МІНІМАЛЬНА ЗАЛИШКОВА ХВОРОБА ПРИ ГОСТРІЙ ЛІМФОБЛАСТНІЙ ЛЕЙКЕМІЇ: МЕТОДИ ВИЯВЛЕННЯ ТА КІЛЬКІСНОГО ВИЗНАЧЕННЯ
Recently, the identification of minimal residual disease (MRD), also known as measurable residual disease, has seen significant advances driven by the evolving technological landscape in this field. Initially based on raw morphology, the field has since evolved to incorporate karyotyping, cytogeneti...
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Experimental Oncology| _version_ | 1874183254537404416 |
|---|---|
| author | Addakiri, Sara Bencharef, Hanaa Harrach, Asmaa Jaddaoui, Samiha Ait-Ichou, Khadija Oukkache, Bouchra |
| author_facet | Addakiri, Sara Bencharef, Hanaa Harrach, Asmaa Jaddaoui, Samiha Ait-Ichou, Khadija Oukkache, Bouchra |
| author_institution_txt_mv | [
{
"author": "Sara Addakiri",
"institution": "Hassan II University of Medicine and Pharmacy, Casablanca, Morocco",
"orcid": "0009-0008-5751-9846"
},
{
"author": "Hanaa Bencharef",
"institution": "Hassan II University of Medicine and Pharmacy, Casablanca, Morocco",
"orcid": "0000-0002-0086-0815"
},
{
"author": "Asmaa Harrach",
"institution": "Hassan II University of Medicine and Pharmacy, Casablanca, Morocco",
"orcid": "0000-0002-7524-0688"
},
{
"author": "Samiha Jaddaoui",
"institution": "Hassan II University of Medicine and Pharmacy, Casablanca, Morocco",
"orcid": ""
},
{
"author": "Khadija Ait-Ichou",
"institution": "IBN ROCHD Hospital Centre, Casablanca, Morocco",
"orcid": ""
},
{
"author": "Bouchra Oukkache",
"institution": "Hassan II University of Medicine and Pharmacy, Casablanca, Morocco",
"orcid": "0000-0003-2022-1938"
}
] |
| author_orcid_str_mv | 0009-0008-5751-9846 0000-0002-0086-0815 0000-0002-7524-0688 0000-0003-2022-1938 |
| author_sort | Addakiri, Sara |
| baseUrl_str | https://exp-oncology.com.ua/index.php/Exp/oai |
| collection | OJS |
| container_end_page | 107 |
| container_issue | 2 |
| container_start_page | 98 |
| container_title | Експериментальна онкологія |
| container_volume | 48 |
| datestamp_date | 2026-08-21T12:36:20Z |
| description | Recently, the identification of minimal residual disease (MRD), also known as measurable residual disease, has seen significant advances driven by the evolving technological landscape in this field. Initially based on raw morphology, the field has since evolved to incorporate karyotyping, cytogenetics, flow cytometry, and other sensitive methods. This updated review discusses key technical considerations for successful MRD detection. We explore the correlation between the results obtained by flow cytometry and molecular genetic techniques. |
| doi_str_mv | 10.15407/exp-oncology.2026.02.098 |
| first_indexed | 2026-08-22T01:00:33Z |
| format | Article |
| fulltext |
98 ISSN 1812-9269. Experimental Oncology 48 (2). 2026
■
C i t a t i o n: Addakiri S, Bencharef H, Harrach A, Jaddaoui S, Ait-Ichou K, Oukkache B. Minimal residual disease assess-
ment in acute lymphoblastic leukemia: Methods for detection and quantification. Exp Oncol. 2026; 48(2): 98-107. https://
doi.org/10.15407/exp-oncology.2026.02.098
© PH “Akademperiodyka” of the NAS of Ukraine, 2026. This is an open access article under the CC BY-NC-ND license
(https://creativecommons.org/licenses/by-nc-nd/4.0/)
Acute lymphoblastic leukemia (ALL) is a clonal di
sease of hematopoietic stem cells of either B- or T-cell
lineage. It manifests as lymphoblastoid cells with
blocked differentiation. Despite being rare, ALL is the
most common type of leukemia in children, affecting
4—5 children per 100,000 annually [1]. Precursor
B‑cell ALL accounting for about 85% to 90% of ALL
cases in children is more common than T-cell precur-
sor ALL. T‑cell precursor leukemia accounts for
10%—15% of cases in children and 25% in adults,
with T-lymphoblastic lymphoma (T-LBL) more prev-
alent than B-LBL [1, 2]. The therapeutic strategy in-
volves three stages, including induction, consolida-
tion, and maintenance [3]. In children with B-ALL,
the five-year disease-free survival rate exceeds 85%,
with a cure rate of 90% [4, 5]. Similarly, in children
with T‑ALL, the ffive-year event-free survival (EFS)
rate has increased from 75% to > 90% [6]. However,
a proportion of patients exhibit more risk factors,
higher relapse probabilities, and, consequently, an
