International Cytometry Certification
Examination Content Outline

I. Instrumentation (20%)

  1. Fluidics (5%)
    1. Hydrodynamic focusing and properties of sheath fluids
    2. Generation of differential pressures (e.g., syringe pump, pressure based)
  2. Optics (9%)
    1. Optical filters (e.g., long pass, band pass, short pass, dichroics, neutral density, polarizing)
    2. Light source (e.g., laser type, laser line, arc lamp, led)
    3. Lenses (e.g., beam shape, collecting, focusing, objective)
    4. Optical pathway (e.g., transmission, reflection, interrogation point, collinear, spatial separation, light scatter)
  3. Electronics (6%)
    1. Amplifiers (e.g., Linear, Logarithmic)
    2. Detectors (e.g., photomultiplier tube, photodiode, CCD camera, avalanche photon detector)
    3. Digital vs. analog systems
    4. Noise
    5. Pulse measurement (e.g., time delay, window extension, area, width, Coulter impedance)
    6. Threshold/discriminator

II. Sample (10%)

  1. Sample source (e.g., beads, microspheres, solid tissue, body fluids, subcellular components, cultured cells, microorganisms, plants, whole organisms)
  2. Sample integrity (e.g., collection, handling, storage viability)
  3. Sample preparation and staining (e.g., disaggregation, lysing agents, aggregates, filtering, fixation, permeabilization)
  4. Cell enrichment (e.g., cell sorting, density gradient isolation, magnetic beads)

III. Data (19%)

  1. Data standards (e.g., image file format, FCS format, metadata, multichannel data, XML, storage requirements)
  2. Signal processing (e.g., binning, compensation, pulse processing, baseline restoration, background correction)
  3. Data display (e.g., types of displays, transformations)
  4. Gating (e.g., hierarchical vs. Boolean gating, gates, regions)
  5. Statistical methods (e.g., central tendency, standard deviation, CV, KS statistics, cluster analysis, principal component analysis, discriminant analysis)
  6. Common data modeling techniques (e.g., DNA ploidy, cell cycle analysis, proliferation, phenotyping, ratiometric)
  7. Quantitative cytometry (e.g., molecules of equivalent soluble fluorochrome [MESF], absolute counts)

IV. Safety (3%)

  1. Biosafety procedures (e.g., biosafety categories, Personal Protective Equipment, specimen transport and preparation precautions, aerosols, decontamination)
  2. Instrument safety (e.g., lasers, electronics)
  3. Chemical safety (e.g., mutagenic agents, cytotoxic agents)
  4. Environmental safety (e.g., waste disposal)

V. Quality Control (10%)

  1. Instrument quality control (e.g., optical alignment, detector calibration)
  2. Reagent quality control (e.g., panel verification, titration, lot to lot variation, storage, handling)
  3. Sample integrity
  4. Appropriate sample quality controls selection (internal, external)
  5. Trend analysis and interpretation

VI. Experimental Design (19%)

  1. Assay Development (6%)
    1. Sample state (e.g., activated, resting, proliferating)
    2. Target (e.g., cell type, subcellular location, molecule)
    3. Assay interpretation (e.g., isotype control, autofluorescence, biological systems control, background measurement controls)
    4. Assay optimization (e.g., appropriate use of limited sample, frequency of target, cell seeding, kinetics, scalability, blocking, signal to noise, statistical design, Z factor)
  2. Reagent Selection (10%)
    1. Fluorochrome issues (e.g., antigen density, protein coexpression, optimal combination, photostability, F/P ratio, spectral overlap and compensation, quenching)
    2. Probe types (e.g., antibodies, viability/DNA dyes, physiological, tracking, fluorescent proteins)
    3. Solutions (e.g., buffers, fixatives, chelators, permeabilizing agents)
  3. Assay Validation (3%)
    1. Method validation (e.g., accuracy, reproducibility/precision, sensitivity, specificity, linearity, reference range, robustness)
    2. Method calibration (e.g., standards, controls)

VII. Theoretical Principles (19%)

  1. Physical Principles (13%)
    1. Properties of light (e.g., refraction, diffraction, polarization, scatter)
    2. Fluorescence (e.g., Stokes shift, excitation and emission, energy transfer, environmental sensitivity [such as pH, polarity, calcium])
    3. Optics (e.g., optical filters, image formation, focal plane, numerical aperture)
    4. Electronics (e.g., signal detection, amplification, processing)
    5. Fluid dynamics (e.g., laminar flow, stream width, turbulence)
    6. Cell sorting (e.g., Jet-in-air, cuvette, droplet formation, drop delay, drop deflection, fanning, charging)
  2. Biological Principles (6%)
    1. Antigen/antibody interaction and antibody structure
    2. Fluorescent proteins structure and properties
    3. Optical properties of cells