Charna Albert
March 2025—Theresa Kinard, MD, knew little about phosphatidylethanol (PEth), a blood-based biomarker of alcohol use, when she noticed that patients who adamantly denied drinking were testing positive in their liver pretransplant evaluations.
“There had been cases where I could see in the clinical notes that [liver] transplant candidacy was denied because of PEth results, where I could see in the documentation in the chart that the patient is resolute that they haven’t been drinking,” says Dr. Kinard, a transfusion medicine specialist in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Arizona.
Dr. Kinard turned to her colleague in the department, Christine Snozek, PhD, D(ABCC), codirector of clinical chemistry. “I asked, ‘How is this test performed and what could possibly cause a false-positive result?’” recalls Dr. Kinard, who is also assistant professor of laboratory medicine and pathology at the Mayo Clinic College of Medicine and Science.
PEth has been widely used in recent years thanks to its long detection window, Dr. Snozek says, which at a half-life of seven days is longer than other alcohol biomarkers such as urine ethyl glucuronide or ethanol itself. In addition, concentrations of other biomarkers can’t be used to gauge how frequently or how much someone is drinking, “whereas [PEth] blood concentrations can,” she says.
When Dr. Kinard asked her about PEth, she says, now a couple of years ago, conventional wisdom held that it wasn’t subject to many preanalytical variables. “Red cell turnover, sure,” she allows. “But it wasn’t recognized that there are other factors.” PEth is formed by reaction of ethanol with phosphatidylcholine, with phospholipase D catalyzing formation of PEth on the red blood cell membrane when exposed to ethanol (Kinard TN, et al. Transfusion. 2024;64[9]:1617–1622). “It accumulates on red blood cells more so than other cells,” Dr. Snozek says. “With some of the other alcohol biomarkers you can get preanalytical issues from things like urinary tract infections. But PEth was thought to be pretty close to immune.”
Then, Dr. Kinard posed a question. “If it’s in the red cells,” Dr. Snozek recounts, “couldn’t it be transferred if a patient got a transfusion?” In other words, if a patient with negative PEth receives a red blood cell transfusion from a donor with positive PEth, could the recipient then test positive? Dr. Snozek thought it unlikely but suggested a study. “Lo and behold, we were wrong—it does happen.”
Dr. Kinard had seen as a consult an adult patient with new onset leukemia who required a transfusion of four packed red blood cell units. The patient was not a regular drinker. “It was a good opportunity in someone who was likely to be negative to see if the red cell units had an impact,” Dr. Snozek says.
“Because of the hospital admission,” Dr. Kinard adds, “we knew there would be no exposure to alcohol.” But whether they would learn anything from the red cell transfusion was a question mark. “We weren’t really shooting in the dark—but sort of,” she laughs.
The findings were stark. Residual blood samples collected at admission showed POPEth and PLPEth—homologues of PEth and the typical targets of most PEth assays—at less than 10 ng/mL, decidedly negative. (Only POPEth has consensus guideline interpretation levels, Dr. Snozek notes, with less than 20 ng/mL compatible with abstinence or low alcohol consumption.) A sample drawn after completion of the fourth transfusion showed POPEth at 57 ng/mL and PLPEth at 38 ng/mL.
“With this particular case,” Dr. Kinard says, “we were just really lucky.”
Previous studies had surmised that PEth could be transmitted through red blood cell transfusion, she says. But to prove it conclusively, they tested citrated packed red blood cell segments from three of the four units the patient received. “Because of the evaluation in real time, we were able to sequester the unit segments to test that,” Dr. Kinard says.
First, however, their colleagues at the Mayo Clinic laboratory in Rochester validated the PEth assay for citrated blood. “It was validated on EDTA-anticoagulated whole blood,” Dr. Snozek says. “That’s the preferred sample type.” But a different anticoagulant is used in citrated red cell units, she says, and because the units are packed, the ratio of red cells to fluid differs from whole blood. “We agreed it would be better to make sure the test was going to work properly in the citrated packed red cells,” she says, “so they validated it before we did anything.”
Segments from two of the units showed both PEth homologues below the limit of quantitation; the third had a POPEth of 585 ng/mL and a PLPEth of 250 ng/mL. (Segments from the fourth unit were unavailable, Dr. Snozek says, potentially because they had already been used for another form of additional testing.)
