PO-01 / PV-02:
Using metabolite profiling to unravel potential quality indicators to assess a delayed sample processing in serum
Sven Heiling | Institut für klinische Chemie und Laboratoriumsdiagnostik | Germany
Background: Omics advancements are influenced by ineffectively characterized and harmonized preanalytical conditions that can be a noteworthy wellspring of fluctuation. This variation causes in between 50-85% of clinical errors in clinical diagnostic testing. Consequently, quality indicators must be examined to survey the nature of body liquids and guarantee reliable biomedical research.
Methods: Human serum was subjected to prolonged incubation at room temperature before centrifugation. An extensive metabolite profiling was performed using a high-resolution mass spectrometry-driven approach followed by pathway and chemical similarity enrichment using the web-based tools MetaboAnalyst and ChemRICH. Furthermore, targeted tandem-MS analyses were used to quantify the selected quality indicators in samples of healthy volunteers and patients with cardiologic and rheumatologic diseases.
Results: We investigated the metabolome of serum samples from a healthy cohort (n=10) after 0.5 and 2 h pre-centrifugation delay. To rapidly select quality indicators we performed a chemical similarity enrichment analysis and discovered several highly promising clusters of chemically similar compounds that belong to the eicosanoid metabolism, as well as blood coagulation factors, purine, and pyrimidine derivatives. Additionally, we applied a pathway enrichment approach on the identified clusters and designed scores for promising metabolic ratios. We calculated diagnostic thresholds such as the HID-Score (Hypoxanthine/Inosine/Dihydroorotate-Score), investigated the eicosanoid metabolism and verified our results using additional cohorts of healthy volunteers and patients (n=50) showing a high prediction accuracy with AUROC-values of 0.92 for TTC < 60 min.
Conclusion: The assessment of the quality of body fluids is challenging in clinical research and diagnostics. To guarantee high-quality biospecimen, quality indicators are of utmost importance, and we conceive that this work will stimulate future research into these potential metabolites/metabolic ratios and their possible role as quality biomarkers in biobanking.
Lactate dehydrogenase activity in serum and heparin plasma samples is affected by filling volume in vacuum tubes
Dr. med. Nick Neuwinger | Charité-Universitätsmedizin Berlin | Germany
BACKGROUND: The activity of lactate dehydrogenase (LDH) is routinely monitored, e.g. for stratification of malignant diseases, but is susceptible to preanalytical influences.
METHODS: Blood was withdrawn by antecubital venipuncture from healthy volunteers and collected in vacuum blood collection tubes (serum, SE; heparin plasma, HP). The tubes were either filled completely (≥90% of volume), or were underfilled to approximately 50%. Thereafter, tubes were centrifuged directly or stored at room temperature for 4 h prior to centrifugation. We systematically analyzed potassium, sodium, chloride, LDH, creatine kinase, total cholesterol, and indices for hemolysis, icterus, and lipemia. In a subset of tubes we also measured blood filling velocity. Finally, in an in-patient cohort of more than 74,000 patients, we analyzed plasma yield and data distribution of LDH by means of the DGKL Reference Limit Estimator (RLE) to reconfirm our findings in healthy volunteers.
RESULTS: The activity of LDH was higher in HP tubes compared to SE. The reduction of the filling volume of tubes was followed by significantly higher LDH values (SE: +21.6%; HP: +28.3%), potassium (SE: +4.2%; HP: +5.3%), and hemolysis index (SE: +260.8%; HP: +210.0%). In contrast, levels of other analytes remained generally stable. The filling velocity of blood in the collection tubes was approx. 3-fold higher in the first compared to the second half with no differences between HP and SE. Notably, the plasma yield in the routine patients cohort also inversely correlated with the activity of LDH. Estimation of reference limits for LDH revealed established intervals only in patients’ tubes that were adequately filled.
CONCLUSIONS: The reduction of filling volume in both SE and HP collection tubes leads to significant increases in LDH activity, both in healthy volunteers and in inpatients. This may be considered in cases of clinically implausible enhanced levels of LDH.
Experiences from 6 Years of Quality Assured Model of End-Stage Liver Disease (MELD) Diagnostics
Pascal Hunold | University Hospital Leipzig | Germany
The MELD (Model of End- Stage Liver disease) score is established for allocation of liver transplants and has to guarantee a sensitive, specific, objective and fair organ allocation. The score is based on three medical laboratory parameters: bilirubin, creatinine and international normalized ratio (INR).
