Heterogeneous Distribution and Absorbed Dose of Radiolabeled Nanoparticles and Colloids in Sentinel Lymph Nodes

Abstract

Background:

For breast cancer staging, radiolabeled colloids and superparamagnetic iron oxide nanoparticles (SPIONs) are used for sentinel lymph node (SLN) imaging. This study characterized the intranodal activity distribution and absorbed dose distribution.

Material and Methods:

Six patients diagnosed with primary breast cancer were intradermally injected with ^99mTc-Nanocoll. The SLNs were resected, weighed, and measured for activity. Three groups of six rats were subcutaneously injected into the hind paw with either ^99mTc-Nanocoll, ^99mTc-SPIONs, or ⁶⁸Ga-SPIONs. Macro- and small-scale dosimetry calculations were performed using autoradiography images of cryosections of SLNs from patients and animals.

Results:

The mean absorbed dose in patient SLNs was 0.5 ± 0.3 mGy/MBq for ^99mTc-Nanocoll and 3.4 ± 1.8 mGy/MBq, assuming a ^99mTc-Nanocoll-based distribution of ⁶⁸Ga-SPIONs. Due to different decay characteristics, the heterogeneity in the absorbed dose differed between ^99mTc-SPIONs and ⁶⁸Ga-SPIONs with a maximum to mean absorbed dose ratio of 2.7 ± 0.3 and 1.6 ± 0.2, respectively.

Conclusions:

This study shows that ^99mTc- and ⁶⁸Ga-SPIONs and ^99mTc-nanocolloids have similar activity distribution in human and animal lymph nodes. Small-scale dosimetry models combined with clinical patient biokinetics may serve as a bridge between organ and tissue dosimetry and the interpretation of intrinsic geometric variation and its uncertainties in absorbed dose.

Introduction

Cancer, primarily in breast, skin, and prostate, disseminates through the lymphatic system, with the first metastases tending to spread from one or two regional, sentinel lymph nodes (SLNs), draining the primary tumor site.^1

–5 The clinical procedure to identify the SLNs and perform staging involves intradermal injection of ^99mTc-labeled colloids in combination with vital blue dye (for intraoperative guidance).^6
–8 Less than 2% of the activity is accumulated in the SLN (3–5 h p.i.) due to the wide range in size of colloid particles.^9,10 Large colloidal particles are usually trapped at the injection site, while small colloidal particles, without any specific binding properties, are transported via the lymph through several lymph nodes and back into the circulatory system.^11,12 Recently, a focus has been on developing more specific agents to identify and localize the SLNs accurately.^13

–18 One of these agent is the Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved ^99mTc-labeled-tilmanocept (Lymphoseek), which is being used for lymphoscintigraphy and guided surgery. The small molecular size of 7 nm and high affinity to CD 206, expressed on the surface of both dendritic cells and macrophages in SLN, make it a promising agent.^19,20 In addition, the FDA has also approved superparamagnetic iron oxide nanoparticles (SPIONs) for SLN imaging.^21
–23 The advantages of SPIONs include a narrow size distribution (10–50 nm) and a large surface-to-volume ratio making them suitable for radiolabeling with high specific activity.

We have previously presented the potential of ^99mTc- and ⁶⁸Ga-SPIONs as translational multimodality molecular imaging agents. When injected subcutaneously in a rat model, these agents accumulate in the SLN, providing a potential for the staging of patients with breast cancer. In addition, the ⁶⁸Ga-SPIONs enable simultaneous positron emission tomography, magnetic resonance, and Cherenkov (PET/MR/Cherenkov) imaging of the lymph nodes.^24
–26

In this study, we used autoradiography to image the distribution of activity within the SLNs and estimated the absorbed dose, both for clinical and preclinical data.

Traditionally, SLN dosimetry is calculated using the medical internal radiation dose formalism to estimate the mean absorbed dose by assuming homogeneous activity distribution within the nodes, an approach reported for common radiopharmaceuticals used in lymphoscintigraphy such as ^99mTc-antimony trisulfide colloid, ^99mTc-sulfur colloid, and ^99mTc-Nanocoll.²⁷ However, the absorbed dose distribution pattern depends on the geometry and radiation emission energy spectrum. As a result, several small-scale dosimetry models have been reported.^28
–30 These include applying S-values at a microstructural level, resulting in a more accurate representation of the mean absorbed dose than utilizing organ-level dosimetry. In addition, autoradiography has the capability of revealing a heterogeneous distribution in the organ from the administered radiopharmaceutical.^31,32 Due to this, we, therefore, in this study, included heterogeneous activity distribution in lymph nodes in the absorbed dose calculations clinical and preclinical data.

