Influence of hemolysis, lipemia and bilirubin on biobank sample quality- origin and interference in the use for extracellular vesicle (EV) and MiRNA analyses.
Author(s): Weber B, Welz W, Schaible I, Han J, Henrich D, Marzi I, Leppik L
Publication: Eur J Trauma Emerg Surg, 2025, Vol. 51, Page 153
PubMed ID: 40140056 PubMed Review Paper? No
Purpose of Paper
This paper compared patient characteristics (injury severity score, hemoglobin levels, triglyceride levels and survival), treatment (blood transfusions, propofol use, intensive care unit time), miR-16-5p levels and extracellular vesicle size and concentration between serum specimens with spectrophotometrically detected hemolysis, lipemia, or bilirubin and unmatched serum without. Serum was collected from polytraumatized patients in the emergency room (ER) and over the course of 10 days of hospitalization.
Conclusion of Paper
Among the 88 serum specimens collected in the ER, spectrophotometric analysis classified 16 as hemolyzed, 4 as lipemic, and 3 as having bilirubin. Hemoglobin concentrations declined over the course of hospitalization, regardless of whether the blood specimen collected in the ER was not hemolyzed. Hemoglobin levels were non-significantly higher in all specimens from patients with hemolyzed ER specimens than when the ER blood specimen was non-hemolyzed. The number of blood transfusions the patient received was higher in patients without a hemolyzed specimen in the ER at all but one timepoint, but the difference was not significant, and the injury severity scores were comparable among patients with and without a hemolyzed blood specimen. Levels of miR-16-5p were significantly higher in hemolyzed specimens than non-hemolyzed specimens (P<0.05). Serum triglyceride concentrations were almost two-fold higher in lipemic than non-lipemic specimens, but the difference was not significant, and miR-16-5p levels were not affected by lipemia. Lipemia was associated with propofol use (r=0.64) but not injury severity score. The mean size of EVs was larger and particle concentrations were higher in specimens spiked with 20% SmofKabiven to mimic lipemia compared to those in plain serum. Patients whose specimens were spectrophotometrically classified as having bilirubin, tended to have a higher injury severity score (difference not significant) and non-survival rate (18.2% versus 15.6%), and the authors report longer stays in the hospital and the intensive care unit than patients whose specimens did not have detectable bilirubin.
Studies
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Study Purpose
This study compared patient characteristics (injury severity score, hemoglobin levels, triglyceride levels and survival), treatment (blood transfusions, propofol use, intensive care unit time), miR-16-5p levels and extracellular vesicle size and concentration between serum specimens with spectrophotometrically detected hemolysis, lipemia, or bilirubin and unmatched serum without. Serum was collected from polytraumatized patients in the emergency room (ER) and over the course of 10 days of hospitalization. Blood was collected from 88 polytraumatized patients at a Level 1 Trauma Center in the ER and on day 1, 2, 3, 5, 7 and 10 following hospital admission and from four healthy volunteers. Specimens were stored on ice until serum separation by centrifugation at 3500 g for 15 min at 4°C. Hemolysis and turbidity were quantified by spectrophotometer. Levels of triglycerides and bilirubin were quantified by an unspecified method. EVs were isolated from 4 specimens with hemolysis, 4 with lipemia and the 4 serum specimens from healthy volunteers by size exclusion chromatography and analyzed by nanoparticle tracking analysis. miRNAs were isolated from EVs using the miRNeasy Serum/Plasma Advanced Kit. miRNAs were quantified using the real-time PCR-based miRCURYLNA miRNA PCR Assays.
Summary of Findings:
Among the 88 serum specimens collected in the ER, spectrophotometric analysis classified 16 as hemolyzed, 4 as lipemic, and 3 as having bilirubin. Hemoglobin concentrations declined over the course of hospitalization, regardless of whether the blood specimen collected in the ER was hemolyzed (P<0.05 for days 2, 3, and 5 versus ER) or not (P<0.05 for all days 1-10 versus ER specimen). Hemoglobin levels were non-significantly higher in all specimens from patients with hemolyzed ER specimens than when the ER blood specimen was non-hemolyzed. The number of blood transfusions the patient received was higher in patients without a hemolyzed specimen in the ER at all but one timepoint, but the difference was not significant, and the injury severity scores were comparable among patients with and without a hemolyzed blood specimen. Levels of miR-16-5p were significantly higher in hemolyzed specimens than non-hemolyzed specimens (P<0.05). Serum triglyceride concentrations were almost two-fold higher in lipemic than non-lipemic specimens, but the difference was not significant, and miR-16-5p levels were not affected by lipemia. Lipemia was associated with propofol use (r=0.64) but not injury severity score. The mean size of EVs was larger and particle concentrations were higher in specimens spiked with 20% SmofKabiven to mimic lipemia compared to those in plain serum (200 nm versus 80nm, P<0.01; and P<0.05, respectively). Over half of the specimens identified as having bilirubin spectrophotometrically were also found to have > 1mg/dL bilirubin by clinical chemistry autoanalyzer. Patients whose specimens were spectrophotometrically classified as having bilirubin, tended to have a higher injury severity score (difference not significant) and non-survival rate (18.2% versus 15.6%), and the authors report longer stays in the hospital and the intensive care unit than patients whose specimens did not have detectable bilirubin.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Other diagnoses
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Lipid Spectrophotometry Lipid Clinical chemistry/auto analyzer Morphology Light scattering Small molecule Spectrophotometry Small molecule Clinical chemistry/auto analyzer Protein Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Anesthesia Propofol
No Propofol
Biospecimen Aliquots and Components Hemolysis Absent
Present
Preaquisition Prognostic factor Injury severity scores compared
Number of blood transfusions compared
Biospecimen Aliquots and Components Biospecimen components Lipemic serum
Serum with bilirubin detected spectrophotometrically
Normal serum