Size referenced electronic leukocyte counting threshold and lysed leukocyte size distribution of common domestic animal species

Using a single channel electronic cell counter and attached particle size analyzer, leukocyte size distribution histograms were determined on canine, feline, bovine, and equine blood diluted with chloride-based diluent and treated with a conventional stromatolysin. Histograms were usually unimodal, but a few were bimodal. Mean values for mean lysed leukocyte particle volume were 49.2, 51.1, 55.4, and 65.0 fl for canine, feline, equine, and bovine blood, respectively. From inspection of histograms, a lower threshold of 30 fl referenced to latex spheres was interpreted to be appropriate for counting leukocytes of these four species simultaneously. Debris below the threshold was seen in many samples and was usually separated from the leukocyte population by a valley touching the histogram baseline at the threshold channel. Debris resulted in a visually detectable threshold failure by extending considerably into the leukocyte size range in 9% of feline, 9% of canine, and 7% of bovine samples. It is recommended that careful establishment of the lower counting threshold will minimize frequency and severity of leukocyte count error associated with failure to exclude debris.     

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.     

Hematologic responses in llamas with experimentally-induced iron deficiency anemia

Hematologic abnormalities consistent with iron deficiency anemia were experimentally induced in two healthy llamas by repeated phlebotomy. Hematologic abnormalities included erythrocyte microcytosis and hypochromia, decreased hemoglobin concentration, hypoferremia, and decreased transferrin saturation. Erythrocyte volume distribution histograms were more sensitive than mean corpuscular volume values for detection of microcytosis. Hypochromia, which was often eccentric, was morphologically observed on Wright-Giemsa-stained blood films. Frequent folded erythrocytes and dacryocytes were also noted on the blood films. Hematologic abnormalities resolved rapidly after cessation of blood removal, without parenteral iron supplementation.     

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.     

Artifactual effects of hypernatremia and hyponatremia on red cell analytes measured by the Bayer H*1 analyzer

Hypernatremia in two cats and hyponatremia in a dog were associated with artifactual changes in red blood cell (RBC) indices and hematocrit (HCT) determined on a Bayer H*1 hematology analyzer. The RBC cytograms and histograms revealed a population shifted towards macrocytic, hypochromic RBC in the hypernatremic cats, and towards microcytic, hyperchromic RBC in the hyponatremic dog. Reference intervals for the difference between manual packed cell volume (PCV) and analyzer-derived HCT in normonatremic dogs, cats and horses were established. The difference between PCT and HCT was outside the reference values for all three patients. Quality control measures, such as measuring PCV, and reviewing cytograms and histograms are essential for detecting spurious changes in automated hematology measurements caused by abnormalities in serum sodium concentration and osmolality.     

Comparative hematology: studies on goats.

Results of blood coagulation and hematologic studies on 6 goats, each tested 3 times, were compared to the values seen in persons. Special blood platelet studies were done on an additional goat. Blood coagulation values in the goats and in persons were similar, with these exceptions: In the goat, activated partial thromboplastin time was shorter and thrombin time was longer; one-stage assays of factors V, VIII, and IX were very high, and platelets aggregated poorly epinephrine and ristocetin. Both platelets and erythrocytes were small. On scanning electron microscopy, the erythrocytes appeared as flat disks or triangles, occasionally having a dimpled center, compared to the deeply dimpled doughnut shape of the larger human erythrocytes. Osmotic fragility of these small erythrocytes was greater than that of their human counterparts. By transmission electron microscopy of ultrathin sections of goat buffy coat, platelets had fine structures similar to those of human platelets. Unlike in human platelets, most of the dense bodies in goat platelets were surrounded by clear vacuoles. Biochemical studies showed higher than human levels of phosphorus, chloride, sodium, alkaline phosphatase, and serum glutamic oxaloacetic transaminase.     

