A Microbiopsy Method for Immunohistological and Morphological Analysis: A Pilot Study (original) (raw)
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Journal of Surgical Research, 2010
The muscle biopsy based on the Bergström needle has been widely used for more than 40 y for diagnosis and experimental studies on muscle. More recently, thinner needles and tru-cut needles have also been introduced. Such techniques have been largely tested on various muscles, including the quadriceps, with few studies on upper limb muscles like deltoid, and no studies on latissimus dorsi muscle (LDM). In this study, we implemented and validated a protocol to collect samples of LDM for experimental purposes, causing minimal discomfort to volunteers. Two main problems were considered: the anatomical localization of the biopsy site and the selection of an appropriate needle.A strict protocol of palpatory anatomy was adopted and validated with ultrasonography to localize the biopsy site in LDM in subjects with various degrees of muscle development. A 14 gauge tru-cut needle was selected as the smallest and still effective device for sampling. Biopsy sampling was performed in 18 subjects without any complications, or complains of pain or functional limitations.Approximately 4 mg of tissue were recovered from each introduction of the inner notched cannula of the needle. With three consecutive samplings, an amount of tissue sufficient to prepare proteins for gel electrophoresis and Western blot and to dissect single fiber segment for functional experiments, was obtained.Taken together, the results suggest that this biopsy technique opens to experimental studies muscles until now never considered accessible.
International journal of molecular medicine, 2011
Needle biopsy is widely used to obtain specimens for physiological, anatomical and biochemical studies of skeletal muscle (SM). We optimized a procedure which we termed tiny percutaneous needle biopsy (TPNB), to efficiently gather good numbers of human satellite cells and single dissociated fibers for the functional study of skeletal muscle; these samples permit isolation of high-quality RNA and sufficient amounts of proteins to allow molecular analysis. Moreover, TPNB showed a clear advantage in that the technique was easier than other procedures used on healthy volunteers in human trials. TPNB is a very safe minor surgical procedure. It is less traumatic than needle aspiration biopsy, and significant complications are improbable. TPNB should become established as an important tool in the investigation of SM and may be employed to study various physiological aspects of SM in human subjects. We suggest that TPNB should also be used in the study of muscle diseases and disorders inclu...
European Journal of Histochemistry, 2010
The aim of the present investigation was to evaluate whether routinely frozen biopsies of human skeletal muscle may be suitable for morphological and immunocytochemical analyses at transmission electron microscopy. The fixation/embedding protocols we successfully used for decades to process fresh mammalian tissues have been applied to frozen muscle biopsies stored for one to four years in liquid nitrogen. After 2.5% glutaraldehyde -2% paraformaldehyde -1% OsO 4 fixation and embedding in epoxy resin, the ultrastructural morphology of myofibres and satellite cells as well as of their organelles and inclusions proved to be well preserved. As expected, after 4% paraformaldehyde -0.5% glutaraldehyde fixation and embedding in LR White resin, the morphology of membrane-bounded organelles was relatively poor, although myofibrillar and sarcomeric organization was still recognizable. On the contrary, the myonuclei were excellently preserved and, after conventional staining with uranyl acetate, showed an EDTA-like effect, i.e. the bleaching of condensed chromatin, which allows the visualization of RNP-containing structures. These samples proved to be suitable for immunocytochemical analyses of both cytoskeletal and nuclear components, whereas the poor mitochondrial preservation makes unreliable any in situ investigation on these organelles.
International journal of molecular medicine, 2013
Skeletal muscle biopsies require transecting the muscle fibers resulting, in structural damage near the cut ends. Classically, the optimal ultrastructural preservation has been obtained by the use of relatively large biopsies in which the tissue fibers are restrained by ligating to a suitable retaining support prior to excision, and by examining regions at some distance from the cut ends. However, these methods require invasive surgical procedures. In the present study, we present and substantiate an alternative approach that allows for the excellent ultrastructural preservation of needle biopsy samples, even the very small samples obtained through tiny percutaneous needle biopsy (TPNB). TPNB represents an advantage, relative to standard muscle biopsy techniques and to other needle biopsies currently in use, as in addition to not requiring a skin incision, it leaves no scars in the muscle and requires an extremely brief recovery period. It is most appropriate for obtaining repeated ...
Journal of Neurology, 2012
Muscle biopsy is required to provide a definitive diagnosis in many neuromuscular disorders. It can be performed through an open or needle technique under local anesthesia. The major limitations of the needle biopsy technique are the sample size, which is smaller than that obtained with open biopsy, and the impossibility of direct visualization of the sampling site. However, needle biopsy is a less invasive procedure than open biopsy and is particularly indicated for diagnosis of neuromuscular disease in infancy and childhood. The biopsied muscle should be one affected by the disease but not be too weak or too atrophic. Usually, in case of proximal muscle involvement, the quadriceps and the biceps are biopsied, while under suspicion of mitochondrial disorder, the deltoid is preferred. The samples must be immediately frozen or fixed after excision to prevent loss of enzymatic reactivity, DNA depletion or RNA degradation. A battery of stainings is performed on muscle sections from every frozen muscle biopsy arriving in the pathology laboratory. Histological, histochemical, and histoenzymatic stainings are performed to evaluate fiber atrophy, morphological, and structural changes and metabolic disorders. Moreover, immunohistochemistry and Western blotting analysis may be used for expression analysis of muscle proteins to obtain a specific diagnosis. There are myopathies that do not need muscle biopsy since a genetic test performed on a blood sample is enough for definitive diagnosis. Muscle biopsy is a useful technique which can make an enormous contribution in the field of neuromuscular disorders but should be considered and interpreted together with the patient's family and clinical history.
Journal of Surgical Research, 2009
Recent analytical methods such as real-time polymerase chain reaction (PCR) and Western blotting have now enabled us to analyze the gene and protein expression from small amounts of tissue. A fine needle muscle biopsy is thus expected to obtain a minimally sufficient amount of skeletal muscle to make a successful analysis. As a result, we used this fine needle muscle biopsy technique to obtain muscle tissue specimens from the vastus lateral muscle in 40 participants. The amount of tissue obtained by the fine needle was 5.2 ± 3.2 mg (mean ± standard deviation). The total RNA extracted was 3.0 ± 1.4 mg and the total protein extracted was 2203 ± 1541 mg. Furthermore, the skeletal muscle tissue specimens obtained by the regular needle technique and blood sample were used as the control. Those specimens were used to measure the gene expression of bmyosin heavy chain slow (b-MHC slow) by real-time PCR and the protein expression of monocalboxylate transporter 1 (MCT-1) by Western blotting. b-MHC slow gene expression was detected in both samples obtained by a fine and a regular needle biopsy, but not in a blood sample. Furthermore, the MCT-1 protein was detected in samples obtained by a fine needle muscle biopsy. These results indicated that the fine needle muscle biopsy is therefore a useful technique to obtain skeletal muscle specimens at least to analyze the gene and protein expression. Ó
The Journal of Histochemistry and Cytochemistry, 1992
A m d e biopsy from the ~s t u s lateralis m d e ofa strengthtrained woman was found to contain an unusual fiber type composition and was analyzed by histochemical, biochemical, and ultrastructural techniques. Special attention was given to the C-fibers, which comprised over 15% of the total fiber number in the biopsy. The mATPase activity of the C-fibers remained stable to varying degrees over the pH range normally used for routine mATPase histochemistry. Although a continuum existed, the C-fibers were histochemically subdivided into three main fiber types: IC, IIC, and IIAC. The IC fibers were histochemically more similar to the Type I, the IIAC were more similar to the Type IIA, and the IIC were darkly stained throughout the pH range. Biochemical analysis revealed that all C-fibers c o e x p r e s s e d myosin heavy chains (MHC) I and IIa in variable ratios. The histochemical staining intensity correlated with the myosin heavy chain composition such that the Type IC fibers contained a greater ratio of MHCIIMHCIIa, the IIAC contained a greater ratio
