The Operation of a Hospital Transfusion Service part I—The Preservation and Transfusion of Whole Human Blood part n—The Processing and Use of Citrated Human Blood Plasma OCD Publication 2220 ♦ March 1944 Medical Division OFFICE OF CIVILIAN DEFENSE Washington 25, D. C. Foreword This manual describes some of the techniques which have proved satisfactory for the operation of hospital blood banks and for the preparation of human plasma. They are based upon the experience of many investigators and have the approval of the Subcommittee on Blood Substitutes of the Division of Medical Sciences, National Re- search Council. The establishment of blood and plasma banks, particularly in large hospitals, has been of inestimable value in saving the lives of patients suffering from shock and burns, in improving the care of surgical pa- tients, and in providing a valuable adjunct in the treatment of certain medical conditions. Although they are designed primarily to supply the normal civilian needs of the hospital, they may be rapidly ex- panded to serve practically any major catastrophe. Unlike many es- tablishments which are necessary in time of war but become obsc escent as soon as peace is declared, the blood bank will continue to be useful. The fact that plasma and other blood derivatives are lifesaving in emergencies must not lead to the conclusion that such can completely replace whole blood transfusions. Although this was never the original intention of the advocates of blood derivatives, the trend has been to regard plasma, serum, etc., as “blood substitutes” rather than blood derivatives, which serve a specific purpose but can- not replace whole blood. In nearly all emergency cases in which trauma (or surgery) is associated with significant blood loss, the transfusion of whole blood is preferable to the use of plasma, pro- vided that it is immediately available. If whole blood cannot be administered at once, plasma (serum, etc.) must be given promptly to meet the emergency and followed later by whole blood as indicated. In addition, there is growing interest in the possible uses of con- valescent plasma (or serum) in the treatment of infectious diseases. The ability of a hospital to prepare and store plasma in the frozen state will enable its staff to make use of the in this field. Contents PART I: THE PRESERVATION AND TRANSFUSION OF WHOLE HUMAN BLOOD A. Clinical and Experimental Background Page Chapter I. Blood Groups: Isohemagglutinins and Isohemolysins 1 Noirrnclatures for blood groups 1 In ince 2 Ch „0 s in titer of agglutinins and agglutinogens during life 2 Neutralization of agglutinins in the body 2 Neutralization of agglutinins in vitro 3 The Rh antibody 3 mpter II. The Donor 4 Physical standards 4 Physiology of controlled hemorrhage 5 pter III. The Recipient 6 Indications for transfusion 6 Contraindications for transfusion 6 Survival of transfused erythrocytes 7 Chapter IV. Transfusion Reactions 7 Pyrogenic reactions 7 Urticarial reactions 8 Hemolytic reactions 8 Cardiovascular reactions 10 Retinal hemorrhages 11 Infections transmissible by transfusion 11 Toxicity of potassium 11 Transfusion of cold solutions 11 Incidence of transfusion reactions 12 Chapter V. Changes in Blood During Storage 12 Red cells 12 Other constituents 13 B. Technique Chapter VI. Operation of a Blood Bank 14 Advantages of a blood bank 14 Types of blood banks 14 Methods of accounting 15 Acquisition of “Capital” 15 Personnel 16 Source of pyrogen-free fluids 16 Equipment 16 IV CONTENTS Chapter VII. Typing and Cross-Matching of Blood 17 Determination of blood groups 18 Equipment 18 Choice of methods 18 The slide method 18 The centrifuge method. 20 Confirmation by testing of plasma 20 Mass grouping 21 Cross-matching 22 The centrifuge method 22 The slide method 22 Rh typing and cross-matching 23 Selection of the universal donor 23 Sources of error in typing and cross-matching 24 Grouping sera 25 Preparation 25 Criteria for potency 26 Identification of Aj, A2, and A2B 27 Anti-Rh testing sera 28 Chapter VIII. Collection of Blood 28 Psychology of the inexperienced donor 28 Donor’s release 28 Selection of donors 29 Use of fasting donors 29 Preparation of the donor 29 Care of the donor during bleeding 30 Technique of collection 30 Care of the donor following bleeding 31 Reactions of the donor 31 Chapter IX. Storage of Blood 32 Chapter X. Transportation of Blood 34 Chapter XI. Administration of Bicod 34 Equipment 35 Rate of administration 35 Selection of a vein 36 Danger of warming 36 Care of recipient 36 Laboratory study of the recipient 36 Chapter XII. Administration of Red Cell Suspensions 37 References for Part I 39 PART II: THE PROCESSING AND USE OF CITRATED HUMAN BLOOD PLASMA Chapter I. General Considerations 43 Essential requirements for plasma production 44 Chapter II. Liquid, Frozen, and Dried Plasma 45 Chapter III. Methods of Plasma Preparation 47 CONTENTS V A. Centrifuge Method Page 1. Employment of commercial vacuum type containers 47 Collection of blood 47 Storage of blood 49 Centrifugation 49 Pooling 50 Culturing 51 Addition of a bacteriostatic agent 52 Filling of final containers; storage as liquid plasma 52 Preparation and use of frozen plasma I 53 Restoring frozen plasma to the liquid state 54 Preparation of dilute liquid plasma 54 2. Employment of reusable equipment 56 Apparatus for collection of blood 56 Collection of blood 58 Separation of plasma 58 Pooling apparatus 58 Pooling of plasma 61 Culturing 62 Addition of a bacteriostatic agent 63 Filling of final containers 63 B. Sedimentation Methods 1. Blood preservation and sedimentation method for the preparation of dilute plasma 64 Advantages for blood banks 64 Blood preservation technique 65 Aspiration of plasma 65 Culturing the plasma 66 Multiple aspiration 66 Addition of a bacteriostatic agent 66 Use as liquid plasma 66 2. Citrate sedimentation technique 67 Chapter IV. Labeling of Plasma 68 Chapter V, Plasma Records 68 Chapter VI. Care of Equipment 69 Preparation of equipment for intravenous use 69 Cleaning and preparation of donor sets 71 Cleaning and preparation of aspirating sets 72 Cleaning of storage bottles 72 Chapter VII. Adverse Reactions from the Administration of Human Plasma. 73 References fob Part II 74 Appendix A Section I: Preparation of 1 percent aqueous merthiolate solution and phenyl mercuric borate or nitrate 76 Section II: Refrigeration of blood 76 Section III: Preparation of new rubber tubing 77 VI contents Appendix B Page Section I; Interpretation of serology test for syphilis 78 Section II; Methods for determination of hemoglobin 78 Section III: Pooling, tests for sterility, storage of plasma 79 Section IV: Fluid thioglycollate medium for the sterility tests 82 Section V: Safety test 83 Section VI: Determination of residual moisture 84 Section VII: Pyrogen test 84 Section VIII; Filter adequate for removal of particulate matter 85 Section IX: N. I. H. requirements for commercial firms 86 Section X; U. S. P. requirements for citrated normal human plasma 87 Appendix C The treatment of shocks and burns 89 Part /• The Preservation and Trans- fusion of Whole Human Hiood A. CLINICAL AND EXPERIMENTAL BACKGROUND Chapter I. Blood Groups: Iso hemagglutinins and Isohemolysins I\omenclatures for Blood Groups In 1907 Jansky suggested the designation of the blood groups by numbers, and a short time later Moss made a similar proposal, but he reversed the Jansky groups I and IV. The confusion between the numberings by Jansky and by Moss is now eliminated by the use of the system called the International Nomenclature. The use of num- bers for the designation of blood groups is to be discouraged. Table 1 shows the relationship between the various nomenclatures. Table 1 Approximate distribution among white individuals in U. 8. A. International Jansky Moss Percent 0 I IV 44 A II II 38 B III III 14 AB IV I 4 All human bloods are divided into four groups according to the agglutinogens contained in the cells. The two agglutinogens A and B may be distributed in four ways as shown in table 2. The four blood groups are therefore known as A, B, AB, and O in the preferred International Nomenclature. The serum or plasma may contain spe- cific agglutinins, each of which reacts against only the A or B agglu- tinogen. The combination of cells containing agglutinogen A with serum or plasma containing anti-A agglutinin will produce permanent clumping or agglutination of the cells. It is obvious, therefore, that no single blood can contain an agglutinogen and its corresponding agglutinin. Group A blood contains anti-B agglutinin. Group B 2 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE blood possesses anti-A agglutinin. Group O blood contains both anti-A and anti-B, and AB blood contains no agglutinins. (See table 2). About 30 percent of fresh bloods having agglutinins contain hemolysins which are specific and react against the same agglutinogen as the agglutinin. The hemolysins are weaker in titer than the agglu- tinins and will act only in the presence of complement. They are therefore not encountered when typing serum is used, but their pres- ence constitutes a source of error in the cross-matching of bloods. Group Agglutinogens in cells Agglutinins in serum or plasma 0 None Anti-A and anti-B A A Anti-B B B Anti-A AB A and B None Tatle 2 Inheritance The individual inherits the combination of group-specific substances w’hich determines his blood group. It follows that the blood group cannot change during the life of the individual. According to Bern- steinin, there are two laws governing the inheritance of blood groups: 1. Agglutinogens A or B do not appear in the blood of the child except when present in one or both of the parents. 2. The combinations, group O parents and AB child, or vice versa, are impossible. The Changes in Titer of Agglutinins and Agglutinogens Dur- ing Life This is of importance in the cross-matching and typing of blood. Agglutinogens appear as early as the thirty-seventh day of fetal life. The titer increases until the twentieth year, after which it remains constant. The erythrocytes of a new-born baby are only one-fifth as sensitive to agglutination as are those of an adult. There is some doubt as to whether agglutinins are present at birth. At any rate, the titer quickly increases and reaches a maximum between 5 and 10 years, after which there is a slow diminution of potency. These facts account for the difficulty sometimes encountered in determining the blood groups of children. Neutralization of Agglutinins in the Body When an agglutinin and a group-specific substance come in contact, a combination is effected which is permanent and results in the neu- tralization of the agglutinin. This phenomenon occurs when incom- patible agglutinins are injected into the body, or when group O blood BLOOD GROUPS 3 is transfused into a recipient of another group. This fact explains the safety with which pooled plasma is employed (31), but there is still some question as to whether the use of blood from a group O or “uni- versal donor” is entirely innocuous. There are many who believe that the use of the universal donor is without danger and attribute the cases of reactions reported in the literature to some other cause. The Rh factor, for example, has only recently been recognized, and the older studies did not consider this possibility. One case has recently been reported, however, in which the anti-Rh factor was definitely excluded (27). The Neutralization of the Agglutinins in Vitro This has been made practical by the preparation of the group-specific A and B substances (agglutinogens) by Witebsky et al. (49). These substances have been prepared in a form safe for intravenous injection. The addition of small amounts to group O blood immediately inacti- vates the anti-A and anti-B agglutinins in the plasma, thus producing a universal blood which is free from theoretical objection. The Rh Antibody The red blood cells of approximately 85 percent of all persons, irre- spective of their blood groups, contain an agglutinogen designated Rh, related to a similar agglutinogen found in the red cells of rhesus monkeys. Apparently there are no naturally occurring anti-Eh ag- glutinins, but the Rh negative individuals (15 percent of the popula- tion) are capable of forming anti-Rh agglutinins. This may occur (1) when repeated transfusions of Rh positive blood are given to an Rh negative subject, or (2) when an Rh negative woman bears an Rh positive fetus (from an Rh positive father). Not all Rh negative individuals develop demonstrable isoagglutinins under these circum- stances, but following such isoimmunization, the Rh negative indi- vidual may suffer severe hemolytic reactions when transfused with Rh positive blood cells. Manifestations of Rh isoimmunization usually do not appear until after several transfusions (a variable number), when reactions, usually mild at first, may occur and increase in severity following each succeeding transfusion with Rh positive blood. Mani- festations of Rh isoimmunization usually do not occur in the first pregnancy of the Rh negative woman. However, in subsequent preg- nancies, the anti-Rh agglutinins increasingly formed by the mother cause in her Rh positive infants a profound hemolytic anemia known as erythroblastosis fetalis. If a 'patient receiving repeated blood transfusions has had reactions to previous transfusions, it is important to rule out Rh isoimmuniza- tion as a cause of the reactions by Rh typing of the recipient and donor 4 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE Should any woman with a history of having home an erythr oblast otic infant require a transfusion, Rh typing of recipient and donor should he done before any transfusion, because in such instances even a first transfusion may provoke a fatal reaction. In such cases Rh negative blood of a compatible group can be given safely. Only such blood shoidd be given in the treatment of infants with erythroblastosis, since onti-Rh agglutinins from the mother's blood are present in their cir- culation for some time. (See page %3 for avoiding Rh incompatibil- ity by special typing or cross-matching.) Chapter II. The Donor Physical Standards It is the general practice to accept as blood donors both men and women who: 1. Assert they are in good health. 2. Are between the ages of 18 and 60. 3. Give no history of: a. Recent asthma. (It is apparently not dangerous to transfuse the blood of a donor having pollen sensitivity outside of the pollen season.) b. Repeated attacks of angioneurotic edema. c. Malaria, (Need not disqualify for preparation of plasma which will be frozen or dried.) d. Recent chancre or positive serologic tests for syphilis. e. Jaundice, occurring within the previous 6 months. (This pre- caution is thought to eliminate the possibility of the transmis- sion of infectious jaundice by transfusion.) 4. Do not show the following physical or laboratory findings: a. Evidence of chancre or positive serologic tests for syphilis. (Inspection of the genitalia should be compulsory in male donors.) b. Elevation of temperature over 99.5° F. by mouth. c. Systolic blood pressure of over 200 mm. Hg. or diastolic of over 120. (If a vascular accident occurred following blood donation, it might be ascribed to the donation of blood.) d. Systolic blood pressure of less than 100 mm. Hg. (Donor reactions very common in this group.) e. Hemoglobin less than 80 percent of normal. THE DONOR 5 Discussion.—In ascertaining that the donor’s health is good, it is advisable to inquire into the presence of a cold or sore throat, any recent illness, any chronic illness (particularly heart disease, pulmo- nary tuberculosis, or diabetes), and symptoms such as shortness of breath, persistent cough, chest pain, etc. A positive answer to one or more of these questions would necessitate further investigation and perhaps consultation with the prospective donor’s physician before acceptance of the person. A history of pulmonary tuberculosis or the existence of diabetes should disqualify a donor unless he presents a written statement from his physician that he may donate his blood. It has been noted, also, that donors with a history of frequent fainting or convulsions are very prone to develop untoward reactions when they give blood. Legally, persons under 21 years of age should be required to present written parental permission before being accepted as blood donors. Physiology of Controlled Hemorrhage Recent physiologic studies (21, 45) have thrown much light on the readjustments which take place in the body after controlled hemor- rhage, such as the donor undergoes. After the withdrawal of from 500 to 1,200 cc. of blood, the pulse rate may either remain normal or be increased, or bradycardia may actually develop. The blood pressure frequently falls to levels of 80 or 90 mm. systolic. Some persons show an exaggerated response to changes in posture, the systolic pressure falling instead of rising when the erect position is resumed. This con- dition may obtain for hours and probably accounts for some of the syncopal attacks which are sometimes encountered when the donor gets off the bleeding table. In bleedings of from 500 to 1,200 cc. it takes between 48 and 72 hours for the blood volume to be restored to nor- mal. During this interval there is a steady increase in the fluid content of the blood, but for the first 2-hour period the added fluid is extremely poor in protein. Thereafter, the fluid has a protein con- centration similar to that of normal plasma. In one study (23) it took about 50 days for males to regenerate the hemoglobin lost in donating blood for one transfusion; females took slightly longer. This interval was shortened to 35 days by the daily administration of 1 gram of iron and ammonium citrate. It is a safe rule that not more than 500 cc. of hlood he withdrawn every 90 days from donors who are not carefully supervised. 