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Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing
Forensic Science International 139 (2004) 151–154
Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing M. Klintschara,*,1, P. Schwaigerb,1, S. Regauerc, S. Mannweilerc, M. Kleibera a
Institute of Legal Medicine, University Halle-Wittenberg, Franzosenweg 1, Halle D-06112, Germany Department of Child and Adolescent Psychiatry, Carl von Basedow Hospital, Merseburg, Germany c Institute of Pathology, University Graz, Austria
b
Received 21 March 2003; received in revised form 6 October 2003; accepted 10 October 2003
Abstract Forensic Y-chromosome typing applies Y-chromosomal polymorphisms to the analysis of male/female mixed stains such as vaginal swabs in rape cases. The sensitivity of this approach exceeds that of cytological techniques combined with autosomal DNA typing. Y-chromosome typing is based on the assumption that Y-chromosomal DNA found in tissue or secretions of women must originate from a male individual, usually the perpetrator. Nevertheless, it was shown recently that fetal cells can migrate into the female body during pregnancy and can persist for decades (‘‘persisting fetal microchimerism’’). The body of a woman after a pregnancy with a male embryo can thus display a small fraction of fetal cells with Y-chromosomes. Using high sensitivity PCR protocols (reamplification with nested primers and up to 60 PCR cycles) fetal cells were previously identified in a number of maternal tissues including skin, blood, muscle and solid organs. It is, however, not clear at present, whether these cells can occur in vaginal secretions, and whether they are capable of producing false positive results in forensic Y-chromosome typing. To evaluate these questions, 66 blood samples of women with at least one son and nine vaginal swabs of women without sexual intercourse in the last 2 weeks were amplified for a stretch of the SRY gene. Eight thyroid gland tissues with already established male fetal microchimerism were used as positive control samples. Blood samples of 10 young girls without history of pregnancy were used as negative controls. Using a PCR with 10 ng of extracted DNA and 30 PCR cycles (‘‘routine sensitivity assay’’) none of the samples yielded positive results. However, in a PCR with 200 ng of extracted DNA and 45 PCR cycles (‘‘high sensibility assay’’), 14% of the blood samples of mothers and 33% of the vaginal swabs amplified for SRY. Our results thus show that increasing the sensitivity of the PCR method and the amount of template DNA produce positive results while protocols used for routine Y-chromosomal typing with small amounts of DNA (approximately 10 ng of DNA) and with a limited number of PCR cycles (approximately 30) can clearly eliminate this peril. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Y-chromosome; DNA Typing; Fetal microchimerism; SRY; Vaginal swab
1. Introduction Y-STRs are short tandem repeat polymorphisms located on the Y chromosome. Since the first description of DYS19 * Corresponding author. Tel.: þ49-345-1591; fax: þ49-345-1587. E-mail address: [email protected] (M. Klintschar). 1 M.K. and P.S. contributed equally to this work.
[1], several of these loci were introduced to forensic stain analysis [2,3], but are also used in paternity testing and population genetics [4]. These polymorphisms are suitable for detecting the male component in male/female mixtures. They are consequently mainly used for vaginal swabs after sexual assaults, in which the inevitable female DNA from vaginal epithelial cells or leucocytes is capable of covering up the male component, especially when the female cells are abundant. Because of the high sensitivity of this approach Y-STRs are also recommended for vaginal swabs with
0379-0738/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2003.10.011
152
M. Klintschar et al. / Forensic Science International 139 (2004) 151–154
cytology negative for spermatozoa [5]. The application of YSTR typing to forensic case work, however, relies on the assumption that female blood, tissue and secretions lack cellular or free male DNA. The validity of this assumption is challenged by recent investigations concerning cellular trafficking between expecting mother and fetus [6,7]. The term ‘‘fetal microchimerism’’ (MCH) thus indicates an organism that contains cell populations derived from different individuals, the minor portion stemming from a fetus. It is thus possible to amplify Y-chromosomal sequences from blood and tissue of women pregnant with boys using Y-specific PCR [8]. Moreover, fetal cells have been found to persist for decades past delivery [9]. Persisting fetal microchimerim can be found in healthy mothers, but is more common in women suffering from autoimmune diseases like scleroderma [10,11] or Hashimoto’s thyroiditis [12]. Thus, all women who have ever been pregnant with a male embryo can show MCH with male fetal cells. This phenomenon obviously is in conflict with the basic assumption of forensic Y-STR analysis, i.e. that all cells from a female body lack Y chromosomes and male cells must stem from an intimate and recent contact with a male individual. Nevertheless, it is not known up to now, whether the minute fraction of fetal cells is capable of producing (false positive) results in forensic Y-STR typing. Moreover, although engrafted fetal cells have been found in a number of organs including the skin, it is not known whether these cells are present in the vagina and, if so, whether they are also found in vaginal secretions. The present study was performed to elucidate these questions.
2. Materials and methods 2.1. Samples and DNA extraction DNA was extracted from the following samples: 200 ml blood of 66 women who had given birth to at least one son, 6 months to 8 years prior to phlebotomy and nine vaginal swabs from women with one or more sons and without intercourse for at least 2 weeks. In 6 of these women, the swabs were taken before autopsy after hospitalization for more than 4 weeks, ensuring that the time without intercourse was at least 4 weeks. No autoimmune diseases are known for any of these women. Thyroid tissue from eight female patients suffering from Hashimoto’s thyroiditis for which fetal MCH was established in an earlier study [12] were used as positive controls. Blood samples of 10 young girls (age 1–10 years) without previous pregnancies or blood transfusions were used as negative controls. DNA from the blood samples was extracted using the QIAgen blood kit. DNA from the vaginal swabs was extracted using the QIAgen tissue kit (QIAgen, Hilden, FRG). DNA from the thyroid samples was extracted using the Puregene kit (Gentra Systems, Minneapolis, MN).
2.2. PCR Every sample was analyzed using 2 PCR protocols amplifying a stretch of the SRY gene using published primers [13]. For the ‘‘high sensitivity assay’’ protocol, 200 ng of DNA were used in a 12.5 ml assay containing 2 ml 10 X PCR buffer, 75 nmol/l of each primer, 200 mmol/l of each dNTP, and 0.5 U Dynazyme DNA polymerase (Finzymes, Espoo, Finland). Cycling conditions were 94 8C for 1 min, 59 8C for 1 min and 72 8C for 1 min 30 s for 45 cycles, followed by 60 8C for 45 min and a 25 8C hold. The amplificates were run on horizontal native polyacrylamide gels as previously described [14], and bands were visualized by silver staining. The ‘‘routine sensitivity assay’’ protocol is in accordance with forensic routine praxis: The sensitivity is relatively low to avoid generating artifacts from samples contaminated with minute amounts of DNA. The only differences to the ‘‘high sensitivity assay’’ are the number of PCR cycles (30) and that only 10 ng of DNA were used. Extreme caution was taken to avoid false positive results. All blood samples were handled by female technicians only. The tissue sections were prepared with meticulous care specifically during DNA extraction and immediately put into Eppendorf tubes. PCR and PAGE were performed in separate rooms, and numerous negative controls were always used in every PCR reaction. 2.3. Restriction analysis The amplified sequence of SRY (as published in genbank) was analyzed using the Webcutter V.2.0 software (http:// www.firstmarket.com/cutter/cut2.html) PstI (restriction site CTGCA/G) was identified to cut a 87 bp fragment off the amplificate. To control the specifity of the PCR selected amplificates were restricted and the length of the fragments was determined electrophoretically.
3. Results and discussion In fetal MCH, the persisting fetal cells are believed to be mostly lymphoid/hematologic stem cells [9]. As lymphoid cells can be found in all parts of the body including vaginal secretions, the question has to be raised whether these cells might be capable of interfering with forensic Y-STR typing: It might be possible that fetal male cells in a vaginal swab from a presumed rape case feign a false positive result. However, the extremely small percentage of chimeric cells is usually studied with special high sensitivity PCR protocols involving reamplifications with nested primers and up to 60 PCR cycles [10,11]. PCRs in forensic laboratories, on the other hand, usually are limited to 30 cycles to reduce the danger of contamination. These protocols might thus not be sufficiently sensitive to amplify the minute amounts of DNA, which in many cases might be no more than one cell per
M. Klintschar et al. / Forensic Science International 139 (2004) 151–154
Fig. 1. A typical gel for the high sensitivity approach. Flanking lanes: size marker (123 bp ladder). Lane A: negative control (Reagents control). Lanes B–Y: 24 blood samples of women with male children. The samples run in lane B, G, K, M, V, W and X were positive. Lane Z: positive control (male sample).
assay [15]. To elucidate this question we applied both a ‘‘high sensitivity assay’’ and a PCR protocol of forensic relevance (‘‘routine sensitivity assay’’) to different samples with varying probability of MCH. Using the high sensitivity protocol, male fetal cells were detected in all control samples of Hashimoto’s thyroiditis (as expected) and a part of the blood samples. The blood samples of young nulliparous girls lacked amplification for SRY. Most interestingly three of the nine vaginal swabs were positive (Fig. 1, Table 1). In all tested samples, the specifity of the PCR was demonstrated by restriction digestion as described above, which produced fragments of the expected length. The presence of fetal cells in vagina and vaginal secretions has not been described so far. Leucocytes/ lymphocytes are abundant in vaginal secretions of healthy women, and the majority of persistent microchimeric cells is considered to be derived from hematologic stem cells [9]. It can thus be assumed that the fetal cells in the vaginal swabs are white blood cells rather than epithelial cells. On the other hand, the increased prevalence of microchimeric cells in the
153
Fig. 2. A typical gel for the normal sensitivity approach. Flanking lanes: size marker (123 bp ladder). Lanes A–P: 15 blood samples of women with male children without amplification. Lane Q: positive control (male sample). Lane R: negative control (reagents control).
vagina compared to blood might be evidence of long term persistence of male lymphoid stem cells from semen (rather than of fetal origin) as proposed recently [16]. The ‘‘high sensitivity assay’’ is a PCR protocol with 45 PCR cycles which is considerably more sensitive than the 30 cycles recommended for forensic investigations. Moreover, we used 200 ng of DNA to increase the chance of including male DNA into the assay. Forensic investigators usually use 1–10 ng of DNA for their analyses. Applying the ‘‘routine sensitivity assay’’ with 30 cycles and 10 ng DNA no Ychromosomal DNA/male cells were detected in any of the samples including the 8 MCH positive Hashimoto’s thyroiditis specimens (Fig. 2, Table 1). The relatively high percentage of positive samples in the high sensitivity assay might alternatively be explained by contamination. However, we believe to be able to exclude this possibility as all negative controls, including the blood
Table 1 Amplification results of PCR with SRY using a high sensitivity approach (45 cycles) and a normal sensitivity approach (30 cycles) No. of samples
Thyroid glands of women with known microchimerism Blood of mothers of boys Vaginal swabs Blood of girls
No. of samples positive for male DNA High sensitivity assay
Routine sensitivity assay
8
8
100.0%
0
0%
66 9 12
9 3 0
13.6% 33.3% 0.0%
0 0 0
0% 0% 0%
154
M. Klintschar et al. / Forensic Science International 139 (2004) 151–154
samples of the young girls were negative and meticulous care was taken to avoid the generation of contamination. Alternatively, the minute amounts of male DNA in the vaginal swabs might be remnants from sexual intercourse. However, as we could establish that the last intercourse was at least 2 weeks ago (in six cases, of which two were positive for MCH, even more than 4 weeks) this explanation appears highly unlikely, given that sperm Y-chromosomes are assumed to be detectable no more than 2–4 days after intercourse [17]. We thus conclude that persisting (male) fetal cells that have engrafted into the body of mothers can be detected in blood and secretions using Y-specific PCR. On the other hand, the percentage of these cells small and high sensitivity PCR protocols are required for detection. We could clearly demonstrate that protocols routinely used in forensic laboratories are not capable of detecting these cells. Persisting fetal MCH therefore is extremely unlikely to produce false positive results in a forensic investigation provided that the number of PCR cycles is confined to 30 and less than 10 ng of DNA, as recommended in published Y-STR multiplex assays [3,18]. We propose that these PCR conditions are strictly obeyed when Y-chromosomal markers are used to detect a male contribution to a mixed stain, especially when the assaulted woman has ever been pregnant and in cases without cytological detection of spermatozoa [5].
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
References [1] L. Roewer, J. Arnemann, N.K. Spurr, K.H. Grzeschik, J.T. Epplen, Simple repeat sequences on the human Y chromosome are equally polymorphic as their autosomal counterparts, Hum. Genet. 89 (1992) 389–394. [2] M. Kayser, A. Caglia, D. Corach, N. Fretwell, C. Gehrig, G. Graziosi, F. Heidorn, S. Herrmann, B. Herzog, M. Hidding, K. Honda, M. Jobling, M. Krawczak, K. Leim, S. Meuser, E. Meyer, W. Oesterreich, A. Pandya, W. Parson, G. Penacino, A. Perez-Lezaun, A. Piccinini, M. Prinz, C. Schmitt, L. Roewer, et al., Evaluation of Y-chromosomal STRs: a multicenter study, Int. J. Legal Med. 110 (1997) 125–133, 141–149. [3] A. Gonzalez-Neira, M. Elmoznino, M.V. Lareu, P. SanchezDiz, L. Gusmao, M. Prinz, A. Carracedo, Sequence structure of 12 novel Y chromosome microsatellites and PCR amplification strategies, Forensic Sci. Int. 122 (2001) 19–26. [4] P. de Knijff, M. Kayser, A. Caglia, D. Corach, N. Fretwell, C. Gehrig, G. Graziosi, F. Heidorn, S. Herrmann, B. Herzog, M. Hidding, K. Honda, M. Jobling, M. Krawczak, K. Leim, S. Meuser, E. Meyer, W. Oesterreich, A. Pandya, W. Parson, G. Penacino, A. Perez-Lezaun, A. Piccinini, M. Prinz, L. Roewer
[13] [14]
[15]
[16]
[17]
[18]
et al., Chromosome Y microsatellites: population genetic and evolutionary aspects, Int. J. Legal Med. 110 (1997) 134–149. I. Sibille, C. Duverneuil, G. Lorin de la Grandmaison, K. Guerrouache, F. Teissiere, M. Durigon, P. de Mazancourt, YSTR DNA amplification as biological evidence in sexually assaulted female victims with no cytological detection of spermatozoa, Forensic Sci. Int. 125 (2002) 212–216. L.A. Herzenberg, D.W. Bianchi, J. Schro¨ der, H.M. Cann, G.M. Iversen, Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting, Proc. Natl. Acad. Sci. 76 (1979) 1453–1455. B. Pertl, D.W. Bianchi, First trimester prenatal diagnosis: fetal cells in the maternal circulation, Semin. Perinatol. 23 (1999) 393–402. M.R. Thomas, R. Williamson, I. Craft, N. Yazdani, C.H. Rodeck, Y Chromosome sequence DNA amplified from periphel blood of women in early pregnancy, Lancet 343 (1994) 413–414. D.W. Bianchi, G.K. Zickwolf, G.J. Weil, S. Sylvester, M.A. DeMaria, Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 705–708. J.L. Nelson, D.E. Furst, S. Maloney, T. Gooley, P.C. Evans, A. Smith, M.A. Bean, C. Ober, D.W. Bianchi, Microchimerism and HLA-compatible relationships of pregnancy in scleroderma, Lancet 351 (1998) 559–562. C.M. Artlett, J.B. Smith, S.A. Jimenez, Identification of fetal DNA and cells in skin lesions from women with systemic sclerosis, N. Engl. J. Med. 338 (1998) 1186–1191. M. Klintschar, P. Schwaiger, S. Mannweiler, S. Regauer, M. Kleiber, Evidence of fetal microchimerism in Hashimoto’s thyroiditis, J. Clin. Endocrinol. Metab. 86 (2001) 2494– 2498. F.R. Santos, A. Pandya, C. Tyler-Smith, Reliability of DNAbased sex tests, Nat. Genet. 18 (1998) 103. M. Klintschar, R. Crevenna, HumCD4—validation of a STR system for forensic purposes in an Austrian Caucasian population sample, J. Forensic Sci. 42 (1997) 907–910. N.C. Lambert, Y.M. Lo, T.D. Erickson, T.S. Tylee, K.A. Guthrie, D.E. Furst, J.L. Nelson, Male microchimerism in healthy women and women with scleroderma: cells or circulating DNA? A quantitative answer, Blood 100 (2002) 2845–2851. S. Aractingi, S. Uzan, J. Dausset, E.D. Carosella, Microchimerism in human diseases, Immunol. Today 21 (2000) 116–118. A. Hall, J. Ballantyne, Novel Y-STR typing strategies reveal the genetic profile of the semen donor in extended interval post-coital cervicovaginal samples, Forensic Sci. Int., in press. J.M. Butler, R. Schoske, P.M. Vallone, M.C. Kline, A.J. Redd, M.F. Hammer, A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers, Forensic Sci. Int. 129 (2002) 10–24.
Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing M. Klintschara,*,1, P. Schwaigerb,1, S. Regauerc, S. Mannweilerc, M. Kleibera a
Institute of Legal Medicine, University Halle-Wittenberg, Franzosenweg 1, Halle D-06112, Germany Department of Child and Adolescent Psychiatry, Carl von Basedow Hospital, Merseburg, Germany c Institute of Pathology, University Graz, Austria
b
Received 21 March 2003; received in revised form 6 October 2003; accepted 10 October 2003
Abstract Forensic Y-chromosome typing applies Y-chromosomal polymorphisms to the analysis of male/female mixed stains such as vaginal swabs in rape cases. The sensitivity of this approach exceeds that of cytological techniques combined with autosomal DNA typing. Y-chromosome typing is based on the assumption that Y-chromosomal DNA found in tissue or secretions of women must originate from a male individual, usually the perpetrator. Nevertheless, it was shown recently that fetal cells can migrate into the female body during pregnancy and can persist for decades (‘‘persisting fetal microchimerism’’). The body of a woman after a pregnancy with a male embryo can thus display a small fraction of fetal cells with Y-chromosomes. Using high sensitivity PCR protocols (reamplification with nested primers and up to 60 PCR cycles) fetal cells were previously identified in a number of maternal tissues including skin, blood, muscle and solid organs. It is, however, not clear at present, whether these cells can occur in vaginal secretions, and whether they are capable of producing false positive results in forensic Y-chromosome typing. To evaluate these questions, 66 blood samples of women with at least one son and nine vaginal swabs of women without sexual intercourse in the last 2 weeks were amplified for a stretch of the SRY gene. Eight thyroid gland tissues with already established male fetal microchimerism were used as positive control samples. Blood samples of 10 young girls without history of pregnancy were used as negative controls. Using a PCR with 10 ng of extracted DNA and 30 PCR cycles (‘‘routine sensitivity assay’’) none of the samples yielded positive results. However, in a PCR with 200 ng of extracted DNA and 45 PCR cycles (‘‘high sensibility assay’’), 14% of the blood samples of mothers and 33% of the vaginal swabs amplified for SRY. Our results thus show that increasing the sensitivity of the PCR method and the amount of template DNA produce positive results while protocols used for routine Y-chromosomal typing with small amounts of DNA (approximately 10 ng of DNA) and with a limited number of PCR cycles (approximately 30) can clearly eliminate this peril. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Y-chromosome; DNA Typing; Fetal microchimerism; SRY; Vaginal swab
1. Introduction Y-STRs are short tandem repeat polymorphisms located on the Y chromosome. Since the first description of DYS19 * Corresponding author. Tel.: þ49-345-1591; fax: þ49-345-1587. E-mail address: [email protected] (M. Klintschar). 1 M.K. and P.S. contributed equally to this work.
[1], several of these loci were introduced to forensic stain analysis [2,3], but are also used in paternity testing and population genetics [4]. These polymorphisms are suitable for detecting the male component in male/female mixtures. They are consequently mainly used for vaginal swabs after sexual assaults, in which the inevitable female DNA from vaginal epithelial cells or leucocytes is capable of covering up the male component, especially when the female cells are abundant. Because of the high sensitivity of this approach Y-STRs are also recommended for vaginal swabs with
0379-0738/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2003.10.011
152
M. Klintschar et al. / Forensic Science International 139 (2004) 151–154
cytology negative for spermatozoa [5]. The application of YSTR typing to forensic case work, however, relies on the assumption that female blood, tissue and secretions lack cellular or free male DNA. The validity of this assumption is challenged by recent investigations concerning cellular trafficking between expecting mother and fetus [6,7]. The term ‘‘fetal microchimerism’’ (MCH) thus indicates an organism that contains cell populations derived from different individuals, the minor portion stemming from a fetus. It is thus possible to amplify Y-chromosomal sequences from blood and tissue of women pregnant with boys using Y-specific PCR [8]. Moreover, fetal cells have been found to persist for decades past delivery [9]. Persisting fetal microchimerim can be found in healthy mothers, but is more common in women suffering from autoimmune diseases like scleroderma [10,11] or Hashimoto’s thyroiditis [12]. Thus, all women who have ever been pregnant with a male embryo can show MCH with male fetal cells. This phenomenon obviously is in conflict with the basic assumption of forensic Y-STR analysis, i.e. that all cells from a female body lack Y chromosomes and male cells must stem from an intimate and recent contact with a male individual. Nevertheless, it is not known up to now, whether the minute fraction of fetal cells is capable of producing (false positive) results in forensic Y-STR typing. Moreover, although engrafted fetal cells have been found in a number of organs including the skin, it is not known whether these cells are present in the vagina and, if so, whether they are also found in vaginal secretions. The present study was performed to elucidate these questions.
