A pocket companion to cell-free DNA (cfDNA) preanalytics.

The cumulative pool of cell-free DNA (cfDNA) molecules within bodily fluids represents a highly dense and multidimensional information repository. This "biological mirror" provides real-time insights into the composition, function, and dynamics of the diverse genomes within the body, enabling significant advancements in personalized molecular medicine. However, effective use of this information necessitates meticulous classification of distinct cfDNA subtypes with exceptional precision. While cfDNA molecules originating from different sources exhibit numerous genetic, epigenetic, and physico-chemical variations, they also share common features that complicate analyses. Considerable progress has been achieved in mapping the landscape of cfDNA features, their clinical correlations, and optimizing extraction procedures, analytical approaches, bioinformatics pipelines, and machine learning algorithms. Nevertheless, preanalytical workflows, despite their profound impact on cfDNA measurements, have not progressed at a corresponding pace. In this perspective article, we emphasize the pivotal role of robust preanalytical procedures in the development and clinical integration of cfDNA assays, highlighting persistent obstacles and emerging challenges.

Next-generation diagnostics for precision oncology: Preanalytical considerations, technical challenges, and available technologies.

Molecular diagnostics as the centrepiece of precision oncology has gone through revolutionary developments over the last decade, becoming tremendously broad, deep and precise with still ongoing advancements. In the majority of scenarios, treatment selection for cancer patients without any type of molecular characterization is no longer conceivable. Considering the impact of sample quality on the reliability of molecular analyses and the importance of the results for the fate of an individual patient, it is surprising how sparsely preanalytical and analytical requirements are addressed scientifically. Standardization and rigorous quality assessment continue to play only a marginal role in the field. Within this review, we will systematically discuss influencing preanalytic parameters and technology setups affecting molecular test results. We will shed light on the specifics of different analytes, technical modalities, and analysis pipelines. The review will have a certain focus on broad molecular genetic tumour testing with next generation sequencing but will go beyond that including other molecular diagnostic modalities and will give a glimpse into the future of molecular testing.

Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

The laboratory journey to become a decision engine: a roadmap for diagnostic transformation.

Laboratories and diagnostic departments are presiding over a massive amount of data they are failing to fully leverage it. Data is the new black gold of healthcare organizations and by extracting insights from it, laboratories could become true decision engines, able to drive action across healthcare. This opinion paper responds three fundamental questions: (1) Where are we (diagnostic parties)? Taking a look at the most significant trends and challenges in healthcare and shedding some light upon the status of diagnostics. (2) Where do we want to be? Reviewing the opportunities for digital health, its role in the healthcare of the future and providing inspiration about what success looks like. (3) What do we need to do? Explaining what Digital Health Solutions (DHS) from Abbott is doing in this regard. This will include information about how DHS can impact the Diagnosis Cycle and how to set a roadmap for laboratories and diagnostic organizations. Diagnosis Cycle means the different steps in the diagnosis process, from the beginning when a patient is seen by a clinician and some tests are ordered, until the results are reviewed by the clinician and the treatment, follow up or discharge is decided.

Preanalytical impact on the accuracy of measurements of glucagon, GLP-1 and GIP in clinical trials.

BACKGROUND: Plasma concentrations of glucagon, GLP-1 and GIP are reported in numerous clinical trials as outcome measures but preanalytical guidelines are lacking. We addressed the impact of commonly used blood containers in metabolic research on measurements of glucagon, GLP-1 and GIP in humans. METHODS: Seventeen overweight individuals were subjected to an overnight fast followed by an intravenous infusion of amino acids to stimulate hormonal secretion. Blood was sampled into five containers: EDTA-coated tubes supplemented with DMSO (control), a neprilysin inhibitor, aprotinin (a kallikrein inhibitor) or a DPP-4 inhibitor, and P800 tubes. Plasma was kept on ice before and after centrifugation and stored at -80 Celsius until batch analysis using validated sandwich ELISAs or radioimmunoassays (RIA). RESULTS: Measures of fasting plasma glucagon did not depend on sampling containers, whether measured by ELISA or RIA. Amino acid-induced hyperglucagonemia was numerically higher when blood was collected into P800 tubes or tubes with aprotinin. The use of p800 tubes resulted in higher concentrations of GLP-1 by RIA compared to control tubes but not for measurements with sandwich ELISA. Plasma concentrations of GIP measured by ELISA were higher in control tubes and negatively affected by P800 and the addition of aprotinin. CONCLUSIONS: The choice of blood containers impacts on measurements of plasma concentrations of glucagon, GLP-1 and GIP, and based on this study, we recommend using EDTA-coated tubes without protease inhibitors or P800 tubes for measurements of glucagon, GLP-1 and GIP in clinical trials.

The Effect of Preanalytical and Physiological Variables on Cell-Free DNA Fragmentation.

BACKGROUND: Assays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, preanalytic and physiological differences between individuals on fragmentomic analysis are poorly defined. METHODS: We analyzed the impact of collection tube, plasma processing time, and physiology on the size distribution of cfDNA, their genome-wide representation, and sequence diversity at the cfDNA fragment ends using shallow whole-genome sequencing. RESULTS: Neither different stabilizing collection tubes nor processing times affected the cfDNA fragment sizes, but could impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication, and chronic conditions) minimally influenced the outcome of fragmentomic methods. CONCLUSIONS: Fragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.

Post-mortem changes of prostanoid concentrations in tissues of mice: Impact of fast cervical dislocation and dissection delay.

Prostanoids are potent lipid mediators involved in a wide variety of physiological functions like blood pressure regulation or inflammation as well as cardiovascular and malign diseases. Elucidation of their modes of action is mainly carried out in pre-clinical animal models by quantifying prostanoids in tissues of interest. Unfortunately, prostanoids are prone to post-mortem artifact formation and de novo synthesis can already be caused by external stimuli during the euthanasia of animals like prolonged hypercapnia or ischemia. Therefore, this study investigates the suitability and impact of fast cervical dislocation for the determination of prostanoids (6-keto-PGF1α, TXB2, PGF2α, PGD2, PGE2) in seven tissues of mice (spinal cord, brain, sciatic nerve, kidney, liver, lung, and spleen) to minimize time-dependent effects and approximate physiological concentrations. Tissues were dissected in a standardized sequence directly or after 10 min to investigate the influence of dissection delays. The enzyme inhibitor indomethacin (10 µM) in combination with low processing temperatures was employed to preserve prostanoid concentrations during sample preparation. Quantification of prostanoids was performed via LC-MS/MS. This study shows, that prostanoids are differentially susceptible to post-mortem artifact formation which is closely connected to their physiological function and metabolic stability in the respective tissues. Prostanoids in the brain, spinal cord, and kidney that are not involved in the regulatory response post-mortem, i.e. blood flow regulation (6-keto-PGF1α, PGE2, PGF2α) showed high reproducibility even after dissection delay and could be assessed after fast cervical dislocation if prerequisites like standardized pre-analytical workflows with immediate dissection and inhibition of residual enzymatic activity are in place. However, in tissues with high metabolic activity (liver, lung) more stable prostanoid metabolites should be used. Moreover, prostanoids in the spleen were strongly affected by dissection delays and presumably the method of euthanasia itself.

Characterization of pre-analytical sample handling effects on a panel of Alzheimer's disease-related blood-based biomarkers: Results from the Standardization of Alzheimer's Blood Biomarkers (SABB) working group.

INTRODUCTION: Pre-analytical sample handling might affect the results of Alzheimer's disease blood-based biomarkers. We empirically tested variations of common blood collection and handling procedures. METHODS: We created sample sets that address the effect of blood collection tube type, and of ethylene diamine tetraacetic acid plasma delayed centrifugation, centrifugation temperature, aliquot volume, delayed storage, and freeze-thawing. We measured amyloid beta (Aß)42 and 40 peptides with six assays, and Aß oligomerization-tendency (OAß), amyloid precursor protein (APP)699-711 , glial fibrillary acidic protein (GFAP), neurofilament light (NfL), total tau (t-tau), and phosphorylated tau181. RESULTS: Collection tube type resulted in different values of all assessed markers. Delayed plasma centrifugation and storage affected Aß and t-tau; t-tau was additionally affected by centrifugation temperature. The other markers were resistant to handling variations. DISCUSSION: We constructed a standardized operating procedure for plasma handling, to facilitate introduction of blood-based biomarkers into the research and clinical settings.

Evaluating the performance of an updated high-sensitivity troponin T assay with increased tolerance to biotin.

OBJECTIVES: Biotin >20 ng/mL may interfere with the Elecsys® Troponin T-high sensitive assay (cTnT-hs; Roche Diagnostics International Ltd). We evaluated the performance of an updated assay, cTnT-hs*, which was designed to reduce biotin interference. METHODS: cTnT-hs* assay performance was assessed using up to two applications (18 min/9 min) on three analyzers (cobas e 411/cobas e 601/cobas e 801). Biotin interference was determined by measuring recovery in an 11-sample series dilution with biotin ranging from 0-3600 ng/mL. Repeatability/reproducibility were evaluated in five serum sample pools (n=75 each). Method comparisons tested: cTnT-hs* vs. cTnT-hs (18 min/cobas e 601); cTnT-hs* assay 18 vs. 9 min (cobas e 601); cTnT-hs* (18 min) on cobas e 601 vs. cobas e 411 and cobas e 601 vs. cobas e 801. Concordance at the 99th percentile decision limit between cTnT-hs* and cTnT-hs (9 min/cobas e 601) was calculated using 300 lithium-heparin plasma samples and a 14 ng/L assay cutoff. RESULTS: cTnT-hs* assay (18 min/cobas e 601) recovery was ≥96% for biotin ≤1250 ng/mL. Across all applications/analyzers, coefficients of variation for repeatability/reproducibility with the cTnT-hs* assay were <5% in most serum sample pools (mean cardiac troponin T: 8.528-9484 ng/L). High correlation (Pearson's r=1.000) was demonstrated for all method comparisons. Concordance at the 99th percentile decision limit was high between the cTnT-hs* and cTnT-hs assays. CONCLUSIONS: The updated cTnT-hs* assay may provide greater tolerance to biotin interference, and shows good analytical and clinical agreement/concordance with the previous cTnT-hs assay.

Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples.

The study was performed to compare real-time PCR after nucleic acid extraction directly from stool samples as well as from samples stored and transported on Whatman papers or flocked swabs at ambient temperature in the tropics. In addition, the possible suitability for a clear determination of likely aetiological relevance of PCR-based pathogen detections based on cycle threshold (Ct) values was assessed. From 632 Tanzanian children <5 years of age with and without gastrointestinal symptoms, 466 samples were subjected to nucleic acid extraction and real-time PCR for gastrointestinal viral, bacterial and protozoan pathogens. Equal or even higher frequencies of pathogen detections from Whatman papers or flocked swabs were achieved compared with nucleic acid extraction directly from stool samples. Comparison of the Ct values showed no significant difference according to the nucleic acid extraction strategy. Also, the Ct values did not allow a decision whether a detected pathogen was associated with gastrointestinal symptoms.

Effect of long-term frozen storage and thawing of stool samples on faecal haemoglobin concentration and diagnostic performance of faecal immunochemical tests.

Background Faecal samples collected and stored frozen over years may be a valuable resource for efficient retrospective evaluation of faecal immunochemical tests (FITs). We aimed to assess how prolonged frozen storage and freeze-thaw cycles might affect measures of faecal haemoglobin (Hb) and diagnostic performance of FITs. Methods From 2005 through 2010, participants of screening colonoscopy (n = 2042) and clinical colorectal cancer (CRC) cases (n = 184) provided faecal samples in stool containers (60 mL). The samples were stored at -80 °C for up to 11 years and underwent three freeze-thaw cycles. Between each cycle, a defined amount of faeces was extracted using the manufacturer's sampling device of one or two FITs (RIDASCREEN, OC-Sensor). Faecal Hb concentration and diagnostic performance were calculated and compared across freeze-thaw cycles. Results For RIDASCREEN and the OC-Sensor, repeat measurements were available for 504 and 551 study participants, respectively. Hb concentrations correlated strongly (0.77 and 0.85, respectively) and diagnostic performance indicators were similar at the repeat measurements among the same FITs. For RIDASCREEN we found even slightly higher Hb levels, sensitivities and area under the curves (AUCs) after the third than after the first freeze-thaw cycle. For the OC-Sensor the Hb levels, sensitivities and AUCs were slightly lower after prolonged storage and one additional freeze-thaw cycle. Conclusions Measures of Hb and diagnostic performance were fairly stable, even after long-term frozen storage and multiple freeze-thaw cycles of raw faecal samples. Faecal samples collected in prospective screening studies and kept frozen at -80 °C before analysis seem useful for timely and efficient retrospective evaluation of FIT performance.

Rapid tissue processing using a temperature-controlled collection device to preserve tumor biomarkers.

Precision tissue diagnostics rely on high quality input specimens so that assay results are not affected by artifact, but advances in collection and processing of tissue specimens have lagged behind innovations in diagnostic assay development. Therefore, we have designed and evaluated a novel surgical tissue collection device that maintains and monitors sample temperature and motion throughout transport so that the major preanalytical variable of tissue temperature can be controlled and measured. This device, in combination with an improved cold-hot tissue fixation protocol affords optimal biomarker preservation in less overall time, thereby simultaneously improving diagnostic quality and turnaround time. We collected 50 primary and metastatic liver tumors using a novel transport device. Tissue was fixed using a rapid cold-hot fixation protocol and immunohistochemical assays were used to assess the performance of the device, in comparison to control tissue preserved using standard clinical fixation protocol. Two pathologists evaluated the IHC studies in a blinded fashion to determine the immunophenotype of each tumor. The observed IHC staining intensities and the clinical impressions of the immunophenotypes did not differ between tissue collected with the novel device and control tissue, while improvements in processing time were achieved. The novel cold transport device and rapid fixation protocol can be successfully and safely combined and used to monitor specimen conditions, thus preserving the diagnostic utility of specimens and improving the overall turn-around time of the diagnostic process.

Variability of cytokine concentration in whole blood serum and bronchoalveolar lavage over time.

Measurement of cytokines in peripheral blood and bronchoalveolar lavage fluid (BALF) is a useful method to assess human immune responses in a large range of pulmonary diseases. One of the major pre-analytical challenges of cytokine analysis is the quality and stability of cytokines in the timeframe between sample collection and the separation of supernatant from cells. To evaluate if the method of storage may affect cytokine quantification, whole blood and BALF were collected, aliquoted, and left at room temperature (RT) to be processed at different time points. In addition, sera and BALF were left either at RT or at 4 °C for 24 h after cell separation to test cytokine variations in the absence of cells. Samples were analysed by a multiple array containing ten cytokines. Most of the cytokines analysed (interleukin (IL)-4, IL-5, IL-6, IL-12p70, IL-13, IL-17A, IL-23, interferon (IFN)-γ, and tumour necrosis factor (TNF)-α) did not show significant variations in whole blood and BALF. Levels of IL-8 however, increased after storage of whole blood and BALF for 24 h at RT. Ex vivo IL-8 production seems to correlate with higher numbers of macrophages in collected BALF. These data demonstrate that many cytokines are stable for a brief time after sample collection. For IL-8, freshly collected whole blood and BALF should be quickly processed and frozen to avoid false positive results.

Preanalytical robustness of blood collection tubes with RNA stabilizers.

Background Efficient blood stabilization is essential to obtaining reliable and comparable RNA analysis data in preclinical operations. PAXgene (Qiagen, Becton Dickinson) and Tempus (Applied Biosystems, Life Technologies) blood collection tubes with RNA stabilizers both avoid preanalytical degradation of mRNA by endogenous nucleases and modifications in specific mRNA concentrations by unintentional up- or down-regulation of gene expression. Methods Sixteen different preanalytical conditions were tested in PAXgene and Tempus blood samples from seven donors: different mixing after collection, different fill volumes and different 24-h transport temperature conditions after collection. RNA was extracted by column-based methods. The quality of the extracted RNA was assessed by spectrophotometric quantification, A260/A280 purity ratio, RNA Integrity Number (Agilent Bioanalyzer), miRNA quantative real time polymerase chain reaction (qRT-PCR) on two target miRNAs (RNU-24 and miR-16), mRNA quality index by qRT-PCR on the 3' and 5' region of the GAPDH gene, and the PBMC preanalytical score, based on the relative expression levels of the IL8 and EDEM3 coding genes. Results When PAXgene RNA and Tempus blood collection tubes were used following the manufacturers' instructions, there was no statistically or technically significant difference in the output RNA quality attributes. However, the integrity of the RNA extracted from Tempus collection tubes was more sensitive to fill volumes and effective inversion, than to storage temperature, while the integrity of RNA extracted from PAXgene collection tubes was more sensitive to effective inversion and storage temperature than to fill volumes. Conclusions Blood collection tubes with different RNA stabilizers present different robustness to common preanalytical variations.

