[Cytometry] Teaching Flow Cytometry

Hirschkorn, Dale dhirschkorn at bloodsystems.org
Fri May 6 13:14:45 EDT 2011


I agree that Howard's comments are not relevant to the individual making
the original post simply because the poster is the exception rather than
the norm.  Way too many folks are not motivated enough to think and to
think critically about the whole process.  I have known too many flow
cytometer users that are just as Howard describes--they want to put a
tube of cells or, heaven forbid, whole blood on a cytometer, push a
button and magically out pops a fully analyzed set of data.  They all
seem to be more interested in what is on that piece of paper at the end
than all of the stuff that must happen in between.  To many of them, all
that stuff is irrelevant and little do they think how relevant proper
cytometer setup, sample quality, sample staining and a properly
maintained cytometer really is.  The more one knows about everything
from sample procurement to data analysis the better off in the cytometry
world one is.  Thank goodness the poster was motivated.  We need more
individuals like him/her willing to learn the basics on their own, use
them efficiently and pass that knowledge on to others.   

Dale Hirschkorn, MT(ASCP)
Blood Systems Research Institute
270 Masonic Ave
San Francisco, CA  94118
(415) 749-6672
www.bloodsystems.org
www.bsrisf.org
 
-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Facs
Sent: Friday, May 06, 2011 8:46 AM
To: Howard Shapiro
Cc: flow list Purdue flow list Purdue (cytometry at lists.purdue.edu)
Subject: Re: [Cytometry] Teaching Flow Cytometry

Howard wrote:

"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."

I think you should rethink this one as the response is not relevant to
the new "motivated" individual who posted the question. 

Ann Atzberger
Flow Cytometry Facility
Institute of Molecular Medicine
Trinity Health Sciences: St James Hospital
Trinity College Dublin
Dublin 8 Ph. 0035318963055
________________________________________
From: cytometry-bounces at lists.purdue.edu
[cytometry-bounces at lists.purdue.edu] On Behalf Of Howard Shapiro
[hms at shapirolab.com]
Sent: 06 May 2011 14:52
To: Dettmering, Till
Cc: hsfc at elist.tufts.edu; flow list Purdue flow list Purdue
(cytometry at lists.purdue.edu)
Subject: Re: [Cytometry] Teaching Flow Cytometry

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.

-Howard



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