The Continued Need for the Routine Assessment of Folate Status

Bremansu Osa-Andrews, Melissa Sanchez, Ibrahim A Hashim, The Continued Need for the Routine Assessment of Folate Status, Laboratory Medicine, Volume 54, Issue 4, July 2023, Pages 424–428, https://doi.org/10.1093/labmed/lmac148

Abstract

The Choosing Wisely initiative recommended cessation of folate measurement, suggesting folate supplementation in macrocytic anemia. This study reviewed the need for continued blood folate testing at a large SafetyNet county teaching hospital.

Red blood cell (RBC) folate, vitamin B₁₂, iron, ferritin, and hemoglobin results were obtained for utilization review.

Of the 593 RBC folate results, 69 (11.7%) were deficient and 30 (5%) had high values. Collectively, 369 (73.9%) had normal vitamin B₁₂ levels, 342 (70%) had low hemoglobin, 184 (62.5) had normal and 57 (19.4%) had low ferritin, 122 (38.2%) had normal and 188 (59%) had low iron levels. A total of 41 (12%) had normal folate, low ferritin, low hemoglobin, and low iron, suggestive of iron deficiency anemia. There were 11 patients who exhibited low folate, low or normal ferritin, low hemoglobin, and low iron levels, suggesting combined folate and iron deficiency anemias.

This study highlights the need for institutions to assess the applicability of national recommendations to their local population.

Nearly 20 years after the US Food and Drug Administration mandated the fortification of all foods with folate, the Choosing Wisely organization of the American Society for Clinical Pathology in 2017 recommended the withdrawal of laboratory measurement of red blood cells (RBC) and serum folate, citing the progressive decline of folate deficiency. 1 This recommendation may have garnered support from National Health and Nutrition Examination Survey (NHANES) data, which indicate a substantial decrease in folate deficiency from 16% to 0.5% before and after fortification, respectively. 2 This NHANES report may not be representative of some patient demographics, which may have higher prevalence of folate deficiency than reported. Moreover, low dietary intake may represent only a partial component of the etiology of folate deficiency. Folate deficiency has been reported during pregnancy, in pediatric and geriatric patients where there is increased demand for folate, and in conditions such as hemolysis, leukemia, and exfoliative dermatitis, which are associated with rapid cellular proliferation. Jejunum disease and short bowel syndrome cause malabsorption, and bacterial overgrowth as well as drugs such as alcohol, anticonvulsants, and oral contraceptives have been shown to cause folate deficiency. 3 In populations with these conditions, supplementing folate without testing may not resolve the underlining conditions responsible for the folate deficiency. Diagnosis of folate deficiency anemia, one of two forms of megaloblastic macrocytic anemia (the other being vitamin deficiency anemia), requires low blood folate, hemoglobin, and macrocytosis (MCV >100 fL). 3 Nonmegaloblastic macrocytic anemia may occur due to hypothyroidism, liver disease, myelodysplastic syndrome, and certain drugs, and many patients with these conditions may have normal folate and vitamin B₁₂ levels. 4 The Choosing Wisely organization further indicated that providers could commence folate supplementation treatment if macrocytic anemia is suspected without testing for folate status. However, peer-reviewed reports of elevated folate-related risks and resurgence of cancers 5 and other morbidities have multiplied. 6–8 Recent data from the National Institutes of Health revealed a strong association between oversupplementation of folate and vitamin B₁₂ deficiency anemia masking with concomitantly serious clinical implications, 9, 10 since folate is required to activate vitamin B₁₂. These situations underline the intricate role of folate in the diagnoses of macrocytic anemia and other folate-associated abnormalities.

The evidence provided above suggests the need for further investigation into the clinical utility of folate assessment. In this article, we lay out the argument for continued laboratory testing of RBC folate by laboratory institutions within the setting of accurate diagnosis and management of macrocytic anemia.

Materials and Methods

Laboratory findings for RBC folate, vitamin B₁₂, iron studies, ferritin, and hemoglobin of all patients being investigated for suspected anemia were obtained from 2020 to 2021 as part of a test utilization review effort at Parkland Memorial Hospital. The demographics of our patient population are 24.2% African American, 37.3% Hispanic, and 52% female; about 25,000 pregnant persons are seen annually. The population is unique in that 21.5% of those assessed for social determinants of health exhibited food insecurities. Over 38,000 patients present annually with gastrointestinal disorders, over 34,000 with eating disorders, and nearly 230,000 with clinical diagnosis of anemia; it is a population clearly at risk of folate deficiency. All measurements were performed in-house except RBC folate, which was sent to a reference laboratory. The reference laboratory where the RBC folate was determined used the quantitative chemiluminescent immunoassay method for testing using 1 mL whole blood sample in a lavender tube. Hemoglobin (a component of complete blood count profile) was measured using a Sysmex automated analyzer (Sysmex). Iron studies and ferritin were performed using cobas automated chemistry analyzers (Roche Diagnostics). Vitamin B₁₂ was measured using the Vitamin B₁₂ II binding assay using the cobas 8000 (e602) (Roche Diagnostics). The data used for this project were retrieved from Beaker EPIC in retrospect. The Excel and GraphPad Prism statistical software tools were used to filter and analyze the raw data, respectively. Of 593 patient results for RBC folate, 553 were analyzed for vitamin B₁₂, 489 for hemoglobin, 294 for ferritin, and 319 for iron. The cut-offs for RBC folate and vitamin B₁₂ used were 1300 ng/mL (based on the Centers for Disease Control and Prevention microbiological assay reference range of 223–1100 ng/mL, given the difference in methodology) 11 and a postulated optimal RBC folate level of 1000 to1500 ng/mL by Knew Health. 12 Reference ranges used for statistical analysis were 13.2 to 16.9 g/dL (male) and 11.2 to 15.7 g/dL (female) for hemoglobin, 20 to 250 ng/mL (male) and 10 to 263 ng/mL (female) for ferritin, and 59 to 160 µg/dL (male) and 37 to 145 µg/dL (female) for iron. Institutional review board approval was obtained (study No. STU 2022-0262).

Results

A total of 593 RBC folate results of persons being investigated for diagnosis of suspected anemia were analyzed. A cut-off value of 366 ng/mL (in use by the reference laboratory) was used to define folate status. Of the total number of 593 patients, 69 (12%) had low RBC folate values, and 30 (5%) had elevated RBC folate values ( Figure 1). Further, 494 patients had normal RBC folate levels, 83% ( Figure 1) of all subjects tested for the vitamin. Of the 494 patients who showed normal RBC folate, 93 had anemia indicated by low hemoglobin. Of the 553 patients assessed using the cut-off of 12, 17 showed low values, 413 had normal levels, and 140 patients had high levels >2000 pg/mL ( Figure 1). The hemoglobin results showed 342 with low values and 147 at normal levels of a total 489 patients tested for the analyte. We found that 342 of 489 patients were anemic with low hemoglobin levels, representing 70% of the population. Of 294 ferritin measurements, 57, 53, and 184 patients had low, high, and normal results, respectively. Of the 319 iron measurements taken, 188 patients showed low values, 9 had high values, and 122 showed normal results ( Figure 2). Additional analysis indicated that 41 patients had normal folate, low ferritin, low hemoglobin, and low iron, representing 12.0% of the anemic group ( Figure 2). There were 52 patients with normal folate, normal ferritin, low hemoglobin, and low iron, making up 15.2% of the anemia group. Interestingly, 4 patients had low folate levels with low ferritin, low hemoglobin, and low iron (1.2%) ( Figure 2). A total of 8 patients showed low folate levels, normal ferritin, low hemoglobin, and low iron (2.3%) ( Figure 2). There were 23 patients with low folate levels, low or normal ferritin, low hemoglobin, and normal iron. Additional results showed that 7 patients had low folate levels with normal ferritin, normal hemoglobin, and normal iron. Of the 30 patients who had high RBC folate, 14 (46.7%) were found to be anemic, representing 4.1% of the total anemic population ( Figure 2). Altogether, 31.3% of the patients who had low hemoglobin also exhibited normal or high folate levels. Those patients would be most at risk of mistreatment ( Figure 2). Three patients had low folate, low vitamin B₁₂, and low hemoglobin, whereas 2 patients had high folate, low vitamin B₁₂, and low hemoglobin levels ( Figure 2). Table 1 summarizes the ranges and median values of the results obtained. The upper limits of the analytical measuring range of RBC folate and vitamin B₁₂ have been normalized as upper reference intervals to establish ranges.

Summary of the Range and Median of Analyte Results

Summary of the Range and Median of Analyte Results

Distribution of partial anemia indices. Uneven distribution of RBC folate, vitamin B12, hemoglobin, ferritin, and iron in patients being investigated for anemia and presenting at a SafetyNet hospital.

with low hemoglobin levels." />

Clinical utility of RBC folate measurement in the investigation of suspected anemia. Histograms showing the observed different permutations of RBC folate levels in the presence of low, normal, or high ferritin, hemoglobin, and iron. The anemic patients (n = 342) represent those with low hemoglobin levels.

