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Clinica Chimica Acta 412 (2011) 1550 – 1553 Contents lists available at ScienceDirect Clinica Chimica Acta

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Clinica Chimica Acta

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Evaluation of the interference of hemoglobin, bilirubin, and lipids on Roche Cobas 6000 assays

Jing Zhang Ji a , b , Qing H. Meng a ,

a Department of Pathology and Laboratory Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, SK, Canada b Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine, Wenzhou Medical College, Wenzhou, China

article info

Article history:

Received 31 March 2011 Received in revised form 27 April 2011 Accepted 28 April 2011 Available online 6 May 2011


Analytical interference




Roche Cobas


Background: Pre-analytical error accounts for major total laboratory errors. We assessed the impacts of hemolysis, icterus, and lipemia on laboratory tests on Roche Cobas 6000. Methods: Various concentrations of hemoglobin, bilirubin, or Intralipid® were added into the plasma to simulate hemolytic, icteric, or lipemic samples. The analytes were then measured on Roche Cobas 6000 and the change of the analyte concentrations was determined. Results: For most of the chemistry assays, our data were in a good agreement with Roche package inserts. However some assays had signi cant interference at lower index values while others were affected at higher index than the Roche package inserts indicated. In addition, we observed the positive interference by hemolysis on ALT, lipase, total protein, potassium, and iron. Negative interference was noted on calcium and CK. Most of the immunoassays were not affected by hemoglobin, bilirubin, and lipids although there were a few exceptions. Several therapeutic drugs were affected either positively or negatively by hemolysis, icterus, or lipemia to a certain extent. Conclusions: We have demonstrated some test interferences which have not been reported previously on the Cobas 6000. The implementation of the cut-off indices on Cobas 6000 would provide more accurate test result reporting.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Analytical interference caused by pre-analytical factors is a signicant source of error in clinical laboratory measurements [1,2]. Analytical interference by hemolysis, bilirubin and lipids with laboratory assays is the most common concern in laboratory medicine. These altered results may lead to repeat tests, incorrect interpretation, wrong diagnosis, and potentially inappropriate inter- vention and unfavorable outcome for the patients [35]. Hemolysis, icterus, and lipemia commonly interfere with spectrophotometric methods with hemolysis being the most common [3,4,6,7]. This interference is mainly caused by components released from the red cells. Although direct spectral interference on chemistry analyzers has been minimized with bichromatic and kinetic analysis, the contents of red cells like potassium and lactate dehydrogenase falsely increase these constituents in plasma or serum. There are other constituents released from red cells that can interfere with test reactions. Finally, analytes can also get diluted in hemolysis [4] . Hemolysis can occur in vivo, but the major problem that clinical laboratories get is that it

Corresponding author at: Department of Pathology and Laboratory Medicine, Royal

University Hospital, University of Saskatchewan, 103 Hospital Drive, Saskatoon, SK, Canada S7N 0W8. Fax: +1 306 655 2193. E-mail address: (Q.H. Meng).

0009-8981/$ see front matter © 2011 Elsevier B.V. All rights reserved. doi: 10.1016/j.cca.2011.04.034

occurs during and after collection of specimens. Bilirubin can interfere with assays spectrally but also chemically in some reactions. Normally, bilirubin concentrations N 35 mmol/l are clinically dened as hyperbilirubinemia, whereas icteric samples have bilirubin con- centrations N 100 mmol/l [3] . A lipemic sample is the result of high concentrations of chylomicrons and very-low density lipoprotein (VLDL). Lipemia may interfere in any assay that is based on the detection of light transmission or scattering or by volume displace- ment [8,9]. The presence of hemoglobin, bilirubin, and lipids in a specimen can cause a positive or negative interference in the measurement result of many analytes [1012]. Depending on the magnitude of this interference, the results may lead to wrong interpretation and inappropriate intervention [5,1315]. Interference on the Beckman system has been well investigated [8,16]. Neverthe- less, the degree of interference varies with the methodology and individual instrumentation and comprehensive interference evalua- tion and published data are not available on Roche Cobas system (Indianapolis, IN).

2. Subjects and methods

To evaluate the impact of an interferent on analytes, the analyte was measured in a sample spiked with increasing concentrations of

J.Z. Ji, Q.H. Meng / Clinica Chimica Acta 412 (2011) 15501553


the interferent, and the relative deviation of the result from the non- spiked baseline value was then calculated.

