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IVD Technology Magazine
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Originally Published July 2000

A workstation approach to bioassays

New technology with built-in quality control uses laboratory space and employee time efficiently and removes sources of testing error.

Manufacturers of IVD instrumentation continue to expand test menus in order to improve the efficiency of medical testing. However, two key areas of laboratory medicine remain problematic.

In the Luminex¹⁰⁰ system, color-coded microspheres pass lasers which illuminate the colors inside and on the surface of each microsphere. Advanced optics and digital signal processing translate the signals into real-time, quantitative data.

One problem area is laboratory compartmentalization. Dedicating particular instruments and technologists to the performance of certain types of testing with limited throughput results in inefficiencies of both labor and capital expenditure. The specimen aliquotting required in such compartmentalized structures can also be a main source of laboratory error. Ideally, laboratory testing should be done via a workstation approach, wherein a single technologist uses the master patient specimen to perform virtually all of the assays ordered by the physician.

A second area of concern is quality control. Laboratories use all available tools in order to maximize the accuracy of patient results, typically employing them on a per-run basis. While this practice provides a good measure of overall assay and instrument performance, it cannot detect either minor day-to-day shifts in a standard curve or the serum effects introduced by individual specimens. Small shifts in the standard curve may result in large numbers of false positives. In certain critical tests, such as tumor markers, this can have a major impact on patient care and health expenditures. Serum effects caused by rheumatoid factor or heterophile antibodies cannot be detected by gross specimen examination yet can adversely affect the test result. Such other conditions as lipemia, hemolysis, or bilirubinemia can be seen and noted, but their effect cannot be measured. Reagent addition failure, while rare, can also have devastating results.

A versatile bioassay technology has been developed that allows immunoassays, nucleic acid testing (NAT), and enzymatic assays to be performed on the same instrument. This multifunctionality minimizes the need for specimen aliquotting, enhances labor productivity, and maximizes return on capital expenditure.

LabMAP from Luminex Corp. (Austin, TX) makes possible simultaneous analysis of up to 100 analytes on a single specimen, allowing internal standard curves and tests for rheumatoid factor and heterophile antibodies to be performed on every patient sample. A system using LabMAP can cost-effectively meet the needs of a laboratory running 100 tests a day or those of a facility carrying 1000 times that work load.

System Technology

LabMAP bioassays are built on the surfaces of polystyrene microspheres that have been encoded by means of a proprietary dyeing process. The core of each sphere is impregnated with two different fluorescent dyes. Each dye can have any of 10 possible levels of fluorescence intensity. When a red diode laser in a specialized analyzer illuminates a dyed microsphere, the sphere's fluorescent signature identifies it as a member of one of the 100 possible sets. A green laser in the analyzer simultaneously excites a third fluorescent dye on the surface of the microsphere which quantifies the bioassay. Digital signal processing provides real-time data acquisition and analysis at up to 5000 microspheres per second, supporting as many as 400,000 analyte measurements a day.

LabMAP technology is performed by means of the Luminex¹⁰⁰ system, which consists of a Luminex¹⁰⁰ analyzer, associated software, fluorescently encoded microspheres, and a sample handler (see Figure 1). The standard front-end sample handler, called the XY platform, offers unattended 96-well capability with a throughput of 4000 samples per day. Anywhere from 1 to 100 analytes can be measured per sample; therefore, daily data points can range between 4000 and 400,000 under full-scale operation.

Figure 1. Using the Luminex¹⁰⁰ analyzer, LabMAP assays can simultaneously analyze up to 100 analytes on a single specimen.

A second liquid-handling system, named the HTS, is being developed for applications requiring higher sample throughput of smaller panels. This front-end handler enables users to sample simultaneously as many as eight wells from 96- or 384-well microplates in a process known as confluent sample analysis (CSA). In CSA, each sample, or well, is effectively labeled by the fluorescent signatures of the microsphere sets used for the bioassays. By sampling up to eight wells at a time, system users can increase the rate of sample analysis from 4000 to 32,000 per day.

Applications

The broad applicability of LabMAP technology is demonstrated by its recent use in allergy testing, screening for Factor V Leiden and cystic fibrosis, and profiling the response to a pneumococcal vaccine.

