[²²⁵Ac]Ac-PSMA Therapy in mCRPC: Early Efficacy and Biomarker Trends in a Selected Patient Population

1. Introduction

Metastatic castration-resistant prostate cancer (mCRPC) presents a significant therapeutic challenge, as it usually shows limited or no durable response to newer treatments such as novel antiandrogen agents, taxane-based chemotherapy, or Radium-223 (²²³Ra) [1,2]. In this context, radioligand therapy (RLT) has emerged as a promising alternative, with clinical studies such as the VISION trial demonstrating favorable outcomes for [¹⁷⁷Lu]Lu-PSMA-617, despite its associated resistance or recurrence rates [3,4,5,6]. These challenges have led researchers to explore the potential of alpha-emitting radionuclides, which offer higher energy transfer rates and shorter path lengths than beta-emitting agents, making them an attractive option for PSMA (Prostate-Specific Membrane Antigen)-targeted RLT in mCRPC [7].

Actinium-225 (²²⁵Ac), with an alpha particle energy range of 5.8–8.4 MeV and a half-life of 9.9 days, has been frequently used in TAT [8,9]. It is often paired with its daughter radionuclide, Bismuth-213, which has a short half-life of 46 minutes, making [²²⁵Ac]Ac-PSMA-617 a promising candidate for mCRPC treatment [10]. Since 2017, numerous reports have confirmed that [²²⁵Ac]Ac-PSMA-617 therapy induces significant declines in prostate-specific antigen (PSA) levels following treatment [11,12,13,14,15,16,17,18]. Furthermore, it is associated with improved progression-free survival (PFS), especially in patients who have previously been refractory to [¹⁷⁷Lu]Lu-PSMA-617. Given the promising yet varied clinical outcomes, a comprehensive clinical analysis is essential to provide a holistic understanding of treatment efficacy, patient outcomes, and associated toxicities. This analysis will offer valuable insights into the therapeutic benefit of [²²⁵Ac]Ac-PSMA-617.

In [²²⁵Ac]Ac-PSMA-617 therapy, the salivary gland is one of the organs at risk (OAR) due to physiological PSMA expression, leading to potential off-target uptake and xerostomia [19]. Quantifying this uptake is crucial for assessing toxicity, optimizing therapy, and evaluating the tumor sink effect, which occurs when tumor reduction alters biodistribution, increasing uptake in non-target tissues. Semi-quantitative PET analysis represents a pragmatic and essential approach for evaluating treatment outcomes and assessing therapeutic efficacy.

Despite encouraging preliminary results, the potential relationship of biomarkers, such as blood biomarkers and also total tumor volume (TTV), with treatment efficacy and overall survival (OS) remains largely unexplored and underreported. This study retrospectively evaluates the treatment efficacy and outcomes of [²²⁵Ac]Ac-PSMA-617 in patients with mCRPC, focusing on the correlation between patient treatment characteristics (e.g., PSA levels and TTV) and OS. Additionally, we investigate potential associations between baseline and post-treatment blood biomarker profiles and OS, an area not addressed in previous studies.

To contextualize and compare our findings, we conduct a systematic meta-analysis to consolidate existing data on the efficacy and safety of [²²⁵Ac]Ac-PSMA-617 therapy in mCRPC. By integrating outcomes from multiple cohorts, the meta-analysis enhances statistical power and offers a comprehensive evaluation of PSA response rates, progression-free survival (PFS), OS, and treatment-related toxicities, including xerostomia, renal dysfunction, and hematologic effects. Through the synthesis of both retrospective findings and pooled evidence, this study provides a robust, evidence-based perspective on therapeutic efficacy, safety, and predictive biomarkers informing future strategies for treatment optimization.

2. Results

2.1. Patients and Treatment Administration

A total of 12 patients with metastatic castration-resistant prostate cancer were selected who underwent [²²⁵Ac]Ac-PSMA-617 targeted alpha therapy (Table 1). The patients had received various prior treatments including local treatments, androgen receptor–targeted therapy (AR-TT), chemotherapy, and [²²³Ra]Ra-dichloride before initiation of [²²⁵Ac]Ac-PSMA-617 TAT. Local treatments included radical prostatectomy (67%), and radiotherapy (42%), both of which remain key in local/locally advanced disease management. Most patients had undergone multiple lines of AR-TT, including abiraterone (75%) and enzalutamide (83%). Recent analyses suggest that AR-TT inhibitors continue to contribute significantly to survival in mCRPC even in patients heavily pretreated by other modalities [20]. Chemotherapy was also widely used: docetaxel (67%), established as first-line chemotherapy in mCRPC [21,22], and cabazitaxel (42%), which showed efficacy after docetaxel failure [23,24]. Notably, one patient (8%) initially referred for [²²³Ra]Ra-dichloride therapy due to extensive osseous metastases demonstrated no response to the therapy. All patients received [¹⁷⁷Lu]Lu-PSMA-617 therapy following multidisciplinary Tumor Board approval, ensuring uniform eligibility criteria and therapeutic intent across the cohort, with a median of 4 cycles (range 2-8). The patients then were transitioned to [²²⁵Ac]Ac-PSMA-617 TAT. The distribution of [²²⁵Ac]Ac-PSMA-617 treatment cycles was as follows: three patients (25%) underwent five cycles, three patients (25%) received three cycles, and another three patients (25%) received two cycles. Additionally, two patients (17%) completed four cycles, and one patient (8%) received 6 cycles. The median injected radioactivity (injected activity (IA)) was 8 MBq, ranging from 5.0 MBq to 11.0 MBq (approximately IA= 10 kBq/kg).

Baseline serum biomarkers and hematologic parameters were assessed in mCRPC patients prior to administration of [¹⁷⁷Lu]Lu-PSMA-617 and [²²⁵Ac]Ac-PSMA-617 therapy. Table 2 summarizes an overview of laboratory values before treatment initiation.

Treatment cycles for each patient are illustrated using a swimmer plot (Figure 1), highlighting the number of [¹⁷⁷Lu]Lu-PSMA-617 and [²²⁵Ac]Ac-PSMA-617 administrations as well as intervals between therapies. Patients received 2 to 8 cycles of [¹⁷⁷Lu]Lu-PSMA-617 and 2 to 6 cycles of [²²⁵Ac]Ac-PSMA-617.

2.2. Safety

Safety evaluations demonstrated stable clinical and biochemical profiles following [²²⁵Ac]Ac-PSMA-617 TAT. No patient exhibited signs or symptoms of urinary tract obstruction, as confirmed by repetition or sequential pre-treatment dynamic renal scintigraphy using [^99mTc]Tc-MAG3.

Hematologic and renal biomarkers, including hemoglobin, white blood cell (WBC) count, platelet count, creatinine, estimated glomerular filtration rate (eGFR), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), remained stable throughout therapy. Comparative analyses between baseline and follow-up values showed no statistically significant changes (Figure 2), suggesting minimal hematologic and renal toxicity associated with [²²⁵Ac]Ac-PSMA-617.

PSA levels were analyzed pre and post RLT and TAT to assess treatment response (Figure 3). In the non-response group, PSA significantly increased from pre- to post- [²²⁵Ac]Ac-PSMA-617 TAT (***p < 0.001), whereas in the response group, PSA significantly decreased (**p < 0.01). Comparisons at other time points, including pre- and post-[¹⁷⁷Lu]Lu-PSMA-617 RLT, did not show statistically significant changes.

Functional status, assessed by the Barthel Index, which measures the patients’ ability to perform activities of daily living, remained preserved with a median score of 100 (100 points: the individual is completely independent in all 10 Activities of Daily Living (ADLs) and no physical assistance is required for any daily activity.) before and after TAT. This indicates that patients maintained full independence in daily living, underscoring the favorable tolerability of therapy.

Regarding salivary gland uptake (SGU), in our cohort, PSMA-SGU decreased by −63.7% after [²²⁵Ac]Ac-PSMA-617, closely matching the findings of Feuerecker et al. [25], who reported a −61% reduction in PSMA-SGU after one cycle of [²²⁵Ac]Ac-PSMA-617. Similarly, SUVmean fell by −45.5% in our data versus −42.4% in the Feuerecker series, supporting a reproducible, large functional impairment of salivary glands after targeted alpha therapy.

2.3. PSA-Based Biochemical Response After [²²⁵Ac]Ac-PSMA-617 Therapy

Figure 4 presents waterfall plots showing individual patients’ changes in PSA levels after the first cycle (panel I) and after the last cycle (panel II) of [²²⁵Ac]Ac-PSMA-617.

