SNP Genotyping and ApoE Genotyping v1

This protocol details the steps for DNA extraction from a human blood sample, quality control, and SNP and APOE genotyping. The protocol has been adapted from the PRoBaND SNP Genotyping and ApoE Genotyping Protocol. The overall protocol for PRoBaND /Tracking Parkinson’s is published: Malek, N., Swallow, D. M. A., Grosset, K. A., Lawton, M. A., Marrinan, S. L., Lehn, A. C., Bresner, C., Bajaj, N., Barker, R. A., Ben-Shlomo, Y., Burn, D. J., Foltynie, T., Hardy, J., Morris, H. R., Williams, N. M., Wood, N., & Grosset, D. G. (2015). Tracking Parkinson’s: Study Design and Baseline Patient Data. Journal of Parkinson’s Disease, 5(4), 947–959. https://doi.org/10.3233/JPD-150662

Large-scale identification of extracellular plant miRNAs in mammals implicates their dietary intake

Extracellular microRNAs (miRNAs) have been proposed to function in cross-kingdom gene regulation. Among these, plant-derived miRNAs of dietary origin have been reported to survive the harsh conditions of the human digestive system, enter the circulatory system, and regulate gene expression and metabolic function. However, definitive evidence supporting the presence of plant-derived miRNAs of dietary origin in mammals has been difficult to obtain due to limited sample sizes. We have developed a bioinformatics pipeline (ePmiRNA_finder) that provides strident miRNA classification and applied it to analyze 421 small RNA sequencing data sets from 10 types of human body fluids and tissues and comparative samples from carnivores and herbivores. A total of 35 miRNAs were identified that map to plants typically found in the human diet and these miRNAs were found in at least one human blood sample and their abundance was significantly different when compared to samples from human microbiome or cow. The plant-derived miRNA profiles were body fluid/tissue-specific and highly abundant in the brain and the breast milk samples, indicating selective absorption and/or the ability to be transported across tissue/organ barriers. Our data provide conclusive evidence for the presence of plant-derived miRNAs as a consequence of dietary intake and their cross-kingdom regulatory function within human circulating system.

Evaluation of possible processing time effects on the global N-glycosylation profile of human blood samples

: The utilization of N-glycan profiling recently gained high importance both in fundamental biomedical and applied clinical research. However, for the time being, no glycan biomarker has been approved for clinical diagnosis by the regulatory agencies due to the lack of verifications on large patient cohorts and suitable analytical technologies. In this paper the effect of human blood sample handling was studied prior to N-glycosylation profiling by capillary electrophoresis coupled with high sensitivity fluorescence detection. Special attention was paid to the preservation of sialylated structures because of their important clinical – biological relevance. Our results suggested that it is adequate to refrigerate and store the collected total blood samples prior to analysis to obtain unbiased results. Furthermore, we report on the good practice of serum sample handling in order to prevent decomposition of the sialylated structures. Our findings may promote procedure standardization and easier clinical translation of diagnostic N-glycosylation profiling in molecular medicinal applications.

Ultrafiltration-based Extraction and LC-MS/MS Quantification of Phenylalanine in Human Blood Sample for Metabolite Target Analysis

Background: Phenylalanine is a significant biomarker for various diseases like phenylketonuria, gastric cancers, and ischemic stroke according to recent studies. Methods: In the present study; a simple, sensitive, selective and novel analytical method was validated by using an ultrafiltration-based extraction and LC-MS/MS quantification of phenylalanine in human plasma using 13C phenylalanine heavy isotope. Amicon® Ultra Centrifugal Filter was used for ultrafiltration. Parameters affecting LC separation and MS/MS detection were investigated and optimized. Chromatographic separation was achieved on a Merck SeQuant ZIC-HILIC (100x4.6 mm, 5 μm) at a column temperature of 40°C using a mobile phase of mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid (50:50 v/v) at a flow rate of 0.35 mL/min. The transitions m/z 167→121 for 13C phenylalanine, m/z 166→120 for phenylalanine itself were monitored using the MRM mode. Result: The assay was linear concentration range of 0.0025 μg/mL to 1.20 μg/mL (R2=0.999). The developed method was validated according to FDA guidelines. The method was found linear, sensitive, precise, accurate, and selective.

Development and Application of a LC-MS/MS Method for Simultaneous Quantification of Four First-Line Antituberculosis Drugs in Human Serum

A simple, rapid, and sensitive liquid chromatography (LC)/mass spectrometry (MS) method was established and validated for simultaneous quantitation of pyrazinamide, isoniazid, rifampicin, and ethambutol in human blood sample. Samples were pretreated by a single-step precipitation with acetonitrile. Chromatographic separation was achieved on XSelecT HSS T3 column by gradient elution with a total run time of 5.0 min. MS detection was performed by a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode with a positive electrospray ionization source. Isotope-labeled internal standard, especially rifampicin-D8, was applied to adjust for the loss during sample treatment. The established LC-MS/MS method showed a wide analytical range (pyrazinamide: 1.02∼60.0 μg/mL, isoniazid: 0.152∼10.0 μg/mL, rifampicin: 0.500∼30.0 μg/mL, and ethambutol: 0.0998∼5.99 μg/mL) and a good linearity (r > 0.99 for the four analytes) with acceptable accuracy and precision (90.15%∼104.62% and 94.00%∼104.02% for intra- and interaccuracy, respectively; RSD%: <12.46% and <6.43% for intra- and interprecision, respectively). It also showed excellent recoveries (79.24%∼94.16% for all analytes) and absence of significant matrix effect. This method was successfully applied to the quantification of four first-line antituberculosis (anti-TB) drugs, suggesting its suitability for therapeutic drug monitoring in the clinical practices.

Estimation of White Blood Cells using Convolutional Neural Network

Normally blood samples contain red blood cells, white blood cells and platelets. White blood cells are also called as leukocytes and they are the cells of immune system. The measure of White Blood Cells is so important for the doctors in diagnosing various diseases like leukemia or tissue damage etc. So, counting of White Blood Cells plays an important role. The manual counting of White Blood Cells in medical laboratories involves a device called Haemocytometer. But this process is extremely monotonous, time consuming, and leads to inaccurate results. In this work, image processing and deep learning mechanisms are used to locate and classify the White Blood Cells based on their categories. The White Blood Cells which are classified are counted and compared with the standard range of the types available in the human blood sample. By comparing the availability of White Blood Cells types, the normal and the abnormal blood samples are predicted accordingly. The dataset of the normal blood sample is obtained from the laboratory in biotechnology department and the datasets used for training in Convolutional Neural Network are attained from the website Leukocyte Images for Segmentation and Classification (LISC). This will increase efficiency and reduce the doctor’s burden as traditional manual counting is dull, tedious, and possibly subjective.

Glucose Concentration Measurement in Human Blood Plasma Solutions with Microwave Sensors

Three microwave sensors are used to track the glucose level of different human blood plasma solutions. In this paper, the sensors are evaluated as glucose trackers in a context close to real human blood. Different plasma solutions sets were prepared from a human blood sample at several added glucose concentrations up to 10 wt%, adding also ascorbic acid and lactic acid at different concentrations. The experimental results for the different sensors/solutions combinations are presented in this work. The sensors show good performance and linearity as glucose level retrievers, although the sensitivities change as the rest of components vary. Different sensor behaviors depending upon the concentrations of glucose and other components are identified and characterized. The results obtained in terms of sensitivity are coherent with previous works, highlighting the contribution of glucose to the dielectric losses of the solution. The results are also consistent with the frequency evolution of the electromagnetic signature of glucose found in the literature, and are helpful for selecting frequency bands for sensing purposes and envisioning future approaches to the challenging measurement in real biological contexts. Discussion of the implications of the results and guidelines for further research and development of more accurate sensors is offered.