Prenatal HLA genotyping of uncultured amniotic fluid samples contaminated with maternal blood.
OBJECTIVE
Allogenic transplantation of umbilical cord blood (UCB) from HLA-identical siblings is a therapeutic concept of increasing importance. Prenatal HLA typing results can provide important information as to whether the UCB should be collected. Therefore, we tested the suitability of two methods based on polymerase chain reaction (PCR) for low-resolution HLA genotyping of uncultured amniocytes contaminated with maternal blood.


METHODS
Amniotic fluid samples (~10 mL, n = 4) were collected and divided into 5 equal sized portions. Subsequently, maternal blood was added to produce a series of varying degrees of artificial contamination ranging from 0% to 5% (vol/vol). Corresponding maternal blood and cord blood samples were collected and served as reference material. After DNA preparation, all samples were subjected to both PCR-SSP (sequence-specific primers) and PCR-SSO (sequence-specific oligonucleotides) typing procedures, designed for low-resolution typing of the highly polymorphic HLA-B locus.


RESULTS
Both PCR tests were able to accurately predict the fetal HLA-B type when pure amniotic fluid was used in all cases. The PCR-SSP method could still determine the fetal HLA type at 0.1% contamination but not at levels above this; in contrast, PCR-SSO failed if any degree of contamination was present.


CONCLUSIONS
Fetal cells from amniotic fluid represent a reasonable source for prenatal testing of the fetal HLA genotype with either the PCR-SSP or the PCR-SSO method. Low levels of contamination with maternal blood in the amniotic fluid are tolerated by the more robust PCR-SSP method. In contrast, the PCR-SSO procedure is more sensitive to any degree of contamination, but it is the method of choice if the amount of DNA is limited because of the very low volume of specimens of amniotic fluid available.
Eichler
Hermann
H
Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg, Mannheim, Germany. h.eichler@blutspende.de
Lese
Andrea
A
Meckies
Jessica
J
Zieger
Wolfgang
W
Klüter
Harald
H
Bugert
Peter
P
D000653:Amniotic Fluid / Q000276:immunology* / Q000378:metabolism*; D001769:Blood / Q000378:metabolism; D005260:Female; D005333:Fetus / Q000502:physiology*; D005544:Forecasting; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics*; D015235:HLA-B Antigens / Q000235:genetics; D006801:Humans; D015335:Molecular Probes; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011247:Pregnancy / Q000097:blood*
D006680:HLA Antigens; D015235:HLA-B Antigens; D015335:Molecular Probes; D009841:Oligonucleotides