The Integration of a Three-Dimensional Spheroid Cell Culture Operation in a Circulating Tumor Cell (CTC) Isolation and Purification Process: A Preliminary Study of the Clinical Significance and Prognostic Role of the CTCs Isolated from the Blood Samples of Head and Neck Cancer Patients.

Conventional positive and negative selection-based circulating tumor cell (CTC) isolation methods might generally ignore metastasis-relevant CTCs that underwent epithelial-to- mesenchymal transition and suffer from a low CTC purity problem, respectively. To address these issues, we previously proposed a 2-step CTC isolation method integrating a negative selection CTC isolation and subsequent spheroid cell culture. In addition to its ability to isolate CTCs, more importantly, the spheroid cell culture used could serve as a cell culture model mimicking the process of new tumor tissue formation during cancer metastasis. Therefore, it is promising not only to selectively isolate metastasis-relevant CTCs but also to test the potential of cancer metastasis and thus the prognosis of disease. To explore these issues, experiments were performed. The key findings of this study demonstrated that the method was able to harvest both epithelial (E)- and mesenchymal (M)-type CTCs without selection bias. Moreover, both the M-type CTC count and the information obtained from the multidrug resistance-associated protein 2 (MRP2) and MRP5 gene expression analysis of the CTCs isolated via the 2-step CTC isolation method might be able to serve as prognostic factors for progression-free survival in head and neck squamous cell carcinoma.

Development of a high sensitivity TaqMan-based PCR assay for the specific detection of Mycobacterium tuberculosis complex in both pulmonary and extrapulmonary specimens.

Tuberculosis (TB) causes a heavy health burden worldwide, especially in developing countries. The need for the rapid and accurate diagnosis of TB has not been satisfied, especially for extra-pulmonary specimens or specimens with acid fast stain (AFS)-negative condition. Development and validation of a novel, sensitive and specific assay for diagnosing TB is essential. We developed IS4 primer/probe based on insertion sequence 6110 (IS6110). A qPCR assay was designed for detecting a specific region in IS6110 by BLAST. The IS4 primer/probe concentration, qPCR efficiency and various of PCR additives were evaluated and optimized. Thirty-four species of commonly isolated microorganisms were used for evaluating the analytical specificity. Moreover, 130 clinical specimens were collected for evaluating the performance versus Cobas TaqMan MTB (CTM) assay kit and culture. The amplification efficiencies of IS4 were 99.61% and 102.61% without and with internal control DNA (Bacteriophage Lambda), respectively. Dimethyl sulfoxide outperformed glycerol or BSA for eliciting the most effective amplification and the lowest limit of detection. In evaluating the clinical performance, various specimen types were collected. IS4 demonstrated a high degree of agreement (kappa = 0.71) with CTM. The clinical sensitivity and specificity of IS4 and CTM were 92.11% (35/38), 82.61% (76/92), 84.21% (32/38) and 95.65% (88/92), respectively. The clinical sensitivity and specificity of IS4 were similar for both pulmonary [92.00% (23/25) and 76.92% (30/39), respectively] and extrapulmonary [92.31% (12/13) and 86.79% (46/53), respectively] specimens. Among AFS-negative cases, the clinical sensitivity and specificity remained 90.48% (19/21) and 83.91% (73/87), respectively, with culture as the gold standard. We concluded that IS4, a new primer/probe pair for TaqMan based qPCR assay, was developed, optimized, and validated for the sensitive and specific detection of TB among various specimen types. The performance was not compromised under AFS-negative conditions.

An Optically Induced Dielectrophoresis (ODEP)-Based Microfluidic System for the Isolation of High-Purity CD45neg/EpCAMneg Cells from the Blood Samples of Cancer Patients-Demonstration and Initial Exploration of the Clinical Significance of These Cells.

Circulating tumour cells (CTCs) in blood circulation play an important role in cancer metastasis. CTCs are generally defined as the cells in circulating blood expressing the surface antigen EpCAM (epithelial cell adhesion molecule). Nevertheless, CTCs with a highly metastatic nature might undergo an epithelial-to-mesenchymal transition (EMT), after which their EpCAM expression is downregulated. In current CTC-related studies, however, these clinically important CTCs with high relevance to cancer metastasis could be missed due to the use of the conventional CTC isolation methodologies. To precisely explore the clinical significance of these cells (i.e., CD45neg/EpCAMneg cells), the high-purity isolation of these cells from blood samples is required. To achieve this isolation, the integration of fluorescence microscopic imaging and optically induced dielectrophoresis (ODEP)-based cell manipulation in a microfluidic system was proposed. In this study, an ODEP microfluidic system was developed. The optimal ODEP operating conditions and the performance of live CD45neg/EpCAMneg cell isolation were evaluated. The results demonstrated that the proposed system was capable of isolating live CD45neg/EpCAMneg cells with a purity as high as 100%, which is greater than the purity attainable using the existing techniques for similar tasks. As a demonstration case, the cancer-related gene expression of CD45neg/EpCAMneg cells isolated from the blood samples of healthy donors and cancer patients was successfully compared. The initial results indicate that the CD45neg/EpCAMneg nucleated cell population in the blood samples of cancer patients might contain cancer-related cells, particularly EMT-transformed CTCs, as suggested by the high detection rate of vimentin gene expression. Overall, this study presents an ODEP microfluidic system capable of simply and effectively isolating a specific, rare cell species from a cell mixture.

