Original Article

Clinical Utility of Multiparameter Flow

Cytometry in the Diagnosis of 1013 Patients
With Suspected Myelodysplastic Syndrome
Correlation to Cytomorphology, Cytogenetics, and Clinical Data

Wolfgang Kern, MD; Claudia Haferlach, MD; Susanne Schnittger, PhD; and Torsten Haferlach, MD

BACKGROUND: The diagnosis and classification of myelodysplastic syndromes (MDS) is based on cytomorphology
(CM) and cytogenetics (CG). Multiparameter flow cytometry (MFC) may add important diagnostic information.
METHODS: To evaluate the potential role of MFC in the diagnostic setting of MDS, the authors analyzed the results
from 1013 patients with suspected MDS by using CM, CG, and MFC in parallel. RESULTS: Concordance between CM
and MFC was 82% for diagnostic results in 788 patients who had unequivocal CM results. An additional 225 patients
had only minor dysplastic features identified by CM, including 51 patients (22.7%) who had clear evidence of MDS by
MFC. Twelve patients who had no indication of MDS identified by CM had MDS-typical CG aberrations; in 6 of those
patients (50%), MFC revealed MDS characteristics. In another 11 of 23 patients (47.8%) who had minor dysplastic fea-
tures identified by CM and MDS-typical CG aberrations, MFC revealed MDS characteristics. The percentages of blasts
determined by CM and by MFC were strongly correlated (P < .001). The frequency of aberrantly expressed antigens
differed significantly between patients rated by CM as MDS (highest frequencies), suspected MDS, and no MDS (low-
est frequencies). In various patients, MFC identified MDS-typical aberrant antigen expression in cell compartments
that were not rated dysplastic by CM. The numbers of aberrantly expressed antigens were correlated with Interna-
tional Prognostic Scoring System scores and overall survival. CONCLUSIONS: The current analysis clearly demon-
strated an increased diagnostic yield with MFC when added to CM and CG in patients with suspected MDS. Cancer
2010;116:4549–63. V C 2010 American Cancer Society.

KEYWORDS: myelodysplasia, flow cytometry, cytomorphology, cytogenetics.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal diseases that originate in malig-
nant hematopoietic stem cells.1 Clinical manifestations of MDS result from peripheral blood cytopenias and include fa-
tigue, infections, and bleeding.1 The diagnosis of MDS relies on the evaluation of dysplastic findings in bone marrow
(BM) cells. Acquired chromosomal aberrations are detected in 50% of patients.2-4
Diagnostic criteria and classification of MDS have been based on the French-American-British (FAB) classification
for 2 decades5 with standardized criteria for dysplasia.6 Cytogenetic (CG) analysis has been included in the standard diag-
nostic workup and classification.7-9 In addition to cytomorphology (CM) and CG, the number of cytopenias and transfu-
sion requirements have been included in the powerful International Prognostic Scoring System (IPSS)10 and World
Health Organization (WHO) classification-based Prognostic Scoring System,11 respectively, for prognosis.
It has been demonstrated that both FAB and the WHO classification identify clinically relevant subgroups.12 In
patients with chromosomal aberrations, CG may contribute substantially to establish a diagnosis of MDS if CM results
are equivocal. However, in a large number of patients with cytopenias, the diagnosis may not be made, and repeat evalua-
tions are recommended.
Because of these diagnostic difficulties, it is worthwhile to test the power of multiparameter flow cytometry (MFC).
MFC repeatedly has demonstrated the ability to identify the aberrant expression of antigens that are considered dysplastic

Corresponding author: Wolfgang Kern, MD, MLL Munich Leukemia Laboratory, Max-Lebsche-Platz 31, 81377 Munich, Germany; Fax: (011) 49-89-990-17-209;
wolfgang.kern@mll-online.com
MLL Munich Leukemia Laboratory, Munich, Germany
DOI: 10.1002/cncr.25353, Received: October 5, 2009; Revised: February 21, 2010; Accepted: February 25, 2010, Published online June 22, 2010 in Wiley Online
Library (wileyonlinelibrary.com)

Cancer October 1, 2010 4549

Original Article

Table 1. Panel of Monoclonal Antibodies Used for Immunophenotyping

Combination FITC PE ECD PC5 PC7

1 CD11b CD13 HLA-DR CD16 CD45
MoAb Bear1 Immu103.44 Immu357 3G8 J33

2 CD71 CD235a CD19 CD2 CD45

MoAb YDJ1.2.2 11E4B-7-6 J3-119 39C1.5 J33

3 CD61 CD5 CD14 CD4 CD45

MoAb SZ21 BL1A RMO52 13B8.2 J33

4 CD15 CD66c CD3 CD64 CD45

MoAb 80H5 B6.2 UCHT1 22 J33

5 CD65 CD56 CD10 CD33 CD45

MoAb 88H7 N901 ALB1 D3HL60.251 J33

6 CD36 CD38 CD34 CD7 CD45

MoAb FA6-152 T16 581 8H8.1 J33

FITC indicates fluorescein isothiocyanate; PE, phycoerythrin; ECD, phycoerythrin-Texas Red; PC5, phycoerythrin-cyanine 5; PC7, phycoerythrin-cyanine 7;
MoAb, monoclonal antibody; CD, cluster of differentiation; CD11b, integrin a M; CD13, alanine aminopeptidase; HLA-DR, human leukocyte antigen-D related;
CD16, human neutrophil antigen 1; CD45, protein tyrosine phosphatase, receptor type, C; CD71, transferrin receptor 1; CD235a, glycophorin A; CD19, protein
encoded by the CD19 B-lymphocyte antigen gene; CD2, lymphocyte function-associated antigen 2; CD61, protein encoded by the integrin beta 3 gene; CD5,
type I transmembrane protein; CD14, monocyte differentiation antigen CD14; CD4, protein encoded by the T-cell surface glycoprotein CD4 gene; CD15, 3-
fucosyl-N-acetyl-lactosamine; CD66c, carcinoembryonic antigen-related cell adhesion molecule 6 (nonspecific cross-reacting antigen); CD3, T-cell coreceptor;
CD64, integral membrane glycoprotein; CD65, fucosylated carbohydrate cell-surface antigen; CD56, neural cell adhesion molecule; CD10, common acute lym-
phocytic leukemia antigen; CD33, 67-kDa membrane glycoprotein; CD36, collagen type 1 receptor; CD38, cyclic adenine diphosphate hydrolase; CD34, cell
surface antigen, glycoprotein 105-120; CD7, glycoprotein 40.

features.13-16 These findings have been described in MDS
and do not occur or occur only infrequently in patients
who have cytopenias that are not considered MDS. The
objective of the current study was to estimate the potential
diagnostic value of MFC.
MATERIALS AND METHODS
Patients
In total, 1013 BM samples from patients with suspected
MDS were sent to MLL Munich Leukemia Laboratory
between August 2005 and January 2009 and were ana-
lyzed in parallel by using CM, CG, and MFC. The suspi-
cion of MDS was raised when the physician sent the
sample and was based on peripheral blood count and dif-
ferential as well as clinical criteria. Patients who had
hematologic malignancies other than MDS were excluded
after the respective diagnosis was confirmed. Thus, in the
current cohort, all patients had in common an initial suspi-
cion of MDS. All 3 diagnostic methods were performed in-
dependently from each other. CM was performed by T.H.,
CG was performed by C.H., and MFC was performed by
W.K. The median patient age was 69.8 years (range, 1.8-
88.7 years), and the ratio of men to women was 539:474.
Median values and ranges for peripheral blood counts were
as follows: median white blood cell count, 4400/lL (range,
600-117,600/lL); median hemoglobin, 10.7 g/dL (range,
4.0-20.2 g/dL); and median thrombocyte count, 157,500/
lL, (range, 2000-1195,000/lL).
Cytomorphology and Cytogenetics
CM assessment was based on May-Grunwald-Giemsa
stains, myeloperoxidase reactions, and nonspecific esterase
using alpha-naphtyl-acetate17-19 and was used for diagno-
sis according to FAB20-22 and WHO7,8 criteria. CG anal-
yses were performed according to standard protocols.
Classification was according to the International System
for Human Cytogenetic Nomenclature.23-25 Complex
aberrant karyotypes were defined by
3 clonal chromo-
some aberrations.8,26 Fluorescence in situ hybridization
was used according to standard procedures27 to clarify all
difficult cases.
Multiparameter Flow Cytometry
Samples were processed by Ficoll gradient centrifugation
followed by ammonium chloride-based erythrocyte lysis to
isolate mononuclear cells. MFC was performed by applying
5-fold stainings and using the antibodies outlined in Table
1 (selected based on previous studies).13-15,28,29 Antibodies
were purchased from Immunotech (Marseilles, France)
except for carcinoembryonic antigen-related cell adhesion
molecule 6/nonspecific cross-reacting antigen (cluster of
differentiation [CD] 66c [CD66c]) (Becton Dickinson,
Heidelberg, Germany). Antibody combinations were

4550 Cancer October 1, 2010


Figure 1. Cluster of differentiation 13 (alanine aminopeptidase) (CD13)/human neutrophil antigen (CD16) expression patterns are
shown (Left) in granulocytes from normal bone marrow (BM) and (Right) in myelodysplastic syndrome (MDS) PC5 indicates phy-
coerythrin-cyanine 5; PE, phycoerythrin.
added to 106 mononuclear cells (volume, 100 lL) and
incubated for 10 minutes. After the addition of 2 mL lys-
ing solution, samples were incubated for additional 10
minutes, then washed twice in phosphate-buffered saline
(PBS), and resuspended in 0.5 mL PBS. FC500 flow
cytometers were used (Beckman Coulter, Miami, Fla),
and 20,000 events were acquired. Cytomics CXP Soft-
ware (Beckman Coulter) was used for data analysis.
Gating Strategy
Protein tyrosine phosphatase, receptor type, C (CD45)-
side scatter (SSC) gating was performed to identify cellu-
lar compartments to allow the separate evaluation of anti-
gen expression for each compartment.30 The lack of
expression of an antigen is indicated by ‘‘,’’ dim expres-
sion is indicated by ‘‘(þ),’’ any expression is indicated by
‘‘þ,’’ and strong expression is indicated by ‘‘þþ.’’ A
broader range of expression is indicated by 2 respective
values separated by a virgule, ie, expression ranging from
‘‘(þ)’’ to ‘‘þ’’ is indicated by ‘‘(þ)/þ.’’ Granulocytes were
identified as SSCþ/(þ)CD45(þ), monocytes were iden-
tified as SSC(þ)CD45þ, myeloid blasts were identified
as SSC([þ])CD45(þ), and erythroid cells were identified
as CD45 glycophorin A (CD235a)þþ. It should be
recognized that, because a DNA-binding dye was not
used, there is no guarantee that the erythroid cells identi-
fied are truly nucleated. By applying back-gating, mono-
cytes also were identified as monocyte differentiation
antigen CD14 (CD14)þ, and blasts were separated from
the protein encoded by the CD19 B-lymphocyte antigen
Cancer October 1, 2010
MFC in MDS/Kern et al
gene (CD19)-positive B-lymphoid progenitors, and in
addition, were demonstrated to express cell surface anti-
gen-glycoprotein 105-120 (CD34), or human leukocyte
antigen-D related (HLA-DR), or both. The percentage of
blasts was calculated by using the total amount of mono-
nuclear cells after sample preparation as the denominator.
According to previous reports,13-15,28,29 the following fea-
tures were evaluated.
Granulocytes were evaluated for SSC signal, abnormal
alanine aminopeptidase (CD13)/human neutrophil antigen
1 (CD16) and integrin a M (CD11b)/CD16 expression
patterns, neural cell adhesion molecule (CD56) coexpres-
sion, 67-kDa membrane glycoprotein (CD33) negativity,
and integral membrane glycoprotein (CD64) negativity.
The CD13/CD16 expression pattern was considered
abnormal if a deviation of normal granulocytic matura-
tion (CD13þ/CD16!CD13[þ]/CD16!CD13/
CD16[þ]!CD13/CD16þ!CD13þ/CD16þþ) was
observed (Fig. 1). Also, the CD11b-/CD16 expression
pattern was considered abnormal if a deviation of normal
granulocytic maturation (CD11b/CD16!CD11bþ/
CD16[þ]!CD11bþ/CD16þ!CD11bþþ/CD16þþ)
was observed. Deviation from normal was defined as a dif-
ference that amounted to at least a half-log signal intensity
in 1 parameter, although it must be recognized that the
assessment is always at least partially subjective.
Monocytes were evaluated for CD11b negativity,
HLA-DR negativity, CD13 negativity, CD16 coexpres-
sion, CD56 coexpression, aberrant lymphocyte function-
associated antigen 2 (CD2) coexpression.
4551
Original Article

Figure 2. (Left) Monocytes in normal bone marrow (BM) revealed no expression of cluster of differentiation 56 (CD56) (neural
cell adhesion molecule). (Right) In myelodysplastic syndrome (MDS), strong CD56 expression was observed. CD33 indicates 67-
kDa membrane glycoprotein; PC5, phycoerythrin-cyanine 5; PE, phycoerythrin.

