Quality assurance programs for vitamin A and E in serum: are we doing enough to assess laboratory performance?

OBJECTIVES: Monitoring serum vitamin A (retinol) and vitamin E (α-tocopherol) concentrations is common practice for assessing nutritional status. Measurement of these vitamins can be challenging due to several factors. Whilst the RCPAQAP Vitamins: Serum Program assists participating laboratories in harmonisation, the materials provided do not contain the analogues of retinol and α-tocopherol that may be present in real patient samples. We aimed to assess participants' capacity to accurately report retinol and α-tocopherol in the presence of the vitamin E analogues tocopherol acetate and γ-tocopherol. METHODS: A supplementary series of a control sample and three matched spiked samples were distributed to each laboratory participating in the Program. Retinol and α-tocopherol results for each spiked sample were compared to the results of the control sample submitted by each participant. Acceptability of retinol and α-tocopherol results was determined based on the RCPAQAP allowable performance specifications (APS). RESULTS: Thirteen participants returned results for the supplementary sample series. Interference from α-tocopherol acetate was observed with results below the APS in 30 % (n=4) of laboratories for retinol quantification and in 23 % (n=3) for α-tocopherol quantification. One laboratory returned results above the APS for α-tocopherol when γ-tocopherol was present. CONCLUSIONS: This supplementary sample series has shown that the presence of vitamin E analogues can lead to the over or under estimation of nutritional status by some participants. Affected laboratories are encouraged to review their analytical procedures. To further assess laboratory competence, EQA providers should consider using patient samples or spiked challenge samples.

Linearity assessment: deviation from linearity and residual of linear regression approaches.

In this computer simulation study, we examine four different statistical approaches of linearity assessment, including two variants of deviation from linearity (individual (IDL) and averaged (AD)), along with detection capabilities of residuals of linear regression (individual and averaged). From the results of the simulation, the following broad suggestions are provided to laboratory practitioners when performing linearity assessment. A high imprecision can challenge linearity investigations by producing a high false positive rate or low power of detection. Therefore, the imprecision of the measurement procedure should be considered when interpreting linearity assessment results. In the presence of high imprecision, the results of linearity assessment should be interpreted with caution. Different linearity assessment approaches examined in this study performed well under different analytical scenarios. For optimal outcomes, a considered and tailored study design should be implemented. With the exception of specific scenarios, both ADL and IDL methods were suboptimal for the assessment of linearity compared. When imprecision is low (3 %), averaged residual of linear regression with triplicate measurements and a non-linearity acceptance limit of 5 % produces <5 % false positive rates and a high power for detection of non-linearity of >70 % across different types and degrees of non-linearity. Detection of departures from linearity are difficult to identify in practice and enhanced methods of detection need development.

Bilirubin measurements in neonates: uniform neonatal treatment can only be achieved by improved standardization.

Measurement of total bilirubin (TBil) concentration in serum is the gold standard approach for diagnosing neonatal unconjugated hyperbilirubinemia. It is of utmost importance that the measured TBil concentration is sufficiently accurate to prevent under treatment, unnecessary escalation of care, or overtreatment. However, it is widely recognized that TBil measurements urgently require improvement in neonatal clinical chemistry. External quality assessment (EQA) programs for TBil assess for differences between laboratories and provide supporting evidence of significant differences between various methods, manufacturers and measurement platforms. At the same time, many countries have adopted or only slightly adapted the neonatal hyperbilirubinemia management guidelines from the USA or UK, often without addressing differences in the methodology of TBil measurements. In this report, we provide an overview of the components of bilirubin that are measured by laboratory platforms, the availability of current reference measurement procedures and reference materials, and the role of EQA surveys in this context. Furthermore, the current status of agreement in neonatal bilirubin against clinical decision thresholds is reviewed. We advocate for enhancements in accuracy and comparability of neonatal TBil measurements, propose a path forward to accomplish this, and reflect on the position of the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Working Group Neonatal Bilirubin (WG-NB) in this matter.

Ensuring quality in 17OHP mass spectrometry measurement: an international study assessing isomeric steroid interference.

OBJECTIVES: Interference from isomeric steroids is a potential cause of disparity between mass spectrometry-based 17-hydroxyprogesterone (17OHP) results. We aimed to assess the proficiency of mass spectrometry laboratories to report 17OHP in the presence of known isomeric steroids. METHODS: A series of five samples were prepared using a previously demonstrated commutable approach. These samples included a control (spiked to 15.0 nmol/L 17OHP) and four challenge samples further enriched with equimolar concentrations of 17OHP isomers (11α-hydroxyprogesterone, 11ß-hydroxyprogesterone, 16α-hydroxyprogesterone or 21-hydroxyprogesterone). These samples were distributed to 38 participating laboratories that reported serum 17OHP results using mass spectrometry in two external quality assurance programs. The result for each challenge sample was compared to the control sample submitted by each participant. RESULTS: Twenty-six laboratories (68 % of distribution) across three continents returned results. Twenty-five laboratories used liquid chromatography-tandem mass spectrometry (LC-MS/MS), and one used gas chromatography-tandem mass spectrometry to measure 17OHP. The all-method median of the control sample was 14.3 nmol/L, ranging from 12.4 to 17.6 nmol/L. One laboratory had results that approached the lower limit of tolerance (minus 17.7 % of the control sample), suggesting the isomeric steroid caused an irregular result. CONCLUSIONS: Most participating laboratories demonstrated their ability to reliably measure 17OHP in the presence of the four clinically relevant isomeric steroids. The performance of the 12 (32 %) laboratories that did not engage in this activity remains unclear. We recommend that all laboratories offering LC-MS/MS analysis of 17OHP in serum, plasma, or dried bloodspots determine that the isomeric steroids are appropriately separated.

Emerging technology: a definition for laboratory medicine.

The term "emerging technology" (ET) is used extensively, and there are numerous definitions offered, but to our knowledge, none specifically encompass the field of laboratory medicine. An ET definition that incorporates the overarching IFCC aim of "Advancing excellence in laboratory medicine to support healthcare worldwide" would clarify discussions. We discuss key aspects of the term "emerging technology(ies)" as it applies to laboratory medicine with a view to laying the foundations for a practical definition for the profession and propose the definition of an ET as "An analytical method or device that by virtue of its stage of development, translation into broad routine clinical practice, or geographical adoption and implementation has the potential to add value to clinical diagnostics".

Method evaluation in the clinical laboratory.

Method evaluation is one of the critical components of the quality system that ensures the ongoing quality of a clinical laboratory. As part of implementing new methods or reviewing best practices, the peer-reviewed published literature is often searched for guidance. From the outset, Clinical Chemistry and Laboratory Medicine (CCLM) has a rich history of publishing methods relevant to clinical laboratory medicine. An insight into submissions, from editors' and reviewers' experiences, shows that authors still struggle with method evaluation, particularly the appropriate requirements for validation in clinical laboratory medicine. Here, we consider through a series of discussion points an overview of the status, challenges, and needs of method evaluation from the perspective of clinical laboratory medicine. We identify six key high-level aspects of clinical laboratory method evaluation that potentially lead to inconsistency. 1. Standardisation of terminology, 2. Selection of analytical performance specifications, 3. Experimental design of method evaluation, 4. Sample requirements of method evaluation, 5. Statistical assessment and interpretation of method evaluation data, and 6. Reporting of method evaluation data. Each of these areas requires considerable work to harmonise the practice of method evaluation in laboratory medicine, including more empirical studies to be incorporated into guidance documents that are relevant to clinical laboratories and are freely and widely available. To further close the loop, educational activities and fostering professional collaborations are essential to promote and improve the practice of method evaluation procedures.

The LEAP checklist for laboratory evaluation and analytical performance characteristics reporting of clinical measurement procedures.

Reporting a measurement procedure and its analytical performance following method evaluation in a peer-reviewed journal is an important means for clinical laboratory practitioners to share their findings. It also represents an important source of evidence base to help others make informed decisions about their practice. At present, there are significant variations in the information reported in laboratory medicine journal publications describing the analytical performance of measurement procedures. These variations also challenge authors, readers, reviewers, and editors in deciding the quality of a submitted manuscript. The International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Method Evaluation Protocols (IFCC WG-MEP) developed a checklist and recommends its adoption to enable a consistent approach to reporting method evaluation and analytical performance characteristics of measurement procedures in laboratory medicine journals. It is envisioned that the LEAP checklist will improve the standardisation of journal publications describing method evaluation and analytical performance characteristics, improving the quality of the evidence base that is relied upon by practitioners.

The LEAP checklist for Laboratory Evaluation and Analytical Performance Characteristics reporting of clinical measurement procedures.

Reporting a measurement procedure and its analytical performance following method evaluation in a peer-reviewed journal is an important means for clinical laboratory practitioners to share their findings. It also represents an important source of evidence base to help others make informed decisions about their practice. At present, there are significant variations in the information reported in laboratory medicine journal publications describing the analytical performance of measurement procedures. These variations also challenge authors, readers, reviewers, and editors in deciding the quality of a submitted manuscript.The International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Method Evaluation Protocols (IFCC WG-MEP) developed a checklist and recommends its adoption to enable a consistent approach to reporting method evaluation and analytical performance characteristics of measurement procedures in laboratory medicine journals. It is envisioned that the LEAP checklist will improve the standardisation of journal publications describing method evaluation and analytical performance characteristics, improving the quality of the evidence base that is relied upon by practitioners.

Current and emerging technologies for the timely screening and diagnosis of neonatal jaundice.

Neonatal jaundice is one of the most common clinical conditions affecting newborns. For most newborns, jaundice is harmless, however, a proportion of newborns develops severe neonatal jaundice requiring therapeutic interventions, accentuating the need to have reliable and accurate screening tools for timely recognition across different health settings. The gold standard method in diagnosing jaundice involves a blood test and requires specialized hospital-based laboratory instruments. Despite technological advancements in point-of-care laboratory medicine, there is limited accessibility of the specialized devices and sample stability in geographically remote areas. Lack of suitable testing options leads to delays in timely diagnosis and treatment of clinically significant jaundice in developed and developing countries alike. There has been an ever-increasing need for a low-cost, simple to use screening technology to improve timely diagnosis and management of neonatal jaundice. Consequently, several point-of-care (POC) devices have been developed to address this concern. This paper aims to review the literature, focusing on emerging technologies in the screening and diagnosing of neonatal jaundice. We report on the challenges associated with the existing screening tools, followed by an overview of emerging sensors currently in pre-clinical development and the emerging POC devices in clinical trials to advance the screening of neonatal jaundice. The benefits offered by emerging POC devices include their ease of use, low cost, and the accessibility of rapid response test results. However, further clinical trials are required to overcome the current limitations of the emerging POC's before their implementation in clinical settings. Hence, the need for a simple to use, low-cost POC jaundice detection technology for newborns remains an unsolved challenge globally.

Multi-omics analysis from archival neonatal dried blood spots: limitations and opportunities.

Newborn screening (NBS) programs operate in many countries, processing millions of dried bloodspot (DBS) samples annually. In addition to early identification of various adverse health outcomes, these samples have considerable potential as a resource for population-based research that could address key questions related to child health. The feasibility of archival DBS samples for emerging targeted and untargeted multi-omics analysis has not been previously explored in the literature. This review aims to critically evaluate the latest advances to identify opportunities and challenges of applying omics analyses to NBS cards in a research setting. Medline, Embase and PubMed databases were searched to identify studies utilizing DBS for genomic, proteomic and metabolomic assays. A total of 800 records were identified after removing duplicates, of which 23 records were included in this review. These papers consisted of one combined genomic/metabolomic, four genomic, three epigenomic, four proteomic and 11 metabolomic studies. Together they demonstrate that the increasing sensitivity of multi-omic analytical techniques makes the broad use of NBS samples achievable for large cohort studies. Maintaining the pre-analytical integrity of the DBS sample through storage at temperatures below -20 °C will enable this important resource to be fully realized in a research capacity.

Validation of steroid ratios for random urine by mass spectrometry to detect 5α-reductase deficiency in Vietnamese children.

OBJECTIVES: The 5α-reductase-type-2 deficiency (5ARD2) is a rare autosomal recessive 46,XY disorder of sex development caused by the mutated 5α-reductase type 2 (SRD5A2) gene. In this disease, defective conversion of testosterone to dihydrotestosterone leads to variable presentations of male ambiguous genitalia during fetal development. We aimed to examine characteristics of patients presenting with 5ARD2 over a 4 year period. METHODS: Random urine samples of control and patients with suspected 5ARD2 were collected and urine steroidomic metabolites were measured by Gas chromatography-mass spectrometry (GC-MS) in the period from 2017 to 2021 at National Children's Hospital, Hanoi Vietnam. 5α- to 5ß-reduced steroid metabolite ratio, 5a-tetrahydrocortisol to tetrahydrocortisol (5α-THF/THF), was reviewed by receive operator characteristics (ROC) curve analysis. Molecular testing was offered to 25 patients who were diagnosed with 5ARD2 by GC-MS urinary steroid analysis. RESULTS: Urine steroidomic profiling was conducted for 104 male controls and 25 patients between the ages of 6 months and 13 years old. Twelve of the twenty-five 5ARD2 patients agreed to undertake genetic analysis, and two mutations of the SRD5A2 gene were detected in each patient, confirming the diagnosis. All patients showed a characteristically low ratio of 5α-THF/THF. There was no overlap of 5α-THF/THF ratio values between control and 5ARD2 groups. The ROC of 5α-THF/THF ratio at 0.19 showed 100% sensitivity and 100% specificity for boys between 6 months and 13 years of age. CONCLUSIONS: Analysis of the urine steroid metabolome by GC-MS can be used to assist in the diagnosis of 5ARD2. We recommend consideration of random urine steroid analysis as a first-line test in the diagnosis of 5ARD2.

Newborn bloodspot screening in the time of COVID-19.

PURPOSE: A COVID-19 pandemic business continuity plan (BCP) was rapidly developed to protect the Victorian newborn screening (NBS) program. Here, we present the outcomes of our COVID-19 BCP and its impact on the Victorian NBS laboratory service. METHODS: Change management principles were used to develop a BCP that included mapping of NBS processes against staff resources, triaging priorities, technology solutions, supply chain continuity, gap analysis, and supporting maternity service providers. The effect was assessed quantitatively by review of key performance indicator data and qualitatively from staff feedback. RESULTS: A four-stage BCP was implemented. Stage 1 split teams into two, which rotated weekly, onsite (laboratory) and offsite (home). At 20 weeks post-implementation the BCP only progressed to stage 1 and the overall turnaround time was maintained. Staff experience indicated benefits from the review of workflow but noted some social impact associated with the change. CONCLUSION: The preparedness and agility of implementation was based on our focus on the newborn babies and their families, our production system, and a continuous improvement mindset. Both our people and technology infrastructure processes are crucial to this success as we continue to adapt to new challenges.

Mechanism of bilirubin elimination in urine: insights and prospects for neonatal jaundice.

Despite a century of research, bilirubin metabolism and the transport mechanisms responsible for homeostasis of bilirubin in serum remain controversial. Emerging evidence on the hepatic membrane transporters and inherited disorders of bilirubin metabolism have contributed to a greater understanding of the various steps involved in bilirubin homeostasis and its associated excretory pathways. We discuss these recent research findings on hepatic membrane transporters and evaluate their significance on the newborn bilirubin metabolism and excretion. New insights gained speculate that a proportion of conjugated bilirubin is excreted via the renal system, as an alternative to the intestinal excretion, even in normal physiological jaundice with no associated pathological concerns. Finally, this paper discusses the clinical relevance of targeting the altered renal excretory pathway, as bilirubin in urine may hold diagnostic importance in screening for neonatal jaundice.

