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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1807-1813
An Assessment of Dried Blood-Spot Technology for Identifying Iron
Deficiency
By
James D. Cook,
Carol H. Flowers, and
Barry S. Skikne
From the Department of Medicine, University of Kansas Medical Center,
Kansas City, KS.
 |
ABSTRACT |
The present study was undertaken to assess the feasibility of using
ferritin and transferrin receptor measurements on dried capillary blood
spots to identify iron deficiency (ID) in public health surveys.
Measurements on serum and blood spots prepared from venous blood were
performed in 71 healthy subjects, 41 of whom were iron-replete and 30 who had ID, either without (n = 20) or with (n = 10) anemia.
Parallel measurements were performed on hemolyzed whole blood and
washed hemolyzed red blood cells to assess the erythrocyte contribution
of ferritin and transferrin receptor to dried blood samples. The
concentration of ferritin in dried blood samples was threefold higher
than serum assays due to the release of ferritin from hemolyzed
erythrocytes, which diminished the usefulness of ferritin measurements
for detecting ID. On the other hand, there was negligible erythrocyte
contribution to the measurement of transferrin receptor in dried blood
spots. The most sensitive parameter in dried blood spots was the ratio of receptor/ferritin, which was suitable for identifying
iron-deficiency anemia (IDA), but less reliable than serum assays for
detecting milder ID without anemia. We conclude that tandem
measurements of serum ferritin and transferrin receptor in dried blood
spots can be used to facilitate the identification of IDA in
epidemiologic studies.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
BASED ON A REVIEW of more than 500 studies published between 1960 and 1983, the global prevalence of
anemia was reported to be approximately 30%, with a range from 8% to
36% depending on the economic status of the region.1
Although the cause of the anemia was not determined in the majority of
studies, the investigators estimated that iron deficiency (ID) was
responsible in approximately 50% of the anemic individuals based on
differences in anemia prevalence in various age and sex categories. In
countries where the prevalence of anemia is highest, there are many
causes of anemia other than ID, such as chronic bacterial infection,
malaria, acquired immunodeficiency syndrome, malnutrition, folate or
vitamin B12 deficiency, and inherited defects in hemoglobin
synthesis, such as sickle cell anemia and thalassemia. The most widely
used intervention strategy to reduce the high prevalence of anemia is
to increase the intake of oral iron, which will be ineffective and
possibly harmful if the anemia is not due to iron deficiency.
Consequently, it is important to incorporate specific measures of iron
status in population studies designed to assess or reduce the high
prevalence of anemia.
There is a large array of laboratory measurements to identify and
assess the severity of ID. Most prior surveys of iron status have
required venous sampling to obtain sufficient blood for these determinations. In recent years, highly sensitive and specific immunoassays of iron status, such as the serum ferritin and serum transferrin receptor, have become available that require only a few
microliters of serum and are therefore suitable for measurements on
blood obtained by capillary sampling. Despite the advantage of
fingerstick sampling, there is still a logistic problem, especially in
rural areas, of processing samples in the field to obtain sera and
transporting the latter to a central laboratory facility under controlled temperature.
The present study was undertaken to examine the feasibility of using
small samples spotted onto filter paper for measurements of ferritin
and transferrin receptor. This technology has been used extensively to
detect inborn errors of metabolism in newborns and more recently in
field studies to identify iodine deficiency.2,3 In the
present study, venous blood from 71 subjects representing a wide
spectrum of iron status was obtained to compare assay results on dried
blood spots eluted from filter paper with measurements on serum.
Additional assays were performed on whole blood (WB) and hemolyzed red
blood cells (RBCs) to assess the erythrocyte contribution of ferritin
and transferrin receptor to blood-spot measurements.
| |
MATERIALS AND METHODS |
Clinical samples.
Blood samples were obtained from 25 men and 46 women ranging in age
from 19 to 71 years (mean, 27.9). All subjects without anemia were
considered to be in good health. ID was defined as a serum ferritin
concentration less than 12 µg/L and anemia as a hematocrit less than
37% in women and 40% in men.4 Based on these criteria,
the participants were further divided for certain statistical analyses
into 41 normal subjects, 20 subjects with ID defined as a low serum
ferritin and normal hematocrit, and 10 subjects with iron-deficiency
anemia (IDA) defined as a low serum ferritin and low hematocrit. All
specimens were obtained according to procedures approved by the Human
Subjects Committee of the Kansas University Medical Center.
