Search

To investigate oxidative erythrocyte damage in dogs treated with different non-steroidal anti-inflammatory drugs.Case-controlled prospective observational study using blood obtained from dogs presenting for lameness examinations or standard surgical procedures to a private referral clinic. Sampling was performed from April 2018 to July 2019. Groups comprised dogs receiving either metamizole (dipyrone) (22 dogs), carprofen (20 dogs) or meloxicam (20 dogs) for a minimum of 10 days. Dogs with gastrointestinal hemorrhage were excluded from the study. A complete hematological, as well as a basic biochemical profile were performed in every dog. Pappenheim stained blood smears were evaluated for eccentrocytes and brilliant cresyl blue stained smears for Heinz bodies. EDTA blood was frozen at -80°C immediately after sampling for measurement of superoxide dismutase and gluthathione peroxidase activity at an external laboratory. Hemoglobin concentration, superoxide dismutase and gluthathione peroxidase activities, reticulocyte count, eccentrocyte and Heinz body numbers were determined prospectively as key parameters for further statistical assessment with Kruskal-Wallis test and Dunn's multiple comparisons test.Dogs receiving metamizole showed a significant increase in eccentrocyte (median 14.5/500 cells vs. 0/500 cells in the other groups, p0.0001) and reticulocyte number (median 191.4 × 10Treatment with metamizole for 10 or more days resulted in decreased hemoglobin concentration, eccentrocytosis and reticulocytosis in dogs in this study. This might be a sign of increased oxidative damage caused by this drug.Prolonged metamizole therapy should be evaluated critically in patients already affected by severe illness or underlying anaemia.Untersuchung des oxidativen Erythrozytenschadens bei Hunden, die mit unterschiedlichen nicht steroidalen Antiphlogistika behandelt wurden.Die prospektive Fall-Kontroll-Studie erfolgte von April 2018 bis Juli 2019 mit Hunden, die zu Lahmheitsuntersuchungen oder für Routineoperationen in einer Überweisungsklinik vorgestellt wurden. Die Studiengruppen bestanden aus Hunden, die entweder Meloxicam (20 Hunde), Carprofen (20 Hunde) oder Metamizol (Dipyron) (22 Hunde) für mindestens 10 Tage erhalten hatten. Hunde mit gastrointestinalen Blutungen wurden ausgeschlossen. Jeder Hund unterlag einer vollständigen hämatologischen sowie klinisch-chemischen Untersuchung. Zur Untersuchung auf Ekzentrozyten dienten mit Pappenheim-Färbung gefärbte und für Heinz-Innenkörperchen mit Brilliantkresylblau gefärbte Blutausstriche. EDTA-Blut wurde sofort nach Probenentnahme bei –80°C eingefroren und zur weiteren Untersuchung der Superoxid-Dismutase- und Glutathion-Peroxidase-Aktivität auf Trockeneis an ein Fremdlabor versendet. Hämoglobinkonzentration, Superoxid-Dismutase-, Glutathion-Peroxidase-Aktivität sowie Retikulozyten-, Ekzentrozyten- und Heinz-Innenkörperchen-Zahl wurden prospektiv als Schlüsselparameter für die statistische Auswertung mittels Kruskal-Wallis-Test und Dunn-Nachtest bestimmt.Hunde, die Metamizol erhielten, hatten eine signifikant höhere Ekzentrozyten- (Median 14,5/500 Zellen vs. 0/500 Zellen in den anderen Gruppen, p0,0001) und Retikulozytenzahl (Median 191,4 × 10Die Studie zeigte, dass eine Behandlung von Hunden mit Metamizol über 10 oder mehr Tage zu erniedrigtem Hämoglobinwert sowie erhöhter Retikulozyten- und Ekzentrozytenanzahl führen kann. Dies könnte ein Hinweis auf durch Metamizol verursachten oxidativen Schaden an Erythrozyten sein.Eine länger andauernde Metamizoltherapie sollte bei anämischen und schwer kranken Hunden kritisch evaluiert werden.

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.

