SCYM Quality Control & Assurance 5 — Questions and Answers
Question 1: A flow cytometer's photomultiplier tube (PMT) voltage is adjusted upward. The primary effect on QC bead data will be:
- A shift of bead populations to higher MFI values (Correct answer)
- A decrease in the CV of the bead populations
- Reduced autofluorescence from cellular samples
- Narrower spectral spillover into adjacent channels
Correct answer: A shift of bead populations to higher MFI values
Increasing PMT voltage amplifies the signal, shifting populations to higher MFI without changing the coefficient of variation proportionally.
Question 2: Which of the following scenarios represents a random error in flow cytometry QC?
- A single QC bead result that is high, followed by results within normal range (Correct answer)
- Consecutive QC results that trend steadily upward over five days
- All QC results for one channel consistently falling below the mean
- A sudden permanent shift to lower MFI after a reagent lot change
Correct answer: A single QC bead result that is high, followed by results within normal range
Random errors produce isolated, non-directional excursions that return to expected range, unlike systematic errors that cause trends or shifts.
Question 3: The purpose of including a viability dye in a cytometry staining protocol relates to QC because:
- It excludes dead cells that non-specifically bind antibodies and inflate false-positive signals (Correct answer)
- It calibrates the forward scatter threshold setting
- It standardizes the staining time for all antibodies
- It eliminates the need for an FMO control
Correct answer: It excludes dead cells that non-specifically bind antibodies and inflate false-positive signals
Dead cells non-specifically bind antibodies, so excluding them with a viability dye improves staining specificity and data quality.
Question 4: Which of the following defines 'precision' as validated in a clinical flow cytometry assay?
- The closeness of repeated measurements of the same sample under defined conditions (Correct answer)
- The agreement of the assay result with the true analyte value
- The lowest detectable concentration of the analyte
- The ratio of true positives to all positive calls
Correct answer: The closeness of repeated measurements of the same sample under defined conditions
Precision (reproducibility) measures how consistently an assay returns the same result when the same sample is tested multiple times.
Question 5: A laboratory discovers that two operators consistently report different CD34+ stem cell percentages on split aliquots. This most likely reflects a problem with:
- Inter-operator variability in gating strategy (Correct answer)
- Instrument laser misalignment
- Inadequate PMT voltage for CD34 detection
- Sample anticoagulant type
Correct answer: Inter-operator variability in gating strategy
Inconsistent results between operators on the same sample typically indicate non-standardized gating strategies requiring harmonization.
Question 6: In flow cytometry, 'accuracy' of a clinical assay is most rigorously established by:
- Comparing results to a certified reference method or reference material with a known value (Correct answer)
- Achieving a low CV across repeated bead measurements
- Optimizing antibody concentrations by titration
- Using the same operator for all patient samples
Correct answer: Comparing results to a certified reference method or reference material with a known value
Accuracy is confirmed by demonstrating agreement between the assay result and a certified reference value or method.
Question 7: Which quality indicator best monitors the pre-analytical phase of a clinical flow cytometry workflow?
- Time from sample collection to staining initiation (sample age) (Correct answer)
- PMT voltage stability across the run
- Number of events acquired per sample
- Degree of spectral overlap in the panel
Correct answer: Time from sample collection to staining initiation (sample age)
Sample age (time from collection to processing) is a key pre-analytical variable that affects cell viability and antigen expression integrity.
SCYM Quality Control & Assurance 5
A flow cytometer's photomultiplier tube (PMT) voltage is adjusted upward.
The primary effect on QC bead data will be: