Isolation and characterization of feline endometrial mesenchymal stem cells.

BACKGROUND: Recently, there has been a growing interest in stem cells for human medicine. Limited feline endometrial mesenchymal stem cell (fEM-MSC) research in veterinary medicine necessitates reporting for future feline disease research and therapy. OBJECTIVES: This study aimed to isolate fEM-MSCs from feline endometrial tissues and evaluate their morphology, proliferative ability, differentiation ability, and immunophenotype. METHODS: Feline endometrial tissues were obtained from the ovariohysterectomies of healthy cats and isolated using an enzymatic method. The morphology and proliferative ability of the isolated cells were assessed using a doubling time (DT) assay from passages 3 to 6 (P3 - P6). We measured pluripotency gene expressions of cells in P2 using quantitative real-time polymerase chain reaction (qRT-PCR). To investigate MSC characteristics, a trilineage differentiation assay was conducted in P4, and cells in P4 were immunophenotyped using flow cytometry. RESULTS: fEM-MSCs showed a typical spindle-shaped morphology under a microscope, and the DT was maintained from P3 to P6. fEM-MSCs could differentiate into adipocytes, osteoblasts, and chondrocytes, and expressed three pluripotency markers (OCT4, SOX2, and NANOG) by qRT-PCR. Immunophenotypic analysis showed that the fEM-MSCs were CD14 -, CD34 -, CD45 -, CD9+, and CD44+. CONCLUSIONS: In this study, the feline endometrium was a novel source of MSCs, and to the best of our knowledge, this is the first report on the isolation method and characteristics of fEM-MSCs.

Immunophenotyping of canine T cell activation and proliferation by combined protein and RNA flow cytometry.

The limited availability of canine-reactive monoclonal antibodies restricts the analyses of immune cell subsets and their functions by flow cytometry. The PrimeFlow™ RNA Assay may serve as a potential solution to close this gap. Here we report a blood immunophenotyping method utilizing combined protein- and RNA-based flow cytometry to characterize canine T cell activation and proliferation within individual cells. In this assay, CD69 expression was detected by an RNA probe and CD25 and Ki67 were detected by antibodies. Canine peripheral blood mononuclear cells (PBMCs) were stimulated with three agents with different modes of action, anti-CD3/CD28 antibodies, phytohemagglutinin, or phorbol myristate acetate /ionomycin. Robust T cell activation (CD25+ and/or CD69+) and proliferation (Ki67+) were detected. Both CD69 and CD25 appear to be robust and sensitive T cell activation markers with early induction and low background expression. Upon stimulation, T cell proliferation occurred later than T cell activation and was associated with CD25 expression. This canine T cell activation and proliferation immunophenotyping method was evaluated in 5 independent experiments using PBMCs from 10 different beagle dogs with satisfactory assay performance. This method can greatly facilitate the evaluation of immune disease pathogenesis and immunotoxicity risk assessment in nonclinical drug development in canine.

Canine T zone lymphoma is a tumor of mature, previously activated αß T cells.

T cell lymphomas are a diverse group of tumors found in both dogs and humans, originating from various normal T cell types. Identifying the origin of neoplastic lymphocytes can offer valuable insights into the pathogenesis and clinical behavior of these tumors. T zone lymphoma (TZL) in dogs is characterized by the absence of CD45 expression, a strong breed predilection, and its association with adult-onset demodicosis-a condition believed to be linked to immunosuppression. In this study, our aim was to employ transcriptomic and functional data to determine the normal counterpart of TZL. Identifying the normal counterpart may help us understand both how these tumors arise and explain their clinical behavior. Gene expression profiling using NanoString and RNA seq was used to compare the transcriptome between neoplastic T zone cells, normal canine T cells and publicly available gene sets using Gene Set Enrichment Analysis. Mitogen, anti-CD3 stimulation and PMA/ionomycin stimulation were used to assess T cell proliferation in vitro, and intracellular cytokine production was measured by flow cytometry. Gene expression profiling revealed that TZL is most likely derived from an activated or memory alpha-beta T cell but the cells do not fall cleanly into an effector subtype. TZL cells express CD4-specific transcription factors GATA3 and THPOK, even though TZL cells more commonly express CD8, or neither CD4 nor CD8. TZL cells produce high levels of interferon gamma and tumor necrosis factor alpha when stimulated, further supporting the hypothesis that they are derived from an antigen experienced T cell. TZL cells do not proliferate when stimulated through the T cell receptor but will divide when the T cell receptor is bypassed with PMA and ionomycin. The observation that these cells are derived from a mature, previously activated T cell is the first step in understanding the genesis of this unique T cell tumor.

