2012 Cheat Sheet

A puzzler from Pam Owens: We recently encountered a patient on warfarin whose INR is 2.22, protein C and S are in the 30–40% range, but her factors VII and IX are normal. What could cause this?

George posed this to colleagues while attending the ASCLS/AACC annual meeting. Nobody had a really good answer, so he’s taking a “scientific wild guess.” Assuming factors II and X were reduced, and that V and VIII were normal, the patient may have recently discontinued warfarin, so that VII and IX, with half-lives of 6 hours and 24 hours, respectively, had recovered whereas II and X, half-lives of 60 hours and 48–52 hours had not. The low II would account for the INR , which should return to normal as their activity levels reach normal. The weakness in George’s argument is that protein C, whose half-life is also around 6 hours, should also have returned to normal!

A July 25 comment from Pam: The protein S antigen was decreased as expected, but her protein C antigen was normal. Makes one think she may have a type 2 deficiency (that would explain that pesky half life issue), but we are recommending the clinician draw no conclusions while she is on warfarin.

July 26: George thinks these results help to strengthen his guess that the warfarin had been recently discontinued, as you would anticipate that protein C would recover at nearly the same rate as factor VII.

From Teresa Lovejoy, Children’s of Alabama: Why is it not important to double-spin D-dimer specimens for PPP when there’s a delay in testing? Is this because D-dimers are not affected by labile factors?

George mentioned that Children’s of Alabama is moving into a magnificent new building that now dominates the Birmingham’s medical center skyline. The purpose for PPP is to ensure no platelet secretions affect coagulation test results. Platelets may become activated in vitro and secrete, for example platelet factor 4, which neutralizes heparin and artifactually shortens the PTT. Platelets also secrete factor V, VIII, and VWF , and release plasma membrane phospholipids that interfere with LA testing. Platelet materials become especially critical when plasma is frozen, as cells become ruptured.  The D-dimer concentration seems to be unaffected by platelets, as D-dimer is a product of fibrin crosslinking and fibrinolysis. Consequently, it is not necessary to prepare PPP for a D-dimer assay; however, there is no harm in it, either. Since most labs prepare PPP for all coagulation testing, most D-dimer assays probably are performed using PPP.

From Thomas Exner, Owner, HAEMATEX RESEARCH, Sydney, Australia: “I read your response to a question about LA mixing tests. I’m a little disappointed that you still recommend prolonged incubation times. Most recommendations now are that LA mixes should be tested immediately. There is no logical reason for time-dependent LA testing since they work by interfering with the phospholipid added in the reagent at the time of testing. Yes, you may see prolongation of an inhibitor effect on PTTs with some test plasmas but it is an artifact due to pH increase. The same problem was noted in FVIII Bethesda assays a while ago and fixed by the Nijmejen people by including a pH buffer. Uncovered unbuffered citrated plasmas drift to pH above 8.5 over an hour at 37ºC and this knocks out a lot of the factor V. Time dependent inhibitors are more typical for FVIII antibodies and should be looked for for that purpose, not for LA. Sorry to disagree with your recommendations in this case.

George thanks Tom for this important update. Several laboratories incubate their mixing studies for the purpose of detecting LAs as well as specific inhibitors such as factor VIII inhibitors, this is an opportunity to disseminate this information.

July 27 from Herb Crown, “SLU.” The PTT mixing study suffers from a number of weaknesses. It seems simple enough, but it does not have a universally recognized standardized procedure and the method of interpretation varies widely. Few laboratories have reference ranges and generally use some type of interpretation that refers back to the patient’s PTT. The PTT mixing study has four uses; a) to aid in the detection of LA , b) to aid in the detection of a factor inhibitor c) to aid in the detection of a factor deficiency and d) to provide direction when working up a patient sample that unexpectedly presents a prolonged PTT.

For a known LA , the PTT mixing study is of limited utility to further define the abnormality. The PTT mixing study is not necessarily sensitive to LA. In our laboratory, about 50% of our LA patients show a normal PTT and a normal PTT mixing study. There are better tests to determine whether or not a patient has an LA.

For a known factor deficiency, a mixing study incorporating an incubation step should be performed to rule out an inhibitor. Most often, when a sample shows a normal mixing study at immediate mix and prolongation after a 60 minute incubation at 37º a factor inhibitor is generally suspected. A normal PTT mix before and after incubation would indicate a factor deficiency.

More often, we see the greatest value of the PTT mixing study in the context of helping to explain a prolonged PTT. After ruling out the presence of heparin we will perform the PTT mixing study with a 60 minute incubation. This will drive the direction of the rest of the work up. If the mixing study is normal before and after incubation, we would work up factors. If the mixing study is abnormally prolonged before and after incubation, we would work up the specimen for LA. If the mixing study is normal before incubation and prolonged after incubation we would look for a factor inhibitor followed with a Bethesda titer.

The PTT mixing study is not 100% specific or 100% sensitive. It is one part of a complete set of tools we use. It is not the “end all” of all things coag. As an isolated test, on a patient with a known disorder, the PTT mixing study is of limited utility. As part of a prolonged PTT workup, the PTT mixing study, with incubation, is a valuable tool to help guide the workup.

This is a basic understanding of the PTT mixing study. There are certainly exceptions to the above discussion. We see those exceptions on a routine basis. One thing predictable about coag is its unpredictability.

From “Maria:” Literature states that the Rosner Index must be validated with each new lot of PTT or PT reagent. Do we also need to validate each new lot of CaCl2? Also, what would be the way to validate the Rosner Index?

George knows of no direct method for validating the Rosner index or any parallel means for establishing correction, instead, you need simply validate the underlying PT and PTT methods. NASCOLA provides a proficiency testing module, and mixing study guidelines are available from the ISTH Subcommittee on Lupus Anticoagulant and Phospholipid-Dependent Antibodies, chaired by Thomas Ortel, MD, of Duke University Medical Center. These may help you to develop your mixing study protocols.

The July issue of Clinical Chemistry presented a well-written case of combined factor V and VIII deficiency (referenced). George noted the recommendation for plasma to replace factor V and asked: As an alternative to FFP , would you recommend platelet concentrate as a means for delivering adequate doses of factor V, in view of the high concentration of factor V in platelets?

From the author: “To our knowledge, platelet transfusions are a standard of care for those who have factor V inhibitors because indeed they do carry a lot of FV in the alpha granules and have an added benefit of protecting it from plasma-based inhibitors. In theory, inhibitors have much less time to react once platelets release factor V at the location of clotting. The importance of platelet factor V is demonstrated by bleeding in patients with Quebec abnormality where platelet factor V gets proteolyzed in the alpha granules. As for treating FV deficiency with platelets, one has to balance the risk of developing HLA antibodies, cost, and efficacy in comparison with FFP. It appears that FFP is still standard of care for FV deficiency without inhibitors. It is also likely the combined FVIII and FV deficiency patients would be less likely to develop inhibitors as they have been exposed to autologous normal FVIII and FV.”

From Donna Lawler, University of Wisconsin: Do you have any references concerning PT/INR reagents? We use a low ISI thromboplastin (close to 1.0) with good factor sensitivities but we are getting pressure from our lab director to switch to a different reagent because the peer group in the CAP proficiency testing is small. What are your thoughts?

George references a recent publication describing thromboplastin standardization. This article describes lot-to-lot reagent validation using a set of reference plasmas, an approach that is mostly unavailable in the US because of FDA restrictions. He suggests that if the reagent performs well, matches well with instrumentation, and meets internal validation requirements, there is no reason to change, just because it is not the reagent with the biggest market share. If there is any reason to be concerned about you reagent’s accuracy, you can also check it by comparing to the chromogenic factor X assay (reference). In fact, there are a few people around who advocate for routine use of the chromogenic factor X in place of the PT , as it is less prone to interference.

