Laboratories use a variety of methods to perform the vast range of tests needed for diagnosis, monitoring treatment and managing your health. Understanding the method used for a test provides a useful context for understanding your test results.
Laboratory methods are based on established scientific principles involving biology, chemistry and physics, and encompass all aspects of the clinical laboratory from testing the amount of cholesterol & glucose in your blood, to investigating your DNA, to growing infectious organisms and to observing cell integrity with a microscope. Pathology testing is one of the most regulated areas of medicine and stringent quality control systems are always in place. Test methods specifically define the procedures or processes that are to be used. The laboratory scientist carefully follows step-by-step procedures until the end product, a test result, is achieved.
Some methods are much more complicated and labour-intensive than others and require varying degrees of expertise. Often, more than one method can be used to test for the same substance. A test may be performed differently by different laboratories, a fact that is crucial when comparing test results.
are proteins produced by the immune system to recognise, bind to, and neutralise foreign substances in the body. Immunoassays are tests based on the very specific binding that occurs between an immunoglobulin (called an ) and the substance that it specifically recognises (the foreign molecule, called an ). Immunoassays can be used to test for the presence of a specific antibody or a specific antigen in blood or other fluids.
When immunoassays are used to test for the presence of an antibody in a blood or fluid sample, the test contains the specific antigen as part of the detection system. If the antibody being tested for is present in the sample, it will react with or bind to the antigen in the test system and will be detected as positive. If there is no significant reaction, the sample tests negative. Examples of immunoassay tests for antibodies include Rheumatoid Factor (which tests for the presence of autoimmune antibodies seen in patients with rheumatoid arthritis) or antibodies made in response to a (such as tests for antibodies to Hepatitis B to assure that the vaccination was successful).
When immunoassays are used to test for the presence of antigens in a blood or fluid sample, the test contains antibodies to the antigen of interest. The reaction of the antigen that is present in the person's sample to the specific antibody is compared with reactions of known concentrations and the amount of antigen is reported. Examples of immunoassay tests for antigens include drug levels (like digoxin, vancomycin), hormone levels (like insulin, TSH, oestrogen), and cancer markers (like PSA, CA-125, and AFP).
(© 2006). Immunoassay Detection Technologies, Chapter 2. Abbott Diagnostics Scientific Resources Learning Guide [On-line information]. PDF available for download through http://www.abbottdiagnostics.com.
Clarke, W. and Dufour, D. R., Editors (2006). Contemporary Practice in Clinical Chemistry, AACC Press, Washington, DC. Harris, N. and Winter, W. Chapter 10, Immunoassays. Pp. 117-119.
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is a laboratory method used for making a very large number of copies of short sections of DNA from a very small sample of genetic material. This process is called "amplifying" the DNA and it enables specific genes of interest to be detected or measured.
DNA is made up of repeating sequences of four bases – adenine, thymine, guanine, and cytosine. These sequences form two strands that are bound together in a double helix structure by hydrogen bonds (like a spiral staircase). Each half of the helix is a complement of the other. In humans, it is the difference in the sequence of these bases on each strand of DNA that leads to the uniqueness of each person's genetic makeup. The arrangement of the bases in each gene is used to produce RNA, which in turn produces a protein. There are about 25,000 genes in a human genome, and expression of these genes leads to the production of a large number of proteins that make up our bodies. The DNA of other organisms such as bacteria and viruses is also composed of thousands of different genes that code for their proteins.
How is the method performed?
PCR is carried out in several steps or "cycles" in an instrument called a thermocycler. This instrument increases and decreases the temperature of the specimen at defined intervals during the procedure.
The first step or cycle of PCR is to separate the strands of DNA into two single strands by increasing the temperature of the sample that contains the DNA of interest. This is called "denaturing" the DNA.
Once the strands separate, the sample is cooled slightly and forward and reverse primers are added and allowed to bind to the single DNA strands. Primers are short sequences of bases made specifically to recognise and bind to the section of DNA to be amplified, which are the very specific sequence of bases that are part of the gene or genes of interest. Primers are called "forward" and "reverse" in reference to the direction that the bases within the section of DNA are copied.
After the two primers attach to each strand of the DNA, a DNA enzyme (frequently Taq polymerase) then copies the DNA sequence on each half of the helix from the forward to the reverse primer, forming two double stranded sections of DNA, each with one original half and one new half. Taq polymerase is an enzyme found in a bacterium (Thermues aquaticus) that grows in very hot water, such as in geysers or hot springs. Polymerases copy DNA (or RNA) to make new strands. The Taq polymerase is especially helpful for laboratory testing because (unlike many other enzymes) it does not break down at very high temperatures needed to do PCR.
When heat is applied again, each of the two double strands separate to make four single strands and, when cooled, the primers and polymerase act to make four double strand sections. The four strands becomes eight in the next cycle, eight become sixteen, and so on.
Within 30 to 40 cycles, as many as a billion copies of the original DNA section can be produced and are then available to be used in numerous molecular diagnostic tests. This process has been automated so that a billion copies of the original DNA can be produced within a few hours.
How is it used?
This method can be used, for example, to detect certain genes in a person's DNA, such as those associated with cancer or genetic disorders, or it may be used to detect genetic material of bacteria or viruses that are causing an infection.
These are just a few examples of laboratory tests that use PCR:
Real-time PCR is similar to PCR except that data are obtained as the amplification process is taking place (i.e., "real time") rather than at a prescribed endpoint and shortens the time for the test from overnight to a few hours. This method is used to measure the amount of DNA that is present in a sample.
