"BANKED BLOOD" A Study in Blocd Preservation by Charles Richard Drew, M.D., ©... From the Surgical Pathology Laherstory oft the Collese of :nyaiclans and Surgeons, ColumbLe University, snd the Devartment of Surgery, Presbyterion Hogs itel. Supmitted in pvartiol fulfillment of the requirements for the degree of Dooter of Medlecl Sclence in the Feeulty of Medicine, Columbie University. AC KNOWLEDGE SENTS The thought, labor, advice anc monetary assistance of many individuals and groups have mde this work possible. -I am particularly grateful to: Dr. allen 0. Whipple, for the privilege of working in the surgery Department and the pleasure of so many fine associations. Dr. John Scudder, who suggested this investigation and has actively participated in or guided nearly every step of it. Dr. a. Purdy Stout, for the many courtesies extended to me while working in the Department of surgical Pathology. Dr. Hans T. Clarke, for valuable instruction in some of the aifficult spots. Mrs. Ethel B. Gutman, for instruction and use of her methods in the magnesium anc phosphorous determinations. Dr. B. Padersky of Palestine, now a xesesrch Fellow at the Mt. Sinai Hospital, for translations of over twenty of the most important Russian works on blood transfusions; likewise, to Mr. and Mrs. John Ivanoff and Miss Olga Mordwin, 1i for great aid in transluting other Hussian articles and checking the dussian bibliography. all of my associates in the laborutory throughout the perioa of study, in particular, Liss Dorothy Corcoran, wiss kunice Thompson, Mrs. Margaret Nelson, ors. Johnette sarnock and hiss xuth Liebhold. To Dr. Margaret smith goes the entire credit for working out the technique of the ammonia determinations and to Miss Elizabeth Tuthill, co-author of the sodium method, especial thanks for bearing the brunt of most of the later chemical determinations. Miss Helen Stoddard, indefatiguable nurse in charge of the “blood bank", and Mr. Josiah Lasell, hematologist. The General Cdueation Board of the Hockefeller Foundation, for the Fellowship which made this period of study possible. Vhe Blood Betterment Aigsociation of New York city, for funcs to carry out much of the experimental work. Miss Mary “. ourgent, without whose constant aid in every phase of its preparation this work would not have been possible. CHAPTER I CHAPTER II Part I Part ITI Pert III Pert IV Part ef q ry SF th Ba ote ara oh, iad hs Pigs ii, ve as poeta 4 we a ve, Such ebso of use, however, wos poet e@ sudden Jump Prom e asuaren to the present. Yhe presiem of blcoed transfusions has intrigued the gureicsi and mechenicel Ineenulty of numbers aA oY mem. With the srowth of aseptic suraery, Carrel (71) in Ai enelnmmann nana emt fe 1906 perfected a methoa of ensatavoeing the ertery of the Gonor to the bela of & recipient, eliminating eontect with all forelen meteriai and oresentins gn unbroken endothelium over which tne blood could pass, thereby greatiy reducing the chences go iT clotting. Whis was « delicate and difficult retion Cc re) © not elweys successful in the hards of even skillful nae SULRCODS, Gut of tae question for «sreral use. a es Crile, i909, mede this operstion easier by using a Small cunpuls through wsich the vein of the reolpient was a@ravn end curred puck, allowlng the artery to be telescopes over the outturned endothelium of the vein to form a con- tinuous lining of intima. Elsberg (1909), Soresi (1911), and Jeger (1913) modified this method but the method fell into disuse because of the delicacy and difficulty of the operation, the danver to the donor, and the inability to estimate the amount of blood transfused (414). (6) Paraffined Containers Other men attempted to gxet better results by paraffining the containers. Chief arong these were David and curtis (1912), Kimpton and Brown. (1913), Satterlee and Hooker (1914), Perey (1915), and Kreuscher (1918). (7) Athrombit Neubauer and Lempert in 1930 introduced a container made of an amber-like condensation product of phenol and formaldehyde called "athrombit." Burkle-de le Camp (67) constructed a flask of this material almost exactly like Percy's modification of the Kimpton-Brown tube and it is this tube wnich is used with- out anticoagulant in Germany on a large scale. (8) Switches and Velves ( Unger (1915) introduced a syringe cannula apparatus with we Baw a fourewsey stopcock arrengement which allowed outlets to the donor, the reciplent, end a bowl of saline for flushing to prevent clots. Innumerable veriaticns on this theme of complex switches and valves have been introduced, among them Brines (60), Feinblatt ' 22), Scannell (347), Soresat (374, Koster (221), Cashman and Baker (72), and Penneli (313). Beck (24), who invented one of the more elaborate machines, has many interesting reporduetions of the clder ones. Most of them heve failed by virtue of their eleborateness and complexity, but still they come. We ~o beck agein for guidance to Hueter who in 1870 said: "Too much stress is laid on the technic as in all operations which are stili in the stage of infancy. Cf all the instruments only the simplest are kept." CHAPTER II Part ¥V Anticoagulants (1) Early Use The prolonged preservetion of donor blocd depends largely on the quality of the anticoagulant. The use of enticoegulants in biecd seems to have had its inception in that stormy four years, 1664-1668, which really mark the beginning cf modern experimental medicine. gd. BD. Major (62), who contested Lower's cleim to fame as the first animel transfusionist, used ammonium sulfate to prevent coagulution of the blood in 1667. Transfusions goon fell inte lll repute and so the method did not grow. Soon after Blundell (1824) revived interest in the operetion, defilbrineted blood ceme into wider usage for transfusicns and was the method of choice until K&8hler in 1877 pointed out the possible dangers of intravascular clotting following its administration (102). Braxton Hicks (1868) successfully used sodium phos- phate in his obstetrical practice but gave up the method because of the apparent toxicity of blood so stabilized. At about the same time or a little later, 187541880, physiological salt solution, introduced by Latte in 1831, practically repleced blocd in the treatment of acute anemia, 4? an henorrhege, and shock. Lendots introduced hirydin as an anticoasulent in Le92, and it proved very effective for investigstion in vitro of blood for it neither chanszes the cell volume nor. the specifie grevity of the plasma, but ita source was 30 limited and its toxicity s0 veriable thet ite use for humans has not srown (1, 345). In en ettenst to get more uniform results with the Capliisry hematocrit of Blix, introduced by Hedin in 1891, “many attempts to find sultable anticoagulents were tried. Dalend (1891) tried 2.5 per cent potassium bichromate, Blernacki (1994) used e mixture of oxelie seid end sodium 9S xalate, while Koeppe (1995) found thet blood could be prevented from clotting for a considerable period if the tube contelned ceder oil, and Herz in 1898 used ccdliver o1l to effect the same end. (2) Priority in the Use of Sodium Citrate Sebattent, in 1900, suggested that sodium citrate hed an edvantase over oxaletes and fluorides as an auticoagulant Decause it simply immobilized the calcium necessary for Clotting insteed of cresting an insclueble precipitate. It reacts in the ratio of three molecules of sodiwa citrate to one atom of caleium. (132). Safe indirect transfusions date from the introduction of sodium citrate on a lerge seale into the praotice of blood transfusicns. ‘The method was thrown into the medical spotileht by the wer (1914) with its attendant demand for effective methods of treating the wounded. Some controversy arose about the honor of pricrity as regarda the introduction of this method. Search of the orlginel papers esteblishes without much doubt that Andre Hustin of Srussels not only suggested the use of citrated blood for transfusion in april of 1914, but in Kay of the same year reported to the belgian Sureieal Soclety experiments testing the rute of coagulation of blood in isotonic saline, in Kinger's solution, in tive per cent glucose solution, in citrate and physiological saline, and in & solution of 5 per cent glucose in saline to which sodium citrate had been added to make «& 2.5 per cent solution. ‘The last he pronounced as best end then reported results on four . types of transfusions using this preservetive. lL. From one dog to another. 2. From one Rabbit to another. 3. From man to dog. 4. From man to man, He listed the advantazes of the method as being; no surgical operation, known amount of blood given, less danger for donor, many donors for game patient, blood could be oxygenated or treated with other therapeutic agents, glucose good for cells, infusion can be given slowly, and coagulation time of reciplent's blood actuully shorteced (189). On Decenber 17, 1914, Hichard Weil of the General Memorial Hospitel cf New York reported at the Aesdemy of Medicine the results of forty-three transfusions given by the indirect method with the ald of 10.0 es. of a 10 per cent solution of sodium citrate in saline fer each 100.8 ec. of blocd. This work appeared in print on Janusry 30, 1915 (403). | For us, in considering the crigins of the idea of preserved blood for transfusion, Weil's worx is of perhaps the greatest importance because he net only atarted the work in America but wea the very first to use blood kept for three to five days, first to crossexzroup blood ahead of time and keep it on hend where the possibility of hemorrhage existed, and first te report the morphology of cells kept for & wesk or more to determine the rapidity of change under such cenditionsa of preservation. At the seme time thet Hustin wes carrying out his work in Selgium and Weill in aAverioa, Agote wes cerrying on in- dependently in Buenos Aires and presented his work in November, 1914. In Janusry, 1915, he published his resulta under the title of "A New Procedure for the Transfusion of Blood." Richard Lewisohn published in New York the first of a mB Qa long list of papers on the cltrate method on January 23, 1915 (259). He knew of Huastin's work and had heard Weil speak. He has, therefore, no claim to priority but to him core then to any otner individusl gces the credit for populsrizging blood transfusions by the so-called "medical procedure,” for simplifying the technique, and putting th practice on a thoroughly workable basis. 8e has been the grestest protagonist for end defender of the citrete method. Fiscner introduced the method in German literature (128). In kareh, 1916, he reported comparative studies on hirudin, scdium citrate, sodium oxslate, peptone and vlucose mixtures with the direct methods in relation to use in the German armies. On « whole, they adhered to direct transfusion. Hédon (1908),under the impression that the toxie sub- stance in blood was carried in the serum, had been the first investigator to remove end then resuspend the cells in physiological saline for transfusions, He, likewise, was one of the firat to point cut the advantuge or using dee fibrineted blood several hours old. After wen licependent studics, ln 1917 he introduced to french iiterature the Citrate method of blood conservation; and Jeanbreau, at that ime Chief of the French Army Ambulance Service, put it into immediate practical use at the army bases. In July, 1917, he reported the results in his first eleven caseg (192). During the same month, Stansfeld (375) advocated the metnod fer the English Aimsy, but the Britiah Medical Com. mittee was 30 interested in the use cf suf sdacla recom- mended py Seyliss (8£}) a5 e substitute for blood in the fe trestment of shook an es heworrhage that this new and aimpler method of transfusions waa overlooked ror e lone time. Since Hustin's first attempt to find the wost suite able method of using citrate, there have been weany mod. fications of the solutions in which it was basic. There have been attempts to buffer the solution by the addition of slishtly acid ssits as grey (1937) aid. The best known of the physiological citrate solutions Ls the Il. B. T. solution balschovexy prepared st the institute for Hematology and Transfusion of Moscow, sometines referred to as the Central Hematology Institute. It conteined sodium citrate, Bodiwu: chloride, potassiua chloride, and magnegium sulfate With the advent of the American forees into the war, Robertson (337) introduced et first the procedure elaborated oy Rous and Turner in 1916 which consisted cf preserving the eslis for later transfusions by adding 100.0 ec. of 2.8 per cénut sodium citrate and 150.0 ec. of 5.0 per eent clucose to each 100.0 ce. cf blood. Hemolysia was minimal in this preservative, but the amount of citrate present in a trans- fusion of any aize precluded its use for immediate transfusion into human heines. The cells were resuspended in saline just before use snd ea small series of transfusions was carried out with these preserved cella. This vreeedure did not work out well in setual practice and Pobertseon chansed to the oltrate method slready in use by the French. Ne gupolied front Line hospltsle of the Anertean Expeditionary Foress with compact aeta econsiatine of a stoppered bottles containing aterile gitrate soluticns end en Ingres syringe for creating either pressure or suction se thet the blood could be received into and dispensed from the same contelner without nandling. On April #8, 1918, his Pirst report of its use in forty-four eeses appesred (f26). (3) Citreted Blood versua Unmedified Blecd With the intreductien of this new method of trensfugion, there srose the guestion of the relative metita of modified blood and unmodified hload. Lewisoin (1916) reported ex- cellent results with the methed. Fis reactiona (19 per cent) were lese then these reported by Lindenan,in 1914, who used the multiple syringe direct method. Garbht felt that 0.25 per cent was surer then 0.2 per cent and declared the method sere. Syvderstricker, Mason end Elvere (1917) confirmed Lewisohn's work and reported 17 per cent resetions with eitrated bloods of eli teres. ALL of the reports, however, were not so favorable, for in the same year Meleney, Stearns, Fortunl, end erry (288), in & report cf two hundred elshty treanafvstors, placed their reactions foliawing treesfusgicns with rultizle syringe method at G4.4 per cent, with almost idertios#sl figures for the eltrate metros, 64.0 per cent. Thev cencluded thet the method played no pert in the occurence of the resections; “thet neither did any good in tre cese of severe infection or bacteremia; thet euell transfusions red less teudency to be followed oy resections and the tiehest incidence was found in cases of repeated tressfusions, espeelesliy if the donors were used repentedly. Drinker end ‘SO per cent resetion and stated: ‘We should count ourselves fortunate eould we reduce reactions from trensfusicns of whole citrated blood te 45 per cent." Unger (1921) whe becane one e » of the method's early ontagonlets llkewise reported an in- Gidence of #0 per cent. Beruheis (iP2l), Br ines {12253), Bacon (1924) added their warcing thet the tethod was dangerous, while Boffmen (1952), Ioancides and Camerson (1924) felt that the danger wes over utressed. cichner (1227) felt that the Limite of safety were even greater than seme of the earlier authors nad indleated. In this country reactions since 1930 have dropped so precipitously from the everage high lnecidence before that time that some factor besides the citrate itself must have played a role in this sudden improvement. All of the older writers had expected chills following transfusions, and when intravenous saline displeced transfusions to a degree no @lern was felt when they too were followed by reactions of varying degree. This attitude i3 epitomized by Crile who says in his book published in 1909 when Speaking of the course following a transfusion; "A chill of ereater or lesa aeverity followed by a corresponding febrile reeetion is to be expected, and usually cceurs. Urdinerliy, it apperently has no more significance than the chill which frequently follows the in- fusion of seline solution." Not until salvarsen was introduced into medicine and intravenous injections became widespread was atte:tion sharply focused on the “water fever" ag Weeiselmann ealle@ it when he first demonstrated in 1911 that the water itself was the cause of the reaction and not the drug. He desonstrated many actuel bacterie and other growths of « fungua-Like nature in the dia tilled water used (402 Hort and Penfold, the same year, demonstrated that “pyrogenic™ substances were often present even when no bacteria were demonstrable. It was shown thet they developed on standing; were Tfilterable; and gave two types of fever, one immediate and transitive, the other delayed but con- tinuous; probably were of bacterial crigin but were not actually bacteria (185). This most importent piece of work wes overlooked until Seibert in 1923 demonstrated that these "pyrogens" were actually the protein end products of certain river water bacteria which were more prevalent in certain seasons than in others. She carefully ruled out bacteria and specific iron effect as the cause and went on to show that # still outfitted with a proper baffle could prevent the carrying over of these substances into the distillate (360). Fantus, reviewing this phese of the work in 1926, pointed out that not only must the water be freshly distilled but sufficient metal could be dissolved from lead, copper, nickel, or gine stills to cause toxic manifestetions. Not only were glags stills preferable; but, quoting Metzensurer, he points out thet enough alkali may be dissolved from ordinary glass to cause toxic reections (119). This work brought into prominence the observations of Busman (68) that the sulfur in certain types of rubber tubing used for intravenous work likewise would cause reactions. These studies removed some cf the odium ~ BG— from the anticcesulant, but there remaina the question of its action on the blood constituents and this becomes of increesgingw interest when the matter of prolonged preservetion is considered. All of this infermation was slowly put into use. It seems certain that it is ag untenable to try to compare the incidence of nonfatal feorile reactions following citrated blood transfusions before 1925 and after 1930 as it ia to compere justiy the incidence of hemolytic crises before and after the discovery of the blood grours. (4) The Action of Sodium Citrate No better explanation of the apecifie toxie aecticn of sodium citrate upon introduction inte the blood stream has been offered then that of Vietinghoff-Scheel. In 1908, he postulated thet the toxicity was due to immobilization of the calcium ion in the tiaggue, in a uenner aimilar to thet ‘in which Sabattend hed shown it is imecbilized in the serum, thereby producing a clinical effect similer to thet produced by a true hypocalcemia. This wes especially true as regsrds the reaction of the nerves and muscles in the presence of an excess of citrate (397). Gros in 1915 observed « slowing of « frog's heart on which sodium eitrete had been poured. Salent and Hecht Bw in 1915 compared the influence of of cltrates, es well as oxalates and tartrates, by perfusion experiments on the isolated hearts of other animals and observed following citrsetes slowing 3imiler to that observed on the frog's heart. Lewisoln, guided by Hustin's and Weilts work, established 0.2 per cent as the smallest dose that would keep blood from clotting for thirty minutes. He established 5 erans for an individual transfusion es the upper linit of wise therapeutic use, 10 grams os definitely in the toxic realm, and 15 to 20 grams a8 probably a fatal dose (260). Garbet (1916) repeated Lewisohn'ts experinent and con« cluded that a 0.25 per cent citrate solution is more likely to prevent coagulation, without incressing the danger. He set the fatal dose for humens as between 15 to 20 grams (135). Salant and Wise (1916),in e cereful inveatigation of citrate and its decomposition in the body, put the fatel dose between 0.4 and 1.5 grams per kilosram; the reauits veryling greatly in the different experimental enimals used and eppeered particularly dependent on the rate of injection. / They concluded | thet citrate disappears rapidly from circulation and is most toxic in those animals in which large quantities were eliminated unchenged. Overdosage caused only ecute poisoning varying in intensity with the rate of oxidation. Wo subseute or chronic effects could be demonstrated (348). = Baw Drinker and Grittingham (105) after testing many types of citrete solutions discarded the idea that the toxie factor in the blood was due to "fibrin ferments"™ in the plaama or "potential coaguletive"™ factors go sdvocated by Satterlee and Hooker (345) for they found "sitrated plasma, thoroughly freed from ali formed elements, is singularly nontoxie.™ | They | felt that aside from the direct toxic action of citrate, the platelets freed a toxic substance when they were, broken down. Unger (1921) ascribed the untavoreble reaction not to changes in platelets alone but to chanvzes in all of the cellular — ™, elements of the blood. from the erythrocytes, he felt, it ‘, \, oA extracted a substance which Ls able to inactivate complement i by the creation of an anticomplementary substance, He reported _ that leukocytes was destroyed, the red cells were made more op fragile, the opsotins were reduced to zero and complement wes | disinished by direct action. Meny of these findings heve been sibdstantiated by subseguent observers (chapter III) but their part in the causation of resections is not a proven one. There were meny who felt thet citrated bloods were dangerous and caused an unnecesssrily tich percentage of reactions (Bernheim, L921; Brines, 1923; Lederer, 1923; Bacon, i924; Kretaler, 1924; Peauchet and Becert, 1924), and while many transfusions were reported in the late 1980's few atteupta were made to elucidate furtuer the mechsanians or offer sugsestions for prevecting the chenges supposedly associated ie witn increasing toxielty. goannides and Cemeron (1984), in an experimentel study on dogs, observed thet overdosege was followed by convulsiona, | respiratory slowing, and cardiec failure; while Sheliing and Maslow (1924) observed death in their rebbits following the feeding ef eltrate in milk by mouth. Minot, bead, end oryan in 1933, having observed the death of eg maerentic child following a citrate transfusion con- cluded thet since in infantlle tetany caleium was reduced, the citrate probabiy further reduced it by forming en unionized complex in the plasma, jana by Llnereasing tne alkalinity of the NS blood in two ways: by direct addition of the very alkaline ‘ solution and by increused bicarbonete formation ss a result of the oaidstion of the preservative. They produced experimentally relative degrees of hypocaloenie by parathyroidectomiginw dogs, injecting quanidine, end by injecting soda bicerbonate. In each Instance the addition of citrated blood or isotonic sodium / * citrate equivelent to the emount usually given in 4 transfusion ve ; ? (10 cc. of 0.5 per cent citrate blocd per pound of dog) cansed { Slowing of pulse and respirations, twitehing, vomiting, and i déath if the infusion wes continued. In each case, if the i f dog at the level of unconscicuaness was given 3 to LO ce, of | i f 10 per cent calcium gluconate rellef was slmoat imrediate..< » These guthors, therefore, recommend the giving of calcium ¥ «= hO— aluconate, prophylactically, when citrated bloods are to be used in states wnich ere likely to be associated with e hypocalcemia (266). AS & good dercustration uf the ever reourrins cyele of things Sheffer and crismon in 1936, hevine observed the peralyzing effeet of citrate on s Prog's heart, perfused Cats end derconstrated that first the vasus becomes paralyzed; the action teking place at the synapses between the pre end post gargliomic fibers, then the heart muscle becomes direetly affected, venou 2 preSsure goes up, arteriel pressure down, and the heart atops in dlestole if ecslcium salts are not quickly edded (362). There is evidence, therefore, thet citrute imrobilizes caleium in the tissues end causes a picture comparable to acute calcium deficiency ss Vietinuhoff-Scheel had pointed out in 19082, (5) Heparin Heperin has beer used @€8 an anticoagulent in two way Si in vitro in a menner similer to sodium cltrete (Skold, 1936; Tretew, 1937; Sehurch, 1938; Clerens, 1934), and in vive by renderins the cells of the blood of the donor inccagulable by intrevenous injections of heparin e short time before drawlnge the blood (Hedenius, 1956, 1937; Knoll and Senlireh, 1989; Seppinzeton, 1939). &% is an active frection ef the naturaliy occurring anticorgulents, first lacleted by MeLeon working in Howell's laboratory at Johns Hopkins Hospital in 1916 snd named by the Latter heparin because it was found in the liver. n The purification of the substance offered gone difficulties, however, and Magou (1924) who tried Lt in transfusions gave it up because cf the reactions. He reported that in doses of 100 millivrens it wes tcaic for men. Howell obtained s purer product in 1928 and reported es good reenlts on ten patient @ » Seat and hoe vestigations with the hope of obtaining aetive fractions in the sane veer (828). Charles and Scott (75) cerried on this investigation at Toronto and in 19323 obtained a orystelline barium galt of uniform potency. The game yoar gohmi tz and Fischer (358) reported rom Copenhagen # purified product and discussed the chemical composition of the substance in some detail. Jorpes in 1955 sugested that the substence wes a mucoltin polysulfurie ester, end noted thet the loss of the sulfuric acid groups seemed to interfere with its activity. further investigetion of Lltg chemicel properties have been carried out im-this-eitwie by Chargefi end OQleen (1927) whe showed thst if heparin ils given to ean abpimal end is rollowed by protomine the effeat of the bepsrin mey be completely En neutralized and the clotting time brought back to normal rapidly. | Under physiological conditions it 1s apcearently an antithrombese (Mellanby, 1934); under certein pathological conditions it may act as an anti prothrombinese (Howell, 1918); and it has been sugeested by Waters, arkowitz, and Jaques (1938) that the increased clotting time seen in various types of shock may be due to inecréased liberation of heparin. This is extremely Important from the point of view of using heparin- ized stored blood for transfusions in such cases. The results, using heparin as a simple anticoagculent in vitro, do not seem striking in elther one way or the other except that it requires much more heparin to keep the blood fluid encugh to be easily transfused then it does just to prevent couguletion. | | Seppington (1989) has pointed out thet it requires 25 to 75 milligrams of heparin to stabllize 500 ec. cf blood and thet this guentity in every cese exceeds the 0.25 milli- grams per kilogram threshold-dose of the reoiplent, hence in every cese the clotting time is incresged from a normel of ten to twelve minutes to periods of from fifty minutes to two hours, The second method of procedure consists cf heparinizing the donor's blood by the injection of about 1.0 milligrans per Kilogram of body weight intravenously (1.5 ec. of the 5.0 per cent solution), waitin, five to ten minutes, then withdrawing the blood and using it immediately for trenafusing the patient. Hedenius hes reported such success with the method thet in Stockholm it nas replaced the citrate method (168). This mey be go, but set the present stage of development the use of heperin as an anticcagulant for routine transfusions is an expensive snd potentiaily danevercus experiment; for certain special types of cases it offers undoubted advantages. The antidote for heparin is protamine, an amine derived from spermatozoa and fish spawi. Jorpes (1959) feelga that it acts by removing the negative slectric charge and causing flocculation. ao (6) Other Preservatives While eltrate in one form or another has been the anti- “coagulant of choice in most inatences, there have been trials with meny other substances. Roberti, Flandin, and Tzanek in 1921 introduced the use 9 or sufarsenol and reported preservetive properties greater than citrate with less toxicity (122). Norton (1924) advocated sodium iodide while Brines (1926) advoceted ammonium ozxelate and arsphenamine, and in the same yeer MecCracen (60) used sodium aulfate successfully. One of the more interesting pieces of work in 1926 was that of ~ b4— Perry who used lithium citrate with the definite idea of preserving the erythrocytes longer (316). Shs, like Rous and Turner, remceved the cells from the praservative at the time of transfusion and resuspended them in normal galine. Since 1935 there have been 6 number of articles from Itely advoceting the use of sodium polyacetyl dioxy sulfonate under the trade name of "Transfusol." Forti (1937) states that it acts ag an antithrombin, does not alter the blo- chemistry, does not precipitate the salctum, does not lose it qualities on storing, is bacteriostatio, atoxic, and always ready for use in sterile vials of 5 cc. for each 100 cc. of blood. There have been reports by Lettes, Pieroni, Ceecie, end Morgarla sustaining these observations and if all of these reports sre true, this is indeed en ideal anticcagulant (131). CHAPTER IT Part VI Cadaver blood (1) Origin of Its Use a Hoy, \8 Professor We. Ke shamov of } ” harkov in 1927 became in-~ terested in the question of correcting defecta in living bodies by the transplantation of tissue from the dead (364). He felt, as many before him hed felt, that death is only the moment of dissolution of en intricate complex of mutual relations between the seperate tissues of an organism which individually retain their vitality for a verying time after the orgenism as a whole ceases to function. In the first series of investivations, he and hia assistant, Kostriukov, aade hundreds of examinetions on tissue and orgens of animals from fifteen minutes to twelve days after dexth when the bodies were sept at O°C. They observed that after ten to twelve days neurly all tissues became infected and guickly deterloreted. All infection seemed to Sweep out~ ward Trom the abdominal cavity, those tissves farthest removed { ee from the intestinal tract remaintre becteria free longest. If there were other foci of infection st the time of death, they —&« served as the starting pointiof 2 wavesof infection paraiier to that from the Intestine. It was observed that when the tissues were kept close to 2ero temperature even the perltoneum often remained free of infection for ten days walle more distant organs, @.