Two domestic shorthair cats were presented with anorexia and dehydration following ingestion of caramelized onions. Shared key findings from a CBC (ADVIA 2120), serum biochemistry, and urinalysis included a spurious, marked leukocytosis with discordant basophil (BASO) channel and peroxidase channel WBC counts, normal manual leukocyte counts, mild, non-regenerative anemia with discrepancies between automated and manual reticulocyte counts, an abundance of large Heinz bodies (HBs), and highly irregular scattergrams. Case 1 also demonstrated a markedly elevated mean corpuscular hemoglobin concentration (MCHC) and discrepancies between RBC hemoglobin indices. Spurious leukocyte results were confirmed through re-analysis of samples (including the acquisition of a new sample, use of an alternate analyzer (Sysmex XT-2000iV; Case 1 only), and evaluation of scattergrams and blood films (Cases 1 and 2). Repeatedly discrepant reticulocyte counts were also identified. In both cases, the erroneous BASO WBC counts, discrepancies in reticulocyte counts and RBC indices, and atypical scattergrams were interpreted to result from various effects of the HBs. These cases emphasize the importance of reviewing blood films, interpreting scattergrams, and the usefulness of duplicate methods for determining various measurands on hematology analyzers.

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.

A 4-month-old male indoor cat was referred for dyspnea, mental dullness and weakness, which appeared two days earlier. The cat had been adopted at 3 months of age. Physical exam showed cyanosis, dyspnea and mild hypothermia. The “spot test” was supportive of methemoglobinemia. Co-oximetry blood gas analysis revealed severe methemoglobinemia (81.40%), severe hyperchloremia and mild hyponatremia. CBC, biochemistry and urinalysis were within normal limits, blood smear showed the presence of Heinz bodies. Treatment was instituted with oxygen therapy, methylene blue 1% solution, ascorbic acid, intravenous fluid therapy. The clinical course was favorable with rapid improvement of cyanosis and methemoglobinemia (4.2%). Acquired methemoglobinemia was hypothesized. Two weeks after discharge the cat was asymptomatic but mild methemoglobinemia (15.60%) and hyperchloremia were evident. Exposure to oxidants contained in drinking water was suspected so the owners were instructed to use bottled water only. One month later the cat was asymptomatic, and methemoglobinemia and chloremia were within normal limits. Even if a congenital form due to cytochrome b5 reductase deficiency cannot be ruled out, drinking water intoxication is the most likely cause of methemoglobinemia in this cat.

Unstable haemoglobinopathies are rare autosomal dominant disorders that lead to haemolytic anaemia with varied severity. Structural defects in globin chains result in instability of the haemoglobin (Hb) tetramer with decreased solubility and intraerythrocytic precipitation (Heinz bodies). Clinically, unstable Hbs may remain undiagnosed either due to asymptomatic/milder phenotypes or may be misdiagnosed due to masquerading of other phenotypes. Laboratory identification depends on Hb high-performance liquid chromatography (HPLC), Hb electrophoresis, presence of Heinz bodies and heat and isopropanol stability tests.1 We report a child with de novo inheritance of Hb Sabine where the initial Hb HPLC was misleading. A 4-year-old boy presented to the paediatric gastroenterology services with a history of progressive severe anaemia, high-coloured urine, waxing-and-waning jaundice, and abdominal distention for the last 2 years. In the past, he had been treated for viral hepatitis at 2 years of age and had an atypical febrile seizure at age 2.5 years. He had required 2 units of blood transfusion once, a year back. The boy was the fifth of six children. All siblings were asymptomatic and there was no family history of severe anaemia. Physical examination revealed growth retardation with a weight of 13 kg (

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.

Background Plants 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. Results Beagles 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

Heinz body formation has been reported in cats repeatedly administered propofol for anesthesia induction, although the resultant changes were deemed of little clinical significance (1, 2). This report suggests repeated propofol administration to some individual cats might induce anemia with clinical signs and cessation of propofol administration may result in rapid resolution. A 9-years-old American Domestic Shorthair cat receiving a 20-fraction radiation protocol for lateral thoracic fibrosarcoma showed lethargy, decreased appetite and activity, and Heinz body (3+ on blood smear examination) anemia (packed cell volume 22%; reference interval 24–45%) after 12 repeated propofol anesthesia inductions. The anesthesia induction protocol was adjusted to exclude propofol. Over the following week, the anemia resolved (packed cell volume, 30%), and the cat's activity level, appetite and attitude improved. The total dose of propofol received over the 12 treatments was 62.4 mg/kg.

