.
Flow Cytometry:
- Start with a sample of the fresh tissue biopsy
- Mince into a cell suspension and aliquot into small tubes- each tube of cells can be labeled with eight different markers
- Cells are labeled with various antibodies marked with different fluorochromes
- Cell samples are placed in the flow cytometer, which interrogates the cells (about 10,000 cells per second)
- The instrument determines which color of the fluorochrome is on which cell, which is dictated by the antibody
- If a cell expresses, for instance, CD10, it will fluoresce in a certain color and a cell which expresses CD20, will fluoresce in another color.
- Will also test for Kappa and Lambda light chain ratios
Example of CD5+/ CD10- B-cell lymphoma, solely expressing Lambda. This is indicative of a mantle-cell lymphoma. Click image to enlarge.
Real Time Polymerase Chain Reaction (RT PCR):
RT PCR is fast becoming one of the most popular techniques for diagnosing lymphoma due to several advantages. One, it has high sensitivity making it possible to detect even a single molecule. This makes diagnostics possible with much smaller tissue samples than required by other techniques. This benefits the patient in that FNA can be used to replace surgical biopsies, which would allow more samples from various areas to be collected and it would not confer as much stress on the patient. Another advantage of RT PCR is that gives quantitative results. This is important in cancer diagnosis because it allows for the possibility of classifying cancers into more distinct groups based on overall expression patterns and amounts. It is not only a good tool for predicting type of cancer but may also predict the survivability of the patient based on commonality of genes present in cancer survivors. The following video demonstrates PCR, which is accompanied by further steps below for an overview of the process.
The following recaps the steps of PCR from the video and includes steps for RT analysis :
- mRNA is copied to cDNA using reverse transcriptase
- cDNA is denatured (by heating to nearly boiling) so the two strands of DNA separate. It is then cooled
- Primers are affixed that are complementary to a site on each strand
- Temperature is raised again (72 degrees) and heat-stable taq DNA polymerase extends the DNA from the primers. The DNA has been duplicated
- It is denatured again and the cycles continue to be performed
- During this process a combination of sequence-specific and non-specific fluorescent labels are used to tag DNA (this provides the real-time analysis)
- Non-specific labels are DNA-binding dyes that fluoresce in proportion to the amount of double-stranded products formed
- Sequence-specific probes are tagged to specific DNA sequences in order to show amplification and expression of the specific genes contained in those regions of DNA
- The fluorescence is interrogated by a computer in between each cycle of DNA duplication (hence, the real time application), displaying not only the expression of genes, but quantity of expression as well
lymphoma 