Evaluation of platelet transfusions from Histocompatibility locus: a matched and nonmatched unrelated donors to thrombocytopenic patients

Prolonged platelet transfusion therapy often tends towards a refractory state in the thrombocytopenic patient. This may be caused by alloimmunization (isoimmunizaytion) to platelet antigens, especially transplantation antigens of the Histocompatibility locus-A (HL-A) system. Histocompatibility between patient and donor is essential for the hemostatic effectiveness of transfused platelets. In this study, platelet transfusions were studied in 19 patients exhibiting different thrombocytopenic states. The number and frequency of platelet transfusions were determined by the recipient's physician(s). All patients that received ABO specific non HL-A matched platelets became refractory after 30 units. Patients that received ABO specific non HL-A matched platelets, bleeding severely and had received blood transfusion six months prior to this study, demonstrated the lowest 20 hour increments of all group investigated. They demonstrated median and mean increments of 1,230 X M2/unit and 890 X M2.unit respectively. Patients that received ABO specific non HL-A matched platelets, not actively bleeding and had not received blood transfusions six months or longer prior to this study, responded with a median and mean 20 hour increments of 19,460 X M2/unit and 21,460 X M2/unit respectively. These values are similar to those of non-sensitized patients receiving non HL-A matched platelets over a short period of time. One patient received ABO, HL-A matched compatible platelets. He did not show the expected response. This was probably due to sensitization to non HL-A antigens and specific platelet antigens from prior numerous blood transfusion. The patient died soon after admission into the hospital. Two patients received both HL-A matched platelets. The median and mean 20 hour increments were 940 X M2/unit and 3,380 X M2/unit with HL-A matched platelets as compared to 200 X M2/unit 400 x M2/unit respectively with non HL-A matched platelets. The feasibility of establishing a volunteer HL-A platelet donor program was also investigated. At this time it appears a 100 percent volunteer donor program is not possible until such time of greater public, physician, and blood transfusion services education, and awareness of the necessity of HL-A matched platelets in prolonged platelet therapy. Utilizing occasional paid donors it was possible to establish a HL-A match platelet transfusion program.

EVALUATION OF PLATELET TRANSFUSIONS FROM HISTOCO~WATIBILITY LOCUS-A MATCHED AND NONMATCHED UNRELATED DONORS TO THROMBOCYTOPENIC PATIENTS by Judith Grace Ow Young A thesis submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the degree of Master of Science in Medical Technology College of Pharmacy University of Utah August 1975 Copyright G) Judith Grace Ow Young 1975 All Rights Reserved LJNIVE1{SITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a thesis submitted by Judi ttl Grace O\'lYoun:� I have read this thesis and have found it to be of satisfactory quality for a master's ()k"t(]k1� [,lr'ccl l'!i:/;J.: : ' . :) . t Chainnan. Supervisory Committee T hav(' read this thesis and havc found it to be of satisfactory quality for a master's degree. � t;cLnley Date :11. D i .;::.rcu.�, . Member, Supervisory Committee T have read this thesis and ha\'c found it to be of satisfactory quality for a master's degree. \ t'/('c f,<� Date / T have read this th('sis and h:1\,(' found it to be of satisfactory quality for degree. ' )1 l"r: l,�� Date , ,,, " �-f-'h·/ ',Ll4' (;f " ' {-l-t�t·.t.. . ,;-.... . . , ' J i Garth Croft, �.f . Member. Supervisory Committee � a master's UNIVERSrIY OF FINAL UTAH GRADUATE READING SCHOOL APPROVAL To the Graduate Council of the University of Utah: I have read the thesis of ,riLe] i.:tt_ G L=-Clg!-� J'J}:«(()U-ri C______ ___ ___ _ final form al l d have found that (I) its format, citations, and bibliographic cOllsisten t and acceptable; (2) its HI style Its are illustrative materials includ ing figures, ta bles, and charts are in plac e ; and (3) the final manuscript is satisfactory to the Supel·;isory submission Commi tt ee and is ready for /r�ecl to the G radu at e SchooL :iiVZLt ;':.�-)., Member, Supervisory Committee Approved for the Graduate Council J; ' / �-'J-- j/ /;'</, - ....:J lil-- _-//1 -,�-/ ---fL _'--L!. - . ), ( ! � terlino: i<:'j. :'1'1.1), ')' l j , _.I i- L? , ) '" r.-;dlurrjc-, I Dean'l'o( thl' Graduate School Il1.D. , ,1- ACKN01JJLEDGEIvIENTS The author wishes to express her appreciation to the following individuals and laboratory personnel: ~~s. Bonnie Wilson and her staff of the Utah Heart Association for their help in contacting potential donors. All donors who participated in this program. The Latter-day Saints Hospital Blood Bank staff of medical technologists, register nurses and secretaries for their help and patience in drawing of plateletpheresis donors and paperwork. Ii/IrS. Loslc Barwick, R. N., IJatter-day Saints Hospi tal Blood Procurement, for composing letters and help in correlating plateletpheresis donors. Donors' HL-A typing were possible through Intermountain Regional Medical Program, project no. 58: Intermountain Regional Platelet Typing. The laboratories of Dr. Paul Terasaki, University of California, Los Angeles, Department of Surgery, and Dr. Charles DeWitt, University of Utah, College of Medicine, for their patience in receiving last minute sample testing. To my committee members, Drs. Fred Miya (chairman), Stanley Marcus, and J. LaMar Robbins and Garth Croft, M.S., for their advice and aid. TABLE OF CONTENTS Page ACKN OvlLEDGEMENTS . . • . LIST OF ILLUSTRATIONS. ABSTRACT . . .. INTRODUCTION . ....... ·... ... ... • • Platelet Platelet ... ..... • • II • • • ·...... Function • . . . . . . . . Therapy. ·...... . . Procurement • . REVIEW OF LITERATURE . Platelet .... Platelet Antibodies • . HL-A System . . . . . . • • • ....... . ·... .. .·... ..... ·... ........ HL-A Platelet Therapy • . . .. Volunteer Donor Recruitment. Random donors . • • HL-A typed platelets • ..... ..... ·. Evaluation of Bleeding in Recipients . • 3 3 5 7 11 13 30 34 ... ·..... . • • • . . . . . . . . .. . ... . • • . . • • • • 29 32 Recipient's Response • • • • . Plateletpheresis Donors • 1 30 Laboratory Test Results •• • • • viii 29 Platelet Procurement. . . . . . • • . . RESULTS. . • . . vii 22 r'IlATERIALS AND METHODS. . • . • • Notes • • iv 35 35 38 39 39 Page Recipient's Response • . . . . .... 40 HL-A matched vs non HL-A matched platelet response • . . . . . 44 Recipient's response vs number of platelet transfusions . . . . . . . . ...... 44 ..... 48 DISCUSSION . . . . . • . . .... HL-A Donor Program. 48 50 Recipient's Benefit HL-A matched platelet transfusion . . .... 50 HL-A vs non HL-A matched platelets • . 51 Prior exposure to HL-A antigens . . . 54 Actively bleeding vs non-actively bleeding . 55 Long term platelet therapy . 56 Conclusion. . .. 57 APPENDIX . 59 REFERENCES CITED . . 68 VITA . . . 75 vi LIST OF ILLUSTRATIONS Page Figures ·. 1. Hemostasis Sequence • • 2. HL-A Chromosome • • • • • • • 3. HL-A Inheritance. 4. Individual Platelet Response •• • • • • • • 43 5. Recipient no. 22 HL-A Matched Platelet Response. • • • • • • • • • • • • • • • • •• 53 1. Antigens on Platelets, Erythrocytes and LeUkocytes • • • • • • • • • • • • • • • ... 12 2. HL-A Antigens . • • • • • • • • • • • • 18 3. Review of Literature Median HL-A Matched vs Unmatched Platelet Response • • • • • • • • . 25 4. Yankee' s Compara ti ve Median HL-A 1\'1a tched vs Unmatched Platelet Response . • • • • • • •• 25 5. Plateletpheresis Donor's Response . • • • • • .... • • • • • 5 ·...... 17 • • • • • • • • • • 18 • Tables ..• 6. Clinical Study of Recipients • • • • ·.... ·.... 39 41 7. HL-A vs Non HL-A Matched Platelet Response. . 45 8. Long Term Platelet Therapy Response • • • • . 47 ABSTRACT Prolonged platelet transfusion therapy often tends towards a refractory state in the thrombocytopenic patient. This may be caused by alloimmunization (isoimmunization) to platelet antigens, especially transplantation antigens of the Histocompatibility Locus-A (HL-A) system. Histocompati- bility between patient and donor is essential for the hemostatic effectiveness of transfused platelets. In this study, platelet transfusions were studied in 19 patients exhibiting different thrombocytopenic states. The number and frequency of platelet transfusions were determined by the recipient's physician(s). All patients that received ABO specific non HL-A matched platelets became refractory after )0 units. Patients that received ABO specific non HL-A matched platelets, bleeding severely and had received blood transfusions six months prior to this ~udy, demonstrated the lowest 20 hour increments of all groups investigated. They demonstrated median and mean increments of 1,2)0 X M2/unit and 890 X M2/unit respectively. Patients that received ABO specific non HL-A matched platelets, not actively bleeding and had not received blood transfusions six months or longer prior to this study, responded with a median and mean 20 hour increments of 19.460 X M2/unit and 21.460 X M2/unit respectively. These values are similar to those of non- sensitized patients receiving non HL-A matched platelets over a short period of time. One patient received ABO, HL-A matched compatible platelets. He did not show the expected response. This was probably due to sensitization to non HL-A antigens and specific platelet antigens from prior numerous blood transfusions. The patient died soon after admission into the hospital. Two patients received both HL-A matched and non HL-A matched platelets. The median and mean 20 hour increments were 940 X M2/unit and 3.380 X rvr 2/unit with HL-A matched platelets as comnared to 200 X M2/unit and 400 X M2/unit respectively with non HL-A matched platelets. The feasibility of establishing a volunteer HL-A platelet donor program was also investigated. At this time it appears a 100 percent volunteer donor program is not possible until such time of greater public, physician, and blood transfusion services education, and awareness of the necessity of HL-A matched platelets in prolonged platelet therapy. Utilizing occassional paid donors it was possible to establish a HL-A match platelet transfusion program. ix INTRODUCTION There is an increasing demand for platelet transfusions for the treatment of thrombocytopenia and for controlling acute hemorrhage in leukemic and aplastic patients. The transfusion of viable platelets that will function at their optimal capacity and survival rate is necessary to adequately treat these patients. The result of increased viability and function of transfused platelets, there is a more rapid correction of bleeding time and peripheral platelet count to normal levels (1,2). This reduces the total number of plate- let transfusions per patient, the cost to the individual patient, and the need for blood transfusion services. With long term or massive transfusions. alloimmunization (isoimmunization) may result causing a drastic decrease in transfused platelet survival rate and even greater thrombocytopenia (),4,5,6). Alloimmunization may involve Histo- compatibility Locus-A (HL-A) antigens, which are found on platelets and leukocytes, and/or platelet specific antigens (PI-A, PI-E, Ko). Platelet specific antigen alloimmunization occurs less frequently than