@., brein, pone marrow, ond heart blood, were cood ufter twelve or more days; whiis st 22° only the bones and warrow sre sterile after twenty-four hours, | He concluded, therefore, that tissues from cadavers of previously heelthy individuals may be used by surgeoas, not only some hours but even deys after death, if the bodies are kept at low temperstures. filetov (1957),taxking this advice, has transplanted - corneas from the dead to the eyes of the living with excellent \- results. | Testes te determine the emount of papers by sokolows~1, Lugkovsel, Marat, and sleanndrowitz gives anu account of their findings (140). Tza.ck, long & greet student of the probles o1 trans- nF Bow fusions, was iate in condoning the use of banked blood but his investigetions stimulated pernaps by his skepticism were instrumental in securately establishing meny of the changes in preserved blood (391). Fe This skepticiam was perhaps merited for in Karavanov's report from Shamov's clinic in 1935, where blocd only seven days cold had been used, there were 67 per sent reactions, 25 per cent of which were severe. Filatoy (1935) reported rexotions in 50 per cent and four deaths in the first six hundred fifty-nine tranafusilons, while Grozdov (1934) hed reactions in 41.2 per cent of one hundred forty-two trans~ fusions. As in ahny new departures, results were not too good eat first. Bagdasserov in 1937 reported his results in over six thousand transfusions end noted reactions in 62 per cent when I. EH. T. serum was used, 62 per cent with glucose-citrate solution, 65 per cent with 6 per cent citrate, and 89 per cent with 3.8 per cent citrate. Gnoinski (140) director of the Blological and Hematological Laboratory of the Red Cross Hospitel st Varsovie geve preserved blood its severest test. In 1938, after man dog experiments, he geve blood which had deen preserved in 6 per cent sodium | citrate for 66, 64, 66, 69, 66, 64, 66, and 54 days respectively. In eéch case there was a sharp rise in teuperature; in most, | severe chills; in four, increased urobilinogen; in four, herovlobinuris; and in tnree, severe pain with dyvpsnea. All recovered and all showed improvement in the blood victure. The avert(6 Gg. ount of blocd wiven was 260 ec. fron this exper- fence he concluded thet preserved Lloud wes innocious and of greet therapeutic value. He, apparently, wes not easily ver- turded by te:.peratures of 104 to 105° ror in ore case in which the paticnt wes civer, blood over elwhty days cld, he mede the note that 611 west well,-v:e tempereture “exzen to fall in eight hours and was juite nomial in two days. On august 119, 1938, Durér Jordb oresuized for the Re- publican Army of Spain,under the neme of the Sarcelone Klood Trenafusion service, the best syste. of collection and dia- tripution cf blood yet deviseds\ Tre vervice at one tir.e had inzediate scoess to 2£,900 donurs end distributed over 9,000 liters of blood before rranco's victory. The hernetically setled conteiners were of an advanced desi«n and coriteined blood from & pool of sinilar types under a »ressure of two atmospheres. He used « citrsate-glucose wixture (1 per cent glucose, 4 per cent sodiun citrate). The blood reedy for use was delivered to the fro:t in refri, ereted trucxs or train OHTS. In America during the years 1925 to 1935, while many individuals undoubtedly took blood from donors and saved it until sometime later before giving the transfusion, no orgenized storage and distribution was noted until about 1827. In the aurvey of "Blood Transfusion in America® by Levine and Ketzin which appeared in 1988 no mention is made of preserved blood in any form. | This work was done under the | auspices of the Blood setterment Association of New York City | by means of a questionnaire to which three hundred fifty hos- | pitels responded. | In Mareh, 1937, Fentus instituted at the Cook County Hospital in Chicago a system built sround the principle of having a centrel depot in the hospital where donors could be sent to have blood drawn and stored for future use. He called this system a “blood bank [enc in his original report stated: be SJust as one cannot drew money from a bank unlesa cne has de- posited some, so the blood preservation department cannot Supply blood unless as much comes in as goes out. The term *slood Bank® 13 not a mere metaphor." by the end of its second year of operation, the orizinal bank, whose long back- ground has been truced, had handled over four thousand trang- fusions. During this period there were three deaths due to the procedure. In the first year there were 12,2 per cent reactions. For the seeond year the reactions were roughly 6 per cant. In 1989, Doctor Sohirmer, full time director of the bank, reported at the Round Table Discussion of the American College of Surgeons that the total had reached over esleht thousand transfusions, that there had been no deseths in the last four thousand, and reactions had been reduced to 1.8 per cent. Just how remarkable a stride this represents can be spprecieted when it is remexbered that in 1986 when the question of facilitating blood transfusions came up in Leningrad e@ record of only forty-two trensfusions could be found. This was in the institution which in a few years was to become the world renowned Institute of Blood Trensfusions (126). In the slow, simetinea stageering, rise of the operation from the stagnant pool of humorel pathology to an accredited place in the rapidly moving stresm cf modern medicine, it has come to a place where evolution mey approach revolutionary proportions. Trenafusions carried by this new found power, the blood bank, mey be overdone; blocd has lost not only the awe it held for the enecients but perhaps some of the respect it deserves from the moderns. Ita limitations must be re~ coenized, its indications orystallized, and its indiscriminate use condemned. Already many studies have been made and more are being made to more clearly define the changed properties of this living tissue, in reapect to the time it has remained cutside the body and its effect on re-introduction. To in- vestigate some of these properties ig the purpose of the Studies to follow. CHAPTER ITI REPORTED CHANGES IN PRESERVED BLOOD Fart I Blochemiesl Chrnges (1) Hemoly sis The most cbvious change in preserved blood ia the aradual hemolysis thut teres plece. Any change in the external medium of the cell is likely to alter the cell membrene. Stewart (1909) offered both histological and physicochemical evidence to show that alter:tions of the superficlal layer cf the envelope plsy an important, often decisive, part in regulating the exchenge between the corpuscles and plasme. He felt that the native blood pigment probably is present in combination with the stroma constituents in the form of « gel. When weter passes intc the erythrocyte through the altered envelope, the hemochrome gel is transformed into an agueous solution of hemoglobin. This aqueous hemoglobin insofer as the cell is concerned is a foreisn body and aa euch is extruded. The envelope is” plement free a:d hes no affinity for the blood vigment. This hemochromolysis, the change of hemoechrome into hemo-~ globin, mey be distinguished from stromstolysis. The former is accompanied by «= relctively amcll, the lstter by 4 rélstively large escape of electrolytes from the corpuscles. To Stewart there seemed evidence enough to sugsest that the ele ctrolytes which escape may be divided into three fractions: a@ portion presumably in solution, which escapes with even the gentlest methods of laking; a portion, hypothetically in loose combination or adsorbed, set free only by more energetic methods; and a portion, more intimately ea part of the cell structure, which is freed only by such strong acticn as incineration. This work was one of the earlier, better attempts to oriticaliy analyze a phenomenon which had long been observed. Observations in this laboratory indicate that there need not be any relation between henoglobin loss ami electrolyte loss from the cells (Chapter IV). The toxicity of hemolyzecd blood has been ascribed to many different factors. aAmberson (1937) in a most complete summary of the subject reviews what have been thought to be the chief etiological factors in this toxicity. They are: (1) strom as Toxte agent. Beginning with Sehmidt in 1875 a great number of workers have ascribed the accelerated clotting of blood upon the addition of hemolyzed blood to the stromate of the laked cells ($75), while Silberman (1866) believed that it was not the stromata of the rea bleed eslls so much as the fibrin ferment released from destroyed leucocytes(367}). It has been sugfestéed that the whole phenomenon my be cue to the mechanical blocking of the smllest blood vessels (93). (2} Improper ion balance. Kronecker (i862) first suggested that the toxic effect of lsked blood might arise from the high potassium content of the hemolyzed cells. Brandenburg (1905) showed that bloods with celis with high concentrations of potassiun such ss found in the human, dog, pig, guinea pig, rabbit and harse were very toxic for the frog heart and that bloods with lower potassium contents such as the celf, sheep, goat and eat, being very low in potassium were not very toxie (55). a (3) anaphylactic Heactions Heidelberger and Lancsteiner (1923) showed that hemo~ globin is a weak antigen, hence a second injection of hemolyzed blood may give anaphylactic shock. (4) Vascconstrictor .ction Many authors have described a vasotonic action. Moldavan (1910) showed that whatever the toxic substance is in freshly defibrinated blood, it has a tendency to diauppear, et least in part, in about one half hour. stevens ana Lee in 1584 showed that whatever the vasotonin is, it is associated with the phenomenon of clotting for there is reletively littie of it in citrated plasms. Goldberger (1932) distinguishes between ~G fa a lablle, rapidiy disappearing substance, and a more permanent stable one; the latter derived from disintegrating cells and failing to appear if the cells ure removed at once, amberson has noted the marked kidney damge in a hemo- lytic crisis following a transfusion or after massive in- Jections of hemoglobin experimentally, when the si bstance collects in the form of crystals or granular casts in cap- sule and tubules, yet he hss shown thet these conditions per se are not lethel or irreversible. Since this crys-~ tallization takes place in an acid urine it is wise to alkalinize the patient or experimental animal (96). The relation between hemolysis anc trauma, especially the type associated with heating of blood, has been stressed by Depp (101). Levine (25%), in a concise experiment, shaved thet the effects on animals given overheated blood were very similar or exactly like the reactions seen after heterclogous transfusions. «after the injection of severely hemolyzed blood death followed as the result of vasospasm ana emboli in the lung tissue. in the anuria following hemolytic crisis with spasm and plugging of the kidney vessels, Hesse and Filatov (1952) advised denervation of the kidney followed by repeated large transfusions of com~ patible blood. From the gathered writings uf many suthors thia may be concluded; thet there is a toxic substance in hemolyzed blood, therefore, hemolyzed blood should not be used for trensfusions, but apparently that toxic substance ig not hemoglobin. {2) Glyeolysis in Preserved Blood. ivans (1922) showed that there is rapid decomposition of glucose with the formation of lactic agid in shed blood. This has a marked effect on the blood in two ways: (1) the pi goes down, the blood becaming more acid; and (2) the CO, tension goes up as the process continues. This observation has been confirmed in the studies on preserved blood by many authors including Bagdassarov (16), Gnoinski (140), Jenneney and Vievoz (199) and Jorda and Diez (208), each of whom has stressed the fact that a glucose sOlution 16 a particularly good medium for the preservation of red blood cella. The following chart expresses at a glance the conmon observation that the glucose value goes down and that of lsotic acid goes up. From Bagdassarov The study of glucolysis led Jorda and Roos to study other ferments. They found that there is no sppreciable change in the ures coneentretion of preserved blood and concluded that the low temperatures at whieh the blood is stored completely destroys the vreolytic diesteses. Amylese, likewlse, is graduully destroyed. The presence of glucose neither retards or saceleretes this process (210). (3) Vital Cupseity Shamov and Kostriukov (1929) in their earliest experi- ments on cadaver blocd established the fact that the vitelity of the red blood cells as measured by ita ability to carry on gaseous exchange remained intact as long es the cells lasted. This work wes reinvestigsted and confirmed by Skudine and Barenboim (1931-1932) in Yudin's laboratory before he used cadaver blood for human transfusions, The Leningred Inatitute in an attempt to preserve the cella added hydrogen peroxide to preaerved blood. This, however, was done not go much to meintaln function ss to prevent infeotion for they hed tried both urotropin and the salyciletes. Hemolysis from any ceuse, but especially in- featicon, greatly inore.:sed the rate cf oxygen consumption (16). Ponder, in @ prélininary report of his studies to the Blood Transfusion Betterment Agsocisation of New York, had ~95— this to suy: In bloods stored three weeks “no hemolysis was oO: served, their resistance to hypotonic saline was unebanged within the limits of experimental error and the oxygei consumption of the cells was unchangec. This last point is cuite remarkable and wholly unexpected. The respiration cf the red cell is a very delicate indication of its state of vitality and we had expected that it would fall off steadily, if for no other reason than that the reticulocytes mature into cells having «a very much smailer oxygen consumption.” Yonder pointed out that in those cases where oxygen consuaption suddenly increased enormously (10 - 100 fold) contamination of the blood by bacteria was always the cause, yet without such a fine test this condition woulda not have been suspected. He concludea that absence of lysis and general satisfactory appeerance of the blcod does not mean that it is not contaminated. Novak (296), to guard against such contamination, has sugested the use of suifanilamide in all preserved blood. ingeniously taking advantage of the fact that the rate of oxygen consumption increases with infection, Jorda (205) enclosed all blood in hermetically sealed containers under two atuospheres of pressure. The amount of oxygen in this quantity of air was sufficient to turn the blood a bright red. -96- If, however, the blood had been contamin: tea, the oxygen consumption increased so rapidly that the blood would turn dark and venous in character, thereby serving as a subtle and automatic indicator of the fitness of the specimen for use, (4) Protein Changes and the irocess of Deamination. Bodanov, Kagan and Depp, in 1934, made rather complete studies on the proteins before and after transfusion of fresh blood. ‘these studies were not carried far encugch to establish complete values for preserved blood but the protein changes in preserved blood apparently did not vary greatly from fresh blood in their results on the protein picture of the patient. . typiesl protocol is as follows: Total ked Cell tlasma serum Fibrin -rrotein frotein irotein Protein Before transfusion 16.55, 12.26 4.29 2.9 1.5 after transfusion 19.80 15.50 4.24 3.4 0.83 Increase per cent 19.6 # 19.58% -0.3% 3.0% 2.84 Knoll (1939) showed considerable variation of the albumen- Elobulin ratio with different preservatives, the inversion being marked in both heparin and vetren (another heparin preparation) in the second week with a normal 7:3 inverted in each case to 2:5. No explanation for this marked change was offered. In “O97 — citrate and in citrate glucose the change was not marked. conway and Cook, after preliminary studies in 1930, presented evidence in 1959 that shed blood rapidly loses ammonia if the carbon dioxide tension is not maintained at @ level sufficient to prevent shift in pH. No actual stuiies heve been reported on stored blood, but the question of deamination in preserved blood is of great interest because of the effect free anuonia has on the permeability of cell membranes ana the redistribution of inorganic elements through this altercca membrane. This phase will be entered into more fully in the section devoted to experiments. (5) Changes in Hefraction and Viscosity. Jeanneney, -angernez, Leynarie (1938) concluded after a careful series of refraction experiments that the index of refraction in the plasma of preserved blood for at least 15 days is essentially the same as that of fresh blood. The values varied between 1.3480 and 1.0366. Knoll (1059) has reported an increase in viscosity in citrated plasma from 1.64 to £2.82 in three weeks, This per- sistent increase in viscosity played a decisive: part in the attitude of the Moscow Hematological Institute which determined the use of their special preservative in equal parts with the blood. ~9G~ (6) wsedistribution of Inorganic }lements Between Plasma and Cells. Duliere in 1831 reported 4 constant enrichment of plasms potassium. This work was confirmed in this labora- tory in april of 1938 and later by De Gowin (1939). In July (1936) Jeanneny anc Servantie reported similar findings and in October reportec that as the potassium went up socium went down. Knoll, ina very extensive study compared the changes in solutions of sodium citrate, heparin, vetren, the Leningrad solution and the iioscos solution. There were cifferences in the various preservatives, glucose citrate giving the best results. These conclusions were as follows: calcium remains almost constant, potassium gracually increases, sodium gradually decreases, chlorides decrease, alkaline reserve as measured by carbon dioxide content gradually diminishes and undeter- mined nitrogen very slowly increases while indican decreases. kalukhovexy and Gingburg have reported a slow rise in plasma phosphates (17). ~ Gi CHAPTER III WORPHOLUG ICaL CHANG! 