Free erythrocyte protoporphyrin assay in the diagnosis of iron deficiency in the anemic aged subject. A prospective study of 103 anemic patients

We determined the relative value of the free erythrocyte protoporphyrin (FEP) assay compared to those of total iron-binding capacity (TIBC) and serum ferritin in the diagnosis of iron deficiency in a population of elderly anemic subjects. One hundred and three patients, 65 to 98 years old (mean +/- SD: 81.5 +/- 8.8), with hemoglobin levels of less than 110 milligrams (mean +/- SD: 97 +/- 12, range 53-109) were included in the study. In the patients with iron-deficiency anemia due solely to chronic bleeding, mean values for the three parameters were highly different from those in patients without chronic bleeding. In the patients with anemia due to an association of chronic bleeding and chronic inflammation, the mean FEP value was very significantly different (p less than 0.001) from that in the patients with chronic inflammation but without bleeding, whereas this was not the case for TIBC or serum ferritin. The sensitivity of FEP in the diagnosis of iron deficiency due to chronic bleeding in this population of anemic subjects was 60% (specificity 90%), compared to 13% (specificity 96%) for TIBC and 20% (specificity 100%) for serum ferritin. The FEP assay thus emerges as being highly suitable for the diagnosis of iron-deficiency anemia in the elderly subject, particularly when bone marrow is not examined.     

Morphological features and secretory gastric function in patients with chronic gastritis against the background of B12 deficiency anemia]

As many as 45 patients with chronic atrophic gastritis against the background of B12 and iron-deficiency anemia were examined. Chronic atrophic gastritis combined with B12 deficiency anemia was found to damage in most cases the antral and fundic parts of the stomach simultaneously, while the gastric process combined with iron-deficiency anemia involved the antral part only in most cases. The disease is accompanied by a considerable reduction of acid, pepsinogen and biermerin formation, especially in cases of chronic atrophic gastritis associated with B12 deficiency anemia.     

Single values of serum transferrin receptor and transferrin receptor ferritin index can be used to detect true and functional iron deficiency in rheumatoid arthritis patients with anemia

OBJECTIVE: To elucidate the use of serum transferrin receptor (sTfR) to distinguish between iron-deficiency anemia (IDA) and anemia of chronic disease (ACD), and to establish an improved scheme to identify functional iron deficiency (FID) in rheumatoid arthritis (RA) patients with anemia. METHODS: We studied 30 anemic RA patients whose iron status was confirmed by bone marrow examination and determination of the sTfR level, serum ferritin level, and sTfR-log ferritin index (TfR-F Index). All patients with diminished or exhausted iron stores (n = 18) received oral iron supplementation. RESULTS: Baseline values of sTfR and the TfR-F Index predicted the response correctly in all patients who received supplementation treatment and were normal in 10 of 11 patients with normal initial iron stores (ACD). CONCLUSION: The results of this study elucidate the roles of sTfR and the TfR-F Index in the differential diagnosis between IDA and ACD and provide direct evidence that these parameters are useful in detecting FID, irrespective of the concurrent iron storage status.     

Serum erythropoietin (EST) titers in anemia

Erythropoietin (ESF) titers were determined in sera from patients with different types of anemia using the fetal mouse liver cell bioassay. An inverse relationship was found between hemoglobin concentration and ESF titer. However, ESF titers differed markedly between patients at comparable degrees of anemia. Several groups of patients were distinguished on the basis of the activity of their erythroid bone marrow. In each of these groups, a significant negative correlation was found between the hemoglobin concentration and the logarithm of the ESF titer. ESF titers in patients with pure red cell aplasia were fourfold higher than those in patients with iron-deficiency anemia and tenfold higher than those in patients with megaloblastic anemia and homozygous sickle cell anemia at comparable hemoglobin concentrations. Following the initiation of specific therapy in patients with pernicious anemia and patients wit iron-deficiency anemia, serum ESF titers were found to decrease prior to any substantial rise in hemoglobin concentrations. In the patients with pernicious anemia, the lowest ESF levels were found 1 day after administration of vitamin B12, whereas in the patients with iron-deficiency anemia, the lowest ESF levels were reached in the second week of oral iron therapy. ON the basis of these data it was concluded that serum ESF titers in anemic patients are not only inversely related to the hemoglobin concentration but also to the activity of the erythroid bone marrow.     

