Jun 04, 2026

Standardized Protocol for Human Milk Collection and Storage in a Multicenter Study on Climate Change and Human Milk Composition

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvjk495gk5/v1
  • 1Federal University of Minas Gerais;
  • 2University of Brasília;
  • 3Canoas University Hospital;
  • 4Federal University of Maranhão
Isabella Costa
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjk495gk5/v1
Protocol Citation: Isabella Costa, Zilma Reis, Artur Nogueira de São José, Paulo de Jesus Hartmann Nader, Silvana Salgado Nader, Márcia Gomes Penido Machado, Marynéa Silva do Vale 2026. Standardized Protocol for Human Milk Collection and Storage in a Multicenter Study on Climate Change and Human Milk Composition. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjk495gk5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: April 28, 2026
Last Modified: June 04, 2026
Protocol  Integer ID: 315863
Keywords: Human milk, Breastfeeding, Lactation, Thermal stress;, Maternal health, standardized protocol for human milk collection, human milk sample, human milk composition this protocol, human milk composition, variations in human milk composition, human milk collection, environmental data in maternity hospital, lactating women, including temperature, harmonization of multicenter maternal, biological sample collection, multicenter maternal, environmental data, involving biological sample collection, standardized multicenter procedure, standardized procedure, child health study, using standardized multicenter procedure, humidity index, investigating thermal stress, freezing condition, data reliability, sample traceability
Funders Acknowledgements:
Fundação de Apoio à Pesquisa do Distrito Federal
Grant ID: 00193-00001402/2024-51
Abstract
This protocol describes standardized procedures for recruitment of lactating women, human milk collection, sample identification, temporary storage, and data collection of clinical, demographic, breastfeeding-related, and environmental data in maternity hospitals participating in a multicenter study investigating thermal stress and human milk composition.
Human milk samples will be manually collected, aliquoted into labeled microtubes, and stored under controlled freezing conditions using standardized multicenter procedures designed to ensure sample traceability and data reliability.
The protocol is expected to support future investigations evaluating associations between environmental conditions, including Temperature-Humidity Index (THI), and variations in human milk composition. In addition, the standardized procedures described may contribute to reproducibility and harmonization of multicenter maternal-child health studies involving biological sample collection and storage.
Guidelines
  • All procedures should be performed by previously trained research personnel following the standardized operational instructions described in this protocol.
  • Participant eligibility criteria must be confirmed before enrollment and sample collection procedures.
  • Human milk collection should preferably occur at least 1 hour after the last breastfeeding session and no earlier than 7 days postpartum.
  • All biological samples must be identified using participant-specific alphanumeric codes before collection and aliquoting procedures.
  • The same identification code must be applied to all aliquot tubes obtained from the same participant to ensure sample traceability.
  • Milk samples should be aliquoted immediately after collection whenever possible to minimize handling variability.
  • Samples must be maintained under controlled freezing conditions between −12°C and −18°C until transportation or further processing.
  • Clinical, demographic, breastfeeding-related, and environmental data should be recorded on the same day as milk collection using the standardized digital platform.
  • Environmental data used for future Temperature-Humidity Index (THI) calculations should correspond to the collection site and collection date.
  • All study procedures must follow institutional ethical requirements and confidentiality standards for research involving human participants.
Materials

Equipment

  • Freezer (−12°C to −18°C) for temporary storage of biological samples

Consumable

  • Eppendorf Safe-Lock‱ tubes, 5.0 mL, clear
  • Sterile universal collection containers with screw caps (50 mL capacity)
  • Disposable plastic Pasteur pipettes (3 mL)
  • Non-sterile examination gloves (size G or equivalent)
  • Disposable fluid-resistant gowns with long sleeves and cuffed wrists
  • Disposable triple-layer face masks with nose clip
  • Waterproof permanent markers for sample labeling
  • Moisture-resistant adhesive labels for biological sample identification
  • Cardboard storage boxes for microtubes (capacity: 100 units)

Sample Handling and Storage Materials

  • Participant-specific sample identification system using unique alphanumeric codes
  • Sample tracking forms for recording collection date, storage conditions, and sample location
  • Standardized clinical and demographic data collection forms
  • Digital spreadsheets or electronic data capture platform for sample registration and tracking

Administrative Materials

  • Secure document folders with plastic sleeves for storage of informed consent forms and study records
  • Participant enrollment logs
  • Printed copies of informed consent forms (ICF)
  • Study checklists and Standard Operating Procedure (SOP) documents
  • Password-protected digital files for study organization and participant tracking

Troubleshooting
Problem
Insufficient milk volume collected
Solution
Allow participant to alternate breasts and provide additional time for manual expression if comfortable
Problem
Milk sample contaminated with blood or purulent secretion
Solution
Discontinue collection and exclude sample according to exclusion criteria
Problem
Incorrect or missing tube identification
Solution
Label all tubes before collection and confirm identification codes before aliquoting
Problem
Sample thawing during storage or transport
Solution
Exclude affected samples and document the occurrence in study records
Problem
Participant does not meet eligibility criteria
Solution
Reassess inclusion and exclusion criteria using the screening checklist before consent
Problem
Difficulty during milk aliquoting
Solution
Prepare all materials before collection and perform aliquoting on a stable surface
Problem
Inconsistency between sample codes and digital records
Solution
Verify participant identification codes immediately after data entry and before sample storage
Safety warnings
Warnings
  • Do not collect milk samples from participants presenting signs of breast infection, mastitis, or nipple injury.
  • Do not include samples visibly contaminated with blood or purulent secretion.
  • Avoid interruptions in the cold chain during sample handling and storage. Samples exposed to room temperature for more than 30 minutes or showing evidence of thawing should be excluded.
  • Ensure that all aliquot tubes are correctly labeled before sample collection to prevent identification errors and loss of traceability.
  • Use disposable collection materials and appropriate personal protective equipment during all collection procedures.
  • Incomplete or inconsistent clinical and environmental data may compromise future analyses and should be reviewed immediately after collection.
  • Access to identifiable participant information must be restricted to authorized study personnel only.
Ethics statement
This protocol involves research with human participants and biological sample collection. The associated research project has been submitted for evaluation by the Research Ethics Committee of the University of Brasília and is currently awaiting approval prior to participant recruitment and study initiation.
All procedures described in this protocol must be conducted in accordance with institutional ethical requirements and applicable national regulations for research involving human participants. Written informed consent must be obtained from all participants before any study-related procedures are performed.
Participant confidentiality and data protection must be maintained throughout all study phases, and access to identifiable participant information must be restricted to authorized study personnel only.
Standard Operating Procedure (SOP)
Participant Recruitment

Eligibility Criteria
Inclusion criteria:
  • Lactating women aged ≥18 years
  • Lactating women ≥7 days postpartum
  • Gestational age ≥34 weeks at delivery
  • No contraindications to breastfeeding
  • No signs of breast infection or nipple injury
Exclusion criteria:
  • Milk samples with cold chain disruption (e.g., thawing or storage outside refrigeration for >30 minutes)
  • Milk samples visibly contaminated with blood or purulent secretion


Screening Checklist
Before enrollment, confirm that:
  1. The participant meets all inclusion and no exclusion criteria
  2. Milk production is established (≥7 days postpartum)
  3. The last breastfeeding session occurred at least 1 hour prior to collection
Study Explanation and Informed Consent
  1. Explain the study objectives and procedures using clear and accessible language.
  2. Present the Informed Consent Form (ICF).
  3. Allow sufficient time for reading and questions.
  4. Answer all participant questions before enrollment.
  5. Obtain written informed consent in duplicate.
  6. Provide one signed copy to the participant.
  7. Store the second signed copy securely in study records.
Human Milk Collection
Preparation for Sample Collection

  1. Prepare the following materials on a clean and stable surface (bench, tray, or support table):
  • Three Eppendorf Safe-Lock‱ tubes (5 mL)
  • One disposable Pasteur pipette (3 mL)
  • One sterile 50 mL collection container

2. Each participant will receive a unique alphanumeric identification code according to the recruitment site and enrollment order. The same identification code must be used for all aliquots obtained from the same participant to ensure sample traceability and linkage with clinical and environmental data.
3. Label the three Eppendorf tubes using a waterproof permanent marker before sample collection. All three tubes corresponding to the same participant must receive identical identification codes.
Participant Identification Code Examples
  • Hospital Sofia Feldman → HSF01, HSF02
  • Hospital da Universidade Luterana do Brasil → ULBRA01
  • Hospital Universitário da Universidade Federal do Maranhão → UFMA01


Participant Preparation
  1. Ensure that the participant is seated comfortably, preferably without holding the infant.
  2. Perform hand hygiene and don a surgical mask, a disposable gown, and disposable gloves.
  3. Inspect both breasts for signs of infection or nipple injury.