3 Fart IT (1) irythrocytes ned célls are well preserved whether they be in cadaver, placenta, or donor blood. There is sone cifference between the rates at which deterioration taxes place but the reports are sc untform from the caifferent clinics that ind ividuel atatistics are of no sdded value (98, 198, 206, 411). In citrate there muy be a loss of 1,000,000 to 1,500,00G in thirty days. Gnoinsky has divided the deterioration into five stages: (1) Stage of onduletion, (2) Stage of crenation.s (3) Stelliform phase. (4) Koribund phase. (5} Ghost cells. Bagdassorov (37) has pointed out that these phases do not hole for blood in glucose for there in the early stages instesd of the usual shrinking in size the cells msy actually become and remain larger for awhile (16). aLl agree that the addition of glucose to the preservative definitely aids in the preservation of red blood cells. (2) Hemoglobin There is no divergence of opinicn in the ob servation that hemoglobin levels remain constant or are augmented slightly -100- by evaporation of fluid. t Popova (1934) measured the resistance of the hemoglobin by messuring the time in seconds it takes for the oxyhemo-~ globin spectrum to disappear after adding sodium hydroxide to the blood. For normal males the average time is 60.5 seconis, for females, 56.2 seconds. Flscental blood has greater resistance than thet of the mothers from which it came ang preserved blood resistance ia increased to 72.5 seconds or higher. (5) Leukocytes Unger (1e22) showed thut the addition of citrate to blood caused marked changes in the form and function of white cells. wJuran corda (204) was among the first students of preserved blood to point out that the leucocytes are gradually destroyed and become arorphous masses by the fifteenth to the twentieth day. The polymorphonuclear leukocytes are the least resistant and disappear in about one week, the eosino- philes most resistant and the lymphocytes intermediate in the length of preservation of normal contour. Glucose did not affect the destruction of these cells. Pzanck and vreyfuss (391) pointed out that the monocytes may completely dise ppear in from three to four hours and this observation wae confirmed by Gnoinski (140). xolmer (230) reported obvious changes in the leucocytes in twenty-four hours. in our iaboratory these ~101- changes were observed before any literature had been seen, (4) Flateletes brinker and Brittingham (1919) felt that one of the chief causes of reactions following transfusions with citrated blood was the destruction of the platelets with the Liberetion of toxic substances. «ll subseguent investigators have concured in the observation but not the conclusion. #y the end of a week, the platelets have fallen to levels between 10,000 and 50,000 and in some preservatives have completely disappeared. (5) #ragility of surythrocytes The red ceils become more fragile as they grow older. Lepp (1934) reported that for the first two days the fragility remains unchanged, approximately 0.44 for partial hemolysis and 6.24 for complete. From the third dsy, there is an in-~ crease which by the slxth day reaches 6.6, the seventh day 0,76-0.80, the tenth day 0.85, and on the eleventh and twelfth day the first signs of spontaneous hemolysis begins to show in blood..stored in citrate. The addition of glucose retards this hemolysis to about the thirty-fifth day. Both shaking and warming increase the fragility. All subsequent Observers have simply confirmed these findings, the results vary ing but little from ccuntry to country. ~LOL- (6) Prothrombin ehile prothrombin in the strictest sense is not a morphologienl element, it is included here es a convenience. Results concerning its changes have not been as consistent as those for the countsble elements. guick, stanley Srown and Beneroft in 1935 showed that this substance or quality is the precursor of thrombin, the active coagulating enzyme; end that it is remarkably constant in fresh normil blood. Brinkhous:, smith and sarner (1937) showed that serious hemorrhage does not occur as a result of prothrombin deficiency until the level sinks below 20 per cent. Khoades and fanzer in 1939 reported a fall of prothrombin to ineffectual levels within a week after storing preserved blood. These investigators used the method of juick in deter~- mining their levels. in vecember, 1959, Lord and Pastore using the Brinkhouse, smith and garner method reparted that banx blood is an adequate souree of vlasma prothrombin for ebout nine days and reaches 61 per cent of normal by the end of the thira week. They were unable to account for the difference between their results and those determined by the .uick m thed, but did stress the importance of good refrigeration as a large factor in preserving the prothrombin content. (7} Sedimentation rate ~1LO3- Jeanneney and Vieroz (1934) determined that the sedi- mentation rate of preserved cells gradually decresses with age. at twenty days it may have difficulty in flowing. Fourestier and Faillas (132) state thet a rapid sedimentation rate in the donor is perhaps a frequent cause of post-trans-~- fusion reactions. «at the Cook County Blood Bank it is re- garaed as one of the most impartant findings in rejecting a donor (287). of particular interest is the theory presented oy Jeanneny, in which he postulates that sedimentation rate Chunges and perhaps some reactioas are due to the electrical charge on the cells; ¢. @. assuming «ll the charges on the cells are positive, trauma in any form might change the charge on the injured cells to negative, then instead of a utuaily repellant foree of like charges in the suspension, there would be attractions of eells lesdine to clumping in the fora of pseudo-agglutinaition or even trus agglutination, with ell of the clinical sequellae consequent to such a eondition (138). (6) Length of life of Lrythrocytes asby (in 191%), after a thorough review of the literature, decided that the published statistics on the length of life of transfused erythrocytes was too short and the methods used te determine thea inaccurate. She attempted to cot wore accurate results by transfusing « patient with plood of ea sroup other “104~ than his own, then by treating specimens of this patient's blood with serum thet agglutinated his cells, leaving the cells of the transfused blood intact, she eould count the unaggelutinated cells and trace the time of their disappearance. She concluded that the life of « fresh donor's erythrocyte in the body of the transfused lives et least thirty days, at times one hundred and ten deys. Martinet (1938) stimulated to eheck the findings of aghby, ofter the severe criticism of Gérl (1926), by means of the agglutinogens & and N, determined that fresh transfused ecils remained in the recipient's cells at least sixty days. He determined this by giving, for instanee, the blood of an ON conor to an ON recipient, then collected two tubes of plood. ‘“o one (1) he added anti-M serum and to the other (2) anti-N serum, In tube (1) the cells of the recipient containing wN are agglutinated, while the donor's cells con~ taining only N iseagglutinogen remain suspended in the scrum and give it a eloudy appearance. The end point is reached at the tine at which the serum becomes clear, indicating the final destruction of all the donor's cells. In tube (2) since both donor and recipient cells are acted on by anti-N serum both are agglutinated and the serum becomes limpid at once. The test lies in following, in this case, the degree and final disappearance of the cloudiness in tube (1). «“lO5~ Philip Levine, using this method, was able to report at the Americen College of Surgeons meeting in Philadelphia, October, 1939, preliminary studies (to be published later) on preserved blood of group M transfused into group N individuals with the followings results: Fresh blood 95+ days Three dsy old blood 80 days Ten day old blood 60 days Fourteen days old blood 20 days ~LO6- CHAPTER III Part III Changes in Immune Properties (1) Coaplement— carly opponents of citrated blood transfusion (Unger 1921) advanced the ob jection that sodium citrate destroys the complement. Kolmer 1939 states: "This, however, docs not appear to be true, indeed the reverse appears to be the case as sodium citrate in 0.35 per cent concentration apparently preserves the complement of human blood in a remarkable degree, analogous to the preservation of guinea pig com- plement of the Wasserman reaction by sodium acetate and sodium chloride." he did find, however, that at the end of 7 - 21 days there was a loss of bactericidal activity for one or more of three test organisms. «hether this was due to gradual inactivation of complement and opsonins, loss of bacteriol- ysins and other none specific leukins or plakins wes not determined. (2) Phagocytosis Phagocytic activity wis rather markedly reduced in seventy-two hours and there was alnost total absence of phagocytosis on and after the seventh day. Here again -107~ Kolmer felt: “the marked changes in the opsonophagocytic activities may have been due in part to deterioration of opsonins but undoubtedly were mainly and most likely entirely due to autolytic changes in the neutrophiles." gorda and Llorach (209) found that preserved blood lost half of its complement by the seventh day and by the twentieth is almost completely destroyed, the loss par- alleling fairly accurately the loss of white cells. Jeanneney, Castanet, and Cator (June, 1939) have made an attenpt to measure accurately the hemobacteriacidal power of the blood. By preparing dilutions of the organism against which the blood is to be tested of from 1,000 to 25,000,000 per cc. and adding to each an equal quantity of blood, hanging drops may be made from a gelatine bouillon after twenty-four hours which will show the bactericidal power in each dilution. This may be recorced in terms of percentage and used as an index from day to day in following the course of a patient. Using this same test he has demonstrated the gradual fall in the bacteriacidal power of preserved blood. -1L08- CHAPTER IV EXPERIMENTAL STVDLES IN BLOGD PRESERVATION Part I Repartition of Potussium in Cells and Plasma It hes been the eommon observation of all investigators of blood preservation that if the blocd is kept long enough it invariably becomes hemolyzed or laked (chapter III). Fron the earliest transfusions (Denis, 1667) severe and fatel re-~ actions heave been associxted in an ill-defined but constant manner with hemolysia in the blood stream of the recipient; Since the use of preserved blood has become more widespread, the degree of hemolysis observed in the donor's blood has been used as @ rough index of the suitability of such blood for transfusion (121, 148, 411} Naunyn in 18868 first deacribed the teoxle properties of leaked blood. His observations heve been reaffirmed by many subsequent investigators beginning with Landois, but the nature of the toxic substance or substances has yet to be re» veeled. The purpose of the experiments thet follow is to throw new light, if possible, on the nature of this substance. Phemister and Hendy reported in 1927 that there is some- thing in slightly laxed bloods which ecauses vesodilatation while more severely traumatized bloods cause vasoconstriction. y ~109- It is not histemine, the product cf adrenaline disintegration, nor the pituitary principle. It is not developed as the result of changes in oxygen, carbon dioxide, or hydrogen ion concen- tration; shifts in temperature; or exposure to light or air, Petroff observed vasoconstriction cf the splenic, renal, and pulmonary vessels following the injeetion of hemolyzed blocad, but did not isolate the causative fectors (319). Amberson has established the feet that the tcxle factor is not heno-« globin (5). Kronecker as long ago a3 1862 augeested thet the toxie effects of laked blood were due to its high potassium content. The report of Duliére, in 1931, of a constant enrichment of the serum potessium in whole blood stored for several deys raises again the question of the relation of this texie sub- Stance to the unfavorable results still encountered too free guently in the operation of blood transfusion. The celis of & healthy human blood in vive contein twenty times as much potassium as the glasaa. Any dislooation from its natural environment introduces the possibiiity of altering this normal Gistribution of the chief bags of the cells. Altered structure is tantamount to altered function. Sanked blocd, therefore, seems worthy of reinvestigcation from the polnt of view of its alterec chealesl composition. ~110- First Series the first group of experiments was designed to check Dulitre's statement. Two semples cf the same human venous blooG were ccolieeted with aseptic precautions and kept in pyrex flasks stoppered with cotton, in the dark, in a re~ frigerator at 4° C. in the following manner. Experiment 1 The dDlood, £50 cc., wes kept under Liquid petroletun, Experiment @ The blood, £50 oc., was mised with £.5 per cent solution of sodium citrete in sufficient quantity to make e mixture conteliing 0.31 Gm. per 100 oc. of blood. At the sane time H Oo oc. Semple was mized with heparin (Connevght) in « centrifuce tube and Spun Yor one hcur. This served es the sample for bese line determinations, Portions of the serum or plesma were pipetted off at twenty-four nour lntervels for five deys and then at approx imetely weekly intervals for a zenth. With each anélysis, materia; for culture was teken and streeked on blood ager pletes. These were ovserved for srowth at the end of twenty~- four and forty-eight hours. Methods Potessium detersinations were done throughout by the argenticobaitinitrite method of Kramer and Tisdall (£81) -ili- as modified by Breh and Gaebler (57) and further refined by Truszkowaki end Zwemer in 1936. The finel readings were made on the Evely photoelectric colorimeter (117), The values given are the mean of two aliquots. Cell volume wes determined by Sanford-MeGeth tubes (343), spun for one hour at 2,000 revolutions per minute. Specific gravity was measured by the method of Barbour end Hamilton (19). The plasma protein content was calculated from the specific gravity, by the formula of Weeoh, Reeves, snd Goettach (416). Results Blood Kept Under 011 Without A Preservative The blood of the donor, Doctor J. 3., was of group B. Procedure! In addition to the determinetions made at the time of the bleeding which served as basic values, eight other sets of analyses were made, and the results are expressed in four wavs (table 1): i. Column 4; actuelly observed; serum potassium as milligrams per hundred cubic centimeters of serum. £. Column 5; by calculation as willigrams of potassium in the serum of 100 oc. of blood. This figure for practical _ purposes gives at a glance the actusl amount of serum potassium in every hundred cubie centimeters of blocd on any given day when preserved in the atated manner. 