Improving laboratory workflow through automated pre-processing of SurePath specimens for human papillomavirus testing with the Abbott RealTime assay.

BACKGROUND: Cervical specimens collected in liquid-based cytology (LBC) are used for cervical screening in the UK. The Abbott RealTime high-risk human papillomavirus (hrHPV) assay for the detection of 14 hrHPV types is approved by the English NHS Cervical Screening Programme (NHSCSP). As per manufacturer's instructions, pre-analytic processing of LBC involves a manual vortex and liquid transfer into a secondary tube. In high-throughput settings, hands-on time for pre-processing is considerable and poses the potential for human error. Implementation of hrHPV primary screening planned for 2019 accompanied by centralisation of services is a major change for the NHSCSP, increasing the demand for availability of automated pre-analytics. This study evaluated a custom-configured work-table setup of the Tecan Freedom EVO designed to automate pre-processing of BD SurePath LBC prior to HPV testing on the Abbott m2000. METHODS: Automatically and manual pre-processed specimen results were compared (primary screening population n = 307; triage population n = 169). RESULTS: Excellent agreement of overall hrHPV results (98.1%; k: 0.95) was observed. On average, it takes approximately 1.5 minutes hands-on-time per sample to process manually compared to 45 minutes to aliquot 48 samples using the Freedom EVO 150. CONCLUSION: The Tecan worktable configuration designed to automate and control pre-analytics required for preparing SurePath LBC samples for HPV testing using Abbott RealTime resulted in assay performance comparable to that following the manufacturer validated manual process. It significantly reduces hands-on-time and allows for complete specimen identification tracking and documentation of process control.

Targeted On-line SPE-LC-MS/MS Assay for the Quantitation of 12 Apolipoproteins from Human Blood.

Laborious sample pretreatment of biological samples represents the most limiting factor for the translation of targeted proteomics assays from research to clinical routine. An optimized method for the simultaneous quantitation of 12 major apolipoproteins (apos) combining on-line SPE and fast LC-MS/MS analysis in 6.5 min total run time was developed, reducing the manual sample pretreatment time of 3 µL serum or plasma by 60%. Within-run and between-day imprecisions below 10 and 15% (n = 10) and high recovery rates (94-131%) were obtained applying the high-throughput setup. High-quality porcine trypsin was used, which outperformed cost-effective bovine trypsin regarding digestion efficiency. Comparisons with immunoassays and another LC-MS/MS assay demonstrated good correlation (Pearson's R: 0.81-0.98). Further, requirements on sample quality concerning sampling, processing, and long-term storage up to 1 year were investigated revealing significant influences of the applied sampling material and coagulant on quantitation results. Apo profiles of 1339 subjects of the LIFE-Adult-Study were associated with lifestyle and physiological parameters as well as establish parameters of lipid metabolism (e.g., triglycerides, cholesterol). Besides gender effects, most significant impact was seen regarding lipid-lowering medication. In conclusion, this novel highly standardized, high-throughput targeted proteomics assay utilizes a fast, simultaneous analysis of 12 apos from least sample amounts.

Hemolysis and IgA-antibodies against tissue transglutaminase: When are antibody test results no longer reliable?

