Pre-analytical Aspects Research Articles

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.

Comparison and commutability study among four faecal immunochemical tests (FIT) systems.

Faecal immunochemical tests for haemoglobin (FIT) are used in colorectal cancer screening programs around the world and increasingly for triage of symptomatic patients. FIT results are currently not traceable to a common reference standard and results obtained on various FIT systems may not be equivalent. The size of the bias between the systems is difficult to quantify due to the complex pre-analytical aspects of FIT. This study aimed to quantify the bias and the correlation between four FIT systems by measuring a panel of 38 faecal samples while limiting the effect of the pre-analytical aspects. In addition, the commutability of seven candidate reference materials (RM) was assessed. Pairwise method comparisons based on faecal samples demonstrated Pearson correlation coefficients ranging between 0.944 and 0.970 and an average proportional bias of-30 to-35 % for one FIT system compared to the other three. The relative standard deviation among biases of the individual samples was around 20 %. Due to these sample specific differences, no decisive conclusions could be drawn in the commutability study. However, twocandidate RMs, prepared in the FIT system-specific storage/extraction buffers, had a better commutable profile than the other five. The use of a common threshold for all FIT systems is currently not possible due to the presence of a proportional bias. We have identified potential commutable RMs to take to further studies on the production of a common calibrator, with the aim being to reduce the analytical bias observed on different FIT systems.

Standardising the preanalytical reporting of biospecimens to improve reproducibility in extracellular vesicle research – A GEIVEX study

AbstractThe standardization of clinical studies using extracellular vesicles (EVs) has mainly focused on the procedures employed for their isolation and characterization; however, preanalytical aspects of sample collection, handling and storage also significantly impact the reproducibility of results. We conducted an online survey based on SPREC (Standard PREanalytical Code) among members of GEIVEX (Grupo Español de Investigación en Vesiculas Extracelulares) to explore how different laboratories handled fluid biospecimens destined for EV analyses. We received 70 surveys from forty‐three different laboratories: 44% focused on plasma, 9% on serum and 16% on urine. The survey indicated that variability in preanalytical approaches reaches 94%. Moreover, in some cases, researchers had no access to all relevant preanalytical details of samples, with some sample aspects with potential impact on EV isolation/characterisation not coded within the current version of SPREC. Our study highlights the importance of working with common standard operating procedures (SOP) to control preanalytical conditions. The application of SPREC represents a suitable approach to codify and register preanalytical conditions. Integrating SPREC into the SOPs of laboratories/biobanks will provide a valuable source of information and constitute an advance for EV research by improving reproducibility and credibility.

Pre-analytical aspects in metabolomics of human biofluids - sample collection, handling, transport, and storage.

Metabolomics is the field of omics research that offers valuable insights into the complex composition of biological samples. It has found wide application in clinical diagnostics, disease investigation, therapy prediction, monitoring of treatment efficiency, drug discovery, or in-depth analysis of sample composition. A suitable study design constitutes the fundamental requirements to ensure robust and reliable results from the study data. The study design process should include a careful selection of conditions for each experimental step, from sample collection to data analysis. The pre-analytical variability that can introduce bias to the subsequent analytical process, decrease the outcome reliability, and confuse the final results of the metabolomics research, should also be considered. Herein, we provide key information regarding the pre-analytical variables affecting the metabolomics studies of biological fluids that are the most desirable type of biological samples. Our work offers a practical review that can serve and guide metabolomics pre-analytical design. It indicates pre-analytical factors, which can introduce artificial data variation and should be identified and understood during experimental design (through literature overview or analytical experiments).

