Influence of sample preparation on estimates of blood fibronectin concentration.

In view of the growing interest in blood concentrations of fibronectin (FN) it is appropriate to sound a note of caution regarding the preparation and storage of specimens for FN measurement. We used blood from 12 volunteers to investigate the effect of using different anticoagulants in plasma preparation, different clotting times in serum preparation, and storage of these samples, on FN concentrations. Material and methods Fibronectin concentrations were determined by single radial immunodiffusion' on 1% agarose supplemented with 3% polyethylene glycol, using a specific antiserum to FN and standards supplied by Behringwerke, Marburg, Lahn, West Germany. The plates were incubated at room temperature for four days to reach completion, and stained with Coomassie blue. Blood was obtained from 12 volunteers, six men and six women. Fibronectin was measured in the plasma or serum obtained after spinning down a portion of whole blood. Portions of whole blood were treated as shown in Table 1. The sera produced by methods 2-4 inclusive were each divided into two portions; one was stored at-20°C, one at-70°C. The FN concentrations were determined immediately after preparation of the plasma or serum, and subsequently after freezing and thawing the samples five times, FN being measured after each thawing. The time interval between each thawing was one week. Results The results are presented in Figs. 1-3 and Table 2. Leaving blood to clot for 5 to 6 h resulted in the same FN concentration, which was lower by 33-69% than that in blood clotted for 1 h. The temperature of storage, whether-20°C or-70°C, made no appreciable difference to FN concentrations (Fig. 1). The effect of storage on serum prepared by clotting blood at 4°C and 37°C (method 1, 5) was similar to that on serum prepared at 21°C (method 2). It was observed that the higher the clotting temperature, the greater the amount of FN retained in serum (Table 2). Although EDTA plasma had the highest FN concentrations when assayed immediately after storage, these became unpredictable in that on sequential freezing and thawing the amount of FN fluctuated extensively and concentrations ranged Table 2 Effects ofsamplepreparation on the initial value of FN Method Initialfibronectin mean ± SD (mg/l)