The aims of this study were, first, to characterize, using autoradiography, the intranodal distribution of ^99mTc-labeled nanocolloids in both humans and rats as well as ^99mTc- and ⁶⁸Ga-labeled SPIONs in rats, and second, to calculate the absorbed dose and absorbed dose distribution within the SLNs.

Materials and Methods

The study, including both patients and animals, was performed in accordance with ethical standards and approval, following all national and local regulations. All patients provided informed consent.

Labeling and quality control of tracers

SPIONs were obtained from Genovis AB (Lund, Sweden). The monodisperse nanoparticles were produced with a narrow size distribution (11 nm ± 2 nm) and a biocompatible coating of polyethylene glycol for further functionalization.²⁴

Radiolabeling of SPIONs with ^99mTc and ⁶⁸Ga was performed as described in previous studies.^24,25 Briefly, ^99mTc-pertechnetate (300–500 MBq) was added to a vial containing the reducing agent 0.5 mg stannous chloride dissolved in sterile water (pH 3.4). The SPIONs were added to the mixture and then incubated for 60 min at room temperature (18°C). ⁶⁸Ga was obtained from a ⁶⁸Ga/⁶⁸Ge-generator (IDB, Holland) by eluting the ⁶⁸GaCl₂ with 0.6 M hydrochloric acid in 0.3–0.4 mL fractions with 40–60 MBq activity. The ⁶⁸Ga-SPIONs were produced via a direct labeling method using ammonium acetate as buffer (1 M, pH 5.5). Labeling efficiency and quality control were ensured by using instant thin-layer chromatography and 0.2 M citric acid as running buffer (ITLC, silica gel impregnated glass fiber sheets, Life Science or TEC-control chromatography strips, Biodex, USA). Imaging of the ITLC strips was performed using a phosphor imager (Perkin Elmer, Welesley, MA, USA). The colloidal stability of ^99mTc-SPIONs and ⁶⁸Ga-SPIONs was measured in vitro using human serum (pH 7, 37°C), 4 h and 24 h, respectively, after radiolabeling.

^99mTc-Nanocoll was prepared using ^99mTc-pertechnetate from a ⁹⁹Mo/^99mTc-generator (DRN 4329 Ultratechnekow FM, The Netherlands) and labeled in accordance with instructions from the manufacturer, including quality control ensured by authorized staff at the local radiochemistry laboratory/Nuclear Medicine Department.

Patient studies

Six patients aged 30–65 years had SLN biopsies that were included in this study. All patients had a histologically confirmed primary breast cancer tumor and were planned for surgical treatment, including SLN mapping. Four periareolar injections of ^99mTc-Nanocoll™ (40 MBq, volume of 0.4–0.6 mL) were performed, 2–4 h before surgery. Planar lymphoscintigraphic images (frontal and lateral, acquisition time 5 min) were performed for each patient using a scintillation camera (Sky Light, Philips or Discovery, GE, USA) equipped with a low-energy, high-resolution collimator. The energy window was 15% centered over the 140 keV photopeak. The uptake of the agent was confirmed in the SLN imaging. Immediately before surgery, a blue dye (Patent V Blue) was administered to visualize the draining lymphatic channels and SLNs. A hand-held γ-probe (Euro Probe3, Capintec, USA) was used to localize the SLN and to confirm that the correct node was being resected. The resected SLNs were weighed and measured for activity content with a radionuclide calibrator (Capintec CRC 25R, UK). The SLNs were cut longitudinally and frozen. Sections that were 10-µm-thick were placed on microscope slides, dried, stained with hematoxylin and eosin (H&E), and imaged using autoradiography. A simplified work scheme is presented in Figure 1.

FIG. 1.

Working scheme to obtain the activity distribution images of human SLNs. SLNs, sentinel lymph nodes; SPIONs, superparamagnetic iron oxide nanoparticles.