Discussion

Following multiple reports of drastically diminished incidences of folate deficiency due to the FDA mandate to fortify all food with folate, the Choosing Wisely campaign recommended cessation of blood folate test orders. In this report, we investigated the continued need for blood folate measurement among our patient population and reasons that clinicians continue to order the test regardless of the aforementioned regulatory recommendation and examined the vital importance of blood folate testing before supplementation.

Although the notion that folate deficiency has markedly dwindled from a broader national perspective, it is not necessarily a reflection of specific local evidence. Moreover, decreased cases of folate deficiency may not be an adequate justification for the discontinuation of the assessment of folate status. Results based on our local patient population showed a 11.5% higher folate deficiency than the 0.5% reported in the 2005 NHANES postfortification data. Further, one of the articles on which the decision of the Choosing Wisely organization was based reported that only 4 out of 560 patients tested at the Health Sciences Center in Canada for blood folate had folate deficiency, representing 0.7% of all patients tested. 13 Data from our study not only shows a higher percentage of folate deficiency but does so based on a slightly higher sample size than the Shojania and von Kuster 13 report. Although folate deficiency can persist with or without anemia, the more clinically relevant folate index is that related to anemia. Folate deficiency anemia is generally considered to be low blood folate level with low hemoglobin and macrocytosis. Our results showed a higher percentage of folate deficiency–induced anemia, 35 of 593 patients, representing 6%, than earlier reports where no anemia cases attributable to low folate were found. The highest percentage of folate deficiency anemia in the Canadian study was 0.2%, recorded at the St Boniface General Hospital. The discrepancy between data from our study compared with the studies that informed the Choosing Wisely campaign’s decision cannot be overemphasized.

Conventionally, folate deficiency has been a greater concern for clinicians and laboratory professionals than elevated blood folate levels. This observation is predominantly due to lack of clinically useful information regarding the consequences of increased blood levels of folate. Results from this study indicate that 5% of patients had elevated RBC folate levels, suggesting not just elevated folate but possibly folate toxicity. More concerning is that recent reports have revealed the association of elevated blood folate with higher risk of certain cancers, including lung, prostate, and small cell carcinomas. According to the Choosing Wisely campaign, clinicians may prescribe folate supplements in any cases of macrocytic anemia without the need to first evaluate folate status. However, this recommendation has the potential of exposing patients to elevated folate levels. Anemia is a multietiological condition that includes but is not limited to folate deficiency. Anemia is also known to be caused by vitamin B₁₂ and iron deficiencies, both of which may present with a normal blood folate level. Normal RBC folate is useful to conclusively rule out folate deficiency anemia when found with low hemoglobin and when either ferritin or iron levels are reduced. Without folate assessment, misdiagnosis of folate deficiency anemia may escalate the risk of folate toxicity. Supplementing folate without testing in every case of macrocytic anemia diagnosis can elevate folate level, thereby compromising patient safety. In the current study, 16% of the 83% of patients who had normal folate were simultaneously diagnosed with anemia with a correspondent low hemoglobin. Persons in this group are at high risk for possible future folate spikes on folate supplementation prescription. It could be suggested that a segment of the 5% of patients who had elevated RBC folate may have been prescribed supplemental folate, or the possible abuse of folic acid supplements may be cited as the cause. However, patients exhibiting normal folate test results with the background of anemia risk a similar hazard should providers comply with the folate supplementation without testing campaign. The risk to patients seems latent, untraceable, gradual, and long term since it correlates indirectly with the risk of cancer and not necessarily to the malignancy itself. 5–7 Also, there is a degree of ambiguity in some of the data implicating the relationship between high folate, when given with vitamin B₁₂, and risk of cancer. Nevertheless, it is reasonable not to trade patient safety for reduction in cost burden to patients.

Mean corpuscular volume >100 fL is a frequently used marker for macrocytic anemia, whereas folate (vitamin B₉) and vitamin B₁₂ deficiencies are markers purposely for megaloblastic anemia. Measurement of homocysteine and methyl malonic acid distinctively distinguishes between vitamin B₁₂ and vitamin B₉ deficiency megaloblastic anemia. The pathological consequences of folate deficiency and vitamin B₁₂ deficiency anemias are similar and their distinctive management regimen are obvious. Supplementing folate without vitamin B₁₂ usually treats the symptoms of anemia but not the neurological pathology. These facts demonstrate the importance of vitamin B₁₂ testing and supplementation within the scope of anemia, especially if vitamin B₁₂ deficiency is prevalent. However, our findings reveal a higher frequency of folate (12%) deficiency than vitamin B₁₂ deficiency (3%) in a patient population presenting with anemia. This may suggest a higher incidence of folate-induced megaloblastic anemia than vitamin B₁₂-deficiency megaloblastic anemia. In contrast, the Choosing Wisely campaign has no documented reservation regarding vitamin B₁₂ testing as it does folate testing. Deficiency of such nutritive analytes could be a function of food security within certain demographics and may vary from population to population. On the other hand, elevated folate causes vitamin B₁₂ masking, which, although it can be resolved when diagnosed early, can go unnoticed without folate testing, leading to serious medical consequences. 14 Since folate is required for the activation of vitamin B₁₂, disproportionally high folate concentration can activate even small amounts of cobalamin, thereby concealing the underlining deficiency until neurological effects of vitamin B₁₂ commence. In this study, only 17% of patients had low vitamin B₁₂ levels. Two of those patients had high folate and low hemoglobin levels, the most at risk of high folate-mediated vitamin B₁₂-deficiency masking. Additionally, three patients had full-blown megaloblastic anemia with indices of low folate, low vitamin B₁₂, and low hemoglobin levels. Given our results, the operationalization and withdrawal of essential nutrient testing should be based on local population experience rather than generalized recommendation, albeit by a credible professional entity whose endorsement drew on alternative results.

Low RBC folate is an independent predictor of folate deficiency and folate deficiency anemia, improbable diagnoses to definitively obtain without the measurement of blood folate, demonstrating the continual need for the retention of the test. The alternative to RBC folate is serum folate, but RBC folate has some comparative advantages. Low serum folate reflects a transient deficiency whereas low RBC folate is a function of stored folate status over approximately 4 months. The nonspecific serum folate 15 may even be low in the presence of normal tissue folate storage, suggesting that low RBC folate indicates a higher risk of anemia does than serum folate. A single RBC folate measurement is therefore a reflection of the patient’s stored folate level but, more than that, cumulative low folate results of a patient population are an indirect measure of the nutritive habits and economic status of the local demographic. However, RBC folate testing is not without inaccuracy. According to internal data from the Mayo Clinic, RBC folate testing is fraught with significant analytical variability and poor precision. The Mayo Proceedings stated that these drawbacks were the predominant informant in their withdrawal of the test, in addition to their comparative studies between serum and RBC folate that showed a strong correlation. 16 These pieces of information indicate that RBC and serum folate are generally comparable, barring methodological differences, strengths, and frailties. The Choosing Wisely campaign’s recommendation for the abolishment of both tests suggests their rationale is not a function of the variability in the type of specimen but on the supposed clinical inexpediency of both tests. Data from the Mayo Clinic study 16 showed strong concordance between serum and RBC folate levels and support the use of RBC folate as the sole specimen type in this study.

Patients with combined folate and iron deficiency anemia do not necessarily show macrocytosis with a concomitantly lower value in the measurement of MCV. The one dependable way to distinguish such patients from other anemia variants is to measure blood folate. However, supplementation of folate without testing in this patient group will be unlikely to lead to clinical consequences. In the present data, only 3.5% of the anemic patient population exhibited this coexistence of folate and iron deficiency anemias, suggesting that it is an uncommon occurrence. Together with the group that had folate deficiency anemia without iron deficiency, only 9.4% of the anemic group would not have been affected by folate prescription without testing. This number is not comparable to the 31.3% of the population who had anemia in the setting of normal to high folate and whose safety might be compromised if folate were given without prior folate measurement. This normal folate anemic group could either have microcytic anemia, vitamin B₁₂ deficiency anemia, or nonmegaloblastic macrocytic anemia, usually caused by hypothyroidism or liver disease. We have shown here that the conditions in which measurement of blood folate is not consequential are also rare. The more common anemic disorders pose realistic but silent risks if folate testing is disregarded. These data connote the necessity for institutions to conduct regionally based research as the major source of operationalization instead of implementing recommendations from regulatory entities without thorough considerations.

Conclusion

This study has shown the expediency of continuing blood folate measurement while creating awareness of the associated risks of supplementing folate without testing. Low RBC folate level is more prevalent than low vitamin B₁₂ in a patient population presenting with anemia even in this era of folate fortification. Normal RBC folate is useful to assertively rule out folate deficiency anemia when hemoglobin and either ferritin or iron levels are decreased. Low RBC folate and hemoglobin levels consistent with folate deficiency anemia persist regardless of folate fortification. The RBC folate measurement remains clinically valuable and improves the diagnosis while reducing the incidence of misdiagnosis of folate deficiency anemia. Our study shows the importance for clinical laboratories to critically assess the applicability of recommendations by professional societies, particularly those referring to test utilization, to the population they serve and from which samples are being received. National recommendations may not be applicable to the local population. Assessment of folate status is useful to assess folate deficiency in patients with microcytic anemia. Higher than normal RBC folate values should be reevaluated for their association with the risk of certain cancers and appropriate intervention prescribed.