Specimen Collection Manual and Test Catalog

2 mL

• If the patient’s hematocrit (HCT) is >55%, the volume of anticoagulant in the tube must be adjusted. Contact a performing location for sodium citrate adjusted tubes or instruction for how to adjust the sodium citrate.
• When possible, use fresh venipuncture.
• Avoid prolong tourniquet time (< 1 minute) and hemolysis during collection as this will alter results.
• If indwelling catheter or butterfly collection device must be used, draw sample from a non-heparinized lumen or flush the line with 5 mL of saline and discard the first 10 mL of blood.
• Tubes should be >90% filled.
• Immediately mix gently after collection by inverting the tube end over end 5 to 6 times. Avoid vigorous mixing or additional inversion. Observe for the presence of clots or hemolysis and recollect if observed.

Centrifuge specimen immediately at designated time and speed to obtain platelet-poor plasma (<10,000/µL). Using a plastic pipette, carefully remove plasma from cells, avoiding platelet/buffy layer by leaving a thin layer of plasma on top the cells. The centrifuged plasma should be aliquoted (1 mL per aliquot) into clearly labeled polypropylene tubes. The number of tests ordered will determine the aliquots needed. Check the residual specimen for clot and if present discard sample and redraw specimen. If plasma is icteric, lipemic or hemolyzed a lab comment should be generated to notify staff. Freeze upright in non-thaw freezer.

Frozen on dry ice

Room temperature (whole blood): 4 hours 
Room temperature (plasma off cells): 8 hours 
Frozen (plasma) -20°C: 30 days 
Frozen (plasma) -70°C: 6 months

Clotted, hemolyzed, improperly filled tubes, improper anticoagulant ratio (HCT >55% and citrate not adjusted), refrigerated specimens, or stability exceeded.

Free and Total Protein S also rejected if grossly icteric or lipemic.

Note: Specimen suspected of thawing in transport, indicated by slant in aliquot or specimen in lid of tube, will have testing performed, and the comment “interpret results with caution as thawing suspected” added to results.

Protein S activity: 65-140% 
Free Protein S: 50-134% (female), 70-148% (male) 
Total Protein S: 60-140%

Anticoagulation Impact on Coagulation Tests

Protein S activity and if abnormal free protein S antigen and total protein S are added to sample.

Functional Protein S, Free Protein S, Total Protein S, PROTSB

Protein S is a vitamin K-dependent protein produced by the liver that acts as a cofactor of activated protein C to prevent blood clot formation. Low protein S levels can be associated with an increased risk for a blood clot. Protein S deficiency is congenital or acquired. Acquired causes include liver disease, pregnancy, vitamin K deficiency, coumadin therapy, proinflammatory state, and disseminated intravascular coagulopathy (DIC).

Protein S circulates in the blood as a free protein (approximately 40%) or it forms a complex with C4b of complement (60% of the protein). Only the free protein S (not bound to C4b) is functional and prevents clot formation while the C4b-bound protein S is not functional.

Therefore, the assessment of protein S includes 3 assays. The screening assay protein S activity, which is a functional measurement of the protein using a clot-based assay based on inhibiting factor Va. If protein S activity is abnormal the circulating protein in the blood/plasma is measured to subclassify the deficiency.

Limitations of assays:
Protein S activity:

• falsely low in patients with factor V Leiden mutation, elevated factor VIII (>150%) and lupus anticoagulants
• overestimated in patients on direct thrombin inhibitors, anti-Xa inhibitors, and supratherapeutic heparin

Protein S Antigen (free and total):  

• overestimation of levels occurs in patients with elevated rheumatoid factor and decreased levels with cloudy plasma (icteric or lipemic specimens)