Prädiktive Immunzytochemie beim nicht-kleinzelligen Lungenkarzinom

Die Immunchemie ist eine zeit-, tumorproben- und kosteneffiziente Methode zur Untersuchung prädiktiver Biomarker bei fortgeschrittenen nicht-kleinzelligen Lungenkarzinomen (NSCLC). Die Immunhistochemie (IHC) an Formalin-fixiertem, Paraffin-eingebettetem (FFPE) Tumorgewebe hat sich für den Nachweis der PD-L1-Expression sowie für die ALK-, ROS1- und neuerdings auch für die NTRK-Untersuchung bewährt. Zytologische Proben als Quelle für prädiktive Markeranalysen sind sehr wichtig, da bis zu 40 % aller NSCLC rein zytologisch diagnostiziert werden.Trotz der etablierten Rolle der Zytologie in der Lungenkarzinomdiagnostik wurden keine kommerziellen IHC-Assays für zytologische Proben validiert.Die prädiktive Immunzytochemie (ICC) ist am einfachsten an FFPE-Zellblöcken (CB) durchzuführen, da für FFPE-Histologie standardisierte Protokolle verwendet werden können. CB sind jedoch nicht immer verfügbar.Nicht als CB verarbeitete zytologische Präparate sind weniger standardisiert als histologische Präparate und weisen eine erhebliche präanalytische Variabilität auf. Daher ist eine strenge zytologiespezifische Optimierung, Validierung und Qualitätskontrolle von ICC-Protokollen erforderlich. Unter dieser Voraussetzung ist die prädiktive ICC, die in der Regel an Papanicolaou-gefärbten Zytologien durchgeführt wird, robust und zuverlässig. Dieses wertvolle zytologische Material sollte für prädiktive Biomarkeranalysen genutzt werden, um Patientinnen und Patienten nicht dem unnötigen Risiko einer erneuten Probenentnahme auszusetzen. Diese Übersichtsarbeit beleuchtet präanalytische, analytische und postanalytische Aspekte, die ICC-Ergebnisse beeinflussen können, und fasst die veröffentlichten Daten zur prädiktiven ICC für PD-L1, ALK und ROS1 bei NSCLC zusammen.

Analytical and pre-analytical aspects of neurofilament light chain determination in biological fluids

Analytical and pre-analytical aspects of neurofilament light chain determination in biological fluids

Preanalytical features of the determination of circulating microRNAs as new specific biomarkers of the body’s response to physical activity

MicroRNAs are small non-coding single-stranded RNAs, 18 to 25 nucleotides long, they regulate gene expression at the post-transcriptional level through specific binding to the target mRNA, leading to its degradation. In recent decades, the development of technologies for determining the expression profiles of miRNAs has become an important part of research projects, and the role of miRNAs as potential highly informative molecular biomarkers of various physiological and pathological processes in the body is actively explored by the scientific community. In particular, physical activity is an important modifying factor for circulating miRNAs. Unlike classical blood biochemical parameters, which can change over time depending on the temperature and storage conditions of the sample, microRNAs remain stable during storage and even after multiple freeze-thaw cycles, which makes them an attractive and easily accessible target for detection. However, the determination of the microRNA expression profile in clinical practice is still difficult due to the high heterogeneity of analytical procedures used for testing. In sports medicine, the preanalytical stage is especially important, since often the conditions for sampling are not standardized and can affect the analysis result. This review shows the role of miRNAs as new sensitive biomarkers of the effectiveness of the training process and regulators of the body’s response to physical activity, and also discusses some preanalytical aspects of the analysis of miRNA expression profiles.

Dried blood spots, an emerging tool for doping control

The presentation will afford information on the possibilities for dried blood spot (DBS) samples as a new tool supporting the control of the abuse of prohibited substances in the world of sport. Based on previous developments in other biological fields, recent years have seen increasing studies devoted to the possible application of dried drops of blood collected on different support materials as a promising tool to detect the presence of prohibited substances in the body of potential doping athletes. Among the studied analytes, there are examples of most of the groups of substances in the extensive prohibited list of the World Antidoping Agency (WADA). In general terms, the mere presence of most of those prohibited substances in sports drug testing showed by DBS could be enough to be considered as proof of external application to the body, and therefore susceptible to plausible sanctions. However, in other cases, the decision on potential positivity should be based on quantitative criteria (the so-called threshold substances), which is somehow more challenging for DBS. Pre-analytical aspects such exact volume of blood collected in the spots, hematocrit derived effects or homogeneity of the spots may be relevant. Examples of DBS testing for some of the more representative compounds (e.g. testosterone esters, stimulants, erythropoietin, human growth hormone, GH secretagogues, …) analyzed by chromatographic, mass spectrometric, electrophoretic or immunological methods will be presented. Also, considerations related to: – different potential matrices to collect the blood spots, such as cellulose based materials or polymers supports; – collection from finger prick or upper arm; – comparison of capillary vs. venous blood; – use of either manual or device-based collecting approaches; – insight into the degree of acceptability of those different possibilities by athletes, will be commented. Given the strict requirements for procedures in doping control (e.g. WADA and ISO 17025 accreditations), other aspects such as rigorous sample collection supervision (sample A for initial analysis and sample B for counter analysis), transport, analytical procedures and storage are to be aligned with the strictness for other usual samples such as urine or liquid blood in sport drug testing. To this end, a comprehensive Technical Document is being developed by a Working Group and to be approved by the WADA. In a first step, qualitative applications are included. A second step, addressed to threshold substances requiring quantitative estimations, is to be subsequently developed. DBS appear as an emerging new tool for prohibited drugs testing in sport, initially supporting more traditional procedures in the field such as urine or liquid blood analyses, but also susceptible of further developments by itself alone. Advantages in terms of easiness of collection, greater analyte stability and simple transport and storage, probably will overcome limitations such as the reduced amount of available sample and consequent limit in the number of substances detectable from a single DBS. In any case, its emerging applicability in sports drug testing will need strict technical controls.

What do we need to obtain high quality circulating tumor DNA (ctDNA) for routine diagnostic test in oncology? – Considerations on pre-analytical aspects by the IFCC workgroup cfDNA

The analysis of circulating cell free DNA is an important tool for the analysis of tumor resistance, tumor heterogeneity, detection of minimal residual disease and detection of allograft rejection in kidney or heart transplant patients. The proper use of this technique is important, and starts with considering pre-analytic aspects. The current paper addresses some important technical considerations to ensure the proper and harmonized use of cfDNA techniques.

Fluoropyrimidine chemotherapy: recommendations for DPYD genotyping and therapeutic drug monitoring of the Swiss Group of Pharmacogenomics and Personalised Therapy.

Fluoropyrimidines (FPs), mainly 5-fluorouracil (5-FU) and its oral prodrug capecitabine (Cap), remain the backbone of the treatment of many different solid tumors. Despite their broad use in clinical routine, 10–40% of patients experience severe, and in rare cases (0.2–0.5%) even lethal, FP-related toxicity in early chemotherapy cycles. Today, there is a plethora of evidence that genetic variants in the gene encoding for the 5-FU catabolising enzyme dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) are predictive of severe FP-related toxicities, and international clinical practice recommendations for DPYD genotype-guided FP dosing and therapeutic drug monitoring (TDM) are available. In spite of this strong evidence and DPYD genotyping becoming standard practice in other countries, it is has not been widely adopted in Switzerland to date. Here, we discuss current guidelines on genotype-guided FP dosing and TDM, and propose recommendations tailored to the situation in Switzerland to facilitate their clinical uptake for the further individualisation of FP chemotherapy. We recommend preemptive testing of four DPYD variants (c.1905+1G>A (rs3918290), c.1679T>G (rs55886062), c.2846A>T (rs67376798) and c.1129-5923C>G (rs75017182, c.1236G>A/HapB3)) in patients with an indication for FP-based chemotherapy, with the costs reimbursed through the compulsory health insurance in Switzerland. Carriers of these variants (6.5% in the Swiss population) have a 40–50% risk of developing severe early-onset toxicity when treated with standard FP doses. In these patients, we therefore recommend the use of a reduced starting dose, based on a dose adjustment scheme provided herein. Furthermore, we recommend the use of infusional 5-FU in patients with a DPYD risk genotype in order to enable TDM-based dose escalation. Only if the use of an infusional 5-FU regimen is not feasible should a slow titration of Cap, starting with the recommended reduced dose and basing further doses on monitoring of toxicity, be considered. Given that several studies have shown that TDM in 5-FU treatment improves not only the therapy’s safety, but potentially also its efficacy, we also include detailed TDM-based dosing guidelines and discuss the pre-analytical aspects of 5-FU TDM.

Recommendations and Best Practices for Standardizing the Pre-Analytical Processing of Blood and Urine Samples in Metabolomics.

Metabolomics can be significantly influenced by a range of pre-analytical factors, such as sample collection, pre-processing, aliquoting, transport, storage and thawing. This therefore shows the crucial need for standardizing the pre-analytical phase with the aim of minimizing the inter-sample variability driven by these technical issues, as well as for maintaining the metabolic integrity of biological samples to ensure that metabolomic profiles are a direct expression of the in vivo biochemical status. This review article provides an updated literature revision of the most important factors related to sample handling and pre-processing that may affect metabolomics results, particularly focusing on the most commonly investigated biofluids in metabolomics, namely blood plasma/serum and urine. Finally, we also provide some general recommendations and best practices aimed to standardize and accurately report all these pre-analytical aspects in metabolomics research.

PREDICT: a checklist for preventing preanalytical diagnostic errors in clinical trials.

Although the importance of guaranteeing a high level of preanalytical quality in routine diagnostic testing has already been largely acknowledged over the past decades, minor emphasis is currently being placed on the fact that accurate performance and standardization of many preanalytical activities are also necessary prerogatives of clinical trials. Reliable evidence exists that clear indications on how to manage the different preanalytical steps are currently lacking in many clinical trials protocols, nor have detailed authoritative documents been published or endorsed on this matter to the best of our knowledge. To fill this gap, the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for Preanalytical Phase (WG-PRE) will provide here a specific checklist for preventing preanalytical diagnostic errors in clinical trials (PREDICT), especially focused on covering the most important preanalytical aspects of blood sample management in clinical studies, and thus encompassing test selection, patient preparation, sample collection, management and storage, sample transportation, as well as specimen retrieval before testing. The WG-PRE members sincerely hope that these recommendations will provide a useful contribution for increasing the success rate in clinical trials.

Preanalytical aspects of investigations of prostate-specific antigen in distillated and deionized water extracts of semen in forensic samples

Preanalytical aspects of investigations of prostate-specific antigen in distillated and deionized water extracts of semen in forensic samples

Therapeutic drug monitoring (TDM) of 5-fluorouracil (5-FU): new preanalytic aspects.

Background 5-Fluorouracil (5-FU) is frequently used for the treatment of gastrointestinal tumors. The pharmacological effect of 5-FU is influenced by genetic polymorphisms as well as differently dosed regimens. Currently, 5-FU is generally administered as a continuous infusion via an implanted port system using a body surface area (BSA)-based dose calculation. In order to optimize treatment, the area under the curve (AUC) can be estimated to allow for individual dose adjustment. A 5-FU AUC range between 20 and 30 [mg×h×L] is recommended. The aim of the current study was to assess if blood for AUC analysis could also be drawn at the side where the port system had been placed. Methods We collected EDTA blood samples of patients receiving infusional 5-FU simultaneously from different sampling points (right/left cubital vein). 5-FU concentrations were measured in a steady-state equilibrium based on nanoparticle immunoassay (My5-FU; Saladax). Results A total of 39 patients took part in this study. About half of the patients did not reach the target 5-FU concentration window (37% were under- and 16% of the patients were overdosed). Calculated median AUC was 23.3 for the right arm (range 5.8-59.4) and a median of 23.4 for the left arm (range 5.3-61.0). AUC values showed no difference between right compared to left arms (p=0.99). Conclusions In all, these results confirm that a high percentage of patients are not treated with 5-FU doses reaching suggested AUC levels of 20-30. The location of venepuncture, however, had no impact on the results of plasma 5-FU concentration.

Investigating the Role of Mitochondria in Type 2 Diabetes - Lessons from Lipidomics and Proteomics Studies of Skeletal Muscle and Liver.

Mitochondrial dysfunction is discussed as a key player in the pathogenesis of type 2 diabetes mellitus(T2Dm), a highly prevalent disease rapidly developing as one of the greatest global health challenges of this century. Data however about the involvement of mitochondria, central hubs in bioenergetic processes, in the disease development are still controversial. Lipid and protein homeostasis are under intense discussion to be crucial for proper mitochondrial function. Consequently proteomics and lipidomics analyses might help to understand how molecular changes in mitochondria translate to alterations in energy transduction as observed in the healthy and metabolic diseases such as T2Dm and other related disorders. Mitochondrial lipids integrated in a tool covering proteomic and functional analyses were up to now rarely investigated, although mitochondrial lipids might provide a possible lynchpin in the understanding of type 2 diabetes development and thereby prevention. In this chapter state-of-the-art analytical strategies, pre-analytical aspects, potential pitfalls as well as current proteomics and lipidomics-based knowledge about the pathophysiological role of mitochondria in the pathogenesis of type 2 diabetes will be discussed.

Sample transportation – an overview

Transportation of blood samples is a major part of the preanalytical pathway and can be crucial in delaying laboratory results to the clinicians. A variety of aspects however makes sample transportation a complex, challenging and often overlooked task that needs thorough planning and dedicated resources. The purpose of this review is to outline the options available for this task and to emphasize the preanalytical aspects that need consideration in this process, e.g. performance specifications for sample transportation as stated in ISO standards 15189 and 20658, quality control of automated transportation systems, monitoring of sample integrity parameters and temperature surveillance in general and for external samplers in particular. All these are tasks that the laboratory must assure on a daily basis in terms of continuous quality control, and simultaneously the laboratory must remain alert to alterations in clinical demands (sample frequency, turn-around-times) and new regulations within this area (e.g. the recent General Data Protection Regulation from the EU).

Preanalytical aspects on short- and long-term storage of serum and plasma.

Following an ordered clinical chemistry plasma/serum test, ideally the venous blood specimen is adequately collected at a health care facility, then swiftly transported to and readily handled, analyzed and sometimes interpreted at a clinical chemistry laboratory followed by a report of the test result to the ordering physician to finally handle the result. However, often there are practical as well as sample quality reasons for short- or long-term storage of samples before and after analysis. If there are specific storage needs, the preanalytical handling practices are specified in the laboratory's specimen collection instructions for the ordered test analyte. Biobanking of specimens over a very long time prior to analysis includes an often neglected preanalytical challenge for preserved quality of the blood specimen and also involves administrative and additional practical handling aspects (specified in a standard operating procedure - SOP) when demands and considerations from academic, industry, research organizations and authorities are included. This short review highlights some preanalytical aspects of plasma/serum short- and long- term storage that must be considered by clinicians, laboratory staff as well as the researchers.

Validated measurements of microbial loads on environmental surfaces in intensive care units before and after disinfecting cleaning.

Preanalytic aspects can make results of hygiene studies difficult to compare. Efficacy of surface disinfection was assessed with an evaluated swabbing procedure. A validated microbial screening of surfaces was performed in the patients' environment and from hands of healthcare workers on two intensive care units (ICUs) prior to and after a standardized disinfection procedure. From a pure culture, the recovery rate of the swabs for Staphylococcus aureus was 35%-64% and dropped to 0%-22% from a mixed culture with 10-times more Staphylococcus epidermidis than S. aureus. Microbial surface loads 30min before and after the cleaning procedures were indistinguishable. The quality-ensured screening procedure proved that adequate hygiene procedures are associated with a low overall colonization of surfaces and skin of healthcare workers. Unchanged microbial loads before and after surface disinfection demonstrated the low additional impact of this procedure in the endemic situation when the pathogen load prior to surface disinfection is already low. Based on a validated screening system ensuring the interpretability and reliability of the results, the study confirms the efficiency of combined hand and surface hygiene procedures to guarantee low rates of bacterial colonization.

Preanalytical issues that may cause misdiagnosis in haemophilia and von Willebrand disease.

von Willebrand disease (VWD) and haemophilia represent common inherited or acquired bleeding disorders, but many laboratories and clinicians continue to struggle with their diagnosis or exclusion. Difficulties in achieving a correct diagnosis or exclusion of VWD or haemophilia might be due to analytical issues. Sometimes assays may generate a wrong result (ie an analytical error) or may have limitations in their dynamic range of measurement and/or their level of low analytical sensitivity. Less well recognized is the influence of preanalytical issues on the diagnosis of VWD or haemophilia. Therefore, this narrative review aims to provide an overview of some important preanalytical aspects that may affect the diagnosis of VWD or haemophilia, as well as a range of solutions that may help in mitigating or abrogating their influence. The review includes discussion of the more commonly noted preanalytical issues, such as haemolysis/icterus/lipaemia, and sample collection, processing and transport. However, we also extensively discuss other less well-recognized preanalytical issues, including clinical requests, anticoagulants and anticoagulant therapy, and laboratory test choices to name a few.

Measuring zinc on the Roche cobas c502 analyzer-Validation, comparison, and pre-analytic aspects.

Characterization of an in vitro diagnostic zinc assay (LT-SYS) on a Roche cobas c502 analyzer and evaluation of the influence of pre-analytic factors on zinc concentration measurements. Imprecision, bias, linearity, limit of blank (LoB), and limit of detection (LoD) were established and method comparisons were performed based on the respective CLSI guidelines. The influence of time elapsed until analysis, the usage of a pneumatic tube delivery system (PTDS) and of special trace element sample tubes was evaluated as well. Estimates of imprecision ranged from 0.9% to 5.0% and bias was low with 1.3% and 1.5% deviation from target value. Linearity was met for the measuring range of 1.15-34.7μmol/L (7.51-226.9μg/dL), LoB and LoD were 0.17μmol/L (1.11μg/dL) and 0.73μmol/L (4.77μg/dL) respectively. The method comparison revealed an average deviation of 8.44% (y=0.542+1.036x). Plasma samples had 7.3% higher zinc values than serum samples on the average. Zinc values of uncentrifuged serum and plasma samples increased 20% in 8hours, while after centrifugation no significant increase could be detected. Usage of PTDS increased zinc values by 17% and usage of trace element sample tubes showed no advantage over normal ones. The LT-SYS zinc assay showed a fully acceptable performance with good degrees of imprecision and bias, no deviation from linearity and both a very low LoB and LoD. Samples for zinc analysis should be centrifuged timely and transport over PTDS should be avoided.