Hemolysis Interference Research Articles

A Biophysical Solution to Prevent Hemolysis Interferences

In-vivo and in-vitro hemolysis, membrane rupture of red blood cells (RBCs) and release of their contents into blood plasma, is one of the major critical issues in medicine. For in-vitro blood-based molecular diagnostics, inhibitors (hemoglobin, potassium, mRNA etc.) released from RBCs due to hemolysis during blood plasma separation can cause serious difficulties in quantitative molecular analysis with sensitivity and selectivity. Here we describe an innovative biophysical solution to prevent hemolysis in blood plasma separation devices for precision molecular diagnostics. In order to avoid biochemical interferences, RBCs are exposed to both fluidic drag force and gravitational force after the computation of transition fluid velocity from vertical up-flow to sedimentation was in the range of 1.3 to 2.5 µm/sec in the design of microfluidic separation device. A membrane filter for filtration was positioned on top of a vertical up-flow channel to reduce clogging of RBCs by gravity-assisted cells sedimentation. As a result, separated plasma volume was increased about 4-fold (2.4 µL plasma after 20 min with 38 % hematocrit human whole blood) and hemoglobin concentration in separated plasma was decreased about 90 % due to the prevention of RBCs hemolysis in comparison to a filter-in-bottom configuration. On-chip plasma contains ∼90 % of protein and ∼100 % of nucleic acids compared to off-chip centrifuged plasma, showing comparable target molecules recovery. This simple and reliable blood plasma separation platform can be easily integrated with downstream detection module for sample-to-answer POC diagnostics.

Harmonization of automated hemolysis index assessment and use: Is it possible?

The major source of errors producing unreliable laboratory test results is the pre-analytical phase with hemolysis accounting for approximately half of them and being the leading cause of unsuitable blood specimens. Hemolysis may produce interference in many laboratory tests by a variety of biological and analytical mechanisms. Consequently, laboratories need to systematically detect and reliably quantify hemolysis in every collected sample by means of objective and consistent technical tools that assess sample integrity. This is currently done by automated estimation of hemolysis index (HI), available on almost all clinical chemistry platforms, making the hemolysis detection reliable and reportable patient test results more accurate. Despite these advantages, a degree of variability still affects the HI estimate and more efforts should be placed on harmonization of this index. The harmonization of HI results from different analytical systems should be the immediate goal, but the scope of harmonization should go beyond analytical steps to include other aspects, such as HI decision thresholds, criteria for result interpretation and application in clinical practice as well as report formats. With regard to this, relevant issues to overcome remain the objective definition of a maximum allowable bias for hemolysis interference based on the clinical application of the measurements and the management of unsuitable samples. Particularly, for the latter a recommended harmonized approach is required when not reporting numerical results of unsuitable samples with significantly increased HI and replacing the test result with a specific comment highlighting hemolysis of the sample.

Hemolysis reflex rules based on the evaluation of hemolysis interference on Abbott ARCHITECT Ci8200 assays

Hemolysis reflex rules based on the evaluation of hemolysis interference on Abbott ARCHITECT Ci8200 assays

