Clinical Laboratory - Molecular Diagnostics

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Genetic Testing Techniques


Karyotyping of cells yields information about chromosome number as well as abnormalities such as large translocations, deletions, and inversions of portions of a chromosome. Cells must be collected and grown in culture, induced to begin mitosis, and then stopped in metaphase where distinct chromosomes can be identified, enumerated, and assessed for morphologic changes.


Amniocentesis is an invasive procedure done during pregnancy in which a needle is passed through the mother's lower abdomen into the fetal amniotic cavity inside the uterus. For prenatal diagnosis, most amniocenteses are performed between 14 and 20 weeks gestation. However, an ultrasound examination always precedes amniocentesis in order to determine gestational age, the position of the fetus and placenta, and determine if enough amniotic fluid is present. Within the amniotic fluid are fetal cells which can be grown in culture for chromosome analysis, biochemical analysis, and molecular biologic analysis.

Risks with amniocentesis are uncommon, but include fetal loss and maternal Rh sensitization. The increased risk for fetal mortality following amniocentesis is about 0.5% above what would normally be expected. Rh negative mothers can be treated with RhoGam. Contamination of fluid from amniocentesis by maternal cells is highly unlikely.

Chorionic Villus Sampling (CVS)

In this procedure during early pregnancy, a catheter is passed via the vagina through the cervix and into the uterus to the developing placenta under ultrasound guidance. Alternative approaches are transvaginal and transabdominal. This allows sampling of cells from the placental chorionic villi. These cells can then be analyzed by a variety of techniques, including chromosome analysis to determine the karyotype of the fetus. The cells can also be grown in culture for biochemical or molecular biologic analysis. CVS can be safely performed between 9.5 and 12.5 weeks gestation.

CVS has the disadvantage of being an invasive procedure, and it has a small but significant rate of morbidity for the fetus; this loss rate is about 0.5 to 1% higher than for women undergoing amniocentesis. Rarely, CVS can be associated with limb defects in the fetus. The possibility of maternal Rh sensitization is present. There is also the possibility that maternal blood cells in the developing placenta will be sampled instead of fetal cells and confound chromosome analysis.


Fluorescence in situ hybridization (FISH) is a technique that utilizes DNA probes that are specific to regions of individual chromosomes. The probe attaches to the spread of chromosomes from a cell, then a fluorescein stain is applied. This "paints" the chromosome so that it is visible with the aid of a fluorescent microscope. In the example diagram below, the chromosome 21 pair have been painted.

Gene amplification

Genetic damage with DNA alterations may include gene reduplication with amplification. These alterations transform proto-oncogenes into oncogenes to drive neoplasia. One example is amplification of the HER2 gene in some breast cancers.

In the diagram below, fluorescence in situ hybridization (FISH) assay for for HER-2 in a breast carcinoma shows that within the cells the probe which marks in red for amplification of the HER-2 gene is in much greater quantity than the green probe marking for a normal component of a pair of chromosomes. HER-2 oncogene overexpression is typically the result of gene amplification (more gene copies).

Comparative Genomic Hybridization (CGH)

CGH analyzes gains or losses in DNA content by mapping copy number changes of cellular DNA. The hybridization is detected with two different fluorochromes. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along target chromosomes. CGH is most often used in analysis of neoplastic cells from cancers.

Quantitative PCR

Quantitative real time polymerase chain reaction (RT-PCR) is used to detect, amplify, and simultaneously quantify a specific DNA sequence. The reaction progresses in real time, compared to standard PCR in which the product of the reaction is detected at the end of the procedure, and only qualitatively (yes-present; no-absent). Real-time PCR provides quantification of an absolute number of specific DNA sequence copies when normalized to DNA input or additional normalizing genes. Real-time PCR is often combined with reverse transcription to quantify mRNA and noncoding RNA in cells.

Direct-to-Consumer (DTC) Genetic Testing

DTC testing may not be performed with the same rigorous quality as medical grade testing for health care institutions. Ultimately, it is the consumer who may become the product, by giving over to unknown potential usage their individual information, including genetic data. Results returned are unlikely to provide useful information for health care decisions. Such testing is not performed to the level of quality needed for medical genetic testing. False positive results may occur, 40% for reported variants in a variety of genes. The risk for false negative tests results is harder to quantify. More ominously, DTC marketing may offer free genetic testing to fraudulently retrieve personal information, to potentially steal identify and perpetrate billing fraud.

Sources of False Positive and False Negatives in Genetic Testing

Attention to detail in performance of molecular diagnostic procedures is essential, because even tiny amounts of DNA or RNA can contaminate samples and lead to false positive results. This is particularly true for microbiologic work, where environmental contamination or cross-contamination from other samples must be avoided.

Attention to sample collection is essential to preserve genetic material and avoid false negative results. RNA degrades very quickly. DNA can persist for millenia in optimal environments, but over time DNA tends to become fragmented, with shorter sequences having less specificity. Samples may contain insufficient genetic material for analysis. Amplification inhibitors may be present in the sample, such as heme compounds.

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