Technetium-99m-pyrophosphate imaging-based computed tomography-guided core-needle biopsy of internal oblique muscle in wild-type transthyretin cardiac amyloidosis

Abstract Background Technetium-99m-pyrophosphate (99mTc-PYP) uptake in the internal oblique muscle (IOM), which is often observed in patients with wild-type transthyretin cardiac amyloidosis (ATTR-CA), indicates amyloid transthyretin (ATTR) deposition. Objective This study aimed to assess the safety and efficacy of 99mTc-PYP imaging-based computed tomography (CT)-guided core-needle biopsy of the IOM as a new extracardiac screening biopsy for confirming the presence of ATTR deposits. Methods Patients with suspected ATTR-CA in whom myocardial tracer uptake was detected on chest- and abdomen-centered images of 99mTc-PYP scintigraphy underwent CT-guided core-needle biopsy at the site with the highest tracer uptake in the IOM between September 2021 and November 2022. Results All 18 consecutive patients (mean age, 86.3 years ± 6.5; 61.1% male) enrolled in the study showed 99mTc-PYP uptake into the IOM. Adequate tissue samples were obtained from all patients except one without serious complications. Immunohistochemical analysis confirmed ATTR deposits in 16/18 (88.9%) patients. In the remaining two patients, ATTR deposits were observed via endomyocardial biopsy. All patients were diagnosed with wild-type ATTR-CA based on transthyretin gene sequence testing results. Conclusion In wild-type ATTR-CA, 99mTc-PYP imaging-based CT-guided core-needle biopsy of the IOM could be used as an extracardiac screening biopsy to confirm the presence of ATTR deposits.


Introduction
Single-photon emission computed tomography (SPECT) with bone-avid tracers, such as technetium-99m-pyrophosphate ( 99m Tc-PYP), has been utilised increasingly in the diagnosis of suspected transthyretin cardiac amyloidosis (ATTR-CA) due to its wide availability, minimal contraindications, and the ability to differentiate ATTR-CA from other cardiomyopathies including immunoglobulin light chain cardiac amyloidosis (CA) with high sensitivity and specificity [1][2][3].Myocardial uptake of bone-avid tracers in the absence of plasma cell dyscrasia can be used to diagnose ATTR-CA.However, confirming the presence of amyloid transthyretin (ATTR) deposits histologically in patients with plasma cell dyscrasia, which is detected in up to 40% of patients with wild-type ATTR-CA (ATTRwt-CA), is essential [4,5].In Japan, tafamidis is prescribed to patients with ATTRwt-CA only after confirming the deposition of nonmutated ATTR by immunohistochemical analysis and genetic testing [6][7][8].Tissue samples can be obtained from the primarily affected organ (e.g.heart) or more easily accessible tissues, with the choice varying between hospitals [9].Endomyocardial biopsy, with a specificity and sensitivity of nearly 100%, remains the gold standard for the detection of ATTR; however, it is invasive and requires technical expertise [10], and sampling of an alternative tissues has minimised its need.Subcutaneous abdominal fat fine-needle aspiration biopsy is currently the most widely used screening biopsy technique in patients with suspected systemic amyloidosis [9].However, its diagnostic sensitivity for detecting ATTR deposition in patients with ATTRwt-CA is low.Incisional biopsy of the subcutaneous abdominal fat has a detection rate of approximately 70% for ATTR deposition, the highest among all minimally invasive extracardiac biopsies [11,12].Nevertheless, the diagnostic sensitivity for the detection of amyloid deposition varies with the amount of sample acquired and degree of amyloid deposition in the sampled tissue [7,12,13].
Some uptake of 99m Tc-PYP in the skeletal muscles of the trunk, particularly the internal oblique muscles, is often detected in patients with ATTRwt-CA, indicating the deposition of ATTR [6][7][8]14].Hutt et al. [15] reported the usefulness of skeletal muscle biopsies in ATTR-CA; however, these results were not described in detail.Therefore, this prospective study aimed to assess the safety and efficacy of performing computed tomography (CT)-guided core-needle biopsy at the site with the highest 99m Tc-PYP uptake in the internal oblique muscle as a new extracardiac screening biopsy to confirm the presence of ATTR deposits.

