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Online Supplemental Material 1
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Validation of fluorescence method to measure serum retinol concentrations
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Method: A first prototype of the fluorescence machine (iCheck) was tested in the field during sample
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collection in 2010. The correlation between serum retinol by HPLC and fluorescence was low (r=0.43
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p<0.01), and sensitivity and specificity of fluorescence to in detecting vitamin A deficiency (defined as
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serum retinol<0.70 μg/L as analyzed by HPLC) were 69% and 55%. Based on our findings and
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recommendations, the manufacturer made adjustments to the machine and provided an improved iCheck
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version in 2011. This supplement shows the methods and results of a validation of the 2011 iCheck in our
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laboratory under controlled conditions
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For this, 15 ml whole blood was obtained from 3 individuals and 3 aliquots of 5 ml were kept in the dark at
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2˚- 8˚ C. Two aliquots were centrifuged at 3,000g for 10 minutes and serum was stored at 2–8 ˚C until
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analyses. For measuring fluorescence, 2×250 µL of whole blood or serum was injected into a sealed glass
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cuvette prefilled with a registered reagent (BioAnalyt IEX tm MILA) comprising a mixture of alcohols and
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organic solvents. The vial was shaken vigorously for 10 seconds and left to settle for 5 minutes in dark
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conditions to allow separation of solvent and blood. The outer surface of the cuvette was cleaned with a
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tissue before it was inserted into the machine. Four readings were taken at 20 sec intervals, each time
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turning the vial by one-quarter. The machine automatically reports the result as the average of these four
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readings. For each sample, we used the average of two measurements at a one-minute interval (hence
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2×4 readings). Data obtained from whole blood was adjusted for hematocrit value, which was estimated
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from hemoglobin levels (Hemocue 301, Ängelholm, Sweden) according to Bioanalyt iCheck guidelines.
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Standard control samples provided by the manufacturer were measured at the beginning and at the end of
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each batch of measurements and were within range.
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Linearity of the relationship between serum retinol concentrations measured by fluorescence and HPLC
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was assessed in duplicate over a range of concentrations (100%, 70%, 50%, 35%, 20% and 0 %) in whole
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blood samples from two individuals diluted with phosphate-buffered saline. The prefilled cuvette used for
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fluorescence requires a 500 µL sample volume. To assess whether a 250 µL sample would provide similar
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results as a 500 µL sample we measured blood samples from two individuals as 500 µL whole blood, 500
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µL serum and 250 µL serum + 250 µL phosphate-buffered saline in duplicate and compared it to HPLC
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concentrations. To correct for the lower serum volume in the latter we multiplied the results by two.
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Retinol concentrations measured by fluorescence could potentially be influenced by storage time,
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temperature of the environment, and light, which are typical factors that can vary in field settings. To
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assess the effect of each of these factors, we first stored whole blood samples of 3 individuals in triplicate
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for 30 minutes, 1 hour, 7 hours and 24 hours at 4 °C in dark conditions, followed by fluorescence
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measurement. Secondly, we exposed 6 samples with varying concentrations to increasing temperatures
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(15 °C, 20 °C, 25 °C, 30 °C and 35 ˚C) in a climate room with a constant humidity of 50%-60% and
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artificial light. Samples and machine were allowed to adapt to the indicated temperatures for 10 minutes,
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starting with 15 ˚C. Lastly, we exposed 6 samples with varying concentrations to three different light
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conditions: 1) in the laboratory, at 4 meters removed from the window with artificial room lights on; 2)
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outside at mid-morning with major overcast conditions and no visible sun; and 3) in the laboratory, close to
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the window with artificial lights on.
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Results: Linearity of retinol concentration measured by fluorescence and HPLC from two individuals was
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good, with high correlation and good association (Figure 1). We found no meaningful difference between
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results derived from whole blood samples and those from serum. At 7h and 24h of storage, concentrations
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were reduced by 3.5% and 12%, respectively. We found no evidence that storage for 1 hour or less
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influenced retinol concentrations. We found no evidence that temperature affected retinol concentrations
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measured by fluorescence (Figure 2). We found a clear effect of light conditions: samples measured
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outside and close to a window gave consistently higher retinol values than those measured inside (Figure
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3).
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Conclusion: Based on our results we conclude that the iCheck method provides good linearity when
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compared to retinol by HPLC. Light has a clear influence on the measurements and therefore the method
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should be used under controlled light conditions.
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Serum retinol concentration, µgl/dL (fluorescense)
80
70
A
60
50
B
40
30
20
y = 1.20 x
A R2 = 1.00
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y = 0.99 x
B R2 = 0.99
0
0
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10
20
30
40
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Serum retinol concentration, µg/dL (HPLC)
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Figure 1: Linearity of retinol concentrations measured by fluorescence and HPLC in µg/dL over a dilution
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range of whole blood from two individuals (A and B)
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1200
Serum retinol concentration µg/dL
1000
Control
Dilutions
0%
20%
35%
50%
70%
100%
800
600
400
200
0
0
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15
25
20
Temperature C
30
35
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Figure 2: Retinol concentrations measured by fluorescence over a temperature range of 15˚ to 30˚ in 6 different
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dilutions
Serum retinol concentration µg/dL (fluorescense)
3000
2500
2000
In a room
Outside in daylight
Next to a window
1500
1000
500
0
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Dilution
Figure 3: Retinol concentrations measured by fluorescence during three different light conditions
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Online supplemental material 2
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Elimination of diagnostic error when estimating the prevalence of vitamin A deficiency
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Let true vitamin A status be defined by a gold standard, and let it be indicated by a diagnostic test with a binary
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outcome. Table 1 shows the cross-tabulated data that might be obtained in a survey.
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Table 1: Cross-tabulated data for true vitamin A status, defined by a gold standard, and diagnostic test results
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Diagnostic test result
Vitamin A deficient
Total
Yes
No
Positive
A
B
P
Negative
C
D
Q
Total
R
T
N
Capital letters in each cell indicate the number of people in that cell
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The true and estimated prevalence of vitamin A deficiency can thus be noted as follows:
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PrevT = R/N
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PrevE = P/N
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We can show that the estimated prevalence is a function of sensitivity, specificity and the true prevalence:
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i.
Sensitivity = A/R, hence A = R ·Sensitivity
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ii.
(1 ‒ Specificity) = B/T, hence B = T · (1 ‒ Specificity)
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Thus: PrevE = P/N = (A + B)/N = [R · Sensitivity + T · (1 ‒ Specificity)]/N = (R · Sensitivity)/N + [(N-R) · (1 ‒
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Specificity)]/N = PrevT · Sensitivity + (1 ‒ PrevT) · (1 ‒ Specificity)
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If Prev = PrevT = PrevE, then it can be derived that:
𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦 = 1 + (1 −
1
) (1 − 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑡𝑦)
𝑃𝑟𝑒𝑣
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5
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Note that the association between Sensitivity and (1 ‒ Specificity) is linear, with Sensitivity = 1 when (1 ‒
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Specificity) = 0, and slope defined by (1 ‒ 1/Prev). This linear association is shown in Figure 5 (main text) for
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different, arbitrarily chosen values of Prev. Thus each line represents an infinite number of paired values for
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Sensitivity and (1 ‒ Specificity), given a true prevalence, that will allow for an unbiased estimation of that
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prevalence.
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