Pre-analytical Treatments Research Articles

Comparison of proteomic approaches used for the detection of potential biomarkers of Alzheimer's disease in blood plasma.

At present, Alzheimer's disease is detected mainly using psychological tests, which can only confirm the disease in its more advanced phases. Therefore, bioanalytical possibilities for detecting this disease earlier are being investigated. To date, the results of analyses, which focus mainly on the study of lipids and proteins either in cerebrospinal fluid or much less often in blood plasma, do not provide satisfactory results. In addition, cerebrospinal fluid sampling is uncomfortable for the patients and involves many health risks. In this work, we deal with proteomic analysis using Matrix-Assisted Laser Desorption/Ionisation-Time of Flight and Liquid Chromatography coupled to tandem Mass Spectrometry of blood plasma with a focus on various ways of preanalytical sample treatments. This should lead to results improvement and facilitate the subsequent evaluation using principal component analysis and partial least squares discriminant analysis. The obtained results indicate the direction of further research, namely the study of interactions between proteins and lipids contained in blood plasma. These substances may be regarded as potential biomarkers allowing for the diagnosis of Alzheimer´s disease even in its early stages.

Ultralow amounts of DNA from long-term archived serum samples produce high-quality methylomes

BackgroundLong-term stored serum is considered challenging for epigenomic analyses: as there are no cells, circulating DNA is scarce, and amplification removes epigenetic signals. Additionally, pre-analytical treatments and storage might introduce biases and fragmentation to the DNA. In particular, starting with low-input DNA can result in low-diversity libraries. However, successful whole-genome bisulphite sequencing (WGBS) of such serum samples has the potential to open biobanks for epigenetic analyses and deliver novel prediagnostic biomarkers. Here, we perform WGBS using the Accel-NGS library preparation kit on ultralow amounts of DNA from long-term archived samples with diverse pretreatments from the Janus Serum Bank.ResultsNinety-four of the 96 samples produced satisfactory methylation calls; an average of 578 M reads per sample generated a mean coverage of 17× and mean duplication level of 35%. Failed samples were related to poor bisulphite conversion rather than to sequencing or library preparation. We demonstrate the feasibility of WGBS on ultralow DNA yields from serum samples stored up to 48 years.ConclusionsOur results show the potential of large serum biobank collections for future epigenomic studies and biomarker discovery.

Effects of Freezing and Thawing Procedures on Selected Clinical Chemistry Parameters in Plasma.

Introduction: Measurements from frozen sample collections are important key indicators in clinical studies. It is a prime concern of biobanks and laboratories to minimize preanalytical bias and variance through standardization. In this study, we aimed at assessing the effects of different freezing and thawing conditions on the reproducibility of medical routine parameters from frozen samples. Materials and Methods: In total, 12 pooled samples were generated from leftover lithium heparinized plasma samples from clinical routine testing. Aliquots of the pools were frozen using three freezing methods (in carton box at -80°C, flash freezing in liquid nitrogen, and controlled-rate freezing [CRF]) and stored at -80°C. After 3 days, samples were thawed using two methods (30 minutes at room temperature or water bath at 25°C for 3 minutes). Ten clinical chemistry laboratory parameters were measured before (baseline) and after freeze-thaw treatment: total calcium, potassium, sodium, alanine aminotransferase, lactate dehydrogenase (LDH), lipase, uric acid, albumin, c-reactive protein (CRP), and total protein. We evaluated the influence of the different preanalytical treatments on the test results and compared each condition with nonfrozen baseline measurements. Results: We found no significant differences between freezing methods for all tested parameters. Only LDH was significantly affected by thawing with fast-rate thawing being closer to baseline than slow-rate thawing. Potassium, LDH, lipase, uric acid, albumin, and CRP values were significantly changed after freezing and thawing compared with unfrozen samples. The least prominent changes compared with unfrozen baseline measurements were obtained when a CRF protocol of the local biobank and fast thawing was applied. However, the observed changes between baseline and frozen samples were smaller than the measurement uncertainty for 9 of the 10 parameters. Discussion: Changes introduced through freezing-thawing were small and not of clinical importance. A slight statistically based preference toward results from slow CRF and fast thawing of plasma being closest to unfrozen samples could be supported.