Processing of blood specimens.
Eight-milliliter venous blood samples were collected into evacuated
tubes containing potassium EDTA as the anticoagulant. After measurement
of the microhemocrit, duplicate 25-µL samples of WB were spotted onto
filter paper (Schleicher & Schuell #903, Keene, NH) and
allowed to dry at room temperature for a minimum of 3 hours. The paper
samples were stored for periods ranging up to 1 year in sealed plastic
bags with 10 g of Drierite (calcium sulfate) (W.A. Hammond Drierite Co,
Xenia, OH) desiccant at 4°C until the day of assay. The
Drierite was contained in a separate porous package.
The remainder of the blood sample was fractionated for ferritin and
transferrin receptor measurements on plasma, WB, and saline-washed RBCs. The latter two fractions were hemolyzed to assess the erythrocyte contribution of ferritin and transferrin receptor in dried blood spots
that are fully hemolyzed when the sample dries. The WB samples were
hemolyzed by adding an equal volume of distilled water and freezing at
 30°C. To obtain the RBC samples, WB was centrifuged at
1,500g for 20 minutes, and after removing the plasma and buffy coat, the RBCs were washed twice with sterile normal saline. The cells
were then lysed by adding 2 vol of distilled water and centrifuging at
10,000g for 30 minutes to remove the RBC membranes from the supernatant. The plasma, WB, and RBC samples were frozen and stored at
30°C until the day of assay.
Elution of blood spot.
Preliminary studies were performed to determine the optimal procedure
for eluting the dried blood spot. Two drops of WB from two normal
volunteers were spotted onto the filter paper in 16 replicate samples
and allowed to dry overnight at room temperature. The blood spots were
cut from the paper, placed in 1.5-mL microfuge tubes containing 1 mL of
the following buffers, and mixed on an end-over-end hematology rotator
for 2 hours at 4°C. Measurements of L-ferritin and transferrin
receptor were used to compare the elution efficiency of four Tris
buffers (Tris pH 8.0 plus 10 mmol/L Chaps, Tris plus
2% sodium dodecyl sulfate [SDS], Tris plus 1% NP40, and Tris plus
1% Brij 35) and four phosphate-buffered saline (PBS) buffers (PBS pH
7.2 plus 0.05% tween-20, PBS plus 10 mmol/L (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate) (CHAPS), PBS plus 2% SDS, and PBS plus 1% NP40). Recovery was markedly reduced with buffers containing SDS, presumably due to protein
denaturation. The Tris buffers also produced lower recoveries. Maximal
recovery was achieved with the PBS-tween buffer, which was then used in
all subsequent studies. Extending the elution time from 2 to 12 hours
or increasing the elution temperature from 4°C to 25°C did not
improve recovery significantly. For the selected elution protocol of 2 hours at 4°C in PBS-tween with end-over-end mixing, the elution
efficiency, defined as the ratio of transferrin receptor or ferritin in
dried blood spots to hemolyzed WB, ranged for 10 samples from 0.93 to
0.97, with a mean of 0.95 ± 0.01.
Effect of storage.
To assess the effect of storage under different conditions, venous
blood from nine different volunteer subjects was collected in vacuum
container tubes containing potassium EDTA. Twenty-five microliters of
WB was used to prepare multiple dried spots from each sample. Six sets
of dried blood spots were placed in each of six separate sealed plastic
storage bags. Two of these bags were stored at 4°C, two at room
temperature, and two at 37°C. One of the bags at each of these
temperatures was stored with desiccant (calcium sulfate), the other
without. At 1, 3, 7, 14, 21, and 28 days of storage, one blood spot was
removed from each bag and assayed for L-ferritin and transferrin
receptor. In a separate study, samples were stored at 4°C with
desiccant for 1 year.
Variability studies.
To determine the intraassay variability of dried blood-spot
measurements, 10 replicate samples of 25 µL from five subjects were
spotted onto filter paper and allowed to dry overnight. The spots were
eluted from the paper and the eluants were assayed in the same
microtiter plate, each for ferritin and transferrin receptor
concentrations. To determine interassay variability, dried blood-spot
samples from seven subjects were measured in duplicate in six separate
assays over a 2-week period.