Effect of iron therapy on serum ferritin levels in iron-deficiency anemia

The level of serum ferritin is a reliable indicator of body iron stores. Exceptions include liver disease, malignant diseases, and treatment of iron-deficiency anemia. The latter was noted in iron- deficient infants who showed a rise of serum ferritin to normal levels in the first week of treatment. To evaluate this in adults, 14 patients with iron-deficiency anemia were studied prior to and after beginning treatment with oral ferrous sulfate in standard dose, 300 mg t.i.d., or double dose, 600 mg t.i.d. Serum ferritin was assayed by radioimmunoassay. No rise occurred in the first 3 wk in 5 patients treated with standard dose, although hematologic response occurred. With double dose, 7 of 9 showed a ferritin rise in 2 days with return to subnormal levels within 6 days of discontinuing iron. This study indicates that standard treatment of iron deficiency anemia in adults does not cause a rise in serum ferritin until hemoglobin levels are normal. The early rise seen with double dose is most likely due to absorption of iron in excess of utilization for erythropoiesis resulting in temporary storage. When iron is discontinued, stores are rapidly depleted as reflected by the prompt decrease in serum ferritin.     

An enzyme-linked immunosorbent assay for erythropoietin using monoclonal antibodies, tetrameric immune complexes, and substrate amplification

We recently reported the development of several monoclonal antibodies (MoAbs) to native human erythropoietin (Ep). In the present study we have used the two antibodies with highest affinity to develop a two- sided or sandwich enzyme-linked immunosorbent assay (ELISA) to measure Ep in human serum. In this assay Ep is incubated in microtiter wells precoated with the first (IgE) anti-Ep antibody. Assay wells are then incubated with the second (IgG1) anti-Ep antibody, which is labeled noncovalently with the enzyme alkaline phosphatase (AP) by means of bispecific tetrameric antibody complexes consisting of IgG1 anti-Ep cross-linked to IgG1 anti-AP using rat MoAbs specific for mouse IgG1. Application of this noncovalent labeling procedure, in combination with substrate amplification, results in a detection sensitivity of 0.5 to 1.0 mU/sample (5 to 10 mU/mL), which makes this assay suitable for measuring normal serum Ep levels. The validity of this ELISA for quantitating Ep in biological fluids was demonstrated by the parallelism obtained between pure recombinant Ep dose-response curves and those obtained with plasma and serum from healthy donors and patients with various hematologic disorders. Normal plasma Ep levels detected with this ELISA ranged from 9 to 101 mU/mL with a mean of 32 +/- 23 (SD) mU/mL. Ep levels in sera from patients with polycythemia vera were in the low to normal range, whereas Ep levels in sera from patients with secondary polycythemia and patients with aplastic anemia were moderately to strongly elevated. These results demonstrate that the Ep-ELISA is a sensitive, reliable, and nonradioactive immunologic method for quantitating Ep levels and should prove useful in a variety of clinical and laboratory settings.     

Diurnal Levels of Immunoreactive Erythropoietin in Normal Subjects and Subjects with Chronic Lung Disease

S ummary . Serum levels of immunoreactive erythropoietin (Ep) were measured in 48 normal male and female volunteers, ages 20-60 years, to establish a control value for Ep of 18·5±5·0 ( SD) mU/ml. Levels of the hormone were also measured sequentially over a 24 h period of time in an additional 17'normal'volunteers with no diurnal variation. Diurnal levels of immunoreactive Ep were also measured in 30 subjects with chronic lung disease. These patients, in contrast to normal subjects, exhibited a diurnal variation in the level of immunoreactive Ep with peak levels occurring at midnight. The only variable measured which correlated with the serum immunoreactive Ep level in subjects with chronic lung disease was the level of carboxyhaemoglobin ( P <0·02).     

