Steps to preventing coag test processing error

Laboratories may want to test two specimens, he says, one hand-transported and the other through the system. “Some studies have encountered a greater than 10 percent difference” in the result, he says. “If the difference is less than 10 percent, it’s considered the same. But you have to do a very good study to make sure you’re not getting erroneous results. You have to pick the tests that are the most fragile, if you will, and make sure they are not disrupted or erroneous in nature.”

A number of post-collection factors can lead to hemolysis. Too much shaking of the sample or getting it too hot or cold may cause damage, and Dr. Marlar believes all tubes should be checked if possible. “Sometimes that’s impractical now that we have very large hospitals, very large labs. But we have to be aware of problems when a PTT sample is short. For example, we try to check every sample that comes in if we can, and I think every lab should try to do that. Unfortunately, that requires popping the top off the tube, which can be a hassle.”

But, he adds, “We don’t have a lot of ability to say there’s a clot present in the sample, without automated instruments that may be able to detect clots. If the sample doesn’t make sense, if it’s shorter than they expected it to be, if it’s a heparinized sample and the PTT should be up in the 90s but it’s normal, the technologists should be checking into what’s going on.”

Hemolysis affects PTT results more often, Dr. Adcock says. “Most factors, factors that are PTT based, have a limited stability compared to PT and that’s also true of hemolysis. It can affect both PTT and PT, although PTT tends to be affected more.”

After the freeze-thaw cycle is when a fibrin clot typically appears, she notes. “A red clot is a typical clot and you often see one in the primary tube. But if you receive your samples as a secondary frozen aliquot, and many do, you can still potentially reject a sample because of a fibrin clot, which is a pale clot. If you see that pale gelatinous clot post-thawing, that’s also cause for rejection.”

Interfering substances or HIL—hemolysis, icterus, and lipemia—“are one of the biggest problems we have,” Dr. Marlar says. One of the nice things about the newer instruments now coming out is that they have a system for detecting the levels of HIL, he notes. “If the instrument says the icterus is above the range that the manufacturer provides, the machine will flag it and you can set it up to ‘pending’ and it won’t run at all. Or it can be run and the technologists can make the decision whether to release the result.”

Adjustments may be needed depending on patient population. Because many liver patients are seen at the University of New Mexico Hospital, “our instrument had a lower threshold than what we would like for icterus,” Dr. Marlar says. “So we validated samples with high icterus levels so we can now report out results that are higher for these liver patients who have much higher bilirubin levels.” It was a big plus for the gastroenterologists and patients to have those formerly flagged samples analyzed, he adds, “so we would be able to report them.”

A relatively new addition to the latest ICSH guidance is platelet function testing. When performing platelet function testing, “you’re no longer dealing with proteins; you’re dealing with cells, which are a lot more finicky than a protein. You can disrupt a platelet much more easily than you would a protein,” Dr. Marlar says.

Most previous guidance documents didn’t discuss platelet processing, he says. “We kind of just ignored that and stuck with coagulation, but now it’s becoming an important testing area and we have to do it right.” Specifically concerning the processing phase of platelet function testing, the ICSH guidance includes recommendations on centrifugation, ambient temperature, and time limits.

For centrifugation generally, the biggest problem is not sufficiently spinning down specimens and having too many platelets, Dr. Marlar says. A sample with 20 to 25,000 platelets per mL in it can sit for three or four hours and the platelets may break and the heparin gets neutralized. “We can have artifacts generated that way too,” and if the centrifuge has a cooling system and it cools the sample down too low, “then we get an underestimation on the heparin sample.”

That’s where the standards come in, he says. “We try to make every sample process the same way. That doesn’t always happen but that’s our goal.”

Some coagulation factors are not stable over time, Dr. Adcock notes. “For instance, if you receive a sample on Monday and the doctor calls you on Friday to ask, ‘Can you add a factor VIII to that sample?’ Well, no, you can’t. Because factor VIII is not stable. It is notorious for decreasing over time. So you have to have stability information in your standard operating procedures. Certain assays can be performed and certain assays can’t be performed, because some factors are stable and some are not.”

If a sample is processed within the laboratory’s validation, or the verification based on the literature, “I think you’re okay using the primary tube for storage up to that point,” Dr. Marlar says. “After that, I am reluctant to go get a sample out of the refrigerator that’s been there for 24 hours and come back and do specialized testing.”

In their conclusion, the authors of the ICSH recommendations call for acceptance/rejection protocols that are balanced. Not all questionable results should go unreleased, Dr. Adcock explains. “Even if a sample doesn’t meet your criteria, there can be exceptions. The lab needs to balance the risk of releasing unsafe or misleading results against the risks associated with rejection. So you could go ahead and release the results in several instances.”

“For example, PTT-based samples are subject to more variation than PT-based results. Say a sample coming to you for PT INR rolled off the lab table and was found on the floor 24 to 48 hours later, so it’s out of stability. The lab tech tests it anyway. The patient’s always been therapeutic and you look at previous results. It may be best just to report that therapeutic result rather than have the patient come in again.”

Another example is antithrombin. “Antithrombin is really stable, and if a sample is eight days old, rather than the stated typical stability of seven days, the antithrombin result is still good. Also, if you know that a sample beyond stability will falsely elevate D-dimer and the D-dimer result is below the laboratory’s or manufacturer’s determined cutoff, you’re still good.” Those types of situations should always be taken to the medical director, she says, so they can make the ultimate decision about reporting.

One could reject a sample that doesn’t meet criteria, Dr. Adcock notes, “but our International Journal of Laboratory Hematology article is saying that there’s always an exception and you have to consider the impact on the patient of performing the assay or rejecting the sample.”

The last recommendation of the ICSH guidance is that all coagulation labs establish written policies on transport, storage, and processing of both primary tubes and secondary aliquots. Dr. Adcock does not see that as having much impact since it is part of good laboratory practice to have such policies. But now that this new guidance is out, she suggests that laboratories review current practices to be certain they are in keeping with the guidance.

The ICSH recommendations are based on evidence, on published, peer-reviewed research by experts, Dr. Marlar says. Clinical labs processing citrated blood samples for coagulation tests need to look at these recommendations, check the accreditation requirements for their country or state, then focus on what they are doing that is different. “Then they need to find out how to change what they’re doing, or they should document that it’s being done correctly and they are not getting potential erroneous results.”

The recommendations are meant to apply in all regions of the world, Dr. Adcock says, and she has found that reaching an international consensus has not been problematic. In working with standards experts in Italy and Australia, she has found that “we speak the same language. The samples are the same, as are things that we’ve verified in our laboratory and they’ve verified in their laboratories. And it was rewarding early on in our interactions because we had the same battles, the same issues, the same resolutions.”

How much adjustment will be needed by laboratories to meet the new ICSH recommendations? “If institutions have well-described processes and if they’re consistent with the current guidance, then all should be good,” Dr. Adcock says.

But, she says, laboratories will have the best results if they don’t make assumptions. To ensure that coagulation processing is up to the mark, she says: “Read the guidance document, look at your processes, and make sure they are being followed as part of good ISO practice. And always do what you say you do.”

Anne Paxton is a writer and attorney in Seattle.