Flow cytometric evaluation of hemophagocytic disorders in canine

Background — Hemophagocytic macrophages in canine bone marrow are observed in malignant histiocytosis as well as benign hemophagocytic histiocytosis. Cytomorphologic evaluation alone may be inadequate to consistently differentiate between benign and malignant forms of hemophagocytic disorders. Objective — The purpose of this study was to evaluate the ability of flow cytometry and immunophenotyping to differentiate between benign and malignant types of hemophagocytic disorders in dogs. Methods — Blood smears and bone marrow differential cell counts were evaluated for 10 dogs with hemophagocytic disorders. Bone marrow samples were labeled with monoclonal antibodies to CD18, MCH class‐II, Thy‐1, CD14, CD3, and CD21. Using flow cytometry, forward‐angle versus side‐angle light scatter plots were analyzed and immunophenotypes were determined. Results — Scatter plots from 3 dogs with a necropsy diagnosis of malignant histiocytosis revealed 2 atypical cell clusters. One cluster contained cells of similar size or larger than immature myeloid cells and metamyelocytes. Cells in the other cluster were highly granular, with granularity similar to or greater than that of metamyelocytes. In bone marrow from dogs with malignant histiocytosis that was labeled with anti‐CD14 antibody, macrophages represented 29–48% of nucleated cells. Seven dogs had a clinical or histopathologic diagnosis of benign hemophagocytic syndrome. Three of the dogs had normal cell distribution in scatter plots. Two dogs had 2 abnormal cell clusters: 1 within the immature myeloid and metamyelocyte gates and the other with granularity similar to or greater than that of metamyelocytes. The remaining 2 dogs had an atypical cell population, mostly within the immature myeloid gate. For dogs with benign hemophagocytic syndromes, 6–17% of cells in the bone marrow were CD14 positive. Conclusions — The cellular distribution in scatter plots and the total number of macrophages in bone marrow may be useful in differentiating malignant histiocytosis from benign hemophagocytic syndromes in dogs.     

Flow cytometric evaluation of canine bone marrow differential cell counts

Abstract: Three flow cytometric techniques were evaluated for determination of differential cell counts on canine clinical bone marrow specimens. Techniques included staining bone marrow specimens with 2'7'-dichlo-rofluorescein (DCF) or 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluation of forward-angle light scatter vs. side-angle light scatter plots. Flow cytometric evaluation of bone marrow cells stained with DCF failed to separate bone marrow cells into distinct cell populations. Staining with DiOC6 resulted in separation of bone marrow cells into populations of mature and immature erythroid cells, mature and immature myeloid cells, and lymphocytes. The scatter plot method resulted in identification of mature and immature erythroid cells, immature myeloid cells, metamyelocytes, and bands and segmenters. Lymphocytes could not be differentiated from mature erythroid cells by the scatter plot method. When the results of the DiOC6 method and the scatter plot method were compared with manual bone marrow differential cell counts, the scatter plot method had more similar mean values and higher correlation coefficients. The scatter plot method has the potential of providing rapid semiquantitative assessment of bone marrow differential cell counts in dogs for specimens that contain low numbers of lymphocytes.     

Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model

Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.     

Flow cytometric evaluation of canine bone marrow based on intracytoplasmic complexity and CD45 expression

BACKGROUND: Because of the complexity, subjectivity, time, and technical skill required for determination of manual bone marrow differential cell counts, an alternative method is needed. Several flow cytometric methods have been described, but all have limitations. OBJECTIVE: The purpose of this study was to evaluate a technique for bone marrow differential cell counting based on flow cytometric evaluation of CD45 expression and intracellular complexity (CD45 scatter plots). METHODS: Bone marrow was obtained from 15 dogs that were being evaluated for hematologic disorders. In preliminary studies, the location of bone marrow subpopulations in the CD45 scatter plots was evaluated by labeling bone marrow with lineage-specific markers. A template was developed to identify these cell populations. Gates were set to identify granulocytes, myeloblasts, monocyte/macrophages, lymphocytes, and nucleated erythroid populations. RESULTS: The CD45 labeling technique accurately quantified granulocytes, myeloblasts, erythroid precursors, and lymphocytes in canine bone marrow. Correlation coefficients with manual counts for granulocytes, myeloblasts, erythroid cells, lymphocytes, and monocyte/macrophages were 0.90, 0.89, 0.96, 0.91, and 0.54, respectively. CONCLUSIONS: The capacity of the CD45 scatter-plot technique to quantify lymphocytes and myeloblasts is an advantage over previously described techniques. The simplicity of the CD45 labeling method and the ease with which batches of samples can be analyzed makes the technique potentially applicable as a routine test in clinical and research laboratories.     

