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The European Myeloma Network www.myeloma-europe.or
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Report of the European Myeloma Network (EMN)
workshops on multi-parametric flow cytometry in
multiple myeloma and related disorders
Andy C. Rawstron, Alberto Orfao, Meral Beksac,
Ludmila Bezdícková, Rik A. Brooimans, Horia
Bumbea, Klara Dalva, Gwenny Fuhler, Jan Gratama,
Dirk Hose, Lucie Kovarova, Michael Lioznov, Gema
Mateo, Ricardo Morilla, Anne K. Mylin, Paola
Omede, Roger G. Owen, Catherine
Pellat-Deceunynck, Martin Perez Andres, Maria
Petrucci, Marina Ruggeri, Grzegorz Rymkiewicz,
Alex Schmitz, Martin Schreder, Carine Seynaeve,
Martin Spacek, Els Van Valckenborgh, Nicky
Weston-Bell, Jesús San Miguel, Pieter Sonneveldt,
Hans E. Johnsen on behalf of the European Myeloma
Network

• 30 participants from 13 countries at two
  workshops undertook a review of procedures and
  data analysis to identify areas of consensus
• Register at www.myeloma-europe.org to participate
  in
• Harmonisation of procedures for diagnosis and
  MRD detection
• Data analysis quality control development

• Clinical Indications
• Differential Diagnosis between Myeloma and other
  Monoclonal Gammopathies
• Demonstration of the presence of phenotypically
  aberrant plasma cells can be used to
  differentiate monoclonal gammopathies from
  reactive conditions
• The ratio between phenotypically normal and
  aberrant plasma cells can be used to predict the
  risk of disease progression in MGUS and
  asymptomatic myeloma
• Minimal Residual Disease Detection
• Multiparametric flow cytometry is a feasible and
  sufficient method for residual disease monitoring
  and evaluation of response to therapy. This
  application of flow cytometry is likely to
  increase, and it will require the development of
  standardized approaches with defined specificity
  and sensitivity, along with suitable quality
  control schemes.

Technical Considerations Plasma cell enumeration
continued use of a morphological assessment of
plasma cell percentage is necessary for
concordance with the current diagnostic criteria.
Discrepancies between flow cytometry and
morphology are primarily due to the sample
quality and flow cytometric enumeration may be
more reliable at predicting outcome than
morphological assessment Sample preparation any
whole blood/marrow approach is suitable for PC
enumeration and phenotyping while density
gradient centrifugation is inappropriate. Primary
gating reagents the use of four or more colour
analysis are recommended. Two-colour
immunophenotypic analyses are not feasible as at
least two antigens are required to gate PC
accurately. At least one tube should incorporate
CD38, CD45 and CD138 in combination, with further
tubes including either CD38 or CD138 (preferably)
or CD38 and CD45. If sufficient detectors are
available, the optimal approach would include
CD38, CD45 and CD138 in all tests. The primary
gate should be set to include CD38CD138
events Controls for gating and immunophenotyping
a control for the gating reagents is not
necessary. Controls for staining should be in
accordance with general consensus (H43-A2,
NCCLS) Number of events to analyse the minimum
number required for accuracy is 100. For a limit
of sensitivity of 0.01, then the minimum number
of total events required is 1,000,000. This can
be acquired over more than one tube Clonality
Assessment assessment of kappa/lambda expression
by flow cytometry is appropriate for the
assessment of a stringent complete remission
according to the Durie criteria. Immunophenotypic
analysis is more sensitive than clonality
assessment by immunohistochemistry and/or flow
cytometry for the detection of residual disease
but combined assessment of clonality with basic
immunophenotype may be useful for screening at
diagnosis and follow-up Antibodies for diagnosis
and MRD it is not possible to perform MRD
assessment using only one test antigen. In
addition to the PC gating markers discussed
above, the minimal test antigens for assessing
MRD analysis are CD19 and CD56. A preferred panel
would incorporate CD20 and CD117. Assessment of
CD28, and CD27 is also recommended. Members of
EMN collaborative studies will assess these
reagents and also CD200 and CD81 Measure of
sample quality the sample is suitable for
quantitative MRD analysis if normal PC
(CD19CD56- and/or polyclonal) are detectable. If
normal PC are not detected the quality of sample
should be assessed by morphology and/or the
presence of normal erythroid, myeloid or
B-progenitors. If there are no marrow elements
present but neoplastic PC are detected, the
sample should be reported as MRD-positive but
note that the sample is unsuitable for
quantitative assessment. If there are no marrow
elements and no PC, the sample should be reported
as unsuitable for analysis.