Owen P. Phillips

Prenatal diagnosis with fetal cells isolated from maternal blood by multiparameter flow cytometry

A long-sought goal of medical genetics has been development of prenatal diagnostic procedures that do not endanger the conceptus. Reliable and universal screening for cytogenetic disorders would require analysis of fetal cells isolated from the maternal circulation. This would be applicable to all pregnant women, irrespective of their ages or histories. In the current study fetal nucleated erythrocytes were flow sorted on the basis of four parameters: cell size, cell granularity, transferrin receptor, and glycophorin-A cell surface molecule. By polymerase chain reaction with oligonucleotide primers flanking single-copy Y-specific deoxyribonucleic acid sequences, male fetuses were correctly identified among flow-sorted samples in 12 of 12 (100%) pregnancies; female fetuses were correctly identified in 5 of 6 (83%) pregnancies. We also achieved the prenatal diagnosis of fetal aneuploidies by use of flow-sorted nucleated fetal erythrocytes and in situ hybridization with chromosome-specific deoxyribonucleic acid probes: one case of trisomy 21 that was detected in maternal blood taken 1 week after chorionic villus sampling and one case of trisomy 18 that was detected in maternal blood taken immediately before chorionic villus sampling. Although our results are promising, additional data on the background sensitivity and specificity of in situ hybridization in flow-sorted fetal cells will be necessary to minimize subjective interpretation and permit clinical application.

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses.

Risk of fetal mosaicism when placental mosaicism is diagnosed by chorionic villus sampling

OBJECTIVE Our purpose was to determine the risk of fetal mosaicism when placental mosaicism is found on chorionic villus sampling. STUDY DESIGN We present a case of mosaic trisomy 22 detected on chorionic villus sampling and subsequently found in the fetus. A review of comprehensive chorionic villus sampling studies with emphasis on follow-up for fetal mosaicism was conducted. RESULTS Among 13 studies reviewed, 469 cases of placental mosaicism are presented; fetal mosaicism was found in 50 (10.7%). Factors associated with fetal mosaicism are (1) mosaicism on mesenchymal core culture and (2) type of chromosome abnormality involved--specifically, marker chromosomes (26.7%) and common autosomal trisomies (19.0%). Amniocentesis predicted fetal genotype in 93% to 100% of cases of placental mosaicism, depending on the cell type in which mosaicism was diagnosed. CONCLUSIONS Although mosaicism is usually confined to the placenta, the fetus is involved in about 10% cases. Patients should be counseled about this risk and the accuracy of follow-up amniocentesis.

Charge flow separation: quantification of nucleated red blood cells in maternal blood during pregnancy.

We set out to ascertain the numbers of fetal cells that enter the maternal blood stream during pregnancy. Samples of 15–16 ml of whole blood were collected from 225 women—mostly 10–18 weeks pregnant—and then processed by charge flow separation, a novel method based on free flow electrophoresis in a buffer counterflow gradient. After their recovery in four different separation instruments, nucleated red blood cells (NRBC) were enumerated histologically. In some cases fetal NRBC were identified and enumerated by fluorescence in situ hybridization with probes for the X and Y chromosomes and fetal haemoglobin mRNA. Recoveries were consistent among the four separation instruments: the median numbers of NRBC obtained were 4190, 1590, 2805 and 3860. Our data show that approximately 30 per cent of those cells were fetal. Thus, recent reports on the separation of fetal NRBC by other methods, give underestimates of their frequency in maternal blood.

A jumping Robertsonian translocation : a molecular and cytogenetic study

Abstract We report a patient with mosaicism for two different Robertsonian translocations, both involving chromosome 21. She carries an unbalanced cell line with an i(21q) and a balanced cell line with a rob(21q22q). She is phenotypically normal but has two children who inherited the i(21q) and have Down syndrome. We demonstrate that both abnormal chromosomes are dicentric and that the proband’s 21/21 rearrangement is an isochromosome formed from a maternally derived chromosome 21. We propose a model in which the i(21q) is the progenitor rearrangement in the proband, which subsequently participated in a nonreciprocal rearrangement characteristic of a jumping translocation. In addition, we review other cases of constitutional mosaicism involving jumping translocations.

Severity of abnormality influences decision to terminate pregnancies affected with fetal neural tube defects

We examined parental decision concerning pregnancy management in women having fetuses with neural tube defects (NTDs) to determine whether severity of defect or method of detection has an impact on the decision making process. Analysis of decisions by 50 women, whose pregnancies were affected by an isolated neural tube defect (NTD) and characterized by a singleton gestation at 24 gestational weeks or less with normal chromosomal complement (46,XX or 46,XY), were assessed. All 23 women carrying fetuses with anencephaly elected to terminate their pregnancies. Of the 27 women carrying fetuses with spina bifida, 21 (77.8%) elected to terminate their pregnancies and 6 (22.2%) elected to continue their pregnancies. Of the 6 pregnancies that were continued, 4 were initially detected by ultrasonography and 2 were ascertained by maternal serum alpha-fetoprotein screening; defects ranged from 2 to 14 vertebral bodies, and none of the defects were craniad to the T9 level. This is in comparison to 5 of the 21 spina bifida cases that were elective pregnancy terminations, which were characterized by fetal lesions craniad to the T9 level. Severity of NTD thus appears to influence the decision to continue or terminate an affected pregnancy.

