Multi-Analyte Profiling Using Suspension Arrays
By Michael Spain, MD
V irtually no disease results in just one laboratory abnormality, nor is there a single laboratory result that is 100 percent sensitive and 100 percent specific for a certain disease. Typically, when using a single marker, one can only increase sensitivity by sacrificing specificity and vice-versa. If one combines multiple tests into a disease algorithm, however, both specificity and sensitivity can be increased simultaneously. Historically, though, increased cost and specimen requirements make this impractical. This will become increasingly problematic as DNA testing is combined with immunoassay and enzymatic assays into logical test combinations.
Luminex Corp. (Austin, TX) has developed an enabling technology that allows up to 100 analytes to be performed simultaneously in the same reaction vessel (tube, microtiter well, etc.). This can be done at a cost comparable to single tests today without increasing sample size or sacrificing sensitivity or specificity.
The system uses fluorescently encoded microspheres (5.6 µm in diameter) that each carry distinct biochemical reactions. One hundred separately addressable microsphere sets can be constructed by using two fluorescent dyes internally within the microspheres at 10 potentially different fluorescent intensities each and then mixing them in all possible combinations. Once constructed, these sets can pass through the Luminex 100 instrument randomly where they travel single file through the two lasers. The first laser excites the internal dyes that are used to classify the microsphere set. The second laser excites a third fluorescent dye that is used to quantitate the biologic reaction taking place on the surface of that particular microsphere set (Figure).
The testing instrument is PC-sized and capable of analyzing any binding event whether immunoassay, nucleic acid-based, enzymatic or receptor-ligand. This degree of flexibility, combined with low cost, allows the clinical laboratory to adopt a workstation approach to testing. A single technologist can perform all ordered testing from the master specimen on a single instrument, thus eliminating the time and error rate associated with aliquotting.
Types of Testing
Autoimmune testing lends itself to Multi-Analyte Profiling (MAPing) particularly well, as logical panels have already been delineated. To perform this type of testing, specific nuclear antigens are coupled to specific microsphere sets; these sets are then mixed with patient sample. If the patient has specific IgG antibodies against any of the antigens, they will bind to that microsphere set. The entire reaction is developed by adding fluorescently labeled anti-IgG. Any antibodies present in the patient will be detected by the surface fluorescence on the distinct microsphere set.
In addition to routine testing, the system offers many advanced capabilities. Optimal capture reagents for the surface of the beads can be identified quickly and simply. For a single analyte, a different capture antibody can be coupled to each of 10 distinct microsphere sets. A limited amount of fluorescently labeled antigen is added to the mixture of the bead sets. The capture antibody with the highest avidity will surpass the other capture antibodies, thus displaying the greatest surface fluorescence. Optimal antibody pairs can be identified in a slightly different manner.
Internal controls can also be built into immunoassays to detect interferences such as Rheumatoid Factor. In addition to the specific assay microsphere sets in the reaction, one would add an extraneous set with non-specific Immunoglobulin G coupled to the surface. The secondary reagent, in addition to specific fluorescently labeled reporter, would also contain labeled anti-immunoglobulin M. Since Rheumatoid Factor is an IgM antibody with specificity against IgG, they will bind to the extraneous microsphere set if any are present and subsequently be detected by the fluorescently labeled anti-IgM. Similar techniques can be used to detect other heterophile antibodies, such as Human anti-Mouse (HAMA) and Human anti-Rabbit (HARA).
Likewise, internal control analysis can be combined with specific analyte analysis in the area of nucleic acid-based testing. If one measures an HIV viral load and uses polymerase chain reaction (PCR) amplification, an internal control must be included to verify that the PCR has worked correctly. Typically, the viral load and internal control are measured separately. With this new technology, these are analyzed simultaneously in the same tube.
Luminex has combined high-speed, digital-signal processing, microspheres and elements from flow analysis into an economical, simple-to-operate, flexible system. This enabling technology will radically alter the approach to laboratory medicine, restoring it to the preeminent diagnostic information center in the field of medicine.
Dr. Spain is medical director of Luminex Corp., Austin, TX.