Heinz Bodies Research Articles

Hb Sun Prairie: A rare cause of chronic hemolysis in an Indian patient

Haemolytic anaemia is a commonly encountered condition in clinical haematology practise. Dissecting the aetiology of haemolytic anaemia is of paramount importance for appropriate management. We describe a 29-years-old lady of Indian origin, who presented with fatigue and recurrent jaundice for 2 years. Examination revealed pallor, mild icterus, and splenomegaly. Blood tests showed anaemia, reticulocytosis, indirecthyperbilirubinemia, and high serum lactate dehydrogenase, consistent with haemolytic anaemia. Peripheral smear showed severely microcytic hypochromic red cells and polychromasia. Heinz bodies and inclusion bodies were seen with supravital staining. Haemoglobin high pressure liquid chromatography showed low HbA2 and normal HbF. Work-up for iron deficiency was negative. Polymerase chain reaction of the genomic DNA failed to identify common deletions in the HBA genes. Sangers sequencing of HBA2 gene revealed a homozygous missense mutation NM_000517.6: c.391G > C (p.Ala131Pro) leading to a highly unstable hemoglobin, Hb Sun Prairie. Mother was heterozygous for the same mutation, and father was unavailable for genetic testing. We highlight the role of sangers sequencing in unravelling the underlying aetiology of haemolytic anaemia. Pathophysiology and existing literature of Hb Sun Prairie has been discussed.

Physiological peculiarities of dog’ blood erythrocytes in conditions of the Northern Black sea coast

Erythrocyte microscopy is an important step in the study of blood smears, which gives the researcher a lot of information and under certain conditions allows one to make a reliable diagnosis. This article presents new scientific data on the physiological characteristics of erythrocytes in dogs, the variability of their size and shape depending on age, sex and season of the year. The experiment was performed on 147 clinically healthy dogs of different ages, breeds and genders. Blood smears were stained by the Romanowski-Gimse method and subjected to light microscopy with oil immersion. It was found that physiological anisocytosis occurs in 34% of dogs, and 86% are unknown poikilocytosis, 10% – moderate and only 4% – severe. If we analyze the changes in cell size, macrocytic anisocytosis was observed more often in the blood smear of dogs (in 19% of cases), slightly less – macrocytic (14%) and only in one case megalocytic anisocytosis was detected. Every third case of anisocytosis was reported in dogs under 6 months of age. In spring, changes in the size of erythrocytes were detected in 59% of cases (in 10 dogs out of 17), in summer and autumn – 17-18% and in winter in 8% of the studied animals. Only 60% of the studied dogs were found to have normochromia by laboratory analysis. Moreover, hypochromia is 4.7 times more common than hyperchromia. Hyperchromia in dogs was more often recorded in spring (18%), hypochromia in autumn and spring (41–45%), and polychromia in summer (24% of the total number of studied dogs). Inclusions in the erythrocytes of dogs were found in 32% of experimental blood smears, with 85% of cases being Jolie bodies, 7.5% – Kebot rings, 5.6% – Heinz bodies and 1.9% – pseudo-inclusions. Physiological poikilocytosis was detected in 47.6% of blood smears of dogs, out of which in 35.4% of samples there was a slight poikilocytosis, in 11.6% – moderate and up to 1% – severe. The ones that were more frequently encountered were target cells and stomatocytes (14% each), acanthocytes (11%), echinocytes (5%), dacrinocytes (4%), spherocytes (3%), keratocytes (2%), schistocytes (2%), crescent-shaped cells (1% of the total number of studied smears). Poikilocytosis in dogs is more common in spring (82% of smears), slightly less in summer (up to 35%) and autumn (21%).

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.

Multimodal detection and analysis of a new type of advanced Heinz body-like aggregate (AHBA) and cytoskeleton deformation in human RBCs.

A new type of aggregate, formed in human red blood cells (RBCs) in response to glutaraldehyde treatment, was discovered and analyzed with the classical and advanced biomolecular imaging techniques. Advanced Heinz body-like aggregates (AHBA) formed in a single human RBC are characterized by a higher level of hemoglobin (Hb) degradation compared to typical Heinz bodies, which consist of hemichromes. The complete destruction of the porphyrin structure of Hb and the aggregation of the degraded proteins in the presence of Fe3+ ions are observed. The presence of such aggregated, highly degraded proteins inside RBCs, without cell membrane destruction, has been never reported before. For the first time the spatial differentiation of two kinds of protein mixtures inside a single RBC, with different phenylalanine (Phe) conformations, is visualized. The non-resonant Raman spectra of altered RBCs with AHBA are characterized by the presence of a strong band located at 1037 cm-1, which confirms that glutaraldehyde interacts strongly with Phe. The shape-shifting of RBCs from a biconcave disk to a spherical structure and sinking of AHBA to the bottom of the cell are observed. Results reveal that the presence of AHBA should be considered when fixing RBCs and indicate the analytical potential of Raman spectroscopy, atomic force microscopy and scanning near-field optical microscopy in AHBA detection and analysis.

Вміст метгемоглобіну та карбоксигемоглобіну в еритроцитах щурів за умов токсичного ураження на тлі аліментарної депривації протеїну

The study of the content of total hemoglobin and its pathological derivates methemoglobin and carboxyhemoglobin in rats’ red blood cell under the toxic damage and alimentary protein deprivation are presented. In order to simulate the low-protein diet of animals for 28 days were kept, an isoenergy diet containing 4.7% protein, 10% fats and 85.3% carbohydrates, which was calculated according to the American Institute of Nutrition recommendations. The simulation of acute toxic damage was performed by per os acetaminophen-administration to experimental animals at doses of 1250 mg/kg of animal body weight. It was found that in experimental animals the 2-fold decrease in erythrocytes counts compared with the values of the control group was observed under the toxic damage after alimentary protein deprivation. In this case, both protein deficiency and the administration of acetaminophen toxic doses have a clear effect on the studied indicator. At the same time, a decrease of erythrocyte counts is accompanied by a simultaneous decrease of total hemoglobin content in rat erythrocyte hemolysate. In animals consuming a low protein diet, this may be due to a deficiency of essential amino acids involved in the synthesis of globin’s polypeptide chains. The maximal increase of methemoglobin content (5-fold compared with control) in rats’ erythrocyte hemolysate was recorded under the administration of acetaminophen toxic doses after alimentary protein deprivation. In these experimental conditions this may be associated with a decrease of methemoglobin reductase activity, which causes methemoglobin accumulates in erythrocytes in the form of Heinz bodies. An increase of the carboxyhemoglobin level in rats’ erythrocyte hemolysate of all the experimental groups compared with the control under the conditions of toxin administration after protein deficiency, probably indicates impaired synthesis and/or degradation of heme-containing proteins.

Heinz body-related interference with leukocyte and erythrocyte variables obtained by an automated hematology analyzer in cats.

Heinz bodies (HBs) are known to interfere with automated hematology in cats, particularly with the white blood cell (WBC) count. We evaluated the influence of feline HBs on the complete blood count (CBC) results obtained using a flow cytometry-based analyzer. We retrospectively selected cats with circulating HBs and reviewed the results of their CBCs, including red blood cell (RBC) indices, basophil/lobularity (Baso) WBC count (WBCB), peroxidase (Perox) WBC count (WBCP), and cytograms. Based on the presence or absence of HB-related artifacts in their Baso cytogram, cats were grouped into Baso-HBs and HBs groups, respectively, for comparison. The WBCB and WBCP were compared to manual counts of WBCs carried out on blood smears at 400× (MC-WBC). We included 32 cats in our study: 9 of 32 were in the Baso-HBs group, and 23 of 32 were in the HBs group. Baso-HBs cats had a significantly increased HB percentage (p < 0.001), WBCB (p < 0.001), difference between WBCB and WBCP (p < 0.001), lymphocyte count (p < 0.001), mean corpuscular hemoglobin concentration (p < 0.001), and difference between calculated and measured erythrocyte hemoglobin concentrations (p < 0.001) compared to HBs cats. In Baso-HBs cats, the WBCB was significantly higher than the WBCP (p = 0.02); no significant difference was detected between the WBCP and the MC-WBC (p = 0.88). Evaluation of automated CBC results raised the suspicion of HB-related interference when using a hematology analyzer in cats; hence, blood smear examination remains essential in routine practice.

