ANESTHESIA AND ANALGESIA . . . Current Researches VOL.54, No. 1, JAN.-FEB.,1975

1

Blood Components in the Treatment of Acute Blood Loss: Use of Freeze-Preserved Red Cells,' Platelets, and Plasma Proteins CAPTAIN C. R. VALERI, USNR (MC)* Chelsea, Massachusetts

To avoid untoward reactions from blood transfusions and to make best use of t h e limited supply of blood, anesthesiologists and surgeons have many newly developed means a t their disposal. Blood components should be separated from whole blood at t h e time of collection and prepared f o r either liquid or freeze-preservation. Citrate-phosphate-dextrose (CPD) blood should be separated into i t s components at room temperature within 4 hours of collection f o r greatest service from each collected unit. Red cell concentrates with hematocrits of 70 volumes percent can be prepared from t h e whole blood at t h e time of collection and frozen either shortly thereafter or after storage a t 4°C. f o r up t o 3 weeks. Redcell levels of 2,3-diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP) can be increased by a rejuvenation process prior t o freeze-preservation with either 40 percent W/V glycerol and storage a t - 8 O O C . or with 20 percent W/V glycerol and storage at - 1 5 0 O C.

While hemorrhagic shock can best be managed with fresh whole blood, such blood is often not available; liquid- and freeze-preserved products serve as best substitutes. When previously-frozen washed red cells a r e used, crystalloid, colloid, coagulation factors, and platelets may also be required. Platelet concentrates stored a t 4 O C. provide platelets t h a t a r e hemostatically effective immediately upon infusion but have poor circulation. Platelet concentrates stored a t 22O C. provide platelets t h a t have good circulation but upon transfusion have impaired hemostatic effectiveness. The coagulation factors and oncotic properties of plasma protein necessary f o r proper treatment of patients in hemorrhagic shock can be met by a n adequate supply of freshfrozen plasma and albumin. When liquid-stored red-cell concentrates or whole blood is given, filters must be used to remove t h e accumulated amorphous material, although t h e actual effects of t h e infused microaggregates a r e not yet known.

banks are responsible for providing blood and blood products to anesthesiologists for use during surgical procedures to replace acute blood loss. This need can be met by having available compatible blood that is free of the Australia antigen (HAA, hepatitis-associated antigen, or HBAg, hepatitis B antigen) , malaria, cytomegalovirus, and other infectious diseases. Ideally, the blood should be collected in heparin and stored at room temperature for no more than 4 hours. Since fresh whole blood often is not available, liquid- and freeze-preserved products serve as the best substitutes.

The practice of storing whole blood in either acid-citrate-dextrose ( ACD) or citrate-phosphate-dextrose (CPD) at 4"C. for up to 3 weeks before transfusion is no longer considered acceptable by many investigators. Although posttransfusion survival values of red cells, albumin, gamma globulin, and fibrinogen are adequately maintained, significant deterioration of factors V and VIII, platelets, granulocytes, and red-cell oxygen (0,) transport function occurs (figs. 1-4).

LOOD

Preparation of Blood Components. Whole blood is used when it is necessary

*Officer in Charge, Naval Blood Research Laboratory, Chelsea, Massachusetts 02150. This work was supported by the U.S. Navy. The opinions or assertions contained herein are those of the author, and are not to be construed as official or reflecting the views of the Navy Department or Naval Service a t large. Paper received: 12/21/73 Accepted for publication: 3/19/74

2

ANESTHESIA AND ANALGESIA . . . Current Researches

z y y

244".

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100

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FIG. 1. 24-hour posttransfusion survival and lifespan of red cells stored at 4" C. in either ACD or CPD for up to 3 weeks, either as whole blood or as red-cell concentrates.'

0

7

14

21

Days Stared at +4C

FIG.2. Schematic of effect of storage of red cells in ACD or CPD at 4" C. on 24-hour posttransfusion survival value and red-cell affinity for O?.

L

0

7 14 21 FIG. 3. Schematic of deterioration of plasma proteins following storage of whole blood in ACD or CPD at 4" C. for up to 21 days.

to maintain blood volume and increase the 0,-carrying capacity of the circulating blood. Whole blood may produce a number of adverse reactions that cannot always be detected with the technics now used for crossmatching. Such reactions may be triggered by antigens in white cells and platelets, by isoagglutinins, by the anticoagulant preserv-

54, NO. 1,JAN.-FEB., 1975

I

21

Days at + 4 C FIG.4. Schematic of deterioration of platelets, granulocytes, and lymphocytes following storage of whole blood in ACD or CPD at 4" C. for up to 2 1 days.

ative, or by protein and nonprotein plasma factors.' To avoid these reactions and to determine the best use of our limited supply of blood, the physiology of red cells and platelets has been examined, and large-scale development of plasma fractionation methods has been undertaken. This concept of component therapy involves separation and storage of cellular and plasma components shortly after collection (fig. 5). Serial or continuous centrifugation is necessary to separate the red cell and platelet concentrates from platelet-poor plasma. Blood collected in either heparin, ACD, or CPD and stored at room temperature (22" C . ) for not more than 4 hours is considered to be "fresh blood," and is the best replacement for acute blood loss. During this storage time there is only minimal damage to red cells, platelets, and plasma proteins. When the blood is collected in a multiple plastic bag system and is not transfused within 4 hours of collection, it can be separated into its components and prepared for freeze-preservation (fig. 5). It is common practice in Europe to collect blood in glass containers, and to use plastic bags for collection and preparation of platelet concentrates and for plasmapheresis of donor blood. In the United States, plastic containers are used for blood collection simply because they make preparation of blood components easier. With a system using three plastic bags, it is simple, though fairly expensive, to separate red-cell concentrates, platelet concentrates, and plasma from whole blood. The plasticizers used in polyvinylchloride bags have been reported to be potentially toxic, Jaeger and Rubin2 having shown that phthalate esters accumulate in blood stored in such bags. One of these phthalate esters,

