Volume 59, Issue 1

Objectives Several studies have shown an inverse association between diabetes mellitus and prostate cancer (PCa). Some researchers suggest that this relationship is due to reduced PCa detection in diabetics due to lower prostate-specific antigen (PSA) levels compared to non-diabetics. Our objective is to analyze the impact of diabetes on PSA in asymptomatic men without known prostate pathology and without prior prostate intervention. Methods We searched Medline (via PubMed), Embase and Scopus. We included studies that reported the relationship between serum PSA levels and diabetes or diabetes treatment in asymptomatic adult men without known prostate pathology, and without prior prostate intervention. Pooled mean differences were compared between diabetics and non-diabetics. Results Of 2,392 screened abstracts, thirteen studies met the inclusion criteria and 8 (62%) reported appropriate measures that could be included in a meta-analysis. Eleven (85%) examined the influence of diabetes on PSA levels and 8 (62%) evaluated the influence of diabetes treatments on PSA levels. Overall diabetics had a significantly lower PSA level compared to non-diabetics (mean difference: −0.07 ng/mL; 95% CI −0.10, −0.04). Conclusions Diabetes and related factors (such as disease duration, severity and treatment) were significantly associated with lower PSA levels among asymptomatic men, yet differences were small and are unlikely to influence PCa detection in a screening setting.

Cell senescence is a fundamental mechanism of aging and appears to play vital roles in the onset and prognosis of cardiovascular disease, fibrotic pulmonary disease, liver disease and tumor. Moreover, an increasing body of evidence shows that cell senescence plays an indispensable role in the formation and development of atherosclerosis. Multiple senescent cell types are associated with atherosclerosis, senescent human vascular endothelial cells participated in atherosclerosis via regulating the level of endothelin-1 (ET-1), nitric oxide (NO), angiotensin II and monocyte chemoattractant protein-1 (MCP-1), senescent human vascular smooth muscle cells-mediated plaque instability and vascular calcification via regulating the expression level of BMP-2, OPN, Runx-2 and inflammatory molecules, and senescent macrophages impaired cholesterol efflux and promoted the development of senescent-related cardiovascular diseases. This review summarizes the characteristics of cell senescence and updates the molecular mechanisms underlying cell senescence. Moreover, we also discuss the recent advances on the molecular mechanisms that can potentially regulate the development and progression of atherosclerosis.

Background Minimum retesting intervals (MRI) are a popular demand management solution for the identification and reduction of over-utilized tests. In 2011 Association of Clinical Biochemistry and Laboratory Medicines (ACB) published evidence-based recommendations for the use of MRI. Aim The aim of the paper was to review the use of MRI over the period since the introduction of these recommendations in 2011 to 2020 and compare it to previous published data between 2000-2010. Methods A multi-source literature search was performed to identify studies that reported the use of a MRI in the management or identification of inappropriate testing between the years prior to (2000–2010) and after implementation (2011–2020) of these recommendations. Results 31 studies were identified which met the acceptance criteria (2000–2010 n=4, 2011–2020 n=27). Between 2000 and 2010 4.6% of tests (203,104/4,425,311) were identified as failing a defined MRI which rose to 11.8% of tests (2,691,591/22,777,288) in the 2011–2020 period. For those studies between 2011 and 2020 reporting predicted savings (n=20), 14.3% of tests (1,079,972/750,580) were cancelled, representing a total saving of 2.9 M Euros or 2.77 Euro/test. The most popular rejected test was Haemoglobin A 1c which accounted for nearly a quarter of the total number of rejected tests. 13 out 27 studies used the ACB recommendations. Conclusions MRI are now an established, safe and sustainable demand management tool for the identification and management of inappropriate testing. Evidence based consensus recommendations have supported the adoption of this demand management tool into practice across multiple healthcare settings globally and harmonizing laboratory practice.

Drug-induced cardiotoxicity is a major clinical problem; cardiotoxic drugs may induce both cardiac dysfunction and myocardial injury. Several recent studies reported that cardiac troponins measured with high-sensitivity methods (hs-cTn) can enable the early detection of myocardial injury related to chemotherapy or abuse of drugs that are potentially cardiotoxic. Several authors have some concerns about the standard definition of cardiotoxicity, in particular, regarding the early evaluation of chemotherapy cardiotoxicity in cancer patients. Several recent studies using the hs-cTn assay indicate that myocardial injury may precede by some months or years the diagnosis of heart failure (HF) based on the evaluation of left ventricular ejection fraction (LVEF). Accordingly, hs-cTn assay should considered to be a reliable laboratory test for the early detection of asymptomatic or subclinical cardiotoxic damage in patients undergoing cancer chemotherapy. In accordance with the Fourth Universal Definition of Myocardial Infarction and also taking into account the recent experimental and clinical evidences, the definition of drug-cardiotoxicity should be updated considering the early evaluation of myocardial injury by means of hs-cTn assay. It is conceivable that the combined use of hs-cTn assay and cardiac imaging techniques for the evaluation of cardiotoxicity will significantly increase both diagnostic sensitivity and specificity, and also better prevent chemotherapy-related left ventricular (LV) dysfunction and other adverse cardiac events. However, large randomized clinical trials are needed to evaluate the cost/benefit ratio of standardized protocols for the early detection of cardiotoxicity using hs-cTn assay in patients receiving chemotherapy for malignant diseases.

To ensure that clinical laboratories produce results that are both accurate and of clinical utility it is essential that only samples of adequate quality are analysed. Although various studies and databases assessing the stability of analytes in different settings do exist, guidance on how to perform and report stability studies is lacking. This results in studies that often do not report essential information, thus compromising transferability of the data. The aim of this manuscript is to describe the C hecklist for R eporting S tability S tudies (CRESS) against which future studies should be reported to ensure standardisation of reporting and easy assessment of transferability of studies to other healthcare settings. The EFLM WG-PRE (European Federation of Clinical Chemistry and Laboratory Medicine Working Group for the Preanalytical Phase) produced the CRESS checklist following a detailed literature review and extensive discussions resulting in consensus agreement. The checklist consists of 20 items covering all the aspects that should be considered when producing a report on a stability study including details of what should be included for each item and a rationale as to why. Adherence to the CRESS checklist will ensure that studies are reported in a transparent and replicable way. This will allow other laboratories to assess whether published data meet the stability criteria required in their own particular healthcare scenario. The EFLM WG-PRE encourage researchers and authors to use the CRESS checklist as a guide to planning stability studies and to produce standardised reporting of future stability studies.

Objectives Young laboratory medicine professionals (YLMPs) are the future of clinical laboratories. Although everyday practice shows significant differences among countries, especially during residency training, most of them face the same challenges. Besides promoting scientific, professional and clinical aspects of laboratory medicine in Europe, the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) should take into consideration YLMPs’ concerns and interests to help them achieve excellence. The aim of this survey was to assess the opinion and expectations of YLMPs about their involvement in the activities of EFLM. Methods An online survey was distributed to YLMPs in Europe through different channels. The questionnaire consisted of 21 items grouped into five sections: demographic questions, opinion about the current status of YLMPs within EFLM, YLMPs network, suggestions and opportunities, and scientific training and exchange. Where appropriate, responses from residents and specialists were compared. Results A total of 329 valid responses were obtained from 53 different countries. Countries with the highest number of participants were Spain, Turkey, Croatia and Romania. A significant percentage would like to know more about EFLM and their activities (86%) and wish EFLM promoted networking and scientific exchanges (95%), for instance by means of a European YLMPs network (93%). EFLMLabX project was widely unknown (75%). Conclusions YLMPs demand better connection to share concerns about daily healthcare duties, to keep updated and to advance professionally. EFLM needs to improve their advertising through national societies to increase YLMPs’ participation. In addition to international meetings and congresses, respondents have emphasized that workshops and other small group activities would significantly help promote laboratory medicine practice in Europe.

In recent years, the formulation of some immunoassays with high-sensitivity analytical performance allowed the accurate measurement of cardiac troponin I (cTnI) and T (cTnT) levels in reference subjects. Several studies have demonstrated the association between the risk of major cardiovascular events and cardiac troponin concentrations even for biomarker values within the reference intervals. High-sensitivity cTnI and cTnT methods (hs-cTn) enable to monitor myocardial renewal and remodelling, and to promptly identify patients at highest risk ofheart failure. An early and effective treatment of individuals at higher cardiovascular risk may revert the initial myocardial remodelling and slow down heart failure progression. Specific clinical trials should be carried out to demonstrate the efficacy and efficiency of the general population screening by means of cost-benefit analysis, in order to better identify individuals at higher risk for heart failure (HF) progression with hs-cTn methods.

Objectives Colorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays. Methods We systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol. Results The yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively. Conclusions Through the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.

Objectives The new European In Vitro Diagnostic (IVD) Regulation 2017/746 (IVDR) restricts the use of lab-developed tests (LDT) after 26th May 2022. There are no data on the impact of the IVDR on laboratories in the European Union. Methods Laboratory tests performed in UZ Leuven were divided in four groups: core laboratory, immunology, special chemistry, and molecular microbiology testing. Each test was classified as Conformité Européenne (CE)-IVD, modified/off-label CE-IVD, commercial Research Use Only (RUO) or LDT. Each matrix was considered a separate test. Results We found that 97.6% of the more than 11.5 million results/year were generated with a CE-IVD method. Of the 922 different laboratory tests, however, only 41.8% were CE-IVD, 10.8% modified/off-label CE-IVD, 0.3% RUO, and 47.1% LDT. Off-label CE-IVD was mainly used to test alternative matrices not covered by the claim of the manufacturer (e.g., pleural or peritoneal fluid). LDTs were mainly used for special chemistry, flow cytometry, and molecular testing. Excluding flow cytometry, the main reasons for the use of 377 LDTs were lack of a CE-IVD method (71.9%), analytical requirements (14.3%), and the fact the LDT was in use before CE-IVD available (11.9%). Conclusions While the large majority of results (97.6%) were generated with a CE-IVD method, only 41.8% of laboratory tests were CE-IVD. There is currently no alternative on the market for 71.5% of the 537 LDTs performed in our laboratory which do not fall within the scope of the current IVD directive (IVDD). Compliance with the IVDR will require a major investment of time and effort.

Objectives The National Center for Clinical Laboratories (NCCL) in China initiated a serum electrolyte trueness verification (ETV) program in 2014 for measurement standardization. Methods Every year, two levels of fresh frozen commutable serum samples determined by inductively coupled plasma mass spectrometry (ICP-MS) reference methods were transported to participating clinical laboratories for the measurement of sodium, potassium, calcium and magnesium. Both samples were measured 15 times in 3 days, and the mean values and coefficient variations (CVs) were calculated from the results. The tolerance limits of trueness (bias), precision (CV) and accuracy (TE) based on the biological variation database were used as the evaluation criteria. The overall trend of the ETV program over 6 years was surveyed by calculating the pass rates of the participating laboratories. The mean bias, inter-laboratory CV, and TE of all laboratory results were analysed. Furthermore, homogeneous and heterogeneous systems were compared, and the bias and CV results of mainstream analysis systems were analysed. Results Pass rates of the three quality specifications increased, and the overall mean bias and inter-laboratory CVs decreased. The homogeneous system was superior to the heterogeneous system for calcium and magnesium measurements. For sodium, potassium, calcium and magnesium, the minimum bias corresponded to Hitachi, Siemens, Beckman AU and Roche, respectively. For inter-laboratory robust CVs, no obvious differences were observed between each peer group. Conclusions The commutable ETV materials assigned via reference methods can evaluate the accuracy and reproducibility of an individual laboratory and the calibration traceability and uniformity between laboratories for measurements.

Objectives External quality assessment (EQA) with commutable samples is used for assessing agreement of results for patients’ samples. We investigated the feasibility to aggregate results from four different EQA schemes to determine the bias between different measurement procedures and a reference target value. Methods We aggregated EQA results for creatinine from programs that used commutable EQA material by calculating the relative difference between individual participant results and the reference target value for each sample. The means and standard errors of the means were calculated for the relative differences. Results were partitioned by methods, manufacturers and instrument platforms to evaluate the biases for the measurement procedures. Results Data aggregated for enzymatic methods had biases that varied from −8.2 to 3.8% among seven instrument platforms for creatinine at normal concentrations (61–85 μmol/L). EQA schemes differed in the evidence provided about the commutability of their samples, and in the amount of detail collected from participants regarding the measurement procedures which limited the ability to sub-divide aggregated data by instrument platforms and models. Conclusions EQA data could be aggregated from four different programs using different commutable samples to determine bias among different measurement procedures. Criteria for commutability for EQA samples as well as standardization of reporting the measurement methods, reagents, instrument platforms and models used by participants are needed to improve the ability to aggregate the results for optimal assessment of performance of measurement procedures. Aggregating data from a larger number of EQA schemes is feasible to assess trueness on a global scale.

Objectives To maintain the consistency of laboratory test results, between-reagent lot variation should be verified before using new reagent lots in clinical laboratory. Although the Clinical and Laboratory Standards Institute (CLSI) document EP26-A deals with this issue, evaluation of reagent lot-to-lot difference is challenging in reality. We aim to investigate a practical way for determining between-reagent lot variation using real-world data in clinical chemistry. Methods The CLSI EP26-A protocol was applied to 83 chemistry tests in three clinical labs. Three criteria were used to define the critical difference (CD) of each test as follows: reference change value and total allowable error, which are based on biological variation, and acceptable limits by external quality assurance agencies. The sample size and rejection limits that could detect CD between-reagent lots were determined. Results For more than half of chemistry tests, reagent lot-to-lot differences could be evaluated using only one patient sample per decision level. In many cases, the rejection limit that could detect reagent lot-to-lot difference with ≥90% probability was 0.6 times CD. However, the sample size and rejection limits vary depending on how the CD is defined. In some cases, impractical sample size or rejection limits were obtained. In some cases, information on sample size and rejection limit that met intended statistical power was not found in EP26-A. Conclusions The CLSI EP26-A did not provide all necessary answers. Alternative practical approaches are suggested when CLSI EP26-A does not provide guidance.

Objectives Drug-laboratory test interactions (DLTIs) are one of the major sources of laboratory errors. Calcium dobesilate (CaD) interference on serum creatinine testing is a widespread problem that has long been ignored in China. A national EQA-based survey was launched to investigate the current status of CaD interference on creatinine routine methods used in China and enhance the education of CaD interference in clinical laboratories. Methods A descriptive survey was developed to characterize the status quo of Chinese laboratory professionals’ cognition to CaD interference. Four of survey samples which were spiked with/without interference additive were shipped to 175 participant laboratories. The target reference values from a reference measurement procedure were compared against the results from participating laboratories to evaluate the CaD interference on serum creatinine measurements using enzymatic method or Jaffé method. Results The lack of knowledge of DLTIs and the barriers to collect information from pharmacological and laboratory data systems had become the main problems on implementing DLTIs education in China. A significant negative influence of CaD on enzymatic method was observed regardless of measurement platforms. Jaffé method was generally free from interaction with CaD but showed poor precision and accuracy at low creatinine concentrations. Conclusions More efforts should be made to enhance the education of DLTIs in clinical laboratories in China.

