2014 Cheat Sheet

Pam Owens wrote that a pediatric group asked if we would do a fibrinogen level on pleural fluids before and during administration of TPA and asked what is the utilization and would you put a viscous sample on an instrument?

George thought that the idea of treating a mucinous pleural effusion with TPA seemed radical, but provided a few references that describe the procedure as safe and effective in children. He found nothing that supports measuring effusion fibrinogen levels, however, but suggests a manual Clauss assay may provide a semiquantitative answer. Dr Vadim Kostousov provided two relevant references. We’ve received no additional responses.

In February 2011, the plasma-derived CLS Behring factor XIII concentrate Corifact™ received US FDA clearance for bleeding prophylaxis for patients with congenital factor XIII deficiency. In January 2013, the FDA extended Corifact’s indication to include management of perioperative bleeding in factor XIII-deficient patients. A copy of the package insert is provided with this entry.

Pamela L. Pool is considering the free protein S latex test and asks for comparison studies among manufacturers’ kits. George provided a reference and Robyn Colemanresponded, “We have used the Stago LIA free S kit and recently changed to Siemens Free S Antigen. Both kits have performed well, were simple to use, had good CVs and performed well in external QAP. The choice depends on your instrumentation. We have a clot based S for very rare occasions when we need to confirm a free S result or a suspect dysfunctional genotype. Dr. Vadim Kostousov provided a relevant CAP survey reference.

Louise Pleiss re-runs the normal pooled plasma (NP ) after a 2 hour mixing study incubation, but is having a difference of opinion about the results. One site says that the PTT prolongs to ~42 seconds due to labile factors. Scientists at her site the result should be near the unincubated result. Do you have any data to support either side?

George suggests the site may be overestimating the degree of prolongation. The NP clotting time should lengthen by 2–3 seconds during incubation as factor VIII activity becomes reduced. The best mixing study approach is to compare the PTT result of the incubated NP-test plasma mix with the PTT result of the incubated NP.

Dr. Ali Sadeghi-Khomami indicates the results depend on the factor VIII sensitivity of the PTT reagent, the instrument, how the NP was prepared, and initial factor levels in the NP. The factor VIII deterioration rate is calculated at 5% per hour at room temperature, so at 37°C after 2 hours the impact would be noticeable.

Dr. Vadim Kostousov agreed with Ali and provides a reference that suggests 20% factor VIII depletion in 2 hours.

Herb Crown added, “I would emphasize the importance of establishing a range for your NP to eliminate the “guess work” of determining what should be seen. Once your range is established, the question changes to, “did the assay perform within the established parameters?”

Irene Regan writes there seems to be no consensus over what coagulation results should be released when there is a suspicion of in vivo hemolysis such as in DIC , when the samples are grossly hemolyzed.

George agrees and asserts that hemolysis implies platelet and coagulation factor activation, generating unreliable results across the board. Additionally, hemolysis interferes with the determination of the clotting endpoint or with chromogenic assays in optical instrumentation.

George suggests employing an electro-mechanical instrument and appending a comment warning of possible interference. He also suggests that when diagnosing DIC , check the D-dimer package insert D-dimer to learn the manufacturer’s claims regarding the degree of hemolysis interference and to do the same for chromogenic assays.

Scott Miller responded, “We found that normal or prolonged PTs are mostly unaffected by even moderate amounts of hemolysis. However, with PTTs we found that there is a positive bias even with slight amounts of hemolysis that worsens with higher results, so that those of us still using APTTs to monitor heparin therapy should take notice. Further, any specimen that appears to be a difficult draw should be rejected for clotting tests, if only to avoid the effects of activated platelets. Hemolysis by itself may not be affecting the test.

Dr. Vadim Kostousov provides a reference and reports his study regarding influence of free hemoglobin and bilirubin influence on the PTT and anti-Xa assay will be soon published in Arch Pathol Lab Med.

Dr. Emmanuel Favaloro comments that distinguishing in vivo from in vitro hemolysis is clinically very important so that those suffering in vivo hemolysis are not denied potentially life saving treatments. In his small test series investigating short PTTs , some 10% of samples seemed to represent in vivo effects. It is clear that at least a portion of samples rejected by laboratories as hemolysed come from patients suffering in vivohemolysis. It is also important to recognize that interferences are not restricted to coagulation tests. Dr. F. provides a book reference.

Kathleen Tobertga reports varied 2-hour NP unmixed control mixing study results between sites. Her site generates a PTT around 40 seconds, which seems to be elevated due to heat labile factors being destroyed. The other site is getting about 32 seconds. Both use the same kit for the NP , but they thaw at 37°C, we at room temperature. Are we correct about the heat labile factors and that the 2 hour incubated control should be prolonged, and do you have any place I can go to find documentation?

George assumes Kathleen is in the same system as Louise Pleiss. He suggests the difference in thawing approaches is the key and that they may want to confirm that everyone is adequately mixing the thawed NP. George provided one reference.

Dr. Ali Sadeghi-Khomami responds that thawing at room temperature increases factor VIII cryoprecipitation, causing the 40 second PTT. He suggests running a sensitive functional assay for fibrinogen and factors V, VII, and VIII to document the difference between the two thawing technique. Thawing frozen plasma at room temperature is not recommended.

Dr. Emmanuel Favaloro agrees that thawing is key. CLSI guidelines are clear, and his experience also indicates that thawing at room temperature or slow thawing leads to gradations in plasma levels of FVIII , high levels at the bottom and low levels at the top, compounded by inadequate mixing. Also the original levels of factors V and VIII in the NP make a difference. If they are closer to 50 U/dL than 100 U/dL, then small losses of labile factors may reduce to levels that prolong the PTT. Thaw at 37°C for 5–10 minutes and mix well before use. Also, although some factor VIII inhibitors only acquire full inhibition at 2 hours or even longer, a 1 hour incubation could provide a reasonable compromise for many cases tested. Dr. F. provides two reviews.