Expression of CD13 and CD26 on extracellular vesicles in canine seminal plasma: preliminary results.

Canine seminal plasma is a complex fluid containing proteins, peptides, enzymes, hormones as well as extracellular vesicles that are involved in many physiological and pathological processes including reproduction. We examined the expression of the extracellular vesicles surface antigens Aminopeptidase-N (CD13) and Dipeptidyl peptidase IV (CD26) by flow cytometry. For this study, third fraction of the ejaculate, from fertile adult male German Shepherd dogs, was manually collected twice, two days apart. FACS analyses revealed that CD13 and CD26 are co-expressed on the 69.3 ± 3.7% of extracellular vesicles and only a 2.0 ± 0.5% of extracellular vesicles express CD26 alone. On the other hand, 28.6 ± 3.6% of seminal EVs express CD13 alone. Our results agree with the hypothesis that CD26 needs to be co-expressed with other signal-transducing molecules, while CD13, can perform functions independently of the presence or co-expression of CD26. The results obtained in normal fertile dogs could represent physiological expression of these enzymes. Therefore, it would be interesting to carry out further studies to evaluate the expression of CD13 and CD26 on extracellular vesicles as biomarker for prostate pathological condition in dogs.

Prognostic value of peripheral blood and bone marrow infiltration assessed by flow cytometry in dogs with de novo nodal peripheral T-cell lymphoma receiving alkylating-rich chemotherapy.

Peripheral T-cell lymphoma (PTCL) is highly aggressive in dogs and demonstrates a poor response to traditional chemotherapy. The aim of this retrospective study was to assess the prognostic significance of peripheral blood (PB) and bone marrow (BM) infiltration evaluated by flow cytometry (FC) in dogs with treatment-naïve and histologically confirmed PTCL. To be included, dogs had to undergo complete staging, including FC on lymph nodes, PB and BM samples. Additionally, dogs had to receive an alkylating-rich protocol and have a complete follow-up. Treatment response was evaluated based on RECIST criteria at each chemotherapy session, and the end-staging was conducted at the completion of treatment. Endpoints were time to progression (TTP) and lymphoma-specific survival (LSS). The relationship between TTP/LSS and the percentage of PB and BM infiltration, categorized as > 1%, > 3%, > 5%, > 10%, > 15% and > 20% was investigated. Fifty dogs were included: based on imaging and FC, 78.0% had stage 5 disease, 14.0% had stage 4, 6.0% had stage 3 and 2.0% had stage 1. By multivariable analysis, the CD4-negative phenotype was the only factor associated with a shorter TTP (P = 0.049), while BM infiltration was significantly associated with LSS (P = 0.037). Dogs with BM infiltration > 5% had shorter median LSS (114 days; 95%CI: 0-240) compared to dogs with BM infiltration ≤ 5% (178 days; 95%CI: 145-211). Lack of complete response (P = 0.039) and administration of corticosteroids before chemotherapy (P = 0.026) also significantly worsened LSS. BM flow cytometric evaluation could be considered an essential part of staging work-up for dogs with PTCL and has prognostic relevance.

Optimising flow-cytometry methods for marine mollusc haemocytes using the pearl oyster Pinctada maxima as a model.

Flow-cytometry has become increasingly popular to assess the haemocytes morphology and functions of marine molluscs. Indeed, haemocytes are the first line of defence of the immune system in molluscs and are used as a proxy for oyster health. Authors publishing in the field of flow-cytometry and molluscs health seemed to utilise the same methods for all model species used, independently of their geographical location in the world (temperate, tropical, etc.). Hence, this paper dived into flow-cytometry methodology and investigated if using different plates, different thresholds, different incubation times and temperatures as well as different fluorochromes concentrations affected the results. This study revealed that the cell count did not change when using different thresholds on the FSC-H parameter of the instrument but was affected by the plate type, the temperature of incubation, and the time of incubation. Indeed, non-adherent plates yielded the highest cell count and lower cell counts were associated with a higher temperature and a longer time of incubation. Furthermore, the haemocytes functions such as the phagocytosis, the lysosomal content, the intracellular oxidative activity, and the mitochondria activity were also affected by the temperature and the time of incubation. An increase in the phagocytosis capacity, lysosomal content and mitochondria activity was observed with a higher temperature. At the exception of the phagocytosis rate, all the other parameters such as the phagocytosis capacity, the intracellular oxidative activity, and the lysosomal content increased with a longer incubation time. We also showed that it is best to optimise the amount of fluorochromes used to avoid unnecessary background or non-specific staining.

B-cell leukemia in an adult sheep.

B-cell leukemia is a rare form of hematologic neoplasia in sheep, especially in adult animals. We present a case report of a 5-year-old WhiteFace Sheep wether with suspected acute lymphoblastic leukemia. The patient, a second-generation relative of ewes experimentally inoculated with atypical scrapie, exhibited acute lethargy and loss of appetite. Laboratory investigation revealed marked leukocytosis, lymphocytosis, and abnormal serum chemistry panel results. Microscopic examination of blood and bone marrow smears exhibited a high percentage of large neoplastic cells with lymphoid characteristics. Histopathologic analysis of the spleen, liver, lungs, and other organs confirmed the presence of widespread tissue infiltration by neoplastic cells. Immunohistochemical labeling demonstrated strong intracytoplasmic labeling for CD20, consistent with B-cell neoplasia. Flow cytometric analysis confirmed the B-cell lineage of the neoplastic cells. Screening for bovine leukemia virus, which can experimentally cause leukemia in sheep, yielded a negative result. In this case, the diagnosis of B-cell leukemia was supported by a comprehensive panel of diagnostic evaluations, including cytology, histopathology, immunohistochemistry, and immunophenotyping. This case report highlights the significance of accurate diagnosis and classification of hematologic neoplasia in sheep, emphasizing the need for immunophenotyping to aid in the diagnosis of B-cell leukemia. It also emphasizes the importance of considering spontaneous leukemia as a differential diagnosis in sheep with lymphoid neoplasia, especially in the absence of circulating infectious diseases.

Determination of optimal storage time and temperature for the detection of red blood cell and platelet surface-associated immunoglobulin by flow cytometry in healthy horses.

Differentiating immune-mediated causes from other causes of anemia and thrombocytopenia can be challenging. Flow cytometry can detect surface-associated immunoglobulin (sIg) on red blood cells (RBC) and platelets (PLT) in dogs and horses. Sample storage parameters for ideal assay performance has not been evaluated in horses. The study objective is to identify optimal storage time and temperature of equine whole blood for the detection of RBC-sIg and PLT-sIg via flow cytometry. Both assays were performed on samples at time 0, 4, 24, 48, and 72 h post collection. RBC-sIg samples were stored at 4 °C and PLT-sIg samples were stored at 4 °C and room temperature. RBC-sIg percentages were stable up to 72 h storage. Platelet surface-associated IgG percent positive platelets increased above baseline at all timepoints and percent positive platelets were inconsistent across timepoints for IgM and IgA. PLT-sIg testing should ideally be performed within 4 h of collection. In instances where this is not feasible, samples should be stored at 4 °C and analyzed no later than 24 h after collection. Whereas cutoff values for RBC-sIg remained similar across timepoints, results for PLT-sIg should be compared to time-specific cutoff or reference intervals established by the laboratory running the test.

