Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.
BACKGROUND
Functional 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.


RESULTS
We 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.


CONCLUSIONS
CVL 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.
Department of Medicine, University of Toronto, Toronto, Canada ; Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya.
Lyle R
LR
McKinnon
Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
Sean M
SM
Hughes
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America ; Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.
Stephen C
SC
De Rosa
Department of Immunology and Microbiology, Rush University Medical Center, Chicago, Illinois, United States of America.
Jeffrey A
JA
Martinson
Department of Immunology and Microbiology, Rush University Medical Center, Chicago, Illinois, United States of America.
Jill
J
Plants
Department of Immunology and Microbiology, Rush University Medical Center, Chicago, Illinois, United States of America.
Kirsten E
KE
Brady
Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
Pamela P
PP
Gumbi
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
Devin J
DJ
Adams
Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
Lucia
L
Vojtech
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
Christine G
CG
Galloway
Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
Michael
M
Fialkow
Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America.
Gretchen
G
Lentz
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Dayong
D
Gao
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
Zhiquan
Z
Shu
Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya.
Billy
B
Nyanga
Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya.
Preston
P
Izulla
Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya.
Joshua
J
Kimani
Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya.
Steve
S
Kimwaki
Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
Alfred
A
Bere
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
Zoe
Z
Moodie
Department of Immunology and Microbiology, Rush University Medical Center, Chicago, Illinois, United States of America.
Alan L
AL
Landay
Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa ; National Health Laboratory Services, Cape Town, South Africa.
Jo-Ann S
JA
Passmore
Department of Medicine, University of Toronto, Toronto, Canada ; Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya ; Department of Medicine, University Health Network, Toronto, Canada.
Rupert
R
Kaul
College of Medicine, University of Illinois, Chicago, Illinois, United States of America.
Richard M
RM
Novak
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America ; Department of Medicine, University of Washington, Seattle, Washington, United States of America ; Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America ; Department of Global Health, University of Washington, Seattle, Washington, United States of America.
M Juliana
MJ
McElrath
Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, United States of America ; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America ; Department of Medicine, University of Washington, Seattle, Washington, United States of America.
Florian
F
Hladik
D000293:Adolescent; D000328:Adult; D001706:Biopsy / Q000379:methods; D002469:Cell Separation; D002470:Cell Survival; D002986:Clinical Trials as Topic; D005260:Female; D015658:HIV Infections / Q000175:diagnosis / Q000517:prevention & control; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000473:pathology*; D015203:Reproducibility of Results; D007507:Therapeutic Irrigation; D014621:Vagina / Q000473:pathology*; D055815:Young Adult