6 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE Chapter III. The Recipient Indications for Transfusion The principal functions of whole blood and plasma transfusions may be classified as shown: Table 3 Indication Whole blood Plasma or serum Choice State (fresh or pre- served) Choice State (fresh liquid, stored liquid, frozen, dried) Shock due to hemorrhage (traumatic shock). First' No preference Second No preference. Shock with hemoconcen- tration (burns, crush syn- drome). Second.. No preference First No preference. Hypoproteinemia Second No preference First No preference. Acute and chronic anemias Imperative No preference . Not indicated. CO poisoning and methe- moglobinemia. Imperative— No preference. - Not indicated. Immune therapy Second No preference First Fresh liquid, frozen or dried. Deficiency of complement.. Either Fresh Either Fresh liquid, frozen or dried. Deficiency of prothrombin. Either Fresh. Either Fresh liquid, frozen or dried. Leukopenia and thrombo- cytopenia. Imperative Fresh Not indicated. Second Fresh liquid, frozen or dried. i The recommendation of first and second choice is made on the assumption that both blood and plasma are immediately available Contra-indications There are few contra-indications for blood or plasma transfusion, but these should be observed with extreme care. The presence of edema of the lungs due to cardiac decompensation is almost always a contra-indication. It has been shown that failure of the left side of the heart may be produced by the intravenous injection of as little as 200 cc. of saline in an individual with borderline compensation (38). In extreme cases edema of the lungs may be produced by the rapid injection of as little as 50 cc. of blood. THE RECIPIENT 7 Survival of Transfused Erythrocytes Of prime interest in blood transfusion is the time of survival of the transfused erythrocytes. The data for fresh blood have shown considerable variation due to the different methods of storage em- ployed. In general, comparisons between fresh and preserved blood are more informative because the same methods have been used throughout. By means of the differential agglutination method of Ashby, it has been shown (7) that blood stored in sodium citrate for 8 days survives in the recipient approximately one-half as long as fresh blood. Blood stored in dextrose-citrate solutions survives for much longer periods (7, 33, 34). When the concentration of dextrose is high, as in the Rous-Turner mixture, 42 percent of the cells stored for 28 days were present in the circulation 1 month after transfusion (35). The survival period of blood stored in dextrose-citrate solutions has re- cently been confirmed by Denstedt (56). Clinical experience with dextrose preserved blood in a large number of hospitals has been entirely satisfactory. Chapter IV. Transfusion Reactions It is of the utmost importance to attempt the differentiation of the various types of transfusion reactions, since the etiology of each type is different. It is no more diagnostic to state unqualifiedly that “the patient had a transfusion reaction” than to state that “the patient had a fever.” Transfusion reactions are frequently difficult to differentiate from the manifestations of the underlying disease. The following categories include the great majority of transfusion reactions. Pyrogenic Reactions Clinical description.—While the transfusion is being received or soon thereafter, the patient may experience a chill. This is usually followed by a rise in temperature to 101° or 102° F. The fever persists for 5 or 6 hours and subsides without treatment. Occasionally there is a definite chill with no elevation of temperature, or there may be no chill preceding the fever. Etiology.—Certain water-borne bacteria may grow in distilled water and give off soluble ultrafilterable products which are not in- activated by temperatures usually used in sterilization. When these substances, which are called pyrogens, are injected intravenously they produce the clinical syndrome described above. Lack of care in the preparation of blood transfusion equipment and of the fluids employed 8 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE is undoubtedly responsible for most of the pyrogenic reactions accompanying transfusion. Prophylaxis.—Proper preparation of transfusion equipment and fluids for intravenous therapy; avoid contamination. Treatment.—Mild reactions require no treatment. If reactions are severe, the transfusion should be discontinued. Different diagnosis.—Onset with severe chills and fever with some dyspnea suggest the possibility of the much graver hemolytic type of reaction. The transfusion should be promptly discontinued, and the typing and cross-matching should be rechecked at once (see “Hemo- lytic Reactions”). Urticarial Reactions Clinical description.—At any time during the course of the trans- fusion or immediately afterward the patient may develop an urticarial eruption which may consist of only a few “hives,” or may become gen- eralized. In some individuals the process takes the form of angio- neurotic edema, in which one part of the body becomes massively edematous. The periorbital tissue and the lips are most commonly involved in this process. The eruption usually subsides in from 6 to 24 hours. There is some danger that in the severe cases edema of the larynx may develop. Etiology.—Little is known about the cause of many of these reac- tions, It has been observed that the blood from an individual with repeated attacks of angioneurotic edema has produced similar manifes- tations in recipients. On the other hand, in many instances neither donor nor recipient gives a history of allergic manifestations. A recipient may react to a certain blood with the first injection and not with subsequent injections. The same blood may produce urticaria in one individual and not in another. Prophylaxis.—A small proportion of reactions may be prevented by disqualifying donors who are subject to angioneurotic edema. The use of blood from fasting donors will tend to diminish this type of reaction. Treatment.—With minor degrees of eruption, the transfusion may be continued, but severe manifestations require discontinuance. The drug of choice is epinephrin hydrochloride, 0.5 cc. (1-1000 solution) subcutaneously. Differential diagnosis.—This offers no difficulty. Hemolytic Reactions Clinical description.—A distinction must be made between the symptoms following the intravenous injection of free hemoglobin and the symptoms occurring after intravascular hemolysis. This prob- ably is largely a matter of the amount of hemoglobin involved, since TRANSFUSION REACTIONS 9 it is possible to release much greater amounts of free hemoglobin by the rupture of cells already in the circulation than can be introduced through a needle in unit time. The intravenous injection of small amounts of hemoglobin causes no symptoms, but when large amounts are given, the patient complains of a feeling of constriction behind the sternum, chilliness, fever and pain in the lumbar region (24). The symptoms are similar when slight degrees of hemolysis have occurred mtravascularly. When red cell destruction has been more extensive, the patient may go into shock, with cold, clammy skin, lowered arte- rial tension, and air hunger. Death may occur at this stage if proper treatment for shock is not immediately instituted. If shock does not appear, or is successfully overcome, another manifestation to be expected is jaundice. This has been observed within six hours after an accident. Blood bilirubin values of from 10 to 20 mg. percent are not uncommon. The blood serum may be col- ored red by free hemoglobin for only a few hours and turn yellow as the hemoglobin is converted into bilirubin. The urine may contain sufficient hemoglobin to appear bright red. On standing it becomes brown. The hemoglobinuria may disappear within 48 hours and re- covery be uneventful. The jaundice fades within a few days. In the exceptional case, however, oliguria or anuria develops, usually during the first 24 hours. This results in progressive azotemia. The low- ering of the alkali reserve due to the development of acidosis is a characteristic feature. Coma supervenes during the last few days of life. Death from uremia usually results on the fourth to nineteenth day after transfusion unless spontaneous diuresis occurs, in which case complete recovery is the rule. Hypertension may develop. Patients dying of hemolytic transfusion reaction show a character- istic pathologic picture. There are no gross lesions except some edema of the parenchyma of the kidneys. Microscopically there is inter- stitial edema of the kidneys with varying degrees of tubular necrosis. Tubular regeneration may be evidenced by an increased number of mitotic figures. The glomerular tufts are normal, and the lumina of the proximal convoluted tubules are usually dilated. The distal convoluted tubules may contain pus cells, cellular debris, and a few casts of yellowish brown pigment, which is thought to be hemoglobin. The interstitial tissue is frequently infiltrated with polymorphonu- clear leukocytes. In some cases there are areas of focal necrosis in the liver (9,10,11,12, 26). Etiology.—The most common cause of intravascular hemolysis is the transfusion of incompatible blood. Experience indicates that the transfusion of 75 to 100 cc. of such blood is necessary to cause a severe reaction. Other causes of hemolysis should also be considered. The blood may have been hemolyzed before injection into the body as a 10 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE result of improper storage, freezing, application of excessive heat, or the addition of distilled water or other hemolytic agents. Intravascu- lar hemolysis may occur spontaneously in malaria, paroxysmal hemo- globinuria, and idiosyncrasy to quinine and sulfonamides. There is a renal threshold for hemoglobin (24, 39). In normal kid- neys hemoglobin appears in the urine when the plasma level reaches about 135 mg. per 100 cc. The threshold is lower in damaged kidneys. Empirically, it has proved a safe rule that injections of free hemo- globin insufficient to exceed the renal threshold are innocuous. Prophylaxis.—The prevention of hemolysis lies in an understand- ing and avoidance of the factors causing it, as outlined under etiol- ogy, and in the care with which the recipient is transfused. The results of some animal studies (10, 11) suggest that routine alkalinization of the urine before transfusion might prevent transfusion anuria. This, however, is not at all certain. Treatment.—If the patient with a hemolytic reaction develops neither shock nor anuria, recovery is spontaneous. If shock develops, prompt transfusion with plasma may be indicated. The immediate administration of alkalis is indicated to forestall or minimize the precipitation of hematin in the renal tubules. If, however, renal insufficiency develops, every effort should be made to reestablish an adequate urinary output. Differential diagnosis.—If the patient is seen within a few hours after receiving the transfusion, a specimen of his blood should be im- mediately withdrawn in dry, clean apparatus and centrifuged. The presence of hemoglobin in the supernatant fluid will indicate hemoly- sis. In the examination of the urine it is important to differentiate between hematuria and hemoglobinuria. Hematuria is not evidence of a blood transfusion reaction. Cardiovascular Reactions Clinical description.—This complication usually occurs in patients suffering from chronic cardiac disease. During or immediately after transfusion, the recipient may become extremely dyspneic and cyan- otic. Fine crackling and coarse rales may be heard in the lungs. The patient may recover spontaneously or die within a few hours. At autopsy the lungs are edematous, and there may be evidence of cardiac dilatation. Etiology.—It is now recognized that even small increases in the blood volume of patients with borderline cardiac compensation may result in left ventricular failure. Frank cardiac decompensation is usually easily recognized, and such patients should almost never be trans- fused. It is in the unrecognized borderline cases that transfusions are most likely to produce circulatory embarrassment. Death has been reported (12) from the injection of 200 cc. of blood into the adult TRANSFUSION REACTIONS 11 recipient. However, it should be borne in mind that cardiac patients who have been severely injured or burned may require transfusion in order to restore blood volume. In such cases, extreme caution is in- dicated and frequent examination of the patient during transfusion to detect overloading of the circulation is imperative. Prophylaxis.—This depends (a) on the clinical acumen used in the diagnosis of the underlying disease and in the assessment of the patient’s cardiovascular status and (b) on the rate of administration of the transfusion. Treatment.—As soon as the condition is recognized, transfusion should be discontinued and phlebotomy should be performed. Tour- niquets may be placed on all four extremities with sufficient pressure to cause venous stasis, but not for longer than 15 minutes. Differential diagnosis.—This offers no difficulty if the possible de- velopment of cardiac failure be kept in mind. Retinal Hemorrhages Retinal hemorrhages have been observed to appear during or shortly after transfusions. They are seen most often in patients with blood dyscrasias or capillary damage. Infections Transmissible by Transfusion Malaria and syphilis have many times been transmitted by the trans- fusion of whole blood. A negative serologic reaction in the donor does not necessarily insure against transfusion syphilis. Several cases are on record in which syphilis was transmitted by the blood of a donor who had primary syphilis but whose serologic reaction had not yet become positive. However, recent work (44) has shown that the Treponema pallidum will not survive over 96 hours in preserved blood. Utilization of blood more than 96 hours old should therefore eliminate transfusion syphilis. The viability of the malarial parasite in preserved whole blood (25) is somewhat longer, but the figures of different observers vary considerably. It has recently been established that infectious jaundice has been transmitted by transfusion of both blood and plasma. Toxicity of Potassium When the fact became known that potassium diffused into the plasma from the erythrocytes during storage, it was feared that the high potas- sium content might prove toxic in tranfusion (40). No evidence of such poisoning by potassium has ever been observed (16). T rans fusion of Cold Solutions It was formerly considered necessary to warm blood or other fluids before intravenous administration. This practice not only resulted 562943°—44 2 12 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE in the loss of valuable time and required the use of special equipment, but it was potentially dangerous in the case of blood transfusion. At least one fatality has been reported (5) from the injudicious heating of blood during a transfusion. It has been shown that fluids may be injected intravenously at relatively low temperatures (15° C.) with- out reaction (IT). This means that blood or plasma may be taken from the refrigerator at 2° to 5° C. and used at once, since a tem- perature of about 15° C. (60° F.) will be attained by the time the solution reaches the recipient’s vein after passing through the intra- venous set. This fact is exceedingly useful in the transfusion of preserved blood or plasma in emergencies, since it allows all possible haste. Incidence of T runs fusion Reactions It is apparently true that within the safe period of storage for any preservative mixture described in this manual the incidence of reactions can be held to a low level. If that period of storage is exceeded, spontaneous hemolysis becomes excessive, and the reaction rate will increase. The incidence of reactions seems quite comparable in clinics where proper precautions are taken. Diggs and Keith (51) reported an incidence of 6.7 percent for stored citrated blood. Rosen- thal et al. (53) had an incidence of 13.4 percent for citrated blood, fresh and stored. If transfusions of blood stored for over 10 days are excluded, their rate wTas 7 to 8 percent. Muether and Andrews, (52) using a dextrose-citrate mixture, had an incidence of 5.3 percent for bloods stored up to 90 days, mostly under 30 days. DeGowin and Hardin (20), in a series of 2.423 transfusions of citrated blood (10-day limit) and dextrose-citrate blood (30-day limit), could find no signif- icant difference between reactions in the two preservative mixtures, either fresh or stored. The incidence of reactions in the entire series was 4.8 percent. Alsever (54) in a series of 1,500 transfusions of fresh and stored dextrose-citrate blood (21-day limit) had a reaction rate of approximately 5 percent. No difference was observed in the use of fresh and stored blood. Chapter V. Changes in Blood During Storage Red Cells Spontaneous hemolysis begins in minute degree as soon as blood is collected. When it is stored at 2° to 5° C. hemolysis proceeds at rates depending upon the preservative mixture employed (13, 14, 36, 37). CHANGES IN BLOOD DURING STORAGE 13 If citrated blood be taken as a standard of reference, the addition of electrolytes, such as sodium chloride, accelerates hemolysis. The addi- tion of dextrose definitely inhibits the rate of destruction of the red cells. The mode of action of the dextrose is not known, but it has been demonstrated that to secure optimum effect, a concentration of about 3 percent dextrose must be attained in the blood mixture. With such conditions, there is less than one-half as much hemolysis in 30 days of storage as occurs in citrated blood at 10 days. Isotonic concentra- tions of solutions should be used in the preservative mixtures. When appreciably hypertonic solutions are employed, the contents of the erythrocytes become hypertonic during storage so that they may be ruptured when they come in contact with the plasma of the recipient. There is slightly less spontaneous hemolysis when blood is stored in a container from which all the air is displaced by the blood mixture. It has been repeatedly demonstrated (20) that human blood with- stands very well the agitation incident to transportation over great distances if the containers are full. Red cells stored in dextrose- citrate mixtures persist longer in the circulation of the recipient than do erythrocytes stored in citrate alone (35). Other Constituents The leukocytes lose viability rapidly during storage, very few sur- viving the fourth day (8). This seems to be independent of the preservative employed. The platelets are even more evanescent (6), disintegrating within a few hours. The plasma proteins are relatively stable (41) and are not denatured to any significant extent. The prothrombin of the plasma disin- tegrates slowly during storage at refrigerator temperatures, being about 70 percent of normal at 21 days and about 30 percent in 30 days (32, 46, 50). It should be emphasized, however, that the prothrombin is only one factor in the clotting mechanism and that blood 1 month old will clot promptly when recalcified. Normally, human red cells contain about 20 times as much potassium as does plasma, while the latter contains sodium to the exclusion of the cells. During storage the red cells lose much of their potassium to the plasma, and sodium diffuses into the cells. This process attains its maximum in about 15 days (15), producing a plasma with a rela- tively high concentration of potassium. Some study of the rate of loss of complement and antibacterial sub- stances has been made, and the reader is referred to the original articles for details (28, 29). B. TECHNIQUE Chapter VI. Operation of a Blood Bank Advantages of a Blood Bank The advantages of a blood bank are: 1. The instant availability of blood or plasma in emergencies. 