Ethical approval
This study was approved by the Ethical Committee of the Yawatahama City General Hospital (20210913-002) and was conducted in accordance with the Declaration of Helsinki.Written informed consent was obtained from the patients before participation and for publication of this report and the accompanying images.

Study patients
Between September 2021 and November 2022, patients with suspected cardiac amyloidosis underwent a comprehensive protocol of evaluation comprising biochemical tests, such as the analysis of cardiac biomarkers (high-sensitivity cardiac troponin I and brain natriuretic peptide levels), serum and urine protein immunofixation electrophoresis and serumfree light chain assay, electrocardiography, echocardiography, and 99m Tc-PYP scintigraphy in the Yawatahama City General Hospital.Among these patients, 29 consecutive patients with myocardial 99m Tc-PYP uptake were potentially eligible for enrolment in this study.
99m Tc-PYP scintigraphy A SPECT camera (Symbia E; Canon Medical Systems Corporation, Japan) equipped with low-energy high-resolution collimators was used for 99 m Tc-PYP scintigraphy.The axial and transaxial fields of view of the detector were 38.7 and 53.3 cm, respectively.Chest-and abdomen-centered SPECT scans were acquired as two-bed acquisitions for 20 min without arm elevation.The acquisition parameters were in accordance with the guidelines [1,2,7,8].
After SPECT acquisition, a low-dose CT (120 keV, 30 mA, and 3.4 s) was performed separately using a dedicated CT scanner (Aquilion ONE; Canon Medical Systems Corporation, Japan) during end-expiratory breath-hold for the fusion of SPECT and CT images.The tracer uptake at the sternum was considered the maximum, whereas the tracer uptake at other sites was expressed as relative values; the images were displayed in colour.Positive myocardial tracer uptake was defined as the confirmation of myocardial uptake in the fused SPECT/CT images.In addition to the tracer uptake in the myocardium, the tracer uptake in the internal oblique muscles was also investigated.

CT-guided core-needle biopsy of internal oblique muscle
At the beginning of this study, CT images were obtained using the standard-dose protocol.CT-guided biopsy using a spring-loaded biopsy needle (18-gauge Fine CoreV R , Dr. Japan Corporation, Tokyo, Japan) was performed at the site of the lesion with the highest 99m Tc-PYP uptake in the internal oblique muscle by an experienced radiologist.The patient was positioned in the supine or supine oblique position on the scanner table, and the first CT images were obtained after applying the GuidelinesV R CT biopsy grid (Beekley Medical, Connecticut, United States) to the skin of the biopsy site to optimise the entry site of biopsy needle and improve first-stick accuracy.The skin, subcutaneous fat tissue, and fascia of the abdominal oblique muscles were anaesthetised with 0.5% procaine hydrochloride after cleansing with an iodine solution, and a set of introducer stylet and cannula were advanced into the internal oblique muscle, with or without a small pre-incision through the skin.Only after confirming the correct orientation and depth on the CT images acquired subsequently, the introducer stylet was retracted, and the biopsy needle was advanced into the cannula left in place.Tissue sampling was then repeated with rotation of the cannula/needle at the same depth as well as at different depths until a minimum of three muscle fragments of approximately !1 mm 3 in size were obtained according to the endomyocardial biopsy to avoid sampling errors that produce false-negative results [16].The cannula and biopsy needle were retracted after a radiologist and a cardiologist determined that adequate specimens had been obtained, and manual compression was applied for several minutes to limit bleeding.No suturing was required.Lastly, CT was performed at the end of the procedure to exclude possible complications.The procured samples were immediately fixed in 10% buffered formalin and embedded in paraffin.The complete procedure has been illustrated in Figure 1.
The procedure time was defined as the duration between the positioning of the patient on the scanner table and end of the final CT scan.The irradiation dose was calculated as the dose-length product (DLP) and effective dose (ED) with ED/DLP of 0.020 mSv/mGycm [17].Internal oblique muscle haematomas were evaluated on CT images after biopsy [18].
At Kumamoto University, a specialised amyloid centre in Japan, the presence of amyloid deposition in the tissue samples was detected via Congo red staining, and subsequently, immunohistochemical analysis was performed to confirm the typing of amyloid fibrils.Patients in whom amyloid deposits were not detected via extracardiac biopsy underwent endomyocardial biopsy of the right ventricle at Yawatahama City General Hospital.

