Methods used in the medical laboratory have both stayed the same and changed drastically and range from requiring no technology to high technology. This article will review some of the methods that are currently used in medical laboratories.
Agglutination
Agglutination is a procedure by which antigen-antibody complexes are detected without the use of a label by visualizing clumping when the antigen and antibody bind and form a complex that is insoluble. Antigen is detected in a patient specimen or on bacterial cells growing on an agarose plate by using an antibody- coated particle, such as bacterial antigen detection in cerebrospinal fluid and streptococcal serotyping. Alternatively, antibody is detected in the patient using antigen-coated particles, such as with the Treponema pallidum-Particle Agglutination assay (Fujirebio, Malvern PA).
Agglutination assays date back to 1896 when Gruber and Durham published their observations of clumping produced when serum was mixed with bacterial cells and formed the basis for the first test to detect typhoid fever.1 Agglutination assays are typically manual tests performed on a card, in a tube, or in microtiter plate wells. They are relatively simple procedures to perform and take from a few minutes to a couple of hours.
Particles used as carriers of antigens or antibodies include latex, gel, bacteria, or erythrocytes. Passive agglutination involves artificially putting an antigen onto a carrier, whereas active or direct agglutination is performed when the antigen is a normal component of the carrier. Staphylococcus aureus is the most common bacterial cell that is used as a carrier because Protein A expressed on the surface of the bacterium naturally binds to the Fc region of immunoglobulin, allowing for the binding of the antibody to the bacterial cell while leaving the Fab region of the immunoglobulin free to bind to the antigen. An agglutination method that uses S aureus as a carrier is called coagglutination. When erythrocytes are used as the carrier, the procedure is called hemagglutination. Blood-typing reactions are examples of direct agglutination because antibodies are used to detect antigens (A and B) that when present on the red blood cell are present naturally. The direct and indirect antiglobulin (or Coombs) tests are other examples of agglutination assays. In these tests though, antibody is being measured as either bound to the red blood cell (direct) or present in the serum (indirect).
Automation is not usually used to measure agglutination reactions, but nephelometry in which the amount of light scatter by particles is measured, has been used to measure agglutination and is then called a particle-enhanced immunoassay.
Enzyme immunoassay
Agglutination reactions have limited sensitivity. Sensitivity can be increased by binding a label to the antigen or antibody. The enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) were developed and first published in 1971.2,3 The development of assays using enzymes as labels was modeled after Yalow and Berson’s radioimmunoassay (RIA) that used radioactive molecules to tag an antigen or antibody. The RIA was first described in 1960 as a method for measuring insulin in plasma.4 Interestingly, the early applications of RIA and EIA were home-brew assays developed by individual researchers who were working ahead of the commercial manufacturers — which is what we see today with molecular-based tests.
Manufacturers have fully automated EIA procedures to the point where a technologist is only needed to accession the specimen into a laboratory information system and put the specimen onto the analyzer for processing and analysis. There are many different formats for the assay including homogeneous, heterogeneous, sandwich, and competitive. Horseradish peroxidase and alkaline phosphatase are the most commonly used enzymes. The substrates converted by the enzymes can either produce a color change measured spectrophotometrically or give off light (chemiluminescence) as measured by a luminometer. Analytes detected by EIA range from antibodies and antigens of microorganisms to human hormones.
Not only are EIAs based in diagnostic laboratories on large, complex instruments, but point-of-care rapid tests use EIA methods. Often, these tests involve use of a membrane in a cartridge and movement of antigens, antibodies, or antigen-antibody complexes across the membrane in a chromatographic flow toward a detection zone where a color change in the membrane indicates a positive result. For example, rapid EIA tests are available for the detection of influenza virus antigens, anti-HIV-1 antibodies, troponin for heart attacks, Streptococcus pyogenes, and human chorionic gonadotropin for pregnancy.
Nucleic-acid amplification
The future growth of diagnostic testing is in the development of assays that detect nucleic acid. For most of these assays, the nucleic acid is amplified and then detected as first described by Kary Mullis in 1987.5 The polymerase chain reaction (PCR) involves replicating DNA in vitro in the same method as the cell replicates DNA in vivo using template DNA, RNA primers, nucleotide bases, and DNA polymerase. To start the process, double-stranded template DNA is denatured to form single strands, primers that are specific for the target gene of interest anneal to the template, DNA polymerase then adds nucleotides that are complementary to the template strand, thereby copying the template and making double-stranded DNA. The process starts again when the double-stranded DNA is denatured. Temperature changes drive the reaction from step to step.
From the original description of PCR have come variations such as:
- real-time or quantitative for measuring the amount of starting DNA present in the sample;
- reverse transcriptase for amplifying RNA;
- multiplex for amplifying more than one target strand of DNA at a time;
- amplification of variable number of tandem repeats (VNTR) and short tandem repeats (STR) is used to analyze a genome and is the basis of DNA fingerprinting databases like CODIS;
- touchdown PCR in which the annealing temperature decreases in later cycles to increase the amount of amplification as the assay proceeds; and
- methylation-specific PCR which is used to measure methylation of the DNA at cytosine-guanine islands.
PCR is an example of target amplification where the amount of DNA in the sample is increased to a level that is detectable. Other target-amplification procedures include transcription-mediated amplification, nucleic-acid sequence-based amplification, and strand displacement amplification.6,7 Another type of amplification method is a signal amplification procedure in which the target nucleic acid is bound to a large chemiluminescent signal that is quantitated. Hybrid capture and branched DNA assays are both types of signal amplification procedures.
When PCR was first described, like other diagnostic assays, it involved several manual steps from isolation of the nucleic acid to detection of the product. The thermal cycler was the first instrument developed for use in PCR reactions that automates the temperature changes. In the last few years, the product detection has been automated by detecting the product as it is made; and, finally, the most laborious part of the procedure, nucleic-acid isolation, has been automated. Now, the whole procedure has been automated from beginning to end to the point where performing a molecular-based assay is similar to performing EIA procedures.
Applications of molecular-based assays are only limited by the genome of the organism being studied. Any gene can be amplified if primers are made that are complementary. Thus, molecular-based tests are being used to detect infectious disease, mutations resulting in cancer or inherited disease, and identity.
Given the potential of molecular-based tests, the question remains if they will replace all other methods available in the medical lab of the future. Maybe yes, maybe no.
Maribeth Laude Flaws, PhD, SM(ASCP)SI, is associate chairman and associate professor of the Department of Medical Laboratory Science at Rush University Medical Center in Chicago, IL.
References
- Gruber M, Durham HE. Eine neue Methode zur raschen Erkennung des Choleravibrio und des Typhusbacillus. Münchener medicinische Wochenschrift. 1896;43:285-286.
- Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971;8:871-874.
- Van Weemen BK, Schuurs AHWM. Immunoassay using antigen-enzyme conjugates. FEBS Letts. 1971;15:232-236.
- Yalow RS, Berson SA. Immunoassay of endogenous insulin in man. Clin Invest. 1960;39:1157-1175.
- Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335-350.
- Ginocchio CG. Life beyond PCR: alternative target amplification technologies for the diagnosis of infectious diseases, part 1. Clin Micro News. 2004;26:121-128.
- Ginocchio CG. Life beyond PCR: alternative target amplification technologies for the diagnosis of infectious diseases, part 2. Clin Micro News. 2004;26:129-136.
