Automated Motion Tracking and Data Extraction for Red Blood Cell Biomechanics

[sb4]

https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpcy.75

Abstract

Red blood cell biomechanics can provide us with a deeper understanding of macroscopic physiology and have the potential of being used for diagnostic purposes. In diseases like sickle cell anemia and malaria, reduced red blood cell deformability can be used as a biomarker, leading to further assays and diagnoses. A microfluidic system is useful for studying these biomechanical properties. We can observe detailed red blood cell mechanical behavior as they flow through microcapillaries using high‐speed imaging and microscopy. Microfluidic devices are advantageous over traditional methods because they can serve as high‐throughput tests. However, to rapidly analyze thousands of cells, there is a need for powerful image processing tools and software automation. We describe a workflow process using Image‐Pro to identify and track red blood cells in a video, take measurements, and export the data for use in statistical analysis tools. The information in this protocol can be applied to large‐scale blood studies where entire cell populations need to be analyzed from many cohorts of donors. © 2020 The Authors.

New Ron Davis paper. Haven't read it yet.

 

[Ben H]

Just about to post (insomnia). Nice catch, and @wigglethemouse .

Quote from paper:

"Our method provides a test that is sensitive enough to track changes in single cell morphologies rapidly with high‐throughput. We hope that clinical assays can be developed using this process as a proof of concept, and the results may be used in diagnostic tests."


B

 

[wigglethemouse]

Just about to post (insomnia). Nice catch, and @wigglethemouse .

I saw @sb4's post first and then posted on social media. Thanks @sb4

This was the ME/CFS presentation from the 2019 Stanford Symposium using similar techniques but for ME/CFS red blood cells.

The paper of this thread just looked at healthy participants with and without a chemical to stiffen the red blood cell. The paper describes use of off-the-shelf software with instructions on how to configure. The presentation in September 2019 described a 16 bit pixel camera wheras here is an 8 bit one, with instructions on how to adjust contrast to maximise image quality of those 8 bits.

At the symposium Santiago presented a single channel micro-fluidic device with 7um channel and null results in ME/CFS vs Healthy Control. His opinion was that a smaller channel was needed. Here Amit Saha designed a 5um channel device with several parallel paths. The software was set to filter out any stuck RBC's in channels so if the experiment is run in parallel mode data can still be collected. (I'm not sure how this would affect velocity parameters). Here is Figure 2, a micro-fluidic device image from the paper showing 4 channels

Here is an image from a microscope that combines 11 different images of the same cell at different time points to show the changing shape as the cell progresses through the channel (Figure 1B in paper). The red line is 20um long.

The red blood cells need to be diluted with PBS (phosphate buffered saline) so that they don't clump together. In the Stanford Symposium the thought was that this cleaning RBC's and then putting them in PBS affected the deformability properties and that some blood would need to be mixed in with the RBC's so that they maintain those properties. The paper describes diluting the red blood cells, but I couldn't see a full preparation recipe.

To validate the tool they took red blood cells + PBS with and without chemical treatment of glutaraldehyde that changes the deformability of the cells, and measured the results. Fig 9 shows the spread of results and the difference between cells.

It's clear that measuring lots of cells in an automated manner gives statistically a better picture - you see that even though cells are treated exactly the same, their properties vary by different amounts. However diluting the cells with PBS so they don't clump may affect cell properties too so I imagine if this work continues that is the next step to understand.

It's nice to see that this work has continued since the September Symposium, and that a method is shared using off the shelf software so others can build on it. Hopefully they will write, or have written, some grant applications to build on this work for ME/CFS patient red blood cell science.

 

[wigglethemouse]

Now I don't know if this is new work or not and how it differs from the 7um channel work described by Santiago in the September 2019 Stanford Symposium.

This conference abstract from June 2019 seems to describe the automated test using ImagePRO software as described in the current paper of this thread on ME/CFS patients using 5um channels.
Red Blood Cell Biomechanics In Chronic Fatigue Syndrome

Thread for conference abstract: https://forums.phoenixrising.me/thr...-in-chronic-fatigue-syndrome-saha-2019.77025/

 

[wabi-sabi]

This looks like good news to me. I think we are making progress.