Pooled normal plasma

The Bethesda assay is used to detect levels of inhibitory antibodies. One Bethesda unit is the reciprocal of the dilution of test plasma at which 50% of normal activity is inhibited. Briefly, the test plasma sample is mixed with an equal volume of pooled normal plasma and incubated at 37 °C for 2 h. Residual activity is measured by aPTT as described above (Herzog et al., 1999 Chao et al., 2001 Fields et al., 2001 Gallo-Penn et al., 2001). 

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. 

Heparin therapy may be monitored by its increases in clotting time in the APTT, although this method for measuring heparin is difficult to standardize. Heparin is more specifically assayed by its effect on factor Xa inactivation by antithrombin. Such factor Xa-based heparin assays usually employ purified factor Xa as a reagent and factor X-deficient substrate plasma as the source of antithrombin. The prolongation of the clotting time that results from the heparin in the patient s plasma is compared with pooled normal plasma that is known to be free of heparin. Many variations of this heparin assay are available. Heparin assays can use thrombin rather than factor Xa, however, the low-molecular-weight heparins are not reliably measured in thrombin-based assays. 

A young African-American male patient is brought to you because of a hemarthrosis sustained after he twisted his knee while running. His PT is normal, but his APTT is 51 s (normal 20-32 s). Mixing equal volumes of the patient s plasma and normal plasma shortens the APTT to 25 s. Specific factor assays show a normal factor IX activity, but a factor VIII activity of 25% of that in pooled normal plasma. Why was his PT normal What else, prior to the factor IX and VIII assay results, might have been responsible for the prolonged APTT ... 

A. General description Prolastin is prepared from pooled human plasma of normal donors it has a molecular weight of 52kDa. To reduce the potential risk of transmission of infectious agents, Prolastin has been heat-treated in solution at 60 + 0.5°C for not less than 10 hours. However, no procedure has been found to be totally effective in removing viral infectivity from plasma fractionation products. 

Fig. 5.2.10 Nondenaturing two-dimensional electrophoresis with agarose gel electrophoresis in the first dimension and gradient polyacrylamide gel electrophoresis in the second dimension combined with anti-apolipoprotein AI (apoA-I) immunoblotting differentiates apoA-I-contain-ing lipoproteins by charge (pre-/ l-LpA-I versus a-LpA-I) and size (HDL2 versus HDL3). In normal plasma the major part of the HDL pool is formed by a-LpA-I, a smaller by pre-/ l-LpA-I (left picture). In the plasma from Tangier patients only some pre-/ l-LpA-I particles are present (right picture)...

The recovery of atropine (hyoscyamine) added to various concentrations to pooled normal human plasma was near 100%. 

Calibration curves were obtained for free carnitine in the range of 5-lOOpmol/L, 2-40pmol/L for acetyl-carnitine and from 0.25-6pmol/L for all other available acyl-camitines by adding standards to a normal plasma pool. All calibration curves were linear (r > 0.99, data not shown). For unsaturated and hydroxylated acyl-camitines an identical response as for their saturated counterparts was assumed. 

In discussing iodide metabolism, we need only be concerned with the fate of inorganic iodides because organic iodides (tyrosine derivatives) are absorbed and enter the normal plasma pool of organic iodides produced by the thyroid. The site and the mechanism of absorption of organic iodides in the intestine are not exactly known, but somehow the dietary iodine is transported from the intestinal lumen into the blood. 

After having demonstrated that a very quick equihbrium of Apo C specific activities takes place in vitro and essentially in vivo in normal plasma, we were obhged to come back to a simple tri-compartmental model (Fig. 2). The central plasma compartment is a black box in which very quick exchanges, compared to the frequency of sampling, take place between VLDL, IDL, and HDL. A schematic representation analogous to the one we have developed has been published in 1978 by Schaefer et al. [1]. There is no evidence for the existence of a free pool of Apo C. 

The findings of Armstrong, Budka, and Morrison were essentially similar. They are reproduced in detail in Table I. They concluded that the contribution of albumin to the total refractive increment of unextracted pooled human plasma proteins, by the electrophoretic method, was 61 1.6 per cent. On the basis of protein nitn n calculations, this figure yielded an estimate of 64 1.6 per cent albumin nitrogen as per cent of total protein nitrogen, in pooled normal human plasma. 

In addition to inadequate separations by the Howe method, there is the further problem of the inadequacy of the conventional factor 6.25, which is generally applied to all fractions to convert from grams protein nitrogen to grams dried protein. The average nitrogen factor found experimentally in the dried proteins of pooled normal human plasma (5) is 6.73. The conversion factor of individual proteins (5) varies from 6.10 to 8.40. 

Details of the process and results of low-temperature-low-saJt-ethanol fractionation have recently been reviewed at length by Edsall (82) and the relevant aspects will be recapitulated here only in brief outline. The method yields the following five principal fractions in pooled normal human plasma ... 

Fig. 4. Distribution of electrophoretic components in fractions obtained by low-temperature - low-salt - ethanol fractionation of pooled normal blood plasma. From Oncley, Scatohard, and Brown (270).

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