Preanalytical Variables Research Articles

Subpar reporting of pre-analytical variables in RNA-focused blood plasma studies.

Extracellular RNA (cell-free RNA; exRNA) from blood-derived liquid biopsies is an appealing, minimally invasive source of disease biomarkers. As pre-analytical variables strongly influence exRNA measurements, their reporting is essential for meaningful interpretation and replication of results. The aim of this review was to chart to what extent pre-analytical variables are documented, to pinpoint shortcomings and to improve future reporting. In total, 200 blood plasma exRNA studies published in 2018 or 2023 were reviewed for annotation of 22 variables associated with blood collection, plasma preparation, and RNA purification. Our results show that pre-analytical variables are poorly documented, with only three out of 22 variables described in over half of the publications. The percentage of variables reported ranged from 4.6% to 54.6% (mean 24.84%) in 2023 and from 4.6% to 57.1% (mean 28.60%) in 2018. Recommendations and guidelines (i.e., BRISQ, ASCO-CAP, BloodPAC, PPMPT, and CEN standards) have currently not resulted in improved reporting. In conclusion, our results highlight the lack of reporting pre-analytical variables in exRNA studies and advocate for a consistent use of available standards, endorsed by funders and journals.

Abstract 6093: Evaluation of preanalytical and physiological variables affecting cfDNA-based multi-cancer early detection test

Abstract Background: The multi-cancer early detection (MCED) tests utilizing blood-based circulating cell-free DNA (cfDNA) have gained significant attention in the market as a promising approach for early cancer detection. However, potential effects of preanalytical and physiological variables, including extraction methods, preservation techniques, and physiological factors such as diet, on the accuracy and reliability of MCED results remains poorly investigated. In this study, we aimed to evaluate the impact of physiological variables during blood collection and preanalytical procedures on the outcomes of a MCED test. Methods: A total of 105 plasma samples from 19 healthy donors was collected and analyzed using a MCED test (MERCURY) which leverages the low-coverage whole-genome sequencing and a set of genome-wide features based on cfDNA fragmentomics. Samples were controlled for preanalytical procedures, including transportation condition and preservation time or physiological conditions (before/after meal or exercise) at blood collection. Results: Repeat blood collection (N=4) from the 5 healthy participants consistently yielded negative results for cancer (PPA: 100.0%; 95% CI [56.6%,100.0%]). Among the 5 healthy participants, plasma samples frozen within 1 year (7 days, 3 months, 9 months) showed the same agreement with the reference condition as non-frozen samples (PPA: 100.0%; 95% CI [56.6%,100.0%]), except for one sample frozen for 1 year, which showed a higher risk score and became a positive signal (PPA:80.0%; 95% CI [37.6%,96.4%]). Transportation conditions within 2 hours, 24 hours, 48 hours, and 96 hours at room temperature or 4℃ did not affect the test outcomes, as all samples remained in agreement with the reference condition as of 2 hours at 4℃ (PPA:100.0%; 95% CI [61.0%,100.0%]). Physiological conditions, including pre- and post-meal as well as pre- and post-exercise states, did not exert any influence on the test results, as all samples exhibited agreement with the reference condition (PPA:100.0%; 95% CI [43.9%,100.0%]). Conclusions: The results of this study suggest that variables such as time of blood collection and plasma freezing time may not significantly affect the accuracy of the MCED test in healthy participants. Transportation conditions and physiological conditions evaluated in this study did not have a notable influence on the test outcomes. Nevertheless, it is advised that the freezing duration of plasma samples should not exceed one year. Citation Format: Hua Bao, Xiaoxi Chen, Xuxiaochen Wu, Wanxiangfu Tang, Min Wu, Shiting Tang, Xue Wu, Yang Shao. Evaluation of preanalytical and physiological variables affecting cfDNA-based multi-cancer early detection test [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6093.

