Erythropoiesis, erythropoietin, and iron metabolism in elective surgery: Preoperative strategies for avoiding allogeneic blood exposure

Erythropoiesis, Erythropoietin, and Iron Metabolism in Elective Surgery: Preoperative Strategies for Avoiding Allogeneic Blood Exposure Mark A. Goldberg,

Preoperative autologous donation (PAD) of blood and administration of recombinant human erythropoietin (Epoetin alfa) are two strategies for increasing red blood cell (RBC) mass preoperatively. The success of PAD depends primarily on the patient’s ability to manufacture new RBCs before surgery to replace those removed during PAD. Red blood cell manufacture depends in turn on adequate supplies of iron and the increased renal production of endogenous erythropoietin following PAD. Successful PAD also requires adequate time for regeneration of predonated RBCs. Parenteral administration of Epoetin alfa causes a dose-dependent stimulation of RBC production. Its use has been studied as an adjunct to PAD and as a method to enhance endogenous erythropoiesis without PAD. Several studies suggest that administration of Epoetin alfa, begun several days before surgery, may stimulate erythropoiesis and help decrease the number of RBC transfusions required postoperatively. The precise role of Epoetin alfa in the surgical setting is not yet established, and optimal dosage regimens have not been determined.

From the Hematology/Oncology Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Requests for reprints should be addressed to Mark A. Goldberg, MD, Hematology/Oncology Division, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115.

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atients undergoing elective surgical procedures frequently, require allogeneic red blood cell (RBC) transfusions to replace blood lost during surgery. Increased frequency of testing and improved methods have greatly lowered the risk of infection from such transfusions. Still, the small chance of exposure to infectious agents, such as hepatitis viruses and the human immunodeficiency virus (HIV), has led to several strategies for avoiding exposure to allogeneic blood altogether. Strategies under active investigation and in routine use are diverse, yet all aim at enabling the patient to maintain a minimally acceptable RBC mass. This may be accomplished by decreasing intraoperative blood loss, increasing RBC mass preoperatively, or both. Intraoperative blood loss can be decreased by surgical techniques that minimize bleeding, adjuncts to hemostasis, intraoperative blood salvage techniques, and isovolemic hemodilution. This review concentrates on two strategies for increasing preoperative RBC mass: preoperative autologous donation (PAD) of blood and the use of recombinant human erythropoietin (Epoetin alfa). ERYTHROPOIETIN, IRON, AND REGULATION OF ERYTHROPOIESIS

To grasp the rationale behind strategies used to increase RBC mass, it is important to understand the key factors involved in regulating erythropoiesis. Erythropoietin (EPO) is a glycoprotein hormone that plays a critical role in the normal regulation of RBC production [1,2]. Hypoxia is, in turn, the chief stimulus for endogenous EPO production [2,3]. When hypoxia is sensed in the kidney and, to a lesser extent, in the liver, EPO gene transcription increases, leading to increased EPO messenger RNA (mRNA) levels and increased production and secretion of EPO protein [3,4]. The secreted hormone travels through the blood to hematopoietic tissues in the bone marrow, where it binds to its receptor on erythroid progenitor cells, stimulating them to proliferate and differentiate into mature RBCs. These in turn increase the blood’s oxygen-carrying capacity and alleviate the hypoxic stimulus, leading to a decrease in endogenous EPO synthesis and secretion. This process provides a complete feedback loop for the regulation of endogenous EPO gene expression and RBC production (Figure 1). Elythropoietin

is an etythropoietic, lineage-specific hematopoietic growth factor that has no known clinically significant effect on thrombopoiesis or any other hema-

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Figure 1. Feedback loop for the regulation of erythropoietin production. BFU-E = burst-forming unit-erythroid; CFU-E = colony-forming unit-erythroid.

