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Updated: Aug 31, 2025
Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry
Published on: October 14, 2021
Isolation and characterization of uterine leukocytes collected using a uterine swab technique
Parveen Parasar1, Matthew Bernard2, Soo Hyun Ahn1
1Department of Pathobiology & Diagnostic Investigation, Children's Hospital Boston, East Lansing, Michigan, USA.
Insights
Collecting uterine leukocytes via surgical gauze post-C-section is a less invasive method. This technique enriches for uterine natural killer (uNK) cells, offering a valuable alternative for pregnancy immune studies.
Area of Science:
- Reproductive Immunology
- Maternal-Fetal Interface Immunology
- Cellular Immunology
Background:
- Studying immune cells at the maternal-fetal interface is crucial for understanding pregnancy.
- Traditional methods like placental bed biopsy are invasive and yield limited tissue.
- A less invasive method is needed to obtain uterine leukocytes for analysis.
Purpose of the Study:
- To purify and characterize leukocytes from peripheral and uterine blood samples.
- To assess the feasibility of a less invasive uterine blood collection method.
- To determine if this method yields enriched uterine leukocytes for ex vivo and in vitro studies.
Main Methods:
- Isolation of peripheral blood mononuclear cells (PBMC) and uterine blood mononuclear cells (UBMC) from surgical gauze post-C-section.
- Immunophenotyping using multi-parameter flow cytometry.
- Staining for T lymphocytes, B lymphocytes, macrophages, regulatory T cells, and natural killer (NK) cells, including CD16-CD56++ NK cells.
Main Results:
- Over 95% of both PBMC and UBMC were identified as CD45+ leukocytes.
- Uterine blood mononuclear cells (UBMC) showed significantly higher percentages of CD16-CD56++ uterine NK (uNK) cells (18.41%) compared to peripheral blood mononuclear cells (PBMC) (2.73%).
- A substantial proportion (49.64%) of CD3-negative cells in UBMC were of peripheral NK cell phenotype (CD16+CD56++), indicating maternal peripheral NK cell infiltration.
Conclusions:
- Intrauterine leukocytes, particularly CD16-CD56++ NK cells, can be collected with increased purity using surgical gauze post-delivery.
- This non-invasive protocol is a useful technique for isolating CD16-CD56++ cells.
- Peripheral blood contamination may reduce the overall NK cell yield compared to more invasive decidual or endometrial sampling.
Problem:
Leukocytes from the maternal-fetal interface are a valuable tool to study local changes in immune function during pregnancy; however, sampling can be challenging due to inadequate tissue availability and the invasive nature of placental bed biopsy. Here, we aim to purify and characterize leukocytes from paired peripheral and uterine blood samples to assess whether a less invasive method of uterine blood collection could yield a population of enriched uterine leukocytes suitable for ex vivo and in vitro analyses.
Method Of Study:
Human peripheral blood mononuclear cells (PBMC) and uterine blood mononuclear cells (UBMC) expressed from surgical gauze post C-section were isolated, and immunophenotypic information was acquired by multi-parameter flow cytometry. PBMC and UBMC were stained for markers used to define T and B lymphocytes, macrophages, regulatory T (TReg ) cells, and natural killer (NK) cells. Prime flow was performed to check expression and analysis of CD16- CD56++ and CD16- CD56++ NK transcripts in PBMC and UBMC samples.
Results:
Immunophenotyping revealed that over 95% of both live PBMC and UBMC consisted of CD45+ leukocytes. Higher percentages of CD16- CD56++ , characterized as uterine NK (uNK) cells, were observed in UBMC samples as compared to PBMC samples (18.41% of CD45+ CD3- vs. 2.73%, respectively), suggesting that CD16- CD56++ cells were enriched in these samples. In UBMC, 49.64% of CD3-negative cells were of peripheral NK phenotype (CD16+ CD56++ ), suggesting infiltration of maternal peripheral NK (pNK) cell in the uterine interface.
Conclusion:
Intrauterine leukocytes, especially CD16- CD56++ NK cells, can be collected in sufficient numbers with increased purity by sampling the uterine cavity postdelivery with surgical gauze. Our results suggest that this non-invasive protocol is a useful sampling technique for isolating CD16- CD56++ cells, however, due to peripheral blood contamination, the NK cell yield could be lower compared to actual decidual or endometrial samples post-partum which is more invasive.
