Adaptation of a Commercially Available Soluble P-Selectin ELISA for Automated Frozen Aliquotting

Employing a commercially available soluble P-Selectin (S-PS) ELISA as a model system, the homogeneity of S-PS in sodium citrate plasma was characterized for various freeze rates and the resulting impact on concentration data via ELISA was assessed. Recommendations on adapting and optimizing the ELISA workflow in order to support an assay format fully reliant on frozen aliquotting was formulated and tested. Heterogeneity of S-PS in the form of vertical sedimentation in frozen plasma samples was effectively mitigated for an off-the-shelf commercial ELISA kit. S-PS concentrations for technical replicates of unknown test samples exhibited CV values below 7.5% and were indicative of a well-performing assay. Overall, the data suggests that bioanalytical assays, such as an ELISA, can be qualified and developed with the intent of integrating frozen aliquotting technology for upstream sample handling and the elimination of unnecessary freeze-thaw exposures. Read more >

Evaluating the Impact of Freezing Biologic Fluids on Drug Distribution and Its Impact on Frozen Aliquotting for Quantitative Drug Analysis

Researchers at CryoXtract investigated the homogeneity of small molecules atenolol (6-16% protein boand) and warfarin (99% protein bound) in frozen serum and EDTA plasma prepared using three protocols: snap freeze in LN2 vapor, -80°C freezing, and -20°C freezing. Frozen cores collected from the perimeter of frozen samples were compared to center cores to assess lateral distribution. 50µL, 100µL, and full-length cores (~150µL) were compared to sample remainders and un-cored controls to assess vertical distribution. There was no strong evidence of a lateral gradient being formed. There was evidence of vertical gradient formation which was associated with an increase in freezing temperature / decrease in freeze rate. Despite the presence of moderate vertical gradients, the reproducibility of drug concentratins from cores suggests that frozen aliquotting can be integrated into the workflow of a validated assay. Read more >

Methodology for Assessing Protein Biomarker Distribution in Frozen Biological Fluids Using Frozen Aliquotting Technology

Researchers at CryoXtract have developed a frozen aliquotting technique for the assessment of protein distribution profiles in a frozen matrix. This technique was used to assess distribution of p-selectin in serum and plasma samples SNAP frozen in LN2 vapor, frozen at -80°C, and frozen at -20°C. Frozen cores collected from the perimeter of frozen samples were compared to center cores to assess lateral distribution. 50µL, 100µL, and full-length cores (~150µL) were compared to sample remainders and un-cored controls to assess vertical distribution. For all conditions, no lateral gradients were observed. SNAP frozen cores correlated well with the un-cored control at all vertical depths. When freezing at -80°C, soluble p-selectin began to settle into the bottom two-thirds of the cryovial. At -20°C, a greater degree of settling was observed. While individual biomarkers should be evaluated for distribution upon freezing and analyte stability, SNAP freezing is strongly recommended in conjunction with frozen aliquotting to preserve sample and analyte integrity. Read more >

The Quality Advantage of Using CryoXtract’s CXT350 to Core Frozen Pediatric Thyroid Tissues

Biorepositories worldwide store frozen tissue sections for future analysis. Researchers at the Children's Hospital of Philadelphia processed ten frozen pediatric thyroid samples collected between 2012 and 2015. Tissues were divided in half and processed using freeze-thaw aliquotting and frozen aliquotting. RNA quality (RIN score) was determined via the Agilent 2100 Bioanalyzer. RT-PCR was performed to determine expression of the BRAF gene and several housekeeping genes. Researchers saw much higher variability between samples for the control data (frozen slicing) and freeze-thaw aliquotting vs. frozen aliquotting. The CXT350/353 enables researchers to access and distribute frozen tissue sections without thawing, improving sample quality and consistency. Read more >

Ex vivo Stabilization of Small Molecule Compounds and Peptides in EDTA Plasma for LC-MS/MS Analysis Using Frozen Aliquotting

Drug development and pharmaceutical research relies heavily on animal and human bio-specimens to generate the vast amounts of data needed to bring new drugs to market. However, the sensitivity of some compounds in response to common storage and handling practices can convolute analytical data and obscure scientific outcomes. Furthermore, it is not uncommon for the development time lines of bioanalytical assays to be extended by weeks, or even months, as a result of efforts to identify stabilization protocols for the target compounds. The CXT 750 Frozen Sample Aliquotter, an automated instrument commercialized by CryoXtract Instruments, can enable an intact cold chain work-flow for bioanalysis of freeze-thaw sensitive compounds and bio-specimens such as plasma.
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Advancing Brain Tissue and CNS Sample Handling and Distribution

Drug development and pharmaceutical research relies heavily on animal and human bio-specimens to generate the vast amounts of data needed to bring new drugs to market. However, the sensitivity of some compounds in response to common storage and handling practices can convolute analytical data and obscure scientific outcomes. Furthermore, it is not uncommon for the development time lines of bioanalytical assays to be extended by weeks, or even months, as a result of efforts to identify stabilization protocols for the target compounds. The CXT 750 Frozen Sample Aliquotter, an automated instrument commercialized by CryoXtract Instruments, can enable an intact cold chain work-flow for bioanalysis of freeze-thaw sensitive compounds and bio-specimens such as plasma.
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A Proof-of-Principle Evaluation for the Utilization of the CXT350 Frozen Sample Aliquotter in Brain Tissue Research

An evaluation was performed in an independent laboratory to provide proof-of-principle data for the utilization of frozen aliquotting in Alzheimer's disease (AD) research. The CXT350 Frozen Sample Aliquotter was used to generate frozen aliquots of both frozen human brain tissue and frozen cerebrospinal fluid (CSF) samples. Quantitative bioanalytical assays were performed to measure the presence of myelin basic protein (MBP) and apolipoprotein E (ApoE) in frozen brain tissue cores and CSF cores respectively.
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Effective Feces Aliquotting Workflow Supports Increased Microbiome Research

