Blood extraction and analysis is a routine practice for a lot of medical tests and disease monitoring. It’s a simple, relatively non-invasive procedure that gives a sufficient volume of sample to work with for whatever tests need to be run for the patient. More recent advances in genomics and particularly immune-based genomic therapies have provided an entirely new avenue for using collected blood samples. The subset of cells called the peripheral blood mononuclear cells (PBMCs) contain all of the non-red blood cells with a round nucleus (i.e. lymphocytes, monocytes, natural killer cells (NK cells) or dendritic cells). These are the cells of interest for these types of therapies as the immune cells will be re-programmed to both study and treat disease.
There are well-established protocols to isolate PBMCs and to remove red blood cells (RBCs) to obtain optimally purified immune cells for downstream purposes. But one critical step is knowing how many cells have been isolated. This is where cell counting comes into play, as this check will determine how to move forward with either patient treatment or research experiments.
For prepping downstream experiments, confidence in the concentration of cells received when counting is vital. Because the patient sample is often a limited resource and multiple counting runs whittle away at the precious sample volume, getting a concentration value that is reliably close to the true mean with the fewest number of replicates possible is ideal.
A hemocytometer has the highest variability between replicates and requires the most time. So while it is a simple procedure using minimal additional technology to get a result, the confidence in your concentration calculation will be low. This comes from the dual problems of counting a very small number of cells (~100) and extrapolating the entire sample’s concentration from it as well as relying on a dye like Trypan blue to identify real cells versus debris. This second problem is made more difficult in PBMC counting because it is very difficult to distinguish between RBCs and immune cells via the naked eye. Because of all of this, manual counting with a hemocytometer is too unreliable for something like PBMC samples.
Image-based cell counters have removed some of this unreliability by automating the counting and AI-based algorithms are better at distinguishing debris from RBCs and from PBMCs. They do still however only count a small number of cells (~300-700) and still rely on a stain and an image to get their numbers. As a result, it is common to obtain still variable and therefore unreliable results even when triplicates are performed on the same sample.
The only way to overcome the challenges that still persist in counting complex cell suspensions like blood samples is to find a way to count more cells and find a way to remove the reliance on an image to distinguish what is a real cell. This is where the Moxi line of products comes in. All three of the instruments (Moxi Z, Moxi V, and Moxi GO II) count the number of cells you want (> 10,000 cells). This means that, according to the law of large numbers, you will be much closer to the true mean even with just one run. While triplicate runs can be done to ensure accuracy, the variance will be a fraction of that obtained using another method.
The need for a stain is also eliminated because of the proprietary blend of Coulter Principle-based technology and microfluidics. Because these measurements are physics-based and are not reliant on an image, no additional dyes or variables are introduced to increase inaccuracy.
A real-world example of a PBMC run on a Moxi machine recorded over 18,000 events (cells + debris) is depicted in the graph below.
However, this concentration is inaccurate because it includes debris particles in its calculation (See red rectangle)
Because the counting technology by default obtains size information for every event and because debris is significantly smaller than real cells, determining what is debris and what is cells is simple:
The peak that appears at < 3 μm (red square) is all of the smaller particles, which in this case is debris.
Using the gating feature (3rd image) on a Moxi system enables the removal of those events from the total counts and ensures that only real cells are included in the concentration calculation.
The significant shift in concentration pre and post-gating highlights how much adding debris in your concentration calculations can affect the concentration. This shows the importance of properly distinguishing between debris and cells and why a Moxi instrument will give a more reliable concentration.
All of the Moxi instruments are equipped with this level of cell counting sensitivity to make sure that cell counting remains reliable no matter which one is chosen. You can view all of the available instruments and what they can do on the products tab of our website: https://www.orflo.com/
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