Assay Design Affects the Interpretation of T-Cell Receptor Gamma Gene Rearrangements
2010; Elsevier BV; Volume: 12; Issue: 6 Linguagem: Inglês
10.2353/jmoldx.2010.090183
ISSN1943-7811
AutoresAllison M. Cushman‐Vokoun, Solomon Connealy, Timothy C. Greiner,
Tópico(s)T-cell and B-cell Immunology
ResumoInterpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9% vs. 96.4%; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5% vs. 56.3%; P = 0.049) when a 2× ratio was used to define clonality. Specificity was improved to 81.3% by the use of a 5× peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay. Interpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9% vs. 96.4%; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5% vs. 56.3%; P = 0.049) when a 2× ratio was used to define clonality. Specificity was improved to 81.3% by the use of a 5× peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay. Evaluation of clonal T-cell receptor γ gene rearrangements (TCRγGR) using various PCR-based methods is useful in the diagnosis of T-cell malignancies. The TRG@ gene is often used to assess T-cell clonality due to its simplistic structure as compared to the TRA@ and TRB@ genes. Within the TRG@ gene locus on chromosome 7p14, there are 11 variable region genes and five joining region genes involved in the formation of gene rearrangements in tumors. No diversity region genes are present. Various methods have been used in the detection of clonal PCR products including agarose and polyacrylamide gel electrophoresis,1Lamberson C Hutchison RE Shrimpton AE A PCR assay for detecting clonal rearrangement of the TCR-gamma gene.Mol Diagn. 2001; 6: 117-124PubMed Google Scholar,2Benhattar J Delacretaz F Martin P Chaubert P Costa J Improved polymerase chain reaction detection of clonal T-cell lymphoid neoplasms.Diagn Mol Pathol. 1995; 4: 108-112Crossref PubMed Scopus (89) Google Scholar denaturing gradient gel electrophoresis (DGGE),3Greiner TC Raffeld M Lutz C Dick F Jaffe ES Analysis of T cell receptor-gamma gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products. Correlation with tumor-specific sequences.Am J Pathol. 1995; 146: 46-55PubMed Google Scholar heteroduplex analysis4Bottaro M Berti E Biondi A Migone N Crosti L Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas.Blood. 1994; 83: 3271-3278PubMed Google Scholar and single strand conformational polymorphism analysis.5Guitart J Kaul K A new polymerase chain reaction-based method for the detection of T-cell clonality in patients with possible cutaneous T-cell lymphoma.Arch Dermatol. 1999; 135: 158-162Crossref PubMed Scopus (37) Google Scholar,6Lynas C Howe D Simple, reliable detection of T cell clones by PCR-LIS-SSCP analysis of TCRgamma rearrangement.Mol Cell Probes. 1998; 12: 41-48Crossref PubMed Scopus (16) Google Scholar The most common detection method involves fluorochrome-tagged primers and capillary electrophoresis,7Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar8Meier VS Rufle A Gudat F Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four-color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates.Am J Pathol. 2001; 159: 2031-2043Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar9Vega F Medeiros LJ Jones D Abruzzo LV Lai R Manning J Dunmire V Luthra R A novel four-color PCR assay to assess T-cell receptor gamma gene rearrangements in lymphoproliferative lesions.Am J Clin Pathol. 2001; 116: 17-24Crossref PubMed Scopus (94) Google Scholar10Luo V Lessin SR Wilson RB Rennert H Tozer C Benoit B Leonard DG Detection of clonal T-cell receptor gamma gene rearrangements using fluorescent-based PCR and automated high-resolution capillary electrophoresis.Mol Diagn. 2001; 6: 169-179PubMed Google Scholar which has good sensitivity and quick turn-around time. While the method of detection is important for accurate testing, proper PCR assay design is of utmost importance in reducing the number of false positive and false negative results. Multiplex versus monoplex reaction(s), product sizes, and the number of targets amplified (common versus rare rearrangements) are issues that must be thoroughly addressed before detection with capillary electrophoresis. The distribution of primers over the gene segments must be considered, for if not enough segments of the gene are amplified, false negative results may occur. For example, the JγP rearrangement has been unnecessarily excluded in some primer sets due to its rare involvement in T-cell neoplasms and the supposed risk of identifying false positive