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Quality Control in Immunophenotyping. Dr. N. Varma Prof. & Head – Hematology Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh. PGIMER, Chandigarh. Quality Control. Department of Hematology, PGIMER: BD FACSCanto II with blue and red lasers

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quality control in immunophenotyping

Quality Control in Immunophenotyping

Dr. N. Varma

Prof. & Head – Hematology

Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh

quality control
Quality Control
  • Department of Hematology, PGIMER: BD FACSCanto II with blue and red lasers
  • High quality flow cytometry is intrinsically linked to:
    • Instrument set up
    • Good sample handling and preparation
instrument set up
Instrument set up
  • BD Cytometer Setup and Tracking (CST) beads define baseline performance of the cytometer (at the time of installation) by measuring median fluorescence intensity (MFI) and percent robust CV (% rCV) for each bead type.
  • Software evaluates for linearity, detector efficiency (Qr), optical background (Br), electronic noise, and laser delays.
  • PMT voltages are then adjusted to maximize population resolution in each detector.
instrument set up1
Instrument set up..
  • CST beads are run on daily basis to track cytometer performance and measure variation from baseline measurements. Laser delays, area scaling factors and PMT voltages are adjusted.
  • Baseline and performance check reports are automatically created.
  • Performance check values plotted on Levy-Jenning charts allows tracking of cytometer performance and noting spot trends.
  • Multicolor immunophenotyping on leukemia cases cannot be carried out without correction of spectral overlap.
  • CST beads do not allow correction of such spill over.
  • BD FACS 7-color set up beads are used for automatic compensation besides adjusting voltages.
However, these are not used on regular basis and compensation is routinely done using appropriate peripheral blood/bone marrow aspirate samples to obtain single-stained cells as compensation control.
  • Capture beads can be used when an antigen is not commonly present on normal cells. These beads can be stained with antibodies much like cells and provide a bright, uniform signal for each antobody.
sample handling
Sample Handling
  • The flow cytometry laboratory in department of Hematology, PGIMER, mostly receives 2 kinds of samples:
    • Peripheral venous blood
    • Bone marrow aspirate
  • Immunophenotyping is carried out on:
    • fresh samples or
    • within 24 hours of collection
appropriate information along with the sample
Appropriate information along with the sample:
  • Patient identification (name, age, gender, registration number)
  • Type of sample
  • Date of sample collection
  • Presumptive diagnosis
  • Test ordered
  • Name of the physician
  • Recent treatment and medications
sample preparation
Sample Preparation
  • “Lyse-stain-wash” protocol is the preferred methodology for all routine samples, lyses being carried out with in-house ammonium chloride lysing solution
  • This validated procedure is suitable to obtain suspension of cells of interest (eliminating erythrocytes), at a concentration optimal for monoclonal antibody staining (0.5-1 x 107/ml)
  • Cell count and viability check (trypan blue dye exclusion) is routinely done
experiment associated controls
Experiment associated controls

Antibody titration:

  • Titration is carried out for every new lot to obtain optimum staining volume of each antibody

Isotype control:

  • Isotype specific antibodies were routinely used earlier to measure non-specific binding of antibody
  • Presently, used along with intracytoplasmic markers

Autofluorescence control:

  • Unstained cells are routinely used along with each sample to measure autofluorescence in each channel
experiment associated controls1
Experiment associated controls..

Flourescence minus one (FMO) control:

  • It provides means to measure effects of spill over from populations in other dye dimensions on a particular channel of interest
  • FMO control experiment is performed whenever there is an attempt to develop a new multicolor panel
projects future collaborations
Projects & Future Collaborations
  • Projects:
    • Immunophenotypic correlates of CG/MG subgroups: AML & B lineage ALL
    • MPAL by EGIL and WHO criteria
    • MRD in ALL by FCM and RQ-PCR
    • FCM for BCR-ABL proteins in CML
  • Future Collaborations:
    • FCM in diagnosis of CLL: CD 38 and Zap-70
    • FCM in diagnosis of MM
    • MRD in ALL