The study, published in the Journal of Pharmacy & Pharmaceutical Sciences, focused on volumetric absorptive microsampling, or VAMS, a technique in which a small absorbent tip wicks up a fixed volume of blood from a finger prick. The device looks almost trivially simple, but the chemistry behind it is not. Once blood dries on the tip, the concentration of a drug measured from the whole sample may not match what clinicians would see in plasma, the matrix on which most therapeutic targets for mycophenolic acid are based. The reason lies in how the drug partitions between plasma and red blood cells, and that partitioning is governed largely by hematocrit, the fraction of blood occupied by red cells.

Tacrolimus and mycophenolic acid sit at opposite ends of this problem. Tacrolimus is a calcineurin inhibitor that suppresses T-cell activation, and it is extensively taken up into erythrocytes, which is why whole blood has always been the recommended monitoring matrix. Mycophenolic acid, the active metabolite of mycophenolate mofetil and mycophenolate sodium, behaves differently. It inhibits an enzyme called inosine monophosphate dehydrogenase, shutting down lymphocyte proliferation, but the molecule itself stays almost entirely in plasma, where roughly 97 percent of it is bound to proteins. Measuring it from whole blood therefore gives a number that is not directly comparable to the plasma concentrations on which decades of clinical experience rest.

To bridge that gap, the researchers enrolled adult transplant recipients, mostly kidney and lung transplant patients, who were already receiving mycophenolate therapy, and collected paired samples of plasma, whole blood, and VAMS microsamples between June and October 2023. Twenty-one paired samples were analyzed with a validated liquid chromatography-tandem mass spectrometry method capable of quantifying both drugs simultaneously from a single microsample. The analytical performance was strong. Calibration curves were linear across ranges of 10 to 20,000 nanograms per milliliter for mycophenolic acid and 0.5 to 500 nanograms per milliliter for tacrolimus, with correlation coefficients exceeding 0.99, and accuracy fell within the accepted 85 to 115 percent window at nearly all quality-control levels.

With the assay validated, the team turned to the harder question: how to convert a VAMS whole-blood concentration into a plasma-equivalent value for mycophenolic acid. They compared five strategies, including two variations of a formula derived from dried blood spot work, one VAMS-specific linear model, a multiple regression model, and a physiologically based population model. The simplest approach, which assumed a fixed hematocrit of 0.45 liters per liter for every patient, performed poorly, achieving agreement within 20 percent in only 19 percent of samples and carrying a mean bias above 30 percent. The regression model that included hematocrit as a covariate but lacked an interaction between hematocrit and concentration also showed marked proportional bias.

The clear winner was an individualized correction that divides the VAMS concentration by one minus the patient’s own measured hematocrit and then multiplies by the plasma protein binding fraction of 0.97. This formula achieved clinical agreement in 86 percent of samples, with a mean Bland-Altman bias of just over 3 percent and tight predictive error metrics. A physiologically derived population model performed nearly as well, and a VAMS-specific model with a concentration-hematocrit interaction term actually produced slightly better numerical statistics, but the researchers judged the individualized formula the best balance of accuracy, interpretability, and ease of calculation for routine clinical use. Deviations from plasma values clustered mainly at very low concentrations, below roughly 488 nanograms per milliliter, a range that lies beneath the therapeutic exposure window and rarely changes clinical decisions.

Tacrolimus told a different story. Because the drug distributes so heavily into red cells, VAMS concentrations correlated strongly with conventional whole-blood values without any hematocrit correction at all. Passing-Bablok regression showed a slope close to unity, and the modest negative bias of about 18 percent remained within predefined acceptance limits. In practical terms, that means a single finger-prick microsample could replace a venous draw for routine tacrolimus trough monitoring, while the same sample simultaneously delivers a mycophenolic acid measurement once the individualized hematocrit adjustment is applied.

The clinical payoff became vivid in two lung transplant recipients who developed leukopenia, a dangerous drop in white blood cells, while taking the drug combination. Using eight-point VAMS sampling across a dosing interval, the team reconstructed full area-under-the-curve profiles for mycophenolic acid. The complete profiles yielded AUCs of 28.9 and 23.1 milligrams per hour per liter, near or below the lower therapeutic threshold of 30, while conventional three-point schemes underestimated exposure by 16 to 35 percent. Notably, the dense sampling captured secondary peaks at six to eight hours after dosing, the signature of enterohepatic recirculation, which sparse sampling misses entirely. Despite these subtherapeutic AUCs, both patients exhibited leukopenia, most likely due to the combined myelotoxic effects of concomitant immunosuppressants and anti-infective agents rather than excessive mycophenolic acid exposure, a distinction that full-profile monitoring can help clinicians make.

