MAY 1941 373 NEWER KNOWLEDGE OF BLOOD TRANSFUSIONS* Joxun Scupper, Cuartes R. Drew, EvizasetH TuTuttz, B.Sc., AND Marecaret E. Smitu, Px.D. @ WBEL' in 1914 reported on combating experimental hemor- rhagic shock in dogs by infusing red cells suspended in Locke’s solution after discarding the plasma. Subse- quently, Rous and Turner? demonstrated that the lon- a f) gevity of the red blood cells could be enhanced if such erythrocytes were kept in a dextrose-citrate solution in a refrigerator. These two experimental observations led Robertson* to use preserved red cells in treating the wounded at the casualty clearing stations of the Third Army of the B. E. F. during the first World War. Twenty-two such red cell transfusions, some fortified with gelatin, were given to twenty individuals. Of these, eleven were discharged to the base hos- pital and nine died. Among the latter, all except one were temporarily benefited by the stimulating effect of the cells. Perry* extended the preservation of erythrocytes by showing that the oxygen-carrying power of the red blood cells was maintained bet- ter in a solution of dextrose and lithium citrate than in one containing sodium citrate. Cells so preserved were transfused to man after removing the supernatant plasma. Prior to this use of preserved cells, Richard Weil,® in 1915, had transfused citrated human blood which had been kept for several days in cold storage. The observations of Yudin* on the transfusion of stored cadaver blood and the experimental wide scale use by Durin Jord4™* of con- served citrated blood during the Spanish Civil War stimulated investiga- tions in many laboratories.*” *® 1 In the present war, plasma or serum is being used in preference to both red cell and preserved blood transfusions. What has led to this complete reversal in current medical thought? ° © Presented before The New York Academy of Medicine, Jan y 1941, in the Friday Afternoon Lecture Series. This study was made possible by a grant the Blood Transfusion Better- ment Associati New York, New York. From the Surgical Entholory Laboratory of the College of Physicians Surgeons, Columbia University, New York, New Yi 374 THE BULLETI ah SSS Se nde ere Tazrz I CHANGES IN CELLULAR ELEMENTS OF PRESERVED BLOOD Over 30 days in refrigerator at 4-6° C. Constituent - Red Blood cells Mean cell diameter (Halometer method) Hemoglobin (Hellige) White blood count Heparin™ Cell counts at constant level for a month 20 per cent decrease in 30 days Total remains constant 15-25% in plasma in 30 days 50% decrease in 24 hours Sodium citrate™ Moderate destruction uf ery- throcytes after 15th day with decrease of 1,000,000 to 1,500,- 000 at end of 80 days Total remains constant 15-25% may diffuse out in 30 days Fall 27% in first 5 days Polymorphonuclear Show earliest and most rapid | Nuclear changes in 24 hours. neutrophiles degenerative changes with nu- | In 48 hrs. 50% decrease. In 15 clei losing shape. 50% decrease | days liquefaction and droplet in 48° formation becoming subse- quent smudges Eosinophiles Show least changes in size, | Well preserved at end of 80 : shape and staining qualities | days over 1 month. Basophiles Well preserved at end of 30 days Lymphocytes Retain shape, size and staining | More resistant than poly- properties r than neutro- | morphonuclears philes. Recognizable at end of | Recognizable at end of 80 days 80 days Monocytes More resistant than neutro- philes - | Difficult to trace Thrombocytes Rapid decrease during first 3 days Early decrease Conprrions or EXPERIMENT Donor Type O Type O Blood (venous) 5.0 cc. 4.5 cc. Anticoagulant 5. mg. dry heparin (Con- | 0.5 cc. 3.5% sodium citrate naught) : solution Test tube Round bottom—Internal di- | Flat bottom—Internal dia- Mixing of sample Time of experiment ameter 1.1 cm. 0.5 cc. plasma removed for K analysis after certrifugation 1 hr. 3000 RPM. B. mixed inverting tube 15 x. Fall, 1938 meter 1.6 cm. Shaking Winter, 1939. Newer Knowledge of Blood Transfusions 375 CELLULAR CHANGES IN HEPARINIZED 8LOOD 6 ERYTHROCYTES MILLIONS & 16 HEMOGLOBIN MEDIAL CELL DIAMETER ° 3 10 15 20 25 30 35 Figure 1 The answer lies in the fact that blood on leaving the vascular system starts to undergo degeneration immediately.*° To appreciate what some of these changes are, will enable one to use those measures which will hinder or retard them, thereby prolonging the usefulness of preserved blood. The following report deals only with the work done in our labora- — tory. It will be treated under three separate heads: 1. Changes which occur in the cellular elements of the blood. 2. Changes which occur in the electrolyte distribution. 3. Changes which appear in the protein patterns.