The Journal of Nutrition
Methodology and Mathematical Modeling
Comparison of Serum and Red Blood Cell
Folate Microbiologic Assays for National
Christine M. Pfeiffer,4* Mindy Zhang,4David A. Lacher,5Anne M. Molloy,6Tsunenobu Tamura,7
Elizabeth A. Yetley,8Mary-Frances Picciano,8,9and Clifford L. Johnson5
4National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341;5National Center for
Health Statistics, Centers for Disease Control and Preventio, Hyattsville, MD 20782;6Institute of Molecular Medicine, Trinity College,
Dublin Ireland;7University of Alabama at Birmingham, Birmingham, AL 35294; and8Office of Dietary Supplements, National Institutes
of Health, Bethesda, MD 20892
Three laboratories participated with their laboratory-specific microbiologic growth assays (MA) in the NHANES 2007–2008
to assess whether the distributions of serum (n = 2645) and RBC folate (n = 2613) for the same one-third sample of
participants were comparable among laboratories. Laboratory (L) 2 produced the highest and L1 the lowest serum and
RBC folate geometric means (nmol/L) in the NHANES sample (serum: L1, 39.5; L2, 59.2; L3, 47.7; and RBC: L1, 1120; L2,
1380; L3, 1380). Each laboratory produced different reference intervals for the central 95% of the population. Pearson
correlation coefficients were highest between L3 and L1 (serum, r = 0.95; RBC, r = 0.92) and lowest between L2 and L1
(serum, r = 0.81; RBC, r = 0.65). Notable procedural differences among the laboratories were the Lactobacillus rhamnosus
microorganism (L1 and L3: chloramphenicol resistant, L2: wild type) and the calibrator [L1: [6S]5-methyltetrahydrofolate
(5-methylTHF), L2: [6R,S] 5-formyltetrahydrofolate ([6R,S] 5-formylTHF), L3: folic acid (FA)]. Compared with 5-methylTHF
as calibrator, the folate results were 22–32% higher with FA as calibrator and 8% higher with 5-formylTHF as calibrator,
regardless of the matrix (n = 30 serum, n = 28 RBC). The use of different calibrators explained most of the differences in
results between L3 and L1 but not between L2 and L1. The use of the wild-type L. rhamnosus by L2 appeared to be the
main reason for the differences in results between L2 and the other 2 laboratories. These findings indicate how assay
variations influence MA folate results and how those variations can affect population data. To ensure data comparability,
better assay harmonization is needed.J. Nutr. 141: 1402–1409, 2011.
Serum and RBC folate concentrations are important nutri-
tional status markers. The NHANES has measured these con-
centrations for over 30 y. The introduction of mandatory folic
acid (FA)10fortification in 1998 contributed to appreciable
increases in blood folate concentrations in the U.S. population
(1). This meant continued monitoring of folate status through
NHANES. Since 1991, that monitoring had been done with the
Bio-Rad QuantaPhase II radioassay (Bio-Rad Laboratories), but
in 2007, the manufacturer discontinued its production. For
NHANES 2007–2008, CDC selected the traditional microbio-
logic growth assay (MA) using Lactobacillus rhamnosus (for-
merly known as L. casei) to measure serum and RBC folate
Important improvements were made to the MA in the 1960s,
such as the development of a chloramphenicol-resistant strain of
the organism, eliminating the need for sterilization or aseptic
addition and enabling the use of disposable labware; the ability
to cryopreserve the inoculum, providing standardized growth
curves for hundreds of assays; and the introduction of auto-
mated microtiter plate technology, miniaturizing the assay and
providing dramatically improved efficiency in absorbance read-
ings (2). These improvements made it possible to use the MA in a
high-throughput routine setting. The MA is sensitive and there-
fore requires only small sample volumes; it is known to measure
all folate vitamers equally (3,4), whereas clinical protein binding
1Supported by the Office of Dietary Supplements, NIH. The findings and
conclusions in this report are those of the authors and do not necessarily
represent the official views or positions of the CDC/Agency for Toxic Substances
and Disease Registry, the NIH, or the Department of Health and Human
2Author disclosures: C. M. Pfeiffer, M. Zhang, D. A. Lacher, A. M. Molloy, T.
Tamura, E. A. Yetley, M-F. Picciano, and C. L. Johnson, no conflicts of interest.
3Supplemental Figures 1 and 2 are available from the “Online Supporting
Material” link in the online posting of the article and from the same link in the
online table of contents at jn.nutrition.org.
10Abbreviations used: FA, folic acid; 5-formylTHF, [6S]5-formyltetrahydrofolate;
L, laboratory; LC-MS/MS, light chromatography tandem MS; MA, microbiologic
growth assay; 5-methylTHF, [6S]5-methyltetrahydrofolate; NIBSC, National
Institute for Biological Standards and Controls; NIST, National Institute of
Standards and Technology; QC, quality control.
* To whom correspondence should be addressed. E-mail: CPfeiffer@cdc.gov.
ã 2011 American Society for Nutrition.
Manuscript received March 18, 2011. Initial review completed April 04, 2011. Revision accepted April 22, 2011.
First published online May 25, 2011; doi:10.3945/jn.111.141515.
assays may have different recoveries for different folate vitamers
(4–7). It is also a comparatively inexpensive assay. Some coun-
tries use the MA in their national nutrition surveys for popula-
tion monitoring and it is of interest to know whether population
results among laboratories can be compared. There is currently
no guidance on using a specific MA protocol or calibrator and
countries implement the MA for their surveys expecting com-
parable results when the same type of methodology is used.
It is widely known that clinical folate assays using mainly a
competitive protein binding format lack good agreement, par-
ticularly for RBC folate (2,8,9). Little information is available
on the comparability of MAs across laboratories. Small sample
sets have shown that mean results produced by different labora-
tories were not always in agreement (8,9). The causes for such
disagreement, however, have remained uninvestigated. It is not
known how method differences affect the distributions of folate
concentrations in the population or the classification of the pop-
ulation regarding folate status. This complicates any attempt to
compare data from different laboratories.
We set out to address the above questions by conducting a
comprehensive method comparison study among 3 expert labo-
ratories using their laboratory-specific MA to analyze serum
and whole blood samples from the same one-third sample of
NHANES 2007–2008 participants. Our study allows for the
first time, to our knowledge, to generate percentile distributions
and reference intervals from 3 different MA to investigate
potential differences among laboratories. To identify potential
sources of differences among these 3 laboratories, calibrators
and quality control (QC) samples were exchanged and available
reference materials were analyzed.
Procedural differences between the folate microbiological assays performed in the 3 laboratories
Chloramphenicol resistant (ATCC 27773)
5-MethylTHF from Eprova
WB diluted 1/11 with 1% ascorbic acid within
1 h of phlebotomy (hemolysate pH, 3.8)
Wild-type (ATCC 7469)
5-FormylTHF (racemic) from Sigma
WB frozen within 1 h of phlebotomy;
at the time of analysis diluted 1/10
with 0.1 mol/L K3PO4(pH 6.3) with 1%
ascorbic acid (hemolysate pH, 4.5)
and incubated for 60 min/378C
7 (0.1 mol/L K3PO4, pH 6.3 with 0.1%
2 Serum QC pools (containing 1%
ascorbic acid), no WB QC
NIST SRM 1955; twice annually
Chloramphenicol resistant (ATCC 27773)
FA from Sigma
WB diluted 1/11 with 1% ascorbic
acid within 1 h of phlebotomy
(hemolysate pH, 3.8); at the time of
analysis incubated for 30 min/RT1
Dilutions/sample (diluent), n 2 (0.5% Na ascorbate) 2 (0.5% Na ascorbate)
Incubation time at 378C
Polynomial regression (3rd order)
3 Serum/3 WB QC pools,
2 serum/2 WB blind QC pools
NIST SRM 1955, NIBSC 03/178, NIBSC 95/528;
minimum twice annually
6.9–8.6% (6.7–49 nmol/L, 59–78 runs)
4 Parameter logistic
4 Serum/4 WB QC pools
Use of international reference materialsNIST SRM 1955; 6 times annually
Serum folate assay between-run
range, n runs)
RBC folate assay between-run
range, n runs)
4.0–9.3% (23–82 nmol/L, 116 runs)5.2–6.8% (7.4–81 nmol/L, 64–73 runs)
6.7–13% (407–1570 nmol/L, 36–105 runs) Data not available6.2–7.7% (397–1110 nmol/L, 64 runs)
1RT, room temperature, WB, whole blood.
