Blood samples, preparation

Yuen PK, Kricka L, Fortina P, Panaro NJ, Sakazume T, Wilding P Microchip module for blood sample preparation and nucleic acid amplification reactions. Genome Res 2001 11 405-12. 

A final area for improvement is test turnaround time. Although many current procedures involve 1-2 hours of sample preparation, 2-3 hours of thermocycling, and 1-12 hours for detection, there is nothing sacred about these times. Some whole-blood sample preparation procedures can be done in 1-15 minutes and several nonisotopic detection methods for amplified DNA have been described... 

NAA has become more common, particularly where multielement analysis is required [57-59]. Some sample preparation is usually necessary. For example, a blood sample preparation will require separation of the serum and plasma followed by freeze-drying of the specimen before irradiation. Afterward samples were wet-ashed and fractionated on various columns before the y spectra from each fraction could be obtained [60]. 

Blood Samples Preparation. Blood samples were collected in EDTA vials and centrifuged for 15 minutes at 1000 x g within 30 minutes. The separated plasma was placed in Eppendorf tubes and transported in ice-packed container within 10 hours to the AMDFs laboratory for further measurements. In the laboratory samples were stored in the freezer at - 20°C and processed at shortest possible time. 

Blood sample preparation can crack cells to extract DNA. 

Immunoassays. Immunoassays (qv) maybe simply defined as analytical techniques that use antibodies or antibody-related reagents for selective deterrnination of sample components (94). These make up some of the most powerflil and widespread techniques used in clinical chemistry. The main advantages of immunoassays are high selectivity, low limits of detection, and adaptibiUty for use in detecting most compounds of clinical interest. Because of their high selectivity, immunoassays can often be used even for complex samples such as urine or blood, with Httle or no sample preparation. 

Two methods of sample preparation were investigated. The former is dilution of blood semm with 0.1% Triton X-100, the latter is aeid mierowave digestion. As evaluated, the most adequate mineralization proeedure for determining the majority of elements in blood semm by ICP AES is aeid mierowave digestion. However, the ICP AES determination of abundant elements (B, Si, Mn), whieh present in semm at 0.001-0.01 ppm levels should be follow sample dilution with Triton X-100. 

The two examples of sample preparation for the analysis of trace material in liquid matrixes are typical of those met in the analytical laboratory. They are dealt with in two quite different ways one uses the now well established cartridge extraction technique which is the most common the other uses a unique type of stationary phase which separates simultaneously on two different principles. Firstly, due to its design it can exclude large molecules from the interacting surface secondly, small molecules that can penetrate to the retentive surface can be separated by dispersive interactions. The two examples given will be the determination of trimethoprim in blood serum and the determination of herbicides in pond water. 

Several methods are available for the analysis of trichloroethylene in biological media. The method of choice depends on the nature of the sample matrix cost of analysis required precision, accuracy, and detection limit and turnaround time of the method. The main analytical method used to analyze for the presence of trichloroethylene and its metabolites, trichloroethanol and TCA, in biological samples is separation by gas chromatography (GC) combined with detection by mass spectrometry (MS) or electron capture detection (ECD). Trichloroethylene and/or its metabolites have been detected in exhaled air, blood, urine, breast milk, and tissues. Details on sample preparation, analytical method, and sensitivity and accuracy of selected methods are provided in Table 6-1. 

Indeed, a bDNA assay for diagnosis of African trypanosomiasis was developed and compared with buffy coat microscopy for detection of T brucei in human blood samples (Harris etal., 1996). Two repetitive DNA sequences found only in the T. brucei complex, a 177-bp satellite repeat and the ribosomal mobile element, were selected as targets in the bDNA assay. The assay used the standard bDNA components capture probes, target probes, amplifier molecules, and alkaline phosphatase-labeled probes. Various blood fractions and sample preparation methods were examined. Ultimately, buffy coat samples resulted in the highest sensitivity. Although typanosomes do not infect leukocytes, they cosediment with them. 

Several additional instrumental techniques have also been developed for bacterial characterization. Capillary electrophoresis of bacteria, which requires little sample preparation,42 is possible because most bacteria act as colloidal particles in suspension and can be separated by their electrical charge. Capillary electrophoresis provides information that may be useful for identification. Flow cytometry also can be used to identify and separate individual cells in a mixture.11,42 Infrared spectroscopy has been used to characterize bacteria caught on transparent filters.113 Fourier-transform infrared (FTIR) spectroscopy, with linear discriminant analysis and artificial neural networks, has been adapted for identifying foodbome bacteria25,113 and pathogenic bacteria in the blood.5... 

Figure 8.2 Sample preparation protocols, currently employed in malaria parasite detection by LDMS. Both protocols require less than 50pl blood, obtained by either finger-stick or phlebotomy.

Figure 8.3 Positive ion LD TOF mass spectrum of blood from a P. vivax infected human patient (only asexual parasites have been observed by microscopy estimated parasitemia approximately 72 parasites/pl). Protocol C is used for sample preparation estimated number of parasites deposited per well is approximately 90. A commercial TOF system is used laser wavelength 337 nm. All one hundred single laser shot spectra, obtained from hnear scanning of an individual well, are averaged (no data smoothing). The characteristic fingerprint ions of detected heme are denoted.

In the first LDMS-based detection of malaria in human subjects (unpublished), lOOpl P. falciparum or P. v/vax-infected blood samples, grouped into three different parasitemia ranges—low (10-150 parasites/pl), mid (2 x 103 parasites/pl), and high (25 x 103-60 x 103 parasites/pl)—have been examined using both sample preparation protocols. Parasitemia levels in these samples were previously determined independently for each sample by optical microscopy examination of blood smears. The LDMS data clearly indicate that... 

One of the most sensitive biological effects of ionizing radiation is to increase the frequency of normally observed chromosome aberrations (but not to induce qualitatively special abnormalities). Peripheral blood lymphocytes are the most feasible cells for chromosome investigations, as blood samples are easy to obtain and the techniques to stimulate the lymphocytes to proliferate within a culture medium and to prepare suitable chromosome slides for microscopic analyzation have their routine protocoil (e. g. Yunis, 1965 Lloyd et al, 1982). 

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