Abstract
Background: Hematopoietic chimerism analysis is important in the follow-up of patients undergoing allogeneic stem cell transplantation. PCR of short tandem repeats is mainly used for monitoring chimerism after transplantation. Validation studies and precision of assay’s performance with respect to different mixed chimerism stages is not fully addressed. The aim of the present study was to assess the impact of several microsatellite analytical parameters in the quantification of hematopoietic chimerism after allogeneic hematopoietic stem cell transplantation and to analyze the overall analytical process through the application of internal quality control procedures.
Methods: Artificial DNA mixtures prepared in known proportions and patients samples were analyzed using three microsatellites, together with amplification of amelogenin gene and fluorescence in situ hybridization (FISH) for X and Y chromosomes. Limit of detection, analytical and clinical sensitivity, stochastic threshold and precision profiling was established. Levey-Jennings charts and Westgard rules were applied for quality control evaluation.
Results: Analytical and clinical sensitivity of the microsatellite markers was between 0.5% and 1.6%. Amelogenin detection and FISH for X and Y chromosomes showed a similar sensitivity. Severe allelic imbalance resulted in up to 50% difference between the calculated and corrected mixed chimerism. Systematic errors were identified using Levey-Jennings charts and Westgard rules.
Conclusions: Analysis of hematopoietic chimerism performance is a critical step to better understand potential intrinsic errors that may impact the final hematopoietic chimerism results. Implementing quality control tools, such as Levey-Jennings charts together with Westgard rules can identify systematic and random errors so corrective actions can be performed.
We are indebted to Prof. Dr. R. Mertelsmann for continuous support and Dr. Milena Pantic for assistance with FISH evaluation. We thank also Ingrid Huber, Sabine Enger and Ewa Samek for excellent technical assistance and the transplantation team at Freiburg University Hospital.
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
References
1. Bader P, Niethammer D, Willasch A, Kreyenberg H, Klingebiel T. How and when should we monitor chimerism after allogeneic stem cell transplantation? Bone Marrow Transplant 2005;35: 107–19.10.1038/sj.bmt.1704715Search in Google Scholar PubMed
2. Antin JH, Childs R, Filipovich AH, Giralt S, Mackinnon S, Spitzer T, et al. Establishment of complete and mixed donor chimerism after allogeneic lymphohematopoietic transplantation: recommendations from a workshop at the 2001 Tandem Meetings of the International Bone Marrow Transplant Registry and the American Society of Blood and Marrow Transplantation. Biol Blood Marrow Transplant 2001;7:473–85.10.1053/bbmt.2001.v7.pm11669214Search in Google Scholar
3. Khan F, Agarwal A, Agarwal S. Significance of chimerism in hematopoietic cell transplantation: new variations on an old theme. Bone Marrow Transplant 2004;34:1–12.10.1038/sj.bmt.1704525Search in Google Scholar PubMed
4. Choi SJ, Lee KH, Lee JH, Kim S, Chung HJ, Lee JS, et al. Prognostic value of hematopoietic chimerism in patients with acute leukemia after allogeneic bone marrow transplantation: a prospective study. Bone Marrow Transplant 2000;26: 327–32.10.1038/sj.bmt.1702504Search in Google Scholar PubMed
5. Butler JM, Buel E, Crivellente F, Mc Cord BR. Forensic DNA typing by capillary electrophoresis using the ABI 310 and 3100 genetic analyzers for STR analysis. Electrophoresis 2004;25:1397–412.10.1002/elps.200305822Search in Google Scholar
6. Lion T. Summary: reports on quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection. Leukemia 2003;17:252–4.10.1038/sj.leu.2402753Search in Google Scholar PubMed
7. Lion T, Watzinger F, Preuner S, Kreyenberg H, Tilanus M, de Weger R, et al. The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation. Leukemia 2012 April 10 [Epub ahead of print].http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=000307650000010&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=b7bc2757938ac7a7a821505f8243d9f310.1038/leu.2012.66Search in Google Scholar
8. Mortera J, Dawid AP, Lauritzen SL. Probabilistic expert systems for DNA mixture profiling. Theor Popul Biol 2003;63:191–205.10.1016/S0040-5809(03)00006-6Search in Google Scholar PubMed
9. Spyridonidis A, Zeiser R, Wäsch R, Bertz H, Finke J. Capillary electrophoresis for chimerism monitoring by PCR amplification of microsatellite markers after allogeneic hematopoietic cell transplantation. Clin Transplant 2005;19:350–6.10.1111/j.1399-0012.2005.00347.xSearch in Google Scholar PubMed
10. Elmaagacli AH, Becks HW, Beelen DW, Stockova J, Bützler R, Opalka B, et al. Detection of minimal residual disease and persistence of host-type hematopoiesis: a study in 28 patients after sex-mismatched, non-T cell depleted allogeneic bone marrow transplantation for Philadelphia-chromosome positive chronic myelogenous leukemia. Bone Marrow Transplant 1995;16:823–9.Search in Google Scholar PubMed
11. Kristt D, Klein T. Reliability of quantitative chimerism results: assessment of sample performance using novel parameters. Leukemia 2006;20:1169–72.10.1038/sj.leu.2404191Search in Google Scholar PubMed
12. Westgard JO, Barry PL, Hunt MR, Groth T. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem 1981;27:493–501.10.1093/clinchem/27.3.493Search in Google Scholar PubMed
13. Appelbaum FR, Forman SJ, Negrin RS, Blume KG, editors. Thomas hematopoietic cell transplantation, 4th ed. Oxford: Blackwell Publishing, 2009:1043.10.1002/9781444303537Search in Google Scholar
14. Liang SL, Lin MT, Hafez MJ, Gocke CD, Murphy KM, Sokoll LJ, et al. Application of traditional clinical pathology quality control techniques to molecular pathology. J Mol Diagn 2008;10:142–6.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=000253874500005&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=b7bc2757938ac7a7a821505f8243d9f310.2353/jmoldx.2008.070123Search in Google Scholar PubMed PubMed Central
15. Baron F, Baker JE, Storb R, Gooley TA, Sandmaier BM, Maris MB, et al. Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2004;104: 2254–62.10.1182/blood-2004-04-1506Search in Google Scholar PubMed
16. Seong CM, Giralt S, Kantarjian H, Xu J, Swantkowski J, Hayes K, et al. Early detection of relapse by hypermetaphase fluorescence in situ hybridization after allogeneic bone marrow transplantation for chronic myeloid leukemia. J Clin Oncol 2000;18: 1831–6.10.1200/JCO.2000.18.9.1831Search in Google Scholar PubMed
17. Walsh PS, Erlich HA, Higuchi R. Preferential amplification of alleles: mechanisms and solutions. PCR Methods Appl 1992;1:241–50.10.1101/gr.1.4.241Search in Google Scholar PubMed
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