Effect of histologic processing on dimensions of skin samples obtained from cat cadavers.

OBJECTIVE To determine changes in dimensions of feline skin samples as a result of histologic processing and to identify factors that contributed to changes in dimensions of skin samples after sample collection. SAMPLE Cadavers of 12 clinically normal cats. PROCEDURES Skin samples were obtained bilaterally from 3 locations (neck, thorax, and tibia) of each cadaver; half of the thoracic samples included underlying muscle. Length, width, and depth were measured at 5 time points (before excision, after excision, after application of ink to mark tissue margins, after fixation in neutral-buffered 10% formalin for 36 hours, and after completion of histologic processing and staining with H&E stain). Measurements obtained after sample collection were compared with measurements obtained before excision. RESULTS At the final time point, tissue samples had decreased in length (mean decrease, 32.40%) and width (mean decrease, 34.21%) and increased in depth (mean increase, 54.95%). Tissue from the tibia had the most shrinkage in length and width and that from the neck had the least shrinkage. Inclusion of underlying muscle on thoracic skin samples did not affect the degree of change in dimensions. CONCLUSIONS AND CLINICAL RELEVANCE In this study, each step during processing from excision to formalin fixation and histologic processing induced changes in tissue dimensions, which were manifested principally as shrinkage in length and width and increase in depth. Most of the changes occured during histologic processing. Inclusion of muscle did not affect thoracic skin shrinkage. Shrinkage should be a consideration when interpreting surgical margins in clinical cases. 945).

Cancer screening tests for small animals.

Cancer is increasingly more common. Several tests for the diagnosis and treatment of cancer in companion animals have been developed. Screening tests discussed include those for lymphoid neoplasia, hemangiosarcoma, and transitional cell carcinoma of the bladder. None of these tests should be used in isolation for diagnosis. Vincristine and doxorubicin are mainstays in the treatment of canine lymphoma. However, it is important and accepted practice to test individuals of predisposed breeds for this mutation before administering these drugs in a lymphoma protocol.

Hypercalcemia of malignancy associated with renal cell carcinoma in a dog.

A 10 yr old castrated male Siberian husky was evaluated for polyuria, polydipsia, a retroperitoneal mass, and urolithiasis. A marked elevation in Ca was noted on initial blood work, and results of additional testing were consistent with hypercalcemia of malignancy, including an elevated parathyroid hormone-related peptide (PTHrp) value. Based on clinical signs, blood work, diagnostic imaging, and cytology results, unilateral renal neoplasia was suspected. Following a complete right nephrectomy and cystotomy, histopathologic examination confirmed a diagnosis of renal cell carcinoma (RCC). Five days postoperatively, the hypercalcemia had nearly resolved and the PTHrp was zero. This is the first reported case of hypercalcemia of malignancy associated with RCC in a dog.

May-Hegglin anomaly in a dog.

An 8-year-old female spayed Pug dog was presented for evaluation of cutaneous lesions occurring secondary to immunosuppressive treatment of presumed immune-mediated thrombocytopenia. Abnormal hematologic findings included persistent thrombocytopenia, macrothrombocytes, and variably shaped, often fusiform, blue cytoplasmic inclusions in neutrophils. May-Hegglin anomaly (MHA) was suspected based on the morphologic appearance of platelets and neutrophils. Examination of cells by transmission electron microscopy revealed normal platelet ultrastructure; neutrophil inclusions had features similar to those reported for inclusions in human MHA. Neutrophil function was within normal limits based on flow cytometric analysis. Thrombelastography indicated a prolonged clotting time (r), and PlateletMapping showed a lack of response to 2 µM ADP compared with a moderate response in the control dog. Immunocytochemical staining of blood smears using 2 commercially available antibodies against MYH9 protein (nonmuscle myosin heavy chain II) yielded negative results. However, genomic DNA sequencing analysis of the dog's MYH9 gene identified a single point mutation, resulting in substitution of lysine for glutamine at the 1841 amino acid position; this mutation is identical to one identified in people with MHA. To our knowledge, this is the first report of an MYH9 mutation in the dog. MHA-associated macrothrombocytopenia may be mistaken for immune-mediated thrombocytopenia.

Resistance of canine lymphoma cells to adenoviral infection due to reduced cell surface RGD binding integrins.

Recombinant adenovirus vectors (Ad) have been recognized as effective in vivo gene delivery vehicles and utilized as gene therapy agents for a number of cancers. The elucidation of viral entry mechanisms has allowed the development of recombinant vectors that exploit existing cell surface receptors to achieve entry into the cell. B lymphocytes are normally resistant to infection by adenovirus 5, likely due to the lack of the Coxsackie and Adenovirus receptor (CAR). Using reverse-transcriptase PCR and flow cytometry, the CD40 receptor has been shown to be expressed on many lymphoma cells. We exploited this finding to develop a gene therapy strategy for treatment of canine B cell lymphoma. Ad5 was targeted to cells expressing CD40 via CD40 ligand (CD40L) and was effective in infecting CD40-expressing control cells; however, both primary canine lymphoma cells and cell lines demonstrated limited evidence of transduction. Following receptor binding, adenovirus entry into cells may require interaction with α(v)ß(3/5) integrins; we demonstrate that canine lymphoma cells are deficient in these integrins. Reduced α(v)ß(3) integrin expression may render these cells incapable of internalizing Ad vectors. Thus, any viral targeting approaches for treatment of canine lymphoma must also take into account the potential lack of internalization signals.

Asparaginase-associated pancreatitis in a dog.

A dog with lymphosarcoma was evaluated for vomiting, lethargy, and abdominal pain 48 h after treatment with L-asparaginase. Based on drug administration, clinical signs, bloodwork, and elevated canine pancreatic lipase immunoreactivity, L-asparaginase-associated pancreatitis was diagnosed. This is an acknowledged toxicity; however, its pathophysiology and incidence rate in veterinary patients are unknown and sparsely documented.

Flow cytometric evaluation of multidrug resistance proteins on grossly normal canine nodal lymphocyte membranes.

OBJECTIVE: To demonstrate efficacy of flow cytometric evaluation of expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) efflux pumps and characterize and correlate their expression and activity in grossly normal canine nodal lymphocytes. SAMPLE POPULATION: Nodal lymphocytes from 21 clinically normal dogs. PROCEDURES: Pump expression was assessed by use of fluorescent-labeled mouse antihuman P-gp (C494) and MRP1 (MRPm6) antibodies and expressed as median values (antibody value divided by isotype control value). The P-gp and MRP activities were assessed by measuring cellular retention of rhodamine 123 and 5(6)-carboxyfluorescein diacetate in the absence and presence of inhibitors (verapamil and PSC833 for P-gp, probenecid and MK-571 for MRP). Protein activity was expressed as median fluorescence of cells with inhibitors divided by that without inhibitors. RESULTS: Expression of P-gp was (mean +/- SEM) 50.62 +/- 13.39 (n = 21) and that of MRP was 2.16 +/- 0.25 (13). Functional activity was 1.27 +/- 0.06 (n = 21) for P-gp and both inhibitors and 21.85 +/- 4.09 (21) for MRP and both inhibitors. Function and expression were not correlated. CONCLUSIONS AND CLINICAL RELEVANCE: Use of flow cytometry effectively assessed P-gp and MRP expression and activity in canine lymphocytes. Optimization of the flow cytometric assay was determined for evaluating activity and expression of these pumps in canine lymphoid cells. Evaluation of expression or activity may offer more meaning when correlated with clinical outcome of dogs with lymphoproliferative diseases. Cell overexpression of P-gp and MRP can convey drug resistance.