Abstract 2292: Impact of delayed processing on cfDNA quantity and quality using STRECK cfDNA tubes: Connect Pilot Study

Abstract Interest in cell-free DNA detection for multi-cancer early detection and tumor monitoring is growing.1 Prospective evaluations of liquid biopsy assays are critical, and knowledge gaps in standard collection and preparation practices must be addressed for adequate assay execution.2 The NCI Connect for Cancer Prevention Cohort Study (Connect) aims to study new approaches in early detection. A pilot study was conducted with the STRECK Cell-Free DNA BCT tube to determine the impact of processing time on cfDNA quantity and quality utilizing two complementary quality control assays. 4 STRECK DNA BCT tubes were drawn from 40 donors. Plasma samples were analyzed, with half processed immediately and the other half stored for five days before processing. Two QC assays were performed to compare results at the two processing time points. The Alu EFIRM assay measured Alu element concentration (copies/mL). The Cell-free DNA ScreenTape assay evaluated Qubit DNA concentration (μg/mL) and %cfDNA. Descriptive statistics and paired sample t-tests were performed to compare mean concentrations at the two processing time points, with and without identified samples with cytolysis. Median, mean, and range were recorded for each assay measure (Table 1). A reduction in cfDNA quantity was observed at the 5-day processing time point. Five 5-day processing samples were identified with high DNA quantity and a high Alu element concentration. The five samples experienced hemolysis and clotting as a result of handling and processing at odds with standard protocol. There was a significant difference between the mean DNA quantities of the processing time points following exclusion of the five samples. There was no change in significance between the mean Alu concentration and %cfDNA following exclusion (Table 2). Both assays successfully detected samples with high nuclear DNA, a major contaminant for cfDNA assays, and identified a reduction in cfDNA at the 5-day processing time point. Proper handling and processing procedures are necessary to avoid cell lysis and maximize cfDNA quality and yield. This pilot establishes a starting point for future evaluations of liquid biopsy tube types, processing delays, and preanalytical cfDNA quality variables. Citation Format: Jazmyn Bess, Domonique Hopkins, Norma Diaz Mayoral, Heather Cooley, Angela de Palatis, David T. Wong, Stephanie Weinstein Oshinsky, Christian Abnet, Marie-Josephe Horner, Amanda Black, Michelle Brotzman, Kathleen Wyatt, Lynn Sorbara, Matthew Young, Mia Gaudet, Nicolas Wentzensen, Mickey Williams, Christopher Karlovich, Eric Greenbank, David Elashoff, Fang Wei, Feng Li. Impact of delayed processing on cfDNA quantity and quality using STRECK cfDNA tubes: Connect Pilot Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2292.

Abstract 5035: Use of Streck nucleic acid BCT with plasma nucleic acid next-generation sequencing workflows

Abstract Introduction: Liquid biopsies, which interrogate body fluids in search of analytes released by cancer cells, are becoming increasingly common in clinical oncology. Foremost among the technologies used are those that employ nucleic acids, such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA). Currently, many NGS-based tests perform near the limit of assay detection, making the identification of critical mutations, fusions, or expression patterns like finding a needle in a haystack. Further hindering successful assay development is the variability in analyte availability and detectability that can occur prior to analysis due to blood collection, transport, and storage. Here, we demonstrate that Nucleic Acid BCT stabilizes draw-time levels of plasma nucleic acids for downstream use with NGS-based assays. Methods: Blood from self-proclaimed healthy donors was drawn into EDTA and Nucleic Acid BCT and stored at ambient temperature for up to 7 days before plasma isolation with a general double-spin protocol and storage at -80 °C. Plasma nucleic acids were isolated using the MAGicBead cfDNA Kit (Zymo, cfDNA) or the QIAamp Circulating Nucleic Acid Kit (QIAGEN, cfRNA). Library prep for cfDNA was carried out with added synthetic mutant spike-in oligonucleotides using the Illumina TruSight Tumor 15 kit (Illumina). cfRNA library prep was done with the NEBNext Ultra II RNA Library Prep Kit for Illumina with upfront rRNA depletion (New England Biolabs). Sequencing was performed using an Illumina NextSeq 500 system, and all data were saved in and analyzed with Illumina BaseSpace applications. Results: When blood samples were stored in EDTA tubes for up to 7 days, spiked mutant alleles (EGFRL858R, EGFRdel19, KRASG12D, and PIK3CAE545K) were no longer detected, likely due to a dilution of the spiked DNA concentration following the release of genomic DNA containing wildtype alleles during white blood cell degradation. In contrast, equivalent blood samples stored in Nucleic Acid BCTs displayed mutant allele frequencies reflective of draw-time levels. Interrogation of plasma RNA expression patterns in samples stored in Nucleic Acid BCT versus EDTA revealed similar results, where differentially expressed plasma transcripts in blood samples stored in EDTA were found at high levels (>1,000 transcripts) versus donor-matched blood samples stored in Nucleic Acid BCT (<50 transcripts). Conclusions: Our data demonstrate that Nucleic Acid BCT maintains mutant allele frequencies and the plasma transcriptome profile of blood samples during ambient temperature storage. This provides precious sample integrity during whole blood storage and transport, offering laboratories and assay developers reduced preanalytical variability for NGS-based analysis of gene mutations, fusions, indels, and the plasma transcriptome. Nucleic Acid BCT is for Research Use Only. Not for use in diagnostic procedures. Citation Format: Nicholas M. George, Lisa Bartron. Use of Streck nucleic acid BCT with plasma nucleic acid next-generation sequencing workflows [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5035.

