Positron emission tomography (PET) with 18F-2-fluoro-2-deoxy glucose (18F-FDG) is emerging as a robust technique for early diagnosis of focal myocardial inflammation and infection, particularly prosthetic material infection. 18F-FDG is a cyclotron-produced glucose analog with a half-life of 110 minutes. Due to its long half-life, 18F-FDG can be shipped as unit doses to sites without a cyclotron on-site. 18F-FDG is a surrogate marker for cellular glucose uptake. Tissue hypoxia is a potent stimulus for increased glucose utilization by the cardiomyocytes. High glycolytic activity from infiltrates of active inflammatory cells also increases glucose utilization in inflammation and infection. GLUT1 synthesis and cell membrane expression of GLUT1 receptors are upregulated in activated macrophages facilitate increased glucose utilization.26 In addition, circulating cytokines and growth factors are thought to increase the affinity of glucose transporters for 18F-FDG.27 Once transported into the cells through GLUT1, GLUT3, and GLUT4 receptors,28 18F-FDG is phosphorylated by hexokinase to 18F-FDG-6-phosphate which is not metabolized any further. The trapped 18F-FDG-6-phosphate within the cell28 provides the imaging signal for metabolically active inflamed tissues. This chapter will focus on imaging of cardiac sarcoidosis and cardiovascular prosthetic infection using 18F-FDG PET. (See Chapter 3 for further review of FDG.)
18F-FDG PET/CT Imaging of Cardiac Sarcoidosis
Sarcoidosis is an idiopathic, multisystem inflammatory disorder characterized by accumulation of noncaseating granulomas. These organized collections of macrophages, epithelioid cells, and lymphocytes are formed in response to inciting antigens and can eventually lead to organ damage. Both cardiac and neurologic sarcoidosis can occur in isolation without involvement of other organs.29
Cardiac sarcoidosis can involve virtually any part of the heart from endocardium to pericardium and both ventricles and atria.30 The myocardium is most frequently affected, in particular, the left ventricular free wall at the base of the heart, followed by the basal interventricular septum.31 Clinical manifestations range from arrhythmias to congestive heart failure to sudden death. Cardiac involvement represents the cause of death in up to 25% of fatal sarcoidosis in the United States. Early diagnosis is important as these complications are potentially preventable with early treatment. Until recently cardiac sarcoidosis was diagnosed using the criteria proposed by the Japanese Ministry of Health and Welfare, that included a histological diagnosis by endomyocardial biopsy or a clinical diagnosis by extracardiac biopsy-proven sarcoidosis in conjunction with major and minor criteria including findings on ECG, echocardiography, myocardial perfusion imaging with 201thallium, 99mtechnetium, 67gallium, and CMR (Table 24-1). 18F-FDG PET/CT, though not included in the Japanese criteria, plays a major role in the contemporary diagnosis and management of cardiac sarcoidosis.32
Table 24-1Japanese Ministry of Health and Welfare Guidelines for Diagnosis of Cardiac Sarcoidosis ||Download (.pdf) Table 24-1 Japanese Ministry of Health and Welfare Guidelines for Diagnosis of Cardiac Sarcoidosis
Histologic diagnosis group
Cardiac sarcoidosis is confirmed when endomyocardial biopsy specimens demonstrate noncaseating epithelioid cell granulomas with histological or clinical diagnosis of extracardiac sarcoidosis.
Clinical diagnosis group
Although endomyocardial biopsy specimens do not demonstrate noncaseating epithelioid cell granulomas, extracardiac sarcoidosis is diagnosed histologically or clinically and satisfies the following conditions and more than 1 in 6 basic diagnostic criteria.
2 or more of the 4 major criteria are satisfied.
1 in 4 of the major criteria and 2 or more of the 5 minor criteria are satisfied.
Advanced atrioventricular block.
Basal thinning of the interventricular septum.
Positive 67gallium uptake in the heart.
Depressed ejection fraction of the left ventricle (<50%).
Abnormal ECG findings: ventricular arrhythmias (ventricular tachycardia), multifocal or frequent PVCs, CRBBB, axis deviation, or abnormal Q-wave.
Abnormal echocardiography: regional abnormal wall motion or morphological abnormality (ventricular aneurysm, wall thickening).
Nuclear medicine: perfusion defect detected by 201thallium or 99mtechnetium myocardial scintigraphy.
