Separating Whole Blood by Multiphase Systems

DANIEL J. KRAMER

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Figure 1: Graphical abstract highlighting major points of research. (a) White blood cells (WBCs) separated from whole blood while simultaneously separated into their mononuclear cell (MNC) and polymorphonuclear cell (PMN) subtypes experience changes in osmotic pressure and pH that damage the cells and make them unviable for further study. (b) Aqueous polymer phases, calibrated with proper densities, osmolalities, and pHs, could be used to separate WBCs from whole blood and directly into their subtypes while maintaining morphological viability. (c) Whole blood effectively separated by density into red blood cells (RBCs), WBCs, and plasma. WBCs appear to be separated into MNCs and PMNs, cell extraction for identification ongoing.

The ability to calibrate changes in density within a single volume of solution brings with it exciting possibilities for improved separation methods of mixtures of biological materials. Aqueous multiphase systems (MuPS)—layers of identical solvent containing different solutes—allow for stable separation of immiscible polymers, which are sorted in the column by density (Figure 2) (Akbulut et al. 2012). Phases are distinct (Figure 3) and maintained over time (Mace et al. 2012), so that density is uniform in each. This produces sharp density changes that can be calibrated to as small as 0.001 g/mL at each interface between phases.

The maintenance of distinct density phases, with small density changes between phases, as well as their low cost and ease of use could make MuPS an extremely valuable tool in point-of-care and clinical diagnostics involving samples of biological materials of varying cellular density. Current work includes diagnostic techniques for sickle cell disease and various forms of anemia. Density fractionation is necessary in these diagnostic techniques because structural deformation of red blood cells (RBCs) and changes in their chemical composition, such as the amounts of iron or hemoglobin, change the density of these cells. When cell density is changed, their separation pattern in the column is also affected.

The goal of my project was to evaluate and improve the use of MuPS in the separation of human blood components. I aimed to separate human leukocytes, commonly known as white blood cells or WBCs, from whole blood. I also hoped to separate, within the same column, the WBCs into their two subtypes: the less dense mononuclear cells (MNCs) and the denser polymorphonuclear cells (PMNs). Each of these cell types plays unique roles in immunity and cellular repair that are of particular interest to clinical and biomedical researchers.4 Current methods exist to isolate WBCs from whole blood. However, the only methods that also separate WBCs into MNCs and PMNs require additional steps with osmotic pressures and pH levels outside the physiological normal range, which would damage the cells and make them unviable for further study (Figure 1).

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Figure 2: Representative phase separation of whole blood in Ficoll/Dextran aqueous multiphase system (MuPS) with image of final separated state. RBCs—denser than both phases—sink to the bottom of the tube, PMNs at intermediate density move in between the phases with mixed plasma, and the less dense MNCs remain above both phases with lighter plasma and cellular debris.

The major benefit of MuPS its environment, which is similar to normal body conditions. MuPS controls for pH and isotonicity, allowing separated components to be extracted and used in further study. The two water phases in our MuPS contained Ficoll and Dextran, two sugar polymers of high molecular weight, neither of which are dangerous or reactive; in fact, they are used in the colored shell coating of M&M’s candies.

The densities of MuPS phases were calibrated to be between the densities of different blood cell types in order to separate cells by their type. The high-density RBCs sank below both phases, most PMNs were observed to stay between the two phases, while MNCs, mixed plasma, and cellular debris appeared to remain above both phases (Figure 3) (Pember, Barnes, Brandt, & Kinkade, 1983). Cell layers are currently being extracted from separated columns to verify cell type and confirm viability. Another benefit of MuPS for cell fractionation is the relatively clear visibility of the separation process. Blood components can be observed moving through the system in real-time (Figure 2). Also, because the phases are clear, distinct and vibrant color patterns appearing in the column are indicative of cells present in that region. Under the bottom phase a dark red can be seen, indicating RBC compaction, while the bottom phase is a light shade of red, indicating RBCs that did not reach the bottom of the tube (Figure 2). As more RBCs reach the bottom of the column, fewer will remain in the Dextran phase, representing a more effective separation. The top phase is typically clear to light yellow, its shade coming from yellow plasma.

The goal of my project was to evaluate and improve the use of MuPS in the separation of human blood components.

As I conducted these experiments to study the use of MuPS, I encountered some issues for future consideration. The PMNs positioned between the phases, while having no distinct color of their own, possess neither the red color of the RBCs below nor the yellow color of the plasma above, producing a distortion in shade between the phases that makes the PMNs appear in a light but distinctly visible band (Figure 3). Finally, upon addition of whole blood to a MuPS of aberrant tonicity, RBCs began to separate themselves into two layers independent of expected separation. This was due to some RBCs losing water, increasing their density and causing them to sink beneath non-dehydrated RBCs (Figure 1c, third panel). This phenomenon made observation of later density-based separation impossible. Properly-calibrated osmotic pressure is not only necessary for experimental use of the cells after separation, but also necessary for MuPS to work properly.

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Figure 3: Replicated MuPS phase separation in I mL Eppendorf tubes suggests presence of PMN-containing layer of intermediate density between phases.

While proper cell types have been observed in each layer, specificity and enrichment levels of cells in between the phases are not yet high enough to offer immediate research benefits. To this end, in the next stage of my work, polymorphonuclear neutrophils—key players in innate immunity and wound healing—will be further isolated by adding lumican from the extracellular matrix (ECM) to the MuPS system (Lee, Bowrin, Hamad, & Chakravarti, 2009). Lumican specifically binds neutrophils’ β2 integrin receptors, causing them to move toward the bottom phase, thus serving as a model for neutrophil migration and cellular adhesion.

Acknowledgements:

Thank you to Dr. George M. Whitesides, TJ Martin, and Melissa LeGrand for hosting me in the laboratory of the Whitesides Research Group. Thank you to Dr. Barbara S. Smith for teaching of essential methods and collaboration on work. AJ Kumar, Matt Patton, and Dr. Jon Hennek provided guidance and vital insight on the experimental process. Greg Llacer enabled me to remain on the Harvard campus as a Summer Research Fellow through the duration of my project. This work was jointly supported by the Harvard College Program for Research in Science and Engineering (PRISE) and by a Pechet Family Fund undergraduate research grant.

References:

1.   Akbulut, O., Mace, C.F., Martinez, A.W., Kumar, A.A., Nie, Z., Patton, M.R., and Whitesides, G.M. (2012). Separation of Nanoparticles in Aqueous Multiphase Systems through Centrifugation. Nano Letters, 12, 4060-4064.

2.   Mace, C.R., Akbulut, O., Kumar, A.A., Shapiro, N.D., Derda, R., and Whitesides, G.M. (2012). Aqueous Multiphase Systems of Polymers and Surfactants Provide Self-Assembling Step-Gradients in Density. Journal of the American Chemical Society, 134, 9094-9097.

3.   Pember, S.O., Barnes, K.C., Brandt, S.J., and Kinkade, J.M. (1983). Density Heterogeneity of Neutrophilic Polymorphonuclear Leukocytes: Gradient Fractionation and Relationship to Chemotactic Stimulation. Blood, 61, 1105-1115.

4.   Lee, S., Bowrin, K., Hamad, A.R., Chakravarti, S. (2009). Extracellular Matrix Lumican Deposited on the Surface of Neutrophils Promotes Migration by Binding to 2 Integrin. The Journal of Biological Chemistry, 284, 23662-23669.

Daniel J. Kramer is a Brevia guest writer.  He can be reached at dkramer@college.harvard.edu.