Application of Flow Cytometry in the Diagnosis of Bovine Epidemic Disease.

As science and technology continue to advance, the use of flow cytometry is becoming more widespread. It can provide important information about cells in the body by detecting and analysing them, thereby providing a reliable basis for disease diagnosis. In the diagnosis of bovine epidemic diseases, flow cytometry can be used to detect bovine viral diarrhoea, bovine leukaemia, bovine brucellosis, bovine tuberculosis, and other diseases. This paper describes the structure of a flow cytometer (liquid flow system, optical detection system, data storage and analysis system) and its working principles for rapid quantitative analysis and sorting of single cells or biological particles. Additionally, the research progress of flow cytometry in the diagnosis of bovine epidemic diseases was reviewed in order to provide a reference for future research and application of flow cytometry in the diagnosis of bovine epidemic diseases.

Biochemical Features of X or Y Chromosome-Bearing Spermatozoa for Sperm Sexing.

This review presents information on biochemical features of spermatozoa bearing X or Y chromosome, enabling production of a sperm fraction with pre-defined sex chromosome. The almost only technology currently used for such separation (called sexing) is based on the fluorescence-activated cell sorting of sperm depending on DNA content. In addition to the applied aspects, this technology made it possible to analyze properties of the isolated populations of spermatozoa bearing X or Y chromosome. In recent years, existence of the differences between these populations at the transcriptome and proteome level have been reported in a number of studies. It is noteworthy that these differences are primarily related to the energy metabolism and flagellar structural proteins. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between the spermatozoa with different sex chromosomes. Sperm sexing is a part of the widespread protocol of artificial insemination of cows with cryopreserved semen, it allows to increase proportion of the offspring with the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.

Lymphocyte immunophenotyping in dogs with lymphopenia of common causes.

Lymphocyte immunophenotyping can be useful for evaluating immune competence and predicting the disease prognosis. It is essential to gain knowledge about canine lymphocyte immunophenotypes in various conditions. The study deals with the characteristics of lymphopenia in dogs, with an emphasis on lymphocyte immunophenotyping by flow cytometry. Blood samples from 44 dogs with lymphopenia were included in the study. All lymphopenias sent from veterinary clinics to the diagnostic laboratory were analyzed. The hematological and biochemical abnormalities were investigated, as well as the effect of the age. Lymphopenias were classified according to the level of C-reactive protein (CRP). The percentage of T cells, B cells, Th cells and Tc cells, and T/B and Th/Tc ratios were determined by flow cytometry. Lymphopenias often occurred in dogs over 7 years of age (79.5 %). The most common were postoperative lymphopenia (31.8 %) and inflammatory diseases (29.5 %), most commonly affecting the gastrointestinal tract. Frequent abnormalities were monocytosis (56.8 %), increased CRP (72.7 %) and decreased albumin/globulin ratio (50.0 %). The percentage of Th lymphocytes was significantly lower in the group with elevated CRP than in the group with basal CRP (P = 0.0329). A negative correlation was found between the level of CRP and the percentage of Th lymphocytes (r = -0.3278, P = 0.0390). This study provided new insights into the appearance, incidence and classification of canine lymphopenia.

Evaluation of the susceptibility of Tritrichomonas foetus to extracts of Lantana camara (Verbenaceae) by flow cytometry.

Bovine Trichomonosis (BT), a sexually transmitted disease endemic in countries with extensive cattle farming and natural service, is one of the most common causes of reproductive failure. 5-nitroimidazoles and their derivatives are used for its treatment, mainly metronidazole. The emergence of drug resistance mechanisms and treatment failures raise the need to investigate the effectiveness of new active compounds that contribute to parasite control. In this regard, extracts of Lantana camara (Verbenacea) have shown high biocidal potential against isolates of Trypanosoma cruzi and Leishmania braziliensis in vitro assays, although their effect on Tritrichomonas foetus has not been demonstrated yet. The available information on in vitro susceptibility of trichomonicidal drugs comes from the use of a diversity of methodologies and criteria, especially the observation of parasite motility under the optical microscope to assess their viability. Recently, in our laboratory, the use of flow cytometry has been described for the first time as a rapid and efficient method to evaluate the viability of T. foetus against metronidazole. The present study aimed to evaluate the cytostatic effect of L. camara extracts against T. foetus isolates by flow cytometry. Under aerobic conditions, IC50 values of 22.60 µg/mL were obtained on average. Under anaerobic conditions, the IC50 oscilated around 29.04 µg/mL. The results obtained allowed describing the susceptibility exhibited by these protozoa, being a valuable information for the development of potential BT treatments.

Use of multi-color flow cytometry for canine immune cell characterization in cancer.

Although immunotherapy is becoming a standard approach of human cancer treatment, only a small but critical fraction of patients responds to the therapy. It is therefore required to determine the sub-populations of patients who will respond to immunotherapies along with developing novel strategies to improve efficacy of anti-tumor immune reactions. Current development of novel immunotherapies relies heavily on mouse models of cancer. These models are important for better understanding of mechanisms behind tumor immune escape and investigation of novel strategies to overcome it. Nevertheless, the murine models do not necessarily represent the complexity of spontaneously occurring cancers in humans. Dogs spontaneously develop a wide range of cancer types with an intact immune system under similar environment and exposure to humans, which can serve as translational models in cancer immunotherapy research. To date though, there is still a relatively limited amount of information regarding immune cell profiles in canine cancers. One possible reason could be that there are hardly any established methods to isolate and simultaneously detect a range of immune cell types in neoplastic tissues. To date only a single manuscript describes characterization of immune cells in canine tumour tissues, concentrating solely on T-cells. Here we describe a protocol for multi-color flow cytometry to distinguish immune cell types in blood, lymph nodes, and neoplastic tissues from dogs with cancer. Our results demonstrate that a 9-color flow cytometry panel enables characterization of different cell subpopulations including myeloid cells. We also show that the panel allows detection of minor/aberrant subsets within a mixed population of cells in various neoplastic samples including blood, lymph node and solid tumors. To our knowledge, this is the first simultaneous immune cell detection panel applicable for solid tumors in dogs. This multi-color flow cytometry panel has the potential to inform future basic research focusing on immune cell functions in translational canine cancer models.

Cross-reactivity of human monoclonal antibodies with canine peripheral blood mononuclear cells.

In drug development, the dog is often used as a model for non-rodent preclinical safety studies. In particular, immunophenotyping in dogs can be important to characterize the toxicological profile of a test item. A wide range of antibodies specific to surface antigens is needed, however, commercially available antibodies to dog are scarce. To date, numerous studies have reported the cross-reactivity of human monoclonal antibodies with canine peripheral blood mononuclear cells (PBMC). In this study, we aimed to increase the number of canine-specific antibodies and took a rather novel approach to further determine cross-reactivity of 378 human recombinant antibodies lacking Fc regions to surface antigens on canine PBMC. The screening resulted in 30 human monoclonal antibodies well reactive to canine PBMC. Sequence homology of the targeted human and canine antigens was analyzed with Basic Local Alignment Search Tool. Thirteen human cross-reactive antibodies of interest were analyzed with cells from canine whole blood in combination with lineage markers. Finally, ten antibodies were identified as useful markers for the application in dog. Except for CD27, the remaining nine antibodies are already commercially available human cross-reactive antibodies. This study provides a new source for all ten antibodies described here.

