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 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 blood 2.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 10 7 /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 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

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

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