Blood Fractionation Protocol for Collection of White Blood Cells

1. Dispense 1.2 ml RNAlater into a 2 ml tube for each sample 
   
   For each sample, dispense 1.2 ml of RNAlater into a 2 ml microfuge tube.
   
   For samples expected to contain a higher-than-normal WBC (white blood cell) content (for example, leukemic blood samples) prepare 2 tubes of RNAlater per sample.
2. Collect 2.5-10 ml blood samples
   
   Collect 2.5-10 ml blood samples according to standard procedures in tubes containing anticoagulant (recommended anticoagulant is EDTA sodium or potassium salt).
3. Centrifuge samples at ~1500-2000  X g for 10-15 min at room temp
   
   Fractionate the whole blood by centrifuging at 1500-2000  X g for 10-15 min at room temperature. This will separate the blood into an upper plasma layer, a lower red blood cell (RBC) layer, and a thin interface containing the WBCs (see Figure 1).
   
   Although the suggested procedure is to fractionate the blood as soon as possible after collection, intact RNA has been recovered from EDTA-anticoagulated blood that was stored at room temp for 1 day before fractionation.
   
   NOTE: In a typical clinical centrifuge 1500-2000 X g is ~3000-3400 rpm. For example, in an IEC CL2 table-top centrifuge using the 236 rotor and the 2092S carrier, centrifugation at 3000 rpm = 1560 X g, and 3400 rpm = 2000 X g.
4. Remove the plasma with a transfer pipet, being careful not to disturb the WBCs
   
   
   
   • Use a disposable, plastic transfer pipet (e.g. Falcon Cat #357524)  to aspirate off the plasma (upper layer) down to ~1 mm from the RBCs (see Figure 1). The plasma may be reserved for other applications (for example recovery of viral particles or for serological studies) or discarded.  When removing the plasma do not disturb the WBC layer, also called the buffy coat, which forms a thin film between the upper plasma layer and the lower layer of packed RBCs. Samples with exceptionally high WBC counts will have a thicker buffy coat.
   • Expel all residual plasma from the transfer pipet before continuing.
   
   
   
   
   
   
   
   
   Figure 1. Appearance of Blood Samples during Recovery of WBCs
   
   1. Whole blood in the collection tube
      
   2. Blood after centrifugation
      
   3. WBCs and RBCs after plasma removal
      
   4. Top view of the WBCs (buffy coat)
   
   5. Top view of sample after WBC removal


5. Recover the WBCs in ≤0.5 ml by aspiration
   
   Use the same transfer pipet to carefully aspirate the exposed WBC layer in a volume of about 0.5 ml or less. Aspirate slowly, using a circular motion, to pull all the visible buffy coat material into the transfer pipet. Some contamination of the WBCs with the underlying RBCs is expected. Alternatively, use a cytology brush to recover the WBCs.


6. Put the WBCs into a tube with 1.2 ml RNAlater and mix well
   
   
   
   • Slowly expel the WBCs into a tube containing 1.2 ml of RNAlater. Avoid introducing bubbles into the sample.
   • Recover any visible remaining WBCs using the same transfer pipet, being careful to keep the total volume <0.5 ml (WBCs in excess of ~0.5 ml will exceed the volume of the tube with RNAlater). If the WBCs occupy more than 0.5 ml, split the sample into 2 aliquots using two tubes of RNAlater.
   
   
   Important: At least 2 volumes of RNAlater are required to stabilize the RNA.    
   
   
   • Aspirate up and down several times in the RNAlater to rinse the pipet. Close the tube and mix the RNAlater and WBCs thoroughly by vortexing or inversion.


7. Store the stabilized WBCs or continue with RNA isolation using Invitrogen RiboPure-Blood Kit

This is a potential stopping point in the procedure; the stabilized WBCs can be stored at this point at ambient temperature (up to about 30°C) for up to about 5 days. Storage for longer than 5 days should be at -20°C.