Comparison of techniques for minimizing interference of bilirubin on serum creatinine determined by the kinetic Jaffé reaction

This study compared the effect of sodium dodecyl-sulfate, potassium ferricyanide, and preincubation technique to continuous-flow analysis and prior deproteinization to correct the negative interference of bilirubin on serum creatinine found by the kinetic Jaffé reaction. Bilirubin increased to 684 μmol/L did not interfere with serum creatinine measured by the methods incorporated with dialysis or deproteinization. Trichloroacetic acid was the best protein precipitant. The reagent incorporated with sodium dodecyl sulfate was more appropriate to minimize bilirubin interference than reagent containing potassium ferricyanide. An increase in potassium ferricyanide concentration resulted in false positive creatinine values. Incorporation of both SDS and potassium ferricyanide in the reagent did not help in minimizing the bilirubin interference over use of each chemical alone. The 10 minutes of preincubation of the sample with alkaline buffer incorporating with either SDS or potassium ferricyanide before starting the Jaffé reaction was the appropriate way to overcome unconjugated bilirubin interference at a level of 342.0 μmol/L. However, the technique did not work uniformly with icteric patient sera containing conjugated, unconjugated, and delta bilirubin. This is a challenging problem that remains to be solved by the clinical chemist. © 1994 Wiley-Liss, Inc.     

选择Jaffe法消除胆红素对肌酐测定的干扰

目的通过调整全自动生化仪测试参数,合理选择比色时间,以消除高胆红素对Jaffe法测定肌酐的干扰。方法使用Jaffe速率法测定血清肌酐,不加入任何化学物品,只通过调整迈瑞BS400全自动生化分析仪的比色周期来消除高胆红素对肌酐测定的干扰。结果试验组(高胆红素组)和对照组(低胆红素组)在44～49比色周期之间读取吸光度变化,可消除高胆红素对肌酐测定的干扰(0.01P0.05)。结论根据仪器特点,合理调整参数可有效排除高胆红素对肌酐测定的干扰,取得与低胆红素含量条件下相同的结果。     

Kinetic serum creatinine assays. II. A critical evaluation and review

We have evaluated the Technicon AutoAnalyzer method, the Beckman Creatinine Analyzer II, DuPont aca, Electro-Nucleonics GEMSAEC, and Instrumentation Laboratory Model 916 kinetic methods for determination of serum creatinine. These methods, which involve the Jaffé reaction, all showed a statistically significant proportional error relative to an ion-exchange pretreatment method for creatinine in 92 serum samples; the GEMSAEC method also showed a bias. The positive interference of several acid metabolites was quantitated in each of the methods. The evidence indicates that bilirubin, although associated with a negative creatinine interference, is not the sole interferent. We determined the magnitude of this interference and studied several methods for minimizing it.     

利用Rate-Blank消除胆红素对肌酐测定的负干扰因素

目的分析苦味酸法测定肌酐时胆红素干扰物质的影响,探讨一种消除胆红素干扰苦味酸法测定肌酐的方法。方法在日立7170A全自动生化分析仪上设定两种苦味酸测量肌酐的方法,一种为RateA,另一种利用带血清信息的空白速率(Rate-Blank)RateA法,参数设定为测定方法RateA,反应时间10分钟,读数点〔19 23 11 15〕。用两种方法分别测定正常体检组、高胆红素组标本血清肌酐的浓度。将以上两种方法测得的结果与酶法测得的结果进行对比分析。结果高胆红素对Rat-eA法测量肌酐有明显负干扰,采用Rate-Blank RateA法后此干扰明显降低。结论采用Rate-Blank RateA可基本消除胆红素对苦味酸测定肌酐的干扰。     

Exogenous interferences with Jaffe creatinine assays: addition of sodium dodecyl sulfate to reagent eliminates bilirubin and total protein interference with Jaffe methods

Background: The study evaluated the impact of interferences on the analytical specificity of three commercial and commonly used creatinine methods (two Jaffe and one enzymatic). Methods: Manufacturer creatinine methods plus modified methods were tested with the following interferences: spiking serum with bilirubin, albumin, glucose, hemoglobin and lipid, and patient sera with maximum concentrations of bilirubin, 1,090 µmol/l and protein, 117.8 g/l. Results: Hemoglobin, 7.5 g/l and lipaemic with triglyceride concentration of 6.27 mmol/l, did not interfere with all assays. Glucose >33.3 mmol/l increased creatinine recovery for Dimension method. Samples spiked with bilirubin imparted a negative bias for Dimension and Architect methods but imparted a positive bias for Vitros assay. However, using patient sera, negative bias with bilirubin was found for all methods, from which Architect method gave the highest effect (R²=0.861), followed by Vitros (R²=0.239) and Dimension (R²=0.163). Protein provided the positive bias for all creatinine measurements that increased with increasing concentration (R² ranging from 0.104 to 0.182, P<0.0001). Addition of sodium dodecyl sulfate (SDS) in alkaline‐picrate reagent reduced the effect of bilirubin and protein for kinetic Jaffe method. Although adding potassium ferricyanide was well effective for eliminating negative interference of bilirubin, it was prone to interference from protein. Conclusions: Endogenous interferences continue to plague creatinine accuracy measurement in both Jaffe and enzymatic methods, and consequentially the estimated glomerular filtration rate. The addition of SDS to the alkaline‐pirate reagent was shown to be effective in reducing bilirubin and protein interferences. J. Clin. Lab. Anal. 24:123–133, 2010. © 2009 Wiley‐Liss, Inc.     

Compensating for the influence of total serum protein in the Schwartz formula

Abstract Background: The Schwartz 2009 creatinine-based revised formula is the only pediatric GFR estimating formula, which is compatible with the recent global creatinine standardization. This formula is only applicable if enzymatic creatinine methods are used. We propose an equation, taking into account the relative bias caused by serum proteins to use Jaffe based creatinine data for GFR estimation. Methods: In a cohort study of 100 pediatric patients, serum creatinine was measured using a kinetic rate-blanked Jaffe assay (modified kinetic alkaline picrate method), a kinetic rate-blanked Jaffe compensated assay for reactive proteins and an enzymatic assay (creatinine plus method). Serum total protein, albumin, urea, uric acid and total bilirubin were measured with the use of commercial agents. Results: The difference in serum creatinine between the enzymatic method and the compensated Jaffe method was mainly dependent on the total protein concentration in serum (r2=0.61, p<0.001). After applying the proposed protein correction, corrected compensated Jaffe results and creatinine clearance values became interchangeable with enzymatic serum creatinine results (r2=0.99, p<0.001; Deming regression: slope: 0.9787, intercept: -0.351) and with the newly proposed Schwartz formula, respectively (r2=0.99, p<0.001; Deming regression: slope 1.004, intercept: 2.16). Conclusions: In this study, we demonstrated the usability of the alkaline picrate method in the Schwartz formula, taking into account the relative bias caused by serum proteins.     

Interferences in current methods for measurements of creatinine

We measured the interference of carbonyl compounds, drugs, and other substances in human serum on the determination of creatinine by the two-point, fixed-time kinetic modification of the Jaffé reaction as well as by four enzymatic methods. We added known concentrations of the interfering substances to a solution of creatinine in water. For bilirubin, we used both pooled normal sera with added bilirubin and icteric patient sera. The magnitude of interferences varies widely from method to method. Carbonyl compounds, dopamine, cephalosporines, and bilirubin interfere with the Jaffé reaction. Bilirubin, creatine, dopamine, ascorbic acid, and sarcosine interfere with the enzymatic methods. We conclude that the elimination of interferences in the determination of creatinine has still not been achieved.     

Enzymatic determination of bilirubin fractions in serum

Novel enzymatic methods using bilirubin oxidase from Myrothecium verrucaria are described for the determination of bilirubin fractions in serum. These fractions include an unconjugated form (Bu), a conjugated form (Bc), and a delta fraction of bilirubin that reacts with direct diazo reagent and is irreversibly bound to serum albumin (Bδ). The determination is based on the different reactivities of the enzyme to bilirubin fractions at different pH in the presence or absence of anionic detergent such as sodium dodecyl sulfate (SDS) or sodium cholate. Thus, in the absence of detergents, Bc and Bδ are oxidized at acidic pH, while Bc is oxidized at alkaline pH; Bu is not oxidized at either acidic or alkaline pH.

The first approach is based on measuring the decreased absorbance of bilirubin colour at 450 nm caused by the enzymatic oxidation. Total bilirubin is measured at pH 8.0 in the presence of SDS. Direct bilirubin is measured at pH 3.7 and Bc is measured at pH 10.0 in the absence of SDS, respectively.

The second approach is made by coupling the diazo reaction with the enzyme reaction. Total and direct bilirubin are determined by a conventional diazo method without prior treatment by the enzyme. Bδ is determined with a direct diazo method after the serum sample is treated at pH 10.0 by the enzyme to oxidize the Bc fraction. The precision and the accuracy of these methods were compared with a precision and the accuracy of these methods were compared with a diazo method, an HPLC method and a slice method, and good results were obtained.     

