Dual-tracer PET/CT Imaging using 18F-AV-45 and 18F-AV-1451 for the Diagnosis of Alzheimer's Disease

Summary

This protocol describes the standardized procedure for dual-tracer PET/CT imaging using ¹⁸F-AV-45 and ¹⁸F-AV-1451 in patients with suspected Alzheimer’s disease (AD). It includes patient preparation, tracer administration, image acquisition, and interpretation criteria, as well as representative imaging features of amyloid and tau pathology.

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by an insidious onset and gradual deterioration. Clinically, it manifests as cognitive decline and behavioral changes rather than only as generalized dementia. Currently, the diagnostic accuracy of AD remains limited due to the nonspecific clinical manifestations and the low specificity of conventional examinations such as neuropsychological assessments, electroencephalography (EEG), computed tomography (CT), and magnetic resonance imaging (MRI). Positron emission tomography/computed tomography (PET/CT) provides detailed structural and functional information along with specific molecular distributions, making it increasingly valuable for the diagnosis and research of AD. We performed PET/CT scans using the radiotracers ¹⁸F-AV-45 (florbetapir) and ¹⁸F-AV-1451 (flortaucipir). The key distinction between these tracers lies in their molecular targets: ¹⁸F-AV-45 binds to β-amyloid (Aβ), whereas ¹⁸F-AV-1451 specifically targets tau protein. This study elaborates on the protocol for the combined use of both tracers in AD diagnosis, encompassing patient preparation procedures, image acquisition techniques, interpretation standards, and the clinical significance of their joint application. This dual-tracer protocol provides a sensitive, comprehensive, and non-invasive method for detecting both amyloid and tau pathology, enhancing early diagnosis, disease staging, and research on disease progression.

Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a slow onset and progressive deterioration of cognitive function. Clinically, it presents with subjective cognitive decline (SCD), mild cognitive impairment (MCI), memory deficits, executive dysfunction, aphasia, and behavioral changes¹. The hallmark pathological features of AD include the deposition of β-amyloid (Aβ) plaques and the formation of neurofibrillary tangles composed of hyperphosphorylated tau protein. These pathological markers are key diagnostic criteria for AD and serve as primary molecular targets for imaging and biomarker studies. The prevalence of AD increases sharply with age. Globally, more than 50 million individuals are affected, and the number of dementia cases is expected to triple by 2050². AD is one of the leading causes of death in the elderly, posing an enormous socioeconomic burden. However, early detection of AD remains a global challenge³. In light of this, there is an urgent need to establish more precise early diagnostic strategies.

Cognitive function assessment is the preferred method for diagnosing AD dementia, but some scales lack consensus on optimal cutoff values and education-adjusted norms. Computed tomography (CT) and magnetic resonance imaging (MRI), as routine neuroimaging modalities, can be employed to detect cerebral atrophy or other structural alterations that may contribute to cognitive dysfunction. Despite the use of cognitive testing, CT, and MRI, early diagnosis remains challenging because structural imaging typically detects changes only in moderate to advanced stages and lacks disease specificity⁴. Positron emission tomography/computed tomography (PET/CT), as a non-invasive molecular imaging technique, enables semi-quantitative analysis of brain metabolism and protein deposition. The combined application of amyloid and tau PET tracers provides complementary information about disease pathophysiology^5,6,7,8.

The aberrant metabolism and deposition of Aβ represent the initiating event and central driving force in the pathological progression of AD. Under the influence of genetic factors (e.g., mutations in APP or PSEN1/2 genes) or age-related factors, the production of Aβ (particularly Aβ42) increases and/or its clearance decreases, leading to elevated concentrations. Soluble Aβ monomers initially aggregate into soluble oligomers, which are widely recognized as the primary neurotoxic species. These Aβ oligomers further polymerize into insoluble fibrils and ultimately deposit extracellularly in the brain parenchyma, forming senile plaques, one of the hallmark pathological features of AD brains^9,10. The deposition and neurotoxic effects of Aβ initiate downstream pathological cascades, notably the hyperphosphorylation of Tau protein leading to neurofibrillary tangles, as well as sustained neuroinflammation, collectively resulting in widespread neuronal loss and cerebral atrophy¹¹. The formation of neurofibrillary tangles due to hyperphosphorylation of tau protein is one of the pathological hallmarks of AD¹², with strong spatiotemporal correlations to the development of neurodegeneration and clinical symptoms. Tau protein, the most abundant microtubule-associated protein in the body, is widely distributed in both the peripheral and central nervous systems. Its pathological accumulation correlates strongly with cognitive impairment severity, making it a promising therapeutic target for AD patients.

