Rubidium-82 PET-CT for quantitative assessment of myocardial blood flow: validation in a canine model of coronary artery stenosis

Purpose

Absolute quantification of myocardial blood flow expands the diagnostic potential of PET for assessment of coronary artery disease. ⁸²Rb has significantly contributed to increasing utilization of PET; however, clinical studies are still mostly analysed qualitatively. The aim of this study was to reevaluate the feasibility of ⁸²Rb for flow quantification, using hybrid PET-CT in an animal model of coronary stenosis.

Methods

Nine dogs were prepared with experimental coronary artery stenosis. Dynamic PET was performed for 8 min after ⁸²Rb(1480–1850 MBq) injection during adenosine-induced vasodilation. Microspheres were injected simultaneously for reference flow measurements. CT angiography was used to determine the myocardial regions related to the stenotic vessel. Two methods for flow calculation were employed: a two-compartment model including a spill-over term, and a simplified retention index.

Results

The two-compartment model data were in good agreement with microsphere flow (y?=?0.84x + 0.20; r?=?0.92, p<0.0001), although there was variability in the physiological flow range <3 ml/g per minute (y?=?0.54x + 0.53; r =?0.53, p?=?0.042). Results from the retention index also correlated well with microsphere flow (y?=?0.47x + 0.52; r?=?0.75, p?=?0.0004). Error increased with higher flow, but the correlation was good in the physiological range (y?=?0.62x + 0.29; r?=?0.84, p?=?0.0001).

Conclusion

Using current state-of-the-art PET-CT systems, quantification of myocardial blood flow is feasible with ⁸²Rb. A simplified approach based on tracer retention is practicable in the physiological flow range. These results encourage further testing of the robustness and usefulness in the clinical context of cardiac hybrid imaging.     

A Rb infusion system for quantitative perfusion imaging with 3D PET

A ⁸²Rb infusion system is described with two important features for imaging with 3D positron emission tomography. First, a generator bypass line is added to flush the patient infusion line at the end of an elution. Second, feedback control is implemented to permit ‘slow-bolus’ constant-activity elutions. A model for the activity eluted from a ⁸²Sr/⁸²Rb generator based on a volume–activity empirical relationship, is used as the basis for performing simulations to demonstrate the efficacy of varying the flow rate through the generator to achieve desired eluted ⁸²Rb activity rate profiles. A ⁸²Rb infusion system was constructed to verify the accuracy of the simulations. The system can deliver accurate constant-activity elutions from 10% to 70% of the total generator activity.     

Clinical cardiac PET using generator-produced Rb-82: A review

Cardiac positron emission tomography (PET) with generator-produced rubidium-82 (Rb-82) provides information not previously available for optimal diagnosis and management of cardiac disease. This new information includes the accurate, noninvasive diagnosis of coronary artery disease in asymptomatic or symptomatic patients, the noninvasive assessment of coronary stenosis severity, myocardial infarct imaging, myocardial viability, collateral function, and cardiomyopathy. Cardiac positron imaging may be carried out economically at the same or less cost as other high-tech diagnostic imaging but provides perfusion and metabolic information quantitatively for routine clinical studies not available by other diagnostic modalities.     

Measurement of cardiac output with first-pass determination during rubidium-82 PET myocardial perfusion imaging

In addition to providing useful clinical information, cardiac output determined during rubidium-82 positron emission tomography (PET) myocardial perfusion studies can be used in the measurement of absolute regional myocardial blood flow using Sapirstein's method. This investigation was conducted to compare cardiac output values obtained by post-processing data acquired in a list mode PET myocardial perfusion study with those obtained using a technetium-99m-labeled red blood cell method on the same patients. Results from 14 patients showed that cardiac output can be accurately measured simultaneously in a⁸²Rb PET myocardial study, allowing determination of multiple perfusion and functional parameters of the heart, thus improving the cost-effectiveness of the⁸²Rb PET study.     

Quantification of myocardial blood flow with <Superscript>82</Superscript>Rb positron emission tomography: clinical validation with <Superscript>15</Superscript>O-water

Purpose

Quantification of myocardial blood flow (MBF) with generator-produced ⁸²Rb is an attractive alternative for centres without an on-site cyclotron. Our aim was to validate ⁸²Rb-measured MBF in relation to that measured using ¹⁵O-water, as a tracer 100% of which can be extracted from the circulation even at high flow rates, in healthy control subject and patients with mild coronary artery disease (CAD).

