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.

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.

Copper toxicosis in a Boer goat.

A 1-year-old female Boer goat was presented with a 1-day history of pigmenturia, anorexia, and shivering. Anemia was not present initially, but progressive hemolytic anemia developed subsequently and was characterized by the finding of Heinz bodies in both intact RBCs and in ghost cells and the presence of atypical fusiform RBCs. Plasma biochemical analysis revealed increased activities of aspartate aminotransferase and gamma-glutamyltransferase, hyperbilirubinemia, and azotemia. Histopathologic examination of a liver biopsy revealed necrosis of individual hepatocytes and intracytoplasmic rhodamine-positive granules, consistent with copper. Copper concentration in ante-mortem hepatic tissue was increased, and a diagnosis of copper toxicosis was made. Despite supportive therapy, the goat continued to decline and was euthanized. Necropsy findings included hepatic necrosis and hemoglobinuric nephrosis. Freshly collected specimens of liver and kidney had markedly increased copper concentrations. The mineral composition of the water, grass hay, and goat chow was evaluated, and toxins and significant mineral imbalances were not found. The underlying cause of the hepatic accumulation and subsequent release of copper remains unclear in this goat. Recently, Boer goats have been recognized as being prone to copper toxicosis and may be more susceptible than other breeds; similar to sheep, Boer goats may experience a hemolytic crisis secondary to copper toxicosis.

Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies.

The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species.

Hb Sitia [beta128(H6)Ala-->Val]: an unstable variant with a substitution in the alpha1beta1 interface.

Hb Sitia [beta128(H6)Ala-->Val] was found in a Greek female with slightly reduced red blood cell indices. The abnormal hemoglobin was indistinguishable from Hb A by electrophoresis but eluted after Hb A on cation exchange high performance liquid chromatography. DNA sequence analysis revealed a GCT-->GTT mutation at codon 128, which is predicted to encode an Ala-->Val substitution. This was confirmed by mass spectrometry analyses of the beta-globin chain. Since alanine at beta128(H6) interacts with several amino acids of the alpha1beta1 contact, its replacement by a larger residue results in a mild instability of the molecule and slight modifications of the oxygen binding properties.

Hb Hammersmith [beta 42(CD1) Phe-->Ser]: occurrence as a de novo mutation in black monozygotic twins with multiple congenital anomalies.

PURPOSE: To present the occurrence of Hb Hammersmith as a de novo mutation in African-American twins with multiple congenital anomalies. METHODS: Standard hematologic methods were used. The presence of an unstable Hb variant was confirmed by brilliant cresyl blue staining and an isopropanol stability test. Hb Hammersmith was confirmed by the sequencing of polymerase chain reaction-amplified beta-globin gene. RESULTS: The presence of Hb Hammersmith was confirmed in female monozygotic twins of African-American origin with congenital Heinz body hemolytic anemia and multiple congenital anomalies. The variant occurred as a de novo mutation in the twins. CONCLUSION: This report describes the occurrence of Hb Hammersmith [B42(CD1)Phe-->Ser] in African-American twins. As with the other reported cases, both twins were female. In addition to Heinz body hemolytic anemia, a low arterial O2 saturation in the proposita was shown by pulse oximetry. Multiple congenital anomalies involving various systems were also found in both twins.

Free radical erythrocyte damage in tumoral disease assessed by He-Ne laser and optical microscope through "Heinz Bodies" method.

Results obtained from blood sample readings by optical microscopy and He-Ne laser (lambda = 630.1 mW), have confirmed the reduction in Heinz Bodies (HB) formation time and the Transmittance Reduction Degree (TRD), in malignancies. The results of spectrometric readings in colorectum polyposis, (TRD = 0.07) and fibrocystic mastopathy (TRD = 0.08) gave results overlapping with controls (TRD = 0.08). In neoplasias, the early HB formation in erythrocytes observed by optical microscope corresponded to TRD increase = 0.17 (P < 0.01). TRD increase was statistically significant (P < 0.01), as well as the reduction in the time of HB appearance (< 0.01). The relationship between optical and laser readings was exponential in tumors, while it was linear in controls, in polyposis and mastopathies. The values of the correlation coefficients obtained by both methods were significant (P < 0.01) for all the studied groups. Moreover, these research data further support the existence, even in the earlier stages of the disease, of the labile state of the red cell membrane due to strong lipid peroxidation by FRs.

Hb Bibba or alpha 2 136(H19)Leu-->Pro beta 2 in a Caucasian family from Alabama.

