Flow cytometric analysis of ovine peripheral blood lymphocytes.

Leukocyte suspensions were prepared from the peripheral blood of 12 sheep three times at two month intervals beginning at 12 months of age. Monoclonal antibodies and flow cytometric analysis were used to characterize the cells. There were no significant differences over time therefore the data from the three time intervals were pooled. The mean percentages and ranges (minimum to maximum) of the major lymphocyte subtypes were: B-cells (29.6%, 11-50), gamma delta T-cells (36.6%, 22-68), CD4+ T-cells (14.1%, 8-22) and CD8+ T-cells (12.0%, 4-22). Lymphocyte subtype percentages appeared less variable within than between individuals. Two populations of B-cells were noted; one population had more cytoplasm and light scatter characteristics similar to monocytes while a second population of B-cells was more typical of small lymphocytes. The nature of the large B-cells requires further study.     

Flow cytometric analysis of T-lymphocyte subsets in cats.

We report a rapid, reliable method for the immunophenotype analysis of feline lymphocytes. Fluorescein isothiocyanate (FITC) conjugated to murine monoclonal antibodies f43, Fel 7 and fCD8 was used to identify phenotypes corresponding to feline T-cells, CD4+ T cells and CD8+ T cells. For isolation of white blood cells, whole blood lysis was faster, less variable and required much less sample than density gradient separation. To identify feline CD4+ and CD8+ cells simultaneously, directly conjugated FITC-fCD8 and phycoerythrin (PE) fCD4 (Fel 7) were used in two-color analysis. The two T cell sub-populations were non-overlapping. Dual-label and single-label values were not significantly different. Mean lymphocyte subset percentages in conventional and specific-pathogen-free (SPF) cats did not differ significantly. These values were: pan T lymphocytes (f43), 54.8%, CD4+ cells (Fel 7), 33.9%, and CD8+ cells (fCD8), 19.1%. Mean CD4/CD8 ratio was 1.9 in normal cats; the range was 1.2-2.6.     

Flow cytometric detection of alpha-1-acid glycoprotein on feline circulating leucocytes

To assess whether alpha‐1‐acid glycoprotein (AGP) can be detected on the membrane of feline circulating leucocytes. Design The presence of AGP on circulating leucocytes was investigated in both clinically healthy cats and cats with different diseases. A group of feline coronavirus (FCoV)‐positive cats, comprising cats with feline infectious peritonitis (FIP) and cats not affected by FIP but seropositive for FCoV, were included in this study because the serum concentration of AGP increases during FCoV infection. Procedure Flow cytometry (using an anti‐feline AGP antibody), serum protein electrophoresis, routine haematology and measurement of the serum AGP concentration were performed using blood samples from 32 healthy cats (19 FCoV‐seropositive), 13 cats with FIP and 12 with other diseases (6 FCoV‐seropositive). The proportion of cats with AGP‐positive leucocytes in the different groups (e.g. controls vs sick; FIP vs other diseases, etc.) or in cats with different intensities of inflammatory response was compared using a Chi‐square test. Results AGP‐positive leucocytes were found in 23% of cats. Compared with controls, the proportion of patients with positive granulocytes and monocytes was higher among sick cats (especially cats with diseases other than FIP) and cats with high serum AGP concentration, but not in cats with leucocytosis or that were FCoV‐seropositive. Conclusion AGP‐positive leucocytes can be found in feline blood, especially during inflammation. Conversely, no association between AGP‐positive leucocytes and FIP was found. Further studies are needed to elucidate the mechanism responsible for this finding and its diagnostic role in cats with inflammation.     

Flow cytometric analysis of thymocyte subpopulations in mice after whole-body X-irradiation.

To determined the cellular kinetics of thymocyte subpopulations in DBA1 mice after whole-body 6.8 Gy X-irradiation, they were analyzed for the expression of several cell surface antigens using flow cytometry. The results show that i) The majority of thymocytes rapidly depleted by irradiation was CD4+8+ cells. ii) radioresistant CD4+8- and CD4-8+ survived 18-48 hr after X-irradiation were considered to be relatively mature type, since they expressed high levels of CD3 and LECAM-1. iii) CD3-positive cells were detected in CD4-8- cells at 72 hr after irradiation.     

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.     

Flow cytometric analysis of bone marrow leukocytes in neonatal dogs

Dogs represent both an important veterinary species and a convenient model for allogeneic hematopoietic stem cell transplantation. Even though anti-canine CD34 antibodies have recently become available, little is known about hematopoietic lineages in dogs, partially because CD34- cells have been ignored in all analyses performed so far. In this study, we have focused on the bone marrow mononuclear compartment to provide an additional piece of information on the phenotype of CD34+ progenitors and to identify the dominant CD34- population. We have shown that, in contrast to the adults, mature lymphocytes are scarce in neonatal dog bone marrow. Using cross-reactive antibodies against CD79alpha we have shown that the B lineage of hematopoiesis strongly prevails. CD34+ cells were shown to be positive for MHC class II and SWC3, a member of the signal regulatory protein family.     

Evaluation of flow cytometric counting procedure for canine reticulocytes by use of thiazole orange.

An automated reticulocyte counting method that used a flow cytometer and the nucleic acid staining dye, thiazole orange, was developed. Anticoagulated (EDTA) blood specimens were suitable for flow cytometric reticulocyte counting when stored at 4 C for 96 hours after collection. Thiazole orange-stained samples were stable for 5.5 hours after staining when stored capped at 20 C and protected from light. Flow cytometric and manual microscopic reticulocyte counts were compared for counts in the 0.27 to 5.32% range (as determined by flow cytometry) and 0.10 to 4.90% range (as determined by 1 technician). Although the results of flow cytometric analysis generally correlated well (r = 0.821) with manual counts, there was poor correlation between the procedures for counts less than or equal to 2.0% (r less than or equal to 0.272). Linearity of flow cytometric counts over the range 0.27 to 14.46% was excellent (r = 0.999). Within-run precision of flow cytometric counts (% coefficient of variation [cv] = 3 to 5) was superior to manual microscopic counts obtained by one technician (% cv = 19 to 23) and to manual microscopic counts, which were an average of counts done by 3 technicians (% cv = 8 to 18). Comparable flow cytometric counts were obtained by counting 50,000 or 100,000 blood cells in the flow cytometer.     

Flow cytometric analysis of an immunodeficiency disorder affecting juvenile llamas

The present study was undertaken to characterize the immune system of llamas and alpacas and establish the basis for an immunodeficiency disorder affecting juvenile llamas. Flow cytometric (FC) analysis of the immune system with a panel of monoclonal antibodies (mAbs) revealed the immune system of llamas and alpacas is similar in leukocyte subset composition to that in ruminants. Peripheral blood mononuclear cells in adults are comprised of surface immunoglobulin (sIg(+)) B-cells (31%+/-8 S.D.), alphabeta T-cells (27%+/-12 S.D.), WC1(+) gammadelta T-cells (16%+/-11 S.D.), and 5-16% monocytes. In contrast to cattle, goats, and sheep, however, the frequency of WC1(+) gammadelta T-cells is not high in juveniles but similar to the frequency in adults. Also, sIg(+) B-cells are present in high concentration in juveniles (43%+/-11 S.D. ). Expression of major histocompatibility class II molecules on resting T-cells was low or absent. Comparative analysis of peripheral blood lymphocyte composition in normal juvenile llamas and llamas presenting with the signs of the juvenile llama immunodeficiency syndrome (JLIDS) revealed the concentration of B-cells is extremely low (1-5%) in affected animals. The findings suggest JLIDS is attributable to an autosomal recessive genetic defect in the development of B-cells.     

Flow cytometric analysis of T cell response in mice infected with Cowdria ruminantium.

A 3-fold increase in the numbers of Lyt-2+ T cells in the circulating blood of mice infected and re-infected with the Welgevonden stock of Cowdria ruminantium, as determined by flow cytometry, is supportive evidence that immunity in heartwater is cell-mediated. The rise in Lyt-2+ cells only after re-infection of the mice is further evidence that the development of immunity in heartwater is dependent on the unhindered and adequate replication of C. ruminantium.     

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 analysis of bronchoalveolar lavage fluid immune dynamics in calves

Understanding the immune dynamics in the respiratory mucosa of calves is necessary for a good management of bovine respiratory disease. Immune dynamics in the respiratory mucosa in humans and experimental animals has been assessed by flow cytometric analysis of bronchoalveolar lavage fluid (BALF); however, few reports have addressed this subject in calves. The aim of this study was to establish a universal method to analyze bronchoalveolar lavage fluid (BALF) by flow cytometry and to obtain basic knowledge of bovine respiratory mucosal immune dynamics. We investigated the immune cell populations in BALF and evaluated the surface antigen expression of alveolar macrophages in calves using flow cytometer. To further analyze the surface antigen variation observed in alveolar macrophages in detail, stimulation assays were performed in vitro. BALF cells were separated into three distinct populations based on their light scatter plot, which were considered to be macrophages, lymphocytes, and neutrophils. In most individuals, most of the BALF immune cells were alveolar macrophages, but an increased proportion of lymphocytes and neutrophils was observed in some individuals. Analysis of each surface antigen expression in alveolar macrophages showed that CD21 and MHC class II expression changed in response to changes in the leukocyte population. Moreover, when alveolar macrophages were stimulated with interferon-γ in vitro, the expression of CD21 was drastically reduced and MHC class II was increased, suggesting that functional changes in alveolar macrophages themselves are involved in the immune dynamics.     

Flow cytometric analysis of nuclear DNA for sex identification in three psittacine species

OBJECTIVE: To evaluate flow cytometric analysis for sex identification in 3 psittacine species, establish reference values for blood cell DNA content for each species, and determine effects of sample storage on DNA content. ANIMALS: 36 orange-winged Amazon parrots, 41 budgerigars, and 39 cockatiels. PROCEDURE: Blood samples were stained and analyzed by use of flow cytometry to measure cellular DNA content. Samples were analyzed immediately after collection and after being stored at 4 C for 48 and 72 hours. RESULTS: Mean DNA content (picograms per cell) was 3.248 for Amazon parrots, 2.702 for budgerigars, and 2.946 for cockatiels; DNA concentrations in samples analyzed immediately overlapped in a male and a female Amazon parrot and among 19 cockatiels. For budgerigars, DNA overlap between sexes was not detected in samples analyzed immediately or after storage for 72 hours. Sex was identified correctly in 94.4% of Amazon parrots, 100% of budgerigars, and 51.3% of cockatiels. For both sexes, DNA content in samples analyzed immediately was significantly different from that of stored samples. CONCLUSIONS AND CLINICAL RELEVANCE: Flow cytometric analysis was accurate for sex identification of Amazon parrots and budgerigars. Sample storage at 4 C for 48 or 72 hours caused variability in DNA content.     

Flow cytometric analysis of the DNA content of bovine and human bone marrow cells

The defence against infection in high-yielding dairy cows is correlated with the number and function of circulating neutrophils and depends on their production in bone marrow. Therefore, the DNA content of isolated bone marrow cell suspensions from 7 calves, 7 cows and 14 humans was assayed by flow cytometry. Bovine sternal bone marrow samples were collected within 30 min of death, and human marrow samples were collected by sternal puncture and aspiration. Mononucleated cells were isolated by gradient centrifugation. In the bone marrow samples from calves and cows, 35 +/- 2.6% and 31.8 +/- 1.5% of the isolated bone marrow cells respectively were in the S/G2/M-phase. The difference between calves and cows was not significant. In the human samples, only 12 +/- 0.8% of the cells were in the S/G2/M-phase. A significant (P < 0.001) difference was observed between the two species. These results indicated that the proliferative, in activity of haematopoietic cells is significantly higher in cattle than in humans.     

Flow cytometric analysis of peripheral blood mononuclear cells induced by experimental endotoxemia in horse

Cellular activation and functional cell surface markers were evaluated during experimentally-induced endotoxemia in healthy horses. Eight healthy adult horses were infused a low dose of endotoxin (lipopolysaccharide from Escherichia coli O26: B6, 30 ng/kg of body weight, IV) and five control horses were given an equivalent volume of sterile saline solution. Venous blood samples were collected for flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and to measure plasma endotoxin concentrations. Clinical signs of endotoxemia were recorded at 10, 20, 30, 40, 50 min, 1, 2, 3, 4, 8, 16, 24 and 48 hr after endotoxin or saline solution administration. Clinical findings characteristic of endotoxemia (tachycardia, tachypnea, increased rectal temperature, and leukopenia) occurred transiently in all horses administered endotoxin; however, plasma endotoxin concentrations were detectable in only 50% (4/8) of the endotoxin-infused horses. The percentage of CD4(+), CD5(+), and CD8(+) cells decreased while the percentage of CD14(+), IgM(+), and MHC class II(+) cells increased significantly after endotoxin infusion. Alterations in the immunophenotype of PBMCs from horses with experimentally-induced endotoxemia were associated with changes in vital signs, indicating that endotoxin altered the immuno balance.     

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 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 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.