Flow cytometric immunophenotype of canine lymph node aspirates

Increasing availability of reagents able to distinguish subtypes of lymphocytes and other leukocytes has enabled greater understanding of lymphocyte biology and pathology in the dog. Lymphocytes in circulation most commonly are subjected to immunophenotypic assessment by flow cytometry, but needle aspirates of lymph nodes can be similarly suitable for immunophenotypic examination. In this investigation, the feasibility of immunophenotyping samples obtained by needle aspiration of lymph nodes from 32 dogs with no physical abnormalities and 6 dogs with lymphoma was determined. In addition, samples from 6 dogs were stored overnight at 4 degrees C and reanalyzed 24 hours later. For each sample, stained smear preparations were examined microscopically for lymphocyte morphology, neoplasia, and the presence of inflammatory cells. Expression of antigens on a corresponding sample of aspirated cells was determined by flow cytometric detection of antibody binding on a minimum of 10,000 events. The distribution of data was determined with Anderson-Darling tests, and reference intervals incorporating the central 95% of values were established. Adequate samples were obtained from 30 of 32 clinically normal dogs. Immunophenotypic results after 24 hours of storage were consistent with those obtained immediately after sampling. Reference intervals for lymphocyte subsets from normal dog lymph nodes were similar to the proportions of CD3+, CD4+, CD8+, and CD21+ lymphocytes found in blood. Aspirates of enlarged lymph nodes from dogs with lymphoma were readily classified by this technique. Aspiration of lymph nodes from dogs for comprehensive analysis by flow cytometry is feasible and applicable to immunophenotyping of lymphoma.     

Flow cytometric evaluation of ki67 for the determination of malignancy grade in canine lymphoma

Ki67 is a nuclear antigen significantly correlated with degree of malignancy in human non‐Hodgkin lymphomas. We wanted to assess the ability of flow cytometric evaluation of Ki67 index (Ki67I) in differentiating the grade of malignancy in canine lymphomas. Ki67I was determined on lymph node aspirates of 90 immunophenotyped lymphomas classified according to the updated Kiel classification: 80 high grade (HG, 62 B cell and 18 T cell) and 10 low grade (LG, 3 B cell and 7 T cell) lymphomas. HG lymphomas showed significantly higher Ki67I compared with LG lymphomas (P < 0.0001). A significant difference in HG lymphomas was detected between B‐ and T‐immunophenotypes. Receiver operating characteristic (ROC) curve highlighted a high accuracy of Ki67I in recognizing HG lymphomas [area under the curve (AUC) = 99.4] and a cut‐off value of 12.2% was established (sensitivity = 96.3% and specificity = 100%). Thus, we suggest the combination of Ki67I flow cytometric determination and immunophenotype as a reliable tool to classify canine lymphomas.     

Flow cytometric analysis of canine umbilical cord blood lymphocytes

Lymphocyte subsets in canine umbilical cord blood were flow cytometrically analyzed and compared with those of the dams' peripheral blood. The proportion of CD3+ T lymphocytes, CD21+CD3- B lymphocytes, and CD3-CD21- non-T non-B lymphocytes in umbilical cord blood was 52.9%, 30.4%, and 16.7%, respectively. T lymphocyte/B lymphocyte ratio was significantly lower in the umbilical cord blood than in the dams' peripheral blood (2.1 +/- 1.4 versus 11.0 +/- 8.1, P < 0.001). In contrast, CD4+ lymphocyte/CD8+ lymphocyte ratio was significantly higher in the umbilical cord blood than in the dams' peripheral blood (7.6 +/- 2.2 versus 1.8 +/- 0.6, P<0.001). These findings clarified the phenotypic characters of canine umbilical cord blood lymphocytes.     

Flow cytometric analysis of lectin-binding to canine blood lymphocytes

The cell surface glycoconjugates of blood lymphocytes from 19 dogs with and without neoplastic disease were quantitated using flow cytofluorometric analysis of the binding characteristics of 3 lectins, namely, wheat germ agglutinin, concanavalin-A, and Lens culinaris agglutinin. The specificity of lectin binding was determined using competitive monosaccharide inhibitors. The results show enhanced binding of concanavalin-A to blood lymphocytes from dogs with lymphosarcoma relative to healthy dogs, or those with a variety of neoplastic and nonneoplastic diseases.     

