One of the concerns about the use of cord blood as a source of hematopoietic stem cells for allogeneic trans- plantation is the possibility of contamination by.
Bone Marrow Transplantation, (1998) 21, 1097–1099 1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt
Detection of maternal DNA in umbilical cord blood by polymerase chain reaction amplification of minisatellite sequences ´ 1 M Briz1, C Regidor , D Monteagudo3, N Somolinos3, C Garaulet3, R Fores1, M Posada4 ´ 1 and MN Fernandez Departments of 1Hematology and 4Epidemiology, Hospital Puerta de Hierro; Departments of 2Hematology and 3Gynecology, Hospital de Mostoles, Madrid, Spain
Summary: One of the concerns about the use of cord blood as a source of hematopoietic stem cells for allogeneic transplantation is the possibility of contamination by maternal cells which could cause life-threatening GVHD. We have assessed cord blood contamination using PCR analysis of several minisatellite regions to detect maternal DNA. Eighty mother–cord pairs were obtained for this study. In one case there were no specific maternal alleles at any loci and, therefore, cord blood could not be evaluated. Thus, there was a total of 79 informative cases for the detection of maternal cells in the fetal circulation. In most cases, the level of detection was between 0.5 and 1%. We detected maternal DNA in the cord blood sample in only one case (1.26%), and the analysis of dilution experiments led to an estimate of 0.5–1% maternal cells. In conclusion, using PCR amplification of hypervariable regions, maternal DNA is very rarely detected in the cord blood collected at birth, although this approach has a relatively low level of sensitivity. Keywords: cord blood; maternal cell contamination; hematopoietic stem cell transplantation; minisatellite repeats; polymerase chain reaction
Umbilical cord blood contains hematopoietic progenitor cells1 that can be used as an acceptable alternative to bone marrow for clinical transplantation.2 Although initial donors were siblings, more recently cord blood transplants from unrelated donors have been used, and international umbilical cord blood banks have already been established in Europe and in the USA. One of the remaining concerns about the use of cord blood in unrelated transplantation is the possibility of contamination by maternal cells; maternal T lymphocytes only partially matched with the histocompatibility antigens of the recipient could cause life-threatening GVHD after transplantation. The objective of this study was to determine the frequency and degree of cord blood contamination by ´ Correspondence: Dr M Briz, Hospital Puerta de Hierro, San Martın de Porres 4, 28035-Madrid, Spain Received 4 November 1997; accepted 3 January 1998
maternal cells; we used polymerase chain reaction (PCR) amplification of highly polymorphic DNA regions (minisatellite sequences) to detect maternal DNA. Materials and methods ´ Pregnant women were recruited before delivery at Mostoles Hospital (Department of Obstetrics and Gynecology). Samples were collected after full-term vaginal delivery from women who had experienced no complications during pregnancy or at the time of delivery. Informed consent was obtained in all cases. Collection of maternal and umbilical cord samples Cord blood was collected during the third stage of labor, before delivery of the placenta. Briefly, the umbilical cord was double-clamped and cut; after removal of the newborn, the umbilical vein of the cord was punctured aseptically and the blood was collected in a standard plastic blood donation bag containing CPD as anticoagulant. To minimize maternal cell contamination, we collected cord blood by gravity in a closed system, without any attempt to flush placental vessels. In all cases a cord tissue sample and 5 ml of EDTA-anticoagulated maternal whole blood were collected at the same time. High molecular weight DNA was extracted from all of these samples by the salting-out procedure.3 PCR The detection method consisted of PCR amplification of genomic hypervariable regions. A panel of six minisatellites (MCT118, YNZ22, HVR 3′-globin, H-ras, COL and Apo B) was used to distinguish between maternal and cord DNAs; by using PCR primers that flank the minisatellite loci the whole allele was amplified and, therefore, the size of the PCR product was determined by the number of tandem repeats. PCR amplification of minisatellite sequences was performed as previously described with minor modifications.4–7 We optimized our amplification conditions with 150 ng of DNA in a total volume of 25 l. PCR products were run on agarose gels stained with ethidium bromide and visualized under UV transillumination. As a first step, we amplified mother and cord DNA to obtain at least one informative locus. Informative minisatellites were sub-
Cord blood maternal cell contamination M Briz et al
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sequently used to assess the presence of maternal DNA in cord blood samples. Sensitivity To determine the sensitivity of the technique, mixing experiments were performed in the first 10 mother–cord pairs; maternal DNA was diluted in cord DNA in different proportions (10, 5, 2.5, 1, 0.5, 0.25 and 0.1%), keeping the total amount of DNA constant. Quantification of maternal contamination In all cases in which maternal DNA was detected, different ratios of cord/mother DNAs were amplified and analyzed in parallel with cord blood samples. The values were estimated by visual comparison of the relative intensity of the bands with the dilution of cord/mother samples. Results Sensitivity of the PCR method Differences in sensitivity were observed when testing these artificially mixed maternal/cord samples; in most cases it was between 0.5 and 1% (seven cases), but it was higher in one case (0.25%) and lower in two cases (2.5%). Genotyping of mother–cord pairs and selection of minisatellite systems Eighty mother–cord pairs were recruited in this study. We amplified DNA from mother and cord samples to choose the most informative minisatellite systems. Only those for which the mother presented a specific allele were eligible. Seventy-nine out of the 80 mother–cord pairs (98.75%) were informative for at least one minisatellite locus. In one case, there were no specific maternal alleles at any locus and, therefore, the study could not be performed. Because shorter fragments are better amplified by polymerase, sensitivity is dependent on the relative sizes of mother and cord fragments. To ensure an optimum sensitivity, we selected those loci which in the mother had the shortest allele to avoid the impairment of sensitivity by the effect of preferential amplification. M
C
Mo
CB
Case 40 (Apo B)
M
C
Detection of maternal DNA sequences in umbilical cord blood DNA Among the 79 informative cases for the detection of maternal cells in the fetal circulation, at least two systems were used to study cord blood in 63 cases, while in six cases there was only one minisatellite region with a maternal-specific allele. We detected maternal DNA in only one cord blood sample (cord blood number 64), giving an incidence of 1.26%. Comparison between the signal intensity obtained with this sample and the result of the dilution experiment performed in the same analysis led to an estimate of 0.5–1% maternal cells. We were unable to detect maternal DNA in any of the other 78 cases (Figure 1).