HL-A alloimmunization because platelet antigens are of higher frequency in the general population (1,7,8.9,10). Once refractory, the recipient's response to non HL-A matched platelets is not improved by splenectomy, corticoids, androgens or any combination of 2 these (11). The prevention of alloimmunization and effective therapy for patients already refractory to non HL-A matched platelets can be accomplished by transfusing HL-A matched compatible platelets (9,10,11,12,13,14,15). The purpose of this study was two fold: first, was it feasible and practical to set up an all volunteer HL-A platelet donor program for Salt Lake City and surrounding areas; and second, will patients benefit from such a program? REVIEW OF LITERATURE Platelet Function Platelets are fragments of megakaryocytes that circulate in the blood stream as cytoplasmic discs with a curvilinear life span of 9.5 ~ 0.6 days (16,17). The role of platelets in maintaining hemostasis depends on three of their properties: endothelial support by adhesion, which is measured by capillary resistance tests; release of lipoprotein material (platelet factor 3), which is measured by the prothrombin consumption time and other coagulation tests; and, formation of hemostatic plugs by aggregation, which are measured by the standard template bleeding time. When endothelial walls are damaged, there is exposure of the underlying basement membrane and collagen and/or subendothelial microfibrils. Passing platelets adhere and flatten themselves over the exposed area in a monolayer fashion. The platelets then become sticky and form surface "spikes" causing adhesion of additional layers of platelets. In normal individuals, platelets also adhere to the lining of the endothelial wall to strengthen them. In thrombocyto- penic patients, insufficient numbers of platelets are available for incorporation into the endothelial cytoplasm and in this weakened state, the slightest trauma may cause a bleeding breakthrough in the endothelial lining (3). 4 During the release reaction of platelets, substances discharged can be divided into three groups: first. those that are released within 20 seconds and primarily consist of ADP, serotonin and calcium; second. a group of rapid released components which are released during the first two minutes and consist of fibrinogen, platelet factor 4 and some hydrolytic enzymes; and third, slowly released platelet factor 3 (18). The mechanism of aggregation is still debated. In 1971, Booyse, et al., as cited by Firkin, 1972 (18), proposed that platelets are coated with thrombasthenin (S Thrombasthenin) which lengthens by uncoiling after exposure to ADP or thrombin. The resulting protuberances link themselves with pro- tuberances from neighboring platelets and a bridge is formed, with subsequent contraction and aggregation. Figure 1 shows a schematic diagram of events leading to primary hemostasis as depicted by Firkin (18). Other functions or roles of platelets have been described by many authors (1,5,19). These include clot retrac- tion, vasoconstriction, aggregation in presence of bacteria for clearance into the reticuloendothelial system for destruction, the absorption of endotoxins from gram negative bacteria, and maintenance of tissue integrity of the gastrointestinal tract to prevent bacteria from entering into the general circulation. 5 Figure 1. Hemostasis Sequence Damaged endothelial wall----,)Exposure to collagen and/or unidentified subendothelial fibrils / J -' - - Platelet adhesion t small Platelet aggregation not ADP dependent amount~ ADP ..... \. 1 ~Primary . Adenosine ~ Platelet refractoriness platelet adhesion I /' Platelet factor 3 and ac tiva ti on 0 f " coagulation cascade Thrombin- ,Release reaction I /~/ - - - - - - large amount ADP ~ J Secondary aggregation General agreement among investigators: Some disagreement among investigators: Platelet Therap:y In 1910, Duke first demonstrated the value of fresh platelet transfusions for the management of hemorrhage in thrombocytopenic patients, as cited by Gardner, 1966 (20). Because of inadequate equipment and knowledge of platelet collection, platelet transfusion lay dormant for forty years. In 1957, Faber, et al. (21) renewed the interest in platelet transfusion by demonstrating in a ten year study the full value and safety of platelet transfusion for controlling bleeding in leukemic and thrombocytopenic patients. Since 6 then numerous studies have confirmed Duke's initial study (16,22,2),24). In 1962, Gaydos (19) attempted to determine the "threshold" peripheral platelet count for spontaneous bleeding. No "threshold" level could be demonstrated. Gaydos and subsequent investigators concluded that counts above 70,000/mm J there is little or no risk of hemorrhage, between 20,000 and 70,000/mmJ spontaneous bleeding but not surgical bleeding can be controlled, and below 20,000/mmJ there is high risk of uncontrolled spontaneous bleeding (2,19.20). It appears that bleeding tendency is a variable phenomenon which involves the clinical state of the individual, for many leukemics and persons with hereditary thrombocytopenia do not show signs of hemorrhage even when platelet counts are between 5,000 and 10,000/mmJ (25). Some investigators believe that when hemorrhage occurs, not only must viable platelets but also young platelets must be transfused (20,22). Young platelets which are synthe- sizing protein are sequestered by the spleen for a short interval; they are then released into the circulation and become hemostatically effective. Other investigators believe that platelet age does not make a great difference and that only circulating platelets are hemostatically effective (6. 26). Cronkite (25) believes that non-circulating platelets are also hemostatically effective. Before the effects of immunization from prolonged platelet therapy were know, it was surmised that platelet trans- 7 fusion would: 1. Increase the post transfusion platelet count. This suggested that an excess number of platelets were transfused. After the needs of the endothelium and hemostatic plug formation were satisfied, the excess platelets remained in the peripheral blood circulation (). 2. No increase in the post transfusion platelet count. This suggested that the transfused platelets did not circulate because: a. Nonviable platelets were transfused and were removed by the reticuloendothelial system. b. Viable platelets were transfused, circulated and incorporated into the endothelial wall of blood vessels. c. Viable platelets were transfused, circulated and incorporated into the endothelial wall and the excess were utilized in the formation of hemostatic plugs. Hypotheses band c have been supported by the studies of many investigators which demonstrated an improvement in vascular resistance without significant changes in the post transfusion platelet count and/or bleeding time (),20,26,27). Platelet Procurement With the undisputed knowledge of the value of platelet therapy, investigators turned to the problem of the collection and storage of platelets. Earlier investigators used silicon coated syringes for the collection of fresh platelets. In 1956, Klein, as cited by Djerassi, 1966 (1), developed the 8 closed system plastic bag. With the advent of plastic bags, platelet collection became easier and resulted in decreased risk of bacterial contamination. It has been suggested recently that Fenwal's PL-146 plastic bags maintain viability of platelets longer than other plastic bags (28,29). Fenwal's satellite bags are made of PL-146 plastic. The anticoagulants used for whole blood collection were applied to platelet collection. Heparin was used but it was soon found that it made platelets extremely adhesive and thus difficult to separate from whole blood via centrifugation. Ethylenediaminetetraacetate (EDTA) was found to preserve platelets as well as heparin though in large amounts it became toxic to platelets and caused renal toxicity to the recipient. In addition, large amounts of EDTA inactivated some of the blood coagulation factors in the recipient, as demonstrated by vitro tests ()o). Platelets collected in EDTA are temporarily sequestered by the liver and spleen and then released into the circulation after a few hours. Platelets collected in Acid-Citrate-Dextrose formula A (ACD-A) were also found to be temporarily sequestered by the spleen soon after transfusion but to a lesser extent than those collected in EDTA. ACD-A collected platelets tended to clump during preparation after centrifugation but were easily resuspended. Platelets collected in Citrate- Phosphate-Dextrose (CPD) also tended to clump during centrifugation and required more time to resuspend than ACD. CPD collected platelets are sequestered to a lesser extent 9 than those in ACD-A. After transfusion of normal individuals, calculated recovery rates have been around 70-75 percent with CPD and 15-35 percent with EDTA and ACD-A (31). Recently the freezing of platelets have been investigated. data show promis Initial results. The last problems of platelet collection involves storage time and temperature prior to transfusion. is still under investigation. This area Investigators are divided into two groups; those advocating 4°c storage temperature and those advocating 22 0 C storage. Numerous studies have demonstrated that upon short exposure to the cold (4°c), platelets change from their normal disc shape appearance to spheres. This is the result of the formation of microfila- ments and microtubules along the axis of rigid projections from the cytoplasm. this change is After two hours of exposure to the cold i~reversible (28,29,32,33,34). Ando (35), demonstrated that platelets either fresh, stored at 4°C for four hours, or stored at 22°C for four hours, did not differ significantly in distribution of membrane polypeptides and glycopeptides, nor in levels of sodium flux and gluthathione. At 4°c not only did the plate- lets form spheres but their electrophoretic mobility, contractile response as well as vivo life span decreased. Those stored at 22 0 C exhibited decreased calcium uptake compared to platelets stored at 4°c for four hours. Also a longer life span in vivo was reported together with decrease cell survival due to a lowering of pH in vitro from lactic 10 acid formation during storage. In 1974. Murphy (29) employed Cr 51 labeled platelets; fresh, stored at 22°C and stored at 4°C for eighteen hours. Immediately after infusion of the fresh platelets, 60-70 percent of the platelets were found in the circulation and they had a mean half-life of 4.3 days. Platelets stored at 22°C. 52 percent were found immediately after infusion in the circulation with a mean half-life of 4.0 days as compared with 40 percent and mean half-life of 1.2 days for 4°C stored platelets. Murphy postulated that if in vitro pH fell below 6.0 there would be decreased survival in vivo due to morphological changes. Platelets would change from their normal disc shape to spheres, similar to the change observed when platelets are exposed to the cold, and this change would result in immediate removal from the circulation. Increased survival was accomplished in his study by maintaining a pH above 6.0 by increasing the amount of resuspending plasma of the concentrate. Many investigators agree that the infusion of 4°C and 22 0 C stored platelets will yield satisfactory post transfusion platelet increments (32,33,34). Platelet increment measures the rise in post transfusion platelet count after the infusion of one unit of platelets. Platelets stored at hOC are able to induce hemostasis more qui~kly, though they have a shorter life span in vivo. It has been suggested that 4°c stored platelets should be infused into actively bleeding patients since the concern is the arrest of hemorrhage and 11 not the life span of platelets in vivo. Platelets stored at 22 0 C survive longer in vivo but appear to have some type of functional defect that requires a twenty-four hour post transfusion period to be "repaired" before becoming effective (28,32,33,34). It has thus been suggested that 22 0 C stored platelets be administered primarily for prophylaxis. The ideal would be to prepare platelets at 22 0 C and transfuse them as soon as possible or within six hours (23,32). Platelet Antibodies With increased platelet therapy, in the 1950's investigators began to report patients receiving long term therapy to have decreased post transfusion platelet counts and platelets with decreased life span. the prevailing explanation. By 1960 alloimmunization was The major antibodies responsible were found to be directed against antigens shared by both platelets and leukocytes, the Histocompatibility Locus-A (HL-A) antigens and platelet specific antigens. The three major systems of antigens on platelets are PI-A, PI-E, and Ko. The frequency of these antigens in the general popUlation are: PI-AI (Zw a ) 92 percent, PI-A 2 (Zw b ) 26 percent, PI-E l 99 percent, PI-E 2 5 percent, Ko a 16 percent, and Kob 99 percent (17). With subsequent investigations on platelet antigens, it was discovered that platelets shared many antigens with white cells as well as with erythrocytes (4,5,7,36). 'Table 1 list some of the antigens common to erythrocytes, platele.ts and leukocytes. 12 Table 1. Antigens on Platelets, Erythrocytes and Leukocytes Antigens on platelets only Antigens on leukocytes only Antigens on platelets and erythrocytes Antigens on leukocytes and erythrocytes A, B, and H A, B, and H Dif- D* C* Cw E c~t- S M and N T· a E~f- (L~b ) C M and N p.r.. T· a (L~b ) Lea (Lu b ) P U JkaJk b I and i Gerbich HL-A K Fya I and i * Disagreement among investigators () Doubtful results only at present time Shulman in 1966 (6) demonstrated that platelet alloimmunization occurs infrequently with the transfusion of less than ten units of whole blood~ approximately 5 percent of such recipients will have detectable antibodies. With increasing number of transfusions, the incidence of platelet specific antibodies increases; 16 percent after 10-25 13 units, 24 percent after 25-50 units, 37 percent after 50-100 units and 80 percent after 100 units. Of the patients Shulman studied, 50 percent had incomplete antibodies and they could not be detected by the standard agglutination tests. Complement fixation testing was found to be the most sensitive test for platelet antibodies. Shulman postulated recipients who demonstrated decreased response to platelets but who had no detectable antibodies did in fact possess a low titer of antibody that could not be detected in vitro. The concentration of these antibodies were sufficient to cause platelet destruction in vivo. A five to ten-fold increase in the titer would be necessary for in vitro detection. Women with gr~ater than three pregnancies have been shown to have platelet specific antibodies with an incidence of 15-30 percent. Neonatal purpura is caused by Anti-PI-AI 50 percent of the time (6). HL-A System The HL-A system is a highly complex genetic system of multiple alleles similar to the Rhesus system in man and the H-2 system in mice. The antigens are present on the cell surfaces of nucleated cells, including most tissues and circulating cellular elements. It was thought HL-A antigens were absent from erythrocytes but recent investigations have shown HL-A antigens or HL-A antigen precursor substances on reticulocytes, nucleated erythrocytes and mature erythrocytes 14 (7,37,38,39). The HL-A system is second in importance to the ABO system in organ transplantation, and in platelet and leukocyte long term transfusion therapy. In human transplantation, the HL-A system is responsible for organ rejection and is analogous to the H-2 locus in mice, Ag-B locus in rats. and the B locus in chickens (40). The observation that platelets and leukocytes shared antigens in common with other tissues was an early step in the elucidation of histocompatibility relationships in man. In 1961. Baldini, et ale and Bosch, et al., as cited by Yankee, 1974 (5), demonstrated that the survival of transfused platelets was shortened in recipients who had previously received skin grafts from the same platelet donor. In blood transfusion services, HL-A antibodies are seen as immune responses associated with febrile transfusion reactions against leukocytes and platelets, and as a lack of response to leukocyte and platelet therapy. Like other blood systems the HL-A system was first suspected to exist and defined after the discovery of its antibodies. As cited by many investigators (41,42.43,44), Doan in 1926 was the first to describe the presence of leukocyte antibodies after blood transfusions. Dausset in 1952 dis- covered similar antibodies in multiply transfused patients and in 1958 demonstrated that the antibodies were allospecific (isospecific) by immunization of volunteer donors with a single individual's leukocytes, as cited by Zmijewski and Fletcher, 1972 (41). Dausset postulated immunization can 15 occur not only as the result of blood transfusion but also from pregnancy. This postulate was confirmed by Payne and Rolfs in 1958 and by van Rood in 1959 (41). In 1964, Jensen (45) found HL-A leukoagglutinins in 2 percent of women after one pregnancy and 16 percent in women after two or more pregnancies. Also those women with erythrocyte antibodies had a higher incidence of leukocyte antibodies than those without erythrocyte antibodies. The majority of the leu- kocyte antibodies were of the incomplete type. The incidence of lymphocytotoxic antibodies following pregnancy are higher than those of leukoagglutinins. 1969 (L~6) Overweg and Engelfriet in were able to detect lymphocytotoxic antibodies as early as the twenty-fourth week of the first pregnancy. Lymphocytotoxic antibodies were present in 10 percent of women during the first pregnancy and 13 percent after the first pregnancy_ In 1967, Goodman and Masaitis, as cited by Mollison (7) detected lymphocytotoxic antibodies ln 25 percent of women after the first pregnancy and in 55 percent of women after a third pregnancy. In 1964 confusion existed concerning the leukocyte antigens. This resulted from investigators employing different techniques, antisera, nomenclature, and theory as to mode of inheritance. The techniques used at that time and that are still employed are: 1. Leukocyte agglutination. van Rood and van Lecuween first described this technique in 63 (47). rently this method is principally applied when Cur- 16 typing polymorphonuclear leukocytes. 2. Lymphocytotoxicity. Walford, et all first des- cribed this technique in 1964 (48) as a modification of Gorer's technique of detecting leukocyte antigens in mice. This method is principally applied when typing lymphocytes. Complement fixation reaction was applied to leukocyte and platelet studies in the latter 1960's. This method was found to be preferable when applied to platelets and not leukocytes (5,7,17,49). Complement fixation studies of plate- lets are often used as a supplement to lymphocytotoxicity testing. Antisera was and is still obtained from multiply transfused individuals, multiparous women, persons having rejected skin grafts and organ transplants, and from voluntarily immunized donors. In 1965 at a histocompatibility workshop held in Leiden (50), investigators tested their own antisera with a panel of cells previously typed by van Rood. The results were computerized and it was shown that many investigators had in the past detected the same antigens and had given them different names. This workshop was the inital step in clarification of the confusion concerning HL-A antigens. Later observations resulted in a 1967 histocompati- bility workshop (51) and through family studies the mode of inheritance of the HL-A antigens was elucidated. It was agre,ed by the World Health Organization to name the area of the chromosome responsible for the antigens to 17 be Histocompatibility Locus-A, or abbreviated HL-A. Anti- gens of the HL-A system are present on two separate but closely linked loci on a single pair of autosomal chromosomes. Chromosome 6 has been implicated recently (43,52). The loci are closely linked since there is less than I percent crossing-over (53). Multiple alleles at each loci are responsible for two separate groups of antigens: first segregant series (old terminology - LA series) and second segregant series (old terminology - Four series or Seven series). The second segregant series can be divided into two groups: 4a and 4b according to their reactivity with 4a and 4b antisera. Schematically the chromosome carrying the information for the HL-A antigens is shown in figure 2 (53). Figure 2. HL-A Chromosome o----~I----~--~I--~I-MLC FOUR AJ LA Analogous to the H-2 locus in mice, the centromere is to the left of the histocompatibility locus. The antigens tested for in this study and the antigens currently widely accepted are listed in table 2 (54). Offspring inherit from each parent one of the two possible chromosomes (haplotype) which confer antigenic determinants of one antigen per series (figure 3). The antigens are co-dominantly expressed and therefore an individual's 18 Table 2. HL-A Antigens First Segregant HL-A HL-A HL-A HL-A 1 2 3 9 Second Segregant (Mac, PLB-l) ( IrJ23 = 9.1) (~v24 9.2) HL-A 10 (~112 5 :: 10.1) (w26 = 10.2) HL-A 11 ~]19 (TE 19) (TE 40) W28 (TE 63) W29 (TE 66) 1;v30 (TE 59) W32 5 7 8 12 (TE (TE HL-A 13 (TE W5 (TE WI0 W14 (TE (TE W15 (TE vJ16 (TE W17 W18 (TE (TE W21 W22 (TE (TE ~v27 HL-A HL-A HL-A HL-A 87 = 12.1) 88 :: 12.2) 50) 60) 54) 55) 64) 57) 58) 61) 51) 52) Prefix vJ represents antigens defined during histocompatibility workshops 1970 and 1972. Prefix TE represents equivalent specificities from Dr. Paul Terasaki's laboratory, UCLA. Figure 3. HL-A Inheritance Genotype: Mother Haplotype: Offsprings genotype : Phenotype: (2,12) (3,13) Father (9,5) (10,8) (2.12) ; (3,13) ; (9,5) ; (10,8) (2,12) (9.5) ; (2,12) (10,8) ; (3,13) (9,5) (3,13) (10,8) 2,9,5,12 ; 2,10,8,12 ; 3,5,9,13 ; 3,10,8,13 19 phenotype can only consist of a maximum of four antigens and a minimum of two antigens. If an individual fails to type for a full complement of four HL-A antigens then family studies must be done to differentiate between homozygosity for one or two antigens, the possibility of a new antigen not yet defined by available antisera, or a known antigen too weak to be detected with available antisera. nicity of the HL-A antigens varies. Antige- HL-A 12 is a weak anti- gen and cannot be detected in 20 percent of those individuals possessing it (5). HL-A 2 antigen is capable of showing dosage effect (49) and HL-A 1 and HL-A 2 stimulates immunization more frequently than HL-A ) or HL-A 12 (5,9,55,56). The number of known antigens increases with time. At present 14 are know1 for the first segregant and 27 for the second segregant. With two loci of multiple alleles, the polymorphism of this system is remarkable. According to Kissmeyer-Nielsen, et al (57), the polymorphism of this system can be shown on a numerical basis. With two loci of 14 and 27 antigens respectively, there would be )78 different haplotypes which would combine into 71,6)1 different genotypes, and 32,384 different phenotypes would result from the combination of 92 different first segregant phenotypes and 352 different second segregant phenotypes. In recent years a possible third locus, AJ, has been discovered (4),57.58,59). With this third locus the number of genotypes would be over 2,000,000 and the number of phenotypes would be over 1,000,000 (43). 20 The biochemistry of the antigens has not been completely elucidated. It is lmown that HL-A 2 is a soluble substance and HL-A 7 is a low densi ty soluble ~ -lipoprotein (41). The soluble HL-A and H-2 antigens are glycoproteins comprising of 92 percent protein and 8 percent neutral sugar (60). Papain soluble HL-A antigens are composed of two polypeptide chains which may be separated under dissociating conditions. One chain has a molecular weight of 30-31,000 and carries the HL-A alloantigenic specificity. chain, a ~-2 The other microglobulin, has a molecular weight of 11-12,000, Rf of 0.47 by disc electrophoresis and appears to be a common characteristic feature of all HL-A antigens (61,62). The f-2 microglobulin may play an important part in immunological reactions (61). Some antigens have been discovered in association with certain diseases such as anklylosing spondylitis, Graves disease, Hodgkins, acute leukemia, and Systemic lupus erythematosus. It has been proposed by Grumet, et all (63), that HL-A genes influence the susceptibility to disease in either of three ways: 1. They represent specific receptor sites for the attachment of virus. 2. They have antigenic receptor sites common with viral or tumor antigens, which result in these antigens to be recognized as self. 3. They are linked to immune response gene(s) similar to Ir-l in mice. 21 On the same chromosome as HL-A there is a locus responsible for the reactivity seen in the mixed lymphocyte culture test. The locus is named after the test as Mixed Lymphocyte Cytotoxicity (MLC). The MLC test is an in vitro measure of the immlli1e response of living lymphocytes when they are stimulated with allogenic lymphocytes (57). Mito- mycin-C-treated cells are mainly employed as the allogenic lymphocytes. Treatment with mitomycin-C prevents lympho- cytes from transforming to blast cells but it does not destroy their antigens and they are thereby still capable of stimulating other lymphocytes. Blast cell formation by the untreated lymphocytes indicates they have recognized a foreign antigen on the treated lymphocyte. The degree of stimulation is determined by the measurement of DNA synthesis using the cell incorporation of radioactive thymidine. At first it was thought that this reactivity was due to immune response by lymphocytes to foreign HL-A antigens. This concept was supported by observations that identical HL-A siblings are non-reactive with each other in culture. This concept may not be true because recent observations have shown some siblings that are HL-A mismatched are MJJC nonreactive. These recent observations may represent another MLC locus closely linked to the HL-A locus but distinct from the first rilLC locus (5). MLC reacti vi ty is of primary concern in organ and tissue transplantation. 22 HL-A Platelet 'l'herapy HL-A antigens and antibodies not only play a major role in the outcome of organ and tissue transplantation but also in the outcome of effectiveness of blood component transfusions. With prolonged blood and/or platelet transfusions, patients develop HL-A antibodies in addition to erythrocyte and platelet specific antibodies. Alloimmunization most frequently occurs with HL-A antigens due to the polymorphism of this system. Also platelet specific antigens have a higher frequency in the general population, therefore a lower occurrence of sensitization. Platelet specific antigen sensitization accounts for approximately 2 percent of these alloimmunization (10). Patients alloimmunized to HL-A antigens display decreased post transfusion platelet counts and a decreased life span of the transfused platelets. The rate and degree of alloimmunization varies and no "threshold" time or number of transfusions has been established. Alloimmuniza- tion may occur as early as after one blood transfusion or after seven to twenty platelet transfusions (10,42). Patients receiving more than five blood transfusions, 50 percent will demonstrate HL-A lymphocytotoxic antibodies (42). Shulman (6) believes it is not the number of trans- fusions given but the period of time over which they are given. Intense therapy over a short period of time would not result in immunization but a prolonged period of low intensity therapy would result in immunization. Howard and 23 Perkins (64) believe previous exposure to HL-A antigens will determine the rate of alloimmunization. They found cyto- toxic antibodies in 53 percent of their patients receiving repeated platelet transfusions. Those patients previously exposed to HL-A antigens developed detectable antibodies as early as four days and those with no history of previous exposure as early as ten days. Patients may receive mul- tiple platelet transfusions over a much protracted period of time and never become immunized. It therefore appears that the rate and degree of alloimmw1ization depends upon: 1. Number of previous transfusions. 2. Length of period transfusions are given. 3. Previous exposure to antigens. Grumet (44) pro- fesses that after the development of HL-A antibodies a second exposure to the antigen(s) will not cause an anamnestic response (secondary response). 4. Clinical status of the recipient. 5. Chemotherapy of the recipient. Patients on immuno- suppressive therapy have a lower tendency to form antibodies (5,8,12,23). 6. HL-A type. Patients positive for HL-A 3 appear to develop antibodies much later than other HL-A types (55 ,56) · Once alloimmunization has occurred, patients become refractory to non HL-A matched platelet transfusions. The recipient's poor response to non HL-A matched platelets cannot be improved with splenectomy, corticoids, androgens, or 24 any combination of these (11). In order to maintain hemo- stasis in refractory patients a larger number of platelets would have to be transfused to compensate for those transfused platelets being destroyed. Grumet and Yankee (11) believe five times the normal number of platelets would be needed. However, this approach has been shown to be of little benefit, for patients develop greater thrombocytopenia after transfusion (3,4,5,6). Shulman, as cited by Dausset, 1965 (4), attributes this response to the non-specific absorption of platelet antigen-antibody complex onto the recipient's ovm platelets thereby making them susceptible to the lytic action of complement. The only prevention of alloimmunization and effective therapy for recipients refractory to non HL-A matched platelets is the transfusion of HL-A matched compatible platelets. One must transfuse platelets without antigens for which the recipient possess antibodies. Response to platelet trans- fusion can be easily compared by relating one and 20 hour post transfusion increments of the recipient's platelet count to body surface area and to the number of units of platelets transfused. Initial HL-A matched platelet therapy was done with siblings. In 1972, Thorsby demonstrated HL-A matched plate- lets from unrelated donors also gave excellent results (15). Subsequent investigations have confirmed Thorsby's findings (8,10). With refractory recipients the median one and 20 hour increment platelet counts vary from investigation to 25 investigation. Table 3 summarize the varied increments reported in literature. Table 3. Review of Literature Median HL-A matched vs Unmatched Platelet Response 1 Hour 20 Hour Matched siblings o - 66.5 f,1atched unrelated o - 30.5 o - 54.5 o - 17.0 Unmatched o - 14 . .5 o- 2.5 In 1974, Yankee (5) compared values obtained from three different investigations of refractory aplastic and leukemic patients. Table 4. His :findings are given in table 4. Yankee's Comparative Median HL-A matched vs Unmatched Platelet Response 1 Hour 20 Hour Matched siblings 15.0 (11.0 - 21.0) 9.0 (0 - 16.0) Matched unrelated 15.5 ( 4.0 - 23.5) 9.5 (0 - 22.0) 2.5 ( Unmatched o - 6.5) 0 (ranges in parenthesis) Some of the differences in increments can be accounted for by the differences in the recipient's clinical status, pres- 26 ence of fever in the recipient, immunosuppressive therapy of the recipient, and the degree of HL-A match of the transfused platelets. The basic agreement is that post transfusion platelet counts are higher in those recipients receiving HL-A matched platelets and that hemostasis is obtained earlier than in those receiving non HL-A matched platelets. st~ted As previously, once a patient is refractory, splenectomy does not change the response to unmatched platelets. Sple- nectomy does aid in the response to HL-A matched platelets. Grumet and Yankee (11) demonstrated that with splenectomy there was an increase in recovery of transfused platelets and the median number of compatible platelet units necessary to maintain hemostasis was 4.4 units/M 2/week as compared with 8.4 units/~/week before splenectomy. Patients receiving non HL-A matched platelets did not exhibit a similar decrease in number of platelet units needed after splenectomy and signs of thrombocytopenia persisted. Yankee (13) demonstrated that the degree of HL-A match also influenced post transfusion platelet counts in refractory patients. firmed this. In 1974, Lohrmann, et ala (10) con- Unrelated donor-recipient compatibility were divided into three groups: A-match Donor and recipient had identical HL-A phenotypes. B-match Donor possessed all the HL-A antigens in the recipient's phenotype, but the donor lacked one (B-1) or two (B-2) of the recipient's 27 antigen (s ) . Mismatch: Donor had HL-A antigen(s) not found in the recipient. In tissue transplantation, the mismatch group further divided into C, D, and E matches (65). C-match Donor has one HL-A antigen not found in the recipient. D-match Donor has two HL-A antigens not found in the recipient and each antigen resides in separate subloci. E-match Donor has two haplotypes different from the recipient. Lohrmann, et ale studied 15 alloimmunized patients refractory to non HL-A matched platelet therapy. They found the infu- sion of A-matched platelets gave responses similar to those of HL-A identical siblings. A-matched platelets responses were higher than B-1 match, B-1 match were higher than B-2 match, and mismatched platelets gave 20 hour increments of zero. When considering platelet transfusion HL-A matched or nonmatched, ABO and Rh (D) type of the recipient and donor o must be considered. A, B, and H antigens have been shown to be on platelets but investigators have disagreed upon its influence on platelet transfusion effectiveness (2,6,8,10, 15,16). Most investigators feel that ABO specific plate- lets should be given if possible, but the HL-A type is the first consideration in choosing a potential donor. 28 The presence of Rho(D) antigen on leukocytes and platelets have also caused a great deal of disagreement among investigators (2,4,7,10,20,42). The majority of the inves- tigators do not demonstrate any adverse effect or Rho(D) antibodies being stimulated when Rho(D) incompatible platelets are transfused. Even if Rh (D) is only an erythrocyte o antigen, Rho(D) negative women of child bearing age or future child bearing age should receive Rh (D) compatible plateo lets since there are always some erythrocytes in the platelet preparation. MATERIALS AND METHODS Volunteer Donor Recruitment The initial goal was to recruit 600 volunteer donors of known ABO, Rh and HL-A types. Announcement of the HL-A plateletpheresing program was made by advertisement in the blood bank donor area, verbal communications and by a short introduction letter (appendix) to utah Heart Association donors and to interested individuals. alone was given to patient's siblings. Verbal information A large percentage, approximately 70 percent, responded by asking for further information. Via a second letter' (appendix) and/or tele- phone conversation, information concerning the actual plateletpheresing process and reasoning for the program were given. The number of persons responding with willingness to join was initially good but soon dropped to a disappointing number. Initially 15 - 20 donors were typed each week. After a few months the number dropped to 0 - 2 per week. initial goal of 600 donors was not obtained. The During a ten month period, 469 donors were enrolled into the program. Persons joining the program had a sample of 15 mI. blood dravm, in which 0.15 mI. sodium heparin (10, 000 units per mI .• beef lung, USP) was added (100 units per mI. blood). Donors' HL-A typing were done by Dr. Paul Terasaki's laboratory, University of California, Los Angeles, by lympho- JO cytotoxicity testing. Patients' and emergency donors' typing were done by Dr. Charles DeWitt's laboratory, University of Utah Medical Center. Dr. DeWitt's laboratory em- ployed lymphocyte microtoxicity trays from Dr. Terasaki's laboratory and the microlymphotoxicity technique outlined in N.I.H. Manual of Tissue Typing Techniques, 1974 (66). Table 2 list those antigens tested. Platelet Procurement Throughout this study two methods of platelet procurement were done: platelets collected from random donors, and platelets collected from plateletpheresis donors. Random Donors. For the purpose of this study random donors refer to donors not HL-A typed. Platelets from ran- dom donors were collected for transfusions when recipients had no siblings, offspring(s), or parents as potential HL-A matched donors; recipients were in need of immediate platelet transfusion and there was insufficient time available for recipient HL-A typing; recipients had lymphocyte counts that Vlere too low to harvest enough cells for HL-A typing; and when recipients' physicians were unaware of our program and/or the benefits of HL-A matched compatible platelet transfusions, therefore the physician did not have their patients HL-A typed nor ordered HL-A compatible platelets. Unfortunately the majority of the recipients fell into this large category of random donor platelet recipient. Platelets were harvested from ABO identical routine 31 whole blood donations. One unit of platelets was obtained from each 450 ± 45 mI. whole blood donation. Blood was collected in Fenwal l double blood pack plastic bags, JF-25N. The main collection bag contained 63 mI. CPD anticoagulant and the satellite bag devoid of any anticoagulant (appendix). The blood units were maintained at room temperature until time of platelet harvest, which was within four hours after blood donation. The unit was centrifuged in an International refrigerated centrifuge model Pr-6 2 at room temperature for four minutes at 2,400 rpm. The platelet rich plasma was expressed into the satellite bag and recentrifuged at room temperature for six minutes at 5,000 rpm. The platelet poor plasma was expressed back into the unit of packed red cells to form platelet poor whole blood and the platelet concentrate was resuspended in 30 - 50 mI. of the plasma and the satellite bag separated from the primary bag. The concen- trate was allowed to remain undisturbed for at least one hour at room temperature prior to agitation to disassociate any aggregated material or clumps of platelets. ABO, Rh, and syphilis testing were performed prior to labelling and transfusion of the platelet concentrate. Hepatitis asso- ciated antigen testing. performed by radioimmunoassay (RIA), and irregular antibody testing were performed the following day even though the platelet concentrate may have already been administered. The satellite bags were stored at room temperature with continuous gentle agitation. The individual units were pooled into one unit bag immediately before trans- 32 fusion. The number of units pooled depended upon the physician's request. Most platelets were transfused within nine hours after donation. Each unit contained a mean value of 0.63 (± 0.22) x lOll platelets as determined by analysis of six different units of platelets. HL-A Typed Platelets. Plateletpheresis was performed on donors for recipients of known ABO and HL-A type, and for recipients of known ABO but unknown HL-A type when ABO specific platelets were not sufficiently available from fresh CPD whole blood donations. Patients typed for HL-A were matched with siblings or unrelated donors of identical ABO and closest HL-A type. If possible, donors differed from the recipient by one or two HL-A antigen(s). As in non HL-A matched (random) donors, specific Rh type was taken into consideration, but was not always matched. As stated previously, the Rh antigen is considered an erythrocyte antigen and probably is not found on platelets. Patients not HL-A typed were matched with siblings or unrelated donors of identical ABO type. One to five days in advance to plateletpheresing, blood samples from selected donors were drawn for ABO group, Rh type, syphilis, total protein and hepatitis testing, and for HL-A crossmatch against recipient's serum. HL-A cross- match was performed by Dr. Charles DeWitt's laboratory, by lymphotoxicity technique outlined in N.I.H. Manual of Tissue Typing Techniques, 1974 (66). Immediately before platelet- pheresing, selected donors completed a standard blood 33 donation history form, had vital signs checked, and gave written consent to be pheresed (appendix). A Blood sample was also drawn immediately before and after pheresing to determine platelet count, complete blood count and white cell differential. Modification of the procedure of Tullis (67) was used for the pheresis of eight units of platelets from each donor on the Haemonetics Latham Blood Processor model 10 3 . Haemonetics pheresis harness 5600 and disposable bowl 5810 or 5510 were used in conjunction with Fenwal Transpaks TA-l or TA-2. Our method involved the use of both arms of a single donor. One arm as the site of phlebotomy and the other arm as the site of blood reinfusion. In this manner withdrawal of the subsequent unit of platelets could be started before the previous unit of platelet poor blood was completely reinfused. This method allowed for the collection of eight units of platelets to be completed in less than 90 minutes. of McGaw Phlebotomy tubing was kept clear by the infusion 4 ACD formula B, USP, or by Fenwal ACD formula A, USP (appendix), immediately after each unit. The reinfusion tubing was kept patent by the infusion of McGaw normal saline (appendix) between each reinfusion ient set, HW-92D. via Fenwal 3-lead recip- For the collection of eight units the donor received 400 - 800 mI. of ACD and 50 - 100 mI. of normal saline. Time intervals between and during each unit were recorded throughout the pheresis (appendix). Throughout pheresing one or two medical technologist(s) 34 and a phlebotomy nurse were present. Most plateletpheresis were uncomplicated with no serious reactions. Primary com- plaints of donors were stiffness due to immobilization, chills. anxiety, thirst and tingling sensation of the tongue. These latter complaints were corrected by the slowing of the infusion of ACD or changing from ACD formula A to ACD formula B. Some donor's anxiety and/or other problems caused pheresis to be discontinued as a preventative measure for the donor's general welfare. Pheresis was also discontinued when there was clotting in the pheresis system. For a period of time the disposable collection bowl was misaligned causing discontinuation of the procedure; however after a minor adjustment no further problems were encountered. Platelet packs were labeled and stored at room temperature until transfused. Transfusion occurred within nine hours after donation. Laboratory Test Results As stated previously, pre and post plateletpheresis hematological testing was done on pheresis donors. Recip- ients were tested for platelet count, complete blood count, and white cell differential at three intervals: previous to transfusion, one hour post transfusion. and 20 hour post transfusion. All hematological tests were performed accord- ing to the protocol set-up by the Latter-day Saints Hospital Clinical Laboratory; CBC by Coulter Electronic Cell Counter model S5, and platelet count by Coulter Electronic Cell 35 Counter model F after a 1:10,000 dilution of the sample. Evaluation of Bleeding in Recipients Template bleeding times were attempted on the first recipient to receive platelets but were not done on subsequent patients due to their physician's reluctance to have further "holes" inflicted on their patients. Hemostasis was then estimated based upon communication with floor nurses and physicians. red cell count. hemoglobin level, hematocrit and usage of blood and blood components. Patients utilizing more than two units of blood (whole, packed, or leukocyte poor packed) per day while also receiving platelets were considered to be actively bleeding. Patients receiving less than two units of blood (whole, packed, or leukocyte poor packed) per day while also receiving platelets were considered non-actively bleeding. Recipient's Response In vitro analysis of the effectiveness of platelet therapy can be performed by measuring the decrease in blood loss from the circulation, measurement of capillary integrity and/or measurement of the increase in post transfusion platelet counts (23,25,26). Measurement of blood loss is easily done in animals, but almost impossible in man. Capillary integrity measured by the standard template bleeding time is useful to measure platelet hemostatic effects when there is no increase in post transfusion platelet levels. As stated previously, template bleeding times were attempted 36 but were discontinued due to recipient's physician request and consideration for the recipient's clinical status. Even though post transfusion platelet counts measure platelets in excess needed for establishing hemostasis it is a useful tool for analysis. Platelet counts were more clinically suitable under the conditions of this study and therefore were employed instead of template bleeding times. Data collected on each recipient was recorded on a work-up sheet (appendix). Effectiveness of each platelet transfusion was calculated by the following formula: Increment = a b c Xd a - post transfusion platelet count/rom 3 . b - pre transfusion platelet count/rom3 . c - number of platelet units transfused. d - body surface area in square meter (M2 ). Body sur- face area was established from the nomogram of Sendroy and Cecchini based on the weight and height of the recipient (68, appendix). One and 20 hour increment counts were calculated and examined for significance, two sided. by the following formula: 37 ~X21. 1 _ (£ Xli )2] + lt nl with Sp .- \ X2 2. 1 - (~X2i)21 n2 nl + n2 - 2 The t-test assumes a normal distribution of two independent or more observations (69.70). As stated previously, one and 20 hour increments measure the ability of one unit of platelets to increase the circulating platelet levels at one and 20 hour post platelet transfusion. , ; 38 Notes 1. Fenwal Laboratory, Division of Travenal Laboratories Morton Grove, Illinois 2. International Equipment Natick, Massachusetts 3. McGaw Laboratories, Division of American Hospital Supply Corporation Glendale, California 4. Coulter Electronics Incorporation Hialeah, Florida RESULTS Plateletpheresis Donors The mean and ranges of the hematological results on 32 plateletpheresis donors are given in table 5. Table 5. Plateletpheresis Donors' Response Hemoglobin 100 mI. Males Pre Post Females Pre Post Total Pre Post 350 (560 - 209) 6.0 (7.6 - 4.4) 15.4 (17.3 - 13.2) 241 (315 - 188) 5.4 (8.1 - 4.4) 14.2 (15.4 - 13.0) 300 (410 - 230) 5.4 (8.4 - 4.4) 13·3 (14.2 - 12.6) 223 (228 - 198) 5·0 (5.7 - 4.4) 13·3 (15.0 - 11.3) 342 (560 - 105) (B.LI- - 5.9 4.4) 14.9 (17.3 - 12.6) 237 (331 - 188) 5·3 (8.1 - 4.4) 14.0 (15.4 - 11.3) (ranges in parenthesis) The above data demonstrate that donor's hemopoietic system was not adversely affected by platelet donation. The re- l}O suIts correlates well with those of Tullis, et ale (67) and Schiffer, et ale (71). Plateletpheresis was confirmed to be a safe and easy method of collecting large numbers of platelets from a single donor. Barr (72) demonstrated platelets drawn on the Latham Blood Processor were viable and underwent minimal damage. Recipient's Response Between April 1, 1974 and November 15, 1974, 24 persons received platelets; 2 received HL-A matched platelets, 20 received non HL-A matched platelets. and 2 received both HL-A and non HL-A matched platelets. Five persons did not have 20 hour post transfusion platelet counts taken and therefore were excluded from the study. This left 19 recipients, ages 22-71, with 1 receiving HL-A matched platelets, 16 receiving non HL-A matched platelets and 2 receiving both HL-A matched and non HL-A matched platelets. A brief summary of the clinical course of each patient is given in table 6. Figure 4 demonstrates increment response by each recipient per platelet transfusion. Recipients receiving random (non HL-A matched) platelets were divided into four groups: A. Received blood (whole, packed, or leukocyte poor packed) during the six months prior to the study and used more than two units of blood per day during platelet therapy. D. Received blood (whole. packed, or leukocyte poor Recipient No. Sex Age Platelet type and BTOUP NOQ platelet units transfused No. days received transfusion t Admission pIt. count (mm 3 ) Meal transfusion n 1 ~tT-4 3 HL-A 32 6 15,000 18 2 ~'-58 non HL-A CD) 18 5 35,000 39, 3 F-34 non CD) 8 1 28,000 28, I 8 1 62,000 62, 12 2 26 oOO 21, 124 31 24,000 19, 22 6 17,000 15, 12 2 .3't 000 55, 225 34 7,000 2Q , 8 1 13,000 13, 4 5 non HL-A \.'<~- 11 ,.-t 64 :.~- 30 ) .. no!! HL-A non HL-A • \11 HL-A a1J.d F-51 6 7 h~-A :I\ ...... A ) non HL-A (D) I no!'). HL-A (A) t I '-' 12 ~-47 non HL-A (D) 9 1 15,000 18, 13 ~-46 non HL-A (C) 6 2 38,000 38, 14 W-22 non HL-A (A) 10 1 60,000 60, 15 tF-31 i non HL-A 24 8 27.000 28, 16 F-72 ! 19 7 34,000 36, 1 ~11-6 1 24 000 24, I """' ) \~ non HL-A CD) non HL-A f"'I v ~---~- .on Mean pre transfusion It. count t Mean -I hour post transfusion It. count Mean. 20 transfuEion hour post It. count 15,000 18,.300 29,000 31,000 35,000 .39,000 - --- 95,000 28,000 28,000 153,000 205,000 62,000 62,000 - --"-- 60,000 26.000 21,000 -------- 30,500 24,000 19,)00 32,000 21,900 17,000 15,000 "'---_.-=- 24,000 .3 1} 000 55,000 3),500 94,500 2:~ ~--'-- t 7,000 , 700 32,700 13,000 1),000 ~-- .. -.-""""'- 33,000 ,000 18,000 ----"--- 95,000 38,000 38,000 - -- 73,000 60,000 60,000 31,000 27,000 28,000 34,000 36,500 24,000 24,000 34,000 ~ 68,000 40,000 139,000 26,000 41,000 1ean '1 hour post lsion nlt. count (mm3 ) 29,000 3,000 32,000 3,500 31,000 34,000 Mean 20 hour post transfusion plt. count (mm3 ) Mean 202hour Inc. X M :junit 31,000 1,400 95,000 11,100 205, 000 22,000 60,000 o 30,500 Red cell transfusion during ulatelet thera~ 6 Leukemia. E] ° ° 10 Purpura. Exp with pIt. cou 3,300 7 Lympho8carcom transfusion. 21,900 1,300 19 Myelomonocyti ulatelet tran 24,000 2,000 4 Expired 1 day 94,500 17,000 4 Liver tumor. 32,700 1,200 18 33,000 4,900 2 Discharged 3 95,000 16,900 4 Bleeding tend transfusion. 73,000 11,200 15 Cardio-vascul transfusion w 68,000 1,400 13 Pre-eclampsia following las after last :rl 40,000 3,900 7 Melanoma. 139,000 21,000 4 Thrombocytope transfusion ITF. Dischar GI bleeder. transfusion. Myeloblastic platelet tran Di 0 26,000 41,000 11,200 6 Bowel obstruc 02 hour M . unit Red cell transfusion durin ulatelet thera Clinical impressions 00 6 Leukemia. 00 o Purpura. Expired 6 days after last platelet transfusion with plt. count 145,000 mm). 00 o ITF. o 10 00 7 00 19 00 4 Expired 1 day after last platelet traYlsfusion. 00 4 l,iver tumor. Expired 2 days after last platelet transfusion. Discharged 16 days after last platelet transfusion. GI bleeder. Discharged 10 days after last platelet transfusion. Lymphoscarcoma. transfusioYl. Expired 1 day after last platelet Myelomonocytic leukemia. , Dlatelet traru::fusion. Expired 11 days after laEt })latelet Myeloblastic leukemia. platelet transfusion. 00 Discharged 16 days after last tra~sfusion. Diecharged 18 da.ys after last 00 2 Discharged J days after last platelet 00 4 Bleeding tendency. transfusion. 00 15 Cardio-vascular surgery. Expired 1 day after last platelet transfusion with plt. count 52,000 mm). 00 13 eclampsia. Tra.Ylsfused)O uni ts of t'·lood over ) days following last platelet transfusion. Discharged 15 days after t p12telet transfusion. 00 7 Melanoma. 00 4 Thrombocytoper:ia. transfusion. Expired 11 days after J ast platelet 00 6 Bowel obstruction. Expired 2 day[' after last platelet t~·ansfusion. Expired 18 days after last platelet Discharged 1 day after last telet transfusion. Table 6. Reci 1)ient rio. Clinical Study of Recipients (cont.) Sex Age t ?latelet type and group I~o. Platelet units transfused IreceivedNo. transfusion days Admission pIt. count (mm 3 1 Me traYlEfusion 19 tF-45 '!"lon HL-A (C) 10 2 67,000 6 20 ~/I-71 non HL-A (C) 23 4 87,000 12 21 n,rr-Ll'O non HL-A (C) 21 2 ,000 6 22 :2-26 232 35 14,000 I EL-A and BL-A :":01']. Mean pre fusion tra~~fusion pIt. count 'J ( rm:: ..; Me~1 transfusicn 1 hour post It. count r.:ean 20 hour PO tra:r:sfu~icn .It. coun 67,000 69 000 32,000 87.000 122,300 131,300 ,000 65,500 125,000 14,000 13,600 13,200 9 fusi en 1 hour post . COLlflt r.~ean 20 hour post traY":sfusicn nl t. count I 222ho~: IYlc. X hl UDl t Red c 1 trar. s fus i on 'latelet theran durin 32,000 5,300 38 Abdominal m transfusion. 131,300 8,800 7 Fracture hi transfusion. 125,000 13,000 ...., J Lymphoscarc transfusion. 600 40 13,200 / Leukemia. 42 ~02hour M unit Red durin c 1 tranEfusion 'latelet theran Clinical imnressions Abdominal maE'E. tranEfufion. 300 )8 300 7 Fracture hip. transfusion. )00 3 Lymphoscarcoma. transfusion. Sao 49 I..;eukemia. Discharged 6 days after JaEt platelet Discharged 9 days after last platelet Dischar~ed 15 days after la~t platelet Exnired day of last platelet tJ'ansfusion. Figure 4. 32.0 24.0 16.0 Individual Platelet Response * ';E- ~!- ·u· * i~ it- ·U· iI- 9.8 iI- 9.0 if- 8.5 7.4 6.6 {~ 5.8 5.0 h.6 ('t"'\ Ii- +' 3.8 3.4 '--" .r; ~ ~ "h 3·0 2.6 :x: 2.2 () !:! H S-.! ::J 0 .,..... .. ~: iE- ~:. .2 ~ 1.8 1.4 1.0 il- ·ll- if- * * * i~ ~f- ifiI- ~t-.;} i:· * ~~ -31- if- ..:r it- it~ - - - - - - ........ 0 if- i} il- * it- - - *il- it- - - - - - - - - - - - - if-- it· 0.8 ,1-* ii- N ·u· 0.6 i~* ii- 0.4 i~ 0.2 0.1 ·'C· ~!. ·n· ~~ ';H} -:1- 0.0 -l~ 1 2 3 ~t.. .. -;( ~HH!- ~(o iHi- 7 11 12 13 1 it- ~t--:t-~f- 17 18 19 20 21 22 Recipient No. i!~ Jt* i:.. *** ~ .j~ iHi- response per transfusion median response for all recipients 44 packed) during the six months prior to the study and used less than two units of blood per day during platelet therapy. C. Received no blood during the six months prior to the study and used more than two units of blood per day during platelet therapy. D. Received no blood during the six months prior to the study and used less than two units of blood per day during platelet therapy. HL-A Matched vs Non HL-A Matched Platelet Response. It was expected that recipients receiving HL-A matched platelets would give a higher 20 hour increment than those receiving random non HL-A matched platelets. our small sample. This did not occur in The one recipient receiving only HL-A matched platelets (recipient's phenotype: 2,12,15 and two donor's phenotype: 11,12,15 and 2,WJ2,12,15 respectively) expired nine days after being admitted into the hospital. He received blood and platelets on days one through eight. Test for HL-A antibodies in the recipient by Dr. Terasaki's laboratory proved to be negative. Increments resulting from HL-A matched and random non HL-A matched platelet therapy are given in table 7. Recipient's Response vs Number of Platelet Transfusions. No definite point was identified when recipients began to show decreased 20 hour increments, the finding of which would have demonstrated alloimmunization. Of the three recipients receiving long term therapy, only one was tested HL-A vs Non HL-A Matched Platelet Response Inc. X M2Lunit ~mm3} No. Studied 1 Hour 20 Hour mean I median mean median Table 7. HL-A Comnatibility I 4.08 (0.0 - 4.28) 1.35 1.53 (0.0 - 2.55) I 1.92 0.57 (0.0 - 6.50) matched 3.38 0.94 (0.0 - 6.50) non matched 0.41 0.20 (0.0 - 3.10) I 9.16 11.16 (0.0 - 39.83) I 3.45 (0.0 - 7.64) 0.89 1.23 (0.0 - 1.44) 1.69 (1.58 - 1.80) 3.89 3.89 HL-A matched 1 HL-A matched and non 2 2.79 16 Non matched A 4 3.12 I B 1 C 5 9.99 11.16 (5.55 - 13.47) D 6 21.87 19.46 (11.05 - 39.33) (ranges in parenthesis) 1.69 I 46 for HL-A antibodies. After 150 platelet uni ts this re-- cipient reacted with 69.1 percent of the cells tested by Dr. Terasaki's laboratory. Lymphocytotoxicity crossmatch against HL-A matched donors remained negative even after she Vias shovnl to possess HL-A antibodies. Twenty hour increments after 10, 20, 25, 50, 100, 200, and )00 units of platelets are shown in table 8. Table 8. HL-A Compatibility Long Term Platelet Therapy e Mean 20 Hour Inc. X M2/unit (mm 3 ) No. ts Transfused No. Studied (10 <20 (2S I (SO <100 (200 I (]OO HL-A matched 1 2.6 2.1 3.3 1.4 --- --- --- HL-A matched and non 2 1.9 1.7 0.3 0.3 0.5 0.6 0.6 matched 3.7 0.1 0·3 0.3 --- --- --- non matched 0.2 1.5 1.2 0.9 0.8 0.6 0.6 Non matched 16 A 4 0.5 1.8 1.6 1.6 0.9 1.4 1.2 B 1 0.0 3.4 --- --- --- --- C 5 9.2 11.6 11.2 --- --- --- ----- D 6 2 0.2 .0 --- --- --- --- --- 1.3 --- I Prolonged therapy recipients No. I i 3 ,, 6 0.0 1.0 1.0 1.3 0.7 No. 11 --- 2.6 2.3 1.8 1.2 1.6 1.2 No. 22 0.2 0.1 0.3 0.3 0.8 i 0.7 0.6 I I DISCUSSION The purpose of this study was to determine if a volunteer HL-A platelet donor program for Salt Lake City and surrounding areas was feasible and if patients benefit from such a program. HL-A Donor Program At this time it appears that a 100 percent volunteer program is not possible, The main objections encountered with potential plateletpheresis donors were: 1. The reluctance of the donor to have his erythrocytes reinfused. The prevailing attitude was "you can take it out, but once you have it, you keep it." 2. The prolonged time necessary for donation; which many times resulted in prolonged absence from employment. Many donors said they would donate if it did not require more than their lunch break. 3. The prolonged time that phlebotomy needles were in the arms as compared with normal whole blood donation. q.. The nonfinancial reimbursement for time and product. With education of the general public and physicians, platelet donation may become as common as whole blood donation. 49 For whole blood donation, the blood transfusion services had to rely upon paid donors for many years. Only in recent years are blood transfusion services beginning to reach 100 percent volunteer blood donation. It appears that platelet donation programs will follow a similar pattern. Until such time arrives, blood transfusion services will have to rely upon the incentives of financial reinbursement and aid to relatives for HL-A matched platelets. Even though 469 donors are enrolled into the program, more than 469 persons had blood samples taken. A small number of potential donors had to be eliminated from the program due to insufficient leukocyte levels for typing and due to an initial inefficient bookkeeping system during donor recruitment. Donors were requested to fill out a card providing their name, home address, home and business telephone numbers, and blood type and group. HL-A specimens were sent out with only social security numbers as identification, as per Dr. Terasaki's request. Unfortunately those cards previously completed did not have social security numbers. When social security numbers were requested for the test specimen labelling, the numbers were not similarly placed on the information card. Request for social security number was then added to the information card and the problem eliminated. Computerization of donors was planned but not executed. A program of under 500 donors is not sufficient to offer a large assortment of HL-A phenotypes and thus a pro- 50 gram involving the whole intermountain area would be of greater usefulness. With over 30,000 genotypes possible the chances of locating an HL-A identical unrelated donor is 1 in 3,000 (44). Even though less than 500 donors were enlisted, the program was feasible as demonstrated by the fact that all patients HL-A typed were matched with HL-A compatible unrelated donors. An unique situation which exist in Utah, that is the presence of large families, did not make it as much of a necessity to have a large random donor pool. The chances of finding an HL-A identical sibling is 1 in 4 (7,44). This situation added to the feasibility of the program. ReciRient's Benefit Most of the recipients in this study did not benefit from the program because of the lack of knowledge of the recipient's HL-A type. In order to correct this situation, physicians and blood transfusion services must be educated concerning potential benefits of HL-A compatible platelets. Physicians must order typings on their patients the moment there is clinical indications for prolonged platelet therapy and the blood transfusion services must help the physician by drawing to their attention HL-A platelet therapy and having such services available. HL-A Matched Platelet Transfusion. The benefits of transfusing HL-A compatible platelets could not be evaluated. One recipient received only HL-A matched platelets but he 51 expired soon after being admitted into the hospital. The responses that did occur in this patient were similar to tho~e of group A non HL-A matched random donor recipients (active bleeding and prior exposure to HL-A antigens). Cytotoxicity testing demonstrated that the recipient did not possess HL-A antibodies. Bucher, et al. (73) attributed poor response to HL-A matched platelets in their patients to specific platelet antibodies even though the patients did not demonstrate any detectable platelet antibodies. The poor responses to HL-A matched platelets in this patient can also be attributed to platelet specific antibodies. Testing for platelet specific antibodies was not done. HL-A vs Non HL-A tmtched Platelets. Recipients re- ceiving both HL-A matched and non HL-A matched platelets did not differ significantly in their responses, with mean 20 hour increment of 3.380 X Mf/unit and 410 X Mf/unit respectively (p=.50). This group was made up of two indi- viduals; recipients nos. 5 and 22. Recipient no. 5 received one transfusion of eight units of HL-A matched and one transfusion of eight units of non HL-A matched platelets. The response to each transfusion was significantly different, with 20 hour increment of 6,500 X M2/unit for HL-A matched platelets and zero with non l{L-A matched platelets. Using one recipient it can not be concluded statistically that HL-A matched platelets are of greater benefit. Recipient no. 22 showed little difference between HL-A matched and non HL-A matched platelets, with mean 20 hour 52 increment of 260 X rvf/unit and 720 X l\12/unit respectively. The recipient initially received HL-A matched platelets and when the response was poor, non HL-A matched platelet therapy was begun. After 200 non HL-A matched platelet units were given, the cytotoxicity testing was positive for 76 out of 110 cells. The recipient was then placed back onto HL-A matched platelets when the lymphocytotoxicity crossmatch was shown to be negative. The response was still poor. Heasons for the poor responses may have been due to sensitization to platelet specific antigens, HL-A ffiltibody titers too low to detect incompatibility in vitro, antigens on donor cells not detected and/or insufficient numbers of platelets transfused to handle the bleeding problem. The most likely explanation is a combination of those listed above. Up to 16 units of platelets were given daily to the recipient but she continued to bleed, though not actively. Therefore the transfused platelets were being removed or destroyed from the circulation at a rapid rate. Individuals not showing a full complement of four HL-A antigens may be homozygous for one or two antigens, possess an antigen not yet defined by currently used antisera, or possess a weak antigen which is difficult to detect. The recipient was typed for a full complement of four antigens and is thereby not homozygous for any antigens or have an wldefined antigen. Recipient's phenotype: 2, vJ24 , 12, 1!JIO Donor's phenotype: 2,J,12,WIO 1) 53 2) 2,W24,12,WIO J) 2,12,VJIO 4) 2,w30,12,WIO Antigens HL-A 3 and WJO are missing in the recipient but present in the donors. These two antigens could have stim- ulated antibody production. HL-A 3 is an infrequent stim- ulator (5,9,55,56) and no information has been published on W30. It is not possible to state if the recipient developed antibodies to either of these two antigens since the phenotypes of the 76 out of 110 cells which reacted in the lymphocytotoxicity test were not reported. Using Lohrmann's (10) study as a reference, donors 1 and 4 are mismatched, donor 2 is an A-match and donor 3 a B-1 match. Platelets from donor 1 and 4 should have resulted wi th the lovlest response and donor 2 the highest. In figure 5, this is not shown to be the case. Figure 5. Recipient no. 22 HL-A Matched Platelet Hesponse 1.3 * 1.1 20 Hour 0.9 Inc. X rf-/ un itO. 7 ') (rnm 3 ) ·n· * 0.5 0.3 * 0.1 Donor **'*~~ 1 * 2 3 * 51+ Clinically the recipient, in addition to bleeding on a low level had a bacterial infection. "As stated previously. one possible function of platelets is to remove bacteria to the reticuloendothelial system for destruction (5). This function can help explain the low response to HL-A matched platelets. Fever usually accompanies bacterial infection and this recipient was febrile. Many investigators have shown febrile patients to have a lower response to platelet transfusion (11,14.74). Grumet and Yankee (11) demon- strated that with fever, the 20 hour increment in HL-A identical platelet transfusions may be zero, but the high one hour increment would indicate that HL-A identical platelets were transfused. Unfortunately this patient had no one hour post transfusion platelet counts taken due to her clinical status. Freireich, et all (74) believes that one cannot assume poor response after transfusion establishes the presence of platelet antibodies or the establishment of a permanent state of refractoriness. Some clinical states can cause thrombocytopenia, not associated with platelet antibodies. Freireich feels that vfhen the clinical status is improved the thrombocytopenia associated with it becomes reversible. This patient expired prior to an improved clinical status, therefore this concept could not be tested. Prior Exposure to HL-A Antigens. Persons receiving blood up to six months prior to this study all showed low responses to non HL-A matched platelets with the very first 55 transfusion. Those not receiving blood up to six months prior to the study had excellent responses to platelet therapy even after 20 units of platelets, with mean 20 hour increment of 15,040 X M2/unit as compared to 2,490 X M2/unit for persons receiving blood up to six months prior (p=.15). This suggest that blood transfusions do stimulate platelet or HL-A specific antibodies. These antibodies may then remain in the circulation for at least six months. Heinrich, et al. (55) discovered that HL-A antibodies induced by multiple transfusions tend to disappear within six months and those induced by pregnancy within one year after delivery. Even though HL-A antibodies usually disappear after one year after pregnancy, Heinrich could only explain some HL-A immunization by pregnancy .3 - 40 years earlier in some patients. Actively Bleeding vs Non-actively Bleeding. Recipients actively bleeding (utilization of more than two units of blood per day during platelet therapy) responded less than those patients not actively bleeding (utilization of less than two units of blood per day during platelet therapy), 2 with mean 20 hour increment of 5,440 X M /unit and 12,880 X M2/unit respectively (p=.50). The difference in response may be attributed to transfused platelets being removed from circulation to form hemostatic plugs and to strengthen endothelial walls in the actively bleeding recipients. Recipients not actively bleeding would, on the other hand, have excess transfused platelets after the needs of hemo- stasis was satisfied and these excess platelets remained to circulate in the peripheral blood. If this explanation is correct then actively bleeding patients should show a gradual increase in response and then show identical response to those of non-actively bleeding patients. After 20 units of platelets there was no significance in response between the two groups, with a mean increment of 6,700 X M2/unit and 9,200 X M2/unit (p=.70) respectively for actively bleeding and non-actively bleeding recipients. Long Term Platelet Therapy. Long term HL-A platelet therapy could not be evaluated in this study. No recipient received long term HL-A platelet therapy and as stated before the one recipient receiving only HL-A matched platelets expired soon after admission into the hospital. Studies have shovm refractory patients respond for well over two years with HL-A matched platelets and non-immunized patients can be HL-A transfused up to three and a half years without alloimmunization (9,15). As stated before, long term non HL-A matched platelet thArapy does lead to alloimmunization. Recipients in this study receiving more than 30 units of platelets did show poor responses. Testing for cytotoxic antibodies in these recipients would have made it possible to determine if the poor responses were due to HL-A alloimmunization, platelet specific alloimmunization, or to a specific clinical state. Unfortunately cytotoxic antibodies were not determined with the exception of recipient no. 22. As stated previously, 57 this recipient reacted to 69.1 percent of the 110 cells tested by Dr. Terasaki's laboratory. Conclusion 1. At this time it is possible and feasible to establish a volunteer HL-A platelet donor program in Salt Lake City and surrounding areas. paid donors. This program must include some A 100 percent volunteer donor program is not possible at this stage of evolution of platelet donation programs. 