Cytology of bone marrow.

Cytologic examination of bone marrow aspirates can provide a wealth of diagnostic information. Practitioners should not hesitate to perform bone marrow aspirates when indicated. This article is designed to assist the practitioner in the evaluation of bone marrow aspiration biopsies. The indications for marrow evaluation, methods of sample collection, sample preparation, and cytologic examination of bone marrow are discussed. Cases are provided to demonstrate accurate interpretation of bone marrow aspirates.     

Bone marrow biopsy and evaluation

Bone marrow evaluation provides valuable diagnostic and prognostic information about neoplastic, metabolic, and inflammatory diseases. Bone marrow biopsies should be done only after examination of peripheral blood, to avoid performing unnecessary biopsies. A blood sample should be taken at the time of the bone marrow biopsy, for complete hematopoietic evaluation. It is preferable to take both an aspiration and core biopsy simultaneously. A good sample is mandatory for accurate evaluation and interpretation. The method of evaluation should be systematic, complete, and cover the following points: adequacy of specimens; estimation of cellularity; identification of number, maturation pattern, and morphology of megakaryocytes, myeloid cells, and erythroid cells; estimation of M:E ratio; and identification of abnormal cells, cellular reactions, infectious agents, or abnormal stromal reactions. Bone marrow findings should be interpreted in conjunction with signalment, history, physical findings, and laboratory results. Reference or institutional laboratories should be contacted for proper handling of bone marrow specimens for special procedures, such as histopathology, cytochemistry, immunopathology, and electron microscopy.     

骨髓间充质干细胞的研究进展

骨髓间充质干细胞是存在于骨髓中的除造血干细胞以外的另一类具有多向分化潜能的干细胞。在一定的诱导条件下 ,这类细胞可定向分化为多种造血以外组织 ,特别是中胚层和神经外胚层来源的组织细胞。例如成骨细胞、成软骨细胞、脂肪细胞、腱细胞、肌肉细胞、神经细胞等。骨髓间充质干细胞具有贴壁生长的特性 ,在体外易分离和扩增 ,还易于外源基因的转入和表达 ,在人类医学上被认为是一种理想的治疗性细胞和基因治疗中的靶细胞。本文针对骨髓间充质干细胞的研究进展和在临床医学上的应用进行综述     

Bone Marrow Cytological Findings in 4 Dogs and a Cat With Hemophagocytic Syndrome

Hemophagocytic syndrome or hemophagic histiocytosis was diagnosed in 4 dogs and 1 cat by evaluation of bone marrow aspirate smears. One of the dogs had a suspected infection with canine parvovirus and a confirmed infection with Salmonella spp, 2 dogs had presumptive diagnoses of myeloproliferative and lymphoproliferative disease, respectively, and 1 dog died without a diagnosis. The cat had hepatic lipidosis and lesions compatible with feline calicivirus infection. All animals had cytopenias involving 2 or more cell lines, and fragmented erythrocytes in the blood, along with mild to moderate increases in the number of macro-phages in the bone marrow. Numerous marrow macro-phages contained phagocytized hematopoietic cells. Other cytological features of the bone marrow were variable in each patient, but the degree of response in the blood was inadequate, even in those with bone marrow hyperplasia. The phagocytosis of hematopoietic elements did not appear to be caused by a primary immune disorder, but rather by the inappropriate activation of normal macrophages secondary to infectious, neoplastic, or metabolic diseases. These findings suggest that hemophagocytic syndrome may be an important factor in the development of cytopenias; the data also support the cytological evaluation of bone marrow aspirates as an aid in the diagnosis of hemophagocytic syndrome. J Vet Intern Med 1996;10:7–14. Copyright © 7996 by the American College of Veterinary Internal Medicine .     

Bone marrow necrosis in a cat infected with feline leukemia virus

A one-year old castrated male cat was admitted to the hospital with vomiting and diarrhea. Laboratory examination revealed pancytopenia and positive for FeLV antigen. A bone marrow examination indicated necrosis of the nucleated cells. Based on these findings, the cat was diagnosed as bone marrow necrosis. Pancytopenia was effectively treated with corticosteroids. Re-examination of the bone marrow confirmed a recovery of normal hematopoietic cells with a infiltration of many macrophages. It is strongly suspected that the bone marrow necrosis in this case could be associated with a bone marrow suppression due to FeLV infection.     

