OBJECTIVES: To determine whether 20 % or 25 % polyethylene glycol 6000 (PEG) is more suitable for macroprolactinemia screening and to investigate methodological factors affecting PEG precipitation. METHODS: Serum samples from five individuals with macroprolactinemia and five hyperprolactinemic controls were analyzed. Gel filtration chromatography quantified the precipitation of individual prolactin (PRL) molecular forms. Nonspecific monomeric PRL precipitation was assessed using an artificial serum matrix. Incubation time, temperature, pH, and PEG solvent were also investigated. RESULTS: In the artificial serum matrix, monomeric PRL precipitation was significantly lower with 20 % than with 25 % PEG (29.3 ± 5.0 % vs. 39.2 ± 6.2 %; p=0.0053). In macroprolactinemic sera, both PEG concentrations precipitated more than 90 % of macroPRL and big PRL, with no significant differences between the two concentrations. However, monomeric PRL precipitation was significantly lower with 20 % than with 25 % PEG (28.5 ± 9.4 % vs. 39.8 ± 11.0 %; p=0.0033). Despite this difference, the proportions of monomeric PRL in the PEG precipitates differed by only 3.0 percentage points. Incubation time and temperature had limited effects on the signal-to-noise ratio. Increasing pH reduced precipitation rates without substantially altering this ratio. A 0.1 mol/L phosphate buffer at pH 7.0 maintained higher precipitation rates in alkalinized, long-term-stored serum samples. CONCLUSIONS: Both PEG concentrations efficiently precipitated macroPRL and big PRL, whereas 20 % PEG reduced nonspecific monomeric PRL precipitation and may be preferable for macroprolactinemia screening. A 0.1 mol/L phosphate buffer at pH 7.0 may be advantageous as a PEG solvent for analyzing alkalinized, long-term-stored serum samples.
Objectives Lower respiratory tract infections (LRTI) remain a leading cause of global morbidity and mortality, with approximately 62 % of cases lacking definitive etiological diagnoses via conventional methods. This study aimed to develop and validate a multiplex PCR-based targeted next-generation sequencing (mp-tNGS) panel for comprehensive pathogen detection in respiratory samples. Methods We conducted a combined retrospective (n=209) and prospective (n=256) study involving 465 bronchoalveolar lavage fluid (BALF) samples from patients with suspected LRTI. The mp-tNGS panel was designed to detect 277 clinically relevant pathogens, integrating simultaneous DNA/RNA co-detection and single-nucleotide polymorphism (SNP)-based contamination control. Diagnostic performance was evaluated against conventional microbiological tests (CMTs) and metagenomic next-generation sequencing (mNGS). Results The mp-tNGS assay revealed a limit of detection (LoD) of 25 copies/mL for bacteria/fungi and 50 copies/mL for viruses, with acceptable precision (intrabatch CV=9.42 %; interbatch CV=26.03 %). A novel SNP-based bioinformatics pipeline achieved exceptional discriminative performance (AUC=0.992) for distinguishing true infections from contamination. Compared with CMTs and mNGS, mp-tNGS showed superior sensitivity (93.59 %, 95 % CI: 90.81–95.73 %), particularly for viruses (AUC=0.92 vs. 0.845). The assay reliably identified fastidious pathogens (e.g., Pneumocystis jirovecii and Legionella pneumophila ) and predicted antimicrobial resistance (97.08 % concordance with phenotypic testing). Conclusions We developed an mp-tNGS panel with diagnostic performance comparable to that of mNGS. The panel integrates broad pathogen coverage, rapid turnaround time, machine learning-based false-positive reduction, and concordance with phenotypic susceptibility testing, positioning it as a promising molecular diagnostic tool for LRTI.
