Blood transfusion: Merits of component therapy

T H E J O U R N A L OF

PEDIATRIC S JANUARY

MEDICAL

1974

Volume 84

Numberl

PROGRESS

Blood transfusion: Merits of component therapy L The clinical use of red cells, platelets, and granulocytes* D. H. Buchholz, M.D., Baltimore, Md.

BLOOD transfusion is frequently required to facilitate delivery of oxygen to tissues, to promote hemostasis, and to correct hypovolemia. Unfortunately, it is not generally appreciated that administration of a specific blood component may be equally or more effective than infusion of whole blood. It is hol~ed that through physician education m a x i m u m appropriate utilization of blood components will be realized. Freshly drawn blood may be easily fractionated into three major components: packed red cells, platelets, and plasma. Plasma may be used as a volume expander, or may be fresh frozen and used to supply coagulation factors such as V and VIII which are relatively labile under conditions of routine blood bank storage. Plasma may also be processed to provide highly concentrated solutions of factor VIII, fibrinogen, albumin, gamma globulin, or the factor II-VII-IX-X complex. Since it is only rarely that all components present in fresh blood are needed in a given clinical situation, the large-scale, From the Cell Support Service, Section o f Medical Oncology, Baltimore Cancer Research Center, National Cancer Institute. *Part II will appear in the FebrUary, 1974, issue of the JOURNAL.

Reprint address: St-. Barnabas Medical Center; Otd Short Hills Rd. Livingston. N. J. 07039

judicious use of specific components would allow harvest of blood fractions ordinarily wasted and would, in turn, allow more effective treatment of greater numbers of patients. This review describes current therapeutic and investigational uses of blood components. Abbreviations used ACD: acid-citrate-dextrose EDTA: ethylenediametetra-acetic acid CPD: citrate-phosphate-dextrose APT: adenosine triphosphate 2,3-DPG: 2,3-diphosphoglycerate RED BLOOD CELL TRANSFUSION The principal indication for the infusion of red blood cells is anemia. Packed red cells are the treatment o f choice unless there is severe c~ncurrent hypovolemia secondary to trauma, major surgery, or shock. Even in these instances, packed cells may be used in conjunction with appropriate volume expanders such as albumin, dextran, plasma, saline, or Ringer's lactate solution. Hydroxyethyl starch has also been suggested for use as a volume expander, 1 although its use is currently investigational. Infants may require only a portion of the red cells from a single unit of blood. A unit of blood can be

Vol. 84, No. 1, pp. 1-15

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Buchholz

divided into two or three aliquots if it is initially collected in a closed, triple-bag system. This technique allows the use of the remaining portion of blood which might be otherwise wasted if only a small volume transfusion is required. Anticoagulants. Most blood in the United States is collected in acid-citrate-dextrose (ACD) solution; salts of ethylenediarnine-tetraacetic acid (EDTA) are no longer used since damage to platelets results upon exposure to the anticoagulant. 2 Heparin, while useful as an anticoagulant for exchange transfusion and open heart surgery, preserves red cells poorly and blood must be used within 24 to 48 hours of collection, since there is no metabolic substrate present to support red cell metabolism.3, 4 The heparin present (4.5 I.U, per rnilliter)is sufficient to prevent coagulation for the up to 48 hours. Whole blood or packed cells collected in A C D can be stored at 4 ~ C. for 21 days with survival of at least 70 per cent of the red cells after transfusion. 5,6 A less acidic anticoagulant, citrate-phosphate-dextrose (CPD), is currently in somewhat limited use in the United States; it m a y provide slightly better red cell survival a n d longer storage with slower loss of 2,3-diphosphoglycerate. s. 6 Both A C D and CPD act as anticoagulants by chelating ionic calcium; massive transfusion or exchange transfusion may lead to transient hypocalcernia in the recipient and occasionally may result in tetany or cardiac arrest. The intravenous administration of calcium (0.5 to 1.0 ml: o f 10 per cent calcium gluconate per 100 mI. infused) may be helpful in preventing severe hypocalcemia when large amounts of blood are rapidly infused. Storage. During storage, red cells undergo metabolic changes which lead to loss of viability and, transiently, of function. Following transfusion, a small proportion of cells are rapidly cleared from the circulation while the remainder survive normally. Cell survival is primarily related to i n t r a c e l l u l a r c o n c e n t r a t i o n of a d e n o s i n e triphosphate (ATP); during 21 day storage in A C D solution, red cell ATP concentrations drop to about 60 per cent of normal. 7 Haradin and associates 8 have shown that loss o f ATP is associated with a change in red cell shape from disc to sphere, a loss of membrane lipid, and a rather striking increase in cellular rigidity. A T P loss occurs even when less acidic anticoagulants such as CPD are used as preservative. Marly investigators agree that blood may be stored in either A C D or CPD for 28 days with 70 per cent cell survival following transfusion; however, present Federal Drug Administration regulations do not allow use of A C D or CPD blood stored for more than 21 days. Only recently has it been appreciated that even though

The Journal of Pediatrics January 1974 cells may survive reasonably well in the recipient circula. tion following transfusion, they may transiently function poorly in delivering oxygen to tissue if stored for more than seven days in A C D or'ten days in CPD. s Valtis and Kennedy 9 first noted that the oxygen dissociation curve o f stored ACD blood was shifted to the left; the net result of this shift is that under a n y given partial pressure Of oxygen, the oxygen is more tightly bound to hemoglobin and is less available t~ tissue. This change in hemoglobin-oxygen dissociation is related to intracellular concentrations of the organic phosphate compounds A T P and, especially, 2 , 3 - d i p h o s p h o g l y c e r a t e (2,3DPG). l~ During storage, 2,3-DPG falls to low concentrations which are not restored to normal until 8 to 24 hours following transfusion. TM12Inverting blood to mix plasma and red cells periodically during storage does little to improve cellular concentrations o f ATP but will delay the depletion of 2,3-DPG, 5 Since the purpose of red cell infusion is to increase the delivery of oxygen to the tissues, the use of 2,3-DPG-: depleted blood for transfusion has been questioned. It has been shown that massive transfusion of blood low in 2,3-DPG is associated with a change in the oxygen dissociation curve and a fall in central venous pressure of the recipient. 13 In a recent study, slight decreases in the Ps0 (partial pressure of oxygen required for 50 per cent saturation of hemoglobin at pH 7.4 and 37 ~ C.) in infants receiving exchange transfusion were noted when 4- to 5day-old blood was used. The Ps0 rose following transfus i o n with blood less than 24 hours old, and although the authors speculated on theoretical grounds that fresh blood was better than that stored for four to five days, no clinical differences in the two transfused groups were reported. 14 Although o f theoretical concern, no clear-cut evidence of harm to recipients has yet been described when 2,3-DPG-deficient blood has been infused in human beings. Investigational blood preservatives. Recently, several additives such as adenine, 15 inosine, 16 dipyridamole, 17 and ascorbic acid 18 have been used in conjunction with A C D or CPD to promote cell viability and maintain function during storage. The addition of adenine allows A T P to be maintained in A C D or CPD blood for up to 35 days with resultant good post-transfusion cell survival; unfortunately, adenine hastens the rate of 2,3-DPG disappearance. 19 Partial restoration o f A T P and 2,3-DPG concentrations can be achieved by incubating stored red cells with pyruvate, inosine, glucose, and phosphate, with or without adenine, for one hofir. 2~Although it requires washing of cells to remove t h e compounds, such a method o f restoring cell function might eventually be

Volume 84 Number 1

necessary if it is determined that transfusion of stored 2,3-DPG-deficient red cells is indeed harmful to the recipient. Frozen red blood cells. Human red cells were first frozen in glycerol and transfused by Mollison and Sloviter21 in 1951. Since then, considerable effort has been expended in developing methods of long-term, low-temperature, red cell preservation. Serveral techniques are currently in limited use. 22"24All require washing of cells to remove the cryoprotective agent after thawing, and some require the use of liquid nitrogen storage temperatures. Excellent in vitro and good in vivo cell recoveries have been documented. 2~ 22-24 There are few leukocytes present in frozen erythrocyte preparations after thawing.20 , 25, 26 Their use may prevent alloimmunization in potential transplant recipients who frequently become sensitized to leukocyte h i s t o c o m p a t i b i l i t y (HL-A) antigens through blood transfusion; such immunization is thought to decrease chances of successful organ engraftment. Some investigators have noted that while few intact leukocytes remain in frozen washed red cell preparations, considerable amounts of white cell fragments remain, and they have questioned the potential of such fragments to act as alloimmunogens. 27 Proponents of frozen red cells have cited their use in 9 preservation of rare cell types for transfusion or investigationai use. In addition, the potential for largev o l u m e autologous cell t r a n s f u s i o n , the i m p r o v e d viability and function of such cells (if frozen fresh or after "rejuvenation" of stored cells2~ the presence of very small numbers of intact leukocytes and small amounts of donor plasma, and, finally, a decreased risk of post-transfusion hepatitis make them theoretically attractive. Others point out the high cost of a unit of frozen b l o o d (two to three times the cost of blood stored conventionally26), the i n c r e a s e d time and technical manipulation necessary to freeze, thaw, and wash cells, and possible risks of bacterial contamination during preparation. Because of this risk, frozen-thawed red cells must be used within 24 hours of thawing. While long-term cryopreservation of red blood cells is undoubtedly clinically useful in certain specific instances, such as the preservation of "rare" blood, it remains unclear whether purported benefits outweigh the added cost and time required if frozen red cells were routinely used for all transfusions. Chaplin 26 has recently reviewed this subject in detail. Autologous red cell t r a n s f u s i o n . The use o f autologous 28 stored or frozen red cells for elective surgical procedures is unfortunately not in widespread use,

Blood transfusion

3

although the technique offers many advantages including avoidance of exogenous blood-borne disease, greatly reduced risk of transfusion reaction, and the lack of exposure to "foreign" blood cells; the latter may be especially important in patients receiving tissue grafts. 29 In the author's experience, the procurement of one or two units of autologous CPD blood prior to exploratory laparotomy for staging of Hodgkin's disease has dramatically reduced the need for transfusion of random donor blood at the time of surgery. Although the size of children might prevent removal of entire units of blood, smaller volumes might be removed and stored or frozen prior to elective surgery. Compatibility testing. The concepts of "universal donor" and "universal recipient" are of limited usefulness: group- and Rh-identical blood should be used whenever possible. The ABO system is unique in that so-called "naturally occurring" antibodies (with specificities directed against A and B antigens not present on host erythrocytes) are nearly always present after about six months of age. Thus, while compatible "universal donor" group O packed cells" may be transfused to persons of any blood group without difficulty, the infusion of the same cells as whole blood could result in the destruction of recipient cells o f Type A, B, or AB by alloagglutinins in the group O plasma. Occasionally, severe hemolytic reactions have been observed following transfusion of "universal donor" whole blood containing high t i t e r s o f anti-A and anti-B. 31 In most transfusion c e n t e r s , the ABO and Rh o (D) type of the recipient are determined prior to transfusion, and attempts are made to provide ABO and Rho-identical blood; however, there are other antigens within the Rh system capable of sensitization, as well as numerous other red cell antigen systems such as Kell, Duffy, Lewis, Lutheran, Kidd, etc. A n y antigenic determinant on donor blood cells not present on recipient cells has the potential for inducing alloimmunization, although fortunately most red cell antigens outside the Rh, Kell, and Duffy systems are relatively poor immunogens. At present. there are more than 260 different red cell antigens known. 32 It is obviously impossible to provide blood identical to the recipient for all known antigens or even for most of them. As a consequence, antibody production occasionally results from transfusion, dependent upon both host ability to respond to alloantigens 33 and the "antigenicity" (potency) of the foreign antigenic determinant. 34 The possibility that alloimmunization against any antigenic determinant may result from a single unit transfusion has been estimated at about one per cen~t.34

4

Buchholz

As the number of units infused increases, so do chances of antibody formation, and recipients of many units (as for example during open heart surgery) frequently become alloimmunized. 35 C r o s s - m a t c h l n g . Compatibility testing of blood should always be performed prior to transfusion except in extreme emergency when the immediate infusion of blood may be lifesaving. Even in this instance, determination of recipient group and Rh can be rapidly done and ABO- and Rh-specific blood used. It should be stressed that the physician infusing blood without crossmatch assumes responsibility for the consequences of transfusion should the unit(s) prove to be incompatible, and careful consideration should be given to the clinical urgency belbre uncross-matched blood is used. Transfusion reactions. Transfusion reactions may result from the infusion of incompatible red cells, plasma, platelets, or leukocytes. Serious hemolytic reactions are most often due to administration of blood to the wrong recipient; great care should be taken to assure the correct identity of both the blood sample sent to the laboratory for cross-matching and the identity of the unit of blood cross-matched for an intended recipient. Recipients should be observed frequently during transfusion and the infusion discontinued if there is evidence of reaction. When frequent transfusions are anticipated, a serum sample should be sent to the blood bank every 48 hours to allow detection antibodies (anamnesiic response) which occasionally appear within a few days following transfusion. Untoward reactions to blood infusion have been estimated to occur in about five per cent of transfusions. 36 Reactions have been broadly classifed as allergic, pyrogenic, bacterial, or hemolytic. Allergic reactions typically produce itching and urticaria unaccompanied by chills and fever but may result in bronchospasm or anaphylactoid reactions. These reactions are poorly understood but are thought to be related to the presence of atopic substances capable of interacting with antibodies present in donor or recipient plasma. Severe reactions have been reported in immunoglobulin A-deficient recipients who have antibody directed against IgA present in donor plasma. 37 In general, the use of antihistamines may ameliorate or prevent allergic reactions, although if they are severe, epinephrine and corticosteroids may be needed. Pyrogenic reactions due to the presence of bacterial lipopolysaccharides are no longer a frequent cause of transfusion reaction since the advent of disposable plastic blood bags. However, the term is frequently used to describe the reactions which occur following incompatible leukocyte or platelet infusion. Both the pyrogenic

The Journal of Pediatrics January 1974

reaction and the leukocyte reaction characteristically begin about an hour after, the start of the transfusion and usually consist of an elevation in blood pressure, a Chilly sensation, and frank shaking chills followed by fever. The use of antipyretic agents may be helpful in controlling fever. Such reactions, if severe, may be prevented either by us!rig frozen blood or by removing granulocytes prior to transfusion by passage of fresh heparinized blood through nylon I~er filters 41 (LeukoPak, Fenwal Laboratories). Repeated washing, alone or in conjunction with centrifugation, has also been used to remove leukocytes as has sedimentation of red cells in dextran or plasma gel. 27Recent data suggest that transfusion frequently serves to initiate both humoral (cyt0toxic leukocyte antibodies and leukoagglutinins) and cellular immunologic response. Schechter and associates 29 have observed increases in atypical lymphocytes and/or increases in in vitro incorporation of tritiated thymidine by blood leukocytes in 15 of 17 patients seven days following transfusiomof whole blood, suggesting host response to transfused alloantigens. Such changes were not observed with frozen washed red cells or with autologous cellsfl 9 The infusion of blood or components contaminated with bacteria may produce a severe reaction characterized by chills, high fever, confusion, marked hypotension, disseminated intravascular coagulation, and, frequently, death. Examination of a Gramstained specimen from the blood bag may reveal the presence of microorganisms, but bacterial contamination should not be ruled out if none is seen. Therapy of bacterial transfusion reactions should include the liberal use of antibiotics and treatment of shock; the occurrence of disSeminated intravascular coagulation may warrant heparinization of the recipient followed by component or fresh blood infusion. Hemolytic transfusion reactions may be associated with virtually any s y m p t o m or combination of symptoms or may, during surgery, be first detected by excessive bleeding. ABO hemolytic transfusion reactions tend to be most severe since cells usually are destrOyed intravascularly; most other blood group antibodies tend to bind to the red cell membrane and the cells are subsequently removed by the reticuloendothelial system, although occasionally intravascular hemolysis may be observed. Since it is impossible to be certain if a reaction is allergic, pyrogenic, bacterial, or hemolytic on the baSis of symptoms of the recipient, it is imperative to evaluate all transfusion reactions to be certain that red cell incompatibility does not exist. If a reaction occurs, the infusion should be stopped immediately, and samples of blood

Volume 84 Number 1

from both the recipient and the blood bag should be sent to the blood bank with a description of the reaction. If intravascular hemolysis is suspectwd, a sample of recipient blood may be centrifuged and the plasma or serum examined for the presence of hemoglobin (after the binding capacity of haptoglobin is exceeded, the presence of free hemoglobin in concentrations of about 20 to 25 mg. per 100 ml. results in a faint pink tinge to the plasma; with concentrations of 100 mg. per 100 ml. the plasma appears red42). A direct antiglobulin (Coombs) test of recipient blood may reveal the presence of antibody bound to donor cells. However, in instances o f ABO hemolysis, the direct antiglobulin test may be negative if all infused cells have been destroyed. Occasionally a delayed hemolytic reaction may occur several days following transfusion due to the development of antibody specific for antigenic determinants on transfused cells. Cell destruction is predominantly extravascular in this instance, and the direct antiglobulin test is usually positive, although the indirect antiglobulin test may remain negative until all offending cells have been removed from the circulation. If intravascular hemolysis has occurred, osmotic diuresis should be attempted using 10 per cent mannitol; good urine output should result unless the recipient is in shock or renal damage has already occured. Severe bleeding secondary to utilization of coagulation factors in disseminated intravascular coagulation may occur, presumably due to the release of thromboplastic substances from red cell membranes. 43 Determination of platelet count, partial thromboplastin timel prothrombin time, and fibrinogen, as well as fibrin degradation products may be helpful in'establishing the diagnosis. Administration of heparin with or without component or fresh blood infusion may be lifesaving. 44The use of antifibrinolytic agents such as epsilon aminocaproic acid remains controversial44; these agents probably should not be used. Intrauterine transfusion. Erythroblastosis fetalis may require transfusion therapy to prevent stillbirth or the consequences of severe anemia in an affected fetus. The infant may be treated in utero or following delivery depending upon the severity of the disease. Amniocentesis is helpful in determining the severity of disease in utero and may indicate the need for early delivery or intrauterine transfusion. Intrauterine infusion of compatible red cells may allow the pregnancy to progress to the point at which delivery is feasible. 45 Liley 46 introduced this technique by taking advantage of the fact that red cells injected into the fetal peritoneal cavity make their way into the fetal circulation. Although not without risk, the infusion of 30 to 120 mt. of packed compatible cells

Blood transfusion

5

may maintain fetal viability until the time of delivery. Although in most instances compatible red cells should be readily available, it is helpful to remember that, in the event of an unusual antibody, maternal cells of necessity lack the antigen responsible for the destruction of fetal cells and may be used for intrauterine transfusion after washing. While of theoretical concern, the transfer of immunocompetent cells to the fetus during intrauterine transfusion or exchange transfusion rarely results in establishment of grafts or graft-versus-host reactions in the recipient, 47 although donor lymphocytes have been detected for long periods of time after transfusion. 48 Exchange transfusion. Exchange transfusion is most often performed to remove damaged red cells and/or bilirubin from newborn infants with erytbr0blastosis fetalis, although it has also been used in the treatment of hepatic encephalopathy, 49,5~ sickle cell anemia crisis, 5l neonatal isoimmune thrombocytopenic purpura, 52 and disseminatedintravascular coagulation. 53 Blood for exchange transfusion is frequently cross-matched with maternal serum prior to infusion. Such a cross-match may result in selection of blood lacking the antigenic determinant resulting in red cell destruction but of a different ABO group than that of the infant. If time permits, the spec!ficity of the maternal antibody should be determined and blood lacking the corresponding antigen but ABO compatible with the infant used. For erythroblastosis due to presumed or proved ABO incompatibility, "low-titer" group O blood with some of the plasma removed should be used for exchange. Blood should be warmed prior to infusion during exchange transfusion. Fresh heparinized blood is probably the agent of choice for exchange transfusion, although ACD blood less than 5 days old is adequate. ACD blood stored longer than 5 days should not be used since it will contain high plasma concentrations of potassium as well as decreased erythrocyte concentrations of 2,3-DPG. Heparinized blood, in contrast to ACD blood, is slightly alkaline and may partially counteract the acidosis usually seen in severe hemolytic disease. Heparin does not chelate calcium, and problems of hypocalcemia in the recipient during exchange are avoided. Protamine sulfate may be used to neutralize heparin remaining in the infant following exchange.The amount of heparin remaining in the infant may be estimated by multiplying the calculated percentage of blood exchanged (see below) by the concentration of heparin present in the donor blood (unitls per milliliter). From 0.5 to 0.75 rag. of protamine should be administered for each 100 I.U. of heparin estimated to be present in the infant. 56

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Buchholz

The Journal of Pediatrics January 1974

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kilogram has been assumed. Percentage based on the equation %R =

In general, a single unit of blood will allow a "'twovolume" exchange of the infant with removal of 75 to 85 per cent of infant red cells. Exchange transfusion is an excellent example of the law o f diminishing returns, for as the volume of exchange increases, progressively poorer exchange efficiency results (Fig. 1). The percentage of blood exchanged may be estimated by the equation: OR = [ (V-S)IN L

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R is the per cent of infant cells remaining in the circula9 tion after exchange, Vis the estimated blood volume of the infant, Sis the syringe size or aliquot volume sequentially removed and infused during exchange, and n is the number of removal/infusion cycles performed 57 (Fig. 2). If removal of bilirubin is the primary goal, it is better to perform exchange transfusion with a single unit of blood on two separate occasions than to use two units of blood during a single exchange; the interval between exchanges allows additional bilirubin to diffuse from the tissues into the bloodstream for removal, during the second exchange. PLATELET

TRANSFUSION

Thrombocytopenia may result from decreased platelet production, increased platelet destruction, or following massive transfusion with stored bank blood. Bleeding secondary to tbrombocytopenia seldom occurs if the platelet count remains above 50,000 per microliter,

although impairment of platelet function unrelated to count has 'been noted in uremia, s8 dysproteinemia, 59 following administration of drugs sug,h as aspirin, 6~ or after dextran infusions. 6~ In the absence of such exogenous factors, serious bleeding episodes generally do not occur unless the platelet count falls below 15 to 20,000 per microiiter. Although platelet transfusion is not generally useful in the management of diseases associated with increased platelet destruction, the temporary or long-term administration of platelets has clearly reduced the morbidity and mortality rates associated with hemorrhage in patients with decreased platelet production. 62There are approximately 1.1 x 1011 platelets in a unit of fresh blood; such cells have little hemostatic function after 48 hour blood bank storage, and therefore they must be harvested from fresh blood if they are to be used for t r a n s f u s i o n . Platelets are p r e p a r e d by low-gravity centrifugation of whole blood with separation of red cells and plasma. The majority of platelets remain in the plasma, which may either be used as platelet-rich plasma or be further processed to prepare platelet concentrate. Although platelet-rich plasma, if infused, should be ABO compatible with the recipient, platelet concentrate offers the advantage of providing large numbers of platelets in reldtively small volumes of plasma which need not be ABO compatible with the recipient. Small losses result during preparation of platelet concentrate from platelet-rich plasma; however, an average of from 0.70 to 0.80 x 10~ platelets should be present in each

Volume84 Numberl

B~odtrans[usion

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. The -actual percentage of blood exchanged was determined by Cr51tagging of 10 ml. of red blood cells prior

to exchange transfusion; washed red cell samples were assayed for radioactivity after each 500 ml. of blood was exchanged and corrected for hematocrit and background. unit of concentrate in order to be effective. Platelet preparation. Methods of collection and preparation may greatly affect platelet function and survival. ACD is currently the most widely used anticoagulant for platelet preparation, although CPD may be equally effective. 63 Other anticoagulants are not useful f o r platelet harvest: E D T A damages cell ultrastructure and results in poor post-infusion circulation, 2 whereas heparin induces clumping of platelets during preparation. Partially reversible clumping is also seen during preparation of platelet concentrate from refrigerated fresh ACD blood, 2 b u t this may be considerably reduced if the blood is processed at ambient temperature. 64Much of the blood used in the United States is collected in bloodmobiles and then transported to blood centers for processing; as a consequence, refrigeration of blood frequently follows collection and interferes with platelet preparation at blood centers. Although the addition of extra A C D to plasma prior to platelet concentrate preparation has lessened the degree of platelet clumping in chilled blood, the added A C D is felt by some to interfere with the subsequent recovery of factor VIII from the plasma. 6s Recently, the addition of very small amounts of prostaglan-

din E 1 has been shown to inhibit cold-induced platelet aggregation66, 67; although clearly investigational at the present time, the future use of such antiaggregating agents may allow routine preparation of nonclumped platelets from freshly drawn but refrigerated blood. Platelet transfusion. Platelets have a lifespan of 9 to 10 days 68 but are probably hemostatically effective for only the first 4 or 5 days. Following production, platelets are preferentially sequestered in a non-exchangeable pool in the spleen for about two days and are then released to the circulation. 69About two thirds remain in the circulation while the rest comprise a freely exchangeable splenic pool. 68 As a result, even under optimal circumstances only about 65 per cent of the platelets infused can be accounted for in the circulation following transfusion. Correspondingly greater recoveries may be observed in splenectomized recipients whereas very low recoveries are seen in patients with hepato- or splenornegaly. 68 Blood-borne disease and'alloimmunization to leukocyte, platelet, and red cell antigens are related to the number of different donor bloods to which a recipient is exposed; thus an appropriate therapeutic goal

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Buchholz

shouldbe established and enough components given to meet but not exceed that goal. The number of platelets needed for transfusion may be estimated as follows: Expected increment = 2/3 [N • 0.75 • 1011/estimated blood volume] where the expected increment following transfusion is expressed in platelets per microliter (ram.3), N is the number of units infused, and the estimated blood volume is expressed in microliters. If it is desired to roughly determine the number of platelets needed, a blood volume of 70 ml. per kilogram (80 ml. per kilogram in young infants) will result in a reasonable approximation of the blood volume. Using the above formula as a guide, it can b e seen that three units of platelets would increase the platelet count of a 25 Kg. child by approximately 85,000 per microliter. The clinical status of the recipient also affects posttransfusion cell recovery, and poor responses are frequently seen in patients with organomegaly,68 fever] ~ 71 and septicemia. 71 It may be worthwhile to attempt to lower the temperature of febrile recipients prior to transfusion. Following repeated infusion of platelets from random donors, alloimmunization frequently occurs so that most transfused cells are rapidly destroyed after infusion. in general, platelets are usually given without regard to ABO group and Rh type. Some workers 72 have suggested that better post-transfusion survival will result if the cells are ABO compatible, but others dispute this. 71 Sincesmall numbers of donor erythrocytes are always present in platelet products, ' consideration should be given to the possibility of recipient sensitization to red cell antigens, especially Rh o (D). Since Rh-negative platelets are often not readily available, it is frequently impossible to avoid giving Rh-positive cells to Rh-negative recipients. Although the ability of the host to respond to immunogenic stimuli may be compromised by disease33or by therapy, it may be worthwhile to consider the concurrent administration of one of the Rh immunoglobulin preparations if only limited platelet transfusion therapy is anticipated, especially in females. Platelets are usually passed through filters, either in blood administration sets or specific platelet administration sets, although(some have questioned the need for filters. 73 A platelet filter is smaller than t h e filter in a blood administration set, and consequently less platelet concentrate will be left in the filter and tubing after the infusion. Filters remove very few platelets if the cells are transfused fresh or after storage at ambient temperature. TM For most effective transfusion, the filter system should be flushed with isotonic saline to allow complete platelet infusion. Evaluation of transfusion response. The effectiveness

The Journal of Pediatrics January 1974

of platelet infusion is frequently assessed on clinical grounds by cessation of bleeding and by post-transfusion recovery and circulation of transfused cells. Unfortunately, no in vitro test accurately predicts post-transfusion cell survival, although many are in use. Drug interactions with platelets. Certain drugs, particularly aspirin, have been shown to prolong the bleeding time of both normal people 6~ and recipients of platelets from donors recently ingesting aspirin, 38 apparently by interfering with platelet "release" of endogenous adenosine diphosphate. 38,75While the infusion of a single unit of"aspirinated" platelets may not be of particular consequence in an adult receiving multiunit platelet transfusion, difficulty might be anticipated in small children receiving only one or two units of platelets, since a large proportion of the total number of cells infused might be nonfunctional. The use of aspirin or aspirin-containing compounds (AIka-Seltzer, Bufferin, Empirin, Anacin, Excedrin, Liquiprin [tablets]) should be avoided in thrombocytopenic patients; acetaminophen (Tylenol) or Bromo-Seltzer may be used if afialgesics are necessary. 76,77(Other drugs known to interfere with platetet function, at least in vitro, include sulfinpyrazone, phenylbutazone, some antihistamines, and dipyridamole). 77 Even maternal ingestion of drugs such as aspirin and promethazine prior to or during labor have been shown to alter neonatal platelet function i n vitro, v8 and a history of maternal drug ingestion should be sought in instances of unexplained neonatal bleeding. Neonatal thrombocytopenia. Thrombocytopenia in newborn infants may possibly be due to maternal ingestion of thiazide diuretics during pregnancy 79 or may be found in association with congenital rubella, toxoplasmosis, cytomegalic inclusion disease, and herpes virus infections. It should be noted that neonatal isoimmune thrombocytopenic purpura is estimated to occur in about 1 in 5,000 births.52 The mechanism is thought to be similar to that which results in erythroblastosis fetalis, and there is apoor response to transfused random donor platelets. Exchange transfusion 52or the use of compatible (washed maternal) platelets 8~may be helpful in controlling bleeding. Post-transfusion purpura, although extremely rare, may also be followed by rapid recovery after exchange transfusion. 81 Histocompatibility and platelets. If long-term platelet support is anticipated, a search for HL-A-identical or compatible family members for use as repeated platelet donors may be worthwhile. Although platelets possess unique antigenic determinants, they also share at least some of the leukocyte HL-A antigens. Grumet and Yankee 7~have shown it possible to provide effective platelet therapy to adults totally refractory to random

Volume 84 Number 1

donor platelets by utilizing repeated plateletpheresis of HL-A-identical family members. Plateletpheresis d i f fers from routine platelet harvest in that following withdrawal of a unit of blood, only the platelets are retained, and plasma and red cells are returned to the donor. If the p l a s m a is r e t a i n e d , the p r o c e d u r e is t e r m e d plasmapheresis. It is routinely possible to harvest four units of platelet-rich plasma per week from a single donor without development of thrombocytopenia or hypoproteinemia in the donor. 82, 83 When the need for platelets is great and the recipient rapidly destroys random donor cells, more intense plateletpheresis o f a single compatible donor may be performed. The author has obtained as many as 24 units of platelet concentrate from a single HL-A-identical donor over a 10 day period without development of donor thrombocytopenia. Techniques of platelet collection utilizing the IBM or Aminco blood cell separator 84 or a Latham centrifuge with disposable bowl have also been described. With the Latham bowl system, six units of platelet concentrate m a y be prepared from a single donor in two hours. 85 The concept of single- or limited-donor platelet support potentially allows effective transfusion therapy without multiple-donor blood exposure and should decrease alloimmunization and disease transmission. Although parents, except in very rare circumstances, will not be HL-A identical with their children, siblings have a 25 per cent chance of being identical. Even if nonidentical siblings or parents are used as donors, posttransfusion platelet survival rate may be better than that with random donors. Yankee and associates 86 have recently shown that it is possible to provide long-term platelet s u p p o r t using unrelated H L - A - i d e n t i c a l donors. The development of large pools of potential donors of known HL-A type may someday allow more widespread use of this method of providing adequate transfusion therapy. Platelet storage. Until relatively recently, platelets were stored at refrigerator temperature (4~ C.). It was felt that such stored platelets were less effective than fresh cells when stored for even a few hours at these temperatures,87, 88 since the ceils displayed shortened halflives after transfusion. In 1969, Murphy and Gardner 88 showed that significantly better post-transfusion recovery and survival resulted if platelets were kept at 22 ~ C. rather than 4 ~ C. Many blood centers now routinely store platelets at room temperature for 48 hours or longer. A potential risk associated with such storage is proliferation of bacteria inadvei'tenfly introduced during phlebotomy or component preparation. Although two investigators found n o evidence of bacterial contamina-

Blood transfusion

9

tion in platelets stored for up to 96 hours at ambient temperature89, 90 two larger studies showed that bacteria were sometimes recovered from platelets stored at room temperature9~, 92 and that occasionally such contaminated platelet prel~arations caused gram-negative septicemia in leukopenic recipients. 91 At least two deaths have been directly associated with infusion of such stored platelets contaminated with bacteria. 93 Recently Handin and Valer~94demonstrated that fresh platelets rapidly corrected aspirin-induced prolongation of bleeding time in normal volunteers but that platelets stored at 22 ~ C. for 24 hours did not, raising the question of whether post-transfusion platelet survival was indica9tive o f effective p l a t e l e t function. It should be emphasized that no in vitro test is predictive of in vivo platelet survival and functional activity. Most studies dealing with storage and post-transfusion survival have been performed using in vitro assays followed by the infusion of autologous or homologous cells into normal volunteers. Whether such information regarding posttransfusion survival and function can be directly applied to trarisfused thrombocytopenic patients has been challenged by Becker and associates 95 who pointed out that in the clinical setting differences in post-transfusion survival between 4 ~ C. and 22 ~ C. stored cells are small, since both are removed from the circulation relatively rapidly. These investigators also suggested platelet function was better preserved at 4 ~ C. storage temperatures than at 22 ~ C. Frozen platelets. Although many investigators have attempted long-term platelet storage in the frozen state, the best of currentlY" available methods provide a maximum of only about 50 to 60 per cent recovery after transfusion compared with fresh platelets. Among the agents that have been used for cryopreservation are glycerol, dimethylsulfoxide, dimethylacetamide, sodium gly.cerol phosphate, and hydroxyethyl starch. 96-1~ Although relatively poor recovery of platelets frozen by current methods has led some to abandon freezing of random donor platelets, they remain useful in emergencies. The.stockpiling of frozen single donor platelets, either HL-A identical or autologous, may proffer benefits outweighing cell loss if prevention of alloimmunization and disease are considered. Platelets obtained from patients with acute leukemia in remission have been frozen by the author and stored at liquid nitrogen temperatures until needed by the patient during relapse. The use of autologous frozen cells may literally be lifesaving in a patient who is unresponsive to random donor platelet infusion (Fig. 3). By means of this technique, several patients at t~he Baltimore Cancer Research Center have received only their own platelets during periods of chemotherapy

10

Buchholz

The Journal of Pediatrics January 1974

200

180

[] [] [] [] 0

160

x

140

MARROW EXAMINATION Day .3 : Markedly Hypocellular No Megakaryocytes

120

Day I0: Hypocellular Occasional Immature Megakoryocytes

Z O s

Plotelet Count Expected PIotelet Count Random Donor Cells Autologous FrozenCells % Of Expected Recovery*

, Based Upon No. of Plotelets Actually Infused. (Approximately 30% of Autologous Plotelets were Not Recovered Following Freezing and Washing)

I00: zi

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60

g 4o

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NiiyiiiiliiiSHiilEili

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78

9 ~oH ~m3~4t5 DAY

Fig. 3. Autologous frozen and fresh random donor platelet transfusion. Expected and actual platelet counts following transfusion in a patient refractory to random donor platelets. Expected counts were based on the number of cells infused and the recipient's estimated blood volume, and were corrected for normal splenic sequestration. The percentage of expected recovery was based on the circulating platelet count 16 hours following transfusion. Autologous cells had been obtained by multiunit plateletphereses and had been frozen in 5 per cent dimethylsulfoxide and stored at -120 ~ C. without the need for random donor cells. Although the size of children might limit the p r o c u r e m e n t of autologous cells, stockpiles of cells from compatible related donors might be used with equal benefit. GRANULOCYTE

TRANSFUSION

9 Marrow hypoplasia with resulting severe leukopenia and thrombocytopenia frequently follows aggressive chemotherapeutic treatment of malignancy. The liberal use of platelet transfusion has clearly reduced the number of deaths due to thrombocytopenic hemorrhage, and infections, especially those caused by gram-negative microorganisms, are now the leading cause of death in these patients. 62Bodey and associates ~~ have shown that the incidence of infection increases significantly as the

granulocyte count falls below 1,000 per microliter. Although newer antibiotics in conjunction with protected environments such as the life island or laminar air flow room may reduce or delay the development of infection, the neutropenic patient remains at significant risk until remission is achieved and marrow function returns. Although it might be expected that granulocyte replacement therapy, like platelet transfusion, would be in wide use, the infusion of such cells has been hampered until recently by lack of methods allowing largescale cell harvest. Unlike platelets which can be easily separated from plasma and red blood cells by differential centrifugation, granulocytes possess a density similar to that of the least 9 red blood cells and

Volume 84 Number 1

Blood transfusion

11

5 . 8 9 xlO I0

Total Leukocytes

12.0

1277J Granulocytes I Bands I~1 Pre -- Donation ,":'~ P o s t - Donation

5.O3xlO m

I0.0 ~" -

•

1.58 xlO m 1,15xlO Io

8.0

1.44x I0 I~

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3.91 x I0 Io

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4 . 9 2 x I0 I~

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I

DAY I

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

II

DAY 7

I

I

II

DAY B

I

DAY 9

DAY IO

Fig. 4. Hematologic changes with repeated granulocyte donation. Effect of repeated single donor granulocyte donation using nylon fiber filters. Although large numbers of granulocytes were collected, the donor maintained normal white blood cell counts. The donor platelet count remained above 150,,000 per microliter at all times with the exception of Day 9 when a postdonation platelet count of 84,000 per microliter was noted. Total granulocytes collected are indicated above the bars indicating the pre- and postdonation leukocyte counts. tend to accumulate in the upper red cell layer during centrifugation, making purification difficult. Cells may be more readily obtained from donors with greatly increased granulocyte counts as in chronic myelogenous leukemia. Freireich and associates 1~ made use of such cells in the treatment of infected granulocytopenic patients with aplastic anemia or acute leukemia. Although only five per cent of the transfused cells could be accounted for in the recipient circulation one hour after transfusion, some i:ecipients responded to the transfusion by lysis of fever and clearance of bacteria from the bloodstream. Impressively, 7 of 13 patients with Pseudomonas septicemia survived, although occasional short-term engraftment 'of donor cells was demonstrated. The apparent efficacy of such cell infusions has subsequently spurred efforts to devise methods of granulocyte harvest from normal donors. The Continuous Flow Blood Cell Separator, developed jointly by the National Cancer Institute and the IBM Corporation, has been used to harvest granulocytes, lymphocytes, and platelets from normal donors with modest effectiveness. 84,104, ]05 During a donation (which typically lasts four hours) blood is removed from one donor vein and subjected to continuous flow centrifugation in a small centrifuge bowl. As plasma/red cell separation occurs, granulocytes are semiselectively skimmed from the upper red blood cell layer and collected for later transfusion; red cells and plasma are returned to the donor via a second vein.

Although from 10 to 20 liters of donor blood may be processed, efficiency is poor (about 15 per cent granulocyte recovery in the author's experience), the equipment expensive, and yields of granulocytes are modest (an average of about 5 to 6 billion per donation). However, yields have been improved by the use of agents which mobilize donor granulocytes, such as cortisone 1~ or etiocholanolonO ~ and/or the addition of rouleaux-inducing agents such as hydroxyethyl starch 1~ to the blood prior to centrifugation. Recently, Djerassi and associates 109 described a simple, inexpensive, and efficent technique for granulocyte harvest using nylon fiber filters (Leuko-Pak, Fenwal Laboratories). W h e n h e p a r i n i z e d blood is passed through the filters, granulocytes are selectively retained by the fibers while lymphocytes, red cells, plasma, and most platelets pass through unhindered. Incorporation of such filters in a continuous-flow system has resulted in considerable improvement in the number of granulocytes which can be collected from normal, nonstimulated donors. In the author's experience, using two filters, an average of approximately 40 billion granulocytes can be collected in a 3 hour period from normal donors, and the same donor may be used repeatedly without development of granulocytopenia or severe thrombocytopenia (Fig, 4). Although the functional efficacy of such collected cells has not been firmly established, initial work appears encouraging. 1~ Granulocyte transfusion, just as platelet or red cell

12

Buchholz

transfusion, may lead to recipient alloimrnunization and render future therapy difficult.H2 In addition to antigens shared with lymphocytes in the HL-A system, granulocytes possess unique antigens not shared with other blood cells. ~13 HL-A-compatible family members may be much more likely than random donors to possess cells compatible with the recipient if leukoagglutinins or lymphocytotoxins directed against most random donor cells are present. Transfusion of granulocytes in the presence of such cell-directed antibody may resuR in serious transfusion reaction112; additionally, recent investigation has suggested that cells may display a diminished phagocytic and bactericidal capability in. the presence of specific antibody. 11~112 Granulocyte transfusion must currently be considered an investigational procedure; however, such cells may eventually play a major role in supportive therapy: The author has seen a patient with acute leukemia and Psuedomonas cellulitis which progressed over a 3 day period despite the liberal use of appropriate antibiotic therapy; clear-cut regression of the lesion followed after sequential administration of granulocytes collected using the nylon fiber filters. Graw and associates TM have shown that normal granulocytes, obtained using either the blood cell separator or the nylon fiber filter, appear to be beneficial in the treatm e n t of septicemia. They noted a 30 per cent survival rate in a control group-of 37 septicemic patients treated with antibiotics alone, and a 46 per cent survival rate in 39 patients given both antibiotics and granulocytes. Although there is some question as to whether these differences in survival are significant, such studies are encouraging; a great deal of additional work is needed to firmly establish the safety and efficacy of granulocyte transfusion and to develop improved methods of cell harvest and preservation. The author is indebted to Miss Margo T. Coady for her patient secretarial assistance and to Peter H. Wiernik, M.D., Head, Section of Medical Oncology, National Cancer Institute, Baltimore Cancer Research Center, for his thoughtful review of the manuscript. REFERENCES 1. Maurer, P. H., and Berardinelli, B.: Immunologic studies with hydroxyethyl starch (HES)--a proposed plasma expander, Transfusion 8: 265, 1968. 2. Aster, R. H., and Jandl, J. H.: Platelet sequestration in man, J. Clin. Invest. 43: 843, 1964. 3. Standards for blood banks and transfusion services, Chicago, 1972, American Association of Blood Banks, p. 9. 4. Mollison, P. L.: Blood transfusion in clinical medicine, Oxford, 1972, Blackwell Scientific Publications, p. 57. 5. Beutler, E.: The maintenance of red cell function during

The Journal of Pediatrics January 1974

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7.

8.

9.

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1'2.

13.

14.

15.

16.

17.

18.

19.

20.

liquid storage, in Schmidt, P. J., editor: Progess in transfusion and transplantation, Chicago, 1972, American Association of Blood Banks, pp. 285-297. Warner, W. L.: Red cell preservation and survival determinations in anticoagulant systems, in Spielmann, W., and Seidl, S., editors: Modern problems in blood preservation, Stuttgart, 1970, Fisher-Verlag, pp. 63-71. de Verdier, C. H., Akerblom, O., Arturson, G., Garby, L., H6gman, C. F., Kreuger, A., and Westman, M.: Maintenance of oxygen transport function of stored blood, in Schmidt, P. ,r., editor: Prog~'ess in transfusion and transplantation, Chicago, 1972, American Association of Blood Banks, pp. 299-315. Haradin, A. R., Weed, R. I., and Reed, C. F.: Changes in physical properties of stored erythrocytes: Relationship to survival in vivo, Transfusion 9: 229, 1969. Valtis, D. J., and Kennedy, A. C.: The causes and prevention of defective function of stored red blood cells after transfusion, Glasgow Med. J. 34: 521, 1953. Chanutin, A., and Curnish, R. R.: Effect of Organic and inoranic phosphates on the oxygen equilibrium Ofhuman erythrocytes, Arch. Biochem. Biophys. 121: 96, 1967. Beutler, E., and Wood, L.: The in vivo regeneration of red cell diphosphoglyceric acid (DPG) after transfusion of stored blood, J. Lab. Clin. Med. 74: 300, 1969. Valeri, C. R., and Hirsch, N. M.: Restoration in vivo of erythocyte adenosine triphosphate, 2, 3-diphosphoglycerate, potassium ion, and sodium con, centrations following the transfusion of acid-citrate-dextrose-stored human red blood cells, J. Lab. Clin. Med. 73: 722, 1969. Sugerman, H. J:, Davidson, D. T., Santi-Vibul, A. B., Delivoria-Papadopoulos, M., Miller, L. D., and Oski, F. A.: The basis of defective oxygen delivery from stored blood, Surg. Gynecol. Obstet. 131: 733, 1970. Delivoria-Papadopoulos, M., Morrow, G., III, and Oski, F. A.: Exchange transfusion in the newborn infant with fresh and "old" blood: The role of storage on 2,3diphosphog.lycerate, hemoglobin-oxygen affinity, and oxygen release, J. PEDIATR.79: 898, 1971. Strumia, M. M., Strumia, P. V., and Eusebi, A. J.: The preservation of blood for transfusion. VII. Effect of adenine and inosine on the adenosine triphosphate and viability of red cells when added to blood stored from zero to seventy, days at 1~ C., J. Lab. Clin. Med. 75: 244, 1970. Akerblom, O., deVerdier, C.-H., Garby, L., and H6gman, C.: Restoration of defective oxygen-transport function of stored red blood cells by addition of inosine, Scand. J. Clin. Lab. Invest. 21: 245, 1968. Gibson, J. G., and Lionetti, F. J.: The effect of dipyridamole on the adenosine triphosphate level of stored human blood, Transfusion 6: 427, 1966. Wood, L., and Beutler, E.: The effect of ascorbic acid on the 2,3-DPG level of stored blood, Clin. Res. 20: 186, 1972. Chanutin, A.: The effect of the addition of adenine and nucleosides at the beginning of storage on the concentrations of phosphates of human erythrocytes during storage in acid-citrate-dextrose and citrate-phosphatedextrose, Transfusion 7: 120, 1967. Valeri, C. R., and Zaroulis, C. G.: Rejuvenation and

Volume 84 Number 1

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22. 23. 24.

25.

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27.

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31. 32.

33.

34. 35.

36.

37.

freezing of outdated stored human red cells, N. Engl. J. Med. 287: 1307, 1972. Mollison, P. L., and Sloviter, H. A.: Successful transfusion of previously frozen human red cells, Lancet 2: 862, 1951.. Huggins, C. E.: Frozen blood, Ann. Surg. 160: 643, 1964. Rowe, A. W., Eyster, E., and Kellner, A.: A low glycerolrapid freeze procedure, Cryobiology 5: 119, 1968. Meryman, H. T., and Hornblower, M.: A method for freezing and washing red blood cells using a high glycerol concentration, Transfusion 12: t45, 1972, Crowley, J. P., and Valeri, C. R.': Factors influencing the reduction of countable leukocytes and platelets in frozen blood, presented at the Twenty-fifth Annual Meeting of the American Association of Blood Banks-Thirteenth International Congress, International Society of Blood Transfusion, Washington, D. C., August 31, 1972. Chaplin, H., Jr.: Fro'Ten red cell storage: Perspectives and potentials, in Schmidt, P. J., editor: Progress in transfusion and transplantation, Chicago, 1972, American Association of Blood Banks, pp. 329-342. Helgeson, M., McCullough, J., Nelson, C., and Polesky, H.: Evaluation of methods for preparation of HL-A poor blood, presented at the Twenty-fifth Annual Meeting of the American Association of Blood Banks-Thirteenth International Congress, International Society of Blood Transfusion, Washington, D. C., August 28, 1972. Milles, G., Langston, H. T., and Dalessandro, W.: Autologous transfusions, Springfield, II1., 1971, Charles C Thomas, Publisher. Schechter, G. P., Soehnlen, F., and McFarland, W.: Lymphocyte response to blood transfusion in man, N. Engl. J. Med. 287: 1169, 1972. Klebanoff, G.: The physiological consequences to be anticipated when intraoperative blood salvage and reinfusion is accomplished using the Bentley ATS 100-disposable autotransfusion system, presented at the Twenty-fifth Annual Meeting of the American Association of Blood Banks-Thirteenth lnterna.tional Congress, International Society of Blood Transfusion, Washington, D. C., August 31, 1972.. Mollison, P. L.: Blood transfusion in clinical medicine, Oxford, 1972, Blackwelt Scientific Publications, p. 529. Issitt, P. D.: Applied blood group serology, Oxnard, Calif., 1970, Spectra Biologicals, Division of Becton, Dickinson and Co., pp. 145-152. Goldfinger, D., and McGinniss, M. H.: Rh-incompatible platele.t transfusionsmrisks and consequences of sensitizing immunosuppressed patients, N. Engl. J. Med. 284: 942, 1971. Giblett, E. R.: A critique of the theoretical hazard of inter-vs, intra-racial transfusion, Transfusion 1: 233, 1961. Lostumbo, M. M., Holland, P. V., and Schmidt, P. J.: Isoimmunization after multiple transfusions, N. Engl. J. Med. 275: 141, 1966. Masouredis, S. P.: Clinical use o f whole blood, in Williams, W. J., Beutler, E., Erslev, A. J., and Rundles, R. W., editors: Hematology, New York, 1972, McGrawHill Book Company, Inc., p. 1314. Vyas, G. N., Perkins, H. A., and Fudenberg, H. H.: Anaphylactoid transfusion reactions associated with antilgA, Lancet 2: 312, 1968.

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13

Stuart, M. J., Murphy, S., Oski, F. A., Evans, A. E., Donaldson, M. H., and Gardner, F. H.: Platelet function in recipients of platelets from donors ingesting aspirin, N. Engl. J. Med. 287:1105, 1972. Kasper, C. K., and Rapaport, S. I.: Bleeding times and platelet aggregation after analgesics in hemophilia, Ann. Intern. Med. 77: 189, 1972. Kaneshiro, M. M., Mielke, C. H., Kasper, C. K., and Rapaport, S. I.: Bleeding time after aspirin in disorders of intrinsic clotting, N. Engl. J. Med. 281: 1039, 1969. Greenwalt, T. J., Gajewski, M., and McKenna, J. L.: A new method for preparing buffy coat-poor blood, Transfusion 2: 221, 1962. Verschoyle, M. J.: Laboratory diagnosis: Biochemical, in A seminar on hemolytic transfusion reactions, presented at the Twentieth Annual Meeting of the American Association of Blood Banks, New York, October 22, 1967. Mollison, P. L.: Blood transfusion inclinical medicine, Oxford, 1972, Blackwell Scientific Publications, pp. 550-552. Rapaport, S. I.: Defibrifiation syndromes, in Williams, W. J., Beutler, E., Erslev, A. J., and Rundles, R. W., editors: Hematology, New York; 1972, McGraw-Hill Book Company, Inc., pp. 1234-1255. Bashore, R. A., and Lecky, J, W.: Intrauterine fetal transfusion in the management of Rh disease, Obstet. Gynecol. 35: 79, 1971. Liley, A. W.: Intrauterine transfusion of foetus in haemolytic disease, Br. Med. J. 2: 1107, 1963. Bowman, J. M., Friesen, R. F., Bowman, W. D., Mclnnis, A. C., Barnes, P. H., and Grewar, D.: Fetal transfusion in severe Rh isoimmunization, J. A. M. A. 207: 1101, 1969. Borrone, C., and Bricavelli, F. D.: Survival of donors' lymphocytes in new-born infants submitted to exchange transfusion, Helv. Paediatr. Acta 24: 192, 1969. Trey, C., Burns, D. G., and Saunders, S. J.: Treatment of hepatic coma by exchange blood transfusion, N. Engl. J. Med. 274: 473, 1966. Schwartz, A. D.: The coagulation defect in Reye's syndrome, J. PEDIATR.78: 326, 1971. Brody, J. 1., Goldsmith, M. H., Park, S. K., and Soltys, H. D.: Symptomatic crises of sickle cell anemia treated by limited exchange transfusion, Ann. Intern. Med. 72: 327, 1970. Pearson, H. A., Shulman, N. R., Marder, V. J., and Cone, T. E., Jr.: Isoimmune neonatal thrombocytopenic purpura--clinical and therapeutic considerations, Blood 23: 154, 1964. Gross, S., and Methorn, D. K.: Exchange transfusion with citrated whole blood for disseminated intravascular coagulation, J. PEDIATR.78: 415, "1971. Garmer, L. M., Snyder, R. N., Chabon, R. S., and Bernstein, J.: Kernicterus: High incidence in premature infants with low serum bilirubin concentrations, Pediatrics 45: 906, 1970. Waters, W. J., and Porter, E.: Indications for exchange transfusions based upon the rol~eof albumin in the treatment of hemolytic disease of the newborn, Pediatrics 33: 749, 1964. ~Huestis, D. W., Bove, J. R., and Busch, S.: Practical blood transfusion, Boston, 1969, Little, Brown & Company, pp. 229-230.

14

57. 58.

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Allen, F. H., Jr., and Diamond, L. K.: Erythroblastosis fetalis, Boston, 1957, Little, Brown & Company. Stewart, J. H., and Castaldi, P. A.: Uraemic bleeding: A reversible piatelet defect corrected by dialysis. Q. J. Med. 36: 409, 1967. Borchgrevink, C. F.: Platelet adhesion in vivo in patients with bleeding disorders, Acta Med. Scand. 170: 245, 1965. Mielke, C. H., Jr., Kaneshiro, M. M., Maher, I. A., Weiner, J. M., and Rapaport, S. 1.: The Standardized normal Ivy bleeding time and its prolongation by aspirin, Blood 34: 204, 1969. Langdell, R. D., Adelson, E. A., Furth, F. W., and Crosby, W. H.: Dextran and prolonged bleeding time, J. A. M. A. 166: 346, 1958. Perry, S.: Supportive care in cancer therapy, in Report of the chemotherapy program, National Cancer Institute, Bethesda, 1972, National Institutes of Health, pp. 2119-2123. Tranum, B. L., and Haut, A.: In vivo survival of platelets prepared in CPD anticoagulant, Transfusion 12: 168, 1972. Mourad, N.: A simple method for obtaining platelet concentrates free of aggregates, Transfusion 8: 48, 1968. Gilchrist, G. S., and Ekert, H.: Reduction of factor VIII activity in cryoprecipitate obtained from acidified plasma, Transfusion 8: 294, 1968. Becker, G. A., and Aster, R. H.: Prostaglandin E 1in preparation and storage of platelet concentrates, Science 175: 538, 1972. Valeri, C. R., Zaroulis, C. G., Rogers, J. C., Handin, R. I., and Marchionni, L. D.: Prostaglandins in the preparation of blood components, Science 175: 539, 1972. Aster, R. H.: Pooling of platelets in the spleen: Role in the pathogenesis of"hypersplenic" thrombocytopenia, J. Clin. Invest. 45: 645, t966. Ebbe, S.: Origin, production, and life-span of blood platelets, in Johnson, S~, editor: The circulating platelet, New York, 1971, Academic Press, Inc., pp. 19-43. Grumet, F. C., and Yankee, R. A.: Long term platelet support of patients with aplastic anemia-effect of splenectomy and steroid therapy, Ann. Intern. Med. 73: 1, 1970. Freireich, E. L, Kliman, A., Gaydos, L. A., Mantel, N., and Frei, E., Ill: Response to repeated platelet transfusion from the same donor, Ann. Intern. Med. 59: 277, 1963. Aster, R. H.: Effect of anticoagulant and ABO incompatibility on recovery of transfused human platelets, Blood 26: 732, 1965. Huestis, D. W., Bore, L R., and Busch, S.: Practical blood transfusions, Boston, 1969, Little, Brown & Company, p. 299. Arora, S. N., and Morse, E..E.: Platelet filters--an evaluation, Transfusion 12: 208, 1972. Weiss, H. J., Aledort, L. M., and Kochwa, S.: The effect of salicylates on the hemostatic properties of platelets in man~ J. Clin. Invest. 47: 2169, 1968. Mielke, C. H., and Britten, A. F. H.: Use of aspirin or acetaminophen in hemophilia, N. Engl. J. Med. 282: 1269, 1970. Schwartz, A. D., and Pearson, H. A.: Aspirin, platelets, and bleeding, J. PEDIATR.78: 558, 1971.

The Journal of Pediatrics Janualy 1974

78. Corby, D. G., and Schulman, I.: The effects of antenatal drug administration on aggregation of platelets of newborn infants, J. PEDIATR.79: 307, 1971. 79. ' Rodriguez, S. U., Leikin, S., and Hiller, M. C.: Neonatal thrombocytopenia associated with antepartum administration of thiazide drugs, N. Engl. J. Med. 270: 881, 1964. 80. Adner, M. M., Fisch, G. R., Starobin, S. G., and Aster, R. H.: Use of "compatible" platelet transfusions in treatment of congenital isoimmune neonatal thrombocytopenic purpura, N. Engl. J. Med. 280: 244, 1969. 8t. Cimo, P. L., "and Aster, R. H.: Post-transfusion purpura--successfu[ treatment by exchange transfusion, N. Engl. J. Med. 287: 290, 1972. 82. Kliman, A., Carbone, P. P., Gaydos, L. A., and Freireich, E: J.: Effects of intensive plasmapheresis on normal blood donors, Blood 23: 647, 1964. 83. Cohen, M. A., and Oberman, H. A.: Safety and long-term effects of plasmapheresis, Transfusion 10: 58, 1970. 84. Graw, R. G., Jr., Herzig, G. P., Eisel, R. J., and Perry, S.: Leukocyte and platelet collection from normal donors with the continuous flow blood cell separator, Transfusion 11: 94, 1971. 85. Tullis, J. L., Tinch, R. J., Baudanza, P,, Gibson, J. G., II, DiForte, S., Conneely, G., and Murthy, K,: Plateletpheresis in a disposable system, Transfusion 11: 368, 1971. 86. Yankee, R., Graft, K, Dowling, R., and Henderson, E. S.: Selection of unrelated compatible platelet donors by lymphocyte HL-A matching, N. Engl. J. Med. 288: 760, 1973. 87. Baldini, M., Costea, N., and Dameshek, W.: The viability of stored human platelets, Blood 16: 1669, 1960. 88. Murphy, S., and Gardner, F. H.: Effect of storage temperature on maintenance of platelet viability-deleterious effect of refrigerated storage, N. Eng[. J. Med. 280: 1094, 1969. 89, Silver, H., Sonnenwirth, A. C., and Beisser, L. D.: Bacteriologic study of platelet concentrates prepared and stored without refrigeration, Transfusion 10: 315, 1970. 90. Katz, A. J., and Tilton, R. C.: Sterility of platelet concentrates stored at 25~ C., Transfusion 10: 329, 1970. 91. Buchholz, D. H., Young, V. M. Friedman, N. R., Reilly, J. A., and Mardiney, M. R., Jr.: Bacterial proliferation in platelet products stored at room temperature--transfusion-induced Enterobacter sepsis, N. Engl. J. Med. 285: 429, !971. 92. Cash, J. D., and Cunningham, M.: Bacterial contamination of platelet concentrates stored at room temperature, presented at the Twenty-fifth Annual Meeting of the American Association of Blood Banks-Thirteenth International Congress, International Society of Blood Transfusion, Washington, D. C., August 31, 1972. 93. Buchholz, D. H., Young, V. M., Friedman, N. R., Reilly, J. A., and Mardiney, M. R., Jr.: Detection and quantitation of bacteria in platelet products stored at ambient temperature, Transfusion 13: 278, 1973. 94. Handin, R. I., and Valeri, C. R.: Hemostatic effectiveness of platelets stored at 22 ~ C., N. Engl. J. Med. 285: 538, 1971. 95. Becker, G. AI, Tuccelli, M., Kunicki, T., Chalos, M. K., and Aster, R. H.: Studies of platelet concentrates stored at 22 ~ C. and 4 ~ C., Transfusion 13:61, 1973.

Volume 84 Number 1

96. Cohen, P., and Gardner, F. H.: Platelet preservation. IV. Preservation of human platelet concentrates by controlled slow freezing in a glycerol medium, N. Engl. J. Med. 274: 1400, 1966. 97. Djerassi, I., Farber, S., Roy, A., and Cavins, J.: Preparation and in vivo circulation of human platelets preserved with combined dimethylsulfoxide and dextrose, Transfusion 6: 572, 1966. 98. Djerassi, I., Roy, A., Kim, J., and Cavins, J.: Dimethylacetamide, a new cryoprotective agent for platelets, Transfusion 11: 72, 1971. 99. Greiff, D., and Mackey, S.: Cryobiology of platelets. II. Effects of freezing and storage at low temperatures on the survival of isolated blood platelets as measured by assays for aminopeptidases, Cryobiology 7: 9, 1970. 100. Weatherbee, L., Starkweather, W. H., Knorpp, C. T., Schinitzer, B., and Spencer, H. H.: ,An approach to the long term preservation of platelets, Cryobiology 8: 393, 1971. 101. Handin, R. I., and Valeri, C. R.: Improved viability of previously frozen platelets, Blood 40: 509, 1972. 102. Bodey, G. P., Buckley, M., Sathe, Y. S., and Freireich, E. J.: Quantitative relationships between circulating leukocytes and infection in patients with acute leukemia, Ann. Intern. Med. 64: 328, 1966. 103. Freireich, E. J., Levin, R. H., Whang, J., Carbone, P. P., Bronson, W., and Morse, E. E.: The function and fate of transferred leukocytes from donors with chronic myelocytic leukemia in leukopenic recipients, Ann. N. Y. Acad. Sci. 113: 1081, 1964. 104. Graw, R. G., Jr., Herzig, G., Perry, S., and Henderson, E. S.: Normal granulocyte transfusion therapy, N. Engl. J. Med. 287: 367, 1972. 105. Buchholz, D. H.: Unpublished observation. 106. Bishop, C. R., Athens, J. W., Boggs, D. R., Warner, H. R.,

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Cartwright, G. E., and Wintrobe, M. M.: Leukokinetic studies. XItI. A non-.steady-state kinetic evaluation of the mechanism of cortisone-induced granulocytosis, J. Clin. Invest. 47: 249, 1968. Kimball, H. R., Vogel, J. M., Perry, S., and Wolff, S. M.: Quantitative aspects of pyrogenic and hematologic responses to etiocholanolone in man, J. Lab. Clin. Med. 96: 415, 1967. Freireich, E. J.: Personal communication. Djerassi, I., Kim, J. S., Suvansri, U., Mitrakul, C., and Ciesielka, W.: Continuous flow filtration--leukopheresis, Transfusion 12: 75, 1972. Herzig, G. P., Root, R. K., and Graw, R. G., Jr.: Granulocyte collection by continuous-flow filtration leukopheresis, Blood 39: 554, 1972. Buchholz, D. H., Wiernik, P. H., and Mardiney, M. R., Jr.: Granulocyte procurement by leukocyte filtration, presented at the Twenty-fifth Annual Meeting of the American Association of Blood Banks-Thirteenth International Congress, International Society of Blood Transfusion, Washington, D. C., August 28, 1972. Goldstein, I. M., Eyre, H. J., Terasaki, P. I., Henderson, E. S., and Graw, R. G., Jr.: Leukocyte transfusions: Role of leukocyte-allOantibodies in determining transfusion response, Transfusion 11: 19, 1971. Lalezari, P., and Bernard, G. E.: Distribution of leukocyte antigens in various blood cells and body tissues, m Balner, H., Cleton, F. J., and Eernisse, J. G., editors: Histocompatibility testing 1965, Copenhagen, 1966, Ejnar Munksgaards Forlag, pp 167-169. Graw, R. G., Jr., Herzig, G., Perry, S., and Henderson, E. S.: Normal granulocyte transfusion therapy--treatment of septicemia due to a gram-negative bacteria, N. Engl. J. Med. 287: 367, 1972.