Personalized and Population-Based Reference Intervals for 48 Common Clinical Chemistry and Hematology Measurands: A Comparative Study.

BACKGROUND: Personalized reference intervals (prRIs) have the potential to improve individual patient follow-up as compared to population-based reference intervals (popRI). In this study, we estimated popRI and prRIs for 48 clinical chemistry and hematology measurands using samples from the same reference individuals and explored the effect of using group-based and individually based biological variation (BV) estimates to derive prRIs. METHODS: 143 individuals (median age 28 years) were included in the study and had fasting blood samples collected once. From this population, 41 randomly selected subjects had samples collected weekly for 5 weeks. PopRIs were estimated according to Clinical Laboratory Standards Institute EP28 and within-subject BV (CVI) were estimated by CV-ANOVA. Data were assessed for trends and outliers prior to calculation of individual prRIs, based on estimates of (a) within-person BV (CVP), (b) CVI derived in this study, and (c) publically available CVI estimates. RESULTS: For most measurands, the individual prRI ranges were smaller than the popRI range, but overall about half the study participants had a prRI wider than the popRI for 5 or more out of 48 measurands. The dispersion of prRIs based on CVP was wider than that of prRIs based on CVI. CONCLUSION: The prRIs derived in our study varied significantly between different individuals, especially if based on CVP. Our results highlight the limitations of popRIs in interpreting test results of individual patients. If sufficient data from a steady-state situation are available, using prRI based on CVP estimates will provide a RI most specific for an individual patient.

Machine Learning-Assisted Pattern Recognition of Amyloid Beta Aggregates with Fluorescent Conjugated Polymers and Graphite Oxide Electrostatic Complexes.

Five fluorescent positively charged poly(para-aryleneethynylene) (P1-P5) were designed to construct electrostatic complexes C1-C5 with negatively charged graphene oxide (GO). The fluorescence of conjugated polymers was quenched by the quencher GO. Three electrostatic complexes were enough to distinguish between 12 proteins with 100% accuracy. Furthermore, using these sensor arrays, we could identify the levels of Aß40 and Aß42 aggregates (monomers, oligomers, and fibrils) via employing machine learning algorithms, making it an attractive strategy for early diagnosis of Alzheimer's disease.

Methods to reduce lipemic interference in clinical chemistry tests: a systematic review and recommendations.

OBJECTIVES: Lipemia is the presence of abnormally high lipoprotein concentrations in serum or plasma samples that can interfere with laboratory testing. There is little guidance available from manufacturers or professional bodies on processing lipemic samples to produce clinically acceptable results. This systematic review summarizes existing literature on the effectiveness of lipid removal techniques in reducing interference in clinical chemistry tests. METHODS: A PubMed search using terms relating to lipid removal from human samples for clinical chemistry tests produced 1,558 studies published between January 2010 and July 2021. 15 articles met the criteria for further analyses. RESULTS: A total of 66 analytes were investigated amongst the 15 studies, which showed highly heterogenous study designs. High-speed centrifugation was consistently effective for 13 analytes: albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, creatine kinase (CK), creatinine (Jaffe method), gamma-glutamyl transferase (GGT), glucose (hexokinase-based method), lactate dehydrogenase (LDH), phosphate, potassium, and urea. Lipid-clearing agents were uniformly effective for seven analytes: ALT, AST, total bilirubin, CK, creatinine (Jaffe method), lipase, and urea. Mixed results were reported for the remaining analytes. CONCLUSIONS: For some analytes, high-speed centrifugation and/or lipid-clearing agents can be used in place of ultracentrifugation. Harmonized protocols and acceptability criteria are required to allow pooled data analysis and interpretation of different lipemic interference studies.

Evaluation of 20 clinical chemistry and 12 immunoassay analytes in terms of total analytical error and measurement uncertainty.

In analytical quality management, target setting models that are selected by the purpose together with the error models that are applied correctly have critical importance. In our study, we aimed to compare the analytical performance characteristics of routine clinical chemistry and immunoassay tests with different target-setting models proposed by various organizations. Our study was performed with internal and external quality control data obtained using Beckman Coulter AU680 for clinical chemistry analytes and Roche Cobas 8000 autoanalyzer for immunoassay analytes. The total analytical error (TAE) was calculated by the formula TAH%=1.65×(CV%)+Bias%. Measurement uncertainty (MU) has been calculated adhering to the Nordtest guideline. Results were compared with BVEFLM, CLIA, RCPA, PRDEQA%, pUEQAS%, and permissible MU (pU%) data to investigate analytical performance qualities. When we compare the results of TAE and MU, MU was found to be higher than TAE for all analytes. ALT, AST, glucose, K, and triglycerides met all target values, showing the best performance. Taken together, our results show that CLIA for total analytical error and PRDEQA% and pUEQAS% for measurement uncertainty can match better than BVEFLM, RCPA and pU%. These test results should be evaluated with measurement uncertainty to avoid misdiagnosis. Appropriate specification limits should be defined for the examination of test methods that meet the objectives for fitness for clinical purposes.

Comparison of regression for blood ALP levels using methods of the Japan Society of Clinical Chemistry and the International Federation of Clinical Chemistry and Laboratory Medicine in bovine, canine, feline, and human testing.

Livestock and companion animal health have a direct impact on human health. Research on clinical laboratory technology for veterinary medicine is as important as that on human laboratory technology. Reagents and analysis equipment for human medical laboratory tests are often used in veterinary medicine. Medical laboratories in Japan utilize the Japan Society of Clinical Chemistry (JSCC) method for blood alkaline phosphatase (ALP) analysis. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) method is used worldwide for ALP catalytic concentration measurement. When the IFCC method is used, human blood ALP activity is approximately one-third of the JSCC method's activity. The JSCC method for ALP measurement was switched to the IFCC method in medical laboratories in Japan in April 2020 for global standardization purpose. It is uncertain whether conventional JSCC method reagents will continue to be supplied. In veterinary medicine, the relationship between the JSCC and IFCC methods in terms of ALP measurement is almost unclear. This study investigated the regression between JSCC and IFCC methods measuring ALP in bovine, canine, feline, and human. The regression formulas for bovine, canine, feline, and human ALP values using the conventional JSCC (x) and IFCC (y) methods are y = 0.379x + 0.124, y = 0.289x + 8.291, y = 0.358x + 0.432, and y = 0.337x + 2.959, respectively. These results suggested that the IFCC method measurement could be estimated by approximately one-third of the JSCC method measurement in animal species such as bovine, canine, and feline. By applying the conversion factors proposed in this study, a very good correlation could be obtained between the two methods for each animal.

Impact of managing affected results in haemolysed samples of an infant-maternity hospital using an unconventional approach.

BACKGROUND: The management of affected results in haemolysed samples (HS) is debated. In an infant-maternity setting, for reporting interfered test results, we provided the result itself, the degree of haemolysis (as free haemoglobin concentration), and a warning recommending sample recollection. We investigated the impact of this approach on sample quality and clinicians' decision-making. METHODS: Free haemoglobin was measured on Beckman Coulter AU680 as haemolytic index. We estimated the total HS number, the clinical wards more affected by HS, the most interfered analytes, and the retesting rate of interfered tests, by comparing data from Apr-Dec 2017, the period just after the introduction of the new policy, vs. Apr-Dec 2018. RESULTS: One year after the new report introduction, a significant HS decrease (5.8% vs. 7.8%, P < 0.001) was detected, together with a reduction of the frequency by which haemolysis affected results. The most affected wards, i.e., Paediatric and Neonatal Intensive Care Units, showed an improvement in sample quality (HS rate, 30.6% to 16.1%, P < 0.001, and 25.2% to 20.9%, P = 0.048, respectively). We noted a significant decrease in retesting after an alerted result for aspartate aminotransferase, magnesium, potassium, conjugated bilirubin, and lactate dehydrogenase. CONCLUSIONS: Our approach led to a HS decrease, suggesting that the provided report could be a driving force for improvement of phlebotomy quality, also helping clinicians in deciding if retesting is essential or not.

Evaluation of the quick-clotting serum separator tube, VQ-Tube™, for clinical chemistry and thyroid hormone assays.

BACKGROUND: The type of blood collection tube affects specimen quality and laboratory results. Because plasma specimens have a shorter processing time compared with serum specimens, emergency biochemistry tests use plasma. However, serum specimens remain stable after centrifugation and show more accurate results than plasma. Therefore, a quick-clotting serum separator tube is expected to be useful for shorter turnaround times and accurate results. We evaluated a new quick-clotting serum separator tube VQ-Tube™ (AB Medical, Korea) for clinical chemistry and thyroid hormone assays. METHODS: One hundred volunteers from four university hospitals were recruited, and peripheral blood samples were collected in quick-clotting serum separator tube VQ-Tubes™ and the commonly used serum separator tube V-Tubes™. The obtained specimens were used for 16 clinical chemistry assays and three thyroid hormone assays. RESULTS: The differences (%) in the test results obtained from the samples in each tube satisfied the allowable difference ranges (19 assays). The differences in the test results between the tubes satisfied the desired specifications for accuracy except for the glucose results (2.75%). The paired t-test revealed significant differences between the results of six assays, but each set of results showed a good correlation. Samples were visually inspected for serum clarity and gel barrier integrity, and incomplete clotting reactions and haemolysed serum were not observed. CONCLUSIONS: The new quick-clotting VQ-Tube™ demonstrated reliable test results compared with the commonly used serum separator tube V-Tube™. This quick-clotting tube will provide fast test results with adequately separated serum specimens, especially for patients who need fast tests.

The Time Has Come for Quantitative Protein Mass Spectrometry Tests That Target Unmet Clinical Needs.

Protein mass spectrometry (MS) is an enabling technology that is ideally suited for precision diagnostics. In contrast to immunoassays with indirect readouts, MS quantifications are multiplexed and include identification of proteoforms in a direct manner. Although widely used for routine measurements of drugs and metabolites, the number of clinical MS-based protein applications is limited. In this paper, we share our experience and aim to take away the concerns that have kept laboratory medicine from implementing quantitative protein MS. To ensure added value of new medical tests and guarantee accurate test results, five key elements of test evaluation have been established by a working group within the European Federation for Clinical Chemistry and Laboratory Medicine. Moreover, it is emphasized to identify clinical gaps in the contemporary clinical pathways before test development is started. We demonstrate that quantitative protein MS tests that provide an additional layer of clinical information have robust performance and meet long-term desirable analytical performance specifications as exemplified by our own experience. Yet, the adoption of quantitative protein MS tests into medical laboratories is seriously hampered due to its complexity, lack of robotization and high initial investment costs. Successful and widespread implementation in medical laboratories requires uptake and automation of this next generation protein technology by the In-Vitro Diagnostics industry. Also, training curricula of lab workers and lab specialists should include education on enabling technologies for transitioning to precision medicine by quantitative protein MS tests.

Verification of the hemolysis index measurement: imprecision, accuracy, measuring range, reference interval and impact of implementing analytically and clinically derived sample rejection criteria.

Automated spectrophotometric measurement of hemolysis index (HI) allows rapid and cost-effective assessment of hemolysis interference. We evaluated the analytical performance of HI on two different platforms. Further, the impact of implementing analytically and clinically derived sample rejection criteria was investigated. Precision profiles were established, and analytical error was assessed by comparison with the reference method for hemoglobin measurement on Architect c8000/c16000 (Abbott) and Cobas c702 (Roche Diagnostics) instruments. The impact of a more analyte-specific cut off based on analytical and biologival variation was examined for five hemolysis-sensitive plasma analyses according to European recommendations. Lastly, a reference interval was established for the HI on Cobas, using the CLSI C28A3c nonparametric method. Imprecision for HI of 0.6-3.0 % for Architect and 1.5-4.5 % for Cobas was considered acceptable, which also applied for trueness in the measuring tange > 2 g/L. If cutoffs based on analytical and biological variation were used to manage results from hemolyzed samples, more potassium, LDH, conjugated bilirubin and phosphate results would be suppressed. Considering RCV only LDS remained sensitive to hemolysis. The Cobas-based HI reference interval was established to 0.01-0.16 g/L. Thorough verification of the HI on two different clinical chemistry analyzers reveals acceptable analytical performance. HI cutoffs suggested by manufacturers may be optimized by clinical laboratories using analytical and/or biological variation. A reference interval for the HI analysis is relevant as the analysis has been suggested as a diagnostic tool in the assessment of in vivo hemolysis.

Regression formula to predict the International Federation of Clinical Chemistry and Laboratory Medicine measure of alkaline phosphatase activity in canine blood based on the Japan Society of Clinical Chemistry reference method.

The Japan Society of Clinical Chemistry reference method (JSCC method) is used to measure alkaline phosphatase (ALP) activity only in Japan. Other countries use the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method to measure ALP activity. Since April 2020, human medical institutions in Japan have been gradually switching to the IFCC method. However, it is unclear whether the supply of reagents required for the JSCC method will be steady in the future. Additionally, the comparison of the performances and accuracies of these two methods for measuring ALP values remains uncertain in several animal species. In this investigation, we measured canine ALP activity using both methods and developed a formula to interconvert the two resulting values. The regression formula for ALP values measured using the modified JSCC (x) and IFCC (y) methods was determined as log10 y=0.960 log10 x-0.395 (r=0.997). However, the correlation between values based on JSCC and IFCC methods can change depending on the composition of ALP isozymes. Therefore, the developed formula can currently serve as a provisional strategy in calculating ALP levels. Nevertheless, this formula might avoid confusion in the clinical field during the transition from the JSCC to the IFCC method when both measurement values co-exist.

Reviewing the Analytical Methodologies to Determine the Occurrence of Citrinin and its Major Metabolite, Dihydrocitrinone, in Human Biological Fluids.

Until now, the available data regarding citrinin (CIT) levels in food and the consumption of contaminated foods are insufficient to allow a reliable estimate of intake. Therefore, biomonitoring configuring analysis of parent compound and/or metabolites in biological fluids, such as urine or blood, is being increasingly applied in the assessment of human exposure to CIT and its metabolite, dihydrocitrinone (DH-CIT). Most studies report urinary levels lower for the parent compound when compared with DH-CIT. A high variability either in the mean levels or in the inter-individual ratios of CIT/DH-CIT between the reported studies has been found. Levels of DH-CIT in urine were reported as being comprised between three to seventeen times higher than the parent mycotoxin. In order to comply with this objective, sensitive analytical methodologies for determining biomarkers of exposure are required. Recent development of powerful analytical techniques, namely liquid chromatography coupled to mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography (UHPLC-MS/MS) have facilitated biomonitoring studies, mainly in urine samples. In the present work, evidence on human exposure to CIT through its occurrence and its metabolite, in biological fluids, urine and blood/plasma, in different countries, is reviewed. The analytical methodologies usually employed to evaluate trace quantities of these two molecules, are also presented. In this sense, relevant data on sampling (size and pre-treatment), extraction, cleanup and detection and quantification techniques and respective chromatographic conditions, as well as the analytical performance, are evidenced.

Proteomic applications in pathology and laboratory medicine: Present state and future prospects.

Clinical mass spectrometry applications have traditionally focused on small molecules, particularly in the areas of therapeutic drug monitoring, toxicology, and measurement of endogenous and exogenous steroids. More recently, the use of matrix assisted laser desorption/ionization time of flight mass spectrometry for the identification of microbial pathogens has been widely implemented. Following this evolution, there has been an expanding role for the measurement of peptides and proteins in pathology and laboratory medicine. This review explores the current state of protein measurement by clinical mass spectrometry and the analytical strategies employed, as well as emerging applications in clinical chemistry, clinical microbiology and anatomical pathology.

Human apolipoprotein CIII levels; evaluation of three assay methods.

Apolipoprotein CIII (apoCIII) is associated with triglyceride (TG)-rich particles like VLDL and exerts an inhibitory effect of lipoprotein lipase. Increased levels are related to cardiovascular diseases and diabetes and therefore apoCIII has been proposed as a useful biomarker. Even if several commercial assays for measuring apoCIII in human plasma/serum are available, data is scarce concerning their reliability and none is used clinically. In the present study a comparative investigation has been done. Two ELISA-based methods (Cusabio Biotech and Assay Pro) and one nephelometric assay (Siemens Healthcare) were investigated. Serum and plasma samples were obtained from healthy volunteers and from samples sent to the Laboratory of Clinical Chemistry, preferably with higher levels of TGs. The Cusabio Biotech assay did not yield any valid results. However, both the methods from Assay Pro and Siemens Healthcare showed good performance with similar dynamic ranges. The latter assay had lower CV and required less work. In healthy individuals, apoCIII levels were not affected by fasting, freezing or thawing, nor did we find any gender differences. Individuals with elevated levels of TG displayed higher apoCIII values. Females with oral intake of contraceptives had higher levels. In conclusion, the nephelometric assay showed the best performance with the lowest CV, was less labor intensive than an assay based on ELISA and could therefore be suitable for clinical use.

Measurement repeatability profiles of eight frequently requested measurands in clinical chemistry determined by duplicate measurements of patient samples.

Measurement uncertainties in clinical chemistry are commonly regarded as heteroscedastic - having a constant relative standard deviation irrespective of the concentration of the measurand. The uncertainty is usually determined at two concentrations using stabilized control materials and assumed to represent the analytical goal. The purpose of the present study was to use duplicates of unselected patient samples to calculate the absolute and relative repeatability component of the intra-laboratory measurement uncertainty from duplicates, using the Dahlberg formula and analysis of variance components. Estimates were made at five different concentration intervals of ALT, AST, Calcium, Cholesterol, Creatinine, CRP, Triglycerides and TSH covering the entire concentration interval of the patient cohort. This partioning allows detailing their repeatability profiles. The calculations of the profiles were based on randomly selected results from sets of duplicates ranging from 12,000 to 65,000 pairs. The repeatability of the measurands showed substantial variability within the measuring interval. Therefore, characterizing imprecision profiles as purely homo- or heteroscedastic or by a single number may not be optimal for the intended use. The present data make a case for nuancing the evaluation of analytical goals and minimal differences of measurement results by establishing uncertainty profiles under repeatability conditions, using natural patient samples.

Repeatability imprecision from analysis of duplicates of patient samples and control materials.

Measurement imprecision is usually calculated from measurement results of the same stabilized control material(s) obtained over time, and is therefore, principally, only valid at the concentration(s) of the selected control material(s). The resulting uncertainty has been obtained under reproducibility conditions and corresponds to the conventional analytical goals. Furthermore, the commutability of the control materials used determines whether the imprecision calculated from the control materials reflects the imprecision of measuring patient samples. Imprecision estimated by measurements of patient samples uses fully commutable samples, freely available in the laboratories. It is commonly performed by calculating the results of routine patient samples measured twice each. Since the duplicates are usually analysed throughout the entire concentration interval of the patient samples processed in the laboratory, the result will be a weighted average of the repeatability imprecision measured in the chosen measurement intervals or throughout the entire interval of concentrations encountered in patient care. In contrast, the uncertainty derived from many measurements of control materials over periods of weeks is usually made under reproducibility conditions. Consequently, the repeatability and reproducibility imprecision play different roles in the inference of results in clinical medicine. The purpose of the present review is to detail the properties of the imprecision calculated by duplicates of natural samples, to explain how it differs from imprecision calculated from single concentrations of control materials, and to elucidate what precautions need to be taken in case of bias, e.g. due to carry-over effects.

Aptamer-gold nanoparticle doped covalent organic framework followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for selective enrichment and detection of human insulin.

In this work, we introduced an aptamer modified Au nanoparticles doped covalent organic frameworks composite (IBAs-AuNPs/COF) to improve the property of selective enrichment of insulin from serum samples. The Au nanoparticles were immobilized on imine-based COF by in-situ reduction reaction via mussel inspired polydopamine coating, and then sulfhydryl-containing aptamers were bonded to the surface of AuNPs through an Au-S linkage. Due to the excellent adsorption property of COF and specific recognition between insulin and IBAs, the IBAs-AuNPs/COF composites show selective and satisfactory extraction property to insulin in serum samples. Excellent specifity was obtained for insulin in the presence of 50-fold interfering substances including human immunoglobulin, lysozyme and biotin. The concentrations of insulin in the range of 1.0 to 50.0 µg L-1 show good linear relationship (R2 = 0.9917) with limit of detection and limit of quantitation of 0.28 µg L-1 and 0.93 µg L-1, respectively. Then, the IBAs-AuNPs/COF composites were applied to enrich insulin in serum samples followed by analysis with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). After the recovery experiment, the developed method shows good recoveries in range of 91.6%-112.4% with low RSD value (2.4%-9.4%, n = 3) for diabetic and healthy serum samples. The developed IBAs-AuNPs/COF composites propose a new perspective for selective and efficient enrichment of biomarkers in serum samples by functionalized COF.

A rapid and non-invasive fluorescence method for quantifying coenzyme Q10 in blood and urine in clinical analysis.

BACKGROUND: Coenzyme Q10 (CoQ10) supplementation can improve cognition in patients with Alzheimer's disease (AD) and AD transgenic model mice. To ameliorate the discomfort that patients with AD suffer after several blood extractions, a non-invasive method for detecting urine CoQ10 levels needs to be established. METHODS: Here, we developed a new technique of fluorescence spectrophotometry with ethyl cyanoacetate (FS-ECA), on the basis of the principle that the chemical derivative obtained from the interaction between CoQ10 and ECA was detected by a fluorescence detector at λex/em  = 450/515 nm. As a standard reference method, the same batches of the clinical samples were analyzed by high-performance liquid chromatography with an ultraviolet detector (HPLC-UV) at 275 nm. RESULTS: The limits of detection (LOD) and limits of quantization (LOQ) (serum: 0.021 and 0.043 mg/L; urine: 0.012 and 0.025 mg/L) determined by the FS-ECA method were similar to that obtained through HPLC-UV (serum: 0.017 and 0.035 mg/L; urine: 0.012 and 0.025 mg/L). More importantly, this new FS-ECA technique as well as the conventional HPLC-UV method could detect a marked difference in urine CoQ10 levels between AD and controls. CONCLUSION: Our findings suggest that this non-invasive method for quantifying urine CoQ10 potentially replaces HPLC to detect blood CoQ10.