Free DNA Fetal Kit® RhD

Non invasive fetal RHD genotyping from RhD-Negative pregnant women plasma DNA

(Real-Time PCR)

Ref. : 502080133

1. Anti-D immunization 

Anti-D immunization is a significant cause (if no prophylaxis) of haemolytic disease of the fetus and newborn (HDFN). This severe affection is most often caused upon fetal RhD-positive red blood cells destruction by anti-D antibodies present in the blood of an immunized RhD-negative mother that have crossed the placenta.

The anti-D antibody production can be prevented by immunoprophylaxis consisting in administration of anti-D immunoglobulins to RhD-negative mothers.The determination of the fetal RHD genotype is essential : 
- to establish whether a non anti-D immunized RhD-negative pregnant woman requires an appropriate immunoprophylaxis,
- for the management of pregnancy at risk : RhD negative pregnant women with anti-D, to assess whether the fetus is at risk of HDFN.

This kit determines the fetal RHD genotype, with two distinct PCR amplifications, by using fetal cell-free DNA from plasma of RhD-negative pregnant women.

2. Test principle

It has been demonstrated that plasma of pregnant women contains increasing concentrations of fetal DNA with the gestational age. This fetal DNA concentration corresponds from some copies in the first trimester of pregnancy up to several hundreds of copies in the third trimester of pregnancy of fetal genome per ml of plasma. In most cases, RhD-negative pregnant women miss the RHD gene in their genome, at least in Caucasians. Consequently, an RHD gene identified by PCR in plasma of pregnant RhD-negative women (Oxford University patent) is of fetal origin. For each fetal RHD genotype analysis, DNA is extracted from maternal plasma. The presence of a fetal RHD gene in the plasma DNA is detected by real-time PCR amplifications of two different segments of the RHD gene (exons 7 and 10), to detect most large number of RHD variants. Each amplicon is revealed with specific hydrolysis probes. 

3. Kits contents

4. Additional equipments and reagents required

The following equipments and reagents are not included but are required to perform the assay:
* Kit for Plasma DNA extraction (with equipment and associated accessories):
“QIAamp ® MinElute ® Virus Vacuum Kit” 50 columns, QIAGEN ref. 57714, 
or
“QIAamp DSP virus kit” (IVD CE) 50 columns, QIAGEN ref. 60704

* Reagent (reactive) real time PCR:
- For LightCycler ®: kit “LightCycler ® Taqman ® Master” Roche®,
ref. 04 535 286 001. (50 reactions), or ref. 04 735 536 001 (480 reactions).
- Other instrument (ABI, MX): kit “Faststart Taqman® ProbeMaster” Roche®,
ref. 04673409 (100 reactions).

* Consumables for real time PCR instrument, with system glass capillaries (20µl), or with multi-well plate.

* Nuclease-DNA free labwares: pipettes and specific tips with filter for PCR.

* Molecular biology grade water.

5. Limits

This test is exclusively validated for the analysis of human plasmas collected on EDTA or ACD anticoagulants. Heparin inhibits the PCR and must not be used with this method. 
It is recommended to perform this test on samples taken from 12 weeks of amenorrhea (sufficient level of circulating fetal DNA).
A negative fetal RHD genotype on a first blood maternal sample must be considered as probable, and has to be confirmed on a second maternal blood sample collected at least 2 weeks later to prevent a false-negative result. 
A positive fetal RHD genotype on a first blood maternal sample can be considered as acquired.
A false positive result is possible by cross contamination, or if the RHD fetal haplotype is a silent variant.

6. Performances

A study concerning 300 samples stemming from plasmas of pregnant women of RhD negative phenotype was made (C. Rouillac-Le Sciellour, P. Puillandre, R. Gillot, C. Baulard, S. Métral, C. Le Van Kim, J-P. Cartron, Y. Colin, Y. Brossard, Large-scale pre-diagnosis study of fetal RHD Genotyping by PCR on Plasma DNA from RhD-Negative pregnant Women, Mol Diagn 2004, 8(1): 23-31).(Read the full article). The results were compared with the phenotype RhD of the child in the birth, as well as in the technique initially developed from the works of C. Rouillac-Le Sciellour and al. in the CNRHP (Paris-France) (C. Rouillac-Le Sciellour, P. Puillandre, R. Gillot, C. Baulard, S. Métral, C. Le Van Kim, J-P. Cartron, Y. Colin, Y. Brossard, Large-scale pre-diagnosis study of fetal RHD Genotyping by PCR on Plasma DNA from RhD-Negative pregnant Women, Mol Diagn 2004, 8(1): 23-31). (Read the full article).
100 % of correlations were obtained between 2 methods.
No negative false result, defined by the absence of amplification with both RHD exons, was found at the child's RhD's positive. All the mothers whose children were positive RhD had a genotyping on their plasma revealing the presence gene RHD.
No amplification of RHD exons 7 and 10 was observed at the mother's whose child was negative RhD.
However, a negative false result cannot be excluded in the absence at present of a universal fetal DNA marker.

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