The Enigma of the Adrenarche: Identifying the Early Life Mechanisms and Possible Role in Postnatal Brain Development.

Dehydroepiandrosterone (DHEA) and its sulfated metabolite (DHEAS) are dynamically regulated before birth and the onset of puberty. Yet, the origins and purpose of increasing DHEA[S] in postnatal development remain elusive. Here, we draw attention to this pre-pubertal surge from the adrenal gland-the adrenarche-and discuss whether this is the result of intra-adrenal gene expression specifically affecting the zona reticularis (ZR), if the ZR is influenced by the hypothalamic-pituitary axis, and the possible role of spino-sympathetic innervation in prompting increased ZR activity. We also discuss whether neural DHEA[S] synthesis is coordinately regulated with the developing adrenal gland. We propose that DHEA[S] is crucial in the brain maturation of humans prior to and during puberty, and suggest that the function of the adrenarche is to modulate, adapt and rewire the pre-adolescent brain for new and ever-changing social challenges. The etiology of DHEA[S] synthesis, neurodevelopment and recently described 11-keto and 11-oxygenated androgens are difficult to investigate in humans owing to: (i) ethical restrictions on mechanistic studies, (ii) the inability to predict which individuals will develop specific mental characteristics, and (iii) the difficulty of conducting retrospective studies based on perinatal complications. We discuss new opportunities for animal studies to overcome these important issues.

Candidate reference method for determination of vitamin D from dried blood spot samples.

Background The current millennium has seen an explosion in vitamin D testing with the overarching aim of requests to clinically stratify patients as replete or deficient in vitamin D. At a population level, dried blood spot (DBS) sampling offers a less invasive and more practical application for assessment of vitamin D status. We have therefore aimed to develop a sensitive and robust DBS vitamin D method that is traceable to serum for use in population-based studies. Methods Blood spots, calibrators and controls were prepared by punching a 3.2 mm DBS from filter paper and placed into a 96-well micro-plate. The DBS disk was eluted with a combination of water-methanol and internal standard (ISTD) solution followed by supported-liquid extraction and derivatisation. The extract was analysed by liquid-chromatography tandem-mass spectrometry in positive electrospray-ionisation mode with 732.5 > 673.4 and 738.4 > 679.4 m/z ion-transitions for derivatised vitamin D and the ISTD, respectively. Vitamin D results were made traceable to the National Institute of Standards and Technology reference material through the inclusion of Chromsystems vitamin D calibrators. Results 25-Hydroxy-vitamin D3 and its related ISTD were detected at a retention time of 7 min. The seven-point calibration-curve consistently demonstrated a coefficient of determination of 0.99 with an experimentally determined reportable range of 0.5-376 nmol/L. Method validation studies using DBS samples demonstrated 12.9% between-assay imprecision at 45 nmol/L, 84% average recovery and high correlation with plasma vitamin D (correlation coefficient = 0.86). Conclusions We have successfully developed an analytical method for vitamin D quantitation from DBSs which will be applied to our population-based vitamin D research study. This approach improves traceability of DBS results and potentially could be used broadly for other DBS measurands that require comparison to serum/plasma for their interpretation.

Vitamin C measurement in critical illness: challenges, methodologies and quality improvements.

