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22 nd International Congress of Clinical Chemistry and Laboratory Medicine

22 nd International Congress of Clinical Chemistry and Laboratory Medicine. Istanbul, Turkey- Istanbul Congress Center- June 22-26, 2014. SYM 19-New advances in prenatal and postnatal testing.

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22 nd International Congress of Clinical Chemistry and Laboratory Medicine

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  1. 22nd International Congress of Clinical Chemistry and Laboratory Medicine Istanbul, Turkey- Istanbul Congress Center- June 22-26, 2014 SYM 19-New advances in prenatal and postnatal testing Non invasive prenatal fetal blood group genotyping in the monitoring of allo-immunized anti-KEL1 pregnant women: experience of CNRHP Agnes MaillouxCentre National de Référence en Hémobiologie Périnatale (CNRHP) Hôpitaux Universitaires de l’Est Parisien – AP-HP - Paris

  2. INTRODUCTION • Hemolyticdisease of the fetus and newborn (HDFN) is a severe disease, resulting from maternal red cell allo-antibodies directed against fetal red cells. • Three antibodies are associated with severe fetal disease: anti-RH1, anti-RH4 and anti-KEL1 • This disease is caracterised by anemia and hyperbilirubinemia which may lead to fetalhydrops, kernicterus or death. • KEL1 antibodies cause supression of KEL1-positive erythroid precursor cells. KEL1 allo-immunization is the second major cause for fetalhemolyticanemia and has still increasing incidence. • Successful management of RBC-alloimmunized pregnancies depends on early detection of fetalanemia and timely intervention by intra-uterine blood transfusions Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  3. AIM OF STUDY • Fetal blood typing is necessary to diagnosis feto-maternal incompatibility for allo-immunized women in order to guide antenatal monitoring • The aim of this presentation is the evaluation of non invasive prenatal fetal genotyping to guide the follow-up of allo-immunized anti-KEL1 pregnant women. Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  4. …AAC CGA ACG CTG AGA… …TTG GCT TGC GAC TCT… …AAC CGA ATG CTG AGA… …TTG GCT TAC GAC TCT… aa193 KEL2 (Thr)/KEL1 (Met) Extracellular Intracellular RBC Gene KEL - 19 exons and 19 introns - code for transmembranglycoprotein (23 antigens) KEL1 (K=Kell) et KEL2 (k=Cellano) -aa193 coded by exon 6 KEL1 (Met) KEL2 (Thr) Mutation of substitution (transversion) Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  5. Since 1995 - Invasive fetal genotyping from amniotic cells or chorionic villus • RFLP-PCR for KellLee et al,Blood, 1995 : …GAATGCN… BsmI …CTTACGN…  site of cuttingofKEL1 antigen 16 Diagnosis of feto-maternal incompatibilityin 2009 Specificantenatal monitoring Indication of this test verylimited : risk of progression of allo-immunization Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  6. Non Invasive fetal genotypingExperience of CNRHP Evidence for fetal DNA presenceintopregnantwomen’s plasma (1 to 6 % of fetal DNA) LO Y.M. et al. Am J Hum Genet, 1998, 62 : 768-775. 1998 - 2000 : Development of fetal RHD genotyping method in maternal plasma from the previous genotyping made on amniotic cells 2000 - 2004 : First clinical trials C. Rouillac- Le Sciellour et col. Large-Scale-prediagnosisstudy of fetal RHD genotyping by PCR on plasma DNA fromRhD-NegativepregnantWomen, Mol Diagn 2004 2004 - 2007 : Collaboration with Jacques Boy Institute and INTS for the creation of a diagnosis genotyping kit 2010 : Development and setting up of a KEL1 genotyping method in maternal plasma Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  7. MATERIAL AND METHODS Test based on 3 points 1-KEL1specificreal time PCR (SyberGreen) perfomedintriplicate(91pb)  To identifie fetalKEL1 DNA 2- exon 7 ABO genereal time PCR (SyberGreen) performed in simplicate(128pb)  To determinematernal DNA quantity 3- DNA tracer Maizereal time PCR (TaqMan)  To validate extraction step To prevent the risk of false negative or positive, all results have to be confirmed on a second sample Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  8. MATERIAL AND METHODS 2-Manual extractions (QIAGEN) Kit QIAampMinEluteVirusVaccum Kit Plasma volume 500 µl Elution volume 40 µl Agnès Mailloux, CNRHP, Hôpital Saint Antoine 3-Real time PCR amplifications 1-Blood samples of patient at least 13 GW -SSP-PCR in triplicate to identifie KEL1fetalallele -Amplification of ABO gene to determine the maternal DNA quantity -Amplification of a DNA tracer (maize) to validate extraction step 3 x 5 ml of blood collected on EDTA and received before 48h Centrifugation 6 x 1 ml plasma ( - 20°C) LightCycler

  9. Patients Patients EXEMPLE OF AMPLIFICATION CURVE Maize KEL1 ABO Tem Neg Tem Pos Blanc Amplification curve Tm=80,8°C ±0,5°C Tm=91,8°C ±1°C Fusion curve Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  10. RESULTS OVER 3 YEARS 124 allo-immunized anti-KEL1 women had non invasive fetalKEL1genotyping More than 78 % of thesepregnant allo-immunized anti-KEL1 womenhadan antibodywith a titerhigherthan 1/32. More than 62% of bloodsamplesreceivedwerecollectedbetween 11 and 18 GW Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  11. RESULTS Specific antenatal monitoring Sensibility : 96% Specificity : 69,2% PPV : 96% NPV : 100 % For 38% of the allo-immunized women, the pregnancy was compatible. Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  12. Monitoring of allo-immunized anti-KEL1 pregnant women in France positive antibodyscreen at first trimester of pregnancy Antibody Identification = Anti-KEL1 Non invasive fetalKEL1genotypingfrommaternalbloodfrom 13 GW Antibody Titration If fetusKEL1negative for 2 samples If fetusKEL1 positive for 2 samples No specificantenatal monitoring Antibodyscreenevery six weeks Anti-KEL≥1/32 Anti-KEL1<1/32 Specificantenatal monitoring -Weekly ultrasound and fetal middle cerebralpeaksystolicvelocity of blood flow (MCAPSV) -Monthly anti-KEL1 titration from 18 GW Specificantenatal monitoring - Normal ultrasound - Monthly anti-KEL1 titration from 18 GW Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  13. CONCLUSION • test accredited NF ISO 15189 since 2014 • Non invasive KEL1fetal genotyping is a powerful tool to diagnose a feto-maternal red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring only to pregnant women carrying incompatible fetus with monthly anti-KEL1 titration and weekly search for signs of fetal anemia (Velocimetry Doppler). We are developping2 new non invasive fetal genotyping in order to diagnosis feto-maternal incompatibility by real time PCR 1-Rhc fetal genotype 2-RhEfetal genotype Agnès Mailloux, CNRHP, Hôpital Saint Antoine

  14. Workdone by N. Da Silva Dr S. HUGUET-JACQUOT Dr C. TOLY-NDOUR Olivier OUDIN Priscilla SAULET Martine OGER UF biologie CNRHP pôle biologie et pathologie : Dr M VAUBOURDOLLE Dr A. CORTEY Pr. B. CARBONNE UF biologie CNRHP pôle de périnatalité : Pr D. MITANCHEZ Agnès Mailloux, CNRHP, Hôpital Saint Antoine

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