Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2.

BACKGROUND: The detection of serum antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a new tool for the coronavirus disease 2019 (COVID-19) diagnosis. Since many coronaviruses are sensitive to heat, heating inactivation of samples at 56°C prior to testing is considered a possible method to reduce the risk of transmission, but the effect of heating on the measurement of SARS-CoV-2 antibodies is still unclear. METHODS: By comparing the levels of SARS-CoV-2 antibodies before and after heat inactivation of serum at 56°C for 30 minutes using a quantitative fluorescence immunochromatographic assay RESULTS: We showed that heat inactivation significantly interferes with the levels of antibodies to SARS-CoV-2. The IgM levels of all the 34 serum samples (100%) from COVID-19 patients decreased by an average level of 53.56%. The IgG levels were decreased in 22 of 34 samples (64.71%) by an average level of 49.54%. Similar changes can also be observed in the non-COVID-19 disease group (n = 9). Of note, 44.12% of the detected IgM levels were dropped below the cutoff value after heating, suggesting heat inactivation can lead to false-negative results of these samples. CONCLUSION: Our results indicate that heat inactivation of serum at 56°C for 30 minutes interferes with the immunoanalysis of antibodies to SARS-CoV-2. Heat inactivation prior to immunoanalysis is not recommended, and the possibility of false-negative results should be considered if the sample was pre-inactivated by heating.

Unique Protein Profiles of Extracellular Vesicles as Diagnostic Biomarkers for Early and Advanced Non-Small Cell Lung Cancer.

Extracellular vesicles (EVs) mediate intercellular communication via transferring proteins and other biological molecules and have been recently investigated as biomarkers of disease. Sensitive and specific biomarkers are required for lung cancer diagnosis and prognosis. The present study screens for abnormal EV proteins in non-small cell lung cancer (NSCLC) using a quantitative proteomics strategy involving LC-MS/MS to identify ideal biomarkers for NSCLC diagnosis. EVs are enriched from the sera of early and advanced NSCLC patients and healthy controls and from cell culture supernatants of lung adenocarcinoma and bronchial epithelial cell lines. In the sera and supernatants, 279 and 632 differentially expressed proteins, respectively, are associated with signaling pathways including extracellular membrane-receptor interaction, focal adhesion, and regulation of the actin cytoskeleton. Thirty-two EV proteins are identified at the intersection of differentially expressed proteins between the NSCLC groups and cell lines. Based on bioinformatics analysis, in silico immunohistochemical, and PRM verification, fibronectin is selected for following in vitro studies and validation with an independent cohort. Fibronectin on EVs is estimated to perform well in the diagnosis of NSCLC patients based on AUC, showing great potential for clinical use and demonstrating the efficacy of this method for EV-associated biomarker screening.

Performance of the digital cell morphology analyzer MC-100i in a multicenter study in tertiary hospitals in China.

BACKGROUND: This study investigated the performance of the MC-100i, a pre-commercial digital morphology analyzer utilizing a convolutional neural network algorithm, in a multicentric setting involving up to 11 tertiary hospitals in China. METHODS: Blood smears were analyzed by MC-100i, verified by morphologists, and manually differentiated. The classification performance on WBCs and RBCs was evaluated by comparing the classification results using different methods. The PLT and PLT clump counting performance was also assessed. The total assay time including hands-on time was evaluated. RESULTS: The agreements between pre- and post-classification were high for normal WBCs (κ > 0.96) and lower for overall abnormal WBCs (κ = 0.90). The post-classification results correlated well with manual differentials for both normal and abnormal WBCs (r > 0.93), except for basophils (r = 0.8480) and atypical lymphocytes (r = 0.8211). The clinical sensitivity and specificity of each RBC abnormality after verification were above 90 % using microscopy reviews as the reference. The PLTs counted by the MC-100i before and after verification correlated well with those measured by the PLT-O mode (r = 0.98). Moreover, PLT clumps were successfully classified by the analyzer in EDTA-dependent pseudothrombocytopenia blood samples. CONCLUSIONS: The MC-100i is an accurate and reliable digital cell morphology analyzer, offering another intelligent option for hematology laboratories.

Comparison of the performance of two automatic cell morphology analyzers for peripheral-blood leukocyte morphology analysis: Mindray MC-100i and Sysmex DI-60.