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different but overlapping normal ranges. As expected, these levels have proved inversely proportional to hematocrit (HCT), provided that renal function is normal [6,7] (Figure 2). Studies have shown, however, that when normal individuals are sequentially phlebotomized up to 1 unit of blood twice a week for 3 weeks, their circulating endogenous serum EPO levels increase only modestly [B,91 (Figure 3). This reveals an important aspect of the natural regulation of erythropoiesis. In response to progressive anemia-hence, increasing degrees of tissue hypoxiathe increase in endogenous EPO production is gradual and graded. The low levels of endogenous EPO that are always present in the plasma appear sufficient to allow for a basal rate of erythropoiesis. Relatively small losses of blood, such as one-unit blood bank donations, stimulate endogenous EPO production only to a small degree. The RBC mass returns to its steady-state level with only a small increase in the rate of erythropoiesis. Only after a major blood loss do endogenous EPO production and the rate of erythropoiesis increase sharply. Such a measured response to varying degrees of anemia makes intuitive sense. It helps prevent the increased rate of erythropoiesis from overshooting and causing polycythemia, with its associated complications. This gradual response suggests a possible role for Epoetin alfa, however, in augmenting the endogenous rate of erythropoiesis and allowing candidates for elective surgery to donate more autologous blood in a shorter period of time. Although endogenous EPO is necessary for the appropriate production of RBCs, it is not the only requisite factor. Adequate supplies of vitamin B12, folic acid, and iron are also essential to normal erythropoiesis. Vitamin B12 and folic acid are required for DNA synthesis, while iron is necessary for hemoglobin (Hb) production. Without adequate supplies of these nutrients, normal RBC production will not occur even when EPO is present in excessive amounts. The vast majority of individuals have plentiful stores of vitamin Bt2, but folic acid and iron are often less abundant. In settings of accelerated erythropoiesis, therefore, folic acid, iron, or both, may become rate limiting. IMPACT OF INFLAMMA TION ON ERYTHROPOIESIS Inflammation of the sort seen in patients with rheumatoid arthritis, cancer, and infection-as well as transient inflammation immediately following surgery-may inhibit erythropoiesis and lead to the so-called anemia of chronic disease or anemia of inflammation [IO,I1]. The pathogenesis of this anemia appears to be multifactorial. Although bone-marrow iron stores are adequate or increased, there is impaired iron reutilization together with low serum iron and iron-binding capacity and normal or elevated serum ferritin levels. Significantly, patients with anemia of chronic disease often have relatively low endogenous serum EPO levels for their degree of anemia [12-141. This relative deficiency in endogenous EPO production is compounded by a 170

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bone-marrow responsiveness to EPO compared with normal bone marrow [IO]. Both the relative

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171. PREOPERATIVE AUTOLOGOUS DONATION OF BLOOD Patients preparing for elective surgical procedures in which significant blood loss is expected often choose to participate in PAD programs. A patient comes to the blood bank, usually once a week for 2-3 weeks, donating a unit of blood per visit. This blood is then returned to the patient as needed during or after surgery. Patients and physicians alike often fail to appreciate fully that the success of PAD, because it depletes the body’s own supply of RBCs, depends primarily on the patient’s ability to manufacture new RBCs prior to surgery. The production of new RBCs depends, in turn, on adequate supplies of iron and on increased renal production of EPO in response to the anemia induced by PAD. This fact can best be illustrated by the following hypothetical example: A 65-year-old woman preparing to undergo elective hip replacement for osteoarthritis goes to the blood bank of her hospital to participate in a PAD program. Her surgeon has requested that she donate 2 units of blood before her surgery, which is scheduled to occur in 3 weeks. She is found to have a HCT level of 36% with a mean corpuscular volume of 80 fL, and her iron studies are consistent with borderline iron deficiency. She is started on oral iron supplementation and proceeds to donate 1 unit of blood.On her return to the blood bank 1 week later, she is found to have a HCT level of 33%. In accordance with the American Association of Blood Banks guidelines-which allow PAD if HCT is ~33%~the patient then donates her second unit of blood without complication. At admission to the hospital for surgery 2 weeks later, she is noted to have hematocrit of 30%. Her estimated blood loss during surgery is 1,300 mL, and postoperatively, her HCT level is found to be 20%.

In this example, although the patient’s endogenous serum EPO levels increased after her phlebotomies, she had inadequate iron stores for generating new RBCs to replace her predonated blood. She subsequently entered surgery with a hematocrit level of 30% and lost 1,300 mL of blood, thus losing 390 mL of RBCs (30% of 1,300) in the operating room. Had she not predonated blood, she wouid have gone into surgery with a hematocrit level of 36%. In that situation, a loss of 1,300 mL of blood would have amounted to 468 mL of RBCs (36% of 1,300). By donating 2 units of blood without successfully generating new RBC production, this patient was no better off than if she had participated in an isovolemic hemodilution protocol. As a result, she saved only 78 mL (468-390 mL) of RBCs-the equivalent of about onethird of a unit of blood. If, on the other hand, the patient had been able to THE AMERICAN JOURNAL OF SURGERY