unfavorable overall condition. In 1993, the National
Cancer Center identified risk factors for childhood
B-ALL for prognostic purposes. These factors in-
clude age, white blood cell count, central nervous sys-
tem involvement, cytogenetic alterations, aneuploidy,
and early response to therapy. These factors reliably
predict patient outcomes, and risk stratification was
developed based on EFS. In contrast, risk factors for
REVIEW
https://doi.org/10.15407/exp-oncology.2026.02.098
Sara Addakiri 1, 2, *, Hanaa Bencharef 1, 2,
Asmaa Harrach 1, 2, Samiha Jaddaoui 1, 2,
Khadija Ait-Ichou 2, Bouchra Oukkache 1, 2
1 Hassan II University of Medicine and Pharmacy,
Casablanca, Morocco
2 IBN ROCHD Hospital Centre, Casablanca, Morocco
* Correspondence: E-mail: saraaddakiri1995@gmail.com
MINIMAL RESIDUAL DISEASE ASSESSMENT
IN ACUTE LYMPHOBLASTIC LEUKEMIA:
METHODS FOR DETECTION AND QUANTIFICATION
Recently, the identification of minimal residual disease (MRD), also known as measurable residual disease, has seen
significant advances driven by the evolving technological landscape in this field. Initially based on raw morphology, the
field has since evolved to incorporate karyotyping, cytogenetics, flow cytometry, and other sensitive methods. This up-
dated review discusses key technical considerations for successful MRD detection. We explore the correlation between
the results obtained by flow cytometry and molecular genetic techniques.
Keywords: MRD, flow cytometry, molecular technique.
https://doi.org/10.15407/exp-oncology.2026.02.098
https://doi.org/10.15407/exp-oncology.2026.02.098
https://creativecommons.org/licenses/by-nc-nd/4.0/
https://doi.org/10.15407/exp-oncology.2026.02.098
mailto:saraaddakiri1995@gmail.com
ISSN 1812-9269. Experimental Oncology 48 (2). 2026 99
Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: Methods for Detection and Quantification
T-ALL are less well-defined [7]. Previous research by
the Pediatric Oncology Group (POG) indicates that,
unlike in B-ALL, advanced age and a high white
blood cell count are not reliable indicators in T-ALL
[8]. However, POG has observed a correlation bet
ween T-cell maturation and patient outcomes. This
discovery has been the focus of multiple studies, in-
cluding the ALL 2000 study at St. Jude Children’s Re-
search Hospital, which provided deeper insights into
this association [9]. A pivotal question in the man-
agement of ALL in children is identifying those who
require more intensive therapy to mitigate the risk of
relapse. Clinical and biological factors, such as white
blood cell count, age, immunophenotype, ploidy,
structural chromosomal abnormalities, and genetic
rearrangements, can be used to stratify treatment, al-
though these factors are not ideal. While some pa-
tients with promising indicators experience recur-
rence, others at a higher risk of relapse receive more
intensive treatment than necessary.
Several studies focused on minimal residual di
sease (MRD) to obtain more reliable estimates of
lymphoblast counts during the clinical remission
phase. MRD serves as an indicator of treatment sen-
sitivity and intensity, enabling the adoption of tai-
lored treatment protocols. The reliable detection of
MRD aims to identify children at high risk of relapse
as early as possible. Additionally, it is used to assess
bone marrow and peripheral blood for the suitability
of autologous hematopoietic stem cell transplanta-
tion. Moreover, MRD plays a crucial role in evalua
ting the clearance process [10]. Finally, MRD allows
for comparing the effectiveness of different chemo-
therapy regimens. This paper aims to provide a com-
prehensive overview of MRD identification tech-
niques. Indeed, one of the major challenges in child-
hood ALL is identifying those who require more
intensive treatment to prevent the risk of relapse.
During the clinical remission phase, MRD assess-
ment contributes significantly to improving the
quantification of the total burden of leukemia cells.
This is crucial as it indicates disease aggressiveness
and treatment sensitivity, thus facilitating the selec-
tion of the most appropriate therapeutic strategy.
MRD detection
and quantification methods
An effective platform for identifying MRD must
possess three key advantages encompassing opti-
mal sensitivity, specificity, and reproducibility [11].
Despite advances in immunohistochemistry testing
methods, morphological assessment remains insuf-
ficient in terms of sensitivity and specificity. Simi-
larly, traditional methods such as karyotyping and
fluorescence in situ hybridization lack the required
sensitivity to detect submicroscopic leukemia cells,
often achieving detection rates as low as 1%—5%
[12]. The primary techniques used to assess MRD
include flow cytometry, which relies on identifying
cells with abnormal immunophenotypes, and mo-
lecular genetic methods such as polymerase chain
reaction (PCR) for identifying tumor-specific anti-
gen receptor sequences. Today, next-generation se-
quencing (NGS) has emerged as another valuable
tool for molecularly identifying these genetic ab-
normalities [13]. The accuracy and scope of MRD
tests are undergoing significant changes, driven by
the development of improved antibodies, upgrades
to flow cytometers, and advancements in comput-
ing [14]. Flow cytometry has revealed significant
correlations between MRD findings, treatment ef-
ficacy, and clinical features, thereby enhancing the
credibility of this approach [15—18]. Flow cytom-
etry, with its accessibility and straightforward con-
cept, stands out as the most readily available tech-
nique for identifying MRD [19]. It directly mea-
sures MRD, simplifying quantification and
enabling more precise measurements [20]. More-
over, flow cytometry can also detect dying cells and
cellular fragments. This is particularly useful for
analyzing fresh samples (e.g., those collected with-
in several hours) because it allows researchers to
identify cell death caused by a lack of survival fac-
tors before spontaneous cell death occurs. Never-
theless, the application of flow cytometry may have
several limitations. To identify a single leukemia
cell among 105 or more normal cells is challenging
with flow cytometry but achievable with PCR. This
level of sensitivity is particularly valuable in MRD
studies involving patients with irregular disease
distribution, as well as in the context of cell collec-
tion for autograft procedures. It is also important
to note that the immunophenotype of leukemia
cells evolves as the disease progresses. However, if
these changes affect the markers used for MRD
surveillance, there is a risk of obtaining false-neg-
ative results [14, 21—23]. This risk is inversely pro-
portional to the number of marker combinations
applicable to each patient. In other words, if cells
100 ISSN 1812-9269. Experimental Oncology 48 (2). 2026
S. Addakiri, H. Bencharef, A. Harrach, S. Jaddaoui, K. Ait-Ichou, B. Oukkache
exhibit multiple suitable phenotypes, the loss of one
phenotypic pattern can be compensated by the per-
sistence of other abnormal patterns. Another limita-
tion of flow cytometry techniques is that their results
rely on the analysis of immunophenotypic charac-
teristics of leukemia, while immunophenotype may
change substantially between diagnosis and relapse.
These frequent changes complicate the use of the
original diagnostic markers for targeted MRD detec-
tion by flow cytometry [23].
Immature T cells are homologs of normal T-cell
lineage ALL cells. Unlike leukemic T-cell lympho-
blasts, which circulate freely throughout the body,
these cells are limited to the thymus. To detect
MRD in patients with T-cell lineage ALL, the focus
is on identifying cells with the immature T-cell
phenotype in bone marrow or peripheral blood. Ef-
fective immunophenotypes for this purpose in-
volve the simultaneous expression of T-cell mar
kers such as CD3, CD5, and either TdT or CD34
[24]. Some studies also suggest that certain combi-
nations of antibodies, such as CD7 and CD3 with
CD2 or CD5, may exhibit abnormal expression in
a subset of T-ALL patients [25, 26]. B cell progeni-
tors, which are the normal counterparts of B-cell
lineage ALL cells, are primarily found in the bone
marrow and in smaller quantities in peripheral
blood [25]. Therefore, MRD studies in the B-cell
ALL lineage require distinguishing between leuke-
mia cells and their normal counterparts, which can
be achieved by examining abnormal levels of ex-
pression of various molecules in leukemia cells
[25—27]. T-ALL MRD backbone panels are set up
to evaluate both the aberrant expression of mature
T/NK-cell antigens (i.e., surface/cytoplasmic CD3,
CD5, CD7, CD2, CD4, CD8, CD45, CD16, and
CD56) and immature markers (CD34, CD1a, TdT,
and CD99) These immature markers are frequent-
ly absent or very dim, and CD45 is commonly quite
bright in the residual leukemic cells after treatment.
It is hard to say whether the immature nature of
residual T-ALL and the “different-from-normal”
strategies are available to identify a T-cell popula-
tion different from normal T cells [28]. In most
cases, the second strategy includes looking for ab-
normal CD4/8 double-negative (DN), CD4/8 dou-
ble-positive (DP), and/or sCD3–cCD3+ cells.
In the context of B-ALL, markers such as CD13,
CD15, CD33, and CD65 myeloid markers, along with
the CD21 marker, associated with mature B cells, have
the potential to be expressed by CD19+CD34+ cells.