Influence of mechanical hemolysis of blood on two D-dimer immunoassays

Although there is broad information about the influence of spurious hemolysis on several laboratory tests, less is known on the bias produced on D-dimer testing. Four different pools were obtained from primary blood tubes, and each of them was divided into four aliquots. The first nonhemolyzed was centrifuged, the plasma was separated and then tested for hemolysis index and D-dimer. The second (hemolyzed aliquot A), third (hemolyzed aliquot B) and fourth (hemolyzed aliquot C) aliquots were mechanically hemolyzed by aspirating whole blood one, two and three times through a fine needle. The plasma was then separated and tested for hemolysis index and D-dimer. D-dimer was quantified by HemosIL AcuStar D-dimer and HemosIL D-dimer HS for ACL TOP. Undetectable hemolysis was present in aliquot nonhemolyzed (<0.5 g/l), whereas the concentration of cell-free hemoglobin significantly increased from hemolyzed aliquot A (5.5-7.0 g/l hemoglobin) to hemolyzed aliquot B (11.5 and 15.0 g/l hemoglobin) and hemolyzed aliquot C (20-22 g/l hemoglobin). The plasma concentration of D-dimer decreased from aliquots nonhemolyzed to hemolyzed aliquot C, achieving clinical significance fromhemolyzed aliquot A and hemolyzed aliquot B when measured with D-dimer HS for ACL TOP and AcuStar D-dimer, respectively. The decrease with AcuStar D-dimer was -5 ± 3% in hemolyzed aliquot A, -7 ± 3% in hemolyzed aliquot B, and -9 ± 3% in hemolyzed aliquot C, whereas the decrease with D-dimer HS for ACL TOP was -5 ± 3% in hemolyzed aliquot A, -8 ± 3% in hemolyzed aliquot B and -9 ± 3% in hemolyzed aliquot C. The similar trend towards decreasing values observed when measuring D-dimer with chemiluminescent and turbidimetric immmunoassays on four heterogeneous plasma pools suggest that the hemolysis interference is more likely to be biological than analytical. The modest bias observed in samples with frank hemolysis (i.e. cell-free hemoglobin of 11.5 g/l) confirms that both methods are robust against this type of interference, so that test results might be released in the majority of mildly hemolyzed samples.

A method for determining the concentration of haptoglobin in cattle blood following haemolysis caused at collection

This study aimed to develop an in-house assay for haptoglobin determination in bovine blood samples, assess the effect of haemolysis on the reported haptoglobin concentration and develop a method to correct for haemolysis interference. The assay developed is highly repeatable (92.3% across plates and 94.8% between assays). A correction equation (Hpcorrected=Hpraw-Hpendogenous activity-Hpdue to Hb; where Hpdue to Hb=0.118×Hbfree+0.015) was developed based around the linear relationship of haptoglobin and haemoglobin (by-product of haemolysis) and endogenous interference, tested and validated for use with haemolysed samples. The method described in this paper allows samples inadvertently haemolysed at collection to be analysed, with the reported haptoglobin concentration being an accurate reflection of the physiological levels in the animal’s blood at the time of collection.

Stability of serum samples and hemolysis interference on the high sensitivity troponin T assay

Stability of serum samples and hemolysis interference on the high sensitivity troponin T assay : Letter to the editor

Stability of plasma adrenocorticotrophic hormone (ACTH): Influence of hemolysis, rapid chilling, time, and the addition of a maleimide

Objectives The aim of this study was to examine the effects of hemolysis, rapid chilling, time, and the addition of a maleimide on the stability of human plasma ACTH measurements. Design and methods Partially hemolyzed EDTA blood ( n = 10), initially at 37 °C, was centrifuged at 4 °C either immediately or after rapid chilling in ice/water. Plasma ACTH was then measured either immediately, or after 1 h at 22 °C with or without the addition of 2 mM N-phenyl maleimide (NPM). Results For 0.2% hemolysis compared to no hemolysis, the mean (±SEM) loss with immediate centrifugation and immediate ACTH measurement was 11 ± 1%. This loss was significantly ( p < 0.002) reduced to 6 ± 1% by an initial rapid chilling of the samples. For analysis after 1 h at 22 °C, the addition of NPM decreased the loss of ACTH from 15 ± 2% to 2 ± 2% ( p < 0.002). Conclusion Rapid chilling, prompt analysis, and addition of NPM can each reduce the interference of hemolysis in the measurement of plasma ACTH concentrations.

Interference of haemolysis and hyperproteinemia on Sodium, Potassium, and Chloride measurements in canine serum samples

Interference of haemolysis and hyperproteinemia on Sodium, Potassium, and Chloride measurements in canine serum samples D. Bernardini & G. Gerardi & B. Contiero & M. Caldin Published online: 28 July 2009 # Springer Science + Business Media B.V. 2009

Multicenter evaluation of the hemolysis index in automated clinical chemistry systems