Ultralow amounts of DNA from long-term archived serum samples produce quality genotypes

While genotyping studies are scavenging for suitable samples to analyze, large serum collections are currently left unused as they are assumed to provide insufficient amounts of DNA for array-based genotyping. Long-term stored serum is considered to be difficult to genotype since preanalytical treatments and storage effects on DNA yields are not well understood. Successful genotyping of such samples has the potential to activate large biobanks for future genome-wide association studies (GWAS). We aimed to evaluate genotyping of ultralow amounts of DNA from samples stored up to 45 years in the Janus Serum Bank with two commercially available platforms. 64 samples, with various preanalytical treatments, were genotyped on the Axiom Array from Thermo Fisher Scientific and a subset of 24 samples with slightly higher yield were genotyped on the HumanCoreExome array from Illumina. Our results showed that about 80% of the serum samples produced call rates with the Axiom arrays that would be satisfactory in GWAS. The mean DNA yield was 5.8 ng as measured with PicoGreen, 3–6% of recommended yield. The failed samples had on average lower input amounts of DNA. All serum samples genotyped on the HumanCoreExome with a standard and FFPE protocol produced GWAS satisfactory call rates, with mean 97.57% and 98.35% call rates, respectively. The mean yield was 10.65 ng, 6% of the recommendations. Successful array-based genotyping of ultralow DNA yields from serum samples stored up to 45 years is possible. These results demonstrate the potential to activate large serum biobank collections for future studies.

NMR for sample quality assessment in metabolomics

The EU Framework 7 project SPIDIA was the occasion for development of NMR approaches to evaluate the impact of different pre-analytical treatments on the quality of biological samples dedicated to metabolomics. Systematic simulation of different pre-analytical procedures was performed on urine and blood serum and plasma. Here we review the key aspects of these studies that have led to the development of CEN technical specifications, to be translated into ISO/IS in the course of the EU Horizon 2020 project SPIDIA4P. Inspired by the SPIDIA results, follow-up research was performed, extending the analysis to different sample types and to the different effects of long-term storage. The latter activity was in conjunction with the local European da Vinci Biobank. These results (which partially contributed to the ANNEX of CEN/TS 16945“MOLECULAR IN VITRO DIAGNOSTIC EXAMINATIONS - SPECIFICATIONS FOR PRE-EXAMINATION PROCESSES FOR METABOLOMICS IN URINE, VENOUS BLOOD SERUM AND PLASMA”) are presented in detail.

Heparin removal by ecteola-cellulose pre-treatment enables the use of plasma samples for accurate measurement of anti-Yellow fever virus neutralizing antibodies

Technological innovations in vaccinology have recently contributed to bring about novel insights for the vaccine-induced immune response. While the current protocols that use peripheral blood samples may provide abundant data, a range of distinct components of whole blood samples are required and the different anticoagulant systems employed may impair some properties of the biological sample and interfere with functional assays. Although the interference of heparin in functional assays for viral neutralizing antibodies such as the functional plaque-reduction neutralization test (PRNT), considered the gold-standard method to assess and monitor the protective immunity induced by the Yellow fever virus (YFV) vaccine, has been well characterized, the development of pre-analytical treatments is still required for the establishment of optimized protocols. The present study intended to optimize and evaluate the performance of pre-analytical treatment of heparin-collected blood samples with ecteola-cellulose (ECT) to provide accurate measurement of anti-YFV neutralizing antibodies, by PRNT. The study was designed in three steps, including: I. Problem statement; II. Pre-analytical steps; III. Analytical steps. Data confirmed the interference of heparin on PRNT reactivity in a dose-responsive fashion. Distinct sets of conditions for ECT pre-treatment were tested to optimize the heparin removal. The optimized protocol was pre-validated to determine the effectiveness of heparin plasma:ECT treatment to restore the PRNT titers as compared to serum samples. The validation and comparative performance was carried out by using a large range of serum vs heparin plasma:ECT 1:2 paired samples obtained from unvaccinated and 17DD-YFV primary vaccinated subjects. Altogether, the findings support the use of heparin plasma:ECT samples for accurate measurement of anti-YFV neutralizing antibodies.

