Effect of immune-mediated erythrocyte agglutination on analysis of canine blood using a multichannel blood cell counting system

Blood from six dogs with in vitro immune-mediated erythrocyte agglutination resulted in analytical errors in directly measured counting and sizing functions on a multichannel blood analysis system with histogram capability. Errors in the directly measured values, mean cell volume (MCV), and erythrocyte count were attributed to agglutinated erythrocyte particles that persisted during the relatively short reagent contact time of the analysis. Agglutinated particles less than 240 fl were visible on erythrocyte histograms and resulted in a false low erythrocyte count and false high MCV. Agglutinated cell particles greater than 240 fl were not present on the histogram scale. Because these latter particles exceeded the upper threshold, they did not influence determination of MCV, but resulted in a further decrease in the erythrocyte count. As a result, the other dependent erythrocyte indices were in error. These included false low hematocrit and false high mean corpuscular hemoglobin concentration (MCHC), when compared to corrected reference blood values. Similar errors occurred when analyzing blood samples that were agglutinated in vitro by incubating erythrocytes with incompatible plasma. The counting and sizing errors observed with electronic counting techniques were eliminated or greatly reduced by incubating blood in cell counting diluent for 10 minutes followed by analysis on a single channel counter with attached particle size analyzer. Error in erythrocyte measurement on a multichannel system may be anticipated if there is overt erythrocyte agglutination in a blood sample, an abnormally high MCHC is reported by the system, or subpopulations of large volume (agglutinated cells) are observed on a volume distribution histogram.     

Erythrocyte macrocytosis in feline leukemia virus associated anemia

Using erythrocyte volume distribution histograms (erythrograms), erythrocyte macrocytosis and anisocytosis were quantitated in 139 cats tested for feline leukemia virus group-specific antigen. Feline leukemia virus-negative cats with non-regenerative anemia or normal packed cell volumes had normal mean corpuscular volume values. Uninfected cats with regenerative anemia had prominent significantly increased macrocytosis and anisocytosis (p less than 0.01). Ninety percent of 62 feline leukemia virus-positive cats had altered erythrograms. Thirty-three feline leukemia virus-positive cats with non-regenerative anemia had marked macrocytosis. Their mean corpuscular volume values (mean 60 fl +/- 2 fl standard error, reference range of 37-49 fl) were significantly greater than those of feline leukemia virus-negative cats except for those with regenerative anemia. Feline leukemia virus-positive, non-anemic cats had significantly increased mean corpuscular volume values of intermediate magnitude. Nine adult cats experimentally infected with feline leukemia virus developed non-regenerative anemia with significant increases in mean corpuscular volume and anisocytosis. However, the macrocytosis observed in these cats was considerably less than in naturally occurring feline leukemia virus-positive cats with non-regenerative anemia. These observations indicate there are events in the pathogenesis of feline leukemia virus-associated anemia other than simple erythroid hypoplasia. We suggest that hemolysis and erythrocyte regeneration occur before erythroid hypoplasia and may partially account for macrocytosis observed in the face of non-regenerative anemia.     

Hematologic features of iron deficiency anemia in llamas.

Iron deficiency anemia was identified and characterized in three 14 to 29-month-old male llamas (llama Nos. 1-3) from separate herds in Colorado. The identification of iron deficiency anemia was based on hypoferremia (serum iron = 20-60 micrograms/dl), erythrocytic features, and hematologic response to iron therapy. The anemia was moderate and nonregenerative and characterized by erythrocyte hypochromia, microcytosis (mean cell volume = 15-18 fl), and decreased mean corpuscular hemoglobin concentration (36.0-41.0 g/dl). Morphologic features unique to llamas with iron deficiency anemia included irregular distribution of hypochromia within erythrocytes and increased folded cells and dacryocytes. The cause of iron deficiency was not determined. The llamas were treated with various doses and schedules of parenteral iron dextran. Two of the llamas were monitored for up to 14 months after the start of iron therapy and experienced increases in hematocrit and mean cell volume values. In one llama, progressive replacement of microcytic cells with normal cells was visualized on sequential erythrocyte volume distribution histograms following iron therapy.     