Discussion The results of the first attempts to test cord blood samples for maternal cell contamination, performed by restriction fragment-length polymorphism or karyotypic analysis, were negative.8–10 More recently, larger numbers of cord blood samples have been analyzed by molecular biological techniques and there have already been a few reports of the identification of maternal cells or maternal DNA sequences in cord blood specimens.11–14 After amplification of hypervariable regions, sensitivity can be increased by transferring PCR products to nylon membranes for hybridizing with locus-specific oligonucleotides,4 although this procedure is time consuming. Another approach is to study a greater number of minisatellite regions and select the one that offers the highest sensitivity. In the present study, using a panel of six minisatellites, the sensitivity in most cases was between 0.5 and 1%; under these conditions, we identified maternal DNA sequences in one out of the 79 umbilical cord blood samples (1.26%). ´ This percentage is similar to that described by Socie et al12 (one in 47) using, as in our report, PCR amplification of minisatellite regions with which they detected 0.1–1% maternal cells in umbilical cord blood. Thus, this study confirms the fact that with PCR amplification of hypervariable regions, maternal DNA is very rarely detected in cord blood collected at birth. Nevertheless, the level of sensitivity of this approach is relatively low because two populations of DNA are coamplified using Mo
CB
Case 57 (MCT118)
M
C
Mo
CB
Case 64 (YNZ22)
Figure 1 PCR amplification of DNA at Apo B, MCT118 and YNZ22 minisatellite loci. Lane M, X174 HaeIII DNA marker; lane C, umbilical cord tissue; lane Mo, maternal peripheral blood; lane CB, cord blood. In cases 40 and 57, cord blood (CB) and cord (C) samples show an identical pattern of bands, with no evidence of maternal DNA (Mo) in the cord blood sample. In contrast, cord blood from case 64 shows a faint band corresponding to maternal DNA contamination.
Cord blood maternal cell contamination M Briz et al
primers that are common to both; although we are searching for maternal DNA, the major population corresponds to cord blood cells, the DNA of which acts as a competitor and, consequently, is preferentially amplified. Thus, this technique may have been of insufficient sensitivity to detect cells present in proportions of less than 0.5–1%. By identification of maternal locus-specific sequences, sensitivity can be increased to detect DNA in amounts equivalent to one maternal cell in 105 neonatal cells; under these conditions some authors have reported a higher contamination rate,11,14 detecting small numbers of maternal cells in about 40% of the umbilical cord blood samples. In summary, the incidence and level of maternal cell contamination reported in cord blood have varied, but the different rates of contamination probably reflect the sensitivity of the respective detection method used. The infusion of maternal lymphocytes into a transplant recipient may potentially cause severe GVHD. However, when maternal cells have been detected, the counts have been very low, and their presence in such low concentrations may play no role in GVHD after cord blood transplantation. This possibility is clinically supported by the fact that the frequency of severe GVHD after unrelated cord blood transplantation is relatively low, and no cases of GVHD secondary to engraftment of maternal cells have been reported to date. Moreover, a recent publication describes the case of a cord blood transplant contaminated with a significant number of maternal T cells in which the recipient developed only minimal GVHD.15 PCR amplification of hypervariable regions is often used to study post-transplantation chimerism;4 it has also occasionally aided in the diagnosis of transfusion-associated GVHD by detection of blood donor DNA.16 Likewise, in cases of severe GVHD after cord blood transplantation, chimerism studies should be done to assess the fetal or maternal origin of the lymphocytes responsible.
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Acknowledgements We are grateful to Carlos Pastor, Nuria de la Fuente and Dolores Sanchez-Mora for their excellent technical assistance and to Martha Messman for her editorial assistance. We also appreciate the ´ collaboration of all the midwives from the Hospital de Mostoles. This work was supported by a grant from the Fondo de Investigaciones Sanitarias (FIS 96/0610).
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