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Non-invasive Fetal RhD Type Prediction by Direct qPCR Open Access


Other title
direct qPCR
non-invasive prenatal testing
Type of item
Degree grantor
University of Alberta
Author or creator
Klein, Kellar B
Supervisor and department
Acker, Jason (Laboratory Medicine and Pathology)
Examining committee member and department
Clarke, Gwen (Laboratory Medicine and Pathology)
Izevbaye, Iyare (Laboratory Medicine and Pathology)
Yanow, Stephanie (School of Public Health)
Laboratory Medicine and Pathology

Date accepted
Graduation date
2016-06:Fall 2016
Master of Science
Degree level
Hemolytic disease of the fetus and newborn (HDFN), characterized by anemia, jaundice, and edema, can lead to fetal/neonatal death in its most severe form. Linked to maternal immune response to the RhD antigen, the disease can be prevented by administering Rh immune globulin (RhIg) to D-negative pregnant women. Current Canadian guidelines recommend an antepartum dose be given at 28 weeks’ gestation. However, up to 40% of these antepartum injections are unnecessary because the fetus is also D-negative and incapable of causing RhD sensitization. Unnecessary RhIg treatment could be avoided if the fetal blood type were known. Some international regions use non-invasive prenatal testing (NIPT), a genetic test that detects the small amount of cell-free fetal DNA that circulates in maternal blood, to predict fetal RhD type and determine RhIg eligibility, but the technique has not been widely adopted in North America. There are many factors that could contribute to this, including cost and availability of technical expertise. NIPT is a quantitative polymerase chain reaction (qPCR) method and current protocols require purified DNA. An inhibitor-resistant polymerase eliminates the DNA purification step and can be used to perform PCR directly from whole blood, plasma or other “dirty” sample types. By eliminating the DNA purification step, direct qPCR (dqPCR) can reduce costs, improve turnaround times and make fetal RhD typing easier to perform. This thesis examined the hypothesis that dqPCR using maternal blood from D-negative women can accurately predict fetal RhD type at or before 28 weeks’ gestation. The work presented in this thesis has provided insight into the impact of sample matrix on whole blood dqPCR and demonstrated that whole blood dqPCR for RhD antigen typing was comparable to traditional qPCR using extracted DNA. Additionally, this work described the development of a plasma-based dqPCR protocol. Finally, this thesis described the first use of dqPCR for RhD NIPT.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
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