Plasma collection procedure

Procedures for the collection of whole blood are similar throughout the world. An interval from at least 8 weeks (United States) to 12 weeks (United Kingdom) is required between a donation of 450 mL blood, which yields about 250 mL plasma. In some countries a smaller volume of blood is collected, eg, 350—400 mL in Italy, Greece, and Turkey and as Httie as 250 mL in some Asian countries (147). Regulations concerning plasmapheresis donations vary more widely across the world eg, up to 300 mL of plasma can be taken in Europe in contrast to 1000 mL in the United States, both on a weekly basis. Consequentiy, both the mode of donation and the country in which it is given can have a profound effect on plasma collection (Table 6). 

The question often arises whether a sample must be analyzed immediately or can be stored, and if so, under what conditions and for how long (B4a, H5a, W9a). Freshly drawn blood maintained anaerobically (A3) at 38 C decreases in pH at the rate of —0.062 unit per hour and in pCOj, at 4.8 1.3 mg Hg per hour. At 0-4°C, the change is minimal — 0.006 0.004 pH unit and 0.6 0.06 mm Hg. There has been controversy concerning the use of minerol oil to maintain specimens for carbon dioxide analysis (G2). Paulsen found that values of total carbon dioxide in plasma collected in stoppered tubes with and without paraflSn oil were identical if the tubes without oil were completely filled to the stopper (P4). The loss of carbon dioxide in tubes stored at room temperature without oil was about 6 mEq/1 in 2.5-4 hours. The problem for the laboratory is unfilled tubes and the storage of separated serum or plasma before analysis and in plastic cups during continuous-flow procedures. 

Serum is the specimen of choice for many assay systems. Sometimes considerable differences may be observed between the concentrations of analytes in serum and plasma as shown in Table 2-3. However, some assay systems require a whole blood or plasma specunen. If so an anticoagulant must be added to the specimen during the collection procedure. A number of anticoagulants are used including heparin, EDTA, sodium fluoride, citrate, oxalate, and iodo acetate. 

Laycock, J.D. Jaramilla, J. Zhang, L. Smithson, A. Mallard, S. Miller, K.J. Optimized Plasma Sample Collection Procedures to Minimize Clot Formation Prior to Robotic Liquid Handling, paper presented at LabAutomatlon2003, February 2-5, 2003, Palm Springs, CA. 

Sometimes a clinical site may deviate from the study protocol-defined sample-collection procedures. For example, instead of collecting blood into heparinized tubes, the sample may be collected into a tube containing another anticoagulant, such as citrate or EDTA. Under these conditions, it is necessary to cross-validate each of the plasma/serum matrices QC samples in the deviant matrix must be prepared and processed along with the deviant samples to ensure that the method is validated in the new matrix. 

Several of the blood collection procedures alter the plasma albumin and total protein concentrations, and these changes should be considered when interpreting toxi-cokinetic data, particularly when perturbations of plasma proteins occur (Hulse et al. 1981 Chou and Levy 1984 Tamura et al. 1990 Proost, Wierda, and Meijer 1996). 

Plasma collection tubes with a variety of additives are commercially available (e.g., heparin, EDTA, sodium citrate). These additives have the potential to cause interference with some analytical assays. Thus, interference testing should be performed prior to implementing sample collection procedures. It should be noted that the collection tubes intended to generate plasma contain the optimal amount of the appropriate additive for the indicated sample volume. If tubes are not filled to this indicated volume, the concentration of additive will be higher than recommended and could pose problems during downstream analysis. After collection, sample tubes should be mixed by gentle inversion several times to ensure proper mixing of additive with the blood. 

Stainless steel needles and plastic catheters used in blood collection contribute negligible amounts of aluminum to the procedure. Standard collection equipments, e.g., Vacutainer and Monovetten , can be used after checking all sources for possible contamination. Separation aids must be avoided. The blood collection procedure in the authors laboratories involves drawing blood with a stainless steel needle into an EDTA-K Monovette (Sarstedt, Numbrecht, ERG). After centrifugation the plasma samples are directly transferred to the vials of the atomic absorption spectrometry (AAS) sampler. 

Initially, whole blood, plasma and serum were the accepted indicators of Se exposure. However, selenium measurements have now been extended to many organs and other body fluids. Publications on Se analysis of biological samples are numerous, but it is evident that there have been incorrect, inaccurate and imprecise results reported. The main reasons for these have often been related to sampling errors, contamination, losses during handling, pretreatment, decomposition and steps in procedure. Vital information on sample collection, procedure, precision and use of certified material is rarely provided [7], Several interlaboratory comparisons of Se analyses have appeared, which focus on the use of certified material and other factors [80-82]. 

Ion exchange chromatography has been used mainly in preparative separations of cobalamin forms and their precursors. The use of Amberlite resins and cellulose derivatives has been reviewed by Pawelkiewicz (38). With these materials it was impossible to separate methyl-, cyano-, ado-, and hydroxocobalamin in one step. Tortolani et al. (93) first described a combination of CM-cellulose and Dowex 50W-X2, but later a one-step separation on SP-Sephadex (94). Subsequently, Gams et al. (95,96) and Gimsing and Hippe (97) used this procedure to separate the various coenzyme forms in L1210 cells, mitochondria, and plasma. Begley and Hall (98) measured the conversion of CN( Co)Cbl into coenzyme forms in cells in tissue culture with this technique. They found that a fifth form of cobalamin, later identified as sulfitocobalamin, almost coeluted with the CN-Cbl peak, but by a small modification of the fraction collection procedure the SOs-Cbl peak was eluted separately from the CN-Cbl peak (Fig. 8). The method is still in use as is apparent from the publication of Anes et al. (99) on the separation of nitrito-and nitrosocobalamin. 

The trial consisted of 6 consecutive periods (3 d of adaptation + 1 d of expired CO2 and plasma collection). On collection day, tracer phenylalanine was given orally in 8 A-hourly meals and expired [ l]-labelled CO2 was quantified. Data was analyzed using the Proc Mixed procedure in SAS (2001) where nesting THR intake within type of THR addition (e.g. free THR or THR in com or barley) gave the change (slope) in PHE oxidation per g of THR for each type of THR addition. The MAof THR in com and barley was calculated by dividing the slope for THR from the respective feedstuff by the slope for free THR. 

Blood components are also collected through apheresis. In apheresis, advanced blood cell separators are used to collect one or more specific blood components from a donor. The cell separators collect blood iato a separation chamber, isolate the desired blood components, and return the blood components not needed to the donor. This procedure is performed on-line within one sterile disposable tubiag set. The two principal components collected through apheresis are plasma and siagle-donor platelets (SDP). 

Blood is collected in 2.7 ml ethylenediaminetetraacetic acid (EDTA) tubes (Sarstedt, Switzerland) containing 0.1% (w/v) dithioerythritol (DTE), immediately centrifuged at 2000 xg for 10 min, and stored at -80°C. Keep fresh or thawed plasma samples on ice during the oxidation procedure. 

Iron for biosynthesis is transported through the bloodstream by the protein transferrin. The following procedure measures the Fe content of transferrin 10 This analysis requires only about 1 p,g for an accuracy of 2-5%. Human blood usually contains about 45 vol% cells and 55 vol% plasma (liquid). If blood is collected without an anticoagulant, the blood clots, and the liquid that remains is called serum. Serum normally contains about 1 pg of Fe/mL attached to transferrin. 

Tissue analysis after the 24 h fecal collection, animals are euthanized and any tissues of interest (e.g., for toxicology analysis, such as plasma, liver, kidney, heart, spleen) are harvested. Alternatively, if the mice are precious, they can be used again since the procedure is noninvasive. If the systemic presence of radiolabeled cholesterol does not compromise the second use, little delay is necessary. However, the mice will continue to excrete radiolabeled sterols at detectable levels for 2-3 weeks so bedding must be collected and segregated as contaminated waste. 

A different application of SPE which needs 100-200 pi of sample has recently been successfully applied to plasma. Micro SPE (pSPE) followed by HPLC-MS/ MS was used by Napoletano et al. [67] to determinate amphetamine, methamphet-amine, MDA, MDE, MDMA, cocaine, BEG, mescaline, ketamine, PCP, psilocy-bine in plasma. In this method 180 pi of plasma were submitted to slight PPT with 20 pi of methanol containing ISTD, 100 pi of supernatant were then collected and passed through C18 tips, adapted to automatic pipettes. Validation results showed pSPE allows to reduce matrix effect (<10 %) keeping satisfactory recoveries. pSPE represent a simple, fast and reliable procedure with extremely reduced solvent consumption it will be discussed in more detail below in its other applications. 

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