Amy Carpenter
July 2025—The variables that affect coagulation testing are plentiful, from preanalytic to postanalytic. In a CAP course last year, the presenters walked through a handful that are central to patient safety.
Andrew Goodwin, MD, and Eric Salazar, MD, PhD, members of the CAP Hemostasis and Thrombosis Committee, studied the CAP’s accreditation checklist and most common inspection deficiencies, scanned package inserts and published recommendations, and queried their committee colleagues about their own laboratory practices.
From specimen stability to interferences to calculations, they highlighted the details that matter and one laboratory’s move to automation. (In part two next month: anticoagulant interference, INR calculations, D-dimer units and magnitude of measurement, and viscoelastic testing.)
The sixth edition of “CLSI H21: Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays,” published last year, contains important updates related to specimen stability, said Dr. Goodwin, professor, Department of Pathology and Laboratory Medicine, University of Vermont Larner College of Medicine, and medical director of the thrombosis and hemostasis section, University of Vermont Medical Center. For fibrinogen, D-dimer, and thrombin time, specimen stability time went from four hours in the fifth edition to 24 hours in the sixth. Other examples: Antithrombin was four hours in the fifth edition and is 72 hours in the sixth, and some factor assays went from four to upward of 48 hours. These updated guidelines are based on peer-reviewed literature, and in Dr. Goodwin’s lab, they are performing local validations in hopes of extending specimen stability for fibrinogen and antithrombin.
Unfractionated heparin stability time did not change; it remains one hour unless the collection tube contains CTAD. “And it’s important that laboratories recognize that heparin stability does drop precipitously after an hour,” Dr. Goodwin said.
An activated platelet releases platelet factor 4, which neutralizes heparin and results in a lowered measured concentration in the laboratory. Whole blood samples should be centrifuged to make platelet-poor plasma within one hour of collection. “And if you’re still using a PTT-based method, and you don’t have it highlighted for heparin monitoring, and your stability is four hours, you’re going to start to affect the heparin levels if you don’t process those PTTs for heparin monitoring in an expedited fashion,” Dr. Goodwin said (Adcock D, et al. Blood Coagul Fibrinolysis. 1998;9[6]:463–470).
Published data show that heparin stability can be extended beyond one hour in some circumstances. His lab performed an in-house stability study to monitor the initial heparin result followed by heparin levels at different points in time. He and colleagues designed such a study for their chromogenic anti-Xa assay and found there was a negligible drop in heparin stability at 75 minutes and a clinically significant drop between 90 minutes and up to two hours.
His advice is to know the most recent specimen stability guidelines, refer to the package insert for stability, develop protocols that reflect best practices for stability, and exercise caution when the lab has many separate stability requirements.
HEM.37175 in the CAP hematology and coagulation accreditation checklist requires the laboratory to measure the actual platelet count of the platelet-poor plasma used for many coagulation tests at least annually and after major centrifuge maintenance or service.
It’s this requirement that is cited most often as a deficiency in coagulation laboratories. “It was cited 32 times in one year, 1.6 percent of labs,” Dr. Salazar said. “It’s the most common deficiency for coagulation labs.”
Platelets can release microparticles/phosphatidylserine, fibrinogen, factors V and VIII, von Willebrand factor, and platelet factor 4, all of which can neutralize heparin and affect other coagulation tests. “So it’s important to get rid of residual platelets if you’re going to move on with coagulation testing,” said Dr. Salazar, associate professor, Department of Pathology and Laboratory Medicine, University of Texas Health San Antonio; medical director of clinical laboratories, UT Health Multispecialty and Research Hospital; and section chief of the hemostasis and thrombosis laboratory at University Hospital.
Platelet-poor plasma is defined as less than 10 × 109/L. Platelet activation can result from a freeze-thaw, a cooling of the sample, hemolysis, or use of a pneumatic tube system. Activated platelets release procoagulant phospholipids that can neutralize lupus inhibitors, which can bind to the procoagulant phospholipids, Dr. Salazar said, noting lupus anticoagulants are antiphospholipid antibodies. “And that can lead to false-negative testing if you have too high a platelet count in your lupus anticoagulant test.”
Clotting factors may have an impact on traditional coagulation assays, Dr. Salazar said, though studies have shown that the APTT, PT/INR, and thrombin time assays performed on fresh samples do not seem to be affected by a platelet count of up to 200 × 109/μL. “This is not true for samples evaluated for heparin therapy or lupus inhibitors,” he said.
The CAP, the CLSI, and others generally recommend a con-servative approach. “Make sure you have platelet-poor plasma in case you’re going to use that sample for this kind of testing,” he said. “Keep your platelet-poor plasma platelet level low and make sure you validate that at appropriate times.”
