Cytogenetic Testing Methods |
Conventional Cytogenetic Testing
Molecular Cytogenetics
Testing (FISH)
Microarray Comparative Genomic Hybridization Testing (aCGH)
Conventional
Cytogenetic Testing
Method Performance Specifications
Methods Overview: Conventional cytogenetic
testing or routine chromosome analysis, sometimes referred to as karyotyping,
is provided for a variety of clinical 
applications. These
types of studies are utilized to detect numerical and/or structural chromosome
abnormalities in metaphase cells. Constitutional studies are employed
for the diagnoses of the commonly known congenital conditions, such as
Down syndrome, but are also employed in numerous other clinical situations,
such as infertility. In contrast, acquired studies are utilized to assess
the status of specific tissue types, such as blood, bone marrow, and
solid tumors, for acquired chromosomal changes associated with neoplastic or
cancer processes.
Routine or conventional chromosome analyses require sterile viable tissue samples (click here for specific specimen requirements). These types of studies generally require some form of cell culture, followed by chromosome harvesting, chromosome banding, microscopic analysis, and karyotype production.
Limitations of Routine or Conventional Cytogenetics
Testing:
Detection of chromosome
abnormalities in metaphase cell preparations by conventional banding methods is limited to those alterations
detectable by conventional oil-immersion light microscopic methods (1000x total mag), following currently accepted cytogenetic
processing and analysis standards.
Depending upon the application (i.e., high resolution studies vs cancer studies), detection of structural chromosome changes resulting in a loss or gain of genetic material, by these methods, has been estimated to be limited to those of approximately 4-6 mb in size.
These types of studies do not rule out other forms of genetic abnormalities,
such as submicroscopic or molecular defects (i.e., gene mutations), uniparental
disomies, subtelomeric rearrangements. In addition, the detection of
low level or tissue-specific mosaicisms is limited.
MOLECULAR CYTOGENETICS TESTING (FISH)
Method Performance Specifications
Methods Overview: Molecular cytogenetic testing, often referred to as FISH
(fluorescence in situ hybridization), may be utilized to address specific focused
clinical questions and is provided for a variety of clinical applications, including
the assessment of both constitutional and acquired chromosomal aberrations.
FISH
testing is basically a method by which an assessment is made for the presence,
absence, relative positioning, and/or the copy number of specific DNA segments
by fluorescence microscopy. Depending upon the application, FISH can be applied
to metaphase chromosome preparations and/or interphase cell nuclei. The laboratory
methodologies employed vary with the type of study requested; therefore, it is
imperative that the clinical reason for the test be provided at the time of sample
submission!
Metaphase FISH studies (aka microdeletion FISH): These forms of FISH studies are designed to detect changes (i.e, microdeletions, microduplications)associated with specific phenotypic findings and, therefore, require specific and focused clinical questions to be addressed via appropriate DNA probe selection. Metaphase FISH testing
is limited to those DNA probes currently available and validated for clinical use by our laboratory (GEN.42020) and is required to characterize gains and losses detected by aCGH testing (see below).
Genetic changes detected in metaphase chromosome preparations by this type of testing are limited to position and copy number changes, primarily losses (deletions) and in some instances gains (duplications), of the specific chromosomal regions for which the DNA probes employed are localized. Repositioning of these DNA probes from their normal sites must determined in metaphase cell preparations (i.e., translocations, insertions, etc). However, these types of study do not rule out other forms of genetic abnormalities, which may include low level or tissue-specific mosaicisms, and/or other forms of molecular alterations (i.e., single base pair mutations, uniparental disomies, etc.).
The biological relevance of rearrangements of all DNA probes
employed has not been fully established and may require additional familial studies
and correlation with clinical data (GEN.42025). Furthermore, unknown familial
genetic polymorphisms may result in false positive or negative FISH results,
which may or may not be of phenotypic consequence (GEN.42030).
Interphase FISH studies: Enumeration or rearrangements involving specific DNA probes is the only information available from interphase FISH studies (GEN.42025). Additional inferences regarding the chromosome constitution of cells utilized are not possible from interphase FISH studies. Aneuploidy screening in interphase FISH studies DOES NOT address structural content of the chromosomes detected. The use of prenatal interphase FISH studies, as an in vitro diagnostic test, has been cleared by the FDA only as an adjunct test to standard cytogenetic analysis. In addition, the use of interphase nuclear observations for the purpose of determining the chromosomal status of a patient is currently prohibited as a stand alone test by the State of Florida.
Dual color/fusion FISH studies utilizes DNA probe systems that are designed to detect well known neoplastic rearrangements affecting two specific loci but may not detect variant, complex, and/or atypical rearrangements involving these loci. Additional secondary clonal rearrangements may not be detected in interphase nuclei. Further characterization of abnormal cell populations by metaphase FISH or conventional cytogenetic methods is highly recommended. Sensitivity of these types of systems is measured against normal control samples (see written report for current and applied reference values).
Dual color/Break-Apart FISH studies utilize DNA probe systems which are designed to detect the involvement of specific loci known to participate in rearrangements involving a variety of translocation partners. These types of DNA probe systems can only identify rearrangements involving a single locus and can not identify other loci which may be involved in the rearrangement, by interphase analyses. Further characterization of abnormal cell populations by metaphase FISH or conventional cytogenetic methods is recommended. Sensitivity of these types of systems is measured against normal control samples (see written report for current and applied values).
The University of Florida Cytogenetics Laboratory, as required
by the CLIA '88 regulations, determines the FISH tests performance characteristics
in use in its laboratory. FISH tests have not been cleared or approved for specific
uses by the US Food and Drug Administration. However, the FDA has determined
that such clearance or approval is not necessary for clinical applications. The
results from such may be used as adjunctive information relative to a conventional
chromosome analysis/interpretation and clinical data.
Microarray Comparative Genomic Hybridization Testing (aCGH)
Method Performance Specifications
To be posted soon
Methods Overview:
Limitations of aCGH Testing:
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