Leukemia (2000) 14, 816–825
 2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00
www.nature.com/leu
BIOMED-1 Concerted Action report: Flow cytometric characterization of CD7ⴙ cell
subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell
acute lymphoblastic leukemia (T-ALL)
A Porwit-MacDonald1, E Björklund1, P Lucio2, EG van Lochem3, J Mazur1, A Parreira2, MWM van den Beemd3,
ER van Wering4, E Baars4, G Gaipa5, A Biondi5, J Ciudad6, JJM van Dongen3, JF San Miguel6 and A Orfao6
1
Department of Pathology, Karolinska Hospital, Stockholm, Sweden; 2Instituto Portugues de Oncologia, Lisboa, Portugal; 3Department of
Immunology, Erasmus University, Rotterdam, and University Hospital, Rotterdam, The Netherlands; 4Dutch Childhood Leukemia Study
Group, The Hague, The Netherlands; 5Centro di Ricerca M Tettamanti, Monza, Italy; and 6Department of Hematology, University Hospital,
Salamanca, Spain
The European BIOMED-1 Concerted Action was initiated in
1994 to improve and standardize the flow cytometric detection
of minimal residual disease (MRD) in acute leukemia (AL).
Three different protocols were defined to identify the normal
subsets of B, T and myeloid cells in bone marrow (BM), and
were applied to the different types of AL in order to study
aberrant immunophenotypes. Using sensitive acquisition
methods (‘live gate’) T cell subsets in normal BM could be
identified with five triple-stains: CD7/CD5/CD3, CD7/CD4/CD8,
CD7/CD2/CD3, CD7/CD38/CD34 and TdT/CD7/surface or cyto-
plasmic (cy)CD3 (antibodies conjugated with FITC/PE/PECy5 or
PerCP, respectively). The identification of T cell subsets in BM
allowed definition of ‘empty spaces’ (ie areas of flow cytometric
plots where normally no cells are found). All studied T-ALL
cases (n = 65) were located in ‘empty spaces’ and could be
discriminated from normal T cells. The most informative triple
staining was TdT/CD7/cyCD3, which was aberrant in 91% of T-
ALL cases. In most cases, two or more aberrant patterns were
found. Apparently the immunophenotypes of T-ALL differ sig-
nificantly from normal BM T cells. This is mostly caused by
their thymocytic origin, but also the neoplastic transformation
might have affected antigen expression patterns. Application
of the five proposed marker combinations in T-ALL contributes
to standardized detection of MRD, since cells persistent or
reappearing in the ‘empty spaces’ can be easily identified in
follow-up BM samples during and after treatment. Leukemia
(2000) 14, 816–825.
Keywords: T cells; bone marrow; acute lymphoblastic leukemia;
immunophenotype; flow cytometry; minimal residual disease
Introduction
It has long been assumed that blasts from T cell acute lym-
phoblastic leukemia (T-ALL) reflect the immunophenotypes of
early stages of T cell differentiation.1–3 Most T-ALL cases dis-
play phenotypes corresponding to various stages of thymocyte
differentiation, which normally are not found in bone marrow
(BM) or peripheral blood (PB).4
The aberrant immunophenotypic characteristics of leu-
kemic T lymphoblasts allow discrimination from T cell subsets
in BM and thus can be used for detection of minimal residual
disease (MRD).5–8 A comprehensive analysis of the immuno-
phenotypic characteristics of T cells and the subsequent
identification of all T cell subsets in normal BM are important
for the reliable distinction between normal and leukemic T
cells.
Previous studies in T-ALL strongly suggest that the use of
Correspondence: A Porwit-MacDonald, Department of Pathology,
Karolinska Hospital, 171 76 Stockholm, Sweden; Fax: 46 851775843
J Mazur was on leave from Department of Epidemiology and Health
Care Planning, Institute Mother and Child, Warsaw, Poland
Received 14 June 1999; accepted 15 December 1999
multiparameter flow cytometry for the investigation of MRD
can reach a high sensitivity and specificity for the identifi-
cation of residual leukemic T lymphoblasts.8–11 A cooperative
study conducted by six European centers involved in the
BIOMED-1 Concerted Action ‘Investigation of Minimal
Residual Disease in Acute Leukemia: International Standardiz-
ation and Clinical Evaluation’ applied multiparameter flow
cytometry in which triple antibody combinations were used
together with sensitive data acquisition methods (such as ‘live
gate’ acquisition). Using this approach we obtained detailed
information on cell subsets that are present in normal BM at
very low frequencies and that may remain undetected using
conventional methods of analysis.
The immunophenotyping methods (staining protocols and
flow cytometry) were carefully standardized in order to
develop a highly reproducible and sensitive approach to
identify both major and minor subsets of T cells present in BM
and their relative distribution according to age. Once normal
T cell subsets were identified, we focused our attention on
the ‘empty spaces’ left in multidimensional flow cytometry dot
plots of each studied antigen combination, as has previously
been reported for precursor B cells.12–14 Subsequently, we
used the same triple antibody combinations and flow cytome-
try protocols to analyze 65 consecutive T-ALL cases diag-
nosed in the six participating laboratories. Our aim was to
assess the incidence of aberrant phenotypes, which can be
used for the follow-up of MRD in T-ALL patients.
Materials and methods
Specimen collection
Normal BM samples: Normal BM samples (n = 35) were
obtained from healthy donors (n = 29) and patients with idio-
pathic thrombocytopenia (ITP) (n = 6), recruited in the six Eur-
opean centers involved in the BIOMED-1 Concerted Action
on MRD. The following centers participated: Portuguese Insti-
tute of Oncology, Lisbon, Portugal; Karolinska Hospital,
Stockholm, Sweden; University of Salamanca, Salamanca,
Spain; Department of Immunology, Erasmus University, Rot-
terdam and University Hospital, Rotterdam, The Netherlands;
Dutch Childhood Leukemia Study Group, The Hague, The
Netherlands; and M Tettamanti Research Center, Monza, Italy.
Informed consent was obtained from all donors or parents
in cases of children less than 15 years old in accordance with
local Medical Ethics Committees. The samples from patients
with ITP were obtained for diagnostic purposes. The median
age of the donors was 15 years, ranging from 2 to 75 years
 BIOMED-1 Concerted Action report
A Porwit-MacDonald et al

old (mean age 24), the male/female ratio was 0.8. Moreover,
16 follow-up BM samples from children receiving chemo-
therapy for B-precursor ALL were included to investigate if T
cell content and distribution changed during cytostatic
treatment.
Leukemic samples: T-ALL samples (n = 65) were obtained
in the participating centers for diagnostic purposes. The mor-
phological diagnosis of ALL was made on the basis of micro-
scopic investigation of May–Grünwald-Giemsa stained BM
smears. The immunologic diagnosis of T-ALL was made
according to well-established criteria.4 The median age of the
patients was 13 years, ranging from 1 to 52 years (mean 16),
and the male/female ratio was 1.8.
817
studies showed that both intracellular staining procedures
yielded similar results.14,15
For standardization purposes, we used triple combinations
of the same Mab clones labeled with identical fluorochromes
(FITC/PE/PECy5 or PerCP) in all immunostainings of normal
BM in all participating centers: CD7/CD5/CD3,
CD7/CD4/CD8, CD7/CD2/CD3, CD7/CD38/CD34 and
TdT/CD7/cyCD3 (Table 1). Isotype-matched immunoglobulins
and a sample stained for CD3-FITC/CD4-PE/CD8-PE/Cy5 were
used as negative and positive controls, respectively. Addition-
ally, expression of CD13, CD33 and CD19 within the CD7+
cell population was studied in BM samples from a total of 24
normal donors in Salamanca. The immunostaining of leu-
kemic samples was performed using the same triple antibody
combinations and sample preparation procedures as for
normal BM.

Sample preparation
Bone marrow aspirates were collected in either heparin or
EDTA anticoagulant and maintained at room temperature (RT)
until processed (not longer than 24 h). The nucleated cell
count was adjusted to 1–2 × 107 cells/ml. For sample prep-
aration, a stain and then lyse/wash technique was used.
Briefly, 100 ␮l of BM cell suspension (at least 1 × 106
nucleated cells) were incubated for 10–15 min (RT, in the
dark) with saturating amounts of five different triple combi-
nations of monoclonal antibodies (Mab, Table 1). After incu-
bation, 2 ml of FACS lysing solution (Becton Dickinson (BD),
San Jose, CA, USA) diluted 1/10 in distilled water was added
and samples were incubated for another 10 min at RT in the
dark. Subsequently, cells were washed and re-suspended in 1
ml of PBS.
Cytoplasmic (cy)CD3 and nuclear terminal deoxynucleoti-
dyl transferase (TdT) were detected after staining for surface
markers (as above), and fixation/permeabilization with Perme-
afix (OPM; Ortho, Raritan, NY, USA) or Fix & Perm (F/P; An
der Grub, Vienna, Austria).15 The reagents were used follow-
ing the manufacturer’s recommendations. After OPM fixation
and during permeabilization with F/P, the cells were incu-
bated with anti-TdT or CD3 Mab (30 or 15 min, respectively)
then washed and resuspended in PBS. Previous comparative
Table 1 Five triple marker combinations used for T cell subset
analysis
Data acquisition and analysis
Data acquisition was performed in all centers in a stan-
dardized manner, using FACScan flow-cytometers (BD),
equipped with either LysisII or CellQuest (BD) software pro-
grams. Instrument set-up and calibration was performed in a
standardized way as previously described.12 For the analysis
of normal BM samples, a two-step acquisition procedure was
used. First, 15 × 103 non-gated events were acquired from the
BM samples. Then a total of 105 to 2 × 106 cells were meas-
ured with a live gate set on the basis of CD7 expression and
low/intermediate side scatter (SSC) characteristics. In this
second step, data acquisition exclusively concerned CD7+
cells with lymphoid scatter generally representing approxi-
mately 10% of BM cells (Table 2). In practice, this meant that
between 5 × 104 and 2 × 105 CD7+ lymphoid BM cells were
acquired for further evaluation. The expression of the other T
cell markers was analyzed within this CD7+ lymphocyte gate
in order to characterize the different CD7+ cell subsets.
Additional live gates were based on CD34+ and TdT+ events
displaying low/intermediate SSC characteristics.
For all leukemic samples, 15 × 103 events were acquired
from the total BM and, whenever appropriate, the expression
of various markers analyzed after gating focused on the leu-
kemic blast cells. The fluorescence intensity of T cell markers
observed in normal T-lymphocytes present in each analyzed
sample was used as the reference value for describing the
intensity of antigen expression in leukemic blasts.

Combination FIT-conjugates PE-conjugates PerCP or PECy5

(Tricolor) Standardization of the procedures and quality control
conjugates
Prior to the study, all procedures used for the immunopheno-
I CD7 CD5 CD3
(Leu9, BD) (Leu1, BD) (Leu4, BD)
typic analysis of the normal compartment of the CD7+ BM
cells were standardized among the six participating centers
II CD7 CD4 CD8
via collaboration in experimental work and regular laboratory
(Leu9, BD) (Leu3, BD) (3B5, Caltag)
meetings. The same antibody clones, fluorochrome conju-
III/IV CD7 CD2 CD3
(Leu9, BD) (Leu5b, BD) (Leu4, BD)
gates, and triple-staining combinations were used throughout
the study in all six participating laboratories. The agreed anti-
V TdT CD7 CD3
(HT6, Dako) (Leu9, BD) (Leu4, BD)
body combinations were those providing the most powerful
discrimination between the T cell subsets under study. Pro-
VI CD7 CD38 CD34
cedures for instrument set-up and calibration were carefully
(Leu9, BD) (Leu17, BD) (HPCA-2, BD)
compared and one common well-standardized method was
FITC, fluoresceine isothiocyanate; PE, phycoerythrin; PerCP, perdi-
selected. The light scatter characteristics and the auto-
din chlorophyll protein; BD, Becton Dickinson (San Jose, CA, USA); fluorescence levels of normal PB lymphocytes were used as
Caltag, Caltag Lab (South San Francisco, CA, USA); Dako, reference values for instrument settings. As described above,
(Dakopatts, Glostrup, Denmark). the same two-step acquisition procedure was used in all lab-

Leukemia
 BIOMED-1 Concerted Action report
A Porwit-MacDonald et al

818
Table 2 Frequency of various T cell subsets present in normal bone marrow

Triple staining T cell subsetsa

combination
(FITC/PE/PercP 1 2 3 4 5 6
alt PE/Cy5)

CD7/CD5/CD3 CD3−/CD5− CD3+/CD5− CD3+/CD5bright CD3dim/neg/CD5bright NA NA

n = 35
I 14.4 (8)/ 1.09 (0.99)/ 83.7 (8)/ 0.7 (0.7)/
1.42 (0.7) 0.14 (0.1) 8.28 (0.7) 0.069 (0.06)

CD7/CD4/CD8 CD4−/CD8− CD4−/CD8dim CD4−/CD8bright CD4dim/CD8bright CD4bright/CD8dim CD4+/CD8−

n = 33
II 14.51 (8.2)/ 8.63 (5.2)/ 34.54 (8.6)/ 0.83 (0.8)/ 1.13 (1.6)/ 40.2 (10.3)/
1.4 (0.8) 0.8 (0.5) 3.3 (0.8) 0.08 (0.07) 0.11 (0.15) 3.9 (0.99)

CD7/CD2/CD3 CD3+/CD2− CD3+/CD2+ CD3−/CD2−/CD7bright CD3−/CD2+/CD7bright CD3−/CD2+/CD7dim NA

n = 26
III/IV 0.37 (0.25)/ 85.83 (8.1)/ 3.8 (3.7)/ 8.1 (4.8)/ 1.63 (2.1)/
0.04 (0.03) 8.5 (3.4) 0.38 (0.46) 0.8 (0.54) 0.16 (0.2)
CD3+/CD7+ (III) CD3−/CD7+ (IV) NA
86.12 (8.3)/8.56 (3.3) 14.12 (7.96)/1.42 (0.96)
CD3+/CD2− b CD3+/CD2+ b CD3−/CD2−/CD7bright c CD3−/CD2+/CD7bright c CD3−/CD2+/CD7dim c
0.45 (0.4) 99.5 (0.45) 25.9 (11.4) 60.73 (13) 12.53 (12.7)

a
See Figure 1, results are expressed as percentages of gated CD7 positive cells and percentages of total BM cells, respectively (mean
(s.d.)).
b
% of CD3+/CD7+ cells.
c
% of CD3−/CD7+ cells.
NA, not applicable.

oratories and data analysis was performed with Paint-a-Gate
Pro (BD) software. A special meeting for personnel of all part-
icipating centers was devoted to the training in calibration,
instrument settings, and data acquisition and analysis.
Quality control of the procedures was performed both prior
to and during the study. For this purpose, triple staining of a
normal PB samples for CD3-FITC (Leu4, BD)/CD4-PE (Leu3,
BD)/CD8-PE/Cy5 (CD8-TRI, Caltag Laboratories) was perfor-
med daily. Finally, the data were centrally re-analyzed at
Karolinska Hospital. Statistical analysis (see below) did not
show any significant differences in data obtained at different
laboratories.
Statistical analysis
All the results are expressed as mean with one standard devi-
ation (s.d.). Correlation between antigen expression and the
age of the control BM donors was investigated using t-test and
Pearson correlation test. In order to assess the statistical sig-
nificance of the differences observed between results from
various groups, the analysis of variance (ANOVA) was used.
P values less than 0.05 were considered to be statistically
significant.
Results
Expression patterns of T cell markers in normal bone
marrow
The mean frequency of CD7+ cells in BM was 10% ± 3.6%
(range 5.2–20.4%). The proportion of CD7+ cells increased
with age: 7.9% ± 1.8% in children under 14 vs 14.1% ± 3.8%
in healthy donors 14 or more years of age (P ⬍ 0.01). No
significant differences between the six participating centers
were found regarding the frequency of CD7+ BM cells (P ⬎
0.05).
Expression patterns of the various T cell marker combi-
nations analyzed within the CD7+ BM cells are exemplified
in Figure 1 and their overall frequencies (expressed both as
percentages of CD7+ cells and of total BM cells) are shown in
Table 2.
No clearly defined populations of CD34+/CD7+ and
TdT+/CD7+/cyCD3+ cells were found in any of the normal BM
samples analyzed. This was true both when ‘live gate’ acqui-
sition was performed on the basis of CD7 expression and for
TdT+ or CD34+ cells. Thus, we can assume that frequencies
of these cells in normal BM were less than 1 in 104 cells in
20 studied samples.
As shown in Figure 1a, CD7/CD5/CD3 triple staining (I)
showed the existence of four distinct CD7+ subsets in BM,
the CD3+/CD5+ subset being dominant (84%); other subsets
included CD3−/CD5− (14%), CD3−/CD5+ (1%) and
CD3+/CD5− (1%) cells (Table 2). Further multiple stainings
(performed at Salamanca) showed that most of the CD3−CD5+
events corresponded to natural killer (NK) cells, while the
CD3+/CD5− cells were TCR␥␦+ T lymphocytes (data not
shown). The relative frequency of the CD3+/CD5− subpopul-
ation expressed as a percentage of total CD7+ cells increased
with age (correlation coefficient (corr coeff) 0.4361, P = 0.01).
However, no significant correlation was found when
CD5−/CD3+/CD7+ cells were expressed as a percentage of the
total number of BM cells. No significant differences were
found in the distribution of the other subsets according to age
or between participating centers (P ⬎ 0.05).
Using the CD7/CD4/CD8 triple combination (II), six subsets
of CD7+ cells were identified (Figure 1b, Table 2). Two minor
subsets of CD7+ cells (maximum 0.2% of the total BM cells)
co-expressed both CD4 and CD8 (populations 4 and 5 in Fig-

Leukemia
 BIOMED-1 Concerted Action report
A Porwit-MacDonald et al

819
a b

c d

Figure 1 Flow cytometric analysis of CD7+ cells in the normal BM. The upper row shows subpopulations revealed after CD7/SSC gating, in
triple staining for CD7/CD5/CD3 (I), CD7/CD4/CD8 (II) and lower row for CD3 positive (III) and negative (IV) subsets in CD7/CD2/CD3 staining,
respectively. The frequencies of various populations are given in Table 2. (a) In the CD7/CD5/CD3 (I) staining the cyan dots illustrate population
(1) CD3−/CD5−, blue dots population (2) CD3+/CD5−, green dots population (3) CD3+/CD5bright and red dots population (4) CD3dim/CD5bright.
(b) In the CD7/CD4/CD8 (II) staining the cyan dots illustrate population (1) CD4−/CD8−, blue dots population (2) CD4−/CD8dim, red dots popu-
lation (3) CD4−/CD8bright, violet dots population (4) CD4dim/CD8bright, black dots population (5) CD4bright/CD8dim and green dots population (6)
CD4+/CD8−. (c) In the CD7/CD2/CD3 staining gated on CD3+ cells (III) the blue dots illustrate population (1) CD3+/CD2− and the green dots
population (2) CD3+/CD2+. (d) In the CD7/CD2/CD3 staining gated on CD3− cells (IV) the green dots illustrate population (3)
CD3−/CD2−/CD7bright, blue dots population (4) CD3−/CD2+/CD7bright and red dots population (4) CD3−/CD2+/CD7dim.

ure 1b, Table 2). In these minor subsets, expression of CD4
was always either higher or lower than CD8 and both antigens
never showed similar intensity of expression (when CD4 was
‘bright’, the CD8 was ‘dim’ and vice versa). The proportion
of the CD4−/CD8− cells decreased with age (corr coeff
−0.4469, P = 0.01), while the percentage of the CD4+/CD8−
subset (corr coeff 0.5363, P = 0.01) increased. This was valid
both when the data was expressed as a percentage of total
CD7+ cells and as a percentage of total BM cells. No signifi-
cant differences were found in the frequency of the various T
cell subsets between the six participating centers, P ⬍ 0.05.
The subsets identified by the CD7/CD2/CD3 staining, were
analyzed separately for CD3+ (III) (two subsets defined, Figure
1c) and CD3− (IV) cells (three subsets defined, Figure 1d),
(Table 2). No significant differences were found regarding the
distribution of these subsets according to age or between part-
icipating centers. Further studies have shown that almost all
cells in the CD7+/CD3− population expressed the CD56
NK-cell associated marker (98% ± 1%).
No CD7+/CD19+ could be detected at frequencies higher
than 10−4. In contrast, CD7+/CD13+ and/or CD7+/CD33+ cells
were detected in 10% of the BM samples at frequencies higher
than 10−4 (mean frequency of 0.06 ± 0.03% and 0.05 ± 0.04%
for CD7+/CD13+ and CD7+/CD33+ cells, respectively). It
should be noted that the intensity of expression of both
myeloid-associated markers was ‘dim’ in all samples.
The total numbers and distribution of various T cell subsets
remained stable during chemotherapy. Table 3 shows the
results obtained in 16 patients with B precursor ALL tested at
various phases of treatment. No significant differences were
found between the patients and healthy donors. No popu-
lations of C7/CD34 or cyCD3/TdT cells were found in these
patients.
Immunophenotypic patterns of T-ALL blast cells
Based on the findings obtained from the analysis of normal
BM samples, we established the potential patterns of aberrant
antigen expression in T-ALL. These aberrant patterns are
defined by their position in the so-called ‘empty spaces’ found
on the multidimensional dot plots of normal BM samples
(Figure 2). Based upon the ‘empty space’ definition, we found
that all 65 investigated T-ALL cases showed aberrant immuno-
phenotypes. The frequencies of each of the various aberrant
phenotypic patterns are given in Table 4, and several aberrant
patterns are exemplified in Figure 2. In most of the cases more
than one aberration was found: 91% of cases had aberrant

Leukemia
 BIOMED-1 Concerted Action report
A Porwit-MacDonald et al

820
Table 3 Frequency of various T cell subsets present in bone marrow under cytostatics treatment

Triple staining T cell subsetsa

combination
(FITC/PE/PercP 1 2 3 4 5 6
alt PE/Cy5)

CD7/CD5/CD3 CD3−/CD5− CD3+/CD5− CD3+/CD5bright CD3dim/neg/CD5bright NA NA

n = 16
I 14.7 (8.6)/ 2.9 (5.16)/ 81 (10.6)/ 1.1 (1.02)/
1.17 (1.3) 0.2 (0.4) 9.1 (9.25) 0.1 (0.13)

CD7/CD4/CD8 CD4−/CD8− CD4−/CD8dim CD4−/CD8bright CD4dim/CD8bright CD4bright/CD8dim CD4+/CD8−

n = 16
II 18.7 (8.6)/ 5.5 (3.1)/ 38.7 (7.0)/ 1.28 (1.05)/ 0.5 (0.98)/ 33.4 (12.9)/
1.6 (1.53) 0.5 (0.47) 4.3 (4.6) 0.1 (0.09) 0.04 (0.06) 4.0 (4.4)

CD7/CD2/CD3 CD3+/CD2− CD3+/CD2+ CD3−/CD2−/CD7bright CD3−/CD2+/CD7bright CD3−/CD2+/CD7dim NA

n = 16
III/IV 1.1 (2.0)/ 83 (7.7)/ 5.1 (4.0)/ 9.5 (5.4)/ 1.55 (1.1)/
0.07 (0.09) 8.6 (8) 0.6 (0.85) 0.9 (1.0) 0.12 (0.1)
CD3+/CD7+ (III) CD3−/CD7+ (IV) NA
83.6 (8.6)/7.8 (8.1) 16.4 (1.6)/1.6 (1.9)
CD3+/CD2− b CD3+/CD2+ b CD3−/CD2−/CD7bright c CD3−/CD2+/CD7bright c CD3−/CD2+/CD7dim c
0.73 (0.64) 99.3 (0.64) 31.14 (12.6) 56.6 (12.5) 11.3 (8.2)

a
See Figure 1, results are expressed as percentages of gated CD7 positive cells and percentages of total BM cells, respectively (mean
(s.d.)).
b
% of CD3+/CD7+ cells.
c
% of CD3−/CD7+ cells.
NA, not applicable.

phenotypes in at least two triple stains, 65% in at least three,
25% in at least four, and 4% of cases were aberrant in all five
antibody combinations.
Table 5 shows a detailed description of the different
aberrant phenotypes found in the 65 T-ALL cases included in
the present study. The most common aberrant pattern was the
co-existence of the TdT together with CD7 and/or cyCD3,
found in 58 of the 64 studied cases (91%). Co-expression of
CD34 with CD7 and other surface T cell-associated antigens
was found in 40% of all T-ALL cases analyzed (23/58). In 36
of 65 cases (55%), a bright expression of CD7 (as compared
to normal T-lymphocytes) was noted. In nine of 65 cases
(14%) the intensity of CD7 expression was higher than in nor-
mal NK cells, and one of these cases co-expressed CD56.
Based on these findings, the most common leukemia asso-
ciated phenotype was an ectopic (thymus associated) antigen
expression (91%). Additionally, asynchronous antigen
expression and rare phenotypes were found in 40% and 72%
of cases, respectively. With regard to cross-lineage antigen
expression, none of the analyzed T-ALL cases expressed the
CD19 B cell-associated marker. ‘Dim’ expression of the
myeloid-associated markers CD13 and CD33 was detected in
eight of 58 (16%) and six of 58 (10.5%) cases, respectively
(four of these showed both markers). Totally, based on the
analysis of myeloid and NK cell-associated markers, only
13.5% of the T-ALL cases showed an aberrant immunopheno-
type.
Discussion
The CD7 antigen has been classically considered the earliest
T cell-associated marker expressed during maturation of
T-lymphocytes. This is based on the presence of
CD45+CD7+CD3−CD4−CD8− cells in human fetal liver at the
gestational age of 7 weeks at which the thymic rudiment is
colonized by T cell progenitors.16 More recent findings have
shown that human multi-potent progenitor cells expressing
CD34 have the capacity to differentiate into T cells once they
are placed in the thymic environment.17 Differentiating CD34+
bone marrow cells that co-express CD7 and display poor
ability to mature into the myeloid lineage can also efficiently
give rise to T cells in culture systems with cytokines
(interleukin 7).18 These lymphoid committed precursors might
be the true thymic repopulating cells. In the thymus, cells with
a similar CD34+CD7+ phenotype include the most primitive
thymocyte precursors. CD34+ thymocytes have no ability to
differentiate into myeloid cells, but may include NK cell pre-
cursors.19,20 In spite of these findings, it is still controversial
whether commitment to the T cell lineage occurs in man
before or after T cell precursors migrate into the thymus.21
CD7 expression is then retained until the last stages of both
T and NK cell differentiation.22,23 The vast majority of T-ALL
cases also express the CD7 antigen.2–4,24 Based on these
observations, we have focused our studies on T cell subsets
in the CD7+ lymphoid population of normal BM. We have
Figure 2 Upper row: flow cytometric plots from staining I–VI (a,
b, c, g, h and i, respectively) illustrate the concept of ‘empty spaces’
where no normal CD7+ cells can be found in normal BM. The blue
dots illustrate normal BM T cells and the circles illustrate patterns of
aberrant expression. The frequency of T-ALL cases showing the pro-
posed patterns of antigen expression are given in Table 4. Lower row:
examples of aberrant patterns of antigen expression in T-ALL. Red dots
represent leukemic blasts and gray dots illustrate normal BM T cells.
The following patterns are exemplified: (d) pattern IB in the
CD7/CD5/CD3 staining; (e) pattern IIA in the CD7/CD4/CD8 staining;
(f) pattern IIIB for CD3 positive cases in the CD7/CD2/CD3 staining;
(j) pattern IVB for CD3 negative cases in the CD7/CD2/CD3 staining;
(k) pattern VA in TdT/CD7/cyCD3 staining; and (l) pattern VIB in
CD34/CD38/CD7 staining.

Leukemia
 BIOMED-1 Concerted Action report
A Porwit-MacDonald et al

821
a b c

d e f

g h i

j k l

Leukemia
 BIOMED-1 Concerted Action report
A Porwit-MacDonald et al

822
Table 4 Frequency of the various patterns of antigen expression found among the 65 T-ALL cases using five studied marker combinations

Marker combinationsa Normal phenotypes Aberrant phenotypes

1 2 3 4 5 6 A B C D

I CD7/CD5/CD3 2 0 5 18 NA NA 0 11b 29b NA

n = 65 (3%) (7.6%) (28%) (17%) (44%)
II CD7/CD4/CD8 24d 10e 0 8e 8d 5f 16d–g 9d,f 0 NA
n = 65 (37%) (15%) (12%) (12%) (7.6%) (24.6%) (13.8%)
III CD7/CD2/CD3 1 2 NA NA NA NA 0 6 8 NA
CD3 dim/pos. (6%) (12%) (35%) (47%)
casesh
n = 17
IV CD7/CD2/CD3 NA NA 2 15c 4 NA 4 2c 5 5
CD3 neg casesh (6%) (45%) (12%) (12%) (6%) (15%) (15%)
n = 33
V TdT/CD7/cyt.CD3 NA NA NA NA NA NA 58
n = 64 (91%)
VI CD7/CD38/CD34 NA NA NA NA NA NA 23
n = 58 (40%)

a
Results are expressed as numbers of cases and percentages. Normal patterns of expression are shown in Figure 1 and Table 2, aberrant
patterns in Figure 2.
b
In two cases patterns B and C were observed.
c
In one case patterns 4 and B were found.
d
In four cases pattern 1 and 2; in one case patterns 1.A and 5; and in three cases pattern 1 and B were observed.
e
In four cases pattern 1 and 2; in one case 2 and A; and in one case 2 and 4 were observed.
f
In one case paterns B and 6 and in one case A and B coexisted.
g
In one case pattern A and C were found.
h
In 12 cases CD3dim and CD3 negative populations coexisted.

used a double-step acquisition strategy in which information Table 5 Most frequent aberrant phenotypes in 65 cases of T ALL
on CD7+ events was selectively obtained. This strategy was
successfully applied for identification of aberrant cells in fol- Type of aberrant antigen expression Number of cases
low-up samples of patients included in this study as exem- found
plified in Figure 3. There were no CD7 negative cases in our (%)
material. An alternative approach for blast cell gating stra-
tegies in CD7 negative or CD7 dim cases would be the use 1 Ectopic antigen expression
of other T cell markers as cyCD3 or CD2 or CD5 (Figure 3b). TdT/CD7/cyt.CD3 58/64 (91%)
Since the results were derived from six different labora- CD34/CD7 23/58 (40%)
tories, our first aim was to standardize the techniques and to CD3dim/CD5dim 11/65 (17%)
compare the results in order to avoid variations due to incon- CD4dim/CD8− 9/65 (14%)
sistencies in methodology or data analysis. Antibodies, sample 2 Asynchronous antigen expression
preparation, staining methods, instrument settings, data acqui-
sition and data analysis were standardized prior to the CD3dim/CD2dim 6/65 (9%)
immunophenotyping of the normal BM and T-ALL samples. 3 Rare phenotypes (⬍1/10−4 in normal
Identical results were obtained in each individual center as BM)
reflected both by the localization of the different cell popu-
CD2dim or neg/CD3−/CD7+++ 10/33 (30%)
lations in the dot plot diagrams and the absence of statistically CD3−/CD5dim 29/65 (44.6%)
significant differences in the percentages of each subset ana-
lyzed. Statistical analysis confirmed the lack of significant 4 Lineage infidelity
inter-laboratory variation of results.
CD13 8/58 (16%)
We have found that the overall percentage of CD7+ cells in CD33 6/58 (10.5%)
normal BM was approximately 10% and increased with age.
Among CD7+ BM cells our interest was initially focused on the 5 Overexpression of antigen
identification of CD34+/CD7+ and TdT+/CD7+ cells. Previous CD7 9/65 (14%)
reports have described the presence of small populations of
TdT+/CD7+ and CD34+/CD7+ T cell precursors in BM.25–27 6 Low expression or lack of antigen
Sorting of human BM CD34+ cells showed that the expression
CD34+/CD7+/CD2− phenotype correlated with the transcrip- CD4dim/CD8dim 16/65 (24.6%)
tion of CD3␨ but not CD3␥ or CD3␦ (a pattern of CD3 gene CD3 (ectopic) 60/65 (92%)
transcription observed in mature blood NK cells but not in T CD5 42/65 (63%)
cells).27 In contrast to the above-mentioned studies, our results CD2 34/54 (62%)
show that the frequencies of CD34+/CD7+ cells and