Descriptive statistics for serum and RBC folate results for the 3 laboratories,
Serum folate, nmol/L, n = 2645 RBC folate, nmol/L, n = 2613
L1 L2L3 L1L2L3
Mean 6 SE
Geomean 6 SE
45.8 6 0.54
39.5 6 0.42
70.0 6 0.85
59.2 6 0.68
55.7 6 0.62
47.7 6 0.53
1210 6 10.5
1120 6 8.73
1570 6 16.2
1380 6 14.0
1500 6 12.7
1380 6 10.9
Methodology and mathematical modeling 1403
Participants and Methods
NHANES 2007–2008 sample. Serum and EDTA whole blood samples
were collected in the NHANES mobile examination center during 2007–
2008 and a one-third sample of participants 1 y and older was used for
this comparison study. An aliquot of EDTA whole blood was either
diluted (1/11) with 1% ascorbic acid solution [for laboratory (L)1 and 3]
or frozen directly (for L2, which added ascorbic acid solution later).
Each laboratory received aliquots of frozen serum (n = 2645) and whole
blood/hemolysate (n = 2613) via dry ice shipments weekly. All respon-
dents gave their informed consent and the NHANES protocol was re-
viewed and approved by the National Center for Health Statistics
Institutional Review Board.
Sample exchange study. To identify potential sources of differences
among these laboratories, in 2008 we conducted a sample exchange
study. Each laboratory provided calibrators and QC samples to the other
2 laboratories and each laboratory analyzed available reference mate-
rials. The calibrators from L1 included several folate stock solutions:
[6S]5-methyltetrahydrofolate (5-methylTHF), 100 mg/L (218 mmol/L),
Merck Eprova (Merck & Cie), used as a calibrator in the MA; [6S]
5-formyltetrahydrofolate ([6S]5-formylTHF), 100 mg/L (211 mmol/L),
Merck Eprova; FA (pteroylglutamic acid), 100 mg/L (227 mmol/L),
Merck Eprova; and FA, 500 mg/L (1133 nmol/L), Sigma (Sigma
Chemicals), 2 different lot numbers. The calibrator from L2 was [6R,S]
5-formylTHF (racemic), 23 mmol/L (Sigma). The calibrator from L3 was
FA, 100 mg/L (227 nmol/L) (Sigma). QC samples included 3 levels each
of serum and whole blood hemolysate pools from L1, 2 levels of serum
pools from L2, and 4 levels each of serum and whole blood hemolysate
pools from L3. The reference materials were from the National Institute
of Standards and Technology (NIST) and the National Institute for
NIST SRM 1955serum material (10),1 levelof the NIBSC 03/178serum
material (11), and 1 level of the NIBSC 95/528 whole blood material
(12). Additionally, 20 randomly selected NHANES serum and whole
blood samples were also analyzed by each laboratory as part of the
sample exchange study.
