Hemolyzed Samples Research Articles

Zero delay plasma vs. Streck cfDNA tubes: Does immediately centrifuging blood at the point of draw improve cell-free DNA signal-to-noise?

e14529 Background: Sample quality is a critical consideration for high fidelity cell-free DNA (cfDNA) testing. Oncological cfDNA tests used for liquid biopsy typically employ specialty blood collection tubes containing chemical preservatives to minimize degradation of samples prior to lab testing. Here we describe a newly developed device, Zero Delay Plasma– a portable centrifuge and disc system designed to immediately isolate cell-free plasma at the point of blood draw – and evaluate its performance against the Streck cfDNA collection tube. Methods: Whole blood was collected, processed, and stored at room temperature for up to 7 days with both the Zero Delay Plasma system and the Streck cfDNA blood collection tube. Sample hemolysis was measured via cell-free hemoglobin. Genomic contamination and cfDNA signal-to-noise were evaluated by qPCR and electrophoresis, comparing signal from target 150-200bp cfDNA to contaminating longer length genomic sequences in the sample. 2 sets of hemolysis experiments, 2 sets of electrophoresis experiments and 4 sets of qPCR experiments were conducted. Results: Plasma processed with the Zero Delay Plasma system yielded ~4X lower hemolysis levels, ~10X lower genomic contamination, and ~20X higher cfDNA signal-to-noise compared to the Streck cfDNA collection tube after 7 days of storage at room temperature. Conclusions: The Zero Delay Plasma system minimizes sample degradation and analytical background signal for cfDNA testing by immediately removing cells and other contaminants at the point of blood collection. Clinical evaluations are in process.

Impact of managing altered results in hemolyzed samples (HS) in an infant-maternity hospital using an unconventional approach

Impact of managing altered results in hemolyzed samples (HS) in an infant-maternity hospital using an unconventional approach

The pitfall of hemolyzed samples: How to manage in vitro hemolysis?

The pitfall of hemolyzed samples: How to manage in vitro hemolysis?

Veris DXn vs Panther - A comparison study

Blood sample hemolysis affects pre-analytical quality and may cause pseudohyperkalemia. We established a statistical model to estimate the corrected potassium (K+) in serum.Serum K+ and H index were analyzed, and blood cell index was obtained from the examined Full Blood Examination (FBE) results. A linear-regression model was developed using hemolysis (H) index, K+ and covariates of blood cell index from 139 cell lysates of blood samples. The model was then validated against 26 in vitro physically hemolyzed serum samples.The final model selected H index, hemoglobin concentration (HGB), and hematocrit (HCT) as important predictors in estimating the K+ content. The model was validated against artificially hemolyzed serum samples, which returned a correlation of 0.942 between observed and predicted net K+ increase by hemolysis. The predictors H index, HCT, and HB contributed 93.7%, 3.5% and 2.8% to the model R2, respectively.In vitro hemolysis induced pseudohyperkalemia could be accurately predicted and restored by our model for clinical application.

An audit of sample hemolysis rate in a Singapore public hospital

The evaluation of quality of care is a complex task that, over the last decades, has been performed using the Donabedian model as its main conceptual framework. Small countries are an ideal setting in which to make innovative, empirical evaluations of the quality of care. In this research, we discussed the challenges and opportunities of assessing hospital performance in Latvia, a small country in the Baltic region of Northern Europe. The study outcomes were 30-day acute myocardial infarction mortality and 30-day ischaemic stroke mortality. We described how indicator specifications, risk-adjustment, data reliability assessment and graphical representations were tailored to the geographic and institutional context of Latvia. By looking at the impact of structural characteristics on hospital performance, we found that cath labs and large caseloads were significantly associated with lower mortality. This approach allows decision-makers at different governance levels to design and implement actions aimed at improving the quality of care. At the health system level, it may help policy-makers adopt proper strategies to tackle poor outcomes; at the hospital level, it may help managers intervene on structural determinants of performance. Because small countries face some relevant issues that have implications for health care, these analyses might be relevant also for larger countries to improve the design of their health-care services

Point-of-care hemolysis detection in blood gas specimens directly at the emergency department

Blood gas analysis represents an important and frequently used clinical decision support. Spurious haemolysis is common at the emergency department (ED) and causes clinically relevant bias to many analyses. Blood gas analysers inability to identify hemolysis can therefore lead to clinical misinterpretations. The objective of this study was to assess the proportion of haemolytic blood gas samples performed at the ED and calculate the predictive values of a novel point-of-care solution for haemolysis detection. Approximately 100 µl of blood from anonymized syringes including venous or arterial blood was, directly after performed blood gas analysis, tested with the POC-Method (Helge H10 s-system, Hemcheck, Karlstad, Sweden) at the ED and result recorded. Thereafter the blood was centrifuged (2000 g) for 10 minutes, 100 µl of plasma put into vials and run in batches of 40 daily on an AU680 analyzer (Beckman Coulter, Brea, California, USA) used as reference method. The threshold for clinically relevant hemolysis was defined as ≥50 mg/dL free haemoglobin. Out of 1270 samples, haemolysis was detected in 7.9% (n = 100). The POC-method identified haemolytic samples with a sensitivity of 80%, specificity of 99%, positive and negative predictive values was 89% and 98% respectively. Spurious hemolysis is frequently present in blood gas samples at the ED and should be taken into consideration when assessed in patients. Errors could be detected directly at the point-of-care with a novel technology for hemolysis detection.

Continual improvement of the pre-analytical process in a public health laboratory with quality indicators-based risk management

Background Quality indicators (QIs) and risk management are important tools for a quality management system designed to reduce errors in a laboratory. This study aimed to show the effectiveness of QI-based risk management for the continual improvement of pre-analytical processes in the Kayseri Public Health Laboratory (KPHL) which serves family physicians and collects samples from peripheral sampling units. Methods QIs of pre-analytical process were used for risk assessment with the failure modes and effects analysis (FMEA) method. Percentages and risk priority numbers (RPNs) of QIs were quantified. QI percentages were compared to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) performance specifications and RPNs were compared to risk level scale, and corrective actions planned if needed. The effectiveness of risk treatment actions was re-evaluated with the new percentages and with RPNs of predefined QIs. Results RPNs related to four QIs required corrective action according to the risk evaluation scale. After risk treatment, the continual improvement was achieved for performance and risk level of "transcription errors", for risk levels of "misidentified samples" and "not properly stored samples" and for the performance of "hemolyzed samples". "Not properly stored samples" had the highest risk score because of sample storage and centrifugation problems of peripheral sampling units which are not under the responsibility of the KPHL. Conclusions Public health laboratories may have different risk priorities for pre-analytical process. Risk management based on predefined QIs can decrease the risk levels and increase QI performance as evidence-based examples for continual improvement of the pre-analytical process.

Relationship between free hemoglobin (hemolysis), potassium and ionized calcium in lithium heparin blood gas samples collected intraoperatively

ObjectiveDevelop sample acceptability rules by determining the relationship between free hemoglobin level (hemolysis) and potassium or ionized calcium in blood gas samples collected intraoperatively. Design and methodsHemolysis was assessed visually or by H index for lithium heparin blood gas samples collected intraoperatively. During periods one and three this was done using two different rules for visual assessment of centrifuged lithium heparin plasma. During period two H index was measured for all visually hemolyzed samples on a Roche Cobas c501 analyzer to determine acceptability. Potassium and ionized calcium were measured in 75 lithium heparin whole blood samples on a Radiometer ABL90 to correlate H index and potassium or ionized calcium. ResultsDuring period one 35 of 5808 (0.6%) blood gas samples had visual hemolysis levels exceeding tolerance for reporting of potassium. By switching to measured H index using a laboratory-established threshold, during period 2 we estimate that 171 of 5396 (3.2%) blood gas samples exceeded the H index threshold for reporting of potassium. In 75 intraoperative blood gas samples with H index and whole blood potassium and ionized calcium measured; we observed no relationship between H index and potassium or ionized calcium. During period 3 we switched to visual assessment of hemolysis with a greater tolerance for hemolysis; with only 3 of 5345 (0.06%) samples exceeding the new visual hemolysis threshold. ConclusionFor blood gas samples collected intraoperatively, there is no relationship between hemolysis and measured potassium or ionized calcium. The results suggest that only grossly hemolyzed intraoperative blood gas samples should be rejected for measurement of whole blood potassium and ionized calcium.