The authors are careful about the limits of their work. The cohort was small, the patients had hematocrit values well below the general population average, and all microsampling was performed by trained professionals rather than by patients themselves. VAMS specimens were also generated from venous whole blood rather than true finger-stick capillary samples, although published data suggest negligible capillary-venous bias for these drugs. The eight-point sampling scheme, while it establishes a high-resolution pharmacokinetic reference, is unlikely to be practical for everyday monitoring; its real value is as a foundation for developing limited sampling strategies and population pharmacokinetic models that could predict full exposure from far fewer time points.

Even with those caveats, the study marks a meaningful step toward decentralizing transplant drug monitoring. A validated microsampling workflow that handles both tacrolimus and mycophenolic acid in one sample, paired with an individualized hematocrit correction that any clinical laboratory can compute, could eventually allow patients to monitor their immunosuppression from home, mailing dried microsamples to the lab. The researchers suggest that future work should evaluate home-based self-sampling, validate simplified sampling strategies derived from VAMS data, and test the approach across broader transplant populations. If those steps succeed, the days of the transplant tightrope walk may become considerably less precarious.

Subject of Research: Hematocrit-adjusted volumetric absorptive microsampling for tacrolimus and mycophenolic acid therapeutic drug monitoring in transplant recipients

Article Title: Evaluation of hematocrit-adjusted conversion strategies for mycophenolic acid and tacrolimus monitoring using volumetric absorptive microsampling in lung and renal transplant recipients

Article References: Juan, L., Cheng, C.-N., Chang, W.-C., Chiu, H.-H., Lu, C.-W., Hsu, H.-H., Kuo, C.-H., & Tseng, Y.-J. (2026). Evaluation of hematocrit-adjusted conversion strategies for mycophenolic acid and tacrolimus monitoring using volumetric absorptive microsampling in lung and renal transplant recipients. Journal of Pharmacy & Pharmaceutical Sciences, 29, Article 16123. https://doi.org/10.3389/jpps.2026.16123

Image Credits: AI Generated

DOI: 10.3389/jpps.2026.16123

Keywords: volumetric absorptive microsampling, mycophenolic acid, tacrolimus, therapeutic drug monitoring, hematocrit, organ transplantation, LC-MS/MS, pharmacokinetics, area under the curve, immunosuppression, enterohepatic recirculation, finger-prick sampling

The research, led by Takashi Shida of the Tokyo Metropolitan Institute for Geriatrics and Gerontology and colleagues, drew on participants from the Itabashi Longitudinal Study on Aging, an ongoing community-based cohort in a residential ward of northern Tokyo. The team analyzed data from 529 adults aged 65 and older who took part in the 2025 survey wave, a group in which 54.3 percent were women. Frailty, the state of heightened vulnerability that leaves older adults at risk of falls, disability, hospitalization, and death, was assessed using the revised Japanese version of the Cardiovascular Health Study criteria, a validated instrument that scores weight loss, exhaustion, low physical activity, weakness, and slowness. Of the 529 participants, 32, or 6.0 percent, met the criteria for frailty.

The central measurement was the concentration of NAD+ in whole blood, quantified in micromoles per liter. Whole-blood NAD+ is a composite signal: it reflects the molecule’s presence inside red blood cells, white blood cells, and platelets, as well as whatever circulates in plasma. Because red blood cells lack mitochondria and rely heavily on NAD+ for glycolysis and antioxidant defense, the blood compartment offers a convenient, if imperfect, window into systemic NAD+ metabolism. The researchers found that the median whole-blood NAD+ concentration was measurably lower among participants with frailty than among those without it, at 24.00 versus 25.00 micromoles per liter, a difference that reached statistical significance with a P value of 0.007.

To move beyond a simple group comparison, the team built multivariable regression models that adjusted for the most obvious confounders: age, sex, and fasting time before the blood draw, the last of which matters because NAD+ levels can fluctuate with recent food intake. After these adjustments, each 1 micromole per liter increase in whole-blood NAD+ concentration was associated with 13 percent lower odds of frailty, with an odds ratio of 0.871 and a 95 percent confidence interval spanning 0.780 to 0.970, and a P value of 0.013. In a cohort where the median sits near 25 micromoles per liter, a one-unit shift is a meaningful fraction of the biological range, which makes the effect size noteworthy rather than trivial.

The investigators then subjected their finding to a battery of robustness checks that reveal how carefully the result was constructed. Restricted cubic spline analysis, a technique that allows the relationship between NAD+ and frailty to bend and curve freely rather than assuming a straight line, showed no clear evidence of nonlinearity, suggesting the association behaves approximately linearly across the observed range. Because frailty was relatively rare in the sample, the team also ran a Firth penalized-likelihood logistic regression, a method designed to produce more stable estimates when outcomes are infrequent and conventional models risk bias. Under this broader covariate adjustment, the association held firm, with an odds ratio of 0.880 and a confidence interval of 0.782 to 0.986.

