Cell vision : the journal of analytical morphology最新文献

In situ Hybridization (ISH), since its introduction in 1969 by Gall and Pardue has found multiple uses in molecular morphology due to its unique capability of visualizing nucleic acid sequences without altering the cell's cytological, chromosomal or histological integrity. Fluorescence in situ Hybridization (FISH) has established itself as a variation of the traditional hybridization process whereby the probes utilized are a fluorescently labeled and produce bright clear signals upon detection. FISH is involved in localizing and exploring chromosomal, genetic and genomic aberrations that are often directly correlated to disease causation and progression. This technique is used as an essential tool not only in research laboratories, but also in prognostics, diagnostics and disease monitoring in medicine. In the past decade, there have appeared multiple modifications to traditional FISH technology allowing a wider variety of practical applications. Some of the latest innovative techniques discussed in this review paper are Forward and Reverse Chromosome Painting, Chromosome in situ suppression hybridization (CISS), Multicolor FISH, Chromosomal bar coding, Micro-FISH, In situ Hybridization to mRNA, in-cell RT-PCR, Fluorescence immunophonotyping and interphase cytogenetics as tool for investigation of neoplasmas (FICTION), Primed in situ DNA synthesis (PRINS), Fiber-FISH, FISHES, and Comparative genomic hybridization (CGH). This paper provides a brief overview of FISH and the techniques exploiting FISH modifications as well as their evaluation and discussion of practical applications.

Although direct in situ PCR is more rapid than indirect method for the in situ identification of low copy number genes, several reports indicate serious nonspecific signals with this method. Some procedures have been reported in an effort to eliminate the nonspecific signals, but the results have not been satisfactory. Exonuclease III can progressively digest blunt or recessed 3' termini of double-stranded DNA whereas DNA with 3' overhanging end is resistant to digestion. DNA fragments amplified by PCR with primers incorporating the recognition site for Sph I generate 3' four bases extensions at both end after digestion. These fragments are expected to be resistant to exonuclease III digestion. It is also expected that nonspecifically incorporated digoxigenin would be released by treatment with exonuclease III thereby reducing background. We succeeded in digesting selectively with the samples after standard PCR by Sph I and exonuclease III treatment. However, we failed to eliminate the nonspecific signals of direct in situ PCR. Southern blotting revealed that the amount of nonspecific incorporation was so huge that exonuclease III was unable to release all of nonspecifically incorporated digoxigenin and maintain specific incorporated digoxigenin simultaneously. Direct in situ PCR is a sensitive method. However, its specificity has a significant problem.

Serological methods of tissue typing have been used for matching patient-donor pairs for transplantation for several decades. Molecular techniques are rapidly being developed that can provide rapid, accurate tissue typing at the DNA sequence level. These techniques can be used to identify the best match pre-transplant and can also be used for the post-transplant monitoring of engraftment of bone marrow transplant patients.

Technical improvements in the application of molecular biology methods to detection and identification of specific nucleic acid sequences have resulted in more widespread incorporation of these techniques into clinical laboratory services. Gene amplification techniques have been refined and automated, making possible the rapid and economical detection of attomolar and smaller quantities of genetic material. Of particular benefit to clinical chemistry applications is the remarkable specificity of a DNA probe for its complementary sequence. Applications of molecular biology techniques in clinical chemistry include diagnosis of infectious, neoplastic, genetic, and hematological diseases. In addition, the use of oligonucleotides as molecular recognition probes may provide new analytical strategies for a wide range of diagnostic applications that currently rely on antibodies.

Calcitonin gene-related peptide (CGRP) is widely distributed in sensory neurons and nerve fibers. It was shown recently in our laboratory that there was CGRP-immunoreactivity (CGRP-ir) in extract of rat lymphocyte of thymus and mesenteric lymph node by radioimmunoassay and reversed-phase HPLC. The aim of this study was to detect the CGRP-ir location in the rat lymphocyte by immunocytochemical method. Single cells isolated from thymus and mesenteric lymph node of male Wistar rat (200-250 g) were suspended in RPMI-1640 medium. After adhesion to plate wall and through nylon wool fiber columns, T cell-riched suspension was obtained. Immunocytochemical ABC method was performed in cell suspension, the specific antiserum (1:200 diluted) was rabbit anti-human CGRP. The cells were examined under light microscope after smeared on glass slide by Shandon Cytospin. The results showed that some lymphocytes were CGRP-ir positive. The positive granules were seen as ring, plaque, cap or spot distributing on the surface or inside the cell. The results indicate that CGRP-ir may be located in some rat T lymphocytes. This work provides new information about the interaction between the nervous and the immune systems. The functional significance of CGRP located in lymphocytes needs to be further studied.

Telepathology is to transform microscopic and gross images of pathological specimen into digital forms and transmit them electronically to remote sites for consultation and collaboration. Many benefits can be derived from telepathology. Although still at its initial developing stage, this technology is beginning to change the way pathological practice is conducted. With the rapid advancement of computer and telecommunication sciences, telepathology is increasingly becoming more feasible and practical for pathologists. This review gives a comprehensive account of the recent development in telepathology, discusses its functionality, applications, limitations and potential, and serves as an introduction to this rapidly evolving field.