title: Application of BIOMED-2 primers in fixed and decalcified bone marrow biopsies: analysis of immunoglobulin H receptor rearrangements in B-cell non-Hodgkin's lymphomas.
abstract: The detection of clonality in lymphomas was recently improved by the BIOMED-2 approach, but analysis of fixed tissues is limited. Here, we adapted the BIOMED-2 protocol for examining immunoglobulin H (IgH) receptor rearrangements in fixed, decalcified bone marrow biopsies (BMBs) for clonality analysis in B-cell non-Hodgkin's lymphomas (B-NHL). The study included 111 decalcified BMBs (12 formalin fixed and 99 glutardialdehyde fixed), with B-NHL (n = 85), T-NHL (n = 8), or reactive infiltrates (n = 18). Initially, IgH FRIII polymerase chain reaction (PCR) analysis of crude DNA extracts from 75 glutardialdehyde-fixed BMBs (B-NHLs) using a standard seminested PCR resulted in clonal peaks in 46 of 75 (61.3%) BMBs compared with 19 of 70 (27.1%) for the original BIOMED-2 protocol. Modifications to both DNA extraction and PCR reaction improved the detection rate to 26 of 36 (72.2%) for BIOMED-2 primers, including 10 of 15 (66.7%) cases not detected by our standard IgH analysis. Moreover, introducing the same modifications for analysis of the FRII region by BIOMED-2 primers revealed clonal peaks in 19 of 36 (52.8%) B-NHLs compared with 5 of 70 (7.1%) for the original BIOMED-2 protocol. Together, analysis of FRII and FRIII regions by the modified BIOMED-2 protocol increased the detection rate to 31 of 36 (86.1%), particularly for BMBs with histological evidence of follicular lymphoma (FRIII, 70%; FRII, 90%). In summary, this study provides an improved protocol for detection of clonality by IgH-specific BIOMED-2 primers in fixed, decalcified bone marrow biopsies.
authors.affiliation: Pathologisches Institut, Universitätsklinikum Freiburg, Albertstrasse 19, 79104 Freiburg. silke.lassmann@uniklinik.freiburg.de
authors.forename: Silke
authors.initials: S
authors.lastname: Lassmann
authors.forename: Uirike V
authors.initials: UV
authors.lastname: Gerlach
authors.forename: Katja
authors.initials: K
authors.lastname: Technau-Ihling
authors.forename: Martin
authors.initials: M
authors.lastname: Werner
authors.forename: Paul
authors.initials: P
authors.lastname: Fisch
mesh_terms: D001706:Biopsy; D001853:Bone Marrow / Q000473:pathology*; D002457:Cell Extracts; D002999:Clone Cells / Q000473:pathology; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics*; D003648:Decalcification Technique; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D050438:Genes, Immunoglobulin Heavy Chain / Q000235:genetics*; D020180:Heteroduplex Analysis; D006801:Humans; D016393:Lymphoma, B-Cell / Q000235:genetics* / Q000473:pathology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D016707:Tissue Fixation
chemical_list: D002457:Cell Extracts; D017931:DNA Primers; D004247:DNA