A Specific HPLC Method to Determine Residual HEPES in [68Ga]Ga-Radiopharmaceuticals: Development and Validation.

Background: Nowadays, in Nuclear Medicine, clinically applied radiopharmaceuticals must meet quality release criteria such as high radiochemical purity and radiochemical yield. Many radiopharmaceuticals do not have marketing authorization and have no dedicated monograph within European Pharmacopeia (Ph. Eur.); therefore, general monographs on quality controls (QCs) have to be applied for clinical application. These criteria require standardization and validation in labeling and preparation, including quality controls measurements, according to well defined standard operation procedures. However, QC measurements are often based on detection techniques that are specific to a certain chromatographic system. Several radiosyntheses of [68Ga]Ga-radiopharmaceuticals are more efficient and robust when they are performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity to be assessed in the QC procedure, prior to clinical use. Thus, Ph. Eur. has introduced a thin-layer chromatography (TLC) method to quantify the HEPES amount that is present in [68Ga]Ga-radiopharmaceuticals. However, this is only qualitative and has proven to be unreliable. Here we develop and validate a new high-performance liquid chromatography (UV-Radio-HPLC) method to quantify the residual amount of HEPES in 68Ga-based radiopharmaceuticals. Method: To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document that was adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. A range of concentrations of HEPES (100, 80, 60, 40, 20, 10, 5, 3 µg/mL) were analyzed. Moreover, to test the validity and pertinence of our new HPLC method, we analyzed samples of [68Ga]Ga-DOTATOC; [68Ga]Ga-PSMA; [68Ga]Ga-DOTATATE; [68Ga]Ga-Pentixafor; and [68Ga]Ga-NODAGA-Exendin-4 from different batches that were prepared for clinical use. Results: In the assessed samples, HEPES could not be detected by the TLC method that was described in Ph. Eur. within 4 min incubation in an iodine-saturated chamber. Our developed HPLC method showed excellent linearity between 3 and 100 µg/mL for HEPES, with a correlation coefficient (R2) for calibration curves that was equal to 0.999, coefficients of variation (CV%) < 2%, and percent deviation value of bias from 100% to 5%, in accordance with acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed and the limit-of-quantification (LOQ) was 3 µg/mL, confirming the high sensitivity of the method. The amount of HEPES that was detected with our developed HPLC method in the tested [68Ga]Ga-radiopharmaceuticals resulted well below the Ph. Eur. limit, especially for [68Ga]Ga-NODAGA-Exendin-4. Conclusions: The TLC method that is described in Ph. Eur. to assess residual HEPES in [68Ga]-based radiopharmaceuticals may not be sufficiently sensitive and thus unsuitable for QC release. Our new HPLC method was sensitive, quantitative, reproducible, and rapid for QCs, allowing us to exactly determine the residual HEPES amount in [68Ga]Ga-radiopharmaceuticals for safe patient administration.

Development and Validation of an Analytical HPLC Method to Assess Chemical and Radiochemical Purity of [68Ga]Ga-NODAGA-Exendin-4 Produced by a Fully Automated Method.

Background: Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in pancreatic islets, especially in ß-cells, and highly expressed in human insulinomas and gastrinomas. In recent years several GLP-1R-avid radioligands have been developed to image insulin-secreting tumors or to provide a tentative quantitative in vivo biomarker of pancreatic ß-cell mass. Exendin-4, a 39-amino acid peptide with high binding affinity to GLP-1R, has been labeled with Ga-68 for imaging with positron emission tomography (PET). Preparation conditions may influence the quality and in vivo behavior of tracers. Starting from a published synthesis and quality controls (QCs) procedure, we have developed and validated a new rapid and simple UV-Radio-HPLC method to test the chemical and radiochemical purity of [68Ga]Ga-NODAGA-exendin-4, to be used in the clinical routine. Methods: Ga-68 was obtained from a 68Ge/68Ga Generator (GalliaPharma®) and purified using a cationic-exchange cartridge on an automated synthesis module (Scintomics GRP®). NODAGA-exendin-4 contained in the reactor (10 µg) was reconstituted with HEPES and ascorbic acid. The reaction mixture was incubated at 100 °C. The product was purified through HLB cartridge, diluted, and sterilized. To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document released by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. Therefore, a range of concentrations of Ga-NODAGA-exendin-4, NODAGA-exendin-4 (5, 4, 3.125, 1.25, 1, and 0.75 µg/mL) and [68Ga]Ga-NODAGA-exendin-4 were analyzed. To validate the entire production process, three consecutive batches of [68Ga]Ga-NODAGA-exendin-4 were tested. Results: Excellent linearity was found between 5-0.75 µg/mL for both the analytes (NODAGA-exendin-4 and 68Ga-NODAGA-exendin-4), with a correlation coefficient (R2) for calibration curves equal to 0.999, average coefficients of variation (CV%) < 2% (0.45% and 0.39%) and average per cent deviation value of bias from 100%, of 0.06% and 0.04%, respectively. The calibration curve for the determination of [68Ga]Ga-NODAGA-exendin-4 was linear with a R2 of 0.993 and CV% < 2% (1.97%), in accordance to acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed using 10 µg of peptide. The mean radiochemical yield was 45 ± 2.4% in all the three validation batches of [68Ga]Ga-NODAGA-exendin-4. The radiochemical purity of [68Ga]Ga-NODAGA-exendin-4 was >95% (97.05%, 95.75% and 96.15%) in all the three batches. Conclusions: The developed UV-Radio-HPLC method to assess the radiochemical and chemical purity of [68Ga]Ga-NODAGA-exendin-4 is rapid, accurate and reproducible like its fully automated production. It allows the routine use of this PET tracer as a diagnostic tool for PET imaging of GLP-1R expression in vivo, ensuring patient safety.

Scale down and optimized automated production of [68Ga]68Ga-DOTA-ECL1i PET tracer targeting CCR2 expression.

BACKGROUND: Recently it has been identified a short peptide that showed allosteric antagonism against C-C motif chemokine receptor 2 (CCR2) expressed on inflammatory monocyte and macrophages. A 7-D-amino acid peptidic CCR2 inhibitor called extracellular loop 1 inverso (ECL1i), d(LGTFLKC) has been identified and labeled to obtain a new probe for positron emission tomography in pulmonary fibrosis, heart injury, abdominal aortic aneurysm inflammation, atherosclerosis, head and neck cancer. Our goal was to develop, optimize and validate an automated synthesis method for [68Ga]68Ga-DOTA-ECL1i to make it available for a broader community. The synthesis of [68Ga]68Ga-DOTA-ECL1i was done using the Scintomics GRP® module with the already estabilished synthesis template for [68Ga]68Ga-DOTATOC/[68Ga]68Ga-PSMA. The radiopharmaceutical production was optimized scaling down the amount of DOTA-ECL1i (from 50 to 10 µg), evaluating synthesis efficiency and relevant quality control parameters in accordance with the European Pharmacopeia. RESULTS: Best results were yielded with 20 µg DOTA-ECL1i and then the process validation was carried out by producing three different batches on three different days obtaining an optimal radiochemical yield (66.69%) as well as radiochemical purity (100%) and molar activity (45.41 GBq/µmol). CONCLUSIONS: [68Ga]68Ga-DOTA-ECL1i was successfully synthesized and it is, thus, available for multi-dose application in clinical settings.

Validation of the HPLC Analytical Method for the Determination of Chemical and Radiochemical Purity of Ga-68-DOTATATE.

Purpose of the Study: Ga-68-DOTA-peptides targeting somatostatin receptors have been assessed as a valuable tool in neuroendocrine tumors imaging using positron emission tomography (PET). A new selective and sensitive high-pressure liquid chromatography (HPLC) method was developed for determining chemical and radiochemical purity of Ga-68-DOTATATE (PET) tracer. The identification of peaks was achieved on a symmetry C18 column 3 µm 120Å (3.0 mm × 150 mm spherical particles) using (A) water + 0.1% trifluoroacetic acid (TFA) and (B) acetonitrile + 0.1% TFA, as the mobile phases at a flow rate of 0.600 mL/min and monitored at 220 nm. The run time was 16 min. Materials and Methods: The method was validated to fulfill International Conference on Harmonization requirements and EDQM guidelines, and it included specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision. Results: The calibration curve was linear over the concentration range from 0.5 to 3 µg/ml, with a correlation coefficient (r2) equal to 0.999, average coefficient of variation (CV%) 2%, and average bias% did not deviate more than 5% for all concentrations. The LOD and LOQ for DOTATATE were 0.5 and 0.1 µg/mL, respectively. The method was considered precise, obtaining coefficients of variation between 0.22% and 0.52% for intraday and 0.20% and 0.61% for interday precision. Accuracy of method was confirmed with average bias% that did not deviate more than 5% for all concentrations. Conclusion: All results were acceptable and this confirmed that the method is suitable for its intended use in routine quality control of Ga-68-DOTATATE to guarantee the high quality of the finished product before release.

