Journal of Clinical Laboratory Analysis最新文献

Background: Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardised protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We compared two methods-in vitro cultivation and membrane integrity assessment-for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate.

Methods: Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions.

Results: Using different types of samples (pure bacterial culture, stool of germ-free and conventionally bred mice, native and heat-treated human stool), we refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in frozen human stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 30.3%, ranging from 7.4% to 60.1%. Conversely, the membrane integrity approach yielded more reproducible results, with a mean coefficient of variance for viable cells of 6.4% ranging from 0.2% to 18.2%.

Conclusion: Bacterial viability assessment in stool filtrate using the membrane integrity method offers robust and precise data, making it a suitable option for faecal material evaluation in FMT. In contrast, the cultivation-dependent methods produce inconsistent outcomes.

Background: Catecholamines (epinephrine; norepinephrine; and dopamine) and their O-methylated metabolites (metanephrine; normetanephrine; and 3-methoxytyramine) are biomarkers for pheochromocytoma and paraganglioma. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was recommended by Endocrine Society for detecting these compounds. The influence of blood collection tubes on the analysis of the six analytes by LC-MS/MS was not thoroughly investigated, which we want to clarify in our study.

Methods: Blood samples of healthy individuals were collected into serum, lithium heparin, and K₂EDTA plasma tubes separately. Samples were subjected to solid phase extraction and then analyzed by LC-MS/MS. The retention behavior and assay performance of the six analytes were assessed for samples from different collection containers. The impacts of potassium and sodium as the counter ions of EDTA on the retention time and matrix effect were also studied.

Results: Compared with O-methylated metabolites, the results for catecholamines were more affected by the collection tubes, especially for norepinephrine, which displayed severely suppressed signal and very low extraction efficiency in K₂EDTA plasma. Changing the counter ion of EDTA from potassium to sodium dramatically changed the retention behavior and matrix effect of norepinephrine.

Conclusions: It is necessary to evaluate blood collection tubes for catecholamines and their O-methylated metabolites analyzed by LC-MS/MS. In addition, attention should also be paid when the anticoagulant counter ion was changed.

Background: Interstitial lung diseases (ILD) is a group of lung disorders characterized by interstitial lung thickening due to inflammatory and fibrotic processes. Krebs von den Lungen-6 (KL-6) is a molecule secreted by damaged type II alveolar pneumocytes in the alveolar space. The goal of the present study was to compare two detection methods of KL-6 in both bronchoalveolar lavage (BAL) and serum from ILD patients at the moment of diagnosis.

Methods: Patients with suspicious of ILD and followed at two Italian referral centres for rare lung diseases were included in the study. BAL fluid and serum were collected and analysed by chemiluminescent enzyme immunoassay (CLEIA) and fluorescent enzyme immunoassay (FEIA) methods provided by Tosoh Biosciences.

Results: A total of 158 (mean age ± standard deviation, 61.5 ± 13.7, 65 females) patients were enrolled. A total of, 36 had diagnosis of idiopathic pulmonary fibrosis (IPF), 74 sarcoidosis, 15 connective tissue disease-ILD (CTD-ILD) and 33 other ILD. Diagnostic agreement between two methods was demonstrated for both BAL (r = 0.707, p < 0.0001) and serum (r = 0.816, p < 0.0001). BAL KL-6 values were lower than serum (p < 0.0001). IPF patients had higher serum KL-6 concentration than other ILDs (p = 0.0294), while BAL KL-6 values were lower in IPF than in non-IPF (p = 0.0023).

Conclusion: This study explored KL-6 concentrations through the CLEIA method in serum and BAL of patients with various ILDs, showing significant differences of biomarkers concentrations between IPF and other non-IPF ILDs. Our findings are promising as they provided further knowledge concerning KL-6 expression across different ILDs and may suggest its utility in differential diagnosis.

Objective: To compare the application of different treatments in the diagnosis of melanoma with severe pigment interference, to solve the problem of pigment interference with immunohistochemical interpretation.

Methods: The pigment-rich melanomas were first depigmented with potassium permanganate using a concentration gradient (0.1%, 0.5%, 1%) and a time gradient (1, 5, 10, 15, 30 min, 6 h), and the optimal concentration and time were found. Then, 12 cases of pigment-rich melanoma tissues were collected, and the tissues were stained with diaminobenzidine (DAB), alkaline phosphatase-fast red (AP red), multiplex immunofluorescence (MIF), and 3-amino-9-ethylcarbazole (AEC), and ferrous sulfate, comparing different methods, positive expression of HMB45, MelanA, S100, SOX10, ki67.

