Red Cell Production Research Articles

A Novel HIFalpha Mutation Associated with Familial Erythrocytosis Supports This Isoform Being the Major Regulator of Erythropoietin in Humans.

Red cell homeostasis is crucial to maintain a sustained tissue oxygen supply. Oxygen is sensed by the kidneys and hypoxia induces renal erythropoietin (Epo) to stimulate red cell production. At the molecular level this process is controlled by the hypoxia inducible factor (HIF) complex, comprised of a labile a-subunit and a constitutively expressed b subunit. Key prolines present in the oxygen-dependent degradation (ODD) domain of the a subunit are hydroxylated by prolyl hydroxylase domain proteins (PHD1-3). The von Hippel Lindau (VHL) protein then binds and targets HIFa to the proteasome. There are 3 isoforms of the a subunit, each of which shows differential tissue distribution. The HIF-1, HIF-2 and HIF-3a subunit, in association with the b subunit, exhibit diverse affinities for the hypoxia response elements (HREs) located in the promoters and enhancers of HIF target genes. It is unclear whether Epo synthesis is under the control of HIF-1 or HIF-2 in humans although recent studies in mice support HIF-2 as the critical regulator. Dysregulation of Epo production can give rise to the red cell disorder of erythrocytosis. Defects in the oxygen sensing pathway are commonly associated with erythrocytosis and mutations in both the VHL and PHD2 genes have been described. Previously, screening the ODD domain of HIF-1a detected a polymorphism, Pro582Ser, that did not impair the function of HIF-1a. Subsequently, when sequencing HIF-2a, a novel heterozygous c.G1609T mutation was uncovered in 3 generations of one family with erythrocytosis. All affected individuals exhibited greatly elevated serum Epo levels. The c.G1609T mutation was absent in other unaffected family members as well as a cohort of 200 normal control samples. The mutation predicts the replacement of Gly by Trp at amino acid 537, which is close to the main site of proline hydroxylation at 531. Functional assays confirmed the Gly537Trp variant was less hydroxylated and exhibited reduced affinity for PHD2. Furthermore, the increased stability in normoxia of the Gly537Trp variant would result in elevated transcription of HIF-2 target genes. Consequently, these findings support a critical role for HIF-2a in control of Epo production in humans.

Thrombopoietin emerges as a new haematopoietic cytokine that confers cardioprotection against acute myocardial infarction

Colony stimulating factors (CSFs), also called haematopoietic growth factors, are circulating cytokines that regulate the bone marrow production of red cells, white cells, and platelets. CSFs have also been reported to act on stem cells to regulate lineage-specific differentiation.1 Erythropoietin (EPO) controls red cell production and has been utilized in recombinant form for the treatment of anaemia in patients with end-stage renal disease since 1988. Granulocyte CSF (G-CSF) acts on haematopoietic stem cells to regulate neutrophil progenitor proliferation and differentiation. G-CSF is routinely used to mobilize stem cells in normal patients for transplantation of cells into patients with haematological malignancies, or in the patients themselves. Several recent reports have suggested that these cytokines possess properties that extend beyond their haematopoietic properties, such as the ability to protect the heart against injury caused by ischaemia and reperfusion (I/R).2,3 Baker et al. 4 now demonstrate for the first time in this issue of Cardiovascular Research that a third haematopoietic growth factor, thrombopoietin (TPO), is able to provide cardioprotection against myocardial I/R injury in a rat model by mechanisms independent of its haematopoietic properties. TPO affects nearly all aspects of platelet production, from self-renewal and expansion of haematopoietic stem cells, through stimulation of the proliferation of megakaryocyte progenitor cells, to support the maturation of these cells into platelet-producing cells.5 Since its purification in 1994, TPO has been used to treat thrombocytopenia caused by many conventional chemotherapy regimens.6 … *Corresponding author. Tel: +1 718 430 2645; fax: +1 718 430 8989. E-mail address : dlefer{at}aecom.yu.edu

Targeting Anemia with Erythropoietin during Critical Illness

The overall goals of clinical research in the intensive care unit (ICU) are to improve clinical outcomes through enhanced understanding of how critical illness develops and how such illness is best prevented, diagnosed, treated, or palliated. Several “normal” physiological measurements and laboratory values have been abandoned as therapeutic targets during critical illness, since in some randomized trials, attempts to normalize these measurements and values have shown either harm or no benefit with respect to clinical outcomes.One such target is the hemoglobin concentration. Anemia is commonly acquired in the ICU owing to hemodilution, blood loss, reduced red-cell production, and enhanced . . .

Decreased femoral diaphyseal mechanical strength mainly due to qualitative impairment of cortical tissue in growing rats with stress erythropoiesis

Stress erythropoiesis (SE) can be defined as a state of increased red cell production in response to an enhanced secretion of erythropoietin. Under normal conditions, erythropoiesis in the adult occurs in the erythropoietic tissue that is confined to the bone marrow within the skeleton. Hypertrophy of the erythropoietic marrow that occurs during SE must be accommodated in a larger space. The resulting expansion of the marrow space may induce bone resorption and could alter the biomechanical performance of bone. The present study was designed to estimate the effect of sustained SE on diaphyseal structure and biomechanics of rat femur by mechanically testing the diaphyseal stiffness and strength and calculating some indicators of bone material properties. Female Sprague–Dawley rats weighing 100.0 ± 5.2 g were divided in a control (C) and an experimental (E) group. E rats were biweekly injected with 60 mg/kg of phenylhydrazyne during 6 weeks to induce a haemolytic state. SE, in response to haemolysis, was estimated by reticulocyte count and erythrokinetic techniques, which were markedly increased. To assess bone mechanical properties, the right femur was tested in three-point bending test. Sections of the left femur were stained with haematoxylin–eosin. Body growth was not altered by treatment. Diaphyseal bone mass (CSA) was 13% lower in E than in C rats, while the cross-sectional bending moment of inertia (xCSMI) was significantly higher. The “load capacity” extrinsic properties of the femoral diaphysis were about 40% decreased in E rats when compared to C ones, while the bone material quality indicators (elastic modulus and yield stress of cortical bone tissue) were 54 and 38% lower, respectively. The histologic sections of E femora exhibited a marked thinning of cortical bone and the presence of woven bone in the medullary compartment. The results obtained in this study indicate that SE caused the impairment of the diaphyseal bone material properties (elastic modulus and elastic stress) but enhanced xCSMI over the control values. Neither this improvement in diaphyseal cross-sectional design nor the formation of woven bone could offset the impairment in the bone material stiffness. It is thus proposed that the biomechanical impact of SE upon the whole-bone stiffness should have been more determined by the negative effect on bone material quality than by the negative changes in the cortical area.

Darbepoetin Alfa Protects the Rat Heart Against Infarction: Dose-Response, Phase of Action, and Mechanisms

Erythropoietin is known to stimulate red cell production and has recently been shown to protect the heart against injury from ischemia/reperfusion. However, it is unknown whether darbepoetin alfa (Dpa), a long-acting analog of erythropoietin, can play a protective role against myocardial infarction. We assessed the potential protective role of Dpa in an in vivo rat model of myocardial ischemia/reperfusion and the underlying mechanisms. We found that a single intravenous Dpa treatment immediately before 30 minutes of regional ischemia reduced myocardial necrosis following 120 minutes of reperfusion in a dose-dependent manner. Optimal protection with Dpa against myocardial infarction was manifest at a dose of 2.5 microg/kg. Dpa conferred cardioprotection when administered after the onset of ischemia and at the start of reperfusion. Dpa (2.5 microg/kg) also reduced infarct size and Troponin I leakage 24 hours after reperfusion. Inhibition of p42/44 MAPK (PD98059), p38 MAPK (SB203580), mitochondrial ATP-dependent potassium (KATP) channels (5-HD), sarcolemmal KATP channels (HMR 1098), but not phosphatidylinositol-3 (PI3) kinase/Akt (Wortmannin and LY 294002) abolished Dpa-induced cardioprotection. Dpa confers immediate and sustained cardioprotection in rats, suggesting a potential therapeutic role of this long-acting erythropoietin analog for the treatment of acute myocardial infarction.

