1 / 20

IMMUNOPHENOTYPING OF HEMATOPOIETIC PROGENITOR CELLS BY MULTIPARAMETER FLOW CYTOMETRY

IMMUNOPHENOTYPING OF HEMATOPOIETIC PROGENITOR CELLS BY MULTIPARAMETER FLOW CYTOMETRY. PROTOCOLS FOR CD34 COUNT Main Features Mulhouse All cells acquired, FSC vs SSC and SSC vs CD34 Milan All cells acquired, FSC vs SSC and SSC vs CD34

aldis
Download Presentation

IMMUNOPHENOTYPING OF HEMATOPOIETIC PROGENITOR CELLS BY MULTIPARAMETER FLOW CYTOMETRY

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. IMMUNOPHENOTYPING OF HEMATOPOIETIC PROGENITOR CELLS BY MULTIPARAMETER FLOW CYTOMETRY

  2. PROTOCOLS FOR CD34 COUNT Main Features Mulhouse All cells acquired, FSC vs SSC and SSC vs CD34 Milan All cells acquired, FSC vs SSC and SSC vs CD34 Nordic All cells acquired, FSC vs SSC and SSC vs CD34 ISHAGE CD45+ cells acquired, CD45 vs SSC, CD34 vs SSC, CD45 vs SSC, FSC vs SSC ProCount CD34 bright cells acquired, DNA vs SSC, CD45 vs SSC, DNA vs CD34

  3. ISHAGE PROTOCOL gated on R1 R1 SSC SSC R2 CD45 CD34 gated on R1 and R2 gated on R1 and R2 and R3 SSC SSC R4 R3 CD45 FSC

  4. MILAN PROTOCOL R2 CD34 FSC R1 SSC SSC

  5. RPCI PROTOCOL gated on R1 AND R2 AND R3 gated on R1 OR R2 R1 SSC R4 R5 R6 CD34 R2 SSC R3 CD45 SSC FSC R1 R2 R4 R5 R6 SSC CD34 SSC R3 CD45 SSC FSC

  6. R1 R1 SIDE SCATTER FORWARD SCATTER

  7. CD34 SSC MOSTLY BRIGHT AVERAGE BRIGHT MOSTLY DIM

  8. Comparison of Gating Strategies • %CD34 positive* • RPCI 1.33 ± 1.44 • ISHAGE 0.87 ± 1.30 • ISHAGE (no CD45 gate) 1.35 ± 1.44 • *n = 80

  9. What CD34 Count is Important? • Protocol to Use • Pre or Post Thaw Count? Any • CD34 bright? or CD34 dim? RPCI • CD45+? CD45-? Any, except ISHAGE

  10. CD34 CD14 threshold CD45 SSC

  11. R3 R3 R3 UNGATED R1 R4 R5 R9 CD34 SSC CD14 R2 R6 R7 R8 FSC CD45 FSC R4 R5 R9 GATED ON R1 GATED ON R1 AND R3 R6 R7 R8 CD14 SSC R10 R11 R15 GATED ON R2 GATED ON R2 AND R3 R12 R13 R14 CD45 FSC

  12. FREQUENCY OF PATIENTS EXPRESSING CD34 BRIGHT HPCs PERCENT OF PATIENTS CD34 BRIGHT (%TOTAL CD34+ HPCs)

  13. CD45 EXPRESSION ON HPCs PERCENT

  14. 100 80 60 40 20 0 Proportion of Subset in CD34+ Population % of Subset in CD34+ Population -/- -/+ +/- +/+ -/- -/+ +/- +/+ CD38 HLADr CD45RO CD45RA

  15. Proportion of Subset Cells in CD34+ Population % of Subset CD34+

  16. Measurement of CD34 Subset Proliferation number number HLADr TC Hoechst Hoechst number number CD38 FITC Hoechst Hoechst

  17. C-KIT RECEPTOR EXPRESSION ON HPC SUBSETS H CD117 CD117 L A D r CD38 CD117 CD117

More Related