title: Monitoring of hematopoietic chimerism by real-time quantitative PCR of micro insertions/deletions in samples with low DNA quantities.
abstract: BACKGROUND
  Sensitive and accurate methods to detect hematopoietic chimerism after hematopoietic stem cell transplantation (HSCT) are essential to evaluate engraftment and to monitor response to therapeutic procedures such as donor lymphocyte infusion. Continuous long-term follow up, however, requires large amounts of pre-HSCT samples limiting the application of many widely used techniques for sensitive chimerism monitoring.
  
  
  METHODS
  DNAs from 42 normal healthy donors and 16 HSCT donor/recipient pairs were employed to validate the use of allele-specific insertion/deletion (indel) quantitative real-time polymerase chain reaction (qPCR) to quantify chimerism in samples with low amounts of DNA. Consequently, indel-qPCR analyses of samples from 16 HSCT patients were compared to short-tandem repeat (STR) specific PCR analyses.
  
  
  RESULTS
  Typing with reduced amounts of input DNA (15 vs. 60 ng) allowed for the reliable distinction of positive (mean threshold cycle (ct) 28.05) and negative (ct >36) signals. The high informativity of primer/probe sets, with 12 out of 19 markers exceeding 20% informativity, was confirmed in our cohort (n = 74). Importantly, a fourfold reduction of input DNA compared to published protocols did not alter PCR efficiencies and allowed for a more sensitive detection of chimerism in 7 of 16 HSCT patients compared to results obtained by STR-PCR.
  
  
  CONCLUSIONS
  Our data suggest that indel-qPCR is a more sensitive technique for the detection of hematopoietic chimerism compared to STR-PCR and works efficiently for samples with low amounts of DNA.
authors.affiliation: Department of Internal Medicine 5, Hematology/Oncology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Bach
authors.affiliation: Department of Internal Medicine 5, Hematology/Oncology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Elmira
authors.initials: E
authors.lastname: Tomova
authors.affiliation: Department of Internal Medicine 5, Hematology/Oncology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Goldmann
authors.affiliation: Department of Transfusion Medicine and Hemostaseology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Weisbach
authors.affiliation: Department of Internal Medicine 5, Hematology/Oncology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Wolf
authors.initials: W
authors.lastname: Roesler
authors.affiliation: Department of Internal Medicine 5, Hematology/Oncology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Mackensen
authors.affiliation: Department of Internal Medicine 5, Hematology/Oncology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Winkler
authors.affiliation: Department of Internal Medicine 5, Hematology/Oncology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Bernd M
authors.initials: BM
authors.lastname: Spriewald
keywords: Chimerism; Molecular diagnostic techniques; Quantitative real-time polymerase chain reaction; Stem cell transplantation