Figure 3. (Left) In normal bone marrow (BM), the majority of erythrocytes had strong expression of cluster of differentiation 71
(CD71) (transferrin receptor 1) with some degree of heterogeneity for dim expression. (Right) In myelodysplastic syndrome
(MDS), negative CD71 expression may be present in the majority of erythrocytes. CD235a indicates glycophorin A; PE, phycoery-
thrin; FITC, fluorescein isothiocyanate.

Myeloid blasts
Myeloid blasts were evaluated for the percentage of
BM myeloid blasts, coexpression of CD11b, type I trans-
membrane protein (CD5), CD56, transferrin receptor
1(CD7), CD15, and CD64 and HLA-DR negativity.
Erythroid cells
Erythoroid cells were evaluated for homogeneously
strong transferrin receptor 1 (CD71) expression and
CD71 negativity. Examples of aberrant antigen expres-
sion are shown in Figures 1, 2, and 3.
Positivity and negativity, respectively, of the respec-
tive antigens were determined by comparison with the re-
spective isotype controls using 20% as the cutoff. The
SSC signal in granulocytes was assessed qualitatively as ei-
ther reduced or not reduced based on the CD45/SSC dot
plot. For a marker with which to describe objectively the
reduced SSC signal in granulocytes, the granulocyte-to-
lymphocyte ratio of the mean SSC signal as acquired on a
linear scale was calculated for each case (the SSC granulo-
cyte:lymphocyte ratio).
Statistical Analysis
Dichotomous variables were compared using chi-square
tests, and continuous variables were analyzed with the

4552 Cancer October 1, 2010

 MFC in MDS/Kern et al

Table 2. Diagnostic Results of Multiparameter Flow Table 3. Diagnostic Results of Multiparameter Flow
Cytometry in Cytomorphologically Defined Myelodysplastic Cytometry in Cytogenetically Defined Myelodysplastic
Syndrome Subgroups Syndrome Subgroups

CM Total No. of Cytogenetic Result Total No. of

Result No. of Patients With No. of Patients With
Patients Results in Patients Results in
Agreement With Agreement With
MDS by MFC MDS by MFC
Independent of Independent of
CM Results (%) CM Result (%)
RA 31 22 (71) Normal karyotype 768 257 (33.5)
RARS 27 16 (59.3) Del(5q) 43 33 (76.7)
RCMD 64 23 (35.9) Aberrations of chromosome 7 14 14 (100)
RCMD-RS 49 32 (65.3) Trisomy 8 30 25 (83.3)
RAEB-1 133 104 (78.2) Del(20q) 21 18 (85.7)
RAEB-2 81 78 (96.3) Complex karyotype 23 19 (82.6)
5q– Syndrome 24 18 (75) Loss of Y-chromosome 43 22 (51.2)
CMML 65 62 (95.4) Other aberrations 71 58 (81.7)
MDS-u 15 12 (80)
MDS/AML 6 6 (100) MFC indicates multiparameter flow cytometry; MDS, myelodysplastic
MDS/MPS 16 9 (56.3) syndrome; CM, cytomorphology; Del, deletion.
Suspected MDS 225 51 (22.7)
Reactive condition 266 13 (4.9)
Normal findings 11 0 (0) lowest percentages of results that were in agreement
CM indicates cytomorphology; MDS, myelodysplastic syndrome; MFC, multi- with MDS by MFC were observed in the MDS sub-
parameter flow cytometry; RA, refractory anemia; RARS, refractory anemia group of patients who had refractory cytopenia with
with ring sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia;
RCMD-RS, refractory cytopenia with multilineage dysplasia and ring sidero-
multilineage dysplasia (RCMD); however, the percen-
blasts; RAEB-1, refractory anemia with 5% to 9% excess blasts; RAEB-2, re- tages still clearly were higher than the respective per-
fractory anemia with 10% to 19% excess blasts; , negative; CMML, chronic
myelomonocytic leukemia; MDS-u; unclassifiable myelodysplastic syndrome;
centages observed in patients with suspected MDS
AML, acute myeloid leukemia; MPS, myeloproliferative syndrome. according to CM results and in patients with no
MDS. CG results are detailed in Table 3. Fluores-
Student t test. Spearman rank correlation was used to ana-
cence in situ hybridization was performed in 443
lyze correlations between continuous parameters. Survival
cases.27
was analyzed using the Kaplan-Meier method,31 and differ-
ences were analyzed using the log-rank test. All calculations
were performed using SPSS software (version 14.0.1; SPSS
Comparison of Diagnostic Results Obtained
Inc., Chicago, Ill). All reported P values are 2-sided. by Cytomorphology and Multiparameter
Flow Cytometry
Study Conduct There was 82% percent concordance between CM and
Patients provided informed consent to participate in the MFC for diagnostic results (646 of 788) in patients with
current diagnostic procedures and the evaluation at MLL definite CM results. In detail, 511 patients were classified
Munich Leukemia Laboratory after they were advised with MDS according to CM, including 382 patients
about the purpose and investigational nature of the study (74.8%) who had antigen expression features according to
and of the potential risks. The study design adhered to the MFC in agreement with MDS. Of 277 patients who had
Declaration of Helsinki. CM results indicating no MDS, only 13 patients (4.7%)
had MDS-typical features according to MFC. The pattern
of these MDS-typical features did not differ from the
RESULTS overall pattern observed in the total cohort. Two hundred
Cytomorphologic and Cytogenetic twenty-five patients had some dysplastic features on CM
Characterization but did not fulfill the diagnostic criteria for MDS; how-
Classification by CM is detailed in Table 2. The ever, 51 of those patients (22.7%) had MDS-typical find-
highest percentages of results that were in agreement ings on MFC. Clinical follow-up of these patients is not
with MDS by MFC were observed in the MDS sub- available to an extent that would allow reasonable clinical
groups with blast counts >10%, as anticipated. The validation.

Cancer October 1, 2010 4553

Original Article

Comparison of Cytogenetics and Diagnostic evidence for MDS on CM, and 50% of those patients had
Results Obtained by Multiparameter Flow an aberrant karyotype. In addition, 5.1% of all patients had
Cytometry MFC results that were in agreement with MDS, whereas
Two hundred forty-five patients had an aberrant karyo- the CM results indicated suspected MDS, and slightly less
type, and 189 (77.1%) of those patients had MDS accord- than 33% of those patients had an aberrant karyotype.
ing to the MFC results (Table 3). Thus, 6.4% of all patients had MFC results that were in
agreement with MDS without a clear diagnosis of MDS by
Comparison of Diagnostic Results Obtained CM, and 33% of those patients had an aberrant karyotype.
by Multiparameter Flow Cytometry,
Cytomorphology, and Cytogenetics
Multiparameter Flow Cytometry in
The numbers of patients who had results that were rated in
Cytogenetically Aberrant Cases Not
agreement and not in agreement with MDS on MFC are Classified as Myelodysplastic Syndrome by
listed separately according to the CM results (no MDS, Cytomorphology
MDS, or suspected MDS), and each category is broken Loss of the Y chromosome was not considered MDS-related,
down according to aberrant CG results and normal CG because it occurs in healthy, older individuals. Thus,
results in Table 4. Overall, 1.3% of patients had MFC although these patients are listed in Table 5, they are not
results that were in agreement with MDS in the absence of included in the totals. In 12 patients, CM gave no indication
Table 4. Diagnostic Results of Multiparameter Flow of MDS, but MDS-typical CG aberrations were present (Ta-
Cytometry in Cytomorphologically Defined Myelodysplastic
Syndrome Subgroups Separated According to the Presence
ble 5). The MFC results from 6 of 12 patients (50%) were in
of Cytogenetic Abnormalities agreement with MDS. In another 23 patients, CM identified
dysplastic features that were not sufficient to diagnose MDS,
MFC Result in
Agreement With MDS but CG revealed aberrations. MFC results from 11 of 23
Independent of patients (47.8%) were in agreement with MDS. It is note-
CM Result: No. of worthy that patients who had loss of the Y chromosome had
Patients (%)
MFC results that were in agreement with MDS only if CM
CM Result No Yes
identified dysplastic features that were not sufficient to diag-
Aberrant cytogenetics nose MDS, whereas all patients who had loss of the Y chro-
No MDS 17 (1.7) 6 (0.6)
mosome and no indication of MDS on CM had MFC
MDS 22 (2.2) 168 (16.6)
Suspected MDS 17 (1.7) 15 (1.5) results that were not in agreement with MDS (Table 5).
Normal cytogenetics
No MDS 247 (24.4) 7 (0.7) Comparison of Myeloid Blasts Counts by
MDS 107 (10.6) 214 (21.1) Cytomorphology and Multiparameter Flow
Suspected MDS 157 (15.5) 36 (3.6) Cytometry
MFC indicates multiparameter flow cytometry; MDS, myelodysplastic syn- The mean  standard deviation percentages of myeloid
drome; CM, cytomorphology. blasts determined by CM versus MFC were 4.67% 
Table 5. Diagnostic Results of Multiparameter Flow Cytometry in Patients With Cytogenetically
Aberrant Results Not Classified as Myelodysplastic Syndrome by Cytomorphology

Suspected MDS by CM No MDS by CM

Cytogenetic Result Total No. No. of Patients Total No. No. of Patients
of Patients With Results in of Patients With Results in
Agreement With Agreement With
MDS by MFC MDS by MFC
Del(5q) 5 3 1 0
Aberrations of chromosome 7 1 1 3 3
Trisomy 8 3 1 3 1
Del(20q) 4 3 1 0
Complex karyotype 1 0 1 1
Loss of Y chromosome 5 4 10 0
Other aberrations 9 3 3 1

MDS indicates myelodysplastic syndrome; CM, cytomorphology; MFC, multiparameter flow cytometry; Del, deletion.

4554 Cancer October 1, 2010

 MFC in MDS/Kern et al

Figure 4. The correlation of blast counts obtained by cyto-

morphology (CM) and by multiparameter flow cytometry
(MFC) is depicted. The height of each column indicates the
numbers of patients. The mean  standard deviation percen-
tages of myeloid blasts determined by CM versus MFC were
4.67%  4.18% versus 3.78%  2.97%, respectively (Spearman
rank correlation, 0.362; P < .001).

4.18% versus 3.78%  2.97% (Spearman rho [r], 0.362;

P < .001) (Fig. 4). A higher blast count on CM in some
patients resulted from the presence of monocytoid cells in
which monocytic characteristics were featured that had to
be included in the blast counts according to WHO criteria
but that did not express progenitor markers according to
the MFC results. However, these cells clearly were mye-
loid/monoblastic or promonocytic blasts that were
detected by CM and cytochemistry (for an example, see
Fig. 5). Accordingly, in patients who had aberrant CD56
expression on monocytes, a higher blast count (as deter-
mined by CM) was observed compared with patients who
did not have aberrant CD56 expression (6.1%  4.6% vs
4.1%  3.9%; P < .001), whereas the respective differen-
ces in blast counts determined by MFC clearly were
smaller (4.1%  3.2% vs 3.6%  2.9%; P ¼ .014).

Characteristics in Patients With
Myelodysplastic Syndromes Defined by
Multiparameter Flow Cytometry
Aberrant antigen expression in myeloid blasts
Aberrant antigen expression in myeloid blasts was
most frequent in patients who had MDS compared with
patients who did not have MDS or who had suspected
MDS according to the CM results (Table 6). In patients
Figure 5. A bone marrow sample from a patient with myelo-
dysplastic syndrome is shown in which multiparameter flow
cytometry yielded (A) a myeloid blast count of 6% (red) and
(A,B) 14% monocytic cells (purple), whereas (C) cytomor-
phology yielded a myeloid blast count of 18%. SSC indicates
side scatter; CD, cluster of differentiation; CD45, protein tyro-
sine phosphatase, receptor type, C; PC7, proprotein conver-
tase 7; CD33, 67-kDa membrane glycoprotein; PC5,
phycoerythrin-cyanine 5; CD56, neural cell adhesion mole-
cule; PE, phycoerythrin.

who had CG aberrations, an aberrant expression of the

Cancer October 1, 2010 4555

Original Article

Table 6. Correlation of Aberrant Antigen Expression in Myeloid Blasts With Blast Count and
Dysplastic Features in Cytomorphologya

Cytomorphologic Findings: No. of

Patients (%)
MFC Findings No MDS, MDS, Suspected P
n5277 n5511 MDS, n5225
CD11bþ 3 (1.1) 27 (5.3) 13 (5.8) .009
HLA-DR 0 (0) 4 (0.8) 0 (0) NS
CD5þ 0 (0) 9 (1.8) 0 (0) .012
CD56þ 0 (0) 17 (3.3) 4 (1.8) .007
CD7þ 1 (0.4) 18 (3.5) 1 (0.4) .002
CD15þ 1 (0.4) 10 (2) 6 (2.7) NS
CD64þ 1 (0.4) 12 (2.3) 6 (2.7) NS
No. of aberrant antigens 0.00.2b,c 0.20.6b 0.10.6c .001b
per patient, meanSD .003c
% Blasts, meanSD 2.81.6b,c 4.63.7b 3.11.7c .001b
.041c

MFC indicates multiparameter flow cytometry; MDS, myelodysplastic syndrome; CD, cluster of differentiation;
CD11b, integrin a M; þ, positive; HLA-DR, human leukocyte antigen-D related; , negative; NS, nonsignificant; CD5,
type I transmembrane protein; CD56, neural cell adhesion molecule; CD7, glycoprotein 40; CD15, 3-fucosyl-N-acetyl-
lactosamine; CD64, integral membrane glycoprotein; SD, standard deviation.
a
Numbers of patients were compared using chi-square tests, and mean values were determined using Student t tests.
b
Comparison of columns 1 and 2.
c
Comparison of columns 1 and 3.

following antigens was observed more frequently com-
pared with their expression in patients who had normal
CG results: CD11b (7.3% vs 3.3%; P ¼ .010), CD56
(4.1% vs 1.4%; P ¼ .018), and CD7 (3.7% vs 1.4%;
P ¼ .036).
Considering CG subgroups, a blast count >5% was
observed more often (compared with the remaining
patients) in those who had aberrations of chromosome 7
and in those who had a complex aberrant karyotype,
respectively (7 of 14 patients [50%] vs 167 of 999 patients
[16.7%]; P ¼ .005 and 14 of 23 patients [60.9%] vs 160
of 990 patients [16.1%]; P < .001). Accordingly, the
mean (standard deviation) blast counts were higher in
the former patients (5.8  3.3 vs 3.8  3.0; P ¼ .039 and
8.3  5.5 vs 3.7  2.8; P < .001). Patients with 20q dele-
tion (del[20q]) or del(5q), respectively, as the sole aberra-
tion had CD11b expression more often than others (4 of
21 patients [19%] vs 39 of 992 patients [3.9%], respec-
tively; P ¼ .010; and 5 of 43 patients [11.6%] vs 38 of
970 patients [3.9%], respectively; P ¼ .010).
Aberrant antigen expression in granulocytes
In granulocytes, aberrant antigen expression was
observed most frequently in patients who had MDS con-
firmed by CM compared with patients who had no MDS
or who had suspected MDS according to the CM results
(Table 7). Aberrant antigen expression was not correlated
strongly with dysgranulopoiesis by CM. Thus, in 406
patients without dysgranulopoiesis according to CM
results, dysplastic features were observed with the follow-
ing frequencies: aberrant CD13/CD16, 104 patients
(25.6%); aberrant CD11b/CD16, 62 patients (15.3%);
CD56 expression, 38 patients (9.4%); CD33 negativity,
44 patients (10.8%); and CD64 negativity, 2 patients
(0.5%). Aberrant expression of
2 antigens in granulo-
cytes was observed in 16 of 31 patients (51.6%) and in 15
of 27 patients (55.6%) with refractory anemia (RA) and
RA with ring sideroblasts (RARS), respectively.
The SSC granulocyte:lymphocyte ratio was lower in
patients with MDS than in patients with no MDS
(6.55  2.32 vs 7.47  1.09, respectively; P < .001). Fur-
thermore, no respective significant differences were
observed between patients with or without cytomorpho-
logically identified dysgranulopoiesis (6.99  2.20 vs
6.98  1.27, respectively; P value not significant.).
In patients who had CG aberrations, aberrant
expression of the following antigens was observed more
frequently compared with patients who had a normal
karyotype: CD13/CD16 (42.9% vs 25.1%, respectively;
P < .001), CD11b/CD16 (24.1% vs 15.4%, respectively;
P ¼ .003), CD56 (20.8% vs 9.4%, respectively; P <
.001), CD33 negativity (12.7% vs 7.7%, respectively;

4556 Cancer October 1, 2010

 MFC in MDS/Kern et al

Table 7. Correlation of Aberrant Antigen Expression in Granulocytes With Cytomorphologya

Cytomorphologic Findings: No. of

Patients (%)
MFC Findings No MDS, MDS, Suspected P
n5277 n5511 MDS, n5225
Abnormal CD13/CD16 25 (9) 219 (42.9) 54 (24) <.001
Abnormal CD11b/CD16 9 (3.2) 143 (28) 25 (11.1) <.001
CD56þ 10 (3.6) 90 (17.6) 23 (10.2) <.001
CD33 18 (6.5) 53 (10.4) 19 (8.4) NS
CD64 0 (0) 14 (2.7) 8 (3.6) .011

No. of aberrant antigens 0.00.2

per patient, meanSD
1 0.20.6 <.001
2 0.10.6 .003
Reduced SSC signal 14 (5.1) 286 (56) 42 (18.7) <.001

SSC ratio of granulocytes 7.471.09

to lymphocytes, meanSD
1 6.552.32 <.001
2 7.381.17 NS

MFC indicates multiparameter flow cytometry; MDS, myelodysplastic syndrome; CD, cluster of differentiation; CD13,
alanine aminopeptidase; CD16, human neutrophil antigen 1; CD11b, integrin a M; CD56, neural cell adhesion mole-
cule; þ, positive; CD33, transmembrane receptor; , negative; CD64, integral membrane glycoprotein; NS, nonsignifi-
cant; SD, standard deviation; SCC, side scatter.
a
Numbers of patients were compared using chi-square tests, and mean values were determined using Student t tests.

P ¼ .020), and CD64 negativity (4.9% vs 1.3%, respec-
tively; P ¼ .002). Patients who had aberrant CG results
had a lower SSC granulocyte:lymphocyte ratio
(6.33  1.26 vs 7.20  2.00, respectively; P < .001).
In patients who had aberrations of chromosome 7
only and in patients who had complex aberrant karyo-
types, distinct aberrant antigen expression was observed
more frequently (for this comparison and for the compari-
sons described below, the remaining patients were used as
comparison groups): CD13/CD16, 9 of 14 patients
(64.3%) versus 289 of 999 patients (28.9%), respectively
(P ¼ .007) and 12 of 23 patients (52.2%) versus 286 of
990 patients (28.9%), respectively, (P ¼ .020).
Patients who had 5q alone also had other specific
markers: CD56 was present in 10 of 43 patients (23.3%)
versus 113 of 970 patients without 5q (11.6%;
P ¼ .031). Among those with trisomy 8, aberrant CD13/
CD16 expression was observed in 16 of 30 patients
(53.3%) versus 282 of 983 patients without trisomy 8
(28.7%; P ¼ .007); aberrant CD11b/CD16 expression
was observed in 13 of 30 patients (43.3%) versus 164 of
983 patients (16.7%), respectively (P ¼ .001); and CD33
negativity was observed in 9 of 30 patients (30%) versus
81 of 983 patients (8.2%), respectively (P ¼ .001). Fur-
thermore, the following CG subgroups had a lower SSC
granulocyte:lymphocyte ratios (trisomy 8, 6.00  1.08 vs
7.02  1.89 without trisomy 8 [P < .001]; 20q alone,
6.25  1.13 vs 7.00  1.89 without 20q alone
[P ¼ .007]; 5q alone, 6.03  1.20 vs 7.03  1.90 with-
out 5q alone [P < .001]; and a complex aberrant karyo-
type, 5.83  1.28 vs 7.01  1.89 without a complex
aberrant karyotype [P < .001]).
Aberrant antigen expression in monocytes
In patients who had both MDS and suspected MDS
according to CM results, an aberrant antigen expression in
monocytes was observed more frequently compared with
patients who had no MDS (Table 8). CD56 coexpression
was observed most often in patients with MDS (43.1%);
however, it also was encountered in patients without CM
evidence of MDS (9.4%). Similar percentages were
observed for CD16 expression (9% vs 3.6%, respectively),
whereas both CD2 coexpression (8.8% vs 0.4%, respec-
tively) and CD13 negativity (8.6% vs 2.2%, respectively)
clearly occurred more often in patients with MDS.
The following analyses were made using the respec-
tive remaining patients in each subgroup for comparisons.
Aberrantly expressed antigens in monocytes were
observed in patients with chronic myelomonocytic leuke-
mia (CMML) more frequently (vs patients without
CMML), including CD13 negativity in 9 of 65 patients
(13.8%) versus 54 of 948 patients (5.7%; P ¼ .015),

Cancer October 1, 2010 4557

Original Article

Table 8. Correlation of Aberrant Antigen Expression in Monocytes With Cytomorphologya

Cytomorphologic Findings: No. of

Patients (%)
MFC Findings No MDS, MDS, Suspected P
n5277 n5511 MDS, n5225
CD11b 1 (0.4) 7 (1.4) 3 (1.3) NS
HLA-DR 3 (1.1) 44 (8.6) 6 (2.7) <.001
CD13 6 (2.2) 44 (8.6) 13 (5.8) .002
CD16þ 10 (3.6) 46 (9) 15 (6.7) .018
CD56þ 26 (9.4) 220 (43.1) 47 (20.9) <.001
CD2þ 1 (0.4) 45 (8.8) 6 (2.7) <.001

No. of aberrant antigens 0.20.5 <.001

per patient, meanSD
1 0.80.9
2 0.40.7

MFC indicates multiparameter flow cytometry; MDS, myelodysplastic syndrome; CD, cluster of differentiation;
CD11b, integrin a M; , negative; NS, nonsignificant; HLA-DR, human leukocyte antigen-D related; CD13, alanine
aminopeptidase; CD16, human neutrophil antigen 1; þ, positive; CD56, neural cell adhesion molecule; CD2, lympho-
cyte function-associated antigen 2; SD, standard deviation.
a
Numbers of patients were compared with chi-square tests, and mean values were determined with Student t tests.

CD56 expression in 53 of 65 patients (81.5%) versus 240
of 948 patients (25.3%; P < .001), and CD2 expression
in 14 of 65 patients (21.5%) versus 38 of 948 patients
(4%; P < .001).
Fifteen of 133 patients (11.3%) who had RA with
excess blasts (RAEB) between 5% and 9% (RAEB-1) were
negative for HLA-DR compared with 48 of 880 patients
without RAEB-1 (5.5%; P ¼ .019); CD13 negativity was
observed in 15 of 133 patients (11.3%) versus 38 of 880
patients (4.3%), respectively (P ¼ .020); CD16 expres-
sion was observed in 16 of 133 patients (12%) versus 55
of 880 patients (6.3%), respectively (P ¼ .027); and
CD56 expression was observed in 51 of 133 patients
(38.3%) versus 242 of 880 patients (27.5%), respectively
(P ¼ .014).
Among the patients who had RAEB with 10% to
19% excess blasts (RAEB-2) (vs patients without RAEB-
2), CD13 negativity was observed in 11 of 81 patients
(13.6%) versus 52 of 932 patients (5.6%) (P ¼ .013);
CD56 expression was observed in 41 of 81 patients
(50.6%) versus 252 of 932 (27%; P < .001); and CD2
expression was observed in 12 of 81 patients (14.8%) ver-
sus 40 of 932 patients (4.3%; P < .001).
In patients with RA (vs patients without RA), CD56
expression in monocytes was observed more often (17 of
31 patients [54.8%] vs 276 of 928 patients [28.1%];
P ¼ .002). In both patients with RA and patients with
RARS, at least 2 aberrantly expressed antigens in mono-
cytes were observed in 6 of 31 patients (19.4%) and in 2
of 27 patients (7.4%), respectively.
In patients with CG aberrations (vs patients without
CG aberrations), CD56 expression was observed more fre-
quently (95 of 245 patients [38.8%] vs 198 of 768 patients
[25.8%]; P < .001). In patients who had MDS with a com-
plex aberrant karyotype (vs patients who had MDS with nor-
mal CG results), HLA-DR negativity was observed in 5 of
23 patients (21.7%) versus 48 of 909 patients (4.8%), respec-
tively (P ¼ .005). In patients who had MDS with trisomy 8
(vs patients who had MDS without trisomy 8), CD13 nega-
tivity was observed more frequently (7 of 30 patients
[23.3%] vs 56 of 983 patients [5.7%]; P ¼ .002), and CD56
expression was observed more frequently (19 of 30 patients
[63.3%] vs 274 of 983 patients [27.9%]; P < .001). Finally,
in patients who had MDS with aberrations of chromosome
7 (vs patients who had MDS without aberrations of chromo-
some 7), CD56 expression was observed in 10 of 14 patients
(71.4%) versus 283 of 999 patients (28.3%; P ¼ .001).
Aberrant antigen expression in erythrocytes
Aberrant CD71 expression was observed signifi-
cantly more often in both in patients who had MDS and
in patients who had suspected MDS (judged by CM)
compared with patients who did not have MDS (Table
9). In patients who had RA, RARS, and RCMD/ringed
sideroblasts, compared with the respective remaining
patients who did not have those findings, homogeneously
strong expression of CD71 was observed in 5 of 31
patients (16.1%) versus 62 of 982 patients (6.3%;
P ¼ .048), in 6 of 27 patients (22.2%) versus 61 of 986
patients (6.2%; P ¼ .007), and in 8 of 49 patients

4558 Cancer October 1, 2010

 MFC in MDS/Kern et al

Table 9. Correlation of Aberrant Antigen Expression in Erythrocytes With Cytomorphologya

Cytomorphologic Findings: No. of

Patients (%)
MFC Findings No MDS, MDS, Suspected P
n5277 n5511 MDS, n5225
CD71 homogeneously strong 3 (1.1) 46 (9) 18 (8) <.001
CD71 negative 6 (2.2) 71 (13.9) 17 (7.6) <.001

No. of aberrant antigens 0.00.2

per patient, meanSD
1 0.30.4 <.001
2 0.20.4 <.001

MFC indicates multiparameter flow cytometry; MDS, myelodysplastic syndrome; CD, cluster of differentiation; CD71,
transferrin receptor 1; SD, standard deviation.
a
Numbers of patients were compared using chi-square tests, and mean values were determined using Student t tests.

