Chemico-physical changes occur in red cells during

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1 THE RATE OF POST-TRANSFUSION LOSS OF NON-VIABLE STORED HUMAN ERYTHROCYTES AND THE RE-UTILI- ZATION OF HEMOGLOBIN-DERIVED RADIO- ACTIVE IRON1 BY JOHN G.GIBSON, 2ND, WENDELL C. PEACOCK, ROBLEY D. EVANS, THEODORE SACK, AND JOSEPH C. AUB (From the Rdioctivity Center, Msschusetts Institute of Technology, Cmbridge, Msschusetts; the Medicl Clinics of the Peter Bent Brighm nd Msschusetts Generl Hospitls; nd the Deprtment of Medicine, Hrvrd Medicl School, Boston, Msschusetts) (Received for publiction August 31, 1946) When humn erythrocytes re trnsfused within few hours of tking in citrte solution, prcticlly ll of them remin intct in the recipient's blood strem (1). Therefter, they dispper from the circultion t rte of bout one per cent per dy (2, 3), nd it is generlly believed tht this is the norml deth rte of homologous erythrocytes. Chemico-physicl chnges occur in red cells during storge in vitro. Depending upon the preservtive vlue of the nticogulnt used, deteriortive chnges my occur grdully nd proceed t reltively constnt rtes for considerble periods, or they my be initited erly in storge nd proceed t ccelerted rtes. Post-trnsfusion vibility of preserved cells is function of the length of storge (1). Thus, the trnsfusion of stored blood, which lwys contins certin quntity of cells in which irreversible chnges hve occurred, necessittes the removl of those ded or dying red cells from the circultion. These cells my rupture in the blood strem nd, through relese of hemoglobin, give rise to hemoglobinemi. It is lso possible tht they my be removed, with membrnes reltively intct, by phgocytosis, or segregtion in the spleen (4), nd subsequently destroyed. In either event lod is imposed upon the mechnism for scvenging the blood strem nd for the hndling of hemoglobin-derived pigments: the reticuloendothelil nd the erythropoietic systems. Hemoglobinemi lso involves renl function since there is plsm level for hemoglobin bove which 1 The work described in this pper ws done under contrct, recommended by the Committee on Medicl Reserch, between the Office of Scientific Reserch nd Development nd the Msschusetts Institute of Technology, in collbortion with the Peter Bent Brighm Hospitl, nd the Msschusetts Generl Hospitl. hemoglobinuri occurs. The degree of this burden will be determined by the quntity of mortlly dmged cells trnsfused (which will in turn depend upon the sie of the trnsfusion, nd, for ny given preservtive solution, the length nd conditions of storge prior to trnsfusion), nd the rte t which the blood is trnsfused. In previous communictions (5) it ws concluded tht the greter prt of the non-vible cells re completely removed from ctive circultion, usully within 24 hours fter trnsfusion, nd tht the reminder of the trnsfused cells resume norml functionl cpcity nd enjoy sojourn in the body equivlent to the reminder of their norml life expectncy. It is therefore only the non-vible portion of the trnsfused red cells tht need be considered in the study of the effects of the trnsfusion of stored blood. In number of experiments in which blood, the cells of which were tgged with rdioctive iron, ws trnsfused, the initil recipient blood smple ws tken within 6 minutes, nd severl subsequent smples within the first 14 hours of the beginning of the trnsfusion. Since little if ny reutilition of hemoglobin-derived rdio-iron cn be expected to occur in this period, the rdioctivity of those blood smples ccurtely mesured the quntity of trnsfused cells removed from the blood strem. These vlues my be expressed in terms of the percentge of totl cells trnsfused or of the contined quntities of hemoglobin or of iron. Such dt were obtined in 9 trnsfusions of stored whole blood nd in 15 trnsfusions of stored resuspended cells. The whole bl6ods were drwn into ACD-1 nd Alsever's solution; they were trnsfused from 8 to 26 dys fter collecting. Five of the bloods in ACD-1 hd been trnsported, by 739

