NIPT Massively Parallel Sequencing (MPS) for Clinically Significant Fetal Antigens

Non-invasive prenatal testing (NIPT) via massively parallel sequencing (MPS) is a molecular blood group genotyping assay for clinically significant fetal antigens. It’s under development in Lifeblood R&D to help predict the blood group phenotype of the fetus. 

We’re developing a MPS assay to detect a panel of clinically significant fetal from cell-free fetal DNA. The advantages of this technique include: 

• Screening for an extended number of clinically significant red blood cell and platelet antigens in one test (see ‘What do we test for?’ below for panel information)
• Inclusion of internal fetal control markers
• Providing fetal genotyping in a single, non-invasive test from 12 weeks gestation

Please note that this testing is not yet accredited. Results are for research use only.

NIPT is performed when the mother is antigen negative and has a corresponding alloantibody, placing the fetus at risk of being affected by HDFN, or if there is concern for FNAIT. 

NIPT uses maternal peripheral whole blood samples. Cell-free DNA, which contains cell-free fetal DNA is extracted from maternal plasma. The cell-free fetal DNA is analysed for the presence of the gene or molecular variation responsible for (or associated with) the blood or platelet group phenotype under investigation.

The testing is performed using a custom QIAGEN panel, which has selected common antigens for the following blood and platelet groups.  Please be aware that the panel does not include all antigens in the listed blood groups, and not all HPA antigens are covered. 

The assay is performed using a custom QIAGEN QIAseq™ Targeted DNA Panel designed to cover red blood cell and human platelet antigen allelic polymorphisms across 12 blood group systems and six platelet systems, respectively, in addition to fetal control markers. The QIAseq 12-index I kit is used to label each sample with adapters containing sequences for sample barcoding. Paired-end sequencing is then performed on an Illumina MiSeq. Maternal genomic DNA (gDNA) samples are tested in tandem with the cell-free DNA (cfDNA) sample. FASTQ files generated from sequencing the cfDNA and gDNA samples are input to a customised bioinformatics workflow using CLC Genomics Workbench software (QIAGEN) to determine the frequency of fetal antigen signals present. Non-specific background reads (commonly associated with massively parallel sequencing methods) are accounted for by subtracting the maternal gDNA allele frequency from the cfDNA allele frequency, forming the ‘subtractive allele frequency’ (SAF). The SAF is used as the threshold for positive and negative fetal antigen predictions during results interpretation, as detailed in 'How are the results interpreted?' below.

The assay has a ≥99.9% positive predictive value and a ≥ 97.1% negative predictive value. The accuracy based on the study data is 98.5% with a 95% confidence interval of 91.9% to 99.9%. A proportion of samples will be reported as inconclusive. In this instance a repeat sample will be requested. 

Final patient cfDNA interpretation takes into account the combination of results obtained for patient cfDNA (blood group allele and housekeeper) and maternal gDNA (blood group allele and housekeeper). Criteria for the subtractive allele frequency (SAF) are shown below.

For more information, see McGowan EC et al. Feasibility for non-invasive prenatal fetal blood group and platelet genotyping by massively parallel sequencing: A single test system for multiple atypical red cell, platelet and quality control markers. Br J Haematol. 2023;00:1–12.

A research report will be generated and emailed, via ‘Secure Send’, to the referring clinician. 

Gestational age must be at least 12 weeks. The concentration of fetal DNA in the mother’s blood increases with the progression of the pregnancy. Any sample collected before 12 weeks gestation can lead to inconclusive or false negative results. 

There is limited data demonstrating the feasibility of NIPT for monozygotic (identical) and/or dizygotic (fraternal) twin pregnancies. We can only report on cases involving monozygotic pregnancies at this stage.

However, we are happy to accept samples from dizygotic pregnancies as the resulting information is valuable for further developing the NIPT system. 

• A minimum of 3 x 6 mL whole blood samples. It is preferred to send samples in 1 X 6 mL EDTA and 2 x 6 mL maternal cell stabilising tubes, either Roche or Streck. (Lithium Heparin samples are not suitable.)
• Roche tubes can be requested from Lifeblood R&D (atypicalNIPT@redcrossblood.org.au).
• It is possible to test a sample sent in 3 x 6 mL EDTA, but this is not preferable. Please contact atypicalNIPT@redcrossblood.org.au to discuss.
• Sample collection should be planned to ensure they are received for processing within 72 hours of collection.
• Samples should be stored at 2-8 °C.
• Samples must be provided with completed ‘Study request form’ with a minimum of 3 identifiers and include the tests requested, referring organisation’s contact details and any relevant clinical information.
• It is very important to include the requesting clinicians’ email for correspondence and reporting.
• Testing cannot be completed without a signed ‘Consent form’, which is also available online at the same location as the ‘Study request form’.  Please send both the study request form and signed consent form together.
• The identifiers on the sample must match the request form exactly. Samples should be sent packaged with a cold ice-brick to the QLD Research and Development Laboratory, via the address below as soon as possible following collection: 
  
  Research and Development, Attention Catherine Hyland
  Australian Red Cross Lifeblood 
  44 Musk Avenue (delivery via Blamey Street)
  KELVIN GROVE
  QLD 4059
  AUSTRALIA 

Sequencing is performed in batches of 5 samples. This can lead to delays in testing if numbers are limited. The average turnaround time is 8 weeks. If there is urgency for testing, please contact atypicalNIPT@redcrossblood.org.au to discuss.

Please direct sample collection and shipment enquiries to the Research and Development team via atypicalNIPT@redcrossblood.org.au.

Please direct all clinical enquiries to the Transfusion Specialist for your state. The contact details are available here: Contact | Lifeblood