Application of Microfluidic Technology to the BIOMED-2 Protocol for Detection of B-Cell Clonality
2011; Elsevier BV; Volume: 14; Issue: 1 Linguagem: Inglês
10.1016/j.jmoldx.2011.07.007
ISSN1943-7811
AutoresAlberto Zamò, Anna Bertolaso, Annemiek W.M. van Raaij, Francesca Mancini, Maria Scardoni, Marina Montresor, Fabio Menestrina, J. Han van Krieken, Marco Chilosi, Patricia J.T.A. Groenen, Aldo Scarpa,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe BIOMED-2 protocol is widely used for detecting clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions, which are analyzed by either capillary electrophoresis (GeneScan analysis) or heteroduplex PAGE analysis. We tested a microfluidic chip-based electrophoresis device (Agilent 2100 Bioanalyzer) for the analysis of B-cell clonality using PCR for the three framework subregions (FR) of the Ig heavy chain gene (IGH) and PCR for two rearrangements occurring in the Ig κ chain gene (IGK-VJ and IGK-DE). We analyzed 62 B-cell lymphomas (33 follicular and 29 nonfollicular) and 16 reactive lymph nodes. Chip-based electrophoresis was conclusive for monoclonality in 59/62 samples; for 20 samples, it was compared with GeneScan analysis. Concordant results were obtained in 45/55 IGH (FR1, FR2, and FR3) gene rearrangements, and in 34/37 IGK gene rearrangements. However, when the chip device was used to analyze selected IGK gene rearrangements (biallelic IGK rearrangements or IGK rearrangements in a polyclonal background), its performance was not completely accurate. We conclude, therefore, that this microfluidic chip-based electrophoresis device is reliable for testing cases with dominant PCR products but is less sensitive than GeneScan in detecting clonal peaks in a polyclonal background for IGH PCR, or with complex IGK rearrangement patterns. The BIOMED-2 protocol is widely used for detecting clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions, which are analyzed by either capillary electrophoresis (GeneScan analysis) or heteroduplex PAGE analysis. We tested a microfluidic chip-based electrophoresis device (Agilent 2100 Bioanalyzer) for the analysis of B-cell clonality using PCR for the three framework subregions (FR) of the Ig heavy chain gene (IGH) and PCR for two rearrangements occurring in the Ig κ chain gene (IGK-VJ and IGK-DE). We analyzed 62 B-cell lymphomas (33 follicular and 29 nonfollicular) and 16 reactive lymph nodes. Chip-based electrophoresis was conclusive for monoclonality in 59/62 samples; for 20 samples, it was compared with GeneScan analysis. Concordant results were obtained in 45/55 IGH (FR1, FR2, and FR3) gene rearrangements, and in 34/37 IGK gene rearrangements. However, when the chip device was used to analyze selected IGK gene rearrangements (biallelic IGK rearrangements or IGK rearrangements in a polyclonal background), its performance was not completely accurate. We conclude, therefore, that this microfluidic chip-based electrophoresis device is reliable for testing cases with dominant PCR products but is less sensitive than GeneScan in detecting clonal peaks in a polyclonal background for IGH PCR, or with complex IGK rearrangement patterns. Detection of clonal lymphoid populations is one of the most significant achievements of diagnostic molecular pathology and is routinely used in many laboratories. Southern blotting analysis has been the gold standard for many years, but PCR-based approaches have been a welcome addition, because they are faster, simpler, and can be performed on fixed and embedded samples.1Achille A. Scarpa A. Montresor M. Scardoni M. Zamboni G. Chilosi M. Capelli P. Franzin G. Menestrina F. Routine application of polymerase chain reaction in the diagnosis of monoclonality of B-cell lymphoid proliferations.Diagn Mol Pathol. 1995; 4: 14-24Crossref PubMed Scopus (134) Google Scholar A limitation of these approaches is the low efficiency of primer annealing, attributable to intrinsic difficulties in primer design and to the presence of somatic hypermutation of Ig genes in some lymphoma categories, such as follicular lymphoma.1Achille A. Scarpa A. Montresor M. Scardoni M. Zamboni G. Chilosi M. Capelli P. Franzin G. Menestrina F. Routine application of polymerase chain reaction in the diagnosis of monoclonality of B-cell lymphoid proliferations.Diagn Mol Pathol. 