“The fact that we were able to demonstrate that it went from a negative pretransfusion sample to a positive post-transfusion sample within the same hospital stay was the first conclusive proof,” Dr. Snozek says.
Adds Dr. Kinard: “And it was the first [study] to test the units, rather than saying only this could be the source. We knew it was the source.”
Their article was published in 2023 (Snozek CLH, et al. Clin Biochem. 2023;120:110651). (Dr. Snozek notes the contributions of coauthors Paul Jannetto, PhD, D(ABCC), MT(ASCP), and Loralie Langman, PhD, who run the laboratory in Rochester where the PEth testing was performed.)
The case made clear not only that artificial elevation of PEth can occur with transfusion, but that transfusion can cause significant elevation, Dr. Snozek says. She recalls thinking that if they saw anything it would be a modest positive, something just above the assay’s lower cutoff. “The segment itself had a relatively high amount of PEth, so it makes sense,” she says. “But it was at a higher concentration than we expected.”
Given the patient’s anemia, Dr. Snozek and her coauthors write, the transfused units represented a substantial fraction of his total red blood cell volume. With the segment from the fourth unit unavailable, “we can’t conclusively state that there wasn’t some PEth in the fourth unit,” she says. But theoretically, a single transfused unit could be enough to push PEth concentrations into clinically relevant ranges. “We could say the math strongly suggests it.”
“The math works out rather well if you calculate the amount of red cell mass transfused versus what would be in a recipient at our transfusion threshold, which is seven grams per deciliter of hemoglobin in most people,” she says. As she and her coauthors explain in the report of the case, a single transfused unit would represent one-fifth to one-sixth of the recipient’s red blood cell volume. Assuming no endogenous PEth in the recipient, a donor PEth concentration above 100 to 120 ng/mL could therefore be enough to elevate the recipient’s post-transfusion PEth concentration to greater than 20 ng/mL.
Thanks to their findings, Dr. Kinard says, Mayo’s transfusion service is now saving additional segments from all packed red blood cell units, which could prove useful for confirming suspect PEth testing. “It’s required that we keep a segment so we can perform ABO confirmation on it when we receive it into the inventory,” she says, and they typically save another from each unit for retrospective investigations, but they’re now saving more. “We decided to keep multiple segments so we can meet our regulatory requirements and still have samples to perform any investigations they ask us to,” she says.
There’s plenty for the laboratory to chew over too, such as the appropriate PEth cutoff for transfusion patients. Dr. Snozek is in favor of the POPEth consensus cutoff of 20 ng/mL, though clinical teams often push for lower thresholds when evaluating PEth. “The consensus guideline cutoff is consistent with abstinence or light drinking,” she says. “Clinical teams want to rule out that light drinking aspect of it, if possible.” But in a recently transfused patient, values between 10 and 20 ng/mL could suggest exogenous PEth. “That higher cutoff is going to give you a little bit of buffer zone,” she says. “So that’s something for labs to consider, at least when phrasing their interpretations. The higher the result, she adds, “the more likely it’s from consumption, as opposed to other sources.”
All that said, she emphasizes this is not a problem with the assay and advises against the use of “false-positive” when referring to PEth-positive results derived from transfusion.
“When we think about false-positives, especially in the toxicology world, we’re saying it truly cross-reacted with something that’s not there. The PEth is there. It’s present. It’s exogenously derived, as opposed to endogenously formed.” She and colleagues prefer “artificial elevation,” which they used for the title of their case report. “I wanted it to be clear that this is not the test picking up something it shouldn’t pick up. It’s just coming from a source that’s not that patient drinking.”
Don’t assume that a healthy blood donor isn’t drinking to “a surprising level of PEth,” Dr. Snozek says.
That’s one upshot from a follow-up study published in 2024, in which she and Dr. Kinard and their coauthors tested for the prevalence and stability of PEth in packed red blood cell units (Kinard TN, et al. Transfusion. 2024;64[9]:1617–1622). In studies they’re doing now, they have seen red cell units with POPEth concentrations greater than 1,000 ng/mL.
The number of units that surpassed 200 ng/mL of PEth—the threshold for what’s considered heavy drinking—came as a surprise, Dr. Snozek says. Yet at least one other study of a blood donor population reported a similar prevalence, she says. “We’re recognizing that more donors might be drinking to higher-than-expected concentrations in an otherwise healthy population.”
