Point-of-care human milk concentration by passive osmosis: comprehensive analysis of fresh human milk samples

Preterm infants require nutrient-rich human milk (HM) for optimal growth, but often need supplementation due to high nutrient needs and limited volume tolerance. Current methods involve fortifying HM with bovine milk-derived fortifiers, which can have negative consequences. This study explores a novel, point-of-care approach: passive osmotic concentration. This method uses a single-use HMC device to selectively remove water from HM, increasing the concentration of nutrients and bioactive components without heat or pressure damage. Prior research demonstrated the efficacy of this device on previously frozen HM, but this study aimed to assess its impact on fresh HM, focusing on the preservation of sensitive components like living cells and enzymes. The hypothesis was that passive osmotic concentration of fresh, never-frozen HM would increase nutrient and bioactive content without damaging sensitive components.

The literature extensively highlights the benefits of mother's own milk (MOM) for preterm infants, demonstrating dose-dependent reductions in morbidity and mortality. MOM improves various aspects of infant development, including brain, vision, microbiome, and immune system development, and reduces the risk of conditions such as bronchopulmonary dysplasia (BPD), retinopathy of prematurity (ROP), necrotizing enterocolitis (NEC), and neonatal sepsis. However, the volume of MOM is often insufficient to meet the high nutrient demands of preterm infants. Current practice involves fortifying MOM or donor human milk (DHM) with bovine milk-derived fortifiers, or less commonly, DHM-derived fortifiers. However, these fortifiers can negatively impact the beneficial components of HM and may be associated with increased risks of certain morbidities, including hypoglycemia. The displacement of MOM by fortifiers further emphasizes the need for methods to increase the nutrient density of MOM.

This study received ethical approval from the New England IRB. Fresh HM samples (60-140 mL) were collected from volunteer donors within 20 hours of expression and stored at 4°C. Samples were gently swirled, and aliquots were taken for baseline analysis before concentration. Passive osmotic concentration was performed using the HMC device, involving incubation with 75 mL of HM at 4°C for 3 hours. Post-concentration, HM volume was measured, and samples were analyzed for various components. Macronutrient analysis (energy, fat, carbohydrates, protein) used a Miris Human Milk Analyzer. Other components analyzed included lactose, sodium, IgA, lactoferrin, various enzymes (lysozyme, PAF acetylhydrolase, catalase, glutathione peroxidase, and bile salt-stimulated lipase (BSSL)), 19 human milk oligosaccharides (HMOs), small molecules (choline, phosphocholine, betaine, phosphatidylcholine, sphingomyelin), fatty acids, pH, and osmolality. Cell viability was assessed using trypan blue exclusion. Statistical analysis used paired t-tests to compare pre- and post-concentration values. Outliers were identified using a robust fit by Huber M-Estimation.

Passive osmotic concentration reduced HM volume by an average of 16.3% ± 3.8%. Ten of the 41 HM components analyzed did not show a significant difference between pre- and post-concentration. Twenty-three components increased within the expected range based on volume reduction, while six increased more than expected, two increased less than expected, and none decreased significantly. The increase in BSSL activity was statistically significant (21.5% ± 39%, p < 0.05). All macronutrients analyzed showed a significant increase, as did sodium, protein, lactose, lactoferrin, total fatty acids, and most small molecules (except phosphocholine). Fourteen of the nineteen HMOs showed significant increases. The mean pH was not significantly different, but osmolality increased significantly (33%). Cell viability was not significantly different between concentrated and unconcentrated samples.

The findings support the efficacy of passive osmotic concentration for enriching fresh HM. The preservation of pH and osmolality within acceptable neonatal feeding parameters indicates good tolerance. The significant increase in a wide range of HM components, including nutrients, enzymes, and HMOs, suggests that this method could offer a valuable alternative to fortifying HM. This point-of-care method avoids heat or pressure damage, preserving delicate components potentially compromised by fortification. The empowerment of mothers in providing enriched MOM without fortifiers may also have positive psychological benefits. Further, the process may enhance the efficiency of NICU feeding preparation workflows.

Passive osmotic concentration of fresh HM using the HMC device effectively concentrates HM components while maintaining acceptable pH and osmolality for neonatal feeding. This method offers a promising alternative to fortification, potentially preserving the integrity of HM and improving preterm infant health outcomes. Further research is needed to validate these findings with larger sample sizes, investigate the effects on other beneficial components, and assess the clinical impact on infant growth and development.

The study's limitations include the relatively small sample size, which was influenced by using fresh HM samples and relying on volunteer donors. The variable storage times prior to analysis could have affected cell viability. The accuracy of the Miris HMA with concentrated samples was not validated; however, the similarity between results of protein measurement using BCA assay and the infrared analysis using the Miris HMA suggests the system is reasonably accurate. The variability in some measurements (e.g., HMOs and fatty acids) might need to be addressed in future studies through optimization of analytical methods. The study also requires confirmation of non-significant changes in pH.