Required section · Section 4 of 6

Work the gate tree

Start with 50,000 acquired events. Exclude debris to retain 44,000 events, then retain 41,800 singlets and 37,620 viable singlets. The hierarchy removes events that do not answer the intended viable-cell question. Each remaining percentage has a different denominator.

Within viable singlets, the CD45-bright lymphocyte gate contains 12,038 events. CD3-positive events are 8,426, or 70.0% of that lymphocyte gate. CD19-positive events are 2,286, or 19.0% of that same gate. The residual 1,326 lymphocyte events are 11.0% and are not a clinical interpretation.

Back-gate the final CD19-positive selection onto preceding plots to check that it maps plausibly to the viable-singlet lymphocyte population. A stable time plot supports acquisition review but does not prove biology. Internal normal populations may support relative staining patterns within a specimen, not a universal antigen-intensity cutoff. Back-gate a consequential final population before releasing a bounded result.

A sequential diagram shows 50,000 acquired events becoming 44,000 nondebris, 41,800 singlets, 37,620 viable singlets, then 12,038 CD45-bright lymphocytes and 2,286 CD19-positive events, with a back-gating plot. — Figure 1The gate hierarchy includes the 12,038-event CD45-bright lymphocyte parent gate before the final CD19-positive population and supports back-gating review.

A line chart with a 0 to 50,000 event axis shows acquired events, debris exclusion, singlets, viable singlets, 12,038 CD45-bright lymphocytes, and the final 2,286 CD19-positive events. — Figure 2Case event counts decline through the gate hierarchy, including 12,038 CD45-bright lymphocytes before the final 2,286 CD19-positive events.

Sequential event counts and stated parent populations
Gate	Events	Percent of named parent	Review context
Acquired	50,000	100.0% of acquired	Minimum event target is assay-specific
Debris-excluded	44,000	88.0% of acquired	Gate location is method dependent
Singlets	41,800	95.0% of debris-excluded	Pulse boundary is validated locally
Viable singlets	37,620	90.0% of singlets	No universal viability cutoff
CD45-bright lymphocytes	12,038	32.0% of viable singlets	Scatter and CD45 pattern are method dependent
CD19-positive	2,286	19.0% of lymphocytes	FMO supports the dim boundary

Knowledge checks

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Knowledge check 1

What is the CD19-positive percentage within the stated CD45-bright lymphocyte gate?

Choose one option.

19.0%, calculated as 2,286 divided by 12,038.

4.6%, calculated as 2,286 divided by 50,000.

6.1%, calculated as 2,286 divided by 37,620.

Knowledge check 2

What does back-gating the final CD19-positive selection primarily check?

Choose one option.

Whether the final selection maps plausibly to the preceding viable-singlet lymphocyte population.

Whether the selection proves a diagnosis.

Whether a universal CD19 intensity cutoff has been met.

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