Quality Control- What, Why, How,

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

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

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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

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Reagent Contribution

• Lot to Lot variation
• ProteinFluorophore ratio
• Tandem conjugate efficiency
• Poor QC on manufacturers part
• Tube variability
• Exposure of fluorochrome to light and air
• Repeat freeze/thaw or cold/warm cycles
• Non Antibody Reagents
• Sheath, Buffers, Media, etc

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User Contribution

• Instrument Parameters
• User doesnt know how to properly use instrument
• Drastically changes voltages settings between
  expts.
• Doesnt understand compensation
• Staining Parameters
• Hasnt done proper optimization of staining
  protocol
• Poor bench skill

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Why?

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

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Why?

• People carelessly report ?MFI without taking into
  consideration above variables.
• People may abort experiments if trial doesnt
  work.

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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)

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Schwartz A, et al Standardizing flow cytometry
a classification system of fluorescence standards
used for flow cytometry. Cytometry 33106-14
(1998)
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Schwartz A, et al Standardizing flow cytometry
a classification system of fluorescence standards
used for flow cytometry. Cytometry 33106-14
(1998)
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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

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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

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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.

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How Often?
6 months or more Monthly Weekly Daily Per Experiment
Change sheath filter Bleach system Check alignment Check alignment Check alignment?
Optimize Laser/Dichroics on analyzers Check tubing/connectors Assess compensation issues Assess comp issues? Run Reference Beads?
Check Linearity Inspect instrument Run Calibration Beads?
Sensitivity

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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

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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!

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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

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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

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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)

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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 QCnecessary?
• How do you make users 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.

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References, Further Reading

• Schwartz A, et al Standardizing flow cytometry
  a classification system of fluorescence standards
  used for flow cytometry. Cytometry 33106-14
  (1998)
• Shapiro, H Practical Flow Cytometry, 4th
  Edition 353-358. 2003.
• Gandler W, Shapiro H. Logarithmic Amplifiers.
  Cytometry 11447-450 (1990).
• Purvis N, Stelzer G. Multi-Plateform, Multi-Site
  Instrumentation and Reagent Standardization.
  Cytometry 33156-165 (1998).
• Whitby L, et al Quality Control of CD4
  T-Lymphocyte Enumeration. Cytometry (Clinical
  Cytometry) 50102-110 (2002).
• Gratama J.W. Flow Cytometric Quantitation of
  Immunofluorescence Intensity Problems and
  Perspectives. Cytometry 33166-178 (1998).
• Bagwell, C.B., A Simple and Rapid Method for
  Determining the Linearity of a Flow Cytometer
  Amplification System. Cytometry 10689-694
  (1989)
• Schmid, I. Conversion of Logarithmic Channel
  Numbers Into Relative Linear Fluorescence
  Intesity. Cytometry 9533-538 (1988).