High Prognostic Value of 68Ga-PSMA PET/CT in Renal Cell Carcinoma and Association with PSMA Expression Assessed by Immunohistochemistry.

In oligo-metastatic renal cell carcinoma (RCC), neither computed tomography (CT) nor bone scan is sensitive enough to detect small tumor deposits hampering early treatment and potential cure. Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein expressed in the neo-vasculature of numerous malignant neoplasms, including RCC, that can be targeted by positron emission tomography (PET) using PSMA-targeting radioligands. Our aim was to investigate whether PSMA-expression patterns of renal cancer in the primary tumor or metastatic lesions on immunohistochemistry (IHC) are associated with PET/CT findings using [68Ga]-PSMA-HBED-CC (PSMA-PET/CT). We then analyzed the predictive and prognostic role of the PSMA-PET/CT signal. In this retrospective single-center study we included patients with renal cancer submitted to PSMA-PET/CT for staging or restaging, with tumor specimens available for PSMA-IHC. Clinical information (age, tumor type, and grade) and IHC results from the primary tumor or metastases were collected. The intensity of PSMA expression at IHC was scored into four categories: 0: none; 1: weak; 2: moderate; 3: strong. PSMA expression was also graded according to the proportion of vessels involved (PSMA%) into four categories: 0: none; 1: 1-25%; 2: 25-50%; 3: >50%. The intensity of PSMA expression and PSMA% were combined in a three-grade score: 0-2 absent or mildly positive, 3-4 moderately positive, and 5-6 strongly positive. PSMA scores were used for correlation with PSMA-PET/CT results. Results: IHC and PET scans were available for the analysis in 26 patients (22 ccRCC, 2 papillary RCC, 1 chromophobe, 1 "not otherwise specified" RCC). PSMA-PET/CT was positive in 17 (65%) and negative in 9 patients (35%). The mean and median SUVmax in the target lesion were 34.1 and 24.9, respectively. Reporter agreement was very high for both distant metastasis location and local recurrence (kappa 1, 100%). PSMA-PET detected more lesions than conventional imaging and revealed unknown metastases in 4 patients. Bone involvement, extension, and lesion number were greater than in the CT scan (median lesion number on PET/CT 3.5). The IHC PSMA score was concordant in primary tumors and metastases. All positive PSMA-PET/CT results (15/22 ccRCC, 1 papillary cancer type II, and 1 chromofobe type) were revealed in tumors with strong or moderate PSMA combined scores (3-4 and 5-6). In ccRCC tissue samples, PSMA expression was strong to moderate in 20/22 cases. The SUVmax values correlated to the intensity of PSMA expression which were assessed using IHC (p = 0.01), especially in the ccRCC subgroup (p = 0.009). Median survival was significantly higher in patients with negative PSMA-PET/CT (48 months) compared to patients with a positive scan (24 months, p= 0.001). SUVmax ≥ 7.4 provides discrimination of patients with a poor prognosis. Results of PSMA-PET/CT changed treatment planning. Conclusions: in renal cancer, positive PSMA-PET/CT is strongly correlated to the intensity of PSMA expression on immunohistochemistry in both ccRCC and chromophobe cancer. PSMA-PET/CT signal predicts a poor prognosis confirming its potential as an aggressiveness biomarker and providing paramount additional information influencing patient management.

Lymph node staging with 68Ga-PSMA PET in patients with intermediate and high-risk prostate cancer suitable for radical prostatectomy managed in a prostate cancer unit.

