Quality Control in Immunophenotyping

Dr. N. VarmaProf. & 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

• High quality flow cytometry is intrinsically linked to:– Instrument set up– Good sample handling and preparation

• 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 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.

Instrument set up..

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

• The flow cytometry laboratory in department of Hematology, PGIMER, mostly receives 2 kinds of samples:1. Peripheral venous blood2. Bone marrow aspirate

• Immunophenotyping is carried out on: – fresh samples or – within 24 hours of collection

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

• “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

Antibody titration:• Titration is carried out for every new lot to obtain

optimum staining volume of each antibodyIsotype control:• Isotype specific antibodies were routinely used earlier to

measure non-specific binding of antibody• Presently, used along with intracytoplasmic markersAutofluorescence control:• Unstained cells are routinely used along with each

sample to measure autofluorescence in each channel

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

Experiment associated controls..

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

Thank You