Volume 63, Issue 7

This scoping review focuses on the evolution of pre-analytical errors (PAEs) in medical laboratories, a critical area with significant implications for patient care, healthcare costs, hospital length of stay, and operational efficiency. The Covidence Review tool was used to formulate the keywords, and then a comprehensive literature search was performed using several databases, importing the search results directly into Covidence (n=379). Title, abstract screening, duplicate removal, and full-text screening were done. The retrieved studies (n=232) were scanned for eligibility (n=228) and included in the review (n=83), and the results were summarised in a PRISMA flow chart. The review highlights the role of healthcare professionals in preventing PAEs in specimen collection and processing, as well as analyses. The review also discusses the use and advancements of artificial intelligence (AI) and machine learning in reducing PAEs and identifies inadequacies in standard definitions, measurement units, and education strategies. It demonstrates the need for further research to ensure model validation, address the regulatory validation of Risk Probability Indexation (RPI) models and consider regulatory, safety, and privacy concerns. The review suggests that comprehensive studies on the effectiveness of AI and software platforms in real-world settings and their implementation in healthcare are lacking, presenting opportunities for further research to advance patient care and improve the management of PAEs.

Although significant progress has been made in recent years, some important questions remain regarding the analytical performance, pathophysiological interpretation and clinical use of cardiac troponin I (cTnI) and T (cTnT) measurements. Several recent studies have shown that a progressive and continuous increase in circulating levels of cTnI and cTnT below the cut-off value (i.e. the 99th percentile upper reference limit) may play a relevant role in cardiovascular risk assessment both in the general population and in patients with cardiovascular or extra-cardiac disease. International guidelines recommend the use of standardized clinical algorithms based on temporal changes in circulating cTnI and cTnT levels measured by high-sensitivity (hs) methods to detect myocardial injury progressing to acute myocardial infarction. Some recent studies have shown that some point-of-care assays for cTnI with hs performance ensure a faster diagnostic turnaround time and thus significantly reduce the length of stay of patients admitted to emergency departments with chest pain. However, several confounding factors need to be considered in this setting. A novel approach may be the combined assessment of laboratory methods (including hs-cTn assay) and other clinical data, possibly using machine learning methods. In the present document of the Italian Study Group on Cardiac Biomarkers, the authors aimed to discuss these new trends regarding the analytical, pathophysiological and clinical issues related to the measurement of cardiac troponins using hs-cTnI and hs-cTnT methods.

The ultimate goal of harmonization, crucial to quality in laboratory medicine, is to improve patient outcomes by providing accurate, actionable laboratory information. Patients and healthcare professionals assume that clinical laboratory tests performed by different laboratories at different times on the same type of sample are comparable, and that results can be reliably and consistently interpreted. In this context the reporting units for tests can have a considerable influence on the numeric result. The harmonization of measurement units in laboratory report, leads to the provision of interchangeable and comparable results, thus maximizing the validity of laboratory information, and assuring a more accurate diagnosis and better treatment for the patient. However, although considerable efforts have been made in recent years, the criticisms continue. This opinion paper, prepared jointly by EFLM Committee Harmonization (C-H) and Committee Postanalytical phase (C-POST), describes the “general pragmatic approach” proposed in the drafting of guidelines for the harmonization of measurement units in reporting results, in order to ensure they are used as widely as possible.

Cancer screening is considered to be a major strategy for combatting cancer. The United States Preventive Services Task Force (USPSTF) recommends screening for five cancers, but the strength of evidence about the effectiveness of screening is limited. To gain insights into the efficacy of early detection requires prospective, blinded, placebo-controlled clinical trials with decades of follow-up and inclusion of millions of participants. Recently, Bretthauer et al. estimated lifetime gained with cancer screening tests by using a meta-analysis of 18 large randomized clinical trials which included more than two million subjects. They asked if cancer screening tests are saving lives and how much life is extended due to commonly used cancer screening tests. Colorectal cancer screening with sigmoidoscopy prolonged lifetime by 110 days, while fecal testing and mammography screening did not prolong life. A modest extension of 37 days was noted for prostate cancer screening with prostate-specific antigen testing and 107 days with lung cancer screening using computed tomography, but these estimates were not statistically significant. The authors concluded that current cancer screening strategies do not significantly prolong life. Based on these data, and the known biological behavior of some cancers, we hypothesized that the current strategies of treating cancer, after detection, could be modified to avoid the side effects of screening, which is a major determinant of the patient’s overall survival.

Simulation-based approaches for setting indirect outcome-based analytical performance specifications (APS) predominantly involve test repetition through analytical reruns or resampling. These methodologies assess the agreement between original and simulated measurement results, determining the APS corresponding to pre-established performance thresholds. For APS related to imprecision and bias, both analytical performance characteristics (APCs) are typically considered in simulations, whereas for APS regarding measurement uncertainty, bias is excluded in alignment with traceability standards. This paper introduces the “APS Simulator,” a novel tool designed to complement the existing APS Calculator by simulating APS under various scenarios involving imprecision, bias, and measurement uncertainty. The APS Simulator facilitates simulations using distinct analytical rerun and resampling models, enabling laboratory professionals to explore a wide range of performance levels for their specific needs. While the APS Simulator provides valuable insights, significant challenges remain in the broader application of indirect outcome-based APS. These include incorporating sources of diagnostic uncertainty, setting appropriate thresholds for performance metrics, validating clinical decision limits, and accounting for population data characteristics. Addressing these limitations will be essential to enhancing the standardization and robustness of APS determination. The source code and desktop application for the APS Simulator are freely available at https://github.com/hikmetc/APS_Simulator, providing a user-friendly platform for researchers and clinicians to further explore these methodologies.

Objectives Detecting point mutations with high sensitivity and specificity can be technically very challenging, but it is crucial for early diagnosis and effective drug treatment of cancers. To enable ultrasensitive and ultraspecific detection of single-base mutations in simple and economical ways, we have developed an artificial base mismatches-mediated PCR (ABM-PCR) detection approach. Methods ABM-PCR was applied to quantitative PCR (qPCR) and droplet digital PCR (ddPCR) detection platforms. The impact of mismatches on the thermodynamic stability of the primer-template duplex and the ability of Taq polymerase to catalyze the extension was examined. Effects of the sequence, position, and the number of mismatches on genotyping performance were characterized. Results As proof of principle, we demonstrated the feasibility of ABM-PCR in detecting epidermal growth factor receptor (EGFR) and B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations that are clinically relevant to diagnosis and prognosis of lung and thyroid cancers. Our ABM-PCR enabled the detection of 0.1 % mutation without amplification of the wild-type DNA strand, even in the presence of a 300 ng human genomic DNA background. It enables ultrasensitive (≥95 %) and ultraspecific (≥95 %) diagnosis of clinical samples for thyroid papilloma and lung cancers. Based on these findings, we have established a set of rules and developed a user-friendly web primer design tool for designing effective ABM-PCR primers. Conclusions This study highlights the impact of primer-template mismatches on PCR amplification and provides insights into rational design of effective ABM-PCR primers for detecting single-base mutations with high specificity and sensitivity. It is highly valuable for clinical diagnosis and prognosis use.

Objectives The Certified Reference Material (CRM) ERM ® -DA477/IFCC is a new polyclonal IgG anti-beta2-glycoprotein I (anti-β2GPI) material for the harmonization of the laboratory diagnosis of antiphospholipid syndrome (APS). We evaluated CRM’s ability to represent the heterogeneity of APS patient anti-β2GPI antibodies and to calibrate IgG anti-β2GPI methods. Methods We characterized CRM for its reactivity against domain-1, using the QUANTA Flash ® β2GPI-domain-1 assay, and against domains-4-5 of β2GPI, and single-domain-deleted β2GPI molecules using in-house ELISAs. We used QUANTA Lite ® ELISA, QUANTA Flash ® CLIA, and EliA™ FEIA methods to evaluate the CRM’s anti-Cardiolipin (anti-CL) activity. Four anti-β2GPI IgG methods (in-house and QUANTA Lite ® ELISA, QUANTA Flash ® CLIA, and EliA™ FEIA) were also used to evaluate the CRM’s calibration efficacy, alongside 133 clinical samples (CSs) and 99 controls. Results The CRM showed high anti-domain-1 activity and no anti-domain-4-5 activity at the recommended assay dilution. The domain-dependent-β2GPI reactivity profiles were comparable with full-blown APS. There was acceptable dilution linearity for anti-CL assays with R 2 ranging from 0.957 to 0.997. For the four anti-β2GPI IgG assays, calibration with the CRM led to a good comparability of the average result of CSs for two of the assays. New cut-offs calculated from this work improved comparability in quantitative results between three of the assays: 85 % concordance with CRM compared to 66 % concordance with assay-specific-calibration. Conclusions The CRM is representative of patient anti-β2GPI/CL heterogeneity and should improve anti-β2GPI IgG method harmonization. However, the level of achievable method harmonization is affected by differences in the selectivity among the assays.

Objectives Detailed technical instructions have been made for harmonization of the prothrombin time (PT) test using the manual tilt tube technique (MTT). The MTT has been proposed as the reference measurement procedure for PT and international normalized ratio (INR). An external quality assessment (EQA) scheme has been developed specifically for calibration laboratories performing the harmonized MTT. Here we report the results of the first 10 surveys of this new EQA scheme and investigate whether there is improvement in performance over time and in comparison with previous studies. Methods Four deep-frozen plasma samples with different PT levels were dispatched to 4 European laboratories. PT’s were determined by eight operators. All operators used the same PT reagent (recombinant human). Various measures of PT variation were defined, i.e. within-operator, within-survey, within-run, between-operator, and between-survey coefficient of variation. Between-operator variation (CV S ) was calculated from the each operator’s mean PT. Results The median within-operator variation of all operators varied from 1.3 to 2.3 %. Some operators improved their performance, others did not. Between-operator CV (CV S ) ranged from 1.0 to 2.2 %. Overall, the between-operator and within-operator variation using the harmonized MTT was lower than in a previously published multicentre calibration study. Overall, the within-operator variation was low and did not change significantly over time. Conclusions within-operator and between-operator variation of the PT measured with the harmonized MTT were low when compared with previous studies. The results suggest that the average within-operator variation of the eight operators in this study is as low as possible.

Objectives Management of phenylketonuria (PKU) relies upon life-long monitoring of phenylalanine (Phe) in dried blood spots (DBS), thus comparability of measurements is important. The lack of harmonisation and standardisation between laboratories, combined with the variable quality of patient-collected DBS specimens, are currently preventing this from being achieved. A traceable, matrix-matched Phe certified reference material, common methodology and means to ensure patient collected DBS specimens are of consistent quality would improve comparability between laboratories. Methods Baseline inter-laboratory (n=15) variation of DBS Phe was determined by triplicate measurement of four DBS materials, on three days. Laboratories prepared and analysed these samples using their routine method of analysis. A sub-set of laboratories (n=5) repeated the process using a common sample preparation and instrument methodology (LC-MS/MS), and three different calibration approaches. Samples prepared on dried blood spot microsampling cards (DBS-MCs) from whole blood, value assigned for Phe concentration by National Measurement Laboratories (NML), were then analysed using the harmonised methodology. Results Inter-laboratory co-efficient of variation (CV) differed with calibration approach; internal calibration 27.7 %; in-house aqueous calibration 4.7 %; centrally distributed aqueous calibration, 2.1 %. Inter-laboratory CV was reduced from 8.7 to 2.1 % by using common sample preparation and LC-MS/MS methodology. No significant difference was observed between consensus and assigned values for Phe in the four materials (p>0.05). Conclusions This study demonstrates a simple approach to harmonising and standardising DBS Phe measurements, traceable to value assigned materials. Combined with the introduction of DBS-MCs to ensure specimen quality, clinical laboratories can achieve comparability of patient results over time.

