Free DNA Fetal Kit ® RhD

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

(Real-Time PCR)

Ref.: 502080233

1 – ANTI-D IMMUNIZATION 

In the absence of prophylaxis, anti-D immunization is a significant cause of hemolytic 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 immunoglobulin 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.

2 – TEST PRINCIPLE

Prenatal determination of the RHD genotype became a reality following two major findings: cloning of the RH (D and CE) genes^1,2, and elucidation of the molecular basis of the RhD-pos/RhD-neg polymorphism³. These studies have shown that the RH locus of RhD-positive individuals is composed of two homologous genes RHD and RHCE, encoding for the RhD and RhCcEe proteins, respectively, and that the RHD gene is deleted in RhD-negative individuals of Caucasian origin. Based on these findings, a first generation of invasive genotyping methods was established by PCR amplification of exon 10 (of the RHD gene) by using amniotic fluid or chorionic villus samples in early pregnancy^4,5. With increasing knowledge on the molecular basis of a large number of RH variants, the discovery of the RHD pseudogene (RHDψ) frequent in Black individuals⁶, and the presence of cell-free fetal DNA in the maternal plasma⁷, a second generation of non invasive PCR methods based on use of several primer pairs for the simultaneous detection of different RHD gene regions (exons or introns) in maternal plasma was designed in many laboratories, mainly in Europe. According to this new approach, most RH variants could be detected (but not identified), thus reducing the risk of potentially harmful false-negative results.

Plasma of pregnant women contains increasing concentrations of fetal DNA with the gestational age. Indeed, fetal DNA concentration (fetal genome per ml of plasma) rise from some copies in the first trimester of pregnancy up to several hundreds copies in the third trimester.

A first generation of RHD genotyping kit, was based on the amplification of two RHD specific regions in exon 10 (the more conserved) and exon 7 (absent in some variants) by real-time PCR^8,9. The second generation now developed in the present kit, includes amplification of a third specific region located in exon 5 that could detect exon 5 from the RHD gene, but not from the RHDψ pseudogene¹⁰. Accordingly, genotyping exon 5D (not Dψ) will allow to genotype the fetuses from mother carrying a RHDψ pseudogene. Thus, in addition to the fetus RHD status (positive or negative) the presence of the main known RHD variants will be detected.

Consequently, the 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, total 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 three different segments of the RHD gene (exons 5, 7 and 10), to detect a most large number of RHD variants. Each amplicon is revealed with specific hydrolysis probes.

[1] Chérif-Zahar B et al., Proc Natl Acad Sci USA 1990, 87:6243-7 ; [2] Le van Kim et al., Proc Natl Acad Sci USA 1992, 89:10925-9 ; [3] Colin et al., Blood 1991, 78:2747-52 ; [4] Bennett et al., N Engl J Med 1993, 329:607-10 ; [5] Lo et al., N Engl J Med 1993, 341:1147-8 ; [6] Singleton et al., Blood 2000, 95:12-8 ; [7] Lo et al., N Engl J Med 1998, 339:1734-8 ; [8] Rouillac et al., Mol Diagn 2004, 8:23-31 ; [9] Rouillac-Le Sciellour et al., Transfus Clin Biol 2007, 14:572-7 ; [10] Finning et al., Transfusion 2002, 42:1079-85.

3 – KIT 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) :

  
* Reagent(reactive) real time PCR :

* 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 taken before 18 weeks of amenorrhea 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 (Rouillac-Le Sciellour C., Sérazin V., Brossard Y., Oudin O., Le Van Kim C.,  Colin Y., Guidicelli Y., Menu M., Cartron JP. Noninvasive fetal RHD genotyping from maternal plasma. Use of a new developed Free DNA Fetal RhD. Trans Clin Biol 2007; 14:572-7) (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 by Rouillac-Le Sciellour C. and al. (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; 8:23-31). (Read the full article).
100 % of correlations were obtained between the 2 methods.
No false-negative result, defined by the absence of amplification of RHD exons, was found from children with a RhD-positive phenotype. All the mothers whose children were phenotypically RhD-positive had a genotyping on their plasma revealing the presence of the fetal RHD gene.
Currently however, a false-negative result cannot be excluded in the absence of a universal fetal DNA marker.

7 – DECISION TREE