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
* 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
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,…
* 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