Genetic diagnosis of band 3 deficiency and sexing in bovine preimplantation embryos

Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3(Bov.Yamagata), is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3(Bov.Yamagata). These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals.     

维生素E缺乏对雏鸡红细胞膜蛋白组分的影响

用低维生素E和添加不饱和脂肪酸的日粮饲喂雏鸡,建立维生素E缺乏实验动物模型。采用SDS-PAGE凝胶电泳法检测雏鸡红细胞膜蛋白组分变化。结果表明:维生素E缺乏组雏鸡红细胞膜蛋白带Ⅰ、Ⅱ含量高于对照组,带Ⅲ、Ⅴ、Ⅵ含量低于对照组,组间差异显著或极显著,带Ⅳ无显著变化。提示VE对维持雏鸡红细胞膜结构起重要作用。     

Studies on vitamin E and selenium deficiency in young pigs. II. The hydrogen peroxide hemolysis test and the measure of red cell lipid peroxides as indices of vitamin E and selenium status.

The usefulness of the hydrogen peroxide hemolysis test and the measure of red cell lipid peroxides as indices of vitamin E and selenium deficiency in swine has been evaluated. Results indicated that although the hydrogen peroxide hemolysis test may be of some indication of the vitamin E status, it is not a reliable index of vitamin E deficiency in swine, at least on an individual basis. In contrast, the measure of red cell lipid peroxides can be considered a reliable test for vitamin E deficiency in swine. The hydrogen peroxide hemolysis test and the red cell lipid peroxides were not significantly affected by selenium deficiency.     

Acute intravascular hemolytic anemia in the black rhinoceros: hematologic and immunohematologic observations

To investigate the syndrome of acute intravascular hemolytic anemia in the black rhinoceros (Diceros bicornis), laboratory techniques used in the differential diagnosis of hemolytic anemias were performed on blood samples from 6 black rhinoceroses: 3 nonrelated healthy rhinoceroses, 1 rhinoceros with iron deficiency anemia, and 2 rhinoceroses with intravascular hemolysis. Osmotic fragility, erythrocyte membrane protein composition, hemoglobin electrophoresis, and hemoglobin stability did not distinguish between healthy and affected (anemia or hemolysis) rhinoceroses. Polyclonal antiglobulin reagents were prepared in rabbits, using whole rhinoceros serum and purified rhinoceros immunoglobulin G. These reagents were nonreactive against erythrocytes of the healthy and iron-deficient rhinoceroses. Reactions with RBC from the rhinoceros with fatal hemolytic anemia indicated increased membrane coating by the third component of complement; this was not evident in a second rhinoceros that survived a hemolytic event.     

In vitro lysis and acute transfusion reactions with hemolysis caused by inappropriate storage of canine red blood cell products

Background: Transfusion of red blood cell (RBC) products carries considerable risk for adverse reactions, including life‐threatening hemolytic reactions. Objective: To report the occurrence and investigation of life‐threatening acute transfusion reactions with hemolysis in dogs likely related to inappropriate blood product storage. Animals: Four dogs with acute transfusion reactions and other recipients of blood products. Methods: Medical records were reviewed from 4 dogs with suspected acute hemolytic transfusion reactions after receiving RBC products at a veterinary clinic over a 1‐month period. Medical records of other animals receiving blood products in the same time period also were reviewed. Blood compatibility and product quality were assessed, subsequent transfusions were closely monitored, and products were diligently audited. Results: During or immediately after RBC product transfusion, 4 dogs developed hemolysis, hemoglobinuria, or both. Two dogs died and 1 was euthanized because of progressive clinical signs compatible with an acute hemolytic transfusion reaction. Blood type and blood compatibility were confirmed. RBC units from 2 blood banks were found to be hemolyzed after storage in the clinic's refrigerator; no bacterial contamination was identified. After obtaining a new refrigerator dedicated to blood product storage, the problem of hemolyzed units and acute transfusion reactions with hemolysis completely resolved. Conclusions: Acute life‐threatening transfusion reactions can be caused by inappropriate storage of RBC products. In addition to infectious disease screening and ensuring blood‐type compatibility, quality assessment of blood products, appropriate collection, processing, and storage techniques as well as recipient monitoring are critical to provide safe, effective transfusions.     