An ultra-performance liquid chromatography-tandem mass spectrometry method to quantify vancomycin in human serum by minimizing the degradation product and matrix interference.

Aim: This study aimed to develop and validate a method for better therapeutic monitoring of vancomycin serum concentration. Methods & results: An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to minimize the interference of crystalline degradation product and matrix. It was compared with chemiluminescence microparticle immunoassay (CMIA)and ultra-performance liquid chromatography with ultraviolet detection (UPLC-UV) in the performance of testing normal, on-dialysis and hemolytic serum samples. For on-dialysis samples, a moderate correlation (r=0.534) was observed between UPLC-UV and UPLC-MS/MS. In testing hemolytic samples, ten (10/85, 11.8%) samples were overestimated by CMIA method. Conclusion: Vancomycin concentration determined by CMIA, UPLC-UV was more affected by various panels of serum samples than UPLC-MS/MS assay, suggesting that UPLC-MS/MS is a more reliable and promising tool for clinical vancomycin therapeutic drug monitoring.

Impact of NABL on Quality Indicators of Pre-Analytical Phase of Testing in Tertiary Care Hospital

Introduction: The laboratory’s compliance to requirements of the standard and its technical competence are assessed by NABL for accreditation. QIs should be part of a coherent and integrated quality improvement strategy implemented according to the specifically developed International Standard for Medical Laboratories Accreditation (ISO 15189: 2012). Pre-analytical errors account for more than 70% of the total number of laboratory errors. The International Federation of Clinical Chemistry (IFCC) and Laboratory Medicine Working Group on Laboratory Errors and Patient Safety (WG-LEPS) has made an important contribution to developing QIs for the preanalytical phase and specifications for those indicators. We selected following QIs pertaining to the key activities of the pre-analytical phase. These were: Hemolyzed samples (in biochemistry; QI-10b); Samples with inadequate quantity (QI-12). Material and Method: QI-10b and 12 were recorded from ‘Sample rejection register’ and ‘Sample transport register’. We calculated the sigma metric for these QIs. First, we calculated the Defect Per Million (DPM) then the DPM rate was converted to a sigma value. Result After NABL accreditation improvement in six sigma values of QI-10b is 4.0-5.0, suggests ‘Good’ level of performance and for QI-12 shows between 3.0-4.0, no significant improvement. Conclusion NABL accreditation does not make any statement about the technical competence of the laboratory. As continual improvement is necessary for the good laboratory practice, we continue to collect data regarding errors to monitor this critical phase of laboratory testing to ensure ongoing satisfactory performance.

European survey on preanalytical sample handling - Part 2: Practices of European laboratories on monitoring and processing haemolytic, icteric and lipemic samples. On behalf of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for the Preanalytical Phase (WG-PRE).

