Hemolyzed Samples Research Articles

Appraisal of six sigma in pre-analytical phase of clinical biochemistry laboratory

Objectives: The aim of the study is to measure the performance of pre-analytical phase of a clinical biochemistry laboratory using sigma metrics and the six sigma scale. Materials and Methods: The study included documented data of blood sample rejection from March 2023 to February 2024 and follow-up data from March 2024 to August 2024. International Federation of Clinical Chemistry and Laboratory Medicine developed Quality Indicators (QIs) used are QI-9 Wrong tubes; QI-10 Hemolyzed samples; QI-11 Clotted samples; QI-12 Insufficient samples; QI-14 Damaged samples in transport; and QI-15 Mislabeled samples. Based on “Six Sigma Quality Design and Control” established by Dr. Westgard, the sigma metric was calculated for the above-mentioned QIs. Statistical analysis: Obtained data were entered and analyzed using Microsoft Excel 2021. Results: Out of 162,380 received samples, 547 samples were rejected as not satisfied with the sample acceptance criteria. The most common pre-analytical error in the observed QIs is hemolyzed samples (458), followed by insufficient sample volume (55). The Sigma score of QI-10 was determined to be 4.81, whereas QI-9, QI-11, QI-12, and QI-15 were well maintained and graded excellent. Following training sessions, the follow-up month revealed a sigma score of 4.98 for QI-10. Conclusions: Six sigma metrics are a competent means to measure the performance of pre-analytical QIs in a clinical biochemistry laboratory. The observed QIs were effectively managed (>4 σ).

Using ELISA to detect pathogen antibodies in wild mammal carcasses: a systematic literature review

Abstract Carcasses of wild mammals provide convenient samples for wildlife disease investigators. Specifically, antibodies can persist in the blood and tissues of animals up to a few days after death and can provide information on exposure to specific pathogens, including microorganisms with zoonotic implications, such as Toxoplasma gondii, Trichinella spp., Brucella spp., and Mycobacterium spp. Although screening samples from decaying wildlife for antibodies is not a new method, an overview of the literature with clear recommendations on best practices is lacking. Here, we present results from a systematic review of 115 studies using enzyme‐linked immunosorbent assay (ELISA) to detect pathogen antibodies in wild mammals across 298 individual research projects. We discuss the range of mammalian hosts and the pathogens most represented in the literature and suggest improvements to consider when using samples from decaying animals for seroinvestigation. Our results indicate that the most examined mammals were artiodactyl ungulates and that antibodies against the bacteria Brucella spp. are those most examined to date, followed by the protozoan Toxoplasma gondii, malignant catarrhal fever virus, Schmallenberg orthobunyavirus, and the coccidian Neospora caninum. The effect of haemolysis in serum samples on ELISA results was considered in only 3.7% of the research projects we reviewed, sample size decisions were not outlined in 83% of research projects, and half of the projects did not examine seroprevalence in different age classes. Nevertheless, these details are important for replicability and accurate interpretation of results. We highlight the benefits and limitations of detecting antibodies against pathogens in carcasses from wild mammalian groups. Wild mammals are reservoirs for a wide range of pathogens including those with zoonotic potential and investigating their carcasses can help wildlife investigators to better understand the epidemiology of many diseases with implications for wildlife, livestock, and human health.

A-343 Novel In-Line Hemolysis Detection on a Blood Gas Analyzer for Capillary Samples