Conversely, normal CD19+CD34+ B cell progenitors
do not express these markers or express them only
weakly [25]. When comparing B-ALL to their nor-
mal counterparts, the expression levels of CD19,
CD10, TdT, and CD34 vary considerably, either be-
ing higher or lower [29]. Leukemia cells often ex-
hibit underexpression of CD38 and CD45. The
EuroFlow consortium is currently developing ad-
vanced protocols for flow cytometry, known as
Next Generation Flow [30]. These protocols aim to
establish standards for MRD detection [31]. The
first step involves a mass lysis procedure to remove
erythrocytes and concentrate leukocytes, thereby
increasing the number of cells available for label-
ing. While traditional four-color flow cytometry
has a sensitivity of 0.01%, this new technique
achieves a sensitivity of 0.001% due to its ability to
label more than 4 × 106 cells [32]. The Children’s
Oncology Group (COG) has a standardized 3-tube
6-color panel for B-ALL MRD detection, which is
used for patients in COG studies. This panel in-
cludes CD34, CD19, CD10, CD20, CD38, CD45,
CD9, CD58, CD13/33, CD71, CD3, and Syto16.
Although it has limitations such as not being able
to detect CD19-negative MRD due to its gating
strategy and consuming more samples, it is highly
standardized with a good sensitivity (10–4) in most
cases. A highly sensitive standardized multipara
meter flow cytometry (MFC) B‑ALL MRD assay
has also been designed by the EuroFlow group,
and it demonstrates the application of a fully
standardized bulk lysis protocol and two stepwise-
designed 8-color tubes (including the backbone
panel plus CD81 and either CD66c/CD123 or
CD73/CD304) [33]. The inclusion of B‑cell mark-
ers other than CD19 has been emphasized in recent
years due to the increasing clinical use of immu-
notherapies targeting CD19 (i.e., blinatumomab
and CAR-T19). CD22 and CD24 are expressed in
B lymphoblasts and can be essential for tracking
B-ALL leukemic cells with downregulation of
CD19 as well as for identification of CD22+ cases
eligible for inotuzumab ozogamicin therapy [34].
In summary, flow cytometry is used in more than
95% of patients and provides valuable information
on the immunophenotypic heterogeneity of leuke-
mia and the cellular state of the bone marrow micro-
environment — aspects that are insufficiently con-
sidered by other MRD techniques [32]. While flow
ISSN 1812-9269. Experimental Oncology 48 (2). 2026 101
Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: Methods for Detection and Quantification
cytometry has lower costs and faster turnaround
time (1 day) compared to the alternative approaches
mentioned below (4 days to weeks), it involves rapid
manipulation due to its execution on living cells. Ad-
ditionally, achieving sensitivity levels above 10–5 re-
quires a significant quantity of cells [30, 35].
Real-time quantitative
PCR (RQ-PCR)
The use of RQ-PCR is an accurate method to iden-
tify MRD, with sensitivity potentially reaching le
vels as low as 10–6. When using PCR to identify
MRD, the genetic marker typically employed to dif-
ferentiate ALL from healthy cells is the rearranged
sequence of the Ig/TCR gene or fusion gene.
Rearrangements of immunoglobulin and T cell
receptor genes can be used to identify a small num-
ber of ALL cells among a large population of normal
lymphocytes expressing different gene rearrange-
ments. In B-cell precursor ALL, incomplete and
complete IGH, IGK, and IGL rearrangements are
more commonly detected and used as MRD targets.
However, sometimes leukemia cells of the B- and
T-lineage display lineage rearrangements, identifi-
able by multipoint PCR and usable for the determi-
nation of MRD [36, 37]. Sequencing of PCR prod-
ucts is performed to determine junctional regions
and acquire allele-specific primers, with successful
cloning confirmation through homo-heteroduplex
analysis [38, 39]. This qPCR technique, using fluo-
rescent marker-labelled probes, is extremely accu-
rate, capable of identifying one leukemia cell among
100,000 healthy lymphoid cells under ideal circum-
stances [40]. The EuroMRD consortium, a dedicat-
ed group of expert laboratories, has played an im-
portant role in establishing the most widely accept-
ed MRD approach. This consortium offers training,
standardization, quality control, and guidelines for
the interpretation of RQ-PCR data [41]. MRD ex-
periments conducted in EuroMRD laboratories can
be reliably replicated, allowing for the comparison
of MRD datasets from different clinical protocols.
However, due to its complexity, the identification of
a small leukemia population using this MRD ap-
proach requires specialized knowledge and exper-
tise [42]. Additionally, technical issues hinder the
identification of MRD targets in 5%–10% of cases
using RQ-PCR-based MRD. This method has a lim-
itation related to the possibility of false-negative
MRD results due to clonal evolution of Ig/TCR re-
arrangements during the disease and relapse. The
study by Szczpansky et al. [43] demonstrated that
22% of leukemia patients lost most or all their Ig/
TCR targets assessed at diagnosis after the onset of
hematological relapse. The amount of diagnostic
DNA available for analysis is another limitation for
this type of MRD assessment. Since it quantifies tu-
mor burden at diagnosis, the initial DNA sample
obtained at the first diagnosis is required for each
MRD experiment and the identification of Ig/TCR
clonal rearrangements [44].
Identification of the fusion gene. Precise identifi-
cation of leukemia subtypes containing fusion genes
is achieved using RQ-PCR. However, this method is
reliable for only 40% of patients with ALL distinct
characteristics and molecular abnormalities. Among
pediatric cases of ALL, four prevalent types of fusion
genes should be distinguished:
• The TEL/AML1 fusion gene (also known as
ETV6-RUNX1) from the t(12;21)(p13;q22) trans-
location. It occurs in 22% of children and 2% of
adults with ALL [45]. This translocation is the most
common in childhood B-ALL. Moreover, it is as-
sociated with an excellent prognosis with intensive
chemotherapy, including asparaginase therapy.
• The TCF3/PBX1 fusion gene from the t(1;19)
(q23;p13) translocation represents 5% of childhood
ALL and 3% of adult ALL and is frequently associ-
ated with the pre-B immunophenotype, in about
25% of cases [45, 46].
• BCR/ABL from the t(9;22)(q34;q11) transloca-
tion is found in approximately 3% of children and
30% of adults, and is associated with an unfavor-
able prognosis [46].
• AF4/MLL is derived from the t(4;11)(q21;q23)
translocation [45,46]. Chromosomal rearrange-
ments of the human MLL gene are the most com-
mon genetic abnormality in the first year of life, but
it occurs in only 8% of children and 10% of adults
with ALL [46].
The first two fusion genes have higher incidence
rates in children with B-ALL. In contrast, the BCR/
ABL fusion gene is more frequently found in
chronic myeloid leukemia and remains rare in pe-
diatric ALL. In more than 2/3 of childhood leuke-
mia cases, the AF4/MLL fusion gene is present. Ac-
cording to Ajuba et al. [47], this fusion gene is as-
sociated with poor prognosis and is predominant
in B-ALL of infants.
102 ISSN 1812-9269. Experimental Oncology 48 (2). 2026
S. Addakiri, H. Bencharef, A. Harrach, S. Jaddaoui, K. Ait-Ichou, B. Oukkache
discovered identifiable clonal rearrangements in
93% of patients in the pretreatment sample [49,
50]. Among MRD-positive cases, the clone most
frequently identified in the pretreatment sample
corresponds to the equally dominant clone on day
29. Like in T-ALL, a subset of MRD patients was
identifiable by NGS but not by flow cytometry.
Patients without clonal rearrangements of the Im-
munoglobulin Superfamily had poorer outcomes
than patients with clonal IGH. Maybe these cases
represent more primitive clones [51]. NGS offers
several advantages over flow cytometry and con-
ventional PCR when examining MRD. It involves
less subjectivity in interpretation compared to
flow cytometry. Once the protocol is established,
the technical requirements are easier than those
of other molecular genetic methods. By using
primers to simultaneously amplify any possible
combination of rearranged IGH or TCR loci, NGS
bypasses the need for patient-specific primers.
This explains the use of HTS because of its speed
in real-time therapeutic decision-making, al-
though it is slower than flow cytometry in practi-
cal applications. However, NGS can only identify
cases with complete rearrangements, leaving be-
hind a limited number of cases without rearrange-
ments or with incomplete rearrangements. In ad-
dition, this method involves matching pre- and
post-processing samples. Several studies demon-
strated that NGS can accurately predict the rate of
recurrence and survival by detecting MRD as
compared to flow cytometry and conventional
PCR [50] and can potentially replace these me
thods in the future [52, 53].
New technologies
Despite significant progress in standardizing RQ-
PCR for MRD detection and ensuring reproduc-
ibility within and between laboratories, there are
still two main concerns that need to be addressed.
One is related to the significant amount of DNA
required for accurate diagnosis. The other pertains
to the risk of non-specific amplification in RQ-PCR
technology, especially in the presence of low levels
of MRD. Indeed, a substantial amount of diagnostic
DNA is essential for the detection of clonal rear-
rangements in MRD genes by IG/TR. This is be-
cause, in addition to early detection of clonal rear-
rangements, a standard dilution curve must be con-
Variations in the detection rates of different fu-
sion genes in different regions are observed. This is
probably a result of genetic diversity among breeds.
Although RQ-PCR has some advantages, such as
simple and fast operation, high specificity, and sen-
sitivity in the detection of fusion genes, it is limited
to specific subtypes of leukemia. The variations in
the expression of fusion genes between individuals
and the potential for false-positive results due to
PCR contamination are additional challenges. Fur-
thermore, RQ-PCR faces difficulties in achieving
accurate ratios between the number of leukemia
cells and PCR products, making it inadequate for
precise cellular-level MRD assessment.
Next Generation Sequencing (NGS)
In the late 1990s, new DNA sequencing tech-
niques emerged. These methods were further de-
veloped into high-throughput sequencing (HTS),
known as NGS, since 2000. This has rapidly
gained adoption by researchers in various fields.
The advantages of NGS are twofold: it can se-
quence large amounts of DNA quickly and has a
sensitivity to detect and quantify very small
clones, such as one cell in a population of 10-6
cells, as well as very low-frequency cells. Re-
searchers have been particularly interested in ex-
ploring the use of NGS for MRD detection. Ini-
tially, they used this method to detect MRD in
T-ALL and then in B-ALL [48, 49].
NGS in the context of T-ALL. Wu et al. [48] used
HTS to study MRD in patients diagnosed with
T‑ALL and compared these results with those ob-
tained by flow cytometry. HTS showed better sen-
sitivity in detecting MRD. Interestingly, among cas-
es without TCR clonal beta rearrangements at di-
agnosis, some had substantial clones identifiable by
flow cytometry on day 29. Almost half of these cas-
es had an immunophenotype of early T-cell precur-
sors (ETP), while the other half had a similar phe-
notype, except for the presence of CD5, which is
referred to as ‘quasi-ETP’. Conversely, none of the
cases with detectable TCR clonal beta rearrange-
ments had an ETP phenotype.
NGS in the context of B-ALL. In line with pre-
vious research on T-ALL, NGS was used for MRD
detection in patients with B-ALL [49]. NGS was
extended to all B-ALL patients, regardless of their
known IGH rearrangements [50]. The authors
ISSN 1812-9269. Experimental Oncology 48 (2). 2026 103
Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: Methods for Detection and Quantification
Detection methods used to monitor MRD in ALL
Method Advantages Disadvantages
FCM (Flow cytometry) Fast
Sensitivity 10–4
Relatively inexpensive
Ability to quantify Aberrant antigen
expression
Variable sensitivity due to similarities
between normal (regenerating) cells and
malignant cells
Limited standardization, no QA
(quantification) results
FCM-DfN (Flow cytometry
different from normal)
Sensitivity 10–3 to 10–5
No diagnostic sample required
Phenotypic shifts will not interfere
with results
Applicable to > 90% of patients
Rapid turnaround time
Significant operator experience required
Limited standardization
RT-PCR of fusion genes Sensitivity 10–4 to 10–5
Simple (uses some primers as used
for diagnosis)
Limited QA rounds
Limited applicability in ALL (absence
of targets in 50% of cases)
Risk of contamination
PCR of Ig/TCR genes Sensitivity 10–4 to 10–5
Applicable to most patients
Standardized guidelines in Europe
Time-consuming
Expensive
Relies on pre-treatment sample
Requires extensive experience and labor
Digital PCR Highly sensitive (10–4 to 10–5)
Rapid and widely applicable
Limited standardization, available only in
a few labs, no approved guidelines
for data analysis, relatively expensive
Next Generation
Sequencing
Very sensitive (10–6)
Applicable to almost all patients
Clone–unbiased (can track multiple
clones and evolution)
Only US FDA-approved assay
(ClonoSEQ)
Data for MRD use in peripheral blood
Expensive
Longer turnaround time than FCM
Requires diagnostic pre-treatment sample
structed for each qPCR MRD experiment, posing
limitations in monitoring patients, especially when
assessing multiple time points. Third-generation
PCR, known as droplet digital PCR (ddPCR),
makes it possible to overcome these limitations of
conventional RQ-PCR. ddPCR technology com-
partmentalizes the sample into thousands of indi-
vidual droplets, each representing an independent
PCR reaction. This allows for endpoint amplifica-
tion and utilizes Poisson statistics [54, 55]. Several
studies on medical diagnosis focused on the use of
ddPCR techniques in oncology, including hemato-
logical malignancies [56, 57]. The recent studies
compared ddPCR and qPCR in adult patients with
mature lymphoid malignancies and adolescent and
young adult patients with ALL, respectively [58],
and reported that ddPCR exhibits sensitivity, ac-
curacy, and reproducibility comparable to qPCR.
Moreover, ddPCR technology made it possible to
determine the absolute amount of specific DNA
molecules in a given sample without the need to
compare to a standard curve. While the final clini-
cal validations of ddPCR are still pending, the first
guidelines for interpreting ddPCR data in a clinical
setting have been published [59].
ddPCR can certainly serve as a viable alternative
to qPCR and as an instrument to preserve DNA
samples. Owing to the extensive partitioning of the
reaction mixture into a large number of individual
compartments, ddPCR can achieve a very high le
vel of analytical precision. Furthermore, ddPCR is
particularly well suited for the detection of low-
abundance targets in the presence of a high back-
ground of non-target DNA. Within partitions that
contain the target molecule, the relative proportion
of target to non-target DNA becomes significantly
higher than in the original sample, thereby facilita
ting detection.
104 ISSN 1812-9269. Experimental Oncology 48 (2). 2026
S. Addakiri, H. Bencharef, A. Harrach, S. Jaddaoui, K. Ait-Ichou, B. Oukkache
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affected by partial PCR inhibition compared to
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Amplification of non-specific, false Ig/TCR rear-
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HTS based on PCR of Ig/TCR gene rearrangements
represents an interesting advance in this context.
HTS identifies clone-specific IG/TR indexing se-
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Several recent studies highlighted the greater spe
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Submitted: September 10, 2025
Сара Аддакірі 1, 2, Ханаа Бенчареф 1, 2, Асмаа Харрах 1, 2,
Саміха Джаддауї 1, 2, Хадіжа Айт-Ічу 2, Бушра Уккаче 1, 2
1 Медико-фармацевтичний факультет университету
Хасана ІІ, Касабланка, Марокко
2 Університетський медичний центр Ібн Рошд,
Касабланка, Марокко
МІНІМАЛЬНА ЗАЛИШКОВА ХВОРОБА ПРИ ГОСТРІЙ ЛІМФОБЛАСТНІЙ
ЛЕЙКЕМІЇ: МЕТОДИ ВИЯВЛЕННЯ ТА КІЛЬКІСНОГО ВИЗНАЧЕННЯ
Ідентифікація мінімальної залишкової хвороби (МЗХ) зазнала значного прогресу завдяки розвитку технологій,
застосованих у цій галузі. Якщо спочатку вона базувалася виключно на морфології, згодом методологія визна-
чення МЗХ зазнала докорінних змін та включила каріотипування, цитогенетику, проточну цитометрію та інші
чутливі методи. В огляді обговорено ключові технічні аспекти успішного виявлення МЗХ i проаналізовано коре-
ляцію між результатами, отриманими за допомогою проточної цитометрії та молекулярно-генетичних методів.
Ключові слова: МЗХ, проточна цитометрія, молекулярно-генетичні методи.
https://doi.org/10.1182/blood-2015-07-655159
https://doi.org/10.1182/blood-2015-07-655159
https://doi.org/10.1373/clinchem.2013.214742
https://doi.org/10.1373/clinchem.2013.214742
https://doi.org/
https://doi.org/10.1007/s00277-016-2623-0
https://doi.org/10.1007/s00277-016-2623-0
https://doi.org/10.3390/cancers15020374
https://doi.org/10.2760/192883
https://doi.org/10.2760/192883
https://doi.org/10.1038/sj.leu.2404586
https://doi.org/10.1038/bmt.2008
https://doi.org/10.1038/bmt.2008
https://doi.org/10.1182/blood-2015-03-580027
https://doi.org/10.1182/blood-2015-03-580027
https://doi.org/10.1038/bmt.2017.16
https://doi.org/10.1182/blood-2015-07-655159
https://doi.org/10.1182/blood-2015-07-655159
https://doi.org/10.1038/leu.2011.89
https://doi.org/10.1038/leu.2013.375
https://doi.org/10.1038/leu.2013.375
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| id | oai:ojs2.ex.aqua-time.com.ua:article-640 |
| institution | Experimental Oncology |
| issn | 2312-8852 |
| keywords_txt_mv | |
| language | English |
| last_indexed | 2026-08-22T01:00:33Z |
| publishDate | 2026 |
| publisher | PH Akademperiodyka |
| record_format | ojs |
| resource_txt_mv | exp-oncologycomua/55/7aa549e98bc4ea79b3b55a1f2d41e055.pdf |
| spelling | oai:ojs2.ex.aqua-time.com.ua:article-6402026-08-21T12:36:20Z MINIMAL RESIDUAL DISEASE ASSESSMENT IN ACUTE LYMPHOBLASTIC LEUKEMIA: METHODS FOR DETECTION AND QUANTIFICATION МІНІМАЛЬНА ЗАЛИШКОВА ХВОРОБА ПРИ ГОСТРІЙ ЛІМФОБЛАСТНІЙ ЛЕЙКЕМІЇ: МЕТОДИ ВИЯВЛЕННЯ ТА КІЛЬКІСНОГО ВИЗНАЧЕННЯ Addakiri, Sara Bencharef, Hanaa Harrach, Asmaa Jaddaoui, Samiha Ait-Ichou, Khadija Oukkache, Bouchra MRD, flow cytometry, molecular technique Recently, the identification of minimal residual disease (MRD), also known as measurable residual disease, has seen significant advances driven by the evolving technological landscape in this field. Initially based on raw morphology, the field has since evolved to incorporate karyotyping, cytogenetics, flow cytometry, and other sensitive methods. This updated review discusses key technical considerations for successful MRD detection. We explore the correlation between the results obtained by flow cytometry and molecular genetic techniques. дентифікація мінімальної залишкової хвороби (МЗХ) зазнала значного прогресу завдяки розвитку технологій, застосованих у цій галузі. Якщо спочатку вона базувалася виключно на морфології, згодом методологія визначення МЗХ зазнала докорінних змін та включила каріотипування, цитогенетику, проточну цитометрію та інші чутливі методи. В огляді обговорено ключові технічні аспекти успішного виявлення МЗХ i проаналізовано кореляцію між результатами, отриманими за допомогою проточної цитометрії та молекулярно-генетичних методів. PH Akademperiodyka 2026-08-21 Article Article application/pdf https://exp-oncology.com.ua/index.php/Exp/article/view/640 10.15407/exp-oncology.2026.02.098 Experimental Oncology; Vol. 48 No. 2 (2026): Experimental Oncology; 98-107 Експериментальна онкологія; Том 48 № 2 (2026): Експериментальна онкологія; 98-107 2312-8852 1812-9269 10.15407/exp-oncology.2026.02 en https://exp-oncology.com.ua/index.php/Exp/article/view/640/470 Copyright (c) 2026 Experimental Oncology https://creativecommons.org/licenses/by-nc-nd/4.0/ |
| spellingShingle | Addakiri, Sara Bencharef, Hanaa Harrach, Asmaa Jaddaoui, Samiha Ait-Ichou, Khadija Oukkache, Bouchra МІНІМАЛЬНА ЗАЛИШКОВА ХВОРОБА ПРИ ГОСТРІЙ ЛІМФОБЛАСТНІЙ ЛЕЙКЕМІЇ: МЕТОДИ ВИЯВЛЕННЯ ТА КІЛЬКІСНОГО ВИЗНАЧЕННЯ |
| title | МІНІМАЛЬНА ЗАЛИШКОВА ХВОРОБА ПРИ ГОСТРІЙ ЛІМФОБЛАСТНІЙ ЛЕЙКЕМІЇ: МЕТОДИ ВИЯВЛЕННЯ ТА КІЛЬКІСНОГО ВИЗНАЧЕННЯ |
| title_alt | MINIMAL RESIDUAL DISEASE ASSESSMENT IN ACUTE LYMPHOBLASTIC LEUKEMIA: METHODS FOR DETECTION AND QUANTIFICATION |
| title_full | МІНІМАЛЬНА ЗАЛИШКОВА ХВОРОБА ПРИ ГОСТРІЙ ЛІМФОБЛАСТНІЙ ЛЕЙКЕМІЇ: МЕТОДИ ВИЯВЛЕННЯ ТА КІЛЬКІСНОГО ВИЗНАЧЕННЯ |
| title_fullStr | МІНІМАЛЬНА ЗАЛИШКОВА ХВОРОБА ПРИ ГОСТРІЙ ЛІМФОБЛАСТНІЙ ЛЕЙКЕМІЇ: МЕТОДИ ВИЯВЛЕННЯ ТА КІЛЬКІСНОГО ВИЗНАЧЕННЯ |
| title_full_unstemmed | МІНІМАЛЬНА ЗАЛИШКОВА ХВОРОБА ПРИ ГОСТРІЙ ЛІМФОБЛАСТНІЙ ЛЕЙКЕМІЇ: МЕТОДИ ВИЯВЛЕННЯ ТА КІЛЬКІСНОГО ВИЗНАЧЕННЯ |
| title_short | МІНІМАЛЬНА ЗАЛИШКОВА ХВОРОБА ПРИ ГОСТРІЙ ЛІМФОБЛАСТНІЙ ЛЕЙКЕМІЇ: МЕТОДИ ВИЯВЛЕННЯ ТА КІЛЬКІСНОГО ВИЗНАЧЕННЯ |
| title_sort | мінімальна залишкова хвороба при гострій лімфобластній лейкемії: методи виявлення та кількісного визначення |
| topic_facet | MRD flow cytometry molecular technique |
| url | https://exp-oncology.com.ua/index.php/Exp/article/view/640 |
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