SAMPLE METHODS SECTION

2. Methods
2.1 Study setting and participants
Adolescent girls and boys aged 14-19 years attending secondary schools were included in the
study. We included public schools that have secondary students (Form 1 (youngest) – Form 6
(oldest)), and for which we had permission from the Ministry of Primary and Secondary
Education at the provincial, district, and school levels. The study was carried out in Harare, the
capital of Zimbabwe, lying in the northeastern part of the country. The participants were
adolescents aged 14 to 19 years with signed informed consent forms and attending secondary
schools in Harare. Ethical approval was obtained from Osaka Metropolitan University (OMU/21-
52) and the Medical Research Council of Zimbabwe (MRCZ/A/2857). This study presents data
that was part of a bigger study entitled: The analysis of related factors of overweight and obesity
among high school students in Harare, Zimbabwe. The purpose was to assess how eating habits,
nutrition knowledge, and physical activity are associated with overweight and obesity among
adolescents (Pencil et al., 2024).

2.2 Sample size and sampling technique

Recruited participants were asked to remain in the classrooms, and they received an in-depth
orientation about the study’s objectives, finger-prick sampling procedure, weight, and height
measurements for BMI calculations, waist and hip circumference measurements, and how long it
would take to fill in the self-administered questionnaire. The participants were informed that no
incentive would be offered for participation, there would be no penalties for dropping out of the
survey and participation in the study was for participants with sign consent forms. This survey
was carried out in accordance with the ethical standards in line with the Helsinki Declaration of
1975, as revised in 2013 (WMA, 2013)
2.3 Research instruments and data collection methods
Blood sampling for anaemia
Capillary blood sampling was done according to the method suggested by (Dhingra, 2010) This
method was selected because it is a minimally invasive sampling technique, routinely used for
point of care testing in home healthcare settings and clinical settings with less pain and
wounding than conventional venepuncture (Hoffman et al., 2023). This method has been proven
to provide accurate results in field work and outpatient work
(Nwankwo et al., 2021; Kuche et al., 2025)
Trained research staff collected data from adolescents at school, whose parents had
provided consent and who had themselves provided assent to participate.

Single-use disposable sterile gloves were used, and a single-use disposable lancet device was
used for each participant. All tests were carried out as per the manufacturer’s instructions. The
research registered nurse measured haemoglobin through the finger-prick technique
(Kuche et al., 2025)
. Haemoglobin status was assessed using a portable battery-operated HemoCue® Hb
301 Analyzer (HemoCue AB, Sweden). Participants’ hands were encouraged to hydrate, and
their hands were warmed to encourage blood flow. Capillary blood samples were taken by
pricking the tip of the adolescents’ middle finger after rubbing it with a cotton swab immersed in
alcohol. The first two drops were wiped away, and the third and fourth drops were used for the
Hb reading (Namaste, Baingana and Brindle, 2024) . Two readings were measured and averaged
to ensure accuracy (Kuche et al., 2025).

Anthropometric measurements were taken twice, according to the WHO protocol. Height was
measured using a Seca 213 Stadiometer (Seca GmbH, Hamburg, Germany) and recorded to the
nearest 0.1 cm. During the measurement, participants stood in the Frankfurt position. Shoes were
taken off, and prominent body parts (occipital, shoulder, buttocks, and heel) touched the
stadiometer. Weight was measured with the Seca 875 weighing scale (Seca GmbH, Hamburg,
Germany) and recorded to the nearest 0.1 kg. Heavy clothes and shoes were taken off. Both the
weighing scale and the length board were calibrated daily.

Structured questionnaire
Dietary data were collected using questionnaires that assessed food habits and nutrition
knowledge. The questionnaires were adapted from validated and reliable sources to ensure
accuracy. The final version consisted of four sections: socio-demographic and anthropometric
information (10 questions), nutrition knowledge (20 questions), food habits (23 questions), and
physical activity (7 questions). Scoring for the questionnaire was adapted as follows:

Nutrition Knowledge
The questionnaire was adapted from (Oz et al., 2016) The nutrition knowledge scores (NKS)
were categorized as either inadequate = 0 (NKS < 50%) or adequate =1 (NKS ≥ 50%). It was
designed to be a practical, easy-to-administer tool with demonstrated reliability for use among
high school students. While the instrument measured general nutrition knowledge, it did not
specifically focus on anaemia-related content. The questionnaire consisted of three subscales:
adequate and balanced nutrition, essential nutrients, and malnutrition-related diseases. Questions
were presented as complete sentences, requiring respondents to identify statements as true, false,
or indicate if they were unsure. The overall reliability of the instrument was strong, with a
Cronbach’s alpha coefficient of 0.85. An example item from the questionnaire is: “Adequate and
balanced nutrition decreases the risk of anaemia?” with responses, Yes/No.

