Correction of patient results for Beckman Coulter LX-20 assays affected by interference due to hemoglobin, bilirubin or lipids: a practical approach.

The influence of interference by hemolysis, icterus and lipemia on the results of routine chemistries may lead to wrong interpretations. On Synchron LX-20 instruments (Beckman Coulter) serum or plasma indices can be used as reliable semi-quantitative measures of the magnitude of such interference. In an article recently published in this journal, we presented the results of a multicenter study carried out in Dutch hospitals in which we determined cutoff indices for analytes above which analytically significant interference exists. Clinically significant interference cutoff indices were also derived for these analytes. In this article, we describe the handling of patient samples with clinically significant interference by hemolysis, icterus or lipemia. We investigated several possible approaches for correction of the result: dilution of the interference; mathematical correction in the case of hemolysis; treatment with ferrocyanide to destroy bilirubin; and removal of lipids in lipemic patient samples. We concluded, that mathematical correction of potassium or lactate dehydrogenase results in hemolytic samples can only be carried out if intravascular hemolysis is ruled out. Hemoglobin quantification in serial patient samples, combined with measurement of haptoglobin, represents a useful tool to rule out in vivo hemolysis. We derived an algorithm for this situation. We do not simply recommend mathematical correction, unless it is clinically acceptable. We present formulas for potassium and lactate dehydrogenase: corrected potassium=measured potassium-(hemolytic index increment x 0.14); corrected lactate dehydrogenase=measured lactate dehydrogenase-(hemolytic index increment x 75). The dilution studies indicated that dilution is only applicable for bilirubin, C-reactive protein and iron. The results of treatment with ferrocyanide were poor, and we do not recommend this method. Removal of lipids using high-speed centrifugation or LipoClear (StatSpin Inc.), a non-toxic and non-ionic polymer, is a very effective approach, although C-reactive protein, creatine kinase-MB (CK-MB) and cholesterol cannot be removed using LipoClear. For all interferants (hemoglobin, bilirubin, lipids), relatively simple algorithms are derived that can easily be implemented in the clinical laboratory.     

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

BackgroundPre-analytical error accounts for major total laboratory errors. We assessed the impacts of hemolysis, icterus, and lipemia on laboratory tests on Roche Cobas 6000.MethodsVarious 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.ResultsFor most of the chemistry assays, our data were in a good agreement with Roche package inserts. However some assays had significant 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.ConclusionsWe 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.     

胆红素和血红蛋白对不同尿白蛋白检测方法结果干扰的研究

目的探讨尿中胆红素(Bil)和血红蛋白(Hb)在不同检测方法中对尿白蛋白检测结果的干扰。方法分别以Bil和Hb作为干扰物,对3种不同检测方法(免疫透射比浊法、免疫散射比浊法和免疫胶体金法)进行尿白蛋白检测的干扰试验。结果不同浓度的Bil和Hb对3种尿白蛋白检测方法的结果均会产生影响,且干扰率会随干扰物浓度的增高而增加。Hb对3种检测方法主要呈负干扰;Bil对免疫透射比浊法和免疫散射比浊法检测结果呈正干扰,而对免疫胶体金法检测结果呈负干扰。当尿液中Hb浓度相当于尿隐血"1+"时,Hb对3种检测方法的干扰率绝对值为6.72%～17.43%;当尿液中Bil浓度相当于Bil"1+"时,3种检测方法的干扰率绝对值为7.89%～22.94%。结论 Bil和Hb均会对尿白蛋白的检测结果造成干扰,在检测可能含有Bil和Hb患者尿液的尿白蛋白时,应考虑这些干扰因素对结果的影响。     

Characterization of bilirubin interference in three commonly used digoxin assays

BackgroundDue to the narrow therapeutic range of digoxin, determining serum/plasma digoxin concentrations is critical for assessing patients with congestive heart failure, atrial fibrillation, and certain types of arrhythmias. However, digoxin quantification by competitive immunoassays is susceptible to interferences that may alter the accuracy of its measurement in patient plasma. This study aimed to characterize the extent of bilirubin interference in three commonly used digoxin immunoassays.MethodsDigoxin concentrations were compared using the Beckman Coulter® Unicel DxI 800, the Vitros® 4600, and the Roche Cobas® 8000 in neat or digoxin-spiked icteric and non-icetric plasma samples. A mixing study was performed to demonstrate how digoxin quantification is affected by bilirubin. An equation was derived that predicts the response of the DxI 800, given known bilirubin and digoxin concentrations.ResultsThe DxI reported detectable concentrations of digoxin in high bilirubin samples with no added digoxin, while the Vitros® 4600 and Cobas® 8000 gave virtually undetectable results. Spiking digoxin into samples with elevated bilirubin concentrations resulted in a higher percent recovery for the DxI 800 when compared to the other two platforms. The mixing study also revealed an increase in the percent recovery in the DxI 800, while the Vitros® 4600 and Cobas® 8000 were comparable to the expected concentration of digoxin.ConclusionsThe DxI 800 is most prone to interference by bilirubin, while the Vitros® 4600 and Cobas® 8000 are relatively unaffected. Icteric samples should be interpreted with caution if digoxin quantification is needed, especially on the DxI 800 assay.     

Multicenter evaluation of five assays for myoglobin determination

BACKGROUND: Lacking assay standardization, different myoglobin methods may produce results that differ significantly. METHODS: A multicenter study was carried out to compare the analytical performance of five commercially available assays for myoglobin measurement. Linearity, imprecision, interferences, and method comparison were studied according to NCCLS guidelines, whereas reference values were determined following IFCC recommendations. RESULTS: The BNA and Opus showed relatively high imprecision (all but one total CV >7.4%). Other assays showed lower CVs, but they varied among laboratories, particularly at a normal myoglobin concentration (Access, 6.0-11%; Hitachi, 3.8-5.8%; Stratus, 3.4-6.5%). Results were lower in anticoagulated samples on the Access, in heparin and citrate samples on the Stratus, and in citrate samples on the BNA and Opus, and increased in heparin and EDTA samples on the Hitachi. Use of separator gel produced results significantly lower (P <0.001) on the Hitachi and higher (P = 0.016) on the Opus. Bilirubin, turbidity, and hemoglobin had no effect on evaluated methods, but rheumatoid factor affected the Access. In method comparisons, high correlation coefficients (>/=0.98) were obtained. The Stratus gave higher results; however, the Access and BNA gave the lowest. The following upper reference limits (microgram/L) for men and women, respectively, were obtained: Access, 70 and 52; BNA, 51 and 49; Hitachi, 67 and 58; Opus, 80 and 50; and Stratus, 86 and 63. CONCLUSION: The possibility of high imprecision and marked disagreement among commercial myoglobin assays should be carefully considered in clinical practice.     

变异血红蛋白和氨基甲酰血红蛋白对糖化血红蛋白测定干扰的评价

目的评价变异血红蛋白和血红蛋白衍生物氨基甲酰血红蛋白对离子交换-高效液相色谱(HPLC)系统和用酶法检测糖化血红蛋白(HbAlc)的影响。方法分别用2种方法同时检测无尿毒症糖尿病患者血红蛋白结构正常的血液样本和含有不同浓度胎儿血红蛋白(HbF)的血液样本及尿毒症患者HbA1c浓度。结果对于血红蛋白结构正常且无尿毒症的糖尿病患者,2种方法的HbA1c检测结果差异无统计学意义(P>0.05)。当HbF<8.75%时,离子交换-HPLC的HbA1c测定结果与预期值无差异;当样本中HbF浓度为8.8%～23.0%时,其HbA1c测定结果要明显低于理论浓度;当HbF浓度达35.0%～70.0%时,离子交换-HPLC无法检测出结果。因尿毒症患者血液中含氨基甲酰血红蛋白,所以离子交换-HPLC的HbA1c检测结果明显高于酶法;而酶法检测HbA1c不受血液中HbF和氨基甲酰血红蛋白的干扰。结论采用离子交换-HPLC检测含HbF和氨基甲酰血红蛋白的样本中的HbA1c时,结果可能受到干扰;而酶法检测HbA1c时几乎不受样本中HbF和氨基甲酰血红蛋白的干扰。     

血红蛋白和胆红素干扰临床化学分析的机理初探

目的　探讨溶血和黄疸对临床化学分析方法的干扰机制。方法　用紫外 可见分光光度计和全自动生化分析仪观察血红蛋白和胆红素的光谱学行为 ;用NCCLSEP7 P方案评价血红蛋白和胆红素的干扰情况。结果　血红蛋白和胆红素自身在溶液中发生吸光特性的改变 ,双波长不能校正二者所造成的干扰。血红蛋白的光谱学干扰方向与EP7 P结果一致 ,可用血清平行空白校正 ;而胆红素的光谱学干扰方向与EP7 P结果不一致 ,并且不能用平行血清空白校正。结论　血红蛋白和胆红素对临床化学的干扰有不同机制 ,临床生化分析除了正确选择纠正方案外 ,有必要报告血清指数以指导临床医生正确诊断     

Multicenter evaluation of assays for estradiol and progesterone in saliva

Blood samples can be difficult to obtain in studies involving serial sampling, especially in developing countries where there may also be logistic, ethical, and cultural constraints that make frequent blood collection impractical. Assays for steroids in saliva may avoid some of these difficulties. A multicenter study involving laboratories in five countries was carried out to compare the results of assays for salivary estradiol and progesterone performed with centrally provided reagents and assay protocols. Concentrations of salivary steroid as obtained by all but one center were comparable with those reported in the literature. We conclude that assays of hormones in saliva are useful adjuncts to those performed on other body fluids.     

Evaluation of hemoglobin interference in capillary heel-stick samples collected for determination of neonatal bilirubin

OBJECTIVE: In this study, we determined the assay performance criteria necessary to produce acceptable results for >or=98% of neonate bilirubin samples collected by capillary heel-stick. STUDY DESIGN AND METHODS: We determined serum free hemoglobin levels in 151 heel-stick serum samples to determine the hemolysis level. We then tested the effect of hemolysis on total bilirubin levels determined by four commercially available assays. RESULTS: The mean level of serum free hemoglobin was 1.62 g/L. Of the serum total bilirubin assays tested, the Total Bilirubin Special (Roche Diagnostics) and the TBILI (Roche Diagnostics) reagents did not show significant interference at the concentrations of free hemoglobin observed in >or=98% of heel-stick samples. The Vitros Bu/Bc slide (Ortho-Clinical Diagnostics) showed significant interference only at normal bilirubin concentrations; while the Bilirubin DPD reagent (Amresco Inc.) showed significant interference starting at hemoglobin concentrations of 1.0 g/L. CONCLUSIONS: Bilirubin assays that are not sensitive to approximately 6 g/L free hemoglobin should provide accurate results for most samples obtained via capillary heel-stick. Of the four assays tested, the Bilirubin DPD reagent (Amresco Inc.) was the most susceptible to the presence of free hemoglobin and will result in a higher rejection rate of neonate capillary heel-stick samples.     

Analytical performance of the Synchron LX20 Pro, BN trade mark II and IMMAGE high sensitivity C-reactive protein assays and concordance in cardiovascular risk stratification

BACKGROUND: C-reactive protein (CRP) can provide valuable prognostic information for risk of cardiovascular events. Several automated high sensitivity CRP immunoassays are currently available for risk assessment. METHODS: The analytical performance of the Synchron LX20 Pro, BN II and IMMAGE high sensitivity CRP assays were evaluated and concordance within cardiovascular risk tertiles was examined for 529 serum samples. RESULTS: All three assays exhibited satisfactory between-run imprecision based on CVs< or =9.0% over a wide range of CRP concentrations. The LX20 Pro and BN II were linear over an extensive measuring range, whereas the IMMAGE exhibited a slight deviation from linearity producing results with a positive bias at CRP levels between 0.7 and 2.6 mg/l. Moderately hemolyzed samples interfered with the LX20 Pro and IMMAGE CRP assays, whereas moderate lipemia interfered with the BN II. Correlation studies revealed that the LX20 Pro and IMMAGE produced results 8.2% lower and 5.1% lower, respectively, compared with the BN II. There was good agreement among methods for cardiovascular risk assessment. CONCLUSIONS: All three CRP assays exhibited acceptable analytical performance for cardiovascular risk assessment. Although results by the LX20 Pro and IMMAGE were lower than the BN II, there was good agreement within each cardiovascular risk assessment tertile.     

