RiaSmart EiaSmart Software for ImmunoAssay Data Reduction By: Max Tyrrell

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1 Gamma Note GN-002 (01/01/89) Introduction RiaSmart EiaSmart Software for ImmunoAssay Data Reduction By: Max Tyrrell ImmunoAssay methodologies have changed dramatically in the last ten years, but RiaSmart and EiaSmart software from Packard have been developed with adaptability in mind. More advanced technology in gamma counting instrumentation and photometrics, and the more sophisticated data reduction available in packages like RiaSmart and EiaSmart, make both RIA and EIA outstanding tools for the nineties. These advances also increase productivity in the laboratory, and provide superior quality control of both the assay and the instrumentation. The significant rise in quality and use of monoclonal antibodies has resulted in a variety of new techniques for assaying analytes. IRMA and the complimentary immunometric ELISA have become increasingly more popular, and the need to plot the data more reliably and accurately have become very important. These new advances in both RIA/EIA and IRMA/ELISA techniques have made it necessary for instrumentation manufacturers to revise many of their data reduction schemes to take advantage of the improved assay sensitivities. RiaSmart and EiaSmart software packages are Packard's response to the laboratory's needs. More than a concept, they are a new way of managing immunoassay data. They incorporate all the criteria needed to report results with speed and reliability, allowing flexible combinations of response calculations, curve fitting, and graph paper needed to best represent the data from any assay. Worklist capabilities provide positive sample-patient ID. This information can be keyed in for each sample prior to processing. The user can also program in a factor for each sample. The assay result is then automatically multiplied or divided, added or subtracted using this factor to produce a final result. From the presentation of the data to the final result, RiaSmart/EiaSmart software provides the user with complete control over what is happening to the assay data. As a rule, most immunoassay data reduction systems can only handle one task at a time. But multi-tasking allows our gamma counters to count and

2 process data, while the user reviews data already processed. And in both RiaSmart and EiaSmart versions, data can be compared, and standard curves can be edited, compared, and overlaid. RiaSmart software turns the on-board computer of a Packard gamma counter into an effective and efficient TM laboratory assistant, while EiaSmart can be used on any laboratory IBM PC for reducing microplate absorbance data. Smart Answers for all of Your Assays RiaSmart and EiaSmart differ only in their references to isotopic and nonisotopic assay idiosyncracies; counting fractions and tubes for RIA; and absorbances and micro-wells for EIA. Both software packages offer the most advanced curve fitting techniques and let the user choose the curve fitting method which best suits the assay. A special MASS TM (Mass Action Smoothed Spline) curve fitting method complements linear interpolation, unweighted and weighted linear regression, logit-log, four parameter logistic (4PL), and smoothed spline curve transformation methods. Curve Fitting These automatic processing software packages use two basic curve fitting methods, regression and interpolation, as well as hybrid techniques which incorporate the advantages of both. Regression Method Regression curve fitting is a method which applies a predefined mathematical function to the data points, such that the square of the difference between the measured values and the values of the function are at a minimum, called the least squares fit. The regression method is best applied empirically, using the experience of the operator to choose an appropriate equation to fit the curve in the best way. The best regression methods are implemented in RiaSmart and EiaSmart as described here. The Logit-Log transformation has received a great deal of attention as a method of plotting RIA/EIA data as first applied by Dr. Rodbard. The Logit function has the ability to linearize sigmoid or S-shaped curves, providing a reasonably linear response over the midrange of equilibrium assays. It will not, however, provide good results for nonequilibrium or multiple binding site assays. Despite modifications to allow this method to fit atypical curves, this technique is not able to fit all assays. In addition, the logit transformation introduces non-uniformity of variance in the standard points, complicating further the regression process. It is recommended that a weighted regression be used to compensate for this predicted heteroscedacticity of the standard points. RiaSmart and EiaSmart offer both weighted and unweighted regression techniques to optimize linear curve fitting with and without transformation of the response.

3 Figure 1 Logit Curve To correct for some of the limitations of the logit-log curve fit, including the loss of the zero standard concentration which restricts the sensitivity of the assay to the lowest standard, the four parameter logistic model has been developed. In this approach, the values of 0 B(RIA) or High Standard (EIA) and the NSB, which are treated as fixed constants in the usual logit-log method, are "adjusted" by Packard's software to give the best possible fit over the entire standard curve. To further improve the performance of logistic calculations for atypical (asymmetrical) curves, such as those found in IRMA and immunometric ELISA's, the RiaSmart and EiaSmart software allows the user to specify the response at maximum binding (B) or a maximum max absorbance, respectively.

4 Figure 2 Four Parameter Logistic Curve While the logit-log and four parameter fitting techniques have become popular regression models to fit immunoassay data, Packard's RiaSmart and EiaSmart programs have a selection of alternative calculation algorithms to fit atypical curves. Interpolation Method The most common alternative curve fitting techniques are based on the interpolation method. In these methods, a function is calculated such that at the point where the standard value is used, the value of the function is exactly the same as the measured value. Between standard points, however, the shape of the curve may vary widely from the actual curve encompassing the points. Linear interpolation and curve fitting by spline function are the most popular interpolation methods used to fit radioimmunoassay curves. Spline interpolation provides a computerized "French-curve." Cubic polynomials are used to connect the standard points. However, this technique is rather unstable when bad data points or outliers are encountered. For this reason, Packard has chosen not to include this curve fitting method in the RiaSmart/EiaSmart series. Better methods such as smoothed cubic spline and

5 Mass Action Smoothed Spline are included. These hybrid techniques incorporate the advantages of both regression and interpolation curve fitting methods. Hybrid Method The smoothed spline function uses two criteria to establish a curve. The area under the second derivative must be at a minimum to eliminate oscillations, and the difference between the data point and the function value must be less than the standard error multiplied by a smoothing factor. By increasing the smoothing factor, the curve is allowed to deviate farther from the standard point. To satisfy the minimum area requirement, the curve will be smoothed to the extent the deviation from the standard points will allow. The smoothing process may go through several iterations before an acceptable curve is reached. After each iteration, the curve is checked for extreme values and conformity to typical immunoassay curve characteristics, e.g., a maximum of one inflection point. If the curve does not meet these characteristics, the smoothing factor is increased, and the curve is reconstructed with the new deviation limits on the standard points. This technique has the advantages of being able to fit all assay types whether equilibrium, nonequilibrium, or multiple binding site assays. It also has the ability to reject outliers of bad precision which cause large standard errors. The disadvantage to the smoothed spline curve fit is that in the process of rejecting the outliers, the curve may also deviate from the good standard points. Here, too, the zero standard is lost through the use of the log scale. Mass Action Smoothed Spline corrects for this problem, by allowing the zero standard to be used.

6 Figure 3 Smoothed Cubic Spline Curve The Mass Action Smoothed Spline fitting technique is unique to Packard's RiaSmart and EiaSmart software. This technique combines the flexibility of the smoothed spline curve fitting method with the kinetic preconditions of the assay system, either equilibrium, nonequilibrium, or multiple binding site assays. Using the same iterative process and immunoassay criteria explained above for smoothed spline, MASS enhances the iterative process by evaluating all points against a stored curve shape for the specific assay type. The kinetics of equilibrium, nonequilibrium, and immunometric equations are stored in the program for comparative purposes. When points of a curve do not compare favorably to the stored theoretical curve, they are identified as outliers, and the standard error for the individual points is artificially increased for the smoothing process. These powerful curve fitting techniques eliminate the necessity to produce standard curves with straight line portions. The use of immunoassay equation models helps ensure integrity of the derived curve and eliminates any "hook effects" that may otherwise result in erroneous interpolative concentrations.

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8 Compare and Overlay of Curves Figure 4 Curve Smoothing With Mass Action Smoothed Spline In addition to the many curve fitting choices, RiaSmart and EiaSmart let the user compare and overlay curves on a high resolution graphics screen. The user can instantly determine the best calculation method for the assay, or compare the curve with a historical curve template. Ria-Smart software even permits the use of decay-corrected curves. On-Screen Curve Editing Figure 5 Curve Comparison (example from RiaSmart) In the unlikely event of a bad standard curve, the on-screen graphics curve editor allows rejection of outliers. Data can then be reprocessed without recounting, or rereading. Patient specimens and costly reagents are not wasted, and neither is valuable time. Immunoassay data management is simplified with RiaSmart and EiaSmart.

9 Processing Elegance Figure 6 Curve Editing (example from EiaSmart) Being able to choose the curve fit best suited for the assay is only one of Packard software's many benefits. RIA - RiaSmart provides a tube protocol display that saves time and reduces errors with the protocol set-up and sample tube positioning, thus ensuring positive sample ID. Whether processing complicated dual label RIA/IRMA assays or simply counting CPM, RiaSmart software adapts to all data reduction needs. Dual label results are reported simultaneously; special programs have been implemented that are applicable to all screening assays. These programs include user definable flagging for positive and negative samples, and clear identification of borderline samples. RiaSmart software easily handles specialized assays such as hepatitis, T3-uptake, inverse ratio, and various RAST assays. The user can link results of different assays to produce final FTI answers, or correct for varying extraction efficiencies and dilution groups. A clear, user-customized report of the assay results saves laboratory time and facilitates interpretation.

10 Figure 7. Tube Protocol Display. Instantaneous verification of protocol set-up and sample tube positioning. EIA - EiaSmart provides a microplate display that allows positioning of blanks, standards, and samples anywhere on the microplate. Sample ID's from the worklist are automatically associated with the well location ensuring positive TM sample ID in reporting. EiaSmart is designed to work with Packard ARGUS microplate readers, or equivalents. Total control of the reader is provided by a push of key from the computer. Absorbance data is retreived and curve fitting established automatically. In addition to the immunometric and competitive curves calculable, simple EIA screening assays employing user defined limits or cutoffs are available. So hepatitis, allergy testing, or even monoclonal antibody screening is easily accomplished with EiaSmart. And all calculation protocols report the results clearly because the reports are user-defined.

11 Figure 8. Microplate display. Both RiaSmart and EiaSmart have worklist capabilities that let the user identify each sample by patient name or number. And dilution factors can be included so end concentration results are reported accurately.

12 Figure 9. Worklist capabilities.

13 Figure 10. Report Output Screen. The user can select 20 parameters for printout, from among the 26 predefined and 16 custom parameters displayed on the report output screen. Example from RiaSmart. EiaSmart reports Absorbance in place of CPM and allows for reprinting of well location. Worklist capabilities let the user identify each sample by patient name or number. Results can be formatted as required by each laboratory. Fast and Accurate Cytotoxicity Results Whether assaying cell mediated cytotoxicity by cytochrome C production and 51 measuring colorimetrically, or radioassaying the release of Cr labeled samples, Packard software can do the processing. Packard's Cobra 5005/5010 gamma counters offer a unique zigzag layout of the detectors minimizing radionuclide crosstalk with 18mm of lead shielding. And the RiaStar counters automatically correct for crosstalk with a normalization procedure. During counting, any counts contributed by adjacent detectors are automatically subtracted. RiaSmart and EiaSmart simply have to calculate the release ratio or absorbance ratio for samples. The Packard gamma

14 counters and microplate readers combined with the RiaSmart/EiaSmart programs make for high sample throughput of cytotoxicity assays. Extend Analytical Capability with Coded Assays A unique Coded Assay TM capability makes RiaSmart and EiaSmart the software systems for all the laboratory testing needs...today and tomorrow. Protocols can be designed (coded) to handle virtually any data reduction requirements, putting any assay or reduction routine at the user s command. Special assays can be processed automatically, such as ft4 and Plasma Renin, or assays with variable NSB or specific screening requirements. No compromises, no manual calculations, no obsolescence: RiaSmart and EiaSmart software with Coded Assay pro-cessing is smart, flexible, and fast.

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16 Figure 11. Printout of RiaSmart Results. The printout of results includes a lab ID header, standard results and curve, and unknowns. 15 Aug 90 15:02 Packard Instrument Company Page #3 Protocol #: 1 Total IgE User : B.P. S# A:ABSORB A:CV_ABS A:CV_CON A:ERROR A:CONC. PAT/ID Seyth, J Auburn, F Range: Hi Ryczak, F Jones, L Bissault, D Westford, H Range: Hi Orley, S Kevick, B Gorette, B Millard, G Tyrrell, M Lyons, N Proulx, R.

17 Figure 12. Printout of EiaSmart Results. Conclusion Packard's software programs for immunoassays, with their many features, appeal to the busy laboratory and meet its needs. From the presentation of the data to the final result, RiaSmart/EiaSmart software provides the user complete control over what is happening with the assay data.