Crystal Azavedo asked if there exists any RCT data that compare bleeding and thrombosis risk in patients receiving UFH monitored with the PTT compared anti-Xa ?

George describes a lab that recently ran parallel PTTs and anti-Xa heparin assays on their UFH patients over several days. In comparing results, in 40% of cases, anti-Xa results that were in range paired with PTT results that were either above or below. Conversely, anti-Xa results that were high or low paired with PTT results that were within range. There were many instances in which patient dosages would have been inappropriately modified based on the PTT results. Dr. Paul Riley, Diagnostica Stago Inc, provided a bibliography of heparin monitoring studies that was linked in this post.

Sara Sneeden, Lee Memorial, said that their hematologists order mixing studies on patients who have been on Pradaxa®. These patients have VMax PT and PTT , and the mixing study is ordered on a subsequent draw. Is there anything to be learned from this study? Is there a better specimen with regard to the timing of this test?

George asserts that there is nothing to be learned from a mixing study while the patient is on dabigatran, and no physician would discontinue A/C therapy simply to get an accurate mixing study. If they were willing to discontinue, the half-life of Pradaxa is short, and they could probably get useful results after the patient is off the anticoagulant for three days. Perhaps they are attempting to identify the type of anticoagulant that is in use, or attempting to monitor its therapeutic effect. Also, if they are looking for a LA , you may be able to help them with the anti-cardiolipin antibody and anti-β-2 glycoprotein 1.

From Monica Truta: Because in Romania we don’t have books, guidance, or procedures about validation I want to know if for fibrinogen validation it is necessary to establish the LLD. If the mfr does not provide CV% for precision and accuracy what can I do?

George recommends that Monica establish LLD, and also precision and accuracy data. She may find everything she needs at the Westgard QC web site. Also, our audio modules 3 and 4, Method Validation 1 and 2, contain the information. Dave McGlasson and I have recently published Quick Guide to Laboratory Statistics and Quality Control, 2012, AACC Press, available from AACC , or Amazon.

Carol Gizzi at Baycare in Florida performs the FVIII assay with a silica-activated PTT reagent to monitor hemophilic patients. Recently she was asked to switch reagent to a PTT reagent using ellagic acid as the activator. We thought the silica-activated reagent was the most sensitive? Are we incorrect?

George knows of no reason to switch from a silica-based to an ellagic acid-based PTT reagent for the routine monitoring of coagulopathies, and in particular for factor VIII. He and participant Dave McGlasson checked with several reagent distributors, and none knew of any reason. However, tt is true that the ISTH recommends silica-based PTT reagents for the detection of lupus anticoagulant, as they are more sensitive than ellagic acid-based reagents, however both should work fine for monitoring factor deficiencies.

In an August 14 comment JLow reference our June, 2012 paper in AJCP (reference provided), where the recommendation was made that the screening PTT reagent should be lupus insensitive. Therefore, if Actin FS was recommended (rather then Actin FSL), this is the rationale for changing PTT reagent for screens and factor assays.

George responded on August 18, yes, an ellagic acid-based PTT reagent would be less sensitive to LA and therefore preferred for routine UFH monitoring and for factor deficiency screens.

On August 19, Dr. Emmanuel Favaloro commented that it is likely that the recommendation for Carol’s lab to switch to an ellagic acid-based PTT reagent is related to the perception that ellagic acid reagents are relatively ‘insensitive’ to LA. Such reagents are now considered to be preferred as a general screening test system to avoid unwanted detection of ‘asymptomatic’ LA positive individuals and the downstream problems this will cause in terms of investigative follow up testing, patient anxiety, and delays to surgery. Moreover, an LA-insensitive PTT reagent may be preferred for factor assay testing to avoid the influence of potential LA on factor assay results. However, it should be cautioned that not all ellagic acid PTT reagents are LA insensitive. In particular, both Actin FS and Actin FSL are ellagic acid based, but Actin FSL is not LA-insensitive. He provided a reference.