* CHANGES IN THE CELLULAR ELEMENTS Methods. Two sets of experiments were carried out. Equal amounts of freely flowing venous blood were collected in each of thirty-five sterile test tubes. The first used dried heparin (Connaught); the second used sodium citrate solution as anticoagulant. The results are tabulated in Table I and a few of the changes are illustrated in Figs. 1-5.27* It is apparent that changes take place with both anticoagulants. The neutrophilic leukocytes show the earliest alterations. * These were carried out under D. A. MacInnes of the Rockefeller Institute for Medical Research.” 376 ' THE BULLETIN a TD VO pS CELLULAR CHANGES IN HEPARINIZED BLOOD PER - TOTAL WHITE COUNT 3900 NEUTROPHILES 3000 LYMPHOCYTES a apao MONOCYTES Figure 2 THROMBOCYTES IN HEPARINIZED BLOOD 140900 120900 Newer Knowledge of Blood Transfusions 377 CELLULAR CHANGES IN CITRATED BLOOD ERYTHROCYTES PLATELETS ° ‘Ss 10 is Figure 4 CELLULAR CHANGES IN CITRATED BLOOD NUCLEATED LEUCOCVTES NEUTROPHILES OTHER WHITE CELLS ° $ 10 15 DAYS Figure 5 378 THE BULLETIN _ Cuances in Evecrrotyte DistrisuTion The Permeability of Erythrocytes to Sodium and Potassium. The exit of the potassium ion from certain plant cells is one criterion of the permeability, for the integrity of the cell is unlikely to be maintained under adverse conditions on account of the steep concentration gradient which exists between the intracellular potassium and that of the external solution.**+*7 Giirber™ reported the impermeability of erythrocytes to sodium and potassium, but Hamburger and Bubanovic” pointed out in 1910 that if the salt concentration of the serum is changed, or the carbon dioxide tension altered, both cations will readily cross the cell membranes. In vivo, disturbances in both the plasma potassium and sodium ion concentrations occur.*° #3, 5, 8, 4,85 In vitro, a rapid diffusion of the potassium ion from the erythrocytes into plasma transpires, *7-***7, 58, 3° With this, there is an associated low- ering of the sodium ion.’* **. + These changes were reinvestigated with the hope of ascertaining some underlying mechanism. Analytical Procedure. Sodium was precipitated as uranyl zinc sodium acetate according to the method of Butler and Tuthill.** Potassium by a modification of the argenticobaltinitrite method.** **.“** Ammonia nitrogen by the isothermal distillation method of Conway.* * Preliminary Analyses. Similar quantities of venous blood were col- lected from the same donor in three separate hematocrit tubes. In the first, there was no anticoagulant. In the second, there was placed exactly 1 milligram of sodium heparin. In the third, 0.5 cc. of 3.5 per cent sodium citrate solution was mixed with exactly 4.5 cc. of blood. RESULTS OF PRELIMINARY ANALYSES FOR SODIUM 1 Serum sodium ..............seeeeeeeceeee _-++| 821.7 mg. % | 189.8 M. eq/T. 2 Heparinized plasma sodium®’................. 317.9 mg. % | 188.1 M. eq/L 8 Citrated plasma sodium*...................-- 828.9 mg. % | 142.8 M. eq/L * Corrected for sodium in anticoagulant. Next, an attempt was made to establish a series of normal values, using each of the anticoagulants. Experiment 1. Blood samples were collected from each of eight nor- mal individuals into hematocrit tubes with an internal diameter varying between 6 and 7mm. and containing exactly 1 milligram of the sodium Newer Knowledge of Blood Transfusions 379 va Taste II PLASMA SODIUM IN NORMAL HEPARINIZED BLOODS Corrected for Sodium in Anticoagulant Number Milligrams Per Cont Milhequivalente l 820.1 189.2 2 819.9 189.1 8 | 323.4 140.6 4 316.0 1874 5 | 816.7 187.7 6 822.0 140.0 7 | 317.9 198.2 8 | 317.9 188.2 319.2 188.8 Tantz IIT PLASMA SODIUM IN NORMAL CITRATED BLOODS Corrected for Sodium Added Number Milligrams Per Cent M iequioalents 1 817.8 188.0 2 818.0 138.8 8 887.8 1469 4 316.8 187.8 5 815.5 187.2 6 809.8 184.7 819.2 188.8 salt of heparin. The results of the plasma sodium analyses are recorded in Table II. These compare closely with the accepted values. 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 III. A COMPARISON OF THE RATE OF CHANGE IN THE PLASMA OF PRESERVED BLOOD OF AMMONIA, SODIUM, AND Tastz IV POTASSIUM ION CONCENTRATION ; Age of Ammonia—Nitrogen Sodium Potassium Number | Donor a : ‘ in Houre | MgPer | wiegst | MoPer | megyt | MRer | M.eg/L 504 A 15 0.34 0.24 299.5 190.2 31.2 8.0 584 J 15 0.28 0.20 318.9 136.5 87.5 9.6 555° Pr 16 0.41 0.29 317.2 137.9 30.9 19 502 A 16 0.82 0.28 317.7 188.1 21.5 5.5 586 Y 18 0.49 0.35 314.8 136.9 34.0 8.7 582 B 18 0.46 0.88 305.2 182.7 82.6 8.3 507 B 19 0.49 0.85 820.7 139.4. 82.6 8.8 509 N 20 0.80 0.21 319.2 188.8 27.4 7.0 583 J 38 0.87 0.41 297.8 129.5 34.0 8.7 542 Cc 68 0.87 0.62 283.5 128.8 55.5 14.2 484 P 69 1.08 0.73 a a 88.7 21.4 592 Ww 98 1.08 077 261.0 118.5 188.0 34.1 541 L 116 0.99 0.71 285.6 1242 182.0 38.8 557 K 140 0.92 0.66 251.9 109.5 95.8 24.5 483 H 140 1.06 0.76 eee eee 128.0 31.5 584 Y 168 1.15 0.82 256.7 111.6 126.0 82.2 611 L 168 0.81 0.58 250.1 108.7 186.0 34.8 549 Ww 18% 1.09 0.78 260.4 118.2 142.0 86.8 598 P 209 1.00 0.71 251.2 109.2 185.0 84.5 w oo ° NILZIINAA ZHL Newer Knowledge of Blood Transfusions 381 Experiment 3. Uniform samples of blood were obtained from each of nineteen flasks at the time of giving the preserved blood transfusions. After centrifuging, the plasma was analyzed for its ammonia, potassium, and sodium content. The corrected results are tabulated in Table IV. Stored blood loses potassium at a coristant rate from the cells. The extreme values recorded show a decrease of 30.7 milliequivalents for plasma sodium during the first week. Sodium, therefore, enters the red blood cells rapidly during the first five days and then approaches at a — steady state. During the same period, there is an increase of 29.3 milli- equivalents of potassium.** With these changes, ammonia nitrogen had increased to 0.58 milliequivalents per liter. A possible explanation may lie in the work of Jacques*? who demonstrated that changes in ammonia concentration alter the per- meability of the sea algae, Valonia macropbysa Kiitz, to both sodium and potassium.”4*7 Conway™:* saw a sharp rise in ammonia concentration occur within the first few minutes after the shedding of blood; this was slowed by collecting blood under COs. The permeability of the erythrocyte protoplasm to cations has been widely investigated. ** ***° Certain factors, however, such as an increase in CO, tension“ or change in fluid medium," or change in pH® will markedly alter this state of “selective permeability.” Maizels™ in 1935 indicated that moderate shifts in pH do not alter the permeability of erythrocytes in respect to sodium and potassium; pronounced changes do affect the permeability, however. It has been noticed that among the different preservatives used in blood storage, the one containing glucose and salt in the proportions sug- gested by Rous and Turner? prevented hemolysis but did not alter the outward diffusion of potassium.** The pH of the plasma which had been preserved with Rous’ solution was 7.1. Others, reporting their results on blood stored in glucose, record a fall in the pH of such specimens. . Sheep’s blood preserved with and without CO, revealed marked dif- ferences in both hydrogen and ammonia ion concentrations.™ To test again the effect of CO, on human blood, the following con- trolled observations were made: In each of the eight experiments, blood was obtained in the usual manner from a different individual, isotonic solution of sodium citrate being used as the anticoagulant. One half of the sample was drawn into Taste V EFFECT OF CARBON DIOXIDE ON SODIUM, POTASSIUM, AMMONIA-NITROGEN, AND pH CHANGES IN THE PLASMA OF PRESERVED BLOOD NH,—-N Na K pH Date air co, elir CO, elir co, 4.6 M M.¢ M M M Air | C0 M. eq. . 6g. M. eq. eq. . eq. . 6g. Mg. % it My. % ihe Mg. % yh My. % 7 My. % Le My. % yk 9/11/89....... 0.10 0.07 0.01 0.01 322.1 140.1 387.4 146.7 14.1 4.4 17.4 4.5 9/12/389....... 0.87 0.26 0.08 0.06 824.0 140.9 336.0 146.1 31.3 8.0 25.4 65 9.76 7A8 9/16739....... : 0.55 0.89 0.18 0.18 302.6 181.6 383.0 144.7 56.4 144 34.6 8.8 7.58 1.22 9/19/89....... 0.17 0.55 0.80 0.21 296.4 128.9 317.0 137.8 73.9 18.9 49.9 28 7.65 7.81 9/26/89....... 0.94 0.67 0.44 0.81 276.8 120.4 312.5 135.9 91.6 28.4 62.1 “18.9 7.69 GAT zgft NILATING FHL Newer Knowledge of Blood Transfusions 383 an atmosphere of carbon dioxide while the control was collected in air. The details of the experiment have been previously reported.** Results. The results of one of these experiments in which determina- tions of the concentrations of ammonia, sodium, and potassium ions were made at intervals during a two-week period is recorded in Table V. It is noted that the concentration of the ammonia ion in the blood taken under carbon dioxide was lower; the rates of potassium and sodium changes slower; and the pH nearer neutral at the end of the experiment. ‘The concentration of ammonia ion in the control, beginning at 0.07 milliequivalents per liter rose to 0.67; in the CO, environment, it began at 0.01 and rose to 0.31. The increase in the concentration of ammonia ion for the two-week period in the blood taken in CO, is, therefore, only 50 per cent of the increase noted in blood taken in air. Plasma sodium values decreased in the control 19.7 milliequivalents per liter; in CO., 10.8, Plasma potassium values increased in the control 19.0 milliequiva- lents per liter; in CO,, 11.4. The plasma pH value in CO2 approached the normal more closely than did the samples taken in air. Hemolysis in the latter was greater than in blood collected in carbon dioxide. ConcLusIons 1. There is a rapid, constant decrease in sodium in the plasma of preserved blood. 