The American Journal of Clinical Nutrition xxx (xxxx) xxx

journal homepage: https://ajcn.nutrition.org/

Original Research Article

Hemoglobin measurement in venous blood compared with pooled and

single-drop capillary blood: a method-comparison study in a controlled and
survey setting in Uganda among children and women
Sorrel ML Namaste 1, *, Rhona Baingana 2, Eleanor Brindle 3
1
The DHS Program, ICF, Rockville, MD, United States; 2 Makerere University College of Natural Sciences, Kampala, Uganda, Africa; 3 The DHS
Program, PATH, Rockville, MD, United States

A B S T R A C T

Background: Standard practice for estimating anemia in population-based surveys is to use a point-of-care device to measure hemoglobin (Hb) in a
single drop of capillary blood. Emerging evidence points to larger than expected differences in Hb concentration depending on the blood source.
Objective: We evaluated use of different blood sources to measure Hb with a HemoCue 201þ analyzer compared with the reference method of venous
blood tested with a Sysmex XN-450 hematology analyzer.
Methods: Hb concentration in venous, pooled capillary, and single-drop capillary blood were collected in controlled (laboratory) and survey (De-
mographic Health Survey-8 pilot) settings in Uganda among children 6–59 mo and nonpregnant women 15–49 y. Venous and capillary blood collected
from the same individual was tested using a HemoCue 201þ analyzer and the venous blood was also measured with a Sysmex XN-450 hematology
analyzer. Agreement between measures was estimated using Lin’s concordance correlation coefficient, Bland–Altman plots, and Deming regression.
Means and prevalences were compared using paired t-tests and McNemar’s tests, respectively.
Results: The limits of agreement between Hb measured using a HemoCue 201þ analyzer and the reference method were lowest for venous (1.1–1.96 g/
dL), followed by pooled capillary (1.45–2.27 g/dL), and single-drop capillary blood (2.23–3.41 g/dL). Mean differences were <0.5 g/dL across com-
parators. There were statistically significant differences in Hb concentration from both types of capillary blood. Anemia prevalence was lower in pooled
capillary blood compared with the reference method.
Conclusions: The variability of Hb measured by capillary blood using the HemoCue 201þ analyzer is higher than venous blood but the extent to which
this impacts the validity of Hb and anemia estimates requires further exploration. Future research is also needed to evaluate the implications of using
venous compared with capillary blood in population-based surveys.
This trial was registered at clinicaltrials.gov (NCT05059457).

Keywords: anemia, capillary, comparative study, Demographic and Health Survey, hemoglobin, measurement, population-based survey, validation,
venous

Introduction World Health Assembly targets and the Sustainable Development

Anemia is associated with increased risk of morbidity and mor-
tality among young children and women of reproductive age [1–4].
It is a disorder that occurs when there are insufficient or impaired
circulating red blood cells for tissue oxygenation, which is caused by
factors that affect the morphology, production, turnover, loss, or
half-life of red blood cells. Estimates of anemia are used to inform
and monitor country programs and track global targets such as the
Goals [5].
The Demographic and Health Survey (DHS) Program is the primary
source of anemia data globally [6]. The DHS Program has measured
hemoglobin (Hb), the biomarker recommended by the WHO, to assess
anemia in populations since the mid-1990s [7,8]. The protocol for DHS
surveys has been to measure Hb concentration using the third single
drop of capillary blood from a finger or heel prick with the HemoCue
201þ analyzer although some surveys have used a different blood drop

Abbreviations: DHS, Demographic and Health Survey; ELCL, Ebenezer Limited Clinical Laboratory; Hb, hemoglobin; IDI, Infectious Diseases Institute; LOA, Limit of
Agreement.
* Corresponding author.
E-mail address: sorrel.namaste@icf.com (S.M. Namaste).