Abstract Objectives Cross-reactivity with high molecular-weight (MW) macroprolactin in prolactin (PRL) immunoassays has been the subject of numerous studies. In contrast, little data are available regarding Big-PRL, a 50–60 kDa PRL isoform. Methods The immunoreactivity of Big-PRL was evaluated on five platforms (Alinity (Abbott), DxI 800 (Beckman Coulter), Liaison XL (DiaSorin), Kryptor compact plus (Thermo Scientific), and iSYS (IDS Immunodiagnostic Systems)) using 10 serum samples characterized by gel filtration chromatography (GFC) and containing between 17 and 80 % of Big-PRL. The spread of the results as a function of Big-PRL percentage was determined. Bland Altman bias between assays was calculated using a panel of GFC-characterised sera containing macroprolactin (n=46) or monomeric PRL (n=45). Results The Big-PRL samples were obtained from nonpregnant women, seven of whom had galactorrhea or oligospaniomenorrhea. All assays recognized Big-PRL in undiluted serum but to a variable extent. The spread of total PRL values correlated positively with Big-PRL percentage (r=0.954, p<0.0001). In samples containing >70 % Big-PRL, the methods ranked in the same order of recognition as that observed with macroprolactin-rich samples. Big-PRL was strongly detected by Alinity and iSYS whereas Kryptor and DxI were less sensitive to Big-PRL, as observed with macroprolactin. Consequently, the risk of false diagnosis of hyperprolactinemia tended to be lower with these two last assays. Conclusions All five immunoassay platforms recognize the Big-PRL component in serum but to a variable extent. A high proportion of Big-PRL, combined with the use of a highly cross-reactive assay, may cause a false diagnosis of hyperprolactinemia.
Objectives Interlaboratory variability in HEp-2 indirect immunofluorescence assay (IFA) interpretation remains a major challenge. Conventional agreement metrics quantify disagreement but do not distinguish discrepancies by diagnostic relevance. We aimed to develop a likelihood ratio (LR)-based framework for clinically informed assessment of interpretation variability. Methods Pattern-specific positive LRs were derived from 33,690 routine HEp-2 IFA records and used to classify patterns into four risk tiers. A pattern discrepancy score (PDS) was developed to quantify discrepancies according to risk-tier displacement. Interlaboratory variability was assessed using a standardized 200-specimen panel tested by 18 laboratories, and diagnostic performance was explored in six laboratories. Results Pattern disagreement rates ranged from 17.5 to 50.0 %. PDS strongly correlated with disagreement rate (Spearman's rho=0.936, p<0.001), but laboratories with similar disagreement rates differed in cross-tier discrepancy distribution. Most discrepancies remained within the same risk tier; among discordant interpretations involving high-risk reference patterns, 20.4 % crossed into another tier. Pattern concordance varied markedly, while titer agreement was moderate to almost perfect (weighted kappa, 0.569-0.821). Across six laboratories, diagnostic performance varied (Youden Index, 0.391-0.617), but was not significantly associated with interpretation consistency. Conclusions HEp-2 IFA interpretation variability differs in both frequency and potential diagnostic relevance. The LR-based framework and PDS complement conventional agreement measures by identifying discrepancies that alter diagnostic risk classification, potentially supporting risk-oriented proficiency assessment and targeted quality improvement.
Objectives To establish age- and sex-specific reference intervals (RI) for complete blood count (CBC) parameters in healthy Vietnamese children aged 0 to <19 years and assess the impact of applying locally derived vs. non-local pediatric RI on abnormal flagging and clinical classification. Methods A community-based cross-sectional study included 3,885 healthy Vietnamese children aged 0 to <19 years recruited through school-based health screening programs between April 2024 and November 2025. Peripheral venous blood samples were analyzed using the Sysmex XN-9000 hematology analyzer under standardized pre-analytical and analytical conditions with internal and external quality control. Outliers were excluded using the Tukey interquartile range method. Age and sex partitions were established through an integrated assessment of visual inspection, the Mann-Whitney U test, Cliff's delta, the Harris-Boyd method, and biological and clinical considerations. Reference intervals were established according to CLSI EP28-A3c using a nonparametric approach (2.5-97.5th percentiles), with 90 % confidence intervals estimated by bootstrap. Results All CBC parameters showed age-related variation, supporting age-specific RI. Sex differences were limited to erythrocyte parameters, including red blood cell count, hemoglobin, and hematocrit, emerging after puberty; sex partitioning was unnecessary for most analytes. Application of the Vietnamese vs. CALIPER reference intervals in an outpatient cohort resulted in bidirectional reclassification, with 0.0-32.6 % of CALIPER-abnormal results reclassified as normal and 0.0-24.4 % of CALIPER-normal results reclassified as abnormal, depending on the analyte. Conclusions This study establishes population-based pediatric CBC RI for Vietnamese children. Non-local pediatric RI may alter abnormal flagging and result interpretation in routine practice.
