Pre-analytical Sample Handling Research Articles

Effects of Preanalytical Sample Collection and Handling on Comprehensive Metabolite Measurements in Human Urine Biospecimens.

Epidemiology studies evaluate associations between the metabolome and disease risk. Urine is a common biospecimen used for such studies due to its wide availability and non-invasive collection. Evaluating the robustness of urinary metabolomic profiles under varying preanalytical conditions is thus of interest. Here we evaluate the impact of sample handling conditions on urine metabolome profiles relative to the gold standard condition (no preservative, no refrigeration storage, single freeze thaw). Conditions tested included the use of borate or chlorhexidine preservatives, various storage and freeze/thaw cycles. We demonstrate that sample handling conditions impact metabolite levels, with borate showing the largest impact with 125 of 1048 altered metabolites (adjusted P<0.05). When simulating a case-control study with expected inconsistencies in sample handling, we predicted the occurrence of false positive altered metabolites to be low (< 11). Predicted false positives increased substantially (≥63) when cases were simulated to undergo alternate handling. Finally, we demonstrate that sample handling impacts on the urinary metabolome were markedly smaller than those in serum. While changes in urine metabolites incurred by sample handling are generally small, we recommend implementing consistent handling conditions and evaluating robustness of metabolite measurements for those showing significant associations with disease outcomes.

Impact of Preanalytical Procedures on Complement Biomarkers in Cerebrospinal Fluid and Plasma from Controls and Alzheimer's Disease Patients.

Studies comparing cerebrospinal fluid (CSF) and plasma complement proteins in Alzheimer's disease (AD) patients versus healthy controls (HC) have yielded inconsistent results. Discrepancies in the preanalytical sample handling could contribute to the heterogeneity in the reported findings. Using qualified immunoassays, we aimed at assessing the impact of preanalytical procedures on complement proteins in blood and CSF from AD patients and HCs. We supplemented HC and AD CSF/plasma with complement stabilizers and measured the complement proteins C4a, C4, C3a, C3, Factor Bb and Factor B by immunoassay. We tested the impact of freeze-thaw (FT) cycles on fluid complement proteins. Most complement proteins were mildly impacted by FT cycles in plasma but not CSF, except for C3a which displayed greater sensitivity to FTs in CSF than in plasma. In CSF, the effect of FTs on C3a was reduced but not prevented by the supplementation with EDTA (±Futhan). Our findings provide recommendations for CSF/plasma sample handling to ensure robust and reproducible complement biomarker analyses in AD.

Challenges for pathologists in implementing clinical microbiome diagnostic testing.

Recent research has established that the microbiome plays potential roles in the pathogenesis of numerous chronic diseases, including carcinomas. This discovery has led to significant interest in clinical microbiome testing among physicians, translational investigators, and the lay public. As novel, inexpensive methodologies to interrogate the microbiota become available, research labs and commercial vendors have offered microbial assays. However, these tests still have not infiltrated the clinical laboratory space. Here, we provide an overview of the challenges of implementing microbiome testing in clinical pathology. We discuss challenges associated with preanalytical and analytic sample handling and collection that can influence results, choosing the appropriate testing methodology for the clinical context, establishing reference ranges, interpreting the data generated by testing and its value in making patient care decisions, regulation, and cost considerations of testing. Additionally, we suggest potential solutions for these problems to expedite the establishment of microbiome testing in the clinical laboratory.

Explaining the Variability of Alzheimer Disease Fluid Biomarker Concentrations in Memory Clinic Patients Without Dementia.