Objectives Pyridoxine-dependent epilepsy (PDE) is a rare genetic disorder characterized by intractable neonatal seizures responsive to pyridoxine. Diagnosis relies on quantification of α-aminoadipic semialdehyde, piperideine-6-carboxylate and pipecolic acid in urine or plasma in patients with overt symptoms. We developed and validated simple and rapid first- and second-tier methods for two recently published biomarkers of PDE (2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) and 6-oxopiperidine-2-carboxylic acid (6-oxoPIP)) in extended newborn screening (NBS) programs from neonatal dried blood spots (DBS). Methods For the first-line test, DBS specimens were collected from 5,405 newborns who underwent routine NBS and analysed by FIA-MS/MS. For the second-tier test, samples were analysed by LC-MS/MS. The neonatal DBS from two patients with genetically confirmed PDE were also analysed. Results The reference values for NBS resulted <0.34 μmol/L for 2-OPP and <4.51 μmol/L for 6-oxoPIP. In the second-tier test, limits of detection were 0.07 μmol/L and 0.14 μmol/L, whereas limits of quantification were 0.25 μmol/L and 0.48 μmol/L, respectively, for 2-OPP and for 6-oxoPIP. The tests provided good linearity, reproducibility, accuracy and precision, with acceptable matrix effect and carry-over, according to international validation criteria. The biomarkers in DBS were stable at room temperature, +4 °C and −20 °C for one month. When assessing these biomarkers in two patients with genetically confirmed PDE, the higher sensitivity of 2-OPP as compared to 6-oxoPIP in discriminating PDE emerged. Conclusions The first-line and second-tier tests developed in this study highlight the potential for including PDE in the NBS panel, early diagnosis and prompt precision treatment initiation.

Objectives Measuring serum 25-hydroxyvitamin D is key in clinical labs, but inter-laboratory variations risk diagnostic errors. This study evaluates the performance of current in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods used in top Chinese clinical laboratories and proposes an optimized method for improving serum 25(OH)D measurement accuracy and reliability. Methods Seven serum pools with different concentrations of 25(OH)D were prepared and sent to 12 participating laboratories for multiple repeat analysis with their current in-house LC-MS/MS methods and then an optimized LC-MS/MS method. Precision was assessed in terms of coefficient of variance (CV), and trueness was assessed in terms of bias referring to the U.S. National Institute of Standards and Technology (NIST) reference measurement procedure (RMP). The analytical performances of the two methods were compared and evaluated. Results Eighty percent and 90 % of the laboratories achieved the defined performance criteria (CV, <12.5 %; mean bias, <8.3 %) with the optimized method for the measurement of 25(OH)D2 and 25(OH)D3, compared with 43 % and 57 % of the laboratories meeting the criteria with their in-house methods, respectively. Precision and trueness improved after applying the optimized method. Although the optimized method didn’t not ensure that all laboratory samples meet the measurement uncertainty (MU) criteria (MU<13.6 %), particularly for low-concentration samples, it significantly reduced the MU compared to the in-house method. Conclusions Precision, trueness and MU improved after applying the optimized method. Nonetheless, more efforts are needed to ensure the reliability and accuracy of 25(OH)D measurements in clinical laboratories in China.

Objectives Bile acid diarrhea is a common but underdiagnosed condition. Because the gold standard test ( 75 SeHCAT) is time-consuming and not widely available, fecal bile acid excretion is typically assessed by chromatography and mass spectrometry. Although enzymatic cycling assays are well established for the rapid and cost-effective analysis of total bile acids (TBA) in serum or plasma, their full potential has yet not been extended to stool samples in clinical routine. Methods The performance of the ‘Total bile acids 21 FS’ reagent (DiaSys) was evaluated in fecal matrix according to CLSI guidelines and EU-IVD Regulations (2017/745), and compared to an established microplate-based kit (IDK ® ) by measuring patient stool samples (n=122). Method agreement was assessed by Passing-Bablok and Bland-Altman analysis. The quantification of eight individual BAs was assessed using HPLC-MS/MS as reference method. Results The DiaSys assay showed linearity between 3.5 and 130 μmol/L, good repeatability, total precision, and reproducibility with CVs of 1.7 %, 3.5 %, and 3.0 %. Limit of blank (LoB), detection (LoD), and quantitation (LoQ) were ≤0.17, ≤0.3, and 3.5 μmol/L, respectively. No significant interference from endogenous substances was observed. The methods showed good correlation up to 140 μmol/L (r=0.988), despite differences in the quantification of individual BAs, with mean deviations of 7 % (DiaSys) and 31 % (IDK ® ), respectively. Conclusions The advantages of enzymatic TBA analysis on fully automated clinical chemistry platforms can be exploited for the routine analysis of stool samples. However, cycling assays may benefit from reference standards that take into account the composition of the fecal BA pool.

Objectives To assess the analytical performances of Snibe Maglumi ® S100 assay and compare it with the Roche Elecsys ® S100B assay in adults with mild traumatic brain injury (mTBI) focusing on reducing unnecessary cranial computed tomography (CT) scans per Scandinavian and French guidelines. Methods Analytical performance of the Maglumi ® S100 kit was assessed using quality controls from both Snibe and Roche, as well as pooled serums. Clinical performances were assessed using serum from 89 adult mTBI patients presenting to the adult emergency department of Clermont-Ferrand University Hospital with a Glasgow Coma Scale score of 14–15. CT scans were performed according to the Elecsys ® S100 measurement, with a decision threshold of 0.10 μg/L. Results Repeatability and reproducibility coefficients of variation determined using Elecsys ® S100B, Maglumi ® S100 controls and pooled serums were below 8 %. Six (7 %) mTBI patients included had clinically relevant intracranial lesions observed on CT scan (CT+), and eighty-three (93 %) patients had no lesions (CT−). S100B medians in CT− and CT+ patients were significantly different: 0.125 (0.085–0.219) vs. 0.368 (0.231–0.489) (p=0.006) for Elecsys ® , and 0.073 (0.046–0.140) vs. 0.327 (0.230–0.353) for Maglumi ® (p=0.004). The areas under the ROC curves for intracranial lesion detection were similar: 0.82 (0.73–0.91; p=0.0084) and 0.83 (0.75–0.92; p=0.0063) for Elecsys ® and Maglumi ® , respectively. Conclusions The Maglumi ® S100B assay can be used in the management of mTBI patients to exclude unnecessary CT scans. Further studies are needed to validate a clinical decision threshold for CT scan decisions.

