The prevalence of COPD in England: An ontological approach to case detection in primary care.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a significant cause of morbidity and mortality in England, however estimates of its prevalence vary considerably. Routinely collected and coded primary care data can be used to monitor disease prevalence, however reliance upon diagnostic codes alone is likely to miss cases. METHODS: We devised an ontological approach to COPD case detection and implemented it in a large primary care database to identify definite and probable cases of COPD. We used this to estimate the prevalence of COPD in England. RESULTS: Use of this approach to detect definite COPD cases yielded a prevalence of 2.57% (95% CI 2.55-2.60) in the total population, 4.56% (95%CI 4.52-4.61) in those aged ≥ 35 and 5.41% (95% CI 5.36-5.47) in ex or current smokers. The ontological approach identified an additional 10,543 definite cases compared with using diagnostic codes alone. Prevalence estimates were higher than the 1.9% prevalence currently reported by the UK primary care pay for performance (P4P) disease register. COPD prevalence when definite and probable cases were combined was 3.02% (95% CI 3.0-3.05) in the total population, 5.38% (95% CI 5.33-5.42) in those aged ≥ 35 and 6.46% (95% CI 6.46-6.40-6.56) in ex or current smokers. CONCLUSIONS: We demonstrate a robust reproducible method for COPD case detection in routinely collected primary care data. Our calculated prevalence differed significantly from current estimates based upon P4P data, suggesting that the burden of COPD in England is greater than currently predicted.

High-throughput microRNAome analysis in human germ cell tumours.

Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non-seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra-embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so-called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa-miR 371-373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high-throughput screen of 156 microRNAs in a series of type II and III GCTs (n = 69, in duplicate) using a quantitative PCR-based approach. After normalization to allow inter-sample analysis, the technical replicates clustered together, and the previous hsa-miRNA 371-373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non-seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs.

Expression microarray analysis of papillary thyroid carcinoma and benign thyroid tissue: emphasis on the follicular variant and potential markers of malignancy.

The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular variant (FVPTC), which is a particularly problematic lesion and can be challenging from a diagnostic viewpoint even in resected lesions. Although fine needle aspiration cytology is very useful in the diagnosis of PTC, its accuracy and utility would be greatly facilitated by the development of specific markers for PTC and its common variants. We used the recently developed Applied Biosystems 1700 microarray system to interrogate a series of 11 benign thyroid lesions and conditions and 14 samples of PTC (six with classic morphology and eight with follicular variant morphology). TaqMan(R) reverse transcriptase-polymerase chain reaction was used to validate the expression portfolios of 50 selected transcripts. Our data corroborates potential biomarkers previously identified in the literature, such as LGALS3, S100A11, LYN, BAX, and cluster of differentiation 44 (CD44). However, we have also identified numerous transcripts never previously implicated in thyroid carcinogenesis, and many of which are not represented on other microarray platforms. Diminished expression of metallothioneins featured strongly among these and suggests a possible role for this family as tumour suppressors in PTC. Fifteen transcripts were significantly associated with FVPTC morphology. Surprisingly, these genes were associated with an extremely narrow repertoire of functions, including the major histocompatibility complex and cathepsin families.

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.

A successful strategy for preimplantation genetic diagnosis of myotonic dystrophy using multiplex fluorescent PCR.

The most common form of inherited muscular dystrophy in adults is myotonic dystrophy (DM), an autosomal-dominant disease caused by the expansion of an unstable CTG repeat sequence in the 3' untranslated region of the myotonin protein kinase (DMPK) gene. Expanded (mutant) CTG repeat sequences are refractory to conventional PCR, but alleles with a number of repeats within the normal range can be readily amplified and detected. Preimplantation genetic diagnosis (PGD) of DM has been successfully applied. However, a misdiagnosis using the reported protocol was recently documented. Two new PGD protocols for DM have been developed which utilise multiplex fluorescent PCR. Ideally a linked polymorphic marker, APOC2, is amplified in addition to the normal DMPK alleles, thus providing a back-up diagnostic result. However, the two couples reported in the present study were not fully informative at the APOC2 locus and so an unlinked short tandem repeat (STR) marker, D21S1414, was substituted. The highly polymorphic nature of the D21S1414, DMPK and APOC2 loci means that a very simple genetic fingerprint can be generated by analyses of these loci. This allows most DNA contaminants to be detected. Contamination is a significant problem for PGD and is the primary reason for the inclusion of D21S1414 and APOC2 in this protocol. This paper reports the first clinical experience and pregnancies following PGD for DM using a multiplex fluorescent PCR protocol.

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.

Red cell indices as predictors of iron depletion in blood donors.

The objective of this study was to investigate whether red cell indices mean cell volume (MCV) and mean cell haemoglobin (MCH) were lower in frequent blood donors and hence, indirectly, able to predict impending iron depletion. Serum ferritin and/or soluble transferrin receptor levels can be used to evaluate iron status but are not practical for routinely screening blood donors prior to donation. Hb, MCV and MCH were measured on venous blood from 886 blood donors using a Sysmex E-5000. Full details were obtained for all donors of each earlier donation over the previous 3 years. MCV and MCH levels were lowest in donors with the highest frequency of previous blood donation. There was a significant negative correlation between MCV and number of donations in males and females and between MCH and number of donations in females, over the 3 year period 1995-97. Similar trends were observed when only the previous year's donations (1997) were considered with all categories showing significant negative correlations and additionally, Hb levels in females showed negative correlation with number of donations in 1997. In conclusion, increased frequency of blood donations is associated with lower MCV and MCH. These red cell indices, or more sophisticated parameters such as percentage hypochromic cells, should be used to monitor early onset of iron depletion in frequent blood donors.

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.

Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation.

Molecular genetic analysis of isolated single cells and other minute DNA samples is limited because there is insufficient DNA to perform more than one independent PCR amplification. One solution to this problem is to first amplify the entire genome, thus providing enough DNA for numerous subsequent PCRs. In this study we have investigated four different methods of whole genome amplification performed on single cells, and have identified a protocol that generates sufficient quantities of DNA for comparative genomic hybridisation (CGH) as well as more than 90 independent amplification reactions. Thus, numerous specific loci and the copy number of every chromosome can be assessed in a single cell. We report here the first reliable application of CGH to single cells from human preimplantation embryos (blastomeres) and to single fibroblasts, buccal cells and amniocytes.

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.

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.

Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification.

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for ensuring the genetic health of offspring born to families affected by inherited disease. This paper sets out to review current protocols for the diagnosis of single gene defects in human preimplantation embryos. These methods, which depend on DNA amplification using PCR, are subject to a variety of pitfalls, such as allele dropout (ADO), contamination and reduced amplification efficiency. Advances in single cell DNA amplification, such as improved multiplex PCR protocols, fluorescent-PCR and whole genome amplification (WGA), can be applied to address some of these problems. Different PGD strategies are discussed in the context of their clinical application.

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.

First trimester prenatal diagnosis using transcervical cells: an evaluation.

Human trophoblastic cells can be retrieved by minimally invasive procedures from the endocervical canal between between 6 and 15 weeks gestation. The incidence with which fetal cells can be detected in transcervical cell (TCC) samples varies according to the method of collection and the molecular techniques employed for their identification. Fluorescence in-situ hybridization (FISH) and polymerase chain reaction (PCR) assays have been successfully used to detect aneuploidies and Y-derived DNA sequences in TCC samples obtained from male fetuses. Chromosome specific polymorphic DNA sequences (small tandem repeats) have also been employed to identify, by quantitative fluorescent PCR, fetal cells in TCC samples. Furthermore, Rh(D) sequences have been amplified in samples retrieved from Rh(D) negative mothers. Preliminary results also suggest that prenatal diagnoses of thalassaemia and sickle cell anaemia can be performed on clumps of cells isolated from TCC samples. Overall systematic studies allow optimism about the possibility of using TCC samples for the prenatal diagnosis of selected inherited disorders.

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.

The 3'-untranslated region of the alpha2C-adrenergic receptor mRNA impedes translation of the receptor message.

We report that two subtypes of alpha2-adrenergic receptors (alpha2A/D- and alpha2C-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3'-untranslated region (3'-UTR) of the alpha2C-AR mRNA that impairs translational processing. NIH-3T3 fibroblasts were transfected with receptor constructs (coding region plus 552 nt, alpha2C-AR; coding region plus 1140 nt, alpha2A/D-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein. 90% of the NIH-3T3 alpha2C-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha2A/D-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3'-UTR of the alpha2C-AR in mRNA processing was determined by generating new constructs in which the 3'-UTR was progressively truncated from 552 to 470, 182, 143, or 74 nt 3' to the stop codon. Truncation of the 3'-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The alpha2C-AR mRNA with the longer 3'-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a key mechanism for regulating alpha2C-AR expression and associated signaling events.

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.

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.