bio-rad laboratories immunohematology free dna fetal kit® rhd labex user meeting november 2012

Bio-Rad Laboratories IMMUNOHEMATOLOGY

Free DNA Fetal Kit® RhD

Labex User Meeting November 2012


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


Ancestral Gallery of RhD

Alexander Solomon Wiener 1907 – 1976

Karl Landsteiner

1868 – 1943

Philip Levine

1900 – 1987


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


History

Transfusion for newborn with “Rh disease” saved the babies live

With this method more than 200.000 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.



Clinical Aspects



• 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 : 768-775



• 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


• 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




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



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



4429632 543Monaco

8395 596493 500Luxembourg

11 22374 8194 450 014Ireland

8 93059 5305 326 314Finland

9 75665 0385 511 451Denmark

11 53576 9007 701 856Switzerland

11 66377 7528 355 260Austria

16 395109 3019 256 347Sweden

15 689104 59410 627 250Portugal

18 749124 99110 750 000Belgium

17 325115 50011 260 402Greece

27 700184 66916 485 787Netherland

77 838518 91745 828 172Spain

86 372575 81060 045 068Italy

119 157794 38361 634 599UK

119 407796 04462 448 977France

102 377682 51482 002 356Germany

15% RhD- mothers Birth 2008 Population 1/1/2009


Method and Kit Content



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 Boyunder 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


Method

Blood samples

Plasma

Plasma DNA

Fetal RHD sequences detected

yes no

Fetal genotype :RHD positive

Fetal genotype:RHD negative

centrifugation

DNA extraction

PCR amplification


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)



Additional equipment and accessories

Extraction• QIAamp DSP Virus Kit (IVD CE) 50 columns, QIAGEN ref. 60704

Amplification• Thermocycler Bio-Rad Dx Real-Time System (IVD CE) ref. 94000 -

centrifuge and microplate

or• LightCycler® Roche and LC Carousel Centrifuge 2.0 Roche



Dx Real-Time System Bio-Rad

LightCycler® Roche:


RHD and RHDΨ


RH LocusRHD-positive individuals may have one or two copies of RHD

RHD gene

RHCE gene

RhD-positive individuals RhD-negative individuals


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


• 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


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


Amplification Curve DΨ


• 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 37- 38 Cq

How to discriminate between fetal and maternal DNA?


Interpretation


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


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


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


ExonRHD Genotype7 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)

+ + + Positive

+ + - DΨ, DV, DBS, DVI

- + + Positive (DIV)

- + - dCes, DBTC. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France)


Interpretation

If there is any doubt – give the pregnant woman the RhD

prophylaxis!

C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France)


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


• 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



• The most widely used fetal DNA marker is a

Y chromosome-specific sequence

• This is just applicable if the fetus is a boy



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



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


Exon 7 Exon 10 Exon 5

Maize

Maize as an internal control


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


• 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)



• 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)




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


Thank you for your attention

QUESTIONS?

bio-rad laboratories immunohematology free dna fetal kit® rhd labex user meeting november 2012

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