The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray

The research field of extracellular vesicles (EVs) is increasing immensely and the potential uses of EVs seem endless. They are found in large numbers in various body fluids, and blood samples may well serve as liquid biopsies. However, these small membrane-derived entities of cellular origin are not straightforward to work with in regard to isolation and characterization.A broad range of relevant preanalytical issues was tested, with a focus on the phenotypic impact of smaller EVs. The influences of the i) blood collection tube used, ii) incubation time before the initial centrifugation, iii) transportation/physical stress, iv) storage temperature and time (short term and long term), v) choice of centrifugation protocol, vi) freeze-thaw cycles, and vii) exosome isolation procedure (ExoQuick™) were examined. To identify the impact of the preanalytical treatments, the relative amounts (detected signal intensities of CD9-, CD63- and/or CD81-positive) and phenotypes of small EVs were analyzed using the multiplexed antibody-based microarray technology, termed the EV Array. The analysis encompassed 15 surface- or surface-related markers, including CD9, CD63, CD81, CD142, and Annexin V.This study revealed that samples collected in different blood collection tubes suffered to varying degrees from the preanalytical treatments tested here. There is no unequivocal answer to the questions asked. However, in general, the period of time and prospective transportation before the initial centrifugation, choice of centrifugation protocol, and storage temperature were observed to have major impacts on the samples. On the contrary, long-term storage and freeze-thawing seemed to not have a critical influence. Hence, there are pros and cons of any choice regarding sample collection and preparation and may very well be analysis dependent. However, to compare samples and results, it is important to ensure that all samples are of the same type and have been handled similarly.

Effect of pre-analytical treatments on bovine milk acute phase proteins.

BackgroundSamples for diagnostic procedures often require some form of pre-analytical preparation for preservation or safe handling during transportation prior to analysis in the laboratory. This is particularly important for milk samples which frequently need preservatives to retain milk composition as close to that found in freshly collected samples as possible.MethodsMilk samples were treated by heating at 56 °C for 30 min or preserved by addition of either potassium dichromate or bronopol respectively. Haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) were measured in the various treatment groups and in control samples which were not treated, using enzyme linked immunoassays. The concentrations of each APP were compared between treated and non-treated groups using the Wilcoxon signed ranks tests.ResultsHeat treatment of samples was found to have a significant lowering effect on milk M-SAA3 and CRP but not Hp. The use of potassium dichromate and bronopol as preservatives in milk had no significant effects on milk Hp and M-SAA3 concentration but lowered milk CRP values compared to controls.ConclusionsThe observed effects of heating and preservative use on milk APP should be taken into consideration when assaying samples which have undergone heat treatment as a result of international transfer regulations involving biological samples or samples needing chemical preservation prior to transport to laboratory.

High variation in hypoxanthine determination after analytical treatment of vitreous humor samples.

In the field of legal medicine the correct determination of the time of death is of great importance because an error in calculating the post-mortem interval (PMI) could be crucial in a criminal investigation. The quantification of hypoxanthine (Hx) concentration in the vitreous humor (VH) as a means to estimate PMI is useful when dealing with a recent time interval of death and has several advantages over other biological matrices. However, due to its viscous nature, VH has to be liquefied prior to its analysis, and the different procedures to liquefy it may cause alterations in Hx concentration, and hence in estimation of PMI. The aim of this study was to determine differences in Hx concentration after applying several pre-analytical treatments to different aliquots of the same VH sample. Enzymatic digestion, sonication, centrifugation and heat were the liquefying methods applied to reduce the viscosity of 105 VH samples. Analysis was performed using solid phase extraction and liquid chromatography in tandem with mass spectrometry. Statistically significant differences in the measured Hx concentrations were found between enzymatic digestion and the other three methods analyzed (sonication, centrifugation, and heat). Additionally, high data dispersion was found under heat treatment but was not observed for centrifugation or sonication, where similar and more moderate results were found. We conclude that high variations in Hx determination are observed when enzymatic digestion or heat treatment is used and we therefore recommend sonication or centrifugation to quantify Hx concentration in the VH.

Global metabolomics characterization of bacteria: pre-analytical treatments and profiling

Pre-analytical treatments of bacteria are crucial steps in bacterial metabolomics studies. In order to achieve reliable samples that can best represent the global metabolic profile in vivo both qualitatively and quantitatively, many sample treatment procedures have been developed. The use of different methods makes it difficult to compare the results among different groups. In this work, E. coli samples were tested by using NMR spectroscopy. Both liquid N2 and cold methanol quenching procedures reduce the cell membrane integrity and cause metabolites leakage. However, liquid N2 quenching affected the cell viability and the NMR metabolites’ profile less than cold methanol procedure. Samples obtained by metabolite extraction were significantly superior over cell suspensions and cell lysates, with a higher number of detectable metabolites. Methanol/chloroform extraction proved most efficient at extraction of intracellular metabolites from both qualitative and quantitative points of view. Finally, standard operating procedures of bacterial sample treatments for NMR metabolomics study are presented.

Development of a chemiluminescence-based quantitative lateral flow immunoassay for on-field detection of 2,4,6-trinitrotoluene

Simple, rapid and highly sensitive assays, possibly allowing on-site analysis, are required in the security and forensic fields or to obtain early signs of environmental pollution. Several bioanalytical methods and biosensors based on portable devices have been developed for this purpose. Among them, Lateral Flow ImmunoAssays (LFIAs) offer the advantages of rapidity and ease of use and, thanks to the high specificity of antigen–antibody binding, allow greatly simplifying and reducing sample pre-analytical treatments. However, LFIAs usually employ colloidal gold or latex beads as labels and they rely on the formation of colored bands visible by the naked eye. With this assay format, only qualitative or semi-quantitative information can be obtained and low sensitivity is achieved. Recently, the use of enzyme-catalyzed chemiluminescence detection in LFIA has been proposed to overcome these problems. In this work, we describe the development of a quantitative CL-LFIA assay for the detection of 2,4,6-trinitrotoluene (TNT) in real samples. Thanks to the use of a portable imaging device for CL signal measurement based on a thermoelectrically cooled CCD camera, the analysis could be performed directly on-field. A limit of detection of 0.2μgmL−1 TNT was obtained, which is five times lower than that obtained with a previously described colloidal gold-based LFIA developed employing the same immunoreagents. The dynamic range of the assay extended up to 5μgmL−1 TNT and recoveries ranging from 97% to 111% were obtained in the analysis of real samples (post blast residues obtained from controlled explosion).

Standard operating procedures for pre-analytical handling of blood and urine for metabolomic studies and biobanks

(1)H NMR metabolic profiling of urine, serum and plasma has been used to monitor the impact of the pre-analytical steps on the sample quality and stability in order to propose standard operating procedures (SOPs) for deposition in biobanks. We analyzed the quality of serum and plasma samples as a function of the elapsed time (t=0-4h) between blood collection and processing and of the time from processing to freezing (up to 24h). The stability of the urine metabolic profile over time (up to 24h) at various storage temperatures was monitored as a function of the different pre-analytical treatments like pre-storage centrifugation, filtration, and addition of the bacteriostatic preservative sodium azide. Appreciable changes in the profiles, reflecting changes in the concentration of a number of metabolites, were detected and discussed in terms of chemical and enzymatic reactions for both blood and urine samples. Appropriate procedures for blood derivatives collection and urine preservation/storage that allow maintaining as much as possible the original metabolic profile of the fresh samples emerge, and are proposed as SOPs for biobanking.