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Acute Blood IASS . . . Valeri

3 REC Liquid stored concentrated RBC( Hct-70%) stored at t 4 C -Frozen with glycerol:

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1) Cryoprecipitote AHG 2 ) Fresh frozen plosmo 3) Albumin 4 ) Gommo globulin

FIG.5. Comparison of preserved whole blood and preserved components of whole blood. Within 4 hours of storage at room temperature (22"C.), platelet concentrates, red-cell concentrates, and plasma protein can be isolated from whole blood, and the components individually preserved using liquid and freezing procedures.

di (2-ethylhexyl) phthalate (DEHP) , has been isolated from liver, lung, and other tissues at necropsies of patients who had received transfusions; however, the same substance has also been found in tissues of patients who had not received transfusions. There are no hard data to show that DEHP actually causes damage to red cells, platelets, granulocytes, or plasma proteins: most of the reported data have been obtained by tissue culture studies. Nevertheless, because of the controversy surrounding the use of these plasticizers, several companies have developed bags of inert materials such as Teflon@, bioriented polyolefin (UCARO), and polyethylene. The purchase price of these is greater, adding to the expense of disposable software and hardware used for collection of blood and separation of components.

transfusions may well be due to embolization of microaggregates of platelets, leukocytes, and amorphous debris from stored blood. After transfusion through a 170-micron (standard) filter, there was a significant stepwise reduction in the screen filtration pressure from central venous, to arterial, to peripheral venous blood drawn simultaneously from these sites. These data suggest that the infused debris was removed mainly by the pulmonary and peripheral microcirculation. Although less debris is infused with small-pore filters, most clinicians prefer the larger-pore ones because they allow more rapid infusion. Solis and Gibbs3 have reported that the standard filter works more efficiently when stored blood is centrifuged before administration. Apparently, centrifugation causes larger particles to form from smaller ones, Detection of Serum Hepatitis.-An at- and these are retained in the standard filter. tempt to curb the transmission of hornoloWhen stored red cells with hematocrit gous serum jaundice has led to regulations values of about 70 volumes percent are not demanding that all blood be tested for the washed before transfusion, they must be adpresence of HAA. The counterelectroosmo- ministered with external pressure either phoresis method, a simple and moderately through a standard filter or through a 40sensitive technic, takes about 2 hours. The micron microaggregate filter. When the unradioimmunoassay, at least 100 times more washed red cells have hematocrit values of sensitive, takes about 3 hours; there is some about 90 volumes percent, they must be doubt as to whether its sensitivity is spe- diluted before transfusion or they will not cific for HAA, Obviously, if the HAA test pass easily through the standard filter. takes longer than 4 hours, the blood must Amorphous material accumulates in blood be kept at 4"C., and at that temperature deterioration of platelets, granulocytes, and during storage at 4" C. This results from deterioration of white cells, platelets, and certain plasma proteins occurs (figs. 3 and lipoproteins. The accumulation of 4). Neither of these tests is ideal, and one plasma amorphous debris in ACD and CPD blood that is simple, sensitive, specific, and rapid during storage at 4"C. was not primarily is being sought. related to the presence of DEHP in the Blood Filters.-Pulmonary insufficiency plastic container. Amorphous material acin patients who have received massive blood cumulates in plastic bags which do not

4

1975 ANESTHESIA AND ANALGESIA . . . Current Researches VOL.54, No. l, JAN.-FEB.,

leech DEHP. McNamara and coworkersJ have suggested that some of this amorphous material passes through the standard filter and is removed through the lung, sometimes producing pulmonary insufficiency. Microaggregate filters may help to solve this problem and are being investigated. In the meantime, if platelets are separated from the blood within 4 hours of collection, this risk may be reduced.

Whole Blood and Blood Components in the Treatment of Acute Blood Loss.-Blood volume often is reduced acutely during surgery. Hypovolemia produces a decrease in perfusion of organs which, in turn, leads to acidosis and tissue hypoxia. Usually, hemostasis is maintained by adequate surgical suturing of bleeding vessels as well as by activation of the platelets and the coagulation system, When clotting is accompanied by acidosis, stasis, and hypoxia as a result of hypovolemia, disseminated intravascular coagulation may occur. Both the degree of blood loss and the duration of the hypovolemic shock influence the changes in coagulation factors and in platelets. Treatment of acute blood loss may include transfusion of red cells to increase the 0,-carrying and releasing capacity of the blood.

bumin as liquid-stored whole blood (fig. 3 ) , but the posttransfusion survival and the 0,-transport function are similar in both. Patients in hemorrhagic shock have been treated successfully with a combination of previously frozen washed red-cell concentrates and crystalloid and colloid plasmavolume expanders.5 Since blood loss in hemorrhagic shock usually is replaced by preserved red cells and not by fresh whole blood, it is imperative that the number of irreversibly damaged red cells produced by the preservation procedure be known. After storage at 4” C. for 1 week, about 90 percent of the red cells remain viable; after 2 weeks, about 80 percent; and after 3 weeks, about 70 percent (fig. 1 ) . Thus, in a patient who has lost 5 units of blood, only 4.5 units will be replaced by the transfusion of 5 units of preserved red cells stored at 4” C. for 1 week, only 4 units will be replaced by the transfusion of 5 units of red cells stored at 4”C. for 2 weeks, and only 3.5 units will be replaced by the transfusion of 5 units of red cells stored at 4” C . for 3 weeks.

