[Levels of immune complexes in fresh and frozen serum and plasma detected by precipitation with polyethylene glycol in patients with malignant diseases].

Introduction: Immune complexes are macromolecules consisting of immunoglobulins (antibodies) bound to different antigens [1]. Determination of circulating immune complexes in patients with malignant diseases can be of some interest for prognosis and follow-up of a disease [2, 3]. According to certain data the immune complexes concentration varies in dependence of disease stage [4] and it is not affected by therapy [5]. Precipitation with polyethylenglycol is a physical method for determination of circulating immune complexes, based on the ability of high molecular polymers to precipitate macromolecules from sera [6]. This mechanism of precipitation is not yet well understood, but it is probably based on steric exclusion of water molecules that affects insolubility of immune complex molecules [7]. Repeatedly frozen sera demonstrated rapid decrease in detected concentration of circulating immune complexes [8] by polyethylenglycol. The presence of complement affects solubility of circulating immune complexes [7]. While there are no data about the influence of other proteins in sera or plasma, the aim of this study was to find out if there are any significant differences between the circulating immune complexes levels, determined by polyethylenglycol, in sera, plasma or in only once frozen sera.

Material And Methods: Eighteen samples of plasma and sera from patients with malignancy (10 males and 8 females) were examined. Eight of them had non-Hodgkin lymphoma, 4 were with Hodgkin lymphoma, 4 with breast carcinoma and 2 with lung carcinoma. All samples were taken before starting chemotherapy. The circulating immune complexes determination was carried out immediately after the separation of plasma and sera and also in sera frozen for 10 days at -35 degrees C. Circulating immune complexes were determined spectrophotometrically. The absorbance (A450) of serum or plasma in 3.75% of polyethylene glycol, polyethylenglycol (M = 6000) solution was used as the measure of the circulating immune complexes level [9]. The standard for circulating immune complexes determination in g/l was aggregated IgG at 36 degrees C for 30 minutes from the serum of healthy volunteers.

Results: The mean value and the range of circulating immune complexes level (A450) are given in Table 1. The values in g/l are presented in Graph 1. The values of circulating immune complexes in plasma were significantly lower than those in fresh sera (t = 2.8125; p < 0.02). There was no significant statistic difference between levels in circulating immune complexes (A450) in fresh and frozen sera (t = 1.3261; p > 0.1).

Discussion: In dependence on its concentration polyethylenglycol shows the ability to precipitate proteins selectively [10]. The selectivity was tested mainly towards immunoglobulins and the complement. Results obtained in this study show statistically significant lower circulating immune complexes level in plasma than in serum or frozen serum. The main difference between sera and plasma is in complete absence of fibrinogen, factors V and VIII in sera and in presence of Ca++ ions. Besides that plasma contains an anticoagulant [11]. It is possible that the presence of fibrinogen and some coagulation factors disturb the polyethylenglycol precipitation mechanism. According to this, it might be, that mechanism, based on steric exclusion of water molecules, selectively influences polyethylenglycol precipitation of circulating immune complexes in plasma. It is difficult to say how much Ca++ ion and anticoagulant, as well as the activity of some plasma enzymes, and possible dissociation of circulating immune complexes influence the formation of precipitate. In any case, there is a significant difference between concentration of circulating immune complexes according to substrate. For that reason, it is necessary to detect circulating immune complexes by polyethylanglycol always in the same medium for exact clinical evaluation. (ABSTRACT TRUNCATED)