Prenatal detection of chromosome disorders by QF-PCR.
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Chromosome Aberrations↗
Biomedical subjects
J Sherlock
Publications and source records attributed to J Sherlock.
Fetal cells in transcervical samples at an early stage of gestation.
Several investigations are in progress with the aim of performing prenatal diagnosis of inherited disorders by noninvasive or minimally invasive techniques. The most important approaches are based on the detection of fetal nucleated cells in maternal blood, the analysis of fetal DNA present in maternal plasma, and the identification and isolation of fetal trophoblastic cellular elements shed into the uterine cavity and the endocervical canal. In this review, we discuss the methods that have been employed for the collection of the transcervical samples at an early stage of gestation and the techniques used for the identification of fetal cells. We also report the results of using endocervical cells for the detection of fetal chromosomal disorders by fluorescent in-situ hybridization and for performing prenatal diagnosis of fetal Rh(D) phenotypes. Recent investigations have also shown that--after the isolation of trophoblastic cells from maternal contaminants by micromanipulation--transcervical samples can be employed for the prenatal diagnosis of single gene defects, such as those causing thalassemia and sickle cell anemia. Although the present results are promising, further investigations are required to demonstrate the feasibility of performing accurate diagnosis of fetal diseases by this minimally invasive approach in all transcervical samples retrieved at an early stage of gestation.
Cell Separation↗
How an audit of epidural patients in a community hospital setting resulted in the development of a formal acute pain management service.
PURPOSE: To describe the results of an audit of patients who received epidural analgesics postoperatively and the subsequent development of a formal acute pain management service in a community hospital. METHODS: To understand how epidural analgesia was being used to treat postoperative pain at the Peterborough Regional Health Centre, Peterborough, Ontario, a retrospective chart review was performed. Audits were performed on 178 patients who had received epidural analgesia postoperatively from October 1994 to May 1995. Data pertaining to demographics, epidural analgesia, pain scores and side effects were collected. RESULTS: Sixty-one per cent of patients received bupivacaine/ fentanyl infusions, and 39% received epidural morphine boluses. More than 60% of patients reported no pain postoperatively. Patients who received bupivacaine/fentanyl were more likely than those who received epidural morphine to also receive co-analgesia and transitional analgesia. Patients who received epidural morphine were more likely than those who received bupivacaine fentanyl to experience respiratory depression, hypotension and pruritus. Patients were followed by the anesthesiologist who provided the anesthetic. Anesthesiologists practised independently, and formal policies and procedures did not exist. CONCLUSIONS: As a result of the audit, an acute pain management service was developed. This included a team that did daily rounds and consisted of a nurse clinician and an anesthesiologist who was assigned to the service on a weekly basis. A committee was created, and formalized policies and procedures were established. Standardized order sheets, data sheets and a computerized database were developed. Reports for administrative and quality improvement purposes were generated monthly. Education programs were developed. Co-analgesia and transitional analgesia are now part of routine care, and epidural catheter placement close to the site of incision is encouraged. A postoperative nausea and vomiting algorithm, and a treatment regimen for pruritus have also been implemented.
Adult↗
Rapid detection of chromosomes X and Y aneuploidies by quantitative fluorescent PCR.
