The study, called PFOCUS, is a prospective, monocentric, observational case-control trial led by Matteo Paolucci and colleagues at the IRCCS Istituto delle Scienze Neurologiche di Bologna in Italy, and it is registered with ClinicalTrials.gov under identifier NCT07349004. Its central ambition is to determine whether patients with migraine with aura who also carry a right-to-left shunt, the abnormal blood flow across the heart that a PFO permits, constitute a biologically distinct subgroup of the disease. Rather than treating migraine and PFO as two coincidental conditions, the researchers hypothesize that shared genetic predisposition and vascular dysfunction may bind them together, shaping both the neurological symptoms and the stroke risk that accompanies them.

The clinical stakes are considerable. Migraine with aura is associated with an increased risk of ischemic stroke, and the association is particularly striking in young people who lack the conventional vascular risk factors, such as hypertension, diabetes or smoking, that usually explain such events. This has left clinicians with an uncomfortable gap in their ability to judge who is genuinely at risk. If PFO-related migraine represents a specific, identifiable phenotype with its own underlying biology, then risk stratification could become far more precise, and preventive strategies, whether medical or procedural, could be tailored to the patients who actually need them rather than applied indiscriminately.

To build that evidence, the team will enroll a total of 240 patients with migraine with aura and sort them into two groups according to whether they show a right-to-left shunt. The classification will rely on contrast-enhanced transcranial Doppler, a non-invasive ultrasound technique in which agitated saline containing microbubbles is injected into a vein while ultrasound probes monitor blood flow in the cerebral arteries. If microbubbles appear in the brain’s circulation within a few heartbeats of the Valsalva maneuver, which raises pressure in the right atrium, this indicates that blood is bypassing the lungs’ filtering system, most commonly through a patent foramen ovale. The technique serves in this study as a surrogate marker of PFO, avoiding the need for invasive cardiac imaging in every participant.

The genetic component of the study is where PFOCUS takes its boldest swing. All participants will undergo whole-exome sequencing, a technique that reads the protein-coding portions of the genome in search of rare variants. The primary endpoint is the difference in the frequency of variants in the NOTCH3 gene between the shunt-positive and shunt-negative groups. The choice of NOTCH3 is deliberate and scientifically loaded: this gene encodes a receptor in the Notch signaling pathway that is crucial for the structural integrity of smooth muscle cells in small blood vessels, and mutations in NOTCH3 cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, a devastating inherited small-vessel disease that frequently produces migraine with aura and stroke. A milder enrichment of NOTCH3 variants in PFO-associated migraine would suggest that vascular wall biology, not merely blood-borne debris, underlies the association.

Secondary endpoints extend the genetic sweep to other genes in a curated panel, and will test whether variant burden correlates with the size of the right-to-left shunt, a parameter that transcranial Doppler can quantify by counting the microbubbles that reach the cerebral circulation. The logic here is that if genetic variants influence the development or persistence of the cardiac defect itself, or the degree to which it allows blood to shunt, then a dose-response relationship between genotype and shunt magnitude would strengthen the case for a genuine biological link rather than statistical coincidence.

Beyond the genome, the study mounts an unusually comprehensive assault on cerebrovascular physiology. Participants will undergo detection of cerebral microemboli, using prolonged transcranial Doppler monitoring to catch silent fragments of clots or debris drifting through brain arteries, events that could indicate subclinical embolism even in patients who have never had a stroke. They will also be assessed for dynamic cerebral autoregulation, the brain’s ability to keep blood flow constant despite swings in blood pressure, and for cerebral vasomotor reactivity, the capacity of brain vessels to dilate in response to a vasodilating stimulus such as carbon dioxide. Both measures probe how well the cerebral circulation copes with physiological stress, and both have shown abnormalities in migraine populations in earlier research. Impaired autoregulation or blunted vasomotor reactivity in the shunt-positive group would point to a vascular phenotype that goes beyond plumbing defects.

The hematological arm of the protocol adds a third dimension: platelet function testing. Platelets are the cellular first responders of the clotting system, and heightened platelet activation has been implicated in both migraine pathophysiology and thromboembolic disease. If patients with migraine with aura and a right-to-left shunt turn out to have measurably more reactive platelets, the paradoxical embolism hypothesis would gain new traction, but with a twist: the problem would lie not only in the cardiac shortcut but in the clot-prone blood traveling through it. Such a finding would also carry direct therapeutic implications, since antiplatelet strategies could be rationally targeted at this subgroup.

Two exploratory aims round out the design and may prove among the most clinically interesting. The study will examine whether different aura phenotypes, for instance purely visual auras versus those involving sensory or language disturbances, are associated with distinct haemodynamic and prothrombotic profiles. It will also assess whether the distribution of lesions seen on brain MRI correlates with these profiles. The underlying idea is that migraine with aura may not be a single entity but a spectrum of conditions with different mechanisms, and that the combination of a heart shunt, vascular dysfunction and clotting tendency could carve out subgroups whose stroke risk and treatment responses differ in ways that current practice cannot distinguish.

If PFOCUS delivers on its design, the consequences could ripple well beyond the headache clinic. Closure of patent foramen ovale has been tested in migraine trials with mixed and contested results, a pattern that many researchers attribute to the inclusion of heterogeneous patient populations in which only a subset truly benefits. Identifying a genetically and physiologically defined phenotype of migraine with aura associated with PFO would give those debates a sharper edge, allowing trials and clinical decisions to focus on patients whose disease is plausibly linked to their cardiac anatomy and vascular biology. The study’s findings may inform stroke risk stratification and, as its investigators put it, open new avenues for personalized preventive strategies in patients with migraine with aura. For the millions of people who experience aura with every attack, and for the young stroke patients whose events have never been adequately explained, the answers that emerge from these 240 carefully phenotyped brains, hearts and genomes could finally turn a long-standing clinical curiosity into actionable medicine.

Subject of Research: Genetic, haemodynamic and platelet determinants of the association between migraine with aura and patent foramen ovale

Article Title: PFOCUS study protocol: a prospective case–control study investigating genetic, haemodynamic, and platelet function in migraine with aura with or without patent foramen ovale

Article References: Paolucci, M., Urbinati, G., Migliaccio, L., Marucci, A., Favoni, V., Mascarella, D., Palombo, F., Carelli, V., Sebastiani, M., Stagni, S., Mancini, R., Cevoli, S., & Zini, A. (2026). PFOCUS study protocol: a prospective case–control study investigating genetic, haemodynamic, and platelet function in migraine with aura with or without patent foramen ovale. Frontiers in Neurology, 17, Article 1952744. https://doi.org/10.3389/fneur.2026.1952744

Image Credits: AI Generated

DOI: 10.3389/fneur.2026.1952744

Keywords: migraine with aura, patent foramen ovale, right-to-left shunt, NOTCH3, transcranial Doppler, whole-exome sequencing, cerebral autoregulation, vasomotor reactivity, platelet function, ischemic stroke, cerebral microemboli, study protocol

The letter, authored by Barina Khan of Karachi Medical and Dental College and published on 3 September 2026 as Volume 29, article number 59 of the journal, is written in direct response to a pilot study by Tran and colleagues that documented dynamic platelet dysfunction in patients undergoing extracorporeal membrane oxygenation. That pilot work, which appeared in the same journal earlier in 2026, used platelet function testing to follow how platelet performance changed over the course of ECMO support and explored whether those changes tracked with clinically significant bleeding. The new letter does not dispute that platelet dysfunction is real and important in ECMO patients. Instead, it questions whether the specific instrument and protocol used can reliably detect it in this population.

The core of the concern lies in what laboratory scientists call a ceiling effect. The PFA-200 measures the time it takes for platelets, under controlled high shear conditions, to form a plug that occludes a microscopic aperture in a cartridge coated with platelet agonists such as collagen and epinephrine or collagen and adenosine diphosphate. When platelet function is severely impaired, the plug simply never forms, and the instrument reports a result at or above the maximum assay limit, typically a value greater than 300 seconds. In profoundly thrombocytopenic or platelet-exhausted patients, which many ECMO patients quickly become, large numbers of test results pile up at this artificial ceiling. Once a value has been censored at the upper limit, the assay can no longer distinguish between moderately severe and catastrophic platelet failure, compressing exactly the range of dysfunction that matters most for predicting bleeding.

Khan’s letter argues that this censoring problem is particularly acute during ECMO, where platelet counts fall progressively, platelets become activated and exhausted through continuous contact with the oxygenator membrane and circuit tubing, and acquired platelet defects accumulate over days of support. If a study correlates closure times with bleeding episodes in such a cohort, the relationship may be flattened or distorted by the many results that read simply as greater than 300 seconds. Statistical associations computed on censored data risk either underestimating the true strength of the link between platelet failure and hemorrhage or, depending on how the ceiling values are handled in the analysis, producing misleading conclusions about when platelet dysfunction begins to matter clinically.

The second confounder the letter highlights is hematocrit, the proportion of blood volume occupied by red blood cells. It is a long-standing observation in hematology that the PFA-100 and PFA-200 are exquisitely sensitive to the hematocrit of the sample. Red cells are not passive bystanders in primary hemostasis; they physically push platelets toward the vessel wall in flowing blood and contribute chemical signals, including adenosine diphosphate released from erythrocytes, that amplify platelet activation. When the hematocrit drops below roughly 30 percent, closure times lengthen even if platelets themselves are functioning normally, and severe anemia can push closure times beyond the assay limit on its own. Conversely, elevating the hematocrit can shorten closure times and mask genuine platelet defects, an effect documented decades ago in studies of uremic and cirrhotic patients evaluated on the platelet function analyzer.

