DIAGNOSIS OF COBALAMIN DEFICIENCY: A REPLY

We read with interest the report by Petit et al (1997) demonstrating the transfer of maternal cells into the fetal circulation during the third trimester of pregnancy. The authors were unable to detect maternal DNA sequences in their single second-trimester sample at a gestation age of 20 weeks. In order to investigate whether there is passage of maternal nucleated cells into the fetal circulation in the second trimester of pregnancy, we studied nine women undergoing elective termination of pregnancy for social indications at the Fetal Medicine Unit, Homerton Hospital, London, U.K. Informed consent was obtained in each case. Ethics approval was obtained from the Ethics Committee at Homerton Hospital. Maternal blood samples were collected from the antecubital fossae of the patients prior to any invasive procedure. Fetal blood sampling was performed under ultrasound guidance immediately before termination of pregnancy as previously described (Abbas et al, 1993). 2– 5 ml of amniotic fluid was taken prior to fetal blood sampling as a control for maternal bleeding during the procedure. Extraction of DNA from fetal and maternal blood samples (Lo et al, 1996) and amniotic fluid samples (Rebello et al, 1991) was as previously described. Three polymorphic systems were used for the detection of maternal DNA sequences in fetal blood, based on the detection of polymorphisms in a region 5 to the human delta-globin gene (the RT region), the glutathione S-transferase M1 locus (GSTM1) and the angiotensin-converting enzyme (ACE) gene (Lo et al, 1996). These polymorphic systems were all bi-allelic: the R and T alleles for the RT region, the presence (GST) or absence (GST) of the gene for the GSTM1 system, and the insertion (I) and deletion (D) of a 287 bp fragment in intron 16 of the ACE gene. To be informative for the detection of maternal DNA sequences in fetal blood, the fetus should be homozygous for at least one of these polymorphisms and the mother heterozygous for the same polymorphism. Detection of maternal DNA was then performed by amplifying the maternal allele which was not possessed by the fetus. Of the nine cases, RT, GSTM1 and ACE genotyping revealed five informative scenarios (Table I: cases 1, 2, 5, 7 and 9). Maternal DNA sequence was detected in the fetal blood sample obtained from case 7 at a gestation age of 13 weeks. The PCR analysis was repeated with the same result. The amniotic fluid sample taken before fetal blood sampling in this case was negative for maternal DNA sequences, thus demonstrating that the result was not an artefact of maternal bleeding during the invasive procedure. Other negative controls, including water blanks and control DNA samples not containing the amplified allele, were consistently negative. These results demonstrate that the prenatal transfer of nucleated maternal cells into the fetal circulation can occur as early as the 13th week of gestation. This represents the earliest documented case of maternofetal cell transfer in humans. This prenatal cell trafficking is a potential mechanism for the mother to influence the development of the immune system of the fetus. This may be one of the explanations why certain individuals exhibit a degree of tolerance to the noninherited maternal HLA antigens (Claas et al, 1988). It also provides a possible route for the prenatal transmission of infectious agents from the mother to the fetus.