Abstract 7631: Protein Plus BCT: A novel blood collection tube for stabilizing plasma proteins of interest in ambient whole blood storage

Abstract Introduction: Many of the soluble plasma proteins (e.g., cytokines) that are crucial for cancer diagnosis, prognosis, and prediction of cancer immunotherapy outcomes are found in blood cells such as platelets and leukocytes. During blood collection and sample storage, ex vivo platelet activation, hemolysis, and blood cell degradation can occur, which causes the release of blood cell-secreted proteins into the plasma. The contamination of these proteins may confound true in vivo levels of plasma proteins of interest, resulting in inaccurate or inconsistent assay results. Protein Plus BCT is a new blood collection tube that maintains accurate plasma protein levels during ambient whole blood storage. Methods: Blood samples from self-proclaimed healthy donors were drawn into Protein Plus BCT, EDTA, and ACD-A tubes. All the samples were stored at ambient temperature for up to 5 days. Plasma was isolated using a general double-spin protocol and frozen at -80 °C until use. Plasma levels of protein biomarkers were measured by Ella Simple Plex Assays (Bio-Techne) and Luminex xMAP® Technology Assays (Bio-Techne, samples tested by LuminexPLORE Lab) following manufacturers’ dilution and processing recommendations. Results: Over 50 key plasma proteins like cytokines, cancer, and immuno-oncology checkpoint markers in samples collected into Protein Plus BCT were analyzed by immunoassays and compared side-by-side to those collected in EDTA and ACD-A tubes. EDTA and ACD-A samples exhibited a substantial elevation in the plasma concentration of many, such as IL-8, IL-1β, HGF, VEGF, MMP-9, CCL5/RANTES and TGF-β1 post draw, indicating platelet activation and blood cell degradation during ambient temperature storage. Samples collected into Protein Plus BCT displayed limited ex vivo platelet activation and minimal contamination by blood cell-secreted proteins, maintaining the draw-time plasma levels of proteins of interest for up to 5 days in whole blood at ambient temperature. Conclusions: The novel Protein Plus BCT maintains draw-time plasma protein levels during extended whole blood storage at room temperature by limiting ex vivo platelet activation, hemolysis, and blood cell degradation. Protein Plus BCT ensures sample integrity and minimizes preanalytical variables, providing cancer researchers and clinical assay developers greater flexibility in sample handling and improved confidence in downstream plasma protein analysis. Protein Plus BCT is for Research Use Only. Not for use in diagnostic procedures. Citation Format: Jing Li, Nicholas M. George, Sama Mehta, Jordan LaRue, Eunju Seong, Nursen Binbuga, Rachel Miller, Karl Bisselou. Protein Plus BCT: A novel blood collection tube for stabilizing plasma proteins of interest in ambient whole blood storage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7631.

Pre-analytical stability of the CEA, CYFRA 21.1, NSE, CA125 and HE4 tumor markers.

For lung cancer, circulating tumor markers (TM) are available to guide clinical treatment decisions. To ensure adequate accuracy, pre-analytical instabilities need to be known and addressed in the pre-analytical laboratory protocols. This study investigates the pre-analytical stability of CA125, CEA, CYFRA 21.1, HE4 and NSE for the following pre-analytical variables and procedures; i) whole blood stability, ii) serum freeze-thaw cycles, iii) electric vibration mixing and iv) serum storage at different temperatures. Left-over patient samples were used and for every investigated variable six patient samples were used and analysed in duplicate. Acceptance criteria were based on analytical performance specifications based on biological variation and significant differences with baseline. Whole blood was stable for at least 6 hours for all TM except for NSE. Two freeze-thaw cycles were acceptable for all TM except CYFRA 21.1. Electric vibration mixing was allowed for all TM except for CYFRA 21.1. Serum stability at 4°C was 7 days for CEA, CA125, CYFRA 21.1 and HE4 and 4 hours for NSE. Critical pre-analytical processing step conditions were identified that, if not taken into account, will result in reporting of erroneous TM results.

Evaluation of Pre-Analytical Variables for Human Papillomavirus Primary Screening from Self-Collected Vaginal Swabs

HPV primary screening is an effective approach to assessing cervical cancer risk. Self-collected vaginal swabs have shown promise to expand testing access, but there are limited data defining analytical performance criteria necessary for adoption of self-collected specimens, especially for those occurring outside the clinic where the swab remains dry during transport. Here, we evaluated the performance of self-collected vaginal swabs for HPV detection using the Roche Cobas 6800. There was insignificant variability between swabs self-collected by the same individual (n=15 participants collecting 5 swabs/participant), measured by amplification of HPV and human β-globin control DNA. Comparison of self-collected vaginal swab and provider-collected cervical samples (n=144 paired collections) proved highly concordant for HPV detection (total agreement=90.3%;PPA=84.2%). There was no relationship between the number of dry storage days and amplification of HPV (n=68, range 4-41days). Exposure of self-collected dry swabs to extreme summer and winter temperatures did not affect testing outcomes. We assessed a second internal control (RNAseP) in a subset and demonstrate that lack of amplification for β-globin from self-collected specimens was consistent with poor, but not absent, cellularity. These data suggest that self-collected vaginal samples enable accurate clinical HPV testing, and that extended ambient dry storage or exposure to extreme temperatures do not influence HPV detection. Further, lack of β-globin amplification in HPV negative samples accurately identified participants that required recollection.

Improving protocols for α-synuclein seed amplification assays: analysis of preanalytical and analytical variables and identification of candidate parameters for seed quantification.

The effect of preanalytical and analytical factors on the α-synuclein (α-syn) seed amplification assay's (SAA) performance has not been fully explored. Similarly, there is limited knowledge about the most suitable assay protocol and kinetic parameters for misfolded α-syn seed quantification. We studied the effect of centrifugation, repeated freeze-thaw cycles (up to seven), delayed freezing, detergent addition, and blood contamination on the performance of the cerebrospinal fluid (CSF) α-syn SAA real-time quaking-induced conversion (RT-QuIC). Moreover, we analysed the inter- and intra-plate variability, the recombinant protein batch effect, and the RT-QuIC parameters' variability when multiple samples were run in controlled conditions. Finally, we evaluated the assay potential of quantifying α-syn seed by assessing kinetic curves in serial CSF dilutions. Among tested preanalytical variables, a≥0.01 % blood contamination and adding detergents significantly affected the RT-QuIC kinetic parameters and the number of positive replicates. Increasing the number of replicates improved result reproducibility. The number of positive replicates in serially diluted CSF samples improved discrimination between samples with high and low seeding activity, and the time to threshold (LAG) was the most reliable kinetic parameter in multiple experiment settings. Preanalytical variables affecting α-syn RT-QuIC performance are limited to blood contamination and detergent addition. The number of positive replicates and the LAG are the most reliable variables for quantifying α-syn seeding activity. Their consistent measurement in serial dilution experiments, especially when associated with an increased number of sample replicates, will help to develop the α-syn RT-QuIC assay further into a quantitative test.

The stability of 65 biochemistry analytes in plasma, serum, and whole blood.

The pre-analytical stability of various biochemical analytes requires careful consideration, as it can lead to the release of erroneous laboratory results. There is currently significant variability in the literature regarding the pre-analytical stability of various analytes. The aim of this study was to determine the pre-analytical stability of 65 analytes in whole blood, serum and plasma using a standardized approach. Blood samples were collected from 30 healthy volunteers (10 volunteers per analyte) into five vacutainers; either SST, Li-heparin, K2-EDTA, or Na-fluoride/K-oxalate. Several conditions were tested, including delayed centrifugation with storage of whole blood at room temperature (RT) for 8 h, delayed centrifugation with storage of whole blood at RT or 4 °C for 24 h, and immediate centrifugation with storage of plasma or serum at RT for 24 h. Percent deviation (% PD) from baseline was calculated for each analyte and compared to the maximum permissible instability (MPI) derived from intra- and inter-individual biological variation. The majority of the analytes evaluated remained stable across all vacutainer types, temperatures, and timepoints tested. Glucose, potassium, and aspartate aminotransferase, among others, were significantly impacted by delayed centrifugation, having been found to be unstable in whole blood specimens stored at room temperature for 8 h. The data presented provides insight into the pre-analytical variables that impact the stability of routine biochemical analytes. This study may help to reduce the frequency of erroneous laboratory results released due to exceeded stability and reduce unnecessary repeat phlebotomy for analytes that remain stable despite delayed processing.

