The research team enrolled patients whose final diagnoses had been confirmed as wild-type transthyretin amyloidosis, hereditary transthyretin amyloidosis, or light-chain AL amyloidosis, the latter driven by abnormal antibody light chains rather than transthyretin. The Bologna cohort comprised 109 patients imaged with DPD, including 70 with wild-type ATTR, 20 with hereditary ATTR, and 19 with AL disease. The Florence cohort comprised 115 patients imaged with HMDP, including 90 with wild-type ATTR, 13 with hereditary ATTR, and 12 with AL disease. Overall, wild-type ATTR accounted for 71 percent of the population, hereditary ATTR for 15 percent, and AL amyloidosis for 14 percent. Both cohorts were strongly male predominant, at roughly 83 percent, and the two centers’ cases were read by teams of three experienced nuclear medicine physicians who underwent joint calibration before the study and were blinded to each patient’s final diagnosis.

Every participant underwent late planar whole-body scintigraphy approximately three hours after intravenous injection of 740 megabecquerels of the respective radiopharmaceutical, following current procedural guidelines. Patients were asked to hydrate well and empty their bladder before scanning to reduce background activity in the pelvis. Imaging used dual-head SPECT/CT gamma cameras with low-energy high-resolution collimators, and dedicated SPECT/CT acquisitions were performed where indicated to confirm the precise cardiac location of uptake and to anatomically characterize any extracardiac findings. The myocardial signal was graded visually with the Perugini score, a four-point scale in which grade 0 means no cardiac activity, grade 1 means uptake weaker than bone, grade 2 means cardiac uptake equal to bone signal, and grade 3 means intense myocardial retention with minimal or absent bone uptake. Beyond the heart, the readers systematically recorded tracer retention in the lungs, the limb musculature, the abdominal viscera, and miscellaneous sites such as the tongue and salivary glands.

The most eye-catching divergence emerged in the visual grading of wild-type ATTR patients. In the DPD cohort, 76 percent of wild-type ATTR scans showed Perugini grade 2, while only 21 percent reached grade 3. In the Florence HMDP cohort the pattern was essentially inverted: 72 percent of wild-type ATTR patients scored grade 3 and just 19 percent scored grade 2, a difference that was highly statistically significant. The authors attribute this skew not to any real difference in disease severity between the two patient groups but to the distinctive whole-body kinetics of the two molecules. DPD has a high affinity for amyloid deposits lodged in the skeletal muscles of the limbs and shoulder and hip girdles. That diffuse soft-tissue retention acts as a competitive background, a so-called soft-tissue sink effect, which dampens the heart-to-body contrast and frequently pins the visual score at grade 2 even when the disease is advanced. HMDP, by contrast, clears rapidly from soft tissues, raising the target-to-background ratio and visually amplifying the myocardial signal, which predisposes scans to a grade 3 classification.

The extracardiac biodistribution data reinforce that interpretation. Among wild-type ATTR patients, appendicular muscle uptake appeared in 84 percent of DPD scans but only 38 percent of HMDP scans, a difference with a p-value below 0.001. A similar trend appeared in the hereditary ATTR subgroup, where muscle uptake was present in 80 percent of DPD scans versus 38.5 percent of HMDP scans. HMDP showed the mirror-image behavior in the lungs: pulmonary tracer retention was observed in 68 percent of wild-type ATTR patients imaged with HMDP versus 46 percent imaged with DPD, and in 61 percent of hereditary ATTR patients imaged with HMDP versus a mere 10 percent imaged with DPD. This distinctive pulmonary signature of HMDP, quantified with the established Janssen Score, matters for daily reporting because unfamiliar readers might otherwise mistake lung retention for a technical artifact or for unrelated interstitial lung disease. Miscellaneous uptake, in sites such as the tongue and salivary glands, was also more frequent with DPD in the wild-type ATTR group, at 89 percent versus 74 percent.

Not every scintigraphic feature depended on the tracer’s chemistry. Visceral uptake, meaning tracer retention in the liver or spleen, was rare in both ATTR subtypes across both centers and showed no significant difference between the two agents. Where it did appear, it was overwhelmingly a hallmark of AL amyloidosis, occurring significantly more often in light-chain patients than in either ATTR group regardless of the tracer used. In multivariate logistic regression, visceral uptake emerged as a powerful negative predictor of ATTR disease, with an odds ratio of 0.016, meaning its presence strongly favored an AL diagnosis. The authors emphasize that this reflects the intrinsic biology of the fibrils rather than radiotracer pharmacokinetics: light-chain amyloid frequently deposits in abdominal organs, and both DPD and HMDP reliably flag that pattern. Neither tracer showed significant myocardial uptake in most AL patients, preserving a robust ability to exclude light-chain cardiac involvement.

The regression models also quantified which imaging findings independently predict an ATTR diagnosis over AL. Across the whole population, a Perugini score of grade 2 or higher carried an odds ratio of 30.49 for ATTR, while miscellaneous uptake carried an odds ratio of 6.35. Muscle uptake, despite its visual prominence on DPD scans, did not reach statistical significance as an independent predictor in the multivariate model, with an odds ratio of 1.32 and a p-value of 0.694, likely because it overlaps with other features. When the cohorts were stratified by tracer, the visual score was significant in both but with a larger odds ratio in the HMDP model, 55.85, than in the DPD model, 15.72, consistent with HMDP’s superior myocardial contrast. Visceral uptake remained a significant negative predictor in both cohorts. Notably, none of the scintigraphic parameters significantly distinguished hereditary ATTR from wild-type ATTR, with wide confidence intervals crossing the line of no effect, a result the authors attribute to the tracers operating at the limits of their pharmacokinetic sensitivity and to differing referral pathways between the two subtypes.

The study’s authors, led by Andrea Di Giorgio and Rachele Bonfiglioli with collaborators including Francesco Cappelli and Simone Longhi, argue that these findings should change how scans are reported. With DPD, skeletal muscle uptake should be actively assessed as a confirmatory sign of systemic amyloid burden, and clinicians should recognize that the agent tends to stabilize the visual score at grade 2, guarding against the misreading of advanced disease as early-stage involvement. With HMDP, pulmonary retention should be recognized as an expected feature rather than a lung pathology, and its absence of muscle signal should not be taken to exclude extracardiac involvement. The team also highlights the practical importance of SPECT/CT fusion imaging, which anatomically localizes the myocardial signal and separates it from overlying soft-tissue activity, resolving ambiguities that planar images alone cannot, particularly when the DPD sink effect suppresses apparent cardiac intensity.

The authors are careful to frame these patterns as probabilistic indicators that complement, rather than replace, the mandatory diagnostic workflow. A positive bone scan, even with a high Perugini grade and characteristic extracardiac uptake, must still be interpreted within the broader clinical picture, and definitive diagnosis requires the biochemical exclusion of monoclonal gammopathy and confirmatory genetic sequencing of the transthyretin gene. Scintigraphic signatures may also appear incidentally on bone scans performed for unrelated orthopedic, oncological, or rheumatological reasons, or in asymptomatic genetic carriers without overt cardiomyopathy. The study carries limitations inherent to its retrospective design, including potential selection bias, the use of different gamma camera models at the two centers without formal phantom-based cross-validation, and a patient population dominated by the Ile68Leu transthyretin variant, which may limit generalizability to other mutations even as it reduces genetic confounding.

The bottom line for clinical practice is that the two tracers remain gold standards for non-invasive identification of transthyretin cardiac amyloidosis, but they are distinct molecular entities whose readings demand tracer-specific expertise. HMDP maximizes the heart-to-bone visual contrast and often drives grades toward 3, while DPD offers a more comprehensive view of the systemic muscular amyloid burden at the cost of a compressed visual scale. Both preserved strong specificity in excluding AL disease, with hepatic and splenic uptake serving as a tracer-independent red flag for light-chain disease. As bone scintigraphy continues to anchor the diagnostic pathway for a disease increasingly targeted by new therapies, the message of this multicenter comparison is that the choice of radiotracer is not a technical detail but a variable that shapes every grade and every extracardiac finding a physician reports.

