Human blood sample preparation

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. 

Magnetic sector thermal Ionization mass spectrometry Is capable of far better precision and accuracy than the other two methods of analysis. A recent comparative study of NAA and Tl/MS zinc analysis In human blood samples demonstrated the marked difference In precision between the two methods (12). The standard deviation of zinc ratio determinations was 0.13% for TI/MS and 8.3% for NAA. It Is likely that, while maximum attainable precision for both methods Is better than this (see Table III), these levels of precision are the best which can be achieved In biological samples. The excellent precision of TI/MS Is offset by several disadvantages. Analysis Is slow, requires considerable skill and experience, and Is expensive. Extensive sample preparation Is required to obtain pure samples pure enough for accurate analysis. However, the high degree of precision attainable by this method make It the only choice when excellent precision and accuracy (e.g. within 1%) are required. 

For biological samples four different contributions to the standard potential Ef can be observed the contribution of the internal standard potential of the reference electrode ERef the diffusion potential over the liquid junction Ej generated between the sample solution and the reference electrode a potential difference (electrical asymmetry) of the ion-selective membrane after preparation and conditioning Eei and a sample-induced asymmetry of the membrane Eas. For measurements in human blood samples directly the adsorption of sample components at the membrane surface creates a drift associated with the affinity of the membrane to lipids as well as proteins. 

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... 

Probably the most effective use of XRF and TXRF continues to be in the analysis of samples of biological origin. For instance, TXRF has been used without a significant amount of sample preparation to determine the metal cofactors in enzyme complexes [86]. The protein content in a number of enzymes has been deduced through a TXRF of the sulfur content of the component methionine and cysteine [87]. It was found that for enzymes with low molecular weights and minor amounts of buffer components that a reliable determination of sulfur was possible. In other works, TXRF was used to determine trace elements in serum and homogenized brain samples [88], selenium and other trace elements in serum and urine [89], lead in whole human blood [90], and the Zn/Cu ratio in serum as a means to aid cancer diagnosis [91]. 

Recently, turbulent flow chromatography (TFC) has shown a great potential for online sample pre-treatment in the analysis of PFCs. Up to now, the use of this technique in food and environmental analysis is scarce, but some successful applications have been developed. Among them, the analysis of PFCs has been carried out in cord blood and also in less invasive human samples, hair and urine. In these works, the main advantages presented were the simplified sample preparation, robustness and sensitivity. In addition in the case of cord blood, a low volume of sample was required. 

Inhibition of COX can be quantified in recombinant or natural enzyme preparations, cellular systems, isolated human cell populations such as platelets (COX-1) and white blood cells (COX-2), or in ex vivo stimulated whole blood samples The closer the experimental system is to the physiological state, the lower the selectivity for most COX-2-inhibitors. The standard test for comparison is considered to be a whole blood assay which mimics in vivo conditions like plasma binding (e.g. Patrignani et al., 1996). It is commonly accepted that reasonable variations occur between different laboratories (see data for Piroxicam). Therefore, whenever possible, data of several compounds generated with a given test system should be compared with each other. 

Howlett and Taylor (1978) used an atomic absorption spectroscopy fitted with a micro-cup assembly (MCAAS) for determining silver levels in human whole blood. The MCAAS technique affords a rapid, precise, and relatively simple method for the measurement of silver in blood. Furthermore, this technique requires no sample preparation prior to analysis except pipetting and drying. A detection limit level of 0.27 pg/100 ml of blood sample was measured. Flowlett and Taylor (1978) noted that repeated measurement of silver in blood using a single nickel cup showed a gradual decrease in sensitivity. 

Machtejevas E, John H, Wagner K, Standker L, Marko-Varga G, Forssmann WG, Bischoff R, Unger KK (2004) Automated multi-dimensional liquid chromatography sample preparation and identification of peptides from human blood filtrate. J Chromatogr B 803 121-130... 

The Human Proteome Project (HUPO, www.hupo.org) initiated the Plasma Proteome Project (PPP) in 2002, and numerous laboratories have contributed to this ambitious project of deciphering all proteins contained in the human plasma. Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins that originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. For the reasons described above, HUPO recommends use of plasma instead of serum, with EDTA (or citrate) for anticoagulation and standardized sample preparation. HUPO proposes combinations of serum depletion, fractionation procedures, and MS/MS technologies, with explicit... 

Whole mesocosm samples, GF/F filtrate and extracts (up to 23 March 2003) were diluted 1 1 with blood cell suspension. For whole-sample transfer, every pipette tip used was clipped with a pair of scissors to have a > 3 mm diameter inlet, making sure to include colonies while sampling. The blood cell suspension was prepared by adding five drops of fresh human blood to 30 ml buffer (Eschbach et al. 2001), centrifugation (5 min, 4500 x g) and addition of the same volume of buffer to the pellet. Standard incubations were done for 24 h in triplicate in test vials (2.5 ml, Eppendorf) at 15°C, 7 gmol photons m 2 s After incubation, intact blood cells and... 

Isolation of Human Platelets The steps described in the subsequent subheading outline the procedure for isolation and purification of platelets from whole blood obtained by venipuncture from human volunteers. Obtain blood sample by venipuncture from an antecubital vein into polypropylene syringes containing either sodium citrate (0.38 % final concentration) or heparin (10 U/ml final concentration). Centrifuge anticoagulated whole blood at 160 x g for 15 min to prepare platelet-rich plasma (PRP). 

Iyer et al. (2001) investigated the metabolism of [14C]omapatrilat in humans with samples collected during a clinical study. Plasma samples were prepared from blood spiked with or without methylacrylate to trap compound free sufhydryl groups which was important for this particular compound. Samples were pooled over the 12 subjects enrolled in the study. Urine was pooled over time to give a 0-24 h pooled urine sample representing 92 % of the radioactivity excreted in urine and a 0-168 h sample. Feces was not analyzed. 

Subjects were 14 healthy men and women who lived in the human metabolic unit of the Department of Human Nutrition and Food Service Management at the University of Nebraska-Lincoln. All meals were prepared and consumed in the metabolic diet kitchen. Subjects made complete urine and fecal collections throughout the study and donated blood samples biweekly. 

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