Assessment of a procedure to determine trace and major elements in atmospheric aerosol

The determination of trace elements in atmospheric particulate is affected by a number of problems that arise from some critical points such as the blank of the filters, sample heterogeneity and pre-analytical treatments. In the framework of a monitoring campaign conducted in the Venice Lagoon the analytical methodology for the determination of 20 trace elements (Al, As, Ca, Cd, Co, Cr, Cs, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Sr, V, Zn) in atmospheric particulate samples by inductively coupled plasma quadrupole mass spectroscopy (ICP-QMS) has been optimized taking into account the individual critical points. Tests were carried out to estimate the blank contributions, and minimize the detection limit (LOD), measurements were also carried out to evaluate the accuracy and the repeatability. To obtain a complete dissolution of aerosol dust material and good recoveries of the elements, the acid mixture and the microwave assisted digestion program were optimized. The blank contributions from membrane filter manipulation and transportation prior to exposure were tested for the slotted and back filters by placing them on the sampling device for some minutes without air flowing to obtain field blanks (FBs). The contribution to the blank values of passive deposition and by contact with the samplers (quoted as campaign blanks, CBs) was measured by exposing the membranes throughout the sampling session (fifteen days) without any air flow. Instrumental ICP-QMS parameters were optimized and calibration curve intervals were selected on the basis of the necessity of simultaneous determination of the elements present at different levels of concentration. The limits of detection for each elements and the investigated method were suitable to determine the 20 elements reported above in the atmospheric aerosol fractionated in 6 classes ranging between 10 to 0.49 microm. It allows the determination of trace elements in aerosol in a large range of concentrations that can be observed in areas characterized by remarkable variability and regions with different levels of contamination.

Effects of sample pre-treatment on the δ13C and δ18O values of living benthic foraminifera

Stable carbon and oxygen isotope ratios ( δ 13C and δ 18O) of the epibenthic foraminiferan Elphidium macellum var. aculeatum were measured and compared both in raw aliquots and after various pre-analytical treatments. We tested the effects of chemical cleaning procedures (oxygen plasma ashing, NaClO and H 2O 2) and physical (ultrasounds) aimed at removing allochthonous particles. We also assessed the effects of using preservatives (formaldehyde and ethanol) and stains (Bengal Rose and Sudan Black B) prior to δ 13C and δ 18O analyses. Although the effects were generally small, three of the pre-treatments had a significant effect on δ 13C or δ 18O values. The use of Sudan Black B for vital identification caused a significant enrichment in the 13C of the samples (0.24‰). In addition, sample oxidation with hydrogen peroxide and ultrasonic cleaning each led to a slight decrease in δ 18O values (– 0.18‰, in both cases). Our results demonstrate that Sudan Black B should be avoided prior to isotopic analysis. Bengal Rose showed no significant effect and should be used instead for staining. Because palaeoclimatic studies are based in small isotopic shifts, we discourage the pre-analysis use of unnecessary harsh cleaning treatments of foraminifera shell samples that may lead to isotopic fractionation and hence confound ecological interpretation.

Hyaluronidase as a Liquefying Agent for Chemical Analysis of Vitreous Fluid

Vitreous humor is a suitable specimen for postmortem clinical chemistry because the analytes remain relatively stable after death and they closely reflect blood levels immediately prior to death. The viscous nature of vitreous fluid, however, presents analytical problems including imprecision and inaccuracy. Various preanalytical treatments, such as boiling, high speed centrifugation, microfiltration and dilution have been used. These techniques are labor intensive and add to imprecision and inaccuracy. Because glycosaminoglycans contribute significantly to the viscosity of vitreous humor, we used hyaluronidase as a liquefying agent. We compared the results of analyses in 33 vitreous humor specimens after hyaluronidase treatment with the results after either no treatment or specimen dilution. Seventeen of the 33 specimens could not be analyzed without dilution. Even after dilution, several analytes still could not be measured. Hyaluronidase treatment negated the need for sample dilution and had no significant effect on the analyses.