BACKGROUND: Antibodies against tissue transglutaminase (TTG) of isotype IgA (IgA-aTTG) represent reliable diagnostic markers to confirm or exclude celiac disease (CD). Hemolysis (HL) is an important pre-analytical factor. HL can be quantified as HL index (HI) correlating with the concentration of free hemoglobin. TTG is abundant in erythrocytes and released upon HL. In immunoassays, the released TTG may interfere with binding of IgA-aTTG to the coated TTG. METHODS: We selected 17 HL-free sera from children with biopsy-confirmed CD: 7 with low-positive (1-5 multiples of upper limit of normal [×ULN]), 5 with intermediate (5-10 × ULN) and 5 with high IgA-aTTG (10-15 × ULN). Sera were spiked with hemolysates resulting in HIs ranging from 12.5 to 800 (12.5-800 mg/dL free hemoglobin). RESULTS: IgA-aTTG values were significantly decreased (>10%) after addition of hemolysates even if HL was invisible (HI <50). This effect is diagnosis-relevant if IgA-aTTG values are measured just below the cut-offs: (i) 0.4-1 × ULN at HI ≥25 (CD not excludable) and (ii) 8.5-10 × ULN at HI ≥200 (diagnosis of CD without biopsy not possible). Antibodies against deamidated gliadin were not influenced by HL. CONCLUSIONS: IgA-aTTG results in sera with HI ≥25 can yield inconclusive results. Therefore, those antibody results should be assessed only under consideration of the HI.

The relationship between vacuum and hemolysis during catheter blood collection: a retrospective analysis of six large cohorts.

BACKGROUND: Blood collection through intravenous (IV) catheters is a common practice at emergency departments (EDs). This technique is associated with higher in vitro hemolysis rates and may even be amplified by the use of vacuum collection tubes. Our aim was to investigate the association of five different vacuum tubes with hemolysis rates in comparison to an aspiration system under real-life conditions and to propose an equation to estimate the amount of hemolysis, depending on the vacuum collection tube type. METHODS: We retrospectively evaluated hemolysis data of plasma samples from our ED, where blood is drawn through IV catheters. Over the past 5 years, we compared 19,001 hemolysis index values amongst each other and against the respective vacuum pressure (Pv) of the collection tubes, which were used within the six observational periods. RESULTS: The highest hemolysis rates were associated with full-draw evacuated tubes. Significantly reduced hemolysis was observed for two kinds of partial-draw tubes. The hemolysis rate of one partial-draw blood collection tube was comparable to those of the aspiration system. Regression analysis of Pv and mean free hemoglobin (fHb) values yielded the formula fHb (g/L)=0.0082*Pv2-0.1143*Pv+ 0.5314 with an R2 of 0.99. CONCLUSIONS: If IV catheters are used for blood collection, hemolysis rates directly correlate with the vacuum within the tubes and can be estimated by the proposed formula. By the use of partial-draw vacuum blood collection tubes, hemolysis rates in IV catheter collections can be reduced to levels comparable with collections performed by aspiration systems.

Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants.

Microvesicles (MVs) are small membrane bound vesicles released from various cell types after activation or apoptosis. In the last decades, MVs received an increased interest as biomarkers in inflammation, coagulation and cancer. However, standardized pre-analytical steps are crucial for the minimization of artifacts in the MV analysis. Thus, this study evaluated the MV release in whole blood samples under the influence of different anticoagulants, storage time and various temperature conditions. Samples were collected from healthy probands and processed immediately, after 4, 8, 24 and 48 hours at room temperature (RT) or 4°C. To identify MV subpopulations, platelet free plasma (PFP) was stained with Annexin V, calcein AM, CD15, CD41 and CD235a. Analysis was performend on a CytoFLEX flow cytometer. Procoagulatory function of MVs was measured using a phospholipid dependent activity and a tissue factor MVactivity assay. Without prior storage, sodium citrate showed the lowest MV count compared to heparin and EDTA. Interestingly, EDTA showed a significant release of myeloid-derived MVs (MMVs) compared to sodium citrate. Sodium citrate showed a stable MV count at RT in the first 8 hours after blood collection. Total MV counts increased after 24 hours in sodium citrated or heparinzed blood which was related to all subpopulations. Interestingly, EDTA showed stable platelet-derived MV (PMV) and erythrocyte-derived MV (EryMV) count at RT over a 48 h period. In addition, the procoagulatory potential increased significantly after 8-hour storage. Based on both, this work and literature data, the used anticoagulant, storage time and storage temperature differently influence the analysis of MVs within 8 hours. To date, sodium citrated tubes are recommended for MV enumeration and functional analysis. EDTA tubes might be an option for the clinical routine due to stable PMV and EryMV counts. These new approaches need to be validated in a clinical laboratory setting before being applied to patient studies. © 2016 International Society for Advancement of Cytometry.