According to the guidelines of the European Medicines Agency (EMA), measurements of β-amyloid (Aβ) peptides and total tau protein levels in cerebrospinal fluid (CSF) can assist in the diagnosis and therapeutic monitoring of Alzheimer’s disease (AD)^13,14. Although this method is relatively low-cost, lumbar puncture is invasive and may cause discomfort or complications. Plasma biomarkers^15,16, including Aβ42/40 ratio, total tau, and phosphorylated tau—offer a non-invasive and cost-effective alternative and are increasingly applied in clinical screening. However, PET imaging remains indispensable for visualizing the spatial distribution of amyloid and tau pathology in vivo, providing detailed topographic information^17,18 that complements fluid biomarkers and improves differential diagnosis and prognostic evaluation¹⁹.

Non-invasive detection of tau protein deposition in specific brain regions holds substantial value for early disease prediction and diagnosis. The application of tau radiotracers enables visual localization of cerebral tau deposition, offering timely and accurate support for differential diagnosis and disease monitoring, while also providing crucial evidence for evaluating experimental clinical interventions.

¹⁸F-AV-45 (florbetapir) exhibits high binding affinity with Aβ protein²⁰. It has been widely utilized in clinical research related to the β-amyloid protein. ¹⁸F-AV-45 PET imaging enables the non-invasive visualization of cerebral Aβ deposition, and, to some extent, reflects the clinical cognitive status of patients with Alzheimer's disease. ¹⁸F-AV-1451 (flortaucipir) is used to study the density and distribution of aggregated Tau neurofibrillary tangles (NFTs) in the brain²¹. As a tau-specific molecular probe for PET imaging, it holds significant potential as a biomarker for effectively detecting cognitive decline and tracking disease progression in AD patients. Although these tracers are well-established individually, reports combining both for simultaneous amyloid and tau assessment remain limited. This protocol introduces a dual-tracer PET/CT imaging workflow designed to offer a more accurate and comprehensive diagnostic model for Alzheimer’s disease.

Protocol

This study was approved by the Medical Ethics Committee of The First Affiliated Hospital, Zhejiang University School of Medicine (Ref. IIT20210007C-R2). All participants provided written informed consent before participation, and the study was conducted in accordance with the Declaration of Helsinki. The reagents and the equipment used are listed in the Table of Materials.

1. Expert qualifications

1. Ensure that the evaluating physician has specialized training in neurology or psychiatry, and that at least 50% of their clinical practice involves the assessment and management of patients with cognitive impairment or dementia, including suspected AD.

2. Patient selection

1. Inclusion criteria: Include patients who meet one or more of the following conditions:
   1. Individuals presenting with subjective cognitive impairment, confirmed by a dementia specialist.
   2. Patients with cognitive impairment of uncertain etiology after comprehensive evaluation.
   3. Patients with mild cognitive impairment (MCI) due to AD, defined according to the NIA-AA criteria²², who may benefit from amyloid or tau imaging to improve diagnostic confidence.
   4. Patients meeting clinical criteria for AD but exhibiting atypical or mixed features.
   5. Individuals with progressive or early-onset dementia (onset before 65 years), requiring clarification of disease subtype.
   6. Patients undergoing imaging for dementia severity assessment.
2. Exclusion criteria
   1. Exclude individuals with vascular dementia, major depressive disorder, traumatic brain injury, psychiatric illness, or other medical conditions that may contribute to cognitive decline.

