Bone marrow aspiration in dogs and cats

This column describes how to obtain a bone marrow sample by way of aspiration from the iliac crest and proximal humerus of the dog and cat for the assessment and diagnosis of bone marrow disease.     

Bone marrow aspiration the posterior iliac crest,an additional safe site

The posterior iliac crest is a readily accessible site for bone marrow aspiration which is safe, psychologically less traumatic, and affords representative samples of bone marrow similar to those obtained from the sternum, the vertebral spine and the anterior iliac crest.     

Bone marrow cells repair cigarette smoke-induced emphysema in rats

The therapeutic potential of stem cells in chronic obstructive pulmonary disease is not well known although stem cell therapy is effective in models of other pulmonary diseases. We tested the capacities of bone marrow cells (BMCs), mesenchymal stem cells (MSCs), and conditioned media of MSCs (MSC-CM) to repair cigarette smoke-induced emphysema. Inbred female Lewis rats were exposed to cigarette smoke for 6 mo and then received BMCs, MSCs, or MSC-CM from male Lewis rats. For 2 mo after injection, the BMC treatment gradually alleviated the cigarette smoke-induced emphysema and restored the increased mean linear intercept. The BMC treatment significantly increased cell proliferation and the number of small pulmonary vessels, reduced apoptotic cell death, attenuated the mean pulmonary arterial pressure, and inhibited muscularization in small pulmonary vessels. However, only a few male donor cells were detected from 1 day to 1 mo after BMC administration. The MSCs and cell-free MSC-CM also induced the repair of emphysema and increased the number of small pulmonary vessels. Our data show that BMC, MSCs, and MSC-CM treatment repaired cigarette smoke-induced emphysema. The repair activity of these treatments is consistent with a paracrine effect rather than stem cell engraftment because most of the donor cells disappeared and because cell-free MSC-CM also induced the repair.     

Diagnosis and subclassification of thymoma by minimally invasive fine needle aspiration directed by endobronchial ultrasound: a review and discussion of four cases

Thymomas have been classified by the World Health Organisation (WHO) into six groups, based on the morphology of epithelial cells and the ratio between epithelial cells and lymphocytes within the tumour. Among 1458 consecutive cases of endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) performed in a tertiary referral centre between February 2008 and February 2012, we have encountered four cases of thymic neoplasms. We discuss the cytomorphological features of three cases of type B thymoma (one each of B1, B2 and B3 subtypes) and one case of thymic carcinoma diagnosed on EBUS-TBNA using cell blocks, immunocytochemistry and flow cytometry which allowed preoperative chemotherapy to be carried out in two cases, diagnosis to be made after unsatisfactory surgical mediastinoscopy in the third and diagnosis of lymph node metastasis of the thymic carcinoma in the fourth. The differential diagnosis and criteria for subclassification of thymomas are discussed; although subclassification of these cases was possible in these cases, and tumours other than thymoma excluded, additional cases would be necessary to assess the potential accuracy of EBUS-TBNA. These, to the best of our knowledge, represent the first cases of thymoma that were diagnosed and subclassified according to WHO criteria using multimodality evaluation of EBUS-derived cytological aspirates.     

Immunohistological assessment of the synovial tissue in small joints in rheumatoid arthritis: validation of a minimally invasive ultrasound-guided synovial biopsy procedure

The aim of the present study was to perform an immunohistological assessment of the synovial tissue from involved small joints in rheumatoid arthritis (RA) and to explore the reliability of a mini-invasive ultrasound (US)-guided technique of small joint synovial biopsy for the histopathological assessment. Synovial tissue collected during arthrotomic surgery of small joints in nine patients served as the gold standard for the validation of the histological assessment. Small hand-joint synovial biopsies from an additional nine patients with erosive RA were obtained by a mini-invasive US-guided procedure, performed percutaneously by the portal and rigid forceps technique. Using digital image analysis, the area fractions of synovial macrophages (CD68 cells), T cells (CD3 cells) and B cells (CD20 cells) were measured in all high-power fields of every sample at different cutting levels. The representative sample was defined as the minimal number of high-power fields whose mean area fraction would reflect the overall mean area fraction within a percentage mean difference of 10%. For each patient, a range of three to five large samples for surgical biopsies and a range of 8–12 samples for US-guided biopsies were collected and analysed. In arthrotomic samples, the analysis of a randomly selected tissue area of 2.5 mm² was representative of the overall value for CD68, CD3 and CD20 cells. US-guided samples allowed histological evaluation in 100% of cases, with a mean valid area of 18.56 mm² (range 7.29–38.28 mm²). The analysis of a cumulative area of 2.5 mm² from eight randomly selected sections (from different samples or from different cutting levels) allowed to reduce the percentage mean difference to less than 10% for CD68, CD3 and CD20 cells. In conclusion, US-guided synovial biopsy represents a reliable tool for the assessment of the histopathological features of RA patients with a mini-invasive approach.     

Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats

Summary Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro. In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow. We have explored development of bone-like tissue in vitro by bone marrow stromal cells. Marrow stromal cells obtained from 40–43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20–30 days. Cells were cultured in either -minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 g/ml ascorbic acid, or the above medium supplemented with either 10 mM Na--glycerophosphate, 10^-8 M dexamethasone, or a combination of both. Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Glaprotein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction. Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both -glycerophosphate and dexamethasone. Cells associated with the nodules exhibited alkaline phosphatase activity. The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Glaprotein were present. X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite. The nodules were also examined for bone morphogenetic protein-like activity. Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats. Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers. This finding provided further support for the bone-like nature of the nodules. The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both -glycerophosphate and, particularly, dexamethasone.     