Characterization of erythrocytic indices and serum iron values in healthy llamas.

An electronic particle counter with attached particle-size analyzer was configured to directly determine concentration, mean cell volume, and volume distribution of erythrocytes in llama blood. Blood from 38 healthy llamas was used to characterize erythrocytic measurements and serum iron values for this species. Volume distribution curves for llama erythrocytes were similar in shape to those of other species. These curves had a unimodal, symmetric shape with a tail skewed to the right. Reference ranges for directly measured mean cell volume, erythrocyte concentration, hemoglobin concentration, and mean cell hemoglobin concentration were 21 to 28 fl, 11.3 x to 17.5 x 10(6) cells/microliters, 12.8 to 17.6 g/dl, and 43.2 to 46.6 g/dl, respectively. Reference ranges for serum iron concentration, total iron-binding capacity, and transferrin saturation were determined to be 70 to 148 micrograms/dl, 230 to 370 micrograms/dl, and 22 to 50%, respectively.     

Comparison of two automated multi-channel blood cell counting systems for analysis of blood of common domestic animals

An automated, multi-channel blood cell counting system (S-Plus) was compared to a reference counting system using blood samples from 187 animals of four species. The standard red cell bath aperture current of 150 volts (V) was used during analysis of 75% of the samples. At this setting, all samples with a Mean Corpuscular Volume (MCV) greater than 50 fl had accurate erythrocyte counts. As the MCV decreased below 50 fl, the severity of false low erythrocyte counts and false high MCV values increased. The remaining 25% of samples were analyzed with the red cell bath aperture current increased to 200 V. At this setting, only 5% or less of erythrocytes from animals with normal MCV values(>36 fl)were below the erythrocyte threshold. The red cell distribution width values provided by the S-Plus indicated that equine and bovine erythrocytes have greater anisocytosis than canine and feline erythrocytes. Leukocyte counts were significantly lower on the S-Plus (p<0.01). Canine and equine samples most frequently had platelet size distribution within the S-Plus platelet counting threshold window. Electronic whole blood platelet counting appeared unsatisfactory in cats due to large platelet size and erythrocyte-platelet size overlap. Small platelet size in cattle indicated that further modifications of the red cell bath aperture current would be required to count and size platelets in this species. Following electronic modifications, this state-of-the-art system appears adaptable to hematologic profiling in most species.     

缺锌对天府肉鸭红细胞免疫功能影响的研究

通过人工复制建立天府肉鸭缺锌病理模型，进行了锌对不同日龄鸭红细胞C3b受体花环(C3bRR)和免疫复合物花环(ICR)测定，并对其机制进行了讨论。结果表明：缺锌明显抑制鸭红细胞免疫活性，具有时间效应。     

Pharmacokinetics of florfenicol in normal and Pasteurella-infected Muscovy ducks

1. Disposition kinetics of florfenicol were studied in Pasteurella-free (control) and Pasturella-infected Muscovy ducks following intravenous and/or intramuscular injection in a single dose of 30 mg/kg body weight. In addition, the tissue distribution and residual pattern of the drug were determined in diseased ducks. 2. The maximum serum concentration of florfenicol in control healthy and infected ducks was reached 1 hour after intramuscular injection but the peak concentration in control ducks was higher than in infected birds. 3. The volume of distribution, total body clearance and systemic bioavailability were higher in infected ducks than in control birds 5.15 l/kg, 10.24 ml/kg/min and 73.03% respectively. Data relating to intravenous injection were analysed using a 2 compartment open model curve fit. 4. Florfenicol was not detected in the serum of infected ducks on the 7th day following intramuscular administration of 30 mg/kg body weight twice daily for 5 successive days but was detected in kidney, bile and liver.     

