The logic behind the advance rests on radiochemistry that is deceptively simple. Since the mid-1970s, clinicians have labeled white blood cells with gamma emitters such as technetium-99m and indium-111, then reinjected them to locate abscesses and inflammatory lesions. Indium-111, with a physical half-life of about 2.8 days, became the workhorse for this purpose because it persists long enough to follow cells for days. Zirconium-89, with a half-life of 3.3 days, offers a nearly identical temporal window but emits positrons, enabling PET imaging, which delivers substantially higher sensitivity and better spatial and temporal resolution than SPECT. The UAB team exploited 8-hydroxyquinoline, known as oxine, a lipophilic chelator that shuttles zirconium-89 across cell membranes, allowing the radionuclide to become trapped inside the leukocytes rather than merely attached to their surfaces.

The trial enrolled four healthy adult women between 37 and 53 years of age, a deliberate choice grounded in radiation protection logic. Extrapolations from decades of indium-111 oxine dosimetry predicted a higher effective-dose coefficient for females, 1.14 mSv/MBq, than for males, 0.855 mSv/MBq, so studying women represented the more conservative test of safety. There was also a clinical rationale: the team’s longer-term goal is to image neuroinflammation in myalgic encephalomyelitis/chronic fatigue syndrome, a condition that disproportionately affects women. Each participant donated roughly 120 milliliters of blood, from which leukocytes were isolated, incubated with zirconium-89 oxine, washed, and returned intravenously within strict time limits. The mean administered activity was 10.9 plus or minus 2.9 MBq, a modest amount by nuclear medicine standards.

Quality control was rigorous. Every labeled cell preparation had to meet predefined release criteria before reinjection, including radiochemical purity of at least 95 percent, radionuclidic purity of at least 99.5 percent, verified leukocyte viability before and after labeling, sterility, and endotoxin testing. The radiolabeling itself was performed at a licensed commercial radiopharmacy under USP Chapter 797 standards, while the zirconium-89 oxine was produced in the UAB Cyclotron PET Production Facility under USP Chapter 823 guidelines. Only products passing every specification were administered, a framework designed to mirror the established clinical workflow for FDA-approved indium-111 oxine labeling and thereby ease eventual regulatory translation.

Imaging followed a demanding schedule. Each participant underwent whole-body PET/CT at five time points: immediately after injection, then at 4 to 6, 24, 48, and 72 hours. The scans captured a dynamic biological story. Early images showed the labeled cells concentrated in the liver, spleen, heart, and blood pool, as would be expected for cells still circulating. By 24 hours and increasingly at 48 and 72 hours, uptake shifted to the liver, spleen, and red marrow, particularly within the pelvis and iliac crests, reflecting the physiological homing of leukocytes to the organs where they are naturally cleared and stored. The pattern was qualitatively similar to that long documented for indium-111 oxine-labeled leukocytes and consistent with prior mouse and non-human primate studies of zirconium-89 oxine labeling.

The dosimetry numbers carry the study’s headline significance. The highest mean absorbed-dose coefficients appeared in the spleen at 3.52 plus or minus 1.38 mGy/MBq, followed by the liver at 2.1 plus or minus 0.45, the adrenals at 1.62 plus or minus 0.24, the uterus at 1.48 plus or minus 0.98, and the kidneys at 1.36 plus or minus 0.12 mGy/MBq. The mean effective-dose coefficient came out to 0.92 plus or minus 0.06 mSv/MBq, numerically lower than the predicted 1.14 mSv/MBq, a difference that reached statistical significance in an exploratory one-sample t-test with a P value of 0.006. The measured biodistribution also diverged from the assumptions used for prediction: roughly 32 percent of activity localized to the liver, 8 percent to the spleen, and 15 percent to red marrow, rather than the 30-30-34 split assumed from indium-111 data. The authors attribute much of the discrepancy to methodological differences, since PET/CT-based whole-organ dosimetry is inherently more direct than the SPECT-only methods used historically.

