PCR Analysis of the Immunoglobulin Heavy Chain Gene in Polyclonal Processes Can Yield Pseudoclonal Bands as an Artifact of Low B Cell Number Kojo S.J.

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PCR Analysis of the Immunoglobulin Heavy Chain Gene in Polyclonal Processes Can Yield Pseudoclonal Bands as an Artifact of Low B Cell Number Kojo S.J. Elenitoba-Johnson, Sandra D. Bohling, Rebecca S. Mitchell, Michael S. Brown, Ryan S. Robetorye The Journal of Molecular Diagnostics Volume 2, Issue 2, Pages 92-96 (May 2000) DOI: 10.1016/S1525-1578(10)60622-8 Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Immunoglobulin heavy chain (IgH) PCR in a monoclonal B cell process (malignant lymphoma). Ten microliters of PCR product were loaded in every lane. Serial dilutions were performed using 50 ng of template DNA as the starting quantity per reaction. Lane 1: polyclonal DNA control showing a smear pattern. Lane 2: undiluted sample at 50 ng/reaction showing a monoclonal band (∼100 bp). Lane 3: 1:10 dilution (5 ng/reaction). Lane 4: 1:100 dilution (0.5 ng). Lane 5: 1:500 dilution (0.25 ng). Lane 6: 1: 1000 dilution (0.05 ng). Lane 7: 1:1500 (0.033 ng). The monoclonal band (∼100 bp) is seen to persist at a dilution of 1:1000. The Journal of Molecular Diagnostics 2000 2, 92-96DOI: (10.1016/S1525-1578(10)60622-8) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 IgH PCR in serial dilutions of DNA from a reactive lymph node. A progressive tendency toward the formation of pseudomonoclonal bands with incremental dilutions is evident. Lane 1: DNA molecular weight marker. Lane 2: negative (H20) control. Lane 3: a polyclonal smear pattern is seen in the undiluted DNA sample (50 ng). Lane 4: 1:10 dilution (5 ng), Lane 5: 1:100 dilution (0.5 ng). Lane 6: 1:200 dilution (0.25 ng). Lane 7: 1:300 dilution (0.17 ng). Lane 8: 1:400 (0.125 ng). Lane 9: 1:500 dilution (0.1 ng). Lane 10: 1:600 dilution (0.083 ng). Lane 11: 1:700 dilution (0.071 ng). Lane 12: 1:800 (0.0625 ng). Lane 13: 1:900 (0.056 ng). Lane 14: 1:1000 (0.05 ng). The Journal of Molecular Diagnostics 2000 2, 92-96DOI: (10.1016/S1525-1578(10)60622-8) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 IgH PCR on microdissected reactive germinal centers. Approximately 50 cells were used per reaction. Lane 1: molecular weight marker. Lane 2: negative (H20) control. Lane 3: positive (monoclonal) control.Lanes 5 and 10 show an oligoclonal pattern.Lanes 4, 6–9, and 11 show a pseudomonoclonal pattern (one or two bands). Lane 12 shows a polyclonal (smear) pattern. Note that lanes 7 and 8 show products of identical size even though the DNA used was extracted from separate germinal centers that were distant from one another. The Journal of Molecular Diagnostics 2000 2, 92-96DOI: (10.1016/S1525-1578(10)60622-8) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 IgH PCR performed on serially increasing numbers of whole sections of paraffin-embedded tissue from a case of reactive gastritis. There is a progressive tendency toward polyclonality as more serial sections putatively contributing more B cells are used for the DNA extraction.Lane 1: molecular weight marker. Lane 2: positive monoclonal (control).Lane 3: negative (H2O) control. Lane 4: pseudomonoclonal pattern (one discrete band) is obtained when using from DNA extracted from three 5-μm sections from the gastric biopsy specimen. Lane 5: a pseudomonoclonal pattern (two bands) is obtained with DNA from four 5-μm sections. Lanes 6 and 7: oligoclonal patterns obtained using five and six 5-μm sections respectively. Lane 8 shows a polyclonal smear pattern obtained using seven 5-μm tissue sections for DNA extraction. The Journal of Molecular Diagnostics 2000 2, 92-96DOI: (10.1016/S1525-1578(10)60622-8) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions