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Background Lung cancer is the leading cause of cancer-related deaths in the world. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases. For lack of conveniently sensitive and specific biomarkers, the majority of patients are in the late stage at initial diagnosis. Long non-coding RNAs (LncRNAs), a novel type of non-coding RNA, have recently been recognized as critical factors in tumor initiation and progression, but the role of exosomal LncRNAs has not been thoroughly excavated in NSCLC yet. Methods We isolated exosomes from the serum of patients with NSCLC and healthy controls. Exosome RNA deep sequencing was subsequently performed to detect differentially expressed exosomal LncRNAs. qRT-PCR assay was then utilized to validate dysregulated LncRNAs in both testing and multicentric validation cohort. Receiver operating characteristic (ROC) curve was used to detect the diagnostic capability of exosomal biomarkers. Furthermore, Kaplan–Meier analysis was applied to evaluate the prognostic values of these molecules. Results On the basis of analysis, we found that novel exosomal LncRNA RP5-977B1 exhibited higher levels in NSCLC than that in the healthy controls. The area under the curve (AUC) value of exosomal RP5-977B1 was 0.8899 and superior to conventional biomarkers CEA and CYFRA21-1 both in testing and multicentric validation cohort. Interestingly, the diagnostic capability of exosomal RP5-977B1 was also validated in early-stage patients with NSCLC. Furthermore, high expression of exosomal RP5-977B1was closely related with worse prognosis in NSCLC (P = 0.036). Conclusions Our results suggested that exosomal RP5-977B1 might serve as a novel “liquid biopsy” diagnostic and prognostic biomarker to monitor NSCLC and improve possible therapy.

BACKGROUND: Due to the high false-positive rate of the high-fluorescence body fluid (HF-BF) cell parameter of the hematology analyzer in BF mode, a novel algorithm based on the Mindray BC-6800 Plus hematology analyzer (BC-6800Plus), with higher diagnostic accuracy compared to that of the traditional HF-BF algorithm, was used to screen for malignant tumor cells in clinical BF samples. In this study, the body fluid mode of BC-6800Plus was applied to investigate the ability of its available parameters and characteristic regional particles in tumor cells screening. METHODS: A total of 220 BF samples (including pleural effusion and ascites) were randomly classified into a training cohort (154 samples) and a validation cohort (66 samples), and detected on the BC-6800Plus in BF mode. Based on the scatter plot analysis of the instrument, a novel gating algorithm, malignant cell algorithm-body fluid (MA-BF), was designed to detect the aggregated cells expressing highest fluorescence (FL) signals and side-scatter (SS) signals than other cells. BF collection and analyses were performed in compliance with the CLSI H56-A guideline. tumor cell-positive samples were defined as greater than or equal to confirIIIb (Papanicolaou class system) by the pathological examination. The diagnostic accuracy of HF-BF and MA-BF were determined by the receiver operating characteristic (ROC) curve analysis. RESULTS: When the cutoff values of the absolute count (HF-BF#) and relative count (HF-BF%) were set as 0.022×10(9)/L and 3.0%, respectively, the area under curve (AUC), sensitivity, and specificity were 0.76, 0.85 and 0.55 for HF-BF#, and were 0.70, 0.85, and 0.49 for HF-BF%, respectively. The new parameters, the absolute tumor cell count (MA-BF#) and relative count (MA-BF%), were established in the training cohort using the novel algorithm. We confirmed the cutoff values of MA-HF# and MA-HF% in BF were set as 0.006×10(9)/L and 0.2% in the training cohort, respectively. In the validation cohort, the AUC, sensitivity, and specificity were 0.89, 0.93, and 0.78 for MA-BF#, and were 0.89, 0.87 and 0.75 for MA-BF%, respectively. CONCLUSIONS: The MA-BF parameters of the novel algorithm output had better diagnostic accuracy for BF tumor cells than the traditional HF-BF parameters.

Xiumei Hu,1,* Changmeng Wu,1,* Bo Situ,1,* Peifu Tian,2,* Taixue An,1 Qiang Li,1 Weilun Pan,1 Ruyi Zhang,1 Biao Yang,3 Dehua Sun,1 Yuhai Hu,2 Qian Wang,1,3 Lei Zheng1 1Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, People’s Republic of China; 2Department of Medicine Laboratory, Hankou Hospital of Wuhan, Wuhan 430010, People’s Republic of China; 3Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510280, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lei Zheng; Qian Wang Email nfyyzhenglei@smu.edu.cn; wangqian@smu.edu.cnObjective: The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now rapidly spreading globally. Serological tests are an important method to assist in the diagnosis of COVID-19, used for epidemiological investigations. In this study, we aimed to investigate the impact of different types of vacuum collection tubes on the detection of SARS-CoV-2 IgM and IgG antibodies, using the colloidal gold immunochromatographic assay (GICA).Patients and Methods: A total of 112 patients with COVID-19 and 200 healthy control subjects with no infection were enrolled in this study. Their serum and plasma were collected into four different types of vacuum blood collection tubes. SARS-CoV-2 IgM and IgG specific antibodies in the plasma and serum were then detected by GICA and chemiluminescence assay (CA), respectively. In addition, the particle sizes of different colloidal gold solutions in the presence of different anticoagulants and coagulants were evaluated by both laser diffraction (Malvern) and confocal laser microscope, respectively.Results: Our results revealed that anticoagulated plasma with EDTA-K2 improved the positive detection rate of SARS-CoV-2 IgM antibodies. Furthermore, our results shown that the detection results by GICA and CA were highly consistent, especially, the results of EDTA-K2 anticoagulated plasma detected by GICA was more consistent with CA results. We confirmed that EDTA-K2 could improve the detection sensitivity of SARS-CoV-2 IgG antibodies by chelating excessive colloidal gold compared with sodium citrate or lithium heparin, these methodologies did not appear to cause false positives. Colloidal gold particles could be chelated and aggregated by EDTA-K2, but not by sodium citrate, lithium heparin and coagulants.Conclusion: GICA is widely used to detect antibodies for the advantages of convenient, fast, low cost, suitable for screening large sample and require minimal equipment. In this study, we found that EDTA-K2 amplified the positive antibody signal by chelating colloidal gold and improved the detection sensitivity of SARS-CoV-2 IgM and IgG antibodies when using the GICA. Therefore, we suggested that EDTA-K2 anticoagulated plasma was more suitable for the detection of SARS-CoV-2 antibodies.Keywords: EDTA-K2, SARS-CoV-2, antibodies, gold immunochromatographic assay; GICA

Highlights • Heating-activation at 56 °C for 30 min does not impair the antibodies of SARS-CoV-2. • Heating-activation would not decrease the diagnostic efficacy of SARS-CoV-2 IgM or IgG antibodies (ELISA-immunoassay). • Sera inactivated by heating should be recommended to minimize the risk of virus contamination of laboratory staff., Background To establish a safe and accurate method for detecting SARS-CoV-2 IgM and IgG, we assessed the impact of sera after heat-inactivation on the SARS-CoV-2 IgM and IgG levels measured by ELISA-immunoassay. Methods The serum samples of 62 patients with COVID-19 and 18 healthy controls were collected in Hankou’s Hospital of Wuhan from February 27 to March 6, 2020. Before and after the samples were inactivated, the levels of IgM and IgG antibodies were measured. Results The indexes of antibodies after inactivated were significantly higher than those in fresh sera, while the positive rates in all participants or in patients with COVID-19 did not change. The positive coincidence rate, negative coincidence rate and total coincidence rate of IgM antibodies before and after inactivation were 100.00% (55/55), 96.00% (24/25) and 98.75% (79/80), respectively (κ = 0.971, P