Safety results were unambiguous. No adverse events, clinically significant laboratory abnormalities, or pharmacological effects were observed in any participant through 72 hours, and follow-up contact 24 hours after protocol completion turned up nothing further. Vital signs remained stable throughout. One intriguing observation warrants mention: activity concentrations in plasma exceeded those in whole blood, especially at early time points, which the researchers suggest could reflect leukocyte lysis, efflux of the radionuclide from cells, or incomplete buffy coat formation during processing. This phenomenon, they note, deserves closer investigation in subsequent studies.

A key technical concern with any intracellular label is stability. Free zirconium-89 has a well-documented affinity for hydroxyapatite and accumulates in mineralized bone, so progressive cortical bone uptake would signal that the label was leaking from cells. To probe this, the team performed an exploratory skeletal analysis comparing marrow-rich regions of the spine and pelvis with cortical tibial bone. Uptake remained consistently higher in the marrow-rich compartment, while cortical tibial activity stayed low with no progressive accumulation, supporting the interpretation that skeletal signal reflects genuine leukocyte homing to red marrow rather than liberated radionuclide. The authors caution that low-level efflux beyond 72 hours cannot be fully excluded and that longer imaging windows will help settle the question.

The study also showcased a modern dosimetry workflow. Organ regions of interest were generated automatically on CT using an in-house deep learning segmentation tool, then manually refined and propagated across time points. This automation reduces interobserver variability and could make dosimetry scalable across larger, multi-institution cohorts. Time-integrated activity coefficients were computed with Simpson’s rule, and doses were calculated in OLINDA 2.1 using standard adult female phantoms and ICRP 103 tissue weighting factors. Beyond the final 72-hour scan, activity was assumed to decay only physically, a conservative assumption that likely overestimates doses for organs still clearing activity biologically.

Looking ahead, the implications stretch well beyond infection imaging. The same labeling strategy could track CAR T-cells and other cell-based therapies as they home to tumors, offering oncologists a real-time window into treatment delivery. It may prove especially valuable in neurology: animal models of stroke, traumatic brain injury, multiple sclerosis, and Alzheimer’s and Parkinson’s diseases all show leukocyte invasion of the brain, but these events have never been directly visualized in living humans because available PET tracers decay too quickly, while the critical window of immune cell infiltration spans three to four days. Zirconium-89’s half-life fits that window precisely. The research team plans to apply the technique to patients with suspected central nervous system inflammation, including multiple sclerosis and ME/CFS, while working to reduce the blood volume needed for labeling and to improve labeling efficiency. Limitations remain, including the small all-female cohort and the absence of functional testing of labeled cells beyond viability, but the foundation is now laid: for the first time, human dosimetry data confirm that PET imaging of a patient’s own immune cells is both safe and feasible, opening a new chapter in molecular imaging of the immune system.

Subject of Research: First-in-human radiation dosimetry and biodistribution of zirconium-89 oxine-labeled autologous leukocytes imaged with PET/CT

Article Title: First-in-human dosimetry and biodistribution of [89Zr]Zr-oxine-labeled autologous leukocytes using PET/CT

Article References: Gultekin, K., Bartels, J. L., Cardenas, C. E., Kumar, Y., Gimblet, G. R., Jones, C. L., Bankston, C., Jeffers, C. D., White, S. L., Lapi, S. E., Younger, J. W., & McConathy, J. E. (2026). First-in-human dosimetry and biodistribution of [89Zr]Zr-oxine-labeled autologous leukocytes using PET/CT. European Journal of Nuclear Medicine and Molecular Imaging. https://doi.org/10.1007/s00259-026-08158-w

Image Credits: AI Generated

DOI: 10.1007/s00259-026-08158-w

Keywords: zirconium-89, PET/CT, leukocytes, cell tracking, dosimetry, radiopharmaceuticals, nuclear medicine, oxine, immunimaging, neuroinflammation, CAR T-cells, first-in-human trial

