Preanalytical Errors in Clinical Laboratory Testing at a Glance: Source and Control Measures.

The accuracy of diagnostic results in clinical laboratory testing is paramount for informed healthcare decisions and effective patient care. While the focus has traditionally been on the analytical phase, attention has shifted towards optimizing the preanalytical phase due to its significant contribution to total laboratory errors. This review highlights preanalytical errors, their sources, and control measures to improve the quality of laboratory testing. Blood sample quality is a critical concern, with factors such as hemolysis, lipemia, and icterus leading to erroneous results. Sources of preanalytical errors encompass inappropriate test requests, patient preparation lapses, and errors during sample collection, handling, and transportation. Mitigating these errors includes harmonization efforts, education and training programs, automated methods for sample quality assessment, and quality monitoring. Collaboration between laboratory personnel and healthcare professionals is crucial for implementing and sustaining these measures to enhance the accuracy and reliability of diagnostic results, ultimately improving patient care.

Pre-Analytical Variables Influencing Stability of Blood-Based Biomarkers of Neuropathology.

BACKGROUND: Sample collection and preanalytical protocols may significantly impact the results of large-scale epidemiological studies incorporating blood-based biomarkers of neuropathology. OBJECTIVE: To evaluate the stability and assay variability of several blood-based biomarkers of neuropathology for common preanalytical conditions. METHODS: We collected serum and plasma samples from 41 participants and evaluated the effect of processing delay of up to 72 h when stored at 4∘C, three freeze-thaw cycles, and a combination of 48-h processing delay when stored at 4∘C and three freeze-thaw cycles on biomarker stability. Using the Simoa assay (Quanterix Inc.), we measured amyloid-ß 40 (Aß40), amyloid-ß 42 (Aß42), neurofilament light (NfL), glial fibrillary acidic protein (GFAP), and phosphorylated tau 181 (p-tau-181). RESULTS: We found that Aß40 and Aß42 levels significantly decreased after a 24-h processing delay in both plasma and serum samples, and a single freeze-thaw cycle (p < 0.0001). Nevertheless, serum Aß42/40 ratio remained stable with a processing delay up to 48 h while plasma Aß42/40 ratio showed only small but significant increase with a delay up to 72 h. Both plasma and serum GFAP and NfL levels were only modestly affected by processing delay and freeze-thaw cycles. Plasma p-tau-181 levels notably increased with a 24-, 48-, and 72-h processing delay, but remained stable in serum. Intra-individual variation over two weeks was minimal for all biomarkers and their levels were substantially lower in serum when compared with plasma. CONCLUSION: These results suggest that standardizing preanalytical variables will allow robust measurements of biomarkers of neuropathology in population studies.

Auto-validation of Routine Coagulation/Hemostasis Assays with Reflex Testing of Abnormal Test Results.

Hemostasis laboratories play a crucial role in the diagnosis and treatment of individuals with bleeding or thrombotic disorders. Routine coagulation assays, including the prothrombin time (PT)/international normalized ratio (INR), and activated partial thromboplastin time (APTT), are used for various purposes. These include as a screen of hemostasis function/dysfunction (e.g., possible factor deficiency) and for monitoring of anticoagulant therapy, such as vitamin K antagonists (PT/INR) and unfractionated heparin (APTT). Clinical laboratories are also under increasing pressure to improve services, especially response (test turnaround) times. There is also a need for laboratories to try to reduce error rates and for laboratory networks to standardize/harmonize processes and policies. Accordingly, we describe our experience with the development and implementation of automated processes for reflex testing and validation of routine coagulation test results. This has been implemented in a large pathology network compromising 27 laboratories and is under consideration for expansion to our larger network (of 60 laboratories). These rules have been custom-built within our laboratory information system (LIS), perform reflex testing of abnormal results, and fully automate the process of routine test validation for appropriate results. These rules also permit adherence to standardized pre-analytical (sample integrity) checks, automate reflex decisions, automate verification, and provide an overall alignment of network practices in a large network of 27 laboratories. In addition, the rules enable clinically significant results to be quickly referred to hematopathologists for review. We also documented an improvement in test turnaround times, with savings in operator time and thus operating costs. Finally, the process was generally well received and determined to be beneficial for most laboratories in our network, in part identified by improved test turnaround times.

