On-the-fly Raman microscopy guaranteeing the accuracy of discrimination.

Accelerating the measurement for discrimination of samples, such as classification of cell phenotype, is crucial when faced with significant time and cost constraints. Spontaneous Raman microscopy offers label-free, rich chemical information but suffers from long acquisition time due to extremely small scattering cross-sections. One possible approach to accelerate the measurement is by measuring necessary parts with a suitable number of illumination points. However, how to design these points during measurement remains a challenge. To address this, we developed an imaging technique based on a reinforcement learning in machine learning (ML). This ML approach adaptively feeds back "optimal" illumination pattern during the measurement to detect the existence of specific characteristics of interest, allowing faster measurements while guaranteeing discrimination accuracy. Using a set of Raman images of human follicular thyroid and follicular thyroid carcinoma cells, we showed that our technique requires 3,333 to 31,683 times smaller number of illuminations for discriminating the phenotypes than raster scanning. To quantitatively evaluate the number of illuminations depending on the requisite discrimination accuracy, we prepared a set of polymer bead mixture samples to model anomalous and normal tissues. We then applied a home-built programmable-illumination microscope equipped with our algorithm, and confirmed that the system can discriminate the sample conditions with 104 to 4,350 times smaller number of illuminations compared to standard point illumination Raman microscopy. The proposed algorithm can be applied to other types of microscopy that can control measurement condition on the fly, offering an approach for the acceleration of accurate measurements in various applications including medical diagnosis.

Correction for Extrinsic Background in Raman Hyperspectral Images.

Raman hyperspectral microscopy is a valuable tool in biological and biomedical imaging. Because Raman scattering is often weak in comparison to other phenomena, prevalent spectral fluctuations and contaminations have brought advancements in analytical and chemometric methods for Raman spectra. These chemometric advances have been key contributors to the applicability of Raman imaging to biological systems. As studies increase in scale, spectral contamination from extrinsic background, intensity from sources such as the optical components that are extrinsic to the sample of interest, has become an emerging issue. Although existing baseline correction schemes often reduce intrinsic background such as autofluorescence originating from the sample of interest, extrinsic background is not explicitly considered, and these methods often fail to reduce its effects. Here, we show that extrinsic background can significantly affect a classification model using Raman images, yielding misleadingly high accuracies in the distinction of benign and malignant samples of follicular thyroid cell lines. To mitigate its effects, we develop extrinsic background correction (EBC) and demonstrate its use in combination with existing methods on Raman hyperspectral images. EBC isolates regions containing the smallest amounts of sample materials that retain extrinsic contributions that are specific to the device or environment. We perform classification both with and without the use of EBC, and we find that EBC retains biological characteristics in the spectra while significantly reducing extrinsic background. As the methodology used in EBC is not specific to Raman spectra, correction of extrinsic effects in other types of hyperspectral and grayscale images is also possible.

Lipid droplet accumulation and adipophilin expression in follicular thyroid carcinoma.

Follicular neoplasms of the thyroid include follicular thyroid carcinoma (FTC) and follicular thyroid adenoma (FTA). However, the differences in cytological findings between FTC and FTA remain undetermined. Here, we aimed to evaluate the accumulation of lipid droplets (LDs) and the expression of adipophilin (perilipin 2/ADRP/ADFP), a known LD marker, in cultured FTC cells. We also immunohistochemically compared adipophilin expression in the FTC and FTA of resected human thyroid tissues. Cultured FTC (FTC-133 and RO82W-1) possessed increased populations of LDs compared to thyroid follicular epithelial (Nthy-ori 3-1) cells. In vitro treatment with phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling inhibitors (LY294002, MK2206, and rapamycin) in FTC-133 cells downregulated the PI3K/Akt/mTOR/sterol regulatory element-binding protein 1 (SREBP1) signaling pathway, resulting in a significant reduction in LD accumulation. SREBP1 is a master transcription factor that controls lipid metabolism. Fluorescence immunocytochemistry revealed adipophilin expression in the LDs of FTC-133 cells. Immunohistochemical analysis of surgically resected human thyroid tissues revealed significantly increased expression of adipophilin in FTC compared with FTA and adjacent non-tumorous thyroid epithelia. Taken together, LDs and adipophilin were abundant in cultured FTC; the evaluation of adipophilin expression can help distinguish FTC from FTA in surgical specimens.

Differentiability of cell types enhanced by detrending a non-homogeneous pattern in a line-illumination Raman microscope.

A line illumination Raman microscope extracts the underlying spatial and spectral information of a sample, typically a few hundred times faster than raster scanning. This makes it possible to measure a wide range of biological samples such as cells and tissues - that only allow modest intensity illumination to prevent potential damage - within feasible time frame. However, a non-uniform intensity distribution of laser line illumination may induce some artifacts in the data and lower the accuracy of machine learning models trained to predict sample class membership. Here, using cancerous and normal human thyroid follicular epithelial cell lines, FTC-133 and Nthy-ori 3-1 lines, whose Raman spectral difference is not so large, we show that the standard pre-processing of spectral analyses widely used for raster scanning microscopes introduced some artifacts. To address this issue, we proposed a detrending scheme based on random forest regression, a nonparametric model-free machine learning algorithm, combined with a position-dependent wavenumber calibration scheme along the illumination line. It was shown that the detrending scheme minimizes the artifactual biases arising from non-uniform laser sources and significantly enhances the differentiability of the sample states, i.e., cancerous or normal epithelial cells, compared to the standard pre-processing scheme.

Mitochondrial Energy Metabolism in the Regulation of Thermogenic Brown Fats and Human Metabolic Diseases.

Brown fats specialize in thermogenesis by increasing the utilization of circulating blood glucose and fatty acids. Emerging evidence suggests that brown adipose tissue (BAT) prevents the incidence of obesity-associated metabolic diseases and several types of cancers in humans. Mitochondrial energy metabolism in brown/beige adipocytes regulates both uncoupling protein 1 (UCP1)-dependent and -independent thermogenesis for cold adaptation and the utilization of excess nutrients and energy. Many studies on the quantification of human BAT indicate that mass and activity are inversely correlated with the body mass index (BMI) and visceral adiposity. Repression is caused by obesity-associated positive and negative factors that control adipocyte browning, de novo adipogenesis, mitochondrial energy metabolism, UCP1 expression and activity, and noradrenergic response. Systemic and local factors whose levels vary between lean and obese conditions include growth factors, inflammatory cytokines, neurotransmitters, and metal ions such as selenium and iron. Modulation of obesity-associated repression in human brown fats is a promising strategy to counteract obesity and related metabolic diseases through the activation of thermogenic capacity. In this review, we highlight recent advances in mitochondrial metabolism, thermogenic regulation of brown fats, and human metabolic diseases.

Transplantation of Chemical Compound-Induced Cells from Human Fibroblasts Improves Locomotor Recovery in a Spinal Cord Injury Rat Model.

The development of regenerative medicine using cell therapy is eagerly awaited for diseases such as spinal cord injury (SCI), for which there has been no radical cure. We previously reported the direct conversion of human fibroblasts into neuronal-like cells using only chemical compounds; however, it is unclear whether chemical compound-induced neuronal-like (CiN) cells are clinically functional. In this study, we partially modified the method of inducing CiN cells (termed immature CiN cells) and examined their therapeutic efficacy, in a rat model of SCI, to investigate whether immature CiN cells are promising for clinical applications. Motor function recovery, after SCI, was assessed using the Basso, Beattie, and Bresnahan (BBB) test, as well as the CatWalk analysis. We found that locomotor recovery, after SCI in the immature CiN cell-transplanted group, was partially improved compared to that in the control group. Consistent with these results, magnetic resonance imaging (MRI) and histopathological analyses revealed that nerve recovery or preservation improved in the immature CiN cell-transplanted group. Furthermore, transcriptome analysis revealed that immature CiN cells highly express hepatocyte growth factor (HGF), which has recently been shown to be a promising therapeutic agent against SCI. Our findings suggest that immature CiN cells may provide an alternative strategy for the regenerative therapy of SCI.

Classifications of moderate to severe asthma phenotypes in Japan and analysis of serum biomarkers: A Nationwide Cohort Study in Japan (NHOM Asthma Study).