This sensitivity is a serious problem in the ECMO population specifically. Patients on extracorporeal support are frequently anemic, whether from hemodilution caused by circuit priming, hemolysis within the circuit, repeated blood sampling, gastrointestinal bleeding, or the suppressed red cell production that accompanies critical illness. In such patients, a prolonged closure time may reflect the red cell deficit rather than intrinsic platelet failure, and the degree of prolongation may bear little relationship to the true functional capacity of the platelets. Khan’s letter suggests that unless hematocrit is either matched across study groups or formally adjusted for in the statistical model, the association reported between PFA-200 closure times and bleeding in ECMO patients may be confounded from the outset, with anemia masquerading as platelet dysfunction.

The letter places these concerns in a broader and somewhat sobering context. The clinical utility of closure times on the platelet function analyzer has been debated for more than two decades. Studies in cardiac surgery patients have repeatedly questioned whether the device predicts blood loss after cardiopulmonary bypass, another setting in which acquired platelet defects, hemodilution and anemia coexist. A comprehensive review in the American Journal of Hematology catalogued the assay’s utility across bleeding disorders while also documenting its susceptibility to hematologic variables, and a worldwide survey of platelet function testing practices conducted under the auspices of the International Society on Thrombosis and Haemostasis underscored how much variability exists in how platelet function is assessed even among expert laboratories. Against that backdrop, applying the PFA-200 to the uniquely harsh environment of extracorporeal circulation demands particular caution about the assay’s known limitations.

None of this means the assay should be abandoned in the ECMO setting, the letter’s argument implies, but rather that its results must be interpreted with an awareness of when the instrument can and cannot see. Practical strategies exist. Researchers can report the proportion of censored results explicitly and use statistical methods designed for data with detection limits rather than treating the ceiling value as a genuine number. Studies can stratify closure times by hematocrit, exclude samples with severe anemia from platelet-focused analyses, or measure closure times only after red cell transfusion has stabilized the hematocrit. Complementary tests that are less dependent on shear, hematocrit and sample handling, such as light transmission aggregometry, flow cytometric markers of platelet activation, or other point-of-care viscoelastic and platelet-mapping devices, can help triangulate the true state of platelet function. The pilot study’s central observation, that platelet dysfunction evolves dynamically during ECMO, remains valuable; the question is whether the PFA-200, as deployed, is measuring that dysfunction or partly measuring the anemia around it.

The stakes are far from academic. Bleeding remains one of the most feared complications of ECMO, occurring in a substantial fraction of patients and driving decisions about anticoagulation intensity, transfusion thresholds and circuit management. If closure times on the PFA-200 are adopted as a bedside guide without accounting for ceiling effects and hematocrit, clinicians risk both false reassurance, when an apparently normal result is actually capped by the assay limit in a patient with profound platelet exhaustion, and false alarm, when a prolonged result in an anemic patient triggers unnecessary platelet transfusion. Given that platelet transfusions carry their own risks, including allergic reactions, transfusion-associated circulatory overload and the theoretical promotion of thrombosis in a patient group already prone to circuit clotting, refining which patients truly need platelet support is a matter of real clinical consequence.

Khan’s letter is a compact methodological intervention, but it lands at a moment when interest in platelet function testing during extracorporeal support is growing rapidly. As devices for measuring closure times, aggregometry and platelet activation migrate from hematology laboratories to the intensive care unit bedside, the letter serves as a reminder that the validity of any such measurement depends on the biology of the sample as much as the precision of the instrument. In ECMO patients, where thrombocytopenia, platelet exhaustion and anemia march together, closure times must be read with both eyes open. Whether future studies can confirm a robust, hematocrit-independent association between PFA-200 results and bleeding in this population will determine whether the assay earns a genuine place in the ECMO management toolkit, or remains a test whose ceiling it cannot see above and whose red cell context it cannot escape.

Subject of Research: The reliability of PFA-200 platelet function closure times for assessing bleeding risk during extracorporeal membrane oxygenation

Article Title: PFA-200 closure times in ECMO: Are ceiling effects and hematocrit confounding the bleeding association?

Article References: Khan, B. (2026). PFA-200 closure times in ECMO: Are ceiling effects and hematocrit confounding the bleeding association?. Journal of Artificial Organs, 29(4), Article 59. https://doi.org/10.1007/s10047-026-01589-0

Image Credits: AI Generated

DOI: 10.1007/s10047-026-01589-0

Keywords: ECMO, PFA-200, platelet function, closure time, bleeding risk, hematocrit, coagulation, extracorporeal membrane oxygenation, platelet dysfunction, anemia, point-of-care testing, Journal of Artificial Organs