Advances in hematology analyzers

The complete blood count is one of the basic building blocks of the minimum database in veterinary medicine. Over the past 20 years, there has been a tremendous advancement in the technology of hematology analyzers and their availability to the general practitioner. There are 4 basic methodologies that can be used to generate data for a complete blood count: manual methods, quantitative buffy coat analysis, automated impedance analysis, and flow cytometric analysis. This article will review the principles of these methodologies, discuss some of their advantages and disadvantages, and describe some of the hematology analyzers that are available for the in-house veterinary laboratory.     

Modifications of a digestion technique for the post-mortem recovery of bursate nematodes of sheep

All automated complement fixation test results and testing procedures carried out at the Central Brucellosis Laboratory for the Brucellosis Eradication Scheme undergo quality control checks. Auto-Analyzer performance is monitored by reagent concentrations, peak heights of the standard positive sera and percent transmission for 0% and 100% lysis of sheep red blood cells. Deviations in the size and form of sample peaks indicate problems with the serum sample or the testing system.

A trial designed to detect inapparent errors found that these constituted 0.6% of the samples surveyed, while apparent errors caused 11.5% of routine serum samples to be retested. The introduction of more stringent quality control measures reduced the number of samples requiring retesting to 3.4% of all samples tested. Continuing education of staff is considered an important means of improving the quality of laboratory work.     

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.     

Morphological evaluation of canine platelets on Giemsa- and PAS-stained blood smears

The morphology of canine platelets (changes in size, shape, staining characteristics, degree of activation and clump formation, distribution of granules, appearance of vacuoles on Giemsa-stained smears) was investigated in 20 healthy control and 181 diseased dogs. In the group of the sick dogs 84 animals suffered from disorders affecting directly the haematological parameters or the haematopoietic organs such as bleeding, thymic haemorrhage, haemolytic disorders, lymphoma, immune-mediated thrombocytopenia, and other 97 dogs were affected by other diseases (hepatopathy, nephropathy, hepatic, splenic or intestinal neoplasm, skin diseases, diabetes mellitus, Cushing's syndrome, sepsis). The alterations found in platelet morphology were not specific for any disorder. The most common platelet abnormalities were polychromasia and the presence of giant platelets. These changes occurred in a high number in disorders accompanied by bleeding or haemolysis. Anisocytosis was the most frequent finding in hepatic, splenic or intestinal neoplasms and in certain endocrinopathies. Microcytosis was observed in immune-mediated thrombocytopenia, hepatic neoplasms and endocrine disorders. Extreme platelet activation was common in haemolysis, hepatopathies, neoplastic diseases and sepsis. Vacuolisation was present in thymic haemorrhage, pancreatitis, diabetes mellitus and Cushing's syndrome. A new morphologic phenomenon, i.e. a ring-like formation of granules, was described in the cytoplasm of the platelets both in healthy and diseased animals. In addition, two forms of pathologic granulation were also described for the first time in Giemsa-stained blood smears: the pseudonuclear and the spot-like formation of granules, which were observed especially in disorders affecting the blood cells. The granulation and morphological characteristics of platelets on smears stained by periodic acid-Schiff reaction (PAS) were also studied. Three localisations of granulation were observed, such as peripheral, eccentric and diffuse. The ratio of PAS-positive and -negative platelets was evaluated in several diseases. Our findings support the diagnostic value of platelet evaluation by light microscopy and help clinicians/clinical pathologists to understand why morphologic changes of thrombocytes might be expected in several diseases.     