Gadolinium-enhanced CMR imaging: delayed enhancement of myocardium.
Endomyocardial biopsy: interstitial fibrosis or monocyte infiltration over moderate grade.
Indications for 18F-FDG PET/CT
Diagnosis of Cardiac Sarcoidosis
The most definitive diagnosis of cardiac sarcoidosis, currently, is based on a positive endomyocardial biopsy. Endomyocardial biopsy is usually a blind procedure with several small samples taken from the right ventricular aspect of the interventricular septum (usual location). As sarcoidosis is a focal disease, endomyocardial biopsy is more prone to sampling error. In sarcoidosis, different parts of the myocardium may harbor granulomas in different stages of inflammation, fibrosis, or an admixture of inflammation and fibrosis. As only a minute portion of the myocardium from the interventricular septum is sampled randomly, assessment of disease activity in the whole heart is limited. 18F-FDG-PET/CT overcomes these limitations. Inflammation is assessed in the entire heart. Furthermore, as shown in Table 24-2, an abnormal 18F-FDG PET/CT clinches a diagnosis of cardiac sarcoidosis, without an endomyocardial biopsy, in individuals with a histological proof of extracardiac sarcoidosis and abnormal cardiac symptoms, examination findings, ECG findings, or LVEF <50%.33
Table 24-2HRS Expert Consensus Recommendations on Criteria for the Diagnosis of Cardiac Sarcoidosis ||Download (.pdf) Table 24-2 HRS Expert Consensus Recommendations on Criteria for the Diagnosis of Cardiac Sarcoidosis
There are 2 pathways to a diagnosis of cardiac sarcoidosis (CS):
Histological diagnosis from myocardial tissue
CS is diagnosed in the presence of noncaseating granuloma on histological examination of myocardial tissue with no alternative cause identified (including negative organismal stains if applicable).
Clinical diagnosis from invasive and noninvasive studies:
It is probablea that is CS if:
(a) There is a histological diagnosis of extracardiac sarcoidosis
(b) One or more of following is present
Steroid +/– immunosuppressant responsive cardiomyopathy or heart block
Unexplained reduced LVEF (<40%)
Unexplained sustained (spontaneous or induced) VT
Mobitz type II 2nd-degree heart block or 3rd-degree heart block
Patchy uptake on dedicated cardiac PET (in a pattern consistent with CS)
Late gadolinium enhancement on CMR (in a pattern consistent with CS)
Positive gallium uptake (in a pattern consistent with CS)
(c) Other causes for the cardiac manifestation(s) have been reasonably excluded
Despite these advantages, when cardiac sarcoidosis is suspected, a CMR study is generally considered before 18F-FDG PET/CT due to its high negative predictive value to exclude sarcoidosis7 and the absence of ionizing radiation. 18F-FDG PET/CT may be considered as the initial test, if CMR is contraindicated (ferromagnetic devices, glomerular filtration rate, GFR <30 mL/min), or if CMR is abnormal and a confirmation of active inflammation may change clinical management or if clinical suspicion of cardiac sarcoidosis remains high despite a normal CMR.32 The sensitivity and specificity of 18F-FDG PET for the diagnosis of cardiac sarcoidosis are summarized in Table 24-3.34 Figure 24-3 outlines a proposed algorithm for CMR and 18F-FDG PET/CT to diagnose cardiac sarcoidosis.
Table 24-3Sensitivity and Specificity of 18F-FDG PET for the Diagnosis of Cardiac Sarcoidosis ||Download (.pdf) Table 24-3 Sensitivity and Specificity of 18F-FDG PET for the Diagnosis of Cardiac Sarcoidosis
|Study ||Year ||Number of Patients ||Cohort ||Diagnosis ||Sensitivity (%) ||Specificity (%) |
|Yamagishi ||2003 ||17 ||Cardiac sarcoid ||Histologic ||100 ||n/a |
|Okumura ||2004 ||22 ||Sarcoid ||Histologic ||100 ||91 |
|Ishimaru ||2005 ||32 ||Sarcoid ||Clinical ||100 ||82 |
|Ohira ||2008 ||21 ||Suspected cardiac sarcoid ||ECG and Holter diagnosis ||88 ||39 |
|Langah ||2009 ||65 ||Suspected cardiac sarcoid ||Suspected sarcoid ||85 ||90 |
|Weighted mean || ||168 || || ||89.9 ||81.4 |
|Mean || ||168 || || ||91 ||75.5 |
Proposed diagnostic algorithm for cardiac sarcoidosis.
*palpitations were defined as "prominent patient complaint lasting > 2 weeks"
**abnormal ECG defined as complete left or right bundle branch block and/or presence of unexplained pathological Q waves in 2 or more leads and/or sustained 2nd or 3rd degree AV block and/or sustained or non-sustained VT
***abnormal echocardiogram defined RWMA and/or wall aneurysm and/or basal septum thinning and/or LVEF <40% (Reproduced with permission from Birnie DH, Sauer WH, Bogun F, et al. HRS expert consensus statement on the diagnosis and management of arrhythmias associated with cardiac sarcoidosis. Heart Rhythm. 2014;11(7):1305–1323.)
Assessment of Response to Treatment
The results of 18F-FDG PET/CT can be useful to initiate, as well as to monitor response to anti-inflammatory therapy (Fig. 24-4). In individuals with ongoing or increasing inflammation an increase in dose or addition of new medications may be considered, while in those with decreased 18F-FDG uptake on subsequent scans, medication dose may be tapered.35 However, the utility of 18F-FDG PET/CT to guide changes in anti-inflammatory therapy has not been validated in prospective clinical trials.
Proposed therapeutic algorithm for cardiac sarcoidosis based on 18F-FDG imaging. (Reproduced with permission from Birnie DH, Nery PB, Ha AC, et al. Cardiac sarcoidosis. J Am Coll Cardiol. 2016;68(4):411–421.)
Cardiac involvement in sarcoidosis carries a poor prognosis. In a study of 118 patients with suspected or known cardiac sarcoidosis, the presence of both a perfusion defect and myocardial 18F-FDG uptake had the strongest association with death or sustained ventricular tachycardia when compared to those with normal studies or perfusion defects alone or myocardial 18F-FDG uptake alone.36 In addition, of the patients with PET/CT abnormalities, those with focal uptake in the right ventricle had an even higher adverse event rate. Cardiac PET/CT imaging of perfusion and metabolism, therefore, has the potential to risk stratify patients with cardiac sarcoidosis.36
Radionuclide imaging protocols for cardiac sarcoidosis comprise of a rest myocardial perfusion imaging study along with cardiac and a limited whole-body 18F-FDG imaging. Myocardial perfusion imaging can be performed with SPECT using 99mTc-sestamibi or 99mTc-tetrofosmin or with a PET using 13N-ammonia or 82rubidium. Table 24-4 outlines a summary protocol for the 18F-FDG PET/CT study for cardiac sarcoidosis. Historically, 67gallium SPECT was the mainstay for inflammation imaging and is included in the Japanese diagnostic criteria for cardiac sarcoidosis. But, due to the poor image resolution, long scan duration and limited sensitivity, in practice, this has largely been supplanted by 18F-FDG PET/CT. PET-only imaging can also be performed on dedicated PET cameras with smaller field of views, but can be challenging to localize focal hotspots of 18F-FDG. Fusion imaging with myocardial perfusion may help localize 18F-FDG uptake. As with PET/CT, images should be reconstructed and displayed as short axis, horizontal long axis, and vertical long axis to correlate with myocardial perfusion imaging.
Table 24-4Radionuclide Imaging Protocol for Cardiac Sarcoidosis ||Download (.pdf) Table 24-4 Radionuclide Imaging Protocol for Cardiac Sarcoidosis
High fat, low to zero carbohydrate diet (see Table 24-5) for at least 2 meals 24 hours prior to the test, followed by overnight fast
Avoid strenuous activity for at least 24 hours
Myocardial perfusion imaging
Rest myocardial perfusion imaging using standard protocols of 99mTc SPECT or 82rubidium PET or 13N-ammonia PET
|18F-FDG PET/CT Imaging |
10 mCi (3D PET) or 15 mCi (2D PET) 18F-FDG administered intravenously
90-minute uptake phase following 18F-FDG administration (keep same uptake period for follow-up scan as at baseline)
Patient should be kept in a quiet room that is shielded for radiation protection
No food intake during the uptake period
No exercise to avoid muscle uptake
Dedicated cardiac 18F-FDG scan: 20-minute (2D) or 10-minute (3D) image acquisition in the cardiac bed position followed by limited whole-body 18F-FDG scan: Base of skull through abdomen, 3 minutes per bed position for 3D PET.
