Contrast-Enhanced CT and Ultrasonography Features of Intracholecystic Papillary Neoplasm with or without associated Invasive Carcinoma

Objective@#To assess the contrast-enhanced CT and ultrasonography (US) findings of intracholecystic papillary neoplasm (ICPN) and determine the imaging features predicting ICPN associated with invasive carcinoma (ICPN-IC). @*Materials and Methods@#In this retrospective study, we enrolled 119 consecutive patients, including 60 male and 59 female, with a mean age ± standard deviation of 63.3 ± 12.1 years, who had pathologically confirmed ICPN (low-grade dysplasia [DP] = 34, high-grade DP = 35, IC = 50) and underwent preoperative CT or US. Two radiologists independently assessed the CT and US findings, focusing on wall and polypoid lesion characteristics. The likelihood of ICPN-IC was graded on a 5-point scale. Univariable and multivariable logistic regression analyses were performed to identify significant predictors of ICPN-IC separately for wall and polypoid lesion findings. The performances of CT and US in distinguishing ICPN-IC from ICPN with dysplasia (ICPN-DP) was evaluated using the area under the receiver operating characteristic curve (AUC). @*Results@#For wall characteristics, the maximum wall thickness (adjusted odds ratio [aOR] = 1.4; 95% confidence interval [CI]: 1.1–1.9) and mucosal discontinuity (aOR = 5.6; 95% CI: 1.3–23.4) on CT were independently associated with ICPN-IC.Among 119 ICPNs, 110 (92.4%) showed polypoid lesions. Regarding polypoid lesion findings, multiplicity (aOR = 4.0; 95% CI: 1.6–10.4), lesion base wall thickening (aOR = 6.0; 95% CI: 2.3–15.8) on CT, and polyp size (aOR = 1.1; 95% CI: 1.0–1.2) on US were independently associated with ICPN-IC. CT showed a higher diagnostic performance than US in predicting ICPN-IC (AUC = 0.793 vs. 0.676; p = 0.002). @*Conclusion@#ICPN showed polypoid lesions and/or wall thickening on CT or US. A thick wall, multiplicity, presence of wall thickening in the polypoid lesion base, and large polyp size are imaging findings independently associated with invasive cancer and may be useful for differentiating ICPN-IC from ICPN-DP

Comparison Between Contrast-Enhanced Computed Tomography and Contrast-Enhanced Magnetic Resonance Imaging With Magnetic Resonance Cholangiopancreatography for Resectability Assessment in Extrahepatic Cholangiocarcinoma

Objective@#To compare the diagnostic performance and interobserver agreement between contrast-enhanced computed tomography (CECT) and contrast-enhanced magnetic resonance imaging (CE-MRI) with magnetic resonance cholangiopancreatography (MRCP) for evaluating the resectability in patients with extrahepatic cholangiocarcinoma (eCCA). @*Materials and Methods@#This retrospective study included treatment-naïve patients with pathologically confirmed eCCA, who underwent both CECT and CE-MRI with MRCP using extracellular contrast media between January 2015 and December 2020.Among the 214 patients (146 males; mean age ± standard deviation, 68 ± 9 years) included, 121 (56.5%) had perihilar cholangiocarcinoma. R0 resection was achieved in 108 of the 153 (70.6%) patients who underwent curative-intent surgery. Four fellowship-trained radiologists independently reviewed the findings of both CECT and CE-MRI with MRCP to assess the local tumor extent and distant metastasis for determining resectability. The pooled area under the receiver operating characteristic curve (AUC), sensitivity, and specificity of CECT and CE-MRI with MRCP were compared using clinical, surgical, and pathological findings as reference standards. The interobserver agreement of resectability was evaluated using Fleiss kappa (κ). @*Results@#No significant differences were observed between CECT and CE-MRI with MRCP in the pooled AUC (0.753 vs. 0.767), sensitivity (84.7% [366/432] vs. 90.3% [390/432]), and specificity (52.6% [223/424] vs. 51.4% [218/424]) (P > 0.05 for all).The AUC for determining resectability was higher when CECT and CE-MRI with MRCP were reviewed together than when CECT was reviewed alone in patients with discrepancies between the imaging modalities or with indeterminate resectability (0.798 [0.754–0.841] vs. 0.753 [0.697–0.808], P = 0.014). The interobserver agreement for overall resectability was fair for both CECT (κ = 0.323) and CE-MRI with MRCP (κ = 0.320), without a significant difference (P = 0.884). @*Conclusion@#CECT and CE-MRI with MRCP showed no significant differences in the diagnostic performance and interobserver agreement in determining the resectability in patients with eCCA.

