Multicenter evaluation of the interference of hemoglobin, bilirubin and lipids on Synchron LX-20 assays.

The influence of interference by hemolysis, icterus and lipemia on the results of routine chemistries may lead to wrong interpretations. The H-, I- and L-indices that can be measured by the Beckman LX-20 instrument (Beckman Coulter) in serum or plasma samples are a reliable semi-quantitative measure of the size of these interferences. A survey carried out in 16 Dutch clinical laboratories on the use of these indices demonstrated that in several of these laboratories, the influence of interferences is largely underestimated. Therefore, a multicenter study was carried out in which we examined the interference of hemolysis, icterus and lipemia on 32 analytes. On the basis of biological variation, we decided on cutoff indices above which analytically significant interference exists. We found analytically significant interference by hemolysis, icterus or lipemia, in 12, 7 and 15 of the 32 analytes studied, respectively. Flagging of results on the basis of analytically significant interference, however, results in too many clinically insignificant comments. On the basis of clinical significance, we conclude that significant interference by hemolysis, icterus or lipemia is present in only 5, 6 and 12 of the analytes studied, respectively. Use of the cutoff indices presented here facilitates optimal use of the LX-20 indices to prevent reporting of wrong results due to interference.     

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.     

Hemoglobin interference from in vivo hemolysis

Laboratory values for specimens from a case of intravascular hemolysis showed that hemoglobin was significantly increased and thus could interfere with the determination of other analytes. We studied this problem by adding increasing amounts of purified hemoglobin (to a maximum concentration of 19.3 mg/L) to aliquots of pooled serum samples. The hemoglobin significantly interfered with the determination of only five analytes: albumin, aspartate aminotransferase, direct bilirubin, and total protein on the SMAC, and creatinine on the Astra. We propose that for cases of proven intravascular hemolysis, values for only the analytes not affected by hemoglobin should be reported. We find lactate dehydrogenase activity useful in assessing the components of in vivo hemolysis; the differences between serum and plasma values for potassium, lactate dehydrogenase, and hemoglobin are related to in vitro hemolysis. Criteria for specimen collection and assessment of type of hemolysis are proposed.     

依据CLSI EP07-A3文件评价血清指数对免疫散射比浊法检测前白蛋白的干扰

目的 探讨溶血、黄疸、脂浊对免疫散射比浊法检测前白蛋白(PA)的干扰效果.方法 以新鲜血清样本作为基础样本,添加干扰物[血红蛋白(Hb)、胆红素及脂质]后,采用免疫散射比浊法检测PA水平.参考美国临床实验室标准化协会(CLSI)EP07-A3文件进行配对差异实验、剂量效应实验.结果 10 g/L Hb(溶血指数为100...     

A component could not be larger than the whole,polyclonal immunoglobulin interference in conjugated/direct bilirubin assay and elimination: A case report

Analytical interferences (hemolysis, icterus, paraproteinemia, and lipemia) are of great concern in laboratory tests. In our case report, the conjugated bilirubin result of a patient was significantly higher than the total bilirubin. We recommend being more cautious with samples that produce indistinguishable results and attempting to resolve the issues before releasing them.     

Unreliable visual estimation of the incidence and amount of turbidity, hemolysis, and icterus in serum from hospitalized patients

We examined the frequency of occurrence for turbidity, hemolysis, or icterus in 2599 serum samples submitted for chemistry testing in an acute-care general hospital. Each specimen was compared visually with full-color photographs of adulterated serum, and designated as either "0" (containing no interferent), or trace, 1+, 2+, 3+, 4+, or 5+. Visible interferents (1+ or greater) were thought to be present in 838 (31%) of the specimens (icterus, 525; hemolysis, 244; lipemia, 69). To assess the accuracy of such visual grading, we determined the concentration of triglycerides, hemoglobin, or bilirubin in the specimens considered to be contaminated. There was little agreement between the actual concentration of each interferent and the assigned grade of turbidity, hemolysis, or icterus, confirming the unreliability of human visual estimation of these potentially interfering substances.     

Influence of hemolysis on clinical chemistry parameters determined with Beckman Coulter tests – detection of clinically significant interference

The aim of this study was to examine the influence of hemolysis on 25 clinical chemistry parameters and to compare the resulting bias with clinically significant differences and the manufacturer’s specifications. Using freeze-thawing of the treated blood aliquot of each subject (N?=?17), four hemolysis levels were prepared with hemolysis index (HI) and hemoglobin concentration as follows: (+)=0.5–0.99?g/L, (2+)=1–1.99?g/L, (3+)=2–2.99?g/L and (4+)=3–4.99?g/L. All analytes were tested on the Beckman Coulter AU480 analyzer using proprietary reagents. It was considered that the interference was detected if the 95% confidence interval for mean differences (%) between hemolyzed and non-hemolyzed samples did not include zero. Clinically significant interference was judged against reference change value (RCV). Hemolysis interference was detected for: alpha-amylase, alkaline phosphatase (ALP), aspartate aminotransferase (AST), total and conjugated bilirubin, creatine kinase (CK), CK-MB, ?-glutamyltransferase (GGT), iron, lactate dehydrogenase (LD), magnesium, potassium, total protein and uric acid at HI=(1+); alanine aminotransferase (ALT) and phosphate at HI=(2+); urea at HI=(3+); albumin and cholinesterase at HI=(4+). Even at the greatest hemolysis degree, HI=(4+), no interference was detected for calcium, chloride, creatinine, C-reactive protein (CRP), glucose and sodium. Clinically significant difference was exceeded for LD at HI=(1+); CK-MB at HI=(2+); AST and potassium at HI=(3+); total bilirubin at HI=(4+). The presented results did not support the manufacturer’s claim for CK and GGT. Establishing HI thresholds for reporting or suppressing test results is the responsibility of each laboratory, taking into account the manufacturer’s data, but also its own investigations.     

Hemolytic anemia

Hemolysis presents as acute or chronic anemia, reticulocytosis, or jaundice. The diagnosis is established by reticulocytosis, increased unconjugated bilirubin and lactate dehydrogenase, decreased haptoglobin, and peripheral blood smear findings. Premature destruction of erythrocytes occurs intravascularly or extravascularly. The etiologies of hemolysis often are categorized as acquired or hereditary. Common acquired causes of hemolytic anemia are autoimmunity, microangiopathy, and infection. Immune-mediated hemolysis, caused by antierythrocyte antibodies, can be secondary to malignancies, autoimmune disorders, drugs, and transfusion reactions. Microangiopathic hemolytic anemia occurs when the red cell membrane is damaged in circulation, leading to intravascular hemolysis and the appearance of schistocytes. Infectious agents such as malaria and babesiosis invade red blood cells. Disorders of red blood cell enzymes, membranes, and hemoglobin cause hereditary hemolytic anemias. Glucose-6-phosphate dehydrogenase deficiency leads to hemolysis in the presence of oxidative stress. Hereditary spherocytosis is characterized by spherocytes, a family history, and a negative direct antiglobulin test. Sickle cell anemia and thalassemia are hemoglobinopathies characterized by chronic hemolysis.     

稀释法在消除溶血对凝血检测干扰中的应用

目的探讨稀释法消除溶血对凝血检测结果影响的可行性。方法选取外观正常凝血标本65例,取35例标本血浆2倍稀释后与未稀释血浆检测凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)值,统计学线性分析并建立直线回归方程。另30例标本注射器反复抽吸使其溶血后,溶血前后凝血检测结果行统计学分析;并对溶血后标本2倍稀释,稀释后结果通过回归方程换算出的结果与溶血前原检测结果进行比较。结果标本稀释前后血浆的PT和APTT两项指标检测值具有线性相关特征。溶血前后两项指标测定结果显示:溶血后测定值高于溶血前,差异有统计学意义(P<0.01)。溶血标本2倍稀释后两项指标检测值经过回归方程换算后所得数据与未溶血的原检测数据相比并无显著统计学差异(P>0.05)。结论临床上对于溶血标本PT和APTT的检测,采用稀释法后能较好消除溶血对检测结果造成的影响。     