In vitro hemolysis, the prevailing cause of preanalytical error in routine laboratory diagnostics, might influence the reliability of several tests, affect the quality of the total testing process and jeopardize patient safety. Although laboratory instrumentation is now routinely equipped with systems capable of automatically testing and eventually correcting for hemolysis interference, to our knowledge there are no reports that have compared the efficiency of different analytical platforms for identifying and classifying specimens with hemolysis. Serum from a healthy volunteer was spiked with varying amounts of hemolyzed blood from the same volunteer, providing a serum free hemoglobin concentration ranging from 0.0 g/L to 2.0 g/L as measured by the reference cyanmethemoglobin assay. The spiked serum samples were shipped to seven separate laboratories and the hemolysis index (HI) was tested in triplicate on the following analytical platforms: Roche Modular System P (n=4) and Integra 400 Plus (n=1), Siemens Dimension RxL (n=3), ADVIA 2400 (n=1) and ADVIA 1800 (n=1), Olympus AU 680 (n=1) and Coulter DXC 800 (n=1). Satisfactory agreement of HI results was observed among the various analytical platforms, despite a trend toward overestimation by the ADVIA 2400 and 1800. After normalizing results according to the instrument-specific alert value, discrepancies were considerably reduced. All instruments except for the Dimension RxL gave values normalized to the instrument-specific alert value, <1.0 for the sample with 0.048 g/L free hemoglobin, and >1.0 for the sample with 0.075 g/L free hemoglobin. The results of the four Modular System P tests were also highly reproducible among the different facilities. When evaluating instruments that provided quantitative HI results, the mean intra-assay coefficient of variation (CV) calculated for the triplicate determinations was always between 0.1% and 2.7%. The results of this multicenter evaluation confirm that efficiency of different analytical platforms to correctly identify and classify unsuitable samples is satisfactory. However, more effort should be placed on the standardization of reporting HI. All the instruments that we tested provide either quantitative or qualitative results that are essentially comparable, but which should always be compared with the instrument-specific alert values to harmonize their efficiency.

Neonatal jaundice: a critical review of the role and practice of bilirubin analysis

Neonatal jaundice is common, and usually harmless, because of physiological jaundice or breast-feeding. In some neonates unconjugated bilirubin concentration, coupled with other risk factors, is sufficient to allow free bilirubin to cross the blood-brain barrier and cause kernicterus. Another subgroup of infants is jaundiced because of elevated conjugated bilirubin; a marker for a number of pathological conditions. Bilirubin measurement must identify those infants at risk. Transcutaneous bilirubin measurement is increasingly used in healthy infants, especially before early discharge or at home, to assess the need for laboratory bilirubin measurement. Transcutaneous measurements are not covered by laboratory quality assessment schemes. Guidelines on management of neonatal jaundice utilize age in hours and other risk factors to define bilirubin action thresholds, which may be as low as 100 micromol/L for sick premature infants, whereas early discharged babies may only present after bilirubin concentrations are extremely high. Hence, there is a requirement for accurate total bilirubin measurement from <100 to >500 micromol/L, with sufficient precision to assess the rate of bilirubin change with time. Babies presenting with late jaundice always require conjugated bilirubin measurement. It is of concern that many total and direct bilirubin automated kit methods suffer from haemolysis interference, while use of in-house methods or modification of commercial methods has virtually disappeared. External quality assessment has a vital role in providing data on different methods' performance, including accuracy, precision and susceptibility to interference. Laboratories should consider whether their adult bilirubin methods are suitable for neonates.

Haemolysis and turbidity influence on three analysis methods of quantitative determination of total and conjugated bilirubin on ADVIA 1650

Plasma bilirubin testing is crucial to prevent the occurrence of neonatal kernicterus. Haemolysis may occur during sampling and interfere with bilirubin determination. Moreover, lipidic infusions may induce plasma lipemia and also interfere with bilirubin measurement. We evaluated the interference of haemolysis and lipemia with three methods of total and direct bilirubin measurement adaptated on an Advia 1650 analyser (Siemens Medical Solutions Diagnostics) : Synermed (Sofibel), Bilirubin 2 (Siemens) and Bilirubin Auto FS (Diasys). The measurement of total bilirubin was little affected by haemolysis with all three methods. The Bilirubin 2 (Siemens) method was the less sensitive to haemolysis even at low bilirubin levels. The measurement of conjugated bilirubin was significantly altered by low heamoglobin concentrations for Bilirubin Auto FS(R) (30 microM or 0,192 g/100 mL haemoglobin) and for Synermed (60 microM or 0,484 g/100 mL haemoglobin). In marked contrast, we found no haemoglobin interference with the Direct Bilirubin 2 reagent which complied with the method validation criteria from the French Society for Biological Chemistry. The lipemia up to 2 g/L of Ivelip did not affect neither the measurement of total bilirubin for all three methods nor the measurement of conjugated bilirubin with the Diasys and Siemens reagents. However, we observed a strong interference starting at 0,5 g/L of Ivelip with the Synermed reagent. Our data suggest that both Siemens and Diasys methods allow to measure accurately total and conjugated bilirubin in hemolytic and lipemic samples, nevertheless, the Siemens methodology is less affected by these interferences.

Influence of hemolysis on routine clinical chemistry testing

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. 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. 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. 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.

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.

Investigation and application of measuring serum total protein by improving biuret double reagent through two point terminal method

目的 研究建立改良双缩脲双试剂二点终点法测定血清总蛋白的方法,以消除标本溶血、黄疸、脂浊、含葡聚糖的干扰.方法采用改良试剂,测定方法的精密度、回收率、检测限、线性范围、干扰试验,以确定所建立方法的基本性能;与Doumas法比较以确定方法的可靠性与消除干扰的效果.结果样品与试剂比例为5∶ 220 μl,线性范围达140 g/L.回归方程:Y=484.0+48.06X,r=0.999 3.批内CV(20次)0.24%,日间CV (20 d)1.07%.回收率95.6%～103%, 平均99.5%.检测限2.36 g/L.血红蛋白(Hb)2.1 g/L以下, 胆红素(Bil)148 μmol/L以下,三酰甘油(TG)28.2 mmol/L以下,葡聚糖30 g/L以下干扰不显著.改良法与Doumas法测定外观正常标本结果高度相关,回归方程:Y=-2.435 4+1.037 4X,r=0.988 2.结果间差异无统计学意义,t=0.156,P=0.877.测定溶血、高脂、黄疸、含葡聚糖标本二种方法间差异有统计学意义.结论采用改良双缩脲双试剂二点终点法测定血清总蛋白,能有效消除溶血、黄疸、高脂和葡聚糖的干扰,提高结果准确性。

The impact of hemolysis on Ortho-Clinical Diagnostic's ECi and Roche's elecsys immunoassay systems

Background: Hemolysis is regularly encountered in clinical specimens and often interferes with a variety of laboratory test methods. Although not widely recognized, immunoassays based on nonisotopic detection systems can also be affected by hemolysis. For this reason, we investigated the effect of differing amounts of hemolysis across a range of values for several immunoassays on the Ortho-Clinical Diagnostics ECi and Roche Elecsys platforms. Methods: Hemolysate was prepared from whole blood and spiked at varying concentrations into pooled patient serum samples for different analytes. Results: Out of the 21 analytes tested, six (28.6%) exhibited significant increases or decreases in measured concentrations with increasing amounts of hemolysis. Conclusions: Although immunoassays are generally thought to be impervious to hemolysis interference, hemolysis can interfere in immunoassay testing platforms. For these reasons, we recommend that laboratories conduct hemolysis interference studies for all laboratory test protocols.

Effects of Haemolysis, Lipaemia, Bilirubinaemia and Fibrinogen on Protein Electropherogram of Canine Samples Analysed by Capillary Zone Electrophoresis

The possible interference of haemolysis, lipaemia, bilirubinaemia and fibrinogen on capillary zone electrophoresis of canine samples were studied. Solutions of haemoglobin, lipid and bilirubin were prepared and mixed with serum aliquots to make up samples containing different concentrations of the putative interferent substance. In addition, samples of serum and plasma were assayed to assess the influence of fibrinogen. Haemolysis and lipids produced a change in electropherogram morphology giving an interference peak located in the beta-2 region when haemoglobin was increased, and in the alpha-2 region when lipids were increased. A rise in concentration of these interferents caused an increase in the beta and alpha-2 fractions respectively, and a decrease in the other fractions. Bilirubin did not alter morphology but gave an increase in the albumin and alpha-1 and a decrease in the alpha-2 and beta-2 fractions. No differences were found between serum and plasma samples, and fibrinogen did not produce any additional peak.

