Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders.

Abstract

The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

Full text

Page 1

DECISION MAKING AND PROBLEM SOLVING
Report of the European Myeloma Network on
multiparametric flow cytometry in multiple myeloma
and related disorders
Andy C. Rawstron*,1Alberto Orfao*,2Meral Beksac,3Ludmila Bezdickova,4Rik A. Brooimans,5
Horia Bumbea,6Klara Dalva,3 Gwenny Fuhler,7 Jan Gratama,5 Dirk Hose,8 Lucie Kovarova,9
Michael Lioznov,10Gema Mateo,2Ricardo Morilla,11Anne K. Mylin,12Paola Omedé,13Catherine Pellat-
Deceunynck,14Martin Perez Andres,2Maria Petrucci,15Marina Ruggeri,13Grzegorz Rymkiewicz,16
Alexander Schmitz,17Martin Schreder,18Carine Seynaeve,19Martin Spacek,4Ruth M. de Tute,1
Els Van Valckenborgh,19Nicky Weston-Bell,20Roger G. Owen,1Jesús F. San Miguel,2Pieter Sonneveld,21
Hans E. Johnsen,22on behalf of the European Myeloma Network23
1The HMDS, Leeds Teaching Hospitals NHS Trust, Leeds, UK;2General Cytometry Service, Department of Medicine and
Cancer Research Center (IBMCC), University of Salamanca/CSIC and Department of Haematology, University Hospital
of Salamanca, Salamanca, Spain;3Department of Haematology, Ankara University, Ankara, Turkey;4Department of
Clinical Haematology, University Hospital Kralovske Vinohrady, Prague and 3rdFaculty of Medicine, Charles University,
Prague, Czech Republic;5Department of Internal Oncology, Erasmus MC, Daniel den Hoed Cancer Center, Rotterdam,
the Netherlands;6Emergency University Hospital, Department of Hematology, "Carol Davila" University of Medicine,
Bucharest, Romania;7Dept. of Cell Biology Section Immunology University Medical Center Groningen, Groningen, The
Netherlands;8Universtitäsklinikum Heidelberg, Medizinische Klinik V, Sektion Multiples Myelom, Multiple Myeloma
Research Laboratory, Heidelberg, Germany;9Dept. of Hematology, University Hospital, Brno, Czech Republic;10University
Medical Centre Hamburg - Eppendorf Clinic for Stem Cell Transplantation, Hamburg, Germany;11Section of Haemato-
Oncology, Institute of Cancer Research, Sutton, Surrey, UK;12Department of Haematology, Rigshospitalet University of
Copenhagen, Denmark;13Flow Cytometry Lab., Divisione di Ematologia dell'Università di Torino, Italy;14Inserm U601
and Laboratoire d'Hématologie, CHU de Nantes, France;15University Rome La Sapienza, Dept. of Cellular Biotechnology
and Hematology, Rome, Italy;16Flow Cytometry Lab, Department of Pathology, the Maria Skodowska-Curie Memorial
Cancer Center and Institute of Oncology, Warsaw, Poland;17Department of Haematology, Aalborg Hospital Science and
Innovation Center, Aarhus University Hospital, Aalborg, and Department for Molecular Biology, Aarhus University,
Aarhus, Denmark;18Wilhelminen-Krebsforschungsinstitut 1stDepartment of Medicine Wilhelminenspital Wien, Vienna,
Austria;19Vrije Universiteit Brussel, Brussels, Belgium;20University of Southampton, Southampton, UK;21Dept of
Hematology, Erasmus MC, Rotterdam, the Netherlands;22Department of Haematology, the Research Laboratory,
Aalborg Hospital Science & Innovation Center, Aarhus University Hospital, Aalborg, Denmark, and 23The European
Myeloma Network (www.myeloma-europe.org) Secretariat, Aalborg Hospital Science and Innovation Center, Aarhus
University Hospital, Aalborg, Denmark
*These authors equally contributed
to this manuscript and should be
considered joint first authors.
Funding: the meetings were
sponsored by the European Myeloma
Network: www.myeloma-europe.org
Manuscript received November 23,
2006.
Manuscript accepted November 6,
2007.
Correspondence:
Hans E.Johnsen,MD,DMSC,
Professor in Hematology,Department
of Hematology,the Research
Laboratory,Aalborg Hospital Science
and Innovation Center,Sdr.Skovvej
15,9000 Aalborg,Denmark.
E-mail: haej@rn.dk
This is an open access paper.
The online version of this article con-
tains a supplemental appendix.
ABSTRACT
The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to
identify specific indications for flow cytometry in patients with monoclonal gammopathies, and con-
sensus technical approaches through a questionnaire-based review of current practice in participat-
ing laboratories.The second aimed to resolve outstanding technical issues and develop a consensus
approach to analysis of plasma cells. The primary clinical applications identified were: differential
diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progres-
sion in patients with MGUS and detecting minimal residual disease.A range of technical recommen-
dations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one
tube for plasma cell identification and enumeration.The primary gate should be based on CD38 vs.
CD138 expression; 2) after treatment,clonality assessment is only likely to be informative when com-
bined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating
a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should
include CD19 and CD56.A preferred panel would also include CD20, CD117, CD28 and CD27; 4)
discrepancies between the percentage of plasma cells detected by flow cytometry and morphology
are primarily related to sample quality and it is, therefore, important to determine that marrow ele-
ments are present in follow-up samples, particularly normal plasma cells in MRD negative cases.
Key words: flow cytometry,myeloma,monoclonal gammopathies of undetermined significance.
Citation: Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA, Bumbea H, Dalva K, Fuhler G,
Gratama J,Hose D,Kovarova L,Lioznov M,Mateo G,Morilla R,Mylin AK,Omedé P,Pellat-Deceunynck C,
Perez Andres M,Petrucci M,Ruggeri M,Rymkiewicz G,Schmitz A,Schreder M,Seynaeve C,Spacek M,
de Tute RM,Van Valckenborgh E,Weston-Bell N,Owen RG,San Miguel JF,Sonneveld P,and Johnsen HE,
on behalf of the European Myeloma Network.Report of the European Myeloma Network on multipara-
metric flow cytometry in multiple myeloma and related disorders.Haematologica 2008 Mar; 93(3):431-
438.doi: 10.3324/haematol.11080.
haematologica | 2008; 93(3) | 431 |

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A.C. Rawstron et al.
| 432 | haematologica | 2008; 93(3)
Introduction
Clinical indications for the use of flow cytometry in
multiple myeloma
At present, immunophenotyping is mandatory for the
diagnosis and monitoring of acute leukemias and chronic
lymphoproliferative disorders.1-4By contrast, in multiple
myeloma, the use of multiparametric flow cytometry in
many clinical diagnostic laboratories is currently restricted
to clinical research studies and the differential diagnosis of
unusual cases.5-7However, the generation and identifica-
tion of markers that allow the unequivocal identification of
plasma cells among other hematopoietic cells (such as
CD138), and the identification of aberrant plasma cell phe-
notypes that enable us to discriminate between normal
and neoplastic plasma cells,8-10means we can identify, char-
acterize and enumerate neoplastic plasma cells even when
few cells are present. This offers several advantages over
other techniques and there is growing evidence in litera-
ture concerning the potential clinical benefit of immuno-
phenotyping plasma cells by flow cytometry in patients
diagnosed and/or suspected of suffering from myeloma or
other plasma cell disorders. The advantages of flow
cytometry in the diagnosis and monitoring of monoclonal
gammopathies can be broadly categorised into three main
topics (Table 1): (i) primary diagnosis of myeloma and
associated disorders, based on the enumeration of plasma
cells in the bone marrow and demonstration that a propor-
tion are phenotypically abnormal, monoclonal and not
reactive. The ability to assess multiple markers in combi-
nation with clonality assessment provides more specific
information than can be obtained by other diagnostic tech-
niques, such as immunohistochemistry; (ii) identification
of independent prognostic markers, in particular those pre-
dicting the risk of progression for patients with MGUS and
asymptomatic myeloma based on the relative proportions
of abnormal and normal plasma cells; (iii) quantitative
evaluation of minimal residual disease (MRD) levels for
assessing efficacy of treatment and prediction of outcome,
as well as the determination of stringent complete remis-
sion as defined by the International Myeloma Working
Group (IMWG).11This article combines a review of the lit-
erature concerning the application of flow cytometry for
the diagnosis of myeloma and other plasma cell disorders,
as well as practical guidelines drawn up from an analysis of
consensus views and group data analysis performed at two
workshop meetings of the European Myeloma Network.
Plasma cell enumeration
Accurate quantitation of the plasma cell burden in bone
marrow is essential for the diagnosis of myeloma.12;13Most
laboratories assess the extent of plasma cell infiltration by
morphological examination of stained bone marrow aspi-
rate samples and trephine sections.14-17The limited use of
flow cytometry in the analysis of myeloma has probably
been due to the well-documented discrepancy in the plas-
ma cell percentage observed between flow cytometry and
conventional microscopy in overall enumeration of plasma
cells in bone marrow samples from myeloma patients.8;18-20
Notably, these discrepancies affect all laboratory investiga-
tions, including cytogenetics/FISH and molecular studies.
The main reason for the discrepancy is the use of a sec-
ondary aspirate for laboratory studies, which is usually of
poorer quality than the primary aspirate taken for morpho-
logical assessment. Counting errors and expression of cer-
tain adhesion molecules may also have an impact.21-23The
causes of the discrepancy are described in detail in the
online appendix. It was emphasized that recent studies
have demonstrated that plasma cell enumeration by flow
cytometry is of greater prognostic value in myeloma
patients than a morphological plasma cell count.20
Plasma cell enumeration consensus
Current diagnostic criteria require morphological
assessment of plasma cell percentage. This is helpful in
providing a global assessment of the sample.
Discrepancies between the plasma cell percentage
detected by flow cytometry compared with morpholog-
ical enumeration are primarily due to the sample quali-
ty and it is likely that the use of first-pull aspirate sam-
ples for immunophenotyping will largely remove the
inconsistency. These discrepancies affect all laboratory
studies, including cytogenetics/FISH and molecular
studies: reducing sampling artefact will benefit all labo-
ratory studies. Flow cytometric enumeration of plasma
cells may be more reproducible and reliable at predict-
ing outcome in myeloma than morphological assess-
ment since larger number of cells are analyzed and there
is less operator bias. However, further studies are
required to confirm this.
Table 1. Consensus medical indications of multiparametric flow
cytometry immunophenotyping in the study of multiple myeloma
and other monoclonal gammopathies.
Clinical applicationParameters measured by flow cytometry
Differential diagnosis
between myeloma,MGUS
and reactive conditions
(i) Plasma cells as a percentage of total
leucocytes.
(ii) Plasma cell immunophenotype
(see Table 2)
(iii) Plasma cell clonality
(iv) Abnormal plasma cells as a percentage
of total plasma cells
Prognostic markers in myelomaExpression of specific antigens by abnormal
plasma cells,e.g.CD45/CD56/CD117/CD28
Prediction of outcome for patients Abnormal plasma cells as a percentage
with MGUS and asymptomatic of total plasma cells
myeloma
Detection of minimal residual
disease in myeloma patients
after treatment and determination κ/λ,as a percentage of either
of a stringent complete response total leukocytes or as a percentage of total
plasma cells; requires high sensitivity
assessment
Abnormal plasma cells,identified by
immunophenotype and cytoplasmic

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EMN: flow cytometry in plasma cell disorders
haematologica | 2008; 93(3) | 433 |
Differential diagnosis of myeloma and other
monoclonal gammopathies
The primary role of flow cytometry in participating lab-
oratories was to demonstrate abnormal and/or monoclon-
al plasma cells as part of the diagnosis of myeloma. A large
body of evidence has been collected to demonstrate that
neoplastic bone marrow plasma cells from myeloma
patients and other monoclonal gammopathies display
aberrant phenotypes10;24-26and restricted immunoglobulin
(Ig) light chain expression at the cytoplasmic9,27and, to a
lesser extent, at the surface membrane level.28Based on
these features, unequivocal identification and enumeration
of aberrant and normal plasma cells co-existing in a bone
marrow sample can be performed.29 These immunopheno-
typical features are described in Table 2 and below in the
section Antigen expression on normal and neoplastic plasma
cells. Therefore, the demonstration of restricted immuno-
globulin coupled with an abnormal immunophenotype
can be used to distinguish between reactive and neoplastic
conditions. Immunophenotyping of plasma cells is recom-
mended in the differential diagnosis between myeloma
and monoclonal gammopathy of undetermined signifi-
cance (MGUS), for the identification of aberrant pheno-
types present in clonal plasma cells at diagnosis that could
be used later during patient monitoring, and for the evalu-
ation of minimal residual disease after therapy. Additional
medical indications of multiparametric flow cytometry
immunophenotypical studies at diagnosis include the dif-
ferential diagnosis of unusual cases. For example,
immunophenotyping can help to distinguish rare cases of
IgM myeloma, where the predominant population will
have the phenotype of abnormal plasma cells, from other
IgM secretory disorders which have distinct phenotype.45
Similarly the demonstration of abnormal plasma cells may
be useful in the diagnosis of patients with non-secretory
myeloma or primary amyloidosis. Immunophenotyping at
diagnosis may also be useful for the identification of
potential therapeutic targets (e.g. CD52 and CD20).46,47
Differential diagnosis: consensus
Demonstration of the presence of phenotypically aber-
rant plasma cells can be used in the differential diagnosis
between MGUS, myeloma and reactive conditions.
Identification of prognostic markers and
immunophenotypical screening of cytogenetic
abnormalities in myeloma and MGUS
The prognostic value of immunophenotyping has not
yet been clearly established.29Several studies have demon-
strated an association between antigenic profile and specif-
ic genetic abnormalities24,27,30,48,49but this is not strong
enough for immunophenotyping to be used to screen for
genetic abnormalities in myeloma. The detection of circu-
lating plasma cells31-33,50,51and the CD45 expression pat-
tern21,34are also reported to be a highly significant prognos-
tic factor but further work is required to define the role of
this assay in routine clinical use. These studies are
described in more detail in the Online appendix.
Arguably one of the most useful prognostic factors that
has been identified is the ratio of abnormal/normal plasma
cells in the bone marrow of patients with MGUS and
asymptomatic myeloma. This affects a large group of
patients whose outcome is currently difficult to predict
from presentation features. The presence of a great major-
Figure 1. Illustrating examples of basic
immunophenotype and clonality assess-
ment to screen for the presence of resid-
ual disease in bone marrow samples from
multiple myeloma patients. The plots
show bone marrow cells from two myelo-
ma patients in morphological remission
prepared using a fixation and permeabi-
lization procedure and demonstrate typical
profiles for CD19, cytoplasmic kappa and
cytoplasmic lambda expression on gated
plasma cells. The plots on the left show
CD19 vs. CD45 expression on the gated
plasma cells: CD19+plasma cells are col-
ored in green and the CD19–plasma cells
in red. Kappa vs. Lambda expression for
CD19+normal plasma cells is shown in the
middle plots and for CD19–/CD45- plasma
cells in the right plots, with the percentage
of gated cells noted in the relevant
regions.
The upper three plots are from a patient with 0.04% total plasma cells at day 100 after high dose therapy: the CD19–plasma cells
are light-chain restricted while a small population of CD19+plasma cells is polyclonal. This demonstrates that low levels of resid-
ual disease can be identified and enumerated using basic immunophenotyping and clonality assessment. The lower three plots
are from a patient in continued complete remission several years after high dose therapy: the majority of plasma cells are CD19+
but some CD19–plasma cells are detectable and both the CD19+and CD19–fraction appear polyclonal. CD19–plasma cells are
present in normal individuals and are not necessarily neoplastic. In this case, extended analysis confirmed that both the CD19+
and CD19–fractions of plasma cells were normal. Screening approaches can only exclude the presence of residual disease if all
the plasma cells are CD19+in a patient known to have CD19–disease.
(a)
48%
52% 2%
66%64%
98%
34%36%
(b)
-1,384 0 103
104
105
CD45 APC-Cy7-A
CD19 PE-A
-837
0
103
104
105
CD19 PE-A
-1,328 0
103
104
105
LAMBDA PE-Cy5-A
-439
0
103
104
105
LAMBDA PE-Cy5-A
-439
0
103
104
105
LAMBDA PE-Cy5-A
-377
0
103
104
105
LAMBDA PE-Cy5-A
-377
0
103
104
105
-1,275 0 103
104
105
CD45 APC-Cy7-A
-78 0 102
103
104
105
KAPPA FITC-A
-78 0 102
103
104
105
KAPPA FITC-A
0101102
103
104
105
KAPPA FITC-A
0101102
103
104
105
KAPPA FITC-A

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A.C. Rawstron et al.
| 434 | haematologica | 2008; 93(3)
ity of abnormal plasma cells (>97% of total bone marrow
plasma cells) is typically seen in myeloma while the pres-
ence of normal plasma cells (>3% of total bone marrow
plasma cells) is more consistent with a diagnosis of MGUS,
although the final distinction between MGUS and myelo-
ma will be dependent on a morphological assessment of
total plasma cell infiltration and on other clinical features.
However, both MGUS and asymptomatic myeloma
patients with a high ratio of abnormal/normal plasma cells
have a greatly increased risk of progression to myeloma
and this is one of the most powerful prognostic factors that
can be identified at presentation.35,36
Prognostic markers: consensus
Several marker combinations, particularly that of CD117
and CD28, show promise in predicting outcome for myelo-
ma; (i) Immunophenotyping is of limited value for the diag-
nostic screening of specific genetic abnormalities in myelo-
ma; (ii) further collaborative studies, including the exchange
of original flow cytometry data files, are required to reach
consensus on the relevance of CD45 expression in myelo-
ma; (iii) the ratio between phenotypically normal and aber-
rant plasma cells can be used to predict the risk of disease
progression in MGUS and asymptomatic myeloma.
Detection of minimal residual disease by flow cytometry
Response assessment using serum or urine paraprotein
assessment can be held back by the long half life of some
immunoglobulin molecules37,52while serum free light chain
is relatively insensitive.53Direct assessment of bone mar-
row tumor load is more predictive of outcome. Allele-spe-
cific oligonucleotide (ASO) PCR is highly sensitive but can
be costly, time-consuming and can have limited applicabil-
ity.38,54-61It is possible to detect neoplastic plasma cells by
flow cytometry above the clinically relevant threshold of
0.01%38,57and this is more informative than conventional
assessment.39,40,62Flow cytometry for residual disease detec-
tion is applicable to almost all patients, more sensitive than
paraprotein or light chain assessment, and considerably
cheaper than PCR analysis. A more detailed comparison of
the different approaches to disease monitoring is provided
in the online appendix.
Minimal residual disease: consensus
Multiparametric flow cytometry is a feasible and ade-
quate method for monitoring residual disease and evaluat-
ing response to therapy. This application of flow cytome-
try is likely to become more widespread, and will require
the development of standardized approaches with defined
specificity and sensitivity, along with suitable quality con-
trol schemes.
EMN consensus approaches and techniques for flow
cytometry in monoclonal gammopathies
The following sections discuss the key issues for
immunophenotyping bone marrow samples at diagnosis
and for detecting residual disease by flow cytometry in
myeloma, with identification of consensus approaches
where available. Unless otherwise stated, the methods
refer to the characterization of plasma cells in bone mar-
row samples and most studies can be undertaken using a
flow cytometer capable of detecting a minimum of three
fluorochromes. Cytometers capable of detecting four or
more colors are to be prefered since they can more easily
identify and reproduce abnormal populations, and can
reduce the time and cost involved in acquiring and analyz-
ing data.
Sample preparation
The majority of participants used a fixative-free erythro-
cyte lysis method for enumeration and phenotypical char-
acterization while a smaller proportion of centres used
whole blood/marrow methods that contain a fixative for
enumeration. Fixation and permeabilization methods were
frequently used in addition to the fixative-free lysis
method for analysis of intracellular immunoglobulin
expression. Two centres used a density gradient centrifu-
gation approach prior to immunomagnetic separation, cell
culture, and/or immunophenotyping. The percentage of
plasma cells detected does not differ significantly between
these approaches except for density gradient centrifuga-
tion which may result in a variable increase or decrease in
the percentage of plasma cells compared with whole
blood/marrow approaches. This approach is not, there-
fore, suitable for enumeration of plasma cells.
Sample preparation: consensus
Any whole blood/marrow approach is suitable for plas-
ma cell enumeration and phenotyping. Density gradient
centrifugation is inappropriate for plasma cell enumeration
but may be suitable for some applications.
Table 2. List of most useful antigens for the detection of aberrant
plasma cells in multiple myeloma.5-10;20-44
Antigen Normal expression profile Abnormal Percentage of Requirement
(percentage expression expression myeloma cases for diagnosis
on normal plasma cells)profilewith abnormal and monitoring
expression
CD19Positive (>70%)Negative 95%Essential
CD56Negative (<15%)Strongly positive 75%Essential
CD117Negative (0%)Positive 30%Recommended
CD20 Negative (0%)Positive30% Recommended
CD28Negative/weak (<15%) Strongly positive 15-45% Recommended
CD27Strongly positive (100%) Weak or negative 40-50% Recommended
CD81Positive (100%)Weak or negative Not Suggested
published
Not
published
CD200Weakly positiveStrongly
positive
Suggested

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Primary gating antibodies
The identification of an accurate gating strategy is a crit-
ical component of a reproducible and sensitive immuno-
phenotypical assay for the analysis of plasma cells. A vari-
ety of approaches based on CD38, CD138 and/or CD45
expression were used. The majority of centers reported a
gating strategy using combined CD38, CD138 and light
scatter characteristics. It was noted that there was no for-
mally published consensus method for gating plasma cells
and this was therefore addressed directly at the Leeds
meeting by experimentally comparing the different gating
strategies. This is described in detail in the Online appendix.
Using CD38 vs. side scatter gives false negative results for
cases with relatively weak CD38 expression on the neo-
plastic plasma cells. Using CD38 vs. CD138 improves the
detection of small plasma cell populations but there is a
high contamination rate which inhibits the ability to
demonstrate an abnormal phenotype. Using CD38 vs.
CD45 reduces contamination but also results in the exclu-
sion of CD45+plasma cells, which can constitute the
majority of abnormal plasma cells. The combined use of
CD38, CD138 and CD45 together with light scatter char-
acteristics provides the optimal detection rate and concor-
dance between different operators. It is critical that the first
gate is set using CD38 vs. CD138 expression rather than
CD38 vs. CD45 expression to ensure that CD45+ plasma
cells are not excluded.
Primary gating antibodies: consensus
It is recommended to use four or more detectors for flow
cytometry analysis. Two-color immunophenotypical
analyses are not feasible as at least two antigens are
required to gate plasma cells accurately. CD138, CD38,
CD45 and light scatter characteristics should all be
assessed simultaneously in at least one tube. If using bi-
variate analysis, the primary gate should be set to include
CD38++CD138+events. For characterization of plasma
cells, further tubes should include at least two markers,
preferably CD38 and CD138, with the optimal combina-
tion identified from the primary gating tube. If sufficient
detectors are available, the optimal approach would
include CD38, CD45 and CD138 in all tests.
Controls for gating and immunophenotyping
The use of suitable controls is essential for any accurate
analysis but there is considerable discussion within the
flow cytometry community about what these should be.
Isotype controls have historically been used but they do
not provide a control for many of the variables that affect
the level of non-specific fluorescence, including antibody
concentration, fluorochrome:antibody ratio, and isoelectric
point. The majority of centers did not use an isotype con-
trol for gating or analysis. It was noted that the gating anti-
bodies should yield a discrete population of plasma cells
and the expression of gating reagents was therefore con-
trolled internally by the remaining leucocytes. For immu-
nophenotypical characterization, centers not using an iso-
type control reported using other leucocyte populations to
define positive/negative limits, or using autofluorescence
alone (i.e. cells labeled with only the gating reagents). The
use of non-isotype controls (such as CD3) was also report-
ed. Guidelines for controlling protein expression analysis
have been published by the Clinical and Laboratory
Standards Institute63and the consensus among participants
was that these guidelines are suitable for flow cytometry
in the diagnosis and monitoring of plasma cell disorders.
Controls for gating and immunophenotyping: consensus
A control for the gating reagents is not required since
these are controlled internally. Controls for staining should
be in accordance with standard flow cytometry proce-
dures.63
Number of events
The detection limit for a typical flow cytometry
immunophenotyping (e.g. MRD) assay is partly deter-
mined by the minimum number of events that can reliably
be used to define a population of neoplastic cells. Among
participants, this varied from 10 to 100 events with the
majority requiring more than 20 events. However, it has
previously been shown that accurate identification of a
population using up to 4-color flow cytometry immuno-
phenotypical approaches requires at least 20 events.64,65If
fewer than 100 neoplastic plasma cell events are counted,
the coefficient of variation of the percent value of neoplas-
tic plasma cells will be greater than 10%, independent of
any biological or experimental variations. Therefore, it is
recommended that at least 100 neoplastic plasma cell
events are acquired. The number of target events need not
be acquired in a single tube but can be made up of the
events identified in several tubes, e.g. two tests with a min-
imum of 50 neoplastic plasma cell events and 500,000 total
events in each test, or four tests with a minimum of 25
neoplastic plasma cell events and 250,000 total events in
each test. This allows counting and biological or experi-
mental errors to be considered simultaneously.
Number of events: consensus
At least 100 neoplastic plasma cell events should be
acquired for accurate enumeration. If an MRD assay is to
have a limit of sensitivity of 0.01%, then the minimum
number of total events required is 1,000,000. If the assay
consists of several individual tests then the minimum
requirements are the sum, not the average, of the individ-
ual tests.
Clonality assessment
Demonstration of plasma cell clonality is important for
diagnostic specimens but the relevance of clonality
assessment in follow-up samples is less clear. As in other
MRD approaches,66the use of κ/λ assessment alone is not
suitable in an MRD setting because restricted light chain
expression only becomes apparent when the monoclonal
population represents more than 30% of the polyclonal

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| 436 | haematologica | 2008; 93(3)
background. Assessment of intracellular heavy chain
expression may also be used in this regard, but relatively
few centers carry out routine analysis of both light and
heavy chains at follow-up. It is possible to combine clon-
ality with basic immunophenotype using six-color analy-
sis to provide rapid detection of abnormal plasma cells at
presentation and follow-up. In many cases, identification
of a clearly defined abnormal population will rule out the
need for extended immunophenotyping. Examples of 6-
color screening panels at Salamanca and Leeds are:
cytIgλ/cytIgk/CD19/CD56/CD38/CD45 and cytIgλ/
CD19/cytIgk/CD138/CD38/ CD45 respectively. Such
approaches can detect neoplastic cells even when they
represent as little as 0.01% of leucocytes.67An example of
clonality assessment is shown in Figure 1.
It is critical that whole marrow samples are washed
twice in a ten-fold excess of buffered saline solution prior
to assessment of cytoplasmic immunoglobulin expression
to remove cytophilic immunoglobulin. During the wash-
ing procedure, the supernatant should be removed by aspi-
ration, not by decanting, in order to avoid excessive cell
loss. Standard commercial fixation and permeabilization
kits were reported to be suitable for assessment of cyto-
plasmic kappa/lambda detection, and participants did not
report any specific advantages or disadvantages of the kits
available from different companies.
Clonality assessment: consensus
Assessment of cytoplasmic κ/λ expression by flow
cytometry is important to demonstrate clonality at presen-
tation and is appropriate for the assessment of a stringent
complete remission according to the IMWG criteria.11
The demonstration of phenotypically abnormal plasma
cells is more sensitive and specific for the detection of
residual disease than clonality assessment by immunohis-
tochemistry and/or flow cytometry. Combined assess-
ment of clonality with basic immunophenotype may be
useful for screening at diagnosis and follow-up.
Antigen expression on normal and neoplastic plasma
cells
The most commonly assessed antigens for the detec-
tion of neoplastic and normal plasma cells from published
literature and workshop participants, apart from the gat-
ing reagents, include CD19, CD56, CD20, CD117, CD28,
CD33, CD27, CD81, CD31, CD39, CD40, CD44,
CyclinD1 and CD34. No single marker has been reported
to systematically differentiate neoplastic plasma cells
from their normal counterparts. There has been no formal
study to identify the minimum requirements for repro-
ducible detection of minimal residual disease and further
investigation is required to identify a common panel.
However, based on reported studies, a panel containing
CD19 and CD56 will be applicable to at least 90% of
patients, with the markers CD20, CD117, CD28 and
CD27 likely to increase this to more than 95% of
patients.5-10,20-29;30-44In addition, several participants had
analyzed CD81 and CD20068 and suggested that these
markers should be assessed further. The proposed anti-
gens for investigation are outlined in Table 2.
Antigen expression on normal and neoplastic plasma
cells: consensus
It is not possible to define plasma cells as being pheno-
typically abnormal, either at diagnosis or after treatment,
using only one test antigen. In addition to the plasma cell
gating markers discussed above, the minimal test antigens
for classifying abnormal plasma cells are CD19 and
CD56. A preferred panel would incorporate CD20,
CD117, CD28 and CD27.
Measure of sample quality
As discussed above, it is known that a lower percentage
of plasma cells is detected by flow cytometry than by
morphology. In most cases, this may be due to the provi-
sion of a blood-diluted sample. This may also be critical
for MRD analysis, since the level of neoplastic plasma
cells will be under-estimated in a blood-diluted sample.
Normal polyclonal CD138+CD19+plasma cells are typi-
cally restricted to the bone marrow and the presence of
such cells has been used to confirm that the sample is rep-
resentative marrow. If only neoplastic cells are present,
then the sample is MRD+but may not be representative
of the marrow. B-cell regeneration is usually rapid after
high-dose melphalan with autologous stem cell rescue,
and if no plasma cells are present, then the sample is
unlikely to be representative. However, there was anec-
dotal evidence from the UK Myeloma IX trial that good
quality bone marrow samples containing neither normal
nor neoplastic plasma cells were observed in a small pro-
portion of patients. It may therefore be necessary to
determine the levels of other cells that are predominantly
restricted to the bone marrow, e.g. erythroid, myeloid
and B-cell progenitors. In cases where marrow elements
are not detectable, it should be stated that the sample is
unsuitable for quantitative MRD analysis.
Measure of sample quality: consensus
The sample is suitable for quantitative MRD analysis if
normal plasma cells (CD19+CD56–and/or polyclonal) are
detectable. If normal plasma cells are not detected, the
quality of the sample should be assessed by morpholog-
ical assessment of a bone marrow smear made from the
same sample used for flow cytometry, and/or additional
flow cytometry for the presence of normal erythroid,
myeloid or B-cell progenitors. If there are no marrow ele-
ments present but neoplastic plasma cells are detected,
the sample should be reported as MRD-positive, but note
that the sample may be unsuitable for quantitative
assessment. If there are no marrow elements and no
plasma cells, the sample should be reported as unsuitable
for analysis.

Page 7

EMN: flow cytometry in plasma cell disorders
haematologica | 2008; 93(3) | 437 |
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