Bilirubin Interference Research Articles

Effects of Bilirubin Interference in the Roche Kinetic Alkaline Picrate Creatinine Assay

The interference of bilirubin in kinetic alkaline picrate creatinine assays (Jaffe) has been widely known and described in the literature (1). Many publications describe a negative interference of bilirubin in Jaffe creatinine assays (2, 3). Falsely decreased creatinine results can have far-reaching implications on the treatment of patients. Patients receiving treatment with aminoglycosides or patients subject to imaging procedures are very dependent on accurate creatinine measurements. Measurements of creatinine clearance and estimation of the glomerular filtration rate are also highly dependent on an accurate creatinine measurement. In this study, we observed a trend of falsely increased creatinine values in the presence of increased bilirubin levels, demonstrated by increasing icterus (I) indices. At our institution, we are using the Roche COBAS 6000, creatinine …

When results matter: reliable creatinine concentrations in hyperbilirubinemia patients.

Background Failure to report a creatinine concentration, especially in icteric patients who are eligible for a liver transplant, can result in a life-threatening situation. We assessed the influence of bilirubin interference on several creatinine assays and investigated ways to circumvent icteric interference without interfering with our normal automated sample logistics. Methods Using icteric patient sera (total bilirubin >255 μmol/L) we determined creatinine concentrations using an enzymatic and Jaffé assay (Roche Diagnostics) in both normal (i.e. undiluted) and decreased mode (i.e. diluted) as well as an enzyme-coupled amperometric assay on a Radiometer ABL837 FLEX analyzer. Creatinine concentrations from the five methods were compared with an in-house developed LC-MS/MS method. Passing and Bablok (proportional and constant bias) as well as difference plot parameters (bias and 95% limits of agreement [LoA]) were calculated. Interferograph-based regression analysis of the enzymatic and Jaffé results was used to investigate if such an approach could be used to report corrected creatinine concentrations in icteric patient sera. Results In icteric patient sera the enzyme-coupled amperometric assay was hardly influenced by icteric interference as shown by a difference plot bias of -1.5% (95% LoA -11.6 to +8.5%). The undiluted Jaffé method had a bias of -1.4% with a very broad 95% LoA (-35.1 to +32.2%) emphasizing the poor specificity of this method. The undiluted enzymatic method had the largest bias (-13.4%, 95% LoA -35.8 to +9.0%). Diluting sera in the enzymatic method did not improve the bias (-10.5%, 95% LoA -25.4 to 4.4%), while diluting the Jaffé method resulted in a bias increase (+11.4%, 95% LoA -14.7 to 37.5%). Using interferograph-based regression analysis we were able to reliably correct enzymatic creatinine concentrations in 97 out of 100 icteric patient sera. Conclusions Analytically, quantifying creatinine in icteric sera using the Radiometer ABL837 FLEX analyzer is the method of choice within our laboratory. However, not all laboratories are equipped with this method and even if available, the limited number of highly icteric patient sera makes this method costly. For those laboratories using the Roche enzymatic method, mathematically correcting an icteric creatinine concentration using an interferograph based on an LC-MS/MS reference method is a suitable alternative to report reliable creatinine results in icteric patients.

Elimination of bilirubin interference on determination of serum N-acetyl-β-hexosaminidase activity

Aim: Elimination interference of bilirubin on determination serum N-acetyl-β-hexosaminidase (HEX) activity. Material and Methods: We determined HEX activity in sera without and with exogenous bilirubin (4.3 mg/dL) using unmodified and modified method. Modification consists in subtraction absorbance of reaction mixture without substrate, from absorbance of reaction mixture incubated with substrate. Results: HEX activity (nKat/L) amounted: 179.7±74.6 without and 291.4±77.9 with exogenous bilirubin (standard method); 160.5±69.7 without and 164.9±76.8 with exogenous bilirubin (modified method). Correlation coefficient between results of HEX determination in serum without bilirubin and with exogenous bilirubin, obtained with modified method was 0.997. Conclusion: Exogenous bilirubin (4.3 mg/dL) significantly overestimates (p ≤0.001) HEX activity in blood serum determined by the standard method. Proposed modification eliminates interference of bilirubin on determination of HEX activity in blood serum.

Icteric human samples: Icterus index and method of estimating an interference-free value for 16 biochemical analyses.