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

International audience; Hb Dompierre [β29(B11)Gly→Arg, HBB: c.88G>C] is a rare β-globin gene variant that was previously described in the heterozygous state in a 24-year-old female patient. It is defined in the HbVar database as being clinically and biologically asymptomatic. A few years after the first description, we had an opportunity of reassessing the index case because she presented with splenomegaly and clinical and biological manifestations of hemolysis. After ruling out the most common causes of hemolysis, further analyses on the variant hemoglobin (Hb) using brilliant cresyl blue staining, indicated that it showed mild instability, which may explain the clinical and biological manifestations. A structural bioinformatic analysis on the Hb variant suggested that the amino acid replacement may be deleterious to the integrity of the Hb. This report confirms the importance of completely characterizing all new Hb variants in order to guide the patients' clinical management and follow-up, as well as to provide the probands and their family members with appropriate genetic counseling.

A 2-month-old kitten was referred for depression and partial anorexia since 3 days and chronic diarrhea lasting for over 3 weeks. General physical examination showed pale and cyanotic mucous membranes. Blood sample was of brownish appearance. Venous blood gas analysis and complete blood count showed 16% methemoglobin level and severe regenerative anemia with Heinz bodies in about 40% of the erythrocytes, respectively. The kitten was transfused with fresh whole blood and treated with supportive care, antimicrobial and antioxidant agents. The kitten totally recovered. To the authors' knowledge, this represents the first case report of severe Heinz body hemolytic anemia and methemoglobinemia with concurrent chronic diarrhea in a young kitten. Diarrhea resolution coincided with Heinz bodies and methemoglobin disappearance. The possibility that diarrhea might have stimulated an inflammatory state causing release of oxygen radicals and prolonged erythrocytes oxidative damage has been discussed.Ein zwei Monate altes Kätzchen wurde wegen Depression und partieller Anorexie seit drei Tagen und chronischem Durchfall während mehr als 3 Wochen überwiesen. Die allgemeine klinische Untersuchung zeigte blasse und zyanotische Schleimhäute. Das Blut war von bräunlicher Farbe. Die venöse Blutgasanalyse und das vollständige Blutbild zeigten 16% Methämoglobin und eine schwere regenerative Anämie mit Heinz-Körpern bei etwa 40% der Erythrozyten. Das Kätzchen wurde mit frischem Vollblut transfundiert, gepflegt und mit Antibiotika und Antioxidanten behandelt. Das Kätzchen erholte sich vollständig. Nach Wissen der Autoren stellt dies den ersten Fallbericht von schwerer Heinz-Körper hämolytischer Anämie und Methämoglobinämie mit gleichzeitig chronischem Durchfall bei einem jungen Kätzchen dar. Die Besserung des Durchfalles fiel mit dem Verschwinden von Heinz-Körpern und Metämoglobin zusammen. Die Möglichkeit, dass Durchfall einen entzündlichen Zustand verursacht haben könnte, der zu Freisetzung von Sauerstoffradikalen und Iänger anhaltenden oxidativen Erythrozyten Schäden führte, wird diskutiert.