Objectives To study the prevalence of trisodium citrate (Na 3 Citrate) contamination in hypernatraemic serum samples by direct measurement of citrate and to evaluate the performance of indirect markers for identification of Na 3 Citrate contamination. Methods Serum citrate was measured in all hypernatraemic serum samples (sodium ≥148 mmol/L) over a three-month period. The performance of serum chloride, sodium-chloride gap, indirect ion selective electrode (ISE)-direct ISE sodium disparity and osmolar gap in identification of Na 3 Citrate contaminated samples was assessed against the ‘gold-standard’ direct citrate measurement. Results In total, 27 Na 3 Citrate contaminated samples were identified based on serum citrate concentration ≥1.5 mmol/L. The prevalence of citrate contamination was 3.1 % of hypernatraemic samples (n=875) and 0.017 % of all samples received for urea and electrolyte analysis (n=153,404). Most contaminated samples were from patients receiving haemodialysis (59.3 %), and the rest from inpatients. Cut-offs to give 100 % sensitivity were chloride ≤105 nmol/L (specificity 93.4 %), sodium-chloride gap ≥47 mmol/L (specificity 95.3 %), indirect ISE-direct ISE sodium disparity ≥3 mmol/L (specificity 81.9 %), and osmolar gap ≥39 mOsm/kg (specificity 2.8 %). Conclusions Trisodium citrate contamination is uncommon. Most contaminated samples were from patients receiving haemodialysis, likely because of contamination with citrate catheter locking solution. Screening with serum chloride or sodium-chloride gap can confidently exclude Na 3 Citrate contamination in over 90 % of hypernatraemic samples, and in nearly all samples with sodium ≥155 mmol/L if metabolic alkalosis has been excluded. In the remaining samples, Na 3 Citrate contamination can only be definitively confirmed or excluded by measurement of serum citrate. We propose algorithms to identify spurious hypernatraemia.

Objectives Chronobiology is the science that studies biological rhythms based on direct methods and empirical time series of individual subjects. In laboratory medicine, the factor of time is often underestimated, and no methods currently exist to study biological rhythms in population databases of point-like, real-world data (RWD). Methods Retrospective databases (24 months, 2022–2023) were extracted for four measurands (sodium, potassium, chloride and leukocytes) from the emergency laboratory. Two different strategies for data grouping were applied: data clouds (with or without outliers) and population-averaged profiles. Cosinor regression analysis was performed on the grouped data to derive circadian parameters. The parameters obtained here were compared to results from the literature, using direct methods and time series. Results A total of 409,719 data points were analyzed. All measurands exhibited symmetrical data distributions, except for leukocytes. The data clouds did not visually display rhythmicity, but cosinor analysis revealed a significant circadian rhythm. The removal of outliers had minimal impact on the results. In contrast, population-averaged profiles showed visible rhythmicity, which was confirmed by cosinor analysis with a better goodness-of-fit compared to the data clouds. Conclusions Population-averaged profiles have advantages over data clouds in characterizing circadian rhythms and deriving circadian parameters. Population chronobiology, based on RWD, is presented as an alternative to classical individual chronobiology, based on time series and overcomes the limitations of direct methods. Utilizing RWD provides new insights into the relationship between chronobiology and clinical laboratory practice.

Objectives To establish the reference intervals (RIs) of serum glial fibrillary acid protein (GFAP) measured by the fully automated Lumipulse system. Methods The study population consisted of 340 healthy individuals, including 251 blood donors and 89 outpatients, with a median age of 56 years. Serum GFAP levels were measured by the Lumipulse G GFAP assay on the fully automated platform Lumipulse G1200 (FUJIREBIO Inc., Tokyo, Japan). GFAP RIs (2.5th and 97.5th percentiles) were calculated for the overall population and stratified by age and sex groups. For the overall population, males, and females partitions, we employed the nonparametric methods, while for the age-and-sex groups we employed the “robust” method, as recommended by CLSI. Results The RI in the whole population was 10.4–92.0 ng/L. When considering sex differences, females showed higher levels of serum GFAP than males across all age groups. A positive correlation was observed between age and GFAP (Spearman’s rho=0.55, p<0.001). Specifically, the biomarker was stable until 60 years, while individuals aged>60 years demonstrated significantly and considerably higher levels than younger age groups. Additionally, in the 50–60 age group, we observed gender-related differences, with females having increased levels than males. Conclusions GFAP levels are influenced by both age and sex. Accordingly, we established RIs of serum GFAP, taking into consideration age and sex-related differences.

Objectives This study aimed to evaluate the performance of PBIA (UIMD, Seoul, Republic of Korea), an automated digital morphology analyzer using deep learning, for white blood cell (WBC) classification in peripheral blood smears and compare it with the widely used DI-60 (Sysmex, Kobe, Japan). Methods A total of 461 slides were analyzed using PBIA and DI-60. For each instrument, pre-classification performance was evaluated on the basis of post-classification results verified by users. Pre- and post-classification results were compared with manual WBC differentials, and the ability to identify abnormal cells was assessed. Results The pre-classification performance of PBIA was better than that of DI-60 for most cell classes. PBIA had an accuracy of 90.0 % and Cohen’s kappa of 0.934, higher than DI-60 (45.5 % accuracy and 0.629 kappa) across all cell classes. The pre-classification performance of both instruments decreased when abnormal cells were observed in manual counts, but PBIA still performed better. PBIA also appeared to show better correlation with manual WBC differential counts, particularly in pre-classification (Pearson’s correlation coefficient: 0.696–0.944 vs. 0.230–0.882 for neutrophils, lymphocytes, monocytes, eosinophils, basophils, and blasts), although the mean differences varied by cell class. For abnormal cells identified in manual counts, PBIA exhibited more false positives for blasts (30.5 vs. 2.3 %), while DI-60 had a higher rate of false negatives (42.1 vs. 6.1 %). Both instruments exhibited high false negative rates for atypical lymphocytes. Conclusions PBIA demonstrated better performance than DI-60, highlighting its clinical utility. Further multicenter studies are required for full validation.

Objectives Risk-based stratification approaches using measurable residual disease (MRD) successfully help to identify T-acute lymphoblastic leukemia (T-ALL) patients at risk of relapse, whose treatment outcomes are very poor. Because of T-ALL heterogeneity and rarity, a reliable and standardized approach for flow cytometry (FC)-based MRD measurement and analysis is often missing. Methods Within the international AIEOP-BFM-ALL-FLOW study group we made a consensus on markers and a standard operating procedure for common 8- and 12-color T-ALL MRD panels. Custom manufactured tubes with dried backbone antibodies were tested in parallel to local FC standards. Results Altogether, 66 diagnostic and 67 day 15 samples were analyzed. We designed two guided MRD gating strategies to identify blast cells in parallel to expert-based evaluation. We proved that the optimized tubes allowed the correct identification of blast cells in all diagnostic samples. Both, expert and guided analysis of day 15 samples correlated to local standard (Spearman R=0.98 and R=0.94, respectively). Only in 2 (3 %) and 4 (6 %) patients expert gating and guided analysis results were substantially discordant from local standard, respectively. The cases that require an individualized approach may be partially identified at diagnosis through a rare immunophenotype or mixed phenotype acute leukemia status. Conclusions Our work shows that standardized operating procedures together with guided analysis are applicable in a great majority of T-ALL cases. Further improvement of MRD detection is needed, as in some cases an individualized analytical approach is still required due to the challenging nature of the T-ALL phenotype.

Objectives In patients with chronic lymphocytic leukemia (CLL), the white progenitor cell (WPC) channel of the Sysmex XN hematology analyzers typically shows a varying proportion of cells in the upper left field of the scattergram, resembling the shape of shark fins. The underlying mechanism causing this phenomenon has not been elucidated so far. In this study we evaluated biological and/or genetic features of CLL cells as a potential background of the “shark fins”. Methods Automated blood counts and WPC scattergrams of 95 CLL patients were analyzed. The scattergrams were examined for the presence, count and percentage of cells in the “shark fins” using a flow cytometry software. Potential influencing factors on the occurrence of the “shark fin” cells were statistically tested by Spearman correlation. Results The lymphocyte count was identified as a highly statistically significant predictor of “shark fins” (p<0.0001). Absence of del(17p) (p=0.02), unmutated TP53 (p=0.01), mutated IGHV (p=0.03) and the percentage of smudge cells in peripheral blood smears (p=0.04) showed a statistically significant positive influence on the percentage of cells in the “shark fins” after adjustment for the lymphocyte count. No significant effect was observed for age, sex, del(13q), del(11q), trisomy 12 and treatment. Conclusions We identified the lymphocyte count, the absence of TP53 mutations and del(17p), a mutated IGHV and the proportion of smudge cells as significant influences on the appearance of “shark fin” cells. Our findings indicate an impact of biological and genetic properties of the leukemic cells on the formation of “shark fins”.