Development of multidrug resistance in a canine lymphoma cell line

New multidrug resistant cell lines developed from the canine B cell lymphoma cell line (GL-1) were characterized in terms of chemosensitivity to some antineoplastics and P-glycoprotein (Pgp) expression. GL-1 was continuously exposed to a culture medium containing gradually increasing levels of doxorubicin and the cells that could grow in the presence of doxorubicin were obtained. Chemosensitivity of these cells to various antineoplastics were investigated with or without verapamil, which reversed Pgp-mediated drug resistance. The expression of Pgp on the cells was also examined by Western blot analysis. As a result, three kinds of resistant cell lines, designated as GL-DOX60, 300, and 4000 were obtained. These cell lines showed stable proliferation in the medium containing 60, 300, and 4000 ng/ml, respectively. These cells were much more resistant to vincristine than doxorubicin. This resistance was strongly reversed by the presence of verapamil. On the other hand, cisplatin was effective enough in killing these derived cells. In the Western Blot analysis, some bands that reacted to the anti-human Pgp monoclonal antibodies were observed in GL-DOX4000. The cells derived from GL-1 have multidrug resistance potential mediated by canine Pgp. The cells produced in this experimental trial are considered to be useful models for various investigations on canine multidrug resistance.     

Flow cytometric evaluation of bound IgG on erythrocytes of anaemic dogs

The detection of red blood cell (RBC)-bound immunoglobulins in case of anaemia with the direct agglutination test (DAT or Coombs test) has been reported to be of low sensitivity. We therefore tested the applicability of flow cytometry for the detection of canine IgG on RBC using two different IgG-specific secondary reagents: goat-anti-dog IgG (GalphaD-IgG) and rabbit-anti-dog IgG (RalphaD-IgG). Membrane staining RBC samples were performed at 4 degrees C. Comparisons of agglutination test at 37 degrees C and 4 degrees C showed, that binding of the secondary antibodies at 4 degrees C was more sensitive compared to agglutination at 37 degrees C and the two antisera differed considerable in their agglutination activity. Binding of GalphaD-IgG and RalphaD-IgG to RBC of healthy dogs (n=15) was low and mean fluorescence intensities were taken to calculate thresholds above which RBC of patients were judged positive. As in agglutination tests, both secondary antisera displayed considerable differences (concentration-dependent binding and histogram profiles) after flow cytometric analysis. Using flow cytometry, with GalphaD-IgG 8 of 17 agglutination-negative patients were positive and RalphaD-IgG was positive with 3 of 3 agglutination-negative RBC samples. Thus, flow cytometric analysis of proved to be a sensitive technique, detecting RBC-bound canine IgG of DAT-negative patients. The results of both techniques, however, are significantly influenced by the used IgG-specific polyclonal reagents.     

Ultrasonographic evaluation of normal canine iliopsoas muscle

The aim of this study was to describe the normal ultrasonographic appearance of the iliopsoas muscle and related landmarks. Hind limbs of five dog cadavers with no history of lameness were evaluated. The origin and mid-body of the psoas major and the common insertion of the iliacus and psoas major on the lesser trochanter of the femur were identified and evaluated. New methylene blue was injected under ultrasonographic guidance at the three sites. Dissection was performed to confirm placement of the dye. The L3 origin, mid-body, and insertion of the muscle were identified in all dogs and were consistent in appearance and compatible with the general appearance of muscle and tendons. The L2 origin was consistently difficult to image. The same ultrasound technique was subsequently applied to four healthy dogs, and consistent images of the iliopsoas muscle and associated landmarks were obtained. In this study, the major structures of the iliopsoas could be identified and in all dogs had a similar appearance. Ultrasound is an important tool for the diagnosis of musculotendinous injury and may be useful for identification of ilipsoas injury as a cause of lameness in the dog.     

Flow cytometric analysis of canine colonic mucosal lymphocytes from endoscopically obtained biopsy specimens

OBJECTIVE: To validate use of canine colonic biopsy specimens obtained via endoscopy as a source of mucosal lymphocytes (ML) for flow cytometric analysis. SAMPLE POPULATION: Mucosal biopsy specimens from 10 adult dogs. PROCEDURE: Mucosal lymphocyte subsets obtained from excised colon were compared with ML subsets obtained from biopsy specimens obtained by use of an endoscopic forceps (6 dogs). Endoscopic colonic biopsy specimens from 4 other dogs were used to define whether obtained ML were predominantly of intraepithelial or lamina propria origin. Mucosal lymphocytes were isolated and labeled, using commercially available monoclonal antibodies directed against canine cell surface antigens. Lymphocyte subsets (cytotoxic or helper T cells; B cells) were determined by use of flow cytometric analysis. RESULTS: A large number of viable ML was obtained after dissociation of the colonic epithelium from excised colon (45.5 + 21.5 X 10(6)) and endoscopic (7.2+/-3.4 X 10(6)) biopsy specimens. Lymphocyte subsets obtained with both methods were identical for each dog and consisted predominantly of intraepithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded a large number of viable lymphocytes from the lamina propria (56.7+/-20.4 X 10(6)), but collagenase digestion of endoscopic biopsy specimens was less rewarding. CONCLUSION AND CLINICAL RELEVANCE: A representative sample of viable intraepithelial ML is obtainable from endoscopic biopsy specimens. Flow cytometric analysis, a minimally invasive technique, can be used to study ML of client-owned animals.     

Flow cytometric characterization of T lymphocyte subsets in the peripheral blood of Chinese rhesus macaques: normal range, age- and sex-related differences

Available data on the normal levels of white blood cell populations in healthy rhesus macaques (Macaca mulatta) originated and living in China is scanty. To obtain such data, blood samples from 150 Chinese rhesus macaques were collected and the normal range of white blood cells and their subsets were analyzed according to age and sex by flow cytometry. CBC data showed that the count of total white blood cells and lymphocytes decreased with age. Phenotypic analysis of CD4 and CD8 expression on CD3+ T lymphocytes showed that the percentage of CD4+ T cells (51.4+/-9.6%), CD4-CD8- T cells (8.5+/-4.1%) and the ratio of CD4+ T to CD8+ T cells (1.26+/-0.55) decreased with age; and the percentage of CD8+ T cells (42.0+/-9.7%), CD4+CD8+ T cells (1.3+/-0.9%) and CD3+ lymphocytes (55.3+/-13.3%) increased with age. However, no statistically significant difference was observed between the male and female groups in most parameters in these monkeys except for the percentage of CD4+CD8+ T cells. This study provided basic information about blood cell count and T lymphocyte subsets in Chinese rhesus macaques. It may be useful for comparative studies using Indian and Chinese rhesus macaques.     

Effects of dietary fish oil and vitamin E supplementation on canine lymphocyte proliferation evaluated using a flow cytometric technique

Lymphocyte proliferation and peripheral blood mononuclear cell (PBMC) production of PGE(2) were assayed in 15 healthy dogs fed a basal diet supplemented with either sunflower oil (Group Sunflower oil), sunflower oil and menhaden fish oil (Group Fish oil), or sunflower oil and menhaden fish oil plus alpha-tocopherol acetate for 12 weeks (Group Fish oil + E). Lymphocyte proliferation was determined by a flow cytometric technique utilizing the fluorochrome carboxyfluorescein diacetate succinimidyl ester (CFSE). The PBMC supernatant PGE(2) concentration was assayed using a competitive enzyme-linked immunoassay. Group Fish oil had a significant decrease in lymphocyte proliferation at week 12. PBMC production of PGE(2) was decreased in all three groups but only significantly reduced in groups receiving fish oil supplementation. Based on these results, this level of fish oil supplementation appears to suppress the lymphoproliferative response in healthy, young dogs but this response can be attenuated by high levels of dietary vitamin E supplementation. Furthermore, fish oil-induced reduction in lymphocyte proliferation appears to manifest through a PGE(2)-independent mechanism and is not associated with increased lipid peroxidation.     

Flow cytometric analysis of lymphocyte proliferative responses to food allergens in dogs with food allergy

Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5- to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (P<0.05), suggesting that lymphocytes against food allergens may be involved in the pathogenesis of canine FA.     

Flow cytometric enumeration of reticulocyte in the peripheral blood from canine infected with Babesia gibsoni

Peripheral blood samples from dogs infected with Babesia gibsoni were analyzed by a flow cytometer for the percentage of reticulocytes after staining with a membrane-permeable fluorochrome, thiazole orange. Though thiazole orange has been reported to stain human reticulocytes and Plasmodium-infected erythrocytes, number of positive cells determined by the flow cytometry did not include that of erythrocytes infected with Babesia gibsoni. Analysis of 51 samples revealed a correlation coefficient of 0.96 as compared to the conventional determination by light microscopy. Separation of reticulocytes from Babesia gibsoni-infected erythrocytes by flow cytometry with or without the stain remained unresolved.     

Flow cytometric techniques for detection of candidate cancer stem cell subpopulations in canine tumour models

The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of ‘CSC’. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell‐associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker‐defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context‐dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria.