From blood transfusions to human recombinant erythropoietin

CH. BIRBILIS[1], I.P. SOFIANOS[2], Κ. KATEROS[2], Β. SAKELLLARIOU[2], G.A. MACHAIRAS[3]
[1]Department of Internal Medicine Livadia General Hospital
[2]Department of Orthopedic Surgery Livadia General Hospital
[3]B' Orthopedic Clinic, 1o Hospital of IKA

 

ABSTRACT
Despite strict donor screening criteria and attentive laboratory testing of every blood unit, allogeneic blood transfusions are associated with certain risks. Complications of blood transfusion may be early or late. Early complications can be haemolytic reactions, fever, allergic reactions, reactions due to infected blood, circulatory overload, clotting abnormalities, and others. The most important late complications include transmission of a wide range of diseases, alloimmunization and immunomodulation.
Concern about transfusion risks has led to numerous alternatives in order to reduce patient exposure to allogeneic blood. The application of lower transfusion "trigger" criteria and the improvements in surgical techniques, such as haemostasis and careful operative planning, the use of pharmacological agents in order to minimize blood loss, the use of blood substitutes and oxygen carriers (currently under investigation), have contributed to a reduction in allogeneic blood requirements. Other alternatives to allogeneic transfusion include preoperative autologous blood donation (PAD), normovolemic haemadilution, intra and postoperative blood salvage, as well as perioperative therapy with recombinant human erythropoietin (rHuEPO).
Erythropoietin (EPO) has been successfully used extensively to treat anemia associated with chronic renal failure and many other diseases. EPO is the primary regulator of red blood cell production. It is a glycoprotein produced primary in the kidneys in response of tissue hypoxia. EPO exerts its biological effects by binding to a specific cell-surface receptor on erythroid-progenitor cells in the bone marrow to promote proliferation, differentiation and survival of those cells. Administration of rHuEPO can be both intravenous and subcutaneous and the dosing regimens widely range from 50 to 600IU/Kg of body weight.
In conclusion rHuEPO increases and maintains haemoglobin levels and reduces transfusion requirements. It is safe, well tolerated and improves quality of life.

Key words: allogeneic transfusion, blood management, erythropoietin, erythropoiesis.

INTRODUCTION
The serious blood loss, which can occur during major orthopedic operations, constitutes perioperative blood management one of the main problems for an orthopedic surgeon is to solve. Orthopedic Surgery is the specialty which uses the biggest number of blood units, both for the elective operations and the urgent ones as well: 2/3 of total transfusions in the USA are associated with major orthopedic operations[20], while the percentage in Europe is proportional[29]. It is estimated that during a primary total knee arthroplasty the blood loss ranges between 600 and 1500ml, and patients are often being transfused with two blood units. During ambilateral primary operation blood loss is twice as much, while during reoperations, blood loss is even more[29]. That means that allogeneic transfusion probability could be as high as 50-60%, especially in patients with a hemoglobin (Hb) value of lower than 10gr/dl[5,10]. In the past, blood transfusion was the main means for the direct increase in surgical patientΥs hematocrit (Hct). Discovery of human immunodeficiency virus (HIV) in 1982, and the knowledge that the virus is transmitted through blood led to a serious skepticism as far as regards cost-benefit relation as well as to a revaluation of blood transfusion criteria, which became stricter. Today, although transfusion remains the only solution for the severe injured patient, in major elective orthopedic operations many blood management techniques have been developed and others are under development, targeting at reducing the risk from allogenic transfusions.