Figure 1. AI-generated education illustration showing common breast and nipple infections thtat may contraindicate humn milk collection, including lactation mastitis, nipple candidiasis (thrush), breast abscess, and fungal skin infection (intertrigo). Image generated using Gemini (Google AI).
4. If signs of infection are identified, discontinue the procedure and thank the participant.
Human Milk Collection Procedure
  1. Instruct the participant to wash her hands thoroughly.
  2. Provide the sterile 50 mL collection container without the lid.
  3. Instruct the participant to perform manual self-expression of breast milk, expressing milk from behind the nipple directly into the collection container.
  4. Request approximately 10 mL of milk, whenever feasible and comfortable for the participant.
  5. If necessary, instruct the participant to alternate between breasts to obtain the desired volume.
Sample Aliquoting
  1. Place the collected milk on a stable surface.
  2. Using a disposable Pasteur pipette, aliquot the milk into the three previously labeled Eppendorf tubes.
  3. Distribute the milk volume equally across the three tubes whenever possible.

Example
  • Total collected volume: 6 mL
  • Aliquot volume: 2 mL per tube
Sample Storage
  1. Place the labeled tubes containing milk samples into a cardboard storage box suitable for microtubes.
  2. Label the storage box with:
  • Study name
  • Responsible researcher
  • “Do Not Handle” notice
3. Store the box containing samples in a freezer at −12°C to −18°C.
Clinical, Demographic, and Environmental Data Collection
Data Collection
  1. Maternal and newborn data will be collected on the same day as human milk collection.
  2. Clinical, demographic, and environmental information will be recorded using a standardized digital data collection platform.
  3. All collected data will be linked to the participant-specific identification code to ensure traceability between biological samples and study records.
  4. Environmental variables relevant for future Temperature-Humidity Index (THI) calculation will also be recorded.
Clinical, Demographic, and Environmental Questionnaire
A structured questionnaire will be used to collect maternal, neonatal, breastfeeding-related, and environmental information associated with human milk collection procedures.
Maternal and Clinical Data
  • Maternal age
  • Educational level
  • Prenatal care history
  • Diabetes, hypertension, and anemia status
  • Mode of delivery
  • Gestational age at delivery
  • Maternal anthropometric data
  • Erythrogram results, when available
Neonatal Data
  • Birth weight
  • Multiple pregnancy status
Breastfeeding and Collection Data
  • Time since last breastfeeding session
  • Breastfeeding type
  • Difficulties or relevant observations during milk collection
Environmental Data
  • Ambient temperature
  • Relative humidity
  • Geographic location of collection site
The complete questionnaire, data collection forms, and data dictionary are available in Supplementary Material 1.
Sample and Data Tracking
1. All collected milk samples will be registered using participant-specific identification codes. 2. Sample tracking records will include:
  • Participant identification code
  • Collection date and time
  • Collection site
  • Storage location
  • Responsible researcher
3. Clinical, demographic, and environmental data will be linked to the corresponding sample identification code through the digital data collection platform.

4. All physical and digital records will be stored securely and accessed only by authorized study personnel
Ethics
Ethical Considerations
The research project associated with this protocol has been submitted for evaluation by the Research Ethics Committee of the University of Brasília and is currently awaiting approval prior to participant recruitment and study initiation.
All study procedures described in this protocol will be conducted in accordance with institutional ethical requirements and applicable national regulations for research involving human participants.
Informed Consent
Participation in this study will be voluntary. Eligible participants will receive verbal and written explanations regarding the study objectives, procedures, potential risks, and confidentiality measures.
Written informed consent will be obtained in duplicate before any study-related procedures are performed. One signed copy will be provided to the participant, and the second copy will be stored securely by the research team.
Confidentiality and Data Protection
Participant confidentiality will be maintained throughout all study phases. Personal identifiers will be replaced by participant-specific identification codes in all biological samples, databases, and study records.
Access to identifiable participant information will be restricted to authorized study personnel only. Secure storage and protection of identifiable participant data will be the responsibility of the study coordinator.
All physical and digital records containing personal information will be maintained in secure storage systems according to institutional and ethical requirements.
Risks and Benefits
Potential risks associated with study participation are minimal and may include mild discomfort during manual milk expression and possible concerns related to confidentiality of personal information.
Although participants may not receive direct individual benefits, the study may contribute to future research investigating environmental influences on lactation and maternal-child health.
Supplementary Material
Supplementary Material 1 – Questionnaire and Data Dictionary
This supplementary material contains the complete clinical, demographic, breastfeeding-related, and environmental questionnaire used in the study, including variable definitions, response formats, and coding instructions for standardized multicenter data collection.
Participant Identification
VariableDefinitionResponse FormatCoding Instructions
Participant identification codeUnique study code assigned to each participantTextUse recruitment-site prefix followed by sequential numbering (e.g., HSF01, ULBRA01, UFMA01)
Medical record numberHospital registration numberNumeric/TextRecord exactly as registered in hospital records
Collection siteParticipating maternity hospitalTextUse standardized institutional abbreviation
Collection date and timeDate and time of milk collectionDD/MM/YYYY HH:MMRecord immediately after collection
Table 1.
Participant identification variables, definitions, response formats, and coding instructions used for sample traceability and data management.
Maternal Demographic Data
VariableDefinitionResponse FormatCoding Instructions
Maternal ageAge at enrollmentNumeric (years)Record completed years
Maternal place of residenceCity of residenceTextRecord municipality name
Educational levelYears of formal education completedNumericRecord total completed years
Maternal weightWeight during hospitalizationNumeric (kg)Record most recent available value
Maternal heightHeight during hospitalizationNumeric (cm)Record from medical records when available
Table 2.
Maternal demographic variables collected through the standardized study questionnaire.


Prenatal and Clinical History
VariableDefinitionResponse FormatCoding Instructions
Prenatal care attendancePrenatal follow-up during pregnancyYes/NoCode Yes = 1; No = 0
Number of prenatal visitsTotal prenatal consultationsNumericRecord total number
Diabetes statusPresence of diabetes diagnosisYes/NoCode Yes = 1; No = 0
Diabetes typeType of diabetes, if applicableTextSpecify gestational, type 1, type 2, or other
Hypertension statusPresence of hypertension diagnosisYes/NoCode Yes = 1; No = 0
Hypertension typeType of hypertension, if applicableTextSpecify chronic, gestational, preeclampsia, or other
Anemia statusPresence of anemia diagnosisYes/NoCode Yes = 1; No = 0
Anemia typeType of anemia, if applicableTextSpecify when available
Erythrogram resultsHemoglobin, hematocrit, and erythrocyte countNumeric/TextRecord laboratory results when available
Table 3.
Prenatal and clinical history variables collected from maternal interview and medical records.
Obstetric and Neonatal Data
VariableDefinitionResponse FormatCoding Instructions
Mode of deliveryType of childbirthCategoricalVaginal = 1; Cesarean = 2
Gestational age at deliveryGestational age at birthNumeric (weeks)Record completed weeks
Multiple pregnancyTwin or higher-order pregnancyYes/NoCode Yes = 1; No = 0
Newborn birth weightBirth weight at deliveryNumeric (g)Record in grams
Breastfeeding and Milk Collection Data
VariableDefinitionResponse FormatCoding Instructions
Time since last breastfeeding sessionInterval between last feeding and milk collectionNumeric (minutes/hours)Record approximate interval
Breastfeeding typeInfant feeding patternCategoricalExclusive = 1; Mixed = 2; Supplemented = 3
Breast discomfort during collectionMaternal discomfort during expressionYes/NoCode Yes = 1; No = 0
Difficulties during milk expressionReported problems during collectionFree textDescribe briefly when applicable
Signs of breast infectionPresence of infection signs during inspectionYes/NoCode Yes = 1; No = 0
Nipple injuryPresence of nipple cracks or lesionsYes/NoCode Yes = 1; No = 0
Milk sample contaminationVisible blood or purulent secretionYes/NoCode Yes = 1; No = 0
Relevant observationsAdditional collection observationsFree textRecord relevant comments
Environmental and Meteorological Data
VariableDefinitionResponse FormatCoding Instructions
Ambient temperatureTemperature at collection siteNumeric (°C)Obtain from nearest meteorological source
Relative humidityHumidity at collection siteNumeric (%)Obtain from nearest meteorological source
Geographic locationCollection site locationTextRecord city and participating center
Collection dateDate used for future THI calculationDD/MM/YYYYMust match sample collection date
Table 6.
Environmental and meteorological variables collected for characterization of collection conditions and future Temperature-Humidity Index (THI) analysis.