Optimizing Viable Leukocyte Sampling from the Female Genital Tract for Clinical Trials: An International Multi-Site Study

Abstract

BackgroundFunctional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear.Methods and FindingsWe enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4+ T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor.ConclusionsCVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.

Increased levels of inflammatory cytokines in the female reproductive tract are associated with altered expression of proteases, mucosal barrier proteins, and an influx of HIV-susceptible target cells

“…45,46 Preliminary data suggest that neutrophils are the most common cell recovered from endocervical cytobrush sampling. 47 MIP-3a, IL-1b, and IL-8 in particular may be critical cytokines that influence neutrophil protease expression, as both were significantly correlated to protease levels. Previous work in cell culture indicates both IL1b and MIP-3a induce neutrophil migration, and that IL-1b is a defining feature of sexually transmitted infections.…”

“…Unfortunately, the lavage specimens used here are typically insufficient for detailed cellular work. 47 Cytobrush samples from the endocervix may contain high concentrations of neutrophils, 47 however, because the matching cytobrush specimen for the current study was used to run the CD4 þ T-cell panel, no cells were available for neutrophil quantitation. In addition, as neutrophils are highly prone to apoptosis, these would best measured in fresh samples collected as part of a new study.…”

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

“…Cervical biopsy, cervical cytobrushes and cervicovaginal lavages (CVL) [10][11][12] are the most commonly used across the literature. CVL collection by PBS lavage is the simplest method and allows the study of immune modulatory proteins but results in extremely low cell yield, and is therefore not suitable for studying the immune cell populations of the FGT 13 . CVL samples are, on the other hand, very useful for evaluating the immune environment of the FGT by measuring the expression of various cytokines, chemokines or antimicrobial factors using methods such as ELISA, cytokine bead array 14 or mass spectrometry 15,16 .…”

“…On the other hand, cervical cytobrushes, despite the lower yield of cells collected, is a less invasive and more convenient method to collect immune cells from the FGT. Both methods can reach the same yield of CD45+ leucocytes, but two sequential cervical cytobrushes are necessary to obtain the same amount of cells contained in one biopsy 13 . Nonetheless, cytobrush sampling still provides an acceptable number of cells (about 5,000 CD45+ cells/cytobrush) for further ex vivo phenotyping by flow cytometry 14 .…”

“…Biopsies are derived from the superior portion of the epithelium and stroma of the ectocervix 12,13 , while cervical cytobrushes sample the cervical os, collecting cells derived from the epithelium of endocervix and presumably the transformation zone. Cytobrush samples therefore sample a region composed of a single layer of columnar epithelium, while biopsies, include a region lined by a squamous stratified epithelium 5 .…”