Cheat Sheet for August-September, 2011

Ultracentrifugation for Lipemia (August 1, 2011)
Tanja Skierka asks about a reference for ultracentrifugation of lipemic samples for PT, PTT, and fibrinogen.
George’s reference for specimen-related questions is CLSI Document H21-A5, which states, “In a small, unpublished study of lipemic samples, PT and APTT assays were decreased and fibrinogen levels were increased by about 10 to 15%. Until published studies are performed or the laboratory has validated the procedure, mechanical and/or electromechanical methods should be utilized when possible for these samples that are icteric, lipemic, or contain substances that interfere with light transmission.”
Most optical coagulometers compensate for moderate lipemia by employing alternate wavelengths, and CLSI excludes ultracentrifugation, so ultracentrifugation is probably not the answer.
Dave McGlasson comments: In 2006 I looked at the effect of hyperlipemic specimens on Stago’s photo-optical LIA D-dimer assay. We assayed specimens before and after ultracentrifugation and found no difference.
Monitor LMWH with PTT? (August 6, 2011)
George responds to a comment that a 2001 CDC survey reported that 72% of US respondents were attempting to use the PTT to monitor Lovenox. The survey referenced Arch Pathol Lab Med 1998;122:799-807, in which CAP recommends the heparin anti-Xa assay for LMWH. The insensitivity of the PTT for LMWH, as well as its non-linearity, renders it useless, though it is used to monitor unfractionated heparin.
Monitoring Dabigatran (August 6, 2011)
Donna Lawler comments on Hyphen BioMed’s Hemoclot Thrombin Inhibitor assay for Dabigatran. Ann Workman of Aniara Diagnostica, LLC indicates the assay, also called the dilute thrombin time, along with its validators and controls, are currently under 510K review at the FDA.
Discard Tubes (August 7, 2011)
Vicki Cardone wants to know what other labs use for the discard tube when collecting coag samples.
George answers that both BD and Greiner market plastic non-additive tubes. Plastic red-closure serum collection tubes, like serum separator tubes, contain negatively charged particulate activator that hastens clotting. The activator can carry over, thus this tube should not be used ahead of a blue-closure tube. Glass red-closure tubes have no additives, however most of us have adopted plastic for its safety. An easier approach is to just use a blue-closure discard tube ahead of the specimen tube. In this approach, cost could be an issue, however a discard tube is only needed when collecting blood using a butterfly set.
Factor XII Deficiency and infections (August 14, 2011)
“Sunny” asks, “Do people with factor XII deficiency suffer constant infection?”
George replies there is no clinical relationship between coagulation factor XII deficiency and infections. Factor XII deficiency is relatively common; Thromb Haemost 71: 68-72, 1994 reported a prevalence of 2%. Although factor XII deficiency markedly prolongs the PTT, it is not associated with bleeding. Several anecdotes propose a relationship with thrombotic risk, however many of these can be explained by the presence of lupus anticoagulant, which prolongs the PTT and produces the artifactual appearance of factor XII deficiency. Through its relationship with prekallikrein and high molecular weight kininogen, factor XII deficiency may potentially suppress inflammatory and complement systems, however, as with coagulation, there appears to be no physiologic effect of factor XII deficiency upon inflammation.
Anti-factor VII Inhibitors? (August 14, 2011)
Dr. Michael Steiner asks, has the development of an inhibitor to factor VII ever been reported after therapy with recombinant coagulation factor VIIa (NovoSeven)?
This seems logical, and George did a PubMed search. Although there are several reviews of recombinant factor VIIa therapy, none reports the development of an anti-factor VIIa inhibitor.
Venous Sinus Thrombosis in a 2-YO (August 19, 2011)
A parent asks about thrombosis risk factors in a 2-YO who had suffered a venous sinus thrombosis. The child was treated and had no lasting effects, but does anyone know of any data that document anti-phospholipid antibody syndrome in pediatric populations? (No responses as of October 3, 2011)
Quick Question Summary: Monitoring Dabigatran (August 22, 2011)
George observes that dabigatran was FDA-cleared in October, 2010 and many in the US have put thought into monitoring. He predicts, based on Samama MM, Guinet C. Laboratory assessment of new anticoagulants. Clin Chem Lab Med 2011;49: 761-72, that no one will stick with the PTT after Stago, BIOPHEN, and others have moved their DTI assays through the FDA process. The various PTT reagent/coagulometer combinations are too variable to monitor dabigatran long-term, whereas the Ecarin clotting time or Ecarin chromogenic assay and the dilute thrombin time appear to be reliable and linear.
Monitoring Heparin with the PTT (August 22, 2011)
“Berrylane” asks if a patient is on IV heparin, what is the procedure for blood collection for the PTT?
In accordance with Chest 2008:133:141S-59S, when monitoring IV unfractionated heparin using the PTT, collect blood below the infusion site or from the arm opposite at least 4-6 hours after the initial bolus, but not more than 24 hours after initiation. The target therapeutic range is the PTT in seconds that corresponds to 0.3-0.7 anti-Xa chromogenic heparin units, and the heparin dosage is adjusted to maintain a PTT result within the target range.
Unexplained Prolonged PTT (August 25, 2011)
Enriqueta Coll reports some pre-op patients without a history of bleeding had PTTs prolonged 5-10 seconds with Actin, but normal using CK Prest.
George responds that Siemens manufactures both Actin FS® and Actin FSL®. Actin FS and Stago’s CK Prest® are formulated with moderate reagent phospholipid and are relatively insensitive to lupus anticoagulant (LA). They are designed to monitor unfractionated heparin and to screen for single or multiple coagulation factor deficiencies. Actin FSL is formulated with low concentration phospholipid and is sensitive to LA.
George assumes Enriqueta is using Actin FSL and that the patients whose PTT results are prolonged when using Actin but normal using CK Prest possess LAs; he suggests switching to LA-insensitive reagent or testing with CK Prest, reserving the Actin FSL for LA detection.
Alternatively, inpatients may be receiving unfractionated heparin. Often nurses flush vascular access devices with heparin, causing a prolonged PTT. A prolonged thrombin time indicates the presence of heparin.
Finally, in any unexpected prolonged PTT result, George suggests a mixing study: mix the patient plasma 1:1 with fresh normal plasma such as Precision BioLogic Inc CRYOcheck^TM, and perform a follow-up PTT on the mix. Correction to within the normal range or to within 10% of the normal plasma PTT is evidence for a coagulation factor deficiency, while lack of correction points to an inhibitor such as LA.
Butterfly Sets for Platelet Function Tests (August 26, 2011)
Dennis Ernst, Center for Phlebotomy Education, reports that someone was told they can’t use a butterfly needle when drawing PFTs. Why?
George responds there is no reason to prohibit a butterfly when collecting for PFT. In fact, platelet aggregometry expert Larry Brace, PhD, insists on collecting specimens using butterflies and syringes to minimize turbulence that can activate platelets.
Christine Hinz commented that her lab requires PFTs be drawn with a 21-gauge needle and not with a 23-gauge butterfly. They contend a smaller diameter needle may cause shear and hemolysis and therefore platelet activation. What is the CLSI recommendation?
George answers that CLSI document H21-A5 does not prohibit the use of a butterfly for PFT. Christine’s lab may have a good reason for prohibiting the butterfly, for instance, they may have run into specimen management problems. However, gauge need not be the issue, as BD markets butterfly sets with 21, 23, and 25-gauge needles. There is no hemolysis problem with a butterfly provided you use a 21g needle.
Combined Protein C and Protein S Deficiency (September 1, 2011)
A FF participant asks, should Lovenox be used for someone with a protein C and protein S deficiency?
George recommends double-checking the diagnosis of protein C and protein S deficiency. The prevalence of protein C deficiency is 1 in 300 and protein S deficiency prevalence is less than 1 in 1,000, so the possibility of someone inheriting both is less than 1 in 300,000. We often err by testing for protein C and protein S deficiency while a patient is on coumadin or when they have had a recent thrombotic event, both of which cause protein C and protein S to be temporarily decreased. To confirm protein C and protein S deficiency, the patient must be off coumadin for at least 14 days and must not have an active clotting episode in progress. Confirm the diagnosis by repeating the test after 12 weeks and testing the patient’s first-degree relatives. There are many instances in which protein C or protein S may be temporarily decreased, such as in inflammation or during pregnancy.
Factor XIII: Urea Solubility (September 1, 2011)
Carol Gizzi asked for a procedure for the factor XIII urea solubility assay. George referenced the procedure from Corriveau DM, Fritsma GA. Hemostasis and Thrombosis in the Clinical Laboratory, Lippincott, 1988. However, George linked his response to Pam Owens factor XIII chromogenic assay question from 2009.
PT Specimens, Centrifuged or Uncentrifuged? (September 13, 2011)
“Cassip” stores PT specimens at room temperature, but they may be delayed up to 8 hours before testing. Do specimens that are held uncentrifuged until testing maintain integrity better than those that are centrifuged immediately and left in the primary tube capped?
George replies that specimens for PTs can be held at RT for up to 24 hours with no deterioration. There are no studies comparing centrifuged versus uncentrifuged specimens for their integrity, according to Dorothy Adcock-Funk, MD, author of CLSI Guideline H21-A5. However, given that the idea has not been tested, Cassip may want to try an experiment using duplicate tubes, one centrifuged and one uncentrifuged.
PTT Controls Drift Upward (September 18, 2011)
From Mary Ann Tara: We recently experienced a problem with our Actin FSL reagent. The abnormal control result drifted up and out of range. We were told by the manufacturer to just adjust the range, as it was a problem affecting only that control and reagent lot. However, we started seeing patients with slightly elevated PTTs but no factor deficiencies, anticoagulation or lupus anticoagulant interference. When we tried a new lot of PTT reagent, there were huge differences, especially as the times got longer, affecting all patients, but especially our patients being anticoagulated. George sent the question on to Carol Shearer at Siemens for clarification.
Carol responded that it looks like Tara really followed our process. We recommend the customer contact Siemens to file a complaint about the reagent or control. We log and file all calls and tally the number of calls by lot number of reagent or control. If there have been no or few calls, we recommend that they adjust the range and we may have them send product into us for testing against non-shipped product. Once testing is complete, a letter of the results is sent to the customer. If we have a documented issue, then we send a letter and replace any product that they have in-house. We do extensive quality testing as products are produced. Siemens stands behind our products and will replace as necessary.
Tara’s also asked what independent resource is available, I (Carol) would say the proficiency testing that they participate in would be considered independent. However, she should not want to use this alone and fail. That is why our LabLink QAP program is used so that customers can see when there is a change in their testing system and investigate before their proficiency samples arrive. Tara does not state whether she participates in the LabLink QAP program, but I highly recommend it. She could review the data to see how her lab, using her instrument, and her reagents and controls compares to others using the same set up. The only difference between her and the other participants could be her reagent lot number. The control lot number would be the same.
Proficiency Testing for Mixing Studies? (September 30, 2011)
Janet Smick is looking for proficiency testing for PT/PTT mix studies other than CAP.
George recommends North American Specialized Coagulation Laboratory Association (NASCOLA), which provides a series of proficiency testing modules, including lupus anticoagulant, factor deficiency, and factor VIII inhibitor modules. Internal control plasmas are available from Precision BioLogic Inc; George King Bio-Medical, Inc; and Aniara.

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