Abstract: Introduction: During routine analysis of chimerism in bone marrow transplant patients pre-transplant genotype of the recipient or the donor might lack. We aimed to develop a new method to analyze DNA results suitable when reference genotypes are not available. Methods: The method was based on the balance between heterozygotes. It was implemented in a standard computer spreadsheet, and considered the hypothetical donor?recipient genotype combinations. Hypotheses with peak height ratios and allele sharing tendency above a critical threshold were accepted. The results were compared with those obtained with prior knowledge of reference genotypes. Results: The algorithm predicted correctly the proportion of donor/recipient chimerism, even in the absence of reference genotypes. In fact, the predicted values were closely correlated (r2 > 0.98) and free of systematic bias (slope 0.98?1.04), in comparison with the reference values obtained with prior knowledge of the donor and recipient genetic profiles. Conclusions: This study constitutes a proof-of-concept of the application of the heterozygote balance for the quantitative study of chimerism. The algorithm computes post-transplant chimerism in an easy and time-efficient way, even when the donor and recipient reference genotypes are unavailable. Therefore, it can be a useful tool for laboratories involved in chimerism analysis.

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