2. This decrease is roughly inversely proportional to the increase in plasma potassium. 3. There is a suggestive evidence that, with an increase in ammonia content of blood plasma, permeability of the erythrocyte protoplasm to these two ions is changed. 4. Blood drawn under carbon dioxide maintains a | plasma pH value _ nearer neutral than blood drawn in air. 5. Changes in the plasma concentrations of ammonia, sodium, and potassium ions in such blood collected in CO, is less than in air. 6. Hemolysis is retarded by collecting blood in an atmosphere of carbon dioxide. CHANGEs IN CaLcruM, MAGNESIUM, AND PHospHorus CoNTENT Of the six minerals commonly present in living matter calcium has the greatest tendency to form insoluble salts. In man, it occurs in the form 384 THE BULLETIN of the phosphate or carbonate and may be divided into two forms: dif- fusible and indiffusible. The former is capable of existing in the ionized state and only a small portion is ever actually dissociated from its very stable salts.“ In the body it exists as tricalcium phosphate, a relatively insoluble compound; but under the influence of carbonic acid of the plasma, it is partly converted to the more soluble calcium bicarbonate and calcium hydrogen phosphate. Cas(PO.)2 + 2H2COs —— > 2CaHPO,. + Ca(HCOs)2 — 2Cat+ + 2HPO.- + Catt + 2HCOs— Methods. Calcium was determined by the method of Clark and Col- lip, the final titration being done against a standard sodium oxalate solution. . Phosphorus, determined as phosphate, was done by an adaptation of the methods of Clark and Collip* and Fiske and Subbarow.% Follow- ing the suggestion of Gamble*’ the base equivalence is 1.8 times the molecular concentration of HPO.. An adaption of several methods*®. * was used in determining magnesium. Procedure. From a voluntary donor, 450 cc. of blood was drawn in air into a bottle containing 50 cc. of 3.5 per cent sodium citrate solution, and then divided into twelve equal portions and kept in refrigerator. On the plasma of one sample, calcium, magnesium, and phosphorus ana- lyses were done on the day of collection and on the following day. Then, on every second day, two tubes were taken. From one, the plasma was immediately removed; from the other, only after i inverting . five times and centrifuging for thirty minutes. Results. The results are presented in Table VI and Table VII and - indicate the changes which took place during nine days. Each is an aver- age of at least two separate determinations by two individuals. Discussion. Joseph and Meltzer® reported in rgro that the toxicity of the chlorides of magnesium, calcium, potassium, and sodium varied in almost inverse proportion to the quantities found in blood, particularly in the plasma. Since plasma potassium concentration is greater than plasma magnesium concentration, significant increases in the latter, ac- cording to this theory, would be more toxic. These results indicate that during the nine day observation period, there is very little outward dif- fusion of magnesium. Newer Knowledge of Blood Transfusions 385 Taszz VI PLASMA CALCIUM AND PHOSPHORUS CHANGES IN PRESERVED BLOOD . Calcium Phosphorus ‘Dee Before Shaking | After Shaking | Before Shaking | After Shaking Mg.% |M.0q./L.| My. % |M.eg./L.| Mg.% | M.eq./L.| Mg.% |M. oq./L. 0 9.2 4.6 . . 3.6 2.1 . 1 8.8 44 8.7 44 3.5 2.0 34 1.97 8 8.9 4.5 8&7 44 84 1.97 3.4 1.97 5 8.6 43 92 4.6 8.5 2.0 8.6 2.1 7 8.7 44 8.7 44 38 2.2 8.6 2.1 9 9.1 4.6 9.1 4.6 8.7 21 4.0 2.8 Tamz VII PLASMA MAGNESIUM CHANGES IN PRESERVED BLOOD ~~ Magnesiom in Milliequivalents per Liter Agein Days Before Shaking After Shaking 0. 28 . 1 28 2.85 3 25 24 5 2.6 24 7 25 2.5 9 24 24 SUMMARY 1. The plasma calcium ion concentration of preserved blood re- mains constant for a period of nine days and is not increased by shaking. 2. There is no definite increase in the plasma phosphorus content. ‘This is not accentuated by shaking, even in the nine-day old blood. 3. Magnesium diffuses out of the erythrocytes of stored blood at a very slow rate, if at all. Shaking apparently does not increase this. 4. The actual increase in magnesium at the end of nine days’ storage appears to be too small to account for any toxic manifestation following transfusions of such preserved bloods. 