Leukocyte Populations in Human Preterm and Term Breast Milk Identified by Multicolour Flow Cytometry

Abstract

Background: Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood.

Methods: Sixty mothers of extremely preterm (<28 weeks gestational age), very preterm (28-31 wk), and moderately preterm (32-36 wk), as well as term (37-41 wk) infants were recruited. Colostrum (d2-5), transitional (d8-12) and mature milk (d26-30) samples were collected, cells isolated, and leukocyte subsets analysed using flow cytometry.

Results: The major CD45+ leukocyte populations circulating in blood were also detectable in breast milk but at different frequencies. Progression of lactation was associated with decreasing CD45+ leukocyte concentration, as well as increases in the relative frequencies of neutrophils and immature granulocytes, and decreases in the relative frequencies of eosinophils, myeloid and B cell precursors, and CD16- monocytes. No differences were observed between preterm and term breast milk in leukocyte concentration, though minor differences between preterm groups in some leukocyte frequencies were observed.

Conclusions: Flow cytometry is a useful tool to identify and quantify leukocyte subsets in breast milk. The stage of lactation is associated with major changes in milk leukocyte composition in this population. Fresh preterm breast milk is not deficient in leukocytes, but shorter gestation may be associated with minor differences in leukocyte subset frequencies in preterm compared to term breast milk.

Fig 1. Overlay of gating strategy applied to peripheral adult blood (red) and to breast milk (blue), based on the method of Faucher et al.

The sequences of gates leading to each panel are shown above each panel. Panel 1: CD45 positive cells were gated as shown in A. Panel 2: CD45+ cells identified in Panel 1 were separated based on CD16 staining and side scatter properties, including a C16+/SSClow gate (B), and two overlapping gates of CD16- cells (C and D), and the CD16+ neutrophil population were identified. Panel 3: CD45+/CD16+/SSClow cells identified in Panel 2 gate B were separated into cytotoxic T and NK lymphocytes and CD16+ monocytes based on CD2/CD294 and CD36 staining properties. Panel 4: CD45+/CD16-/SSClow–intermediate cells (gate C) were separated based on CD2/CD294 positive (gate E) or negative (gate F) populations. Panel 5: From Panel 4, CD2 and/or CD294 positive cells (gate E) were gated into non-cytotoxic T cells or basophils using side scatter and CD45 staining properties. Panel 6: From Panel 4, CD2/CD294- cells (gate F) were gated into CD19+/CD36- cells (gate H) or CD19- cells (gate G). Panel 7a: Cells gated in G in Panel 6 with CD45high and CD36+ were identified as CD16 negative monocytes. Panel 7b: From Panel 6 gate G, CD45low cells with low side scatter were identified as myeloid precursor cells. Panel 8: From Panel 6, CD19 positive cells (gate H) were discriminated into B cells or B cell precursors based on CD45 staining though both populations displayed low side scatter properties. Panel 9: From Panel 2, CD16- cells with intermediate to high side scatter (gate D) and CD45low staining were identified as immature granulocytes, and those with intermediate to high CD45 staining properties and high side scatter were separated into gate I. Panel 10: From Panel 9, cells in gate I were identified by positive CD2/CD294 staining as eosinophils.

Fig 2. Comparison of the total cell concentrations obtained from milk samples with different methods.

Figure shows scatterplot of total cell counts obtained through microscopy on x-axis, compared with total CD45+ leukocyte concentrations obtained with flow cytometry on the y-axis. All data are shown on a log scale.

Fig 3. Leukocyte concentrations in breast milk samples.

Boxplots showing the total concentration of leukocytes (cells/mL) on a log scale in colostrum (C, n = 41), transitional milk (TM, n = 47), and mature milk (MM, n = 46), in extremely preterm (white), very preterm (light grey), moderately preterm (dark grey) and term (striped) donors.

Fig 4. Changes to the composition of breast milk through the first month of lactation.

Line shows the median value for that milk sampling time point. Data show A) concentrations of total CD45+ cells in colostrum (C), transitional milk (TM) or mature milk (MM); B-L) frequencies of leukocyte subsets in colostrum (C), transitional milk (TM) and mature milk (MM) from all donors. *p<0.05 in post-test comparing different stages of lactation; for each comparison, number of symbols indicates p-value (*p<0.05, **p<0.01, ***p<0.001).