2 TABLE I Quntities of whole blood (or resuspensions) nd red cells trnsfused, storge periods, nd percentges of non-vible red cells removed from circultion t successive intervls from beginning of trnsfusion Exp. no. Solution Dys stored Trnsfused Time intervls in minutes from strt of trnsfusion Totl Cells Per cent cells removed in intervl mi. 69 ACD , ACD , ACD , ACD , ACD-I , Alsever's , ACD , Alsever's , ACD , , Citrte-buffered ,26 92 with dded globulin , , , , citrte-dextrose ,5 ph , , , , , per per cent cent corn corn syrup ,44 mi. syrup ~~~ , ,

3 POST-TRANSFUSION FATE OF NON-VIABLE STORED RED CELLS on% WV' 2 -J O 7 m -i : 6 o 4 DISAPPEARANCE OF NON-VIABLE ERYTHROCYTES FROM THE BLOOD STREAM hi. w MINUTES FROM BEGINNING OF TRANSFUSION FIG. 1. DISAPPEARANCE OF NON-VIABLE ERYTHROCYTES FROM THE BLOOD STREAM The percentge of totl trnsfused tgged cells stored s whole blood in ACD-1 nd Alsever's solution remining in circultion during the first 1,5 minutes fter beginning trnsfusion. ir, under constnt refrigertion, from Boston, Msschusetts to Oklnd, Cliforni, nd bck (5 dys elpsed), nd subsequently were stored t 4 C. Two bloods in ACD-1 nd 3 in Alsever's solution hd been trnsported by ir, without constnt refrigertion, from Boston to Pris, Frnce (11 dys elpsed), nd subsequently were stored t 4 C. The pcked cells were resuspended in: 1 per cent corn syrup; citrte-buffered citrte-dextrose solution of ph 5.; nd citrte-buffered citrte-dextrose solution contining 3 per cent Frction IV-3, 4, Lot 31 2 ( nd # globulin). The cells in the globulin-fortified solution hd been in depot storge t 4 C. until trnsfused. The cells in corn syrup nd in the cidified-citrte-dextrose solution were trnsported by truck, under controlled refrigertion (4 to 1 C.), from Boston to New York, nd trnsfused into ptients on the wrds of the New York Hospitl.3 2Prepred in the Pilot Plnt of the Deprtment of Physicl Chemistry, Hrvrd Medicl School, under the direction of Dr. E. J. Cohn. 3 These experiments were crried out, under request from medicl uthorities of the U. S. Nvy, represented by Lt. (s.g.) Henry Blke, U.S.N.R., in collbortion with Dr. Rlph G. Stillmn, of the New York Hospitl, nd Dr. Willim Thlhimer. The trnsfused quntities of whole blood in ACD nd Alsever's solution rnged from 315 to 525 ml. (121 to 253 ml. of cells); cell resuspensions in corn syrup nd in the citrte-buffered citrte-dextrose rnged from 315 to 52 ml. (147 to 256 ml. of cells); nd cell resuspensions in the globulin-fortified resuspension solution, from 42 to 55 ml. (171 to 215 ml. of cells). The verge of the 24 trnsfusions ws 194 ml. Time required for trnsfusion vried from 13 to 65 minutes, nd the rte of flow from 8 ml. to 36 ml. per minute, verging 18 ml. per minute. The hemtocrits of whole blood were from 28 to 46; nd of cell resuspensions from 34.7 to 5.5; the verge hemtocrit of ll the trnsfusions ws Survivl during the first 24 post-trnsfusion hours rnged from 92 to 8 per cent for whole bloods nd from 95 to 32 per cent for the cell resuspensions. These quntities of whole blood nd cells re well within the rnges encountered in single clinicl trnsfusions; hence the dt my be treted s whole in pplying the findings to trnsfusion prctice. The verge rte of inflow, 18 ml. per minute, is perhps more rpid thn tht of the usul routine trnsfusion. The quntity of whole blood or resuspension, cells trnsfused, rte of inflow, nd percentge of totl trnsfused cells lost from circultion t suc-

4 742 J. G. GIBSON, 2ND, W. C. PEACOCK, R. D. EVANS, T. SACK, AND J. C. AUB 1 DISAPPEARANCE OF NON-VIABLE ERYTHROCYTES FROM THE BLOOD STREAM 2rt -j Z. I. -I w '.j w MINUTES FROM BEGINNING OF TRANSFUSION FIG. 2. DISAPPEARANCE OF NON-VIABLE ERYTHROCYTES FROM THE BLOOD STREAM The percentge of totl trnsfused tgged cells drwn s whole blood in 4 per cent sodium citrte nd resuspended in 3 preservtive solutions, remining in circultion during the first 1,5 minutes fter beginning trnsfusion. cessive intervls from the beginning of the trnsfusion re shown in Tble I. The percentges of cells remining in circultion t intervls up to 1,5 minutes fter trnsfusion re shown in Figures 1 nd 2. The post-trnsfusion behvior of both the cells stored s whole blood nd the cells in resuspension ws similr. The rte of loss of non-vible cells from trnsfusions in which the eventul survivl ws better thn 8 per cent ws slow nd reltively constnt. At 8 per cent survivl, or better, the greter prt of the loss occurred during the first 3 to 5 minutes from the strt of the trnsfusion, there being, in generl, little further loss during the reminder of the first 24 hours. In trnsfusions, the survivl of, which ws less thn 8 per cent, the rte of loss of non-vible cells ws rpid nd tended to vry directly with the eventul survivl. The rte of loss ws gretest during the first 6-minute period, becoming progressively less during the next 24 hours. The w hi -i -I6. RATE OF SISAPEARANCE OF NON-VIABL9 ERYTHROCYTES I. 2 -o on IL r A. I.- = PERCENT SURVIVAL FIG. 3. RATE OF DISAPPEARANCE OF NON-VIABLE ERYTHROCYTES The verge percentges of totl trnsfused tgged red cells removed from circultion during successive 6-minute intervls from beginning of trnsfusion, in reltion to the percentge of survivl.

5 POST-TRANSFUSION FATE OF NON-VIABLE STORED RED CELLS mximum loss ws not reched during the first 5 hours, but the rte of loss during the ensuing 19 hours ws considerbly less thn during the first 5-hour period. It should be emphsied tht even in the poorest trnsfusions the 24-hour dt still mesure disppernce uncomplicted by cell regenertion. It is therefore pprent tht the rte of disppernce of non-vible cells is function of percentge of survivl, which in turn is function of time of storge. It is possible, from the time survivl curves presented in Figures 1 nd 2, to determine, for the entire series of experiments, the verge rte of disppernce of cells t given intervls fter the beginning of trnsfusion for ny percentge of eventul survivl. The fmily of curves representing these rtes t 6-minute intervls up to 5 hours RATE OF REMOVAL OF NON-VIABLE STORED ERYTHROCYTES FROM CIRCULATION fter trnsfusion re shown, in reltion to survivl, in Figure 3. It is evident tht the loss of non-vible cells begins very soon fter the dministrtion of blood nd, in bdly deteriorted bloods, my begin during dministrtion. Regrdless of the survivl, the rte of loss is most rpid during the first 6 minutes' post-trnsfusion period nd decreses considerbly during the ensuing four 6-minute periods. This fct is more clerly evident in Figure 4, in which the verge rtes of loss of non-vible cells during the first 5 hours fter the beginning of trnsfusion, expressed s per cent of cells lost per minute, re shown for trnsfusions rnging from 1 to 5 per cent survivl. In the sme figure, the totl quntity of hemoglobin tht would be obtined from immedite U) -j ' U) * I~-. cw 1 -i 5 4 Ito -j w. 2 ' 1 o e~ w HOURS FROM BEGINNING OF TRANSFUSION FiG. 4. RATE OF REMOVAL OF NON-VIABLE STORED ERYTHROCYTES FROM CIRCULATION The verge percentges of loss of the totl trnsfused tgged red cells per hour during the first 5 hours from beginning trnsfusion, in bloods rnging from 5 to 1 per cent survivl (indicted by figures in circles). The totl mount of hemoglobin, in grms per hour (ssuming immedite nd complete intrvsculr hemolysis occurs), liberted from non-vible red cells following trnsfusion of blood surviving from 5 to 1 per cent, is shown in the first column on the right. The theoreticl plsm hemoglobin level, in n individul with plsm volume of 3,5 ml. resulting from complete retention of the liberted hemogloblin, is shown in the column on the extreme right.