An in vitro evaluation of storage media for the preservation of canine packed red blood cells

The effect of four different red blood cell storage media on in vitro parameters of stored canine red blood cells was studied. The storage media included citrate-phosphate-dextrose-adenine (CPDA-1), two additive solutions, and an additive solution modified by the addition of plasma. Biochemical and hematologic parameters, including red cell adenosine triphosphate (ATP); 2,3-diphosphoglycerate (2,3-DPG); pH; percent hemolysis; and supernatant sodium, potassium, and glucose were assessed immediately following preparation of the red cell concentrate and after 35 and 42 days of storage at 4 degrees C. All parameters changed significantly (p < 0.05) during storage. Significant differences due to effect of the storage media were also seen at each time period. After 35 days and 42 days of storage, CPDA-1 maintained the highest pH, potassium, and sodium values, and had the lowest 2,3-DPG, ATP (p=0.052), and glucose values. No differences were seen in hemolysis after 35 days of storage. No additional benefit was noted from the addition of plasma to the additive solution. The additive solutions compared favorably with CPDA-1.     

Surface ultrastructure of pyruvate kinase-deficient erythrocytes in the Basenji dog.

The scanning electron microscope was used to study the surface morphologic features of erythrocytes from a Basenji dog with hereditary hemolytic anemia due to a pyruvate kinase-deficient erythrocytes (RBC). Cells from this dog were compared with RBC from normal dogs and from dogs with regenerative anemias unrelated to pyruvate kinase deficiency. Demonstration of spherical RBC with uniform spicules on their surface (spheroechinocytes) may provide a morphologic explanation for the shortened erythrocytic life-span previously reported in congenital hemolytic anemia of Basenji dogs. Spherical, spiculated RBC were not found in blood from normal dogs or from anemic dogs with reticulocytoses. The surface of reticulocytes from all dogs with regenerative anemia was roughened, with pronounced folding of the cell membrane.     

两种鲫鱼血液红细胞测定与分析

为了解普安鲫鱼和草海鲫鱼血液生理特征,试验采用血涂片和血细胞计数方法分别测定其红细胞及核大小、红细胞数量和形态。结果表明:草海鲫鱼血液中存在一定数量核正处于分裂期的长椭圆红细胞;草海鲫鱼红细胞长径、短径和长短径比均明显大于普安鲫鱼,而红细胞核的短径则显著小于普安鲫鱼,虽然其红细胞的体积、核的长径和核的长短径比略大于普安鲫鱼,但差异不显著;2种鱼红细胞数量分别为(1.31±0.12)×1012个/L和(1.02±0.37)×1012个/L,草海鲫鱼红细胞数量大于普安鲫鱼。综合分析可知,2种鱼所处地理环境不同,是造成其血液红细胞的形态、大小和数量存在差异的根本原因。本研究结果可为2种鲫鱼血液生理、血细胞及其免疫学、遗传育种提供基础资料。     

Exit of Anaplasma marginale from bovine red blood cells.

Incubation of Anaplasma marginale-infected bovine red blood cells in a flow culture system resulted in a decrease of observable marginal bodies. The decrease was twice the degree of hemolysis, indicating that marginal bodies may leave red blood cells without concomitant lysis of the host cell.     

The life span of glutathione-deficient red cells in Tasmanian Merino sheep.

59FeCl3 was used to measure red cell life span in GSH-low type sheep to the Tasmanian Merino breed, whose GSH deficiency is due to a diminished activity of gamma-glutamyl cysteine synthetase. The 59Fe survival slopes fell within normal limits, indicating that red cells with this type of GSH deficiency do not have a shortened life span.     

