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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  • Thank you chairman for giving me the opportunity to present our latest results of our research group, good afternoon ladies and gentlemen. First I would like to give a short introduction to our work and afterwards I will elaborate about the quality assessments we have been conducting.
  • The macrophage method was first described by Herwig et al from Innsbruck Austria in 2005 and we adapted and refined this method in Heerlen and performed a pilot study with prostate cancer patients which we published in 2008. Afterwards we translated the method to colorectal cancer, performed a pilot study in 45 patients and found the ability to detect CRC already in early stages.
  • In the prostate study we confirmed the existence of macrophages containing PSA. The result can be seen here. Cyto-centrifuging peripheral blood samples showed multilobulated cells. The ability for phagocytosis is shown when staining internally for PSA . The PSA-positivity means one can assume these cells are macrophages.
  • Also in CRC we were able to detect internalized tumormarker inside macrophages. Again we cytocentrifuged the samples we also measure ifor the new assay and we found a similar picture as for PSA. The picture shows a bluish staining for the nuclei and a brownish staining for internalized CEA in small vesicles.
  • In short I will show you how the assay functions. The tumor marker detection method we use starts by drawing 3ml peripheral EDTA blood, which we prepare by a SOP-based preparation method. Afterwards a flow cytometric analysis using a parental gating strategy is performed. The parental gating is shown in the upper right corner. first we select mononuclear cells using FSC and SSC. CD14 positivity selects the population of monocytes and a CD16 step filters the activated monocytes. Within this population we further select the tumormarker-positive fraction by tm-positivity compared to a negative control. By at least analyzing 10000 mononuclear cells we make sure enough cells in the subpopulations are analyzed When performing backgating from the CD14-CD16 plot to the FSC-SSC in the top-left corner we can see a clear population with higher SSC and FSC properties, which means the cells are larger and irregular in shape.
  • An example of the outcomes is as follows: The x axis shows the combined tube numbers we measured and on the Y-axis the % CV is shown. The green bar shows the area of CV which is 15% or lower. Most of the datapoints are below 15% CV value.
  • This table shows the addressed populations, their median CV and CV range and the reproducibility within 15% and even 10% range. Lowest reproducibility percentages were found in the Cytokeratin 18 populations as shown in red And the highest reproducibility percentages were found in the monocyte populations
  • Further we measured the stability in time in healthy persons to assess the physiological behaviour of the cell populations we use in our assay
  • We found the CEA-CM analysis in healthy persons over a time period of one year to be variable but in the end returning to baseline values. Multiple measurements in time provide this baseline. This finding is in line with the established way of working with tumormarkers, where multiple measurements in time with elevations of more than 50% in 2 consecutive measurements is considered clinically relevant.
  • What was left for us to assess were the Intra and inter-assay variability and the durability. I will walk you through our intra-assay and inter-assay assessment first.
  • The experiment setup was as you see here, we included 90 patients with CRC, drew 90 samples and prepared the EDTA samples for assessing intra-assay variability. Of 30 patients we were able to obtain a second EDTA-sample for the inter-assay assessment. One negative control-tube and four tumormarker-tubes were provided by each sample, measured on the flow cytometer and analysed according to our standardized gating strategy.
  • In this graph the original measurement of each sample is plotted against the duplicate one. Most measurements align on the 45 degree axis showing correlation within the 90 samples.
  • On both axes the duple tube outcomes have been plotted. The correlation here is less strong than in the duplicate assessments but significant correlation is present.
  • After concluding normality is not present Calculating Spearman r shows a much lower value than in the duplicates. But the correlation still is significantly present. The graph showed earlier showing measurements in time did already show us the variability intra-person. This might mean that also in this type of assay we could need consecutive measurements of tumormarkers. The new days might not be so different from the old days
  • We found the CEA-CM analysis in healthy persons over a time period of one year to be variable but in the end returning to baseline values. Multiple measurements in time provide this baseline. This finding is in line with the established way of working with tumormarkers, where multiple measurements in time with elevations of more than 50% in 2 consecutive measurements is considered clinically relevant.
  • Shown here a result of the fluorochrome durability one patient with on the x-axis the time from venipuncture and on the y-axis the median fluorescence intensity. One can notice the APC signal to rise and fall over time and the FITC signal is climbing slightly over time. The PE-signal in this case slightly decreases. In the next slide all patients are visible in one sheet.
  • Remembering the colour setup from last slide we have positioned all patients in one slide to provide an overall idea of the fluorochrome durability. In these seven patients the fluorochrome durability shows various scenarios, APC and FITC signal vary clearly over time. There is no general increase in aspecific binding of the external fluorochromes in these 7 patients. And by using the standardized gating strategy the qualitative separation of the populations is not affected. One of the good things here to see is that the internal cell staining with PE which is providing the quantitative analysis shows consistency over time.
  • 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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