Insufficient mixing of thawed serum samples leading to erroneous results - experience from a field study and use of a correction procedure.
Author(s): Ekström U, Apelqvist J, Hansson E, Bodin T, Wegman DH, Abrahamson M, Jakobsson K
Publication: Scand J Clin Lab Invest, 2019, Vol. , Page 1-7
PubMed ID: 31847598 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of the method of mixing serum specimens after frozen storage and storage of gel and non-gel tubes on levels of creatinine, sodium, cystatin C, and albumin.
Conclusion of Paper
A gradient in creatinine, sodium, cystatin C, and albumin levels was observed when specimens were not mixed or were only vortexed for 2-3 seconds (Standard Vortex Method) and in sodium, creatinine, and cystatin C in one of five specimens vortexed for 10 sec, but no gradient was observed in specimens inverted 10 times with or without additional vortexing. While specimens collected without gel only displayed a gradient when not mixed, those collected with gel and incubated at room temperature or 50°C displayed a gradient in analyte levels in three of four specimens subjected to the Standard Vortex Method. Importantly, as all analytes were similarly affected, it was possible to apply a correction resulting in differences of <1% between the sampling locations.
Studies
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Study Purpose
This study investigated the effects of the method of mixing serum specimens after frozen storage on levels of creatinine, sodium, cystatin C, and albumin. Venous blood was obtained from five fasting healthy volunteers into six serum tubes without gel. One tube was immediately analyzed (control) and the remaining five were incubated for 1 h at room temperature before centrifugation for 10 min at 2000 x g to obtain serum which was refrigerated for 2 h and then stored at -20°C for 4 days followed by storage at -80°C for 1.5 days. One tube was thawed 4 h, inverted 10 times, vortexed for 10 sec, and centrifuged at 2200 x g for 10 min (Optimal Method). The second tube was thawed for 1–2 h at what, mixed by manual inverting 10 times, then centrifuged at 2200 x g for 10 min (Manual Mixing Method). The third tube was thawed for 1–2 h, mixed by vortexing for 10 sec, then centrifuged at 2200 x g for 10 min (Extended Vortex Method). The fourth tube was thawed for 1–2, mixed by vortexing until a swirl was observed (2-3 sec), then centrifuged at 2200 x g for 10 min (Standard Vortex Method). The final tube was thawed 1-2 h and then centrifuged at 2200 x g for 10 min (No Mixing Method). After the mixing procedure, aliquots were obtained from the top, middle, and bottom. Levels of sodium, creatinine, cystatin C, and albumin were measured on a Cobas 701 clinical chemistry instrument.
Summary of Findings:
A gradient was observed for all four analytes in each of the five specimens that were not mixed and those that were vortexed for only 2-3 seconds (Standard Method) with higher levels of each analyte found at the bottom of the tube than the middle or top. Levels of analytes were also higher in the middle versus top in all specimens that were not mixed or that were subjected to standard vortexing except one standard vortex specimen that had the same level of creatinine and cystatin C in the middle and top and higher levels of sodium and albumin in the middle than top. One of the five specimens subjected to vortexing for 10 sec showed a similar gradient in sodium, creatinine, and Cystatin C but not albumin. No gradients were observed in the specimens processed using the Optimal or Manual Mixing methods.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Clinical chemistry/auto analyzer Electrolyte/Metal Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Sample mixing Inverted 10 times and vortexed for 10 sec (Optimal Mixing)
Inverted 10 times (Manual)
Vortexed 10 sec (Extended Vortexing)
Vortexed until swirl (Standard Vortexing)
Not mixed
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Study Purpose
This study investigated the effects of gel in the serum tube and storage of gel tubes at room temperature or 50°C and mixing after thawing using different strategies on levels of creatinine, sodium, cystatin C, and albumin. Blood was collected from four healthy volunteers into BD tubes with and without gel preincubated at room temperature or 50°C. One specimen was immediately analyzed following the clinical laboratory instructions (details not specified). Specimens without gel were kept at room temperature for 1 h and those with gel at room temperature or 50°C for 24 h before centrifugation. The serum from each type of tube was poured into three tubes stored at 4°C for 2 h, transferred to -20°C, mailed on dry ice, and then stored at -80°C. After thawing for 1-2 h, specimens were mixed by the Optimal Method (10 inversions and 10 sec of vortexing), Standard Vortex Method (until a swirl was observed, 2-3 sec), or left unmixed and then centrifuged at 2200 x g for 10 min. After centrifugation, aliquots were obtained from the top, middle, and bottom. Levels of sodium, creatinine, cystatin C, and albumin were measured on a Cobas 701 clinical chemistry instrument.
Summary of Findings:
While specimens collected without gel only displayed a gradient when not mixed, those collected with gel and incubated at room temperature or 50°C displayed a gradient in analyte levels in three of four specimens subjected to the Standard Vortex Method. Regardless of tube type and temperature, a gradient was observed in specimens that were not mixed after thawing and a gradient was observed when specimens were inverted 10 times and vortexed for 10 sec after thawing (Optimal Method). Importantly, since all analytes were similarly affected, it was possible to apply a correction resulting in differences of <1% between the sampling locations.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Clinical chemistry/auto analyzer Electrolyte/Metal Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Gel tube
Non-gel tube
Storage Storage temperature Room temperature
50°C
Analyte Extraction and Purification Sample mixing Inverted 10 times and vortexed for 10 sec
Vortexed until swirl
Not mixed