J. Biomedical Science and Engineering, 2011, 4, 173-179 JBiSE
doi:10.4236/jbise.2011.43024 Published Online March 2011 (http://www.SciRP.org/journal/jbise/).
Published Online March 2011 in SciRes. http://www.scirp.org/jour nal/JBiSE
A comparison of flow cytometry detection of minimal residual
disease and chimerism kinetics in chronic lymphocytic
leukemia patients after allogeneic hematopoietic stem cell
Adriana Plesa1, Xavier Thomas2, Quoc Hung Le2, Anne-Sophie Michallet4, Valérie Dubois3, Charles
Dumontet1, Mauricette Michallet2
1Laboratory of Hematology, Hospices Civils de Lyon, Edouard Herriot Hospital, Lyon, France;
2 Hematology Department, Hospices Civils de Lyon, Edouard Herriot Hospital, Lyon, France;
3 Laboratory of Histocompatibility, Etablissement Français du Sang, Lyon, France;
4 Hematology Department, Hospices Civils de Lyon, Lyon-Sud Hospital, Pierre-Bênite, France.
Email: xavier.thomas@chu-lyon.fr
Received 29 September 2010; revised 16 October 2011; accepted 18 October 2010.
Determination of minimal residual disease (MRD)
remains crucial for the follow-up after therapy in
chronic lymphocytic leukemia (CLL) patients. Chi-
merism was assessed by short tandem repeat (STR)-
PCR and single nucleotide polymorphisms (SNP)-
PCR, and MRD by a multicolor flow cytometric ap-
proach in 12 consecutive patients with CLL after they
received allogeneic stem cell transplantation (SCT).
Overall, 11 patients achieved MRD flow negativity
[10 had full donor chimerism (FDC) and one had
mixed chimerism (MC)]. Only one patient remained
with MRD flow positivity and displayed MC. Fifty-
six samples were concomitantly studied by both chi-
merism and MRD flow. A significant correlation was
observed between MRD flow data and chimerism in
both PB and BM by using a mixed effect linear re-
gression (p < 0.001). Flow cytometry approach of
MRD can be easily combined with chimerism during
the follow-up post-allogeneic SCT. Both techniques
appeared complementary for guiding post-transplant
Keywords: Chronic Lymphocytic Leukemia, Allogeneic
Stem Cell Transplantation, Minimal Residual Disease,
Important advances have been made over the past two
decades in the prognosis and treatment of chronic lym-
phocytic leukemia (CLL) [1]. New therapeutic ap-
proaches aim to induce molecular remission. The better
quality of response, provided by novel agents and new
therapeutic approaches, requires necessarily more sensi-
tive tools for precise remission assessment [2-7]. The
evaluation of response to treatment in CLL is currently
based on the National Cancer Institute (NCI) criteria [8].
Several studies have demonstrated that CLL patients
achieving response at a molecular level without any de-
tectable minimal residual disease (MRD) have a longer
survival [5-9]. Using a very high sensitive flow cytome-
try technique, several groups have shown that patients in
complete remission (CR) with detectable MRD have an
increased risk of early relapse [3,6]. In this setting, bone
marrow biopsy appeared to be less sensitive than flow
cytometry [10]. Conversely, similar results have been
shown with real-time quantitative (RTQ) polymerase
chain reaction (PCR) and MRD flow monitoring in
terms of leukemia cell clearance kinetics and timing for
reaching MRD negativity [6].
Because of the relatively advanced age of patients
with CLL, no consistent efforts have been made to con-
duct stem cell transplantation (SCT) with use of alloge-
neic donors. However, allogeneic SCT remains the only
curative treatment for CLL in younger adults. In the set-
ting of allogeneic SCT, molecular techniques for chi-
merism determination are routinely used for engraftment
follow-up and early prediction of post-transplant relapse.
They have been routinely used in parallel with MRD
analysis by flow cytometry. Furthermore, allogeneic
SCT after reduced intensity conditioning (RIC) has been
recently developed as a safer approach for older patients
and has therefore emerged as the treatment of choice for
older high-risk CLL patients requiring transplantation
[11]. In this setting, the significance of chimerism and
A. Plesa et al. / J. Biomedical Science and Engineering 4 (2011) 173-179
Copyright © 2011 SciRes. JBiSE
MRD flow cytometry needs to be reevaluated. The aim
of the present study was to compare chimerism and
MRD flow cytometry level after allogeneic SCT in order
to fully document CR and to guide immunomodulation
by donor lymphocyte infusions (DLI).
2.1. Patient Characteristics
Between 2000 and 2007, 12 CLL patients (9 males, 3
females) with a median age of 51 years (range 31 – 66
years) underwent allogeneic SCT in our institution. At
the time of diagnosis, 2 patients were in stage A, 6 in
stage B and 4 in stage C according to Binet’s classifica-
tion [12]. Matutes’ score was noted at 5 for 7 patients, 4
for 2 patients and 3 for the last 3 patients. Before al-
logeneic SCT, one patient received one line of conven-
tional therapy and 11 received more than 2 lines: 11 pa-
tients received fludarabine-based therapeutic regimens, 6
received immunotherapy with rituximab and/or alemtu-
zumab, 3 have previously been auto-transplanted, and
one has received a first allogeneic SCT. At the time of
allogeneic transplant, 3 patients were in CR, while 7
were in partial remission (PR) and 2 in progressive dis-
ease. Response criteria were defined according to NCI
guidelines for CLL [8].
2.2. Transplant Procedure
Patients underwent SCT from HLA identical sibling do-
nors after signing a written informed consent. Transplant
characteristics are summarized in Table 1. Regarding
hematopoietic stem cell source, 9 patients received PB
stem cells and 3 patients BM stem cells. One patient was
allografted twice. For the second allogeneic transplant,
he received PB stem cells. Regarding conditioning regi-
men, 2 patients received a myeloablative regimen com-
bining total body irradiation (TBI) (12 grays) with
Table 1. Transplant characteristics of the 12 CLL patients.
status at
Tr a ns-
regimens HSC Sex
matc h e d
sis to
N of pre-
MRD vs
chimerism at
1 year after
Outcome at last
P # 1 36 B CR CYT 120 + 12
TBI BM R F/D M3.3 2 No No ND/FDC Alive, 82+ months,
(MRD neg / FDC)
49 B PR CYT 200 + ATG PBSC R M/D F2 2 Grade 1Exten-
sive ND/FDC Alive, 80+ months,
( MRD neg / FDC)
P # 3 38 B PR CYT 120 + 12
matched 1 1 Grade 2Limited ND/FDC
Alive, 77+ months,
( MRD neg / FDC)
P # 4 49 C PD
(Relapse) Fluda + 2 TBI PBSC Sex
matched 4.1 2 AutoGrade 1Limited ND/FDC
Alive, 74+ months,
(MRD neg/FDC )
P # 5 52 B CR Fluda + 2 TBI PBSC Sex
matched 4.4 2 Grade 3Exten-
sive Neg/FDC Alive, 53+ months,
(MRD neg/FDC)
P # 6 50 C PR Fluda + 2 TBI PBSC Sex
matched 5.7 5 AutoGrade 2Limited Neg/FDC
Alive, 52+ months,
(MRD neg/FDC)
P # 7 55 B CR Fluda + 2 TBI PBSC R M/D F7.9 3 No Exten-
sive Neg/FDC Death, 28+months,
(MRD neg/FDC)
P # 8 54 C PD Fluda + Busulfan
+ ATG PBSC R M/D F13 7 AlloGrade 3Exten-
sive Neg/FDC Alive, 24+ months,
(MRD neg/FDC)
P # 9 59 A PR CYT 200 + ATG PBSC Sex
matched 5 2 Grade 2Limited
Alive, 29+ months,
(MRD neg/MC
P # 10 66 A PR Fluda + 2 TBI PBSC RM/D F8.9 5 Grade 1No Neg/FDC Dead, 3+ months,
(MRD neg / FDC)
P # 11 60 B PR Fluda + 2 TBI BM Sex
matched 5.8 3 AutoGrade 3ND Neg/FDC
Alive, 3+ months,
(MRD neg/FDC)
P # 12 51 C PR CYT 200 + ATG PBSC Sex
matched 12.3 5 No ND Pos/MC
Alive, 4+ months,
(MRD pos/MC)
Abbreviations: CR = complete response, PR = partial response, PD = progressive disease, CYT 120 = cyclophosphamide (60 mg/kg x 2 days), CYT 200 =
cyclophosphamide (50 mg/kg x 5 days), 12 TBI = total body irradiation (12 Grays), 2 TBI = total body irradiation (2 Grays), 6TBI = total body irradiation (6
Grays), SCT = stem cell transplantation, ATG = anti-thymoglobulins, BM = bone marrow, PBSC = peripheral blood stem cells, R = recipient, D = donor, F =
female, M = male, GVHD = graft-vs-host disease, FU = follow-up, FDC = full donor chimerism, MC = mixed chimerism, ND = not done, pos = positive, neg =
negative, MRD = minimal residual disease, Auto = autologous SCT, Allo = allogeneic SCT.
A. Plesa et al. / J. Biomedical Science and Engineering 4 (2011) 173-179
Copyright © 2011 SciRes. JBiSE
cyclophosphamide (60 mg/kg/day × 2 days) and 10 pa-
tients received a RIC constituted of either fludarabine
(30 mg/m2/day × 3 days) and TBI (2 grays) (n = 6), or
anti-thymoglobulines (ATG) (2.5 mg/kg/day × 5 days) or
fludarabine (30 mg/m2/day × 5 days), busulfan (4
mg/kg/day × 2 days) and ATG (2.5 mg/kg/day × 2 days)
and cyclophosphamide (50 mg/kg/day × 4 days) (n = 3),
(n = 1). Graft-versus-host disease (GVHD) prophylaxis
consisted of either cyclosporine alone (n = 1), or cyc-
losporine and mycophenolate mofetil (MMF) (n = 7), or
cyclosporine and methotrexate (n = 4). Five patients
were sex-mismatched with the recipient (4 females to
males, and one male to female) and for the second trans-
plant the male recipient received PB stem cells from
another HLA identical sister. Regarding cytomegalovirus
(CMV) status, 4 pairs were CMV mismatched (1 D/R+,
3 D+/R), 4 pairs were CMV positive (D+/R+) and 4 pairs
were CMV negative (D/R). In the case with a second
allogeneic SCT, the pair was D+/R+. Four patients pre-
sented an ABO incompatibility (3 minor and one major).
2.3. Graft-versus-Host Disease
After transplant, 6 patients developed a grade 2 (n = 3)
or 3 (n = 3) acute GVHD and 8 patients developed a
chronic GVHD (4 limited and 4 extensive). The patient
allografted twice presented after the second SCT, a grade
2 acute GVHD followed by a limited chronic GVHD. At
the time of the last follow-up [median, 53 months (range:
3 – 82 months)], 2 patients had died from infections,
while 10 were still alive.
2.4. Chimerism Analyses
Chimerism was documented on days 30, 60, 90, and on
months 6 and 12 during the first year following SCT,
and then twice a year. Chimerism was assessed on total
PB cells, total BM cells, selected CD3+ cells, and se-
lected CD19+ cells by short tandem repeat (STR)-PCR
(sensitivity [S]: 5%) or by single nucleotide polymer-
phisms (SNP) real-time (RT)-PCR (S: 0.2%) [13,14].
Overall, 192 PB chimerisms and 59 BM chimerisms
were performed with a median of 16 PB chimerisms
(range: 3 – 41) and 5 BM chimerisms (range: 1 – 16) per
patient. Chimerism was assessed by STR-PCR from
2000 to 2005 and by RT-PCR afterwards. The percent-
age of recipient cells (RC) in each sample was deter-
mined by using STR analysis with Promega multiplex
kits (CTTV, FFFL and gamma STR) based fluorescent
analysis of repetitive sequences peak areas. Mixed chi-
merism was defined by the presence of at least 5% RC.
The RT quantitative PCR chimerism assay, using the
Taqman technology and ABI 7700 (Applera), has been
previously described [14]. Here, mixed chimerism was
defined by the presence of at least 0.1% of RC.
2.5. MRD Flow Cytometry Analysis
MRD flow cytometry was assessed, every 3 months
during the first year following transplant, and every 6
months afterwards, on PB and/or BM cells by an inter-
national standardized multicolor approach (S: 0.01%) [7].
Overall, 65 PB MRD flows, and 23 BM MRD flows
were performed with a median of 5 PB MRD analyses
(range: 1 – 20), and 2 BM MRD analyses (range: 1 – 5)
per patient. Patients were considered MRD flow positive
if the detected MRD CLL levels were 10–4. Specificity
and sensibility of the technique were validated by using
a series of 10 normal PB and BM samples. MRD was
studied by using quadruple antigenic combinations:
CD20-FITC/ CD79b-PE/ CD19-PerCP-Cy5.5/ CD5-APC,
CD43-FITC/ CD79b-PE/ CD19-PerCP-Cy5.5 CD5-APC,
and CD22-FITC/ CD5-PE/ CD19-PerCP-Cy5.5/ CD38
APC. In BM samples, the combination containing CD38
was used to better discriminate CLL cells from B-cell
precursors (hematogones). In addition to daily calibrations
using Calibrite beads (Becton Dickinson) and FAC-
SComp v4.2 software (Becton Dickinson), isotype con-
trols were used to optimize light scatter, amplification
and threshold. For our study, at least 50 CLL cells form-
ing a population in light scatter in at least two of the
three MRD tests (excluding K/L antigens) were required
as evidence for MRD (absolute specificity threshold).
The result was classified as positive if the level was
above the mean number of the false positive cells in
healthy controls. A median of 300,000 leucocytes/samples
(range 50,000 and 600,000) depending on time point was
acquired using CellQuest Pro software (Becton Dickin-
son) and analyzed using Paint-A-Gate software (Becton
Dickinson). The complete gating strategy for identifica-
tion of CLL specific phenotype (MRD flow assay) has
been previously described, using a CD43+ as fourth cri-
terion for CLL cells [5].
2.6. Statistical Analysis
Kaplan Meier product-limit estimates were used to as-
sess the probability of overall survival (OS). The rela-
tionship between chimerism and MRD flow assay was
evaluated by a linear mixed effects model [15]. The
model included MRD as the resulting variable, chimer-
ism as the fixed effect and patients as the random effect.
An analysis was performed for BM and a second analy-
sis was performed for PB. Significative effects were
defined by p values of less than 0.05. All analyses were
performed using Splus 6.2 (Insightful, Seattle).
At the time of study, the median follow-up was 53
months. Two patients had died from infection, while 10
A. Plesa et al. / J. Biomedical Science and Engineering 4 (2011) 173-179
Copyright © 2011 SciRes. JBiSE
were still alive. The 2 patients who died displayed MDR
negativity and had FDC. Among alive patients, 9 were
with MRD flow negativity (8 had FDC and one had MC),
while one was with MRD flow positivity and displayed
MC. Two hundred and fifty-one samples were tested for
chimerism (192 from PB and 59 from BM) and 88 for
MRD flow cytometry (65 from PB and 23 from BM).
Only 56 samples were studied at the same time point,
and were then used for comparing quantitative MRD
flow cytometry and chimerism.
3.1. Chimerism Analysis
Comparison of results obtained from PB and BM sam-
ples showed 93% of concordance. The conversion of
mixed chimerism (MC) to full donor chimerism (FDC)
at Day 30 post-transplant was observed in 5 cases. The
conversion was observed between Day 30 and Day 120
in 3 cases and after Day 120 in one case. Three patients
never converted to FDC (patient # 12 had not enough
follow-up, patient # 9 remained in stable MC at 3
months post-transplant, and patient # 8, who relapsed
after a first RIC SCT, underwent then a second alloge-
neic SCT from another HLA identical sister allowing a
durable FDC). Eleven patients had their chimerism done
on selected cells, of whom 4 with MC (Table 2).
3.2. MRD Flow Cytometry Analysis
Comparison of results obtained from PB and BM sam-
ples showed 100% of concordance. Because of the re-
cent introduction of MRD flow assay in the clinical rou-
tine, longitudinal data during the first year post-trans-
plant were only available for 8 patients. They showed
MRD flow negativity at Day 30 in one case, at Day 90 in
another case, at Day 120 in 3 cases, and at 12 months
post-transplant in 2 cases. One patient remained positive,
but displayed decreasing MRD flow levels. Regarding
the 4 patients without optimal longitudinal follow-up, all
patients achieved MRD negativity but were only as-
sessed at time points for which samples were available
(on months 30, 36, 42, and 48 post-transplant, respect-
tively). Patient # 8 never achieved MRD negativity after
the first transplant, but showed a low MRD level at 12
months and MRD negativity 24 months after the second
3.3. Comparison of Chimerism and MRD
Results are summarized in Table 2. Regarding kinetics
Table 2. Chimerism and MRD kinetics (12 patients).
# Days +21 to +56 (1-2 months) Days+100 (3 months) Days + 180 to + 270 (6-9 months)Days +360 (1 year)
flow Chimerism MRD
flow Chimerism MRD
flow Chimerism MRD
2 ○ ○
○ ○○
○ ○○ /ND /
○ ○○ / /ND
7 ND/ /ND ○ ○ / /
SCT 1 ND/ /ND ● ● /ND /ND
SCT 2 / ND/■ ●●○ / /
9 ND/ ND/■ ● / /
10 /
11 / /
12 ND/ /
complete donor chimerism (< 5% donor cells by STR and < 0,2% donor cells by SNP RT-PCR); mixed chimerism; negative MRD flow (CLL cells < 1 x
10-4 by MRD flow); positive MRD; ND, not done.
A. Plesa et al. / J. Biomedical Science and Engineering 4 (2011) 173-179
Copyright © 2011 SciRes. JBiSE
of disappearance of a detectable disease, a strict concor-
dance was established between MRD flow assay and
chimerism in 50 of the 56 available samples (89%).
Analyses, using a mixed effect linear regression to ac-
count for repeated measures for each patient, showed a
significant correlation between MRD flow and chimer-
ism (p < 0.001) tested on PB samples and between MRD
flow and chimerism (p < 0.001) tested on BM samples.
The 6 non concordant samples were all harvested in a
single patient. In this case, a decreasing percentage of
RC was observed, but remained stable until the sixth
month post-transplant [MC < 1% (range: 0.2 – 0.9)].
During the same time, MRD flow negativity persisted
from 12 months post-transplant until the time of last
Important advances have been made in the prognosis and
treatment of CLL over the past two decades [1]. With
conventional therapy, a large proportion of patients can
achieve CR [16,17]. However, allogeneic SCT remains
the only curative therapy [18,19]. The number of alloge-
neic SCT performed in this disease is therefore suscepti-
ble to significantly increase, mainly due to the recent
development of RIC allogeneic SCT in older patients
[20,21]. New technologies such as 4-color flow cytometry
and RT quantitative PCR have identified the persistance
of a detectable disease in patients considered in CR ac-
cording to international guidelines [5]. The absence of
detectable leukemia by these sensitive tests seems to be
an important prognostic factor [9,22] , since it has been
shown correlated with a longer survival. A strong con-
cordance has been described between these two tech-
niques for MRD evaluation, with a limit of detection of
one malignant cell per 10.000 total cells and 100% ap-
plicability of MRD flow assay.
The present series, summarizing our experience in the
setting of allogeneic SCT in CLL, tends to confirm pre-
vious reports. All patients, except one, showed MRD
negativity when tested by flow cytometry. Ten patients
obtained FDC, while 2 only achieved MC. We first es-
tablished a positive concordance between PB and BM
for both chimerism and MRD flow. Secondly, a positive
concordance (89%) was confirmed between determina-
tion of residual disease by both flow cytometry and chi-
merism. Two types of conversion after transplantation
were described for both chimerism and MRD flow: an
earlier conversion following transplant and a later con-
version. The kinetics of conversion to FDC was faster
than kinetics of disappearance of detectable disease as
observed by using flow cytometry: 42% of documented
cases converted to FDC at Day 30, while at the same
time only 12% of assessable cases were MRD negative
by flow cytometry. Flow cytometry detection of MRD
seems therefore more sensitive. Interestingly, detection
of MRD by flow cytometry seemed also more acurate
than total cell chimerism evaluation among the non
concordant samples coming from one single patient.
However, when considering lineage specific chimerism
by selecting CD19+ cells for instance, a full donor pro-
file quite concordant with MRD flow negativity was
However, as no relapses were reported in the follow-
up of our series, after reaching negativity, it is difficult to
judge the relevance of the suggested use of MRD instead
of chimerism in the long-term follow-up. The two tech-
niques must certainly be considered as complementary
for documenting MRD in CLL patients after allogeneic
SCT and guiding immunomodulation.
The quantitative techniques used for the assessment of
MRD and chimerism seemed therefore interesting tools
for documenting disease and transplant status in CLL, as
it has been also previously demonstrated in other hema-
tological malignancies [23].
In the transplant setting for CLL patients, MRD detec-
tion should be considered as a surrogate marker for dis-
ease eradication. Our results are in agreement with pre-
viously published data showing the importance of MRD
eradication assessed by flow cytometry during the post-
transplant follow-up in CLL [2,3,6,10]. However we
reported only on a small series heterogeneous in terms of
conditioning regimens, and chimerism results could be
different after myeloablative and non-myeloablative
conditioning. MRD flow appeared more specific than
total cell chimerism evaluation. MRD flow assay also
appeared as a faster and less expensive technique for
guiding adoptive immunotherapy or treatment abstention
after allogeneic transplantation. Nevertheless, both tech-
niques appeared complementary, most specially when
studying lineage specific documentation. Another im-
portant point is to consider a strict schedule of evaluation.
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Copyright © 2011 SciRes. JBiSE
ATG, anti-thymoglobulines;
BM, bone marrow;
CLL, chronic lymphocytic leukemia;
CMV, cytomegalovirus;
CR, complete remission;
FDC, full donor chimerism;
DLI, donor lymphocyte infusions;
GVHD, graft-versus- host disease;
MC, mixed chimerism;
MMF, mycophe- nolate mofetil;
MRD, minimal residual disease;
NCI, National Cancer Institute;
OS, overall survival; PB, pe- ripheral blood;
PCR, polymerase chain reaction; PR, par- tial remission;
RC, recipient cells; RIC, reduced intensity conditioning;
RTQ, real-time quantitative; SCT, stem cell transplanta-
SNP, single nucleotide polymorphism;
STR, short tandem repeat;
TBI, total body irradiation.