Animal studies

Three groups of six white Wistar male rats (mean body weight, 200 g) were lightly anesthetized with a mixture of isofluran/O₂ (2.5%–3%, 0.2 L/min) and injected subcutaneously with ^99mTc-Nanocoll (∼7 MBq, 0.05 mL), ^99mTc-SPIONs (∼24 MBq, 0.05–0.07 mL), or ⁶⁸Ga-SPIONs (∼7.4 MBq, 0.05–0.07 mL) in the right hind paw. The animals were imaged with preclinical dual modality single-photon emission computed tomography (SPECT)/CT or positron emission tomography (PET)/CT (NanoSPECT-CT or NanoPET-CT, Bioscan, USA) 1, 2, 3, and 5 h postinjection. These images were only used for localization of the SLNs. Animals from the first group (injected with ^99mTc-Nanocoll and ^99mTc-SPIONs) were euthanized: one after the first imaging session (1 h p.i.), one after the second imaging session (2 h p.i.), two after the third imaging session (3 h p.i.), and two after the last imaging session (5 h p.i.). Animals injected with ⁶⁸Ga -SPIONs were due to the short half-life of ⁶⁸Ga, euthanized after 3 h p.i.

The dissected SLN were weighed and measured for activity using an NaI(Tl) well-type detector (1282 CompuGamma CS; LKB Wallac, Turku, Finland). Frozen sections (100 µm thick) of popliteal (SLN) and iliac lymph nodes were prepared, mounted on sample slides, and imaged for activity distribution with autoradiography.

Autoradiography

To determine the ^99mTc-Nanocoll and ^99mTc-SPIONs intranodal activity distribution in human and animal SLNs, microscope slides containing 1–3 SLN sections were imaged using autoradiography. An imaging system (Biomolex 700 Imager; Biomolex AS, Oslo, Norway) based on a double-sided silicon strip detector with an intrinsic 50 µm spatial resolution was used.^33,34 Each slide was imaged for 10–12 h. The acquired images were then assembled from list-mode data, using an in-hose developed code (IDL 6.4, L3 Harris Technologies, Melbourne, FL, USA), corrected for dead pixels by interpolation and for radioactive decay. No images were acquired for samples containing ⁶⁸Ga-SPIONs due to the short half-life of ⁶⁸Ga.

Dosimetry

The absorbed dose rates in SLNs were calculated from the activity image of autoradiography convolved with a point-dose kernel. The point-dose kernels for ^99mTc and ⁶⁸Ga were simulated with the Monte Carlo code MCNP5 1.60 (Los Alamos National Laboratory, New Mexico), using the new electron sampling logic option and a voxel size of 50 µm, corresponding to the voxel size of the autoradiography system.^34,35 Because data from adjacent tissue were missing, the same image was stacked to get the absorbed dose rate contribution from adjacent tissue. To determine the absorbed dose, the time-integrated activity coefficient, TIAc, (residence time) was determined from the dissected SLNs in rats (n = 6) at 0, 1, 2, 3, and 5 h. Activities were then decay corrected to the time of injection. Exponential fitting was performed on the first 5 h of the uptake curve, during which the maximum specific uptake was reached. For the time beyond 5 h, the uptake was considered constant, with no particles being released from the SLN. After the fitting, the physical decay was added, and the TIAc was calculated. Absorbed-dose images were obtained by multiplying the TIAc with the dose-rate images. These were then filtered using a Gaussian smoothing filter (σ = 1 voxel). Three thresholds (ratio of maximum)-based region of interests (ROIs) were derived, Region1 [0.12–0.25], Region 2 [0.25–0.4], and Region 3 [0.4–1.0]. These ROIs were then applied to the dose-rate images, and the absorbed dose in these regions was obtained. The calculated small-scale absorbed dose distribution depends on the assumption that the activity does not relocate inside the lymph node during the 5h experiment. In addition, dose–volume histograms (DVH), cumulative DVH (cDVH), and maximum to mean absorbed dose ratios were derived (Fig. 5). The short half-life of ⁶⁸Ga (T_1/2 = 68 min) resulted in that tissue samples containing ⁶⁸Ga-SPIONs that could not be imaged using autoradiography. Therefore, we assumed similar activity distribution for ⁶⁸Ga-SPIONs and ^99mTc-Nanocoll in patients as well as ^99mTc-SPIONs in rats.