Statistical analyses
All statistical analyses were performed using EZR (Saitama Medical Centre, Jichi Medical University, Saitama, Japan) [19], which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).The normality of the data was assessed using the Shapiro-Wilk test.Normally distributed variables are presented as the mean ± standard deviation, whereas non-normally distributed variables are reported as median and interquartile range.Categorical data are reported as frequencies and percentages.

Study patients
Among the 29 potentially eligible patients with significant 99m Tc-PYP uptake in the myocardium, all patients demonstrated some uptake of 99m Tc-PYP into the internal oblique muscles.However, five patients who did not wish to undergo any histopathological tests, including CT-guided core-needle biopsy of the internal oblique muscle were excluded.In addition, another five patients with ATTR deposition confirmed by subcutaneous abdominal fat pad fine-needle aspiration biopsy, first performed as an extracardiac biopsy at Yawatahama City General Hospital, were also excluded from the study.Lastly, one patient who requested to underdo endocardial myocardial biopsy without CTguided core-needle biopsy of internal oblique muscle was also excluded from the study.Thus, 18 patients (mean age, 86.3 years ± 6.5; 61.1% male) were included in the study.
These 18 patients, to whom 99m Tc-PYP scintigraphy was subsequently applied, were suspected to have CA based on 'red flag' signs/symptoms in their clinical profiles, including a history of heart failure and orthopaedic diseases, such as carpal tunnel syndrome; results of serum cardiac biomarkers; and electrocardiographic/echocardiographic findings.Eight (44.4%) of the 18 patients visited Yawatahama City General Hospital because of new-onset heart failure.Among the remaining 10 patients, 3 (16.7%)with other heart diseases, such as old myocardial infarction and chronic atrial fibrillation, were suspected to have CA based on follow-up electrocardiographic and echocardiographic findings.Another four (22.2%) patients were incidentally suspected to have CA based on evaluations, including electrocardiography and echocardiography, performed before surgery, such as total knee arthroplasty.The final three (16.7%)patients underwent electrocardiography and echocardiography during hospitalisation for another illness, such as pneumonia and cerebral infarction, because the patient had a history of heart failure with a causative aetiology not confirmed at another hospital or because chest radiographs taken at the time of hospitalisation showed pulmonary congestion, resulting in the suspicion of CA.
Among the 18 patients included in this study, 5, 11, and 2 had New York Heart Association (NYHA) Class I, II and III symptoms, respectively.Among the 13 patients with NYHA Class II and III, the median time from onset of symptom of heart failure to biopsy was 2.5 (0.3-24.0) months.Four (22.2%) of the 18 patients were diagnosed with plasma cell dyscrasia.The mean heart-to-contralateral lung ratio on 99m Tc-PYP scintigraphy was 1.73 ± 0.21.In addition, one patient had grade 2 uptake and the remaining 17 had grade 3 uptake according to the semiquantitative visual scoring of cardiac retention (grade 0, no cardiac uptake; grade 1, mild uptake lower than that in bone; grade 2, moderate uptake equal to that in bone; and grade 3, high uptake higher than that in bone).The characteristics of the 18 patients are summarised in Table 1.

