2,3.Diphosphoglycerate (2,3-DPG) Levels in Stored Blood. By L. A. Wood and E. Beutler. Bicarbonate-Adenine-Glucose-Phosphate-Mannitol (BAGPM) Solution

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From www.bloodjournal.org by guest on June 14, 218. For personal use only. The ffect of Periodic Mixing on the Preservation of 2,3.Diphosphoglycerate (2,3-DPG) Levels in Sred Blood By L. A. Wood and.

1 From by guest on June 14, 218. For personal use only. The ffect of Periodic Mixing on the Preservation of 2,3.Diphosphoglycerate (2,3-DPG) Levels in Sred Blood By L. A. Wood and. Beutler Blood from six donors was sred in two and 2,3-DPG levels were measured at varexperimental blood preservatives, PD- ious times during srage in both the adenine-sodium ascorbate and bicarbon- mixed and unmixed bags. Mixed bags ate - adenine - glucose- phosphate - mannil. maintained higher levels of glucose, ATP, ontrol aliquots were sred in PD- and 2,3-DPG throughout srage. Frequent adenine and AD-adenine preservatives. mixing was shown be essential for the Half of each donor s blood was sred maintenance of 2,3-DPG with these without mixing and half was mixed vigor- preservatives. orously 5 days each week. Glucose, ATP, F OR TH PAST several years, we have been studying experimental blood preservatives and have noted that aliquots of blood that had been subjected periodic mixing during srage maintained higher adenosine tnphosphate (ATP) levels than identical aliquots that had not been agitated. More recently, we have become aware that periodic agitation has a much more dramatic effect on 2,3-diphosphoglycerate (2,3-DPG) maintenance during blood srage. We have developed two new blood preservatives that permit 2,3-DPG be maintained at high levels for 4-6 wk, if the blood-preservative mixture is subjected periodic agitation. This work is a report of a systematic investigation of the role of agitation of sred blood on maintenance of 2,3-DPG and ATP levels. MATRALS AND MTHODS Blood was drawn from normal hospital personnel and was sred in either of two experimental blood preservatives: Bicarbonate-Adenine-Glucose-Phosphate-Mannil (BAGPM) Solution Forty-milliliter aliquots of blood were drawn in 5-ml Fenwal plastic bags containing either 1 ml of acid-citrate-dextrose-adenine (AD-Ad) solution, or 1 ml of acid-citratedextrose, NH formula B, (AD) solution. The concentration of adenine in the AD-Ad solution was 2.5 mm. Four aliquots of blood were collected in AD-Ad preservative and eight aliquots were collected in AD solution. All were centrifuged at 4 g for 1 mm. Following centrifugation, the bags containing AD-Ad were mixed and placed in srage. The bags collected in AD solution were placed in a plasma extracr; the plasma was removed, and a volume of BAGPM solution calculated be equivalent the volume of From the Division of Medicine, Department of Hemalogy, ity of Hope Medical enter, Duarte, alif Submitted Ocber 2, 1972; revised November 2, 1972; accepted December 2, Supported in part by Army ontract DADAX Lee A. Wood, M.D.: Department of Hemalogy, ity of Hope Medical enter, Duarte, alif rnest Beutler, M.D.: hairman, Division of Medicine, ity of Hope Medical enter, Duarte, alif. 911, and linical Professor of Medicine, University of Southern aiforni Los Angeles, alif by Grune & Stratn, nc. Blood, ol. 42, No. 1 (July),

From www.bloodjournal.org by guest on June 14, 218. For personal use only. 18WOOD AND BUTLR the remaining erythrocytes was added. BAGPM is a solution containing 11.4 mm sodium bicarbonate, 14.