Results

Labeling and quality control of tracers

The radiolabeling efficiency for ^99mTc-Nanocoll was >95% for both patients and animals. ^99mTc-SPIONs and ⁶⁸Ga-SPIONs were labeled with an efficiency of 99% and 97%, respectively. The radiolabeled nanoparticles remained at the origin of the ITLC strips, whereas the free ^99mTcO₄ ⁻ or free ⁶⁸Ga migrated with the solvent front with R_f values of 0.7 and 0.9, respectively. On analyzing the colloidal stability of ^99mTc-SPIONs and ⁶⁸Ga-SPIONs in human serum, the amount of free activity was <1% after 24 h (^99mTc-SPIONs) and 4 h (⁶⁸Ga-SPIONs).

Assessment of the SLN of patients

Patient SLN, resected for biopsy, weighed between 1 and 3 g with an average of 1.52 ± 0.94 g and 10–20 mm in size, measured along the longest axis. The mean activity accumulated in the lymph nodes was 1.31 ± 0.5 MBq (3.31% ± 1.44% IA), 3–5 h postinjection. The maximum and mean uptake in SLN is presented in Table 1.

Table 1.

The Maximum and Mean Uptake of Different Tracers in Patient (p) and Animal (a) Sentinel Lymph Nodes, Respectively

Tracer	Time(hours postinjection)	% Injected activity
Tracer	Time(hours postinjection)	Maximum	Mean
^99mTc-Nanocoll (p)	3–5	6.02	3.3 ± 1.4
^99mTc-Nanocoll (a)	5	0.64	0.6 ± 0.2
^99mTc-SPIONs (a)	5	7.39	2.2 ± 2.1
⁶⁸Ga-SPIONs (a)	3	6.47	1.2 ± 2.5

Assessment of the SLN of animals

The small animal SPECT-CT and PET-CT images clearly visualized popliteal lymph nodes in rats injected with ^99mTc-Nanocoll, ^99mTc-SPIONs, or ⁶⁸Ga-SPIONs.

Rat SLNs had a mean weight of 0.01 ± 0.001 g, a size of 2–5 mm, and were bean-shaped with an off-white appearance, almost transparent color. The highest uptake in rat SLNs was 7.39% injected activity (IA) for ^99mTc-SPIONs and 6.47% IA for ⁶⁸Ga-SPIONs, 5 h respectively 3 h postinjection. For ^99mTc-Nanocoll, the maxim uptake was 0.64% IA in rats and 6.02% IA in patients, 3–5 h postinjection. The maximum and mean uptake in SLN for each agent is presented in Table 1.

Autoradiography

Autoradiography imaging revealed a heterogeneous distribution of activity related to the anatomy, in both patient and rat lymph node sections. Despite higher uptake (activity content) in lymph nodes from rats, the activity distribution patterns were very similar when comparing the different agents for patient and rat lymph nodes.

Representative images are presented in Figure 2. The activity was primarily observed most proximal to afferent lymphatic vessels, in subcapsular and medullary sinuses, in both human and animal SLNs.

FIG. 2.

Autoradiography images presenting a comparison of activity distribution in human and rat lymph nodes. (A) Human SLNs after injection of ^99mTc-Nanocoll. (B) Rat lymph nodes after injection of ^99mTc-Nanocoll and (C) rat lymph nodes after injection of ^99mTc-SPIONs, 3 h postinjection. A heterogenous activity distribution can be observed with high concentration in subcapsular and medullary sinuses.

Dosimetry

Despite the fact that ⁶⁸Ga emits much more energy from charged particles than ^99mTc, thus having a higher S-factor, the long accumulation time and the shorter half-life result in lower TIA and, subsequently, also a lower absorbed dose. The maximum absorbed-dose rate in the SLN of patients and rat lymph nodes was 6.4 ± 4.2 mGy/h and 774 ± 830 mGy/h, respectively, using ^99mTc-Nanocoll. Assuming the same biodistribution as ^99mTc-Nanocoll in patients for ⁶⁸Ga-SPIONs, the maximum absorbed dose rate was 136 ± 83 mGy/h in SLNs. Representative absorbed dose images for patients are presented in Figure 3 and for animals are presented in Figure 4. The mean absorbed dose for ^99mTc-Nanocoll in SLNs of patients was 1.8 ± 1.6 mGy.

FIG. 3.

(A) Autoradiography image of activity distribution in SLN in humans. (B) ROI corresponding to thresholds of 12%, 25%, and 40% of max activity in image A, absorbed doses calculated from image A are shown for (C) for ^99mTc-Nanocoll, respectively (D) for ⁶⁸Ga-SPIONs.

FIG. 4.

(A) Autoradiography image of activity distribution in first lymph node in rats, (B) presenting the different regions of interest, (C) absorbed dose ^99mTc-Nanocoll, (D) respectively ⁶⁸Ga.

The absorbed dose calculated from the activity distribution in the autoradiography images for the different regions (Region 1 [0.12–0.25], Region 2 [0.25–0.4], and Region 3 [0.4–1.0]) is presented in Table 2.

Table 2.

The Mean Absorbed Dose in the Different Activity Regions in Rat and Patient Lymph Nodes

Region(% of maximal activity)	Mean absorbed dose per administered activity in sentinel lymph nodes (mGy/MBq)
	Rats			Human
	^99mTc-Nanocoll	^99mTc-SPIONs	⁶⁸Ga-SPIONs	^99mTc-Nanocoll	⁶⁸Ga-SPIONs (estimated)
1 [0.12–0.25]	13 ± 9	630 ± 660	99 ± 66	0.3 ± 0.1	2.8 ± 1.4
2 [0.25–0.4]	24 ± 18	1860 ± 2030	153 ± 110	0.6 ± 0.3	4.1 ± 2.3
3 [0.4–1.0]	39 ± 31	4190 ± 4510	186 ± 140	0.9 ± 0.6	4.9 ± 2.9
Whole SLN 1 + 2 + 3	19 ± 14	1120 ± 1050	122 ± 86	0.5 ± 0.3	3.4 ± 1.8

If ⁶⁸Ga-SPIONs would be used instead of the ^99mTc-Nanocoll, the mean absorbed dose would be as presented in the table, based on the nanocoll biodistribution. SLN, sentinel lymph node; SPIONs, superparamagnetic iron oxide nanoparticles.

Examples of DVH and cDVH for human and rat lymph nodes using ^99mTc-Nanocoll, ^99mTc-SPIONs, and ⁶⁸Ga-SPIONs are presented in Figure 5. For comparison, the DVHs obtained for ⁶⁸Ga-Nanocoll (Fig. 5B and D) are presented together with ^99mTc-Nanocoll and ⁶⁸Ga-SPIONs.

FIG. 5.

The figure shows the dose–volume histograms (DVH) and cumulative dose–volume histograms (cDVH) from single-section autoradiography dosimetry. The DVH has been normalized to the highest bin, and the x-axis is displayed as the absorbed dose to the mean absorbed dose ratio, where the red line visualizes the mean absorbed dose. The y-axis of the cDVH shows the percent of the volume having at least the absorbed dose ratio on the x-axis. The two columns represent ^99mTc and ⁶⁸Ga, respectively, and the upper row represents Nanocoll human (A, B), the middle row is for Nanocoll in rat (C, D), and the lower row (E, F) is for SPIONs in rat.

Absorbed dose heterogeneity differed between ^99mTc- and ⁶⁸Ga-SPIONs, with a maximum-to-mean absorbed dose ratio of 2.7 ± 0.3 and 1.6 ± 0.1. This difference originates from the different decay characteristics of the radionuclides. Compared with ^99mTc, ⁶⁸Ga has a shorter half-life and higher energy of the emitted particles, resulting in energy deposition over a larger volume.

Discussion

γ-camera or PET images have a low spatial resolution, making it difficult to obtain detailed activity distribution within organs such as, e.g., lymph nodes. Therefore, dosimetry calculations based on these imaging modalities may overestimate or underestimate the absorbed doses in organ subregions.

In the present study, autoradiography images made it possible to measure heterogeneous activity distribution and calculate the corresponding absorbed dose distribution of radiolabeled colloids/nanoparticles in lymph nodes.

^99mTc-SPIONs, ⁶⁸Ga-SPIONs, and ^99mTc-Nanocoll present a similar pattern of activity distribution within normal lymph nodes because it primarily depends on SLN anatomy. Animal and human lymph nodes differ in size, number of afferent lymphatic vessels, and lymphatic flow rate. The radiolabeled colloids and SPIONs enter through the afferent lymphatic vessels, often located at the tip of the semiovoid-shaped nodes, and accumulate in the subcapsular and medullary sinuses. Certain particles flow through efferent vessels into the subsequent node in the chain, which can result in hot regions, as shown in autoradiography images in Figure 2. These observations indicate that small-scale dosimetry can reveal the absorbed dose distribution.