Ultrastructural and clinicopathological studies on the toxicity of cationic acrylamide-based flocculant to rainbow trout

Rainbow trout (Salmo gairdneri) of two age groups were exposed to a cationic acrylamide-based flocculant at various concentrations in static bioassay chambers. At lethal concentrations the flocculant produced severe gill alterations in all fish. The principal alterations were necrosis and separation of the respiratory epithelial cells covering secondary lamellae. Many necrotic chloride cells were also seen, their apical plasma membrane was destroyed, and mitochondria were swollen with separated cristae. An influx of a large amount of fluid into the interstitial spaces caused partial or complete separation of subepithelial spaces from the covering epithelial cells and basement membranes of underlying blood vessels. Clinicopathological alterations included marked decreases in blood pH, partial pressure of oxygen, bicarbonate and plasma sodium, and chloride concentrations. Hematocrit, total protein, and blood glucose were increased. Fish exposed to sublethal concentrations had gill alterations characterized by hypercellularity and thickening of the secondary lamellae. These were due to undifferentiated cell proliferation and macrophage and lymphocyte infiltration between the covering epithelial cells and the underlying blood vessels. Macrophages and undifferentiated cells had large phagolysosomes containing cytoplasmic organelles, an indication of cell injury and increased turnover.     

Hemolysis Associated With Water Administration Using a Nipple Bottle for Human Infants in Juvenile Pygmy Goats

A 4-month-old, 6.8-kg, castrated male pygmy goat was examined for recurrent episodic fever and red urine of 7 days' duration. A second, 3-month-old, 7-kg, intact female pygmy goat was presented for similar clinical signs. The red discoloration of the urine in each case was determined to be due to hemolysis with subsequent hemoglobinuria. In both cases, hemolysis and hemoglobinuria were closely associated with the goats consuming large volumes of water from a human infant's nipple bottle. A diagnosis of water intoxication-induced hemolysis and hemoglobinuria was made. Episodes of hemoglobinuria in the first case were consistently associated with dilute (specific gravity < 1.010) urine. Water intoxication has been associated with bottle-feeding in human infants and is also widely reported in human psychiatric patients. The small erythrocytes in goats appear to be the most sensitive of the domestic species to hypotonicity-induced hemolysis.     

Rates of chlorpromazine-induced hemolysis in seven species of animals

Chlorpromazine (CPZ) affects the hemolytic process in vitro. Hemolysis rates induced by CPZ at 10^-3 M concentration were measured in blood from humans, monkeys, horses, cattle, goats, rats and dogs. Hemolysis data were plotted in the form of Lineweaver-Burke plots and the kinetics considered. The canine red cell was markedly more resistant than the other species as represented by a smaller V_max. Basal glutathione peroxidase levels of the red cell were assayed in an attempt to correlate the enzyme activity with the haemolysis rates in the species studied. A strong correlation would have explained the dog's high resistance and would have strongly indicated lipid peroxidation as the mechanism of CPZ-induced in vitro hemolysis. No such correlation was found.     

Cytologic interpretation of canine cerebrospinal fluid samples with low total nucleated cell concentration,with and without blood contamination

Background: Cerebrospinal fluid (CSF) is potentially altered by iatrogenic blood contamination at the time of sampling due to the addition of blood‐associated leukocytes and protein. Objectives: The objective of this study was to assess whether protein concentration, neutrophil percentage, and the presence of activated macrophages, reactive lymphocytes, or eosinophils in CSF samples with low total nucleated cell concentration (TNCC) are affected by blood contamination or associated with central nervous system (CNS) disease. Methods: Case records from the Royal Veterinary College Diagnostic Laboratory were searched retrospectively for dogs with CSF having ≤5 TNCC/μL. TNCC, RBC, and protein concentrations; neutrophil percentage; and the presence of activated macrophages, reactive lymphocytes, and eosinophils were recorded. Results of magnetic resonance imaging (MRI) also were recorded as a marker of CNS disease. Results: Of 906 cases evaluated, 106 (12%) had blood contamination (>500 RBCs/μL) in CSF. Protein concentration and neutrophil percentage were significantly higher and the presence of eosinophils was more likely in blood contaminated vs noncontaminated samples. Non‐blood‐contaminated samples with activated macrophages or reactive lymphocytes had higher protein concentrations and neutrophil percentages, and those with activated macrophages were more likely to have a positive finding on MRI. Conclusions: Protein concentration, neutrophil percentage, and the presence of eosinophils are significantly affected by blood contamination in canine CSF having low TNCC. Activated macrophages and reactive lymphocytes are not affected by blood contamination, however, and may be useful in identifying dogs with CNS abnormalities.     