Posttransfusion Survival and 0, Transis considered satisfactory when the red cells circulate and Patients in hemorrhagic shock are treated function immediately upon transfusion. with either colloid or crystalloid solutions “Preservation injury” produces a certain initially to restore plasma volume. Colloid number of severely and irreversibly damaged solutions such as albumin, plasma, dextran, cells that are removed from the circulation and gelatin solutions have been recommend- at an accelerated rate, usually during the ed because of their ability to maintain fluid transfusion and in the 24 hours afterward.6 within the intravascular space. Such crys- The only way to estimate the number of talloid solutions as sodium chloride and lac- these nonviable red cells is to measure the tated Ringer’s solution have also been used, 24-hour posttransfusion survival in vivo. although their osmotic effects are transient. Preserved red cells that remain in the cirRestoring the plasma volume and the 02- culation 24 hours after transfusion have the carrying and releasing capacity of the blood potential for normal long-term survival must be the initial goal in treating hypo- (fig. 1). But the lifespan may be adversely volemia. Additional treatment with platelets affected by the recipient’s intravascular enand coagulation factors may be required, vironment, especially if certain immunologic, depending upon the magnitude of blood loss, chemical, and mechanical factors are presthe duration of shock, and the solutions and ent. blood products used. O2 Transport Function.-Preservation inDuring a surgical procedure, patients may jury not only makes some cells nonviable require red cells, platelets, or plasma pro- but also alters the ability of the surviving teins to correct red-cell-mass deficits, throm- cells to deliver 02.Valtis and Kennedy7 bocytopenia, plasma oncotic pressure, or reported that the functional defect produced coagulation factors. Liquid or previously during storage of red cells at 4” C. for about frozen red-cell concentrates can be used in 1 week resulted in increased affinity for 0,. combination with crystalloid or colloid solu- This defect has also been shown to be retions to replace blood loss. Liquid-stored lated to deterioration of the red cell organic concentrated red cells with hematocrits of phosphate compound, 2,3-DPG.*.D A corre70 volumes percent contain only one-third lation has been found between 2,3-DPG and as much fibrinogen, gamma globulin, and al- the affinity for 0, of preserved red cells. p o r t Function.-Preservation

Acute Blood Loss . . . Valeri

5

During storage of blood in either ACD or CPD at 4" C., the level of 2,3-DPG decreased substantially, while there was only a slight decrease in ATP (fig. 6 ) . Valtis and Kennedy7 also reported that within 24 hours of transfusion of red cells with this increased O2 affinity, the patient's oxyhemoglobin dissociation curve was restored to normal. The rate at which these cells are restored in vivo is determined by the quantity and quality of the transfused cells and by the patient's metabolic condition. Valeri and Collins'o transfused preserved red cells with an increased O2 affinity into nonsurgical patients who were normotensive and anemic. The effect of the transfusion on systemic 0, consumption and cardiac output were studied, as well as the difference in systemic arteriovenous 0, content, redcell 2,3-DPG, and A T P levels, and the oxyhemoglobin dissociation curves. Transfusion of 3 to 5 units of red cells that were depleted of 2,3-DPG and had high affinity for 0, following storage at 4" C. for 2 to 3 weeks had no effect on systemic 0" consumption (fig. 7 ) . The cardiac index increased immediately after the transfusion, and within 4 hours of the transfusion had returned to normal (fig. 7 ) . The difference in 0, content in blood obtained from the femoral artery and pulmonary artery decreased immediately after the transfusion, and within 4 hours after transfusion it was within normal limits (fig. 7 ) . During the

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FIG.6. Red-cell levels of ATP and 2,3-DPG, and plasma phosphorus levels in ACD- and CPD-collected whole blood stored for up to 30 days at 4"C.

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FIG. 7. Oxygen consumption, arteriovenous difference in O2 content, and cardiac output in stable anemic patients before and after transfusion of 3 to 5 units of preserved red cells with low 2,3-DPG levels and high affinity for 0 2 . ' 0

4-hour posttransfusion period, the increase in 0, extraction from the systemic circulation was associated with an increase in redcell 2,3-DPG (fig. 8 ) . Kopriva and his collaborators11 have reported data on severely injured battle casualties who received at least 12 units of ACD blood that had been stored at 4" C. for 14 days (fig. 9). Their red-cell 2,3-DPG level (4.8 f 2.8 micromoles/gm. Hb, mean 5 S.D.) was about half the normal value (11.9 2 2.2 micromoles/gm. Hb) after transfusion; within 12 hours of the initial sampling, it had increased to 8.9 2 2.8 micromoles/gm. Hb, approximately 75 percent of the normal value. Forty-eight hours after the initial sampling, it was within normal limits, and was significantly above normal 5 days after the initial sampling. The rapid increase during the first 12 hours was associated with an increase in venous blood pH. The rate of restoration of red-cell 2,3-DPG observed in this study appeared to be faster than that previously reported by Valeri and Hirsch,12 and similar to that reported by Beutler and Wood.13 These differences can be explained principally by the physical condition of the patients, that is, their acid-base status, degree of anemia, cardiorespiratory function, and plasma inorganic-phosphate level.

6

ANESTHESIA AND ANALGESIA . . . Current Researches VOL.54, No. 1, JAN.-FEB., 1975

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FIG.8. Mean (2S.D.) oxyhemoglobin dissociation curve Pj, value, red-cell 2,3-DPG level, venous 0, content, Po, of mixed venous blood, arterial blood pH, and hematocrit before and after transfusion of 3 to 5 units of preserved red cells with low 2,3-DPG levels and high affinity for O,.'O

TIME

(nM)

TIME (Horn)

FIG.9. Effect of transfusing a t least 12 units of fresh and stored blood to patients with severe injuries. Measurements of hematocrit, red-cell 2,3-DPG, venous blood pH, inorganic phosphorus, and lactic acid levels were made at time periods indicated."