Leukemia

a the CD7/CD2/CD3 triple staining (75% aberrant cases) fol-
b showed a presence of CD4+/CD8+ double positive blasts simi-
Figure 3 Illustration of flow cytometric detection of MRD using
‘live-gate’ approach in follow-up BM samples obtained from two
patients with T-ALL in morphological complete remission on day 29
(after induction treatment). (a) Triple staining combination
TdT/CD7/cyCD3. The ‘live’ gate was set on CD7/SSC. The percentage
of CD7+ cells in the BM was 7.04%, 30 308 CD7+ lymphoid cells
were acquired, the total of 430 756 cells have passed the flow cyto-
meter and 278 TdT/CD7/cyCD3 triple positive lymphoid cells were
found. MRD was calculated as 0.06% of BM cells. (b) Triple staining
TdT/CD5/cyCD3. The diagnostic sample showed dim CD5 expression
on leukemic blasts. The ‘live gate’ was set on cyCD3/SSC. The
percentage of cyCD3+ cells in the BM was 8.8%, 10482 cyCD3+
lymphoid cells were acquired, a total of 118 880 cells passed the flow
cytometer and 70 triple positive TdT/CD5 dim/cyCD3 cells were
found. MRD was calculated as 0.04% of BM cells.
TdT+/CD7+ cells in childhood and adult BM generally are
lower than 1/104 BM cells. Therefore the observation of
CD34+/CD7+ phenotypes, especially if co-expressed together
with cyCD3 and/or TdT at frequencies higher than 10−4 can
be considered as leukemia associated.
Most T-ALL in our series (91%) co-expressed TdT and
cyCD3 and/or CD7. This makes the TdT/CD7/cyCD3 triple
labeling a powerful and broadly applicable tool for MRD stud-
ies, in accordance with previous early reports.25,28 Twenty-
three of 58 analyzed T-ALL cases (40%) co-expressed CD34
and CD7. It should be noted that in most CD34+ T-ALL cases
the expression of this marker was only seen on a subset of the
CD7+ leukemic blast cells. In the CD7/CD38/CD34 labeling,
the information provided by CD38 was marginal, thus limiting
the use of this triple antigen combination for MRD studies.
The most informative of the other marker combinations was
BIOMED-1 Concerted Action report
A Porwit-MacDonald et al
823
lowed by the CD7/CD5/CD3 staining (58% aberrant cases).
Aberrant phenotypes were also detected in 38% of T-ALL
cases analyzed with the CD7/CD4/CD8 triple staining. Thus,
the combined use of only a few common triple stains would
allow the flow cytometric follow-up of leukemic cells in all
T-ALL cases. Our results are in agreement with previously
reported data on the incidence of aberrant phenotypes in
T-ALL.6,8,9
In most T-ALL cases three or more aberrant immunopheno-
typic patterns were found, mainly reflecting ectopic thymic
phenotypes. The most frequent ectopic phenotype was
characterized by either dim or absent surface CD3 expression
and double expression of cyCD3/TdT.28 In seven of 65 studied
T-ALL cases (10.7%) leukemic blasts were CD3−/CD4−/CD8−/
CD34+. This phenotype was consistent with the earliest stages
of thymic differentiation. Twenty-five of 65 T-ALL cases (38%)
lar to CD4+/CD8+ thymocytes. These ectopic thymic immuno-
phenotypes are found neither in normal BM nor in PB of ALL
patients during and after treatment.29
By comparison to normal BM, T and NK cells, aberrant (low
or high) antigen expression was detected in 60% of the cases
and was usually related to the dim expression of CD3, CD5
or CD2. Over-expression of CD7 was detected in 14% of the
cases and abnormally low expression of CD7 or CD5 was
detected in six (9%) and 29 (45%) of the cases, respectively.
Our results show that only 12% of analyzed cases were
CD13 and/or CD33 positive. We have taken into account only
cases with strong expression of the myeloid markers on T cell
blasts that could be used for MRD investigation. This may
explain the relatively low incidence found in the present study
using sensitive CD33-PE and CD13-PE conjugates, as
compared to some other reports in the literature.11,30–33
It is obvious from our study that the immunophenotypes of
T-ALL differ significantly from normal BM T cells and NK
cells. For a major part this is caused by their thymocyte origin
(see ectopic antigen expression, Table 5). However, the neo-
plastic transformation might also have affected antigen
expression patterns (Table 5), as has been previously observed
in B precursor ALL34,35 and in acute myeloid leukemia.36,37
Furthermore, it should be noted that T-ALL also differ from
each other displaying unique immunophenotypic patterns.
In summary, our results show that the use of only five triple
marker combinations allows detection of ectopic thymic
phenotypes in all T-ALL cases. In addition, standardization of
the methods for cell staining, instrument calibration, data
acquisition and data analysis provides an objective and
reproducible basis for application of flow cytometric MRD
detection in T-ALL patients. In this way, flow cytometric
MRD detection provides a relatively inexpensive and rapid
alternative to PCR-based MRD detection via rearranged
immunoglobulin or T cell receptor genes.6,8,10,38
Acknowledgements
This study was supported by European BIOMED-1 grant
BMH1-CT94-1675, Swedish Cancer Society, Dutch Cancer
Society/Koningin Wilhelimina Fonds (grant EUR 94-852),
grant of the Associazione Italiana per la Ricerca sul Cancro,
by Fondazione M Tettamanti, and a grant from ‘Liga Portug-
uesa contra o Cancro’. We thank Dakopatts (Glostrup,
Denmark) and Caltag Lab (San Francisco, CA, USA) for pro-
viding charge-free reagents for the study and Mr Lewis G
Edgel for editorial assistance.
Leukemia
 BIOMED-1 Concerted Action report
A Porwit-MacDonald et al
824
References
1 Janossy G, Bollum FJ, Bradstock KF, Ashley J. Cellular phenotypes
of normal and leukemic hemopoietic cells determined by analysis
with selected antibody combinations. Blood 1980; 56: 430–441.
2 van Dongen JJ, Krissansen GW, Wolvers-Tettero IL, Comans-Bitter
WM, Adriaansen HJ, Hooijkaas H, van Wering ER, Terhorst C.
Cytoplasmic expression of the CD3 antigen as a diagnostic marker
for immature T-cell malignancies. Blood 1988; 71: 603–612.
3 Ludwig WD, Harbott J, Bartram CR, Komischke B, Sperling C,
Teichmann JV, Seibt-Jung H, Notter M, Odenwald E, Nehmer A.
Incidence and prognostic significance of immunophenotypic sub-
groups in childhood acute lymphoblastic leukemia: experience of
the BFM study 86. Rec Res Cancer Res 1993; 131: 269–282.
4 van Dongen JJ, Adriaansen HJ. Immunobiology of leukemia. In:
Henderson ES, Lister TA, Greaves MF (eds). Leukemia, 6th edn,
WB Saunders: Philadelphia, 1996, pp 83–130.
5 Campana D, Coustan-Smith E, Behm FG. The definition of
remission in acute leukemia with immunologic techniques. Bone
Marrow Transplant 1991; 8: 429–437.
6 van Dongen JJ, Breit TM, Adriaansen HJ, Beishuizen A, Hooijkaas
H. Detection of minimal residual disease in acute leukemia by
immunological marker analysis and polymerase chain reaction.
Leukemia 1992; 6 (Suppl. 1): 47–59.
7 Drach J, Drach D, Glassl H, Gattringer C, Huber H. Flow cyto-
metric determination of atypical antigen expression in acute leuke-
mia for the study of minimal residual disease. Cytometry 1992;
13: 893–901.
8 Campana D, Pui CH. Detection of minimal residual disease in
acute leukemia: methodologic advances and clinical significance
(see comments). Blood 1995; 85: 1416–1434.
9 Orfao A, Ciudad J, Lopez-Berges MC, Lopez A, Vidriales B,
Caballero MD, Valverde B, Gonzalez M, San Miguel JF. Acute
lymphoblastic leukemia (ALL): detection of minimal residual dis-
ease (MRD) at flow cytometry. Leuk Lymphoma 1994; 13 (Suppl
1): 87–90.
10 van Dongen JJ, Szczepanski T, de Bruijn MA, van den Beemd
MW, de Bruin-Versteeg S, Wijkhuijs JM, Tibbe GJ, van Gastel-
Mol EJ, Groeneveld K, Hooijkaas H. Detection of minimal residual
disease in acute leukemia patients. Cytok Mol Ther 1996; 2:
121–133.
11 Ciudad J, San Miguel JF, Lopez-Berges MC, Vidriales B, Valverde
B, Ocqueteau M, Mateos G, Caballero MD, Hernandez J, Moro
MJ, Mateos MV, Orfao A. Prognostic value of immunophenotypic
detection of minimal residual disease in acute lymphoblastic leu-
kemia. J Clin Oncol 1998; 16: 3774–3781.
12 Lucio P, Parreira A, van den Beemd MW, van Lochem EG, van
Wering ER, Baars E, Porwit-MacDonald A, Bjorklund E, Gaipa G,
Biondi A, Orfao A, Janossy G, van Dongen JJ, San Miguel JF. Flow
cytometric analysis of normal B cell differentiation: a frame of ref-
erence for the detection of minimal residual disease in precursor-
B-ALL. Leukemia 1999; 13: 419–427.
13 Weir EG, Cowan K, LeBeau P, Borowitz MJ. A limited antibody
panel can distinguish B-precursor acute lymphoblastic leukemia
from normal B precursors with four color flow cytometry: impli-
cations for residual disease detection. Leukemia 1999; 13: 558–
567.
14 van Lochem EG, Groeneveld K, te Marvelde JG, van den Beemd
MW, Hooijkaas H, van Dongen JJ. Flow cytometric detection of
intracellular antigens for immunophenotyping of normal and
malignant leukocytes: testing of a new fixation-permeabilization
solution (letter). Leukemia 1997; 11: 2208–2210.
15 Groeneveld K, te Marvelde JG, van den Beemd MW, Hooijkaas
H, van Dongen JJ. Flow cytometric detection of intracellular anti-
gens for immunophenotyping of normal and malignant leukocytes.
Leukemia 1996; 10: 1383–1389.
16 Barcena A, Muench MO, Roncarolo MG, Spits H. Tracing the
expression of CD7 and other antigens during T and myeloid cell
differentiation in the human fetal liver and thymus. Leuk Lym-
phoma 1995; 17: 1–11.
17 Barcena A, Galy AH, Punnonen J, Muench MO, Schols D, Roncar-
olo MG, de Vries JE, Spits H. Lymphoid and myeloid differen-
tiation of fetal liver CD34+ linkeage− cells in human thymic organ
culture. J Exp Med 1994; 180: 123–132.
18 Schmitt C, Ktorza S, Sarun S, Verpilleux MP, Blanc C, Deugnier
Leukemia
MA, Dalloul A, Debre P. CD34-positive early stages of human T-
cell differentiation. Leuk Lymphoma 1995; 17: 43–50.
19 Res P, Martinez-Caceres E, Cristina JA, Staal F, Noteboom E,
Weijer K, Spits H. CD34+CD38dim cells in the human thymus can
differentiate into T, natural killer, and dendritic cells but are dis-
tinct from pluripotent stem cells. Blood 1996; 87: 5196–5206.
20 Spits H, Blom B, Jaleco AC, Weijer K, Verschuren MC, van
Dongen JJ, Heemskerk MH, Res PC. Early stages in the develop-
ment of human T, natural killer and thymic dendritic cells. Immu-
nol Rev 1998; 165: 75–86.
21 Blom B, Res P, Noteboom E, Weijer K, Spits H. Prethymic CD34+
progenitors capable of developing into T cells are not committed
to the T cell lineage. J Immunol 1997; 158: 3571–3577.
22 Lazarovits AI, Osman N, Le Feuvre CE, Ley SC, Crumpton MJ.
CD7 is associated with CD3 and CD45 on human T cells. J Immu-
nol 1994; 153: 3956–3966.
23 Ginaldi L, Farahat N, Matutes E, De Martinis M, Morilla R, Catov-
sky D. Differential expression of T cell antigens in normal periph-
eral blood lymphocytes: a quantitative analysis by flow cytometry.
J Clin Pathol 1996; 49: 539–544.
24 van Wering ER, Beishuizen A, Roeffen ET, van der Linden-
Schrever BE, Verhoeven MA, Hahlen K, Hooijkas H, van Dongen
JJ. Immunophenotypic changes between diagnosis and relapse in
childhood acute lymphoblastic leukemia. Leukemia 1995; 9:
1523–1533.
25 van Dongen JJ, Hooijkaas H, Comans-Bitter M, Hahlen K, de Klein
A, van Zanen GE, van’t Veer MB, Abels J, Benner R. Human bone
marrow cells positive for terminal deoxynucleotidyl transferase
(TdT), HLA-DR, and a T cell marker may represent prothymocytes.
J Immunol 1985; 135: 3144–3150.
26 Bender JG, Unverzagt KL, Walker DE, Lee W, Van Epps DE, Smith
DH, Stewart CC, To LB. Identification and comparison of CD34-
positive cells and their subpopulations from normal peripheral
blood and bone marrow using multicolor flow cytometry. Blood
1991; 77: 2591–2596.
27 Long H, Gaffney P, Mortari F, Miller JS. CD3 gamma, CD3 delta,
and CD3 zeta mRNA in adult human marrow hematopoietic pro-
genitors correlates with surface CD2 and CD7 expression. Exp
Hematol 1996; 24: 1402–1408.
28 Campana D, Thompson JS, Amlot P, Brown S, Janossy G. The
cytoplasmic expression of CD3 antigens in normal and malignant
cells of the T lymphoid lineage. J Immunol 1987; 138: 648–655.
29 Caver TE, Slobod KS, Flynn PM, Behm FG, Hudson MM, Turner
EV, Webster RG, Boyett JM, Tassie TL, Pui CH, Hurwitz JL. Pro-
found abnormality of the B/T lymphocyte ratio during chemo-
therapy for pediatric acute lymphoblastic leukemia. Leukemia
1998; 12: 619–622.
30 Pui CH, Rubnitz JE, Hancock ML, Downing JR, Raimondi SC, Riv-
era GK, Sandlund JT, Ribeiro RC, Head DR, Relling MW, Evans
WE, Behm FG. Reappraisal of the clinical and biological signifi-
cance of myeloid-associated antigen expression in childhood
acute lymphoblastic leukemia. J Clin Oncol 1998; 16: 3768–
3773.
31 Uckun FM, Sather HN, Gaynon PS, Arthur DC, Trigg ME, Tub-
ergen DG, Nachman J, Steinherz PG, Sensel MG, Reaman GH.
Clinical features and treatment outcome of children with myeloid
antigen positive acute lymphoblastic leukemia: a report from the
Children’s Cancer Group. Blood 1997; 90: 28–35.
32 Babusikova O, Glasova M, Konikova E, Kusenda J, Cap J, Gyarfas
J, Koubek K. Phenotypic heterogeneity and aberrant markers
expression in T-cell leukemia. Neoplasma 1998; 45: 128–134.
33 Boldt DH, Kopecky KJ, Head D, Gehly G, Radich JP, Appelbaum
FR. Expression of myeloid antigens by blast cells in acute lym-
phoblastic leukemia of adults. The Southwest Oncology Group
experience. Leukemia 1994; 8: 2118–2126.
34 Ryan D, Kossover S, Mitchell S, Frantz C, Hennessy L, Cohen H.
Subpopulations of common acute lymphoblastic leukemia anti-
gen-positive lymphoid cells in normal bone marrow identified by
hematopoietic differentiation antigens. Blood 1986; 68: 417–425.
35 Hurwitz CA, Loken MR, Graham ML, Karp JE, Borowitz MJ, Pullen
DJ, Civin CI. Asynchronous antigen expression in B lineage acute
lymphoblastic leukemia. Blood 1998; 72: 299–307.
36 Reading CL, Estey EH, Huh YO, Claxton DF, Sanchez G, Ter-
stappen LW, O’Brien MC, Baron S, Deisseroth AB. Expression of
 BIOMED-1 Concerted Action report
A Porwit-MacDonald et al

unusual immunophenotype combinations in acute myelogenous
leukemia. Blood 1993; 81: 3083–3090.
37 Terstappen LW, Safford M, Unterhalt M, Konemann S, Zurlutter
K, Piechotka K, Drescher M, Aul C, Buchner T, Hiddemann W.
Flow cytometric characterization of acute myeloid leukemia: IV.
Comparison to the differentiation pathway of normal hematopo-
ietic progenitor cells. Leukemia 1992; 6: 993–1000.
38 Pongers-Willemse MJ, Seriu T, Stolz F, d’Aniello E, Gameiro P,
825
Pisa P, Gonzalez M, Bartram CR, Panzer-Grumayer ER, Biondi A,
San Miguel JF, van Dongen JJ. Primers and protocols for stan-
dardized detection of minimal residual disease in acute lymphobl-
astic leukemia using immunoglobulin and T cell receptor gene
rearrangements and TAL1 deletions as PCR targets: report of the
BIOMED-1 CONCERTED ACTION: investigation of minimal
residual disease in acute leukemia. Leukemia 1999; 13: 110–118.

Leukemia