When the floor runs out of blue tops and asks if I can “make some more”, sure let me just get a light blue sticky note and tape it to a red top. In all seriousness, coagulation samples are especially susceptible to pre-analytical errors. The frequency of pre-analytical errors has been estimated to be as high as 5% of all blood collections for coagulation testing, with notable variations among medical disciplines.1 Spurious hemolysis can be prevalent in emergency departments, while clotted samples can be common in neonatal and pediatric settings. Blood collection technique, needle gauge, blood flow, prevention of venous stasis, ratio of blood to sodium citrate, and overall sample quality are especially important pre-analytical factors for coagulation.
In order to improve the quality of patients results, sample collection and handling should follow the CLSI guideline H21-A5; 2008. The recommended needle size for coagulation blood sampling is 19 - 22 gauge to ensure that blood flows quickly and evenly. If collection is too slow, venous stasis can take place, however if it is too fast platelet damage can occur. Collection staff should avoid probing the vein with the needle as it causes vascular injury with release of tissue factor, may initiate clotting and shorten PT & PTT results. Traumatic venipuncture may also increase the following acute phase reactants FVIII, fibrinogen and von Willebrand factor (vWF). Blue top 3.2% sodium citrate tubes must have anticoagulant in ratio of 9 parts of blood to 1 part anticoagulant (9:1 ratio). Sodium citrate tubes require at least 90% fill of vacutainer, gently mixed with blood immediately and thoroughly according to the tube’s manufacturer instructions. The incorrect ratio of sodium citrate to blood in an underfilled tube results in a dilutional effect, excess calcium binding and falsely prolonged clotting times. Of note, 2 partially filled blue tops cannot be combined to make 1 full tube. Overfilling sodium citrate tubes theoretically risks clotting, as the ratio of additive anticoagulant to whole blood may be insufficient.
Coagulation testing requires platelet poor plasma, defined as a <10,000/µl platelet count, which is critical for frozen plasma aliquots. A high platelet count will neutralize heparin activity, lower PTT and/or anti-Xa results and mask the presence of a lupus anticoagulant causing a false negative. Remember, no coagulation centrifugation in refrigerated centrifuges. RPM’s differ at how many g’s they are by different centrifuge manufacturers. Be careful with Stat Spins because platelets diffuse back into the plasma. Have written procedures about how centrifuges will be validated including all centrifuges where coagulation samples are being spun and aliquoted. Secondary tubes used for plasma-based coagulation assays should be composed of non-activating material, such as polypropylene, and not polystyrene.
As a closing note, know the limitations of your assays when it comes to hemolysis, icterus, and lipemia. Other pre-analytical factors to take into consideration concerning laboratory automation is the possibility that red blood cells and other cellular elements may contaminate citrated plasma when samples travel at high speed, long distances, and/or pass by other elements of an automation track such as other tubes or racks. Remember there is no stable separation after centrifugation by gel or other physical barriers between citrated plasma and red blood cells.2
When a sample is compromised the test result may reflect the status of the sample, but not reflect the clinical status of the patient!
References
- Lippi G. Preanalytical Issues Specific to Coagulation Testing. lecture presented at the: Trainee Council in English; March 15, 2023. https://www.myadlm.org/science-and-research/clinical-chemistry-trainee-council/trainee-council-in-english/pearls-of-laboratory-medicine/2014/preanalytical-issues-specific-to-coagulation-testing. Accessed March 15, 2023.
- Lippi G, Da Rin G. Advantages and limitations of total laboratory automation: a personal overview. Clin Chem Lab Med. 2019;57(6):802-811. doi:10.1515/cclm-2018-1323