Quality Control in Immunophenotyping

1. Quality Control in Immunophenotyping Dr. N. Varma Prof. & Head – Hematology Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh

2. PGIMER, Chandigarh

3. 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

4. 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.

5. 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.

10. 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.

12. 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.

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. Thank You