Measurement of thrombopoietic activity through the quantification of megakaryocytes in bone marrow cytology and reticulated platelets

Reticulated platelets are considered as marker for bone marrow thrombopoiesis. The aim of the study was evaluate the role of reticulated platelets as markers of thrombopoiesis in dogs. Reticulated platelets analysis by flow cytometry and megakaryocyte quantification by bone marrow cytology were determined in 29 healthy adult dogs (control group), 14 dogs with thrombocytopenia without megakaryocytic hypoplasia (group A) and 14 dogs with thrombocytopenia which presented megakaryocytic hypoplasia (group B), detected by bone marrow aspiration cytology. Blood samples were collected and the platelet rich plasma was obtained for reticulated platelets quantification in flow cytometry. Megakaryocytes were quantified in aspiration cytology by two techniques in marrow particles, and correlated to reticulated platelets counts. There are no differences between megakaryocyte quantification. Although there is no correlation between reticulated platelet values and megakaryocyte in bone marrow cytology, the interpretation of reticulated platelet values can be based both on absolute or relative corrected values.     

Identification of acute myeloid leukemia in dogs using flow cytometry with myeloperoxidase, MAC387, and a canine neutrophil-specific antibody

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.     

Generation of anti‐porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis

T cell‐mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti‐porcine CD69 mAb‐producing mouse hybridomas, 01‐14‐22‐51 (IgG2b–κ) and 01‐22‐44‐102 (IgG2a–κ), both showing fine reactivity with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin‐stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti‐interferon‐γ mAb and anti‐tumor necrosis factor‐α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.     

Clinical, histopathological and immunohistochemical findings in a case of megakaryoblastic leukemia in a dog.

The clinical, hematological, and histopathologic features of megakaryoblastic leukemia (M7) were investigated in a 10-year-old female Shih-Tzu dog. Megakaryoblastic leukemia was diagnosed using anti-human platelet glycoprotein (GP IIIa) and anti-human von Willebrand factor (vWF) antibodies. The expression of CD antigen on megakaryoblasts was also assessed using a CD79a monoclonal antibody. Immunological markers allowed visualization of neoplastic megakaryocytes. Antibodies against platelet GP IIIa were demonstrated to be the most useful for the diagnosis of megakaryoblastic leukemia of paraffin-embedded canine tissues. Hematological and histological data coupled with immunohistochemical reactivity for platelet GP IIIa, vWF, and CD79a antigen in blast cells confirmed a diagnosis of M7 megakaryoblastic leukemia.     

Clinical features of canine nodal T‐cell lymphomas classified as CD8+ or CD4−CD8− by flow cytometry

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

Essential thrombocythaemia in the dog and cat: a report of four cases

The occurrence of essential thrombocythaemia in the dog and cat is recorded. In four cases of anaemia profound elevation of platelet numbers was noted. Bone marrow biopsy revealed megakaryocyte proliferation, with excessive platelet production and abnormal megakaryocyte morphology. This condition should be considered in the differential diagnosis of anaemia.     

Hematologic abnormalities and flow cytometric immunophenotyping results in dogs with hematopoietic neoplasia: 210 cases (2002–2006)

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.     

Canine cutaneous epitheliotropic T‐cell lymphoma: a review

Cutaneous epitheliotropic T‐cell lymphoma in the dog is a rare neoplastic condition with unknown aetiology. The dermatitis is characterized by infiltration of neoplastic T lymphocytes with a specific tropism for the epidermis and the adnexal structures. The different clinical and histological forms (mycosis fungoides, pagetoid reticulosis and Sézary syndrome) are reviewed. The disease in the dog resembles the human syndrome, but in 80% of the canine cases, neoplastic cells are CD4^?/CD8⁺ versus CD4⁺/CD8^? in 90% of the human patients. Prognosis is poor with a survival time from few months to 2 years. Treatments frequently have a low efficacy. New protocols using lomustine may improve the poor prognosis of the disease.     