Factors that modulate platelet reactivity as measured by 5 assay platforms in 3429 individuals

BackgroundAssessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing “one-size-fits-all” approach in the interpretation of measures of platelet reactivity, with arbitrary cutoffs often derived from healthy volunteer responses. ObjectivesOur aim was to compare well-used platelet reactivity assays. MethodsBlood and platelet-rich plasma obtained from the Framingham Heart Study (N = 3429) were assayed using a range of agonists in 5 platelet assays: light transmission aggregometry, Optimul aggregometry, Multiplate impedance aggregometry (Roche Diagnostics), Total Thrombus-Formation Analysis System, and flow cytometry. Using linear mixed-effect models, we determined the contribution of preanalytical and technical factors that modulated platelet reactivity traits. ResultsA strong intra-assay correlation of platelet traits was seen in all assays, particularly Multiplate velocity (r = 0.740; ristocetin vs arachidonic acid). In contrast, only moderate interassay correlations were observed (r = 0.375; adenosine diphosphate Optimul Emax vs light transmission aggregometry large area under the curve). As expected, antiplatelet drugs strongly reduced platelet responses, with aspirin use primarily targeting arachidonic acid–induced aggregation, and explained substantial variance (β = −1.735; P = 4.59 × 10−780; variance proportion = 46.2%) and P2Y12 antagonists blocking adenosine diphosphate responses (β = −1.612; P = 6.75 × 10−27; variance proportion = 2.1%). Notably, female sex and older age were associated with enhanced platelet reactivity. Fasting status and deviations from standard venipuncture practices did not alter platelet reactivity significantly. Finally, the agonist batch, phlebotomist, and assay technician (more so for assays that require additional sample manipulation) had a moderate to large effect on measured platelet reactivity. ConclusionCaution must be exercised when extrapolating findings between assays, and the use of standard ranges must be medication-specific and sex-specific at a minimum. Researchers should also consider preanalytical and technical variables when designing experiments and interpreting platelet reactivity measures.

Technical optimization of spatially resolved single-cell transcriptomic datasets to study clinical liver disease

Single cell and spatially resolved ‘omic’ techniques have enabled deep characterization of clinical pathologies that remain poorly understood, providing unprecedented insights into molecular mechanisms of disease. However, transcriptomic platforms are costly, limiting sample size, which increases the possibility of pre-analytical variables such as tissue processing and storage procedures impacting RNA quality and downstream analyses. Furthermore, spatial transcriptomics have not yet reached single cell resolution, leading to the development of multiple deconvolution methods to predict individual cell types within each transcriptome ‘spot’ on tissue sections. In this study, we performed spatial transcriptomics and single nucleus RNA sequencing (snRNAseq) on matched specimens from patients with either histologically normal or advanced fibrosis to establish important aspects of tissue handling, data processing, and downstream analyses of biobanked liver samples. We observed that tissue preservation technique impacts transcriptomic data, especially in fibrotic liver. Single cell mapping of the spatial transcriptome using paired snRNAseq data generated a spatially resolved, single cell dataset with 24 unique liver cell phenotypes. We determined that cell–cell interactions predicted using ligand–receptor analysis of snRNAseq data poorly correlated with cellular relationships identified using spatial transcriptomics. Our study provides a framework for generating spatially resolved, single cell datasets to study gene expression and cell–cell interactions in biobanked clinical samples with advanced liver disease.

Body composition assessment in 6-month-old infants: A comparison of two- and three-compartment models using data from the Baby-bod study.