Serum ultrafiltration for the elimination of endogenous interfering substances in creatinine determination

Serum, at neutral pH, was submitted to a simple filtration, using centrifugation in the disposable Centrisart. The ultrafiltrate was similar to serum in its creatinine content but was virtually free from proteins, including protein-bound bilirubin, haemoglobin and lipoproteins. The creatinine concentrations of anicteric serum specimens and the corresponding ultrafiltrates as determined with Jaffé and enzymic procedures show a high correlation and are convertible. With icteric sera the negative interference effect of bilirubin in a particular analytical procedure can be quantified using ultrafiltrate as the reference. It is suggested that ultrafiltration is useful in selected cases for eliminating elevated concentrations of bilirubin, haemoglobin and turbidity, which would interfere in the direct creatinine determination. Relative to the continuous flow methods with dialysis of the analyte, direct methods for creatinine are more susceptible to interference by endogenous factors like hyperbilirubinaemia, hypertriglyceridaemia and haemolysis (1). The negative interference by bilirubin is of special importance, since it interferes in some modifications of the kinetic Jaffé method (2) and in the chromogenic enzymatic method (3). As a simple alternative, we evaluated the use of serum ultrafiltrate for the accurate determination of creatinine by the Jaffé and enzymatic methods, free from interfering by the high-molecular serum matrix and compounds bound to it.     

酶法与碱性苦味酸法测定肌酐清除值的差异情况研究

目的研究酶法和碱性苦味酸法测定肌酐清除值（CrCl）的差异,探讨酶法CrCl采用国外参考区间是否合适。方法分别用肌氨酸氧化酶法（简称酶法）和碱性苦味酸速率法（简称苦味酸法）测定266名患者的血肌酐、尿肌酐以及CrCl,并以苦味酸法CrCl水平分为4组,第1-4组CrCl分别为〈25、26-50、51-80和〉81 mL/min。分析两法测定血肌酐、尿肌酐和CrCl结果的差异以及各组两法差异的变化。结果酶法血肌酐值和尿肌酐值低于苦味酸法（P〈0.001）,酶法CrCl除第1组外均高于苦味酸法（P〈0.001）。第1组两法血肌酐相对差值较小,且与两法尿肌酐相对差值接近,使CrCl在两法间无差异;第2-4组血肌酐相对差值逐渐增大,尿肌酐相对差值基本不变,导致CrCl在两法间出现差异且差异越来越大（P〈0.001）。结论CrCl〈25 mL/min时,两法CrCl没有差异;CrCl〉26 mL/min时,两法CrCl出现差异且差值逐渐增大;CrCl正常组两法差值很大,酶法CrCl采用国外参考区间不合适。     

样本中胆红素浓度对不同方法测定血糖结果的影响

目的：研究样本中不同浓度胆红素对干化学法和湿化学法测定血结果的影响,分析两种方法所获结果之间的线性关系及相关程度。方法：临床血清标本97份,分别用干化学法（葡萄糖氧化酶法）和湿化学法（己糖激酶法）测定血糖浓度,分析两种方法的相关性。结果：（1）胆红素浓度低于62.7μmol/L时,2种方法测定血糖结果无明显差异;（2）胆红素浓度高于65.9μmol/L时,2种方法测定血糖结果有统计学差异,P〈0.01;（3）2种方法所得结果差值与胆红素浓度高度相关,相关系数r=0.8942。结论：样本中胆红素浓度低水平时,干化学法与湿化学测定血糖无统计学差异,样本中胆红素浓度较高时,两种方法结果差异较大,应注意进行去除胆红素处理再测定血糖浓度,以免引起临床误诊。     

血清肌酐两种测定方法偏倚的比较

目的分析肌氨酸氧化酶法（简称酶法）和苦味酸速率法（Jaffe′s法）测定血清肌酐结果的偏倚。方法依据美国国家临床实验室标准协会EP9-A文件,每天取临床样本8份,分别用两种方法测定血清Cre含量,共测定5d,记录结果,去除离群点,计算线性回归方程和相关系数,进行偏倚估计。结果在测定患者新鲜血清样本Cre时,酶法和Jaffe′s法测定结果的预期相对偏倚在Cre浓度小于100μmol/L时,两种方法的预期偏倚不超过-1.5%,酶法的测定结果低于Jaffe′s法;当Cre浓度大于300μmol/L时,两种方法的预期偏倚不低于6.4%,酶法的测定结果高于Jaffe′s法;当Cre浓度大于500μmol/L时,两种方法的预期偏倚不低于7.9%。结论预期偏倚在中低浓度时随肌酐浓度增高而降低,高浓度时则随浓度增高而增大,但Jaffe′s法测定结果较酶法低。临床实验室在换用不同仪器或试剂时必须对肌酐测定的不同方法建立不同的参考值范围。     