386 THE BULLETIN LL a ee yt SS =—qxqxq—~—*—*—*—*_«—~~—e~*~*K*—~—e—~*»K*K=K#—=E=—=&«&{&F=>{{&2{—_E____ Tastz VIII ACID-BASE COMPOSITION OF FRESH BLOOD PLASMA (Expressed in Milliequivalents per Liter) Base Acid Ion ‘ Gamble Gutman Ton Gamble Gutman Na’ 142 142 HCO,’ 27 28 K! 5 5: clr 108 104 Cal’ 3° 5 HPO,’’ 2 2 Mg'’ 3 2 ; SO,"7 1 Org. Ac. 6 1 Protein 16 18 Total 155 154 155 154 CHANGES IN THE ToTaL ELECTROLYTE STRUCTURE OF THE PLASMA OF Preservep BLoop With these observed alterations in the potassium, sodium, ammonia, calcium, phosphorus, magnesium, and hydrogen ion concentrations, the status of total ionic balance in aging blood needs investigation. . To this end, freshly drawn blood was set aside at approximately monthly intervals for four months. The cations were determined as in the previous section; and of the anions, the bicarbonate, chloride, phos- phate, and hydrogen ions were analyzed. The sulphates, organic acids, and proteins were omitted, as there is not a complete unanimity of opinion concerning the equivalent values to be assigned to the organic acid and protein components on the acid side of the equation. The chlorides were determined by the method of Van Slyke’ and the carbon dioxide and oxygen by the method of Van Slyke and Neill.” The pH measurements were carried out by means of the glass electrode potentiometric method of MacInnes and Longsworth.” The bloods which were examined on the fifth, sixty-eighth, and one hundred seven- teenth days of preservation had been stored in narrow-waisted dumb- bell-shaped flasks which contained 50 cc. of 3.5 per cent sodium citrate and 450 cc. of blood. The interface diameter between the settled cells Newer Knowledge of Blood Transfusions 387 Tastz IX CHANGES IN TOTAL CATION STRUCTURE IN THE PLASMA OF PRESERVED BLOOD (Expressed in Milliequivalents per Liter) Cats N. l Age in Days fons Gutman é are | 68 gos* 1mm =| 117+ Na’........ 142 122.4 106.8 119.0 89.7 105.2 120.7 | 5 28.2 85.0 29.2 54.2 40.6 28.8 Ca’... 5 5.8 5.5 5.7 5.7 5.8 54 Mg?!....... 2 1.9 2.5 2.2 24 2.7 2.1 Total....... 154 157.8 149.8 156.1 152.0 158.8 157.0 CHANGES IN ANION COMPOSITION IN THE PLASMA OF PRESERVED BLOOD#t «Anions HCO,’..... 27 16.6 11.9 15.4 12.4 10.7 16.5 Cl’........ 103 99.3 99.7 100.5 80.0 99.7 107.0 HP,O’’.... 2 19 | 6.0 5.2 8.2 75 3.4 * Commercial bottle ** Undisturbed plasma *** Wide-mouth flask Interface 10.4 cm. t Interface 3.5 cm. Interface 8.6 cm. and plasma was 3.5 cm. All but one of these flasks were inverted to mix thoroughly the cells and plasma, before the sample was centrifuged and the plasma removed for analyses. The values in the last column were obtained from the plasma of a blood one hundred seventeen days old, which had not been disturbed during the entire period. The twenty-one day old blood was kept in a wide-mouthed flask (interface 8.6 cm.) while the ninety-three day old blood (column six from the left) was collected in a commercial bottle (interface 10.4 cm.) which contained 7e cc. of 2.5 per cent sodium citrate in physiologic saline under vacuum of 24-26 inches of mercury. All values are corrected for dilution and added sodium or chloride. _ Discussion. The greatest changes are seen in the sodium and po- tassium ion concentration, particularly in the bottle in which the blood tt We are indebted to A. B. and E. B, Gutman for permission to use their figures for normal human serum. 388 THE BULLETIN was collected under vacuum. Calcium in these bloods stored for longer periods, acted similarly to that in bloods stored for shorter periods and showed relatively little change; nor did mixing a one hundred seventeen day old blood increase the plasma calcium content. Magnesium, as the second largest constituent of the cells, might have been expected to show a greater outward diffusion. The total average number of milliequivalents in the six bloods amounts to 154.3 compared with the control normal value of 154.0. The alkali reserve of the plasma as measured by the CO2 combining power decreases with age. The chloride ion concentration decreases, but not to the extent of the sodium ion. The plasma chloride concentration of the blood which had been collected in a commercial vacuum bottle and which contained an additional 70 cc. of normal saline was strikingly low when compared with the high chloride values in the samples taken under atmospheric pressure without the addition of saline. The plasma phosphate concentration gradually increases in the blood with increasing age, but never as great as that of potassium. The pH of stored blood after mixing with the plasma varied between 7.1 and 7.34. The quantity of the determined anions in these six bloods ranged from 100.6 to 126.9 milliequivalents per liter, with an average of ap- proximately 117. These figures are exclusive of the sulphate, protein and organic acid anions. Summary. In the plasma of bloods stored in an electric refrigerator thermostated at 4°C. for a period ranging from five days to four months, the following changes were observed: 1, Potassium increases. Sodium decreases. Calcium remains practically constant. Magnesium shows little change. Bicarbonate decreases. Phosphate increases, particularly following agitation. Chlorides decrease in plasma intimately mixed with the cells: remain constant or slightly increased when left undisturbed. 