Abstract Background Point-of-care (POC) blood gas and electrolyte analyzers utilizing whole blood as the predominant sample type are blinded by the presence of hemolysis until today. Moreover, capillary samples are particularly vulnerable to in-vitro hemolysis due to excessive force applied during sample collection. Here, we introduce the GEM Premier 7000 with iQM3 (GEM 7000) which incorporates automatic, in-line hemolysis detection in capillary blood gas samples without a need for additional sample volume or reagents. Methods The hemolysis detection module which is integrated into the disposable GEM cartridge uses acoustophoretic technology and photometric measurements to detect hemolysis in whole blood. In GEM 7000, hemolysis of ≥ 116 mg/dL was found to have significant interference with potassium results. A hemolysis flag is presented as the degree of hemolysis: none (0 - 50 and 51 - 115), mild (116 - 200 and 201 - 300), moderate (301 - 400), and gross (≥401). Due to volume limitations with native capillary samples and challenges in assessing hemolysis levels with a comparator assay, venous blood spiked volumetrically with hemolysate and transferred to capillary devices was used to verify the hemolysis flag performance. An agreement analysis was performed using the Roche cobas hemolysis value. The device's (GEM 7000) ability to flag hemolyzed capillary samples is demonstrated in a simulated clinical setting using native and contrived fingerstick capillary samples. Results Hemolysis verification with transferred capillary samples demonstrated that 98.2% (274/279) of samples were correctly flagged based on the cobas hemolysis value of spiked hemoglobin. Hemolysis in the native capillary samples (N = 235) was primarily distributed in the 0-50 mg/dL range. Additionally, native capillary samples altered using vigorous flea mixing (> 1 minute) showed hemolysis distribution over a wide hemolysis degree (none to gross), and their potassium results were flagged accordingly. Conclusions By providing integrated hemolysis detection in capillary blood sample measurements, the GEM Premier 7000 with iQM3 system can provide quality and confidence in the clinical interpretation of critical results such as potassium.

A-362 Hemolysis Detection and Flagging of Potassium Results on GEM Premier 7000 with iQM3

Abstract Background Blood gas analyzers that report whole blood potassium (K+) are vulnerable to inaccurate results due to in vitro hemolysis. GEM Premier 7000 with iQM3 (GEM 7000) automatically detects hemolysis (plasma free hemoglobin) and flags impacted K+ results. Here, we report the hemolysis impact on K+ measurements and the flagging approach used in the GEM 7000 system. Methods Hemolysis interference was assessed according to CLSI EP07c at low (3.8 mmol/L) and high (5.3 mmol/L) K+ levels. Ultrasonically-lysed whole blood was used to generate a high test pool (100% = 1000 mg/dL hemoglobin). This was mixed volumetrically with a low pool (0% = native blood) to create additional test pools (25%, 50%, and 75%) and analyzed on three (3) GEM 7000 systems. K+ bias at each test pool was calculated from the low pool to generate hemolysis interferograms. Linear regression was performed to determine the hemolysis level that exceeds the interference limit of 7%. Results Hemolysis exceeding the interference limit was ≥ 116 mg/dL for the low K+ and ≥ 147 mg/dL for the high K+ level. These correspond to an elevation of 0.27 mmol/L and 0.37 mmol/L of K+, respectively. Based on this study, the GEM 7000 triggers a hemolysis flag when the detected hemolysis exceeds 115 mg/dL. By default, GEM 7000 hemolysis flags are categorized into mild (116 -300 mg/dL), moderate (301 - 400 mg/dL), and gross (≥401 mg/dL) hemolysis. Conclusions The magnitude of the observed increase in K+ (0.277 - 0.295 mmol/L for every 100 mg/dL of hemoglobin) on the GEM 7000 due to in vitro hemolysis is consistent with previously published reports in the literature. Automated hemolysis detection and flagging of K+ results in GEM 7000 overcome the limitation of the inability to detect hemolysis in blood gas samples.

A-098 Reducing Hemolyzed Specimen Rejection for Aspartate Aminotransferase (AST): A Quality Improvement Initiative to Further Optimize Concentration-Specific H-index Thresholds

