Tips and Tricks: Working with Blood Samples

8 Tips and Tricks for Working with Blood Samples 

Red and white blood cells

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 white blood cells 

1. In humans, the concentration of RBCs is approximately 3.9–5.7 x 10¹² cells/L, and the concentration of WBCs is approximately 3.7–11.1 x 10⁹ cells/L, which constitutes a ~1,000-fold difference.

2. Centrifuge the blood at 1,500–2,000 x g for 15 min to separate plasma from RBCs.

3. Centrifuge the blood at 400 x g for 30–40 min for a Ficoll-Paque™ medium cushion.

4. Mononuclear cells (lymphocytes and monocytes) are in the layer over the cushion.

5. Granulocytes (neutrophils, eosinophils, and basophils) are in the layer under the cushion (but above the RBCs).

6. The Invitrogen™ 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).

7. RNA can be extracted directly from the buffy coat fraction, using standard methods such as Invitrogen™ TRIzol™ reagent, RNAqueous™ kits, or the mirVana™ miRNA Isolation Kit.

8. Often there is still a noticeable amount of globin mRNA, which can be removed using Invitrogen™ GLOBINclear™ technology.

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