Exosomes are cell-derived vesicles and are abundant in biological fluids; they contain RNA molecules which may serve as potential diagnostic biomarkers in 'precision medicine'. To promote the clinical application of exosomal RNA (exoRNA), many isolation methods must be compared and validated. Exosomes in cell culture medium (CCM) and serum may be isolated using ultracentrifugation (UC), ExoQuick or Total Exosome Isolation Reagent (TEI), and exoRNA may be extracted using TRIzol-LS, SeraMir, Total Exosome RNA Isolation (TER), HiPure Liquid RNA/miRNA kit (HLR), miRNeasy or exoRNeasy. ExoRNA was assessed using NanoDrop, Bioanalyzer 2100, quantitative polymerase chain reaction and high-throughput sequencing. UC showed the lowest recovery of particles, but the highest protein purity for exosome isolation. For isolation of exoRNA, we found that combinations of the TEI and TER methods resulted in high extraction efficiency and purity of small RNA obtained using CCM. High yield and a narrow size distribution pattern of small RNA were shown in exoRNA isolated by exoRNeasy from serum. In RNA profile analysis, the small RNA constituent ratio, miRNA content and amount varied as a result of methodological differences. This study showed that different methods may introduce variations in the concentration, purity and size of exosomes and exoRNA. Herein we discuss the advantages and disadvantages of each method and their application to different materials, therefore providing a reference according to research design.

Surface protein patterns of tumor-derived exosomes could be promising noninvasive diagnostic biomarkers for liquid biopsy. However, a convenient and cost-effective platform for exosomal protein profiling is still lacking. Herein, a facile fluorescent aptasensor is developed to assess exosomal tumor-associated proteins, combining aptamers, aggregation-induced emission luminogens (AIEgens), and graphene oxide (GO) as recognition elements, fluorescent dye, and the quencher, respectively. Specifically, numberous TPE-TAs could bind one aptamer and form aggregates rapidly, resulting in an amplified fluorescence signal. In the absence of tumor-derived exosomes, GO absorbs the TPE-TAs/aptamer complex, allowing fluorescence quenching. When the target exosomes are introduced, the aptamer preferentially binds with its target. Thus the TPE-TAs/aptamer complexes detach from GO surface, followed by the appearance of a “turn-on” fluorescent signal. Under the optimized conditions, the linear range of target exosomes is estimated to be 4.07 × 105 to 1.83 × 107 particles/μL (0.68–30.4 pM) with a detection limit of 3.43 × 105 particles/μL (0.57 pM). This strategy demonstrated great performance in differentiating prostate cancer from healthy individuals (AUC: 0.9790). Furthermore, by profiling three tumor-associated protein markers including epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (HER2) on exosomes in a breast tumor cohort, this sensing platform diagnoses breast tumors with high efficiency (AUC: 0.9845) and exhibits a high sensitivity of 97.37% for distinguishing malignant breast cancers, where the stage I cases were detected with 92.31% sensitivity. Therefore, this aptasensor provides a promising strategy to profile tumor-derived exosomal proteins for early diagnosis in liquid biopsy.

A novel quantum dot-doped polystyrene nanoparticles-based lateral flow test strips (QPs-LFTS) system was developed to simultaneously detect a cytokeratin-19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA) in human serum to aid the diagnosis and prognosis of lung cancer. Quantum dot-doped carboxylate-functionalized polystyrene nanoparticles (QPs) were prepared and introduced as fluorescent reporters in QPs-LFTS. The detection was based on a sandwich immunoassay and performed on lateral flow test strips, with an assay time of 15 min. The strips were read by a fluorescence strip reader to obtain the fluorescence peak heights of the test lines (HT) and the control line (HC). The ratio of HT/HC was used for quantitation. The QPs showed excellent photoproperties and good performance. Under optimal conditions, the QPs-LFTS system exhibited a wide linear range for CYFRA 21-1 (1.3–480 ng/mL) and CEA (2.8–680 ng/mL). The detection limits for CYFRA 21-1 and CEA were 0.16 and 0.35 ng/mL, respectively. The recovery and reproducibility of the method were satisfactory. Furthermore, excellent correlations (n =120, R2 =0.9862, P