(P17-009-24) Red Blood Cell (RBC) Folate Forms Showed Good Correspondence in Washed and Unwashed Blood Sample Types

Objectives: To simplify processing of RBCs, we compared RBC folate forms in washed and unwashed blood samples.

Methods: We obtained fresh EDTA blood (10 blood bank donors; 3 tubes/donor) and generated RBCs (refrigerated centrifugation at 1500xg, 10 min) using no wash (with second centrifugation step), 1x wash, and 3x wash (conventional procedure). Cold saline (1:1) was used for the 1x and 3x wash conditions. We diluted RBCs (unwashed and washed) with saline (1:1), mixed well, prepared lysates (1:11 dilution) with 1% ascorbic acid, and stored samples at -70°C for 1 week. At the time of analysis (2 replicates/sample type over 2 days), we treated RBC lysates with recombinant exo ɣ-glutamyl hydrolase for folate polyglutamate deconjugation and conducted sample clean-up by solid phase extraction prior to HPLC-MS/MS analysis. We measured hemoglobin (Hb) in the same RBC lysates to correct for residual moisture in the packed RBCs and normalized the concentration of each folate form to Hb using the mean corpuscular Hb content.

Results: Concentrations (mean±SD, nmol/L) of folate forms for no wash or 1x wash vs conventional showed excellent correlation (Pearson r ≥0.985) and correspondence for 5-methyltetrahydrofolate (5-methylTHF: 774±270 or 766±260 vs 761±264), non-methyl folate (53±68 or 55±69 vs 51±69), and MeFox (164±39 or 158±36 vs 159±38), respectively. The Deming slope (95% CI) was not significantly different from 1 for 5-methylTHF (1.02 [0.95-1.09] or 0.99 [0.95-1.03]), non-methyl folate (0.98 [0.95-1.01] or 0.99 [0.89-1.09]), and MeFox (1.04 [0.90-1.17] or 0.94 [0.82-1.06]) and the Deming intercept was not different from 0. The mean relative difference (% [95% CI]) for no wash or 1x wash vs conventional was mostly small: 5-methylTHF (1.8% [-0.2% to 4%] or 0.82% [-1.54% to 3.2%]), MeFox (3.3% [0.4% to 6.2%] or 0.11% [-2.8% to 3.0%]), and non-methyl folate (13% [1.63% to 24%] or 14% [3.1% to 24%]), respectively.

Conclusions: RBC folate form concentrations corresponded well across wash procedures but were slightly higher in no wash samples for minor folate forms.

Funding Sources: This work was supported by direct appropriations from US Congress.

The findings and the conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.