Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood, 2017, McBride et al

Discussion in 'Research methodology news and research' started by Hutan, May 3, 2024.

  1. Hutan

    Hutan Moderator Staff Member

    Aotearoa New Zealand
    Link to paper


    NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user's errors or because of the sensitivity of NK cells to experimental manipulation.

    To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3'-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells).

    This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data's integrity.
    Sean, SNT Gatchaman and Kitty like this.
  2. Hutan

    Hutan Moderator Staff Member

    Aotearoa New Zealand
    I thought this was an interesting read for a couple of reasons.

    One is the information about how to determine the cytotoxicity of natural killer cells. There's been a long history of dispute about whether there is something wrong with NK cell function in ME/CFS. These authors say that the evaluation needs to be done within 4 hours of blood draw.
    I'm currently looking at a NCNED paper where they assess NK cell function. They use blood samples gathered from quite a distance away and don't appear to have determined NK cell function in anything like the 4 hour window. Although we might expect delays to equally affect samples from patients and healthy controls, when the sample size is small (as it always is at NCNED), then experimental results may not mean much.

    The other reason is the data about how natural killer cell numbers and function changes in response to exercise. Here's a chart for 4 runners' NK cell numbers, with the drop at 4 hours post exercise.
    Screen Shot 2024-05-04 at 4.45.35 am.png
    And here's the chart for the NK cell cytotoxicity. One of the runners has a very steep drop in NK cell cytotoxicity 4 hours after exercise, followed by an increase. All four runners have higher NK cell cytotoxicity after exercise.
    Screen Shot 2024-05-04 at 4.47.26 am.png

    I would love to see this careful work done with a group of people with ME/CFS and controls, before during and after some exercise. Perhaps it has been done already?
    Last edited: May 3, 2024
    Sean, Michelle, alktipping and 4 others like this.
  3. Jonathan Edwards

    Jonathan Edwards Senior Member (Voting Rights)

    London, UK
    Very interesting plots.

    Circulating cells are always difficult to interpret because the numbers and behaviour can be affected both by uptake into tissues and new production.

    So in the first graph the apparently odd behaviour of runner 1 may just mean that their NK cells are taken up into muscle a bit quicker than the others. Exercise is likely to stimulate both increased uptake into tissues and maybe a bit later release of new cells from bone marrow.

    NK function tests like K562 are pretty rubbish because NK function depends on receptors that vary from person to person. The target should really be autologous cells. But a profile after exercise should even things out even if you cannot compare one person with another. It looks as if fresh new NK cells come out at about 24 hours with good function.

    Of course my theory would suggest that NK or CD-1+ or MAIT T cells were too busy rather than low on numbers or function. But if they are busy then circulating numbers may be lowish at times because they are being invited into tissues all the time. They might even show low function for K562 because they are distracted by picking up the wrong antibodies on CD16 although that is pretty unlikely.

    It may be worth noting that chasing functional abnormalities of T and related cytotoxic cells may be something nobody has really got the hang of yet, even in diseases where they are very likely misbehaving. I do not know of any clear data on functional changes in blood cells in psoriasis or reactive arthritis for instance - although there may be now.
    shak8, Sean, Michelle and 3 others like this.

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