Preanalytical Variables Research Articles

Abstract A024: Evaluating the effect of plasma-related preanalytical variables on the robustness of the liquid biopsy early CRC detection Signal-C assay

Abstract Liquid biopsies and cell-free DNA (cfDNA) analysis have emerged as novel non-invasive diagnostic tools in oncology. Analysis of cfDNA methylation status has been proposed as promising approach for several diagnostic purposes, and robust methylation-detecting technologies are of primary importance for the clinical implementation of novel diagnostic assays. Signal-C is an accurate NGS-based methyl-sensitive assay detecting colorectal cancer (CRC) currently under clinical validation. In this study we assessed the influence of different preanalytical variables influencing plasma stability, as the storage time of plasma samples and the tube type used for blood collection, on Signal-C assay results to determine its robustness. 958 samples with 3ml to 4ml plasma were used for cfDNA extraction and run through Signal-C assay following UDX protocol. In brief, 8 to 20ng cfDNA were used as an input for methylation- specific library preparation, which was run according to manufacturer’s instructions. Hybrid- capture was performed to enrich libraries for genomic regions included in Signal-C assay and sequenced on Illumina Novaseq machines. Relevant sequencing QC metrics, such as depth filtered, read yield from raw to filtered and PCR duplicate proportion were calculated, together with cancer predictions according to Signal-C test. Pearson coefficient and Pearson's Chi- squared test with Yates' continuity correction have been used to assess correlations, and Wilcoxon test has been used to measure the significance of the differences between quality metrics with respect to the tube type. We have analyzed the influence of different plasma-related preanalytical variables on sequencing metrics and cancer predictions of Signal-C assay. Plasma age, calculated as the time elapsed between blood collection and cfDNA quantification, shows a small negative correlation with YIELD_RAW_FILTERED (R = - 0.08, p=0.013) and a small positive correlation with PCR_DUP_PROP (R = 0.098, p=0.0024), while it doesn't influence DEPTH_FILTERED values (p>0.05). Use of different blood collection tubes (EDTA or Streck tubes) influenced in a small but significative manner the YIELD_RAW_FILTERED and the PCR_DUP_PROP (p<0.001), while it doesn’t affect the DEPTH_FILTERED values (p>0.05). Despite the sequencing QC metrics being subject to small but statistically significant differences, the overall cancer classification performance of Signal-C assay was not affected by the changes in plasma age (Pearson's product-moment correlation, sensitivity p=0.87, specificity p=0.091) nor in tube type used (Pearson's Chi-squared test with Yates' continuity correction, p=0.219). Here we evaluated the effect of plasma’s storage time and the type of blood collection tube on Signal-C assay performance. While signal processing features show small differences between the tested variables, this does not affect overall clinical performance of the test, supporting its robustness. Additional studies to analyze the effect of other preanalytical variables on Signal-C performance are underway to establish its robustness. Citation Format: Fernando Trincado Alonso, Pol Canal Noguer, Kristi Kruusmaa, Arianna Bertossi. Evaluating the effect of plasma-related preanalytical variables on the robustness of the liquid biopsy early CRC detection Signal-C assay [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A024.

Abstract A034: Addressing the challenge of pre-analytical variables in circulating protein biomarker analysis for liquid biopsy

Abstract While liquid biopsy using plasma proteins is a promising avenue for early cancer detection, diagnosis, prognosis, and disease monitoring, the clinical translation of plasma proteomics has been limited due to the complexity of the proteome and the impact of pre-analytical variation on its integrity. In recent years, advancements in plasma proteomic technologies, such as Olink, have helped address the challenge of proteome complexity. However, challenges with pre- analytical variables have persisted. During blood collection and whole blood storage, ex vivo blood cell activation and lysis can occur, releasing proteins into the plasma and obscuring relevant in vivo abundances. This is especially true for cancer where many key biomarkers are expressed in white blood cells and platelets. To address these issues, Streck developed Protein Plus BCT, which stabilizes blood cells and minimizes plasma proteome changes following prolonged whole blood storage. To demonstrate its functionality and compatibility with Olink assays, blood was collected from 5 donors into EDTA, ACD-A, and Protein Plus BCT. Plasma was isolated at draw time (< 2 hours) and following 24, 72, and 120 hours of ambient whole blood storage using a double spin protocol (per Instructions for Use). Plasma samples were analyzed using an Olink Explore 384-plex panel. After just 24 hours of whole blood storage, plasma isolated from EDTA or ACD-A had 4.2 and 2.2 times more proteins that are significantly elevated in abundance relative to draw time, respectively, than plasma isolated from Protein Plus BCT. For the key cancer biomarker IL-8, which is associated with worse clinical outcomes and poor response to immunotherapy, Protein Plus BCT maintains draw-time levels such that the mean NPX difference relative to draw time never exceeds -0.18, or a 0.88-fold change for up to 120 hours. Comparatively, samples collected into EDTA and ACD-A show dramatic increases in IL-8 levels with mean NPX differences of 6.34 and 6.62, respectively, corresponding to 80.1- and 98.3-fold increases after 120 hours of whole blood storage. For Galectin-3, an inflammatory biomarker elevated in many cancer types (i.e., lung, breast, and ovarian), the mean NPX difference relative to draw time for samples drawn into EDTA is 1.46 after 24 hours and increases to 2.27 after 120 hours of whole blood storage (a 2.75-fold and 4.8-fold increase, respectively). In samples collected into Protein Plus BCT, the mean NPX difference, relative to draw time, for Galectin-3 never exceeds 0.05 (a 1.04-fold increase) even after 120 hours of whole blood storage. Taken together, these data suggest that Protein Plus BCT extends the storage of whole blood at ambient temperature without compromising the integrity of the sample or the proteomic results, providing researchers and assay developers valuable flexibility in the transport, storage, and processing of their samples. Protein Plus BCT is for Research Use Only. Not for use in diagnostic procedures. Protein Plus BCT should only be used for research or the development of new assays. Citation Format: Rachel M Miller, Jing Li. Addressing the challenge of pre-analytical variables in circulating protein biomarker analysis for liquid biopsy [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A034.

Challenges of preanalytical variables in erythrocyte sedimentation rate: a CUBE 30 touch evaluation

The erythrocyte sedimentation rate (ESR) is a widely used diagnostic test, influenced by all physiological and pathological conditions that can bias blood rheology by interfering factors. This study aimed to evaluate the performance of the CUBE 30 touch ESR analyzer in samples with preanalytical variables, as lipemia, hemolysis, and icterus or in presence of fibrinogen., Moreover we focused to define the maximum time limits to ensure a reliable ESR measure. Accuracy, intra-run and inter-run precision, and stability studies were performed. Moreover, hemolytic, jaundiced, lipemic samples and fibrinogen sensitivity were analyzed for interference study. Statistical analyses were performed. CUBE 30 touch and Westergren method comparison showed no statistical differences (Spearman Coefficient, R2=0,95). In the intra-run precision, the CV% mean obtained on samples with normal ESR level was 8,9%; with middle ESR level was 5,9% and with high ESR level the CV% was 4,3%. Inter-run precision test showed CV% of for single samples and overall samples in the range (12,3% for normal level and 4,8% for abnormal level). The samples stored at 4 °C showed good stability up to 3 h from collecting time. ESR samples showing lipemia, hemolysis or jaundice showed good correlations with the gold standard method (R2 0,901, 0,940, 0,911; p < 0,0001), however, Westergren tests were more sensitive than CUBE 30 touch to fibrinogen additions. The high comparability with the Westergren method, both in normal and interfering samples, and the good precision, support the usefulness of CUBE 30 touch in the clinical routine laboratory.