BACKGROUND: Asthma is a heterogeneous disease, and phenotyping can facilitate understanding of disease pathogenesis and direct appropriate asthma treatment. This nationwide cohort study aimed to phenotype asthma patients in Japan and identify potential biomarkers to classify the phenotypes. METHODS: Adult asthma patients (n = 1925) from 27 national hospitals in Japan were enrolled and divided into Global Initiative for Asthma (GINA) steps 4 or 5 (GINA 4, 5) and GINA Steps 1, 2, or 3 (GINA 1-3) for therapy. Clinical data and questionnaires were collected. Biomarker levels among GINA 4, 5 patients were measured. Ward's minimum variance hierarchical clustering method and tree analysis were performed for phenotyping. Analysis of variance, the Kruskal-Wallis, and chi-square tests were used to compare cluster differences. RESULTS: The following five clusters were identified: 1) late-onset, old, less-atopic; 2) late-onset, old, eosinophilic, low FEV1; 3) early-onset, long-duration, atopic, poorly controlled; 4) early-onset, young, female-dominant, atopic; and 5) female-dominant, T1/T2-mixed, most severe. Age of onset, disease duration, blood eosinophils and neutrophils, asthma control questionnaire Sum 6, number of controllers, FEV1, body mass index (BMI), and hypertension were the phenotype-classifying variables determined by tree analysis that assigned 79.5% to the appropriate cluster. Among the cytokines measured, IL-1RA, YKL40/CHI3L1, IP-10/CXCL10, RANTES/CCL5, and TIMP-1 were useful biomarkers for classifying GINA 4, 5 phenotypes. CONCLUSIONS: Five distinct phenotypes were identified for moderate to severe asthma and may be classified using clinical and molecular variables (Registered in UMIN-CTR; UMIN000027776.).

Potential of the prognostic nutritional index to determine the risk factor for severe infection in elderly patients with rheumatoid arthritis.

OBJECTIVE: To investigate the influence of nutritional status on severe infection complications in patients with rheumatoid arthritis (RA). METHODS: This retrospective cohort study on 2108 patients with RA evaluated the prognostic nutritional index (PNI) as an index of nutritional status. Patients were classified into the high or low PNI group according to the cutoff PNI value (45.0). Based on propensity score matching analysis, 360 patients in each group were selected for comparing the incidence of serious infection, clinical findings, and PNI scores. RESULTS: The incidence of infection was significantly higher in the low PNI group than in the high PNI group (p < 0.001). The occurrence rate of infectious complication at 104 weeks was significantly higher in the low PNI (<45.0) group than in the high PNI group (p < 0.001). The incidence of infection was particularly high in elderly patients (≥65 years) with a low PNI, but the incidence in elderly patients with a high PNI was similar to that in nonelderly patients with a high PNI. CONCLUSIONS: Patients with RA and malnutrition had a higher incidence of severe infection; thus, evaluating and managing nutritional status is necessary for the appropriate and safe treatment of elderly patients with RA.

Improvement of spatial resolution in photoacoustic microscopy using transmissive adaptive optics with a low-frequency ultrasound transducer.

Maintaining a high spatial resolution in photoacoustic microscopy (PAM) of deep tissues is difficult due to large aberration in an objective lens with high numerical aperture and photoacoustic wave attenuation. To address the issue, we integrate transmission-type adaptive optics (AO) in high-resolution PAM with a low-frequency ultrasound transducer (UT), which increases the photoacoustic wave detection efficiency. AO improves lateral resolution and depth discrimination in PAM, even for low-frequency ultrasound waves by focusing a beam spot in deep tissues. Using the proposed PAM, we increased the lateral resolution and depth discrimination for blood vessels in mouse ears.

5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence Imaging for Tumor Detection: Recent Advances and Challenges.

5-Aminolevulinic acid (5-ALA) is a natural amino acid and a precursor of heme and chlorophyll. Exogenously administered 5-ALA is metabolized into protoporphyrin IX (PpIX). PpIX accumulates in cancer cells because of the low activity of ferrochelatase, an enzyme that metabolizes PpIX to heme. High expression of 5-ALA influx transporters, such as peptide transporters 1/2, in cancer cells also enhances PpIX production. Because PpIX radiates red fluorescence when excited with blue/violet light, 5-ALA has been used for the visualization of various tumors. 5-ALA photodynamic diagnosis (PDD) has been shown to improve the tumor removal rate in high-grade gliomas and non-muscular invasive bladder cancers. However, 5-ALA PDD remains a challenge as a diagnostic method because tissue autofluorescence interferes with PpIX signals in cases where tumors emit only weak signals, and non-tumorous lesions, such as inflammatory sites, tend to emit PpIX fluorescence. Here, we review the current outline of 5-ALA PDD and strategies for improving its diagnostic applicability for tumor detection, focusing on optical techniques and 5-ALA metabolic pathways in both viable and necrotic tumor tissues.

Accumulation of Uroporphyrin I in Necrotic Tissues of Squamous Cell Carcinoma after Administration of 5-Aminolevulinic Acid.

5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is widely used for the intraoperative detection of malignant tumors. However, the fluorescence emission profiles of the accompanying necrotic regions of these tumors have yet to be determined. To address this, we performed fluorescence and high-performance liquid chromatography (HPLC) analyses of necrotic tissues of squamous cancer after 5-ALA administration. In resected human lymph nodes of metastatic squamous cell carcinoma, we found a fluorescence peak at approximately 620 nm in necrotic lesions, which was distinct from the PpIX fluorescence peak at 635 nm for viable cancer lesions. Necrotic lesions obtained from a subcutaneous xenograft model of human B88 oral squamous cancer also emitted the characteristic fluorescence peak at 620 nm after light irradiation: the fluorescence intensity ratio (620 nm/635 nm) increased with the energy of the irradiation light. HPLC analysis revealed a high content ratio of uroporphyrin I (UPI)/total porphyrins in the necrotic cores of murine tumors, indicating that UPI is responsible for the 620 nm peak. UPI accumulation in necrotic tissues after 5-ALA administration was possibly due to the failure of the heme biosynthetic pathway. Taken together, fluorescence imaging of UPI after 5-ALA administration may be applicable for the evaluation of tumor necrosis.

5-ALA-assistant automated detection of lymph node metastasis in gastric cancer patients.

BACKGROUND: 5-aminolevulinic acid (5-ALA) has been utilized for cancer diagnosis as a fluorescence probe. We have reported the feasibility of 5-ALA-induced protoporphyrin IX (PpIX) fluorescence for detecting lymph node (LN) metastasis in gastrointestinal malignancies. However, a major barrier to the fluorescence diagnosis has been that the evaluation has been highly dependent on the observers. In this study, we examined the validity of a developed device for automated detection without subjectivity. METHODS: Gastric cancer patients who received oral administration of 5-ALA (20 mg/kg) prior to surgery were enrolled. For a total of 323 LNs obtained from 64 patients, the diagnostic results of the device were compared to those of conventional histopathological examination based on hematoxylin-and-eosin-stained slides. The accuracy with the device was compared to that of stereoscopic detection with conventional fluorescence microscopy for 211 LNs from 42 patients. We used two types of image processing that we previously developed to eliminate autofluorescence of background tissues: differential and ratio methods. RESULTS: For detection of metastasis in 323 LNs, the areas under the receiver operating characteristic curves with the differential method and ratio method were 0.921 and 0.909, respectively. The sensitivity, specificity, and accuracy with the differential method were 78.0%, 96.8%, and 94.4%; while those with the ratio method were 78.0%, 96.1%, and 93.8%, respectively. In 211 LN analysis, the diagnostic accuracy with the device was comparable to that of stereoscopic examination. CONCLUSION: Our device for automated detection of LN metastasis using 5-ALA can be a useful tool for intraoperative diagnosis.

Crosstalk between tumor necrosis factor-alpha signaling and aryl hydrocarbon receptor signaling in nuclear factor -kappa B activation: A possible molecular mechanism underlying the reduced efficacy of TNF-inhibitors in rheumatoid arthritis by smoking.

OBJECTIVES: To examine the influence of smoking on biologics treatment against different therapeutic targets, such as TNFα, IL-6, and T cell, in rheumatoid arthritis (RA) and elucidate the underlying molecular mechanism. METHODS: The association between drug-discontinuation due to poor therapeutic response and smoking status was analyzed individually in biologics against different therapeutic targets by a multivariable logistic regression analysis using the "NinJa" Registry, one of the largest cohorts of Japanese RA patients. In vitro enhancement of TNFα-induced NF-κB activation and subsequent proinflammatory cytokine production by cigarette chemical components was examined by RT-PCR, qPCR, ELISA, and western blotting using an immortalized rheumatoid synovial cell line, MH7A. RESULTS: The rate of drug-discontinuation due to poor therapeutic response was higher in the current smoking group than in the never- or ever-smoking groups (the odds ratio of current/never smoking: 2.189, 95%CI; 1.305-3.672,P = 0.003; current/ever: 1.580, 95%CI; 0.879-2.839,P = 0.126) in the TNF inhibitor (TNFi) treatment group. However, this tendency was not observed in either the IL-6 or T cell inhibitor treatment groups. Cigarette smoke chemical components, such as benzo[α]pyrene, known as aryl hydrocarbon receptor (AhR) ligands, themselves activated NF-κB and induced proinflammatory cytokines, IL-1ß and IL-6. Furthermore, they also significantly enhanced TNFα-induced NF-κB activation and proinflammatory cytokine production. This enhancement was dominantly inhibited by Bay 11-7082, an NF-κB inhibitor. CONCLUSIONS: These results suggest a crosstalk between TNFα signaling and AhR signaling in NF-κB activation which may constitute one of the molecular mechanisms underlying the higher incidence of drug-discontinuation in RA patients undergoing TNFi treatment with smoking habits.

How to adjust endoscopic findings to histopathological findings of the stomach: a "histopathology-oriented" correspondence method helps to understand endoscopic findings.

BACKGROUND: Endoscopic findings have now become nearly as detailed as histopathological findings. Thus, one-to-one correspondence and precise feedback of histopathological findings is very desirable but may be very difficult to accomplish. We describe a systematic process called the Kyoto One-to-One Correspondence Method (the KOTO Method) that allows detailed adjustments of endoscopic findings to match histopathological findings. METHODS AND RESULTS: By comparing endoscopic and stereoscopic images of the gastric mucosa, we could obtain one-to-one correspondence between endoscopic images and equivalent histology in 44 of 47 fields. CONCLUSIONS: The histological structure of gastric cancers of the same histological subtype may not be similar. One-to-one correspondence between endoscopic images and gastric mucosal histology (histopathology-oriented correspondence) will improve endoscopic diagnosis and provide more useful information for pathological diagnosis.

Highly efficient direct conversion of human fibroblasts to neuronal cells by chemical compounds.

Direct conversion of mammalian fibroblasts into induced neuronal (iN) cells has been attained by forced expression of pro-neural transcriptional factors, or by combining defined factors with either microRNAs or small molecules. Here, we show that neuronal cells can be converted from postnatal human fibroblasts into cell populations with neuronal purities of up to >80% using a combination of six chemical compounds. The chemical compound-induced neuronal cells (CiNCs) express neuron-specific proteins and functional neuron markers. The efficiency of CiNCs is unaffected by either the donor's age or cellular senescence (passage number). We propose this chemical direct converting strategy as a potential approach for highly efficient generation of neuronal cells from human fibroblasts for such uses as in neural disease modeling and regenerative medicine.

Early therapeutic intervention with methotrexate prevents the development of rheumatoid arthritis in patients with recent-onset undifferentiated arthritis: A prospective cohort study.

OBJECTIVES: To examine whether or not earlier therapeutic intervention with methotrexate (MTX) prevents the development of rheumatoid arthritis (RA) in patients with recent-onset undifferentiated arthritis (UA) showing high anti-citrullinated peptide antibody (ACPA) titers. METHODS: The patients were divided into two groups, one was treated with MTX (MTX+ group, n = 29), and the other was treated without MTX (MTX- group, n = 19), and other disease-modifying anti-rheumatic drugs were not permitted in the two groups before the primary endpoint was met. The primary endpoint is the occurrence of definite RA, and it was compared in the two groups after 1 year. RESULTS: The percentage of patients who developed definite RA in the MTX+ group (17.2%) was significantly lower than that in the MTX- group (78.9%) (log-rank test, P < 0.001, n = 48); adjusted hazards ratio: 0.028 [95% confidence interval (CI): 0.003-0.250, P = 0.001, n = 39]. Treatment effectiveness was not decreased by major risk factors of RA onset such as smoking habits and human leukocyte antigen-DRB1 shared epitope (SE) (smoking habit, odds ratio [OR]: 0.041 [95% CI: 0.007-0.246] P < 0.001; SE, OR: 0.022 [95% CI: 0.002-0.204] P < 0.001). The safety issues were comparable between the two groups. CONCLUSIONS: This suggests that early therapeutic intervention with MTX could safely prevent the development of RA in patients with recent-onset UA showing high ACPA titers.

Label-free evaluation of myocardial infarction and its repair by spontaneous Raman spectroscopy.

Raman spectroscopy, which provides information about molecular species and structures of biomolecules via intrinsic molecular vibrations, can analyze physiological and pathological states of tissues on the basis of molecular constituents without staining. In this study, we analyzed Raman spectra of myocardial infarction and its repair in rats using the hypothesis that the myocardium in the course of myocardial infarction and its repair could be recognized by spontaneous Raman spectroscopy on the basis of chemical changes in myocardial tissues. Raman spectra were acquired from unfixed frozen cross sections of normal and infarcted heart tissues upon excitation at 532 nm. Raman spectra of the infarcted tissues were successfully obtained at characteristic time points: days 2, 5, and 21 after coronary ligation, at which the main components of the infarcted region were coagulation necrosis, granulation tissue, and fibrotic tissue, respectively. The latent variable weights calculated by a multivariate classification method, partial least-squares-discriminant analysis (PLS-DA), revealed fundamental information about the spectral differences among the types of tissues on the basis of molecular constituents. A prediction model for the evaluation of these tissue types was established via PLS-DA. Cross-validated sensitivities of 99.3, 95.3, 96.4, and 91.3% and specificities of 99.4, 99.5, 96.5, and 98.3% were attained for the discrimination of normal, necrotic, granulation, and fibrotic tissue, respectively. A two-dimensional image of a marginal area of infarction was successfully visualized via PLS-DA. Our results demonstrated that spontaneous Raman spectroscopy combined with PLS-DA is a novel label-free method of evaluating myocardial infarction and its repair.

Photoacoustic microscopy using ultrashort pulses with two different pulse durations.

We propose photoacoustic microscopy using ultrashort pulses with two different pulse durations in the range from femtoseconds to picoseconds. The subtraction of images for longer-pulse excitation from those for shorter-pulse excitation extracts two-photon photoacoustic images effectively, based on observation that the intensity ratio of two-photon to one-photon absorption-induced photoacoustic signals depends on the pulse duration in the same manner as the intensity ratio of two-photon and one-photon fluorescence signals. Two-photon photoacoustic microscopy using this subtraction method enables precise observation of the cross-sections of silicone hollows filled with the mixture of one-photon and two-photon absorption solutions.

Definite familial multiple system atrophy with unknown genetics.

Multiple system atrophy (MSA) is an oligodendrogliopathy of presumably sporadic origin, characterized by prominent α-synuclein inclusions with neuronal multisystem degeneration, although a few Mendelian pedigrees have been reported. Here we report two familial cases of MSA of unknown genetic background. One patient was diagnosed as a possible MSA-C (cerebellar dysfuntion) case, and the other as clinically possible MSA-P (parkinsonism), which turned out to be definite MSA, based on a detailed autopsy. The neuropathology showed extensive deposition of α-synuclein in the glia as well as in the neurons located in the cerebral cortices and hippocampal systems, although neither multiplication of the SNCA gene or mutations in COQ2 gene were identified in the family concerned.

An Autopsy Case of Severe COVID-19 Pneumonia Complicated by Intrapulmonary Thrombosis in Myelodysplastic/Myeloproliferative Neoplasm With Ring Sideroblasts and Thrombocytosis.

Patients with coronavirus disease 2019 (COVID-19) pneumonia are prone to intrapulmonary thrombosis owing to excessive inflammation and platelet activation. Myelodysplastic/myeloproliferative neoplasm (MDS/MPN) with ring sideroblasts and thrombocytosis (RS-T) is a rare disease in MDS/MPN overlap entities. Patients with MDS/MPN RS-T are known to be at a high risk of thrombosis, and platelet count control with drug therapy does not necessarily reduce this risk. Here, we report the autopsy case of an older male patient with MDS/MPN RS-T and severe COVID-19 pneumonia complicated by intrapulmonary thrombosis. His platelet count had been controlled in the normal range after treatment with hydroxyurea and 5-aza-2'-deoxycytidine. On admission day, he rapidly developed respiratory distress and tested positive on a polymerase chain reaction test for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). After admission, he received supplemental oxygen and was administered remdesivir and dexamethasone; however, his respiratory and circulatory status did not improve. The patient died on day 4 of illness. Autopsy findings revealed massive thrombi within blood vessels and diffuse alveolar damage in both lungs, which were determined to be the cause of death. In patients with MDS/MPN RS-T combined with COVID-19 pneumonia, clinicians may need to pay close attention to the risk of pulmonary thrombosis.