Isolation and characterization of feline dental pulp stem cells.

OBJECTIVES: The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency, immunophenotype and cytogenetic stability. METHODS: Dental pulp was isolated by explant culture from two cats <1 year old at post mortem. Their clonogenicity was characterized using a colony-forming unit fibroblast assay, and their proliferative ability was quantified with a doubling time assay in passages 2, 4 and 6 (P2, P4 and P6, respectively). Multipotency was characterized with an in vitro trilineage differentiation assay in P2, and cells were immunophenotyped in P4 by flow cytometry. Chromosomic stability was evaluated by cytogenetic analysis in P2, P4 and P6. RESULTS: The fDPSCs displayed spindle and epithelial-like morphologies. Isolated cells showed a marked clonogenic capacity and doubling time was maintained from P2 to P6. Trilineage differentiation was obtained in one sample, while the other showed osteogenic and chondrogenic differentiation. Immunophenotypic analysis showed fDPSCs were CD45-, CD90+ and CD44+. Structural and numerical cytogenetic aberrations were observed in P2-P4. CONCLUSIONS AND RELEVANCE: In this study, fDPSCs from two cats were isolated by explant culture and immunophenotyped. Cells displayed clonogenic and proliferative ability, and multipotency in vitro, and signs of chromosomic instability were observed. Although a larger study is needed to confirm these results, this is the first report of fDPSC isolation and in vitro characterization.

TLR7/8 agonist (R848) inhibit bovine X sperm motility via PI3K/GSK3α/ß and PI3K/NFκB pathways.

Sex-control technology have great economic value and is one of the hot topics in livestock research. To produce more milk, dairy farmers prefer female offspring. X/Y sperm separation is an effective method for offspring sex control. Currently, the major commercial production method for sperm separation is flow cytometry sorting in cattle. However, flow cytometry requires expensive equipment and long sorting times. So, a simple and inexpensive method for producing a higher number of dairy cows is required. In this study, R848 activates toll-like receptor 7/8 (TLR7/8), thereby separating X from Y sperm. The results showed TLR7/8 is expressed in the tail of X sperm. Immunofluorescence (IF) of testes, epididymis, and ejaculate shows that the number of TLR7+/8+ sperm cells is up to 50 %. Furthermore, TLR7/8 agonist (R848) affects mitochondrial function through the PI3K/GSK3α/ß/hexokinase and PI3K/NFκB/hexokinase signalling pathways, inhibiting X sperm motility, while the motility of Y-sperm remains unchanged. The difference in sperm motility causes Y sperm (with high motility) to move to the upper layer and X-sperm (with low motility) to the lower layer allowing the separation of X and Y sperm. Based on this study, we reveal a simple and effective method for enriched X/Y sperms from cattle.

Flow Cytometric Identification and Enumeration of Monocyte Subsets in Bovine and Water Buffalo Peripheral Blood.

Monocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions. The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14++ CD16-/low ), intermediate (intM; CD14++/+ CD16+ ), and non-classical (ncM; CD14-/low CD16++ ) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no-centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no-wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14+/++ /CD16+ . These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC. Basic Protocol: Absolute count of cM, intM, and ncM without compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In-house monoclonal antibody labeling using a Pacific Blue™ kit Support Protocol 2: In-house monoclonal antibody labeling using an Alexa Fluor® 647 kit Support Protocol 3: Titration of fluorochrome-conjugated antibodies.

Cryopreservation effect on sperm viability, mitochondrial membrane potential, acrosome integrity and sperm capacitation of alpaca spermatozoa detected by imaging flow cytometry.

