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Cheat Sheet for September, 2012

Preparing PPP (September 7, 2012)

Rebecca Jones, Trinity Medical Center, Birmingham, asked: What is your feeling about performing a “hard spin” on coagulation specimens? We do a “quick spin” for 2–3 minutes and then do PT, PTT, fibrinogen, and D-dimer. The only reference in CLSI documents states that the recommendation is a slower spin to have PPP.

George replies that desk-top centrifuges such as the Stat-Spin Express produce PPP, but because they are angle-head centrifuges, the platelets tend to adhere to the side of the tube and slowly release back into the plasma. You can’t let them stand more than an hour before testing.

Desktops have limited capacity, however, so you may want a larger centrifuge for big batches. Choose a centrifuge with swinging heads that leaves a level interface. If you have a centrifuge capable of reaching 8000 RPM, you can prepare PPP in 2–3 minutes.

For special coagulation testing, most require double-spinning. Spin 10 minutes at 2500 g, separate the plasma, and spin the plasma at 2500g for an additional 10 minutes. Also double-spin if you plan to freeze the specimen.

Jlow” commented (September 10): I am not sure what effect PF4 release from platelets centrifuged at high speed might have on the PTT of heparinized specimens. Also, fast spin with small numbers of tubes might be suitable for a dedicated stat lab where one person can be responsible for the centrifugation and then making sure that testing is done within a certain time to prevent platelets sliding back into the plasma—10 minutes in paper by Kao (J Biomed Lab Sci 2010 22, 23.). It might not work so well logistically in a lab where one operator is responsible for stats and routine and special haemostasis processing.

From Ali Sadeghi-Khomami, PhD, Sr. Research Scientist at Precision BioLogic Inc (September 13): JTH 2009, 1737 states: “In order to achieve <10,000 platelet/µL (PPP), first we need to do 2000 g 15 min followed by >2500 g for 10 min. Surprisingly, they referenced CLSI H21-A5 but these numbers are not actually recommended in that document.

VerifyNow Update (September 7, 2012)

Also from Rebecca Jones (in the same message as the one about PPP), we are still using the VerifyNow for Plavix. We gave the physicians a percent inhibition guide when releasing the new PRU value and we’ve received only three phone calls so far with physicians needing help interpreting. We are considering moving Plavix to the TEG and adding PFA and other testing. We have a TEG on site and are currently monitoring heparin in surgery with it.

Validating POC PTs (September 11, 2012)

Here is an Alan Neal comment appended to an April 26 post about POC: why is there a requirement to check POC INR’s with laboratory results? If local evaluations are performed and give good correlation and interferences are excluded, for instance high HCT or presence of LA, then if INR is raised, surely intervention is required based on clinical findings and INR value checked by repeat analysis. We find that INR values at supratherapeutic levels frequently give poor interlaboratory correlation with main analysers due to different thromboplastin sources and their different sensitivities. We frequently see recombinant thromboplastins performing differently to tissue extract thromboplastins, with INR’s varying as much a 2 units with higher result around 8, though the ISI’s of the reagents are both around 1.0–1.1. Which result is correct? I would also suggest that POC INR analysers using thromboplastins with ISI’s of approximately 2 should be reviewed, as many POC INR systems with ISI’s of approximately 1.0 have better EQA performance than the main laboratory’s, per UK NEQAS information.

George adds that practice standards require that point of care (POC) instruments in anticoagulation clinics be calibrated to the main lab’s plasma-based assay, and indeed, many POC instrument results are mathematically modified by internal circuitry to more closely match reference method results even though they typically operate on whole blood. In our institution, the POC instruments are backed up by the main lab in the sense that an unexpected result is confirmed by what is perhaps the more reproducible system, so we try for reasonable compatibility between the methods. Any time the INR rises to above 5 or 6, however, all bets are off. You would have difficulty finding agreement between any two unlike reagent-instrument combinations, be they POC or plasma-based. I would advocate that clinicians recognize the need for intervention any time the INR exceeds 4, and that a 6, 8, or 10 all be regarded as having the same clinical significance.

Hybrid Curve for Anti-Xa (September 12, 2012)

Crystal Azevedo, Eastern Main Healthcare Systems, is setting up a hybrid curve for heparin monitoring using the Stago’s Rotachrom Heparin reagent and hybrid calibrator and asks which control material should be used?

George invited a reply from the hybrid curve developer, Dave McGlasson: Stago now has a new liquid heparin assay with three calibrators that include UFH and LMWH. I used for the STA-Rotachrom anti-Xa assay the following calibrators from Stago: for the UFH I used the STA-Hepanorm H calibrators (product #00683) 0, 3 and 6 with UFH concentrations 0.0, 0.33, 0.49 IU/mL. The LMWH used the HBPM/LMWH calibrators (product # 00686) 0, 9, 18 (LMWH concentrations 0.0, 0.86, 1.85 IU/mL). See also McGlasson DL. Lab Medicine 2005;36:297–9.

PTT Reagent LA Sensitivity (September 13, 2012)

Marcelo Luide Pereira Gonçalves, MD, clinical pathologist from the Hematology Department of Hermes Pardini Laboratory, Brazil asks what kind of PTT reagents should be used in mixing studies, high or low responsiveness to LA?

George references Fritsma GA, Dambitzer, FR, Randhawa A, Marques MB, Van Cott EM, Adcock-Funk D, Peerschke EI. Am J Clin Pathol 2012; 137: 904-8. We recommend using low- or intermediate-sensitivity PTT reagents when performing routine laboratory screening for coagulopathies or when monitoring unfractionated heparin, and reagents that possess high LA sensitivity when performing mixing studies. Two that are available in the US are Siemens’ Actin FSL and IL’s HemosIL aPTT-SP.

Anyone Using Six Sigma? (September 13, 2012)

Claudia E. Escobar, Diagnostica Stago, Area Manager—Latin America asked, are there any laboratories that are using Six Sigma to monitor QC in coagulation? We know these concepts may be used in the chemistry lab as explained by Dr. James Westgard during the Mayo meeting, but I don’t know of any coagulation lab using the same concepts.

George doesn’t know of any coagulation laboratories that use Six Sigma and asked participants for a response. He added this comment on November 1, 2012: I spoke with Chloe Scott, Quality Assistance, Inc, in Phoenix on September 21. Chloe knows of no coagulation laboratories using Six Sigma, although some do attempt to use LEAN principles. This was confirmed by Holly Weinberg at the Intermountain States Seminar in Jackson, Wyoming, September 26.

Factor Assays; How to Define Parallel Results (September 13, 2012)

From Maria Franco, Orlando Health: When running factor assays (1:10, 1:20, etc), is the agreement between results to r/o inhibitory activity all calculated from the original result (1:10)? I have heard to find the mean of all results and then try to calculate the deviation.

George recommends to compare the result from each dilution with the original (1:10) dilution. If a subsequent dilution, for instance, 1:20, 1:40, or 1:80 produces a result that exceeds the 1:10 dilution result by more than a set percentage, suspect an inhibitor. Once you suspect an inhibitor, the next step is a Bethesda titer.

Added September 14: George spoke with Patti Tichenor and Laura Taylor in the UAB Hospital special coagulation lab, and they reminded him that you should also watch for rising values. For instance, if you see a result of 10% factor VIII in the 1:10 dilution,13% in the 1:20 dilution, 19% in the 1:40, and 24% in the 1:80, that is stronger evidence than say 10%, 13%, 14%, and 15%, even though in the latter example, the change from the 1:10 dilution to the 1:20 jumped by 30%.

Herb Crown, St. Louis University Hospital Coagulation Reference Laboratory, responded on the same day, the major inhibitors are LAs and heparin, and to a lesser prevalence, factor inhibitors. The factor results on a plasma contaminated with heparin will look very much like a patient who has LA with an increase in factor levels over dilution, but the 1:10 dilution rarely is less than 20%. A thrombin time will help sort out the difference between a LA and heparin. On the other hand, a factor inhibitor will be significantly lower, for instance, 1-10% activity and will show increase over dilutions.