Erythrocyte volume distribution in rainbow trout

Reference mean corpuscular volume and volume heterogeneity values for erythrocytes of 23 rainbow trout were measured, using an electronic particle counter, and were analyzed, using a particle-size analyzer and microcomputer. The mean (+/- SD) erythrocyte count was 1.5 X 10(6) +/- 0.16 X 10(6) cells/microliter, the mean corpuscular volume was 346 +/- 25 fl, and the mean coefficient of variation of erythrocyte volumes was 29 +/- 2.3%. All erythrogram curves were skewed to the right, representing a mean of 15.6 +/- 3.5% of the total cells counted.     

Electronic measurement of erythrocyte volume and volume heterogeneity in horses during erythrocyte regeneration associated with experimental anemias

Anemia was induced in three groups of horses by moderate or severe acute hemorrhage, or by acetyl phenylhydrazine-induced hemolysis (Groups I, II, and III, respectively). Serial hemograms were done on a multichannel automated blood cell counter with histogram capability. Changes in hematocrit, mean cell volume, erythrocyte number, red cell distribution width (RDW), and standard deviation of erythrocyte volume were examined over time. Significant increases in mean cell volume were first detectable by days 17, 20, and 14 and reached maximum by days 43, 41, and 29, in Groups I, II, and III, respectively (P less than 0.05). Increased mean cell volume was interpreted as reflecting accelerated erythrocyte regeneration; however, not all horses with accelerated regeneration had changes in mean cell volume. Estimated erythrocyte production rate correlated poorly with hematocrit nadir and change in mean cell volume (r = 0.37 and r = 0.36, respectively, P greater than 0.05). In some horses effective regeneration occurs without development of macrocytosis. Mean cell volume remained increased after other parameters returned to control values, suggesting that mean cell volume values may provide retrospective evidence of altered erythrocyte turnover. Anisocytosis as indicated by significant increases in the standard deviation was greatest during the early part of the regenerative response, reaching maximum values on days 30, 28, and 21 in Groups I, II, and III, respectively, and began to decrease as homogeneous repopulation with macrocytes occurred. Red cell distribution width increased significantly only in severe hemorrhage and hemolysis groups, reaching mean maximum values of 24.3 on day 20 and of 26.4 on day 21 in Groups II and III, respectively (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of single doses of digoxin administered intravenously to ducks, roosters, and turkeys

A single dose of digoxin was injected, IV, into 5 mature male turkeys (0.066 mg/kg of body weight), 8 male ducks (0.066 mg/kg), and 6 roosters (0.33 mg/kg). Twenty-three serial venous blood samples were collected before (baseline) and after the administration of digoxin to turkeys, ducks, and roosters. Plasma concentrations of digoxin were determined in duplicate by a radioimmunoassay that was validated for avian species. The plasma concentrations were best fitted by a 3 (turkeys, ducks)- and 2 (roosters)-compartment open model, with first-order elimination from the central compartment. Significant (P less than 0.05) kinetic differences were determined among species. Mean half-life (t1/2) for ducks, roosters, and turkeys were 8.30 +/- 2.70 (mean +/- SD), 6.67 +/- 3.50, and 23.7 +/- 4.8 hours, respectively. The volume of distribution at steady state (Vss) was 14.7 +/- 2.9, 3.13 +/- 0.49, and 2.27 +/- 0.36 L/kg, and total body clearance (CL) of drug was 1.54 +/- 0.43, 0.461 +/- 0.187, and 0.136 +/- 0.022 L/h/kg for ducks, roosters, and turkeys, respectively. The mean residence time was 10.3 +/- 3.9, 8.37 +/- 4.97, and 16.8 +/- 2.2 hours, respectively. Volume of distribution at steady state and CL in ducks were several fold higher than that in turkeys. The terminal half-life of digoxin determined for ducks and roosters in this study was considerably shorter than those previously reported for several mammalian species.     

Sizing of animal erythrocytes using sulfate based diluent

Sulfate based cell counting diluent and human erythrocyte size standards were evaluated for electronically sizing erythrocytes of common domestic species. Mean differences between calculated and human cell referenced electronic mean corpuscular volume values were 0, 0.4, 0.8, and 2.1 fl for blood of dogs, horses, cats, and cows, respectively. These differences were statistically significant only for the cat (p=0.029) and cow (p=0.0002). Electronic mean corpuscular volume was measured on multiple lots of human cell control material following calibration with human cells and cells of each of the four species. There were no significant differences between assigned assay values and direct measurements at each calibration (F=0.14, df=29). Sulfate based diluent, used on some automated cell counting systems, appears suitable for sizing animal erythrocytes and commercially available human cell standards are appropriate for calibration of certain systems used in veterinary hematology.     

Heparin-induced agglutination of erythrocytes in horses

Heparin was administered subcutaneously 2 times a day for 4 days to 5 horses. An additional group of 5 horses was used as time-matched controls. Significant decreases in PCV, erythrocyte count, and hemoglobin concentration were observed during heparin therapy. The mean corpuscular volume (MCV) of the heparin-treated horses increased to a peak value of 66.1 fl on the last day of treatment. Erythrocyte creatine concentration and glucose 6-phosphate dehydrogenase activity increased moderately during the treatment. These data indicated that the rapid, profound increase in MCV during heparin therapy was not primarily a result of release of large immature erythrocytes from the bone marrow. A second experiment was subsequently performed, using 3 horses. These horses were given heparin 2 times a day, as was done in the first experiment. Saline wet mounts of erythrocyte suspensions were examined once a day for the presence of agglutination. Cell suspensions were examined with or without exposure to a dilute trypsin solution, and erythrocyte counts were done on each suspension, using an electronic cell counter. Agglutination of erythrocytes was evident on the first day of treatment and became more pronounced as treatment progressed. Exposure to trypsin solution reversed the agglutination. The apparent erythrocyte count decreased and MCV increased sharply in the samples processed normally, but there was little change in those suspensions exposed to trypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythrocyte volume distribution analysis and hematologic changes in two horses with immune-mediated hemolytic anemia

Immune-mediated hemolytic anemia was diagnosed in two horses on the basis of regenerative anemia, increased erythrocyte fragility in hypotonic saline, autoagglutination, and a positive direct antiglobulin (Coomb's) test. During steroid therapy partial resolution of the anemia was indicated by rising packed cell volume, macrocytosis, and bone marrow erythroid hyperplasia. Using erythrocyte volume distribution histograms (erythrograms), the regenerative response was characterized by analysis of macrocytic and normocytic erythrocyte subpopulations. In both horses, a gradual net increase of about 2 X 10(6) macrocytes/microliter occurred over a four- to five-week period. Over the same interval there was a gradual decrease in the number of normocytes. We suggest that the macrocytes remained large through this period rather than contributing to normocyte population growth. Erythrograms may provide an additional means of evaluating erythrocyte regeneration in horses.     

The effects of Escherichia coli and its endotoxin on amyloidogenesis in ducks.

To compare the effects of two inocula on the induction of amyloidosis in normal and thymectomized ducks, 180 normal and 50 thymectomized ducks were injected intravenously weekly for up to 16 weeks with either crude endotoxin or crude whole cell extract of a virulent strain of Escherichia coli (O78), and another 60 ducks were injected with normal saline as study control. During the first 5 weekly injections, the initial doses of inducing agents were the smallest and then adjusted upward to the maximum study doses (1 or 2 mg/bird of crude endotoxin and 15-18 x 10(8) bacteria/bird of crude whole cell extract), which were then maintained over the course of the study. The incidence of amyloid deposition were: 50.00% (25/50) for thymectomized ducks that received 1 mg/bird/week of crude endotoxin, 61.67% (37/60) for intact ducks that received 15 x 10(8)-18 x 10(8) bacteria (crude whole cell extract)/bird/week, 53.33% (32/60) for intact ducks received 2 mg/bird/week of crude endotoxin, and 63.33% (38/60) for intact ducks received 1 mg/bird/week of crude endotoxin. The results suggest that crude whole cell extract and crude endotoxin induced amyloidosis in ducks at similar rates and that, in ducks, thymectomy has no appreciable effect on the occurrence of amyloidosis.     

