Development and Validation of Measurement Traceability for In Situ Immunoassays

Area of Science:

• Biomedical Science
• Metrology
• Analytical Chemistry

Background:

• Immunoassays for in situ protein analysis face significant interlaboratory disparities, impacting patient care.
• Lack of standardization tools like reference standards and traceable units hinders immunohistochemical (IHC) testing.
• Previous efforts to create these standardization tools faced technical challenges.

Purpose of the Study:

• To introduce a novel metrology concept, linked traceability, for standardizing IHC testing.
• To develop and validate a new method for quantifying protein analyte concentration in situ.
• To address the need for analytic standardization in the $2 billion laboratory testing industry.

Main Methods:

• Developed a method measuring an attached fluorescein, traceable to NIST Standard Reference Material 1934.
• Utilized newly developed estrogen receptor (ER) calibrators for validation.
• Assessed laboratory performance using ER standards and a breast cancer tissue array in a quality assessment program.

Main Results:

• The tissue array confirmed substantial discrepancies in ER test results among laboratories.
• New ER calibrators demonstrated a wide range of analytic sensitivity (7,310 to 74,790 molecules).
• Demonstrated a quantitative correlation between variable test results and analytic sensitivity.

Conclusions:

• A reference standard enables precise interlaboratory alignment of IHC test sensitivity for in situ protein measurement.
• The introduction of a reference standard and traceable units for protein expression is a significant advancement.
• This work provides a foundation for improved accuracy and reliability in protein analysis assays.