With advancements in genetic diagnostics and genotype-based therapeutics, the demand for germline genetic testing in post-hematopoietic stem cell transplantation patients is increasing. Due to genetic chimerism, blood samples can no longer be used for germline testing after transplantation. This study aims to identify the most suitable tissue for germline analysis following stem cell transplantation by investigating alternative tissue sources. Buccal swab, eyebrow hair, and nail samples were analyzed for donor-derived DNA using next-generation sequencing and short tandem repeat analysis, with linear regression used for evaluation. Factors such as HLA match, transplantation type, sex, and time after transplantation were also evaluated for their effect on donor-derived DNA share. Buccal swab and nail samples exhibited 25% and 22% higher proportions of donor-derived DNA compared to eyebrow hair follicles, respectively. The median donor DNA share in eyebrow hair follicles was 1% for NGS and 3% for STR. Factors such as matched related donors, higher HLA match, different donor-recipient sex, and longer time post-transplantation correlated with lower donor DNA shares. Eyebrow hair follicles are a promising tissue for accurate germline genetic testing in post-SCT patients. Patient characteristics like donor relatedness, HLA match, sex match, and time after transplantation should be considered.

Germline genetic testing plays a critical role in diagnosing inherited predispositions and increasingly guides therapeutic and surveillance choices—but becomes technically challenging after allogeneic hematopoietic stem cell transplantation (HSCT), when donor-derived DNA contaminates host tissues. To address this, we compared donor-derived DNA across three accessible tissues—buccal swab, nail, and eyebrow follicles—in recipients after hematopoietic stem cell transplantation using two orthogonal assays (34-SNP next-generation sequencing and a 27-marker short tandem repeat panel) and modeled clinical covariates that influence chimerism. Eyebrow follicles showed consistently low donor DNA (median 1% by NGS; 3% by STR) whereas buccal swabs and nails carried substantially higher donor fractions (+25 and +22 percentage points versus eyebrow, respectively; both p < 0.01). Across methods, STR yielded on average ≈6 percentage points higher donor fractions than NGS at low-level chimerism. Several transplant covariates correlated with chimerism: matched-related donors and a perfect HLA match (10/10) were each associated with lower donor DNA (≈12–14 and 15–20 percentage points, respectively); longer times since hematopoietic stem cell transplantation correlated with lower levels for nail samples, and donor–recipient sex match correlated with higher donor DNA (~7–8 percentage points). Even low-level chimerism can distort germline variant interpretation. We propose a pragmatic protocol for post-hematopoietic stem cell transplantation germline testing that prioritizes eyebrow follicles as the default tissue. An SNP-based quality control assay is used to flag unsafe donor fractions (≥ 5–10%) before comprehensive germline analysis, reducing the risk that chimeric donor DNA distorts germline variant interpretation.