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Limitations of clonality analysis of B cell proliferations using CDR3 polymerase chain reaction
M A Hoeve1, A D Krol, K Philippo
1Department of Pathology, Leiden University Medical Centre, The Netherlands.
Insights
Polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) rearrangements can detect lymphoma clonality. Optimal sensitivity requires sufficient cells and DNA, with frozen samples yielding better results for lymphoma diagnosis.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- Polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) rearrangements is a promising alternative to Southern blotting for lymphoma diagnostics.
- Understanding the limitations and sample size requirements of PCR in clonality analysis is crucial for accurate diagnosis.
Purpose of the Study:
- To determine the minimum cell and tissue requirements for PCR-based clonality analysis.
- To evaluate the sensitivity and specificity of high-resolution complementarity determining region 3 (CDR3) PCR for various B-cell lymphoproliferative disorders.
- To compare PCR performance on fresh cells versus formalin-fixed, paraffin-embedded tissues.
Main Methods:
- High-resolution CDR3 PCR was employed to assess clonality.
- Minimum cell numbers and DNA amounts for polyclonal detection were investigated using fresh and formalin-fixed, paraffin-embedded tissues.
- Sensitivity and specificity were evaluated on 76 B-cell lymphoproliferative disorder samples and various reactive tissues.
Main Results:
- Approximately 20 ng of high molecular weight DNA (6.5-9 x 10^3 B cells) or 1 mm^3 of tonsillar tissue is sufficient for polyclonal PCR detection.
- Pseudoclonality can be induced with insufficient sample material.
- PCR sensitivity varied by lymphoma type and tissue fixation, with lower sensitivity for follicular lymphoma (FL) and mucosa-associated lymphoid tissue (MALT) lymphomas compared to chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL).
- PCR specificity was high, reaching 96% for frozen and 100% for paraffin-embedded tissues.
Conclusions:
- Minimum template requirements for CDR3 PCR are established, but caution is needed to avoid pseudoclonality with low cell counts.
- Duplicate or triplicate testing is recommended to prevent misinterpretation.
- PCR offers high specificity, but sensitivity is influenced by lymphoma type and tissue fixation; frozen samples are preferred for optimal results, particularly for FL and MALT lymphomas.
Background/Aims:
Detection of clonal immunoglobulin heavy chain (IgH) rearrangements by the polymerase chain reaction (PCR) is an attractive alternative to Southern blotting in lymphoma diagnostics. However, the advantages and limitations of PCR in clonality analysis are still not fully appreciated. In this study, clonality was analysed by means of PCR, focusing in particular on the sample size requirements when studying extremely small samples of polyclonal and monoclonal lesions.
Materials/Methods:
High resolution complementarity determining region 3 (CDR3) PCR was used to investigate the minimum number of cells and the amount of tissue required for the detection of a polyclonal population, both for fresh cells and formalin fixed, paraffin wax embedded tissue. Subsequently, frozen and paraffin wax embedded samples of 76 B cell lymphoproliferative disorders, 43 of which were tested by means of Southern blotting, were analysed to establish the sensitivity of this assay. These specimens included 12 chronic lymphocytic leukaemias (CLLs), nine mantle cell lymphomas (MCLs), 10 follicular lymphomas (FLs), and 45 mucosa associated lymphoid tissue (MALT) lymphomas. The specificity was tested on reactive lymph nodes (n = 19), tonsils (n = 4), peripheral blood lymphocyte fractions (n = 4), and biopsies with gastritis (n = 21).
Results:
In reactive tissue, 20 ng of high molecular weight DNA derived from 6.5-9 x 10(3) B cells was sufficient to obtain a polyclonal PCR result. With smaller amounts "pseudoclonality" could be induced. When using paraffin wax blocks, undiluted DNA isolated from tonsillar tissue of at least 1 mm2 was necessary to obtain a polyclonal pattern. The sensitivity required to detect clonality in paraffin wax embedded and frozen tissue by PCR for FL (40% and 60%, respectively) was lower than that for MALT lymphomas (60% and 86%, respectively), CLL (78% and 89%, respectively), and MCL (88% and 100%, respectively). PCR specificity was 96% and 100% for frozen and paraffin wax embedded tissue, respectively.
Conclusion:
The minimum amount of template for CDR3 PCR is approximately 20 ng of high molecular weight DNA or 1 mm3 of B cell rich paraffin wax embedded normal tonsillar tissue, but care has to be taken to avoid pseudoclonality when low numbers of B cells are present. Duplicate or triplicate tests should be performed to avoid misinterpretation. The specificity of the PCR assay is almost 100%, whereas sensitivity depends on a combination of factors, such as lymphoma type and tissue fixation. Because frozen samples yield better results, obtaining fresh material for the PCR assay is recommended, especially when analysing FL and MALT lymphomas.
