Tips for Working with Blood Samples | Thermo Fisher Scientific

Blood can be used as source material for a variety of biological investigations. The nature of the study dictates how the blood sample should be handled.

If you are attempting to identify pathogens or metastasizing tumor cells, extracting whole blood might be the only choice if there is no method to specifically capture the cells of interest. Red blood cells (RBCs), which can add a confounding factor, can be lysed using an isotonic NH4Cl solution (J Immunol Methods 1:273 (1972)), and the other cells present may be pelleted in a low-speed centrifugation step. This procedure has also been used for white blood cells (WBCs), but there is some question regarding how it affects WBC metabolism. For DNA investigations, this is probably of no consequence, but if the experiment is aimed at analyzing modulations in immune function, it would be advantageous to isolate buffy coat cells or specific white blood cells as soon after the blood draw as possible. For experiments looking at RNA contained in smaller carriers (e.g., protein complexes or exosomes), plasma or serum is the appropriate blood component to work with.

General Considerations When Working With Blood

Whole (and Stabilized) Blood	The small quantity of RNA in blood is coincident with a very large mass of protein. Phenol-based organic extractions are the best way to clear this protein load. For various reasons, including the presence of contaminating globin mRNA, the RINs tend more in the 7–9 range. The use of RNAlater tissue storage reagent (described in the optional protocol for the RiboPure™-Blood Kit ) causes virtually all protein to precipitate (along with the entire cell load). The resulting pellet will be roughly equivalent in volume to the original blood sample. Human and rodent blood are qualitatively different, probably due to the fact that the RBC sizes are roughly the same (even though there is a dramatic difference in overall blood volume in humans vs. rodents). Expected RNA yields from whole blood are 4–8 µg/mL for human, 60–110 µg/mL for mouse, and 36–80 µg/mL for rat. Since blood is often collected at sites remote from where processing will occur, preservation of the blood for a period of time is often desired. The blood RNA profile can be preserved at room temperature for a few days using RNAlater tissue storage reagent (described in the optional protocol for the RiboPure™-Blood Kit) (i.e., add 2.6 or more volumes of RNAlater reagent). Another method to preserve blood is to use specialized tubes (containing a stabilizing agent) to collect the blood. Two examples are Tempus Blood RNA Tubes and PAXgene Blood RNA Tubes. While these stabilize RNA, they also destroy the blood cells, so subsequent fractionation is not possible. In addition, there are MagMAX™ for Stabilized Blood Tubes RNA Isolation Kits for each of these. Freezing blood is not recommended, as it inevitably produces cell damage. One freeze-thaw may potentially be tolerated (if thawing is at 15–30°C), but two freeze-thaws is generally thought to render the sample unusable for molecular studies (freeze-thaw cycles lyse cells, and then cells release nucleases). Storage at 4°C is commonly used for short periods (≤24 hr). The changes in WBC gene expression that occur during this storage are not well characterized. In general, speed is of the essence, and RNA extraction should Although mature RBCs have eliminated both RNA and DNA during their differentiation, there is a significant presence of globin mRNA from immature RBCs. The GLOBINclear™ Kit can be used to remove this unwanted material.
White Blood Cells	In humans, the concentration of RBCs is approximately 3.9–5.7 x 10¹²cells/L, and the concentration of WBCs is approximately 3.7–11.1 x 10⁹ cells/L, which constitutes a ~1,000-fold difference. Centrifuge the blood at 1,500–2,000 x g for 15 min to separate plasma from RBCs. Centrifuge the blood at 400 x g for 30 to 40 min for a Ficoll-Paque medium cushion. Mononuclear cells (lymphocytes and monocytes) are in the layer over the cushion. Granulocytes (neutrophils, eosinophils, and basophils) are in the layer under the cushion (but above the RBCs). The LeukoLOCK™ Total RNA Isolation System can also be used to isolate WBCs for extraction, by using a proprietary filter to retain WBCs (that allows RBCs to pass through). RNA can be extracted directly from the buffy coat fraction, using standard methods such as TRIzol reagent, RNAqueous kits , or the mirVana™ miRNA Isolation Kit. Often there is still a noticeable amount of globin RNA, which can be removed using GLOBINclear™ technology.
Plasma/Serum	Plasma is from anticoagulated whole blood (cells are removed by centrifugation). Serum is the supernatant from whole blood that has clotted. In addition to cells, clotting removes coagulation factors and fibrinogen, substantially decreasing viscosity. RNA can be extracted from either fluid using the mirVana™ PARIS™ Kit . Although volume expansion is kept to a minimum with this kit, there is still a several-fold increase in volume during the process. Yields are very low, in the range of 0.2 to 1 µg RNA per mL of plasma or serum RNA can be extracted from microvesicles purified by ultracentrifugation or other means, by any of the general methods mentioned above. Storage is less of a consideration, since none of the components present are biologically active. However, it is still advisable to avoid multiple freeze-thaw cycles.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.