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22 nd International Congress of Clinical Chemistry and Laboratory Medicine PowerPoint PPT Presentation


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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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22 nd international congress of clinical chemistry and laboratory medicine

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


Introduction

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


Aim of study

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


22 nd international congress of clinical chemistry and laboratory medicine

…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


Since 1995 invasive fetal genotyping from amniotic cells or chorionic villus

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


Non invasive fetal genotyping experience of cnrhp

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


22 nd international congress of clinical chemistry and laboratory medicine

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


Material and methods

MATERIAL AND METHODS

2-Manual extractions

(QIAGEN)

Kit QIAampMinEluteVirusVaccum Kit

Plasma volume500 µl

Elution volume40 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


22 nd international congress of clinical chemistry and laboratory medicine

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


Results over 3 years

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


Results

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


Monitoring of allo immunized anti kel1 pregnant women in france

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


Conclusion

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


22 nd international congress of clinical chemistry and laboratory medicine

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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