The influence of various sample storage conditions and sample bacterial contamination on concentrations of routine biochemical parameters.
Author(s): Gojković A, Vladimirov S, Antonić T, Bogavać-Stanojević N, Novović K, Spasojević-Kalimanovska V, Filipić B
Publication: J Med Biochem, 2024, Vol. 43, Page 413-423
PubMed ID: 39139161 PubMed Review Paper? No
Purpose of Paper
This paper compared the stability of five biochemical analytes (total cholesterol, glucose, triglycerides, urea, and albumin) and the presence of bacterial contamination in pooled EDTA and sodium-fluoride/potassium oxalate (NaF/KOx) plasma during 5 days of storage at 4˚C and during 5 days of storage at -20˚C with daily freeze-thaw cycling; the study included plasma from ten healthy volunteers.
Conclusion of Paper
While urea levels were unaffected by storage of EDTA or NaF/KOx plasma at either 4°C or -20°C (with a daily freeze-thaw cycle) relative to controls that were analyzed after 1 d of storage, urea levels did differ between storage temperatures and collection tubes; urea levels were significantly higher in EDTA plasma stored at -20°C (with freeze-thawing) compared to storage at 4°C for 2 days (P=0.041) and were higher in EDTA than NaF/KOx plasma after 5 days and five freeze-thaw cycles at -20˚C (P=0.001). Compared to levels on day 1, albumin levels were lower in EDTA plasma stored at 4˚C or -20˚C (with daily freeze-thaw) for 2 (P<0.001, both) or 4 days (P<0.001 and P=0.022, respectively) and in NaF/KOx plasma stored at 4˚C for 2 days (P=0.037) or at -20˚C with daily freeze-thaw events for 2 or 4 days (P<0.001 and P=0.004, respectively). Compared to EDTA plasma stored at the same temperature, NaF/KOx plasma had higher albumin levels after storage for 2 days at 4°C (P=0.041). Triglyceride levels were unaffected by storage of NaF/KOx plasma at either temperature, or EDTA plasma at 4°C but increased after storage of EDTA plasma for 3 or 5 days at -20˚C with daily freeze-thaw events (P=0.022, and P=0.10, respectively). Total cholesterol was higher in EDTA plasma on day 4 than on day 1 of storage at 4˚C or -20˚C (with a daily freeze-thaw event) (P=0.014 and P=0.018) and on day 2 of storage of NaF/KOx plasma at -20˚C with a daily freeze-thaw event (P=0.044). Glucose levels were higher in EDTA plasma stored at 4˚C on day 5 than on day 1 (P=0.008), higher in NaF/KOx plasma than EDTA plasma stored for 3 days at -20˚C with a daily freeze-thaw event (P=0.042), and higher in EDTA than NaF/KOx on day 3 of storage at 4°C (P=0.012).
Bacterial growth was only detected in EDTA plasma on day 4 (Staphylococcus epidermidis) and 5 (Staphylococcus epidermidis and Staphylococcus hominis) of storage at 4°C; bacterial growth was not detected in NaF/KOx plasma or in any plasma stored at -20˚C (with a daily freeze-thaw event).
Studies
-
Study Purpose
This study compared the stability of five biochemical analytes (total cholesterol, glucose, triglycerides, urea, and albumin) and the presence of bacterial contamination in pooled EDTA and NaF/KOx plasma during 5 days of storage at 4˚C and during 5 days of storage at -20˚C with daily freeze-thaw cycling. Blood was collected from ten healthy volunteers (five men and five women) into EDTA and NaF/KOx Vacutainer tubes. Plasma was separated by centrifugation at 3000 rpm for 10 min, pooled and aliquoted. Plasma aliquots were stored at 4˚C or -20˚C for 5 days. Each day of the storage period, aliquots were placed at room temperature until both reached room temperature, analyzed, and returned to the refrigerator/freezer for storage. Cholesterol, glucose, triglycerides, urea and albumin were quantified using a Ilab 300+ biochemical analyzer. At each timepoint, an aliquot of plasma was inoculated in Tryptic Soy Agar media and incubated at 35˚C for 18-20 h. Positive cultures were subcultivated, 16S rRNA was amplified by PCR, gel purified and sequenced by Macrogen Sequencing Services. Bacterial contamination was considered positive when cultures were positive on two consecutive days.
Summary of Findings:
EDTA plasma stored at 4˚C had significantly lower levels of albumin on day 2 and 4 (P<0.001 and P=0.008, respectively), higher total cholesterol on day 4 (P=0.014) and higher glucose levels on day 5 (P=0.008) compared to EDTA plasma stored at 4˚C for 1 day, but no significant differences in levels of urea or triglycerides were observed. When EDTA plasma was stored at -20˚C with daily freeze-thaw cycling, triglyceride levels were higher on day 3 and 5 (P=0.022 and P=0.010, respectively), total cholesterol was higher on day 4 (P=0.018), and albumin levels were lower on day 2 and 4 (P<0.001 and P=0.022, respectively) compared to EDTA plasma stored at -20˚C for 1 day, but levels of urea and glucose were not significantly affected. The only difference that occurred when the two storage temperatures were compared was a significantly higher concentration of urea in EDTA plasma stored at -20˚C with freeze-thaw cycling than 4˚C on day 2 (P=0.041). Storage of NaF/KOx plasma at 4˚C did not affect levels of urea, triglycerides, glucose, and total cholesterol, but albumin levels were significantly lower in NaF/KOx plasma stored at 4˚C for 2 days versus 1 day (P=0.037). When NaF/KOx plasma was stored at -20˚C with daily freeze-thaw cycling, cholesterol levels were higher on day 2 (P=0.044), glucose levels were higher on day 5 (P=0.042), and albumin levels were lower on day 2 and 4 (P<0.001 and P=0.004, respectively) compared to day 1; levels of urea and triglyceride were not significantly affected by storage of NaF/KOx plasma at -20˚C with daily freeze-thaw cycling. Compared to EDTA plasma stored at the same temperature, NaF/KOx plasma had higher albumin after storage for 2 days at 4°C (P=0.041), lower glucose after storage for 3 days at 4°C (P=0.012), and lower urea after 5 days at -20˚C with daily freeze-thaw cycling (P=0.001), but comparable levels of triglyceride and total cholesterol.
Bacterial growth was detected in EDTA plasma on day 4 (Staphylococcus epidermidis) and 5 (Staphylococcus epidermidis and Staphylococcus hominis) of storage at 4°C; bacterial growth was not detected in NaF/KOx plasma or in plasma stored at -20˚C with daily freeze-thaw cycling.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Next generation sequencing Carbohydrate Clinical chemistry/auto analyzer Protein Clinical chemistry/auto analyzer Small molecule Clinical chemistry/auto analyzer Steroid Clinical chemistry/auto analyzer Cell count/volume Microbiological assay Lipid Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant EDTA
Sodium fluoride/potassium oxalate
Storage Storage temperature 4°C
-20°C (with daily freeze-thaw)
Storage Storage duration 1 day
2 days
3 days
4 days
5 days
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
4 cycles
5 cycles