Dysregulated monocyte iron homeostasis and erythropoietin formation in patients with anemia of chronic disease

Anemia of chronic disease (ACD) is frequently found in patients with chronic immune activation. Since most studies on ACD pathophysiology were performed with cell culture or animal models but not in humans, we examined 37 ACD patients suffering from autoimmune diseases or infections, 10 subjects with iron-deficiency anemia (IDA), 10 anemic patients with hereditary spherocytosis (HS), and 27 age-matched controls. Although hemoglobin concentrations were comparable between ACD and IDA patients, the latter presented with significantly higher serum erythropoietin concentrations than ACD patients. The significant negative correlation between erythropoietin and hemoglobin levels observed in IDA patients was also found in a group of anemic but not hypoferremic hereditary spherocytosis subjects, but not in ACD patients. Increased serum concentrations of the hepcidin precursor prohepcidin were paralleled by a decreased expression of the iron exporter ferroportin in circulating monocytes of ACD patients. In the latter cells, increased amounts of the iron storage protein ferritin and a reduced activity of iron-regulatory protein indicated monocyte iron accumulation. Our data indicate that hypoferremia in ACD may result from downregulation of ferroportin expression by hepcidin and cytokines with subsequent iron retention in monocytes. Together with a diminished erythropoietin formation, the impaired iron recirculation from monocytes may be central in the pathophysiology of ACD in humans.     

Measurement of erythropoietin in chloroleukemic rats.

Erythropoietin (Ep) levels were measured in Shay chloroleukemic rats at various stages of anemia. Serum Ep was shown to increase logarithmically as the anemia became more severe. This increase in Ep levels was similar to that observed in normal rats subjected to acute blood loss. Significant levels of Ep were also demonstrated in ascitic fluid extracted from the peritoneal cavity of leukemic rats. These results indicate that the anemia of this disease is not due to a diminished production of Ep.     

Serum erythropoietin and erythropoiesis in high- and low-fetal hemoglobin beta-thalassemia intermedia patients

Clinical data suggest that in beta-thalassemia-intermedia patients, higher levels of circulating fetal hemoglobin (HbF) are associated with greater disease severity at comparable degrees of anemia. We assessed the influence of the amount of circulating HbF on serum erythropoietin (s-Epo) levels and on serum transferrin receptor, a measure of erythropoiesis, in 30 beta-thalassemia-intermedia patients. Twenty-four showed more than 40% HbF (21 of whom with beta (0)-thalassemia) and 6 presented lower HbF levels (beta(+)-thalassemia). The two groups of patients did not differ in age (15.3 v 19 years, respectively) or degree of anemia (Hb = 8.8 g/dL in both groups). Log (s-Epo) was correlated inversely with Hb (r = -0.47; P < .01), and directly with HbF (r = .55; P < .001). Multivariate regression analysis showed that Hb and HbF were independently correlated with s-Epo levels. High-HbF patients had greater s-Epo values at the same Hb level than low-HbF patients. Considering that iron-deficiency anemia control patients represented the predicted physiologic response of s-Epo to anemia, the observed/predicted s-Epo ratio in low-HbF thalassemic patients was no different from controls, but was increased in the high-HbF group. High- HbF patients also showed an expansion of erythropoiesis as much as four to nine times the normal value at the same Hb level as low-HbF patients. We conclude that HbF exerts an independent regulatory effect on erythropoietin production and erythropoiesis that is detectable only when HbF levels exceed 40%.     

Ret-Y a measure of reticulocyte size: a sensitive indicator of iron deficiency anemia

In this study the size of reticulocytes was measured, reticulocyte-Y (Ret-Y), to distinguish iron deficiency anemia from the anemia of chronic disease using a Sysmex XE2100 cell counter. We evaluated this parameter prospectively in 100 patients seen for the evaluation of anemia. A clinical diagnosis of iron deficiency anemia or anemia of chronic disease was made on the basis of a complete blood count, examination of the peripheral smear, and serum ferritin along with a history and physical examination. We analyzed the sensitivity and specificity of the Ret-Y in relationship to the clinical diagnosis. We also measured serum transferrin receptor levels to use as the gold standard laboratory test for iron deficiency against which we compared the Ret-Y. In 40 normal individuals with normal serum ferritin and transferrin receptor levels the mean Ret-Y was 1874 +/- 178 (1 SD). The mean Ret-Y in the anemia of chronic disease group (n=62) was 1722 +/- 162, not significantly different from normal. The mean Ret-Y value among iron-deficient patients (n=38), was 1407 +/- 136 (P <0.01 vs. the anemia of chronic disease group's Ret-Y value). Receiver operator curves showed that Ret-Y correlated closely to the serum transferrin receptor and was superior to the mean corpuscular volume, and ferritin level, in differentiating the type of anemia. The Ret-Y parameter has the highest overall sensitivity and specificity of the panel of tests routinely used in differentiating iron deficiency anemia from anemia of chronic disease.     

Erythroid marrow activity and functional anemia in patients with the rare interaction of a single functional a-globin and beta-globin gene

BACKGROUND AND OBJECTIVES: The degree of globin chain imbalance and tissue hypoxia are important determinants of clinical severity in thalassemia syndromes. Thus phenotypic expression may be modified by interaction of alpha- and beta-thalassemia defects, level and type of hemoglobin synthesized and oxygen release to the tissues. We evaluated hematology, erythroid marrow activity and functional anemia in patients with the rare interaction of a single a-globin gene and heterozygous beta-thalassemia (HbH/beta-thal trait). DESIGN AND METHODS: In 7 patients characterized by DNA analysis to have HbH disease genotypes with beta-thalassemia trait, we assessed hematologic findings, serum transferrin receptor (sTfR), serum erythropoietin (Epo), red cell 2,3-disphosphoglycerate (2,3-DPG) and whole blood oxygen releasing capability. RESULTS: Patients with HbH/beta-thal trait had moderate anemia, marked hypochromasia and microcytosis, normal or raised HbA2, and no electrophoretically/chromatographically detectable HbH. Epo and sTfR levels were significantly higher than in beta-thalassemia heterozygotes, but lower than in patients with HbH disease; 2,3-DPG levels were highest in HbH/beta-thal trait. Oxygen binding studies and simulations showed reduced oxygen affinity (P50) in HbH/beta-thal trait, resulting in increased oxygen release (O2R). INTERPRETATION AND CONCLUSIONS: Hematologic findings and bone marrow activity in patients with HbH/b-thal trait were consistent with the modified globin chain imbalance and hemoglobin synthesis expected from interaction of HbH disease with heterozygous b-thalassemia, although this rare complex genotype may elude diagnosis based on hematology alone. Significantly higher red cell 2,3-DPG levels were an unexpected finding, and the consequent increase in oxygen release capability resulted in a compensated functional anemia relative to hemoglobin levels.     

Iron deficiency in inflammatory bowel disease

The prevalence of iron-deficiency anemia was defined in 105 patients with inflammatory bowel disease and an appraisal made of the diagnostic value of serum ferritin for the assessment of iron stores. Iron deficiency, defined by the absence of bone-marrow hemosiderin was found with anemia in 36% of 41 patients with ulcerative colitis (UC) and 22% of 64 patients with Crohn's disease (CD). Iron deficiency without impaired erythropoiesis was detected in an additional 32% of patients with UC and 2% with CD. Anemia with plentiful bone-marrow iron was present in 33 (51%) of patients with CD, only one of whom had vitamin B₁₂ deficiency. Red blood cell morphology, RBC indices, serum iron, and percent transferrin saturation correlated poorly with stainable marrow iron. Serum ferritin, assayed in samples from 45 patients, was <18 ng/ml in 4/12 with iron-deficiency anemia and 0/5 with absent marrow iron and a normal hemoglobin level; values >55 ng/ml were invariably associated with the presence of marrow hemosiderin. Based on a lower normal limit of 18 ng/ml, the serum ferritin had an excellent predictive value (100%) but a high predictive error (32%) in the diagnosis of iron deficiency in inflammatory bowel disease. Serum ferritin >55 ng/ml ruled out iron deficiency as the basis for anemia.     

Severe anemia. Clinical observations in 100 patients with very low hemoglobin levels.

One hundred consecutive patients with hemoglobin concentration less than 3.5 g/dL (hematocrit reading, less than 10%) were admitted to the University of Baghdad Teaching Hospital, Iraq, during a 30-month period. Twenty-eight patients had aplastic anemia, 27 had leukemia or other hemopoietic malignancies, 16 had chronic renal failure, eight had iron-deficiency anemia, eight had hemolytic anemia, seven had thalassemia major, and six had other conditions. Twenty-three patients died within seven days of admission, mostly due to the underlying disease or complications thereof. Heart failure developed in ten patients, and five had retinal exudates and hemorrhages attributed to severe anemia. Arrhythmias and ECG abnormalities were noted in 20 of 68 patients. Blood transfusion was instituted in all but three patients, whose anemia was corrected with specific therapy without blood transfusion. The tolerance of the 100 patients to such severe anemia was remarkable.     

Celiac disease presenting as iron-deficiency anemia in northern India.

BACKGROUND: Adult celiac disease is infrequent in India. Iron-deficiency anemia as its presenting manifestation is still rarer. METHODS: We investigated patients with refractory iron-deficiency anemia attending the hematology clinic of a tertiary-care hospital for celiac disease. The diagnosis of celiac disease was based on histology, serology and response to treatment. RESULTS: Of 19 patients with refractory iron-deficiency anemia seen from April 1998 to March 2000, 11 were diagnosed to have celiac disease. Four of these had abnormal D-xylose test and 3 had fat malabsorption. All 11 patients responded to gluten-free diet with improvement in hematological parameters. CONCLUSION: Patients with refractory iron-deficiency anemia of unknown cause should be investigated for subclinical celiac disease.     

IS SERUM TRANSFERRIN RECEPTOR USEFUL FOR DETECTING IRON-DEFICIENCY IN ANAEMIC PATIENTS WITH CHRONIC INFLAMMATORY DISEASES?

We investigated whether determination of serum transferrin receptor(TfR) is useful for detecting iron-deficiency in patients withchronic inflammatory diseases and for differentiating betweeniron-deficiency anaemia and anaemia of inflammation. Using animmunofluorometric assay, serum TfR was measured in 34 anaemicpatients Of these patients, 23 had a chronic rheumatic disease,13 with both inflammation and iron-deficiency and 10 with anaemiaof inflammation only; the other 11 patients had iron-deficiencyanaemia and no evidence of inflammation. Serum TfR concentrationswere lower in patients with anaemia of inflammation (2.6 ±0.2 mg/l, mean ± S.E.M.) than in patients with iron-deficiencyanaemia (6.7 ± 1.1 mg/l, P<0.01) or those with bothinflammation and iron deficiency (5.8 ± 1.0 mg/l, P<0.01).Among patients with inflammatory disease, correlations betweenTfR and ferritin concentrations (r = –0.62, Pc0.05) andTfR and erythropoietin concentrations (r = 0.69, P<0.001)were observed in iron-deficient subjects only. TfR, though notsuperior to serum ferritin, can help to distinguish betweenanaemia of inflammation and iron-deficiency anaemia and to identifyiron-deficiency in subjects with chronic inflammation. KEY WORDS: Transferrin receptor, Anaemia, Iron-deficiency, Rheumatoid arthritis     

Anemia of chronic disease is the more frequent type of anemia seen in patients with chronic idiopathic neutropenia of adults

This study describes the frequency and the type of anemia seen in patients with nonimmune chronic idiopathic neutropenia of adults (NI-CINA). We found that NI-CINA patients had low hemoglobin levels and increased serum concentrations of erythropoietin (EPO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). The hemoglobin levels correlated positively with the number of circulating neutrophils and inversely with the levels of EPO and TNF-alpha but not of IL-1beta. Anemia, defined as the reduction of the hemoglobin below 12.0 g/dl for women and 13.3 g/dl for men, was found in 23 out of 148 patients studied, a proportion of 15.5%. Two of the anemic patients had iron deficiency anemia (8.7%), 11 had anemia of chronic disease (ACD; 47.8%) presenting with normal or slightly reduced erythrocytic indices, low serum iron, and increased serum ferritin, and the remaining ten had anemia of undefined pathogenesis (AUP; 43.5%) with normal or slightly decreased erythrocytic indices, serum iron ranging from 43 to 88 microg/dl, and ferritin values ranging from 12 to 50 ng/ml. We conclude that ACD is the more frequent type of anemia seen in patients with NI-CINA, and that pro-inflammatory cytokines, notably TNF-alpha, may be involved in the pathogenesis of both ACD and AUP, given that serum levels of the cytokine were significantly increased and that the EPO response to anemia was blunted in these patients. These findings further support our previously reported suggestion for the possible existence, in NI-CINA patients, of an unrecognized low-grade chronic inflammatory process that may be involved in the pathogenesis of the disorder.     

