Tuesday, 7 February 2017

Flow Cytometer the Immunology Machine that goes Ping

For those of you not old enough to know or care, the machine that goes ping is from a comedy sketch by Monty Pythons flying Circus (a so called comedy programme from the 1970s).

Anyway a flow cytometer is a tool used by immunologists to look for the expression of markers on a cell and so you can work out populations of cells say in a blood sample. 

You take a cell suspension and get them to get sucked into the machine in single-file and then you shine a laser on them and the laser gets bent depending on the size and granularity of the cell. You can also add a glow in the dark marker, shine the laser light and it glows and there are detectors to count the intensity and other sensors to do the counting.


In the example above you have a positive and negative population (top right) and in bottom right it is activated T or T regs CD25+, CD4+.

What you do is you draw "gates" where the negative population is
and then you can work out the percentage.  These are seen as lines on the graph)

Mostly people use flourescent beads to do this or negative cells, but sadly some people don;t use common sense when they do this.

In the picture below, bottom left, you see an example. It is stained with two things the further to the left or the further to the top the more markers the cells express.

I have put a gate (black lines) to shown 4 populations a negative an single positive along bottom and on the left hand side another. There is double population with is top right. 

Here was can say that is 10% of the total. So in this population there are 10% of cell that express both markers.

Now in the right hand picture we show "drawing a gate by numbers" such as would occur when you use the beads to define the negative populations and when you use no intuition. 

So now the gate cut through what is obvious a single population of cells and so there are now 20% cells in top left. Same cells but rubbish analysis!


Don't believe me......Next time you look at a paper with flow cytometry look to see if you can see this. 

Send it in we can give them an altmetric they won't want:-).

However,the important thing is to remember the analysis is a based on a proportion. Is this important. Yes it is Read below

5 comments:

  1. MD is it right that you take this attitude to devalue your peers

    ReplyDelete
    Replies
    1. I get pulled up sometimes for being outspoken and shooting from the hip, but at the end of the day.....bad science is bad science and hopefully anyone who reads the blog and does this type of work may remember this when they analyse their own data. It is a constructive comment

      Will I make enemies....maybe....nobody likes being called out.

      However, we want you to be able to interpret the data/papers and we don't want you to pick up bad habits.

      I would be lying if I said all that is out there was great....it isn't. But you already know that.

      Can you critise what we do...sure you can...the referees do it all the time, so perhaps this is a exercise of venting the frustration...whilst performing education as I see it.

      Delete
    2. It is right that bad practice is exposed. Setting flow cytometry gates in the wrong place to presumably give the proportion desired is a common practice that us old hands in the game see again and again. It makes what should be quantitative data into subjective data. We include referees in our criticism, this is clearly obvious and so often is allowed through in the refereeing process.

      Delete
  2. Replies
    1. Too cryptic for me to understand I'm afraid:-(

      Delete

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