Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Hepatology and Nutrition
Creamatocrit Analysis of Human Milk Overestimates Fat and Energy Content When Compared to a Human Milk Analyzer Using Mid-infrared Spectroscopy
O’Neill, Edward F.; Radmacher, Paula G.; Sparks, Blake; Adamkin, David H.
Neonatal Nutrition Research Laboratory, University of Louisville, Louisville, Kentucky.
Address correspondence and reprint requests to Paula G. Radmacher, PhD, Neonatal Nutrition Research Laboratory, 511 S Floyd St, Room 107, University of Louisville, Louisville, KY 40292 (e-mail: firstname.lastname@example.org).
Received 16 August, 2012
Accepted 17 December, 2012
The authors report no conflicts of interest.
Background and Objective: Human milk (HM) is the preferred feeding for human infants but may be inadequate to support the rapid growth of the very-low-birth-weight infant. The creamatocrit (CMCT) has been widely used to guide health care professionals as they analyze HM fortification; however, the CMCT method is based on an equation using assumptions for protein and carbohydrate with fat as the only measured variable. The aim of the present study was to test the hypothesis that a human milk analyzer (HMA) would provide more accurate data for fat and energy content than analysis by CMCT.
Methods: Fifty-one well-mixed samples of previously frozen expressed HM were obtained after thawing. Previously assayed “control” milk samples were thawed and also run with unknowns. All milk samples were prewarmed at 40°C and then analyzed by both CMCT and HMA. CMCT fat results were substituted in the CMCT equation to reach a value for energy (kcal/oz). Fat results from HMA were entered into a computer model to reach a value for energy (kcal/oz). Fat and energy results were compared by paired t test with statistical significance set at P < 0.05. An additional 10 samples were analyzed locally by both methods and then sent to a certified laboratory for quantitative analysis. Results for fat and energy were analyzed by 1-way analysis of variance with statistical significance set at P < 0.05.
Results: Mean fat content by CMCT (5.8 ± 1.9 g/dL) was significantly higher than by HMA (3.2 ± 1.1 g/dL, P < 0.001). Mean energy by CMCT (21.8 ± 3.4 kcal/oz) was also significantly higher than by HMA (17.1 ± 2.9, P < 0.001). Comparison of biochemical analysis with HMA of the subset of milk samples showed no statistical difference for fat and energy, whereas CMCT was significantly higher than for both fat (P < 0.001) and energy (P = 0.002).
Conclusions: The CMCT method appears to overestimate fat and energy content of HM samples when compared with HMA and biochemical methods.
Human milk (HM) is the preferred method of enteral feeding for all infants, including the very-low-birth-weight (VLBW) infant (1–12). HM provides preterm infants important protection against infection (6,13,14) and necrotizing enterocolitis (2,7,15); however, HM may have an insufficient concentration of protein, energy, and minerals to support the rapid growth of the VLBW infant (16–21). Consequently, most HM fed to VLBW infants is fortified with either powder or liquid products. This fortification is based on general assumptions of energy and protein content of the milk (22,23). One method to estimate the energy content of an individual milk sample was first published by Lucas et al in 1978 called the “creamatocrit” (24) and also reported by Lemons et al (25). Much like the familiar hematocrit, this method uses the percentage of cream to total volume, which is then substituted into an equation to yield energy (kcal/oz). The equation is based on assumptions of constant protein and carbohydrate content, with fat being the only variable nutrient. The contributions of protein and carbohydrate are assumed based on values from the literature (26–28). Meier (29) and Valentine (30) showed that the creamatocrit (CMCT) could be used by nursing staff in the clinical setting to take advantage of the natural variations within a milk expression to engineer the milk a mother provided for her infant to contain a higher fat content and, consequently, higher energy. Mothers, too, could perform the measurement to participate in “lactoengineering” their own milk.
Although inexpensive and easy to do, the CMCT-derived energy calculation is directly related to fat content only; it cannot provide information about nonfat nutrients. This limitation may be less important for the term newborn but could have important ramifications for the VLBW infant, whose mother's milk changes significantly during the first weeks of lactation and who has increased growth requirements (17,31–35).
Infrared (IR) spectroscopy has long been used in the dairy industry to analyze bovine milk quality (36). This technology has been adapted and calibrated to measure fat, protein, and lactose in HM (37), and there are now multiple devices that use mid- or near-IR wavelengths for analysis (37–40). Concentrations of these macronutrients (g/dL) are then used to calculate energy content (kcal/oz). The present study compares fat and energy results in discrete HM samples by CMCT and human milk analyzer (HMA).
Aliquots of HM (n = 51) left over from daily preparation were obtained. All had been previously frozen and thawed on the same day as analyzed. No mother's milk was sampled more than once. All milk was prewarmed to 40°C for 10 minutes before analysis and was analyzed within minutes.
