White cell preparation

The technique involves the separation of white cells from the patient s blood and re-injection after labeling in vitro with an appropriate radiolabel (14). For the majority of clinical applications, mixed white cell preparations are labeled, but for research purposes in humans and animal models, it is feasible to separate the white cells further and to label only the granulocyte fraction or monocytes. The granulocyte fraction can itself be separated into neutrophils and eosinophils. The extreme radiosensitivity of lymphocytes renders them unsuitable for ex vivo labeling. 

The full name of these cells is neutrophilic polymorphonuclear leukocytes, but the terms neutrophil and, less-commonly now, polymorph are generally used to describe this cell (Fig. 1.1a). In fact, most preparations of neutrophils contain about 95-97% neutrophils, the remainder being largely eosinophils, because the commonly-used separation techniques do not efficiently separate these cell types. Neutrophils are the most abundant white cell in the blood, accounting for 40-65% of white blood cells, and are found at concentrations usually in the range 3-5 x 106 cells/ml blood. This number can increase dramatically (up to tenfold) in cases of infection. They have a relatively short half-life in the circulation (estimated at about 8-20 h), but this may be extended to up to several days if the cells leave the circulation and enter tissues - although it is difficult to measure the lifespan of a tissue neutrophil. Because of the large numbers of neutrophils in the circulation and their relatively short lifespan, vast numbers of neutrophils enter and... 

Leukocytes are prepared from EDTA-blood (approximately 3-5 ml) by the density gradient method. EDTA-blood is mixed by inversion and 5 ml is slowly added to 1 ml dextran solution. The blood and dextran solution are carefully mixed so that formation of foam is avoided. The mixture is allowed to stand for 1 h. If further time for sedimentation is required, it has to be noted as it may affect the resulting enzymatic activities. The time needed for proper sedimentation depends on sample quality and should not exceed 3 h. The upper phase including the white cells is transferred to another tube and spun at 600-1000 g for 10 min. The supernatant is... 

Progress in the development of on-chip sample preparation has been extensive and there are already devices that can isolate white cells from whole blood and isolate specific DNA sequences. In addition, the first step to integrating sample preparation with other analytical procedures, e.g. PCR, has been achieved, thus demonstrating the potential of totally integrated microfabricated analyzers. 

A more systematic study of the acid phosphatases of erythrocytes and of human prostate was undertaken in 1949 by Abul-Fadl and King (A4). The preparations were crude, the prostatic phosphatase being obtained by grinding human prostate with a 5-fold volume of 0.9% NaCl. The erythrocytic phosphatase consisted of centrifuged red cells, separated from white cells, washed twice with 0.9% NaCl and hemolyzed in 9 volumes of water. The buffer-substrate mixture consisted of equal volumes of acetate buffer (concentration not stated) and 0.02 M disodium phenyl phosphate. 

As was noted above, a preparation obtained from patients peripheral blood in which 98% of the leukocytes were reticulum cells, showed only one isoenzyme. No. 5, of acid phosphatase activity (L8). In patients with a differential white cell count with lesser numbers of reticulum cells and greater numbers of neutrophiles and lymphocytes, isoenzymes 1, 2, 3, and 4 were also evident. For example, in a case with 54% reticulum cells, 20% neutrophiles, 1% monocytes, and 25% lymphocytes, the relative isoenzyme activities were No. 0, 0% No. 1, 30.8% No. 2, 18.8% No. 3, 9.7% No. 3B, 8.0% No. 4, 10.8% No. 5, 21.9% (L8). 

The cytotoxic hypoxia of acute CN intoxication affects the energy-dependent processes controlling cellular ionic homeostasis and the ionic disequilibrium normally maintained between the intracellular and extracellular fluid compartments (Maduh et al., 1993). In isolated cell preparations, the cellular ionic disruption results in marked cellular acidosis and accumulation of cytosolic Ca++ (Bondy and Komu-lainen, 1988 Li and White, 1977 Nieminen et al., 1988). This may result in disturbances of Ca++-activated lipolytic enzyme activity, peroxidation of membrane phospholipids, changes in transmitter release and metabolism and effects on other Ca++-modulating cell signaling systems. Johnson etal. (1986) found that CN significantly... 

Sweeney JD, Holme S, Heaton WAL, Nelson E (1995) White cell-reduced platelet concentrates prepared by in-line filtration of platelet-rich plasma. Transfusion 35 131-136 Tatum JL, Burke TS, Hirsh JI et al (1983) Pitfall to modified in vivo method of technetium-99m red blood cell labelling. lodinated contrast media. Clin Nucl Med 8 585-587 Thakur ML, Welch MJ, Malech HL (1981) Indium-111 labelled human platelets improved method, efficacy and evaluation. J Nucl Med 22 381-385... 

In order to purify the RBCs, the plasma and huffy coat (white cells) are removed for further purification (see above). RBCs are then resuspended and washed three times in a physiological salt solution [PSS in mM, 4.7 KCl, 2.0 CaCh, 140.5 NaCl, 12 MgS04,21.0 tris(hydroxymethyl)aminomethane, 11.1 dextrose with 5% bovine serum albumin (final pH 7.4)]. Cells should generally be prepared and studied on the day of use within 8 h of removal from animal or human subjects. 

White cells at rest are spherical. The surfaces of white cells contain many folds, projections, and microvilli to provide the cells with sufficient membrane area to deform as they enter capillaries with diameters much smaller than the resting diameter of the ceU. (Without the reservoir of membrane area in these folds, the constraints of constant volume and membrane area would make a spherical cell essentially undeformable.) The excess surface area of the neutrophil, when measured in a wet preparation, is slightly more than twice the apparent surface area of a smooth sphere with the same diameter [Evans and Yeung, 1989 Ting-Beall et al, 1993]. It is interesting to note that each type of white cell has its own unique surface topography, which allows one to readily determine if a cell is, for example, either a neutrophil or monocyte or lymphocyte [Hochmuthet al., 1995]. 

Human erythrocytes were prepared from blood freshly drawn in heparin and washed twice in 0.9% NaCl with removal of white cells by aspiration. In order to enrich erythrocytes with PRPP, the packed cells were preincubated in an equal volume of a medium containing 50 mM Tris-HCl (pH 7.4), 5 mM glucose, 0.12 mM Na phosphate, and the appropriate amount of NaCl to give isotonic solution and incubated up to 90 min at 37°C. The cells, washed twice in a medium not containing phosphate (50 mM Tris-HCl, 5 mM glucose, 12.8 mM NaCl, pH 7.4), were transferred in an equal volume of the last medium containing 5 pM l C hypoxanthine. The labeled purine base was incorporated within 2 min and IMP was the only radioactive substance present in detectable amount in the cells . ... 

The studies of Klein and Harris (1938) on different tissues of the rabbit indicated that acetylation was perhaps localized to the liver. Blon-dheim (1955) showed, however, that both red cells and white cells of peripheral blood had the capacity to convert sulfanilamide and p-amino-benzoic acid to their acetylated derivatives. With the development of more sensitive assay techniques making use initially of acetyl coenzyme A generating systems, and later of purified preparations of this cosubstrate, enzymic acetylating activity could be demonstrated in many other mammalian tissues. More recently, tissue distribution studies in animals and man have shown that both the pattern and type of arylamine drug-acety-lating activity varies from one tissue to another within an individual and also between individuals (Hearse and Weber, 1970), in support of pro-... 

Packed red cells are prepared from whole blood. These are collected ia blood coUectioa units having integrally attached transfer packs. The red cells are sedimented by centrifugation, and the plasma and huffy coat are expressed from the bag. Further processiag of the packed red cells may be needed for a number of clinical indications. To reduce the white blood cell (WBC) contamination in a red cell product, two separation techniques are used. 

Stannous Sulfate. Stannous sulfate (tin(Il) sulfate), mol wt 214.75, SnSO, is a white crystalline powder which decomposes above 360°C. Because of internal redox reactions and a residue of acid moisture, the commercial product tends to discolor and degrade at ca 60°C. It is soluble in concentrated sulfuric acid and in water (330 g/L at 25°C). The solubihty in sulfuric acid solutions decreases as the concentration of free sulfuric acid increases. Stannous sulfate can be prepared from the reaction of excess sulfuric acid (specific gravity 1.53) and granulated tin for several days at 100°C until the reaction has ceased. Stannous sulfate is extracted with water and the aqueous solution evaporates in vacuo. Methanol is used to remove excess acid. It is also prepared by reaction of stannous oxide and sulfuric acid and by the direct electrolysis of high grade tin metal in sulfuric acid solutions of moderate strength in cells with anion-exchange membranes (36). 

Xylose-rich pectic polysaccharide was extracted from defatted and protein-free cell wall preparation (5) using HCl solution (pH 1.6) at 85° C for 4 h. The extract was adjusted to pH 5.0 with ammonia, concentrated on a rotary evaporator under reduced pressure at 40°C, and precipitated with 5 volumes of 96% ethanol. After washing twice with 80% ethanol and drying in an air circulated oven at 40°C for 2 h, the pellet was ledissolved with distilled water and then precipitated with 4 vols 96% ethanol. Before the pellet was gently ground, the precipitated pellet was washed twice with 70% ethanol and dried at 40 ° in an air circulated oven for 16 h. The resultant white powder was labelled "xylose-rich pectic polysaccharide" and stored in a refrigerator. 

---