Evaluation of the appropriateness of frozen plasma usage after introduction of prothrombin complex concentrates: a retrospective study.

BACKGROUND: Prothrombin complex concentrates (PCCs) can be used instead of frozen plasma (FP) transfusion to reverse the effect of warfarin. Audits have demonstrated over usage of FP transfusions even before the introduction of PCC. The objective of this study was to determine the appropriateness of current FP transfusion practice in the current era since the introduction of PCCs. METHODS: A retrospective cohort study of consecutive patients receiving FP over 3 months was carried out. Each episode of FP use over a 24-h period was adjudicated independently by two reviewers as appropriate (consistent with Canadian/AABB guidelines), appropriate but inconsistent with guidelines or inappropriate. Discrepancies were resolved by a third reviewer. Use of FP to reverse warfarin was considered inappropriate. FP usage from previous years was assessed as baseline. RESULTS: During the study period, 111 FP transfusions were administered. 74.8% of FP usage occurred in the ICU. The proportion of FP transfusions that were deemed appropriate, inconsistent yet appropriate or inappropriate were 33/89 (37.1%), 16/89 (18.0%) and 40/89 (44.9%), respectively, when use of FP for therapeutic plasma exchange was excluded. The most common reasons for inappropriate use were the absence of bleeding with an increased INR or warfarin reversal. CONCLUSION: Our study is the first to audit FP transfusions in the post-PCC era in Canada. FP usage remains inappropriately high in INR prolongation without another indication or to reverse warfarin. Targeted interventions to reduce FP usage in the future should focus on the ICU and on education about warfarin reversal.

Treatment with tirofiban for acute coronary syndrome (ACS): a systematic review and network analysis.

OBJECTIVE: To assess the efficacy of tirofiban in comparison to usual care or other GPIIb/IIIa antagonists (eptifibatide and abciximab). Results were analysed by drug administration with planned percutaneous coronary intervention (PCI) or as medical management without planned PCI, and separately for STEMI or NSTE ACS patients. RESEARCH DESIGN AND METHODS: A systematic review was performed of randomized controlled trials of tirofiban, abciximab, eptifibatide or usual care given to patients with acute coronary syndrome. Nine databases were searched up to March 2010. Pair-wise meta-analysis was used to combine all available direct comparisons; indirect comparisons and network analysis were performed when this was not possible. The primary outcome was MACE (major adverse cardiac event). RESULTS: The search yielded 8, 119 records and 50 trials were included (total number of patients = 52,958). Compared to usual care, high and medium-dose tirofiban (25 and 10 µg/kg/min) administered with planned PCI reduced MACE at 30 days for patients with STEMI (RR 0.67, 95% CI 0.45, 0.99; RR 0.28, 95% CI 0.10, 0.80), but was not effective as a medical management. Medium-dose tirofiban (10 µg/kg/min) administered with planned PCI or low dose (0.4 µg/kg/min) as medical management reduced the risk of MACE for patients with NSTE ACS (RR 0.39, 95% CI 0.21, 0.75; RR 0.58, 95% CI 0.41, 0.83) in comparison to usual care, but at the expense of increased thrombocytopenia (RR 3.26, 95% CI 1.31, 8.13). Evidence from RCTs and network analysis indicated tirofiban and abciximab were equally effective and safe. Comparing tirofiban and eptifibatide treatment by indirect and network analysis produced inconclusive results. CONCLUSIONS: Tirofiban was more effective than usual care for STEMI and NSTE ACS patients receiving planned PCI, and NSTE ACS patients receiving medical management. Tirofiban and abciximab were equally effective. Comparisons of tirofiban and eptifibatide were inconclusive.

Analysis of Brca1-deficient mouse mammary glands reveals reciprocal regulation of Brca1 and c-kit.

Disruption of the breast cancer susceptibility gene Brca1 results in defective lobular-alveolar development in the mammary gland and a predisposition to breast tumourigenesis in humans and in mice. Recent evidence suggests that BRCA1 loss in humans is associated with an expansion of the luminal progenitor cell compartment in the normal breast and tumours with a luminal progenitor-like expression profile. To further investigate the role of BRCA1 in the mammary gland, we examined the consequences of Brca1 loss in mouse mammary epithelial cells in vitro and in vivo. Here, we show that Brca1 loss is associated with defective morphogenesis of SCp2 and HC11 mouse mammary epithelial cell lines and that in the MMTV-Cre Brca1(Co/Co) mouse model of Brca1 loss, there is an accumulation of luminal progenitor (CD61(+)CD29(lo)CD24(+)) cells during pregnancy. By day 1 of lactation, there are marked differences in the expression of 1379 genes, with most significantly altered pathways and networks, including lactation, the immune response and cancer. One of the most differentially expressed genes was the luminal progenitor marker, c-kit. Immunohistochemical analysis revealed that the increase in c-kit levels is associated with an increase in c-kit positivity. Interestingly, an inverse association between Brca1 and c-kit expression was also observed during mammary epithelial differentiation, and small interfering RNA-mediated knockdown of Brca1 resulted in a significant increase in c-kit mRNA levels. We found no evidence that c-kit plays a direct role in regulating differentiation of HC11 cells, suggesting that Brca1-mediated induction of c-kit probably contributes to Brca1-associated tumourigenesis via another cellular process, and that c-kit is likely to be a marker rather than a mediator of defective lobular-alveolar development resulting from Brca1 loss.

Differential azole antifungal efficacies contrasted using a Saccharomyces cerevisiae strain humanized for sterol 14 alpha-demethylase at the homologous locus.

Inhibition of sterol-14 alpha-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of Saccharomyces cerevisiae that was humanized with respect to the amino acids encoded at the CYP51 (ERG11) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to >1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14 alpha-methyl-ergosta-8, 24(28)-dien-3beta-6 alpha-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.

Clinical, enzymatic and molecular characterization of nine new patients with malonyl-coenzyme A decarboxylase deficiency.

We report nine new patients with malonic aciduria associated with enzyme-confirmed malonyl-CoA decarboxylase (MCD) deficiency in eight. Clinical details were available on eight, and molecular genetic characterization was obtained for nine. As for 15 previously described patients, cardinal clinical manifestations included developmental delay and cardiomyopathy; metabolic perturbations (e.g. acidosis) and seizures, however, were infrequent or not observed in our patients. For all, detection of elevated malonic acid in urine (+/- increased C3DC acylcarnitine by analysis employing tandem mass spectrometry) led to pursuit of enzyme studies. MCD activities (nmol/h PER mg protein) revealed: control (n = 22), 16.2 +/- 1.8 (SEM; range 5.7-46.2); patients (n = 8, assayed in duplicate), 1.7 +/- 0.3 (10% of parallel control; range 0.6-2.8). Molecular characterization by DNA sequence analysis and multiplex ligation-dependent probe amplification revealed nine novel mutations (c.796C>T; p.Gln266X, c.481delC; p.Leu161CysfsX18, c.1367A>C; p.Tyr456Ser, c.1319G>T; p.Ser440Ile, c.1430C>T; p.Ser477Phe, c.899G>T; p.Gly300Val, c.799-1683_949-1293del3128, and two other large genomic deletions comprising exons 1 or the complete gene) and two known mutations in the MLYCD gene. Our findings increase the number of enzyme-confirmed MCD-deficient patients by >50%, and expand our understanding of the phenotypic and molecular heterogeneity of this rare disorder.

A randomized clinical trial comparing 'one-step' and 'two-step' orthodontic bonding systems.

OBJECTIVE: The primary objective of this prospective clinical trial was to assess the clinical bond failure rates of orthodontic brackets bonded using a self-etching primer (SEP), compared with brackets bonded using a conventional acid-etched technique with control adhesive (Transbond). A secondary aim was to investigate whether characteristics of the operator, patient or tooth bonded had any influence on bracket failure. DESIGN: Single-centre randomized controlled clinical trial. Thirty-four patients were bonded, each being randomly assigned to either the test or control adhesive. SETTING: NHS Hospital Orthodontic Department, Chester, UK. SUBJECTS: Orthodontic patients requiring fixed appliance treatment. MAIN OUTCOME MEASURES: Bond failure. MAIN OUTCOME RESULTS: Failure rates over the initial 6-month period were 2.0% (Transbond) and 1.7% (SEP) with no statistically significant difference between the two groups. Over the duration of the fixed appliance treatment, bond failure rates increased, but remained acceptable at 7.4 % (TB) and 7.0% (SEP), respectively. When operator, patient and tooth characteristics were analysed, only the bracket location was found to be significant. Maxillary brackets were more likely to fail than mandibular brackets (RR 0.47%; 95% CI 0.22, 1.03). The failure rate for brackets in our study was low when compared with previous studies. CONCLUSIONS: Both the acid-etched control and self-etching primer in combination with adhesive pre-coated brackets were successful for clinical bonding. Their combined failure rate was lower than that reported in similar trials.

Decreased cholesterol synthesis as a possible aetiological factor in malformations of trisomy 18.

We report a series of neonates and foetuses with trisomy 18 and abnormally low cholesterol levels and propose that down regulation of cholesterol synthesis in trisomy 18 is, in part, responsible for its phenotype. Cholesterol is a major structural lipid of cell membranes, as well as the precursor of steroid hormones and bile acids. Several human malformation syndromes have been identified biochemically as disorders of cholesterol biosynthesis. Trisomy 18, a multi-system malformation syndrome, has clinical features that overlap with those of disorders of cholesterol biosynthesis and dysregulation of this pathway may have a role in the developmental pathology.

Biochemical, clinical and molecular findings in LCHAD and general mitochondrial trifunctional protein deficiency.

General mitochondrial trifunctional protein (TFP) deficiency leads to a wide clinical spectrum of disease ranging from severe neonatal/infantile cardiomyopathy and early death to mild chronic progressive sensorimotor poly-neuropathy with episodic rhabdomyolysis. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency resulting from the common Glu510Gln mutation usually gives rise to a moderately severe phenotype with multiorgan involvement with high morbidity and mortality. However, isolated LCHAD deficiency can also be consistent with long-term survival in patients identified and treated from an early age. We present biochemical, clinical and mutation data in 9 patients spanning the full spectrum of disease. Fibroblast acylcarnitine profiling shows good correlation with clinical phenotype using the ratio C18(OH)/(C14(OH)+C12(OH)). This ratio shows a gradation of values, from high in four patients with severe neonatal disease (2.5+/-0.8), to low in two neuromyopathic patients (0.35, 0.2). Fibroblast fatty acid oxidation flux assays also show correlation with the patient phenotype, when expressed either as percentage residual activity with palmitate or as a ratio of percentage activity of myristate/oleate (M/O ratio). Fibroblasts from four patients with severe neonatal disease gave an M/O ratio of 4.0+/-0.6 compared to 1.97 and 1.62 in two neuromyopathic patients. Specific enzyme assay of LCHAD and long-chain 3-ketothiolase activity in patient cells shows lack of correlation with phenotype. These results show that measurements in intact cells, which allow all determinative and modifying cellular factors to be present, better reflect patient phenotype. Mutation analysis reveals a number of alpha- and beta-subunit mutations. Peripheral sensorimotor polyneuropathy, often as the initial major presenting feature but usually later accompanied by episodic rhabdomyolysis, is a manifestation of mild TFP protein deficiency. The mild clinical presentation and relative difficulty in diagnosis suggest that this form of TFP is probably underdiagnosed.

Mutation and biochemical analysis in carnitine palmitoyltransferase type II (CPT II) deficiency.

Carnitine palmitoyltransferase type II (CPT II) deficiency has three basic phenotypes, late-onset muscular (mild), infantile/juvenile hepatic (intermediate) and severe neonatal. We have measured fatty acid oxidation and CPT II activity and performed mutation studies in 24 symptomatic patients representing the full clinical spectrum of disease. Severe and intermediate phenotypes show a clear correlation with biochemical indices and genetic analysis revealed causative mutations in most patients. Studies of mild phenotypes suggest a more complex interaction, with higher residual fatty acid oxidation, a wider range of CPT II activity (10-60%) but little evidence of genotype-phenotype correlation. Residual CPT II mutant protein from myopathic patients shows thermal instability at 41 degrees C. The common 'polymorphisms' V3681 and M647V are strikingly overrepresented in the myopathic patients, the implication being that they may significantly influence the manifestation of clinical disease and could therefore potentially be considered as a susceptibility variants. Among myopathic individuals, males comprised 88% of patients, suggesting increased susceptibility to clinical disease. A small number of symptomatic patients appear to have significant residual CPT II activity (42-60%) The synergistic interaction of partial deficiencies of CPT II, muscle adenosine monophosphate deaminase and possibly other enzymes of muscle energy metabolism in the aetiology of episodic myopathy deserves wider consideration.

Fish odour syndrome with features of both primary and secondary trimethylaminuria.

We report a patient with the fish odour syndrome who has both primary and secondary trimethylaminuria. The diagnosis was made using biochemical and genetic analysis in the apparent absence of any characteristic smell. Differentiation of primary and secondary trimethylaminuria is usually made on urinary analysis of trimethylamine and its metabolite trimethylamine N-oxide, with different, characteristic patterns of both compounds in primary and secondary trimethylaminuria. Our patient had biochemical analysis consistent with a diagnosis of secondary trimethylaminuria, while analysis of the flavin-containing mono-oxygenase 3 gene, the causative gene in primary trimethylaminuria, demonstrated three sequence polymorphisms, two of which are known to reduce enzyme activity. The patient showed temporary clinical and biochemical response to treatment with metronidazole and neomycin. It is important to be aware of this diagnosis in patients without obvious clinical signs, and of the subjective benefits of treatment.