He advises following a platelet-poor plasma verification protocol, such as the one in CLSI H21. “You take a capped tube centrifuged at a speed and time that results in a platelet-poor plasma, <10 × 109/L platelets.” The laboratory must establish the speed and time and it will vary depending on the centrifuge in use. The most common centrifugation protocol (and the one cited in H21) is 1,500 g for 15 minutes. Other published protocols have a shorter centrifuge time at a faster speed (4,400 g).
There’s discussion on the CAP Hemostasis and Thrombosis Committee and with others about “how high we can go with centrifugation speed and g-forces,” Dr. Salazar said, “but right now we don’t have a good understanding of the acceptable limits of centrifuge force.” A study is underway. “Maybe that leads to a new standard in the future.”
The platelet count should be verified with an automated cell counter, he said. “If the residual count is not achieved, consider a double centrifugation, transport the plasma to a second tube, and centrifuge again.”
CAP checklist requirements address platelet function studies. HEM.38350 on specimen handling says blood specimens for platelet aggregation and platelet function studies must be handled at room temperature before testing. HEM.38300 Platelet Function Studies requires platelet aggregation or initial platelet function tests to be performed within an appropriate period after venipuncture.
“Specimen transport times cannot exceed specimen stability,” Dr. Salazar said. “And for most platelet function tests, that’s usually within four hours, but follow your manufacturer’s instructions.”
CLSI “H58-A Platelet Function Testing by Aggregometry; Approved Guideline” says hand delivery is the preferred transportation mode for these specimens.
Dr. Salazar cited a couple of studies, one of which found that PFA-100 and aggregometry parameters were prolonged by pneumatic tube transport in patients on aspirin (Dyszkiewicz-Korpanty A, et al. J Thromb Haemost. 2004;2[2]:354–356). The other found that TEG clot formation time was shorter in samples transported via pneumatic tube (Wallin O, et al. Clin Chem Lab Med. 2008;46[10]:1443–1449).
For platelet function testing, he said, “if you are using a pneumatic tube system for specimen transport, consider validating its use.” And as hospitals grow and tube systems are expanded, “you might want to revalidate.” For routine coagulation tests, tube systems tend to be acceptable.
Among the sample issues related to optical analysis are hemolysis, icterus, and lipemia interferences, and some newer coagulation analyzers have an automated detection system that provides objective HIL measurements,” Dr. Goodwin said.
The photo-optical method is fairly sensitive to HIL interference, particularly hemolysis, he said. “Anything that interferes with the photo-optical component becomes problematic and might result in less accurate results.” The electromechanical method is less vulnerable to HIL analytic interference, Dr. Goodwin said, “though hemolysis might indicate suboptimal specimen quality.”
The CLSI H21 guideline advises referring to the manufacturer’s package insert for specific interference limits for each coagulation analyte. “Guidelines and literature have substantive discussions of hemolysis,” Dr. Goodwin said, “and they indicate not only is it a potential optical interference, but it calls into question the specimen integrity.”
CLSI H21 also says that if hemolysis is generated because of a traumatic blood draw or a violent pneumatic tube ride, or for another reason, “that is an indication that you might have activated the coagulation system,” Dr. Goodwin said, or done something to compromise the specimen. It identifies the assays most impacted by evidence of hemolysis that could be due to trauma to the specimen, including fibrinogen, D-dimer, antithrombin, anti-Xa, and lupus anticoagulant.
The International Council for Standardization in Haematology recommends that plasma for coagulation testing that has been prepared from citrated blood samples be checked for the presence of in vitro hemolysis, preferably using an automated system for consistency (Kitchen S, et al. Int J Lab Hematol. 2021;43[6]:1272–1283). It also recommends that the APTT not be performed on samples with hemolysis that has occurred in vitro during sample collection, transport, and processing. “And they say the PT doesn’t appear to be quite as impacted,” Dr. Goodwin said.
Another ICSH recommendation says the possibility that hemolysis has occurred in vivo should be considered and that samples from patients with in vivo hemolysis for the determination of coagulation tests can be accepted and tested. “So they’re indicating that the etiology of the hemolysis should be one of your decision points in whether to accept that specimen,” Dr. Goodwin said. “That can be challenging, particularly for a reference laboratory.”
For lipemia, many laboratories have an ultracentrifugation process and will ultracentrifuge if they have high amounts of triglycerides or lipemia in the specimen, Dr. Goodwin said. In their own study, he and colleagues found that ultracentrifugation had an impact on von Willebrand factor and factor VIII levels. vWF activity results were an average of 31.9 percent lower, vWF antigen results were an average of 12.1 percent lower, and FVIII levels were an average of 23.2 percent lower. The impact on APTT was minimal.
“These are large proteins, and they spin out in ultracentrifugation. So we no longer report vWF or FVIII results on specimens that were ultracentrifuged for lipemia.”
The effects may not necessarily be clinically relevant, he said. “But if a von Willebrand activity is slightly above 50 and reported below 50, it crosses the clinically relevant threshold from normal to abnormal,” Dr. Goodwin said.
For icterus, some laboratories have developed dilution protocols to minimize interference. For factor assays, “we do multiple dilutions to identify interfering substances, and if we can dilute out that interfering substance, we will report a result,” he said, noting it’s a practice some laboratories have adopted. (HEM.37980 addresses factor assay criteria.)
In sum, Dr. Goodwin advises understanding the instrument’s interferences, reviewing the literature for the best standardized approach to handling HIL specimens and ensuring the literature is inclusive of your lab’s particular instrument and/or reagent, plus possibly investigating the etiology of hemolysis, particularly when it’s consistent in a single patient. His lab recognizes certain patient populations are subject to in vivo hemolysis, such as those patients undergoing intravascular mechanical thrombolysis or circulatory support.
“In our laboratory, if we detect hemolysis below the threshold, we will issue a comment that says hemolysis was detected but we release the result. If it is above the threshold listed in the IFU, we reject the specimen,” Dr. Goodwin said. In patients who have in vivo hemolysis, a protocol is used whereby if the instrument provides a result, it will be given to the provider in a comment with an explanation that it may be inaccurate. He makes an attempt to cite the literature and provide the specific impact hemolysis may have on the test result(s).
“In our laboratory, the cutoff is 325 mg/dL for hemolysis, and when it gets above that level it flags it. We often will not release that result,” Dr. Goodwin said. “But we will release the result with a disclaimer when we determine in vivo hemolysis, and the key indicator to investigate for in vivo hemolysis is when we’ve tried twice to collect yet the hemolysis persists.” Lastly, it is important to review the instrument information, such as the clot curve along with baseline and change in the milli-absorbance readings.
The common D-dimer interferences are well recognized: free hemoglobin, hyperbilirubinemia, hyper-triglyceridemia, and rheumatoid factor (though many reagent systems add a rheumatoid factor blocking agent to mitigate the impact). Dr. Goodwin tells the story of one less likely to be known.
The D-dimer assay is a latex-enhanced immunoassay, and when the specimen containing the D-dimer is added to the reagent system, the D-dimer will bind to and agglutinate the latex beads, which is reflected in the change in milli-absorbance measured by the instrument. Interfering heterophile antibodies, for example, bind to the beads and cause agglutination. “Even though you have a low D-dimer concentration,” Dr. Goodwin said, “these antibodies appear to cause latex bead agglutination and may overestimate the D-dimer.”
Dr. Goodwin’s laboratory ran a test for a patient with a very high D-dimer concentration. “When you have an output with an initial very accelerated slope [for change in milli-absorbance] that then plateaus, that’s an indication you have antigen excess, and it’s often an indication you have D-dimer above your AMR. At least in our laboratory, you get an ‘above AMR indication’ on the report,” he said.
Often when a result exceeds the analytical measurement range, an onboard or other dilution step is performed with the expectation that the diluted measurement falls within the AMR (the reported result is corrected for the dilution).
In the sample in question, “the result goes from above the AMR to below the AMR, and our dilutions are not set up to do that. It didn’t make sense to us,” Dr. Goodwin said.
The laboratory did another collection on the patient and an extensive investigation with the manufacturer. They did a D-dimer split sample and sent it to another laboratory. (The patient was normal on a different assay method and mildly elevated by the different laboratory with the same instrument using a different reagent.) “We wondered if the patient had antibodies impacting the monoclonal antibody on the latex beads,” Dr. Goodwin said. The manufacturer did multiple studies and identified what it labeled as a “heterophile antibody” that was not anti-mouse, anti-goat, or anti-rabbit but was interfering with the assay.
Now, in addition to the common D-dimer interferences, his laboratory has in its protocol a heterophile antibody where it sees initially excess antigen (above the AMR, and then upon dilution it drops below the AMR). “It’s happened in a handful of patients since 2016,” he said, and in these samples the laboratory reports: “Unable to assay due to interference.”
The laboratory recommends the ordering provider carry out the next step in their protocol. “You can send it to another laboratory that uses a different monoclonal antibody in its system,” Dr. Goodwin said, noting his lab does that upon request.
His advice: Understand the laboratory’s AMRs for D-dimer and look for samples that are initially above and then drop below post-dilution. And be aware that heterophile antibodies are another possible source of D-dimer interference.
Drs. Salazar and Goodwin opened their discussion of middleware and automation with a brief look at reflex rules.
Traditional factor assays require multiple dilutions and interpretation, Dr. Salazar noted. HEM.37980 Factor Assay Criteria requires that three or more dilutions be plotted for each functional factor activity assay to detect nonparallelism, and, if detected, that the nonparallelism be reported. HEM.37984 Inhibitor Interference requires the laboratory to report the highest factor activity apparent with dilution if nonspecific inhibitor interference is apparent in a factor activity assay.
Nonparallelism suggests an inhibitor, Dr. Salazar said, citing as an example data from a 2017 article (Riley PW, et al. J Pathol Inform. 2017;8[1]:25). In coagulation factor activity testing, corrected (post-dilution) calibration and patient results are parallel and show a consistent result on every dilution.
“On the other hand, if you dilute your patient and start to recover more and more activity, that can suggest some kind of inhibitor,” he said. Multiple interferences in a one-stage clotting factor assay can lead to this, he said, pointing to nonspecific inhibitors such as lupus anticoagulants, as well as pharmaceutical anticoagulant inhibitors such as heparin, direct oral anticoagulants, or direct thrombin inhibitors.
Most specific factor inhibitors, such as those seen in acquired hemophilia, do not usually cause nonparallelism, Dr. Salazar said. “Usually these are thought of as nonspecific factor VIII inhibitors.”
“Running these three dilutions and [determining] which value to report can be challenging in the setting of nonparallelism,” he said, “but middleware can make things easier.” And these middleware rules can be “high impact” on laboratory operations.
Riley, et al., reported in their 2017 article in the Journal of Pathology Informatics several advantages of implementing autoverification expert rules as applied to coagulation factor activity testing: ease of training, minimization of required technologist time, reduction in staff fatigue, and better consistency and quality, among others. “Implementation of middleware rules reduced overall labor expenditure by about four percent,” Dr. Salazar said.
At the University of Vermont Medical Center, Dr. Goodwin and colleagues took the coagulation laboratory from a manual to an automated line in June 2023.
The laboratory’s annual specimen volume is 53,000, but it receives 73,060 specimens (holds/spin and freeze). Twenty-three FTE technologists run the hematology and coagulation laboratory sections. UVMMC is a regional hemophilia treatment center, a level one trauma center, and a platinum-level stroke center, and the laboratory supports these services as well as a busy Thrombosis and Hemostasis Program.
Dr. Goodwin and colleagues learned in 2021 that the Windows XP software on the laboratory’s three analyzers for routine and specialized coagulation testing would no longer be supported. They were forced into a decision: “Obviously upgrade our instruments, but it raised the question: Do we want to consider automation at this point?”
To make the case for automation, he focused on the finances, staffing, and scope of services and the potential for improvement in all three.
The laboratory leadership invited feedback from those who do the testing. The manual process is inefficient, they said, and comes with ergonomic problems.
The view of the specimen receiving group: “Everything is fine.”
“But phlebotomy felt rejection rates were high and that it was often subjective,” Dr. Goodwin said.
A workflow analysis counted 38 process steps and 10 decision points in the laboratory’s manual workflow. “That’s a lot because 10 decision points offer 10 opportunities to make the wrong decision, and 38 process steps is 38 times the specimen can be handled incorrectly.”
Streamlining the workflow to improve efficiency, mitigating overuse and repetitive use injuries, and bringing objectivity to specimen evaluation for HIL and fill volume, for example, were the main objectives, as were minimizing redraws, test cancellations, and reducing or eliminating interferences and suboptimal specimens.
Dr. Goodwin and colleagues calculated that in seven years of using an automated line the laboratory would save $1.233 million in expenses, mostly in manual labor, making it possible to expand the scope of the laboratory’s services. “‘We can bring in more testing; we can deliver testing quickly to patients,’ we explained to UVMMC leaders and other ROI stakeholders.”
Post-automation, the workflow went from 38 process steps and 10 decision points to 10 process steps and one decision point. “It was a huge change,” he said. “And we essentially eliminated biohazard exposure for the technologists because they no longer had to uncap specimens for routine testing.”
The technologists use the automated line 98 percent of the time, “which is huge, and there’s no difference in what they put on there,” he said. “You would think maybe they’re putting all the stats on there—they’re not.” While some specimens require frontloading, “most are put on the automated line.”
The approach to automation wasn’t top-down, he said. The technical staff designed the automation line and were in charge of implementation. Coagulation laboratory technical specialist Kristin Lundy, MA, CLS, was the change management leader and worked with the lab staff to create five automation line implementation teams: workflow, agility, procedure, competency, and oversight.
There has been a significant positive impact on the second- and especially third-shift staff, Dr. Goodwin said. “They don’t have to interact nearly as much with the coag specimens.”
The scope of the laboratory’s services is expanding with new testing: apixaban and rivaroxaban, diagnostic HIT, direct thrombin inhibitor measurements (LDT), activated protein C resistance, free protein S, and chromogenic protein C.
Turnaround time is down, workflows are simpler, HIL monitoring is leading to better insight, and job satisfaction is up. “The middleware is extremely powerful and offers the ability to add customizable reflex rules in the middleware system,” Dr. Goodwin said.
Amy Carpenter is CAP TODAY senior editor.