3. Tracer preparation and radiation dose

1. Synthesize ¹⁸F-AV-45 and ¹⁸F-AV-1451 according to established protocols^23,24. Ensure radiochemical purity exceeds 95%.
2. Administer 370–400 MBq (10 ± 1 mCi) of each tracer intravenously at a controlled rate of 1–2 mL/s.
3. Flush the intravenous line with normal saline to minimize residual radioactivity.
4. Record the exact injected dose, injection time, and injection site.

4. Participant preparation

1. Pre-examination instructions
   1. Provide an information sheet outlining study procedures and safety precautions.
   2. Instruct participants to avoid alcohol, caffeine, and strenuous physical activity for 24 h before the scan.
   3. Maintain a stable physiological state to ensure imaging consistency.
   4. Allow a normal diet and hydration prior to imaging.
   5. Discontinue psychotropic or neuroactive medications (e.g., antidepressants, antiepileptics, sedatives) 24–48 h before the scan.
   6. Ensure each participant is accompanied by a caregiver and brings relevant medical and medication records.
2. Day-of-scan procedures
   1. Verify the requisition form and patient identification.
   2. Obtain a signed informed consent form prior to tracer administration.
   3. Record height, weight, vital signs, and any medications taken.
   4. Establish intravenous access using a 20–22 G catheter.
   5. Verify patient information before tracer injection.

5. PET/CT imaging procedures

1. ¹⁸F-AV-45 PET/CT Imaging
   1. Inject ¹⁸F-AV-45 (370–400 MBq) intravenously at 1–2 mL/s.
   2. After injection, flush the line with saline and compress the site for 5 min to prevent extravasation.
   3. Instruct the participant to rest quietly in a dimly lit room (temperature ~26 °C) for 50–70 min, avoiding talking, eating, or movement.
   4. Ask the participant to empty the bladder before scanning.
2. PET/CT image acquisition
   1. Positioning
      1. Ask the participant to lie supine on the PET/CT table with the orbitomeatal baseline perpendicular to the table.
      2. Align the midline laser with the nasal midline.
      3. Use a headrest or foam pads to minimize motion.
      4. If the patient is unable to remain still, administer mild sedation as approved by a neurologist.
   2. Scout and CT acquisition
      1. Acquire low-dose CT scout images for positioning verification.
      2. Conduct a CT scan using 120 kV, 300 mA, slice thickness 3.0 mm, pitch 0.8, and axial FOV of 26 cm (from foramen magnum to vertex).
   3. PET data acquisition
      1. Acquire static PET data for 20 min immediately following the CT scan.
      2. Maintain patient immobility throughout the acquisition.
3. Image reconstruction
   1. Reconstruct PET data using OSEM + PSF + TOF algorithm with 6 iterations and 5 subsets.
   2. Matrix size: 440 × 440; voxel size: 2 × 2 × 3 mm³.
   3. Fuse PET and CT images for anatomical reference.
   4. Use CT data solely for attenuation correction.
   5. Archive all raw and reconstructed datasets.

6. ¹⁸F-AV-1451 PET/CT imaging

1. Schedule ¹⁸F-AV-1451 PET/CT within 1 week after ¹⁸F-AV-45 imaging (minimum 24-h interval).
2. Repeat identical preparation and imaging steps as for ¹⁸F-AV-45.
3. Use an uptake period of 80–100 min, approximately 30 min longer than for ¹⁸F-AV-45.
4. Inject 370–400 MBq of ¹⁸F-AV-1451 intravenously at 1–2 mL/s, followed by saline flush.
5. Maintain the same PET/CT scanning parameters as previously described (step 5).
6. Ensure that all images are anonymized prior to interpretation.

7. Image interpretation and analysis

1. Verify image quality for motion artifacts and alignment.
2. Blind all patient information before review.
3. Perform independent interpretation by two nuclear medicine physicians, with consensus from a senior reviewer.
4. Analyze images visually using consistent grayscale scaling.
5. Evaluate six brain regions: frontal, parietal, temporal, occipital lobes, posterior cingulate cortex, and cerebellum.
6. Ensure the following interpretation criteria:
   1. Negative:
      1. Ensure minimal or absent uptake in gray matter, lower than in adjacent white matter.
      2. Ensure clear contrast between gray and white matter with well-defined borders.
      3. Ensure that the interhemispheric fissure is wide and irregular.
   2. Positive:
      1. Ensure that one or more brain regions show gray matter uptake equal to or greater than white matter.
      2. Ensure symmetric uptake across hemispheres, often involving the lateral temporal, frontal, posterior cingulate, precuneus, and parietal lobes.
      3. Ensure that the cortical Aβ deposition primarily involves the lateral temporal lobe, frontal lobe, posterior cingulate gyrus, precuneus, and parietal lobe, while sparing or minimally affecting the sensorimotor cortex and visual cortex.
         NOTE: The interhemispheric fissure is not visible; if visible, it appears as a thin, regular line. The PET image analysis of ¹⁸F-AV-1451 was conducted using the following methodology: Abnormal tau tracer uptake observed in any brain region across three consecutive slices.

8. Timing and workflow summary

1. Pre-examination preparation: Conduct pre-examination preparation 1–3 days before the PET/CT scan. Ensure that all eligibility checks, informed consent procedures, and tracer scheduling are completed during this period.
2. Day-of-scan preparation: Perform patient preparation 1–2 h before tracer injection, including fasting verification, removal of metallic objects, and patient positioning procedures.
3. ¹⁸F-AV-45 uptake phase: Allow a tracer uptake period of 50–70 min after intravenous injection of ¹⁸F-AV-45 before image acquisition.
4. ¹⁸F-AV-1451 uptake phase-Allow a tracer uptake period of 80–100 min after intravenous injection of ¹⁸F-AV-1451 before image acquisition.
5. PET/CT acquisition: Acquire PET/CT images for approximately 20 min per tracer using the standardized scanning protocol.

Results

Typical image features of amyloid PET
Typical amyloid PET–negative imaging features include the “ridge sign” (the gradient phenomenon formed by the uptake difference between gray and white matter, most evident in the temporo-occipital lobes on axial images), the “double convex lens sign” (a spindle-shaped phenomenon caused by the separation of the high-uptake white matter in the centrum semiovale by adjacent low-uptake gray matter, observed at the level of the centrum semiovale on axial images), and ...

Discussion

Alzheimer’s disease (AD) represents the most common cause of dementia worldwide. Conventional diagnostic approaches, including cognitive testing, CT, and MRI, often lack specificity, particularly during the early stages of disease progression. Dual-tracer PET/CT imaging provides a molecular imaging strategy that allows for the in vivo visualization of the key pathological proteins of AD—β-amyloid and tau—offering improved accuracy in disease characterization and staging.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Audiovisual isolation (earplugs, eye mask)	3M Company	1100 Earplugs; generic eye mask	Reduce sensory input during the rest period.
Foam pads and restraining straps	Qfix	RT-4542	Minimize the motion of head and limbs.
Headrest	CIVCO Radiotherapy	MTAP100	Head immobilization during the scan.
Image analysis software	Beijing MedEx	MEMRS-NM-C	Semi-quantitative analysis and VOI-based assessments.
Image reconstruction workstation	Siemens Healthineers	syngo.via	PET/CT reconstruction and image fusion.
IV extension set / 3-way stopcock	BD	20045	Secure the injection line and perform a saline flush.
Peripheral IV catheter (20–22G)	BD	Insyte Autoguard 20G	Establish peripheral venous access.
PET/CT scanner	Siemens Healthineers, Germany	Biograph Vision	Acquisition of PET and CT images.
Sterile syringes (5 mL / 10 mL)	BD	Ref. 309604	Radiotracer injection.
Thermometer/hygrometer	Testo	608-H1	Maintain room temp (~26°C) during uptake.