Determination of myoglobin: comparative evaluation of the new automated VIDAS R assay with two other immunoassays

Objectives: Myoglobin provides the earliest indication of acute myocardial infarction. In this study, the new myoglobin assay for the VIDAS system (bioMérieux) was evaluated. Design and methods: This assay, using an enzyme-linked fluorescent immunoassay (ELFIA) method, was compared with the Olympus R immunoturbidimetric method and with another immunometric method (Immulite turbo R) using an enzyme-linked chemiluminescent immunoassay (CLIA). Results: The CVs for within-and between-run reproducibility are very similar for the tested methods and acceptable linearity ranges were obtained. No significant interference of hemolysis, turbidity and icteria was observed. In the whole cohort, we obtained decreased values over the entire range of the assay with VIDAS and Immulite turbo methods compared to the Olympus assay; this is probably mainly linked to differences in standards used due to the absence of international standardization of the myoglobin determination. Conclusions: The new VIDAS myoglobin automated assay provides biologists with a rapid, accurate and reliable determination of myoglobin in plasma samples collected during cardiac workup.

Bleomycin-detectable Iron Assay for Non-Transferrin-bound Iron in Hematologic Malignancies

A microwell modification of the bleomycin assay for determining non-transferrin-bound iron (NTBI) was evaluated and compared with a chelation method. The bleomycin assay reagent and sample volumes were halved, and measurements were done in microwell plates. Samples from patients treated for hematologic malignancies were studied. The chelation method was based on mobilization of NTBI with a chelator and measurement of the ultrafiltered iron-chelator complex. NTBI results were also compared with transferrin saturation and the distribution of transferrin iron forms by urea-polyacrylamide gel electrophoresis. The bleomycin assay intraassay imprecision (CV) was 7.7% and 8.2% and the interassay imprecision was 18% and 9.8% for a low (0.2 micromol/L) and a high (1.5 micromol/L) contrtrol, respectively. Hemolysis increased measured NTBI. A detection limit of 0.1 micromol/L was established based on the interference of nonvisible hemolysis and on accuracy studies. In patient samples, NTBI exceeded the detection limits only when transferrin saturation was >80%. Compared with the chelation method, the bleomycin assay gave clearly lower NTBI concentrations. The chelation method also gave positive results at <80% transferrin saturation. The recovery of iron added as ferric nitrilotriacetate to serum was 33% by the bleomycin assay and 64% by the chelation assay. The microwell version of the bleomycin assay is reproducible. When hemolyzed samples were excluded, bleomycin-detectable iron was found only when the transferrin saturation was >80%, suggesting high specificity. Bleomycin-detectable iron constitutes only a portion of the NTBI measured by the chelation method.

Study of hemolysis interference with a peak rate nephelometric assay of lipoprotein(a)

Lipoprotein(a) [Lp(a)] is considered an independent risk factor in atherosclerosis and thrombosis. Its measurement in clinical laboratories necessitates the use of reliable methods and a strict control of preanalytical conditions. We studied here the potential interference of hemolysis on a peak rate nephelometric method. Serum samples were spiked with up to 500 μmol/L hemoglobin (Hb), and Lp(a) was measured with a Beckman Array 360 analyzer. Hb supplementation induced a significant decrease of Lp(a) values: −15% and −25% (p < 0.01) for Hb concentrations 200 and 500 μmol/L, respectively. Similar results were obtained when Hb addition was done before and after the manual step of sample predilution (1/5, v/v) in PEG-containing buffer. No significant difference of Hb interference was noticed according to apo(a) phenotypes. Our results indicate that hemolysis may significantly alter Lp(a) values obtained with this method, probably because of the low-dilution rate of samples. Even if this interference is of limited extent, it should be taken into account when interpretating results close to decisional values used for risk assessment. © 2000 John Wiley & Sons, Inc. Lab Robotics and Automation 12:23–26, 2000

Study of hemolysis interference with a peak rate nephelometric assay of lipoprotein(a)

Study of hemolysis interference with a peak rate nephelometric assay of lipoprotein(a)