Panoptic Stain Research Articles

Feline sporotrichosis: occurrence in the Metropolitan Region of the municipality of Recife and anatomical distribution of lesions on cats’ body

Sporotrichosis is a fungal infection of worldwide importance caused by a saprophyte fungus of the genus Sporothrix. The objective of this study was to report the occurrence of feline sporotrichosis in the Metropolitan Region of the city of Recife and the anatomical distribution of lesions on the bodies of cats. Stray cats (n=100) with suggestive sporotrichosis lesions were examined. Material from the lesion was collected from all patients using an imprint and/or sterile swab for cytology. The material was placed on a glass slide and stained using rapid panoptic staining. Data on the localization of each skin lesion were recorded, and cigar-shaped yeast structures were searched at the microscopical examination. A heat map graphically represented the anatomical distribution of each lesion. In 70% (70/100) of samples were visualized fungal structure compatible with Sporothrix spp. There was a predominance of positive males (?2=6.691; p=0.0097). Skin lesions related to Sporothrix spp. infections were unevenly distributed throughout the animal's body. Based on the interpolation of data, the chance for detecting lesions was higher on the nose, anterior paw, and ears than on other body sites (H=40.0043; p=0.0000). Because of the high positivity in these cats, measures of health vigilance are imperative to reduce the risk of animal and human infection.

Morphological and morphometric sperm analysis of dwarf sperm whale (Kogia sima)

Nowadays, an increase in the number of marine species at risk of extinction has been observed. Therefore, studies on the reproductive characteristics of these animals are essential. This animal is considered relatively rare, and there is scarce information regarding its reproductive biology and physiology. Thus, this study aims to describe the morphology and morphometry of sperm obtained from dwarf sperm whale. The material used in this work was collected during the necropsy of a dwarf sperm whale specimen. Thus, seminal samples were fixed and stained with panoptic stain. In morphometric analysis, the results obtained were: acrosome length of 0.83±0.01 μm, head length of 1.5±0.02 μm, intermediate part of 0.4±0.00 μm and total length of 27.3±0.51 μm. In terms of morphology, the defects observed were double head, heavily curled tail, abnormal small head, simply bent tail, piriform head, heavily bent tail and specimens within the normal range. In this context, the morphometric and morphological sperm analysis of Kogia sima described in this study can assist future studies regarding the reproductive physiology of these animals.

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function.

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although havinglimitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab's resources and demands.

Morfología de las células en los tumores moxoides

We describe a morphologic study of myxoid cells in myxoid tumors or with myxoid regions: myxoid fibrosarcoma, myxoma, myxoid liposarcoma, liposarcoma embryonal rhabdomyosarcoma, chondroma, chondrosarcoma, myxoid leiomyosarcoma, schwannoma, and odontoameloblastoma; and we compare with fibroblasts of umbilical cord, embryonal mesenquima and loose conective tissue in inflamatory conditions. Histologic techniques: H-E-PASS, Masson's tichroom, and Del Río Hortegas's panoptic silver staining. Histologically Del Río Ortegas's techniques reveals bipolar fibrobasts with loh processes.

FNAB AS A DIAGNOSTIC TOOL ON CYSTICERCOSIS AFFECTING SUBMANDIBULAR AREA

The purpose of this work is to report the usefulness of fine needle aspiration biopsy in a 17-year-old female patient with swelling in the left submandibular region. On extraoral examination, skin eruptions in the lower limbs and a nodule on the left submandibular region were observed. The cervical nodule was painless, measuring 1 cm × 1 cm, with a firm consistency and 4 months of evolution. Intraorally there were no clinical changes. Cervical ultrasound was requested and showed a hypoechoic area near the submandibular gland. Fine needle aspiration cytology was done and revealed structures compatible with <i>Taenia</i> sp. including body parts (panoptic stain). The diagnosis of cysticercosis was proposed. The treatment consisted of praziquantel 600 mg (8/8 h) for 15 days with partial remission and improvement of clinical complaints. An excisional biopsy of the lesion was performed, showing a lymph node–like material with suggestive areas of <i>Taenia</i> sp. residual body.

Effect of benzophenone-3 on the blood cells of zebrafish (Danio rerio)

Benzophenone-3 (BP-3) is a common component of organic sunscreen widely used that can affect especially aquatic ecosystems health, including fish. To verify the biological effects of low concentrations of BP-3 on blood cells, one hundred and forty zebrafish (D. rerio) were used and then randomly divided into five groups: control group (water), solvent group (alcoholic water), and BP-3 group (BP-3 at 7 µg L−1, BP-3 at 70 µg L−1, and BP-3 at 700 µg L−1). The blood slices were stained with Panoptic stain and with Giemsa solution for the hematological analysis. During the exposure to BP-3, no behavioral changes were observed. Although no significant difference in total leukocytes occurred, an increase in neutrophils and a reduction of lymphocytes at the highest concentration on both 7th and 14th days were detected. The total and cytoplasmic area of erythrocytes on the 7th day at the highest concentration were reduced. In addition, alterations on the erythrocyte nuclear morphology in fish exposed to BP-3 were usually visualized, mainly when considered the occurrence of blebbed nucleus and micronucleus, indicating that BP-3 exhibits cytotoxic and mutagenic effects. The results indicate that BP-3 can interfere with the morphophysiology of aquatic organisms.

Immunomonitoring of Human Breast Milk Cells During HCMV-Reactivation

BackgroundBreast milk leukocytes may play a role in protecting the infant from pathogens. The dynamics and the role of lymphocytes in human cytomegalovirus (HCMV)-seropositive mothers shedding HCMV into breast milk during the first months postpartum (p.p.) are mostly unclear.MethodsBreast milk cells were analyzed by Pappenheim panoptic and alpha-naphthyl acetate esterase staining as well as by imaging and polychromatic flow cytometry to simultaneously establish their morphological and phenotypic properties. The latter were characterized in HCMV-seropositive and seronegative mothers´ breast milk cells at different time points p.p.ResultsPanoptic staining of breast milk cells revealed the presence of monocytes/macrophages, granulocytes and lymphocytes. Imaging flow cytometry data combining phenotypic and morphological analysis identified NKT-like cells, NK cells, epithelial cells, T cells and monocytes/macrophages. HCMV-seropositive but not -seronegative mothers had significantly higher T cell frequencies in mature milk.ConclusionsThe presence of lymphocyte subsets in breast milk may be more influenced by the HCMV-seropositivity of the mother than previously recognized.

Morphohistometric Evaluation of Embryonic Development of Spleen in Chicken

The aim of this study was to evaluate the morphohistometric development of chick spleen by considering specific embryonic periods. For the study, spleens obtained from 18 Babcock White Leghorn chick embryos on the 13th, 16th and 21st days of incubation were used. The sections were stained with Crossmon’s trichrome stain and Pappenheim’s panoptic stain and differential leukocyte counts were made in the blood smears. In the measurements, an increase in the spleen volume, embryo weight and vitellus sac weight were determined. There was an increase between the 13th and 16th21st days in spleen volume. The highest heterophil granulocytes (74.83%) and lowest lymphocyte ratio (23%) were found on the 21st day. On the 13th day, there were very few lymphocytes around the vessels. On the 16th day, arteria centralis were frequently encountered and periarteriolar lymphoid tissue formation with lymphocyte accumulations around them started to develop in the spleen parenchyma. The red and white pulp areas could be easily distinguished in splenic parenchyma on the 21st day. It was concluded that the structures characterised by lymphocyte infiltrations in the spleen parenchyma were formed and caused changes in the number of lymphocytes in peripheral blood during the embryonal period.

An audit of cytopathology in the oral and maxillofacial region: 18years of experience.

The aim of this study was to perform an audit of oral and maxillofacial specimens submitted for cytological diagnosis to verify the importance of this complementary examination. A retrospective analysis of our institutional cytopathology database was performed over an 18-year period. Clinical information and cytological data were collected. Associations between independent variables and outcomes were assessed using the Pearson χ2 test or Fisher's test, with a 5% significance level. When available, the histological diagnosis was compared with cytological diagnosis to identify the percentage of agreement and the specificity, sensitivity and accuracy of cytology in identifying malignant neoplasms. A total of 1082 cases were identified, which included 65 different cytological diagnoses. Exfoliative cytology (EC) was performed in 312 cases (29.1%) and fine needle aspiration cytology (FNAC) in 770 cases (70.9%). EC was mainly employed to diagnose oral infectious diseases (P<0.001) and FNAC to diagnose neoplasms, cystic, reactive and miscellaneous lesions (P<0.001). Cell-block was performed in 555 FNAC cases (51.3%). Panoptic, Papanicolaou and haematoxylin-eosin staining were performed in FNAC and periodic acid-Schiff in EC (P<0.001). In 211 cases (19.5%), the histological diagnosis was available and the percentage agreement with the cytological diagnosis was 41.2%. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy to identify malignant neoplasms were 84.6%, 100%, 100%, 77.8% and 90.0%, respectively. EC was mainly performed for diagnosis of infectious diseases and FNAC for diagnosis of salivary gland tumours, odontogenic lesions, reactive lesions and cervical metastasis.

Diagnóstico laboratorial da esporotricose felina em amostras coletadas no estado do Rio de Janeiro, Brasil: limitações da citopatologia por imprint

INTRODUCAO: O estado do Rio de Janeiro vem passando por uma epidemia de esporotricose, considerada doenca negligenciada, de transmissao zoonotica pelo felino domestico (Felis catus). Apesar do isolamento de fungos do complexo Sporothrix schenckii em cultura micologica ser o padrao-ouro no diagnostico, a citopatologia por imprint das lesoes e rotineiramente utilizada na pratica da clinica veterinaria. OBJETIVO: Avaliar o desempenho da citopatologia como metodo diagnostico da esporotricose em gatos com suspeita dessa micose, provenientes de diferentes areas geograficas do estado do Rio de Janeiro, Brasil. MATERIAIS E METODOS: Apos avaliacao clinica, o swab do exsudato e lâminas via imprint da lesao foram coletados de 196 felinos domesticos, com posterior semeadura em Agar Sabouraud Dextrose e Mycosel e coloracao das lâminas por Panotico Rapido. RESULTADOS: Dos 196 animais, 102 (52,0%) foram diagnosticados com esporotricose. Para 50,0% da populacao, os resultados da cultura e da citopatologia foram discordantes (p 0,05). CONCLUSAO: A dosagem de itraconazol ≥ 100 mg/dia, independente da duracao do tratamento, reduz a sensibilidade diagnostica da citopatologia. Tal resultado representa um alerta para o uso dessa ferramenta como recurso unico para a confirmacao diagnostica da esporotricose felina na rotina da clinica veterinaria.

HEMOPARASITOS DE PIRANHAS VERMELHAS PYGOCENTRUS NATTERERI (KNER, 1858) (CHARACIFORMES: CHARACIDAE) CAPTURADAS NA REGIÃO DO MÉDIO RIO ARAGUAIA, ESTADO DE GOIÁS, GO

This work aimed to evaluate blood smears of 128 red piranha specimens Pygocentrus nattereri (Kner, 1858) (Characiformes: Characidae) as for the presence of hemoparasites and blood cells that were visualized. Samples were collected in Rio Vermelho (Red River) (15°10'44.73''S and 51°09'55.83W''), in the Brazilian state of Goias, between April 2012 and August 2013, including ebb, full and dry phases. After identificating and numbering the piranhas, we collected approximately 0.5 mL of blood from each animal, from the caudal vein or through intracardiac puncture, and two blood smears were done and stained with May GrunwaldGiemsa and Fast Panoptic stains. After euthanasia, the body mass and biometrics of every fish were measured. Abiotic factors of the river water were also analyzed, such as temperature and transparency. In general, the biometric parameters were higher in the full phase of the river, and the values obtained on water quality were similar in relation to the average temperature of the water, which was around 82,4°F. The average transparency of the water varied, being greater at low tide. Erythrocytes, thrombocytes, neutrophils, eosinophils, monocytes, heterophile and lymphocytes were identified as blood cells of red piranhas and hemogregarines have been found in about 25% (32/128) of the blood smears.

Alterações nos parâmetros hematológico durante a gestação e no pós-parto de cabras da raça Saanen criadas no sul do Espírito Santo

Resumo: O objetivo deste estudo foi avaliar o perfil hematológico de cabras da raça Saanen em diferentes fases da gestação e no pós-parto. Para sua realização foram utilizadas 24 cabras gestantes e 10 não gestantes da raça Saanen, criadas em condições intensivas no município de Alegre, Espírito Santo. As amostras foram coletadas dos 60 aos 135 dias de gestação, com intervalos de 15 dias entre as coletas. Após a parição foram realizadas mais duas coletas, também com intervalos de 15 dias. As amostras de sangue coletadas foram refrigeradas até a chegada ao laboratório, onde foi realizado o eritrograma e a leucometria global em analisador automático e as contagens diferenciais em esfregaço sanguíneo. Após análise dos resultados pode-se observar que, nas cabras avaliadas, houve influência do período de gestação, do pós-parto e da ordem de parto sobre os valores do eritrograma. Todos parâmetros avaliados, exceto VGM, diminuiram com o avançar da gestação até os quinze dias pós-parto, sendo que as maiores alterações ocorreram no final da gestação. A leucometria global (LG) e específica também apresentaram diferenças significativas de acordo com o estado fisiológicos das fêmeas. Os valores médios de LG foram de 10.579 a 15.836 leucócitos/mm3. Fêmeas não gestantes apresentaram maiores valores de LG, diminuindo com a gestação até os 15 dias do pós-parto. Trinta dias pós-parto o valor de LG aumentou, assemelhando-se aos valores das cabras não gestantes. Os valores absolutos de neutrófilos segmentados seguiram a mesma tendência da LG. A quantidade de linfócitos apresentou diminuição a partir dos 120 dias de gestação, aumentando a partir dos trinta dias pós-parto. A quantidade de eosinófilos não sofreu alteração de acordo com o estado fisiológico e a quantidade de basófilos foi maior em fêmeas não gestantes. Para monócitos os resultados foram em geral superiores nas cabras não gestantes e com 120 a 135 dias de gestação. Com relação a ordem de parição observou-se que fêmeas com mais de quatro partos apresentaram menores valores de VG, não havendo diferenças nos outros parâmetros do eritrograma. A LG foi maior na cabras com quatro partos, principalmente devido ao aumento de neutrófilos, enquanto que a quantidade de linfócitos foi menor nestas cabras. Os resultados encontrados evidenciaram a influência da gestação, do pós-parto e da ordem de parto das fêmeas da raça Saanen estudadas sobre os parâmetros hematológicos e também a necessidade da realização de estudos para se determinar os valores de referência do hemograma dos caprinos para cada tipo de criação, estado fisiológico e raça.

Quality control of bone marrow aspirates: Additional steps toward a safer and more efficient procedure

We read with great interest the article by Odejide et al.1 Bone marrow aspiration and biopsy play a central role in the diagnosis and follow-up of many hematological and nonhematological disorders.2, 3 Obtaining adequate specimens for pathology analysis is crucial, because nondiagnostic samples often require repetition of the procedure, leading to patient discomfort, familial distress, and increased costs. The causes of bone marrow aspirate failure are diverse and occasionally complex or multifactorial. They may include factors related to the disease (eg, solid tumor infiltration, aplastic anemia, bone marrow fibrosis, or hypocellularity), the patient (eg, radiation therapy, anatomic variation, obesity, agitation, or anxiety), or the operator (eg, anxiety, improper needle location, or experience).4 In addition, it is important to keep in mind that the pathology team can also misclassify an adequate sample as nondiagnostic.4 We would like to contribute to the discussion by reporting the results of our quality-control process of morphologic assessment of bone marrow aspirates during a 6-month period between April 2013 and September 2013. At our institution, we have a team of 26 hematologists who are responsible for bone marrow aspirates and biopsies collected from children and adults in 5 outpatient units and 11 general hospitals in the São Paulo and Recife metropolitan areas of Brazil. Hematologists who performed the procedure also prepared the blood smears; they were assisted by a nurse technician in most cases. As a standard technique, we use 20-mL syringes for aspiration and obtain approximately 0.5 mL of bone marrow aspirate for the preparation of the smears for cytomorphological assessment. We also encourage operators to take advantage of a rapid panoptic stain in cases in which the macroscopic aspect of the smears is put into question. In accordance with our process, if the operator obtains an improper smear (eg, one that is devoid of bone marrow elements), and it is identified by the rapid panoptic stain, it is possible to perform a new procedure before the patient is dismissed. In addition, it is possible to discuss the addition of a bone marrow biopsy with the attending physician who had initially ordered only a bone marrow aspirate. Bone marrow smears were classified according the dilution in peripheral blood as adequate (good number of bone spicules with little dilution), suboptimal (moderate dilution), and nondiagnostic. The number of procedures per operator varied widely, with a median of 22 procedures (range, 3-110 procedures) performed over the period of the study. The majority of those aspirations included several other tests, such as immunophenotyping, karyotyping, fluorescence in situ hybridization analysis, molecular biology tests, and bone marrow trephine biopsy. The preferred site for the aspirate procedure was the posterior iliac crest at the lateral decubitus; however, puncture at the sternum, although discouraged at our institution, was performed in a few challenging cases. We performed a total of 1126 bone marrow aspirations for morphologic analysis over the 6-month period of the study. Patients had a median age of 48 years (range, birth to 96 years); 555 of the patients (49.2%) were female. We determined a rate of 16.1% suboptimal samples, and 1.3% nondiagnostic samples (samples were too diluted and/or devoid of bone marrow elements). In such circumstances, a new specimen was collected for analysis. In an effort to sustain low rates of suboptimal and nondiagnostic samples, we perform a monthly quality-control evaluation for which each team member completes a form with data regarding the tests collected and the specific conditions of the procedures that were performed. Data from those forms are audited by the leader of the hematology team and a monthly report is compiled detailing individual and team rates of suboptimal and nondiagnostic samples. For professionals with higher rates on those indicators, we offer an opportunity for recycling and one-on-one retraining. These monthly reports are sent to both the hematology and pathology teams, and we hold a regular forum for literature review and technical discussions. In conclusion, our results are similar to those reported by Odejide et al,1 with a trend toward a lower rate of nondiagnostic samples in our series. We believe that the introduction of regular quality audits and reports and a permanent forum for discussion can greatly benefit patients by reducing specimen recollection rates. The authors made no disclosures. Gustavo Loureiro, MD, PhD, Hematology Edgar G. Rizzatti, MD, PhD, Hematology Alex F. Sandes, MD, PhD, Hematology Maria de Lourdes Chauffaille, MD, PhD, Hematology Fleury Medicine and Health Sao Paulo, Brazil

Myonecrosis by Clostridium septicum in a dog, diagnosed by a new multiplex-PCR

Clostridial myositis is an acute, generally fatal toxemia that is considered to be rare in pet animals. The present report describes an unusual canine clostridial myositis that was diagnosed by a new multiplex-PCR (mPCR) designed for simultaneous identification of Clostridium sordellii, Clostridium septicum, Clostridium perfringens type A, Clostridium chauvoei, and Clostridium novyi type A. A ten-month-old male Rottweiler dog, that had displayed lameness and swelling of the left limb for 12h, was admitted to a veterinary hospital. The animal was weak, dyspneic and hyperthermic, and a clinical examination indicated the presence of gas and edema in the limb. Despite emergency treatment, the animal died in only a few minutes. Samples of muscular tissue from the necrotic area were aseptically collected and plated onto defibrinated sheep blood agar (5%) in anaerobic conditions. Colonies suggestive of Clostridium spp. were submitted to testing by multiplex-PCR. Impression smears of the tissues, visualized with Gram and also with panoptic stains, revealed long rod-shaped organisms, and specimens also tested positive using the fluorescent antibody technique (FAT). The FAT and mPCR tests enabled a diagnosis of C.septicum myonecrosis in the dog.

Histological and enzyme histochemical investigation of the hemal nodes of the hair goat

We investigated the structure of the hemal node in six healthy hair goats using histological and enzyme histochemical methods. After processing, tissue sections were stained with Crossman's trichrome, Gordon-Sweet's silver and Pappenheim's panoptic stains. Alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) were demonstrated in frozen sections. Hemal nodes were encapsulated by connective tissue and few smooth muscle cells. Several trabeculae originated from the capsule and extended into the hemal node. A subcapsular sinus was present beneath the capsule and was continuous with the deeper sinuses. Subcapsular and deep sinuses were filled with erythrocytes. The parenchyma consisted of lymphoid follicles, diffuse interfollicular lymphocytes and irregular wide lymphoid cords. Cortical and medullary regions were not distinct. ANAE (+) and ACP-ase (+) cells were located mainly in the germinal centers of the lymphoid follicles and also were scattered equally in the interfollicular region and lymphoid cords. Monocytes, macrophages and reticular cells displayed a diffuse positive reaction, whereas localized granular positivity was observed in lymphocytes. We demonstrated that the general structure of the hair goat hemal nodes is similar to that of other ruminant species.

Pelger-Huët anomaly in two related mixed-breed dogs

A 6-month-old male mixed-breed dog weighing 12.6 kg weight was presented for evaluation of a subcutaneous nodule on the dorsum. The medical history indicated trimethoprim-sulfamethoxazole treatment 2 months before presentation at the veterinary hospital. The initial complete blood cell count (CBC) results included an apparent left shift. Microscopic examination of a blood smear (Panoptic stain) revealed granulocytes with hyposegmented nuclei, coarse mature chromatin, and a nuclear shape varying from round to bilobed (pince-nez) or slightly indented. Occasional neutrophils and eosinophils had typical segmentation of nuclei. Abnormalities were not present in limited serum biochemical testing. The CBC was repeated 17 and 120 days later, and the results were similar to those observed in the first examination. The parents of the patient were located, and a CBC was performed on both animals. The dam, but not the sire, had nuclear hyposegmentation of granulocytes, confirming the diagnosis of Pelger-Huët anomaly.

