Cell3™ Direct: Fetal RhD Blood Group Genotyping Kit

Fetal RhD diagnosis using cell-free fetal DNA

With no cffDNA extraction required, Cell3™ Direct reduces cost and saves time in fetal Rhesus D diagnosis.

Results in under 3 hours

With no cffDNA extraction required and a simple qPCR protocol, it takes under 3 hours from sample receipt to result.

Less material required

Using less than <0.25ml of plasma sample, this test leaves plenty of sample for other prenatal tests.

Flexible and fully validated

A validated kit and flexible tube format enables laboratory processing on any qPCR machine.

Multi-target assay

A multi-target approach delivers an assay suitable for all populations and intra assay concordance for increased sensitivity.

Non-invasive fetal Rhesus D (RhD) blood group testing

Non-invasive prenatal testing (NIPT) of cell free fetal DNA (cffDNA) in maternal blood during pregnancy can be used to determine fetal Rhesus D blood group status. This means that RhD negative pregnant women can avoid receiving antenatal anti-D immunoglobulin if they are carrying a RhD negative baby and are not at risk of Hemolytic Disease of the Fetus and Newborn (HDFN).

Current practice is to provide antenatal anti-D prophylaxis at 28-30 weeks gestation, which means that about 40% of healthy RhD-negative pregnant women are exposed to a pooled human blood product that they do not need as their baby is also RhD negative1.

The Cell3™ Direct Fetal RhD Blood Group Genotyping kit predicts fetal RhD status with high accuracy and not only improves care for RhD-negative women but allows health services to apply a targeted approach to anti-D prophylaxis and conserve the use of an expensive product that can be in short supply.

A 'direct from plasma' option for fetal RhD testing

Nonacus have developed the first commercially available, direct from plasma, non-invasive prenatal diagnosis (NIPD) kit for fetal RhD genotyping.

With no cffDNA extraction required and a simple real-time qPCR protocol, the Cell3™ Direct Fetal RhD Blood Group Genotyping kit delivers results direct from plasma in under three hours reducing technician time and providing a quicker, more cost-effective assay than previously available.

If, however, you already have high-throughput processing for extracted cffDNA set up in your laboratory or you are testing at 10-11 weeks when you need to maxmise sensitivity, the Cell3 Direct Fetal RhD Genotyping kit can be still be used with extracted cffDNA.

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Meets NICE guidelines on fetal Rhesus D status

Non-invasive prenatal testing to identify fetal Rhesus D status is recommended by NICE for pregnant women who are Rhesus D negative as a cost-effective option to guide antenatal prophylaxis with anti-D immunoglobulin (anti-D Ig) within the UK NHS2.

The Cell3™ Direct Fetal RhD Blood Group Genotyping kit meets all of the NICE requirements for this test.

 

Multi-target approach for a multi-ethnic assay

There are now known to be several genetic causes for an RhD-negative phenotype, and these vary according to ethnic origin. In Caucasians a homozygous deletion of the RhD gene is the predominant cause, but in black Africans the RhD gene is intact but non-functional. This is known as the RhD-pseudogene (RhD-Psi) and it contains a 37 base pair insert in exon 4 and a nonsense mutation in exons 5+6, which may introduce stop codons preventing translation3. For an assay to be accurate across all populations, it must therefore target multiple exons in the RhD gene.

The Cell3 Direct Fetal RhD genotyping kit targets sequences specific for exons 5, 7 and 10 of the RhD gene and can distinguish between RhD positive, RhD negative and RhD PSI genotypes making it suitable for any population.

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Sensitive qPCR screening for fetal RhD

The technical sensitivity of our assay was demonstrated using RhD positive genomic DNA spiked into an RhD negative background at 10%,  and 1% ratios (equivalent to 30 and 3 genomic equivalents) to reproduce realistic fetal fractions.

Amplification of all targets across all replicates was observed even at the lowest 1% spike-in fraction, with RhD exon 5 assay capable of discriminating the RhD PSI variant.

Direct from plasma vs extracted cff DNA

Our direct from plasma protocol was compared with extracted cfDNA and results were demonstrated to be comparable using the same plasma sample (RhD positive, 24 weeks’ gestation).

Amplification plots of direct from plasma testing (see below) shows robust amplification of all targets: RhD exon 5 (blue), RhD exons 7 and 10 (green) and CCR5 (black).

DNA amplification profiles-landscape

Product Support

If you have a query relating to any of our products please fill in the support request form here and one of our team will get back to you as soon as possible.

A full compatibility matrix is available here or alternatively can be found in the protocol for a specified product, all protocols can be downloaded in the product specific resource section.

Cell3™ Direct qPCR technology is extremely sensitive, as few as 1-3 copies can be amplified. Amplification in the NTCs or negative controls that is random in nature is likely due to low level contamination. We recommend the following actions:

  • Use clean working practises to minimise the potential of template contamination.
  • Where possible use separate areas for PCR mix preparation, template addition and qPCR reaction running.
  • Set up qPCR reaction in a PCR or biological safety cabinet using sterile / PCR-clean equipment and consumables only.

Each qPCR machine is different and therefore we recommend validation of our kit in each laboratory. However, we have provided some guidelines that in most instances provide a robust analysis approach.

Cell3™ FS Direct Analysis Guidelines v1.2

Cell3™ RHD Direct Analysis Guidelines v1.3

An inconclusive result is where there are not enough markers to be certain that the sample is positive, but some replicates have been amplified and therefore it is not clearly a negative result. This could be for a number of reasons including low level contamination, poor PCR efficiency or there is simply not enough cell free fetal DNA to amplify reliably. If an inconclusive result is obtained we recommend re-running the assay direct from plasma or alternatively extracting cell free DNA from the remaining plasma and running using the cell free DNA extracted protocol option.

References

  1. Diagnostic accuracy of routine antenatal determination of fetal RHD status across gestation: population based cohort study: BMJ 2014; 349: g5243 https://doi: 10.1136/bmj.g5243
  2. NICE recommends test to identify fetal rhesus D status: BMJ 2016;355:i6106 BMJ 2016;354:i3944
  3. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype: Blood 2000 95:12-18; Singleton et al. https://doi.org/10.1182/blood.V95.1.12