Membrane capacitance of thousands of single white blood cells.

As label-free biomarkers, the electrical properties of single cells are widely used for cell type classification and cellular status evaluation. However, as intrinsic cellular electrical markers, previously reported membrane capacitances (e.g. specific membrane capacitance Cspec and total membrane capacitance Cmem) of white blood cells were derived from tens of single cells, lacking statistical significance due to low cell numbers. In this study, white blood cells were first separated into granulocytes and lymphocytes by density gradient centrifugation and were then aspirated through a microfluidic constriction channel to characterize both Cspec and Cmem Thousands of granulocytes (ncell = 3327) and lymphocytes (ncell = 3302) from 10 healthy blood donors were characterized, resulting in Cspec values of 1.95 ± 0.22 µF cm-2 versus 2.39 ± 0.39 µF cm-2 and Cmem values of 6.81 ± 1.09 pF versus 4.63 ± 0.57 pF. Statistically significant differences between granulocytes and lymphocytes were located for both Cspec and Cmem In addition, neural network-based pattern recognition was used to classify white blood cells, producing successful classification rates of 78.1% for Cspec and 91.3% for Cmem, respectively. These results indicate that as intrinsic bioelectrical markers, membrane capacitances may contribute to the classification of white blood cells.

Application of optically-induced-dielectrophoresis in microfluidic system for purification of circulating tumour cells for gene expression analysis- Cancer cell line model.

Circulating tumour cells (CTCs) in a blood circulation system are associated with cancer metastasis. The analysis of the drug-resistance gene expression of cancer patients' CTCs holds promise for selecting a more effective therapeutic regimen for an individual patient. However, the current CTC isolation schemes might not be able to harvest CTCs with sufficiently high purity for such applications. To address this issue, this study proposed to integrate the techniques of optically induced dielectrophoretic (ODEP) force-based cell manipulation and fluorescent microscopic imaging in a microfluidic system to further purify CTCs after the conventional CTC isolation methods. In this study, the microfluidic system was developed, and its optimal operating conditions and performance for CTC isolation were evaluated. The results revealed that the presented system was able to isolate CTCs with cell purity as high as 100%, beyond what is possible using the previously existing techniques. In the analysis of CTC gene expression, therefore, this method could exclude the interference of leukocytes in a cell sample and accordingly contribute to higher analytical sensitivity, as demonstrated in this study. Overall, this study has presented an ODEP-based microfluidic system capable of simply and effectively isolating a specific cell species from a cell mixture.

DNA detection using commercial mobile phones.

This study investigates the feasibility of using mobile phones cameras for DNA detection. DNA amplification uses the convective polymerase chain reaction (cPCR) technique due to its simple mechanism, which requires no thermal cycling control. Fluorescence increment analysis and information entropy analysis are employed separately to determine whether the test samples contain target DNA (Positive) or not (Negative). The fluorescence increment method uses the brightness of the captured images before and after DNA amplification to calculate ΔF. ΔF values above a threshold level indicate that the test sample is positive. The information entropy method defines the probability, P(C/X), which indicates whether the fluorescence image tends towards a specific shape. If a DNA template is successfully amplified, the captured fluorescence image should be a perfect circle. P(C/X) provides a threshold of 0.5 to identify a circle and values above 0.5 indicate the test sample is positive. Experimental results show that P(C/X) is more effective than ΔF for determining DNA detection results. The information entropy analysis method is applied to ten mobile phones of three different brands equipped with camera sensors, which have pixel numbers ranging from 120 M to 800 M. The clinical evaluation study (n = 60) for screening hepatitis B virus (HBV) plasmid samples shows that the accuracy rate of all models of mobile phones ranges from 85% to 100%. This illustrates that successful DNA detection can be achieved using the most widely deployed electronic device.

Rapid DNA amplification in a capillary tube by natural convection with a single isothermal heater.

Herein we describe a simple platform for rapid DNA amplification using convection. Capillary convective PCR (CCPCR) heats the bottom of a capillary tube using a dry bath maintained at a fixed temperature of 95°C. The tube is then cooled by the surrounding air, creating a temperature gradient in which a sample can undergo PCR amplification by natural convection through reagent circulation. We demonstrate that altering the melting temperature of the primers relative to the lowest temperature in the tube affects amplification efficiency; adjusting the denaturation temperature of the amplicon relative to the highest temperature in the tube affects maximum amplicon size, with amplicon lengths of ≤500 bp possible. Based on these criteria, we successfully amplified DNA sequences from three different viral genomes in 30 min using CCPCR, with a sensitivity of ~30 copies per reaction.

Hepatitis B virus quantification and detection of YMDD mutants in a single reaction by real-time PCR and annealing curve analysis.

BACKGROUND: Long-term antiviral therapy, although effective for treatment of hepatitis B, might select the emergence of drug-resistant hepatitis B virus (HBV) mutants. Detection of HBV mutants and determination of viral titre are two crucial parameters for monitoring treatment response and occurrence of mutants. In this study, we take lamivudine resistance as an example to develop a method that can determine both parameters in a single-tube PCR reaction. METHODS: The method contained two consecutive steps: in the first step, real-time PCR was used for quantification; in the second step, a novel annealing curve analysis was used for detecting YMDD mutants. For accurate quantification, PCR primers and hybridization probes were chosen from highly conserved regions to ensure the equivalent amplification of all HBV genotypes. Within the sensor probe, there were signature nucleotide polymorphisms that could effectively differentiate YMDD mutants from wild type by distinct melting temperatures (Tm) values. The clinical applicability of the assay was tested in serial samples from 90 patients receiving lamivudine treatment. RESULTS: This assay could readily differentiate YMDD, YIDD and YVDD mutants by their distinct Tm values. The quantification results showed great consistency in a linear range from 10(3) to 10(11) copies/ml. Moreover, this assay could detect YMDD mutants accounting for < or = 10% of the total viral population. Its clinical feasibility has been verified in primary specimens. CONCLUSIONS: The newly designed YMDD detection method is simple, sensitive, cost-effective, time-saving and provides a useful tool for follow-up of patients treated with lamivudine or other antiviral drugs.

Mitochondrial DNA 4,977-bp deletion in paired oral cancer and precancerous lesions revealed by laser microdissection and real-time quantitative PCR.

Oral cancer is the fourth leading cause of cancer deaths among men in Taiwan and is closely associated with areca quid chewing habits. Recent studies showed that mitochondrial DNA (mtDNA) mutations occur in various tumors, including oral cancers, and that the accumulation of mtDNA deletions could be an important contributor to carcinogenesis. Using laser microdissection, we have analyzed mtDNA deletions by pairwise comparisons in oral cancer, precancerous cells, and their adjacent submucosal stoma tissues in 12 patients with areca quid chewing history. Real-time quantitative polymerase chain reaction (RTQPCR) was performed to detect and quantify mtDNA with the 4,977-bp deletion in the histologically defined specified cell groups. Quantitative analysis of 60 samples by RTQPCR revealed that the average proportions of 4,977-bp deleted mtDNA over total mtDNA were 0.137%, 0.367%, and 0.001% in cancer, precancer cells, and lymphocytes of lymph node biopsies, respectively. Pairwise analysis of the proportion of mtDNA deletion in cancer, precancer, and their stroma tissues revealed a consistent trend among these patients. All of the patients (12/12) presented a higher proportion of mtDNA with 4,977-bp deletion in the lesions than in the lymphocytes, with average increases of 198-fold in cancer and 546-fold in precancer cells. A decrease in the proportion of deleted mtDNA was observed in 8 of 12 patients when the disease progressed from precancer to cancer lesions. Interestingly, 7 of 12 cancer tissues and 8 of 12 precancer lesions exhibited an average of 6.3-fold and 17.4-fold increases in the proportion of 4,977-bp deleted mtDNA in the stromal cells than in the lesion cells, respectively. The observation that the proportion of 4,977-bp deleted mtDNA in all oral lesions was higher than normal and consistently decreased during cancer progression from precancer to primary cancer suggests that accumulation and subsequent cytoplasmic segregation of the mutant mtDNA during cell division may play an important role in oral carcinogenesis. This study also demonstrates that laser microdissection combined with RTQPCR is an efficient and sensitive tool to gain insight into the role that mtDNA mutation may play in carcinogenesis.