The radiolabeled colloids and SPIONs were not homogenously distributed through the entire SLN, and the mean activity concentration was 2–3 times higher in some regions compared with other regions (see Figs. 3 and 4). The mean absorbed dose calculated for the entire patient lymph node was concurrent with those reported in the product specifications for ^99mTc-Nanocoll. The absorbed doses were in accordance with those for individual lymph nodes (0.49 mGy/MBq) after subcutaneous injection of ^99mTc-SbSC (antimony sulfide colloid; 40 MBq) by Bergqvist et al.¹²

The absorbed dose depends on the radionuclide emission properties. The conversion electrons from ^99mTc deposit 90% of energy within 0.15 mm in SLNs in comparison with high-energy positrons emitted from ⁶⁸Ga, having a mean range of 2.6 mm. Therefore, in human lymph nodes, the absorbed dose per IA is slightly higher if ⁶⁸Ga-SPIONs were to be used instead of ^99mTc-SPIONs or ^99mTc-Nanocoll (Table 2), and less heterogeneous. To our knowledge, no other studies have attempted to estimate the absorbed dose using small-scale dosimetry of ^99mTc-Nanocoll or ⁶⁸Ga-labeled SPIONs for SLNs. In animal experiments, the absorbed dose per IA was higher than that in patients, due to the smaller size of the SLNs (Table 2).

The results of the heterogenous absorbed dose distribution in SLNs with up to a factor of 3 (maximum/mean absorbed dose) emphasize the importance of small-scale dosimetry, when a comprehensive radiobiological evaluation is needed.

As has been outlined in several publications, the radiobiological response in a tissue is dependent on the homogeneity of the absorbed dose. For radiopharmaceuticals, the uptake in a tissue is dependent on its cellular components. A recent publication by Pouget et al. states that the mean absorbed dose alone is insufficient to predict treatment response or toxicity.³⁶ One such factor to consider is the absorbed dose heterogeneity, and we have in this study shown a heterogeneity of absorbed dose in SLN and a deviation from mean absorbed dose. Future studies of the radiobiological response from radionuclides should, therefore, focus on small-scale dosimetry.

Conclusions

The DVH of SLNs clearly show that large volumes of the tissue will experience either a higher or lower absorbed dose than the reported mean, and therefore different biological effects. Our study also clearly shows the importance of dosimetry evaluation in organs both on a macro and small scale. When evaluating dose effects for optimization studies, small-scale dosimetry can ensure optimal dose while minimizing risks.

Footnotes

Acknowledgments

This study was performed with generous support from the Swedish Cancer Foundation, the Swedish Science Council, Mrs. Berta Kamprad’s Foundation, Gunnar Nilsson’s Foundation, and the ALF Foundation of the Medical Faculty of Lund University. The SPIONs were provided as a generous gift from Genovis AB, Sweden. The authors also want to thank Lund University Bioimaging Center (LBIC), Lund University, for providing experimental resources.

Authors’ Contributions

R.M.: Conceptualization, patient and animal data curation, formal analysis, methodology, project administration, and writing original draft preparation. E.L.: Conceptualization, data curation, formal analysis, software (dosimetry), and writing—reviewing. A.Ö.: Conceptualization, data curation, formal analysis, software (autoradiography), and writing—reviewing. C.I.: Conceptualization, patient data curation, project administration, resources, and writing and reviewing. D.G.: Conceptualization, formal analysis, investigation, methodology, and visualization (microscopy). L.K.: Conceptualization, supervising, and writing—reviewing. S-E.S.: Conceptualization, funding acquisition, methodology, supervising, and writing—reviewing.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

References

Leong

, Pissas

, Scarato

, et al. The lymphatic system and sentinel lymph nodes: Conduit for cancer metastasis. Clin Exp Metastasis, 2022; 39(1):139–157; doi: 10.1007/s10585-021-10123-w

Giammarile

, Vidal-Sicart

, Paez

, et al. Sentinel lymph node methods in breast cancer. Semin Nucl Med, 2022; 52(5):551–560; doi: 10.1053/j.semnuclmed.2022.01.006

Albertini

, Lyman

, Cox

, et al. Lymphatic mapping and sentinel node biopsy in the patient with breast cancer. JAMA, 1996; 276(22):1818–1822.