2. This study was done coincidently with patient's treatment during hospitalization. This approach made it difficult to control variables such as: time and number of laboratory tests, medication, type of platelets transfused (HL-A matched or non HL-A matched), additional platelets given as whole blood, and time and number of platelet transfusions. As the result of these non-ideal research conditions, the conclusions are tenuous. Any future research along these lines should be conducted under better controlled conditions. 3. In this study the benefits of HL-A matched platelets could not be completely shovm. 4. Previous exposure to HL-A antigens apparently does influence the effectiveness of non HL-A matched platelet therapy. 5. Bleeding recipients will demonstrate poor response to platelet therapy, as measured by post transfusion peripheral platelet counts. 6. Long term non HL-A matched platelet therapy does lead to alloi~munization. APPENDIX Announcement Letter Dear Volunteer Donor: The Latter-day Saints Hospital jointly with the University Medical Center are planning to develop a program for transfusions of blood platelets to recipients in need of this blood component. For this program we will need approximately 1,000 volunteer donors who will be willing to be HL-A typed in addition to their ABO and Rh type. charge wi No be made for the necessary tests for this program to those who wish to participate. It expected that the recipients will do better clinically if the platelets are more compatible in the HL-A system. If you are interested in this program, additional information will be sent to you.. Please fill out the enclosed card and return it to the blood bank in the enclosed envelope. Thank you, 60 Additional Information Letter Dear Volunteer Donor: Thank you for returning the card for additional information regarding the HL-A program. The Latter-day Saints Hospital and the University Medical Center has an apparatus called a Processor, which assists us in obtaining up to eight platelet packs per donor. two to three hours. The total time involved is Your plasma and red cells are returned to you thus allowing you to donate platelets at least once every seven days. Since your red cells are returned to you, you are still eligible to donate whole blood in Cardiovascular programs as the same frequency as before, five times a year. There are no charges for the necessary tests for this program. Kindly let us know if you would be willing to participate. We will answer any further questions for information. Your interest in helping others in need of more progressive medical care is greatly appreciated. Thank you, 61 Additional Information Letter Supplement HL-A stands for Human Leukocyte is for tissue typing. Antibody~}; this test Dr. Terasaki, a nationally known researcher, in Los Angeles, is conducting these tests. Every l\Ionday, rruesday, and t'Jednesday, between 8 a.m. and 12 noon a speciman will be dra\m from 20-40 people that would like to participate in the HL-A program. be made through the Blood Bank. Appointments should These specimens will be sent by Air Express to Los Angeles and the results are returned within two days. Platelets are the cells that normal healthy individuals have that control hemorrhaging or bleeding. Patients that have leukemia, aplastic anemia and other diseases which eliminate the production of platelets need these cells from a healthy donor. The patients will also be HL-A typed. The donor's platelets are replaced almost immediately after being drawn. With the return of his/her red cells and plasma, a donor can be plateletpheresed at least once a week. This does not prevent a donor from donating whole blood five times a year. To participate in this program we will need your name, address, telephone number, blood type and social security number; unfortunately this was eliminated on the original cards that were sent out. It will be used for identification only for the donor on the computer. ~:. If you are interested HL-A does not stand for Human Leukocyte Anti body, but for ease of the layman donor this misnomer was used. 62 in being a platelet donor please call for an appointment to have a speciman drawn, giving the above information for our records. Thank you, Fenwal CPD Double Blood Pack JF-25N 206 mg. Citric acid (Hydrous) 1.66 g. Sodium citrate (Hydrous) 140 mg. Sodium biphosphate 1.61 g. Dextrose (Hydrous) Saline Q.S. to 63 mI. McGaw ACD Formula B, USP 1.47 g. Dextrose (Hydrous) USP 1.32 g. Sodium citrate (Hydrous) USP 0.44 g. Citric acid (Anhydrous) USP Saline Q.S. to 100 mI. Fenwal ACD Formula At USP 2.45 g. Dextrose (Hydrous) USP 2.20 g. Sodium citrate (Hydrous) USP 0.73 g. Citric acid (Anhydrous) USP Saline Q.S. to 100 mI. McGaw Normal Saline 0.90 g. Sodium chloride Water Q.S. to 100 mI. Electrolyte level: 154 meq/l. sodium 154 meq/l. chloride 64 Plateletpheresis Release I hereby grant permission to the Latter-day Saints Hospital Blood Bank to 'pherese me, removing approximately 450 mI. of whole blood each time and allowing them to reinfuse my plasma with my red cells after the desired components have been removed. This procedure is to be done on the Latham Blood Processor and I have been informed and understand the procedure. Si~1ature Date Witness 'l:.1 DATE: DorWR: MEDICAL TEC~":OlOGI~ Tt PHY~ REGI~, -- \-I ICLl\ -1: TERED P> c:+ '!UR~E: (D \-I CBC U~'IT~ D!{Al:J~: FIR~T SECOND 'rHIRD trOURTH FIFTH ~IXTH k-,EVEi':TH !EIGHT PRE (D POST c:+ d ::t PHLEBOTO~Y ~TARTED (D t1 (D r!'LACHPIE STAHTED m ...... ' CJl PLASMA FLO ','J EEGIrJS ~ o PLATELETS BEGIn Ii ?ii' MACHINE STOPPED INFU~ IO~I Ul ::1 E TARTED (D (D c:+ INFUSION STOPPED ,GE LOT # r.:ATERIALS USED PHERESIS HARNE~S TOTAL FLUIDS Sf\LI:,rE US ED PHERES IS Bm'/L ACD SOLUTION U5ED RECIPIE:~T PLASf,'!,A [';OT RETURNED SET ACD SOLUTIO:! WEIGHTS BAG #1 LEUKOCYTES ~ALBE BAG #2 PLATELETS PLASMA TRA~JSFER SET (2 BAG #3 PLASMA TANDEM SET (-nR!P CHAlV'J3ER) TRANSPAKS 0\ V"t :::r: t""1 I :t> ,.,1-3 Patier.t: _ _ _ _ _ _ _ _ _ _ _ _ _ HL-A TypLng: lit. t'INCRE~lE;>IT ---- Wt. § Body Surface Area: (Scc Nor-.op;ar:1) en s:; H:l X llody Surf.lce .\rea· l};CP.l:::.::::.-r CALCULATION: CIl PUlE::T I:~rOR.\l"TIO~{ - LJb Value!':: !-I" o (Ab!"olute) ::s Pr.:! T.""":f ... ,,, .0n Date I !'-Be GRA~lULO 1 1 r. Post Transfusion Donor IdL'ntiflcation B.T. PLTs. Name of Donor nate of Ix. Units Given WBC GRANULO PLIs. B. T. 20 IIrs true Post: JIJll'lS us ion PUs. GRA!;ULO B.T. ~ *n:CRE~:r.:;! (20 Hr.) ,.,o ?;' I (/'} iI I ::r I CD CD c+ I I 1 I I I 1:;'::':'1. r:::::i?-::,\TIO;: - [..1.0 V:Jlues: Dono :-:-.e of O(,>:10r Iel :ifi Sec Sec - (flbsolute) P tti, ~Io. - HL-A Typ1n~ -- Dati: , - Pas t-I"h lebotomy 'Ph leb HBC I CRANt'LQ PLTs. D:lte wnc GR-\~ULO PLTs Unit'i DraIJn I I 0\ 0\ 67 Nomogram for Calculating the Body Surface Area of Adults ].00 i' SlZJJ 2~O l.\(} ZSo 2'0 I~O ISS 'd' - LCO '?Ii "~ Ul 0:: ISo ~ IQS en 0:: 140 f.xl 8 ~ ~ H E-I 8 f!:l ,.=:, ,,' 125 . 6 10" W 0 12.0 Z H H ItS :z:: "~a H ~ 8 lOS 110- 1,90 9S lOO I.~ ,~O 1.7S 1.70 180 1.35 1.30 r:a :r:: l.ZS 1.10 z H 1.15 1.)0 <C r:a 0:: <C 170 ,,0 I./,O 1.55 1.60 1.4S 1.40 M ...:=. 130 ~ Z 100 I.QS ,.(,~ ISS 115 220 I.~~ 1'0 E-I P:1 HO 1.10 leo -110 f,,' 130 ls:;t> 2'10' l.bO lO5 200 t9S " '40 .3CO l.IS '0· tSO 310 1.05 1.00 ::'15 ISO (l}J40 0 Z ;::=l .30 75 -10 - ~~ ~ ~120 5!i ~ 50 110 3' ,0 .?5 !S H ~ 100 CJ E-t ~ H P::I CJ ::: .'30 H P::I :;: 70 .'10 60 .65 ZS 80 .60 so 15 ::c::: 2: 8 .&5 .80 CJ 0 H H .90 'IS ~~ ,:-60 <: 0:: .. 55 20 .SO 40 REFERENCES CITED 1. Djerassi, I. 1966. The Role of Platelet Administration in a Blood Transfusion Service. Transfusion 6:55. 2. Huestis, D., J. Bove and S. Busch. 1969. Practical Blood Transfusion. Little, Brown and Co., Boston. 3. Johnson, S., D. Van Horn, H. Pederson, et ale 1966 The Function of Platelets: A Review. Transfusion 6:). 4. Dausset, J. and J. Tangun. 1965. Leucocyte and Platelet Groups and Their Practical Significance. Vox Sang. 10:641. 5. Yankee, R. A. 1974. HL-A Antigens and Platelet Therapy. in Platelets: Production, Function, Transfusion, and storage. M. Baldini and S. Ebbe eds. Grune and Strattone Inc., New York. 6. Shulman, N. R. 1966. Immunological Considerations Attending Platelet Transfusion. Transfusion 6:39. 7. l\1011ison, P. L. 1972. Blood Transfusion in Clinical Medicine. Blackwell Scientific, London. 5th edt 8. Yankee, R. A., K. S. Graff, R. Dowling, et all 1973. Selection of Unrelated Compatible Platelet Donors by Lymphocyte HL-A Matching. New Eng. J. Med. 288:760. 9. Yankee, R. A. 1971. Importance of Histocompatibility in Platelet Transfusion Therapy. Vox Sang. 20:415. 10. Lohrmann, H., M. Bull, J. Decter, et ale 1971~. Platelet Transfusion From HL-A Compatible Unrelated Donors to Alloimmunized Patients. Ann. Internal Med. 80:9. 11. Grumet, F. and R. A. Yankee. 1970. Long Term Platelet Support of Patients with Aplastic Anemia: Effect of Splenectomy and Steroid Therapy. Ann. Internal Med. 71:1. 12. Slichter, S. J., P. J. Kane, R. Storb, et al. 1973. Platelet Transfusion Sensitization Modification: By Donor Selection or Recipient Immunosuppression. Clin. Res. 21:654. 13. Yankee, R. A., F. C. Grumet and G. N. Rogentine. 1969. Platelet Transfusion Therapy: The Selection of Compatible Platelet Donors for Refractory Patients by Lymphocyte HL-A Typing. New Eng. J. IVIed. 281: 1208. 14. Reich, L. and H. Almondhiry. 1974. The Use of HL-A Compatible Platelet Transfusion in Patients Resistant to Random Platelets. Transfusion 14:502. 15. Thorsby, E., A. Helgesen and T. Gjemdal. 1972. Repeated Platelet Transfusions from HL-A Compatible Unrelated and Sibling Donors. Tissue Antigen 2: 397. 16. Gardner, F. H. and P. Cohen. Transfusion 6:23. 17. Harker, L. 1972. Platelet Kinetics. in Progress in Transfusion and Transplantation. P. Schmidt edt American Association of Blood Banks, Chicago. 18. Firkin, B. 1972. Hemostatic Functions and Disorders. in Progress in Transfusion and Transplantation. P. Schmidt edt American Association of Blood Banks, Chicago. 19. Gaydos, L., E. Freireich and N, Matel. 1962. The Quantitative Relation Between Platelet Count and Hemorrhage in Patients with Acute Leukemia. New Eng. J. Med. 266:905. 20. Gardner, F. H.. 1974. Platelet Transfusion. in Platelets: Production, Function, Transfusion and Storage. M. Baldini and S. Ebbe eds. Grune and Strattone Inc., New York. 21. Faber, S. and E. Klein. 1957. The Nature and Control of Bleeding in Acute Leukemia and Other Thrombocytopenic States: A Review of a Ten Year Program of Research. Ann. Peadiat. Fenn. 3:348. 22. Roy, A. J., N. Jaffe and I. Djerassi. 1973. 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