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Coagulation Corner

Monday, March 6, 2017

What Policies Do We Need In The Coagulation Laboratory?

Written By Donna Castellone, MS, MT (ASCP) SH | LinkedIn

Rules, regulations, polices can in many cases can complicate what we do in the laboratory. But there are many policies that need to be part of ensuring quality in the coagulation laboratory. They should be part of standard operating procedure to ensure optimal testing and cover pre-analytical, analytical and post analytical phased of testing. Check and make sure these processes are in place!

Up to 64% of errors in coagulation occur in the pre-analytical phase. Everything from how the sample is collected, the ratio of blood to anticoagulant, and the stability of the sample. Gone are the days when the laboratory can say, I can't control that, so I have to accept it. Let's look at sample stability. Most laboratories follow CLSI guidelines, which give you that 4 hour stability for aPTT testing and 24 hours for PT- unspun and at room temperature. What happens if you can't meet that 4 hour window? Test your instrument reagent combination for results across the reportable range, and see what happens, many publications are now seeing a longer window of stability on patients not on heparin- up to 8 hours. Make sure you have a robust study to demonstrate this. Same with overfill and underfilled tubes. By looking at the same sample collected at different tube volumes can justify what results best reflect truth. This can provide testing personnel with a clear cut understanding of what volumes can really impact patient results.

Testing must be performed on platelet poor plasma. Platelet poor plasma is defined as plasma with a platelet count <10 X 109/L. We know that platelets are a source of phospholipids and can interfere with coagulation reagents and falsely shorten results. It has been shown that for routing testing platelet counts of up to 200 x 109/L do not impact results. However, if that is your laboratories policy a purposeful study must be done to implement that, in particular since most manufacturers' package insert state their FDA approval was granted based on using platelet poor plasma. In order to demonstrate that plasma used for coagulation testing is platelet poor, centrifuges should be checked twice/year.

All coagulation studies must be performed in a sodium citrated (3.2%) anticoagulated tube. The proportion of the blood to anticoagulantis 9:1. Inadequate filling of the tube, may cause inaccurate results. This is problematic in patients with a HcT that is >55%, which will alter the ratio due to excess red blood cells.

If a patient has a high Hct, the amount of citrate may be too high for the amount of plasma and may falsely prolong the aPTT. To adjust the amount of citrate for a high Hct, the following formula is used:
C= volume of NaCitrate
V= volume of whole blood
H= Hct %

To collect 2.7mls of blood from a patient with a HcT of 64% formula is:
C = (1.85 x 10) -3 x (100-64) x 2.7mls
0.0185 x 38 x 2.7= 0.18 mls of citrate.
The citrate in the collection tube should be adjusted for the high hematocrit. This will adjust the ratio to be in correct proportion, and eliminate a falsely prolonged result.

Coagulation tubes should have a 90% acceptable fill rate, that is 10% over 2.7 ml or 10% less. But what about those samples that fall just shy of that- do we cancel? Do we know if we perform the test and the result is 11.9 and maybe would have been 12.2- maybe that is statistically significant but is it clinically significant? Are you cancelling too many routine tests? Do a study, look at the same donors/patients collected at different tube volumes. This will provide you with information coagulation results are impacted by volume, for your instrument/reagent combination.

Coagulation samples should be visually inspected to check for the presence of clots. If a coagulation sample has a clots present, it may impact the outcome of coagulation results. Using applicator sticks have the potential to activate the plasma coagulation cascade and most probably platelets as well, either by direct contact or as a secondary consequence of plasma activation. If you run a sample and the results are questionable, checking the sample with applicator sticks may provide information on the integrity of the sample.

What do you do with those samples after you get them? Do you store them at room temperature? What about samples for heparin levels are they tested in a timely manner? Or do the cells sit on the buffy coat? Remember the platelets release PF4 which will neutralize heparin, and your anti-Xa levels and your aPTT will be effected. If you need to be convinced, take an aPTT sample from a patient on unfractionated heparin- that is about 4 hours old, and re-run the aPTT, you will be amazed at the difference in the aPTT result. Think of the ramifications. If clinicians are using this to monitor UFH, and the results are much shorter than expected, they might increase the amount of heparin they are giving to the patient, putting them at a risk for bleeding! Also, remember FV and FVIII are heat labile, they don't last long, and FVII can be activated by the cold. Coagulation factors are inversely proportionate to screening tests, low factor levels will prolong PT or aPTT testing, while if a factor is elevated or activated, the PT or aPTT will be shortened. All laboratorians should understand how storage conditions can influence outcomes.

Do you aliquot samples? What kinds of tubes? Do you freeze at -20 or -80? Are the tubes 12 x 75 or are they put into specific aliquot tubes, that are smaller to match the volume that is being stored. If you have too much empty space, when you freeze those samples, if ice forms on the inside, upon thawing it can dilute your sample. Speaking of thawing, it should be done in a water bath which is at 37 degrees for 5 minutes. Samples should not be thawed by sitting on the lab bench.

So that is a lot of stuff, and we haven't even considered the testing process- equipment. Reagents and sensitivity. All of these scenarios can impact coagulation testing results. While policies may be difficult to keep up with, and there always seems to be another regulation around the corner, these are important practices that should be adhered to in every coagulation laboratory. Your results depend on it.



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