A case series highlighting structured hematological, biochemical and molecular approach to clinical oral iron refractoriness in children: A pressing need for a 3-tier system for classification of variants in TMPRSS6 gene.

In current study, we discuss clinical oral iron refractoriness cases and highlight need for a classification system to define TMPRSS6 gene variants. Out of 231 cases of microcytic hypochromic anemia screened (Sept 2019-Dec 2020), 17 cases (7.35%) with unexplained iron refractoriness (URIDA) phenotype were enrolled after ruling out secondary causes and compliance related issues. 11 (65%) had absent/negligible response (0-0.4 g/dl Hb rise) while 6 (35%) partial (0.5-0.9 g/dl Hb rise) response to initial iron trial at 4-8 weeks. Of these 17 cases, inappropriate hepcidin levels (normal-high) were noted in 11/15 (73%) tested. TSAT/Hepcidin ratio was low in 13/15 (87%). Genetic analysis of TMPRSS6 gene by NGS revealed variations in 15/17 (88%) cases. 10/15 cases with variations harbored a common splice site INDEL that was noted to be pathogenic SNP (MAF-0.19) on case-control association study in combination with other known missense SNPs with an odds ratio of 6.38 and relative risk 2.66 (p- < 0.01).

Dizygotic twins with prolonged jaundice and microcytic, hypochromic, hemolytic anemia with pyropoikilocytosis.

Dizygotic twin males, born at 34 weeks gestation, had prolonged jaundice, microcytic, hypochromic anemia, FABarts hemoglobin, elevated end-tidal CO, and blood films consistent with hereditary pyropoikilocytosis. DNA sequencing revealed both had a heterozygous alpha spectrin (SPTA1) mutation (c.460_462dup) inherited from their asymptomatic mother, plus a 3-base pair duplication in alpha globin (HBA2) (c.364_366dupGTG) inherited from their asymptomatic father.

Reticulocyte hemoglobin and hypochromic erythrocytes in the study of erythropoiesis in patients with inflammatory bowel disease.

Anemia due to lack of iron (absolute or functional deficiency) is a common complication of inflammatory bowel disease (IBD). We investigated the potential utility of reticulocyte hemoglobin content (Ret-He) and percentage of hypochromic red cells (Hypo-He) in the assessment of erythropoiesis. We recruited 123 anemic outpatients with IBD. Biochemical tests for iron deficiency were carried out. Full blood counts were performed on a XN 20 system (Sysmex Diagnostics). Differences among groups were studied using analysis of variance and post-hoc tests, considering p < .05 to be significant. Receiver operating characteristic analysis was used to assess the diagnostic performance of Ret-He and Hypo-He for detecting iron-deficient erythropoiesis. The gold standard used for diagnosing iron deficiency was soluble transferrin receptor (sTfR), with a cut-off of >52 nmol/L. Overall, 60 patients had iron deficiency anemia (IDA), 27 anemia of chronic disease (ACD) and 36 mixed ACD + IDA. Ret-He showed the best performance, with an area under curve (AUC) of 0.858 (95% CI 0.816-0.952), considering a cut-off of 30.0 pg, sensitivity of 76.8% and specificity of 99.8% (vs. AUC 0.727 [95% CI 0.624-0.814], considering a cut-off of 4.0%, sensitivity 72.0% and specificity 72.5% for Hypo-He). Ret-He and Hypo-He can be used to assess iron supply for erythropoiesis in patients with IBD, to evaluate long-term (Hypo-He) and short-term (Ret-He) periods.

Loss of Dynamin 2 GTPase function results in microcytic anaemia.

In a dominant mouse ethylnitrosurea mutagenesis screen for genes regulating erythropoiesis, we identified a pedigree with a novel microcytic hypochromia caused by a V235G missense mutation in Dynamin 2 (Dnm2). Mutations in Dnm2, a GTPase, are highly disease-specific and have been implicated in four forms of human diseases: centronuclear myopathy, Charcot-Marie Tooth neuropathy and, more recently, T-cell leukaemia and Hereditary Spastic Paraplegia, but red cell abnormalities have not been reported to date. The V235G mutation lies within a crucial GTP nucleotide-binding pocket of Dnm2, and resulted in defective GTPase activity and incompatibility with life in the homozygous state. Dnm2 is an essential mediator of clathrin-mediated endocytosis, which is required for the uptake of transferrin (Tf) into red cells for incorporation of haem. Accordingly, we observed significantly reduced Tf uptake by Dnm2+/V235G cells, which led to impaired endosome formation. Despite these deficiencies, surprisingly all iron studies were unchanged, suggesting an unexplained alternative mechanism underlies microcytic anaemia in Dnm2+/V235G mice. This study provides the first in vivo evidence for the requirements of Dnm2 in normal erythropoiesis.

Clinical Value of Hypochromia Markers in the Detection of Latent Iron Deficiency in Nonanemic Premenopausal Women.

BACKGROUND: Iron deficiency (ID) is the most common cause of anemia in fertile women and hemoglobin (Hb) within the reference interval does not exclude ID. The consequence of an imbalance between the iron requirements and supply is a reduction of red-cell Hb content, which causes hypochromic cells. The aim of this study was to assess the reliability of new parameters low Hb density (LHD%), reticulocyte Hb equivalent (RetHe), and percentage of hypochromic erythrocytes (%HypoHe) in the detection of latent ID (LID), defined as depletion of iron stores without anemia. METHODS: Two hundred fifty consecutive nonanemic women in fertile age (18-40 years, mean 33.5 years), whose analyses had been requested by general practitioners, were included. Independent samples t-test, receiver operating characteristic (ROC) curve analysis (gold standard for detecting LID ferritin <30 µg/l), and Cohen's kappa index were applied. RESULTS: One hundred fifty-three women had ferritin within the reference range and Hb >120 g/L; 97 (38.8%) had LID. The results were as follows: %HypoHe-AUC 0.934, cutoff 1.6%, sensitivity 85.7%, specificity 92.1%; RetHe-AUC 0.914, cutoff 29.9 pg, sensitivity 86.8%, specificity 85.7%; LHD%-AUC 0.898, cutoff 5.0%, sensitivity 85.9%, specificity 84.1%. Applying those cutoffs, agreement between ferritin and %HypoHe was κ 0.61 and 0.56 for RetHe and LHD%. CONCLUSIONS: LHD%, %HypoHe, and RetHe emerge as reliable tests for the investigation of LID and could improve the ability to detect ID before anemia is present.

Diagnosis of iron-deficient states.

The diagnosis of iron deficiency anemia is typically straightforward, especially when classic biochemical and hematological changes are present in a subject at risk. It can be challenging in the presence of diseases or when it is due to inherited defects of iron metabolism. The identification of iron deficiency prior to anemia development is also difficult. New hematological parameters such as reticulocyte Hb content have expanded the classic ones such as MCV, MCH and MCHC. A variety of hematology analyzers now provide novel parameters to assess cellular hypochromia and microcytosis in both reticulocytes and mature red blood cells. The repertoire of biochemical markers has also been expanded, with iron, transferrin and ferritin being supplemented by circulating transferrin receptor and hepcidin. Molecular identification of functional variants of key iron metabolism determinants has provided explanations for the heritability of some iron metabolism biomarkers. Genetic defects in some of these molecules are responsible for hereditary microcytic anemias, also called atypical microcytic anemias. In this review, we examine the most significant hematological and biochemical markers for iron metabolism, as well as relevant genetic polymorphisms and defects affecting iron handling.

[Prognostic value of hemoglobin concentration in the course of chemoradiotherapy of patients with oropharyngeal squamous cell carcinoma].

There was performed a comparative analysis of the indicators of immediate and long-term results of chemoradiotherapy in 342 patients with squamous cell oropharyngeal carcinoma depending on hemoglobin level before and after treatment. In patients with normal level of hemoglobin a rate of response to treatment was almost two times higher than that of patients with anemia (75,3% vs. 23,5%) and complete regression of tumors was detected by more than three times often (65,4% vs. 17,6%). The overall five-year survival of patients with anemia was significantly worse than that of patients who had normal hemoglobin level (50,7% vs. 67,7%). Patients who had normal hemoglobin level at the time of discharge demonstrated a five-year overall survival of 75,7%, while those with a hemoglobin level below normal, but more than 80 g/l,--only 57,8%. Hemoglobin concentration was a significant prognostic factor for survival of patients with squamous cell carcinoma of the oral cavity and oropharynx. Low baseline of hemoglobin was also a negative prognostic factor for tumor response to treatment, especially to chemotherapy.

DMT1-mutant erythrocytes have shortened life span, accelerated glycolysis and increased oxidative stress.

BACKGROUND/AIMS: Deficiency of the divalent metal transporter 1 (DMT1) leads to hypochromic microcytic anemia. We have previously shown that DMT1 deficiency impairs erythroid differentiation and induces apoptosis of erythroid cells. Here we analyzed metabolic processes and survival of mature erythrocytes in order to address potential involvement of erythrocyte defect in the pathophysiology of the disease. METHODS: FACS analysis was used to determine the half-life of erythrocytes (CFSE fluorescence), phosphatidylserine exposure (Annexin V binding), cytosolic Ca(2+) (Fluo3/AM fluorescence) and reactive oxygen species (ROS; DCF fluorescence). Enzyme activities were determined by standard biochemical methods. The concentration of ATP and ADP was measured on HPLC-MS/MS. RESULTS: We observed an accelerated clearance of CFSE-labeled DMT1-mutant erythrocytes from circulating blood when compared to wild-type erythrocytes. In vitro, DMT1-mutant erythrocytes showed significantly increased Annexin V binding after exposure to hyperosmotic shock and glucose depletion. Despite exaggerated anti-oxidative defense, higher ROS levels were present in DMT1-mutant erythrocytes. Accelerated anaerobic glycolysis and reduced ATP/ADP ratio detected in DMT1-mutant erythrocytes indicate enhanced demand for ATP. CONCLUSIONS: We propose that DMT1 deficiency negatively affects metabolism and life span of mature erythrocytes; two other aspects of defective erythropoiesis which contribute to the pathophysiology of the disease.

Aceruloplasminemia in a Turkish adolescent with a novel mutation of ceruloplasmin gene: the first diagnosed case from Turkey.

Aceruloplasminemia is a rare autosomal recessive disease that affects the iron metabolism of the body. When there is a lack of ceruloplasmin ferroxidase activity, iron accumulates, especially in the brain, pancreas, liver, and retina. The first symptom is generally a persistent hypochromic microcytic anemia with a mild high-serum ferritin level. The affected patients are usually recognized at later ages, when the neurological symptoms appear. The neurological outcome has an adverse effect on the prognosis, which may result in fatality. Therefore, early diagnosis and intervention may prevent a devastating neurological damage. Here, we report a case of aceruloplasminemia in a teenage girl with hypochromic microcytic anemia.

Hematological parameters in patients of cleft lip and cleft palate with special reference to eosinophil counts.

INTRODUCTION: Birth abnormalities like cleft lip and cleft palate account for about 1.4 per 1000 live births in India. These are seen to be associated with a high incidence of eosinophilia which delays the surgical management of these patients. AIMS: The aim of this paper is to study the hematological parameters in patients of cleft lip and cleft palate. PATIENTS AND METHODS: A total of 223 cases of cleft lip and cleft palate were taken up for the study. Hematological parameters including hemoglobin, total leukocyte count, differential leukocyte count, absolute eosinophil count, and red cell indices were studied. RESULTS: Anemia was found in 182/223 (81.63%) cases which was most commonly of microcytic hypochromic type. Eosinophilia was seen in 46/223 (20.60%) cases. CONCLUSIONS: Many cleft lip and cleft palate patients show high eosinophil counts. Absolute eosinophil count was found to be a better parameter for assessment of eosinophils.

Homozygosity for HBA1: c.179G > A: Hb Adana in an infant.

Hb Adana (HBA1: c.179G > A) is a very rare, unstable form of α-globin variant that results from deficient synthesis of functional α chains. We present a 2-month-old boy with hypochromic microcytic anemia, and remarkable anisocytosis, target cells and basophilic stippling on his peripheral blood smear. α-Globin gene analysis of the patient determined homozygosity for HBA1: c.179G > A, a mutation known as Hb Adana. On his follow-up visit, hemoglobin (Hb) levels were stable at 9.0-9.5 g/dL and mean corpuscular volume (MCV) was 62.2-62.5 fL without the need for a blood transfusion. Clinical and hematological findings of our case were comparable to Hb H (ß4) or ß-thalassemia intermedia (ß-TI)-like phenotypes, despite the fact that he carried an α1 gene mutation. Heterozygosity for the HBA1: c.179G > A mutation may also lead to microcytosis only as seen in his parents. According to our current knowledge, this is the first described case with homozygosity for the Hb Adana mutation, carried on the α1 gene. The relatively mild presentation of the case highlights the milder phenotypic consequences of nondeletional α mutations in the α1 vs. the α2 gene.

[A simple algorithm for anemia]. / Az anaemia egyszeru algoritmusa.

The author presents a novel algorithm for anaemia based on the erythrocyte haemoglobin content. The scheme is based on the aberrations of erythropoiesis and not on the pathophysiology of anaemia. The hemoglobin content of one erytrocyte is between 28-35 picogram. Any disturbance in hemoglobin synthesis can lead to a lower than 28 picogram hemoglobin content of the erythrocyte which will lead to hypochromic anaemia. In contrary, disturbances of nucleic acid metabolism will result in a hemoglobin content greater than 36 picogram, and this will result in hyperchromic anaemia. Normochromic anemia, characterised by hemoglobin content of erythrocytes between 28 and 35 picogram, is the result of alteration in the proliferation of erythropoeisis. Based on these three categories of anaemia, a unique system can be constructed, which can be used as a model for basic laboratory investigations and work-up of anaemic patients.

Concordance between platelet counts by impedance and optical technique during microcytic anemia: towards a threshold value of the mean corpuscular volume.

INTRODUCTION: Platelet count is crucial for clinical decision. In cases of microcytosis, platelet count based on impedance technique (PLT-I) may overestimate platelet count. AIM: To compare PLT-I with platelet count using the optical technique (PLT-O) and establish a Mean Corpuscular Volume (MCV) threshold for considering PLT-O. METHODS: A prospective analytical study conducted over two months involved blood samples collected in standard K2 EDTA tubes for complete blood count analysis, revealing microcytosis (MCV<80 fL). PLT-O analysis in channel-Ret mode was performed using the Sysmex-XN1000 (Sysmex Corporation, Kobe, Japan). Percentage of fragmented red cells (FRC%) and percentage of microcytic red cells (Micro-R%) were recorded. Blood smears stained with May-Grünwald-Giemsa were examined for potential interfering particles. RESULTS: A strong correlation was observed between the two techniques for all platelet values as well as for PLT <150 x 109/L (correlation coefficient r = 0.971, 95% CI: [0.956-0.982]; P<10-3 and r = 0.90, 95% CI: [0.79-0.95]; P< 10-3). The Bland-Altman plot revealed a bias of 16.53 x 109/L between the two methods, with agreement limits between -55.8 and 88.8 x 109/L. A threshold MCV value indicating the use of the optical method, with a cut-off at 72.9fL, demonstrated promising performance consistent with literature findings. However, less favorable performance was observed with Micro-R%. CONCLUSION: Impedance could be employed in routine practice. However, for MCV<72.9 fL or in the presence of schizocytes, the hemogram validation procedure may incorporate the use of PLT-O.

A novel type of congenital hypochromic anemia associated with a nonsense mutation in the STEAP3/TSAP6 gene.

STEAP3/TSAP6 encodes a ferrireductase that is involved in the acquisition of iron by developing erythroblasts and steap3/tsap6 null-mice display severe microcytic anemia. We report a family in which 3 siblings born to healthy parents display transfusion-dependent hypochromic anemia. A nonsense STEAP3/TSAP6 was identified in the siblings at the heterozygous state. This mutation was inherited from their father while no mutation was found in their mother. A large variability of expression was found between normal alleles in a control population, confirming a previous report that STEAP3/TSAPS6 is an expressed quantitative trait locus (e-QTL). Determination of the relative allele expression showed that the "normal" allele was expressed at a significantly higher level in the father than in the affected siblings relative to the shared mutated allele. The blood level of STEAP3/TSAP6 mRNA was severely reduced in the siblings, while both parents were in the lower range of normal controls. The STEAP3/TSAP6 protein was also reduced in lymphocytic cell lines from the patients. Collectively, our data support the hypothesis that STEAP3/TSAP6 deficiency leads to severe anemia in the affected siblings and results from the combination of a mutated allele inherited from their father and a weakly expressed allele inherited from their mother.

[Treatment with beta-erythropoietin in lung cancer--effectiveness and quality of life improvement in clinical practice]. / Eritropoetin-béta-kezelés tüdôrákban--hatékonyság és életminôség-javulás a klinikai gyakorlatban.

Anemia is common among patients with malignant tumors, due to the disease and chemotherapy. Anemia decreases patient's quality of life, and worsens the dose-intensity of chemotherapy. The aim of this retrospective data-analysis was to evaluate quality of life and hemoglobin levels in 19 consecutive lung cancer patients receiving beta-erythropoietin, due to chemotherapy induced anemia. A self developed, patient source data based quality of life questionnaire was used. The mean pre-erythropoietin hemoglobin concentration of the patients was 96.31±6.72 g/L (mean±SD), the post-treatment hemoglobin concentration 111.63±14.05 g/L (p<0.05). During the chemotherapy of the 19 patients with lung cancer, transfusion was given only four times. The mean quality of life total score of the patients increased significantly during erythropoietin treatment that was resulted by the improvements of scores determining dizziness, tachycardia, and fatigue. Main limitations of this real life data analysis are low patient number and the lack of validation in the used questionnaire. In summary, according to our experiences, the use of beta-erythropoietin in patients with lung cancer results improved quality of life and a low rate of transfusions.

Application of HbA2 levels and red cell indices-based new model in the differentiation of thalassemia traits from iron deficiency in hypochromic microcytic anemia Cases.

INTRODUCTION: Thalassemia traits and iron deficiency anemia are the most common types of hypochromic microcytic anemia with similar clinical and laboratory features. It is vital to establish a new screening model based on HbA2 levels and red cell indices for the differentiation of TT from IDA in hypochromic microcytic anemia cases. METHOD: The data comprised of the red blood cell indices and HbA2 prenatal diagnostic test results of 810 individuals who were identified to conform to the following criteria: MCV < 80 fl or MCH < 26 pg. We launched a new model consisting mainly of significative red cell indices and HbA2 levels, as well as proposing cutoff values by using decision trees and logistic regression analyses. Next, we evaluated our new method by comparing the sensitivity, specificity, positive, and negative predictive values with those of the previous formulas. RESULTS: We put forward a new model and compared it with 5 efficient formulas. The new model exhibited the highest accuracy (0.918), with its sensitivity and specificity calculated as 0.917 and 0.921, respectively. Our new model's Youden index was 0.838, which is higher than the other formulas' Youden indices. CONCLUSIONS: The new screening model, based on HbA2 levels and red cell indices, is suitable for the screening of thalassemia patients in the hypochromic microcytic anemia group and has the best efficiency in distinguishing TT and IDA.

Zinc protoporphyrin in the erythrocytes of patients with lead intoxication and iron deficiency anemia.

The fluorescent porphyrin in the erythrocytes of patients with lead intoxication or with iron deficiency anemia is zinc protoporphyrin that is bound to globin moieties, probably at heme binding sites.