Use of either serum or plasma for folate and vitamin B12 determinations.

Kubasik et al. (1) reported that the literature regarding proper specimencollection procedures for substances measured by radioassay or radioimmunoassay is poorly defined. Specifically, folate values for ethylenediaminetetraacetate(EDTA)-containing plasma were reported to be only one-third as large as those obtained by analyzing the corresponding serum or heparmn-treated plasma by radioassay. We have further investigated the effect of various anticoagulants on the values obtained for folate and vitamin B12 with simultaneous radioassay (24). Venous blood samples were collected from 30 healthy individuals, 25 to 45 years old. Five types of evacuated tubes were used to collect the blood samples: (a) Becton Dickinson no. 3206U, 5-ml capacity, no anticoagulant (red top); (b) Becton Dickinson no. 3206Q, 5-ml capacity, 6mg of disodium EDTA (lavender top); (c) Monoject no. 00866, 4-ml capacity, 0.3 ml of 3.8% solution, equivalent to 10.8 mg of sodium citrate (blue top); (d) Monoject no. 00607, 5-mI capacity, 0.5 ml of 0.1 mol/liter solution, equivalent to 6.7 mg of sodium oxalate (black top); (e) Monoject no. 00647, 2.5-mi capacity, 286 USP units of sodium heparin (green top). We attempted to obtain a specimen for each tube from each individual, but this was not possible for all the subjects. The blood with no anticoagulant was allowed to clot for 15 mm. All tubes were then centrifuged for 20 mm at 1240 X g. Plasma or serum was separated from packed erythrocytes and stored at -20 #{176}C until assayed. The Simu1TRAC Radioassay Kit, Vitamin B12 (57Co)-Folate 1 I (Schwarz/Mann, cat. no. 225517, lot CN4034A) was used to determine vitamin B12 and folate. A Packard Model 5230 dual-channel gamma counter was used to determine the radioactivity with discriminator limits for I and 57Co set so that in either case the spillover into the other channel was <1%. The reagents were brought to room temperature before the assay and the samples were analyzed in duplicate. No more than 50 tubes were run within a single assay. Two commercial controls were analyzed with each assay. Tubes containing clinical samples (100 jl) or standards were added to buffer and the tubes were heated at 100 #{176}C (boiling water bath) for 45 mm. The tubes were cooled to 20-25 #{176}C, tracers and binders were added, and the tubes left at room