A dog with myelodysplastic syndrome: chronic myelomonocytic leukemia

An eleven-year-old female pug was referred to Yamaguchi University Animal Hospital for evaluation of anemia and thrombocytopenia. The cytological examination of the peripheral blood showed some giant monocytic lineage blast cells. A few granulocytes and platelets had dysplastic features. On day 7, in addition to increasing the monocytic lineage cells, the dysplastic features of the blood had also increased compared to the initial examination. We performed bone marrow aspiration upon her death. The bone marrow revealed dysplastic features in all three hematopoietic cell lines, and an increase in the monocytic cell line. Based on the features of the bone marrow and the peripheral blood, this case was confirmed to be myelodysplastic syndrome--Chronic myelomonocytic leukaemia (MDS-CMML).     

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.     

Long-term bone marrow culture systems: normal and cyclic hematopoietic dogs.

A continuous long-term liquid culture in both a micro and macro system that incorporates bone marrow cells from normal and cyclic hematopoietic dogs is described. An adherent layer composed of fibroblasts, endothelial cells, mononuclear phagocytic cells, and fat-containing cells is essential for continuous hematopoiesis. Hematopoiesis was measured by the recovery of the nonadherent cells and the generation of committed granulocyte-monocyte progenitor cells for a period of seven weeks. Optimum growth factors include the use of horse serum, fetal bovine serum, dog serum, hydrocortisone, a 33 degrees C incubation temperature and feeding twice a week. As is true for both human and murine marrow liquid cultures, horse serum and hydrocortisone are essential for development and maintenance of fat-containing cells in the described systems. Both factors are important in hematopoiesis but their respective roles have not been defined. Normal and cyclic hematopoietic dogs bone marrow cells are comparable in their ability to establish long-term cultures. The micro-method (Linbro-well culture) gave similar results in maintaining hematopoiesis as did a macromethod (flask culture).     

Myeloproliferative disorders

Myeloproliferative disorders are uncommon in the dog and may be classified as chronic or acute. Excessive proliferation of mature cells leads to an overproduction of terminally differentiated blood cells (chronic MPD). Inability of cells to mature results in the accumulation of poorly differentiated blast cells in the peripheral blood and bone marrow (acute MPD). Because the lesion appears to be at the level of the hematopoietic stem cell, all cell lines in the bone marrow may be affected. Diagnosis depends upon the accurate identification of neoplastic cells and the absence of other diseases associated with bone marrow hyperplasia. The prognosis for chronic MPD is guarded, whereas for acute MPD it is grave. Accurate identification of these disorders in animals is important. Investigation and greater understanding of the pathophysiologic mechanisms may lead to more lasting therapeutic successes in the future.     

Emperipolesis of hematopoietic cells within megakaryocytes in bone marrow of the rat

Emperipolesis of hematopoietic cells within megakaryocytes was found in rats. The incidence was less than 0.3% in young rats (2 to 12 months old) but increased to 2-5% among the aging rats (18 to 24 months old). The incidence increased markedly in hyperplastic bone marrow secondary to chronic suppurative or neoplastic lesions. Mature neutrophils appeared to be the most common marrow cell engulfed by megakaryocytes. By light microscopy, engulfed cells were separated from megakaryocyte cytoplasm by a narrow pericellular space. By electron microscopy, marrow cells engulfed by megakaryocytes were located in the open canalicular system. Cell membranes of both engulfed cells and megakaryocytes were intact, and there was no fusion of cell membranes or phagosome formation in the megakaryocytes.     

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.     

