IVD Technology Magazine
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Originally published January, 1998

Multiplexed testing for disease-marking synergies

Michael Spain

A high-tech combination of microspheres, flow cytometry, and high-speed digital processing is making multiple analyte quantitation a reality.

In the Flowmetrix system, a single sample can contain up to 64 discrete microsphere sets differentiated by their unique red-orange fluorescence. Each set carries the reactants of a distinct bioassay. Illustration by Keith Kasnot

In the human body, an effective immune response to even the simplest antigen requires the properly orchestrated activity of tens, if not hundreds, of distinct molecules including cytokines, enzymes, and eventually antibodies. This complex system of biochemical control employs thousands of biomolecules to coordinate the many processes necessary for proper function. Disease, however, alters the balance of these control elements, and it is this deviation from normal that forms the basis of much of today's diagnostic laboratory medicine.

Current laboratory methods often look at only one piece of this complex biochemical puzzle. Rarely, though, are diseases confined to a single, isolated molecular abnormality. The predictive value of diagnostic testing is increased significantly when it supplies many relevant pieces to the puzzle. For example, maternal alpha-fetoprotein (AFP) was initially used as a screen for fetal neural tube defects. It was subsequently discovered that AFP could identify approximately 20% of fetuses with Down's syndrome. Unfortunately, a high false-positive rate and low predictive value made reliance on AFP alone of questionable benefit. When combined with human chorionic gonadotropin and free estriol however, approximately 70% of fetuses with Down's syndrome could be identified prenatally.¹ It is likely that other markers will be discovered which could raise this percentage even higher.

Similar examples of synergistic testing exist throughout laboratory medicine, and more will be developed as additional biochemical interrelationships are described. However, the economic environment of today's clinical laboratory clearly demands cost-efficient testing. Future in vitro diagnostics must deliver the advantage of advanced multiple analyte quantitation in a simple, cost-effective format. To meet this demand, Luminex Corp. (Austin, TX) has developed a flow cytometry—based approach to multiplexing assays, called the Flowmetrix system.

System Description

Flowmetrix is a unique platform currently capable of performing 64 different assays in a single specimen aliquot using a flow cytometer and advanced digital signal processing hardware and software.^2—4 The system depends on the signal processor's ability to classify polystyrene beads (microspheres) dyed with distinct proportions of red and orange fluorophores when illuminated by the flow cytometer (see Figure 1). The reagents of each separate assay are allocated to the individual sets of microspheres, all 5.5 µm in diameter. A green fluorescent reporter molecule is used for quantitation of the analyte. The flow cytometer is gated at 5.5 µm so that only fluorescence associated with the bead is measured, eliminating the need for wash steps. This also eliminates any interferences caused by icteric, hemolytic, or lipemic samples. In fact, assays performed in whole blood are not affected by cellular elements, since few components will enter the 5.5-µm gate and none will have the unique combination of different fluorescences that identify the unique bead sets.

Figure 1. The surface of each microsphere contains multiple carboxyl groups that function as sites for covalent ligand attachment. In their interior, precise proportions of red and orange fluorophores identify discrete microsphere sets.

Flowmetrix differs from other microsphere-based multiplex systems in that only a single size of microsphere is used. Classification is based solely on the fluorescence of multiple dyes within the beads. The number of possible bead sets is established by the number of lasers exciting different dyes in the microspheres. For instance, the 64 assays currently performed by the Becton Dickinson Facscan increases to several hundred performed simultaneously on the dual-laser Facscalibur. This number could reach into the thousands and beyond with this year's introduction of a solid-state benchtop analyzer designed specifically for Flowmetrix applications. By combining small diode lasers with inexpensive digital signal processors and microcontrollers, it is expected that this instrument will perform rapid, multicolor analysis for about one-fifth the cost of conventional flow cytometers ($20,000 compared to $100,000).

The reagent components of the Flowmetrix system are also economical. Fluorescent dyes and microspheres are relatively inexpensive, and reagent usage on these very small microspheres is commonly 1% or less than that of microwell-based assays. Preanalytical specimen preparation is also markedly decreased, resulting in lower costs for labor and consumables. With these advantages, multiplexed analysis can be performed for the cost of most single immunoassays, enabling physicians to make use of the predictive power of synergistic testing for potential disease-causing agents (see Table I).

Table I. Comparison of intralaboratory cost factors for performing a cardiac marker panel on the Flowmetrix system versus doing so using other methods. For nonmultiplexed tests, costs increase in direct proportion to the number of analyses performed.

Synergistic Panel for Myocardial Infarction

An example of the multiplexed testing made possible by the Flowmetrix system can be seen in the analysis of biochemical markers for myocardial infarction. These markers have traditionally been evaluated individually rather than as synergistic groups. However, by combining the sensitivity of myoglobin with the specificity of troponin I and the general utility of creatine kinase-MB (CK-MB), the positive and negative predictive value of the group far surpasses that of any of the individual tests.^5—7 Since digoxin toxicity can mimic myocardial infarction, digoxin was also added to the panel.⁸ Despite the different molecular sizes and reference ranges of these analytes, all are measured simultaneously from a single aliquot and reported in real time (see Figure 2). If other markers, such as fatty acid binding protein, glycogen phosphorylase BB, or atrial natriuretic peptide demonstrate clinical utility, they could be easily added to the panel.

Figure 2. Laser illumination of microspheres (A) elicits red-orange internal fluorescence (B and C), identifying each microsphere set. Reporter molecules added with the patient sample quantitate the reactions by the intensity of their associated green fluorescence (D).

The system could also allow simultaneous measurement of biomolecules that regulate blood flow and pressure (e.g., aldosterone, renin, prorenin, antidiuretic hormone), since these analytes may have heretofore unrecognized diagnostic and predictive value. Since all of these analytes could be assayed at once, a previously unrecognized clinical efficacy could be unveiled.

Multiplexed Flowmetrix assays begin with the separate development of each component assay. For the cardiac panel, distinctly colored bead sets were coupled to the appropriate ligand through either chemical or avidin-biotin linkages. All reactants were then titrated to generate standard curves covering the physiologically significant ranges of analyte concentrations. Sensitivity and range can be balanced by altering the degree of saturation of capture ligate on an individual bead set, as well as by the number of microspheres used in the assay development. Regardless of format, a typical coupling to a microsphere set uses 2 ng of reactant per assay. Approximately 50 ng of green fluorescent reporter molecule is required per assay.

Each assay was designed to provide limits of detection significant to confirm or disprove occurrence of myocardial events as well as to cover physiologically significant ranges for each ligate (see Table II). For the most sensitive quantitative results, Flowmetrix assays require incubation times typical of immunoassays (approximately 15 minutes). However, semiquantitative data can be available in less than 5 minutes due to the near-liquid phase kinetics of soluble microsphere-based reactions. Multiple results on each individual patient require less than 10 seconds instrument analysis time. Correlation with predicate technologies has been excellent on every application attempted, including cardiac markers, hormones, tumor markers, allergy testing, deoxyribonucleic acid (DNA)—based assays, and others. The system is a rapid, powerful, and flexible method for analyzing multiple biomolecular events in real time.

Table II. Functional limits of detection and dynamic range for a cardiac marker panel on the Flowmetrix system.

With the Flowmetrix system, extensive risk-stratification panels could be developed. For example, a test for an individual tumor marker is now quite expensive. As components of a multiplexed Flowmetrix test, however, entire panels could be delivered in a cost-effective manner. Markers such as free and bound prostate-specific antigen (PSA) could easily be combined with other tests such as CK-BB to increase predictive value. In addition, cost constraints have made it difficult for many patients to have their coronary risk properly categorized. A cholesterol test alone is inadequate for risk stratification. The Flowmetrix system will allow complete coronary risk analysis, including high-density lipoprotein, low-density lipoprotein, apolipoproteins, lipoprotein a, and other markers to be performed simultaneously, at a reduced cost. As other important analytes are identified, such as C-reactive protein, they can be easily added with no system modification. In fact, virtually any other test grouping is possible, including cytokines, DNA-based testing, allergy, autoimmunity, or combinations thereof.

How it works: Flowmetrix assay development

The Flowmetrix system can perform multiplexed analysis of up to 64 different reactions simultaneously by using a flow cytometer and digital signal processor to perform real-time analysis of multiple microsphere-based assays. There are three major components of the system: a benchtop flow cytometer, microspheres, and computer hardware and software.

Figure 3. Preparing for a capture-sandwich format assay on the Flowmetrix system: (A) microsphere preparation; (B) reporter preparation; (C) positive sandwich assay result recorded by the flow cytometer.