Laboratory MA protocols. We allowed the expert laboratories to use
their laboratory-specific supplies, calibrators, and MA protocols, be-
cause this would be the case if different countries conducted their na-
tional nutrition surveys. Table 1 summarizes the procedural differences
among the 3 laboratories. L1 and 3 performed a 96-well plate assay
using chloramphenicol-resistant cryo-preserved L. rhamnosus (ATCC
27773 or NCIB 10463) (3,13). L1 used 5-methylTHF as a calibrator
(concentration verified spectrophotometrically at 290 nm). L3 used FA
(concentration verified spectrophotometrically at 283 nm). Both labo-
ratories prepared an 11-point calibration curve covering the range of
0–1.0 nmol/L with 8 replicates/point in each assay. L1 used polynomial
regression (3rd degree), whereas L3 used 4-parameter logistic curve
fitting. Both laboratories used a robotic work station to dilute and
dispense samples and reagents into the 96-well plate. L1 prepared 4
replicates/sample at 2 dilutions using 0.5% sodium ascorbate (1/100 and
1/200 for serum and 1/140 and 1/280 for whole blood hemolysate). L3
prepared 4 replicates per sample at 2 dilutions using 0.5% sodium
ascorbate (1/160 and 1/320 for both serum and whole blood hemolysate
samples). But L3 analyzed each blood sample twice, so that 8 estimates
contributed to the final reported value. L3 also incubated the thawed
whole blood hemolysate for 30 min at room temperature before per-
forming the dilutions. L1 omitted this step; it was shown previously not
to influence the results (14). L1 and 3 incubated sealed 96-well plates for
42–45 h at 378C and measured turbidity at 590 nm using a microplate
reader. L2 performed a 96-well plate assay using wild-type, cryo-
protected L. rhamnosus (ATCC 7469) and 5-formylTHF as a calibrator
(concentration verified spectrophotometrically at 282 nm) (15). Because
the microorganism is not antibiotic-resistant, sterile labware was used
and reagents were filtered through a 0.22-mm microfilter to prevent bacte-
rial contamination. A 7-point calibration curve (logit-log fit) was prepared
for every 5 unknown samples covering the range of 0.04–0.27 nmol/L,
with 2 replicates/point (n = 14). Sample dilutions and dispensing were
performed manually using 8-channel pipettes. Samples were tested at 7
dilutions from 1/40 to 1/2560 with 2 replicates/dilution. Results from 2
or more consecutive dilutions with acceptable agreement (,20% CV)
were used in the calculation of the final reported value. L2 prepared
hemolysates from thawed whole blood by 1/10 dilution with 0.1 mol/L
potassium phosphate buffer (pH 6.3) containing 1% ascorbic acid and
incubated the hemolysates for 60 min at 378C. A shorter incubation time
of 16–20 h at 378C was sufficient for the growth of the wild-type L.
rhamnosus. Turbidity was measured at 600 nm.
Statistical analysis. For each laboratory, we calculated descriptive
statistics (mean 6 SE, geometric mean 6 SE, selected percentiles) for the
one-third NHANES sample. We calculated the prevalence of low or high
folate values for each laboratory using various cutoff levels. We deter-
mined the Pearson correlation coefficients and assessed agreement among
the laboratories by Deming regression and Bland-Altman difference plot
analysis by using Microsoft Excel, with a clinical statistical analysis plug-
in (Analyze-it; Analyze-it-Software). All statistical comparisons were eval-
uated at a significance level of a = 0.05.
NHANES 2007–2008 sample. Both serum and RBC folate
geometric means (nmol/L) were highest in L2 and lowest in L1
(Table 2). The same was true for selected percentiles for serum
folate. For RBC folate, L2 produced the lowest values of the 3
laboratories at lower percentiles and the highest values at and
above the median. The central 95% reference intervals (2.5th
and 97.5th percentiles, nmol/L) for serum folate were different
for each laboratory: 12.6–107 (L1), 18.0–178 (L2), and 14.5–
138 (L3). The same was true for RBC folate: 506–2600 (L1),
460–3660 (L2), and 617–3370 (L3).
Proportions of low or high folate concentrations for
the 3 laboratories, NHANES 2007–2008
Serum folate results, n = 2645
,12.61nmol/L, % (n)
,27.82nmol/L, % (n)
,40.63nmol/L, % (n)
.56.94nmol/L, % (n)
.1075nmol/L, % (n)
RBC folate results, n = 2613
,5061nmol/L, % (n)
,8732nmol/L, % (n)
,11203nmol/L, % (n)
.14304nmol/L, % (n)
.26005nmol/L, % (n)
1.2 (33)0.4 (11)
18.1 (479) 10.6 (281)
63.9 (1691)75.2 (1990)
41.4 (1096)54.2 (1434)
6.1 (162)14.8 (391)
0.7 (18) 3.8 (99)
11.8 (308)17.6 (461)
71.7 (1873)69.8 (1823)
45.1 (1179) 49.4 (1290)
5.9 (153)9.9 (258)
597.5th percentile. Based on data from L1.
1404 Pfeiffer et al.
Few NHANES participants had serum and RBC folate levels
below the traditional cutoff value that could indicate deficiency
(17): 2, 1 and 2 of a total of 2645 participants had serum folate
values , 7 nmol/L when analyzed by L1, 2, and 3, respectively;
318 nmol/L, respectively.
concentrations were different across laboratories (Table 3). For
example, at the 2.5th percentile for L1, the proportions for low
serum folate levels were one-half and one-fifth for L2 and 3,
respectively; the proportions for low RBC folate values were one-
third and one-and-a half for L2 and 3. This difference also appears
in the frequency distribution curves obtained by the 3 laboratories
for serum and RBC folate (Fig. 1). The distribution curves for L2
were widest and most positively skewed.
Pearson correlation coefficients were highest between L3 and
1 and lower between L2 and 1 and between L2 and 3 (Table 4).
Deming regression analysis showed proportional bias between
L3 and 1 and proportional and constant bias between L2 and
1 and between L2 and 3 (Table 4; Supplemental Figs. 1 and 2).
Bland-Altman analysis showed a similar relative bias for serum
and RBC folate between L3 and 1 (;20%) but different relative
biases between L2 and 1 or L2 and 3 for serum and RBC folate.
Sample exchange study. Similar to the NHANES 2007–2008
participants, L2 produced the highest and L1 the lowest serum
folate results with the QC samples and the NIST SRM 1995 and
to-sample variability appeared in the ratio of serum results from
the 2 laboratories (1.19–2.95) (Table 5). L2 produced both lower
and higher results compared with L1 for RBC folate (0.84–1.54).
L3 produced variably higher serum folate (1.04–1.80) and con-
sistently higher RBC folate (1.10–1.34) results compared with L1.
To compare the response of the chloramphenicol-resistant L.
rhamnosus for different folate compounds, L1 generated mul-
tiple calibration curves in the same assay using 7 folate cali-
brators from each laboratory (Fig. 2). All 4 FA calibrators (3
from L1 and 1 from L3) produced overlapping response curves.
The 2 reduced folate calibrators, 5-methylTHF from L1 and
5-formylTHF from L1 and 2, produced comparable response
curves, but the response was higher than for FA, particularly at
higher folate concentrations. L3 had similar findings when it
generated multiple calibration curves in the same assay: over-
lapping response curves for 2 FA calibrators (Fig. 3A) and higher
response curves for 5-methylTHF and 5-formylTHF compared
with FA (Fig. 3B).
In the same assay in which L1 generated 7 calibration curves,
it measured 20 serum and whole blood samples each from
NHANES, QC samples from each laboratory, and all available
reference materials for a total of 30 serum samples and 28 whole
blood samples. Folate results calculated with each of the 4 FA
calibrators were consistently 22–33% higher than results calcu-
lated with the 5-methylTHF calibrator for serum and RBC folate
(Table 6). Results calculated with each of the 2 5-formylTHF
calibrators were within 10% of results calculated with the
5-methylTHF calibrator (8% lower to 8% higher).
whole blood hemolysates could explain the differences in results
between the laboratories. Using freshly collected EDTA whole
not differ regardless of whether the hemolysate was prepared
according to the procedure used by L1 and 3 (1/11 dilution with
1% ascorbic acid; hemolysate pH of 3.8) and incubated for up to
0.1 mol/L potassium phosphate buffer containing 1% ascorbic
acid; hemolysate pH of 4.5) and incubated for up to 2 h at 378C
(0 h: 352, 1 h: 349, 2 h: 370, respectively).
folate (B) results from NHANES 2007–2008.
Frequency distribution curves for serum (A) and RBC
Comparison of serum and RBC folate results
between the 3 laboratories, NHANES 2007–2008
n = 2645
n = 2613
Pearson correlation coefficient (r), P-value
Deming regression slope (95% CI)
Deming regression intercept (95% CI)
Bland-Altman relative bias (95% limits of agreement)
29.43 (217.9 to 20.95)
211.2 (216.4 to 26.04)
2512 (2643 to 2381)
Methodology and mathematical modeling 1405
Monitoring of biochemical indicators over time as part of
population surveys requires stable laboratory methods. To allow
comparability of population survey data among countries,
assays used in population monitoring must achieve comparable
results. Earlier studies on small sample sets indicated, however,
that MA performed in different laboratories produced inconsis-
tent results (8,9). This is the first investigation to our knowledge
that used a large sample set to assess the magnitude of differences
in distributions of serum and RBC folate results among labora-
knowledge to identify potential sources for the differences be-
Using the NHANES 2007–2008 one-third sample, we ob-
served distinct differences in serum and RBC folate levels, means
and reference intervals, among the 3 laboratories. L3 produced
from L1 but ~20% higher across the entire concentration range
(Table 4); 95% of serum and RBC folate samples analyzed by L3
produced results between 10% lower to 50% higher than results
produced by L1 (limits of agreement). The correlation of folate
for serum and ;0.7 for RBC). Although L2 generally produced
the highest results of the 3 laboratories, this was not the case at
lower RBC folate concentrations. This different response by
matrix was also seen in the different relative bias for serum and
RBC folate between L2 and 1 (39 and 21%, respectively) or L2
and 3 (21 and 1%, respectively). The limits of agreement on re-
lative bias estimates for comparisons that included L2 were much
wider than for the comparison of L1 and 3, reflecting the wider
scatter of points.
The increased folate status of the U.S. population since the
serum or RBC folate levels indicative of deficiency, making a com-
When we used folate concentrations at selected percentiles from L1
low or high folate levels (Table 3). Without doubt, differences in
assays can lead to differences in folate status interpretation.
The 2 most distinguishing procedural differences among the
3 laboratory MAwere the L. rhamnosus microorganism and the
folate calibrator. Smaller differences between the 3 assays, not
expected to be as influential on folate results, were the number
of dilutions/sample, the number of replicates/dilution, and the
curve-fitting algorithm. Common features of all 3 assays were
the similar assay imprecision, the use of multi-level matrix-based
QC samples for daily internal QC, and the periodic use of ref-
Older reports found an equivalent response of L. rhamnosus
to different folate vitamers (3,4). More recently, small differences
have been observed in the response of the chloramphenicol-
resistant L. rhamnosus to different folate vitamers (17), with the
Comparison of serum and RBC folate results from the sample exchange study
between the laboratories
Folate concentration, nmol/L Ratio
L1 L2L3 L2:L1L3:L1L2:L3
Serum QC pools
L1 – Low QC
L1 – Medium QC
L1 – High QC
L2 – Low QC
L2 – High QC
L3 – Low QC
L3 – Medium QC
L3 – High QC
L3 – Ultrahigh QC
Whole blood QC pools
L1 – Low QC
L1 – Medium QC
L1 – High QC
L3 – Low QC
L3 – Medium QC
L3 – High QC
L3 – Ultrahigh QC
NIBSC 03/178 (serum)1
NIBSC 95/528 (whole blood)2
NIST SRM 1955 (serum)3
5.11 6 0.814
11.3 6 1.334
39.0 6 4.144
6.08 6 0.545
14.3 6 0.525
47.4 6 4.215
5.30 6 0.574
11.7 6 1.694
41.1 6 3.944
1Assigned content is 12.1 nmol/L and was obtained by LC-MS/MS.
2Assigned content is 28.3 nmol/ampoule and is a consensus value obtained from laboratories using microbiological assays or radioassays.
3Assigned content is 5.28 nmol/L (level 1), 14 nmol/L (level 2), and 44 nmol/L (level 3) and represents orientation values obtained by the
4Mean 6 SD (n = 40).
5Mean 6 SD (n = 5).
1406Pfeiffer et al.
microorganism responding slightly better to reduced folates
compared with FA. The sample exchange study confirmed and
extended the recent observation. It showed that different stock
produced overlapping calibration curves (Fig. 2). In addition, dif-
ferent stock solutions of 5-methylTHF and 5-formylTHF (pre-
pared in different laboratories) produced comparable calibration
curves. But those curves were slightly higher compared with the
FA calibration curves, regardless of whether the assay was con-
ducted in L1 or L3.
Higher calibration curves will result in lower calculated
folate concentrations. The proportional difference (;20%) in
serum and RBC folate between L1 (using 5-methylTHF as a
calibrator) and 3 (using FA as a calibrator) may therefore be due
mainly to the differences in the calibrators (Table 6). Use of
different calibrators in L1 (5-methylTHF) and 2 (5-formylTHF),
however, did not appear to explain the differences in serum and
RBC folate results (Table 6). The different procedure to prepare
whole blood hemolysates for L2 also did not seem to explain the
differences in folate level compared with L1 and 3. The use of
the wild-type L. rhamnosus by L2 appeared to be the main
reason for differences in results between L2 and the other 2
The chloramphenicol-resistant L. rhamnosus yields a higher
response to reduced folate forms than to FA. Also, the wild-type
microorganism seems to respond differently to serum and RBC
folate than does the chloramphenicol-resistant microorganism,
particularly as folate concentrations increase. There have been at
least 2 known changes that could contribute to these effects: the
properties of the microorganism could have been slightly changed
in the process of rendering it antibiotic resistant and subculturing
the wild-type microorganism in L2 over the last 40 y could have
changed its properties. Tamura et al. (18) have shown that the
form of folate used during the development of methotrexate
resistance affects the response of the assay organism to folates. A
similar phenomenon could have occurred with the chloramphen-
icol-resistant microorganism, making it respond slightly better
to reduced folates. Further research will be required to find the
reasons for the responses of microorganisms to folate species. The
current data cannot be used to conclude whether one organism
should be regarded as superior to the other. Forty years ago, when
the chloramphenicol-resistant strain was evaluated as a tool for
assessing folate status, blood folate concentrations generated
with the new organism were generally comparable to those ob-
tained by the standard method using the wild-type microorgan-
ism; however, there was heterogeneity in the data, probably as a
result of the variability of the assay (19–22).
The measurement of available reference materials as part
of the sample exchange study (Table 5) was of limited value,
because the assigned values for each material were derived from
different assays and there is currently no serum or whole blood
reference material available that has certified concentrations for
total folate assigned by a higher-order liquid chromatography-
tandem MS (LC-MS/MS) reference measurement procedure.
