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1.
Flow cytometry is useful to study lymphoid malignancies since it allows both immunophenotyping of neoplastic cells and quantification of antigen expression. CD18 and CD45 are commonly exposed membrane antigens with different levels of expression on blood leukocyte and neoplastic cells. The aim of this retrospective study was to semi-quantitatively evaluate the expression of CD18 and CD45 in dogs with different lymphoid malignancies with blood involvement and to compare results with those from healthy dogs and dogs with reactive diseases. Blood samples from 13 dogs with precursor lymphoid malignancies, 20 with mature neoplasms (either chronic lymphocytic leukaemia or lymphoma), of different immunophenotypes, were compared with 24 healthy dogs and 12 dogs with different reactive diseases. The median fluorescence intensity (MFI) for CD18 and CD45 was recorded on lymphoid and granulocytic populations using dual colour flow cytometry, and the ratio between MFI for lymphoid and granulocytic populations (L/N ratio) was calculated to compare the results obtained in different sessions using an internal control (granulocyte fluorescence intensity). Significant decreases in the L/N ratio were detected in neoplastic samples for both CD18 (either precursors or mature versus controls) and CD45 (either precursors or mature versus control), while using MFI only slight differences were detectable in CD45 between precursors and controls. Neoplastic cells often exhibited lower expression of the L/N ratio for CD18, and mainly for CD45, most likely due to a less mature pattern than normal cells and/or to an aberrant quantitative expression of surface antigen. Moreover, more than 50% of neoplastic lymphoid cells exhibited L/N ratios that were not within the values observed in controls for at least one antigen. Altered L/N ratios, in particular decreases of CD45, were mainly observed in precursor neoplasms and in T-cell neoplasms. Detection of altered expression of common antigens, and in particular a L/N ratio for CD45 lower than a value of 103% may be useful as a confirmation of pseudo-clonality thus helping in differentiating reactive and neoplastic lymphocyte expansions.  相似文献   

2.
Seventeen cutaneous and oral tumours with light microscopic features of plasmacytomas from 16 dogs were studied. Clinically, most neoplasms were benign, although three recurred after excision and three were locally invasive. Tumours most often arose on the pinnae, digits, gingiva, and inguinal regions near areas of chronic inflammation and exhibited variable degrees of plasmacytic differentiation microscopically. Diagnosis of plasmacytoma was confirmed in paraffin-embedded tissues with a panel of leukocyte differentiation antigen markers that included cross-reactive antibodies for Mb-1 (CD79a), CD3, and vimentin and canine-specific antibodies for CD45RA and CD18. Immunoreactivity for Mb-1 and CD45RA, including staining of multinucleate cells and cells with karyomegaly, confirmed a B-cell origin of neoplasms, while staining for CD3 and CD18 revealed an extensive network of infiltrative T-cells and dendritic cells in tumours suggestive of a directed immune response. These findings (i) demonstrate the value of using a panel of antibodies for leukocyte antigens to differentiate plasmacytomas from other cutaneous and oral round cell tumours, and (ii) suggest that immune recognition and responsiveness within tumours may play a role in the behaviour of plasmacytomas in dogs by affecting tumour cell growth and differentiation.  相似文献   

3.
The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix ‘w' which will lead to ‘wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.  相似文献   

4.
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.  相似文献   

5.
The aim of this study was to identify magnetic resonance (MR) signs that aid differentiation of neoplastic vs. non-neoplastic brain diseases in dogs and cats. MR images of 36 dogs and 13 cats with histologic diagnosis of intracranial disease were reviewed retrospectively. Diagnoses included 30 primary and three metastatic brain tumors, 11 infectious/inflammatory lesions, three vascular, one degenerative disease, and one developmental malformation. Upon univariate analysis of 21 MR signs, there were seven that had a significant association with neoplasia: single lesion (P = 0.004), shape (P = 0.015), mass effect (P = 0.002), dural contact (P = 0.04), dural tail (P = 0.005), lesions affecting adjacent bone (P = 0.008), and contrast enhancement (P = 0.025). Increasing age was also found to be associated with neoplasia (P = 0.0001). MR signs of non-neoplastic brain diseases in dogs and cats were more variable than those of brain neoplasia.  相似文献   

