Detection Of Cross-contamination Research Articles

An artificial intelligent platform for live cell identification and the detection of cross-contamination

BackgroundAbout 30% of cell lines have been cellular cross-contaminated and misidentification, which can result in invalidated experimental results and unusable therapeutic products. Cell morphology under the microscope was observed routinely, and further DNA sequencing analysis was performed periodically to verify cell line identity, but the sequencing analysis was costly, time-consuming, and labor intensive. The purpose of this study was to construct a novel artificial intelligence (AI) technology for “cell face” recognition, in which can predict DNA-level identification labels only using cell images.MethodsSeven commonly used cell lines were cultured and co-cultured in pairs (totally 8 categories) to simulated the situation of pure and cross-contaminated cells. The microscopy images were obtained and labeled of cell types by the result of short tandem repeat profiling. About 2 million patch images were used for model training and testing. AlexNet was used to demonstrate the effectiveness of convolutional neural network (CNN) in cell classification. To further improve the feasibility of detecting cross-contamination, the bilinear network for fine-grained identification was constructed. The specificity, sensitivity, and accuracy of the model were tested separately by external validation. Finally, the cell semantic segmentation was conducted by DilatedNet.ResultsThe cell texture and density were the influencing factors that can be better recognized by the bilinear convolutional neural network (BCNN) comparing to AlexNet. The BCNN achieved 99.5% accuracy in identifying seven pure cell lines and 86.3% accuracy for detecting cross-contamination (mixing two of the seven cell lines). DilatedNet was applied to the semantic segment for analyzing in single-cell level and achieved an accuracy of 98.2%.ConclusionsThe deep CNN model proposed in this study has the ability to recognize small differences in cell morphology, and achieved high classification accuracy.

Sample tracking in microbiome community profiling assays using synthetic 16S rRNA gene spike-in controls

Workflows for microbiome community profiling by high-throughput sequencing are prone to sample mix-ups and cross-contamination due to the complexity of the procedures and large number of samples typically analyzed in parallel. We employed synthetic 16S rRNA gene spike-in controls to establish a method for tracking of sample identity and detection of cross-contamination in microbiome community profiling assays based on 16S rRNA gene amplicon sequencing (16S-seq). Results demonstrated that combinatorial sample tracking mixes (STMs) can be reliably resolved by Illumina sequencing and faithfully represent their sample of origin. In a single-blinded experiment, addition of STMs at low levels was shown to be sufficient to unambiguously identify and resolve swapped samples. Using artificial admixtures of individually SMT-tagged samples, we further established the ability to detect and quantify cross-contamination down to a level of approximately 1%. The utility of our technique was underscored through detection of an unplanned case of cross-contamination that occurred during this study. By enabling detection of sample mix-ups and cross-contamination throughout 16S-seq workflows, the present technique thus assures provenance of sequence data on a per-sample basis. The method can be readily implemented in standard 16S-seq workflows and its routine application is expected to enhance the reliability of 16S-seq data.

Challenges in Additive Manufacturing of Space Parts: Powder Feedstock Cross-Contamination and Its Impact on End Products.

This work studies the tensile properties of Ti-6Al-4V samples produced by laser powder bed based Additive Manufacturing (AM), for different build orientations. The results showed high scattering of the yield and tensile strength and low fracture elongation. The subsequent fractographic investigation revealed the presence of tungsten particles on the fracture surface. Hence, its detection and impact on tensile properties of AM Ti-6Al-4V were investigated. X-ray Computed Tomography (X-ray CT) scanning indicated that these inclusions were evenly distributed throughout the samples, however the inclusions area was shown to be larger in the load-bearing plane for the vertical specimens. A microstructural study proved that the mostly spherical tungsten particles were embedded in the fully martensitic Ti-6Al-4V AM material. The particle size distribution, the flowability and the morphology of the powder feedstock were investigated and appeared to be in line with observations from other studies. X-ray CT scanning of the powder however made the high density particles visible, where various techniques, commonly used in the certification of powder feedstock, failed to detect the contaminant. As the detection of cross contamination in the powder feedstock proves to be challenging, the use of only one type of powder per AM equipment is recommended for critical applications such as Space parts.

Species identification in cell culture: a two-pronged molecular approach

Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the "barcode region" by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts.

Detection of cross-contamination in feedstuffs: presence of constituents of animal origin.

Detection of cross-contamination in feedstuffs: presence of constituents of animal origin.

Characterization of lactate dehydrogenase isozyme pattern and morphology of three marine fish cell lines

Three continuous marine fish cell lines of FG (i.e., Flounder Gill) from flounder (Paralichthys olivaceus) gill, SPH (i.e., Sea Perch Heart) from sea perch (Lateolabrax japonicus) heart and RSBF (i.e., Red Sea Bream Fin) from red sea bream (Pagrosomus major) fin, were characterized by lactate dehydrogenase (LDH) isozyme and morphological analysis. The LDH isozyme patterns of these three cell lines and their corresponding tissues of origin were investigated and compared. The results showed: (1) No difference was found in the LDH isozyme patterns of FG and flounder gill tissue. However, the LDH isozyme patterns of SPH and RSBF were significantly different from their corresponding tissues of origin; (2) LDH isozyme patterns of FG, SPH and RSBF were markedly different from each other and could serve as genetic markers for species identification and detection of cross contamination. Morphological change analysis of these three cell lines in comparison to their original tissues indicated that FG cells still appeared epithelioid without morphological transformation. However, morphological changes were found in SPH and RSBF compared to their original tissues. Therefore, the cellular morphology was still plastic in the relatively stable culture conditions, and it was possible that change of LDH patterns was related to morphological changes of fish cellsin vitro.

Rapid screening and detection of cellular cross-contamination in cell cultures: a new application of cytochemistry.

Cellular cross-contamination is a serious problem that can potentially affect all who use cell culture systems in research and production work. Reports from national testing services indicate that frequencies as high as 17-36% of the lines examined may be incorrectly identified by source laboratories, and intra/interspecies contamination was identified in 11-25% of cell cultures examined (1). National cell banks employ a variety of techniques to attempt cell line authentication, i.e., fluorescent antibody staining, isoenzymology, cytogenetics, and, more recently, DNA fingerprinting via Southern blotting and PCR methodologies (2). Each of these procedures has advantages and disadvantages, and none is capable on a routine basis of detecting very low levels, e.g., 0.5% or less, of cross-contamination of one cell line with another from the same species. In March of 1994, a histiocyte lymphoma cell line U-937 (AKC CRL 1593) was found to be cross-contaminated with a myeloid cell line K-562 (AKC CCL243). An extraordinary, cumbersome, and expensive effort, including PCR and cytogenetics studies, was required to solve this problem. Initial stocks, after deposit, contained as little as 0.4% of the offending K-562 line (3). After proliferation for 4 weeks and many passages, the &nding cell line emerged more apparently. The U-937 line was established by C. Sundstrom and K. Nilsson in 1976 and has been extensively used to study biology of human lymphoma cells and hematological malignancies in vitro (4). In search of a rapid, reliable, and inexpensive method to screen the U-937 cell line routinely for purity and to detect early andlor minor contamination with K-562, we applied a series of cytochemical and immunocytochemical methods. We found that esterase cytochemistry is very helpful in this case and will serve in quality control for purity of large production batches. Alpha-naphthyl acetate esterase and naphthol AS-D chloroacetate esterase were present in both cell types in different amounts, as assessed by image analysis. However, alpha-naphthyl butyrate esterase was positive in U-937 and negative in K-562 cell line cells. A clear distinction on slides can be made between green-bluish (counterstain, methylene blue) K-562 cells and brown-red (esterase activity deposit) U-937 cells (Figure 1). This technique allows detection of cross-contamination with K-562 cells below 0.1%. Morphological criteria, based on Wright-Giemsa modified staining, were used in addition. The entire procedure (preparation of cells and reagents, fixation, cytochemical visualization of enzyme activity, and counterstaining) can be performed within 2 hr (5.6). We recommend the alpha-naphthyl butyrate esterase cytochemical assay as a screening procedure for detecting K-562 cell cross-contamination in U-937 cell line. This test has been applied at ATCC to a stock of U-937 now available as CRL1593.2(4). Contaminant K-562 cells have not been detected in this population. A battery of cytochemical tests can be applied on many cell lines, and specific identification characteristics can bc identified. Once such identification cards are created, cytochemistry can be used as quality control in every cell culture laboratory. This type of assessment of cell cross-contamination will save researchers from possible disappointments and will increase their confidence in the purity of cell lines. However, this application has not yet been sufficiently exploited. We hope that our letter will motivate researches to explore further applications of cytochemistry in cell culture studies.

Detection of Mixtures of Lager Yeast Strains by the Use of Selective Inhibitors in Solid Media

The use of more than one lager yeast strain in a brewery poses the risk of cross-contamination with consequent problems in beer quality. In this work, conditions are reported that allow the detection of cross-contamination among three lager yeast strains used in this company's breweries by the use of selective inhibitors in solid media. The inhibitors used were bromocresol green, copper sulfate, basic fuchsin, streptomycin, and Actidione (cycloheximide). Contamination levels as low as 0.01% (one in 10,000) were detected in some cases.

Lymphoid cell line identification and the detection of cross contamination.

Four lymphoid cell lines, presumably recent derivatives from non-neoplastic human tonsils, were identified in actuality as established Burkitt lymphoma and lymphoblastoid cell lines (LCL) when tested by chromosome banding and typing for HLA antigens. Additionally, the morphology, growth characteristics, tumorigenicity, expression of Epstein-Barr virus antigens and surface membrane immunoglobulins (SmIg) of the four lymphoid cell lines confirmed the correct identifications. In determining the possibility of cross contamination, the most reliable criteria for the identification of lymphoid cell lines were found to be HLA antigenic profile, karyotype, cellular morphology, and growth characteristics.