Abstract Background Serum aspartate aminotransferase (AST) is a commonly ordered test useful for diagnosing and monitoring hepatocellular damage. The hemolysis (H) index interference threshold for the Roche Cobas AST reagent is H=20, which is the lowest of all tests on Roche chemistry platforms (Cobas c701, c502, and e801). Previous optimization studies supported the implementation of an AST concentration-specific tiered hemolysis interference threshold schema: AST<100 U/L (H=50), AST=100-200 U/L (H=100), AST=201-300 U/L (H=200) and AST>301 U/L (H=500). This schema significantly reduced redraws; however, a significant number of rejected samples were noted to have AST results within the reference interval (RI). The aim of this study was to establish an H-index threshold for samples with AST results within the RI. Methods Twelve pools of residual serum samples were created with AST concentrations ranging from 17-66 U/L. Each pool was split into 2 aliquots, receiving either 10% of a lab-produced hemolysate solution generating H-indices from 62-508 or 10% of a 0.9% saline control. Aliquots were mixed in equal portions to create a series of 5 samples with increasing hemolysis according to CLSI guideline EP-07 and analyzed in EP Evaluator. Median(interquartile range (IQR)) H-index thresholds were calculated for error limits beginning at +5 U/L and increasing incrementally (+1 U/L) to +10 U/L. Clinical cases with an H-index slightly above the hemolysis interference threshold (50≤H≤70) and results within the RIs (Male:8-48U/L; Female:8-43U/L) were reviewed by the Chemistry Resident/Fellow on-call (n=68) over a period of 8 months. Results were released with a comment if risk of clinical misinterpretation due to a falsely elevated result was low. Previous, non-hemolyzed AST results were compared within 45 days, if available, by paired-Wilcoxon test (Graphpad Prism). Results The median(range) of initial AST results in the serum pools was 20(17-66) U/L. The calculated H-index thresholds (median(IQR)) using error limits of +5, +6, +7, +8, +9, +10 U/L were 49(43-53), 56(52-63), 66(61-78), 78(72-81), 91(84-91) and 101 (96-107), respectively. All slightly hemolyzed AST results (50≤H≤70) within the RI were released with a comment upon chart review. Previously reported AST values (n=23) were compared to current (hemolyzed) concentrations and found to be statistically significantly increased relative to previous results from the same patient (median(range) difference: 3.7(-3.6-11)U/L, p=0.009). However, the median difference was <5U/L or 10%, the laboratory’s criteria used to define a clinically significant difference. Conclusions A median H-index limit of up to 100 can be applied to samples when AST is within the RI. The risk of misinterpretation for results with mild-moderate hemolysis was low in the cases reviewed. Sample rejection rates and reporting delays can be diminished by implementing this practice. Interpretation can be further enhanced by appending the following comment to hemolyzed samples: “Specimen was received with hemolysis slightly above the accepted threshold. This degree of hemolysis is associated with a false increase of approximately 5-10 U/L. Interpret in conjunction with other laboratory and clinical findings.”

A-209 Preanalytical Phase Errors Constitute the Vast Majority of Errors in Clinical Laboratory Testing

Abstract Background Laboratory errors can occur during the preanalytical, analytic, or post-analytic phase of testing and may pose a considerable threat to patient safety. Studies have estimated that pre-analytical error encapsulates ∼45-68% of laboratory errors and occurs in up to 1.52% of specimens. However, limitations of previously published studies include the use of manual error detection, reliance on manual transcription, small study sizes, and exclusion of common errors. Herein, we aim to identify the types and frequency of errors occurring in each phase of clinical laboratory testing using a large retrospective dataset across a broad range of errors detected in clinical laboratories. Methods This study was performed in the Barnes-Jewish Hospital core laboratory which provides testing for a 1,400-bed tertiary care hospital and included all tests performed for clinical chemistry, special chemistry, toxicology, automated hematology, blood gas testing, coagulation, non-infectious serology, infectious disease serology, urinalysis, and send out testing between January 2022 and May 2023. Errors were compiled from two separate sources. The first source of errors was captured from the laboratory information system (LIS, Cerner). These errors were flagged manually by laboratory staff in real time. The second source of error was a daily report manually curated by a trained medial laboratory scientist as part of routine, laboratory operating procedures and standard workflow. Results There were ∼40,000,000 individual results from ∼11,000,000 specimens during the study period and 87,317 errors observed (∼0.8% of specimens). Of these errors, specimens that were hemolyzed sufficiently to impact result but still reportable were the majority (41,047, 47.2%). When excluding minimal hemolysis, there were 46,270 errors documented. Among these, 44,847 (97.0%) were in the preanalytical phase, 451 (1.0%) were in the analytical phase, and 972 (2.0%) occurred in the postanalytical phase. Subcategorization of preanalytical errors revealed that most of the errors were related to hemolyzed samples requiring results to be suppressed (n=19,701), quantity not sufficient samples (n=8,068), clotted specimens (n=5,840), collection errors (e.g., wrong tube type, n=5,780), not on ice (n=1,369) and IV fluid contamination (n=1,122). In the analytical phase, the causes of error were interfering substances (n=279), QC out of range (n=65), instrument problems (n=58) and reagent issues (n=49). Among the postanalytical errors, 581 were comment errors, 216 were results entry errors, 131 were comments errors with potential impact on patient care, and 44 were dilution errors. Conclusions This study demonstrates that the vast majority of lab errors (∼97.0%) occurring during the preanalytical phase. The high incidence of preanalytical errors, especially errors related to sample integrity implies a need for enhanced tools and training to minimizing errors from specimen collections, maintenance of sample stability, transportation, and processing.

Циркулирующие микроРНК тромбоцитарного происхождения как потенциальные биомаркеры STEMI у пациентов с гипертонической болезнью

Background: Circulating extracellular platelet-derived microRNAs have been actively studied over the last decade as promising biomarkers of cardiovascular disease (CVD), since platelet activation plays a key role in the pathogenesis of CVD. The aim of the study: To compare platelet-derived microRNA profiles in patients with uncomplicated hypertension (HT) and in patients with HT and acute myocardial infarction with ST-segment elevation (STEMI). Materials and methods: The study included 20 patients with uncomplicated HT and 15 patients with HT and STEMI. At least 90% of the participants in each group received antiplatelet therapy. A blood sample was collected from each patient in a CTAD anticoagulant tube and blood plasma was obtained by two-step centrifugation. The degree of hemolysis of the plasma samples was determined spectrophotometrically. MicroRNAs were isolated from plasma samples and used for reverse transcription and quantification of relative levels of eight main circulating extracellular platelet-derived microRNAs (miR-223-3p, miR-126-3p, miR21-5p, miR-24-3p, miR-320a-3p, miR-191-5p, miR-150-5p and miR-23a-3p) by quantitative PCR using the TaqMan Advanced technology (Thermo Fisher Scientific, USA). Normalized relative expression levels for each miRNA were calculated. Differences were considered statistically significant at p < 0.05 (Mann-Whitney test). Results: Patient groups did not differ in age, BMI, platelet count, lipid profile and degree of hemolysis of plasma samples. No differences were found between the ratio of platelet-derived and erythrocyte-derived microRNA between patient groups. No statistically significant differences were found in the relative levels of circulating microRNA between the study groups. Principal component analysis and heatmap analysis of microRNA expression in the samples showed a division of the samples into groups based more on the degree of hemolysis than on the presence/absence of STEMI. Conclusion: In the studied sample of hypertensive patients, the effect of hemolysis of plasma samples on circulating platelet-derived microRNA profiles was more significant than the presence of STEMI.

Haemolysis overestimates plasma oxidative stress biomarkers in free-ranging roe deer

Quantifying oxidative stress has garnered extensive interest in evolutionary ecology and physiology since proposed as a mediator of life histories. However, while the theoretical framework of oxidative stress ecology is well-supported by laboratory-based studies, results obtained in wild populations on oxidative damage and antioxidant biomarkers have shown inconsistent trends. We propose that red blood cell lysis could be a source of bias affecting measurements of oxidative stress biomarkers, distorting the conclusions drawn from them. Using an experimental approach consisting of enriching plasma from roe deer with lysed red blood cells, we show that the values of commonly used oxidative stress biomarkers linearly increase with the degree of haemolysis – assayed by haemoglobin concentration. This result concerns oxidized proteins (carbonyls) and lipids (TBARS), as well as enzymatic (superoxide dismutase) and non-enzymatic (trolox assay, OXY assay) antioxidant markers. Based on 707 roe deer blood samples collected in the field, we next show that the occurrence of haemolysis in plasma samples is negatively related to age. Finally, we illustrate that considering the variance explained by age-related haemolysis improves explanatory models for inter-individual variability in plasma oxidative stress biomarkers, without substantially altering the estimates of the parameters studied here. Our results raise the question of the veracity of the conclusions if the degree of haemolysis in plasma is not considered in animal models such as roe deer, for which the occurrence and severity of haemolysis vary according to individual characteristics. We recommend measuring and controlling for the degree of haemolysis be considered in future studies that investigate the causes and consequences of oxidative stress in ecophysiological studies.

Novel In-Line Hemolysis Detection on a Blood Gas Analyzer and Impact on Whole Blood Potassium Results.

Preanalytical error due to hemolyzed blood samples is a common challenge in laboratory and point-of-care (POC) settings. Whole blood potassium (K+) measurements routinely measured on blood gas analyzers are particularly susceptible to hemolysis, which poses a risk for incorrect K+ results. The GEM Premier 7000 with IQM3 (GEM 7000) blood gas analyzer provides novel integrated hemolysis detection within the sample measurement process. Therefore, the GEM 7000 can detect and flag hemolyzed whole blood samples at the POC, warning the operator of potentially erroneous results. Heparinized venous or arterial whole blood samples were used for K+ interference studies and assessed for hemolysis agreement utilizing either a traditional volumetric method or chemistry analyzer serum index measurements with the Roche cobas c311 or Abbott Alinity c. Hemolysis interference studies performed at 2 different K+ concentrations (3.8 and 5.3 mmol/L) identified that a plasma free hemoglobin ≥116 mg/dL can impact K+ results on the GEM 7000. Hemolysis agreement studies demonstrated an excellent agreement of >99% with the volumetric method, 98.8% with cobas H index, and 96.4% with Alinity H index. GEM 7000 K+ results were correctly flagged for both native and spiked samples. GEM 7000 hemolysis detection provides a novel technology to detect hemolysis in whole blood samples. Moreover, the GEM 7000 demonstrates excellent agreement with traditional laboratory hemolysis detection methods and offers an integrated technological solution for assuring the quality of whole blood K+ results in POC settings.

Method comparison between two hemostasis analyzers: STARMax3 and Cobast511

The STARMax3 (Stago, France) and Cobas t511 (Roche Diagnostics, Germany) are two automated hemostasis analysers that can be used to perform a wide range of tests. The STARMax3 uses a mechanical clot detection system to measure coagulation times, while the Cobas t511 uses optical detection. The aim of this study was to compare the analytical performance of these two analysers using fresh plasma samples with or without a risk of interference due to hemolysis or lipemia. For plasma samples without interference, acceptable agreement was observed for prothrombin time (PT) (n =55), activated partial thromboplastin time (APTT) (n =56), fibrinogen (n =56) and factor II (n =43) with R² of 0.907, 0.963, 0.979 and 0.968 respectively. For factor V (n =43) and D-dimers (n =45), agreement was less acceptable, with respective R² of 0.756 and 0.887, but with no clinical impact. For hemolysed samples, acceptable results were observed for PT, fibrinogen and D-dimers, but three results were clinically discordant for APTT, and STARMax3 offered greater robustness for processing highly lipemic samples.

Bridging the gaps: A prospective analysis of root causes for rejection and incompleteness in blood requisition forms

IntroductionBlood request form (BRF) stands as a pivotal document in ensuring safe and effective blood transfusions within healthcare settings. Incomplete or erroneous data on BRF can heighten risk of adverse reactions and compromise patient safety. Aim of study was to assess level of completion of BRFs by clinicians and to evaluate root cause analysis (RCA) of incompleteness of BRFs and factors leading to their rejection. Materials and MethodsThis prospective study was carried out from February 2024 to April 2024 on BRFs received in the blood centre. They were audited and RCA for factors leading to their incompleteness and rejection were analysed. ResultsTotal number of BRFs received in blood centre was 14,468. 13,358 (92.3%) BRFs were accepted and 1,110 (7.7%) BRFs were rejected. 12,804 (95.85%) of accepted BRFs were incomplete. Weight was the most common missing parameter (89% {n = 11403}) while name of the requesting clinician was least common (2.5% {n-318}). 3.52% n = 510) BRFs were rejected due to mismatch in name and patient registration number on BRF and samples. 0.14% n = 21) BRFs were rejected due to hemolysed samples. RCA for incompleteness of BRFs showed that main reason was manpower (61–83%) while environment was least common (17–67%). RCA for rejection of BRFs showed that environment was most common cause (13.3–80.15%) while manpower was least common (9–19.85%). ConclusionRegular audits and personnel training, and quality assurance measures can help identify and address deficiencies in BRF completion to enhance patient safety and reduce incidence of transfusion-related errors and complications.

The polychromatism of postmortem cerebrospinal fluid.

Based on the assumption that postmortem cerebrospinal fluid (CSF) is contaminated depending on the chosen sampling technique in the forensic setting resulting in bloody or at least hemolytic CSF samples, we systematically documented a total of 183 postmortem CSF samples. These samples were all assessed for their quality and color, regardless of the cause of death or the postmortem interval. The investigations were carried out through subjective assessment of color and turbidity, as well as objective measurements of the optical density (OD) of the CSF supernatants after centrifugation of each sample, with standardized photographic documentation. The observations revealed that in 28 cases the CSF was absolutely (crystal-) clear and transparent. Most of our samples showed color changes ranging from xanthrochromic to rose. Intensive staining of the supernatants was only found in a small proportion of the examined collective. We found that postmortem CSF has no uniform appearance but rather a diverse range of color spectra, and the color, as well as the OD of the CSF, correlates significantly with the postmortem interval (p < 0.001) when sampled using the proposed standard procedure.

Phosphorylated tau 181 (p-tau181) as an innovative, fast and robust biomarker for cerebrospinal fluid leaks.

Cerebrospinal fluid (CSF) leaks can lead to serious complications if left untreated, making rapid and accurate diagnosis essential. Biomarkers such as β2-transferrin (B2TRF) and β-trace protein are used to detect CSF leaks, but their limitations warrant the exploration of alternative markers. This study investigates the potential of phosphorylated tau at threonine 181 (p-tau181) as a biomarker for CSF leaks. Samples from 56 subjects were analyzed for B2TRF and p-tau181 using immunoaffinity blotting and chemiluminescent enzyme immunoassay, respectively. Data analysis included Mann-Whitney test to assess the overall difference in median p-tau181 concentrations between B2TRF positive and negative patients and a receiver operating characteristic (ROC) curve analysis to determine optimal p-tau181 cutoff values for predicting B2TRF positivity. p-tau181 levels were significantly higher in B2TRF positive samples compared to negative samples (p < 0.001). ROC analysis showed high diagnostic performance for p-tau181, with an optimal cutoff of 13.22 pg/mL providing 92.0% sensitivity and 93.1% specificity. Excluding hemolyzed samples improved further the diagnostic performances, maintaining high sensitivity (90.9%) and achieving perfect specificity (100.0%). This study highlights the potential of p-tau181 as a valuable biomarker for the detection of CSF leaks due to its high diagnostic accuracy and practical advantages over the current biomarkers. The characteristics of p-tau181 assay being both quantitative and rapid, with high diagnostic accuracy, suggest that it could be a valuable tool for the detection of CSF leaks. Further research are now needed to validate these findings.

Effect of hemolysis on D-dimer testing measured with the Improgen kit: is all manufacturer information correct?

D-dimer is a fibrin degradation product and its measurement is affected by hemolysis. This study was designed to reveal the value of hemolysis affecting D-dimer in our laboratory. In this study, hemolysate samples obtained by both mechanical and freezing methods were used. D-dimer levels of all plasmas were measured with Improgen Diagnostic kit by immune-turbidimetric method. Numerical change in hemolyzed samples was evaluated by calculating the percentage difference, and clinically significant differences were evaluated by calculating the maximum acceptable bias (MAB). In the hemolysate study prepared by both freeze-thaw and mechanical methods, it was observed that low D-dimer levels did not exceed the total allowable error (TAE) (30%) up to +2 hemolysis (corresponds to hemoglobin = 1.01-2 g/l) and did not exceed the MAB (65%) even at +4 hemolysis (corresponds to hemoglobin = 1.01-2 g/l). High D-dimer levels did not exceed the limit values of both TAE (30%) and MAB (68%) even in +4 hemolysis. The D-dimer test was affected by lower levels of hemolysis compared to both other studies and the values in the kit insert (hemoglobin >5 g/l corresponds to +4 hemolysis index). We verified the hemolysis interference in the D-dimer test, which we thought was not compatible with the kit insert, under our own laboratory conditions. This is the first hemolysis interference study performed with the Improgen brand d-dimer kit. In samples with a hemolysis rate of +2 and above, it would be more accurate to reject the D-dimer result as a 'hemolyzed sample'.

Clinical Application of ISO and CEN/TS Standards for Liquid Biopsies-Information Everybody Wants but Nobody Wants to Pay For.

Liquid biopsies are emerging as valuable clinical biomarkers for cancer monitoring. Although International Organization for Standards (ISO) and Technical Specifications from the European Committee for Standardization (CEN/TS) standardized workflows exist, their implementation in clinical practice is underdeveloped. We aimed to assess the applicability of ISO and CEN/TS standards in a real-world clinical setting, with a particular focus on evaluating the impact of preanalytical parameters and hemolysis on liquid biopsy analysis. We evaluated 659 peripheral blood samples from advanced prostate cancer patients against ISO and CEN/TS standards and documented all essential criteria, including tube draw order, filling level, temperature, and time tracking from blood draw to storage. We assessed hemolysis and its effect on circulating tumor DNA (ctDNA) and circulating tumor cell (CTC) analysis. Our results demonstrated a high compliance rate, with 96.2% (634/659) of samples meeting essential ISO and CEN/TS criteria. We did not observe a significant impact on ctDNA or CTC detection rates between hemolytic and nonhemolytic samples. Hemolysis was identified in 12.9% (40/311) of plasma samples from our advanced prostate cancer cohort, and within the draw order of 5 blood collection tubes, hemolysis did not significantly increase from tube 1 to 5. In total, 83.8% (552/659) of blood collection tubes had high fill levels above 80% of nominal filling level. Our study demonstrates the feasibility and benefits of adhering to ISO and CEN/TS standards in a clinical liquid biopsy study. The standards revealed that hemolysis occurred frequently but did not impair downstream ctDNA and CTC analysis in our cohort of advanced prostate cancer patients.

Analytical and Clinical Interference of Sample Hemolysis in Evaluating Blood Biochemical and Endocrine Parameters in Cows.

Hemolysis is a common cause of errors in laboratory tests as it affects blood parameters and leads to a positive or negative bias. This study aims to examine the relationship between the level of hemolysis (expressed as cell-free hemoglobin concentration, g/L) and the variability of metabolic and endocrine parameters and to determine the threshold level of hemolysis that causes an analytically and clinically significant bias for the twenty most frequently examined blood parameters in cows. Paired blood samples of 10 mL each were obtained from 30 cows. One was subjected to mechanical trauma and plasma was extracted directly from the other. Hemolyzed and non-hemolyzed samples from the same animal were mixed to obtain final samples with cell-free hemoglobin concentrations of 0, 1, 2, 4, 6, 8, and 10 g/L. Metabolic and endocrine parameters were measured in the samples and their deviation and the linear equation between the level of hemolysis and the deviation were determined. The following threshold values of hemolysis were determined, which correspond to the acceptable analytical (lower value) and clinical (upper value) levels of parameter variability: BHB 0.96 and 4.81; NEFA 0.39 and 3.31; GLU 0.38 and 3.90; ALB 1.12 and 6.11; TPROT 1.40 and 6.80; UREA 6.62 and 20.1; TBIL 0.75 and 5.65; AST 0.11 and 2.18; GGT 1.71 and 8.90, LDH 0.01 and 0.11, ALP 0.97 and 2.95; TGC 1.56 and 15.5; CHOL 1.29 and 8.56; Ca 5.68 and 25.7; P 0.57 and 8.43; Mg 1.10 and 8.47; INS 1.15 and 3.89; T3 8.19 and 15.6; T4 8.97 and 18.5; and CORT 2.78 and 11.22 g/L cell-free hemoglobin. Three decision levels are available for each metabolic and endocrine parameter: if hemolysis is below the lower (analytical) threshold value, results can be reported without restriction; if hemolysis is between the lower and upper thresholds, the results can be issued with guidance in the form of corrective linear equations; and if hemolysis is above the upper (clinical) threshold, the results and sample must be discarded. This method contributes to an optimal approach to hemolysis interference with metabolic profile parameters in blood samples from cows.

Accuracy of screening for hemolysis in plasma samples using a commercial urine dipstick

Objectives: Identification and quantification of hemolysis in serum or plasma samples is an important requirement in laboratory diagnostics; however, this is not always possible automatically, and visual sample inspection is not sufficiently accurate. We have planned this study to determine whether a commercially available urine dipstick with a hemoglobin-reactive pad could be used for this purpose. Materials and Methods: Sixty-five routine plasma samples, whose hemolysis index (HI) was previously determined on Roche Cobas 8000, were assayed with a commercial urine dipstick to obtain semi-quantitative data on plasma hemoglobin. Plasma was diluted 1:1000 in water to enter the dipstick’s range of hemoglobin measurement, and 10 µL of this dilution was applied to the hemoglobin pad. Results were visually interpreted within 60 s by comparing the pad color with that on the dipstick box label. Statistical Analysis: Semi-quantitative test results obtained with the urine dipstick were directly correlated with those of the same samples assayed on Roche Cobas 8000 using non-parametric Spearman’s correlation and agreement. Results: The sample size consisted of 40 non-hemolyzed (HI ≤0.3 g/L) and 25 hemolyzed (HI &gt;0.3 g/L) plasma samples. Spearman’s correlation between Cobas HI and dipstick hemoglobin concentration was r = 0.96 (95% confidence interval, 0.93–0.97; P &lt; 0.001). The concordance of hemolysis detection was 95.4%, with 1.00 sensitivity, 0.93 specificity, 1.00 negative predictive value, and 0.89 positive predictive value compared to the reference HI measurement on Cobas. The cumulative agreement between Cobas HI and the various plasma hemoglobin thresholds obtained with the dipstick was 75.4%. Conclusions: We have demonstrated here that plasma hemoglobin assessment with commercially available urine dipsticks may generate semi-quantitative test results accurate enough to influence decision-making regarding sample quality and its suitability for testing.

