Determination of Prealbumin in Serum of Newborns

Manual and automated immunonephelometric tests (1, 2) have been developed for determination of serum prealbumin (transthyretin, PA). This methodology is more precise and rapid than radial immunodiffusion, but lipoproteins and immunoglobulins interfere with immunonephelometric methods (2,3). We describe here an immunonephelometric method for PA in serum from the newborn. We took special care to remove endogenous lightscattering particles, which frequently are present in thawed sara from newborns, because they alter the lower limit of detection and the precision and accuracy of nephelometry (4). For nephelometry we used a monospecific antibody (Hyland, Division of Travenol Laboratories, Plaisir, France) on a laser “PDQ” nephelometer (Hy.. land). The antiserum was diluted fourfold with a solution containing 40 g of polyethylene glycol 600 (PEG; Merck, Hohenbrunnen, F.R.G.) and 150 mmol of sodium chloride per liter. The mixture was then filtered through a 0.22m pore-size filter (Millipore Corp., Molsheim, France), and 1 mL of the ifitrate was mixed with 60 tL of either a serum sample diluted 40-fold with isotonic saline, or a control serum (Immunotrol; Biomerieux Corp., Lyon, France), or a calibration solution (Hyland) previously diluted with saline to give six concentrations of PA ranging from 37 to 448 mgfL. The mixtures were left at room temperature for 60 mm before nephelometry. About 30% of the thawed sara exhibited blank values corresponding to 50 to 160% of the test values measured in the presence of PEG-antiserum solution, so we have compared two different pretreatments on 12 thawed sara from newborns in one of two ways. First, the serum samples were vigorously mixed with an equal volume of “Lipoclean” (Behring Corp., Marburg/ Lahn, F.R.G.), then centrifuged for 10 mm at 18000 X g. Each supernate was then assayed the same as the untreated specimens. Second, sara were mixed with an equal volume of a solution containing 80 g of PEG and 150 mmol of sodium chloride per liter. After 30 mm at room temperature, the mixtures were centifuged (10 mm, 18000 x g), and the supernates were diluted 20-fold with isotonic saline. Essentially, treatment with PEG made the blank values negligible for all our specimens. An advantage of using PEG pretreatment is that blank determinations can thus be omitted. To explore the possibility of precipitation of PA by PEG, we treated 500 1zL of each serum with PEG in the same way as before. After centrifugation, we redissolved the pellets in 100 ,LL of saline and determined PA by radial immunodiffusion (M. Partigen plates, Behnng). No detectable PA was found in the various pellets, in agreement with the findings of Iverius and Laurent (5). When PA was assayed by inununonephelometry in the above-mentioned supernates, all calculated concentrations were slightly more than expected (Table 1). To check whether this effect would be related to the nephelometric procedure, we also assayed supernates by radial immunodiffusion. The diameters of the precipitation rings were measured independently by two persons. Again, the results were higher than those obtained with nontreated specimens. Both procedures had about the same increase: + 14% for radial immunodiff’usion and + 18% for immunonephelometry. Furthermore, the variations in blank values were correlated negatively with those of the test reactions: n = 12, r = 0.857, slope = 0.643, and yintercept = -0.922, where y is the variation of relative intensity of scattered light produced by PEG pretreatment. PEG pretreatment did not alter the relative intensity of scattered light of mixtures containing calibration solutions or control serum plus the antiserum solution. Thus PEG is a nonspecific agent that both precipitates some endogenous components of the specimens and enhances the antigen-antibody reaction. We believe that some of these precipitated components may limit the antigen-antibody reaction to some extent. Lipoclean pretreatment of the same Table 1. Influence of Pretreatment on lmmunonephelometric Determination of PA in Serum from Newborns