The myocardium is a metabolic omnivore that consumes glucose or fatty acids for its metabolic needs, depending on the substrate abundance. The goal of dietary preparation is to suppress normal myocardial glucose utilization to be able to detect abnormal signal within the myocardium. This is accomplished by shifting myocardial metabolism to fatty acid use. Several approaches exist including prolonged fasting, specific diet (a high fat, low to zero carbohydrate, protein permitted diet), and intravenous unfractionated heparin (Table 24-5).
Table 24-5Summary of Different Approaches to Patient Preparation ||Download (.pdf) Table 24-5 Summary of Different Approaches to Patient Preparation
|Patient Preparation ||Rationale ||Advantages ||Disadvantages |
|Prolonged fasting (5–18 hours) ||In the fasting state, free fatty acids account for up to 90% of oxygen consumption in normal myocytes35 ||Easy to instruct patient ||Variable myocardial suppression |
|High fat, zero- to low-carbohydrate diet (24 hours prior to the study) followed by fasting after midnight ||Fatty acid loading suppresses glucose metabolism36 ||More reproducible myocardial suppression ||Compliance can be difficult |
|Intravenous unfractionated heparin (15–50 IU/kg administered 15 minutes prior to 18F-FDG injection) ||Activates lipoprotein and hepatic lipases ultimately increasing plasma free fatty acid levels37 ||No special preparation necessary ||May be contraindicated in some patients |
A retrospective study37 from the Netherlands compared three methods: (1) fasting alone (>6 hours), (2) low carbohydrate, protein and fat permitted diet for 12 hours followed by 12-hour fasting, and (3) low carbohydrate diet, protein and fat permitted diet for 12 hours followed by 12-hour fasting and IV unfractionated heparin 50 IU/kg, 15 minutes prior to injection of 18F-FDG. This study showed that dietary preparation (method 1) outperformed fasting alone while the addition of IV unfractionated heparin to diet and fasting (method 3) outperformed diet and fasting (method 2) in terms of suppression of myocardial glucose utilization. However, this study did not include a high fat diet. A high fat, low to zero carbohydrate, protein permitted diet for two meals on the day prior followed by an overnight fast prior to the 18F-FDG PET examination currently remains the standard protocol for 18F-FDG PET for suppressing physiological myocardial glucose utilization for sarcoid, infection/inflammation protocol 18F-FDG PET.15 Table 24-6 lists some sample foods for the sarcoid 18F-FDG PET diet.
Table 24-6Sample Foods for High Fat, High Protein, Low Carbohydrate Diet ||Download (.pdf) Table 24-6 Sample Foods for High Fat, High Protein, Low Carbohydrate Diet
|Foods Allowed ||Food to Avoid |
|Coffee or tea WITHOUT milk/sugar/Splenda || |
|Sweet'n low (saccharin), Nutra-sweet (aspartame) or Equal (aspartame) is ok ||Sugar (glucose, fructose, sucrose, etc.), Splenda (sucralose) |
|Fatty, unsweetened foods without breading (chicken, turkey, fish, red meat, bacon, fried eggs, meat only sausage) ||Milk, cheese |
|Butter and oils ||Carbohydrates (bread, rice, pasta, bagels, cereal, cookies, crackers) |
|Diet Pepsi or Diet Coke || |
Nuts, peanut butter
Candy, gum, mint, cough drops
Any kind of sauce other than pure oil
Sausage with additives other than meat (e.g., casings containing carbohydrates)
Interpretation of the perfusion and 18F-FDG integrated study involves four steps: (1) interpretation of perfusion images, (2) interpretation of cardiac 18F-FDG PET/CT, (3) integration of perfusion and 18F-FDG PET/CT data, and (4) interpretation of limited whole-body 18F-FDG PET/CT. Images should be interpreted in conjunction with clinical data, ECG findings, and other results of imaging studies such as echocardiography and CMR. Interpretation of perfusion images is discussed separately in this book. For the purposes of cardiac sarcoidosis, perfusion imaging is approached the same way as for coronary artery disease.