The morphology of erythrocyte Packed Red Cells (PRC) is subject to changes during storage at 2 o –8 o C. In maintaining their viability, erythrocytes require adenosine triphosphate (ATP). The decrease in intracellular ATP level results in damaged membrane lipid, accumulation of intracellular Sodium and Calcium, decreased intracellular potassium and water, cell dehydration, rigidity of membrane and changes in its shape from a disc into crenated red cells, spherocytes and bite cells. This study was aimed to observe the presentation of erythrocyte crenated cells, spherocytes and bite cells of stored PRC at day 3 (control), day 7, day 14 and day 21 from the blood collection date of the blood bag. An observational study with cohort study approach was conducted in August 2015 at the Blood Bank of Dr. Wahidin Sudirohusodo Hospital Makassar. The total sample was 30 blood bag that resulted in 120 slide blood smears. From 30 samples of blood bags comprising blood group A 26.6%, B 13.3%, AB 16.6% and O 43.3%, the increased percentages of crenated cells, spherocytes and bite cells were observed at day 3, day 7, day 14 and day 21. Day 3 of storage was taken as control. Data were processed by Fiedman test and Wilcoxon test with significance level at p

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is known to cause haemolysis in patients with Hansen's disease receiving dapsone containing multidrug-therapy (MDT). However, sparse recent data are available on this topic from India. Methods: All patients aged over 10 years with Hansen's disease receiving MDT who presented with clinical deterioration to our tertiary care teaching hospital in Moradabad, Uttar Pradesh, India, were included in the study. G6PD levels were estimated in all of them. Once G6PD deficiency was confirmed dapsone was stopped and rescue therapy was initiated. Results: Between March 2015 and June 2016, 50 patients (mean age 34 ± 13.3 years) were included. G6PD deficiency was found in 14.3% patients. Peripheral blood smear showed Heinz bodies and bite cells in all of them. Mean pretreatment G6PD levels were 19.5 (range 17.5 – 25) units/g haemoglobin, mean serum bilirubin was 3.3 (range 1.6-9.2) mg/dL. Nineteen patients had lepra reactions (type 1 = 5 type 2 = 14). Ten patients required packed red blood cell transfusion. Conclusions: Our observations suggest that checking for G6PD levels before initiating dapsone containing MDT can be helpful in reducing the occurrence of haemolytic complications.

BACKGROUND In 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. OBJECTIVES To analyze the NADH-cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency. ANIMALS Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls. METHODS Methemoglobin 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. RESULTS Methemoglobin 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 IMPORTANCE This 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.

Exposure to crude oil during spill events causes a variety of pathologic effects in birds, including oxidative injury to erythrocytes, which is characterized in some species by the formation of Heinz bodies and subsequent anemia. However, not all species appear to develop Heinz bodies or anemia when exposed to oil, and there are limited controlled experiments that use both light and electron microscopy to evaluate structural changes within erythrocytes following oil exposure. In this study, we orally dosed zebra finches (Taeniopygia guttata) with 3.3 or 10 mL/kg of artificially weathered Deepwater Horizon crude oil or 10 mL/kg of peanut oil (vehicle control) daily for 15 days. We found that birds receiving the highest dosage experienced a significant increase in reticulocyte percentage, mean corpuscular hemoglobin concentration, and liver mass, as well as inflammation of the gastrointestinal tract and lymphocyte proliferation in the spleen. However, we found no evidence of Heinz body formation based on both light and transmission electron microscopy. Although there was a tendency for packed cell volume and hemoglobin to decrease in birds from the high dose group compared to control and low dose groups, the changes were not statistically significant. Our results indicate that additional experimental dosing studies are needed to understand factors (e.g., dose- and species-specific sensitivity) and confounding variables (e.g., dispersants) that contribute to the presence and severity of anemia resulting from oil exposure in birds.

Copper toxicity is thought to be a rare condition in horses. However, the number of cases diagnosed in Brazil is growing. This article aims to describe cases of copper toxicity involving horses from different geographic locations and discuss findings of physical examinations, differential diagnoses and potential causes. Five cases referred from 4 different properties where at least 15 other horses were affected were described. Hemolytic anemia and hemoglobinuria, presence of Heinz bodies and elevated aspartate aminotransferase and gamaglutamil transferase levels were detected in all cases. The diagnosis was based on clinical history and signs, laboratory tests results, copper level determination in feed and/or soil and histopathological findings. Two horses progressed to acute death; remaining horses responded to clinical management with or without blood transfusion, depending on disease severity. However, one of these horses, after several returns to the veterinary hospital, was euthanized due to complications. One horse was treated with ammonium tetrathiomolybdate. Two horses had several recurring episodes over the course of several months, an uncommon presentation in ruminants suffering from copper toxicity. Excess copper was associated with soil fertilization with poultry litter or treatment of previous or neighbor crops with copper-containing products. It can be concluded that copper toxicity does occur in horses and may arise from several sources and/or be associated with predisposing dietary factors. Given the growing number of cases, the condition should be included in the differential diagnosis list and proper preventive dietary and pasture fertilization measures adopted.

