Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research.
Author(s): Sens A, Rischke S, Hahnefeld L, Dorochow E, Schäfer SMG, Thomas D, Köhm M, Geisslinger G, Behrens F, Gurke R
Publication: J Mass Spectrom Adv Clin Lab, 2023, Vol. 28, Page 35-46
PubMed ID: 36872954 PubMed Review Paper? No
Purpose of Paper
This paper compared metabolite and lipid levels in case-matched plasma from blood stored at room temperature or on ice for up to 240 min before centrifugation and in specimens stored at room temperature or on ice for up to 240 min after plasma separation. The combined effects of pre- and post- centrifugation storage were analyzed by summing the effects of the two different storage periods on a given analyte.
Conclusion of Paper
More of the 489 analytes were affected by pre-centrifugation storage of blood than post-centrifugation storage of plasma and when storage occurred at room temperature rather than on ice. When plasma (post-centrifugation) was stored on ice for 20 min only 10 analytes were affected (>20% change), but 21 analytes were affected when plasma was stored for 240 min on ice, and 7 were affected at room temperature. When blood was stored on ice or at room temperature for 240 min before centrifugation, 38 and 87 analytes were affected, respectively (22 and 31, respectively when stored for 20 min). The scope of effects were dependent on analyte class, with only one analyte affected in some analyte classes and all analytes affected in others. Importantly, the findings were generally concordant with a prior study (κ = 0.701). Based on the original study, the authors added the expected fold-change of each analyte when stored pre- and post-centrifugation to obtain combined values for specimens that were stored for 20 min, 60 min or 240 min both pre- and post- centrifugation at room temperature or on ice (the same duration and temperature was used for both pre- and post-centrifugation storage periods). Only 28 analytes were calculated to be unstable (>20% fold change at one of the storage durations or >30% fold change when both storage periods were combined) when stored on ice for 20 min pre-centrifugation and again post-centrifugation. When the effects of storage for 60 min pre- and post- centrifugation were combined, 46 analytes were considered to be unstable when stored on ice and 58 when stored at room temperature. Only 50 and 53 analytes were calculated to be unstable when specimens were stored on ice for either 120 or 240 min for both pre- and post- centrifugation. In contrast, 113 analytes were calculated to be unstable when specimens were stored at room temperature for 240 min pre-centrifugation and again post-centrifugation, and 72 analytes were unstable when specimens were stored at room temperature for 120 min pre-centrifugation and again post-centrifugation. The authors concluded that while many analytes are affected by centrifugation delays and/or plasma storage, others are stable even when specimens are stored at room temperature for up to 4 h pre- and post-centrifugation.
Studies
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Study Purpose
This study compared metabolite and lipid levels in case-matched plasma from blood stored at room temperature or on ice for up to 240 min before centrifugation and in specimens stored at the same temperature for the same duration after plasma separation. The combined effects of pre- and post-centrifugation storage were analyzed by summing the effects of the two different storage conditions on a given analyte. Sixteen tubes of non-fasting K3EDTA blood were collected from each of 9 healthy volunteers (5 men and 4 women) and plasma was obtained by centrifugation 2000 g for 10 min at 4°C. To investigate the effects of delayed centrifugation, case-matched blood was stored for 20, 60, 120 or 240 min on ice or at room temperature before centrifugation and immediate storage of the plasma at < -70°C. To investigate the effects of plasma storage, plasma was obtained immediately after blood draw and matched aliquots were stored for 0, 20, 60, 120 or 240 min on ice or at room temperature before snap-freezing on dry ice and storage <-70°C. Plasma was thawed at 4°C and endocannabinoids, oxylipins, sphingolipids, Lysophosphatidic acids, tryptophan-like metabolites, lipids and polar metabolites were extracted and quantified by targeted LC-MS/MS. Stability was defined as a fold change between 0.8 and 1.2 or the sum of the fold changes for plasma and blood storage between 0.7 and 1.3.
Summary of Findings:
Of the 1070 analytes (25 sphingolipids, 68 oxylipins, 12 endocannabinoids, 7 Lysophosphatidic acids, tryptophan, 6 tryptophan metabolites 665 lipids and 276 polar metabolite), 468 were excluded because levels were below the limit of quantification and 199 were excluded based on acceptance criteria, leaving 489 analytes. More of the 489 analytes were affected by pre-centrifugation storage of blood than post-centrifugation storage of plasma and when storage occurred at room temperature rather than on ice. When plasma (post-centrifugation) was stored on ice for 20 min only 10 analytes were affected (>20% change), but 21 analytes were affected when plasma was stored for 240 min on ice, and 7 were affected at room temperature. When blood was stored on ice or at room temperature for 240 min before centrifugation, 38 and 87 analytes were affected, respectively (22 and 31, respectively when stored for 20 min). While all molecules in some analytes classes (such as ethanolamides) were affected by storage of blood or plasma, in other analyte classes, only one analyte was affected. Generally, the 14 ceramides were considered stable with changes limited to 1 ceramide when whole blood was stored on ice. One of the most affected classes of analytes was sphingoid bases, with all three affected by storage of blood at room temperature for ≥20 min or plasma for 240 min, but none affected when plasma or blood was stored in ice water. All of the 11 endocannobinoids were affected by storage of whole blood on ice for ≥20 min or at room temperature for 240 min (10 affected after ≥60 min), but only 2 endocannobinoids were affected by storage of plasma in ice water and up to 5 were affected by storage of plasma at room temperature. Importantly, the findings were generally concordant with a prior study (κ = 0.701). Based on the original study, the authors added the expected fold-change of each analyte when stored pre- and post-centrifugation to obtain combined values for specimens that were stored for 20 min, 60 min or 240 min both pre- and post- centrifugation at room temperature or on ice (the same duration and temperature was used for both pre- and post-centrifugation storage periods). Only 28 analytes were calculated to be unstable (>20% fold change at one of the storage durations or >30% fold change when both storage periods were combined) when stored on ice for 20 min pre-centrifugation and again post-centrifugation. When the effects of storage for 60 min pre- and post- centrifugation were combined, 46 analytes were considered to be unstable when stored on ice and 58 when stored at room temperature. Only 50 and 53 analytes were calculated to be unstable when specimens were stored on ice for either 120 or 240 min for both pre- and post- centrifugation. In contrast, 113 analytes were calculated to be unstable when specimens were stored at room temperature for 240 min pre-centrifugation and again post-centrifugation, and 72 analytes were unstable when specimens were stored at room temperature for 120 min pre-centrifugation and again post-centrifugation. The authors concluded that while many analytes are affected by centrifugation delays and/or plasma storage, others are stable even when specimens are stored at room temperature for up to 4 h pre- and post-centrifugation.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Lipid LC-MS or LC-MS/MS Small molecule LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Aliquots and Components Blood and blood products Whole blood
Plasma
Storage Storage conditions As plasma
As blood
Storage Storage temperature Room temperature
On ice
Storage Storage duration 0 min
20 min
60 min
120 min
240 min