INTRODUCTION: To compare the morphological classification ability of peripheral-blood leukocytes of the automatic cell morphology analyzers MC-100i and DI-60. METHODS: (1) MC-100i and DI-60 were used to analyze leukocytes in 432 venous blood samples collected from three tertiary hospitals across China. The preclassification results were compared with the results reported by senior morphological experts (postclassification results) to evaluate the accuracy, sensitivity, specificity, and consistency of leukocyte preclassification for both instruments. (2) In 200 of the 432 blood samples, morphological experts conducted manual microscopic examination for various types of leukocytes. The correlation between the MC-100i and DI-60 leukocyte postclassification results and the expert microscopist results were analyzed. RESULTS: (1) MC-100i preclassified leukocytes and nucleated red blood cells (RBCs). Compared with the postclassification results, the total leukocyte preclassification accuracy of MC-100i was 97.16%, while that of DI-60 was 87.24%. The sensitivity of MC-100i to abnormal cells (including blasts, promyelocytes, neutrophilic myelocytes, neutrophilic metamyelocytes, reactive lymphocytes, abnormal promyelocytes, plasma cells, abnormal lymphocytes and nucleated RBCs) was 90.24%, which was significantly higher than the 50.72% sensitivity of DI-60. (2) Comparing the postclassification results with manual microscopy, except for reactive lymphocytes and basophils, the MC-100i and DI-60 results had good correlations with various leukocyte types and nucleated RBCs (r > 0.85), and MC-100i was better than DI-60 in the recognition of basophils. CONCLUSION: Both MC-100i and DI-60 have good detection ability for five normal types of leukocytes in peripheral blood. MC-100i has significantly better detection sensitivity for abnormal cells in peripheral blood than DI-60.

A PTPN22 promoter polymorphism -1123G>C is associated with RA pathogenesis in Chinese.

The minor allele of the non-synonymous single nucleotide polymorphism (SNP) +1858C>T within the PTPN22 gene has now been unequivocally confirmed as conferring susceptibility to RA in population from Europe and America, but not in population from Asia. The aim of this study was to jointly address and integrate these separate findings to further elucidate the association between the PTPN22 gene and RA in Chinese Hans of Guangdong province. Four hundred and ninety-four cases with RA and 496 healthy controls were randomly selected, their SNPs at position -1123G>C (rs2488457), +1858C>T (rs2476601), +788G>A (rs33996649), and rs1310182 were genotyped using PCR-RFLP, followed by agarose gel electrophoresis. +1858C>T (rs2476601) and +788G>A (rs33996649) are not polymorphic in Chinese Hans. Meanwhile, our result reveals that the degree of association between the promoter polymorphism, -1123G>C and RA, was analogous to that observed in Japanese reports (odds ratio [OR] = 1.517, 95% CI = [1.154-1.995], P = 0.003). Expression study also indicated a tendency for association between -1123G>C and PTPN22 gene expression. Our study underpins that the promoter polymorphism, -1123G/C, may be a causal SNP for RA in Asian.

A deep learning-based system for assessment of serum quality using sample images.

BACKGROUND: Serum quality is an important factor in the pre-analytical phase of laboratory analysis. Visual inspection of serum quality (including recognition of hemolysis, icterus, and lipemia) is widely used in clinical laboratories but is time-consuming, subjective, and prone to errors. METHODS: Deep learning models were trained using a dataset of 16,427 centrifuged blood images with known serum indices values (including hemolytic index, icteric index, and lipemic index) and their performance was evaluated by five-fold cross-validation. Models were developed for recognizing qualified, unqualified and image-interfered samples, predicting serum indices values, and finally composed into a deep learning-based system for the automatic assessment of serum quality. RESULTS: The area under the receiver operating characteristic curve (AUC) of the developed model for recognizing qualified, unqualified and image-interfered samples was 0.987, 0.983, and 0.999 respectively. As for subclassification of hemolysis, icterus, and lipemia, the AUCs were 0.989, 0.996, and 0.993. For serum indices and total bilirubin predictions, the Pearson's correlation coefficients (PCCs) of the developed model were 0.840, 0.963, 0.854, and 0.953 respectively. Moreover, 30.8% of serum indices tests were deemed unnecessary due to the preliminary application of the deep learning-based system. CONCLUSIONS: The deep learning-based system is suitable for the assessment of serum quality and holds the potential to be used as an accurate, efficient, and rarely interfered solution in clinical laboratories.

Screening for malignant tumor cells in serous effusions with an automatic hematology analyzer using a novel diagnostic algorithm.

