Preanalytical Issues Research Articles

Platelet Function Tests.

Traditionally developed for diagnosis of bleeding disorders, platelet function assays have become increasingly used in basic research on platelet physiology, in phenotype-genotype associations in bleeding disorders, in drug development as surrogate endpoints of efficacy of new antiplatelet therapy, and to an extent, in the monitoring of antiplatelet therapy in clinical practice to predict thrombotic and bleeding risk. A multiplicity of platelet function assays is available to measure the level of platelet activity in various settings. These include assays that are restricted to a specialized laboratory as well as point-of-care instruments meant to investigate platelet function at patient bedside. Unlike tests that determine a defined quantity or measurement of a clinical biomarker (e.g., cholesterol or blood pressure), platelet function testing assesses the dynamics of living cells, which immediately presents a series of unique problems to any laboratory or clinic. This article presents currently used platelet function assays and discusses important variables to take into account when performing these assays, including preanalytical issues and difficulties in interpreting platelet function test results.

HIL Interferences on Three Hemostasis Analyzers and Contribution of a Preanalytical Module for Routine Coagulation Assays

Preanalytical issues are a major part of routine coagulation laboratory errors. Automation in detection of preanalytical problems, including hemolysis, icterus and lipemia (HIL), improper tube fill volume, and undue clotting, has recently been implemented on specific hemostasis instruments. The aim of this study was to assess the added value of a new preanalytical module integrated into hemostasis analyzers compared to visual inspection of samples. The detection of preanalytical issues was performed by visual or manual inspection of the samples and by the new preanalytical module integrated on the ACL TOP 550. Additional tests were done to evaluate the interference of hemoglobin (Hb), bilirubin or triglycerides (TG). Plasma pools containing the interference substance were tested for routine coagulation assays on the STA-R Evolution, CS-5100, and ACL TOP 550. Visual or manual inspection detected statistically less samples with preanalytical issues than the new preanalytical module integrated on the ACL TOP 550 (3.5% vs. 6.6%, p < 0.001). The majority of the samples were rejected for poor filling. HIL interferences appeared on assays when the concentration of Hb, bilirubin or TG exceeded a certain threshold that was analyzer and reagent dependent. Automatic and standardized check of routine coagulation samples by ACL TOP 550 increased the accuracy and consistency in detection of preanalytical issues as compared to visual inspection only. The main advantages were the detection of insufficient filled tubes and icteric samples that are not detected visually.

Impact of Clotting Condition on the Measurement of Circulating MicroRNAs in Serum.

Circulating microRNA (miRNA) biomarkers have attracted attention as possible blood-based indicators of disease. However, pre-analytic technical issues including serum preparation remain problematic for the clinical translation of such technology. This study investigated the effect of decreased sample preparation time on miRNA measurement. Blood samples obtained from healthy donors were drawn into blood collection tubes with or without clot activator, and sera were obtained at different preparation time points. The expression levels of miR16 and miR21 were examined. The miR-16 expression levels were significantly higher in sera separated after 5 minutes compared to ones separated after 10 and 60 minutes, whereas no significant difference of miR21 expression was detected in association with the serum separation conditions. Serum miRNA expression levels should be interpreted carefully with consideration of the characteristics of collection tubes and clotting status of serum separation.

In-vitro hemolysis and its financial impact using different blood collection systems

Abstract Background: Hemolytic specimens are among the most challenging preanalytical issues in laboratory diagnostics. The type of blood collection tube in use is claimed to influence in vitro hemolysis. We aimed to examine this hypothesis and estimate the respective financial impact, evaluating routine blood samples from the past 4 years. Methods: A total of 47,820 hemolysis index (HI) values from five different time intervals (IV1–IV5) were compared against each other, representing the following tubes: IV1-Sarstedt Monovette; IV2-8 mL/16×100 mm Greiner BioOne (GBO) Vacuette; IV3/IV4-5 mL/16×100 mm GBO Vacuette; IV5-4.5 mL/13×75 mm GBO Vacuette. For estimation of the economic impact, material, personnel and analytical costs were calculated. Results: HI mean values in time interval IV2 were significantly higher than in all other intervals, while mean values amongst all other intervals were comparable. The number of moderately and severely hemolyzed samples increased with incrementing vacuum. Overall comparable costs between intervals IV1 and IV5 were €11,370, €14,045, €12,710, €11,213 and €8138 per 10,000 samples, respectively. Conclusions: Aspiration tubes and low vacuum tubes show comparable hemolysis rates. Increasing vacuum levels are associated with higher hemolysis rates. By decreasing in vitro hemolysis, financial savings up to €5907 per 10,000 samples could be gained.