IntroductionNo guideline currently exists on how to detect or document haemolysis, icterus or lipemia (HIL) in blood samples, nor on subsequent use of this information. The EFLM WG-PRE has performed a survey for assessing current practices of European laboratories in HIL monitoring. This second part of two coherent articles is focused on HIL.Materials and methodsAn online survey, containing 39 questions on preanalytical issues, was disseminated among EFLM member countries. Seventeen questions exclusively focused on assessment, management and follow-up actions of HIL in routine blood samples.ResultsOverall, 1405 valid responses from 37 countries were received. A total of 1160 (86%) of all responders stating to analyse blood samples - monitored HIL. HIL was mostly checked in clinical chemistry samples and less frequently in those received for coagulation, therapeutic drug monitoring and serology/infectious disease testing. HIL detection by automatic HIL indices or visual inspection, along with haemolysis cut-offs definition, varied widely among responders. A quarter of responders performing automated HIL checks used internal quality controls. In haemolytic/icteric/lipemic samples, most responders (70%) only rejected HIL-sensitive parameters, whilst about 20% released all test results with general comments. Other responders did not analysed but rejected the entire sample, while some released all tests, without comments. Overall, 26% responders who monitored HIL were using this information for monitoring phlebotomy or sample transport quality.ConclusionStrategies for monitoring and treating haemolytic, icteric or lipemic samples are quite heterogeneous in Europe. The WG-PRE will use these insights for developing and providing recommendations aimed at harmonizing strategies across Europe.

Portable Device for Measuring Blood Test Hemolyzed Samples Based on Computer Vision and Neural Network

Hemolysis is a challenging problem and still represents a frequent source of errors in blood test laboratory practice. Due to the broad and heterogeneous bias induced in the measurement of several parameters by hemolysis, inaccurate results may be reported, and the patient may be required to repeat sample collection, delaying diagnosis. Existing automated laboratory devices including hemolysis detection are not suitable for lower volume and smaller sample collection sites. In many situations, hemolysis is still detected by visual inspection of the sample after centrifugation, during the blood test pre-analytical stage. Visual inspection is highly dependent on a qualified workforce, subjective to interpretation discrepancies, and thus difficult to standardize. The paper aims to describe the design and performance of a portable device for measuring hemolyzed samples based on computer vision and neural network. The results indicate that the device provides hemolysis indexes with sufficient accuracy to guide laboratory decision in the blood test pre-analytical stage.

Influence of hypertriglyceridemia, hyperbilirubinemia and hemolysis on thrombin generation in human plasma.

Background Although accumulating evidence suggests that the hemostatic balance is impaired in patients with hypertriglyceridemia, hyperbilirubinemia or hemolytic anemias, little is known on the underlying biological mechanisms. This experimental study was aimed at exploring whether increasing values of triglycerides, bilirubin or cell-free hemoglobin promote thrombin generation in plasma. Methods Three different pools were prepared from three different sets of 20 normal routine plasma citrate samples. The native pools were spiked with increasing amounts of exogenous triglycerides (up to 8.8 mmol/L), bilirubin (up to 350 μmol/L) or autologous hemolyzed blood (up to 3.5 g/L cell-free hemoglobin). Using the fully-automated thrombin generation analyzer ST Genesia, we measured the following parameters: lag time (LT), time to peak (TP), peak height (PH) and endogenous thrombin potential (ETP). Results A sustained increase of PH and ETP was found in parallel with increasing triglyceride concentrations, peaking in the aliquot with 8.8 mmol/L. Conversely, LT and TP displayed an opposite trend, reaching a maximum decrease in the 8.8 mmol/L aliquot. Increasing bilirubin concentrations promoted remarkable increases of PH and ETP and decreases of TP and LT, up to 211 μmol/L. After this threshold, all parameters tended to return towards baseline values. A constant increase of PH and ETP was also noted in hemolyzed samples, peaking in the 3.5 g/L cell-free hemoglobin aliquot, whereas the TP and LT remained unchanged in all hemolyzed aliquots. Conclusions Our findings suggest that hypertriglyceridemia, hyperbilirubinemia and hemolysis may promote a hypercoagulable state in human plasma.

Pre-analytical quality control in hemostasis laboratories: visual evaluation of hemolysis index alone may cause unnecessary sample rejection

Abstract Background Visual inspection is the most widespread method for evaluating sample hemolysis in hemostasis laboratories. The hemolysis index (HI) was determined visually (visual index, VI) and measured on an ACL TOP 750 (IL Werfen) system with a hemolysis-icterus-lipemia index (HIL) module. These values were compared with those measured on clinical chemistry systems Unicel DXC600 and AU680 and with quantitation of free-hemoglobin (Hb) performed by a spectrophotometric measurement method (SMM). Methods The HI was measured in 356 sodium citrate plasma samples, 306 of which were visibly hemolyzed to varying degrees and 50 were not hemolyzed. The analytical performance of each method was evaluated. Results Linear regression analysis, calculated between SMM and the other systems in the study, returned coefficients of determination r2 = 0.853 (AU680), r2 = 0.893 (DXC600) and r2 = 0.917 (ACL TOP 750). An r2 = 0.648 was obtained for linear regression analysis between VI and ACL TOP 750. In addition, ACL TOP 750 showed an excellent correlation in multivariate analysis (r2 = 0.958), showing good sensitivity (0.939) and specificity (0.934) and a diagnostic accuracy of 94%. By comparison, DXC600 and AU680 showed coefficients of determination of 0.945 and 0.923, respectively. A cut-off was set at 0.15 g/L free-Hb, as determined by the automated method, such that any hemostasis samples measuring above this threshold should not be analyzed. Based on this criterion, samples were classified as accepted or rejected, and the number of samples discarded during VI or ACL TOP 750 measurements was compared. Conclusions This study confirmed that hemostasis laboratories should consider introducing an objective, automated and standardized method to check samples for hemolysis. By relying solely on visual inspection, up to 50% of samples could be unnecessarily rejected. The ACL TOP 750 system demonstrated a satisfactory analytical performance, giving results comparable to those of the reference method.

Hemolyzed Specimens: Major Challenge for Identifying and Rejecting Specimens in Clinical Laboratories.

Pre-analytical quality in clinical chemistry testing is as important as analytical and post-analytical quality. The most prevalent pre-analytical interference and a major source of error producing unreliable laboratory test results is hemolysis of blood samples. In vitro hemolysis may be due to the blood withdrawal technique or sample handling whereas in vivo hemolysis can originate from acquired, hereditary, or iatrogenic conditions and is not technique dependent. Interpreting in vivo or in vitro hemolysis requires clinicians to supply reliable clinical history and findings. Even then, to reject or release the result with interpretation is still under debate. Thus, hemolyzed specimens are a serious pre-analytical problem calling for well-designed and strictly implemented laboratory guidelines. The aim of this non-systematic review (addressed to healthcare professionals) was to highlight the challenges in identifying and rejecting hemolysis specimens.

The Hemolyzed Sample: To Analyse Or Not To Analyse.

Preanalytical errors constitute about 40-65% of laboratory errors, of which 60% are due to hemolysis. This leads to imprecise reporting and misinterpretation of the actual concentration of analytes. Hence the aim of this study was to estimate the extent of different degrees of interference by visible hemolysis. 25 hemolysed samples along with their fresh unhemolysed sample were studied. Hemolyzed serum was mixed with unhemolyzed serum in predefined serial ratios from 100%, 70%, 50%, 30% and 10% to achieve different grades of hemolysis. Each dilution was analysed for BUN, creatinine, uric acid, phosphorus, Na, K, total protein, amylase, lipase, LDH, tacrolimus and methotrexate. Percentage difference of each dilution of the hemolyzed sample as compared to the unhemolyzed sample was calculated and considered acceptable only if less than TEa. It was observed that Percentage difference of BUN, creatinine, amylase and lipase in all dilutions of hemolyzed samples were within TEa while phosphorus, Na, K, total protein and LDH were beyond the acceptance criteria. Hence It was concluded that it may be safe to analyse a hemolysed sample for BUN, creatinine, amylase, lipase, tacrolimus and methotrexate while uric acid may be estimated in a moderately hemolysed sample. Phosphorus, sodium, potassium, total protein and LDH must never be analyzed in any hemolysed sample.

Evaluation of management strategy results of laboratory studies in serum samples with hemolysis

There is an ongoing debate about what the laboratory should do with hemolyzed samples. Several strategies are proposed for managing the results obtained in such samples. The safest option from the analytical and clinical points of view is to perform a study of a new sample without hemolysis. Another approach is to carry out a test irregardless, but at the same time indicate a limit on the clinical interpretation of the result, by making a comment on possible hemoglobin interference. The choice of strategy should be based on a comparison of the risk of negative consequences in the absence of a test result and the likelihood of harm due to the transfer of the result with high uncertainty to the clinician.

Preanalytical challenges - time for solutions.

The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for the Preanalytical Phase (WG-PRE) was originally established in 2013, with the main aims of (i) promoting the importance of quality in the preanalytical phase of the testing process, (ii) establishing best practices and providing guidance for critical activities in the preanalytical phase, (iii) developing and disseminating European surveys for exploring practices concerning preanalytical issues, (iv) organizing meetings, workshops, webinars or specific training courses on preanalytical issues. As education is a core activity of the WG-PRE, a series of European conferences have been organized every second year across Europe. This collective article summarizes the leading concepts expressed during the lectures of the fifth EFLM Preanalytical Conference "Preanalytical Challenges - Time for solutions", held in Zagreb, 22-23 March, 2019. The topics covered include sample stability, preanalytical challenges in hematology testing, feces analysis, bio-banking, liquid profiling, mass spectrometry, next generation sequencing, laboratory automation, the importance of knowing and measuring the exact sampling time, technology aids in managing inappropriate utilization of laboratory resources, management of hemolyzed samples and preanalytical quality indicators.

Evaluation of hemolysis effect on hemoglobin measurement by capillary electrophoresis

BackgroundHemoglobin (Hb) is a hemeprotein with two linked pairs of globin chains. Each chain is connected to a heme residue in its center. Hemoglobinopathies are divided into quantitative and qualitative defects in globin synthesis. Hemolysis is a pre-analytical problem that reduces quality of sample for measurement of many analytes.MethodsBlood samples from 311 female and 189 male subjects with normal and abnormal Hb electrophoresis patterns were studied. Three milliliters of whole blood was obtained from all subjects and transferred into EDTA tubes. To analyze hemolysis, 1.5 ml of blood from each tube was aliquoted and frozen, and the remaining blood was stored at 4 °C for 24 h. Hemoglobin was measured in both hemolyzed and non-hemolyzed samples by capillary electrophoresis.Results and discussionData was analyzed with linear regression. The results were linear for the lower limit of detection of 9, 0.5, and 0.1% up to at least 99.5, 6.6, and 99.4% for HbA, HbA2, and HbF, respectively. Method comparison demonstrated good agreement between non-hemolyzed and hemolyzed conditions for hemoglobin measurement.ConclusionUse of hemolyzed samples had no effected on hemoglobin measurements.

The effect of automated hemolysis index measurement on sample and test rejection rates

Abstract Objective Vast majority of laboratory errors occurs in preanalytical phase and in vitro hemolysis is the most common among preanalytical errors. Automated serum index measurement is being used in routine biochemical analysis in Antalya Public Health Care Laboratory, since June 2014. Our aim in this study is to reveal the impact of serum index usage on rejected samples and rejected test rates due to hemolysis. Materials and methods Hemolysis, icterus and lipemia (HIL) spectral interference reagent and program have been used in our laboratory since June 2014. In the current study, the number of samples and tests that were rejected due to hemolysis in June–August 2014 were compared with those rejected in the same period of 2013. Results In 2014, the sample rejection rate was 2.53% and the rejected test rate was 0.48%. In 2013, the sample rejection rate was 0.56% and the rejected test rate was 0.55%. When compared two periods, statistically significant increase in rejected sample number due to hemolysis in 2014 is result of, visually undetectable hemolyzed samples previously can be identified by HIL method (p<0.05). Conclusion Usage of hemolysis index program in automated systems for detecting hemolysis was evaluated as a method which is standardized, semi-quantitative, with high reproducibility and allows test based rejection.