Poor Quality Samples Research Articles

Assessment of the microbiological quality and safety of minimally processed vegetables sold in Piracicaba, SP, Brazil

The present study shows the results of the microbiological quality and safety of minimally processed vegetables sold in supermarkets and grocery stores located in the city of Piracicaba, SP, Brazil. A total of 100 samples were collected and submitted to enumeration of total coliforms, coliforms at 45°C and generic Escherichia coli using the standard most probable number (MPN) method, in addition to enumeration of total Enterobacteriaceae by plating on MacConkey agar. Moreover, colonies of Enterobacteriaceae were randomly selected and submitted to identification on a MALDI-TOF MS Biotyper™. Samples were also tested for Salmonella spp. according to the ISO 6579:2002 method. The mean count of total coliforms was 2·9±0·5 log MPN per g. For coliforms at 45°C, 20 samples were positive (mean 1·5±1·0 log MPN per g). Generic E. coli was detected in 16 samples (mean 1·4±0·9 log MPN per g) and only one was positive for Salmonella. The mean count of total Enterobacteriaceae was 6·5±1·2 log CFU per g and the most frequent genera identified by MALDI-TOF were Enterobacter (25·9%), Pantoea (9·6%) and Rahnella (9·0%). Overall, results point to poor microbiological quality of a few samples, indicating hygiene failure during their processing. This can pose health risks to consumers, mainly because these products were labelled as sanitized and marketed as ready-to-eat. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the microbiological quality and safety of minimally processed vegetables sold in the city of Piracicaba, SP, Brazil, were evaluated through the detection of Salmonella spp., generic Escherichia coli and other Enterobacteriaceae. The results obtained point to poor microbiological quality and safety of a few samples, as generic E. coli was detected in 16 out of 100 samples and Salmonella was isolated from one of them. This indicates hygiene failures during their processing and health risk to consumers, since these products are usually sold as ready-to-eat and typically require no further heat treatment before consumption.

Evaluation of a Hybrid Capture–Based Pan-Cancer Panel for Analysis of Treatment Stratifying Oncogenic Aberrations and Processes

Stratification of patients for targeted and immune-based therapies requires extensive genomic profiling that enables sensitive detection of clinically relevant variants and interrogation of biomarkers, such as tumor mutational burden (TMB) and microsatellite instability (MSI). Detection of single and multiple nucleotide variants, copy number variants, MSI, and TMB was evaluated using a commercially available next-generation sequencing panel containing 523 cancer-related genes (1.94 megabases). Analysis of formalin-fixed, paraffin-embedded tissue sections and cytologic material from 45 tumor samples showed that all previously known MSI-positive samples (n=7), amplifications (n=9), and pathogenic variants (n=59) could be detected. TMB and MSI scores showed high intralaboratory and interlaboratory reproducibility (eight samples tested in 11 laboratories). For reliable TMB analysis, 20 ng DNA was shown to be sufficient, even for relatively poor-quality samples. A minimum of 20% neoplastic cells was required to minimize variations in TMB values induced by chromosomal instability or tumor heterogeneity. Subsequent analysis of 58 consecutive lung cancer samples in a diagnostic setting was successful and revealed sufficient somatic mutations to generate mutational signatures in 14 cases. In conclusion, the 523-gene assay can be applied for evaluation of multiple DNA-based biomarkers relevant for treatment selection.

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing.

Poor quality of biological samples will result in an inaccurate analysis of next-generation sequencing (NGS). Therefore, methods to accurately evaluate sample integrity are needed. Among methods for evaluating RNA quality, the RNA integrity number equivalent (RINe) is widely used, whereas the DV200, which evaluates the percentage of fragments of >200 nucleotides, is also used as a quality assessment standard. In this study, we compared the RINe and DV200 RNA quality indexes to determine the most suitable RNA index for the NGS analysis. Seventy-one RNA samples were extracted from formalin-fixed paraffin-embedded tissue samples (n = 30), fresh-frozen samples (n = 25), or cell lines (n = 16). After assessing RNA quality using the RINe and DV200, we prepared two kinds of stranded mRNA sequencing libraries. Finally, we calculated the correlation between each RNA quality index and the amount of library product (1st PCR product per input RNA). The DV200 measure showed stronger correlation with the amount of library product than the RINe (R2 = 0.8208 for the DV200 versus 0.6927 for the RINe). Receiver operating characteristic curve analyses revealed that the DV200 was the better marker for predicting efficient library production than the RINe using a threshold of >10 ng/ng for the amount of the 1st PCR product per input RNA (cutoff value for the RINe and DV200, 2.3 and 66.1%; area under the curve, 0.99 and 0.91; sensitivity, 82% and 92%; and specificity, 93% and 100%, respectively). Our results indicate that NGS libraries prepared using RNA samples with the DV200 value > 66.1% exhibit greater sensitivity and specificity than those prepared with the RINe values > 2.3. These findings suggest that the DV200 is superior to the RINe, especially for low-quality RNA, because it is a more consistent assessment of the amount of the 1st NGS library product per input.