CT-guided core-needle biopsy of the internal oblique muscle
Among the 18 patients included, 13 (72.2%)were receiving antiplatelet or anticoagulant medications for atrial fibrillation or coronary artery disease; however, the antiplatelet or anticoagulant medications were not discontinued before the biopsy in these patients.The biopsy was performed in an outpatient setting for 6 (33.3%) patients, and the remaining 12 were hospitalised.The median interval between 99m Tc-PYP scintigraphy and CT-guided core-needle biopsy of the internal oblique muscle was 17  days.The median thickness of the internal oblique muscle at the biopsy site on CT images was 11.0 (7.9-13.2) mm, and the median distance of the biopsy access path was 26.5 (14.8-31.5)mm.
The puncture was made from the posterolateral side rather than the anterior side in one patient (patient no. 1) who could not be placed in the supine position due to kyphosis, (Figures 2(G-J)).The puncture was made from the anterolateral side in the remaining patients.Notably, the biopsy could be performed from the left internal oblique muscle without complication in a 96-year-old man (patient no.16) with a colostomy in the left rectus abdominis muscle (Figures 2(D-F)).Exhaustive scanning was required in some cases to adapt the direction of the needle tip.The mean DLP and mean ED were 2300.4 mGycm ± 1294.1 and 46.0 mSv ± 25.9, respectively.We considered the radiation exposure to be high in the initial 9 patients (patient nos.1-9) who underwent biopsy using the standard-dose CT protocol (median tube current setting, 318.4 mA/s).The CT scan area was narrowed in the subsequent patients (patient nos.10-18), and the tube current was reduced to 50 mA/s in the last two patients (patient nos.17 and 18).
The procedure time for each biopsy session ranged from 25 to 58 min (mean time, 40 min ± 10).Punctures were performed 3-17 times (mean number, 7 ± 3), and 3-5 (median, 4) cores sized approximately !1 mm 3 were obtained.Thus, the amounts of muscle tissue obtained from all patients were considered sufficient.The presence of amyloid deposits was confirmed by Congo red staining in 17 (94.4%) of the 18 patients, and 16 (88.9%)had diagnostic immunohistochemistry results suggestive of ATTR (Figure 3).The tissues collected comprised mostly fat, fibrous components, and clots, with only a small amount of muscle in one patient with negative amyloid deposition (patient no.18); therefore, we considered the sample inadequate.ATTR deposits were not observed via this biopsy in two patients (patient nos. 4 and 18); however, ATTR deposits were observed via endomyocardial biopsy.One patient (patient no.8) had a small extra-muscular haematoma that did not require treatment (Figures 2(K-N)).A vasovagal reflex occurred just after the procedure in one patient (patient no.13).Intravenous atropine sulphate was administered, and no sequelae were observed in this patient.Puncture site pain was mild in all patients.The biopsy summary of each patient is shown in Table 2 and Supplementary Table 1.A chart showing the patient flow through the study is presented in Figure 4.All enrolled patients were diagnosed with ATTRwt-CA based on transthyretin gene sequence testing results.

Discussion
We selected the internal oblique muscle with a high 99m Tc-PYP uptake as the biopsy site and used 18-gauge core needle and CT guidance to show that this biopsy method had a high ATTR detection rate.Moreover, no significant complications, such as intramuscular haematoma, were observed in any of the patients despite 13 of the 18 patients receiving anticoagulants or antiplatelet agents.
Wild-type ATTR amyloidosis is a systemic disease characterised by the presence of amyloid deposits in most organs, including those that do not show signs of disease [20].An immunohistopathological study confirmed that the deposition of amyloid varies among organs; however, the reason underlying this heterogeneity remains unclear [21].A previous study reported that among 11 skeletal trunk muscles, such as pectoralis major, abdominal oblique, and glutaeus muscles, the muscle with the highest rate of 99m Tc-PYP uptake is the abdominal oblique, where uptake is observed in 34 of 36 patients [8].In particular, tracer uptake was detected in the internal oblique muscle (Figures 1 and  2).Moreover, abnormal 99m Tc-PYP uptake into soft tissue, such as subcutaneous abdominal fat pad and skeletal trunk muscles, including the internal oblique muscle, is reportedly indicative of amyloid deposition [7,14].Therefore, we selected the internal oblique muscle as the biopsy site.These muscles are in close proximity to the body surface.In addition, other than the peritoneal cavity, no organs are present in the proximity of these muscles that could result in serious complications if punctured accidentally.
The biopsies obtaining a sufficient amount of tissue with high amyloid deposition would result in a high amyloid detection rate [12,13].However, biopsy-induced complications, especially organ damage and haemorrhagic complications, must be avoided.The risk of bleeding increases with thicker needle size, the use of cutting needles, and the vascularity of the organ/lesion selected as the biopsy site [22][23][24][25].Therefore, we selected an 18-gauge core-needle biopsy with a coaxial system, which would allow multiple biopsyneedle passes through a stationary cannula, to obtain a sufficient amount of tissue for histologic, but not cytologic, evaluation and minimise complications.Core-needle biopsies are associated with a lower risk of bleeding than incisional biopsies.Similarly, CT guidance was selected to improve the accuracy of targeting the lesion.CT-guided percutaneous needle biopsy is an effective and safe procedure with high accuracy for diagnosis in many clinical settings [13].In this study, CT-guided core-needle biopsy of the internal oblique muscle had a high amyloid detection rate without significant complications, such as intramuscular haematoma identified on CT photographs, even when antiplatelet or anticoagulant medications were not discontinued.However, it must be noted that abdominal internal oblique muscle haematoma can occur as a result of non-traumatic injuries, such as overcontraction or overstretching of the abdominal muscles due to coughing, sneezing, twisting, or vomiting, in rare cases [18].Biopsies can cause major bleeding, especially in elderly patients who are unable to rest during the procedure due to cognitive impairment or pain in the back or extremities [23].
Compared with previous reports on the biopsies of other tissues, we considered the radiation exposure in the initial 9 patients (patient nos.1-9) who underwent biopsy using the standard-dose CT protocol (median tube current setting, 318.4 mA/s) to be high.Therefore, we narrowed the CT scan area in patient nos.10-16; however, a sufficient reduction in radiation exposure was not achieved.Consequently, based on previous reports showing that the success rate of the procedure and incidence of complications at lower doses are the same compared with those of standard doses [26,27], we reduced the tube current to 50 mA/s in the last two patients (patient nos.17 and 18).Thus, we were able to achieve the results reported in previous studies [27,28].Ultrasound-guided biopsy with no radiation exposure may be acceptable instead of CT-guided biopsy in cases with diffuse 99m Tc-PYP uptake in the internal oblique muscle (Figure 2(H)), especially if the operators are more familiar with the ultrasound-guided biopsy [25].
In this study, the target site for CT-guided biopsy was the site with the highest tracer uptake in 99m Tc-PYP scintigraphy.However, whether this would also apply to 99m Tc-hydroxymethylene diphosphonate (HMDP) and 99m Tc-3,3-diphosphono-1,2-propanedicarboxylic acid (DPD), which are used as frequently as 99m Tc-PYP in the diagnosis of ATTR-CA, remains unknown.99m Tc-PYP is available for use in the United States and Japan, whereas 99m Tc-HMDP and 99m Tc-DPD are available in Europe.The COVID-19 pandemic has caused unprecedented disruptions in industrial supply chains worldwide.In particular, labour shortages and social lockdown due to the surge of COVID-19 have resulted in delays in raw material procurement, production lines, and shipment processing [29].Consequently, there have been unexpected challenges in obtaining 99m Tc-PYP in the United States and Japan.In 2022, the Food and Drug Administration in the United States and Ministry of Health, Labour and Welfare in Japan approved the use of 99m Tc-HMDP in place of 99m Tc-PYP for the evaluation of suspected CA.Thus, it will be necessary to test whether uptake of 99m Tc-HMDP into the skeletal trunk muscles, such as the internal oblique muscles, is observed in patients with ATTRwt-CA.Hutt et al. reported that the 99m Tc-DPD uptake rate within soft tissues, predominantly the muscles of the shoulder, abdominal wall, or glutaeal region, was approximately 90% in patients with ATTRwt-CA [30].Although 99m Tc-HMDP and 99m Tc-DPD show comparable myocardial uptake intensity and can be used alternatively for the diagnosis of ATTR-CA, no reports on the uptake of 99m Tc-HMDP into the skeletal trunk muscles are available [31,32].The average age of the 18 patients in our study was 86.3 years, with seven being among the oldest-old, aged !90 years [33].According to the State of World Population 2022 report published by the United Nations Population Fund [34], the average life expectancy of Japanese men and women is 82 and 88 years, respectively.This makes Japan one of the countries with the longest life expectancy in the world.Over 50,000 individuals reside in the area surrounding Yawatahama City General Hospital, and 40% of these individuals are aged !65 years.A certain number of Japanese individuals are robust, active, and motivated even after 90 years of age [35].They are among the fittest for their age.On the other hand, many patients live with frailty.In our study, these frail patients and their families did not wish them to undergo biopsies.A previous study showed that use of tafamidis was effective in reducing all-cause mortality and cardiovascular-related hospitalisations as compared to use of non-tafamidis, but the difference was not statistically significant until after about 18 months of treatment [36].Moreover, the cost of this drug is very high [37].It is therefore a drug that should be considered in patients with a reasonable expected survival duration [38].However, there are no specific age restrictions on its use.Furthermore, since tafamidis significantly reduces the decline in functional capacity, and a difference is first observed 6 months after initiation of treatment [36], even the oldest-old patients, particularly those with no to mild frailty, may be prescribed tafamidis.
This study had some limitations.First, it was conducted at a single centre with a limited sample size.Second, patients with no 99m Tc-PYP uptake in the internal oblique muscle were not included in this study.Deposition of slight amyloid fibrils below the resolution of the SPECT imaging system may occur [7,8,12], and obtaining an appropriate amount of tissue would lead to diagnostic immunohistochemistry results for ATTR, even in this setting.Third, no patients with systemic amyloidosis other than wild-type ATTR amyloidosis were included in this study.Fourth, as shown in Figures 1 and 2, there was misregistration or blurring of the 99m Tc-PYP SPECT/CT fusion images, mainly due to patient movement and breathing.Thus, using the fusion image as a reference may result in a deviation from the exact puncture site.Lastly, no direct comparison was made between CT-guided core-needle biopsy of the internal oblique muscle and other biopsy methods, such as incisional biopsy of the subcutaneous abdominal fat.
In conclusion, approximately 90% of patients with ATTRwt-CA showed ATTR deposition via CT-guided coreneedle biopsy at the site with the highest 99m Tc-PYP uptake in the internal oblique muscle.Although associated with the disadvantage of radiation exposure, 99 m Tc-PYP imagingbased CT-guided core-needle biopsy of the internal oblique muscle could be used as a minimally invasive extracardiac screening biopsy for the confirmation of ATTR deposits in patients with suspected ATTR-CA and plasma cell dyscrasia.However, refining the procedure to reduce radiation exposure and shorten the procedure time is necessary, and further studies with larger sample sizes and multi-centre studies are warranted.