An appreciation of infant body composition is helpful to understand the 'quality' of growth in early life. Air displacement plethysmography (ADP) using PEA POD and the deuterium dilution (DD) technique are commonly used body composition approaches in infants. We evaluated the comparability of body composition assessed using both techniques with two-compartment (2C) and three-compartment (3C) models in 6-month-old infants. Infant fat mass (FM) and percent fat mass (%FM) obtained from a 2C model using PEA POD (2C-PP) and a 2C model using the deuterium dilution technique (2C-DD) were compared to those derived from a 3C model, and to each other, using Bland-Altman analysis and Deming regression. Measurements were available from 68 infants (93% Caucasian, 53% male). The mean biases were not significant between any of the method comparisons. However, significant constant and proportional biases were identified in 2C-DD vs 3C and 2C-PP vs 2C-DD, but not in the 2C-PP vs 3C comparison. Furthermore, we observed significant associations between the mean differences and infants' percent total body water (%TBW). While no significant between-method mean differences were found in body composition estimates, some comparisons revealed significant constant and proportional biases and notable associations between the mean differences and %TBW were observed. Our results emphasise the importance of method choice, ensuring methodological uniformity in long-term studies, and carefully considering and regulating multiple pre-analytical variables, such as the hydration status of the participants.

Building pathology capacity in sub-Saharan Africa to improve breast cancer diagnosis and treatment: training laboratory technicians in high-quality manual immunohistochemistry

BackgroundTo address the need for a skilled workforce in breast cancer (BC) pathology in sub-Saharan Africa (SSA), we implemented an education program to train laboratory technicians in manual immunohistochemistry (IHC).MethodsA quality improvement education project was developed. Interactive webinars were held every six months with didactics and presentations from African experts with experience in IHC. We conducted knowledge assessments and surveys on current practice, equipment, and human resources. A digital mentorship platform (DMP) was created for discussions, sharing SOPs, and networking. For one year (2022–2023), we followed developments in pathology capacity, practice changes, and educational needs. A paired t-test was used to calculate the significance of changes in knowledge immediately after the webinar and comfort level with topics 35 days after the webinar.ResultsTwo hundred and sixty six participants from 10 SSA countries attended the first webinar, a series of six lectures on IHC theory, methods, and practice. Ninety-five participants from nine SSA countries provided a baseline assessment of pathology capacity and feedback. Mean knowledge increased by 17.4% immediately after the webinar (from 41.8% pre-webinar to 59.2% post, p = < 0.0001). Self-reported comfort level in topics 35 days after the webinar increased by 11.3%, but this was not statistically significant (mean 3.36 pre- to 3.74 post, p = 0.1). Over six months, recordings were accessed 412 times. After six months, the second webinar had 93 participants from eight SSA countries. Membership in the DMP increased from 64 to 172; recordings were viewed 412 times in six months; and 113 participants from nine SSA countries completed surveys. Among 74 respondents who perform IHC, 43.5% reported moderate or significant positive practice changes such as improved antigen retrieval techniques and optimization of preanalytical variables. Over half (52.7%, n = 39) reported the quality of slides had moderately or significantly improved. After one year, a third webinar had 98 participants from eight SSA countries. Thirty-eight completed surveys, DMP membership increased to 199, and 1 reported launching IHC in a lab in Nigeria.ConclusionsOur program 1) reached hundreds of participants and provided a baseline assessment of pathology capacity across nine SSA countries; 2) created a novel mechanism to build pathology capacity and assess progress with this cohort; and 3) improved practices and the preparation of slides for over half performing manual IHC. After one year, interest was sustained. Tracking impact on diagnosis and treatment of BC in the region is needed long-term.

A Retrospective Observational Study to Assess the Quality Management System in a Molecular Diagnostic Laboratory of a COVID-19 Dedicated Hospital in Delhi, India