2 From by guest on June 14, 218. For personal use only. 18WOOD AND BUTLR the remaining erythrocytes was added. BAGPM is a solution containing 11.4 mm sodium bicarbonate, 14.3 mm sodium carbonate, 55 mm glucose, 1 mm adenine, 2 mm sodium phosphate, and 27.4 mm mannil. Two bags collected in AD-Ad and four bags sred in BAPM solution were placed in the refrigerar at 4#{176} and remained unuched until analyzed. The other six bags were placed in the refrigerar and were removed 5 consecutive days each week and mixed thoroughly for 1-2 mm. All bags were sred in the vertical position. At 7 and 15 days, one mixed and one unmixed bag of blood sred in AD-Ad were removed for analysis. After 7, 15, 28, and 42 days in srage, one mixed and one unmixed bag of blood sred in BAPM solution were removed for analysis. The bags were placed upright in the plasma extracr, and a 2-gauge needle attached three-way spcock was inserted in the injection site in the p of the bag. From one opening of the three-way spcock a plastic tubing with a male fitting on each end was connected an empty plastic bag. The other orifice of the three-way spcock was used extract aliquots of plasma or red cells during the transfer of the blood-preservative mixture from the original bag the empty bag. The entire contents of the bag used during srage were pressed over in the empty bag, and aliquots of 1-3 ml were removed from the plasm from the first 2-3 ml of packed red cells that came through the spcock and from the final 2-3 ml that were pressed over in the empty bag. After the entire contents of the original bag (minus the aliquots removed) had been placed in the new bag, the bag was mixed vigorously, and an aliquot was removed for analysis. This aliquot will be referred as tal. The first few milliliters of red cells pressed over are designated p and the final few milliliters are referred as botm. Plasma was assayed for ph and glucose; ATP, 2,3-DP, ph, and glucose were assayed in the p, botm, and tal aliquots. rythrocytes from an additional three donors were sred in BAGPM solution, as outlined above, and after 24-hr srage, viability studies were performed by a method described previously.1 ATP, ADP, AMP, and 2,3-DPG were measured in fresh erythrocytes and in erythrocytes sred 24 hr. itrate-p hosphate-d extrose-aclenine-sodium Ascorbate (PD-Ad-NaA) PD-Ad-NaA solution consists of PD solution supplemented with sufficient adenine and sodium ascorbate give final concentrations in the blood-preservative mixture of.5 mm and 5.5 mm (1 mg/1 ml), respectively. Blood from each of three donors was collected in four bags of PD solution and six bags of PD-Ad-NaA solution. Two bags from each donor collected in PD and four bags collected in PD-Ad-NaA were sred and were unuched until the time of analysis. Duplicate samples were agitated vigorously for 1-2 mm 5 consecutive days each week. At 15 and 21 days, one mixed and one unmixed bag sred in PD solution were analyzed as described in the preceding section. At 15, 21, and 28 days, one mixed and one unmixed bag sred in PD-Ad-NaA were subjected similar investigation. n another experiment, blood from two donors was sred in AD-Ad. Two bags were drawn from each donor, and each had a final volume of 15 ml. One bag was sred in the upright position, and the other was sred horizontally. The bags were not moved until after 42-days srage. At this time, a spinal needle with stilette in place was inserted in the upright bag, and successive aliquots from the upper, middle, and lower third of the red cell layer were removed for analysis. Without changing the position of the bag, a spinal needle was inserted in the horizontal bag, and aliquots from the p and botm layers of red cells were removed. Between the successive withdrawals of various fractions, the stilette was in place. After the needle had moved each new layer of the blood,.5 ml of erythrocytes was removed and discarded before the aliquot used for analysis was drawn. After successive fractions were withdrawn, the bags were mixed thoroughly, and aliquots were drawn for viabiity studies. n one patient, a red cell viability was done on successive days using blood from the upright and horizontal bags. Glucose was estimated using the glucose oxidase method. Blood ph was measured at #{176} in a Beckman blood ph meter. ATP, ADP, AMP, and 2,3-DP were determined as previously described.2 Purified adenine was obtained from Dr. Grant Bartlett, San Diego,

3 From by guest on June 14, 218. For personal use only. PRSRATON OF 2,3 DPG LLS N STORD BLOOD 19 alif. All other chemicals were obtained from commercial sources and were of analytic grade. Blood was sred in plastic bags obtained from Fenwal Laboraries, Morn Grove, ll. or Mcaw Laboraries, Glendale, alif. These bags were specially made contain no more than 75 ml. BAGPM RSULTS rythrocytes from three donors were sred in this preservative, as outlined above, and in AD-Ad. Aliquots of the plasm the p layer of erythrocytes, botm layer of erythrocytes, and the entire mixture (tal) were assayed for ATP, 2,3-DPG, ph, and glucose at 7, 15, 28, and 42 days. Results are presented in Table 1 and Fig. 1. rythrocytes sred in AD-Ad rapidly lost their 2,3-DPG. 2,3-DPG levels were negligible in the unmixed aliquot at 1 wk and in the mixed aliquot at 2 wk. There was better maintenance of 2,3-DPG in BAGPM solution, but even here 2,3-DPG levels were diminished in unmixed bags. A significant MXD -Ad NOT MOD - #{149}--#{149} 8 MOD - BAG-PM NOT MOD --.s 7.-f S 3 1\ 3 \\ 2 f,, / L J \ N..% Fig. 1. Blood sred in AD-Ad and BAGPM, mixed and unmixed for 42 days. Mean and standard error for three donors. (A), 2,3-DPG; (B), PH; (), ATP; (D), glucose.

4 From by guest on June 14, 218. For personal use only. 2 WOOD AND BUTLR 8) 1 )11, F- 1 1 X t )X OF.F-F- X () >X ( c,j a- (!J a) to ) ) l) a), (), (f a- a.!, a- ) csj (), >,., - 8) 88 O,._ ) ( o n. 1 o. 8) > 1 8) () 8) >.g 1L 11fl-L()F-O)O%J,- 11,-1 )1-F- O 1 X,- l) ) O a X O () Q O 1 F.F- O,1,-OO.g,-j - O O 1 X 4 X F- 1 F. F- F- F. F. F- F. F. F- F- F- F- F-F.F-F-F- F- F. F- F. 5. F-. F. F- F- F- F. F- F- F- F. F. F- F. F- F- F- F- F- XJX, ) D O F- (X X ci ) X O )( v.v ( < <1< z D ) D ) (, D SJ a U) () D F- U- a) l).!, a a- ci v) ( 4 c.j. o ) (,) ( = <.! 1.3. (.. -. U). N, X )(t) )( ;;t:; F-8) J ) ( X 11 O O ) 1 O X1 to ( ( X ( 41 O O JX X X JO )F- OOF-) (1( X F- O O X X X X ) F-ma O O -,-)1F- - X v- i2 -j F-F-F-F. SF-F- U. ;t; F- XU) )O) 1 X 1) gf.. F- 1 -a i! F-F. F-F-F-F- F-F-F-F- F-O F-F-F-F- F-F-F-F- F-F- F- 4)F- X J )1 O. * -t).-.- z F-F-F-O 4- J 883 F-F- cio v- j :s -.- z i X F-F-F-F- F-F-F-F- c t) fl F- #{149}- D z

From www.bloodjournal.org by guest on June 14, 218. For personal use only.

5 From by guest on June 14, 218. For personal use only. PRSRATON OF 2,3 DPG LLS N STORD BLOOD 21 difference between the p and botm layers of erythrocytes was observed on days 28 and 42. n AD-Ad solution, ph fell more rapidly in the unmixed than in the mixed bags and the ph in both cell layers was more acid than that of the plasm n BAGPM solution, the initial ph was 8.3 and the final ph in both mixed and unmixed bags was approximately the same. However, it can be seen that the ph in both p and botm layers in unmixed bags fell more rapidly than that of the medium. Glucose levels fell more rapidly in unmixed bags of erythrocytes sred in either AD-Ad or BAGPM, as seen in Fig. 1D. ATP levels were well maintained at 1 and 2 wk in AD-Ad. n BAGPM solution, ATP fell low levels in unmixed bags after 42 days and the AT! of the botm layer was significantly less than that of the p layer of erythrocytes at 28 and 42 days. n the mixed BAGPM bag levels, the ATP fell low levels at 7 days and then steadily increased throughout the rest of the srage period. n other experiments, we have observed that the ATP level in erythrocytes sred in this preservative reaches its nadir at 24 hr and then gradually increases. n the blood from one donor sred in this manner, the initial ATP level of fresh erythrocytes was 3.55 jtmoles/g Hb. After 24-hr srage, the ATP had fallen 1.33 prnoles/g Hb and at 2 and 5 days the ATP was 1.74 and 1.81 /Lmoles/g Hb, respectively. The initial ph of the blood-preservative mixture was 7.92 and the ph had fallen 7.75 by day 5. t is probable that the low levels of ATP observed after 24-hr srage are due the high ph of the blood-preservative mixture, which results in reduction of NAD NADH and inhibition of glycolysis at the glyceraldehyde phosphate dehydrogenase step. Since ATP levels are closely related posttransfusion viability in erythrocytes sred in conventional blood preservatives, we estimated the viability of cells sred for 24 hr in BAGPM solution. Red cells from three donors sred in BAGPM solution were reinfused after 24-hr srage. rythrocyte viability was greater than 95% in each of the three donors, despite the fact that ATP levels had fallen markedly during srage. AD? levels had increased an average of 34%, and AMP levels had increased an average of 35% during the 24-hr srage. Thus, there was sufficient adenine nucleotide for formation of AT? after reinfusion. PD-Ad-NaA Blood from three donors was collected in either PD or PD-Ad-NaA. Half was not mixed and half was mixed vigorously five times a week during srage. At 15, 21, and 28 days, the bags were fractionated and analyzed as in the BAGPM experiments. The mean values of three donors are presented in Table 2 and the mean and standard error of the Total columns of Table 2 are presented graphically in Fig. 2. The results are, in many respects, similar those found in the BAGPM experiment. n unmixed PD bags, 2,3-DPG fell low levels by 14 days and was absent at 21 days. Mixed PD bags had better 2,3-DPG maintenance, but the 2,3-DPG was essentially gone at 21 days.

6 From by guest on June 14, 218. For personal use only. 22 WOOD AND BUTLR 8 - MXD PD - NOT MXD #{149}--#{149} PO-Ad-NoA MXD - NOT MXD S--#{149} Fig. 2. Blood sred in PD and PD-Ad-NaA, mixed and unmixed, for 28 days. Mean and standard error for three donors. (A), 2,3-DPG; (B), ph; (), ATP; (D), glucose. Blood sred in PD-Ad-NaA had higher levels of 2,3-DPG throughout the srage period, and at 28 days the mixed sample had levels half that of fresh blood, while unmixed bags had 1%-2% of normal levels. The ph of both PD and PD-Ad-NaA mixed bags remained higher throughout the 28-day srage period than that of unmixed bags. ATP levels remained higher in PD-Ad-NaA bags throughout srage and, after 14 days, AT? levels in the unmixed PD-Ad-NaA bags fell much more rapidly than that in mixed bags. After 21 days, the glucose level appeared drop more rapidly in unmixed PD-Ad-NaA bags than in mixed bags, and the glucose level in the botm layer of the unmixed bags was significantly different than that of the p layer. Bags of blood from two donors were sred in AD-Ad in the vertical and horizontal position. The bags remained unuched throughout srage and, after 42 days, aliquots were taken from the p, middle, and botm thirds of the red cell layer in the upright bag and from the p and botm levels of the erythrocyte layer in the horizontal bag. AT?, ph, and glucose were

From www.bloodjournal.org by guest on June 14, 218. For personal use only. PRSRATON OF 2,3 DPG LLS N STORD BLOOD 23 Table 3.

7 From by guest on June 14, 218. For personal use only. PRSRATON OF 2,3 DPG LLS N STORD BLOOD 23 Table 3. ATP, ph, and Glucose in Top, Middle, and Botm Thirds of rythrocyte Layer of AD-Ad Blood Sred Unmixed for 42 Days in ertical and Horizontal Postions ertical Horizontal Donor Top Middle Botm Top Botm ATP (moles/g Hb) W.S PP ph W.S P.P Glucose (mg/1 ml) W.S PP measured and are presented in Table 3. n the bag sred vertically, AT? levels fell progressively from the p botm. n the horizontal bag there was little difference in AT? between the p and botm layer of cells. The ph tended be higher in the botm than in the p layer of red cells in the bags sred vertically. The ph was more uniform in the erythrocyte layers of the hbrizontal bags. The glucose level was lower in the botm layer of erythrocytes in vertical bags than in those sred horizontally. After 42 days, a viability study was performed, using blood from the vertical bag of donor P.?. These cells were 57.5% viable. On the following day, a viability study was done on the blood sred in the horizontal bag, and this was 71.5%. DSUSSON Dern et al.3 have shown that AT? levels and erythrocyte viability are higher in blood that is agitated during srage than in unmixed blood. The present work confirms this finding and also shows that periodic mixing during srage is essential if 2,3-D?G levels are be maintained. When blood or blood preservative mixtures are sred without agitation, the cells rapidly fall the botm of the container. ells at the p of the red cell layer have a different metabolic environment from those at the botm. ells in the Upper layer are in contact with the plasma or preservative medium, and the potassium lost and byproducts of their metabolism, such as lactic acid, can diffuse in this relatively large, nonmetabolizing volume. The cells at the botm of the container, on the other hand, are surrounded by actively metabolizing red cells. The lactic acid they produce diffuses in the small amount of plasma in the immediate vicinity of the cell, and the glucose level falls rapidly. n contrast, cells at the p of the erythrocyte layer have much more glucose available them. A gradient is formed between the p and botm layers of red cells, and as shown by the present experiments, ATP, 2,3-DPG, and glucose all fell lower levels in the botm layer. The greater the distance between the p and the botm of the erythrocyte layer, the greater the gradient.

From www.bloodjournal.org by guest on June 14, 218. For personal use only. 24 WOOD AND BUTLR Since levels of 2,3-D?G, AT?

8 From by guest on June 14, 218. For personal use only. 24 WOOD AND BUTLR Since levels of 2,3-D?G, AT?, and glucose are diminished in the lower levels of the red cell layer when compared the upper levels, it might be expected that ph would also be diminished at this site. t is not clear why this could not be demonstrated experimentally. With all preservatives tested, ph levels remained higher throughout srage in mixed bags than in unmixed bags. Plastic blood bags are permeable 24 and it is possible that periodic mixing permitted more efficient diffusion of O2 through the bags. rythrocyte 2,3-D?G levels, known be very ph dependent,5 tend diminish with decreasing ph. t is of interest that the unmixed cells manifested an increased rate of glucose consumption in spite of the lower ph. As has been shown in this laborary6 and by others,7 2,3-D?G inhibits glycolysis by affecting the rates of the hexokinase, phosphofruckinase, aldolase, and glyceraldehyde phosphate dehydrogenase reactions. Thus, the 2,3-D?G depleted erythrocytes apparently are able convert more glucose lactic acid in spite of the slightly lower ph. t is possible that the late decrease in red cell AT? levels from unmixed sampies may be due local exhaustion of glucose. When cells are mixed frequently during srage the ph remains high, and this favors the continued maintenance of 2,3-D?G levels. Therefore, less glucose is used and less lactic acid is produced. Frequent mixing throughout blood srage leads better maintenance of AT?, 2,3-D?G, and ph. The two experimental blood preservatives used here have been described elsewhere. rythrocytes sred in?d-ad-naa have a viability greater than 7% after 28-days srage,8 and red cells sred in BAGPM preservative have a viability greater than 7% after 42 days.9 Although AT? levels are markedly diminished after 24-hr srage in this preservative, viability is not adversely affected. We have achieved better 2,3-D?G levels with both of these preservatives in other experiments because we routinely sred bags in the horizontal position. We sred these bags in the vertical position so that the depth of the red cell layer would more closely approximate that found in whole units under standard methods of srage. Bottles and bags of blood are commonly sred in the upright or vertical position. This is so with bottles because the botm of blood bottles is commonly the flattest part of the container. With the help of various types of cardboard containers, blood bags are also sred in the upright position because this obviates the need for centrifugation if packed red cells, instead of whole blood, should be called for. The present experiments show that this is the least advantageous way in which sre blood. Although the cells used in these experiments were mixed 1-2 mm 5 consecutive days each week, for most of each 24-hr period they were stationary. More frequent mixing, several times a day and every day, might well be desirable. Modification of existing blood bank refrigerars for periodic mixing could probably be carried out relatively easily. Frequent mixing has been shown be necessary for the maintenance of 2,3-D?G levels in erythrocytes sred in these experimental preservatives but mixing produced little effect on erythrocytes sred in AD-adenine or in?d.

9 From by guest on June 14, 218. For personal use only. PRSRATON OF 2,3 DPG LLS N STORD BLOOD 25 n most clinical situations requiring blood transfusion, the level of 2,3-DPG in sred red cells is of little significance. n patients with massive blood loss, however, the 2,3-DPG level of transfused erythrocytes may be quite important, since several hours are required following transfusion for 2,3-D?G attain normal levels and since 2,3-DPG-depleted erythrocytes have an increased affinity for oxygen. Such patients, rapidly given several units of 1-3-wk-old AD blood, may suffer tissue oxygen deprivation despite the presence of an adequate hemacrit. Several studies 3 have shown that this may occur. AKNOWLDGMNT We wish thank Miss Judy Metro and Mrs. rnestine Williams for their technical assistance. RFRNS 1. Wood LA, Beutler : The viability of human blood sred in phosphate adenine medi Transfusion 7:41, Beutler : Red ell Metabolism: A Manual of Biochemical Methods. New York, Grune & Stratn, 1971, pp Dern R, Wiorkowski JJ, Matsuda T: Studies on the preservation of human blood.. The effect of mixing anticoagulated blood during srage on the post-srage erythrocyte survival. J Lab lin Med 75:37, Beutler, Wood LA: Preservation of red cell 2,3-DPG and viability in bicarbonate-containing medium: The effect of blood-bag permeability. J Lab lin Med 8:723, Beutler, Meul A, Wood LA: Depletion and regeneration of 2,3-diphosphoglyceric acid in sred red blood cells. Transfusion 9:19, Srivastava SK, Beutler : The effect of normal red cell constituents on the activities of red cell enzymes. Arch Biochem Biophys 148:249, Keitt AS: Metabolic characteristics of 2,3-DPG-rlch red blood cells. lin Res 19:4, Wood L, Beutler : The effect of ascorbic acid on the 2,3-diphosphoglycerate (2,3-DPG) level of sred blood. lin Res 2:186, Wood L, Beutler : Srage of erythrocytes in artificial medi Transfusion 11:123, Beutler, Wood L: The in vivo regeneration of red cell 2,3-diphosphoglyceric acid (DPG) after transfusion of sred blood. J Lab lin Med 74:3, Mconn R, Derrick JB: The respirary function of blood: Transfusion and blood srage. Anesthesiology 36:119, aleri R, ollins FB: Physiologic effects of 2,3-DPG-depleted red cells with high affinity for oxygen. J App Physiol 31 :823, Broennle AM, Tung K, Buchman B, Layer MB: Oxyhemoglobin dissociation folowing massive transfusion in man. Fed Proc 29:329, 197

From www.bloodjournal.org by guest on June 14, 218. For personal use only. 1973 42: 17-25 The ffect of Periodic Mixing on the Preservation of 2,3-Diphosphoglycerate (2,3-DPG) Levels in Sred Blood L.

10 From by guest on June 14, 218. For personal use only : The ffect of Periodic Mixing on the Preservation of 2,3-Diphosphoglycerate (2,3-DPG) Levels in Sred Blood L. A. Wood and. Beutler Updated information and services can be found at: Articles on similar pics can be found in the following Blood collections nformation about reproducing this article in parts or in its entirety may be found online at: nformation about ordering reprints may be found online at: nformation about subscriptions and ASH membership may be found online at: Blood (print SSN , online SSN ), is published weekly by the American Society of Hemalogy, 221 L St, NW, Suite 9, Washingn D 236. opyright 211 by The American Society of Hemalogy; all rights reserved.

with their viability and resistance to hemolysis ,19

with their viability and resistance to hemolysis ,19 A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e, V o l. 1, N o. 2 C o p y r i g h t 1 9 7 1, I n s t i t u t e f o r C l i n i c a l S c i e n c e In Vitro Parameters of the Integrity