Post-analytical Issues in Hemostasis and Thrombosis Testing: An Update.

There are typically three phases identified as contributing to the total testing process. The preanalytical phase starts with the clinician and the patient, when laboratory testing is being considered. This phase also includes decisions about which tests to order (or not), patient identification, blood collection, blood transport, sample processing, and storage to name a few. There are many potential failures that may occur in this preanalytical phase, and these are the topic of another chapter in this book. The second phase, the analytical phase, represents the performance of the test, which is essentially covered in various protocols in this book and the previous edition. The third is the post-analytical phase, which is what occurs after sample testing, and is the topic of the current chapter. Post-analytical issues are generally related to reporting and interpretation of test results. This chapter provides a brief description of these events, as well as guidance for preventing or minimizing post-analytical issues. In particular, there are several strategies for improved post-analytical reporting of hemostasis assays, with this providing the final opportunity to prevent serious clinical errors in patient diagnosis or management.

Blood Collection Processing and Handling for Plasma and Serum Proteomics.

The plasma and serum proteome has enormous potential as a tool for understanding the health of a number of physiological systems. Despite this potential, the use of plasma and serum proteomics clinically and for research is limited, and there are no strict guidelines on how samples should be collected and prepared for proteomic analysis. Given the sensitivity of proteomic analysis, there are a number of pre-analytical variables that should be considered and determined prior to undertaking proteomics-based methodologies.In this chapter, we provide an example of a blood processing protocol and highlight major considerations for pre-analytical variables involving the collection, processing, and handling of blood samples for plasma and serum proteomics. We provide comprehensive notes on aspects of the protocol that must be considered before commencing sample collections for a proteomic study as well as a thorough checklist to be used when designing new proteomic studies.

The effect of pre-analytical variables on downstream application and data analysis of human endometrial biopsies.

STUDY QUESTION: What are the effects of pre-analytical variables on the downstream analysis of patient-derived endometrial biopsies? SUMMARY ANSWER: There are distinct differences in the protein levels of the master regulator of oxygen homeostasis, hypoxia-inducible factor-1-alpha (HIF1α), and the protein and mRNA levels of three related genes, carbonic anhydrase 9 (CA9), vascular endothelial growth factor A (VEGFA) and progesterone receptor (PR) in human endometrial biopsies, depending on the pre-analytical variables: disease status (cancer vs benign), timing of biopsy (pre- vs post-hysterectomy) and type of biopsy (pipelle vs full-thickness). WHAT IS KNOWN ALREADY: Patient-derived biopsies are vital to endometrial research, but pre-analytical variables relating to their collection may affect downstream analysis, as is evident in other tissues. STUDY DESIGN SIZE DURATION: A prospective observational study including patients undergoing hysterectomy for endometrial cancer (EC) or benign indications was conducted at a large tertiary gynaecological unit in the UK. Endometrial biopsies were obtained at different time points (pre- or post-hysterectomy) using either a pipelle endometrial sampler or as a full-thickness wedge biopsy. PARTICIPANTS/MATERIALS SETTING METHODS: The changes in HIF1α, CA9, VEGFA and PR protein levels were measured by semi-quantitative analysis of immunostaining, and the expression levels of three genes (CA9, VEGFA and PR) were investigated by quantitative real-time PCR, in endometrial biopsies from 43 patients undergoing hysterectomy for EC (n = 22) or benign gynaecological indications (n = 21). MAIN RESULTS AND THE ROLE OF CHANCE: An increase in HIF1α immunostaining was observed in EC versus benign endometrium (functionalis glands) obtained pre-hysterectomy (P < 0.001). An increase in CA9 immunostaining was observed in EC versus benign endometrial functionalis glands at both pre- and post-hysterectomy time points (P = 0.03 and P = 0.003, respectively). Compared with benign endometrial pipelle samples, EC samples demonstrated increased mRNA expression of CA9 (pre-hysterectomy P < 0.001, post-hysterectomy P = 0.008) and VEGFA (pre-hysterectomy P = 0.004, post-hysterectomy P = 0.002). In benign uteri, HIF1α immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.009), VEGFA immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.01) and VEGFA mRNA levels (P = 0.008) were increased in matched post-hysterectomy versus pre-hysterectomy samples. Similarly, in EC, an increase in VEGFA immunoscores (epithelial and stromal) and VEGFA mRNA expression was observed in the matched post-hysterectomy versus pre-hysterectomy biopsies (P = 0.008, P = 0.004 and P = 0.018, respectively). Full-thickness benign post-hysterectomy endometrial biopsies displayed increased VEGFA (P = 0.011) and PR (P = 0.006) mRNA expression compared with time-matched pipelle biopsies. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This descriptive study explores the effect of pre-analytical variables on the expression of four proteins and three hypoxia-related genes in a limited number of endometrial biopsies from patients with EC and benign controls. Due to the small number, it was not possible to investigate other potential variables such as menstrual cycle phase, region-specific differences within the endometrium, grade and stage of cancer, and surgical technicalities. WIDER IMPLICATIONS OF THE FINDINGS: Careful consideration of the effects of these pre-analytical variables is essential when interpreting data relating to human endometrial biopsies. A standardized approach to endometrial tissue collection is essential to ensure accurate and clinically transferrable data. STUDY FUNDING/COMPETING INTERESTS: The authors have no conflicts of interest to declare. The work included in this manuscript was funded by Wellbeing of Women project grants RG1073 and RG2137 (D.K.H.), Wellbeing of Women Entry-Level Scholarship ELS706 and Medical Research Council MR/V007238/1 (A.M./D.K.H.), Liverpool Women's Hospital Cancer Charity (M.A.) and University of Liverpool (L.B., L.R. and E.N.).

Determining the optimal storage time and temperature for performing platelet function assays and global hemostasis assays.

Platelet function assays and global haemostasis assays are essential in diagnosing bleeding tendencies, with light transmission aggregometry (LTA) as golden standard. The Multiple Electrode Aggregation (Multiplate), platelet function assay (PFA) and rotational thromboelastometry (ROTEM) are mostly used as whole-blood screening tests. Currently, patients have to travel to specialized laboratories to undergo these tests, since specific expertise is required. Pre-analytical variables, like storage time and temperature during transport, are still considered to be the most vulnerable part of the process and may lead to discrepancies in the test results. We aim to give a first impression on the stability of blood samples from healthy volunteers during storage and investigate the effect of storage time (1, 3, 6 and 24 hours) and temperature (4°C, room temperature and 37°C) on the Multiplate, PFA, ROTEM and LTA test results. Our data indicated that, for the PFA, whole blood can be stored for 3 hours at room temperature. Whole blood used for the Multiplate and ROTEM can be stored for 6 hours of storage. For LTA, PRP and whole blood were stable up to 3 hours at 4°C or room temperature and 6 hours at room temperature, respectively.

A Physically Active Status Affects the Circulating Profile of Cancer-Associated miRNAs.

Circulating miRNAs are ideal diagnostics and prognostics biomarkers in cancer since altered levels of specific miRNAs have been associated to development/progression of several cancers. Physical activity is a recognized preventive strategy against several cancers, but it may also modify the baseline levels of cancer-associated miRNAs and, hence, may act as a confounding pre-analytical variable. This study aimed at understanding whether physical activity-dependent changes in cancer-associated circulating miRNAs profile could act as a confounding variable. A panel comprising 179 miRNAs was assayed in plasma from 20 highly trained and 10 sedentary men. RT-qPCR data were analyzed with the 2-2ΔΔCT methods and normalized on hsa-miR-320d, as determined by bioinformatics analysis. miRNAs associated with the diagnosis of the most prevalent cancers were considered. Only those miRNAs, relevantly associated with cancers, found ≥2-fold up- or downregulated in highly trained subjects compared to sedentary were disclosed. The results reveal that chronic physical activity determined modifications altering the baseline level of several cancer-associated miRNAs and, hence, their diagnostic and prognostic potential. In conclusion, based on our results, a physically active status emerges as an important pre-analytical variable able to alter the basal level of circulating miRNAs, and these alterations might be considered as potentially misleading the analytical output.