2. The privilege extended to donors of trading their blood for that of the proper type in the bank. 3. Economy to hospital and patients. Each bank may be modified to suit local conditions, since an organi- zation which is admirable for one set of circumstances would not fit another. An attempt will be made to outline the principal variations and to indicate some of the advantages of each. Types of Blood Banks Complete hlood and plasma hank.—This type of organization is suitable for a hospital having over 25 to 30 transfusions per week. Donors of all types are accepted as the laws of chance present them. The bloods are kept as whole blood until the limit of storage is at- tained (determined by the preservative mixture used). If the whole blood reaches the outdating period without being called for, the super- natant plasma is then recovered by aspiration from the cell layer, and the red cells are discarded. This citrated plasma should preferably be stored at room temperature in the liquid state or at minus 15° to 20° C. in the frozen state. Proof of sterility is required before it may be administered. If the transfusion service is large, the turn-over of bloods will be relatively rapid, and there will be few which are ulti- mately converted into plasma, unless an excess is collected. Where the service is small, the amount of plasma accumulated will be cor- respondingly increased. Blood hanks with limited hlood types.—It is the usual experience that most of the out-dated bloods in a bank belong to the rarer types B (14 percent of population) and AB (4 percent). Since the incidence of group A in the population is about 38 percent and that of group O is 44 percent, a blood bank accepting only those two groups could use 82 percent of the donors presenting themselves. Group O bloods could be given to the recipients of all groups, and group A bloods could be given to groups A and AB. Such an arrangement would merely re- quire the determination of the donor’s group before the blood is drawn. This type of a bank wdll operate successfully for hospitals with smaller transfusion requirements than 25 to 30 per week. Bank employing group 0 hlood only.—A satisfactory blood bank may be operated for small hospitals by the use of only group O blood. OPERATION OF A BLOOD BANK 15 If these are treated with the group-specific A and B substances (see page 3), they become truly “universal donor” bloods and can be used more safely than untreated group O blood, at least theoretically. This is probably the most satisfactory type of blood bank for the very small hospital. Plasma hank.—When no attempt is made to store and use whole blood because the transfusion service is too small to make it feasible, blood may be collected at irregular intervals, regardless of type, and the plasma separated from the cells either by sedimentation or centrifuga- tion. The plasma is kept primarily for the emergency treatment of traumatic shock, the treatment of the dehydration and shock accom- panying severe burns, and the treatment of other conditions as pre- viously discussed. In the treatment of shock due to hemorrhage, the plasma is usually employed as an emergency measure until a suitable blood donor can be found. There is no limit on the smallness of the plasma bank, but the overhead cost increases and could become pro- hibitive for each unit of plasma processed. Methods of Accounting Several methods have been devised by which the accounts of the bank may be kept. In a large hospital where the medical specialties are organized into services, it may be convenient to carry an account in the bank for each department. When a donor is sent in from a particular service, the account carries a credit of blood in cubic centi- meters. When a patient on that service receives a transfusion from the bank, the account is debited by the amount of blood given. This system possesses the convenience of placing responsibility on one house officer on each service, wTho in turn guards the interest of his service by supervising the interns in the procurement of donors. Although donations are requested on the basis of transfusions given to patients in whom the donors are interested, a surplus of blood frequently accumulates in the bank. With service accounts, the occasional pa- tients requiring transfusion but unable to procure donors may be served from the departmental surplus. This is extremely hard to manage under any other type of accounting system. In a smaller hospital it may be desirable to have each intern carry an account in the bank. When he procures a donor, he receives credit for the volume of blood, which he may then transfuse into one of his patients. Or the individual physician or the individual patient may have to be credited and debited with blood as the local conditions suggest. Many variations of the foregoing scheme are possible. Acquisition of “Capital” After the blood bank has been established, the bloods are collected and dispensed on some definite trading arrangement, but it is necessary 16 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE to have a stock of bloods with which to initiate operations. The most practical method for a community hospital is for the members of service clubs, lodges, or churches to contribute blood with which to begin the bank. The practice has been widely adopted and has proved popular. It can be explained that it is a community enterprise and that the mere existence of such a bank is a form of protection for the members of the community. Personnel It is essential that the blood bank be under the direction of a labora- tory worker who is thoroughly familiar with all the procedures in the typing, and cross-matching of blood and is capable of carrying out aseptic technique in the collection of blood and in the processing of plasma. The amount of the technician’s time consumed by the opera- tion of a blood and plasma bank will, of course, depend upon the magnitude of operations. It is excellent policy to restrict to a mini- mum the number of persons responsible for handling the blood. The cross-matching and typing should be done only by experienced labora- tory technicians. Too frequently, these important procedures have not been well handled when made the responsibility of interns. Source of Pyrogen-Free Fluids Before attempting to establish a blood or plasma bank, one should be satisfied that there is a source of pyrogen-free fluids available in the hospital with which to prepare equipment for intravenous use. The lack of this precaution will tend to discredit operation of the entire bank, since it is the common inclination to ascribe pyrogenic reactions to the blood and plasma rather than to improperly pre- pared equipment. Most small hospitals very properly employ com- mercially prepared parenteral fluids which are pyrogen-free. The commercial firms unfortunately cannot control the preparation of the equipment used to give their solutions, and this is frequently the source of pyrogenic reactions. A thorough understanding of the prep- aration of the equipment and centralization of its preparation in one place is the sine qua non of a proper transfusion service. The reader is referred to pages 69-72 of the manual for a description of the proper methods to insure maximum freedom from pyrogenic reactions. Equipment The most expensive item required for the operation of the blood bank is a mechanical refrigerator. For small banks a standard do- mestic model electric or gas refrigerator is satisfactory. Most models have thermostats which are adequate to control the temperature be- tween 2° and 5° C. A continuously recording thermometer is advis- able. If a stock of whole blood is to be kept on hand, consideration should be given to substitute arrangements while the refrigerator is TYPING AND CROSS-MATCHING 17 being periodically defrosted. (For detailed description of refrigera- tion see p. 76.) A centrifuge is necessary for the cross-matching and typing of blood; this may be a relatively small, electrically driven, angle-type machine. If it is desired to separate plasma from erythro- cytes by centrifugation in the processing of the plasma, a larger, more expensive type of centrifuge with the proper accessories is required. A laboratory microscope will be used in the compatibility tests. Microscope slides, serologic type of test tubes, medicine droppers, and other laboratory accessories will be required. Facilities for steam and dry-heat sterilization will, of course, be necessary. There is a great variety of transfusion apparatus available, and standard acceptable equipment should be employed. The advantages are great of equip- ment designed to collect blood and process plasma in a completely “closed system” of the type provided by commercially available vac- uum bottles. Completely closed systems are difficult to improvise from ordinary laboratory equipment. If the plasma is to be kept in the frozen state, a small commercial freezing unit maintaining tem- peratures below minus 15° C. will be required. These are ordinarily employed for the storage of ice cream in retail establishments. It is desirable to have a suite of at least two adjoining rooms in which to conduct the procedures of the blood bank. One room should be reserved for the collection of blood from donors. This can be divided into several cubicles, each containing an examining table and a small side table. The examining tables should be fitted with arm rests. The side tables should be supplied with iodine, acetone-alcohol mixture, sterile gauze, adhesive tape, and tourniquets. A sphyg- momanometer makes an excellent tourniquet. The other room should be fitted for laboratory procedures and the keeping of records; the refrigerator may be conveniently placed there. If the donors must wait their turns, it is desirable to have a waiting room from which the operations of the blood bank cannot be observed. Chapter VII. Typing and Cross-Matching of Blood There is no laboratory jwocedure in which the results of erroneous technique or interpretation are more disastrous than in the typing and cross-matching of hlood. The direct result of a mistake may be fatal. For this reason it is extremely desirable that these procedures be in the hands of an experienced individual. Most physicians have not troubled to obtain the information nor the experience needed to conduct these tests properly. The printed directions for carrying out these procedures are deceptively simple and give a false sense of security. 18 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE Determination of Blood Groups 1. Equipment.—The only reagents required for blood grouping are specific and potent agglutinating sera. With satisfactory sera, fairly complete agglutination should be visible to the naked eye within 15 to 20 seconds. The activity of grouping sera should be checked at weekly intervals against known A and B cells in order to avoid the use of deteriorated sera which may become too weak to group all bloods properly. Equipment required: a. Glass slides. b. Wooden applicators or tooth picks. c. Wax pencils. d. Capillary pipettes fitted with rubber bulbs or hypodermic syringes fitted with 24-gage needles. e. Small test tubes, such as 8 by 75 mm. f. Four percent sodium citrate (dihydric). g. Physiological saline solution (0.85 percent sodium chloride). h. Microscope. i. Centrifuge. 2. Choice of methods.—Blood grouping and cross-matching can be carried out by a slide method or a centrifuge test tube method. It is desirable when possible to use the centrifuge test tube method. The slide method is described in detail for blood groupings, the centrifuge method for cross-matching, in order to present both in a minimum of space. 8. The slide method.—The test is made as follows: a. Divide a slide equally with a wax pencil, b. Place the subject’s initials or number in the lower right-hand corner of the slide, the letter “A” in the upper left, and the letter “B” in the upper right-hand corner. c. The blood should be collected preferably by venipuncture (5 cc.). If this is not feasible, 0.5 to 1.0 cc. may be obtained by deep puncture of a finger or ear-lobe, after cleansing the site with alcohol and allow- ing it to dry. The blood is placed in tubes containing citrate solution in approximately the proportion y5 to y10 of the volume of blood drawn. d. A red cell suspension is prepared by mixing one drop of the citrated blood with about 1 cc. of saline solution. With normal blood this gives a cell suspension of approximately 2 percent. In the case of anemic patients, more blood should be added, to make a suspension matching in color that of the donor’s. Mark the tubes containing the recipient’s blood RC (recipient’s cells), and that containing the donor’s blood DC. Save the remainder of the blood collected for cross-matching. . TYPING AND CROSS-MATCHING 19 e. Place one drop of cell suspension on each half of the marked glass slide. f. Place a drop of A (anti-B) serum on the left side of the slide and drop of B (anti-A) serum on the right side of the slide. g. Mix well with a wooden applicator or toothpick (separate end for each side), rock the slide manually 5 to 10 seconds to insure thorough mixing, then allow to stand for 5 to 10 minutes, tilting a few times, about once every minute. h. In warm climates where the slide preparation is apt to dry up, it should be kept in a moist chamber during the period of observation. (A moist chamber can be made by placing pledgets of wet cotton under petri dishes or glass trays. If the use of a moist chamber is imprac- ticable, the addition of a drop of saline solution to each side of the slide after about 5 to 10 minutes’ standing will usually prevent drying.) i. The reactions are read with the naked eye and under the low power of the microscope, if one is available. In a positive reaction the cells are stuck together in clumps usually visible to the naked eye. The interpretation of the grouping tests is shown below: GROUP 0 GROUP A GROUP B GROUP AB Figure 1.—Blood grouping. Fine dots represent no clumping (negative re- action). Massed dots represent agglutination (positive reaction). (Aftei Army Technical Manual.) 20 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE 4. Centrifuge method.—a. Prepare two small test tubes, one marked with the letter “A” and the subject’s initials, the other with the letter “B” and the subject’s initials. b. Blood is collected and the red cell suspension prepared as pre- viously described. c. Place one drop of cell suspension in each tube. d. Place a drop of A (anti-B) serum in the tube marked “A,” and a drop of B (anti-A) serum in the tube marked “B.” e. The tubes are now centrifuged and the reactions read as described in paragraph c, under centrifuge method, page 22. 5, Confirmation of grouping by the testing of plasma.—When time permits, and preferably as a routine, the following confirmatory test on the plasma of the individual being grouped should be carried out. This is essential for the certification of universal donors. a. Divide a slide in halves with a wax pencil and mark the left side “AC” (group A cells) and the right side “BC” (group B cells). b. On the left half of the slide place a drop of fresh known group A cell suspension; on the right half place a drop of known group B cell suspension. c. Add two drops of the subject’s plasma to each side of the slide and mix each by stirring with a separate applicator or toothpick. d. Observe the slide for at least 20 minutes, tilting it back and forth at 2- or 3-minute intervals, and then examine for agglutination. e. This test may also be done by the centrifuge method. f. If it is difficult to distinguish between true agglutination and rou- leaux formation, stir again with an applicator. This will usually break up rouleaux into a uniform suspension. g. The scheme of identification of blood groups from the reaction of unknown plasma and known cells is given in the right half of table 4, Table 4 Identification of blood groups Grouping of un- Grouping of un- known cells with known plasma known sera Agglutinogens in cells with known cells Agglutinins in plasma Group A B A B + + o + A + A + B B + + AB TYPING AND CROSS-MATCHING 21 6. Mass grouping.