Kathleen ultimately found that the dry incubator at her site was slightly above 37°C. George King Biomedical, her supplier, recommends thawing in a circulating water bath at 37°C.

Crystal Azevedo asks, is there a relationship between rivaroxaban and an elevated plasma homocysteine level?

George found no publications linking rivaroxaban with homocysteinemia, nor with any of the metabolites in the folic acid pathway. The Janssen Pharmaceuticals package insert doesn’t mention folic acid interactions. George asks, “could some of your NOAC patients be on antifolate chemotherapy?” Though anticoagulant therapy may be indicated in some forms of cancer, for instance, acute promyelocytic leukemia, there’s no current cancer indication for rivaroxaban. This post attracted no responses so far.

The FDA advisory committee unanimously withheld clearance for rivaroxaban’s acute coronary syndrome indication. This is the third time that Janssen’s application has been denied for this indication. Rivaroxaban is cleared for stroke prevention in atrial fibrillation, thrombosis prevention following total hip or knee replacement surgery, and treatment following venous thromboembolism.

Lori Pinelli’s PT mixing study procedure recommends only unincubated patient plasma and NP 1:1 mix but she has been asked to perform an incubated PT mix on two patients with normal PTTs and prolonged PTs. Both were factor VII deficient and corrected upon addition of NP. She was asked to perform the incubated mix to rule out an inhibitor and asks if there are time/heat-dependent factor VII inhibitors?

George distributed Lori’s question to several colleagues. Dave McGlasson and Dr. Larry Smith confirmed that there is no value in performing an incubated PT mixing study. If both the PT and PTT are prolonged, perform only the PTT mixing study. If the immediate mix corrects, perform the incubated PTT mixing study to determine if a specific inhibitor, usually anti-factor VIII, is present. Additionally, some weak LAs show up after incubation. If only the PT is prolonged, the likelihood of an LA is low because the phospholipid concentration of the PT reagent exceeds the phospholipid concentration of the PTT reagent and is consequently less sensitive to LA. According to both Larry and Dave, the greatest likelihood is a factor VII deficiency, which requires no incubation.

In the past, anti-prothrombin and anti-factor V antibodies would be detected in patients who had been treated with bovine thrombin-based fibrin glue in a prior surgical procedure. The antibodies arose as cross-reaction to the bovine thrombin and could generate severe bleeding as they neutralized most available factor V and prothrombin. We see fewer of these now as distributors have switched to human thrombin. The anti-factor II and V antibodies are detected in either the PTT and in the PT mixing study without incubation. Larry further suggests that when the PTT is normal and the PT is prolonged, avoid the PT mixing study altogether and go straight to a factor VII assay for the best and most timely results.

Dr. Ali Sadeghi-Khomami reports that 10–30% of LAs are temperature/time-dependent and their inhibitory effect appears after incubation at 37°C. Some believe the dilute PT (DPT) detects a sub-population of Las that target extrinsic pathway factors that PTT or DRVVT assays cannot such as tissue factor, factor VII, or TFPI. Considering the heterogeneity of inhibitors, there may be merit to the DPT and an incubated DPT. Further he suggests the DPT may assess if there is an inhibitor induced by rFVIIa (NovoSeven). Ali adds there an argument against incubation at 37°C, reasoning that pH changes introduce an artifact.

Chris Ferrell has not incubated PT mixes, but recently Stago gave a webcast by an Esoterix pathologist who discovered an inhibitor using an incubated mix on the PT. It may have been a temperature and time dependent LA that affected the PT and not the PTT. As far as inhibitors directed at the extrinsic system, none of them have been temp/time dependent. Chris has detected both bovine and human thrombin inhibitors. The thrombin inhibitor also had a very strong LA that balanced out the bleeding and the clotting issues. She’s also seen factor V inhibitors associated with a necrotizing insect bite. All of them have been immediate acting. If someone asked Chris to incubate a PT mix, I would ask them what they were looking for and what literature are they reading so I could also educate myself.

Dr. Karen Moser responds, “I am the pathologist who gave that recent lecture. The case I presented was that of an EDTA plasma submitted for factor V activity and inhibitor studies, including PT mixing studies. As it turned out, the entire order was one big error as the physician actually intended to order factor V Leiden mutation analysis.

Since we had the PT mixing study data, I included them because I thought the pattern of prolongation in the incubated mix as compared to the incubation control for our EDTA plasma was interesting. Factor V inhibitors have been reported to show time dependence in some cases (references given). In the case I presented, you would have to consider the possibility of the wrong sample type, and then you would need some basic chemistry tests (potassium, calcium) to make a diagnosis of EDTA plasma. Admittedly, this is not a typical problem in hospital laboratories dealing with primary tubes, but it does come up from time to time in reference laboratories dealing with aliquots.”

Dave McGlasson worked up a patient who had a Keflex-induced anti-factor V. Dave attached a copy of his paper about this patient with some unusual PT mixing study results that increased upon incubation. The paper reports a platelet neutralization procedure (PNP ) and the kaolin clotting time (KCT) tests, but later follow-up testing (unpublished) showed it to be STACLOT-LA negative but DRVVT positive.

George also posted this comment on behalf of a local colleague: “We just incubate all our mixes. Most of the time, we’re mixing both PT and PTT , so it’s not worth doing an immediate mix and then turning around and incubating it. One of the patients I can think of who did have a factor V inhibitor–which I’m pretty sure was because of bovine thrombin–we happened to do an immediate mix on and neither side corrected. But even if it’s just a PT mix and the PTT is normal, we still incubate it.

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