Pre-analytical variables in haemostasis: Findings from the United Kingdom National External Quality Assessment scheme for Blood Coagulation (UK NEQAS BC) haemolysis exercise.

INTRODUCTION: Haemolysis is considered one of the major contributors of nonconformities and sample rejection in coagulation testing. MATERIALS AND METHODS: Two lyophilized plasmas were distributed to 800 centres registered for prothrombin time (PT), activated partial thromboplastin time (APTT) and either Clauss fibrinogen or thrombin time (TT) in the UK NEQAS BC programme. The same pool of normal plasma was used to prepare both samples, to one of which red blood cell haemolysate was added to mimic haemolysis at 3 g/L haemoglobin concentration. Participants were asked to complete a questionnaire about their laboratory approach to dealing with haemolysed samples, including strategies used to deal with different levels of haemolysis. RESULTS: Results for tests performed did not show great differences between the two samples. It should be noted that artificially constructed haemolysed samples may not behave in the same way as patient samples (ie, may not be commutable). However, the possibility of carrying out a large multicentre study for detection of haemolysis was demonstrated. Inconsistency in practice was observed with 226/551 (41%) of centres indicated they reject haemolysed samples solely on visual checks, and 163 (30%) using initial visual checks with further sample rejection evaluation by analyser flags. Furthermore, 333 (72%) of centres indicated that the level of haemolysis affects sample rejection decisions, while 132 (28%) stated it did not. CONCLUSION: Variability of responses for dealing with haemolysed samples reflects a lack of clear consistency in the pre-analytical area of sample processing.

Effect of fasting status and other pre-analytical variables on quantitation of long-chain fatty acids in red blood cells.

Long-chain fatty acids (LCFAs), including omega-3 and omega-6 fatty acids, play essential roles in health maintenance and outcomes. Insufficient intake or the inability to absorb LCFAs from the diet can cause a number of health problems. Evaluation of fatty acid profiles in plasma, serum or red blood cells (RBCs) is routinely used to monitor patients at risk of developing deficiency. Quantitation of LCFAs in RBCs offers advantages over serum/plasma due to low intra-individual variability. Fatty acid composition in RBCs also reflects long-term dietary intake, providing additional information about the patient's nutritional status. However, the literature does not currently address the impact of pre-analytical factors (conditions of RBC collection, sample handling and short-term storage) on LCFA measurements. This study evaluated the effect of several anticoagulants, interferents, different storage conditions and fasting status on quantitation of the twenty-one most abundant LCFAs in RBCs by gas chromatography negative chemical ionization-mass spectrometry (GCNCI-MS). LCFA results were assessed quantitatively (nmol/mL) or as a percent of total. Most common tube types (containing citrate, sodium heparin or EDTA) were all appropriate for blood collection. Whole blood and lysed RBCs were stable at least 24 h at room temperature and up to 7 days refrigerated. Lysed RBCs were also stable for up to three freeze/thaw cycles. The presence of icterus or lipemia did not affect results. LCFAs concentrations in RBCs did not change ~4 h after high-fat intake when the lipid concentration in circulation reaches a peak, while plasma levels of most fatty acids increased up to 40% in response. In summary, RBCs are a reliable sample type for LCFA quantitation in the clinical laboratory. In contrast to plasma or serum, RBCs isolated from non-fasting, hemolyzed or lipemic whole blood specimens are all acceptable for testing. Therefore, RBCs might be a preferable sample type for evaluation of nutritional status of young pediatric patients and in patients with conditions associated with hemolytic anemia or hyperlipidemia.