(16.3%) versus 59 of 964 patients (6.1%; (P ¼ .012),

respectively. In patients with RAEB-1 and RAEB-2, com-
pared with the respective remaining patients without
those findings, CD71 negativity was observed in 21 of
133 patients (15.8%) versus 73 of 880 patients (8.3%;
P ¼ .009) and in 14 of 81 patients (17.3%) versus 80 of
932 patients (8.6%; (P ¼ .015), respectively.
In the presence of CG abnormalities (vs normal CG
findings), greater frequencies of homogeneously strong
CD71 expression (27 of 245 patients [11%] vs 40 of 768
patients [5.2%]; P ¼ .003) and of CD71 negativity (32 of
245 patients [13.1%] vs 62 of 768 patients [8.1%];
P ¼ .023) were observed. In particular, in patients who had
5q as a sole CG abnormality, CD71 negativity was pres-
ent in 9 of 43 patients (20.9%) versus 85 of 970 patients
without 5q as a sole abnormality (8.8%; P ¼ .014).

Correlation of the Total Numbers of

Aberrantly Expressed Antigens With
Cytomorphology
The median total numbers of aberrantly expressed anti-
gens in blasts, granulocytes, monocytes, and erythrocytes
were 3 (range, 0-11), 1 (range, 0-8), and 0 (range, 0-7) in
patients from each group with CM results of MDS, sus-
pected MDS, and no MDS, respectively (P < .001) (Fig.
6). Using the CM result as the diagnostic gold standard,
different total numbers of aberrantly expressed antigens
were analyzed with regard to the sensitivity and specificity
of MFC in reproducing CM results. Table 10 demon-
strates that, although specificity remains at similar levels Figure 6. The total numbers of aberrant antigen expression in
blasts, granulocytes, monocytes, and erythrocytes are illus-
when applying higher numbers of aberrantly expressed trated for patients who were diagnosed by cytomorphology
antigens, a gradual loss of sensitivity is observed. with (A) no myelodysplastic syndrome, (B) myelodysplastic
syndrome, or (C) suspected myelodysplastic syndrome. The
A cutoff value of 6.3 for the SSC granulocyte:lym- height of each column indicates the numbers of patients.
phocyte ratio was identified as the best discriminate

Cancer October 1, 2010 4559

Original Article

Table 10. Sensitivity and Specificity of Multiparameter Flow Cytometry in Reproducing Cytomorphologic Diagnostic Results

Aberrantly Expressed Aberrantly Expressed Aberrantly Expressed

Antigens Only, % Antigens or Blast Antigens or Blast
Count > 5%, % Count > 5% or SSC
Ratio (Granulocytes:
Lymphocytes) > 6.3, %
MFC Criteria: Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity
No. of Aberrantly
Expressed Antigens

2 75.5 92.6 79.6 91.3 85.3 86.3

3 57.5 93.6 65.2 92 77.3 86.6

4 32.9 95.5 46.8 92.6 66.7 85.9

5 16.9 94.5 35 91.3 61.6 85.4

SSC indicates side scatter; MFC, multiparameter flow cytometry.

Table 11. Relation Between the Total Number of Aberrantly

Expressed Antigens in All Cell Lineages and International
Prognostic Scoring System Scorea

IPSS No. of No. of Aberrantly

Score Patients Expressed Antigens,
Mean6SD
0 99 2.261.65
0.5 123 2.781.93
1.0 85 2.991.82
1.5 47 3.191.39
2.0 56 3.341.81
2.5 14 4.002.08
3.0 7 2.431.27

IPPS indicates International Prognostic Scoring System; SD, standard

deviation.
a
Data on IPSS scores were evaluable for 431 patients.

Figure 7. This Kaplan-Meier plot illustrates overall survival for

between results that were rated as MDS and no MDS by
CM. Taking into consideration an SSC granulocyte:lym-
phocyte ratio of >6.3 and a blast count by MFC of >5%
and combining these parameters with the total number of
aberrantly expressed antigens resulted in improved sensi-
tivity (Table 10).
Relation of Aberrant Antigen Expression to
the International Prognostic Scoring System
The relation of MFC results to IPSS was assessed in 431
patients with MDS who had IPSS scores available. Table
11 demonstrates that the total number of aberrantly
expressed antigens gradually increases with increasing
IPSS score (Spearman: r ¼ 0.409; P < .001).
Relation of Aberrant Antigen Expression
to Overall Survival
In 257 patients who had clinical follow-up, the relation
between MFC findings and overall survival (OS) was ana-
patients who had any of the 3 parameters 1)
3 aberrantly
expressed antigens, 2) a blast count >5%, or 3) a side scatter
ratio (granulocytes:lymphocytes) >6.3 (solid line) versus
patients who had none of those parameters (dotted line;
10-year over survival rate, 68% vs 100%, respectively; P ¼.008).
lyzed. OS, as determined from the date of a diagnosis or a
suspected diagnosis of MDS to death, amounted to 74%
at 6 years. The number of aberrantly expressed antigens
had a trend toward a correlation with OS (relative risk,
1.2; P ¼ .09). Three parameters that were related to OS in
univariate analyses were combined for an overall analysis
of the relation between MDS-related findings by MFC
and OS: Those parameters were
3 aberrantly expressed
antigens, a blast count >5% in MFC, and an SSC granu-
locyte:lymphocyte ratio >6.3. Separating patients accord-
ing to the presence of either versus none of these
parameters resulted in significant differences in the 2-year
OS rate (86% vs 100%; P ¼ .008) (Fig. 7).

4560 Cancer October 1, 2010


DISCUSSION
Standard BM evaluation in suspected MDS includes CM,
cytochemistry, and iron staining as well as a CG analysis for
classification according to WHO8 and estimation of prog-
nosis.10,11 However, many patients who have inconclusive
CM results and a normal karyotype remain9,32,33 for
whom an MDS cannot be diagnosed or ruled out. MFC is
capable of identifying dysplastic features in BM samples
and, thus, was supposed to be added to the diagnostic
workup in patients who have suspected MDS.13-16,32,34
In the current study, several aspects regarding the
specificity and sensitivity of MFC for diagnosing MDS
have been elucidated in agreement with previous stud-
ies.15,16 In the current series and in the data reported by
van de Loosdrecht et al,16 this also is true for the identifi-
cation of dysplastic features in cell lineages that were not
rated dysplastic by CM alone.
The enumeration of BM blasts has major prognostic
impact10 and also guides the WHO classification.8 We
observed a significant degree of correlation and concord-
ance; however, patients who had counts that differed
between both methods also were encountered. Although a
difference in sample quality always may be considered as a
cause, methodologic aspects also have to be considered.
First, smears for CM investigation and BM samples after
Ficoll processing for MFC, per se, are different sources.
Second, it is suggested that myeloid blasts should be iden-
tified in MFC by using multiple parameters, including
CD45/SSC signal and the expression of HLA-DR and
CD34, together with myeloid markers.35 It has to be
taken into consideration, however, that cells counted as
myeloid blasts by CM may fall into the monocyte cate-
gory in MFC and, thus, may be identified as dysplastic
monocytes, which are not easily identified by CM and
need at least nonspecific-esterase evaluation. Along this
line, we observed that higher percentages of blasts were
determined by CM in samples that had aberrant antigen
expression in monocytes. Therefore, despite the high
degree of concordance, the percentages of blasts deter-
mined by CM and by MFC should be considered sepa-
rately. Consequently, the prognostic value of blast counts
determined by flow cytometry should be evaluated in pro-
spective multicenter studies.
Although the majority of previous reports focused
on patients with proven MDS and, in part, added control
samples from patients who clearly did not have a malig-
nancy,13-16 in the current study, a large series of 1013
patients with suspected MDS was the focus of our analy-
ses. None of these patients had undergone a prior proof or
Cancer October 1, 2010
MFC in MDS/Kern et al
rule out of MDS. Thus, the current series represents a per-
fect cohort for the assessment of the potential of MFC in
the diagnostic workup of patients with unclear cytopenias
and a differential diagnosis of MDS. For the first time to
our knowledge, the current study allows an evaluation of
the diagnostic usefulness of MFC in MDS in the context
of a large cohort of patients characterized by CM and CG.
In patients who had unequivocal CM findings, we
observed a high degree of concordance (82%) between
CM and MFC results. It is noteworthy that, in patients
who had dysplastic features that were insufficient to diag-
nose MDS using CM alone, aberrant antigen expression
was observed with MFC, although at lower frequencies
compared with the frequencies observed in patients with
clear-cut MDS. Furthermore, 13 of 277 patients (4.7%)
who had no evidence of MDS by CM were considered in
agreement with MDS by MFC, posing the question of the
specificity of MFC findings or the diagnostic role of MFC
in addition to CM. A definite proof of MDS could be
accomplished in 6 such patients in the current series based
on MDS-typical CG aberrations, arguing in favor of the
significance of MFC findings (which have been debated
during the revision of the WHO classification).9
Thus, the current data suggest that, in patients who
have normal karyotypes, MFC also may identify individu-
als with MDS who have CM results that indicate either
no evidence of MDS or dysplastic characteristics that are
not sufficient to clearly diagnose MDS. Consequently,
although the diagnosis of MDS is based on CM results
according to the WHO classification,8 it is noteworthy
that CM per se does not have 100% sensitivity; therefore,
an analysis of the sensitivity and specificity of MFC in
diagnosing MDS using CM as the gold standard is ham-
pered, and the respective results should be interpreted
accordingly. This was proven in the current analysis at
least in some of the patients who had positive MFC
results, negative CM results, and CG results that revealed
MDS-typical chromosomal aberrations. This scenario
further supports the need for clinical validation of the
diagnostic use of MFC in MDS, which should be eval-
uated together with patient outcomes.36
It is also clear, however, that, in the current series,
129 patients who had MDS clearly proven by CM did not
have dysplastic features identified by MFC. Thus, the
results of this crude analysis are suggestive of a combined
use of CM and MFC in the diagnostic evaluation of
patients with suspected MDS.
Although we were able to reproduce some previously
reported findings, like the aberrant CD56 expression in
4561
Original Article
monocytes in CMML,37 the current study adds important
data on the capability of MFC to identify cell compart-
ments affected by dysplasia that are not identified by CM.
This was reported previously by van de Loosdrecht et al16
and was reproduced here by the identification of an aber-
rant antigen expression in granulocytes in patients who
had no dysgranulopoiesis according to the CM results.
One important aspect of handling data generated by
MFC in MDS is the definition of criteria that are useful
to define MDS by MFC. Wells et al demonstrated that
the diagnostic specificity of MFC increases together with
an increase in the number of aberrant markers detected,
although at the cost of decreasing sensitivity.15 We made a
similar observation, although a specificity of 100% could
not be reached in our dataset. This difference may be
because, in the current series, some patients without evi-
dence of MDS in CM analysis had MDS proven in CG
analysis, as discussed above. Furthermore, it has to be
stressed that the context of the current analysis is unique,
in that a large number of patients with yet unclear cytope-
nias and with a differential diagnosis of MDS was assessed
in parallel by the 2 gold-standard diagnostic methods, ie,
CM and CG, complemented by MFC. Thus, we were
able to analyze both the specificity and the sensitivity of
MFC findings in a routine diagnostic setting. These
results are in line with a previous report on the diagnostic
value of MFC in MDS that was based on a smaller series
of 124 retrospectively analyzed patients in which both
CM and CG were used to validate MFC.38
In the current series, a correlation between an
increasing number of aberrantly expressed antigens and
an increasing IPSS score was observed, and this finding
confirmed previous reports in smaller series.16 In addi-
tion, in a subset of 257 patients, OS was inferior if MDS-
typical findings by MFC were present. This also is in line
with previous data on patients with MDS who underwent
stem cell transplantation.15
In conclusion, the current results demonstrate that,
in a large series of patients who had a differential diagnosis
of MDS, MFC in combination with CM and CG may
add important diagnostic information. Patients who have
aberrant antigen expression can be identified although
their CM results may indicate no features of MDS. Cell
compartments that are affected by dysplasia and are not
identified by CM can be identified by MFC criteria. The
further comparison based on CG and clinical data
strengthens the significance of the current MFC findings.
Because the sensitivity of both CM and MFC for identify-
ing MDS is <100%, we suggest considering CM and
4562
MFC in a combined approach to optimize the overall
diagnostic yield. Thus, the current gold standard for diag-
nosing MDS, ie, CM, should be used as such but with the
awareness that other methods (ie, CG and MFC) may
identify patients with MDS who have results that are not
rated MDS by CM. Accordingly, as the most reasonable
approach to diagnosing MDS, we suggest a combination
of CM, CG, and MFC. Further studies should be per-
formed to confirm these results and especially to define
standards for the application of MFC in the diagnostic
workup of MDS.34,35
CONFLICT OF INTEREST DISCLOSURES
The authors made no disclosures.
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