Fractionnement plasmatique international : état des lieux

From 22 to 25 million liters of plasma are fractionated yearly in about 70 fractionation plants, either private or government-owned, mainly located in industrialized countries, and with a capacity ranging from 50 000 to three million liters. In an increasingly global environment, the plasma industry has recently gone through a major consolidation phase that has seen mergers and acquisitions, and has led to the closure of a number of small plants in Europe. Currently, some fifteen countries are involved into contract plasma fractionation programs to ensure a supply of plasma-derived medicinal products. The majority of the plasma for fractionation is obtained by automated plasmapheresis, the remaining (recovered plasma) being prepared from whole blood as a by-product of red cell production. Plasma for fractionation should be produced, and controlled following well established procedures to meet the strict quality requirements set by regulatory authorities and fractionators. The plasma fractionation technology still relies heavily on the cold ethanol fractionation process, but has been improved by the introduction of modern chromatographic purification methods, and efficient viral inactivation and removal treatments, ensuring quality and safety to a large portfolio of fractionated plasma products. The safety of these products with regards to the risk of transmission of variant Creutzfeldt-Jakob disease seems to be provided, based on current scientific data, by extensive removal of the infectious agent during certain fractionation steps. The leading plasma product is now the intravenous immunoglobulin G, which has replaced factor VIII and albumin in this role. The supply of plasma products (most specifically coagulation products and immunoglobulin) at an affordable price and in sufficient quantity remains an issue; the problem is particularly acute in developing countries, as the switch to recombinant factor VIII in rich countries has not solved the supply issue and has even led to an increase of the mean price of plasma-derived factor VIII to the developing world. In the last few years, the plasma fractionation industry has improved greatly, and should remain essential in the years to come for the procurement of many essential medicines.

HIF-1 and HIF-2: working alone or together in hypoxia?

Erythropoietin (EPO) is the hormonal regulator of red cell production and provided the paradigm for oxygen-regulated gene expression that led to the discovery of hypoxia-inducible factor (HIF). In this issue of the JCI, Rankin and colleagues show, using targeted gene inactivation, that induction of Epo expression in murine liver is dependent on the integrity of HIF-2alpha, and not HIF-1alpha (see the related article beginning on page 1068). These results demonstrate distinct functions for different HIF-alpha isoforms that could potentially be exploited in therapeutic approaches to anemia.

Zinc deficiency depresses red cell production in rats

Zinc (Zn) deficiency occurs worldwide and a very important public health problem. To investigate the physiological role of Zn in hematopoiesis, forty 4-week-old male Sprague- Dawley rats were assig...

Use of isotopes in the diagnosis of hematopoietic disorders

Isotopes of iron have been paramount in clinical tracer studies of hematopoiesis. Kinetics of disappearance from plasma, appearance in circulating erythrocytes, and localization in general areas of bone marrow, spleen, and liver were early revealed by gamma-/beta-emitting Fe-59 with note of characteristic differences of kind and degree of dysfunction among various anemias. Findings have helped in management (e.g., decisions about splenectomy). Positron/gamma-emitting Fe-52 (better for imaging) has provided more detail on marrow expansion and erythropoietic relocation. Other gamma-emitting tracers of hematopoiesis have been In-111 (linked to transferrin) and technetium 99m (Tc-99m) colloids (localizing to reticuloendothelial cells, which have close association with blood cell progenitors), but Fe-52 has proved to be more accurate for recognition of erythropoiesis. Occasional diverse heterotopic sites of hematopoiesis beyond spleen and liver in states of bone marrow disease have posed diagnostic challenges and also raised questions about migration and/or activation of stem cells. Studies of granulopoiesis utilize Tc-99m--labeled leukocytes or Tc99m-labeled antibodies to circulating and progenitor white cells. Iron-deficiency anemias due to malnutrition, malabsorption, blood loss, or special need are explored by dual study (oral vs intravenous) with radioactive (Fe-59, Fe-55) or stable (Fe-54, Fe-57) iron isotopes, which can guide dietary supplementation. Tests for B-12 deficiency in pernicious anemia or malabsorption with radioisotopes of cobalt (Co-57, Co-58) have been upgraded in sensitivity and scope. Rates of oxidation to expired carbon dioxide from particular carbon (radioactive C-14 or stable C-13)--labeled compounds can test B-12 or folic acid deficiencies or gastric infestation as causes of megaloblastic anemias.

