Folate Forms in RBC and Whole-Blood Lysates Appear Stable When Stored Frozen for 2 Years.

ABSTRACT

BACKGROUND: The use of RBC lysate (RBC-Lys) eliminates the need for serum folate and hematocrit (Hct) measurement to calculate RBC folate. Information on the long-term frozen storage stability of RBC-Lys is missing. OBJECTIVES: We aimed to assess the comparability of RBC folate forms in whole-blood lysate (WB-Lys) and RBC-Lys and the folate stability in both matrices. METHODS: We prepared conventional WB-Lys (111 dilution with 1% ascorbic acid) and RBC-Lys (111 dilution of washed and saline-diluted RBCs with 1% ascorbic acid) from EDTA blood (n = 60 adult donors) and stored lysates at -70°C until analysis at baseline (1 wk), 3, 6, 12, and 24 mo. Before analysis by HPLC-tandem MS, we incubated the WB-Lys (4 h at 37°C) and treated the RBC-Lys with human recombinant γ-glutamyl hydrolase for folate polyglutamate deconjugation. We analyzed RBC-Lys samples for hemoglobin (Hb) (same aliquot) to normalize for the preanalytical dilution; Hb-folate was converted to RBC folate for each folate form using the mean corpuscular Hb concentration. We analyzed Hct as well as folate forms in matching serum samples for traditional RBC folate calculation. We conducted descriptive data analyses (correlation, Bland-Altman plot, Deming regression). RESULTS: At baseline, results for RBC folate forms derived from WB-Lys compared with RBC-Lys samples showed excellent correlation (Pearson r ≥ 0.97). Mean ± SD concentrations compared well for total folate (WB-Lys 886 ± 255 compared with RBC-Lys 899 ± 271 nmol/L), 5-methyltetrahydrofolate (WB-Lys 831 ± 258 compared with RBC-Lys 843 ± 276 nmol/L), and nonmethyl folate (WB-Lys 53.3 ± 74.4 compared with RBC-Lys 52.9 ± 70.7 nmol/L), but were 17% higher in RBC-Lys for pyrazino-s-triazine derivative of 4α-hydroxy-5-CH3-H4folate (MeFox) (WB-Lys 147 ± 44.1 compared with RBC-Lys 172 ± 53.5 nmol/L). Frozen storage of WB-Lys and RBC-Lys samples for ≤24 mo showed ≤5%, ≤5%, ≤13%, and ≤11% change in total folate, 5-methyltetrahydrofolate, nonmethyl folate, and MeFox, respectively. CONCLUSIONS: Erythrocyte folate forms appear to be stable in RBC-Lys samples stored frozen at -70°C for ≤2 y. The relatively small changes in folate concentrations over time were comparable between RBC-Lys and conventionally prepared WB-Lys samples.