TTI Test (September 13, 2012)

From the CLS Educators’ list: We are trying to figure out the principle and procedure for the tissue thromboplastin inhibition (TTI) test. We are aware that it is no longer performed, but feel there is a good chance that the ASCP could ask about it on their certification exam. Can anyone clarify this test for us?

George responds that the TTI test is synonymous with the dilute prothrombin time (DPT) test and quoted from Corriveau DM, Fritsma GA. Hemostasis and Thrombosis in the Clinical Laboratory. Lippincott, 1988:

“In the TTI test, PT reagent is diluted 1:50 or 1:500 with saline. The dilution is warmed, then 0.1 mL of dilute reagent is mixed with 0.1 mL of the test plasma and the mixture is incubated 3 minutes. CaCl2 is forcibly added and a timer started. PNP is tested at the same time, and the results are compared. If the ratio of patient plasma interval to PNP interval is 1.3 or greater, LA is presumed to be present. A ratio of 1.1 or less is considered to be normal, and LA is absent.”

The TTI was developed in Dr. Triplett’s laboratory, and is now usually called the DPT. Some still swear by it, although in 2009 the ISTH discouraged its use as being poorly reproducible in Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, De Groot PG, J Thromb Haemost. 2009;7:1737-40.

Herb Crown, St. Louis University Hospital Coagulation Reference Lab added that it is a lab-developed test used in the detection of LA. We routinely perform this test even though the ISTH does not recommend it any longer.  LAs rarely affect the extrinsic pathway and most facilities are unable to recognize them because our commercially available kits do not test for them. The DPT has an important role to play in conjunction with a complete LA work up as long as one understands its major weakness, false positives in specimens contaminated with heparin. Without understanding this, and not evaluating the specimen for heparin, I can understand this would be one of the reasons the assay is reported as “being poorly reproducible”. A complete LA workup should evaluate the common, intrinsic, and extrinsic pathways.

Reduce Blue-Top Tube Volume (September 19, 2012)

From Maria E. Martinez. Is anyone re-adjusting anticoagulant levels in blue tops? We at Orlando Health have a large NICU and collecting a 2 mL blue top sometimes can be a huge demand. I have heard of re-adjusting the 200 uL anticoagulant in the 2 mL blue top to 100 uL and instructing the nurse to draw up to 1 mL instead. Is this something that would need to be validated before implementing?

George supports collecting smaller volumes for the health of the patient, however he doesn’t recommend adjusting the anticoagulant volume of the 2 mL plastic BD Vacutainer tube. You’ll manage to accurately reduce the volume of anticoagulant, but you will have difficulty controlling the degree of vacuum in the tube. If you take off the top, the phlebotomist will have to use a syringe and transfer to the tube. Either way, it will be difficult to collect an accurate volume.

It appears that Greiner’s VACUETTE® series includes a 1 mL blue-top evacuated tube, catalog number 454320. This may be the answer! Geo.

Ellagic Acid-based PTT Reagent (September 25, 2012)

Kelly Rose at Novartis is interested in comparing different PTT reagents on a Stago Sta-R. Currently we use the silica-based (STA PTT-Automate) and kaolin-based STA C. K. Prest? We would like to try ellagic acid. What kit would you recommend?

George recommends either IL SynthAFax or Dade Actin FS, distributed by Siemens. Both can be adapted to the Stago instrument.

Emmanuel Favaloro, PhD, Australia, adds on September 27, it depends on whether you want an LA ‘sensitive’ or ‘insensitive’ reagent. Actin FS is fairly insensitive, but Actin FSL is fairly sensitive; for more info, refer to: Kershaw G, Suresh S, Orellana D, Nguy YM. Laboratory identification of lupus anticoagulants. Semin Thromb Hemost. 2012;38:375-84.

Vanessa Chan, from Sick Kids in Toronto, adds (October 9), we use Actin FS on our STA-R but it tarnishes needle #2 which means we need to change that needle every PM.

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