Measurement of CSF core Alzheimer disease biomarkers for routine clinical diagnosis: do fresh vs frozen samples differ?

BACKGROUND: Cerebrospinal fluid (CSF) amyloid-beta (Aß) 42/40 ratio, threonine-181-phosphorylated-tau (p-tau), and total-tau (t-tau) represent core biomarkers of Alzheimer disease (AD). The recent availability of automated platforms has represented a significant achievement for reducing the pre-analytical variability of these determinations in clinical setting. With respect to classical manual ELISAs, these platforms give us also the possibility to measure any single sample and to get the result within approximately 30 min. So far, reference values have been calculated from measurements obtained in frozen samples. In this work, we wanted to check if the values obtained in fresh CSF samples differ from those obtained in frozen samples, since this issue is mandatory in routine diagnostic work. METHODS: Fifty-eight consecutive CSF samples have been analyzed immediately after lumbar puncture and after 1-month deep freezing (- 80 °C). As an automated platform, we used Lumipulse G600-II (Fujirebio Inc.). Both the fresh and the frozen aliquots were analyzed in their storage tubes. RESULTS: In fresh samples, a mean increase of Aß40 (6%), Aß42 (2%), p-tau (2%), and t-tau (4%) was observed as compared to frozen samples, whereas a slight decrease was observed for Aß42/Aß40 ratio (4%), due to the higher deviation of Aß40 in fresh samples compared to Aß42. These differences are significant for Aß40, Aß42/Aß40 ratio, p-tau, and t-tau. Nevertheless, the Aß42/Aß40 ratio showed a lower variability (smaller standard deviation of relative differences) with respect to Aß42. With respect to the AD profile according to the A/T/(N) criteria for AD diagnosis, no significant changes in classification were observed when comparing results obtained in fresh vs frozen samples. CONCLUSIONS: Small but significant differences have been found for Aß40, Aß42/Aß40 ratio, p-tau, and t-tau in fresh vs frozen samples. Importantly, these differences did not imply a modification in the A/T/(N) classification system. In order to know if different cutoffs for fresh and frozen samples are required, larger, multi-center investigations are needed.

Determining the maximal storage time of centrifuged citrated samples for performing add-on routine coagulation tests.

BACKGROUND: Several routine coagulation tests have been developed to give insight in the coagulation pathway. The laboratory diagnostical process consists of 3 phases, the pre-analytical, analytical and post-analytical phase; however, the pre-analytical phase is most sensitive to errors. The amount of time blood is stored, can affect the measurements of these coagulation tests and result in an incorrect conclusion. Therefore, we performed experiments to determine the maximal storage time, centrifuged blood samples can be reliably measured. METHODS: Citrated whole blood from hospital patients, who were tested for routine coagulation, was collected in 2.7 mL citrate tubes. These whole blood samples were centrifuged and the plasma was stored on top of the cell pellet at room temperature. After 2 h, 4 h, 6 h, 12 h and 24 h, the prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen concentration, antithrombin activity, D-dimer concentration and thrombin time were measured using Sysmex CS2100 coagulation analysers. RESULTS AND CONCLUSION: Analytical evaluation of routine coagulation tests resulted in various significant differences and large variations between the various time intervals. Our results indicated that the PT and INR can be measured up till 24 h of storage. Centrifuged blood for measuring the fibrinogen concentration, antithrombin activity, D-dimer concentration and thrombin time can be stored up to 4 h, while 2 h of storage might already be too long for obtaining reliable aPTT measurements.

Impact of specific preclinical variables on coagulation biomarkers in cancer-associated thrombosis.