Frequency of nucleated red blood cells in maternal blood during the different gestational ages

We wished to determine the time of pregnancy at which optimal numbers of nucleated red blood cells (NRBC) are present in maternal blood. Because 30% of the NRBC in maternal blood are fetal, there are implications for prenatal screening and diagnosis. Samples of whole blood were collected from each of 225 women at various times during pregnancy. The samples were processed by charge flow separation (CFS), the NRBC enumerated, and the numbers compared on a week-to-week basis. To quantify the relationship between week of pregnancy and actual and log-transformed numbers of NRBC recovered, Pearson product moment and Spearman correlation coefficient were estimated for each of four CFS instruments and for the four instruments combined. When the data were analyzed, we found no relationship between stage of pregnancy and numbers of NRBC recovered. Even after logarithmic transformation, variability among the women, estimated by standard deviation, was large and relatively stable across the different stages of pregnancy. The number of NRBC recoverable by CFS appears to be constant between 7 and 25 weeks.

Sister chromatid exchange frequency in directly prepared cytotrophoblasts : demonstration of in vivo deoxyribonucleic acid damage in pregnant women who smoke cigarettes

Assessing frequency of sister chromatid exchange is a sensitive method of monitoring exposure to clastogens, mutagens, and other substances that induce deoxyribonucleic acid damage. Aware that cigarette smoke is associated with increased sister chromatid exchange in many cell types, we sought to determine whether an in vivo effect of cigarette smoke could be demonstrated by study of sister chromatid exchange in chorionic villus cells. Directly prepared cytotrophoblasts and cultured mesenchymal core cells were analyzed. Mean sister chromatid exchange frequency in cytotrophoblasts from smoking subjects (8.87 sister chromatid exchanges per cell) was significantly greater than in nonsmoking subjects (5.81 sister chromatid exchanges per cell; p less than 0.001); however, no significant difference in cultured mesenchymal core cells was found. Our results demonstrate that maternal exposure to cigarette smoke results in direct placental deoxyribonucleic damage, which in turn could explain deleterious effects of smoking on pregnancy. Increased sister chromatid exchange frequency was observed only in directly prepared cytotrophoblasts, showing the necessity of using this cell type to evaluate the effects of clastogens on placentas.

Discordant direct and culture results following chorionic villus sampling and the diagnosis of a third cell line in the fetus

We report a case of discordant non‐mosaic karyotypes following chorionic villus sampling (CVS). A 45,XX,der(21;21)(q10;q10) karyotype was found on direct preparation of cytotrophoblasts and 46,XX was found on long‐term culture of mesenchymal core cells. Analysis of amniotic fluid cells and fetal tissue revealed a third karyotype: 46,XX,+21,der(21;21)(q10;q10). Had only culture analysis been performed, follow‐up studies might not have been undertaken. This case demonstrates the importance of direct CVS preparation in helping to identify fetal abnormalities, and the need for follow‐up of discordant CVS results.

Unexplained elevated maternal serum α-fetoprotein is not predictive of adverse perinatal outcome in an indigent urban population

OBJECTIVE The null hypothesis of this study is that in an urban, indigent obstetric population at high risk for adverse perinatal outcome, unexplained elevations of maternal serum alpha-fetoprotein are not an additional predictor of adverse perinatal outcome. STUDY DESIGN Perinatal outcomes of 72 patients from a clinic for indigent patients with unexplained elevated maternal serum alpha-fetoprotein levels were compared with those of matched controls from the same population with normal maternal serum alpha-fetoprotein levels. Subjects and controls were matched for age, race, parity, and presence or absence of Hollister risk factors. The frequency of adverse perinatal outcome in the two groups was subjected to matched-pair chi 2 analysis. RESULTS Adverse perinatal outcome occurred in 38.9% (28 of 72) of subjects with unexplained elevated maternal serum alpha-fetoprotein levels greater than or equal to 2.5 multiples of the median, compared with 31.9% (23 of 72) of controls with normal maternal serum alpha-fetoprotein levels (p = 0.5). No statistically significant difference in adverse perinatal outcomes was found. CONCLUSIONS Elevated maternal serum alpha-fetoprotein levels offer little if any additional predictive value for adverse perinatal outcome in populations already at high risk for such outcomes on the basis of obstetric or socioeconomic criteria.