, D. V. CLATANOFF, AND R. F. SCHLLNG 6 - Q j So S EOREAT A.H.A. NORM"L A.H.A. WTHOUT FAVORABLE POOR HEMOLYTC RESPONSE TO RESPONSE TO ECHANSS SPLENECTOKY SPLENECTOMY FG. 1. CORRELATON OF RESPONSE TO SPLENECTOMY WTH AvERAcz SPUEN: LiVER RADo- ACmTVT RATios N AcQunw HEMOLYTC ANEMA Ech br represents the verge spleen: liver rdioctivity rtio for single study. The number t the top of ech br represents the Cr' red cell hlf-life in dys. A.H.A. = Acquired nei. * See text. expnding spleen red cell volume in n enlrging spleen. An even greter ccumultion of splenic rdioctivity with grossly reduced red cell survivl occurred fter infusion of the ptient's own lbelled red cells. A negtive fmily history nd conventionl lbortory dt excluded congenitl (Tble ). The cid hemolysis test for proxysml nocturnl hemoglobinuri ws negtive. Further typing of the donor's nd the ptient's red cells, however, reveled difference in Rh genotypes. We were unble to repet study using truly homologous erythrocytes, but the observtions mde suggested n Rh typespecific. Prior to lbelled red cell studies, our lbortory dt in cquired did not provide criteri for predicting response to splenectomy. This observtion is in ccord with tht recently reported by Chertkow nd Dcie (26). Gross splenomegly, however, ws common to ll ptients with cquired showing ' evidence for splenic ccumultion of Cr5l-lbelled red cells. Ptients with cquired nd splenic sequestrtion of lbelled red cells did not hve spherocytosis in the peripherl blood s judged by osmotic frgility studies. SUMMARY The rtio spleen: liver rdioctivity s determined by externl scintilltion counting fter the infusion of Cr51-lbelled erythrocytes hs been used to determine orgn locliztion of red cells in seven control subjects nd 14 ptients with. As nticipted, the spleen ccumulted rdioctivity rpidly following the trnsfusion of lbelled spherocytes. Some ptients with cquired showed gross evidence of trpping of rdioctive red cells in their spleen. Removl of the spleen in five such ptients ws beneficil in ech. One ptient who showed no evidence for splenic trpping of red cells ws splenectomized without beneficil effects on the

16 SPLENC TRAPPNG OF RED CELLS N HEMOLYTC STATES process. The method described is considered useful id in the selection of ptients whose process will be benefitted by splenectomy. ACKNOWLEDGMENT The uthors pprecite the coopertion of Ky Strutz, Virgini Loy, nd Frncis Morris. REFERENCES 1. Emerson, C. P., Jr., Shen, S. C., nd Cstle, W. B., The osmotic frgility of the red cells of the peripherl nd splenic blood in ptients with congenitl jundice trnsfused with norml red cells (bstrct). J. Clin. nvest., 1946, 25, Young, L. E., Pltzer, R. F., Ervin, D. M., nd zzo, M. J., Hereditry spherocytosis.. Observtions on the role of the spleen. Blood, 1951, 6, Weismn, R., Jr., Hurley, T. H., Hrris, J. W., nd Hm, T. H., Studies of the function of the spleen in the hemolysis of red cells in hereditry spherocytosis nd sickle cell disorders. J. Lb. & Clin. Med., 1953, 42, Hm, T. H., nd Cstle, W. B., Studies on the destruction of red blood cells. Reltion of intrvsculr stsis nd of bnorml frgility of erythrocytes to the mechnism of hemolysis in certin s. Yer Book, Am. Philosoph. Soc., Phildelphi, 1939, p Hm, T. H., nd Cstle, W. B., Studies on destruction of red blood cells. Reltion of incresed hypotonic frgility nd of erythro-stsis to the mechnism of hemolysis in certin s. Proc. Am. Philosoph. Soc., 194, 82, Hm, T. H., nd Cstle, W. B., Mechnism of hemolysis in certin s: Significnce of incresed hypotonic frgility nd of erythrostsis (bstrct). J. Clin. nvest, 194, 19, Hm, T. H., nd Cstle, W. B., Reltion of incresed hypotonic frgility nd of erythrostsis to the mechnism of hemolysis in certin s. Tr. A. Am. Physicins, 194, 55, Weismn, R., Jr., Hm, T. H., Hinz, C. F., Jr., nd Hrris, J. W., Studies of the role of the spleen in the destruction of erythrocytes. Tr. A. Am. Physicins, 1955, 68, Emerson, C. P., Jr., Shen, S. C., Hm, T. H., Fleming, E. M., nd Cstle, W. B., Studies on the destruction of red blood cells. X. Quntittive methods for determining the osmotic nd mechnicl frgility of red cells in the peripherl blood nd spleen pulp. The mechnism of incresed hemolysis in hereditry spherocytosis (congenitl jundice) s relted to the functions of the spleen. Arch. nt. Med., 1956, 97, Micheli, F., Unmittelbre effekte der splenektomie bei einem fll von erworbenem hiimolytischen 1485 splenomeglischen ikterus typus nyem-widl. Wien. klin. Wchnschr., 1911, 24, Huff, R. L., Elmlinger, P. J., Grci, J. F., Od, J. M., Cockrell, M. C., nd Lwrence, J. H., Ferrokinetics in norml persons nd in ptients hving vrious erythropoietic disorders. J. Clin. nvest., 1951, 3, Jndl, J. H., Greenberg, M. S., Yonemoto, R. H., nd Cstle, W. B., Clinicl determintion of the sites of red cell destruction. Clin. Reserch Proc., 1955, 3, Jndl, J. H., Greenberg, M. S., Yonemoto, R. H., nd Cstle, W. B., Clinicl determintion of the sites of red cell sequestrtion in s. J. Clin. nvest., 1956, 35, Korst, D. R., Cltnoff, D. V., nd Schilling, R. F., Externl scintilltion counting over the liver nd spleen fter the trnsfusion of rdioctive erythrocytes. Clin. Reserch Proc., 1955, 3, Hm, T. H., Ed., A syllbus of lbortory exmintion in clinicl dignosis. Criticl evlution of lbortory procedures in the study of the ptient. Cmbridge, Hrvrd University Press, Mlloy, H. T., nd Evelyn, K. A., The determintion of bifirubin with the photoelectric colorimeter. J. Biol. Chem., 1937, 119, Ducci, H., nd Wtson, C. J., The quntittive determintion of the serum bilirubin with specil reference to the prompt-recting nd the chloroform-soluble types. J. Lb. & Clin. Med., 1945, 3, Dcie, J. V., The Hemolytic Anemis. Congenitl nd Acquired. New York, Grune & Strtton, Wtson, C. J., Studies of urobilinogen.. An improved method for the quntittive estimtion of urobilinogen in urine nd feces. Am. J. Clin. Pth., 1936, 6, Wtson, C. J., Schwrtz, S., Sborov, V., nd Bertie, E., Studies of urobilinogen. V. A simple method for the quntittive recording of Ehrlich rection s crried out with urine nd feces. Am. J. Clin. Pth., 1944, 14, Red, R. C., Studies of red-cell volume nd turnover using rdiochromium. Description of new "closed" method of red-cell-volume mesurement. New Englnd J. Med., 1954, 25, Ebugh, F. G., Jr., Emerson, C. P., nd Ross, J. F., The use of rdioctive chromium 51 s n erythrocyte tgging gent for the determintion of red cell survivl in vivo. J. Clin. nvest., 1953, 32, Strumi, M. M., Tylor, L., Smple, A. B., Colwell, L. S., nd Dugn, A., Uses nd limittions of survivl studies of erythrocytes tgged with Cr'. Blood, 1955, 1, Schloesser, L. L., Unpublished dt. 25. Korst, D. R., Unpublished dt. 26. Chertkow, G., nd Dcie, J. V., Results of splenectomy in uto-immune hemolytic nemi. Brit. J. Hemt., 1956, 2, 237.