胆红素测定校准物二牛磺酸胆红素的研究

对合成的二牛磺酸胆红素的部分理化性质，吸收光谱及重氮试剂反应的特点做了探讨，结果表明，二牛磺酸胆红素作为总胆红素测定时的校准物，效果与用非结合胆红素作校准物一致，而作为直接胆红素测定时的校准物则优于Ｂｕ，当前在没有葡萄糖醛酸胆红素商品的情况下，二牛磺酸胆红素是胆红素测定时，尤其是ＤＢＩＬ测定的理想校准物。     

胆红素氧化酶法测定血清结合胆红素

介绍使用国产胆红素氧化酶和二牛磺酸胆红素校准物建立血清结合胆红素测定方法。测定缓冲液为０．１ｍｏｌ／Ｌ柠檬酸盐缓冲液（ｐＨ＝５．０），胆红素氧化酶浓度５００Ｕ／Ｌ，反应（终点）时间１５分钟。本法线性范围可达２４０μｍｏｌ／Ｌ，批向不精密度＜１０％，回收率为９８．３％～１０７．１％，与重氮方法比较，回归方程为Ｙ（酶法）＝０９５６Ｘ（重氮法）－０．５１３。本法测定人血清结合胆红素参考值２．５７±２．５     

胆红素氧化酶法测定血清结合胆红素

介绍使用国产胆红素氧化酶和二牛磺酸胆红素校准物建立血清结合胆红素测定方法。测定缓冲液为０．１ｍｏｌ／Ｌ柠檬酸盐缓冲液（ｐＨ＝５．０），胆红素氧化酶浓度５００Ｕ／Ｌ，反应（终点）时间１５分钟。本法线性范围可达２４０μｍｏｌ／Ｌ，批内不精密度＜１０％，回收率为９８．３％～１０７．１％，与重氮方法比较，回归方程为Ｙ（酶法）＝０．９５６Ｘ（重氮法）－０．５１３。本法测定人血清结合胆红素参考值２．５７±２．５６μｍｏｌ／Ｌ（ｎ＝９５）。     

血清结合胆红素的酶法测定

目的 建立用胆红素氧化酶（ＢＯＤ）作为催化剂，选择性地测定血清结合胆红素（ＣＢ）的新方法。方法 测定条件为：０．１ｍｏｌ／Ｌ甘氨酸缓冲液、ｐＨ１０．０，ＢＯＤ活性０．５～０．８Ｕ／反应管，反应时间２分钟。结果 线性至少可达到２２０μｍｏｌ／Ｌ；精密度为批内ＣＶ１．７１～３．９８％、批间ＣＶ８．０～９．７２％；正常参考值范围为０～３．１４μｍｏｌ／Ｌ；与偶氮法所测结果相关性较好。结论 本文所建立的Ｃ     

结合胆红素校准血清的制备及初步应用

直接胆红素测定是临床对肝、胆疾病鉴别诊断的重要项目。可是，多年来，这个项目的检测结果可比性很差．八十年代起，国外试用牛磺酸胆红素（DTB）为直接睑红素测定的核准品。国内虽有研制的报道，但尚未用于测定结合阻红素的校准血清。本文经过努力制备成冻干型的结合胆红素校     

用胆红素氧化酶两点法测定血清直接胆红素

用胆红素氧化酶两点法测定血清直接胆红素瞿卫（南京市第一医院检验科，南京２１０００６）张瑞生，任宏英（南京市临床检验中心）在不同ｐＨ条件下，胆红素氧化酶（Ｂｉｌｉｒｕｂｉｎｏｘｉ－ｄａｓｅ，ＢＯＤ）催化各种胆红素的氧化反应不同。在ｐＨ８．０～８．２时，...     

风湿性心脏病心力衰竭血清胆红素水平与近期预后关系…

观察风湿性心脏病合并心力衰竭（风心病心衰）患者的血清胆红素（ＢＩＬ）水平，探讨其与近期预后的关系。结果：９６例患者中２６例血清ＢＩＬ增高，死亡８例，病死率为３０．８０％；非高ＢＩＬ组７０例，死亡６例，病死率８．５７％，两组差异非常显著（ｘ＾２＝７．６６，Ｐ〈０。０１）。提示：风心病心衰患者血清ＢＩＬ升高者比正常者预后差。作者认为：对风心病心衰患者常规做血清ＢＩＬ和肝功能检查有利于了解病情和治疗效果     