Methods

MBF was measured at rest and during adenosine-induced hyperaemia with ⁸²Rb and ¹⁵O-water PET in 33 participants (22 control subjects, aged 30?±?13 years; 11 CAD patients without transmural infarction, aged 60?±?13 years). A one-tissue compartment ⁸²Rb model with ventricular spillover correction was used. The ⁸²Rb flow-dependent extraction rate was derived from ¹⁵O-water measurements in a subset of 11 control subjects. Myocardial flow reserve (MFR) was defined as the hyperaemic/rest MBF. Pearson’s correlation r, Bland-Altman 95% limits of agreement (LoA), and Lin’s concordance correlation ρ _c (measuring both precision and accuracy) were used.

Results

Over the entire MBF range (0.66–4.7 ml/min/g), concordance was excellent for MBF (r?=?0.90, [⁸²Rb–¹⁵O-water] mean difference?±?SD?=?0.04?±?0.66 ml/min/g, LoA?=??1.26 to 1.33 ml/min/g, ρ _c?=?0.88) and MFR (range 1.79–5.81, r?=?0.83, mean difference?=?0.14?±?0.58, LoA?=??0.99 to 1.28, ρ _c?=?0.82). Hyperaemic MBF was reduced in CAD patients compared with the subset of 11 control subjects (2.53?±?0.74 vs. 3.62?±?0.68 ml/min/g, p?=?0.002, for ¹⁵O-water; 2.53?±?1.01 vs. 3.82?±?1.21 ml/min/g, p?=?0.013, for ⁸²Rb) and this was paralleled by a lower MFR (2.65?±?0.62 vs. 3.79?±?0.98, p?=?0.004, for ¹⁵O-water; 2.85?±?0.91 vs. 3.88?±?0.91, p?=?0.012, for ⁸²Rb). Myocardial perfusion was homogeneous in 1,114 of 1,122 segments (99.3%) and there were no differences in MBF among the coronary artery territories (p?>?0.31).

Conclusion

Quantification of MBF with ⁸²Rb with a newly derived correction for the nonlinear extraction function was validated against MBF measured using ¹⁵O-water in control subjects and patients with mild CAD, where it was found to be accurate at high flow rates. ⁸²Rb-derived MBF estimates seem robust for clinical research, advancing a step further towards its implementation in clinical routine.

     

Absolute quantitation of myocardial blood flow with <Superscript>201</Superscript>Tl and dynamic SPECT in canine: optimisation and validation of kinetic modelling

Purpose ²⁰¹Tl has been extensively used for myocardial perfusion and viability assessment. Unlike ^99mTc-labelled agents, such as ^99mTc-sestamibi and ^99mTc-tetrofosmine, the regional concentration of ²⁰¹Tl varies with time. This study is intended to validate a kinetic modelling approach for in vivo quantitative estimation of regional myocardial blood flow (MBF) and volume of distribution of ²⁰¹Tl using dynamic SPECT. Methods Dynamic SPECT was carried out on 20 normal canines after the intravenous administration of ²⁰¹Tl using a commercial SPECT system. Seven animals were studied at rest, nine during adenosine infusion, and four after beta-blocker administration. Quantitative images were reconstructed with a previously validated technique, employing OS-EM with attenuation-correction, and transmission-dependent convolution subtraction scatter correction. Measured regional time–activity curves in myocardial segments were fitted to two- and three-compartment models. Regional MBF was defined as the influx rate constant (K ₁) with corrections for the partial volume effect, haematocrit and limited first-pass extraction fraction, and was compared with that determined from radio-labelled microspheres experiments. Results Regional time–activity curves responded well to pharmacological stress. Quantitative MBF values were higher with adenosine and decreased after beta-blocker compared to a resting condition. MBFs obtained with SPECT (MBF_SPECT) correlated well with the MBF values obtained by the radio-labelled microspheres (MBF_MS) (MBF_SPECT = −0.067 + 1.042 × MBF_MS, p < 0.001). The three-compartment model provided better fit than the two-compartment model, but the difference in MBF values between the two methods was small and could be accounted for with a simple linear regression. Conclusion Absolute quantitation of regional MBF, for a wide physiological flow range, appears to be feasible using ²⁰¹Tl and dynamic SPECT.     

Protocol for measuring myocardial blood flow by PET/CT in cats

Purpose  The aim of this study was to establish a protocol for measuring myocardial blood flow (MBF) by PET/CT in healthy cats. The rationale was its future use in Maine Coon cats with hypertrophic cardiomyopathy (HCM) as a model for human HCM. Methods  MBF was measured in nine anaesthetized healthy cats using a PET/CT scanner and ¹³NH₃ at rest and during adenosine infusion. Each cat was randomly assigned to receive vasodilator stress with two or three adenosine infusions at the following rates (μg/kg per minute): 140 (Ado 1, standard rate for humans), 280 (Ado 2, twice the human standard rate), 560 (Ado 4), 840 (Ado 6) and 1,120 (Ado 8). Results  The median MBF at rest was 1.26 ml/min per g (n = 9; range 0.88–1.72 ml/min per g). There was no significant difference at Ado 1 (n = 3; median 1.35, range 0.93–1.55 ml/min per g; ns) but MBF was significantly greater at Ado 2 (n = 6; 2.16, range 1.35–2.68 ml/min per g; p < 0.05) and Ado 4 (n = 6; 2.11, 1.92–2.45 ml/min per g; p < 0.05). Large ranges of MBF values at Ado 6 (n = 4; 2.53, 2.32–5.63 ml/min per g; ns) and Ado 8 (n = 3; 2.21, 1.92–5.70 ml/min per g; ns) were noted. Observed adverse effects, including hypotension, AV-block and ventricular premature contractions, were all mild, of short duration and immediately reversed after cessation of the adenosine infusion. Conclusion  MBF can be safely measured in cats using PET. An intravenous adenosine infusion at a rate of 280 μg/kg per minute seems most appropriate to induce maximal hyperaemic MBF response in healthy cats. Higher adenosine rates appear less suitable as they are associated with a large heterogeneity in flow increase and rate pressure product, most probably due to the large variability in haemodynamic and heart rate response.     

Optimization of temporal sampling for <Superscript>82</Superscript>rubidium PET myocardial blood flow quantification

Background

Suboptimal temporal sampling of left ventricular (LV) blood pool and tissue time-activity curves (TACs) may introduce bias and increased variability in estimates of myocardial blood flow (MBF) and flow reserve (MFR) from dynamic PET myocardial perfusion images. We aimed to optimize temporal sampling for estimation of MBF and MFR.

Methods

Twenty-four normal volunteers and 32 patients underwent dynamic stress/rest rubidium-82 chloride (⁸²Rb) PET imaging. Fine temporal sampling was used to estimate the full width at half maximum (FWHM) of the LV blood pool TAC. Fourier analysis was used to determine the longest sampling interval, T _S, as a function of FWHM, which preserved the information content of the blood phase. Dynamic datasets were reconstructed with frame durations varying from 2 to 20 seconds over the first 2 minutes for the blood phase and 30 to 120 seconds for the tissue phase. The LV blood pool and tissue TACs were sampled using regions of interest (ROI) and fit to a compartment model for quantification of MBF and MFR. The effects of temporal sampling on MBF and MFR were evaluated using clinical data and simulations.

Results

T _S increased linearly with input function FWHM (R = 0.93). Increasing the blood phase frame duration from 5 to 15 seconds resulted in MBF and MFR biases of 6-12% and increased variability of 14-24%. Frame durations <5 seconds had biases of less than 5% for both MBF and MFR values. Increasing the tissue phase frame durations from 30 to 120 seconds resulted in <5% biases.

Conclusions

A two-phase framing of dynamic ⁸²Rb PET images with frame durations of 5 seconds (blood phase) and 120 seconds (tissue phase) optimally samples the blood pool TAC for modern 3D PET systems.

     

PET心肌灌注显像测定心肌血流量及冠状动脉血流储备的研究进展

PET心肌灌注显像可绝对定量测定局部心肌血流量（MBF）和冠状动脉血流储备（CFR）。由于显像剂半衰期短,允许在短时间内重复进行PET心肌灌注显像,获得静息态、冷加压试验和药物负荷试验等不同状态下的MBF,进而评价冠状动脉血管内皮依赖性和非依赖性的CFR功能。在早期诊断冠心病,准确诊断冠状动脉多支病变,评价微血管病变,早期检测冠状动脉内皮细胞功能异常及CFR功能的异常,估测预后,帮助临床治疗方案的制定以及检测疗效等方面,PET心肌灌注显像有重要的临床价值。该文将介绍PET心肌灌注显像相关知识及其在心血管领域的主要应用。     

Parametric renal blood flow imaging using [<Superscript>15</Superscript>O]H<Subscript>2</Subscript>O and PET

Purpose  The quantitative assessment of renal blood flow (RBF) may help to understand the physiological basis of kidney function and allow an evaluation of pathophysiological events leading to vascular damage, such as renal arterial stenosis and chronic allograft nephropathy. The RBF may be quantified using PET with H₂ ¹⁵O, although RBF studies that have been performed without theoretical evaluation have assumed the partition coefficient of water (p, ml/g) to be uniform over the whole region of renal tissue, and/or radioactivity from the vascular space (V _A. ml/ml) to be negligible. The aim of this study was to develop a method for calculating parametric images of RBF (K ₁, k ₂) as well as V _A without fixing the partition coefficient by the basis function method (BFM). Methods  The feasibility was tested in healthy subjects. A simulation study was performed to evaluate error sensitivities for possible error sources. Results  The experimental study showed that the quantitative accuracy of the present method was consistent with nonlinear least-squares fitting, i.e. K _1,BFM=0.93K _1,NLF−0.11 ml/min/g (r=0.80, p<0.001), k _2,BFM=0.96k _2,NLF−0.13 ml/min/g (r=0.77, p<0.001), and V _A,BFM=0.92V _A,NLF−0.00 ml/ml (r=0.97, p<0.001). Values of the Akaike information criterion from this fitting were the smallest for all subjects except two. The quality of parametric images obtained was acceptable. Conclusion  The simulation study suggested that delay and dispersion time constants should be estimated within an accuracy of 2 s. V _A and p cannot be neglected or fixed, and reliable measurement of even relative RBF values requires that V _A is fitted. This study showed the feasibility of measurement of RBF using PET with H₂ ¹⁵O.     

Perfusion CT compared to H<Subscript>2</Subscript><Superscript>15</Superscript>O/O<Superscript>15</Superscript>O PET in patients with chronic cervical carotid artery occlusion

INTRODUCTION: The purpose of this study was to compare the results of perfusion computed tomography (PCT) with those of (15)O(2)/H(2) (15)O positron emission tomography (PET) in a subset of Carotid Occlusion Surgery Study (COSS) patients. MATERIALS AND METHODS: Six patients enrolled in the COSS underwent a standard-of-care PCT in addition to the (15)O(2)/H(2) (15)O PET study used for selection for extracranial-intracranial bypass surgery. PCT and PET studies were coregistered and then processed separately by different radiologists. Relative measurement of cerebral blood flow (CBF) and oxygen extraction fraction (OEF) were calculated from PET. PCT datasets were processed using different arterial input functions (AIF). Relative PCT and PET CBF values from matching regions of interest were compared using linear regression model to determine the most appropriate arterial input function for PCT. Also, PCT measurements using the most accurate AIF were evaluated for linear regression with respect to relative PET OEF values. RESULTS: The most accurate PCT relative CBF maps with respect to the gold standard PET CBF were obtained when CBF values for each arterial territory are calculated using a dedicated AIF for each territory (R (2) = 0.796, p < 0.001). PCT mean transit time (MTT) is the parameter that showed the best correlation with the count-based PET OEF ratios (R (2) = 0.590, p < 0.001). CONCLUSION: PCT relative CBF compares favorably to PET relative CBF in patients with chronic carotid occlusion when processed using a dedicated AIF for each territory. The PCT MTT parameter correlated best with PET relative OEF.     

<Superscript>11</Superscript>C-acetate PET imaging of lung cancer: comparison with <Superscript>18</Superscript>F-FDG PET and <Superscript>99m</Superscript>Tc-MIBI SPET

Recently carbon-11 acetate (AC) positron emission tomography (PET) has been reported to be of clinical value for the diagnosis of cancer that is negative on fluorine-18 fluorodeoxyglucoce (FDG) PET. We investigated the uptake of AC in lung cancer to determine whether this tracer is of potential value for tumour detection and characterisation, and to compare AC PET imaging with FDG PET and technetium-99m sestamibi (MIBI) single-photon emission tomography (SPET). Twenty-three patients with 25 lung cancers underwent AC and FDG PET. Twenty of 23 patients were also investigated with MIBI SPET. Dynamic images were acquired for 26 min after the injection of 555 MBq of AC. Standardised uptake values (SUVs) and/or tumour to non-tumour activity ratios (T/N) for each tumour were investigated at 10–20 min after AC administration, 40–60 min after administration of 185 MBq FDG and 15–45 min after administration of 555 MBq MIBI. Twenty lung cancers were resected surgically, and the degree of tracer uptake in the primary lesion was correlated with histopathological features (cell dedifferentiation and aggressiveness) and prognosis. Rapid uptake of AC followed by extremely slow clearance was observed. For the purpose of tumour identification, AC PET was inferior to FDG PET in 8 of 25 (32%) lung cancers, and the T/N of AC was lower than that of FDG. However, AC PET was superior to FDG PET in the identification of a slow-growing tumour (bronchiolo-alveolar carcinoma). There was a positive correlation between AC uptake (T/N) and MIBI uptake (T/N) (r=0.799, P<0.0001). A positive correlation was not observed between either AC or MIBI uptake and the degree of cell dedifferentiation in lung adenocarcinomas, whereas FDG uptake did correlate with the degree of cell dedifferentiation. In lung adenocarcinoma, there was a weak correlation between aggressiveness and FDG uptake, but no correlation was evident for AC and MIBI. In addition, a positive correlation was not observed between AC or MIBI uptake and postoperative recurrence in lung adenocarcinoma, whereas FDG uptake did correlate with postoperative recurrence. Thus, the greater the FDG uptake, the higher the malignant grade. In conclusion, for the purpose of tumour identification, AC PET was inferior to FDG PET but superior to MIBI SPET. Neither AC nor MIBI uptake reflects the malignant grade in lung adenocarcinoma, whereas FDG uptake does. AC PET is less diagnostically informative than FDG PET in patients with lung cancer. However, AC PET may play a complementary role in the identification of low-grade malignancies that are not FDG avid.     

Measurement of myocardial blood flow with oxygen-15 labelled water: comparison of different administration protocols

Positron emission tomography (PET) in conjunction with C¹⁵O₂ or H₂ ¹⁵O can be used to measure myocardial blood flow (MBF) and tissue fraction (TF), i.e. the fraction of the tissue mass in the volume of the region of interest. However, with C¹⁵O₂ inhalation, the tissue fraction in the septum is overestimated. Bolus injection of H₂ ¹⁵O together with arterial cannulation gives very precise results but is invasive. The purpose of this study was to develop a method which circumvents these problems. A four-parameter model with parameters for MBF, TF and spill-over fractions from both left and right ventricular cavities was developed. This method was compared with a three-parameter model (no right ventricular cavity spill-over) in both septal and non-septal regions of interest for three different administration protocols: bolus injection of H₂ ¹⁵O, infusion of H₂ ¹⁵O and inhalation of C¹⁵O₂. It was found that MBF can be measured with intravenous administration of H₂ ¹⁵O without the requirement for arterial cannulation. The four-parameter protocol with bolus injection was stable in clinical studies. The four-parameter model proved essential for the septum, where it gave highly significantly better fits than did the three-parameter model (P<0.00003 in each of 15 subjects). Administration of H₂ ¹⁵O together with this four-parameter model also circumvented the problem of overestimation of TF in the septum seen with C¹⁵O₂ inhalation. In addition, the radiation dose of H₂ ¹⁵O protocols is lower than that of C¹⁵O₂ inhalation. Using a left atrial input curve instead of a left ventricular cavity input curve gave the same mean MBF and TF. Received 30 January and in revised form 8 April 1998     

<Superscript>11</Superscript>C-Acetate PET imaging for renal cell carcinoma

Purpose  In this study, we investigated the effectiveness of positron emission tomography (PET) with ¹¹C-acetate (AC) for evaluation of renal cell carcinoma. Methods  Enrolled in the study were 20 patients with suspected renal tumour, one of whom had three renal lesions. In all, 22 renal lesions were evaluated. Following administration of 350 MBq (10 mCi) of AC, whole-body PET images were obtained. Based on these PET findings, kidney lesions were scored as positive or negative. The PET results were correlated with the CT findings and histological diagnosis after surgery. Results  In 18 patients, 20 tumours were diagnosed as renal cell carcinoma. Lesions in the remaining two patients were diagnosed as complicated cyst without malignant tissue. Of the 20 renal cell carcinomas. 14 (70%) showed positive AC PET findings; 6 were negative. The two patients with complicated cyst had negative AC PET findings. Of the 20 renal cell carcinomas, 19 were clear-cell carcinoma and 1 was a papillary cell carcinoma. This papillary cell carcinoma showed high AC uptake. Conclusion  AC demonstrates marked uptake in renal cell carcinoma. These preliminary data show that AC is a possible PET tracer for detection of renal cancer.     

Comparison of the myocardial blood flow response to regadenoson and dipyridamole: a quantitative analysis in patients referred for clinical <Superscript>82</Superscript>Rb myocardial perfusion PET

Background  

Regadenoson is a novel selective A_2A adenosine receptor agonist, which is administered as an intravenous bolus at a fixed dose. It is currently not clear if the absolute flow increase in response to this fixed dose is a function of distribution volume in individual patients or if it is generally comparable to the previous standard agents dipyridamole or adenosine, which are dosed based on weight. We used quantitative analysis of clinical ⁸²Rb PET/CT studies to obtain further insights.     

Pathophysiologic correlates of <Superscript>82</Superscript>Rb biodistribution in cardiac PET/CT

Purpose  

PET perfusion imaging with ⁸²Rb is a powerful tool for evaluating coronary artery disease (CAD). Little is known about normal patterns or significance of ⁸²Rb lung distribution in the setting of heart disease. Herein, PET/CT hybrid imaging was used to obtain insights into the frequency and potential radiomorphologic correlates of altered ⁸²Rb distribution.     

Comparison of <Superscript>18</Superscript>F-FLT PET and <Superscript>18</Superscript>F-FDG PET for preoperative staging in non-small cell lung cancer

Purpose The nucleoside analog 3′-deoxy-3′-¹⁸F-fluorothymidine (FLT) has been introduced for imaging cell proliferation with positron emission tomography (PET). We prospectively compared the diagnostic efficacy of FLT PET with that of 2-deoxy-2-¹⁸F-fluoro-d-glucose (FDG) PET for the preoperative nodal and distant metastatic staging of non-small cell lung cancer (NSCLC). Methods A total of 34 patients with NSCLC underwent FLT PET and FDG PET. PET imaging was performed at 60 min after each radiotracer injection. The PET images were evaluated qualitatively for regions of focally increased metabolism. For visualized primary tumors, the maximum standardized uptake value (SUV) was calculated. Nodal stages were determined by using the American Joint Committee on Cancer staging system and surgical and histologic findings reference standards. Results For the depiction of primary tumor, sensitivity of FLT PET was 67%, compared with 94% for FDG PET (P = 0.005). Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for lymph node staging on a per-patient basis were 57, 93, 67, 89, and 85%, respectively, with FLT PET and 57, 78, 36, 91, and 74%, respectively, with FDG PET (P > 0.1 for all comparisons). Two of the three distant metastases were detected with FLT and FDG PET. Conclusion In NSCLC, FLT PET showed better (although not statistically significant) specificity, positive predictive value and accuracy for N staging on a per-patient basis than FDG PET. However, FDG PET was found to have higher sensitivity for depiction of primary tumor than FLT PET.     

PET imaging of apoptosis with <Superscript>64</Superscript>Cu-labeled streptavidin following pretargeting of phosphatidylserine with biotinylated annexin-V

Purpose In vivo detection of apoptosis is a diagnostic tool with potential clinical applications in cardiology and oncology. Radiolabeled annexin-V (anxV) is an ideal probe for in vivo apoptosis detection owing to its strong affinity for phosphatidylserine (PS), the molecular flag on the surface of apoptotic cells. Most clinical studies performed to visualize apoptosis have used ^99mTc-anxV; however, its poor distribution profile often compromises image quality. In this study, tumor apoptosis after therapy was visualized by positron emission tomography (PET) using ⁶⁴Cu-labeled streptavidin (SAv), following pre-targeting of apoptotic cells with biotinylated anxV. Methods Apoptosis was induced in tumor-bearing mice by photodynamic therapy (PDT) using phthalocyanine dyes as photosensitizers, and red light. After PDT, mice were injected i.v. with biotinylated anxV, followed 2 h later by an avidin chase, and after another 2 h with ⁶⁴Cu-DOTA-biotin-SAv. PET images were subsequently recorded up to 13 h after PDT. Results PET images delineated apoptosis in treated tumors as early as 30 min after ⁶⁴Cu-DOTA-biotin-SAv administration, with tumor-to-background ratios reaching a maximum at 3 h post-injection, i.e., 7 h post-PDT. Omitting the administration of biotinylated anxV or the avidin chase failed to provide a clear PET image, confirming that all three steps are essential for adequate visualization of apoptosis. Furthermore, differences in action mechanisms between photosensitizers that target tumor cells directly or via initial vascular stasis were clearly recognized through differences in tracer uptake patterns detecting early or delayed apoptosis. Conclusion This study demonstrates the efficacy of a three-step ⁶⁴Cu pretargeting procedure for PET imaging of apoptosis. Our data also confirm the usefulness of small animal PET to evaluate cancer treatment protocols.