PCR and the future of molecular testing.
DRAMATIC CHANGES in medical science have occurred during the last 10 years. One of the greatest of them has been the movement of molecular biology from the research to the clinical lab and even to the patient's bedside.
This shift from the exotic and speculative to the routine and practical has placed the clinical laboratory in a pivotal position: not only does it help diagnose diseases but in many cases it now actually defines their basic natures.
One development of the past decade, polymerase chain reaction (PCR), offers to revolutionize a vast array of scientific disciplines ranging from the study of prehistoric man to the rapid diagnosis of pathogenic organisms and disease states that are hard to target with conventional assays. This article explores the factors that have led us to this watershed point, reviews molecular techniques and their uses in and out of the clinical laboratory, and looks forward to the not-too-distant millennium.
* Double helix. Molecular pathology has its basis in the Nobel Prize-winning work of Watson and Crick, which was published in 1953. The knowledge of the structure of deoxyribonucleic acid (DNA), the "twisted rope ladder" that carries the genetic makeup and, with it, the destiny of every living thing, was followed by an explosion of new discoveries. Two of them are the synthesis of hormones that previously were obtainable only through painstaking tissue extraction, and the revelation of the molecular basis of a variety of diseases.
Today we can identify, in certain of those diseases, the actual molecular change that causes the visible clinical syndrome. An excellent example is the simple base substitutions--subtle alterations in the rungs of the familiar DNA ladder--that cause sickle cell anemia. Current progress is being made in identifying the genetic mutations that cause these changes. The goal is the use of gene therapy to cure or ameliorate sickle cell anemia and, eventually, other previously incurable conditions.
* What is disease? The changes being wrought by molecular biology are both dramatic and subtle, involving as they do the very definition of disease. For generations, diabetes has been defined by the abnormal glucose tolerance that is a result, rather than the cause, of pancreatic insufficiency.
Cystic fibrosis can be described by the clinical conditions that are associated with it and can be diagnosed by an abnormal sweat test that, again, reveals a consequence, not the cause, of the disease. Using today's molecular techniques, however, an individual can actually be tested to determine whether he or she carries the gene that causes the conditions observed. In that way we are able to move a number of steps inward--from the outward manifestations of an illness to the actual molecular cause. Thus informed, new therapies and diagnostic tools can be developed. Unfortunately, new ethical dilemmas will also develop; among them, the troubling social and psychological consequences of learning that you are destined to develop a potentially lethal disorder.
* Detection system. What is the basis of molecular tests? Typically they involve the isolation of specific strands of DNA or its cellular workhorse, ribonucleic acid (RNA). The DNA of Chlamydia, for example, would stand out as being quite different from that of an infected human host. Once localized and, in the case of the most promising new tests, amplified, some type of system is used to precisely identify the target material. "So what?" you say; you can identify pathogens now. But can you do it when the target specimen is one strand of DNA? A snippet of a strand? A snippet of a strand from a 5,000-year-old, mummified stone-age man? To cite a more practical example, what about using a piece of DNA from a drug-resistant strain of Mycobacterium to get, a few hours later, the diagnosis of an organism that would take weeks to culture otherwise?
At first glance, the new tests can appear exceedingly complicated. On the contrary, they are no more difficult to understand than RIA, ELISA, or other common laboratory methods. The actual techniques involved are familiar to most laboratorians. They include electrophoresis, fluorescence detection (using a microscope), spectroscopy, protein separation, and radiography. In molecular methods, multiple techniques are used for each analysis. Nonetheless, an automated test kit for Chlamydia that went on sale in June will seem familiar to any laboratory staff member who has used the aforementioned methods.
* Accessibility. Some tests are so complex or so infrequently ordered that they are performed only in research laboratories, greatly limiting their utility. The occasional arcane assay notwithstanding, more and more tests are being designed and packaged in a manner that allows them to be used by most clinical laboratories. This packaging involves the standardization of techniques to limit operator dependence and the use of a certain amount of automation to make the assays financially feasible.
