Sylvia Bunting, Children’s Healthcare of Atlanta, does an immediate and incubated mixing study. She asks, what criteria do you use to decide that the PTT was prolonged after incubation?
George replies, if the PTT immediate mix corrects, prepare a new patient plasma-normal control plasma mix and incubate. Simultaneously incubate an aliquot of the normal control plasma. Perform a PTT on the incubated mix and the incubated control plasma. If the mix result is prolonged by more than 13% beyond the incubated control plasma, the result is uncorrected, indicating a time-dependent inhibitor. Less than 10% difference indicates correction and no inhibitor. Repeat, ensuring the patient plasma was platelet poor, if the ratio is between 10 and 13% longer than the control.
A colleague’s relative is 35 and is currently about 20 weeks pregnant. She has 2 healthy children, but has also had two miscarriages in the past 3 years. She is a double heterozygote for MTHFR C677T and A1298C. Does the double heterozygosity increase her chances for fetal loss?
George responds no, that several publications link MTHFR double heterozygosity with chronic homocysteinemia, but none has found a link to increased risk of thrombosis. However, if her OB physician is convinced her previous losses were caused by thrombotic events, it may make sense to treat her with enoxaparin or fondaparinux even if she has no inherited risk factor.
FD Patel, MD has a patient who was tested for both FDP and D-dimer. Her D-dimer was mildly elevated at 0.79 but her FDP assay result was undetectable. Since the D-dimer is a type of fibrin split product, how can you explain this discrepancy? I’m thinking it has something to do with what each test measures.
George explains that quantitative D-dimer assays are considerably more sensitive than FDP assays. You may wish to consider deferring the FDP assay in the future, considering it is the less sensitive of the two and they measure the same thing. If your patient develops disseminated intravascular coagulation (DIC), both will be strongly positive.
Vanessa Chan, Sick Kids Hospital in Toronto asks for information on how PEG-ylated drugs affect clotting assays.
George has heard that PEG can affect the phospholipid structures in PT and PTT reagents and he has seen some unexpected laboratory results that have caused us to suspect PEG, but can find no references that confirm or reject the idea. He discussed this with Jeff Dlott, MD of Quest, who has had the same experience.
Scott Miller, St. Mary’s of Michigan Medical Center, asks, is there any consensus on whether APAs like LA affect PT/INRs? We have a patient who has been tested positive in the past for LA, and her INRs consistently run a bit higher in the lab (2.2) vs. a point-of-care (POC) device (1.7). If the presence of LA is an issue, are there any guidelines on how a physician is to monitor therapy in a LA positive patient, especially considering the tendency for LA titers to rise and fall over time?
George agrees that some PT reagents are sensitive to LA, those that are synthetic and employ relatively small concentrations of phospholipid. LA sensitivity may vary from lot to lot as well as among reagents from various manufacturers. While plasma-based assays and POC results may remain consistently 0.5 units apart, they may change with a change in reagent lot or POC cartridge lot. See Balaban M, Stanrić V, Rincić G, et al. Acta Clin Croat 2010;49:469–77. The Balaban article makes reference to the chromogenic factor X assay (CFX) as an appropriate substitute when the PT/INR is unreliable. For more CFX documentation, check McGlasson DL, Romick BG, Rubal BJ. Blood Coag Fibrinolys 2008;19:513–7, and also Rosborough TK, Shepherd MF. Pharmacotherapy. 2004;24:838–42. The only company that provides an FDA-cleared CFX is DiaPharma, Inc. Their assay correlates nicely with the standard clot-based factor X assay.
Madan Verma wonders if there are any correlation studies about whether switching hemodialysis patients from heparin to 4% sodium citrate will affect the PT or PTT.
George found over 500 PubMed references, most of which show that citrate anticoagulation in dialysis confers a lower risk of bleeding than heparin. One example is Morabito S, Pistolesi V, Tritapepe L, et al. Crit Care. 2012;16):R111. While heparin prolongs the PTT, no reference indicates an effect of citrate on routine coagulation screening assays. George also checked with Jill Adamski, MD, pathologist at University of Alabama at Birmingham Hospital who mentioned that we routinely use citrate for anticoagulation during pheresis. There is no lasting effect on any plasma parameters once off the pump. Thus citrate used in dialysis should have no effect on the PT or PTT.