Hemolysis, Icterus, and Lipemia constituting the HIL index, are the most common causes of interference with accurate measurement in biochemistry. This study focuses on bilirubin interference, aiming to identify the analyses impacted and proposing a way to predict nominal interference-free analyte concentrations, based on both analyte level and Icterus Index (Iict ). Sixteen common analytes were studied: alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), amylase (AMY), aspartate aminotransferase (AST), total cholesterol (CHOLT), creatinine (CREA, enzymatic method), fructosamine (FRUC), gamma-glutamyl transferase (GGT), HDL cholesterol (HDLc), total iron (Iron), lipase (LIP), inorganic phosphorus (Phos), total protein (PROT), triglycerides (TG), and uric acid (UA). Both the traditional 10% change in concentrations from baseline and the Total Change Level (TCL) were taken as acceptance limits. Nineteen pools of sera covering a wide range of values were tested on the Cobas® 6000 (Roche Diagnostics). Iict ranged from 0 to 60. Eight analytes increased (FRUC and Phos) or decreased (CHOLT, CREA, HDLc, PROT, TG, and UA) significantly when Iict increased. FRUC, HDLc, PROT, and UA showed a linear relationship when Iict increased. A non-linear relationship was found for TG, CREA, and for CHOLT; this also depended on analyte levels. Others were not impacted, even at high Iict . A method of estimating an interference-free value for FRUC, HDLc, PROT, Phos, UA, TG, and CREA, and for CHOLT in cases of cholestasis, is proposed. Iict levels are identified based on analytical performance goals, and equations to recalculate interference-free values are also proposed.

Total Antioxidant Capacity in beta-thalassemia: A systematic review and meta-analysis of case-control studies.

Total Antioxidant Capacity (TAC), a biomarker measuring the antioxidant potential of body fluids, including redox synergistic interactions, is influenced by the presence of products of catabolism such as bilirubin (BR) and uric acid (UA). Hyperuricaemia and increased BR levels were observed in thalassemia. In order to evaluate the differences in TAC values between thalassemic patients and healthy subjects, we performed a systematic review and meta-analysis of case-control studies. After the exclusion of data deemed unsuitable for meta-analysis inclusion and a study imputed of bias by Trim-and-fill analysis, mean difference (MD) and confidence intervals 95% (CI 95%) were calculated by the random effect model for beta-thalassemia major (BTM) (1351 subjects: 770 thalassemic and 581 controls, from 15 studies) and Trait (BTT) or Hemoglobin E (BTE) (475 subjects: 165 thalassemic and 310 controls, from 5 studies). Despite the differences in clinical symptoms and severity, similar decreased levels of TAC were found in BTM [MD -0.22 (-0.35 -0.09) p<0.001] and BTT or BTE [MD -0.22 (-0.44 -0.01) p<0.05]. In conclusion, UA and BR interference on TAC suggests that corrected TAC and in particular the UA-independent TAC, considering the prominent influence of UA, might be the better approach to evaluate body antioxidant status.

Eltrombopag interference in routine chemistry testing.

High doses of Eltrombopag have been previously reported to cause bilirubin interference. Following receipt of a sample from a patient receiving high-dose Eltrombopag therapy, the laboratory decided to investigate the effect of this drug on routine chemistry testing. Interference studies were performed by spiking Eltrombopag into aliquots of a serum pool to give concentrations ranging from 0 to 500 µg/mL. The following analytes, namely albumin, alkaline phosphatase, alanine transaminase, aspartate transaminase, Urea, total calcium, cholesterol, triglycerides, glucose, high-density lipoprotein cholesterol, iron, magnesium, inorganic phosphate, creatinine, bicarbonate, transferrin, ferritin, electrolytes, total and direct bilirubin and serum indices (hemolysis, icterus and lipaemia) were then measured on the Roche Cobas 6000 chemistry analyzer (Roche, Indianapolis, USA). Eltrombopag interference (>10% change of the baseline value) was observed for total cholesterol, triglycerides, inorganic phosphate and high-density lipoprotein cholesterol. Clinical significant interference was observed for total cholesterol, inorganic phosphate and high-density lipoprotein cholesterol Presence of high Eltrombopag concentrations in blood samples has been demonstrated to cause interference in the measurement of certain spectrophotometric-based assays on the Roche Cobas 6000 analyzer.

A paraprotein interference and its management in clinical laboratory / Bir paraprotein interferansı vakasının klinik laboratuvarda yönetimi

Abstract In the present study we describe a patient who has interference due to paraproteinemia in her labaratory results. In a patient with a total protein concentration of 10.8 g/dL, a direct bilirubin result higher than total was detected. She also had discordant results in her whole blood count parameters. Further investigation was performed on this patient. Presence of any cold agglutinin and cryoglobulin was tested and excluded first. After 2-mercaptoethanol (2-ME) treatment, patient was idendified as Ig-M Kappa monoclonal gammapathy on immunofixation electrophoresis (IFE). Direct bilirubin interference disappeared after removal of the paraprotein by polyethylene glycol (PEG) precipitation. Laboratory specialist should know paraprotein interference and be able to manage it.

Bilirubin Interference in Serum Creatinine Estimation by Jaffe'skinetic Method and Its Rectification in Three Different Kits.

The negative interference by bilirubin in serum creatinine estimation by Jaffe's kinetic method is well known. Several approaches have been suggested to overcome this interference. In this article three different creatinine kits (Jaffe's kinetic method) have been tested for bilirubin interference and its rectification using two simple approaches. The performance of three kits (A, B and C) supplied by three different manufacturers was tested using IQC and EQAS sera and pooled serum with added bilirubin. To overcome the bilirubin interference two approaches viz. NaOH preincubation and TCA precipitation were used. Bilirubin did interfere in creatinine estimation after a certain level (2.3mg/dl). However, both NaOH preincubation and TCA precipitation approach rectified this interference. The performance of kit A was better than kit B and C. All the three kits showed bilirubin interference upon increasing the bilirubin concentration but kit A performed better than kit B and C. However, NaOH incubation and TCA precipitation methods overcame this interference to a great extent.

Evaluation of the In Vitro Interference of Hemoglobin, Lipids, and Bilirubin on the Measurement of Plasma Advanced Oxidation Protein Products

The pre-analytical phase is the most vulnerable and contributes to the majority of errors in laboratory assays. Therefore, the aim of this study was to evaluate in vitro interference of hemoglobin, lipids, and bilirubin on measurement of plasma advanced oxidation protein products (AOPPs). AOPPs were measured in a sample spiked with increasing concentrations of hemoglobin, Intralipid® 20% or bilirubin. Then, the relative deviation of the result from the nonspiked baseline value was calculated. We found that all interferents analyzed influenced AOPPs baseline value significantly. Hemoglobin produced a non-linear negative bias, with a maximum decrease of 21.1%. Otherwise, lipids and bilirubin produced a positive bias, ranging from 20.0 to 90.9%, and 12.1 to 33.2%, respectively. The addition of interferents produced a significant bias in the measurement of AOPPs and in different degrees at the majority of concentrations added.

HIL Interferences on Three Hemostasis Analyzers and Contribution of a Preanalytical Module for Routine Coagulation Assays

Preanalytical issues are a major part of routine coagulation laboratory errors. Automation in detection of preanalytical problems, including hemolysis, icterus and lipemia (HIL), improper tube fill volume, and undue clotting, has recently been implemented on specific hemostasis instruments. The aim of this study was to assess the added value of a new preanalytical module integrated into hemostasis analyzers compared to visual inspection of samples. The detection of preanalytical issues was performed by visual or manual inspection of the samples and by the new preanalytical module integrated on the ACL TOP 550. Additional tests were done to evaluate the interference of hemoglobin (Hb), bilirubin or triglycerides (TG). Plasma pools containing the interference substance were tested for routine coagulation assays on the STA-R Evolution, CS-5100, and ACL TOP 550. Visual or manual inspection detected statistically less samples with preanalytical issues than the new preanalytical module integrated on the ACL TOP 550 (3.5% vs. 6.6%, p < 0.001). The majority of the samples were rejected for poor filling. HIL interferences appeared on assays when the concentration of Hb, bilirubin or TG exceeded a certain threshold that was analyzer and reagent dependent. Automatic and standardized check of routine coagulation samples by ACL TOP 550 increased the accuracy and consistency in detection of preanalytical issues as compared to visual inspection only. The main advantages were the detection of insufficient filled tubes and icteric samples that are not detected visually.

