Comparison to other techniques Chromogenic in situ hybridization

a) direct fish detection. fluorescent labels attached probe hybridize target dna strand. b) indirect fish detection. biotin, example, attached probe. streptavidin linked fluorescent tag binds biotin high specificity. c) indirect cish detection. again, biotin attached probe. streptavidin linked horseradish peroxidase binds biotin high specificity. horseradish peroxidase converts diaminobenzidine brown precipitate.


compared fish

fish considered gold standard detection of chromosomal abnormalities because sensitive , has high resolution. other techniques developed detect chromosomal abnormalities compared sensitivity , specificity of fish see how measure up. example, compared fish, cish has been shown have sensitivity of 97.5% , specificity of 94% detection of her-2/neu gene amplification. concordance rate between fish , cish 94.8%, showing cish comparable technique fish. other sources agree , report equal performance on gene amplification assays fish , cish. however, cish shows lower sensitivity low level amplifications.

cish has advantages on fish in reagents , equipment uses. noted above, cish cheaper , easier use because uses bright-field microscopes instead of fluorescence microscopes. in addition, cish reagents more stable fish reagents possible store samples , examine same sample multiple times. fish reagents fade overtime due photobleaching sample can examined once. apart expensive fluorescence microscope, fish requires high-resolution digital camera capture micrographs of sample before fluorescence fades. advantage of using bright-field microscopy tissue or cell sample whole can visualized through cish whereas cell morphology difficult assess using fluorescence microscopy in fish.

cish differs fish in probes used in overall method. there many different types of fish probes available, such repeat probes, probes detect specific genes or telomeres, , probes detect chromosomal abnormalities. in contrast, there limited variety of commercially available cish probes, including probes bind centromere of chromosomes 3, 7, 8, 9, 10, 11, 17, 18, x, , y gene-specific probes cancer-related genes, such her-2, egfr, myc, , top2a. despite limited variety of available cish probes, more cost-effective fish probes. regard overall method, fish can performed using direct labelling—fluorochromes attached probes—or indirect labelling—the probes labelled biotin or digoxigenin detected using fluorescently-labelled streptavidin or antibodies, respectively. cish performed using indirect labelling in antibodies or streptavidin conjugated enzymes such hrp or alkaline phosphatase (ap).

compared ihc

cish , ihc similar in both used same purpose (mainly detect her-2/neu amplification) , both use enzyme reactions (hrp/ap) measure amplification. cish , ihc different in ihc measures protein expression whereas cish measures dna amplification. difference particularly useful her-2/neu because has been found gene amplification of higher prognostic value protein expression.

a disadvantage of ihc is not possible identify false-negative , false-positive results. in cish, if there no signal reference probe, assay has failed.

for low , high protein overexpression/gene amplification, cish , ihc show concordance of on 86% , on 89%, respectively. has been shown monoclonal antibodies better polyclonal antibodies detection in both ihc , cish bind more specifically, leads higher concordance rate.

for medium protein overexpression/gene amplification concordance varies, higher when monoclonal antibodies used when polyclonal antibodies used. variable concordance due fact gene amplification not strictly correlate protein expression , tumor heterogeneity can make difficult detect protein overexpression in tissue.