Objective: To develop a technique that would permit the identfication 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 purfication using a Ficoll density gradient.
Conclusion: 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.
1. Villar J, Ezcurra EJ, Gurtner de la Fuente V, Campodonico L. Pre-term delivery syndrome: The unmet need. Res Clin Forums1994;16:9-38.
2.
2. Lamont RF, Fisk NM. The role of infection in the pathogenesis of preterm labour. Progr Obstet Gynaecol1993;10:135-158.
3.
3. Lettieri L, Vintzileos AM, Rodis JF, Albini SM, Salafia CM. Does ‘idiopathic’ preterm labour resulting in preterm birth exist?Am J Obstet Gynecol1993;168:1480-1488.
4.
4. Yoon BH, Jun JK, Romero R, et al. Amniotic fluid inflammatory cytokines (interleukin-6, interleukin-1 beta, and tumor necrosis factor-alpha), neonatal brain white matter lesions, and cerebral palsy. Am J Obstet Gynecol1997;177:19-26.
5.
5. Romero R, Gomez R, Mazor M, Ghezzi F, Yoon BH. The preterm labor syndrome. In: Elder MG, Lamont RF, Romero R, eds. Preterm Labour. New York: Churchill Livingstone, 1997:29-49.
6.
6. Ghezzi F, Gomez R, Romero R, et al. Elevated interleukin-8 concentrations in amniotic fluid of mothers whose neonates subsequently develop bronchopulmonary dysplasia. Eur J Obstet Gynecol Reprod Biol1998;78:5-10.
7.
7. Gomez R, Romero R, Ghezzi F, Yoon BH, Mazor M, Berry SM. The fetal inflammatory response syndrome. Am J Obstet Gynecol1998;179:194-202.
8.
8. Yoon BH, Romero R, Park JS, et al. Fetal exposure to an intraamniotic inflammation and the development of cerebral palsy at the age of three years. Am J Obstet Gynecol2000;82:675-681.
9.
9. Bruning T, Daiminger A, Enders G. Diagnostic value of CD45RO expression on circulating T lymphocytes of fetuses and newborn infants with pre-, peri- or early post-natal infections. Clin Exp Immunol1997;107:306-311.
10.
10. Duggan PJ, Maalouf EF, Watts TL, et al. Intrauterine T-cell activation and increased proinflammatory cytokine concentrations in preterm infants with cerebral lesions. Lancet2001;358:1699-1700.
11.
11. Banchereau J. Immunobiology of dendritic cells. Annu Rev Immunol2000;18:767-811.
12.
12. Holt PG. Primary allergic sensitisation to environmental antigens: Perinatal T cell priming as a determinant of responder phenotype in adulthood J Exp Med1996; 183:1297-1301.
13.
13. Cohen SBA, Morgan CL, Perez-Cruz I, Perandin F, Martinez B, Madrigal JA. Cord blood serum does not increase lymphocyte responses in comparison to adult serum. Hum Immunol2000;61:111-114.
14.
14. Robinson SP, Patterson S, English N, Davies D, Knight SC, Reid CD. Human peripheral blood contains two distinct lineages of dendritic cells. Eur J Immunol1999;29:2769-2778.
15.
15. Kleihauer E, Braun H, Betke K. Demonstration of fetal haemoglobin in erythrocytes of a blood smear. Klin Wochenschr1957;35:637-642.
16.
16. Szabolcs P, Park K-D, Reese M, Marti L, Broadwater G, Kurtzberg J. Absolute values of dendritic cell subsets in bone marrow, cord blood, and peripheral blood enumerated by a novel method. Stem Cells2003;21:296-303.
17.
17. Macey MG, McCarthy DA, Vogiatzi D, Brown KA, Newland AC. Rapid flow cytometric identification of putative CD14- and CD64-dendritic cells in whole blood. Cytometry1998;31:199-207.
18.
18. Sorg RV, Kögler G, Wernet P. Identification of cord blood dendritic cells as an immature CD11c- population. Blood1999;93:2302-2307.
19.
19. Vuckovic S, Gardiner D, Field K, et al. Monitoring dendritic cells in clinical practice using a new whole blood single platform TruCOUNT assay. J Immunol Methods2004;284:73-87.
20.
20. Cai Z, Pan ZL, Pang Y, Evans OB, Rhodes PG. Cytokine induction in fetal rat brains and brain injury in neonatal rats after matemal LPS administration. Pediatr Res2000;4:64-72.
21.
21. Balasingam V, Tejada-Berges T, Wright E, Bouckova R, Yong VW. Reactive astrogliosis in the neonatal mouse brain and its modulation by cytokines. J Neurosci1994;14:846-856.
22.
22. Gilles FH, Leviton A, Kerr CS. Endotoxin leukoencephalopathy in the telencephalon of the newborn kitten. J Neurol Sci1976;27:183-191.
23.
23. Holt PG, Jones CA. The development of the immune system during pregnancy and early life. Allergy2000;55:688-697.
