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Quality Assurance in a Flow Cytometry Lab Dr Kunal Sehgal, M.D Associate Consultant Hematopathology, Department of Laboratory Medicine, P.D.HINDUJA Hospital.

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Presentation on theme: "Quality Assurance in a Flow Cytometry Lab Dr Kunal Sehgal, M.D Associate Consultant Hematopathology, Department of Laboratory Medicine, P.D.HINDUJA Hospital."— Presentation transcript:

1 Quality Assurance in a Flow Cytometry Lab Dr Kunal Sehgal, M.D Associate Consultant Hematopathology, Department of Laboratory Medicine, P.D.HINDUJA Hospital and MRC

2 Diagnosis of Leukemias and Lymphomas Multidisciplinary Approach Adequate Clinical History and Examination Laboratory work up - CBC, ESR, LDH, B 2 microglobulin etc. Radiological Evaluation - PET Scan, CT scan etc. Molecular Studies Morphology - H & E Immunophenotyping - for diagnosis, subtyping, prognosis

3 Quality Assurance in FCM Why should we care? Assurance that flow cytometry results you are generating are due to the properties of the sample and not changes in: Instrument Assay performance Reagents Operator variation

4 How do I achieve good QA in a FCM Lab? Plan It ! Make A SOP and QC manual Implement It ! Systematically follow the procedures laid down by you in your manual Document It ! Prove that you have done what you planned to do

5 PreanalyticalAnalyticalPostanalytical Staff Safety policies Waste Disposal Sample transport Sample storage Sample handling Sample viability Sample Cell Count Reagents Instrument maintenance Internal Quality Controls Pipette calibration Temperature charting Sample processing Acquisition Data templates Analysis Interpretation Result Reporting Data Backup Record retention Clerical error policies EQAS and Proficiency testing Quality improvement program Quality Assurance

6 Flow Cytometry Assay

7 What sample do I need to collect for doing a FCM assay? Clinical History CBC Bone Marrow slides – Air Dried Bone Marrow Biopsy and Imprint smears Sample for Flowcytometry - EDTA/Heparin Sample for Cytogenetics - Heparin

8 Sample Storage Peripheral Blood and Bone Marrow 1.Unprocessed bone marrow and peripheral blood can be stored as is, at room temperature overnight. 2.Suspected Burkitt's Lymphoma should be refrigerated at 2-8°C. Because of an increase in proliferation of these cells, they die quickly. Fluids and Fine Needle Aspirates 1.Samples should be set up the same day, as cells die quickly. 2. Samples can be refrigerated (2-8°C) Storage Media: RNG = RPMI 1640 with 10% Newborn calf serum, glutamine, and Gentamicin.

9 OPERATING TECHNICIAN A basic science graduate is a must One year training/work experience in a busy flow cytometry lab is ideal Exposure to workshops and CME Participation in quality assurance program of the lab is essential

10 Viability Trypan blue Add one part of 0.4% Trypan Blue to one part cell suspension (Example 50ul : 50ul). Incubate for 1-2 minutes. Place one drop (20ul) on hemacytometer. Count one out of a total of 9 large squares on hemacytometer. The viable cells are refractile; dead cells will take up the blue stain. % viability = live cells x 100 (live + dead cells) Notify consultant/supervisor of any specimens with poor viability. Report may indicate that results may be affected by low viability. NCCLS. Clinical Applications of Flow Cytometry: Immunophenotyping of Leukemic Cells; Approved Guideline. NCCLS document H43-A, Bauer, Duke, Shankey, Clinical Flow Cytometry Principles and Application, Williams and Wilkins, Baltimore, 1993.

11 Viability 7AAD CD45 vs. SSC plot and FSC vs. SSC Plot Cell Counters

12 Slide Morphology and Panel Selection It is ideal to make slides from all samples received in the laboratory for a given patient Morphology based panels - –Acute leukemia panel –CLPD panel

13 Antibody Panel Design Weak Antibody - Strong Fluorochrome Strong Antibody - Weak Fluorochrome Weak Antigens - B antigens and Myeloid antigens Strong Antigens - T cell markers Strong Fluorochromes - PE, APC, PE-Cy7 Weak Fluorochromes - FITC, PerCP

14 Choosing a Fluorochrome

15 Reagents Purchase policy Stock maintenance Labeling policy Storage Titration of Antibodies Antibody Cocktails

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17 Titration of Antibodies

18 INSTRUMENT Basic Knowledge about the configuration of the machine is essential It is important to know basic start up and shut down procedures along with routine maintenance processes MAINTENANCE log has to be maintained

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21 Instrument What all is to be monitored – Optics – Fluidics – Electronics How do you monitor – Manual methods – Automated methods QC beads are very useful for monitoring and evaluating instrument performance