Allergy testing today is at best semiquantitative, with results reported in artificial units rather than concentrations of allergen-specific IgE. In a quality control application involving LabMAP, six different amounts of IgE are coupled to six distinct microsphere sets so that, when fluorescently labeled anti-IgE is added, the microsphere sets act as an internal standard curve (see Figure 2). The microspheres are part of the reagents added to every patient sample. Results are thus reported in units of IgE. Also, if a nonspecific serum effect is caused by a particular patient sample, resetting the standard curve will compensate for it.

Figure 2. Six-point internal standard curve for IgE.

In order to detect specific serum effects, other sets of microspheres are coupled with nonspecific human IgE, mouse IgE, rabbit IgE, and so on. Fluorescently labeled anti-IgM and IgG are added along with the labeled anti-IgE. If the sample contains rheumatoid factor, then the microsphere set having human IgG on its surface will fluoresce. If human antimouse or antirabbit antibodies are present, then those prepared microsphere sets will fluoresce.

These quality control measures would typically be used to detect the presence of such interferents rather than to quantitate them, although quantitation is certainly possible. Two distinct sets of microspheres carrying no biologic activity would be used to verify that capture and reporter reagents have been added, while an albumin assay could verify addition of the patient sample (see Figure 3).

Figure 3. Schematic of internal quality control for detection of rheumatoid factor, human antimouse antibodies, and patient-sample addition, as well as a one-point internal standard for prostate-specific antigen (PSA).

LabMAP facilitates high-throughput allergy testing. At a very large allergy testing laboratory, a panel of 11 inhalant allergens accounts for almost 80% of the test volume. Using the LabMAP system, these 11 allergens were each coupled to a distinct microsphere set and combined in a single master reagent lot. An aliquot of the master lot was mixed with patient sample and incubated. Allergen-specific IgE bound to the microspheres was detected with a fluorescently labeled anti-IgE reporter antibody. The degree of fluorescence on each microsphere was directly proportional to the amount of allergen-specific IgE in the patient's serum (see Figure 4). It is expected that the HTS system now in development will be able to generate more than 150,000 allergy assay data points per day.

Factor V Leiden is a single-point mutation leading to the incorporation of an arginine rather than a glutamine at amino acid position 506 (R506Q) in the Factor V protein. This amino acid substitution prevents the cleavage of the peptide bond at amino acid 506 by activated protein C that would normally inactivate the coagulation factor. Patients who carry the Factor V Leiden mutation in either heterozygous or homozygous form are at higher risk for deep venous thromboembolism. Factor V Leiden is the most common cause of inherited thrombophilia and, as such, ought to be a routine in the molecular diagnostic workup. But because current screening methods tend to be labor intensive as well as time intensive, routine screening for Factor V Leiden is uncommon.

A LabMAP-based rapid screening assay for Factor V Leiden was developed, and it provides genotype results within 15 minutes after amplification. Patient DNA is amplified by the polymerase chain reaction to produce labeled amplicons representing the mutation site in the Factor V gene. The amplified material is then hybridized to LabMAP microspheres bearing oligonucleotide capture probes specific for the mutant Factor V Leiden and normal alleles of the gene and analyzed in the Luminex¹⁰⁰ system. Genotype results are clearly distinguished (see Figure 5).

Figure 5. Patient samples and controls typed for Factor V Leiden and normal sequence. A total of 50 samples of known genotype were typed in triplicate on separate days at a national laboratory. Results were 100% concordant with those achieved
with an established
gel-based method.

To investigate the utility of the LabMAP approach with respect to a more complex system, a multiplexed assay to detect normal and mutant DNA sequences in the cystic fibrosis transmembrane conductance regulator (CFTR) gene was constructed. The mutations selected consisted of both single-base changes and small deletions. They are the five most common cystic fibrosis (CF) mutations found in North America, representing more than 73% of CF cases in this population.

Regions of subject DNA containing the mutation sites were amplified by the polymerase chain reaction and hybridized to 10 capture probes complementary to the five mutant and five corresponding normal sequences on 10 different microsphere sets. After five minutes of incubation, genotype results were produced by analysis of the microsphere mixture using the Luminex¹⁰⁰ system. The assay correctly identified the CFTR genotype for all patient DNA samples tested (see Figure 6). Cystic fibrosis affects approximately 1 in 3800 newborns. Routinely screening infants for CF mutations by means of a LabMAP system would create opportunities for early intervention, which in turn could reduce CF morbidity and mortality and the healthcare costs associated with patient treatment.