Introduction: The purpose of this study was to investigate the therapeutic effect of hydrogen on the therapy of onion poisoned dogs. Material and Methods: A total of 16 adult beagle dogs were divided into two groups (control and hydrogen) and all were fed dehydrated onion powder at the dose of 10 g/kg for three days. The dogs of the experimental group were given subcutaneous injection of 0.2 mL/kg of hydrogen for 12 days after making the poisoned model successful. Blood samples were collected before feeding onions, one day before injecting hydrogen, and 2 h after the injection of hydrogen on days 1, 3, 5, 7, 9, and 12. Control dogs were not treated with hydrogen. Results: The levels of leukocyte production, anaemia, red blood cell degeneration which was reflected by the values of Heinz body count, haemolytic ratio, and oxidative products in hydrogen treated group were lower than in control dogs on some days. The capacity of medullary haematopoiesis that was based on reticulocyte counts, and the antioxidation in hydrogen group were higher compared with control group. However, the differences in renal function were not obvious in both groups. Conclusion: Accordingly, it was concluded that subcutaneous injection of hydrogen could alleviate the symptoms in onion poisoned dogs.

Introduction Hemoglobinopathies are considered a major public health problem in many countries, including Brazil, a fact explained by their frequency, genetic diversity and clinical importance. The unstable hemoglobin group is associated with congenital non-spherocytic hemolytic anemia due to instability of the hemoglobin molecule which can lead to the formation of characteristic inclusion bodies or Heinz bodies. Hb Koln beta 98(FG5) Val>Met is the result of the substitution of the amino acid valine for methionine at position 98 of the Beta chain of hemoglobin. Hemoglobin Koln reports are concentrated in Europe and in some parts of the Orient, such as northern Europe, among Jews, Czechoslovaks, Japanese, and also Koreans. A case was recently reported in India and, in Brazil, some cases have been known since 1993. This hemoglobinopathy is widely distributed, however, its frequency is extremely low when compared to the most frequent variants such as hemoglobin S (HbS) and C (HbC) in Brazil. Patients with Hb Koln usually present with mild hemolytic anemia characterized by reticulocytosis, splenomegaly and bilirubin, jaundice, and persistent dark urine excretion. In addition, some case reports describe Koln patients with priapism or persistent thrombocytopenia. Case description We report a case of Koln's Hemoglobin in a 54-year-old female patient with hemolytic anemia under investigation. Osmotic instability tests were performed with a NaCl solution, hemoglobin electrophoresis in cellulose acetate at alkaline pH in which the presence of a band migrating between A2 and S hemoglobins was observed, when the hemolysate was performed with 1% saponin, search for Heinz bodies with a 1:1 brilliant cresyl blue solution incubated at 37°C for 30 minutes, after which the bodies were identified in the blood smear. Upon suspicion of unstable hemoglobin, other standard laboratory procedures were performed, such as High Performance Liquid Chromatography (HPLC) with the Trinity®Premier Resolution Analytical Column equipment, in which a dosage of 3.18% HbA2 and 0.16% HbF was observed. The results corroborate the electrophoretic and chromatographic profile suggestive of heterozygosity for Hb Koln. Discussion It is known today that there are 155 unstable hemoglobins identified worldwide and, of these, approximately 102 lead to hemolysis or abnormal oxygen affinity. In contrast the other variants do not show hematological abnormalities, but show instability in in vitro test. The phenotype of unstable variants of hemoglobin is diverse and dependent on the position of the mutation, which can lead to changes in the structure of the globin chain or of the hemoglobin molecule. Hb Koln is the most common of the unstable variants of hemoglobin and was first described by Pribilla in 1962. One of the main characteristics of unstable hemoglobins is the presence of Heinz bodies, which was curiously observed in this patient's red blood cells, corroborating data already described in the literature. Thus, a reliable detection of Hb Koln together with genetic counseling and prenatal diagnosis are essential to improve the well-being of patients.