However, efforts to generate such a serum material by NISTare
underway. This is the first study to our knowledge to investigate
the magnitude of differences across the entire distribution of
serum and RBC folate results among laboratories using MA and
to conduct systematic investigations to identify potential sources
for the differences. Using FA as a calibrator produced ~25%
higher serum and RBC folate results than using 5-methylTHF
as a calibrator. The majority of folate in blood is in the form
of 5-methylTHF. Unmetabolized FA appears in serum after a
larger bolus dose of FA from foods or supplements and its con-
centration in fasting individuals is usually small compared with
the total folate (23). Thus, using 5-methylTHF as a calibrator
is expected to produce more accurate results than the FA cali-
brator and is recommended. However, the laboratory has to pay
generated by L1 using 7 folate calibrators from the 3 laboratories.
L. rhamnosus polynomial (3rd order) calibration curves
generated by L3 using 2 different FA calibrators (A) or using 2 reduced
folate calibrators (5-methylTHF and 5-formylTHF) and FA (B).
L. rhamnosus polynomial (3rd order) calibration curves
Methodology and mathematical modeling 1407
attention to the purity of this compound, expeditiously handle
the preparation of stock solutions, and use appropriate antiox-
idants and store stock solutions in a 2708C freezer to ensure
their stability (2,17).
The question whether the wild-type or the chloramphenicol-
resistant microorganism produces more accurate results cannot
be satisfactorily settled until serum- and whole blood-based
reference materials with certified values for total folate by
LC-MS/MS are available. In the interim, NHANES continues to
monitor folate status employing an MA that uses the chloram-
phenicol-resistant microorganism and 5-methylTHF as a cali-
brator and an LC-MS/MS assay for additional information on
folate species. Other countries that wish to compare their folate
population data to that of the US can harmonize their MA by
using the same microorganism and calibrator.
We thank the following laboratory members: Donna LaVoie,
Bridgette Haynes, Neelima Paladugula, and Daniel Rabinowitz
(CDC’s National Center for Environmental Health), Regina
Dempsey (Trinity College), and Kelley E. Johnston (University
of Alabama). C.M.P., D.A.L., E.A.Y., M.F.P. (recently deceased),
and C.L.J. designed the overall research project; C.M.P., M.Z.,
A.M.M., T.T., and D.A.L. conducted most of the research;
C.M.P., M.Z., and D.A.L. analyzed the majority of the data;
C.M.P. wrote the initial draft, which was modified after feed-
back from all coauthors; and C.M.P. has primary responsibility
for content. All authors read and approved the final manuscript.
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FA, Sigma lot 1
FA, Sigma lot 2
Serum samples (n = 30)
Deming slope (95% CI)
Deming intercept (95% CI)
Pearson correlation coefficient,
Whole blood samples (n = 28)
Deming slope (95% CI)
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1.24 (1.19, 1.29)
20.33 (21.36, 0.70)
1.31 (1.26, 1.35)
0.21 (20.68, 1.11)
1.33 (1.28, 1.37)
0.17 (20.73, 1.07)
1.22 (1.18, 1.26)
0.77 (20.09, 1.62)
0.92 (0.92, 0.93)
0.42 (0.21, 0.63)
1.08 (1.07, 1.09)
0.16 (0.00, 0.32)
1.25 (1.22, 1.28)
212.2 (222.8, 21.53)
1.31 (1.28, 1.33)
20.91 (210.2, 8.36)
1.33 (1.30, 1.36)
22.31 (212.7, 8.11)
1.23 (1.22, 1.23)
8.40 (4.56, 12.3)
0.93 (0.91, 0.95)
5.50 (21.73, 12.7)
1.08 (1.08, 1.09)
1.88 (0.81, 2.94)
1FA, 100 mg/L (227 mmol/L), Merck Eprova.
2FA, 500 mg/L (1133 nmol/L), Sigma (Sigma Chemicals), 2 different lot numbers.
3FA, 100 mg/L (227 nmol/L), Sigma.
45-formylTHF (racemic), 23 mmol/L, Sigma.
55-formylTHF, 100 mg/L (211 mmol/L), Merck Eprova.
1408Pfeiffer et al.
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Methodology and mathematical modeling1409