Several members of a large Caucasian family who presented with a congenital Heinz body hemolytic anemia were found to be carriers of the unstable Hb Bibba or alpha 2 136(H19)Leu-->Pro beta 2. Identification by protein analysis was hampered by the instability of the variant which complicated its isolation from shipped blood samples. Moreover, the detection of the CTG-->CCG mutation at codon 136 of the alpha 2 gene required the substitution of dGTP by dITP during the DNA sequencing process to prevent the occurrence of secondary structures and compressions in the sequencing gel. The first Hb Bibba heterozygote, characterized in 1968 (1), is believed to be a member of this family. The clinical expression of the disease is surprisingly variable.

Comparative toxicity of alkyl-1,4-naphthoquinones in rats: relationship to free radical production in vitro.

2-Methyl-1,4-naphthoquinone causes haemolysis in vivo. This toxic effect is believed to result from oxidative damage to erythrocytes by "active oxygen" species formed via one-electron reduction of the naphthoquinone by oxyhaemoglobin. In the present investigation, seven 2-alkyl-1,4-naphtoquinones have been studied with regard to their haemolytic activity in rats, their ability to cause oxidative damage in erythrocytes in vitro, and their reactivity toward oxyhaemoglobin. A close correlation was observed between the in vivo and in vitro parameters, suggesting that the proposed mechanism of toxicity of 2-methyl-1,4-naphthoquinone is correct and is also applicable to other alkylnaphthoquinones.

An outbreak of Heinz body positive hemolytic anemia in chronic hemodialysis patients.

During the four month period, from December 1988 to March 1989, there was an outbreak of Heinz body positive hemolytic anemia in 34 patients undergoing hemodialysis in a 500-bed hospital, Seoul, Korea. The episodes of hemolysis were not reduced by changing the charcoal column and reverse osmosis system, or by adding ascorbic acid to the dialysate. The concentrations of nitrate, copper, aluminum and zinc in the treated water were all within the standards for hemodialysis. The chloramine concentration of the treated water was over 0.6 mg/L, markedly exceeding the allowable level of 0.1 mg/L. This high level of chloramine was proved to be due to the contamination of the water source by raw sewage. After we changed the source of water supply to another, no more episodes of hemolytic anemia occurred. It is concluded that chloramine is one of the major contaminants causing dialysis-induced hemolytic anemia and regular determinations are necessary, especially during winter and dry seasons.

[Superoxide dismutase activity of erythrocytes and their osmotic fragility]. / Aktywnosc dysmutazy ponadtlenkowej erytrocytów a ich opornosc osmotyczna.

Blood samples were obtained from 15 diabetic patients (type I) and 10 healthy subjects. Erythrocyte superoxide dismutase activity, Heinz bodies and osmotic fragility were determined. Our results suggest that decreased activity of erythrocyte SOD predisposes to denaturation of haemoglobin (Heinz bodies) and haemolysis (increased osmotic fragility).

Phosphine-mediated Heinz body formation and hemoglobin oxidation in human erythrocytes.

In this work the in vitro reactions of phosphine with intact red blood cells and membrane-free hemoglobin extracts are reported. We demonstrate that phosphine or phosphine derivatives induce dense aggregates of denatured hemoglobin known as 'Heinz bodies' in intact red blood cells. The reaction products include irreversible hemichrome formation. We further demonstrate an oxygen requirement for these effects. PH3 appears to act as a novel type of O2 radical chain initiator or propagator with heme proteins.

Erythrocyte pathology and mechanisms of Heinz body-mediated hemolysis in cats.

Despite the frequency of both oxidant drug-induced and spontaneous Heinz body formation in cats, the cellular and biochemical mechanisms by which Heinz bodies result in red blood cell (RBC) destruction and hemolytic anemia in this species remain unknown. Feline spleens are non-sinusoidal and inefficient at removing Heinz body-containing RBC from the circulation; therefore, alternative mechanisms must be involved in accelerated RBC destruction. Propylene glycol (PG) ingestion causes dose-dependent Heinz body formation and decreased RBC survival in cats. We investigated several aspects of Heinz body-containing RBC from three cats ingesting diets that provided 8.0 g PG/kg body weight for up to 3 weeks, in order to characterize cellular lesions that are associated with the presence of Heinz bodies and that might contribute to chronic, accelerated RBC destruction, as well as to gain insight into the mechanism by which PG induces Heinz body formation. Erythrocytes with PG-induced Heinz bodies had decreased levels of reduced glutathione and adenosine triphosphate and reduced deformability. There was no change in hemoglobin isoelectric focusing or membrane lipid peroxidation. Electrophoretic patterns of Heinz body-containing RBC membranes were significantly altered, and membrane surface charge distribution was disturbed. Progressively protruding Heinz bodies suggested that extrusion of Heinz bodies may be a means of cell healing and/or destruction in the absence of splenic pitting. When compared to results obtained using RBC from cats treated with the oxidant drug phenylhydrazine, significant differences were noted in packed cell volume, turbidity index, membrane heme, and morphologic appearance of Heinz bodies. Our results indicate that multiple cellular abnormalities develop in RBC with PG-induced Heinz bodies that do not cause acute hemolysis but that may shorten RBC survival. Propylene glycol-induced Heinz bodies provide an ideal model for studying the chronic effects of Heinz bodies on RBC structure and function and may be useful in understanding the mechanisms of formation and the consequences of endogenous Heinz bodies in cats.

[Does induced Heinz body formation lead to the liberation of IgG receptors on the erythrocyte membrane?]. / Führt induzierte Heinz-Körper-Bildung zur Freisetzung von IgG-Rezeptoren an der Erythrozytenmembran?

Immunofluorescence microscopical and biochemical studies led other authors to the conclusion that the formation of membrane-bound Heinz bodies at these parts of the plasmalemma of erythrocytes leads to clustering of band 3-protein and increased binding of IgG. We failed to detect immunocytochemically an increased IgG binding over phenylhydrazine induced membrane-bound Heinz bodies in otherwise intact erythrocytes using transmission electronmicroscopy and we also did not notice any clustering of intramembraneous particles over the numerous Heinz bodies by means of freeze etching. Especially at higher phenylhydrazine concentrations the erythrocytes show formation of vesicles and hemolysis accompanied by increased IgG binding and clustering of the intramembraneous particles. Photographs of such cells obtained by immunofluorescence microscopy are very similar to the pictures known from the literature.

Sensitivity of erythrocytes to oxidant stress in uremia.

The erythrocytes from 19 chronic hemodialysis patients were examined for Heinz bodies and their sensitivity to oxidant stress. Heinz bodies were found in 63% of patients and an elevated level of oxidized hemoglobin in 36%. When exposed to acetylphenhydrazine oxidant stress, 84% had a normal response and 95% had stable reduced glutathione levels. Ascorbic-acid-induced oxidant stress was tolerated by 84%. The activities of enzymes associated with the hexose monophosphate shunt were examined and found to be intact. This study demonstrates an increased number of Heinz bodies in hemodialysis patients. However, this is not due to an increased sensitivity to oxidant stress. Other mechanisms must be sought to explain the presence of Heinz bodies in these patients.

An unusual Heinz body anaemia in a cat.

A severe anaemia was diagnosed in a 7-month-old female cat. Most erythrocytes contained unusually large Heinz bodies and showed marked distortion. Six weeks later, the cat had recovered from the anaemia, but the erythrocytes still contained smaller inclusions. By light microscopy, these inclusions did not stain as typical Heinz bodies, but, by electron microscopy, Heinz bodies and autophagocytic vacuoles were identified. It is suggested that this cat had a pre-existing defect in its haemoglobin which made it more susceptible to damage by an unknown oxidizing agent.

Impaired antioxidant defense in hemoglobin E-containing erythrocytes: a mechanism protective against malaria?

Red blood cell (RBC) antioxidant defense was investigated in eight individuals with hemoglobin E (Six EE and two E-B(+) thalassemia) and compared to that in six individuals with thalassemia and ten normal subjects. Individuals with hemoglobin E had increased incubated Heinz body formation (68% +/- 18%; p less than 0.001) compared to normal and thalassemic RBC (10% +/- 2% and 11% +/- 5%, respectively). Stimulated pentose phosphate shunt activity was increased in the thalassemic and decreased in the hemoglobin E RBC as compared to normal. The 2,3-diphosphoglycerate (DPG) content of the EE RBC was increased to 5.59 +/- 0.69 mumol/ml RBC as compared to normal (4.51 +/- 0.77; p less than 0.001). In the EE RBC, there was a direct correlation between Heinz body formation and DPG content (r = 0.73). Ascorbic and dehydroascorbic acid (0.1 and 1.0 mM) were able to decrease the degree of Heinz body formation in the hemoglobin E RBC. Ascorbic acid (0.1 mM) prolonged the response of the pentose shunt. Thus impaired antioxidant defense may account for the persistence of the hemoglobin E gene in areas where malaria is endemic. Oxidant medications should be used with caution in individuals of Southeast Asian origin.