Figure 1 .
Figure 1.Chest-and abdomen-centered single-photon emission computed tomography (SPECT)/computed tomography (CT) fusion images of technetium-99mpyrophosphate ( 99m Tc-PYP) Scintigrams obtained 2 h after injecting the radiotracer and CT images obtained during CT-guided core-needle biopsy of the internal oblique muscle in an 80-year-old Japanese male patient with wild-type transthyretin amyloidosis (patient no.13).Panels A and B show the 18-gauge Fine Core V R (Dr.Japan Corporation, Tokyo, Japan) spring-loaded semi-automatic biopsy needle and introducer cannula with a length of 10 cm, respectively.The tip of the biopsy needle is magnified (upper part of panel A).The successive steps of the biopsy are as follows: a) the GuidelinesV R CT biopsy grid (Beekley Medical, Connecticut, United States) is applied to the skin of the biopsy site (C); b) a set of introducer stylet and cannula are advanced into the internal oblique muscle and the introducer stylet is retracted (D); c) the biopsy needle is advanced into the introducer cannula left in place (E); d) and the muscle specimen is obtained (F).Panel G shows multiple small cores of tissue taken with the diameter of approximately 1 mm.SPECT/CT fusion images, showing horizontal plane of the (H) heart, (I) abdomen, and (J) coronal plane, indicating the uptake of 99m Tc-PYP in the myocardium (red arrows) and internal oblique muscles (white arrows).The yellow and green arrows indicate the GuidelinesV R CT biopsy grid applied to the biopsy site, and the introducer cannula left in the right internal oblique muscle, respectively (K and L).No haematoma is observed after retracting the biopsy needle and introducer cannula, although air is present in the right internal oblique muscle (orange arrows) (M).