Evaluation of postoperative pain and toxicological aspects of the use of dipyrone and tramadol in cats.

The aim of this study was to evaluate the effects of dipyrone and tramadol, used for 5 days, on postoperative pain, hematological and biochemical parameters, and oxidative markers on erythrocytes. Twenty-eight healthy cats underwent ovariohysterectomy and were randomly allocated to four groups (each n = 7), according to the postoperative treatment administered intravenously: control (saline 1 ml q8h), DIP1 (dipyrone 25 mg/kg q24h), DIP2 (dipyrone 25 mg/kg q12h) and DIP3 (dipyrone 25 mg/kg q8h). All animals received tramadol (2 mg/kg q8h). Pain was assessed by visual analog (VAS), multidimensional UNESP and Glasgow pain scales for cats preoperatively and at 3, 6, 12, 24, 36 and 48 h after extubation. Venous blood was collected daily for 5 days, and on day 10, to perform a complete blood count (CBC) and determine the percentage of Heinz bodies (HBs). Serum biochemistry was evaluated preoperatively and on days 5 and 10; superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO) and lipoperoxidation were evaluated preoperatively and on days 3, 5 and 10. Control cats had higher pain scores than DIP3 cats by UNESP (P = 0.0065), and DIP2 (P = 0.0035) and DIP3 cats (P = 0.0108) by VAS 3 h postoperatively. Rescue analgesia was required by two animals in the control group and one each in the DIP1 and DIP2 groups. There was no difference in SOD or CAT among groups. On day 5, MPO was more active in DIP2 than in DIP3 cats (P = 0.0274). No difference in lipoperoxidation among treatment and control cats was found. CBC remained constant and without statistical difference among groups. Control, DIP2 and DIP3 cats presented a similar percentage of HBs on day 10. Biochemical variables were similar among groups and times. The administration of dipyrone in cats, when used in combination with tramadol, did not ensure better analgesia than tramadol alone. Dipyrone did not significantly affect biochemical variables and oxidative markers, despite minimal, clinically irrelevant, hematological differences between groups.

Acute Ramson (Allium ursinum) Toxicosis in Captive Tortoises

The present case report documents acute ramson (Allium ursinum) toxicosis in tortoises. A group of four captive tortoises, including one Aldabra giant tortoise (Aldabrachelys gigantea) and three African spurred tortoises (Centrochelys sulcata), was fed a large quantity of ramson foliage. Approximately 12 hr later, the giant tortoise and two spurred tortoises developed acute and pronounced lethargy, anorexia, and palor of the oral and conjunctival mucosae. The remaining spurred tortoise showed palor of the oral mucosa but did not display other clinical signs. At initial presentation, approximately 24 hr after being fed with ramson, one spurred tortoise had died, and supportive care as well as the administration of activated charcoal was initiated in the remaining tortoises. While the giant tortoise died 5 days following initial presentation, the other two tortoises recovered. Gross pathological, histopathological, blood biochemistry, and hematological findings largely supported what has been documented in other species with Allium sp. toxicosis. Heinz bodies, however, do not seem to be a consistent finding in the blood of tortoises fed with ramson. Although the development of toxicosis due to the ingestion of ramson in tortoises seems to be dose dependent based on the present report, clinical practitioners and reptile pet owners should be aware of the potential detrimental effects of feeding this herb to tortoises. To the authors' knowledge, this is the first report of toxicosis caused by an Allium sp. in reptiles.

Poisons affecting the blood

Blood circulates as a liquid containing cells and numerous chemicals; it functions to transport nutrients, chemicals, oxygen and waste products to and from cells, and is involved in defence and wound repair to tissues. Numerous toxic substances can disrupt the normal function of the blood through various mechanisms resulting in clinical signs of poisoning. Some of the substances that commonly cause adverse effects on the blood in companion animals are described. These include onions and related food plants which cause oxidative damage to red blood cells and the formation of Heinz bodies, and zinc which causes haemolytic anaemia. Haemoglobin is also subject to oxidative damage from paracetamol metabolites resulting in the formation of methaemoglobin which is non-functioning, resulting in tissue hypoxia. Disruption of the clotting cascade by anticoagulant rodenticides results in delayed-onset haemorrhage and the drug 5-fluorouracil disrupts the formation of blood cells causing bone marrow depression.

Hb Milano [α109(G16)Leu→Pro (CTG>CCG); HBA1: c.329T>C]: A Novel Variant on the α1-Globin Gene in an Italian Family

Interest in α-globin point mutations has increased in the past few years because nondeletional variations can affect protein function and stability, giving rise to hemoglobin (Hb) variants that present a wide spectrum of phenotypes, from asymptomatic forms to hemolytic anemia. We describe a novel α1-globin gene variant, which we have named Hb Milano [α109(G16)Leu→Pro (CTG>CCG); HBA1: c.329T>C]. We performed high performance liquid chromatography (HPLC) to carry out Hb analysis, capillary electrophoresis (CE) for Hb separation and quantitation of Hb subtypes, two tests on stroma-free lysates for evaluating Hb stability, multiplex ligation-dependent probe amplification (MLPA) to detect deletions/duplications within the α gene cluster and Sanger sequencing of the α-globin genes. No abnormal Hb variants were identified by HPLC and CE. Isopropanol and stability tests were negative. The peripheral blood film showed no inclusions such as Hb H or Heinz bodies. Multiplication ligation-dependent probe amplification of the α-globin gene cluster detected a heterozygosity for the −α3.7 (rightward) deletion. Direct sequencing of the α-globin genes identified the Hb Milano variant on the HBA1 gene. No mutations were found on the HBA2 gene. The clinical consequences of the Hb Milano variant differ based on the genotype: according to our study, the hematological parameters range from a marked microcythemia with mild anemia if the variant is coinherited with an α gene deletion, to mild microcytosis when the variant is not associated with α gene deletions.

Phenylhydrazine and its salts – calculated on phenylhydrazine. Documentation of proposed values of occupational exposure limits (OELs)

Phenylhydrazine and its salts – calculated on phenylhydrazine. Documentation of proposed values of occupational exposure limits (OELs)

Safety and efficacy of aged garlic extract in dogs: upregulation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and Nrf2-regulated phase II antioxidant enzymes

BackgroundPlants of Allium spp., including garlic (A. sativum) and onions (A. cepa), are known to be oxidatively toxic to canine erythrocytes resulting in Heinz body hemolytic anemia in dogs. In humans, these plants have been used as medicinal agents for multiple diseases since ancient times. Especially, fresh garlic extracted over a prolonged period produces less irritative and odorless aged garlic extract (AGE), containing unique and beneficial organosulfur compounds that can help prevent many kinds of diseases. In this study, the safety and efficacy of long-term oral administration of AGE is evaluated in dogs. The objectives are to confirm the safe dosage for long-term use and beneficial functions of AGE for dogs and to plan and design a canine health supplement or a preventive agent for multiple diseases based on the data of this study.ResultsBeagles were orally administered AGE (45 or 90 mg/kg body weight once a day) or an equivalent amount of water as control for 12 weeks. In AGE-treated groups, at 12 weeks post-administration at a dose of 90 mg/kg, there were no observable changes in the clinical signs, complete blood count, and serum biochemical parameters. Heinz bodies and eccentrocytes, the markers of oxidative damage in erythrocytes, did not appear in blood smear examination. In order to further evaluate the beneficial effects of AGE on health of dogs, the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) gene (NFE2L2) and Nrf2-regulated phase II antioxidant enzyme genes (NQO1, GCLM, HMOX1, and SOD2) were determined in whole blood between pre- and post-AGE administration. The expression of NFE2L2 gene was significantly upregulated in the AGE-treated groups [45 (p < 0.05) and 90 mg/kg (p < 0.01), 8 weeks] as compared to in the control group. Among the Nrf2-regulated enzymes examined, the expressions of NQO1 [45 (p < 0.05) and 90 mg/kg (p < 0.01), 8 weeks] and GCLM [45 (p < 0.05) and 90 mg/kg (p < 0.01), 12 weeks] genes were significantly upregulated.ConclusionThe long-term oral administration of AGE at a dose of 90 mg/kg/day for 12 weeks did not show any adverse effects in dogs. Furthermore, the administration of AGE upregulated the gene expressions of canine Nrf2 and Nrf2-regulated phase II antioxidant enzymes. These results suggest that AGE might safely contribute to the health of dogs provided that the appropriate dosage is used.

Poisonous plants part 2

In this second article more toxic plants are discussed and a table of other potentially toxic plants is provided for reference. Poisoning with Allium species, including onions and garlic, is characterised by Heinz body anaemia in both cats and dogs. Conkers and acorns are commonly eaten in the autumn, particularly by dogs, and generally only cause gastrointestinal signs, although there is a potential risk of obstruction. Numerous plants contain cyanogenic glycosides that may potentially release cyanide in the gut (depending on the circumstances); however, gastrointestinal signs are the main feature of poisoning in cats and dogs. Severe laburnum (Laburnum anagyroides) and yew (Taxus spp.) poisoning in cats and dogs is also rare, although deaths have been reported in the literature. Laburnum can cause central nervous system stimulation and yew can result in cardiac arrhythmias which may be difficult to manage. The castor oil plant (Ricinis communis) contains ricin, which is extremely toxic and causes multi-organ failure, but severe cases in dogs are generally only associated with ingestion of chewed seeds or castor oil bean meal fertiliser. Hops (Humulus lupulus), which are commonly eaten as spent hops from brewing, cause severe pyrexia in dogs, which requires aggressive treatment and is associated with a high mortality.

Haematological and morphological evaluation of feline whole blood units collected for transfusion purposes.

Despite the increasing availability of feline blood collected and stored for transfusion purposes, few studies have been performed on feline blood units. The aim of this prospective in vitro study was to evaluate haematological and morphological changes in feline blood cells in whole blood units between collection and end of storage. Haematological examination (red blood cells [RBCs], haemoglobin, haematocrit, red cell distribution width, mean cell volume, mean cell haemoglobin concentration, mean cell haemoglobin, white blood cells [WBCs] and platelet [PLT] count) was performed on 40 non-leukoreduced feline whole blood units at the time of collection (day[D]0) and after storage (D35). The blood was collected into citrate-phosphate-dextrose-adenine anticoagulant-preservative solution using an open system in a veterinary blood bank and stored for 35 days at 4 ± 2°C. Twenty of these feline whole blood units were also analysed for blood cell morphology (normal RBCs, macrocytes, echinocytes, spherocytes, schistocytes, lysed RBCs, RBCs with Heinz bodies and recognisable WBC and PLT count). Differences between the two examination times were statistically analysed. There was a statistically significant decrease in WBC and PLT counts after storage at D35 (P <0.0001 for both). The most significant cellular morphological changes after storage were an increase in echinocyte count (P = 0.0001), and lysed RBCs (P <0.0001), and a decrease in normal RBCs (P <0.0001). Recognisable WBCs - mainly lymphocytes - were present at the end of storage. This study showed that significant morphological changes occur in RBCs in feline blood units during storage for 35 days. In vivo studies are required to establish if these changes could affect the ability of stored RBCs to circulate and provide adequate oxygen delivery after transfusion.

Protective Role of Selenium Against Hemolytic Anemia Is Mediated Through Redox Modulation

Selenium (Se), an essential trace element and potent nutritional antioxidant, exerts its biological effects through incorporation into selenoproteins like glutathione peroxidase (GPx). Modest decrement in the levels of GPx could be partly responsible for peroxidation of RBCs, which results into hemolytic anemia. Therefore, it is hypothesized that dietary Se, as selenoproteins (GPx), can maintain the homeostasis in RBCs and regulate the erythropoiesis by preventing oxidative stress-mediated hemolysis. Se-deficient (0.01ppm), Se-adequate (0.1ppm sodium selenite), and Se-supplemented (0.5ppm sodium selenite) status were created in Balb/c mice by feeding yeast-based diets for 8weeks and established by measuring Se levels in plasma and activities, expressions of Se-dependent selenoproteins. Fifty percent of mice from each differential Se group were treated with phenylhydrazine (PHZ, 20mg/kg, i.p.) to induce hemolytic anemia. Results indicated that PHZ-treated Se-deficient animals demonstrated increased hemolysis, abnormal RBC morphology, increase in Heinz bodies and reticulocytes, and denaturation of hemoglobin to globin precipitates and methemoglobin. Se supplementation protected against these hemolytic changes and makes RBCs less fragile. These findings were consistent with dietary Se concentration-dependent changes in activity and expression of GPx indicating that ROS-mediated oxidative stress is integral to hemolysis. Protective effects of Se supplementation against increased levels of ROS, protein carbonyls, and peroxide damage to membrane lipids and enzymatic antioxidants validated these observations. In conclusion, dietary Se supplementation protected the RBCs against hemolysis by mitigating ROS-mediated oxidative stress.

Para-tertiary butyl catechol induces eryptosis in vitro via oxidative stress and hemoglobin leakage in human erythrocytes

Exposure of human population to industrial chemicals is believed as a significant contributing factor to the outgrowth of occupational diseases especially in developing countries due to improper safety measures and sanitary conditions. Para-tertiary butylcatechol (PTBC) widely employed in petrochemical, thermofax and phototypesetting industries, induces melanocytotoxicity and contact dermatitis leading to occupational leukoderma/vitiligo. Few vitiligo patients were reported for oxidative stress-induced hemolytic anemia and thrombocytopenia, however its impact on blood components is still not clear. Erythrocytes are the major cell population in circulation and play a prominent role in various diseases. In this work, the effect of PTBC on human erythrocytes is evaluated in vitro. PTBC induces oxidative stress-mediated eryptosis (erythrocyte death) causing detrimental changes such as depleted antioxidant levels, altered surface morphology, hemoglobin denaturation and heinz body formation. These findings validate that PTBC could induce toxic effects on human erythrocytes. Exposure of humans to toxic chemicals constitutes an important issue in various industries; one such issue is the exposure of PTBC at work place resulting in a spectrum of dermal complications. Therefore, it is imperative to appraise the long-term toxicities in order to further delineate the mechanisms of resultant disorders associated with PTBC and to establish the therapeutic interventions.

Effect of aluminum (Al) speciation on erythrocytic antioxidant defense process: Correlations between lipid membrane peroxidation and morphological characteristics

Al contamination becomes a growing problem in human society. Accumulation of Al in blood could destroy the structure and disorder function of erythrocyte, and finally cause blood diseases. In the present study, AlCl3 and Al(malt)3 are respectively used in the erythrocyte system, in order to investigate the comparative toxic effect on erythrocyte fragility, the influence on cellular biochemical components and lipid peroxidation level. We find that the osmotic fragility, the number of Heinz bodies, the content of MDA and advanced oxidation protein product of the AlCl3 treated erythrocytes were higher than the Al(malt)3 treated erythrocytes at the same concentrations of Al(Ⅲ). The morphological and membrane protein changes of the AlCl3 treated group show superior to the Al(malt)3 treated group. In summary, we conclude that the comparative effect on the erythrocyte between organic aluminum and inorganic aluminum is significantly different, and the prime comparative difference between the toxic effects of both the compounds is oxidative stress. Further research should focus on in vivo experiments to confirm the differential toxicity and to elucidate the molecular mechanisms underlying Al-induced erythrocyte toxicity in order to prevent hematological disorders.

Study of Glucose-6-Phosphate Dehydrogenase Deficiency: 5 Years Retrospective Egyptian Study.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide that causes a spectrum of diseases including neonatal hyperbilirubinemia, acute and chronic hemolysis after exposure to oxidative stress. This five years retrospective study was carried out to study the demographic, clinical and laboratory data of 1000 patients with G6PD deficiency anemia registered in Hematology Unit, Pediatric Department, Tanta University Hospital. Data were collected from patient's files, from November 2011 to November 2016, using the pre-designed questionnaires to obtain the complete history, clinical presentation and laboratory investigations including the complete blood count, red blood cells morphology, liver and renal functions and quantitative assay of G6PD enzyme activity by spectrophotometric method. Males were more commonly affected than females (932 males versus 68 females). The highest prevalence of hemolytic crisis in G6PD deficiency patients was found within the age group of 1-3 years (920 patients; 92%) with mean age of the first presentation of 22.8±15.54 months. Patients presented mainly with pallor (1000 patients; 100%), dark red urine (896 patients; 89.6%) and jaundice (878 patients; 87.8%) after 24-72 hours of exposure to the precipitating factors (mean: 36±17.73 hours). Diets were the most common precipitating factor of hemolysis in patients with G6PD deficiency (834 patients; 83.4% of studied cases) especially fava beans (326 patients; 32.6%) and falafel (194 patients; 19.4%) which were the most common precipitating food products causing hemolysis followed by chick pea (108 patients; 10.8%), broad bean (76 patients; 7.6%), green pea (44 patients; 4.4%), pea nuts (38 patients; 3.8%), lentil (28 patients; 2.8%), and lastly black eyed peas (20 patients; 2 %). Infections were the 2nd most common cause of hemolysis (124 patients; 12.4%) including pneumonia (34 patients; 3.4%), tonsillitis (32 patients; 3.2%), typhoid fever (28 patients; 2.8%), hepatitis A (18 patients; 1.8%) and urinary tract infection (12 patients; 1.2%). Drugs were the least common cause of hemolysis (42 patients; 4.2%) including diclofenac sodium (24 patients; 2.4%), ibuprofen (8 patients; 0.8%), acetylsalicylic acid (4 patients; 0.4%), co-trimoxazole (4 patients; 0.4%) and nitrofurantion (2 patients; 0.2%). There was normocytic normochromic anemia with reticulocytosis and Heinz bodies in pre-transfusion complete blood picture in all studied cases. G6PD assay show marked decrease in enzyme level at time of presentation in all cases with the commonest G6PD enzyme level of 3-4 U/gm Hb (592 patients; 59.2%). G6PD deficiency anemia presented mainly with pallor, dark red urine and jaundice after exposure to certain diets, drugs and diseases and therefore patients with G6PD deficiency should avoid exposure to these precipitating factors of hemolysis. We can also recommend large neonatal screening programs to detect cases of G6PD deficiency before the occurrence of acute hemolysis and molecular studies to detect G6PD enzyme variant in Egypt.

A Coincidental Discovery of a New Stable Variant (Hb Hachioji or HBB: c.187C>T) in a Patient with Chronic Hemolytic Anemia of Unexplained Origin

We report a new hemoglobin (Hb) variant, Hb Hachioji (HBB: c.187C>T), which was detected in a 32-year-old male with hemolytic anemia. The proband had undergone splenectomy in his childhood after being diagnosed with hereditary spherocytosis (HS) with no clinical improvement. A recent study showed that Heinz bodies were frequently observed in his red cells, however, no abnormal band was separated by isoelectric focusing (IEF), and the isopropanol (instability) test was negative. Direct sequencing revealed that the proband was a heterozygous carrier of a novel mutation (GCT>GTT) at codon 62 of the β-globin gene, leading to an alanine to valine substitution. This variant was named Hb Hachioji. Characterization at the mRNA level by cDNA sequencing detected βHachioji mRNA, as well as βA mRNA. Subsequently, study of the proband’s family indicated that his father was a carrier of this Hb variant, although unexpectedly, the father was asymptomatic and clinically healthy. Oxygen affinity measurement of total Hb showed no alteration in the P50 and oxygen equilibrium curve. The presence of Hb Hachioji was confirmed by mass spectrometry (MS). Hb Hachioji comprised approximately 50.0% of the total Hb and was a stable variant. The phenotypic discrepancy between these two carriers suggests that Hb Hachioji may not be associated with the hemolytic involvement in the proband. P4.2Nippon, which is the primary cause of most cases of Japanese HS, was absent in the proband’s parents. The coexistence of glucose-6-phosphate dehydrogenase (G6PD) deficiency was ruled out. Thus, the cause of hemolytic involvement in this patient remains unclear.

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH-Cytochrome B5 Reductase Gene.

BackgroundIn veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.ObjectivesTo analyze the NADH‐cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency.AnimalsThree Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls.MethodsMethemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing.ResultsMethemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu).Conclusions and Clinical ImportanceThis dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH‐binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.