BACKGROUND: Prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT) is coming up as a superior imaging tool for prostate cancer (PCa). However, its use in primary staging is still debated. The aim of this study was to assess accuracy of 68Ga-PSMA PET/CT in staging patients with intermediate and high risk PCa candidates to radical prostatectomy managed in the Prostate Cancer Unit of our institution. METHODS: We retrospectively evaluated patients with biopsy-proven PCa staged through PSMA PET/CT before undergoing radical prostatectomy (RP) with extended pelvic lymph node dissection (ePLND). PET findings were categorized with respect to primary tumor (T), nodal (N) and distant metastasis (M). We analyzed the correspondence between PSMA PET/CT and final histopathological examination. RESULTS: We evaluated 42 men with high and intermediate risk PCa submitted to RP with ePLND. Mean age was 65.5 years (range, 49-76 years) and median preoperative prostate-specific antigen (PSA) was 13 ng/mL (IQR, 8.1-20 ng/mL). Patients in the high-risk group were 23 (54.7%), and the remainders were in the intermediate risk group. The mean risk of lymph node involvement (LNI) using the Memorial Sloan Kettering Cancer Center (MSKCC)-nomogram was 20%. The most common International Society of Urological Pathology (ISUP) grade was 3 (26.19%) after prostate biopsy. PSMA PET/CT showed focal prostatic uptake in 28 patients [mean value of maximum standardized uptake value (SUVmax) 18.5] and detected pelvic lymph node metastases in 6 cases (14.3%) with a median value of SUVmax 4.5 (IQR, 2-6.9). Histopathological examination detected lymph node metastases in seven patients (16.6%). In the only patient with negative PSMA PET/CT pathology revealed the presence of micrometastasis. After histopathological confirmation, sensitivity, specificity, positive and negative predictive values of pre-operative 68Ga-PSMA PET/CT were 85.7%, 100%, 100% and 97%, respectively. CONCLUSIONS: In our series, 68Ga-PSMA PET/CT holds high overall diagnostic value for lymph node staging in patients with intermediate and high risk PCa. Accuracy may depend on lymph node size.

Feasibility of a Scale-down Production of [68Ga]Ga-NODAGA-Exendin-4 in a Hospital Based Radiopharmacy.

BACKGROUND: Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in ß-cells, but it is highly expressed in human insulinomas and gastrinomas. Several GLP-1 receptor- avid radioligands have been developed to image insulin-secreting tumors or to provide a quantitative in vivo biomarker of pancreatic ß-cell mass. Exendin-4 is a high affinity ligand of the GLP1- R, which is a candidate for being labeled with a PET isotope and used for imaging purposes. OBJECTIVE: Here, we report the development and validation results of a semi-manual procedure to label [Lys40,Nle14(Ahx-NODAGA)NH2]exendin-4, with Ga-68. METHODS: A68Ge/68Ga Generator (GalliaPharma®, Eckert and Ziegler) was eluted with 0.1M HCl on an automated synthesis module (Scintomics GRP®). The peptide contained in the kit vial (Radioisotope Center POLATOM) in different amounts (10-20-30 µg) was reconstituted with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethansulfonic acid (HEPES) solution and 68GaCl3 (400-900 MBq), followed by 10 min incubation at 95°C. The reaction solution was then purified through an Oasis HLB column. The radiopharmaceutical product was tested for quality controls (CQs) in accordance with the European Pharmacopoeia standards. RESULTS: The synthesis of [68Ga]Ga-NODAGA-Exendin-4 provided optimal results with 10 µg of peptide, getting the best radiochemical yield (23.53 ± 2.4%), molar activity (100 GBq/µmol) and radiochemical purity (91.69%). CONCLUSION: The study developed an imaging tool [68Ga]Ga-NODAGA-Exendin-4, avoiding pharmacological effects of exendin-4, for the clinical community.

Performance and long-term consistency of five Galliform 68Ge/68Ga generators used for clinical Ga-68 preparations over a 4 year period.