Liquid biopsy-based technologies: a promising tool for biomarker identification in her2-low breast cancer patients for improved therapeutic outcomes

The molecular classification of breast cancer plays a pivotal role in developing personalized treatment strategies, with the aim of improving therapeutic outcomes. Despite significant advancements, current diagnostic workflows are hindered by several challenges, including pre-analytical variability, interpretive ambiguity, and inconsistencies in threshold definitions, particularly in cases involving human epidermal growth factor receptor 2 (HER2)-low breast cancer. In this context, liquid biopsy technologies have emerged as promising tools for refining breast cancer diagnostics. Techniques such as circulating tumor DNA and circulating tumor cell analysis provide a non-invasive approach to assessing tumor-associated biomarkers. These methodologies are particularly advantageous for analyzing low-abundance materials, such as formalin-fixed paraffin-embedded samples and liquid biopsies, thus enhancing the precision of molecular classification and informing more targeted therapeutic decisions for breast cancer patients. This review aims to explore the potential of liquid biopsy in addressing the limitations of current diagnostic practices, with a specific focus on its application in HER2-low breast cancer. Furthermore, it advocates for a transition toward high-throughput RNA-based screening and quantification, which may address critical unmet clinical needs.

Haematological and Biochemical Reference Intervals Towards a Proactive Health Monitoring Approach in Norwegian Atlantic Salmon Farming.

Haematological and plasma parameters are used in veterinary medicine as a diagnostic tool, allowing for the early detection of potential health and welfare issues. For the implementation of monitoring strategies, reference intervals (RIs) are needed to know when deviations in those parameters become clinically relevant. Here, haematological and plasma biochemical RIs of Norwegian Atlantic salmon (Salmo salar Linnaeus) are presented for 29 biomarkers. Two subgroups were defined from the reference population based on water type, referred to as pre-adult (freshwater, 166.8 ± 118.4 g, N = 979) and adult (seawater, 2655 ± 2883 g, N = 4941), collected at 93 fish farms along the Norwegian coast. While the influence of various pre-analytical variables on RIs is well documented, aquaculture practitioners cannot always control those variables in the field. Here, RIs were calculated from blood samples collected based on local pre-analytical practices and then further processed under standardised conditions (analytical phase). RIs are therefore considered highly relevant for the application in the field. Finally, when taking blood samples in the field, it is advised to centrifuge whole blood as soon as possible, while plasma parameters investigated were stable for 168 h at 4°C or 72 h at room temperature.

Fast processing of gynecologic cancer tissue in Danish Cancer Biobank makes them well-suited for biomarker studies.

Gynecologic cancers remain a frequent and deadly diagnosis. Historically, treatment has focused on a "one size fits all" approach, but there is an urgent need for more personal medicine. Hence, to enhance personal medicine, new biomarkers are needed. Samples from the Danish Cancer Biobank (DCB) may be well-suited for biomarker studies, as the biobank contains samples from more than 100.000 cancer patients, and the samples are annotated with pre-analytical variables. The aim of this study was to investigate if the recorded pre-analytical variables indicate the gynecologic tissue in DCB are suited for biomarker studies. Data on processing time, transport time, and registration- and verification status were extracted from all patients with a gynecologic tissue sample collected between 2020 and 2022 in DCB. The mean processing time across centers was found to be 1.03 h (SD = 0.71), and the mean transport time was found to be 0.32 h (SD = 0.70). In total, 69% of the tissue samples were pathologically examined, and 91.5% of the pathologically examined samples were found to be concordant with the patient's final diagnosis. While differences were observed, 98% of the samples were processed within 3 h, indicating the majority of gynecologic tissue samples in DCB are of high quality and optimal for biomarker studies.

Review: Current Laboratory and Point-of-Care Pharyngitis Diagnostic Testing and Knowledge Gaps.

Pharyngitis is an inflammatory condition of the pharynx and/or tonsils commonly seen in both children and adults. Viruses and bacteria represent the most common encountered etiologic agents-yeast/fungi and parasites are infrequently implicated. Some of these are predominantly observed in unique populations (eg, immunocompromised or unvaccinated individuals). This manuscript (part 2 of 3) summarizes the current state of laboratory and point-of-care diagnostic testing and highlights the expanding role of nucleic acid amplification in the expedited diagnosis and management of patients with acute pharyngitis. It discusses preanalytical, analytical, and postanalytical variables that impact the performance of culture, rapid antigen, and nucleic acid amplification testing. Finally, it sets the stage for part 3, which discusses the emerging role of biomarkers in the management of individuals with acute pharyngitis.

Assay variables and early clinical evaluation of low-angle light scattering for platelet function analysis.

The recently developed platelet aggregation technique based on low-angle light scattering (LaSca) in diluted platelet-rich plasma (PRP) requires only a small sample volume and provides information about platelet aggregation and shape change. This study aimed to investigate the influence of preanalytical and analytical variables and to validate the method in a real-life pediatric hematology hospital setting. Platelet aggregation was induced by ADP in diluted PRP in the presence of 2mM calcium at 23°C. The study included healthy adults (n = 30), healthy children (n = 20), and pediatric patients with suspected or diagnosed platelet function abnormalities (n = 25). The assay parameters were stable for at least 3h after isolation of PRP and were sensitive to plasma dilution in the range of 2-8%. The initial aggregation velocity was significantly reduced in pediatric patients compared with healthy children (p < 0.05). ADP-induced light transmission amplitude was moderately correlated with LaSca amplitude of aggregation in healthy children (p = 0.52, p < 0.05) but not in pediatric patients. We standardized the protocol for platelet aggregation assessment by LaSca and characterized the influence of preanalytical and analytical variables on it.

Laboratory medicine and sports: where are we now?

Laboratory medicine in sport and exercise has significantly developed during the last decades with the awareness that physical activity contributes to improved health status, and is present in monitoring both professional and recreational athletes. Training and competitions can modify concentrations of a variety of laboratory parameters, so the accurate laboratory data interpretation includes controlled and known preanalytical and analytical variables to prevent misleading interpretations. The paper represents a comprehensive summary of the lectures presented during the 35th Annual Symposium of the Croatian Society of Medical Biochemistry and Laboratory Medicine. It describes management of frequent sport injuries and sums up current knowledge of selected areas in laboratory medicine and sports including biological variation, changes in biochemical parameters and glycemic status. Additionally, the paper polemicizes sex hormone disorders in sports, encourages and comments research in recreational sports and laboratory medicine. In order to give the wider view, the connection of legal training protocols as well as monitoring prohibited substances in training is also considered through the eyes of laboratory medicine.

A-162 Improved Laboratory Workflow using Automated Detection of Hemolysis, Icterus, and Lipemia for Coagulation Testing

