April 1, 1997

11 Min Read
True Multiplexed Analysis by Computer-Enhanced Flow Cytometry

Medical Device & Diagnostic Industry Magazine | MDDI Article Index

An MD&DI April 1997 Feature

IN VITRO DIAGNOSTICS

A personal computer and a novel assay format convert a common laboratory instrument into a state-of-the-art diagnostic device.

New and improved in vitro diagnostics (IVDs) can detect disease at the molecular level. These increasingly sophisticated assays are becoming indispensable to the diagnosis of many medical conditions, including cancer, autoimmune disorders, allergies, and cardiovascular disease.

However, the clinical impact of available IVDs may be curtailed by the current cost constraints in the health-care industry. Health-care payers often consider use of an assay justified only when the test results are presumed critical, not merely useful. The best interests of the patient are served when all relevant diagnostic tests are performed while at the same time costs are minimized to satisfy payers.

This article describes the design and mechanics of a diagnostic system tailored with these requirements in mind. The FlowMetrix system (The Luminex Corp., Austin, TX) allows simultaneous quantitation of multiple analytes with a minimum of reagents and time.

HOW IT WORKS

The FlowMetrix system incorporates three familiar, mature technologies--bioassays, microspheres, and flow cytometry--with novel hardware and software. Both immunoassays and nucleic acid­based tests are compatible with this platform.

Figure 1. Classification of 64 FlowMetrix microspheres by color. Microsphere sets are classified by their distinct red and orange emission signals. Each of the 64 sets comprises thousands of individual microspheres with a characteristic, identifying red-orange emission profile.

For years, IVD developers have been trying to achieve multiplexed analysis, but all systems developed previously functioned by splitting the sample into minute volumes, one for each test. The FlowMetrix system leaves the sample intact and adds microspheres, each of which carries an individual assay. Panels are created by combining up to 64 different microsphere-based assays into a single sample test. Multiplexed assays can be run on sample volumes as small as 5 µl.

Each assay is individually constructed around a single microsphere set with its own identifying fluorescent color. Each set of microspheres is manufactured with unique relative proportions of red and orange fluorescent dyes (Figure 1). During analysis, which takes place in a conventional flow cytometer enhanced with proprietary hardware and software, the microsphere components of each of the 64 different assays are classified according to these unique colors. The emission spectra are so tight in wavelength that, while the human eye cannot distinguish one set of microspheres from another, the processor can discern the ratio of red to orange signals from each individual microsphere.

At the same time, the relative intensity of a green fluorescent dye is also measured. This green color, associated with soluble reporter molecules bound to reagents at the surface of the 64 distinct microsphere sets, provides the qualitative and quantitative assay results. Because the intensity of a reporter molecule is read only at the surface of each microsphere, any reporter molecules remaining in solution do not affect the assay value, which makes the no-wash assay format possible.

The potential of this system can be seen in the example of a proposed emergency room test panel. A single sample of blood from a critically ill patient, added to a well containing the appropriate FlowMetrix reagents and microspheres, could be assayed for bloodborne viruses, cardiac markers, therapeutic drugs and drugs of abuse, allergic sensitivities, and hormones, as well as toxic substances (see Table I). Results would be available after a 15-minute incubation and a 10- to 20-second analysis in the enhanced flow cytometer.

Cardiac Markers

Drugs of Abuse

TherapeuticDrugs

Toxic Substances

Hormones

TumorMarkers

Infectious Diseases

Enzymes

Allergiesand Anaphylaxis

Blood Types

Creatine kinase (CK)

SGOT

CK-MB

Troponin

CK-MB isoforms

Opiates

Benzodiazepines

Barbiturates

Cocaine

Amphetamines

Propoxyphene

Digoxin

Phenytoin

Theophylline

Procainamide

Tricyclics

Neurontin

Isoniazid

Carbamazepine

Acetaminophen

Salicylates

Alcohol

Phenothiazines

Cholinesterase

Hypervitaminosis A & D

Thyroid

hCG

Catecholamines

PSA

Acid phosphatase

Ca-125

HIV I and HIV II

Hepatitis A, B, and C

Sepsismarkers

Amylase

Lipase

Antibiotics

Stinging insects

A, B, O

Rh

Table I. Proposed test panel for emergency room patient screening.

Urgent genetic analysis is another possible application. Potential organ donors can be screened rapidly for the major transplantation antigens.

ASSAY DEVELOPMENT

As described above, each assay in a test panel must be individually developed on a uniquely colored set of microspheres. For instance, an assay for serum levels of human chorionic gonadotropin (hCG) has been developed by covalently attaching a "capture" monoclonal anti-hCG antibody to a uniquely colored microsphere set. After a short incubation with patient serum, a second anti-hCG antibody tagged with a green fluorescent reporter molecule is added (see Figure 2). This "sandwich" technique allows a sensitivity of 10 mIU/ml, comparable to that of other serum assays for hCG.

A similar process is repeated for each assay component of each panel. The assay type is indicated by bead color, and the assay result is determined by the intensity of the green reporter molecules. This format is applicable to both immunometric and competitive immunoassay methods, and both formats may be performed simultaneously in the same tube.

Figure 2. Immunometric "capture-sandwich" assay for human chorionic gonadotropin. Figure 3. Competitive DNA hybridization as a multiplexed tissue-typing tool.

Assays for serum analytes of widely differing concentrations are multiplexed using a single sample preparation dilution. Both the sensitivity and the dynamic range of each assay can be adjusted by controlling the number of target microspheres, the density of target on each microsphere, and the brightness or concentration of the green color attached to the reporter molecule. When assays for analytes of widely differing concentrations are multiplexed, the concentration of reactants for each assay is optimized separately.

For example, a competitive inhibition assay measures serum levels of IgG, IgA, and IgM in the same tube. The primary parameter used for multiplexing was the concentration of reporter for the IgG, which is about 10 times greater than that for IgA or IgM because in serum the concentration of IgG is about 10 to 20 times greater than that of IgA or IgM.

Sensitivity and specificity for the multiplexed assays are comparable to results from conventional immunoassays. For a ToRCH assay, five microsphere sets are conjugated with antigens to Toxoplasma gondii (toxo), rubella togavirus, cytomegalovirus (CMV), and herpes simplex virus (HSV) types 1 and 2. Human serum is buffered at a 1:400 dilution in phosphate-buffered saline for 15 minutes, then reacted with green fluorescent goat-anti-human IgG for 15 minutes, and analyzed with the FlowMetrix system in about 15 seconds. The system counts 100 microspheres of each microsphere set. Specificity was demonstrated in a competitive format by the addition of each soluble antigen, resulting in the corresponding decrease of the appropriate bead-based signal. Human serum calibrators and controls from commercial sources were used to define the limits of the assay sensitivities. Human calibrators negative for all ToRCH antigens were negative on all microsphere sets. Human calibrator sera positive for all the ToRCH antigens were positive on all microsphere sets. A serum control (Blackhawk Biosystems) defined to have only minimal reactivity to each of the five ToRCH antigens was used to compare the sensitivity of this system to that of conventional analyzers. This control was tested using the IMX machine for toxo, rubella, and CMV, and the DiaMedix machine for HSV. At a 1:400 dilution, all five microsphere sets demonstrated reactivities of 3­6 standard deviations above background. The ranges of reactivities for this same control serum run on these two enzyme-based diagnostic instruments ranged from 1.1 to 2.7 times above the limit of detection. These results indicate that the FlowMetrix system ToRCH assay is as sensitive as the IMX and DiaMedix ToRCH assays.

For development of nucleic acid­based tests, the principle is the same as that underlying immunoassays. In these tests, however, the ligand-ligate interaction occurs not between an antigen and an antibody but between oligonucleotides in a hybridization event. This reaction follows normal DNA or RNA amplification, which may be accomplished by any commercially available process.

In a test for organ transplant compatibility, different colored beads are conjugated with distinct oligomers representing different alleles for each histocompatibility gene. Oligonucleotides complementary to 14 different allelic sequences of the HLA-DQA1 locus were covalently attached to 14 different microsphere sets. Fourteen green-labeled oligonucleotides complementary to the 14 allelic sequences were also included in the reaction mixture. In the absence of inhibitor, hybridization yielded 14 bright green fluorescent microspheres. The polymerase chain reaction (PCR)-amplified patient DNA competes most effectively for binding to the microsphere bearing the complementary capture probe, displacing the reporter molecule and thereby lowering the intensity of green fluorophore at the microsphere surface (see Figure 3). Sequence identity, and thus histocompatibility allele, are thereby determined.

