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PBMC Isolation Protocol: Methods for the Isolation of White Blood Cells From Whole Blood

April 2021

Peripheral Blood Mononuclear Cells

Within the human body, an interconnected system of cells works together to carry out a functional immune response. When presented with a harmful pathogen, a variety of lymphocytes and signaling molecules activate to combat and eradicate the threat. Scientists actively study this process to gain insight into possible routes for immunological research. One way to study the immune response is to look at peripheral blood mononuclear cells (PBMCs).

PBMCs include lymphocytes, monocytes, and any other white blood cell (WBC) with a round nucleus. They are isolated directly from whole blood through the use of different cell separation techniques. PBMCs exist in peripheral blood, where they act as the body’s front line of defense. This requires these cells to have built-in mechanisms to defend the body against disease. Researchers can use their combative behaviors to better understand disease progression, how the body fights off different pathogens, and even to develop advanced therapeutics and treatments.

PBMC Applications

Studying PBMCs has multiple applications in toxicology research, including:

  • New compound toxicity assessment – One critical use for PBMCs is to investigate the possible effects of a new drug compound on the human immune system. By studying how PBMCs react to a potential drug, the developing company can discover whether or not it’s safe for widespread human use, as toxicity to PBMCs can be life threatening. These cells also help to determine the appropriate dosing for new drugs.
  • Side-by-side comparisons – To get a better understanding of what a drug or disease does, researchers can compare normal PBMCs to infected PBMCs side-by-side. This allows for an in-depth analysis of which pathways are impacted. Knowing the specific effects of foreign chemicals on PBMCs can provide useful information to drive future research.
  • Chemotherapy toxicity impact studies – PBMCs help doctors to study the levels of toxicity induced by chemotherapy treatment for certain types of cancer. These studies can reveal which populations may be more negatively affected by cytotoxic treatments, and therefore help to weigh the risks and determine which treatment route an individual should take.
  • Personalized medicine – Every individual has unique DNA, and genetic differences can directly contribute to how the human body reacts to different treatments and therapeutics. Two people can be impacted in dramatically different ways by the same medicine, depending on their genes. Studying PBMCs can allow doctors to make treatments more personalized by developing a genetic immune profile, which enables medicine that is both more specific and more effective.
  • Occupational exposure research – PBMCs can also help to study the effects of exposure from a job or environment. Testing peripheral cells for their toxicity can indicate high levels of exposure to different toxic compounds, such as heavy metals or benzene.

PBMCs and white blood cells are integral to toxicology research, but they require processing before they can be studied in these ways.

PBMC Isolation

The PBMC isolation protocol involves directly separating lymphocytes from whole blood. Whole blood is the blood that flows through the human body — the raw fluid with no components removed or separated. This is composed primarily of plasma, platelets, red blood cells, and PBMCs. Overall, PBMCs make up a small percentage (about 1%) of a whole blood sample, and are extremely difficult to study when crowded by other substances. Primary white blood cell isolation from whole blood must be performed before researchers can perform toxicology experiments.

Three of the most common methods for white blood cell separation include:

  • Density-gradient centrifugation
  • Fluorescence activated cell sorting (FACS)
  • Magnetic-activated cell sorting (MACS)

Each of these methods have their own advantages and disadvantages.

Centrifugation of White Blood Cells

Density-gradient centrifugation relies on physical characteristics such as size and density to sort cell populations. By placing a sample into a centrifuge, a device that can rotate at high speeds, the different cell types will sort themselves out by grouping with particles of a similar density. More dense particles will fall to the bottom or the outside, and less dense particles will stay toward the center or rise to the top. Centrifugation of a standard whole blood sample will separate the general layers of the blood components, concentrating the red blood cells (roughly 45% of total volume) at the bottom of the tube, the buffy coat layer – containing the various white blood cells and platelets (roughly 1% of total volume) in the middle layer, and the blood plasma – containing the watery liquid that allows blood to flow, along with the various proteins and dissolved nutrients & gasses (roughly 55% of total volume) at the top of the tube.

WBC Isolation With FACS

One technique for immune cell isolation involves the use of a flow cytometer, a machine that uses fluorescent light to label different particles based on characteristics such as size, shape, and radiance. This method, called fluorescence activated cell sorting (FACS), allows the sample to flow through a tube that then sorts the components into distinct groups.

FACS requires expensive equipment and properly trained personnel. While this technique can be useful when sorting diverse cell samples that need many different populations sorted, it is incredibly time consuming to use this method for WBC isolation. Typically, the sample will first be “prepared” – or cleaned up – to isolate a specific subset of the original sample’s contents before being processed using flow cytometry. This greatly reduces the time required for cell sorting, as the machine will be processing a much smaller and more concentrated sample volume. It also can result in a larger population of healthy, viable cells at the end, as less natural cell death will occur when processing time is greatly reduced.

WBC Isolation With MACS

Another method binds magnetic beads to target cells and flows the sample through a magnetic field, suspending the cells with magnets attached. Magnetic-activated cell sorting (MACS) is faster and cheaper than the two previously mentioned methods, but it has the highest cell loss of the three. The harsh magnetic fields can rupture cells and cause fractured organelles to clutter the sample. This extracellular debris can cause clumping and blockage which results in a lower throughput.

WBC Isolation With BACS

Buoyancy Activated Cell Sorting, or BACS, leverages the power of gravity to enable cell sorting. This method uses microbubbles that are functionalized to capture analytes of interest and float them to the top of the sample container. The microbubbles are simply mixed into the sample, where they bind to the contaminating cells and float them to the top for removal, leaving the cells of interest isolated at the bottom – untouched and ready for use. The microbubbles carry out their function without any blunt physical forces or harsh magnetic fields, adequately maintaining cell health and physiology for downstream applications. Immune cell isolation kits aimed at B cell and/or T cell enrichment use a fast, easy, and gentle 30-to-45-minute workflow, and depletion kits like the human red blood cell (RBC) depletion product can remove over 97% of residual blood cells from a sample in as little as 10 minutes. There is no expensive equipment or extensive training required, and this fast and gentle workflow maintains cellular physiology, increasing throughput and resulting in a highly enriched, viable population of cells of interest.

Akadeum’s Microbubbles

If your process of lymphocyte separation for toxicology research could benefit from the fast, gentle microbubble workflow, be sure to explore Akadeum’s suite of products. We also partner with companies and institutions who are looking to leverage the microbubble platform for next-generation solutions, if you’re interested in learning more about partnering with Akadeum, we’d love to hear from you.

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