Fortification of grain products with folic acid was mandated in order to add about 0.1 mg folate to the daily intake of an adult. Some studies have shown that the resulting increase, in daily folate intake, is about 0.2 mg [10, 11]. Even if a patient has no other source of folate intake, a daily intake of 0.1 mg folic acid is more than enough to prevent folate deficiency anemia. In fact, patients with folate deficiency megaloblastic anemia can be treated with as little as 0.025 mg  or 0.050 mg  of folic acid daily.
Only 11 of 2154 (0.5%) SF assayed at HSC during 2001 were subnormal. In none of these patients, was the SF as low as is seen in patients with folate deficiency anemia. Similarly, in none of the patients seen at SBGH after 1998, could the anemia or macrocytosis be attributed to folate deficiency. In the National Health and Nutrition Examination Survey (NHANES) of 1999-2000 in USA [6, 7], the prevalence of low SF (<6.8 nmol/L) was 0.5% (compared to 16% in NHANES III for the period of 1988-1994) . The prevalence of low SF and low RF reported by a large private Laboratory in Canada, during the period of February 1, 1999 to March 31, 2000 were 0.22% and 0.41% respectively . Latif and colleagues  from Cleveland Clinic found 77 of 4985 (1.6%) SFs during 2001 were lower than normal. The MCV of these 77 patients was not different from those with normal SF. Only 39 of 74 patients with low SFs (0.9% of clinically suspected folate deficient patients) were actually prescribed folic acid. They commented that true folate deficiency is very rare; and questioned whether there is a role for folate determinations in clinical practice in the USA . The prevalence of subnormal SF at HSC during 2001 (0.5%) was similar to prevalence of low SF in random population survey 1999-2000 of NHANES (0.5%) . Joelson et al  reviewed the red cell folate data of 3 USA county hospitals with large cohort of indigent patients, during the years 1997, 2000 and 2004. Cutoffs for RF in these hospitals were 362 and 213 nmol/L. Using a RF cutoff 362 nmol/L, the combined incidence of folate deficiency decreased from 4.8% in 1997 to 0.6% in 2004. At a cutoff of 213 nmol/L, the incidence dropped from 0.98% to 0.09% . They concluded that routine folate measurements for patients with anemia and/or macrocytosis is difficult to justify.
Problems with Folate Assays and Normal Range
There are numerous methods for folate assays, each giving different result for assay of the same sample. Gunter et al  reported on interlaboratory comparison of SF and RF performed by 20 research laboratories (8 using L. Casei microbiological assays and 12 using competitive binding methods). Aliquots of six normal serum and blood were sent to each of 20 laboratories to perform duplicate assays on each sample daily for three days. There were a two to nine-fold differences in reported folate concentrations between the methods. In proficiency testing of the College of American Pathologists for folate assays , when aliquots of one sample of serum were sent to 1233 laboratories, using 12 different competitive binding methods of folate assays, the mean SF for the laboratories that were using the same method of folate assay ranged from 6.95 to 18.75. At SBGH where the same microbiological assay of folates had been performed since 1966, the normal RF was >225 nmol/L; and in folate deficiency megaloblastic anemias, RF was <100 nmol/L. It is for these reasons that we have commented that, in our study, in none of the subjects the anemia could be attributed to folate deficiency.
The normal range of SF and RF varies depending on the geographical location, the season during which the samples were obtained for determining the normal range and the average folate intake of the population. When SF or RF of a patient is low, it does not necessarily mean that the patient is folate deficient. It only means that the patient is consuming or absorbing less folate, compared to normal subjects in the area. It is recommended that laboratories not only report the normal range for SF and RF, but also to indicate the expected range of SF and RF for truly folate deficient individuals . Most of the laboratories in US or Canada, are no longer able to provide a reference range for truly folate deficient individuals because they do not have access to sufficient number of samples of blood from truly folate deficient subjects. In the Herbert study , the reference range for L. Casei microbiological assay of SF was 15.9-36 nmol/L and folate deficient subjects had SF of <6.8 nmol/L.
Relation of low folate assays and anemia due to folate deficiency
Many physicians order serum folate, B12 and ferritin for the investigation of anemia. If SF is low they assume that the anemia is due to folate deficiency. This is not a correct assumption. Anemia due to folate deficiency is megaloblastic. No anemia should be attributed to folate deficiency, unless there are megaloblastic changes in the peripheral blood or in the bone marrow aspirate. Even when there are megaloblastic changes in the bone marrow, the low SF or RF does not necessarily mean that the patient is folate deficient. About 2-19% of patients with pernicious anemia have low SF and, 23-62% have low RF . Serum folate falls below normal within a short period of reduced dietary intake of folate. In the past, low SF was found in 9-75% of probably normoblastic hospitalized patients .
In the sequence of events leading to folate deficiency anemia, the fall of SF is the earliest manifestation of folate deficiency and anemia is a late manifestation of folate deficiency. When a normal man was placed on a folate deficient diet, his SF fell below normal by 3 weeks, RF fell below normal by 17 weeks, macocytic red cells appeared in the blood smear by 18 weeks and anemia and megaloblastic bone marrow developed at 19 weeks . By the time that anemia had developed, his SF was 1.0 nmol/L (normal range 15.9-36 nmol/L) and his RF was 22 nmol/L.
As the result of fortification of grain products with folic acid, dietary folate deficiency has become very rare. Consequently, there is no longer any justification in ordering folate assays to evaluate the folate status of the patients. There are still some patients who may be folate deficient (very small premature babies before they are able to start taking grain products, pregnant women who are not taking multivitamins and who also avoid eating grain products, malnourished alcoholics or those with celiac disease or tropical sprue who are avoiding taking grain products). However, these patients could be easily identified by their medical history and could be placed on prophylactic folic acid therapy without testing for folate deficiency, as long as it is determined that their SB12 is normal.
In taking a patient's dietary history to assess dietary intake of folates, physicians should note that, unlike the past when fresh green leafy vegetables and fruits were the major source of dietary folate, now, grain products are the major source of dietary folate. In the last NHANES, bread rolls and crackers were the single largest contributor of total folate to the American diet .