6 744 J. G. GIBSON, 2ND, W. C. PEACOCK, R. D. EVANS, T. SACK, AND J. C. AUIB intrvsculr hemolysis of non-vible cells is shown in reltion to the per cent of trnsfused cells destroyed. These quntities re lso plotted s mgm. of hemoglobin liberted per hour per 1 ml. of plsm, in n individul with plsm volume of 3,5 ml. Hemoglobinuri ws observed only in those of our subjects who received full-sied trnsfusions of blood the survivl of which ws less thn 5 per cent, nd then ws trnsient ffir, the urine being cler within few hours fter trnsfusion. Even in these subjects the rise in serum totl bilirubin did not exceed 2 mgm. per 1 ml. of plsm. It is cler tht mny ded cells must be removed reltively intct from the blood strem. The role of the spleen in this regrd is too well known to elborte on. The gretest burden of disposing of cell brekdown products is imposed within the first 2 hours fter trnsfusion hs begun. The extent of this lod will vry inversely with the sie of the trnsfusion, percentge of non-vible cells, nd speed of inflow. It would pper to be sfe prctice to set the lower limit of cceptble stored blood t 7 per cent post-trnsfusion survivl. This certinly provides mrgin of sfety which might not be present were less good survivl ccepted. This concept ffords bsis for the ssignment of upper dting limit of bloods in the severl solutions t present in use in certin blood bnks. Reference hs been mde (5) to the re-utilition of iron derived from broken down cells in the synthesis of new hemoglobin. It is of interest to know to wht extent this blood iron is economied. Twenty-six experiments were completed in which recipient's blood smples were tken t frequent intervls, up to 21 dys fter trnsfusion, thus per- TABLE II Re-utilition of rdio-iron derived from non-vible stored erythrocytes Trnsfused Per cent of rdioctive dt Solution Exp. Dys no. stored Whole Hct. Cells Retined Avilble Regen. ml. ml McGill II ACD reu6 Cells Cells resuspended In sline Cells resuspended in per cent corn syrup DeGowin De.owin Prprt's ACD

7 POST-TRANSFUSION FATE OF NON-VIABLE STORED RED CELLS REUTILIZATION'OF IRON FROM NON-VIABLE STORED ERYTHROCYTES N 8 - L'W * I- 1- hi 6 44 to * /~* ~~~~~~~~~~~4 ~~~~~~~~~~~~~~~'~ 1 U, PERCENT OF IRON AVAILABLE FIG. 5. RE-UTILIZATION OF IRON FROM NON-VIABLE STORED ERYTHROCYTES The percentge of rdio-iron derived from hemoglobin from liberted non-vible tgged trnsfused erythrocytes found in intct circulting red cells (re-utilied) in reltion to the percentge of totl trnsfused cells removed from circultion (vilble). About 6 per cent of the vilble iron is economied. Symbols refer to the following blood preservtives: = ACD-1 = De Gowin's solution O = McGill solution * Cells in 1 per cent corn syrup o = Prprt's solution = Cells in.85 per cent sline. hitting mesurement of mximum re-utilition. Significnt dt from these experiments re shown in Tble II. In 5 of the series the cells were trnsfused s whole blood; in 3 s resuspended cells. The quntity of cells given ws smll, rnging from 19 to 68 ml., verging 41 ml., the contined hemoglobin, verging bout 15 grms. Individul circulting red cell volumes were from 1,78 ml. to 2,52 ml., verging 2,214 ml. Survivl ws from 1 to 17 per cent. The method of computing the percentge of trnsfused cells retined, in circultion nd regenerted, hs been described elsewhere (5). The percentge of iron vilble for resynthesis is tken s the rithmetic difference between 1 per cent nd the percentge retined. These vlues re listed in Tble II, together with the percentge of vilble iron ctully re-utilied. The percentge of iron re-utilied is plotted ginst the per cent vilble, in Figure 5. It is pprent tht bout % of the iron does eventully return to the blood strem in the hemoglobin of

8 746 J. G. GIBSON,. 2ND, W. C. PEACOCK, R. D. EVANS, T. SACK, AND J. C. AUB new circulting red cells nd tht this percentge is reltively constnt within the rnge of the dosge given. Forty ml. of cells contin bout 5 mgm. of iron. Hence the iron dministered s defunct cells rnged from to 4 mgm. Hhn, et l (6) hve demonstrted tht the bsorption of rdioctive iron from the norml gstrointestinl trct, s mesured by the mounts detectble in circulting red cells, is extremely smll. Cru (7) lso found tht rdio-iron liberted from hemoglobin from destroyed red cells ws utilied nerly quntittively even in the presence of norml iron reserves. It is of considerble interest tht iron given intrvenously, s hemoglobin in non-vible cells, is utilied pproximtely 2 times s efficiently s when iron is given orlly. Sufficient dt re not vilble t present to determine the efficiency of utilition when lrger mounts of hemoglobin iron re mde vilble. In mny experiments, however, in which full trnsfusions of very much deteriorted cells were given, the recipient's circulting red cell rdioctivities hve shown utilition of from 1 to 3 per cent of the vilble iron on the fifth post-trnsfusion dy. Since %/2 of the per cent vilble utilied is usully present on the fourth to seventh post-trnsfusion dy, this would suggest excellent economy of quntities t lest 1 times those delt with in the bove experiments. CONCLUSIONS (1) Non-vible stored humn erythrocytes re rpidly removed from the blood strem fter trnsfusion. (2) The rte of removl of non-vible cells vries inversely with the percentge survivl of the trnsfused tgged cells. (3) At or bove 8 per cent survivl, non-vible cells re completely removed in 24 hours; below this survivl level, loss of non-vible cells my continue into the second post-trnsfusion dy. (4) At ny survivl level, the mjority of nonvible cells re removed from the blood strem during the first 2 hours fter trnsfusion. (5) On theoreticl nd prcticl grounds 7 per cent retention of ll trnsfused cells my be considered the lowest sfe survivl level. (6) The bodily economy of iron derived from the hemoglobin of non-vible cells is such tht the utilition is bout 2 times tht of equivlent mounts of iron given orlly. BIBLIOGRAPHY 1. Gibson, J. G., 2nd, Evns, R. D., Aub, J. C., Sck, T., nd Pecock, W. C., The post-trnsfusion surivivl of preserved humn erythrocytes stored s whole blood or in resuspension, fter removl of plsm, by mens of two isotopes of rdioctive iron. J. Clin. Invest., 1947, 26, Hwkins, W. B., nd Whipple, G. H., The life cycle of the red blood cell in the dog. Am. J. Physiol., 1938, 122, Shemin, D., nd Rittenberg, D., Studies on the formtion of heme nd on the verge life time of the humn red cell. Federtion Proceedings, 1946, Vol. 5, Prt II, p Singer, K., nd Weis, L., The life cycle of the erythrocyte fter splenectomy, nd the problems of splenic hemolysis nd trget cell formtion. Am. J. M. Sc., 1945, 21, Gibson, J. G., 2nd, Aub, J. C., Evns, R. D., Pecock, W. C., Irvine, J. W., Jr., nd Sck, T., The mesurement of post-trnsfusion survivl of preserved stored humn erythrocytes by mens of two isotopes of rdioctive iron. J. Clin. Invest., 1947, 26, Hhn, P. F., Ble, W. F., Ross, J. F., Blfour, W. M., nd Whipple, G. H., Rdioctive iron bsorption by gstrointestinl trct. Influence of nemi, noxi nd ntecedent feeding distribution in growing dogs. J. Exper. Med., 1943, 78, Cru, W. O., Hhn, P. F., nd Ble, W. F., Hemoglobin rdioctive iron liberted by erythrocyte destruction (cetylphenylhydrine) promptly reutilied to form new hemoglobin. Am. J. Physiol., 1942, 135, 595.