8. The total cation concentration remains constant, despite great variations in the plasma content of individual cations. 9. The observed loss of determined anions suggested that balance Dar hoe py Newer Knowledge of Blood Transfusions 389 Effect of shaking on potassium escape from red bicod cells Potassium as mg. per cent Days Figure 6 is maintained by a gradual increase in the organic acid ion com- ponent and a decrease in albumen. 10. ‘The pH changes of the whole blood after mixing are slight. Some Factors GovERNING TRANSPORT OF BLoop In a previous report trauma to blood in the form of shaking caused loss of potassium from the cells and rapid laking.** During the winter of 1938-1939, the blood from ninety-six volun- tary donors at the Mt. Sinai Hospital* was collected in mason jars con- taining 2.5 or 3.0 per cent sodium citrate. From these, five to six cubic centimeters were removed and stored in identically shaped centrifuge tubes. These tubes were transported at once to the Presbyterian Hos- pital, a distance of six miles. There was no gross hemolysis in these tubes containing freshly shed blood, whereas old blood transported at the same time in partially filled _ mason jars revealed striking hemolysis. At varying intervals, the blood was removed from the refrigerator and mixed by inverting the tube 20 times. The results are depicted in Fig. 6. _* We express our gratitude to N. Rosenthal and his staff for their codperation in this inves. tigation and to the Mt. Sinai Hospital for permission. 390 THE BULLETIN Tanz X INCREASING EFFECT OF TRAUMA WITH INCREASING AGE OF BLOOD Plasma Potassium Plasma Ammonia Nitrogen Time i Milli Milliequival wae saa: Weeks| PerCent =| Per Liter Per Cont Moka Before After Before | After Before { After Before | After 0...... 25.4 26.0 6.5 6.7 0.09 0.151 0.064 | 0.11 Ll... 68.1 149.2 16.1 38.2 0.58 0.126 0.41 0.90 2...... 73.6 172.6 18.8 4.1 0.87* 1.45* 0.62 1.04 * Volume of blood, 25 cc. All other experiments, volume of blood, 50 cc. Discussion. The curve representing the diffusion of potassium in the unshaken blood is the same order of magnitude as previously reported for citrated blood.** In each instance, agitation caused both potassium and hemoglobin to leave the cells. During the first few days, this was noc as great as later. As the plasma potassium concentration approached that within the cells (i.e., decrease in concentration gradient) shaking dislodged less. To check these findings a controlled experiment was set up. Method. The blood of two voluntary donors, type A and type B, was drawn at weekly intervals and placed in 50 cc. colorimeter tubes with an internal diameter of 2 cm. containing 5 cc. of 3.5 per cent sodium citrate solution. At the end of rwo weeks following the drawing of the last blood, the previously drawn samples were removed from the refrigerator where they had been kepr, stoppered and sealed, at a temperature of 5 to 6° C. A small sample of the plasma was taken from each of the six tubes for potassium and ammonia determinations. After this, all of the tubes were rotated end over end on a specially devised piece of apparatus for twenty minutes, centrifuged, and from each, samples were taken for potassium and ammonia determinations after the rotating. The diffusion of potassium from red blood cells to plasma follow- ing trauma increases rapidly with increasing age of the blood. This sug- gests that if transportation of blood is contemplated, it should be done while the blood is fresh for the damage incurred by the cells is less at this time than when the blood is older. Newer Knowledge of Blood Transfusions 391 i The effects of shaking can be greatly reduced by filling the con- tainer completely with blood. Duran Jorda’® employed this principle during the recent Spanish Civil War in which preserved blood was used on an extensive scale. It would appear, then, that in the transportation of preserved blood, factors which would minimize the loss of intracellular substances, such as: decreasing the interface between cells and plasma; obviating any interface between liquid and gas by filling the container completely; should be also considered in addition to proper refrigeration, etc. Summary. 