Introduction: A molecular diagnostic laboratory is the cornerstone of Coronavirus Disease-2019 (COVID-19) disease diagnosis, as the patient’s treatment and management protocol depend on molecular results. Therefore, the laboratory conducting these tests must adhere to quality management process to increase the accuracy and validity of the generated reports. Rajiv Gandhi Super Speciality Hospital established its molecular diagnostic set-up at the beginning of the pandemic. Hence, this study aims to generate quality management data to help improve weak points. Aim: To assess the quality management system for COVID-19 diagnosis. Materials and Methods: This retrospective observational study was conducted at Rajiv Gandhi Super Speciality Hospital in Delhi, India. A total of 14,561 samples were collected over six months, from February 2021 to July 2021. Data from all samples received during this period for COVID-19 ReverseTranscriptase Polymerase Chain Reaction (RT-PCR) testing were included. Data were retrospectively collected from the electronic Laboratory Information Management System (LIMS). Quality variables were analysed over six months from July to December 2021 and classified into preanalytical, analytical, and postanalytical variables. Quality Indicators (QIs) were selected from a common model of QIs set by the International Federation of Clinical Chemistry and Laboratory Medicine. The results were presented in percentages, and descriptive statistics were analysed using Statistical Package for Social Sciences (SPSS) software. Results: During the six-month study period, the molecular laboratory received 14,561 samples. Among the preanalytical variables, sample leakage was the most common cause of sample rejection (134 samples, 0.92%), followed by the non generation of Specimen Referral Form (SRF) identification (76 samples, 0.52%), and non compliance with triple packaging (44 samples, 0.3%). Other preanalytical aspects assessed included incomplete patient identification (17 samples, 0.11%), insufficient sample quantity (12 samples, 0.08%), missing forms/samples (7 samples, 0.04%), samples in the wrong vials/ empty Viral Transport Media (VTM) tubes (5 samples, 0.03%), and incomplete LIMS entry (2 samples, 0.01%). Internal Quality Control (QC) was not obtained in 55 samples (0.37%), and two incidents of cross-contamination resulted in false-positive results. Among the postanalytical factors, 11 samples (0.07%) could not be dispatched within the stipulated time frame. Conclusion: The assessment of the quality management system revealed some areas for improvement, emphasising the importance of adhering to QC processes for the smooth operation of diagnostic laboratories, especially those involved in critical reporting. The assessment of QIs helped monitor laboratory parameters effectively.

A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879).

Alterations in some hematological parameters of feline blood samples preserved at different temperatures for a duration of up to 48 hours

Hematological parameters serve as vital diagnostic tools in veterinary medicine, aiding in the identification of various diseases and metabolic conditions in animals. However, their reliability can be influenced by a multitude of factors, including storage conditions and duration. Pre-analytical errors, which account for up to 70% of laboratory diagnostic errors, primarily stem from issues such as sample collection, transportation, and storage. To address this, the European Federation of Clinical Chemistry and Laboratory Medicine-Working Group for the Preanalytical Phase conducted a survey to understand pre-analytical practices among human laboratories. Despite significant interest, diversity in monitoring approaches was noted among European laboratories. The complete blood count (CBC) stands as a frequently requested test by veterinarians, but its accuracy can be affected by pre-analytical variables like storage temperature and delay in analysis. This study aimed to assess the stability of hematological parameters in feline blood samples stored at different temperatures (4°C and 20°C) for up to 48 hours. Ten clinically healthy cats were included in the study, with blood samples collected and analyzed immediately and at various time points over 48 hours. Results showed stability in hemoglobin concentration for 48 hours at both temperatures. However, RBC and PCV values increased significantly after 24 hours at 20°C. MCV, MCH, and MCHC values remained relatively stable over time, with minor fluctuations. Notably, WBC counts declined significantly after 48 hours at 20°C, while no significant changes were observed at 4°C. These findings underscore the importance of prompt analysis and appropriate storage conditions in maintaining the integrity of hematological parameters in veterinary practice. In conclusion, blood samples from cats stored at 4°C remain reliable for up to 48 hours for most hematological parameters, while samples stored at 20°C are suitable for up to 24 hours. These findings provide valuable insights for veterinarians and laboratory professionals, emphasizing the need for standardized pre-analytical practices to ensure accurate diagnostic results in veterinary hematology.

Sodium fluoride preserves blood metabolite integrity for biomarker discovery in large-scale, multi-site metabolomics investigations.

Background: Metabolite profiling of blood by nuclear magnetic resonance (NMR) is invaluable to clinical biomarker discovery. To ensure robustness, biomarkers require validation in large cohorts and across multiple centres. However, collection procedures are known to impact on the stability of biofluids that may, in turn, degrade biomarker signals. We trialled three blood collection tubes with the aim of solving technical challenges due to preanalytical variation in blood metabolite levels that are common in cohort studies. Methods: We first investigated global NMR-based metabolite variability between biobanks, including the large-scale UK Biobank and TwinsUK biobank of the general UK population, and more targeted biobanks derived from multicentre clinical trials relating to inflammatory bowel disease. We then compared the blood metabolome of 12 healthy adult volunteers when collected into either sodium fluoride/potassium oxalate, lithium heparin, or serum blood tubes using different pre-processing parameters. Results: Preanalytical variation in the method of blood collection strongly influences metabolite composition within and between biobanks. This variability can largely be attributed to glucose and lactate. In the healthy control cohort, the fluoride oxalate collection tube prevented fluctuation in glucose and lactate levels for 24 hours at either 4 °C or room temperature (20 °C). Conclusions: Blood collection into a fluoride oxalate collection tube appears to preserve the blood metabolome with delayed processing up to 24 hours at 4 °C. This method may be considered as an alternative when rapid processing is not feasible.

Red Cell Histograms and Spurious Parameters in Anemia – Study of Diagnostic Utility of Autoanalyzer Results

BACKGROUND: Red blood cell (RBC) histograms generated by automated hematology analyzers have shown characteristic patterns useful in identifying the different red cell populations and give diagnostic clues. Although the analyzer results are precise, spurious results have been reported owing to preanalytical and analytical variables which warrant the evaluation of validity of other parameters. AIMS: The study was meant to evaluate the diagnostic utility of red cell indices and histograms in the morphological diagnosis of anemia and to interpret the spurious values generated by autoanalyzers. SETTINGS AND DESIGN: A retrospective cross-sectional study was undertaken including 917 cases of anemia. MATERIALS AND METHODS: The histograms and the red cell indices generated by autoanalyzer Mindray CAL-6000 were studied in all cases and correlated with peripheral smear. STATISTICAL ANALYSIS USED: Analysis was done using Mann–Whitney U-test for medians and interquartile range of numerical variables and Chi-square test/Fisher’s exact test for categorical variables. RESULTS: Characteristic patterns were observed in histograms such as left shift, right shift, and bimodal peaks in microcytic hypochromic anemia, macrocytic anemia, and dimorphic anemia, respectively. Spurious parameters in the study comprised high red cell indices, low RBC count, hemoglobin, and hematocrit mismatch in cases of autoimmune hemolytic anemia. Spuriously high mean corpuscular volume was associated with reticulocytosis. In vitro hemolysis was identified by factitious thrombocytosis with abnormal platelet and RBC histograms. CONCLUSION: Histograms when combined with red cell indices are of practical utility in differentiating the various morphologic types of anemia and are speculative in alerting the hematologists about the spurious parameters, forming an indispensable diagnostic tool.

The Coagulology Section of the Polish Societyof Laboratory Diagnostics recommendations on the plateletreactivity test. 2023

Platelet reactivity testing is used for a wide variety of clinical purposes; firstly, to study plateletdysfunction in acute bleeding and diagnosis of platelet disorders in patients with long-term bleeding tendency, and secondly, to study the effectiveness of antiplatelet therapy in patientswith increased risk of thromboembolism. A new direction of research is platelet phenotyping.To provide an overview of contemporary platelet function testing, this review summarizes themost commonly used tests in Poland, including their advantages, disadvantages, and clinicalutility. The review also discusses preanalytical variables that should be considered whenexamining platelet function. Finally, we concluded future directions for the study of plateletfunction for clinical or research purposes.

Stability of circulating miRNA in saliva: The influence of sample associated pre-analytical variables

Background and aimsIncreasing evidence supports the practicability of salivary cell-free (cf) miRNA as liquid biopsy markers in cancers. Its successful translation in the clinical setting requires reproducible approaches for saliva manipulation, in order to control for pre-analytical variables influencing miRNA stability. This study aims to define the optimal conditions to maintain the integrity of saliva during collection, transport and processing with respect to cf-miRNA quantification. Materials and methodsSaliva was collected from 20 healthy subjects and 8 oral cancer patients. Two sampling methods were tested and different storage temperatures and times were evaluated. Salivary expression level of target miRNAs was quantified by qPCR. Comparison between group mean values at specific conditions were performed using paired t-tests. Agreement between measurements was evaluated using a Bland-Altman plot. ResultsDifferent collection methods revealed comparable levels of salivary miR-484 and miR-106b-5p in both subject cohorts. MiRNAs were stable for up to 48 h at 4 °C in saliva supernatant, showing significant alteration after 96 h. Mid-term storage of supernatant at −20 °C decreased miRNA stability significantly compared to standard −80 °C. ConclusionsCf-miRNA in saliva were slightly altered by collection methods and storage conditions, both in healthy and in pathological contexts, and remained stable for a period of time compatible with main clinical routine needs.