6.
This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs.  相似文献   

7.
The aim of this study was to evaluate the utility of three monoclonal antibodies (mAbs), and two anticanine IgG and one anticanine IgM polyclonal antibodies (pAbs) for the immunohistochemical diagnosis of canine autoimmune skin diseases. Skin biopsies from 11 cases of pemphigus (7 foliaceus, 3 vulgaris and 1 erythematosus), 12 cases of discoid lupus erythematosus (DLE) and 12 cases of chronic hyperplasic dermatitis were used. The CA4E7 mAb (IgG1 + IgG2) showed similar sensitivity, but higher specificity and lower background than the two anti-IgG pAbs for the immunohistochemical diagnosis of pemphigus and DLE. The CA4F1 mAb (IgG2) and CA3H1 mAb (IgG2) showed moderate and low interepithelial reactivity, respectively, in autoimmune skin diseases, but strong staining of the cytoplasm of plasma cells of the inflammatory infiltrates. These results suggest that the CA4E7 mAb may be valuable in the immunohistochemical diagnosis of such disorders.  相似文献   

8.
Background: Growing interest in veterinary oncohematology has facilitated the recent development and advancement of new techniques, such as flow cytometry, for immunophenotyping hematopoietic neoplasia in animals. Objective: The aim of this retrospective study was to characterize hematologic abnormalities and flow cytometric immunophenotyping (FCI) results in cases of hematopoietic neoplasia in dogs. Methods: Signalment, CBC data, and FCI results were obtained for 210 dogs with blood samples submitted to our laboratory. Immunophenotyping was carried out using an Epics XL‐MCL flow cytometer and a panel of 10 antibodies (CD45, CD3, CD4, CD8, CD79, CD21, CD14, CD34, CD41/61, CD61). The prevalence and severity of hematologic abnormalities was determined for the different types of hematopoietic neoplasms. Results: Based on cell morphology and phenotype, cases were classified as: acute lymphoblastic leukemia (ALL, n=51), acute myeloid leukemia (AML, n=33), chronic lymphocytic leukemia (CLL, n=61), and leukemic high‐grade lymphoma (L‐HGL, n=65). Most cases of ALL (47/51) and L‐HGL (41/65) had a B‐cell phenotype, while most cases of CLL (54/61) had a T‐cell phenotype, with a high prevalence of the large granular lymphocyte subtype (49/61). Anemia was found in 85% of all cases and was significantly more severe in ALL and AML compared with CLL and L‐HGL. Neutropenia was seen in 64–78% of acute leukemias (AML and ALL) in contrast to no cases of CLL and 11% of L‐HGL. Thrombocytopenia was seen in 88–90% of acute leukemias in contrast to 15% of CLL and 40% of L‐HGL. Thrombocytopenia was more prevalent (71% vs 22%) and significantly more severe in T‐cell vs B‐cell L‐HGL. Conclusion: A standard CBC is useful in suggesting the type of hemoproliferative disorder and may also help to predict the phenotype, especially in cases of L‐HGL.  相似文献   

9.
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10.
In this retrospective study, radiographically enlarged sternal lymph nodes (LNs) were evaluated in 71 dogs and 13 cats for average size, location, and most representative radiographic view. Concurrent clinical diagnoses were also noted and grouped into one of three following categories: neoplastic, inflammatory, or hematologic. There were no statistically significant differences in LN size between lateral views within each species. Enlarged sternal LNs were more cranially positioned in dogs than cats. No statistical difference was noted between right and left laterals, as to on which projection the enlarged sterna lymph nodes was seen best. Neoplastic disease (78.9%) was the most prevalent condition seen in association with LN enlargement in dogs, followed by primary infectious or inflammatory diseases (14.1%) and various hematologic conditions (7.0%). In cats, neoplasia was also most common (69.2%), followed by inflammatory diseases (30.8%). No hematologic conditions were noted in cats. The most common etiologic agent seen concurrently with enlarged sternal LNs in both dogs (33.8%) and cats (38.5%) was malignant lymphoma. The results of this study provide a clinically useful representation of the average size and location of radiographically enlarged sternal LNs for dogs and cats. The diseases represented demonstrate the wide spectrum of potential causes of sternal lymphadenopathy.  相似文献   

11.
BACKGROUND: Flow cytometry may be used to determine immunophenotype or lineage of leukemic cells, but few antibodies are available that are specific for cells of monocytic and granulocytic lineage. OBJECTIVE: The purpose of this study was to evaluate the flow cytometric staining patterns of 3 commercial monoclonal antibodies for monocytes and granulocytes in clinically healthy dogs and in dogs with acute myeloid leukemia (AML). METHODS: Mouse antihuman macrophage antibody (MAC387), mouse anti-human myeloperoxidase (MPO), and a canine neutrophil-specific antibody (NSA) were evaluated using flow cytometry on blood from 6 clinically healthy control dogs, and on blood (n = 7) and/or bone marrow (n = 2) from 8 dogs with AML. A diagnosis of acute leukemia was confirmed by >30% blasts in bone marrow or >30% blasts in peripheral blood, together with bi- or pancytopenia, circulating CD34-positive blast cells, and clinical signs of disease. Leukemic samples also were evaluated using a wide panel of monoclonal antibodies. RESULTS: MAC387 stained neutrophils and monocytes from control dogs, although the staining profiles for the 2 cell types differed. MPO and NSA resulted in strong positive staining of neutrophils; MPO also stained monocytes weakly. Lymphocytes did not stain with any of the antibodies. One case was classified as AML of granulocytic lineage (AML-M1), 6 cases were classified as acute monocytic leukemia (AML-M5), and 1 case was classified as acute myelomonocytic leukemia (AML-M4). Neoplastic myeloblasts in the dog with granulocytic AML were positive for MPO, NSA, MAC387, and CD4. All monoblasts from the dogs with AML-M5 were positive for CD14, 5 of 6 were positive for MAC387, and 2 were positive for MPO. NSA staining was negative in the 2 dogs with AML-M5 in which it was evaluated. In the dog with AML-M4 variable percentages of blast cells were positive for CD14, MPO, MAC387, CD4, and NSA. CONCLUSIONS: Antigens identified by antibodies to MAC387, MPO, and NSA were expressed not just by normal mature neutrophils and monocytes, but also by neoplastic myeloblasts and monoblasts. These 3 antibodies may be useful as part of a wider panel for immunophenotyping AML in dogs.  相似文献   

12.
The myeloid panel of monoclonal antibodies (mAbs) submitted to the Third Swine CD Workshop were analysed for reactivity with bone marrow haematopoietic cells (BMHC). Using single and triple immunofluorescence labelling by flow cytometry (FCM), the mAbs were grouped according to their capacity to recognise myeloid cell populations and/or maturation stages. Group 1 consisted of mAbs labelling the majority of myeloid BMHC, including neutrophilic, eosinophilic and monocytic cells. The ligands for SWC3 and CD11b-like mAbs of group 1 showed a maturation-dependent intensity of expression. The other antibodies of group 1 reacted with BMHC to give a sharp, single peak. Group 2 mAbs reacted only with monocytic cells. The anti-human CD49e mAb Sam-1 was the only mAb detecting the majority of monocytic cells, but not other BMHC. The mAbs in group 3 recognised antigens expressed on granulocytes, but not monocytes. The previously identified SWC8 in this group proved to be useful in differentiating major population of BMHC when cells were double labelled with the pan-myeloid SWC3. Other mAbs within group 3, such as MIL4 and TMG6-5 (an anti-human CD11b), only recognised subsets of neutrophils and eosinophils. Group 4 mAbs reacted with the more mature subpopulations of neutrophils and monocytes. Some of these antibodies might prove useful for assessment of cell maturity, such as anti-CD14 and the anti-human CD50 mAb HP2/19.  相似文献   