In 2012 the University Hospital Leipzig created and established a quality ensured lab-MELD-Diagnostic. Every MELD score is directly requested at the laboratory system, calculated and specifically validated by laboratory physicians. Within this process the three parameters as well as the MELD score are checked for plausibility in the context of pre-values and other patients results. This study analyses the experiences since implementation of the laboratory medical quality assurance system for MELD-Diagnostics.
Since 2012, 7270 MELD scores were requested. The majority of these requests could be reported to Eurotransplant without any hint of limited validity. However, 202 scores could not be reported initially, 145 of these cases could not have been reported to Eurotransplant at all for various reasons. In 34 cases the dialysis status did not meet the requirements and 1 case of unstated intake of oral anticoagulation could be determined and corrected within the process of validation.
The laboratory medical quality assurance system for MELD-Diagnostics was effective in identification of results with limited validity. Therefore, it should be applied to transplant centers to prevent diagnostic errors with possible adverse effects for the patients.
Reduction of hemolysis rate by using VACUETTE® HOLDEX® Single-Use Holder or VACUETTE® SAFELINK blood collection device
Dr. Sirid Griebenow | Greiner Bio-One GmbH | Austria
Background: Hemolysis is one of the main sources for sample rejection in a clinical laboratory as it can strongly influence the reliability of results. Using an intravenous line for blood collection frequently causes hemolysis. The target of two studies was to show the performance of the HOLDEX® single use holder and SAFELINK in reducing the hemolysis rate by preventing erythrocyte ruptures, whereby these holders have potential to reduce shear stress caused by differences of pressure between veins and evacuated tube systems. A flash chamber which reduces the flow rate results in a softer draw.
Methods: Blood was drawn from 60 ED patients in Italy into VACUTAINER SST II Plus tubes with a 20G catheter and using a conventional holder (VACUTAINER One Use Holder, Becton Dickinson) or VACUETTE® HOLDEX (Greiner Bio-One). All tubes were centrifuged according to the manufacturers’ instructions. Serum was tested for potassium, lactate dehydrogenase (LDH) and hemolysis index (HI). In an additional second study, 30 healthy volunteers’ blood were drawn to determine 24 chemistry and immunochemistry analytes in lithium-heparin plasma (on Cobas 8000, Roche), hematology parameters in EDTA plasma (on Sysmex XN, Sysmex, Japan) and hemostasis parameters in citrate plasma (on ACL Top 700, Instrumentation Laboratory, USA) using either HOLDEX or SAFELINK in paired specimens. Blood collection was based on two sequential venipunctures, on one arm by using a 19 gauge straight luer needle attached to a HOLDEX and on the other arm using a 19 gauge straight needle attached to SAFELINK. All subjects provided informed consent and approval by ethical committee was given.
Results: In samples collected with VACUTAINER One Use Holder, concentrations of potassium (4.25 vs. 4.16 mmol/L), LDH (498 vs 459 U/L) and cell-free hemoglobin (fHb) (0.42 vs. 0.22 g/L) were higher than with HOLDEX. The frequency of samples with fHb >0.5 g/L was not statistically significant, whilst that of grossly hemolyzed samples was significantly higher for samples collected with VACUTAINER One Use holder. The additional comparison study of HOLDEX and SAFELINK did not result in significant differences. Potassium was slightly higher (4.38 vs. 4.49 mml/L), but the percent bias remained within quality specifications. Of 30 samples, the rate of samples with HI >3 was lower when blood was collected with SAFELINK than with HOLDEX.
Conclusion: The results confirm the reduction in hemolysis rate when drawing blood from an intravenous line and using the holder HOLDEX or SAFELINK, as the specimen quality for parameter analysis is comparable when blood is collected by either of them. Both holders have potential to decelerate the blood flow pressure from veins into an evacuated blood collection tube, thus increasing the effectivity in the laboratory work flow by reducing the number of specimens requested repeatedly due to hemolysis.
Combination of informative biomarkers in small pilot studies and estimation of the sample size for extended studies
Amani Al-Mekhlafi | Helmholz Centre for Infection Research, Microbial Immune Regulation | Germany
Recently, Biomarkers are used widely in medical researches to detect the disease early which nowadays plays an important role in modern clinical and preventive medicine. According to the published data by GVK BIO Online Biomarker , over one hundred thousand biomarkers have been found, and abundance of biomarkers are discovered every year. As consequence, so many biomarkers can be studied when there is a new challenge. To avoid the waste of money and time, it is suggested to control the number of patients strictly at the beginning. For this reason, the pilot studies are commonly did in such studies as a necessary first step. The high AUC abundance (HAUCA) curves is a method that can show the high correlation of biomarkers with the disease exceed the pure random effect. By comparing the number of biomarkers that exceed specific value of AUC in the real data to the random data we can know if the data is worthy to be extended with bigger sample size. Expanding pilot studies sample size allows the researchers to determine at which size the selected markers are highly likely to have performance that is at or above the target level and the probability of finding the difference between the groups just by chance is controlled. However due to the small number of observation and high number of biomarkers, we expect high AUC values just by chance. By Wilcoxon-Mann-Whitney-U test, we compute the probability that an attribute with n values randomly assigned to two classes with a given prevalence exceeds a given AUC value . To remain the probability of getting at least one significant result just due to chance below the certain significant level, the multiple testing procedures are used (Bonferroni correction). The new sample size has been estimated by choosing a specific value of AUC to be validated and we assume the prevalence will be the same in the extended study as in the pilot. We increase n (No. of observations) and n+ (No. of positive class) gradually until we get the same AUC value with a significant corrected p-value. In the medical research, even if the performance of the feature is very high, is still not sufficient to provide sufficient accuracy [4,5]. For that, the researchers hope to achieve higher performance value and better efficiency of the statistical analysis by the improvement in the performance measure that obtained from the combination of biomarkers. Different modeling strategies have been proposed to select and combine biomarkers. However the ROC curve has turned out to be a standard tool for evaluating the diagnostic accuracy of a classifier [6,7]. In our study, we evaluate features by calculating the AUC of each single feature and the k top-ranked features with maximum AUC are selected. Furthermore, we address an important approach that are not widely explored in the literatures which is the correlation of the biomarkers within the groups. Bootstrapping has been used to measure the confidence interval for this combination.
Reliable Minimal Difference of Glucose concentration measurements based on ten year data
PD Dr. Astrid Petersmann | Universitätsmedizin Greifswald | Germany
Diagnosis of diabetes mellitus is based on cut-off values e.g. plasma glucose concentrations. Even though a measurement result is only complete when the attached measurement uncertainty is also reported, imprecision in laboratory measurements is rarely considered in patient care. Lately, the German Diabetes Society guideline on definition, classification and diagnosis on diabetes mellitus recommended to measure plasma glucose with an imprecision given as a minimal difference of 0.7 mmol/L, or less, at a concentration of 7.0 mmol/L. The MDcut-off describes the smallest analytical difference between a measurement and a cut-off which would be regarded as statistically significant different at a 95 % level of confidence, if a coverage factor (k) of 2 is used.
Material and Methods:
Two approaches were included to obtain imprecision: 1. Long-term imprecision based on Rili-BAEK based 24/7 patient care internal quality control (IQC) data (four concentrations) over a period of ten years and 2. detailed short-term imprecision assessed by hourly measurements of control materials (three concentrations) on three measuring systems connected to a laboratory automation used for glucose concentration measurement in the investigating laboratory. Minimal Difference for evaluation of a measurement result in relation to a cut-off value was 〖MD〗_(cut-off)=k×SD = 2 ×SD (k=2). To obtain representative values suitable for use in the diagnosis of diabetes mellitus the 95 % percentiles of the MDcut-off distributions for each concentration were calculated and used to derive a linear equation that allowed calculating MDcut-off for further glucose concentrations.
Results & Discussion:
Based on ten years of IQC data MDcut-off at the diagnostic cut-off of 7.0 mmol/L was found to be 0.45 mmol/L. In the short-term experiment MDcut-off at 7.0 mmol/L was 0.38 mmol/L. MDcut-off remained below the recommended limit of 0.7 mmol/L in both experiments. The variability introduced by slight performance differences within and between instruments can be covered by reporting the long term MDcut-off across all connected instruments. This procedure is recommended when multiple analysers are used in parallel, because the measurement uncertainty should be calculated for the complete laboratory and expressed per analyte as one MDcut-off value.
In this study setting stable results for MDcut-off were obtained after about thirty independent control cycles of MDcut-off. In the study setting this number was reached after one year, because the data from three analysers were combined. Medical laboratories may use IQC of an appropriate time interval to provide a reliable MDcut-off along with glucose concentration results used for diagnosis of diabetes mellitus.
Patient blood management: Single tube technology
PD Dr. Astrid Petersmann | Universitätsmedizin Greifswald | Germany
Patient blood management has become an important focus in providing laboratory results in patient care (1). The intention is to only conduct as few blood collections with as little blood volume as possible to avoid unnecessary blood loss for the patient. So far different tubes are needed for clinical chemistry, hematology and coagulation tests, each with a minimum volume and a dead volume. A new blood collection tube STTMP (Single tube technology multi parameters; Kabe, Nümbrecht, Germany) enables laboratories to conduct all tests in only one tube reducing the blood volume per draw by approximately 60% (4.5 ml Plasma, 2.7 ml Citrate Plasma, 2 ml EDTA whole blood vs. 3.5 ml). The German Rili-BAEK, as a document representing legal requirements, “aims to ensure, in particular, that Influencing factors and in-vitro effects during the pre-analytical phase are minimized […]” (2). Therefore the study evaluates whether the test results obtained from blood samples collected in STTMP are comparable to results collected in other currently commercially available tubes.
Blood from ten healthy volunteers was obtain by venous puncture. About 50 basic clinical chemistry, hematology and coagulation analytes were measured in primary tubes from Sarstedt and Becton Dickinson and compared to the new collection tube. Ethical approval and informed consent were provided prior to sampling. Analysers used in this study were: Dimension Vista 1500 for Clinical Chemistry and the Sysmex CS 2500 for coagulation (both Siemens Healthcare, Eschborn, Germany), TOSOH G8 for HbA1c (TOSOH Bioscience Europe, Griesheim, Germany), and Sysmex XN for hematology (Sysmex, Norderstedt, Germany).
Results & Discussion:
Significant differences of results between the established manufacturers Sarstedt and BD occurred in 10% of the analytes. About 50% of all investigated analytes did not differ significantly from either of the established primary tubes. For use in clinical settings a set of analytes could be identified that can be measured using the investigated new tube. Further analytes could be included in this set, if reference intervals were determined specifically for this material.
The new blood collection tube represents a promising approach to promote patient blood management and lean processes in modern medical laboratories and for the clinical partners. Further development will be needed to include more analytes.
1. Meybohm P, Richards T, Isbister J, Hofmann A, Shander A, Goodnough LT, et al. Patient Blood Management Bundles to Facilitate Implementation. Transfusion medicine reviews. 2017;31(1):62-71.
2. Revision of the “Guideline of the German Medical Association on Quality Assurance in Medical Laboratory Examinations – Rili-BAEK” (unauthorized translation). J Lab Med 2015;39(1):26-69.
10-year rejection rates due to hemolysis from a maxium care hospital
PD Dr. Astrid Petersmann | Universitätsmedizin Greifswald | Germany
Hemolysis is a very common problem associated with drawing and processing of diagnostic blood samples and may occur in all parts of the pre-analytical phase, i.e. handling, interim storage and transport of samples. Hemolysis monitoring is often conducted only intermittently or for short period of times up to a few years or restricted to parts of a given hospital, e.g. emergency room. In this study we monitored hemolysis rates in a maximum care hospital over a period of ten years.
Material and Methods:
Hemolysis is noticed by the clinician when samples are rejected. Among important clinical analytes sensitive to hemolysis are potassium and INR. Here we evaluated hemolysis based on sample rejections for those two analytes. Rejection of samples with orders for potassium was based on HIL index determined by the Dimension Vista (Siemens Healthcare GmbH, Eschborn, Germany). Rejection of samples with orders for INR assessment in citrate samples was based on visual inspection until 2014 for the BCS and on HIL index measured by the CS (both also Siemens Healthcare GmbH). Data on rejection rates (number of analysed and rejected orders) due to hemolysis was retrieved from the laboratory information system for the years 2007-2016. We report yearly 95% percentiles and medians of hemolysis rates: overall, for different ward types (intensive care, normal, ambulatory, and emergency) and frequency of blood draws, respectively.
Over two million measurements were included in the study: 1 156,119 for potassium and 921,464 for INR. Of 150 wards 42 had data for the whole study period accounting for 1,909,996 (92%) of the measurements. Median rejection rates due to hemolysis continuously increased over the study period. At the end of the study period median (95% percentile) rejection rates for potassium and INR were on average 1% (3.6%) and 1.9% (7.1%), respectively. There was no relationship between average number of blood draws and rejection rates with one exception: median INR was lower on wards with less than one blood draw per day. Also differences between ward types were observed with highest rejection rates in emergency rooms and lowest in intensive care units. The introduction of a new pneumatic tube system did not coincide with increased rejection rates whereas the introduction of a new coagulation instrument did.
Discussion and Conclusion:
Hemolysis remains an important pre-analytical issue. Emergency rooms are at high risk for rejection of samples due to hemolysis. The observed overall increase of rejection rates is accompanied with increasing numbers of samples, thus indicating that the workload for blood drawing staff increased during the study period with possible implication for hemolysis rates. Also stricter pre-analytical requirements for instruments may lead to higher rejection rates. Our data also demonstrate that the introduction of a new pneumatic tube system did not lead to an increase in hemolysis based rejections rate.