Quantitative fluorescent polymerase chain reaction (QF-PCR) assays and small tandem repeat (STR) markers have been successfully employed for the rapid detection of major numerical aneuploidies affecting human autosomes. So far, the analysis of chromosomes X and Y disorders has been hampered by the rarity of highly polymorphic markers which could distinguish normal female homozygous PCR patterns from those seen in patients with Turner's syndrome. A new marker (X22) of the X/Y chromosomes has been identified which maps in the Xq/Yq pseudoautosomal region PAR2; used together with the HPRT it allows the rapid diagnosis of numerical aneuploidies of the sex chromosomes. Blood samples from normal male and female subjects and from patients with X and Y chromosome disorders (45,X and 47, XXY) have been tested by QF-PCR with the X22 polymorphic pentanucleotide (12 alleles) together with the HPRT and P39 markers. The samples were also tested by multiplex QF-PCR with STRs specific for chromosomes 21,18,13 and amelogenin (AMXY). Tested by QF-PCR, all samples from normal females were heterozygous for either the X22 or the HPRT marker with fluorescent peak ratios near 1:1, thus allowing a correct, rapid diagnosis of their chromosome complement. Turner's patients (45,X) showed only one X22 and one HPRT fluorescent peak, thus documenting the presence of a single X chromosome. Turner's patients with mosaicism showed a major fluorescent peak for the X22 and HPRT markers and a minor peak revealing the presence of a second minor population of cells. Two 47, XXY cases could also be diagnosed. Multiplex analyses can be performed using simultaneously STR markers for chromosomes 21,18,13 X and Y. The diagnostic value of a third X-linked marker (P39) was also investigated. These results suggest that rapid diagnosis of major numerical anomalies of the X and Y chromosomes can be performed using QF-PCR with a new highly polymorphic X-linked marker, X22, which maps in the Xq/Yq pseudoautosomal region PAR 2. Multiplex QF-PCR tests-using the X22 STR in association with HPRT and, in rare cases, a third P39 marker-allow the rapid diagnosis of major aneuploidies affecting chromosomes 21, 18, 13, X and Y. The X22 marker can also be employed for the detection of fetal cells present in maternal peripheral blood or the endocervical canal.
Aneuploidy↗
Transcervical cells and the prenatal diagnosis of haemoglobin (Hb) mutations.
Prenatal diagnoses of haemoglobin (Hb) mutations were performed using transcervical cells, retrieved by aspiration from the endocervical canal of ten selected pregnant women at about 10 weeks of gestation, prior to chorionic villus sampling (CVS). Both parents were carriers of haemoglobinopathies (thalassaemia or HbS). Clumps of fetal cells were isolated by micromanipulation under an inverted microscope and aliquots of the extracted DNA tested separately for the presence of paternally derived chromosome markers and Hb mutations by quantitative fluorescent polymerase chain reaction (PCR). The correct prenatal diagnosis of Hb diseases, using selected single clumps of trophoblastic cellular elements free of maternal contaminating cells, was achieved in six out of ten cases.
Anemia, Sickle Cell↗
Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells.
We have refined polymerase chain reaction (PCR) assays for the detection of sickle cell anaemia, the delta F 508 deletion causing cystic fibrosis, and the IVS1-110 mutation leading to beta thalassaemia, allowing them to be successfully performed upon single cells using fluorescent primers. We have also assessed the possibility of detecting aneuploidies of chromosomes 13, 18 and 21 using a quantitative fluorescent polymerase chain reaction (QF-PCR) with primers flanking polymorphic short tandem repeat (STR) markers. Trisomies were readily diagnosed by the detection of tri-allelic patterns. However some heterozygote normal and trisomic diallelic patterns did not produce the expected ratios of amplified PCR products due to preferential DNA sequence amplification. Total allelic drop out (ADO) did not occur with any of the cells tested. Multiplex QF-PCR assays can be performed on a single cell in under 6 h and simultaneously provide diagnosis of single gene defects, sex determination and an indication of selected chromosome aneuploidy.
Anemia, Hemolytic, Congenital↗
Prenatal detection of Hb mutations using transcervical cells.
Prenatal diagnoses were performed on six selected pairs of parents known to be carriers of Hb mutations by testing transcervical cells (TCCs) retrieved, prior to chorionic villus sampling (CVS), by aspiration of the cervical mucus from the pregnant mothers at 10-12 weeks of gestation. A concordance between the results of testing chorionic villus cells and isolated clumps of trophoblastic cellular elements was observed in four of the six cases.
Cervix Uteri↗
Quantitative fluorescence polymerase chain reaction for the rapid prenatal detection of common aneuploidies and fetal sex.
OBJECTIVE: We have developed a quantitative fluorescence multiplex polymerase chain reaction assay for the rapid detection of sex and aneuploidies involving chromosomes 21, 18, and 13. STUDY DESIGN: Samples of deoxyribonucleic acid (n = 85) extracted from amniotic fluid, fetal tissues, and blood were investigated by multiplex polymerase chain reaction amplification of polymorphic small tandem repeat markers specific for chromosomes 21, 18, 13, and X. RESULTS: Quantitative analysis of the polymerase chain reaction products allowed us to distinguish between normal samples and samples with autosomal trisomies while sexing was performed simultaneously. From 85 samples only three produced unsatisfactory results with one of the two chromosome 13-specific markers. In these three cases the amplification of the other chromosome 13 marker always resulted in a correct normal pattern. CONCLUSION: Quantitative fluorescence multiplex polymerase chain reaction is a reliable and rapid method that allows prenatal diagnosis of the major numeric chromosomal abnormalities to be performed within 24 hours.
Amniocentesis↗
Prenatal detection of fetal aneuploidies using transcervical cell samples.
In the course of an investigation aimed at detecting the presence of trophoblastic cells in the endocervical canal of pregnant women between 7 and 17 weeks of gestation, several cases of aneuploidies were observed using a fluorescent in situ hybridisation (FISH) assay. The cases include fetal chromosome 21 and 18 trisomies, triploidy and sex chromosome aneuploidies. The results were confirmed by testing placental tissues obtained after termination of pregnancy (TOP). In two of these cases, clumps of cells with the morphology of trophoblasts were isolated from the transcervical cell (TCC) samples using micromanipulation. FISH and fluorescent polymerase chain reactions (PCR), performed on these clumps, showed them to be exclusively of fetal origin. These results show that prenatal diagnoses of major aneuploidies can be performed by FISH using whole TCC samples, or on isolated clumps of cells by FISH and PCR assays.
Adult↗
Rapid detection of aneuploidies by microsatellite and the quantitative fluorescent polymerase chain reaction.
Several studies have been performed to assess the diagnostic value of using small tandem repeat (STR) markers and quantitative fluorescent polymerase chain reaction (QF-PCR) assays for the rapid detection of aneuploidies involving chromosomes 21, 18, 13 (Mansfield, 1993; Pertl et al., 1994, 1996; Adinolfi et al., 1995a). The results of these investigations have documented the diagnostic advantages of this approach to perform prenatal tests using amniotic and chorionic samples, or fetal nucleated cells retrieved from peripheral maternal blood or endocervical samples. The use of two or more STR markers for each autosome facilitates the diagnosis of aneuploidies, while avoiding the need to employ internal non-polymorphic markers. Multiplex quantitative fluorescent analyses can be performed in about six hours from the collection of the samples and, although targeted to specific abnormalities, they can exclude the presence of the most frequent chromosomal disorders. QF-PCR can be exploited to analyse DNA present in single or clumps of cells and thus to perform prenatal diagnoses on maternal peripheral blood or transcervical cell samples and on preimplantation embryos.
Aneuploidy↗
Rapid detection of trisomies 21 and 18 and sexing by quantitative fluorescent multiplex PCR.
Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise. In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities.
Aneuploidy↗
Prenatal detection of fetal RhD DNA sequences in transcervical samples.
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Base Sequence↗
Rapid detection of selected aneuploidies by quantitative fluorescent PCR.
Selected aneuploidies can be rapidly diagnosed by the analysis of fluorescent polymerase chain reaction (PCR) products of chromosome-specific and highly polymorphic small tandem repeats (STRs). The quantitative STR patterns obtained from samples of normal individuals are markedly different from those seen when patients with aneuploidies involving chromosome X, or trisomies of chromosomes 21 and 18, are tested. For example, while samples from normal subjects--tested with a chromosome 21-derived STR (D21S11)--show two fluorescent PCR peaks with similar activities in a 1:1 ratio, the analysis of samples from patients with trisomy 21 reveals the presence of either three peaks (ratio 1:1:1), or two peaks with a ratio of 2:1. The use of an internal non-polymorphic marker allows identification of trisomic samples with three copies of the same allele. This rapid approach (24 hours) is particularly valuable when applied to prenatal diagnosis of chromosomal abnormalities since it reduces the time of anxiety of the parents waiting for the results of the conventional cytogenetic tests, which require several weeks.
Aneuploidy↗
Molecular evidence of fetal-derived chromosome 21 markers (STRs) in transcervical samples.
Transcervical cells (TCCs), collected by flushing or aspiration at 8-13 weeks of gestation, were analysed for the presence of fetal-derived DNA sequences. DNA extracted from maternal peripheral blood, TCC samples, and placental tissue was amplified by the polymerase chain reaction (PCR) to detect small tandem repeat (STR) markers specific to chromosome 21. STR products of fetal origin could be clearly observed in four TCC samples. TCC samples collected by flushing or aspiration were also analysed by fluorescent in situ hybridization (FISH) using X and Y probes simultaneously: 46,XY cells could be detected in all TCC samples obtained from mothers with male fetuses.
Base Sequence↗
Methods for the transcervical collection of fetal cells during the first trimester of pregnancy.
Before termination of pregnancy, four techniques for retrieving fetal cells transcervically were investigated: uterine lavage, endocervical lavage, cytobrush and mucus aspiration. The yield of fetal cells in these samples was studied and found to be somewhat better after uterine lavage. A preliminary assessment of the safety of mucus aspiration was carried out before transcervical chorionic villus sampling (CVS) in continuing pregnancies. No difference in outcome to a control group having only CVS was found.
Biopsy↗
Detection of fetal cells in transcervical samples and prenatal diagnosis of chromosomal abnormalities.
Transcervical samples collected by lavage, aspiration, and cytobrush from women between 6 and 13 weeks of gestation were tested for the presence of fetal cells using fluorescence in situ hybridization (FISH) with probes for chromosomes X, Y, 1, and 21, and by polymerase chain reaction (PCR) amplification of DNA sequences derived from chromosomes X, Y, and 21. With a few exceptions, a good correlation was observed between the results of sexing the fetuses using FISH or PCR on transcervical cell (TCC) samples retrieved by lavage and those obtained by testing fetal (placental) tissue. In a comparative study between TCC samples collected by lavage or cytobrush, the sex of the fetus was correctly diagnosed by PCR amplification of a Y-derived DNA sequence. Variable results were observed with samples obtained by aspiration, mainly because this procedure was found to be more prone to failure to remove thick mucus without previous injection of physiological saline. Chromosome 21-derived small tandem repeats (STRs) of fetal origin were successfully detected in about 40 per cent of TCC samples recovered by lavage. Two cases of chromosomal abnormalities, one of trisomy 21 and one of triploidy, were detected in TCC samples in the course of our investigations.
Cell Separation↗
Isolation of fetal cells from transcervical samples by micromanipulation: molecular confirmation of their fetal origin and diagnosis of fetal aneuploidy.
Transcervical cell (TCC) samples have been shown to contain fetal cells amenable to molecular analysis. However, the presence of 'contaminating' maternal cells limits their use for prenatal diagnoses. In this report we show that clumps of fetal cells can be isolated from transcervical samples by micromanipulation and tested by fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). Out of 129 clumps, isolated from mucus aspirates and transcervical lavages from 29 patients, 29 clumps from 11 patients were found to be exclusively of fetal origin as judged by the detection of chromosome 21-specific polymorphic DNA markers and Y-derived DNA sequences by PCR and FISH. One case of a male triploid fetus, diagnosed by the analysis of TCC samples obtained by mucus aspiration and lavage, was confirmed by testing clumps of cells isolated by micromanipulation.
Aneuploidy↗
Detection of trophoblast cells in transcervical samples collected by lavage or cytobrush.
OBJECTIVE: To compare two methods of obtaining fetal cells from the endocervical canal for prenatal diagnosis during the first trimester: lavage with physiologic saline, and the endocervical passage of a cytobrush. METHODS: Fetal cells were identified morphologically using conventional and immunohistochemical staining. Y-specific sequences were detected using polymerase chain reaction (PCR) and fluorescent in situ hybridization. The presence of fetal DNA was also demonstrated by PCR amplification of a polymorphic short tandem repeat marker. RESULTS: Syncytiotrophoblast cells were identified in four of 11 lavage samples and in one of 11 brush samples. Sex determination using fluorescent in situ hybridization was achieved in all 11 lavage samples and in nine of 11 cytobrush samples; no Y sequences were located in two brush samples from male embryos. Sex determination was achieved successfully in all 22 samples using PCR of X- and Y-specific DNA sequences. Small tandem repeat pattern analysis indicated the presence of fetal DNA in five of 11 lavage samples and in one of eight cytobrush samples, which were informative because the maternal and fetal small tandem repeat patterns differed. CONCLUSION: Although lavage retrieves more trophoblast cells than does brushing, both methods are potentially suitable for molecular prenatal diagnosis.
Female↗