Immunophenotyping is being routinely used for the diagnosis of leukemias. With the advent of specific markers for plasma cells, it has become possible to differentiate between benign and malignant plasma cells based on their immunophenotypic profile. The enhanced use of immunophenotyping in plasma cells dyscrasias may help to categorize the borderline cases which can not be done with morphology alone. Also the immunophenotypic profiling of plasma cells would help in Minimal Residual Disease (MRD) evaluation of patients on treatment of multiple myeloma (MM).

1. ROLE OF FLOW CYTOMETRIC IMMUNOPHENOTYPING IN PLASMA
CELL DYSCRASIAS

2. Review Article
INTRODUCTION
Multiple Myeloma (MM) is a neoplastic plasma cell
dyscrasia (PCD) characterized by marrow plasmacytosis, a
monoclonal protein in the serum or urine, abnormal bone
radiographs, bone pain, anaemia, hypercalcemia and renal
insufficiency or failure. Multiple myeloma comprises 10%
of all haematological malignancies and 1% of all
neoplastic disorders. Based on data collected from 6
population based cancer registries in India, MM incidence
varies from 0.3 to 1.9 per 100,000 for men and 0.4 to 1.3
per 100,000 for women [1]. It has an incidence of
approximately 4.5 per 100,000 per year in the United
States [2]. Monoclonal Gammopathy of Undetermined
Significance (MGUS) has a frequency of approximately
1% in healthy persons over 50 years of age and it increases
to 3% among those over 70 [3]. The prevalence of MGUS
in Africans and African-Americans is double that of
whites. The prevalence of MGUS increases with
advancing age. Also the men have higher frequency of
MGUS than women compared to that of MM [4].
The morphological evaluation of bone marrow (BM)
aspirate sometimes fails to distinguish between the benign
and malignant plasma cells, in cases where morphological
atypia is not prominent. Also due to the non-uniform
distribution of plasma cells (PCs) on bone marrow
aspiration the calculation of percentage of BM PCs may
not be very accurate. Also, the cases that have a borderline
percentage of plasma cells or that do not have a definite M
spike on serum protein electrophoresis; it is a diagnostic
dilemma for the treating physician.
At present, immunophenotyping is mandatory for the
diagnosis and monitoring of acute leukaemias and chronic
lympho-proliferative disorders. The generation and
identification of markers, that allow the unequivocal
identification of PCs and the identification of aberrant
plasma cell phenotypes, may enable us to identify
neoplastic plasma cells disorders from reactive plasma cell
proliferation [5].
The identification of a malignant clone is the first and
the most important step in the characterization of the
disease [6]. Co-expression studies with CD19, CD 56,
CD138, CD38 and CD45, can help to identify the distinct
expression of malignant vs. benign plasma cells [7-9].
While expression of CD56 distinguishes malignant from
benign plasma cells, myeloma without CD56 expression
might be associated with more aggressive disease and
extra-medullary dissemination [8].
IMMUNOPHENOTYPING IN MM & MGUS CASES
Even before the application of flow cytometry,
phenotypicanalysisofmyelomacellswasbeingattempted.
In one such study by Duperray, et al, they compared the
phenotype of human myeloma cell lines with those of
normal lymphoblastoid cell lines. They classified human
myeloma cell lines on the basis of presence or absence of
cytoplasmic (c ) chain. The c + cells had a phenotype
close to pre-B or B cells while c - had a phenotype close to
fresh myeloma cells [10].
In 1990, Benjamin Van Camp found an unexpected
observation during the analysis of immunophenotyping of
ROLE OF FLOW CYTOMETRIC IMMUNOPHENOTYPING IN PLASMA CELL DYSCRASIAS
Pranav Dorwal*, Rashmi Thakur** and Sangita Rawat**
*DNB student, #
Senior Consultant, Department of Hematology, Indraprastha Apollo Hospitals, Sarita Vihar,
New Delhi 110076, India
Corresponding Author: Dr Sangita Rawat, Senior Consultant, Hematology, Indraprastha Apollo Hospitals,
Sarita Vihar, New Delhi 110 076, India
E-mail: sangita_rawat@yahoo.com
Immunophenotyping is being routinely used for the diagnosis of leukemias. With the advent of specific
markers for plasma cells, it has become possible to differentiate between benign and malignant plasma cells
based on their immunophenotypic profile. The enhanced use of immunophenotyping in plasma cells
dyscrasias may help to categorize the borderline cases which can not be done with morphology alone.Also the
immunophenotypic profiling of plasma cells would help in Minimal Residual Disease (MRD) evaluation of
patients on treatment of multiple myeloma (MM).
Key words: Plasma cell dyscrasias, Flow cytometry.
Apollo Medicine, Vol. 7, No. 3, September 2010 176

3. Review Article
177 Apollo Medicine, Vol. 7, No. 3, September 2010
bonemarrowsamplesfrompatientswithmultiplemyeloma
(MM) and monoclonal gammopathy of undetermined
significance (MGUS). The study was an analysis of the
phenotypic pattern of lymphocytes, natural killer cells, and
monocyte/macrophages in myeloma bone marrows as a
basisforthepotentialuseofinterleukin-2(IL-2)andrelated
forms of immunotherapy in patients with plasma cell
dyscrasias. The plasma cells in patients with Multiple
MyelomawerefoundtobestronglypositiveforCD56[11].
The application of immunophenotyping by flow
cytometrytoplasmacelldyscrasiasgotoffrelativelylate(in
the early 1990s). Billadeu, et al in 1993 used FCM to sort
bone marrow samples into CD38+/CD45- and CD38-/
CD45+ populations. End stage plasma cells lost all CD45
expression and majority of multiple myeloma tumour cells
were CD38+ and CD45- [12].
Laterthesameyear,Harada,etal,studiedthedistinction
between normal and myeloma cells; with focus on CD19
and CD56. Most myeloma cases were CD19-/CD56+,
while normal plasma cells were CD19+/CD56-. Myeloma
cells were found to be strongly CD38 positive. These cells
were sorted and stained with Wright’s stain. Cells sorted
from CD38++ were all plasma cells morphologically [13].
This was a pioneering study which paved way for
exploration of immunophenotyping in Plasma Cell
Dyscrasias.
In1994,Zandecki,etal,workingonMGUScasesfound
that CD19 was expressed in MGUS cases, in variable
proportions, but not in the MM cases. CD38 was expressed
onallplasmacellswhileCD56wasfoundin36%patientsof
MGUS patients [14].
A C Rawstron, et al, studied the variable expression of
CD19 and CD56 in the MM cases; Majority of the patients
had CD19-/CD56+ plasma cells, while few were CD19-/
CD56 (low) and CD19+/CD56+. Normal plasma cells
expressed CD19+/CD56(low). They also found that the
peripheral blood plasma cells had a significantly lower
expression of CD56 [15].
By the late 1990s, the work on the imunophenotypic
characterization of the plasma cells had gained pace. In
1998 Ocqueteau, et al, demonstrated two different
populations of plasma cells in MGUS patients (Population
A and Population B). One of which was CD38+/CD19+/
CD56-, while the other one was CD38 (low)/CD19-/
CD56+. They concluded that the PCs in MGUS had
two populations, one expressing phenotypic similarity to
normal plasma cells and other to myelomatous plasma
cells [9].
The lucid report of “1st Latin American Consensus
Conference for Flow Cytometric Immunophenotyping of
Leukemias” held in 1996 recommended a minimum panel
consisting of antibodies against CD38, CD19 and CD56.
This report also said that neoplastic plasma cells showed
dim expression of CD38 compared to their normal
counterparts and the presence of CD19 is characteristic of
normalplasmacells,whereasincreasedexpressionofCD56
was found on neoplastic but not on normal plasma cells
[16].
Alexanian,etal,in1999,whilestudyingthedifferential
diagnosis of monoclonal gammopathies, mentioned that
malignant plasma cells usually express CD28 and CD56,
while the B cell antigens, i.e. CD19, CD20 and CD23 are
lost [3].
In the new millennium, Sezer, et al, worked on
distinguishing MM and MGUS on the basis of their
phenotypic characteristics. In normal individuals, plasma
cellswereCD19-/CD56+,whileMMpatientswereCD19-/
CD56+. In contrast, all the MGUS patients had two
different sub-populations of plasma cells with normal and
MM phenotype. They concluded that the single parameter
that distinguishes myeloma from MGUS was the
percentage of immunophenotypically normal PCs to total
plasma cells on flow cytometer [7]. Similar study was
performed by Manzanera, et al [17].
A C Rawstron, et al concluded that analysis of CD38 vs
CD138 provided the best separation of PCs from other
leucocytes. Also, the immunophenotypic analysis of
neoplastic plasma cells was found to be more sensitive than
immunofixation in majority of cases and it may help to
identify which patients may benefit from additional
treatment strategies at an early stage after transplantation
[18].
Kara and Sahin recommended the panel of CD38/
CD138/CD45 for the identification of MM cells. They
recommended that the identification of the malignant clone
is the first and the most important step in the
characterization of the disease, determining prognosis and
detection of residual disease [6].
In one of the most comprehensive studies on MM by
Lin, et al, a set of 306 patients was taken. PCs were
identified using light scatter, CD38, CD138 and CD45.
Plotting of CD38 vs CD138 showed strong staining,
separating the majority of PCs from other cellular
components. Better results were obtained with CD138 than
withCD38.CD38andCD138expressionwasseeninallthe
cases, while 17.6% showed CD45 positivity. They found
that CD138 could detect myeloma cells with weak or
moderate CD38 reactivity and also those with moderate to
brightCD45expression;whichwasmissedonCD38/CD45

4. Review Article
Apollo Medicine, Vol. 7, No. 3, September 2010 178
gating. They concluded that inclusion of all PCs is
importantforcompleteimmuno-profilingofthepopulation
of interest. Also it was observed that use of ammonium
chloride resulted in relatively lesser loss of CD138+ cells,
which was frequently observed with FACSLyseTM. The
samples that were stored in refrigerator or in which
processing was delayed, loss of CD138 expression was
observed. Different chemotherapy was recommended for
multiplemyelomawithdifferentphenotypicexpression[8].
In the same year, while studying CD45 expression,
Kumar, et al, observed that CD45 expression was higher
among those with early disease (MGUS and Smouldering
MM) as compared to those with advanced disease [19].
Kabayashi, et al in 2006, used a panel of CD19, CD56,
CD45 and CD38. The plasma cells were gated using CD38
vs CD45 expression. They found that normal plasma cells
could be distinguished from myeloma plasma cells by the
expression of CD19 and CD56. The normal plasma cells
were CD19+CD56- or CD56dim while the neoplastic
plasma cells were CD56+ and CD19-/+. Post-treatment the
phenotype of the myeloma cells changed from CD19-
CD56+ to CD19-CD56-. It was also demonstrated that 4-
colour flow cytometry (CD38/CD45/CD19/CD56) is more
sensitive than that of 3-colour flow cytometry (CD38/
CD45/cytoplasmic Ig). Also the expression of CD138 was
higherinmyelomaplasmacellsascomparedtonormalPCs.
A phenotypic change was observed after transplantation
[20]. Morice, et al and de Tute, et al have used 6-color
immunophenotypic assays to identify all plasma cells
aberrant phenotype [21, 22].
Another antigen that has been studied in myeloma cells
is CD52, which was expressed in a significant number of
multiplemyelomapatientsinthestudyconductedbyCarlo-
Stella, et al. Alemtuzumab was postulated as a potential
targeted therapy in these subset of patients [23].
In the comprehensive and exhaustive report of
“European Myeloma Network on Multi-parametric Flow
CytometryinMultipleMyelomaandrelateddisorders”,the
consensus said that, (i) CD38, CD138 and CD45 should be
includedinatleastonetubewithprimarygatewithCD38vs
CD138, (ii) flow cytometry is suitable for demonstrating
complete remission and (iii) CD19 and CD56 should be
included in the panel to demonstrate abnormal PCs [5].
In 2007, while studying MGUS and smouldering MM
cases, Perez-Persona, et al, showed that the proportion of
atypical PCs to the total BM PCs was the most important
criterion for the progression of disease. Patients with
atypical plasma cell to total bone marrow plasma cell
percentage more than 95% have significantly higher risk of
disease progression [24].
Jerez,etalperformedaretrospectivecohortandacross-
sectional study to analyze the prognostic value of aberrant
(CD38+ +CD138+ CD19-CD45weak) to normal phenotype
(CD38+ +CD138+ CD19+CD45+) bone-marrow plasma
cells ratio (A/N ratio) for the development of a plasma cell
dyscrasia and the association with the presence of a chronic
immune disorder. They found that values of 4 or higher
define a group at high risk of progression whileA/N values
of 0.20 or lower are associated with immune disorders or
chronic infections [25].
Recently, Olteanu, et al analyzed bone marrow
specimens from 32 MGUS patients and 32 plasma cell
myeloma patients. All MGUS patients had two
subpopulations of plasma cells, one with a “normal”
phenotype[CD19+,CD56-,CD38(bright+)]andotherone
with an aberrant phenotype [CD19-/CD56+ or CD19-/
CD56-].Also they found that patients lacking CD19 on the
majority of their bone marrow plasma cells as being more
likely to show progression of disease. They recommended
thatflowcytometricevaluationofCD19expressioninbone
marrowplasmacellscouldbeincorporatedasanadjunctive
test in the current patient stratification schemes [26].
IDENTIFICATION OF CIRCULATING PLASMA
CELLS
Multiple studies have been done on the presence of
circulating PCs. Schneider, et al studied the phenotype of
circulating normal and malignant plasma cells using CD45
andCD38.CD45expressionseparatedtheperipheralblood
plasma cells into two subsets of which CD45- were found
only in myeloma patients [27].
In another such study by Nowakowski, et al, the
circulating PCs were gated as CD38+/CD45- cells.As their
number was small, the number of events acquired was
50,000. The patients with 10 or fewer circulating PCs had a
significantly longer survival than the patients with more
than 10 circulating PCs [28]. Detection of circulating
myeloma cells in patients with multiple myeloma indicates
active disease. The monoclonal myeloma cells were
identifiedbygatingcellsthatexpressedhighlevelsofCD38
but showed weak expression of CD45 [29].
A C Rawstron, et al, demonstrated that the CD56
expressioncorrelatedwiththedistributionofplasmacellsin
bone marrow and peripheral blood. The extent of bone
marrow infiltration on trephine biopsy correlated directly
with CD56 expression. Similarly, inverse correlation was
seenbetweenCD56expressionandcirculatingplasmacells
[30].
Bataille, et al, while reviewing the data on myeloma
plasmacells,foundthataberrantCD56expressionisalways

5. Review Article
179 Apollo Medicine, Vol. 7, No. 3, September 2010
observed in MM (over-expression of CD56), while
circulating PCs lack CD56 expression. Also lack of CD45
expression is associated with poor prognosis [31].
MINIMAL RESIDUAL DISEASE (MRD)
EVALUATION IN MM USING FLOW CYTOMETRY
Many authors have work on MRD evaluation in MM
cases. Almeida, et al, used highly sensitive immuno-
phenotyping and DNA ploidy studies for the investigation
of Minimal Residual Disease in Multiple Myeloma. They
used multiple monoclonal antibodies and two step
acquisition procedure using side scatter, CD38 and CD138.
Theyfoundthisapproachtobehighlysensitiveandsuitable
for diagnosing MRD in MM cases [32].
Rawstron, et al studied MRD and found that three
months post autologous bone marrow transplantation,
patients with detectable neoplastic plasma cells had a
significantly lower median progression-free survival (20
months), as compared to those without detectable plasma
cells (34.5 months) [18].
Lin, et al recommended that identification of unique
myeloma immunophenotype for each patient would permit
optimized detection of minimal residual disease [8].
Sarasquete, et al, classified MRD positive (>=1
myelomatouscellper105 residualhematopoieticcells)into
two categories, with cells between 1 in 104 and 1 in 105 as
low MRD and cells more than 1 in 104 as high-MRD, and
they were found to have significantly different disease free
survival [33].
The phenotypic profiling of malignant plasma cells
using a limited panel of CD38, CD138, CD19 and CD56
will help in identifying MRD in MM cases that are on
treatment.
CONCLUSION
Immunophenotypic profiling of plasma cells can be
very helpful in categorizing the borderline cases that is a
handicap with morphological evaluation. CD38 vs CD138
provides the best separation of PCs from leucocytes. These
plasmacellscanthenbeidentifiedtobemalignantorbenign
on the basis of expression of CD19 and CD56. In MM cases
CD19 expression is lost while CD56 expression is gained.
MGUS cases are seen to have two distinct benign (CD19+/
CD56-) and malignant (CD19-/CD56+) subpopulations.
The ratio of benign and malignant plasma cells in MGUS is
an important criterion for disease progression. CD138,
althoughaveryspecificmarkerforPCs,losesitsexpression
with the delay in processing or acquisition of data; and is
affected by the type of lysing agent used. CD56 is a marker
for homing of PCs in BM; and the loss of CD56 expression
in MM patients is associated directly with the percentage of
circulating PCs. The minimum panel that is recommended
forplasmacelldyscrasiasincludesCD38,CD19andCD56.
Hence, immunophenotyping of PCs has a role in
categorization of plasma cell dyscrasis and monitoring of
MM patients on treatment, when routine BM examination
and serum protein electrophoresis fail to provide a definite
answer.
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