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1.
Biopreserv Biobank ; 2023 Oct 25.
Article in English | MEDLINE | ID: mdl-37878356

ABSTRACT

Development of novel biomarkers for diagnosis of disease and assessment of treatment efficacy utilizes a wide range of biospecimens for discovery research. The fitness of biospecimens for the purpose of biomarker development depends on the clinical characteristics of the donor and on a number of critical and potentially uncontrolled pre-analytical variables. Pre-analytical factors influence the reliability of the biomarkers to be analyzed and can seriously impact analytic outcomes. Sample quality stratification assays and tools can be utilized by biorepositories to minimize bias resulting from samples' inconsistent quality. In this study, we evaluated the quality of biobanked specimens by comparing analytical outcomes at 1, 5, and 10 years after collection. Our results demonstrate that currently available assays and tools can be used by biobank laboratories to support objective biospecimen qualification. We have established a workflow to monitor the quality of different types of biospecimens and, in this study, present the results of a qualification exercise applied to fluid samples and their derivatives in the context of urological diseases.

2.
N Biotechnol ; 78: 13-21, 2023 Dec 25.
Article in English | MEDLINE | ID: mdl-37730172

ABSTRACT

Analysis of circulating cell-free DNA (ccfDNA) isolated from liquid biopsies is rapidly being implemented into clinical practice. However, diagnostic accuracy is significantly impacted by sample quality and standardised approaches for assessing the quality of ccfDNA are not yet established. In this study we evaluated the application of nucleic acid "spike-in" control materials to aid quality control (QC) and standardisation of cfDNA isolation for use in in vitro diagnostic assays. We describe an approach for the design and characterisation of in-process QC materials, illustrating it with a spike-in material containing an exogenous Arabidopsis sequence and DNA fragments approximating to ccfDNA and genomic DNA lengths. Protocols for inclusion of the spike-in material in plasma ccfDNA extraction and quantification of its recovery by digital PCR (dPCR) were assessed for their suitability for process QC in an inter-laboratory study between five expert laboratories, using a range of blood collection devices and ccfDNA extraction methods. The results successfully demonstrated that spiking plasmid-derived material into plasma did not deleteriously interfere with endogenous ccfDNA recovery. The approach performed consistently across a range of commonly-used extraction protocols and was able to highlight differences in efficiency and variability between the methods, with the dPCR quantification assay performing with good repeatability (generally CV <5%). We conclude that initial findings demonstrate that this approach appears "fit for purpose" and spike-in recovery can be combined with other extraction QC metrics for monitoring the performance of a process over time, or in the context of external quality assessment.


Subject(s)
Cell-Free Nucleic Acids , Cell-Free Nucleic Acids/analysis , Liquid Biopsy/methods , Quality Control , DNA , Polymerase Chain Reaction/methods
3.
N Biotechnol ; 72: 29-37, 2022 Dec 25.
Article in English | MEDLINE | ID: mdl-36049650

ABSTRACT

An annual External Quality Assurance (EQA) program has been provided to processing laboratories over the last ten years, allowing them to assess the performance of their processing methods, such as nucleic acid extractions or peripheral blood mononuclear cell (PBMC) isolation and cryopreservation. The objective of this study was to perform a global analysis on almost 1000 EQA scheme/participant data in order to assess (i) the impact of critical preanalytical factors on quantitative or qualitative attributes of different types of specimens and (ii) laboratory performance pattern over time. Statistical analysis was performed within each EQA scheme based on categorized preanalytical data provided by the participants and on centralized measurements of relevant quality attributes of the produced specimens (z-scores): DNA, cell-free (cf)DNA or RNA extraction from blood, DNA or RNA extraction from formalin fixed tissue, DNA or RNA extraction from frozen tissue, DNA extraction from saliva or stool, viable PBMC isolation and cryopreservation. The most critical preanalytical factors in nucleic acid extraction schemes were the nucleic acid extraction method and kit, the elution buffer, the enzymes used during extraction, the input material quantity and the storage temperature. Several indications of laboratory performance improvement over time could be seen. The conclusions are that EQA for processing methods provides unique evidence-based insights into the impact of preanalytical factors and the comparative performance of different processing methods and kits, while supporting laboratories in validating their processing methods.