Berger

DMS

, van den Berg

, van der Noort

, et al. Technologic (R)Evolution leads to detection of more sentinel nodes in patients with melanoma in the head and neck region. J Nucl Med, 2021; 62(10):1357–1362; doi: 10.2967/jnumed.120.246819

Morton

, Wen

, Wong

, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg Surg1992; 127(4):392–399; doi: 10.1001/archsurg.1992.01420040034005

Giammarile

, Alazraki

, Aarsvold

, et al. The EANM and SNMMI practice guideline for lymphoscintigraphy and sentinel node localization in breast cancer. Eur J Nucl Med Mol Imaging, 2013; 40(12):1932–1947; doi: 10.1007/s00259-013-2544-2

Bluemel

, Herrmann

, Giammarile

, et al. EANM practice guidelines for lymhoscintigraphy and sentinel lymph node biopsy in melanoma. Eur J Nucl Med Mol Imaging, 2015; 42(11):1750–1766; doi: 10.1007/s00259-015-3135-1

Veronesi

, Paganelli

, Viale

, et al. Sentinel lymph node biopsy as a staging procedure in breast cancer: Update of a randomised controlled study. Lancet Oncol, 2006; 7(12):983–990; doi: 10.1016/S1470-2045(06)70947-0

Tsopelas

. Particle size analysis of ^99mTc-labeled and unlabeled antimony trisulfide and rhenium sulfide colloides intended for lymphoscintigraphic application. J Nucl Med, 2001; 42(3):460–466.

10.

Mariani

, Moresco

, Viale

, et al. Radioguided sentinel lymph node biopsy in breast cancer surgery. J Nucl Med, 2001; 42(8):1198–1215.

11.

Strand

S-E

, Persson

BRR

. Quatitative lymphoscintigraphy. 1. Basic concepts for optimal uptake of radiocolloids in the parasternal lymph nodes in rabbits. J Nucl Med, 1979; 20(10):1038–1046.

12.

Bergqvist

, Strand

, Persson

BRR

. Particle sizing and biokinetics of the interstitial lymphoscintigraphyc agents. Semin Nucl Med, 1983; 13(1):9–19; doi: 10.1016/s0001-2998(83)80031-2

13.

Pallares

, Mottaghy

, Schulz

, et al. Nanoparticle diagnostics and theranostics in the clinic. J Nucl Med, 2022; 63(12):1802–1808; doi: 10.2967/jnumed.122.263895

14.

O’Melia

, Thomas

. Lymphatics drain nanoparticles from tumours. Nat Mater, 2023; 22(11):1287–1288; doi: 10.1038/s41563-023-01701-2

15.

Mitchell

, Billingsley

, Haley

, et al. Engineering precision nanoparticles for drug delivery. Nat Rev Drug Discov, 2021; 20(2):101–124; doi: 10.1038/s41573-020-0090-8

16.

Janjua

, Cao

, Yu

, et al. Clinical translation of silica nanoparticles. Nat Rev Mater, 2021; 6(12):1072–1074; doi: 10.1038/s41578-021-00385-x

17.

Doughton

, Hofman

, Eu

, et al. A first-in-human study of ⁶⁸Ga-nanocolloid PET/CT sentinel lymph node imaging in prostate cancer demonstrates aberrant lymphatic drainage pathways. J Nucl Med, 2018; 59(12):1837–1842; doi: 10.2967/jnumed.118.209171

18.

, Wang

, Lin

, et al. Clinical evaluation of ^99mTc-Rituximab for sentinel lymph node mapping in breast cancer patients. J Nucl Med, 2016; 57(8):1214–1220; doi: 10.2967/jnumed.115.160572

19.

Vidal-Sicart

, Rioja

, Prieto

, et al. Sentinel lymph node biopsy in breast cancer with ^99mTc-tillmanocept: A multicenter study on real-life use of a novel tracer. J Nucl Med, 2021; 62(5):620–627; doi: 10.2967/jnumed.120.252064

20.

Den Toom

, Mahieu

, van Rooij

, et al. Sentinel lymph node detection in oral cancer: A within patient comparison between [99mTc]Tc-tilmanocept and [99mTc]Tc-nanocolloid. Eur J Nucl Med Mol Imaging, 2021; 48(3):851–858; doi: 10.1007/s00259-020-04984-8

21.

Mirzaei

, Wärnberg

, Zaar

, et al. Ultra-low dose of superparamagnetic iron oxide nanoparticles for sentinel lymph node detection in patients with breast cancer. Ann Surg Oncol, 2023; 30(9):5685–5689; doi: 10.1245/s10434-023-13722-x

22.

Mirzaei

, Katsarelias

, Zaar

, et al. Sentinel lymph node localization and staging with a low-dose of Superparamagnetic Iron Oxide (SPIO) enhanced MRI and magnetometer in patients with cutaneous melanoma of the extremity- The MAGMEN feasibility study. Eur J Surg Oncol, 2022; 48(2):326–332; doi: 10.1016/j.ejso.2021.12.467

23.

Aldenhoven

, Frotscher

, Körver-Steeman

, et al. Sentinel lymph node mapping with superparamagnetic iron oxide for melanoma: A pilot study in healthy participants to establish an optimal MRI workflow protocol. BMC Cancer, 2022; 22(1):1062; doi: 10.1186/s12885-022-10146-w

24.

Madru

, Kjellman

, Olsson

, et al. ^99mTc-labelled superparamagnetic iron oxide nanoparticles for multimodality SPECT/MRI of sentinel lymph node. J Nucl Med, 2012; 53(3):459–463; doi: 10.2967/jnumed.111.092437

25.

Madru

, Tran

, Axelsson

, et al. ⁶⁸Ga-labeled Superparamagneticiron Oxide Nanoparticles (SPIONs) for multimodality PET/MRI/Cherenkov luminescence imaging of sentinel nodes. Am J Nucl Med Mol Imaging, 2014; 4(1):60–69.

26.

Evertsson

, Kjellman

, Cinthio

, et al. Combined magnetomotive ultrasound, PET/CT, and MR imaging of ⁶⁸Ga-labelled superparamagnetic iron oxide nanoparticles in rat sentinel lymph nodes in vivo. Sci Rep, 2017; 7(1):4824; doi: 10.1038/s41598-017-04396-z

27.

Bergqvist

, Strand

, Persson

, et al. Dosimetry in lymphoscintigraphy of ^99mTc antimony sulfide colloid. J Nucl Med, 1982; 23(8):698–705.

28.

Roeske

, Aydogan

, Bardies

, et al. Small-scale dosimetry: Challenges and future directions. Semin Nucl Med, 2008; 38(5):367–383; doi: 10.1053/j.semnuclmed.2008.05.003

29.

Stenvall

, Larsson

, Strand

, et al. A small-scale anatomical dosimetry model of the liver. Phys Med Biol, 2014; 59(13):3353–3371; doi: 10.1088/0031-9155/59/13/3353

30.

Larsson

, Meerkhan

, Strand

, et al. A small-scale anatomic model for testicular radiation dosimetry for radionuclides localized in the human testes. J Nucl Med, 2012; 53(1):72–81; doi: 10.2967/jnumed.111.095133

31.

Makrigiorgos

, Ito

, Baranowska-Kortylewicz

, et al. Inhomogeneous deposition of radiopharmaceuticals at the cellular level: Experimental evidence and dosimetric implications. J Nucl Med, 1990; 31(8):1358–1363.

32.

, Bouvier-Capely

, Saldarriaga Vargas

, et al. Heterogeneity of dose distribution in normal tissue in case of radiopharmaceutical therapy with alpha-emitting radionuclides. Radiat Environ Biophys, 2022; 61(4):579–596; doi: 10.1007/s00411-022-01000-5

33.

Örbom

, Ahlstedt

, Serén

, et al. Characterization of a double-sided silicon strip detector autoradiography system. Med Phys, 2015; 42(2):575–584; doi: 10.1118/1.4905049

34.

Peng

, Nadia

, Wen

, et al. Blind image restoration enhances digital autoradiographic imaging of radiopharmaceutical tissue distribution. J Nucl Med, 2022; 63(4):591–597; doi: 10.2967/jnumed.121.262270

35.

X-5 Monte Carlo Team. MCNP - A General Monte Carlo N-Particle Transport Code, Version 5, Volume I: Overview and theory, Los Alamos National Laboratory report LA-UR-03-1987; 2003.

36.

Pouget

, Gabina

, Herrmann

, et al; EANM Radiobiology Working Group. EANM expert opinion: How can lessons from radiobiology be applied to the design of clinical trials? Part I: Back to the basics of absorbed dose-response and threshold absorbed doses. Eur J Nucl Med Mol Imaging, 2025; 52(3):1210–1222; doi: 10.1007/s00259-024-06963-9