Dr. Rich Cambereri wrote, “I have a cluster of “alleged” LA patients, prompting me to revisit the DRVVT. One 48 y/o man has had DVTs, PEs, and an IVC filter. He has been on LMWH. The DRVVT showed the initial mix corrected after the screen was prolonged. This, as I understand it, is incompatible with an inhibitor as normal plasma corrects the prolongation. The lab proceeded with the confirmatory step of phospholipid neutralization and the ratio compared to control was 1.6, consistent with LA. If the initial mix corrects, doesn’t this mean a deficiency of V or X? Why would one proceed to the confirmatory step?
George responds that, given the comments on Ms. Vanessa Chan’s post from Herb Crown and Larry Brace, because the effect of a weak LA can be diluted by the normal plasma in a mixing study, it may be necessary to continue to the confirmatory high phospholipid reagent step. This raises the question, “Why do the mixing study at all?”
Scott Miller, St. Mary’s of Michigan, wonders about the utility of a mixing study as part of LA screening. If the accuracy of a DRVVT-based screen/confirm is in question for some reason, it seems like one would simply go directly to a hexagonal study. How old are the ISTH guidelines? Are there any recent studies to support using one set of tests over another?
Herb Crown, St. Louis University, responded to Scott that in addition to questioning the 50/50 mix component of the DRVVT, we also include a 50/50 mix as an integral part of the popular hexagonal phospholipid LA test system. That seems to me to automatically introduce, by default, a dilution of the antibody. Performing the hex-phase LA study without the 50/50 mixing component could be a more valid study. In this scenario, since an alleged factor deficiency would be present in both the buffer tube and the hexagonal tube to the same extent, the NPP adds nothing to the study.
Catherine Coop asked about stability for anti-Xa specimens. The Stago Rotachrom package insert says to perform testing within two hours of collection. We have noticed some specimen stability issues after one hour if the plasma is not removed from the cells and frozen. Also, would there be any difference if the patient is on LMWH vs. UFH?
George refers to CLSI H21-A5, which states that specimens for UFH must be centrifuged to produce PPP within one hour of collection because of the potential for heparin neutralization by PF4. Once centrifuged, the PPP must be separated and tested within four hours of collection. PF4 is a heparin-neutralizing protein expressed by platelets as they become activated subsequent to specimen collection, falsely reducing plasma heparin concentration in vitro. There is no reference confirming that PF4 also neutralizes LMWH, but it probably does. If the laboratory cannot test the PPP within four hours, it must be frozen. Manufacturer specifications may supersede the guideline, and it appears that Stago has actually shortened the limit for their heparin assay from four to two hours. Please refer to our audio PowerPoint module #17, Hemostasis Blood Specimens, for further detail on coagulation specimen management.
Bob Gosselin, Univ. of California: Hey GF, re POC INRs (March 14), a high HCT should not matter for a system that employs finger-stick whole blood, since there is no citrate:plasma ratio issue. George agrees. He took the 25-55% comment (March 14) from the cartridge package inserts for the Coag-U-Chek and the iStat, but suspects the manufacturers set limits based on current plasma-based standards. Bob also speculates that HCT could associate with viscosity.
Another post from Bob Gosselin: GF, re dabigatran clearing in 24 h: This is not necessarily so, as we have seen “dabi” trough levels (q12) of ~150 ng/ml. Bob suggests using TTs to assess clearance, as most go to >150 secs at 50-75 ng/ml. TT should be used in concert with PTT for assessing drug level, especially if determining whether drug level is low enough for interventional or thrombolytic therapy. George mentions that Hyphen has developed their Hemoclot Thrombin Inhibitor Kit for dabigatran. All the new DTI assays, including Stago’s Ecarin time and Ecarin chromogenic assay are RUO.
An anonymous inquirer has large oval-shaped platelets and a PLT count averaging 40,000/uL. She has normal coagulation times and no bleeding or bruising. Her condition is seen in some first-degree relatives. She wonders if the size of the needle could damage platelets because they are so large, resulting in a lower number? She was treated with IVIG and steroids while pregnant with no changes.
George replies, “The next time you see a physician, you may suggest she look for one of the MYH9-related thrombocytopenia syndromes. The most common MYH9 syndrome is May-Hegglin anomaly, an asymptomatic, inherited (autosomal dominant) thrombocytopenia with giant platelets. The hallmark of May-Hegglin is the presence of Dohle bodies, light-blue areas in the cytoplasm of granulocytes. Dohle bodies are identified by skilled laboratory scientists reviewing blood films, often ordered as a part of the CBC.”
“You may ask your physician to follow up with platelet aggregometry, electron microscopy, and molecular testing. This effort is largely academic, but may have some minimal value as it distinguishes May-Hegglin from the more rare Fechtner syndrome, Sebastian syndrome, and Epstein syndrome. These syndromes do not produce Dohle bodies and are occasionally associated with mild bleeding, renal (kidney) failure, hearing loss, and cataracts.
It is a good idea to let your laboratory scientist know about your thrombocytopenia and large platelets, and to suggest a manual platelet count. A small portion of your platelets could rupture, both during blood collection and the slide-making process, and if they are large enough, some instruments could miss-classify them as red blood cells, falsely elevating your red blood cell count.”
An anticoagulation clinic coordinator asked: What value would you use to reflex a high finger-stick INR to a venous blood PT/INR and why?
George answers that POC instruments provide rapid PT/INR turnaround with reasonable precision. The precision and linearity of plasma-based PT/INRs is superior to POC and the managers of anticoagulation clinics choose to reflex the assay to the lab for confirmation if there is a significant change from a patient’s previous PT/INR result or if a result is unexpectedly high. George recommends INR 4.0 based on published data that show the risks of a serious bleed begin to rise dramatically at 4.0. There exist no published data that delineate the benefits and adverse outcomes of choosing 4.0 vs. 5.0 as the reflex value for POC, nor do the operators’ manuals of the various POC instruments provide a conclusion. At 4.0 you inconvenience patients and incur greater expense, at 5.0 you risk sending home a patient with a bleeding risk.
Comment from Scott Miller, St. Mary’s of Michigan: As with most laboratory tests where two or more systems are used, it is common to validate less robust systems by correlation with a reference system. I am not sure if relying on poll results to establish linearity would turn out to be a good idea when the inspectors come around! We check linearity of INRs on POC analyzers by paired testing against our hospital analyzers. We find that linearity gets much worse around an INR of 3.5-4.0. Our protocol is to have those POC patients with INRs above 3.5 get a venous draw that is run at the hospital. POC INR analyzers generally have high ISIs, often approaching 2.0. Larger analyzers are usually around 1.0. Poor linearity with higher INRs would be expected with anything using high ISIs.
Summary of our April Quick Question: The US FDA is planning guidelines for regulating laboratory-developed tests (LDTs). What plans have you made in response?
a. We are discontinuing all LDTs: 3 (15%)
b. We didn’t know this is happening: 11 (55%)
c. None, this will only apply to reference laboratory LDTs: 1 (5%)
d. We are improving on, and recording our LDT validations: 5 (25%)
We have had a low response to this question, indicating a general lack of concern. It is likely LDT regulation will first affect reference laboratories and tertiary-care institutions that are providing advanced testing, such as molecular diagnosis. The FDA may not have the resources to enforce these new regulations within individual institutions, however the bullets are likely to produce “powder burns” that will motivate us to beef up our LDT validations.
Laurel Veek at Mayo Labs noticed her ristocetin aggregations have a curve that begins to aggregate after 2-3 minutes or generates an abnormal slope if it aggregates before 3 minutes. She spoke to the company and they said some lots of ristocetin cause nonspecific aggregation but ristocetin should react within 30 seconds, and that reactions observed after the first three minutes are not related to the VW protein. Have others noted this?
George checked with the special coagulation staff at the UAB lab, who report no problems with their current lot. He recommends not using this lot of ristocetin, as it seems like you run the risk of generating some false positive results for VWD.
“Danagah” asks how to handle a sample for a chromogenic heparin anti-Xa assay if a patient is experiencing in vivo hemolysis. They reject all markedly hemolyzed samples for any hemostasis testing but ask if George reject a sample with any visible hemolysis?
George reports that anti-Xa assay manufacturers claim no interference up to 2 mg/mL plasma hemoglobin. Plasma hemolysis first becomes visible at 0.5 mg/mL, and CLSI H21-A5 recommends rejecting “severely hemolyzed” specimens. “Danagah” is thus in agreement with CLSI to reject markedly hemolyzed specimens, but what does “severely,” or “markedly” mean? In reality, we assume any visible hemolysis implies platelet activation, releasing PF4, which neutralizes heparin. Consequently, the heparin anti-Xa assay is likely to generate a falsely reduced plasma heparin concentration. Consequently, George recommends rejecting all coagulation specimens with visible hemolysis, especially when monitoring heparin, be it by chromogenic substrate or clot-based assay. Most hemolysis is caused by collection variations, especially collection through a vascular access device. In the case of in vivo hemolysis, however, you have no choice but to perform the assay and append a comment describing the hemolysis and explaining it could affect all test results and could falsely reduce the reported heparin concentration.
Crystal Azevedo asked if any labs are using the ecarin clotting time to test for dabigatran level? She has trialed the ECA-T kit from Diagnostica Stago with Aniara calibrators/controls. George notes that reference labs Esoterix, Machaon, and Quest offer the ECT but doesn’t know how many hospital labs are offering it. In a follow-up comment, “Jlow” uses Hemoclot, a TT-based test which is very easy to set up, works well and is available through Hyphen BioMed. Calibrators and controls are also available.
Dr. Shabnam Roohi is using an STA Compact and would like to validate the fibrinogen reportable range, 60-1200 mg/dl. George went to Dr. Ali Sadeghi-Komami, Precision BioLogic Inc. He reports that PBI offers Cryocheck Normal Reference Plasma with known fibrinogen. It could be used for establishing the calibration curve after a serial dilution and goes up to approximately 11-12 g/L (12 mg/dL) fibrinogen. Further, the usual fibrinogen assay protocol requires a 1:10 dilution of patient plasma with a buffered diluent, so there is actually no need to find a reference with ultra-high fibrinogen to establish such a wide range (0.6-12 g/L fib) calibration curve. Usually the serial dilution does the trick, beginning with an undiluted specimen.