The flow cytometer analyzes individual microspheres by size and fluorescence, simultaneously distinguishing three fluorescent colors: green (530 nm), orange (585 nm), and red (>650 nm). Microsphere size, determined by 90° light scatter, is used to eliminate microsphere aggregates from the analysis. Red and orange fluorescences are used for microsphere classification, and green fluorescence is used for analyte measurement. The Flowmetrix system is currently configured for the Becton Dickinson Facscan, a multiparameter flow cytometer that uses a single 488-nm excitation laser (Becton Dickinson Immunocytometry Systems, San Jose).

To prepare a multiplexed assay, individual sets of microspheres are conjugated with the target molecules required for each reaction (see Figures 3 and 4). For example, one microsphere set might contain 15% red and 85% orange fluorescence whereas a different set might contain the exact opposite ratio. Targets may be antigens, antibodies, oligonucleotides, receptors, peptides, enzyme substrates, or other types of molecules.

Figure 4. A competitive inhibition format assay on the Flowmetrix system: (A) microsphere preparation; (B) reporter preparation; (C) negative result shows reporters binding to the microsphere; (D) positive result shows reporters binding to competitive target molecules.

A fluorescent reactant is then prepared for each target molecule. A reactant may be any molecule that will bind to the target molecule, including oligonucleotides, antigens, antibodies, receptors, and so on. After optimizing the parameters of each assay separately, the assays can be multiplexed by simply mixing together the different sets of microspheres. The fluorescent reactants are also mixed to form a cocktail for the multiplexed reactions.

The microspheres are then reacted with serum, for example, followed by the cocktail of fluorescent reactants. After a short incubation period, the mixture of microspheres—now containing varying levels of green fluorescence on their surfaces—are analyzed with the flow cytometer. Data acquisition, analysis, and reporting are performed in real time on all microsphere sets included in the multiplex. As each microsphere is analyzed by the flow cytometer, the microsphere is classified into its distinct set, based on red and orange fluorescence, and the green fluorescence value is recorded (see Figure 2). One hundred individual microspheres of each set are analyzed and the mean value of the green fluorescence is reported. Using a typical standard curve, true quantitative results can be achieved, with precision enhanced by the analysis of 100 microspheres per data point.

Conclusion

Luminex has miniaturized and multiplexed the long-established microsphere assay format, markedly reducing reagent and consumable costs as well as sample requirements. Because the significant computing power driving Flowmetrix is also essentially free, the only unaffected costs associated with diagnostic testing are those involving sample collection, transport, and handling. In practice, therefore, it will be important to maximize the information derived from each sample. This will enable practitioners not only to avoid repetition of the expensive preanalytical costs, but also to take full advantage of the diagnostic power of synergistic testing.

As is the case with other technologies that are being driven by rapid developments in the microelectronics industry, significant performance gains should be a predictable component of the Flowmetrix system.

References

1. Haddow JE, Palomaki GE, Knight GJ., et al., "Reducing the Need for Amniocentesis in Women 35 Years of Age or Older with Serum Markers for Screening," New Engl J Med, 16:1151-c, 1994.

2. McDade RL, and Fulton RJ, "True Multiplexed Analysis by Computer-Enhanced Flow Cytometry," Med Dev Diag Indust, 19(4):75—82, 1997.

3. Fulton RJ, McDade RL, Smith PL, et al., "Advanced Multiplexed Analysis with the Flowmetrix System," Clin Chem, 43(9):1749—1756, 1997.

4. Gordon RF, and McDade RL, "Multiplexed Quantification of Human IgG, IgA, and IgM with the Flowmetrix System," Clin Chem, 43(9):1799— 1801, 1997.

5. Antman EM, Tanasijevic MJ, Thompson B, et al., "Cardiac-Specific Troponin I Levels to Predict the Risk of Mortality in Patients with Acute Coronary Syndromes," New Engl J Med, 18:1342— 1349, 1996.

6. McLaurin MD, Apple FS, Voss EM, et al., "Cardiac Troponin I, Cardiac Troponin T, and Creatine Kinase MB in Dialysis Patients without Ischemic Heart Disease: Evidence of Cardiac Troponin T Expression in Skeletal Muscle," Clin Chem, 43(6):976—982, 1997.

7. Wu AHB, Feng YJ, Contois JH, et al., "Comparison of Myoglobin, Creatine Kinase MB,
and Cardiac Troponin I for Diagnosis of Acute Myocardial Infarction," Ann Clin Lab Sci, 26: 291—300, 1996.

8. Harrison's Principles of Internal Medicine, 13th ed, Isselbacher, Braunwalk, Wilson, et al. (eds), New York, McGraw-Hill, 1994.

Michael Spain, MD, is medical director at Luminex Corp. (Austin, TX).