RISKS FROM TRANSFUSIONS
Despite blood donors' strict selection and the detailed check of every blood unit, allogenic blood transfusion, although safer today than in the past, continues to have risks. Besides the tragic human error of incompatible blood transfusion, which will lead to acute hemolytic reaction and death, reactions from transfusions are classified according to the time of their development in early and late, while according to their cause in immune and non immune. The most serious reactions are[2,9,18,30]:
1.a. Early immune reactions
– Acute hemolysis, caused by ABO blood group system incompatibility.
– Allergic reactions, caused by donor's plasma proteins.
– Fever reactions, caused by donor's proteins, leukocytes or platelets.
– Hemorhagic syndromes like disseminated intravascular clotting (DIC) following a massive transfusion.
1.b. Non immune early reactions
– Septic shock caused by bacterial superinfection of transfused blood, usually with Gram(-) bacteria.
– Cisrculatory overload, especially with elderly and children.
– Hyperkalemia.
– Non immune hemolysis caused by, for example, very warm or very cold blood.
2.a. Late immune reactions
– Delayed hemolytic reaction, caused by a secondary development of antibodies against red blood cells, about other systems except ABO. It is difficult to be diagnosed, because it occurs 5 to 10 days after transfusion.
– Alloimmunization, caused by development of antibodies against red blood cells, as well as against leukocytes and/or plasma proteins. It is common, usually without great significance, having as a result difficulty in crossing blood and/or the emergence of delayed hemoytic reaction.
– Thrombocytopenia (purpura), caused by development of antibodies against platelets.
– Graft-Versus-Host Disease (GVHD). GVHD is very rare but very serious complication. It appears in immunodeficient persons, in premature infants, in-patients with congenital deficiencies, in cancer patients, or in bone marrow transplants recipients. It is caused because of bone marrow's incorporation and colonization by transfused lymphocytes, which reject the host. Also, it could occur in-patients who have received blood from first degree relatives with a strong histocompatibility. To prevent this kind of complication, blood should be irradiated with low dose radiation[9,21].
2.b. Late non immune reactions
– Diseases transmitted from viruses, bacteria, rickettsiae, parasites e.t.c. The most common virus is CMV, considering that 70-80% of the general population has antibodies against CMV and 1-4% could transmit the infection. Epstein-Barr Virus (EBV) transmission is frequent as well. Infection probability by hepatitis B (HBV) and C (HCV) has been reduced the last years in 1/63.000 and 1/103.000 respectively. Of course, infection by HIV and Human T-cell Leukemia/Lymphoma Virus (HTLV1-2) which is endemic in Japan and Carribean, is more serious, and transmission probability is 1/493.000 and 1/641.000 respectively. It is obvious that in the future new infectious agents will be discovered[21,27]. If in Europe and the USA there is a special concern about retroviruses and hepatitis viruses, in other geographic regions other infections constitute a great danger. Malaria transmission is a huge problem for under developed third world countries: in some regions in Africa more than 15% of donors are infected. In addition, other parasites which constitute a danger are Trypanosoma cruzi, Babesia, e.t.c[21].
– Iron overload in multi-transfused patients[2].
– Finally, there is a discussion about the possibility for multiple transfusions to have an immunosuppressive action, caused both by cellular and humoral factors[29]: In patients with renal transplants, it has been observed a lower incidence of transplant rejection, when they were transfused, an effect which is dose related. Moreover, in Orthopedic Surgery it seems that multiple transfusions are connected with a higher incidence of postoperative infections, mainly urinary infections and pneumonitis. There is also a discussion about neoplasms relapse probability, negative effect in fractures' porosis and joints' prostheses[21,29]. In any case it seems that negative immunomodification manifestations of transfusions are caused by leukocytes' presence as well. Blood administration with leukapheresis, which withhold leukocytes[29], reduces the possibility of such reactions.

Figure 1. Transfused patients percentage in proportion to the initial Hb value.