Objectives The risk of sample confusion continues to be a challenge for the pre-analytical part of the overall testing process. We here describe a novel system to track samples based on a chemical code labeling of test tubes with unique combinations of indicator compounds, which are naturally not present in specimens of human origin. As part of the sample vessel filling, the liquid specimens are permanently labeled with the compound code that can be tracked back to the primary tube. Methods As a proof of concept we used 10 stable-isotope-labeled derivates of medical drugs as indicator substances to create a combinatory 10-digit binary number ID for individual test tubes, i.e. presence/absence of the respective compound. For this purpose, combinations of indicator compounds were provided in evaporated form in polypropylene tubes prior to filling with anonymized patient whole blood and corresponding plasmas subjected to liquid chromatography tandem-mass spectrometry designed to detect the 10 indicator compounds. Results In the blind analysis, we correctly identified 307 different whole blood samples by readout of a 10-digit binary number ID based on the detection of indicator compounds with respect to their presence and number. Conclusions We have demonstrated the feasibility of an internal labeling procedure for diagnostic samples with mass spectrometry-based readout of dissolved indicator compound combinations as a binary number ID. With an increasing number of coding compounds (≫10) a vast number of combinations for sample labeling can be realized beyond the proof of concept setting studied herein.

Objectives A matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method (Mass-Fix) as a replacement for gel-based immunofixation (IFE) has been recently described. To utilize Mass-Fix clinically, a validated automated method was required. Our aim was to automate the pre-analytical processing, improve positive specimen identification and ergonomics, reduce paper data storage and increase resource utilization without increasing turnaround time. Methods Serum samples were batched and loaded onto a liquid handler along with reagents and a barcoded sample plate. The pre-analytical steps included: (1) Plating immunopurification beads. (2) Adding 10 μl of serum. (3) Bead washing. (4) Eluting the immunoglobulins (Igs), and reducing to separate the heavy and light Ig chains. The resulting plate was transferred to a second low-volume liquid handler for MALDI plate spotting. MALDI-TOF mass spectra were collected. Integrated in-house developed software was utilized for sample tracking, driving data acquisition, data analysis, history tracking, and result reporting. A total of 1,029 residual serum samples were run using the automated system and results were compared to prior electrophoretic results. Results The automated Mass-Fix method was capable of meeting the validation requirements of concordance with IFE, limit of detection (LOD), sample stability and reproducibility with a low repeat rate. Automation and integrated software allowed a single user to process 320 samples in an 8 h shift. Software display facilitated identification of monoclonal proteins. Additionally, the process maintains positive specimen identification, reduces manual pipetting, allows for paper free tracking, and does not significantly impact turnaround time (TAT). Conclusions Mass-Fix is ready for implementation in a high-throughput clinical laboratory.

Objectives Congenital disorders of N-glycosylation (CDG) are a large group of rare metabolic disorders caused by defects in the most common post-translational modification of proteins. CDGs are often difficult to diagnose as they are manifested with non-specific symptoms and signs. Analysis of serum transferrin (TRF) isoforms, as the classical procedure used to identify a CDG patient, enables to predict pathological steps in the N-linked glycosylation process. Methods We devised a new strategy based on liquid chromatography-mass spectrometry (LC-MS) for the analysis of TRF isoforms by combining a simple and fast sample preparation with a specific chromatographic cleanup/separation step followed by mass-spectrometric measurement. Single TRF isoform masses were obtained through reconstruction of multiply charged electrospray data collected by quadrupole-MS technology. Hereby, we report the first analyzed serum samples obtained from 20 CDG patients and 100 controls. Results The ratio of desialylated isoforms to total TRF was calculated for patients and controls. CDG-Type I patients showed higher amounts of bi-sialo isoform (range: 6.7–29.6%) compared to controls (<5.5%, mean percentage 3.9%). CDG-Type II pattern showed an increased peak of tri-sialo isoforms. The mean percentage of tri-sialo-TRF was 9.3% (range: 2.9–12.9%) in controls, which was lower than that obtained from two patients with COG5-CDG and MAN1B1-CDG (18.5 and 24.5%). Intraday and between-day imprecisions were less than 9 and 16%, respectively, for bi-sialo- and less than 3 and 6% for tri-sialo-TRF. Conclusions This LC-MS-based approach provides a simple, sensitive and fast analytical tool for characterizing CDG disorders in a routine clinical biochemistry while improving diagnostic accuracy and speeding clinical decision-making.

Objectives Faecal immunochemical tests (FIT) for haemoglobin (Hb) are being used in the investigation of colorectal cancer. These tests use antibodies raised to the globin moiety of human Hb. Here, four automated quantitative FIT systems (HM-JACKarc, NS-Prime, OC-Sensor PLEDIA and SENTiFIT 270) are evaluated analytically to confirm whether the performance of the systems meet the manufacturers’ claims. Methods Assessment of the analytical performance of the FIT systems was undertaken using Hb lysates, real patient samples and external quality assessment (EQA) samples. This analytical assessment focused on detection characteristics, imprecision, linearity, prozone effect, recovery and carryover. Results All four methods demonstrated good analytical performance, with acceptable within- and between-run imprecision, good recovery of f-Hb and limited carryover of samples. They also all show good linearity across the range of concentrations tested. The results of EQA samples showed different variations from the target values (−52 to 45%), due to the absence of standardisation across the different methods. Conclusions All four systems are fit for purpose and have an analytical performance as documented by their manufacturers.

Objectives It is important to select proper quality specifications for laboratories and external quality assessment (EQA) providers for their quality control and assessment. The aim of this study is to produce new total error (TE) specifications for lymphocyte subset enumeration by analyzing the allowable TE using EQAS data and comparing them with that based on reliable biological variation (BV). Methods A total of 54,400 results from 1,716 laboratories were collected from China National EQAS for lymphocyte subset enumeration during the period 2017–2019. The EQA data were grouped according to lower limits of reference intervals for establishing concentration-dependent specifications. The TE value that 80% of laboratories can achieve were considered as TE specifications based on state of the art. The BV studies compliant with Biological Variation Data Critical Appraisal Checklist (BIVAC) were used to calculate the three levels of TE specifications. Then these TE specifications were compared for determining the recommended TE specifications. Results Four parameters whose quality specifications could achieve the optimum criteria were as follows: the percentages of CD3+, CD3+CD4+ (high concentration) and CD3–CD16/56+ cells, and the absolute count of CD3–CD16/56+ cells. Only the TE specifications of CD3–CD19+ cells could achieve the minimum criteria. The TE specifications of remaining parameters should reach the desirable criteria. Conclusions New TE specifications were established by combining the EQA data and reliable BV data, which could help laboratories to apply proper criteria for continuous improvement of quality control, and EQA providers to use robust acceptance limits for better evaluation of EQAS results.

Objectives Regulators of circadian rhythm, including melatonin, influence fundamental biological processes. Measuring the melatonin metabolite 6-sulfatoxymelatonin in urine can estimate melatonin production. 6-sulfatoxymelatonin is mainly analyzed by immunoassays, but these methods are hampered by cross-reactivity and poor reproducibility when used to analyze small molecules. Therefore, we validated a high-throughput liquid chromatography with tandem mass spectrometry (LC–MS/MS) method to quantify 6-sulfatoxymelatonin in urine. We evaluated age-dependent 24-h excretion of 6-sulfatoxymelatonin into urine and the biological variation of urinary excretion in healthy individuals. Methods The online solid phase extraction method combined with LC–MS/MS was validated according to international guidelines, and used to measure the excretion of 6-sulfatoxymelatonin into urine of 240 healthy individuals. Biological variation of 6-sulfatoxymelatonin excretion was examined in 10 healthy individuals. Results Urinary 6-sulfatoxymelatonin results were well within the validation criteria (interassay coefficient of variation: <5.4%, quantification limit: 0.2 nmol/L). There was an age-related decrease in 6-sulfatoxymelatonin excretion into 24-h urine [F(5, 234)=13.9; p<0.001]. Within-subject variation of 6-sulfatoxymelatonin was 39.2% in day urine, 15.1% in night urine, and 12.2% in 24-h urine. Between-subject variation was 39.1% in day urine, 37.9% in night urine, and 36.8% in 24-h urine. Conclusions This MS-based method enables straightforward, reproducible, and sensitive quantification of 6-sulfatoxymelatonin in urine. Urinary 6-sulfatoxymelatonin levels decreased with age. Biological variation of 6-sulfatoxymelatonin excretion into urine was high between subjects and lower within subjects, indicating that repeated measurements of 6-sulfatoxymelatonin in 24-h urine are needed in future studies.

