How we diagnose and treat iron deficiency anemia.

It is estimated that one-third of the world's population is anemic, the majority being due to iron deficiency (ID). In adults, ID is associated with fatigue in the absence of anemia, restless legs syndrome, pica and, in neonates, delayed growth and development. In adolescents, ID is associated with decrements in learning and behavioral abnormalities. In the absence of a clear cause, search for a source of bleeding is indicated. No single test is diagnostic of ID unless the serum ferritin is low or the percent transferrin saturation is low with an elevated total iron binding capacity. Oral iron is considered front line therapy except for conditions such as gastric bypass, heavy uterine bleeding, inflammatory bowel disease, and hereditary hemorrhagic telangiectasia. Oral iron has many unpleasant side effects, resulting in low patient adherence. For patients intolerant of, or unresponsive to, oral iron, intravenous (IV) administration is the preferred route. While early formulations were associated with a high incidence of serious adverse events (SAEs), newer formulations are much safer with SAEs occurring very infrequently. Full replacement doses can be administered in a matter of minutes to a few hours. Nevertheless, there remains a reluctance to use IV iron due to a misunderstanding of the safety of the available formulations. IV iron is safe and effective in all clinical circumstances including pregnancy. The preponderance of published evidence suggests IV iron therapy is underutilized and we believe that IV iron should be moved forward in the treatment of ID and iron deficiency anemia (IDA).

Ironing out fatigue.

Women who are not anemic but who suffer from fatigue may benefit from iron supplementation. In this issue of Blood, Krayenbühl et al provide strong evidence that women complaining of fatigue who were not anemic but who had reduced or absent iron stores were symptomatically improved after receiving parenteral iron. Given the numbers of women who are iron deficient, the findings could find broad application, but work needs to be done to refine the approach to this common problem.

Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.

Although advances have been made in understanding cell differentiation, only rudimentary knowledge exists concerning how differentiated cells form tissues and organs. We studied liver organogenesis because the cell and tissue architecture of this organ is well defined. Approximately 60% of the adult liver consists of hepatocytes that are arranged as single-cell anastomosing plates extending from the portal region of the liver lobule toward the central vein. The basal surface of the hepatocytes is separated from adjacent sinusoidal endothelial cells by the space of Disse, where the exchange of substances between serum and hepatocytes takes place. The hepatocyte's apical surface forms bile canaliculi that transport bile to the hepatic ducts. Proper liver architecture is crucial for hepatic function and is commonly disrupted in disease states, including cirrhosis and hepatitis. Here we report that hepatocyte nuclear factor 4alpha (Hnf4alpha) is essential for morphological and functional differentiation of hepatocytes, accumulation of hepatic glycogen stores and generation of a hepatic epithelium. We show that Hnf4alpha is a dominant regulator of the epithelial phenotype because its ectopic expression in fibroblasts induces a mesenchymal-to-epithelial transition. Most importantly, the morphogenetic parameters controlled by Hnf4alpha in hepatocytes are essential for normal liver architecture, including the organization of the sinusoidal endothelium.

In vitro and in vivo evaluation of apheresis platelets stored for 5 days in 65% platelet additive solution/35% plasma.

BACKGROUND: In the United States, apheresis platelets (PLTs) are suspended in autologous plasma. PLT additive solutions, long used in Europe, decrease recipient allergic reactions and may reduce the risk of transfusion-related acute lung injury. We evaluated Amicus-collected PLTs stored in platelet additive solution (PAS) III (InterSol) for 5 days. STUDY DESIGN AND METHODS: In Study 1, 71 subjects donated two products on a single day-one each stored in 100% plasma or 65% PAS III/35% plasma. Products underwent standard in vitro testing on Days 1 and 5. In Study 2, 43 additional subjects provided Amicus products stored for 5 days in 65% PAS III/35% plasma for in vivo radiolabeled recovery and survival determinations. The effect of approximately 2500cGy Day 1 gamma irradiation was evaluated in a subset of products. RESULTS: PAS III PLTs (n=70) had a median Day 5 pH(22°C) of 7.2 (lower 95%, 95% tolerance limit, 6.9). Mean Day 5 recovery and survival of radiolabeled PAS III PLTs (n=33) were, respectively, 80.5 and 72.1%, of fresh autologous PLTs. With 95% confidence, these values were at least 66% of fresh PLT recovery and 58% of survival. All in vitro variables remained within ranges seen in licensed products for irradiated and nonirradiated PAS III PLTs. CONCLUSION: Leukoreduced Amicus PLTs stored in 65% PAS III/35% plasma in PL-2410 containers maintained pH ≥6.9 throughout 5 days' storage. Radiolabeled PLT recovery and survival values met US Food and Drug Administration statistical criteria. Gamma-irradiated PAS III PLTs demonstrated no significant adverse effects due to irradiation in in vitro testing.