13.
Ligand blotting of Pasteurella haemolytica leukotoxin (LKT) susceptible BL3 bovine lymphoma cell membranes with LKT detected two putative receptors with Mr of 95 and 100 kDa, whereas no LKT binding to membrane proteins was detected for LKT non-susceptible human leukemic cells. Anti-bovine CD18 and CD11a/CD18 mAb recognized 95 and 100kDa bands from BL3 cell membranes. CD18 isolated from BL3 cell membranes bound LKT. Pre-incubation of BL3 cells with anti-bovine CD18 or CD11a/CD18 mAb caused partial inhibition of LKT-induced leukolysis. Therefore, we propose that bovine CD18 acts as a species-specific leukocyte receptor for P. haemolytica LKT.  相似文献   

14.
Background: Cytoplasmic fragments derived from fragile neoplastic lymphocytes are common in samples of lymph nodes collected from dogs with lymphoma. These cytoplasmic fragments interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membranes. Objective: The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments from flow cytometric analysis of canine lymph nodes in which lymphoma was present. Methods: Single‐cell suspensions of neoplastic cells were prepared from biopsy samples and fine‐needle aspirates of lymph nodes from 23 dogs with lymphoma. Suspensions were stained using a violet laser‐excitable (405 nm) membrane‐permeable DNA‐binding fluorescent dye (DyeCycle Violet [DCV]), incubated with antibodies against CD3, CD5, CD21, CD22, and CD45, and then stained with 7‐amino‐actinomycin D (7‐AAD), an argon‐excitable (488 nm) membrane‐impermeable DNA‐binding fluorescent dye. Multiparameter flow cytometry was used for analysis based on selective uptake and laser‐activated fluorescence of these dyes. Results: Cytoplasmic fragments, which were DCV‐negative and CD45‐positive, and dead cells, which were positive for 7‐AAD, were efficiently separated from neoplastic cells. Conclusion: Staining with DCV is a useful method to improve flow cytometric gating methods and quantitative analyses of lymph node samples from dogs with lymphoma.  相似文献   

15.
After initial evaluation of the 176 new and 19 control monoclonal antibodies (mAb) submitted to the Second International Swine CD Workshop, 57 were assigned to the T-cell/activation marker subgroup. These 57 mAb were further analyzed using flow cytometry on whole blood lymphocytes, splenocytes, Peyer's patch lymphocytes, in vitro cell lines, broncho-alveolar lavage cells, Con A and PHA blasts, fetal cell populations, and by 2-color flow cytometry against mAb to porcine CD2, CD4, and CD8. Finally, the molecular weights of the target antigens were characterized when possible. As a result of these analyses, 23 mAb were distributed into 7 CD clusters. Newly confirmed mAb assignments included: two CD2; one CD4; two CD5; one wCD6; and one wCD25. Three new mAb were found that reacted with wCD8, one of which defined a new epitope, wCD8c. For the first time, mAb against porcine CD3 were identified, including 6 mAb that reacted with three different epitopes. Several new mAb reacted with antigens whose expression varied depending on the activation state of the test cell. These will require further characterization in order to assign a CD number.  相似文献   

16.
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17.
OBJECTIVE: To assess the potential clinical relevance of seroreactivity to Bartonella henselae antigens in dogs. ANIMALS: 40 dogs seroreactive to B henselae and 45 dogs that did not seroreact to B henselae. PROCEDURE: A case-control study was conducted. Clinical and clinicopathologic findings were extracted from medical records of each dog. RESULTS: Statistical differences were not detected between dogs seroreactive or nonseroreactive to B henselae when analyzed on the basis of disease category or results of hematologic, biochemical, urine, or cytologic analysis. However, seroreactivity to B henselae antigens was detected in 2 of 4 dogs with a clinical diagnosis of granulomatous meningoencephalitis, 3 of 4 dogs with immune-mediated hemolytic anemia, 3 of 4 dogs with infective endocarditis, 2 of 3 dogs with lymphoid neoplasia, and 5 of 10 dogs with polyarthritis. Additionally, seroreactivity to B henselae antigens was detected in 18 of 34 thrombocytopenic dogs and 14 of 27 dogs with neutrophilia. CONCLUSIONS AND CLINICAL RELEVANCE: Significant associations were not detected between seroreactivity to B henselae and various diseases. Prospective epidemiologic studies investigating specific diseases, such as meningoencephalitis or polyarthritis, and specific hematologic abnormalities, such as immunemediated hemolytic anemia or thrombocytopenia, should be conducted to further define the potential clinical relevance of antibodies against B henselae in dogs. IMPACT FOR HUMAN MEDICINE: Bartonella organisms are increasingly reported as pathogens that induce are increasingly reported as pathogens that induce chronic infections in humans and dogs. Dogs may serve as natural candidates for future study of the disease in humans.  相似文献   

18.
Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections with inherent poor tissue morphology, and are not readily adapted to formaldehyde fixed and paraffin embedded tissue with well preserved morphology. Seven well characterised monoclonal antibodies against porcine leukocyte antigens were tested on neutral buffered paraformaldehyde fixed and paraffin embedded porcine tissue sections using the highly sensitive tyramide signal amplification system. Combining this method with different antigen retrieval techniques enabled us to detect CD2, CD3, CD4, CD8 and SWC3 antigen expressing cells in porcine lymphoid tissue. Thus, we describe herein methods for the detection of several major cell types of the porcine immune system in fixed tissue with optimal preservation of histological details.  相似文献   

19.
Alterations in lymphocyte subpopulations and in other hematologic variables have been documented in people with heart failure. The purpose of the current study was to compare flow cytometric and hematologic variables in dogs with congestive heart failure (CHF) to healthy controls. CD4+ peripheral blood mononuclear cells (PBMC) and CD8+ lymphocytes were analyzed by flow cytometry, and white blood cell count, platelet count, hematocrit, and hemoglobin were determined by a complete blood count. Twenty-five dogs with CHF (International Small Animal Cardiac Health Council [ISACHC] class 2 [n = 12] and ISACHC class 3a [n = 13]) and 13 healthy controls were enrolled in the study. Compared with the controls, dogs with CHF had markedly lower percentages of CD4+ PBMC, CD8+ lymphocytes, hematocrit, and hemoglobin, but markedly higher leukocytes, neutrophils, and platelets. There were no differences in these variables between dogs with dilated cardiomyopathy (n = 6) and those with chronic valvular disease (n = 19). Dogs in ISACHC class 3a had a markedly lower total lymphocyte number, CD4+ and CD8+ cells, and hematocrit, but markedly higher leukocyte and neutrophil numbers relative to the control group. CD4+ and CD8+ subpopulations and other blood cell variables are altered in dogs with CHF. Future studies to determine possible clinical implications of these changes are warranted.  相似文献   

20.
OBJECTIVE: To develop an efficient and reliable method that accurately differentiates bovine lymphocytes from monocytes in leukograms. SAMPLE POPULATION: Blood samples from 30 healthy cows and 1 calf with bovine leukocyte adhesion deficiency. PROCEDURE: Flow cytometric analysis of intracellular complexity and CD45 expression on bovine leukocytes was compared with results for conventional light microscopy methods. Verification of leukocyte subpopulations determined by intracellular complexity and CD45 expression was conducted, using 2-color phenotypic analysis with selected monoclonal antibodies. RESULTS: The CD45 and side-scatter properties of bovine leukocytes clearly differentiated cell types, including neutrophils, eosinophils, monocytes, and lymphocytes. CONCLUSIONS AND CLINICAL RELEVANCE: This is a rapid assay that is simple to use. More importantly, it is more accurate than the conventional method that involves the use of blood slides and light microscopy, because of the ability of the assay to readily distinguish bovine monocytes and lymphocytes. Rapid preparation of samples and short analysis times allow for efficient and reliable examination of a large number of samples, and the task of viewing slides by light microscopy is eliminated. The labor-savings benefit of this procedure is most apparent in research environments that require frequent processing of batches of blood samples.  相似文献   

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