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Abstract
Background
In vitro levels of complement C3 and C4 proteins are sensitive to storage conditions. To avoid in vitro complement activation when testing is delayed, serum should be frozen at -20℃ within 2 hr of venipuncture. However, this is impractical in routine laboratory work. Therefore, we investigated alterations in C3 and C4 levels in refrigerated specimens over time and derived formulae to estimate initial levels of complement concentrations in delayed testing.
Methods
Ten fresh specimens were measured for C3 and C4 concentrations and were refrigerated at 4℃. We measured C3 and C4 levels in refrigerated samples daily for 4 days using an automated nephelometer (Beckman Coulter Inc., USA).
Results
C3 and C4 levels were significantly increased over time in refrigerated specimens (P<0.001, P<0.001, respectively). The increments in C3 and C4 levels were described by the equations: C3 (mg/dL)=3.55x+87.18 (r=0.9909), and C4 (mg/dL)=0.72x+22.3 (r=0.9395), where x=the number of days samples were refrigerated before testing. Increases in C3 and C4 concentrations were described on a percentage basis by the equations: ΔC3 (%)=4.14x+1.07 (r=0.9903), and ΔC4 (%)=3.57x+2.48 (r=0.9405).
Conclusions
As the measured C3 and C4 concentrations increased by 3.55 mg/dL (4.1%) and 0.72 mg/dL (3.6%) per day in refrigerated specimens, the levels of C3 and C4 should be adjusted in delayed testing. We proposed that the formulae presented be used to back-calculate initial levels of C3 and C4 concentrations.
Figures and Tables
Fig. 1
Alterations of complement C3 (A) and complement C4 (B) levels in refrigerated specimens over time, expressed as mg/dL.
Fig. 2
Alterations of complement C3 (A) and complement C4 (B) levels in refrigerated specimens over time, expressed as % changes.
Table 3
Comparisons of C3 and C4 concentrations in fresh and refrigerated specimens
*Statistical analyses were performed using ANOVA and Bonferroni corrections to compare C3 or C4 concentrations in the fresh specimen group and the groups of samples refrigerated for the indicated days; †ANOVA and Bonferroni correction analyses were performed to compare C3 and C4 levels between groups at each indicated day and the preceding day.
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