Canine CD4+ T‐cell lymphoma identified by flow cytometry exhibits a consistent histomorphology and gene expression profile

T‐cell lymphomas (TCL) are a diverse group of neoplasms with variable diagnostic features, pathophysiologies, therapeutic responses and clinical outcomes. In dogs, TCL includes indolent and aggressive tumours such as T‐zone lymphoma (TZL) and peripheral T‐cell lymphoma (PTCL), respectively. Delineation of molecular subtypes and investigation into underlying pathophysiologies of aggressive TCLs remains inadequate. We investigate the correlations between flow cytometry and histopathology of 73 cases of nodal TCL. The majority of cases (82.2%) were characterized as CD4⁺ TCL by flow cytometry. Fewer cases were classified as CD8⁺ TCL (6.8%) or CD4^?CD8^? TCL (11.0%). All cases, regardless of immunophenotype, exhibited conserved histologic features consistent with the WHO classification of PTCL. Histologic subsets of PTCL corresponding to immunophenotypic features were not identified. Neoplastic cell size determined by flow cytometry correlated significantly with mitotic rate. RNA‐seq was performed on a subset of CD4⁺ PTCL cases (n = 6) and compared with sorted control CD4⁺ T‐cells. The gene expression pattern of CD4⁺ PTCL was similar between all cases regardless of breed. PTCL was enriched in pathways representing G‐coupled protein receptor signalling, extracellular matrix remodelling and vascular development, immune signalling and mitotic activity. Furthermore, global gene expression changes were consistent with downregulation of PTEN signalling and upregulation of the MTOR‐PI3K‐ATK axis. In this study, we evaluated the correlations between flow cytometry, histopathology and gene expression within a large cohort of nodal TCLs. We further demonstrate the ability of flow cytometry to identify a subtype of T‐cell lymphoma, CD4+ PTCL, with a uniform histomorphology and gene expression profile.     

In vitro effects of the activity of novel platinum (II) complex in canine and human cell lines

The anticancer activity of novel platinum derivative, a complex of platinum with tris(2‐carboxyethyl)phosphine (Pt‐TCEP), has been evaluated in canine (D‐17) and human osteosarcoma (U2‐OS) cell lines. Viability of cells after incubation for 24 or 72 hours with increasing concentrations (0.625, 1.25, 2.50, 5, 10 and 20 μM) of Pt‐TCEP was tested in an MTT assay and compared to effect of cisplatin. Longer‐term effect of Pt‐TCEP was evaluated in the colony‐forming unit assay after 24 hours exposure to the Pt‐TCEP (2 and 3 μM) and subsequent incubation for 2 weeks. The influence of the compound on the cell cycle was measured after 24 hours treatment with Pt‐TCEP (3 μM). Its pro‐apoptotic activity was examined after 24 hours treatment with Pt‐TCEP (1.25, 2.50, 5, 10 and 20 μM) using flow cytometry. Expression of main proteins involved in apoptosis was measured after exposure for 24 hours to 3 or 5 μM Pt‐TCEP in Western Blot. The compound much more effectively decreased cell viability than cisplatin in case of both cell lines. IC₅₀ of Pt‐TCEP was 5.93 ± 0.12 in D‐17 and 3.45 ± 0.14 in U2‐OS cell lines after 24 hours, and 1.77 ± 0.14 in D‐17 and 1.53 ± 0.11 in U2‐OS after 72 hours (P < .05). The compound arrested cells in the G2/M phase and inhibited the ability of cells to form colonies. Pt‐TCEP induced caspase‐dependent apoptosis. The expression of the anti‐apoptotic Bcl‐X_L protein was decreased after Pt‐TCEP treatment in both cell lines. The results confirmed anti‐cancer activity of Pt‐TCEP against canine and human osteosarcoma cell lines.     

Alteration of integrin-associated protein (CD47) on experimental porcine eperythrozoonosis

To evaluate the alteration of CD47 on RBCs of pigs infected with M. suis, we induced the experimental porcine eperythrozoonosis and collected the blood samples at the different time points. The result of analysis by flow cytometry after reaction with mouse-anti-human CD47 and caprine-anti-mouse IgG-FITC reagents indicated that the CD47 quantity on RBCs changed correlatively with the course of PE. The lowest value of M1 (percentage of the positive cells in fluorescence intensity) occurred on day 7 post inoculation at the peak of parasitemia and decreased 82% compared with the control sample. And then, the values of M1 on day 14 and 21 rised slowly but were still significantly different with the controls (p < 0.01). These suggested that the quantity of CD47 on RBCs altered progressively with the phases of the PE disease. The decrease of CD47 on RBCs maybe weaken the inhibitory CD47-SIRPalpha interaction and provide positive signals for phagocytosis of macrophages resulting in the removal of the RBCs from the circulation. In conclusion, CD47, a marker on RBCs, maybe play an important role on the mechanism of hemolysis caused by infection of M. suis.     

Reactivity of monoclonal antibodies to human CD antigens with cells from mink

Three hundred and seventy six monoclonal antibodies (mAbs) raised against human leukocyte surface antigens were analyzed by flow cytometry for cross reactivities against mink leukocytes. We found 53 mAbs (14%) to cross react. This study defined cross reactions to the following human markers: CD1a, CD9 (4 mAbs), CD10, CD11a (2 mAbs), CD14 (3 mAbs), CD18 (5 mAbs), CD20 (atypical reaction), CD21, CD25 (atypical reaction), CD29 (3 mAbs), CD32, CD41, CD42a, CD44 (4 mAbs), CD45, CD45RO, CD47 (2 mAbs), CD49d (3 mAbs), CD61 (2 mAbs), CD62P, CD66abcd, CD71, CD75s, CD79b (2 mAbs), CD86, CD88, CD104 (atypical reaction), CD172a, CD236R (glycophorin C, (atypical reaction)), Xg(a) carbohydrate antigen, Rhesus antigen and two unspecified PAN-reactive mAbs. In order to characterize the molecular mass of the corresponding cross reacting mink markers, the mAbs were used to immunoprecipitate the surface antigens. Fourteen mAbs out of the 53 mAbs reactive with mink leukocytes gave reproducible IP findings. The masses of the precipitated antigens were generally in good agreement with those of the homologous human markers. We also performed immunohistochemical staining analyses on formalin fixed, paraffin embedded mink tissue from lymph node and spleen, and found 7 out of 22 mAbs to give a positive signal. Generally, the immunohistological analyses resulted in expected staining patterns.     

Cutaneous T‐cell lymphoma – Sézary syndrome in a Boxer

A 7‐year‐old male Boxer with a 3.5‐year history of atopy and food hypersensitivity was presented with multiple poorly circumscribed nodules and maculae of the skin and tongue, and jaundiced mucosal membranes. Cytologic and histopathologic examination of the skin lesions revealed cutaneous epitheliotropic lymphoma. Cells were CD3⁺ and CD8⁺ in flow cytometry. The CBC showed a moderate leukocytosis with 16% atypical lymphocytes with irregularly cleaved nuclei. Flow cytometric phenotyping of peripheral blood showed an elevated proportion of the CD8⁺ T‐lymphocyte subpopulation, indicating a malignant population of T‐cell origin, and the electropherogram of the PCR antigen receptor rearrangement produced a monoclonal peak for TCRγ. Liver enzyme activities were markedly increased and abdominal ultrasound examination showed increased echogenicity of the liver and enlarged abdominal lymph nodes. Fine‐needle aspirates of the liver confirmed infiltration with lymphocytes exhibiting the same morphology as the cells detected in skin and peripheral blood. Treatment was induced with L‐asparaginase, lomustine, and prednisone. Partial clinical remission of the skin and tongue lesions was achieved within 10 days, and hematologic abnormalities resolved. Despite further treatment with L‐asparaginase and lomustine, the dog relapsed within one month and was euthanized. Presence of malignant lymphocytes in skin, peripheral blood, and liver indicate a rare variant of leukemic cutaneous T‐cell lymphoma, equivalent of Sézary syndrome in a dog. This case report describes the use of flow cytometry as a complementary tool for lymphocyte characterization of skin lesions for the first time.     

Exclusion of cytoplasmic fragments in flow cytometric analysis of lymph node samples from dogs with lymphoma using membrane-permeable violet laser-excitable DNA-binding fluorescent dye (DyeCycle Violet)

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.     

Progression of cutaneous plasmacytoma to plasma cell leukemia in a dog

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

Molecular cloning and characterization of Duck CD25

The IL-2Ralpha chain (CD25, Tac) is an essential component of high affinity IL-2Rs, playing critical role for the immune specificity of antigen-activated T-cell clonal expansion. Up to now, no duck cytokine receptor has been described. Here, the cDNA segment of a duck cytokine receptor (duCD25), encoding a 226 aa precursor protein with a 20 aa signal peptide, was isolated. Then a novel mouse monoclonal antibody (mAb) was generated using the prokaryotically expressed duCD25 protein as immunogen. Using this mAb, the endogenous duCD25 molecule was localized on the surface of duck lymphocytes, and the duck IL-2-induced lymphocyte proliferation was further inhibited. Furthermore, flow cytometry analysis showed that duCD25 positive cells were upregulated in ducks infected with avian influenza virus (H9N2). Our findings confirm that duCD25 is a receptor of duck interleukin-2, and duCD25 positive cells play a potential role in H9N2 virus infection.     