In a preliminary trial, this HLA-DQA1 tissue-typing system detected the single base differences between alleles with 100% accuracy. The test was run on 34 homozygous and heterozygous samples of all alleles of the DQA1 locus purchased as individual clones from the UCLA tissue-typing repository. The absolute DNA sequences of the different allelic pieces of DNA were provided by UCLA. The sequences had in most cases been determined by restriction fragment length polymorphism (RFLP) analysis. The FlowMetrix results corresponded to the UCLA data in all cases.

ADVANTAGES

Responding to the current economic forces in the diagnostic industry, the FlowMetrix system is inexpensive: hardware and software necessary to adapt a conventional flow cytometer to the FlowMetrix system costs less than $5000. The system is completely controlled by a personal computer provided with the system and requires no modification of the flow cytometer. A single switch transfers normal instrument function to the FlowMetrix mode. The FlowMetrix system consists of a Becton Dickinson FACScan interfaced through a supplied switch box to a Pentium-based personal computer running in the Windows 95 or NT environment. The PC is loaded with a proprietary signal-processing module board and software that provides both control of all FACScan functions and real-time data acquisition and analysis of the microsphere-based assays.

Operating costs are also low, because microspheres are inexpensive and the microassay format requires very low reagent volumes, typically 100 to 1000 times less than enzyme-linked immunosorbent assays (ELISAs). For example, 500 µg of a mouse monoclonal antihuman IL2 is sufficient to coat 1440 ELISA wells. If these wells are developed in triplicate, as is common practice, about 500 data points result. The same quantity of antibody would coat 5 × 108 microspheres for use in a FlowMetrix assay. Using 1000 microspheres per data point, as is normally required, would generate 500,000 data points in this system.

Figure 3. Competitive DNA hybridization as a multiplexed tissue-typing tool.

Another advantage of the multiplexed format is that cross-reactions or interference can be easily identified and corrected. During assay development, each reporter molecule is individually tested against the complete panel of microspheres constituting the multiplexed assay, leaving out the microsphere known to be reactive with the reporter molecule. Green fluorescence detected at the surface of any microsphere indicates a degree of cross-reactivity.

Interfering substances that can affect an assay result are also easily identified. For example, human antimouse antibodies (HAMAs) present in patient serum in response to monoclonal antibody therapy can be detected by incorporation of a microsphere bearing an irrelevant mouse immunoglobulin.

GETTING STARTED

Installation begins with connection of a specially configured personal computer via cable to the digital interface port of the flow cytometer. Windows-based software then guides the user through the process of assay design and analysis. Procedures are described for creating antigen-antibody, small-molecule, and nucleic acid­based assays from development of the first assay to full-panel development. Luminex supplies the microspheres; customers create the assays. Complete panels will also soon be commercially available.

CLINICAL APPLICATIONS

Flow cytometers are found--and often underutilized--in more than 5000 clinical laboratories and biomedical research departments. With the FlowMetrix system, this instrument can perform most immunodiagnostic tests as well as the increasingly important nucleic acid­based tests now coming onto the market.

The system's speed derives not only from its ability to simultaneously perform multiple assays but also from its rapid throughput. A typical cycle time is approximately 30 seconds. The large menu of possible tests, randomly accessed, allows hospitals as well as smaller labs to function more effectively and efficiently. Point-of-care testing could also bring these advanced capabilities to the smallest clinic. Allergists, for example, could test patients for serum IgE and IgG against a regionally specific panel of suspected allergens.

PERFORMANCE SPECIFICATIONS

A number of multiplexed assays are being developed for the clinical market, including ToRCH, nucleic acid­based tissue typing, serum proteins and hormones, and a fertility panel. Quantitation at the femtomolar level and a dynamic range of 3­4 logs have already been achieved. Intra- and interassay precision of 4­5% is easily achieved because each assay point is a calculated average of over 100 data points representing the number of microspheres in each color set counted per assay.

CONCLUSION

With an inexpensive computer enhancement, the technology described above enables a standard laboratory instrument--the flow cytometer--to perform most common immunodiagnostic and nucleic acid­based assays. In the microassay, no-wash format, reagent costs and sample handling are reduced. The high throughput inherent in the system can be further expanded with off-the-shelf sample delivery instrumentation. Perhaps most importantly, assays can be bundled into panels and performed simultaneously, with the results reported in real time. This system responds directly to the needs of the IVD industry and the increasingly difficult economic and technical environment in which it operates.Ralph L. McDade, PhD, is vice president, development, and R. Jerrold Fulton, PhD, is vice president, research, for The Luminex Corp. (Austin, TX).

Copyright © 1997 Medical Device & Diagnostic Industry

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