Enrichment of circulating fetal nucleated red blood cell for non-invasive prenatal diagnosis with a new polyclonal antibody specific to fetal hemoglobin

Abstract

Objective To investigate the feasibility of a new polyclonal antibody specific to fetal hemoglobin (HbF) and its application in enrichment of circulating fetal nucleated red blood cell(NRBC) for non-invasive prenatal diagnosis. Methods A polyclonal antibody against a synthetic peptide comprising residues 69-78 of the γ-chain of HbF was prepared and conjugated to carrier protein KLH as the immunogen according to the specific antigenic determinant. The peptide-KLH solution was mixed with freund's complete or incomplete adjuvant and immunized goat to prepare specific polyclonal antibody against the γ-chain of fetal hemoglobin. After purification with protein G, maternal blood was obtained from 32 pregnant women at 22 to 39 weeks of gestation. NRBCs were separated and then stained with antibody against the γ chain of HbF. All the positive cells were collected by micromanipulator under microscopic observation, and whole genome was amplified by improved primer extension preamplification (PEP). Multiplex polymerase chain reaction amplification at nine different polymorphic short tandem repeat (STR) loci was also used to determine origin of the positive cells isolated from maternal blood. Results NRBCs stained with antibody against the γ chain of HbF were found in all of the blood from the 32 cases. Attached positive cells with anti-HbF staining have unique morphological characteristics, low nucleus-to-cytoplasm ratio, brown cytoplasm and blue dense nucleus after hematoxylin counterstain under microscopic observation, which can distinguished NRBCs with other cells. A total of 183 NRBCs were found in all of 32 pregnant women at a range of 0.6～1.8 cell/ml venous blood. The accurate rate was 90.6% by the STR genotype identification. Conclusion The antibodies specific to fetal γ-chain of fetal hemoglobin with synthetic peptide technology may have wide clinical utility in identification of fetal NRBCs from maternal circulation for non-invasive prenatal genetic diagnosis. Key words: Fetal hemoglobin; Erythroblasts; Prenatal diagnosis; Peptide biosynthesis