Abstract:

Objective To study the matrix effect of o-methyl benzidine method （classic method）in determining plasma free hemoglobin （Hb）and the eliminating methods. Methods Hb storage solution diluted 1 000 times by normal saline （concentration was 103.1g/L）was used as standard,while the standard diluted by Hb-free plasma 500,1 000,2 000 and 4 000 times（theoretical concentrations were 206.20,103.10,51.55 and 25.78 mg/L）was determined by classic method for plasma free Hb. The results were compared with theoretical values. Hb storage solution diluted 1 000 times by normal saline（classic method）, Hb-free plasma（eliminating method 1） and mixed plasma（eliminating method 2）was used as standard. The equation of classic method and eliminating method 1 was plasma free Hb（mg/L）=test tube absorbance（A）× concentration of standard ÷ standard tube A. The equation of eliminating method 2 was plasma free Hb（mg/L）= test tube A×concentration of standard ÷ （standard tube A× mixed plasma A）. The equation of eliminating method 3 was plasma free Hb（mg/L）= test tube A× concentration of standard ×2.35÷standard tube A. A total of 40 specimens were determined,and the results of different eliminating methods were compared. Hb of 206.20, 103.10 and 51.55 mg/L were added into plasma,and the recovery rate of each method was determined. Adding 0.5 mL of 1.031 g/L standard,in 4.5 mL of vitamin C solution of 6.25,12.50,25.00 and 50.00 μg/L, the recovery rate was determined by classic method. Results The theoretical values of plasma free Hb were 87.85, 43.16, 21.90 and 10.99 mg/L for standard diluted by Hb-free plasma 500,1 000,2 000 and 4 000 times. As to 40 specimens,plasma free Hb concentrations of classic method, eliminating method 1,eliminating method 2 and eliminating method 3 were（7.99±4.90）,（18.61±11.42）,（19.18±11.77） and（18.77±11.52）mg/L,the results of eliminating method 1,eliminating method 2 and eliminating method 3 were higher than that of classic method（P<0.01）,and there was no statistical significance among the 3 eliminating methods（P>0.05）. The average recovery rates of classic method, eliminating method 1 ,eliminating method 2 and eliminating method 3 were 42.54%, 98.33%, 100.98% and 97.56%. The recovery rates of vitamin C solution of 6.25,12.50,25.00 and 50.00 μg/L were 98.57%,98.17%,97.17% and 95.16%. Conclusions There is matrix effect of o-methyl benzidine method in determining plasma free Hb, and the 3 eliminating methods are effective.

Key words: Free hemoglobin, Matrix effect, o-Methyl benzidine method