2.1. Subjects

Pooled lithium heparinized plasma free of interferent was prepared as test sample. Hemolysate was prepared by washing packed red cells (0.9% saline and distilled water) and then freezing, and thawing. Various concentrations of hemolysate were added into the plasma to simulate different degrees of hemolysis. Icteric samples were obtained with the addition of unconjugated bilirubin (Sigma- Aldrich, St. Louis, MO) to specimens to achieve different concentra- tions of bilirubin. Because of poor solubility of unconjugated bilirubin, bilirubin was prepared in a stock solution in dimethyl sulfoxide [6,17]. The proportion of bilirubin in serum or plasma between conjugated and unconjugated is not a signicant factor for interference [3] . Lipemic samples were prepared by adding Intralipid® (20%) (Baxter, Deereld, IL). The analytes were then measured on Roche Cobas 6000 (c501 and e601) and the change of the analyte concentrations was compared with baseline samples.

2.2. Chemistry analyzer

The multi-channel Roche Cobas 6000 analyzer is a fully automated, computer-controlled system designed for the analysis of routine chemistry assays, immunoassays, and therapeutic drugs. The Cobas 6000 uses spectrophotometry to perform kinetic, end-point and non- linear reactions. To a certain extent, the system, similar to most modern analyzers, reduces spectral interference effects by application of two-reagent procedures and bichromatic spectrophotometry. The Cobas 6000 analyzer is able to detect hemolysis, icterus, and lipemia in samples and can generate quantitative index values for the major interfering substances of hemoglobin, bilirubin, and lipids expressed as H-index (hemolysis), I-index (icterus), and L-index (lipemia). The relationship of each HIL index with the SI unit or conventional unit is listed in Table 1.

2.3. Analytes investigated

The following tests were investigated: α1-antitrypsin (A1AT), acetaminophen, α-fetoprotein (AFP), albumin (Alb), alcohol, alkaline phosphatase (ALP), alanine aminotransferase (ALT), ammonia (Amm), aspartate aminotransferase (AST), β2-microglobulin (β2MI), β human chorionic gonadotropin (βHCG), complement C 3 (C 3 ), complement C 4 (C 4 ), calcium (CA), carbamazepine, carcinoembyronic antigen (CEA), ceruloplasmin (Cerulo), cholesterol (Chol), creatine kinase (CK), chloride (Cl), bicarbonate (CO 2 ), cortisol (Cort), creatinine (Creat), high-sensitivity C-reactive protein (hsCRP), direct bilirubin (D. Bili), digoxin, ferrous iron (Fe 2+ ), ferritin (FER), free triiodothy- ronine(FRT3), free thyroxine (FRT4), follicle-stimulating hormone (FSH), gentamicin, γ-glutamyltransferase (GGT), glucose, haptoglobin, high density lipoprotein cholesterol (HDL-C), homocysteine, immu- noglobin A (IgA), immunoglobin G (IgG), immunoglobin M (IgM), potassium (K + ), lactate dehydrogenase (LD), luteinizing hormone (LH), lipase, magnesium (Mg 2+ ), myoglobin (Myo), sodium (Na + ), pre-albumin (PreALB), phenobarbital, phosphorus (Phos), prolactin (PRL), prostate-specic antigen (PSA), phenytoin, rheumatoid factor (RHF), salicylate, total bilirubin (T.Bili), total bilirubin in cord blood

Table 1 Relationship between interferent concentration and the corresponding index.





Conventional units

1 mg/dl


1 (without units)

SI units


0.621 μmol/l

1 mg/dl 17.1 μ mol/l

Lipemic index is determined by the turbidity qualitatively with no units.

Table 2 List of interference on chemistry assays by HIL.


Hemolysis interference at index level

Icterous interference at index level

Lipemic interference at index level

Na +

NS Increased (H 200) NS Decreased (H 130) NS NS

NS NS NS Decreased (I 14) NS NS

NS NS NS NS Increased (L 1000) NS ND NS Decreased (L 150) Decreased (L 50) Decreased (L 179) NS NS Increased (L 50) NS Decreased (L 1200) NS NS Increased (L 233) NS Decreased (L 1200) NS Increased (L 1200) Decreased (L 1200) ND Decreased (L 1200) NS NS Increased (L 50) NS NS Increased (L 500) Increased (L50) NS Decreased (L 180) NS NS

K +


CO 2




Decreased(H200) ND


Decreased (H 235) Increased (H 235) Increased (H 200) Increased (H 40) NS NS NS Increased (H 600) Increased (H 400) NS NS Decreased (H 40) Increased (H 40) Decreased (H 600) NS Decreased (H 118) NS NS Increased (H 41) Increased (H 300) NS Increased (H 400) Increased (H 200) Decreased (H 200) Increased (H 80) ND Increased (H741) Increased (H 50) NS NS