5. Column 6; by calculation es milligrams of potassium ye ~112- given off Into the serum by each hundred eubic centimeters of cells. This figure is theoretically more convenient for comparative purposes, as it obviates the differences created by bloods whose cé@li volumes may vary markedly from normal, 4. Column 7; as percentage of potasaium which has a@iffused out of the cells. Hach day's velues repregent the gum of the inorements for thet particular day, care being taken throughout to estimate the amount of potassium removed in the various test semples. Basic Value: Hematocrit reading D4e5 per cent Cells 45.7 per vent Serum Plesme potessium 21.0 me. per cent Whole blood potassium 202.0 mg. per cent Cell potassium (calculated) 354.0 me. per cent Plesma specific gravity 1.0274 Plasma proteins &.87 Gm. per cent Calculation: (a) For base line or zero velues Blood sample 250.0 oC. Serum volume 250 x .457 (per « cent serum) = 114.4 oo, Determined serum potassium 21.0 me.eper cent Total potassium in original serum 24.0 ma. (b) For first day's increment. 2 ec. sample taken. Serum potessium (observed value) 40.0 mg.per cent Potassium in 2 ce. sample 0.8 ma. Potassium in residusl serum 114.4-2x.4 = 45.0 me. Total potassium in serum efter 24 hours 45 + .6*45.8 mg. Increase in total potessium in serum in 24 hours 45.8 ~ 84,0 (originally present) = 21.8 me, -1l1L3- Increase in serum potassium of 100 cc. blood in first 24 hours 21.8 x 100 = &.7 mg. (ce.) Amount of potassium given off in first 24 hours by each 100 cc. cells 21.8 x 100 16.0 mg. 250 (Tec. biood) x .54@ (% cells) Percentage of cell potessium diffused out into serum in first 24 hours 16( amount lost) = 4.5 354 (original cell potassium) In a similar manner each day's results were determined and expressed as the sum of the increments. The results are shown in teble 1 and fievre l. Experiment @: The same steps were repeated in experiment (2) end the results are grephicelly represented in figure l. In each of the two flesk there was a steedy rise in serum or plasms potussium. In the hematoerit tube, however, for the few deys observed, the increment was less and sugsested either heparin was a markedly superior preservative or the Slow diffusion was the result of some other factor, In each instance, there was discernable discoloration of the supernatant fluid by the fifth day and obvious hemolysis by the fourteenth. Discussion The findings in table 1 sugvest that blood kept under oll at a constant temperature loses in the first week at least 25 per cent of its cell potassium, at the end of three weeks about 40 per cent and diminishing quantities from thet Table l Experiment 1 Blood Kept Under Oil Without Preservative 1 2. 3 4 5 6 7 8 Date Sample Days Milligrams of Potassium Percentage Henolysis . Observed Value Sum of Increments of Cell K (Observed) Per 100 Cc. Per 100 Cc. From 100 Cc. Diffused Out of Serum* of Blood of Cells 3/27/38 1 1 40.0 8.7 16.0 4.5 0 3/28/38 2 2 68.6 él.l 39.2 11.¢ 0 3/29/38 o 3 83.0 27.428 51.0 14.2 0 3/30/38 4 4 100.8 35.1 64.7 18.3 0 3/31/38 5 5 133.2 48.6 89.7 25.8 0 4/ 9/38 6 14 200.0 76.1 140.5 59.6 ++4+ 4/15/38 7 20 206.4 78.6 145.2 40.9 F+++ 4/29/38 8 34 225.0 86.0 158.0 44.8 +++ *Values uncorrected for sample removed. “VEtt~ FIGURE 1 Potassium as mq °/o 144 Hours Data from experiment 1, table 1 Heparinized blood kept in centrifuge tube. The other bloods were stored in Erlenmeyer flasks. -~114- time on. Stated in more practioel terms, guch biood «st the end of the first week contains in the serum cf each hundred cubic centimeters cf bloed at least 50 me. of potassium and at any time after two weeks at least 75 me., the simount eredueally increasing Second Series The second group of experiments was deaigned: (1) to check the results of the first series, (2) to ascertain the effect of trauma (such es shearing) on the rete of loas .of potessium from cells, and (3) to see what effeet the siape of the container had on the rete of diffusion. The dlocd of the donor, Doctor G. S., was cf group 0. Five nundred ce. of blood was collected and placed in three pyrex flasks. Experiment 3 The blood, 150 ce. was -_ -_ gooi1g 30 5001 NI W VWNSV 1d 15 20 25 30 DAYS 10 CITART- BAPER- NO PTH tds Each point on the chert represents a determination done on a different sample each day and represents the actual amount of potassium in the plasma of 100 ec. of blood. FIGURE 9 us Z a t co WwW ” - > 0 o Ww a O _ O O > o QOwWUg O < 5 Soe S ras Set rT 5 OF Go + — ° LW oO eo aT O 5 q 2 7 no a 0 nu a > W - °o N Z W Wl i © Z <{ Tr 0 2 ao _ O ln J O Lt x O I F = ° Yr uJ oO n ° on + oO Oo oO “ + = | ” oO w qe hk wo OQ oO OQ 1 iS a & S € e a que0 aed ‘bur se wintssei0g Days In fresh blocd shaking causes only slight increase, in older bloods & more merked increase, in still older bloods such a large percentage of potassium has already left the cells that the increase is less merked. ~145- set up. Method ahe blooc of two woluntery donors, ¢. Hh. DBD. and Jd. &., was arawn at weekly intervals and placed in 50 ec. colori- meter tubes containing $ ec. of 3.5 per cent sodium eitrate. at the end of two weeks following the drawing of the lust two acmples, the previcusly drawn samples were removed froma the refrigerator were they hac been kept at a tempera- ture of 5° and 6° ¢. since the time of the phlebotomy. » Siill sample of the plasma was taken frou each of the six tubes for potassiun determinstion. Then all of the tubes were rotated end over end on 4 specially devised piece of apparatus for twenty minutes, centrifuged, and from each Samples were tuken for potassium determinations after the rotating. Hesults Since only comparisons between the Levels before ro- tating anc efter roveting were sought, no attempt haa been made to correct the figures for dilution caused by the anti- eougulant, or in the figures obtained after rotating for the Plasin rodioved tu test the vesting samples, fter rotating Vidues are, thereror, svout twenty per cent too high but -146- since they are codpared only to esech other, the percentage change will indicete true differences. Results are tebulated in table 1%. Discussion This experiment was unsatisfactory in several respects. the results, however, are obvious. In one blood there was an increase of 8.5 per cent in the plasma potassium after rotating on the day it was taken compared with a %%.5 per cent inerease at the end of two weeks. in the other blooa the difference was greater 1.2 per cent when fresh to 152 per cent two weeks later. vonclusion vhe diffusion of potassium from red blood cells to the plasma folloxing trauma inereases rapidly with increasing age of the blood. This suggests that any transporting of blood should be done as soon as possible after its with- drawal from the donor to a place near the intended recipient. This suggests thet blood should be drawn and immediately transported to a plsece near the intended recipient. Table 19 Inereesing Effect of Traume with Inerssxaing Age of Blood Time in Cc. R. DD. J. &§. _ Weeks Plasms Potassium Changes rlesme Potassiug Chenge as Miliigrems in as Milligrams in Per Cent Per Cent Per Cent Per Cent Before After Before After 0 14.1 15.3 2.5 20.8 21.3% 1.2 1 45.8 24.0 Bh. 58.1 21.0 132.1 60.1 118.0 “99.5 60.3 140.0 132.2 nS ~“VOVT~- ~147~- CharPten LV Fart VI Changes in ireserved Blood as 4 Function of the Composition and Shape of the Container when Hergz (1898) attempted te use the recently intro- auced .edin-Blix (169; hematcerit tube for estimeting cell voluxe, he found his results much more uniform when the plpette hea cod liver oil blown through it before use. Koeppe (229) used cedar oil and Freund (135) vaseline. Bordet anda Gengou (1901) used praffin to prevent corzgulation during some of their now classical experiments in immunity and in 191i Gurtis and iuvia applied this principle to the practice of transfusion, a method which reached its peak in the 11/23/37 F.F. 58 526868 Perforated duodensl ulcer None 31.9 8.2 ! M 2/24/38 D.P. 25 542008 Paraganglioma of adrenal Partiel re- 34.1 8.7 F cortical tissue section. Op- erative death 6/ 5/38 M.L 45 374569 Chronic cholecystitis, Cholecystectomy, 28.6 7.3 F cholelithiasis, subphrenic incision and and subhepatic abscesses drainage ab- scesses 9/12/38 J.Mc. 70 556217 Carcinome of colon with Exploratory 26.3 6.7 M metastases to liver Tinh i; as Laie 20 Plasma Potassium of Cardiac Blood at Death (continued) Date In- Age, Hospital Diagnosis Operetion Plasma itiasals Sex Number Potassium Me. per Meeq./L Cent 9/27/38 M.K. 26 550662 Intestinal obstruction Ileostomy 28.9 7.4 F complicating pregnancy ll/ 4/38 L.W. 31 949037 Mesenteric thrombosis Enterectomy 38.0 9.7 F lly 7/38 E.Mec. 62 564918 Acute pancreatitis None | 31.6 8.1 F. 12/ 13/38 L.V. 73. 860067 Diabetic gangrene Amputation 52.6 8.3 ¥ 2/17/39 L.H. 58 566522 Carcinoma of breast Mastectomy 26.1 6.7 F . 2/24/39 A.M. 55 572568 Pneumotiia, type IIl 27.9 7.1 u Average ee eae e eee oe tee ee Bs me eevee seve ee es *# 6 © # © @ e+e 8 FP Oe aeeanee wee Ge ee ee eo ee ee HE woes cneve ceeded 7.6 Standard devietion from the mean 3.6 mg. Coefficient of variation 14.5 per cent. -dect~ « in dogs, following intravencus injections of isotonic potassiun chloride in lethal doses, the concentration amounted to 59.5 me. per cent (15.2 M.eq./L.) in man the average at death was 29.6 me. per cent (7.6 M.eq./L}) compared to an average of 17.2 mgs per cent (4.4 m. eq./L) in the venous bloods of sixty health human adults. ~160- whiabToa IV rart Iz Serua Fotassilumg of Gadaver Blood Cadavers were the first suurce of blood utilized on a large scale for storage and later use (Charter i}. Cut cf this practice grew the present wide spread use of lving donors, as a source of supply for storage Gepots. In preceding experiments 1+ has been shown that living aonors' cells gradually disintegrate as they are stored anc any form of traume will hasten this pro- cess of deteriors tion. It seems logical, therefore, to suppose that cells already subjected to the injurious forces snd changes incident to the death of a patient would have suffered some harm quring the process, and would probably show wore rapid changes when preservea after withdrawal. 20 test this presunption cardiac bloods were ob- tainea fron twenty-seven cudavers ut autopsy and the plasma potavsium content aetermined as in previous experi- ments to get 2 baseline for future studies on stored cadaver blood. Yhe results are tebulated in table 26. Table 28 Serum Potassium of Cadsever Cerdise Blood Obtained at Autopsy Number Initiels, Autopsy Findings Hours Postmortem Serum Potassium Hospital Blood serum Ne.per M.eq./L Number Otteained Separated Cent 1 JH. Rheumetie endocarditis 13 46 54.7 13.1 534998 2 T.G. Carcinoma of stomach 5 1/2 le 59.2 15.4 531679 3 J.G, Tuberculous leptomeninsitis 2 24 60.9 15.5 37890 : 4 C.V. Glioblastome of brain 12 -- 64.5 16.5 37862 5 M.A. Hyperthyroidisn & -- 70.0 17.9 547187 6 ML. Congenital heart disease 9 -< 70.4 128.0 538189 7 H.Lac. Fracture of skull a L5 72.0 18.4 11647 8 C.0'K. Cerebral sbscesses 8 8 73.5 16.7 37736 9 A.R. Uremia . 8 14 76.6 19.6 467166 -YOST- 10 il 12 16 17 18 19 20 MF. . £89643 I.B. 548449 G.H. 545832 ROR. 433652 B.T. 552685 W.R. 367456 H.R. 451845 S.F. 250743 WH. 284144 — M.S. 37623 Table <6 Chronic myeloid leucemia Carcinoma or breast Placenta praevia Cercinome of stomach Uremia Gengrenous appendicitis Carcinome of sigmoid Lymphossrccnme Urenia Rheumetic endocarditis Meningiomna 46 Ll ll 12 12 1/4 1/2 1/2 1/2 12 96 19 a8 77.4 77.7 80.8 B72 110.0 118.0 118.0 19.8 19.8 20.6 22.38 24.7 25.0 25.1 £5.35 -doet~ Teble <6 GM. - Subacute bacterial endocarditis 14 1/2 34 118.6 30.3 520112 ° J.S. Carcinome of cclon 7 1/2 ~— 128.0 $2.7 543474° VE. Carcinoma of stomach 21/2 Ze 141.0 36.6 552258 R.R. Rheumatic endocarditis é a 160.0 40.9 £94654 , K.S. Cerebral thrombosis . 8 1/2 155 168.0 42.4 549302 K.B. Venous angioma of brain . 18 67 175.0 44.9 946147 R.M. Carcinoms of kidney 22 -- 180.0 46.0 545415 “909T- Average 1 Sé 3/4 101.0 "5.8 -~lél- Conme nt when the results are compared with the normal Valués ana those cbserved in cardiac blood at death (table £7) the average is seen to be about six times greater than the former ané 3,5 times grester than the concentration found in the latter. a Glance at Sigure © for comparison willshow that the rate of diffusion is much sore rapia in cadaver blood. . Several cxuses for such inereased rate of changes Bugrest theaselves: 1. ‘the temperature of the body et death and the fsilure to immediately refrigerate which means thet the blood for a pe ied is storec at room temperature or above, not at 2 - 4°C, the temperature ut which blood is usually readucec 45 soon as it is withdrawn for storage. oheaimov (1¥69) in his earliest work pointed out the marked effect the temperature has on the blood and other tissues. &. the natural autolysis that takes place in dead tissus. 3. The formation of amsonia as the result of break~- down of body protoins. Table 26A Plasme Potassium cf Normal Venous Blood* Sixty Donors Average (mean) 17.2 me. per cent Median 17.2 me. per cent Range 13.5-21.5 me. per cent Standard devistion 0.33 me. per cent Coefficient of verlation 1.9 per cent ee cree Ot it te 8 AIR St Sl a AF Sn SN Ma Sl AR ON NI ON OE SA SN “A Oe mR ee mh cm mes A om Se Sm ee in AON he A em ne I See SAR Ek Yam ek ee MORE: A wees ey, SR *This group comprised 50 males and 10 females. Each value represents the meat of two aliguots cf the original sample, 0.5 ml.of plasna being used. ~VI9T- Plasme Potessium of Human Blood* Number of Source of semple Plasme potassium expressed es samples Milligrams Milliequivalents analyzed per cent per liter Aversge Range Average Range 72 Venous blocé from normal 13 Cardiac blood removed at 27 Cardiac plood removed at autopsy** 101.0 54.7~180.0 25.8 14.0-46.0 *Centrifuged at 2,000 r.p.m. for 1 hour after which the plasus or serum wa3 immediately separated from the cells. **Te average time of separating the serum from the cells after death was 31.5 hours. ~atot- The figure for each determinstion represents the mean of 2 aliquot samples. l. conclusions ine rate of cell disintegration of blood in a cadaver as weagured by the rate of potassium aitfusion is much greater than that of fresh blood stored at once at 4°, Glood from cadavers gay present changes at the time ef withdrawal comparable to those founda in fresh stored blood 3-5 days old. -163- 05 Fart x Atmaonia Chenges in freserved Slood The high potassium values found in the observations on gadaver blood raised two questions. ‘The first: are these valucs true? The second: if true, why se high? it has becn known for a Long time thet in the cobal- tinitrite method of potsesiun determination any ammonia present would be quantitatively precipitated as amsoniun calbaltinitrite along with the potassium. in a deed body, where autolysis goes on at a&@ rapid pace even at low temperatures, the possibility of breakdown of protein sub- stances with release of ummonis is not only plausible but very probable. 