2. Materials and methods 2.1. Samples and DNA extraction DNA was extracted from the following samples: 200 ml blood of 66 women who had given birth to at least one son, 6 months to 8 years prior to phlebotomy and nine vaginal swabs from women with one or more sons and without intercourse for at least 2 weeks. In 6 of these women, the swabs were taken before autopsy after hospitalization for more than 4 weeks, ensuring that the time without intercourse was at least 4 weeks. No autoimmune diseases are known for any of these women. Thyroid tissue from eight female patients suffering from Hashimoto’s thyroiditis for which fetal MCH was established in an earlier study [12] were used as positive controls. Blood samples of 10 young girls (age 1–10 years) without previous pregnancies or blood transfusions were used as negative controls. DNA from the blood samples was extracted using the QIAgen blood kit. DNA from the vaginal swabs was extracted using the QIAgen tissue kit (QIAgen, Hilden, FRG). DNA from the thyroid samples was extracted using the Puregene kit (Gentra Systems, Minneapolis, MN).
2.2. PCR Every sample was analyzed using 2 PCR protocols amplifying a stretch of the SRY gene using published primers [13]. For the ‘‘high sensitivity assay’’ protocol, 200 ng of DNA were used in a 12.5 ml assay containing 2 ml 10 X PCR buffer, 75 nmol/l of each primer, 200 mmol/l of each dNTP, and 0.5 U Dynazyme DNA polymerase (Finzymes, Espoo, Finland). Cycling conditions were 94 8C for 1 min, 59 8C for 1 min and 72 8C for 1 min 30 s for 45 cycles, followed by 60 8C for 45 min and a 25 8C hold. The amplificates were run on horizontal native polyacrylamide gels as previously described [14], and bands were visualized by silver staining. The ‘‘routine sensitivity assay’’ protocol is in accordance with forensic routine praxis: The sensitivity is relatively low to avoid generating artifacts from samples contaminated with minute amounts of DNA. The only differences to the ‘‘high sensitivity assay’’ are the number of PCR cycles (30) and that only 10 ng of DNA were used. Extreme caution was taken to avoid false positive results. All blood samples were handled by female technicians only. The tissue sections were prepared with meticulous care specifically during DNA extraction and immediately put into Eppendorf tubes. PCR and PAGE were performed in separate rooms, and numerous negative controls were always used in every PCR reaction. 2.3. Restriction analysis The amplified sequence of SRY (as published in genbank) was analyzed using the Webcutter V.2.0 software (http:// www.firstmarket.com/cutter/cut2.html) PstI (restriction site CTGCA/G) was identified to cut a 87 bp fragment off the amplificate. To control the specifity of the PCR selected amplificates were restricted and the length of the fragments was determined electrophoretically.
3. Results and discussion In fetal MCH, the persisting fetal cells are believed to be mostly lymphoid/hematologic stem cells [9]. As lymphoid cells can be found in all parts of the body including vaginal secretions, the question has to be raised whether these cells might be capable of interfering with forensic Y-STR typing: It might be possible that fetal male cells in a vaginal swab from a presumed rape case feign a false positive result. However, the extremely small percentage of chimeric cells is usually studied with special high sensitivity PCR protocols involving reamplifications with nested primers and up to 60 PCR cycles [10,11]. PCRs in forensic laboratories, on the other hand, usually are limited to 30 cycles to reduce the danger of contamination. These protocols might thus not be sufficiently sensitive to amplify the minute amounts of DNA, which in many cases might be no more than one cell per
M. Klintschar et al. / Forensic Science International 139 (2004) 151–154
Fig. 1. A typical gel for the high sensitivity approach. Flanking lanes: size marker (123 bp ladder). Lane A: negative control (Reagents control). Lanes B–Y: 24 blood samples of women with male children. The samples run in lane B, G, K, M, V, W and X were positive. Lane Z: positive control (male sample).
assay [15]. To elucidate this question we applied both a ‘‘high sensitivity assay’’ and a PCR protocol of forensic relevance (‘‘routine sensitivity assay’’) to different samples with varying probability of MCH. Using the high sensitivity protocol, male fetal cells were detected in all control samples of Hashimoto’s thyroiditis (as expected) and a part of the blood samples. The blood samples of young nulliparous girls lacked amplification for SRY. Most interestingly three of the nine vaginal swabs were positive (Fig. 1, Table 1). In all tested samples, the specifity of the PCR was demonstrated by restriction digestion as described above, which produced fragments of the expected length. The presence of fetal cells in vagina and vaginal secretions has not been described so far. Leucocytes/ lymphocytes are abundant in vaginal secretions of healthy women, and the majority of persistent microchimeric cells is considered to be derived from hematologic stem cells [9]. It can thus be assumed that the fetal cells in the vaginal swabs are white blood cells rather than epithelial cells. On the other hand, the increased prevalence of microchimeric cells in the
153
Fig. 2. A typical gel for the normal sensitivity approach. Flanking lanes: size marker (123 bp ladder). Lanes A–P: 15 blood samples of women with male children without amplification. Lane Q: positive control (male sample). Lane R: negative control (reagents control).
vagina compared to blood might be evidence of long term persistence of male lymphoid stem cells from semen (rather than of fetal origin) as proposed recently [16]. The ‘‘high sensitivity assay’’ is a PCR protocol with 45 PCR cycles which is considerably more sensitive than the 30 cycles recommended for forensic investigations. Moreover, we used 200 ng of DNA to increase the chance of including male DNA into the assay. Forensic investigators usually use 1–10 ng of DNA for their analyses. Applying the ‘‘routine sensitivity assay’’ with 30 cycles and 10 ng DNA no Ychromosomal DNA/male cells were detected in any of the samples including the 8 MCH positive Hashimoto’s thyroiditis specimens (Fig. 2, Table 1). The relatively high percentage of positive samples in the high sensitivity assay might alternatively be explained by contamination. However, we believe to be able to exclude this possibility as all negative controls, including the blood
Table 1 Amplification results of PCR with SRY using a high sensitivity approach (45 cycles) and a normal sensitivity approach (30 cycles) No. of samples
Thyroid glands of women with known microchimerism Blood of mothers of boys Vaginal swabs Blood of girls
No. of samples positive for male DNA High sensitivity assay
Routine sensitivity assay
8
8
100.0%
0
0%
66 9 12
9 3 0
13.6% 33.3% 0.0%
0 0 0
0% 0% 0%
154
M. Klintschar et al. / Forensic Science International 139 (2004) 151–154
samples of the young girls were negative and meticulous care was taken to avoid the generation of contamination. Alternatively, the minute amounts of male DNA in the vaginal swabs might be remnants from sexual intercourse. However, as we could establish that the last intercourse was at least 2 weeks ago (in six cases, of which two were positive for MCH, even more than 4 weeks) this explanation appears highly unlikely, given that sperm Y-chromosomes are assumed to be detectable no more than 2–4 days after intercourse [17]. We thus conclude that persisting (male) fetal cells that have engrafted into the body of mothers can be detected in blood and secretions using Y-specific PCR. On the other hand, the percentage of these cells small and high sensitivity PCR protocols are required for detection. We could clearly demonstrate that protocols routinely used in forensic laboratories are not capable of detecting these cells. Persisting fetal MCH therefore is extremely unlikely to produce false positive results in a forensic investigation provided that the number of PCR cycles is confined to 30 and less than 10 ng of DNA, as recommended in published Y-STR multiplex assays [3,18]. We propose that these PCR conditions are strictly obeyed when Y-chromosomal markers are used to detect a male contribution to a mixed stain, especially when the assaulted woman has ever been pregnant and in cases without cytological detection of spermatozoa [5].
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
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