This is a Phase I SBIR proposal request to develop an assay for the identification of fetal blood group antigens using a DNA based assay method that would allow the use of cells obtained at amniocentesis. The Investigator indicates approximately 16% of all multigravida pregnancies in the United States occur in women who are Rh negative and 84% of the male population is Rh positive, approximately 13%of fetuses are at risk for development of a hemolytic diseases in the newborn due to Rh incompatibility. Although less than one percent of fetuses actually develop full blown erythroblastosis fetalis, this outcome is so severe that serial amniocentesis and percutaneous umbilical blood sampling was adopted for use in all multigravida Rh pregnancies by the American College of Obstetricians and Gynecologists. This is required because all women at risk, even though approximately 16% of the total women at risk are bearing Rh negative fetuses and thus are at no risk for hemolytic disease of the newborn.In order to achieve the goals of desired, the Investigator plans to develop a commercial diagnostic test. His Specific Aims in accomplishing this goal are (1) to use PCR and DNA sequencing for molecular characterization of multiple Rh forms obtained from clinical partners, (2) correlate the DNA sequence information with serological data, (3) development of a DNA heteroduplex screen that will allow rapid identification of multiple alleles,and (4) screening, identification and clinical correlation of Rh isoforms from aminocenteses to demonstrate the utility of the test in predicting hemolytic disease in the newborn. Thus, during Phase I of this proposal, the Investigator plans to identify the major Rh isoforms and by demonstrating that DNA based technology can be adapted to amniotic specimens. Subsequently during Phase II, he plans to convert to laboratory procedures for Rh isoforms into a kit format that can be used by any laboratory performing PCR amplifications of DNA samples. He recognizes that the specific type of assay procedure for identifying isoforms will be governed to some extent by the number of alleles that are identified during phase I. If the allele number is large, heteroduplex method will be employed and converted to amplified kit formats for direct analysis on amniotic samples. He indicates that his laboratory has demonstrated that mutant alleles from a variety of genetic disorders can be identified in this manner and has a patent pending on the method of using heteroduplex patterns for identification of specific alleles. He plans to include all the components in kits needed for performance of the tests. This will include small precast gels, all necessary primers,buffers, a inexpensive gel apparatus and a visual allele pattern recognition template. If there is a low number of alleles, they plan to adopt an allele specific oligonucleotide assay based on a microtiter plate format. This would incorporate fluorescently labeled probes, along with a commercially available microtiter plate reader.