 

TRANSFUSION CRITERIA
For decades hemoglobin (Hb) value of lower than 10gr/dl was the traditional transfusion criterion. Today, it is known that simple lab measure of Hb and Hct is not adequate as a criterion. This happens because transfusion does not take place for the increase of the Hb lab value, but for better oxygenation of tissues. Stabilized patients, without heart failure, fever and stress have low demands in oxygen and can tolerate well enough a Hb value of 7gr/dl[29], while the limit of 10gr/dl is taken into consideration only in patients with heart failure or other cardio-pulmonary diseases. So, the need for transfusion depends on the patient and its condition[29], and it is safer the decision about transfusing a patient to based on clinical criteria which are valuated in every patient separately[23]. Such clinical criteria are hypotension, tachycardia, hypoxemia, dyspnea, angina, fatigue, weakness e.t.c
In spite of all this, preoperative Hb value (baseline Hb) is an important prognostic factor for a transfusion probability in surgical patients[4]. That means that the lower the preoperative Hb value is, the higher transfusion probability is 12. It has been observed in many studies[1,7,11] that patients with Hb value higher than 10gr/dl and equal or lower than 13gr/dl have doubled the possibility to be transfused than patients with Hb value higher than 13gr/dl (figure 1). In addition, in another study Nuttall et al.[26] have shown that patients with preoperative Hb value of higher than 15gr/dl had essentially 0% probability to be transfused, as opposed to patients with Hb value of equal or lower than 11gr/dl, who had almost 100%.

BLOOD MANAGEMENT
Revision of transfusion criteria has led to reduction of allogenic transfusions, considering that today lower values of Hb are accepted in surgical patients who do not have other health problems. However, several techniques about pre-, peri-, and postoperatively blood management have been developed, aiming at a further reduction of transfusions[20]. In these techniques are included:
1. Improvement of surgical techniques and surgical operationsΥ replanning, targeting at reducing blood loss and minimizing the need for allogenic transfusions. During operation, blood saving could result from better hemostasis, as well as from implementation of special techniques by anesthesiologists, like hypotensive anesthesia
2. Use of pharmaceutical agents, targeting at reducing blood loss. These agents are classified into two categories: in localized acting agents like thrombin, collagen and fibrin, and in agents with systematic action like aprotinin, tranexamic acid and minocaproic acid, which are administered perioperatively and postoperatively intravenously with safety[20].
3. Oxygen carriers, another class of pharmaceutical agents, which are under research in clinical trials phase ΙΙ and ΙΙΙ. Two categories of chemical substances are researched: blood substitutes, chemical compounds having hemoglobin as a base, and transient oxygen carriers, perfluorocarbons, which are organic compounds where gases including oxygen and carbon dioxide, show very high solubility[20].
4. Autologous transfusion, which is blood collection from the patient himself and its use to cover his needs. This method is popular enough and is applied in parallel with the above mentioned blood management techniques. Autologous transfusion or self Πtransfusion is classified in:
a. Preoperative autologous donation (PAD). During the weeks before elective operation, blood is collected from the patient with one or more donations. One to four units can be collected in between five to seven days each. Last donation takes place 72 hours before operation. Autologous predonation is allowed in patients with Hb values of 11gr/dl or higher, but it will be ideal in patients with Hb values of 13gr/dl or higher, because with these values predonation reduces the chances of allogenic transfusion. During PAD iron per os coadministration is obligatory for the erythopoiesis enhancement, while in the last years it is combined with erythropoietin administration (EPO), especially in patients with inappropriate endogenous erythopoiesis. Preoperative autologous donation was a popular practice in 80s, but since 1992 it has been observed a significant decline in its use16. Causes of this decline are mentioned as following: Inappropriate erythropoiesis, which has as a result patients with serious anemia to be operated. Thus, making allogenic transfusion, high cost of predonation, the need for frequent visits of the patient and the disposal of non usable blood units unavoidable.
b. Preoperative hemodilution. With this technique, either directly preoperatively or perioperatively, blood is taken from patient and it is replaced with an equal volume of colloid or crystalloid solutions. So patient loses fewer erythrocytes and blood flux in microcirculatory is improved. Blood is readministered to the patient during or after operation and after hemostasis has been completed. This method is contraindicated in patients with renal, cardiac, pulmonary or hepatic failure.
c. Perioperative and postoperative blood salvage. Via special devices blood is collected from operational field or/and postoperatively from surgical drainages, is centrifuged, filtered, washed (not necessarily) and is readministered to the patient. This technique is more efficient when it is combined with other methods, and especially with preoperative autologous donation[9,16,18,29].
5. Human recombinant erythropoietin alpha (rHuEPO) accelerates erythropoiesis and increases reticulocytes, Hct and Hb. This has as a result the increase in oxygen delivery and tissuesΥ oxygenation amplification. Its effectiveness has been proved by a great number of clinical trials[1,7,11] among patients who received placebo or rHuEPO, and was especially effective on patients with Hb values higher than 10 and equal or lower than 13gr/dl, who are at the greatest risk to be transfused. rHuEPO has the same structure with endogenous ΕΡO. It can be used alone or in combination with the other techniques, for reducing allogenic transfusion probability. For a more extensive understanding of the role, action and rHuEPOΥs use, a more extensive report about EPO and erythropoiesis follows.