CMCT was performed in triplicate, according to the method of Lemons et al (25). Well-mixed milk samples were drawn into a standard capillary tube, sealed at 1 end with clay, then centrifuged for 15 minutes at 3500 rpm. The cream layer and the total volume were each measured to the nearest 1 mm; the percentage of the total volume of the sample represented by the lipid layer was calculated. The CMCT (percent fat) was then substituted into the following equation: kcal/dL = 38.7 + (5.9 × CMCT [%])/3.33 to estimate the energy content (kcal/oz).
The HMA (Calais Human Milk Analyzer, Metron Instruments, Solon, OH) uses mid-IR spectroscopy (36). IR energy with wavelength 3 to 10 μm radiates from an incandescent source lamp and passes through 6 filters mounted on a disk. This allows readings at 6 wavelengths (3 for fat, 1 each for protein, lactose, and solids). Absorption at each wavelength is most sensitive to a particular component in the sample to determine the amount of protein, fat, and carbohydrate based on the absorption of IR energy by specific chemical bonds: CH groups in fatty acid chains of fat molecules (3.48 μm), carbonyl groups in ester linkages of fat molecules (5.723 μm), peptide linkages between amino acids of protein molecules (6.465 μm), and OH groups in lactose molecules (9.610 μm). A detector converts the IR energy into an electrical signal processed by a computer to generate a value for each macronutrient.
The HMA device has been adapted from use in the dairy industry and is specifically calibrated to HM with a series of 6 pooled HM samples with varying concentrations of each compound. Nutrient content of the calibrators was assayed by a certified reference laboratory (DQCI, Mounds View, MN) by the following methods: protein (Kjeldahl) (41), carbohydrate (high-performance liquid chromatography) (42), and fat (Mojonnier) (43). Those values are then entered into a computer model, which was used to derive the results of subsequent samples. The energy content is a mathematical function, multiplying the protein and carbohydrates by 4 kcal/g and the fat by 9 kcal/g. A control milk was included in daily runs as an additional check on instrument performance. This is a pooled HM sample that has been analyzed similarly to the calibration samples and comes with an established range of expected results for each compound. Additionally, 10 random samples of milk were analyzed locally by both methods and then sent to the reference laboratory for analysis.
Descriptive statistics are provided. The results of fat and energy analysis were compared by paired t test and 1-way analysis of variance. Statistical significance was set at P < 0.05.
Table 1 displays the mean ± SD values and ranges for fat content and energy by the 2 methods for both discrete samples and controls. CMCT results exceeded HMA in all analyses. Overall, mean fat content of HM samples was, on average, 80% higher by the CMCT method compared with the HMA. Mean energy content of those samples was, on average, 26% higher by the CMCT method when compared with the HMA. When comparing fat content results from the control milks, the mean CMCT method exceeded the HMA results by 46% and the energy content by 16%. Three strata of CMCT values were created posthoc and the CMCT was compared with the HMA value (Fig. 1). There was a significant difference between the ratio for <4% stratum and the >6% stratum for both fat and energy. The mean protein content in the HM samples, which cannot be determined from the CMCT method, was 1.42 ± 0.41 g/dL as measured by the HMA.
Table 2 displays results for 10 random milk samples, which were analyzed locally by HMA and CMCT and then sent to the certified reference laboratory for analysis. Laboratory results for fat and energy showed no statistically significant difference from HMA. CMCT results for fat were significantly higher than laboratory or HMA (P < 0.001) and, on average, 74% to 76% higher. Energy results from the CMCT calculation were significantly higher than laboratory or HMA (P = 0.002) and, on average, 23% to 24% higher.
Extrauterine growth failure is common in the ELBW (extremely-low-birth-weight; <1000 g birth weight) infant, and may be associated with long-term growth failure and neurodevelopmental deficit (4,44–46). HM is often not sufficient on its own to provide the nutrients needed for adequate growth by the ELBW infant, and therefore must be fortified to increase the energy, protein, and minerals. HM is routinely fortified with the assumption it has 20 kcal/oz of energy and 2.1 g/100 kcal of protein. As our study shows, this is not always the case. The macronutrient content is known to vary from mother to mother, within an expression period, and from week to week in lactation (26–28). Individualized fortification for the extremely small premature infant based on an accurate assessment of macronutrient content in HM allows for standardized intakes of macronutrients.
Point of care analysis of HM has been largely limited to a method called the CMCT, developed in the late 1970s by Lucas et al (24) and subsequently used by others in clinical settings (29,30,47,48). This is a simple and inexpensive tool to estimate energy content based on measurement of the percent fat in an HM sample, but assuming constant amounts of protein and lactose. The lipid component in HM has the highest degree of variability within a single feeding/expression among the major macronutrients, and therefore, is the major component in the milk energy calculation by the CMCT method. When planning fortification of milk for the VLBW infants, the provision of adequate protein is one of the most important considerations. The CMCT method cannot provide this information.
We compared fat and energy content as measured by the CMCT and HMA on discrete HM samples and “control” milks. The CMCT method is an extremely gross measurement based on the gravimetric differences between lipid and nonlipid components in the milk sample and visual quantification. The HMA employs measurements that are specific to the chemical bonds of the lipid (as well as other components) and are independent of human estimation. We demonstrated an increasing deviation between the methods for fat and energy as the CMCT increased, sometimes as much as 2-fold. This resulted in much higher estimates of energy content by CMCT when compared to the HMA and laboratory-based results. The use of an external control milk in our protocol checked proper instrument performance. The laboratory analysis confirmed the accuracy and reliability of the HMA results.
Although it was not the purpose of the present study to include measurement of other nutrients, the HMA analyzer provides information about protein content, which is often the limiting nutrient in postnatal growth for this population. Having this information in real time contributes to the ongoing nutritional management of these infants.
The mid-infrared HMA is an excellent option for real-time analysis of the lipid, protein, and carbohydrate content in HM. It is easy to use, and has a process for calibration and instrument reliability. It provides information that can be used to individualize HM fortification for infants at high risk for extrauterine growth failure. The present study demonstrated its greater accuracy versus the CMCT, which overestimates fat and energy content of HM.
1. Whitfield JM, Hendrikson H. Prevention of protein deprivation in the extremely low birth weight infant: a nutritional emergency. Proc Bayl Univ Med Cent
2. Sisk PM, Lovelady CA, Dillard RG, et al. Early human milk feeding is associated with a lower risk of necrotizing enterocolitis in very low birth weight infants. J Perinatol
3. Simmer K. Aggressive nutrition for preterm infants--benefits and risks. Early Hum Dev
4. Morley R, Fewtrell MS, Abbott RA, et al. Neurodevelopment in children born small for gestational age: a randomized trial of nutrient-enriched versus standard formula and comparison with a reference breastfed group. Pediatrics
5. Bertino E, Giuliani F, Occhi L, et al. Benefits of donor human milk for preterm infants: current evidence. Early Hum Dev
2009; 85 (10 suppl):S9–S10.
6. Lepage P, Van de Perre P. The immune system of breast milk: antimicrobial and anti-inflammatory properties. Adv Exp Med Biol
7. Maayan-Metzger A, Avivi S, Schushan-Eisen I, et al. Human milk versus formula feeding among preterm infants: short-term outcomes. Am J Perinatol
8. McGuire W, Anthony MY. Donor human milk versus formula for preventing necrotising enterocolitis in preterm infants: systematic review.[see comment]. Arch Dis Child Fetal Neonatal Ed
9. Schanler RJ. The use of human milk for premature infants. Pediatr Clin North Am
10. Wight NE. Donor human milk for preterm infants. J Perinatol
11. Simmer K. Agressive nutrition for preterm infants—benefits and risks. Early Hum Dev
12. Neville MC, Anderson SM, MacManaman JL, et al. Lactation and neonatal nutrition: defining and refining the critical questions. J Mammary Gland Biol Neoplasia
13. Jantscher-Krenn E, Bode L. Human milk oligosaccharides and their potential benefits for the breast-fed neonate. Minerva Pediatr
14. Blaymore Bier JA, Oliver T, Ferguson A, et al. Human milk reduces outpatient upper respiratory symptoms in premature infants during their first year of life. J Perinatol
15. Kosloske AM. Breastmilk decreases the risk of neonatal necrotizing enterocolitis. Adv Nutr Res
16. Heird WC. Determination of nutritional requirements in preterm infants, with special reference to ’catch-up’ growth. Semin Neonatol
17. Wilson DC, McClure G. Energy requirements in sick preterm babies. Acta Paediatr Suppl
18. Schanler RJ, Shulman RJ, Lau C. Feeding strategies for premature infants: beneficial outcomes of feeding fortified human milk versus preterm formula. Pediatrics
19. Darby MK, Loughead JL. Neonatal nutritional requirements and formula composition: a review. J Obstet Gynecol Neonatal Nurs
20. Canadian Medical Association. Nutrient needs and feeding of premature infants. Nutrition Committee, Canadian Paediatric Society. CMAJ
21. Rose J, Gibbons K, Carlson SE, et al. Nutrient needs of the preterm infant. Nutr Clin Pract
22. Atkinson SA, Bryan MH, Anderson GH. Human milk: differences in nitrogen concentration in milk from mothers of term and premature infants. J Pediatr
23. Neville MC, Keller RP, Seacat J, et al. Studies on human lactation. I. Within-feed and between-breast variation in selected components of human milk. Am J Clin Nutr
24. Lucas A, Gibbs JA, Lyster RL, et al. Creamatocrit: simple clinical technique for estimating fat concentration and energy value of human milk. Br Med J
25. Lemons JA, Schreiner RL, Gresham EL. Simple method for determining the caloric and fat content of human milk. Pediatrics
26. Hibberd CM, Brooke OG, Carter ND, et al. Variation in the composition of breast milk during the first 5 weeks of lactation: implications for the feeding of preterm infants. Arch Dis Child
27. Lemons JA, Moye L, Hall D, et al. Differences in the composition of preterm and term human milk during early lactation. Pediatr Res
28. Weber A, Loui A, Jochum F, et al. Breast milk from mothers of very low birthweight infants: variability in fat and protein content. Acta Paediatr
29. Meier PP, Engstrom JL, Murtaugh MA, et al. Mothers’ milk feedings in the neonatal intensive care unit: accuracy of the creamatocrit technique. J Perinatol
30. Valentine C, Hurst NM, Schanler R. Hindmilk improves weight gain in low-birth-weight infants fed human milk. J Pediatr Gastroenterol Nutr
31. Hay WW Jr. Assessing the effect of disease on nutrition of the preterm infant. Clin Biochem
32. Krugman SD, Dubowitz H. Failure to thrive. Am Fam Physician
33. Anderson DM. Feeding the ill or preterm infant. Neonatal Netw
34. Huysman WA, de Ridder M, de Bruin NC, et al. Growth and body composition in preterm infants with bronchopulmonary dysplasia. Arch Dis Child Fetal Neonatal Ed
35. Adamkin DH. Issues in the nutritional support of the ventilated baby. Clin Perinatol
36. Official Method 972.16. Fat, lactose, protein, and solids in milk. Mid-infrared spectroscopic method. In: Horitz W, ed. Official Methods of Analysis
. 17th ed. Gaithersburg, MD: AOAC International; 2003.
37. Menjo A, Mizuno K, Murase M, et al. Bedside analysis of human milk for adjustable nutrition strategy. Acta Paediatr
38. Corvaglia L, Battistini B, Paoletti V, et al. Near-infrared reflectance analysis to evaluate the nitrogen and fat content of human milk in neonatal intensive care units. Arch Dis Child Fetal Neonatal Ed
39. Michaelsen KF, Pedersen SB, Skafte L, et al. Infrared analysis for determining macronutrients in human milk. J Pediatr Gastroenterol Nutr
40. Casidio YS, Williams TM, Lai CT, et al. Evaluation of a mid-infrared analyzer for the determination of the macronutrient composition of human milk. J Hum Lact
41. Protein/nitrogen tests. In: Wehr M, Frank JF, eds. Standard Methods for the Examination of Dairy Products
. 17th ed. Washington, DC: American Public Health Association; 2004:480–509.
42. Lactose and galactose tests. In: Wehr M, Frank JF, eds. Standard Methods for the Examination of Dairy Products
, 17th ed. Washington, DC: American Public Health Association; 2004:434–9.
43. Fat determination methods. In: Wehr M, Frank JF, eds. Standard Methods for the Examination of Dairy Products
, 17th ed. Washington, DC: American Public Health Association; 2004:408–33.
44. Morley R, Lucas A. Nutrition and cognitive development. Br Med Bull
45. Clark RH, Thomas P, Peabody J. Extrauterine growth restriction remains a serious problem in prematurely born neonates. Pediatrics
46. Dusick AM, Poindexter BB, Ehrenkranz RA, et al. Growth failure in the preterm infant: can we catch up? Semin Perinatol
47. Wang CD, Chu PS, Mellen BG, et al. Creamatocrit and the nutrient composition of human milk. J Perinatol
48. Meier PP, Engstrom JL, Zuleger JL, et al. Accuracy of a user-friendly centrifuge for measuring creamatocrits on mothers’ milk in the clinical setting. Breastfeed Med
creamatocrit; human milk; mid-infrared spectroscopy
© 2013 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,
What does "Remember me" mean?
By checking this box, you'll stay logged in until you logout. You'll get easier access to your articles, collections,
media, and all your other content, even if you close your browser or shut down your
To protect your most sensitive data and activities (like changing your password),
we'll ask you to re-enter your password when you access these services.
What if I'm on a computer that I share with others?
If you're using a public computer or you share this computer with others, we recommend
that you uncheck the "Remember me" box.
Highlight selected keywords in the article text.
Data is temporarily unavailable. Please try again soon.
Readers Of this Article Also Read