Human blood fractionation is a huge industry whose end-products are valued at around $25 billion annually. Centrifugation, the main process through which various blood products are isolated, separates blood into three main fractions. The densest layer consists of blood cells, neutrophils, and eosinophils, followed on top by a region containing white blood cells and platelets. The top layer consists of plasma, the largest blood fraction in terms of volume.
Peripheral blood mononuclear cells (PBMCs) are blood cells from peripheral blood containing a round nucleus. PBMCs include lymphocytes (e.g. T cells, B cells, natural killer cells), which comprise 70–90% of all cells present, and monocytes. Through their ability to activate T and B immune-system cells, lymphocytes are critical components of both humoral and cell-mediated immunity. CD3+ T cells make up approximately 40–70% of lymphocytes and exist mostly in an antigen-naive state, as do CD19+ B cells. When activated by antigens CD3+ T and CD19+ B cells convert to memory cells, from which they initiate, respectively, cell-mediated and humoral immune responses.
Immune system activation, over-activation, and inhibition are implicated in dozens of serious human diseases. Natural and vaccine-induced immunity has taken center stage with the emergence of the coronavirus pandemic. But interest in vaccines was high before anyone had heard of Covid-19, particularly for preventing communicable diseases like seasonal influenza. Vaccines for HIV, tuberculosis, and even cancer have thus far eluded developers, but not for lack of effort. Similarly, immune system activation in autoimmune diseases has been an intense area of study.
PBMC isolation is the prelude to a range of downstream processes and analytics, including flow cytometry, cell proliferation assays, cell culture, toxicology, immune response assays, DNA/RNA isolation, proteomics/metabolomics, and many others. None of these investigations are possible without access to PBMCs.
Standard protocols for isolating PBMCs all involve density gradient centrifugation using Ficoll Paque. Ficoll is a neutral, soluble, highly branched, high-molecular weight polysaccharide that serves as the gradient medium. In a typical isolation, Ficoll-Paque is pipetted into a centrifugation-suitable blood collection tube, and the blood is layered on top. After centrifugation the blood separates as described above, with the intermediate-density PBMC layer sitting above the red blood cells and below the plasma.
PBMCs may be further purified, either specific lineages or as a whole, through further ultracentrifugation or by pulling target cells out with the help of magnetic bead methods.
Figure 1. Comparison of PBMC fractionation in standard and ZenBio EZPREP collection tubes. The indicated bracketed regions represent the PBMC layers in both tubes.
Many factors affect the purity, integrity, and homogeneity of the PBMC fraction during normal collection and isolation. Investigators must add an anti-coagulant immediately after blood collection, and time is of the essence in all isolation and centrifugation steps to avoid PBMC degradation or association with non-PBMC cells, including plasma cells that begin dying as soon as blood is withdrawn.
Figure 1 compares PBMC layer collection in a standard collection/centrifugation tube (left) to isolation in a ZenBio EZPREP centrifugation tube (right). The fraction containing PBMCs is clearly visible in both tubes, but is much less well-defined in the standard tube.
EZPREP centrifugation tubes also use a Ficoll gradient but are shaped like an hourglass, with the narrower-diameter neck of the hourglass positioned optimally for collecting and concentrating the PBMC layer. Instead of spreading across a broad, thin region typical for standard centrifugation tubes, blood samples separate cleanly in EZPREP tubes, making PBMCs easier to isolate. EZPREP essentially contracts or compacts the band of interest, resolving its margins cleanly instead of allowing them to spread.
Yields and purity of PBMC fractions collected with EZPREP are high compared with standard tubes, in large part because the clean physical separation of fractions facilitates complete and exclusive removal of the intermediate layer. The higher-density fraction just below the PBMC contains neutrophils (e.g. basophils, granulocytes), which begin to die off as soon as blood leaves the body. Dead neutrophils become sticky and adhere to PBMCs, inactivating them complicating their enumeration and characterization.
EZPrep tubes have been shown to provide superior yields, viability, and purity compared with standard blood centrifugation tubes. Table 1 shows slightly lower migration of leukocytes into the PBMC fraction and an improvement of 2.3 percentage points in PBMC viability and in 1-hour viability of around 5%. Even more significant are yield improvements as measured by cell counts, calculated at 58% immediately after centrifugation and close to 100% for EZPrep. Higher viable cell yields translate directly to fewer blood draws and conservation of rare or precious patient samples.
Figure 2. EZPREP flow cytometry PBMC population distribution
Isolating PBMCs involves care and adherence to protocols designed to maintain sample integrity, purity, and homogeneity. Researchers learn these techniques almost by rote, but until now the last step in the process—isolating the PBMC layer from adjacent fractions—was the weak link. EZPREP tubes eliminate concern with isolating pure, viable PBMCs, and afford researchers the full benefits of blood cell isolation methods they have spent years perfecting.
Table 1. Comparison of cell count, viability, and purity for PBMC isolation using conventional blood centrifugation tubes (top row) and EZPrep tubes (bottom).
CLAS Automation, an EZPrep customer, has noted that the tubes “give us a more concentrated and purer PBMC population with the additional processing advantages of easier and faster layering and harvesting steps. The tube configuration has also allowed us to automate key steps in a liquid handler to minimize hands-on time for this usually time-demanding procedure.”
The EZPREP Peripheral Blood Mononuclear Cell Isolation Kit is available from ZenBio for research purposes only.