RT – PCR (Reverse Transcriptase PCR)
This method uses PCR to amplify RNA. RNA is a single stranded nucleic acid molecule and needs to be made into DNA before it can be amplified. The addition of a new strand that is the complement of RNA is achieved by the enzyme called Reverse Transcriptase (RT) and an antisense (reverse) primer. The primer binds to the single stranded RNA and the enzyme RT copies the RNA strand to make a single stranded DNA, which it then copies to make a double stranded DNA molecule. The double stranded molecule can now be amplified by PCR. Detection can also be by real-time methods.
Here are two examples of laboratory tests that use RT-PCR:
(February 27, 2012). Polymerase Chain Reaction (PCR). National Human Genome Research Institute [On-line information]. Available online at http://www.genome.gov/10000207 through http://www.genome.gov. Accessed August 2012.
Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Burtis CA, Ashwood ER, Bruns DE, eds. St. Louis: Elsevier Saunders; Fifth edition, 2011, Pp 1412-1413.
Clarke, W. and Dufour, D. R., Editors (2006). Contemporary Practice in Clinical Chemistry, AACC Press, Washington, DC. Pp 135-137.
Fluorescence in situ Hybridisation (FISH)
Fluorescence in situ hybridisation (FISH) is a molecular testing method that uses fluorescent probes to evaluate genes and/or DNA sequences on chromosomes.
Humans normally have 23 pairs of chromosomes: 22 pairs of non-sex-determining chromosomes (autosomes) and 1 pair of sex chromosomes (XX for females and XY for males). Chromosomes are made up of DNA, repeating sequences of four bases that form the thousands of genes that direct protein production in the body and determine our physical characteristics. DNA consists of two strands bound together in a double helix structure (like a spiral staircase). Each half of the helix is a complement of the other.
For a FISH test, a sample of a person's cells containing DNA is fixed to a glass slide. Samples can include blood, bone marrow, amniotic fluid, or tumour cells, depending on the clinical indication. The slides with the "target" (person's) DNA are heated to separate the double strands of DNA into single strands. Fluorescent probes are then added to the sample. Fluorescent probes are sections of single-stranded DNA that are complementary to the specific portions of DNA of interest. The probe, which is labeled with a fluorescent dye, attaches to the specific piece of DNA. When the slides are examined using a special microscope, the genes that match the probe can be seen as areas of fluorescence, which will appear as bright spots on a dark background.
This technique can be used to show the presence of extra gene copies (duplicated or amplified genes), and genetic sequences that are missing (gene deletions) or have been moved (translocated genes). Increased numbers of chromosomes, as seen in certain genetic disorders, are also diagnosed using FISH technologies (trisomy 21 or Down syndrome, for example). The targeted area(s) or sequences of DNA are determined by the probes that are used. Multiple targeted areas in the DNA can be assessed at the same time using FISH probes labeled with a number of different fluorescent dyes.
The following photographs show cells that have been evaluated using the FISH methodology. These are just a few examples of the use of FISH technique.
In the figure below, FISH testing is applied to cells in amniotic fluid, obtained from a pregnant woman carrying a baby suspected of having Down syndrome (trisomy 21). Three copies of chromosome 21 are observed (red signals). The green signals (two copies) are for chromosome 13; these are for control purposes and show that the test is working properly. FISH supports a clinical diagnosis of trisomy 21. The doctors and genetic counsellors will work with the woman to help her understand the results of the test.
In the figure below, FISH is used to assess breast tumour cells for the presence of an amplified gene, HER-2/neu (red signals). In approximately 25% of breast cancers, HER-2/neu is amplified. Women with amplified HER-2/neu tumours are treated with a drug (Herceptin) that targets the protein that is the product of the abnormal gene. If a woman is NOT positive for HER-2/neu amplification, she is not likely to receive any therapeutic benefit from Herceptin therapy and other drugs are considered.
The figure below, shows FISH used in a particular type of chronic leukaemia, chronic myelogenous leukaemia (CML). The specific probes used in this case detect BCR-ABL, an abnormal gene sequence formed by the translocation of a portion of chromosome 22 (BCR, a green probe) with a portion of chromosome 9 (ABL1, a red probe). The areas of yellow fluorescence signify the abnormal, fusion gene (joining of red and green probes). Finding the BCR-ABL fusion confirms a diagnosis of CML. BCR-ABL positive patients receive benefit from molecular-targeted drugs, such as imatinib.
(August 16, 2010) Fluorescence In Situ Hybridization (FISH). National Human Genome Research Institute [On-line information]. Available online at http://www.genome.gov/10000206 through http://www.genome.gov. Accessed March 2011.
(August 16, 2010) Frequently Asked Questions about Genetic Testing. National Human Genome Research Institute [On-line information].Available online at http://www.genome.gov/19516567 through http://www.genome.gov. Accessed March 2011.
(March 6, 2006) Genetics Home Reference. Fluorescent in situ hybridization. Available online at http://ghr.nlm.nih.gov/glossary=fluorescentinsituhybridization through http://ghr.nlm.nih.gov. Accessed March 2011.
(June 29, 2011) Hiller B, Bradtke J, Balz H and Rieder H (2004). CyDAS Online Analysis Site. Available online at http://www.cydas.org/OnlineAnalysis/ through http://www.cydas.org. Accessed July 2011.