新生儿血清游离胆红素的测定及临床应用价值探讨

Determination of Serum Unbound Bilirubin and Its Clinical Application Value in Neonates

[目的]探讨过氧化物酶(POD)法检测新生儿血清游离胆红素(UB)的临床适用性及应用价值。[方法]向胆红素标准品溶液(1、2、3μmol/L)加入H_(2)O_(2)(0.33 mol/L)和三种不同终末浓度(0.019、0.038、0.075μg/mL)的辣根过氧化物酶(HRP)以获得标准化的HRP一级速率常数Kp;新生儿血清上样量25μL,稀释倍数41.6,于37℃且避光条件下用2.4、4.8μg/mL HRP检测血清UB水平,并验证该方法学的准确性、精密度以及稳定性;收集33例黄疸患儿的一般情况及临床资料,主要包括血清总胆红素(TSB)、间接胆红素(IDB)、白蛋白(ALB)、胆红素与白蛋白摩尔比值(BAMR)等;用Graphpad Prism 8.0统计分析实验数据。[结果]在pH值为7.4±0.2,反应温度37℃且避光条件下,标准化Kp为(7.20±1.08)mL·μg^(^(-1))·min^(-1)。HRP活性和UB水平分别于-20℃冻存3周和1周内无明显下降。不同UB水平血清样品的平均日内、日间变异系数均小于10%。本研究用POD法检测33例黄疸患儿(胎龄≥35周)的血清UB范围为(0.32~1.20)μg/dL,与TSB、IDB及BAMR均显著相关。有5例UB水平≥1.0μg/dL,强光疗者占60%(3/5)。[结论]采用POD法结合常见设备分光光度计检测新生儿血清UB水平不仅操作简易、检测快速,且试剂成本低,具有良好的准确性和精密度,便于临床开展。血清UB的测定对新生儿黄疸的临床管理有一定的指导意义。

[Objective]To investigate the clinical appropriateness and application value of the peroxidase(POD)method for the detection of unbound bilirubin(UB)in neonatal serum.[Methods]Hydrogen peroxide(0.33 mol/L)and three different final concentrations(0.019,0.038,0.075μg/mL)of horseradish peroxidase(HRP)were added to stan⁃dard bilirubin solution(1,2,3μmol/L)to obtain a standardized HRP primary rate constant Kp.Then 25μL of neonatal serum was diluted by 41.6 fold,and measured with 2.4 and 4.8μg/mL HRP at 37℃under the dark,to determine the UB concentration.The accuracy,precision,and stability of the methodology were validated.The clinical characteristics of 33 jaundiced neonates were collected,including total serum bilirubin(TSB),indirect bilirubin(IDB),albumin(ALB),bil⁃irubin to albumin molar ratio(BAMR),etc.The experimental data were analyzed by Graphpad Prism 8.0.[Results]A standardized Kp of(7.20±1.08)mL·μg^(-1)·min^(-1) was determined at pH 7.4±0.2,37℃in the dark.The HRP activity and UB concentrations remained stable at-20℃for 3 weeks and a week,respectively.The mean intra-day and inter-day coef⁃ficients of variation of the serum samples with different UB concentrations were less than 10%. In this study, the UB con⁃ centrations in 33 jaundiced neonates (gestational age ≥35 weeks) were measured by the POD method in the range of (0.32 ~1.20) μg/dL, which was positively correlated with TSB, IDB and BAMR. Of the five infants whose UB concentrations measured more than 1 μg/dL, three received intensive phototherapy (60%).[Conclusions] The POD method combined with a standard equipment spectrophotometer to detect serum UB concentrations in neonates is easy to operate, rapid to de⁃ tect, and low cost. This method has good accuracy and precision, which is convenient for clinical implementation. More⁃ over, the measurement of serum UB may assist us in better management of neonatal jaundice in clinical practice.