0, Transport Function of Liquid-Stored Red Cells.-Blood can be collected in one of several liquid preservatives or stabilizers -ACD, CPD, ACD plus adenine, CPD plus adenine, with or without inosine, guanosine, or pyruvate. CPD is believed to be the better anticoagulant because it maintains the oxyhemoglobin dissociation curve and the levels of 2,3-DPG longer (fig. 6 ) . Several investigators have suggested that CPD-collected blood can be stored at 4" C. for 28 days instead of 21. However, we found no significant difference in the 24-hour posttransfusion survival between red cells stored

as whole blood in ACD and those stored at 4"C. in CPD for up to 28 days (fig. 10A and B) Storageability characteristics were similar for ACD- and CPD-collected cells stored at maximal (about 90 volumes percent) or submaximal (about 70 volumes percent) hematocrit (fig. 1OB). The 24-hour posttransfusion survival of ACD or CPD-preserved red cells is not affected by the plasma in which they are stored (fig. 10A and B) . Many investigators believe that red-cell levels of A T P and 2,3-DPG can be

Acute Blood Loss . . . Valeri

7

freeze-thaw technic. When heparinized red cells and ACD-collected red cells were frozen within 5 hours of collection (high glycerol concentration, slow freeze-thaw) they had normal oxyhemoglobin dissociation characteristics after thawing, deglycerolization, and resuspension.

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FIG. 10. A. 24-hour posttransfusion survival of red cells stored in ACD as whole blood or as concentrated red cells with about 70 volumes percent hernato~rit.'~ B. 24-hour posttransfusion survival of red cells stored with 45 to 90 percent hematocrit in ACD or CPD.14

maintained better when the ACD and CPD solutions are supplemented with adenine or inosine at various times during storage at 4"C. However, the amount of inosine that would be infused into a patient receiving 3 or 4 units of such blood would probably produce a hyperuricemia that would persist for about 24 hours. A further cause for concern is the possible renal toxicity of 2,8-dioxyadenine, a metabolite of adenine.

0, Transport Function of Previously Frozen Red Cells.-Organic phosphate levels are maintained in human red cells preserved with high concentrations of glycerol and the slow freeze-thaw technic or with low concentrations of glycerol and the rapid

The following variables can be expected to affect the levels of organic phosphates in washed, previously frozen red cells: the anticoagulant used for collection, the length and temperature of storage prior to freezing, the pH of the glycerolizing solution, the pH of the wash solutions, the composition and pH of the resuspension medium, and the length of time the washed red cells are stored at 4" C. before transfusion. Higher red-cell 2,3-DPG levels can be obtained by increasing the pH levels of the preservative and wash solutions from 5.5 to 7.5; however, these higher pH levels adversely affect A T P levels. CPD-collected red cells can be stored at 4" C. for 3 to 5 days before freeze-preservation.lj Alternatively, the red cells can be stored at 4" C. for as long as 3 weeks, at which time they can be rejuvenated with a solution containing pyruvate, inosine, glucose, phosphate, and adenine, to restore the 2,3-DPG and A T P levels and to decrease the red cell affinity for 0 2 The . rejuvenated red cells can be freeze-preserved either with 40 percent W/V glycerol and storage at -80" C., or with 20 percent W/V glycerol and storage at -150" C. The wash procedure employed to reduce the glycerol concentration to less than 1 gm. percent, in addition, reduces the concentrations of additives used during the rejuvenation procedure, as well as the products of hemolysis, 99 percent of the protein, and 98 percent of the white cells and platelets, and significantly reduces HAA. 1,16-18

Red Cells with 1% to 2 Times Normal 2,3-DPG Levels and Decreased Affinity for O,.-Patients with hypoxic or anemic hypoxia who have normal or elevated blood pH levels usually have peripheral red cells with decreased affinity for 02,and 2,3-DPG levels that are increased to about twice normal.19 When the red-cell 2,3-DPG level is increased to this level and when O2 consumption by the tissue k constant, cardiac output is usually reduced. Treating patients in hemorrhagic shock with preserved red cells with increased 2,3-DPG levels may help to reduce the cardiac work for 2 or 3 days after the transfusion.

AXESTHESIA AND ANALGESIA . . . Current Researches VOL.54, No. 1, JAN.-FEB.,1975

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FIG 11. 24-hour posttransfusion survival and lifespan values of red cells stored in CPD at 4' C. for 2 days prior to rejuvenation and freeze-preservation with 40 percent W/V glycerol at -80' C. In 3 patients, the red cells were transfused within 4 hours of washing. The red-cell 2,3-DPG and ATP levels and the P;,, values are reported for each patient.'?