An 11-month-old Border collie presented collapsed and continued to deteriorate rapidly despite supportive treatment. The dog had a history of failure to thrive and recurring respiratory infection. Laboratory abnormalities included neutrophilic leucocytosis, Heinz body anaemia, hyperammonaemia, hyperbilirubinaemia, proteinuria and hypocobalaminaemia. Post-mortem examination revealed multi-focal necrosis within the heart, kidneys, pancreas, liver, meninges and cerebral cortex. Fungal hyphae in lesions were identified as Scedosporium prolificans following culture. Subsequent genotyping confirmed that the dog carried the CUBN:c.8392delC mutation in a homozygous state, verifying hereditary cobalamin deficiency (a.k.a. Imerslund-Grasbeck syndrome). Cobalamin deficiency may have been a predisposing factor for the development of systemic fungal infection in this dog.

In some hemoglobins variants substitution has altered the ability of the hemoglobin to remain in solution in the red cell. The precipitate material is detectable by supravital stain as dark aggregates called Heinz bodies. These intracellular inclusions reduce the life expectancy of the red cell and generate a hemolytic syndrome of varied severity called the congenital Heinz body hemolytic syndrome. Although the formation of methemoglobin might not be the only pathway by which hemoglobin denatures, it is certainly the most common, with the process actually beginning at the level of the iron or at the level of the globin. Heinz bodies, the hallmark of the congenital Heinz body hemolytic syndrome that we are discussing, are the product of hemoglobin denaturation. Hemoglobin is a tight globular protein, which means that the secondary structural arrangements characterizing each subunit must be packed into a solid sphere.

Zinc toxicosis is commonly associated with ingestion of metallic zinc objects. This report documents an uncommon presentation of zinc toxicosis, secondary to prolonged ingestion of a zinc oxide cream. A 5.6 kg 6-year-old ovariohysterectomised female Poodle cross presented as a transfer to an emergency practice for severe anaemia, pigmenturia, weakness, and inappetence. She had a history of vomiting and nine days of diarrhoea. Amongst other supportive treatments, a zinc oxide cream had been applied to her hind end due to severe diarrhoea scalding, and in hindsight, the owners realised she had ingested large quantities of this cream. She developed a severe Heinz body haemolytic anaemia, along with spherocytosis, left-shift neutrophilia, prolonged activated partial thromboplastin time, and mildly elevated blood urea nitrogen. The serum zinc concentration was markedly elevated. She was treated supportively and made a full recovery. This case illustrates the importance of a thorough medical history. Zinc toxicosis can have a good prognosis when diagnosed and treated promptly.Keywords: Canine, Haemolytic anaemia, Heinz bodies, Toxicosis, Zinc

We report a case of a 91-year-old Caucasian woman with a history of chronic lymphocytic leukaemia who developed acute hypoxic respiratory failure (AHRF) requiring intubation for less than 24 hours after receiving rasburicase. Laboratory workup was significant for methemoglobinemia and acute anaemia, and blood film demonstrated evidence of oxidative haemolysis with bite cells. The patient was given a presumptive diagnosis of glucose-6-phosphate dehydrogenase (G6PD) deficiency and was managed conservatively with successful resolution of AHRF and stabilisation of haemoglobin level. Seven days after admission, she passed away due to subsequent complications; hence, follow-up G6PD level could not be obtained. Haemolytic anaemia and methemoglobinemia in the setting of recent rasburicase administration should raise clinical suspicion for G6PD deficiency. In non-emergent cases, patients should be screened prior to receiving rasburicase regardless of risk factors. Because rasburicase is often needed emergently, patients at high risk of tumour lysis syndrome should be screened early for G6PD deficiency.

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H2O2, but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H2O2 or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.