Histochemical characterization of masseter muscle fibres in a biopsy study of normal young women
Archives of Oral Biology, 1992
Muscle fibres from biopsies of the anterior part of the masseter muscle (pars superficialis) were histochemically characterized in 13 healthy female students. They were 21-28 yr old with a full complement of teeth, and normal facial and occlusal relations. Before surgery, normal masseter muscle function was demonstrated by bite-force measurements and recordings of electromyographic activity. After staining for myosin ATPase activity, the relative mean areas of muscle fibres were: type I 82.9%, type IM 9.5% and type II 8.3%. The intraindividual (18-155% of 2) and interindividual (O-216% of .f) variation of the fibre size was large. The type I fibres had the largest diameter (IO-80 pm, P: 39 pm). the type II fibres (647Rm, .f: 21 pm) and the IM fibres (8-54pm. 2: 28pm) the smallest. The biopsy technique and the histochemical characterization will be suitable for reference in women with functional changes or diseases of the masseter muscle.
Muscle histopathology in today’s era of molecular genetics: Role and limitations
Introduction: Past few decades have seen an increasing application of techniques like electron microscopy, western blotting, and molecular genetics in the evaluation of muscle diseases. However, due to their limited availability, histopathological interpretation of muscle biopsies still remains an important component of diagnostic approach to muscle diseases. A systematic methodology is required in the evaluation and interpretation of muscle biopsies. This study was undertaken to analyze the histopathological spectrum of 164 muscle biopsies and to assess the diagnostic yield of basic histopathological procedures in the work up of muscle biopsy. Materials and Methods: Retrospective analysis was done for 164 cases of muscle biopsies. Step-wise approach was adopted to assess the efficacy of routine stains, enzyme histochemistry, and immunohistochemistry. Based on hematoxylin and rosin (H and E) appearance, biopsies were broadly categorized into destructive, nondestructive but myopathic, and inflammatory morphology. Role of special stains, enzyme, and immunohistochemical stains in each category was then evaluated. Results: On the basis of histopathological features, 164 muscle biopsies were broadly categorized into biopsies with abnormal histopathological features (115) and biopsies with normal histopathology (49). Abnormal muscle biopsies were further categorized into destructive morphology (56.5%), nondestructive but myopathic morphology (30.5%), and inflammatory pathology (13%). A near definitive diagnosis could be made in 115 cases out of 164 muscle biopsies on the basis of routine histopathology, enzyme histochemistry, and immunohistochemistry. Conclusion: Though advanced techniques like electron microscopy, western blotting, and molecular genetics are essential for confirmatory diagnosis, a substantive diagnostic yield can be offered with the basic armamentarium of routine (frozen) stains, enzyme histochemistry, and immunohistochemistry
Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies
Histochemistry, 1990
Muscle fiber typing is conventionally performed using mATPase enzyme histochemistry on cryostat sections. After pre-incubation of sections at pH 4.3, 4.6 and 10.3, based on the pattern of enzyme reactivity, the fibers can be classified in types I, II (subtypes A, AB and B) and the intermediate C (I and II) fibers. We have attempted to perform fiber typing of human psoas muscle by immunohistochemistry, using monoclonal antibodies R11D10 (specific for cardiac and type I skeletal myosin) and MY-32 (specific for fast muscle fibers) on cryostat as well as on paraffin sections. Staining of consecutive cryostat sections showed that type I fibers are R11D10 reactive whereas type II fibers are MY-32 reactive. Subtyping of type II fibers could not be performed by immunohistochemistry. Quantitative analysis of type I and II fibers showed that enzyme histochemical and immunohistochemical analysis are in close agreement.