1995; 4: 14-24Crossref PubMed Scopus (134) Google Scholar The BIOMED-2 protocol was developed as a European multicenter effort to optimize and standardize clonality detection in lymphoproliferative disorders.2van Dongen J.J.M. Langerak A.W. Brüggemann M. Evans P.A.S. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H.J.M. Droese J. Gonz lez D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. 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 protocol aims at detecting any possible immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangement, as well as the most common chromosomal translocations. The complete protocol requires 14 multiplex PCR reactions: eight for Ig gene rearrangements and six for TCR rearrangements). However, a protocol with five PCR amplifications for Ig genes (three for IGH VH-JH and two for IGK rearrangements) and five for TCR (three for TCRB and two for TCRG), is recommended as the most informative, economical, and efficient alternative.2van Dongen J.J.M. Langerak A.W. Brüggemann M. Evans P.A.S. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H.J.M. Droese J. Gonz lez D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. 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 analytic phase for PCR products includes either high-resolution fragment analysis by capillary electrophoresis (GeneScan; Applied Biosystems, Foster City, CA), which exploits heterogeneity in size, or heteroduplex analysis on polyacrylamide gels (HDA-PAGE), which exploits heterogeneity in both size and composition of the amplified PCR fragments.2van Dongen J.J.M. Langerak A.W. Brüggemann M. Evans P.A.S. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H.J.M. Droese J. Gonz lez D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. 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 GeneScan analysis has high resolution, and is very sensitive and fast, so it is the preferred technique for detecting clonality; however, it requires a specialized CE platform. HDA-PAGE is performed after denaturation and reannealing of PCR fragments. Noncomplementary products form heteroduplexes with lower electrophoretic mobility compared with homoduplexes; thus, background polyclonal bands that might impair detection of the clonal peak are eliminated. Chip-based microfluidic systems were developed in the 1990s3Woolley A.T. Mathies R.A. Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips.Proc Natl Acad Sci USA. 1994; 91: 11348-11352Crossref PubMed Scopus (576) Google Scholar and were immediately recognized as a viable alternative to classical CE analysis.4Effenhauser C.S. Bruin G.J. Paulus A. Integrated chip-based capillary electrophoresis.Electrophoresis. 1997; 18: 2203-2213Crossref PubMed Scopus (206) Google Scholar These systems are based on miniaturized parallel CE structures that can be mass-produced by a technology derived from electronics. They tolerate high electric fields, and this, together with a short separation distance, allows a fast separation on a time scale of seconds.4Effenhauser C.S. Bruin G.J. Paulus A. Integrated chip-based capillary electrophoresis.Electrophoresis. 1997; 18: 2203-2213Crossref PubMed Scopus (206) Google Scholar Movement of fluids through the network of microchannels is regulated by electrokinetic effects. These devices offer numerous advantages, including fast run time, low cost, extreme versatility, and finally a planar structure that allows easy automation and parallel analysis. These chip-based systems can potentially perform many tasks, including sample preparation, PCR amplification, electrophoresis of different samples (DNA, RNA, proteins) with good size discrimination,5Zamò A. Bertolaso A. Franceschetti I. Weirich G. Capelli P. Pecori S. Chilosi M. Hoefler H. Menestrina F. Scarpa A. Microfluidic deletion/insertion analysis for rapid screening of KIT and PDGFRA mutations in CD117-positive gastrointestinal stromal tumors: diagnostic applications and report of a new KIT mutation.J Mol Diagn. 2007; 9: 151-157Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar and cytofluorimetry. Moreover, the instruments have very few moving parts and therefore require only little maintenance. Because chip-based electrophoresis systems seemed highly suitable for analysis of complex PCR samples (such as those obtained from the BIOMED-2 protocol) in a much shorter time than conventional systems, and at a very competitive cost, we tested whether a commercially available apparatus6Mueller O. Hahnenberger K. Dittmann M. Yee H. Dubrow R. Nagle R. Ilsley D. A microfluidic system for high-speed reproducible DNA sizing and quantitation.Electrophoresis. 