Detection of Clonally Restricted Immunoglobulin Heavy Chain Gene Rearrangements in Normal and Lesional Skin Minakshi Nihal, Debra Mikkola, Gary S. Wood

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Detection of Clonally Restricted Immunoglobulin Heavy Chain Gene Rearrangements in Normal and Lesional Skin Minakshi Nihal, Debra Mikkola, Gary S. Wood The Journal of Molecular Diagnostics Volume 2, Issue 1, Pages 5-10 (February 2000) DOI: 10.1016/S1525-1578(10)60609-5 Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Diagramatic representation of various regions of the immunoglobulin heavy chain (IgH) gene. The various framework regions (FR) within the variable region of the IgH gene are shown along with the positions of the oligonucleotide primers used in the different PCR assays. The Journal of Molecular Diagnostics 2000 2, 5-10DOI: (10.1016/S1525-1578(10)60609-5) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Agarose gels showing positive and negative results for B-cell clonality in a variety of samples using three different IgH PCR assays whose upstream primers are targeted to consensus sequences in the IgH gene variable region known as FR1, FR2, and FR3. Lanes containing a dominant clonal IgH gene rearrangement exhibit a discreet band (lanes marked +), whereas lanes containing only polyclonal IgH gene rearrangements show only a diffuse smear (lanes marked −). B, blank; P, positive control B cell lines; C, carry-over negative control; J, Jurkat T cell line negative control; T, tonsil polyclonal control; S, normal skin control; CBCL, cutaneous B-cell lymphoma; NBCL, nodal B-cell lymphoma; M, marker. The Journal of Molecular Diagnostics 2000 2, 5-10DOI: (10.1016/S1525-1578(10)60609-5) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 The impact of the number of PCR cycles on the detection of dominant clonality. The B cell lymphoma cell line LTR228 was diluted in reactive tonsil. The FR3 assay was used. Each lane contains 6 μl of radiolabeled PCR product. The proportion of LTR228 DNA is listed as a percentage above each lane. The three panels represent 30, 40, and 50 PCR cycles. The Journal of Molecular Diagnostics 2000 2, 5-10DOI: (10.1016/S1525-1578(10)60609-5) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Detection of restricted IgH gene rearrangements in normal skin. Triplicate analyses of three normal skin samples (A-C) are shown using the FR1 assay. The lanes marked “mix” contain pooled PCR products from the three preceding individual PCR reactions. Each lane contains 6 μl of radiolabeled PCR product. Daudi is a positive control cell line (one band). Tonsil and blood are polyclonal B cell controls that show a complex smeared ladder of many bands. CO is the carry-over lane that contains all reagents except DNA. The pseudo-monoclonal band patterns seen in some lanes (one or two bands) are converted to a polyclonal complex band pattern in the lanes containing the triplicate mixes. The Journal of Molecular Diagnostics 2000 2, 5-10DOI: (10.1016/S1525-1578(10)60609-5) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Titration of normal blood in the squamous cell carcinoma cell line SCC 13. The pattern of IgH gene rearrangements using the FR1 assay is shown at various concentrations of blood. Each lane has 6 μl of radiolabeled PCR product. At lower percentages of blood corresponding to sparse B cells, a pseudo-monoclonal band pattern emerges. The Journal of Molecular Diagnostics 2000 2, 5-10DOI: (10.1016/S1525-1578(10)60609-5) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Combined immunopathological/molecular biological algorithm for the analysis of IgH gene rearrangements in extranodal tissues such as the skin. This approach should help avoid false positive results secondary to sparse B cells. The Journal of Molecular Diagnostics 2000 2, 5-10DOI: (10.1016/S1525-1578(10)60609-5) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions