The cell tube block technique and an immunohistochemistry panel including Wilms tumor 1 to assist in diagnosing cavitary effusions in dogs and cats.

BACKGROUND: Cell blocks and immunohistochemistry (IHC) are increasingly recognized as being complementary tools for cytologic diagnostics, especially for neoplastic diseases. OBJECTIVES: The study aimed to evaluate the utility of cell tube block (CTB) IHC for refining the diagnosis of effusions in dogs and cats. METHODS: Cavitary effusions (n = 25) from dogs and cats classified by cytology as reactive, neoplastic, borderline (suspicious of neoplasia), and chylous were studied. CTB sections were stained with H&E, and immunostained with PAX-5, CD3, pancytokeratin (CK), vimentin, and Wilms tumor 1 protein (WT1) antibodies, according to the cytologic diagnoses. A histologic case series of confirmed normal, reactive, and neoplastic mesothelium and several different carcinomas were included to test the utility of WT1 as a marker of mesothelial cells. RESULTS: CTBs had a layered appearance with reduced background staining. CD3 and PAX5 immunolabeling allowed immunophenotype assessment in all of the lymphoma cases. In carcinomatous effusions, neoplastic cells were CK-positive, WT1-negative, and vimentin-negative (except for two cases). Wilms tumor 1 protein was positive in the nuclei of normal, reactive, and neoplastic mesothelial cells, and ovarian carcinomatous cells. Other carcinomas and lymphomas were negative. CONCLUSIONS: CTBs are valuable tools to assist in making a diagnosis of cavitary effusions in dogs and cats, and WT1 is a promising marker to differentiate mesothelial from carcinomatous cells.

Feline Sporothrix spp. detection using cell blocks from brushings and fine-needle aspirates: Performance and comparisons with culture and histopathology.

BACKGROUND: Sporotrichosis is an emerging zoonotic mycosis that presents as a cutaneous lymphatic or disseminated disease, caused by fungi from the Sporothrix schenkii (S schenkii) clinical clade. Its importance is growing, primarily due to an outbreak that occurred in Brazil, affecting mainly cats and people. OBJECTIVES: In Brazil, an S schenkii diagnosis is often made using cultures, which allows genus identification and sufficient growth to perform molecular biology testing. Despite its advantages, fungal cultures are slow to develop and can delay public health measures, highlighting the importance of developing additional diagnostics techniques. METHODS: Cell block cytology (CBLC) is an older method that regained importance after liquid-based cytology (LBC) was introduced, and it has been previously and successfully applied to veterinary diagnostics. We aimed to standardize and compare CBLC from cervical brush exfoliation of open wounds and fine-needle aspirates with culture and immunohistochemistry of skin biopsies for sporotrichosis in cats, as a novel method. RESULTS: For this purpose, we selected 40 cats with skin lesions suspected of having sporotrichosis in Guarulhos city, São Paulo state, Brazil. We achieved 97.5% and 95% positivity using CBLC and culture, respectively, and 100% of feline skin biopsies were positive for Sporothrix spp on histopathology/immunohistochemistry. CONCLUSIONS: Cell block cytology is an efficient and rapid tool to diagnose sporotrichosis in cats, particularly during epidemics.

Cell tube block: a new technique to produce cell blocks from fluid cytology samples.

BACKGROUND: Cell blocks are widely used in human cytopathology. Several techniques have been proposed to convert fluid specimens into solid material, which after embedding in paraffin can be used for histochemistry, immunohistochemistry, or molecular testing. In contrast, only in the last few years, have cell blocks begun to be used in the veterinary field. OBJECTIVES: The purpose of the study was to present the production and features of cell tube blocks (CTB) with veterinary liquid samples. METHODS: Liquid samples including cerebrospinal fluid, cutaneous cyst fluid, pericardial and pleural effusions, bronchoalveolar lavage, urine, and blood were centrifuged in a microhematocrit centrifuge. Capillary tubes were broken at the liquid-solid interface and fixed in 10% formalin for 24 hours. After paraffin embedding, sections of CTB were used for different stains including immunohistochemistry. RESULTS: The morphology and cellular detail in CTB sections were comparable to conventional histologic sections and other existing cell block techniques. The use of special stains such as Gram, Giemsa, alcian blue, and periodic acid-Schiff was straightforward, and immunohistochemistry results with antibodies to pan-cytokeratin, PAX-5, and CD3 were considered good. CONCLUSIONS: The CTB method was easily implementable under practice conditions (up to the fixation of the microhemtocrit tube), analogous to surgical biopsy submission for histology. Cell tube blocks can increase diagnostic accuracy when the technique is used in tandem after the cytologic evaluation, and the technique allows storage of fluids. Other advantages of CTB were the simplicity, low cost, and separation of erythrocytes from the nucleated cells, which was helpful in hemodiluted samples.