Purvi Jariwala, Texas Children’s Hospital alerts us to a CAP checklist requirement published 7/31/2012. Heme.38009 AMR validation: “Validation of the analytical measurement range (AMR) is performed with matrix-appropriate materials, which include the low, mid and high range of the AMR.” Purvi wants to see how other laboratories are addressing this requirement.
Joe Lamb, October 29, wrote, the CAP standard was revised again 7/25/12. Some of the verbiage has been deleted and changed. Your instrument or method manufacturer should be your first resource.
Kim Kinney, October 30, said IU Health is also just starting to look at these new questions. She wonders what materials are available and if patient samples can be used, similar to a fibrinogen linearity check.
Joe Lamb, same day, wrote that clot-based assays are exempt from the CAP standard. If you calibrate more often than once every 6 months, you don’t have to do a separate calibration verification and the calibration itself may be the AMR validation.
Results of our September, 2012 Quick Question; Historically, what assay was used to monitor heparin?
a. Bleeding time: 14 (17%)
b. Recalcification time: 7 (9%)
c. Lee-White clotting time: 51 (63%)
d. Thromboplastin generation time: 9 (11%)
George suggests that participants must be well-read, or “of a certain age” to answer this one. Bleeding time is incorrect. The Duke, Ivy, or Mielke template BT may still be used (with little success) to identify vascular disorders, von Willebrand disease, or platelet function disorders. The recalcification time was once used to monitor heparin by a few of us. This was a simple test in which calcium was added to citrated (or in those days, oxalated) plasma and the interval to clotting was recorded. The “recal time” grew to be first the partial thromboplastin time, then the present-day activated partial thromboplastin time (PTT, APTT). However, most of us used the Lee-White clotting time. We pipetted fresh, unanticoagulated blood to a 10X75 or 12X75 mm glass tube, attempted to keep it at 37°C and tipped it every 30 seconds until it clotted, recording the time. The thromboplastin generation time was too cumbersome for routine heparin monitoring, we used it mainly to detect coagulation factor deficiencies.
David Summers asked if following lab results represent systemic lupus erythematosus (SLE).
- · PTT LA 52 seconds, reference limit 40 seconds
- · DRVVT screen 44 seconds, reference limit 42 sec
- · Antithrombin antigen 39 mg/dL, reference interval 18–33 mg/dL
- · Protein C activity 13%, reference interval 70–180%
- · There is a negative anti-nuclear antibody (ANA) test.
George’s answer is no, based on the negative ANA test result, the person doesn’t have SLE.
Mr. Summers’ question highlights a problem that plagues laboratory medicine. We do a poor job of naming our laboratory tests and medical disorders. SLE is a chronic inflammatory disease of connective tissue, part of a family that includes rheumatoid arthritis, scleroderma, and Sjogren syndrome. SLE is confirmed using the ANA test profile.
Conversely, LA is plasma antibody that reacts with phospholipid-bound proteins, especially a plasma protein called β-2-glycoprotein I. Though LAs are detected in a little less than 50% of SLE sufferers, they are also found in other conditions or may arise separately from any primary condition. SLE is rare, however LAs are found in 1–2% of the general population. Most LAs are transient, but chronic LAs are associated with strokes, TIAs, AMIs, PAD, VTE, and recurrent spontaneous abortions. Chronic LAs are “bad actors,” and hemostasis labs put a lot of effort into detecting and confirming that they are present or, more happily, ruling them out.
Of the laboratory results listed, the PTT-LA and the DRVVT relate to LA, however they are incomplete. Because both are prolonged, lab scientists would say they are presumptively positive for LA, but require confirmation using specially designed, readily available high-phospholipid reagents. It would be a mistake to report the presence of LA without these confirmatory test results, and the patient’s laboratory is probably equipped to perform the tests.
The elevated AT has no clinical significance. A chronically reduced AT level would associate with many of the disorders that are associated with LA, especially venous thromboembolic disease.
The reduced protein C could be very significant, as low protein C also associates with VTE. This test, however, is affected by Coumadin, and must be confirmed by a repeat test performed when the person is in good health and not taking any drugs or dietary supplements. LA, if present, also interferes with the protein C test. We never conclude that someone is protein C deficient without confirmatory testing at a time when we can be assured there are no interfering circumstances.
Kim Kinney at IU Health is trying to collect information from TEG users. She is in the process of validation to support Berlin Heart transplants for their pediatric population. She is wondering what other sites use TEG for, who runs the samples, who interprets and what results are entered into the computer system.