It is possible to prepare red cells with increased 2,3-DPG levels by using a combination of liquid and freeze-preservation procedures.I8 After red cells have been stored in C P D for 2 to 3 days, they can be incubated at 37" C. for 1 hour with a solution containing pyruvate, inosine, glucose, phosphate, and adenine PIGPA). During incubation the 2,3-DPG level increases to about 20 micromoles/gm. Hb (normal value 12 micromoles/gm. H b ) , the ATP level to about 6 micromoles/gm. H b (normal value 4 micromoles/gm. H b ) , and the P,,, value increases to about 40 mm. Hg (normal value 28 mm. H g ) . Then the red cells are concentrated by centrifugation and the supernatant solution is removed. With freezepreservation with either 40 percent W/V glycerol and storage a t -80" C. or 20 percent W/V glycerol and storage a t -150" C., the red cells can be stored in the frozen state for at least 2 years. After thawing and washing they can be kept at 4" C. in a sodium chloride-glucose-phosphate medium for up to 24 hours before transfusion, at which time they will have 1y2 to 2 times normal 2,3-DPG and ATP levels and decreased affinity for oxygen.lS These red cells have recovery values in vitro of about 90 percent, and 24-hour posttransfusion survival values of about 85 percent (fig. 11).The lifespan values vary, depending upon the recipient's intravascular environment. I n 2 patients, red cells with 2,3-DPG levels of 23 to 26 micromoles/gm. Hb, A T P levels of about 6 micromoles/gm. Hb, and in vitro Pjo values of about 40 to 45 nun. Hg had increased Pj, values both

in vivo and in vitro during the 24 to 72 hours posttransfusion (fig. 12A and B ) . The in vitro P,, values were measured by the I L Co-oximeter and the Bellingham and Huehns method. These systems use different pH and Pco? conditions. Whole blood is used in the Co-oximeter, and washed red cells are used in the Bellingham and Huehns method. Measurements of percent saturation were similar with the I L Co-oximeter and the Lex O2 CON machines (fig. 12A and B ) . The in vivo Pzovalue was estimated from the Po, and percent saturation in a n anaerobically-collected peripheral venous blood sample and an assumed slope of n=2.7 of the oxyhemoglobin dissociation curve. The venous blood must be collected without stasis from the arm a t rest. This measurement reflects the in vivo effects of the following factors on red cell affinity for 0,: venous blood pH, red cell pH, venous blood Pco,, carboxyhemoglobin, and red-cell ATP, 2,3-DPG, and inorganic phosphorus levels (fig. 13). An increase in the red cell affinity in vivo usually is associated with increased cardiac output, while a decrease usually is associated with a decrease in cardiac output. The advisability of using red cells with elevated 2,3-DPG levels in the treatment of patients in hemorrhagic shock is obvious (fig. 12A and B) . Studies are in progress to determine the physiologic effect of the transfusion of red cells with 1% to 2 times normal 2,3-DPG levels on myocardial function following extracorporeal circulation. Transfusion of preserved red cells with increased affinity for 0, to patients may produce a demand for increased blood flow or greater extraction of O2 from the red cells, or both. Since it is difficult to assess Po, in tissue, mixed venous blood Po, is usually measured. The combined effects of blood flow, red-cell mass, red cell affinity for O?, and 0, consumption by tissue are reflected in the mixed Pvo, measurement. Patients who require transfusion usually have elevated 2,3-DPG levels (fig. 14). Thus, when improvement of O2 transport during the 24 to 72-hour posttransfusion period is desired without a n accompanying demand for increased blood flow, red cells with 11/2 to 2 times normal 2,3-DPG levels should be used. As many a s 6 units of red cells with lyz to 2 times normal 2,3-DPG levels have been transfused to a single patient.

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in a 23-year-old man with injuries. Red cells were stored in ACD for 2 days, rejuvenated with a solution containing PIGPA, frozen with 40 percent W/V glycerol and stored a t -80" C., washed in an IBM Blood Processor with 2.2 L. NaCl solution and stored a t 4 " C. for 24 hours in a NaC1-glucose-phosphate solution before transfusion.'' B. Effects of transfusion of 4 units of red cells with 2,3-DPG level of 23 micromoles/gm. H b and in vitro P,,, value of 40 mm. Hg on: red-cell mass red-cell P,, value in vitro and in uiuo, whole blood and red-cell pH, red-cell potassium, plasma inorganic phosphorus, red-cell ATP and 2,3-DPG, and carboxyhemoglobin levels in a 59-year-old man with injuries. Red cells were stored in ACD for 2 days, rejuvenated with a solution containing PIGPA, frozen with 40 percent W/V glycerol and stored a t -80" C., washed in the Fenwal Elutramatic Washer with 2.2 L. NaCl solution, and stored a t 4" C. for 24 hours in a NaClglucose-phosphate solution before transfusion."

P,, value in viuo and in uitro, whole blood and red cell pH, plasma inorganic phosphorus, red-cell 2,3-DPG and A T P levels, Pco,, and carboxyhemoglobin level

FIG.12. A. Effects of transfusion of 3 units of red cells with 2.3-DPG levels of 26 micromoles/gm. Hb, and in uitro P,,, of 45 mm. Hg on: red-cell mass, red-cell

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ANESTHESIA AND ANALGESIA . . . Current Researches VOL.54, NO.1, JAN.-FEB., 1975

10 ATP

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temperature for at least 2 minutes, after which they are diluted with 450 to 500 ml. of 1.6 percent sodium chloride (fig. 15A). With this two-step dilution process, the glycerol concentration is reduced to about 18 gm./100 ml.

7

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Increased Affinity for Oxygen

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temperature,

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ATP, and inorganic phosphorus levels on red cellhemoglobin 0, affinity.