Classification of microvascular invasion of hepatocellular carcinoma: correlation with prognosis and magnetic resonance imaging

Background/Aims@#The microvascular invasion (MVI) of hepatocellular carcinoma (HCC) involves a wide histological spectrum, and it is unclear whether the degree of MVI correlates with patient prognosis or imaging findings. Here, we evaluate the prognostic value of MVI classification and analyze the radiologic features predictive of MVI. @*Methods@#Using a retrospective cohort of 506 patients with resected solitary HCCs, the histological and imaging features of MVI were reviewed and correlated with clinical data. @*Results@#MVI-positive HCCs invading ≥5 vessels or those with ≥50 invaded tumor cells were significantly associated with decreased overall survival (OS). The 5-year OS, recurrence-free survival (RFS), and beyond Milan criteria RFS rates were significantly poorer in patients with severe MVI compared with those with mild or no MVI. Severe MVI was a significant independent predictive factor for OS (odds ratio [OR], 2.962; p<0.001), RFS (OR, 1.638; p=0.002), and beyond Milan criteria RFS (OR, 2.797; p<0.001) on multivariable analysis. On MRI, non-smooth tumor margins (OR, 2.224; p=0.023) and satellite nodules (OR, 3.264; p<0.001) were independently associated with the severe-MVI group on multivariable analysis. Both non-smooth tumor margins and satellite nodules were associated with worse 5-year OS, RFS, and beyond Milan criteria RFS. @*Conclusions@#Histologic risk classification of MVI according to the number of invaded microvessels and invading carcinoma cells was a valuable predictor of prognosis in HCC patients. Non-smooth tumor margin and satellite nodules were significantly associated with severe MVI and poor prognosis.

Volumetric CT Texture Analysis of Intrahepatic Mass-Forming Cholangiocarcinoma for the Prediction of Postoperative Outcomes: Fully Automatic Tumor Segmentation Versus Semi-Automatic Segmentation

Objective@#To determine whether volumetric CT texture analysis (CTTA) using fully automatic tumor segmentation can help predict recurrence-free survival (RFS) in patients with intrahepatic mass-forming cholangiocarcinomas (IMCCs) after surgical resection. @*Materials and Methods@#This retrospective study analyzed the preoperative CT scans of 89 patients with IMCCs (64 male; 25 female; mean age, 62.1 years; range, 38–78 years) who underwent surgical resection between January 2005 and December 2016. Volumetric CTTA of IMCCs was performed in late arterial phase images using both fully automatic and semi-automatic liver tumor segmentation techniques. The time spent on segmentation and texture analysis was compared, and the first-order and second-order texture parameters and shape features were extracted. The reliability of CTTA parameters between the techniques was evaluated using intraclass correlation coefficients (ICCs). Intra- and interobserver reproducibility of volumetric CTTAs were also obtained using ICCs. Cox proportional hazard regression were used to predict RFS using CTTA parameters and clinicopathological parameters. @*Results@#The time spent on fully automatic tumor segmentation and CTTA was significantly shorter than that for semiautomatic segmentation: mean ± standard deviation of 1 minutes 37 seconds ± 50 seconds vs. 10 minutes 48 seconds ± 13 minutes 44 seconds (p < 0.001). ICCs of the texture features between the two techniques ranged from 0.215 to 0.980. ICCs for the intraobserver and interobserver reproducibility using fully automatic segmentation were 0.601–0.997 and 0.177– 0.984, respectively. Multivariable analysis identified lower first-order mean (hazard ratio [HR], 0.982; p = 0.010), larger pathologic tumor size (HR, 1.171; p < 0.001), and positive lymph node involvement (HR, 2.193; p = 0.014) as significant parameters for shorter RFS using fully automatic segmentation. @*Conclusion@#Volumetric CTTA parameters obtained using fully automatic segmentation could be utilized as prognostic markers in patients with IMCC, with comparable reproducibility in significantly less time compared with semi-automatic segmentation.