Distinct roles of phosphoinositide-3 kinase and phospholipase Cgamma2 in B-cell receptor-mediated signal transduction.

During B-cell receptor (BCR) signaling, phosphoinositide-3 kinase (PI3K) is thought to function upstream of phospholipase Cgamma2 (PLCgamma2). PLCgamma2 deficiency specifically impedes transitional type 2 (T2) to follicular (FO) mature B-cell transition. Here, we demonstrate that PI3K deficiency specifically impaired T2-to-FO mature B-cell transition and marginal zone B-cell development. Furthermore, we investigated the functional relationship between PI3K and PLCgamma2 using PI3K-/-, PLCgamma2-/-, and PI3K-/- PLCgamma2-/- B cells. Interestingly, PLCgamma2 deficiency had no effect on BCR-mediated PI3K activation, whereas PI3K deficiency only partially blocked activation of PLCgamma2. Moreover, whereas PI3K-/- PLCgamma2-/- double deficiency did not affect hematopoiesis, it resulted in embryonic lethality. PI3K-/- PLCgamma2-/- fetal liver cells transplanted into B-cell null JAK3-/- mice failed to restore development of peripheral B cells and failed to progress through early B-cell development at the pro-B- to pre-B-cell transition, a more severe phenotype than was observed with either PI3K or PLCgamma2 single-deficiency B cells. Consistent with this finding, BCR signaling was more severely impaired in the absence of both PI3K and PLCgamma2 genes than in the absence of either one alone. Taken together, these results demonstrate that whereas PI3K functions upstream of PLCgamma2, activation of PLCgamma2 can occur independently of PI3K and that PI3K and PLCgamma2 also have distinct functions in BCR signal transduction.

Renal disease and anemia in the elderly.

As individuals age, the incidence of both renal insufficiency and anemia increases. Some of the changes in renal function can be traced to age-related alterations in the renal vasculature that may dissociate renal blood flow from glomerular filtration. In addition, there appear to be paradoxical changes in renal production of erythropoietin (Epo), in that Epo levels actually increase over time with aging, suggesting that the erythroid marrow may become less sensitive to Epo stimulation. These factors, together with the confounding effects of possible nutritional deficits and the disproportionate effect of early-stage diabetes mellitus on renal endocrine function and Epo production, all can contribute to anemia in the elderly. As anemia is an independent predictor of poor outcomes with many diseases and at any age, recognizing and correcting the underlying cause of the anemia is an important aspect of medically managing the elderly patient.

Synthetic erythropoietic proteins: tuning biological performance by site-specific polymer attachment.

Chemical synthesis in combination with precision polymer modification allows the systematic exploration of the effect of protein properties, such as charge and hydrodynamic radius, on potency using defined, homogeneous conjugates. A series of polymer-modified synthetic erythropoiesis proteins were constructed that had a polypeptide chain similar to the amino acid sequence of human erythropoietin but differed significantly in the number and type of attached polymers. The analogs differed in charge from +5 to -26 at neutral pH and varied in molecular weight from 30 to 54 kDa. All were active in an in vitro cell proliferation assay. However, in vivo potency was found to be strongly dependent on overall charge and size. The trends observed in this study may serve as starting points for the construction of more potent synthetic EPO analogs in the future.

Assessment of pure red cell aplasia in US dialysis patients: the limits of the Medicare data.

BACKGROUND: A report of neutralizing antibodies to recombinant human erythropoietin (rHuEPO) in European dialysis patients raised concerns that US dialysis patients may be at similar risk. We investigated the frequency of diagnosis codes that include pure red cell aplasia (PRCA) in the Medicare end-stage renal disease data. METHODS: Medicare claims data were used to identify incident dialysis patients from 1995 through 1999 aged 67 years or older who did not have a diagnosis code for aplastic anemia (AA; which includes PRCA) or were not administered rHuEPO before dialysis therapy initiation. Patients were assessed for complicating conditions, rHuEPO dose, hematocrit level, blood transfusion, bone marrow testing, and AA diagnosis (maximal follow-up, 13 months). RESULTS: Of 101,782 patients eligible for study, 9,896 patients had diagnosis codes for AA after dialysis therapy initiation, 3,894 patients underwent bone marrow tests (required for PRCA diagnosis), 19 patients had diagnosis codes for AA based on bone marrow examination and no other complicating conditions, and 5 patients were administered blood transfusion in 1 or more months during follow-up, of whom only 1 patient had persistent low hematocrit levels while being administered rHuEPO and blood transfusion. This latter patient was identified from a total of 101,782 patients, or 70,706.75 person-years of rHuEPO exposure. CONCLUSION: Diagnosis codes in the Medicare data are inadequate for the conclusive study of PRCA in US dialysis patients. Despite limitations of this study, it appears that few cases of rHuEPO-associated PRCA have occurred in US Medicare dialysis patients. Additional investigation is needed to determine whether apparent differences between US complication rates and those elsewhere result from differences in the manufacturing and/or use of erythropoietin products.

Iron, erythropoietic stimulating agents, and anemia in cancer.

Anemia commonly accompanies malignancies in patients and may contribute to morbidity. Correction of the anemia with erythropoietic stimulating agents, while possible, may be associated with complications such as tumor progression and adverse cardiovascular events. This review examines the data behind these concerns and raises the strategy of using iron, alone, in the setting of cancer-associated anemia to reduce exposure to transfusions in these patients and to potentially improve their quality of life.

Iron metabolism in man.

Iron metabolism in man is a highly regulated process designed to provide iron for erythropoiesis, mitochondrial energy production, electron transport, and cell proliferation. The mechanisms of iron handling also protect cells from the deleterious effects of free iron, which can produce oxidative damage of membranes, proteins, and lipids. Over the past decade, several important molecules involved in iron homeostasis have been discovered, and their function has expanded our understanding of iron trafficking under normal and pathological conditions. Physiologic iron metabolism is strongly influenced by inflammation, which clinically leads to anemia. Although hepcidin, a small circulating peptide produced by the liver, has been found to be the key regulator of iron trafficking, molecular pathways of iron sensing that control iron metabolism and hepcidin production are still incompletely understood. With this review, we provide an overview of the current understanding of iron metabolism, the recently discovered regulators of iron trafficking, and a focus on the effects of inflammation on the process.

Iron deficiency and heart disease: ironclad evidence?

Patients with heart failure have elevated levels of circulating inflammatory cytokines and commonly have iron deficiency anemia or anemia of chronic inflammation. Clinical trials in patients with congestive heart failure and iron deficiency have demonstrated that intravenous iron treatment appears to improve subjective and objective outcomes. Most patients in these trials were not anemic or only had mild anemia, and hemoglobin concentration rose only slightly after treatment with iron. Experimental evidence demonstrates that iron is a cofactor for muscle function, which could explain the improvement in clinical outcomes. Many questions remain to be answered to understand the role of iron therapy in patients with congestive heart failure.

Hyporesponsiveness to erythropoiesis stimulating agents in chronic kidney disease: the many faces of inflammation.

The vast majority of patients with chronic kidney disease (CKD) receive an erythropoietic-stimulating agent (ESA) to partially correct the almost inevitable anemia associated with renal insufficiency. However, a minority of CKD patients will require much higher than usual doses of ESA in order to maintain a reasonable hemoglobin concentration. In many instances, these patients will have either an obvious or a clinically unapparent inflammatory process to account for the hyporesponsiveness to ESA replacement therapy. This review focuses on the mechanisms by which inflammation interferes with erythroid marrow function and particularly the new information about the role of inflammatory cytokines and the small peptide, hepcidin, on altered iron metabolism.

The anemia of inflammation/malignancy: mechanisms and management.

Anemia is a common complication in patients with inflammatory diseases of many kinds, including cancer. The mechanisms that have captured the most attention include cytokine-mediated changes in both the production of and the response to erythropoietin (Epo), as well as important alterations in iron metabolism. The last is brought about by the relatively recently recognized peptide hormone, hepcidin. The availability of recombinant human Epo and its derivatives (known by class as Erythropoietic Stimulating Agents, ESAs) has dramatically changed anemia management in patients with cancer but, in the process, has raised as many issues as have been answered. This chapter reviews the mechanisms resulting in anemia in inflammation, including cancer, and focuses on the controversies around management with the ESAs and the adjuvant use of iron in anemia management.

Design and chemical synthesis of a homogeneous polymer-modified erythropoiesis protein.

We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.