Effects of fixation and demineralization on histomorphology and DNA amplification of canine bone marrow.

Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA amplification is incompletely understood. In this study, 2 fixatives and 3 demineralization methods were tested on canine BM samples. Twenty replicate sternal samples obtained within 24 hours of death were fixed overnight in either acetic acid-zinc-formalin (AZF) or 10% neutral-buffered formalin (NBF) and demineralized with formic acid for 12 hours. Another 53 samples were fixed in AZF and demineralized with hydrochloric acid for 1-hour, formic acid for 12 hours, or ethylenediamine tetraacetic acid (EDTA) for 24 hours. Histologic sections were scored by 4 raters as of insufficient, marginal, good, or excellent quality. In addition, DNA samples extracted from sections treated with the different fixation and demineralization methods were amplified with 3 sets of primers to conserved regions of T cell receptor gamma and immunoglobulin heavy chain genes. Amplification efficiency was graded based on review of capillary electrophoretograms. There was no significant difference in the histomorphology scores of sections fixed in AZF or NBF. However, EDTA-based demineralization yielded higher histomorphology scores than demineralization with hydrochloric or formic acid, whereas formic acid resulted in higher scores than hydrochloric acid. Demineralization with EDTA yielded DNA amplification in 29 of 36 (81%) samples, whereas demineralization with either acid yielded amplification in only 2 of 72 (3%) samples. Although slightly more time-consuming and labor-intensive, tissue demineralization with EDTA results in superior morphology and is critical for polymerase chain reaction (PCR) amplification with the DNA extraction method described in this article.

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes.

Introduction: The domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. Methods: Peripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5' reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. Results: Unsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. Discussion: Our study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Antemortem cytologic diagnosis of pulmonary Langerhans cell histiocytosis in a cat.

Feline pulmonary Langerhans cell histiocytosis (FPLCH) is a rare histiocytic proliferative disease of middle-aged to older domestic cats. Langerhans cells in the terminal airways proliferate and infiltrate the interstitium and the airways to a lesser degree, widely effacing normal parenchyma. Historically, definitive diagnosis has required postmortem evaluation where pulmonary lesions have a classic gross and histologic morphology. Here, we present the first documented antemortem diagnosis of FPLCH using bronchoalveolar lavage (BAL) cytology and immunocytochemistry (ICC) in a 9-year-old British shorthair mix. The cat had a 3-month history of respiratory difficulty that was refractory to steroids and antimicrobials. Pulmonary radiographs had marked diffuse changes with a complex bronchointerstitial and micronodular pattern. BAL cytology revealed neutrophilic inflammation and markedly increased histiocytes with morphology distinct from typical pulmonary macrophages. ICC characterized histiocytes as CD1a+ /E-cadherin+ /CD11b- /PanCK- , consistent with a Langerhans cell phenotype. The cat was humanely euthanized due to poor prognosis and presented for necropsy. Gross, histopathologic, immunophenotypic, and ultrastructural findings confirmed a diagnosis of FPLCH. Proliferative cells were E-cadherin+ /Iba-1+ /CD18+ /CD1a+ /CD5+ /MHCII+ /CD204- /CD4- ; transmission electron microscopy identified the presence of Birbeck granules in the proliferating histiocytes, consistent with previous reports of FPLCH.

Large T-cell extradural lymphoma with concurrent marked cerebrospinal fluid eosinophilia in a dog.

A 3-year-old male pit bull terrier was presented for a 4-day history of progressive tetraparesis and cervical pain. Magnetic resonance imaging confirmed an extradural mass within the left lateral vertebral canal extending from caudal C5 to mid-T2. Lumbar cerebrospinal fluid (CSF) demonstrated marked (90%) eosinophilic inflammation. A C6-7 dorsal laminectomy and C7-T2 left hemilaminectomy were done, with gross disease remaining. Histopathology revealed a large T cell lymphoma with marked eosinophilic infiltration. The dog underwent CHOP-based chemotherapy with resolution of clinical signs, with a similar course of therapy performed at recurrence 37 months after initial presentation. The dog was euthanized 39 months after presentation for multiorgan failure secondary to neutropenic sepsis and aspiration pneumonia. This represents a positive long-term response to multimodal treatment of extradural T-cell lymphoma within the vertebral canal associated with a marked CSF eosinophilia.

Oral cytarabine ocfosfate pharmacokinetics and assessment of leukocyte biomarkers in normal dogs.

