STR typing of skin swabs from individuals after an allogeneic hematopoietic stem cell transplantation

The proportion of recipient and donor alleles was calculated based on the peak area ratios across all systems.

Statistical calculations were performed using IBM® SPSS® Statistics Version 25 software.

Since cells newly produced by the bone marrow after successful transplantation should exclusively show the donor’s alleles, blood samples were taken from the patients in order to determine them. For 18 tested persons, STR typing of such blood samples revealed single person profiles that differed from the respective buccal swab results (complete chimerism). Eight blood samples showed mixtures (mixed chimerism), which indicate a possible recurrence. In seven samples, isolated recipient alleles were detected, whereas one sample showed the complete recipient’s DNA profile as a minor component (assignment of donor alleles according to the respective buccal swabs). Two blood samples resulted in balanced two-person-mixtures, whereby the donor characteristics could be derived for one of the samples based on the alleles obtained for the corresponding buccal swab. The donor alleles could not be derived for the other sample (patient 15).

Only two buccal swabs (reference sample for the recipient’s DNA profile) yielded individual profiles that did not match the blood samples’ alleles, including one sample with a weakly positive preliminary blood test. However, this person showed a complete chimerism in the blood sample. Thus, the presence of the patient’s blood on the according buccal swab, e.g., due to bleeding gums, would have resulted in a mixture of recipient and donor alleles. Consequently, the Combur test result may have been falsely positive, possibly triggered by other substances on the swab, such as food residues. All other buccal swabs showed mixtures in STR typing. For 16 of these samples, however, clearly definable main components could be derived, which in turn did not correspond to the respective alleles of the blood samples. Analysis of the buccal swabs of the remaining ten persons examined resulted in more or less balanced allele mixtures, whereby partial recipient DNA profiles could be derived regarding the donor alleles obtained from the corresponding blood samples. For one individual (patient 15), the recipient alleles could not be deduced due to the mixed genotype in the corresponding blood sample. In all ten buccal swabs that tested positive for blood and showed mixtures, the admixture of donor alleles can be explained by the additional blood cells. For the remaining 16 swabs with visible allele mixtures, the preliminary blood test was negative. This means that either the test showed false negative results or that cells with donor alleles were found on the swabs lacking the admixture of erythrocytes (for further discussion, see the “Determination of ratio of recipient’s to donor’s alleles” section).

Since neither the blood sample nor the buccal swab from patient 15 revealed individual DNA profiles or main components, neither donor nor recipient characteristics could be determined for this individual. Consequently, the skin swabs of this person could not be evaluated with regard to the proportion of recipient or donor alleles and were assigned to category A. Only the quantification results of these samples were included in the evaluation below. All other results exclusively refer to the remaining 27 patients.

The quantification of human DNA resulted in less than 0.5 pg/µl for 11 swabs (8%). These samples were not further examined. Fourteen percent of the samples contained DNA amounts of 0.5–1 pg/μl, 53% amounts of 1–10 pg/µl, and 22% contained a DNA amount of 10–100 pg/µl. Four samples (3%) contained an amount greater than 100 pg/µl DNA (see Table 1). The highest yield of DNA was obtained from a swab from one person’s neck with 570 pg/µl. The calculated median values of DNA amounts across all collection sites per subject resulted in a range from 0.2–82.1 pg/µl (see Table 1), showing a very large variability in the amount of DNA obtained from different individuals. The tendency of certain people to leave more (good shedder) or less (poor shedder) DNA in skin contact traces has already been observed many times. In addition to skin characteristics [4‐7], a person’s age as well as the activity before contact, such as washing hands, can cause varying results regarding one and the same person [4, 5, 8‐11].

The results also show that some persons shed widely varying amounts of DNA from different parts of the body. Swabs from the neck showed DNA amounts greater than 0.5 pg/µl for all patients and provided the greatest DNA yield in 19 of 28 tested individuals. Three of the remaining nine patients left the greatest DNA amount on swabs of each, the right or left hand, two on swabs of the back of their hand and one on the swab taken from the upper arm (see Table 1). Statistical comparison of the different swabbing sites regarding the DNA amounts obtained (Kruskal–Wallis test, significance level 5%) resulted in a p-value of 0.000 and thus in a significant difference. However, pairwise comparison of DNA amounts obtained from the skin swabs of the various sampling sites (Mann–Whitney U test, significance level 5%) showed no significant differences between samples taken from the right or left hand, the back of the hand, and the upper arm (data not shown). Only the swabs taken from the neck showed a significant difference compared to the samples from the right palm (p-value = 0.004), left palm (p-value = 0.007), back of the hand (p-value = 0.001), and upper arm (p-value = 0.000). Thus, as already described by Kamphausen et al. [12], for most people, the neck area provides more DNA material than other body regions.