Food habits
The food habits questionnaire was adapted from (Johnson et al., 2002) . The food habits score
(FHS) is a metric, often from a questionnaire, that quantifies dietary patterns by assigning points
for healthy eating behaviors (like eating fruits/veg, limiting fats/sugars) and unhealthy ones,
resulting in a total score indicating overall diet quality, with higher scores meaning healthier
habits, helping assess nutritional intake beyond just calories.
FHS = (Number of healthy responses × 23) ÷ Total number of items completed
Scores were categorized as inadequate if FHS was below 50%, and adequate if FHS was 50% or
higher. One example of a food habits question is: “I try to ensure I eat at least one portion of
fruits and vegetables per day.” With responses, Yes/No.

Physical activity
The physical activity questionnaire was adapted from (Silva et al., 2018) . The Physical Activity
Score (PAS) was calculated based on responses, which were structured differently depending on
the specific questions. Overall, scores were classified as inadequate (PAS < 50%) or adequate
(PAS ≥ 50%). The questionnaire demonstrated acceptable internal reliability, with a Cronbach’s
alpha of 0.71. An example question from this section is: “Do you usually practice any form of
physical activity?”

Variables categorization
Food habits, nutrition knowledge, and physical activity were dichotomized after total scores were
calculated according to the instructions prescribed (Johnson et al.,2002; (Oz et al., 2016) and
(Silva et al., 2018) respectively. The data satisfied the conditions, for instance defining cut off
pints based on clinical guideline, prior research etc. These are conditions under which
dichotomization is considered justifiable, as highlighted by (Iacobucci et al., 2015) and
(Garcia, et al., 2015)

Adolescents anaemia status was considered as the outcome variable and was defined according
to WHO age- and sex- specific cutoffs (the gold standard for assessing anaemia) as individual
haemoglobin levels between 11 and 11.9 g/dl, 8–10.9 g/dl, and lower than 8 g/dl as mild,
moderate and severe respectively, for children 12–14 years and non-pregnant females over 15
years (WHO, 2011) . Independent variables considered as potential factors associated with
anaemia in this study were identified by reviewing previous literature
(Chauhan et al., 2022; Habtegiorgis et al., 20
. Hemoglobin concentrations
were adjusted for Harare altitude, 1,483 meters above sea level (UN, 2022) according to methods
stated by (WHO, 2024b) Adjustments to haemoglobin concentrations are recommended when
diagnosing anaemia in individuals and populations to account for the effect of residential altitude
on haemoglobin levels (Robalino et al., 2016).

Nutritional status was evaluated following the WHO standard procedures for children aged 5–19
years (WHO, 2016). Body Mass Index (BMI, kg/m²) was converted to z-scores using WHO.
BMI-for-age z-scores were classified as underweight (<−2 SD), normal (≥ −2 to ≤ +1 SD),
overweight (≥ +1 to +2 SD), and obese (> +2 SD).
2.4 Data Analysis
Data was analysed using IBM SPSS version 23. After data cleaning and removal participants
data with missing variable, data from 386 participants remained and was analysed further. The
normality of continuous variables was assessed using the Shapiro-Wilk test. Normally distributed
variables were summarized as mean ± standard deviation (SD), while non-normally distributed
data were presented as median ± interquartile range (IQR). Associations between categorical
variables were evaluated using Pearson’s chi-square (χ²) test.

Although anaemia severity was originally classified into three ordered categories (mild,
moderate, and severe), the outcome variable was (Not Anaemic = 0 and Anaemic =1) with
demographic variables, food habits, and nutrition knowledge for regression analysis. This
approach was adopted because the primary objective of the study was to identify factors
associated with the presence of anaemia as done by (Getie et al., 2025), rather than determinants
of its severity. Collapsing severity categories improved model stability and interpretability,
particularly because some severity categories contain relatively small numbers of observations.
Therefore, the use of binary logistic regression in this study aligns with the research objectives
and established epidemiological studies (Bharati et al., 2008; Getie, et al., 2025) . Statistical
inference was based on a significance threshold of p < 0.05. Regression estimates are presented
alongside 95% confidence intervals (CIs).

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