International Journal of Legal Medicine

, Volume 127, Issue 1, pp 49–54 | Cite as

Chimerism in DNA of buccal swabs from recipients after allogeneic hematopoietic stem cell transplantations: implications for forensic DNA testing

  • Burkhard Berger
  • Roswitha Parson
  • Johannes Clausen
  • Cordula Berger
  • David Nachbaur
  • Walther Parson
Short Communication

Abstract

We attempted to quantitatively determine the chimeric state in a total of 162 buccal swabs from 77 adult recipients aged 19–74 (median 50 years) after allogeneic hematopoietic cell transplantation by estimating the chimeric recipient/donor DNA ratios through analysis of 15 autosomal short tandem repeat markers. From each individual between one and nine, buccal swabs were taken at known time intervals after transplantation, ranging from 17 to 3,361 days (median 394 days). In buccal cells, the determined recipient/donor DNA ratios turned out to be highly variable between individuals and also within an individual. Relative donor chimerism levels (%Ch) between 0 and 100 % were detected with maximal frequencies between 10 and 30 %. Blood was always found to show the donor's genotype while hair samples in all cases gave the recipient's genotype. We examine chimerism levels with respect to age, gender, and posttransplantation period and discuss the results in the context of forensic identity testing.

Keywords

Chimerism STRs Allogeneic hematopoietic cell transplantations Forensic 

Introduction

The analysis of polymorphic short tandem repeat markers (STRs) has become the method of choice for human identity testing. Individuals can be distinguished by an appropriate number of STR markers (exception: identical twins), and DNA mixtures from two or more contributors can be discriminated. The STR profile of one person is independent of the DNA source (e.g., blood, saliva, and hair), sampling date, and the age of the involved persons. Besides other rare cases such as microsatellite instability and loss of heterozygosity, chimerism is known to lead to violation of these advantageous properties of STR analysis. Nowadays, chimerism is most commonly acquired through therapeutic applications such as blood stem cell transplantation. Allogeneic hematopoietic cell transplantation (allo-HCT) has become a successful and regularly applied medical treatment for a number of hematologic malignancies. After a chemotherapy and/or radiotherapy to suppress the blood formation and the immune system, the administration of stem cells derived from a human leukocyte antigen compatible donor mediates the reconstitution of hematopoiesis in the recipient. While circulating blood cells and their derivatives develop the donor genotype after transplantation other cells and tissues should theoretically remain those of the recipient.

However, there is increasing evidence that hematopoietic stem cells can differentiate into various cell types other than blood [1, 2, 3, 4, 5, 6, 7, 8] including buccal epithelia [9, 10, 11]. From a forensic point of view, the simple fact that chimeric epithelial tissues do exist is of special interest. The collection of buccal cells has been established as a forensic standard to obtain reference genotypes for DNA analysis. Thus, it can easily be conceived that an unexpected observation of the donor genotype in the samples of the recipient can cause misinterpretation of DNA evidence. This has been shown in individual cases. So far, a systematic investigation and discussion in the forensic context is still missing.

Material and methods

Sample collection

The study was carried out in cooperation with the Department of Haematology and Oncology of the University Hospital Innsbruck and was reviewed and approved by the ethics commission of the Innsbruck Medical University (UN3121, session ID 262/4.10 of February 28, 2008). Adult recipients of hematopoietic stem cell transplantations participated under written consent. Blood samples were taken from recipients and donors prior the medical intervention. Buccal swabs, blood samples, and if feasible, hair roots were sampled from 2008 to 2010 in the course of the routine follow-up examinations at different posttransplantation intervals (PTI). For collecting buccal cells, Omni Swabs (Whatman, Springfield Mill, UK) were used. In the majority of cases, buccal swabs were taken randomly from the patients' left or right interior cheek side. In some cases, two or three samples were collected at the same time from different locations and denoted as cheek side 1 and cheek side 2 (corresponding to left and right), lower lip, and tongue.

DNA analysis and chimerism calculations

Total human genomic DNA was isolated from blood using the NucleoSpin Dx Blood kit (Macherey-Nagel, Düren; Germany) according to the manufacturer's protocol. DNA of buccal swabs was extracted by applying the Chelex method [12]. DNA from hair roots was extracted using a BioRobot M48 workstation (Qiagen, Hilden, Germany) following the manufacturer's protocol and the Qiasoft M Operating System software (ver.2.0E001). The quantity of nuclear DNA in hair roots was determined as described in [13, 14] using a 7500 Fast real-time PCR system (Applied Biosystems, AB, Foster City, CA, USA) with standard temperature ramp speed settings.

STRs were amplified using the AmpFlSTR Identifiler PCR amplification kit (AB) following the manufacturer's protocol on a GeneAmp PCR System 9700 (AB). The amplicons were analyzed on an ABI PRISM 3100 Genetic Analyzer using a 36 cm array, POP-6 (both AB) and injection conditions of 2 kV and 7 s. Data analysis was performed using a GeneScan software v3.7 (AB) and GeneMarker HID V1.7 (SoftGenetics, Inc. State College, PA, USA).

The relative donor chimerism levels (%Ch) was calculated from informative STR markers according to [15]. An informative marker must exhibit at least one different allele between the donor and recipient. The %Ch for each informative marker was calculated separately to obtain a mean ± STD.

Samples with more than 90 %Ch were classified as complete chimerism (100 %Ch); samples which gave less than 10 %Ch were classified as no chimerism (0 %Ch). We discern the following chimerism classes: 20, 40, 60, and 80, where 20 represents %Ch between 10 and 30 and so forth.

Results

Adult recipients (n = 77) of hematopoietic cell transplantations that suffered from different malignant diseases participated in this study. Sample numbers and clinical details are listed in Table 1. The age of the recipients at the date of the transplantation ranged from 19 to 74 years (median 50). The PTI ranged from 17 to 3,361 days (approximately 9 years). The median and mean PTI amounted to 403 and 751 days, respectively.
Table 1

Quantitative summary of sampling and clinical details of the patients

Category

n

%

Number of recipients

  

  Total

77

100

  Male

46

60

  Female

31

40

Number of samples

  

  Buccal swabs

162 (from 77 recipients)

 

  Blood

136 (from 76 recipients)

 

  Hair roots

88 (from 62 recipients)

 

Recipient/donor relationship

  

  Related

48

62

  Not related

29

38

Disease

  

  Acute myelogenous leukemia

29

38

  Chronic myelogenous leukemia

11

14

  Acute lymphoblastic leukemia

10

13

  Myelodysplastic syndrome

7

9

  Multiple myeloma

5

6

  Secondary acute leukemia

5

6

  Aplastic bone marrow

4

5

  Other

6

8

Conditioning

  

  Myeloablative

50

65

  Dose-reduced

27

35

Number of transplantations

  

  1x

67

87

  2x

7

9

  3x

3

4

Origin of stem cells

  

  Peripheral blood

70

91

  Bone marrow

7

9

An average of 11 informative STR markers was observed, when no direct familial relationship between donor and recipient existed. In the latter case, a mean of six informative markers provided the basis for chimerism calculations.

All 136 blood samples from 76 patients gave exclusively the donor genotype without any trace of minor recipient contributions. On the other hand, no indication of the donor genotype was found in all 88 hair samples, and only recipient STR profiles were detected.

The majority of the recipients (56 %) were sampled several times (Table 2) resulting in 162 buccal swabs. In most cases, the swabs were collected from uninjured epithelial tissue of the interior cheek (152 swabs), the tongue, or the lower lip. In five cases, injuries like plaques, lesions, or ulcers were observed in the sampling area. In 120 samples (74 %), a mixture of donor and recipient genotypes was found, 35 (22 %) showed no chimerism, and only in 7 samples (4 %) a complete donor chimerism was detected. The median value of 23 % indicates that the recipient STR profile was the major component in the mixtures in the majority of the cases (mean %Ch 30 ± 27). Only 33 samples (20 %) gave the donor genotype as major component (%Ch > 50 %).
Table 2

Buccal swab sampling regime: between one and 9 buccal swabs were taken from each of the 77 recipients (R)

Sampling per R

n (R)

1x

34

2x

20

3x

12

4x

7

5x

3

9x

1

Buccal swabs were taken from each of the 77 recipients (R). A total of 162 swabs were collected

The distribution of mean chimerism levels found in the buccal epithelia of the 77 recipients is shown in Fig. 1a. The maximal frequency was found in the class between 10 and 30 %. In Fig. 1b, the distribution of the male and female subsamples shows a slight tendency to higher chimerism levels in males. This was also reflected by the corresponding median values of 18.3 % for females and 31.6 % for males. Further allocations concerned the family relationship between recipients and donors and the conditioning regime (myeloablative vs. dose-reduced). Very similar distribution patterns were found in both cases with highest frequencies in the class between 10 and 30 % (Fig. 1c, d).
Fig. 1

Frequencies of chimerism classes in buccal swabs of all 77 recipients (a), within the female (black) and the male (gray) subsamples (b), in recipients with (black) and without (gray) a recipient/donor family relationship (c), and with a reduced-dose (black) or a myeloablative (gray) conditioning regime (d)

Fig. 2

Relative donor chimerism levels of 162 buccal swabs (mean of all informative STR markers ± STD) depending on the age of the recipients at the time of the transplantation

No correlation of chimerism levels found in buccal swabs was observed with the age of the patients at the time of transplantation (Fig. 2). Furthermore, there was no detectable influence of the PTI on the chimerism state (Fig. 3).
Fig. 3

Relative donor chimerism levels of 162 buccal swabs (mean of all informative STR markers ± STD) depending on the posttransplantation interval

Fig. 4

Individual examples showing contrasting developments of the relative donor chimerism levels over the sampling period expressed as days after transplantation

Individual recipients that were sampled more than once showed all possible %Ch developments. Examples outlined in Fig. 4 comprise recipient #840 that showed a nearly constant chimerism level of almost 30 %; whereas recipient #845 showed an increase from 0–29 %, while the %Ch of recipient #684 decreased from 72 to 20 %.

The %Ch of samples from different locations in the oral cavity taken at the same time from the same individual were found to differ significantly, and a consistent trend could not be determined (Table 3). Some recipients showed high differences in %Ch levels between the different sampling locations, such as recipients #521 and #809. Others gave similar values, e.g., #417 (cheek and tongue) or #771 with a complete chimerism occurring at both sides of the cheek.

Discussion

Findings of mosaicism and chimerism in forensic cases have been reported occasionally [16, 17, 18, 19], but very likely is the number of unrecorded cases be higher, as this special genetic constellation cannot be distinguished from regular mixed DNA profiles without additional information, e.g., recordings of medical interventions or supplemental DNA data from other tissues or from relatives. However, such information is often not available or is not requested, because the possibility of chimerism is not considered.

In this study, three source tissues typically sampled for forensic DNA analysis—buccal swabs, blood, and hair roots—were collected systematically from recipients of allo-HCT and were investigated by STR analysis. Special emphasis was put on the potential occurrence of chimerism in reference material derived from buccal swabs as this sample type represents the starting point for typical forensic investigations, including intelligence searches via national DNA databases that usually contain high numbers of DNA profiles from this source.

A typical observation in this study was that totally discrepant results were obtained from the tested sample types. We commonly observed two completely different single-source DNA profiles and one mixture: the blood samples showed exclusively the STR profiles of the donor, the hair roots exhibited only the genotype of the recipient, and in the buccal swabs, every possible ratio between donor- and recipient-derived DNA was found.

Generally, our results confirm earlier findings that in the buccal epithelium, donor-derived DNA can be found [6, 9, 20, 21], but our data support the fact that this is the rule rather than the exception for recipients of allo-HCT. All mixtures could be explained by the alleles found in the genotypes of the donors and recipients prior to transplantation. Therefore, spontaneous somatic mutations due to the malignant disease or the intensive chemotherapy or radiotherapy can be excluded as causes for the additional alleles. The biological cause of donor-derived DNA in buccal epithelia of the recipients has not been clarified [20], but it is unlikely that the migration of hematopoietic cells into the buccal epithelia is responsible for the observed chimerism levels as the buccal swabs almost exclusively consist of epithelial cells [20, 22].

We observed huge differences in chimerism levels of buccal swabs ranging from 0 and 100 % between individuals (Fig. 1), and more surprisingly, within individuals at different sampling dates (Fig. 4 and Table 3). A reliable attribution of the profiles to donor and recipient would be impossible without additional knowledge. The fact that the recipient's genotype was the major component in 133 out of 162 tested individual samples does not provide sufficient evidence but could at best serve as a supporting hint.
Table 3

Relative donor chimerism levels (%Ch) of simultaneously collected samples from different locations of the oral cavity

Patient no.

PTI (days)

Location

%Ch

STD

835

237

Cheek side

33.1

4

 

Tongue

100

314

Cheek side

49.3

4

 

Tongue

100

890

251

Cheek side 1

45

12.2

 

Cheek side 2

82.3

4.9

386

3,361

Cheek side 1

54.7

4.5

 

Cheek side 2

37

6.6

 

Lower lip

71.8

7.1

521

1,879

Cheek side 1

66.3

4.4

 

Cheek side 2

13.7

3.7

2,400

Cheek side 1

77.5

7

 

Cheek side 2

20

3.2

565

1,695

Cheek side

0

 

Tongue

17.8

6.6

2,144

Cheek side 1

18.5

2.1

 

Cheek side 2

17.3

4.1

657

1,172

Cheek side

45.9

6.2

 

Tongue

65.9

1.7

634

1,727

Cheek side 1

24.7

9.1

 

Cheek side 2

47.2

9.6

720

1,345

Cheek side 1

14.3

2.8

 

Cheek side 2

0

771

881

Cheek side 1

100

 

Cheek side 2

100

809

704

Cheek side 1

12

2.2

 

Cheek side 2

22.2

7

866

442

Cheek side 1

13.5

2.9

 

Cheek side 2

22.1

9.5

417

2,564

Cheek side

88.5

3.6

 

Tongue

88.7

2.3

489

2,683

Cheek side 1

0

 

Cheek side 2

19.9

4.3

Cheek side “1” refers to left and “2” to right, respectively

PTI posttransplantation interval

The observed differences in buccal swabs raise the question of possible factors influencing chimerism levels. We analyzed effects of some forensically relevant aspects, such as gender and age, and also included medical factors like the PTI, the conditioning regime or the donor/recipient relationship. None of those factors were found to be correlated with the chimerism level (Figs. 1, 2 and 3) and thus added no useful information. This trend was even extended by the high spatial diversity of chimerism levels of buccal swabs collected simultaneously from different locations of the oral cavity from a recipient (Table 3).

Finally, buccal swabs are not useful for separating donor and recipient fractions. However, as mentioned earlier, the identification of chimerism itself is often the critical point. For this purpose, buccal swabs can serve as useful indicators as they show mixed DNA profiles in most cases of our study and are usually not prone to contamination. Other specimens can be used to infer the recipients' profile and to add evidence to the existence of chimerism in the buccal swabs. These include blood samples that are useful for identifying the donor genotype and hair roots, which are reliable indicators of the recipient genotype. In that way, the problem of unexpected chimerism can be solved in most cases by analyzing a restricted set of samples collected from buccal tissue, blood, and hair roots.

Notes

Acknowledgments

We are grateful to all recipients participating voluntarily in this study and to the staff of the University Hospital of Internal Medicine V for their assistance in sample collection. We thank Nicole Krug for her contribution with routine data analysis, and Bettina Zimmermann and Lorenz Decristoforo for their useful discussions.

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Burkhard Berger
    • 1
  • Roswitha Parson
    • 2
  • Johannes Clausen
    • 2
  • Cordula Berger
    • 1
  • David Nachbaur
    • 2
  • Walther Parson
    • 1
  1. 1.Institute of Legal MedicineInnsbruck Medical UniversityInnsbruckAustria
  2. 2.Department of Hematology and OncologyUniversity Hospital of Innsbruck, Internal Medicine VInnsbruckAustria
  3. 3.3rd Medical DepartmentParacelsus Medical University SalzburgSalzburgAustria

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