results.11van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2505) Google Scholar The risk however, is based more on assay design, as will be seen later in the results. JγP T-cell receptor gamma primer alignments are available at (http://www.unmc.edu/media/pathology/jp.pdf, last accessed on June 29, 2010). Secondly, the number of primers in a single tube reaction may also affect results. It has been suggested that specific combinations of primers may result in competitive inhibition of the formation of products.11van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2505) Google Scholar Finally, the number of size distributions of amplicons may also make interpretation difficult. Multiple protocols have been described for the detection of TCRγGR using capillary electrophoresis. The protocols vary from those that may contain one normal distribution of TCRγGR with a single fluorochrome to protocols with multiple tubes that have multiple polyclonal distributions (eg, BIOMED-2).11van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2505) Google Scholar If there are multiple ranges of product sizes, then there will be a reduced number of events to produce polyclonal Gaussian distributions for each individually labeled gene segment. As a result, it may be difficult to accurately evaluate small subclonal peaks in a reduced polyclonal background with the uncommonly used variable or joining gene segments. Most laboratories use labeled joining region primers in capillary electrophoresis. There are three possible approaches to evaluate TCRγGR using multiple fluorescent labeled joining region primer sets. The first approach consists of a complete multiplex assay using multiple variable and joining region primers within a single tube as previously described.7Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,12Lawnicki LC Rubocki RJ Chan WC Lytle DM Greiner TC The distribution of gene segments in T-cell receptor gamma gene rearrangements demonstrates the need for multiple primer sets.J Mol Diagn. 2003; 5: 82-87Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar In this tri-color approach, each primer in a set of joining region primers is labeled with a different fluorochrome tag (Figure 1A). The second complete multiplex approach is similar; however, instead of a different fluorescent label on each primer, the joining primers are labeled with the same fluorochrome tag (one-color, eg, NED) (Figure 1B).7Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar The third approach involves a partial multiplex reaction using a single fluorochrome-labeled primer for each joining gene segment in separate tubes (Figure 1C). The first goal of this report is to show, in the figures that follow, how assay design can heavily influence the likelihood of generating pseudoclonal peaks that can result in a false positive interpretation of a TCRγGR. The second goal of this work is to compare the performance of two assays with divergent designs; a one-tube assay with one size distribution versus the two-tube TCRG Gene Clonality Assay based on the BIOMED-2 design.11van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2505) Google Scholar Clinical cases were selected, that have not had nor developed a diagnosis of T-cell lymphoma, but which did contain small subclonal peaks, consisting of either a small JγP1/JγP2 (five cases) or JγP (five cases) gene segment, arising within a larger, higher amplitude, polyclonal or oligoclonal background of Jγ1/Jγ2 TCRγGR. DNA was derived from eight fresh tissue cases (peripheral blood, frozen tissue, or bone marrow) and two biopsies from formalin-fixed paraffin-embedded tissue. Three patients had reactive large granular lymphocytosis, three patients had an isolated cytopenia (neutropenia, anemia, or thrombocytopenia) of unknown cause, two patients had atypical lymphoid hyperplasia, one patient had a benign inflammatory process in the skin, and one patient had hairy cell leukemia. PCR reactions were performed as outlined in Figure 1. First, the cases were amplified in a single multiplex tri-color assay using five unlabeled variable region primers (Vγ2, Vγ3, Vγ9, Vγ10, Vγ11) and three separately labeled joining region primers (Jγ1/Jγ2-NED, JγP1/JγP2-JOE, JγP-FAM) as previously described by Lawnicki et al (Figure 1A).12Lawnicki LC Rubocki RJ Chan WC Lytle DM Greiner TC The distribution of gene segments in T-cell receptor gamma gene rearrangements demonstrates the need for multiple primer sets.J Mol Diagn. 