Handling Of Biological Samples Research Articles

Delineating monoclonal antibody specificity by mass spectrometry.

The inoculation of animals with complex biological samples is aiming at the discovery of novel disease biomarkers, present in the biological specimens, as well as the production of rare reagents that will facilitate the ultra-sensitive analysis of the biomolecules' native form. In the present study, we initially propose a general workflow concerning the handling of biological samples, as well as the monoclonal antibody production. Furthermore, we established protocols for the reliable and reproducible identification of antibody specificity using various immuno-affinity purification techniques coupled to mass spectrometry. Our data suggest that processed biological fluids can be used for the production of mAbs targeting proteins of varying abundance, and that various immuno-MS protocols can offer great capabilities for the mAb characterization procedure.

Impact of Collection and Storage of Lung Tumor Tissue on Whole Genome Expression Profiling

Gene expression profiling could assist in revealing biomarkers of lung cancer prognosis and progression. The handling of biological samples may strongly influence global gene expression, a fact that has not been addressed in many studies. We sought to investigate the changes in gene expression that may occur as a result of sample processing time and conditions. Using Illumina Human WG-6 arrays, we quantified gene expression in lung carcinoma samples from six patients obtained at chest opening before and immediately after lung resection with storage in RNAlater [T1a((CO)) and T1b((LR))], after receipt of the sample for histopathology, placed in RNAlater [T2a((HP))]; snap frozen [T2b((HP.SF))]; or snap frozen and stored for 1 week [T2c((HP.SFA))], as well as formalin-fixed, paraffin-embedded (FFPE) block samples. Sampling immediately after resection closely represented the tissue obtained in situ, with only 1% of genes differing more than twofold [T1a((CO)) versus T1b((LR))]. Delaying tissue harvest for an average of 30 minutes from the operating theater had a significant impact on gene expression, with approximately 25% of genes differing between T1a((CO)) and T2a((HP)). Many genes previously identified as lung cancer biomarkers were altered during this period. Examination of FFPE specimens showed minimal correlation with fresh samples. This study shows that tissue collection immediately after lung resection with conservation in RNAlater is an optimal strategy for gene expression profiling.

What additional factors beyond state-of-the-art analytical methods are needed for optimal generation and interpretation of biomonitoring data?

BackgroundThe routine use of biomonitoring (i.e., measurement of environmental chemicals, their metabolites, or specific reaction products in human biological specimens) to assess internal exposure (i.e., body burden) has gained importance in exposure assessment.ObjectivesSelection and validation of biomarkers of exposure are critical factors in interpreting biomonitoring data. Moreover, the strong relation between quality of the analytical methods used for biomonitoring and quality of the resulting data is well understood. However, the relevance of collecting, storing, processing, and transporting the samples to the laboratory to the overall biomonitoring process has received limited attention, especially for organic chemicals.DiscussionWe present examples to illustrate potential sources of unintended contamination of the biological specimen during collection or processing procedures. The examples also highlight the importance of ensuring that the biological specimen analyzed both represents the sample collected for biomonitoring purposes and reflects the exposure of interest.ConclusionsBesides using high-quality analytical methods and good laboratory practices for biomonitoring, evaluation of the collection and handling of biological samples should be emphasized, because these procedures can affect the samples integrity and representativeness. Biomonitoring programs would be strengthened with the inclusion of field blanks.

Dispensing of Very Low Volumes of Ultra High Viscosity Alginate Gels: A New Tool for Encapsulation of Adherent Cells and Rapid Prototyping of Scaffolds and Implants

We present a tool for dispensing very low volumes (20 nL or more) of ultra high viscosity (UHV) medical-grade alginate hydrogels. It uses a modified piezo-driven micrometering valve, integrated into a versatile system that allows fast prototyping of encapsulation procedures and scaffold production. Valves show excellent dispensing properties for UHV alginate in concentrations of 0.4% and 0.7% and also for aqueous liquids. An optimized process flow provides excellent handling of biological samples under sterile conditions. This technique allows the encapsulation of adherent cells and structuring of substrates for biotechnology and regenerative medicine. A variety of cell lines showed at least 70% viability after encapsulation (including cell lines that are relevant in regenerative medicine like Hep G2), and time-lapse analysis revealed cells proliferating and showing limited motility under alginate spots. Cells show metabolic activity, gene product expression, and physiological function. Encapsulated cells have contact with the substrate and can exchange metabolites while being isolated from macromolecules in the environment. Contactless dispensing allows structuring of substrates with alginate, isolation and transfer of cell-alginate complexes, and the dispensing of biological active hydrogels like extracellular matrix-derived gels.

Analysis of acute myelogenous leukemia: preparation of samples for genomic and proteomic analyses.

During the last decade, several large clinical studies have demonstrated that analysis of chromosomal abnormalities is an essential basis for therapeutic decisions in patients with acute myelogenous leukemia (AML), and cytogenetic studies should now be regarded as mandatory both for routine treatment and as a part of clinical investigations in AML. However, new techniques for detailed genetic characterization and analysis of gene expression as well as protein modulation will become important in the further classification of AML subsets and the development of risk-adapted therapeutic strategies. In this context, we emphasize the importance of population-based clinical studies as a basis for future therapeutic guidelines. Such studies will then require the inclusion of patients at small clinical centers without specialized hematological research laboratories. To document a high and uniform quality of the laboratory investigations, it will be necessary to collect material for later analysis in selected laboratories. In this article, we describe current methods for collection of biological samples that can be used for later preparation of DNA, RNA, and proteins. With the use of gradient-separated AML cells, it should be possible to establish the necessary techniques for collection and handling of biological samples even at smaller centers, and complete collections from all included patients should then be possible even in population-based clinical studies.

Prepper, PhD: DNA purification robotics system

This article describes the motivation and engineering trade-offs in the development process of a robot to greatly speed the handling of biological samples. The robot is called Prepper, PhD, (see H.R. Gamer et al., Scientific Computing and Automation, vol.9, no.4, p.61-8, 1993) named after the biological slang term-pop the process to extract and purify DNA from cells. The development of the Prepper, PhD, system started in 1991 with the construction of an alpha prototype that was merely a proof-of-principle. Since then, the author has advanced the system into a beta test unit that is being operated in the field. Soon, a full blown commercial unit will be introduced. The software is fault tolerant and capable of completely automatic operations as well as manual operations. The principle goal of the design is to perform biological manipulations rather than advance the state of the art of robotics. A study of the biological protocols involved was made prior to constructing any hardware. They were redesigned to integrate well on a robot and were simplified without any performance degradation. Simply integrating commercial robotics components would lead to an unacceptably expensive system. By limiting the motion of each component to only one direction, mechanical and control complexity was greatly reduced, yielding acceptable costs. >

Pranoprofenのヒトにおける体内動態

The pharmacokinetics of pranoprofen, 2-(5H-[1]benzopyrano[2,3-b]pyridin-7-yl) propionic acid (I) in humans were examined. 1-O-Acylglucuronide of I (II) and its isomer (III) were isolated from the human urine after oral administration of I. The stability of II was tested in order to establish the suitable conditions for the storage and handling of biological samples. Maximum stability of II was found at pH 3-4. Unchanged drugs, II and III were detected in the human plasma after oral administration of I. Plasma concentrations of these compounds reached the maximum at 1-2 h after the administration and thereafter decreased biphasically. From the urinary sample, 1-O-acylglucoside of I (IV) was detected in addition to unchanged drugs, II and III. Within 24 h after dosing, 1.3, 84.0, 7.0 and 0.6% of the dose were excreted as I, II, III and IV, respectively and the total urinary excretion amounted to 92.9% in term of unchanged drug.

High-performance liquid chromatographic determination of tolmetin, tolmetin glucuronide and its isomeric conjugates in plasma and urine

A rapid and sensitive analytical procedure is described for the simultaneous measurement of tolmetin (T), tolmetin glucuronide (1β-TG) and the isomers of tolmetin glucuronide in plasma and urine. A reversed-phase liquid chromatographic system is used with an ion-pairing mobile phase of methanol-tetrabutylammonium hydrogensulfate buffered to pH 4.5 and kept at a constant temperature of 50°C. Detection is by UV at 313 nm. Plasma (0.5 ml) and urine (0.1 ml) are collected in pre-cooled containers and immediately adjusted to pH 3.0 to minimize TG isomerization and hydrolysis. Samples are then deproteinated with acetonitrile, the supernatant is evaporated to dryness and reconstituted in an acetate buffer (pH 4.5), and 50 μl are injected onto the system. Using zomepirac as the internal standard, the measurable, linear concentration ranges are 0.05–50 μg/ml for T in plasma and 0.025–50 μg/ml for T in urine. Chromatographic peaks representing T,1β-TG and three isomers of TG were identified, all with retention times less than 10 min. The need for special handling of biological samples is discussed.

Handling of biological samples in the determination of the anti-neoplastic drug mitomycin C

A study to ascertain suitable conditions for handling biological samples from patients, treated with the antibiotic mitomycin C (MMC), with the objective of improving the accuracy and reliability of the determination is described. Situations frequently occurring in medical practice are simulated to optimize procedures for reliable and reproducible sampling, sample treatment and determination of MMC. Continuation of drug partitioning in whole blood after sampling can be prevented by immediate cooling in ice before the separation of plasma from cells. The adjustment of the pH of urine samples is shown to be particularly important since a low urinary pH causes decomposition of MMC; moreover, it may decrease extraction recovery. Furthermore, long-term exposure of samples to daylight induces drug decomposition. Frozen storage of plasma and urine samples for periods greater than 3 weeks is to be avoided as this results in a considerable drop in MMC concentration. Repeated cycles of freezing and thawing are shown to have no effect upon the analytical results (6 cycles tested). The analysis of extracts of biological samples may take place up to at least 24 h after their preparation without measurable loss of analyte.

Practical methods for biological NMR sample handling

The ability of NMR parameters T 1 and T 2 to be used to distinguish one tissue from another and diseased from normal tissues has wide application in diagnostic medicine. Measurement of such values in vitro on excised tissues and fluids was the basis for fundamental discoveries which provided the impetus for the development of in vivo clinical whole body NMR imagers. Therefore, as in vivo imaging grows, the need for screening and testing of new ideas in vitro will grow. The success of data collection in vitro depends greatly on the care and understanding with which biological samples are processed. This paper summarizes proven methods for handling of soft and firm biopsy material, blood components, body fluids, and culture cells. The effects of temperature variation and storage parameters are discussed for animal and human samples. The paper is a practical guide to the hows and whys of biological sample handling for NMR.

The collection and handling of biological samples for prostaglandin analysis

The collection and handling of biological samples for prostaglandin analysis