Comparison of MicroFlow Imaging with color and power Doppler imaging for detecting and characterizing blood flow signals in hepatocellular carcinoma

Purpose@#The purpose of this study was to compare the sensitivity of MicroFlow Imaging (MFI) with that of color and power Doppler imaging (CDI and PDI, respectively) in detecting the vascularity of hepatocellular carcinomas (HCCs). @*Methods@#This prospective study enrolled 51 patients diagnosed with HCC between August 2018 and December 2018. CDI, PDI, MFI, and contrast-enhanced ultrasound (CEUS) were performed. Two radiologists evaluated the presence and pattern of tumoral vascularity on CDI, PDI, and MFI. Vascular presence was graded on a 5-point scale (0, absent; 4, >50% of the tumor). The vascular pattern was chosen from following categories: basket, vessels in tumor, spot, detouring, mixed, or others. Two additional radiologists assessed CEUS images for the presence and pattern of tumoral vascularity, which served as the reference standard. If the tumoral vascular pattern on each examination matched that of the CEUS images, the Wilcoxon test and McNemar test, respectively, were used to compare the sensitivity for detecting tumoral vascularity between MFI and CDI, and between MFI and PDI. Logistic regression analysis was performed to identify factors associated with MFI detectability of tumoral vascularity. @*Results@#CEUS demonstrated tumoral vascularity in 98.0% (50 of 51) of patients. MFI (58.0%, 29 of 50) demonstrated a higher sensitivity than CDI (14.0%, 7 of 50) or PDI (14.0%, 7 of 50) (P<0.001 for both) in detecting tumoral vascularity, provided that the vascular pattern was correctly depicted. Only tumor depth was associated with the MFI detectability of tumoral vascularity. @*Conclusion@#The sensitivity of MFI was higher than that of CDI or PDI in detecting the vascularity of HCCs when the vascular pattern was considered. MFI better detected the vascularity of shallow tumors.

Prognostic Value of Tumor Regression Grade on MR in Rectal Cancer: A Large-Scale, Single-Center Experience

Objective@#To determine the prognostic value of MRI-based tumor regression grading (mrTRG) in rectal cancer compared withpathological tumor regression grading (pTRG), and to assess the effect of diffusion-weighted imaging (DWI) on interobserveragreement for evaluating mrTRG. @*Materials and Methods@#Between 2007 and 2016, we retrospectively enrolled 321 patients (male:female = 208:113; meanage, 60.2 years) with rectal cancer who underwent both pre-chemoradiotherapy (CRT) and post-CRT MRI. Two radiologistsindependently determined mrTRG using a 5-point grading system with and without DWI in a one-month interval. Two pathologistsgraded pTRG using a 5-point grading system in consensus. Kaplan-Meier estimation and Cox-proportional hazard models wereused for survival analysis. Cohen’s kappa analysis was used to determine interobserver agreement. @*Results@#According to mrTRG on MRI with DWI, there were 6 mrTRG 1, 48 mrTRG 2, 109 mrTRG 3, 152 mrTRG 4, and 6 mrTRG 5.By pTRG, there were 7 pTRG 1, 59 pTRG 2, 180 pTRG 3, 73 pTRG 4, and 2 pTRG 5. A 5-year overall survival (OS) was significantlydifferent according to the 5-point grading mrTRG (p= 0.024) and pTRG (p= 0.038). The 5-year disease-free survival (DFS)was significantly different among the five mrTRG groups (p= 0.039), but not among the five pTRG groups (p= 0.072). OSand DFS were significantly different according to post-CRT MR variables: extramural venous invasion after CRT (hazard ratio= 2.259 for OS, hazard ratio = 5.011 for DFS) and extramesorectal lymph node (hazard ratio = 2.610 for DFS). For mrTRG, kvalue between the two radiologists was 0.309 (fair agreement) without DWI and slightly improved to 0.376 with DWI. @*Conclusion@#mrTRG may predict OS and DFS comparably or even better compared to pTRG. The addition of DWI on T2-weightedMRI may improve interobserver agreement on mrTRG.

Two-Dimensional-Shear Wave Elastography with a Propagation Map: Prospective Evaluation of Liver Fibrosis Using Histopathology as the Reference Standard

Objective@#The aim of this study was to prospectively evaluate whether liver stiffness (LS) assessments, obtained by twodimensional (2D)-shear wave elastography (SWE) with a propagation map, can evaluate liver fibrosis stage using histopathology as the reference standard. @*Materials and Methods@#We prospectively enrolled 123 patients who had undergone percutaneous liver biopsy from two tertiary referral hospitals. All patients underwent 2D-SWE examination prior to biopsy, and LS values (kilopascal [kPa]) were obtained. On histopathologic examination, fibrosis stage (F0–F4) and necroinflammatory activity grade (A0–A4) were assessed. Multivariate linear regression analysis was performed to determine the significant factors affecting the LS value.The diagnostic performance of the LS value for staging fibrosis was assessed using receiver operating characteristic (ROC) analysis, and the optimal cut-off value was determined by the Youden index. @*Results@#Reliable measurements of LS values were obtained in 114 patients (92.7%, 114/123). LS values obtained from 2D-SWE with the propagation map positively correlated with the progression of liver fibrosis reported from histopathology (p < 0.001). According to the multivariate linear regression analysis, fibrosis stage was the only factor significantly associated with LS (p < 0.001). The area under the ROC curve of LS from 2D-SWE with the propagation map was 0.773, 0.865, 0.946, and 0.950 for detecting F ≥ 1, F ≥ 2, F ≥ 3, and F = 4, respectively. The optimal cut-off LS values were 5.4, 7.8, 9.4, and 12.2 kPa for F ≥ 1, F ≥ 2, F ≥ 3, and F = 4, respectively. The corresponding sensitivity and specificity of the LS value for detecting cirrhosis were 90.9% and 88.4%, respectively. @*Conclusion@#The LS value obtained from 2D-SWE with a propagation map provides excellent diagnostic performance in evaluating liver fibrosis stage, determined by histopathology.

Radiologic intervention due to delayed gastric emptying after pylorus preserving gastrectomy for gastric cancer does not affect pyloric function

Purpose@#The purpose of this study is to evaluate whether radiologic intervention in the pylorus decreases its function for delayed gastric emptying (DGE) patients after pylorus preserving gastrectomy (PPG) for gastric cancers and to determine the optimal interventional algorithm. @*Methods@#PPG patients who underwent intervention for DGE from January 2013 to December 2017 and a control group using propensity score matching were identified. Pyloric function was compared by subjective symptoms, postoperative upper gastrointestinal series at 3 months (short-term function), and esophagogastroduodenoscopy findings at 12 months (long-term function). Serum albumin levels and body weight change, 6 months and 12 months postoperatively, were compared to evaluate nutritional status. Interventional success rate, mean hospital stay, and recurrence of DGE were analyzed to determine the optimal intervention plan. @*Results@#Fifty-one out of 677 patients (7.53%) received intervention. There was no difference in pyloric function and nutritional status between the intervention and control groups. The interventional success rate for first-time balloon dilatation was 41.7% (20/48). If a second intervention was required and balloon dilatation was done, the success rate was 45.5% (5/11). However, if stent insertion was done, the success rate was 100% (17/17). Subsequent stent insertion after balloon dilatation resulted in a shorter mean hospital stay. Intervention including stent insertion had a lower recurrence of DGE than balloon only intervention (1.96% vs. 5.88%, P=0.041). @*Conclusion@#Radiologic intervention did not decrease long-term pyloric function. For treating DGE, if at first balloon dilatation fails, retrievable stent insertion can be considered as a second choice.

Two-Dimensional-Shear Wave Elastography with a Propagation Map: Prospective Evaluation of Liver Fibrosis Using Histopathology as the Reference Standard

Objective@#The aim of this study was to prospectively evaluate whether liver stiffness (LS) assessments, obtained by twodimensional (2D)-shear wave elastography (SWE) with a propagation map, can evaluate liver fibrosis stage using histopathology as the reference standard. @*Materials and Methods@#We prospectively enrolled 123 patients who had undergone percutaneous liver biopsy from two tertiary referral hospitals. All patients underwent 2D-SWE examination prior to biopsy, and LS values (kilopascal [kPa]) were obtained. On histopathologic examination, fibrosis stage (F0–F4) and necroinflammatory activity grade (A0–A4) were assessed. Multivariate linear regression analysis was performed to determine the significant factors affecting the LS value.The diagnostic performance of the LS value for staging fibrosis was assessed using receiver operating characteristic (ROC) analysis, and the optimal cut-off value was determined by the Youden index. @*Results@#Reliable measurements of LS values were obtained in 114 patients (92.7%, 114/123). LS values obtained from 2D-SWE with the propagation map positively correlated with the progression of liver fibrosis reported from histopathology (p < 0.001). According to the multivariate linear regression analysis, fibrosis stage was the only factor significantly associated with LS (p < 0.001). The area under the ROC curve of LS from 2D-SWE with the propagation map was 0.773, 0.865, 0.946, and 0.950 for detecting F ≥ 1, F ≥ 2, F ≥ 3, and F = 4, respectively. The optimal cut-off LS values were 5.4, 7.8, 9.4, and 12.2 kPa for F ≥ 1, F ≥ 2, F ≥ 3, and F = 4, respectively. The corresponding sensitivity and specificity of the LS value for detecting cirrhosis were 90.9% and 88.4%, respectively. @*Conclusion@#The LS value obtained from 2D-SWE with a propagation map provides excellent diagnostic performance in evaluating liver fibrosis stage, determined by histopathology.

Radiologic intervention due to delayed gastric emptying after pylorus preserving gastrectomy for gastric cancer does not affect pyloric function

Purpose@#The purpose of this study is to evaluate whether radiologic intervention in the pylorus decreases its function for delayed gastric emptying (DGE) patients after pylorus preserving gastrectomy (PPG) for gastric cancers and to determine the optimal interventional algorithm. @*Methods@#PPG patients who underwent intervention for DGE from January 2013 to December 2017 and a control group using propensity score matching were identified. Pyloric function was compared by subjective symptoms, postoperative upper gastrointestinal series at 3 months (short-term function), and esophagogastroduodenoscopy findings at 12 months (long-term function). Serum albumin levels and body weight change, 6 months and 12 months postoperatively, were compared to evaluate nutritional status. Interventional success rate, mean hospital stay, and recurrence of DGE were analyzed to determine the optimal intervention plan. @*Results@#Fifty-one out of 677 patients (7.53%) received intervention. There was no difference in pyloric function and nutritional status between the intervention and control groups. The interventional success rate for first-time balloon dilatation was 41.7% (20/48). If a second intervention was required and balloon dilatation was done, the success rate was 45.5% (5/11). However, if stent insertion was done, the success rate was 100% (17/17). Subsequent stent insertion after balloon dilatation resulted in a shorter mean hospital stay. Intervention including stent insertion had a lower recurrence of DGE than balloon only intervention (1.96% vs. 5.88%, P=0.041). @*Conclusion@#Radiologic intervention did not decrease long-term pyloric function. For treating DGE, if at first balloon dilatation fails, retrievable stent insertion can be considered as a second choice.

Quantitative Evaluation of Hepatic Steatosis Using Normalized Local Variance in a Rat Model: Comparison with Histopathology as the Reference Standard

OBJECTIVE: To evaluate the diagnostic performance of the normalized local variance (NLV) ultrasound technique in the assessment of hepatic steatosis, and to identify the factors that influence the NLV value using histopathological examination as the reference standard. MATERIALS AND METHODS: Forty male Sprague-Dawley rats were fed a methionine-choline-deficient diet for variable periods (0, 2, 4, 6, 8, 10, or 12 days or 2, 3, or 4 weeks; four rats per group). At the end of each diet duration, magnetic resonance spectroscopy (MRS) and NLV examination were performed. Thereafter, the rats were sacrificed and their livers were histopathologically evaluated. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic capability of the NLV value in the detection of varying degrees of hepatic steatosis. Univariate and multivariate linear regressions were used to determine the factors associated with the NLV value. RESULTS: The areas under the ROC curve for the detection of mild, moderate, and severe hepatic steatosis were 0.953, 0.896, and 0.735, respectively. The NLV value showed comparable diagnostic performance to that of MRS in the detection of ≥ mild or ≥ moderate hepatic steatosis. Multivariate linear regression analysis revealed that the degree of hepatic steatosis was the only significant factor affecting the NLV value (p < 0.001). CONCLUSION: The NLV value of ultrasound demonstrated satisfactory diagnostic performance in the assessment of varying degrees of hepatic steatosis. The degree of hepatic steatosis was the only significant factor that affected the NLV value.

Cone-Beam CT Virtual Navigation-Guided Percutaneous Needle Biopsy of Suspicious Pleural Metastasis: A Pilot Study

OBJECTIVE: To evaluate the diagnostic performance of cone-beam computed tomography (CBCT) virtual navigation-guided percutaneous pleural biopsy for suspected malignant pleural disease. MATERIALS AND METHODS: This study enrolled 59 patients (31 males and 28 females; mean age, 63.4 years) with suspected malignant pleural disease diagnosed with CBCT from December 2010 to December 2016. Sixty-three CBCT-guided biopsies were performed using a coaxial system with 18- or 20-gauge cutting needles. Procedural details, diagnostic performance, radiation exposure, and complication rates were investigated. RESULTS: The mean diameter perpendicular to the pleura of 51 focal and 12 diffuse pleural lesions was 1.53 ± 0.76 cm. The mean distance from the skin to the target was 3.40 ± 1.51 cm. Mean numbers of CT acquisitions and biopsies were 3.21 ± 0.57 and 3.05 ± 1.54. Total procedure time and coaxial introducer indwelling time were 11.87 ± 5.59 min and 8.78 ± 4.95 min, respectively. The mean dose area product was 12013.61 ± 7969.59 mGym². There were 48 malignant, 10 benign, and 5 indeterminate lesions. Sensitivity, specificity, and diagnostic accuracy were 93.8% (45/48), 100% (10/10), and 94.8% (55/58), respectively. Positive and negative predictive values for malignancy were 100% (45/45) and 76.9% (10/13), respectively. Four patients (6.8%) with benign pathology during initial biopsy but still showing a high suspicion of malignancy underwent repeat biopsy and three of them were finally diagnosed with malignant pleural disease. There were three cases of minimal pneumothorax and no grave procedure-related complications. CONCLUSION: Cone-beam computed tomography-guided biopsy is an accurate and safe diagnostic technique for suspected malignant pleural lesion with reasonable radiation exposure and procedure time.

A portable high-intensity focused ultrasound system for the pancreas with 3D electronic steering: a preclinical study in a swine model

PURPOSE: The aim of this animal study was to evaluate the safety and feasibility of a portable, ultrasonography-guided, high-intensity focused ultrasound (USg-HIFU) system to treat the pancreas. METHODS: Eight swine were included. Using a portable HIFU device (ALPIUS 900, Alpinion Medical Systems), ablations were performed on the pancreas in vivo. Different acoustic intensities were applied (1.7 kW/cm2 or 1.5 kW/cm2, n=2 [group A for a pilot study]; 1.5 kW/ cm2, n=3 [group B]; and 1.2 kW/cm2, n=3 [group C]). Magnetic resonance imaging (MRI) was performed immediately (group A) or 7 days (groups B and C) after HIFU treatment. In groups B and C, serum amylase and lipase levels were measured on days 0 and 7, and performance status was observed every day. Necropsy was performed on days 0 (group A) or 7 (groups B and C) to assess the presence of unintended injuries and to obtain pancreatic and peripancreatic tissue for histological analysis. RESULTS: Ablation was noted in the pancreas in all swine on MRI, and all pathologic specimens showed coagulation necrosis in the treated area. The mean ablation areas on MRI were 85.3±38.1 mm2, 90.7±21.2 mm2, and 54.4±30.6 mm2 in groups A, B, and C, respectively (P>0.05). No animals showed evidence of complications, except for one case of a pseudocyst in group B. CONCLUSION: This study showed that pancreas ablation using a portable USg-HIFU system may be safe and feasible, and that coagulation necrosis of the pancreas was successfully achieved with a range of acoustic intensities.

The Influence of Glutaraldehyde Concentration on Electron Microscopic Multiple Immunostaining

The present study was aimed to evaluate the influence of glutaraldehyde (GA) concentration on multiple electron microscopic (EM) immunostaining using pre-embedding peroxidase and post-embedding immunogold method. Influence of various concentrations of GA included in the fixative on immuoreactivity was assessed in the multiple immunostaining using antisera against anti-transient receptor potential vanilloid 1 (TRPV1) for peroxidase staining and anti-GABA for immunogold labeling in the rat trigeminal caudal nucleus. Anti-TRPV1 antiserum had specificity in pre-embedding peroxidase staining when tissues were fixed with fixative containing paraformaldehyde (PFA) alone. Immunoreactivity for TRPV1 was specific in tissues fixed with fixative containing 0.5% GA at both perfusion and postfixation steps, though the immunoreactivity was weaker than in tissues fixed with fixative containing PFA alone. Tissues fixed with fixative containing 0.5% GA at the perfusion and postfixation steps showed specific immunogold staining for GABA. The results of the present study indicate that GA concentration is critical for immunoreactivity to antigens such as TRPV1 and GABA. This study also suggests that the appropriate GA concentration is 0.5% for multiple immunostaining with peroxidase labeling for TRPV1 and immunogold labeling for GABA.