Use of highly sensitive mitochondrial probes to detect microchimerism in xenotrans- plantation models Maddalena Soncini, 1,2Ã Patrizia Bonassi Signoroni, 1,Ã Marco Bailo, 1,2 Daniela Zatti, 1 Alessandra Gregori, 3 Guerino Lombardi, 3 Alberto Albertini, 4 Georg S. Wengler 1 and Ornella Parolini 1 1 Centro di Ricerca ‘‘E. Menni’’, Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy, 2 Universita ` degli Studi di Milano, Dipartimento di Scienze e Tecnologie Biomediche, Milano, Italy, 3 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy, 4 Istituto di Tecnologie Biomediche, CNR, Milano, Italy Soncini M, Bonassi Signoroni P, Bailo M, Zatti D, Gregori A, Lombardi G, Albertini A, Wengler GS, Parolini O. Use of highly sensitive mitochondrial probes to detect microchimerism in xenotransplantation models. Xenotransplantation 2006; 13: 80–85. r Blackwell Munksgaard, 2006 Abstract: Chimerism, defined as the co-existence of cells of different origin within the same organism, has received much attention in hematopoietic cell and organ transplantation because of the strict relationship between its establishment and the induction of specific tolerance. Traditional methods applied for chimerism detection, such as immunohistochemistry, cytogenetics, fluorescent-activated cell sorter analysis, and serological and biochemical testing, are limited by their sensitivity.We have established a highly sensitive molecular approach based on the amplification of the mitochondrial cytochrome B gene and tested its specificity and sensitivity level in six different mammalian species, including human, pig, mouse, rat, sheep and rabbit. Increased sensitivity of detection of specific amplification products was obtained by the non-radioactive Southern blot technique. This novel approach allows the detection of one cell against the background of 1 to 4 Â 10 6 xenogenec cells and will be helpful for high-sensitivity analysis of donor cell engraftment after xenotransplantation procedures in these animal models.                                                                                                             Introduction After solid organ transplantation, circulating donor cells can be found in different tissues of the host [1]. The co-existence of cells of different genetic origins in the same organism is defined as chimerism, or microchimerism in the case of less than 1% donor cell engraftment [2]. The presence of chimerism after transplantation is a reflection of the host’s immunological tolerance towards donor cells. Whether this might be a pre- requisite for long-term acceptance of the graft is an issue of ongoing debate [3,4]. Chimerism has been assessed in xenogeneic and allogeneic transplantation procedures, both in hu- man clinical settings [5] and in animal models [6,7], to demonstrate engraftment of donor cells. Different approaches to study chimerism after transplantation have been described that are based on detection of intact donor cells by immunohysto- chemistry [8] or flow cytometry [9]. The presence of functioning donor cells has been substantiated by detection of specific molecules produced by such cells using serology and biochemistry tests [10]. Fi- nally, molecular evidence of donor cell chimerism has been obtained by detection of chromosome- specific regions with cytogenetic [11] or fluorescent in situ hybridization (FISH) analyses [12]. Recently, polymerase chain reaction (PCR) amplification of Xenotransplantation 2006: 13: 80–85 Printed in Singapore. All rights reserved doi: 10.1111/j.1399-3089.2005.00256.x Copyright r Blackwell Munksgaard 2006 XENOTRANSPLANTATION Ã Contributed equally. Key words: animal model – cytochrome B gene – human – microchimerism – mitochondrial probe – mouse – pig – rabbit – rat – sheep – xenotransplantation Address reprint requests to Ornella Parolini, Centro di Ricerca ‘‘E. Menni’’, Fondazione Poliambulanza Istituto Ospedaliero, Via Bissolati, 57 I-25124 Brescia, Italy (E-mail: ornella.parolini@tin.it) Received 20 March 2005; Accepted 12 October 2005 80