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regenerate all her predonated RBCs and enter the operating room with a HCT level of 36%, she would have received the full benefit of her 2-unit autologous blood donation. Optimal generation of new RBCs to replace her predonated blood would have made the autologous procedure approximately six times more effective. Clearly, optimal blood generation depends on adequate time between blood donation and surgery, on an adequate increase in endogenous EPO production in response to the mild anemia induced by phlebotomy, and on adequate stores of total body iron, folate, and vitamin B12. EPOETIN

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Figure 4. Changes in hematocrit (A), hemoglobin concentration (B), and absolute reticulocyte counts (C) in three groups of normal mate volunteers receiving Epoetin aifa. Group 1 (0) received 300 U/kg Epoetin alfa on days 1,4,7, and 10; group 2 (0) received 400 U/kg Epoetin alfa on days 1,5, and 9; group 3 (0) received 600 U/kg Epoetin alfa on days 1 and 10. (Reprinted from [32] .)

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strated in patients with anemia secondary to renal failure [18-201, in a subset of anemic HIV-infected patients with low endogenous serum EPO levels receiving zidovudine [221,22],and in cancer patients undergoing myelosuppressive chemotherapy [23]. Pharmacokinetics: A clear understanding of the pharmacokinetics and bioavailability of Epoetin alfa is crucial to its effective use. Its pharmacokinetics following both intravenous (IV) and subcutaneous (SC) administration have been documented in healthy volunteers and in patients with chronic renal failure [24,25]. Because of the potential convenience of subcutaneous injection in ambulatory patients, the pharmacokinetits and efficacy of subcutaneous versus intravenous routes assume considerable importance. When the drug is administered intravenously, peak serum levels are attained within minutes, and mean serum half-life is 4-9 hours [26]. After subcutaneous administration, however, the pharmacokinetic profile is quite different. Peak serum levels are markedly lower-only about 5% of those following comparable intravenous doses-and time required to reach peak levels is 5-24 hours. Once attained, this peak serum concentration is followed by a very gradual decline, with a half-life > 24 hours. Recent evidence strongly suggests that a given dose of Epoetin alfa may be more effective when administered in frequent, small subcutaneous injections than in less frequent large intravenous doses. Perhaps this is because the more sustained serum level achieved with frequent dosing more closely mimics the physiologic response to hypoxia. Proper attention to route and frequency of administration will be critical for studying the optimal efficacy of Epoetin alfa in the elective surgical setting. Epoetin alfa in the elective surgery setting: The hypothetical case study discussed above demonstrates the importance of RBC production for maximizing the effectiveness of autologous blood donation. Now that Epoetin alfa is available for the pharmacologic enhancement of erythropoiesis, several studies have been conducted and others are currently in progress to determine how this agent can best be used in the elective surgery setting to increase RBC production and decrease exposure to allogeneic blood. Two clinical strategies are being studied extensively. In the first, Epoetin alfa is used as adjunctive therapy to enhance the patient’s ability to undergo PAD. Goodnough eta1 [271studied nonanemic, iron-replete patients preparing for elective orthopedic procedures in which the attending physician anticipated that 2 3 RBC transfusion units would likely be required. Patients came to the blood bank twice weekly for 3 weeks before surgery. At each visit, in accordance with the American Association of Blood Banks policy then in effect, patients with HCT levels ~34% donated 1 unit of blood for autologous use in the perioperative period. These patients were randomized to receive intravenous Epoetin alfa (600 U/kg) or placebo at each visit. 170