Immunoassays.
Ferritin is composed of 24 subunits of two main types designated H and
L, which are encoded by genes on separate chromosomes.5-7 The L subunit predominates in liver and spleen ferritin and produces a
basic isoferritin profile, whereas the ferritin in heart tissue and
tumors contains predominantly H subunits and results in a more acidic
isoferritin profile. Serum ferritin is rich in L subunits and is
consequently measured by clinical assays developed against purified
liver or spleen ferritin. In the present report, ferritin measured by
such an assay is referred to as L-ferritin or simply ferritin, whereas
the ferritin measured by an assay sensitive to the H-rich ferritin
contained in heart is designated H-ferritin. Erythrocytes contain a
preponderance of H-ferritin, which changes less with variations in body
iron status than the smaller component of L-ferritin.8
H-ferritin assays were performed in the present study in an attempt to
assess the erythrocyte contribution of L-ferritin to measurements in
dried blood spots.
Measurements of H-ferritin, L-ferritin, and transferrin receptor were
performed by enzyme-linked immunoadsorbent assay (ELISA) using double
monoclonal antibodies. The assay of L-ferritin was made by adding 200 µL of sample to duplicate microtiter wells precoated with monoclonal
antibody in 0.2 mol/L carbonate buffer at pH 9.6 and incubating for 2 hours at room temperature as previously described.9 After
washing the wells with PBS-tween, 200 µL of horse radish peroxidase
(HRP)-conjugated antiferritin monoclonal antibody was added. After an
additional 2-hour incubation at room temperature, the wells were again
washed with PBS-tween, and 200 µL of o-phenylenediamine
dihydrochloride substrate in citrate-phosphate buffer pH 5.0 was added.
After 30 minutes of incubation in the dark, the color reaction was
terminated by adding 50 µL of 2.5-mol/L sulfuric acid. The optical
density was measured at 492 nm in a microplate reader and the ferritin
values of the samples were determined from a standard curve of known
concentrations of recrystallized human liver ferritin plotted against
optical density. The concentration of ferritin standards was calibrated
with the international World Health Organization (WHO) reference
standard obtained from the National Bureau of Standards.10
The assay of H-ferritin was developed using purified human heart
ferritin obtained by ultracentrifugation and gel filtration on
Sepharose 6B as the ferritin source.11 Monoclonal
antibodies were used for both the solid-phase and indicator reagent.
The assay was performed by the same procedure used for the measurement of L-ferritin. Recrystallized human liver ferritin was nonreactive in
the H-ferritin assay at all concentrations. On the other hand, 6% of
the purified H-ferritin could be detected with the L-ferritin assay.
This percentage is assumed to represent the minimal L-subunit composition of H-ferritin.12,13 The transferrin receptor
was assayed as previously described using monoclonal antibodies against receptor purified from human placenta.14 The assay protocol was nearly identical to that used for the measurement of H- and L-ferritin.
Statistical analysis.
Because of the skewed distribution of L-ferritin in WB and plasma and
the transferrin receptor/ferritin ratio, these data were converted to
logarithms for certain statistical analyses and the results reconverted
to antilogarithms. The degree of separation between the three
categories of iron status, (normal, ID, and IDA) was assessed for
various laboratory parameters by analysis of variance. The correlation
between any two measurements was evaluated by Pearson's product
moment. Statistical calculations were performed using the Abstat
statistical program (Anderson Bell, Parker, CO).
| |
RESULTS |
The normal subjects included 18 women and 23 men who had hematocrits of
40.6% ± 1.9% (mean ± 1 SD) and 45.4% ± 2.6%, and geometric mean plasma ferritin concentrations of 33.5 (16.4 to 68.3, ± 1 SD) and 53.1 (29.8 to 94.6) µg/L, respectively. The
mean hematocrits in the 20 subjects with ID and the 10 subjects with
IDA were 40.3% ± 1.6% and 30.5% ± 3.8%, respectively. All 30 subjects in the latter two groups were women.