She and coauthors obtained 102 whole blood and 78 apheresis units with corresponding donor EDTA samples from the blood donation center at Mayo Clinic in Rochester and tested for PEth with limit of quantitation 10 ng/mL. More than 40 percent of apheresis and whole blood donors had PEth of 10 ng/mL or above. The maximum observed in donors in this study was 587 ng/mL.
Testing the native patient blood before it was processed allowed them to examine how the manufacturing process affects PEth concentrations. As whole blood units were processed into component packed red blood cells, PEth concentrations increased, becoming higher than donor whole blood levels prior to collection (maximum observed 711 ng/mL). The reason? “We’re creating a concentrated product,” Dr. Kinard says, with a higher percentage of hematocrit. The study findings suggest that processing might increase the risk of artificial PEth elevation in recipients, she and her coauthors write. Of note, she says, the PEth assay does not account for individual variation in hematocrit.
The assay reports a concentration of the PEth homologues that are being measured, Dr. Snozek elaborates. “It does not factor in whether the person has 35 percent hematocrit, 40 percent hematocrit, 55 percent—you’re just going to get a number, even though there may be more or fewer red cells contributing to that number.” Some have suggested the assay be normalized to hematocrit, she says. “There’s been at least one group to suggest that might be a better way of reporting it. If it’s present on the red cells and that’s primarily where you’re measuring it from, accounting for variability between individuals in terms of how much of their blood is made up of red cells versus other components makes sense.”
Plus, the transfusion recipient is likely anemic. “They’re going to have a low red count or red cell mass to begin with, and that transfusion is going to make up a relatively large percentage of their red cell mass. Since we aren’t normalizing for it,” she says, “the test may be disproportionately recognizing the PEth present in the transfused cells.”
To assess the stability of PEth in storage, they saved six segments from 20 randomly chosen packed red blood cell units and after five weeks tested for PEth. Though a prior study reported potential de novo synthesis of PEth with prolonged storage, Dr. Kinard says, she and her coauthors saw no evidence of PEth formation in the stored samples.
Dr. Snozek notes that prior study had its limitations. “The authors themselves stated they could not quantify PEth, so what they saw might well have been negligible background noise,” she says. “When we tested the segments out to 42 days, whether the samples were positive or negative to begin with there was no evidence of any increase. Everything decreased or stayed negative.” (Storage for up to five weeks post-donation resulted in a mean 17.3 percent decrease in the positive units.)
The only instance in which PEth has been proven to form in storage is when a donor with ethanol in their system provides a blood sample, she says. “And then you’re just getting ex vivo formation of it. In terms of a blood donor population, I find it unlikely that anybody’s going to show up with ethanol still on board.”
“I suppose it’s not impossible,” she adds. “But in the absence of that, it’s unlikely to be a risk for the donated blood cell.”
Irradiation, another variable they studied, had little impact on PEth concentrations. That finding was expected, Dr. Kinard says. “The test is just measuring PEth on a membrane. With irradiation we’re damaging the integrity of the cells and potentially reducing its lifespan when it’s transfused, but it’s still there,” she says, referring to the PEth in the sample. “If it’s in the sample, it’s going to be picked up in testing.” It isn’t clear if irradiation affects PEth half-life, she says. “If we had enough of those data points, we would be able to tease out whether irradiation impacts the half-life in a recipient.”
Artificially elevated PEth is the common concern in the setting of liver transplant candidacy. But PEth concentrations can also decline with transfusion of negative packed red blood cell units.
“We had an abstract at AABB where we demonstrated a patient who got massively transfused,” Dr. Kinard says (Alegria KN, et al. Abstract presented at: AABB Annual Meeting; Oct. 17, 2024, Houston, Tex.). Before transfusion, patient PEth was less than 10 ng/mL. In the massive transfusion, the patient received seven red blood cell units. Donor segments were not available for PEth testing, but post-transfusion, patient PEth was 28 ng/mL.
Over the next 24 hours, the patient received eight more units. Donor segment PEth levels ranged from undetectable to 161 ng/mL. Patient PEth remained stable after two additional units were received despite the moderately high PEth concentrations in those units, reflecting the patient’s active bleeding. “Units with high PEth would increase it, and then depending on the rate of bleeding it could either stabilize or go down,” Dr. Kinard says. “But we could also see it drop precipitously with units that were negative.” Patient PEth declined below 10 ng/mL with transfusion of five PEth-negative units. A final unit with PEth of 128 ng/mL elevated patient PEth to 18 ng/mL. An artificially lowered result, then, would depend on the recipient’s rate of blood loss and their having received many PEth-negative units, Dr. Kinard says.
