How many cells do you need to reliably detect a population of interest?

The answer depends on the confidence you need, and the variability you can accept. Variability is expressed by the coefficient of variation (CV) is simply the standard deviation divided by the mean. The higher this number, the more “variable” the measurement. The lower the number, the less “variable” the measurement. Intuitively, a population that appears a lot, say 10% of the time, needs fewer cells than a population that occurs 0.001% of the time.

For purposes of this answer, let’s consider two populations, intermediate Monocytes (inMono) and T cells. These figures are derived from our mass cytometry validation report: https://teiko.bio/technology/, however we believe these reference ranges are applicable regardless of the instrument type (spectral cytometry or mass cytometry).

Cell PopulationMedianInter-run CV%Intra-run CV%
T cells37.28%0.39%1.27%
Table 1: Reference Range Values for PBMCs from Healthy Subjects, % of non-granulocytes

Luckily, this question has been addressed in the work of Keeney et al. To wit, “for cell-based assays such as flow cytometry, a simple calculation can be used to determine the size of the database/sample that will provide a given precision: r = (100/CV)2; where r is the number of events meeting the required criterion, and CV is the coefficient of variation of a known positive control.”

We’ve adapted Keeney’s table below:

Desired Coefficient of Variation (%)151020
r = number of events of interest10,00040010025
When occurring at a frequency of:
Fraction1:nTotal number of events which must be collected
Table 2: “Determination of database/sample size that will provide a given precision in rare event analysis”, Keeney, et. al

Now, let’s couple that with the instruments we have at our disposal: mass cytometry and spectral flow cytometry. Based on our experience, mass cytometry and spectral flow have recovery rates of 50% and 90%, respectively.

Drawn from Patient (mL)Cells per mL (M)Number of Cells in a Vial (M)InstrumentRecovery RateResulting Events (M)
31.85Mass Cytometry50%2.63
Spectral Flow90%4.73
Table 3: Estimated events for Peripheral Blood Mononuclear Cell collection

Drawn from Patient (mL)Cells per mL (M)Number of Cells in Whole Blood (M)% GranulocytesApproximate number of Granulocytes (M)Non-Granulocytes (M)InstrumentRecovery RateResulting Events (M)Resulting non-granulocyte events (M)
251050%55Mass Cytometry50%5.002.50
55Spectral Flow90%9.004.50
Table 4: Estimated events for Whole Blood collection

For PBMCs, you can get 0.5M – 10.8M events, depending on the volume of blood collected. In the table, we just show the average ranges, i.e. 2.63M – 4.73M. And for whole blood, you can get 4M – 10.8M total events. Since at least 50% of the cells will be granulocytes, you’ll get to ~2M – ~5.4M non-granulocytes. For readability, we’re showing only a smaller range, from 2.5M – 4.5M.

Now, let’s come back to the two populations from Table 1, inMono and T-cells.

Desired CV1%5%
Teiko observed inMono Median % of non-Granulocytes0.47%
Total Number of events that must be collected, based on Keeney Table1,000,00040,000
Estimated inMono Population needed to achieve CV4,700188
Actual Teiko collected inMono events302
Above Threshold?NoYes
Actual Teiko Intra-Run CV4.63%
Actual Teiko Inter-Run CV16.09%
Industry Standard CV Acceptance Criteria25-30%
Table 5: Desired CV for inMono

For a 5% CV for a 0.47% population, you would need about 188 events to achieve this CV. And We collected 302, so we were above the threshold. Turns out, we were right in line with inter-run CVs, at 4.63%, and Inter-run CVs at 16.09%.

Desired CV1%5%
Teiko observed T-cell Median % of non-Granulocytes37.28%
Total Number of events that must be collected, based on Keeney Table100,0004,000
Estimated T-cell Population needed to achieve CV37,2801,491
Actual Teiko collected T-cell events39,210
Above Threshold?YesYes
Actual Teiko Intra-Run CV1.27%
Actual Teiko Inter-Run CV0.39%
Industry Standard CV Acceptance Criteria25-30%
Table 6: Desired CV for T-cells

Now, T-cells are much more populous. In our precision study, we collected 39,210 events, beyond the 37,280 and 1,491 cells we need to achieve a good coefficient of variation. And, as it turns out, we clocked 1.27% CVs for intra-run and 0.39% CVs for inter-run.

So, hopefully this gives you an intuition for “How many cells do you need to reliably detect a population of interest?” Interested in capturing a specific population?

Contact us to discuss further!