Modification and evaluation of a multichannel blood cell counting system for blood analysis in veterinary hematology

A multichannel, semiautomated, blood cell counting system (Coulter Counter Model S550) was modified for use in veterinary hematology by increasing both the erythrocyte and leukocyte aperture currents to 225 V and 195 V, respectively, followed by calibration with human blood. It was evaluated by use of 350 samples from dogs, cats, horses, and cows. Values for leukocyte count, erythrocyte count, mean corpuscular volume, and hematocrit generated by the S550 were compared with values generated by an automated multichannel counter with histogram capability and other reference procedures when appropriate. Mean differences for values between S550 and reference values were less than calibration tolerance limits for the instrument. Correlation coefficients were excellent for all values of each species. To assess behavior of leukocytes of the different species with respect to the counting threshold, leukocyte size distribution histograms were generated for all samples analyzed on the S550. Means for mean leukocyte volumes in diluent and lysing reagents were 55.5, 56.6, 67.4, and 72.8 fl for dogs, cats, horses, and cows, respectively. Canine leukocyte counts, because of small leukocyte size, were an average of 14% less for 5 samples analyzed on the unmodified instrument, compared with analysis after increasing the leukocyte aperture current. Leukocyte threshold failures attributable to interfering particles, resulting in falsely high counts, were recognized in 14%, 10%, 8% and 0% of feline, bovine, canine, and equine samples, respectively. The magnitude of error in these samples averaged 5% for cows and dogs, but was considered not important. However, leukocyte counts of feline samples in this group averaged 44% falsely high.     

Microcytosis and Iron Status in Dogs With Surgically Induced Portosystemic Shunts

Microcytosis is common in dogs with congenital portosystemic shunts (PSS) and acquired liver disease. The objective of this study was to determine if microcytosis could be induced in normal dogs by surgical creation of PSS, and to characterize the changes in hematology and iron status. Hematocrit, mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration decreased linearly from 45.5%. 69.1 fL, 22.8 g/dL and 33.1% to 39.5%. 55.9 fL, 17.8 g/dL and 31.9%. respectively, 18 weeks after creation of PSS. The erythrocyte count did not change, but red cell distribution widths indicated a shift to a heterogenous population with decreased volume. Mean cell volume and mean cell hemoglobin decreased rapidly after induction of PSS and were significantly ( P < .05) different from presurgery values within 2 weeks. Serum iron and copper concentrations and total iron binding capacity were decreased in dogs with PSS. Liver iron concentration doubled after creation of PSS, with the majority of stainable iron located in Kupffer cells. The changes in erythrocyte indices and measures of iron status in dogs with surgically induced PSS were similar to those in dogs with congenital PSS. Microcytosis developed rapidly in dogs after induction of PSS. These results indicate that iron deficiency was not the cause of microcytosis in these dogs.     

Pharmacokinetics of cyclosporine in woodchucks and Pekin ducks

The purpose of this study was to evaluate the pharmacokinetics of cyclosporine (Cy) in woodchucks ( Marmota monax ) and Pekin ducks. These data are needed to design rational dosing regimens. Pharmacokinetic parameters were calculated from blood concentration-time data obtained following intravenous (i.v.) administration of 10 mg/kg body weight to woodchucks and Pekin ducks. Whole blood samples were collected in EDTA and assayed using a commercially available radioimmunoassay kit that employs a monoclonal antibody specific for Cy. The blood concentration-time profile best Dtted an open, two-compartmental model in Pekin ducks. Compartmental analysis of data in woodchucks did not adequately describe the data. When non-compartmental pharmacokinetic analysis of the data was performed, the resulting mean (± SD) pharmacokinetic parameters in woodchucks and Pekin ducks, respectively, were as follows: volume of distribution at steady-state, 2.9 (± 0.8) and 2.7 (± 0.2) L/kg; systemic clearance, 10.2 (± 2.8) and 28.6 (± 6.1) mL/kg/min; mean residence time, 4.8 (± 1.1) and 1.6 (± 0.3). These data suggest that Pekin ducks clear Cy at a faster rate than do woodchucks and that a greater dose of Cy should be administered to Pekin ducks in order to achieve adequate immunosuppression.     

Erythrogram and red cell distribution width of Equidae with experimentally induced anemia

The erythrogram (erythrocyte histogram) and red cell distribution width (RDW) were evaluated in 5 purebred horses and 1 pony of mixed breeding with experimentally induced anemia. Four horses were studied for 6 weeks after 20% of their estimated blood volume was removed on each of 2 consecutive days (40% total blood loss; acute blood-loss group). Two horses were given acetylphenyl hydrazine IV daily, until acute Heinz body hemolytic anemia was induced; the 2 horses were then evaluated for 6 weeks. One horse and the pony had 20% of their estimated blood volume removed via phlebotomy once each week for 8 weeks to induce iron-deficiency anemia (chronic blood-loss group); the horse had been partially depleted of iron before the study began. Weekly blood samples were examined for changes in the erythrogram, RDW, mean cell volume (MCV), and erythrocyte glucose-6-phosphate dehydrogenase activity. Fourteen days after acute blood loss, mild increases were seen in the MCV, which persisted to day 42. The RDW was increased at day 14 and remained increased until day 42; however, the percentage increase was double that of the MCV at days 14, 21, and 28. Erythrograms had mild extensions of the right slope at days 14 to 28. Mean erythrocyte glucose-6-phosphate dehydrogenase activity increased in all 3 groups, but individual concentrations were erratic. In the 2 horses with acute hemolytic anemia, modest increases of similar magnitude were seen in RDW and MCV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of tobramycin in ducks and sex-related differences

The aims of the present study were to determine the disposition of tobramycin after single intravenous (IV) and intramuscular (IM) injections in ducks, and to establish any sex-related differences. Tobramycin sulfate was administered as a 2.5% water solution in a cross-over design at a dose of 5 mg/kg to 12 healthy ducks (six males and six females). Concentrations of the drug in serum were determined by a microbiological assay. The serum pharmacokinetic values for tobramycin were best represented using a one- or two-compartment open model, depending on the method of administration. Non-compartment analysis was also performed after IV administration. Tobramycin had a low degree of distribution and a relatively fast elimination. The mean volume of distribution in ducks (males and females) was higher than that reported in pigeons but lower than in chickens, with a slower rate of elimination. The IM injection resulted in a fast and complete absorption. The rate of elimination after IM administration was about twice as slow as in other avian species. Sex-related variations in tobramycin pharmacokinetics were similar to those reported for kanamycin and apramycin in hens and roosters.     

In vitro increase of mean corpuscular volume difference (dMCV) as a marker for serum hypertonicity in dogs

Spurious increase in erythrocyte mean corpuscular volume (MCV) on automated cell analyzers is a well-characterized lab error in hypertonic patients. A difference between automated and manual MCV (dMCV) greater than 2 fl has been shown to predict hypertonicity in humans. The purpose of this study was to investigate dMCV as a marker for serum hypertonicity in dogs and to examine the relationship between dMCV and three methods of estimating serum tonicity: measured (OsM_M), calculated (OsM_C), and calculated effective (OsM_CE) osmolalities. OsM_C, OsM_CE, and dMCV were calculated from routine blood values and OsM_M was directly measured in 121 dogs. The dMCV of hypertonic dogs was significantly larger than that of normotonic dogs for all three osmolality methods. dMCV predicted hypertonicity as estimated by OsM_M better than it predicted hypertonicity as estimated by OsM_C and OsM_CE. A cutoff of 2.96 fl yielded the best sensitivity (76%) and specificity (71%) for hypertonicity estimated by OsM_M.