The basis of this method was to use low speed centrifugation to concentrate the fat globules into a cream layer, followed by the adjustment of the underlying skim milk and then resuspension of the cream layer. In order to accurately adjust the fat content of whole milk to a specified fat amount, an equation was developed for calculating the amount of skim milk to be either removed or added. Given the relatively low RCF used for this procedure it is likely that some fat would remain in the skim and it was therefore necessary to account for the amount of fat remaining in the skim when performing this calculation.
The samples chosen for the proof of concept studies had relatively low variation of 19.3% between samples, which was reduced to 2.6% by employing the method described. For these studies, an assumed value skim milk fat content of 17 g/l was used, which was derived from the average of 72 skim milk samples centrifuged at optimal RCF. Measurement of the fat content of the skim milk would decrease the variation between samples, but was not considered to be clinically important for either the milk bank or NICU. Nonetheless, assuming a skim milk fat content did result in a large decrease in the variability of fat between samples and contributed to less than 2.2% error in the final fat content of the reconstituted milk.
The effect that centrifugation and resuspension of the milk had on the fat globule size distribution was also investigated. Low temperatures are recommended for preventing microbial growth in human milk , however it is not known how low temperatures affect the solidity of the cream layer and the ease of which the fat globules can be resuspended. Centrifuging at temperatures between 4°C and 10°C did not appear to affect the resuspension process. Subsequently, later centrifugation procedures were performed at 4°C to minimize any microbial growth. Fat globule size also may be important to infant gastric emptying and ability to absorb fat from the gut .
Centrifugation results in concentration of the fat globules into a dense cream layer at the top of the vessel, leading to the possibility of coalescence occurring and in turn altering the milk fat globule distribution. To test this hypothesis, samples of fresh and frozen milk, before and after manipulation were analyzed using laser diffraction particle sizing, a technology that has been succesfully used for studying the particle distribution of bovine milk . Results were similar to that of previous findings for human milk [26, 27] which employed more classical techniques such as coulter counters. Centrifugation and resuspension of milk did not alter the fat globule distribution, suggesting that coalescence does not occur under these conditions. The effect of centrifugation on milk proteins would be insignificant because much higher centrifugal forces are required for casein sedimentation  and the separation of small molecules (eg. lactose, oligosaccharides, peptides, hormones) from complex biological solutions cannot be achieved using centrifugation alone.
The method presented here has the potential for incorporation with current human milk banking protocols. While this study used a spectrophotometric assay for quantifying fat content of human milk, it was also demonstrated that the results from the simpler and quicker creamotocrit method correlated well with those derived from the more advanced spectrophotometric method. It is unlikely that most milk banks or NICUs would have access to a spectrophotometer, and the creamotocrit is an accurate and cost-efficient alternative for determining fat content of human milk. Using a creamotocrit it would be possible to determine milk fat content in the NICU or milk bank, standardize the fat content of the milk prior to pasteurization, followed by appropriate fortification.
The validation of this method involved using assumed values of protein and lactose. Human milk composition is challenging to quantify outside the laboratory environment. Consequently, the concentrations of nutritional components in human milk are often assumed, contributing to inaccurate nutrition of the preterm infant. In recent years, human milk analysis equipment such as the MilkoScan (FOSS International) have become available that simultaneously determines protein, lactose and fat content in human milk. The equation presented here can be expanded to include these components in relation to total energy of the milk. Ideally all the variables including fat, protein, lactose and specified energy content can be inputted into the expanded equation and in combination with current fortification regimes, a standardized fortified human milk of known energy and protein content can be prepared that precisely meets the infant's nutritional recommendations. The method is also versatile, allowing for batch processing by employing a large capacity centrifuge or alternatively, for prescriptive use for standardizing the fat content of donor milk or mother's own milk to meet the needs of a particular infant. Finally, the simplicity of this method ensures that with minimal training, non-laboratory trained staff can utilize it to standardize the energy content of breast milk for use in the NICU.