https://doi.org/10.1016/j.ajcnut.2023.12.025
Received 13 May 2023; Received in revised form 3 December 2023; Accepted 28 December 2023; Available online xxxx
0002-9165/© 2024 The Authors. Published by Elsevier Inc. on behalf of American Society for Nutrition. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
S.M. Namaste et al.
or hemoglobinometer [8]. Until recently, this method has been
considered an acceptable measure of Hb concentration [9]. However,
substantial differences in anemia prevalence estimates have been
observed using venous compared with capillary blood when comparing
surveys matched by country and time [6,10].
Recent reviews have identified several preanalytical and analytical
factors that can affect Hb concentration estimates [11–14]. Much of the
evidence contributing to these reviews is based on data from studies not
designed to answer these questions or on studies conducted in non-
survey settings. This limits our ability to isolate the factors that result in
meaningful differences in Hb concentration and to ascertain the extent
to which these factors compromise the use of anemia data.
Blood sample source has emerged as a potential factor affecting
Hb concentration [12]. Capillary blood samples were adopted as a
field-friendly alternative to venous blood, but can be compromised by
squeezing or “milking” the finger, poor lancet technique, not allowing
a sufficient blood drop to form, or touching the microcuvette to the
finger or heel when filling the microcuvette [11]. There is some
indication of interindividual (for example, sex and age) and intra-
individual inherent drop-to-drop Hb variability from capillary blood
[13,15]. Pooled capillary blood samples, rather than a single drop,
may provide greater within-subject reliability but evaluations of this
approach are scarce [16].
In this study, we evaluated the impact of blood sample collection
technique (venous, pooled capillary, and single-drop capillary blood)
and Hb testing method (HemoCue 201þ analyzer compared with
Sysmex XN-450 hematology analyzer) on Hb concentration among
children and women of reproductive age in a controlled setting and in a
population-based survey setting to inform Hb data collection proced-
ures and improve the interpretation of anemia data.
Methods
Study site and design
Ebenezer Limited Clinical Laboratory (ELCL) conducted the blood
collection in the controlled setting in Kampala, Uganda. A convenience
sample of apparently healthy children aged 6–59 mo and nonpregnant
women aged 15–49 y was recruited from the community. All 3 blood
sources (venous, pooled capillary, and single-drop capillary blood)
were collected from each individual.
In the survey setting, blood collection was nested within the DHS-8
questionnaire pilot designed to test select content of the new DHS-8
standard questionnaires and modules. The pilot was designed to
mimic an actual DHS survey and was implemented by the Uganda
Bureau of Statistics in communities outside of Kampala, Uganda.
Recruitment was also a convenience sample and blood was collected at
participants’ households in conjunction with DHS survey questionnaire
data. To minimize participant burden and risk, individuals were
randomly assigned to provide either pooled or single-drop capillary
blood and venous blood was collected from all individuals.
Blood collection was performed by 2 phlebotomists in the
controlled setting and 7 phlebotomists in the survey setting. In the
controlled setting, the venous blood was collected first and tested,
followed by the single drop of capillary blood and then the pooled
capillary blood using a different finger. In the survey setting, the venous
blood was collected first and tested, followed by only 1 of the 2 types of
capillary blood. The median time between venous and capillary blood
collection in the controlled and survey setting was 3 and 5 min,
respectively. Participants remained seated between measurements. In
both settings, Hb concentration was measured immediately on all
2
The American Journal of Clinical Nutrition xxx (xxxx) xxx
samples with the HemoCue 201þ analyzer. Same day testing on all
venous blood samples, whether collected in a controlled or survey
setting, was conducted using a Sysmex XN-450 hematology analyzer.
The protocol was for samples to be delivered within 6 h to the labo-
ratory and for samples to be tested no more than 8 h after data
collection. The data collection period was from August 23, 2021, to
September 26, 2021.
Blood collection procedures
The phlebotomists collected 3 mL of venous blood in a BD Vacu-
tainer® containing EDTA as an anticoagulant (Becton, Dickinson).
They slowly inverted the tube 10 times to prevent clot formation and
then used a small plastic pipette to transfer 2–3 large drops of whole
blood (minimum ~50 μL) onto parafilm. The phlebotomists then filled
a HemoCue 201þ microcuvette and immediately tested the Hb con-
centration in the HemoCue 201þ analyzer.
The capillary sample was drawn from the finger (or heel in the case
of children 6–11 mo) using a high-flow BD Microtainer® contact-
activated lancet (Becton, Dickinson). For the single drop of capillary
blood, the first 2 blood drops were wiped away, and the third drop of
blood was placed in the microcuvette directly from the finger or heel
and Hb concentration was immediately tested in the HemoCue 201þ
analyzer. For pooled capillary blood, the first drop was wiped away and
then blood was captured directly in a BD Microtainer tube (250–500
μL) containing EDTA. The microtainer tube was slowly inverted 10
times, and the same procedures were followed to transfer and test the
blood as the venous samples.
The remaining venous blood in both settings was conveyed to
ELCL. In the controlled setting, venous specimens were placed in a
cold box immediately after being tested with the HemoCue 201þ
analyzer and the cold boxes were hand-delivered to the laboratory. In
the survey setting, the venous specimens were placed in a cold box
while in the household and then transferred to a portable refrigerator for
transport to the laboratory. The target temperature range for the entire
cold chain was 2 C–8 C, but samples were considered viable if the
temperature was between 0 C and 25 C and did not exceed 20 C for
>2 h [17,18]. Temperature determination was based on Elitech RC5þ
data logger information when available and manual records on trans-
mittal sheets.
Laboratory methods and quality control
ELCL conducted same day testing on all venous blood specimens
using a Sysmex XN-450 hematology analyzer. The ELCL is accredited
by the South African National Accreditation System and undergoes
periodic testing of samples provided as part of that program. ELCL
conducted daily quality control checks using Sysmex quality control
materials at 3 levels. To provide additional internal imprecision esti-
mates, a subset of venous blood specimens was randomly selected and
tested 3 additional times by ELCL on the same day they were collected.
For external quality control, the same 3 specimens selected for repeat
testing were sent by courier to the accredited Infectious Diseases
Institute (IDI) laboratory for triplicate testing on the same day. The IDI
is accredited by The College of American Pathologists’ Laboratory
Accreditation Program.
Quality control checks for the HemoCue 201þ analyzer were per-
formed prior to data collection and daily during data collection using a
set of 3 levels of HemoTrol controls. Phlebotomists were instructed to
clean the microcuvette holder daily and the optics unit of the HemoCue
201þ analyzer weekly. A single HemoCue 201þ analyzer was
assigned to each phlebotomist. To investigate whether analyzers
S.M. Namaste et al. The American Journal of Clinical Nutrition xxx (xxxx) xxx

[19,20]. On the basis of 80% power, a P value of 0.05, and inflation of

the sample size by 1.25, the samples size was 140 participants for each
comparison, amounting to a total sample of 840 (n ¼ 280 for the
controlled setting and n ¼ 560 for the survey setting) [20].

FIGURE 1. Comparisons between sample type, test method, and both in the
controlled and survey setting.
performed similarly and whether there were variations between the
phlebotomist, we analyzed Bland–Altman bias by the instrument.
Sample size
Because the primary aim of the study was to determine the per-
formance of the methods, the sample size estimation was based on the
Bland–Altman agreement assessment [19,20]. The following pre-
defined values were derived from previous studies: 1) an expected
mean difference of Hb of 0.9 g/dL and 2) SD of mean difference of 0.07
Statistics
Stata version 17.0 was used for all statistical analyses. To mimic the
standard analysis used for DHS surveys, we adjusted Hb concentration
for altitude among children and women. In the survey setting, we used
a centroid of each cluster, and from that obtained the elevation using the
digital elevation model. In the laboratory setting, we identified the
longitude and latitude in a gazetteer on the basis of the town or village
and district where the participant resides and obtained the elevation
using the digital elevation model. An adjustment of 0.2 g/dL was
applied to all participants (altitude ranged from 1132 to 1257 m).
Adjustments were not made for smoking status because the quantity
was below the cutoff for adjustment with only 6 smokers. There were
no implausible Hb values (defined as Hb concentrations outside of
4–18 g/dL after adjusting for altitude) [21]. We removed 9 extreme
outliers defined as a 3 g/dL difference between the reference method
(Sysmex XN-450 hematology analyzer, venous) and comparators as
these likely indicated data entry errors rather than analytic variability.
We tested for demographic differences in participant groups using
chi-square test. In the event of a statistically significant global differ-
ence, we considered an absolute residual of >3 to contribute to the
significant difference.

TABLE 1
Sample size and demographic characteristics of participant groups among children and women
Controlled Survey pooled capillary Survey single-drop capillary P value
Children 6–59 mo
N
Eligible participants1 143 363 370
Consent granted 143 168 162
Sufficient volume2 142 144 139
Cold chain maintained 142 121 114
Included in analysis3 138 119 114
Male (%) 42 49 54 0.19
Age (mo, %) 0.05
6–11 94 1 3
12–23 22 19 18
24–35 20 29 29
36–47 22 24 26
48–59 27 27 24
Women 15–49 y
N
Eligible participants1 139 515 467
Consent granted 139 216 204
Sufficient volume2 138 187 199
Cold chain maintained 138 142 147
Included in analysis3 135 142 147
Age (y, %) 0.33
15–19 28 24 22
20–29 41 51 52
30–39 21 17 21
40–49 10 8 5
1
Excludes children and women who did not meet the eligibility requirements
2
Children: evacuated tube blood volume < 2.5 mL (n ¼ 28) and unknown blood volume (n ¼ 11); pooled capillary blood < 250 μL (n ¼ 4), >500 μL (n ¼ 1),
and unknown blood volume (n ¼ 7); women: evacuated tube blood volume < 2.5 mL (n ¼ 2) and unknown blood volume (n ¼ 11); pooled capillary blood < 250
μL (n ¼ 2), >500 μL (n ¼ 13), and unknown blood volume (n ¼ 7); note can have insufficient blood volume and/or unknown blood volume for both evacuated
tube and microtainer.
3
Outliers removed if there was 3 g/dL difference between the reference method (Sysmex XN-450 hematology analyzer, venous) and comparators. Total of 9
outliers removed.
4
Residual >3.

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S.M. Namaste et al. The American Journal of Clinical Nutrition xxx (xxxx) xxx

For each individual, we assessed the agreement between paired Hb
concentration of the reference method (Sysmex XN-450 hematology
analyzer, venous blood) against blood type measured on a HemoCue
201þ analyzer (venous, pooled capillary, and single-drop capillary
blood) (Figure 1). We also assessed the agreement using the HemoCue
201þ analyzer with venous against either pooled or single-drop
capillary blood; for these comparisons, we refer to the HemoCue
201þ analyzer as the field method (Figure 1). These comparisons allow
for the evaluation of differences attributable to analyzer and attributable
to blood source.
We generated Bland–Altman plots to assess whether there was
agreement between paired measurement and whether there was any
concentration-dependent bias. An acceptable limit of agreement (LOA)
was defined as 95% of the data points lying within 1.96 SD [22]. We
calculated the average mean difference [average(reference methodobs 
comparatorobs)]. We also took the absolute values of the difference and
calculated the average mean difference {average[abs(reference meth-
odobs  comparatorobs)]} to assess variations from 0 in either direction.
The same approach was used for the field method.
We used a paired t-test to test differences in means for each blood
source measured on the HemoCue 201þ analyzer compared against the
reference method and capillary blood sources measured on the
HemoCue 201þ analyzer compared with the field method, and a
McNemar’s chi-square statistics test to compare anemia prevalence
estimates. We calculated receiving operating curve area, sensitivity,
specificity, and percentage correct on the basis of any anemia. Anemia
for children was defined as Hb < 11 g/dL and for women as Hb < 12 g/
dL). Statistical significance was defined as P < 0.05 before applying
the Bonferroni corrections to correct for multiple comparisons (P ¼
0.05  k, where k equals the number of comparisons).
To show the deviation from perfect agreement between paired
measurements, we calculated Lin’s concordance correlation co-
efficients and visually examined Lin's concordance correlation plots
[23]. We also performed a Deming regression to assess bias.
Ethics
This study was approved by the ICF Institutional Review Board, the
Research and Ethics Committee, School of Health Sciences, Makerere
University (MAKSHSREC-2021-106), and the Uganda National
Council for Science and Technology (HS1529ES). This study was
registered at www.clinicaltrials.gov (NCT05059457). Informed con-
sent was provided prior to study participation.
Results
Quality control
All daily internal Sysmex XN-450 quality control checks performed
at ECLC were found to be within range [mean (SD) g/dL: 6.1 (0.1),
12.2 (0.1), 16.4 (0.1) and CV (N ¼ 21): 2.09%, 1.52%, and 1.49% for
the low, medium, and high controls, respectively]. In the subsets of
venous blood selected at random for repeat testing, intralaboratory CVs
calculated across the 3 replicate measures for a single sample ranged
from 0.00% to 1.35% [mean (SD) g/dL: 12.6 (1.4) and average CV:
0.51%]. For Sysmex XN-450 external quality control, a total of 84

FIGURE 2. Bland–Altman plots, hemoglobin g/dL: Sysmex XN-450 hematology analyzer compared with HemoCue 201þ analyzer by blood source in the
controlled setting.1
1
Bland–Altman plots of difference in hemoglobin concentration between (A) venous (HemoCue 201þ)-venous (Sysmex XN-450) in children, (B) pooled
capillary (HemoCue 201þ)-venous (Sysmex XN-450) in children, (C) single-drop capillary (HemoCue 201þ)-venous (Sysmex XN-450) in children, (D)
venous (HemoCue 201þ)-venous (Sysmex XN-450) in women, (E) pooled drop capillary (HemoCue 201þ)-venous (Sysmex XN-450) in women, and (F)
single-drop capillary (HemoCue 201þ)-venous (Sysmex XN-450) in women. Y axes: difference between paired measurements; X axes: average of the mea-
surements. The horizontal lines represent 95% confidence intervals around the mean difference between the hemoglobin concentration comparisons. Average
difference (SD) presented in bottom right of each plot. N ¼ 138 children and N ¼ 135 women.

4
S.M. Namaste et al. The American Journal of Clinical Nutrition xxx (xxxx) xxx

samples were tested at IDI. This resulted in similar mean and SDs for
the Hb concentration, and the low interlaboratory CVs indicated good
reproducibility [mean (SD) g/dL and average CV: 12.8 (1.4) and 0.45%
at IDI compared with 12.8 (1.4) and 0.78% at ELCL]. The mean
agreement between the results from ELCL and IDI was 99.48% (SD:
0.44%). All external quality control check sample results from ELCL
were within 4% of the IDI value.
Daily quality control checks for the HemoCue 201þ analyzer
showed that 1 out of 148 quality control tests was outside the stated
range of the control materials and for the 1 out of range value on
subsequent days, values were all in range [mean (SD): 8.0 (0.2), 11.9
(0.2), 15.9 (0.3) and CV percentages: 1.19%, 1.72%, and 2.10%, or-
dered from lowest to highest level). However, Bland–Altman plots of
results from a single phlebotomist using a single analyzer showed no
differences in Hb bias between instruments (Supplemental Table 1).
Given that a HemoCue 201þ analyzer was assigned to each phlebot-
omist, these results also indicate that the analyzers performed similarly.
Participant characteristics
Matched venous Hb concentration on the Sysmex XN-450 hema-
tology analyzer and from the different blood sources on the HemoCue
201þ analyzer was available for 371 children aged 6–59 mo and 424
women aged 15–49 y (Table 1 and Supplemental Figure 1). There were
fewer younger children than older children in the study sample. The
demographic information did not differ between the subsets of par-
ticipants who contributed to the study groups, except that there were
more children aged 6–11 mo in the controlled setting than those in the
survey setting (Table 1).
Comparison of reference method to HemoCue 201þ
analyzer by blood source
Bland–Altman plots
Mean differences between each blood source measured on the
HemoCue 201þ analyzer and the reference method (Sysmex XN-450
hematology analyzer, venous) were plotted against the average be-
tween the 2 measurements in a Bland–Altman plot depicted in
Figures 2–4. In these plots, the average bias and LOA was used to
evaluate the agreement between different blood sources in the Hemo-
Cue 201þ analyzer compared with the reference method. The
consistent distribution of differences (Y axis values) across the range of
Hb concentration (X axis) indicated that the bias was not concentration
dependent for any comparators.
The average mean difference between Hb measured using a
HemoCue 201þ analyzer against the reference method was lowest for
venous blood (0.1 to 0.16 g/dL), followed by single-drop capillary
blood (0.21 to 0.34 g/dL), and pooled capillary blood (0.25–0.48 g/
dL); this indicated a bias in both directions depending on the participant
group for venous blood and single-drop capillary blood, and a positive
bias for pooled capillary blood measured on the HemoCue 201þ
analyzer (Figures 2–4). In contrast, when taking the absolute value of
the differences and then examining the average mean difference, the

FIGURE 3. Bland–Altman plots, hemoglobin g/dL: Sysmex XN-450 hematology analyzer compared with HemoCue 201þ analyzer by blood source in the
pooled capillary survey setting.1
1
Bland–Altman plots of difference in hemoglobin concentration between (A) venous (HemoCue 201þ)-venous (Sysmex XN-450) in children, (B) pooled
capillary (HemoCue 201þ)-venous (Sysmex XN-450) in children, (C) venous (HemoCue 201þ)-venous (Sysmex XN-450) in women, and (D) pooled drop
capillary (HemoCue 201þ)-venous (Sysmex XN-450) in women. Y axes: difference between paired measurements; X axes: average of the measurements. The
horizontal lines represent 95% confidence intervals around the mean difference between the hemoglobin concentration comparisons. Average difference (SD)
presented in bottom right of each plot. N ¼ 119 children and N ¼ 142 women.

