Quality Control- What, Why, How, & How Often

1. Quality Control-What, Why, How, & How Often

2. What? • Quality control encompasses • Instrumentation • Reagents • Users • Method(s) used to ensure reproducibility of data over time.

3. Inter/Intra Instrument Contribution • Instrumentation is variable • An instrument varies over time • Alignment drift • PMT/Log Amp degredation • Laser changes • Fluidic problem (salt crystals, debris, microorganisms) • Instruments from different manufacturers vary • Analog/Digital electronics • “Sensitivity” • Filter sets used

4. Reagent Contribution • Lot to Lot variation • Protein:Fluorophore ratio • Tandem conjugate efficiency • Poor QC on manufacturer’s part • Tube variability • Exposure of fluorochrome to light and air • Repeat freeze/thaw or cold/warm cycles • Non Antibody Reagents • Sheath, Buffers, Media, etc…

5. User Contribution • Instrument Parameters • User doesn’t know how to properly use instrument • Drastically changes voltages settings between expts. • Doesn’t understand compensation • Staining Parameters • Hasn’t done proper optimization of staining protocol • Poor “bench skill”

6. Why? • For a Core • Instrumentation must work properly • Providing a service that you charge for • For clinicians • Diagnosis/Prognosis based %-ages and expression

7. Why? • People carelessly report ΔMFI without taking into consideration above variables. • People may abort experiments if “trial” doesn’t work.

8. How? • Beads, of course. • Schwartz, et al1 established a taxonomy of bead standards used for cytometer quality control and quantitative flow cytometry • Alignment Particles (Type I) • Instrument Set-up Particles (Type II) • Quantitative/Calibration Particles (TypeIII)

9. Schwartz A, et al: Standardizing flow cytometry: a classification system of fluorescence standards used for flow cytometry. Cytometry 33:106-14 (1998)

10. Schwartz A, et al: Standardizing flow cytometry: a classification system of fluorescence standards used for flow cytometry. Cytometry 33:106-14 (1998)

11. Instrument Performance • Facility/owner is responsible • Use Type I beads • make sure laser alignment/power is stable • Dichroics and stream are aligned • No partial clogs/obstructions in stream • On the whole, system is performing well • Bead Properties • Uniform in size and intensities (low CVs-use linear scale) • Very bright in all channels and excited by many laser lines • Stable over time (dye contained in bead) • Not effected greatly by environment

12. Instrument Performance & Experimental Control • Facilty and/or User is responsible • Use Type II beads • Ensure compensation electronics are functioning • Establishing “window of analysis” • Setting voltages and compensation (e.g. FACSComp) • Bead Properties • Sizes and intensities vary (use Log scale) • Typically are same size as lymphs • Can be environmentally sensitive, when fluorochrome-matched

13. Instrument Performance & Quantitative Flow • Responsibility of Facility and/or user • Use Type III beads • Measure linearity of Log amps • Dynamic Range of analysis • Detection Threshold or Resolution (“Sensitivity”) • Used for Calibration in Quantitative Flow • Bead Properties • Uses actual fluorochrome on bead instead of dye • Sensitive to environment and handling • Short “shelf-life” • Typically come in a set including particles with several fluorescence intensities.

14. How Often?

15. Case Study • IACF Flow Cytometry Facility at the University of Chicago • Instrumentation • 2 FACSCans – Analog Electronics, 1 laser, 5 detector • 1 FACSCalibur – Analog Electronics, 2 lasers, 6 detectors • 1 LSR II – Digital Electronics, 3 lasers, 10 detectors • 1 FACStar Plus – Analog Electronics, 2 lasers, 7 detectors • 1 Cyan LX – “Digilog” Electronics, 3 lasers, 11 detectors • 1 MoFlo - “Digilog” Electronics, 3 lasers, 11 detectors

16. Cell Sorter QC • Daily and/or Per Experiment Alignment • Includes laser steering, stream, and dichroics • Monthly Bleaching of system • Twice yearly laser alignment- or sooner!

21. Benchtop Analyzers • Weekly alignment check • Flow Check for 488nm • Molecular Probes 633nm beads • Molecular Probes UV beads • SpectrAlign Beads (good for all laser lines)* • Weekly Compensation Check • Calibrite Beads (FITC, PE, PerCP, APC) • Use FACSComp when available • Monthly Bleaching • Twice yearly, laser alignment/dichroic check • Yearly, linearity, sensitivity check, or as needed

22. User Responsibility • Run Reference beads (Type II) if doing longitudinal study • Run Calibration beads (Type III) if doing quantitative analysis • Problem • Do users know they should be doing this type of quality control??? • Probably not

23. Record Keeping • Using Type I beads • Record MFI at specified voltage • Record CV • Chart both over time • Using Type II beads • Run compensation controls (FACSComp) • Set up “acceptable” values for all (before service is called)

25. Discussion Topics • What is the responsibility of the Core Facility/Non-Clinical Lab as far as quality control is concerned? • How do clinical lab protocols differ? • How often should routine alignment QC be performed? Linearity? Sensitivity? • What type of beads are most useful for alignment? • Compensation QC…necessary? • How do you make user’s aware of potential problems with instruments • How do you inform users of the necessity of running Type II or III beads when doing inter-experimental comparisons.

26. References, Further Reading Schwartz A, et al: Standardizing flow cytometry: a classification system of fluorescence standards used for flow cytometry. Cytometry 33:106-14 (1998) Shapiro, H: Practical Flow Cytometry, 4th Edition 353-358. 2003. Gandler W, Shapiro H. Logarithmic Amplifiers. Cytometry 11:447-450 (1990). Purvis N, Stelzer G. Multi-Plateform, Multi-Site Instrumentation and Reagent Standardization. Cytometry 33:156-165 (1998). Whitby L, et al: Quality Control of CD4+ T-Lymphocyte Enumeration. Cytometry (Clinical Cytometry) 50:102-110 (2002). Gratama J.W. Flow Cytometric Quantitation of Immunofluorescence Intensity: Problems and Perspectives. Cytometry 33:166-178 (1998). Bagwell, C.B., A Simple and Rapid Method for Determining the Linearity of a Flow Cytometer Amplification System. Cytometry 10:689-694 (1989) Schmid, I. Conversion of Logarithmic Channel Numbers Into Relative Linear Fluorescence Intesity. Cytometry 9:533-538 (1988).