Objectives Reference materials are important in the standardization of autoantibody testing and only a few are freely available for many known autoantibodies. Our goal was to develop three reference materials for antibodies to PML bodies/multiple nuclear dots (MND), antibodies to GW bodies (GWB), and antibodies to the nuclear mitotic apparatus (NuMA). Methods Reference materials for identifying autoantibodies to MND (MND-REF), GWB (GWB-REF), and NuMA (NuMA-REF) were obtained from three donors and validated independently by seven laboratories. The sera were characterized using indirect immunofluorescence assay (IFA) on HEp-2 cell substrates including two-color immunofluorescence using antigen-specific markers, western blot (WB), immunoprecipitation (IP), line immunoassay (LIA), addressable laser bead immunoassay (ALBIA), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation–mass spectrometry (IP-MS). Results MND-REF stained 6–20 discrete nuclear dots that colocalized with PML bodies. Antibodies to Sp100 and PML were detected by LIA and antibodies to Sp100 were also detected by ELISA. GWB-REF stained discrete cytoplasmic dots in interphase cells, which were confirmed to be GWB using two-color immunofluorescence. Anti-Ge-1 antibodies were identified in GWB-REF by ALBIA, IP, and IP-MS. All reference materials produced patterns at dilutions of 1:160 or greater. NuMA-REF produced fine speckled nuclear staining in interphase cells and staining of spindle fibers and spindle poles. The presence of antibodies to NuMA was verified by IP, WB, ALBIA, and IP-MS. Conclusions MND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.

Objectives Newborn screening (NBS) for β-thalassemia is based on measuring the expression of the hemoglobin A (HbA) fraction. An absence or very low level of HbA at birth may indicate β-thalassemia. The difficulty is that the HbA fraction at birth is correlated with gestational age (GA) and highly variable between individuals. We used HbA expressed in multiples of the normal (MoM) to evaluate relevant thresholds for NBS of β-thalassemia. Methods The chosen threshold (HbA≤0.25 MoM) was prospectively applied for 32 months in our regional NBS program for sickle cell disease, for all tests performed, to identify patients at risk of β-thalassemia. Reliability of this threshold was evaluated at the end of the study. Results In all, 343,036 newborns were tested, and 84 suspected cases of β-thalassemia were detected by applying the threshold of HbA≤0.25 MoM. Among the n =64 cases with confirmatory tests, 14 were confirmed using molecular analysis as β-thalassemia diseases, 37 were confirmed as β-thalassemia trait and 13 were false-positive. Determination of the optimum threshold for β-thalassemia screening showed that HbA≤0.16 MoM had a sensitivity of 100% and a specificity of 95.3%, whatever the GA. Conclusions NBS for β-thalassemia diseases is effective, regardless of the birth term, using the single robust threshold of HbA≤0.16 MoM. A higher threshold would also allow screening for carriers, which could be interesting when β-thalassemia constitutes a public health problem.

Objectives Synthetic B-type natriuretic peptide (BNP) is employed in most clinical testing platforms as a raw material of calibrator. Characterization of impurities with structures similar (BNP strimp compounds) to that of BNP is a reasonable way to decrease clinical measurement errors and improve drug safety. Methods A novel quantitative method targeted towards BNP strimp compounds was developed. First, the peptide samples were separated and identified using ultra-performance liquid chromatography, coupled with high-resolution mass spectrometry (MS). To evaluate biological activity further, BNP strimp immunoaffinity was investigated using western blot (WB) assays. Second, a quantitative label-free data-independent acquisition (DIA) MS approach was developed, and the internal standard peptide (ISP) was hydrolyzed. Absolute quantification was performed using an isotope dilution MS (ID-MS) approach. Third, method precision was investigated using the C-peptide reference material. Results Seventeen BNP strimp compounds were identified in synthetic BNP, and 10 of them were successfully sequenced. The immunoassay results indicated that deaminated, oxidized, and isomerized BNP strimp compounds exhibited weaker immunoaffinity than intact BNP 1-32 . The mass fraction of the synthetic solid ISP 1-16 , quantified by ID-MS, was 853.5 (±17.8) mg/g. Validation results indicated that the developed method was effective and accurate for the quantitation of the well-separated BNP impurities. Conclusions The developed approach was easy to perform, and it was suitable for the parallel quantification of low-abundance BNP strimp compounds when they performed a good separation in liquid chromatography. The quantitative results were comparable and traceable. This approach is a promising tool for BNP product quality and safety assessment.

Objectives Hemoglobin (Hb) variant is one of the most common monogenic inherited disorders. We aimed to explore the prevalence and hematological and molecular characteristics of Hb variants in southern China. Methods We collected blood samples from all patients with suspected variants found during HbA 1c measurement via a cation-exchange high-performance liquid chromatography system (Bio-Rad Variant II Turbo 2.0) or a capillary electrophoresis method (Sebia Capillarys). Hematological analysis, Sanger sequencing, and gap-PCR were performed for these samples. Results Among the 311,024 patients tested, we found 1,074 Hb variant carriers, including 823 identified using Capillarys and 251 using Variant II Turbo 2.0, with a total carrier rate of 0.35%. We discovered 117 types of Hb variants (52 HBB , 47 HBA , and 18 HBD mutations) containing 18 new mutations. The most common variant found was Hb E, followed by Hb New York, Hb J-Bangkok, Hb Q-Thailand, Hb G-Coushatta, Hb G-Honolulu, Hb G-Taipei, and Hb Broomhill. Most heterozygotes for the Hb variant exhibited normal hematological parameters. However, most patients with compound heterozygotes for the Hb variant and thalassemia showed varied degrees of microcytic hypochromic anemia. Conclusions The prevalence of hemoglobin variants remains high and exhibits genetic diversity and widespread distribution in the population of southern China.

Objectives Hemoglobin (Hb) variants remain an important cause of erroneous HbA 1c results. We present an approach to overcome the interference of Hb variants on HbA 1c measurements using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods Samples containing or not containing Hb variants were analyzed for HbA 1c using an MALDI-TOF MS system (QuanTOF) and a boronate affinity comparative method (Ultra 2 ). For QuanTOF, two sets of HbA 1c values were obtained through α- and β-chain glycation. Results A robust correlation between the glycation degrees of the α- and β-chains was found, and HbA 1c values derived from α- and β-chain glycation correlated well with the Ultra 2 results. Statistically significant differences (p<0.01) were found for all the Hb variants tested. When using the conventional β-chain glycation to determine HbA 1c , clinically significant differences were only found among samples containing β-chain variants detected by QuanTOF (i.e., Hb J-Bangkok, Hb G-Coushatta, and Hb G-Taipei). In contrast, based on α-chain glycation, no clinically significant differences were found for these three variants. Conclusions In addition to conventional β-chain glycation, α-chain glycation can be used to calculate HbA 1c values. The interference of Hb variants on HbA 1c quantification can be overcome by employing the glycation of the globin chain without a genetic variant to estimate HbA 1c values.