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Lymphoma diagnosis in dogs and cats is continually evolving as new subtypes and human correlates are being recognized. In humans, T-cell lymphomas with MUM1 expressed and plasma cell neoplasia or B-cell lymphomas with CD3 expressed aberrantly are reported only rarely. We report here a case series of tumors in dogs and cats with CD3 and MUM1 co-expressed as determined by immunocytochemistry or immunohistochemistry. Lineage was assigned for these tumors by 3 board-certified pathologists and a veterinary immunologist based on review of clinical and cellular features and the results of ancillary testing including PCR for antigen receptor rearrangements, flow cytometry, and serum protein electrophoresis with immunofixation. In cats, 7 of 7 tumors, and in dogs, 3 of 6 tumors with CD3 and MUM1 co-expressed had clonal rearrangement of the immunoglobulin gene or serum monoclonal immunoglobulin, consistent with a diagnosis of a plasma cell neoplasia or myeloma-related disorder with CD3 expressed aberrantly. Disease was often disseminated; notably, 3 of 7 feline cases had cutaneous and/or subcutaneous involvement in the tarsal area. In dogs, 3 of 6 cases had a clonal T-cell receptor gamma result and no clonal immunoglobulin gene rearrangement and were diagnosed as a T-cell tumor with MUM1 expressed. The use of multiple testing modalities in our series of tumors with plasma-cell and T-cell antigens in dogs and cats aided in the comprehensive identification of the lymphoproliferative disease subtype.

Canine acute leukaemia is a heterogeneous neoplasm with multiple phenotypes. Criteria to subtype acute leukaemia by flow cytometry have not been validated. The goal of this study was to develop a panel of antibodies and objective antigen expression criteria for the assignment of lymphoid or myeloid lineage by flow cytometry. We isolated mRNA from the blood of 45 CD34+ acute leukaemia cases and measured expression of 43 genes that represent lymphoid and myeloid lineages using NanoString technology. We determined differentially expressed genes between major groups identified by unsupervised hierarchical clustering. We then evaluated the expression of antigens by flow cytometry to determine if cases could be assigned to a lineage. Two groups were identified by gene expression. Group 1/LYMPH overexpressed lymphoid-associated genes (ex. DNTT) and had a higher percentage of CD5 + CD3- cells by flow cytometry. Group 2/MYELO overexpressed myeloid-associated genes (ex. ANPEP/CD13) and had a higher percentage of class II major histocompatibility complex (MHCII)- CD14+ and/or CD18 + CD4- cells. We proposed that12.5% CD5 + CD3- cells in the blood was indicative of lymphoid lineage, and 3.0% CD14 + MHCII- cells or 18% CD18 + MHCII-CD4- cells was indicative of myeloid lineage. 15/15 cases that met the proposed criteria for acute lymphocytic leukaemia were in LYMPH group and 12/15 cases that met the proposed criteria for acute myeloid leukaemia were in MYELO group. The majority of CD34+ cases that did not meet either immunophenotyping lineage criterion (12/13) clustered within the LYMPH group. In conclusion, currently available antibodies can be useful for determining canine acute leukaemia subtypes.

Supplementary Tables 1-2 from Distinct B-Cell and T-Cell Lymphoproliferative Disease Prevalence among Dog Breeds Indicates Heritable Risk

Immunophenotypes in lymphoproliferative diseases (LPD) are prognostically significant, yet causative factors for these conditions, and specifically those associated with heritable risk, remain elusive. The full spectrum of LPD seen in humans occurs in dogs, but the incidence and lifetime risk of naturally occurring LPD differs among dog breeds. Taking advantage of the limited genetic heterogeneity that exists within dog breeds, we tested the hypothesis that the prevalence of LPD immunophenotypes would differ among different breeds. The sample population included 1,263 dogs representing 87 breeds. Immunophenotype was determined by the presence of clonal rearrangements of immunoglobulin heavy chain or T-cell receptor γ chain. The probability of observing the number of B-cell or T-cell tumors in a particular breed or breed group was compared with three reference populations. Significance was computed using χ2 test, and logistic regression was used to confirm binomial predictions. The data show that, among 87 breeds tested, 15 showed significant differences from the prevalence of LPD immunophenotypes seen across the dog population as a whole. More significantly, elevated risk for T-cell LPD seems to have arisen ancestrally and is retained in related breed groups, whereas increased risk for B-cell disease may stem from different risk factors, or combinations of risk factors, arising during the process of breed derivation and selection. The data show that domestic dogs provide a unique and valuable resource to define factors that mediate risk as well as genes involved in the initiation of B-cell and T-cell LPD.

BACKGROUND T-cell leukemia/lymphoma accounts for roughly 30% of all types of lymphoproliferative neoplasia in dogs. Two forms of T-cell lymphoma (T-zone and peripheral T-cell lymphoma) exhibit breed-specific predilections. During the course of routine immunophenotyping, we observed a breed-specific presentation of a unique form of T-cell leukemia in young English bulldogs. OBJECTIVE To describe the clinical presentation and outcome of a novel T-cell leukemia in English bulldogs and determine the frequency of this neoplasm in other breeds. METHODS The Clinical Hematopathology database, containing immunophenotyping data from peripheral blood of nearly 11,900 dogs, was queried for the phenotype observed in young English bulldogs: CD45+ CD4- CD8- CD5+ CD3+ class II major histocompatibility complex (MHC)ᶫ° T-cell leukemia. Clinical presentation, treatment, and survival data were collected for a subset of cases. RESULTS Fifty-five English bulldog cases and 64 cases of other breeds were identified. No other breed was represented by >5 cases. Complete medical records were obtained for 50 bulldogs. Median age at diagnosis was 3 years and 76% of cases were male. Median lymphocyte count was 44,286 lymphocytes/ul (range, 1,800-317,684/ul) and lymphocytes were described as small to intermediate-sized. Many dogs were thrombocytopenic and had liver and spleen involvement, but not lymphadenopathy. Bulldogs that received multi-agent chemotherapy had longer median survival times (83 days) compared to dogs that received no treatment (6 days) or less aggressive therapy (15 days) (p=0.001). Non-bulldogs had similar outcomes. CONCLUSION CD4- CD8- class II MHCᶫ° T-cell leukemia has an aggressive clinical course and predilection for young English bulldogs. Breed-specific presentation suggests an underlying genetic cause. This article is protected by copyright. All rights reserved.