Coagulation biomarkers are being actively studied for their diagnostic and prognostic value in patients with venous thromboembolism and cancer, as well as in the study of pathogenic mechanisms between cancer and thrombosis. For the results of such studies to be accurate and reproducible, attention must be paid to minimize sources of error in all phases of testing. The pre-analytical phase of laboratory testing is known to be fraught with the majority of errors. Coagulation testing is particularly susceptible to conditions during collection, processing, transport and storage of specimens which can lead to clinically significant errors in results. In addition, changes in pre-analytical conditions can impact different biomarkers differently. Therefore, research studies investigating coagulation biomarkers must carefully standardize not just the analytical phase, but also the pre-analytical phase of testing to ensure accuracy and reliability. We briefly review the impact of pre-analytical conditions on coagulation testing in general, and on specific biomarkers in cancer and thrombosis. In addition, we provide recommendations to reduce pre-analytical errors by developing and sharing standard operating procedures that specifically target standardization of methodologies for collecting specimens and measuring current and emerging coagulation biomarkers in cancer studies.

Targets, pitfalls and reference materials for liquid biopsy tests in cancer diagnostics.

Assessment of cell free DNA (cfDNA) and RNA (cfRNA), circulating tumor cells (CTC) and extracellular vesicles (EV) in blood or other bodily fluids can enable early cancer detection, tumor dynamics assessment, minimal residual disease detection and therapy monitoring. However, few liquid biopsy tests progress towards clinical application because results are often discordant and challenging to reproduce. Reproducibility can be enhanced by the development and implementation of standard operating procedures and reference materials to identify and correct for pre-analytical variables. In this review we elaborate on the technological considerations, pre-analytical variables and the use and availability of reference materials for the assessment of liquid biopsy targets in blood and highlight initiatives towards the standardization of liquid biopsy testing.

Pre-analytical variables evaluation in laboratory tests of patients attended at the Vitória da Conquista Central laboratory, Bahia, Brazil / Avaliação de variáveis pré-analíticas em exames laboratoriais de pacientes atendidos no Laboratório Central de Vitória da Conquista, Bahia, Brasil

ABSTRACT Introduction: Laboratory tests are intended to provide information necessary to clarify diagnoses or specific situations that cannot be elucidated by other means. Laboratory analyses are subject to several interferences. Even healthy individuals could suffer variations in laboratory tests due to biological factors, which may lead to misinterpretation of the results. Objective: Assess the pre-analytical variables in patients' laboratory tests at the Vitória da Conquista Central Laboratory, Bahia, Brazil. Material and method: This is a cross-sectional study with a descriptive approach and the use of a questionnaire as a research instrument. Data were collected through individual interviews, carried out at the laboratory. Results: The sample consisted of 425 patients, with female predominance (76.5%). Regarding patient instructions for laboratory tests, 94.6% had not been given any previous guidance, and 68.5% used some kind of medication, which could produce alterations in the exams. The use of the antihypertensive agents captopril, enalapril, hydrochlorothiazide or propranolol was associated with increased serum urea [odds ratio (OR) = 7.2, confidence interval (CI) = 95%, 1.87-27.58; p = 0.002], and smoking was associated with increased red blood cells count (OR = 3.7, CI = 95%, 0.86-15.75; p = 0.02). Conclusion: The findings indicate that pre-analytical variables remain an important focus for attention to the clinical laboratory. The correct data collection of patients' conditions is critical to support the post-analytical phase with useful information for the production of reliable exams and serve as a useful interpretation to diagnosis.

RESUMEN Introducción: Las pruebas de laboratorio tienen como objetivo proporcionar información necesaria a la aclaración de diagnósticos o situaciones específicas que no se pueden esclarecer por otros modos. Los análisis de laboratorio están sujetos a diversos interferentes. Incluso individuos sanos pueden presentar variaciones en las pruebas derivadas de factores que pueden crear equivocaciones en la interpretación de los resultados. Objetivo: Evaluar la influencia de variables preanalíticas en pruebas de laboratorio de pacientes atendidos en el Laboratório Central de Vitória da Conquista, Bahía, Brasil. Material y método: Estudio transversal con enfoque descriptivo y empleo de cuestionario como herramienta de investigación. Los datos fueron recogidos mediante entrevistas individuales, realizadas en el establecimiento investigado. Resultados: La muestra se compuso por 425 pacientes, con predominio de mujeres (76,5%). En cuanto a instrucciones para realización de las pruebas, el 94,6% de los pacientes no recibieron ningún tipo de orientación previa y el 68,5% utilizaban algún tipo de medicamento que puede producir alteraciones en pruebas. El uso de los antihipertensivos captopril, enalapril, hidroclorotiazida o propranolol estuvo asociado con el aumento de la urea sérica [oportunidad relativa (OR) = 7,2; intervalo de confianza (IC) = 95%, 1,87-27,58; p = 0,002]. El tabaquismo se ha asociado al aumento de eritrocitos (OR = 3,7; IC = 95%, 0,86-15,75; p = 0,02). Conclusión: Los hallazgos clínicos indican que variables preanalíticas son importante causa de atención para el laboratorio clínico. La correcta recogida de datos acerca de las condiciones de los pacientes es esencial para instrumentar la fase postanalítica con información relevante para la realización de pruebas fiables y que sean útiles para el proceso del diagnóstico.

RESUMO Introdução: Os exames laboratoriais têm como objetivo fornecer informações necessárias para o esclarecimento de diagnósticos ou situações específicas não passíveis de serem elucidadas por outros meios. As análises laboratoriais estão sujeitas a diversos interferentes. Mesmo indivíduos saudáveis podem apresentar variações nos exames decorrentes de fatores que podem gerar equívocos na interpretação dos resultados. Objetivos: Avaliar a influência de variáveis pré-analíticas em exames laboratoriais de pacientes atendidos no Laboratório Central do município de Vitória da Conquista, Bahia, Brasil. Material e método: Trata-se de um corte transversal com abordagem descritiva e utilização de questionário como instrumento de pesquisa. Os dados foram coletados por meio de entrevistas individuais, realizadas na instituição pesquisada. Resultados: A amostra foi composta por 425 pacientes, com predomínio de indivíduos do sexo feminino (76,5%). No que se refere a instruções para realização dos exames laboratoriais, 94,6% dos pacientes não receberam nenhum tipo de orientação prévia e 68,5% utilizavam algum tipo de medicamento que pode produzir alterações em exames. O uso dos anti-hipertensivos captopril, enalapril, hidroclorotiazida ou propranolol esteve associado ao aumento da ureia sérica [odds ratio (OR) = 7,2; intervalo de confiança (IC) = 95%, 1,87-27,58; p = 0,002]. O tabagismo associou-se ao aumento de eritrócitos (OR = 3,7; IC = 95%, 0,86-15,75; p = 0,02). Conclusão: Os achados indicam que variáveis pré-analíticas são importante fonte de atenção para o laboratório clínico. A correta coleta de dados acerca das condições dos pacientes é fundamental para instrumentar a fase pós-analítica com informações relevantes para a produção de exames fidedignos e que sejam úteis ao processo do diagnóstico.

In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue Biospecimen Research Working Group of the Spanish Biobank Network.

The purpose of the present work is to underline the importance of obtaining a standardized procedure to ensure and evaluate both clinical and research usability of human tissue samples. The study, which was carried out by the Biospecimen Science Working Group of the Spanish Biobank Network, is based on a general overview of the current situation about quality assurance in human tissue biospecimens. It was conducted an exhaustive review of the analytical techniques used to evaluate the quality of human tissue samples over the past 30 years, as well as their reference values if they were published, and classified them according to the biomolecules evaluated: (i) DNA, (ii) RNA, and (iii) soluble or/and fixed proteins for immunochemistry. More than 130 publications released between 1989 and 2019 were analysed, most of them reporting results focused on the analysis of tumour and biopsy samples. A quality assessment proposal with an algorithm has been developed for both frozen tissue samples and formalin-fixed paraffin-embedded (FFPE) samples, according to the expected quality of sample based on the available pre-analytical information and the experience of the participants in the Working Group. The high heterogeneity of human tissue samples and the wide number of pre-analytic factors associated to quality of samples makes it very difficult to harmonize the quality criteria. However, the proposed method to assess human tissue sample integrity and antigenicity will not only help to evaluate whether stored human tissue samples fit for the purpose of biomarker development, but will also allow to perform further studies, such as assessing the impact of different pre-analytical factors on very well characterized samples or evaluating the readjustment of tissue sample collection, processing and storing procedures. By ensuring the quality of the samples used on research, the reproducibility of scientific results will be guaranteed.

Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles.

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids ("liquid biopsies"). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.

Towards a unified protocol for handling of CSF before ß-amyloid measurements.

BACKGROUND: Widespread implementation of Alzheimer's disease biomarkers in routine clinical practice requires the establishment of standard operating procedures for pre-analytical handling of cerebrospinal fluid (CSF). METHODS: Here, CSF collection and storage protocols were optimized for measurements of ß-amyloid (Aß). We investigated the effects of (1) storage temperature, (2) storage time, (3) centrifugation, (4) sample mixing, (5) blood contamination, and (6) collection gradient on CSF levels of Aß. For each study participant, we used fresh CSF directly collected into a protein low binding (LoB) tube that was analyzed within hours after lumbar puncture (LP) as standard of truth. Aß42 and Aß40 were measured in de-identified CSF samples using EUROIMMUN and Mesoscale discovery assays. RESULTS: CSF Aß42 and Aß40 were stable for at least 72 h at room temperature (RT), 1 week at 4 °C, and 2 weeks at - 20 °C and - 80 °C. Centrifugation of non-blood-contaminated CSF or mixing of samples before the analysis did not affect Aß levels. Addition of 0.1-10% blood to CSF that was stored at RT without centrifugation led to a dose- and time-dependent decrease in Aß42 and Aß40, while Aß42/Aß40 did not change. The effects of blood contamination were mitigated by centrifugation and/or storage at 4 °C or - 20 °C. Aß levels did not differ between the first to fourth 5-ml portions of CSF. CONCLUSIONS: CSF can be stored for up to 72 h at RT, 1 week at 4 °C, or at least 2 weeks at either - 20 °C or - 80 °C before Aß measurements. Centrifugation of fresh non-blood-contaminated CSF after LP, or mixing before analysis, is not required. In case of visible blood contamination, centrifugation and storage at 4 °C or - 20 °C is recommended. After discarding the first 2 ml, any portion of up to 20 ml of CSF is suitable for Aß analysis. These findings will be important for the development of a clinical routine protocol for pre-analytical handling of CSF.

Development and implementation of an expert rule set for automated reflex testing and validation of routine coagulation tests in a large pathology network.

INTRODUCTION: Haemostasis laboratories play a critical role in diagnosis and treatment of individuals with bleeding or thrombotic disorders. Routine coagulation assays such as prothrombin time (PT)/ international normalized ratio (INR), and activated partial thromboplastin time (APTT), are used in monitoring of anticoagulant therapy, as provided for treatment/prevention of thromboembolic disease, and also inform on potential haemostasis dysfunction. Increasing pressure is applied on clinical laboratories to improve response (test turnaround) times, reduce error rates and standardize policies. To this end, we describe our experience with the development and implementation of an automated process for reflex testing and validation of routine coagulation test results in a large pathology network compromising 27 laboratories. METHODS: Custom-built expert rules were created to perform reflex testing and fully automate routine test validation. These rules were developed/implemented over a 15-month period, including 6 months for development/ testing and 9 months for training/implementation of >100 personnel at 27 sites. RESULTS: These rules have enabled adherence of standardized pre-analytical (sample integrity) checks, automated reflex decisions, automated verification and overall alignment of network practices. In addition, clinically significant results are immediately referred to haematologists. We report an improvement in test turnaround times, also reflecting savings in operator time. CONCLUSION: The process was generally well received and generally beneficial to most laboratories in the network.