Abstract P6-11-20: Capacity-building for molecular analyses of formalin-fixed paraffin-embedded breast cancer samples from Rwanda

Abstract Background: The global burden of cancer is rising exponentially in countries with the least resources to manage it. Breast cancer mortality is higher in African women than European women, but neither race nor access to care completely account for this discrepancy. Molecular profiling and methylation analysis could be informative but is expensive and the feasibility of using archived samples from low-resource settings is not known. We present a retrospective tissue collection study of breast cancer patients diagnosed at the University of Rwanda to investigate the quality of DNA from FFPE. Methods IRB approval and Material Transfer Agreement were obtained. Fifty tumor blocks and eight sets of FFPE slides from 2014-2018 were transported from Rwanda to USA in 2 batches. Pathology was reviewed for concordance. DNA was extracted using the QIAamp DNA FFPE Tissue kit (Qiagen). Batch 1: DNA quality and quantity was assessed using the Qubit 3.0 fluorometer (Life Technologies). Batch 2: DNA quality was assessed using Kapa hgDNA pPCR QC Assay. Bisulfite conversion and array hybridization protocols for the Illumina EPIC methylation platform to detect 2.5% methylated DNA target were used. DNA methylation data generated using the Infinum MethylationEPIC BeadChip (Illumina, Inc.) were pre-processed using minfi (v1.30.0), prior to quality control using Enmix (v1.20.2). A detection P-value threshold of 1E-06 was used to identify poorly detected probes, and a maximum threshold of 5% low quality data was used to identify poorly performing samples and CpG loci. Outliers based on beta-value distributions were also flagged as poor-quality samples. Mean bisulfite intensity values were calculated using Infinium type I and II control probes. DNA methylation beta-values were calculated to assess the proportion of methylated alleles at each individual CpG locus. Results: Histology was 82% concordant. All samples in Batch 1 had >5% low quality CpG’s. FFPE processing was reviewed and buffered formalin was not used. For Batch 2 10% neutral buffered formalin was transported and delivered to Central University Hospital of Kigali in March 2019. Eight additional patient samples were obtained and 6 out of 8 had <5% low quality CpG’s. Conclusion: Complex molecular studies are feasible with international collaboration. Optimal reagents and processing are crucial for successful analysis. Quality checkpoints must be done early in the process to maximize the potential for interpretable results. Capacity-building to improve DNA quality is the first step toward optimizing cancer treatment and minimizing disparities as molecular profiling increasingly directs cost-effective treatment.These results confirm methods appropriate for future molecular research on breast cancer in low resource settings. Citation Format: Mary Dickinson Chamberlin, Victoria Forbes, Germaine Dushimimana, Arminja Kettenbach, Kristen Muller, Emile Musoni, Eric Rutaganda, Owen M. Wilkins, Fred W. Kolling, Brock Christensen. Capacity-building for molecular analyses of formalin-fixed paraffin-embedded breast cancer samples from Rwanda [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-11-20.

Study on Factors Delaying Lead Time in the Apparel Supply Chain Management: A Case Study on the Order of Execution Strategies of a Garment Factory in Bangladesh

Bangladesh garments industry is searching out for ways to reduce lead time during apparel order execution for many rational reasons among which buyer's satisfaction, achieving target shipment date, and building a stable sector are some mentionable motives. One way to abridge lead time is to identify and analyze the root causes to delay in the approval period. Lead time refers to the time in between receiving order and dispatching to buyer. A number of reasons are responsible for delayed lead time. For instance, factory capacity, poor work force, raw material supply, wrong understanding about product details, miscommunication among inter-departments, longer approval period, lack of proper planning, submission of poor quality items, re-work, and re-submissions, etc. Among all of these reasons listed, longer approval period, submission of poor quality materials, re-work, and raw materialsupply hold the utmost importance. This study was based on the investigation of the abovementioned factors that reasonably delay lead time. A compliant garments industry was selected that receives order from a renowned buying office located in Germany. The ultimate customer of that buying office is also located in Germany who operates a number of lucrative stores in several countries in the globe. The study was carried out for an order with a quantity of 73,890 pcs. Results evinced that, due to longer approval period for submitted material (i.e., metal button, first styling sample), up to 24 days’ extension are required to extend for order execution. Besides, submission of poor quality sample and re-work (i.e., print strike-off, size set, and lab dip sample) lead up to 22 days extension. The raw material (i.e., fabric and trims) in-house period also delayed the total period. For the total order shipment, it required 45 days’ extension with respect to the given shipment date.