Bone marrow cryopreservation: biological aspects

Ten bone marrow suspensions have been cryopreserved by a Programmed Freezer Planer R 201. Total cellularity, viability, differential myelograms, cytochemical pattern and CFU-GM growth "in vitro", have been evaluated on the cellular suspensions both before and after 1 and 18 months of storage in liquid nitrogen. Total cellular recovery and viable cell recovery were satisfactory, cellular loss being due, almost entirely to death of the more mature cells. NASDA reaction did not vary after freezing, on the contrary peroxidase reaction and overall PAS reaction showed respectively a slight and an almost complete disappearance. LAP reaction was not valuable, after freezing, because of the more mature myeloid cell loss. CFU-GM recovery was satisfactory and clusters and colonies growth in methylcellulose appeared quite similar before and after 1 and 18 months of storage at very low temperature. Our cryopreservation technique cannot prevent some cellular loss or some qualitative cellular damage, but colonizing ability is almost completely preserved.     

Myelopeptides: Bone marrow regulatory mediators

Bone marrow cells of various animal species and men produce a group of bioregulatory peptides called myelopeptides (MPs). A highly purified MP fraction and some individual molecules have been isolated from the supernatant of porcine bone marrow cell cultures by reverse phase chromatography.MPs have a wide spectrum of functional activities: immunoregulatory, differentiating and opiate-like. They evoke 2–5-fold stimulation of antibody production to various antigens. They correct some immune defects in MRL/lpr mice with spontaneous autoimmune disorders that results in 2-fold prolongation of the life span of these mice. MPs influence the differentiation of bone marrow and peripheral blood cells derived from healthy and leukemic donors. They induce terminal differentiation in the leukemic human HL-60 cell line. MPs also show an effect on pain sensitivity.A new immunocorrective drug Myelopid has been developed on the basis of MP mixtures. This drug is effectively used in Russia both in medicine and veterinary practice for prophylaxis and treatment of diseases accompanied by immunodeficiency.Two individual MPs were isolated and identified: Phe-Leu-Gly-Phe-Pro-Thr (MP-1) and Leu-Val-Val-Tyr-Pro-Trp (MP-2). MP-1 displays immunoregulatory activity; MP-2 abolishes the inhibitory effect of leukemic cells on T-lymphocyte functional activity.MPs seem to provide not only immunoregulation but also to participate in complex interactions between different systems in the organism.     

Bone marrow dosimetry for mice: exposure from bone-seeking 89,90Sr

Studies of radiobiological effects in murine rodents exposed to internal radiation in the wild or in laboratory experiments require dosimetric support. The main problem of bone marrow (BM) dosimetry for bone-seeking β-emitters is dosimetric modeling, because the bone is a heterogeneous structure with complex microarchitecture. To date, there are several approaches to calculating the absorbed dose in BM, which mostly use rough geometric approximations. Recently, in the framework of studies of people exposed to ⁹⁰Sr in the Urals, a new approach (SPSD) has been developed. The aim of the current study was to test for the first time the possibility of extension of the SPSD approach elaborated for humans to mice. For this, computational phantoms of femur bones of laboratory animals (C57BL/6, C57BL/6 J, BALB/c, BALB/cJ) aged 5–8 weeks (growing) and?>?8 weeks (adults) were created. The dose factors DF_Sr-90(BM?←?TBV?+?CBV) to convert the Sr isotope activity concentration in a bone tissue into units of dose rate absorbed in the bone marrow were 1.75?±?0.42 and 2.57?±?0.93 μGy day^?1 per Bq g^?1 for growing and adult animals, respectively, while corresponding values for DF_Sr-89(BM?←?TBV?+?CBV) were 1.08?±?0.27 and 1.66?±?0.67 μGy day^?1 per Bq g^?1, respectively. These results are about 2.5 times lower than skeleton-average DFs calculated assuming homogenous bone, where source and target coincide. The results of the present study demonstrate the possibility of application of the SPSD approach elaborated for humans to non-human mammals. It is concluded that the study demonstrates the feasibility and appropriateness of application of the SPSD approach elaborated for humans to non-human mammals. This approach opens up new prospects for studying the radiobiological consequences of red bone marrow exposure for both laboratory and wildlife mammals.

     

Design of a multifunctional compliant instrument for minimally invasive surgery

A new multifunctional compliant instrument has been designed for use in minimally invasive surgery. The instrument combines scissors and forceps into a single multifunctional device. The main advantage of using multifunctional instruments for minimally invasive surgery is that instrument exchanges can be reduced, thus reducing procedure time and risk of inadvertent tissue injury during instrument exchanges. In this paper, the length, width, and thickness of the multifunctional compliant mechanism tool tip is optimized to maximize the jaw opening and the grasping force. The optimized design is then modeled to simulate the stresses encountered in the scissors mode. A 5.0 mm diameter stainless steel prototype is fabricated using electro-discharge machining and is shown to grasp and cut successfully.