Assay validation in flow cytometric analysis

Assay validation in flow cytometric analysis

• 1.
  Flow cytometry- Introduction andassay validation Atia Ali Mughal Technologist- Immunology Department of Pathology, PKLI
• 2.
  Contents  Introduction  Principle Components  Analytes, fluorochromes  Data analysis  Applications  Assay validation  Performance assessment  Challenges  Internal QC  External QC
• 3.
  Introduction Flow cytometer isa sophisticated instrument measuring multiple physical characteristics of a single cell passing through fluid stream. It analyzes and differentiates the cells on the basis of:  Size  Granularity/complexity  Fluorescent features derived from dyes/antibodies
• 4.
  Principle  A fluorescentcompound has a range of specific wavelengths at which it absorbs light energy.  This absorption of light causes an electron to rise from a ground state to a higher energy level (excited state).  The excited electron quickly goes back to its ground state while giving the excess energy as a photon of light.  This transition of energy is called fluorescence.  This fluorescence is captured by specific detectors.
• 5.
  Components The main componentsof flow cytometers are:  Fluidics- responsible for directing liquid containing particles to the focused light source  Optics- focuses the light source on the cells/particles while collection optics transmits the light scatter or fluorescent light of the particle to an electronic network  Electronic network- detects the signal and converts it to digital data for analysis
• 6.
  Analytes  Antigens- membrane,cytoplasmic and nuclear  Whole cells and microorganisms  Cellular components- organelles, nuclei, cytokines, hormones and protein content  Nuclear material- DNA, RNA, chromosomes  Cell proliferation  Cell cycle  Calcium flux and membrane potentials
• 7.
  Dyes Used inFlow Cytometry Fluorochrome Ex WL EmWL Application R-Phycoerythrin Green Fluorescent Protein YO-PRO-1 Fluorescein diacetate Alexa488 Sytox Green SNARF-1 Fluo-3 dsRED PE-CY5 (Tricolor, Cychrome) PE-Cy7 Texas Red TO-PRO-3 Alexa 647 APC-Cy7 Allophycocyanin (APC) Phenotyping Reporter molecule Apoptosis Cell viability Pehnotyping DNA analysis pH measurement Calcium Flux Reporter molecule Phenotyping Phenotyping Phenotyping DNA analysis Phenotyping Phenotyping Phenotyping 480 488 488 488 488 488 488 488 596 488 643 488 488 650 647 647 588 578 510 510 530 670 530 770 530 550 530 615 661 667 660 774
• 8.
  Data analysis  Gatesand regions  Single parameter analysis  Two parameter analysis
• 9.
  Applications of flowcytometry  Phenotypic characterization of blood cells  Tumor cell phenotyping  Diagnosis / classification of leukemias and lymphomas  Determination of clonality of Ig-bearing cells  To assess prognosis of cancers  Measurement of apoptosis markers  Cell viability  Detection of plasma membrane changes  Detection of active caspase-3 activity  DNA fragmentation  Intracellular cytokine detection  DNA ploidy  Leukocyte cross-matching in Tx
• 10.
  Assay Validation A widevariety of data outputs can be reported:  Characteristics of cells, or cell subsets  Percentage of positive events  Absolute counts  Median fluorescence intensity  Quantitative antigen expression levels  Ratiometric indices  Markers coexpression  Relative nucleic acid content
• 11.
  Quality assurance  Internalquality control (instrument, assay validity, reagents)  External quality control/proficiency testing  Procedures/records  Inspection/audits  Staff training  Quality improvement
• 12.
  Instrument QC Schedule:  Electronicstandardization (PMT voltages)- daily  Check references through reference beads- daily  Optical alignment through CST beads- daily  Compensation- daily  Sensitivity and Linearity- monthly, after lot change  Multiple instrumentation- bi-annually
• 13.
  Measures of assayvalidation 1. Accuracy  Not applicable for most cellular assays  Unavailability of cellular reference material  CAP, FDA- cleared assays are consensus based 2. Method comparison  Panel designing (CD8 counts, CD4:CD8)  Correlate with other lab results (hematology, viral serology, viral loads) 3. Clinical validation  Necessary when assay serves as a diagnostic test  Medication history (ART), infection history
• 14.
  4. Inter laboratorycomparison  split sample can be used 5. Specificity  Gating strategy  Isotype controls 6. Sensitivity and Linearity  Using reference beads  Serial dilutions, spiking  LOD/ LOB  FMO tube
• 15.
  Reagent QC  Antibodies,Non-antibody reagents  To validate/ verify the reagent reactivity when the lot is changed  Healthy donor samples, cultured cells and commercially available controls can be used  Determine LJs, SD, %CV for percentage of cells, absolute counts and MFIs
• 16.
  External QC (proficiencytesting)  Three times/ year for each area  Accreditation programs  QASI  CAP  UK-NEQAS for immunophenotyping  Validation guidelines  CLSI guidelines  ICSH and ICCS