Collection tubes are typically 10 mL; coagulation tubes, used to collect samples for coagulation determination, are typically 4 mL.1 “There is some good news here,” says Kaufman. “Advances in the tests and assays themselves have allowed smaller and smaller volumes for testing, into the range of nanograms.” That should translate to smaller and smaller blood volumes in the coming years.

Written protocols or lab manuals should be reviewed prior to a blood draw for specific requirements associated with a given test. Given the rapid accumulation of new biomarkers and tests, the updating of lab manuals represents a conundrum, cautions Kaufman.

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“Typically, the lab manual does not require IRB [Institutional Review Board/Human Subjects Review Committee] review and can be changed relatively easily,” she notes. “That’s fine when administrative details need to be updated. However, if something in the lab manual changes that affects the protocol or budget—volume of blood, ultrasound guidance for a biopsy—then it should be subject to review.”

Depending on the intended analysis, the blood collection tubes will contain anticoagulants or preservatives, as indicated by color-coded caps.1 Red tube caps typically indicate no additives, such as those used for identifying antibodies, immune proteins, or lipids; whole-blood coagulation tubes have light blue caps; lavender or royal blue caps contain clot-preventing EDTA for complete blood counts and DNA extraction for genetic mutation analysis; gray-capped tubes contain sodium fluoride to preserve glucose and allow electrolyte analysis; and green-capped tubes contain heparin to prevent clotting for rapid blood chemistry labs.1 Yellow/purple (bicolored tiger-striped) tube caps usually represent collection tubes for circulating tumor cell (liquid biopsy) analyses of cancer genomes and genetic mutations.1 Close attention to collected volumes is necessary, particularly as a collection tube’s expiration date approaches and tube vacuums grow weaker, resulting in incomplete filling.1

Blood collection supplies should be organized and reviewed before placing the tourniquet, Kaufman and coauthors emphasize.1 “(T)he longer the tourniquet is in place, the higher the risk of hemoconcentration of nonfilterable elements such as proteins in the blood,” they explain.1 “If the blood must be drawn from the same arm as an intravenous access site, then the tourniquet must be placed several inches above the site and only tight enough to restrict superficial venous flow. Tourniquets should not be left in place longer than 2 minutes before being loosened.”

Once filled, blood collection tubes are completely inverted gently and repeatedly, typically five to 10 times. One inversion involves turning the tube completely upside-down (180˚) and back to cap-end-up1 (Table 1). If the protocol calls for clot formation within the tube, the tube is left at room temperature for a minimum of 30 minutes.1

TABLE 1. Tube handling for blood collections1 

Tube/syringe order of draw Number of inversions
Plain syringe None
Royal blue (plain metal) 5
Special coag (screw cap) 3-4
Blue 3-4
Black 3-4
Gold (calcium & other SST)
Mint green
Royal Blue (EDTA metal) 8-10
Black/tan tiger (sequenom)
Cellsave (purple/yellow) 8-10
Gray 8-10
Yellow 8-10
Pyruvate (screw cap) Shake vigorously 10 times
QTB tubes (3) Immediately after filling tubes, shake 10 times just firmly enough to coat tubes
PAXgene (RNA) 10
Lithium heparin syringe Mix the sample horizontally for 30 seconds and then invert several times
KEY: CAI, free ionized calcium; EDTA, ethylene diamine tetra acetate; RNA, ribonucleic acid; SST, serum separation tubes.

Processing collected blood samples may also include centrifuge spinning at specific speeds and durations to precisely fractionate the sample into readily visible layers, with red blood cells at the bottom, above which will be found white blood cells and plasma on the top, respectively.1 Carefully following written protocols’ specific guidance on centrifuge timing and speed is crucially important to avoid degrading the blood sample, as incorrect timing can leave blood components inadequately separated or damage blood cells.1 After a sample is centrifuged, the layers are collected with a pipette into separate tubes for subsequent analysis. Naturally, each sample must be carefully documented.