Results: First, the concentration of 0.5% potassium permanganate after 15 min treatment of the pigment significantly faded, and the intensity of antibody positivity was better than other concentrations and time. Second, after depigmentation treatment, the antibody positivity rate was 41.7%-66.7% for DAB, 66.7%-91.7% for AP red, 83.3%-100% for multiplex immunofluorescence, 25%-33.3% for AEC, and 33.3% for ferrous sulfate.

Conclusion: AP red staining and mIF are more suitable for the diagnosis of melanoma with severe pigment interference, and AP red staining is more economical and practical.

Background: The current investigation aims to analyze the occurrence of thalassemia in patients who participated in hemoglobin A1c (HbA1c) testing in clinical laboratory showing high hemoglobin F (HbF) level (≥ 1.5%) or abnormal Hb peak and predict the main influence factors by using different statistical models.

Methods: The current investigation is a single-center retrospective cohort study. HbA1c concentration was detected by using TOSOH HLC-723G8 glycated hemoglobin analyzer. SNaPshot SNP (Single Nucleotide Polymorphism) typing and AccuCopy technology were employed to detect mutations in thalassemia-related pathogenic genes.

Results: A total of 126 patients endured high HbF levels or abnormal Hb peak during HbA1c detection, and 66.7% of subjects (n = 84) showed thalassemia mutations. Three heterozygosity mutations, including c.52A>T (p.K18*), c.-78A>G, and c.126_129delCTTT(p.F42Lfs*19) present in HBB gene, were also identified. --^SEA/αα mutation demonstrated the youngest ages (p < 0.001). 17 M (p < 0.001) and 41/42 M (p < 0.01) mutations with β-thalassemia showed higher HbF levels compared with patients without thalassemia mutations. Except for -α^3.7, mutations in thalassemia showed lower levels of mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) compared with patients without thalassemia mutations. Patients with thalassemia mutations showed younger age (p < 0.001), lower Hb (p < 0.001), MCV and MCH levels (p < 0.001), higher red blood cell (RBC) count (p < 0.001), and platelet distribution width (PDW) level (p = 0.007) than patients without thalassemia mutations. Three statistical models indicate MCV is the most valuable independent factor for predicting thalassemia and ROC (receiver operating characteristic) curves analysis of AUC (Area Under the Curve) of 0.855 (95% CI [0.787-0.923], p < 0.001) with MCV.

Conclusion: High HbF level (≥ 1.5%) or abnormal Hb peak present in HbA1c testing indicated high incident rate of thalassemia. MCV is the most valuable independent predicting factor for subjects having thalassemia.

Background: Circulating tumor deoxyribonucleic acid (ctDNA) is increasingly applied in clinical practice. This study aimed to explore clinical utility of a minimal invasive and sensitive way of ctDNA for next-generation sequencing in non-small cell lung cancer (NSCLC) with inadequate tumor samples.

Methods: Targeted DNA sequencing was performed on tissue biopsies and matched plasma samples from 60 patients with NSCLC.

Results: A total of 13 driving genes were detected in 60 matched tissue DNA (tDNA) and ctDNA samples. Overall concordance rate was 75.47%, with 77.55% sensitivity and 50% specificity. Epidermal growth factor receptor (EGFR) mutations were the most common in both tDNA and ctDNA samples. Among other mutated genes were tumor protein p53 (TP53), erb-b2 receptor tyrosine kinase 2 (ERBB2), anaplastic lymphoma kinase (ALK), cyclin-dependent kinase inhibitor 2A (CDKN2A), ros proto-oncogene 1, and receptor tyrosine kinase (ROS1). Mutations in b-raf proto-oncogene, serine/threonine kinase (BRAF), cluster of differentiation 274 (CD274), neurotrophin receptor tyrosine kinase 1 (NTRK1), and rearranged during transfection (RET) occurred only in plasma. The majority of mutations in both samples were single-nucleotide variants. Deletions were found in EGFR, BRAF, and TP53 in ctDNA, whereas in tDNA, deletions were only found in EGFR. In ALK, single nucleic acid-site amplification occurred simultaneously in tissue and plasma, but insertions and copy number variations were detected only in plasma.

Conclusions: Identifying ctDNA mutations by targeted sequencing in plasma is feasible, showing the clinical value of ctDNA-targeted sequencing in NSCLC patients when tumor tissue sampling is insufficient or even impossible.

