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V1c 97 03 Hstm 2011 Hamburg 20110526 Dj

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Presentation given 31st of May in Hamburg at HSTM 2011 (www.hstm.de)

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V1c 97 03 Hstm 2011 Hamburg 20110526 Dj

  1. 2. Biological and technical validation of an experimental macrophage-assay; the necessary steps to take D . Japink 1 , M.N. Sosef 1 , J.Coy 4 , M. Nap 2 , M.P.G. Leers 3 Dept. Surgery 1 , Pathology 2 and Clinical chemistry 3 , Atrium MC Parkstad Heerlen, the Netherlands, Tavarlin AG 4 , Darmstadt, Germany
  2. 3. Macrophage Assay
  3. 4. <ul><li>Enriched cytocentrifuged peripheral blood samples: </li></ul>Immunohistochemical analysis of Cytospin I macrophages with internal PSA-staining in prostate cancer patient
  4. 5. CEA in activated macrophages Immunohistochemical analysis of Cytospin II
  5. 6. Tumor marker detection in macrophages Stopping gate (SG)  10000 MNC
  6. 7. Validation ? <ul><li>A new method </li></ul><ul><ul><li>Improving best practice </li></ul></ul><ul><li>Validation </li></ul><ul><ul><li>Reproducibility </li></ul></ul><ul><li>Applicability in follow-up? </li></ul><ul><ul><li>Needs assessment </li></ul></ul>
  7. 8. Reproducibility-assessment <ul><li>Reproducibility checks </li></ul><ul><li>1) Transferable? </li></ul><ul><ul><li>Reproducible outcomes on 2 machines </li></ul></ul><ul><li>Stable in time in healthy persons? </li></ul><ul><ul><li>Physiological variability in humans in one year </li></ul></ul><ul><li>Intra-assay and inter-assay variability? </li></ul><ul><ul><li>Duplicate and duple measurements </li></ul></ul><ul><li>Durable? </li></ul><ul><ul><li>Within what time should the samples be assessed </li></ul></ul>
  8. 9. Reproducibility-assessment <ul><li>Reproducibility checks </li></ul><ul><li>1) Transferable? </li></ul><ul><ul><li>Reproducible outcomes on 2 machines </li></ul></ul><ul><li>Stable in time in healthy persons? </li></ul><ul><ul><li>Physiological variability in humans in one year </li></ul></ul><ul><li>Intra-assay and inter-assay variability? </li></ul><ul><ul><li>Duplicate and duple measurements </li></ul></ul><ul><li>Durable? </li></ul><ul><ul><li>Within what time should the samples be assessed </li></ul></ul>
  9. 10. Transferability: results Reproducibility: %CV < 15% 15%
  10. 11. Transferability: statistics Stopping gate (SG)  10000 MNC Fractions of all events Median %CV of %CV Range of Reproducibility (%CV) < 15.0% < 10.0% Quadruplicate measurements Lymphocytes 6.65 % 0.3 – 15.2 % 97.7 % 73.3% Mononuclear cells (MNC) 7.0 % 0.0 – 27.1 % 82.2 % 68.9% Monocytes 2.1 % 0.02 – 11.7 % 100.0% 86.7% Activated Macrophages 2.5 % 0.07 – 32.6 % 88.9 % 75.6% Duplicate measurements CEA – positive events 9.9 % 0-37.3 % 71.1 % 55.5% TKTL1 – positive events 8.9 % 0.5-40.0 % 80.0 % 55.5% CK18 – positive events 11.1 % 0.05-37.6 % 71.1 % 44.4%
  11. 12. Reproducibility-assessment <ul><li>Reproducibility checks </li></ul><ul><li>1) Transferable? </li></ul><ul><ul><li>Reproducible outcomes on 2 machines </li></ul></ul><ul><li>Stable in time in healthy persons? </li></ul><ul><ul><li>Physiological variability in humans in one year </li></ul></ul><ul><li>Intra-assay and inter-assay variability? </li></ul><ul><ul><li>Duplicate and duple measurements </li></ul></ul><ul><li>Durable? </li></ul><ul><ul><li>Within what time should the samples be assessed </li></ul></ul>
  12. 13. Stability in time healthy persons
  13. 14. Reproducibility-assessment <ul><li>Reproducibility checks </li></ul><ul><li>1) Transferable? </li></ul><ul><ul><li>Reproducible outcomes on 2 machines </li></ul></ul><ul><li>Stable in time in healthy persons? </li></ul><ul><ul><li>Physiological variability in humans in one year </li></ul></ul><ul><li>Intra-assay and inter-assay variability? </li></ul><ul><ul><li>Duplicate and duple measurements </li></ul></ul><ul><li>Durable? </li></ul><ul><ul><li>Within what time after venipuncture should the samples be assessed? </li></ul></ul>
  14. 15. Reproducibility-assessment <ul><li>Reproducibility checks </li></ul><ul><li>1) Transferable? </li></ul><ul><ul><li>Reproducible outcomes on 2 machines </li></ul></ul><ul><li>Stable in time in healthy persons? </li></ul><ul><ul><li>Physiological variability in humans in one year </li></ul></ul><ul><li>Intra-assay and inter-assay variability? </li></ul><ul><ul><li>Duplicate and duple measurements </li></ul></ul><ul><li>Durable? </li></ul><ul><ul><li>Within what time after venipuncture should the samples be assessed? </li></ul></ul>
  15. 16. <ul><li>Intra-assay </li></ul><ul><li>90 single EDTA samples </li></ul><ul><li>SOP preparation </li></ul><ul><ul><li>Provides: </li></ul></ul><ul><ul><ul><li>1 negative control (FMO) </li></ul></ul></ul><ul><ul><ul><li>4 marker-tubes </li></ul></ul></ul><ul><ul><ul><li>M30, CEA, TKTL1,CK </li></ul></ul></ul><ul><li>Each single tube measured twice </li></ul><ul><li>Standardized gating by single observer </li></ul><ul><li>Inter-assay </li></ul><ul><li>30 double EDTA samples </li></ul><ul><li>SOP preparation </li></ul><ul><ul><li>Provides: </li></ul></ul><ul><ul><ul><li>1 negative control (FMO) </li></ul></ul></ul><ul><ul><ul><li>4 marker-tubes </li></ul></ul></ul><ul><ul><ul><li>M30, CEA, TKTL1,CK </li></ul></ul></ul><ul><li>Each duple tube measured once </li></ul><ul><li>Standardized gating by single observer </li></ul>Intra- and interassay variability Experiment Setup
  16. 17. Intra-assay reproducibility <ul><li>Duplicate measurements of 90 samples </li></ul>
  17. 18. <ul><li>Intra-assay </li></ul><ul><li>n=90 </li></ul><ul><li>No normality </li></ul><ul><li>Spearman r: 0.9157 </li></ul><ul><li>CI = 0.8989 – 0.9298 </li></ul><ul><li>p = <0,0001 </li></ul><ul><ul><li>alpha = 0.05 </li></ul></ul><ul><li>Interpretation </li></ul><ul><li>Within tube: </li></ul><ul><ul><li>Reproducibility accurate </li></ul></ul><ul><li>Correlation </li></ul><ul><ul><li>Correlation significant </li></ul></ul>Intra-assay variability Experiment outcome
  18. 19. Inter-assay reproducibility <ul><li>Duple measurements of 30 samples </li></ul>
  19. 20. <ul><li>Inter-assay </li></ul><ul><li>n=30 </li></ul><ul><li>No normality </li></ul><ul><li>Spearman r: 0.3366 </li></ul><ul><li>CI = 0.1777 – 0.4783 </li></ul><ul><li>p = <0.0001 </li></ul><ul><ul><li>alpha = 0.05 </li></ul></ul><ul><li>Interpretation </li></ul><ul><li>Within experiment </li></ul><ul><ul><li>Reproducibility acceptable </li></ul></ul><ul><li>Correlation significant </li></ul><ul><ul><li>But less than intra-assay </li></ul></ul>Inter-assay variability Experiment outcome
  20. 21. Stability in time healthy persons
  21. 22. Reproducibility-assessment <ul><li>Reproducibility checks </li></ul><ul><li>1) Transferable? (presented on ISOBM) </li></ul><ul><ul><li>Reproducible outcomes on 2 machines </li></ul></ul><ul><li>Stable in time in healthy persons? (presented on ISOBM) </li></ul><ul><ul><li>Physiological variability in humans in one year </li></ul></ul><ul><li>Intra-assay and inter-assay variability? </li></ul><ul><ul><li>Duplicate and duple measurements </li></ul></ul><ul><li>Durable? </li></ul><ul><ul><li>Within what time after venipuncture should the samples be assessed? </li></ul></ul>
  22. 23. Durability <ul><li>“ What is max bench-time of an EDTA-sample for providing proper outcomes using our assay?” </li></ul><ul><li>Experiment setup </li></ul><ul><li>6 CRC-patients </li></ul><ul><li>3 EDTA tubes at once </li></ul><ul><li>Preparation start: </li></ul><ul><ul><li>1) directly </li></ul></ul><ul><ul><li>2) after 6 hours bench-time at RT </li></ul></ul><ul><ul><li>3) after 24 hours bench-time at RT </li></ul></ul>
  23. 24. Results – one patient
  24. 25. Results - 7 patients
  25. 26. Conclusion <ul><li>Reproducibility checks </li></ul><ul><li>1) Transferable? </li></ul><ul><ul><li>Reproducible outcomes on 2 machines </li></ul></ul><ul><li>Stable in time in healthy persons? </li></ul><ul><ul><li>Physiological variability in humans in one year </li></ul></ul><ul><ul><li>“ In accordance with regular tumormarker assays” </li></ul></ul><ul><li>Intra-assay and inter-assay variability? </li></ul><ul><ul><li>Duplicate measurements provide good variability </li></ul></ul><ul><ul><li>Duple measurements provide acceptable variability </li></ul></ul><ul><li>Durable? </li></ul><ul><ul><li>Assessing samples within 24 hours shows stable intracellular staining and no excessive aspecific staining </li></ul></ul>
  26. 27. Future steps <ul><li>Electron-microscopy </li></ul><ul><ul><li>Assessment exact CEA localization </li></ul></ul><ul><li>Follow-up applicability </li></ul><ul><ul><li>Assessment usability in CRC follow-up </li></ul></ul><ul><li>Improvements to address </li></ul><ul><ul><li>Direct labeling of primary antibodies. </li></ul></ul>
  27. 28. Acknowledgements ProFaCo-team
  28. 29. Thank you

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