A total of 38 of the remaining 129 skin swabs examined were assigned to category A. All samples of person four were among those 38 samples. In ten of the remaining 26 subjects, all skin swabs produced could be classified in category B. In the remaining 16 patients, usable results were obtained for only 1 to 4 swabs.

In 76 of the total of 91 (83%) samples assigned to category B, alleles of other persons could be detected in addition to the recipient and donor alleles. The affected samples showed between two and 31 additional alleles in the 16 autosomal STR systems examined. Most frequently, one to ten additional signals were found (data not shown). No extraneous material was detectable on the remaining 15 swabs. All four swabs with DNA amounts greater than 100 pg/µl were among those samples. The remaining samples in this group showed a DNA amount of 1–10 pg/µl (four samples) and 10–100 pg/µl (seven samples), respectively. A correlation of the DNA amounts in all samples and the results regarding the number of additionally obtained alleles shows a Spearman-Rho correlation coefficient of − 0.202 with a p-value of 0.055 (significance level 5%) and consequently gives a very low negative, but non-significant correlation. This, together with the fact that no additional features were found on the four swabs with a DNA amount greater than 100 pg/μl, provides at least an indication that less foreign material can be detected on swabs with a higher DNA yield. It is well known that as the amount of DNA decreases, the number of drop-in events increases, which could explain at least part of these results. Moreover, this fact was also previously described by Goray et al. [13], where good shedders turned out to leave more of their own DNA on objects than foreign alleles and, in turn, poor shedders release less of their own DNA to their environment than foreign DNA. According to Goray et al. [13], this could possibly be due to the fact that poor shedders take up similar amounts of foreign DNA as good shedders but do not overlay it with their own DNA. Since STR typing only reliably detects mixtures up to a ratio of 1:20, foreign material is likely to be present on swabs with a high DNA yield, but it is not detectable in STR typing due to unbalanced mixture ratios.

All 91 samples assigned to category B showed both recipient and donor alleles in different proportions or mixture ratios. For nine of the skin swabs, mixtures could be obtained in which all recipient alleles were detectable. The donor’s alleles were detected almost completely or only partially (one to five missing alleles). On the other hand, in 22 samples, donor’s alleles were completely present in the mixtures and those of the recipient only incompletely (one to ten missing alleles). Donor’s as well as recipient’s complete DNA profiles were found in the mixtures of 34 samples in total. In the remaining 26 samples, neither the recipient nor the donor alleles could be completely detected.

Twenty-one skin swabs with complete profiles of the recipient and/or donor (three samples with the complete recipient’s profile, 14 with the complete donor’s profile, and four samples which showed complete profiles of both persons) showed admixtures caused by additional persons, observable either due to too many alleles per locus or unusually high signal intensities. These samples as well as the 26 samples in which neither the recipient’s nor the donor’s alleles could be completely detected were not further evaluated. The remaining 44 skin swabs were assigned to category C and the ratio of recipient to donor alleles was determined.

The 44 samples assigned to category C consisted of eight swabs from the right palm, ten from the left palm, seven from the back of the hand, four swabs from the upper arm, and 15 samples from the neck from a total of 18 different patients. Sixteen of these samples contained a DNA amount of 1–10 pg/µl, 24 between 10 and 100 pg/µl, and four of the swabs more than 100 pg/µl. The proportion of recipient’s to donor’s alleles of these samples was determined considering the peak areas. This showed a range of variation from 12–81% for recipient alleles and consequently from 19–88% for donor alleles (see Table 2). These results show that cells with donor alleles can be found on the prepared skin swabs. However, the preliminary test for blood was negative for all samples, suggesting that the samples contain other cells formed by the new bone marrow of the donor.

Since skin swabs were collected from the epidermis’ surface, which consists mainly of keratinocytes in this area, this result is somewhat surprising. Only the so-called Langerhans cells (LC) located in the lower stratum spinosum and first described by Paul Langerhans in 1868 should be mentioned as cells of the epidermis with a myeloid background while dermal dendritic cells are located beyond the basal membrane, in the dermis [14, 15]. While the function of LC is still being discussed, their myeloid origin is proven in mice [16‐18] as well as in humans [19, 20]. As antigen-representing cells of the epidermis, they react to a stimulus by differentiating and migrating to the local lymph nodes [21] and play a role in coordinating an immune response [16, 22, 23]. Remarkably, they appear to renew themselves locally via mitosis rather than upon migration of hematopoietic cells under normal conditions [24]. Under inflammatory conditions, on the other hand, the neonatal, long-lived LC are supplemented or replaced by fresh, short-lived LC, which mature from the pool of monocytes [24‐28]. Since those monocytes develop from the bone marrow, they exhibit donor alleles after alloHSCT. The extent to which either this or the different subclasses are clinically relevant, e.g., with regard to the occurrence of graft versus host disease, has not been finally clarified. Graft versus host disease (GvHD) is a crucial factor for mortality and morbidity after alloHSCT and was first described in mice by Barnes and Loutit [29] and defined by Billingham [30]. Acute GvHD (aGvHD) primarily infests the skin but also the colon and liver [31]. In contrast, chronic GvHD (cGvHD), which occurs later, is a syndrome that shows significantly greater variability regarding the organs involved [32].

The wide range of variation between 19–88% of donor alleles and 81–12% of recipient alleles obtained in samples collected at least 180 days after alloHSCT and examined here shows that different amounts of recipient’s cells and cells with donor’s alleles were found on the prepared swabs. Assuming that after this time all LC have been replaced by donor cells [33, 34], the recipient portion would consist of an admixture of DNA from non-myeloid cells such as keratinocytes. Consequently, this would be an indication that not all individuals shed an equal number of skin cells or immune cells located in the skin upon contact.

However, the wide variation can also be explained by an incomplete chimerism of these cells. Divergent observations regarding a chimerism of the skin were made based on different models. Thus, complete donor chimerism of the LC was described after 84–100 days after dose-reduced conditioning, whereas a study by Perreault et al. [35] showed that LC of the host could be detected up to a year after transplantation. The time span in which this exchange takes place also seems to depend on the amount of transplanted T-cells from the donor. Merad et al. [36] showed that in mice transplanted with allogeneic bone marrow or with purified hematopoietic stem cells, Langerhans cells of the donor could not be detected in the skin even 18 months after transplantation. The exchange of these immune cells only took place after donor T cells were added to the transplanted bone marrow. Thus, both the post transplantation time span and the proportion of T-cells in the transplant may be responsible for the variability of donor allele to recipient allele ratios among the tested patients. Potential GvHD may also have an impact. The association of antigen-presenting cells [37] or specific LC [35] with GvHD was postulated early on, but is not yet well understood. Whereas Merad et al. [36] state that skin GvHD can be prevented by LC depletion, other results suggest that GvHD does not require classic LC [38], aGvHD is triggered by donor monocytes [39], and that skin chimerism can occur regardless of cGvHD [40]. The impact of different conditioning regimes or the immunosuppressive treatment prior to transplantation on skin chimerism is yet another issue demanding further clarification.

In only three cases (patients 8, 19, and 27), all skin swabs could be assigned to category C and the calculation of recipient to donor allele ratio could subsequently be carried out (see Table 3). Patient 27 showed allele mixtures with identically dominant components in all samples that could be assigned to the donor. The proportion of recipient alleles in these samples was only 12–28%. With 18–57% of recipient alleles in patient 8, typing showed either balanced allele mixtures of recipient and donor alleles or mixtures with a main component of donor alleles. Of the three subjects, person 19 showed the largest variation of recipient alleles from 26–81% and thus of donor alleles from 19–74%. In swabs of the right and left palms, 81% of recipient alleles could still be detected, which was reflected in derivable dominant alleles in the respective mixture. Typing the swabs of the neck and the back of the hand resulted in balanced allele mixtures with a percentage of 59% and 64% of recipient alleles, respectively. Finally, a dominant proportion of donor alleles were found in the swab of the upper arm with a minor component of 26% of recipient alleles (see Fig. 1). Thus, the distribution between recipient cells and cells with donor DNA not only differs between individual patients, but also within one and the same person. Depending on the body region, large variations between the proportions of recipient and donor alleles could occur. This could also be related, as already mentioned above, to the fact that different compositions of delivered skin cells and cells with donor alleles are found in swabs of the different collection sites.

However, the samples collected could also vary regarding proportions of donor and recipient LC. This may be due to a variable kinetics with which recipient cells are replaced by donor cells. This means that the exchange does not take place evenly but at a different pace or at different times in different skin parts. The type of transplantation may also play a role here. According to Merad et al. [36], transplantations without donor T cells might initially not lead to an exchange of immune cells. The recipient’s LC would accordingly remain in the skin until an exchange of cells is initiated by inflammatory reactions. Because an inflammatory stimulus mostly occurs locally, differences in the distribution of immune cells of the recipient and donor would result in the different skin areas. A local or generalized GvHD or different conditioning regimes may well play a role here, as well.