Wash Systems.--In an attempt to simplify red cell washing, the Naval Blood Research Laboratory (NBRL) has modified the established wash systems. Dilution of the glycerolized red cells after thawing reduces both the volume of wash solution and the preparation time required (fig. 15A and B ) . The 40 percent W/V glycerolized red cells are diluted with 150 ml. of 12 percent sodium chloride and equilibrated at room

Any of these washing systems can be used: ( 1) the IBM Blood Cell Frocessoran automated serial centrifugation system using disposable collapsible polyvinyl-chloride plastic bags; (2) the Fenwal Elutramatic Cell Washer-an automated continuous-flow system using collapsible polyvinylchloride plastic bags held in centrifuge cups; i3 ) the Haemonetics Model 15-a nonautomated continuous-flow wash system using rigid disposable polycarbonate bowls. Figure 15A shows the number of units that can be washed in each machine at one time, the length of time required for washing, and the total volume of wash solution. More sodium chloride wash solution (2.2 to 3.2 L./unit of glycerolized red cells) is required to reduce the final glycerol concentration to less than 1 percent W/V in the 40 percent W/V glycerolized red cells than in the 20 percent W/V glycerolized red cells ( 1.5 to 2.5 L./unit) . The small residual amount of glycerol produces no adverse effect either on the red cells or in the recipient, The initial glycerol concentration also determines the time required for washing: about 30 minutes for red cells containing 40

Acute Blood If i s s . . . Valeri

11

RED ELLS CONTAINING 40% W/V GLYCEROL

Mshing M i n e :

Type of Washer :

IEM EUITRAMATIC

AlllONATB

SERIAL ENTRIFMTIEU

AUWTMIITEO

myTlNUOUS FLW

(XYTRIFUUTW

MODEL15

W-AUWATB GRAVITI WYTIYWWS M COITRIWATW

No.of Units Washed In Each Machine :

p"--4

1

Set Up Tlme ( M i d :

-35

-

1

2 8-10

-3

-20

-20 -25

-30 2.7

3.2

MUTED GLYGEROLIZED RED CELLS CONTAINING -WH%W/VGLYGERX

units wst!ad In Eaeh Machine

No. of

=;-=--.:*-..-,

==C--z =

--

-

ADDITION OF ZW-YYJ~II OF 3.2% NacL WITH AGITATIDN

Set Up Time ( M i d

Washing Tim, Win):

z-t= Totol Time (Min) : Mlume of NoCL s o l u t i (Liters)

:

1 "3-5

-

-

16

2 *8-0

-

12

20

"20

I.5

I.7

1 -3

-

I5

-20 2.5

FIG.15. A. Dilution of 40 percent W/V glycerolized red cells first with 150 ml. of 12 percent NaCl and then with 450 to 500 ml. of 1.6 percent NaCl solutions, prior to recovery and washing in one of the three systems described in text. B. Dilution of 20 percent W/V glycerolized red cells with 250 to 500 ml. of 3.2 percent NaCl solutions prior to recovery and washing in one of the three systems described in text.

percent WIV glycerol, and about 20 minutes for red cells containing 20 percent W/V. A continuous-flow cell-washing system with rigid disposable polycarbonate bowls is now commercially available.* This system employs a Model 15 processor, which holds the disposable bowl. Solutions are added to the spinning bowl by gravity. This system can be used to wash liquid whole blood, liquid concentrated red cells, low concentration glycerolized red cells (20 percent WIV) ,and high concentration glycerolized red cells (40 percent W/V). Only 1 unit of blood can be washed at a time in the bowl. However, as many as 5 units of ABOand Rh-identical liquid whole blood or concentrated red cells, and 2 units of ABO- and Rh-identical previously-frozen red cells

__ *Haemonetics Corporation, Natick, Massachusetts.

(preserved with either high or low glycerol concentrations) can be washed in succession in the same bowl. We recommend that no more than 2 units of previously-frozen glycerolized red cells be washed in each bowl. Fenwal'f has introduced a disposable polyvinyl-chloride plastic bag that can be held in a centrifuge cup in an RC-3 Sorvall centrifuge. A special programmer is attached to control the volume and rate at which the wash solutions are added. This continuousflow system can be used to wash whole blood, concentrated red cells, and red cells containing either 20 percent or 40 percent W/V glycerol. While the rotary seal used with the Haemonetics disposable bowl can be spun "dry," that used with the Elutramatic system requires a primer (a flow of

-

tFenwal Laboratories, Chicago, Illinois.

12

ANESTHESIA AND ANALGESIA . . . Current Researches VOI..54, NO. 1, JAN.-FEB., 1975

solution through the seal to maintain its integrity). Two units of 40 percent W/V glycerolized red cells can be washed at one time in 30 minutes using 2.7 L. of sodium chloride solution for each unit, or 2 units of 20 percent W/V glycerolized red cells can be washed within 20 minutes using 1.7 L. of sodium chloride solution for each unit. IBM"' has introduced an automated serial (batch) washing system. This system can be used to wash liquid blood, as well as 20 percent W/V and 40 percent W/V glycerolized red cells. When red cells containing 40 percent W / V glycerol are diluted with 150 ml. of 12 percent sodium chloride ( NaCl) , equilibrated at room temperature ( 22" C.) for 2 minutes, and then diluted with 450 to 500 ml. of 1.6 percent NaCl solution, the glycerol concentration is reduced to 18 to 20 percent. The diluted glycerolized red cells are recovered in a polyvinyl-chloride washing bag and then washed with additional NaCl solution. With a volume of 2.2 L. of NaCl solution, a single unit of 40 percent W / V glycerolized red cells can be washed within 20 minutes. The final wash solution contains NaC1, glucose, and phosphate. After resuspension in this solution, the red cells can be stored for a t least 24 hours a t 4" C. Red cells containing 20 percent W / V glycerol are diluted with 500 ml. of 3.2 percent NaCl solution. Using the IBM wash system, the diluted red cells are recovered and washed with a total of 1.5 L. of NaCl solution within 16 minutes before resuspension in the same NaC1-glucose-phosphate solution as described above (fig. 15B). Tullis and coworkers16 have reported that postthaw washing of red cells significantly reduces the incidence of posttransfusion hepatitis. It also removes most of the adenine and inosine that might prove harmful after multiple-unit transfusions. On the other hand, if the red cells were frozen immediately after collection there would be no need for purine nucleoside supplementation. The other components separated from the whole blood could be stored individually by the most satisfactory technic. An additional benefit of the freeze-thaw-wash procedure is that it removes at least 95 percent of the white cells. When nonfrozen whole blood or red cell concentrates are washed, only 80 to 85 percent of the white cells are removed.' Platelet Circulation and Function.-Platelets can be separated from whole blood with__ *IBM Corporation, Princeton, New Jersey.

in 4 hours of collection and prepared for liquid preservation a t 22" C. or a t 4" C.; or for freeze-preservation a t -80" C. or -150" C. Numerous investigators have studied the survival and hemostatic effectiveness of preserved platelets. Platelet survival can be determined by measuring the increase in platelet count in thrombocytopenic recipients or by labeling the preserved platelets with :*Cr. Hemostatic effectiveness can be determined in various ways. The ability of platelets to correct bleeding induced by a standardized incision in the skin or from surgical wounds, and the aggregation of platelets to ADP, epinephrine, and collagen, are some indications. Another is the ability of platelets to correct an aspirin-induced thrombocytopathy in healthy volunteers. Some investigators have reported that platelet concentrates stored a t 22" C. for 72 hours have acceptable posttransfusion survival. In patients with thrombocytopenia, restoration of platelet glycogen and platelet aggregation occurred within 24 hours after the transfusion of platelet concentrates that had been stored a t 22" C. for 24 hours. Other investigators have reported that platelets were maintained better a t 4" C. storage than at 22" C. I n a study from om laboratory, 8 units of platelet concentrates that had been stored a t room temperature for 24 hours did not correct a n aspirin-induced prolonged bleeding time in healthy volunteers within 2 hours of the transfusion, but the bleeding time and aggregation patterns returned toward normal within 24 hours. In another study, platelets stored at 22" C. for 24 hours had ZICr in vivo recovery of about 50 percent, and lifespan values of 8 days, but 1 unit could not correct the aspirin-induced bleeding time 2 hours after transfusion in a healthy valunteer.20 However, in some volunteers, the bleeding time 24 hours after transfusion was similar to that observed before aspirin ingestion (fig. 16). Although 1unit of platelet concentrate stored at 22" C. for 24 hours had good circulation, t h e e platelets usually did not reduce the prolonged bleeding time within 24 hours of infusion.20 On the other hand, platelets stored a t 4" C. for 24 hours had jlCr recovery of platelet radioactivity of only about 38 percent, and lifespan values of about 3 days, but they were able to reduce the bleeding time significantly within 24 hours of infusion. This is because these platelets are activated during the 24 hours of storage a t

Acute Blood Loss

. . . Valeri

13

1 Bleeding Time I

I5'crSurvival I

-

STORAGE TIME

1 1

< 4firs. at 22C (Fresh/

24 Hrs. at 22C 2 4 H n . at 4C 81eedtag Tima Control

STORAGE TIME

-

ZQHn. at -fWC with 5%OMSO -wOC wlm 6 % Bleeding T/ma Cantrol

I---. 24Hrs. at

1-'

I

0

2

I

I

4

6

8

1

0

DAYS

control

Re-224 48 72 Tx Harn pat-Tx 2

FIG.16. 5'Cr survival of platelet concentrates isolated from C P D blood and transfused to J.F. as: (1) platelet concentrates stored at 22" C. for less than 4 hours (fresh platelet concentrates); (2) pIatelet concentrates stored a t 22°C. for 24 hours; (3) platelet concentrates stored a t 4°C. for 24 hours. Thefresh platelet concentrate was a homologous transfusion, while concentrates stored at 4"C. and 22" C. were autologous transfusions. Twenty-four hours after aspirin ingestion, 1 unit of the platelet concentrate was administered to determine its effect on bleeding time, measured before and for 96 hours after ingestion of 650 mg. of aspirin. In addition, bleeding time was measured over a 96-hour period after aspirin ingestion when no platelet transfusion was given (control). W r survival of platelet concentrates isolated from CPD blood and transfused to F.C. as: (1) platelet concentrates stored a t 22" C. for less than 4 hours (fresh platelet concentrates); (2) platelet concentrates frozen with 6 percent DMSO and stored a t -80" C. for 24 hours: (3) platelet concentrates frozen with 5 percent DMSO and stored a t -150" C. The fresh platelet concentrate was a homologous transfusion, while freeze-preserved, washed-platelet concentrates were autologous transfusions. Twenty-four hours after ingestion, 1 unit of the platelet concentrate was administered to determine its effect on bleeding time, measured before and for 96 hours after ingestion of 650 mg. of aspirin. In addition, bleeding time was rneasused over a 96-hour period after aspirin treatment when no platelet transfusion was given (control).

4" C., so they have poor in uivo circulation but increased hemostatic effectiveness (fig.

16). Fresh platelets, on the other hand, have excellent circulation, but 4 units are required to reduce an aspirin-induced prolonged bleeding time. Excellent results have been obtained with the transfusion of human platelets preserved using 5 percent DMSO, a controlled rate of freezing a t 1" C./min., and storage a t

-150" C . for up to 8 weeks. These platelets can be washed by a single dilution and centrifugation procedure to remove at least 90 percent of the DMSO. In a recent study at the NBRL, we chose to arbitrarily increase the DMSO concentration to 6 percent and to freeze the platelets by placing them in a -80" C. mechanical freezer. We found this method to be far simpler than the one employing 5 percent DMSO and a controlled rate of freezing.

14

AXESTHESIAAND ANALGESIA . . . Current Researches VOL.54, NO.1,JAN.-FEB.,1975

Both the 5 percent DMSO and the 6 percent DMSO platelets had "Cr recovery in vivo of about 45 percent, and lifespan values of 8 days. But 1 unit of the 6 percent DMSO platelet concentrate reduced the aspirin-induced bleeding time within 2 hours of infusion, whereas 1 unit of 5 percent DMSO platelet concentrate did not (fig. 16). Human platelets freeze-preserved with 6 percent DMSO and stored at -80" C. for up to 6 weeks have excellent circulation and increased hemostatic effectiveness. These platelets not only reduce the aspirin-induced bleeding time, but also have normal lifespan of 8 days, both significant factors in the treatment of thrombocytopenia. Although excellent results have been obtained using DMSO to freeze human platelets, the potential toxicity of the residual DMSO in the washed platelet concentrate continues to be a problem. Platelet transfusions are indicated for the treatment of a bleeding diathesis associated with thrombocytopenia or thrombocytopathy, and for the prophylactic treatment of severe thrombocytopenia. Active bleeding associated with thrombocytopenia should be treated with platelets having increased hemostatic effectiveness, whereas platelets with the best posttransfusion survival should be used when an increase in platelet count is desired. Only by studying preserved platelets in patients with thrombocytopenia will we be able to state unequivocally which method of platelet preservation yields the most satisfactory product. Plasma Proteins.-Plasma proteins should be separated from blood shortly after collection and prepared for preservation (fig. 5 ) . This would make cryoprecipitate, fresh-frozen plasma, and the plasma proteins, albumin, gamma globulin, and antihemophilic globulin, readily available for transfusion when needed to restore both the plasma oncotic pressure and coagulation factors.

REFERENCES 1. Valeri CR: Recent advances in techniques for freezing red cells. Crit Rev Clin Lab Sci 1:381425, 1970

2. Jaeger RJ, Rubin R J : Plasticizers from plastic devicesextraction, metabolism, accumulation by biological systems. Science 170:460-462, 1970 3. Solis RT, Gibbs MD: Filtration of the microaggregates in stored blood. Transfusion 12:245-250, 1972

4. McNamara J J , Molot MD, Stremple J F :

Screen filtration pressure in combat casualties. Ann Surg 172:334-341, 1970 5. Moss GS, Valeri CR, Brodine CE: Clinical experience with the use of frozen blood in combat casualties. New Eng J Med 278:747-752, 1968 6 . Valeri CR: Viability and function of preserved red cells. New Eng J Med 284:81-89, 1971

7. Valtis DJ, Kennedy AC: Defective gas transport function of stored red blood cells. Lancet 1:119-124. 1954

8. Benesch R, Benesch RE: The effect of organic phosphates from the human erythrocyte on the allosteric properties of hemoglobin. Biochem Biophys Res Commun 26:162-167, 1967 9. Chanutin A, Curnish RR: Effect of organic and inorganic phosphates on the oxygen equilibrium of human erythrocytes. Arch Biochem 121:96-102, 1967 10. Valeri CR, Ccllins FB: Physiologic effects of 2,3 DPG depleted red cells with high affinity for oxygen. J Appl Physiol 31:823-827, 1971 11. Kopriva CJ, Ratliff J L , Fletcher J R , e t al: Biochemical and hematological changes associated with massive transfusion of ACD-stored blood in severely injured combat casualties. Ann Surg 176: 585-589, 1972

12. Valeri CR, Hirsch NM: Restoration in viuo of erythrocyte adenosine triphosphate, 2,3 diphosphcglycerate, potassium ion, and sodium ion concentrations followinn the transfusion of acid-citratedextrose-stored human red blood cells. J Lab Clin Med 73:722-733, 1969 13. Beutler E, Wood L: The in viuo regeneration of red cell 2,3 diphosphoglyceric acid (DPG) after transfusion of stored blood. J Lab Clin Med 74: 300-304, 1969 14. Valeri CR, Szymanski 10, Zaroulis CG: 24hour survival of ACD and CPD stored red cells. 1. Evaluation of nonwashed and washed stored red cells. Vox Sang 22:289-308, 1972 15. Valeri CR: 24-hour posttransfusion survival and oxygen transport function of red cells frozen with 40c/, W/V glycerol and stored a t -80" C for up to 2?4 years. Transfusion 14:l-15, 1974

16. Tullis JL, Hinman J, Sproul MT, e t al: Incidence of posttransfusion hepatitis in previously frozen blocd. JAMA 214: 719-723, 1970 17. Valeri CR, Zaroulis CG: Rejuvenation and freezing of outdated stored human red cells. New Eng J Med 287:1307-1313, 1972 18. Valeri CR: Metabolic regeneration of depleted erythrocytes and their frozen storage. The Human Red Cell in Vitro. Greenwalt TJ, Jamieson GA Editors, Grune and Stratton, Inc. 1974, pp 281-321 19. Valeri CR, Fortier NL: Red cell 2,3 diphosphoglycerate and creatine levels in patients with red cell mass deficits or with cardiopulmonary insufficiency. New Eng J Med 281:1452-1455, 1969 20. Valeri CR: Hemostatic effectiveness of liquidpreserved and previously frozen human platelets. New Eng J Med 290:353-358, 1974

Blood components in the treatment of acute blood loss: use of freeze-preserved red cells, platelets, and plasma proteins.

To avoid untoward reactions from blood transfusions and to make best use of the limited supply of blood, anesthesiologists and surgeons have many newl...
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