Background: The clinical value of procalcitonin (PCT) in infection diagnosis and antibiotic stewardship is still unclear. This study aimed to investigate the association between serum PCT and different clinical conditions as well as other infectious/inflammatory parameters in different septic patients in order to elucidate the value of PCT detection in infection management.

Methods: Chemiluminescence immunoassay was used for serum PCT analysis. Hematology analysis was used for complete blood cell count. Digital automated cell morphology analysis was used for blood cell morphology examination. Blood, urine, and stool cultures were performed according to routine clinical laboratory standard operating procedures. C-reactive protein (CRP) was analyzed by immunoturbidimetry. Erythrocyte sedimentation rate test was performed using natural sedimentation methods.

Results: Outpatients, ICU patients, and patients under 2 years of age with respiratory infections had higher serum PCT levels. Septic patients had the highest-serum PCT levels and other infection indexes. PCT levels in the blood, urine, and stool culture-positive patients were significantly higher than in culture-negative patients. The neutrophil granulation and reactive lymphocytes were observed together with the PCT-level increments in different septic patients, and these alterations were lessened after treatment. There was no significant change in monocyte morphology between pre- and posttreatment septic patients.

Conclusions: Serum PCT is associated with neutrophil cytotoxicity and lymphocyte morphology changes in sepsis; thus, the combination of neutrophil and lymphocyte digital cell morphology evaluations with PCT detection may be a useful examination for guiding the clinical management of sepsis.

Background: Oral candidiasis (OC) is one of the most common mucosal infections in those afflicted with HIV/AIDS. This study aimed to provide detailed information on the phenotype, genotype, antifungal susceptibility, and biofilm formation ability of oral Candida albicans isolated from HIV-infected patients with OC.

Methods: A total of 25 C. albicans isolates were collected from oral lesions of HIV-infected patients referred to Behavioral Diseases Counseling Center affiliated with Ahvaz Jundishapur University of Medical Sciences, Iran. The antifungal susceptibility testing was done according to CLSI M27 guideline (fourth edition). The crystal violet method was used to evaluate the biofilm formation ability of isolates. Different phenotypes were identified on yeast extract-peptone-dextrose agar medium supplemented with phloxine B. Genotyping analysis of the isolates was performed using high-resolution melting (HRM) assays and ABC genotyping.

Results: The highest and lowest susceptibility of the C. albicans isolates was found for fluconazole 24 (96%) and ITC 18 (72%), respectively. Forty-eight percent of the isolates had high biofilm formation ability and exhibited gray cell type. The most common genotype was genotype B (52%). HRM analysis of HIS3, EF3, and CDC3 markers showed three, four, and five different groups, respectively.

Conclusion: Investigating the phenotype, antifungal susceptibility and biofilm formation ability of the C. albicans isolates obtained from oral lesions of HIV-infected patients revealed that the dominant genotypes in the current research could cause more serious infections from the oral source. We recommend further research with a larger sample size to determine the molecular epidemiology of C. albicans among HIV patients in Iran.

Background: We developed a fully automated quantitative immunoassay for the detection of prostaglandin E-major urinary metabolite (PGE-MUM). In this study, we evaluated the analytical performance of this assay.

Methods: Sensitivity, within-run reproducibility, correlation with radioimmunoassay (RIA), cross-reactivity, dilution linearity, spike recovery performance, analyte stability, and effects of coexisting substances were evaluated. The assay was also used to measure PGE-MUM in 211 healthy people.

Results: The limit of detection and quantification were 1.0 and 1.3 ng/mL, respectively. When the assay was performed six times in a single run, the coefficient of variation ranged from 1.4% to 2.2%. The coefficient of correlation with a preceding RIA method was 0.970 with a correlation slope of 0.88. There was no cross-reactivity with PGE-MUM analogs. Linearity of dilution was confirmed at up to 16-fold dilution with assay results within 100 ± 20% of the theoretical values calculated based on the undiluted sample. Spike recovery was good and ranged from 94% to 101%. Analyte stability was tested by storing samples at 25°C for 6 days, 10°C for 1 month, and by performing up to five freeze-thaw cycles. Assay results were all within 100 ± 10%, the values measured before storage and before the freeze-thaw process. Assay results in healthy people ranged from 3.1 to 162.7 ng/mL (mean: 35.8 ng/mL). After correction for creatinine, the 95% confidence interval was 8.68-42.25 μg/g creatinine.

Conclusion: The assay precisely detects PGE-MUM.