Quality Control of Serum and Plasma by Quantification of (4E,14Z)-Sphingadienine-C18-1-Phosphate Uncovers Common Preanalytical Errors During Handling of Whole Blood.
Author(s): Liu X, Hoene M, Yin P, Fritsche L, Plomgaard P, Hansen JS, Nakas CT, Niess AM, Hudemann J, Haap M, Mendy M, Weigert C, Wang X, Fritsche A, Peter A, Häring HU, Xu G, Lehmann R
Publication: Clin Chem, 2018, Vol. , Page
PubMed ID: 29567661 PubMed Review Paper? No
Purpose of Paper
This paper identified sphingosine-1-phosphate (S1P-d18:2) and lactate as biomarkers for blood processing delays in plasma and investigated the effects of delayed centrifugation, exercise, and patient condition on these biomarkers. The effects of clot time, clot activator, post-clot storage, patient condition, and use of a specialized collection team on levels of S1P-d18:2 in serum were also investigated.
Conclusion of Paper
UPLC-MS analysis identified a total of 188 ion masses that could be used as biomarkers for delayed processing at room temperature of which sphingosine-1-phosphate (S1P-d18:2) and lactate were the most promising candidates. Both S1P-d18:2 and lactate in plasma increased with storage of blood at room temperature for 2 h or 4 h and lactate levels increased with storage at 4˚C for 4 h. The authors found threshold values of ≤0.085 µg/mL of S1P-d18:2 and ≤1.306 µg/mL of lactate to be indicative of high quality plasma. Although S1P-d18:2 levels were unaffected by exercise and remained within the expected ranges for high quality plasma for patients undergoing extreme physiologic stress, lactate levels were higher than the threshold in some ICU specimens and in healthy patients after circuit training to exhaustion. Levels of S1P-d18:2 did not differ between plasma stored for 6 months and that stored for 5 years.
Levels of S1P-d18:2 were significantly higher in serum collected without clot activator that was allowed to clot for 60 min than in serum collected with clot activator and allowed a 30 min clot time. In specimens collected without clot activator, storage for 2 or 4 h after the initial 60 min clot time resulted in a significant increase in S1P-d18:2 levels. Comparison of S1P-d18:2 levels in serum specimens in a biobank showed that those levels of S1P-d18:2 were lower and more consistent when collected by a specialized team than when collected by the clinician. Importantly, in the 869 serum specimens collected at eight hospitals for seven different biobanks using the same SOP had similar levels of S1P-d18:2 and levels were comparable among the patient diagnoses.
Studies
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Study Purpose
This study identified lactate and S1P-d18:2 as biomarkers of delayed plasma processing and investigated the effects of patient condition and exercise on the suitability of these biomarkers. Blood was drawn into 10 mL EDTA tubes from 109 healthy patients, 6 patients with severe sepsis, 6 patients with liver cirrhosis, 1 patient with liver cirrhosis and sepsis, and 4 patients who had undergone cardiopulmonary resuscitation. Specimens were immediately split into 5 aliquots and stored for 2 or 4 h at room temperature (22˚C) or on ice (4˚C). After storage, plasma was obtained by centrifugation at 3100 x g at 4˚C, aliquoted, and stored at -80˚C. Proteins were identified by UPLC-Triple Quadrupole MS. Biomarkers were identified using the blood from 30 healthy patients and validated using the blood from 79 healthy individuals. The effects of extreme physiological condition were investigated using specimens from patients with severe sepsis and/or liver cirrhosis and patients who had undergone cardiopulmonary resuscitation. The effect of exercise was investigated by collecting specimens from seven healthy patients before and immediately after exercise to complete exhaustion and from ten patients before and after 1, 2, and 3 h of cycling at 50% maximum oxygen volume. The ability of S1P-d18:2 to serve as a biomarker for processing delay was further tested using plasma from three different biobanks, two of which processed blood in accordance with an SOP that required immediate cooling of blood to 4˚C followed by plasma isolation within 2 h (197 specimens total) and the third biobank which specified plasma isolation as soon as possible (99 specimens). The effects of long term storage of plasma were evaluated in 50 plasma specimens isolated within 2 h of collection and stored at -80˚C for 5 years.
Summary of Findings:
Pre-centrifugation storage at room temperature for 2 h and 4 h affected the levels of 9.2% and 16.1% of identified ion masses, respectively, compared to specimens processed immediately. In contrast, pre-centrifugation storage at 4˚C affected the levels of only 0.5% and 1.8% of ion masses after 2 and 4 h, respectively. Analysis showed that there were a total of 188 ion masses that could be used as biomarkers for delayed processing at room temperature, of which, S1P-d18:2 and lactate were the most promising candidates. Further investigation of these biomarkers in the plasma of 79 healthy individuals showed an expected significant increase in S1P-d18:2 and lactate with storage at room temperature for 2 h or 4 h (P<0.0001, all), but also found a significant increase in lactate levels when specimens were stored at 4˚C for 4 h (P<0.002). The authors identified the cut-off values for good quality plasma to be ≤0.085 µg/mL of S1P-d18:2 and ≤1.306 µg/mL of lactate. Although S1P-d18:2 levels remained below the cut-off values for good plasma in patients who had undergone extreme physiologic stress and in healthy patients, post-exercise; lactate levels were higher than the cut-off in some ICU patient specimens and specimens from individuals collected post-circuit training to exhaustion, making it a poor biomarker for specimen processing. In all 199 plasma specimens from the two biobanks that processed blood within 2 h, S1P-d18:2 levels were below the established cut-off value, indicating high quality, but many plasma specimens from the biobank in which processing delays were as long as 9 h had levels of S1P-d18:2 above the quality threshold. Importantly, there were no differences in S1P-d18:2 levels between plasma stored 6 months and that stored for 5 years. S1P-d18:2 levels were lower in ICU patients than in healthy patients. S1P-d18:2 levels were modestly correlated with hematocrit (r2=0.45, P=0.003), indicating that lower levels of S1P-d18:2 in ICU patients could be attributed to anemia. Levels of S1P-d18:2 in the NIST standard reference for plasma were below the threshold, indicating high quality.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Cirrhosis
- Other diagnoses
Platform:
Analyte Technology Platform Protein LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 22˚C
4˚C
Storage Storage duration 2 h
4 h
<4 h
6-9 h
6 months
5 years
Preaquisition Diagnosis/ patient condition Severe sepsis
Liver cirrhosis
Liver cirrhosis and sepsis
Underwent cardiopulmonary resuscitation
Healthy
Biospecimen Acquisition Time of biospecimen collection Before exercise
After exercise to exhaustion
After 1 h cycling
After 2 h cycling
After 3 h cycling
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
This study investigated the effects of clot activator, processing delays, training of individual collecting serum, and liver disease on S1P-d18:2 levels in serum. Blood was collected from 50 patients into tubes with coagulation enhancer (S-Monovette with Kaolin beads) and allowed to clot for 30 min before centrifugation. Blood was also collected from 43 patients into tubes without the enhancer and allowed to clot for 60 min and then stored at 4˚C for 0, 2, or 4 h before serum isolation. Serum was obtained by centrifugation at 3100 x g for 7 min, aliquoted, and then frozen at -80˚C. To investigate the effects of experience of the person collecting the specimen, levels of in S1P-d18:2 were compared between 113 specimens obtained by a specialized team and 80 specimens collected by doctors and nurses as part of routine care and stored in a biobank. To determine the consistency of S1P-d18:2 across specimens and investigate the effects of liver disease, S1P-d18:2 levels were compared among more than 800 serum specimens collected during a research trial on liver disease (hepatitis, hepatocellular carcinoma, cirrhosis) using an SOP that specified a 30 min clot time.
Summary of Findings:
S1P-d18:2 levels were significantly higher in specimens without clot activator that were allowed to clot for 60 min than in specimens with clot activator that were allowed a 30 min clot time (P<0.0001). In specimens collected without clot activator, storage for 2 or 4 h after the initial 60 min clot time resulted in a significant increase in S1P-d18:2 levels (P<0.0001, both). Comparison of S1P-d18:2 levels in serum specimens in a biobank showed that those levels of S1P-d18:2 were lower and more consistent when collected by a specialized team than when collected by the clinician. Importantly, in the 869 serum specimens collected at eight hospitals and stored in seven different biobanks using the same SOP had similar levels of S1P-d18:2 and levels were comparable among the patient diagnoses. Finally, levels of S1P-d18:2 in the NIST serum reference were within the range for good quality serum.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Cirrhosis
- Hepatitis
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution S-Monovette with Kaolin beads
S-Monovette without clot activator
Preaquisition Diagnosis/ patient condition Hepatitis
Hepatocellular carcinoma
Cirrhosis
Healthy
Storage Storage duration 30 min
60 min
2 h
4 h
Biospecimen Acquisition Method of fluid acquisition Staff group performing phlebotomy compared
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated