[Cytometry] Teaching Flow Cytometry

Howard Shapiro hms at shapirolab.com
Fri May 6 09:52:45 EDT 2011

Till Dettmering wrote:

> I'm a PhD student for a year now and I'm working with flow cytometry for 2.5 years. Since there is no real dedicated FACS-'guru' in our group who I could have learned from, I had to teach practically everything about flow cytometry to myself, including maintenance of the device (a Partec PAS III). I find myself more often in the situation in which I have to teach new students who have never seen a flow cytometer how to work with it and how to interpret the data. Although it works out quite well, I'm very interested on your experiences on how to best teach new people flow cytometry. What do you start with? Do you have something like curriculum you follow when teaching it? What did you start with when you learned it? What should one pay special attention to when teaching?
> I'm very interested in hearing your experiences!

Problem number one these days seems to be motivating the new people to think; too many of them want to mix samples with magic juice, put them in the magic box, press the magic buttons on the box and do the necessary magic mouse clicks, keystrokes, etc. on the computer, and save the PowerPoint presentation and the manuscript that will be submitted online to one prestigious journal or another. Then it's back to social media.

Those of us who write flow books and vent on this Mailing List have tried to spell out the basics. Cytometry, as I have often said, begins with the cell, even at the level of the word itself. You have to know what information you want from the cells in your samples, and what reagents you need to use and parameters you need to measure to get that information, and it helps to consider alternatives when they are there. 

The cell identification game is like "Where's Waldo (his name is Wally in some places)?" - The game wouldn't be any fun if Waldo were the only person in the crowd wearing magenta and chartreuse; the intellectual challenge comes in picking out physical features and the patterns of the colors on his clothing and distinguishing them from those on everybody else in the crowd among which he is hidden. Generally speaking, one can always identify a cell from a given species by detecting a specific nucleic acid sequence, or a combination of antigens. In unlysed blood, CD45 is good for discriminating white blood cells from red cells but, in cases where you know you won't encounter nucleated red cells, it's much simpler to use a permeant DNA dye. Simpler is always better.

One can learn these kinds of tricks from books and material available online. It's harder to learn how to set up an instrument, verify that it is working properly, and deal with it if it isn't. The theoretical part of that is accessible in books, but which knobs you turn and/or how you set up the software depend entirely on the specific cytometer you're using. Manufacturers offer training courses, and provide manuals, but, as is the case with computers, you can sometimes RTFM and find that it doesn't really tell you all you'd need to set up a flow lab in a postapocalyptic venue. Oral tradition only goes so far; even prehistoric hunters seem to have needed seminars with presentations painted on cave walls.,

When TFM leaves you in the dark, you can get computer questions answered online, sometimes for nothing, or you can buy something like the books in David Pogue's "the missing manual(R)" series, published by O'Reilly.

What we need in cytometry are equivalents. Practical Flow Cytometry had its genesis in my "Building and Using Flow Cytometers: The Cytomutt Breeder's and Trainer's Manual," and the material from that on building and using instruments, which appeared in the first couple of editions of PFC, has apparently been sufficient to enable dozens of people to put together instruments and keep them running for decades without access to service from outside. 

It does not strike me as unreasonable that manufacturers should support their user groups in generating similar documentation, but the users could get organized to do this without the manufacturers' help, which would minimize the likelihood of problems being swept under the rug. The material, which should cover software from both manufacturers and third parties as well as apparatus, should be kept online in a presumably neutral site such as this one. 

I've seen people with little or no lab experience get up to speed, at least with relatively simple flow cytometers, in weeks, and people with decades of experience fail to do so in months to years, and I can't always explain either.  There hasn't been a Mozart in cytometry yet, and even he started small. If you can't hear a sonata playing in your head, don't try for a symphony, and, if you can't do two-color fluorescence and two-angle scatter, don't try running a 4-way sorter with 7 beams and 24 colors. I spent several years training in surgery, and would have had to spend several more before anybody let me do any significant part of a heart operation; it is, admittedly, harder to kill people by running a flow cytometer incompetently than it is to do so when you are waving a scalpel around vital organs, but the malpractice of adding junk to the medical and scientific literature has its own adverse consequences.

So let's get organized. 

I'll be at my 50th College Reunion during ISAC, which declined my offer to give a plenary talk by remote video, but catch up to me online if you're willing to help.


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