Laboratories use normal reference ranges (NRRs) to identify whether a test result is within the normal range or outside this range (and to thus identify an abnormal result). The relative false positive to true positive rate increases substantially for rare disorders and is a particular problem with congenital disorders such as protein C, protein S, and antithrombin, especially when patient cohorts are inappropriately selected for testing (Favaloro, Emmanuel J, 2012). Age, gender, ethnicity, and blood group might influence reference values for certain parameters of laboratory hemostasis, and/or generate variable test results for some tests. Since there are several factors that can affect lab results, our Director of Laboratory Operations & Product Development, Barbara Young, has shared three insightful tips:
Dry ice is perfect for keeping plasmas frozen during transportation, however, it is recommended to have your plasmas removed from dry ice for at least four hours prior to conducting testing. Removing the plasma from the dry ice allows for the CO2 to dissipate, returning the plasma to its original state. If the plasma is not removed for the recommended amount of time, those sample results will be skewed, possibly exhibiting elevated protime results.
The best practice is to remove your plasmas from the dry ice and then place them carefully into a -70 degrees centigrade freezer overnight. The plasma can be stored in a -20 to -69 degree centigrade freezer for up to five days as long as the freezer is NOT self-defrosting. After 4 hours in the freezer, you may remove the plasmas and begin thawing them for testing.
While there are different methods available for thawing plasma, it is recommended to thaw for three to five minutes in a 37 degree centigrade water bath. A water bath will thaw the plasma rapidly and evenly, unlike a heat block where the heat is concentrated. Sample integrity may be compromised if samples are not completely thawed or if kept too long in a water bath. Also ensure that water bath temperatures are no higher than 37 degrees centigrade as you may start to see deterioration of coagulation factor activities.
Platelets (thrombocytes) are colorless blood cells that help blood clot. Platelets stop bleeding by clumping and forming plugs in blood vessel injuries. However, platelets tend to cause more harm than good when evaluating a patient’s plasma for clotting abnormalities. Any residual platelets can lyse upon thawing, releasing their phospholipid contents which can cause shortening of some clotting assays. In addition, different instrument-reagent combinations may be more susceptible to interference from platelets.
The good news? This can be avoided by double spinning the plasma! It is important that the plasma is completely removed after the first spin, making sure to use plastic pipets and tubes as glass with activate the clotting cascade. Spinning the plasma twice in the same tube is not a good practice because the platelets may return into the plasma. Be sure to spin the plasma, remove it from the tube and then spin it a second time. Feeling dizzy yet?
The International Normalised Ratio (INR) is the PT ratio of a test sample compared to a normal PT (derived from the geo mean normal prothrombin time of 20 or more normal donors) corrected for the sensitivity of the thromboplastin used in the test. The INR is reagent and instrument specific.
Each manufacturer assigns an International Sensitivity Index (ISI) value for any tissue factor they manufacture. The ISI value indicates how a particular batch of tissue factor compares to an international reference tissue factor.
The main reason for re-establishing the mean of the normal range when you change lot numbers of thromboplastin is because the ISI value is likely to change from lot to lot. The ISI reflects the sensitivity of the PT reagent to decreased levels of vitamin K-dependent coagulation factors. A change in sensitivity will affect the calculation of the INR value. Failure to re-evaluate the INR may result in a patient’s Coumadin® dosage being changed unnecessarily.