22 Flow Cytometry Beads Spherotech Beads Cytometer Setup and Tracking (CS&T) beads Seven colour setup beads Calibrite Beads Flow-Check Fluorospheres IMMUNO-BRITE Standards Kit Flow-Check Pro Fluorospheres Cell Counting Beads Antibody Capture beads

23 Key Performance Factors in High Quality Flow Cytometry Data Relative measured values of fluorescence Linearity and accuracy Resolution of subpopulations, including dim subpopulations Sensitivity Reproducibility of results and cytometer performance Tracking Comparison of results across time and among laboratories Standardization

24 CS&T Functions of the CS&T beads Fully characterize the cytometers performance –Linearity, –Detector efficiency (Qr); –Background fluorescence (Br); –Electronic noise (SDEN); and –laser alignment (rCV) Optimize cytometer settings –Laser delays; –Area scaling factors; –PMT voltages Track cytometer performance and Detect component failures and alterations Provide graphical representations of performance trends over time

25 Know your beads Seven colour setup beads - Instrument monitoring with compensation CS&T beads - Instrument monitoring without compensation

26 SEVEN COLOUR SETUP BEADS REPORT

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28 Why monitoring is important Changes in parameters can indicate cytometer problems –Increases in Electronic Noise (SDEN) bad PMT connections or other electronic problem –Decreases in Detector Efficiency (Q) low laser power, dirty flowcell, alignment or filter issue –Increases Optical Background (B) fluorescent contaminant, failing laser or filter problems

29 Compensation How to do it –Using Cells –Antibody Capture Beads How often to do compensation Is it important to keep a check on the same

30 Optimisation Compensation settings established with beads need to be optimized to the cells used in the experiment. Eg: The 7 colour setup compensation settings may not always be the most accurate for your experiment and will require optimization and fine tuning for the cells used in the experiment

31 Verification and Validation Wikipedia definition Verification and Validation, in engineering or quality management systems, it is the act of reviewing, inspecting or testing, in order to establish and document that a product, service or system meets regulatory or technical standards. Validation – Are you building the right thing? Verification – Are you building it right?

32 Verification and Validation Lot to Lot Antibody VERIFICATION Setting up a new permeabilisation and fixing procedure using home brewed reagents - Assay validation

33 Flowcytometry Assay Verification Confirm specifications established by the manufacturer Accuracy Precision Reportable range of results Verify of manufacturers normal range Validation In-house, home brewed assay Accuracy Precision Analytical sensitivity Analytical specificity Reportable range Reference range Calibration procedures Control procedures Sample stability

34 Sample processing Stain lyse wash Lyse wash stain Ficoll Hypaque

35 Cell counting Required Concentration: x 10 6 cells/ tube Eg: If your target is 0.5 million (0.5 x 10 6 ) cells per tube (per 100ul sample volume) 0.5 x 10 6 / 100ul (x10) = 5 x 10 6 / 1000ul = 5 x 10 6 / 1 ml You require a concentration of 5 x10 6 /ml or a CBC count of 5 x 10 3 /ul ( 5000/cmm) In clinical scenario, Antibody staining is volume dependent and time dependent Target (100ul/ tube) 0.1 x 10 6 cells 1 x 10 6 cells Per ml 1 x 10 6 cells/ml 10 x 10 6 cells/ml CBC Count 1x 10 3 /ul10x10 3 /ul

36 Stain-Lyse-Wash 100 ul of whole blood (0.1-1 million cells)+ Antibodies Vortex Incubate at RT in the dark for minutes. 2.0 ml Lysing Reagent- vortex- let sit in dark for 6-10 minutes at RT Vortex and centrifuge at 1400 rpm for 5 minutes. Carefully discard supernatant from cell pellet and vortex pellet Wash with 2 ml of PBS-BSA. Centrifuge at 1400 rpm for 5 min. Carefully aspirate supernatant and resuspend pellet in 0.5 ml of PBS-BSA and vortex.

37 Acquisition and Analysis –No of events to acquire –Gating Strategies –Provision for checking internal controls using normal cells –Doublet exclusion is ideal –Ideal to have all possible permutations and combination plots –Fixed templates help in reproducibility and consistency of data –Data backup

38 FCM Reporting Consolidated Report –Clinical History in short –Morphology findings if Available –FCM data –Cytogenetic and Molecular correlations –Advise

39 FCM Report Format

40 Signatory Authority MD/DNB/DM Pathology or Hematology Six months – one year training in a busy hematology-flow cytometry lab is ideal Exposure to workshops and CME Participation in quality assurance program of the lab is essential

41 Quality Improvement Indicators TAT of FCM reports Number of samples requiring Repeat processing Number of samples requiring additional markers and evaluation of these markers Error reporting

42 Proficiency Testing Participation in any PT program is ideal and valuable Inter lab Comparison program - TMH CAP RCPA Wet samples or Dry challenges

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