When blood spots were stored in sealed plastic bags at 4°C with or
without desiccant, there was no significant decline in ferritin or
transferrin receptor assays over 28 days (Fig
1). When stored at room temperature with
desiccant, the immunoreactivity in both assays remained constant, but
there was a slight decline in activity when stored at room temperature
without desiccant. Ferritin and transferrin receptor values decreased
by approximately 10% when stored at 37°C for 28 days if the storage
bag contained desiccant. However, without desiccant, there was a
dramatic reduction in the concentration of both proteins. Thus, the
combination of ambient humidity and high temperature resulted in a
rapid deterioration in the immunoreactivity of both ferritin and
transferrin receptor.
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| Fig 1.
Stability of WB spots under different storage conditions.
Individual points represent the total of six measurements on samples
dried and stored in sealed bags at 4°C ( ,  ), room temperature
(#,  ), and 37°C (*,  ). These were stored with dessicant (,
#, *) and without dessicant (, , ).
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In a second study in which dried blood spots were stored at 4°C with
desiccant for 1 year, there was no significant loss of immunoreactivity
for either L-ferritin or transferrin receptor. In nine such
measurements, values ranged from 95% to 107% of the original, with a
mean of 98.4% ± 0.04%.
The intraassay variability for 10 measurements on each of five blood
samples was 4.48%, with a range of 3.11% to 7.3%. The interassay
variability for duplicate measurements on seven dried blood-spot
samples measured over a period of 2 weeks averaged 5.55%, with a range
of 2.7% to 9.6%.
The immunoassay data for L- and H-ferritin and transferrin receptor in
the normal, ID, and IDA subgroups of iron status and in the composite
group of 71 subjects are listed in Table 1. The L-ferritin concentrations in WB, RBC, and paper samples were similar, and more than twofold higher than the plasma concentration, which reflects the contribution of L-ferritin from hemolyzed
erythrocytes. The latter was more pronounced in subjects with ID. Thus,
the WB and paper L-ferritin values were only two to three times greater than the serum concentration in normal subjects, but 10- and 20-fold higher in the ID and IDA groups, respectively (Fig
2). The difference in the concentration of
L-ferritin in WB and dried blood spots in the composite group was less
than 5%, reflecting complete elution of L-ferritin from the dried
blood spots.
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| Fig 2.
Relationship between L-ferritin values determined on
serum and on dried blood spots (paper). Interrupted line is the line of
identity. Correlation coefficient is significant (r = .723,
P < .001).
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Both L-ferritin and H-ferritin are released from hemolyzed
erythrocytes, with the proportion of H-ferritin being considerably higher than L-ferritin.8 Compared with serum, L-ferritin
measurements on hemolyzed samples (WB, RBC, and paper specimens in this
study) are elevated, due both to the contribution of L-ferritin from erythrocytes and the fact that the L-ferritin assay measures a small
percentage of H-ferritin. Assays of H-ferritin were performed to
determine whether the erythrocyte contribution to the paper L-ferritin
concentration could be estimated from the H-ferritin concentration.
However, the relationship between L- and H-ferritin in the RBC samples
varied with iron status. For example, the L-ferritin in RBC samples was
121.1 µg/L in normals as compared with 67.3 µg/L in ID, whereas the
H-ferritin concentration was 599.3 and 530.2 µg/L, respectively
(Table 1). As a result, when a constant ratio for L-/H-ferritin in
erythrocytes of 0.18 based on values in the composite group
(100.8/549.4) was used to estimate the erythrocyte contribution of
L-ferritin to WB values, the calculated serum L-ferritin concentration
was negative in more than half of the subjects with ID or IDA. When
corrections were based on a lower L-/H-ferritin ratio of 6%, which
represents the cross-reactivity of purified H-ferritin in the
L-ferritin assay, the L-ferritin concentration in WB samples was not
reduced appreciably. Consequently, we were unable to use the
measurement of H-ferritin in WB or paper to derive a satisfactory
estimate of serum L-ferritin concentration.
An impressive reciprocal change in serum transferrin receptor
concentration with increasing ID has been reported
previously15,16 and was observed again in the present
study. The mean serum receptor concentration in IDA of 16.09 mg/L was
threefold higher than the mean of 5.66 mg/L in normal subjects (Table
1). In contrast with ferritin measurements, the concentration of
transferrin receptor in RBC was negligible regardless of iron status.
In the composite group of 71 subjects, the mean WB transferrin receptor
concentration of 4.43 mg/L was similar to the serum receptor mean of
7.44 mg/L when the following correction was made for the volume
displaced by RBCs. Based on the composite mean fractional hematocrit of 0.41, the serum transferrin receptor concentration predicted from the
WB value was 4.43/0.59 0.05 = 7.46 mg/L, where 0.59 is the fractional plasmatocrit and 0.05 is the RBC receptor concentration. When this correction was applied, the paper values of transferrin receptor were 24%, 27%, and 11% lower than WB values in normal subjects, ID, and IDA, respectively. A highly significant correlation was observed between WB and paper receptor values (Fig
3).
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| Fig 3.
Relationship between measurements of transferrin receptor
performed on serum and on dried blood spots (paper). Interrupted line
represents the line of identity. Correlation is highly significant
(r = .968, P < .001).
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An important aspect of any new measurement or combination of
measurements for assessing iron status in prevalence studies is the
extent to which ID and IDA can be distinguished from iron-replete individuals. Because of the significant and variable contribution of
L-ferritin in erythrocytes to the paper L-ferritin values, the
distinction between ID or IDA and normal subjects was less satisfactory
than with serum ferritin measurements (Fig
4). On the other hand, the separation
between normal and IDA based on transferrin receptor measurements was
similar for serum and paper assays (Fig 5).
With both paper and serum determinations, only one of 10 subjects with
IDA had transferrin receptor concentrations in the normal range.
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| Fig 4.
Values of (A) dried paper samples of blood L-ferritin and
(B) serum L-ferritin. Values are shown separately for normal subjects
(N), subjects with ID, and subjects with IDA.
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| Fig 5.
Values of (A) dried paper samples of blood transferrin
receptor and (B) serum transferrin receptor. Values are shown
separately for normal subjects (N), subjects with ID, and subjects with
IDA.
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One of the more difficult problems in identifying IDA in population
studies conducted in developing countries is the presence of chronic
illnesses or infections, which falsely elevate the serum ferritin and
lessen its diagnostic utility. Recent clinical studies have indicated
that the serum transferrin receptor is typically elevated in IDA
regardless of associated illness and that the ratio of
receptor/ferritin is more efficient for identifying IDA than either the
transferrin receptor or ferritin alone.16,17 Although none
of our subjects had associated illnesses, it was of interest to compare
the diagnostic efficiency of serum and paper measurements of
receptor/ferritin ratio.
The geometric mean receptor/ferritin ratio varied widely depending on
iron status from a mean of 128 in normal subjects to 936 in ID and
3,839 in IDA (Table 1). Because of the erythrocyte contribution of
L-ferritin to the paper values, the mean ratio with the paper samples
was only one tenth that observed with serum measurement in the
composite group of subjects, although the degree of separation and
extent of overlap between the three study groups was similar for serum
and paper measurements (Fig 6). When
evaluated by analysis of variance, the separation between normal, ID,
and IDA using the logarithm of the receptor/ferritin ratio was similar for serum and paper (F = 132.8 and 105.3, respectively;
P < .001). There was no overlap between normal and IDA with
either paper or serum determinations.
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| Fig 6.
Values of receptor/ferritin ratio (R/F ratio) from (A)
serum and (B) dried paper samples of blood. Values are shown separately
for normal subjects (N), subjects with ID, and subjects with IDA.
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| |
DISCUSSION |
There has been a marked increase in recent years in the use of dried
serum or blood spots collected on filter paper for a variety of
laboratory measurements. The earliest and most extensive use of this
technology was in screening cord blood for neonatal metabolic disorders
such as phenylketonuria18,19 and congenital hypothyroidism.20,21 With the availability of increasingly sensitive immunologic and molecular probes, blood-spot technology has
been extended to a broad range of medical applications. The technique
is used in screening for infectious diseases such as Toxoplasma
gondii,22 human immunodeficiency virus,23
and Pseudomonas aeruginosa,24 and it has been used
successfully to monitor drug therapy such as cyclosporine25
and phenytoin.26 In nutritional screening programs, dried
blood-spot determinations of thyrotropin have been used for the
detection of iodine deficiency in developing countries.2
There are several advantages in using dried blood spots. Perhaps the
most important is in eliminating the need for venous blood sampling,
which is often a technical challenge in infants and young children.
