Search

Establishing reliable phenotypic data sets from the analysis of peripheral blood lymphocytes of normal animals is required to assess disease states. The rhesus macaque animal model is well established with respect to adult animals, but limited data are available that characterizes lymphocyte subsets in normal neonates. To address this, we used four-color flow cytometric analysis to follow phenotypic changes in 29 normal rhesus animals through their first ten months of life. From birth to 44 wk of age, the white cell count and absolute lymphocyte count were both elevated compared to adults. CD4+ cells constituted over 80% of all T cells at birth, a percentage that declined gradually over the first 12 wk of life, coincidental with increases in the percentages of CD8+ T cells, CD3-8+ natural killer cells and CD20+ B cells. This difference in relative frequency of CD4 and CD8 results in a significant skewing of CD4:CD8 ratio from 0.7:1 in adults to 3.5:1 in neonates. In addition, the predominant population of T lymphocytes consisted of CD45RA+CD62L+ naive cells. This subset continues to be the predominant phenotype for at least the first year of age. After birth the expression of activation markers (CD25) increased particularly on CD4+ T cells, although these levels generally reached a frequency similar to that observed in adults between 12 and 20 weeks after birth. These results are similar to those seen in humans and further confirm the reliability of the rhesus macaque animal model to study human diseases.

Fetal nucleated erythrocytes (FNRBC) flow sorted from maternal peripheral blood, using monoclonal antibodies (mAb) that bind fetal cell surface antigens, are a noninvasive source of fetal DNA for prenatal diagnosis. These mAbs, however, also bind antigens shared by maternal cells. In sorted populations, this results in maternal cell contamination and low fetal cell purities, which complicates genetic analysis by fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). Fetal hemoglobin, (α2γ2), has been proposed as a useful fetal marker. To improve fetal cell enrichment from maternal blood, we developed an intracellular staining protocol that combines anti-γ mAb with Hoechst 33342 to identify and flow sort FNRBC. Artificial mixtures of male umbilical cord cells (as a source of fetal hemoglobin) and female adult, nonpregnant peripheral blood mononuclear cells were stained and flow sorted using this protocol. FISH analysis was performed using chromosome X and Y specific probes. Fetal cell purities were calculated by microscope confirmation of anti-γ staining and counting the number of X and Y signals present after FISH. Results from microscope analyses showed a fetal cell yield of 39–100% and fetal cell purities of 59–73%. These purities are significantly higher than the .001–4.8% previously reported by us in maternal samples using cell surface staining protocols. FISH results demonstrated that 83–100% (mean = 98%) of anti-γ positive cells were male, whereas 82–100% (mean = 92.5%) of anti-γ negative cells were female. These results confirmed that the anti-γ mAb is highly fetal specific. When applied to maternal blood samples, this protocol should lead to increased sensitivity for prenatal diagnosis. © 1996 Wiley-Liss, Inc.

Three major methods have been described for the isolation of fetal cells from maternal blood : fluorescence-activated cell sorting (FACS), immunomagnetic beads, and magnetic-activated cell sorting (MACS). To date, no study has directly compared fetal cell recovery using each of these methods. Here we describe out system using a 'model' male fetal cell mixed into female peripheral blood mononuclear cells. Fetal cell yields and purities were assayed by a quantitative polymerase chain reaction (qPCR) using chromosomes Y- and 7-specific sequences. Fetal cell recovery was investigated by selection of CD71 + cells or depletion of CD45 + cells. Out data demonstrated variation in fetal cell recovery for all methods tested, although CD71 + selection by FACS gave the best and most consistent results.

OBJECTIVE: The aim of this study was to determine the accuracy of noninvasive fetal RhD genotyping by fetal cell isolation from maternal blood. STUDY DESIGN: Candidate fetal cells from 18 pregnant women (one twin gestation) were flow-sorted. Polymerase chain reaction amplification of a 261 bp fragment of the RhD gene was performed on sorted fetal cells. The presence of amplified product was considered predictive of the RhD-positive genotype in the fetus. RESULTS: Sixteen of the 19 fetal RhD genotypes were correctly predicted in fetal cells isolated from maternal blood (10 were Rh positive, 6 were Rh negative). In 3 cases no amplification products were detected in RhD-positive fetuses. The association between presence of the fragment and RhD-positive genotype was significant ( p = 0.003, Fisher's exact test). CONCLUSIONS: Noninvasive prenatal diagnosis of the fetal RhD genotype is feasible. Absence of amplification products in the reaction requires confirmation that fetal material is present. Improvements in fetal cell purity and yield should increase diagnostic accuracy, although the current protocol has a positive predictive value of 100% and a negative predictive value of 67%. (AM J OBSTET GYNECOL 1996;174:818-22.)

Rare nucleated fetal cells circulate within maternal blood. Noninvasive prenatal diagnosis by isolation and genetic analysis of these cells is currently being undertaken. We sought to determine if genetic evidence existed for persistent circulation of fetal cells from prior pregnancies. Venous blood samples were obtained from 32 pregnant women and 8 nonpregnant women who had given birth to males 6 months to 27 years earlier. Mononuclear cells were sorted by flow cytometry using antibodies to CD antigens 3, 4, 5, 19, 23, 34, and 38. DNA within sorted cells, amplified by PCR for Y chromosome sequences, was considered predictive of a male fetus or evidence of persistent male fetal cells. In the 32 pregnancies, male DNA was detected in 13 of 19 women carrying a male fetus. In 4 of 13 pregnancies with female fetuses, male DNA was also detected. All of the 4 women had prior pregnancies; 2 of the 4 had prior males and the other 2 had terminations of pregnancy. In 6 of the 8 nonpregnant women, male DNA was detected in CD34+CD38+ cells, even in a woman who had her last son 27 years prior to blood sampling. Our data demonstrate the continued maternal circulation of fetal CD34+ or CD34+CD38+ cells from a prior pregnancy. The prolonged persistence of fetal progenitor cells may represent a human analogue of the microchimerism described in the mouse and may have significance in development of tolerance of the fetus. Pregnancy may thus establish a long-term, low-grade chimeric state in the human female.

The close phylogenetic relationship of macaques to humans has resulted in their widespread use as a preclinical model for bone marrow transplantation and stem cell gene therapy. To facilitate further use of this model, we undertook analysis of hematopoietic cells using multiparametric flow cytometric analysis. Rhesus CD34+CD38- cells displayed a number of characteristics of primitive hematopoietic cells, including low forward and orthogonal scatter and the lack of expression of lineage-specific markers or human lymphocyte antigen-DR. Four-color flow cytometric analysis demonstrated that rhesus CD34+CD38- cells were heterogenous with respect to Thy-1 expression and were CD59dim. Quantitative limiting dilution long-term culture-initiating cell (LTC-IC) analysis demonstrated that CD34+CD38- cells were approximately 150-fold enriched for LTC-IC as compared with unfractionated bone marrow, and occurred at a frequency similar to that previously reported in humans. Thus, as in humans, the CD34+38- population of rhesus macaque bone marrow is enriched for primitive, multipotent hematopoietic progenitor cells.

Human and simian immunodeficiency virus (HIV and SIV) replicate optimally in activated memory CD4+T cells, a cell type that is abundant in the intestine. SIV infection of rhesus monkeys resulted in profound and selective depletion of CD4+T cells in the intestine within days of infection, before any such changes in peripheral lymphoid tissues. The loss of CD4+T cells in the intestine occurred coincident with productive infection of large numbers of mononuclear cells at this site. The intestine appears to be a major target for SIV replication and the major site of CD4+T cell loss in early SIV infection.

Objective: The detection of fetal aneuploidy and gene mutations by analysis of fetal cells in maternal blood has demonstrated the feasibility of noninvasive prenatal diagnosis. Fetal cells are rare in the maternal circulation; all current methods used for their isolation also yield maternal cells. We developed a method that permits a quantitative assessment of the relative numbers of fetal and maternal cells. Study Design: Samples from 40 pregnant women were flow sorted with different monoclonal antibodies. Deoxyribonucleic acid was subsequently purified from candidate fetal cells; polymerase chain reaction was performed with synthetic primers specific for sequences on chromosomes Y and 7. Results: The maximum number of fetal cells detected was 52 in 1080 maternal cells. Fetal cell purity ranged from 0.001 % to 4.8%. Fetal cells were detected with antibodies to CD71, CD36, and glycophorin A. Conclusion: Quantitative polymerase chain reaction enables the determination of the purity and yield of fetal cells remaining after isolation from maternal blood, facilitating rapid comparisons between different cell separation techniques.