Negative interference of bilirubin and hemoglobin in the MEIA troponin I assay but not in the MEIA CK-MB assay

Troponin I is a sensitive and specific marker for the diagnosis of myocardial infarction. Several commercially available immunoassays measure the concentration of troponin I in serum. The microparticle enzyme immunoassay (MEIA) for troponin I (Abbott Laboratories, Abbott Park, IL) is widely used in clinical laboratories, including our hospital laboratory. We studied the effect of bilirubin and hemolysis on the MEIA for troponin I and compared our assay with a newly available chemiluminescent assay (CLIA) for troponin I (Bayer Diagnostics, Tarrytown, NY). We also measured CK-MB concentration using the MEIA CK-MB assay. One serum pool was prepared by combining several specimens of one patient with elevated troponin I and with a diagnosis of myocardial infarction. Other serum pools were prepared by combining sera with similar troponin I values. All serum pools showed normal bilirubin concentrations and had no hemolysis. Then we supplemented aliquots of serum pools with various concentrations of bilirubin (5.0, 10.0, 15.0, and 20.0 mg/dL). After supplementation, troponin I concentrations were measured again using the MEIA and CLIA. We observed a statistically significant decrease in troponin I concentration in the presence of bilirubin with the MEIA. For example, in serum pool 1, the troponin I concentration was 16.3 (bilirubin: 0.8 mg/dL). In the presence of 5.0, 10.0, 15.0 and 20.0 mg/dL of added bilirubin, the cardiac troponin I concentrations were 13.9, 13.4, 13.3 and 13.0 ng/ml respectively. We observed similar negative interference of bilirubin in troponin I measurement by the MEIA in other pools. The troponin I value decreased slightly (not statistically significant) in one pool and did not change in two other pools in the presence of bilirubin when we measured troponin I concentration using the CLIA. Interestingly, bilirubin did not interfere with the MEIA CK-MB assay. Moderate hemolysis did not have any effect on the troponin I assay using either the MEIA or CLIA. However, gross hemolysis (hemoglobin > 40 mg/dL) interfered with both assays for troponin I.     

Prevention of hemoglobin interference on the formazan reaction

BACKGROUND: Hb interference induces false test results and thus hinders the establishment of further applications that use the formazan method. We attempted to eliminate hemoglobin (Hb) interference on the formazan reaction. METHODS: Total serum bile acid (TBA) reagent was constructed to elucidate the mechanism of Hb interference. We determined the interaction between formazan reagents and Hb using spectral analysis and investigated the optimal condition of the reagent to establish a practical technique for its prevention. RESULTS: Hb caused overestimation during TBA measurement, and its influence was attributed to the errors of both the sample and reagent blank signals. These errors were triggered by the reaction between Hb and formazan reagents. The main finding was that the addition of both imidazole and sodium nitrite in the reagent considerably accelerated the oxidation of Hb, even at neutral pH. The overestimation of TBA concentration was reduced to <3 micromol/l even with Hb concentrations >or=500 mg/dl. CONCLUSIONS: We elucidated the mechanism of Hb interference on the formazan reaction and succeeded in preventing its influence. This will help to solve the technical difficulties associated with utilizing formazan reactions as routine diagnostic tools.     

Performance evaluation of automated assays for beta-CrossLaps, N-MID-Osteocalcin and intact parathyroid hormone (BIOROSE Multicenter Study).

INTRODUCTION: Biochemical markers of bone metabolism have been mainly determined manually until now and the precision and accuracy of these methods have not always been satisfactory. This has been shown in several external quality assessment schemes (EQAS). OBJECTIVE AND STUDY DESIGN: A study named BIOROSE was undertaken to evaluate new automated assays for serum markers of bone metabolism. The main focus was to evaluate the assay performance in a multicenter setting with 20 laboratories participating in Germany. The evaluation consists of a familiarization phase to determine precision and accuracy and an EQAS to evaluate the comparability between laboratories. MATERIALS: The parameters beta-CrossLaps (CTX), N-MID-Osteocalcin (OC) and intact parathyroid hormone (PTH) were measured with reagents including calibrators and control sera obtained from Roche Diagnostics, Mannheim, Germany, with electrochemiluminescence immunoassays (ECLIA) on the automated analyzer Elecsys 2010. RESULTS: We calculated for the control samples, PCB 1-3, the mean and median values from the measured values of all participating laboratories and used these as target values. From these target values, a recovery range for the participating laboratories was calculated for beta-CrossLaps, OC and intact PTH of better than 80-126% for PCB 2 and PCB 3, and for PCB 1 (low concentration range) for beta-CrossLaps 79-129%, OC 90-120% and intact PTH 78-126%. The between-day imprecision was 2.4-7.2% for beta-CrossLaps, 1.1-5.9% for OC and 1.7-5.5% for intact PTH in the elevated range (sample PCB 2). In the EQAS, the inter-laboratory imprecision for beta-CrossLaps in the sample with a value of 0.8 ng/ml (above the upper limit of normal, which is 0.6 ng/ml) was 9.8% on day 1 and 9.7% on day 2. CONCLUSION: The performance evaluation of automated assays for beta-CrossLaps, N-MID-Osteocalcin and intact parathyroid hormone in the BIOROSE multicenter study showed that the participating laboratories had no problems in setting up these methods and they yielded results for precision and accuracy that are superior to results achieved in external quality assessment schemes for manually performed methods. In addition, at the clinically important decision level of the upper limit of the normal range, all three tested analytes gave precise results that improved medical decisions.     

Multicenter evaluation of the first automated Elecsys sFlt-1 and PlGF assays in normal pregnancies and preeclampsia

ObjectivesPerformance evaluation of Elecsys® sFlt-1 and PlGF assays.Design and methodsWithin-, between-run, total imprecision, functional sensitivity, inter-laboratory comparison, method comparison and lot-to-lot reproducibility were evaluated.ResultsWithin- and between-run CVs were below 4% for sFlt-1 > 60 and PlGF > 20 pg/mL. Total imprecision CVs were below 4.3%. Functional sensitivity was < 5 pg/mL. Inter-laboratory CVs were < 5%. Elecsys correlated well with Quantikine VEGF-R1 (r = 0.960) and PlGF (r = 0.968). Lot-to-lot comparisons yielded highly correlated results (r > 0.999).In healthy pregnancies, the median levels of sFlt-1 remained constant in first (1107 pg/mL) and second trimesters (1437 pg/mL) but increased in the third trimester (2395 pg/mL), while median PlGF levels increased in the first (30 pg/mL) and second trimesters (279 pg/mL) and peaked at 29 to 32 weeks (626 pg/mL) and decreased thereafter (340 pg/mL). The sFlt-1/PlGF ratio is highest in the first trimester (median: 28) but remained constant in the second (median: 4.7) and third trimesters (median: 5.1).In PE/HELPP samples matched for gestational age the sFlt-1 levels were significantly higher (6894–34,624 pg/mL), whereas PlGF levels were lower (9.2–80 pg/mL) and the median sFlt-1/PlGF ratio is much higher (461; range: 121–2614) than in apparently healthy pregnancies (3.6; range: 0.3–105).ConclusionThe new Roche Elecsys sFlt-1 and PlGF immunoassay showed excellent precision and reliability. There was a clear difference in the Elecsys sFlt-1/PlGF ratio between samples obtained from women with apparently normal pregnancy at the time of blood collection and those diagnosed with PE/HELLP at the same age of gestation.