The measurement of dendritic cells in umbilical cord blood: a novel technique using small volumes of whole blood.
OBJECTIVE
To develop a technique that would permit the identification of dendritic cells (DC) in small volumes of preterm neonatal cord blood in order to investigate neonatal immune response in relation to infection and preterm labor.


METHODS
We used three-color flow cytometry and a fixed quantity of fluorospheres to determine absolute cell numbers. Four red cell lysis techniques, sample dilution, time delay experiments, and a comparison with traditional Ficoll cell separation techniques were performed. Absolute numbers of DC recovered using each technique were calculated and compared.


RESULTS
With increasing time delay, there was a statistically significant reduction in the numbers of leukocytes in adult blood. In contrast, there was a significant increase in leukocytes in umbilical cord blood. Sample dilution did not significantly affect the total number of leukocytes or DC. The use of the reagent Optilyse B (Immunotech, Oxford, UK) combined with an additional washing step produced the best discrimination of all populations based on light scatter properties. For the same blood sample, antibody labeling of whole blood resulted in a greater recovery of DC when compared to prior cell purification using a Ficoll density gradient.


CONCLUSIONS
Using traditional Ficoll cell separation, cell manipulation requires large volumes of blood and leads to cell loss and alteration in phenotype. We have validated a novel method using small volumes of whole blood, diluted if necessary, and using red cell lysis to enable analysis of small volumes of preterm neonatal cord blood. This may permit further analysis of the contribution of the fetal immune response in the development of spontaneous preterm labor and preterm birth due to infection.
Mason
M Ruth
MR
Department of Obstetrics and Gynaecology, Northwick Park and St Mark's NHS Trust, Imperial College, London, United Kingdom.
Stagg
Andrew J
AJ
Knight
Stella C
SC
Lamont
Ronald F
RF
D000328:Adult; D002452:Cell Count; D003713:Dendritic Cells; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007231:Infant, Newborn; D007234:Infant, Premature; D007239:Infections; D007752:Obstetric Labor, Premature / Q000276:immunology; D011247:Pregnancy