—When large numbers of individuals are to be grouped in a relatively short space of time (500 or more per day), certain modifications of the technique just described are necessary. a. For the sake of expediency the test should be done on glass slides. b. The slides should have the left and right halves marked “A” and “Bv in advance, as indicated in the description of the slide method. c. A team of three persons should work simultaneously at a table. The personnel to be grouped file past one by one. Accurate blood grouping can be done at the rate of 60 to 90 per hour. The three members of the grouping team may be designated as X, Y, and Z. d. Member X cleanses and punctures the finger as described. He places a small drop of whole blood, the size of a pinhead, on each side of one of the slides by touching the slide to the drop as it forms. The use of too large a drop may obscure and delay the agglutination reaction. He numbers the slides serially with a wax pencil. To the left side he adds one drop of A serum (anti-B), and to the right side, one drop of B serum (anti-A). He mixes each with a toothpick or applicator, rocks the slide for 10 to 20 seconds, and makes a prelimi- nary reading which is recorded by Z. e. The individual being grouped has in the meantime passed to member Y, who independently repeats the test, but uses grouping sera from different bottles. He also makes a preliminary reading, which is likewise recorded by Z. f. Both X and Y pass their slides to Z, who rocks them a few times, about once every 5 minutes, and retains each slide until 30 minutes have elapsed. This is important in order to insure that weak subgroups of A, particularly of AB, do not escape detection. g. In case of discrepancy between the results of the tests carried out by X and Y, or between the readings and the group determined for the individual previously, if any, he should be recalled for re- grouping later, when enough blood will be taken for plasma. Then both cells and plasma should be tested. h. Member Z discards the old slides for washing after the lapse of 30 minutes, at the same rate that new ones accumulate. He keeps records in a book ruled with seven columns: individual’s name and number, previous grouping (if any), preliminary reading by mem- ber X, final reading of X’s slide by Z, preliminary reading by Y, final reading of Y’s slide by Z, and final grouping. Note.—The method described above is reliable only if (1) the sera are of high potency, (2) the size of the drop of blood is small, about the size of a pinhead, and (3) the grouping sera are added before the blood has a chance to dry. 22 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE C ross-Matching After a donor belonging to the same blood group as the patient has oeen selected, the cross-matching test must be performed before the transfusion is given. In rare instances the bloods of donor and recipient, even though of the same group, are not compatible; that is, there will be some agglutination or hemolysis of the donor’s cells by the patient’s serum or plasma or of the patient’s cells by the donor’s serum or plasma. (See pp. 2T-25.) 1. The centrifuge method.—The test is performed as follows: a. For the test, use the citrated blood samples obtained from the donor and recipient. b. Separate the plasma and cells of both donor and recipient by cen- trifugation or sedimentation. c. Prepare two small test tubes, bearing the subject’s initials, one marked “DP/RC” and the other “RP/DC.” In the first, place with a capillary pipette, or syringe and needle, one drop of the donor’s plasma (DP) and one drop of the recipient’s cell suspension (RC), using a different pipette (or syringe) for each reagent. (If only a single pipette is available, it should be rinsed twice with saline before taking up another reagent.) In the other tube place one drop of the recip- ient’s plasma (RP) and one drop of the donor’s cell suspension (DC). Mix, centrifuge at low speed for exactly 1 minute, observe for hemol- ysis, and then resuspend by gentle shaking. Even if the cells appear to resuspend to an even suspension, examine a drop on a slide under the low power of the microscope for agglutination. Agglutination or appreciable hemolysis should disqualify the donor. 2. The slide method.—If no centrifuge is available, the cross-match- ing may be done on a slide by the following alternative procedure: a. Divide a clean slide as for the standard grouping test and mark the left side “DP/RC” and the right side “RP/DC,” plus the subject’s initials or number. b. Place one drop of donor’s plasma (DP) on the left side and one drop of recipient’s plasma (RP) on the right, using different capillary pipettes (or syringes and needles) for each transfer. c. Mix one drop of recipient’s cells (RC) with the donor’s plasma (DP) and one drop of donor’s cells (DC) with the recipient’s plasma (RP), using different capillary pipettes for each transfer. d. The remainder of the test is done in the same manner as for the standard grouping, except that it is necessary to observe the tests for a longer time. Any agglutination or appreciable hemolysis evident within 20 to 30 minutes should disqualify the donor, and others should be tried until one is found whose blood gives no trace of agglutination or hemolysis. This is especially true when there is any agglutination of the donor’s cells by the recipient’s plasma. Since the slide cross- TYPING AND CROSS-MATCHING 23 matching test requires long observations, precautions to avoid drying should be observed. Rh Typing and Cross-matching The indications for testing to rule out Eh incompatibility are given in italics on page 8. It is preferable to eliminate this danger by typing so that only Eh negative blood is used. If no typing serum is available, Eh incompatibility can be demonstrated by special cross- matching. 1. Typing.—The tests are set up in small test tubes, following essen- tially the procedure described for grouping by the test tube method. Small narrow Kahn tubes of inside diameter 7 to 8 mm. are satisfactory. a. One drop of a fresh 2 percent blood suspension in saline is mixed with one drop of Eh testing serum in a small test tube which is placed in a water bath or air incubator at 37° C, for 1 hour. b. The reaction is read by gross inspection of the undisturbed sedi- ment in each tube, noting whether the sediment is smooth and compact or rough and diffuse, and by gross and microscopic inspection of the cell suspension after gentle shaking. The tubes are then centrifuged 1 minute at low speed, after which the sediments are again examined in the same way for gross evidence of agglutination, and the results rechecked by microscopic examination of a drop of the gently resus- pended cells on a slide. c. Control tests should be set up, using suspensions of known Eh negative and Eh positive blood cells. In any laboratory where these tests are done, the personnel should be tested in order to have immediately available blood cells of known Eh type for the control tests and for prospective Eh-negative donors. 2. Cross-matching.—a. To demonstrate Eh incompatibility, a cross-match, prepared as directed on page 22, should be incubated at 37° C. for 1 hour. b. Agglutination (EP/DC) indicates Eh incompatibility. c. Controls should be set up as described in 1c above. Selection of Universal Donor 1. Donors belonging to group O are often used as universal donors because their cells are not ordinarily agglutinated by the serum of any of the other three groups. Rarely, however, group O individuals are encountered with such potent isoagglutinins that the dilution of their serum in the patient’s circulation may not suffice to prevent a hemolytic reaction. Preferably, those group O individuals should be used as universal donors whose sera have been shown not to have excessively high titer isoagglutinins by actual titration. In an emergency, any donor certified as belonging to group O, as proved by complete tests 24 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE on cells and plasma, may be used, provided that the blood is transfused slowly. 2. Test to establish acceptability of a “universal donor.” a. Prepare a 1:50 dilution of donor’s plasma by adding 0.1 cc- of plasma to 4.9 cc. of saline solution. b. Place one drop of the diluted plasma on a slide and add one drop of the patient’s cell suspension. Mix well with a wooden applicator or toothpick and observe the mixture for about 10 minutes, tilting the slide about once a minute. c. If no or only weak agglutination occurs within 10 minutes, the titer of incompatible agglutinins is not excessively high, and this donor may therefore be used. If agglutination visible to the naked eye occurs, the donor should not be used. It is known that group O in- dividuals who have previously received transfusions of pooled plasma occasionally develop a dangerously high isoagglutinin concentration. The use of a universal donor does not obviate the need for cross- matching tests, although in an emergency these tests may be omitted if proved group O blood is used. This procedure is more apt to be safe if the donor is known to have weak isoagglutinins in his plasma and is Rh negative. Sources of Error in Typing and Cross-Matching False negative reactions are most often due to the following: 1. Weak titer of sera. 2. Insufficient time of observation of test. 3. Cell suspensions too heavy (the agglutinins may be absorbed by excess of cells). 4. Low sensitivity of agglutinogens. a. In children. b. The A agglutinogen in AB blood. c. The cells of stored blood. 5. The presence of a powerful hemolysin (cell clumps quickly hemolyzed). Fresh plasma may, in rare instances, give hemolysis instead of agglutination, especially in warm climates. Hemolysis is more frequently observed when fresh serum is employed. Care should be taken not to read this as a negative reaction. 6. Errors in labeling. T. Erroneous recombination of cells and serum from the same blood in cross-matching. False 'positive reactions are usually due to the following: 1. Rouleaux formation (pseudoagglutination). This can usually be differentiated by microscopic appearance; in case of doubt, dilute with saline; rouleaux will break up, true agglutination will not. 25 TYPING AND CROSS-MATCHING In many patients with rapid sedimentation rate due to severe sepsis or other causes, rouleaux formation may be confused with true agglutination when the patient’s plasma is grouped with known cells or cross-matched with the donor’s blood cells. Rouleaux formation can often be recognized under the high dry power of the microscope by the appearance of loose clumps of red cells with their flat surfaces in contact so as to resemble stacks of coins. Pseudoagglutination should be suspected whenever unexpected clumping is encountered and is almost certain if the patient’s cells ‘ suspended in his own plasma show a similar phe- nomenon. However, rouleaux formation is usually broken up by stirring, a procedure which as a rule intensifies true agglutination. Pseudoagglutination can be prevented by repeating the test with 1:2 or 1:3 dilution of the plasma, a dilution usually insufficient to weaken true agglutination. 2. “Cold agglutination” due to agglutinins which react only in cold; very few react at room temperature and none at 37° C. 3. Contaminated sera, 4. Panagglutination may occur in certain diseases (test cells against group AB serum). 5. Autoagglutination (test cells against serum of same blood). In most individuals the serum contains a weak agglutinin capable of acting on the individual’s own cells and also on all other human bloods. Normally, auto- agglutination can be observed only at low temperature. In certain diseases, e. g., hemolytic anemias, trypanosomiasis, virus pneumonia, etc., or in the course of sulfonamide therapy, the reaction may also occur at room temperature. Auto- agglutination can be recognized by its reversibility at body temperature and by its nonspeciflcity; i. e., agglutination occurs with every human blood, regardless of the blood group, including the blood of the individual from whom the serum is derived. The phenomenon does not, as a rule, affect the outcome of a blood trans- fusion and is mainly important as a source of error in blood grouping. When the recipient’s plasma agglutinates cells of prospective donors of the same group: 1. Rule out rouleaux formation. 2. Prove autoagglutination by testing the recipient’s plasma against his own cells. 3. Carry out the cross-matching at 37° C., since the phenomenon does not usu- ally occur at body temperature. 4. If agglutination is still present, a new cell suspension should be prepared as follows: Separate the recipient’s plasma (or serum) from the cells at 2 to 5° C., since the autoagglutinins are absorbed by the cells at low temperature. Wash the cells at 37° C. and repeat the cross-matching as in 3. 5. If no agglutination occurs under conditions 3 or 4, it may be presumed that the agglutination phenomenon occurring at lower temperatures was due to autoagglutinins. 6. False agglutination of blood from umbilical cord. Grouping Sera 1. Preparation.—a. General.—Strong B serum is difficult to obtain because of the scarcity of B donors in general. It is wise, therefore, always to have an up-to-date list of donors with a note as to the strength of the reaction in each case. Choose a person of the desired group, known to have a potent serum, and take the blood by venipunc- 26 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE ture. Enough serum can be obtained from 30 to 50 cc. of blood to last over almost any emergency. Serum which must be used immediately or within 1 to 7 days after collection should be inactivated by heating in a water bath at 56° C. for 30 minutes, to avoid hemolysis which may mask agglutination. Reasonable care should be taken to maintain asepsis while drawing the blood and separating the serum, because sterile serum will retain its full strength for a long time. b. Collecting the hlood.—It has been found best to collect blood in sterile stoppered centrifuge tubes or bottles. Allow the blood to clot and then shake the containers gently but sufficiently to break up the clot so that the greatest possible yield of serum can be obtained. c. Separating serum.—Centrifuge the containers of blood at 1,500 to 2,000 r. p. m. to separate the serum from the broken clot, or allow to separate in the refrigerator overnight. Decant or pipette off the clear serum into sterile containers; then recentrifuge, if necessary, to get rid of remaining red cells, and decant into sterile containers. d. Preservation of serum.—When possible, it is desirable to add chemical preservatives to the sera, although in emergencies they may be kept for some time without preservatives, especially if kept cold. The addition of the dyes and the preservative solution to the sera serves both to minimize bacterial growth and to facilitate their identi- fication. If the dyes and mercurial preservatives are not available, the sera may be preserved by adding tricresol to give a final concentra- tion of 0.5 percent. (1) Preserving and coloring group A serum.—(a) Have ready a 1 percent aqueous solution of neutral acriflavin and a 1 percent aqueous solution of phenyl mercuric nitrate, phenyl mercuric borate, or merthiolate. (b) For each cubic centimeter of clear A serum add 0.015 cc. of the acriflavin solution and 0.01 cc. of the preservative solution. (c) Mix thoroughly. Store in 2 or 5 cc. sterile vials sealed wdth rubber stoppers. Keep in refrigerator when not in use. (2) Preserving and coloring group B serum.—(a) Have ready a 1 percent aqueous solution of brilliant green, (b) For each cubic centimeter of clear B serum add 0.01 cc. of the brilliant green solution and 0.01 cc. of the preservative solution. (c) Mix and store as described for the A senim. If long preservation is desired, store the sera in the frozen state in small quantities, e. g., 1 to 5 cc. The frozen sera are then thawed as needed. 2, Criteria for the selection of potent grouping sera.—a. General.— The criteria for selection of potent grouping sera depend upon bio- logical reactions and are consequently subject to considerable variation. TYPING AND CROSS-MATCHING 27 Several methods are employed in various laboratories for selecting grouping sera. All of these methods are subject to: (1) Variations in sensitivity of test cells. (2) Probable variations in properties of the agglutinins in the serum. (3) Protein concentration employed. (4) Intrinsic stability of the preparations. (5) Probably other factors still unknown. b. The following technique for testing is recommended as one satisfactory method for the selection of potent grouping sera. (1) Group A serum \anti-B).— (a) Minimal titer. Prepare a 1:16 dilution of the serum by mixing 0.1 cc. of serum with 1.5 cc. of saline. Mix one drop (0.05 cc.) of the diluted serum oh a slide with a drop (0.05 cc.) of a group B fresh cell suspension, prepared as directed on page 18. If possible, set up a parallel test with a cell suspension from a second individual of group B. Mix with an applicator or toothpick; agitate by rocking the slide to and fro at intervals of 1 minute. The titer of the serum is satisfactory if agglutination readily visible to the naked eye appears in less than 10 minutes with both bloods. (b) Speed and intensity of agglutination (avidity). Set up a test similar to that described in (a), using undiluted serum, and rock the slide continuously. Agglutination must be visible to the naked eye within 15 seconds, and should be complete within 30 seconds. (c) Specificity. It is recommended that the serum be used to test at least 50 bloods taken at random, in parallel with a known serum, with satisfactory results, before being considered acceptable. 2. Group B serum {anti-A).