2003; 5: 82-87Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar These reactions yield products of 190 ± 20 nucleotides in the usual normal distribution. In evaluating for clonal populations with capillary electrophoresis, we used our previously described formula: ratio of peak to background (RPB) must be greater than two times the highest polyclonal peak height, which defines that at least a 2% clonal population is present.7Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar The height of peaks outside the normal distribution must first be added to the height of the distribution before calculating the ratio.7Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Second, the DNA was then amplified with five upstream variable region primers and all three downstream joining primers (Jγ1/Jγ2-NED, JγP1/JγP2-NED and JγP-NED) each labeled with the same fluorochrome (NED) in a single tube (one-color multiplex reaction, Figure 1B). Finally, to illustrate how design flaws can cause interpretation problems, we subsequently analyzed these cases with the individual joining primer in separate tubes (partial multiplex reactions). This third amplification contained all five variable region primers and either a single labeled JγP1/JγP2-NED or JγP-NED primer in separate tubes (Figure 1C). PCR reaction mixes were composed of Perkin Elmer PCR buffer I (Perkin Elmer, Waltham, MA), 100 μmol/L dNTPs, 0.6 μmol/L each Vγ primer, 0.6 μmol/L each Jγ primer (in both complete multiplex or single multiplex assay), and 1.25 U Platinum Taq (Invitrogen, Carlsbad, CA). Amplification was performed on the Omnigene (Omnigene Bioproducts, Woodburn, MA) or MJ Dyad thermocyclers (BioRad, Hercules, CA) using the same protocol for all three approaches. The initial denaturing step (9 minutes at 94°) was followed by 30 cycles with the following time and temperature settings: denaturing, 94°C (75 seconds); annealing, 60°C (75 seconds); extension, 72°C (10 seconds plus 1 second for each cycle). After the reaction, the products were diluted 1:10 with water, and were analyzed using capillary electrophoresis on an ABI 3130xL instrument (Applied Biosystems, Foster City, CA). DNA was obtained from 44 clinical cases that had morphological diagnoses and had TRB@ Southern blot results previously performed with a constant region probe. The cases did not have the diagnosis established with either of the PCR methods compared in this manuscript. The series included 28 cases of T-cell malignancy, of which 26 cases had a TRB@ gene rearrangement (Table 1A). One of the two TRB@ negative cases of peripheral T-cell lymphoma had a complex cytogenetic abnormality. The remaining case was regarded as containing lymphoma below the detection limits of Southern blotting as a second biopsy showed definitive peripheral T-cell lymphoma. The series also included eight cases of B-cell malignancy and eight benign specimens, of which six of eight cases had negative TRB@ Southern blot results in each group (Table 1A). B-cell malignancies were included because amplification of small numbers of intermixed T-cells can sometimes result in interpretations of “clonal” TCRγGR that do not represent an aberrant TCRγGR in the B-cell tumor itself. Concurrent IgH and TCRγGR are known to often occur in lymphoblastic lymphomas. The most common source of tissue was from lymph node biopsies (Table 1B).Table 1APathologic DiagnosesT-cell malignanciesN = 28Peripheral T-cell lymphoma, not otherwise specified18Peripheral T-cell lymphoma, angioimmunoblastic2Large granular lymphocytic leukemia2Anaplastic large cell lymphoma1Lymphoblastic lymphoma1Subcutaneous panniculitis-like1Prolymphocytic leukemia1Acute lymphoblastic leukemia1Mycosis fungoides1B-cell malignanciesN = 8Follicular lymphoma3Diffuse large cell lymphoma2Lymphoblastic lymphoma1Chronic lymphocytic leukemia1Hodgkin's lymphoma1Benign disordersN = 8Atypical lymphoid hyperplasia4Reactive lymphocytosis2Follicular hyperplasia1Negative marrow1Table 1B. Tissue sources N = 44Lymph node27Peripheral blood8Bone marrow4Skin2Other-uterus, pheresis product, and pleural fluid3 Open table in a new tab Archival DNA (1 μg) was amplified for TCRγGR in a single tube with the tri-color multiplex PCR assay [Jγ1/Jγ2 NED (black), JγP1/JγP2 JOE (green), and JγP FAM (blue) labeled primers and five unlabeled variable region primers].7Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar A one-color method with the same NED label on the same three Jγ primers was also performed as it is easier to interpret.7Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Secondly, the BIOMED-2-based TCRG Gene Clonality Assay was used to amplify 1 μg of DNA per the manufacturer's directions in a 50 μl reaction using the following cycling program: 95°C 7 minutes, 35 cycles of 95°C 45 seconds, 60°C 45 seconds, 72°C 90 seconds; final 72°C 90 seconds. Whereas the exact primer sequences are proprietary information, the assay is based on the BIOMED-2 primers described by van Dongen et al.11van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2505) Google Scholar The products, which were expected to range from 80 to 200 nucleotides and 145 to 255 nucleotides in the two tubes, were analyzed by capillary electrophoresis on an ABI 3130XL instrument using 3130 Performance Optimized Polymer-4 (Applied Biosystems, CA). A positive result was defined as a peak height with a ratio greater than two times (2×) the polyclonal background peak height (RPB) for the one-tube methods.7Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Two RPB methods of greater than two (2×) or five times (5×) were used for the TCRG Gene Clonality Assay. Oligoclonal results, defined as cases with three or more peaks present, were regarded as negative for the calculations of sensitivity and specificity. The assay results were then compared with the original morphological diagnoses to determine sensitivity and specificity. The presence of pseudoclonal spikes was defined for each method as: One-tube method, peaks between 1× and 2× RPB; TCRG Gene Clonality Assay, peaks between 2× and 5× RPB. The Chi Square Goodness of Fit Test statistic was calculated to assess the significance of the results. Examples of positive clonal results with Jγ1/Jγ2, JγP1/JγP2 and JγP using the one-tube, tri-color method are identified in Figure 2A. For a negative polyclonal result, peripheral blood lymphocyte DNA from a normal patient sample demonstrates polyclonal distributions in both the tri-color and one-color approaches (Figure 2B). An expected polyclonal background is identified with the separate JγP1/JγP2 assay (Figure 2B). However, the separate JγP assay shows a more prominent peak in the central portion of the curve, that is now easily more than two times the height of the polyclonal background, which may incorrectly suggest a clonal rearrangement (Figure 2B). This JγP peak, which likely represents canonical rearrangements, corresponds to the same small blue peak in the tri-color assay, wherein the tri-color assay clearly shows it does not exceed the overall Jγ1/Jγ2 polyclonal distribution and therefore contains far less than a 2% population necessary for interpreting it as a clonal result. This limited JγP distribution observed is due to the known rarity of JγP rearrangements used by T-cells, thereby resulting in the low number of events in the JγP polyclonal distribution. Ten clinical cases are shown in which insignificant small green JγP1/JγP2 (Figure 3A, first column) or blue JγP (Figure 3B, first column) peaks are present in the tri-color assay. While the cases are confirmed as polyclonal by the one-color multiplex reactions (second column), the cases give prominent peaks in separate assays using only the single joining region primer (third column). Using the standard definition for clonality described in the methods (2× RPB), these insignificant peaks would become false positive pseudoclonal peaks with this separate primer approach (Figure 3, A and B; third columns). The sensitivity between both methods was not significantly different (χ2 0.35, P = 0.55). Clonal TCRγGR results were seen in 92.9% (26 of 28) of the cases of T-cell malignancies by the One-tube method whereas 96.4% (27 of 28) were positive by the TCRG Gene Clonality assay, regardless of whether a RPB of 2× or 5× was used for assessing the peaks in the latter assay. A JγP rearrangement, that was seen with the one-tube method in a case of peripheral T-cell lymphoma, was not identified with the TCRG Gene Clonality Assay, as the JγP primer is not included in the design of the assay (Figure 4D). The case status (clonal) was not affected as a biallelic TCRγGR with a common joining gene, which occurs about 35% of the time, was detected by both methods (Figure 4D). Specificity was assessed in the cases of B-cell malignancy combined with benign tissues. The specificity of the one-tube method was 87.5% (14 of 16 cases) whereas the TCRG Gene Clonality Assay had a specificity of only 56.3% (9 of 16 cases) when using the RPB of 2× (χ2 3.86, P = 0.049). Specificity was improved to an equivalent 81% (13 of 16 cases) when an RPB of 5× was used in the TCRG Gene Clonality Assay (χ2 0.237, P = 0.626). There was greater specificity in the one-tube method because there were fewer spikes outside the polyclonal background. Seven percent of the cases had spikes (3 of 44 cases) in the one-tube assay, whereas the TCRG Gene Clonality Assay had spikes in 38.6% of cases (17 of 44 cases) (χ2 10.6; P = 0.001). Tube B in the TCRG Gene Clonality Assay had the greatest percentage (88%) of the spikes in 15 of 17 cases. In the analysis of peripheral blood lymphocytes (PBLs) one can see the difficulty of using a uniform peak height ratio rule for two differently designed assays. While there are no prominent peaks in PBL with the one-tube assay, a prominent peak (spike) could be called positive with a RPB ratio of greater than 2 times the height of the polyclonal background in the TCRG Gene Clonality Assay using the same DNA (Figure 4A). In a case of TCRβ Southern blot negative diffuse large B-cell lymphoma, multiple spikes are seen in both IVS tubes that would be regarded as oligoclonal, thus, there would be no adverse clinical outcome (Figure 4B). However, in a case of reactive lymphocytosis that is negative by Southern blot and negative with the one-tube assays, there are two prominent spikes between 2× and 5× RPB in the TCRG Gene Clonality Assay that could result in a clonal interpretation by a lab using a 2× RBP threshold (Figure 4C). T-cell receptor gamma gene rearrangement analysis is useful as an ancillary test in the diagnosis of T-cell neoplasms. However, interpretation can be complicated by small, but observable, peaks, which may be incorrectly interpreted as clonal rearrangements. This phenomenon is most likely to occur in assays where multiple tubes, size distributions and colors are used. The number of primers, size distributions and reaction tubes varies between methods used for the evaluation of TCR-γGR. Table 27Greiner TC Rubocki RJ Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.J Mol Diagn. 2002; 4: 137-143Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar8Meier VS Rufle A Gudat F Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four-color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates.Am J Pathol. 2001; 159: 2031-2043Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar9Vega F Medeiros LJ Jones D Abruzzo LV Lai R Manning J Dunmire V Luthra R A novel four-color PCR assay to assess T-cell receptor gamma gene rearrangements in lymphoproliferative lesions.Am J Clin Pathol. 2001; 116: 17-24Crossref PubMed Scopus (94) Google Scholar10Luo V Lessin SR Wilson RB Rennert H Tozer C Benoit B Leonard DG Detection of clonal T-cell receptor gamma gene rearrangements using fluorescent-based PCR and automated high-resolution capillary electrophoresis.Mol Diagn. 2001; 6: 169-179PubMed Google Scholar11van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2505) Google Scholar12Lawnicki LC Rubocki RJ Chan WC Lytle DM Greiner TC The distribution of gene segments in T-cell receptor gamma gene rearrangements demonstrates the need for multiple primer sets.J Mol Diagn. 2003; 5: 82-87Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar13Lee SC Berg KD Racke FK Griffin CA Eshleman JR Pseudo-spikes are common in histologically benign lymphoid tissues.J Mol Diagn. 2000; 2: 145-152Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar,15Dippel E Assaf C Hummel M Schrag HJ Stein H Goerdt S Orfanos CE Clonal T-cell receptor gamma-chain gene rearrangement by PCR-based GeneScan analysis in advanced cutaneous T-cell lymphoma: a critical evaluation.J Pathol. 1999; 188: 146-154Crossref PubMed Scopus (97) Google Scholar,22Kuo FC Hall D Longtine JA A novel method for interpretation of T-cell receptor gamma gene rearrangement assay by capillary gel electrophoresis based on normal distribution.J Mol Diagn. 2007; 9: 12-19Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar23Patel KP Pan Q Wang Y Maitta RW Du J Xue X Lin J Ratech H Comparison of BIOMED-2 versus laboratory-developed polymerase chain reaction assays for detecting T-Cell receptor-gamma gene rearrangements.J Mol Diagn. 2010; 12: 226-237Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar24Lukowsky A Richter S Dijkstal K Sterry W Muche JM A T-cell receptor gamma polymerase chain reaction assay using capillary electrophoresis for the diagnosis of cutaneous T-cell lymphomas.Diagn Mol Pathol. 2002; 11: 59-66Crossref PubMed Scopus (33) Google Scholar25Munro NJ Snow K Kant JA Landers JP Molecular diagnostics on microfabricated electrophoretic devices: from slab gel- to capillary- to microchip
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