Development of a rapid means of estimating the haemoglobin F content of candidate fetal cells isolated from maternal blood using HPLC.
Prenatal diagnosis of genetic disorders in nucleated fetal red blood cells present in maternal blood requires methods to detect and enrich for such cells. Here we describe a rapid high performance liquid chromatography (HPLC) method that allows one to determine as few as 100 cells containing haemoglobin F (HbF) in the presence of a vast excess of haemoglobin A (HbA)-producing cells. The HPLC separations of haemoglobins were performed with a weak cation exchange column-silica gel-bound asparaginic acid-and ammonium phosphate buffer as the mobile phase. Separations were carried out in 7 min. When applied to estimation of the recovery of fetal cells from maternal blood, the HPLC method indicates in a timely manner whether or not to proceed with further techniques (i.e., FISH or PCR). Several current techniques such as Ficoll gradients and fluorescence (FACS) or magnetic (MACS) activated cell sorting were thus evaluated. Unexpectedly, our method indicates high cell losses with both single gradient and triple density Ficoll pre-enrichment methods. Less than 20 per cent of the nucleated red blood cells can be recovered in the most optimal setting. Lysis of erythrocytes may be an alternative technique that leaves nucleated red blood cells of all maturation stages intact. Thus, any further improvements in the technology for fetal cell recovery may be aided by monitoring the yield with HPLC.
Huber
K
K
Department of Laboratory Medicine, Donauspital SMZ-Ost, Vienna, Austria.
Wolf
H
H
Van Lindern
M
M
Worofka
B
B
Rosen
A
A
Hafner
E
E
Beug
H
H
Philipp
K
K
Bauer
K
K
D002469:Cell Separation; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005319:Fetal Hemoglobin / Q000032:analysis*; D005434:Flow Cytometry; D006801:Humans; D018189:Immunomagnetic Separation; D011247:Pregnancy
D005319:Fetal Hemoglobin