After the first cycle, 9 out of 12 patients (75%) exhibited a measurable decline in PSA levels, including 6 patients (50%) with reductions exceeding 30% and 3 patients (25%) surpassing the 50% threshold. Conversely, 3 patients (25%) showed either stable PSA levels or biochemical progression.

At the end of treatment, 8 patients (67%) maintained PSA reductions, with 5 patients (42%) showing > 50% decreases. Notably, 6 out of 7 patients with initial PSA declines ≥ 30% sustained their responses, suggesting durability of biochemical benefit.

The PSA kinetics offer critical insight into early therapeutic efficacy and align with molecular imaging outcomes (see Section 2.4), reinforcing the potential of TAT for disease control in patients refractory to prior [¹⁷⁷Lu]Lu-PSMA therapy.

2.4. Molecular Imaging Response Following [²²⁵Ac]Ac-PSMA-617 Therapy

Molecular treatment response was evaluated using [⁶⁸Ga]Ga-PSMA-11 PET/CT, focusing on changes in total tumor volume (TTV) and tracer uptake. Among the cohort:

• (7/12) exhibited partial molecular response.
• (3/12) demonstrated stable disease.
• (2/12) showed progressive disease.

Across the 32 treatment cycles administered:

• (24/32) resulted in partial molecular response,
• (4/32) in stable disease,
• (4/32) in progression.

These imaging outcomes mirrored PSA trends, reinforcing the concordance between biochemical and radiographic response. The disease control rate (DCR) was calculated as:

• 85.7% per cycle,
• 83.4% per patient, reflecting partial response or stable disease in 10 of 12 patients.

Figure 5 and Figure 6 illustrate representative [⁶⁸Ga]Ga-PSMA-11 PET/CT images, including individuals previously refractory to [¹⁷⁷Lu]Lu-PSMA-617 therapy who demonstrated near-complete molecular and biochemical responses after three cycles of TAT. Others who had progressed after treatment pauses showed disease stabilization following [²²⁵Ac]Ac-PSMA-617 TAT.

2.5. Survival Outcomes

Progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan-Meier methodology (Figure 7). The observed survival metrics were:

• Median PFS: 27 weeks (95% CI: 23–31 weeks)
• Median OS: 53 weeks (95% CI: 36–70 weeks)
• Probability of OS >100 weeks: 16%

Univariate analysis revealed that elevated PSA, ALP, and LDH, along with low hemoglobin, platelet count, and creatinine, were associated with poorer OS (Table 3).

Multivariate modeling (Table 4) further identified key predictors:

• LDH (p = 0.0024) and WBC count (p = 0.0013) were independently associated with reduced OS.
• Conversely, higher creatinine (p = 0.0058), hemoglobin (p = 0.0141), and eGFR (p < 0.0001) were linked to improved survival outcomes.

These findings suggest that integrating blood biomarkers alongside PSA and imaging parameters can offer enhanced prognostic insight and support tailored therapeutic strategies in mCRPC.

Kaplan-Meier plots showing (a) progression-free survival (PFS) and (b) overall survival (OS) in mCRPC patients treated with [²²⁵Ac]Ac-PSMA-617 TAT.

Median PFS was 27 weeks (95% CI: 23–31 weeks).

Median OS was 53 weeks (95% CI: 36–70 weeks).

Survival probabilities were:

-

31% for PFS >30 weeks

-

50% for OS >50 weeks

-

16% for OS >100 weeks

The dark lines represent Kaplan-Meier survival curves, while lighter lines indicate the upper and lower bounds of the 95% confidence intervals.

2.6. Meta-Analysis: Contextualizing [²²⁵Ac]Ac-PSMA-617 Efficacy and Safety Across Studies

To place our retrospective findings within a broader clinical framework, we conducted a meta-analysis of 8 published studies encompassing 675 patients treated with [²²⁵Ac]Ac-PSMA-617 for metastatic castration-resistant prostate cancer (mCRPC) [7,10,12,13,14,15,16,17]. This synthesis aimed not to replicate prior results, but to validate and contrast our cohort outcomes against pooled international data, particularly regarding PSA response rates. A meta-analysis of median PFS and OS was not performed because hazard ratios (and their associated variances) were not available in the pooled data. Figure 8 compares the outcomes of our retrospective cohort (n = 12) against pooled estimates from a meta-analysis (n = 675) across eight clinical measures. The meta-analytic pooled effect size was 0.56 (95% CI: 0.41–0.71), with a statistically significant overall effect (z = 7.50, p < 0.0001), indicating a meaningful proportion of events across studies.

Figure 9 presents the pooled PSA response rate for patients achieving a > 50% decline (ES = 0.60, 95% CI: 0.51–0.70) and includes a funnel plot showing no evidence of publication bias (Begg’s test p = 0.811, Egger’s test p = 0.782). The funnel plot illustrates that studies with larger sample sizes tend to have smaller standard errors and appear higher on the plot.

Pooled Efficacy Outcome

The meta-analysis revealed a pooled PSA response rate of 0.60 (≥ 50% decline). The result supports the clinical relevance of [²²⁵Ac]Ac-PSMA-617, particularly in heavily pretreated or refractory to [¹⁷⁷Lu]Lu-PSMA-617 therapy mCRPC patients. The pooled PSA response rate aligns with our findings, though our cohort’s lower response may reflect more advanced disease burden prior [¹⁷⁷Lu]Lu-PSMA RLT.

Study-Level Insights

Table 5 presents current strategies for the prevention of TAT-induced xerostomia, while Table 6 summarizes individual study outcomes. Notably, the study by Alan-Selcuk et al. [14] was excluded due to statistical heterogeneity, reinforcing the importance of methodological consistency in pooled analyses.

4. Materials and Methods

4.1. Patients and Ethics

This retrospective study included consecutive patients with mCRPC who received [²²⁵Ac]Ac-PSMA-617 therapy after demonstrating resistance to prior treatments. Patients were considered treatment refractory if they no longer responded to androgen deprivation therapy (ADT), chemotherapy, radiotherapy, and multiple cycles of [¹⁷⁷Lu]Lu-PSMA-617 RLT (median [min-max]: 3 [2,3,4,5,6,7,8] cycles). Other resistance was determined by a sustained increase in serum PSA levels, progression of existing disease, or the appearance of new metastases despite castrate testosterone levels. To be included in the analysis, patients had to have complete follow-up data available at least 6 weeks after each cycle. Eligibility for [²²⁵Ac]Ac-PSMA-617 TAT required progression on or resistance to [¹⁷⁷Lu]Lu-PSMA-617 RLT, or the presence of extensive metastasis despite prior treatments.

Treatment decisions were approved by a tumor board after a comprehensive review of each patient’s medical history and current status. The study was conducted at the Department of Nuclear Medicine, Philipps University Hospital, Marburg, Germany, between July 2021 and January 2024.

Treatment was administered on a compassionate use basis in accordance with the German Medical Products Act (Arzneimittelgesetz – AMG §13 (2b)). To be eligible, patients must have had disease progression despite other treatments such as chemotherapy, radiotherapy, or second-generation anti-androgen therapy, or the tumor board determined that these interventions were contraindicated. All patients were informed of the experimental nature of [²²⁵Ac]Ac-PSMA-617 therapy and signed consent forms.

This retrospective study was approved by the Ethics Committee of the School of Medicine at Philipps University of Marburg (Ethics vote reference number: 24-109 RS, approval date: 25/04/2024).

4.2. [²²⁵Ac]Ac-PSMA-617 Administration

The PSMA-617 peptide was labeled with [²²⁵Ac]AcCl₃ (ITM Isotope Technologies, Garching, Germany), and after radiosynthesis and quality control, it was administered intravenously. The administered activity was 100 kBq/kg of [²²⁵Ac]Ac-PSMA-617, an activity proposed by Kratochwil et al. [11] as a balanced amount to enhance therapeutic efficacy while minimizing adverse effects. Details of the labeling process are described in Appendix A.

To potentially reduce salivary gland perfusion, radiopharmaceutical uptake, and toxicity, patients’ salivary glands were covered with cool packs 30 minutes before and up to 4 hours after radiopharmaceutical administration. In the event of post-injection nausea, patients were treated with Granisetron.

In compliance with the German Radiation Protection Act, [²²⁵Ac]Ac-PSMA-617 therapy was administered on an inpatient basis, with patients remaining in the nuclear medicine ward for at least 48 hours post-administration before being discharged when deemed medically appropriate. A minimum of two cycles of therapy were scheduled, administered at 6-week intervals. Subsequent dosage, cycle number, and interval were adjusted individually based on the disease burden, patient condition, and logistical factors.

4.3. Safety, Efficacy, and Treatment Outcome Evaluation

4.3.1. Safety Monitoring

Safety was evaluated based on laboratory data, including blood biomarkers (hemoglobin, white blood cell count, platelet count, creatinine level, estimated glomerular filtration rate (eGFR), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and renal function tests). These were assessed at baseline, after each subsequent cycle, and during follow-up. Bone marrow, and renal toxicities—common side effects of this treatment modality—were graded using laboratory data. Renal toxicity was graded based on changes in eGFR, and hematological adverse effects (e.g., anemia, leukopenia, thrombocytopenia) were also tracked.

4.3.2. Salivary Gland Uptake

Quantitative parameters, including SUV_max and SUV_mean, were extracted for the salivary glands as organs at risk (OAR) through segmentation of [⁶⁸Ga]Ga-PSMA-11 PET/CT images. Segmentation was performed using HERMES software (HERMES Medical Solutions, Sweden), which enabled precise delineation of salivary glands for accurate measurement and analysis.

4.3.3. Renal Function

Each patient underwent dynamic renal scintigraphy using [^99mTc]Tc-MAG3 prior to each cycle to detect any potential urinary tract obstruction [35].

4.3.4. Functional Status

The Barthel-Index was calculated to evaluate the patient’s functional status at baseline and after treatment. This index assesses a patient’s ability to perform daily activities, with scores ranging from zero (complete dependence) to 100 (complete independence) [36]. The index was administered by trained clinicians at our institution.

4.3.5. Efficacy and Treatment Outcome Evaluation

It was characterized using four endpoints:

• Biochemical response was defined based on the percentage change in PSA from baseline, with the criteria of the Prostate Cancer Working Group 3 (PCWG3) guidelines [37]. A biochemical response is defined as a decline in PSA levels after treatment. In this study, we considered a decline > 30% in PSA level as a positive sign of therapeutic efficacy. A partial response was defined as a decline of > 50%, while biochemical disease progression was defined as an increase of > 25% in PSA from baseline.
• Molecular tumor response after every two cycles of TAT was categorized based on changes from baseline in TTV as reflected by [⁶⁸Ga]Ga-PSMA-11 uptake values extracted from PET/CT images, which were most often acquired after every 2 cycles of TAT:
  • Molecular partial response: > 30% decrease.
  • Molecular progressive disease: > 20% increase.
  • Molecular stable disease: ≤ 30% change, (changes < 30% decrease or < 20% increase)

TTV segmentation was performed using HERMES software. Disease progression was evaluated using Response Evaluation Criteria in Solid Tumors (PERCIST 1.0) [38].

• Disease control rate (DCR) was calculated by dividing the number of patients exhibiting a partial response or stable disease on molecular imaging by the total number of patients.
• PFS was defined as the interval between the initial TAT administration and the first documentation of biochemical or molecular disease progression on imaging, or the date of last contact (if no progression was observed). OS was defined as the interval between the first TAT administration and the patient’s death or the date of last clinical visit.

4.4. Meta-Analysis

A meta-analysis was conducted to assess the efficacy and safety of [²²⁵Ac]Ac-PSMA TAT in patients with mCRPC. A comprehensive clinical literature search was performed using PubMed, Scopus, and Web of Science databases to identify relevant studies published up to 2024. Inclusion criteria encompassed prospective and retrospective studies reporting at least one clinical outcome, including OS, PFS, PSA response, and treatment-related toxicities such as xerostomia, hematologic toxicity, and renal toxicity.

Using meta-analysis, the pooled PSA response rate (PSA decline < 50%) across eligible studies was estimated. Relevant data were extracted from each study, including the number of patients achieving a PSA response as defined by study-specific criteria. A random-effects meta-analysis and 95% confidence intervals were conducted using the Restricted Maximum Likelihood (REML) estimation method to account for potential heterogeneity among studies. The effect size was defined as the proportion of events (prop), with standard errors (se) provided by each study.

Heterogeneity was assessed using the following metrics:

• τ² (tau-squared) = 0.0055, indicating low between-study variance.
• I² = 29.76%, suggesting moderate heterogeneity.
• H² = 1.42, indicating the presence of some inconsistency across studies.

The test for homogeneity yielded a non-significant result (Q(1) = 1.42, p < 0.10), suggesting that the observed heterogeneity is not statistically significant. The statistical significance of the overall effect was evaluated using the Z-test. All analyses were performed using STATA software, version 18 (StataCorp LLC, College Station, TX, USA).

4.5. Statistics

No formal sample size calculation was made for this retrospective observational study. PSA levels and other laboratory markers following [²²⁵Ac]Ac-PSMA-617 TAT were compared with baseline using a one-way analysis of variance (ANOVA). Kaplan-Meier survival curves were generated for both PFS and OS. Univariate analysis was performed to assess the association between hematologic biomarkers and OS. Each biomarker was analyzed individually to determine its potential prognostic significance. Threshold levels were established based on the median values obtained after two cycles of therapy. Statistical significance was determined using p-values, with a threshold set at p < 0.05. Following univariate analysis, a multivariate analysis was conducted to evaluate the independent impact of blood biomarkers on OS. Results were expressed as beta coefficients (β) to quantify the strength and direction of associations. All statistical analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, Boston, MA, USA).

5. Conclusions

This study regards TAT with [²²⁵Ac]Ac-PSMA-617 as a treatment option for mCRPC patients who had developed resistance to [¹⁷⁷Lu]Lu-PSMA-617 therapy. [⁶⁸Ga]Ga-PSMA-11 PET imaging and blood biomarkers may serve as useful predictors for assessing treatment efficacy and outcomes in mCRPC patients receiving [²²⁵Ac]Ac-PSMA-617 TAT. Although PSA level monitoring is a widely used approach to evaluate treatment response, incorporating TTV measurements and blood biomarkers could provide additional insights into therapeutic effectiveness. In addition, the findings in our patient population suggested that a > 50% decrease in PSA levels following the first two cycles of TAT can serve as a predictive marker for favorable treatment outcomes in subsequent cycles. Interestingly, pre-therapeutic biomarkers extracted from blood tests such as PSA, hemoglobin, creatinine, and platelet count may help as the prognostic biomarkers for survival outcomes and efficacy evaluation after TAT. Tumor uptake, biodistribution, and TTV are promising parameters for evaluating the efficacy and outcomes of TAT. The tumor sink effect could be considered in subsequent [²²⁵Ac]Ac-PSMA-617 therapy cycles, as tumor burden reduction may increase salivary gland uptake, potentially worsening toxicity, and optimizing therapy with semi-quantitative PET analysis can balance efficacy and safety. Post-treatment dosimetry and the estimation of absorbed doses for organs at risk following TAT present a novel challenge due to the suboptimal quality of post-treatment [²²⁵Ac]Ac-PSMA-617 SPECT and planar images. It is proposed that [⁶⁸Ga]Ga-PSMA-11 PET images may be employed as a predictor of absorbed dose for post-TAT dosimetry evaluation in organs at risk.

Appendix A.1. Radiopharmaceutical Preparation

Vipivotide tetraxetan (PSMA-617, 10µg/GBq) was added to 1 mL of 0.1M sodium ascorbate stock solution. The mixed solution was added into the vial containing ²²⁵AcCl₃ (ITM Isotope Technologies, Garching, Germany), then was shaken gently. Then, the vial was heated at 90 ˚C for 25 minutes. After cooling to room temperature, the [²²⁵Ac]Ac-PSMA-617 product was diluted with 5 mL of NaCl and filtered through a sterile filter.

Instant radio thin-layer chromatography (rTLC) was performed for quality control at room temperature by pipetting 5µL of the final product onto an iTLC-SA strip using Natrium-Citrate-buffer and an iTLC-SG strip using Ammonium acetate (MeOH-NH₄Ac) buffer. The radiochemical yield was determined using a TLC scanner Marita Star (Elysia Raytest) ≥ 95%, and the pH was 7–8. Additionally, bacterial endotoxin level was tested <10 EU/mL using he Endosafe nexgen-PTS (Charles River) before releasing for patient application.