Everything You Need to Know About the Three Isolation Techniques for PBMCs

May 15, 2019

A peripheral blood mononuclear cell, otherwise known as a PBMC for short, is a term used to describe any peripheral blood cell that also has a round nucleus. Lymphocytes fall under this category, which themselves include not only T cells but also NK cells and B cells as well. Monocytes are also examples of this idea throughout life.

Within the human body, the major role that these blood cells play involves both fighting infection and adapting to intruders. They give very precise, selective responses to the immune system during periods of distress and are therefore considered to be some of the most critical cells in the entire body.

When it comes to isolation techniques for PBMCs, there are three main options to be aware of, all of which bring with them their own advantages and disadvantages that are worth exploring.

Density Centrifugation with Ficoll-Paque

Beginning in 1968, density centrifugation with Ficoll-Paque was regularly used to isolate PBMCs for a wide range of research purposes – though mainly to evaluate various immune responses within patients. After blood samples are obtained from healthy donors, a gradient medium (with a density of 1.077 g/ml) is then used to separate that whole blood into two distinct fractions.

The PBMCs remain in the upper fraction, otherwise known as the “low density” fraction. Red blood cells and PMNs (polymorphonuclear neutrophils) have a higher density and are thus relegated to the lower fraction.

The major problem with this is that Ficoll-isolated PBMC samples can sometimes be contaminated with red blood cells or PMNs during the density centrifugation process. Because of that, a number of alterative options have been developed that aimed to A) simplify the procedure considerably in terms of the time necessary and samples required, and B) to avoid that contamination with other cell types.

Isolation via Cell Preparation Tubes

Another commonly used method for isolating PBMCs comes by way of cell preparation tubes, or CPTs for short. In this scenario, which differs significantly from Ficoll-Paque, a density gradient is present underneath a gel barrier. Blood samples are drawn directly into the tube and at that point, no further dilution is needed. Centrifugation only takes about 30 minutes, with dramatically limits sample handling times as well.

The other major benefit of this is that most cell preparation tubes not only allow for cell separation during a single step, but that sample then does not have to be removed from the container during transportation. The included gel forms a very stable barrier between each of the layers of a cell, thus avoiding any interaction with some of the denser blood components that are also present.

SepMate Tubes with Lymphoprep

For this technique heparinized blood samples are diluted with phosphate-buffered saline before being layered on top of Lymphoprep. Lymphoprep is a density gradient medium that is often recommended for the isolation of mononuclear cells.

The key difference between this and the Ficoll-Paque method in particular is that SepMate tubes include an insert that actually makes the layering process easier. Not only can blood be layered over the density gradient medium very quickly, but the tube’s design also prevents those layers from mixing. With a SepMate tube, consistent PBMC isolation can happen in as little as 15 minutes with repeatable, predictable results. After that, isolated PBMCs are transferred into a fresh tube for further analysis.

In one study comparing all three of these methods, recovery levels were actually the highest for those samples isolated using cell preparation tubes, then the Ficoll method, then SepMate tubes with Lymphoprep. In that experiment, the cell viability of all three methods was then assessed using a trypan blue exclusion. All three of the techniques saw a cell viability rating of 100% immediately after isolation had taken place.

Regardless of the isolation technique used, there are still a number of important factors that can ultimately affect the outcome of any research being conducted. Nutritional status, hormone levels and even current levels of inflammation will all influence the way immune cells react in a given situation, thus the composition of PBMCs will always depend on the donor. In all three of these methods, experts agree that the ability at which they can reproduce results with cells from multiple donors is ultimately what supports the results they’ve found to begin with.