With great interest we read a recent report by Endler and coworkers.1 In this paper, the authors show that DNA derived from mouthwashes after allogeneic blood stem cell transplantation frequently displays a chimeric or complete donor genotype and is thus not suitable to obtain information on the pre-transplantation allelotype of the recipient. Although we agree with the results obtained in this study, we would like to report our experiences on buccal epithelial cells as a source for chimerism analysis.
DNA-based assessment of chimerism after allogeneic blood stem cell transplantation (BSCT) using highly polymorphic DNA sequences, such as short tandem repeat (STR) markers, has become a widely used method because of the sensitivity, the ease of performance and the independence of sex mismatch between donor and recipient. A limitation frequently imposing uncertainties in the interpretation of the results is that a pre-transplantation sample has to be analyzed for donor and recipient, in order to define informative STR combinations. Although this should not be difficult in theory, our experience shows that in 10–15% of all patients, the corresponding pre-transplantation sample is missing. The oral mucosa represents an attractive source of appropriate material, since it is easily accessible and should theoretically be derived from the recipient. However, Endler et al1 found that mouthwashes performed after BSCT frequently contain a high proportion of donor cells and sometimes the DNA derived from this material completely reflects the donor STR profile. We have employed a different approach, aiming to directly use epithelial cells derived from buccal swabs to obtain the pre-transplantation STR genotype of the recipient. This technique is standard in forensic medicine. However, after allogeneic transplantation the situation is complicated, since granulocytes and lymphocytes, which are trafficking through the normal human mucosa and can be found in the saliva, are usually derived from the donor. There is highly divergent information on the number of leukocytes in the normal saliva, ranging from 2 to 136000 cells/ml, and in patients with inflammation of the oral cavity, this number was found to be up to 1.1 × 106/ml.2 Thus, in order to remove as many non-epithelial cells as possible, patients were asked to thoroughly rinse their mouth twice with 5–10 ml of water. The first portion of rinsing fluid was used as mouthwash sample (see below). Then, a sterile cytological brush (Cytosoft Cytology Brush, Medical Packaging, Camarillo, CA, USA) was employed to collect epithelial cells from the buccal mucosa on both sides of the oral cavity. Cells were immediately recovered by immersion in 200 μl PBS. DNA was subsequently extracted from this sample and an aliquot of the mouthwash sample using a silica-based procedure (Qiagen DNA Blood Kit; Qiagen, Hilden, Germany). The DNA was eluted in 40–50 μl of TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8), quantified and diluted to a final concentration of 0.125 ng/μl in TE buffer. Usually, the DNA derived from the buccal swabs was too dilute to be accurately quantified. In these cases, a 1/10 dilution of the original sample was used. Quantitative chimerism analysis was performed using a recently developed procedure based on a multiplex PCR with coamplification of nine STRs labeled with fluorescent primers.3 PCR, gel electrophoresis and calculation of the donor/recipient ratio were performed as described.
This procedure has been successfully used in our institution in more than 20 cases. In order to compare the amount of donor DNA present in buccal swabs and in mouthwash samples, we analyzed both materials in 13 patients with known profiles for the donor and the recipient. These samples were taken 1–24 months after allogeneic transplantation. The clinical data and the results are summarized in Table 1. As shown, our results support the findings by Endler,1 in that mouthwash samples contain high amounts of donor DNA, and are sometimes almost completely derived from the donor (Table 1, patients 1 and 12). DNA samples derived from buccal swabs still contained donor DNA. However, the amount was much lower than in the mouthwash samples (median 21% in the buccal swab samples compared to 74% in the mouthwash samples). In 11/13 samples, the donor peaks accounted for 1/3 of the total signal or less. In all these cases, the recipient profile could be easily identified using our protocol (Figure 1). Similar results were obtained in only 2/13 mouthwash samples.
Thus, we conclude that although mouthwash DNA usually represents a poor source to obtain a pre-transplantation profile of the recipient, buccal swab DNA can be used for this purpose in the majority of the cases. This method appears considerably easier than procedures using magnetic beads to enrich epithelial cells. It is still not clear which factors define the number of donor cells present in mouthwashes as well as buccal swab samples. We looked for the association with several clinical factors, including the time after transplantation, the graft source or the presence of GVHD. Among these, local GVHD might be an important factor, since four of the five patients with donor chimerism values above 30% in the buccal swabs had clinical manifest inflammation of the oral mucosa. However, in the light of the high number of variables potentially involved, our results in a small group of patients have to be interpreted with caution.
Endler G, Greinix H, Winkler K et al. Genetic fingerprinting in mouthwashes of patients after allogeneic bone marrow transplantation Bone Marrow Transplant 1999 24: 95–98
Burnett GW, Scherp HW . Oral Microbiology and Infectious Disease Williams & Wilkins Co: Baltimore 1962 pp 322–323
Thiede C, Florek M, Bornhäuser M et al. Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection Bone Marrow Transplant 1999 23: 1055–1060
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Thiede, C., Prange-Krex, G., Freiberg-Richter, J. et al. Buccal swabs but not mouthwash samples can be used to obtain pretransplant DNA fingerprints from recipients of allogeneic bone marrow transplants. Bone Marrow Transplant 25, 575–577 (2000). https://doi.org/10.1038/sj.bmt.1702170
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