Background B‐cell chronic lymphocytic leukemia (BCLL) in dogs generally is considered an indolent disease, but previous studies indicate a wide range in survival times. Objectives We hypothesized that BCLL has a heterogeneous clinical course, similar to chronic lymphocytic leukemia in humans. We aimed to assess presentation and outcome in dogs with BCLL and evaluate the prognostic relevance of clinical and flow cytometric factors. Animals One hundred and twenty‐one dogs with BCLL diagnosed by flow cytometry. Three breed groups were represented: small breed dogs (n = 55) because of increased risk of BCLL; Boxers (n = 33) because of preferential use of unmutated immunoglobulin genes; and other breeds (n = 33). Methods Retrospective study reviewing signalment, clinicopathologic data, physical examination findings, treatment, and survival of dogs with BCLL. Cellular proliferation, determined by the percentage of Ki67‐expressing CD21+ B‐cells by flow cytometry, was measured in 39 of 121 cases. Clinical and laboratory variables were evaluated for association with survival. Results The median survival time (MST) for all cases was 300 days (range, 1‐1644 days). Boxers had significantly shorter survival (MST, 178 days) than non‐Boxers (MST, 423 days; P 40% Ki67‐expressing B‐cells) had significantly shorter survival (MST, 173 days) than did cases with 60 000 lymphocytes/μL) or clinical signs at presentation had significantly shorter survival. Conclusions and Clinical Importance B‐cell chronic lymphocytic leukemia had a variable clinical course and Boxer dogs and cases with high Ki67 had more aggressive disease.

Routine electrophoresis [agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE)] and species-specific immunofixation (IF) can be used alone or in combination to detect immunoglobulin paraprotein (M-protein) and diagnose secretory myeloma-related disorders (sMRD).We aimed to evaluate the performance of AGE, CZE, CZE plus IF (CZE-IF), and AGE plus IF (AGE-IF) for detecting canine serum M-proteins.One hundred canine cases that had AGE, CZE, and routine IF performed on serum, and where B-cell lineage neoplasia (such as B-cell lymphoma and plasma cell tumors) had been diagnosed or excluded, were evaluated. Routine IF protocols targeted IgG-FC, IgA, and IgM heavy chains and light chains. IgG4 IF and free light chain IF were also performed. B-cell lineage neoplasms with an M-protein detected, using any available method, were classified as sMRD. Datasets from AGE, CZE, IF, CZE-IF, and AGE-IF (electrophoretograms, gel images, and fraction concentrations) were composed and reviewed. The sensitivity, specificity, and Youden's index for M-protein detection were determined for each dataset.The combination of AGE-IF or CZE-IF was more sensitive (82.9%) than CZE alone (72.0%) or AGE alone (64.6%) and more specific (66.1%, 48.3%, 51.7%, respectively). Immunofixation could be used alone to detect M-proteins (sensitivity 82.9%, specificity 61.9%), but there were technical challenges that complicated the performance and evaluation of the test. Myeloma with free light chains only was found in 5/41 cases of sMRD.Adding routine IF to routine electrophoresis increases the ability to accurately identify M-proteins; however, there is still room for further diagnostic performance improvements.

Background Current recommendations for monitoring disease progression and response to treatment in humans with multiple myeloma include evaluation of serum paraprotein (M-protein) concentration. Densitometry, species-specific radial immunodiffusion (RID) and ELISA methods can be used to quantify M-proteins. Objective Retrospectively evaluate use of the International Myeloma Working Group (IMWG) response criteria for humans in dogs with multiple myeloma. Animals Sixteen dogs with a diagnosis of multiple myeloma, M-protein documented by serum protein electrophoresis (SPE) and immunofixation (IF) in an initial sample and subsequent electrophoretic evaluation of serial samples. Methods Retrospectively, densitometric M-proteins, RID and globulins were measured and characterized according to IMWG criteria. Available clinical history was reviewed. Overall survival time (OST) was calculated from initial electrophoretic evaluation to death or last contact. Results All cases received some form of nonstandardized chemotherapy. Complete response (CR), a lack of detectable M-protein by SPE and IF, was documented in 1 case. Median survival was longer for dogs that attained ≥90% densitometric M-protein reduction (630 days) than for those that did not attain at least 50% reduction in densitometric M-protein (284 days; log rank P = .006). Five dogs were defined as having progressive disease (M-protein increase of >25% and at least 0.5 g/dL from nadir), which correlated with concurrent or subsequent clinical deterioration. Response criteria categorized by serum globulins or RID was not correlated with OST or clinical findings. Conclusions and clinical importance Densitometric M-protein characterized using IMWG response criteria correlated with OST and clinical findings. Densitometric M-protein detection should be used to monitor dogs with multiple myeloma.

Background English bulldogs disproportionally develop an expansion of small B-cells, which has been interpreted as B-cell chronic lymphocytic leukemia (BCLL). However, clonality testing in these cases has often not been supportive of neoplasia. Hypothesis English bulldogs have a syndrome of nonneoplastic B-cell expansion. Animals Eighty-four English bulldogs with small-sized CD21+ B-cell lymphocytosis in the blood as determined by flow cytometry. Methods This is a retrospective study. We characterized this syndrome by assessing B-cell clonality, clinical presentation, flow cytometric features, and immunoglobulin gammopathy patterns. We identified 84 cases with CD21+ lymphocytosis among 195 English bulldogs with blood samples submitted to the Colorado State University-Clinical Immunology laboratory for immunophenotyping between 2010 and 2019. Flow cytometry features were compared to normal B-cells and BCLL cases. PCR for antigen receptor rearrangements (PARR) by multiple immunoglobulin primers was performed to assess B-cell clonality. A subset of cases with gammopathy were examined by protein electrophoresis, immunofixation, and immunoglobulin subclass ELISA quantification. Results Seventy percent (58/83) of cases had polyclonal or restricted polyclonal immunoglobulin gene rearrangements, suggesting nonmalignant B-cell expansion. The median age of all dogs in the study was 6.8 years and 74% were male. The median (range) lymphocyte count was 22 400/μL (2000-384 400/μL) and B-cells had low expression of class II MHC and CD25. Splenomegaly or splenic masses were detected in 57% (26/46) of cases and lymphadenopathy in 11% (7/61). Seventy-one percent (52/73) of cases had hyperglobulinemia and 77% (23/30) with globulin characterization had IgA ± IgM polyclonal or restricted polyclonal gammopathy patterns. Conclusions and clinical importance Polyclonal B-cell lymphocytosis in English bulldogs is characterized by low B-cell class II MHC and CD25 expression, splenomegaly and hyperglobulinemia consisting of increased IgA ± IgM. We hypothesize that this syndrome has a genetic basis.

