Analytical precision and accuracy of commercial immunoassays for specific IgE: establishing a standard

P. Brock Williams, PhD, James H. Barnes, PhD, Sheryl L. Szeinbach, PhD, Timothy Sullivan, MD

Journal of Allergy and Clinical Immunology, June 2000

Study Background
Different lab assays are used to detect and measure specific IgE antibodies, but no standard exists to assess the analytical performances of these different assays. The objective of this study was to analyze reported specific IgE results from different labs using different testing technology with the same serum samples and testing for accuracy and precision. Six labs using 5 different test procedures were sent identical samples on 3 different occasions, resulting in 12,708 specific IgE test results on the same 26 serum samples and analyzed for 17 different allergens.

Assays/Labs in Study

Conclusion
ImmunoCAP performed very close to the ideal. Extensive variability was observed in the other 4 lab/assay systems. For some specific allergens, they were not able to detect serial dilutions of the same sample. These assays demonstrated substandard overall performance with multiple instances of poor precision and accuracy, particularly for certain allergens such as weeds and molds.

Editorial Highlights

• Reliable and accurate assessment of specific IgE antibodies is critical to determine allergy and identify causative allergens to make appropriate treatment decisions including allergen avoidance, selection of medications, and immunotherapy.

• Steady procedural modifications to the original IgE serological tests over the last 25 years have resulted in a number of currently available test formats using different chemistries, reagents, tests, and scoring systems. These tests do not perform equally, and not all are accurate.

• While FDA licensing and guideline requirements for manufacturers and labs have assisted in assuring more consistent results, they do not address accuracy or precision parameters in a comprehensive fashion. There is a need to identify and accept a well-recognized
  in vitro allergy test standard.

• An ideal immunoassay for specific IgE should meet certain analytical performance criteria, including slope of unity over the useful measurement range for all allergens, low variation across replicate measurements, statistically determined limits of detection, a practical cut-off, and defined saturation points. For an assay to be considered a standard, it would require close approximation to these criteria.

• Standardized analysis of the same diluted samples revealed significant differences between labs and methods, as well as significant differences across different allergens. Only the results from 2 labs using ImmunoCAP technology did not differ significantly from the ideal distribution.

• The average standardized slope coefficients from best to worst were S-CAP (0.98), A-RAST (0.71), L-RAST (0.70), C-RAST (0.68) and
  A-STAT (0.65). I-CAP was the standardized slope.

• Slope data, along with the standard error for each individual allergen, were used to calculate the confidence intervals for each allergen. Confidence intervals should encompass a value of 1.0 with small confidence intervals. Only ImmunoCAP (I-CAP, S-CAP) achieved this. A-STAT confidence intervals encompassed the value of 1.0 but had very wide confidence intervals. The remainder did not fulfill this requirement.

• Given the dilution range used in this study, values of R-Square below approximately 0.80 generally reflect poor performance in the ability to correctly detect levels of specific IgE antibodies. Only ImmunoCAP had consistently acceptable R-Square values, suggesting good performance in their ability to correctly detect the concentrations of specific IgE antibodies across the different samples and allergens. ImmunoCAP (I-CAP, S-CAP) had 1 R-Square value below 0.80 (1/17 = 6%). L-RAST had 7 (7/17 = 41%), A-RAST had 13 (13/17 = 76%), A-STAT had 14 (14/17 = 82%), and C-RAST had 16 (16/17 = 94%).

• ImmunoCAP performed extremely close to the ideal. A-STAT results revealed a reasonable comparison with the ideal for 6 allergens out of 17. The modified RAST procedures exhibited notably poor overall results and probably indicate a saturation effect reflecting the assay's inability to differentiate higher levels of specific IgE for most allergens (14 of 17). This indicates the inability of these assays to accurately quantitate different concentrations of specific IgE.

• This study indicates that ImmunoCAP is capable of measuring specific IgE antibodies over a large range with precision and accuracy for the allergens studied and supports its use as a current standard for quantitative (kU_A/L) measurements of specific IgE antibodies.

• There has been a long-standing debate regarding the clinical value of serological tests for specific IgE antibodies, such as those analyzed in this study. The results of this study indicate that data from different assay systems are not interchangeable. The Pharmacia CAP System performed well when compared to the standard of an ideal assay, while other assays often did not perform up to this standard.

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