2000; 21: 128-134Crossref PubMed Scopus (165) Google Scholar could be used in the analytical phase of the simplified BIOMED-2 protocol for the detection of B-cell clonality. The use of this device in combination with laser-capture microdissection for the detection of T-cell clonality according to a different PCR protocol7McCarthy K.P. Sloane J.P. Kabarowski J.H. Matutes E. Wiedemann L.M. A simplified method of detection of clonal rearrangements of the T-cell receptor-gamma chain gene.Diagn Mol Pathol. 1992; 1: 173-179PubMed Google Scholar has been addressed previously.8Yakirevich E. Jackson C.L. Meitner P.A. MacKenzie D. Tavares R. Robinson-Bostom L. DeLellis R.A. Resnick M.B. Analysis of T-cell clonality using laser capture microdissection and high-resolution microcapillary electrophoresis.J Mol Diagn. 2007; 9: 490-497Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar For clonality detection, 62 frozen samples were analyzed. This panel of B-cell lymphomas comprised 33 follicular lymphomas (FL) and 29 non-FL lymphomas. The non-FL samples comprised B-cell chronic lymphocytic leukemia (n = 18), diffuse large B-cell lymphoma (n = 6), splenic marginal zone lymphoma (n = 1), lymphoplasmacytic lymphoma (n = 1), extranodal marginal zone lymphoma (n = 1), hairy cell leukemia (n = 1), and Hodgkin's lymphoma (n = 1). Frozen samples of reactive lymphadenopathy (n = 16) were used as polyclonal controls. To test whether microfluidic chip-based electrophoresis (which has lower resolution than GeneScan analysis) enables detection of complex clonal patterns with small Gaussian curves (characterized by a narrow span of peaks), because of the limited junctional regions, we selected DNAs from 10 frozen samples with complex IGK clonality profiles that had been previously detected by GeneScan analysis specifically for the evaluation of IGK-VJ and IGK-DE rearrangement profiles. Complex patterns were defined as falling into three types, as follows: i) cases that clearly had clonal products but were difficult to interpret because of multiple clonal peaks in one of the PCRs; ii) cases that were difficult to interpret because weak clonal signals were detected (in duplicate) in a background considered to be polyclonal irregular; and iii) cases that had with multiple (biallelic) IGK gene rearrangements differing by 5 to 10 nt in amplicon size (which we expected to be difficult to evaluate from the Bioanalyzer chip-based readout). DNA quality was checked for all samples using control gene PCR as in the BIOMED-2 protocol. All DNAs amplified a 400-bp amplicon, demonstrating the good quality of the samples. DNA (100 ng) was amplified by PCR amplification according to the PCR BIOMED-2 protocol for detection of B-cell clones. The recommended five-PCR BIOMED-2 protocol includes three Ig heavy chain amplifications (IGH VH-JH FR1, FR2 and FR3, corresponding to BIOMED-2 IGH tubes A, B, and C) and two Ig κ light chain amplifications (IGK VK-JK and KDE, corresponding to BIOMED-2 IGK tubes A and B). For microfluidic analysis of the PCR products, nonlabeled high performance liquid chromatography-purified primers were used (MWG Biotech, Ebersberg, Germany), and PCR reactions were performed on a GeneAmp 9700 instrument (Applied Biosystems). For GeneScan analysis, fluorescently labeled primers were purchased from Invivoscribe Technologies (San Diego, CA) and from Invitrogen (Carlsbad, CA), and PCR was performed on a PTC-200 thermocycler (MJ Research; Bio-Rad, Waltham, MA). DNA from the MAVER-1 cell line9Zamò A. Ott G. Katzenberger T. Adam P. Parolini C. Scarpa A. Lestani M. Menestrina F. Chilosi M. Establishment of the MAVER-1 cell line, a model for leukemic and aggressive mantle cell lymphoma.Haematologica. 2006; 91: 40-47PubMed Google Scholar was mixed with DNA from one reactive lymph node. DNA concentration was measured by a fluorimetric method (Qubit; Invitrogen). The two samples were mixed so that the percentage of DNA from neoplastic cells was 100%, 20%, 10%, 5%, and 1% of total DNA. We chose these percentages because many of our samples were already composed of approximately 20% neoplastic cells and 80% reactive B cells and/or T cells (as evaluated by flow cytometry). PCR products were analyzed both by microfluidic chip-based electrophoresis and also by fluorescence fragment analysis by CE (GeneScan). For each PCR, 1 μL of reaction mixture was separated on a 2100 Bioanalyzer microfluidic chip-based platform (Agilent Technologies, Waldbronn, Germany),6Mueller O. Hahnenberger K. Dittmann M. Yee H. Dubrow R. Nagle R. Ilsley D. A microfluidic system for high-speed reproducible DNA sizing and quantitation.Electrophoresis. 