EXPLANATION OF CYTOGENETIC TESTS
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Fetal skin cells are sloughed as a normal part of fetal growth, so the amniotic fluid (AF) contains cells that can be grown in culture. Typically, amniocentesis is performed at 16-20 weeks gestational age and 20-30 ml of fluid is obtained. The fluid is centrifuged to concentrate the cells, which are then diluted in culture medium. The cell suspension is plated out onto coverslips (5-7 coverslips depending on sample size) and a backup flask, where the cells will settle down and grow. As the cells divide, they form colonies where all the cells are descended from the original cell that settled down. After 6-8 days there are usually sufficient colonies with actively dividing cells for harvest. The AF's are harvested in-situ so that cells from independent colonies can be analyzed. Seven cells/colonies are fully analyzed for chromosome number and structure and an additional 13cells/colonies are checked for chromosome number and sex chromosomes.
Cytogenetic analysis of amniotic fluid is useful for pregnancies at risk due to maternal age, abnormal AFP or triple screen, ultrasound abnormalities, parental chromosome rearrangement carrier, previous trisomy, previous history of miscarriage or infertility, past x-ray exposure, etc.
If initial results suggest mosaicism, an additional 15 colonies and/or 50 cells will be analyzed; in case of unusual variants it may be necessary to study parents’ blood chromosomes to clarify fetal chromosome results.
CVS allows for prenatal karyotype at an earlier gestational age (10-12 weeks) than amniocentesis, useful for pregnancies at risk due to maternal age, parental chromosome rearrangement, past X-ray exposure, etc. A small biopsy of the developing placenta (chorionic villi) is obtained. Working under a microscope, all signs of maternal contamination are removed. The cleaned villi are treated with trypsin (a digestive enzyme) and colcemid (a mitotic inhibitor) to remove the outer layer of cells (trophoblast) for a direct harvest. The remaining mesenchymal cells are dissociated with collagenase, diluted with culture medium and plated out onto coverslips (4-6 coverslips depending on sample size) and backup flask, where the cells will settle down and grow. As the cells divide, they form colonies where the cells are descended from the original cells that settled down. After 6-8 days, there are usually sufficient colonies with actively dividing cells for harvest. The CVS's are harvested in-situ so that cells from independent colonies can be analyzed Seven cells/colonies are fully analyzed for chromosome number and structure and an additional 13 cells/colonies are checked for chromosome number and sex chromosomes.
The chromosome changes in human leukemia are usually confined to the leukemic cells. Therefore, chromosome analysis is most reliably done by examining the cells in bone marrow. The preferred technique is to do a direct harvest of the bone marrow. Since not all samples will provide sufficient metaphases for analysis, additional cultures are grown for 24-48 hours and then harvested. Twenty metaphases are examined for chromosome number and structure.
Cytogenetic studies are becoming more important in hematologic disorders for diagnosis and determination of the prognosis of the patient. In addition to the first chromosome analysis at the time of diagnosis, it is often necessary to repeat the analysis to follow the course of the disease and therapy. Also, repeated analyses should be done after bone marrow transplants to confirm the success of the transplant and to check for relapse.
High resolution banding is a technique for obtaining longer, less condensed chromosomes where more sub-bands can be visualized.
After a 72-hour growth period, an intercalating agent, Ethidium bromide, is added to the blood cultures. The molecules of Ethidium bromide become inserted between the base pairs of the DNA in the chromosome and inhibit condensation of the chromosomes during the mitotic cycle. When colcemid is added to block the cells during metaphase, the chromosomes are still in an uncondensed stage similar to late prophase.
This technique results in the identification of approximately 650-800 bands in a haploid karyotype instead of 400 bands. With this technique one can detect subtle loss or gain of genetic material that may be missed otherwise. The main purpose of prometaphase technique is that it gives us a better chromosome resolution; therefore, minor abnormalities are detectable. This test is useful for: syndrome identification, atypical development, atypical sexual development, infertility, history of pregnancy loss, family studies, etc.
The chromosome changes in human leukemia are usually acquired and confined to the leukemic cells. When it is not possible to obtain a bone marrow sample, testing may be performed on peripheral blood if there are sufficient mitotic immature granulocytes present. The blood is cultured without any mitogen so that only spontaneously dividing cells will be analyzed. Cultures are harvested after 24 and 48 hours. A 48 hr. PHA-stimulated culture is set up for examination of the constitutional karyotype. Twenty metaphases (when possible) are analyzed for chromosome number and structure.
Cytogenetic studies are becoming more important in hematologic disorders for diagnosis and determination of the prognosis of the patient. In addition to the first chromosome analysis at the time of diagnosis, it is often necessary to repeat the analysis to follow the course of the disease and therapy. Also, repeated analyses should be done after bone marrow transplant to confirm the engraftment of the transplant and to check for relapse.
The chromosomal changes in cancer are usually acquired and confined to the cancerous cells. A biopsy of the tumor is mashed and finely minced to dissociate the cells. A portion of the cells is harvested directly to look for actively dividing cells. The remainder is set up for short-term culture. Cultures are harvested as early as possible to minimize the outgrowth of normal cells. Twenty metaphases (when possible) are analyzed for chromosome number and structure.