—The tests are performed much as for A, except that account must be taken of weakly reacting A agglutino- gens (A2 and A2B). (a) Minimal titer. Test as described for A serum. The 1:16 dilu- tion should agglutinate A2 cells within 10 minutes. (b) Speed and intensity of reaction (avidity). Test as described for A serum against two suspensions of cells, at least one of them of subgroup A2. Distinct clumping should be visible within 60 seconds with the A2 cells and within 15 seconds with the Ax cells. (c) Specificity. Carry out tests similar to the foregoing with 50 bloods taken at random, and include if possible at least one blood of subgroup A2B. 3. Identification of Ai, A2, and A2B bloods.—The simplest method is to use a commercial absorbed B serum (anti-Ax), if obtainable. This agglutinates bloods of subgroups Aj and AiB, but not those of sub- groups A2 and A2B. The next best procedure is to test a series of known A and AB bloods with one or two weak group B sera. Usually 562943°—44 3 28 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE some of the bloods are definitely more weakly agglutinated than others. These weakly reacting cells are used as A2 (or A2B) in the foregoing tests. 4. Anti-Rh testing sera.—Potent sera may be obtained from the blood of mothers who have borne babies with erythroblastosis fetalis. How- ever, only about 1 in 50 of these mothers yield serum of a titer high enough to be satisfactory for testing purposes. Before distribution for use, the testing serum should have had its anti-A and anti-B ag- glutinins (if present) neutralized by the addition of group-specific substances.1 Chapter VIII. Collection of Blood Psychology of the Inexperienced Donor The inexperienced blood donor must be treated with due regard for his psychological state when he presents himself at the blood bank. The immense amount of publicity released in the last 5 years has already acquainted the average layman with the facts of the four blood groups and the question of incompatibility of bloods in transfusion. He also knows something about the operation of blood banks. If he has never actually given blood, however, he may have certain apprehen- sions which can easily be enhanced to the point where untoward symptoms may develop if he is made to wait too long before bleeding is actually performed, or if he is carelessly allowed a glimpse of a flask of blood or an intimate view of the blood-letting of other donors. Tact in meeting the prospective donor and promptness in dealing with him will prevent considerable distress to him and embarrassing delay or failure for the operator. Donor’s Release It is a moot question whether to require the donor to sign a release stating that he had been fully informed as to the use to be made of his blood and that he accedes to the arrangement. Legal opinion is not agreed as to the value of such a document or as to its necessity. Many hospital administrators prefer not to request a written agree- ment, believing that this only accentuates the importance of the pro- cedure in the eyes of the donor. However, it is the usual practice at present to require a release. 1 Antl-Rh testing serum is now being prepared in most of the large medical centers. Lim- ited supplies are now obtainable from Dr. Louis K. Diamond, Children’s Hospital, Boston, Mass.; Dr. Philip Levine, Newark Beth Israel Hospital, Newark, N. J.; and Dr. Alexander S. Wiener, Jewish Hospital, Brooklyn, N. Y, COLLECTION OF BLOOD 29 A reasonably satisfactory form of release is as follows: “I, the donor described herein, voluntarily donate my blood to Hospital, to be used as determined by said hospital. I am giving this blood at my own risk, and agree that neither the hospital nor any members of its staff shall be in any way responsible to me or my heirs for any consequences resulting to me from this procedure.” Many institutions use only a statement similar to that in first sen- tence suggested here. Selection of Donors Donors must be selected by means of the standards (age, history, physical examination, and laboratory tests) described in part I, chap- ter II, The Donor, page 4. The Use of Fasting Donors The use of fasting donors is not absolutely essential, but is desirable. Blood from nonfasting donors may produce a “milky” or fatty plasma. Such plasma produces no untoward reaction in the recipient except an occasional instance of urticaria. It is recommended that donors have no fatty food for 12 hours prior to bleeding, but they may have any desired quantity of carbohydrate and protein food. A minimum period of 4 hours of fasting should be observed in any event. It is desirable to bleed donors during the forenoon if possible. The breakfast should consist of fruit juices, dry toast, coffee without cream, fruits, etc. Preparation of the Donor 1. Place the donor in the recumbent position while the blood is being collected. 2. Determine the arm vein most suitable for venipuncture. 3. Bare the arm to the shoulder. A blood pressure cuff, folded to one-half its width, is applied to serve as a tourniquet. It is advisable to apply the cuff in the reverse position so that the tubing will be away from the site of venipuncture. 4. Prepare the skin with acetone-alcohol mixture 2 and follow by 7 percent iodine. Remove the iodine with acetone-alcohol mixture. 5. Saturate a sterile gauze pad with the acetone-alcohol mixture and apply it over the area selected for venipuncture. 6. Raise the pressure in the cuff to 40-60 mm. of mercury. 7. Using a small syringe and a 26- or 27-gage needle, raise a small intradermal wheal at the site of venipuncture with a suitable local anesthetic, such as 1 percent solution of procaine. After making the injection, replace the gauze pad saturated with acetone-alcohol mixture * Acetone-alcohol mixture: 10 cc. of acetone and 90 cc. of 70 percent alcohol. 30 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE over the wheal and prepare the blood bottle and donor set. If there is a delay in making the venipuncture, the pressure in the blood pressure cuff should be released, as prolonged pressure of the cuff produces pain and has a poor psychological effect on the donor, thereby increasing the tendency to fainting. (See first paragraph, ch. Ill, p. 47.) Care of the Donor During Bleeding 1. If the donor pales and the flow of blood decreases, give aromatic spirits of ammonia, drams 2 in water, by mouth. 2. Waiting donors should be kept out of the bleeding room until their turn and should be segregated from the donors who have pre- ceded them. The Technique of Collection If a commercial vacuum apparatus is employed, the closure of the evacuated flask containing the anticoagulant is uncovered aseptically, and the needle of the special donor valve is inserted through the closure, the valve being completely closed. A 15-, 16-, or 17-gage needle is then inserted in the donor’s vein; as soon as it is evident that this has been accomplished, the donor valve is opened gradually allowing the vacuum in the flask to give the proper rate of flow of blood. The flask is preferably held in an inverted position and is gently moved so that the contents are set in motion, thoroughly mix- ing the blood with the preservative mixture. In the meantime, the donor has been instructed to open and close his fist slowly and repeat- edly to aid the flow of blood. When sufficient blood has been collected, the donor valve is closed, the tourniquet is released, the needle is with- drawn from the donor’s vein while a gauze sponge is pressed over the site of the puncture, and the donor is requested to open the hand and elevate the arm. A pressure bandage is placed on the antecubital fossa for a few hours. (See also pp. 47-49, 56-58.) The donor valve is now withdrawn from the closure of the flask and the blood in the rubber tubing is utilized as follows: Two or three drops are allowed to flow into a small test tube containing 3 to 5 cc. of the preservative mixture, and the remainder is drained into a serology tube. Before the latter is used for the serologic tests, two or three capillary pipettes (drawn from glass tubing) are filled with serum, sealed, and placed in the tube containing the cell suspension. This is now clearly marked and attached to the flask of blood as the “pilot tube,” to be used later for typing and cross-matching. The flask of blood mixture should be appropriately labeled, thoroughly mixed by repeated inversion of the bottle for 1 minute, and promptly placed in the refrigerator. It is possible to collect blood satisfactorily in a commercial vacuum flask without the use of a donor valve. This may be accomplished COLLECTION OF BLOOD 31 in two ways. In one method a length of heavy-walled rubber tubing is employed, in both ends of which are inserted 17-gage needles. One needle is inserted into the donor’s vein and, as soon as the blood is flowing into the tubing, the other needle is thrust aseptically through the stopper of the vacuum bottle. The gage of the needles is such as to properly control the rate of flow. The collection of blood is discontinued by first withdrawing the needle from the flask. In the other method, the flow of blood is controlled with a screw clamp. Therefore, larger gage needles may be used if desired, since the clamp serves the same purpose as the donor valve. When the preservative mixture “4” is used (see p. 33), the collecting flask and contents should be ice-cold before the blood is drawn into it (14). This procedure is extremely important to obviate extensive hemolysis from the diffusion of dextrose into erythrocytes. If outside temperatures are high, the procedure is also advised when blood is collected in any preservative mixture. Care of the Donor Following Bleeding 1. The donor should be allowed to rest in the recumbent position for 15 to 30 minutes. 2. It is advisable that a physician examine the donors before they are permitted to leave the bleeding room. 3. The donor has given blood voluntarily and should be shown every possible consideration. It has been found that a high percent- age of donors volunteer their blood again if they have been treated well on their first visit. 4. Coffee and crackers, or similar food, should be given to the donor after the bleeding. This often prevents delayed syncopal reactions. (Orange juice or other sugar-containing drink given before the col- lection of blood is also very helpful.) Reactions of the Donor If the donor is experienced, he may be allowed to leave the room almost immediately. Unpleasant symptoms may be expected in 1 to 5 percent of the inexperienced donors. They may feel well until they attempt to resume the erect position, when extreme pallor develops, sometimes followed by syncope. The pulse is frequently slow. Occa- sionally, there is nausea and vomiting. A rest of 30 minutes in the horizontal position will prevent these symptoms in most instances. The administration of very hot or very cold drinks is ill advised, be- cause this increases the incidence of reactions. However, it is recom- mended that some food and fluid be given after blood letting. Donor reactions may also be caused by too rapid a withdrawal of blood (a rate in excess of 100 cc. per minute is not recommended). 32 the operation of a hospital transfusion service A few individuals seem to retain for some hours a tendency to lowered systolic blood pressure when the erect position is assumed. Accidents have occurred when donors have been released after a 30- minute period of observation. There is some danger that if the donor leaves the range of observation too soon he may develop syncope and sustain injuries, such as skull fracture. Two other types of reaction have been noted occasionally in large series of bleedings. The donor may develop transitory, generalized convulsions during the collection of blood. Another rare type of reaction is the occurrence of tetany with carpopedal spasm and a positive Chvostek’s sign. Chapter IX. Storage of Blood The whole blood is stored in the refrigerator with temperatures usu- ally ranging from 2° to 5° C.3 A little latitude may be allowed in the upper limit of temperature, but reduction below 0° C. will, of course, result in hemolysis. The blood flasks should be shielded from direct sunlight, since most of the rays of the spectrum exert some hemolytic effect. Within 24 to 48 hours the erythrocytes have settled considerably, and the cell layer is covered by a thin gray layer termed the “buffy coat.” This consists of leukocytes, platelets, and debris. Above this is the supernatant layer of plasma. This varies in color and turbidity in different bloods. The presence of lipemia is insignificant, but it im- parts to the plasma a uniform cloudiness which is quite characteristic. Increase in turbidity over a period of a few days is an indication for culture. In the course of 5 to 10 days of storage, the supernatant citrated plasma develops a heavy cloud which has been variously called fibrin, fibrinogen, and gamma globulin. This is not noted, or is very slight, in plasma diluted with a dextrose solution. As hemolysis pro- ceeds, a layer of free hemoglobin diffuses upward through the plasma, coloring it red. The rate of this hemolysis defends on the preservative mixture employed. The following preservatives are recommended for specific purposes with their limitations stipulated: 1, Blood-Citrate Composition : 500 cc. of blood. 70 cc. of 2.5 percent or 50 cc. of 4 percent sodium citrate (dihydric) in distilled water. Recommended purposes: a. For storage as whole blood for 5 days, during which it may be used for whole blood transfusions. 8 See Appendix A, Section II. STORAGE OF BLOOD 33 b. For conversion to plasma by centrifugation within 48 hours after collection, or by sedimentation for 4 to 7 days. Comment: a. Inferior for storage of whole blood, since it does not inhibit hemolysis. b. There is not sufficient dilution to allow recovery of plasma eco- nomically by sedimentation. 2. Blood-Dextrose-Gitrate (dilution ratio 1 to 0.5) (56) Composition: 500 cc. of blood. 150 cc. of 5.4 percent dextrose in distilled water. 100 cc. of 8.2 percent sodium citrate (dihydric) In distilled water. Recommended purposes: a. For the storage of whole blood suitable for transfusion for 18 days. b. For the transportation of blood over long distances. Comment: a. Minimum added bulk, but a greater tendency to precipitation of fibrin during storage than with solutions 3 and 4. 3. Blood-Saline-Dextrose-Citrate (dilution ratio 1 to 1) (4) Composition: 500 cc. of blood. 500 cc. of solution containing per liter; 18.66 gm. dextrose (anhydrous). 4.18 gm. sodium chloride. 8.0 gm. sodium citrate (dihydric). Recommended purposes: a. For the storage of whole blood suitable for transfusion for 21 days. b. For the transportation of whole blood over long distances. c. For conversion to plasma by aspiration of the supernatant dilute plasma after 16 days of sedimentation or after the whole blood has been outdated. Comment: a. The bulk might be considered disadvantageous for some purposes. b. Optimum plasma recovery. 4. Blood-Dextrose-Citrate (dilution ratio 1 to 2.5) (13, 14) Composition: 500 cc. of blood. 650 cc. of 5.4 percent dextrose in distilled water. 100 cc. of 3.2 percent sodium citrate (dihydric) in distilled water. Recommended purposes: a. For the storage of whole blood suitable for transfusion for 30 days. b. For the transportation of whole blood over long distances. 34 the operation of a hospital transfusion service c. For conversion to plasma of outdated blood by the aspiration of the supernatant dilute plasma. Comment ; a. The bulk might be considered disadvantageous for some purposes. b. When blood is collected in this mixture, the flask and preservative must be ice cold, in order to avoid the danger of spontaneous hemolysis. Solutions 2, 3, and 4 all employ dextrose in an effective final concen- tration (0.5 to 3 percent.) The duration of adequate preservation in vitro depends upon the dilution employed. Clinical experience indi- cates that, within the storage limits prescribed for each solution, the life of the stored red cells after transfusion compares very favorably with 5-day-old citrated blood. The available experimental data con- firm this. Present evidence would indicate that the best recovery of dilute plasma is obtained from the 1 to 1 dilution (solution 3). Chapter X. Transportation of Blood It has already been noted that the agitation incident to transporta- tion has little effect on human erythrocytes. However, the blood- preservative mixture should fill the container used, preferably with displacement of practically all the air. This permits only a minimal amount of shaking and an optimal carbon dioxide tension for pre- servation. When preserved whole blood is contained in flasks with watertight closures, it may be transported most easily by immersing the flasks in ice water. The water-ice mixture will maintain an even temperature as long as unmelted ice is present. The ice may be replenished as often as necessary in the course of the trip. A 10-gallon milk can will accommodate 10 commercial 1-liter flasks and sufficient cracked ice to maintain low temperatures for nearly 24 hours during summer heat, provided an insulated shipping jacket is employed (20). Special precautions are necessary if dry ice (carbon dioxide) is used, since there is danger of freezing the blood. When water-ice mixtures are used, care should be taken not to lower the freezing point by the addition of sodium chloride, sugar, or other substances. Waterproof labels must, of course, be provided if the flasks are to be immersed in water. This type of refrigeration can be used as an emergency measure when the mechanical refrigeration of the hospital fails. Chapter XL Administration of Blood When a transfusion is required, the blood group of the recipient is determined and his blood is cross-matched with a blood of suitable ADMINISTRATION OF BLOOD 35 group in the bank. The specimen in the “pilot tube” is used for this purpose. Equipment Two items of equipment are needed in the administration of pre- served blood: a device for the initiation of the transfusion, and a filter for the blood. The initiation of the injection of blood presents the problem of excluding the donor’s blood from the needle until the back- flow of the recipient’s blood indicates that the needle is in the vein. Several types of procedures are used to accomplish this. The needle may be attached originally to a Luer syringe which is detached after receiving the backflow; the needle is then attached to the line of flow by an adapter. Another method is the use of a Kaufmann side-arm Luer syringe. The backflow is then pulled into the barrel of the syringe up to the side-arm, where the donor blood then enters and reverses the flow. Another procedure is the employment of an acces- sory line of physiologic saline, which is first run through the system until the successful insertion of the needle in the vein is demonstrated; the donor’s blood is then shunted in, either by means of a Y-tube and pinch clamps or by a two-way stopcock. All of the methods mentioned so far are adapted to the administration of blood through a “closed” or nearly “closed” system. When an “open” method is employed, a single line of tubing is attached to a receptacle such as a Kelly bottle or a salvarsan tube. The saline may be poured into the flask and the infusion satisfactorily initiated; then the blood mixture may be added to the infusion. In the administration of both fresh and preserved blood, a filter should always be employed to remove the particulate matter. Small shreds of fibrin and debris from the leukocytes, which might plug the needle or serve as emboli, are more numerous in preserved blood. When an open system of administration is used, the blood may be easily and adequately filtered through three or four thicknesses of sterile surgical (washed) gauze. Filtration in the closed system offers more difficulties since the limited filtration area frequently be- comes completely occluded, and it becomes necessary to use more than one filter. It is hoped that more adequate filters will soon be com- mercially available. Rate of Administration The chief factor governing the rate of administration of the trans fusion is the caliber of needle employed. For the adult recipient, a needle of 18-, 19-, or 20-gage may be used. The blood is usually deliv- ered from a height of about 3 feet above the recipient’s arm. With such a system and an 18-gage needle, the maximum volume of delivery is about 25 cc. per minute with citrated blood and about 36 cc. per 36 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE minute with blood-dextrose-citrate. The rate may be diminished by constriction of the tubing with a screw clamp. These or faster rates may be advisable in the treatment of shock. The average rate of ad- ministration of 10 to 20 cc. (150 to 300 drops) per minute should not ordinarily be exceeded. Selection of a Vein The selection of a vein for the insertion of a needle is a matter of experience, and further discussion is probably valueless. It goes without saying that the ability to place a needle in a vein is the sine qua non of all transfusion procedures. Occasionally dissection and cannulation of the vessel may be necessary. Attention is directed to the fact that femoral vein injection can usually be performed when all other veins are inaccessible because of severe burns, anasarca, obesity, or extreme shock. Danger of Warming As has been mentioned previously, blood, plasma or other fluids may be transfused without preliminary warming. This is necessary when preserved blood is employed, since it not only saves valuable time but excessive hemolysis may be caused by too rapid warming. Care of Recipient The recipient should he attended dwring the transfusion, not only so that the apparatus may be regulated but also in order that the transfusion may be discontinued at the first sign of a severe reaction of the urticarial, the hemolytic, or the circulatory type. It is known that hemolytic transfusion reactions, as a rule, are evidenced by severe symptoms before 75 to 100 cc. of blood have been administered, and, furthermore, that fatal reactions can almost always be avoided if an incompatible transfusion is stopped as soon as symptoms first appear. It is recommended, therefore, that the first hundred cubic centimeters of blood be administered slowly and under the supervision of a physician. It is also recommended that a physician remain in attendance during the entire transfusion in cases where a patient with cardiovascular disease must be transfused. There is great danger of overloading of the vascular system in such patients. Laboratory Study of the Recipient Transfusion reactions should be dealt with as indicated in chapter IV. If circumstances permit, it is desirable to examine the urine just prior to the transfusion and ascertain whether it is alkaline or acid in reaction. The result of such a test in the presence of cystitis is, of course, no true indication as to the reaction of the urine as it conies from the kidneys. The urine may be alkalinized by the oral admin- RED CELL SUSPENSIONS 37 istration of sodium salts such as bicarbonate or citrate. This may take several hours. Sodium r-lactate may be used intravenously with al- most immediate effect. This is thought to minimize the danger from hemolytic transfusion reactions. In the event of a transfusion reaction the urine voided for 24 hours after the transfusion should be saved, and the volume output should be recorded, since this may be the only indication that oliguria or anuria has developed in a severely ill patient. The urine should also be tested for the presence of hemoglobin. Chapter XII. Administration of Red Cell Suspensions The preparation of plasma by centrifugation has made available an inexpensive means of obtaining red blood cells for the treatment of anemic patients. In making use of such therapy, one should, of course, be sure that a patient’s need will be wholly, or in a large part, met by the transfusion of red cells alone. Reports of this procedure have recently appeared in the literature (57, 58, 59, 60, 61). 1. (Titrated whole blood, from which the plasma has been removed, can be used for a red cell transfusion up to 96 hours after collection of the blood, provided the cells are stored at 5° to 10° C. 2. If no plasma is available for a check on the cell typing and for cross-matching, the cells should be typed with two different lots of grouping sera. 3. Unless the red cell residue can be positively identified with the sample in the pilot tube, cells must be removed from the centrifuged residue for typing and subsequent cross-matching with the recipient’s serum. 4. Resuspend the cells in 100 to 250 cc. of known pyrogen-free nor- mal salt solution immediately after the plasma has been aspirated. It seems likely that the use of one of the dextrose preservative solutions as the diluent would permit the 96-hour storage limit to be prolonged. a. If commercial vacuum-type containers are used, the integrity of the closure of the bottle is usually destroyed when the plasma is aspirated. Therefore, the red cell residue should at once be aspirated into a fresh bottle containing the desired amount of diluent in order to assure continued sterility of the cells. A sample for typing and cross-matching can be obtained from the aspirating set. b. With reusable equipment, the technique differs. Here the bleed- ing stopper is removed aseptically after the plasma has been aspirated, THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE and the desired amount of diluent poured into the bottle. It is then immediately closed aseptically with a sterile solid stopper, after removal of a sample for typing and cross-matching. 5. If appreciable hemolysis or change in the color of the cells occurs before use, the bottle should be discarded. (A violaceous discoloration develops with contamination.) 6. Sterility tests on resuspended cells should be carried out routinely while the technique is being established, and periodically thereafter as a check on the procedure. The risk of contamination is minimized if a closed system is employed. Samples for testing should be taken at the time of resuspension. 7. Like whole blood, red cell suspensions should not be warmed and must be filtered either immediately before or during administration. REFERENCES FOR PART I Books 1. Kilduffe, R. A., and DeBakey, Michael: The Blood Bank and the Technique and Therapeutics of Transfusion, C. V. Mosby Company, St. Louis, 1942. 2. Riddell, V. H.: Blood Transfusion, 1939, Oxford University Press, London. 3. Wiener, A. S.: Blood Groups and Blood Transfusion, 3d Edition, 1943, Charles C. Thomas, Springfield, 111. Articles 4. Alsever, J. B., and Ainslie, R. B.: A new method for the preparation of dilute blood plasma and the operation of a complete transfusion service, N. Y. State J. Med. 41:126-135 (Jan. 15) 1941. 5. Baker, S. L.: Urinary suppression following blood transfusion, Lancet 1: 1390-1394 (June 12) 1937. 6. Belk, W. P.; Henry, N. W., and Rosenstein, Florence: Observations on hu- man blood stored at 4 to 6 degrees Centigrade, Am. J. M. Sc. 198: 631-633 (Nov.) 1939. 7. Bushby, S. R. N.; Kekwick, A., Marriott, H. L., and Whitby, L. E. H.; Sur- vival of stored red cells after transfusion, Lancet 2 : 414 (Oct.) 1940. 8. Crosbie, Andrew, and Scarborough, Harold: Studies on stored blood. II. The leukocytes in stored blood, Edinburgh M. J. 47:553-556, 1940. 9. Daniels, W. P.; Leonard, B. W., and Holtzman, Saul: Renal insufficiency fol- lowing transfusion, J. A. M. A. 116:1208-1215 (Mar. 22) 1941. 10. DeGowin, E. L.; Osterhagen, H. F., and Andersch, Marie: Renal insufficiency from blood transfusion. I. Relation to urinary acidity, Arch. Int. Med. 59:432-444 (Mar.) 1937. 11. DeGowin, E. L.; Warner, E. D., and Randall, W. L. Renal insufficiency from blood transfusion. II. Anatomic changes in man compared with those in dogs with experimental hemoglobinuria, Arch. Int. Med. 61:609-630 (April) 1938. 12. DeGowin, E. L.: Grave sequelae of blood transfusions. A clinical study of 13 cases occurring in 3500 blood transfusions, Ann. Int. Med. 11: 1777-1791 (April) 1938. 13. DeGowin, E. L.; Harris, J. E., and Plass, E. D.: Changes in human blood pre- served for transfusion, Proc. Soc. Exper. Biol. & Med. 40:126-128 (Jan.) 1939. 14. DeGowin, E. L.; Harris, J. E., and Plass, E. D.: Studies on preserved human blood. I. Various factors influencing hemolysis, J. A. M. A. 114: 850-855 (Mar. 9) 1940. 15. DeGowin, E. L.; Harris, J. E., and Plass, E. D.: Studies on preserved human blood. II. Diffusion of potassium from the erythrocytes during storage, J. A. M. A. 114: 855-857 (Mar. 9) 1940. 40 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE 16. DeGowin, E. L.; Hardin, R. 0., and Harris, J. E.: Studies on preserved human blood. III. Toxicity of blood with high plasma potassium tranfused into human beings, J. A. M. A. 114: 858-859 (Mar. 9) 1910. 17. DeGowin, E. L.; Hardin, R. C., and Swanson, L. W.: Studies on preserved human blood. IY. Transfusion of cold blood into man, J. A. M. A. 114; 859-861 (Mar. 9) 1940. 18. DeGowin, E. L., and Hardin, R. C.: Reactions from the transfusion of pre- served blood, Brit. M. J. 2: 1 (July 6) 1940. 19. DeGowin, E. L., and Hardin, R. C.: A plan for the collection, transportation, and administration of whole blood and of plasma in warfare, War Med. 1: 326-341 (May) 1941. 20. DeGowin, E. L., and Hardin, R. C.: Studies on preserved human blood. VI. Reactions from transfusion, J. A. M. A. 115: 895-898 (Sept. 14) 1940. 21. Ebert, R. V.; Stead, E. A., Jr., and Gibson, J. G., II; Response of normal sub- jects to acute blood loss (with special reference to the mechanism of restoration of blood volume), Arch. Int. Med. 68: 578-591 (Sept.) 1941. 22. Ebert, R. V., and Stead, E. A., Jr.; The effect of the application of tourniquets on the hemodynamics of the circulation, J. Clin. Investigation 19: 561-567 (July) 1940. 23. Fowler, W. M., and Barer, A. P.: Rate of hemoglobin regeneration in blood donors, J. A. M. A. 118: 421-427 (Feb. 7) 1942. 24. Gilligan, D. R.; Altschule, M. D., and Katersky, E. M.: Studies of hemoglo- binemia and hemoglobinuria produced in man by the intravenous injection of hemoglobin solutions, J. Clin. Investigation 20: 177-187 (Mar.) 1941. 25. Gordon, E. F.: Accidental transmission of malaria through the administra- tion of stored blood, J. A. M. A. 116: 1200-1202 (Mar. 22) 1941. 26. Goldring, William, and Graef, Irving: Nephrosis with uremia following transfusion with incompatible blood, Arch. Int. Med. 58: 825-845 (Nov.) 1936. 27. Kleendshoj, N. C., and McNeil, Crichton : A transfusion reaction following use of universal blood, J. A. M. A. 118 : 528-529 (Feb. 14) 1942. 28. Kolmer, J. A.: Preserved citrated blood “banks” in relation to transfusion in the treatment of disease with special reference to the Immunologic aspects, Am. J. M. Sc. 197: 442-452 (April) 1939. 29. Kolmer, J. A., and Howard, Mary; Studies on the preservation of human blood, Am. J. M. Sc. 200: 311-321 (Sept.) 1940. 30. Landsteiner, Karl, and Wiener, A. S.: Studies on an agglutinogen Rh in human blood reacting with antirhesus sera and with human isoantibodies, J. Exper. Med. 74 : 309-320 (Oct.) 1941. 31. Levinson, S. O., and Cronheim, Anny: Suppression of isoagglutinins and the significance of this phenomenon in serum transfusions, J. A. M. A. 114: 2097-2098 (May 25) 1940. 32. Lord, J. W., Jr., and Pastore, J. B.: Plasma prothrombin content of bank blood, J. A. M. A. 113: 2231-2232 (Dec. 16) 1939. 33. Mollison, P. L., and Young, I. M.: Survival of the transfused efythrocytes of stored blood, Lancet 2: 420 (Oct. 5) 1940. 34. Mollison, P. L., and Young, I. M.: On the survival of the transfused erythro- cytes of stored blood, Quart. J. Exper. Physiol. 30: 313-327, 1940. 35. Mollison, P. L. and Young, I. M.: Failure of in vitro tests as a guide to the value of stored blood, Brit. M. J. 2: 797-800 (Dec. 6) 1941. 36. Muether, R. O., and Andrews, K. R.: Studies on “stored” blood, Am. J. Clin. Path. 11: 307-328 (April) 1941. REFERENCES FOR PART I 41 37. Muether, R. O., and Andrews, K. R.; Studies on the uses of stored blood and plasma, South. M. J. 34 : 453-462 (May) 1941. 38. Murphy, F. D.; Correll, Howard, and Grill, J. C.: The effects of intravenous solutions on patients with and without cardiovascular defects, J. A. M. A. 116: 104-108 (Jan. 11) 1941. 39. Ottenberg, Reuben, and Fox, C. L., Jr.: The rate of removal of hemoglobin from the circulation and its renal threshold in human beings, Am. J. Physiol. 123: 516-525 (Aug.) 1938. 40. Scudder, John; Drew, C. R.; Corcoran, D. R., and Bull, D. C.: Studies in blood preservation, J. A. M. A. 112 : 2263-2271 (June 3) 1939. 41. Scudder, John: Studies in blood preservation. The stability of plasma proteins, Ann. Surg. 112: 502-519 (Oct.) 1940. 42. Seibert, F. B.: Fever-producing substances found in some distilled waters, Am. J. Physiol. 67: 90-104 (Dec.) 1923; Ibid. 71: 621-651 (Feb.) 1925. 43. Thalhimer, William, and Myron, S. A.: Globulin fractions of A and B agglu- tinating serums for blood typing, J. A. M. A. 118: 370-372 (Jan. 31) 1942. 44. Turner, T. B., and Diseker, T. H.: Duration of infectivity of Treponema pallidum in citrated blood stored under conditions obtaining in blood banks, Bull. Johns Hopkins Hosp. 68: 269-279 (Mar.) 1941. 45. Wallace, John; Sharpey-Schiifer, E. P., and Pincock, A. C.: Blood changes following controlled hemorrhage in man, Lancet 2: 393 (Oct. 4) 1941. 46. Warner, E. D.; DeGowin, E. L., and Seegers, W. H.: Studies on preserved human blood. V. Decrease in prothrombin titer during storage, Proc. Soc. Exper. Biol, and Med. 43: 251-254, 1940. 47. Wiener, A. S., and Peters, H. R.: Hemolytic reactions following transfusions of blood of homologous group, with three cases in which the same agglutino- gen was responsible, Ann. Int. Med. 13 : 2306-2322 (June) 1940. 48. Wiener, A. S.: Hemolytic reactions following transfusion of blood of the homologous group, Arch. Path, 32: 227-250 (Aug.) 1941. 49. Witebsky, Ernest; Klendshoj, N. C., and Swanson, Paul: Preparation and transfusion of safe universal blood, J. A. M. A. 116 : 2654-2656 (June 14) 1941. 50. Ziegler, E. R.; Osterberg, A. E., and Hovig, Mildred: The prothrombin changes in banked blood, J. A. M. A. 114: 1341-1342 (April 6) 1940. 51. Diggs, L. W., and Keith, A. J.: Problems in blood banking, Am. J. Clin. Path. 9 : 591-605, 1939. 52. Muether, R. O., and Andrews, K. R.: Studies on “stored blood,” Am. J. Clin. Path. 11: 321-328, 1941. 53. Rosenthal, N.; Wasserman, L. R.; Abel, N.; Basson, F., and Vogel, P.: The organization of the blood bank at Mount Sinai Hospital, J. Mt. Sinai Hosp. 8 : 210-231, 1941. 54. Alsever, John B.: Unpublished data. 55. Denstedt, O. F.; Osborne, Dorothy E.; Roche, Mary N., and Stansfleld, Hugh: Problems in the preservation of blood, Canad. M. A. J. 44: 1941. 56. Denstedt, O. F.; Osborne, Dorothy E.; Stansfleld, H., and Rochlin, I.: The survival of preserved erythrocytes after transfusion, Canad. M. A. J. 48: 477- 486, 1943. 57. Williams, G. E. O., and Davie, T. B.: Preparation and use of concentrated red cell suspensions in treatment of anemia, Brit. M. J. 2: 641-644, (Nov. 8) 1941. 58. Transfusions of derivatives of blood, Report of the Royal Society of Medicine, Lancet 1: 178-179 (Feb. 8) 1941. THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE 59. Watson, Leslie: Red-cell suspension transfusions, Lancet 1: 107-110 (Jan. 23) 1943. 60. MacQuaide, D. H. G., and Mollison, P. L.: Treatment of anemia by transfusion of concentrated suspensions of red cells, Brit. M. J. 2: 555-556 (Oct. 26) 1940. 61. Murray, Clifford K.; Hale, Donald E., and Sliaar, C. M.: The preparation and use of red blood cell suspensions in treatment of anemia, J. A. M. A. 122: 1065-1067 (Aug. 14) 1943. Part If. The Processing and Use of Citrated Human Blood Plasma . Chapter I. General Considerations Experimental and clinical observations have established the thera- peutic value of citrated normal human plasma. Plasma has been popularly regarded as a blood substitute. This is improper because not all of the functions of whole blood are possessed by plasma, and, conversely, plasma is successfully employed in some clinical condi- tions in which the injection of whole blood is neither indicated nor therapeutically suitable. It is proper, therefore, to regard blood and plasma as having different fields of application as shown in table 3, page 6. Detailed discussion of the therapeutic use of plasma in shock may be found in OCD publication 2212, “The Clinical Rec- ognition and Treatment of Shock,” and in burns in OCD publica- tion 2203-1, “The Treatment of Bums and Prevention of Wound Infections.” Plasma can be prepared at small expense, may be transported with- out risk of deterioration, and may be stored for long periods of time. No serious reactions follow its administration even in large and repeated doses, and it is available for immediate administration. In an appraisal of the value of any therapeutic agent, first consid- eration must be given to the possibility of its harmful effects. Human plasma, properly prepared from citrated blood collected from healthy donors, can be administered intravenously to patients without regard to blood grouping and without reactions, save for occasional mild urticarial manifestations. To serve its purpose fully, blood plasma should be available in a form requiring a minimum of time for its administration, as well as in a form most nearly meeting the require- ments of the circumstances under which it is to be used or transported. In the development of methods of plasma preparation, it must be remembered that liquid plasma is a good culture medium and that bacterial contamination occurs with comparative ease. In addition, certain unstable plasma proteins have a tendency to flocculate or lose their specificity. Pyrogens and bacterial contaminations are appar- ently responsible for the febrile reactions which have been reported following the administration of plasma. Contamination can be 562943’—44 4 44 the operation of a hospital transfusion service greatly reduced by the use of aseptic surgical technique during bleed- ing and the employment of a closed system throughout the processing procedure. Reactions due to pyrogens may be reduced to a minimum by scrupulous care in the preparation of the fluids and equipment used in the preparation and administration of plasma. If it is desired to preserve the unstable protein fractions, the sterile plasma must be promptly fixed by bringing it to the frozen state within a minimum of time after bleeding. Essential Requirements for Plasma Production Irrespective of the agency which undertakes to prepare citrated normal human blood plasma intended for intravenous administration, there are certain essential minimum requirements which must be ob- served without variation. These requirements include the protection of the donor, the method of drawing and processing the blood, quali- fications of the laboratory personnel and its medical responsibility as required by law, and the storage of the finished product. These mini- mum requirements are itemized as follows: 1. The donor must be in such physical condition that the taking of the desired amount of blood will not endanger his health. (See p. 4.) 2. The donor must be free from any diseases transmissible by blood transfusion, as determined by those methods of examination which are considered adequate by competent authority within the jurisdiction of the processing laboratory. (See p. 4.) 3. The bleeding must be done in an adequately equipped bleeding center which conforms to such municipal, State, or Federal laws as are applicable. 4. The bleeding must be under the immediate supervision of a quali- fied doctor of medicine, assisted by the necessary trained personnel, 6. The bleeding and all the subsequent steps involving the plasma fraction, until it is injected into the recipient, must be carried out in a closed system. (A closed system is defined as an apparatus which will permit nothing to be drawn into the system at any point except the liquid under transfer and the air required for replacement when negative or positive pressure is applied at the proper place. All air for replacement must first pass through a suitable antibacterial filter.) 6. The blood must not have undergone excessive hemolysis. This can be determined by the color of the plasma following centrifugation. A hemoglobin content of 50 mg. percent4 is considered to be within safe limits. Directions for preparing a satisfactory color standard for comparison are found in paragraphs 1 and 2 of section II, ap- pendix B, page 78. * 25 mg. percent is the maximum allowed by the National Institute of Health regulations governing commercial preparation of plasma. LIQUID, FROZEN, AND DRIED PLASMA 45 7. All glassware coming in contact with the blood or plasma should be clear glass and of good quality (preferably high quality ampoule glass). All rubber stoppers should be of high grade “sulfur-free,” nonoxidizing rubber and suitable for stoppering biological products having a high protein content. Each piece of equipment coming in contact with either the blood or the plasma must have been made scrupulously clean by washing in suitable cleaning solutions followed by adequate rinsing with pyrogen-free distilled water or physiological solution of sodium chloride. All exposed parts must be adequately covered by suitable wrappings or inserted into stoppered test tubes. All equipment coming in contact with either the blood or the plasma must have been sterilized in the autoclave at 121.5° C. (15 pounds pressure) for at least 20 minutes (i. e., each part of the material to be sterilized must attain this temperature for at least the full 20 minutes). 8. The plasma must be sterile, as determined by suitable sterility tests. 9. If the final product is retained in the liquid or the frozen state, it must be placed in a container made of good quality glass, com- pletely sealed by a rubber stopper which will permit the entrance of the necessary needles or trochar for administration to the recipient, and the container must be properly labeled. 10. Explicit instructions should accompany each unit of liquid, frozen, or dried plasma, pointing out the necessity of placing into the lumen of the tube leading from the plasma reservoir to the vein of the recipient a filter adequate for the removal of all particles which are of such size as to be dangerous for intravenous administration. (See “Filter Adequate for the Removal of Particulate Matter,” p. 85.) Chapter II. Liquid, Frozen, and Dried Plasma The degree to which human plasma should be processed depends primarily upon the degree of stabilization of the component parts desired, upon the storage facilities available, upon the amount of han- dling and transportation involved before the plasma reaches the recipient, and upon the interval likely to elapse before use. For the most frequent usages, namely, the replacement of blood volume in shock, hemorrhage, and burns, the three forms of human plasma (liquid, frozen, and dried) must be considered equal in therapeutic value within the limits of the dating periods allowed. A definite expi- ration or dating period of not more than 2 years 5 has been recognized 11 year is the present dating period approved by the National Institute of Health. 46 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE for liquid plasma and not more than 5 years for dried plasma. An expiration date for the frozen product has not been officially set, since experience with this form is more limited. However, 3 years is the provisional expiration date, provided the plasma is kept continuously at or below the temperature range permitted. It is anticipated that this time limit will be extended. (See p, 54, par. 7.) Liquid plasma is the most economical to produce and may be made relatively stable by the addition of dextrose as approved by the U. S. P. XII. It is recommended that sterile 50 percent dextrose solution be added in sufficient quantity to obtain a final dextrose concentra- tion of 6 percent. Assurance must be had that the dextrose is free from pyrogenic substance. Liquid plasma with added dextrose will usually remain free from fibrin clot for the entire dating period, pro- vided it is stored at the prevailing room temperature but not below 13° C. (55° F.). If prepared and stored in the liquid state, it soon loses the unstable protein elements, such as prothrombin and comple- ment, and more gradually any specific antibodies. Frozen plasma is somewhat less economical to process and requires suitable cold storage facilities for its preservation, which add slightly to its cost. It is superior to liquid plasma in that it has a longer period of usefulness, the freezing fixes the unstable protein elements, and the risk of contamination is greatly reduced. It is desirable to process this with the addition of dextrose so that the fibrinogen will remain stable at room temperature after the plasma has been thawed. Frozen plasma may be the product of choice in large hospitals, particularly where production needs are large but restricted to process- ing at infrequent intervals, and also where the demand is irregular. The storage of plasma in the frozen state is particularly advantageous when convalescent plasma is to be used for the treatment of infectious diseases. At the present time it is the product of choice to meet the needs of communities where the exact time of need cannot be pre- determined and where this unexpected need may be excessively heavy. This is particularly true in large industrial areas or other places where comm unity-wide catastrophes are apt to occur. Dried plasma is the most expensive form of plasma to produce because of the time involved, the equipment needed for processing and bottling, and also because of the number and qualifications of the laboratory personnel. It has the advantage of stability under the most unfavorable circumstances and over a very long dating period. It is easily and quickly restored to the liquid state, provided it has been properly prepared and bottled. Contrary to the prevailing opinion among the uninformed, however, the preparation of dried plasma of U. S. P. quality or better is an exacting task which calls not only PLASMA PREPARATION 47 for skill on the part of the operator but, even more, the use of drying apparatus capable of accomplishing the task properly. At present, it should be undertaken only by the large laboratory having adequate staff, financial resources, and a distribution area extending over a very large territory, or where other special factors make the preparation of dried plasma advisable. Chapter III. Methods of Plasma Preparation Several methods of preparing plasma are detailed on the following pages, including the employment of both commercial and reusable equipment. The method or methods chosen by a particular laboratory should depend upon its needs and facilities. The reader will appre- ciate that adequate substitutions can be made for many of the indi- vidual pieces of apparatus to be described and for the individual steps taken without affecting the quality of the finished product. A. Centrifuge Method 1. EMPLOYMENT OF COMMERCIAL VACUUM-TYPE CONTAINERS The bottles must be sterile and of the proper size to fit into the standard centrifuge cup. In addition, the bottle used must provide a closed system for the collection of blood, a self-contained vacuum, and a stopper which will maintain an airtight seal after puncture with a 15-gage needle (37). (It is suggested that the incidence of reactions following bleeding will be reduced if the following procedure is adopted: Collect 500 to 600 cc. if the donor weighs more than 150 pounds, 250 to 300 cc. if less than 150.) Collection of Blood The collection of blood should be under the direct supervision of a trained physician. (See pp. 29-32 for preparation and care of the donor.) 1. Expose the rubber stopper aseptically. (This bottle should not contain an air tube unless the collection may possibly be used as whole blood.) 2. Apply 7 percent Tr. iodine to the top of the bottle, leaving an alcohol sponge over the stopper until ready to insert the valve needle. 3. Unpack the sterile donor set and dose the donor valve. 4. Wipe off excess iodine and alcohol from the stopper of the bottle. 48 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE 5. Remove the protective glass tube from the donor valve needle and insert the needle through the rubber stopper. Rotate the bottle so that all surfaces will be bathed by the citrate solution. 6. Reapply pressure on the blood pressure cuff (40-60 mm. mer- cury). The pressure should be maintained near the donor’s diastolic pressure during the period of collecting the blood. 7. Remove the glass tube covering the donor needle and insert the needle into the vein selected. 8. Open the valve and allow the blood to run into the bottle. The donor should be instructed to open and close his hand slowly to in- crease the flow of blood. It is preferable to have the bottle in the inverted position so that the blood will be mixed adequately with the anticoagulant. The bottle should be shaken gently and constantly during the collection of the blood and for at least 1 minute after the collection has been completed. This further insures mixture of the blood with the sodium citrate solution. Failure to agitate the bottle properly during the period of collection may result in clotting. If during the collection period there is a fluttering sound at the donor needle, close the valve for 2 or 3 seconds and then reopen it slowly. The fluttering is caused by attempting to draw the blood faster than the vein can be filled. The flutter valve action is unde- sirable, as it has a definite tendency to hemolyze the blood. The rate of flow should not exceed 100 cc. per minute. After a little experience the operator can judge the desired rate of flow. 9. If two 300 cc, containers are to be filled with blood, prepare the second bottle as in step 5, then shut off the donor valve and transfer the valve to second bottle. Open the valve and proceed as in step 8. 10. After obtaining the desired amount of blood, close the valve of the donor set, release the pressure on the cuff, and remove the donor needle from the vein. The bottle should be below the level of the arm when the needle is removed from the vein; this prevents leakage of blood from the donor needle. 11. Maintain pressure with a sterile gauze pad over the site of veni- puncture for at least 5 minutes after withdrawing the needle. The arm should be raised for the first 1 or 2 minutes. 12. Withdraw the needle from the stopper of the bottle. 13. Allow 2 or 3 drops of blood from the donor set to flow into a test tube containing 3 cc. of sodium citrate, 2y2 percent, in normal saline (if blood typing is desired). 14. Remove the donor needle from the rubber tubing, open the donor valve and allow the remainder of the blood to run into the serology tube. (See p. 65 for material for cross-matching when collection may be used as whole blood.) PLASMA PREPARATION 49 15. The donor set must be cleaned immediately after use. (See instructions for cleaning and preparing donor set, p. 71.) Storage of the Blood 1. Place the blood in an icebox (2° to 5° C.) preferably vy'ithin 1 hour after collection. This temperature must be maintained until the blood is ready for centrifugation. The blood container must not be opened from the time of the bleeding until the final preparation of the plasma. Twelve to twenty-four hours of storage is preferable before centrifugation. 2. Do not attempt to pool ivhole blood, as it produces an undue amount of hemolysis. 3. A preservative (bacteriostatic agent) must not be added to whole blood at any time. 4. Freezing of whole blood must be avoided, as it p-roduces marked hemolysis. Centrifugation of Whole Blood Any rapidly spinning object must be perfectly balanced in order to rotate freely. One of the most important steps in the preparation of plasma is to have the spinning objects (blood, bottles, and trunnion cups) in perfect balance during the period of centrifugation. If this step is performed in a hasty, careless manner, excessive vibration de- velops, and there is danger of breaking the bottles and damaging the centrifuge. The trunnion cups and bottles must be 'perfectly balanced for the satisfactory separation of plasma. 1. Balancing of Cups and Bottles Before Centrifugation.—A good torsion balance is essential for this step. Each cup should be filled with water up to the shoulder of the bottle. This reduces the danger of breakage. a. First method.—If the cups and bottles are weighed against one another, it is absolutely essential that the torsion balance be perfectly level. After balancing by this method, reverse the position of the cups on the torsion balance. If the cups still balance, they may be placed opposite each other in the centrifuge. If the cups fail to balance when reversed, the torsion balance is not level and must be made so before the balancing proceeds. b. Second method.—Another method of balancing is to place weights on one pan of the torsion balance and then adjust the slide beam weight until the cup and weights balance perfectly. Considerable time can be saved if the operator selects the bottle containing the greatest amount of blood for the first balancing; then the other cups and bottles must be balanced to weigh the same as the master bottle. 50 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE c. Achieving balance.—This should be accomplished by adding rub- ber bands of various sizes to the balance tray containing the lighter cup and bottle. After balance has been achieved in this fashion, the rubber bands should be placed around the neck of the bottle. This places the additional weight in the correct location on the bottle, which is essential for high speed centrifuging. 