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1.
Cytometry A ; 95(8): 885-897, 2019 08.
Article in English | MEDLINE | ID: mdl-30852843

ABSTRACT

Isolation of circulating tumor cells (CTCs) has generated clinical and academic interest due to the important role that CTCs play in cancer metastasis and diagnosis. Here, we present a PDMS and glass prototype of a microfluidic device for the immunomagnetic, immiscible phase filtration based capture, and isolation of MCF-7 breast cancer cells, from various sample matrices including PBS-based buffer, blood plasma, and unprocessed whole blood. Following optimization of surface energy of an oil-water interface, microfluidic geometry, and bead-binding kinematics, our microfluidic device achieved 95 ± 4% recovery of target cells from PBS-based buffer with 95% purity, 90 ± 3% recovery of target cells from blood plasma and recovery of ~70 ± 5% from unprocessed whole blood with purity >99% with 1 ml blood samples with 1,000 spiked target cells. From quantitative studies to assess the nonspecific carryover of contaminants from whole blood, we found that our system accomplishes a >175 fold depletion in platelets, >900 fold depletion in erythrocytes, and >1,700 fold depletion in leukocytes with respect to unprocessed whole blood, enabling us to avoid sample pre-processing. In addition, we found that ~95% of the isolated target cells were viable, making them suitable for subsequent molecular and cellular studies. We quantify and propose mechanisms for the carryover of platelet, erythrocyte, and leukocyte contamination in purified samples, rather than relying on sample pre-processing. These results validate the continued study of our platform for extraction of CTCs from patient samples and other rare cell isolation applications. © 2019 International Society for Advancement of Cytometry.


Subject(s)
Cell Separation/methods , Cell-Free Nucleic Acids/blood , Microfluidics/methods , Neoplastic Cells, Circulating/metabolism , Cell Count , Erythrocytes/cytology , Humans , Leukocytes/metabolism , MCF-7 Cells
2.
J Mol Diagn ; 14(3): 187-91, 2012.
Article in English | MEDLINE | ID: mdl-22414609

ABSTRACT

In recent years, the mutational status of the KRAS oncogene has become incorporated into standard medical care as a predictive marker for therapeutic decisions related to patients with metastasized colorectal cancer. This is necessary, because these patients benefit from epidermal growth factor receptor (EGFR)-targeted therapy with increased progression-free survival only if the tumor does not carry a mutation in KRAS. Many different analytical platforms, both those commercially available and those developed in house, have been used within pathology laboratories to assess KRAS mutational status. For a testing laboratory to become accredited to perform such tests, it is essential that they perform reliability testing, but it has not previously been possible to perform this kind of testing on the complete workflow on a large scale without compromising reproducibility or the mimicry of the control sample. We assessed a novel synthetic control for formalin-fixed, paraffin-embedded (FFPE) tumor samples in a blind study conducted within nine laboratories across Europe. We show that FFPE material can, at least in part, mimic clinical samples and we demonstrate this control to be a valuable tool in the assessment of platforms used in testing for KRAS mutational status.


Subject(s)
Colorectal Neoplasms , DNA Mutational Analysis/standards , Genes, ras , Molecular Diagnostic Techniques/standards , Proto-Oncogene Proteins/genetics , ras Proteins/genetics , Cell Line, Tumor , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , ErbB Receptors/antagonists & inhibitors , Fixatives , Formaldehyde , Humans , Mutation , Paraffin Embedding , Proto-Oncogene Proteins p21(ras) , Quality Control , Reproducibility of Results
3.
Clin Chem ; 57(8): 1108-17, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21677092

ABSTRACT

Results between different clinical laboratory measurement procedures (CLMP) should be equivalent, within clinically meaningful limits, to enable optimal use of clinical guidelines for disease diagnosis and patient management. When laboratory test results are neither standardized nor harmonized, a different numeric result may be obtained for the same clinical sample. Unfortunately, some guidelines are based on test results from a specific laboratory measurement procedure without consideration of the possibility or likelihood of differences between various procedures. When this happens, aggregation of data from different clinical research investigations and development of appropriate clinical practice guidelines will be flawed. A lack of recognition that results are neither standardized nor harmonized may lead to erroneous clinical, financial, regulatory, or technical decisions. Standardization of CLMPs has been accomplished for several measurands for which primary (pure substance) reference materials exist and/or reference measurement procedures (RMPs) have been developed. However, the harmonization of clinical laboratory procedures for measurands that do not have RMPs has been problematic owing to inadequate definition of the measurand, inadequate analytical specificity for the measurand, inadequate attention to the commutability of reference materials, and lack of a systematic approach for harmonization. To address these problems, an infrastructure must be developed to enable a systematic approach for identification and prioritization of measurands to be harmonized on the basis of clinical importance and technical feasibility, and for management of the technical implementation of a harmonization process for a specific measurand.


Subject(s)
Clinical Laboratory Techniques/standards , Quality Assurance, Health Care , Biomarkers/analysis , Humans , International Cooperation , Practice Guidelines as Topic , Reference Standards , Sensitivity and Specificity
4.
J Gen Virol ; 91(Pt 5): 1213-7, 2010 May.
Article in English | MEDLINE | ID: mdl-20053823

ABSTRACT

Here we report on a segment in the genomic 3' non-translated region (3'NTR) of bovine viral diarrhea virus (BVDV) that is accessible for the insertion of foreign sequence elements such as the 5'NTR of hepatitis C virus. Recombinant viruses exhibited replication kinetics similar to those of the parental strain, and characterization of RNA species after several passages revealed that foreign inserts had the same genetic stability as the BVDV 3'NTR. The generation of such BVDV recombinants is relevant for several applications.


Subject(s)
Hepacivirus/genetics , Pestivirus/genetics , RNA, Viral/genetics , Recombination, Genetic , 3' Untranslated Regions , 5' Untranslated Regions , Animals , Base Sequence , Cattle , Cell Line , Molecular Sequence Data , Nucleic Acid Conformation
5.
Clin Chem ; 55(9): 1701-10, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19574467

ABSTRACT

BACKGROUND: Viral load testing for cytomegalovirus (CMV) is an important diagnostic tool for the management of transplant recipients and immunocompromised individuals; however, inconsistency among laboratories in quantitative measurements of viral load limits interinstitutional comparisons. These inconsistencies stem from the lack of assays cleared by the US Food and Drug Administration, the absence of international standards, the wide variety of CMV-extraction and -detection methods, and differences in materials used for calibration. A critical component of standardization is the use of calibrators that are traceable and commutable. METHODS: Bland-Altman plots and prediction ellipses were used to test the commutability of 2 CMV calibrators for 2 different quantification methods. RESULTS: Tests with 2 methods showed 1 calibrator to be commutable and the other to be noncommutable. The results for the commutable calibrator were within the 95% prediction interval of the clinical samples in the Bland-Altman plot and within the 95% prediction ellipse for a simulated commutable calibrator, whereas the results for the noncommutable calibrator were not within these prediction intervals. When used to calibrate patient results, only the commutable calibrator, the OptiQuant CMV(tc) Calibration Panel, significantly improved the comparability of viral loads for the 2 different measurement methods. CONCLUSIONS: This study demonstrates that an important goal in the effort to improve healthcare for patients with CMV-related disease is the establishment of traceable and commutable reference materials, including both calibrators and controls. .


Subject(s)
Cytomegalovirus Infections/blood , Cytomegalovirus Infections/virology , Cytomegalovirus/isolation & purification , Viral Load/methods , Viral Load/standards , Calibration , DNA, Viral/analysis , Humans
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