Global study of the human microbiome has been accelerating due to the growing understanding of how various microbes affect our health and play a role with various diseases. Fecal samples, the primary biosample of relevance in many microbiome studies, are proving to be rich sources of data key to understanding the relationship between the microbial community that lives within the human gut and a broad variety of health and disease indications, such as diabetes, obesity, celiac disease, and autism.
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Overcoming the Challenges of Fecal Sample Handling

The gut microbiome has become an increasingly important system for scientific study with a potentially broad application base in drug development, nutritional research, and preventative and diagnostic medicine. However, the logistics of working with fecal samples pose significant operational and health and safety challenges, and physical handling of these samples is often undesirable. Due to the semi-solid nature of the samples, they are not readily amenable to manual or automated liquid handling methodologies, making it difficult to scale from small pilot studies to larger studies requiring high throughput capabilities.
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Demonstration of a Frozen Sample Aliquotter to Prepare Plasma and Serum Aliquots Without Thawing Frozen Parent Samples

Human biospecimens represent invaluable resources to advance molecular medicine, epidemiology, and biomarker discovery/validation, among other biomedical research. Biobanks typically cryopreserve biospecimens to safeguard their biochemical composition. However, exposing specimens repeatedly to freeze/thaw cycles can degrade their integrity in unforeseen ways. Those biobanks storing liquid samples, thus, regularly make a fundamental compromise at collection time between freezing samples in many small volumes (e.g., 0.5 mL or smaller) or in fewer, larger volumes (e.g., 1.8 mL). The former eliminates the need to expose samples to repeated freeze/thaw cycling, although increasing up-front labor costs, consumables used, and cold storage space requirements. The latter decreases up-front labor costs, consumables, and cold storage requirements, yet exposes samples repeatedly to damaging freeze/thaw cycles when smaller aliquots are needed for analysis. The Rhode Island BioBank at Brown University (RIBB) thoroughly evaluated the performance of an original technology that minimizes a sample’s exposure to freeze/thaw cycling by enabling the automated extraction of frozen aliquots from one single frozen parent sample without thawing it. A technology that eliminates unnecessary sample exposures to freeze/thaw cycles could help protect sample integrity, extend its useful life, and effectively rectify and eliminate the aforementioned need to compromise. This report presents the results of the evaluation, and conclusively demonstrates the technology's ability to extract multiple uniform frozen aliquots from a single cryotube of never-thawed frozen human plasma, which faithfully represent the parent sample when analyzed for typical biochemical analytes, showing a coefficient of variability lower than 5.5%.
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Carryover Test in Frozen Serum Cored by an Automated Frozen Sample Aliquotter

A cleaning protocol for the CryoXtract Automated Frozen Sample Aliquotter (AFSA) was tested for its effectiveness in eliminating sample-to-sample carryover during hands-free frozen sample aliquotting. The instrument’s test platform was used to extract a statistically significant number of frozen cores from frozen bovine serum samples spiked with human DNA and from identical DNA-free frozen controls, processed in alternating succession, with a proprietary cleaning protocol performed in between samples. Measurements of DNA content by a highly sensitive real-time quantitative polymerase chain reaction assay revealed no traces of DNA in the frozen control cores, whereas they revealed the expected DNA amounts in all DNA-containing frozen cores. Analysis of high-copy DNA demonstrated that CryoXtract’s AFSA’s cleaning protocol effectively reduced DNA carryover by more than a million-fold.
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Improved utilization of fresh frozen tissue specimens using Frozen Sample Aliquotter

Frozen human bio-specimens are invaluable resources that advance translational research, molecular medicine, and biomarker discovery. The quality and yield of target analytes, such as RNA, from frozen tissue samples can be directly impacted by the stress and exposure incurred through handling and processing procedures. The novel Frozen Tissue Aliquotter addresses the above issue by maintaining the tissue at an ultralow temperature, thereby extending the usability of the banked tissues. This proprietary instrument allows for the mounting of the specimen to a frozen (e.g. -80°C) fixture, cutting of frozen slides from the frozen sample, and the ability to repeatedly access and remove frozen aliquots from specific regions (e.g. normal, tumor, margin) of the primary tissue in a consistent and safe manner that ensures the preservation of the parent sample integrity. This presentation describes the materials, methods, and data obtained in an independent comparative study, performed by The Collaborative Human Tissue Network (Eastern Division), to evaluate the feasibility of utilizing frozen aliquotting in an application involving the extraction of total RNA from human uterine tissue.
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Methodology for Assessing Protein Biomarker Distribution in Frozen Biological Fluids Using Frozen Aliquotting Technology

Researchers at CryoXtract have developed a frozen aliquotting technique for the assessment of protein distribution profiles in a frozen matrix. This technique was used to assess distribution of p-selectin in serum and plasma samples SNAP frozen in LN2 vapor, frozen at -80°C, and frozen at -20°C. Frozen cores collected from the perimeter of frozen samples were compared to center cores to assess lateral distribution. 50µL, 100µL, and full-length cores (~150µL) were compared to sample remainders and un-cored controls to assess vertical distribution. For all conditions, no lateral gradients were observed. SNAP frozen cores correlated well with the un-cored control at all vertical depths. When freezing at -80°C, soluble p-selectin began to settle into the bottom two-thirds of the cryovial. At -20°C, a greater degree of settling was observed. While individual biomarkers should be evaluated for distribution upon freezing and analyte stability, SNAP freezing is strongly recommended in conjunction with frozen aliquotting to preserve sample and analyte integrity. Read more >