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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.


Compensation
Compensation

  • 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 compensation is routinely done using appropriate peripheral blood/bone marrow aspirate samples to obtain single-stained cells as compensation control.

  • 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: compensation is routinely done using appropriate peripheral blood/bone marrow aspirate samples to obtain single-stained cells as compensation control.

  • 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 compensation is routinely done using appropriate peripheral blood/bone marrow aspirate samples to obtain single-stained cells as compensation control.

  • “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 compensation is routinely done using appropriate peripheral blood/bone marrow aspirate samples to obtain single-stained cells as compensation control.

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.. compensation is routinely done using appropriate peripheral blood/bone marrow aspirate samples to obtain single-stained cells as compensation control.

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 compensation is routinely done using appropriate peripheral blood/bone marrow aspirate samples to obtain single-stained cells as compensation control.

  • 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


Thank You compensation is routinely done using appropriate peripheral blood/bone marrow aspirate samples to obtain single-stained cells as compensation control.


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