Specific biological effects of an anti-rat PMN antiserum intraperitoneally infected into f344/n rats

Neutropenia can be produced with antimitotic chemicals, but this method lacks specificity. An alternative is to use antibody-dependent cytotoxicity to produce neutropenia; however, this method has not been completely evaluated with respect to efficacy, specificity, and potential collateral damage, especially to constituents of bone marrow. This study used in vitro and in vivo methods to evaluate specific biological effects of a commercially available rabbit anti-rat neutrophil (PMN) antiserum in F344/N rats. The viability of rat pulmonary alveolar macrophages (PAMs), PMNs, and lymphocytes in vitro was quantified using a trypan blue dye exclusion test. Amounts of antiserum in vitro that rendered PMNs 100% nonviable did not decrease the viability or phagocytic ability of the PAMs and did not decrease the viability of the lymphocytes. Intraperitoneal (IP) injection of the antiserum into rats resulted in complete depletion of the PMNs and about a 50% depletion of the lymphocytes in circulating blood within 24 hours. The numbers of both cell types remained lowered for 5 days, but returned to control values by Day 6 after the IP injection. The antiserum had no effect on the numbers of PAMs or lymphocytes in the pulmonary alveolar airspaces, as determined by quantifying the numbers of these cell types in bronchoalveolar lavage fluid (BALF). The numbers of PMNs in BALF, however, decreased on Days 3 and 4 after IP injection of antiserum, but were not different from control values by Day 5. The viability of the PAMs in BALF of treated rats was not different from control values at any time point. There were no morphological indications that the injected antiserum damaged lung tissue or stem cells in bone marrow. Results demonstrate that the anti-rat PMN antiserum administered IP to F344/N rats depletes circulating PMNs and partially depletes lymphocytes for a period of about 6 days without adversely affecting the precursors of red or white blood cells in bone marrow. We concluded that the antiserum is a relatively specific way to temporarily render rats neutropenic without damaging precursor cells in bone marrow.     

Characterization of CD34+ cells from canine umbilical cord blood, bone marrow leukocytes, and peripheral blood by flow cytometric analysis

Characterization of CD34+ cells in canine bone marrow, umbilical cord blood, and peripheral blood was performed by flow cytometric analysis. The ratio of CD34+CD45hi cells, which are absent in human blood, was high in the CD34+ cell fraction, but 98% of these was suggested B-cells. The remaining CD34+CD45lo cells may comprise canine hematopoietic progenitor cells, and these cells accounted for 0.23 +/- 0.07% of the fraction in cord blood, 0.30 +/- 0.07% in bone marrow, and 0.02 +/- 0.01% in peripheral blood.     

Flow cytometric evaluation of canine bone marrow based on intracytoplasmic complexity and CD45 expression

BACKGROUND: Because of the complexity, subjectivity, time, and technical skill required for determination of manual bone marrow differential cell counts, an alternative method is needed. Several flow cytometric methods have been described, but all have limitations. OBJECTIVE: The purpose of this study was to evaluate a technique for bone marrow differential cell counting based on flow cytometric evaluation of CD45 expression and intracellular complexity (CD45 scatter plots). METHODS: Bone marrow was obtained from 15 dogs that were being evaluated for hematologic disorders. In preliminary studies, the location of bone marrow subpopulations in the CD45 scatter plots was evaluated by labeling bone marrow with lineage-specific markers. A template was developed to identify these cell populations. Gates were set to identify granulocytes, myeloblasts, monocyte/macrophages, lymphocytes, and nucleated erythroid populations. RESULTS: The CD45 labeling technique accurately quantified granulocytes, myeloblasts, erythroid precursors, and lymphocytes in canine bone marrow. Correlation coefficients with manual counts for granulocytes, myeloblasts, erythroid cells, lymphocytes, and monocyte/macrophages were 0.90, 0.89, 0.96, 0.91, and 0.54, respectively. CONCLUSIONS: The capacity of the CD45 scatter-plot technique to quantify lymphocytes and myeloblasts is an advantage over previously described techniques. The simplicity of the CD45 labeling method and the ease with which batches of samples can be analyzed makes the technique potentially applicable as a routine test in clinical and research laboratories.     