20 rule out this probability as a factor several Cadaver bloods were @xamined for their ammonia content. ihe method was imperfectly worked out at the time but with crude méthodsa the highest ammonia value found was 1.3 me. per 100 ce.. This small quantity even if it were falsely read with the potussium precipitate, could not, it was con- cluced ana this conclusion was eoncurred in by Doctor Jacques, account directly for the markec inerease in cadaver plasma potassium values over the Levels found just at death. ~164= ammonia, even in minute quantities might, however, heve the same effect on the potassium diffusion of a red blood cell fin plasma es it does on the Vealonia in sea water. Jacques (190) has shown that if the con- centration of amnonia in sea water is raised by G.GOCL molav there occurs 6 rapic exit of potassium from the Valonia as thoush the cell were injured. ‘There has been a very strenous effort on the part of nature it seems to prevent s concentration even this great in human plasma for Conway (1955) has shown that there is less than one part of amionia in 30,000,000 parts of blood. tO determine just whet part ammonia docs play in the chunges observed in stored blocd a series of experiments were planned. Experiment (1) M6 thoa 30 ascertain the effect of ammonia on potassium aiffusion in vitro « relatively stable amaonium compound was chosen to becin with, NHgCL, and the bloods were kept &t &2bOuL vody temperature to simulate a Little closer the actual state of a patient at death. to one of two similar portions of citrated blood 0.133 eraas of ammonium chloride was auced to Mase a concentration -165- of approximately 0.G1 molar solution. ‘hese flasks were suspended in a water bath kept at S8°c throughout the experiment and were undisturbed except for the taking of the samples. The results ere tabulated in tuble £3. Interpretation One obvious fact is that the contr] blood cells gave up potassium at a markedly increased rate when compared to bloods stored at 4°(part I). On the other hand, these fisures offer some difficulty when an interpretation is attempted in regard to the amnonis-potassium relationship. If the ammonium chloride diffused equally throughout the entire fluid mass approximately 10 to 12 milligrams of ammonia nitrogen were added to each 100 ca. of plasma. If this quantity was precipitated as potaasium then the difference recorded between the eontrol and mmoniated blood is que to precipitation of the ammonia as cobaltinitrite, and not due to an increased rate of loss of potassium from the cells. If this be sa, the difference due to ammonia nitrogen actually present would remain relatively constant, therefore any increase in the difference between the control plasma potassium values ana that in the amnoniated blood, which is greater than that cf the first aeterminations would seen to be due to a truc increase in diffusion. wT m *1 Effect oF fenonte on the kate of Chanves in Preserved blood a Temperature 38°C. Date Time in hours Potassium 63 milligrams per cent from phlebotomy tin plasze of citrated blood Control Amnonisted Difference blood between specimens 3/18/39 0 20.9 -- -- 2 19.6 50.6 11.0 4 20.0 29.0 9.0 6 20.3 31.7 11.4 3/19/39 8 21.9 33.2 11.3 15 23.0 40.0 17.0 18 27.2 43.9 16.7 23 30.9 52.8 21.9 3/20/39 31 40.4 55.7 15.3 39 79.9 9ALL 18.2 3/21/39 71 121.0 120.0 1.0 -166- she next question remains: Is there a gradually in- ereasing amount of aamonis in stored blood; and if so, wheat levels does it reach on suecessive days? | TO answer this question, DOr. Margaret T. Smith in this laboratory spent several months working out an accurate technique for determining the minute amounts of anmonia-~ nitrogen in human blood, based on the method of conway (1935). The latter was able to show thet the normal content of free NH. in venous blood was either zero or below the analytical level, but when shed into an open vessel « rapid rise cccurred in the first five minutes. In a later publication (1939), he diviaed this free Su, into three categories, namely: (1) the “alpha” _ which originates immediately after shedding am apparentlygrises from adenosine; (2) the "beta” NH, which was shown to be derived after a series of stages from adenyl- pyrophosphoric scid; and (3) the "ganuma” NH, which appears to come either directly or indirectly from adenyldeoxyribonucleotide, or vegetable adenylic acid. He concluded that from available evidence it appears that most, if not all, of the Ng forming in sterile blood collected by open s edding is derived finally from adenosine. Experiment (2) Lie thod Slood was collected in air at room temperature with no -167- special precautions from ten healthy donors and its ammonia- nitrogen content determined as quickly as possible after with- drawal. «snalysis was carried out by the “method of suspended absorption, with the special micro-diffusion unit” (85). ‘The results are tabulated in table 29. This range is comparable to that reported by Conway and Gooke (84) for this age of plaswsa after shedding and will be used as baseline values for the following experiments: Experiment (<4) To determine the rate of increase in the ammonia of blood stored for actual transfusions, samples were taken fron twenty flasks at the time the blood was given the patients and analyzed. Results The bloods varied in age from 15 to 20¢ hours (1 to ¥ days) and in milligrams of ummonia ni: rogen per 100 cc. of plasma from 0.31 to 0.82. There wus 4 sharp rise for the first ninety-three hours (four days), then an almost constant level for the next five days. These results are eraphically presented in figure 16. Discussion In this series of experiments no attempt wos made to ~L67A- Table 29 Plages Ammonie Content of Recently Shed Venous Blood Number Age in Kiliierems Miliiequivalents Hours Per Cent Per Liter 504 L 0.081 9,058 502 2 0.131 0.094 m4. z 0.169 0.078 533 2 0.074 6.053 O82 2 0.085 6.061 S57 z 0.082 0.016 242 2 0.18. 0.094 509 3 0.059 0.6428 554 3 0.076 0.054 507 3 0.042 0.050 FIGURE 16 SS 0 o © ox o + “ ° =O ° ° ° ° IN PRESERVED BLOOD AMMONIA-NITROGEN CHANGES MGS.% 105 090 0.75 060 045 0.30 0.15 90 HOURS 20 30 40 50 60 70 80 10 0.00 2.—T15—138 CIIART PAPER NO Data from table 32 Human blood in vivo contains less than one part of ammonia in 32,000,000. When shed in sir there is a rapid increase in the first few minutes, then a slower rise. -168- follow the precaution suggested by Conwy to the effect that all bloods for ammonia determinations shoulé be drawn under carbon dioxide to prevent the rapid loss of this gas when blood is drawn into an cpen flask. The purpose was rather to establish the valuesunder actual operating con- ditions in a hospital transfusion service and check, if possible, the effect of the ammonia of blood so drawn on the rate of cell disintegration as measured by the rate of potsssium diffusion. The results from the data presented are rather inconclusive. «ll that can be saic is that there was 6 gradusl rise in the anmonia-nitrogen content of the plasma of citrated blood as the blood became older. Conclusions le. Oitrated bloods kept at 38°C show changes in its plasma in three days comparable to those observed in blood stored at 4°c on the fifteenth day. 2. There is a constant rise in the plasme ammonie of preserved blooc, murked in the first few minutes, continuing at a rapid rate the first four days to reach s level of approximately one milligram per cent at which level it remains until the tenth day. oe Blood, kept at body heat, to which ammonia chloride -169- has been added shows « greater rate of potassium diffusion for the first three days of storage. In the presence of ammonia, the possibility of false.high potassium values as a result of precipitation of ammonia cobaltinitrite alone with the potassium cobaltinitrite is pointed cut. -~170- CHaPTin IV Part XI Sodium Changes in Freserved Slood In the intact humin organism, when some disturbance causes a rise in potassium, there is often seen & con- conuitant Lowering of sodium values. uch 4 condition has its best illustration perhaps in severe addison's disease (4124). In preservec bloo: it has been demonstrated that there is a rapid diffusion of potassium from the cells into the plasm. If this is interpreted to mean that the permeability of the cell membrane has changed sufficiently to allow one cation to pass out, it seems reasonable to expect another to pass in. Yo test this concept, a series of analyses was rum to Getcrmine the changes in the sodium content of preserved blood. ¥reliminary analyses approximately similar quantities of fresh venous blood were collected from the sume donor in three separate hematocrit tubes. In the first tube there was no anticoagulant; the blood promptly clotted. In the second tube there was placed exactly oné milligram of sodium heparin. In the third tube, 0.5 ce. of -171- 5.5 per cent sodium citrate was mixed with exactly 4.5 oe. of blood. Sodium determinations were then done on the serum from the first tube and the plasma of the second and third tubes after centrifugation at 2,500 r. p. m. for one hour. wet hod wod lum was precipitated as uranyl 2ine sodium acetate accoruing to the method of Butler and Tuthill (68) with certain refinements added by bilas Tuthill, now a member of this laboratory staff. Results of rreliminary analyses {1} Serum Na 321.7 me. % 139.8 M.eq./Le (2) Heparinizea plasma Na 624.5 " 4% 141.6 " " (3) Citrated plasma Na 398.4 " " 169.0 ” . +O correct fer the sci ium added in the form of sodium heparin it was necessary to run analyses on the heparin and actually dete mine the amount of sodium in one milligram of the substance since the formula is not known accurately enough to calculate its content. Checks were excellent and the averuged results showed 0.172 milligrams of sodium in each milligram of sodium hepsrin. The hematocrit shoved a cell volume of 47.4 per cent, plasna volume of 52.6 per cent and total volume of 4.81 ec... Flasma wlume, therefore, equals -172- ee es G.526 x 4.81 equals £2.55 ce.. the sodium added to the plasma @quals 0,172 x 100 equals 6.8 milligrams per cent. TO correct for the sodium added in the form of sodium citrate, the following steps were necessary. Na gC glig 09-5 .5H,0 has a molecular weight of 357 of which sodiuu forms 69 or 18.3 per cent. ’ Of the total 5.0 ce. in the tube, 4.5 cc. was bloed, 2.20 0c. Plusma and 0.5 ca. OF 3.5 per cent sodium cl trate or which 17.5 x .193 or 3.56 milligram were sodium. This guantvity in 6.75 oc. of plasma citrate mixture equals 3.38 x 100 equals 1l2e me. per eent sodium added to the sodium al ready present. ihis smount subtracted from the observed resding e and corrected for diluticn is as follows: 38% - 122 x 275 equals ; BES 328.9 milligrems per cent or 142.8 milliequivalents. Corrected vaiues, thereforc,on the same blood were as follows: (1) serua sodium 321.7 mz. % 139.8 M.Eq./L. (2) epsrinized plasma ia 517.9 " * 138.1 * " {3) Gitrated plasma $28.9 * ” 142.8 " "* These observations showed that fairly accurate results coula be obtained using pissms instead of serum. an attempt was made to establish a series of normal values, using each of the anticoagulants. -175- Uxperiment (1) Blood samples were collected from each of eight normal individuals in henutoerit tubes which contained exactly one mililgram of the soclum salt of heparin. Plosma sodium wis determined as in the preliminary anulysis. The results sre recorded in table 3. This range of values cospsres closiy with those obtained in the seaical Laboratories in « long series of serum sodium ageterminations. experiment (2) in a manner similar to experiment (1) the range of normal values was checked on citrated blood plasma. The results are listed in table Sl. Experiment (3) a Sample of blood was obtained from each of nineteen transfusion flasks .t the time of the transfusi on. an attempt was made in co.lectinge the blood to obtain accurately one part of 3.5 per cent aodium citrate to nine parts of blood. at tines t:is was not quite possible. The plasma was amlyzed from each flask for ammonia, potassium, and sodium content. she corrected results are tabulated in table 52. Figure 17 -1735A= Table SO Plasre Sodium in Kormal Heparinized Bloods Humber Milligrears Per Cent Hillitequivealents Cbserved Corracted Per Liter L 329.0 ERO.L L39.1 2 S26. 319.9 189.0 3 SE8.9 S23.4 140.5 4 3E2.9 216.0 137.5 5; S25. S15.7 137.8 6 3£8.9 382.0 140.2 7 324.5 217.9 LBE.2 8 324.5 217.9 188.2 ~173B- Table 1 Plasme sodium in Normel Citrated Blooda Number Mililerars Per Cent Millilequivelents Observed Corrected Per Liter 1 380.2 «-B17.8 | 137.8 z 080.5 316.0 156.0 3 596.6 537.8 146.6 4 379.6 616.8 137.6 5 378,5 O15.5 137.0 6 873.9 309.8 134.5 A Table 32 Compserison of the Rate of Change in Plasme of Amvonie, Sodium, end Potassium Serial Initial Age of Amuonie-Nitrocen Sodium Potessium Humber Donor Blood Me. Per .ege/l Ug. Per h.eg/L Ug. per W.eq./h in Hours Cent Cent Cent 504 A 15 0.34 0.84 299.5 129.7 21.2 8.0 584 Jd 15 0.28 0.20 $15.9 136.3 37.5 3.6 555 Pp 16 0.41 0.29 217.8 137.7 30.9. 7.9 502 A 16 0.52 0.83 217.7 137.9 61.45 §.5 586 Y 18 0.49 G.25 214.8 136.7 34.0 8.7 532 B 18 0.46 6.33 305.8 182.5 32.6 8.3 507 B 19 0.49 O.35 320.7 139.2 32.6 8.3 509 N 20 0.30 0.22 219.2 138.8 2744 7.0 536 J 38 0.57 o.41 2397.8. 129.3 34.0 e.7 842 c 68 6,87 0.62 283.5 ig3.1 55.5 14.2 4B4 P 69 1.048 0.75 — =-- BS.7 21.4 592 Ww 93 1.08 0.77 261.0 113.2 133.0 34.1 541 L 116 0.99 0.71 225.6 124.0 132.6 335.8 557 K 140 G.92 0.66 £51.9 109.4 98.6 24.5 4&3 H 142 1.06 0.76 -- ~< 123.0 21.5 554 Y 1638 1.15 0.82 256.7 111.4 126.0 ge.1 6ll L 163 0.61 0.58 250.1 108.6 136.9 24.7 549 ¥ Le4é 1.09 0.78 260.4 113.0 142.0 36.4 593 P £0 1.00 0.72 261.2 109.6 135.90 34.4 “0S 4T~ FIGURE 17 M.EQ, PER LITER 150 140 145 135 130 DECREASE IN PLASMA SODIUM OF PRESERVED BLOOD MGS. PER oO °o ©o oO oO b 2 t+ “” N - °o uJ ~- “) oO “” ” ~m y CHART PAPER XO) 2 Data from table 32. This curve was constructed from figures which were uncorrected for the sodium in the preservetive. The true range of values was from 320.7 to 250.1 milligrams per cent, or 139.2 to 108.6 milli- equivalents. -174- was drawn from results which have been uncorrected for sodium in the citrate solution. viseussion whe question of the permeability of the red blood célis to soluble anions and cations has long been ea burning and unsettled one. Under normal conditions it is practically impossible for fons to eross this meabrane but it has been shown that an inerease in VU, tension or change in fluid medium (275) or change in pH (290) wili markedly change this State of selective permeability. Blood stored in anti- coagulants which have a certain toxic action do lose potassium from the cells at # constant rate end the above series of observations suggests that sodiwn enters the cells at a very rapid rate for about five days and then reaches e state of equilibrium. the extreme values recorded show ea difference of 30 miili-eguivalents of sodium in one week. This, apparently, has gone into the cells. There seems no other wsy to account for its loss. During this same period there was a difference of 29.4 milliequivalents of potassium. ‘The ammonia nitrogen increased approximately 30 per cent. oodium and potassium, although very similar from a physical and chemical viewpoint, functionally are so different in the numan being that at times they seem to be actual -170- antagonists. «hat part these fonic chenges play in the destruction of the vitelity of the cell in vitre or in viw, ig not Known. It docs seem -;lausibie, hawver, to think that & substance which coulc cdeerease the permeability of the cell wali without injury would greatly prolong the usefulness of preserved blood. i. oe ConeLusions ahere is a4 repid, coustant decrease of sodium in the plasma of preserveu blood. the rate of decrease in sojiua is roughly inversely proportional to the rate of increase of plasma potassium. inere is suggestive evidence that es the ammonia content of the blood increases, permeability of the cell membranes is changed. This asllows freer passage of ions and they have « tendency to establish an equilibrium on the two sidea of the membrane in place of the marked gradient which exists in perfeatly normal eellis. -176- CHAPTER IV Part XII The &ffect of pli on the Permeation of Frythrocytes by Cations The leakage cf cation from cells suspended in salt sOluticns is a very stringent criterion of cell permeability, for the integrity of the cell is unlikely to be maintained for long under such conditions. Girber (273) originally reported the impermeability of erythrocytes to sodium and potassium, but Hamburger and Bubanovic (162) pointed out in 1910 that if the salt con- centration of the serum is changed or the earbon dioxide tension is sltered both eations wiil readily cross the cell mem brane S. Mona (1028) working with the celis of oxen showed that marked changes occurred only when the pli hed risen to at least 8, and considered ali the conclusions drawn from previous work on permenbility where accurate account of the pH changes had not been mace as open to considerable doubt. Gonway (1955) showed that only by collecting venous blood under CG, could the true ammonia content be determined, for 2 there i186 a sharp rise in the amncnisa content of the plasma which terminates st the end of about 5-5 minutes, then passes -177- into a slow ascent for several days. This increased aumonia increases the pi of the blood, Jacques (1956) hes shown that even minute quantities of ammonis in sea waiter will greatly change the permeability of the ceil membrane of the see alga, Valonis machrophysia, Kutz, to sodium and potassium. Malzels (1935) has shown thet moderate shift in pH causes increased permeation, probably by physical changes, but marked shift either side of 7.0 causes changes in per- meablility ss a result of actual eceli destruction. He also pointed out that in glucose solutions, CO at first diffuses out of the celi sore rapidly than base bicarbonate, hence renaers the external solution acid; thereafter potassium and bicarbonate Gciffuse out and return the fluid to a neutral or slightly alkaline condition. The addition of swali cuantities of sodium chloride to the solution, it is thought, delay de- adsorption or maintain the charge on the ¢ell surface and so prevent Loss of caution. ‘Yhe question of CO, transport was well reviewed by Houghton in 1955. ail of these findings are of extreme importence in the setusl practice of transfusion of preserved blood for only through an understanding of such an interplay of forces can one hope to prepsre eventually an anticoagulant which will “&intain osmotic balance, constant pk and biolociesl properties of such biood. -178- In Pert I it was noticed that among ell the pre- servatives tested only that containing glucose and anslt in the proportions sugeested by Rous and Turner (19165) com- pletely prevented hemolysis, at the time it was recorded that the ph of blood kept in such 4 solution was lower, neurer neutral than in the other bloods, but no significanee was attached to it. chen coctor Scudder, 25 a result cf his association with Loctors Osterhout and Jacques, became greatly interested in the effect of U0, on the blood, it was noticed that the ph of those bloods taken under COy likewise had values at times lower then those of freshly drawn blood. all investigators who have reported results on blood stored in glucose have shown a sharp fall in the pE of the blood (chapter I1) and each has noted the superiority of such solutions in preventing hemolysis. in this luboratory tests run on distilled water shused « pil of 5<6.5, the ten per cent glucose solutions were found to have « ph of 3.75 to 4.25; the 5S per cent glucose solutions, 4.5 - 5; and the sodium oi trate, an average of 7.6. some conuercial preparations of sodiuu citrate have been observed to have a pk of over 8, espéeciaily in the more concentrated forus. »xpecting to find a complete pamillel between the degree of Lémolysais and the pi, a series of determinations was done on bloods which happened to be on ham et the time. ahs results were as follow: -179- Blood Hemolyaise pil in Lithium citrate + 7 old In Seciuna Citrate ++ 7.93 feTec. in oitrete 7 7.81 Placental blood, = 7 7.81 rg re Vv terre 7.86 " " G G 7.59 In sodium citrate +++ 7.73 it is obvious that with such uncontroiled material and With so dittle correlation in the résults, mo gonclusion can be Grewnh. wore sontrolled work was planned as foilows; 6 thoas dn cach of aight experiments, bleod was obtained fron & Gifferent individual in the usial manner; one lielLf of the Sam@pie Was Gvawh into an ai mosphere of carbon dioxide while Whe control was collected in air. On the Sumples so taken, trou four to six determinations of the aomonia, pa asyium and soclum were asde at intervals during 4 two-week period. In each experiment, ten 40 ec, centrifuge tubes with a Gisaeter of &.5 centiseters were used es containers, Carbon Gicxide from a cyhinder, filtered through sterile cotton, was introduced into the bottanm of five centrifuge tubes to dis~ pisce the sir. To exch were added £.5 ce. a 3.5 per cent sodium citrate and 22.5 ce. of blood. For the carbon adloxide Cxperiments, the bloou was acmitte. directiy tothe bottom of the centrifuge tubes; in the controls, the blood was allowed -150- to flow in st the top. both sets were closed with sterile rubber stoppers, sealed with pereffin, and piacea in a refrigerator at 4° centigrade. at intervals during a two-weex period, a tube anc its control were re- moved frou the refrirerator and centrifuged. The super- natent plasma was then drawn off for amlyses, she ammonia was determined by the method of Conway (85), the potassium by a modificstion (389) of the ar- eenticobaltinitrite method of Brek and Guebler (57), and the sodium by the method of Sutber and Tuthill (69). De- terminations of pi were made on six of the ten samples, using the glass electrode of MueInnes (272). sessuremen ts of aumonia, sodium, ph, and the development of color in the potassium determinations were done in a constant tempera ture room, 20.8° € i° centigrade. xesults The results in ell of the experiments were consistent. In each, the concentration of ammonia in the blood taken under carbon dioxide waa lower, the rates of potessium and sodium changes slower and the pil nearer neutral at the end of the experiment. a typical set of findings is tabulsted in table 33 and graphically shown in figure 18. Discussion The concentration of ammonia in the control beginning Table 33 Effect of Carbon Lioxide on Sodium, Potassium, Ammonia-nitrogen, and pH Changes in Preserved Blood Date NHe - NO Na K ee pH Air CO Air COs Air COs Air Ng.% Weeq. Eg.% Uleq. Nese Eveq. He.% Uieq. Heck Mveq. Mg.%” Eve. {ls /Le fle {le fi. /L. i i ' 1 { ' ( ! ! ! ! | ! 3 9/11/34 0.101 0.07} 0.01 10.01 | 322.11150.8] 537.41146.5] 17.1! 4.4 | 17.41 4.5 - | ! ! ! ! ! ! ! 9/12/38 0.87 0.27 6.08 10.06 324.01140.7 336.0 1145.9 51.3 | 8.9 2544! 6.5 7.76 | 9/16/39 0.551 0.39] 0.16 10.13 | 302.61131.4] 533.0!144.6] 56.4114.4 | 34.6! 8.9 7.56 | ! ! | 3 | ! | . ' i { 9/19/38 0.77 0.55} 0.30 10.21 296.41128.7 17.0 1137.6] 73.9 18.9 49.9 112.8 7.65 | 9/26/39] 0.94 10.67 | 0.44 10.31 | 276.8'120.2| 312.51135.7| 91.6123.4 | 62.1115.9 7.69 ! ~YOST~ FIGURE 18 << z 160 150 140 4130 120 15 DAYS 10 15 Na, K, AND NHsN CHANGES In BLOOD DRAWN uNDER CO, 10 fo) ” ° w) Q Ss NS nu “ = “IZ6 32 2 YSLIT Yad SLNAIVAINDAITTIIN NI SANIWA ATV < oO Date from table 33. The scdium values as presented are uncorrected for the scdium in the preservetive. Range: Sodium in control 322.1 to 276.8 milligrens per cent; in carbon dioxide, 337.4 to 312.5 milligrams per cent. All changes are less marked in carbon dioxide than in air. pH in control 7.76 to 7.69; in carbon dioxide, 7.48 to 7.17. ~161- at 07 ailliequivalents per liter rose to G.67, an increase of 60; in the CUy environment it began at 0.01 and rose to QO.S51, an increase of exactly 50 per cent of thet seen in the control. Jodlum values decreased in the control 19.6 milli- equivalents; in CO. , 10.68 or 55.1 per oent as much. Fotassiua values inereased in the control 19.0 milli- equivalents; in CO,, 11.4 or 58.4 per cent as much. The ph velues in Co, approached a normel value of seven more closely than they did when taken in air. No mau surements of the amounts of hemoglobin lost in the plasma were done in these experiments but hemolysis in the sumples taken da CO, was less than in those taken in air. conclusions Blood druwn under carbon dioxide maintains a pii value nearer neutrai then blood drawn in air and is effective in retarding changes in the concentrations of ammonia, sodiun, and potassium. ~18Z- CHaPTER IV Fart ZIT Calcium and Phosphorous Changes in Preserved Blood Of the six metals commonly present in living matter, calcium is the one with the greatest tendency to form insoluble salte (282). In man, for the most part, it is found to be present in the form of the phosphate or carbonate and my be divided into two forms (1) diffusible and (2) indiffusible. The diffusible portion alone is capable of existing in the lonized state and cnly a small portion is ever actually dissociated from its very stable salts (408). In the blood it is found in the form of tricalcium phosphate, a relatively insoluble compound, but under the lafluence of carbonic acid of the plasma, it is pertly con- vertea to the more soluble calcium bicarbonate and caleium hydrogen phosphate, which may ionize to some degree in the following manner: Cas(FUg)e | 2lipco., RCaHPO, , Ca(HCO,,)» 2ca’* + BHPOg + cut? + PHOO when carbonic scid tension is reduced, reprecipitation ~183- of the lonized sctive forms will teke place. Howlend (408) has excressed this interdependence in the following menner: (Cat+) x (HCO,”) x (HPO ,) - x R* It cen be seen that the concentration of any one ion will at once produce changes in the concentration of the other ions. A reluction of CO, tension in the blood will lower the H lon concentretion, therefore, an equivalent diminution in the calcium, bicsrbonate and phosphate ilona must fellow. Clinically this condition is geen in alkalemia. In preserved blocd it hus been observed in the pre- ceding, experiment that there is - tendency for GO, to escape, thereby chensing the H ton concentration of the blood end oauging « change in the pertition coefficienta for sodiurn, potassium «and ammonia. Ideally the bicarbonste value on the seme blood should be done st the seme time the calelum ond »hcesphorous content is done. In this particuler experiment mechaniesl and technical aifficulties nade this impossible, but the changes in the lstter two sre cof interest. Methods Calcium wes determined by the zethod of Clarke end Collip (78), the finel titration being dose ageinst sa stendard sodium oxalsete solution. -~184- Fhosphorous, cetermined as nPU ys was done by an s@aptation of the methods of Gollip and Clarke (78) and Fish and subbarow (L2G). Following Gamble'ts (134) suggestion the valence of the HPO, radicle is considered 1.8. Procedure Frog a voluntery donor 450 ¢c. of olood was drawn in 5 LB. 45.0 P9.2 24.2 40.6 PAB = Catt 5 5.3 2.5 o.7 o.7 O28 ved Nett ° 1.9 2.5 2.2 £44 2.7 2.1 “Total 164 187.8 «149,5—~=—«a19.0S1SELOS 163.8 157.0 _ Charres in Anion Composition in the Flesre of Preserved islood Anions HCO! 27 16.6 11.9 15.4 12.4 10.7 18.5 Cit 103 99.5 o°.7 100.5 69.0 90.7 107.0 HPO,g'? 2 1.9 6.0 D.2 8.2 7.5 3.4 pH 7.3 7,21 7.3 7.2 7.10 7.25 7.24 4K * Sexter Transfuso-vec. ** Undisturbed olssus. Vide moutned tlesk. -1S1- potassium value the highest, as though the added sodium hed increased the concentration wradient and forced more of the cations into the cells when the permeability had been chanred sufficiently to allow free passage. Calcium in these bloods stored for longer pe:ilods acted Similarly to that in bloods stored for sherter periods and showed relatively little change, nor did thoroughly mixing a one hundred am seventeen day old blood increase the plasma calcium content. magnesium, as the second largest constituent of the cells, might have been expeetea to show e greater outward diffusion. apperently, it forms compounds within the cell which are more atable, hence less readily ionized and slower to diffuse cut. The average number of milliecuivalents of the totals in the six bloods studied siounte to 155.8 compsred to the control normel value of 154.0. Very true indeed is Claude sernerd's conception of the constancy of the milieu interieur (29). The alkalie reserve of the plasma as measured by the We combining power of the blood decresses with age. No exact relation can be established from these dsta to conclude that 1t varies cirectly with the pH. The chloride content diminishes but not to the extent the sodium does. Two things are striking in the table, the . *l92~ the low value st the enc of ninety-three days in the flask wnieh contained 70 cc. of normal saline at the time the blood was taken, and the high value of the plasne chloride at the end of four months in the flask which was left undisturbed. The phosphate gradually increased in the plasma with the increasing uge of the blood even in the undisturbed state, more so when agitated but never so great as the rate at wiich polassium is lost from the cells. The ph of stored blood, though quite variable in the early dsys of its storage as shown by previous experiments, bas a tendency to equalize conflicting forces and ep proaches its normal level after several months. The total quantity of ne gative jons in these six bloods ranged from one hundred to one hundred twenty-six milliequiva- lents per liter, an average of approximately one hundred and sixteen. Having shown that the total positive tonic content remains constant, thut the pa varied very Little, that the protein changes very iittle (056), the quantity of negative ions necessury to complete the acid-base balance must be supplied py increxses in the organic acid ion content. There isa evi- dence (Chapter I1L) that a purt of this may cote from the increesing quantities of lactic acid formed by the process of glycolysis. This is a fuector yet to be integrated with -1lygs- the above facts to camplete the picture of the canplete adid-base composition of the plasma of stored blood. summary In the plasma of bloods stored from five days to four months, the following changes were observed: l. Fotassiua increases. 2. sodium decreases. 5. Caleium remains practically constant. 4, i-agnesium increases slowly. 5. Bicarbonate decreases, 6. Fhosphate increases, purticularly following trauma. 7. Chlorides decrease in plasm intimately mixed with the cells, remain constant or alightly increase when left undisturbed. 