T-cell leukemia/lymphoma accounts for roughly 30% of all types of lymphoproliferative neoplasia in dogs. Two forms of T-cell lymphoma (T-zone and peripheral T-cell lymphoma) exhibit breed-specific predilections. During the course of routine immunophenotyping, we observed a breed-specific presentation of a unique form of T-cell leukaemia in young English bulldogs. To describe the clinical presentation and outcome of a novel T-cell leukaemia in English bulldogs and determine the frequency of this neoplasm in other breeds. The Clinical Hematopathology database, containing immunophenotyping data from peripheral blood of nearly 11 900 dogs, was queried for the phenotype observed in young English bulldogs: CD45

Background The diagnostic performance of routine electrophoresis (agarose gel electrophoresis [AGE] and capillary zone electrophoresis [CZE]) and species-specific immunofixation (IF) for the detection of immunoglobulin paraproteins (M-proteins) and diagnosis of secretory myeloma-related disorders (sMRD) can be improved. Available canine IF targets were IgG-FC, IgA, IgM, light chain (LC), IgG4, and free LC (fLC) antibodies. Objective We aimed to review specific features associated with the presence of M-proteins in canine serum samples and the common features causing inaccurate reporting of M-proteins to improve the diagnostic performance of routine electrophoresis and IF for the detection of M-proteins. Methods Features found in AGE, CZE, routine IF, IgG4 IF, and fLC IF of 100 canine serum samples from Part 1 of this study were evaluated by simple and multivariate logistic regression to identify factors associated with the presence of M-proteins. Cases falsely called negative or positive for M-proteins were reviewed to identify the common features that could be used to increase the diagnostic performance of SPE and IF for M-protein detection. Results The presence of hypogammaglobulinemia or any peak taller than albumin was associated with an M-protein. Total protein concentrations, globulin concentrations, or peaks wider than albumin were not associated with an M-protein. Free LC sMRD cases were not diagnosed by SPE and routine IF. Cases with infectious and inflammatory etiologies had a restricted polyclonal gammopathy with multiple γ-globulin restrictions resulting in some false-positive results. SPE combined with all available IF results and the specific features identified in this study had an estimated sensitivity of 95.1% and specificity of 81.4%. Conclusions The identified criteria of this study increase the diagnostic performance of the electrophoretic evaluation for M-proteins.

Virtual microscopy (VM) using scanned slides and imaging software is increasingly used in medical curricula alongside instruction in conventional microscopy (CM). Limited reports suggest that VM is useful in the veterinary education setting, and generally well-received by students. Whether students can apply knowledge gained through VM to practical use is unknown. Our objective was to determine whether instruction using VM, compared to CM, is a successful method of training veterinary students for the application of cytology in practice (i.e., using light microscopes). Seventy-one veterinary students from Colorado State University who attended a voluntary 3-hour cytology workshop were randomized to receive the same instruction with either VM ( n = 35) or CM ( n = 36). We compared these students to a control group ( n = 22) of students who did not attend a workshop. All students took a post-workshop assessment involving the interpretation of four cases on glass slides with CM, designed to simulate the use of cytology in general practice. Students also took an 18-question survey related to the effectiveness of the workshop, providing their opinions on cytology instruction in the curriculum and their learning preference (VM or CM). The mean assessment score of the VM group (14.18 points) was significantly higher than the control group (11.33 points, p = .003), whereas the mean of the CM group (12.77 points) was not statistically significantly different from controls ( p = .170). Not only is VM an effective method of teaching cytology to veterinary students that can be translated to a real-world case scenario, but it outperformed CM instruction in this study.

Key Clinical Message An 11‐year‐old female spayed Golden Retriever presented for an incidentally found liver mass. The hepatic mass and intra‐abdominal lymph nodes had a marked heterogeneous T‐cell population and far fewer numbers of small clonal B cells. This T‐cell‐rich small B‐cell lymphoma had a unique histological pattern and indolent clinical course.

Canine T-zone lymphoma (TZL) is a subtype of T-cell lymphoma characterized by unique histologic pattern and cytomorphology, immunophenotypic loss of CD45 expression, and an indolent clinical behaviour. Dogs with TZL typically present with 1 or more enlarged lymph nodes and/or lymphocytosis. We describe a novel extranodal presentation of TZL involving the tongue. Twelve dogs with tongue masses were diagnosed with lingual TZL based on a variable combination of immunophenotyping via flow cytometry, cytology, histopathology, immunohistochemistry and/or PCR for antigen receptor rearrangement (PARR) assay. Eleven dogs exhibited concurrent lymphocytosis and/or lymph node enlargement. Three cases were initially diagnosed as plasma cell tumours based on histology alone, thereby revealing a potential diagnostic challenge. Seven dogs achieved clinical remission and 4 achieved stable disease following variable treatment, consistent with the indolent nature of typical TZL involving the lymph nodes and peripheral blood. In 1 case the TZL resulted in progressive disease and failure to respond to treatment. In this case, the TZL exhibited histologic features of a higher grade neoplasm. This case series highlights a unique presentation of TZL and identifies a new differential diagnosis for lingual neoplasia. In this study, we characterize the clinical presentation, diagnostic features and patient outcomes of 12 dogs with lingual TZL.

A 5-year-old male neutered Bernese Mountain Dog was presented for cutaneous plasmacytoma, which was treated by surgical excision. Four months later, the dog developed multiple skin masses, hyphema, pericardial and mild bicavitary effusions, myocardial masses, and marked plasmacytosis in the peripheral blood. Circulating plasma cells expressed CD34 and MHC class II by flow cytometry. Immunocytochemistry demonstrated that these cells were strongly positive for multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM-1) and weakly to moderately positive for Pax5. The dog was hypoglobulinemic but had a monoclonal IgA gammopathy detected by serum immunofixation electrophoresis. The PCR analysis of antigen receptor gene rearrangements (PARR) by fragment analysis using GeneScan methodology revealed that plasmacytoid cells in the original cutaneous plasmacytoma and peripheral blood had an identical immunoglobulin heavy chain gene (IgH) rearrangement, indicating that both populations were derived from the same neoplastic clone. Canine cutaneous plasmacytoma rarely progresses to a malignant form and plasma cell leukemia is rarely diagnosed in the dog. This report describes a case of cutaneous plasmacytoma progressing to plasma cell leukemia with a rapid and aggressive clinical course. This report also highlights the utility of flow cytometry, immunocytochemistry, immunofixation electrophoresis, and PARR by fragment analysis using GeneScan methodology in the diagnosis of this hematopoietic neoplasm.

Cytology is commonly used to diagnose lymphoma and leukemia. Frequently, a diagnosis of lymphoproliferative disease can be obtained via cytology, and some of the common subtypes of canine lymphoma and leukemia can have characteristic cytologic features. Flow cytometry is a critical tool in the objective diagnosis and further characterization of lymphoma and leukemia. Features of the immunophenotype, such as expression of certain cell surface proteins or cell size, can provide important prognostic information. This review describes the cytologic features, flow cytometry immunophenotype, and immunophenotypic prognostic information for 6 major types of canine lymphoma and leukemia.

Canine T-cell lymphoma (TCL) encompasses a heterogeneous group of diseases with variable clinical presentation, cytomorphology, immunophenotype, and biologic behaviour. The most common types of TCL in dogs involving peripheral lymph nodes include indolent T-zone lymphoma (TZL) and biologically aggressive peripheral T-cell lymphoma (PTCL). TCL phenotypes can be categorized by expression of the surface antigen molecules CD4 and CD8. The majority of TCL cases are CD4+ , with far fewer cases being CD8+ or CD4- CD8- . The clinical features of CD4+ TCLs have been previously described. The less common TCL phenotypes, however, are poorly characterized with little to no information about prognosis. In this retrospective study, we describe and correlate the presenting clinical signs, flow cytometry, and outcomes of 119 dogs diagnosed with nodal, non-TZL, CD8+ or CD4- CD8- TCL by flow cytometry. Skin lesions present at the time of diagnosis were more commonly observed in the CD8+ TCL group. Mediastinal enlargement and/or hypercalcemia were more commonly seen in the CD4- CD8- TCL group. Dogs with either CD8+ or CD4- CD8- TCLs had aggressive clinical disease with median overall survival (OS) times of 198 days and 145 days, respectively. In both groups, neoplastic cell size determined by flow cytometry ranged from small to large, and large cell size was associated with shorter OS times (median OS = 61 days). Cases classified as small cell had a median OS of 257 days. Expression levels of major histocompatibility complex (MHC) class II and CD5 were highly variable among cases but were not prognostically significant in this group of patients.

Background Lymphocytosis is relatively common in cats, but few studies describe lymphocyte populations or the clinical course associated with different immunophenotypic expansions. Hypothesis/objectives We hypothesized that cats frequently develop non-neoplastic lymphocytosis and that different neoplastic immunophenotypes have variable prognoses. We aimed to characterize the lymphocyte expansions in a large population of cats with lymphocytosis and to assess clinical presentation and outcome in a subset. Animals Three cohorts of cats older than 1 year with lymphocytosis (>6000/μL) were examined to define immunophenotypic categories (n = 146), evaluate outcome (n = 94), and determine prevalence of immunophenotypes (n = 350). Methods Retrospective study of cats with blood submitted for flow cytometry. Medical records (n = 94) were reviewed for clinical data, treatment, and survival information. Results Five major immunophenotypic categories were identified: B cell, heterogeneous (≥2 lineages expanded), CD4+ T cell, CD4-CD8- (double negative [DN]) T cell, and CD5-low-expressing T cell. B-cell and heterogeneous phenotypes were more consistent with a non-neoplastic process, having polyclonal antigen receptor gene rearrangements, younger age at presentation, lower lymphocyte counts, and prolonged survival. The neoplastic phenotypes, CD4+ T cell, DN T cell, and CD5 low T cell, had different median survival times (752 days [n = 37], 271 days [n = 7], 27.5 days [n = 12], respectively). Among CD4+ T-cell cases, cats with abdominal lymphadenopathy, intestinal involvement, or both and females had shorter survival. Among 350 cats with lymphocytosis, CD4+ T-cell lymphocytosis was most common, followed by heterogeneous and B-cell phenotypes. Conclusions and clinical importance Neoplastic CD4+ T-cell lymphocytosis is common in cats and has a prolonged clinical course compared to aberrant T-cell phenotypes. Cats with heterogeneous and B-cell lymphocyte expansions commonly have non-neoplastic disease.

Digital microscopy (DM) has been employed for primary diagnosis in human medicine and for research and teaching applications in veterinary medicine, but there are few veterinary DM validation studies. Region of interest (ROI) digital cytology is a subset of DM that uses image-stitching software to create a low-magnification image of a slide, then selected ROI at higher magnification, and stitches the images into a relatively small file of the embedded magnifications. This study evaluated the concordance of ROI-DM compared to traditional light microscopy (LM) between 2 blinded clinical pathologists. Sixty canine and feline cytology samples from a variety of anatomic sites, including 31 cases of malignant neoplasia, 15 cases of hyperplastic or benign neoplastic lesions, and 14 infectious/inflammatory lesions, were evaluated. Two separate nonblinded adjudicating clinical pathologists evaluated the reports and diagnoses and scored each paired case as fully concordant, partially concordant, or discordant. The average overall concordance (full and partial concordance) for both pathologists was 92%. Full concordance was significantly higher for malignant lesions than benign. For the 40 neoplastic lesions, ROI-DM and LM agreed on general category of tumor type in 78 of 80 cases (98%). ROI-DM cytology showed robust concordance with the current gold standard of LM cytology and is potentially a viable alternative to current LM cytology techniques.

Background Differentiation between neoplastic and reactive lymphocytic proliferations can be challenging in cats. PCR for antigen receptor rearrangements (PARR) testing is a useful diagnostic tool to assess clonality of a lymphoid population. Previous feline PARR studies evaluated clonality of complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma (TRG) gene rearrangements. Objectives We aimed to evaluate the sensitivity and specificity of feline PARR primers targeting complete IGH-VDJ and TRG rearrangements, as well as incomplete IGH-DJ, kappa deleting element (Kde), and immunoglobulin lambda light chain (IGL) gene rearrangements in defined feline neoplasms and nonneoplastic controls. Methods Fluorescently labeled PCR primers were designed to amplify complete IGH-VDJ, incomplete IGH-DJ, Kde, IGL, and TRG gene rearrangements in two multiplexed PCR reactions, and PCR products were analyzed by fragment analysis. Fresh tissue samples from 12 flow cytometrically confirmed B-cell lymphomas, 26 cytologically confirmed gastric and renal lymphomas of presumed B-cell origin, 30 flow cytometrically confirmed T-cell leukemias, and 11 negative control cats were tested. Results Using four immunoglobulin primer sets (IGH-VDJ, IGH-DJ, Kde, and IGL), clonal immunoglobulin rearrangements were detected in 87% (33/38) of the presumed B-cell neoplasms. The IGH-VDJ reaction alone only detected clonality in 50% (19/38) of these cases. TRG rearrangements were clonal in 97% (29/30) of the T-cell leukemia cases. All negative control samples had polyclonal immunoglobulin and TRG rearrangements. Conclusions The PARR assay developed in this study is useful for assessing clonality in feline lymphoid neoplasms. Clonality assessment of incomplete IGH-DJ, Kde, and IGL rearrangements helped identify clonal B-cell neoplasms not detected with complete IGH-VDJ PARR alone.

Protein electrophoresis and immunotyping can be a useful adjunct to the standard biochemical techniques for characterizing serum and urine proteins. This paper reviews currently available and commonly used methods for diagnostic protein electrophoresis, including both agarose gel and capillary zone electrophoretic techniques and total protein assessments. Immunofixation and immunosubtraction methods for identification of immunoglobulin location and class are also presented. Practical application of quality assurance and quality control strategies in compliance with American Society of Veterinary Clinical Pathology (ASVCP) best practices are discussed. Commonly encountered serum and urine electrophoretic diagnostic patterns, including electrophoretically normal, acute-phase protein responses, polyclonal gammopathies, restricted polyclonal/oligoclonal gammopathies, paraproteinemias (monoclonal or biclonal gammopathies), and Bence-Jones proteinurias are also reviewed using relevant case material. Cases in which immunofixation electrophoresis are particularly useful are highlighted, and methodologies to more accurately quantify serum monoclonal proteins (M-proteins), monitoring tests commonly used in human medicine, are discussed.

Diffuse large B-cell lymphoma (DLBCL) is the most common haematopoietic malignancy in dogs. Recently, MYC and BCL2 expression levels determined with immunohistochemistry (IHC) were found to be prognostic in people with DLBCL. We hypothesized that canine DLBCL can be similarly subdivided into prognostic subtypes based on expression of MYC and BCL2. Cases of canine DLBCL treated with CHOP chemotherapy were retrospectively collected and 43 dogs had available histologic tissue and complete clinical follow-up. Median values of percent immunoreactive versus immunonegative cells were used to determine positive or negative expression status. Completion of CHOP was significantly associated with a positive outcome. Compared with human patients, our canine DLBCL patients had high IHC expression of both MYC and BCL2, and relative expression levels of one or both markers were not associated with clinical outcome.

Cytology is commonly used to diagnose lymphoma and leukemia. Frequently, a diagnosis of lymphoproliferative disease can be obtained via cytology, and some of the common subtypes of canine lymphoma and leukemia can have characteristic cytologic features. Flow cytometry is a critical tool in the objective diagnosis and further characterization of lymphoma and leukemia. Features of the immunophenotype, such as expression of certain cell surface proteins or cell size, can provide important prognostic information. This review describes the cytologic features, flow cytometry immunophenotype, and immunophenotypic prognostic information for 6 major types of canine lymphoma and leukemia.

Case Description—A 9-year-old castrated male mixed-breed dog and a 7-year-old spayed female Boston Terrier, with clinical histories of a liver mass (dog 1) and bloody vomitus, diarrhea, and weight loss (dog 2), respectively, were referred for further evaluation. Clinical Findings—At the time of referral, each dog had differing laboratory abnormalities; however, the serum total protein and globulin concentrations were within reference range in both dogs. Cytologic examination of fine-needle aspirates obtained from affected organs (a liver mass [dog 1] and enlarged submandibular lymph node [dog 2]) revealed 2 main nucleated cell types: atypical lymphoid cells and lesser numbers of Mott cells. With the use of serum immunofixation electrophoresis and serum immunoglobulin quantification, a monoclonal immunoglobulin protein was identified in both dogs and a final diagnosis of secretory B-cell lymphoma with Mott cell differentiation (MCL) was made. Treatment and Outcome—Both dogs received chemotherapy for their disease. The first dog was euthanized 8.5 months after diagnosis because of acute respiratory distress of unknown etiology, and the second was euthanized 7 days after diagnosis for worsening clinical disease and quality of life. Clinical Relevance—To our knowledge, this report is the first of a secretory form of MCL in dogs. Findings indicate that in dogs with suspect MCL, even in patients that lack characteristic hyperproteinemia or hyperglobulinemia, serum protein content should be fully evaluated for the presence of a monoclonal immunoglobulin protein. Such an evaluation that uses immunofixation electrophoresis and immunoglobulin quantification will aid in the diagnosis of MCL in dogs.

Background: Class II major histocompatibility complex (MHC) is an independent predictor of outcome in human B-cell lymphoma. We assessed class II expression together with other markers for their impact on prognosis in canine B-cell lymphoma. Hypothesis: Low class II MHC expression, large cell size, and expression of CD34 will predict a poorer outcome in canine B-cell lymphoma. Expression of CD5 and CD21 on tumor cells also may be associated with outcome. Animals: One hundred and sixty dogs with cytologically confirmed lymphoma. Methods: Patient signalment, treatment type, and flow cytometry characteristics were analyzed for their influence on outcome. A multivariable predictive model of survival was generated using 2/3 of the patients and validated on the remaining 1/3 of the dataset. Results: Class II MHC expression had a negative association with mortality and relapse. Treatment type also influenced relapse and mortality, whereas cell size and patient age was only associated with mortality. CD34, CD21, and CD5 expression was not associated with disease outcome. The constructed model performed variably in predicting the validation group's outcome at the 6-month time point. Conclusions and Clinical Importance: Low levels of class II MHC expression on B-cell lymphoma predict a poor outcome, as in human B-cell lymphoma. This finding has implications for the use of dogs to model human lymphomas. Class II expression, cell size, treatment, and age can be combined to predict mortality with a high level of specificity.

Two dogs, an 8.5-year-old intact male Golden Retriever and a 10-year-old spayed female English Springer Spaniel, each with varied clinical histories, were referred to the Colorado State University Veterinary Teaching Hospital for evaluation of hypercalcemia and severe anemia, respectively. In each dog, serum total protein and globulin concentrations were within reference intervals. Cytologic examination of bone marrow aspirates from both dogs revealed moderate to marked numbers of atypical lymphoid cells with plasma cell features. Using serum immunofixation and serum immunoglobulin (Ig) quantification, a monoclonal Ig protein was identified. In conjunction with other clinicopathologic and molecular findings, IgA secretory neoplasms, B-cell lymphoma with plasmacytoid features and multiple myeloma (MM), were diagnosed. To our knowledge, these cases represent the first descriptions of IgA-secreting neoplasms in dogs that lacked hyperglobulinemia. In cases of suspected B-cell lymphoma or MM in dogs, serum proteins should be fully evaluated for the presence of a monoclonal Ig even in dogs that lack characteristic hyperproteinemia or hyperglobulinemia. This evaluation will aid in the diagnosis of secretory B-cell lymphoma or MM leading to appropriate clinical and therapeutic case management.

Impaired dendritic cell (DC) function is thought to be central to human immunodeficiency virus-associated immunodeficiency. In this study, we examined the effect of chronic feline immunodeficiency virus (FIV) infection on DC cytokine production in response to microbial and T-cell stimulation. Cytokine production after either Toll-like receptor (TLR) or CD40 ligation in bone marrow-derived DCs (BM-DCs) was measured in naïve and chronically FIV-infected cats. The BM-DCs were stimulated with ligands to TLR-2, -3, -4, -7 and -9 or cocultured with 3T3 cells expressing feline CD40 ligand. Ligation of TLR-4 and TLR-9 in BM-DCs from infected cats resulted in a significant decrease in the ratio of interleukin-12 (IL-12) to IL-10. Conversely, TLR-7 ligation produced a significant increase in the IL-12 : IL-10 ratio in BM-DCs from infected cats. No difference was noted for TLR-3 ligation. RNA expression levels of TLR-2, -3, -4, -7 and -9 were not significantly altered by FIV infection. CD40 ligation significantly elevated both IL-10 and IL-12 messenger RNA production but did not alter the IL-12 : IL-10 ratio. Chronic FIV infection alters the ratio of immunoregulatory cytokines produced by BM-DCs in response to certain pathogen-derived signals, which is probably relevant to the increased risk of opportunistic infections seen in lentiviral infection.

Canine lymphoproliferative disease often presents with lymphocytosis and is immunophenotypically diverse.Immunophenotype predicts prognosis in canine lymphoproliferative disorders involving circulating lymphocytosis.Dogs that had peripheral blood evaluation performed by flow cytometry by the Clinical Immunology Service at Colorado State University between 2003 and 2005.Outcome data regarding treatment and survival were sought on patients with lymphocytosis comprising a single lymphocyte subset. Ninety-six patients that met the inclusion criteria had sufficient follow-up information to be included in the study.Four main phenotypic classifications were found: CD8+ T-cell, CD21+ B-cell, CD4-8-5+ (aberrant T-cell phenotype), and CD34+ (undifferentiated progenitor). Expression of CD34 predicted poor outcome with median survival of 16 days (P.0001) compared with other phenotypes. Within the CD8+ phenotype, dogs presenting with a lymphocytosis30,000 lymphocytes/muL had significantly shorter median survival (131 days) than those presenting with30,000 lymphocytes/muL (1098 days, P.0008). Within the T-cell leukemias, there was no difference in outcome between dogs with CD4-8-5+ leukemia and dogs with the CD8+ T-cell phenotype nor was the loss of expression of the pan-leukocyte marker CD45 associated with decreased survival time. A CD21+ lymphocytosis composed of large cells was associated with shorter survival time (129 days) than those with smaller circulating cells (median survival not reached, P.01).Immunophenotyping provides an objective method for determining prognosis in lymphoproliferative disorders characterized by lymphocytosis.

Case Description—A 16-week-old female Boxer that had been treated for 5 weeks with trimethoprim-sulfamethoxazole and chloramphenicol because of aspiration pneumonia was evaluated for bilaterally symmetric masses in the subcutaneous tissues of the ventral neck, in the region of the larynx. Clinical Findings—Fine-needle aspirates were obtained from the neck masses; cytologic examination revealed well-differentiated thyroid epithelial tissue. A blood sample was collected for serum biochemical and thyroid function analyses. Mild hyperphosphatemia, severe hypercholesterolemia, mild hyperkalemia, and a mild increase in creatine kinase activity were identified. Serum concentration of total thyroxine was less than the lower reference limit, and that of thyroid-stimulating hormone was greater than the upper reference limit. Findings were consistent with a diagnosis of clinical hypothyroidism in a skeletally immature dog. Treatment and Outcome—Treatment with trimethoprim-sulfamethoxazole was discontinued. The dog was reevaluated 3 weeks later, at which time the neck masses were markedly decreased in size. Serum concentrations of cholesterol and potassium were lower; serum concentrations of total thyroxine and thyroid-stimulating hormone were near or within respective reference ranges. Age-appropriate increases in serum phosphorus concentration and serum alkaline phosphatase activity were also detected. Clinical Relevance—To the authors' knowledge, this is the first report of antimicrobial-induced goiter in a dog. Cytologic examination of fine-needle aspirates and interpretation of data from serum biochemical and thyroid function analyses were needed to obtain a definitive diagnosis. Practitioners should include goiter among the differential diagnoses for ventral neck swellings in young dogs receiving potentiated sulfonamide antimicrobials.

