1. Labex User Meeting November 2012
Free DNA Fetal Kit® RhD
Labex User Meeting November 2012

2. Content of this presentation
History of the D
Clinical aspects
Method and Kit content Free DNA Fetal Kit® RhD
RHD and RHDΨ
Interpretation
Internal Fetal DNA Control Marker
Results from Evaluations and Studies

3. Ancestral Gallery of RhD
Karl Landsteiner
1868 – 1943
Philip Levine
1900 – 1987
Alexander Solomon Wiener 1907 – 1976

4. 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. 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. Free DNA Fetal Kit® RhD
Clinical Aspects

7. 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. 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. Free DNA Fetal Kit® RhD 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

10. 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. 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
30-40 % 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. Free DNA Fetal Kit® RhD Sweden 9 256 347 109 301 16 395 Denmark
Population 1/1/2009
Birth 2008
15% RhD- mothers
Germany
France UK
Italy
86 372
Spain
77 838
Netherland
27 700
Greece
17 325
Belgium
18 749
Portugal
15 689
Sweden
16 395
Austria
77 752
11 663
Switzerland
76 900
11 535
Denmark
65 038
9 756
Finland
59 530
8 930
Ireland
74 819
11 223
Luxembourg
5 596
839
Monaco
32 543
296 44

13. Method and Kit Content

14. 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. Fetal RHD sequences detected
Method
Blood samples
centrifugation
Plasma
DNA extraction
Plasma DNA
PCR amplification
Fetal RHD sequences detected
yes no
Fetal genotype :
RHD positive
Fetal genotype:
RHD negative

16. Free DNA Fetal Kit RhD PRODUCT 1 Kit = 87 tests REF number 060001
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. 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. Free DNA Fetal Kit® RhD Dx Real-Time System Bio-Rad
LightCycler® Roche:

19. RHD and RHDΨ

20. RH Locus RHD gene RHCE gene RhD-positive individuals
RHD-positive individuals may have one or two copies of RHD
RHD
gene
RHCE
gene
RhD-positive individuals
RhD-negative individuals

21. 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. RHD neg and RHDΨ
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

23. 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. Amplification Curve DΨ

25. How to discriminate between fetal and maternal DNA?
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
32-34 Cq
The RHD DNA from the fetus is amplified after
35 Cq at about Cq

26. Interpretation

27. 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. 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. 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. RHD silent maternal gene (ser. neg)
Exon
RHD Genotype 7 10 5
Cq > 35
(fetus) +
Positive -
DΨ, DV, DBS, DVI
Negative (always control on a 2nd blood sample)
Negative (DHAR)
Positive (DIV)
dCes, DBT
RHD silent maternal gene (ser. neg)
Cq < 35
(mother)
C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France)

31. If there is any doubt – give the pregnant woman the RhD prophylaxis!
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. Internal fetal DNA control marker

33. 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. Internal fetal DNA control marker
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

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

36. Internal fetal DNA control marker
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

37. 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. Maize as an internal control
Exon 7
Exon 10
Exon 5
Maize

39. Results from Evaluation and Studies

40. 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. Results from Evaluation and Studies
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)

42. Results from Evaluation and Studies
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)

43. 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. Thank you for your attention
QUESTIONS?