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Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Labex User Meeting November 2012.

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Presentation on theme: "Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Labex User Meeting November 2012."— Presentation transcript:

1 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Labex User Meeting November 2012

2 Bio-Rad Laboratories IMMUNOHEMATOLOGY Content of this presentation History of the D Clinical aspects Method and Kit content Free DNA Fetal Kit® RhD RHD and RHD Ψ Interpretation Internal Fetal DNA Control Marker Results from Evaluations and Studies

3 Bio-Rad Laboratories IMMUNOHEMATOLOGY Ancestral Gallery of RhD Alexander Solomon Wiener 1907 – 1976 Karl Landsteiner 1868 – 1943 Philip Levine 1900 – 1987

4 Bio-Rad Laboratories IMMUNOHEMATOLOGY History  Wiener and Landsteiner discovered the Rh factor in 1937/1940  The importance of the Rh factor was the better blood finger print for criminal matters  M, N, or P factors where known and Rh factor was just an additional one  Later it was recognized that the new Rh factor was associated with problem in transfusions  Between 1940 to 1946 Philip Levine discovered the close association between RhD factor and HDN

5 Bio-Rad Laboratories IMMUNOHEMATOLOGY History  Transfusion for newborn with “Rh disease” saved the babies live  With this method more than lives could be saved  1961 Finn et al. demonstrated that the administration of anti-D accelerates the clearance of Rh+ RBC’s  1965 the first post partum administration of anti-D was given to a mother.  1977 the ante- post partum anti-D prophylaxis lead to almost 100% protection of the HDN due to anti-D  1997 the first publication about cell free fetal DNA by Lo et. al.

6 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Clinical Aspects

7 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD The test detects fetal RHD gene sequences in maternal plasma The plasma of pregnant woman contains in the course of the pregnancy an increasing concentration of cell free fetal DNA beginning with a few copies in the first trimester several hundred copies in the third trimester Clearance of ffDNA is 48 hours after the delivery Lo Y.M. et al. Am J Hum Genet, 1998, 62 :

8 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD But, studies show that cell-free fetal DNA concentration varies between pregnant women In some pregnancies insufficient fetal DNA might be obtained according to known data this occurs in 0.2% of all pregnancies In some pregnancies the amount of fetal DNA can be very high E.g. if the child is suffering from a CMV infection

9 Bio-Rad Laboratories IMMUNOHEMATOLOGY By using the "Free DNA Fetal Kit® RhD" A very early detection of the fetal RHD genotyping is possible starting with the 12th gestational week In some rare cases even earlier The detection is proceeded via PCR amplification with three different segments of the RHD genes Exon 5, 7 and 10 This allows the greatest possible coverage of all RhD variants Free DNA Fetal Kit® RhD

10 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Further aspects for fetal genotyping Restrict the use of antenatal Anti-D prophylaxis To be given only to RhD neg women carrying a RHD positive fetus For only about 60% of pregnant women For the monitoring of pregnancies at risk RhD – negative pregnant women with natural anti-D Assess whether the fetus is at risk of HDFN

11 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Facts: In the 17 European countries: approximately 4`400`000 pregnancies every year 650`000 are from RhD negative women % of them have an RhD negative fetus. Why not continue the old way? RhD immunoglobulin is becoming increasingly expensive Many women today receive unnecessary injections of human blood products Human material is a precious source

12 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Monaco Luxembourg Ireland Finland Denmark Switzerland Austria Sweden Portugal Belgium Greece Netherland Spain Italy UK France Germany 15% RhD- mothers Birth 2008 Population 1/1/2009

13 Bio-Rad Laboratories IMMUNOHEMATOLOGY Method and Kit Content

14 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD What is this kit about?  A non invasive fetal RHD genotyping of plasma DNA from RhD - negative pregnant women  Legal manufactured by Institut de Biotechnologie Jacques Boy under the label of Bio-Rad  Using Real Time PCR  Determination of the RHD status of the fetus through maternal blood, by a non invasive blood sampling  Allows to prevent the risk of fetal anemia and haemolysis when the mother is serologically RhD neg and the fetus is RHD pos

15 Bio-Rad Laboratories IMMUNOHEMATOLOGY Method Blood samples Plasma Plasma DNA Fetal RHD sequences detected yesno Fetal genotype : RHD positive Fetal genotype: RHD negative centrifugation DNA extraction PCR amplification

16 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit RhD PRODUCT 1 Kit = 87 tests REF number RHD positive (+) Control:6 x 1000 µL (red top) RHD negative (-) Control:6 x 1000 µL (green top) [100X] Maize DNA Control (not ready to use):3 x 14 µL (yellow cap insert) Maize exon IVR2 primers sense/antisense + probe:3 x 38 µL (green cap insert) RHD exon 5 primers sense/antisense + probe:3 x 38 µL (purple cap insert) RHD exon 7 primers sense/antisense + probe:3 x 38 µL (white cap insert) RHD exon 10 primers sense/antisense + probe:3 x 38 µL (red cap insert)

17 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Additional equipment and accessories Extraction QIAamp DSP Virus Kit (IVD CE) 50 columns, QIAGEN ref Amplification Thermocycler Bio-Rad Dx Real-Time System (IVD CE) ref centrifuge and microplate or LightCycler® Roche and LC Carousel Centrifuge 2.0 Roche

18 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Dx Real-Time System Bio-Rad LightCycler® Roche:

19 Bio-Rad Laboratories IMMUNOHEMATOLOGY RHD and RHD Ψ

20 Bio-Rad Laboratories IMMUNOHEMATOLOGY RH Locus RHD-positive individuals may have one or two copies of RHD RHD gene RHCE gene RhD-positive individualsRhD-negative individuals

21 Bio-Rad Laboratories IMMUNOHEMATOLOGY RH gene The antigens of the Rh system are encoded by a pair of paralogous genes on chromosome 1 RHD RHCE These genes each have 10 exons They share 94% of its sequence identity

22 Bio-Rad Laboratories IMMUNOHEMATOLOGY Today we know there are several genetic causes for the RhD-negative phenotype. Caucasians: In serologically RhD negative individuals the RHD gene is nearly always absent Black Africans: only 18% of serologically D-negative black Africans are homozygous for an RHD deletion 66% of D-negative black Africans have an inactive RHD gene, called RHDΨ RHDΨ does not produce any D epitopes RHD neg and RHDΨ

23 Bio-Rad Laboratories IMMUNOHEMATOLOGY RHDΨ RHD genes producing variant D antigens should give a positive result and this is being achieved by exons 7 and 10 But - exons 7 and 10 are not suitable for testing any population containing people of African origin, as they will give false-positive results when the fetus has RHDΨ A testing including exon 5 will give a negative reaction with RHDΨ Exon 7 and 10 will amplify the pseudogene RHDΨ When the mother has the RHDΨ, an early positive reaction is given by the maternal DNA

24 Bio-Rad Laboratories IMMUNOHEMATOLOGY Amplification Curve DΨ

25 Bio-Rad Laboratories IMMUNOHEMATOLOGY The RHD genotyping is performed only in plasma from RhD – negative phenotyped pregnant women It is expected that any RHD positive result is from the fetus Separation of fetal DNA from maternal DNA has remained unachievable The mothers RHD DNA is amplified at about Cq The RHD DNA from the fetus is amplified after 35 Cq at about Cq How to discriminate between fetal and maternal DNA?

26 Bio-Rad Laboratories IMMUNOHEMATOLOGY Interpretation

27 Bio-Rad Laboratories IMMUNOHEMATOLOGY The analysis is interpretable if: Test validation according to controls The RhD negative (-) Control, and the blank control: No amplification detected with exon 5, 7 and 10 The RhD positive (+) Control: Amplification signals for exon 5, 7 and 10 (Cq constantly < 38 cycles) Amplification signals for Maize DNA Control (internal control of extraction) allows to validate the efficiency of the extraction the absence of PCR inhibitors for each tested sample If Maize Cp is > 36: repeat the test

28 Bio-Rad Laboratories IMMUNOHEMATOLOGY The analysis is interpretable if: Patient interpretation The positive results will present a value of Cp between 34 and 40 cycles varying according to the gestational week The awaited values of Cp could be rather different depending on PCR Instrument used. A validation for the Cycler in use must be performed The results with Cp beyond 40 cycles are not to be considered as definitive, they must be checked

29 Bio-Rad Laboratories IMMUNOHEMATOLOGY Interpretation If discordant results between the three PCR amplifications are observed it can be due to: a problem of a technical issues a problem of sensitivity difference between the three exons due to the presence of a very small DNA quantity Exon 5 is the most sensitive, then exon 7 and 10 A none coding or coding variant for the fetal RhD antigen which can be identified later by the RhD phenotyping of the newborn

30 Bio-Rad Laboratories IMMUNOHEMATOLOGY Exon RHD Genotype 7105 Cq > 35 (fetus) +++Positive ++-DΨ, DV, DBS, DVI ---Negative (always control on a 2nd blood sample) --+Negative (DHAR) -++Positive (DIV) -+-dCe s, DBT +++RHD silent maternal gene (ser. neg) Cq < 35 (mother) +++Positive ++-DΨ, DV, DBS, DVI -++Positive (DIV) -+-dCe s, DBT C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France)

31 Bio-Rad Laboratories IMMUNOHEMATOLOGY Interpretation If there is any doubt – give the pregnant woman the RhD prophylaxis! C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France)

32 Bio-Rad Laboratories IMMUNOHEMATOLOGY Internal fetal DNA control marker

33 Bio-Rad Laboratories IMMUNOHEMATOLOGY Internal fetal DNA control marker Several strategies have been proposed to confirm the presence of fetal DNA in the maternal plasma, in the following slides some of them will be shortly described

34 Bio-Rad Laboratories IMMUNOHEMATOLOGY WHO has made an international WHO standard by diluting freeze-dried plasma of an RhD-positive man in plasma of an RhD-negative woman This human DNA is not from a fetus and therefore is not suitable to use as an internal control Maternal cell-free DNA in plasma of pregnant women consists of longer fragments than in non pregnant women Fetal fragments are much shorter than maternal fragments Internal fetal DNA control marker

35 Bio-Rad Laboratories IMMUNOHEMATOLOGY The most widely used fetal DNA marker is a Y chromosome-specific sequence This is just applicable if the fetus is a boy Internal fetal DNA control marker

36 Bio-Rad Laboratories IMMUNOHEMATOLOGY Hyper- and Hypomethylation The promoter of the RASSF1A gene is: hypermethylated in fetal DNA hypomethylated in maternal DNA It is discussed as not being a good candidate for universal internal control for fetal DNA The technique is time-consuming Exhibits too low sensitivity and specificity compared to the fetal RHD genotyping Internal fetal DNA control marker

37 Bio-Rad Laboratories IMMUNOHEMATOLOGY Maize as an internal control Exogenous DNA (maize) is provided as: Extraction/amplification control because an universal control for fetal DNA is not yet available Amplification of the maize DNA added to each plasma and control provides a control for: adequate DNA extraction PCR amplification

38 Bio-Rad Laboratories IMMUNOHEMATOLOGY Exon 7 Exon 10 Exon 5 Maize Maize as an internal control

39 Bio-Rad Laboratories IMMUNOHEMATOLOGY Results from Evaluation and Studies

40 Bio-Rad Laboratories IMMUNOHEMATOLOGY Results from Evaluation and Studies In France more than 5000 fetal RHD genotypings from maternal blood have been performed the results obtained confirmed the procedure of the Free DNA Fetal Kit® RhD

41 Bio-Rad Laboratories IMMUNOHEMATOLOGY A study concerning 300 samples pregnant RhD negative women was made by using Exon 7 and 10 The results were compared with the phenotype RhD of the child after the birth 100 % of correlations were obtained between the 2 methods However, a false negative result cannot be excluded in the absence of a universal fetal DNA marker C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France) Results from Evaluation and Studies

42 Bio-Rad Laboratories IMMUNOHEMATOLOGY A second retrospective evaluation of 120 plasma samples was performed using the Free Fetal DNA Kit® RhD (by using exon 5, 7 and 10) These samples were from pregnant woman having an RHD-negative phenotype, five were carriers of a silence gene. The results were compared to the phenotypes of the children at birth with a correlation of 100%. C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France) Results from Evaluation and Studies

43 Bio-Rad Laboratories IMMUNOHEMATOLOGY Free DNA Fetal Kit® RhD Conclusion Earliest detection of cell-free DNA from plasma of RhD negative women No impact on pregnancy High specificity due to real-time PCR test method Standardized and reliable Avoids unnecessary prophylaxis treatments Primers for RHD Exon 5, 7 and 10 The Free DNA Fetal Kit® RhD is CE marked

44 Bio-Rad Laboratories IMMUNOHEMATOLOGY Thank you for your attention QUESTIONS?


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