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Moore G.W.,GSTS Pathology
Seminars in Thrombosis and Hemostasis | Year: 2014

The International Society on Haemostasis and Thrombosis (ISTH) and the British Committee for Standards in Haematology (BCSH) have recently updated their lupus anticoagulant (LA) detection guidelines. The Clinical and Laboratory Standards Institute (CLSI) subsequently will publish its first LA guideline. General agreement exists on issues such as sample preparation, the use of dilute Russell viper venom time (dRVVT) in diagnostic repertoires, the use of normalized ratios, calculations to demonstrate phospholipid dependence, calculations to demonstrate inhibition, and interpretive reporting. The ISTH recommendation to employ only dRVVT and activated partial thromboplastin time is not mirrored in the BCSH and CLSI documents. The potential for false negatives in mixing tests is acknowledged by all panels, yet they remain mandated by ISTH as there are occasions when they are crucial to diagnostic accuracy. BCSH indicates that a negative mixing test need not exclude the presence of a LA, and CLSI reprioritizes test order to screen-confirm-mix, the latter being considered unnecessary in specific circumstances. Opinions in the guidelines differ on setting cutoff levels (i.e., 97.5th vs. 99th percentile for normally distributed data). All guidelines cover testing of anticoagulated patients, more detail being given by BCSH and CLSI, who suggest that Taipan snake venom time is a useful adjunct test in patients receiving vitamin K antagonists. Although complete agreement is not apparent, the guidelines represent significant moves toward engendering common practices. © 2014 by Thieme Medical Publishers, Inc. Source

Mann K.,GSTS Pathology | Ogilvie C.M.,Guys and St Thomas NH Foundation Trust
Prenatal Diagnosis | Year: 2012

Quantitative fluorescent polymerase chain reaction has been in diagnostic use in the UK for over 10years and has proved to be a cost-effective, robust and accurate rapid prenatal test for common aneuploidies. Specific advantages include detection of triploidy, mosaicism and maternal cell contamination. Its application at our centre is described, with developments including stand-alone testing and improvements in strategies for the preparation and testing of chorionic villus biopsies. © 2012 John Wiley & Sons, Ltd. Source

Patel Y.M.,Kings College London | Lordkipanidze M.,University of Birmingham | Lowe G.C.,University of Birmingham | Nisar S.P.,University of Bristol | And 7 more authors.
Journal of Thrombosis and Haemostasis | Year: 2014

Summary: Background: The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY12 mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y12 receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. Objectives: To determine the functional consequences of the R122C substitution for P2Y12 function. Patient/methods: We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y12 stably expressed in cells was also performed. Results: ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y12 activity in the patient and family members. Cell surface R122C P2Y12 expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. Conclusions: Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait. © 2014 International Society on Thrombosis and Haemostasis. Source

Scriven P.N.,GSTS Pathology
Journal of visualized experiments : JoVE | Year: 2011

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting. Source

Scriven P.N.,GSTS Pathology | Scriven P.N.,Center for Preimplantation Genetic Diagnosis | Bossuyt P.M.M.,University of Amsterdam
Human Reproduction | Year: 2010

BACKGROUND: The aim of this study was to develop and use theoretical models to investigate the accuracy of the fluorescence in situ hybridization (FISH) technique in testing a single nucleus from a preimplantation embryo without the complicating effect of mosaicism. METHODS: Mathematical models were constructed for three different applications of FISH in preimplantation genetic testing (sex determination for sex-linked diseases, two-way reciprocal translocations and sporadic chromosome aneuploidy). The input values were the degree of aneuploidy (initially set at 3 per chromosome for sporadic aneuploidy) and the accuracy per probe (initially set at 95), defined as the proportion of normal diploid nuclei with a normal signal pattern. The primary statistic was the predictive value of the test result. RESULTS: Testing two chromosome pairs to determine sex chromosome status or detect unbalanced translocation products had high predictive value: at least 99.5 for a normal test result (95 CI: 99-100), and 90 for an abnormal test result (95 CI: 88-92). However, the predictive value of an abnormal test result testing five chromosomes for sporadic chromosome aneuploidy was 41 (95 CI: 36-46); 90 would be achieved with an aneuploidy rate per chromosome of 20.3 (equivalent to 99.5 prevalence for 23 chromosomes) rather than 3, or with an accuracy per probe of 99.6 rather than 95, or when testing 23 chromosome pairs, rather than 5 pairs, with either 8.3 aneuploidy (86.4 prevalence) or 99.5 accuracy. CONCLUSIONS: Testing a single cell using the FISH technique has the potential to achieve acceptable analytical performance for sex determination and two-way reciprocal translocations, but is unlikely to achieve adequate performance testing for sporadic chromosome aneuploidy. New techniques for detecting the copy number of every chromosome are emerging, but it remains to be seen if the high accuracy required will be achieved. © 2010 The Author. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. Source

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