NS NS Increased (I 1) NS NS Decreased (I 7) NS NS Decreased (I 14) Decreased (I 14) ND NA ND Decreased (I 14) NS NS Decreased (I 25) NS NS NS NS NS NS ND NA ND Decreased(I 14) Decreased (I 3)




β 2MI

CA 2+






D. Bili

Fe 2+








Mg 2+










Uric acid


NS Decreased (I 25)

The index value listed indicates that signi cant interference was observed at this level. NA: not applicable, NS: not signicant, ND: not determined.

(TBICB), theophylline, tobramycin, total protein (TP), Troponin T (TnT), thyroid stimulating hormone (TSH), unsaturated iron binding capacity (UIBC), urea, uric acid, vancomycin, vitamin B12 (VB12), and valproic acid.

2.4. Statistical analysis

Identify the measured hemolytic, icteric or lipemic index

that corresponds to the point where the test analyte differs by N 10% from the baseline. The percent change was calculated as:

Interference%= 100 × (measured value true

where the measured value is the apparent analyte concentration in the presence of interferent, and the true value is the analyte concentration in the baseline without any addition of interferent. Signicant interference was de ned when the change of the analyte value exceeded 10% of the baseline value [13,17].

value)/true value,

3. Results

3.1. Interferences of hemoglobin, bilirubin, and lipids on

chemistry assays

The overall interferences of hemoglobin, bilirubin, and lipids on chemistry assays are listed in Table 2. For most of the chemistry assays, our data were in a good agreement with what Roche reagent package inserts indicated. However some assays were affected


J.Z. Ji, Q.H. Meng / Clinica Chimica Acta 412 (2011) 15501553

signicantly at lower indices in our study in comparison with those listed in Roche package inserts. These assays included ceruloplasmin, HDL-C, phosphorous, RHF, Total bilirubin, UIBC, and uric acid. Others were affected by interferents at higher indices such as A1AT, CK, CO 2 , GGT, and haptoglobin than those indicated by Roche. In addition, we observed interferences on some assays which have not been reported by the manufacturer or in the literature. For example, we observed positive interference by hemoglobin on ALT, lipase, total protein, potassium, ferrous iron and negative interference on calcium and CK.

3.2. Interferences of hemoglobin, bilirubin, and lipids on immunoassays

As expected, most of the immunoassays were not affected by hemoglobin, bilirubin, and lipids with only a few exceptions ( Table 3 ). Beta HCG and IgG were negatively interfered by bilirubin at I 13 and I 24, respectively. Free T3 level was falsely increased by bilirubin at I 13 while troponin T level was decreased by hemoglobin.

3.3. Interferences of hemoglobin, bilirubin, and lipids on therapeutic


Signicant interferences on drugs by hemoglobin, bilirubin, and lipids are listed in Table 4 . Acetaminophen levels were falsely increased by hemoglobin and bilirubin. Digoxin, valproic acid, and vancomycin levels were decreased by hemoglobin. Bilirubin falsely increased levels of gentamicin, phenobarbital, and theophylline while it decreased tobramycin levels. Blood levels of phenytoin, salicylate, theophylline, and vancomycin were signicantly decreased by lipids. We also observed that the degree of interference is dependent on the initial drug concentration.

4. Discussion

The interferences of hemolysis, icterus, or lipemia with various analytes on Roche Cobas 6000 analyzer were assessed. Our data indicate that some index values are not fully concordant with those provided by the manufacturer. Moreover, we demonstrate additional assay interference ndings not reported by the manufacturer or published in the literature. The release of hemoglobin and other cellular components in hemolysis can have impacts on laboratory results. We observed the

Table 3 List of interferences on immunoassays by HIL.


Hemolysis interference at index level

Icterus interference at Index level

Lipemic interference at index level



ND Decreased (I 13) ND ND ND ND ND NS Increased (I 13) ND ND NS Decreased (I 24) NS ND ND ND ND







































Decreased (H 290) NS



The index value listed indicates that signi cant interference was observed at this level. NA: not applicable, NS: not signi cant, ND: not determined.

Table 4 List of interferences on drugs by HIL.


Hemolysis interference at index level

Icterous interference at index level

Lipemic interference at index level


Increased (H 40) Inconclusive Decreased (H 100) NS NS NS NS NS NS Decreased (H 80) Decreased (H 600)

Increased (I 40) ND Inconclusive Increased (I 2) Increased (I 5) ND NS Increased (I 2) Decreased (I 3) ND NS

NS NS NS NS NS Decreased (L 112) Decreased (L 100) Decreased (L 50) NS NS Decreased (L 50)









Valproic acid


The index value listed indicates that signi cant interference was observed at this level. NA: not applicable, NS: not signicant, ND: not determined.

changes (positive or negative) of analytes such as potassium, LDH, ammonia, AST, ALT, CK, Fe 2+ , acetaminophen, ethanol, either due to interference by hemoglobin or increased release of the cellular components from erythrocytes during hemolysis [7,1820] . Hemo- globin begins to absorb around 340 nm and thus affects non-kinetic methods based on the absorbance properties of NADH or NADPH [21,22] . Similarly, bilirubin interference arises from its spectral properties and its ability to react chemically with other reagents resulting in decreased analyte values such as creatinine concentra- tions. Although the enzymatic creatinine assay is reportedly to be less interfered by bilirubin, negative interference of creatinine assay by bilirubin was still observed on Roche Cobas 6000. This is consistent with what was observed on Roche Modular system [17] . This interference was presumed to be attributed to the consumption of peroxide in the initial reaction mixture [23]. In lipemic samples, chylomicrons and VLDL particles scatter light and produce turbidity. Thus, lipemia may interfere in any assay that uses the transmission of light as part of the detection scheme [9]. In this study, different lipemia index had positive or negative effect on measured concen- trations of various chemistry analytes such as A1AT, ALT, ammonia, ceruloplasmin, prealbumin, and bilirubin. We did not observe interference of albumin by lipemia as stated in the Roche package insert. For most of the immunoassays analyzed on Cobas, no signicant interference by hemoglobin, bilirubin, or lipid was observed. This is expected since the assay is two monoclonal antibodies directed sandwich chemiluminescent assay with extensive washing and nal measurement by electrical methods. In comparison with the electro- immunoassays, interference was observed for some drugs measured by turbidometric method but there is an improvement from the old version of Cobas [13]. This is likely due to the similarity of the methodology used for drugs and chemistry assays. We have also observed that the degree of interference can be different depending on the initial analyte concentrations in the specimen. It should be apparent to the readers that a constant interference in any assay but measured at either a low or high analyte concentration may result in different percentage changes. It should be noted that the in vitro results may not completely support what would happen in vivo. For example, in vivo hemolysis may be more complicated than the in vitro addition of hemoglobin. In vivo hemolysis results in all the intracellular components released into circulation. However, the incidence of samples with in vivo hemolysis is extremely low compared with the samples that have hemolysis introduced during and after the collection procedure. For simplicity, we used unconjugated bilirubin to mimic icteric samples instead of both conjugated and unconjugated bilirubin [3] . Future studies should be conducted to determine if there is a difference in terms of interference by different bilirubin species. Although

J.Z. Ji, Q.H. Meng / Clinica Chimica Acta 412 (2011) 15501553


Intralipid is the universally acceptable substance used for in vitro interference study, it may not be completely comparable with in vivo lipemic samples [24] . Intralipid is not the same as VLDL and chylomicrons in terms of the spectrum of particle size. Lipemic specimens are far more complex than Intralipid and there is a poor relationship between triglyceride concentration and the lipemic indices of absorbance. Thus, the heterogenous lipemic sample cannot be easily simulated by addition of Intralipid® [9]. Therefore the degree of interference observed may not exactly represent what would occur endogeneously [18]. Nevertheless, these data still provide useful information as to the degree of analyte change in uenced by hemolysis, icterus, and lipemia. Generally, the visual judgment on these factors is variable and not reliable which can affect the interpretation on the degree of interference [3,25,26]. With implementation of automation and high demand for fast turnaround time, it is not practical to inspect the samples visually. We believe the implementation of HIL indices on the Cobas 6000 will greatly improve the accuracy and the quality of the test results. Comments based on our data will be added to results when the interference is clinically signi cant to help clinicians interpret the results appropriately. In conclusion, this study has produced useful information on analyte interference by hemoglobin, bilirubin, and lipids on Roche Cobas 6000. The use of the cut-off indices in the instrument instead of visual inspection will provide more accurate information as to which analyte is to be affected and to what degree. This automatic detection system and corresponding comment on potential interference will reduce error when reporting results.


We thank Ms. Lana Link, Ms. Cheryl Booth, and Ms. Cheryl Brenaut for their technical assistance. We also thank Dr. John Krahn for reviewing the manuscript.


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