# Human Milk Sampling Protocols: Is the Mean Macronutrient Composition of Pre- and Post-Feed Samples the Average of One Entire Feed?

## TL;DR

This study examines how well different milk sampling methods estimate the nutrient content of a full breastfeed, finding that some methods are more accurate than others.

## Contribution

The study evaluates the accuracy of three human milk sub-sampling protocols for estimating macronutrient concentrations in a complete feed.

## Key findings

- The mean of pre- and post-feed samples is suitable for high-fat milk when measurement errors are larger than the bias.
- Pre- or post-feed samples alone are not accurate for estimating fat concentration in a complete feed.
- One sample during a breastfeed is sufficient to estimate protein and carbohydrate concentrations.

## Abstract

Human milk sub-sampling protocols are used in lactation research to estimate milk composition, while minimizing interference with normal breastfeeding. However, macronutrient concentrations in human milk can be highly variable, and the accuracy of sub-sampling protocols for a single breastfeed is currently unknown.

We investigated the accuracy of three milk sub-sampling protocols for estimating the macronutrient concentrations of a complete feed: the mean of pre- and post-feed samples, pre-feed samples only, and post-feed samples only.

In this observational study, macronutrient concentrations from each sub-sampling protocol were compared to the volume-weighted average of the complete pumping session, based on foremilk, bulk milk, and hindmilk samples from 15 mothers. Macronutrient concentrations of each milk fraction were measured with a human milk analyzer. Additionally, correlations between macronutrient concentrations and lactation characteristics were studied.

Macronutrient concentrations from each sub-sampling protocol were strongly correlated with the volume-weighted average reference concentrations. Significant biases were found for the fat concentration (between -1.3 and +1.9 g/dl, depending on the protocol), but not for protein and carbohydrate concentrations.

For the fat concentration, none of the three sub-sampling protocols was accurate. The mean of pre- and post-feed samples could, however, be used for high-fat milk samples where measurement errors exceed the bias (+0.3 g/dl). Pre-feed or post-feed samples only should not be used to estimate the fat concentration of a complete feed. For proteins and carbohydrates, one milk sample taken at any moment in a breastfeed is sufficient to represent the concentration in the complete feed.