Table 1.

Summary of experiments to study folate stability during suboptimal preanalytical conditions

Experiment	Design	Samples	Laboratory analysis1	Statistical analysis
1. Delayed processing of whole blood	Blood tubes stored at 32°C for 1, 2, and 3 d prior to processing and freezing at −70°C; reference: serum from promptly processed whole blood frozen at −70°C	Whole blood from 21 volunteer blood donors from CDC	1 replicate/condition batch analyzed by HPLC-MS/MS	1-way (time) ANOVA
2. Delayed freezing of serum	Serum stored at 11°C for 2, 7, 10, and 14 d prior to freezing at −70°C; reference: serum from promptly processed whole blood frozen at −70°C	Serum from 21 volunteer blood donors from CDC	1 replicate/condition batch analyzed by HPLC-MS/MS	1-way (time) ANOVA
3. Short-term exposure of serum to elevated temperatures	Serum stored at 22°C and 37°C for 2, 4, 6, and 24 h prior to freezing at −70°C; reference: serum stored at −70°C	Low, medium, and high serum QC pools	4 replicates/condition batch analyzed by HPLC-MS/MS	2-way (time, pool level) ANOVA
4. Repeated freeze/thawing of serum (closed vials)	Serum exposed to up to 3 freeze/thawing cycles (2 h/cycle at room temperature in closed vials); reference: serum stored at −70°C	Low, medium, and high serum QC pools	4 replicates/condition batch analyzed by HPLC-MS/MS	2-way (time, pool level) ANOVA
5. Repeated freeze/thawing of serum (open vials)	Serum from same vial exposed to up to 4 analyses (2 h/analysis at room temperature in open vials); reference: 1st analysis	30 pooled samples from serum from a large CDC study	1 replicate/condition batch analyzed by HPLC-MS/MS	1-way (time) repeated measures ANOVA
6. Long-term frozen storage of serum	Serum stored at −20°C for 3, 6, 9, and 12 mo prior to freezing at −70°C; reference: serum stored at −70°C	Low, medium, and high serum QC pools	6 replicates/condition batch analyzed by HPLC-MS/MS	2-way (time, pool level) ANOVA
7. Long-term refrigerated storage of serum	Serum stored at 5°C for 6 and 12 mo prior to freezing at −70°C; reference: serum stored at −70°C	Low, medium, and high serum QC pools	6 replicates/condition batch analyzed by HPLC-MS/MS	2-way (time, pool level) ANOVA

¹HPLC-MS/MS method used for experiments 6 and 7 did not separate the two isobaric compounds MeFox (pyrazino-s-triazine derivative of 4α-hydroxy-5-methyltetrahydrofolate) and 5-formyltetrahydrofolate