Mid-term outcomes of right ventricular papillary muscle approximation for severe functional tricuspid regurgitation.

OBJECTIVES: Recurrence of tricuspid regurgitation (TR) after tricuspid annuloplasty can occur in cases where a dilated right ventricle exists and subsequent leaflet tethering follows. We previously reported a new technique of the right ventricular papillary muscle approximation (RV-PMA) for functional TR associated with leaflet tethering. The objective of this study is to elucidate the mid-term outcomes and evaluate the durability of RV-PMA. METHODS: Between January 2014 and March 2023, we applied RV-PMA in 20 patients of advanced functional TR with severe leaflet tethering. The indication of the technique was severe TR with leaflet tethering height >8 mm, and/or a right ventricular end-diastolic diameter >45 mm. The patients were followed up with echocardiography before discharge and at annual interval thereafter. RESULTS: There was no perioperative mortality. In the echocardiography performed before discharge, TR was decreased to mild or less in 85%, and a significant improvement in right ventricular end-diastolic diameter and tethering height were achieved (53-45 mm and 11.1-4.4 mm, respectively). Furthermore, during the median 3-year follow-up period, TR was kept controlled mild or less in 80% of the cases. CONCLUSIONS: RV-PMA is considered to be a safe, effective and durable technique as an additional approach for tricuspid annuloplasty.

Reduced chemokine C-C motif ligand 1 expression may negatively regulate colorectal cancer progression at liver metastatic sites.

Colorectal cancer (CRC) liver metastasis, albeit a stage-IV disease, is completely curable by surgical resection in selected patients. In addressing the molecular basics of this phenomenon, differentially expressed genes at primary and liver metastatic sites were screened by RNA sequencing with the use of paraffin-embedded surgical specimens. Chemokine C-C motif ligand 1 (CCL1), a chemotactic factor for a ligand of the chemokine C-C motif receptor 8 (CCR8), was isolated as one of the differentially expressed genes. Histological analysis revealed that the number of CCL1-positive cells, mainly tumour associated macrophages (TAMs) located in the stroma of CRC, decreased significantly at liver metastatic sites, while the expression level of CCR8 on CRC remained unchanged. To explore the biological significance of the CCL1-CCR8 axis in CRC, CCR8-positive CRC cell line Colo320DM was used to assess the effect of the CCL1-CCR8 axis on major signalling pathways, epithelial mesenchymal transition induction and cell motility. Upon stimulation of recombinant CCL1 (rCCL1), phosphorylation of AKT was observed in Colo320DM cells; on the other hand, the corresponding significant increase in MMP-2 levels demonstrated by RT-qPCR was nullified by siRNA (siCCR8). In the scratch test, rCCL1 treatment significantly increased the motility of Colo320DM cells, which was similarly nullified by siCCR8. Thus, the activation of the CCL1-CCR8 axis is a positive regulator of CRC tumour progression. Reduced CCL1 expression of TAMs at liver metastatic sites may partly explain the unique slow tumour progression of CRC, thus providing for a grace period for radical resection of metastatic lesions.

Dorsal approach in laparoscopic extended left hemi-hepatectomy: A case series.

RATIONALE: The utility of the dorsal approach has been reported for laparoscopic left hemi-hepatectomy. PATIENT CONCERNS: The aim of the present study is to show the usefulness of the dorsal approach for laparoscopic extended left-hemi-hepatectomy while ensuring safe identification of hepatic veins and dissection of the dorsal tumor margin. DIAGNOSES: Tumors requiring extended left hemi-hepatectomy. INTERVENTIONS: After mobilization of the lateral sector and division of the Arantius plate, parenchyma above the Arantius plate is removed to expose the root of the middle hepatic vein and left hepatic vein. Each of these veins can be isolated separately either intra- or extra-hepatically. After removing the parenchyma on the cranial side of the left Glissonean pedicle continuous with the exposed hepatic veins, the left Glissonean pedicle is isolated using the Glissonean pedicle transection method. After division of the left hepatic vein and Glissonean pedicle, segment 4 (in which the main part of the tumor is commonly located) is dissected from the anterior plane of the paracaval portion of the caudate lobe by the dorsal approach, along with the hepatic hilum. Following dissection of the dorsal side of the tumor, and division of parenchyma from the anterior edge of the liver, the anterior Glissonean branches and middle hepatic vein are divided safely and the specimen is resected. OUTCOMES: Three patients underwent laparoscopic extended left hemi-hepatectomy, with no open conversions. Operative time and blood loss were 331 (concomitant with another partial hepatectomy), 277, and 315 minutes; and 200, 100, and 100 g, respectively. The postoperative courses were uneventful. LESSONS: The dorsal approach maximizes the advantages of laparoscopic extended left hemi-hepatectomy and can be performed safely.

Prolonged warm ischemia time in the recipient is associated with post-transplant biliary stricture following living-donor liver transplantation.

PURPOSE: Post-transplant biliary stricture (PBS) is a common and important complication following orthotopic liver transplantation (LT). This study clarified the incidence of PBS and identified its risk factors. METHODS: We retrospectively reviewed the medical records of 67 patients who underwent living-donor LT (LDLT) at our institute between June 2010 and July 2022 and analyzed their clinical characteristics, prognosis, and risk factors for PBS. RESULTS: Of the 67 patients, 26 (38.8%) developed PBS during the observation period. Multivariate analyses revealed the following independent risk factors for PBS formation: increased red cell transfusion volume per body weight (> 0.2 U/kg; hazard ratio [HR], 3.8; P = 0.002), increased portal vein pressure (PVP) at the end of LT (> 16 mmHg; HR, 2.88; P = 0.032), postoperative biliary leakage (HR, 4.58; P = 0.014), and prolonged warm ischemia time (WIT) (> 48 min; HR, 4.53; P = 0.008). In patients with PBS, the cumulative incidence of becoming stent free was significantly higher in patients with a WIT ≤ 48 min than in those with a WIT > 48 min (P = 0.038). CONCLUSION: Prolonged WIT is associated with intractable PBS following LDLT.

C-Reactive Protein-to-Albumin Ratio to Predict Tolerability of S-1 as an Adjuvant Chemotherapy in Pancreatic Cancer.

Adjuvant chemotherapy (AC) with S-1 after radical surgery for resectable pancreatic cancer (PC) has shown a significant survival advantage over surgery alone. Consequently, ensuring that patients receive a consistent, uninterrupted S-1 regimen is of paramount importance. This study aimed to investigate whether the C-reactive protein-to-albumin ratio (CAR) could predict S-1 AC completion in PC patients without dropout due to adverse events (AEs). We retrospectively enrolled 95 patients who underwent radical pancreatectomy and S-1 AC for PC between January 2010 and December 2022. A statistical analysis was conducted to explore the correlation of predictive markers with S-1 completion, defined as continuous oral administration for 6 months. Among the 95 enrolled patients, 66 (69.5%) completed S-1, and 29 (30.5%) failed. Receiver operating characteristic curve analysis revealed 0.05 as the optimal CAR threshold to predict S-1 completion. Univariate and multivariate analyses further validated that a CAR ≥ 0.05 was independently correlated with S-1 completion (p < 0.001 and p = 0.006, respectively). Furthermore, a significant association was established between a higher CAR at initiation of oral administration and acceptable recurrence-free and overall survival (p = 0.003 and p < 0.001, respectively). CAR ≥ 0.05 serves as a predictive marker for difficulty in completing S-1 treatment as AC for PC due to AEs.

Three new acridone alkaloids from Glycosmis lanceolata (Blume) D.Dietr. and their cytotoxic effects on tumour cell lines.

We separated and structurally elucidated three new acridone alkaloids (glycomontamine A (1), B (2), and C (3)), together with three known compounds (glycofoline, kokusaginine and dictamnine) from the acetone extract of Glycosmis lanceolata (Blume) D.Dietr. branches collected in Thailand. The compounds were assayed for cell viability using human lung adenocarcinoma cell line A549, breast adenocarcinoma cell line T47D, cervix epithelioid carcinoma cell line Hela, acute lymphoid leukaemia B cell line NALM-6, and human dermal fibroblasts. The viability of Hela cells treated with compound 1 (IC50 17.6 µM) and T47D cells treated with compound 2 (IC50 17.4 µM) decreased dose-dependently. Both compounds also showed cytotoxicity against NALM-6 cells (IC50 16.5 and 9.3 µM). Additionally, compound 1 decreased the mitochondrial membrane potential of Hela cells, whereas compound 2 did not change the mitochondrial membrane potential in T47D cells.

Clamp-crushing Pancreatic Transection in Minimally Invasive Distal Pancreatectomy.

BACKGROUND: Minimally invasive distal pancreatectomy has become a widely accepted procedure for tumors located in the pancreatic body or tail. However, pancreatic transection by linear stapler is generally avoided for pancreatic body tumors located above the portal vein because the surgical margin width is narrowed after taking into account the cutting allowance for insertion of the stapling device. Herein, we report a parenchymal clamp-crushing procedure that provides a sufficient surgical margin in pancreatic transection. METHODS: Two patients with suspected early pancreatic cancer underwent pancreatic transection using the clamp-crushing procedure. The planned pancreatic transection line was set just to the left of the gastroduodenal artery in both cases. Robotic and laparoscopic distal pancreatectomy were performed in 1 patient each. Patients were positioned supine with split legs. Parenchymal transection was performed with crushing by VIO 3 (ERBE Elektromedizin) operated in softCOAG Bipolar mode with Effect 2/modulation 50. After crushing, remnant tissue was cut in autoCUT Bipolar mode operated by VIO 3 with Effect 2/modulation 50, or cut after secured by clipping. RESULTS: The surgical duration was 253 and 212 minutes, and estimated blood loss was 0 and 50 mL in the 2 patients, and both were discharged with uneventful courses. Pathologic examination confirmed a negative surgical margin in both patients. CONCLUSION: Clamp-crushing pancreatic transection for distal pancreatectomy might be a suitable treatment option for achieving sufficient surgical margin in pancreatic body tumors located close to the portal vein.

Impact of the inferior vena cava morphology on fluid dynamics of the hepatic veins.

We reported previously that a large vertical interval between the hepatic segment of the inferior vena cava (IVC) and right atrium (RA), referred to as the IVC-RA gap, was associated with more intraoperative bleeding during hemi-hepatectomy. We conducted a computational fluid dynamics (CFD) study to clarify the impact of fluid dynamics resulting from morphologic variations around the liver. The subjects were 10 patients/donors with a large IVC-RA gap and 10 patients/donors with a small IVC-RA gap. Three-dimensional reconstructions of the IVC and hepatic vessels were created from CT images for the CFD study. Median pressure in the middle hepatic vein was significantly higher in the large-gap group than in the small-gap group (P = 0.008). Differences in hepatic vein pressure caused by morphologic variation in the IVC might be one of the mechanisms of intraoperative bleeding from the hepatic veins.

Staining, magnification, and algorithmic conditions for highly accurate cell detection and cell classification by deep learning.

OBJECTIVES: Research into cytodiagnosis has seen an active exploration of cell detection and classification using deep learning models. We aimed to clarify the challenges of magnification, staining methods, and false positives in creating general purpose deep learning-based cytology models. METHODS: Using 11 types of human cancer cell lines, we prepared Papanicolaou- and May-Grünwald-Giemsa (MGG)-stained specimens. We created deep learning models with different cell types, staining, and magnifications from each cell image using the You Only Look Once, version 8 (YOLOv8) algorithm. Detection and classification rates were calculated to compare the models. RESULTS: The classification rates of all the created models were over 95.9%. The highest detection rates of the Papanicolaou and MGG models were 92.3% and 91.3%, respectively. The highest detection rates of the object detection and instance segmentation models, which were 11 cell types with Papanicolaou staining, were 94.6% and 91.7%, respectively. CONCLUSIONS: We believe that the artificial intelligence technology of YOLOv8 has sufficient performance for applications in screening and cell classification in clinical settings. Conducting research to demonstrate the efficacy of YOLOv8 artificial intelligence technology on clinical specimens is crucial for overcoming the unique challenges associated with cytology.

Diagnostic value of quantification of cell-free DNA for suspected gallbladder cancer.

Background and Aim: An accurate preoperative diagnosis as the basis for deciding the most appropriate surgical procedure is essential for patients with suspected gallbladder cancer (GBC). The aim of this study was to investigate the usefulness of cell-free DNA (cfDNA) for the preoperative detection of ≥T2 invasion in patients with suspected GBC. Methods: Twenty-four patients who underwent resection for suspected GBC were enrolled. The concentration of cfDNA obtained from blood samples preoperatively was measured and evaluated in two distributions. The first peak (less than 200 base pairs) of cfDNA distribution was defined as the shorter fragment cfDNA, considered to originate mainly from apoptosis; and the second peak (200 base pairs or more) was defined as the longer fragment cfDNA, originating mainly from necrosis. Results: Pathological analysis identified benign disease in 12 patients and GBC in 12 patients, of whom 6 patients had ≥pT2 GBC. Carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 were significantly higher in the ≥pT2 GBC group than in the benign/

Living Donor Liver Transplantation in Patients with Preformed Donor-Specific Anti-Human Leukocyte Antigen Antibodies Using Preoperative Desensitization Therapy According to Intensity of Donor-Specific Antibodies: A Single-Center Study.

BACKGROUND In liver transplantation (LT), preoperative desensitization therapy is considered necessary in patients positive for donor-specific anti-human leukocyte antigen antibodies (DSAs). However, the relationship between DSA intensity and the necessary desensitization therapy is unclear. MATERIAL AND METHODS A total of 37 adult living donor (LD) LTs performed between January 2016 and March 2022 were examined. Mycophenolate mofetil (MMF) was administered preoperatively in DSA-positive cases with positive lymphocyte cross-matching who underwent LDLT. In those with strongly positive DSA (mean fluorescence intensity 10 000), rituximab was administered 2 weeks before LDLT in addition to MMF. Cross-reactive epitope group antigen (CREG)-alone-positive cases were also treated with preoperative MMF when lymphocyte cross-matching was positive. RESULTS Of the 37 patients, 9 were DSA-positive, 7 were CREG-alone-positive, and the others were double-negative. Of 9 DSA-positive cases, desensitization therapy was performed in 7, among which rituximab administration was performed in 3 strongly DSA-positive cases. Of 7 CREG-alone-positive cases, 2 were lymphocyte cross-match-positive and underwent desensitization therapy. The 1-year survival rate was 100% in both DSA- and CREG-alone-positive cases. The frequency of T-cell mediated rejection in DSA-positive, CREG-alone-positive, and double-negative cases was 22%, 43%, and 29%, respectively, with no significant difference. Antibody-mediated rejection occurred in only 1 patient, who was strongly DSA-positive and blood-group incompatible. There was also no significant difference among the 3 groups in terms of the frequency of biliary complications or 90-day mortality. CONCLUSIONS Satisfactory LDLT results were achieved in DSA- and CREG-alone-positive cases following desensitization therapy.

Clinical Classification of Variants in the Valosin-Containing Protein Gene Associated With Multisystem Proteinopathy.

Background and Objectives: Pathogenic variants in the valosin-containing protein (VCP) gene cause a phenotypically heterogeneous disorder that includes myopathy, motor neuron disease, Paget disease of the bone, frontotemporal dementia, and parkinsonism termed multisystem proteinopathy. This hallmark pleiotropy makes the classification of novel VCP variants challenging. This retrospective study describes and assesses the effect of 19 novel or nonpreviously clinically characterized VCP variants identified in 28 patients (26 unrelated families) in the retrospective VCP International Multicenter Study. Methods: A 6-item clinical score was developed to evaluate the phenotypic level of evidence to support the pathogenicity of the novel variants. Each item is allocated a value, a score ranging from 0.5 to 5.5 points. A receiver-operating characteristic curve was used to identify a cutoff value of 3 to consider a variant as high likelihood disease associated. The scoring system results were confronted with results of in vitro ATPase activity assays and with in silico analysis. Results: All variants were missense, except for one small deletion-insertion, 18 led to amino acid changes within the N and D1 domains, and 13 increased the enzymatic activity. The clinical score coincided with the functional studies in 17 of 19 variants and with the in silico analysis in 12 of 19. For 12 variants, the 3 predictive tools agreed, and for 7 variants, the predictive tools disagreed. The pooled data supported the pathogenicity of 13 of 19 novel VCP variants identified in the study. Discussion: This study provides data to support pathogenicity of 14 of 19 novel VCP variants and provides guidance for clinicians in the evaluation of novel variants in the VCP gene.

Demethylation in promoter region of severely damaged hepatocytes enhances chemokine receptor CXCR4 gene expression.

The liver is known to possess remarkable regenerative potential, but persistent inflammation or severe acute injury can lead to liver fibrosis and incomplete regeneration, ultimately resulting in liver failure. Recent studies have shown that the axis of two types of CXCL12 receptors, CXCR4 and CXCR7, plays a crucial role in liver fibrosis and regeneration. The present study aimed to investigate the regulatory factors involved in CXCR4 expression in injured liver. Immunohistochemical screening of liver tissue samples collected during liver transplantation revealed a reciprocal expression pattern between CXCR4 and MeCP2. An in vitro system involving cultured cell lines and H2O2 treatment was established to study the impact of oxidative stress on signaling pathways and epigenetic alterations that affect CXCR4 mRNA expression. Operating through distinct signaling pathways, H2O2 treatment induced a dose-dependent increase in CXCR4 expression in both hepatocyte- and intrahepatic cholangiocyte-derived cells. Treatment of the cells with trichostatin and azacytidine modulated CXCR4 expression in hepatocytes by modifying the methylation status of CpG dinucleotides located in a pair of TA repeats adjacent to the TATA box of the CXCR4 gene promoter. Only MeCP2 bound to oligonucleotides representing the TATA box region when the cytosine residues within the sequence were methylated, as revealed by electrophoretic mobility shift assay (EMSA). Methylation-specific PCR analysis of microdissected samples revealed a correlation between the loss of CpG methylation and the upregulation of CXCR4 in injured hepatocytes, replicating the findings from the in vitro study. Besides the conventional MEK/ERK and NF-κB signaling pathways that activate CXCR4 in intrahepatic cholangiocytes, the unique epigenetic modifications observed in hepatocytes might also contribute to a shift in the CXCR4-CXCR7 balance towards CXCR4, leading to irreversible liver injury and fibrosis. This study highlights the importance of epigenetic modifications in regulating CXCR4 expression in liver injury and fibrosis.

Epstein-Barr Virus-Associated Latent Malignant Lymphoma With Acute Exacerbation After Living Donor Liver Transplantation: Case Report.

Concomitant malignant lymphoma at the time of transplantation is usually considered a contraindication to liver transplantation (LT). We report a case of Epstein-Barr virus (EBV)-associated malignant lymphoma that was latent preoperatively and rapidly became aggravated after LT. A 69-year-old man was referred to our hospital with an exacerbation of abdominal distension due to polycystic liver. As cystic infection, ascites, and deteriorated liver reserve function occurred after hepatic artery embolization, he underwent living-donor LT with his daughter as the donor. His respiratory condition worsened, and he was moved to the intensive care unit on postoperative day 34. Histopathologic examination of the excised liver returned around the same time revealed findings suggestive of EBV-associated malignant lymphoma in lymph nodes near the gallbladder. Subsequent computed tomography scans showed apparent neoplastic lesions in the abdominal cavity and worsening pleural effusion and ascites. Numerous atypical lymphocytes were observed in the pleural effusion and ascites, and the patient was diagnosed with exacerbation of EBV-associated malignant lymphoma. He was treated unsuccessfully with rituximab and died 66 days after LT. Caution should be exercised in elderly immunocompromised transplant candidates who may have comorbid EBV-associated lymphoproliferative disease.

Early Postoperative Varicella-Zoster Virus Encephalitis After Adult ABO-Incompatible Living Donor Liver Transplantation: A Case Report.

There have never been any reports of adult varicella-zoster virus (VZV) encephalitis cases. Here, we report a case of VZV encephalitis after adult ABO-incompatible living donor liver transplantation (LDLT). A 38-year-old man with decompensated liver cirrhosis caused by the hepatitis C virus was referred to our hospital as an LDLT candidate. Rituximab was administered 3 weeks before the operation, and immunosuppression agents were administered 1 week before the LDLT. Plasma exchange was performed 3 times before the LDLT. The right lobe from his mother's liver was used for the ABO-incompatible LDLT. On postoperative day (POD) 9, vascular stenting for intraabdominal bleeding from the common hepatic artery was performed by interventional radiology and was followed by re-laparotomy for abdominal drainage of the hematoma. However, there were various degrees of continued bleeding thereafter. On POD 12, due to a convulsion seizure with loss of consciousness, the patient was started on anticonvulsant therapy. On POD 15, there was an increased frequency of convulsion attacks and a prolonged loss of consciousness. A lumbar puncture was performed on POD 20 due to the appearance of shingles. The positive polymerase chain reaction of the VZV-DNA from the cerebrospinal fluid was detected, and he was diagnosed with VZV encephalitis. He rapidly regained alertness, and there were no further observed convulsion attacks after administration of a steroid pulse and acyclovir. Brain magnetic resonance imaging performed on 2 subsequent postoperative months showed findings that matched with VZV encephalitis. He was discharged as he had recovered and was ambulatory 3 months after LDLT.

Effect of liquid-based cytology fixing solution on immunocytochemistry: Efficacy of antigen retrieval in cytologic specimens.

BACKGROUND: Immunocytochemistry (ICC) is an indispensable technique to improve diagnostic accuracy. ICC using liquid-based cytology (LBC)-fixed specimens has been reported. However, problems may arise if the samples are not fixed appropriately. We investigated the relationship between the LBC fixing solution and ICC and the usefulness of antigen retrieval (AR) in LBC specimens. METHODS: Specimens were prepared from five types of LBC-fixed samples using cell lines and the SurePath™ method. ICC was performed using 13 antibodies and analyzed by counting the number of positive cells in the immunocytochemically stained specimens. RESULTS: Insufficient reactivity was observed using ICC without heat-induced AR (HIAR) in nuclear antigens. The number of positive cells increased in ICC with HIAR. The percentage of positive cells was lower in CytoRich™ Blue samples for Ki-67 and in CytoRich™ Red and TACAS™ Ruby samples for estrogen receptor and p63 than in the other samples. For cytoplasmic antigens, the percentage of positive cells for no-HIAR treatment specimens was low in the three antibodies used. In cytokeratin 5/6, the number of positive cells increased in all LBC specimens with HIAR, and the percentage of positive cells in CytoRich™ Red and TACAS™ Ruby samples was significantly lower (p < .01). For cell membrane antigens, CytoRich™ Blue samples had a lower percentage of positive cells than the other LBC-fixed samples. CONCLUSION: The combination of detected antigen, used cells, and fixing solution may have different effects on immunoreactivity. ICC using LBC specimens is a useful technique, but the staining conditions should be examined before performing ICC.

The impact of cystic duct tube on the onset time of postoperative bile leakage after hepatectomy: A propensity score-matched analysis.

BACKGROUND: The cystic duct tube (C-tube) was used to reduce bile leakage (BL) incidence after hepatectomy. Nevertheless, delayed BL is sometimes experienced even using C-tube. This study investigates the impact of C-tube use on the onset time of post-hepatectomy BL. METHODS: Data from 455 consecutive patients who underwent hepatectomy without biliary reconstruction between November 2007 and July 2020 were analyzed retrospectively. A C-tube was used for intraoperative biliary injury or in consideration of BL risk. BL was divided into two groups according to the postoperative onset time: early onset and late onset. To assess the association between C-tube use and BL, propensity score matching in a 1:1 ratio was performed to match BL risk factors between the C-tube and no-C-tube groups. RESULTS: BL occurred in 30 (6.6%) of the 455 included patients. C-tubes were used in 51 patients (11.2%) with open hepatectomy, high-risk hepatectomy, massive blood loss, long operation time, or prophylactic drain placement. After propensity score matching, BL occurred in 17 of 102 patients (16.7%). Early-onset BL occurred significantly less frequently in the C-tube group than in the no-C-tube group (3.9% vs. 15.7%, p = 0.046); however, late-onset BL was more common in the C-tube group (9.8% vs. 3.9%, p = 0.24). Six of seven patients (85.7%) with BL with C-tube use developed BL after C-tube removal. CONCLUSION: C-tube drainage may reduce early-onset BL in cases having risk factors for BL. Conversely, since late-onset BL often occurs after C-tube removal, attention should be paid to those cases.

Relationship between a deep learning model and liquid-based cytological processing techniques.

OBJECTIVE: Artificial intelligence (AI)-based cytopathology studies conducted using deep learning have enabled cell detection and classification. Liquid-based cytology (LBC) has facilitated the standardisation of specimen preparation; however, cytomorphology varies according to the LBC processing technique used. In this study, we elucidated the relationship between two LBC techniques and cell detection and classification using a deep learning model. METHODS: Cytological specimens were prepared using the ThinPrep and SurePath methods. The accuracy of cell detection and cell classification was examined using the one- and five-cell models, which were trained with one and five cell types, respectively. RESULTS: When the same LBC processing techniques were used for the training and detection preparations, the cell detection and classification rates were high. The model trained on ThinPrep preparations was more accurate than that trained on SurePath. When the preparation types used for training and detection were different, the accuracy of cell detection and classification was significantly reduced (P < 0.01). The model trained on both ThinPrep and SurePath preparations exhibited slightly reduced cell detection and classification rates but was highly accurate. CONCLUSIONS: For the two LBC processing techniques, cytomorphology varied according to cell type; this difference affects the accuracy of cell detection and classification by deep learning. Therefore, for highly accurate cell detection and classification using AI, the same processing technique must be used for both training and detection. Our assessment also suggests that a deep learning model should be constructed using specimens prepared via a variety of processing techniques to construct a globally applicable AI model.

Effect of Specimen Processing Technique on Cell Detection and Classification by Artificial Intelligence.

OBJECTIVES: Cytomorphology is known to differ depending on the processing technique, and these differences pose a problem for automated diagnosis using deep learning. We examined the as-yet unclarified relationship between cell detection or classification using artificial intelligence (AI) and the AutoSmear (Sakura Finetek Japan) and liquid-based cytology (LBC) processing techniques. METHODS: The "You Only Look Once" (YOLO), version 5x, algorithm was trained on the AutoSmear and LBC preparations of 4 cell lines: lung cancer (LC), cervical cancer (CC), malignant pleural mesothelioma (MM), and esophageal cancer (EC). Detection and classification rates were used to evaluate the accuracy of cell detection. RESULTS: When preparations of the same processing technique were used for training and detection in the 1-cell (1C) model, the AutoSmear model had a higher detection rate than the LBC model. When different processing techniques were used for training and detection, detection rates of LC and CC were significantly lower in the 4-cell (4C) model than in the 1C model, and those of MM and EC were approximately 10% lower in the 4C model. CONCLUSIONS: In AI-based cell detection and classification, attention should be paid to cells whose morphologies change significantly depending on the processing technique, further suggesting the creation of a training model.

A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system.

We evaluated the efficacy of simultaneous multiple-gene knockout in human culture cells. By simple co-transfection of HeLa cells with a mixture of pX330-based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin-resistant cells, Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co-transfection of up to seven targeting plasmids for p38α, p38ß, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA-induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co-transfection-based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene-knockout cell lines.