The test employed in the new and burgeoning field of molecular pathology use methods that vary according to application. "Molecular pathology" is an umbrella term. Like "immunoassay," it covers a multitude of methods. There are a wide variety of formats, but each uses a common principle. Let's examine how some typical test formats work.
* Nucleic acid probes. One of the most common techniques involves the use of nucleic acid (NA) probes. DNA consists of two strands of nucleic acid that contain complementary pairs of bases. Think again of DNA as a ladder wherein each base pair is a rung. The strands match perfectly as long as the base pairs are complementary.
Nucleic acid probes are short segments of DNA or RNA that precisely match the bases in the strand of DNA or RNA that is their target. Probes will only combine with RNA or DNA that they match precisely. Send in a probe for, say, Chlamydia. If it hooks up with a target base pair, you can detect the combination and identify your piece of DNA or RNA as being from that bacterium.
* Slow-growing pathogens. One area where NA probes are used is in microbiology, where specimens are tested looking for certain known pathogens. Tuberculosis is a perfect example; the organism is very slow growing and can require weeks to identify using traditional growth methodology. By the time a traditionally based diagnosis has been made, the patient might have been released, perhaps to infect others.
A nucleic acid probe is much, much faster. The NA probe for tuberculosis will combine only with the RNA segment of TB that it matches. Since the likelihood of matching to RNA that is not from TB is quite low, specificity is high. The specificity of the tests can be further improved by carefully selecting the segment of nucleic acid that is used. Sensitivity also is high since one is able to detect a very small amount of bacterial RNA.
Once combined with the target DNA or RNA, some system of visualization is required. This is typically done by chemoluminescence, by fluorescent methods, or by the use of certain colorimetric compounds. Radioactive compounds have also been used, but the techniques have limited usefulness in the clinical lab.
* Hard to automate. What are the advantages of nucleic acid probes? For slow-growing organisms like TB, NA probes can cut weeks off the amount of time required for diagnosis by conventional methods. In other cases, like Chlamydia or gonorrhea, NA probes can identify an organism that is difficult to grow in many laboratories.
Why aren't probes used for all microbiology tests? For one thing, they are unnecessary in the many cases in which conventional methods are adequate. Furthermore, probe technology is far more expensive to perform so it is at a competitive disadvantage unless, as mentioned previously, time or growth difficulties are significant factors in the cost equation. Since they are not easily automated, NA probes are relatively difficult to perform and can require considerable skill to interpret. And the presence of DNA or RNA does not necessarily mean that the individual has an active infection, only that there is evidence of the organism. Nonetheless, nucleic acid probe technology is already in the lab and will play an increasingly important role as it is applied to more diseases.
* Polymerase chain reaction. Molecular pathology techniques have considerable advantage in the microbiology lab when it comes to looking for organisms that are found in extremely low numbers. This is because it is possible to use amplification methods that can take the DNA found in a single cell or virus and multiply it thousands of times.
Figure 1 lists the advantages of one amplification technique, polymerase chain reaction, which is the nearest thing recent science has produced to an Archimedean burst of excitement. And while maverick inventor Kary B. Mullis, Ph.D., didn't cry "eureka," he did pull his Honda Civic to the side of the winding, northern California mountain road that had inspired him, to make some calculations and ponder what he had wrought. The results of Mullis's 1983 midnight drive was sold by his employer, Cetus Corp. (Emeryville, Calif.), to Hoffmann-La Roche (Nutley, N.J.) in 1991 for $300 million.
Roche Molecular Systems (Branchburg, N.J.) was just granted FDA clearance to market its Amplicor PCR test kit for the detection of Chlamydia. In development are kits for infectious diseases, including tuberculosis, Chlamydia/gonorrhea, HIV-1 quantitative, and hepatitis C, and genetic disorders, including cystic fibrosis. Clinical trials are under way for a PCR test kit for the detection of HIV-1.
Simply put, PCR splits a portion of DNA's twisted rope ladder down the middle of the rungs, then builds new halves onto each side. Where at first there was one ladder, now there are two. Then PCR repeats the process, over and over, until there is a veritable forest of identical ladders. It takes 3 minutes to complete the first cycle and go from one to two strands of DNA. At that rate it takes less than 3 hours to complete 30 cycles and have in hand more than 100 million identical slices of DNA--more than enough specimen to study conclusively by any of the methods of molecular biology.
Figure 3 shows how the speed at which PCR operates has been reduced in its transition from the research to the clinical lab. Enough DNA is produced so that high-sensitivity radioactive probes aren't required; nonradioactive probes or stains suffice.
* Increased sensitivity. Amplification has many advantages. Since it can greatly increase the number of copies of the target DNA, quantities as small as a single viral particle can potentially be detected. Because of this, sensitivity is dramatically increased to levels unachievable with conventional detection methods.
Paradoxically, this sensitivity is also one of the disadvantages of amplification. Contaminants present in the specimen in minute quantities can be amplified to high levels, making it appear as if they are inordinately represented. This is usually not a problem with non-amplification techniques or conventional methods. To guard against contamination, labs often prepare and amplify DNA in separate areas. PCR assays that have been automated for the clinical lab suggest a one-way workflow covering three distinct lab areas and include an enzyme that erases contamination.
* Uses of PCR. Amplification techniques can be exceedingly useful. Figure 4 lists viral, bacterial, fungal, and parasitic diseases currently or potentially detectable with PCR.
One excellent example of the polymerase chain reaction at work is its ability to detect infection with HIV. The most commonly used tests detect antibody to the human immunovirus. Since antibodies develop at a variable time period after infection, there is a lag between infection and detection. Additional tests exist that can detect the presence of the HIV antigen. So doing reduces the lag, but it remains present.
Because it is exceedingly sensitive, amplification technology reduces the lag even more. That is especially important in the diagnosis of HIV infection in the newborn children of HIV-positive mothers. Early detection allows early intervention. In the case of neonates, PCR has an additional attraction: extremely low specimen volume.
Hepatitis B and C are candidates for this technology in the near future. Clearly, the capabilities of PCR are not needed for all or many diseases. They can be particularly helpful in specific instances.
While Roche's PCR technology currently dominates the field of DNA amplification, several other diagnostic firms are preparing their own versions. Becton Dickinson Diagnostic Instrument Systems (Sparks, Md.) is developing strand displacement amplification (SDA), while Abbott Diagnostics (Abbott Park, Ill.) is readying ligase chain reaction (LCR). Like SDA, the self-sustained sequence reaction system (SSSR) under development at Baxter Diagnostics (McGaw Park, Ill.), operates under isothermal conditions. Q-beta Replicase, under development at Gene-Trak Systems (Framingham, Mass.) is also isothermal.
While SDA and LCR are closer to clinical application than other alternatives to PCR, they remain several years away. Expect to see many more tests using amplification techniques, especially as automation enters the marketplace.
* In-situ hybridization. Nucleic acid probes are not only instrumental in the amplification of DNA; they are also useful in surgical pathology. One technology that may play a growing role is in-situ hybridization.
In this method, an NA probe is allowed to react with a tissue specimen on a specially prepared tissue slide. Fluorescent and colorimetric labels are most popular since they can be visualized directly. This allows the pathologist to examine the specimen and localize the area where the probe has combined.
How is this technique used? One use is the detection of the human papilloma virus (HPV) in cervical specimens. When the probe is applied to the slide preparation, it will combine with the virus if that organism is present. Direct visualization is then used to determine if HPV is the target organism. Another good example is the detection of cytomegalovirus (CMV) in liver biopsies from transplant patients.
Current practice involves looking at cells for a characteristic appearance. Using in-situ hybridization allows the pathologist to determine conclusively if the virus is present. If CMV is present, it will appear as a clearly defined spot in the cell. This allows the pathologist to directly correlate the appearance of the cells and the presence of the virus.
* Direct visualization. The major advantages of this method are quite clear. It allows direct visualization of the DNA in the tissue in which it is present, giving the most relevant information to the pathologist. In-situ hybridization is time-consuming, however, and requires special equipment and tissue preparations.
Its use is expanding greatly nonetheless. One popular area of expansion will be in genetics. Fluorescent in-situ hybridization has become increasingly useful in identifying specific areas of chromosomes. As the human genome is mapped, probes will be developed for these genes, leading to their localization on chromosomes. From that information, other tests can flow.
* Can't-do-without technology. Molecular pathology has already begun to provide the laboratory with the capabilities needed in the forthcoming decades. Their utility will be measured not in the number of assays performed but rather in the fact that certain diagnoses could never be made without the technology.
How widespread will molecular pathology become? That will depend on the degree of automation that is introduced and the types of tests that are available. Probe technology is clearly no longer just a research laboratory curiosity, but has become a bona fide clinical laboratory procedure. Yes, these tests are more costly today; expense limits their application to low volume, specialty settings. But this limitation will evaporate as volume increases and procedures are simplified. Such was the experience with immunoassays and therapeutic drug monitoring. History will likely repeat itself.
Will most microbiology tests be probe based? Conventional wisdom says no, but this may change because DNA probes hold the promise of being far more sensitive and rapid. Laboratorians have the unique experience of being--yet again--on the cutting edge of a technology that is revolutionizing medicine. The position will further enhance the role of the laboratory in the provision of patient care.
Advantages of PCR
* Rapid diagnosis. Results are available in as little as 2 hours. Speed is especially important when hard-to-culture organisms are involved. Drug-resistant tuberculosis can be determined before the patient is released back into the population.
* Extreme sensitivity. For example, PCR allows the differentiation between active hepatitis infections and those that are resolved. It also can differentiate between strains of a pathogen, for example, between Mycobacterium tuberculosis and M. avium, M. intracellulare, and other opportunistic organisms.
* Direct detection. PCR gives a reliable positive or negative result independent of the antibody response.
* Supplemental method. PCR can detect HIV in an asymptomatic, nonreactive patient specimen or in a culture-negative patient whose Western blot assay is indeterminate.
* Status monitoring. PCR enables clinicians to monitor cancer patients in remission because the technology can detect the changes in the genetic code responsible for transforming a normal cell into a malignant one.
* Tissue typing. PCR can search a specimen for a specific genetic sequence and amplify it, allowing the determination of tissue compatibility between donor and recipient. The same information can help determine parent-child relationships.
* Catalyst for improvement of existing diagnostic methods. PCR can be used to establish baseline levels of sensitivity against which all other methods can be measured.
* Minute specimen volume. This is especially important in the case of neonates and in forensics (testing blood, hair, and semen specimens, for example). Missing persons have been identified through PCR.
* Fragmented or trace specimens. Entomologists recently used PCR to amplify tiny remnants of DNA taken from a 30 million-year-old termite fossilized in amber. By comparing the ancient insect with its modern descendant, scientists may be able to correlate specific aspects of body size and shape with DNA sequences. This summer's moviegoers will remember that a fictional leap of imagination from such research produced the reconstituted dinosaurs in Jurassic Park. PCR can also target trace amounts of the genetic material of pathogens in blood, cells, water, food, and other clinical and environmental specimens.
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De Cresce is director of clinical laboratories, Rush-Presbyterian-St. Luke's Medical Center, and assistant professor of clinical pathology, University of Illinois, Chicago. Lifshitz is director of laboratories, New York University Medical Center, and clinical associate professor of pathology, NYU School of Medicine, New York City. The authors publish a monthly newsletter, "The Instrument Report."
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|Title Annotation:||polymerase chain reaction|
|Author:||De Cresce, Robert P.; Lifshitz, Mark S.|
|Publication:||Medical Laboratory Observer|
|Article Type:||Cover Story|
|Date:||Aug 1, 1993|
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