Microdissection molecular copy-number counting (microMCC)--unlocking cancer archives with digital PCR.

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.

Point-of-care testing of neonatal coagulation.

The aim of this validation study was to compare prothrombin time (PT) and activated partial thromboplastin time (APTT) results from a point-of-care testing (POCT) device (Rapidpoint Coag) with those from standard laboratory tests. The subjects were newborn infants needing coagulation screen for any clinical indications within a regional neonatal intensive care unit. The level of agreement between POCT and laboratory measurements of PT and APTT was determined. For PT: the bias was from -7.6 to 12.4 s and precision was 5.0 s. For the detection of prolonged PT at a level of 16 s, the sensitivity was 0.70, specificity was 0.57 and the positive predictive value (PPV) was 0.62. For APTT: the bias was from -39.1 to 23.7 s, and precision was 15.7 s. For the detection of prolonged APTT at a level of 55 s, the sensitivity was 0.80, specificity was 0.95 and the PPV was 0.80. The POCT device tested has limited utility as a cot-side device for screening for a prolongation of the APTT in the newborn but is not sensitive for screening for prolongation of the PT.

Effects of transfusion in anemia of prematurity.

The authors aimed to test the hypothesis that blood transfusions depress hematopoiesis in healthy infants with anemia of prematurity (AOP). They also set out to find markers that predict recovery from AOP. Thirty-nine premature babies underwent weekly and post-transfusion measurements of hemoglobin concentrations, reticulocyte counts (RCC), and erythropoietin levels (EPO). RCC and EPO dropped significantly 7 days after a blood transfusion but had normalized after 14 days. Elevated RCC or EPO levels were not predictive of an increase in hemoglobin. Postnatal HbFg/dL was higher in babies who had received transfusions. The authors conclude that blood transfusions depress erythropoiesis in infants with AOP and stimulate HbF synthesis but this effect is not sustained. Reticulocyte counts and erythropoietin levels are unhelpful in predicting recovery from AOP.

Clinical decision support systems for neonatal care.

BACKGROUND: Clinical decision support systems (CDSS) are computer-based information systems used to integrate clinical and patient information to provide support for decision-making in patient care. They may be useful in aiding the diagnostic process, the generation of alerts and reminders, therapy critiquing/planning, information retrieval, and image recognition and interpretation. CDSS for use in adult patients have been evaluated using randomised control trials and their results analysed in systematic reviews. There is as yet no systematic review on CDSS use in neonatal medicine. OBJECTIVES: To examine whether the use of clinical decision support systems has an effect on 1. the mortality and morbidity of newborn infants and 2. the performance of physicians treating them SEARCH STRATEGY: The standard search method of the Cochrane Neonatal Review Group was used. Searches were made of the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 1, 2004), MEDLINE (from 1966 to August 2004), EMBASE (1980-2004), CINAHL (1982 to August 2004) and AMED (1985 to August 2004). SELECTION CRITERIA: Randomised or quasi-randomised controlled trials which compared the effects of CDSS versus no CDSS in the care of newborn infants. Trials which compared CDSS against other CDSS were also considered. The eligible interventions were CDSS for computerised physician order entry, computerised physiological monitoring, diagnostic systems and prognostic systems. DATA COLLECTION AND ANALYSIS: Studies were assessed for eligibility using a standard pro forma. Methodological quality was assessed independently by the different investigators. MAIN RESULTS: Two studies fitting the selection criteria were found for computer aided prescribing and one study for computer aided physiological monitoring.Computer-aided prescribing: one study (Cade 1997) examined the effects of computerised prescribing of parenteral nutrition ordering. No significant effects on short-term outcomes were found and longer term outcomes were not studied. The second study (Balaguer 2001) investigated the effects of a database program in aiding the calculation of neonatal drug dosages. It was found that the time taken for calculation was significantly reduced and there was a significant reduction in the number of calculation errors.Computer-aided physiological monitoring: one eligible study (Cunningham 1998) was found which examined the effects of computerised cot side physiological trend monitoring and display. There were no significant effects on mortality, volume of colloid infused, frequency of blood gases sampling (samples per day) or severe (Papile Grade 4) intraventricular haemorrhage. Published data did not permit us to analyse effects on long-term neurodevelopmental outcome. AUTHORS' CONCLUSIONS: There are very limited data from randomised trials on which to assess the effects of clinical decision support systems in neonatal care. Further evaluation of CDSS using randomised controlled trials is warranted.

Detection and identification by real time PCR/RFLP analyses of Cryptosporidium species from human faeces.

AIMS: To detect a wide range of Cryptosporidium species from human faeces by analysis of the Cryptosporidium oocyst wall protein gene by PCR. METHODS AND RESULTS: The nested-assay comprised an initial amplification using a conventional thermocycler followed by real time PCR using a LightCycler with SYBR Green I for the characterization of the amplicons. The technique uses four sets of primers composed of five to six oligonucleotides with one to six base differences corresponding to the inter-species sequence differences of the gene fragment. Restriction fragment length polymorphism analysis identified Cryptosporidium hominis and C. parvum. The assay was evaluated using DNA extracted from purified material and faecal specimens containing a range of potential pathogens (including Cryptosporidium). The assay was specific, sensitive, reproducible and rapid. CONCLUSIONS: This unique technique enables the rapid detection of a range of polymorphic COWP gene sequences directly from faeces using real time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a novel approach to identification of Cryptosporidium species and the identification of C. hominis and C. parvum. The technique may be especially useful for the analysis of environmental samples which are likely to contain heterogeneous mixtures of Cryptosporidium species.

A mathematical model of CO2 variation in the ventilated neonate.

A mathematical model of the variation of partial pressure of carbon dioxide in the arterial blood of a ventilated neonate is developed. The model comprises alveolar, arterial, pulmonary, venous and tissue compartments, with gas exchange in the lung determined by inspiration and expiration terms. Gas exchange is modelled through diffusion and convective transfer. Carbon dioxide is produced in the tissue by a metabolic term. Shunting is modelled by allowing blood flow to bypass the pulmonary compartment in which diffusion takes place. The model predicts changes in the carbon dioxide partial pressures that occur following abrupt changes in the ventilation settings, and show broad agreement with actual data obtained from novel sensing technology.

Sequence of Plasmodium falciparum chromosomes 1, 3-9 and 13.

Since the sequencing of the first two chromosomes of the malaria parasite, Plasmodium falciparum, there has been a concerted effort to sequence and assemble the entire genome of this organism. Here we report the sequence of chromosomes 1, 3-9 and 13 of P. falciparum clone 3D7--these chromosomes account for approximately 55% of the total genome. We describe the methods used to map, sequence and annotate these chromosomes. By comparing our assemblies with the optical map, we indicate the completeness of the resulting sequence. During annotation, we assign Gene Ontology terms to the predicted gene products, and observe clustering of some malaria-specific terms to specific chromosomes. We identify a highly conserved sequence element found in the intergenic region of internal var genes that is not associated with their telomeric counterparts.

Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces.

An assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping of Giardia duodenalis on the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containing G. duodenalis and other parasites, and their corresponding immunofluorescence-stained fecal smears on glass microscope slides. The assay was specific and discriminated between G. duodenalis assemblages A and B. RFLP analysis further distinguished two groups (designated groups I and II) within assemblage A. Among 35 DNA samples extracted from whole feces from patients with confirmed sporadic giardiasis, the tpi gene was amplified from 33 (94%). Of these, nine (27%) samples contained assemblage A group II, 21 (64%) contained assemblage B, and 3 (9%) contained a mixture of assemblage A group II and assemblage B. The tpi gene of G. duodenalis assemblage B was amplified from 21 of 24 (88%) DNA samples extracted from whole feces from patients with confirmed cases of infection in a nursery outbreak. No amplification was detected from the remaining three DNA samples. Overall, analysis of DNA extracted from material recovered from stained microscope slides identified identical G. duodenalis genotypes in 35 (65%) of the 54 samples for which a genotype was established with DNA from whole feces. The heminested PCR method developed is sensitive, simple, and rapid to perform and is applicable for the analysis of other intestinal pathogens.

Central venous catheter tip in the right atrium: a risk factor for neonatal cardiac tamponade.

Fatal cardiac tamponade is a well recognised complication of the use of central venous catheters in neonatal patients. There is controversy over optimum catheter tip position to balance catheter performance against risk of adverse events. We report a series of five cases of tamponade occurring in one neonatal unit over a 4-year period, related to catheter tip placement in the right atrium. Right atrial catheter angulation, curvature or looping (CA) was present in all five cases on plain radiograph. It was frequently seen in other patients over the same period. Review of the literature indicates that CA was present in 6 of the 11 previous cases where the presence or absence of CA can be determined. Where right atrial catheter tip placement is accepted, clinicians should be aware of this characteristic catheter configuration, which is a major risk factor for cardiac tamponade. We recommend that catheter tips should not be placed in the right atrium to avoid risk of tamponade.

A high-resolution HAPPY map of Dictyostelium discoideum chromosome 6.

We have made a high-resolution HAPPY map of chromosome 6 of Dictyostelium discoideum consisting of 300 sequence-tagged sites with an average spacing of 14 kb along the approximately 4-Mb chromosome. The majority of the marker sequences were derived from randomly chosen clones from four different chromosome 6-enriched plasmid libraries or from subclones of YACs previously mapped to chromosome 6. The map appears to span the entire chromosome, although marker density is greater in some regions than in others and is lowest within the telomeric region. Our map largely supports previous gene-based maps of this chromosome but reveals a number of errors in the physical map. In addition, we find that a high proportion of the plasmid sequences derived from gel-enriched chromosome 6 (and that form the basis of a chromosome-specific sequencing project) originates from other chromosomes.

Effect of changes in oscillatory amplitude on PaCO(2) and PaO(2) during high frequency oscillatory ventilation.

AIMS: To describe the relation between oscillatory amplitude changes and arterial blood gas (ABG) changes in preterm infants receiving high frequency oscillatory ventilation, using a multiparameter intra-arterial sensor (MPIAS). METHODS: Continuous MPIAS ABG data were collected after amplitude changes and stratified according to FIO(2): high (> 0.4) or low (< 0.3). For each amplitude change, the maximum change (from baseline) in PaCO(2) and PaO(2) over the following 30 minutes was determined. In total, 64 oscillatory amplitude changes were measured in 21 infants (median birth weight 1040 g; gestation 27 weeks). RESULTS: All amplitude increases produced PaCO(2) falls (median -0.98 and -1.13 kPa for high and low FIO(2) groups respectively). All amplitude decreases produced PaCO(2) rises (median +0.94 and +1.24 kPa for high and low FIO(2) groups respectively). About 95% of the change in PaCO(2) was completed in 30 minutes. Amplitude changes did not affect PaO(2) when FIO(2) > 0.4. When FIO(2) < 0.3, amplitude increases produced a PaO(2) rise (median = +1.1 kPa; P < 0.001) and amplitude decreases a fall (median = -1.2 kPa; P < 0.001). CONCLUSIONS: After oscillatory amplitude changes, the speed but not the magnitude of the PaCO(2) change is predictable, and a rapid PaO(2) change accompanies the PaCO(2) change in infants with mild lung disease and a low FIO(2).

SEL1L, the human homolog of C. elegans sel-1: refined physical mapping, gene structure and identification of polymorphic markers.

We have cloned the human full-length cDNA SEL1L, which is highly similar to the C. elegans sel-1 gene, an important negative regulator of the "notch" pathway which acts as a key regulator of the cellular proliferation and specification processes in both vertebrates and invertebrates. The SEL1L gene maps to 14q24.3-31 and here we report its fine localization by HAPPY mapping, which determines its molecular distance to microsatellite markers isolated in the region. We have found two new polymorphic (CA)n microsatellites located in the gene, and have identified the exon-intron boundaries. The gene is composed of 21 exons spanning 70 kb of genomic DNA. Human SEL1L protein exhibits a high degree of similarity compared to the mouse and nematode homologs.

Genomic and functional map of the chromosome 14 t(12;14) breakpoint cluster region in uterine leiomyoma.

A translocation involving chromosomes 12 and 14 [t(12;14)(q15;24.1)] is commonly seen in benign smooth muscle tumor as uterine leiomyoma (UL). A contig of P1-derived artificial chromosome and bacterial artificial chromosome clones on chromosome 14, encompassing a t(12;14) breakpoint cluster region (BCR) in UL, was generated principally using the recently developed HAPPY map of chromosome 14 as a framework (P. H. Dear et al., 1998, Genomics 48: 232-241). Three UL t(12;14) breakpoints have been localized within this contig, showing that a BCR of at least 400 kb exists on chromosome 14. Other studies of tumors with t(12;14) rearrangements similarly show breakpoints within a 475-kb multiple aberration region on chromosome 12. Thus t(12;14) is an example of a translocation in which the breakpoints are located within a BCR on both chromosome 12 and chromosome 14, justifying the identification of expressed sequences that are altered in these BCR regions. A total of four expressed sequences were identified in the BCR on chromosome 14. Two of these were novel cDNAs (D14S1460E and D14S1461E). The chromosome 14 cDNAs were expressed in multiple adult tissues. The identification of a large breakpoint cluster region on chromosome 14 suggests that translocations in this region mediate their effects at a distance and also that elements that predispose this region to recurrent chromosomal translocation may be widely distributed.

