Upload
hall
View
63
Download
1
Tags:
Embed Size (px)
DESCRIPTION
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 - PowerPoint PPT Presentation
Citation preview
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