BACKGROUND: Cytosine arabinoside (Ara-C) is a nucleoside analog prodrug utilized for immunomodulatory effects mediated by its active metabolite Ara-CTP. Optimal dosing protocols for immunomodulation in dogs have not been defined. Cytarabine ocfosfate (CO) is a lipophilic prodrug of Ara-C that can be administered PO and provides prolonged serum concentrations of Ara-C. OBJECTIVES: Provide pharmacokinetic data for orally administered CO and determine accumulation and functional consequences of Ara-CTP within peripheral blood leukocytes. ANIMALS: Three healthy female hound dogs and 1 healthy male Beagle. METHODS: Prospective study. Dogs received 200 mg/m2 of CO PO q24h for 7 doses. Serum and cerebrospinal fluid (CSF) CO and Ara-C concentrations were measured by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Complete blood counts, flow cytometry, and leukocyte activation assays were done up to 21 days. Incorporation of Ara-CTP within leukocyte DNA was determined by LC-MS/MS. RESULTS: Maximum serum concentration (Cmax ) for Ara-C was 456.1-724.0 ng/mL (1.88-2.98 µM) and terminal half-life was 23.3 to 29.4 hours. Cerebrospinal fluid: serum Ara-C ratios ranged from 0.54 to 1.2. Peripheral blood lymphocyte concentrations remained within the reference range, but proliferation rates poststimulation were decreased at 6 days. Incorporation of Ara-CTP was not saturated and remained >25% of peak concentration at 13 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Oral CO may produce prolonged serum Ara-C half-lives at concentrations sufficient to induce functional changes in peripheral leukocytes and is associated with prolonged retention of DNA-incorporated Ara-CTP. Application of functional and active metabolite assessment is feasible and may provide more relevant data to determine optimal dosing regimens for Ara-C-based treatments.

Effect of time and autologous serum addition on the analysis of cerebrospinal fluid in horses.

BACKGROUND: Cerebrospinal fluid (CSF) is highly labile and delayed processing might alter results of analysis. HYPOTHESIS/OBJECTIVES: To determine the effects of time and addition of autologous serum on cytological evaluation of CSF. ANIMALS: Ten client-owned adult horses requiring euthanasia. METHODS: Prospective study. Serum and CSF were collected from each horse before and within 10 minutes after euthanasia. CSF samples were divided into 15 aliquots (2 mL each); 1 aliquot was submitted for routine CSF analysis within 60 minutes of collection. Four drops of autologous serum were added to 7 of the aliquots, and stored at 4°C (serum group); the remaining 7 samples were stored unaltered at 4°C (control group). Total nucleated cell count (TNCC) and cell morphology score were done at T4, T8, T12, T24, T48, T72, and T96 hours after collection. Protein concentration was measured in the control group at T0 and T96 hours. RESULTS: The cell morphology scores were significantly different in the control group at T48 (median 2, range 0-4), T72 (2, 0-4), and T96 (3, 0-4) in comparison to T0 (1). No change was observed in the serum group. TNCC remained stable over time in both groups. No statistically significant difference in CSF protein concentration was found between T0 and T96. CONCLUSIONS AND CLINICAL IMPORTANCE: The addition of autologous serum to an aliquot of CSF sample before shipping improves the preservation of cell morphology up to 96 hours after collection.

Metal reactivity is present in dogs with tibial plateau leveling osteotomy and total hip replacement implants.

OBJECTIVE: Determine whether dogs with well-functioning orthopedic metal implants can develop metal reactivity. SAMPLE: Client-owned dogs that had tibial plateau leveling osteotomy (TPLO) or total hip replacement (THR) implants for 12 months or more and control dogs with no implants. PROCEDURES: Lymphocyte transformation testing was performed by exposing peripheral blood lymphocytes to nickel (Ni), chromium (Cr), cobalt (Co), or a combination of these metals. Lymphocyte proliferation was assessed with flow cytometry. Lymphocyte stimulation indexes (SIs) were calculated. A SI > 2 was considered reactive. Median SIs of dogs in response to metal exposure were compared statistically. RESULTS: Samples from 10 dogs with TPLO, 12 dogs with THR, and 7 control dogs were analyzed. Six dogs out of 22 with metal implants had a reactive SI to 1 or more metals, while 2 of 7 control dogs had a SI > 2 when exposed to nickel only. When all metals were considered, no differences in metal reactivity were found between TPLO, THR, and control groups. CLINICAL RELEVANCE: Metal reactivity is present in dogs and can be identified using lymphocyte transformation testing. Reactivity to Ni is present in dogs with and without metal implants. Reactivity to Co and Cr occurs in some dogs with metal implants.

Validation and method comparison for a point-of-care lateral flow assay measuring equine whole blood insulin concentrations.

The Wellness Ready Test (WRT) is a lateral flow, stall-side assay that measures equine insulin in whole blood and requires validation before recommending clinical use. We evaluated intra- and inter-assay precision and linearity and compared the WRT with a radioimmunoassay (RIA). Tested concentrations ranged from <139 to >695 pmol/L (<20 to >100 µIU/mL). For 20 replicates at each insulin level, intra-assay CVs of the WRT for insulin were 13.3%, 12.9%, and 15.3% at low (139-278 pmol/L; 20-40 µIU/mL), intermediate (278-417 pmol/L; 40-60 µIU/mL), and high (>417 pmol/L; >60 µIU/mL) concentrations, respectively. For 10 replicates at each level (3 assay lots), inter-assay CVs were 15.9%, 11.0%, and 11.7%, respectively. In the weighted linear regression of 5 measured insulin concentrations against expected concentrations, R2 = 0.98, slope = 1.02, and y-intercept = 14.4 pmol/L (2.08 µIU/mL). The Spearman correlation coefficient (rs) was 0.90 (95% CI: 0.85-0.94) between the WRT and RIA; the WRT = f(RIA) Passing-Bablok regression yielded the fit, y = 1.005x + 24.3 pmol/L (3.50 µIU/mL). The WRT result averaged 10.4% higher than the RIA result, with targeted bias of 25.9, 26.1, and 26.7 pmol/L (3.74, 3.76, and 3.84 µIU/mL) for cutoffs used to diagnose insulin dysregulation of 312, 347, and 451 pmol/L (45, 50, and 65 µIU/mL). Assay clinical sensitivities, specificities, and accuracies determined at the 3 selected clinical cutoffs and using the RIA as gold standard were 87-95%, 92-96%, and 91-95%, respectively (n = 99 samples). Observed total error was 28.4-30.4%. The WRT had acceptable precision, excellent linearity, and good association with the RIA.

Pericardial effusion in a dog due to T-cell lymphoma of granular lymphocyte type.

Pericardial effusions in dogs are most often diagnosed as haemorrhagic and idiopathic. Pericardial effusions secondary to an underlying neoplastic process are infrequently diagnosed, as neoplastic cells are rarely observed in a sample of the effusion. In the present report, we describe a 9-year-old dog with pericardial effusion due to T-cell lymphoma of granular lymphocyte type. Immunophenotyping and molecular clonality PCR were performed to confirm the cytologic diagnosis. To our knowledge, this is the first report of pericardial effusion in a dog due to T-cell lymphoma of granular lymphocyte type.

Clinical and pathological findings of rabbits with lymphoma: 16 cases (1996-2019).

OBJECTIVE: To determine the clinical and pathological findings of rabbits diagnosed with lymphoma. ANIMALS: 16 rabbits. PROCEDURES: The medical and pathology records database of the Veterinary Medical Teaching Hospital at the University of California, Davis was searched for rabbits diagnosed with lymphoma from 1996 to 2019. RESULTS: Mean age of the 16 rabbits was 8 years (range, 4.5 to 12 years). Immunophenotyping was performed in 14 cases. Diffuse, large, B-cell lymphoma was most common (n = 7) followed by epitheliotropic, T-cell lymphoma (2); type II enteropathy-associated, T-cell lymphoma (2); marginal-zone, B-cell lymphoma (1); peripheral, T-cell lymphoma not otherwise specified (cutaneous nonepitheliotropic lymphoma; 1); primary, mediastinal (thymic) large B-cell lymphoma (1), and unclassified (cytology only with no immunophenotyping; 2). Multiple chemotherapy protocols were used on the basis of each individual animal's disease state. Initial clinical improvement was reported for most rabbits receiving chemotherapy (5/6), with diffuse B-cell lymphoma responding most favorably to treatment. The 11 rabbits included in the survival analysis had a median survival time of 60 days (range, 1 to 480 days), and those diagnosed with B- and T-cell lymphoma had a median survival time of 8 and 36 days (range, 1 to 150 and 1 to 90 days), respectively. CLINICAL RELEVANCE: Rabbits develop a range of lymphoma subtypes and, similar to humans and dogs, diffuse large B-cell lymphoma appears to be the most common. Chemotherapy treatments followed multiple protocols, which were mostly well tolerated and had a highly variable response. Further research into chemotherapy protocols is needed to optimize treatment of lymphoma in rabbits.

Novel clonality assays for T cell lymphoma in cats targeting the T cell receptor beta, T cell receptor delta, and T cell receptor gamma loci.

BACKGROUND: T cell clonality assays in veterinary medicine currently target only the T cell receptor gamma (TRG) locus. Existing assays have suboptimal sensitivity because of insufficient primer coverage of all possible rearrangements. OBJECTIVE: Develop higher sensitivity clonality assays targeting the TRG, delta (TRD), and beta (TRB) loci in cats. ANIMALS: Cats with histopathologically confirmed lymphoma (n = 89), non-lymphoma (n = 35), and possible hepatic small cell lymphoma (n = 31). METHODS: Molecular clonality assay development utilizing our recently reported topology and expressed repertoire data of the T cell receptor loci in cats. Determination of clonality status of lymphoma, non lymphoma, and possible hepatic small cell lymphoma samples, and calculation of assay sensitivity and specificity. RESULTS: The new multiplex TRG assay yielded the highest sensitivity (95.5%). All assays yielded 100% specificity except for the new multiplex TRG assay (97.3%). The combination of the new TRG and TRB assays yielded sensitivity of 98.9% and specificity of 97.0%. The new TRG assay detected clonality in 17/31 possible small cell lymphoma livers, whereas an existing TRG assay detected clonality in 6/31 livers. CONCLUSIONS AND CLINICAL IMPORTANCE: The assessment of multiple T cell loci compensates for the potential shortcomings of individual assays. Using a combination of molecular clonality assays will increase the overall sensitivity for the diagnosis of T-cell lymphoma in cats, especially intestinal, and hepatic small cell lymphoma. Hepatic small cell lymphomas detected by the new TRG assay utilized rarely expressed V and J genes not recognized by previous assays, likely indicating unique biology of hepatic small cell lymphoma in cats.

Treatment of indolent cutaneous T-cell lymphoma with hypofractionated radiation therapy in three cats.

BACKGROUND: Feline indolent cutaneous T-cell lymphoma (ICL) is an uncommon neoplastic disease. There is currently no consensus on treatment recommendations for ICL. OBJECTIVE: To report the clinical outcome of three cats with ICL treated with hypofractionated electron-beam radiotherapy (RT). ANIMALS: Three privately owned cats with ICL. MATERIALS AND METHODS: Medical records and client surveys were reviewed. A diagnosis of probable ICL was based on history, clinical presentation and histopathological findings, and confirmed using CD3 immunohistochemical analysis and PCR for antigen receptor gene rearrangement (PARR). All cats were treated with hypofractionated RT (four fractions of 8 Gy). RESULTS: All cats presented with skin lesions characterised by erythema and alopecia that were refractory to previous treatment with systemic glucocorticoids. Before hypofractionated RT treatment, lesions were histologically described as having diffuse infiltration of the dermis with CD3+ T cells. Molecular clonality analysis revealed clonal T-cell receptor gamma gene rearrangement. After RT, two cats showed histological improvement defined by decreased infiltration of lymphocytes, with cellular infiltrate present only in the deeper dermis; one cat had near complete histological resolution of lesions with only minimal residual lymphocytes. One cat was determined to have a complete clinical response while the other showed partial responses. No acute adverse effects of radiation were observed; chronic effects included leukotrichia, partial alopecia and mild fibrosis. All clients reported improvement in quality of life for their cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical and histological improvement in these cats suggests that hypofractionated RT can be a useful treatment modality for cats with ICL.

Correlations between clinical signs and corneal cytology in feline eosinophilic keratoconjunctivitis.