Cells depleted for RPS19, a protein associated with Diamond Blackfan Anemia, show defects in 18S ribosomal RNA synthesis and small ribosomal subunit production

The gene encoding the small subunit ribosomal protein 19 (RPS19) is mutated in about 25% of cases of the bone marrow failure syndrome Diamond Blackfan Anemia (DBA), a childhood disease characterized by failure of red cell production. In these cases DBA is inherited as an autosomal dominant trait and RPS19 haploinsufficiency is thought to cause the disease. To study the molecular pathogenesis of DBA we used siRNA to decrease the level of RPS19 in two human cell lines, HeLa cells and U-2 OS osteosarcoma cells. Cells with reduced RPS19 levels showed a dramatic reduction in the amounts of small 40S ribosome subunits and mature 80S ribosomes and an excess of large 60S subunits. These cells were defective in 18S rRNA production and accumulated 21S and 20S nuclear pre-rRNA molecules, suggesting that RPS19 is required for specific steps in rRNA processing. RPS19 depletion produced a reduction in steady-state levels of RPS6 and RPS16 via a post-transcriptional mechanism while the levels of RPL7 and RPL26 were unaltered, indicating that levels of ribosomal proteins are determined by subunit assembly. This has interesting implications for the pathogenesis of DBA suggesting that deficiency of any of the RPS proteins might have a similar effect and thus may be responsible for causing DBA. Finally in cell lines from DBA patients with mutations we find increased levels of 21S rRNA precursors but no abnormality in the ribosome profile on sucrose gradients or in the steady-state levels of RPS19 suggesting that some cells can partially compensate for the loss of one allele of RPS19. We conclude that defects in ribosome biogenesis may underlie the pathology of Diamond Blackfan Anemia.

307: Erythropoeitin may improve long term preservation of the donor heart

Purpose: Erythropoietin (Epo) is well known to stimulate red cell production and has recently been shown to protect the heart against injury from ischemia/reperfusion. The University of Wisconsin (UW) solution is an effective preservative for cold storage of the cardiac allograft. The efficacy of Epo in the setting of cardiac preservation for transplantation is unknown. We evaluated the effect of Epo on preservation of the donor heart in an animal transplant model using UW solution.

A survey of the demographics of blood use

Epidemiological information was obtained by a series of questions to experts in the field of epidemiology of transfusion from the United States, England, Australia and Denmark. Although it became clear that the methods for collecting the data had differed between the countries, useful information was obtained for all questions. The data highlighted some major differences between the countries: the incident rate for red cell transfusion varied from 44.7 to 54.1 units, for platelets from 2.0 to 6.0 units and for plasma from 4.8 to 13.8 units transfused per 1000 population per year. Age and sex distribution of transfused patients was similar in all countries. Most of the red cell products are transfused to older recipients, and the distribution between men and women is approximately equal. The distribution for platelets is over a wider age range, and the difference between men and women is marked, with men predominating in all countries. The distribution for plasma is also directed to the elderly, and there is a predominance of men. The relationship between the disease or surgical procedure and the use of blood products was similar between countries. The use of red cells in cardiovascular surgery predominated. Neoplasms and digestive disorders were also prevalent. Neoplasms, including those relating to haematology, were the main use for platelets, but cardiovascular surgery was also important. In all countries, plasma is largely used in cardiovascular surgery. Two countries provided data relating to the number of units per transfusion episode including information relating to massive transfusion. In Australia, red cell use of >or=50 units per episode was largely associated with multiple traumas. In Denmark, it was associated with gastrointestinal bleeding and various medical requests.

Americans Sweep Nobel Prizes: Thinking Inside and Outside the Box Wonder — is not precisely Knowing And not precisely Knowing not—

Americans Sweep Nobel Prizes: Thinking Inside and Outside the Box Wonder — is not precisely Knowing And not precisely Knowing not—

Epo-Independent Proliferation and Differentiation of Genetically Modified Erythroid Progenitor Cells Using a Chemical Inducer of Dimerization.

Recombinant human erythropoietin (Epo) revolutionized the care of anemia in cancer. In light of emerging (albeit controversial) roles for Epo in supporting tumor growth, it may be useful to develop Epo-independent methods for regulating red blood cell production. Previous studies in this laboratory established that marrow transduced with conditional derivatives of fibroblast growth factor receptor-1 (F36VFGFR1) and the thrombopoietin receptor (F36VMpl) can support the chemical inducer of dimerization (CID)-dependent production of erythroid cells in transplanted mice. In the current study, we used human CD34+ cord blood (CB) cells to test whether CID-regulated red cell production might require collaboration between CID-initiated signals and those provided by Epo. CD34+ CB cells cultured in the absence of Epo did not proliferate and retained expression of the myeloid marker CD33. Epo (5U/mL) promoted proliferative expansion (66.2-fold in 12 days) and CD33 expression was lost as the cells differentiated to mature, glycophorin A+ erythroid cells (92.9 +/− 1.8%, n=3). Addition of a soluble human Epo receptor extracellular domain (shEpoR) at a concentration of 3ug/mL completely blocked Epo-dependent proliferation and similar to cells cultured without Epo, they retained expression of CD33. Addition of CID (100nM AP20187) to F36VMpl-transduced CD34+ CB cells in the absence of Epo promoted proliferative expansion (89.8-fold in 12 days) and differentiation as glycophorin A+ erythroid cells (77.2 +/− 4.8%, n=3). These CID responses were not significantly affected by the addition of shEpoR, as CID induced both mitogenic expansion (84.5 fold in 12 days) and differentiation as glycophorin A+ erythroid cells (74.2 +/− 6.7%, n=3) in the presence of concentrations of shEpoR that blocked Epo responses. These data suggest that F36VMpl does not require Epo signaling to support the proliferation and differentiation of human erythroid progenitor cells, and further define an Epo-independent method of erythroid cell production.

Erythropoietin Modulation of Podocalyxin within an Erythroblast Niche.

Epo's capacity to promote red cell production is ascribed largely to anti-apoptotic effects on erythroid progenitor pools. Via gene profiling of bone marrow- derived erythroblasts, Epo is now revealed to down-modulate chemokine receptor-4 (Cxcr4) and integrin alpha-4 (Itga4), and to selectively up-modulate the expression of growth differentiation factor 3 (Gdf3), oncostatin-M (Onco-M), and the sialomucin podocalyxin like-1 (PODXL). Epo dose-dependent induction of PODXL was approximately 20 fold; was discovered in KitposCD71high erythroblasts; and peaked at subsequent KitnegCD71high and Ter119pos stages. As studied using minimal knocked-in Epo receptor (EpoR) alleles, efficient PODXL expression depended upon a proximal EpoR PY343 Stat5 binding site. In mice expressing a phosphotyrosine-null EpoR-HM allele, compromised PODXL expression further correlated with an abnormal representation of anucleated red cells in marrow (and with defective Epo-induced production of PODXLpos reticulocytes). By selectively modulating Gdf3, Onco-M, Cxcr4, Itga-4 and PODXL expression, Epo is proposed to modify a unique erythroid niche, mobilize early-stage erythroblasts, and promote reticulocyte emigration to blood.

SI16 Standardising the Red Cell Dose: Pros and Cons

Unlike other prescription medicines, blood components are issued in variable doses. For example, last year's SNBTS data indicates a mean 55 g haemoglobin in additive red cell product but with a range from 36 to 88 g. With the advent of metered anticoagulant addition it is possible to collect donations to a given dose of Hb rather than a fixed volume using various apheresis equipment or the Macopharma ABC machine. Hogman and others have advocated that there would be advantages in providing red cells as a standard dose product, while Arslan has shown it may be possible to match red cell dose to patient size. Since patients as well as product vary in size at present pursuing a standard dose may have less impact than hoped. The usual rule of thumb that a single unit of red cells transfused to an adult should result in an increment in circulating haemoglobin of 1 g dL‐1 will be assessed against a range of patient blood volumes and the likely impact of a reduction in dose variation discussed.References Hogman C.F. & Meryman H.T. (2006) Red blood cells intended for transfusion: quality criteria revisited. Transfusion, 46, 137–42.Arslan O, Toprak S, Arat M & Kayalak Y. (2004) Hb content‐based transfusion policy successfully reduces the number of RBC units transfused. Transfusion, 44, 485–8.

Intravenous Iron Following Cardiac Surgery Does Not Increase the Infection Rate

Intravenous iron (FeIV) has been used increasingly, alone or in combination with recombinant erythropoietin, to promote red cell production as part of a blood conservation program. Given the important role that iron plays in the growth of bacteria, it has been hypothesized that this use of FeIV may promote surgical site infection. However, this hypothesis has not yet been tested appropriately. To assess this hypothesis, postoperative infection rates in patients undergoing cardiothoracic surgery were analyzed. Data were collected on 863 patients undergoing cardiopulmonary bypass surgery in 2001. Patients were either enrolled voluntarily in a blood conservation program in which they received either postoperative FeIV and erythropoietin (n=302), as indicated, or blood transfusions and no FeIV (n=561), as indicated, to correct postoperative anemia. Infections were defined according to the U.S. Centers for Disease Control and Prevention guidelines. Thirty-nine infections developed. The overall infection rate was 4.52%, with an infection rate of 3.97% in the iron-treated group (n=12) and a rate of 4.81% in the untreated group (n=27). When the impact of gender, age, diabetes mellitus, operating time, type of surgery, and blood transfusions were controlled for, FeIV did not increase the risk of infection (odds ratio of 1.031 for each increment of 125 mg of FeIV; 95% confidence interval 0.908, 1.170; p=0.64). There was no impact of FeIV on the subsequent infection rate in a cardiac surgery patient cohort, indicating its safety for use in the postoperative setting.

Recent Advances in Erythropoietic Agents in Renal Anemia

Red cell production in chronic kidney disease is usually too low to maintain a normal haemoglobin, and thus anaemia develops in a large proportion of patients. The ability to stimulate erythropoiesis in the bone marrow by the use of therapeutic agents has only been possible in the last 20 years, initially with recombinant human erythropoietin (epoetin), and later darbepoetin alfa. Many new agents are, however, in clinical development, and these include CERA, Hematide, and HIF stabilisers, in addition to the imminent launch of biosimilar epoetins. The main issue with biosimilars is the unknown risk of immunogenicity. CERA is a large molecule, approximately twice the size of epoetin, which was created by integrating a single polymer chain into the erythropoietin molecule. CERA has a much prolonged half-life, and Phase II and III clinical trials have investigated administration of CERA every 3 or 4 weeks. Hematide is derived from original research on the erythropoietin-mimetic peptides, and is in Phase II of its clinical trial programme. Again, this compound is being investigated as a once-monthly administration. The HIF stabilizers are orally-active inhibitors of the enzyme that degrades hypoxia-inducible factor (prolyl hydroxylase), and this leads to upregulation of erythropoietin gene expression. Other strategies for stimulating erythropoiesis, briefly described in this review, are at an earlier stage of development. This is an exciting and rapidly developing area of scientific and translational research.

Increased serum erythropoietin but not red cell production after 4 wk of intermittent hypobaric hypoxia (4,000–5,500 m)

This study tested the hypothesis that athletes exposed to 4 wk of intermittent hypobaric hypoxia exposure (3 h/day, 5 days/wk at 4,000-5,500 m) or double-blind placebo increase their red blood cell volume (RCV) and hemoglobin mass (Hbmass) secondary to an increase in erythropoietin (EPO). Twenty-three collegiate level athletes were measured before (Pre) and after (Post) the intervention for RCV via Evans blue (EB) dye and in duplicate for Hbmass using CO rebreathing. Hematological indexes including EPO, soluble transferrin receptor, and reticulocyte parameters were measured on 8-10 occasions spanning the intervention. The subjects were randomly divided among hypobaric hypoxia (Hypo, n = 11) and normoxic (Norm, n = 12) groups. Apart from doubling EPO concentration 3 h after hypoxia there was no increase in any of the measures for either Hypo or Norm groups. The mean change in RCV from Pre to Post for the Hypo group was 2.3% (95% confidence limits = -4.8 to 9.5%) and for the Norm group was -0.2% (-5.7 to 5.3%). The corresponding changes in Hbmass were 1.0% (-1.3 to 3.3%) for Hypo and -0.3% (-2.6 to 3.1%) for Norm. There was good agreement between blood volume (BV) from EB and CO: EB BV = 1.03 x CO BV + 142, r2 = 0.85, P < 0.0001. Overall, evidence from four independent techniques (RCV, Hbmass, reticulocyte parameters, and soluble transferrin receptor) suggests that intermittent hypobaric hypoxia exposure did not accelerate erythropoiesis despite the increase in serum EPO.

Automated red cell collection – quality and value

Automated red cell collection was initially used largely for therapeutic purposes. New technology has rendered the procedure safer for donors and easier for machine operators. Optimal additive solution can be automatically added and the red cells filtered to provide a leucodepleted product. Two units of red cells may be collected during a single procedure from individuals who have a high enough red cell mass, whilst a single unit of red cells plus platelets or plasma can be collected from smaller donors. In vitro studies suggested that red cells collected by automated methods would be of better quality than those collected by gravity. This was not confirmed in vivo, but red cells collected by automated methods have the major advantage of consistency in terms of haemoglobin content, volume and haematocrit, compared with red cells collected by gravity. This standardised product is of particular value for transfusion dependent patients as the patient's haemoglobin can be maintained within narrow limits. The use of a double dose red cell product for transfusion to a single patient also confers benefit in terms of reduction in donor exposure.