Evaluation of bilirubin interference and accuracy of six creatinine assays compared with isotope dilution–liquid chromatography mass spectrometry

ObjectivesThe aim of this study was to estimate bilirubin interference and accuracy of six routine methods for measuring creatinine compared with isotope dilution–liquid chromatography mass spectrometry (ID–LC/MS). Design and methodsA total of 40 clinical serum samples from 31 patients with serum total bilirubin concentration >68.4μmol/L were collected. Serum creatinine was measured using two enzymatic reagents and four Jaffe reagents as well as ID–LC/MS. Correlations between bilirubin concentration and percent difference in creatinine compared with ID–LC/MS were analyzed to investigate bilirubin interference. Bias estimations between the six reagents and ID–LC/MS were performed. Recovery tests using National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 967a were also performed. ResultsBoth the enzymatic methods showed no bilirubin interference. However, three of the four Jaffe methods demonstrated significant bilirubin concentration-dependent interference in samples with creatinine levels <53μmol/L, and two of them showed significant bilirubin interference in samples with creatinine levels ranging from 53.0 to 97.2μmol/L. Comparison of these methods with ID–LC/MS using patients' samples with elevated bilirubin revealed that the tested methods failed to achieve the bias goal at especially low levels of creatinine. In addition, recovery test using NIST SRM 967a showed that bias in one Jaffe method and two enzymatic methods did not achieve the bias goal at either low or high level of creatinine, indicating they had calibration bias. One enzymatic method failed to achieve all the bias goals in both comparison experiment and recovery test. ConclusionsIt is important to understand that both bilirubin interference and calibration traceability to ID–LC/MS should be considered to improve the accuracy of creatinine measurement.

Interference of Bilirubin in Creatinine Value Measurement by Jaffe Kinetic Method

BACKGROUND: Creatinine measurement in icteric sample is a major but unresolved problem. Bilirubin causes negative interference in creatinine value measurement using general techniques. The objective of this study was to find differences in creatinine value by Jaffe Kinetic method pre-incubation and without pre-incubation with NaOH. METHODS: This was a cross sectional, descriptive study carried out in 71 samples with different level of bilirubin concentration. We took blood samples of 71 different patients, 48 males and 23 females, from two different hospitals of Kathmandu village. Both creatinine and bilirubin concentration in serum samples were measured by using Staxfax 3300 semi auto analyzer in the hospital. In the laboratory creatinine value was measured by kinetic method and bilirubin measured by Jendrassik/ Grof method using commercial kits. Statistical analysis of quantitative data was done by using SPSS version 16.0. RESULTS: The results shows differences in creatinine values with respect to methods and extent of bilirubin concentration. It was found that the creatinine obtained by pre-incubation with NaOH has greater value than without pre-incubation (i.e. by direct estimation using working reagent). It was also shown that the high bilirubin cause the interference in greater extent. The significant interference was seen in the sample with bilirubin concentration greater than 20 mg/dl i.e. creatinine value after treatment with NaOH prior to dispensing picric acid is significantly increases, P&lt;0.01 at 99% confidence level. CONCLUSION: This shows that the bilirubin has negative interference in creatinine value measurement by ordinary laboratory practices and interference increases with higher concentration of bilirubin in blood sample. DOI: http://dx.doi.org/10.3126/acclm.v1i1.12311 Ann. Clin. Chem. &amp; Lab. Med. 1(1) 2015: 25-28

Interference of Bilirubin in the Determination of Magnesium with Methyl Thymol Blue.

Introduction:Jaundice is a disease named for the yellow color of the skin. This color is the result of elevated levels of bilirubin in the blood serum. In Roma from Krusevac region in the last few years have seen the emergence of jaundice.Material and methods:In 80 of them (40 suffering and 40 from control group) were performed tests of numerous parameters in the laboratories of the Health Center in Krusevac. Magnesium was determined by spectrophotometry with methyl thymol blue, titanium yellow and blue xylidene. Bilirubin was determined by Jandrešek Grofov’s method.Results:The results were within the expectations, in addition to magnesium which was determined with methyl thymol blue. In all patients suffering from jaundice concentration of magnesium (0.67 ±0.14 mmol/l) statistically was significantly lower than tested of the control group (0.91± 0.059 mmol/). There is no theoretical data to reduce the concentration of magnesium in serum as a result of jaundice. That’s why we determined magnesium both in the control group and in sufferings with two methods as the titanium yellow, and xylidene blew. With these two methods we obtained results that were examined were within normal limits.Conclusion:This has led us to conclude that the determination of bilirubin interferes with magnesium methyl thymol blue.

Bi-variate approach to negative interference of bilirubin towards an acetaminophen assay

BackgroundIt is well known that enzymatic assays for acetaminophen are positively interfered by bilirubin. The effect on acetaminophen not only depends on the concentration of bilirubin but also on that of acetaminophen. We demonstrated a negative interference instead of a positive one in a commonly used routine analyzer and investigated the recovery of acetaminophen in an enzymatic assay by a bi-variate regression. MethodsCommercially available blank serum specimens were spiked with acetaminophen and bilirubin at various concentrations, and were analyzed in the Beckman Coulter AU5822 analyzer. The specimens were run in duplicates. The results were then analyzed by least-square analysis and was built into a bi-variate quadratic model. ResultsThe recovery of acetaminophen in this experiment ranged from 38.9% to 100% throughout a range of 23μmol/L to 2052μmol/L (for acetaminophen) and 19μmol/L to 570μmol/L (for bilirubin). A contour map, as well as a bi-variate equation was established, describing the relationship between acetaminophen recovery, acetaminophen concentration, and bilirubin concentration. ConclusionIt was shown that the degree of bilirubin interference in a commercially available acetaminophen assay is dependent on both bilirubin and acetaminophen concentrations. There was a decrease in the apparent acetaminophen concentration by an average of 30% at a bilirubin concentration of 420μmol/L in the Beckman Coulter AU5822 analyzer. The complex relationship can be modeled by mathematical means. This allows the laboratory staff to estimate the recovery of acetaminophen when bilirubin level is concurrently measured.

Differences in bilirubin and biliverdin interferences in an enzymatic acetaminophen assay

Differences in bilirubin and biliverdin interferences in an enzymatic acetaminophen assay

Monoclonal and polyclonal immunoglobulin interference in a conjugated bilirubin assay.

Monoclonal proteins can interfere with the conjugated bilirubin assay on the Beckman Coulter AU5400 and AU2700 instruments and produce spurious results. Protein depletion eliminates the interference, suggesting that monoclonal proteins cause the problem. To determine the interference rate with the Beckman Coulter AU5400 and AU2700 conjugated bilirubin assay and to identify the interfering substance. Beckman Coulter bilirubin results from 33,720 samples analyzed during 6 months were evaluated for interference. On a subset of samples, protein G columns were used to specifically remove immunoglobulin G (IgG) to determine the cause of the interference. Another 117 samples containing a (known) monoclonal protein were selected to determine the interference rate. From 26 different patients, 103 samples had conjugated bilirubin results greater than the total bilirubin for a false-positive rate of 0.3%. Removal of IgG from a subset of those samples with IgG monoclonal protein and increased polyclonal IgG eliminated the conjugated bilirubin interference. In separate, selected samples containing monoclonal proteins, the false-positive interference rate was 7.7% (9 of 117) and was greatest when the monoclonal protein concentration was greater than 4 g/dL. Another 11 of the 117 samples (9.4%) with monoclonal proteins exhibited assay interference by producing false-negative results. The overall interference rate, therefore, was 17.1%. The false-positive interference rate for the Beckman Coulter conjugated bilirubin assay was 0.3% for routinely analyzed serum/plasma samples. In addition to monoclonal proteins, we found that polyclonal immunoglobulins can also interfere with the conjugated bilirubin assay. The overall interference rate was 17.1% for samples with a monoclonal protein.

Pseudo‐hypoproteinemia in a hyperbilirubinemic dog with immune‐mediated hemolytic anemia

A 10-year-old spayed female Miniature Poodle was presented to the University of Georgia veterinary teaching hospital for evaluation of lethargy, vomiting and anorexia of 4 days' duration. Physical examination, history and a minimum database led to a diagnosis of immune-mediated hemolytic anemia accompanied by marked hyperbilirubinemia. Refractometric protein determination was within the reference interval, whereas the biuret method indicated hypoproteinemia. This discrepancy was attributed to interference of bilirubin and biliverdin with the spectrophotometric read-out of the biuret total protein assay. The albumin concentration, determined by bromcresol green, and refractometric total protein were less affected by this interference.

Evaluation of the in vitro interference of bilirubin and lipids in the measurement of the plasma glutathione reductase activity.

The pre-analytical phase is the most vulnerable to errors, and some of the most common interferents in laboratory routine are bilirubin and lipemia. Therefore, the aim of this study was to evaluate the in vitro interference of bilirubin and lipids in the measurement of the activity of glutathione reductase (GR) in plasma samples. The evaluation of the in vitro interference of bilirubin was performed by addition of bilirubin to a plasma pool at the following final concentrations: 0.9, 1.9, 3.8, 7.5, 15, and 30 mg/dL. The turbidity of lipemia was simulated by the addition of Intralipid to the plasma pool at the following final concentrations: 0.67, 1.25, 2.5, 5, and 10 mg/dL. GR activity was measured on a Cobas MIRA automated analyzer. Plasma GR activity was significantly affected by bilirubin and lipids. At the concentrations of 0.9 to 30 mg/dL of bilirubin added, the decrease of GR activity ranged between 22.9 to 45.4%. At the concentrations of 0.67 to 10 mg/dL of Intralipid added, the decrease of GR activity ranged between 22.4 to 36.5%. The addition of bilirubin and lipids in plasma samples interferes negatively in the measurement of GR activity, since GR values are reduced in the presence of these in vitro interferents.

Does bilirubin interfere with capillary electrophoresis of serum proteins?

Capillary electrophoresis of serum proteins is a fast, reliable and simple technique, but many interference exist. The objective of our work is to study the interference of bilirubin on this technique; 70 icteric sera were analysed on Capillarys ™ (Sebia). A second electrophoresis was performed on 40 samples after bilirubin photodegradation. The bilirubin and serum proteins were determinated respectively by Jendrassik and Grof and biuret methods on Konélab 20i ™ (Thermo Electron Corporation). We found abnormal spreading of the albumin fraction of the anode side wich constitute sometimes an isolated fraction in the traditional area of pre-albumin migration. This fraction varies from 2.0 ± 2.0% (0.0 to 7.3%) or 0.98 ± 1.53 g/L (0 to 5.3 g/L) and it seems to be related to the direct bilirubin since, following overloading sera with a solution of bilirubin, no further fraction was recovered. An average decrease of bilirubin after photodegradation of 58 ± 17% (26-89%) is followed by a decrease in the same order 64 ± 38% (10-100%) of the additional fraction. Acetate cellulose electrophoresis of the same samples showed no variation. The high bilirubin levels seem modify slightly the electrophoretic profile. However the impact of the interference on the interpretation of electrophoretic trace is negligible.

Interferencia de la bilirrubina en la medición del paracetamol por el método del indofenol en el analizador Dimension® EXL™

BackgroundBlood levels of acetaminophen (paracetamol) are measured routinely in large acetaminophen overdoses or when poisoning is suspected, in order to predict the likelihood of hepatotoxicity. However some cases of spurious acetaminophen levels have been reported in jaundiced patients. Material and methodsThe interference of bilirubin in acetaminophen measurements was studied on the Dimension® EXL™ by preparing a primary mixture of bilirubin at 800 mg/dL, which was spiked to a quality control solution with high acetaminophen concentration in order to get a bilirrubin concentration of 40 mg/dl on the mixture. Acetaminophen concentration was measured in the mixture with bilirubin and in reference solution without bilirubin. Interference was assessed and quantified by diluting the bilirubin solution to achieve final concentrations of 20, 10, 5, 2.5, 1.25 and 0.625mg/dl (342, 171, 85.5, 42.75, 21.37 and 19.69μmol/l). A graph of the interference was obtained showing the bias of acetaminophen concentrations with reference solution vs. increasing bilirubin concentrations. ResultsAcetaminophen concentrations in both solutions followed Gaussian distributions. The t test showed significant negative interference at bilirubin concentrations higher to 5mg/dl (85.5μmol/l), which means that blood acetaminophen concentrations must be interpreted with caution in cases of hyperbilirubinemia.