Francesco Cian

Stability of immunophenotypic lymphoid markers in fixed canine peripheral blood for flow cytometric analysis

BACKGROUND Flow cytometric analysis of blood samples for immunophenotyping lymphoproliferative diseases has become popular in veterinary medicine. Unfortunately, the use of this technique has been limited by the necessity to test samples within a short time frame after blood collection. A possible solution to this problem is the use of fixative products to preserve the stability of lymphoid antigens. OBJECTIVES The aim of this study was to evaluate the expression of 5 lymphoid surface markers (CD3, CD4, CD8, CD21, and CD45) in blood samples collected in K3-EDTA and Cyto-Chex BCT tubes from healthy dogs. METHODS Blood from 8 dogs was collected in K3-EDTA and Cyto-Chex BCT tubes and analyzed by flow cytometry at 6 hours, one day, 3 days, and 7 days after collection. Lymphocyte percentage, lymphocyte mor-phology, expression of lymphoid surface markers and mean fluorescence intensity (MFI) were recorded at each time point and compared. RESULTS Lymphocyte percentage and morphology were preserved up to 3 days in samples collected in Cyto-Chex BCT, and lymphocyte percentage was mildly decreased on day 7. CD4, CD8, and CD21 were stable in Cyto-Chex BCT up to 7 days, whereas CD3 and CD45 showed a significant decrease in expression from day 3, with a decrease on average of 21% and 2.4%, respectively, on day 7. MFI was significantly decreased on day 7 for all markers except CD21. CONCLUSIONS These findings indicate that storage of samples in Cyto-Chex BCT affects lymphoid marker expression and caution should be exercised when interpreting data produced on such samples.

Immunocytochemical demonstration of feline infectious peritonitis virus within cerebrospinal fluid macrophages

A 4-month-old female entire domestic shorthair cat presented with an acute onset of blindness, tetraparesis and subsequent generalised seizure activity. Haematology and serum biochemistry demonstrated a moderate, poorly regenerative anaemia, hypoalbuminaemia and hyperglobulinaemia with a low albumin:globulin ratio. Serology for feline coronavirus antibody was positive with an elevated alpha-1 acid glycoprotein. Analysis of cisternal cerebrospinal fluid (CSF) demonstrated markedly elevated protein and a mixed, predominately neutrophilic pleocytosis. Immunocytochemistry for feline coronavirus was performed on the CSF, with positive staining observed inside macrophages. The cat was subsequently euthanased, and both histopathology and immunohistochemistry were consistent with a diagnosis of feline infectious peritonitis. This is the first reported use of immunocytochemistry for detection of feline coronavirus within CSF macrophages. If this test proves highly specific, as for identification of feline coronavirus within tissue or effusion macrophages, it would be strongly supportive of an ante-mortem diagnosis of feline infectious peritonitis in cats with central nervous system involvement without the need for biopsy.

DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia, modelling human myeloid leukaemia

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C-terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs.

The use of flow cytometry for immunophenotyping lymphoproliferative disorders in cats: a retrospective study of 19 cases

Flow cytometric immunophenotyping is a useful step in the diagnosis of lymphoproliferative malignancies in human and veterinary medicine. The purpose of this study was to assess the usefulness of this technique for the diagnosis of lymphoproliferative disorders in cats. Nineteen cats were retrospectively enrolled in this study and allocated into two groups. Group 1 consisted of 13 cats with lymphoma, whereas group 2 consisted of 6 cats with non-neoplastic lymphoproliferative disorders. Fine-needle aspiration biopsies were analysed by flow cytometry in order to evaluate the immunophenotype. Flow cytometric analysis identified a neoplastic lymphoid population in 12 of the 13 cats of group 1, confirming the diagnosis of lymphoma and further characterizing it. The six cats in group 2 showed a mixed lymphoid population, which was not suggestive of a neoplastic disorder. Flow cytometry is a valuable and powerful tool for refining the diagnosis of feline lymphoproliferative disorders.

Use of Six Sigma Worksheets for assessment of internal and external failure costs associated with candidate quality control rules for an ADVIA 120 hematology analyzer

BACKGROUND Quality control (QC) validation is an essential tool in total quality management of a veterinary clinical pathology laboratory. Cost-analysis can be a valuable technique to help identify an appropriate QC procedure for the laboratory, although this has never been reported in veterinary medicine. OBJECTIVE The aim of this study was to determine the applicability of the Six Sigma Quality Cost Worksheets in the evaluation of possible candidate QC rules identified by QC validation. METHODS Three months of internal QC records were analyzed. EZ Rules 3 software was used to evaluate candidate QC procedures, and the costs associated with the application of different QC rules were calculated using the Six Sigma Quality Cost Worksheets. The costs associated with the current and the candidate QC rules were compared, and the amount of cost savings was calculated. RESULTS There was a significant saving when the candidate 1-2.5s, n = 3 rule was applied instead of the currently utilized 1-2s, n = 3 rule. The savings were 75% per year (£ 8232.5) based on re-evaluating all of the patient samples in addition to the controls, and 72% per year (£ 822.4) based on re-analyzing only the control materials. The savings were also shown to change accordingly with the number of samples analyzed and with the number of daily QC procedures performed. CONCLUSIONS These calculations demonstrated the importance of the selection of an appropriate QC procedure, and the usefulness of the Six Sigma Costs Worksheet in determining the most cost-effective rule(s) when several candidate rules are identified by QC validation.

A case of giant cell tumor of soft parts in a horse

A 12-year-old British Warmblood mare was examined by the referring veterinarian for evaluation of a cutaneous lesion on the dorsal thorax to the right of the midline. Cytologic examination of fine-needle aspirates from the mass was supportive of a giant cell tumor of soft parts (GCTSP). Laser surgical excision and postoperative methyl aminolevulinate (MAL) photodynamic therapy (PDT) were performed. Histologic examination of the mass confirmed the cytologic diagnosis. At 8 months from surgery, no evidence of recurrence has been observed. Giant cell tumors of soft parts are rare cutaneous neoplasms, observed in several domestic species, including the horse where they commonly appear as superficial cutaneous lesions without aggressive biologic behavior. Previously classified as giant cell variant of malignant fibrous histiocytoma, these superficial tumors have now been designated as giant cell tumors of soft tissue or giant cell tumors of low malignant potential within the category of fibrohistiocytic neoplasms.

What is your diagnosis? Swelling of the left antebrachium and carpus in a horse

An 8-month-old Thoroughbred colt was presented to Newmarket Equine Hospital with a 6-week history of swelling of the left antebrachium and carpus. On admission, the horse was bright and alert. Temperature, pulse, and respiration rate were normal. The animal exhibited marked left forelimb lameness. There was marked tense distension of the carpal sheath of the extensor carpi radialis with swelling extending proximally beyond the musculotendinous junction. Carpal flexion was reduced to approximately 10° only. Radiographic examination demonstrated periosteal new bone on the dorsodistal radius, bordering the extensor carpi radialis grooves. Ultrasonography revealed extensive disruption of the extensor carpi radialis muscle and displacement of its tendon of insertion. CBC and serum biochemical profile were unremarkable. At tenoscopy under general anesthesia, the extensor carpal radialis tendon sheath was filled with organized hemorrhagic tissue which extended also proximally, infiltrating themuscle.Multiple small incisional biopsies and impression smears were taken from the lesion. The impression smears were stained with Wright–Giemsa (Figure 1A and B). A

What is your diagnosis? Cerebrospinal fluid from a dog

An 8-year-old female neutered Springer Spaniel dog was presented as an emergency case for investigation of progressive ataxia of all 4 limbs, staggering and behavioral changes. Neurologic signs started 2 weeks before referral with further acute deterioration. The dog had a solid mammary carcinoma surgically removed one year previously, which recurred 6 months later andwas also resected. On physical examination, the dog was tachypneic with hyperemicmucousmembranes. The dog was obtunded, nonambulatory tetraparetic with decreased postural reactions and increased spinal cord segmental reflexes in all 4 limbs. The neurologic examination was indicative of multifocal brain localization. Hematology, biochemistry, electrolytes, and urinalysis were unremarkable and there were no abnormalities on thoracic radiographs and in an abdominal ultrasound examination. Magnetic resonance imaging (MRI) revealed a mass lesion with perilesional edema in the left temporal lobe that was hyperintense on the T2-weighted and fluid-attenuated inversion recovery (FLAIR) images. The mass was enhanced after contrast administration. A cerebrospinal fluid (CSF) sample was collected aseptically from the cisterna magna immediately after the MRI study (Figure 1). A