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

Link to paper

Abstract

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.

 

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.

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.


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?