Figure 6. Four DNA samples of known cystic fibrosis genotype were analyzed for five mutant and five normal DNA sequences using the Luminex¹⁰⁰ system. Sample number one was heterozygous at two alleles. The other three samples were heterozygous at a single allele.

Figure 7. Analysis of the immune antibody response to the 23 capsular polysaccharide antigens present in a multivalent pneumococcal vaccine. The immunization index was determined by dividing the postimmunization response by the preimmunization response.

Another LabMAP application for discussion here involves Streptococcus pneumoniae, which continues to be a leading cause of bacterial meningitis and pneumonia in high-risk individuals in the United States. Immunization against S. pneumoniae with the licensed pneumococcal vaccine containing 23 serotype-specific pneumococcal capsular polysaccharides (PPSs) has been demonstrated to induce protective levels of anti-PPS antibody; various vaccine efficacy studies have found vaccine-induced protective immunity to be about 60 to 75%.¹ Laboratory methods now used to measure anti-PPS antibody include radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Although both of these techniques provide a quantitative measurement of this antibody, they require that all of the serotype-specific anti-PPS antibodies be determined individually —a total of 23 individual tests for each serum sample.

Unique microsphere sets are colorcoded using a blend of fluorescescent intensitites of two dyes to provide a matrix of 100 possible bead sets.

A multianalyte profile was developed for LabMAP, to detect anti-PPS antibodies to 23 pneumococcal serotypes simultaneously in a single sample. Each PPS was coupled to a distinct microsphere set and reacted with pre- and postimmunization sera from patients (see Figure 7). An immunization index was calculated for each PPS by dividing the postimmunization response by the preimmunization response. Results are readily obtained for all serotypes of PPS currently present in the vaccine.

Conclusion

The workstation approach to laboratory testing described in this article, in which a single technologist performs virtually all testing on a single instrument, can maximize operational efficiency while minimizing testing errors. Since NAT and immunoassay patient results derive from a single station, better data correlation can be achieved. Quality control built into every assay as opposed to every run should improve patient care while lowering overall healthcare costs.

Other LabMAP applications described elsewhere include screening for single nucleotide polymorphisms (SNPs); measuring of cytokines, and thyroid hormone levels; cystic fibrosis screening genetic human lymphocyte– antigen (HLA) typing; and kinase testing.^2–9 These diverse procedures have all been performed on the Luminex¹⁰⁰ system characterized above, demonstrating the throughput potential and flexibility of LabMAP technology.

References

1. Physicians' Desk Reference, 53rd ed. (Montvale, NJ: Medical Economics, 1999).

2. M Iannone et al., "Multiplexed Single Nucleotide Polymorphism Genotyping by Oligonucleotide Ligation and Flow Cytometry," Cytometry 39 (2000): 131–140.

3. J Chen et al., "A Microsphere-Based Assay for Multiplexed Single Nucleotide Polymorphism Analysis Using Single Based Chain Extension," Genome Research 10 (YEAR): 549–557.

4. D Vignali et al., "Simultaneous Quantitation of 15 Cytokines Using a Multiplexed Flow Cytometric Assay," Journal of Immunological Methods 227 (1999): 41–52.

5. K Kellar, "Multiplexed Fluorescent Bead–Based Immunoassays for Quantitating Human Cytokines in Serum and Culture Supernatants" (poster presented at the 14th annual meeting on Clinical Applications of Cytometry, Palm Springs, CA, September 1999).

6. R Colinas et al., "Multiplexed Genotyping of Beta-Globin Variants from PCR-Amplified Newborn-Blood-Spot DNA by Hybridization with Allele-Specific Oligonucleotides Coupled to an Array of Fluorescent Microspheres," Clinical Chemistry 46, no. 7 (2000): (in press).

7. S Dunbar, "Aplication of the Luminex LabMAP in Rapid Screening for Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Gene" (poster presented at the AACC Oak Ridge Conference, Boston, May 2000).

8. RJ Fulton et al., "Advanced Multiplexed Analysis with the Flowmetrix System," Clinical Chemistry 43, no. 9 (1997): 1749–1756.

9. K Oliver et al., "The Luminex Lab MAP System: A Rapid Homogeneous, Multianalyte Platform" (poster presented at the Society for Biomolecular Screening Meeting, Edinburgh, UK, September 1999).

Michael Spain, MD, is medical director and Ralph McDade, PhD, is vice president for scientific affairs at Luminex Corp. (Austin, TX).