BACKGROUND: Gallium-68 is a positron emitter for PET applications that can be produced without cyclotron by a germanium (Ge-68) chloride/gallium (Ga-68) chloride generator. Short half-life (67.71 min) of Ga-68, matching pharmacokinetic properties of small biomolecules, facilitates isotope utilization in compounding radiopharmaceuticals for PET imaging. The increasing cost of good manufacturing practice-compliant generators has strengthened the need for radionuclide efficient use by planning specific radiopharmaceutical sessions during the week, careful maintenance of the generator and achievement of high labeling yield and radiochemical purity (RCP) of the radiolabeled molecules. METHODS: The aim of this study was to evaluate the annual performance of five consecutive 68Ge/68Ga generators used for small-scale preparations of 68Ga-radiopharmaceuticals. To assess the long-term efficiency of isotope production we measured the weekly elution yield. To assess process efficiency we measured elution yield, labeling yield and RCP of four radiopharmaceutical preparations (68Ga-DOTATOC, 68Ga-PSMA-HBED-CC, 68Ga-PENTIXAFOR and 68Ga-DOTATATE). RESULTS: The annual mean elution yield of the generators was 74.7%, higher than that indicated by the manufacturer, and it never went below 65%. The Ge-68 level in the final products was under the detection limits in all the produced batches (mean value 0.0000048%). The RCP of radiopharmaceuticals determined by high-performance liquid chromatography was 98 ± 0.22%. The mean yield of radiolabelling was 64.68, 68.71, 57 and 63.68% for 68Ga-DOTATOC, 68Ga-PSMA-HBED-CC, 68GaPENTIXAFOR and 68Ga-DOTATATE. CONCLUSION: The ability to prepare in the hospital radiopharmacy high-purity and pharmaceutically acceptable 68Ga-radiolabeled probes on a routine basis facilitates patient access to precision imaging for clinical and research aims.

A Rapid and Specific HPLC Method to Determine Chemical and Radiochemical Purity of [68Ga]Ga-DOTA-Pentixafor (PET) Tracer: Development and Validation.

BACKGROUND: Due to its overexpression in a variety of tumor types, the chemokine receptor 4 (CXCR4) represents a highly relevant diagnostic and therapeutic target in nuclear oncology. Recently, [68Ga]Ga-DOTA-Pentixafor has emerged as an excellent imaging agent for positron emission tomography (PET) of CXCR4 expression in vivo. Preparation conditions may influence the quality and in vivo behaviour of this tracer and no standard procedure for the quality controls (QCs) is available. OBJECTIVE: The developed analytical test method was validated because a specific monograph in the Pharmacopoeia is not available for [68Ga]Ga-DOTA-Pentixafor. METHOD: A stepwise approach was used based on the quality by design (QbD) concept of the ICH Q2 (R1) and Q8 (Pharmaceutical Development) guidelines in accordance with the regulations and requirements of EANM, SNM, IAEA and WHO. RESULTS: The purity and quality of the radiopharmaceutical obtained according to the proposed method were found to be high enough to safely administrate it to patients. Excellent linearity was found between 0.5 and 4 µg/mL, with a correlation coefficient (r2) for calibration curves being equal to 0.999, the average coefficient of variation (CV%) < 2% and average bias% that did not deviate more than 5% for all concentrations. CONCLUSION: This study developed a new rapid and simple HPLC method of analysis for the routine QCs of [68Ga]Ga-DOTA-Pentixafor to guarantee the high quality of the finished product before release.

18F-FDG and 68GA-Prostate-Specific Membrane Antigen PET/CT Perform Better Than CT Alone in Restaging Papillary Renal Cell Carcinoma Recurrence.

ABSTRACT: Renal cell carcinoma (RCC) shows variable FDG uptake; recently, PET/CT with prostate-specific membrane antigen (PSMA)-target radiotracers was demonstrated to be a promising tool in staging and restaging of RCC patients. We describe the case of a 77-year-old man with a lung metastasis of papillary RCC missed by CT scan who successfully underwent [18F]FDG PET/CT restaging. Targeted therapy with sunitinib was administered. A [68Ga]PSMA PET/CT performed during follow-up demonstrated, among the already known lesions, also a bone marrow metastasis, missed by previous CT scans. This case demonstrates that PET/CT molecular imaging with [18F]FDG and [68Ga]PSMA is superior to conventional imaging in RCC restaging and in assessing therapy response.

Synthesis, validation and quality controls of [68Ga]-DOTA-Pentixafor for PET imaging of chemokine receptor CXCR4 expression.

BACKGROUND: Chemokine receptor-4 (CXCR4) is involved in tumor growth and progression in several types of human cancer. Recently, [68Ga]-DOTA-Pentixafor has been assessed as an excellent imaging probe targeting CXCR4-expression using positron emission tomography (PET). Here we report on the entire production cycle of [68Ga]-DOTA-Pentixafor, including quality control development and process validation. METHODS: Synthesis of [68Ga]-DOTA-Pentixafor was validated via three independent and consecutive production runs using an automated synthesis system. All validation runs must pass the pre-set quality control (QC) limits. Validation was performed for established QC tests to ensure that methods were reproducible and reliable in routine use. Germanium-68 breakthrough was determined for each sample. Production yield was calculated for each synthesis to assess the performance and efficiency of the radiolabeling process. The quality of the final product was determined by ITLC and HPLC methods after each synthesis. RESULTS: The average ITLC-measured radiochemical purity was above 98.5% and HPLC-measured radiochemical purity was 99.86%, 99,83% and 100% in the three validation runs. Germanium breakthrough was 4.8*10-5%, 4.9*10-5% and 4.7*10-5% of total activity, far below the recommended level of 0.001%. Residual ethanol resulted 5.22%, 5.58% and 5.32%V/V; spot of HEPES impurity was not more intense than spot of reference solution (200µg/V). Endotoxin level resulted <17.5EU/ml. pH of the final product was 7 in all samples. CONCLUSION: [68Ga]-DOTA-Pentixafor fit requirements of the pre-set quality parameters of purity, efficacy and safety in the batches considered for this study  and fulfilled all the acceptance criteria for injectable radiopharmaceutical products. The results demonstrated a batch-to-batch reproducibility providing high radiochemical purity.

Validation of Quality Control Parameters of Cassette-Based Gallium-68-DOTA-Tyr3-Octreotate Synthesis.

PURPOSE OF THE STUDY: Gallium (Ga)-68-DOTA peptides targeting somatostatin receptors have been assessed as a valuable tool in neuroendocrine tumor imaging using positron emission tomography. However, at the moment, a specific monograph in the European Pharmacopoeia (Ph. Eur.) does exist only for Ga-68-edotreotide (DOTATOC) injection. Here, we report on the validation process of Ga-68-DOTA-Tyr3-octreotate (DOTATATE) cassette-based production and quality control (QC). MATERIALS AND METHODS: Preparation of Ga-68-DOTATATE was performed according to the current European Union-good manufacturing practices, the current good radiopharmacy practice, the Ph. Eur., and the guidelines on validation of analytical methods for radiopharmaceuticals. Process was validated via three consecutive production runs to ensure that the methods are reproducible and reliable in routine use. The QC tests for Ga-68-DOTATATE were radiochemical purity (RCP - high-pressure liquid chromatography [HPLC]), radiochemical impurities 68Ga3+ (HPLC and instant thin layer chromatography [ITLC]), chemical purity (HPLC and gas chromatography [GC]), pH (pH-strips), radionuclidic purity (principal γ-photon), germanium-breakthrough (68Ge-content), Ga-68 half-life (γ-ray spectrometry), and sterility/endotoxin assay. RESULTS: Radiolabeling procedure of Ga-68-DOTATATE fits all the applicable Ph. Eur. specifications. RCP measured via ITLC was >99% in the three validation batches. HPLC-measured RCP resulted 99.45%, 99.78%, and 99.75%. Germanium-breakthrough was far below the recommended level established in the Ph. Eur. Ga-68-DOTATOC injection (#2482). Residual ethanol tested with GC was less than 10%. All the batches were tested for endotoxin content, which always resulted lower than 17.5 EU/ml. All preparations passed the sterility tests. pH of the final product was 7 in all samples. CONCLUSION: Ga-68-DOTATATE fulfilled all the pre-set QCs and release criteria in the batches considered for this validation study. The results demonstrated a batch-to-batch reproducibility, ensuring that synthesis process leads to the expected final product in terms of yield, quality, reliability, safety, and efficacy.

SPECT/CT with 99mTc labelled heat-denatured erythrocyte to detect thoracic and abdominal splenosis.