1. The diffusion of intracellular substances (potassium and hemoglobin) is accelerated by shaking and factors which limit this should be employed in blood preservation. 2. Transportation of preserved blood adequately refrigerated in suitable containers completely filled should be done early after shedding. PLASMA These studies indicate that degenerative changes occur as soon as blood leaves the vascular system, and progress with age. The large changes in the electrolyte composition might indicate its unsuitableness in those pathological states which are known to be associated with dis- turbances in the mineral metabolism, such as dehydration,** ** adrenal insufhiciency,**** and traumatic and hemorrhagic shock.***° Should large amounts of old blood be given rapidly in these conditions, dan- gerous sequelae might ensue. Since the work of Bowditch” in 1871, increasing attention has been directed to both serum and plasma as possible substitutes for whole blood transfusion.» ** 7 Amberson™ in his review on this subject has pointed out some of the advantages of plasma. Using the electrophoretic method of Tiselius,”? as modified by Longsworth,*® the stability of the various protein components in plasma has been investigated.” These observations confirmed the previ- ous work of Knoll®* who reported a decrease in albumin, a change in the albumin-globulin ratio, and an increase in gamma globulin. To ascertain more exactly the magnitude of these alterations, blood was drawn from a single donor at varying intervals, and the plasma sepa- rated from the cells on the same day. These values indicate a decrease in the albumin and a shift in the Tantz XI ELECTROPHORETIC PATTERN OF PLASMA FROM SAME INDIVIDUAL*® TYPE A Aye of Composition Mobilities, U w 10-* Blood dD ays Globulin Albumin! 4/@ a/jA p/A $/A /A pH | Albumin Renurks Per Cent a & s Y Fresh 3.96 2.27 0.08 0.18 0.08 0.18 7.72 5.95 4.2 2.9 1.6 0.2 Citrate 12 3.81 1.92 0.11 0.21 0.07 0.20 rece | 6.15 4.3 2.9 1.6 0.1 Citrate 20 3.66 1.72 0.11 0.21 0.08 0.26 7.78 6.25 4.4 3.2 17 0.2 Citrate 28 2.89 1.54 0.12 0.27 0.08 0.26 1.48 6.18 4.5 3.1 1.7 0.8 Citrate CONDITIONS OF EXPERIMENT In brief, a four times diluted portion of plasma is dialyzed in a bag made from cellophane tubing constructed in such a manner as to give a large surface to volume relationship. The buffer with a pH at 7.8 to 25° C. consisting of 0.025 M. lithium diethyl barbiturate, 0.025 M. diethyl barbituric acid, and 0.025 M lithium chloride is used. ‘The dialysis is carried out from 48 to 72 hours in a two liter flask containing fresh buffer at a temperature between 0° and 2° C. in a thermostatically controlled electric refrigerator. During the dialysis some precipitate separates out. It is, therefore, necessary to clear the protein solution in an angle centrifuge operated at 0° C. before its introduction ‘into the electrophoresis cell. The pH measurement is determined with the glass electrode of MacInnes and Longsworth.” The conductivity cell is of special design as well as the screened bridge used for the measurement of electrolytic conductance.” The es- tablishment of a Donnan equilibrium is assumed when further dialysis produces no change in conductance of the protein solution and the outside solution has the conductance of the original buffer. The manner of obtaining the protein patterns and of computing the different mobilities of the protein. constituents has been published by Longsworth, Shedlovsky, and MacInnes.” z6e NILGAITAG AHL Newer Knowledge of Blood Transfusions 393 albumin-globulin ratio, accompanied by an increase in gamma globulin in the supernatant plasma of stored blood. In order to compare two common methods of desiccating plasma, blood was drawn from the same individual and the plasma of one portion was dried under vacuum from the frozen state, while the second por- tion was dried at body temperature as has been suggested by Edwards, Kay, and Davie.™ Electrophoretic patterns of the former gave a sharper picture than the one derived from the latter, indicating that the plasma reconstituted from the frozen state appears to be more normal.?* Electrophoretic studies on liquid plasma preserved for five weeks, and in one instance for a year, showed evidence of some alterations, particularly in the beta globulin region. Practica, CONSIDERATIONS Healthy donors, free from communicable diseases, are to be chosen. Bluod obtained from cadavers is to be rejected, both on account of marked electrolytic changes and degradation products.”+ ** In the collection of blood, strict surgical asepsis is to be observed. Cleansing the skin is the most important step on account of the danger of contamination. The skin of the antecubital fossa should be scrubbed with soap for two minutes. This is removed with 70 per cent alcohol. Three and a half per cent tincture of iodine is painted over the area and allowed to dry. Venipuncture. Prior to the venipuncture, the skin is again swabbed with 70 per cent ethyl alcohol. A wheal is raised over the selected vein by injecting 1 per cent novocain, in the center of which a small nick is made by using a number eleven blade. A large needle of number thir- teen or fifteen gauge is used for the phlebotomy. The blood is collected by the closed system. This is superior to an open one for not only are chance contaminations decreased but also the loss of COs is lessened. The keeping qualities of blood are further enhanced by its collection in an atmosphere of carbon dioxide. As chemical changes are a function of temperature, the nearer zero the blood is stored, the slower will be these changes. Freezing is to be avoided as it causes rupture of the red cells. As chemical reactions are also a function of the surface area, it is natural that blood kept in spe- cially designed bottles in which the interface between the plasma and the cells is small, will enhance its keeping qualities. 394 THE BULLETIN | | | 6 ve Fresh 12 day ald Alb. day ald 28 day old. Figure 7—Electrophoretic patterns of preserved plasma from same donor. Four b! samples tuken at different times; collected in 35 per cent sodium citrate and stored in four tubes in electric refrigerator at 4° C. Type A blood. In the preparation of plasma, either the settling or the centrifuge method may be employed. With the latter, a bottle capable of contain- ing the whole donation of blood (500 cc. in one bottle) is ideal for two reasons: 1) this halves the chance of contamination, and 2) the yield of plasma is greater. (Number three International General Electric cen- trifuge bottle—62 per cent citrated plasma yield vs. 46 per cent yield by settling seventy-two hours in a dumbbell shaped flask.) In the removal of plasma, the procedure should be carried out in a dustproof, air-conditioned room, the air of which has been sterilized by ultraviolet light radiation. This will prevent possible airborne con- taminants that have been found in plasma. The removal of the plasma should be carried out in a cabinet, thus minimizing further chance of infection. Pooling and culture. The plasma from six to eight donor bottles is Newer Knowledge of Blood Transfusions 395 siphoned off by suction and pooled in a two-liter flask. Cultures, both aerobic and anaerobic, are taken. Final container. The plasma is not considered suitable for final pro- cessing until a two-week negative report has been received. The pool is then broken down into the final containers, 500 cc. of plasma may be mixed with 500 cc. of saline. The last portion of the pool is collected in a pilot bottle so that the concentration of the plasma mixed with saline is similar to that of the larger bottles. This material serves as a test on the sterility of the final container as well as furnishing another check on the sterility of the plasma. Filtration: The safety of the plasma is enhanced if it has been passed through a clarifying and sterilizing filter. This step may be carried out after the initial pooling in a Seitz filter. Dried Plasma: As proteins are more stable in a dried state, the keep- ing qualities of the plasma may be enhanced if it is reduced to such a condition by a suitable lyophile process. The dried plasma can then be dispensed in glass sealed ampoules. Use: Dried plasma is turned into the liquid state by the addition of distilled water. It may be reconstituted in either an isotonic or hyper- tonic form, depending upon the amount of diluent added. One abnormality, however, of such plasma is its extreme alkalinity. REFERENCES 1. Abel, J. J., Rowntree, L. G. and Tur- ner, B. B. Plasma removal with return ‘of corpuscles (plasmaphaeresis), J. Pharmacol. & Exper. Therap., 1918-14, 5:625. . Rous, P. and Turner, J. R. The pres- ervation of living red blood cells in vitro; methods of preservation, J. Eaper. Med., 1916, 28:219. . Robertson, O. H. Transfusion with preserved red blood cells, Brit. M. J., 1918, 7:691. Perry, M. C. Preservation of blood for transfusion, Wisconsin M. J., 1926, 25: 128. . Weil, R. Sodium citrate in the transfu- sion of blood, J.4.M.A., 1915, 64:425. . Yudin, S. S. Transfusion of stored cadaver blood; the first one thousand cases, Lancet, 1987, 2:861. . Jordé, F. D. 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