Assay Development

Steps in Assay Development, as applied to CTEP studies

1. Provide logistic plan and project budget for review and approval.
2. After final approval, the histology project will commence.
3. Antibodies and assay kits will be procured for the study.
4. The project electronic database will be prepared.
5. CTEP validation will be initiated for perspective biomarkers.
6. Validation studies will be completed within 90 days and the “Immunohistochemical (IHC) Marker Template for Integral Markers in Clinical Trials” and the “Study Checklist for CTEP-Supported Early Phase Trials with Biomarker Assays” will be submitted for each assay developed to the project PI.Ultimately, documents will be sent by the PI to CTEP along with the laboratory SOP (Standard Operating Procedure).
7. Upon receipt, project specimens will be logged into the electronic database.
8. Specimens are then processed using an automated tissue processor and embedded in paraffin blocks.
9. Sections are prepared from blocks and placed on charged glass slides for staining and analysis.
10. Bar code labels are prepared and affixed to slides and the slides stored in a secure temperature and humidity-
    controlled room until staining.
11. Slides are then transferred to the laboratory histotechnologist for staining using either the Ventana Benchmark XTor Dako Link-48 clinical autostainers as per the validated method for each biomarker. For Laboratory Derived Tests (LDT) staining is optimized using both positive and negative controls and by adjusting specimen pretreatment and antigen retrieval, primary antibody dilution, contact time and temperature as well as signal amplification.
12. Stained slides are returned to the laboratory study coordinator and are logged into the database.
13. Stained slides are then transferred to the board-certified laboratory pathologist for scoring. Specific scoring methods are used for each biomarker and may include use of the H-score method and data input to the electronic database for each specimen and the slides returned to the study coordinator.
14. Scoring QC is performed by a return of 10% of the specimens by the study coordinator to the original pathologist for re-scoring. Data is reviewed by the study coordinator and discrepancies are assessed.
15. Additional scoring QC is performed by the laboratory’s external pathologist who scores 10% of the study specimens.
16. All data contained in paper documents and electronic spreadsheets is reviewed for accuracy by the laboratory’s quality assurance technician.
17. Data is then compiled into a report for review by the study PI. The laboratory team is available for meetings and teleconferences with the study principals on an as-needed basis.
18. A final report is prepared following receipt of comments from the draft report by the study principals.