Cardiac 18F-FDG PET/CT images are reviewed in the standard cardiac imaging planes of short axis (SA), vertical long axis (VLA), and horizontal long axis (HLA) as well in the standard radiological imaging planes of axial, sagittal, and coronal planes. 18F-FDG uptake in the right ventricle (RV) and atria are evaluated on the fused 18F-FDG emission and the CT transmission images. In individuals with adequate suppression of physiological myocardial glucose uptake, focally increased 18F-FDG uptake with or without corresponding perfusion defects may represent myocardial inflammation. Lack of focal myocardial 18F-FDG uptake may represent noninflamed tissue, including normal myocardium and scar. Patterns of myocardial perfusion and 18F-FDG uptake and their interpretations are summarized in Figure 24-5. In a multivariate analysis, Blankstein et al.36 found that a pattern of both abnormal perfusion and abnormal FDG had the strongest association with death or VT (Fig. 24-6).
Normal perfusion and metabolism (Category 1), abnormal perfusion or metabolism (Category 2), abnormal perfusion and metabolism (Category 3). FDG, 18F-fluorodeoxyglucose. (Reproduced with permission from Blankstein R, Osborne M, Naya M, et al. Cardiac positron emission tomography enhances prognostic assessments of patients with suspected cardiac sarcoidosis. J Am Coll Cardiol. 2014;63(4):329–336.)
Survival free of death or ventricular tachycardia (VT) in individuals with suspected sarcoidosis stratified by cardiac 18F-FDG PET and myocardial perfusion imaging results. (Reproduced with permission from Blankstein R, Osborne M, Naya M, et al. Cardiac positron emission tomography enhances prognostic assessments of patients with suspected cardiac sarcoidosis. J Am Coll Cardiol. 2014;63(4):329–336.)
Notably, hibernating myocardium could cause a similar pattern of perfusion–metabolism mismatch. Hence, significant epicardial coronary artery disease needs to be excluded, preferably, by coronary angiography (CT or invasive) or by rest and stress imaging. Indeed, local tissue hypoxia from microvascular perfusion abnormalities related to endothelial dysfunction or microvascular compression from sarcoid granulomas may account for some of the increased myocardial 18F-FDG signal in active sarcoidosis. The limited whole-body 18F-FDG PET/CT can be a valuable adjunct by providing evidence of active extracardiac sarcoidosis (or lack thereof) and potentially identifying a biopsy site. Individuals with sarcoidosis have a twofold higher risk of malignancy38; hence, the whole-body images are evaluated also for coexisting undiagnosed malignancy (Fig. 24-7). Knowledge of the normal biodistribution of 18F-FDG is essential to understand normal variants and identify pathological uptake on the whole-body images. The spectrum of extracardiac 18F-FDG PET/CT findings in sarcoidosis is beyond the scope of this text and readers are referred to an excellent review on this topic.39
A 55-year-old man with known pulmonary sarcoidosis was referred for 18F-FDG PET/CT imaging to evaluate for cardiac sarcoidosis activity. The 18F-FDG PET/CT images demonstrated intense uptake in the lungs, myocardium and a focus of intense FDG uptake in the left subareolar region (black arrow). Biopsy of the subareolar lesion confirmed active sarcoid granulomas along with breast cancer.
Assessing Response to Treatment
Due to the serious side-effect profile with long-term use of anti-inflammatory medications (steroids, methotrexate, or the novel TNF alfa inhibitors), 18F-FDG is frequently repeated several months after initiation of anti-inflammatory therapy to tailor medication dose. Interval decrease or complete resolution of 18F-FDG uptake in the myocardium, compared to baseline study, could either mean resolution of inflammation or progression to fibrosis. Comparison with myocardial perfusion imaging is very important as a concomitant improvement in perfusion would support resolution of inflammation while worsening perfusion indicates a scar.40 It is important to recognize that the images are normalized to the hottest pixel and hence a purely visual assessment could be potentially misleading. Semi-quantitative metrics of standardized uptake value (SUV) (defined as the radioactivity activity concentration [kBq/mL] measured by the PET scanner within an ROI divided by decay-corrected amount of injected radiolabeled 18F-FDG [kBq] per unit patient weight [g]), offer the advantage of absolute quantitation of tissue uptake. That being said, semi-quantitative analyses using SUVmax or SUV mean, though widely used clinically, are influenced by a number of factors,41 and need further evaluation. With studies performed at the same institution with the same protocol, suggested methods include comparing mean (SUV mean) and maximum SUVs (SUVmax), measuring volume of inflamed tissue using an SUV threshold, and measuring target-to-background ratio comparing myocardial SUV to blood pool or cerebellum as outlined in Table 24-7.
Table 24-7Semi-Quantitative Methods for Assessment of 18F-FDG Uptake ||Download (.pdf) Table 24-7 Semi-Quantitative Methods for Assessment of 18F-FDG Uptake
|Reference ||Method ||Advantage |
|SUVmax38 ||Measure SUVmax in the myocardium using ROI tool ||Maximal intensity of inflammation independent of extent |
|Volume of metabolically active myocardium ||VOI tool tissue with uptake above a predefined threshold of SUV39 ||Extent of inflammation can be estimated |
|Target-to-background ratio || |
Myocardium to blood pool40 (left atrium, aorta)
Myocardium to liver
Myocardium to cerebellum
|No need for SUV measurements |
Several artifacts need to considered in interpreting 18F-FDG PET/CT scans for cardiac sarcoidosis. A major potential pitfall in interpretation is insufficient suppression of physiological glucose utilization by normal myocardium. Diffuse myocardial 18F-FDG uptake is more likely due to insufficient suppression of physiological uptake rather than cardiac sarcoidosis. Likewise, focal increase in 18F-FDG uptake in the lateral wall, especially with normal perfusion in the lateral wall, may represent a normal variant.36 In these scenarios, one should investigate possible dietary noncompliance, remedy accordingly and repeat the study if needed. Otherwise, only data from perfusion images and any extracardiac evidence of sarcoidosis on the 18F-FDG PET/CT portion of the study can be used for interpretation. As mentioned above, there is evidence that the addition of IV unfractionated heparin to dietary preparation could result in improved suppression and the study can be redone using this protocol, if there are no contraindications to heparin.
Patient motion during image acquisition is another potential cause of artifact and care must be taken to ensure proper motion correction and registration to ensure accurate attenuation correction. Focal hotspots may pose a problem particularly in individuals with intracardiac devices, especially ICD. In those cases both the attenuation-corrected and non–attenuation-corrected images need to be reviewed or metal artifact reduction applied.
Some of these patients may be on corticosteroid therapy which could alter the biodistribution of 18F-FDG. In this case, using target-to-background ratio or ratio of SUV uptake in different organs can be helpful.
Other factors that alter biodistribution such as hyperglycemia and hyperinsulinemia should also be taken into consideration during interpretation. In general, patients with blood glucose >120–200 mg/dL based on various guidelines42,43 should be rescheduled if possible as altered biodistribution from hyperglycemia could decrease the diagnostic specificity of 18F-FDG PET/CT.
The report should include all relevant clinical information (including prior echocardiography, CMR, 18F-FDG PET/CT results, dose and type of anti-inflammatory medications), technique (including imaging protocol, dose of 18F-FDG and uptake period, interval between injection and scan), the quality of the study (including adequacy of suppression of physiological glucose utilization), results of perfusion (including LV size, RV size, tracer uptake, perfusion defects), gated SPECT findings (including rest LVEF, LV volumes, wall motion, RV function), 18F-FDG PET/CT cardiac and extracardiac findings as well as a final impression (presence of myocardial inflammation and concordance with perfusion defects, change from prior, LV function, evidence of extracardiac sarcoidosis).44
18F-FDG PET/CT plays a critical role in the diagnosis and treatment of individuals with suspected or known cardiac sarcoidosis. Diagnosis of active inflammation and assessment of response to treatment are the two primary indications for 18F-FDG PET/CT in cardiac sarcoidosis. While 18F-FDG PET/CT is sensitive to detect myocardial inflammation, it is not specific; other noninflammatory or neoplastic processes that increase myocardial glucose utilization or even normal myocardium may exhibit increased 18F-FDG uptake. Further studies are warranted to optimize patient preparation methods to suppress physiological myocardial 18F-FDG uptake and improve the specificity of 18F-FDG PET/CT in cardiac sarcoidosis. Finally, several studies are underway to evaluate more specific radiotracers including somatostatin receptor–binding agents, as well as novel more specific radiotracers for inflammation (11C-PBR28).