For the clinical team interpreting a post-transfusion PEth result, it would be advisable to have the pretransfusion PEth levels on hand, Dr. Kinard says, though she’s aware that with most laboratories sending out for PEth testing, “it would have to take some foresight to obtain that sample.”
But even if it’s a few days after the fact, it’s worth querying the laboratory to see if pretransfusion samples are still available for add-on testing, she says.
The blood bank also may have pretransfusion segments available for testing, Dr. Snozek notes, though it may not always be able to spare them. Then, too, most laboratories have not validated citrated blood for PEth testing. “Labs doing this testing might want to consider that if it is a particularly relevant question for their patient population,” she says.
Questions arise when there are no pretransfusion samples to guarantee clinical proof of abstinence. For instance, is there a significant difference in PEth half-life when it’s acquired from a transfused unit? Knowing that, Dr. Kinard says, would aid some investigations. “If the clinical team were to come to us and say, ‘What’s the possibility that this is from a transfused unit, five days ago?’” for example.
Says Dr. Snozek, “We’ve always had conversations with our liver transplant team.” But since the findings on PEth and transfusion have become known, they’ve been fielding more questions: How likely is it to happen? How high can the results get? How long does the effect last? “We’ve been keeping them in the loop as we’ve been continuing to do additional studies to work on answering these questions, or at least getting preliminary answers.” One of those studies has been submitted for publication; others are in process, she says.
She can already say with confidence that when it comes to the exceedingly high concentrations, it’s safe to rule out transfusion as the source. “If a recent transfusion recipient comes in and their post-transfusion PEth is 1,000, that’s not from transfusion,” she says. “It might be in there as well, but they came in with some on board.”
Another question: Does receiving a unit with elevated PEth have clinical impact for recipients?
“I suspect it doesn’t,” Dr. Kinard says. “The hematologic derangements that we have seen or that have been reported in the literature are in patients with severe alcoholic liver disease, to the point where they wouldn’t be eligible to donate blood.” Could it affect specific sensitive populations? “Perhaps,” she concedes. “It’s to be determined if there’s any type of difference where it could affect specific populations that could be more sensitive to those changes, or to the reduced lifespan, perhaps.”
With everything that’s known about the potential for artificial elevation of PEth from transfusion, should donors be assessed for alcohol consumption?
“My opinion is it’s unlikely this would be introduced into the process for collecting,” Dr. Kinard says. “It would have huge implications for donor eligibility, when we don’t have scientific proof that it impacts the clinical benefits of having a blood transfusion.”
Dr. Snozek agrees. “There’s already enough difficulty in getting people willing to donate, and alcohol can be a touchy subject,” she says. “Plenty of studies in the literature show people aren’t always forthcoming about their alcohol use even when it isn’t at crazy, college-party-binge-drinking-type levels.” Definitive proof of negative clinical implications for the recipient would have to be established, she says, “before that could be incorporated and expected that people give an honest answer about it.”
“I don’t think it’s likely to have a huge impact on the function of the red cell,” she adds. “But it’s a question.”
Dr. Kinard, for her part, emphasizes all that can be accomplished through communication. “At centers like ours, we’re doing a lot of testing for a specific population where we’re hyperaware of these implications. Just by having this knowledge and having a good relationship with the lab, we can try to catch these things.”
In the absence of pretransfusion samples, can the laboratory provide other data to round out the clinical picture?
That’s a question the clinical teams at Mayo Clinic have asked, Dr. Snozek says, and one they’re attempting to answer. “We can always go back and pull the segments in our facility,” she says. “But if the transfusion was in an outside hospital, for example, where you’re not going to be able to get to the samples, what information can we provide to help them interpret in a way that doesn’t discredit the patient unfairly, but also tries to interpret as accurately as possible and discern what might be real alcohol consumption versus a preanalytical factor out of the patient’s control?”
“There’s still a lot to be figured out from the standpoint of a provider stuck with a result,” she says.
Charna Albert is CAP TODAY associate contributing editor.