二甲亚砜双试剂法测定血清胆红素

血清标本的色泽及脂浊度对胆红素测定结果有较大干扰，用双试剂两点终点法是消除此类干扰的理想方法。二甲亚砜（ＤＭＳＯ）是胆红素的优良溶剂，可破坏非结合胆红素的氢键，加速胆红素与重氮试剂的反应。通过实验我们建立了双试剂测定血清胆红素的自动分析方法，具有除干扰能力强，重复性好，与改良Ｊ－Ｇ法有较好的一致性，试剂配制简单，稳定期长等优点，现介绍如下。１材料和方法１．１仪器日立７１７０全自动生化分析仪，ＵＶ－７５４分光光度计。１．２试剂配制１．２．１总胆红素（ＴＢ）测定试剂配制试剂Ｉ（Ｒ１）：量取ＤＭＳＯ …     

风湿性心脏病心力衰竭血清胆红素水平与近期预后关系的临床观察

观察风湿性心脏病合并心力衰竭（风心病心衰）患者的血清胆红素（ＢＩＬ）水平，探讨其与近期预后的关系。结果：９６例患者中２６例血清ＢＩＬ增高，死亡８例，病死率为３０．８０％；非高ＢＩＬ组７０例，死亡６例，病死率８．５７％，两组差异非常显著（Ｘ￣２＝７．６６，Ｐ＜０．０１）。提示：风心病心衰患者血清ＢＩＬ升高者比正常者预后差。作者认为：对风心病心衰患者常规做血清ＢＩＬ和肝功能检查有利于了解病情和治疗效果。     

新生儿高胆红素血症血清酶谱的变化

为了探讨血清酶谱在高胆红素血症患者中的意义,我们对５６例高胆红素血症患者作了血清ＡＳＴ、ＨＢＤＨ、ＬＤＨ、ＣＰＫＭＢ测定,报告如下。１　对象和方法１．１　对象　新生儿高胆红素血症５６例,男３０例,女２６例,年龄１～２１天,黄疸时间１～１６天,其中败血症１６例,脐炎１０例,新生儿肺炎８例,ＡＢＯ溶血５例,头颅血肿５例,脓疱疹４例,母乳性黄疸４例,围产期因素（催产素应用等）４例。按血清总胆红素高低分成２组：轻度组３０例,ＢＩＬＩＴ＜２５６．５μｍｏｌ／Ｌ;重度组２６例,ＢＩＬＩＴ≥２５６．５μｍｏ…     

Chemical nature of a synthetic bilirubin conjugate and its reactivities in the total and direct reactions by the Jendrassik-Gróf method

Using liquid chromatography, nuclear magnetic resonance spectroscopy, and elemental analyses, we verified that a commercially available synthetic bilirubin conjugate is predominantly a ditaurobilirubin (DTB) disodium salt. The Jendrassik-Gróf total bilirubin (TBIL) method quantitatively measures unconjugated bilirubin (Bu) and the Bu-equivalent content in DTB, from which we infer that the azopigments of Bu and DTB have identical molar absorptivities, which do not change in the presence of either serum or serum albumin of human or bovine origin. However, based on the ratio of direct bilirubin (DBIL) to TBIL, the DBIL reaction in dilute HCI is incomplete (even up to 20 min), with lower yields in a matrix of bovine serum albumin than in human serum. By contrast, the DBIL reaction in acetate buffer (pH 4.75) is quantitative for DTB in human serum (or its albumin), but less so in bovine serum (or its albumin). DTB is water soluble, is relatively stable when lyophilized with human serum, and is determined with such high precision in the above-mentioned endpoint assays that it may be a suitable surrogate calibrator for conjugated bilirubin.     

Use of a synthetic soluble bilirubin derivative to assess interference in creatinine measurements

In assessing interference from bilirubin, the use of a synthetic soluble derivative (ditaurobilirubin, DTB) is recommended as a surrogate for the natural conjugates (Bc). We compared the interference effect of unconjugated bilirubin (Bu), Bc, and DTB, using six mechanized methods for serum creatinine measurement. No significant interference was noted in methods that include removal of proteins or in an enzymatic method involving NADH oxidation. Heavy (negative) interference was observed in an alkaline picrate method, and in direct enzymatic methods based on hydrogen peroxide measurement: interference was always more pronounced in the presence of the two soluble derivatives (Bc and DTB), whose interference was of the same magnitude. These results point out the utility of testing for bilirubin interference by using soluble derivatives, in addition to Bu, and suggest the feasibility of using DTB as a surrogate for Bc for this purpose.     

Effects of serum bilirubin on determination of uric acid by the uricase-peroxidase coupled reaction.

We examined the possible effect of different bilirubin species, including unconjugated (Bu), sugar-conjugated (Bc), and authentic delta bilirubin (Bd) isolated from serum, on the direct enzymatic measurement of uric acid by using the uricase (EC 1.7.3.3) and peroxidase (EC 1.11.1.7) coupled reaction. Bc, isolated from human bile, produced the most interference with uric acid determination. Although the presence of human serum albumin reduced interference by Bu and Bc, albumin-bound Bc complex still produced greater interference than Bu. Interference with the peroxidase reaction by Bc covalently bound to serum albumin (Bd) was less than that by Bu. We examined this phenomenon by using serum-isolated Bd obtained by ammonium sulfate precipitation and ultrafiltration and by using commercially available ditaurobilirubin (DTB) and chemically synthesized Bd (via Woodward's reagent, WBd) as surrogates for bile-isolated Bc and natural Bd, respectively. DTB produced the same amount of interference as natural Bc, but the interference produced by DTB in the presence of serum albumin was not the same as that produced by natural Bc with albumin. Our synthetic Bd appears to be a reliable surrogate for natural Bd in testing for bilirubin interference.     

Performance of bilirubin determinations in US laboratories--revisited

BACKGROUND: The diagnosis and management of hyperbilirubinemia in the newborn requires accurate and precise bilirubin determinations. We evaluated the current status of bilirubin measurements in US laboratories by examining data submitted by laboratories participating in the College of American Pathologists (CAP) Neonatal Bilirubin (NB) and Chemistry (C) Surveys. METHODS: We analyzed specimens from the CAP NB and C Surveys by the reference method for total bilirubin in three laboratories. The reference method bilirubin values were compared with bilirubin values reported by Survey participants. RESULTS: The imprecision (CV) for all instruments combined (CAP-All Instruments) ranged from 4.7% to 5.6% at the bilirubin concentrations tested. The CVs of the four most commonly used instruments were smaller, ranging from 1.9% to 4.5%. Differences between bilirubin values by the reference method and mean values from the four most common instruments ranged from -21.6% to 10.9%. When the same specimens from the NB Surveys were used in the C Surveys, the Vitros values were strikingly different from those of the NB Surveys. The use of different methods in the NB and C Surveys coupled with the presence of a nonhuman protein base and ditaurobilirubin (DTB) in the Survey specimens accounted for the discrepant results. CONCLUSIONS: The evaluation of accuracy is impossible from the CAP Surveys because the specimens consist of bovine serum containing a mixture of unconjugated bilirubin and DTB. For the evaluation of accuracy, we recommend that Survey specimens consist of human serum enriched with unconjugated bilirubin.     

Enzymatic assay for conjugated bilirubin (Bc) in serum using bilirubin oxidase (BOD).

We described an automated enzymatic assay for conjugated bilirubin (Bc) in serum using the Iatro D-Bil kit, with bilirubin oxidase (EC 1.3.3.5 BOD) from Myrothecium species. The specificity of the enzyme in the Iatro D-Bil kit was examined by analyzing unconjugated bilirubin (Bu), delta bilirubin (Bdelta), and Bc with high-performance liquid chromatography (HPLC), before and after the enzymatic reaction using BOD. The within-assay coefficients of variation (CV) of this method were 0.58 to 5.00% (n = 20) at 1.4 to 155.8 micromol/L. Day-to-day Cvs ranged from 1.61 to 7.14% at 1.2 to 182.1 micromol/L. The analytical recovery was 96 to 101%. The presence of ascorbic acid, reduced glutathione, L-cysteine, uric acid, urea, creatinine, glucose, lipemic material, anticoagulants, hemoglobin, or human serum albumin did not affect this assay system. The correlation coefficient between values obtained with the Iatro D-Bil kit (y) and HPLC method as reference for conjugated fractions (x) was; r = 0.983, y = 0.952x + 8.851 micromol/L, Sy/x = 11.97 (n = 56). We studied serum Bc levels, not including Bu and Bdelta, in patients with hepatic diseases or autoimmune hemolytic anemia. Levels of Bc obtained by the proposed method changed more rapidly than did those of direct bilirubin (D-Bil) obtained by diazo-dye method during the course of the diseases.     

Rapid detection of pulmonary tuberculosis using the BDProbeTEC ET Mycobacterium tuberculosis Complex Direct Detection Assay (DTB)

The ability to rapidly detect tubercle bacilli in respiratory secretions was determined for the BDProbeTEC ET Mycobacterium tuberculosis Complex Direct Detection Assay in comparison with the acid-fast smear (AFS). A total of 267 respiratory specimens obtained from 89 patients were evaluated. The DTB assay was positive in 70 of 78 culture positive specimens (89.7%) and 12 of 177 culture negative specimens (6.8%). The AFS was positive in 33 of 78 culture positive specimens (42.3%) and 3 of 186 culture negative specimens (1.6%). The sensitivity, specificity, positive predictive value, and negative predictive value of DTB assay were 89.7%, 93.7%, 85.4%, and 95.7%, respectively. The sensitivity of a single DBT (74.4%) was 2.1-times greater than three AFS (35.9%). The greater cost of the DTB assay compared to the AFS was compensated by its valuable information for the diagnosis and control of tuberculosis. These results demonstrated the clinical usefulness of the DTB assay for the rapid diagnosis of tuberculosis in respiratory specimens.     

In vitro effects of light on serum bilirubin subfractions measured by high-performance liquid chromatography: comparison with four routine methods.

The effects of light on serum bilirubin subfractions in vitro were investigated by HPLC and four routine methods for bilirubin analysis. By HPLC, the rate of photodegradation of unconjugated bilirubin (Bu) was nearly twice that of monoconjugated bilirubin (mBc) and threefold that of diconjugated bilirubin (dBc); delta bilirubin (Bd) was most stable against photoirradiation. In the diazo method, the rate of photodegradation of direct bilirubin was almost the same as that of the sum of mBc, dBc, and Bd determined by the HPLC method. However, the rate of photodegradation of indirect bilirubin was significantly lower (P < 0.001) than that obtained by HPLC, because approximately 30% of the bilirubin photoproducts reacted with the diazo reagent as indirect bilirubin. The rate of photodegradation of total bilirubin determined by the direct spectrometric method was lower than that determined by the diazo method, but equal to that of the total peak areas of HPLC. In the Ektachem method, bilirubin photoproducts affected total bilirubin negligibly, and Bc and Bu positively, so that the value of Bd decreased. In the bilirubin oxidase method, bilirubin photoproducts were oxidized enzymatically by both the total and direct bilirubin reagents. We re-emphasize the importance of shielding serum from light to avoid generating bilirubin photoproducts that interfere with the accurate determination of serum bilirubin subfractions. We also recommend HPLC analysis as a standard method for bilirubin measurement.     

Methotrexate interferes with determinations of conjugated bilirubin with the Kodak Ektachem 400

Immediately after intravenous infusion of a high dose (concentration in serum greater than 1000 mumol/L) of methotrexate, the apparent conjugated bilirubin (Bc) concentrations in serum of two osteosarcoma patients, as measured by the Kodak Ektachem 400 analyzer, were greater than the corresponding total bilirubin concentrations, but decreased as the concentrations of methotrexate in serum decreased. In an interference study we found that methotrexate added to sera containing a wide range of basal Bc concentrations increased the measured Bc concentration in a linear and dose-related fashion. Methotrexate also interfered negatively with measurements of unconjugated bilirubin (Bu). The source of the interference appears to be an overlap in the absorption spectrum of methotrexate with Bc and Bu at 400 nm.     

Evaluation of the BDProbeTec ET DTB assay(1) for direct detection of Mycobacterium tuberculosis complex from clinical samples

The purpose of this study was to evaluate the ability of BDProbeTec ET DTB system to detect Mycobacterium tuberculosis complex directly from clinical specimens. A total of 628 specimens (553 respiratory and 75 non respiratory specimens) were collected from 478 patients. These samples were tested with the BDProbeTec ET DTB assay and results were compared with acid fast microscopy and culture. Sixty eight out of 77 culture positive M. tuberculosis complex samples were detected with overall sensitivity and specificity of 89.5% and 98.2% respectively. Overall sensitivity was 100% in smear positive samples and 79% in smear negative samples. After resolution of discrepant results, sensitivity and specificity for respiratory samples were 91.6% and 98.7% respectively. BDProbeTec ET DTB assay demonstrated to be a rapid, sensitive and specific method for detection of M. tuberculosis complex.     

Analysis of unconjugated bilirubin in serum by reversed-phase high performance liquid chromatography.

A high performance liquid chromatographic method for the analysis of unconjugated bilirubin in neonatal serum is presented. Bilirubin was dissociated from protein with caffeine reagent and extracted with chloroform. An isocratic, reversed-phase HPLC system based on a C18 column was used. Bilirubin was detected at 450 nm. Bilirubin SRM 916a from National Institute of Standards and Technology, USA was the primary calibrator. An average recovery factor of 0.996 (SD = 0.018; N = 16) was obtained for bilirubin added to neonatal cord sera. The measurement range extended from 25 to 500 mumol l-1 L-1. The method is proposed as a reference method for unconjugated bilirubin in neonatal sera.