The rationale for targeting macrophages directly is rooted in their complicated role in cancer biology. Macrophages are versatile immune cells that maintain tissue homeostasis and defend against pathogens, but within the tumor microenvironment they are notoriously plastic and heterogeneous, adopting functions that range from pro-inflammatory to strongly immunosuppressive. Dense infiltration of tumors by CD68-positive macrophages has been linked in breast cancer to larger tumor size, higher histologic grade, hormone-receptor negativity, lymphovascular invasion, and reduced overall and progression-free survival. Existing imaging probes have struggled to capture this picture accurately. Some tracers, such as those targeting CD163 or CD206, preferentially label the immunosuppressive M2-like subset, while probes aimed at the translocator protein TSPO are not exclusive to macrophages and are affected by genetic polymorphisms that vary between individuals. A copper-64-labeled CD68-Fc fusion protein has been explored, but it targets scavenger receptors indirectly rather than macrophages themselves.

To fill this gap, the researchers turned to phage display, a technique that screens enormous libraries of antibody fragments for those that bind a chosen target. They used a synthetic Fab library, designated Library F, that was deliberately designed without lysine residues in its complementarity-determining regions, the loops that make contact with the antigen. Lysines were replaced with arginines, which preserve the basic character of the loops but lack the reactive nucleophilic amine that would otherwise be chemically modified during radiolabeling. Framework lysines elsewhere in the molecule were retained to allow controlled conjugation to a chelator without disturbing the antigen-binding surface. Screening this library against immobilized recombinant human CD68 yielded 14 unique sequences, of which five, named Fab3, Fab4, Fab7, Fab8, and Fab14, could be expressed successfully in bacterial cultures and purified on Protein A columns.

Characterization of the five candidates revealed important differences. Enzyme-linked immunosorbent assays showed that all five bound human CD68 with high significance compared with a bovine serum albumin control, but Fab3 also bound strongly to BSA itself and cross-reacted with mouse CD68, so it was eliminated. The remaining four Fabs bound human CD68 in a concentration-dependent manner with half-maximal effective concentrations ranging from 1.7 to 15 nanomolar, an affinity range well suited to immunoPET probes, and showed no detectable binding to BSA or mouse CD68. Production metrics then became the deciding factor. Fab4 and Fab7, despite their favorable affinities, expressed inconsistently and lost purity during storage, retaining only 71 and 75 percent monomer after 60 days at 4 degrees Celsius. Fab14 expressed at high yields but with purities of only 60 to 70 percent. Fab8, by contrast, delivered consistent yields of about 1.7 milligrams per liter, the highest purity of 80 to 90 percent straight from Protein A purification, and 96 percent monomer after two months of storage, making it the clear lead candidate.

Converting Fab8 into a PET tracer required attaching a chelator that could hold a positron-emitting radionuclide. The team selected p-SCN-Bn-deferoxamine, or DFO, and zirconium-89, a pairing with a well-established chemistry and a track record of clinical translation. After optimizing the conjugation stoichiometry to a 4:1 ratio of DFO to Fab, which minimized free chelator without inducing protein aggregation, the researchers radiolabeled the conjugate at a specific activity of 7.4 kilobecquerels per nanomole, achieving a radiochemical yield of 99 percent. Stability testing showed that the resulting tracer, [89Zr]Zr-DFO-Fab8, remained more than 95 percent intact monomer in phosphate-buffered saline over 48 hours, and about 80 percent intact in human serum at 37 degrees Celsius over the same period, with the remainder of the radioactivity associated with serum proteins.

In vitro experiments confirmed that the tracer behaved as intended on real cells. The human monocytic leukemia line THP-1 was differentiated into macrophages, which expressed human CD68 on roughly 25 percent of cells compared with about 4 percent of undifferentiated THP-1 cells, as measured by flow cytometry. When fixed macrophages were incubated with the tracer, binding reached about 10 percent of the added activity, five times higher than an irrelevant radiolabeled Fab control and roughly three times higher than in undifferentiated THP-1 cells. Importantly, neither the unlabeled conjugate nor the radiolabeled tracer compromised macrophage viability at the concentrations tested, with only a modest 19 percent decrease at the highest radiotracer concentration of 125 nanomolar, far above the estimated nanomolar circulating levels in the animal studies.

The first in vivo test used a cleverly simple model. Because Fab8 does not cross-react with mouse CD68, the team implanted subcutaneous plugs of Matrigel mixed with either recombinant human CD68 protein or BSA into opposite shoulders of immunocompetent BALB/c mice. PET/CT imaging at 1 and 4 hours after tracer injection showed significantly higher uptake in the human CD68 plugs than in the BSA control plugs, with plug-to-muscle ratios of 11 and 13 versus roughly 5 and 6, respectively. Voxel-level histogram analysis confirmed that the spatial distribution of tracer within the antigen plugs differed significantly from the controls over time. Biodistribution measurements correlated strongly with the imaging data, with a Pearson correlation coefficient of 0.97, and showed the expected renal dominance typical of antibody fragments of this size.

The more biologically demanding test came next. The researchers grew bilateral MDA-MB-231 triple-negative breast cancer xenografts, a tumor line that forms highly vascularized tumors and does not express CD68, in immunodeficient NSG mice. Once the tumors had developed vasculature, they injected differentiated THP-1 macrophages directly into one tumor and undifferentiated THP-1 cells into the other, then administered the tracer systemically. PET imaging at 1 and 6 hours revealed significantly greater uptake in the macrophage-containing tumors, with tumor-to-muscle ratios of about 5.2 and 5.9 compared with 2.7 and 3.6 in the control tumors. Immunofluorescence staining of the excised tumors provided the decisive validation: macrophage-injected tumors contained roughly 660 CD68-positive cells per area versus only 16 in the controls, and the CD68 density correlated with the PET standardized uptake values with a Pearson coefficient of 0.9663. This is the study’s central achievement, a direct, quantitative bridge between a noninvasive PET signal and the histopathological gold standard for pan-macrophage burden.

The authors are candid about the limitations that must be addressed before clinical translation. Kidney uptake was high, as expected for a 50-kilodalton Fab, and about 10 percent aggregation in serum may have contributed to elevated liver and spleen signal through sequestration by the reticuloendothelial system. Because the target uptake in the xenograft model was inherently low, background signal was comparatively prominent, and tumor-to-heart ratios below one suggested incomplete blood clearance at the early imaging timepoints used. Future work will explore polyethylene glycol modification to reduce renal retention, an approach already validated clinically by the approved drug certolizumab pegol, along with better animal models of human macrophage infiltration. Even so, the study establishes a complete pipeline, from phage display discovery through radiopharmaceutical engineering to correlated PET and pathology, for a first-generation human CD68 immunoPET tracer. Such a probe could eventually help select patients for macrophage-targeting therapies, monitor treatment response, and predict resistance to immunotherapy, all from a single noninvasive scan.

Subject of Research: Development of a zirconium-89-labeled anti-CD68 Fab immunoPET tracer for imaging human macrophages

Article Title: A FABulous approach to image human macrophages

Article References: Roohani, B., Vaughn Embs, A., Kumar, R., Lee, S., Katz, S. R., La Prairie, C., Nash, D., Dangarwala, M., Kluger, H. M., Klein, D. E., Zhang, P., Larimer, B. M., Nelson, B., & Marquez-Nostra, B. (2026). A FABulous approach to image human macrophages. European Journal of Nuclear Medicine and Molecular Imaging. https://doi.org/10.1007/s00259-026-08180-y

Image Credits: AI Generated

DOI: 10.1007/s00259-026-08180-y

Keywords: CD68, macrophages, immunoPET, phage display, Fab, zirconium-89, tumor-associated macrophages, PET imaging, triple-negative breast cancer, molecular imaging, radiopharmaceutical, tumor microenvironment