Cytogenetic studies are becoming more important in cancer for diagnosis and determination of the prognosis of the patient.
Skin tissue may be used for chromosome analysis in special cases when the results from peripheral blood are inconclusive, e.g. suspected mosaicism, confirmation of a new chromosome disorder, or special dermatological disorders. Tissue samples of skin, kidney, lung, or placenta may also be used when blood cultures are not available, such as on stillbirths. In the case of stillbirths, chromosome studies are performed to determine if a chromosomal abnormality is present that may have been inherited from the parents, putting them at risk for future pregnancy losses and/or children with birth defects.
The tissue sample is finely minced and set up in culture flasks with growth media. The cultures are incubated and checked for signs of growth. When there are sufficient numbers of growing cells in the flasks, the cell cultures are sub-divided and harvested using colcemid to obtain chromosome preparations that are then used for karyotype analysis. Harvests are done on at least two different culture flasks to allow for the identification of cultural artifacts and to distinguish between pseudo and real mosaicism.
Cultured fibroblasts from skin tissue may be used for testing other than cytogenetics, such as biochemical analysis for errors in metabolic pathways.
The skin biopsy is finely minced and set up in culture flasks with growth medium. The cultures are incubated and checked daily for signs of growth. When there are sufficient numbers of growing cells in the flasks, the cells are dissociated, subcultured, and allowed to re-grow. When there are sufficient cells, they can be sent to the appropriate lab for further testing.
Prader-Willi Syndrome (PWS) is associated with paternal deletion and imprinting errors of regions of chromosome 15q11.2-q13 and maternal disomy of chromosome 15. Conversely, maternal deletions of this region and paternal disomy of chromosome 15 are associated with Angelman Syndrome (AS).
A CpG island at the 5 ' end of the SNRPN gene is completely methylated on the maternal chromosome, while the paternal chromosome is completely unmethylated. Extensive methylation of CpG islands is associated with transcriptional inactivation of regulatory regions of imprinted genes such as those associated with PWS/AS. MSPCR detects abnormal gene methylation by employing a deamination reaction (known as bisulfite modification) followed by amplification designed to distinguish methylated from unmethylated DNA. MSPCR for PWS/AS amplifies a differentially methylated site present at the CpG island of the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN).
Cytogenetic studies and FISH cannot detect uniparental disomy (UPD) or an abnormality of the imprinting process that causes a lack of gene expression while MSPCR can. However, MSPCR alone cannot distinguish between a chromosomal deletion, UPD, or a lack of gene expression. MSPCR does not detect mutations of the UBE3A gene.
Fragile X syndrome results from mutations in the FMR-1 gene. The gene product of FMR-1 is diminished or absent in patients with Fragile X syndrome. The mutations disrupting FMR-1 gene function include expansion of a CGG tandem repeat sequence within the FMR-1 gene and methylation of cytosine residues in a CpG promotor region 5’ to FMR-1. The number of CGG repeats in normal alleles of the FMR-1 gene is polymorphic and ranges from 6 to 45 triplets with the most common allele containing 29. In the majority of transmitting males and carrier females, the repeat number is in the range of 56 to 200 (premutation range). The range of 45 to 55 repeats is considered a gray zone between normal and premutation. Normal alleles and small premutations can be identified by PCR. In individuals with the Fragile-X phenotype, (full mutation) the repeat number usually exceeds 200 copies. Full mutations are evaluated using Southern hybridization.
PCR is used to amplify a highly GC-rich, 250-350 bp region of the FMR-1 gene. Amplification is followed by agarose gel electrophoresis and fragment sizing to determine the number of a triplet repeats in this region.
RNA is used as a template for the synthesis of complimentary DNA (cDNA) using the enzyme reverse transcriptase. The resultant cDNA provides a template for PCR amplification. This process is useful for the examination of chromosomal translocations in which DNA breakpoints are varied and occur over large distances giving rise to fusion messenger RNAs (mRNAs.)
Qualitative multiplex reverse transcriptase polymerase chain reaction (RT-PCR) is designed to detect fusion mRNAs that result from translocations or chromosomal rearrangements and have been found to be specific markers for particular subtypes of leukemia. This test is designed to detect 28 different translocations or chromosomal rearrangements, including more than 80 breakpoints or splice variants.
An internal control fragment is amplified in each assay. Amplification of this region checks for RNA integrity and the presence of PCR inhibitors. A positive and negative (reagent) control are included in each run.
FISH is the detection of highly specific DNA probes that have been hybridized to either interphase or metaphase chromosomes using fluorescence microscopy.
DNA for probe use is labeled with fluorescent (direct method) or non-fluorescent molecules that are then detected by fluorescent antibodies (indirect method). The probes bind to a specific region or regions on the target chromosome. The chromosomes are then stained using a contrasting color, and the cells are viewed using a fluorescence microscope.
Uses for FISH include: simple determination of aneuploidy (monosomies, trisomies), sex determination, gene amplification, derivative chromosome or marker determination, micro-deletion detection and tracking translocations in interphase cells for use in diagnosis and treatment follow-up in tumors and hematologic disorders.