Objectives Despite its utility, interpreting multiparameter flow cytometry (MFC) data for hematologic malignancy remains time-intensive and complex. This study evaluated the applicability of two dimensionality reduction (DR) algorithms, t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP), to MFC data of hematologic malignancy. Methods A total of 237 samples were re-analyzed by t-SNE- and UMAP-based gating: 80 with acute leukemia orientation tube panel, 42 with B-cell lymphoma (BCL) panel, 45 with multiple myeloma (MM) panel, 40 and 30 with measurable residual disease (MRD) panels for B-cell acute lymphoblastic leukemia (B-MRD) and MM (MM-MRD), respectively. Each result was compared to the manual gating, and sensitivity and precision were assessed using BCL and B-MRD panels. Results Compared to manual gating, DR-based gating demonstrated agreements over 95.0 % for all MFC panels, and quantitative correlations ( ρ ) exceeded 0.94. Both t-SNE- and UMAP-based gating showed a sensitivity and negative predictive value of 100 %. Also, in one sample each from the BCL and MM-MRD panels, DR-based gating identified populations that were missed by manual gating. Sensitivity evaluation showed that both t-SNE- and UMAP-based gating successfully identified MRD populations down to the lowest MRD level of 10 −5.30 when applying primary-gating strategy for CD19-positive population. Precision evaluation showed coefficient of variation below 10 % across all levels. Conclusions This study shows that DR-based gating streamlines data interpretation and minimizes overlooked populations, demonstrating significant potential as a valuable tool in MFC analysis for hematologic malignancies.

Objectives We aimed to evaluate sex-specific 0-h high-sensitivity cardiac troponin T (hs-cTnT) thresholds for risk stratification in patients with suspected non-ST-segment elevation myocardial infarction (NSTEMI). Methods This retrospective study investigated a derivation cohort of 6,691 (44.4 % female and aged 65 (57–72) years) and a validation cohort of 6,589 (48.4 % female and aged 63 (50–71) years) patients with suspected NSTEMI who visited the emergency department. Uniform and sex-specific thresholds were derived and validated, and their performances were compared. Results In the derivation cohort, 11.3 % of males and 5.4 % of females were diagnosed with adjudicated NSTEMI. As male-specific thresholds, 0-h hs-cTnT <10 ng/L ruled out 30.1 % of patients with an NPV of 99.6 % [95 % CI (99.0–99.9 %)], and 0-h hs-cTnT ≥65 ng/L ruled in 12.3 % of patients with a PPV of 70.5 % [95 % CI (66.0–74.6 %)]. As female-specific thresholds, 0-h hs-cTnT <9 ng/L ruled out 39.2 % of patients with an NPV of 99.9 % [95 % CI (99.4–100.0 %)], and 0-h hs-cTnT ≥45 ng/L ruled in 6.7 % of patients with a PPV of 71.7 % [95 % CI (64.8–77.8 %)]. The validation cohort showed similar results. Sex-specific and uniform thresholds did not significantly affect rule-in performance, whereas sex-specific thresholds increased female sensitivity by 1 % (99.7 vs. 98.7 %). Patient follow-up did not change when sex-specific thresholds were applied. Conclusions Sex-specific 0-h hs-cTnT thresholds could slightly improve the safety in ruling out NSTEMI in females, but their role in terms of efficiency, rule-in performance and prognosis was similar to that of uniform thresholds.