National and international initiatives and approaches for the establishment of reference intervals in pediatric laboratory medicine

Abstract Thoroughly validated laboratory medicine reference intervals for children of all ages groups have been published increasingly during the last years. The aim of this review is to present a number of these studies and the various approaches to the generation of reference intervals. Population-based data obtained in large cohorts of healthy children in Germany, Northern Europe, North America, and selected other countries as well as patient-derived data collected in many areas of the world are discussed. Additionally, special research aspects such as reference intervals for preterm neonates, preanalytical issues, intraindividual variation of analytes, or follow-up studies that are covered in many of these studies are presented.

Biobanking for the future, how to prepare for the next generation of Next Generation Sequencing

The technology watch of the BBMRI-LPC 2015 will focus on the pre-analytical, ethical and data management issues that may prevent biobanks from providing samples that fully take advantage of the next generation of sequencing technology. Biobanks are a long term commitment and by preparing for the next generation of technology, biobanks provide researchers with immediate access to material for testing scientific theories that would otherwise take years or decades to evaluate.

Relevance of EDTA carryover during blood collection.

The order of draw is regarded as a preanalytical issue to prevent carryover of additives during blood collection. Our objective was to prove the theory of ethylenediaminetetraacetic acid (EDTA) carryover for a closed vacuum system and the influence of EDTA on concentrations of selected biomarkers. To test the carryover of EDTA, a blood collection with tripotassium EDTA (K3EDTA) and subsequent non-additive tubes was simulated using distilled water as substitute for blood. EDTA concentrations were measured by tandem mass spectrometry. Then we added increasing concentrations of EDTA to heparinized blood and measured routine biomarkers, thereby simulating a carryover of EDTA whole blood and pure EDTA, respectively. Additionally, we tested for EDTA contamination and biomarker alteration in samples collected from 10 healthy volunteers by a syringe with subsequent transfer into sample tubes. No EDTA contamination was detected in samples collected subsequent to a K3EDTA tube when adhering to guidelines of blood sampling. Magnesium, calcium, and potassium levels were altered by artificial K3EDTA whole-blood contamination as well as when adding 1 μL pure K3EDTA. Iron values were altered at EDTA concentrations of 4.4 mmol/L. All other parameters remained unaffected. A slight EDTA carryover was observed in syringe collection and subsequent transfer into EDTA and heparin tubes, however, without any biomarker alteration. An EDTA carryover during blood collection using a closed vacuum system is highly unlikely. Even if carryover of EDTA whole blood occurs, an absolute volume larger than 10 μL would be necessary to alter test results. However, contamination of samples with preloaded pure K3EDTA solution by severe neglect of current recommendations in blood collection may significantly alter testing results.

Peptidomics of Urine and Other Biofluids for Cancer Diagnostics

Cancer is a leading cause of death worldwide. The low diagnostic sensitivity and specificity of most current cancer biomarkers make early cancer diagnosis a challenging task. The comprehensive study of peptides and small proteins in a living system, known as "peptidomics," represents an alternative technological approach to the discovery of potential biomarkers for the assessment of a wide variety of pathologies. This review examines the current status of peptidomics for several body fluids, with a focus on urine, for cancer diagnostics applications. Several studies have used high-throughput technologies to characterize the peptide content of different body fluids. Because of its noninvasive collection and high stability, urine is a valuable source of candidate cancer biomarkers. A wide variety of preanalytical issues concerning patient selection and sample handling need to be considered, because not doing so can affect the quality of the results by introducing bias and artifacts. Optimization of both the analytical strategies and the processing of bioinformatics data is also essential to minimize the false-discovery rate. Peptidomics-based studies of urine and other body fluids have yielded a number of biomolecules and peptide panels with potential for diagnosing different types of cancer, especially of the ovary, prostate, and bladder. Large-scale studies are needed to validate these molecules as cancer biomarkers.

Currently Available Biomarkers and Strategies for the Validation of Novel Candidates for Neurochemical Dementia Diagnostics in Alzheimer’s Disease and Mild Cognitive Impairment

The number of people afflicted with Alzheimer’s disease (AD) and other types of dementing conditions has grown exponentially in the last decades. This review focuses on the diagnostic role of the classic cerebrospinal fluid (CSF) biomarkers of neurochemical dementia diagnostics (NDD) and critically discusses potential strategies for the development and validation of novel potential candidates. In some countries, NDD is already established as a routine diagnostic tool, used for the evaluation of patients with cognitive impairments. On the other hand, preanalytical and technical issues, partly discussed in this paper, prevent NDD from the general acceptance worldwide. Currently, two groups of biomarkers in the CSF are considered in NDD: amyloidβ(Aβ) peptides and Tau proteins, including the hyperphosphorylated forms of the latter (pTau). The analyses of these two groups of biomarkers can reveal pathologic alterations as early as twenty years before the onset of clinical symptoms. In mild cognitive impairment (MCI), NDD can reliably predict which individuals are at risk of converting to AD. The roles of biomarkers of amyloidβdeposition in the brain tissue (including the CSF concentration of Aβ42) and biomarkers of neurodegeneration (including the CSF concentrations of Tau/pTau proteins) are reflected in the currently proposed diagnostic criteria for AD and MCI.

Laboratory testing issues for protein C, protein S, and antithrombin.

Thrombophilia is a complex disease process, which clinically expresses as venous thrombosis. The presence of a genetic defect in one of the major contributing components (protein C [PC], protein S [PS], and antithrombin [AT]) to thrombophilia can be determined by clinical laboratory assays. However, understanding the limitations and problems associated with assays is paramount to an accurate analysis of the genetic status. This review will discuss the major analytical issues and provide recommendations for assaying PC, PS, and AT in plasma. Recommendations are also made about pre-analytical and postanalytical issues clinically affecting these assays.

Stability of coagulation parameters: review of available data

Samples for hemostasis testing are vulnerable to the effects of preanalytical variables and quality standards for sample processing and storage must be defined to insure accurate results. This article presents the available informations on these preanalytical issues.

Algorithmic Approaches to Hemostasis Testing

There are many unique issues that may make a pathologist's consultation helpful in hemostasis testing. Besides the rapidly expanding knowledge of both bleeding and thrombotic disorders and a wide test menu, hemostasis testing is very sensitive to preanalytical issues (hemolysis, fill volume, time, temperature, storage conditions) and the interference of many commonly prescribed drugs. The pathologist can serve an important role in the evaluation of a patient for a bleeding or thrombotic disorder. Using defined algorithms, hemostasis testing can proceed in a logical fashion and be reported using patient-specific comments that take into account clinical history and medication therapy. This approach can improve the diagnostic process, preventing misdiagnoses and leading to a decreased time to diagnosis and an improved utilization of laboratory resources.

Preanalytical Phase – an updated review of the current evidence

European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working group for preanalytical phase (WG-PRE) has recently launched a series of conferences on preanalytical phase. First such meeting was held in Parma (Italy) in the spring of 2011 and the second one took place in Zagreb (Croatia) during 1–2 March 2013. The Zagreb audience had the opportunity to enjoy a focused and challenging scientific program as well as to participate actively in interactive e-voting sessions. Speakers of the conference have kindly accepted the invitation to submit their review articles covering topics presented during the meeting. This Journal issue comprises a number of high quality review articles providing an updated overview of the current evidence on the various preanalytical issues. The first article in this series is presented by prof. Walter G. Guder who has prepared a historical overview of the milestones in the development of the key concepts in the field of preanalytical phase (1). Bowen and Remaley in their article provide an excellent update on the most important blood collection tube additives and their components which may affect laboratory test results (2). De Carli and her colleagues have compiled the existing evidence of healthcare workers risk of sharps injuries and subsequent infections and review respective prevention strategies (3). Several subsequent articles are dedicated to some preanalytical sources of interferences such as lipemia (4), physical activity (5) and medical contrast media (6). Delanghe and Speeckaert have reviewed current pre-analytical problems and requirements for the most common urinary analytes, with a special highlight of the importance of the preanalytical phase in urine toxicology and metabolomics (7). Last, but not the least, this article series also covers the recent advances in the harmonization of preanalytical quality indicators (8) and External Quality Assessment Schemes for the pre-analytical phase (9). We hope that this series shall contribute to the better understanding and recognition of various important preanalytical phase variables. As this body of evidence grows, we will keep you updated by providing high quality reviews and original research addressing the current and future preanalytical challenges.

Pleural, peritoneal and pericardial effusions – a biochemical approach

The pathological accumulation of serous fluids in the pleural, peritoneal and pericardial space occurs in a variety of conditions. Since patient management depends on right and timely diagnosis, biochemical analysis of extravascular body fluids is considered a valuable tool in the patient management process.The biochemical evaluation of serous fluids includes the determination of gross appearance, differentiation of transudative from exudative effusions and additional specific biochemical testing to assess the effusion etiology. This article summarized data from the most relevant literature concerning practice with special emphasis on usefulness of biochemical tests used for the investigation of pleural, peritoneal and pericardial effusions. Additionally, preanalytical issues concerning serous fluid analysis were addressed and recommendations concerning acceptable analytical practice in serous fluid analysis were presented.

Preanalytical requirements of urinalysis.

Urine may be a waste product, but it contains an enormous amount of information. Well-standardized procedures for collection, transport, sample preparation and analysis should become the basis of an effective diagnostic strategy for urinalysis. As reproducibility of urinalysis has been greatly improved due to recent technological progress, preanalytical requirements of urinalysis have gained importance and have become stricter. Since the patients themselves often sample urine specimens, urinalysis is very susceptible to preanalytical issues. Various sampling methods and inappropriate specimen transport can cause important preanalytical errors. The use of preservatives may be helpful for particular analytes. Unfortunately, a universal preservative that allows a complete urinalysis does not (yet) exist. The preanalytical aspects are also of major importance for newer applications (e.g. metabolomics). The present review deals with the current preanalytical problems and requirements for the most common urinary analytes.

Predicting OncoDx Recurrence Scores With Immunohistochemical Markers

Recent reports suggest that immunohistochemistry (IHC) markers can be used to give prognostic information in breast cancer that is similar to that contained in the Genomic Health Inc. OncoDx recurrence score (Onco-RS). The goal of this study is to examine the potential prognostic value of a score derived from results of a simple set of IHC tests in the prediction of the Onco-RS. A score (IHC-RS) was derived to predict the Onco-RS using IHC-based quantitative and semiquantitative results from a subset of markers selected from those used in the generation of an Onco-RS score. The patient population consists of a retrospectively identified cohort of 158 women with ER-positive, HER2neu-negative breast cancer who completed OncoDx testing. A predictive model was developed to generate the IHC-RS using stepwise multiple regression incorporating Ki67 percentage and semiquantitative ER and PR scores. Using only these 3 IHC markers, the IHC-RS predicted 62% of the Onco-RS variability (adjusted R=0.624, P=0.004). In addition, analysis of outliers in the correlation between the IHC-RS and the Onco-RS reveals the possibility of sampling error as a drawback of the Onco-RS. This is contrasted against potential interlab and intralab variability and preanalytic issues that may negatively impact the implementation of an IHC-RS.

D-dimer testing for suspected venous thromboembolism in the emergency department. Consensus document of AcEMC, CISMEL, SIBioC, and SIMeL

D-dimer testing is currently considered a cornerstone in the diagnostic approach of patients with suspected venous thromboembolism (VTE) across different health-care settings, including the emergency department (ED). Nevertheless, inappropriate or incorrect activities developing throughout the total testing process may substantially impair the clinical usefulness of this test and delay or even challenge the fast rule out or diagnosis of VTE. The leading problem of D-dimer is represented by the poor specificity for diagnosing VTE, wherein a minority of patients with a positive D-dimer are finally diagnosed with VTE, and even more importantly, the specificity further decreases with ageing, thus contributing to increase the overcrowding in short stay units such as the ED. Due to the large heterogeneity that characterizes the use of D-dimer in the emergency room, three Italian societies of laboratory medicine (Italian Committee for Standardization of Hematology and Laboratory Methods, Italian Society of Clinical Biochemistry and Molecular Biology, and Italian Society of Laboratory Medicine), along with the Academy of Emergency Medicine and Care, have developed a consensus document about the use of D-dimer testing for diagnostics of patients with suspected VTE in this health-care setting. The evidence-based indications contained in this document will cover the leading preanalytical, analytical, and postanalytical issues that may impair the clinical efficacy of D-dimer testing in the ED.

Validation of cell‐based fluorescence assays: practice guidelines from the ICSH and ICCS – part II – preanalytical issues

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part II - Preanalytical issues. © 2013 International Clinical Cytometry Society.

Nitrogen biobank for cardiovascular research.

Biobanks play a crucial role in "-Omics" research providing well-annotated samples to study major diseases, their pathways and mechanisms. Accordingly, there are major efforts worldwide to professionalize biobanks in order to provide high quality preservation and storage of biological samples with potentially greater scientific impact. Biobanks are an important resource to elucidate relevant disease mechanisms as well as to improve the diagnosis, prognosis, and treatment of both pediatric and adult cardiovascular disease. High-quality biological sample collections housed in specialized bio-repositories are needed to discover new genetic factors and molecular mechanisms of congenital heart disease and inherited cardiomyopathies in order to prevent the potential risk of having a fatal cardiac condition as well as to facilitate rational drug design around molecular diseases (personalized medicine). Biological samples are also required to improve the understanding the environmental mechanisms of heart disease (environmental cardiology). The goal of this paper is to focus on preanalytical issues (informed consent, sample type, time of collection, temperature and processing procedure) related to collection of biological samples for research purposes. In addition, the paper provides an overview of the efforts made recently by our Institute in designing and implementing a high-security liquid nitrogen storage system (-196°C). We described the implementations of reliable preservation technologies and appropriate quality control (the right temperature, the right environment, fully traceable with all possible back-up systems) in order to ensure maximum security for personnel as well as the quality and suitability of the stored samples.

Current practices of bleeding time in a developing country: an alert for noncompliance with the standard procedures

Bleeding time is still widely performed in many developing countries including Thailand. To generate an accurate result, the procedure should be complied with standard recommendations such as those from Clinical and Laboratory Standards Institute (CLSI) and World Federation of Hemophilia (WFH). The authors surveyed the current practices of bleeding time in Thailand in order to verify the practices that did not comply with the accepted standard. The questionnaires were sent to hospitals participating Thailand National External Quality Assessment Scheme (NEQAS) for blood coagulation. Items in the questionnaire comprised information about preanalytical, analytical, and postanalytical issues of bleeding time. From a dispatch of 201 questionnaires, 155 (77.1%) were returned. The common noncompliance with standards observed in this survey included inappropriateness of indication, e.g. use for preoperative screening (95 of 126, 75.4%), use of devices other than standard template (130 of 132, 98.5%), and inappropriate reference range (125 of 127, 98.4%). The noncompliance shown in this survey can affect the accuracy of bleeding time results. The authors would like to address these problems as an alert for other laboratories especially in the developing countries where the standard templates are not widely available.