Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink.     

Expression of CD34 mRNA and protein in cattle

CD34 is a transmembrane glycoprotein expressed by hematopoietic progenitors and endothelial cells. It is used widely in the clinic for purification of human hematopoietic stem cells transplants, and as an endothelial marker for several species. The aim of this study was to produce an anti-bovine CD34 antibody and to characterize the expression of CD34 mRNA and protein in cattle tissues. The bovine CD34 cDNA was cloned by RT-PCR, and the expression of bovine CD34 mRNA investigated by RT-PCR and in situ hybridization. Polyclonal antibodies were raised against CD34 polypeptide fragments expressed in Escherichia coli, and affinity purified. Alternative splicing of bovine CD34 mRNA was observed. Both splice variants were readily observed in endothelium, while the variant encoding a truncated cytoplasmic domain was mostly undetectable in bone marrow mononuclear cells. A polyclonal antibody against an extracellular fragment of the CD34 polypeptide was characterized using Western blots, cytocentrifuge preparates, and paraffin sections. CD34 immunoreactivity was enriched in lineage-depleted bone marrow cells. The antibody labelled most blood vessel endothelia in fetal and adult cattle, with highest intensity in capillaries. Newly forming capillaries in granulation tissue were also stained. Lymphatic vessels and the endothelium of liver sinusoids were negative.     

Histiocytic sarcoma of macrophage origin in a cat: case report with a literature review of feline histiocytic malignancies and comparison with canine hemophagocytic histiocytic sarcoma

Mild nonregenerative anemia was detected in a 9-year-old neutered male domestic shorthair cat during a routine examination. Bone marrow core biopsy revealed erythroid hyperplasia; however, a specific cause was not identified. Over the next 8 months the anemia progressed, eventually becoming mildly regenerative, and moderate thrombocytopenia developed. On ultrasonographic examination, marked splenomegaly, mild hepatomegaly, and abdominal lymphadenopathy were found. Cytologic evaluation of splenic aspirates revealed increased numbers of mildly to moderately pleomorphic histiocytes that frequently had phagocytosed RBCs, leukocytes, and occasionally platelets. Histopathologic examination of the spleen and liver revealed effacement of splenic architecture by a histiocytic sarcoma (HS), and neoplastic histiocytes in hepatic sinusoids. A second bone marrow aspirate revealed neoplastic infiltration by similar cells. The histiocytes in all tissues were mildly to moderately pleomorphic and markedly erythrophagocytic. The immunophenotype of histiocytes in the spleen was CD1c(-)/CD11b(+)/CD18(+)/MHC-II(+), supporting a macrophage cell lineage. The clinical, pathologic, and immunophenotypic findings in this cat were similar to those in hemophagocytic HSs in dogs. To our knowledge, this is the first report of a HS of purported macrophage phenotype in a cat.     

Evaluation of monoclonal antibodies for identification of subpopulations of myeloid cells in bone marrow obtained from dogs

OBJECTIVE: To evaluate monoclonal antibodies that may be useful for immunophenotyping myeloid cells in bone marrow of dogs. SAMPLE POPULATION: Bone marrow specimens obtained from 5 dogs. DESIGN: Specimens were labeled with monoclonal antibodies that detected CD18, major histocompatability antigen class-II (MHC class-II), CD14, and Thy-1. Cells labeled with each of the antibodies were isolated by use of a fluorescence-activated cell sorter. Differential cell counts of sorted cells were used to determine cells that were labeled by each of the various antibodies. RESULTS: Myeloid cells labeled with anti-CD18 antibody included granulocytes, lymphocytes, and monocytes-macrophages. Immature and mature granulocytes were labeled. Lymphocytes, monocytes-macrophages, and eosinophils were labeled with anti-Thy-1 antibody. Cells labeled with anti-MHC-class II antibody included approximately 9% of bone marrow cells, which consisted almost exclusively of lymphocytes and monocytes-macrophages. Approximately 4% of bone marrow cells were labeled with anti-CD14 antibody, with > 90% of sorted cells being monocytes-macrophages. CONCLUSIONS AND CLINICAL RELEVANCE: Four monoclonal antibodies for use in detecting subpopulations of canine bone marrow cells were evaluated. These antibodies should be useful in differentiating the origin of leukemic cells in dogs.     

Detection of feline immunodeficiency virus infection in bone marrow of cats.

Natural or experimental feline immunodeficiency virus (FIV) infection in cats is often associated with hematologic abnormalities which are similar to those observed in human immunodeficiency virus (HIV) infected patients. To determine if cells in bone marrow are infected with FIV and whether severity of hematopoietic disorder is correlated with the level of viral infection, bone marrow tissues from ten experimentally and two naturally FIV infected cats were examined by in situ hybridization for presence of FIV RNA. Seven of the 12 FIV infected cats were also naturally or experimentally coinfected with feline leukemia virus (FeLV). FIV RNA was detected mainly in megakaryocytes and unidentified mononuclear cells in the bone marrow of cats that were sick and had marrow hypercellularity and immaturity. These included all cats in the acute phase of FIV infection and two of seven long term FIV infected cats. One long term FIV infected cat with lymphosarcoma was also positive for FIV RNA in bone marrow cells. The other four long term FIV infected cats were relatively healthy, with normal bone marrow morphology, and were negative for FIV infected cells. Bone marrow from three non-infected and two cats infected with FeLV alone were also negative for FIV RNA by in situ hybridization. We concluded that megakaryocytes and mononuclear cells were targets of the viral infection and that the presence of FIV RNA in cells of the bone marrow correlated with marrow hypercellularity and immaturity, and severity of illness.     

Purification and adhesion receptor phenotype of ovine bone marrow-derived haemopoietic colony-forming cells.

Ovine haemopoietic progenitor cells that form colonies (CFC) in soft agar cultures were compared to more mature bone marrow cells for their level of expression of the adhesion receptor molecules ovine (ov) CD44, ov CD11a (LFA-1) and ov CD58 (LFA-3) as well as the 175-antigen using specific monoclonal antibodies. Ov CD44, ov CD11a and ov CD58 were expressed on all CFC of the myeloid (non-erythroid) series, whereas ov CD44 and ov CD11a expression was very low or absent from a small number of blast and erythroid series CFC. Within the mature non-erythroid population of myeloid cells, neutrophils retained a low level of expression of ov CD11a. Most CFC representing all lineages strongly expressed the ov CD44 antigen. In contrast, the majority of CFC lacked the 175-antigen, as did bone marrow lymphocytes, basophils and mast cells. This property of CFC was exploited in a negative selection technique using panning and immunomagnetic beads to select CFC from other bone marrow cells with a 116-125-fold enrichment, 12-14% purity and 29-40% yield. These results demonstrate that ovine CFC express some of the molecules necessary to allow adhesion to haemapoietic stromal cells and vascular endothelium in the tissues. Future studies will concentrate on the function of the adhesion receptor molecules in medullary and extra-medullary haemopoiesis and inflammatory cell development in sheep.     

Erythrophagocytic multiple myeloma in a cat

A 7-year-old neutered male domestic shorthair cat was presented to the Veterinary Medical Teaching Hospital at the University of Georgia for further evaluation of a suspected osteolytic lesion of the left 10th rib. Results of a CBC and biochemistry profile revealed mild nonregenerative anemia, hyperproteinemia, hyperglobulinemia, and hypercalcemia. Serum protein electrophoresis was consistent with a monoclonal gammopathy. Marked proteinuria with an increased urine protein to creatinine ratio was found. Cytologic examination of the liver, spleen, and bone marrow revealed numerous plasma cells, many of which were erythrophagocytic. Within the bone marrow, the plasma cells contained phagocytosed metarubricytes in addition to phagocytosed erythrocytes. A diagnosis of erythrophagocytic multiple myeloma was made and treatment with prednisone and melphalan was begun. Four weeks after presentation, the cat was euthanized due to clinical deterioration. A complete necropsy was performed. The distal one-third of the left 10th rib was completely absent. Histologically, there was no evidence for osteolysis or neoplastic cells in the remaining portion of the rib. However, large sheets of plasma cells were found infiltrating the spleen and bone marrow. Moderate erythrophagocytosis by the plasma cells was observed in both organs. The plasma cells, including the erythrophagocytic cells, were positive for CD79alpha by immunohistochemical staining. Erythrophagocytosis by plasma cells as a cause of anemia is uncommon in people with multiple myeloma and is rare in animals. To our knowledge, this is the first report of erythrophagocytic plasma cells in a cat with multiple myeloma.     

Characterization of bovine haemopoietic progenitor cells using monoclonal antibodies and fluorocytometry

Monoclonal antibodies against bovine leucocyte cell surface differentiation antigens were used in combination with a fluorescence activated cell sorter to enrich bovine haemopoietic progenitor cells present in bone marrow cell populations prior to in vitro culture. After two sequential centrifugations of the bone marrow cell suspension through Ficoll-Paque, the interface fraction was stained with a cocktail of monoclonal antibodies directed against mature monocytes/macrophages, granulocytes and lymphocytes. Using appropriate electronic window settings on a FACStar Plus, cells with a high 90 degrees light scattering property (granular cells), a low forward light scattering property (erythrocytes and reticulocytes) and cells positive for monoclonal antibodies specific for lineage-restricted leucocyte markers were removed and the negative cell fraction collected. These negatively-selected cells were stained with monoclonal antibodies specific for a pan-leucocyte or a MHC class II marker and the positive cell population was collected in a second sort and subsequently submitted to culture. All erythroid and granulocyte/macrophage colony forming cells expressed MHC class II antigens, as well as the pan-leucocyte antigen. These same progenitors did not bind any of a variety of monoclonal antibodies directed against lineage-specific antigens on lymphocytes, granulocytes or monocytes/macrophages, although they did bind monoclonal antibodies recognizing MHC class I antigens. Between 85% and 91% of the isolated cells seeded were capable of forming erythroid or granulocyte/macrophage colonies within 5 to 10 days, thus increasing the plating efficiency of these cell types in bone marrow populations by at least 60 fold.     

传染性法氏囊病病毒超强毒感染SPF鸡免疫器官中CD4+和CD8+T淋巴细胞动态分布

利用免疫组织化学染色对传染性法氏囊病病毒（IBDV）超强毒LX株感染SPF鸡免疫器官中CD4^ 和CD8^ T淋巴细胞的动态分布进行了研究。超强毒LX株接种2周龄SPF雏鸡，在其法氏囊、脾脏、胸腺、盲肠扁桃体、骨髓和哈氏腺中均可检出IBDV抗原的存在和CD4^ 与CD8^ T淋巴细胞的数量改变。在法氏囊中，CD4^ 淋巴细胞主要存在于淋巴滤泡间隙和滤泡皮质，而CD8^ T淋巴细胞则丰在于整个淋巴滤泡和滤泡间隙，并且CD8^ T淋巴细胞数量明显多于CD4^ T淋巴细胞，在接种后14d仍未见CD4^ 和CD8^ T淋巴细胞数量减少。脾脏中CD4^ T淋巴细胞主要存在于外周小动脉淋巴鞘或散在，而CD8^ T淋巴细胞则多存在于外周小动脉淋巴鞘和红髓。接种后胸腺中CD4^ 和CD8^ T淋巴细胞在皮质中减少，但在髓质增多，尤其是CD8^ T淋巴细胞数明显多于CD4^＋T淋巴细胞。盲肠扁桃体中CD4^ 和CD8^ T淋巴细胞主要存在于发生中心，尤其是CD8^ T淋巴细胞数比CD4^ T淋巴细胞明显多。骨髓和哈氏腺中也可见CD4^ 和CD8^ T淋巴细胞，而且CD8^ T淋巴细胞更多。在这些淋巴器官中，病毒损伤部位出现CD4^ 和CD8^ T淋巴细胞的迁入聚集，表明T淋巴细胞可能参与IBDV超强毒的免疫致病过程。     

Comparison of rabbit monoclonal and mouse monoclonal antibodies in immunohistochemistry in canine tissues.

Rabbit monoclonal (RM) antibodies appear to have higher affinity for antigens than mouse monoclonal (MM) antibodies. However, RM antibodies have not been used in veterinary diagnostic immunohistochemistry. The authors compared reactivities of RM and MM antibodies on formalin-fixed, paraffin-embedded canine tissues, targeting 11 different antigens: CD3, CD79a, calcitonin, calretinin, chromogranin A, COX-2, estrogen receptor, Ki67, progesterone receptor, synaptophysin, and vimentin. Paraffin-embedded tissue sections were processed by 1 of 2 antigen-retrieval methods: 1) proteinase K digestion or 2) steam heat in citrate buffer. An additional set of slides did not receive antigen retrieval. Immunostaining was performed using an automated stainer, and scores were assigned to the different dilutions and antigen-retrieval methods on the basis of staining intensity and number of positive cells. Steam heat was usually the best antigen-retrieval method. The optimal dilution for each antibody was that which resulted in the highest specific staining and the lowest nonspecific (background) staining. The RM or MM antibodies yielded a specific reaction for all antigens examined except calretinin. The RM and MM antibodies yielded a specific reaction for 4 antigens only: COX-2, Ki67, synaptophysin, and vimentin. Three antigens (CD3, chromogranin A, and progesterone receptor) were detected only with RM antibodies, whereas the other 3 (CD79a, calcitonin, estrogen receptor) were detected only with MM antibodies. The results of this study differed from those reported for human tissues by the manufacturers of the antibodies. These results emphasize that, regardless of manufacturers' recommendations, each antibody must be individually standardized and validated before routine use in canine tissues.     

Isolation and characterization of pediatric canine bone marrow CD34+ cells

Historically, the dog has been a valuable model for bone marrow transplantation studies, with many of the advances achieved in the dog being directly transferable to human clinical bone marrow transplantation protocols. In addition, dogs are also a source of many well-characterized homologues of human genetic diseases, making them an ideal large animal model in which to evaluate gene therapy protocols. It is generally accepted that progenitor cells for many human hematopoietic cell lineages reside in the CD34+ fraction of cells from bone marrow, cord blood, or peripheral blood. In addition, CD34+ cells are the current targets for human gene therapy of diseases involving the hematopoietic system. In this study, we have isolated and characterized highly enriched populations of canine CD34+ cells isolated from dogs 1 week to 3 months of age. Bone marrow isolated from 2- to 3-week-old dogs contained up to 18% CD34+ cells and this high percentage dropped sharply with age. In in vitro 6-day liquid suspension cultures, CD34+ cells harvested from 3-week-old dogs expanded almost two times more than those from 3-month-old dogs and the cells from younger dogs were also more responsive to human Flt-3 ligand (Flt3L). In culture, the percent and number of CD34+ cells from both ages of dogs dropped sharply between 2 and 4 days, although the number of CD34+ cells at day 6 of culture was higher for cells harvested from the younger dogs. CD34+ cells harvested from both ages of dogs had similar enrichment and depletion values in CFU-GM methylcellulose assays. Canine CD34+/Rho123lo cells expressed c-kit mRNA while the CD34+/Rhohi cells did not. When transplanted to a sub-lethally irradiated recipient, CD34+ cells from 1- to 3-week-old dogs gave rise to both myeloid and lymphoid lineages in the periphery. This study demonstrates that canine CD34+ bone marrow cells have similar in vitro and in vivo characteristics as human CD34+ cells. In addition, ontogeny-related functional differences reported for human CD34+ cells appear to exist in the dog as well, suggesting pediatric CD34+ cells may be better targets for gene transfer than adult bone marrow. The demonstration of similarities between canine and human CD34+ cells enhances the dog as a large, preclinical model to evaluate strategies for improving bone marrow transplantation protocols, for gene therapy protocols that target CD34+ cells, and to study the engraftment potential of various cell populations that may contain hematopoietic progenitor cell activity.     

Use of monoclonal antibodies to refine flow cytometric differential cell counting of canine bone marrow cells

OBJECTIVES: To evaluate use of monoclonal antibodies to increase accuracy of flow cytometric differential cell counting of canine bone marrow cells. SAMPLE POPULATION: Bone marrow specimens from 15 dogs. PROCEDURES: Specimens were labeled with monoclonal antibodies that detected CD18, major histocompatability antigen class-II (MHC class-II), CD14, and Thy-1. Location of fluorescent and nonfluorescent cells within gates of a template developed for canine bone marrow differential cell counting was determined, the template was revised, and 10 specimens were analyzed by use of the old and revised templates and by labeling cells with anti-MHC class-II and anti-CD14. RESULTS: Data confirmed the presumptive location of marrow subpopulations in scatter plots, permitted detection of lymphocytes and monocytemacrophages, and was used to revise the analysis template used for differential cell counting. When differential cells counts determined by the original and revised templates were compared with results of manual differential cell counts, the revised template had higher correlation coefficients and more similar mean values. Labeling cells with anti-MHC class-II and anti-CD14 permitted identification of lymphoid and monocyte-macrophages cells in bone marrow specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the revised flow cytometric analysis template combined with anti-CD14 and anti-MHC class-II antibody labeling provides reliable differential cell counts for clinical bone marrow specimens in dogs. These techniques have potential applications to clinical bone marrow examination and preclinical toxicity studies.     

Nonsecretory Multiple Myeloma in a Dog: Immunohistologic and Ultrastructural Observations

A 12-year-old, female spayed Chihuahua was diagnosed with nonsecretory multiple myeloma on the basis of multiple osteolytic lesions, histological evidence of plasma cell infiltrate on a bone biopsy, and absence of a monoclonal protein on serum and urine electrophoresis. A 6-week course of prednisone therapy resulted in no clinical improvement and the dog was euthanized 2 weeks after presentation because of progressive neurological impairment. Bone marrow specimens were processed and stained for ultrastructural and immunohistologic evaluation. Staining with antisera to immunoglobulin (Ig) G, IgM, and IgA was negative. Tumor cells in both the pelvic and rib masses displayed prominent reactivity with an antibody specific for a canine β1 integrin similar to VLA-4; however, the tumor cells failed to stain with antibodies known to react predominantly with antigens on B-lymphocytes (major histocompatibility complex class II, CD45RA, and CD21) or T-lymphocytes (Thy-1). The tumor cells also failed to stain with an antibody specific for the β-subunit (CD18) of the leukocyte integrins (CD11/CD18). Ultrastructural studies performed on bone marrow specimens revealed a pleomorphic population of plasma cells with moderate amounts of rough endoplasmic reticulum, erythrophagocytosis, and lack of crystalline inclusions.     

Detection of type II avian adenoviral antigen in tissue sections using immunohistochemical staining.

An immunohistochemical staining technique for the detection of marble spleen disease (MSD) viral antigens and other type II avian adenoviral antigens was developed using a mixture of monoclonal antibodies produced against hemorrhagic enteritis (HE) virus and a commercial streptavidin-biotin peroxidase indicator system. This technique was applied to both frozen and formalin-fixed paraffin-embedded tissue sections. The immunohistochemical staining technique was used on tissues from pheasants with experimental MSD, on tissues from a pheasant with natural MSD, and on tissues from turkeys with natural HE. Staining results were compared with routine hematoxylin-and-eosin (H&E) staining. Additional viral inclusions, not detected with H&E, were found in the liver, lung, bone marrow, and kidney sections using the immunohistochemical technique. The immunohistochemical technique was highly specific and sensitive for the detection of type II adenoviral antigen, and it appears to be useful for studying the pathogenesis of these diseases and for retrospective evaluation of routinely processed diagnostic tissue samples.     

Chronic myelomonocytic leukemia in a cat

A 1-year-old spayed domestic short-haired cat was referred with anorexia and weight loss. Hematologic findings indicated nonregenerative anemia, severe neutropenia and monocytosis. The feline leukemia virus (FeLV) antigen test was positive reaction by enzyme-linked immunosorbent assay. Dysgranulopoiesis with slight increase in blast cells were observed in bone marrow smears. On the basis of blood and bone marrow findings, the cat was diagnosed as chronic myelomonocytic leukemia (CMMoL), which possibly corresponds to a kind of the subtypes in human myelodysplastic syndrome (MDS).     

FIV-infected cats respond to short-term rHuG-CSF treatment which results in anti-G-CSF neutralizing antibody production that inactivates drug activity

The hematological and virological effects of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) were evaluated in feline immunodeficiency virus (FIV)-infected cats. Six age-matched, FIV-infected cats used in this cross-over study were injected subcutaneously with 5 microg/kg of rHuG-CSF daily for 3 weeks, while six control cats received a placebo. Five of six rHuG-CSF-treated cats had significant increases in neutrophil counts that peaked on days 11-21 of treatment. All rHuG-CSF-treated cats exhibited an increase in myeloid:erythroid ratios of the bone marrow cells without significant changes in lymphocyte, CD4 counts, CD4/CD8 ratios, RBC counts, FIV antibody titers, and FIV loads in peripheral blood, and without clinical and hematological toxicities. Five of six rHuG-CSF-treated cats developed antibodies to rHuG-CSF by 14-21 days of treatment, which correlated with decreasing neutrophil counts and increasing neutralizing antibodies to rHuG-CSF. Three cats re-treated with rHuG-CSF rapidly developed neutralizing antibodies to rHuG-CSF, while one cat also developed neutralizing antibodies to recombinant feline G-CSF (rFeG-CSF). Overall, rHuG-CSF treatment increased neutrophil counts in FIV-infected cats without affecting the infection status of cats. However, long-term use of rHuG-CSF is not recommended in cats because of the neutralizing antibody production to rHuG-CSF that affects the drug activity. In addition, a preliminary finding suggests that repeated treatment cycle can also induce cross-neutralizing antibodies to rFeG-CSF, which may potentially affect the homeostasis of endogenous FeG-CSF.     

Transmission of feline leukaemia virus in the milk of a non-viraemic cat

The possibility of the transmission of feline leukaemia virus (FeLV) from latently infected cats was studied. Five female cats with latent infections were examined for evidence of transmission of the virus to their kittens. One of the cats infected members of four consecutive litters of kittens which subsequently became persistently viraemic and transmitted the virus to other susceptible kittens by contact. Shortly after birth its kittens were apparently FeLV-free since neither viral antigen nor infectious virus was detected in their blood and no virus was found in cell cultures made from aspirates of bone marrow. The kittens became viraemic from 45 days of age onwards at a time when their passively acquired colostral FeLV neutralising antibodies were no longer detectable. Transmission of the virus occurred via the milk since both FeLV antigen and infectious virus were found in milk samples taken six weeks after kittening and the virus was transmitted to a fostered kitten. Eleven weeks after the birth of the fourth litter the cat became viraemic. The intermittent presence of FeLV antigens detected by the Leukassay F test, but not infectious virus, in the plasma of this cat over the previous months and a low level of serum neutralising antibodies distinguished it from four other latently infected queens which did not transmit infection to their kittens. These factors may indicate a risk of milk transmission and reactivation of latent virus.     

Morphological aspects of prenatal haematopoietic development in the cat

The appearance of haematopoietic cells and the development of haematopoiesis in certain embryonic organs of the cat were studied using light microscopic (cytological smears and paraffin embedded tissues) and transmission electron microscopic methods. Primitive erythropoiesis occurred predominantly in the yolk sac whereas definitive erythropoiesis occurred in the yolk sac, liver, bone marrow and, to a lesser extent, in the spleen. In the yolk sac, erythropoiesis was predominantly intravascular whereas in the liver and bone marrow it was usually extravascular. Granulopoiesis occurred mainly in the liver and bone marrow. In the liver it was predominantly extravascular and occurred around vessels, bile ducts and in perisinusoidal spaces. Megakaryocytopoiesis occurred in the yolk sac, liver, bone marrow and spleen. The megakaryocytic line of cells were similar to those occurring in adult cats except that in the yolk sac unusual small, mononuclear to binuclear thrombocytogenic megakaryocytes were present also. The relative contributions of the embryonic organs of the cat to haematopoiesis were similar to those described for man and certain other mammals but the results for the sites of development and the appearance of early forms in the erythrocytic, granulocytic and megakaryocytic lines varied at times from those reported for man and other mammals.     

Immunohistological demonstration of erythroid cells in canine bone marrow

In an immunohistological/cytological study of canine bone marrow, the aim was to demonstrate canine erythroid cells with the help of various commercially available antibodies against human antigens (monoclonal antibody against glycophorin A, polyclonal antibodies against haemoglobin and spectrin). In order to preserve possible cross-reacting epitopes various fixation methods (cross-linking, precipitating and dehydrating fixing agents, partly in combination with unmasking measures), decalcification techniques [acid or ethylenediaminetetraacetic acid (EDTA) decalcification] and tissue-embedding methods (paraffin embedding, cryostat sectioning technique) were used. Alternative methods, such as the preparation of cell smears and immunoblotting, were also employed. The only result that was of use for routine diagnostic procedures (paraffin sections) was that obtained by using polyclonal antibodies against haemoglobin. Best results were achieved when tissue was fixed in a formaldehyde-glutaraldehyde mixture, decalcified in EDTA and treated with microwave irradiation. The primary antibody was used in a dilution of 1:500 and incubated for 16 h. With the exception of mature red blood cells and proerythroblasts, different stages of erythrocytopoietic cells in canine bone marrow were shown to be arranged in erythrons. The polyclonal antibody against spectrin also showed clear cross-reactivity, but was only employable in other systems (immunoblotting). The monoclonal antibody against glycophorin A reacted only when used on human tissue or cells.