Evaluation of various methods of maternal placental blood collection for immunology studies

Abstract

The collection of maternal placental intervillous blood (IVB), without contamination of fetal blood and with an accurate mononuclear cell profile, is essential for immunological studies of placental malaria and other infectious diseases of the placenta. We have compared five documented methods of IVB collection: perfusion, incision, biopsy, tissue grinding, and puncture (prick) for fetal blood contamination and mononuclear cell profiles using flow cytometry. Twenty-five placentas were obtained from Plasmodium falciparum and human immunodeficiency virus-negative primigravid and secundigravid women delivering at Nyanza Provincial Hospital in Kisumu, western Kenya. Each of the five methods was performed on the same placenta. Fetal red blood cell contamination was significantly lower for the prick and perfusion methods (4.1% and 8.3%, respectively) than for incision (59.5%), biopsy (42.6%), and tissue grinding (19.9%). Significant variation was noted among the five methods in the percentages of monocytes, total T cells, CD4+ and CD8+ T cells, B cells, and NK cells. Further, a pairwise comparison of prick and perfusion, the two methods with low fetal blood contamination, showed that they were not different for fetal red blood cell contamination levels; however, prick yielded significantly higher percentages of CD4 T cells and CD4 memory T cells than perfusion. Collection by prick was determined to be the best method of intervillous blood collection for immunology studies, and perfusion represented the next best method of choice due to high sample volume yield. Overall, in considering the advantages/disadvantages of the two methods with low fetal cell contamination, we conclude that a combination of prick and perfusion is most suitable for immunology studies.

FIG. 1.

Chronology of sampling events. Within approximately 2 min of placenta expulsion, an intervillous blood sample by the prick method was collected from a region appropriately chosen for the technique. Perfusion was carried out next in a separate quadrant; perfusate was obtained within 20 to 25 min of placental expulsion. Blood collection by incision was carried out next, followed by biopsy within at least 5 min of each other, and finally, approximately 45 min after placental expulsion, blood by tissue grinding was collected. On average, all techniques were carried out within a maximum period of 45 min after placenta expulsion.

FIG. 2.

Fetal hemoglobin staining dot plot analysis for a representative placenta sample. (A) FSC-SSC gating of red blood cells; (B and C) 0 and 10% controls, respectively; (D) placental prick (5.5%); (E) perfusion (12%); (F) incision (71.6%); (G) biopsy (50.5%); and (H) tissue grinding (24.1%).

FIG. 3.

Immunophenotyping FSC-SSC dot plot analysis for a representative placenta sample. Peripheral blood is used as a reference (A), and IVB was obtained by prick (B), perfusion (C), incision (D), biopsy (E), and tissue grinding (F).