Subject of Research: Comparative biodistribution of 99mTc-DPD and 99mTc-HMDP radiotracers in scintigraphic diagnosis of cardiac amyloidosis

Article Title: The role of extracardiac radiotracer biodistribution in the evaluation of cardiac amyloidosis: a retrospective multicenter study on [99mTc]Tc-DPD and [99mTc]Tc-HMDP

Article References: Di Giorgio, A., Battisti, G., Bonazzi, N., Prisco, S. E., Starace, M., Longhi, S., Fanti, S., Berti, V., Cappelli, F., & Bonfiglioli, R. (2026). The role of extracardiac radiotracer biodistribution in the evaluation of cardiac amyloidosis: a retrospective multicenter study on [99mTc]Tc-DPD and [99mTc]Tc-HMDP. European Journal of Nuclear Medicine and Molecular Imaging. https://doi.org/10.1007/s00259-026-08153-1

Image Credits: AI Generated

DOI: 10.1007/s00259-026-08153-1

Keywords: cardiac amyloidosis, transthyretin amyloidosis, bone scintigraphy, DPD, HMDP, radiotracer biodistribution, Perugini score, SPECT/CT, AL amyloidosis, extracardiac uptake, nuclear medicine, multicenter study

The study, a short communication by Hao Zhang of Onestep Technologies Inc. in Vancouver, evaluates a stabilized single-reagent method based on tetramethylbenzidine, commonly abbreviated TMB. TMB is a chromogenic substrate that reacts with free chlorine to form a colored oxidation product, allowing the chlorine concentration to be quantified by measuring how strongly the solution absorbs light. In this work, absorbance was read at a wavelength of 650 nanometers using a controlled benchtop fixed-time readout taken 30 seconds after reaction. The choice of a single, pre-stabilized reagent is the central design idea: instead of requiring operators or automated analyzers to mix multiple reagents in precise sequence, the entire chemistry is packaged in one solution that can be stored sealed and deployed directly.

To appreciate why that matters, it helps to consider how free chlorine is measured today. The dominant routine technique relies on N,N-diethyl-p-phenylenediamine, or DPD, a dye that free chlorine oxidizes to produce a pink-colored species measured photometrically. DPD methods are codified in international standards, including ISO 7393-2 for routine control purposes and the U.S. Environmental Protection Agency’s Method 334.0 for online chlorine analyzers, and they are enshrined in Standard Methods for the Examination of Water and Wastewater. DPD works well, but the reagent system is notoriously fussy. DPD solutions degrade on exposure to air and light, reagent blanking is often needed, and multi-reagent fluidic handling adds pumps, valves, and failure points to online instruments. For continuous monitors installed across a distribution network, every additional reagent and fluidic step translates into maintenance burden, consumable cost, and opportunities for error.

TMB has long been recognized as an alternative. It was proposed as a colorimetric chlorine reagent as early as the 1990s, and subsequent studies demonstrated its use for hypochlorite detection in tap water and for spectrophotometric sequential injection analysis of free chlorine in waters. TMB has also been promoted as a multi-colorimetric indicator whose response can be aligned with health guideline values. What has limited its practical adoption in online monitoring is the same problem that afflicts many chromogenic reagents: instability in solution. The new work addresses this directly by using a stabilized single-reagent formulation, and it backs the reagent-handling rationale with sealed storage observations described in the study.

The analytical core of the paper is a head-to-head quantitative comparison between the TMB method and DPD. The dataset comprised 32 paired measurements. Twenty of these were real tap-water or finished-water samples, while the remaining twelve were controlled matrix–concentration conditions: three defined mineral-background matrices, each evaluated at four free-chlorine levels. This design is important because drinking water is not chemically uniform. Mineral content, hardness, and background ions vary widely between sources and across distribution systems, and a monitoring chemistry that performs well in one water can drift in another. By deliberately spanning multiple defined mineral backgrounds, the study probes whether the TMB chemistry is robust to realistic matrix variation rather than performing only in clean laboratory buffers.

The comparison protocol was deliberately tight. TMB and DPD measurements were performed within five minutes of each other and in triplicate, minimizing the chance that chlorine decay between measurements would masquerade as analytical disagreement. Agreement was assessed with Deming regression, a statistical technique appropriate when both methods carry measurement uncertainty, rather than ordinary least squares which assumes the reference method is error-free. The results were strikingly close: the Deming regression yielded a slope of 0.985 and an intercept of 0.007 milligrams per liter expressed as chlorine. In practical terms, a slope near unity means the two methods scale almost identically across the concentration range, and an intercept near zero means there is essentially no systematic offset between them.

The paired-difference statistics reinforce that picture. The mean paired difference between TMB and DPD readings was just +0.001 milligrams per liter as chlorine, meaning the methods were unbiased relative to one another on average. The mean absolute difference was 0.013 milligrams per liter, a figure comfortably below the discrimination levels typically relevant to regulatory and operational decisions about residual disinfectant. Precision, assessed from the triplicate TMB readings, produced a median relative standard deviation of 2.4 percent and a mean relative standard deviation of 3.6 percent. For a simple fixed-time absorbance readout at 30 seconds, that level of repeatability indicates the reaction chemistry and the readout timing are well controlled, since kinetic colorimetric methods are sensitive to reaction time if the color development is not sufficiently stable or reproducible.

The author frames the method as a measurement layer for simplified online monitoring, and the study’s logic points toward a compact instrument in which a single sealed reagent reservoir feeds a mixing point, a short reaction delay, and an optical detector reading at 650 nanometers. Eliminating multi-reagent fluidics would reduce the number of pumps and valves, shrink the mechanical footprint, and simplify the reagent supply chain for distributed sensors. This matters because continuous residual monitoring is increasingly seen as essential for managing biological stability in distribution systems, a theme emphasized in guidance from Health Canada on monitoring drinking water biological stability, and because chlorine decay depends on factors such as pipe service age, as documented in studies of transmission and distribution networks. Reviews of continuous chlorine detection have repeatedly identified reagent handling and fluidic complexity as barriers to wider deployment of online analyzers, which is precisely the problem a single-reagent format attacks.

Several caveats deserve attention. This is a short communication based on a controlled benchtop evaluation, not a full validation across the enormous chemical diversity of real distribution systems, and the author notes that the results provide a quantitative basis for further translation into a compact monitoring platform rather than a finished field-proven instrument. The dataset, while well designed, is modest in size, and the competing-interest declaration is explicit: the author is the founder of a company developing stabilized TMB reagents and prototype online chlorine analyzers, so readers should weigh the promising numbers with that affiliation in mind. Independent replication, longer-term reagent stability data, and interference testing across a broader set of water chemistries would be the natural next steps before utilities adopt the approach.

Even with those qualifications, the study offers a tidy demonstration of how rethinking reagent packaging, not just detection physics, can advance environmental monitoring. Free chlorine sits at the intersection of public health and water infrastructure management, and the tools used to measure it have changed little in decades. A chemistry that agrees with DPD to within a hundredth of a milligram per liter on average, achieves a few percent repeatability with a 30-second readout, and arrives in a single stabilized bottle is exactly the kind of unglamorous but consequential engineering that could bring denser, cheaper, and more reliable residual monitoring to the pipes that deliver drinking water. The work suggests that the next generation of online chlorine analyzers may be simpler than the last, built around one reagent, one wavelength, and one short, well-timed wait.

Subject of Research: Colorimetric free chlorine monitoring in drinking water using a stabilized single-reagent TMB method compared with DPD

Article Title: A stabilized single-reagent TMB method for free chlorine monitoring in drinking water–related matrices

Article References: Zhang, H. (2026). A stabilized single-reagent TMB method for free chlorine monitoring in drinking water–related matrices. Environmental Monitoring and Assessment, 198(11), Article 1142. https://doi.org/10.1007/s10661-026-15979-7

Image Credits: AI Generated

DOI: 10.1007/s10661-026-15979-7

Keywords: free chlorine, drinking water, TMB, DPD, colorimetric analysis, online monitoring, Deming regression, water treatment, residual disinfectant, repeatability, analytical agreement, environmental monitoring