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Although Epoetin alfa significantly stimulated erythropoiesis and facilitated PAD in this study, there was no significant difference in allogeneic blood exposure between the Epoetin alfa group and the placebo group. It remains to be seen in which precise clinical settings a combination of Epoetin alfa treatment and PAD might prove more efficacious and cost-effective than an aggressive PAD program alone. It may well be, as Mercuriali et al [28] have recently shown, that those who will benefit most from Epoetin aifa therapy are chronically anemic patients facing elective surgery. In the second strategy, Epoetin alfa is used to enhance endogenous erythropoiesis without PAD. In this approach, Epoetin alfa is administered to increase RBC mass before elective procedures in patients who would probably otherwise require 1-3 units of RBC transfusions. If RBC mass can be increased safely and adequately before surgery, the RBCs remaining after blood loss occurs should be sufficient to avoid allogeneic blood exposure. In essence, such patients would serve as their own blood banks. As Figure 3 shows, endogenous serum EPO levels increase only slightly after several units of blood are donated. By using Epoetin alfa instead of PAD, markedly increased serum EPO levels can be achieved. These levels should provide a greater stimulus for increasing the patient’s rate of erythropoiesis. Findings from several studies suggest that the administration of Epoetin alfa begun several days before surgery-without the cost and complexities of PADmay stimulate erythropoiesis and help decrease the number of RBC transfusions required in the postoperative period. A large, randomized, placebo-controlled study by the Canadian Orthopedic Perioperative Erythropoietin Study Group has demonstrated the safety and efficacy of perioperative Epoetin alfa administration (300 U/kg SC daily, from 10 days pre- through 3 days postsurgery) versus placebo in decreasing RBC transfusion requirements of patients undergoing elective hip replacement [29]. The mean number of RBC transfusions in the Epoetin alfa group was 0.52 compared with 1.14 in the placebo group. Only 23% of the Epoetin alfa-treated patients required RBC transfusions compared with 44% of the placebo group (P = 0.007). No increased frequency of venous thrombosis was seen. Kyo et al [30] used IV Epoetin alfa two or three times weekly at relatively low doses-3,000, 6,000, and 9,000 IU per IV dose-for 2 weeks pre- and 2 weeks postoperatively in patients scheduled for cardiac surgery. Intravenous iron was given simultaneously. Only those receiving the highest dose three times weekly showed a significant increase in preoperative Hb. Of those treated with Epoetin alfa, 41% (29/71) received allogeneic RBC transfusions compared with 58% (15/26) of controls. D’Ambra et al [32] systematically studied perioperative Epoetin alfa administration without concurrent PAD in a double-blind, placebo-controlled study using 8 consecutive days of perioperative subcutaneous Epoetin alfa treatment in patients undergoing coronary artery bypass graft surgery. These patients received either 300 THE AMERICAN JOURNAL OF SURGERY

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Figure 5. (A) Correlation between baseline serum ferritin (ng/ ml) and maximum hematocrit (HCT) increase in 24 subjects. Correlation parameters are y = -3.051 log (x), r = 0.682, P < 0.001. (B) Changes in serum ferritin level as a function of time in the three groups studied. (C) Saturated iron binding capacity on days 1, 13, and 24 in the three groups studied. (Reprinted from [32].)

U/kg or 150 U/kg of Epoetin alfa from the fifth day preceding surgery through the second day following surgery. Significantly more of the placebo-treated patients (5/11) needed allogeneic RBC transfusions than did those receiving Epoetin alfa (l/25). Despite their low rate of RBC transfusion, the Epoetin alfa patients had at discharge a mean HCT level equal to or higher than that of the placebo group. In preparation for our own studies of short-term Epoetin alfa treatment as an alternative to PAD, we investigated dosage regimens in healthy male volunteers VOLUME

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[32,33]. Earlier studies had suggested that subcutaneous administration of Epoetin alfa was more effective than the IV route [34]. We therefore compared three different subcutaneous dosage schedules to determine which were both effective and practical for routine perioperative stimulation of erythropoiesis as assessed by Hb concentration, HCT, and absolute reticulocyte counts. In view of previous work suggesting that mobilization of iron stores might limit response to Epoetin alfa [35,36], we also studied the effect of accelerated erythropoiesis on iron indices and hemoglobinization of individual RBCs. A randomized trial was performed on 24 healthy, iron-replete males. Subjects received a total dose of 1,200 U/kg SC Epoetin alfa in one of three dosage schedules: group l-300 U/kg on days 1, 4, 7, and 10; group 2400 U/kg on days 1, 5, and 9; group 3-600 U/kg on days 1 and 10. All subjects were given 300 mg of oral elemental iron daily for 10 days. Complete blood counts, absolute reticulocyte counts, serum ferritin, serum iron, and serum total iron-binding capacity (TIBC) were measured periodically during the 24day study. Reticulocyte characteristics were examined with a flow cytometry method that allowed measurements of individual reticulocyte Hb content. No significant adverse events were observed. All groups showed a statistically significant increase in HCT levels, Hb content, and absolute reticulocyte count (Figure 4). There was no significant difference in Hb or HCT response among the three groups. Mean maximum increases in HCT levels were 5.4 2 1.7 in group 1, 6.0 + 2.1 in group 2, and 7.2 + 2.6 in group 3. Increases in HCT levels correlated positively with log baseline ferritin (r = 0.682, P 100 ng/mL (8.1 -C 1.7% vs 5.4 2 1.9%, P ~0.005). Administration of Epoetin alfa was associated with a highly significant (P I 0.0005) 74% decrease in serum ferritin as well as a marked decrease in percent saturation of TIBC, from 39 2 14% to 14 * 4% (P ~0.0005; Figure 5B and SC), even though subjects lost <250 mL of blood as a result of venipunctures during the entire course of the study. As noted above, subcutaneous administration of Epoetin alfa has been associated with a significant increase in reticulocyte production. However, many subjects who had relatively low iron stores (as estimated by baseline serum ferritin) produced reticulocytes with decreased Hb content [33]. In fact, the production of reticulocytes with reduced Hb content correlated inversely with the log value of baseline serum ferritin (r = -0.82; P
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tiveness of erythropoiesis in response to Epoetin alfa therapy. A recent study by Mercuriali et al [371 supports this practice. They gave intravenous iron saccharate to candidates for elective surgery who were receiving Epoetin alfa to enhance PAD. These patients, whose preoperative serum ferritin levels were ~52 ng/mL, displayed a greater increase in reticulocyte counts and number of autologous donations-and thus lower exposure to allogeneic blood--than did similar patients receiving an oral form of iron supplementation. ROLE OF EPOETIN ALFA IN THE SURGICAL SETTING: FUTURE DIRECTIONS The precise role of Epoetin alfa in the surgical setting is still under investigation. Several Epoetin alfa dose schedules appear to provide an effective regimen for increasing RBC mass prior to elective surgery. Even with oral iron supplementation, however, normal iron stores for basal erythropoiesis may not always be sufficient for the accelerated erythropoiesis associated with acute Epoetin alfa administration. In subjects whose iron stores are in the low-normal range, there is evidence that Epoetin alfa-accelerated erythropoiesis may lead to the production of iron-deficient reticulocytes. These findings provide support, however, for further study of Epoetin alfa treatment, perhaps in combination with intravenous iron administration, as an alternative to PAD in the elective surgery setting. Optimal dose, route and frequency of administration, and duration of therapy before surgery remain to be determined. Which subsets of patients stand to benefit most from Epoetin alfa therapy, particularly with regard to basal RBC mass and type of surgical procedure, also awaits further definition. The safety, efficacy, and cost-effectiveness of Epoetin alfa versus PAD have yet to be studied in a properly controlled clinical trial. REFERENCES 1. Krantz SB. Erythropoietin. Blood 1991; 77: 419-34. 2. Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev 1992; 72: 449-89. 3. Porter DL, Goldberg MA. Regulation of erythropoietin production. Exp Hematoll993; 21: 399-404. 4. Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that oxygen sensor is a heme protein. Science 1988; 242: 1412-5. 5. Sherwood JB, Goldwasser E. A radioimmunoassay for erythropoietin. Blood 1979; 54: 885-93. 6. Spivak JL, Hogans BB. Clinical evaluation of a radioimmunoassay (RIA) for serum erythropoietin (EPO) using reagents derived from recombinant erythropoietin (rEP0). Blood 1987; 7O(suppll): 143a. 7. Erslev AJ, Wilson J, Caro J. Erythropoietin titers in anemic, nonuremic patients. J Lab Clin Med 1987; 109: 429-33. 8. Kickler TS, Spivak JL. Effect of repeated whole blood donations on serum immunoreactive erythropoietin levels in autologous donors. JAMA 1988; 260: 65-7. 9. Goldberg MA, Schneider TJ, Khan S, et al. Clinical validation of an RIA for natural and recombinant erythropoietin in serum and plasma. Clin Biochem 1993; 26: 183-9. IO. Means RT Jr, Krantz SB. Progress in understanding the pathogenesis of the anemia of chronic disease. Blood 1992; 80: 163H7.

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11. &hilling RF. Anemia of chronic disease: a misnomer? Ann Intern Med 1991; 115: 572-3. 12. Baer AN, Dessypris EN, Goldwasser E, et al. Blunted erythropoietin response to anaemia in rheumatoid arthritis. Br J Haemato1 1987; 66: 559-64. 13. Hochberg MC, Arnold CM, Hogans BB, et al. Serum immunoreactive erythropoietin in rheumatoid arthritis: impaired response to anemia. Arthritis Rheum 1988; 31: 1318-21. 14. Miller CB, Jones RJ, Piantadosi S, et al. Decreased erythropoietin response in patients with the anemia of cancer. N Engl J Med 1990; 322: 1689-92. 15. Faquin WC, Schneider TJ, Goldberg MA. Effect of inflammatory cytokines on hypoxia-induced erythropoietin production. Blood 1992; 79: 1987-94. 16. Jelkmann W, Fandrey J, Wiedemann G. Immunoreactive erythropoietin in the anemia of nonrenal chronic diseases. Biomed Biochim Acta 1990; 49: S265-70. 17. Means RT Jr, Krantz SB. Inhibition of human erythroid colony-forming units by gamma interferon can be corrected by recombinant human erythropoietin. Blood 1991; 78: 2564-7. 18. Eschbach JW, Egrie JC, Downing MR, et al. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin: results of a combined phase I and II clinical trial. N Engl J Med 1987; 316: 73-8. 19. Eschbach JW, Abdulhadi MH, Browne JK, et at. Recombinant human erythropoietin in anemic patients with end-stage renal disease: results of a phase III multicenter clinical trial. Ann Intern Med 1989; 111: 992-1000. 20. Lim VS, DeGowin RL, Zavala D, et af. Recombinant human erythropoietin treatment in predialysis patients: a double-blind placebo-controlled trial. Ann Intern Med 1989; 110: 108-14. 21. Fischl M, Galpin JE, Levine JD, et al. Recombinant human erythropoietin for patients with AIDS treated with zidovudine. N Engl J Med 1990; 322: 1488-93. 22. Henry DH, Beall GN, Benson CA, et al. Recombinant human erythropoietin in the treatment of anemia associated with human immunodeficiency virus (HIV) infection and zidovudine therapy. Ann Intern Med 1992; 117: 739-48. 23. Abels RI. Use of recombinant human erythropoietin in the treatment of anemia in patients who have cancer. Semin Oncol 1992; 19: 29-35. 24. McMahon FG, Vargas R, Ryan M, et al. Pharmacokinetics and effects of recombinant human erythropoietin after intravenous and

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subcutaneous injections in healthy volunteers. Blood 1990; 76: 1718-22. 25. Salmonson T. Pharmacokinetic and pharmacodynamic studies on recombinant human erythropoietin. Stand J Urol Nephrol Suppl1990; 129: l-66. 26. Erslev AJ. Erythropoietin. N Engl J Med 1991; 324: 1339-44. 27. Goodnough LT. Rudnick S, Price TH, er al. Increased preoperative collection of autologous blood with recombinant human erythropoietin therapy. N Engl J Med 1989; 321: 1163-8. 28. Mercuriali F. Gualtieri G, Sinigaglia L, et al. Use of recombinant human erythropoietin to assist autologous blood donation by anemic rheumatoid arthritis patients undergoing major orthopedic surgery. Transfusion 1994; 34: 501-6. 29. Canadian Orthopedic Perioperative Erythropoietin Study Group. Effectiveness of perioperative recombinant human erythropoietin in elective hip replacement. Lancet 1993; 341: 1227-32. 30. Kyo S, Omoto R, Hirashima K. et al. Effect of human recombinant erythropoietin on reduction of homologous blood transfusion in open-heart surgery: a Japanese multicenter study. Circulation 1992; 86: B-413-18. 31. D’Ambra MN, Lynch KE, Boccagno J, et al. The effect of perioperative administration of recombinant human erythropoietin (r-HuEPO) in CABG patients: a double-blind, placebocontrolled trial. Anesthesiology 1992; 77: A159. 32. Rutherford CJ, Schneider T, Dempsey H, et ul. Efficacy of different dosing regimens for recombinant human erythropoietin in a simulated perisurgical setting: the importance of iron availability in optimizing response. Am J Med 1994; 96: 139-45. 33. Brugnara C, Collela GM, Cremins J, et al. Effects of subcutdneous recombinant human erythropoietin in normal subjects: development of decreased reticulocyte hemoglobin content and irondeficient erythropoiesis. J Lab Clin Med 1994; 123: 660-7. 34. Bommer J, Ritz E, Weinreich T, et al. Subcutaneous erythropoietin. (Letter.) Lancet 1988; ii: 406. 35. Brugnara C, Chambers LA, Malynn E, et nl. Red blood cell regeneration induced by subcutaneous recombinant erythropoietin: iron-deficient erythropoiesis in iron-replete subjects. Blood 1993; 81: 95664. 36. Van Wyck DB. Iron management during recombinant human erythropoietin therapy. Am J Kidney Dis 1989; 14: 9-13. 37. Mercuriali F, Zanella A, Barosi G, et ~1. Use of erythropoietin to increase the volume of autologous blood donated by orthopedic patients. Transfusion 1993; 33: 55-60.

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