Skip to content

Why Recovery, NOT Purity, Is The Best Measure Of A Cell Sorting Performance

Written by Tim Bushnell, PhD

One of the best perks of being a part of the flow cytometry community is the opportunity to attend one of the many regional flow meetings.

The chance to network with colleagues that do what you do is incredibly energizing. 

It’s also a great opportunity to see what new research ideas they’re involved in before they’re published, and to talk with them directly about their ideas, and the research they’re involved in. I was recently at the Australasian Cytometry meeting and had a chance to talk to Dr. Rui Gardner, the Director of Flow Cytometry at the Gulbenkian Institute of Science in Lisbon, Portugal.

Rui gave a great talk on the concept of Rmax, which I’ll be talking about here.  I want to make sure and acknowledge the other colleagues also involved in this work, who are Andy Riddell, Flow Cytometry Manager at Wellcome Trust-MRC Stem Cell Institute, Centre for Stem Cell Research University of Cambridge;  Alexis Perez Gonzalez, Manager Flow Cytometry Core Facility, EMBL in Heidelberg; and Lola Martinez, Head of the Flow Cytometry Facility at CNIO, Madrid, Spain.

You can find this procedure in more detail here:

Measuring Cell Sorter Performance

There seems to be quite a bit of confusion about how to measure the performance of a cell sorter, so I want to begin by defining a few terms, and then I want to discuss some of the variables that affect sort performance.

As a core manager for 30 years, I went through quite a transformation, as I learned that the sorter’s performance was very dependent on the sample preparation.

This is quite difficult to explain to the customer, though, so hopefully we can clear up some misconceptions. 

One of the most important metrics to customers is the sort purity.

Purity is defined as :

Purity Formula | Expert Cytometry | FlowJo SPICE Analysis

Unfortunately, purity is NOT a good measure of instrument performance.

The instrument can be performing well, and the purity will be good, and the instrument can NOT be performing well, and the purity will also be good.

If the drop delay is not calculated correctly, the purity can be good, but the recovery will suffer. 

There have been a few publications and posters on this, but it has gone largely unnoticed that measuring purity is not the best way to measure sort performance or to calculate the drop delay, but it is the method that most commercial sorters use to do this.

How do we do a better job?

Improved Recovery Formula | Expert Cytometry | FlowJo SPICE Analysis


Yield Formula | Expert Cytometry | FlowJo SPICE Analysis

Calculating Recovery, Yield & Purity

If we want to calculate the actual yield, we have a lot of counting methods, like cell counters and Neubauer chambers, but these all come with a fairly high level of associated error.

The best method would be independent of this error.

As long as we sort similar particles, which show the same Poisson behavior when sorted, the following holds.

At any given time, after a fraction α of the original sample is sorted, the absolute number of target particles (t) in this fraction (α.Ot) is distributed between the sorted tube (St) and the center stream catch (Ct) collected during the sort:

Cell Sort Formula | Expert Cytometry | FlowJo SPICE Analysis

The same applies for non-target particles where:

Non-Target Particles | Expert Cytometry | FlowJo SPICE Analysis

We can estimate the maximum recovery for a given set of sort conditions:

Max Recovery Sort Condition | Expert Cytometry | FlowJo SPICE Analysis

If sort purity approaches 100%, this simplifies to:

Cell Sort Purity Formula | Expert Cytometry | FlowJo SPICE Analysis

The first time I saw the above equation, I was thinking this was going to be a complicated experiment, but Rui and colleagues have given us all of the tools to make it really easy to do.

The experiment is outlined in the protocol I linked above.

Adjusting Drop Delay For Purity Versus Recovery

You set up a sort with two different particle types, and measure the proportions of each in the sort tube and the waste.

The data are entered into the spreadsheet provided, and the maximum recovery can be predicted.

By comparing the recovery and purity at the drop delay calculated by the instrument, and by sorting slightly above and below this value, you can see that the calculated drop delay is rarely the ideal for recovery.

As you can see from the graphs below, from two different instruments, the calculated delay works very well for purity, but for maximal recovery, the delay should be shifted.

BOP Delay | Expert Cytometry | FlowJo SPICE Analysis

In sum, recovery is much more sensitive to the correct calculation of the drop delay than the purity, and we should be sure we choose the correct metric for measuring sort performance.

To learn more about cell sorting and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on Mastery Class wait list.

Tim Bushnell, PhD


Advanced Microscopy

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.
flow cytometry tablet eBook cover

Modern Flow Cytometry

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more. 

Advanced 4-10 Color Compensation

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.

Top 40 Networking Scripts For PhDs

If you want to get replies from top employers and recruiters, this ebook is for you. These networking scripts will show you the exact words ...

Informational Interviews For PhDs

If you want to learn how to set up and execute informational interviews with PhDs working in industry, this ebook is for you. Here, you ...

Industry Resume Guide For PhDs

If you have a PhD and want to create the perfect industry resume to attract employers, this ebook is for you. Here, you will get ...

Top 20 Industry Jobs For PhDs

If you want to learn about the top 20 industry careers for PhDs regardless of your PhD background, this ebook is for you. Here, you ...

Salary Negotiation For PhDs

If you have a PhD and want to learn advanced salary negotiation strategies, this ebook is for you. Here, you will learn how to set ...

Top 20 Transferable Skills For PhDs

If you want to learn the top 20 transferable skills the industry employers ranked as most important for PhDs to include on their resumes and ...

Related Posts You Might Like

We Tested 5 Major Flow Cytometry SPADE Programs for Speed – Here Are The Results

Written By: Tim Bushnell, PhD As a follow-up to our post on tSNE where we compared the speed of calculation in leading software packages, let’s ...
Read More

5 FlowJo Hacks To Boost The Quality Of Your Flow Cytometry Analysis

Written By: Tim Bushnell, PhD Primary data analysis, that is the analysis at the sample or tube level, is where the populations of interest are ...
Read More

Statistical Challenges Of Rare Event Measurements In Flow Cytometry

Written by Tim Bushnell, PhD To conclude our series on rare event analysis, it is time to discuss the statistics behind rare event analysis. The ...
Read More

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

Written by Tim Bushnell, PhD “Not everything that can be counted counts and not everything that counts can be counted.” — William Bruce Cameron (but ...
Read More

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

Written by Tim Bushnell, PhD Next to the flow cytometer itself, the most important component of a flow cytometry experiment comes down to the antibodies. ...
Read More

How To Achieve Accurate Flow Cytometry Calcium Flux Measurements

Written by Tim Bushnell, PhD Most flow cytometry experiments work with antibodies conjugated to a fluorochrome for some variation on immunophenotyping. However, any fluorochrome that ...
Read More