Patients' comorbidities can affect Alzheimer disease (AD) blood biomarker concentrations. Because a limited number of factors have been explored to date, our aim was to assess the proportion of the variance in fluid biomarker levels explained by the clinical features of AD and by a large number of non-AD-related factors. MEMENTO enrolled 2,323 individuals with cognitive complaints or mild cognitive impairment in 26 French memory clinics. Baseline evaluation included clinical and neuropsychological assessments, brain MRI, amyloid-PET, CSF (optional), and blood sampling. Blood biomarker levels were determined using the Simoa-HDX analyzer. We performed linear regression analysis of the clinical features of AD (cognition, AD genetic risk score, and brain atrophy) to model biomarker concentrations. Next, we added covariates among routine biological tests, inflammatory markers, demographic and behavioral determinants, treatments, comorbidities, and preanalytical sample handling in final models using both stepwise selection processes and least absolute shrinkage and selection operator (LASSO). In total, 2,257 participants were included in the analysis (median age 71.7, 61.8% women, 55.2% with high educational levels). For blood biomarkers, the proportion of variance explained by clinical features of AD was 13.7% for neurofilaments (NfL), 11.4% for p181-tau, 3.0% for Aβ-42/40, and 1.4% for total-tau. In final models accounting for non-AD-related factors, the variance was mainly explained by age, routine biological tests, inflammatory markers, and preanalytical sample handling. In CSF, the proportion of variance explained by clinical features of AD was 24.8% for NfL, 22.3% for Aβ-42/40, 19.8% for total-tau, and 17.2% for p181-tau. In contrast to blood biomarkers, the largest proportion of variance was explained by cognition after adjustment for covariates. The covariates that explained the largest proportion of variance were also the most frequently selected with LASSO. The performance of blood biomarkers for predicting A+ and T+ status (PET or CSF) remained unchanged after controlling for drivers of variance. This comprehensive analysis demonstrated that the variance in AD blood biomarker concentrations was mainly explained by age, with minor contributions from cognition, brain atrophy, and genetics, conversely to CSF measures. These results challenge the use of blood biomarkers as isolated stand-alone biomarkers for AD.

Optimized Pre-analytical Handling Protocol for Blood-Based Biomarkers of Alzheimer's Disease.

The therapeutic management of patients with Alzheimer's disease (AD) has been hindered by poor diagnostic accuracy. As such, there is an unmet clinical need for tools that can detect and diagnose the disease in its early stages. Compared with cerebrospinal fluid (CSF)-based biomarkers or positron emission tomography(PET), the use of reliable blood-based biomarkers could offer an accessible and minimally invasive method of streamlining diagnosis in the clinical setting. However, the influence of pre-analytical processing and sample handling parameters on the accurate measurement of protein biomarkers is well established, especially for AD CSF-based biomarkers. In this chapter, we provide recommendations for an optimal sample handling protocol for the analysis of blood-based biomarkers specifically for amyloid pathology in AD.

B-378 Cell-free Plasma Preparation Workflow Solution

Abstract Background Liquid biopsy for the study and monitoring of various cancers has become a growing area of study, pre-analytical sample handling can impact the quality and profile of the circulating tumor nucleic acid. Preparing samples like cell-free plasma for downstream assays is imperative to maintaining quality in downstream applications for clinical research in oncology. To obtain cell-free plasma, blood samples must undergo centrifugation at 2000g for 10 min at 4C followed by a second spin for 30 min at 4C. This two-step spin process presents an issue with labs that don’t have a refrigerated centrifuge suitable for higher speeds. Methods Here, we perform a series of studies to investigate the impact of various spin conditions on cell-free nucleic acid recovery and quality. Four plasma centrifugation methods encompassing variables attributing to speed, temperature, and number of centrifugations steps, were evaluated from whole blood tubes obtained from six donors in K2EDTA upfront of nucleic acid isolation with MagMAX™ cell-free TNA nucleic acid isolation chemistry on a KingFisher™ magnetic particle processor. Yield, quality, and functional performance was evaluated from the extracted cell-free nucleic acid and compared across all plasma spin-down methods. Result Study results indicated that centrifugation of plasma at lower speeds for a shorter period at room temperature had no negative impact to nucleic acid recovery and integrity. Conclusion Here, we offer a plasma spin workflow solution to mid- to high- throughput clinical research labs to allow versatility within the laboratory setup. Reducing the need for refrigerated centrifugation allows clinical researchers to obtain high-quality cfNA suitable for their downstream applications in clinical oncology and genomics research.

Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research

The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.

Impact of pre-analytical sample handling factors on plasma biomarkers of Alzheimer's disease.

An unmet need exists for reliable plasma biomarkers of amyloid pathology, in the clinical laboratory setting, to streamline diagnosis of Alzheimer's disease (AD). For routine clinical use, a biomarker must provide robust and reliable results under pre-analytical sample handling conditions. We investigated the impact of different pre-analytical sample handling procedures on the levels of seven plasma biomarkers in development for potential routine use in AD. Using (1) fresh (never frozen) and (2) previously frozen plasma, we evaluated the effects of (A) storage time and temperature, (B) freeze/thaw (F/T) cycles, (C) anticoagulants, (D) tube transfer, and (E) plastic tube types. Blood samples were prospectively collected from patients with cognitive impairment undergoing investigation in a memory clinic. β-amyloid 1-40 (Aβ40), β-amyloid 1-42 (Aβ42), apolipoprotein E4, glial fibrillary acidic protein, neurofilament light chain, phosphorylated-tau (phospho-tau) 181, and phospho-tau-217 were measured using Elecsys® plasma prototype immunoassays. Recovery signals for each plasma biomarker and sample handling parameter were calculated. For all plasma biomarkers measured, pre-analytical effects were comparable between fresh (never frozen) and previously frozen samples. All plasma biomarkers tested were stable for ≤24 h at 4°C when stored as whole blood and ethylenediaminetetraacetic acid (EDTA) plasma. Recovery signals were acceptable for up to five tube transfers, or two F/T cycles, and in both polypropylene and low-density polyethylene tubes. For all plasma biomarkers except Aβ42 and Aβ40, analyte levels were largely comparable between EDTA, lithium heparin, and sodium citrate tubes. Aβ42 and Aβ40 were most sensitive to pre-analytical handling, and the effects could only be partially compensated by the Aβ42/Aβ40 ratio. We provide recommendations for an optimal sample handling protocol for analysis of plasma biomarkers for amyloid pathology AD, to improve the reproducibility of future studies on plasma biomarkers assays and for potential use in routine clinical practice.

Pre-analytical processing of plasma and serum samples for combined proteome and metabolome analysis.

Metabolomic and proteomic analyses of human plasma and serum samples harbor the power to advance our understanding of disease biology. Pre-analytical factors may contribute to variability and bias in the detection of analytes, especially when multiple labs are involved, caused by sample handling, processing time, and differing operating procedures. To better understand the impact of pre-analytical factors that are relevant to implementing a unified proteomic and metabolomic approach in a clinical setting, we assessed the influence of temperature, sitting times, and centrifugation speed on the plasma and serum metabolomes and proteomes from six healthy volunteers. We used targeted metabolic profiling (497 metabolites) and data-independent acquisition (DIA) proteomics (572 proteins) on the same samples generated with well-defined pre-analytical conditions to evaluate criteria for pre-analytical SOPs for plasma and serum samples. Time and temperature showed the strongest influence on the integrity of plasma and serum proteome and metabolome. While rapid handling and low temperatures (4°C) are imperative for metabolic profiling, the analyzed proteomics data set showed variability when exposed to temperatures of 4°C for more than 2h, highlighting the need for compromises in a combined analysis. We formalized a quality control scoring system to objectively rate sample stability and tested this score using external data sets from other pre-analytical studies. Stringent and harmonized standard operating procedures (SOPs) are required for pre-analytical sample handling when combining proteomics and metabolomics of clinical samples to yield robust and interpretable data on a longitudinal scale and across different clinics. To ensure an adequate level of practicability in a clinical routine for metabolomics and proteomics studies, we suggest keeping blood samples up to 2h on ice (4°C) prior to snap-freezing as a compromise between stability and operability. Finally, we provide the methodology as an open-source R package allowing the systematic scoring of proteomics and metabolomics data sets to assess the stability of plasma and serum samples.

Pitfalls in complement analysis: A systematic literature review of assessing complement activation

The complement system is an essential component of our innate defense and plays a vital role in the pathogenesis of many diseases. Assessment of complement activation is critical in monitoring both disease progression and response to therapy. Complement analysis requires accurate and standardized sampling and assay procedures, which has proven to be challenging. We performed a systematic analysis of the current methods used to assess complement components and reviewed whether the identified studies performed their complement measurements according to the recommended practice regarding pre-analytical sample handling and assay technique. Results are supplemented with own data regarding the assessment of key complement biomarkers to illustrate the importance of accurate sampling and measuring of complement components. A literature search using the Pubmed/MEDLINE database was performed focusing on studies measuring the key complement components C3, C5 and/or their split products and/or the soluble variant of the terminal C5b-9 complement complex (sTCC) in human blood samples that were published between February 2017 and February 2022. The identified studies were reviewed whether they had used the correct sample type and techniques for their analyses. A total of 92 out of 376 studies were selected for full-text analysis. Forty-five studies (49%) were identified as using the correct sample type and techniques for their complement analyses, while 25 studies (27%) did not use the correct sample type or technique. For 22 studies (24%), it was not specified which sample type was used. A substantial part of the reviewed studies did not use the appropriate sample type for assessing complement activation or did not mention which sample type was used. This deviation from the standardized procedure can lead to misinterpretation of complement biomarker levels and hampers proper comparison of complement measurements between studies. Therefore, this study underlines the necessity of general guidelines for accurate and standardized complement analysis.