Ribonuclease k6: chromosomal mapping and divergent rates of evolution within the RNase A gene superfamily.

We have localized the gene encoding human RNase k6 to within approximately 120 kb on the long (q) arm of chromosome 14 by HAPPY mapping. With this information, the relative positions of the six human RNase A ribonucleases that have been mapped to this locus can be inferred. To further our understanding of the individual lineages comprising the RNase A superfamily, we have isolated and characterized 10 novel genes orthologous to that encoding human RNase k6 from Great Ape, Old World, and New World monkey genomes. Each gene encodes a complete ORF with no less than 86% amino acid sequence identity to human RNase k6 with the eight cysteines and catalytic histidines (H15 and H123) and lysine (K38) typically observed among members of the RNase A superfamily. Interesting trends include an unusually low number of synonymous substitutions (Ks) observed among the New World monkey RNase k6 genes. When considering nonsilent mutations, RNase k6 is a relatively stable lineage, with a nonsynonymous substitution rate of 0.40 x 10(-9) nonsynonymous substitutions/nonsynonymous site/year (ns/ns/yr). These results stand in contrast to those determined for the primate orthologs of the two closely related ribonucleases, the eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), which have incorporated nonsilent mutations at very rapid rates (1.9 x 10(-9) and 2.0 x 10(-9) ns/ns/yr, respectively). The uneventful trends observed for RNase k6 serve to spotlight the unique nature of EDN and ECP and the unusual evolutionary constraints to which these two ribonuclease genes must be responding. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF037081-AF037090.]

A high-resolution metric HAPPY map of human chromosome 14.

We have mapped 1001 novel sequence-tagged sites on human chromosome 14. The mean spacing between markers is approximately 90 kb, most markers are mapped with a resolution of better than 100 kb, and physical distances are determined. The map was produced using HAPPY mapping, a simple and widely applicable in vitro approach that is analogous to linkage or to radiation hybrid mapping, but that circumvents many of the difficulties and potential artifacts associated with these methods. We show also that the map serves as a robust scaffold for building physical maps using large-insert clones.

A HAPPY map of Cryptosporidium parvum.

We have constructed a HAPPY map of the apicomplexan parasite Cryptosporidium parvum. We have placed 204 markers on the 10.4-Mb genome, giving an average marker spacing of approximately 50 kb, with an effective resolution of approximately 40 kb. HAPPY mapping (an in vitro linkage technique based on screening approximately haploid amounts of DNA by the polymerase chain reaction) is fast and accurate and is not subject to the distortions inherent in cloning, meiotic recombination, or hybrid cell formation. In addition, little genomic DNA is needed as a substrate, and the AT content of the genome is largely immaterial, making it an ideal method for mapping otherwise intractable parasite genomes. The map, covering all eight chromosomes, consists of 10 linkage groups, each of which has been chromosomally assigned. We have verified the accuracy of the map by several methods, including the construction of a >140-kb PAC contig on chromosome VI. Less than 1% of our markers detect non-rDNA duplicated sequences.

Life-threatening transient neonatal Behçet's disease.

This case report describes transient neonatal Behçet's disease, with life-threatening complications in the neonate. Male Baby R developed blood-streaked diarrhoea 5 days after birth, followed by recurrent severe scarring orogenital ulceration and vasculitic skin lesions. In this sixth week of life, he developed stridor leading to a respiratory arrest and necessitating assisted ventilation. No infective cause was isolated. Baby R responded well to i.v. and subsequent oral steroid therapy. At 8 weeks old he had fully recovered and remains well. Baby R's mother was not previously known to have Behçet's disease. During the pregnancy, she began to suffer orogenital ulceration, associated with skin lesions typical of Behçet's disease. Mild orogenital ulceration has become recurrent.