Oxidative stress is thought to contribute to the pathogenesis of HIV infection in humans. For example, CD4+ T cells are particularly affected in HIV patients and oxidative stress may also contribute to impairment of neutrophil function in HIV/AIDS patients. Since cats infected with FIV develop many of the same immunological abnormalites as HIV-infected humans, we investigated effects of acute FIV infection on oxidative stress in cats. Cats were infected with a pathogenic strain of FIV and viral load, changes in neutrophil number, total blood glutathione, malondiadehye, antioxidant enzyme concentrations, and reduced glutathione (GSH) concentration in leukocytes were measured sequentially during the first 16 weeks of infection. We found that superoxide dismutase and glutathione peroxidase concentrations in whole blood increased significantly during acute FIV infection. In addition, neutrophil numbers increased significantly during this time period, though their intracellular GSH concentrations did not change. In contrast, the numbers of CD4+ T cells decreased significantly and their intracellular GSH concentration increased significantly, while intracellular GSH concentrations were unchanged in CD8+ T cells. However, by 16 weeks of infection, many of the abnormalities in oxidative balance had stabilized or returned to pre-inoculation values. These results suggest that acute infection with FIV causes oxidative stress in cats and that CD4+ T cells appear to be preferentially affected. Further studies are required to determine whether early treatment with anti-oxidants may help ameliorate the decline in CD4+ T cell number and function associated with acute FIV infection in cats.

We designed and tested, in vitro, an adenoviral construct containing the feline interleukin-12 (IL-12) gene under control of the heat-inducible promoter HSP70B. This construct, AdhspfIL12, was used in a phase I trial in feline soft tissue sarcomas. During the course of our experiments, we noted that IL-12 was being produced in the transfected Crandell Feline Kidney (CrFK) cells under certain conditions even in the absence of hyperthermia. This observation was further explored to identify the cause of this unintended HSP70B induction.We used real-time PCR as a sensitive method to quantitatively detect the presence of even small amounts of IL-12 mRNA. This served as a surrogate indicator of HSP70B induction. Various conditions were tested to induce the heat shock promoter, including nutritional deprivation, radiation and changes in pH.Nutritional stresses, specifically the absence of glucose and glutamine, could induce the heat shock promoter, thus, resulting in production of the downstream gene product. Other factors known to trigger the heat shock response, pH change, and reactive oxygen species production were also studied but were not found to contribute to heat shock promoter induction in our setting.The human heat shock promoter (HSP70B) is reported to be an efficient and tightly regulated promoter. We discovered, using sensitive real-time PCR techniques, that it can also be induced in response to cellular nutrient stresses. The pros and cons of this phenomenon and its implications for cancer gene therapy are discussed.

Currently most dendritic cells (DC) for in vitro study are generated from bone marrow or peripheral blood by culture in high concentrations of GM-CSF and other cytokines. However, in mice it is also possible to generate DC from spleen cells using long-term stromal cell cultures. To determine whether tissue DC could be also be generated from cats, we established stromal cell cultures from a number of different tissues of newborn cats. We found that stromal cell cultures from spleen, lung, liver, kidney, brain, and lymph node tissues were all capable of spontaneously generating DC over long periods of time (months), without requiring the addition of exogenous cytokines. The tissue DC generated from these stromal cell cultures could be readily isolated at high purity by simple mechanical detachment. The feline tissue DC expressed high levels of CD11c, CD11b, and MHC Class II and variable levels of CD80 and CD14 and exhibited high levels of spontaneous macropinocytosis. Moreover, DC from spleen stromal cell cultures, but not DC from lung or liver stromal cell cultures, stimulated mixed-lymphocyte reactions. The DC generated from the stromal cell cultures were relatively independent of GM-CSF for survival and proliferation, indicative of a dependence on other growth factors produced by the stromal cells. These results suggest that tissues of young cats contain a population of resident DC progenitor cells that under appropriate conditions are capable of spontaneous proliferation and generation of immature DC.

Oxidative stress and abnormal glutathione metabolism is thought to play an important role in various diseases of cats. However, current assays for the reduced form of glutathione (GSH) are time-consuming and semi-quantitative and do not allow assessment of GSH concentrations in individual cell populations. Therefore, we developed a flow cytometric assay for rapid determination of intracellular GSH concentrations in feline blood leukocytes. The assay was based on the ability of the non-fluorescent substrate monochlorobimane (mBCl) to form fluorescent adducts with GSH in a reaction catalyzed by the enzyme glutathione-S-transferase. Using flow cytometry, we found that mBCl was sensitive and specific for intracellular detection of the reduced form of GSH in feline leukocytes. Intracellular GSH concentrations were also stable for at least 24h in EDTA preserved whole blood samples stored at 4 degrees C. Neutrophils and monocytes from normal cats had significantly higher intracellular concentrations of GSH than T cells and B cells. The effects of FIV infection on intracellular GSH concentrations in cats were assessed using flow cytometry. We found that neutrophils from FIV-infected cats had significantly increased GSH concentrations, whereas intracellular GSH concentrations were significantly decreased in CD4(+) and CD8(+) lymphocytes from FIV-infected cats, compared to age-matched control animals. We conclude that a flow cytometric assay based on mBCl may be used to accurately and rapidly assess the effects of various disease states and treatments on GSH concentration in cat leukocytes and to help assess intracellular oxidative stress.

Understanding the early cytokine response to lentiviral infections may be critical to the design of prevention and treatment strategies. By using the feline immunodeficiency virus (FIV) model, we have documented an interleukin 10 (IL10)-dominated response in lymphoid tissue CD4+and CD8+T lymphocytes within the first 4 weeks after mucosal FIV infection. This profile coincided with the period of high tissue viral replication. By 10 weeks postinfection, tissue viral levels decreased significantly, and gamma interferon (IFNγ) production in CD8+T cells had increased to restore the IL10/IFNγ ratio to control levels. Concurrently, increased production of IL6 and viral RNA was detected in macrophages. These temporal associations of viral replication with cytokine balance in tissues suggest roles for IL10 in the permissive stage of infection and IFNγ in the subsequent down modulation of lentiviral infection.