Background There is renewed interest in high-dose vitamin C interventions in clinical medicine due to its antioxidant properties, safe use and cost-effectiveness. Yet, randomised control trials (RCTs) employing these interventions are failing to include robust analytical methodology and proper sample handling and processing techniques. Consequently, comparisons between studies becomes impossible as there is no metrological traceability and results may be prone to pre-analytical errors. Content Through published vitamin C stability studies, method comparison papers and data from vitamin C external quality assurance programs, an assessment was made on the functionality of current methods for critically ill patient samples. Summary Data was obtained from two external quality assurance programs, two papers assessing sample stability and interlaboratory agreement and a publication on vitamin C method comparisons. A shift from spectrophotometric and enzymatic methodologies to high performance liquid chromatography (HPLC) greatly improved the variability and interlaboratory agreement. Therefore, the current analytical performance of vitamin C HPLC methodologies are acceptable for the requirements of a high-dose vitamin C RCTs. Outlook Recommendations across the total testing process of vitamin C have been provided to improve the quality of the results. The harmonisation of sample handling and processing procedures will further improve the reliability of current analytical methodologies.

Influence of isotopically labeled internal standards on quantification of serum/plasma 17α-hydroxyprogesterone (17OHP) by liquid chromatography mass spectrometry.

Objectives Our recent survey of 44 mass spectrometry laboratories across 17 countries identified variation in internal standard (IS) choice for the measurement of serum/plasma 17α-hydroxyprogesterone (17OHP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The choice of IS may contribute to inter-method variations. This study evaluated the effect of two common isotopically labeled IS on the quantification of 17OHP by LC-MS/MS. Methods Three collaborating LC-MS/MS laboratories from Asia, Europe and Australia, who routinely measure serum 17OHP, compared two IS, (1) IsoSciences carbon-13 labeled 17OHP-[2,3,4-13C3], and (2) IsoSciences deuterated 17OHP-[2,2,4,6,6,21,21,21-2H]. This was performed as part of their routine patient runs using their respective laboratory standard operating procedure. Results The three laboratories measured 99, 89, 95 independent samples, respectively (up to 100 nmol/L) using the 13C- and 2H-labeled IS. The slopes of the Passing-Bablok regression ranged 0.98-1.00 (all 95% confidence interval [CI] estimates included the line of identity), and intercept of <0.1 nmol/L. Average percentage differences of -0.04% to -5.4% were observed between the two IS materials, which were less than the optimal bias specification of 7% determined by biological variation, indicating no clinically significant difference. The results of 12 Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) proficiency samples (1-40 nmol/L) measured by the laboratories were all within the RCPAQAP analytical performance specifications for both IS. Conclusions Overall, the comparison between the results of 13C- and 2H-labeled IS for 17OHP showed good agreement, and show no clinically significant bias when incorporated into the LC-MS/MS methods employed in the collaborating laboratories.

Determination of haemoglobin derivatives in aged dried blood spot to estimate haematocrit.

Introduction Dried blood spot (DBS) sample applications now encompass analytes related to clinical diagnosis, epidemiological studies, therapeutic drug monitoring, pharmacokinetic and toxicokinetic studies. Haematocrit (Hct) and haemoglobin (Hb) at very high or low concentrations may influence the accuracy of measurement quantification of the DBS sample. In this study, we aimed to predict the Hct of the punched DBS through primary spectrophotometric estimation of its haemoglobin-derivative (Hb-drv) content. Methods Formic acid solution was used to elute Hb-drv content of 3.2 mm spotted blood from its dry matrix. Direct spectrometry measurement was utilised to scan the extracted Hb-drv in the visible spectrum range of 520-600 nm. The linear relationship between an individual's Hct percentage and Hb-drv concentration was applied to estimate the Hct level of the blood spot. De-identified whole blood samples were used for the method development and evaluation studies. Results The Hb-drv estimation is valid in samples >2 months old. Method validation experiments DBS demonstrate linearity between 82.5 and 207.5 g/L, average coefficient of variation of 3.6% (intra-assay) and 7.7% (inter-assay), analytical recovery of 84%, and a high positive correlation (r=0.88) between Hb-drv and the original whole blood Hct. The Bland-Altman difference plot demonstrates a mean difference of 2.4% between the calculated DBS Hct and the directly measured Hct from fresh whole bloods. Conclusions We have successfully developed a simple Hb-drv method to estimate Hct in aged DBS samples. This method can be incorporated into DBS analytical work-flow for the in-situ estimation of Hct and subsequent correction of the analyte of interest as required.