Figure 2. Relation between EPO plasma levels and Hb values.

Figure 3. Main steps of erythropoiesis

ERYTHPOIETIN
ΕΡO is a glucoprotein which belongs to the cytokinesΥ complicated system. Cytokines are glucoproteins, which resemble hormones and are produced from am impressive large variety of tissues and neoplastic cells. Hemopoietic growth factors (HGF) and interleukins (IL)32 belong to these. ΕΡO is an HGF and constitute the first and more important modulator of erythropoiesis. ΕΡO was the first HGF which was standardized and cloned in the middle of 80s. It was isolated for the first time in 1977 in a patient's urine with aplastic anemia, but the first indications for a factorΥs existence which was modulating hemopoiesis was shown in 1906. In 1987 the first clinical trials with rHuEPO in uremic patients are reported[3].
Since then and especially during the last decade, rHuEPO is used successfully worldwide for the treatment of anemia in many diseases, and the possibility of its use in some others is under research[3]. So, except for its initial use in anemia of chronic renal failure, it has been used in chronic anemia of neoplastic diseases and chemotherapy induced anemia and/or radiotherapy, and in many hematologic malignancies as well, in rheumatoid arthritis, on HIV-therapy induced anemia, e.t.c[3,32]. In the last years rHuEPO is also used for correction and/or therapy of orthopedic patients' perioperative anemia who are submitted to elective orthopedic surgical operations[10,16].

STRUCTURE OF ERYTHROPOIETIN
Blood circulated ΕΡO is constituted of 165 amino acids and is strongly glucosylated, up to 40%. Its molecular weight is 30.4Kd. There are two types of rHuEPO available for use: α-type glucosylated about 39%, and β-type about 24%. Between these types there are no pharmacological differences except for pharmacokinetics[3]. Glucosylation is not necessary for EPO's biological action, but for extending its stay in plasma and delaying its clearance from the liver[25]. Non glucosylated EPO is taken from the liver and removed from the circulation in a few minutes. rHuEPO's glucosylation positions are proportional to these of physiological human ΕΡO[31].

PRODUCTION OF ERYTHROPOIETIN
Kidney and especially peritubular interstitial cells are the main area of EPO production in adults, in percentage of about 90%, while the other 10% is produced mainly in liver. During serious hypoxia or intensive stress, liver can take up to 33% of EPO's total production in adult, while brain, spleen and testes produce small amount of EPO as well. In infanst, liver is the main organ of EPO production, and "changing" from liver to kidney takes place gradually from 4th -5th month of pregnancy and is completed within 40 days after birth[3].

MECHANISM OF PRODUCTION
Tissue hypoxia is a primary stimulation for EPO's production. Tissue hypoxia may result from a reduction in oxygen concentration, like in Chronic Obstructive Pulmonary Disease or living in high altitude, from a reduction in HbΥs binding ability for oxygen, like in several hemoglobinopathies, and also from a reduction in oxygen delivery because of blood loss or hemolysis.[3,13].
It seems that there is an additional mechanism in EPO's production, due to the fact that it has been observed that precursor erythroid cellsΥ mass in bone marrow and serum EPO values are inversely proportional. In addition, it is worth mentioned that EPO plasma level is remarkably stable when Hb values are in normal range and increases rapidly when Hb falls below 12gr/dl (figure 2). These findings from recent clinical trials show that possibly the excellent biological effect of administered rHuEPO is achieved when Hb falls below 12gr/dl[13].

BIOLOGICAL ACTION OF ERYTHOPOIETIN
ΕΡO acts on precursor erythroid cells and controls red cellsΥ production. It promotes proliferation, maturation and differentiation of[3,24] precursor cells of red blood cell line and protects cells from apoptosis, that is programmed cell death. ΕΡO acts by binding to special receptors on the surface of target cells[13]. Cells which express EPO's receptors are mainly the Colony Forming Unit - Erythroid (CFU - E), unipotential cells binded for erythroid line, and erythroblasts (proerythroblast and more mature types) which express almost 1000 receptors per cell.
The less mature unipotential cell of erythroid line, Burst Forming Unit - Erythroid (BFU - E), expresses very few receptors, while the reticulocyte one not at all[3].

ERYTHROPOIESIS
Erythropoiesis is a very accurate procedure which leads to production of mature red cells. Stem cell gives birth to BFU - E cell, which in its turn gives birth to CFU - E cell[6,24]. This cell is divided further into proerythroblast, which after four divisions[13] (erythroblast, basophil erythroblast, polychromatofil erythroblast, oxyphilic erythroblast)[24] ends up at last to oxyphilic or orthochromatic erythroblast, which is not divided any more, it only matures and by expelling its nucleus, is transformed to reticulocyte (figure 3). Finally, starting from a proerythroblast, 16 mature erythrocytes are created[19].
ΕΡO exerts action from CFU - E cell till oxyphilic erythroblast. From BFU - E stage to reticulocytes 14 days are required, while in two days inside bone marrow and in the circulated blood as well, reticulocytes mature further to erythrocytes[6].

TREATMENT - ADMINISTRATION
ΕΡO has fluctuations in plasma during the day and its value is higher in the morning and lower in the evening. Normal plasma values are between 7,8 and 30mIU/ml8. Its administration depends on the disease, in doses from 50 till 600IU/Kgr, with several administration regimens, once, twice or three times per week. Multiple administration doses of rHuEPO (150IU/Kgr 3 times per week or 600IU/Kgr 1 or 2 times per week) are more effective than stat administration[8,10,16]. In addition, it seems that biological response to rHuEPO is achieved with doses till 1800IU/Kgr, possibly because of the receptors' saturation[8].
rHuEPO can be administered subcutaneously or intravenously and is equally effective. Iron deficiency is the most common cause of treatment failure, thus it is necessary the coadministration of at least 200mgr elementary iron daily. In the non anemic patient who receives iron, the increase of reticulocytes is detectable from the third day, while blood unit equivalent is produced on the seventh day, and the equivalent of five blood units on the 28th day[16].

SUITABILITY - ADVERSE EFFECTS - BENEFITS FROM rHuEPO USE
rHuEPO is suitable for equal or lower than 11gr/dl Hb values and it can be co-administered with chemotherapies. It is not recommended in cases of non controlled hypertension, iron deficiency, bone marrow malignancies and in patients with life expectancy less than 6 months[14].
Adverse effects which have been reported in about 10% of patients are feeling of cold and pain in long bones, difficulty in controlling hypertension, thrombotic episodes, fever, constipation, nausea, exanthemas, neutropenia, leukopenia, fatigue e.t.c[3,16,32].
In conclusion, rHuEPO generally is safe and well tolerated. It increases and maintains Hb and Hct levels, resulting in reduction of allogenic transfusions and in improving quality of life[14,17,28].

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