Extreme lymphocytosis with myelomonocytic morphology in a horse with diffuse large B‐cell lymphoma

An 11‐year‐old, 443‐kg Haflinger mare was presented to the North Carolina State University Veterinary Teaching Hospital with a 2‐week history of lethargy and a 3‐day duration of anorexia, pyrexia, tachycardia, and ventral edema. Severe pitting edema, peripheral lymphadenopathy, and a caudal abdominal mass were noted on physical examination. An extreme leukocytosis (154.3 × 10³/μL) and microscopic hematologic findings suggestive of myelomonocytic leukemia were observed. Serum protein electrophoresis revealed a monoclonal gammopathy and urine protein electrophoresis revealed a monoclonal light chain proteinuria. Necropsy and histopathology confirmed widespread neoplastic infiltration in many organs with a heterogenous population of cells; there was no apparent evidence of bone marrow involvement. Immunohistochemistry confirmed presence of a majority of B cells with a limited antigen expression, admixed with a lower number of T cells. Molecular clonality analysis of IgH2, IgH3, and kappa‐deleting element (KDE, B cell) on whole blood and KDE on infiltrated tissues revealed clonal rearrangements, and the KDE intron clones that amplified in blood and in infiltrated tissue were identical. In contrast, the clonality analysis of T‐cell receptor γ revealed no clonality on blood cells and infiltrated tissues. In conjunction with the histopathologic changes, the lesion was interpreted to be composed of neoplastic B cells with a reactive T‐cell population. Polymerase chain reaction testing for equine herpes virus 5 was negative. The final diagnosis was diffuse large B‐cell lymphoma with a marked hematogenous component.     

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.     

樗树皮提取物对血液性质的影响

采用樗树皮三种不同提取物对血浆复钙时间、血液凝固时间以及溶血作用进行研究,以观察樗树皮提取物对上述各指标的影响.实验组与对照组相比较,提取物能缩短血液凝固时间（Ⅰ、Ⅲ：P＜0.001）,明显缩短血浆复钙时间（Ⅰ、Ⅲ：P＜0.001,Ⅱ：P＜0.01）,提取物Ⅱ、Ⅲ能引起红细胞轻度溶血,提取物Ⅰ不引起溶血.结果提示,提取物Ⅰ、Ⅲ能够改善凝血因子,并且有较强的凝血、止血作用,提取物Ⅰ可作为注射剂应用于临床.     

California mastitis test scores as indicators of subclinical intra-mammary infections at the end of lactation in dairy cows

Insect-borne diseases exact a high public health burden and have a devastating impact on livestock and agriculture. To date, control has proved to be exceedingly difficult. One such disease that has plagued sub-Saharan Africa is caused by the protozoan African trypanosomes (Trypanosoma species) and transmitted by tsetse flies (Diptera: Glossinidae). This presentation describes Trypanosoma evansi (T. evansi) which causes the disease known as trypanosomosis (Surra) or trypanosomiasis in which several attempts have being made to unravel the clinical pathogenic mechanisms in T. evansi infections, yielding various reports which have implicated hemolysis associated to decrease in life span of erythrocytes and extensive erythrophagocytosis being among those that enjoy prominence. T. evansi generates Adenosine Triphosphate (ATP) from glucose catabolism which is required for the parasite motility and survival. Oxidation of the erythrocytes induces oxidative stress due to free radical generation. Lipid peroxidation of the erythrocytes causes membrane injury, osmotic fragility and destruction of the red blood cell (RBC) making anemia a hallmark of the pathology of T. evansi infections.     

