Whole Blood Stored at −70°C for 10 Years Appears to Provide the Comparable Stability to Folate Forms as Frozen Hemolysate with Ascorbic Acid
Authors: Paladugula Neelima , Fazili Zia , Jabbar Shameem , Zhang Mindy , Pfeiffer Christine 
Published: November 2023

ObjectiveLong‐term storage of whole bloodsamples can negatively affect folate stability and thus the accuracy of statusassessment. To our knowledge there is no information available on the long‐termfrozen storage stability of hemolysates prepared with ascorbic acid compared to whole blood (WB).MethodsWe stored several aliquots of undiluted WB and hemolysates (WB diluted 1/11 with 1% ascorbic acid) from 15 blood donors (n=10 with MTHFR C/C and n=5 with T/T genotype) for 10 y at‐70°C. In 2016, we thawed 1 aliquot each of frozen WB for ~0.5 h at room temperature and prepared fresh hemolysates. Similarly, we thawed 1 aliquot each of frozen hemolysate for ~0.5 h at room temperature. The fresh (2016) and the previously prepared (2006) hemolysates were incubated for 4 h at 37°C to deconjugate folate polyglutamates before we extracted and analyzed the samples for 6 folate forms by LC‐MS/MS. We calculated the sum of folate forms as total folate by LC‐MS/MS (TFOLLC). We also measured total folate by microbiologicassay (TFOLMA) from the same fresh and previously made hemolysates, however without incubating the hemolysates. Each experiment was carried outover 2 days and we calculated mean concentrations from the 2 analytical methods.ResultsWe found good agreement between whole blood folate concentrations (mean±SD, nmol/L) for stored WB vs. stored hemolysates for both genotypes and methods: TFOLLC 408±147 vs. 400±147 (C/C) and 418±82 vs. 409±62 (T/T); TFOLMA 381±161 vs. 385±162 (C/C) and 417±96 vs. 423±76 (T/T). The LC‐MS/MS folate form concentrations also compared reasonably well for stored WB vs. stored hemolysates: 5‐methyltetrahydrofolate(5‐methylTHF): 330±126 vs. 319±125 (C/C) and 314±70 vs. 308±51 (T/T); MeFox(an oxidation product of 5‐methylTHF): 59±15 vs. 56±15 (C/C) and 65±14vs. 56±10 (T/T); and non‐methyl folate (sum of 5‐formylTHF, THF, and 5,10‐methenylTHF): 17±7.1 vs. 24±7.6(C/C) and 37±18 vs. 43±18 (T/T). The mean relative differences (%) between stored WB and stored hemolysate for TFOLLC,5‐methylTHF, and MeFox, were 2.3%, 4.1%, and 6.2% in C/C subjects and 1.6%, 1.0%, and 15.8% in T/T subjects, respectively. The differences for non‐methyl folate were larger (‐29% in C/C and −17% in T/T subjects). The differences between stored WB and stored hemolysate for TFOLMA were small (‐0.5% in C/C and −1.9% in T/T subjects). For stored WB, the TFOLLC produced on average 8.7% (C/C) and 0.8% (T/T) higher concentrations than the TFOLMA, while for stored hemolysates, TFOLLC concentrations were on average 5.6% (C/C) higher and 3.0% (T/T) lower than TFOLMA concentrations. The Pearson correlation between these 2 methods was >0.97 both for stored WB and stored hemolysates.ConclusionIn this small sample set of MTHFR C/C and T/T WB samples, long‐term (10 y) storage of undiluted WB vs. hemolysates at −70°C showed TFOLLC and TFOLMA concentrations that were comparable for these 2 sample types. Concentrations of folate forms were also similar for these 2 sample types, however non‐methyl folate seemed to favor the hemolysate storage while methyl folate seemed to favor the WB storage. Both methods produced TFOL results that were comparable within ±10%.