2010 Cheat Sheet

Bill Chamlee at the Cleveland VA comments that he uses two applicator sticks to check for clots after centrifuging and running assays. He contends sticks act as an activator and shortens the PTT and that you often can’t identify a clotted specimen by looking at the test results, for instance, a normal-looking PTT could really be prolonged. With his method he has seen plenty of clotted specimens that had generated plausible results. He notes that you can’t visually inspect for clots when numerous labels cover the tubes, and without the labels you still can’t see small clots. This post generated six comments including one from Erwin A Vogler, PhD, Professor of Materials Science and Engineering and Bioengineering, The Pennsylvania State University discussing the activating effect of various materials and another from Dave McGlasson, Wilford Hall USAF Hospital, San Antonio who would like to study platelet activation by applicator sticks. (March 3, 2010)

Elpidio Pena, MD sent this request through my friend and colleague Marisa Marques, MD, Medical Director of Special Coagulation and Transfusion Medicine at UAB: “We are trying to set up the anti-Xa for low molecular weight heparin. We are having a hard time finding another laboratory to do the initial correlation. We use a BCX instrument, a straight curve for the anti-Xa for unfractionated heparin and exogenous antithrombin in the test system. Do you know of any lab that uses a similar system or is there an alternative for the correlation?” As of April 15, 2010, we’ve received no response. (March 9, 2010)

From Richard Larsen, US Army: I have a 33 yo HIV positive non-compliant female patient with chronic kidney disease.

Labs: PT , 13.4s (11.7-14.2); PTT , 47s (23-33), fibrinogen, 300 mg/dL ; D-dimer, 1.61 FEU (0.5); thrombin time 27s (15-20); reptilase time 38s (20-25); DRVVT 38s (30-40).

1:1 PTT corrected to 33 s; 1:1 thrombin time did not correct, 24s; reptilase time did not correct, 35s; PTT post-hepzyme 45s.

Why did mixing studies correct the PTT but not the TT or reptilase times and where do I go from here?
George responds the thrombin time may be caused by an anti-bovine thrombin inhibitor. Also it may be fibrin degradation products are interfering with the reptilase and thrombin time results. It would be interesting to track the D-dimer results with the TT and RT over a few days to see if both resolve. (March 9, 2010)

From Mr. Peter Clement, MLS Student at Brigham Young University in Provo, Utah
Is there a way to distinguish between Fitzgerald factor and factor XII deficiencies without assays? Apparently, you can differentiate prekallikrein (PK , Fletcher) deficiency by incubating for a longer period with kaolin and you can differentiate factor XI deficiency because it’s associated with bleeding tendency. However, my research indicates that assays and perhaps family history are the only ways to differentiate HMWK from XII deficiency. Also, have they found anything that indicates why people with factor XII deficiency do not experience bleeding tendency?
George answers the only way to distinguish HMWK factor deficiency from factor XII deficiency is by a factor assay, and lists sources of materials. He avers Peter can detect PK deficiency by incubating plasma with kaolin 10-15 m. The PTT is prolonged before incubation but corrects to normal as PK becomes activated.
PK , HMWH, and XII have no in vivo procoagulant function, though they may be involved in fibrinolysis. That is why people with deficiencies have no bleeding tendency. (March 13, 2010)

Julie Tange, Developmental Technologist at Mayo Medical Laboratories asks whether companies are developing local INR calibrator sets to be sold commercially. Beckman has their verification and calibration set, but only for their instruments. George answers that Beckman-Coulter, Inc has the only FDA-cleared local INR calibration kit on the market, and confirms it is only cleared for use with their instruments and reagents. This is a source of frustration for coagulation laboratories everywhere, especially as local calibration is routine in Europe. Two companies have brought kits with acceptable technical parameters to the FDA but both have failed to clear. This post elicited two “warm” responses describing Precision’s effort to bring local calibrators to market. (March 18, 2010)

From Bob Dahms, Coagulation Technical Specialist, Long Beach Memorial is looking for literature to reduce performance of factor VIII inhibitor screening and Bethesda titers. Hw restricts orders for both unless the patient has a factor VIII <20%. A second area of interest is factor IX inhibitor screening. Most reports seem to suggest either 15 minutes incubation at 37 degrees or immediate testing. George responds that in July, 2009 we ran a Quick Question about factor VIII activity level cutoffs for ruling out a Bethesda titer. There is little consensus, laboratory practitioners use cutoffs from 20 to 50%. He also refers to a comment from Dr. Larry D. Brace, who performs a 1:19 dilution screen. While we use such screens, they are not validated and should be treated as preliminary results to be followed with a Bethesda titer. Factor IX inhibitors neutralize the factor within five minutes, so you can perform the Bethesda titer assay for factor IX with no incubation. (March 20, 2010)

ML Drejka asks, “Will citrated blood from a heparinized port be acceptable for a euglobulin clot lysis test?” George answers no ; one should collect the specimen from a site separate from the vascular access device to avoid heparin contamination. If forced to collect through a device, flush first with 5 mL of saline, then draw and discard a volume of blood that is equal to or exceeds 6 times the dead space volume of the tube before collecting a specimen. (March 23, 2010)

Paul Riley, PhD, Manager of Research Use Products, Diagnostica Stago, Inc., said Stagois now partnering with the advanced research and development company BioCytex to market ready to use flow cytometric assays for hemostasis diagnosis. They can now identify P2Y12 occupancy via the downstream marker VASP, platelet glycoprotein IIb/IIIa occupancy, platelet PAIg, and RBC paroxysmal nocturnal hemoglobinuria (PNH) markers CD 55 and CD 59. This is an exciting new venture for Stago and helps open the door to more extensive platelet activation analysis. (March 23, 2010)

Kim Kinney at Clarian asked about screening for suspected prekallikrein (PK , Fletcher factor) when the PTT is prolonged. George suggests you can incubate the plasma with PTT reagent for 10–20 minutes and repeat the PTT. If the PTT result corrects to normal, it probably means PK deficiency caused the initial prolongation. This works when the PTT reagent uses kaolin or Celite, but not ellagic acid as its activator. George speculates that the kaolin or Celite activates factor XII upon prolonged incubation and bypasses the need for PK.
Kim also noticed that people with high molecular weight kininogen (HMWK , Fitzgerald factor) deficiency seem to have PK deficiency. This discussion was left open. April 7

Subscriber Dkaguni asks, “Historical ranges are often used to determine the range for coagulation assays. Are there general guidelines as to how large these ranges can be?” George assumes the question addresses internal assay precision, expressed as coefficient of variation (CV%). Per Dr. John Olson, there is no historical range for an acceptable CV%. Some may be as small as 4%, other assays are clinically effective at &gt ;10%. The key issue, as recommended by Dr. Olson, is that the laboratory perform and record local (in-house) precision studies to confirm manufacturer’s claims. Methods for in-house precision studies are presented in audio modules 3 and 4. For preparation of reference intervals (normal ranges), the standard approach is to assay aliquots from a well-defined cohort of normal subjects and compute the mean and standard deviation. The typical reference interval is ± 2 SDs. Methods for computing reference intervals are provided in audio modules 5 and 6. April 13