Prenatal diagnosis of coarctation of the aorta improves survival and reduces morbidity.

OBJECTIVE: To investigate whether antenatal diagnosis of coarctation of the aorta results in reduced mortality and improved preoperative haemodynamic stability compared with postnatal diagnosis. DESIGN: Retrospective review of all cases of coarctation of the aorta presenting to a tertiary fetal and neonatal cardiology service from January 1994 to December 1998. METHODS: Prenatal, postnatal, and necropsy records were reviewed to determine survival in the two groups. Markers of preoperative illness severity were recorded, including presence of femoral pulse, collapse, left ventricular function, ductal patency on echocardiography, coagulation status, duration of intensive care unit and total hospital stay, heart rate, respiratory rate, plasma creatinine, plasma potassium, and right upper limb blood pressure. A univarate and multivariate analysis was conducted on all variables and a cumulative score was created and subjected to logistic regression analysis. RESULTS: Both collapse and death were more common in the postnatally diagnosed group (p < 0.05). Femoral pulses were more likely to be palpable and there was echocardiographic evidence of duct patency in the antenatally diagnosed infants (p < 0.001 and p < 0.05, respectively). An increased respiratory rate was associated with postnatal presentation (p < 0.05). Infants with haemodynamic instability preoperatively were more likely to have been diagnosed postnatally (p < 0.01). CONCLUSIONS: Antenatal diagnosis of coarctation of the aorta is associated with improved survival and preoperative clinical condition.

2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency in a 23-year-old man.

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.178) deficiency is a recently described defect of isoleucine catabolism. The disorder is characterized by normal early development followed by a progressive loss of mental and motor skills. Deterioration may be rapid or may follow a slower decline with a possible stabilization of the disorder on a low-protein diet and appropriate medication. We report a 23-year-old man with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency with a very mild clinical course. He had apparently normal early development and remained relatively well until the age of 6 years, when he contracted measles. Following this illness, his motor skills and school progress deteriorated. At 15 years he had significant dysarthria, and generalized rigidity with some dystonic and unusual posturing. He was then treated with a low-protein high-carbohydrate diet with a good response in terms of balance and gait. At 18 years he was given benzhexol (Artane), increased slowly from 2 mg to 6 mg daily, resulting in improvement in tremor and dystonia. At 23 years he can dress himself and works in sheltered employment but remains severely dysarthric.

Plant sterol 14 alpha-demethylase affinity for azole fungicides.

Azole fungicides were thought to have much greater affinity for the fungal cytochrome P450 enzyme, sterol 14 alpha-demthylase (CYP51) than the plant orthologue. Using purified CYP51 from the plant Sorghum bicolor L Moenech, a direct comparison of the sensitivity to the fungicides triadimenol and tebuconazole has been carried out. S. bicolor CYP51 was purified to homogenity as determined by SDS--PAGE and specific heme content. Addition of the azole fungicides triadimenol and tebuconazole induced type II spectral changes, with saturation occurring at equimolar azole/P450 concentrations. Inhibition of reconstituted activities revealed only a threefold insensitivity of the plant CYP51 compared to a fungal CYP51, from the phytopathogen Ustilago maydis, as judged by IC(50) values. The implications for fungicide mode of action and application are discussed.

Features of carnitine palmitoyltransferase type I deficiency.

Carnitine palmitoyltransferase type I (CPT I) is unique among long-chain fatty acid oxidation enzymes in that there are two tissue-specific isoforms, 'hepatic' and 'muscle', which are encoded by two separate genes. The 'hepatic' isoform is expressed in liver, kidney and fibroblasts and at low levels in the heart, while the other isoform occurs in skeletal muscle and is the predominant form in heart. Reported patients with CPT I deficiency lack activity of the hepatic isoform and present before 30 months of age with hypoketotic hypoglycaemia, hepatomegaly with raised transaminases, seizures and coma. We discuss four new cases in three families showing, variously, renal tubular acidosis, transient hyperlipidaemia and, paradoxically, myopathy with elevated creatinine kinase or cardiac involvement in the neonatal period as additional features that deserve wider recognition.

Treatment and outcome of serious structural congenital heart disease.

Serious structural congenital heart disease usually presents to the neonatal paediatrician, although increasingly these conditions are being diagnosed before birth. It is, therefore, important that those dealing with these fetuses and infants have some knowledge of their natural and modified history. The vast majority of lesions can either be corrected or given symptomatic palliation and this review discusses treatment options and provides up-to-date outcome information to enable fetal and neonatal staff to anticipate and to complement information given to families by paediatric cardiologists.