A peroxidase-coupled kinetic enzymatic procedure evaluated for measuring serum and urinary creatinine

We evaluated an enzymatic procedure for the automated determination of serum and urinary creatinine. The procedure was sensitive and precise for serum creatinine concentrations as low as 3 mg/L, and the standard curve was linear to 200 mg/L. Measurements of creatinine in clinical serum and urine specimens agreed with those obtained by two other enzymatic methods and four picric acid methods. There was no interference from picrate-reactive substances tested. Among other substances, bilirubin (50-100 mg/L) interfered negatively with the assay, decreasing apparent creatinine concentrations by as much as 6 mg/L. The advantages of specificity, speed, and convenience make this enzymatic procedure an attractive primary or secondary method for creatinine determinations in clinical laboratories.     

酶法和Jaffe速率法测定血清肌酐的方法学比较

目的：了解酶法和Jaffe速率法测定血清肌酐结果的偏倚大小，为室间评估结果分组提供依据。方法：按照美国全国临床实验标准委员会（NCCLS）EP9－A文件进行评估，将测得的数据进行相关回归分析。计算两方法间的偏倚。结果．；酶法和Jaffe速率法测定肌酐，其回归方程式Y＝1．005X＋17．9（Y为Jaffe速率法，X为酶法），相关系数r=0.998，有明显恒定偏倚。结论：必须对不同方法原理的肌酐室间评估结果作分组处理。     

Comparison of creatinine as determined with the Ames Seralyzer and by three Jaffé-based methods

We compared results for urinary creatinine, serum creatinine, and creatinine clearance, as determined with the Ames Seralyzer, with results determined with the Beckman ASTRA, the DuPont aca, and Technicon's AutoAnalyzer and SMAC. Results for urinary creatinine from the Seralyzer differed significantly (p less than 0.05) from those obtained with the ASTRA and AutoAnalyzer, but not with the aca. The Seralyzer results for serum creatinine were at least 1.0 mg/L higher (p less than 0.05) than by the other three methods. Results for creatinine clearance from the Seralyzer were 8 to 11 mL/min lower (p less than 0.05) than results by the other three methods. These differences are related to the positive interference by bilirubin in the Seralyzer creatinine method. We also evaluated 23 other compounds for interference with these methods for creatinine.     

Falsely-elevated serum creatinine values in diabetic ketoacidosis -- clinical implications

Unusual elevations of serum creatinine (S-CR) out proportion to increases in serum urea nitrogen (S-UN) are frequently observed in patients with diabetic ketoacidosis when S-CR is measured by the Jaffe end-point reaction. This has been ascribed to interference from acetoacetate but this is not however observed with kinetic DuPont ACA methodology. Eighteen patients with diabetic ketoacidosis were studied: SCR measurements were done using the end point Technicon SMA 6/60 method (Group a, 10 patients) or the kinetic DuPont ACA method (Group B, 8 patients). The values for S-CR in Group A patients (mean value and S-D were 3.3 +/- 1.1 mg/dl) were significantly different from Group B patients (1.6 +/- 0.24 mg/dl) (P less than 0.01). A significant positive correlation was obtained between the "excess anion gap" and S-CR in Group A patients (r = + 0.81, p less than 0.01). The results from two patients in whom serial measurements of S-UN, S-CR and the anion gap were carried out further demonstrate the analytical interference. The study demonstrates that in diabetic ketoacidosis elevated creatinine values measured by an end-point method should not necessarily be interpreted as evidence of significant renal impairment and if possible should be verified by a kinetic method which is free of "ketone" interference.     

酶法与碱性苦味酸法测定肌酐清除值的差异情况研究

目的研究酶法和碱性苦味酸法测定肌酐清除值(CrCl)的差异,探讨酶法CrCl采用国外参考区间是否合适。方法分别用肌氨酸氧化酶法(简称酶法)和碱性苦味酸速率法(简称苦味酸法)测定266名患者的血肌酐、尿肌酐以及CrCl,并以苦味酸法CrCl水平分为4组,第1~4组CrCl分别为<25、26~50、51~80和>81 mL/min。分析两法测定血肌酐、尿肌酐和CrCl结果的差异以及各组两法差异的变化。结果酶法血肌酐值和尿肌酐值低于苦味酸法(P<0.001),酶法CrCl除第1组外均高于苦味酸法(P<0.001)。第1组两法血肌酐相对差值较小,且与两法尿肌酐相对差值接近,使CrCl在两法间无差异;第2~4组血肌酐相对差值逐渐增大,尿肌酐相对差值基本不变,导致CrCl在两法间出现差异且差异越来越大(P<0.001)。结论CrCl<25 mL/min时,两法CrCl没有差异;CrCl>26 mL/min时,两法CrCl出现差异且差值逐渐增大;CrCl正常组两法差值很大,酶法CrCl采用国外参考区间不合适。     

利福平对血清胆红素测定的干扰探讨

目的 探讨利福平对血清胆红素测定的干扰。方法 用亚硝酸钠氧化法测定血清总胆红素（TBIL）、直接胆红素（DBIL）浓度。结果 利福平对TBIL、DBIL测定有正相干扰作用，干扰的程度与加入血清中的利福平的浓度呈线性关系。结论 利福平对亚硝酸钠氧化法测定TBIL、DBIL可产生光学干扰。     

Multicentre evaluation of an enzymatic method for creatinine determination using a sensitive colour reagent

A new enzymatic colorimetric method for the determination of creatinine in serum and urine (Creatinine PAP, Cat. No. 839434 and 836885, Boehringer Mannheim GmbH, Mannheim, FRG) was evaluated in 16 clinical chemistry laboratories and the manufacturer's testing laboratory. The test is based on the enzymatic degradation of creatinine and its reaction products by creatininase, creatinase and sarcosine oxidase. The H2O2 produced by the oxidation of sarcosine is determined using a modified Trinder reaction. The test can be carried out either manually or in mechanized analysers which enable the pipetting of a starter reagent to be made. The following results were obtained: Depending on the analyte concentration (range 40 to 1240 mumol/l), medians for the coefficients of variation were: 4.6-0.9% within-run and 6.4-2.8% between-day. At 546 nm the linear measuring range extended from 13 mumol/l (detection limit) to 1780 mumol/l, at 510 nm from 9 to 890 mumol/l. Recoveries in aqueous and human serum based standards as well as method comparisons with Fuller's earth methods and an enzymatic UV test indicate a high accuracy of this new enzymatic method in serum and urine. No interference was observed with haemolysed and lipaemic sera. This also applied to anticoagulants and to 36 drugs at therapeutic concentrations, with the exception of calcium dobesilate, which led to decreased values. Icteric samples containing 120-310 mumol/l bilirubin invariably led to decreased creatinine values (10-50 mumol/l lower). In a collaborative study substantially better interlaboratory agreement was observed with the new colorimetric enzymatic test than with the comparison methods (enzymatic UV test and various Jaffe procedures). In conclusion, this new enzymatic colorimetric test permits a precise and specific determination of creatinine in serum and urine. It makes a considerable contribution to improving the interlaboratory comparability of creatinine determinations and is suitable for routine use.     

注射用头孢替安对钒酸盐氧化法和干化学法测定的总胆红素浓度的影响

目的探讨头孢替安对钒酸盐氧化法、干化学法测定总胆红素的干扰。方法收集总胆红素浓度小于20μmol/L的样本20例,总胆红素浓度介于150～220μmol/L的样本20例,总胆红素浓度介于350～410μmol/L的样本20例,共计60例,每例加入等体积不同浓度的头孢替安,配制成头孢替安终浓度分别为300、150、75mg/L的血清样本作为实验组,以加入等体积纯水血清样本作为对照组,分别采用干化学法和钒酸盐氧化法测定加入不同浓度头孢替安和纯水的血清样本中总胆红素浓度,比较头孢替安对两种方法测定的总胆红素浓度的影响,所有数据用统计学软件SPSS13.0进行处理。结果干化学法中,加入头孢替安的实验组与加入纯水的对照组比较,实验组总胆红素浓度较对照组升高,差异有统计学意义(P0.05),在同一总胆红素浓度,随着头孢替安浓度的降低,实验组总胆红素浓度的升高率逐渐降低。在钒酸盐氧化法中,总胆红素浓度在150～220μmol/L时,实验组总胆红素浓度较对照组升高,差异有统计学意义(P0.05)。结论注射用头孢替安对干化学法测定总胆红素浓度的干扰作用较强,且随着药物浓度的升高,影响越明显,而对钒酸盐氧化法测定总胆红素浓度几乎无影响。