Eighty-five sperm samples were cryopreserved and SYBR14/PI, MitoTracker Deep Red FM, FITC-PSA/PI and chlortetracycline were used for imaging flow cytometry evaluation of sperm viability, mitochondrial membrane potential (MMP), acrosome integrity and sperm capacitation, respectively. Sperm motility was also registered. Sperm motility (46.1 ± 7.7 vs. 24.1% ± 6.5%), sperm viability (49.8 ± 11.5 vs. 32.3% ± 9.6%) and high MMP (49.8% ± 12.4% vs. 34.9% ± 9.9%) decreased significantly (p < .05) during cryopreservation process, in contrast to acrosome-reacted in viable spermatozoa (1.0% ± 1.6% vs. 1.0% ± 1.0%) and sperm capacitation (10.0 ± 9.8 vs. 8.2% ± 12.4%) that were similar (p > .05) before and after cryopreservation. Positive correlations were found between sperm motility versus high MMP (r = .63), sperm motility versus sperm viability (r = .67) and sperm viability versus high MMP (r = .88). In conclusion, cryopreservation of alpaca spermatozoa is related to a decrease in sperm motility, sperm viability and high MMP, meanwhile acrosome integrity and sperm capacitation are not affected.

Evaluation of a novel moving threshold gating strategy for assessment of reticulated platelets in dogs using the ADVIA 2120 analyzer.

BACKGROUND: A novel method using a moving threshold (r-PLTmt) to determine canine r-PLTs (reticulated platelets) has been introduced for ADVIA 2120 software v6.11.7. OBJECTIVES: We aimed to evaluate absolute (ar-PLTmt) and percent (%r-PLTmt) prior to and after visual inspection of scattergrams (ar-PLTmtv, %rPLTmtv) compared with flow cytometry (flow) and to determine reference intervals (RIs) in 120 dogs. METHODS: For method comparison, 42 blood samples of healthy and thrombocytopenic dogs were included. Calculation of Spearman's rho, Bland-Altman, and Passing-Bablok analysis was performed. Coefficients of variation (CVs) were determined for three concentration levels. RESULTS: Moderate correlations between %r-PLTmt and %r-PLTmtv (rs 0.75-0.76) were seen compared with flow cytometry. The CV for medium %r-PLTs counts assessed with flow cytometry was 12.9%. Comparable CVs were obtained for ar-PLTmt (14.4%) and %r-PLTmt (15.7%), and ar-PLTmtv and %r-PLTmtv (10.9% and 12.9%, respectively). At low and high concentration levels, CVs for % and absolute r-PLTmt/rPLTmtv ranged between 23%-30% and 15%-20%. In patients with microcytic hypochromic erythrocytes, CVs for ar-PLTmt and %r-PLTmt were 36%-66%. Visual inspection of scattergrams resulted in a marked decrease in CV ranging between 15% and 20%. A proportional bias of 10.8% between %r-PLTmt and flow cytometry became lower (9.7%) after visual validation of scattergrams. Passing-Bablok analysis showed proportional and constant error. RIs for r-PLTmt and r-PLTmtv were 0.2%-3.8% and 0.6-10.2 × 109 /L and 0.3%-4.5% and 1.1-10.3 × 109 /L, respectively. Median values for %r-PLTmtv were higher in young adults (≤2 years) than in older dogs (P = 0.03). CONCLUSIONS: r-PLTmt and r-PLTmtv were moderately correlated with flow cytometry. Visual inspection of scattergrams is recommended.

Flow Cytometry in Veterinary Practice.

This article summarizes the current applications of flow cytometry in clinical veterinary medicine, which is largely restricted to the diagnosis of hematopoietic neoplasms (lymphomas and leukemias) of domestic dogs, cats, and horses. A brief background on the technique of flow cytometry and fundamentals of data interpretation are included. Major emphasis is placed on clinical indications for flow cytometry, principles of sample collection and submission, and awareness of diagnostic and prognostic utility. Expectations regarding both the benefits and limitations of flow cytometry in a clinical setting, and its complementary nature with other types of testing, are also reviewed.