Minimal Residual Disease(MRD) in B cell pediatric ALL patient

Significance of Detection of Minimal
Residual Disease in treatment of
Paediatric ALL & Prediction of
Relapse &its experience in tertiary
care centre in Eastern India
Dr Kalyan Kusum Mukherjee
MBBS, MD,FCCM,ECMO
H.O.D Medical Oncology and Clinical & Translational Research
Chittaranjan National Cancer Institute, Kolkata

Research Team
• Kalyan K Mukherjee
• Debasish Banerjee
• Anjan Das
• Subham Halder
• Dattatreya Mukherjee
• Mili Das
• Surya Kanta Roy
• Chinmoy Kumar Panda
• Utpal Choudhury

Two Papers Published based on this research
• 1 Correlation of Minimal Residual Disease Detection in Paediatric B
Cell All Patient with Their Overall Survival and Prognosis-
Experience in Tertiary Care Centre in Eastern India, IOSR-JDMS,Vol
19, Series5 (1) 1-8
• 2. Significance of minimal residual disease detection by flow
cytometry in Paediatric B cell ALL treatment in tertiary care
centre in Eastern India, IOSR-JDMS, VOL 19, SERIES 4 (2) 11-15

POINTS TO BE DISCUSSED
• INTRODUCTION AND METHOD OF DETECTING MINIMAL RESIDUAL DISEASE (MRD)
• SIGNIFICANCE OF DETECTION OF MRD IN THE HAEMATOLYMPHOID MALIGNANY
• SIGNIFICANCE OF DETECTION OF MRD IN ALL SPECIFICALLY IN PADIATRIC GROUP
• EXPERIENCE OF MRD BASED TREATMENT PADIATRIC B CELL ALL IN TERTIARY CARE
CENTRE IN EASTERN INDIA

POINTS TO BE DISCUSSED

Introduction:
• A stage in leukaemia treatment when patient is in remission,
symptoms of the disease are absent but small number of leukemic
cells are still present in the body.
• Minimal residual disease (MRD) refers to the presence of disease in
cases deemed to be in complete remission by conventional pathologic
analysis.
• Nowadays, minimal residual disease (MRD) is accepted as the
strongest independent prognostic factor in acute lymphoblastic
leukemia (ALL).
• The first report of minimal residual disease (MRD) in
leukemia was published nearly 4 decades ago.

Techniques for Measuring MRD
• The term minimal residual disease (MRD) is used to describe the low-level disease
which is not detectable by conventional cytomorphology . Compared with the
classical microscopic detection of residual leukemic cells, MRD is assessed by
sensitive molecular and flow cytometric methods to more precisely monitor
disease kinetics during and after treatment.
• Morphology
• Clonogenic Assays
• Immunophenotype Analysis
• Karyotype Analysis
• FISH
• PCR

Why MRD is Important?
• Many trials have confirmed that MRD is the independent strongest prognostic
factor in both children and adults with ALL.
• MRD detection is not only useful for the assessment of initial treatment response
and subsequent definition of MRD-based risk groups, but also to monitor disease
burden in the setting of stem cell transplantation (SCT), for early recognition of
impending relapse, and as potential end point in clinical trials.
• MRD is used for guiding clinical decisions in current treatment protocols. This
review highlights relevant methodologic aspects for correct interpretation of
MRD results and the most important data on the significance of MRD
quantification and its application to tailoring treatment in padiatric ALL.

Technique of Measuring MRD
• Morphology
• FISH
• PCR

Techniques: MORPHOLOGY
• 1 to 100 cells are identified as malignant
• Tough to make difference between immature cancer cell and early
regenerating cells
• Low sensitivity

Technique of Measering MRD
• Morphology
• FISH
• PCR

Technique: Clonogenic Assay:
• In vitro technique where bone marrow samples are grown under
condition favourable for stimulation of leukemic cell
• Advantage is Cancer malignant cells are easily identified and their
biological Charecteristics and growth requirement can be easily
studied.
• Disadvantage is dependence of growth rates of leukemic
Progenitor cell.

Technique Measuring MRD
• Morphology
• FISH
• PCR

Immunophenotyping Assay
• Use of monoclonal antibodies by means of flowcytometry or
fluorescence microscopy to detect nuclear, cytoplasmic, surface antigen
that are expressed by malignant cells can be fast and reliable.
• Sensitivity of detection range is as high as 1 abnormal cell per 10000 to
100000 normal cell using double or triple color immunofluorescence
technique.
• Standard four color FCM, CD used are CD22, CD45, CD58 and CD97 in
combination with CD10, CD19, CD34
• MRD level <0.01% was considered negative, while positive MRD was
classified according to its level into 2 groups: ≥ 0.01- <0.1 and ≥ 0.1,

Technique: Flowcytometry (FCM)
• A flow cytometry as a useful diagnostic modality and for the assessment of treatment
response in hematopoietic neoplasms.
• Prognostication and disease monitoring is also being recognized increasingly as one of the
important process.
• Specimens include fluid specimens from serous cavity effusions and samples from solid
tissues like lymph nodes, reticulo-endothelial tissue, central nervous system tissue, etc.
Flow cytometry technique provides a unique blend of rapidity, high sensitivity and
specificity compared to cyto-morphology and conventional immunohistochemical staining.
• Remarkable for simultaneous analysis of more than one marker on the cells.
• Evaluation of limited samples such as cerebrospinal fluid or fine needle aspiration samples
makes Flow cytometry a valuable tool.
• DNA ploidy analysis and assessment of pediatric non-hematopoietic neoplasms by Flow
cytometry has envisaged the utility vista of this technique.
• This review is aimed at providing an insight into the applications of flow cytometry in
pediatric malignancies.

FCM-MRD
• The most predictive prognostic levels of postindunction disease as
detected by FCM which in both ALL and AML appear to lie between 0.01%
and 0.035% leukaemic cell involvement of the morphologically normal
appearing bone marrow. We must keep in mind, however that to reliably
detect 0.01% of leukaemic cells, FCM is pushed to its extreme limits which
may not be reached in the every clinical routine laboratory due to
variability In technical expertise

USAGE OF FCM
• Multiparameter flow cytometric immunophenotyping has been primarily
used to provide rapid diagnosis and help classify most of the hematological
malignancies, including pediatric leukemias and lymphomas, where there is
blood and/or marrow involvement, as well as body fluid samples like
cerebrospinal fluid, pleural fluid and peritoneal fluid.

Disadvantage of using FCM
• The lack of antigen Specificity for malignant cells as these cells represent the
counterparts of normal cells with in many cases identical or similar antigen
profiles.
• The existence of several subpopulation, some of them as minor clones, that are
difficult to identify.
• The inability to identify phenotypic switch, a phenomenon that may occur at
relapse.
• flow cytometry technique has limited its use for fluid samples, primarily blood
and bone marrow.

Karyotype Analysis
Karyotype is the arrangement of chromosome from large size to small size
• Spectral Karyotype (SKY) analysis is an important tool in risk stratification
• By this malignancy specific markers are identified and detect cytogenetic signs of
clonal evolution and relapse.
• The use of SKY substantially improves the precision of karyotype analysis of
malignant cells, which in turn leads to a more accurate assessment of the genotypic
abnormalities in those cells.
• The translocations and Chromosomal abnormality is easily detected by Karyotype
analysis.
• Despite its specificity, the sensitivity of the method is low and does not exceed that
of morphologic assessment of marrow smears.

Karyotype Analysis
• Structural and numerical aberration can be easily detected through Karyotype
analysis.
• Structural aberration: Deletion, Duplication, Inversion, ring, transposition
• Numerical aberration: Polyploidy, Aneuploidy ( Monosomy, Trisomy), Euploidy
• Transposition of 5p to 6q, ABL-BCR translocation, RARA-PML translocation can
be identified by Karyotype analysis

FISH- Fluorescence In Situ Hybridization
• FISH is the detection of highly specific DNA probes which has been hybridized to
either interphase or metaphase chromosome using fluorescence microscopy.
• FISH has emerged in the recent years as a promising new tool for the identification
on a molecular level of both Chromosomal aberration and malignancy specific DNA
sequence.

FISH
• Although 100 times more sensitive than standard cytogenetics, the sensitivity level
is achieved by FISH (one cell in 1000) is markedly below that desired for MRD
detection (one cell in 10000)
• The main advantage of FISH, a large amount of cells can be easily studied in a
time efficient manner
• FISH allows analysis of both metaphase and no dividing interphase cells.
• Interphase FISH can even be performed on peripheral blood samples, thus avoiding
the need for marrow aspiration.

