Image-Based Morphometric Analysis of Human Milk Fat Globules Versus Laser Diffraction

Human milk fat globules (MFGs)' size characterization is key for evaluating milk quality and processing effects. Laser diffraction (LD) is widely used for particle size analysis but provides limited morphological information. This study applied image-based morphometric analysis (IBMA) to characterize MFGs' size and shape distributions in human milk and compared the results with LD measurements. Milk samples from 12 women delivering term and preterm infants were analyzed. LD was performed using a Mastersizer 3000 (Malvern Panalytical, Malvern, UK) and IBMA using a Morphologi 4 (Malvern Panalytical, Malvern, UK), acquiring 2D images at 20× magnification covering particle sizes from ~1.5 to 130 µm. IBMA classified MFGs as individual particles (IP) (HS circularity ≥ 0.920; circle equivalent diameter < 25 µm) or agglomerates (HS circularity < 0.920; solidity < 0.970), extracting descriptors including circularity, elongation, and solidity. IP predominated, while agglomerates represented ~15% of particles. Number-mean diameters (D[1,0]) were 4.91 µm (total), 4.36 µm (IP), and 8.00 µm (agglomerates). Volume-weighted particle diameters (D[4,3]) were 7.21 µm for IP and 14.02 µm for agglomerates. The highest level of agreement between methods was observed for IP D[4,3], although minor differences may be clinically relevant. IBMA and LD provide complementary information; however, IBMA uniquely enables the characterization of MFG structural organization, including the identification of agglomerates, which cannot be resolved by LD. This added level of structural detail may have important implications for understanding the digestibility of human milk, particularly in preterm populations.