Effect of hemolysis on nonesterified fatty acid and beta-hydroxybutyrate concentrations in bovine blood.

Nonesterified fatty acid (NEFA) and beta-hydroxybutyrate (BHB) assays are used for evaluating dairy herds for negative energy balance and subclinical ketosis, respectively. Hemolysis is a common artifact in samples submitted to diagnostic laboratories. The effect of hemolysis on NEFA and BHB in bovine serum was determined. Hemolysis was introduced into 26 serum samples by adding serial dilutions of a red cell hemolysate, prepared by repeated freeze-thawing of EDTA-anticoagulated bovine blood. NEFA, BHB, and degree of hemolysis (hemolytic index) were measured by an automated chemistry analyzer. Two endpoint assays that differed by inclusion of a sample blank were used for NEFA measurement. A kinetic enzymatic assay with 2 reagent sources was used for BHB measurement. The assessed methods yielded similar NEFA or BHB results in baseline, nonhemolyzed samples (median NEFA: 0.25 mEq/L, median BHB: 3 mg/dL, median hemolytic index: 8 units). NEFA results were adversely affected by hemolysis, with values increasing significantly with higher degrees of hemolysis. Median values increased above a critical medical decision limit (0.40 mEq/L) at a hemolytic index of 506 units (marked hemolysis). This increase was prevented by inclusion of a sample blank. Result interpretation was affected in individual animals when samples were moderately hemolyzed (median hemolytic index: 258 units). In contrast, BHB results were unaffected by hemolysis with either reagent source. Thus, assays for measuring NEFAs should include a sample blank and NEFA results should not be interpreted in moderately to markedly hemolyzed bovine samples, because result accuracy cannot be assured.     

Evaluation of Canine Red Blood Cells Stored in a Saline, Adenine, and Glucose Solution for 35 Days

An additive solution for the storage of red blood cells was evaluated for use in dogs. Blood collected from 6 dogs was processed into packed red blood cells and stored for 35 days in the additive solution Nutricel (Miles, Inc, Pharmaceutical Division, West Haven, CT). Packed red blood cells stored in citrate-phosphate-dextrose-adenine (CPDA-1; Fenwal Laboratories, Baxter Health Care Corp, Deerfield, IL) also were evaluated for comparison. Red blood cell 2,3-diphosphoglycerate (2,3-DPG) concentration, adenosine triphosphate (ATP) concentration, percentage hemolysis, and pH were determined. The red blood cell post-transfusion viability (PTV) after 35 days of storage was assessed with both single-labeled chromium 51 (⁵¹Cr) and double-labeled technetium 99m/chromium 51 (^99mTc/⁵¹Cr) techniques. Mean ATP concentration and percentage hemolysis of the cells stored in Nutricel were 1.1 μmol/g hemoglobin (Hb) and 0.28% respectively and did not differ significantly (P < .05) from the values of 1.0 μmol/g Hb and 0.33% from the CPDA-1-stored red blood cells. The mean pH of red blood cells stored in Nutricel was 6.19, which was significantly lower than the pH of 6.47 for cells stored in CPDA-1. The mean 2,3-DPG concentration of red blood cells stored in Nutricel was significantly higher at 10.1 μmol/g Hb than the 2,3-DPG concentration of 3.4μmol/g Hb for cells stored in CPDA-1. The mean PTV of canine red blood cells stored in Nutricel for 35 days was 85% with ⁵¹Cr and 90% with ^99mTc/⁵¹Cr. This was significantly higher than the mean PTVs of 38% and 36% for the CPDA-1 stored cells as assessed with ⁵¹Cr and ^99mTc/⁵¹Cr techniques, respectively. It was concluded that 35-day-old canine red blood cells stored in Nutricel are of acceptable quality for transfusion purposes.