A 4-year-old, female spayed Domestic Shorthair cat was presented for a 2-day history of lethargy, anorexia, and vomiting. Diagnostic assessments revealed a severe, regenerative anemia and the presence of a solitary, 3 × 4 cm splenic mass. Cytologic examination of fine-needle aspirates of the splenic mass showed sheets of pleomorphic histiocytic cells with numerous intracellular and extracellular, negatively staining to deeply basophilic, branching structures. These structures were typically of thin uniform thickness with random angled branching that formed geometrical shapes and fragments, occasionally with sharpened ends and a crystalline appearance. Septal-like divisions were irregularly spaced along some of the linear branches. The cytology was interpreted as a probable histiocytic inflammatory response to the unidentified foreign material with histiocytic sarcoma as a differential diagnosis. Histopathologic evaluation of the splenic mass resulted in a diagnosis of histiocytic sarcoma. The branching material stained positively for both iron and calcium, indicating that the material was compatible with Gamna-Gandy bodies present secondary to tumor-associated hemorrhage and the formation of calcium-iron complexes.

Background Canine T-cell lymphoma (TCL) is conventionally considered an aggressive disease, but some forms are histologically and clinically indolent. CD4 TCL is reported to be the most common subtype of TCL. We assessed flow cytometric characteristics, histologic features when available, and clinical outcomes of CD4+ TCL to determine if flow cytometry can be used to subclassify this group of lymphomas. Objective To test the hypothesis that canine CD4+ T-cell lymphoma (TCL) is a homogeneous group of lymphomas with an aggressive clinical course. Animals Sixty-seven dogs diagnosed with CD4+ TCL by flow cytometry and treated at 1 of 3 oncology referral clinics. Methods Retrospective multivariable analysis of outcome in canine CD4+ TCL including patient characteristics, treatment, and flow cytometric features. Results The majority of CD4+ TCL were CD45+, expressed low class II MHC, and exhibited an aggressive clinical course independent of treatment regimen (median survival, 159 days). Histologically, CD4+ TCL were classified as lymphoblastic or peripheral T cell. Size of the neoplastic lymphocytes had a modest effect on both PFI and survival in this group. A small number of CD4+ TCL were CD45− and class II MHC high, and exhibited an apparently more indolent clinical course (median survival not yet reached). Conclusions and Clinical Importance Although the majority of CD4+ TCL in dogs had uniform clinical and flow cytometric features and an aggressive clinical course, a subset had a unique immunophenotype that predicts significantly longer survival. This finding strengthens the utility of flow cytometry to aid in the stratification of canine lymphoma.

Background Canine T-cell lymphoma (TCL) is clinically and histologically heterogeneous with some forms, such as T-zone lymphoma (TZL), having an indolent course. Immunophenotyping is an important tool in the classification of TCL in people, and can be equally useful in dogs. Hypothesis/Objectives We hypothesized that loss of expression of the CD45 antigen is a specific diagnostic feature of TZL. Animals Twenty dogs with concurrent histology and immunophenotyping by flow cytometry were studied in depth. An additional 494 dogs diagnosed by immunophenotyping were used to characterize the population of dogs with this disease. Methods Lymph node biopsies from 35 dogs with TCL were classified by 2 pathologists using WHO criteria. Twenty lymph nodes were from dogs with CD45− TCL and 15 were from CD45+ TCL. The pathologists were blinded to the flow cytometry findings. Outcome information was sought for the 20 dogs with CD45− lymphoma, and population characteristics of the additional 494 dogs were described. Results All 20 CD45− cases were classified as TZL. The 15 CD45+ cases were classified as aggressive TCL and are described in an accompanying paper. TZL cases had a median survival of 637 days. Examination of 494 additional dogs diagnosed with TZL by immunophenotyping demonstrated that 40% of cases are in Golden Retrievers, are diagnosed at a median age of 10 years, and the majority have lymphadenopathy and lymphocytosis. Conclusions TZL has unique immunophenotypic features that can be used for diagnosis.

Two dogs, an 8.5-year-old intact male Golden Retriever and a 10-year-old spayed female English Springer Spaniel, each with varied clinical histories, were referred to the Colorado State University Veterinary Teaching Hospital for evaluation of hypercalcemia and severe anemia, respectively. In each dog, serum total protein and globulin concentrations were within reference intervals. Cytologic examination of bone marrow aspirates from both dogs revealed moderate to marked numbers of atypical lymphoid cells with plasma cell features. Using serum immunofixation and serum immunoglobulin (Ig) quantification, a monoclonal Ig protein was identified. In conjunction with other clinicopathologic and molecular findings, IgA secretory neoplasms, B-cell lymphoma with plasmacytoid features and multiple myeloma (MM), were diagnosed. To our knowledge, these cases represent the first descriptions of IgA-secreting neoplasms in dogs that lacked hyperglobulinemia. In cases of suspected B-cell lymphoma or MM in dogs, serum proteins should be fully evaluated for the presence of a monoclonal Ig even in dogs that lack characteristic hyperproteinemia or hyperglobulinemia. This evaluation will aid in the diagnosis of secretory B-cell lymphoma or MM leading to appropriate clinical and therapeutic case management.

Dendritic cells (DC) are potent antigen presenting cells which initiate and coordinate the immune response making them central targets of and attractive candidates for manipulation in chronic lentiviral infections. Emerging evidence suggests that DC immune function is disrupted during both acute and chronic infection with human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Despite some early promising data, the use of DC for lentiviral immunotherapy has not fulfilled its expected potential and has been complicated by the large number of variables involved in DC harvesting, purifying, and antigen loading. Pre-clinical studies aimed at identifying successful strategies for DC augmentation of current HIV treatment protocols are needed. Over the past two decades, the FIV model for HIV infection has increased the understanding of retroviral pathogenesis, and studies have begun using the FIV model to study DC dysfunction and DC-mediated immunotherapy. Careful consideration of the many variables involved in DC function and therapy should help develop protocols to explore the potential of DC vaccine-based therapies for lentiviral infection.