STANDARD OPERATING PROCEDURE

B-ALL analysis on BD FACSCanto II

Issue Date: Aug 2021
SOP03.V.Fl
ZUHL

Zagazig University Hospitals laboratories

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 STANDARD OPERATING PROCEDURE
B-ALL analysis on BD FACSCanto II
Issue Date: Aug 2021
SOP03.V.Fl
ZUHL

Table of modifications

No. Date Item/Page No. Text Cancelled in brief Requester

Principle. When whole blood is added to the reagent (labeled

monoclonal antibodies) the reagent binds specifically to
specific cell antigens. The stained samples are treated with
FACS Lysing Solution to lyse erythrocytes. During acquisition,
the cells travel past the laser beam and scatter the laser light.
The stained cells fluoresce. These scatter and fluorescence
signals, detected by the instrument, provide information about
the cells’ sizes, internal complexity, and relative fluorescence

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 STANDARD OPERATING PROCEDURE
B-ALL analysis on BD FACSCanto II
Issue Date: Aug 2021
SOP03.V.Fl
ZUHL
intensity. Reagents employ fluorescence triggering, allowing
direct fluorescence gating of the cell population to reduce
contamination by unlysed or nucleated red blood cells in the
gate. This allows analysis of a relatively cleaner population of
cells.
Clinical significance of the The significant of this test is to:
test • Identification of a cell lineage e.g. B cells, T cells,
myeloid cells, etc.
• Identification of immature and mature cells e.g. blasts,
lymphocytes, granulocytes etc.
• Differentiation of normal versus abnormal cells e.g.
hematogones vs leukemic blasts
Identification of all major cell population in the given
specimen.
Patient Preparation Patient should not receive drugs that may alter the cell shape
and surface markers expression as corticosteroid or
chemotherapy
Specimen Type Whole blood samples (PB or BM)
Sample Precautions:
PB or BM is collected on EDTA anticoagulant (2ml is
sufficient for this assay).
Vacutainers or other collection tubes should be filled properly
to account for the amount of anticoagulant.
Collection and Storage PB or BM is collected in EDTA anticoagulant (2ml is sufficient
for this assay).
Vacutainers or other collection tubes should be filled properly
to account for the amount of anticoagulant.
All specimens should be regarded as potentially infectious;
universal precautions for blood collection and handling should
be properly followed.

Stability of the samples:

Specimens should be maintained at room temperature (18°C–
25°C) during transportation and storage.
Testing must be performed within 24 hours from the time of
collection.
Performance PRECISION
Characteristics Refer to Method Performance Log record
Method Comparison
Refer To Method Performance Log Record
Uncertainty Refer To Method Performance Log Record/ Results Of
Uncertainty Record
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 STANDARD OPERATING PROCEDURE
B-ALL analysis on BD FACSCanto II
Issue Date: Aug 2021
SOP03.V.Fl
ZUHL
Criteria for acceptability Acceptable criteria:
and rejection of the  EDTA is the most widely used anticoagulant
sample.  Heparin and ACD have also been used
 Sample age: Up to 24h
 Rejection criteria:
 Inadequate sample volume
 Samples more than 24h old
 Samples with hemolysis
Coagulated samples
Reagents and equipment Monoclonal antibodies Labeled with Fluorochromes ( BD
used Biosciences):
o cyMPO FITC/ cyCD79a PE/ CD34 PerCP-cy5.5/ CD19 PE-CY7/
CD7 APC/ CD3 APC-H7/ cyCD3 V-450/ CD45 V-500
o TDT FITC/ CD13 PE/ CD34 PerCP- cy5.5 / CD19 PE-CY7/ CD10
APC/ CD20 V-450/ CD45 V-500
o CD22 FITC/ CD33 PE/ CD34 PerCP- cy5.5/ CD19 PE-CY7/
CD10 APC/ HLA-DR V-450/ CD45 V-500
o Anti-Kappa FITC/ Anti-Lambda PE/ CD34 PerCP- cy5.5 /
CD117 PE-CY7/ SIgM APC/ CD20 V-450/ CD45 V-500(when
needed)
• FACS Lysing solution 10X (BD Biosciences)
• COMB Beads (BD Biosciences) for calibration
• BD FACSFlow Sheath fluid, (BD Biosciences)
Reagent-grade (distilled or deionized) water.
Warnings and Precautions
For in-vitro diagnostic use.
Storage and Stability:
Keep at 2-8°C in the dark until use
Do not freeze

Test procedure Whole blood surface staining

• According to the ALL panel used; the first tube (ALOT) has
been prepared, and on the basis of the result, the rest of
the tubes are prepared.
• 100µl of PB or BM was added in labeled tube.
• 10 µl from each Monoclonal antibody were added mix
gently and incubated for 20min
• a ratio of 1:10 of sample to lysing solution is prepared
• add 0.5 ml of the lysing solution in a each tube to lyse
RBCs.
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 STANDARD OPERATING PROCEDURE
B-ALL analysis on BD FACSCanto II
Issue Date: Aug 2021
SOP03.V.Fl
ZUHL
• Incubate for 6–8 minutes at room temperature.
• Centrifuge for 2 minutes at 400xg at room temperature to
remove red cell debris.
• Discard the supernatant.
• Gently tap the pellet to re-suspend the cells and add 2 ml
of PBS.
• Wash by centrifuging for 2 minutes at 400xg at room
temperature to remove the left over lysing solution.
• Discard the supernatant and re-suspend in PBS to get the
• Final cell suspension.
NB: A given panel may have four to six tubes per panel with
each tube containing 2 to 8 antibodies (colors) based on
cytometer configuration. One can add individual
antibodies or pre-prepared antibody cocktails based on
the sample work-load and the laboratory policy. It is
highly recommended to use pre-prepared antibody
cocktails to avoid the pipetting errors.
For cytoplasmic and nuclear staining:
Cells are fixed and permeabilized before antibody staining
and such reagents may be prepared in-house or procured
commercially.

Acquisition and Analysis:

 Make sure you have filtered and vortexed your samples
well.
 Analysis was done using FACS Diva software
 Create a folder with patient name.
 Create a new experiment or import a previous
experiment template.

 Draw all needed graphs and prepare the worksheet.

 Place your sample tube on the SIT arm.
 Activate the tube in Diva software and press “Acquire”.
Maximum event count is 20'000 events.
 Adjust the voltage for FSC & SSC to observe your cells on scale.
Set your gates and the cell number you would like to measure and
press “Record”. Use the “low” speed to avoid big CVs.

Interpretation • It should study adequate markers to establish the

diagnostic and prognostic classification of hematological
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 STANDARD OPERATING PROCEDURE
B-ALL analysis on BD FACSCanto II
Issue Date: Aug 2021
SOP03.V.Fl
ZUHL
neoplasm or document the absence of the disease.
• Markers that are differentially expressed with
maturation within a particular lineage should be combined to
identify synchronous maturational patterns of antigen
expression. Abnormalities in the patterns of maturation are
common and are extremely useful for the recognition of
hematopoietic neoplasms.

For example in the B cell lineage, the combination of CD10,

CD20 and CD45 is useful to follow early B cell maturation
pattern.

• Before releasing patient results check that the assay

control values are within the expected ranges.
• Patient results should not be released until verified by
the laboratory supervisor. Submit both the completed
requisition form and the physician report printout for each
patient sample to the physician.
• CD19, cCD22, CD79a and HLA-DR almost always
positive. TDT usually positive. CD45 may be weakly
expressed or negative
• Characteristics marker for pro B ALL is CD10 negative
• Characteristics marker for Common B ALL is CD10
positive
• Characteristics marker for pre B ALL is Cyt μ+,
• Characteristics marker for mature B ALL is SmIg+

• Standard Safety precautions for handling blood samples.

• Wear disposable gloves and lab coat.
• The lasers in the FACSCanto II emit laser beams which can
Safety be harmful to the eyes. Avoid contact with naked eyes
when the top cover of the FACSCanto II is opened.

Results Reporting
• The report should include gating strategies and percentage of
gated cells examined.
• It should also include descriptive information about the
immunophenotype of the abnormal cells, if identified, and
comments necessary to facilitate the interpretation.
• Stress should be laid on interpreting the intensity of positivity and
not only the percentages. While interpreting the intensity of
positivity as moderate, bright or dim, the abnormal population
should be evaluated against known normal leukocyte populations.
• Final impression should be clearly stated along with a differential
diagnosis if required.
• Comments and suggestions regarding useful follow up test or

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 STANDARD OPERATING PROCEDURE
B-ALL analysis on BD FACSCanto II
Issue Date: Aug 2021
SOP03.V.Fl
ZUHL
other ancillary techniques should be added.
It is done by using Comp beads (BD) to optimize cytometer set up
and successful results are saved on Diva software
Control: By running CS&T daily. The correct results are verified
Calibration and Control
Procedures

Corrective Actions Repeat the test system’s calibration procedure. If after recalibration
there are still issues, the laboratory must take corrective action and
document this activity
Biological Reference Results of this test should always be interpreted in conjunction with
Intervals/Clinical the patient’s medical history, clinical presentation and other
Decision Values. findings
Limitations In • presence of autofluorescent events
Methodologies • Analysis is limited by factors such as hypocellularity and poor cell
Including Interfering viability due to necrosis or improper specimen collection and
Substances. handling.
• Flow cytometric results cannot be used alone to diagnose
malignancy. Results of this test should always be interpreted in
conjunction with Morphology, the patient’s medical history,
clinical presentation and other ancillary tests for a definitive
diagnosis.
Reference Interval 20%; if at least 50 000 events was collected revealed abnormal
lineage expression; 10% if collected cells expressed immature marker
revealed presence of blast cells
Interference, • Flow cytometric analysis is limited by factors such as
Cross Reactions hypocellularity and poor cell viability due to necrosis or improper
And Source Of specimen collection and handling.
Variations • Flow cytometric results cannot be used alone to diagnose
malignancy. Results of this test should always be interpreted in
conjunction with Morphology, the patient’s medical history,
clinical presentation and other ancillary tests for a definitive
diagnosis.

References 1) Clinical flow cytometric analysis of neoplastic

hematolymphoid cells, approved guideline- sections 6 –9, Clinical
lab Med 27 (2007) 607 – 707
2) Clinical and Laboratory Standards Institute. Clinical Flow
cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved
Guideline—Second Edition. CLSI document H43-A2 Clinical and
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 STANDARD OPERATING PROCEDURE
B-ALL analysis on BD FACSCanto II
Issue Date: Aug 2021
SOP03.V.Fl
ZUHL
Laboratory Standards Institute, 940 West Valley
1) Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA,
2006.
2) Infection Control and Hospital Epidemiology. 1996;
17(1):5380), MMWR 1987; 36 [suppl 2S] 2S-18S), and (MMWR
1988; 37:377-382, 387-388).
3) Clinical Laboratory Medicine From beginning to end, the most
complete, current, and clinically oriented information in the field.
Source: Lippincott Williams & Wilkins (LWW) Author(s):
McClatchey, Kenneth D.; Alkhan, Serhan; Hackel, Emmanuel;
Keren, David F.; Lewandrowski, Kent; Lee-Lewandrowski,
Elizabeth; Oberman, Harold A.; Triplett, Douglas A.; Van Dyke,
Daniel L.; Ward, Patrick; Woods, Gail L. 27 (2007) 607 – 707.
4) Vos JA, Simurdak JH, Davis BJ, Myers JB, Brissette MD. Vortex
disaggregation for flow cytometry allows direct histologic
correlation: a novel approach for small biopsies and inaspirable
bone marrows. Cytometry. 2003; 52B(1):20-31.
5) Carter PH, Resto-Ruiz S, Washington GC, et al. Flow
cytometric analysis of whole blood lysis, three anticoagulants, and
five cell preparations. Cytometry. 1992; 13(1):68-74.
6) Brunialti MK, Kallás EG, Freudenberg M, Galanos C, Salomao
R. Influence of EDTA and heparin on lipopolysaccharide binding
and cell activation, evaluated at single-cell level in whole blood.
Cytometry (Clinical Cytometry) 50:14–18 (2002).
7) Carter PH, Resto-Ruiz S, Washington GC et al. Flow cytometric
analysis of whole blood lysis, three anticoagulants, and five cell
preparations. Cytometry. 1992; 13(1):68-74.
8) Standardization of flow cytometry in myelodysplastic
syndromes: report from the first European Leukemia Networking
conference on flow cytometry in myelodysplastic syndromes.
Haematologica 2009; 94:1124-1134.
9) Optimal cellular preservation for high dimensional flow
cytometric analysis of multicentre trials. Journal of Immunological
Methods 385 (2012) 79–89.
10) Laboratory Guidelines for enumerating CD4 T Lymphocytes in
the context of HIV/AIDS. WHO Regional Office for South- East Asia,
New Delhi, June 2007.

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 STANDARD OPERATING PROCEDURE
B-ALL analysis on BD FACSCanto II
Related Pre-examination Procedure (PD01.V.T)
Issue Date: Aug 2021
Documents Process Control process (PR01.V.T)
SOP03.V.Fl
Work instruction BDFACSCANTO II (WI 02.III.T)
ZUHL
Post Examination, Reporting and (PD04.V.T)
Release Results (PD02.V.T)
Examination procedure (PD03.V.T)
Ensuring Quality of Examination (ZUHL.LM/
Results ZUHL.LM/ar )
Laboratory Service Manual (ED42.Q)
Material Safety Data Sheet
Related Records Method Performance Log record (F03PD02.V.T)
Method Evaluation record (F01PD02.V.T)
Results of uncertainty record (F02PD02.V.T)

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