Immunohistochemical analysis of low-temperature methylmethacrylate resin-embedded goat tissues.

Goats are frequently used as a suitable animal model for tissue engineering. Immunohistochemistry can be helpful in improving the understanding and evaluation of the in vivo tissue responses at a molecular level. Several commercially available antibodies (KI67, vimentin, CD31, core-binding factor alpha-1, osteocalcin, alkaline phosphatase, MAC387, CD3, CD20, CD20cy, CD79 and CD45) were evaluated on Technovit 9100 New embedded goat tissues. Only vimentin, osteocalcin, MAC387 and CD3 revealed positive staining. These antibodies can be routinely used to evaluate goat tissues at molecular level. The use and development of alternative antibodies might further supplement and complete the possibilities for immunohistochemical analysis of goat tissue samples.

Effectiveness of a computer-based tutorial for teaching how to make a blood smear.

BACKGROUND: Computer-aided instruction (CAI) was developed to teach veterinary students how to make blood smears. This instruction was intended to replace the traditional instructional method in order to promote efficient use of faculty resources while maintaining learning outcomes and student satisfaction. OBJECTIVES: The purpose of this study was to evaluate the effect of a computer-aided blood smear tutorial on 1) instructor's teaching time, 2) students' ability to make blood smears, and 3) students' ability to recognize smear quality. METHODS: Three laboratory sessions for senior veterinary students were taught using traditional methods (control group) and 4 sessions were taught using the CAI tutorial (experimental group). Students in the control group received a short demonstration and lecture by the instructor at the beginning of the laboratory and then practiced making blood smears. Students in the experimental group received their instruction through the self-paced, multimedia tutorial on a laptop computer and then practiced making blood smears. Data was collected from observation, interview, survey questionnaires, and smear evaluation by students and experts using a scoring rubric. RESULTS: Students using the CAI made better smears and were better able to recognize smear quality. The average time the instructor spent in the room was not significantly different between groups, but the quality of the instructor time was improved with the experimental instruction. CONCLUSIONS: The tutorial implementation effectively provided students and instructors with a teaching and learning experience superior to the traditional method of instruction. Using CAI is a viable method of teaching students to make blood smears.

Effective antigen-retrieval method for immunohistochemical detection of abnormal isoform of prion proteins in animals.

For immunohistochemistry of the prion diseases, several pretreatment methods to enhance the immunoreactivity of human and animal abnormal proteinase-resistant prion protein (PrP(Sc)) on the tissue sections have been employed. The method of 121 degree C hydrated autoclaving pretreatment or the combination method of 121 degree C hydrated autoclaving with a certain chemical reagent (formic acid or proteinase K, etc) are now widely used. We found that an improved hydrated autoclaving method at 135 degrees C, more effectively enhanced PrP(Sc) immunoreactivity for the antibodies recognizing the linear epitope. In addition, this method was more effective for the long-term fixation samples as compared with other previous methods. However, this modified method could not retrieve PrP(Sc) antigenic epitopes composed of conformational structures or several discontinuous epitopes. We describe the comparative studies between our improved method and other antigen-retrieval procedures reported previously. Based on the differences of reaction among the antibodies, we also discuss the mechanisms of the hydrated autoclaving methods to retrieve PrP(Sc) immunoreactivity.

An inexpensive sedimentation chamber for the preparation of cytologic specimens of cerebrospinal fluid.

An inexpensive sedimentation chamber to obtain cytologic specimens of cerebrospinal fluid (CSF) is described. The device, which has a total cost of about $5.00 can be built in few minutes. The device permits the cytologic study of specimens of CSF in clinics, where because of economic constraints, a cytocentrifuge is not available. The device permits the study of the CSF cells on either air-dried or wet smears even under field conditions, and the results obtained are consistent. Also, the device permits to retrieve the cell-free fluid for its use in chemical or immunologic procedures.

Observations of aspergillosis in the brains of turkey poults using different histopathological staining techniques.

Different staining methods were evaluated for studying aspergillosis of the central nervous system (CNS). The pathological changes and fungal elements in cerebrum and cerebellum of 17 turkey poults with aspergillosis were examined and described. Tissue sections were stained with hematoxylin-eosin (HE), Kluver-Barrera's and Grocott's methods, and periodic acid-Schiff (PAS). Focal granulomatous reactions with central necrosis were observed in the HE stained slides. Fungal hyphae were easily demonstrated using Grocott's method and PAS. These two methods, however, were not suitable for describing detailed histopathological changes. The Kluver-Barrera method was used to demonstrate the neural tissue reaction. Neurons were found to be sensitive to aspergillosis, in contrast to glial cells that showed fewer pathological changes. The fungal elements were clearly visible with the Kluver-Barrera method, resulting in better information about the interactions of neural tissue, the inflammatory response, and the fungus. The use of the Kluver-Barrera method for this purpose has not been documented previously.