Red blood cell contaminants from source material

RBC contamination ultimately comes from the selected starting material. Manual isolation of the often thin buffy coat from whole blood carries the greatest risk for error. RBC lysis buffers and washing steps can help reduce contamination from heterogenous starting materials. However, these methods add to the total reagent and labour costs.

Downstream impact

Once cells are isolated, typical cultures require activation and expansion. It is in these steps where RBC contamination has the greatest impact.

Peripheral blood mononuclear cells (PBMCs) must be properly enumerated to determine the concentration of activation and isolation reagents. Appropriate cell density is also a critical determinant for the long-term health of a cell culture. Lingering RBCs and platelets can make accurate lymphocyte enumeration difficult and disrupt flow cytometry.1 Inaccurate enumeration methods may therefore impact activation kinetics, isolation purity and expansion times if the cell culture is overly diluted.

Expansion must be sustained throughout the duration of a culture, so cytokine growth factors are frequently added to cell culture media. In T-cell cultures, IL-2 synthesis is suppressed by RBC-exposed T-cells, and the addition of exogenous IL-2 does not rescue proliferative capabilities.2 This can increase expansion times, adding to associated labour and media costs.

Open, manual operations to reduce RBC contamination increase the number of touchpoints, thus increasing the risk of microbial contamination. It may take several days of culture expansion for these errors to be detected, resulting in wasted supplies and effort. Closed system technologies exist that can reduce this risk, but the consumables for these devices are often several times more expensive than the price differential between whole blood and purified cells as a starting material.

Upstream mitigation

While the temptation exists to source PBMCs from whole blood or LRS cones, any cost savings may quickly disappear due to contamination. Leukapheresis products have long been the gold standard for starting material within the cell therapy field. However, for R&D applications, isolated immune cell subsets can reduce your processing time and guarantee a high level of starting material consistency.

References
[1]?Fesnak, A., Lin, C., Siegel, D. L., & Maus, M. V. (2016). CAR-T Cell Therapies From the Transfusion Medicine Perspective. Transfusion medicine reviews, 30(3), 139-145.
[2]?Long, K., Meier, C., Bernard, A., Williams, D., Davenport, D., & Woodward, J. (2013). T-cell suppression by red blood cells is dependent on intact cells and is a consequence of blood bank processing. Transfusion, 54(5), 1340-1347.