Extensive epigenetic and transcriptomic variability between genetically identical human B-lymphoblastoid cells with implications in pharmacogenomics research

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The impact of delayed sample handling and type of anticoagulant on the interpretation of dysplastic signs detected by flow cytometry

Introduction: A growing body of evidence supports the usefulness of dysplastic signs detected by flow cytometry in the diagnosis of myelodysplastic syndromes (MDS). Our aim was to assess the impact of pre-analytical variables (delayed sample handling, type of anticoagulant, and different clones of antibody) in the interpretation of flow cytometric results. Material and methods: Bone marrow samples were labelled and analysed immediately after aspiration and on two consecutive days. The effect of anticoagulant type was evaluated in 16 bone marrow samples. Thirty-seven different immunophenotypic variables were recorded after eight-colour staining. Furthermore, 8 normal peripheral blood samples collected in K3-EDTA and Na-heparin were examined with different clones of CD11b antibodies and four parameters were recorded with both anticoagulants on two consecutive days. Results: Fourteen significant differences were detected in the initial immunophenotype of fresh samples collected in K3-EDTA and Na-heparin. Regardless of the anticoagulant type, eleven parameters remained stable despite delayed sample handling. Due to delayed sample processing, more alterations were detected in the samples collected in K3-EDTA than in the samples collected in Na-heparin. The type of CD11b clone influenced the reduction of fluorescence intensity only in samples collected in K3-EDTA, where the alterations were contrary to the changes observed in Na-heparin. Conclusions: Delayed sample processing causes considerable immunohenotypic alterations, which can lead to false interpretation of the results. If delayed sample evaluation is unavoidable, markers that remain more stable over time should be considered with more weight in the diagnosis of MDS

Ritka öröklött és szerzett haemorrhagiás diathesisek molekuláris elemzése, a rendellenességek strukturális és funkcionális következményei = Molecular evaluation of rare inherited and acquired bleeding diatheses, structural and functional consequences of the disorders

Ritka trombocita funkciós zavarok és koagulopátiák vizsgálata molekuláris genetikai és fehérje szinten. Az alábbiakban a projekt három legfontosabb eredményét soroljuk fel: 1/Két új mutációnak a felfedezése a trombocita glikoprotein (GP) IIb-ben, melyek II-es típusú Glanzmann thrombastheniát okoztak. A mutáns fehérjék transzfektált sejtekben történő analízise rávilágított: a) a "thigh" domén szerepére a GPIIIa-val való komplex képződésben, b) a "calf-2" domén fontoságára a fehérje érési folyamatában és intracelluláris vándorlásában. 2/ A factor X (FX) génjében bekövetkezett homozigóta c.730G>A/p.Gly204Arg mutáció következtében a FX antigén hiányát észleltük egy súlyos vérzékenységben szenvedő gyermekben. Az aminósav csere destabilizálta a FX két láncát összetartó diszulfid hidat. A mutáns fehérje eltérül a normális szekréciós úttól, megakad a transz Golgi-késő endoszóma szinten, s így nem szekretálódik. 3/ Egy SLE-ben szenvedő betegen életet veszélyeztető vérzés alakult ki. A beteg vizsgálata a szerzett faktor XIII (FXIII) deficiencia egy új, eddig nem leírt formájának a felismeréséhez vezetett. A beteg plazmájában FXIII aktivitás, FXIII komplex, FXIII A és B alegység antigén nem volt kimutatható, míg trombocitákban a FXIII aktivitás és FXIII-A antigén normál volt. A súlyos vérzést egy autoantitest okozta, mely a komplexben lévő és szabad FXIII-B-hez egyaránt kötődött és nagyon kifejezetten meggyorsította eliminációjukat a keringésből. | Rare platelet function disorders and coagulopathies were investigated at molecular genetic and/or protein level. Below the three most important results of the project are described: 1/ Investigation of a patient with type II Glanzmann thrombasthenia led to the discovery of two novel causative mutations in the platelet glycoprotein (GP) IIb gene. Analysis of the mutant proteins in transfected cells shed light a) on the role of thigh domain in the complex formation with GPIIIa, b) on the involvement of calf-2 domain in the maturation and intracellular trafficking of the protein. 2/ Due to a homozygous novel c.730G>A/p.Gly204Arg mutation in the factor X (FX) gene no FX antigen was found in the plasma of a child with severe bleeding diathesis. The amino acid replacement destabilized the disulfide bond that holds the two FX chains together. The mutant protein could not be secreted; it was diverted from the normal secretory pathway and retained at the trans Golgi-late endosome level. 3/ In an SLE patient with life threatening bleeding a new form of acquired factor XIII (FXIII) deficiency was discovered. The patient had undetectable FXIII activity, FXIII complex, FXIII A and B subunit antigen levels in the plasma, while in platelets FXIII activity and FXIII-A antigen level was normal. It was revealed that the patient developed an autoantibody that bound to FXIII-B both in complex and free form and highly accelerated their elimination from the circulation

A véralvadás XIII-as faktora = Blood coagulation factor XIII.

I. A XIII-as faktor (FXIII) struktúrája és funkciója Az aktivációs peptid nélkül a FXIII-A instabil. A FXIII aktivációt a plazmában a fibrin polimerizáció determinálja, a FXIII-A Val34Leu polimorfizmusnak csak moduláló hatása van. A FXIII A és B alegységének kapcsolódását gátló monoklonális antitestek előállítása. A celluláris FXIII szerepet játszik a monocyták/macrophagok phagocytosisában. Az alvadékban aktiválódó granulocytákból felszabadult proteázok lebontják a FXIIIa-t. II. Klinikai FXIII kutatások Új típusú módszer a FXIII (ill. más transzglutaminázok) mérésére, ill. a FXIII-A Val34Leu polimorfizmus kimutatására. Az intracelluláris FXIII-A áramlásos citometriás kimutatására új módszer, mely kiválóan alkalmazható az acut myleoid leukemiák diagnosztikájában. Csontvelő abláció során szignifikánsan csökken a plazma FXIII szintje, magas thrombocyta számmal járó myeloproliferatív megbetegedésekben viszont emelkedik. Nőkben az emelkediett FXIII szint több mint kétszeresére növeli a myocardiális infarctus rizikóját nőkben. Krónikus bronchoalveoláris megbetegedésekben jelentősen emelkedik a bronchoalveoláris mosófolyadékban a FXIII mennyisége. 4 új mutációt írtunk le FXIII hiányos betegekben, s analizáltuk ezek fehérje biokémiai következményeit. FXIII hiányíos transzgén egereken igazoltuk, hogy a FXIII szükséges a normális sebgyógyuláshoz. | I. Structure and function of factor XIII (FXIII) The absence of activation peptide makes FXIII-A instable. The activation of FXIII in the plasma is determined by fibrin polymerization; FXIII-A Val34Leu polymorphism only modulates activation. Production of monoclonal antibodies with inhibitory effect on the association of FXIII A and B subunits. Cellular FXIII plays a role in the phagocytosis by monocytes/macrophages. Proteases released from granulocytes in the clot break down activated FXIII. Clinical studies on FXIII New methods on the measurement of FXIII activity, and on the detection of FXIII-A Val34Leu polymorphism. Method on the detection of intracellular FXIII-A and its application to the diagnosis of acute myeloid leukemias. Bone marrow ablation results in the significant decrease of plasma FXIII level; in myeloproliferative diseases with high platelet count plasma FXIII is elevated. In women elevated FXIII level increases the risk for myocardial infarction by more than two-folds. In chronic bronchoalveolar inflammation the amount of FXIII in the lavage fluid is significantly increased. Description of four new mutations in FXIII deficient patients, and protein structural analysis of their consequences. It was demonstrated on FXIII deficient transgene mice that FXIII is required for normal wound healing

Coagulation FXIII-A protein expression defines three novel sub-populations in pediatric B-cell progenitor acute lymphoblastic leukemia characterized by distinct gene expression signatures

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An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia : An I-BFM-FLOW-Network Report

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.publishersversionPeer reviewe