Quantification and characterization of plasma cells by ...

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John Cazale. In The Godfather I/II/III. Peter Boyle in Taxi Driver. Jim Davis. In Dallas. Paul Kent. In Star Trek II. What do have these. (male) actors in common?
Quantification  and  characterization  of   plasma  cells  by  flow  cytometry:  principles   and  pitfalls   N.  Bailly  –  Alex  Dromelet  –  Pr  François  Mullier   –  Pr  Bernard  Chatelain  

MACS  FlowDay  –  October  2,  2014   Leiden  -­‐                    

Intro  

Roy  Scheider   In  Jaws  

John  Cazale  

In  The  Godfather  I/II/III  

Peter  Boyle   in  Taxi  Driver  

What  do  have  these   (male)  actors  in   common????  

Jim  Davis   In  Dallas  

Paul  Kent   In  Star  Trek  II  

What  is  a  multiple  myeloma?   *  Multiple  Myeloma  =  Kahler’s  disease.   *  Etimiology:   *  myelo  =  marrow   *  oma  =  tumor  

*  Hemopathology  where  plasma  cells  are   involved   Otto  Kahler  

What  is  a  multiple  myeloma?   *  Plasma  Cell  neoplasm  

*  Monoclonal  Gammopathy  of  Undetermined  Significance  (MGUS)   *  Plasma  Cell  Myeloma   *  Multiple  Myeloma   *  Variant  

*  Asymptomatic  myeloma   *  Non-­‐secretory  myeloma   *  Plasma  Cell  Leukemia  

*  Immunoglobulin  deposition  diseases  

*  Primary  amyloidosis   *  Systemic  light  and  heavy  chain  deposition  diseases  

*  Osteosclerotic  myeloma  (POEMS  syndrome)  

*  Result  from  expansion   of  a  clone  of  a   monoclonal  Ig-­‐ secreting  terminally   differentiated  B  cells.  

Interest  of  Flow  Cytometry  in   plasma  cells  disorders  

Interest  of  Flow  Cytometry  in  plasma   cell  disorders   *  Diagnosis  and  differential  diagnosis  of   plasma  cell  disorders  

*  Reactive  Plasmocytosis  or  plasma  cell   leukemia   *  MGUS  or  benign  plasmocytes   *  MM  IgM  or  Waldenström   *  Reactive  plasmocytosis  or  relapsed  MM  post   allotransplant  

*  Risk  assessment  

*  Transformation  or  relapse  

*  Targeted  Therapies  

Interest  of  Flow  Cytometry  in  plasma   cell  disorders   *  Differenciation  between   myelomatous  and  benign   plasma  cells   *  Benign:     *  Immunophenotype:  CD38+++,   CD56-­‐,  CD19+  &  CD45+   *  Myelomatous:     *  Cytology:  Anarchic  criterias:   anisocytosis,  anisokaryosis,   nucleoles,  multiple  nucleus,…   *  Immunophenotype:  CD38++,   CD56+++,  CD19-­‐  &  CD45-­‐  

Benign  PC  

Multiple  Myeloma  PC  

CD138  

++  

+++  

CD38  

+++  

++  

CD19  

+  

-­‐  (>80%)  

CD28  

-­‐  

+  (36-­‐49%)  

CD56  

-­‐  

+  (60-­‐78%)  

CD27  

+  

+  ou  -­‐  (50%)  

CD45  

+/-­‐  

-­‐/+  

Pitfalls  &  Keypoints  for  plasma   cell’s  characterization  by  FCM  

Keypoint  1:  the  sample   Flow  Cytometry  è  required  individualized  cells.  

*  Bone  marrow     *  Cells  are  run  through  a   needle  (21G)  

*  Whole  blood   *  Cells  already  in  suspension  

*  Red  Blood  Cell’s  Lysis  is   required  

*  Plasmocytes  could  be   considered  as  rare  events   *  Enrichment  required  

*  Ficoll,  Magnetic  Beads  è   Only  to  characterize,  not   counting  

*  Washing  Steps?  

*  Could  not  be  performed  for   cell  counting   *  Cell  losts:  ~20%  for  each   washing  steps  

Keypoint  2:  The  quantification  

*  (relative)  Quantification:   *  Morphology  >  Flow  Cytometry   *  Paiva  et  al.  Haematologica  2009   *  Rawtron  et  al.  Haematologica   2008  

Keypoint  2:  The  quantification   *  Why?   *  Heterogenous  distribution  of  plasmocytes  in  Bone   Marrow   *  Aspirate  sample  used  in  FCM:  often  a  second-­‐draw  bone   marrow  aspirate   *  Contamination  by  peripheral  blood   *  Selection  of  area  with  a  lot  of  plasma  cells  by  the   cytologist   *  Adhesion  to  lipids  (Nadav  et  al.  BJH  2006)   *  Quantification  on  biopsy:  probably  more  accurate  but   standardization  is  needed  

Keypoint  2:  The  quantification   *  Solution  to  the  discordance  between  morphology  and  FCM:   *  Loken  et  al  (Cytometry  Part  B  2009):     *  Normalized  blasts  %=  (80%/%  neutros  dimCD16)  *  %  blasts  

*  Brooimans  et  al  (Cytometry  Part  B  2009):       *  BM  purity:  [1-­‐(RBCBM/RBCPB)*  ((WBCPB/WBCBM)]*100%  

*  Frébet  et  al  (Cytometry  Part  B  2010):     *  plasma  cells%/  precursors  (dimCD45  CD117+CD34+)  

*  Monoclonal  hemopathology  

*  Reminder:  Result  from  expansion  of  a   clone  of  a  monoclonal  Ig-­‐secreting   terminally  differentiated  B  cells.  

*  Requires  the  analyzis  of  intracellular   light  chains  

On  Bone  Marrow  

Keypoint  3:  Expression  of  Light   Perfix  NC  Permeab   Chains  

*  Use  of  permeabilizing  solution  

*  Some  of  them  require  RBC  to  perform   permeab.  (i.e.:  Beckman  Coulter  PerFix   NC)   *  Could  NOT  be  performed  on  separated   cells,  lymph  nodes,…  

On  PBMC  

*  Kappa   *  Lambda  

Keypoint  4:  The  gating  strategy   *  Usually  

*  Use  of  CD45  to  select  WBC  

*  But…  

*  Myelomatous  plasma  cells:  CD38++,  CD56+++,  CD19-­‐  &  CD45-­‐   *  Benign  plasma  cells:  CD38+++,  CD56-­‐,  CD19+  &  CD45+  

*  Adapt  your  gating  strategy  

*  Don’t  use  CD45  as  primary  discriminant  antigen   *  Use  relevant  markers   *  Take  care  of  some  variations  (some  plasmoblasts  are  CD138  neg)  

CD138   CD38   CD19   CD28   CD56   CD27   CD45  

Benign  PC  

Multiple  Myeloma   PC  

++   +++   +   -­‐   -­‐   +   +/-­‐  

+++   ++   -­‐  (>80%)   +  (36-­‐49%)   +  (60-­‐78%)   +  ou  -­‐  (50%)   -­‐/+  

*  Many  antibodies  are   available.   *  The  B-­‐B4  clone  from   Miltenyi  seems  to   give  the  best  Signal/ Noise  ratio   *  The  choice  of  the   clone  could  have  an   impact  on  the  result  

S/N  

Keypoint  5:  Choice  of  the  clone  

A.Rawstron, Utility of FCM fior MRD Monitoring in Myeloma, ESCCA - Budapest 2012

Keypoint  6:  Patients  under  treatment  

*  New  treatment:  Daratumumab®   *  Anti-­‐CD38  

*  Usually  

*  Plasma  cells  are  defined  as  CD138+  and  CD38+   *  By  the  way,  CD38+  isn’t  «  expressed  »  in  flow  cytometry  for   Daratumumab  treated  patients  

*  Solution:  use  CD138  and  other  aberrant  markers  to  identify   plasmocytes  (CD56+/CD19-­‐/CD45-­‐  and  eventually  the  light  chains)   *  Don’t  forget  to  ask  the  clinician  about  the  treatment  to  avoid  any   nasty  surprises  

Take-­‐home  message!  

*  Flow  Cytometry  is  an  useful  tool  to  characterize   plasmocytes  and  diagnoses  multiple  myeloma  (vs   other  hematopathologies).   *  There  are  some  pitfalls  to  take  in  consideration   *  Each  of  them  as  an  appropriate  solution  

Acknowledgments  

Thanks  for  your  attention   *  Pr  Bernard  Chatelain   *  Pr  François  Mullier   *  Alex  Dromelet