Scintigraphy with 99mTc labelled heat-denatured erythrocyte (DRBC) allows non invasive diagnosis of heterotopic splenic tissue implantation (splenosis) following splenic trauma or surgery. Single-photon emission computed tomography (SPECT) combined with computed tomography (CT) improves diagnostic accuracy of planar imaging through a more precise localization of functional findings. We report about two cases of splenosis occurring many years after splenectomy. 99MTc-DCRBC scintigraphy was used for differential diagnosis of metasttic disease in one case and to assess an incidental finding at bone scan in the second one. SPECT/CT increased specificity of planar imaging, expecially revealing combined (thoracic and abdominal) splenosis.

Unknown SARS-CoV-2 pneumonia detected by PET/CT in patients with cancer.

INTRODUCTION: In January 2020, the coronavirus disease 2019 (COVID-19) outbreak in Italy necessitated rigorous application of more restrictive safety procedures in the management and treatment of patients with cancer to ensure patient and staff protection. Identification of respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was a challenge during the pandemic owing to a large number of asymptomatic or mildly symptomatic patients. METHODS: We report 5 patients with unknown SARS-CoV-2 infection undergoing positron emission tomography (PET)/computed tomography (CT) with radiopharmaceuticals targeting different tumor processes: 18F-FDG, 18F-choline (FCH), and 68Ga-PSMA. RESULTS: In all patients, PET/CT showed increased tracer uptake in the lungs corresponding to CT findings of SARS-CoV-2 pneumonia. Quantitative assessment of tracer uptake showed more elevated values for the glucose analogue 18F-FDG (mean SUVmax 5.4) than for the other tracers (mean SUVmax 3.5). CONCLUSIONS: Our findings suggest that PET/CT is a sensitive modality to hypothesize SARS-CoV-2 pneumonia in patients with cancer, even when asymptomatic. More data are needed to verify the correlation among immune response to SARS-CoV-2 infection, clinical evolution, and PET results. Under the strict safety measures implemented at the PET center, the number of potentially SARS-CoV-2-positive patients undergoing PET/CT was very low (1.6%), and no staff member has been diagnosed with infection as of April 30, 2020.

Validation of in vitro labeling method for human use of heat-damage red blood cells to detect splenic tissue and hemocateretic function.

BACKGROUND AND AIM: Selective imaging of the splenic tissue is obtained with heat-damaged, or heat-denatured, red blood cells (RBCs) of the patient labeled with 99mTc in a variety of clinical scenarios. Aim of the study was to validate the process used for labelling heat-damaged red blood cells "totally in vitro", after blood sample collection, before re-inject labeled RBCs to the patient. Moreover, we assessed efficacy of the staff training programme in order to guarantee repeatibility and method standardization in the clinical routine. METHODS: The validation process of the labeling procedure was performed in three different patients during three consecutive days. After collection of a blood sample using a heparinized syringe, we isolated erythrocytes from other blood components by centrifugation and washing steps. Then, we added the stannous pyrophosphate (PYP) to the erythrocytes pellet, after  pH control. The 'pretinning' of RBCs was necessary to reduce Tc-99m once pertechnetate was entered them. After the labeling reaction with 130 MBq of 99mTc-pertechnetate, the erythrocytes were denatured in a water bath at a temperature of 49°-50°C, for 10 min. Radioactivity of blood aliquotes was measured with a dose calibrator and labelling efficiency (LE%) was determined. The labelling purity was measured using a gamma counter  and calculated using the formula: counts of pellet/counts of pellet+(counts of surnatant)*100.Training program was evaluated using a Learning Questionnaire (LQ). with a grading score from 6 ("") to 1 ("nothing") for each operator (n=3). RESULTS: We didn't observed the presence of macroaggregates during the entire process, until the final sample. The labelling efficiency resulted at very high values in the three consecutive measured aliquotes (mean value 73.67%) as well as the labelling purity (>95.22%). In our instituion, we use splenic imaging with labelled heat-damaged RBCs to detect ectopic spleen, splenosis, extramedullary hematopoiesis. We performed 3 procedures with  heat-damaged labeled RBCs with a  mean labelling efficiency 73.67%.Training and learning programmes were scored by key objective areas with a mean value of 5. CONCLUSIONS: Our in vitro labeling process of heat-damaged RBCs is simple and safe, providing a useful technique  easy to implement in clinical routine for splenic imaging Learning outcome of the training programme was scored as effective by all the operators with evidence of high-efficiency-reproducible procedure mantained over time.

Novel approach for quality assessment and improving diagnostic accuracy in cell-based infection imaging using 99mTc-HMPAO labeled leukocytes.

BACKGROUND AND AIM: Labeled leukocytes with 99mTc-HMPAO are routinely used for infection imaging. Although cell labeling with 99mTc-HMPAO represents an imaging probe to detect infection sites, the diagnostic efficiency of the probe is largely influenced by cell manipulation, multidisciplinary interventions (i.e., biologist, technicians) and available technology (i.e., SPECT, SPECT/CT). The aim of the study was to assess in vitro and in vivo accuracy of a comprehensive approach for quality assessment (QA) of all steps of the procedure. METHODS: Radiochemical purity (RCP), pH, labeling efficiency (LE) were measured in 320 procedures. White Cell Viability Factor (WVF) was determined in consecutive blood samples. Images (490 studies) were scored using a 5-point scale. Training program was evaluated using a Learning Questionnaire and a score system. RESULTS: Pre/post-labelling WVF was 0.99% (max value 1%) in all blood samples. LE (mean value 72%) and RCP (>80% until 55 minutes) yielded considerably high values. The vast majority of images were scored as diagnostic by three independent observer (90% with score ≥4). CONCLUSIONS: This method appears highly reproducible and easy to use in clinical routine for leukocyte labeling, especially when standardized training and total QA system are implemented.

Development and Validation of a High-Pressure Liquid Chromatography Method for the Determination of Chemical Purity and Radiochemical Purity of a [68Ga]-Labeled Glu-Urea-Lys(Ahx)-HBED-CC (Positron Emission Tomography) Tracer.

Background: Prostate-specific membrane antigen (PSMA) has gained high attention as a useful biomarker in the imaging evaluation of prostate cancer with positron emission tomography (PET) during recent years. [68Ga]-labeled Glu-urea-Lys(Ahx)-HBED-CC ([68Ga]-PSMA-HBED-CC) is a novel PSMA inhibitor radiotracer which has demonstrated its suitability in detecting prostate cancer. Preparation conditions may influence the quality and in vivo behavior of this tracer, and no standard procedure for the quality control (QC) is available. The aim of this study was to develop a new rapid and simple high-pressure liquid chromatography method of analysis for the routine QCs of [68Ga]-PSMA-HBED-CC to guarantee the high quality of the radiopharmaceutical product before release. Methods: A stepwise approach was used based on the quality by design concept of the International Conference of Harmonisation Q2 (R1) and Q8 (Pharmaceutical Development) guidelines in accordance with the regulations and requirements of European Association of Nuclear Medicine, Society of Nuclear Medicine, International Atomic Energy Agency, World Health Organization, and Italian Association of Nuclear Medicine and Molecular Imaging. The developed analytical test method was validated because a specific monograph in the pharmacopoeia is not available for [68Ga]-PSMA-HBED-CC. Results: The purity and quality of the radiopharmaceutical obtained according to the proposed method resulted high enough to safely administrate it to patients. An excellent linearity was found between 0.8 and 5 µg/mL, with a detection limit of 0.2 µg/mL. Assay imprecision (% CV) was <2%. Conclusions: The developed method to assess the radiochemical and chemical purity of [68Ga]-PSMA-HBED-CC is rapid, accurate, and reproducible, allowing routinely the use of this PET tracer as a diagnostic tool for imaging prostate cancer and also assuring patient safety.

Role of molecular imaging in the management of patients affected by inflammatory bowel disease: State-of-the-art.

AIM: To present the current state-of-the art of molecular imaging in the management of patients affected by inflammatory bowel disease (IBD). METHODS: A systematic review of the literature was performed in order to find important original articles on the role of molecular imaging in the management of patients affected by IBD. The search was updated until February 2016 and limited to articles in English. RESULTS: Fifty-five original articles were included in this review, highlighting the role of single photon emission tomography and positron emission tomography. CONCLUSION: To date, molecular imaging represents a useful tool to detect active disease in IBD. However, the available data need to be validated in prospective multicenter studies on larger patient samples.