Researchers studying chronic lymphocytic leukemia (CLL) observe that in some patients the disease is indolent, with limited growth over more than a decade, whereas in other patients the disease is more aggressive and becomes symptomatic early on. They find that genetic abnormalities can be detected in a majority of patients with CLL and may play a role in determining the difference between the 2 disease courses. A clinical study is conducted with the hypothesis that individuals with CLL with trisomy of chromosome 12 have a more favorable course than individuals with other genetic changes. Which of the following laboratory techniques would be most useful for identifying patients with this genetic abnormality?
Malignant cells typically contain a number of chromosomal translocations, deletions, or duplications that result in deregulation of gene expression and production of novel fusion proteins. Cytogenic analysis of tumor cells often demonstrates characteristic chromosomal abnormalities that may aid diagnosis, treatment selection, and estimation of prognosis.
Fluorescence in situ hybridization (FISH) is a highly sensitive cytogenetic test that can identify the presence of chromosomal duplications and large deletions and translocations. FISH probes are single-stranded DNA segments that are a few hundred kilobases in length; they are added to the cell of interest and anneal to complementary regions of the cell chromosome, which allows the area of interest to be identified visually.
FISH is far more sensitive than traditional karyotyping (chromosome banding) and can be applied to both dividing (metaphase) and nondividing (interphase) cells. It is often used to identify large structural chromosomal abnormalities such as the BCR-ABL1 fusion of t(9;22) in chronic myeloid leukemia. However, small chromosomal insertions and deletions cannot be identified by FISH (because the probe size is somewhat large), and chromosomal inversions will be missed because FISH can only identify the presence or absence of a chromosomal segment, not the order in which it is coded.
(Choice A) Enzyme-linked immunosorbent assay (ELISA) is used to quantify antibodies or antigens in a fluid.
(Choice B) Flow cytometry is often used in the diagnosis of leukemia. However, this laser-based test evaluates surface markers (eg, CD5) on individual cells, not chromosomal duplications.
(Choice D) Northern blotting is a technique that size-fractionates RNA in a gel and then targets a specific mRNA sequence of interest. It can determine the quantity and size of a particular mRNA section.
(Choice E) Western blotting is a technique that size-fractionates proteins in a gel and then targets a specific protein using an antibody with a radiotracer/enzyme. It is often used to confirm a positive ELISA test in the setting of HIV or Lyme disease infection.
Educational objective:
Fluorescence in situ hybridization (FISH) can identify specific chromosomal translocations, duplications, or deletions using a single-stranded, complementary DNA segment that is tagged with a radiotracer. FISH is rapid, highly sensitive and specific, and can be used on tissues with low mitotic rates. However, the targeted chromosomal abnormality must be somewhat large in order to be identified.