Abstract Background Managing preanalytical variables is critical to providing quality laboratory test results. However, this can be a challenge for laboratory staff, particularly with manual detection of interfering substances and complicated workflows. For coagulation testing, we assess specimen integrity and acceptability by visual checks and quantification of interfering substance on a chemistry analyzer. To improve this subjective process, we evaluated the technical performance of a new model of coagulation analyzer with automated preanalytical specimen integrity detection. Our objective was to determine if we could reduce the number of specimens requiring visual inspection/off-analyzer interference testing in order to simplify our testing workflow. Methods Citrated plasma specimens received in the Hospital Clinical Laboratory at Mayo Clinic (Rochester, MN) were tested on the TOP ACL 500 or TOP ACL 550 Series Coagulation analyzers (Werfen, Boston, MA) according to manufacturer’s instruction. Specimens tested on the TOP 500 were visually inspected for hemolysis, icterus, and lipemia (HIL). If the technologist detected a possible interferent, an aliquot of plasma was analyzed on our cobas c501 chemistry analyzer (c501) (Roche Diagnostics, Indianapolis, IN) to obtain HIL values. Laboratory-established or manufacturer-defined interference thresholds were applied in determining specimen acceptability. For samples tested on the TOP 550, specimen integrity was assessed by the instrument’s automated interference detection module. The analyzer reported HIL as an “interference cloud” or estimated range, rather than a single value like the c501. HIL values were estimated by visual assessment of the low end of the “interference cloud”. Values were confirmed on the c501 only for specimens with an “interference cloud” at/exceeding the test-specific interference thresholds. We analyzed data from a 2-month period pre- (TOP 500) and post-implementation (TOP 550). The percent of specimens requiring quantitation/confirmation on the c501 was calculated. Statistical significance was calculated using the Chi-squared test where significance was defined as p&amp;lt;0.01. Correlation between c501 and TOP 550 estimated HIL values was evaluated. Results A total of 2.2% (171/7605) and 1.3% (113/8983) of specimens tested on the TOP 500 and TOP 550, respectively, required HIL measurements on the c501 (p&amp;lt;0.001). Of those samples, 8.2% (14/171) and 14.2% (16/113) from the TOP 500 and TOP 550, respectively, had HIL values above the interference thresholds and were rejected by the laboratory. Comparison of the estimated HIL values from the “interference cloud” and from the c501 resulted in slopes (Pearson correlation coefficients) of: H 1.56 (0.77), I 0.67 (0.97), and L 1.35 (0.99). Conclusions In comparison to our workflow with visual assessment of interference, use of the automated interference detection functionality on the TOP 550 instrument resulted in a lower percentage of specimens requiring HIL measurements on the chemistry analyzer. Agreement between interference measurements obtained on the c501 and the TOP 550 was moderate where TOP 550 overestimated the degree of hemolysis and lipemia and underestimated icterus. Implementation of automated interference detection on the TOP 550 analyzer improved our workflow by eliminating the need for tedious and subjective visual checks and reducing the percentage of specimens requiring additional HIL measurements.

Analysis of Gliomas DNA Methylation: Assessment of Preanalytical Variables

Precision oncology is driven by biomarkers. For glioblastoma multiforme (GBM), the most common malignant adult primary brain tumor, O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation is an important prognostic and treatment clinical biomarker. Time-consuming preanalytical steps such as biospecimen storage, fixation, sampling, and processing are sources of data irreproducibility, and all these preanalytical variables are confounded by intratumor heterogeneity of MGMT promoter methylation. To assess the effect of preanalytical variables on GBM DNA methylation, tissue storage/sampling (CryoGrid), sample preparation multisonicator (PIXUL), and 5-methylcytosine DNA immunoprecipitation (Matrix-MeDIP-qPCR/seq) platforms were used. MGMT promoter methylation status assayed by MeDIP-qPCR was validated with methylation-specific PCR. MGMT promoter methylation levels in frozen and formalin-fixed paraffin-embedded sample pairs were not statistically different, confirming the reliability of formalin-fixed paraffin-embedded for MGMT promoter methylation analysis. Warm ex vivo ischemia (up to 4 hours at 37 °C) and 3 cycles of repeated sample thawing and freezing did not statistically impact 5-methylcytosine at MGMT promoter, exon, and enhancer regions, indicating the resistance of DNA methylation to common variations in sample processing conditions that might be encountered in research and clinical settings. Twenty-six percent to 34% of specimens exhibited intratumor heterogeneity in the MGMT DNA promoter methylation. These data demonstrate that variations in sample fixation, ischemia duration and temperature, and DNA methylation assay technique do not have a statistically significant impact on MGMT promoter methylation assessment. However, intratumor methylation heterogeneity underscores the value of multiple biopsies at different GBM geographic tumor sites in the evaluation of MGMT promoter methylation status. Matrix-MeDIP-seq analysis revealed that MGMT promoter methylation status clustered with other differentially methylated genomic loci (eg, HOXA and lncRNAs) that are resilient to variation in the above preanalytical conditions. These observations offer new opportunities to develop more granular data-based epigenetic GBM biomarkers. In this regard, the high-throughput CryoGrid-PIXUL-Matrix toolbox could be useful.

Choice of blood collection methods influences extracellular vesicles counts and miRNA profiling.