Subject(s)
Laboratories , Leukocytes, Mononuclear , Humans , DNA , RNA
5.
N Biotechnol ; 54: 52-61, 2020 Jan 25.
Article in English | MEDLINE | ID: mdl-31398512

ABSTRACT

Next-generation sequencing (NGS) analyses on DNA derived from archived Formalin-Fixed Paraffin-Embedded (FFPE) clinical material can provide a powerful tool in oncology research and clinical diagnostics. Although several studies have established that NGS can be performed using DNA from FFPE tissue, the accuracy and reproducibility of such analyses, as well as their robustness to the biomolecular quality of the samples used, remains a matter of debate. Excellent reviews have recently been published, providing evidence-based best practices for FFPE DNA extraction. Alternative fixatives exist, although their implementation in clinical practice is difficult. In this article, we present (i) a review of fixed tissue DNA preanalytics with a special focus on DNA extraction and fixed tissue sample qualification and (ii) results from comparisons between different methods of DNA extraction from tissue samples that have been fixed or stabilized by different methods, in terms of NGS metrics and different DNA quality metrics.


Subject(s)
DNA/analysis , DNA/isolation & purification , High-Throughput Nucleotide Sequencing/methods , Pre-Analytical Phase/standards , Tissue Fixation , DNA/genetics , High-Throughput Nucleotide Sequencing/standards , Humans , Quality Control
6.
J Immunol Methods ; 465: 13-19, 2019 02.
Article in English | MEDLINE | ID: mdl-30496732

ABSTRACT

BACKGROUND: Uncontrolled preanalytical variables can reduce the accuracy and reproducibility of downstream analytical results from peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs were isolated from EDTA and citrate-anticoagulated blood samples, obtained from healthy subjects and patients with inflammatory and infectious conditions. PBMC-derived RNA samples were examined for gene expression changes induced by extended blood pre-centrifugation delays at 4 °C and RT. We used Taqman RTqPCR to evaluate the combination of two target genes for their "diagnostic performance" in identifying EDTA and citrate-anticoagulated PBMC samples with extended pre-centrifugation times. RESULTS: We established the PBMC preanalytical score, a gene expression metric to asses the PBMC quality related to the pre-centrifugation delay at room temperature for different anticoagulants. The PBMC preanalytical score measurement can identify: CONCLUSION: The proposed PBMC preanalytical score may enable objective PBMC sample qualification for downstream applications, which may be influenced by blood precentrifugation delays.


Subject(s)
Calcium-Binding Proteins/biosynthesis , Gene Expression Regulation , Interleukin-8/biosynthesis , Leukocytes, Mononuclear/metabolism , alpha-Mannosidase/biosynthesis , Adult , Aged , Female , Humans , Leukocytes, Mononuclear/cytology , Male , Middle Aged
7.
Biopreserv Biobank ; 16(6): 467-476, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30234371

ABSTRACT

Although there are millions of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown fixation conditions. We analyzed FFPE tissue biospecimens as part of the NCI Biospecimen Preanalytical Variables (BPV) program to identify microRNA (miRNA) markers for fixation time. miRNA was extracted from kidney and ovary tumor FFPE blocks (19 patients, cold ischemia ≤2 hours) with 6, 12, 24, and 72 hours fixation times, then analyzed using the WaferGen SmartChip platform (miRNA chip with 1036 miRNA targets). For fixation time, principal component analysis of miRNA chip expression data separated 72 hours fixed samples from 6 to 24 hours fixed samples. A set of small nuclear RNA (snRNA) targets was identified that best determines fixation time and was validated using a second independent cohort of seven different tissue types. A customized assay was then developed, based on a set of 24 miRNA and snRNA targets, and a simple "snoRNA score" defined. This score detects FFPE tissue samples with fixation for 72 hours or more, with 79% sensitivity and 80% specificity. It can therefore be used to assess the fitness-for-purpose of FFPE samples for DNA or RNA-based research or clinical assays, which are known to be of limited robustness to formalin overfixation.


Subject(s)
RNA, Small Nucleolar/analysis , Tissue Banks/standards , Tissue Fixation/methods , Female , Fixatives , Formaldehyde , Humans , Kidney/chemistry , MicroRNAs/analysis , MicroRNAs/genetics , MicroRNAs/standards , Oligonucleotide Array Sequence Analysis/methods , Ovarian Neoplasms/chemistry , Ovarian Neoplasms/genetics , Paraffin Embedding , Quality Control , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/standards , Tissue Fixation/standards
8.
Alzheimers Res Ther ; 10(1): 87, 2018 08 28.
Article in English | MEDLINE | ID: mdl-30153863

ABSTRACT

BACKGROUND: In this study, we tested to which extent possible between-center differences in standardized operating procedures (SOPs) for biobanking of cerebrospinal fluid (CSF) samples influence the homogeneity of the resulting aliquots and, consequently, the concentrations of the centrally analyzed selected Alzheimer's disease biomarkers. METHODS: Proficiency processing samples (PPSs), prepared by pooling of four individual CSF samples, were sent to 10 participating centers, which were asked to perform aliquoting of the PPSs into two secondary aliquots (SAs) under their local SOPs. The resulting SAs were shipped to the central laboratory, where the concentrations of amyloid beta (Aß) 1-42, pTau181, and albumin were measured in one run with validated routine analytical methods. Total variability of the concentrations, and its within-center and between-center components, were analyzed with hierarchical regression models. RESULTS: We observed neglectable variability in the concentrations of pTau181 and albumin across the centers and the aliquots. In contrast, the variability of the Aß1-42 concentrations was much larger (overall coefficient of variation 31%), with 28% of the between-laboratory component and 10% of the within-laboratory (i.e., between-aliquot) component. We identified duration of the preparation of the aliquots and the centrifugation force as two potential confounders influencing within-center variability and biomarker concentrations, respectively. CONCLUSIONS: Proficiency processing schemes provide objective evidence for the most critical preanalytical variables. Standardization of these variables may significantly enhance the quality of the collected biospecimens. Studies utilizing retrospective samples collected under different local SOPs need to consider such differences in the statistical evaluations of the data.


Subject(s)
Alzheimer Disease/cerebrospinal fluid , Amyloid beta-Peptides/cerebrospinal fluid , Laboratory Proficiency Testing/standards , Peptide Fragments/cerebrospinal fluid , Albumins/cerebrospinal fluid , Biomarkers/cerebrospinal fluid , Humans , Phosphorylation , Reproducibility of Results , tau Proteins/cerebrospinal fluid , tau Proteins/metabolism
9.
Clin Chem Lab Med ; 56(7): 1054-1062, 2018 06 27.
Article in English | MEDLINE | ID: mdl-29425105

ABSTRACT

BACKGROUND: Longer pre-centrifugation times alter the quality of serum and plasma samples. Markers for such delays in sample processing and hence for the sample quality, have been identified. METHODS: Twenty cytokines in serum, EDTA plasma and citrate plasma samples were screened for changes in concentration induced by extended blood pre-centrifugation delays at room temperature. The two cytokines that showed the largest changes were further validated for their "diagnostic performance" in identifying serum or plasma samples with extended pre-centrifugation times. RESULTS: In this study, using R&D Systems ELISA kits, EDTA plasma samples and serum samples with a pre-centrifugation delay longer than 24 h had an IL16 concentration higher than 313 pg/mL, and an IL8 concentration higher than 125 pg/mL, respectively. EDTA plasma samples with a pre-centrifugation delay longer than 48 h had an IL16 concentration higher than 897 pg/mL, citrate plasma samples had an IL8 concentration higher than 21.5 pg/mL and serum samples had an IL8 concentration higher than 528 pg/mL. CONCLUSIONS: These robust and accurate tools, based on simple and commercially available ELISA assays can greatly facilitate qualification of serum and plasma legacy collections with undocumented pre-analytics.


Subject(s)
Interleukin-16/blood , Interleukin-8/blood , Adult , Arthritis, Rheumatoid/blood , Biomarkers/blood , Blood Chemical Analysis/methods , Centrifugation , Cohort Studies , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Pancreatitis/blood , ROC Curve , Specimen Handling , Temperature , Time Factors , Young Adult
10.
Am J Clin Pathol ; 146(1): 25-40, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27402607

ABSTRACT

OBJECTIVES: To evaluate the PAXgene tissue fixation system. METHODS: Clinical biospecimens (n = 46) were divided into PAXgene-fixed paraffin-embedded (PFPE), formalin-fixed paraffin-embedded (FFPE), and fresh-frozen (FF) blocks. PFPE and FFPE sections were compared for histology (H&E staining) and immunohistochemistry (14 antibodies) using tissue microarrays. PFPE, FFPE, and FF samples were compared in terms of RNA quality (RNA integrity number, polymerase chain reaction [PCR] amplicon length, and quantitative reverse transcription PCR), DNA quality (gel electrophoresis and methylation profiling) and protein quality (liquid chromatography-mass spectrometry [LC-MS/MS]). RESULTS: PFPE protocol optimization was required in most cases and is described. RNA extracted from PFPE sections was considerably less degraded than that from FFPE sections but more degraded than that from FF blocks. Genomic-length DNA was extracted from PFPE and FF biospecimens, and methylation profiling showed PFPE and FF biospecimens to be almost indistinguishable. Only degraded DNA was extracted from FFPE biospecimens. PFPE sections yielded peptides that were slightly less amenable to LC-MS/MS analysis than FFPE sections, but FF gave slightly better results. CONCLUSIONS: While it cannot be envisaged that PAXgene will replace formalin in a routine clinical setting, for specific projects or immunodiagnostics involving biospecimens destined for immunohistochemical or histologic staining and DNA or RNA analyses, PAXgene is a viable option.


Subject(s)
Gene Expression Profiling/methods , Tissue Fixation/methods , Acetic Acid , Adult , Aged , Carcinoma/diagnosis , Colonic Neoplasms/diagnosis , Ethanol , Female , Humans , Immunohistochemistry , Lung Neoplasms/diagnosis , Male , Methanol , Middle Aged , Paraffin Embedding , Polymerase Chain Reaction , Proteomics/methods , Pulmonary Aspergillosis/diagnosis , Tissue Array Analysis
11.
J Alzheimers Dis ; 52(1): 51-64, 2016 03 01.
Article in English | MEDLINE | ID: mdl-26967210

ABSTRACT

BACKGROUND: Assay-vendor independent quality control (QC) samples for neurochemical dementia diagnostics (NDD) biomarkers are so far commercially unavailable. This requires that NDD laboratories prepare their own QC samples, for example by pooling leftover cerebrospinal fluid (CSF) samples. OBJECTIVE: To prepare and test alternative matrices for QC samples that could facilitate intra- and inter-laboratory QC of the NDD biomarkers. METHODS: Three matrices were validated in this study: (A) human pooled CSF, (B) Aß peptides spiked into human prediluted plasma, and (C) Aß peptides spiked into solution of bovine serum albumin in phosphate-buffered saline. All matrices were tested also after supplementation with an antibacterial agent (sodium azide). We analyzed short- and long-term stability of the biomarkers with ELISA and chemiluminescence (Fujirebio Europe, MSD, IBL International), and performed an inter-laboratory variability study. RESULTS: NDD biomarkers turned out to be stable in almost all samples stored at the tested conditions for up to 14 days as well as in samples stored deep-frozen (at - 80°C) for up to one year. Sodium azide did not influence biomarker stability. Inter-center variability of the samples sent at room temperature (pooled CSF, freeze-dried CSF, and four artificial matrices) was comparable to the results obtained on deep-frozen samples in other large-scale projects. CONCLUSION: Our results suggest that it is possible to replace self-made, CSF-based QC samples with large-scale volumes of QC materials prepared with artificial peptides and matrices. This would greatly facilitate intra- and inter-laboratory QC schedules for NDD measurements.


Subject(s)
Clinical Chemistry Tests/standards , Dementia/blood , Dementia/cerebrospinal fluid , Amyloid beta-Peptides/blood , Amyloid beta-Peptides/cerebrospinal fluid , Animals , Anti-Bacterial Agents/pharmacology , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Cattle , Humans , Peptide Fragments/blood , Peptide Fragments/cerebrospinal fluid , Quality Control , Reference Standards , Serum Albumin, Bovine/analysis , Sodium Azide/pharmacology , Time Factors , Tissue Preservation/methods , tau Proteins/blood , tau Proteins/cerebrospinal fluid
12.
Biopreserv Biobank ; 14(2): 89-98, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26886348

ABSTRACT

Several approaches to the preservation of biological materials at ambient temperature and the relative impact on sample stability and degradation are reviewed, with a focus on nucleic acids. This appraisal is undertaken within the framework of biobank risk, quality management systems, and accreditation, with a view to assessing how best to apply ambient temperature sample storage to ensure stability, reduce costs, improve handling logistics, and increase the efficiency of biobank procedures.


Subject(s)
Biological Specimen Banks/organization & administration , Nucleic Acids , Preservation, Biological/methods , Temperature , Quality Control
13.
Biopreserv Biobank ; 13(4): 296-8, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26186497

ABSTRACT

This biospecimen research case study illustrates the importance of a neglected pre-analytical factor, the polypropylene type of storage tubes. We measured amyloid ß1-42 peptide and showed that a non-irradiated, homopolymer type of polypropylene has the lowest adsorption properties.


Subject(s)
Alzheimer Disease/cerebrospinal fluid , Amyloid beta-Peptides/cerebrospinal fluid , Polypropylenes/chemistry , Specimen Handling/methods , Adsorption , Alzheimer Disease/diagnosis , Amyloid beta-Peptides/chemistry , Biological Specimen Banks , Biomarkers/chemistry , Diagnostic Errors , Enzyme-Linked Immunosorbent Assay , Humans , Polymers/chemistry , Temperature
14.
Electrophoresis ; 36(17): 2072-81, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25998866

ABSTRACT

The analytical and clinical validity of analyses of RNA samples destined for clinical diagnosis and therapeutic management is directly impacted by RNA quality. RNA is affected by heat, enzymatic degradation, and Ultraviolet (UV) light. RNA from three eukaryotic cell lines was degraded by heat, RNase, or UV light. RNA integrity values obtained with the benchmark Agilent Bioanalyzer 2100 system were compared with those from the more recent QIAxcel Advanced system. The application of this novel method has allowed us to unravel differences between RNA biophysical and biochemical degradation modes. Agilent RNA integrity number (RIN) and QIAxcel RIS were comparable in heat-degraded and RNase III-degraded RNA. Agilent RIN and QIAxcel RIS were comparable at a RIN decision level of 7 in UV-degraded RNA but not overall. The QIAxcel RIS method was more precise than Agilent RIN for RIN<8, while the inverse was true for RIN≥8. Greater degradation of mRNA and rRNA in UV-damaged samples hampered the Agilent RIN calculation algorithm. Overall, RIS was more robust than RIN for assessing RNA integrity. The ΔΔCt-values for heat- and UV-degraded RNA samples showed slightly higher correlation with RIS than with RIN. RNA integrity can be used to categorize RNA samples for suitability for downstream gene expression analyses, independently of the RNA degradation mechanism. The new method QIAxcel is more robust and therefore allows more accurate categorization of compromised RNA samples.


Subject(s)
RNA/analysis , RNA/radiation effects , Electrophoresis, Capillary , HeLa Cells , Humans , Jurkat Cells , Quality Control , RNA/chemistry , RNA/standards , Ultraviolet Rays
15.
Biopreserv Biobank ; 13(3): 152-63, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25830476

ABSTRACT

BACKGROUND: This article is part of a series of publications providing formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report the optimization and validation for fitness-for-purpose of automated and manual protocols for isolating peripheral blood mononuclear cells (PBMCs) from whole blood, and compare the two methods. METHODS: The manual method was optimized for whole blood centrifugation speed, gradient type (Ficoll, Leucosep, CPT), and freezing method (Mr Frosty, Controlled Rate Freezing). Various parameters of the automated protocol using a CPT gradient on a Tecan liquid handler were optimized. Optimal protocols were validated in parallel for reproducibility and robustness. Optimization and validation were assessed in terms of cell yield, viability, recovery, white blood cell (WBC) subpopulation distribution, gene expression, and lymphoblastoid cell line (LCL) transformation. RESULTS: An initial centrifugation of whole blood at 2000 g was considered optimal for further processing, allowing isolation of plasma and PBMCs from a single sample. The three gradients gave similar outcomes in terms of cell yield, viability, and WBC subpopulation distribution. Ficoll showed some advantages and was selected for further evaluations. Optimization of the automated protocol script using a CPT gradient gave 61% cell recovery. No significant differences in quality, quantity, and WBC subpopulation distribution were seen between the two freezing methods, and Mr. Frosty was selected. The manual and automated protocols were reproducible in terms of quantity, recovery, viability, WBC subpopulation distribution, gene expression, and LCL transformation. Most (75%-100%) of the 13 robustness parameters were accepted for both methods with an 8 h pre-centrifugation delay versus 38%-85% after 24 h. Differences identified between the automated and manual methods were not considered consequential. CONCLUSIONS: We validated the first fully automated method for isolating viable PBMCs, including RNA analysis and generation of LCLs. We recommend processing within 8 h of blood collection.


Subject(s)
Cell Separation/methods , Leukocytes, Mononuclear/cytology , Automation , Blood Cell Count , Cell Survival , Freezing , Humans , Leukocytes/cytology , Reproducibility of Results
16.
Biopreserv Biobank ; 12(3): 206-16, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24955735

ABSTRACT

The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed, in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature, dry ice, and liquid nitrogen) and in different preservation media (serum with cryoprotectant, commercial cryopreservation solution, and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion, flow cytometry, Cell Analysis System cell counting (CASY)), and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However, we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions, satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells.


Subject(s)
Blood Preservation/methods , Blood Specimen Collection/methods , Cryoprotective Agents/pharmacology , Nitrogen/pharmacology , Cell Survival , Humans , Jurkat Cells , Leukocytes, Mononuclear/cytology , Quality Control , Temperature
17.
Biopreserv Biobank ; 12(2): 151-3, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24749883

ABSTRACT

This case study illustrates the usefulness of the DNA fingerprinting method in biobank quality control (QC) procedures and emphasizes the need for detailed and accurate record keeping during processing of biological samples. It also underlines the value of independent third-party assessment to identify points at which errors are most likely to have occurred when unexpected results are obtained from biospecimens.


Subject(s)
Biological Specimen Banks/standards , DNA Fingerprinting , Case-Control Studies , DNA/analysis , DNA/isolation & purification , Electrophoresis, Agar Gel , Humans , Quality Control
18.
Biopreserv Biobank ; 11(5): 316-8, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24835263

ABSTRACT

Proteomic research requires high-quality, standardized samples. Quality control (QC) biomarkers, which are sensitive to the collection, processing or storage conditions, would be useful tools to identify compromised samples. This study evaluates the usefulness of renal lithostatine as a QC tool for urine sample processing in daily biobank work. Four factors (pre-analytical variations) were examined for their effect on renal lithostatine as measured by ELISA: time from sample collection to centrifugation, number of specimen freeze-thaw cycles, specimen preservation with protease inhibitors, and the inclusion or exclusion of urinary sediment.


Subject(s)
Biomarkers/urine , Lithostathine/urine , Biological Specimen Banks , Centrifugation , Freezing , Humans , Male , Quality Control , Specimen Handling/methods
19.
Biopreserv Biobank ; 11(3): 161-5, 2013 Jun.
Article in English | MEDLINE | ID: mdl-24850093

ABSTRACT

Preanalytical conditions applied during sample collection and processing can affect the detection or quantification of unstable phosphoprotein biomarkers. We evaluated the consequences of tissue stabilization and protein extraction methods on phosphoprotein analysis. The effects of stabilization techniques (heat stabilization, snap-freezing) and time on the levels of phosphoproteins, including phospho-Akt, p-ERK 1/2, p-IkBα, p-JNK, and p38 MAPK, were evaluated using a BioPlex phosphoprotein assay. Additionally, two different protein extraction protocols, using different extraction buffers (8 M urea buffer, or Bio-Rad buffer without urea) were tested. For snap-frozen samples, protein extraction yields were comparable with the two buffer systems. For heat-stabilized samples, total protein yields were significantly lower following extraction in non-urea buffer. However, the concentrations of specific phosphoproteins were significantly higher in heat-stabilized samples than in the corresponding snap-frozen samples, indicating that this tissue processing method better preserved phosphoproteins. Significant differences were found between the measured phosphoprotein levels in heat-stabilized and snap-frozen tissue, suggesting that alterations occur very rapidly after tissue excision. Our results suggest that heat stabilization can be used as a tissue processing method for subsequent phosphoprotein analyses, but also suggest that the BioPlex phosphoprotein assay could be used as a possible quality control method to assess tissue sample integrity.


Subject(s)
Phosphoproteins/analysis , Phosphoproteins/isolation & purification , Specimen Handling/methods , Tissue Preservation/methods , Animals , Biomarkers/analysis , Brain , Buffers , Male , Mice , Mice, SCID , Reference Standards , Temperature
20.
Bioelectrochemistry ; 73(2): 151-4, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18585980

ABSTRACT

Changes of the intracellular Ca2+ content in human red blood cells (RBCs) in glycerol-containing solutions and after freeze-thawing the cells with glycerol and subsequent deglycerolization were investigated with the Ca2+-sensitive fluorescent dye fluo-4 using fluorescence microscopy. In the glycerol-containing solutions the Ca2+ content increased when compared with a physiological medium (Hepes buffered saline solution (HBSS)). This effect was most likely a result of an inhibition of the Ca2+ pump. After inhibiting the Ca2+ pump using o-vanadate, the Ca2+ uptake was not significantly different in the cells in glycerol-containing and physiological medium. Freeze-thawing and deglycerolization of RBCs resulted in a more pronounced increase in the Ca2+ content. Also in this case, the Ca2+ pump seemed to play a major role.


Subject(s)
Calcium/metabolism , Cryoprotective Agents/pharmacology , Erythrocytes/drug effects , Erythrocytes/metabolism , Glycerol/pharmacology , Intracellular Space/drug effects , Cation Transport Proteins/metabolism , Erythrocytes/cytology , Fluorescent Dyes/metabolism , Freezing , Humans , Incubators , Intracellular Space/metabolism , Mannitol/pharmacology , Microscopy, Fluorescence , Sensitivity and Specificity , Vanadates/pharmacology
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