Question 8 of 20 Fundamentals Practical

Why do you exclude doublets in flow cytometry, and how do you gate them out?

Short answer

Two cells stuck together pass the laser as one event and combine their signals, which can create false double-positive cells or a false G2 peak. Exclude them by plotting a pulse's area against its height or width, where single cells fall on a tight diagonal and doublets have greater width for their height.

Why

The instrument records each passage through the laser as a pulse with a height, a width and an area. A single cell gives a pulse whose area and height rise together. Two cells in a row give a longer pulse, so width and area increase while height increases much less.

Plotting forward scatter area against height, and often side scatter as well, puts singlets on a diagonal and doublets off it. Doublets matter because they fake biology. A T cell stuck to a B cell appears to express CD3 and CD19 at once.

In DNA content analysis, two G1 cells together carry the same DNA as one G2 cell. The Cossarizza guidelines note that on some sorters forward scatter width alone misses many doublets, and recommend both forward and side scatter plots. A practical check is to sort a negative fraction.

If it is very pure while the positive fraction is not, undetected doublets are the likely cause. Good sample preparation reduces the problem, including filtering, EDTA and DNase for sticky samples, and a modest event rate.

A strong answer covers

Pulse area, height and width behave differently for doublets
Doublets create false double positives and false G2 cells
Use forward and side scatter singlet gates together
Prevent clumps with filtering, EDTA and moderate event rates

Common mistakes

Skipping singlet gating because the sample looked like a single-cell suspension
Reporting a rare double-positive population without checking it against the singlet gate
Using one forward scatter singlet gate on an instrument where it misses rotated doublets

The interviewer might ask next

How would you tell a genuine double-positive cell from a cell-cell conjugate in imaging data?