POLYMERISE CHAIN REACTION (PCR)
• Methods for MRD quantification are based either on the discrimination of ALL cells
from normal physiological counterparts and the identification of the leukemia-
associated immunophenotype(LAIP)by multiparametric flowcytometry(MFC) the
detection of leukemia-specific rearrangements of immunoglobulin and T-cell
receptor (IG/TR) genes and/or fusion gene transcripts by real-time quantitative
polymerase chain reaction (RT-qPCR).
• IG/TR RT-qPCR can be used for MRD detection in.95% of patients with ALL.
Sensitivity is determined separately for each assay and routinely reaches 10^24 to
10^25 (1 leukemic cell in 10 000 to 100 000 healthy cells).

PCR
• Drawbacks of PCR lies in its extraordinary sensitivity sample contamination and false
positive result can occur.
• RT-PCR measures transcript expression, a malignant clone that transiently does not
express its diagonostic transcript would evade detection by this technique.

POINT TO BE DISCUSSED

Use Of MRD In The Detection Of ALL
• ALL is a heterogeneous disease affected by many patient and disease related
factors including age, immunologic subtype, clinical, genetic, molecular feature.
• Most Children, adolescents and young adults with ALL in first complete
remission(CR1) have excellent prognosis with multi agent chemotherapy in
induction, consolidation, re induction and maintenance therapy.
• There is a subset of patient with a more guarded prognosis using this approach,who
may benefit from haematopoitic allogeneic stem cell transplantation(alloHSCT)
• Targets: t(9,22) BCR-ABL, t( 12,21) ETV 6-RUNX1 (TEL-AML1), Patient specific
assays for immunoglobulin and T cell receptor genes.
• Use: Chromosomal Translocation MRD detection is used as a standard clinical
practice. Practice specific assay are gaining acceptance

ACUTE MYELOID LEUKAEMIA (AML)
• MRD is an important prognostic test in AML.
• Favourable and intermediate cytogenetics.
• Targets: t(15,17) PML- RARA, t(8,21) AML1-RUNX1T1 (AML-ETO), inv(16)
• Uses: Chromosomal translocation MRD detection used as a standard clinical
practice.

Chronic Lymphoblastic Leukaemia
• Targets: Cell Surface protein, Patient specific assay for immunoglobulin and T cell
receptor Gene
• Uses: Ig method are gaining wider useas more advanced FCM are utilized for
clinical testing. Pt. Specific assays are still generally only used in research
protocols.

Chronic Myeloid leukaemia
• Target: t(9,22) BCR- ABL
• Use: MRD detection of the t(9,22) is the considered standard of the care for all
patients with CML and is extremely valuable for patient being treated with imatinib
masilate.

Follicular Lymphoma
• Target: t(14,18) IgH/BCL2, Patient specific assays for
immunoglobulin and T CR gene
• Use: The t(14,18) is regulary used for MRD detection. Patient
specific assays are still generally only used in research protocols.

Mantle Cell Lymphoma
• Targets: t(11,14) IgH/CCND1 (IgH/BCL1), Patient specific assays
for immunoglobulin and T cell receptor genes.
• Uses: The t (11,14) is regularly used for MRD detection, but the
assay can only reliably detect 40-60% of the t(11,14)
translocations. Patient specific assays are still generally only used
in research protocols.

Multiple Myeloma
• Targets: M- Protien levels in blood, Patient specific assays for
immunoglobulin and T cell receptor genes.
• Uses: M- protein level in the blood is standard of the care and is used
for almost all patient with multiple myeloma. Patient specific Assays
are still generally only used in research protocols.
• Bens Jones Protien
• HyperCalcemia
• Multiple Myeloma Kidney

SOLID TUMORS
• Research into MRD detection of several Solid tumors such as breast cancer and
neuroblastoma has performed. These assays have been used to sample lymph nodes
and blood for residual or metastatic tumor cells. Applicable targets for MRD
detection have been more difficult to determine in solid tumor and the use of MRD
in solid tumors is much less advanced than the use in leukaemia and lymphoma.

Significance of MRD
• Several large studies in both childhood and adult ALL have shown that the initial
MRD response is a highly relevant prognostic factor, and therefore MRD has been
used for the refinement of initial treatment stratification.
• Analysis of the largest cohort of adult ALL data so far was performed by the
German Multicenter Study Group for Adult ALL (GMALL), which assessed MRD in
Philadelphia chromosome–negative (Ph–) patients with standard-risk and high-risk
features.
• Molecular response to standard induction and consolidation treatment was the
only significant prognostic factor for remission duration and survival throughout
both risk groups in a multivariable analysis
• Patients with molecular failure undergoing SCT in
firstcompleteremission(CR1)hadsignificantlybetterprobability of continuous CR
than those without SCT (66% vs 11%).

Significance of MRD
• The French Group for Research on Adult ALL (GRAALL),the Northern Italian Study Group
(NILG), and the Spanish Programa Español de Tratamientos en Hematologıa (PETHEMA)
study group confirmed the strong and independent prognostic impact of MRD after
induction and early consolidation treatment.
• MRD negativity was associated with a significant improvement in disease-free survival
(DFS) and overall survival (OS) at all time points.
• Early achievement of MRD negativity during induction therapy correlated with a particular
good outcome in adult ALL. Earlier studies identified a small subset of ALL patients with a
very rapidtumor clearance (low-level or undetectable MRD after 2 weeks of therapy)
andanexcellentprognosis,27 which is in line with reports on childhood ALL.28 In an MFC-
based trial, high levels or disease at day 14 of treatment(.30%)identified a small subgroup
of 10% of patients with aparticular lypoorprognosis and median event-freesurvival(EFS)and
OS of only 9and21months,respectively.However,MRD at this early time point lost its
independent prognostic impact when MRD at later time points was incorporated into a
multivariable analysis.

Significance of MRD
• The level of MRD at a certain time in treatment, is a useful guide, to the patient
prognosis.
• MRD testing could predict outcome
• MRD is a powerful and independent prognostic indicator
• Monitoring people for early sign of recurring leukaemia
• By Checking MRD we can diagnose the relapse of the disease early, before
symptoms come back.
• This means regular blood or bone marrow samples. This is being explored mainly in
CML, where we can study the leukaemia in blood, which is easier to sample
regularly than bone marrow.

Significance of MRD
• It identifies patients individual risk of relapse and can theoretically allow them to
receive just enough treatment to prevent it.
• Treatment intensification for patients with slow clearance of Leukaemia and
persistent or resurgent MRD is well supported now.
• MRD monitoring has redefined remission in ALL. Numerous studies have
demonstrated the strong association between MRD levels and treatment outcome
in childhood ALL supporting the concept that MRD during the initial phases of
chemotherapy provides a reliable measurement of the drug sensitivity of
leukemic lymphoblasts. This realization has profoundly refined risk-directed
therapy, with MRD being applied in virtually all major protocols for pediatric ALL to
guide treatment decisions.

Significance of MRD
• It is also used in to detect the quality of graft, the efficacy of
procedures quality of grafts, the efficacy of procedures to remove
malignant cells from auto grafts and to determine its clinical
significance.
• It has been shown that the detection of High level of MRD before
transplantation was universally associated with relapse.

MRD in Stem Cell Transplantation
• Relapse still remains the major cause of treatment failure after SCT in
ALL, even in patients who received transplantation during hematologic
remission. Several studies showed the prognostic relevance of
pretransplantation MRD in adults. Bassan et al highlighted the fact that
MRD is a quantitative variable and MRD levels correlate with post-SCT
outcome. Patients with MRD levels of >= 10^-3. at week 16 and/or week
22 had a worse posttransplantation outcome with a 6-year relapse
incidence of 64% compared with 23% in patients with MRD levels <10^-3.

Current Research And Controversy
• Clinical Usefulness of MRD test
• It’s a new process, test is done in few patients. So there is less evidence available
• Controversy is about best time to test and best procedure of the test
• There is also controversy on the safety level of MRD
• The goal of MRD treatment may be to reduce it to safe level, not to eradicate it
completely.

IS MRD TESTING USEFUL FOR PATIENT?
• Some types of Leukaemia are difficult to treat. In these, it is not clear how MRD
testing would help. The patient may not do well on current treatment, but
sometimes it is not clear what other treatment but sometimes it is not clear what
other treatment, if anything , might be better. There is thus an argument that as
the test is not necessary: it might involve an additional procedure for the patient, it
will contribute no useful information on treatment, it is not necessary.

How MRD is done?
• Bone marrow (2-3 ml) with heparin anticoagulation was taken from pediatric patients
in the initial diagnosis before chemotherapy, and the karyocytes were separated
routinely and counted.
• Then the karyocytes were stained using CD45-FITC grading, CD10-PE, CD34-Percp and
CD19-APC monoclonal antibodies; the antibody combination was effective if the
leukemia cells and normal bone marrow cells could be distinguished.
• At 33 days and 12 weeks after induction chemotherapy, the bone marrow samples were
taken from pediatric patients, and the mononuclear cells were separated using Ficoll,
followed by four-color fluorescence labeling detection (CD58, CD66c, CD38, CD123 and
TdT were added based on CD34/CD19/CD10); the original data in the initial diagnosis
and screening were called up; in the two-parameter diagram, if there were cells in the
original area of leukemia cells, it indicated the minimal residual leukemia; the
proportion of these residual cells in total bone marrow mononuclear cells was the
result of monitoring MRD.

MRD Testing by Hospital and Other Labs
• MRD is not a routine test yet, it is carried out in all place.
• Currently most MRD testing in leukaemia research is done during clinical trials and
would be funded as a part of the trial, for patients enrolled in the trial. The test is
specialised, so sample are usually sent to a central reference lab in each region and
country. The test are not mostly done in routine lab.

COST
• As it is not a routine test so in a limited amount of lab offer this test. Technically
complex and demanding MRD is a costly test. As this is done during clinical trial so
the cost is fulfilled by the study authority.
• Cost in Republic Of India: INR 30000
• Cost in USA:$3660
• MRD testing saved $1156600 over patients remaining life time. In addition health
outcomes slightly favoured MRD testing over no testing with an increase in Quality
adjusted life years (QALY) of 0.01.The researchers suggested that MRD testing
resulted in an improved QALY because there was lower risk of adverse events from
continued treatment when it was not needed.

Point To Be Discussed

Objects
PRIMARY OBJECTIVES:
• To interpret the MRD data obtained from Flow cytometry (FCM).
• Bone Marrow samples and peripheral blood samples obtained from children with B-
lineage ALL during treatment and Corroboration of MRD results with the relapse
time from the completion of the maintenance treatment.
SECONDARY OBJECTIVES:
• MRD based risk stratification of childhood B-cell ALL under treatment.
• Precise MRD levels and optimal sampling time points in treatment protocols used in
our institution.

Materials And Method
Patients and cells: No. of patients with B cell ALL will be taken 50. No. of
peripheral and bone marrow sample will be 200(4 samples from each patient).Age of
patient >7 to 12 yrs. Patient will be chemonaive B cell ALL.
All samples will be collected in aseptic method and will be preserved in sodium
heparin and processed at earliest time of collection.
Samples will be tested for all cell viability by fluorescent viability dyes in a FCI
panel. Samples <71oh viability will be rejected.
Specimen will be processed for flow cytometric immunophenotyping (FCI) and
Polymerase Chain Reaction (PCR) methods. This study will be done with the approval
from IEC of CNCI & IHTM. Informed Consent Form will be obtained from the parents
and guardians of each child.

8 color FCM used in the study
In B-ALL the markers combination to be used for MRD studies are followings:
Markers used
CD10+
CD19/ CD34/CD10/
CD58/ CD45/CD13/ anti-
TdT and CD33
CD 10-
CD19 /
CD34/CD15/CD133/CD10

PATIENTS AND METHODS:
• Study Population:
• Children, age I to younger than 12 years, with newly diagnosed B-cell ALL,
will be enrolled in the study, , Samples requiring a bone marrow aspirate on
day 15, in addition to aspirate on day 29 and post consolidation. Overall
samples for FCM MRD are available for 50 patients; Informed consent to
participate in the study was obtained for all patients by parents or legal
guardians.

Inclusion Criteria
• The subjects who qualified for the study should meet the following inclusion criteria.
• Subjects of either sex aged between > 1 to 12 years.
• Subjects of B-cell Acute Lymphoblastic Leukemia proved from peripheral blood smear &
bone marrow aspiration sample for morphology, cytochemistry & immunophenotyping
• Subjects screening and baseline laboratory test as defined by Haemoglobin ≥ 9 gm%.
• Serum SGPT & SGOT ≤1.5x UNL.
• Serum total bilirubin ≤ 1.5 x UNL
• Serum Creatinine ≤ UNL
• Subjects are willing and able to adhere to the study visit schedule and other protocol
requirements as evidence by signed written informed consent.

Exclusion Criteria
• Subjects with Ph+ chromosome positive B-cell ALL.
• The subjects which who are taking olsalazine, mesalazine or sulphasalazine,
warfarin, thioguanine and drugs whose primary or secondary toxicity is
myelosuppression.
• Anemia (Hemoglobin) < 9 gm%.
• Subjects with inherited deficiency of the TPMT enzyme.
• Subjects who cannot adhere to the protocol due to drug allergy & sensitivity.

Exclusion Criteria
• Subjects who have current signs and symptoms of severe, progressive, or
uncontrolled renal, hepatic ,hematologic, endocrine, pulmonary, cardiac, neurologic
or cerebral disease.
• Subjects who are participating in any other clinical study or who have received
treatment with any investigational drug or device within one month prior to
screening.
• Any other condition that in investigational judgement might increase the risk to the
subject or decrease the chance of obtaining satisfactory information needed to
achieve the objectives of the study.
• Subjects who have a known infection with human immunodeficiency virus (HIV) and
or Hepatitis B or Hepatitis C.

Diagnostic Study
• Bone marrow samples were sent to Institute laboratory on that day. The
diagnosis of ALL was based on standard morphologic, immunophenotypic and
genetic analysis.
• Bone marrow aspirate was examined on day 15 (time point1 [TPl]) and day 29
(TP-2) to assess complete remission (CR),defined as no physical signs of
leukaemia, bone marrow with active haematopoiesis and fewer than 5%
leukemic blast cells, normal CSF.
• Patients who failed to achieve CR were assigned to high - risk group; those who
did not achieve CR after phase IB and consolidation will be defined resistant
and counted as treatment failures.

Risk Group
• The high-risk group included patients with any of the following criteria: t (a;ll) or
MLL /AF4; prednisone poor response (> 10^-3) of PCR-MRD at TP2.
• The standard-risk group included patients who lacked high risk criteria and
tested negative by PCR-MRD performed by using two sensitive markers (> 1* l0-4)
at both TPI and TP2.
• The intermediate-risk group included the remaining patients, and those not
evaluated by PCR MRD.

Flow Cytometry- Methods
• Sample collection:
Venepunture
Blood Smears
Bone marrow aspiration
• Staining: Leishman-Giemsa stain
The air dried films are placed on a staining rack and Leishman Strain is poured
over them to cover the film completely. Double volume of giemsa Phosphate buffer
is used and washed in the tap water and dried.

Acceptance Criteria of FCM
• Properlly lebelled specimen
• Specimen collected in EDTA vacutainers as per guidelines and well preserved until
tested
• Specimen submitted alongwith complete request form
• Adequate specimen volume by protocol
• Adequately prepared patient with respect to test requirements
• Specimen to be sent to the lab at correct time

Rejection Criteria
• Data incomplete
• Insufficient quantity
• Specimen collected in wrong container
• Contamination suspected
• Inappropriate transport
• Unknown time delay

Immunophenotyping-Acute Leukemia
Method
• Purpose of the Examination:
In vitro diagnosis of acute leukaemia in human blood and bone marrow by
immunophenotyping.
It is rapid, objective and quantitative method for
1. Lineage assignment
2. Maturation characterization of Blast cell
3. Detection of Clonality
4. Heterogenesity and aberrant feature of malignant cell population

Principle and Method
• The test is based on the ability of specific monoclonal antibody to bind to the
antigenic determinants exposed by leukocytes
• Specific staining of leukocytes is performed by incubating the samples
• The red cell are removed, lysed and analized by FCM
• CD45 staining
• Other scatter plots combining two of the different parameters available on the
cytometer are also used in gating stage
• The cell population thus gated is subdivided into subpopulations using two other
fluorescence
• Thus the positively stained cells are distinguished from unstained cells
• The results finally exposed as a percentage of fluorescent cells in relation to all
the events acquired by gating

Method
• Performance Charecteristics: 8 Compensation files
• Primary Sample: Blood+ PBS/ BM aspiration+ Asp smear
• Type of container: EDTA
• Equipments and Reagent:
• Flow cytometer, Navois software, 8 color analysis dual laser configuration
• Venous bood/BM samples in sterile tube containing an EDTA salt as anticoagulant
• Automatic pipettes with tips to take 5,10,20,100,500 microL
• Plastic haemolysis tube
• Lysing reagent
• Fixation Reagent
• Buffer, Fluorochrome conjugated antibody mixture, automatic agitator

Panel used for marker
• 1. CMPO-FITC/cCD79a-PE/cCd3ECD
• 2. CD20-FITC/cCD10-PE/cCd19ECD
• 3. CD34-FITC/cCD117-PE/cCd45 ECD/CD2 PC5
• 4. CD15 FITC/CD33PE/CD45ECD
• 5. CD14 FITC/CD13 PE/CD45 ECD
• 6. HLADR FITC/CD7 PE/CD45 ECD
• 7. TdT FITC/CD45 ECD (IF CD 34 NEG)
• 8. CD58 FITC/CD 45 ECD (IF BOTH CD34 AND TdT NEG)

Minimal Residual Disease(MRD) in B cell pediatric ALL patient

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