Sample Pooling Approach Research Articles

Serum methylation of GALNT9, UPF3A, WARS, and LDB2 as noninvasive biomarkers for the early detection of colorectal cancer and advanced adenomas

BackgroundEarly detection has proven to be the most effective strategy to reduce the incidence and mortality of colorectal cancer (CRC). Nevertheless, most current screening programs suffer from low participation rates. A blood test may improve both the adherence to screening and the selection to colonoscopy. In this study, we conducted a serum-based discovery and validation of cfDNA methylation biomarkers for CRC screening in a multicenter cohort of 433 serum samples including healthy controls, benign pathologies, advanced adenomas (AA), and CRC.ResultsFirst, we performed an epigenome-wide methylation analysis with the MethylationEPIC array using a sample pooling approach, followed by a robust prioritization of candidate biomarkers for the detection of advanced neoplasia (AN: AA and CRC). Then, candidate biomarkers were validated by pyrosequencing in independent individual cfDNA samples. We report GALNT9, UPF3A, WARS, and LDB2 as new noninvasive biomarkers for the early detection of AN. The combination of GALNT9/UPF3A by logistic regression discriminated AN with 78.8% sensitivity and 100% specificity, outperforming the commonly used fecal immunochemical test and the methylated SEPT9 blood test.ConclusionsOverall, this study highlights the utility of cfDNA methylation for CRC screening. Our results suggest that the combination methylated GALNT9/UPF3A has the potential to serve as a highly specific and sensitive blood-based test for screening and early detection of CRC.

High-throughput screening of LogD by using a sample pooling approach based on the traditional shake flask method

A new approach for screening LogD is presented. The method is based on the shake flask method combined with rapid generic LC-MS/MS bioanalysis by using a sample pooling approach that enables high-throughput screening of LogD or LogP in the drug discovery stage. The method is evaluated by a comparison of measured LogD between single and pooled compounds for a test set of structurally diverse compounds with a wide range of LogD values (from −0.04 to 6.01). Test compounds include 10 commercially available drug standards along with 27 new chemical entities. A good correlation (RMSE = 0.21, R2 = 0.9879) of LogD between the single and pooled compounds was obtained, suggesting that at least 37 compounds can be simultaneously measured with acceptable accuracy. The sample pooling method significantly reduced the number of bioanalysis samples as compared to the single compound measurement by the conventional shake flask method. The impact of DMSO content on LogD measurement was also investigated and the result demonstrated that at least 0.5% DMSO was tolerated in this method. The current new development will facilitate the drug discovery process by more rapidly assessing the LogD or LogP of drug candidates.

Proposal of RT-PCR–Based Mass Population Screening for Severe Acute Respiratory Syndrome Coronavirus 2 (Coronavirus Disease 2019)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing has lagged in many countries because of test kit shortages and analytical process bottlenecks. This study investigated the feasibility and accuracy of a sample pooling approach for wide-scale population screening for coronavirus disease 2019. A total of 940 nasopharyngeal swab samples (934 negative and 6 positive) previously tested for SARS-CoV-2 were deidentified and assigned random numbers for analysis, and 94 pools of 10 samples each were generated. Automated RNA extraction, followed by RT-PCR, was performed in a 96-well plate. Positive pools were identified, and the individual samples were reanalyzed. Of the 94 pools/wells, four were positive [Ct values: N (22.7 to 28.3), ORF1ab (23.3 to 27.2), and internal control (34.4 to 35.4)]. The 40 samples comprising the four pools were identified and reanalyzed individually; six samples were positive, with Ct values of N gene, ORF1ab, and internal control comparable to their respective wells. Additional experiments were performed on samples with high Ct values, and overall results showed 91.6% positive and 100% negative agreement compared with individual testing approach. Thus, 940 samples were tested in 148 reactions compared with 940 reactions in routine screening. The sample pooling strategy may help catch up with testing needs and minimal turnaround times and facilitate enormous savings on laboratory supplies, extraction, and PCR kits currently in short supply.

Brain Tissue Binding of Drugs: Evaluation and Validation of Solid Supported Porcine Brain Membrane Vesicles (TRANSIL) as a Novel High-Throughput Method

Estimating the unbound fraction of drugs in brain has become essential for the evaluation and interpretation of the pharmacokinetics and pharmacodynamics of new central nervous system drug candidates. Dialysis-based methods are considered to be accurate for estimating the fraction unbound in brain; however, these techniques are hampered by a low throughput. In this study, we present a novel, matrix-free, high-throughput method for estimating the unbound fraction, based on a sample pooling approach combining the TRANSIL brain absorption assay with liquid chromatography-mass spectrometry. The base measurement of the TRANSIL approach is the affinity to brain membranes, and this method is used directly to predict the free fraction in brain. The method was evaluated by comparing the free fraction of drugs in brain [f(u,brain) (%)] obtained using the TRANSIL brain absorption assay and equilibrium dialysis methods for a test set of 65 drugs (27 marketed and 38 proprietary drugs). A good correlation (r(2) > 0.93) of f(u,brain) (%) between the TRANSIL brain absorption assay and equilibrium dialysis was observed. Moreover, we compared the lipid composition of rat and porcine brain and analyzed the influence of the brain albumin content on brain tissue binding measurement. The comparison of the lipid composition indicated only minor differences between rat and porcine brain, and albumin appears to have a low impact on brain tissue binding measurements. The TRANSIL brain absorption assay with sample pooling methodology not only significantly reduces the biological matrix required but also increases the throughput, compared with the conventional dialysis methods.

Deletion of the late cornified envelope LCE3B and LCE3C genes as a susceptibility factor for psoriasis

Psoriasis is a common inflammatory skin disease with a prevalence of 2-3% in individuals of European ancestry. In a genome-wide search for copy number variants (CNV) using a sample pooling approach, we have identified a deletion comprising LCE3B and LCE3C, members of the late cornified envelope (LCE) gene cluster. The absence of LCE3B and LCE3C (LCE3C_LCE3B-del) is significantly associated (P = 1.38E-08) with risk of psoriasis in 2,831 samples from Spain, The Netherlands, Italy and the United States, and in a family-based study (P = 5.4E-04). LCE3C_LCE3B-del is tagged by rs4112788 (r(2) = 0.93), which is also strongly associated with psoriasis (P < 6.6E-09). LCE3C_LCE3B-del shows epistatic effects with the HLA-Cw6 allele on the development of psoriasis in Dutch samples and multiplicative effects in the other samples. LCE expression can be induced in normal epidermis by skin barrier disruption and is strongly expressed in psoriatic lesions, suggesting that compromised skin barrier function has a role in psoriasis susceptibility.

Proteome‐ and microarray‐based expression analysis of lymphoma cell lines identifies a p53‐centered cluster of differentially expressed proteins in mantle cell and follicular lymphoma

We used a standardized electrophoresis protocol to identify differentially expressed proteins based on a sample pooling approach comparing three follicular lymphoma and three mantle cell lymphoma-derived cell lines. One hundred and seventy-five consistently differentially expressed proteins were identified out of more than 1600 protein spots per gel. Of these 175 protein spots, 38 of the 41 most highly expressed proteins were identified by MS analysis (MALDI-TOF), involving different cellular programs such as DNA repair (Rad50), cell cycle control (Mad1L1), transcription (SAFB), and apoptosis (Luca-15 protein). Expression data were confirmed by Western blot analysis of identified proteins and 2-D gel hybridization of proteins with known overexpression (G1/S-specific cyclin-D1, apoptosis regulator Bcl-2). Comparison of proteome analysis to RNA expression array data revealed only a modest correlation of RNA and protein level emphasizing the relevance of post-translational regulation in lymphomagenesis (p = 0.36). Most interestingly, additional data bank search identified 13 out of 17 referenced proteins (76%) as members of a TP53-dependent network of cell regulation.

High-Throughput Screening of Drug−Brain Tissue Binding and in Silico Prediction for Assessment of Central Nervous System Drug Delivery

A high-throughput method for rapid screening of in vitro drug-brain homogenate binding is presented. The method is based on a straightforward sample pooling approach combining equilibrium dialysis with liquid chromatography mass spectrometry (LCMS). A strong correlation of fraction unbound in brain (fu) between single compound measurements and 25-pooled compounds (R2 = 0.906) was obtained for a selection of structurally diverse CNS compounds with a wide range of fractions unbound. Effects of brain homogenate dilution and dialysis time were investigated. To the best of our knowledge, it was the first time that we have demonstrated consistent fraction unbound in mouse and rat brain homogenate, revealing the drug-tissue partitioning mechanism predominated by hydrophobic interaction. On the basis of this finding, a generic approach to estimate drug binding to various tissues is proposed. A robust and interpretable QSAR for fu prediction is also presented by statistical modeling.

Parallel optimization and genotyping of multiple single‐nucleotide polymorphism markers by sample pooling approach using cycling‐gradient CE with multiple injections

Increasing importance of single-nucleotide polymorphisms (SNPs) in determination of disease susceptibility or in prediction of therapy response brings attention of many molecular diagnostic laboratories to simple and low-cost SNP genotyping methodologies. We have recently introduced a mutation detection technique based on analysis of homo- and heteroduplex PCR fragments resolved in cycling temperature gradient conditions on a conventional multicapillary-array DNA sequencer. The main advantage of this technique is in its simplicity with no requirement for sample cleanup prior to the analysis. In this report we present a practical application of the technology for genotyping of SNP markers in two separate clinical projects resulting in a combined set of 44 markers screened in over 500 patients. Initially, a design of PCR primers and conditions was performed for each SNP marker. Then, optimization of CE running conditions (limited just to the proper selection of temperature cycling) was performed on pools of 20 DNA samples to increase the probability of having each of the two allele types represented in the sample. After selecting the optimum conditions, screening of markers in patients was performed using a multiple-injection approach for further acceleration of the sample throughput. The rate of successful optimization of experimental conditions without any pre-selection based on the SNP sequence or melting characteristics was 80% from the initial SNP marker candidates. By studying the failed markers, we attempt to identify critical factors enabling successful typing. The presented technique is very useful for low to medium sized SNP genotyping projects mostly applied in pharmacogenomic research as well as in clinical diagnostics. The main advantages include low cost, simple setup and validation of SNP markers.

High-throughput screening of protein binding by equilibrium dialysis combined with liquid chromatography and mass spectrometry

A new approach for screening plasma protein binding is presented. The method is based on equilibrium dialysis combined with rapid generic LC–MS bioanalysis by using a sample pooling approach enabling high-throughput screening of protein binding in the drug discovery phase. The method is evaluated by a comparison of measured unbound free fractions f u (%) between single and pooled compounds for a test set of structurally diverse compounds with a wide range of unbound fractions. Test compounds include 1 acidic and 10 basic drug standards along with 36 new chemical entities. A good correlation ( R 2 > 0.95) of f u (%) between the single and pooled compounds is found, suggesting that at least 10 compounds can be simultaneously measured with acceptable accuracy. A simplified drug–protein binding model is applied to calculate the f u (%) of drugs at various drug and protein concentrations and this is applied to elucidate the applicability of the sample pooling approach from a theoretical standpoint. Moreover, pH shifts in the plasma were observed after dialysis when using different types of buffers and the impact of that on the f u is illustrated in association with their physicochemical properties, in particular the ionization state of compounds by the profile of effective mobility as a function of pH. A new buffer is proposed being able to minimize the pH shift of plasma during the dialysis. In addition, the application of the proposed buffer does not necessarily require adjusting plasma pH before the dialysis and utilizing a CO 2 incubator during the dialysis. The effect of the ionic strengths of different buffers on MS signals is investigated with regard to ion suppression. The sample pooling method not only significantly reduces the plasma volume required but also the number of bioanalysis samples as compared to the single compound measurements by a conventional approach. The new proposed approach is especially beneficial for measuring in vitro protein binding in matrices such as mouse plasma where plasma is available only in limited amounts. The current new development will facilitate the drug discovery process by more rapidly assessing the protein binding potential of drug candidates.

A high throughput approach for simultaneous estimation of multiple synthetic trioxane derivatives using sample pooling for pharmacokinetic studies

The present study describes the application of concept of sample pooling to increase the throughput of pharmacokinetic screening at drug discovery and development stage. An HPLC-UV method for the simultaneous estimation of three synthetic antimalarial compounds 99/357, 99/408 and 99/411 has been developed and validated in rat serum with internal standard for pharmacokinetic profiling. Drug compounds in serum were extracted by two-step liquid–liquid extraction with 2% isopropyl alcohol in n-hexane and quantitated using a validated gradient HPLC-UV method, which was made feasible for all compounds using gradient elution scheme. The method was validated in terms of HPLC reproducibility, linearity, specificity, recovery, accuracy and precision, freeze thaw stability and long-term storage stability. Excellent linear relationships ( r > 0.99) were obtained for calibration as well as analytical standards over a concentration range of 25–1000 ng/ml for three analytes. Recoveries were fond to be >85% for 99/408 and 99/357 and >70% for 99/411. The method developed for three analytes was found to be accurate and precise as bias and percent relative standard deviation (% R.S.D.) values were within limits (<20%). By employing sample pooling approach, plasma level – time profile following single intravenous dose of all three compounds were obtained in a fraction of the time required by conventional single compound dosing and analysis.

Single-Nucleotide Polymorphism Allele Frequencies Determined by Quantitative Kinetic Assay of Pooled DNA

The identification of genes for complex traits relies on the detection of coinheritance of chromosomal regions in related individuals or the sharing of functional or marker alleles among phenotypically similar individuals. Single-nucleotide polymorphisms (SNPs) comprise the most abundant and accessible class of sequence polymorphisms in the genome and are valuable for high-throughput genotyping (1). Genotyping individual DNA samples is expensive in time, reagents, labor, and DNA. These limitations can be overcome by applying a sample-pooling approach for allele frequency determination before genotyping (2)(3). For quantitative allelotyping using pools, equivalent amounts of DNA from multiple individuals are combined in a single-tube assay. Most sample-pooling approaches involve post-PCR processing (4)(5)(6)(7). One pooled allelotyping method uses kinetic allelespecific PCR coupled with SYBR Green I fluorescence detection (8). This method requires two separate PCR reactions for each DNA pool, each of which needs to be performed in parallel. Another pooled allelotyping method uses a bioluminometric assay coupled to a modified primer extension reaction. This method also involves multiple procedures (9). Allelotyping by hybridization to DNA chips has been described (10), but its quantitative accuracy has not been fully evaluated. The 5′ nuclease assay (TaqMan®) uses fluorogenic allele-specific detection probes that allow PCR amplification and allele detection in a single procedure (11). To discriminate alleles, each fluorogenic probe is labeled with a different fluorescent reporter dye. The reaction is kinetically monitored in “real time” to quantify the ratio of the two alleles (12). Taking advantage of the specificity and sensitivity of kinetic sequence detection, we developed a method for estimating SNP allele frequencies in pooled DNA samples. Accuracy was evaluated using constructed pools in which allele frequencies were incrementally varied and by comparing the allele frequencies determined from pooled DNA with allele frequencies determined by individual …

Sample pooling to enhance throughput of brain penetration study

The advent of combinatorial synthesis and high throughput screening in pharmaceutical research has inevitably given rise to a large number of interesting prelead compounds that requires rapid analytical throughput for kinetic characterization. The traditional approach of one-compound-at-a-time bioanalysis has not been able to meet the demand for high productivity of pharmacokinetic screening. This report demonstrates the application of sample pooling in expediting the pharmacokinetic screening, including assessment of brain penetration, of six NK 1 receptor antagonists in rats: CAM 6108 (C1), CAM 6122 (C2), CAM 6178 (C3), CAM 5825 (C4), CAM 6182 (C5), and CAM 6121 (C6). The approach was adopted to avoid complications associated with cocktail dosing where multiple compounds are administered to one animal. The present investigation features individualized dosing (one compound per animal), followed by sample pooling of brain and plasma and bioanalysis via a conventional LC-fluorescence method. Rats were dosed intravenously with each of the six NK 1 receptor antagonists and blood and brain samples were harvested at suitable post-dose time intervals. Plasma or brain homogenate samples from the same time points were pooled into two groups (C1–C3 and C4–C6) for assay. Drug compounds in plasma or brain were extracted by protein precipitation and quantitated using a validated gradient HPLC/fluorescence method, which was made feasible for both groups of compounds with a modification in gradient scheme. Plasma assay precision and accuracy for C1–C6 were ≤4.7% and within ±9.8%, respectively. Brain homogenate assay accuracy for C1–C6 was within ±7.0%. Brain penetration of these compounds was evaluated as the AUC of brain and plasma and their respective brain/plasma AUC ratio. The sample pooling approach helped to quickly identify C1 as the NK 1 receptor antagonist with the greatest extent of brain penetration, followed by C2, C6, C4, C5, and C3 in that order. By employing sample pooling approach, pharmacokinetic parameters and brain penetration of all six compounds were obtained in a fraction of the time required by conventional single compound dosing and analysis.

Sample pooling to expedite bioanalysis and pharmacokinetic research

In the progression from drug discovery to development, not only pharmacokinetic (PK) characterization needed for lead compound selection often becomes a rate-limiting step, but also high volume of routine sample analysis ensued from numerous required biodisposition studies for the lead compounds and their back-ups often place a burdensome hurdle to the throughput of IND and NDA development phases. Higher throughput of PK screening via cocktail dosing has been reported to accelerate PK screening in the discovery phase. However, concerns on drug-drug interactions and other limitations associated with the cocktail M-in-One dosing (multiple compounds per dose per animal) has prompted the present investigation of sample pooling alongside One-in-One dosing strategy (one compound per dose per animal) as an alternative to the cocktail dosing approach. Using traditional HPLC for bioanalysis as an example, the present study illustrate the concept and usefulness of sample pooling that could facilitate the throughput of PK screening and characterization in both discovery and development phases. Six proprietary dopamine D 4 receptor antagonist preleads representing three different chemical classes, used as model compounds (C1–C6), were administered orally to rats. One rat received one compound and three rats were used for each compound. Six unknown plasma samples from six different rats at each time point were pooled. The pooled plasma samples were extracted by a one-step liquid–liquid extraction and concentrations of the six preleads were quantitated simultaneously. By sample pooling, a substantial amount of PK information was obtained at the same time for the six preleads, which requires much less workload than when bioanalysis is dealt with one compound at a time. For the first time in one aspect of innovative bioanalysis, the present investigation has demonstrated that sample pooling following One-in-One dosing can be utilized to enhance the throughput rate in PK screening in discovery phase. The sample pooling approach is likely to be useful in enhancing the throughput of PK characterization in development phase. With the advent of LC-MS and its becoming user-friendly, where separation of drug compounds is no longer an issue, the uniqueness of sample pooling may also pose a new way of thinking in regard to the old ways of handling bioanalysis for traditional PK research.