The stability of 65 biochemistry analytes in plasma, serum, and whole blood.
Author(s): Zhou J, Fabros A, Lam SJ, Coro A, Selvaratnam R, Brinc D, Di Meo A
Publication: Clin Chem Lab Med, 2024, Vol. 62, Page 1557-1569
PubMed ID: 38443327 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of 8 or 24 h delay to centrifugation at a room temperature, a 24 h delay to centrifugation at 4°C, and storage of plasma/serum for 24 h on levels of 65 analytes in serum/plasma collected and stored in serum separator (SST), lithium (Li) heparin, K2EDTA, and sodium fluoride/potassium oxalate (NaFl/KOx) vacutainers.
Conclusion of Paper
When blood was stored for 8 or 24 h at room temperature or 24 h at 4°C before centrifugation the percent deviation in levels of ammonia, aspartate aminotransferase (AST), chloride, carbon dioxide (CO2), glucose, lactate dehydrogenase (LDH), magnesium, phosphorous, potassium, sodium, calcium, calcitonin, creatinine, ferritin, insulin like growth factor 1 (IGF-1), lactate, and testosterone exceeded the maximal permissible instability (MPI) at one or both timepoints, regardless of tube type. Additionally, homocysteine peptide, free thyroxine (fT3), free tri-iodothyronine (fT4), C-peptide and intact parathyroid hormone (PTH) were affected by storage of blood for 24 h at room temperature before centrifugation, but the effect was dependent on tube type. Hemolysis, icterus, and lipemia remained below the cut-off values for each analyte after storage of blood for 8 h at room temperature, indicating that the effects of delayed centrifugation on analytes was unlikely to be attributable to hemolysis, lipemia or icterus. Deviations in the levels of ammonia, CO2, creatinine, fT3, glucose, homocysteine, IGF-1, lactate, LDH, potassium, and sodium after pre-centrifugation storage of blood at 4°C for 24 h exceeded the MPI, regardless of tube type. Effects of storing blood at 4 °C prior to centrifugation had tube type-dependent effects on β2-microglobulin, 25-hydroxy vitamin D, fT4 and intact PTH. Storage of blood at room temperature or 4°C for up to 24 h did not significantly affect levels of albumin, aldosterone, alkaline phosphatase (ALP), alpha fetoprotein (AFP), alanine aminotransferase (ALT), amylase, bilirubin (direct and total), beta-natriuretic peptide (BNP), cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), cholesterol, chromogranin A, cortisol, creatinine kinase (CK), C-reactive protein (CRP), dehydroepiandrosterone sulfate (DHEA-S), follicle stimulating hormone (FSH), gamma glutamyl transferase (GGT), hemoglobin A1c (HbA1c), high density lipoprotein (HDL), insulin, iron, luteinizing hormone (LH), lipase, N-terminal pro B-type natriuretic peptide (NT-proBNP), prolactin, renin, sex hormone binding globulin (SHBG), soluble transferrin receptor (sTFR), thyroglobulin, transferrin, triglycerides, thyroid stimulating hormone (TSH), urea, uric acid, or vitamin B12, regardless of tube type. Additionally, regardless of tube type, there was no effect of storage at room temperature for 24 h on levels of 25-hydroxy vitamin D, β2-microglobulin, or total protein; or storage at 4°C for up to 24 h on levels of AST, C-peptide, calcitonin, calcium chloride, ferritin, magnesium, phosphorous, or testosterone.
Storage of serum/plasma at room temperature for 24 h led to deviations in levels of ammonia, calcitonin, CO2, fT4, IGF-1, LDH, potassium and total bilirubin that exceeded the MPI. Effects of storage of plasma/serum on levels of β2-microglobulin, glucose, homocysteine, and intact PTH were dependent on tube type. When serum/plasma was stored for 24 h at room temperature before analysis, deviations in 25-hydroxy vitamin D, albumin, aldosterone, ALP, AFP, ALT, amylase, AST, direct bilirubin, BNP, C-peptide, CA-125, calcium, CEA, chloride, cholesterol, chromogranin A, cortisol, CK, creatinine, DHEA-S, ferritin, FSH, GGT, insulin, iron, lactate, LH, lipase, magnesium, NT-proBNP, phosphorous, prolactin, renin, SHBG, sodium, sTFR, testosterone, thyroglobulin, total protein, transferrin, triglycerides, TSH, urea, uric acid, and vitamin B12 remained less than the MPI limit.
Studies
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Study Purpose
The purpose of this study was to investigate the effects of storing blood in different tube types (SST, Li heparin, K2EDTA, or NaFl/KOx) for 8 or 24 hours at room temperature or for 24 h at 4°C prior to plasma/serum separation on the levels of 65 clinical chemistry analytes. Blood was collected from thirty healthy volunteers via a 21-gauge butterfly needle into five SST, Li-heparin, K2EDTA, or NaFl/KOx tube (ten volunteers per tube type). Case-matched blood tubes were centrifuged at 2,107 g for 10 min at 20°C immediately (after 30 min for SST) or after 8 or 24 h at room temperature or 24 h at 4°C. Analytes were quantified immediately in plasma/serum using an Abbott Alinity analyzer (57 analytes), a Diasorin Liaison XL (1,25-Dihydroxy vitamin D, aldosterone, beta 2 microglobulin, growth hormone, IGF-1 and renin), a BRAHMS KRYPTOR (chromogranin A) a BioRad D-100 (HbA1c) or a Roche cobas e411 (intact PTH in EDTA plasma) instrument. Importantly, intact PTH was analyzed using two different analyzers in EDTA plasma (Abbott Alinity and Roche cobas e411) and only the Abbott Alinity for Li heparin plasma. To investigate possible interference from hemolysis, icterus, and lipemia, blood was collected from five healthy volunteers into Li heparin vacutainers and matched tubes were centrifuged after 0 and 8 h at room temperature, respectively, and analytes (calcium, chloride, glucose, potassium, lactate, pCO2, pO2 and sodium were analyzed on a Werfen GEM Premier analyzer. For each analyte, deviations in levels were compared to the maximal permissible instability (MPI) limit calculated using both the intra- and inter-individual biological variation.
Summary of Findings:
When blood was stored for 8 h at room temperature before centrifugation, the percent deviation in levels of ammonia, AST, chloride, CO2, glucose, LDH, magnesium, phosphorous, potassium, and sodium exceeded the maximal permissible instability (MPI) limit, regardless of tube type. While the deviations in calcium, calcitonin, creatinine, ferritin, IGF-1, lactate, and testosterone were less than the MPI limit after an 8 h centrifugation delay at room temperature in all vacutainers tested, they exceeded the MPI limit when blood was stored for 24 h at room temperature before centrifugation. Effects of storing blood prior to centrifugation on homocysteine peptide, fT3, fT4, C-peptide and intact PTH were dependent on tube type. After storage at room temperature for 8 h, deviations exceeding MPI limits were observed for homocysteine peptide, fT3, and fT4 in Li heparin tubes and homocysteine peptide in EDTA tubes; but homocysteine peptide, fT3 and fT4 were not affected by storage of blood in SST tubes. Deviations exceeding the MPI limit occurred in C-peptide after storage at room temperature for 24 h in Li heparin, but not SST or Na-fluoride/K-oxalate, tubes. Deviations in intact PTH exceeded the MPI limit when Li heparin blood was stored for ≥8 h at room temperature but were within the MPI limit when EDTA blood was stored for 24 h (either analyzer). To investigate if hemolysis, icterus, and lipemia were a likely source of the observed biases, hemolysis, icterus, and lipemia were quantified after storage of blood for 0 or 8 h at room temperature. Hemolysis, icterus, and lipemia remained below the cut-off values for each analyte after 8 h at room temperature, and the deviations were small, indicating that the effects of delayed centrifugation on some analytes is not likely attributable to hemolysis, lipemia or icterus. Storage of blood at room temperature for up to 24 h did not significantly affect levels of 25-hydroxy vitamin D, albumin, aldosterone, ALP, AFP, ALT, amylase, β2-microglobulin, bilirubin (direct and total), BNP, CA-125, CEA, cholesterol, chromogranin A, cortisol, CK, CRP, DHEA-S, FSH, GGT, HbA1c, HDL, insulin, iron, LH, lipase, NT-proBNP, prolactin, renin, SHBG, sTFR, thyroglobulin, total protein, transferrin, triglycerides, TSH, urea, uric acid, or vitamin B12, regardless of tube type.