Biomarkers for the diagnosis of cognitive impairment - Recommendations from the Swiss Memory Clinics

Biomarkers for the diagnosis of cognitive impairment - Recommendations from the Swiss Memory Clinics Abstract. Molecular cerebrospinal fluid (CSF) biomarkers of neurodegenerative diseases are now part of the established diagnostic tools for the clinical investigation of cognitive disorders in the elderly. Biomarkers allow for earlier and more accurate differential diagnosis, and are recommended by the Swiss Memory Clinics as an additional investigation based upon individual indication. Information and counselling are needed both before and after biomarker-supported diagnosis. The procedures for diagnostic lumbar punctures and pre-analytical sample handling should follow published recommendations. The results must be interpreted in the context of the other available history and assessment outcome. Thanks to recent research progress, blood-based biomarkers and other non-invasive markers are expected to become available for clinical practice in the near future. This trend will likely lead to a much broader utilisation of biomarkers and may accelerate the development of effective and individually tailored prevention and treatment approaches. This review article provides an overview over the current state of biomarkers and provides the recommendations of the Swiss Memory Clinics for their use in clinical practice.

Defining blood gas analysis stability limits across five sample types

Accurate reporting of blood gas samples is dependent upon following proper preanalytical sample handling requirements though there is variation for sample acceptability criteria across institutions. We examined five common sample types (arterial, venous, umbilical arterial, umbilical venous and capillary) stored at either room temperature or on crushed ice in a time series (0, 15, 30, 45, 60, 90, 180, 240 min) and applied local regulatory and/or institutional allowable performance limits to determine the need for cold preservation and/or maximum stability time for pH, pO2, pCO2, glucose, lactate, sodium, potassium, chloride, and ionized calcium where applicable in each sample type. Although changes in sample pO2 and/or lactate values were responsible, in part or in whole, for surpassing the allowable limits in nearly all sample types analyzed, this was not uniformly observed across sample types within the typical time limits that are referenced in literature. Furthermore, we demonstrated that cold preservation may not ubiquitously provide longer stability for blood gas specimens and this is dependent on the sample type and analyte in question. Nevertheless, these results demonstrate the known instability of pO2 and lactate and suggest that it may be possible to simplify the monitoring of preanalytical conditions by first evaluating pO2 and lactate in patient blood gas samples if applicable.

A systematic review and meta-analysis of preanalytical factors and methodological differences influencing the measurement of circulating vascular endothelial growth factor.