OBJECTIVE: To assess correlations between clinical and cytological features of feline eosinophilic keratoconjunctivitis at the time of cytological diagnosis. ANIMALS STUDIED: Fifteen client-owned, domestic breed cats (18 eyes) examined between 2007 and 2019. PROCEDURES: An electronic search and medical record review of cats diagnosed with feline eosinophilic keratitis or keratoconjunctivitis (FEK) based on clinical examination findings and eosinophils detected on corneal cytology were conducted. Clinical severity was graded using a modified version of a previously validated semiquantitative preclinical ocular toxicology scoring (SPOTS) system. Clinical grades were assigned following review of clinical images and medical record descriptions, and cytological grades were assigned following review of archived corneal cytology slides. Correlations were analyzed for significance using Spearman's rank correlation coefficient. RESULTS: Higher total corneal scores correlated with higher total conjunctival scores, but not with total fluorescein scores. Small lymphocyte scores correlated negatively with scores for collagen degeneration or mineralization. Globule leukocytes, a unique cell type not previously described in ocular cytology, were identified in 4 of 18 cytological samples. Higher globule leukocyte scores were correlated with higher scores for mast cells or plasma cells. Specimens with lower eosinophil scores had higher globule leukocyte scores. CONCLUSIONS: Large variability was detected in the cytological characteristics and clinical features of FEK-affected cats. This is the first report of globule leukocytes being identified in ocular cytology from any species. The role of globule leukocytes in the etiopathogenesis and progression of FEK remains unknown and warrants further investigation.

Clinicopathologic and radiographic features in 33 cats with aspiration and 26 cats with bronchopneumonia (2007-2017).

BACKGROUND: Aspiration pneumonia (AP) and bronchopneumonia (BP) are poorly characterized diseases in cats that share clinical similarities to inflammatory airway disease (IAD). OBJECTIVES: Describe clinicopathologic, radiographic, and microbiologic features in cats with AP and BP and compare findings to those in cats with IAD. ANIMALS: Thirty-three cats with AP and 26 with BP; 44 cats with IAD. METHODS: Retrospective case-control study. Results extracted for all cats included signalment, physical examination findings, historical details, and potential risk factors for aspiration. Diagnostic test results were summarized including CBC, bronchoalveolar (BAL) fluid analysis and microbial culture. Radiographs were reviewed in masked fashion and scored for severity. Results of BAL fluid analysis were assessed for evidence of septic inflammation. RESULTS: Cats with AP were less likely to be presented for evaluation of cough (P < .001) and more likely to be hypothermic (P = .01) than were cats with IAD or BP. Median duration of signs was significantly shorter in cats with AP (12 days) compared to cats with BP or IAD (270 and 180 days; P = .01). Radiographically, cats with AP were more likely to have an alveolar pattern and higher total score than were cats with BP or IAD. Mycoplasma spp. were the organisms most commonly cultured from BAL fluid in cats with BP, but were not cultured from any cats with AP. CONCLUSION AND CLINICAL IMPORTANCE: Pneumonia must be distinguished from IAD in cats with cough and AP should be considered in cats with acute onset of tachypnea.

Unusual splenic B-cell lymphoma in two related Sumatran tigers (Panthera tigris sumatrae).

CASE DESCRIPTION: A 14-year-old 120-kg (264-lb) sexually intact male Sumatran tiger (Panthera tigris sumatrae) and its 10-year-old 130-kg (286-lb) sexually intact male offspring were housed separately and evaluated independently after experiencing weeks of ongoing malaise, weight loss, and anorexia. CLINICAL FINDINGS: Both animals were immobilized and anesthetized for physical examinations and diagnostic testing. Complete blood counts revealed leukopenia and anemia in both tigers. Splenomegaly was identified on abdominal ultrasonography. Cytologic examination and immunohistochemical staining of splenic samples confirmed intermediate to large B-cell lymphoma; no evidence of lymphoma in surrounding organs was noted. TREATMENT AND OUTCOME: The sire was treated with lomustine and prednisolone. This tiger was euthanized 21 months after initiation of treatment because of chronic progressive renal disease. The male offspring was treated with l-asparaginase but did not respond to the treatment. A splenectomy was performed, and malaise and anorexia resolved. No further chemotherapy was administered, and the male offspring was instead maintained on a low dose of prednisolone. Thirty-two months after diagnosis, the male offspring was still considered to be in remission. CLINICAL RELEVANCE: To our knowledge, this was the first known report of the diagnosis and management of a splenic B-cell lymphoma in a tiger. Both tigers achieved positive clinical responses and long-term survival by means of different treatment modalities. The finding of such an unusual neoplasm in a male tiger and its male offspring was noteworthy, raising the possibility of a genetic predisposition for this lymphoma type.

Inter-Institutional Collaboration for the Development of a Local Peer Observation Process to Enhance Teaching.

Local peer observation of teaching is considered an important mechanism for instructors to improve the quality and effectiveness of their teaching, but there is an absence of uniformity to establish a best practice for this process in veterinary curricula. The Regional Teaching Academy (RTA) of the Consortium of Western Colleges of Veterinary Medicine is comprised of educational advocates from five western veterinary colleges with a common goal of enhancing the quality and effectiveness of education in veterinary medical curricula. Members of the RTA recognized this deficit in best practices for local peer observation (LPO) and formed a working group called "Local Peer Observation of Teaching." The goal was to meet a critical need for the enhancement of individual teaching skills by using a scholarly approach to develop robust methods for peer observation of teaching. Two rubric-based instruments were developed: one for large-group/didactic settings, and the second for small-group/clinical settings. Each is accompanied by pre- and post-observation worksheets which are considered instrumental to success. Results of a qualitative survey of instrument users' experiences are shared. Both observers and observees view the experiential learning from faculty peer colleagues very positively and the meaningful feedback is appreciated and incorporated by observees. Suggestions for implementation of the peer observation process are discussed, considering strengths and challenges. The purpose of this article is to describe in depth, the development process and output of the efforts of the Local Peer Observation of Teaching working group as a potential best practice guideline for peer observation.

Serum immunoglobulin E responses to aeroallergens in cats with naturally occurring airway eosinophilia compared to unaffected control cats.

BACKGROUND: Eosinophilic airway disease in cats is sometimes described as allergic in origin, but controversy exists in the documentation of allergy in cats and the utility of allergy testing for respiratory tract diseases. OBJECTIVE: To examine serum immunoglobulin E (IgE) response to aeroallergens in cats with airway eosinophilia. ANIMALS: Fifteen cats with idiopathic eosinophilic airway inflammation and 9 control cats. METHODS: Prospective, case-control study. Surplus serum from cats with airway eosinophilia documented by bronchoscopic bronchoalveolar lavage was submitted for IgE measurement using ELISA polyclonal antibody methodology. Responses for regional allergens (fungal organisms, weeds, grasses, trees, mites, insects) were assessed. Results were reported as ELISA absorbance units with scores 0 to 79 considered negative, scores between 80 and 300 considered intermediate, and scores >300 considered positive. RESULTS: Cats with airway eosinophilia had significantly more positive serum IgE responses (25/720) than did healthy controls (5/432, P = .02); however, the number of cats with positive IgE responses (5/15) did not differ from controls (1/9, P = .35). The allergen that most commonly resulted in positive serum IgE response in cats with airway eosinophilia was dust mite (n = 4) followed by 2 types of storage mites (n = 3 each). No control cat tested positive for these allergens. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum IgE production against aeroallergens was found in some cats with eosinophilic airway inflammation, but the number of affected cats with positive results did not differ from controls. Further investigation in cats with eosinophilic, mixed, and neutrophilic airway disease in comparison to control cats is warranted.

CD4 and CD8 double-negative immunophenotype of thymoma-associated lymphocytes in a dog.

Persistent small-cell lymphocytosis in dogs with a concurrent mediastinal mass has been associated with both thymoma and small-cell lymphoma. In thymomas, neoplastic thymic epithelial cells induce overproduction and release of polyclonal lymphocytes, whereas thymic lymphoma results in thymic effacement by a clonal expansion of neoplastic lymphocytes and subsequent leukemic phase of lymphoma. Flow cytometry has been used to differentiate these 2 entities by immunophenotyping mediastinal mass aspirates. It has been reported that cases with mediastinal masses in which ≥ 10% of the associated small-cell lymphocytes were double positive for CD4 and CD8 were thymomas, whereas masses associated with < 10% were suggestive of lymphoma. We report a unique case of thymoma-associated lymphocytosis lacking the classic CD4+CD8+ immunophenotype. Our findings suggest that there may be more diversity in the thymoma-associated lymphocyte immunophenotype than has been identified previously; immunophenotyping alone might not be sufficient to differentiate thymic small-cell lymphoma from thymoma-associated lymphocytosis. In dogs with mediastinal masses and peripheral lymphocytosis, employing a variety of testing modalities to avoid misdiagnosis is prudent. These modalities include cytologic and/or histologic evaluation, immunophenotyping, and clonality assessment.