Plasma calprotectin - preanalytical stability and interference from hemolysis.
Author(s): Blavnsfeldt AG, Parkner T, Knudsen CS
Publication: Scand J Clin Lab Invest, 2022, Vol. , Page 1-7
PubMed ID: 35822403 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate if delayed centrifugation of blood, storage temperature and duration of K2EDTA plasma, or hemolysis affect quantified levels of calprotectin protein.
Conclusion of Paper
The coefficient of variance (CV) for same day measurements of calprotectin was 2.7-3.5% and the mean CV for duplicates was 2.3-3.1%. Storage of specimens at 5-8°C resulted in an average loss of 5.5% of calprotectin protein levels, but storage at -20°C for 4 weeks led to a mean increase of 10.9%; however, the two specimens stored for 4 months at -20°C had a mean decrease of 1.6%. Delayed centrifugation of blood for 9.5 h, led to a 3.6% increase in mean calprotectin levels compared to controls processed immediately. Calprotectin protein levels increased as the concentration of hemolysate increased, but the magnitude of the increase depended on initial calprotectin levels, with the largest increases observed in specimens with the lowest initial levels of calprotectin.
Studies
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Study Purpose
The purpose of this study was to assess the effects of delayed centrifugation of blood, storage temperature and duration of K2EDTA plasma, and hemolysis on quantified levels of calprotectin protein. Blood was collected into K2EDTA vacutainer tubes from patients (diagnosis not specified) undergoing blood sampling for other reasons. Specimens were transported by hand within 30 min of blood collection. Plasma was separated by centrifugation at 3000 g for 5 min, and aliquoted into Vacuette SECONDARY SIMPLEX BC Tubes. Protein levels of circulating calprotectin were quantified using the Phadia 250 EliA Calprotectin flouroenzymimmunoassay. Assay precision was evaluated by measuring calprotectin levels in two pooled plasma specimens (38 ng/mL and 124 ng/mL calprotectin) 4 times daily over 5 days with storage at 5-8°C between measurements. The effects of storage were investigated by evaluating aliquots of 20 plasma specimens that were stored for 0 h, 1, 2, 3, or 4 days at 5–8°C and for 2, 3, or 4 weeks at -20°C before analysis. Effects of delayed centrifugation were investigated by collecting 5 tubes of blood from 20 patients and storing them at room temperature for 0, 2.5, 5.5, 7.5, and 9.5 h before centrifugation, followed by storage of the isolated plasma aliquots at 5°C for 3 days before analysis in one batch. Two additional aliquots of plasma (240 and 3070 ng/mL) were stored at -20°C for 4 months before reanalysis. To investigate if hemolysis affected the calprotectin assay, hemolysate was added to a plasma pool with low calprotectin levels and one with high calprotectin. Effects of hemolysis were also investigated by by adding 0.001, 0.02, 0.03, 0.06, 0.12, and 0.24 mmol hemoglobin to plasma pools with low (21 and 44 ng/mL), moderate (100 and 134 ng/mL), and high (337 and 927 ng/mL) concentrations of calprotectin protein.
Summary of Findings:
The coefficient of variance (CV) for same day measurements of calprotectin protein was 3.5% for the pooled plasma specimen with a calprotectin protein level of 124 ng/mL specimen and 2.7% for the pooled plasma specimens with a calprotectin level of 38 ng/mL specimen. The between-day CV in calprotectin measurements were 6.0% and 3.7% for the for pooled plasma specimens with a calprotectin level of 124 ng/mL and 38 ng/mL, respectively. The mean CV for duplicates ranged from 2.3% for specimens with >200 ng/mL calprotectin to 3.1% for specimens with <50 ng/mL calprotectin. Storage of plasma specimens at 5-8°C resulted in an average loss in calprotectin of 5.5%, but storage at -20°C led to an increase in calprotectin levels, with a mean increase of 10.9% observed after 4 weeks; however, the two specimens stored for 4 months at -20°C had a mean decrease of 1.6%. Delayed centrifugation of blood for 9.5 h, led to a 3.6% increase in mean calprotectin levels. In the initial analysis, addition of hemolysate led to a doubling in calprotectin levels in the low calprotectin specimen pool, but only a 7% increase in the high calprotectin specimen pool. Further analysis showed that calprotectin levels increased with as the concentration of hemolysate added was increased, but the magnitude of the increase was dependent on initial calprotectin levels. Increases in measured calprotectin ranged from 114% (for specimens with an initial calprotectin level of 21 ng/mL) to <5% (specimens with initial calprotectin ≥337 ng/mL) when 0.01 mmol/L hemolysate was added. However, at the highest concentration of hemolysate evaluated (0.24 mmol/L), the increase in calprotectin protein levels ranged from 69% (for specimens with an initial calprotectin concentration of 927 ng/mL) to 2124% (for specimens with an initial calprotectin concentration of 21 ng/mL).
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Hemolysis Hemolysate added
No hemolysate added
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage conditions As blood
As plasma
Storage Storage duration 0 h
1 day
2 days
3 days
4 days
2 weeks
3 weeks
4 weeks
4 months
Storage Storage temperature 5–8°C
-20°C
Storage Time at room temperature 0 h
2.5 h
5.5 h
7.5 h
9.5 h