Pooled plasma

The adrenal glands and pituitary glands have the highest tissue concentration of ascorbic acid. The brain, Hver, and spleen, however, represent the largest contribution to the body pool. Plasma and leukocyte ascorbic acid levels decrease with increasing age (152). Elderly people require higher ascorbic acid intakes than children to reach the same plasma and tissue concentration (153). 

Plasma protein fractions are used to treat hypovolemic (low blood volume) shock that occurs as the result of bums, trauma, surgery, and infections, or in conditions where shock is not currently present but likely to occur. Plasma protein fractions are also used to treat hypoproteinemia (a deficiency of protein in the blood), as might be seen in patients with nephrotic syndrome and hepatic cirrhosis, as well as other diseases or disorders. As with human pooled plasma, blood type and crossmatch is not needed when plasma protein fractions are given. 

Factor IX Replacement Hemophilia B therapy may include recombinant (produced via transfection of mammalian cells with the human factor IX gene) or plasma-derived (concentrate from pooled plasma) factor IX (see Table 64-2). Guidelines for choosing the factor-concentrate formulation for hemophilia B are similar to the guidelines for hemophilia A. However, older-generation factor IX concentrates containing other vitamin K-dependent proteins (e.g., factors II, VII, and IX), called prothrombin complex concentrates (PCCs), have been associated with thrombogenic side effects. Consequently, these products are not first-line treatment for hemophilia B.11... 

Immunoglobulin obtained from pooled plasma obtained from hepatitis B and HIV negative donors is used as an aspecific immunostimulant in immunodeficiency diseases, idiopathic thrombocytopenia, autoimmune hemolytic anemias, Kawasaki syndrome and to prevent infections in immune compromised patients with leukemia or multiple myeloma. Adverse effects include potentially severe hypersensitivity reactions. 

The lower detection limit in plasma of the aromatic amino acids was at a level of 1 pmol/1, whereas those for alanine and glycine were 5 pmol/1 and 10 pmol/1, respectively. The interassay variation was established by analyzing the pool plasma on seven consecutive days. The coefficient of variation ranged from 1.9% for phenylalanine (at a level of 50 pmol/1) to 11.9% for glycine at a level of 230 pmol/1. 

A pool of plasma is composed by mixing residual amounts of plasma left over from analyses, thereby ensuring the anonymity of the pool sample. Each series of analyses should contain at least one pool plasma sample. The results of 8-10 selected amino acids of the pool are plotted in a Shewhart plot to make deviations from the previously established target values visible. Actions following these deviations will have to be devised by the laboratory staff according to standard quality control rules. 

Pooled plasma collected from healthy subjects is used for standards preparation to avoid matrix effects and for internal control. Pooled plasma is kept in 0.5-ml aliquots at -20 C until required. 

Calibration is performed using three standards selected to cover the range expected in the sample batch (usually 4.2, 8.4 and 33.6 pmol/1), together with pooled plasma to allow for any matrix effect, plus a pooled plasma blank. 

Quality assessment is performed by including pooled plasma as an internal control in every run. External quality control is achieved using samples obtained from the European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) Special Assays Scheme, run according the scheme schedule. 

The reduction reaction is carried out in 1.5-ml Eppendorf tubes. Frozen samples are thawed out, thoroughly mixed and, if necessary, centrifuged to remove particulate material. For samples and the pooled plasma blank, 150 pi plasma and 50 pi internal standard in 0.1 mol/1 borate buffer are added to 20 pi of 10% tri-n-butylphosphine. For each Hey standard, 150 pi of pooled plasma and 50 pi of Hey standard (internal standard is included) are mixed with 20 pi of tri-n-butylphosphine. Tubes are left to stand on ice for 30 min. Samples are deproteinised by the addition of 125 pi of... 

A minimum of 2 ml EDTA blood is placed immediately on ice and centrifuged within 30 min at 2000 xg for 5 min at 4°C. Plasma is immediately deproteinised by adding 1 ml to 0.625 ml of 10% 4 and mixing thoroughly by vortex. The mixture is frozen and stored at -70°C until analysis. 1-ml aliquots of pooled plasma collected from healthy subjects are prepared and stored in the same way and are used to prepare AdoMet and AdoHcy calibration standards to allow for matrix effects and also as an internal quality control. CSF is collected in plain tubes and kept at -70°C until analysis. 

For both AdoMet and AdoHcy, calibration is performed using three standards 53, 106 and 210 nmol/1 for AdoMet and 40, 80 and 160 nmol/1 for AdoHcy, each added to pooled plasma supernatant fluid, plus a pooled plasma blank. Higher concentrations can be used if sample levels are expected to be high. 

Neither AdoMet nor AdoHcy have so far been included in any external quality control scheme. Control samples are not commercially available. A pooled plasma sample is included in each batch as internal quality control. 

A quality control (QC) standard mixture, a normal control and an abnormal control are run with each batch. The normal control is made by aliquoting 100-pl portions of a normal range pooled plasma into screw-cap vials. The abnormal control is made by spiking 50 ml of normal pooled plasma with defined concentrations of unlabeled acylcarnitine standards spanning the mass range covered by the analysis (see Reagents and Chemicals). The concentration for each standard should be chosen based on the upper limit of the reference range for the respective acylcarnitine species. The... 

A mixture of EDTA-plasma samples of left-over blood of a large number of patients. Samples of 150 pi plasma are stored in Eppendorf vials at -20°C for a maximum of 1 year. Every series of analyses includes one pool plasma sample. Ten fatty acid results are plotted on an electronic Shewhart chart. 

A pool of a large number of left-over plasma samples of patients is thoroughly mixed. Aliquots of 150 pi are put into Eppendorf vials and stored at -20°C. These samples are stable for 1 year. Each series of analyses has one pool plasma sample. The concentrations of the five analytes are introduced into an electronic Shewhart chart, which is renewed each year. 

The intra- and inter-assay variation is determined in tenfold analyses of a pool plasma sample. Table 3.4.2 shows the results of these measurements. The linearity of this method should be assessed for all analytes. Pristanic acid and the C26 0 fatty acid were linear up to 16 pmol/1, phytanic acid to 100 pmol/l and the C22 0 and C24 0 fatty acid to 200 pmol/1. The lower detection limit for all analytes was at a level of less than 0.01 pmol/l, for the lower reporting levels (LOQ), an analysis of a blank solution was taken into account. The blank levels of the analytes phytanic acid, pristanic acid, and fatty acids C22 0, C24 0 and C26 0 were 0.04, 0.01, 0.41, 0.68 and... 

As internal control for sterol analysis in plasma, serum, and liquor, each series of samples includes a 150 pi aliquot of a large batch of control plasma samples in which cholestanol and cholesterol concentrations are determined and compared with previously determined concentrations. The values for these should fall within 2 standard deviations. The control plasma sample is composed of pooled plasma samples of different individuals stored as 150-pi aliquots at - 18°C. 

Hsieh Y et al (2002) Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 767 353-362... 

For sample processing, pooled plasma samples (1 and 6 h, 2 ml) were mixed with acetonitrile (6 ml), vortexed vigorously, and centrifuged. The precipitates were again extracted with acetonitrile (2x) and the supernatants from previous extraction were combined. The pooled extracts were evaporated at room temperature under a stream of nitrogen and the residues were reconstituted in HPLC mobile phase. A portion of the sample was used for radioactivity determination, and the remaining sample used for HPLC profiling. 

Passive immunity can be obtained by i.m. injection of globulin containing antibody to the virus normal immunoglobulin prepared from pooled plasma from known immune donors) which confers temporary protection for travellers visiting areas where the virus is endemic. Active immunisation with Hepatitis A vaccine is now preferable protective antibody takes about two weeks to develop. 

Hepatitis B immunoglobulin (pooled plasma selected for high titres of antibodies to the virus) provides passive immunity for post-exposure prophylaxis e.g. after accidental needlestick injury. 

Immunoglobulin preparations are concentrated protein solutions derived from the pooled plasma of adults or animals. They contain specific antibodies in proportion to the infectious and immunization experience of the population from whose plasma they are prepared (1). Large numbers of donors (at least 1000 donors per lot of final product) are used, in order to ensure inclusion of a broad spectrum of antibodies. Intravenous immunoglobulin is also derived from the pooled plasma of adults, but the alcohol-fractionation procedure is modified to a product suitable for intravenous use. The use of intravenous immunoglobulins in selected immunodeficiency and autoimmune diseases has been reviewed (2). 

Albumin solutions are manufactured from pooled plasma. They can contain other plasma constituents, such as heme compounds, prekalli-krein, endotoxins, bradykinin and bilirubin, in sufficient concentrations to cause clinical effects (Pulimood Park 2000). Some batches of human albumin have been shown to induce the expression of adhesion molecules on endothelial cells in vitro (Nohe et al 1999), which could result in activation of the immune system in vivo. Despite appropriate precautions during the manufacturing process, there is always a theoretical chance of transmission of disease with any biological product. 

Antibodies can be targeted more or less specifically, either against a single or a variety of antigens. An example of a broad-spectrum antibody therapy is anti-Rhesus antigen antibody (WinRho) which has been used postpartum for many years to prevent rhesus immunization of an Rh— mother by an Rh+ neonate. There are at least 60 known epitopes of the rhesus D antigen. The product is made from pooled plasma of Rh— male volunteers who have been deliberately challenged with small... 

Plasma samples of 1.0 ml were alkalinized (NaOH), the internal standard (mepivacaine) added and the mixture extracted with chloroform. The chloroform solution was evaporated and the residue redissolved in methanol (25 yl). 1.5 yl of the methanol solution was injected for the gas chromatographic analysis on a packed column of 3 % OV-17 on Chromosorb P. A typical chromatogram is given in Figure 20.5. Peak height ratio measurements produced linear standard curves in the 0.25-10.0 yg/ml range. Absolute sensitivity from a 1.0 ml plasma sample was 0.1 yg/ml. The relative deviation of a 2.0 yg/ml pooled plasma standard curve (done repeatedly over several months) was 5.2 5 . 

Specific factor assays are variations on the APTT or PT tests. In the APTT and PT, dilutions of the patient s plasma are made into a deficient, or depleted, substrate plasma. The assays are then performed in the usual way. The clotting times are compared with those obtained from dilutions of pooled normal plasma, commonly 1 10, 1 20, 1 50, and 1 100. A graph of the logarithm of the clotting time (y axis) versus the logarithm of the concentration as percentage of normal x axis) is used to determine the amount of the factor activity in the patient s plasma. The normal pooled plasma is conventionally assigned a value of 100% activity. Many variations exist for specific factor assays, e.g., the venom of Vipera russellii and phospholipids may be substituted for thromboplastin in a PT-like assay. An enzyme in Russell s viper venom rapidly and relatively specifically activates factor X. In conjunction with a factor X-deficient substrate plasma, this provides a specific factor X assay. 

Hsieh, Y Bryant, M.S. Brisson, J.M. Ng, K. Korfmacher, W.A. Direct Cocktail Analysis of Drug Discovery Compounds in Pooled Plasma Samples Using Liquid Chromatography-Tandem Mass Spectrometry, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 767(2), 353-362 (2002). 

---