Venous sampling is also more costly than capillary sampling in
epidemiologic surveys because of the disposable sampling equipment
required for the former. Another important advantage of using dried
blood spots is the ease in transporting and storing specimens before
analysis. Even if the temperature of paper samples must be controlled,
the cost of transporting specimens is considerably lower for paper
samples than for larger volumes of WB or serum.
The most efficient method for blood-spot tests is to place a drop or
two of capillary blood directly onto the filter paper. If a
quantitative measurement is required, a paper punch can be used to
obtain a constant amount of the spotted blood sample for elution. We
used this approach initially, cutting 8-mm paper discs from the center
of a larger dried blood spot. However, when discs were cut at varying
distances from the center of the spot, we noted significant differences
in both the protein and hemoglobin concentrations, presumably due to
variations in the rate of diffusion in the paper. In subsequent
studies, we therefore elected to use a precisely measured 25-µL blood
spot and elute the entire sample. This approach requires the use of
heparinized microhematocrit tubes or specialized microvette collection
tubes containing potassium EDTA. The former are especially convenient
in field studies if anemia is determined by microhematocrit. The use of
a precisely measured volume of blood also requires disposable pipettes
for pipetting devices and a certain degree of technical skill, but neither of these constraints outweighs the advantage of the increased accuracy and reproducibility achieved by using a precisely measured sample volume.
A major consideration in designing the present study was the published
evidence that sufficient amounts of L-ferritin are contained in
erythrocytes to influence assays in specimens containing hemolyzed RBCs
such as dried WB samples.8,12 Our attempts to derive a
suitable correction for the erythrocyte contribution of L-ferritin
using measurements of H-ferritin proved unsuccessful because of
variations in the ratio of H-/L-ferritin in erythrocytes with
differences in iron status. Ferritin measurements alone in dried blood
spots are unsuitable for distinguishing between normal subjects and
individuals with either ID or IDA (Fig 4).
In contrast to ferritin, the concentration of transferrin receptor in
erythrocytes was negligible. Consequently, measurements of transferrin
receptor in dried blood samples were equivalent to serum measurements
when a correction was made for the plasma displaced by RBCs. However,
clinical studies to date do not support the use of isolated
measurements of transferrin receptor to identify ID or IDA. The major
value of receptor measurements is to identify IDA when the serum
ferritin is falsely elevated because of ongoing inflammation or chronic
infection. Because the serum transferrin receptor remains normal with
inflammation, but increases with the onset of tissue ID, recent
clinical studies have shown that the ratio of transferrin
receptor/serum ferritin can be used to detect IDA even in the presence
of other chronic infection or inflammatory disease.16,27 An
important finding in the present study was that the receptor/ferritin
ratio in dried blood spots was as useful in identifying IDA as
measurements on serum. With both paper and serum determinations,
complete separation between normal and IDA was achieved using the
receptor/ferritin ratio. Thus, the combined determination of ferritin
and transferrin receptor in dried blood samples could be potentially
useful if our present findings are confirmed in future studies
performed under field conditions.
A reasonable alternative is to use serum or plasma samples spotted on
filter paper, rather than WB. Our recent studies indicate that the data
obtained with this approach is equivalent to measurements of ferritin
and transferrin receptor performed directly on serum.28 In
a recent study, a measured volume of serum, spotted and dried on filter
paper, was found to give values comparable to traditional serum
measurements.29 The disadvantage with this approach is it
requires centrifugation of blood samples, which often poses a
significant logistic problem in a field setting. The advantage of using
plasma is the ability to identify both ID and IDA, which cannot be
accomplished with dried blood spots. The choice between measurements on
whole serum and either dried blood or plasma spots will depend to a
large extent on the location of the survey and local laboratory
facilities.
| |
FOOTNOTES |
Submitted February 19, 1998;
accepted May 4, 1998.
Address reprint requests to James D. Cook, MD, Department of Medicine,
University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, KS
66160-7233.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
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Abstract
Introduction
Abstract