A simple algorithm based on complete blood count and haemolysis index to enable potassium determination in haemolysed samples

A simple algorithm based on complete blood count and haemolysis index to enable potassium determination in haemolysed samples

Effect of Hemolysis on Serum Markers of Liver Function

Introduction Samples received in the laboratory are often rejected because of visible hemolysis. Every laboratory has protocols on whether to accept or reject hemolyzed samples based on tests ordered. The objective of the study was to observe and analyze the variations seen in association with hemolyzed samples in liver function test parameters and electrolytes measurement. MethodsThis cross-sectional observational study was carried out in 88 laboratory samples in the Clinical Biochemistry Laboratory. Hemolysis was induced by mechanical mixing. This study compared reported values of bilirubin, protein, albumin, aminotransferases (ALT and AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), sodium and potassium between hemolyzed and non-hemolyzed samples. ResultsMean values of total bilirubin (TB) and direct bilirubin (TB), ALT, ALP, GGT and sodium (Na) were found to be significantly decreased post hemolysis (p&lt;=0.001) with mean variances (σ2) at 0.30 (TB), 0.38 (DB), 0.56 (ALT), 0.18 (ALP), 0.01 (GGT) and 0.001 (Na). In contrast, mean values of AST (σ2= -0.9, p&lt;0.001) and potassium (σ2 = -0.3, p&lt;0.001) were found to be significantly elevated post hemolysis. Total protein and albumin were also found to be elevated in hemolyzed samples but the average variance was not statistically significant. Degree of hemolysis had highly varying effects in some parameters like AST and total protein, but less varying and more consistent effects on other measured parameters. ConclusionFree hemoglobin estimation should be considered before rejecting a clinical sample because of hemolysis. Mathematical equations cannot be expected to reproduce corrected values of the analytes in question because of the large variances, so these tests need to be repeated on non-hemolyzed samples for the analytes that are affected.Keywords

Potential medical impact of unrecognized invitro hypokalemia due to hemolysis: a case series.

The destruction of red cells during blood collection or with the processing of the sample continues to occur at a high rate, especially among emergency department (ED) patients. This can produce pre-analytical laboratory errors, particularly for potassium. We determined the incidence of hemolyzed samples and discuss the potential medical impact for hypokalemic patients who potassium level is artificially normal (pseudoeukalemia). Potassium results were obtained for a 6-month period. Using a measured hemolysis index (HI), hemolysis was present in 3.1 % for all potassium ordered (n=94,783) and 7.5 % for ED orders (n=22,770). Most of these samples were reported as having high normal result or were hyperkalemic. There were 22 hemolytic samples with a potassium of <3.5 mmol/L, and 57 hemolytic samples with a potassium in lower limit of normal (3.5-3.8 mmol/L). From this group, we examined the medical histories of 8 selected patients whose initially normal potassium levels were subsequently confirmed to have a potassium values that were below, at, or just above the lower limit of normal due to hemolysis. The primary complaint for these patients were: necrotizing soft tissue infection, pancreatitis, volume overload from heart failure with reduced ejection fraction, hypertension treated with hydrochlorothiazide, and presence of a short bowel syndrome. A subsequent non-hemolyzed sample was collected demonstrating hypokalemia in all of these patients. Within these cases, there was a potential for harm had hemolysis detection not been performed. We demonstrate the medical importance of detecting hemolysis for patients who have pseudoeukalemia. This is relevant because the HI cannot be obtained when electrolytes are tested using whole blood samples, and a normal potassium may lead to inappropriate patient management.