A multicenter evaluation of the Boehringer Mannheim/Hitachi 717 system

Analytical performance of the Boehringer Mannheim/Hitachi 717 system was evaluated in a multicenter study involving seven different laboratories. Fifty-five methods including end point chemistries, enzymes, ISE, TDM, DAU, and specific protein assays were assessed over a 7 month period. Methods on the analyzer exhibited excellent precision with CVs less than 2% for within run precision, and CVs less than 3% for between day precision for most analytes; linearity, which met or exceeded manufacturer's claims; minimal sample and reagent carryover, and no significant interference from hemolysis; icterus; and lipemia. Recovery of the assigned value for 10 analytes in SRM 909 was acceptable. Comparison of methods with other BM/Hitachi analyzers resulted in slopes close to unity (0.93-1.06); comparison to other clinical chemistry analyzers yielded slopes of 0.88-1.07. Excellent performance and diverse method applications make the BM/Hitachi 717 analyzer a suitable instrument for work station consolidation.     

Hemolysis, lipaemia and icterus in specimens for arterial blood gas analysis

ObjectiveTo assess prevalence of interference from hemolysis, lipaemia, icterus in arterial blood gas analysis (ABG).DesignSerum indices (SI) were assessed in the plasma of ABG samples over a 2-month period.ResultsOut of a total of 478 ABG specimens, we identified 17 hemolyzed samples (4%), 52 (11%) with lipaemia, and 63 (13%) with icterus.ConclusionTest results on a considerable number of ABG specimens might be unreliable due to presence of interference.     

内源性干扰物质对酶法测定钾钠氯结果的影响

目的评价黄疸、溶血、脂血、尿毒症等异常标本中内源性干扰物质对酶法测定血清中钾钠氯结果的影响。方法依据美国国家临床实验室标准化委员会(NCCLS)的EP 7-P文件,分别用间接离子选择性电极(ISE)和酶法测定正常对照组(非黄疸、溶血、脂血)、黄疸组、溶血组、脂血组、尿毒症组标本中钾钠氯,对结果进行统计学分析。结果正常对照组,酶法钠氯均高于ISE(P<0.05),钾在两种方法间无差异;溶血、黄疸对酶法测定钾钠氯均无干扰(P>0.05);脂血(TG<15mm o l/L)对酶法测定钾氯无干扰,钠呈负偏差,但酶法不能测定严重脂血标本(TG>15 mm o l/L)中钾钠氯,脂血标本中钠偏倚与甘油三酯、胆固醇浓度不相关(r=0.06,r=0.10);尿毒症组样品中钾与对照组呈负偏差(P<0.05),与CREA、BUN的浓度间弱相关(r=0.31,r=0.26),而尿毒症组标本中钠氯较正常对照组呈正偏差(P<0.05),与CREA、BUN浓度间无相关性(r=0.07,r=0.05)。结论溶血、黄疸、轻中度脂血对酶法测定钾钠氯无干扰,但不能用于分析严重脂血标本、尿毒症标本的钾钠氯测定。     

Effect of in vitro hemolysis on 25 common biochemical tests.

Clinical chemists frequently encounter hemolyzed samples. Our study examines the effects of hemolysis on the results of 25 common biochemical tests. We collected 60 15-mL blood samples from inpatients and outpatients and mechanically hemolyzed 10 mL of the samples in a two-step procedure. We classified serum from these samples as being nonhemolyzed, moderately hemolyzed, or severely hemolyzed and then performed 25 common biochemical tests. Statistical analysis of the results showed that hemolysis had the greatest effect on the lactate dehydrogenase, acid phosphatase, and potassium tests.     

Microangiopathic hemolytic anemia as the initial manifestation of porcine valve failure

We compare the findings in a patient whose microangiopathic hemolytic anemia was the initial sign of porcine valvular dysfunction, with those of six other patients with porcine valve failure seen at Tripler Army Medical Center and with those of 12 patients with hemolytic anemia and porcine valve failure whose cases have been reported in the literature. Total bilirubin and serum lactic dehydrogenase were directly related to the degree of anemia in patients with porcine valve dysfunction. Echocardiography confirmed valve dysfunction in only four of eight patients, while cardiac catheterization confirmed valve failure in 16 of 17 patients. Laboratory evidence for hemolysis was minimal in 31 patients who had normal, functioning porcine valves. Microangiopathic hemolytic anemia in the presence of a porcine prosthetic heart valve, and in the absence of other causes, warrants a thorough evaluation to rule out valve dysfunction. Routine laboratory screening for hemolysis may aid the early diagnosis of porcine valve failure.     

Influence of hemolysis on routine clinical chemistry testing.

BACKGROUND: Preanalytical factors are the main source of variation in clinical chemistry testing and among the major determinants of preanalytical variability, sample hemolysis can exert a strong influence on result reliability. Hemolytic samples are a rather common and unfavorable occurrence in laboratory practice, as they are often considered unsuitable for routine testing due to biological and analytical interference. However, definitive indications on the analytical and clinical management of hemolyzed specimens are currently lacking. Therefore, the present investigation evaluated the influence of in vitro blood cell lysis on routine clinical chemistry testing. METHODS: Nine aliquots, prepared by serial dilutions of homologous hemolyzed samples collected from 12 different subjects and containing a final concentration of serum hemoglobin ranging from 0 to 20.6 g/L, were tested for the most common clinical chemistry analytes. Lysis was achieved by subjecting whole blood to an overnight freeze-thaw cycle. RESULTS: Hemolysis interference appeared to be approximately linearly dependent on the final concentration of blood-cell lysate in the specimen. This generated a consistent trend towards overestimation of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, creatine kinase (CK), iron, lactate dehydrogenase (LDH), lipase, magnesium, phosphorus, potassium and urea, whereas mean values of albumin, alkaline phosphatase (ALP), chloride, gamma-glutamyltransferase (GGT), glucose and sodium were substantially decreased. Clinically meaningful variations of AST, chloride, LDH, potassium and sodium were observed in specimens displaying mild or almost undetectable hemolysis by visual inspection (serum hemoglobin < 0.6 g/L). The rather heterogeneous and unpredictable response to hemolysis observed for several parameters prevented the adoption of reliable statistic corrective measures for results on the basis of the degree of hemolysis. CONCLUSION: If hemolysis and blood cell lysis result from an in vitro cause, we suggest that the most convenient corrective solution might be quantification of free hemoglobin, alerting the clinicians and sample recollection.     

溶血和脂血标本对凝血指标检测的影响因素探讨

目的探讨溶血和脂血标本对凝血指标检测结果的影响,为规范分析前过程提供实验依据。方法随机收集本院I临床溶血标本40例、脂血标本32例,进行血浆血凝测定。以血红蛋白每下降10g／L为标准,分为轻度、中度、重度溶血3个等级,同时测定3次凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、凝血酶时间（TT）和纤维蛋白原（Fbg）;以检测血浆甘油三脂每升高3mmol／L为标准,分为轻度、中度、重度3个等级,同时测定3次PT、APTT、TT、Fbg。对溶血和脂血的干扰因素进行统计学分析。结果轻度溶血组PT、Fbg检测结果的差异具有统计学意义（P〈0．05）;中度和重度溶血组PT、Fbg、TT检测结果的差异具有统计学意义（P〈0．05）。轻度、中度和重度脂血组PT、APTT、Fbg、TT检测结果的差异均具有统计学意义（P〈0．05）。结论（1）临床检测中溶血标本对APTT检测结果影响较小;而对于PT、TT、Fbg干扰较大,建议临床重抽复测。（2）临床检测中脂血标本对PT、APTT、TT、Fbg测定结果有影响,均建议患者禁食12h后重抽复测。     

溶血、脂血、黄疸对同型半胱氨酸测定的影响

目的观察溶血、脂血、黄疸对同型半胱氨酸(homocysteine,Hcy)测定的影响。方法 10例正常血清用血红蛋白液配制成溶血程度为5、10、15、20、25、30g/L的系列血清,10例正常血清用脂肪乳配制成三酰甘油(TG)为2、4、8、16mmol/L的系列血清,10例正常血清用胆红素标准品配制成总胆红素(TBIL)为20、40、80、160μmol/L的系列血清,采用全自动生化分析仪检测上述30例标本Hcy的变化,并与正常血清Hcy水平进行比较。结果溶血程度为5、10、15、20、25、30g/L时,Hcy降低程度依次为0.9%(0.2%～3.4%)、2.3%(0.4%～12.2%)、8.6%(1.8%～29.4%)、66.2%(47.8%～90.0%)、88.0%(82.2%～96.7%)、102.9%(97.7%～116.3%)(此时大多数Hcy检测值为负值)。不同浓度TG和TBIL下的Hcy检测值差异无统计学意义(P>0.05)。结论溶血对Hcy的测定有显著影响,脂血、黄疸对Hcy的测定无明显影响。     

Insufficient filling of vacuum tubes as a cause of microhemolysis and elevated serum lactate dehydrogenase levels. Use of a data-mining technique in evaluation of questionable laboratory test results.

Experienced physicians noted unexpectedly elevated concentrations of lactate dehydrogenase in some patient samples, but quality control specimens showed no bias. To evaluate this problem, we used a "latent reference individual extraction method", designed to obtain reference intervals from a laboratory database by excluding individuals who have abnormal results for basic analytes other than the analyte in question, in this case lactate dehydrogenase. The reference interval derived for the suspected year was 264-530 U/L, while that of the previous year was 248-495 U/L. The only change we found was the introduction of an order entry system, which requests precise sampling volumes rather than complete filling of vacuum tubes. The effect of vacuum persistence was tested using ten freshly drawn blood samples. Compared with complete filling, 1/5 filling resulted in average elevations of lactate dehydrogenase, aspartic aminotransferase, and potassium levels of 8.0%, 3.8%, and 3.4%, respectively (all p<0.01). Microhemolysis was confirmed using a urine stick method. The length of time before centrifugation determined the degree of hemolysis, while vacuum during centrifugation did not affect it. Microhemolysis is the probable cause of the suspected pseudo-elevation noted by the physicians. Data-mining methodology represents a valuable tool for monitoring long-term bias in laboratory results.     

Quantification of nonenzymically glycated albumin and total serum protein by affinity chromatography

We have evaluated an affinity-chromatographic procedure for determination of glycated albumin (GA) and glycated total serum protein (GSP). Recovery of these analytes was inversely related to free glucose concentration, thus necessitating removal of free glucose. For this we used molecular-exclusion chromatography on G-25 Sephadex, or dialysis, the latter procedure resulting in significantly (p less than 0.05) lower concentrations of GSP and GA. Total protein concentration and percent glycation are also inversely related, and so protein concentrations must be standardized before the assay. Within- and between-run CVs for both GSP and GA were less than 6.5 and 18%, respectively, the determination of GA being generally the more precise of the two. Labile glycated fractions, lipemia, icterus, hemolysis, and type of anticoagulant did not affect the results, but assay temperature did. Diabetic subjects showed substantially higher concentrations of GA and GSP than did normal subjects. Because of the life span of these analytes in circulation, their measurement may provide a short-term index of glycemic control.     

Pediatric reference intervals for 28 chemistries and immunoassays on the Roche cobas® 6000 analyzer—A CALIPER pilot study

ObjectiveThe aim of this study was to determine age- and sex-specific pediatric reference intervals for 28 analytes on the Roche cobas® 6000 analyzer.Design and methodsThe study was conducted at the Hospital for Sick Children in Toronto, Canada. Approximately 600 outpatient samples from a pediatric population deemed to be metabolically stable were subdivided into five age classes ranging from 0 to 20 years of age and further partitioned by gender. Reference intervals were established, after removal of samples significantly affected by hemolysis, icterus and lipemia and outlier exclusion, using the Robust statistical method to obtain the 2.5th and 97.5th percentiles.ResultsAge (birth to 20 years of age) and gender-appropriate pediatric reference intervals for 28 analytes are reported.ConclusionsThese reference intervals provide the basis for clinical interpretation of laboratory results using the Roche cobas® 6000 analyzer or related instrumentation/methods, provided adequate reference interval verification studies are performed.