Fibrinogen Estimation by Paper Electrophoresis

Abstract

Apart from the work of Eerkes and Karas (1) and Antonini and Piva (2) little attention has been directed to the estimation of fibrinogen by paper electrophoresis. This may be due to the numerous difficulties which are attendant on the estimation of the serum protein fractions by electrophoresis and to the existence of other simple and relatively accurate techniques.In a recent study of the plasma protein changes following surgery, however, Birch and Jepson (3) noted an increased fibrinogen &dquo;peak&dquo; on the electrophoretogram. This observation, and in particular the occurrence of an increasingly high peak in a patient who developed a coronary thrombosis on the ninth postoperative day, led them to suggest, as did Margulis and his associates (4), that this fibrinogen response merited further study as a factor in the pathogenesis of thromboembolism, and as the possible basis of a routine prognostic test.As a preliminary step to investigation of this suggestion further study of the electrophoretic properties of fibrinogen was deemed necessary and in particular determination of the quantitative significance of the fibrinogen peak. METHODSFifty men, aged 20 to 65, undergoing elective major abdominal surgery under general anesthesia were selected for study. From each patient 5-ml samples of serum, heparinized plasma (2.5 mg/5 ml), and oxalated plasma (20 mg potassium oxalate/5 ml) wee obtained prior to operation and on the fifth postoperative day.An EEL3 horizontal electrophoresis tank and power pack were used. With this tank a wide separation of the fibrinogen band from the starting-line can be obtained and for this reason it was preferred to the vertical type used by Birch and Jepson (3). Thus any tendency of the fibrinogen to become denatured and adhere to the paper could be easily recognized.Three buffers were compared, the current being adjusted in each case to produce optimum separation: 1) Barbiturate-acetate pH 8.6 (3); Current-0.4 mA/cm. 2) Borate pH 9.0 (5); Curremt-0.1'7 mA/cm. 3) Phosphate pH 9.2 ( 1, 6) ; Current-0.4 mA/cm. Two hundredths ml of each sample was applied as a line 4 cm in length with a m icropipette to strips of Whatman 3MM paper (5 cm in width) which had been dipped in the appropriate buffer and then blotted. The strips were run in parallel for 16 hours, dried at 110°C for 30 minutes and stained for 10 minutes in 0.5 per cent Bromophenol Blue in a saturated ethanolic solution of mercuric chloride. They were washed, using the acetic acid-sodium acetate sequence of at NANYANG TECH UNIV LIBRARY on June 14, 2015 ang.sagepub.com Downloaded from