Perfil citológico dos líquidos amniótico e alantoidiano nos terços inicial, médio e final da gestação na vaca

The cytology of extraembrionic fluids in cows in the first, second, and third trimester of pregnancy was evaluated. For each trimester, 10 pregnant uteri, collected in the slaughterhouse were used. A volume of 20mL of amniotic and allantoic fluids was collected, and centrifuged at 200g for 10 minutes. The sediment was taken and processed on a slide for cytology examination after staining. The results showed that the total cells number of the amniotic fluid increased during gestation, with the panoptic staining method being efficient to evaluate the amniotic fluid cytology in crossbreed Holstein x Zebu cows.

Standardization of sample preparation, staining and sampling methods for automated sperm head morphometry analysis of boar spermatozoa

The accuracy of computer-assisted sperm morphometry analysis (CASMA) depends on the careful preparation, fixation and staining of spermatozoa. The efficiency of CASMA may be enhanced by developing optimized protocols. The aim of the present study was to evaluate the influence of sperm washing and the use of three staining techniques [rapid Panoptic, Hemacolor and Harris's Haematoxylin (HH)] on image-processing accuracy and boar sperm head morphometry. Sperm washing had a significant effect on samples stained with rapid Panoptic, increasing the percentage of correctly binarized sperm heads and the contrast between cells and background. However, rapid Panoptic yielded the lowest percentage of properly digitized sperm heads. HH provided the highest cell/background contrast, and also greater sperm head staining intensity, but discrimination of sperm midpieces was considered insufficient. Hemacolor occupied an intermediate position, providing acceptable colour intensity and satisfactory cell/background contrast. Use of different staining procedures prompted dimensional differences in sperm head morphometry. Significant differences between animals were observed for all morphometric parameters. Low within-animal variation coefficients reflected a homogeneous sperm head population. Between-animal variation coefficients were relatively high for Hemacolor and HH, and significantly high for the rapid Panoptic stain. Using Panoptic and HH, stable morphometric measurements required at least 100 properly digitized sperm heads rather than 200, while Hemacolor required only 50 spermatozoa. These results indicate that both washing of semen and staining procedures significantly affect the accuracy of image processing and sperm head dimensions. Hemacolor and HH proved to be the best staining techniques for evaluating sperm head dimensions in boar.

Morphometry of canine cutaneous mast cell tumors.

Twenty-four canine cutaneous nodules, diagnosed as mast cell tumors by fine-needle aspiration biopsy and confirmed by histopathologic analysis by staining with hematoxylin and eosin (HE) and toluidine blue, were analyzed by computerized nuclear morphometry on panoptic- and HE-stained cytopathology slides. Two hundred nuclei per lesion were examined. The morphometric parameters investigated were nuclear area, mean diameter, perimeter, regularity factor, and ellipticity factor. Lesions were graded as I (well differentiated), II (intermediate differentiation), or III (poorly differentiated) according to the following morphologic features: invasiveness, cellularity and cellular morphology, mitotic index, and stromal reaction. Nuclear morphometric results were then compared with histopathologic grades. Values of nuclear area, mean diameter, and perimeter increased with increase in histopathologic grade, but statistical analysis revealed significant differences only between grades II and III and between grades I and III when HE was used (P < 0.01) and between grades I and III with panoptic stain (P < 0.05). The ellipticity factor and regularity factor did not reveal significant differences between histopathologic grades. The results indicate that nuclear morphometric analysis, in combination with the rapid and inexpensive cytopathology technique, can help in mast cell tumor grading, thus contributing to the establishment of a more precise prognosis and treatment.

A new stain for identification of avian leukocytes

Differential staining of avian leukocytes was achieved within 6 min following brief fixation in a methanolic solution of C.I. acid red 360 followed by immersion in a mixture containing C.I. basic blue 41, C.I. basic blue 141, and C.I. acid red 52. Heterophils contained black angular and punctate granules. Eosinophils contained bright purple granules. Lymphocytes displayed red nuclei and blue cytoplasm. Monocytes contained red-brown nuclei and lavender cytoplasm. Basophils showed red-orange granules. Thrombocytes stained deep purple. Compared to traditional panoptic stains like Wright's or Giemsa's, the new staining method provides brighter colors, more precise details of cellular structures, and shorter staining time. Significantly, it facilitates identification of avian leukocyte species based on differences in color as well as differences in size and shape.