Cytogenetic Testing Methods

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. These types of studies generally require some form of cell culture, followed by chromosome harvesting; chromosome banding; microscopic analysis; and karyotype production.

KaryotypingLimitations of Routine or Conventional Cytogenetics Testing

Detection of chromosome abnormalities in metaphase cell preparations by conventional banding methods is limited to those alterations that are detectable by conventional oil-immersion light microscopic methods (1000x total magnification), following currently accepted cytogenetic processing and analysis standards.

Depending on the application (i.e., high-resolution 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 or subtelomeric rearrangements. Additionally, the detection of low-level or tissue-specific mosaicisms is limited.


Molecular Cytogenetics Testing via Fluorescence in-Situ Hybridization (FISH)


Method Performance Specifications


Methods Overview

Fish StudyMolecular cytogenetic testing, otherwise known as fluorescence in-situ hybridization ( FISH), 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 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 (Microdeletion FISH)

Metaphase FISH studies are designed to detect changes (i.e, microdeletions and/or microduplications) associated with specific phenotypic findings and require specific and focused clinical questions to be addressed via appropriate DNA probe selection. Metaphase FISH testing is limited to those DNA probes that are currently available and validated for clinical use by our laboratory (GEN.42020) and is required to characterize gains and losses detected by aCGH testing.

Genetic changes that are 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 employed DNA probes are localized. Repositioning of these DNA probes from their normal sites must be 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 employed DNA probes 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 are the only information available from interphase FISH studies (GEN.42025). Additional inferences on 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. Additionally, the use of interphase nuclear observations for the purpose of determining the chromosomal status of a patient is currently prohibited as a standalone test by the state of Florida.

Dual-Color/Fusion FISH Studies

This form of testing 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.

Dual-Color/Break-Apart FISH Studies

These studies utilize DNA probe systems that 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 cannot identify other loci that 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.

UF PathLabs, as required by the CLIA 1988 regulations, determines the FISH test performance characteristics in use at its laboratory. FISH tests have not been cleared or approved for specific uses by the U.S. Food and Drug Administration; however, the FDA has determined that such clearance or approval is not necessary for clinical applications. The results from such determination may be used as adjunctive information relative to a conventional chromosome analysis/interpretation and clinical data.

Microarray Comparative Genomic Hybridization (aCGH) Testing


Method Performance Specifications


Methods Overview

Microarray comparative genomic hybridization (aCGH) testing is useful for the detection of small genetic imbalances (gains or losses of chromosomal material), also known as genomic copy number changes, which may not be detectable by conventional cytogenetic and/or FISH techniques.

Microarray comparative genomic hybridization (aCGH) testing can also be useful for the identification of specific genes involved and in sizing a chromosomal abnormality detected by conventional cytogenetics or FISH techniques.

The design of the array is critical in terms of the sensitivity of the test and will include loci of common microdeletion/duplication syndromes, as well as numerous subtelomeric and pericentromeric regions. Subtelomeric locations are sites known to be commonly involved by DNA copy number alteration. aCGH testing utilizes short DNA sequences corresponding to known chromosomal loci spanning the genome that are fixed to a solid surface. To conduct the test, fluorescently labeled DNA from both the patient and a control is hybridized to the array. Different fluorescent probes are used for the patient and control. After hybridization, the signals are detected and software-assisted interpretation of the generated data is performed to determine any copy number change between control and patient DNA.


Note: This array format will replace the previous ISCA 180k format as the default testing modality utilized for all new and pending congenital aCGH testing requests. The 180k and 44k oligonucleotide formats may be employed for direct comparison purposes for additional family members previously studied by these designs, where necessary. Contact the Laboratory before ordering to request for testing to be performed by an alternate design or platform.


Resolution and Method Performance Specifications:The SurePrint G3 Human CGH+SNP© was built using a commercial platform (Agilent Technologies), and it contains ~120k oligonucleotide features (~60mers) that represent coding and non-coding human sequence in the genome and ~60k SNP features (content sourced from the UCSC GRCh37/hg19 human genome; Feb. 2009).

This array contains high-density, genome-wide coverage of clinically relevant deletion/duplication syndromes and the subtelomeric and pericentromeric regions with an average probe spacing of ~25kb. It is designed to detect gains and losses at a minimum of ~500 kb across the genome or smaller when occurring within the ~500 targeted regions of known microdeletion/duplication syndromes or gene-rich areas. Resolution for segmental AOHs is limited to contiguous stretches of 5 mb or greater but may be reported for smaller sizes when detected in regions currently known or implicated in imprinting conditions associated with AOH. 

Call the laboratory at (352) 265-9900, if you have additional questions regarding the availability or applications of specific microarray platforms.

Limitations of aCGH Testing

Microarray comparative genomic hybridization (aCGH) testing may include false-negatives if the patient has a copy number change not covered by the specific array used in the laboratory. Balanced chromosomal rearrangements (i.e., translocations, inversions, etc.) cannot be detected. Mutational changes (base pair substitutions, methylation status, point mutations, etc.) and duplications/deletions less than those that can be resolved by the particular array cannot be detected. Not all copy number changes are clinically significant and, when detected, need to be classified as benign, pathogenic or of unknown significance.

Good practice includes confirmation of any copy number change by FISH analysis, or some other proven technology, and detection of a genomic imbalance frequently requires follow-up testing of a parental sample.

Cytogenetics