Standardization in Coagulation- Or the lack of it- What’s A Laboratory to do?

Where do we get our guidelines in coagulation? There are several agencies that provide guidelines. The Clinical Laboratory Standards Institute (CLSI –formally NCCLS – National Committee for Clinical Laboratory Standards.) provides many publications regarding everything from the PT, APTT, Factor Assays, Fibrinogen, How to validate reference ranges and instruments, collection of specimens and platelet aggregations. The College of American Pathology (CAP) provides checklists that cover everything from adequate space to proficiency testing, heparin and direct thrombin inhibitor contamination, performing heparin therapeutic ranges and criteria for factor assays. Let us not forget CLIA and JCAHO providing safety initiatives, quality control and validation guidelines. With all of these institutions and their regulations, it is still difficult to decide by a standardized format if a mixing study has corrected!

Coagulation itself provides many challenges. Regardless of the guidelines and attempts at standardization, there are many variables. Pre-analytical variables account for up to 64% of errors, many of which are not controllable by the laboratory. (blood collection, pouring from one tube into the next, heparin contamination, tubes floating around the pneumatic system, and of course the sample that sits on a desk unlabeled and then gets a label smacked on it- any of these sound familiar?) The next issue is the instrument/reagent combination – there are many possibilities, just look at a CAP survey! Reagents have different sensitivities, dilutions can be made in saline, or owrens buffer, lyophilized plasma or fresh frozen deficient plasmas can be used, and what about the standard that is used? It is important that a standard be calibrated against a WHO standard. You should also know how that standard is tested. Is it on a different analyzer; with a different reagent? You might want to test it several times on your reagent/analyzer system to determine a more comparable value for your system.
Also, running independent standards as a patient, can give insight as to how your analyzer is performing. If you feel that you have an instrument bias that you can’t seem to resolve on a test, you can use a trusted standard to implement a correction factor. For example, lets say you always run slightly high on your factor VIII’s, this is confirmed by your proficiency testing. If you run a standard, several times and note that you are consistently higher that the value, even after making all the attempts to correct this ( new reagents, maintenance, water, standard curve, and whatever other tricks you have up your sleeve!) it might be helpful to adjust those values. If the standard is assayed at 85% and you are getting 100%, you are too high, and need to bring those values down.

You would apply the following formula:

• 85% (Value of the Standard)
• 100% ( Value Obtained) =0.85 would be the multiplier to bring the value down.
  

If your result was 120% x .85=102%. You need to really make sure this is the issue, you need to have everyone on board to do this, or your values will be all over the place. You should verify this over a period of time.

So what about correcting that mixing study, well there are no clear cut guidelines. If you ask 10 labs you might get all different answers. Some labs use a correction into the normal range, others use within 1-2 seconds of the pooled normal plasma (PNP), while still others use a formula called the Index of Rossner. Other non standardized practices include: Do you perform an incubated mix, how long do you incubate, do you use a 1:2 dilution or a 1:4 dilution? Too many questions- so how does a laboratory judge what is best? It is important to define what you use and to implement standard operating procedures, so that the work that comes out of your laboratory is consistent, and becomes standard for your coagulation laboratory and the clinicians can count on that when they evaluate patient results.

With all of these issues how do you ensure good results? The coagulation laboratory needs to minimize and control what they can to provide good outcomes. Relying on proficiency testing and peer group evaluations from quality control groups provide laboratories with a lot of knowledge as to how they are performing. So what can you do if your results are not within the state limits?

You have several options:

1. Rerun the sample in question.
2. Rerun old proficiency test samples to see how the results are in compared to what they were assayed at, this material is gold and provides a ton of information.
3. Look at running independent controls to see where they are running.
4. Determine if there was a change in reagents, plasma or a new standard curve was performed.
5. What about maintenance on the analyzer, was one due?
6. Water can always be a problem, try using sterile water.
7. Pipettes, are they calibrated? Regulations state to calibrate within a stated period of time, but if they are used frequently and are important in your assays, twice a year may not be sufficient.
8. Run a precision study in the range in question, this may demonstrate carryover or an analyzer drift.
   

Another issue that plagues coagulation laboratories is setting acceptable limits between dilutions for factor assays. This is important in determining if an inhibitor is present or if the dilutions need to be rerun. First, it is very important for all results to be on the curve; an extrapolated result should not be used. After this criteria is met, an allowable acceptable limit between dilutions should be determined by the laboratory. If the manufacturer has guidelines, they should be adhered to, if not, keeping good clinical practices in mind, the laboratory should determine their own. Classifying an inhibitor can be tricky, what information are you giving the clinician? If the value is within the normal - high range, and is a slight inhibitor, other than being a nuisance to the physician, might be better averaging the results. A true inhibitor going from the abnormal to the normal range can be reported out at the low dilution, and the comment of the increasing percentage of activity at the highest dilution.

The International Society of Hemostasis and Thrombosis subcommittees meet each year. For 2008, they will meet in Vienna and discuss everything from Lupus anticoagulants to thrombin generation. They publish the minutes from their meetings and their progress to implement standardization. Their website is www.isth.org and can provide insight to their ongoing mission for standardization.

Adhering to good clinical practice and standard operating procedures within your own laboratory and striving to provide clinicians with good results is the first step in standardization. The ultimate goal is to provide good consistent patient results.