BackgroundIntraocular treatment with antibodies targeting vascular endothelial growth factor (anti-VEGF) inhibits pathological vessel growth in adults and preterm infants. Recently, concerns regarding the impact of anti-VEGF treatment on systemic VEGF levels in preterm infants have been raised. Earlier studies suggest that preanalytical and methodological parameters impact analytical VEGF concentrations, but we have not found a comprehensive systematic review covering preanalytical procedures and methods for VEGF measurements.ObjectiveThis review aimed to evaluate the most critical factors during sample collection, sample handling, and the analytical methods that influence VEGF levels and therefore should be considered when planning a prospective collection of samples to get reproducible, comparable results.Material and methodsPubMed and Scopus databases were searched 2021/Nov/11. In addition, identification of records via other methods included reference, citation, and Google Scholar searches. Rayyan QCRI was used to handle duplicates and the selection process. Publications reporting preanalytical handling and/or methodological comparisons using human blood samples were included. Exclusion criteria were biological, environmental, genetic, or physiological factors affecting VEGF. The data extraction sheets included bias assessment using the QUADAS-2 tool, evaluating patient selection, index-test, reference standard, and flow and timing. Concentrations of VEGF and results from statistical comparisons of analytical methods and/or preanalytical sample handling and/or different sample systems were extracted. The publications covering preanalytical procedures were further categorized based on the stage of the preanalytical procedure. Meta-analysis was used to visualize VEGF concentrations among healthy individuals. The quality of evidence was rated according to GRADE.ResultsWe identified 1596 publications, and, after the screening process, 43 were considered eligible for this systematic review. The risk of bias estimation was difficult for 2/4 domains due to non-reported information. Four critical steps in the preanalytical process that impacted VEGF quantification were identified: blood drawing and the handling before, during, and after centrifugation. Sub-categorization of those elements resulted in nine findings, rated from moderate to very low evidence grade. The choice of sample system was the most reported factor. VEGF levels (mean [95% CI]) in serum (n = 906, 20 publications), (252.5 [213.1–291.9] pg/mL), were approximated to ninefold higher than in plasma (n = 1122, 23 publications), (27.8 [23.6–32.1] pg/mL), based on summarized VEGF levels with meta-analysis. Notably, most reported plasma levels were below the calibration range of the used method.ConclusionWhen measuring circulating VEGF levels, choice of sample system and sample handling are important factors to consider for ensuring high reproducibility and allowing study comparisons. Protocol: CRD42020192433

Pre-analytical stability of serum biomarkers forneurological disease: neurofilament-light, glial fibrillary acidic protein and contactin-1.

Neurofilament-light (NfL), glial fibrillary acidic protein (GFAP) and contactin-1 (CNTN1) are blood-based biomarkers that could contribute to monitoring and prediction of disease and treatment outcomes in neurological diseases. Pre-analytical sample handling might affect results, which could be disease-dependent. We tested common handling variations in serum of volunteers as well as in a defined group of patients with multiple sclerosis (pwMS). Sample sets from 5pwMS and 5 volunteers at the outpatient clinic were collected per experiment. We investigated the effect of the following variables: collection tube type, delayed centrifugation, centrifugation temperature, delayed storage after centrifugation and freeze-thawing. NfL and GFAP were measured by Simoa and CNTN1 by Luminex. A median recovery of 90-110% was considered stable. For most pre-analytical variables, serum NfL and CNTN1 levels remained unaffected. In the total group, NfL levels increased (121%) after 6h of delay at 2-8 °C until centrifugation, while no significant changes were observed after 24h delay at room temperature (RT). In pwMS specifically, CNTN1 levels increased from additional freeze-thaw cycles number 2 to 4 (111%-141%), whereas volunteer levels remained stable. GFAP showed good stability for all pre-analytical variables. Overall, the serum biomarkers tested were relatively unaffected by variations in sample handling. For serum NfL, we recommend storage at RT before centrifugation at 2-8 °C up to 6h or at RT up to 24h. For serum CNTN1, we advise a maximum of two freeze-thaw cycles. Our results confirm and expand on recently launched consensus standardized operating procedures.

Pre-analytical challenges for the quantification of endocannabinoids in human serum

Endocannabinoids (ECs) are potent lipid mediators with high physiological relevance. They are involved in a wide variety of diseases like depression or multiple sclerosis and are closely connected to metabolic parameters in humans. Therefore, their suitability as a biomarker in different (patho-)physiological conditions is discussed intensively and predominantly investigated by analyzing systemic concentrations in easily accessible matrices like blood. Carefully designed pre-analytical sample handling is of major importance for high-quality data, but harmonization is not achieved yet. Whole blood is either processed to serum or plasma before the onset of analytical workflows and while knowledge about pre-analytical challenges in plasma handling is thorough they were not systematically investigated for serum.Therefore, the ECs AEA and 2-AG, and closely related EC-like substances 1-AG, DHEA, and PEA were examined by LC-MS/MS in serum samples of nine healthy volunteers employing different pre-analytical sample handling protocols, including prolonged coagulation, and storage after centrifugation at room temperature (RT) or on ice. Furthermore, all analytes were also assessed in plasma samples obtained from the same individuals at the same time points to investigate the comparability between those two blood-based matrices regarding obtained concentrations and their 2-AG/1-AG ratio.This study shows that ECs and EC-like substances in serum samples were significantly higher than in plasma and are especially prone to ex vivo changes during initial and prolonged storage for coagulation at RT. Storage on ice after centrifugation is less critical. However, storage at RT further increases 1-AG and 2-AG concentrations, while also lowering the already reduced 2-AG/1-AG ratio due to isomerization. Thus, avoidance of prolonged processing at RT can increase data quality if serum as the matrix of choice is unavoidable. However, serum preparation in itself is expected to initiate changes of physiological concentrations as standard precautionary measures like fast and cooled processing can only be utilized by using plasma, which should be the preferred matrix for analyses of ECs and EC-like substances.

Biomarkers for the diagnosis of cognitive impairment - Recommendations from the Swiss Memory Clinics

Established cerebrospinal fluid (CSF) biomarkers allow for earlier and more accurate etiological diagnosis of cognitive impairment. Information and counselling are needed both before and after biomarker-supported diagnosis. The procedures for diagnostic lumbar punctures and pre-analytical sample handling should follow published consensus recommendations. The results must be interpreted in the context of the other available history information and assessments. Blood-based biomarkers and other non-invasive markers are expected to become available for clinical practice soon. Consequently, a broader usage of biomarkers is expected and may accelerate the development of individually tailored prevention and treatment approaches. This article provides the recommendations of the Swiss Memory Clinics for the use of biomarkers in clinical practice.

Non-Invasive Fetal K Status Prediction: 7 Years of Experience

Introduction: In the Kell blood group system, the K and k antigens are the clinically most important ones. Maternal anti-K IgG antibodies can lead to the demise of a K-positive fetus in early pregnancy. Intervention can save the fetus. Prenatal K status prediction of the fetus in early pregnancy is desirable and gives a good basis for pregnancy risk management. We present the results from 7 years of clinical experience in predicting fetal K status as well as some theoretical considerations relevant for design of the assay and evaluation of results. Methods: Blood was collected from 43 women, all immunized against K, at a mean gestational age of 18 weeks (range 10–38). A total of 56 consecutive samples were tested. The KEL*01.01/KEL*02 single nucleotide variant that determines K status was amplified from maternal plasma DNA by PCR without allele specificity. The PCR product was sequenced by NGS technology, and the number of sequenced KEL*01.01 and KEL*02 reads were counted. Prediction of the fetal K status was based on this count and was compared with the serologically determined K status of the newborns. Results: All fetal K predictions were in accordance with postnatal serology where available (n = 34), using our current data analysis. Conclusion: We have developed an NGS-based method for the non-invasive prediction of fetal K status. This approach requires special considerations in terms of primer design, stringent preanalytical sample handling, and careful analytical procedures. We analyzed samples starting at GA 10 weeks and demonstrated the correct prediction of fetal K status. This assay enables timely clinical intervention in pregnancies at risk of hemolytic disease of the fetus and newborn caused by maternal anti-K IgG antibodies.

Effects of EDTA on chemiluminescent immunoassay measurement of ACTH, cortisol, and thyroid hormones in dogs

When measuring blood hormones, pre-analytical sample handling can impact the quality of the results. Previous studies have shown improved stability of canine cortisol in ethylenediaminetetraacetic acid (EDTA) plasma compared to serum and interchangeability of serum and plasma when cortisol is measured by radioimmunoassay. Additionally, cortisol samples were also interchangeable when measured by chemiluminescent immunoassay if the EDTA concentration was consistent with that of optimally filled tubes, whereas excess EDTA interfered with the measurement of cortisol and serum and EDTA plasma were not interchangeable when measuring total thyroxine (TT4). The main limitation of these studies was that they were performed by spiking pooled serum samples with EDTA or in previously collected blood samples submitted to a clinical pathology laboratory. The purpose of the present study was to evaluate the effect of EDTA on the measurement of adrenocorticotropic hormone (ACTH), cortisol, TT4, free thyroxine (FT4), and thyroid stimulating hormone (TSH) in healthy dogs using the Siemens IMMULITE 1000. Whole blood from forty dogs was aliquoted into three Monoject sample tubes: no additive, completely filled EDTA tube, and 50% filled EDTA tube. Handling and storage conditions were identical, and all samples were analyzed on the same day. Bland-Altman plots and Passing-Bablok regression were used to assess agreement and risks for error, respectively. Proportional errors were found between serum and plasma samples for ACTH, cortisol, TT4, FT4, and TSH; systematic errors were also found for FT4. There was poor agreement and clinically significant differences between the measured concentrations of all hormones in serum and plasma, proving that these sample types are not interchangeable. Incompletely filled EDTA tubes were associated with significantly lower ACTH concentrations compared to completely filled EDTA tubes. When measured by chemiluminescent immunoassays that utilize alkaline phosphatase at the reporter enzyme, serum should be used for cortisol, TT4, FT4, and TSH, while plasma from completely filled EDTA tubes should be used for ACTH.

The technical reliability and stability of ATN quantification using the Fujirebio Lumipulse G1200 fully‐automated immunoassay instrument in cerebrospinal fluid

AbstractBackgroundFujirebio’s Lumipulse G1200 is designed to perform fully‐automated, rapid, random access immunoassays for biomarker quantification in biofluids. The Lumipulse instrument was used for quantification of the core Alzheimer’s Disease (AD) cerebrospinal fluid (CSF) biomarkers for Amyloid, Tau, and neurodegeneration (ATN): amyloid β 1–40 (Aβ40), amyloid β 1–42 (Aβ42), phosphorylated Tau 181 (pTau181), and total Tau (tTau). This study evaluated the reliability of the Lumipulse assays by measuring intra‐ and inter‐batch coefficients of variation (CV), and the correlation between Lumipulse values and the clinically used Athena ADmark Aβ42, pTau181 and tTau assays.Method423 CSF samples collected from diagnostic lumbar punctures between 2015 and 2020 were obtained from the MIND biorepository. Aβ40, Aβ42, tTau, and pTau were measured using the Lumipulse G1200 instrument in batches of 50‐60 samples. We tested 60 samples in duplicate to compare the intrabatch stability using the CVs between each pair. Each batch was run with a new set of calibrator controls at the start of each assay to ensure stability for each biomarker run. Finally, we correlated values from the Lumipulse with results from the clinical evaluation using the Athena ADmark test.ResultWe observed low variability in calibrator and control signals between batches with an overall calibrator signal CV of &lt;5% for Aβ40 and tTau, and &lt;10% for Aβ42 and pTau. Control signal CV values for all analytes were &lt;5%. The mean duplicate CV across all batches was similarly stable at &lt;10% for Aβ40, Aβ42, and tTau, and &lt;5% for pTau. Subjects with CV &gt;20% were seen &lt;5% of the time. The correlations between Lumipulse and clinical ADmark results were high for pTau and tTau (R2&gt;0.8 for both analytes), but lower for Aβ42 (R2∼0.5).ConclusionOur results demonstrated that the Lumipulse G1200 is a reliable, easy to use platform for ATN quantification with low sample to sample variability, and generally high correlation to established clinical standards. Inconsistent preanalytical sample handling of clinical send‐outs for Athena ADmark Aβ42 likely resulted in a poor correlation with Lumipulse G1200. The performance of the calibrators and controls proved that the standardization in each batch was consistent.

Characterization of pre-analytical sample handling effects on a panel of Alzheimer's disease-related blood-based biomarkers: Results from the Standardization of Alzheimer's Blood Biomarkers (SABB) working group.

IntroductionPre‐analytical sample handling might affect the results of Alzheimer's disease blood‐based biomarkers. We empirically tested variations of common blood collection and handling procedures.MethodsWe created sample sets that address the effect of blood collection tube type, and of ethylene diamine tetraacetic acid plasma delayed centrifugation, centrifugation temperature, aliquot volume, delayed storage, and freeze–thawing. We measured amyloid beta (Aβ)42 and 40 peptides with six assays, and Aβ oligomerization‐tendency (OAβ), amyloid precursor protein (APP)699‐711, glial fibrillary acidic protein (GFAP), neurofilament light (NfL), total tau (t‐tau), and phosphorylated tau181.ResultsCollection tube type resulted in different values of all assessed markers. Delayed plasma centrifugation and storage affected Aβ and t‐tau; t‐tau was additionally affected by centrifugation temperature. The other markers were resistant to handling variations.DiscussionWe constructed a standardized operating procedure for plasma handling, to facilitate introduction of blood‐based biomarkers into the research and clinical settings.