Molecular cloning and expression of bottlenose dolphin CD34

In terrestrial mammals, the surface molecule CD34 is used as a marker to identify hematopoietic progenitor cells. To clarify whether CD34 expression can be used to confirm the undifferentiated state of hematopoietic-like cells isolated from the bone marrow of bottlenose dolphin, Tursiops truncates, we determined in this study the sequence of dolphin CD34 cDNA and analyzed its mRNA expression. Dolphin CD34 cDNA can be expressed as two forms, one that encodes a full-length version and a variant, truncated version of the gene. Both forms were detected in bone marrow mononuclear cells and in various tissues using RT-PCR. The truncated form was not detected in peripheral blood mononuclear cells, and neither form was detected in polymorphonuclear leukocytes. This is the first report on CD34 in marine mammals and our results suggest that dolphin CD34 may be a useful marker to identify hematopoietic progenitor cells.     

Histologic features of the healing of bone graft donor sites in dogs

Healing of cancellous bone graft donor sites in the proximal tibial metaphysis of 12 healthy adult dogs was studied histologically. Cancellous bone was curetted from the metaphysis of the proximal end of the tibia, via a 1-cm diameter circular opening in the medial cortex. A hematoma and fibrovascular tissue filled the bone defect at 2 weeks. At 4 and 8 weeks, endosteal callus, composed initially of cartilage and woven bone and later of lamellar bone, filled the marrow cavity. At 12 weeks, the normal structural arrangement of lamellar bone and hematopoietic marrow was reestablished in the marrow cavity. The medial cortex defect was filled only with lamellar trabecular bone. It was concluded that, in adult dogs, a second cancellous bone graft could be collected from the proximal portion of the tibial metaphysis 12 weeks or more after an initial collection.     

Bone Marrow Mesenchymal Stem Cells: From Characterization of Neural Differentiation to a Possible Therapeutic Use in Neurodegeneration

Bone marrow derived stromal cells are of mesenchymal origin and precursor cells for skeletal tissue components such as chondroblasts and osteoblasts. Furthermore, under experimental conditions, a differentiation potency into myogenic and neuronal cells could be demonstrated. Because of their multipotency these cells represent a population of non-haematogen stem cells that can be regarded as an alternative to human embryonic stem cells for future autologous cell replacement therapies. For a closer look at the differentiation capacity of these cells, rat and human bone marrow stromal cells were isolated from the femur bone and kept in the cell culture applying different cultivation protocols. In a cultivation medium with a serum content of 20%, the majority of these cells express a variety of neuronal markers such as ß-III Tubulin and NeuN as well as the astrocyte marker GFAP, while a minority of about 20% express the marker for neural precursor cells nestin. Cultivation in a chemically defined serum free medium results in the differentiation of a markedly higher percentage of nestin positive neural precursor-like cells. Using bFGF in combination with B27 these cells can be forced to form three dimensionally organized spheres. In order to elucidate a possible therapeutical potency of the bone marrow derived cells the synthesis of neurotrophic factors such as BDNF and NGF were analysed using the ELISA technique. Furthermore, they can be infected using a third generation adenoviral vector with high efficiency and show migratory activity in vitro . After injection of bone marrow derived mesenchymal stem cells into the lateral ventricle of adult rats they adhere to the ependymocytes and pass the ependymal barrier in order to settle in the subventricular space.     

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.     

Spontaneous nonthymic T cell lymphomas in young Wistar rats

Spontaneous acute lymphomas and related leukemias, occurring in three of 2,974 male Wistar rats used as controls in toxicity studies during the last 14 years (1974-1987), were examined by light and electron microscopy and by using immunohistochemistry. At autopsy, conspicuous hepatosplenomegaly was noted. Morphologically, tumor cells of all three rats were medium-sized lymphocytes with many mitotic figures proliferating mainly in the spleen, liver, and bone marrow. Virus-like particles were not detected. Immunohistochemically, almost all tumor cells were positive for thy-1 antigen but negative for hematopoietic and differentiation markers such as W3/13, W3/25, OX4, OX8, and OX12. The results suggest that the lymphomas in these three rats were derived from T cells.