2000; 21: 128-134Crossref PubMed Scopus (165) Google Scholar using the Agilent DNA1000 chip and reagents. Electronic data were analyzed using the manufacturer's software (Agilent 2100 Expert version B.02.05.SI360), which automatically provides peak sizing and quantitation. A sample was considered positive if one or two peaks (corresponding to one or two rearranged alleles) were detected in the expected size range in at least one reaction2van Dongen J.J.M. Langerak A.W. Brüggemann M. Evans P.A.S. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H.J.M. Droese J. Gonz lez D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. 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 (IGH tube A, 310 to 360 bp; IGH tube B, 250 to 295 bp; IGH tube C, 100 to 170 bp; IGK tube A, 120 to 160 bp, 190 to 210 bp, and 260 to 300 bp; IGK tube B, 210 to 250 bp, 270 to 300 bp, and 350 to 390 bp). For IGK evaluation, a comparison with the polyclonal pattern of bands was necessary for final interpretation, which is similar to what is done in CE analysis. IGH (FR1, FR2, and FR3) and IGK (VJ and DE) gene rearrangements were analyzed in duplicate using the BIOMED-2 PCR protocol and multiplex primer sets. In addition to the samples, both a monoclonal control having one or more monoallelic or biallelic clonal rearrangement of the particular target and a polyclonal control were used in each PCR experiment. Monitoring of clonality was performed by fluorescent fragment analysis (GeneScan analysis) on an ABI 3730 DNA analyzer platform (Applied Biosystems) and analyzed by Genomapper software version 4.0. Evaluation of B-cell clonality by Southern blot using Ig heavy chain and κ light chain probes was available for 22 samples: chronic lymphocytic leukemia (n = 10), diffuse large B-cell lymphoma (n = 3), splenic marginal zone lymphoma (n = 1), lymphoplasmacytic lymphoma (n = 1), and FL (n = 7). The analysis was performed on DNA extracted from frozen samples as described previously.10Scarpa A. Bonetti F. Menestrina F. Menegazzi M. Chilosi M. Lestani M. Bovolenta C. Zamboni G. Fiore-Donati L. Mediastinal large-cell lymphoma with sclerosis Genotypic analysis establishes its B nature.Virchows Arch A Pathol Anat Histopathol. 1987; 412: 17-21Crossref PubMed Scopus (58) Google Scholar Probes were labeled with digoxigenin and detected by chemoluminescence (Boehringer Mannheim DIG luminescent detection kit; Roche Diagnostics, Indianapolis, IN). We assessed the efficiency of microfluidic chip-based analysis of PCR amplifications for detection of B-cell clonality using the BIOMED-2 protocol in 62 lymphomas (33 FL and 29 non-FL lymphomas) and in 16 non-neoplastic lymph nodes. To check the accuracy limit of the chip device, 10 previously identified complex IGK rearrangements were selected for chip analysis. Electropherograms showed one or more clonal products in 58/62 cases: in 30/33 FL samples [90.9%; 95% confidence interval (CI) = 76% to 98%] and in 28/29 non-FL samples (96.5%; 95% CI = 82% to 99%) (see Supplemental Table S1 at http://jmd.amjpathol.org). The IGH (FR1, FR2 and FR3) electropherograms were almost always easy to interpret; however, difficulties were often encountered with IGK (KVJ and KDE) electropherograms, for which direct comparison with polyclonal reference electropherograms was necessary for evaluation of the sample profile. Microfluidic chip-based electrophoresis for the final diagnosis of the entire Ig gene clonality profile (ie, clonal or polyclonal Ig gene rearrangements) were concordant with Southern blot data in 19/22 cases (86%; 95% CI = 65% to 97%). Three samples were polyclonal in the five Ig PCR reactions analyzed by microfluidic chip analysis but were clonal in the Southern blot analysis; notably, two cases (case 5, diffuse large B-cell lymphoma, and case 53, FL) showed clonal IGH rearrangements and one case (case 54, FL) had clonal IGH and IGK rearrangements on Southern blot (see Supplemental Table S1 at http://jmd.amjpathol.org). The clonality results for the IGK gene rearrangements demonstrated concordance of results for the PCR analysis (both IGK-VJ and IGK-DE gene rearrangements; chip-based electrophoresis readout) and the Southern blot analysis in 14/21 cases assessed (67%; 95% CI = 43% to 85%). Clonality of the IGH gene shows that microfluidic CE and Southern blot showed concordant results for the complete IGH gene rearrangements in 17/22 cases (77%; 95% CI = 55% to 92%). A first assessment of sensitivity, obtained by comparing the percentage of neoplastic cells estimated by flow cytometry, showed that a sample containing 17% neoplastic cells was readily detected by the microfluidic apparatus. A more precise determination was obtained by mixing DNA from a mantle cell lymphoma cell line with DNA from a lymph node reactive sample. Microfluidic chip-based electrophoresis had a sensitivity limit at 10% for all IGH tubes (FR1, FR2, and FR3), and all IGK tubes (VJ and DE rearrangements). GeneScan analysis had a sensitivity of 1% for IGH tube B (FR2) and tube C (FR3) and 5% for IGH tube A (FR1) and for IGK tubes A and B. In this experiment, the sensitivity was determined for a single IGH, a single IGK-VJ, and a single IGK-DE rearrangement (see Supplemental Figure S1 at http://jmd.amjpathol.org). To gain experience in polyclonal pattern recognition with the microfluidic device, we analyzed 16 polyclonal lymph nodes for all five PCR reactions. Representative electropherograms of polyclonal samples are shown in Figure 1, Figure 2. The electropherograms of the IGH (FR1, FR2, and FR3) PCR products always showed an easily interpreted Gaussian curve. In contrast, however, the electropherograms of the IGK (KVJ and KDE) products showed slightly variable patterns among the polyclonal controls; sometimes they presented two and sometimes three peaks with narrow baselines, compared with the Gaussian curves of the IGH polyclonal samples.Figure 2Comparison of IGK rearrangement electropherograms acquired by microfluidic chip-based electrophoresis and by GeneScan analysis for 3 of the 10 additional samples with complex patterns. Case numbering corresponds to that of Table 2. Detection of complex IGK-VJ rearrangements was poorly successful using the microfluidic chip-based electrophoresis readout. This is evident for IGK tube A especially when the sample has a low-intensity signal, which is frequently interpreted as polyclonal.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We assessed 20 samples for IGH and IGK rearrangements using the BIOMED-2 approach, analyzed by chip-based electrophoresis and by GeneScan CE, and compared the results (Table 1 and Figure 1). Despite occasional differences in interpretation of single reactions, the final correlation was good: 19/20 samples (95%; 95% CI = 75% to 99%) scored concordantly (either mono- or polyclonal) for the final diagnosis of the sample as clonal or polyclonal. Notably, one reactive lymph node yielded discordant results for all PCRs, with clonal rearrangements in the IGH and IGK genes by GeneScan analysis and polyclonal signals by chip-based electrophoresis. The histology was then checked, and the diagnosis was confirmed as reactive lymphadenopathy (although large germinal centers were present).Table 1Microfluidic Chip Electrophoresis and GeneScan AnalysesID no.IGH tube AIGH tube BIGH tube CIGK tube AIGK tube BFinal clonality assessmentHereTable S1⁎See Supplemental Table S1 at http://jmd.amjpathol.org.DxGSChipGSChipGSChipGSChipGSChipGSChip12LPLPPMMPPPPPPMM25DLBCLPPPPPPPPPPPP320CLLMMMMMMMMMMMM423CLLPPPPPPMMPPMM524FLMMM†Discordant PCR results.P†Discordant PCR results.PPMMMMMM631FLMMMMM†Discordant PCR results.P†Discordant PCR results.MMMMMM736FLM†Discordant PCR results.P†Discordant PCR results.MMMMMMPPMM839FLPPPPPPMMPPMM951FLMMMMMMM†Discordant PCR results.P†Discordant PCR results.PPMM1052FLPPM†Discordant PCR results.P†Discordant PCR results.M†Discordant PCR results.P†Discordant PCR results.MMMMMM1155HLPPPPMMPPPPMM1256HCLMMMMMMMMMMMM1357FLMMMMPPPPPPMM1458DLBCLPPPPPPPPMMMM1559DLBCL//MM//////MM1660EMZLMMMMPPPPMMMM1761DLBCL//////MM//MM1862FLM†Discordant PCR results.P†Discordant PCR results.M†Discordant PCR results.P†Discordant PCR results.PPMMMMMM19RLNPPPPPPPPPPPP20RLNM†Discordant PCR results.P†Discordant PCR results.M†Discordant PCR results.P†Discordant PCR results.M†Discordant PCR results.P†Discordant PCR results.M†Discordant PCR results.P†Discordant PCR results.M†Discordant PCR results.P†Discordant PCR results.M†Discordant PCR results.P†Discordant PCR results.Discordant PCR (n/N)3/184/193/182/191/181/20Chip, microfluidic chip electrophoresis; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma; Dx, diagnosis; EMZL, extranodal marginal zone lymphoma; FL, follicular lymphoma; GS, GeneScan analysis; HCL, hairy cell lymphoma; HL, Hodgkin's lymphoma; LPL, lymphoplasmacytic lymphoma; M, monoclonal; P, polyclonal; RLN, reactive lymphadenopathy; /, not evaluable. See Supplemental Table S1 at http://jmd.amjpathol.org.† Discordant PCR results. Open table in a new tab Chip, microfluidic chip electrophoresis; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma; Dx, diagnosis; EMZL, extranodal marginal zone lymphoma; FL, follicular lymphoma; GS, GeneScan analysis; HCL, hairy cell lymphoma; HL, Hodgkin's lymphoma; LPL, lymphoplasmacytic lymphoma; M, monoclonal; P, polyclonal; RLN, reactive lymphadenopathy; /, not evaluable. There were a few discordances in scoring the individual PCRs, both for the IGH FR PCRs and for the IGK PCRs (VJ and DE) (Table 1). High-intensity signals always received the same evaluation under the two techniques, and most of the discordant results involved low-intensity signals. Representative clonality profiles for IGH (FR1, FR2, and FR3) and IGK (KVJ and KDE) as analyzed by GeneScan CE and by microfluidic chip-based electrophoresis are shown in Figure 1. The sizing resolution of the microfluidic chip-based electrophoresis was lower than that of GeneScan analysis (see sample 12 in Figure 1). We therefore tried to assess whether microfluidic chip-based electrophoresis enabled the detection of complex clonal patterns with small Gaussian curves (characterized by a narrow span of peaks) because of the limited junctional regions, or detection of biallelic rearrangements and clonal rearrangements in a background of polyclonal B cells. For this purpose, we decided to test additional cases with complex IGK rearrangements, because IGK gene rearrangements can be difficult to interpret even in GeneScan analysis. (Our definition of “complex” is given under Materials and Methods.) We selected 10 additional IGK clonality profiles that were interesting either for multiple IGK clonal signals or for low-intensity IGK clonal signals detected by GeneScan analysis. Low-intensity signals are common in cases with suboptimal DNA quality, or in cases with low percentage of lymphoid cells suspected for malignancy.9Zamò A. Ott G. Katzenberger T. Adam P. Parolini C. Scarpa A. Lestani M. Menestrina F. Chilosi M. Establishment of the MAVER-1 cell line, a model for leukemic and aggressive mantle cell lymphoma.Haematologica. 2006; 91: 40-47PubMed Google Scholar Microfluidic chip-based analysis and GeneScan analysis for both IGK-VJ (tube A) and IGK-DE (tube B) were compared for these 10 cases (Table 2 and Figure 2). Detection of complex IGK-VJ rearrangements was poorly successful using the microfluidic chip-based electrophoresis readout: of clonal results from GeneScan analysis, only 1/9 (11%; 95% CI = 0.28% to 48%) could be detected by microfluidic chip-based electrophoresis. The detection of IGK-DE rearrangements was better; the chip-based analysis correctly detected 7/10 (70%; 95% CI = 35% to 93%) cases determined as clonal by GeneScan CE. Based on comparison of the two readouts, GeneScan analysis proved more sensitive.Table 2Comparison of Clonality of IGK Rearrangements by Microfluidic Chip Electrophoresis with GeneScan Analysis in Selected Samples with Complex PatternsIGK tube A (KVJ)IGK tube B (KDE)SampleGSChipGSChipAC199 + PCBPC279/283C285/2910BC153PC278/282C285/290CPPC239 + PCBP/dubDC196/147PC285/275C287EC143/149PCw240 + PCBPFC152/284C293C276/286C289/299GO148/197/285/291PO278/282/288/365C289/296HC148/290P/dubC283C295IC191/196PC281C293LC/C149/283 + PCBPC/C242/376 + PCBP/dubDiscordant PCR [n/N (%)]8/10 (80)3/10 (30)The sizes of the clonal gene rearrangements are given, there can be two rearrangements, or multiple rearrangements.C, clonal; Chip, microfluidic chip electrophoresis; Cw, a small clonal peak; GS, GeneScan analysis; O, oligoclonal; P/dub, polyclonal or dubious result; PCB, polyclonal background. Open table in a new tab The sizes of the clonal gene rearrangements are given, there can be two rearrangements, or multiple rearrangements. C, clonal; Chip, microfluidic chip electrophoresis; Cw, a small clonal peak; GS, GeneScan analysis; O, oligoclonal; P/dub, polyclonal or dubious result; PCB, polyclonal background. In comparison with costs for GeneScan analysis and HDA-PAGE, costs for the chip-based instrument were less than one-fifth of GeneScan analysis, but three to four times that of a HDA-PAGE system. PCR costs were similar for HDA-PAGE and chip-based analyses; the GeneScan analysis system had a slightly higher cost, because it uses fluorochrome-labeled primers. The GeneScan analysis system had the highest costs for analytical consumables (estimated at 10€/sample in Italy, including capillary cost, gel, and buffers); the chip-based system came next (estimated at 1.5€/sample). HDA-PAGE had the lowest cost if home-made gels were used; otherwise the cost per sample was similar to or slightly higher than for the chip-based system. In terms of time, the chip-based system required ∼2.5 minutes for each run, 300 bp. The main artifact of the Agilent analysis technique is the presence of occasional electrical spikes; these are easily discriminated in electropherograms, but may yield the impression of a band in the pseudo-gel image. These spikes are usually due to instrument vibration, which can be easily be prevented by using an appropriate instrument support. Lab-on-a-chip technology is continuously evolving and has become an important research field, with many promising applications. The coverage is highly relevant to a variety of industrial and academic sectors, including pharmaceuticals, medicine, analytical science, biotechnology, physics, engineering and bioengineering, and electronics. Applications to the biomedical field are abundant and promise major breakthroughs in miniaturized, automated sample preparation and analysis.12Holmes D, Gawad S: The application of microfluidics in biology. Methods Mol Biol, 583:55–80Google Scholar, 13Ohno K. Tachikawa K. Manz A. Microfluidics: applications for analytical purposes in chemistry and biochemistry.Electrophoresis. 2008; 29: 4443-4453Crossref PubMed Scopus (325) Google Scholar Lab-on-a-chip technology may provide the key to powerful new diagnostic instruments, especially suited to poorly equipped clinics and to countries with few health care resources. In the context of our experiment, chip-based microfluidic electrophoresis offered promise of many of the advantages of GeneScan analysis (including automation, sensitivity, and size discrimination) at competitive cost, because i) the instrument itself has a lower cost, ii) nonlabeled primers can be used in the PCR reaction, iii) consumables are less expensive, iv) maintenance is minimal, v) chip preparation is very fast (∼10 minutes), and vi) exposure to dangerous chemicals is minimal. Moreover, a complete chip run with 12 samples requires only 32 minutes (Table 2). Compared with home-made HDA-PAGE analysis, however, the chip-based technique has a higher cost in terms of materials. Nonetheless, based on our evaluation of the data, we conclude that GeneScan CE outperforms microfluidic analysis in terms of sensitivity in the detection of weak PCR products, especially in the context of a polyclonal background (Figure 1) and for IGK gene rearrangements (Figure 2). In conclusion, microfluidic chip-based analysis proved to be a reliable device for IGH clonality analysis when a high clonal signal is present. Nonetheless, for the application of the BIOMED-2 clonality protocol, it was easily outperformed by GeneScan analysis in detection of low-intensity clonal signals and complex patterns. Because such situations can occur in a clinical setting, we deem the tested microfluidic device not sufficiently sensitive. Future improvements in chip resolution and sensitivity might make this type of instrument potentially usable for clinical applications. Download .pdf (.18 MB) Help with pdf files Supplemental Figure S1Bioanalyzer and GeneScan electropherograms of the sensitivity experiments for the IGH tubes (FR1, FR2, and FR3) and IGK tubes (VJ and DE rearrangements) obtained by mixing DNA from a mantle cell lymphoma cell line with DNA from a lymph node-reactive sample. The microfluidic chip-based electrophoresis approach had a sensitivity of 10% for all IGH and IGK tubes. GeneScan analysis had a sensitivity of 1% for IGH tube B (FR2) and tube C (FR3) and a sensitivity of 5% for IGH tube A (FR1) and for IGK tubes A and B. Download .xls (.04 MB) Help with xls files Supplemental Table S1
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