Adding Chinese herbal medicine to probiotics for irritable bowel syndrome-diarrhea: A systematic review and meta-analysis of randomized controlled trials

Abstract Objective This study assessed whether Chinese herbal medicine (CHM) combined with probiotics/synbiotics for irritable bowel syndrome - diarrhea (IBS-D) was more effective and safer than probiotics/synbiotics alone. Methods Ten databases were searched for randomized control trials (RCTs) of IBS-D as diagnosed by Manning or Rome criteria. Trials comparing probiotics and probiotics with CHM were included. The Cochrane risk of bias (ROB) was evaluated for each trial. RevMan 5.3 was used to conduct a meta-analysis. Results Twenty-six RCTs were included (25 Chinese, 1 English), involving 2045 participants. Meta-analysis was conducted on two outcomes: overall symptom improvement and relapse. CHM combined with live Bifidobacterium and Lactobacillus preparations reduced relapse rate (RR 0.28, 95%CI 0.15–0.52, 3 trials, n = 205) compared with probiotics alone. The subgroup analysis showed the benefit of CHM prescriptions based on soothing liver and invigorating spleen (1.28, 1.14–1.44, 3, 244), invigorating spleen and resolving dampness (1.20, 1.03–1.41, 2, 128), or warming and invigorating spleen and kidney formulae (1.27, 1.09–1.46, 2, 210) combined with triple Bifidobacterium preparations than the same probiotics alone which improved overall symptoms for IBS-D. There was unclear bias in almost domains of ROB. Most studies had a high risk of bias due to lack of blinding of investigator and participants, and selective reporting. Conclusions This study showed that CHM combined with probiotics may reduce relapse rate by 72%, and improve overall symptoms of IBS-D (as diagnosed by Rome II and III) compared to probiotics alone. From the limited subgroup analysis, only soothing liver and invigorating spleen formulae, represented by Tongxie Yaofang, added to triple Bifidobacterium preparations may be superior to the single preparations in terms of overall symptoms. However, due to the poor methodological quality and small sample size of the trials, these findings must be interpreted with caution.

Species discrimination and total polyphenol prediction of porcini mushrooms by fourier transform mid-infrared (FT-MIR) spectrometry combined with multivariate statistical analysis.

The plateau specialty agricultural products, wild porcini mushrooms, have great value both as a superb cuisine and as a potential medication. Due to quality different between species added with the fraud behavior in sales process, make poor quality or poisonous sample inflow into the market, which pose a health risk for consumers, but also disrupted the mushroom market. Traditional analysis way is time‐consuming and laborious. Therefore, the aim of this study is to develop a way using fourier transform mid‐infrared (FT‐MIR) spectrometry and data fusion strategies for the fast and accurate species discrimination and predict amount of total polyphenol in four porcini mushrooms. The t‐distributed stochastic neighbor embedding based on mid‐level data fusion showed two species of Boletus edulis and B. umbriniporus have been identified. The order of correct rate of PLS‐DA models was mid‐level data fusionq (100%) > mid‐level data fusione (97.06%) = mid‐level data fusionv (97.06%) = stipes (97.06%) > low‐level data fusion (94.12%) > caps (91.18%). The order of correct rate of grid‐search support vector machine models was low‐level data fusion (100%) > caps (94.12%) > stipes (91.18%), and the order of particle swarm optimization support vector machine was low‐level data fusion (100%) > caps (97.06%) > stipes (88.24%). The mid‐level data fusionq and low‐level data fusion had best discrimination accuracy (100%) allowing each mushroom classed into its real species, which could be used for accurate discrimination of samples. B. edulis mushrooms had highest total polyphenol, with 14.76 mg/g dw and 17.33 in caps and stipes mg/g dw, respectively. The phenols were easier to accumulate in the caps in Leccinum rugosiceps (1.03) and B. tomentipes (1.19), and the opposite phenomenon is observed in B. edulis (0.85) and B. umbriniporus (0.95). The correlation coefficient and residual predictive deviation of best prediction model were 86.76% and 2.40%, respectively, indicating that that there is good relevance between FT‐MIR and total polyphenol content, which could be used to predict roughly polyphenols content in mushrooms.

Elements in surface and well water from the central North China Plain: Enrichment patterns, origins, and health risk assessment

The principal aim of this study was to understand the enrichment patterns of elements in water from typical coal mine and irrigation areas. For this study, samples of surface water, shallow water, and deep water were collected from Handan, Jining, and Heze cities and their surrounding areas in the central North China Plain. The results showed that the hydrochemical characteristics were dominated by Ca–Mg–Cl and Ca–HCO3. Elements in the studied surface water, including strontium, iron and boron, were anomalously enriched at levels more than 654, 294 and 134 times their global river water averages, respectively. The concentrations of elements in the studied area were influenced by both natural processes and anthropogenic sources, but the dominant origins of the anomalous enriched elements were bedrock weathering and soil leaching. The deep well water quality in the Handan coal mining area was good, while the poor-quality water samples in the study area were mainly distributed in the alluvial plain, which is characterized by Neogene-Quaternary sediments and aquifers. The measured hazard quotient and hazard index values indicate that the arsenic and nickel in the studied samples could pose a noncarcinogenic risk to the health of local residents, especially children. The leading source of the high arsenic levels is influenced by natural process. Monitoring plans for arsenic, iron, manganese, nitrate and other potentially harmful elements in surface water and groundwater and effective health education on pollution by these elements are essential.

Tensile Strength Modeling of Limestone Rocks in Sulaymaniyah City, Iraq Using Simple Tests

Tensile strength of rocks is one of the mechanical properties of intact rock that is a significant parameter for designing geotechnical structures includes dam foundations and tunnels. The tensile strength can be determined indirectly using Brazilian indirect test procedure that is mentioned in the International Society for Rock Mechanics suggested methods. The availability of rock samples is needed to perform the Brazilian indirect test so as to determine their tensile strength which is expensive, time-consuming, and cost-effective especially for weak quality rock formations. Therefore, non-destructive methods for predicting the tensile strength of the rock are crucially needed during the poor quality of rock samples. Non-destructive tests can be correlated with indirect tests to predict Brazilian tensile strength (BTS) of rocks such as ultrasonic pulse velocity and Schmidt hammer. These methods are simple and can be easily conducted in the field. This study is focused on the tensile strength of limestone rocks for three main formations of Sulaymaniyah city. The samples were obtained using a standard core barrel. Statistical analysis including minimum, maximum, mean, standard deviation, variance, and coefficient of variance for the results was conducted. Single and multiple correlations between BTS and each of ultrasonic pulse velocity and Schmidt hammer rebound number of limestone rocks were created. Reasonable empirical equations were developed to predict the tensile strength of limestone rocks. In addition, the point load strength index was correlated with BTS. The comparison between proposed equations from this study and equation from the literature was also investigated.

Reliable cell and tissue morphology-based diagnosis of endemic Burkitt lymphoma in resource-constrained settings in Ghana

BackgroundEndemic Burkitt lymphoma (eBL) is an aggressive B-cell lymphoma, which is a common childhood cancer in areas with intense transmission of Plasmodium falciparum parasites. Early and accurate diagnosis is a prerequisite for successful therapy, but it optimally involves advanced laboratory investigations. These are technologically demanding, expensive, and often difficult to implement in settings where eBL is prevalent. Diagnosis is thus generally based on clinical assessment and morphological examination of tumour biopsies or fine-needle aspirates (FNAs).MethodsThe purpose of the present study was to assess the accuracy of eBL diagnosis at two tertiary hospitals in Ghana. To that end, we studied FNAs from 29 eBL patients and 21 non-eBL lymphoma patients originally diagnosed in 2018. In addition, we examined 111 archival formalin-fixed and paraffin-embedded (FFPE) biopsies from Ghanaian patients originally diagnosed as eBL (N = 55) or non-eBL (N = 56) between 2010 and 2017. Availability-based subsets of samples were subjected to haematoxylin-eosin or Giemsa staining, C-MYC immunohistochemistry, and fluorescence in situ hybridisation (FISH) analysis of c-myc rearrangements.ResultsWe found a good correlation between original diagnosis and subsequent retrospective assessment, particularly for FNA samples. However, evidence of intact c-myc genes and normal C-MYC expression in samples from some patients originally diagnosed as eBL indicates that morphological assessment alone can lead to eBL over-diagnosis in our study area. In addition, several FFPE samples could not be assessed retrospectively, due to poor sample quality. Therefore, the simpler FNA method of obtaining tumour material is preferable, particularly when careful processing of biopsy specimens cannot be guaranteed.ConclusionWe conclude that the accuracy of eBL diagnostic tools available in Ghana is generally adequate, but could be improved by implementation of additional pathology laboratory investigations. Improved attention to adequate preservation of archival samples is recommended.

Aspicular Bone Marrow Aspiration: A Common, but Not a Minor Problem

Bone marrow (BM) aspiration plays an important role in hematologic malignancies diagnosis. Access and cost of diagnostic flow cytometry remains a problem in low and middle-income countries. In this context, morphological diagnosis by BM smear often represents the only means to rapidly diagnose our patients. Therefore, in this context obtaining the highest quality sample possible during the procedure is paramount. Despite being a well-known problem, evidence-based recommendations to improve BM aspirate quality are few, with studies evaluating factors associated with poor quality samples lacking. Objectives To determine factors associated with poor quality BM aspirates defined by an aspicular or hemodiluted sample in a hematology referral center. Materials and methods We conducted a retrospective study in our University Hospital and analyzed the BM smear samples stored in our center performed from October 2014 to December 2018. We collected and analyzed data based on diagnosis, age, gender, recent chemotherapy, and the variables of a complete blood count performed just before each BM aspiration. The quality of the BM smear was defined in any of the following: aspicular (without spicules), pauciaspicular (1-3 spicules), spicular (> 3 spicules), defining aspicular BM smear as non-diagnostic samples. Univariate analysis was performed looking for diferences between operators (in a 3-year residency program). In the other hand, in the multivariate analysis we seek to reveal the factors associated with obtaining hemodiluted (aspicular) bone marrow aspirate-smears. Results A total of 1,073 BM aspirates were evaluated. Hematology fellows performed 97% of BM aspirates; the remaining 3% were performed by attending physicians. In our analysis, 301 aspirates were aspicular, constituting 28.1% of the total number of aspirate smears. Most BM aspirates were performed for a diagnostic evaluation (66.3%) with the rest of the procedures for subsequent hematologic malignancy response assessments. In the univariate analysis, no differences were observed between operators. In a multivariate analysis the presence of an age >65 years (OR 3.1, 95% CI 2.3 to 4.1) and hemoglobin <6.0 g/dL (OR 2.7, 95% CI 1.4 to 4.5) at the time of the procedure were significantly associated to obtaining a non-diagnostic sample. Diagnosis, WBC count, platelet count, operator experience or other variables did not show statistical relevance. In our center, 18.81% of diagnostic patient samples that had acute leukemia were diagnosed without flow cytometry and through BM aspirate morphology alone. A second procedure to reach a diagnosis was necessary in 7.97% of the patients due to aspicular samples, obtaining a mean of diagnostic-treatment delay of 18.3 days (±5.7 days). Conclusions We found no differences between operators, emphasizing that there are other factors to consider in addition to a correct BM aspiration technique or operator experience. We believe this is crucial to recognize, especially in developing countries where morphological diagnosis remains the only means for the diagnosis or response evaluation of our patients. BM aspirate sample quality is multifactorial, being age and hemoglobin important factors. In addition, obtaining aspicular or hemodiluted samples represents not only a diagnostic challenge, but also delays the treatment of our patients. Disclosures Gomez-Almaguer: Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Teva: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

2179. Detection of Group A Streptococcus in the Saliva of Children Presenting With Pharyngitis Using the cobas®LIAT® PCR System

BackgroundCLIA waived polymerase chain reaction (PCR) has recently become available as a point of care test for Group A Streptococci (GAS) in individuals presenting with pharyngitis, enabling rapid and accurate diagnosis. However, swabbing the pharynx results in discomfort and is often dreaded by young children which may result in poor quality sampling.ObjectiveIn order to assess the viability of saliva as a sample specimen for GAS, this study compared saliva samples with pharynx swabs of children with sore throat, using swabs inoculated by children sucking on them as they would a lollipop in the context of newly available very sensitive techniques.MethodsWe enrolled children ages 5–15 years presenting with sore throat and known to have a positive rapid streptococcal antigen detection test (RADT) performed on a posterior pharyngeal swab, at the discretion of the primary care provider. The RADT used was the SureVue® (Fisher Scientific) system. A second swab was obtained by having the child suck on the swab in the anterior mouth for 30 seconds and a third swab was obtained from the posterior pharynx. PCR was performed on these two additional swabs using the cobas®LIAT® (Roche) system according to the manufacturer’s instructions.ResultsSeventeen children were enrolled in the study between January and April 2019. The mean age of enrollment was 9.6 years (range 6–15). By design all children were known to have a positive RADT for GAS. The LIAT posterior pharynx swab was positive in all 17 subjects. In addition, the LIAT saliva swab was positive in all 17 subjects.ConclusionIn this small pilot study, there was 100% concordance between the RADT for GAS and both the posterior pharyngeal and saliva swab using the cobas®LIAT® PCR system. Performing saliva swabs will result in less discomfort and distress to children who are tested for GAS. Further study is needed to determine the sensitivity and specificity of saliva swabs for the detection of GAS in children presenting with acute pharyngitis.Disclosures All authors: No reported disclosures.

Small Steps for a Big Change: Attention towards Preanalytical Error

Aims: The study aimed to evaluate the preanalytical errors in the Indoor patient department in tertiary care Hospital. To calculate the percentage of preanalytical errors in the Indoor patient department in our Hospital and to recommend standard operative interventions to improve quality of results. To test the effectiveness of attention by continuous educational action at reducing preanalytical errors and improving patient care.
 Study Design: An observational study.
 Place and Duration of Study: The work was done from July 2014 to July 2015 at a tertiary care Hospital India.
 Methodology: We retrospectively reviewed the samples and test request forms received at Biochemistry laboratory for one month. The outcome measures were incomplete laboratory forms, mislabeling samples, inappropriate tests, wrong container, poor quality of samples and transportation problems. Two weeks of interventions in the form of continuous educational training and education regarding standard operative procedures were given to stakeholders to raise awareness towards the preanalytical phase. Two weeks later, data was monitored again for one month.
 Results: 2330 and 2130 samples and request forms were monitored before-after intervention respectively from wards for one month each. Of the total chances of preanalytical errors, 22.17% were due to inappropriate tests, 81.5% were related to incomplete patient information, 97% lacking clinical information, 18.8% errors related to specimen information, 3.5% errors were of the deranged quality of the specimen, and in4.5% transportation problems were observed. Subsequently, these were reduced to 10%, 20%, 16.4%, 7.5%, 2.3%, 3.1% respectively. A significant difference in percentage change was observed in all the above errors after the one-month interventions for the reduction in preanalytical errors.
 Conclusion: The results of the present study revealed that taking small steps in the form of implementing standard operative procedures for collection, storage and transport facilities and continuous educational training of stakeholders would reduce big errors occurring due to human factors in preanalytical phase. We need good interdepartmental communication and cooperation to achieve good laboratory results and patient well being. This study improved the quality of test results and patient care.

1886P - Prospective pathological experience with research biopsies in the context of clinical trials at Vall d’Hebron Institute of Oncology

1886P - Prospective pathological experience with research biopsies in the context of clinical trials at Vall d’Hebron Institute of Oncology

P5712Research Network in Africa (RNA): quality assessment of antihypertensive generic drugs in 10 African countries

Abstract Introduction Hypertension results in more deaths than any other risk factor and has been on the rise in Sub-Saharan Africa over the past few decades. Generic drugs have helped improve accessibility and affordability of antihypertensive therapy in developing countries. However, assessment of quality standards of these products is important. Purpose We performed a quality assessment of five commonly used antihypertensive generic drugs in ten Sub-Saharan African countries and studied the impact of price on quality. Methods Drug samples were prospectively collected using standardized methods between 2012 and 2014. We developed a validated reversed-phase liquid chromatography with tandem mass spectrometry method to accurately quantify the active ingredient in a certified public laboratory. Quality was defined based on the percentage ratio of measured to expected dosage of active ingredient. Results A total of 1185 samples were assessed, of which 70.0% were generic (n=830). Among the generic drugs, the percentage of poor quality drugs was 24.3% (n=202/830). The percentage ratio of measured to expected dosage of active ingredient ranged from 49.2% to 111.3%; the majority (81.7%) of the poor quality samples had insufficient quantity of the active ingredient. Moreover, poor quality was not associated with purchase price of the drug. Conclusion In this study from ten Sub-Saharan African countries, nearly one-quarter of the available generic antihypertensive drugs were found to be of poor quality. Concerted measures to improve the quality of antihypertensive drugs could lead to major improvements in hypertension control with attendant reduction of its deleterious consequences in low and middle income countries. Figure 1. Percentage of poor quality in generic antihypertensive drugs according to the country of purchase. Acknowledgement/Funding Grant AAP-2014-042, ANSM (Agence Nationale Sécurité du Médicament

Blood sample quality using Greiner Bio-One HOLDEX® Single-Use Holder and VACUETTE® SAFELINK holder with male luer lock: a comparative study

Background: Hemolysis is the most frequent and potentially one of the most harmful causes of poor sample quality. Many strategies have been attempted for minimizing the risk of generating spurious hemolysis during blood collection, such as using the HOLDEX ® single-use holder, which features a chamber that decelerates blood flow pressure before entering evacuated blood tubes. We have hence designed a study to establish whether the new VACUETTE ® SAFELINK, which shares different structure but similar strategy affecting blood flow pressure, may ensure a comparable sample quality as using HOLDEX ® single-use holder. Methods: A total number of 24 clinical chemistry, hematologic and hemostasis parameters were measured in paired blood samples collected from 30 healthy volunteers using either HOLDEX ® single-use holder or VACUETTE ® SAFELINK. Test results were compared with paired Student’s t-test, Pearson’s correlation and Bland-Altman plots. Results: All test results were non-significantly different in blood samples collected with HOLDEX ® single-use holder or VACUETTE ® SAFELINK except potassium, whose values were marginally higher in plasma collected with VACUETTE ® SAFELINK. All Pearson’s correlation coefficients were excellent except potassium, chloride and H-index. In this latter case, the correlation did not reach statistical significance. The percent bias of different parameters in samples collected with HOLDEX ® single-use holder or VACUETTE ® SAFELINK was minimal, comprised between −4.5% and +1.6%. In all cases the percent bias was comprised within the quality specifications tests. The rate of plasma samples with H-index >3 was eventually lower when blood was collected using SAFELINK than with HOLDEX ® single use holder (odds ratio, 0.52; 95% confidence interval, 0.17–1.60). Conclusions: The results of this comparative study suggest that sample quality is thoughtfully comparable using HOLDEX ® single-use holder and VACUETTE ® SAFELINK, thus translating into the concept that VACUETTE ® SAFELINK may also be an effective means for reducing spurious hemolysis, especially when drawing blood from catheters.

Abstract 3393: Altered immune response in the transcriptome of patients with lung cancer

Abstract Introduction The immune system is critical to surveying and eradicating abnormal cells, but tumor cells develop ways to escape immunosurveillance and induce an immunosuppressive state. We previously developed and validated gene expression (GE) signatures measured in the normal airway-epithelial brushings in patients undergoing bronchoscopy for suspicion of lung cancer (LC). In this study, we seek to understand if the immunosuppressive environment extends to the airway field of injury via profiling of endobronchial biopsies from the central airway containing a broader range of cell types, including immune cells. Methods Endobronchial biopsies from normal-appearing regions of the central airway were collected from ever smokers undergoing workup of indeterminate pulmonary nodules (7-30 mm in diameter) suspicious for LC at military and VA hospitals within the DECAMP consortium. Initially, total RNA from the biopsies (n=44, discovery-set) were isolated and sequenced. Reads were aligned to hg19 using STAR and gene level counts were quantified with RSEM. Poor quality samples were removed using FASTQC and RSEQC. Differential GE associated with cancer status was identified using edgeR, adjusting for smoking-status, COPD and sample quality. RNA from additional endobronchial biopsies (n=49, validation-set) were isolated and preprocessed similarly. Genes differentially expressed with LC status in the discovery-set were tested in the validation-set using gene set variation analysis (GSVA). Functional enrichment of cancer associated genes was explored using Enrichr. Comparison of cancer signatures identified in previously published LC studies was investigated using GSEA. CIBERSORT, xCell, TIMER software and single-cell RNA sequencing data generated from airway brushings were used to deconvolute the immune cell content of the bulk biopsy samples. Results We identified a GE signature associated with LC which was significantly and concordantly enriched in the validation set of biopsies and two previously published studies of LC-associated GE in airway brushings. Genes decreased in LC patient biopsies were enriched for genes involved in immune-related pathways, including cytokine interactions, the inflammatory response and neutrophil degranulation. Computational deconvolution and comparison with single-cell RNAseq data predicts a decrease in neutrophils in the airway of LC patients. Conclusion We identified LC-associated GE alterations in smokers presenting with indeterminate pulmonary nodules. Down-regulated genes in LC subjects are strongly associated with immune system function, specifically neutrophil biology. Subjects with LC appear to have an immunosuppressive environment directed towards myeloid cell populations, and this could have implications for the future development of immunoprevention therapies. Citation Format: Kahkeshan Hijazi, Julian Lel, Ehab Billatos, Elizabeth Moses, Christopher S. Stevenson, Matthew V. Lorenzi, Gang Liu, Joshua D. Campbell, Yusuke Koga, Jiarui Zhang, Fenghai Duan, Helga Marques, Marc E. Lenburg, Avrum E. Spira, Jennifer Beane. Altered immune response in the transcriptome of patients with lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3393.

Abstract 2896: Impact of intratumoral heterogeneity on gene expression profiles and known biomarkers in FFPE tumor biopsies from early stage breast cancer patients

Abstract Background: Cancer is a dynamic disease that becomes more heterogeneous during its course, with cellular subpopulations with distinct molecular profiles across different regions. Because this intratumoral heterogeneity may result in varying levels of gene expression patterns and sensitivity to treatment across different regions of the tumor, it is important to understand patterns of heterogeneity to develop effective biomarkers. We conducted multiregion sampling within single breast tumor biopsies to evaluate the effect of intratumoral heterogeneity on expression of known and novel biomarkers. Methods: We sampled three sequential regions of breast tumor biopsies to compare gene expression patterns determined by a novel biomarker panel (BA355), 5 known biomarkers (ERS1, ERBB2, PGR, AR, and MKi67) and a novel predictive classifier (BA100). RNA expression levels were measured using a proprietary custom designed nanoString nCounter assay. We investigated formalin-fixed paraffin-embedded (FFPE) breast tumor biopsies from a cohort of 40 early stage breast cancer patients (T1 N0 M0) collected prospectively. RNA was extracted from upper, middle, and lower regions of each biopsy, resulting in a total of 120 samples quantified and QC using standard procedures. Each layer consisted of 40 micrometers, with an interval of 50 micrometers of tissue. BA100 linear regression models were used to generate prediction scores. Analyses were done in R. Results: Eighteen of the 120 samples had poor data quality (sample median = 0 or inter quantile range > mean + 2*sd). Further analyses only included 102 good quality samples corresponding to 34 FFPE biopsies. Among 33 of 34 samples, gene expression patterns between the three regions of the biopsies were highly correlated, with correlation coefficients ranging from 0.90-0.99 (p-values ranged from 2.07e-19 to 8.45e-319). Among 31 of 34 samples, the three regions were clustered together in cluster analysis. The coefficient of variation (CV) for ESR1 ranged from 0.37%-15.5%, with a majority (32 of 34) of the samples having CV <10%. While the CVs of ERBB2, PGR, and AR were within the same range as ESR1, MKI67 had much larger variation (CV ranged from 0.99%-173%). BA100 scores yielded consistent predictions for the three regions in 33 of 34 samples. Conclusion: Examination of gene expression patterns across multiple regions of breast tumor biopsies did not reveal a significant impact of intratumoral heterogeneity on expression patterns using a novel gene signature panel. However, at the individual gene level, while a number of genes including ESR1, ERBB2, PGR, and ARdisplayed consistent gene expression, genes such as MKI67 showed significant variation, indicating that tumor heterogeneity may impact individual gene biomarkers. Further studies are ongoing to evaluate intratumoral heterogeneity. Citation Format: Lucas Delmonico, Joan Chen, Trushna Desai, Lisa Barnett, Aladdin Shadyab, John Obenauer, Gilda Alves Brown, Mauricio Magalhaes Costa, Marcia Fournier. Impact of intratumoral heterogeneity on gene expression profiles and known biomarkers in FFPE tumor biopsies from early stage breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2896.

Abstract 4227: Salvage genetic testing on buccal swab samples using liquid in situ amplification identifies genetic mutations from previous test failures

Abstract Background: NCCN guidelines version 2.2019 address BRCA1/2 testing for men with a personal history of prostate cancer limited to Gleason ≥7 and specific family history features. Inherited genetic mutations have been identified in up to 12% of metastatic prostate cancer, primarily in DNA repair genes. Here we report liquid in situ amplification (LISA) technique followed by hereditary testing on buccal swab samples that had previously failed to yield a genetic testing result. Methods: Previously failed buccal swab samples from another CLIA-certified laboratory were re-prepared using LISA. Qubit quantification following LISA revealed goodDNA concentrations even for these poor-quality swab samples ranging from 5 to 70 ng/uL, which is sufficient for next-generation sequencing analysis (NGS). Coding regions of ATM, BRCA1, BRCA2, CHEK2, PALB2, and RAD51D genes were amplified by a panel of specific primer pools followed by massively parallel sequencing for the identification of germline mutations. Minimal sequencing coverage was set as 50X, and pathogenic, likely pathogenic, and variants of unknown significance (VUS) were classified and reported. Results: Buccal swab samples from 29 men with at least intermediate-risk prostate cancer were received as low-yield, low-quality DNA (n=12), pre-cut swabs in preservation solution (n=6), and aged swab samples (n=11). LISA and subsequent NGS testing was able to salvage results for 24/29 (82.8%) (failed samples included 4 aged swab and 1 pre-cut swab). 1 patient had a pathogenic BRCA1 mutation (c.4309delT) and 3 patients had VUS (BRCA1 c.4776C>G, BRCA1 c.1846_1848delTCT, and RAD51D c.268G>A). The remaining 20/29 (69.0%) samples have either benign or likely benign variants. Conclusions: LISA applied to these previously failed samples was able to salvage results for over 80% of cases, including the identification of a pathogenic BRCA1 mutation which led cascade hereditary testing for immediate family members. It also provided confirmation for nearly 70% of cases about the absence of hereditary risk for the genes on the panel. Citation Format: Glen J. Weiss, Andrew Ford, Charmaine Brown, Elizabeth Caver, Matthew Vallejo, Claribel Adeyemi, Forrest Collins, Chen-Hsiung Yeh. Salvage genetic testing on buccal swab samples using liquid in situ amplification identifies genetic mutations from previous test failures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4227.

Erratum: Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification.

Erratum: Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification.