Infantile pyknocytosis, a neonatal hemolytic anemia with Heinz bodies: A cohort study.

Infantile pyknocytosis (IP) is a rare, probably misestimated, cause of non-immune neonatal hemolytic anemia evolving in two phases: an initial phase with severe jaundice, followed by a second phase with hemolytic anemia, which may require neonatal intensive care. The diagnosis of IP is based on the transient presence on blood smear of hyperdense, contracted, and/or spiculated red blood cells (pyknocytes), associated with the spontaneous resolution of clinico-biological features and the exclusion of other causes. If the etiology remains undetermined, some contributing factors, such as oxidative stress, have been proposed. We report the description of 16 patients with IP aiming at clarifying the circumstances associated with the development of this acquired disorder. In the acute phase, the mean hemoglobin nadir and pyknocyte count were 7.8 g/dL and 11%, respectively, and strikingly, Heinz bodies were evident in 50% of the newborns, but in 100% after prolonged incubation (4 hours). A high proportion of Mediterranean or African ancestry was noted in newborns, as well as a significant number of peripartum events, such as respiratory distress. If the etiology of IP is certainly multifactorial, our series reinforces the role of oxidative stress, which may, at least in part, find origin in desaturation episodes in newborns.

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.

Severe Heinz body anemia and methemoglobinemia in a kitten with chronic diarrhea.

INTRODUCTION: 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.

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies.

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil-injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

DNA damage: beta zero versus beta plus thalassemia.

BACKGROUND: ß thalassemia results in an increase in the α to non-α chain ratio. Iron released from unpaired α chains in RBCs and that ensuing from regular transfusions is the major cause of cellular damage. The use of iron chelators to counter the iron overload is accompanied by side-effects. The extent of iron toxicity could vary from one patient to another and could help in determining the optimal chelator dose for each patient. AIM: To observe the pro-oxidant/antioxidant disturbance and the extent of DNA damage in ß thalassemia patients with different ß globin gene anomalies. METHODS: The formation of Reactive Oxygen Species (ROS ) was observed by incubation of cell suspensions with 2',7', dichlorofluorescin-diacetate (DCFH DA) and DNA damage was demonstrated by single cell gel electrophoresis. Heinz bodies were observed by staining blood smears. SUBJECTS: The study group comprised 50 regularly transfused beta thalassemia patients and 40 non thalassemic controls. RESULTS: While Heinz bodies and nucleated RBCs were seen in all the patients, oxidation of DCFH and DNA damage were seen to be associated with the ß globin gene defect. DNA damage was found to be greater in ß(0) homozygotes as compared to the ß(+) homozygotes, and was maximum in patients presenting with the 619 base pair deletion. CONCLUSION: In the present study, iron toxicity, as indicated by DNA damage, has been seen to vary in the patients. Thus, monitoring of the dose of iron chelators, according to the type of mutation in the beta globin gene, may help improve the compliance of beta thalassemics to chelation therapy and prevent side-effects in patients with beta plus mutations.

Hb Grand Junction (HBB: c.348_349delinsG; p.His117IlefsX42): a new hyperunstable hemoglobin variant.

Hyperunstable hemoglobinopathy (HUH) [dominantly inherited ß-thalassemia (ß-thal)] is a relatively rare form of congenital hemolytic anemia in which mutations occur in the genes encoding for α and ß chains, or both chains of the hemoglobin (Hb) molecule. We describe two Hispanic adolescents with a new unstable Hb variant (HBB: c.348_349delinsG; p.His117IlefsX42), resulting from a frameshift mutation at codons 115/116 of the ß-globin gene. Both patients also have a 3.7 kb deletion on one α gene, leading to a decreased imbalance between α and ß chain formation, and subsequently a milder phenotype than that seen in other hyperunstable Hb variants.

Hemolytic anemia and methemoglobinemia in a preterm baby as a complication of antenatal intraamnial injection of toluidine blue.

A late preterm infant was born 4.5 h after intraamniotic injection of 90 mg of Toluidine blue to confirm premature rupture of membranes. Due to the fetal exposition to the dye, the entire body of the patient was blue stained and the baby suffered from methemoglobinemia, Heinz' body positive hemolytic anemia and hyperbilirubinaemia requiring exchange transfusion. These complications underline that antenatal exposition of toluidine blue may result in considerable postnatal infant morbidity. Therefore intraamniotic application of toluidine blue should be discouraged.

Feline Heinz bodies interfering in standard bicarbonate analysis by the ABL90 FLEX.

Venous blood gases were analyzed with ABL90 FLEX in two cats with Heinz bodies in approximately 60% of the erythrocytes. The instrument demonstrated an inability to correctly report standard bicarbonate (stHCO3 - ), hematocrits, and hemoglobin concentrations by indicating an OXI spectrum mismatch alarm (ie, the spectrum of measured hemoglobin forms differed from the spectrum of calculated forms). Actual bicarbonate (aHCO3 - ) did not indicate any errors. The ABL90 FLEX uses spectrophotometry to measure hemoglobin, and the presence of Heinz bodies interfered with the measurement in these cases. Because hemoglobin is included in the formula for calculating stHCO3 - , the instrument gave an alarm for stHCO3 - . At follow-up, Heinz bodies were present in only 2%-3% of the erythrocytes, and the ABL90 FLEX did not indicate any alarm messages. To the authors' knowledge, these are the first cases reported that have interference in stHCO3 - measurements due to Heinz body formation using the ABL90 FLEX, a common blood gas instrument used in both veterinary and human critical care. The methodology used for evaluating acid-base status should be taken into consideration, and caution is needed when interpreting acid-base results in cats with Heinz bodies.

Copper-associated hepatic cirrhosis in a Friesian horse.

A 6-year-old Friesian stallion was examined because of signs of exercise intolerance, stiff gait and symmetrical hind weakness, and increased serum liver enzymes. On presentation, the horse showed muscle atrophy of the hindquarters. Neurological investigation showed no abnormalities. Laboratory findings revealed a prolonged prothrombin time and increased levels of alkaline phosphatase (AF), aspartate aminotransferase (ASAT), gamma-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), and bile acids. Histological evaluation of the liver revealed severe cirrhosis and intracytoplasmic greyish brown granules in almost all hepatocytes, sinusoidal Kuppfer cells, and macrophages. These granules stained strongly for copper. Treatment to slow hepatic fibrosis was advised and included oral prednisolone administration for at least 1 month. A diet to support liver function was formulated by a nutritional specialist, and vitamin E was advised as dietary supplement to support neuromuscular function. Soon after diagnosis, the animal showed signs of intravascular haemolysis, with the presence of Heinz bodies in peripheral blood smears, and haemoglobinuria. On the basis of this haemolytic crisis and the poor prognosis of the chronic hepatic disease, the horse was euthanized at the owners' request. Although we could not establish the cause of the hepatic copper accumulation, this case report highlights that excessive copper in the liver should be considered in the differential diagnosis of hepatic cirrhosis and Heinz body anaemia in the horse.

Copper Toxicity in Horses: Does it Exist?

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.

Partial glutathione reductase deficiency as a cause of diverse clinical manifestations in a family with unstable hemoglobin (Hemoglobin Haná, ß63(E7) His-Asn).

Hemoglobin Haná [ß63(E7) His-Asn] is an unstable hemoglobin variant that was described in a Czech proband and her sister with Heinz body hemolytic anemia. The mother bearing the same mutation was asymptomatic; nevertheless, all three carriers had the same proportion of the mutant globin chains. Assessment of several erythrocyte antioxidant parameters revealed that both symptomatic children, unlike their asymptomatic mother, had significantly decreased glutathione reductase (GR) activity. Their GR activities were restorable in vitro by flavin adenine dinucleotide. The riboflavin supplementation improved their glutathione metabolism and ameliorated their hemolysis. Pre- and post-treatment assessment of the B(2) vitamers indicated suboptimal pre-treatment vitamin B(2) status in both children. This study provides evidence that partial GR deficiency may alter the clinical manifestation of an unstable hemoglobinopathy.

Molecular biosensing mechanisms in the spleen for the removal of aged and damaged red cells from the blood circulation.

Heinz bodies are intraerythrocytic inclusions of hemichrome formed as a result of hemoglobin (Hb) oxidation. They typically develop in aged red cells. Based on the hypothesis that hemichrome formation is an innate characteristic of physiologically normal Hb molecules, we present an overview of our previous findings regarding the molecular instability of Hb and the formation of hemichrome, as well as recent findings on Heinz body formation within normal human erythrocytes. Human adult Hb (HbO(2) A) prepared from healthy donors showed a tendency to produce hemichrome, even at close to physiological temperature and pH. Recent studies found that the number of Heinz bodies formed in red cells increased with increasing temperature when freshly drawn venous blood from healthy donors was subjected to mild heating above 37 °C. These findings suggest that Hb molecules control the removal of non-functional erythrocytes from the circulation via hemichrome formation and subsequent Heinz body clustering. In this review, we discuss the molecular biosensing mechanisms in the spleen, where hemichrome formation and subsequent Heinz body clustering within erythrocytes play a key role in the removal of aged and damaged red cells from the blood circulation.

Spurious, marked leukocytosis in 2 cats with Heinz body hemolytic anemia.

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.

Heinz-body hemolytic anemia from the ingestion of crude oil: a primary toxic effect in marine birds.

Hemolytic anemia developed in young herring gulls and Atlantic puffins given daily oral doses of a Prudhoe Bay crude oil. Anemia developed 4 to 5 days after the initiation of oil ingestion and was accompanied by Heinz-body formation and a strong regenerative response. The data evince a toxic effect on circulating red blood cells involving an oxidative biochemical mechanism and the first clear evidence of a primary mechanism of toxicity from the ingestion of crude oil by birds.

Virus-induced hemolysis in erythrocytes deficient in glucose-6-phhosphate dehydrogenase.

Red cells from individuals deficient in glucose-6-phosphate dehydrogenase undergo increased autohemolysis when incubated in the presence of influenza-A virus. Normal red cells, but not those from individuals deficient in glucose-6-phosphate dehydrogenase, show increased activity of the hexose monophosphate shunt in the presence of the virus. This increase in shunt activity appears to be related to oxidation of cellular sulfhydryl groups.