2. Centrifuging.—a. After all bottles have been accurately balanced, place them in the centrifuge in the proper location and start the centri- fuge slowly. If excessive vibration develops, stop the centrifuge, reweigh the cups and start the centrifuge as before. All centrifuges develop some vibration at the critical speed, 500 to 750 r. p. m. b. Centrifuge at 2,000 to 2,500 r. p. m. for 1 hour. c. When centrifuging is complete, turn down the rheostat gradually, and when the speed of the centrifuge approximates 800 r. p. m., turn off the switch and disengage the brushes. If the centrifuge is stopped too rapidly, there will be an undue amount of red cells in the plasma, and the cell pack will be greatly disturbed. Do not use the centrifuge brake at any time. d. Remove the bottles from the centrifuge and place in a refrigerator preferably for 6 to 24 hours. The plasma should not be aspirated im- mediately after spinning if swirling of cells occurs following centri- fuging, but should be allowed to stand for at least 6 hours to allow the red cells to settle out completely. If swirling does not occur, it is permissible to aspirate the plasma immediately. Pooling of Citrated Liquid Plasma It is the consensus that undiluted liquid plasma should be pooled in order to reduce the titer of the agglutinins present. (A 2,000 cc. flask containing 200 cc. of 50 percent dextrose is the minimum size recom- mended and will average six to eight bleedings.)6 1. Check the laboratory tests and make sure that only serologically negative blood is used. 2. Prepare the blood bottles in the following manner: Expose the stopper aseptically and apply 7 percent Tr. iodine to its top. (Be sure any indentations are thoroughly cleaned with iodine.) Leave an alcohol sponge on the top of the bottle until ready to start aspirating. 3. Prepare the pooling bottle in the following manner: a. Remove the protective covering from the top of the bottle. b. Apply iodine to the stopper. Leave an alcohol sponge on the top of the bottle until ready to insert the valve needle. 4. Unpack the sterile aspirating set. The aspirating needle should be covered by penrose tubing. (See pp. 60 and 72.) • A minimum of eight bleedings per pool is required by the National Institute of Health. PLASMA PREPARATION 51 5. Insert the needle of the airway filter through the rubber stopper of the blood bottle and release the vacuum. 6. Close the valve on the aspirating set and plunge the valve needle through the stopper of the pooling bottle. 7. Insert the aspirating needle through the stopper of the blood bottle and into the supernatant plasma. 8. Open the valve and start aspiration. The aspirating needle must be kept beneath the surface of the plasma to avoid loss of the vacuum in the pooling bottle. 9. A strong beam of artificial light should be focused at the junction of the buffy coat and plasma during the aspiration. This serves as an aid in detecting cellular elements that might be drawn into the aspirating needle. Care must be taken not to aspirate any of the red cells. Do not attempt to aspirate all of the plasma, or a number of red cells will be pulled over into the final container. Aspirate the last 30 or 40 cc. of plasma very slowly to prevent lifting and drawing over the cells. Do not attempt to aspirate the last 10 cc. of plasma. After the plasma from one bottle has been aspirated, leave the aspirat- ing needle in place until the next bottle is prepared for aspiration. Handle the blood bottles with care to avoid agitation of the red cells. 10. Continue the aspiration as described until the pooling bottle has been filled or until all plasma has been aspirated. 11. Allow the vacuum to pull over the plasma remaining in the as- pirating set before disconnecting the set from the last bottle. Culturing the Pool Ordinarily, culturing should not be done until the pool has been allowed to stand for 24 to 48 hours at room temperature. This mini- mizes the chance of obtaining falsely negative cultures. 1. Aseptically remove the protective covering and apply 7 percent Tr. iodine to the stopper of the pooling bottle. 2. Prepare a sterile glycerinated 50 cc. syringe sfnd attach an 18- gage needle. Aspirate approximately 40 cc. of plasma. 3. Use four tubes of Brewer’s medium. Inoculate each tube with 10 cc. of plasma. Each tube (25 by 150 mm.) contains 20 cc. of Brew- er’s sodium thioglycollate medium. (See p. 62 for additional method.) 4. Additional plasma may be withdrawn for protein determination if desired. 5. Place two culture tubes in an incubator at 37° C. and keep the other two at room temperature (20° to 25° C.). 6. Observe the cultures over a period of 10 days. (For interpreta- tion of positive cultures, see p. 80, par. 16.) 7. If pool and pilot bottle cultures are negative the plasma may be released for use. (See p. 53, par. 10-12.) 8. Keep an accurate, permanent record of all cultures. 52 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE Addition of a Bacteriostatic Agent This procedure, although required of commercial firms by the Na- tional Institute of Health, is optional in hospital practice. If it is desired to add a bacteriostatic agent, this should be done after aspiration of the sample for culturing. Merthiolate, or phenyl mercuric borate (or nitrate), has been found to be fairly satisfactory for this purpose. It must he remembered that these agents are only an additional precaution in the preparation of plasma and 'will not render contaminated plasma fit for use. Actu- ally, they are effective only against small numbers of certain types of bacteria. Administration of plasma containing a mercurial preserva- tive, in amounts exceeding 2,000 cc. per 24 hours, may conceivably lead to renal damage. 1. Apply 7 percent Tr. iodine to the stopper, 2, Using a glycerinated sterile syringe, add 1 cc. of a 1 percent aqueous solution of merthiolate per 100 cc. of plasma in the pool bottle. This gives a 1:10,000 final concentration of merthiolate. If phenyl mercuric borate (or nitrate) is used, add 0.6 cc. of a 1 percent aqueous solution per 100 cc. of plasma in the pool bottle. This gives a 1:16,000 final concentration of phenyl mercuric borate (or nitrate). Filling of Final Containers; Storage as Liquid Plasma 1. At any time after the cultures have been taken, the plasma may be aspirated into the final containers. 2. Clean the stopper of the pool bottle with 7 percent iodine and leave an alcohol sponge over the top of the bottle until ready to start aspiration. (Be sure any indentations are thoroughly cleaned with iodine.) 3. Prepare the final container in the manner indicated in items 1 and 2 under “Collection of Blood.” (See p. 47.) 4. Unpack sterile aspirating set and close the donor valve. 5. Puncture the rubber stopper of the final container with the needle of the valve set. 6. Pierce the stopper of the pooling bottle with the air-filter needle to release the vacuum. 7. Insert the aspirating needle through the stopper. The aspirating needle should be inserted well below the surface of the plasma. 8. Open the valve of the donor set and start aspirating into the final container. When the first container is filled, close the donor valve. 9. Prepare the next final container, remove the donor valve needle from the first final container, and insert through the stopper of the next container to be filled. Open the donor valve and start aspira- PLASMA PREPARATION 53 tion as outlined in the preceding paragraphs. (The donor valve needle is left in place until the next bottle is prepared, in order to prevent undue exposure and possible contamination of the needle.) 10. After the last bottle has been filled, aspirate the remainder of the plasma in the pool into a final container. This plasma, usually 50-150 cc., should be retained as the pilot bottle for this lot of plasma. (It is desirable to include in this sample 20 to 25 cc, of plasma from the top of the pool bottle.) 11. The pilot bottle is allowed to remain at room temperature for 24 hours and then cultured in the manner indicated under “Cul- turing the Pool,” except that a larger quantity of medium is neces- sary if a bacteriostatic agent has been added. (See appendix B, sec. IV.) The remainder of the bottles of this lot are labeled and stored in a cool (ordinary room temperature), dark room. The preferable temperature range is 15.5° to 26.6° C. (60° to 80° F.); maximum limits are 13° to 37.8° C. (55° to 100° F.). 12. If the pilot bottle of the lot shows no evidence of contamination at the end of 10 days, this lot of plasma is ready for issue. It is advisable to retain the pilot bottle as a control. That is, it may be retained until all reports have been returned on this particular lot of plasma. As the pilot bottles thus accumulate, they may be pooled, cultured, and issued for use. In addition to its use for the final ste- rility test, the pilot bottle permits the physician in charge of the plasma unit to have a bottle of plasma from each lot for study at any time desired. (See p. 81, par. 20 and 21; par. 18 does not apply.) 13. Liquid plasma, containing a 5-percent concentration of dextrose, may be safely and satisfactorily stored at room temperature for as long as 2 years. 14. Should liquid plasma prepared by this or other methods be stored in an ordinary refrigerator, this temperature will cause pre- cipitation of large amounts of fibrin, frequently sufficient to make it practically impossible to administer the plasma through the standard types of filters used in administration sets. This will also be true when plasma without added dextrose is stored at room temperature. Preparation and Use of Frozen Plasma The technique for preparing frozen plasma differs slightly from that for liquid plasma. The steps outlined below have been found to be satisfactory. 1. Plasma is pooled as before. 2. Cultures should be taken immediately if it is desired to preserve the maximum content of prothrombin and complement during stor- age in the frozen state. If, on the other hand, it is desired to place maximum reliance on the test of sterility so that the plasma may later 54 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE be thawed and stored for longer than 24 hours in the liquid state, the pool should be allowed to stand 24 to 48 hours at room temperature (60° to 80° F.) prior to taking the cultures. 3. The addition of the bacteriostatic agent is optional. (See p. 52.) 4. Aspirate plasma into the final containers. (See “Filling of Final Containers.”) If plasma is to be frozen, the final container should probably not be filled more than three-fourths to five-sixths full, in order to reduce the danger of breakage. 5. The plasma should be frozen promptly after the final containers are filled. The pilot bottle of the lot should be cultured and stored as indicated in items 10 to 12 on page 53. The plasma should not be used until all cultures have been reported negative at the end of 10 days. 6. It is preferable to place bottles in a frame which tilts them slightly so that more surface area is available to accommodate expan- sion of the plasma during freezing. 7. A special low temperature cabinet should be used. These units are available commercially and are ordinarily used for quick-freezing and storage of frozen foods, etc. The plasma is stored in this freezing chamber until ready for use. A temperature range of minus 15° to minus 20° C. is required. Within this temperature range, it is possi- ble that plasma may be stored indefinitely. Above minus 16° C., labile constituents of plasma will slowly disappear. Plasma should be com- pletely frozen within 4 to 6 hours after it is placed in the cabinet. Certain cabinets now available incorporate a quick-freezing device, such as a fan to circulate the air, or a means of placing the bottles containing plasma in direct contact with the refrigerating walls. If such a device is not built into the cabinet, quick freezing may be accomplished by immersing the bottles in a container filled with a substance (e. g., alcohol) remaining liquid at 20° C. Restoring Frozen Plasma to the Liquid State Place the container in a regulation water bath adjusted to 37° C. It requires 20 to 30 minutes for the frozen plasma to thaw. If plasma has been rapidly frozen and is thawed at 37° C., there is no fibrinogen precipitation. It is desirable to have plasma available for immediate use. This may be accomplished by thawing one or several bottles of frozen plasma and retaining them as liquid plasma. All plasma should be filtered while being administered. This reliquefied plasma should be stored at room temperature until used. It now has the same storage characteristics as described previously for liquid plasma. Preparation of Dilute Liquid Plasma Considerable evidence exists that pooling is unnecessary in the preparation of dilute plasma (50 percent plasma and 50 percent PLASMA PREPARATION 55 diluent). Plasma prepared by this method is low in protein content and is not as satisfactory for the treatment of some conditions as is undiluted plasma. (See p. 64, “Advantages for Blood Banks.”) The diluent commonly employed is 5 or 10 percent dextrose in normal saline. While this method is probably less desirable than pooling after centrifugation, its simplicity and the minimum amount of equip- ment needed may prove advantageous to the small plasma unit doing only a few bleedings per week. The technique for this method is essentially the same as that described under “Pooling of Citrated Liquid Plasma,” except that individual final containers are used in- stead of pooling bottles. The bacteriological cultures may be made by withdrawing plasma from the final container or by taking a sample during aspiration. Use 10 cc. from each container and place into two culture tubes. Incubate one at 37° C. and the other at room tem- perature. (See instructions for “Addition of a Bacteriostatic Agent.”) Before aspirating the plasma, check the serologic tests to make sure that they are negative. 1. Follow the instructions for opening the bottle containing the blood and preparing the final container (substituting in this case a 600 cc. bottle, containing 250 cc. of diluent, for the pooling bottle). There are given in items 1 thorough 5 on page 50. 2. Insert the needle of the airway filter through the rubber stopper of the blood bottle and release the vacuum. 3. Close the donor valve and plunge the donor valve needle through the stopper of the final storage container. 4. Insert the aspirating needle through the stopper and into the supernatant plasma. 5. Open the valve and start aspiration. The aspirating needle must be kept beneath the surface of the plasma to avoid loss of the vacuum in the final container. 6. A strong beam of artificial light should be focused at the junction of the bulfy coat and plasma during the aspiration. This serves to aid in the detection of cellular elements that might be drawn into the aspirating needle. Care must be taken not to aspirate any of the red cells. Do not attempt to aspirate all of the plasma, or a number of red cells will be pulled over into the final container. Aspirate the last 30 to 40 cc. of plasma very slowly to prevent lifting and drawing over the cells. Do not attempt to aspirate the last 10 cc. of plasma. It is possible to use the aspirating set more than once, and specimens for culture may be taken from the aspirating set during transfer to the next container. (See p. 66, “Culturing the Plasma” and “Multiple Aspiration.”) Handle bottle with care to avoid agitation of the red cells. 56 THE OPERATION OF A HOSPITAL TRANSFUSION SERVICE 2. EMPLOYMENT OF REUSABLE EQUIPMENT While some of the equipment now commercially available is entirely satisfactory for the production of plasma, many hospitals prefer to prepare their own equipment. This is particularly desirable in in- stitutions having well equipped laboratories, in view of the fact that it makes possible a considerable saving. The technique described here employs gravity for collection of the blood; it is simple and economical; and it requires minimal replace- ment of critical rubber parts. It can be used for separation of plasma by centrifugation or sedimentation, and maintains a closed system throughout the process (38, 39, and 40). For selection, preparation, and care of the donor during bleeding, and the storage of blood and of plasma, follow the instructions given elsewhere in this manual. (See pp. 4-5, 28-34, 52-65.) Apparatus for Collection of Blood The following parts are required for the assembly of the apparatus (see fig. 2) for collection of blood: 1. A glass bottle made of high grade hard glass, 9.2 cm. in diameter (3% in.), 16.6 cm. in height (6% in.), and with a capacity of approxi- mately 650 cc. This bottle will fit standard centrifuge cups, and is so built as to withstand high speed centrifugation (2,000 to 2,500 r. p. m.). The neck is short and has an inside diameter of 26 mm. 2. A hooded, two-hole rubber stopper, fitting the bottle described above. 3. Two pieces of glass or stainless steel tubing (B and G) 7 mm. outside diameter, 3 and 4 cm. long respectively. 4. Two pieces of transparent amber rubber tubing, 8 cm. long with an outside diameter of 0.48 cm. and a wall thickness of 0.16 cm. (G and E) connected to tubes B and G. 5. One air filter (F) consisting of a glass tube 6 mm. outside diameter and 5 cm, long, with both ends slightly closed by flaming. This tube is filled with cotton, and connected with rubber tube {G). 6. One glass window {H), consisting of a glass tube 5 cm. long and 6 mm. outside diameter, connected with rubber tube E. 7. One piece of rubber tubing (/) of the same size mentioned under 4 above, 60 cm. long, connected to glass window (77). 8. One bleeding needle (/), 5.62 cm. long in.), gage 15, with a round hub to fit rubber tube (/). The needle is protected by a 15 by 100 mm. glass tube firmly fitted with cotton around the hub of the needle (