A hematological study on thirteen cats with myelodysplastic syndrome

Hematological abnormalities were investigated in 13 cats with myelodysplastic syndrome (MDS). Examination of the peripheral blood samples from the 13 cats revealed anemia in 11 cats, leukopenia in 9 cats, and thrombocytopenia in 9 cats. Four cats had pancytopenia (30.8%) and 9 cats had bicytopenia (69.2%). Dysplastic changes of erythrocytes, neutrophils, and platelets in the peripheral blood were found in 5, 10 and 8 cats, respectively. Bone marrow examination of the 13 cats revealed that ratios of blast cells to all nucleated cells (ANC) ranged from 0 to 20%. Ratios of erythroid progenitor cells to ANC were more than 50% in 3 cats and less than 50% in 10 cats. Eosinophils accounted for more than 5% of non-erythroid cells in 10 cats. Dysplastic changes in the granurocytic, erythrocytic, and megakaryocytic cells in the bone marrow were found in 11, 7 and 5 cats, respectively. Dysplastic changes in these cats included giant neutrophils, ring-nucleated neutrophils, binuclear myelocytes, hypersegmented and hyposegmented neutrophils, megaloblastoid erythroblasts, multinucleated erythroblasts, micromegakaryocytes, and segmented multinucleated megakaryocytes. Virological examination indicated the presence of feline leukemia virus antigen in the peripheral blood from all of the 13 cats with MDS. The peripheral blood cytopenias and dysplastic changes in each blood cell lineage in the bone marrow were shown to be important for the diagnosis of MDS in cats.     

Hemophagocytic syndrome in a cat with multiple myeloma

An 11‐year‐old, castrated male, Domestic Medium Hair cat was presented to the University of Florida Small Animal Hospital with a 2‐week history of upper respiratory infection and increased serum globulins, as reported by the referring veterinarian. Physical examination was unremarkable other than melanosis of the left iris, with no evidence of ocular, nasal, or respiratory disease. Laboratory abnormalities included moderate nonregenerative anemia, mild leukopenia, mild hyperfibrinogenemia, severe hyperglobulinemia, mild hypoalbuminemia, and hypocholesterolemia. Abdominal radiographs and ultrasonographic examination revealed mild splenomegaly with no other abnormalities. Thoracic radiographs revealed no abnormalities. Cytologic evaluation of fine‐needle aspirates from the spleen, liver, and bone marrow revealed numerous plasma cells and many vacuolated macrophages exhibiting marked phagocytosis of mature erythrocytes and platelets, occasionally metarubricytes and leukocytes, and rarely plasma cells. The cytologic interpretation was multiple myeloma and associated hemophagocytic syndrome (HPS). Serum protein electrophoresis revealed a monoclonal gammopathy, providing further evidence for a multiple myeloma. To the authors' knowledge, this is the first report of HPS secondary to neoplasia in a cat.     

Hematology without the numbers: in-clinic blood film evaluation.

Technical advances have made it possible for many private veterinary practices to purchase reasonably priced automated hematology instruments to perform in-clinic blood analyses. Although these instruments can quickly provide "numbers" to the clinician, evaluation of a well-made blood film can often provide information critical to the interpretation of those numbers. Blood film review is essential to identify important abnormalities such as neutrophilic left shifts and toxic change, neoplastic cells, hemoparasites, and erythrocyte morphologic changes that may suggest the cause of an anemia. Additionally, the blood film provides an important quality control measure for the automated hematology results. This article outlines a simple method of blood film evaluation, highlights the most common clinically important abnormalities, and reinforces the importance of blood film evaluation as a quality control measure.     

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.     

Detection of antibodies to platelets and erythrocytes during infection with haemorrhage-causing Trypanosoma vivax in Ayrshire cattle

Ayrshire cattle, which were infected with a stock of Trypanosoma vivax from Galana, Kenya, which produced haemorrhagic disease, were examined for the presence of antibodies to erythrocytes and platelets. Antibodies to normal erythrocytes and platelets were detected in the plasma of infected animals using the enzyme-linked immunosorbent assay (ELISA). The antibodies were detectable following the first peak of parasitaemia (10-15 days after infection) and antibody activity was maximal 30-35 days after infection. Plasma from cattle, taken 32 days after infection, precipitated radiolabelled proteins from autologous platelets and, less efficiently, from autologous erythrocytes. Fluorescence-activated cell sorter (FACS) assays demonstrated that erythrocytes and platelets from infected cattle bound IgM and IgG in vivo, and that both normal blood cell types could adsorb these antibodies following incubation in plasma from infected animals. Complement (C3) was similarly adsorbed to erythrocytes during infection. Antibodies adsorbed to infected erythrocytes could be eluted and the eluted antibodies bound to normal erythrocytes, as detected by immunofluorescence, but they did not react with the infecting trypanosome. It is hypothesised that although anti-blood cell antibodies may not be the primary cause of the severe anaemia and thrombocytopaenia which accompany the haemorrhagic syndrome, they could play an important role in the maintenance of these signs of disease, adversely affecting the outcome of T. vivax-associated haemorrhagic disease in the field.     

Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels

Background: Many Cavalier King Charles Spaniel (CKCS) dogs are affected by an autosomal recessive dysplasia of platelets resulting in fewer but larger platelets. The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). We hypothesized that CKCS both with and without hereditary macrothrombocytosis would have a normal plateletcrit and that the QBC results would better identify the total circulating volume of platelets in CKSC than methods directly enumerating platelet numbers.
Objectives: The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs.
Methods: Platelet counts were determined in fresh EDTA blood from 27 adult CKCS dogs using the QBC, Sysmex XT-2000iV (optical and impedance), CELL-DYN 3500, blood smear estimate, and manual methods. Sysmex optical platelet counts were reanalyzed following gating to determine the number and percentage of normal- and large-sized platelets in each blood sample.
Results: None of the 27 CKCS dogs had thrombocytopenia (defined as <164 × 10⁹ platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum.
Conclusions: The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass.     

A monoclonal antibody against chicken thrombocytes reacts with the cells of thrombocyte lineage

A new mouse monoclonal antibody (mAb), HUKT was raised against chicken peripheral blood thrombocytes. The mAb HUKT appeared to detect a specific marker on the surface of chicken thrombocytes. Flow cytometry (FCM) analysis revealed that it did not react with cells from the normal thymus, bursa of Fabricius, six kinds of chicken cell lines, chicken erythrocytes or human platelets. In addition, HUKT(+) cells in peripheral blood leukocytes (PBL) were CD45(low), Bu-1a(-) and CD3(-) cells. Immunoblotting analysis showed that the molecule recognized by HUKT is a monomer with an apparent molecular weight of 150 kDa under non-reducing and reducing conditions. Tissue distribution studies revealed that only cells of thrombocyte lineage in bone marrow and embryonic blood cells were stained by HUKT. The HUKT mAb presented here may be useful for both ontogenetic studies of thrombocyte lineage and immunological studies in the chicken.     

Microtechnique for quantitative platelet isolation from blood enabling electronic counting and sizing of animal and human platelets

A technique for rapidly separating platelets from small quantities of whole blood is described. The isolation method allowed for counting and sizing platelets by electronic means, and counts with this new method were closely correlated with those obtained by phase microscopy. Platelet sizing was possible because of the inert and isotonic nature of the density-gradient medium used in the procedure. The technique was applicable to samples of blood from persons, horses, cattle, sheep, goats, pigs, dogs, mice, rats, guinea pigs, and rabbits. The isolation of platelets from whole blood of various species of animals was necessary for electronic counting because of the great variation in sizes of platelets and erythrocytes and the variable sedimentation properties of animal blood.     

In vitro cultivation of Anaplasma marginale: invasion of and development in noninfected erythrocytes

Ovine erythrocytes infected with attenuated Anaplasma marginale organisms were cultured in a suspension of normal ovine erythrocytes and normal bovine erythrocytes for 42 days. In each system, the organism showed an initial period of rapid growth followed by a gradual decrease in the percentage of parasitized erythrocytes accompanied by cyclic peaks. The percentage of infection of ovine erythrocytes were not different when normal ovine or bovine erythrocytes were added to the cultures. In vitro transmission of the organism from infected ovine cells to normal bovine cells was demonstrated by use of a two-step direct fluorescent antibody method, which allowed for specific identification of the two cell types and the organism.