Indicators of iron metabolism in adolescents with tuberculosis]

Iron metabolic parameters were studied in a sample of 25 adolescent patients with tuberculosis who had been followed up. ++Criteria were examined for assessing the data obtained which differ from those found in iron-deficiency anemia. A number of parameters (hemoglobin, total serum iron-binding capacity, erythrocytic ferritin) have been proposed, which should be considered in detecting iron-deficiency anemia in a tuberculosis process.     

Relationship of iron-deficiency anemia with esophagitis and hiatal hernia: hospital findings from a prospective, population-based study

OBJECTIVE: Iron-deficiency anemia is sometimes attributed to esophagitis and hiatal hernia; however, because these GI conditions are so common, such an association could be coincidental. We examined prospectively whether esophagitis and hiatal hernia increased the risk of iron-deficiency anemia in a national, population-based study. METHODS: The study population comprised 5069 adult participants in the first National Health and Nutrition Examination Survey, who were free of GI hemorrhage and anemia at baseline examination in 1971-1975 and who were hospitalized at some point during nearly 20 yr of follow-up. Rates of hospitalization with iron-deficiency or unspecified anemia were compared between patients with a hospital diagnosis of esophagitis or hiatal hernia and those who had not yet had a diagnosis of these disorders. Adjusted rate ratios were calculated using time-dependent, multivariable, proportional hazards analysis. RESULTS: During follow-up, 59 patients were hospitalized with esophagitis alone, 140 with hiatal hernia alone, and 70 with both diagnoses. A total of 102 participants were hospitalized with iron-deficiency anemia and 256 with unspecified anemia. Compared to those without a diagnosis of esophagitis or hiatal hernia, patients with a diagnosis of hiatal hernia had higher rates of subsequent hospitalization with iron-deficiency anemia. The hazard rate ratio (HRR) for hiatal hernia was 2.9 (95% confidence interval, 1.5-5.5). A trend was found for esophagitis with a HRR of 2.2 (95% confidence interval, 0.79-6.0). Results were similar with unspecified anemia as the outcome. CONCLUSIONS: Hiatal hernia should be considered as a possible cause of iron-deficiency anemia. The relationship of esophagitis with iron-deficiency anemia requires further study.     

Hepcidin as a predictive factor and therapeutic target in erythropoiesis-stimulating agent treatment for anemia of chronic disease in rats

Anemia of chronic disease is a multifactorial disorder, resulting mainly from inflammation-driven reticuloendothelial iron retention, impaired erythropoiesis, and reduced biological activity of erythropoietin. Erythropoiesis-stimulating agents have been used for the treatment of anemia of chronic disease, although with varying response rates and potential adverse effects. Serum concentrations of hepcidin, a key regulator of iron homeostasis, are increased in patients with anemia of chronic disease and linked to the pathogenesis of this disease, because hepcidin blocks cellular iron egress, thus limiting availability of iron for erythropoiesis. We tested whether serum hepcidin levels can predict and affect the therapeutic efficacy of erythropoiesis-stimulating agent treatment using a well-established rat model of anemia of chronic disease. We found that high pre-treatment hepcidin levels correlated with an impaired hematologic response to an erythropoiesis-stimulating agent in rats with anemia of chronic disease. Combined treatment with an erythropoiesis-stimulating agent and an inhibitor of hepcidin expression, LDN-193189, significantly reduced serum hepcidin levels, mobilized iron from tissue stores, increased serum iron levels and improved hemoglobin levels more effectively than did the erythropoiesis-stimulating agent or LDN-193189 monotherapy. In parallel, both the erythropoiesis-stimulating agent and erythropoiesis-stimulating agent/LDN-193189 combined reduced the expression of cytokines known to inhibit erythropoiesis. We conclude that serum hepcidin levels can predict the hematologic responsiveness to erythropoiesis-stimulating agent therapy in anemia of chronic disease. Pharmacological inhibition of hepcidin formation improves the erythropoiesis-stimulating agent’s therapeutic efficacy, which may favor a reduction of erythropoiesis-stimulating agent dosages, costs and side effects.