Figure 2 .
Figure 2. The remaining four examples of computed tomography (CT)-guided core-needle biopsy of the internal oblique muscle in patients with wild-type transthyretin cardiac amyloidosis, an 88-year-old woman with thick subcutaneous abdominal fat (A-C; patient no.14), a 96-year-old man with a colostomy in the left rectus abdominis muscle (D-F; patient no.16), a 90-year-old woman with kyphosis (G-J; patient no.1), and a 91-year-old man with a haematoma between the internal and external oblique muscles that formed during the biopsy (K-N; patient no.8).Single-photon emission computed tomography/CT fusion images, showing horizontal plane of the (A, D, G, and K) heart,(B, E, H, and L) abdomen, and (I) sagittal plane, indicating the uptake of technetium-99m-pyrophosphate in the myocardium (red arrows) and internal oblique muscles (white arrows).In a patient who could not be placed in the supine position due to kyphosis (I), the puncture was made from the posterolateral side rather than from the anterior side (J).The yellow and green arrows indicate the GuidelinesV R CT biopsy grid (Beekley Medical, Connecticut, United States) applied to the biopsy site, and the introducer cannula left in the internal oblique muscles, respectively.In addition, the yellow and brown arrowheads indicate a colostomy and haematoma between the internal and external oblique muscles formed during the biopsy, respectively.

Figure 3 .
Figure 3. Histopathological images of computed tomography-guided core-needle biopsy of the internal oblique muscle in a patient with wild-type transthyretin cardiac amyloidosis (the same patient as Figure 1).Congo red staining performed at Kumamoto University, a specialised amyloid centre in Japan, shows amyloid deposits with red-orange stain under a light microscope (A) and apple-green biorefringence under a cross-polarized light microscope (B), which are characteristics of amyloid deposits (brown and yellow arrows, respectively).In immunohistochemical staining performed subsequently at Kumamoto University using a panel of type-specific antibodies against the most common amyloidogenic proteins-anti-transthyretin 115-124 (polyclonal rabbit anti-human prealbumin, custom made) (C), anti-kappa light chain 116-133 (polyclonal rabbit anti-human kappa light chains, custom made) (D), anti-lambda light chain 118-134 (polyclonal rabbit anti-human lambda light chains, custom made) (E), and anti-amyloid A (monoclonal mouse anti-human amyloid A, DAKO) (F)-after performing the formic acid treatment for kappa light chain, lambda light chain, and transthyretin antibodies because of antigen retrieval, the antibody raised against the anti-transthyretin 115-124 is positive (green arrows), and the antibodies raised against other amyloid proteins are negative.The scale bars in all panels indicate 200 lm.

Table 2 .
Summary of the computed tomography-guided core-needle biopsy results of the internal oblique muscle biopsy of each patient with wild-type transthyretin cardiac amyloidosis.

Table 1 .
Clinical characteristics of the study patients with wild-type transthyretin cardiac amyloidosis.
Data are presented as the mean ± standard deviation or as n (percentage).a Including one patient with right ventricular pacing rhythm.b Including one patient with atrial fibrillation.c Two patients with right ventricular pacing rhythm and one with complete left bundle branch block were excluded from the analysis.Abbreviations: ATTRwt-CA: wild-type transthyretin cardiac amyloidosis; CHF: congestive heart failure; GLS: global longitudinal left ventricular peak systolic strain; hs-cTnI: high-sensitivity cardiac troponin I; NYHA: New York heart association; PYP ¼ technetium-99m-pyrophosphate.