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 0.022×109/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 0.006×109/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.

Zinc­finger E­box­binding homeobox 1 alleviates acute kidney injury by activating autophagy and the AMPK/mTOR pathway.

Zinc­finger E­box­binding homeobox 1 (ZEB1) is involved in epithelial­mesenchymal transition. In the present study, the protective effect of ZEB1 on acute kidney injury (AKI) was explored. The cecal ligation and puncture (CLP) method was performed to establish the AKI model in rats. ZEB1 expression, blood urea nitrogen (BUN) and serum creatinine (SCr) levels, inflammation [interleukin (IL)­1ß, IL­6, and tumour necrosis factor­α], phosphorylated AMP­activated protein kinase (p­AMPK) and phosphorylated mammalian target of rapamycin (p­mTOR) expression, and histopathological changes in CLP­induced AKI rats were assessed. AMPK inhibitor dorsomorphin (DM) was intraperitoneally injected to determine the effect of ZEB1 on AKI and the regulatory mechanism involving the AMPK/mTOR pathway. CLP downregulated ZEB1 expression, increased BUN and SCr levels, promoted inflammation and apoptosis, and increased the acute kidney score in the kidney tissues of CLP­induced AKI rats. Autophagy and the AMPK/mTOR pathway were blocked in CLP­induced AKI rats. ZEB1 overexpression inhibited inflammation and apoptosis, reduced BUN and SCr levels, and activated autophagy and the AMPK/mTOR pathway in CLP­induced AKI rats. The protective effect of ZEB1 overexpression on AKI was reversed by DM. Thus, ZEB1 was revealed to alleviate CLP­induced AKI by activating autophagy and the AMPK/mTOR pathway.

EDTA-K2 Improves the Detection Sensitivity of SARS-CoV-2 IgM and IgG Antibodies by Chelating Colloidal Gold in the Immunochromatographic Assay.

OBJECTIVE: 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.

A rapid and accurate DHPLC assay for determination of apolipoprotein E genotypes.

The variants of apolipoprotein E (apoE) are closely related to hyperlipidemia III, Alzheimer's disease (AD), coronary artery disease (CAD) and many other human lipid metabolism-related problems. A rapid and accurate genotyping method specific for polymorphisms of the APOE gene is needed for population screening as well as diagnosis of apoE-related diseases in both the research and clinical setting. A polymerase chain reaction (PCR) method was designed to generate a 191-bp amplicon, which contains two common polymorphisms located in codons 112 and 158 of exon 4 of the APOE gene. The PCR amplicons for each sample were subjected to denaturing high-performance liquid chromatography (DHPLC) analysis, which was performed under partially denaturing conditions as determined by profiling the mixture of a tested sample and a homozygous standard control amplicon at the given ratio. A total of 297 DNA samples from Chinese population were enrolled to evaluate the specificity of the assay. A blinded validation study was then performed on 130 samples randomly selected from each of the six genotype groups as determined by DHPLC profiling. The genotypes obtained with the DHPLC method were in full agreement with those obtained by direct sequencing (130/130). We have developed a PCR/DHPLC genotyping assay capable of simultaneously determining all six genotypes of APOE gene in unknown test samples at one time.

[Prognostic value of changes of peripheral blood T cell subsets after thermoradiotherapy for nasopharyngeal carcinoma].

OBJECTIVE: To assess the prognostic value of changes of peripheral blood T cell subsets after thermoradiotherapy for nasopharyngeal carcinoma (NPC). METHODS: Peripheral blood T cell subsets in 20 normal subjects (control group), 30 NPC patients undergoing thermoradiotherapy, and 20 NPC patients undergoing radiotherapy were detected by flow cytometry. RESULTS: The percentages of CD3+CD4+ and CD8+CD28+ cells were decreased and the percentages of CD3+CD8+ and CD8+CD28- cells increased as compared with the measurements in normal persons. One month after thermoradiotherapy, the percentages of CD3+CD4+ and CD8+CD28+ cells further decreased and the percentages of CD3+CD8+ and CD8+CD28- cells further increased, which continued to worsen 3 months after the treatment and appeared to be related to the survival of the patients. CONCLUSION: T cell subsets of NPC patients are abnormal and their immune functions depressed in NPC patients within a long period after thermoradiotherapy. CD8+CD28+ and CD8+CD28- T cell subsets can be significant for prognostic assessment in these patients after thermoradiotherapy.