5
S.M. Namaste et al. The American Journal of Clinical Nutrition xxx (xxxx) xxx

FIGURE 4. Bland–Altman plots, hemoglobin g/dL: Sysmex XN-450 hematology analyzer compared with HemoCue 201þ analyzer by blood source in the
single-drop survey setting.1
1
Bland–Altman plots of difference in hemoglobin concentration between (A) venous (HemoCue 201þ)-venous (Sysmex XN-450) in children, (B) single-drop
capillary (HemoCue 201þ)-venous (Sysmex XN-450) in children, (C) venous (HemoCue 201þ)-venous (Sysmex XN-450) in women, and (D) single-drop
capillary (HemoCue 201þ)-venous (Sysmex XN-450) in women. Y axes: difference between paired measurements; X axes: average of the measurements.
The horizontal lines represent 95% confidence intervals around the mean difference between the hemoglobin concentration comparisons. Average difference
(SD) presented in bottom right of each plot. N ¼ 114 children and N ¼ 147 women.

largest was for single-drop capillary (0.54–0.69), followed by pooled
capillary blood (0.43–0.52), and the lowest for venous blood
(0.20–0.29).
The variation was higher in capillary blood than in venous blood.
For single-drop capillary blood, the SD of the mean difference ranged
from 0.57 to 0.87 g/dL and the SD of the absolute mean difference
ranged from 0.42 to 0.58 g/dL. For pooled capillary blood, the SD of
the mean difference ranged from 0.50 to 0.58 and the absolute mean
difference ranged from 0.39 to 0.48 g/dL. For venous blood, the SD of
the mean difference ranged from 0.29 to 0.50 g/dL and the SD of the
absolute mean difference ranged from 0.21 to 0.44 g/dL.
The LOA range was the lowest for venous blood (1.1–1.96 g/dL),
followed by pooled capillary blood (1.45–2.27 g/dL), and the highest
for single-drop capillary (2.23–3.41 g/dL). The LOA 95% confidence
interval of the Bland–Altman plot encompassed all but 3.4%–6.7% of
the observations across settings and participant groups.
Mean Hb and anemia differences
Mean Hb differences were 0.5 g/dL between the reference method
and all comparisons (Table 2). A paired t-test showed a small statisti-
cally significant mean difference between the reference method and
both sources of capillary blood measured with the HemoCue 201þ
analyzer, whereas venous blood was statistically significantly different
for half of the comparisons.
A comparison of any anemia prevalence estimates between the
reference method and each blood source is provided in Table 2. The
anemia estimates when using pooled capillary blood were statistically
significantly lower than those in the reference method among children
in the survey setting and among women in the controlled setting. There
were no other statistically significant differences between prevalence
estimates comparing the different blood techniques to the reference
method. Classification bias for anemia is provided in Supplemental
Tables 2–6.
Lin’s concordance correlation
Lin’s concordance correlation plots measured on the HemoCue
201þ analyzer using venous, pooled capillary, and single-drop capil-
lary blood compared with the reference method are provided in Table 3
and Supplemental Figures 2–4. The coefficients of agreement were the
strongest for venous blood (0.89–0.97), followed by pooled capillary
blood (0.81–0.91), and single-drop capillary blood (0.76–0.88)
(Table 3). The Deming regression analysis is provided in Supplemental
Table 7.
Comparison of field method by blood source
Bland–Altman plots
Bland–Altman plots comparing blood source measured on the
HemoCue 201þ analyzer are provided in Figures 5 and 6. The bias was
not concentration dependent for any comparators. The average mean
difference ranged from 0.25 to 0.31 g/dL for pooled capillary
compared with venous blood, 0.17–0.29 g/dL for single-drop capillary

6
S.M. Namaste et al. The American Journal of Clinical Nutrition xxx (xxxx) xxx

TABLE 2
Hemoglobin concentration and anemia prevalence of participant groups among children and women1
Venous Sysmex XN-450 Venous HemoCue 201þ Pooled capillary HemoCue 201þ Single drop capillary HemoCue 201þ
Controlled setting: Children 6–59 mo, n ¼ 138
Hb mean (SE) 11.2 (1.2)a,b 11.2 (1.2)c,d 11.5 (1.2)a,c,e 11.0 (1.4)b,d,e
Hb median 11.4 11.3 11.6 11.1
Hb minimum, maximum 7.7, 14.1 7.4, 14.1 7.8, 15.1 7.3, 14.5
Hb < 11.0 g/dL (CI) 39.9 (21.6, 48.5) 40.6 (32.3, 49.3)a 31.9 (24.2, 40.4)a,b 45.7 (37.2, 54.3)b
Survey setting pooled capillary group: children 6–59 mo, n ¼ 119
Hb mean (SE) 11.2 (1.0)a,b 11.3 (1.1)a,c 11.6 (1.1)b,c —
Hb median 11.4 11.4 11.7 —
Hb minimum, maximum 7.8, 13.6 7.5, 13.7 7.4, 14.3 —
Hb < 11.0 g/dL (CI) 35.3 (26.8, 44.6)a 31.9 (23.7, 41.1)b 21.8 (14.8, 30.4)a,b —
Survey setting single-drop capillary group: children 6–59 mo, n ¼ 114
Hb mean (SE) 10.9 (1.4)a 11.0 (1.4)b — 11.2 (1.5)a,b
Hb median 11.2 11.2 — 11.5
Hb minimum, maximum 6.4, 13.4 6.3, 13.6 — 6.2, 13.9
Hb < 11.0 g/dL (CI) 41.2 (32.1, 50.8) 40.4 (31.3, 49.4) — 35.1 (26.4, 44.6)
Controlled setting: women 15–49 y, n ¼ 135
Hb mean (SE) 12.8 (1.1)a,b,c 13.0 (1.1)a,d 13.3 (1.1)b,d 13.1 (1.3)c
Hb median 12.9 13.0 13.3 13.2
Hb minimum, maximum 7.5, 15.5 7.5, 15.6 7.4, 16.0 7.3, 16.2
Hb < 12.0 g/dL (CI) 18.5 (12.4, 26.1)a 13.3 (8.1, 20.3) 8.9 (4.7, 15.0)a,b 17.8 (11.7, 25.3)b
Survey setting pooled capillary group: women 15–49 y, n ¼ 142
Hb mean (SE) 12.6 (1.3)a 12.7 (1.3)b 12.9 (1.4)a,b —
Hb median 12.7 12.8 13.0 —
Hb minimum, maximum 8.2, 15.6 8.1, 15.8 7.4, 16.3 —
Hb < 12.0 g/dL (CI) 23.9 (17.2, 31.8) 23.2 (16.6, 31.1) 19.7 (13.5, 27.2) —
Survey setting single-drop capillary group: women 15–49 y, n ¼ 147
Hb mean (SE) 12.8 (1.3)a,b 12.9 (1.3)a,c — 13.2 (1.4)b,c
Hb median 13.0 13.0 — 13.3
Hb minimum, maximum 8.0, 15.4 7.8, 15.6 — 7.6, 16.5
Hb < 12.0 g/dL (CI) 20.4 (14.2, 27.8) 19.7 (13.6, 27.1) — 15.0 (9.6, 21.8)

Abbreviation: CI, confidence interval.

1
The statistical significance of each participant group was compared with each other. A common lowercase letter indicates result pairs with a significant
difference, P < 0.05 (adjusted using Bonferroni correction).

compared with venous blood, and 0.15 and 0.46 for pooled capillary
compared with single-drop capillary blood. The average mean difference
based on absolute values ranged from 0.38 to 0.48 for venous compared
with pooled capillary blood, from 0.43 to 0.67 for venous compared with
single-drop capillary blood, and was 0.72 and 0.60 for single-drop
capillary compared with pooled capillary blood.
The SD of the mean difference (range: 0.41–0.62 g/dL) and the
absolute mean difference (range: 0.33–0.47 g/dL) was lowest for
venous compared with pooled capillary blood in most comparisons.
The LOA range for pooled capillary compared with venous blood was
lower for most groups (1.61–2.43) than single-drop capillary compared
with venous blood (2.08–3.29), or pooled capillary compared with
single-drop capillary blood (2.94–3.33). For capillary blood compared
with venous blood, the percentage of data outside of the LOA 95%
confidence interval of the Bland–Altman plot ranged from 2.96% to
6.34% across settings and participant groups. For pooled capillary

TABLE 3
Lin's concordance correlation coefficients between participant groups among children and women.
Venous Sysmex XN-450 Venous HemoCue 201þ Single-drop
capillary HemoCue 201þ
Venous Pooled capillary Single-drop Pooled Single-drop Pooled
HemoCue 201þ HemoCue 201þ capillary capillary capillary capillary
HemoCue 201þ HemoCue 201þ HemoCue 201þ HemoCue 201þ
Children 6–59 mo
Controlled setting: n ¼ 138 0.93 (0.01) 0.87 (0.02) 0.76 (0.03) 0.92 (0.01) 0.78 (0.03) 0.75 (0.04)
Survey setting pooled capillary 0.91 (0.02) 0.81 (0.03) — 0.81 (0.03) — —
group: n ¼ 119
Survey setting single-drop capillary 0.96 (0.01) — 0.88 (0.02) — 0.92 (0.01) —
group: n ¼ 114
Women 15–49 y
Controlled setting: n ¼ 135 0.89 (0.02) 0.82 (0.03) 0.76 (0.04) 0.90 (0.02) 0.81 (0.03) 0.81 (0.03)
Survey setting pooled capillary 0.97 (<0.01) 0.91 (0.01) — 0.93 (0.01) — —
group: n ¼ 142
Survey setting single-drop capillary 0.96 (0.01) — 0.88 (0.02) — 0.90 (0.02) —
group: n ¼ 147

7
S.M. Namaste et al. The American Journal of Clinical Nutrition xxx (xxxx) xxx

FIGURE 5. Bland–Altman plots, hemoglobin g/dL: HemoCue 201þ by blood source in the controlled setting.1
1
Bland–Altman plots of difference in hemoglobin concentration between (A) pooled capillary (HemoCue 201þ)-venous (HemoCue 201þ) in children, (B)
single-drop capillary (HemoCue 201þ)-venous (HemoCue 201þ) in children, (C) pooled capillary (HemoCue 201þ)-single-drop capillary (HemoCue 201þ) in
children (2 outliers not shown), (D) pooled capillary (HemoCue 201þ)-venous (HemoCue 201þ) in women, (E) single-drop capillary (HemoCue 201þ)-venous
(HemoCue 201þ) in women, and (F) pooled capillary (HemoCue 201þ)-single-drop capillary (HemoCue 201þ) in women. Y axes: difference between paired
measurements; X axes: average of the measurements. The horizontal lines represent 95% confidence intervals around the mean difference between the he-
moglobin concentration comparisons. Average difference (SD) presented in bottom right of each plot. N ¼ 138 children and N ¼ 135 women. Note that pooled
refers to pooled capillary blood and single drop refers to single-drop capillary blood. Note: target on the Y and X axes refers to either the field method (venous
blood) or single-drop capillary blood and the comparator refers to pooled capillary or single-drop capillary blood as specified in each plot.

compared with single-drop capillary blood, the percentage of data Discussion

outside of the LOA 95% confidence interval of the Bland–Altman plot
was 3.6% and 3.7%.
Mean Hb and anemia differences
Mean Hb differences were 0.3 g/dL or less between venous and
pooled or single-drop capillary blood for all comparisons measured on
the HemoCue 201þ analyzer, and this was statistically significant for
nearly all comparisons (Table 2). The mean difference for pooled
capillary and single-drop capillary blood was 0.5 g/dL for children and
0.2 g/dL and was statistically significant.
Any anemia was statistically significantly different between pooled
capillary compared with venous and single-drop capillary blood for
children and between pooled capillary and single-drop capillary blood
for women (Table 2). Classification bias for anemia is provided in
Supplemental Tables 2–6.
Lin’s concordance correlation
Lin’s concordance correlation plots are provided in Table 3 and
Supplemental Figures 5 and 6. The coefficients of agreement between
venous with pooled and with single-drop capillary blood were similar
(0.81–0.93 and 0.78–0.92, respectively) (Table 3); comparisons be-
tween single-drop and pooled capillary blood were 0.75 and 0.81. The
Deming regression analysis is provided in Supplemental Table 7.
We compared Hb results from 3 blood sources tested with the
HemoCue 201þ analyzer to a reference method, venous blood tested
using a Sysmex XN-450 hematology analyzer. Venous blood showed
the lowest bias, least variability, and highest concordance compared
with the reference method, and pooled capillary blood performed
marginally better than single-drop capillary blood. Results were similar
comparing Hb results between blood sources using the field method,
HemoCue 201þ analyzer, to isolate the effects of the analyzer type
from blood source. In our study, it appears that the error was random for
single-drop capillary blood whereas for pooled capillary blood, the
random error appeared to be smaller but biased in the positive di-
rections. Whether this would be robust in other settings requires further
exploration.
The Bland–Altman LOA was selected as the primary outcome of
our study for evaluating differences attributable to blood sources.
Although an acceptable LOA Hb range has not been established for
population surveys, LOA Hb ranges from a laboratory quality assur-
ance program were consistent with our findings for venous and pooled
capillary blood and narrower to what we observed for single-drop
capillary blood. In studies similar to ours, we found narrower and
wider LOA Hb ranges for venous blood measured with various ana-
lyzers [24,25]. LOAs were narrower, similar, or wider measured on

8
S.M. Namaste et al. The American Journal of Clinical Nutrition xxx (xxxx) xxx

FIGURE 6. Bland–Altman plots, hemoglobin g/dL: HemoCue 201þ by blood source in the survey setting.1
1
Bland–Altman plots of difference in hemoglobin concentration between (A) pooled capillary (HemoCue 201þ)-venous (HemoCue 201þ) in children, (B)
single-drop capillary (HemoCue 201þ)-venous (HemoCue 201þ) in children, (C) pooled capillary (HemoCue 201þ) -venous (HemoCue 201þ) in women, and
(D) single-drop capillary (HemoCue 201þ)-venous (HemoCue 201þ) in women. Y axes: difference between paired measurements; X axes: average of the
measurements. The horizontal lines represent 95% confidence intervals around the mean difference between the hemoglobin concentration comparisons.
Average difference (SD) presented in bottom right of each plot. N ¼ 119 children and N ¼ 142 women for pooled capillary blood comparison and N ¼ 114
children and N ¼ 147 women for single-drop capillary comparison. Note that pooled refers to pooled capillary blood and single drop refers to single-drop
capillary blood.

pooled capillary blood [25–27] and single-drop capillary [12,13,25,28]
compared with venous blood (analytical method varied).
In absence of criteria for LOA, studies have defined an acceptable
Hb difference as <1.0 g/dL or 7% of the target Hb mean concen-
tration [24,28,29]. For clinical laboratories, the allowable variation
from target values was recently revised from 7% to  4% (approx-
imately equivalent to an Hb difference of 0.5 g/dL for values within
the normal range) [30]. Although this is the currently available
benchmark, it was established as a quality standard for individual
diagnostic testing, not for accuracy of population-level estimates. In
our study, all comparisons to the reference method met the criteria of
4% (on the basis of an average percent difference that approximates
how the criteria are applied for laboratory accreditation). Similarly, a
systematic review by Whitehead et al. [29] comparing venous or
single-drop capillary blood measured on a HemoCue 201þ analyzer
against an automated hematology found that 25 of 26 comparisons
were within the 7% variation bias. However, at the individual sample
level, we found that results that exceed the criteria for the acceptable
variation were higher for capillary (14.0% pooled and 23.8% single
drop at the 7% threshold; 34.8% pooled and 46.3% single drop at the
4% threshold) than venous blood (4.7% and 11.9% at the 7% and
4% thresholds, respectively).
We note that these comparisons include differences due to the in-
strument (analytic variation) and by sample type (preanalytic varia-
tion). If venous blood Hb measured using the HemoCue 201þ analyzer
is the target value, the average percentage difference was within the
4% variation bias for pooled and single-drop capillary blood but a
large percentage of individual results exceed the criteria (31.6% pooled
and 46.4% single-drop capillary at the 4% threshold, and 11.6%
pooled and 22.7% single drop at the 7% threshold). In the same re-
view by Whitehead et al. [29], when comparing pooled capillary and
single-drop capillary blood at the aggregate level, all comparisons were
within the 7% variation bias, but only included adults.
Small differences in mean Hb concentration have been found to
result in large differences in anemia prevalence and misclassification of
the burden of anemia [12,13,24,28,31,32]. We found small but statis-
tically significant differences in mean Hb concentration between most
of the comparisons. This translated into statistically significantly lower
anemia prevalence using Hb measured in pooled capillary blood only.
This is likely because differences in Hb concentration tended to be
more unidirectional for pooled than single-drop capillary blood. For
other comparisons, we found nonsignificant differences in anemia of
5% or less. This study focused on Hb measurement in surveys in which
this magnitude of difference is unlikely to meaningfully impact policies
and programs as it might for other uses (for example, responsiveness to
an intervention).
We hypothesized that pooled capillary blood may mitigate chal-
lenges seen in single-drop capillary blood collection, but in our study,
pooled capillary blood performed only marginally better. Previously,
Conway et al. found that less experienced technicians had reduced

9
S.M. Namaste et al.
variability using pooled compared with single-drop capillary blood,
and pooled capillary blood performed well in a recent study in
Mexico [16,25]. The most effective approach to collecting pooled
capillary blood has not been examined. Anecdotally, the small blood
volume in a microtainer might make it difficult to solubilize the
anticoagulant, potentially leading to microclots and an inconsistent
blood-to-anticoagulant ratio (D. Killilea, personal communication).
In addition, loading the microcuvette using an intermediate surface
(as in our study) may allow technicians to see more clearly when a
microcuvette is properly filled than collecting directly from the
microtainer or finger (in the case of a single drop) and has performed
better for single-drop capillary blood [28].
Our findings do not align with previous observations of lower
anemia prevalence in children (but not women) among surveys where
Hb was measured in capillary instead of venous blood [6,10]. These
previous observations might be explained by the difficulty of collecting
high-quality capillary samples under challenging field conditions,
especially in children. A study in Mexico found that a single drop rather
than pooled capillary or venous blood was less accurate and precise for
children [25]. In the present study, patterns were similar between
children and women, across settings, and between phlebotomists. This
may be because phlebotomists were well trained, and some had prior
experience collecting capillary blood, and although the survey setting
was designed to mimic a DHS-like survey, implementation was rela-
tively simple (for example, near capital, fewer data collectors, shorter
collection period, and exclusion of anthropometry).
A unique contribution of this study is that it mirrors a real survey
context because it was part of a DHS pilot. Other strengths include
comparing Hb concentration against a reference method on the same
individuals, and the observation of a range of Hb concentrations.
Limitations were high refusal rates and loss of samples because of cold
chain issues; although the study population may not be fully repre-
sentative (for example, wealth, age, and health status), this likely did
not affect the results because groups were randomly assigned. A barrier
to using our and others’ validation results to inform recommendations
is a lack of acceptability standards in population surveys. Evaluation of
the evidence should include an examination of techniques and mate-
rials used to determine differences that can be attributed to blood source
from insufficient skills of the technicians or factors such as lancet type
[33]. Respondent variables may also influence results (for example,
age, sex, and hemoglobinopathies) [11,32]; for example, a recent study
suggests iron status, body composition, and inflammation may
contribute to differences [34]. Lastly, we used the HemoCue 201þ;
there are other HemoCue models (301, 801) that may yield different
results [6].
Accurate, yet simple and inexpensive methods are vital to Hb
measurement at the population level. The current procedure that
meets these criterion—point-of-care testing using a single drop of
capillary blood—may result in too much variation to estimate anemia.
Further research is needed to confirm this (for example, additional
testing in survey contexts), along with research on whether pooled
capillary blood is an acceptable alternative to single-drop capillary
blood. The benefit of improved quality using venous blood should be
weighed against risk of Hb being excluded from surveys because of
increased logistics and costs. Research is also needed on unintended
consequences of switching to venous blood, such as introduction of
selection bias if there are large differences in refusal rates by location,
wealth, age, or health status [35]. A benefit of using venous blood is
the ability to include more biomarkers, although extensive logistics
10
The American Journal of Clinical Nutrition xxx (xxxx) xxx
are required for laboratory-based testing. Further exploration is
needed to determine an effective approach to measuring Hb in pop-
ulation surveys.
Acknowledgments
We would like to thank the phlebotomists, the Ebenezer Limited
Clinical Laboratory staff, and the Uganda Bureau of Statistics for their
contributions in collecting and testing results. We would also like to
thank Shonda Gaylord and Peter Aka for co-leading the biomarker
training of trainers. We would like to dedicate this articles to the,
memory of Kama Bakunda who co-led the biomarker training for this
study and who made many contributions to the field throughout her
career. We thank The DHS Program pilot team, Yodit Bekele, Trevor
Croft, Joy Fishel, Joanna Lowell, and Keith Purvis, for providing
oversight support for the pilot implementation and data processing. The
protocol was informed by the USAID Advancing Nutrition “Protocol
for Comparative Evaluation of Blood Sampling Methods and Analyt-
ical Devices in the Measurement of Hemoglobin in Population Surveys
– A Laboratory Study” developed under the guidance of the HEmo-
globin MEasurement (HEME) working group. Eric Ohuma contributed
to the development of the statistical analysis through the USAID
Advancing Nutrition Project. Dean Garrett contributed to the early
design of the protocol.
Author contributions
The authors’ responsibilities were as follows – SMLN designed the
research, performed the statistical analysis, drafted the manuscript, and
was responsible for the final content. RB conducted the research and
reviewed the manuscript. EB contributed to the design of the research,
provided input on the statistical analysis, and contributed to the drafting
of the manuscript. All authors reviewed and approved the final
manuscript.
Conflict of interest
The authors report no conflicts of interest.
Funding
The study was implemented with support from the United States
Agency for International Development (USAID) and the Bill &
Melinda Gates Foundation through The DHS Program (#720-OAA-
18C-00083). The views expressed are those of the authors and do not
necessarily reflect the views of USAID or the United States
Government.
Data availability
Data described in the manuscript, code book, and analytic code will
be made available upon request to the corresponding author.
Disclaimers
The findings and conclusions in this report are those of the authors
and do not necessarily represent the official position of the United
S.M. Namaste et al.
States Agency for International Development or reflect the views of the
Bill & Melinda Gates Foundation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ajcnut.2023.12.025.
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