But the story took a more complicated turn when the researchers added hematocrit, the proportion of blood volume occupied by red blood cells, to the model. With that adjustment, the association weakened and lost statistical significance, with the odds ratio drifting to 0.911 and the confidence interval crossing unity at 0.800 to 1.034. This attenuation is not a failure of the study; it is one of its most informative results. It suggests that part of the apparent link between blood NAD+ and frailty may be explained by blood cell composition itself. Frail older adults often have altered hematological profiles, including tendencies toward anemia and shifts in red cell indices, and since the vast majority of whole-blood NAD+ resides within blood cells, differences in cell counts and packing could masquerade as, or at least substantially contribute to, differences in NAD+ concentration.

This caveat places the new findings within a lively and sometimes contentious scientific debate. NAD+ has become one of the most hyped molecules in the longevity world, with supplements such as nicotinamide riboside and nicotinamide mononucleotide marketed aggressively on the premise that restoring youthful NAD+ levels can slow aging. Human evidence, however, has been mixed. Some studies have reported that whole-blood NAD+ contents decline with age, while others, including a recent analysis published in Nature Metabolism, found that human whole-blood NAD+ levels do not vary with age or with lifestyle interventions. Blood NAD+ has also been reported to be reduced in very old patients hospitalized for heart failure, hinting that the molecule may track with disease severity rather than chronological age per se. The Itabashi study adds a crucial piece: a community-dwelling, non-hospitalized population in which the association with frailty, a clinical syndrome rather than a single disease, was detectable.

The study’s design imposes important limits on interpretation. As a cross-sectional analysis, it captures a single moment in time and cannot determine whether low NAD+ contributes to causing frailty, whether frailty and its metabolic consequences deplete NAD+, or whether both are driven by a shared underlying process such as chronic inflammation, mitochondrial dysfunction, or poor nutrition. The frailty group was also small, with only 32 affected participants, which limits statistical power and explains the team’s recourse to penalized regression. And the whole-blood measurement, while practical and increasingly standardized, cannot distinguish between NAD+ dynamics in muscle, brain, immune cells, and other tissues where the molecule’s age-related decline is thought to matter most. The authors themselves emphasize that further studies using cell- and tissue-specific measurements are needed to clarify the relationship between NAD+ metabolism and frailty.

Those caveats notwithstanding, the significance of the work lies in its grounding. Much of the NAD+ literature rests on cell culture experiments, animal models, or small and highly selected human samples. Here, the association emerges from ordinary older adults living at home in a Tokyo ward, measured with a validated frailty instrument and adjusted for the mundane but essential variables of age, sex, and fasting status. If blood NAD+ does prove to be a genuine marker of physiological resilience, even one partly confounded by blood cell composition, it could eventually help clinicians identify which older patients are silently sliding toward frailty before falls and disability make it obvious. Biomarkers of frailty are a recognized public health priority, and most current candidates, from inflammatory cytokines to metabolomic signatures, remain far from clinical deployment.

The road ahead will require longitudinal follow-up within cohorts like Itabashi, where repeated NAD+ measurements can be tested as predictors of incident frailty, and intervention studies in which NAD+-boosting compounds are evaluated against hard clinical endpoints rather than blood chemistry alone. Recent work comparing different NAD+ precursors has shown that they exert differential effects on circulating NAD and microbial metabolism in humans, underscoring how much remains unknown about how these interventions behave in the body. Exercise training, for its part, has been shown to reverse the age-dependent decline in NAD+ salvage capacity in human skeletal muscle, pointing to lifestyle approaches that may act on the same pathway without a pill. For now, the Itabashi findings should be read as a carefully qualified signal rather than a prescription: a molecule central to energy metabolism and cellular signaling appears to run low in the blood of frail older adults, and untangling whether that deficit is a driver, a passenger, or a mirror of declining resilience is now one of the most compelling questions in the biology of aging.

Subject of Research: Association between whole-blood NAD+ concentration and frailty in community-dwelling older adults

Article Title: Association between whole-blood NAD+ concentration and frailty in community-dwelling older adults: the Itabashi Longitudinal Study on Aging

Article References: Shida, T., Hatanaka, S., Kojima, N., Osuka, Y., & Sasai, H. (2026). Association between whole-blood NAD+ concentration and frailty in community-dwelling older adults: the Itabashi Longitudinal Study on Aging. GeroScience. https://doi.org/10.1007/s11357-026-02545-9

Image Credits: AI Generated

DOI: 10.1007/s11357-026-02545-9

Keywords: NAD+, frailty, aging, older adults, whole blood, biomarkers, energy metabolism, sirtuins, hematocrit, GeroScience, Itabashi Longitudinal Study, geriatrics

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