8. The pl decreases at first then gradually approaches the level of normal blood. 9. The total positive jfonic content remains constant, in spite of great variations in the plasma content of the various cations. 10. ‘the observed loss of total negative ions suggests thet balance is maintained by a gradual increase in the orgenic acid ion component. This factor is to be determined. -194- CHAPTER IV Fart ZVI General Suimary and Interpretation of =xperimental studies i. Plasma fotassium Increase There is a daily increase in the amount of potassium present in the plasma of preserved blood, beginning at the time the blood is drawn, mounting to twenty-five per cent of the total potassium content of the red blood cells at the end of ten days, and reaching fifty per cent at the end of thirty days, at which time the concentration begins to become equalized inside and outside of the cells. This suggests that bloods stored over long periods of time should be withheld or carefully given in elinical con- ditions associated with hyperpoteassemia such «s is found in cholera (351), intestinal obstruction (353), severe burns (557), renal (50) and hepatic (41) insufficiency, typhoid fever, influenza, pneumonia (252), hypoparathyroia tetany (155), the collapse state of sddison's Disease (3, 124, 275, 388) and in other diseases of the endocrine system associated with disturbances in salt metabolism. (21, 165, 166, 266, 275, 276). There is evidence to sugesst that its inconsiderate use may be dangerous in hemorrhage and sock (356), the field of its greatest uscfulness. ~195- Be Hemolysis as a Criterion of Toxicity The degree of hemolysis cannot be used as an index of potassium loss or toxicity of blood. hemolysis may be completely preventec by the use of a glucose-saline-citrate preservative but no preservative yet found will prevent loss of intracellular ele otrolytes. These chsnges sre the most sensitive indicators of the chunges in permeability of the cell membrane ané of the vitality of the cell. No good practical index of toxicity is known. De Toxioi ty of Potassium The potussium ion has an almost specific action on the heart and peripheral vascular tree, producing on injection disturbances varying from diminished cardiac out-put to imaediate arrest, depending on the rate of injection. Reason- in by analogy from data obtained from animal experimentation, it would require between 3,000 and 5,000 ec. of thirty-day old blood, given at a fairly rapid rate of injection, to kill a healthy man of average size. This suggests that preserved blood is safe if ordinary clinical judgement is exercised. 4. Hed Blood Cells and Hemoglobin Erythrocytes remain almost constant in number for fifteen days in any of the usual preservatives, diminish ~196- about twenty per cent by the end of the first month in citrate solution, and remain intece for periods exceeding a month in oi trate-saline-glucose mliaztures with mei ntenance of full oxygen carrying ability. They becone gradually more fragile. First sigme of hemolysis under good storage conditions appear between the twelfth and twentieth dey in citrace, only efter thirty days in glucose citrate. Hemo- globin content remains constant in the totel specimen, be ing found in measurable quantities in the plasma stout the twentieth day sand asounting to fifteen to twenty per cent of the cell content at the end cf thirty days, a decrease which, in part, is reflected in the twenty per cent loss in the mean cell dismeter of the red cells. Banked blood, therefor, theoretically may be used from the point of view of erythrocyte and hemoglobin content for fifteen days with prospect of excellent results, for thirty days if stored in glucose with fair results. Becuuse of the increasing fragility of the cells anc the possibility of jaundice, it is advisable to use only bloods of less than ten days' storage. 5. shite Blood Cells Leucocytes as a whole disintegrate rapidly, the poly- morphoneuclear neutrophiles, in particular, so that st the end of two or thres days it ia doubtful whether any are capable -197- of function. Because of the close associstion of the white cells with the phagocytic, bacterioidal and iamune © properties of the blooas, it is sugsested that where trans- fusions are indicated because of their antibody content fresh blooa be used or at least blood stored for not over three days. 6. Prothrombin the prothrombin eotent is not markedly reduced for at least ten days and may remain at Levels higher than those associated with bleeding tendencies for four months. Blood stored for many days, therefore, may be used in cases of pro- thrombin deficiency with the expectation of food results. serlier reports from thia clinic showing rapid loss of pro- thrombin concentration were due to the use of old brain exe- tract in carrying out the .uick test. when the work was re- peated with freshly extracted rabbit brain as the source of thromboplastin substances, the fall in prothrouwbin levels was very gradual. 7. Platelets the thrombocytes fall rapidly to a level between 16,000 and 80,000 within the first three days of storage and then remain fairly constant depending on the anticoagulent used. In cases of essential thrombocyt ic purpure, it would -198- seem advisable to use fresh blood or blood which had been stored for less than three days to obtain maximal thera- peutic results. 8. Effect of Trauma The effects of trauma on blood cells are magnified by increasing age. Blood which is to be exposed to the trauma of transportation should be sent to its eventual des- tination as soon after withdrawal as possible, in order to insure the least possible damage to the incressingly fragile cells. Be ideal Vontainer tor Blood The rate at which diffusion of intraceliluler electro- lytes takes place into the plasma varies approximately as the diameter of the container at the inverface of the con- tainer. Blood, therefore, ghould be stored in a flask with a narrow waist to diminish the rate of diffusion, a narrow | neck to prevent changes in the plasma over 4 widely ex osed area, and the air space st the top should be at a minimum to prevent splashing and damage to the cells on movement. The material should be heat resistant, alkalie free and should not be covered by such foreign substances as paraffin for prolonged storage. such a container has been designed and tried out in this laboratory with gratifying results. -199- 10. Placental Blood whe call voluse of placental bloods is approximately fifty per cent higher than that of the mothers at term and twenty-five per cent higher than that of the everage healthy adult. its protein content is lower. dhe cells disintegrate ate pate similer to that of adult bloois. The danger of contuminetion is greater and the airounts obtained from each placenta seem to vary with the technique of the operator, but once obtained, upon experimental grounds, it is egml to or better than adult blood when compared whime for volume. It must be grouped and cross-matched with the same care as blood obtained from eny other source. ii. Cardiac Blood at beath she concentration of potassium in the hearts' blood of animals obtained at death following the intravenous in- Jection of lethal desea of potassium chloride, or following induced shock, was uniformly higher than thet found in human beings at death. this suggests that there is danger in translating the dats from animal experimentation in terms of human re- sistance to a toxic substance. Man may be more sensitive to the toxic action of a poison like potassium than dogs or cuts. In using bloods stored for long periods with marked aeteriorative changes, a large margin of safety should be allowed. ~200~ lz. Gaduver Blood Blood from cadavers say present changes ut the time of collicction coupsrable to thogze found in donor plood stored for thiave to five days. The rate of cell Gisintegretion 2s measured by the rate of potassium diffual on is #ereatily in excess of fresh donor blood stored under similar conditions. There is an element of safety in the use of cadaver blood stored without anticosgulant. Those bloods collected from persons dying of severe in- fections or wasting diseases will clot, wile only bloods from relatively healthy individuals who have died acutely will undergo fibrinolysis anc be suitable for use. where are certain psychic inhibitions to the use of such blood but the matter is worthy of real consideration. Ls. zffect of seat Citrated plood kept at 36°C. shows changes in three days, comparable to those observed in blood stored at 4a°c. after fifteen days. ‘The danger of infection is greatly in- creased. ull bloods should be reguced to a temperature of three to five degrees C., 48 soon us drawn and stored in & refrigerator which is free from mechanicsl shaking or moverent, used for storage of blood alone. ~201- 14. ammonia anc Cell rermeability Freshly drawn blood rapidly loses carbon dioxide and allows a sharp rise in the ammonia content of the plasma. This, in turn, inereases the permeability of the céll membrane and allows more rapid leakage of cellulsr electrolytes, a condition detrimental to the vital pro- perties of the blood. | Blood drawn under carbon dioxide does not show these rapid changes, therefore, is kept in a better state of preservation for longer periods of time. ‘hen bloods are to be used soon after drawing the added precaution of collecting it under incoressed carbon dioxide tension does not seem worthwhile; but if bloods are to be stored for longer pe:iods, especially if they are to be transported, the extra precaution seems well worthwhile. Li. Flasmea wodium Decres se There is a constant decrease in the sodium content of the plaama of preserved blood. ‘The rate at which this takes place is roughly inversely proportional to the rate at which the plasma potassium increases. Both are evidence of a break-down in the selective permeability of the cell menbranes. There is no wey of completely preventing this shift of electrolytes in preserved blocd at the present time. -202- 16. shifts in pi concentration The permeability of cell membranes is markedly affected by wide shifts of pH from that of the blood in the bocy. some citrate solutions ere too alkaline and cause repic change in pH with consequent increased rate of eleatrolyte dispiacement. ‘shen carbon dioxide tension is maintained, the py changes are less. then glucose is aaded to the preservetive, there is at first s tendency for the Pliuia to become sore acid due to glycolytic processes, when this process slows down there is a shift back towards the alkaline side and a restoration of the status guo ante. Bloeé drawn under carbon dioxide end preserved in glucose aaintains a ph value close to neutral anc prevents the changes which follow marked shifts in hydrogen ion con- centraticn. 17. Calcium and Fhosphorous The ealcium oontent of preserved blood remains at practically a constant level for periods as long us four months. Phosphorous shows a very slow but steacy increase in the plasma anc this is accentuated by truunua. The changes in neither appear to te of great practical significance. ~205- is. lotal Cation ~tructure Potassium goes steadily am rather rapidly up in the plasms of preserved blood and megneaium inereasea much more slowly, while the phosphorous content increases 80 slowly thet it muy be considered just a tendency. Sodium values steadily fall and calcium remeins constant. The pum of all of the positive ions when expressed as equiva~ lents ct hydrogen remain constant, always equalling the total for fresh plasma in spite of the very marked difference in distribution between the cclls and the plasma. 1@. anion Changes The alkalie reserve a8 measured by the carbon dioxide combining power decreases with the increasing age of the blood, while the phosphate which to a large degree 1s an intracellular constituent, gradually increases as the in- creased permeability of the celi membrane allows easier passage of both cations and anions. The chlorides diminish but not to the extent the sodium does. BG General vonciusion Blood preserved for periods not exceeding ten days, insofar as could be determined in these investigations, is sefe ani for most purposes should give results when used for transfusions comparable to those following the use of fresh blood. Its rapid loss of white cells precludes its use in -204- cases where bactericidal properties ar antibodies usually associsted with white cells are desired. It would seem less effective in cases of thrombocytemenic purpura and bleeding due to prothrombin deficiency. Its use in large quantities would seen contraindicated in patients suffering with diseases associ.ted with hyperpotassemia but even in such cases its noderate and intelligent use should lecd to no untoward effects. -205 = CHAPTER V PRESBYTERIAN HOSpiTaL BLOOD BANK Part I The Establianment of the Bank Followlng preliminary diseussicns between Mr. John bush, Superintendent of the Hospital, and Doctor Fordyce 8. St. John, Doetor David C. bull, and Doetor John Scudder, on November 26, i956, the Chairman of the Medical Board, Doctor J. Lentley Squier, at the request of Mr. Busch, agced the board to endorse the eppointment of « committee to investigete the advisability of the use cf a blood bank; the comaitte to be composed of: Doctor Yordyce B. St. John, Chairman Deetor A. KH. Dochez Dector Devid C. Bull At © meeting on Janusry 30, 1959, the comolttee reported their findings and recommendations to the Medical Boerd. A brief resumé of that report is as follows: The committee appointed to study the problem presented by the use of stored blood for transfusions has epproached ita review by: 1. 8 _—. O ax2’ O | oa ana O J 4 i CUBICLES a) - (os . N 5 - = WORKROOM —.. | L T Basis re . rr xs 2x05! eee ] 1 toh VBL DL. oe ae , Stavile time: Sets Lee Box aa Cart 7 7 to '4 3° 12! | O O § ao} jo os BANK I J a 7 T (3) O Ken 3’ Oo ] J dese 1O Fehon ns 7 Sve ns a fram cing Toke ls oat) CL) 6.5 x Mx’ 0 DONORIUM C4 } O 2axa" 7 T Table (a Al sees Zinen Ol er : oO O FAN o__|p | i J (3/ vy) ‘ 7 T LJ a |X © 5] |xXly Cc’) =“ x ]es z Vig WD uJ “ J a J (6/ = ___| ___| Lj — t ] 7 _, IN O | This plan consists of two units. One contains the bank, workroom, end donorium and it should be cen- trally located in the hospital. The other, with six cubicles and benches, need not be immediately ad- jacent to the central unit but should be used for the “blood bank clinic" where large numbers of donors are handled. i. Be 4 5. 6. B. 9. 10. il. BIBLIOGRAPHY Abel, J. J., Kowntree, L. G., and Turner, B. B.: Plasma Removal with Return cf Corpuscles (Plasmepheeresia). Jour. Pharmacol, and Exper. Therap., 5: 625-641, 1913-1914. 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Landsteiner, K.: Zur senntnis der antifermentativen, lytiachen und eg#elutinierenden Wirkungen des Elute serums und der Lymphe. Centralbl. f. Bakterlol., £7: Oo7e SBE, LOO, Landateiner, Ko: Ueber Agelutinationsaerschelnungen nornalen mensenlishen Blutesg. Wlen. klin. Wohnachr., 14: 1L1lSeé-11%4, 1901, 7 . : “yp af ’ ‘ . ft Lancstéeiner, A.i Bamagegiutination und Hamolyse. Gppenneiger, C.: Handbuch der Eilochemie des Fenschen und der Tlere. dene, G. Fisehear, 2: 395-541, 1910. Landsteiner, Ke: The Human Bleod Groups. Jordan, E. O., and Falk, I. 5.: The Newer Knowledge of Bacteriology and Imsunclogy. Chicago, The University of Chicago Press, AQZ-908, 1924. & Landsteiner, Ke: Individusl Differences in Human Blood, Selence, 75: 463-409, 1931, Lanudsteiner, K., and Levine, P.; further Observations on Individual Differences of Human Blood. Prea. Soe. Paper. 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