title: Combination of Complement-Dependent Cytotoxicity and Relative Fluorescent Quantification of HLA Length Polymorphisms Facilitates the Detection of a Loss of Heterozygosity.
abstract: Loss of heterozygosity (LOH) is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of LOH requires a high purity of recipient target cells. FACS is time consuming and also frequently prevented by rather nonspecific or unknown immune phenotype. The approach for recipient cell enrichment is based on HLA targeted complement-dependent cytotoxicity (CDC). Relative fluorescent quantification (RFQ) analysis of HLA intron length polymorphisms then allows analysis of HLA heterozygosity. The approach is exemplified in recent clinical cases illustrating the detection of an acquired allele loss. As illustrated in one case with DPB1, distinct HLA loci in donor and patient were sufficient for both proof of donor cell removal and evaluation of allele loss in the patient's leukemic cells. Results were confirmed using HLA-B RFQ analysis and leukemia-associated aberrant immunophenotype (LAIP) based cell sort. Both results confirmed suspected loss of HLA heterozygosity. Our approach complements or substitutes for FACS-based cell enrichment; hence it may be further developed as novel routine diagnostic tool. This allows rapid recipient cell purification and testing for loss of HLA heterozygosity before and after allogeneic HSCT in easily accessible peripheral blood samples.
authors.affiliation: Laboratory of Immunogenetics, Department of Transfusion Medicine, Cell Therapeutic Agents and Hemostaseology, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Klaus
authors.identifier: 0000-0002-7038-4033
authors.initials: K
authors.lastname: Witter
authors.affiliation: Department of Internal Medicine III, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Reibke
authors.affiliation: Department of Internal Medicine III, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Marion
authors.initials: M
authors.lastname: Subklewe
authors.affiliation: Laboratory of Immunogenetics, Department of Transfusion Medicine, Cell Therapeutic Agents and Hemostaseology, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Zahn
authors.affiliation: Laboratory of Immunogenetics, Department of Transfusion Medicine, Cell Therapeutic Agents and Hemostaseology, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Teresa
authors.initials: T
authors.lastname: Kauke
authors.affiliation: Department of Internal Medicine III, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Spiekermann
authors.affiliation: Laboratory of Immunogenetics, Department of Transfusion Medicine, Cell Therapeutic Agents and Hemostaseology, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Spannagl
authors.affiliation: Department of Internal Medicine III, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Tischer
authors.affiliation: Department of Internal Medicine III, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hiddemann
authors.affiliation: Laboratory of Immunogenetics, Department of Transfusion Medicine, Cell Therapeutic Agents and Hemostaseology, University Hospital of Munich, Ludwig Maximilians University, Max-Lebsche-Platz 32, 81377 Munich, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Dick