Deviations in the levels of ammonia, CO2, creatinine, fT3, glucose, homocysteine, IGF-1, lactate, LDH, potassium, and sodium after storage of blood for 24 h at 4°C prior to centrifugation exceeded MPI limits, regardless of tube type. Effects of storing blood at 4°C prior to centrifugation had tube type-dependent effects on β2-microglobulin, 25-hydroxy vitamin D, fT4 and intact PTH. Deviations in β2-microglobulin exceeded the MPI limit when blood was stored at 4°C for 24 h in SST, but not Li heparin tubes. Conversely, deviations in 25-hydroxy vitamin D and fT4 exceeded MPI limits when blood was stored for 24 h at 4°C in Li heparin tubes but not SST. Similar to room temperature storage, deviations in intact PTH exceeded the MPI limit when Li heparin blood, but not EDTA blood (measured with either analyzer) was stored at 4°C for 24 h before centrifugation. Storage of blood for up to 24 h at 4°C did not significantly affect levels of albumin, aldosterone, ALP, AFP, ALT, amylase, AST, bilirubin (direct and total), BNP, C-peptide, CA-125, calcitonin, calcium, CEA, chloride, cholesterol, chromogranin A, CK, cortisol, CRP, DHEA-S, ferritin, FSH, GGT, HbA1c, HDL, insulin, iron, LH, lipase, magnesium, NT-pro BNP, phosphorous, prolactin, renin, SHBG, sTFR, testosterone, thyroglobulin, transferrin, triglycerides, TSH, urea, uric acid, or vitamin B12, regardless of tube type.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Carbohydrate Clinical chemistry/auto analyzer Electrolyte/Metal Clinical chemistry/auto analyzer Gas Clinical chemistry/auto analyzer Glycoprotein Clinical chemistry/auto analyzer Lipid Clinical chemistry/auto analyzer Lipoprotein Clinical chemistry/auto analyzer Peptide Clinical chemistry/auto analyzer Protein Clinical chemistry/auto analyzer Small molecule Clinical chemistry/auto analyzer Steroid Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Lithium heparin
Sodium fluoride/potassium oxalate
Potassium EDTA
None
Biospecimen Acquisition Type of collection container/solution SST
Li heparin tube
K2EDTA tube
NaF/KOx tube
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage duration 0 h
8 h
24 h
Storage Storage temperature Room temperature
4°C
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Study Purpose
This study investigated the effects associated with the storage of plasma/serum for 24 h at room temperature on the levels of 65 analytes; serum/plasma were isolated from blood collected and stored in serum separator tube (SST), lithium (Li) heparin, K2EDTA, and sodium fluoride/potassium oxalate (NaFl/KOx) vacutainers. Blood was collected from thirty healthy volunteers via a 21-gauge butterfly needle into five SST, Li heparin, K2-EDTA, or NaFl/KOx tube (ten volunteers per tube type). Matched blood tubes were centrifuged immediately (after 30 min for SST) at 2,107 g for 10 min at 20°C. Plasma and serum were stored in the original tubes and analyzed immediately or after 24 h at room temperature. Analytes were quantified using an Abbott Alinity analyzer (57 analytes), a Diasorin Liaison XL (1,25-Dihydroxy vitamin D, aldosterone, beta 2 microglobulin, growth hormone, IGF-1 and renin), a BRAHMS KRYPTOR (chromogranin A), a BioRad D-100 (HbA1c), or a Roche cobas e411 (intact PTH in EDTA plasma) instrument. Importantly, intact PTH was analyzed using two different analyzers in K2EDTA plasma (Abbott Alinity and Roche cobas e411) and only the Abbott Alinity for Li heparin plasma. For each analyte, deviations in levels were compared to the maximal permissible instability (MPI) limit calculated using both the intra- and inter-individual biological variation.
Summary of Findings:
Storage of serum/plasma for 24 h at room temperature led to deviations in the levels of ammonia, calcitonin, CO2, fT4, IGF-1, LDH, potassium, and total bilirubin that exceeded the respective MPI limit. Effects of storing plasma/serum for 24 h at room temperature on levels of β2-microglobulin, glucose, homocysteine, and intact PTH were dependent on tube type. β2-microglobulin was affected by the storage of serum but not Li heparin plasma, while FT3 was affected by storage of Li heparin plasma but not serum. Glucose was stable in NaF/KOx plasma and in serum but not in EDTA or Li heparin plasma. Homocysteine was affected by storage of EDTA plasma, but not Li heparin plasma. Intact PTH was affected by storage of Li heparin plasma, but not by storage of EDTA plasma (using either assay). Deviations in 25-hydroxy vitamin D, albumin, aldosterone, ALP, AFP, ALT, amylase, AST, direct bilirubin, BNP, C-peptide, CA-125, calcium, CEA, chloride, cholesterol, chromogranin A, cortisol, CK, creatinine, DHEA-S, ferritin, FSH, GGT, insulin, iron, lactate, LH, lipase, magnesium, NT-proBNP, phosphorous, prolactin, renin, SHBG, sodium, sTFR, testosterone, thyroglobulin, total protein, transferrin, triglycerides, TSH, urea, uric acid, and vitamin B12 remained less than the corresponding MPI limit when serum/plasma was stored for 24 h at room temperature before analysis.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Carbohydrate Clinical chemistry/auto analyzer Electrolyte/Metal Clinical chemistry/auto analyzer Gas Clinical chemistry/auto analyzer Glycoprotein Clinical chemistry/auto analyzer Steroid Clinical chemistry/auto analyzer Lipoprotein Clinical chemistry/auto analyzer Peptide Clinical chemistry/auto analyzer Protein Clinical chemistry/auto analyzer Small molecule Clinical chemistry/auto analyzer Lipid Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Lithium heparin
Potassium EDTA
Sodium fluoride/potassium oxalate
None
Biospecimen Acquisition Type of collection container/solution SST
Li heparin tube
K2EDTA tube
NaF/KOx tube
Storage Time at room temperature 0 h
24 h