Breed-Specific Hematological Phenotypes in the Dog: A Natural Resource for the Genetic Dissection of Hematological Parameters in a Mammalian Species

Remarkably little has been published on hematological phenotypes of the domestic dog, the most polymorphic species on the planet. Information on the signalment and complete blood cell count of all dogs with normal red and white blood cell parameters judged by existing reference intervals was extracted from a veterinary database. Normal hematological profiles were available for 6046 dogs, 5447 of which also had machine platelet concentrations within the reference interval. Seventy-five pure breeds plus a mixed breed control group were represented by 10 or more dogs. All measured parameters except mean corpuscular hemoglobin concentration (MCHC) varied with age. Concentrations of white blood cells (WBCs), neutrophils, monocytes, lymphocytes, eosinophils and platelets, but not red blood cell parameters, all varied with sex. Neutering status had an impact on hemoglobin concentration, mean corpuscular hemoglobin (MCH), MCHC, and concentrations of WBCs, neutrophils, monocytes, lymphocytes and platelets. Principal component analysis of hematological data revealed 37 pure breeds with distinctive phenotypes. Furthermore, all hematological parameters except MCHC showed significant differences between specific individual breeds and the mixed breed group. Twenty-nine breeds had distinctive phenotypes when assessed in this way, of which 19 had already been identified by principal component analysis. Tentative breed-specific reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis. This study represents the first large-scale analysis of hematological phenotypes in the dog and underlines the important potential of this species in the elucidation of genetic determinants of hematological traits, triangulating phenotype, breed and genetic predisposition.     

Nondiscrete heterogeneity of human erythrocytes: comparison of Coulter-principle flow cytometry and Soret-hemoglobinometry image analysis

In the blood of normal subjects, the volumes of single erythrocytes are distributed with a coefficient of variation (CV) of 10.8 +/- 1.8%; while in hemoglobinopathies, CV increases proportionately to the degree of anemia produced. Using single cell Soret-band hemoglobinometry and focused-aperture impedance counting, we compared the distribution of red cell volume, area, hemoglobin content, and hemoglobin concentration in normals and subjects with anemic disorders. The CV, nondiscrete heterogeneity, is first, a general characteristic of biologic measurement, second, a sensitive indicator of abnormality of erythropoiesis, and third, consistently less for hemoglobin concentration than for volume, area, or hemoglobin content of the same cells.     

Renal extraction of glutamine from plasma and whole blood: studies in dogs and rats

The change in plasma and blood cell pools of L-glutamine during a single pass through the kidney was studied in dogs and rats. It was shown that the glutamine content of blood cells does not change following one passage through the renal vascular bed in normal or acidotic dogs. Furthermore, an infusion of L-glutamine elevating by 10-fold the plasma concentration of this amino acid only minimally changed the blood cells' glutamine content. Therefore within the time frame of acute experiments, the dog blood cells can be assumed to be impermeable to glutamine in vivo. Accordingly, renal glutamine extraction can be measured using either whole blood or plasma arteriovenous difference in this species. However, the latter value is larger and therefore can be measured more accurately. In normal rats, no net renal glutamine extraction is measured. In contrast, a considerable renal glutamine uptake occurs in acidotic rats, 23% of the extracted glutamine coming from the blood cell pool. A load of glutamine in vivo significantly elevates both the plasma and the blood cell concentration. It is concluded (i) that the renal extraction of glutamine is best estimated using plasma arteriovenous difference in the dog, especially when the renal extraction is small; (ii) that whole blood measurements should be obtained in the rat.     

In vitro hydrolysis of RR,SS-threo-methylphenidate by blood esterases--differential and enantioselective interspecies variability

Enantioselective in vitro hydrolysis of methylphenidate (MPH) by the blood esterases of seven mammalian species is reported. The species included rats, rabbits, dogs, cattle, horses, monkeys, and humans. In vitro incubations up to 8 h were carried out in plasma, red blood cells, and whole blood of the various species. Enantioselective differences were evident among the different species on comparison of the data obtained from the three biological fluids. The esterases present in plasma appeared to show greater activity in the hydrolysis of MPH in all species where comparison with the other two biofluids was possible. Only in the case of humans did esterases present in plasma and red blood cells demonstrate opposite enantioselectivity in the hydrolysis of MPH. Thus after 8 h incubation, the RR-MPH/SS-MPH ratios in plasma and red blood cells were 0.31 and 1.16, respectively.     

Dielectric Properties and Ion Mobility in Erythrocytes

The impedance of erythrocytes of man, cattle, sheep, dog, cat, rabbit, and chicken was measured in the range from 0.5 to 250 Mc. The dielectric constant of the red cell interior is 50 at 250 Mc, varies but little with species, and can readily be accounted for by the cells' hemoglobin content. The electrical conductivity of the red cell interior was determined between 70 and 100 Mc. The values differ from species to species within the rather limited range from 4.4 to 5.3 mmho/cm. Removal of the cell membranes does not affect the conductivity. Hence, the cell interior behaves, from an electrical point of view, like a highly concentrated hemoglobin solution. A theoretical value for the electrical conductivity of erythrocyte interiors, which is calculated on the basis of the salt content of the cell, ion mobility, and the volume concentration of the hemoglobin, is roughly twice as large as the measured value. This discrepancy is typical not only of the red blood cell. Pertinent measurements show that it is probably caused by hydrodynamic and possibly by electrostatic effects also, which lower the mobility of the ions. From the lower electrical mobility it appears that a lowered diffusion constant of the electrolytes and nonelectrolytes within the cell is indicated.     

Lactate-transport activity in RBCs of trained and untrained individuals from four racing species

In red blood cells (RBC) of horses, both lactate-transport activity and lactate accumulation during races vary interindividually. To study whether similar variation in lactate transport is apparent also in RBCs of other racing species, blood samples were collected from 21 reindeer, 40 horses, 31 humans, and 38 dogs. Total lactate-transport activity was measured at 10 and 30 mM concentrations, and the roles of the monocarboxylate-transporter (MCT) and the inorganic anion-exchange transporter (band-3 protein) were studied with inhibitors. In the reindeer and in one-third of the horses, lactate transport was low and mediated mainly by band-3 protein and nonionic diffusion. In the humans, dogs, and the remaining two-thirds of the horses, lactate transport was high and MCT was the main transporter. No correlation existed between MCT activity and the athleticism of the species. In the horses and humans, training had no effect on lactate transport, but in the reindeer and sled dogs, training increased total lactate transport. These results show that among the racing species studied, only in horses was the distribution of lactate-transport activity bimodal, and the possible connection between RBC lactate and performance capacity, especially in this species, warrants further studies.     

Comparative morphology and morphometry of alveolar macrophages from six species

Pulmonary alveolar macrophages (PAM) were collected from normal, healthy mice, rats, dogs, cynomolgus monkeys, chimpanzees, and humans and evaluated for morphologic and morphometric characteristics. The PAM of mice, rats, and dogs were morphologically similar and had statistically similar frequency distributions for size. The cell size distribution for these three species was relatively homogeneous. The PAM of nonhuman primates and humans were morphologically heterogenous with sometimes prominent cytoplasmic vacuolation, irregular cell outlines, and increased numbers of multinucleated cells as compared to the PAM of rodents and dogs. The mean size of human PAM was statistically greater than that for all other species evaluated, including nonhuman primates. These data indicate that significant differences in PAM morphology and size exist among species.     

Relations among variations in human red cell volume,density, membrane area,hemoglobin content and cation content

It is shown how variations in different properties of red cells can be inter-related provided relations exist among these properties at the single cell level. On the basis of the cell density dependence on cell volume and hemoglobin content, and the assumed volume dependence on red cell cation and hemoglobin content, nine relations among the variations in red cell volume, density, membrane area, hemoglobin content and cation content, and their correlations are derived. Values of seven correlation coefficients are theoretically predicted and are shown to be consistent with the experiments performed by density fractionated red blood cells. The cell volume dependence on cation and hemoglobin content obtained from relations among variations is compared with the predictions obtained by the existing model about the osmotic behavior of the red blood cell. Furthermore, it is shown that data on the variations of the red cell properties indicate the existence of the relation among cation content, hemoglobin content, and membrane area at the level of a single cell.     

Specific features of blood cell composition and structure in fishes from the pelagial and coastal zones of Lake Baikal

Differences in the proportions and ultrastructure of peripheral blood cells between the Baikal cisco and roach have been revealed. Both species contain all main cell types involved in the immune response in higher vertebrates. However, cells mediating the innate immune response are represented in the Baikal cisco only by monocytes and pseudogranulocytes, whereas the blood of roach additionally contains true granulocytes. Significantly greater numbers of leukocytes and thrombocytes per unit volume in the roach are evidence for mobilization of the immune system in this species. The peripheral blood of both species contains lymphocytes, which are responsible for adaptive immunity. The content of hemoglobin in erythrocytes is lower in the roach than in the Baikal cisco. The state of blood in the two species may be considered adapted to typical conditions in their habitats.     

Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.     

Profile of hematological values in hybrid hairless dogs

The purpose of this paper is to describe some fundamental physiological data in F 1 hybrids bred from a Mexican hairless dog and beagle cross. These F 1 hybrids numbered 5 hairless dogs and 12 haired dogs. The hematological profile of these offspring was assessed via an automated cell counter and compared with those of healthy beagles. In hairless dogs, red blood cell count, hemoglobin concentration and packed cell volume tended to be higher than in beagles. White cell distribution curves in hairless dogs and beagles yielded a single peak, while in haired dogs one or two peaks were present. Red blood cell and platelet distribution curves revealed few differences among the 3 kinds of dogs.     

Hemolysis and ATP Release from Human and Rat Erythrocytes under Conditions of Hypoxia: A Comparative Study

Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat erythrocytes. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5- and 2.5-fold, respectively. In rat erythrocytes a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL 67156. In these conditions we observed a positive correlation (R² = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia-induced ATP release from rat erythrocytes. The obtained results indicate a key role of hemolysis in the ATP release from red blood cells.     

The structure and function of plant hemoglobins.

Plants, like humans, contain hemoglobin. Three distinct types of hemoglobin exist in plants: symbiotic, non-symbiotic, and truncated hemoglobins. Crystal structures and other structural and biophysical techniques have revealed important knowledge about ligand binding and conformational stabilization in all three types. In symbiotic hemoglobins (leghemoglobins), ligand binding regulatory mechanisms have been shown to differ dramatically from myoglobin and red blood cell hemoglobin. In the non-symbiotic hemoglobins found in all plants, crystal structures and vibrational spectroscopy have revealed the nature of the structural transition between the hexacoordinate and ligand-bound states. In truncated hemoglobins, the abbreviated globin is porous, providing tunnels that may assist in ligand binding, and the bound ligand is stabilized by more than one distal pocket residue. Research has implicated these plant hemoglobins in a number of possible functions differing among hemoglobin types, and possibly between plant species.     

Interrelations among Na and K content,cell volume,and buoyant density in human red blood cell populations

Summary This study establishes a method for determining the concentration of Na and K in single red blood cells from electron probe microanalysis of a cell's Na and K content. To this end, red blood cells were separated into subpopulations according to their buoyant density by means of bovine serum density gradient centrifugation. Cell water and Na+K contents were then determined in each fraction by conventional analytic methods with cell volume estimated from measurements of hematocrits and cell number. It was found that an inverse relationship obtains between the mean cell volume and buoyant cell density since cells increased in size as density decreased. Although the amount of hemoglobin per cell was found to slightly increase as cell density decreased, hemoglobin concentration showed the inverse relationship, indicating that buoyant cell density differences are primarily the result of differences in hemoglobin concentration. In confirmation of Funder and Wieth (Funder, J., Wieth, J.O. 1966.Scand. J. Lab. Invest. 18:167–180) cell water and cell volume was found to vary directly with the summed content of Na+K. Finally, by means of electron probe microanalysis of single cells, the cellular concentration of hemoglobin was found to vary inversely with the Na+K content, providing a quantitative basis for directly estimating cell volume, and thus ionic concentration, with this technique.     

Co-expression of differentiation markers in hybrids between friend cells and lymphoid cells and the influence of the cell shape

We have studied the regulation of differentiation within the hemopoietic system by fusing mouse Friend cells (which can be induced to undergo red blood cell differentiation) to various mouse lymphomas and myelomas which express characteristic T and B lymphocyte surface antigens. Our results show that both erythroid and lymphoid differentiation markers can be co-expressed within the same cell. To determine whether this result applies to other differentiation states, we fused suspension Friend cells to three adherent fibroblast cell lines, and isolated both adherent and suspension hybrids. In fact, suspension hybrid clones were inducible for hemoglobin, whereas adherent clones were not. No obvious differences in overall chromosome balance were evident between the adherent and suspension hybrids. A similar correlation between suspension morphology and inducibility of hemoglobin was found in hybrids between suspension Friend cells and an adherent lymphoma line. These results show that different developmental programs can be coexpressed within the same hybrid cell; but the strongly adherent type of morphology is inconsistent with expression of the red blood cell phenotype, both in hybrid cells derived entirely from hemopoietic parental cells and in cells from widely different lineages.     

Appraisal of the methodology and applications for measurement of the zinc content of blood components as indicators of zinc status

Methods for the measurement of the zinc content of blood components (plasma, erythrocytes, platelets, mononuclear leukocytes, and polymorphonuclear neutrophils) as indicators of the zinc status of animals have been reviewed and evaluated. The values of plasma or serum zinc concentrations as indicators of zinc status in experimental animals or humans is questionable. Consequently, the zinc content of blood cellular components has been suggested as better indicators of zinc status. Methodological problems, such as incomplete cellular separation or zinc contamination, occur in some of the procedures applied to the quantitation of blood cellular component zinc. Specialized microprobe procedures involving, for example, proton-induced X-ray fluorescence, scanning ion technology, mass spectrometery, or laser microprobe mass analysis might be used to measure the absolute zinc level in individual cells subjected to minimal handling. Then, more conventional methods can be tested against the true standard. Nevertheless, there appears to be species differences in regard to the response of blood cellular zinc concentrations to dietary zinc deficiency. Blood cellular component zinc is conserved during severe zinc deficiency in the rat. In contrast, the zinc content of blood cellular components in humans may reflect the whole body zinc status.     

Preparing for winter: divergence in the summer-autumn hematological profiles from representative species of the squirrel family

We examined hematological parameters in four related sciurid species in the late summer-autumn to assess the role of habitat, elevation, body size, and behavior in shaping these parameters. Red squirrels (Tamiasciurus hudsonicus) and Arctic ground squirrels (Spermophilus parryii) were sampled in southwestern Yukon, yellow-pine chipmunks (Tamias amoenus) in southern Alberta, and the eastern grey squirrel (Sciurus carolinensis) in southern Ontario. We obtained whole blood samples from each species and compared glucose levels, red blood cell characteristics (hematocrit, red blood cell count, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration), and white blood cell counts (neutrophils, lymphocytes, monocytes, eosinophils, basophils) across species. We found species differences in glucose and red blood cell characteristics that may be a function of activity levels, phylogeny, or elevation, but not of body size, body condition, or adaptations to a semi-fossorial habitat. We also found species differences in white blood cell counts that remain unexplained by any single simple explanation and may be more useful for comparison of individuals within a given species than for interspecies comparisons.     

Study of maturation of membrane transport function in red blood cells by X-ray microanalysis

Summary Red blood cells of certain species of animals, such as dogs and cats, contain low potassium and high sodium, whereas the erythropoietic stem cells giving rise to these cells are of high potassium type. This paper examines the sequence of membrane transport changes during erythropoiesis by analyzing the K, Na and Fe in single bone marrow cells, reticulocytes and mature red blood cells with X-ray microanalysis. The relationship between K/Na ratios and Fe/(K+Na) ratios were examined by X-ray microanalysis. The K/Na ratios give a measure of the membrane cation transport function. The Fe/(K+Na), which is analogous to hemoglobin concentration, gives an index of maturation stage. The relationships between K/Na and Fe/(K+Na) in the marrow cells of normal adult dog and those of a phenylhydrazine-injected dog with accelerated erythropoiesis show that the modification of cation composition occurs after the initiation of hemoglobin synthesis but before its completion. Similar relationships in the reticulocytes obtained from phenylhydrazine-injected dogs as well as from newborn dogs show a consistent decrease in K/Na with increased Hb, indicating a drastic change in cation composition during the maturation of the reticulocytes. Therefore the modification in membrane transport function must have occurred before or during the formation of reticulocytes.     

Collection of hematopoietic stem cells from canine peripheral blood and their ability to regenerate hematopoiesis]

A procedure is presented for the collection of a large number of hemopoietic stem cells from the peripheral blood of dogs by means of a single leukapheresis using the NCI-IBM Blood Cell Separator. In the course of a leukapheresis of about 285 min duration a mean of 23 x 10-9 leukocytes is collected from the blood. The hemopoietic stem cells among such separated leukocytes initiate repopulation of bone marrow within 10 days after whole body X-irradiation with 1200 R. The cell numbers in a defined histological section of femoral bone marrow are evaluated 9 to 10 days after irradiation and subsequent autologous transfusion of 6.72 x 10-9 separated mononuclear leukocytes. The results indicate that the bone marrow cell numbers of transfused dogs are significantly greater than in dogs given only 1200 R and reach a level of approximately 49% of the normal value. Possible ways of increasing the yield of hemopoietic stem cells from the peripheral blood will be considered.