目的：研究邻-甲联苯胺法（经典方法）测定血浆游离血红蛋白（Hb）的基质效应及消除方法。方法以用生理盐水1000倍稀释的 Hb 储存液（浓度为103.1 g/L）作为标准液，将此 Hb 标准液再用无 Hb 血浆分别稀释500、1000、2000、4000倍（游离 Hb 理论浓度分别为206.20、103.10、51.55、25.78 mg/L），然后用经典方法测定游离 Hb 浓度，比较与理论值的差异。将 Hb 储存液分别用生理盐水（经典方法）、无 Hb 血浆（消除方法1）、混合血浆（消除方法2）1000倍稀释作为标准液。经典方法和消除方法1的计算公式为血浆游离 Hb（mg/L）=测定管吸光度（A）值×标准液浓度÷标准管 A 值；消除方法2的计算公式为血浆游离 Hb（mg/L）=测定管 A 值×标准液浓度÷（标准管 A 值×混合血浆 A 值）；消除方法3操作同经典方法，计算公式为血浆游离 Hb（mg/L）=测定管 A 值×标准液浓度×2.35÷标准管 A 值。测定40份标本，比较各消除方法的结果。在血浆中分别加入206.20、103.10、51.55 mg/L Hb，测定各消除方法的回收率。分别取6.25、12.50、25.00、50.00μg/L 维生素 C 溶液4.5 mL，各加1.031 g/L Hb 标准液0.5 mL，用经典方法测定回收率。结果采用无 Hb 血浆500、1000、2000、4000倍稀释的 Hb 标准液的游离 Hb 测定结果分别为87.85、43.16、21.90、10.99 mg/L。40份标本采用经典方法、消除方法1、消除方法2、消除方法3测定的游离 Hb 浓度分别为（7.99±4.90）、（18.61±11.42）、（19.18±11.77）、（18.77±11.52）mg/L；消除方法1、消除方法2、消除方法3的结果均高于经典方法（P 均<0.01），但3种消除方法之间差异均无统计学意义（P 均>0.05）。经典方法、消除方法1、消除方法2、消除方法3的平均回收率分别为42.54%、98.33%、100.98%、97.56%。采用经典方法检测Hb 在6.25、12.50、25.00、50.00μg/L 维生素 C 溶液中的回收率分别为98.57%、98.17%、97.17%、95.16%。结论邻-甲联苯胺法测定血浆游离 Hb 存在基质效应，3种消除方法均可消除基质效应影响。%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.
展开▼
