Protocol for the collection, packaging, and transportation of preterm newborn stool samples

Costa, Jessica Santos Passos; Brandao, Heli Vieira; Amaral, Mara Viana Cardoso; Santos, Gabriela Cintra dos; Martins, Camilla da Cruz; Ramos, Michelle de Santana Xavier; Vieira, Tatiana de Oliveira; Benevides, Raquel Guimarães; Vieira, Graciete Oliveira

doi:10.1016/j.clinsp.2024.100531

Background: The newborn’s intestinal microbiota can vary with gestational age. Reliable analyses of stool samples require establishing the steps of collection, packaging, transportation, and storage.

Objective: To describe the development of a protocol and test an algorithm for the sequence of actions and procedures for the collection, packaging, transportation, and storage of stool samples from Preterm Newborn (PTNB) admitted to a Neonatal Intensive Care Unit (NICU).

Materials and methods: This is a descriptive, observational study linked to a non-randomized controlled clinical trial conducted in the NICU of two public hospitals covenanted to the Unified Health System of a large city in inland northeastern Brazil. The methodology adopted in its development followed the Brazilian Ministry of Health Preparation Guide recommendations.

Results: The flow of actions and procedures was consistent with scientific evidence. The sequence of work process steps to implement the proposed protocol was tested and resulted in a graphical representation of an algorithm compatible with the reality of Brazilian public hospitals.

Conclusion: Following the protocol steps with the description of the rules of conduct and recommendations regarding the collection, packaging, transportation, and storage of stool samples from PTNBs ensured the preservation and integrity of the bacterial DNA in the stools sample. The details of the recommendations will allow their reproducibility and improvement by professionals and researchers with similar study objects.

Trial registration: World Health Organization (WHO) under Universal Trial Number (UTN) code U1111–1266–2295, under register RBR-3mm7cs in the Brazilian Registry of Clinical Trials (REBEC).

Keywords:
Microbiota; Colostrum; Preterm newborn; Clinical protocols

Collection	At birth ‒ Fix the notice on the incubator, signaling to the team that the newborn was included in the research. With each collected stool, an “X” must be marked on the notice attached to the incubator with a permanent pen, indicating that collections have been performed. Definition of surgical drape cutout ‒ The tissue can be any brand, as long as it is fenestrated and sterile. Surgical tissue is sold in different sizes; however, the specifically recommended cutout must be made by the team of researchers and adapted to the size of the diaper used in the NICU routine. Thus, for the dimension tests, the researchers will require using personal protective equipment (cap, glove, mask, and apron), besides metal scissors duly disinfected with 70 % alcohol. The cutouts must be stored in airtight-sealed ZipLock plastic bags (disinfected with 70 % alcohol) to avoid contamination. Recommended field dimensions – The tissue should be cut 15 cm long and 5 cm wide. Although this protocol recommends specific dimensions, the cutout can be adapted to the size of the diaper used in the sector. Care when placing the surgical drape ‒ Recommended sizes should be followed to prevent the tissue from sticking out of the diaper or that it is too small, not ensuring the collection of the entire sample. Another recommendation is that the side of the tissue in contact with the newborn should be the toweled one (since the fabric has two sides, plastic and toweled). Spatulas – A sterile plastic Ayre spatula individually packaged and opened only at the time of use is recommended. Microtubes ‒ Sterile Eppendorf centrifuge tube, 1.5 mL. Rack box for tubes ‒ A rack box to store the microtube with a capacity of 100 cryogenic tubes from 1.5 mL to 2.0 mL with a hinged lid is recommended. Collection time ‒ After defecation, the collection must occur within a maximum of 3 h (following the flow of the hospital) so as not to occur total absorption of the sample by the diaper. Personal protective equipment (PPE) – Only professionals wearing PPE (cap, gloves, mask and hospital gown) should change diapers to ensure the quality of the material collected. Collection ‒ The diaper must be opened on a clean surface properly disinfected with 70 % alcohol (such as a counter for example). The rack containing the microtubes and the Ayre spatula must be opened similarly. Then, the fecal material is collected and deposited in the microtube. The tube must be sealed and sanitized (70 % alcohol), encoded (with a permanent pen), and sent to the domestic freezer (253,15k) within 10 min. Research Box – The neonatal sector should have a plastic box with a lid 25.8 cm long, 17.8 cm wide, and 8.5 cm high to store the material for the fecal collection described above.
Packaging	Freezer ‒ A (vertical or horizontal) freezer with a freezing capacity of up to 253,15k should stand next to the neonatal unit to store the samples (for a maximum of 12 h). Thermometer ‒ The freezer temperature must be controlled using a digital thermometer with maximum and minimum temperatures with an external sensor and alarm. Rack box for tubes – A rack box for microtube storage inside the freezer should be available to prevent the tubes from turning over.
Transportation	Thermal box ‒ The transportation of the (frozen) tube from the hospital freezer to the UEFS ultra-freezer must occur in an isothermal, hygienic, and waterproof container, with a digital thermometer with an extension cord attached to the box to control the temperature of the transported material, thus preserving temperature from 263,15k to 253,15k. 5–6-liter plastic cooler 20.8 cm long, 21.3 cm wide, and 28.3 cm high should be used. Gelox – Hard reusable ice (gelox) was used for transport to preserve the cooling of stool samples at low temperatures, thus avoiding sample. Gelox was kept in a freezer for approximately 30 h before being used for transportation. Two 400 mL, 17 cm long, 10 cm wide, and 2.7 cm high gelox units were used to transport the samples. Rack box for tubes – A rack box for storing the microtubes inside the thermal box should be available to prevent the tube from turning over. PPE should always be used for handling the tube.
Storage	Ultra-freezer ‒ Permanent freezing should be realized in an ultra-freezer at 193,15k. These precautions preserve the genetic material. Rack box for tubes – A rack box for storing the microtubes inside the ultra-freezer should be available to prevent the tubes from turning over. PPE should always be used when manipulating the Eppendorf tube.