Circulating RNAs have been investigated systematically for over 20years, both as constituents of circulating extracellular vesicles (EVs) or, more recently, non-EV RNA carriers, such as exomeres and supermeres. The high level of variability and low reproducibility rate of EV/extracellular RNA (exRNA) results generated even on the same biofluids promoted several efforts to limit pre-analytical variability by standardizing sample collection and sample preparation, along with instrument validation, setup and calibration. Anticoagulants (ACs) are often chosen based on the initial goal of the study and not necessarily for the later EV and/or exRNA analyses. We show the effects of blood collection on EV size, abundance, and antigenic composition, as well on the miRNAs. Our focus of this work was on the effect of ACs on the number and antigenic composition of circulating EVs and on a set of circulating miRNA species, which were shown to be relevant as disease markers in several cancers and Alzheimer's disease. Results show that while the number of plasma EVs, their relative size, and post-fluorescence labeling profile varied with each AC, their overall antigenic composition, with few exceptions, did not change significantly. However, the number of EVs expressing platelet and platelet-activation markers increased in serum samples. For overall miRNA expression levels, EDTA was a better AC, although this may have been associated with stimulation of cells in the blood collection tube. Citrate and serum rendered better results for a set of miRNAs that were described as circulating markers for Alzheimer's disease, colon, and papillary thyroid cancers.

Stability of SARS-CoV-2 antibody in serum under various usage and storage conditions.

We investigated the effect of two preanalytical variables, temperature change and freezing-thawing of serum samples, on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG levels. Serum samples were collected from patients who had coronavirus disease 2019 (COVID-19) prior to vaccination. Six serum samples were included, two each with high positivity (HP), low positivity (LP), and a level of close-to-detection limit (CDL) for SARS-CoV-2 IgG. Each of these six samples was divided into three tubes and placed in refrigerators at 4-8 °C, -20 °C, and -70 °C; removed from the storage temperature once per day for 20 consecutive days; and assayed for SARS-CoV-2 IgG level. The coefficient of variation of all the remaining serum samples were within 95% except for CDL-1 serum at -70 °C, HP-2 serum at 4-8 °C, HP-2 serum at -20 °C, and HP-2 serum at -70 °C. The levels increased significantly when the temperature in the samples with CDL was reduced. The values in samples with LP at -20 °C and -70 °C were significantly higher than those at 4-8°C. In the case of samples with HP, the values of samples at -20 °C were higher than those in samples at 4-8 °C. There was no positive-negative change during any of the freeze-thaw cycles. Antibody value in the samples at 4-8 °C remained stable throughout the 20 freeze-thaw cycles. The antibody value of the samples at -20 °C and -70 °C tended to elevate.

Initial development of a rapid, portable, stall-side ELISA for the measurement of equine adrenocorticotropic hormone.

Pituitary pars intermedia dysfunction (PPID) is a neurodegenerative disease of senior horses. Loss of dopaminergic inhibition of the melanotropes of the pars intermedia leads to increased concentrations of pro-opiomelanocortin (POMC)-derived peptides. Diagnosis is challenging due to pre-analytical variables, such as sample storage, handling, and time to analysis. Our objective was to develop an ELISA for ACTH measurement, which could ultimately form the basis for a stall-side equine ACTH test. We selected 2 ACTH-specific monoclonal antibodies, CBL57 and EPR20361-248, based on the recognition of separate epitopes, strong and rapid color change, and minimal background interference, including no cross-reactivity with themselves, each other, and the test reagents. CBL57 was chosen as the detection antibody (or secondary antibody). EPR20361-248, functionalized on superparamagnetic iron oxide beads, was chosen as the capture antibody (or primary antibody) to bind ACTH in plasma. The incorporation of magnetic beads marks the initial stage in establishing a platform that could potentially be utilized in the field, similar to other stall-side tests. The concentrations of antibodies, magnetic beads, and incubation durations were optimized. Our immunoassay detected unglycosylated rat recombinant ACTH. Further studies are ongoing to optimize and validate our assay using equine plasma and serum samples.

MIBLood-EV: An Online Reporting Tool to Facilitate the Standardized Reporting of Preanalytical Variables and Quality Control of Plasma and Serum to Enhance Rigor and Reproducibility in Liquid Biopsy Research.

Pre-analytical variability significantly impacts the reproducibility of liquid biopsy research, which is critical for precision medicine and biomedical research. This report highlights the challenges and variability in the pre-analytical processes of liquid biopsies, especially regarding extracellular vesicles (EVs), which are crucial for diagnostics in oncology. The MIBlood-EV initiative aims to standardize the reporting of pre-analytical variables and the quality control of plasma and serum samples to enhance reproducibility in EV research. By providing a comprehensive and flexible reporting framework, MIBlood-EV seeks to improve the reliability of EV studies and facilitate the development of evidence-based protocols, ultimately advancing the field of liquid biopsy research.

Urine Sample Handling in Laboratory—Process Reengineering is the Need of the Hour

Background and Aims: Monitoring and a continual improvement of turnaround time (TAT) in urine examination and reporting is essential for improved management of laboratory quality as well as ensuring patient satisfaction. TAT can be impacted by multiple factors, some of which appear to be beyond the control of laboratory, especially the preanalytical phase of urine examination such as specimen collection, transportation and sample preparation. The aim of the study was to compare the preanalytical variables like TAT for transportation and incidences of spill between our current urinalysis workflow process and the new urinalysis workflow process using a pneumatic chute-compatible closed urine collection system. Materials and Methods: This observational prospective study was conducted on 4/9/23 from 9 am to 4 pm at the laboratory of Apollo Main Hospital, Chennai. Pneumatic chute compatible closed urine collection cup was given to the patients in Group-A and normal screw cap containers which we are currently using were given to the patients in Group-B. Once the urine samples were collected in the respective containers, they were transported to the laboratory using pneumatic chutes for Group-A samples and manual carry for Group-B samples. Analysis of transportation time using the new collection method (Group-A) when compared with the current method of practice (Group-B) was done. The incidence of spillage was also monitored. Results: A significant improvement was noticed in the transportation process and spill incidence with closed collection devices through the pneumatic chute. Conclusion: To conclude, the implementation of pneumatic chute compatible closed collection tubes for urine improves the transportation and processing time, eliminates the manual carry of urine samples, eliminates the incidences of leakage and spillage, reduces errors and reduces the exposure of urine to healthcare workers, thereby leading to significant improvement in patient care, patient experience and staff satisfaction.

A false reactive hepatitis B surface antigen result due to dislodged gel from Vacutainer

Abstract: Preanalytical variables play a key role in the correctness of laboratory test results. We report a false reactive result for hepatitis B surface antigen (HBsAg) on serological testing with a relatively low OD. The pilot sample was collected in yellow top BD Vacutainer® (SST™ II Advance 5 mL tube) with separator gel and was marked for serological testing. It was centrifuged at 7000 rpm for 30 min. Serological testing was performed by chemiluminescence on Abbott Architect Plus i1000SR with Abbott kits for HBsAg, anti-HIV, and anti-HCV. This particular sample tested reactive for HBsAg with S/CO of 1.91, S/CO for HBsAgQ2NC (Negative kit control) being 0.19 and that of HBsAgQ2PC (Positive kit control) being 3.65. Repeat testing from the same Vacutainer was also reactive for HBsAg with S/CO of 2.10. Nucleic acid testing was performed and was found to be nonreactive. We repeated serological testing with a sample from fresh frozen plasma of the same donor unit. It was nonreactive. Cross contamination was ruled out by repeat testing of two serial samples before and after this unit number. The serum showed an unusual appearance on visual inspection after centrifugation. By exclusion, we concluded that the gel probably got partially dislodged into the serum, giving a relatively low reading and false reactive result for HBsAg. This highlights the possibility of inert gel not always being inert, the importance of repeat testing from a different sample in case of low reading, the importance of visual inspection, a need for manufacturers and laboratory personnel to consider such preanalytical variables which, though not much mentioned in literature, can affect test results. These results have a multi-fold impact on the donor or patient’s mindset, treatment outcome and/or fate of blood donation. These are definitely preventable if more such guidelines are made available for handy reference in handling such results.

RNA-Seq Analysis in Non-Small Cell Lung Cancer: What Is the Best Sample from Clinical Practice?

RNA-based next-generation sequencing (RNA-seq) represents the gold standard for detecting gene fusion in non-small cell lung cancer (NSCLC). Despite this, RNA instability makes the management of tissue samples extremely complex, resulting in a significant number of test failures with missing data or the need to switch to other techniques. In the present study, we analyzed pre-analytical variables in 140 tumor tissue samples from patients affected by NSCLC to detect features that increase the chances of successful RNA-seq. We found that the success rate of the analysis positively correlates with the RNA concentration and fragmentation index. Interestingly, small biopsies were more suitable samples than surgical specimens and cell blocks. Among surgical specimens, wedge resections demonstrated better results than lobectomy. Moreover, samples stored for less than 30 days (1 month) had a better chance of success than older samples. Defining the role of pre-analytical variables in RNA-seq allows the detection of more suitable samples for analysis and more effective planning of molecular-based diagnostic approaches in NSCLC.

Association of Serum Metabolites with Serum Indices and Preanalytical Factors of Biobanked Serum Samples.

Background: Serum indices (hemolysis, icterus, and lipemia; HIL) are known to impact clinical chemistry assay results. This study aimed to investigate the impact of HIL indices on serum metabolite profiles and the association of serum metabolite levels with pre-analytical factors of serum samples. Methods: A cohort of serum samples (n = 12,196) from the Korean Genome and Epidemiology Study (KoGES) was analyzed for HIL indices and the pre-analytical variables (SPRECs) which were generated in the process of serum collection. We further performed targeted metabolomics on a subset comprising hemolyzed (n = 60), icteric (n = 60), lipemic (n = 60) groups, and a common control group of non-HIL samples (n = 60) using the Absolute IDQ p180 kit. Results: We found 22 clinical chemistry analytes significantly associated with hemolysis, 25 with icterus, and 24 with lipemia (p < 0.0001). Serum metabolites (n = 27) were associated with all of hemolysis, icterus, and lipemia (p < 0.05). The PC ae C36 2 had exhibited a significant association with pre-analytical factors corresponding to the third (pre-centrifugation delay between processing) and sixth (post-centrifugation) elements of the SPREC. Conclusions: This study showed the association of the serum index and pre-analytical factors with serum metabolite profiles. In addition, the association of pre-analytical factors with serum metabolite concentrations would corroborate the utility of SPRECs for the quality control of biobanked serum samples.

Time-dependent characteristics of analytical measurands.

Biological variation is a relevant component of diagnostic uncertainty. In addition to within-subject andbetween-subject variation, preanalytical variation also includes components that contribute to biological variability. Among these, daily recurring, i.e.,diurnal physiological variation is of particular importance, as it contains both a random and a non-random component ifthe exact time of blood collection is not known. We introduce four time-dependent characteristics (TDC) of diurnal variations for measurands to assess the relevance and extent of time dependence on the evaluation of laboratory results. TDC address (i)a threshold for considering diurnality, (ii) the expected relative changes per time unit, (iii) the permissible time interval between two blood collections at different daytimes within which the expected time dependence does not exceed a defined analytical uncertainty, and (iv) a rhythm-expanded reference change value. TDC and their importance will be exemplified by the measurands aspartate aminotransferase, creatine kinase, glucose, thyroid stimulating hormone, and total bilirubin. TDCs are calculated for four time slots that reflect known blood collection schedules, i.e.,07:00-09:00, 08:00-12:00, 06:00-18:00, and 00:00-24:00. The amplitude and the temporal location of the acrophase are major determinates impacting the diagnostic uncertainty and thus the medical interpretation, especially within the typical blood collection time from 07:00 to 09:00. We propose to check measurands for the existence of diurnal variations and, if applicable, to specify their time-dependent characteristics as outlined in our concept.