Cross-reactivity of mAbs to human CD antigens with cells from cattle

A panel of 377 commercially available mAbs were submitted to the animal homologue section of the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies on different animal species. In this study we describe the results of testing the mAbs on cattle cells by flow cytometry and Western blot. Eight commercial suppliers participated, providing mAbs to a total of 144 CD antigens plus controls. Fifty-two mAbs were identified as potentially staining cattle cells in the first round screen. In the second phase, 38 mAbs were confirmed as staining cattle cells. This included some that may recognise polymorphic determinants and others with atypical distribution patterns compared to humans. mAb to human CD9, CD11a, CD14, CD18, CD21, CD23, CD29, CD44, CD45R, CD47, CD49d and CD172a cross-reacted with bovine cells and mAb to CD22, CD88, CD119 and CD163 stained CD antigens that have not previously been identified in cattle.     

Acute megakaryoblastic leukemia in a German Shepherd dog

Abstract: An 11‐year‐old spayed‐female German Shepherd dog was presented to the Veterinary Medical Teaching Hospital at Kansas State University with a history of weight loss, anorexia, depression, and lethargy for 2–3 weeks. Radiographic examination revealed a mass in the spleen and several round radiodense foci in the liver. CBC results included normocytic normochromic anemia, marked thrombocytopenia, and low numbers of neoplastic cells that frequently had cytoplasmic projections or blebs. A bone marrow aspirate contained about 80% neoplastic megakaryoblasts with the same microscopic features as those observed in peripheral blood. Using flow cytometry, cells of large size were identified in peripheral blood that expressed CD41/61, CD45, CD61, and CD62P (P‐selectin) and were negative for markers of T cells, B cells, monocyte/macrophages, and dendritic cells. Because of the poor prognosis, euthanasia and subsequently necropsy were performed. On histopathologic examination, neoplastic megakaryoblasts were identified in spleen, liver, mesenteric lymph node, and the pulmonary vasculature. Using immunohistochemistry, the neoplastic megakaryoblasts weakly expressed von Willebrand factor. Based on microscopic and immunophenotypic findings, a diagnosis of acute megakaryoblastic leukemia (AMegL) was made. To our knowledge, this is the first report of AMegL in a domestic animal in which immunophenotyping by flow cytometry and a panel of antibodies against CD41/61, CD61, and CD62P were used to support the diagnosis.     

May-Hegglin anomaly in a dog

An 8-year-old female spayed Pug dog was presented for evaluation of cutaneous lesions occurring secondary to immunosuppressive treatment of presumed immune-mediated thrombocytopenia. Abnormal hematologic findings included persistent thrombocytopenia, macrothrombocytes, and variably shaped, often fusiform, blue cytoplasmic inclusions in neutrophils. May-Hegglin anomaly (MHA) was suspected based on the morphologic appearance of platelets and neutrophils. Examination of cells by transmission electron microscopy revealed normal platelet ultrastructure; neutrophil inclusions had features similar to those reported for inclusions in human MHA. Neutrophil function was within normal limits based on flow cytometric analysis. Thrombelastography indicated a prolonged clotting time (r), and PlateletMapping showed a lack of response to 2 μM ADP compared with a moderate response in the control dog. Immunocytochemical staining of blood smears using 2 commercially available antibodies against MYH9 protein (nonmuscle myosin heavy chain II) yielded negative results. However, genomic DNA sequencing analysis of the dog's MYH9 gene identified a single point mutation, resulting in substitution of lysine for glutamine at the 1841 amino acid position; this mutation is identical to one identified in people with MHA. To our knowledge, this is the first report of an MYH9 mutation in the dog. MHA-associated macrothrombocytopenia may be mistaken for immune-mediated thrombocytopenia.     

Chicken seminal fluid lacks CD9- and CD44-bearing extracellular vesicles

The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals.