Semireannealing, single-stranded conformational polymorphism: a novel and effective tool for the diagnosis of T-cell clonality.

Single-stranded conformational polymorphism (SSCP) is often used for the diagnosis of T-cell clonality in lymphoproliferative disorders. We introduce a semireannealing SSCP (SR-SSCP) protocol that is rapid, reproducible, and effective. By denaturing and reannealing the polymerase chain reaction (PCR) product before high-resolution polyacrylamide gel electrophoresis, it is possible to generate a diagnostic fingerprint for each case with clonal T-cell receptor-gamma (TCR-gamma) gene rearrangement detected after PCR with TCR-gamma specific consensus primers. Discrete and distinct denatured single-stranded DNA band profiles characterize the rearranged TCR-gamma clones. In the same gel, the clone size may be estimated in the reannealed double-stranded PCR DNA and can be assessed down to the 2% clonal T-cell population level. Eighty-four cases, including 37 T-cell neoplasms, 29 B-cell neoplasms, and 18 reactive lymph node samples were analyzed by SR-SSCP. Clonal TCR-gamma rearrangement was diagnosed in 32 out of 37 T-cell neoplasms but in none of the B-cell tumors or reactive lymph node samples corresponding to sensitivity and specificity of 86.5% and 100%, respectively. We compare the results of SR-SSCP to those obtained by capillary electrophoresis and direct sequence analysis with 100% correlation. This novel method is applicable to any system for identification and quantitation of microheterogeneity in PCR products.     

Detection of concurrent/recurrent non-Hodgkin's lymphoma in effusions by PCR

A subset of patients with non-Hodgkin's lymphoma (NHL), present with or subsequently develop lymphocytic effusions. Differential diagnosis between reactive lymphocytosis and recurrent low-grade NHL is difficult by cytology alone. We studied the use of polymerase chain reaction (PCR)-based techniques to detect concurrent/recurrent NHL. Both primary tumors and atypical lymphocytic effusions of 12 low-grade B-NHL patients and 4 T-NHL patients were studied. Six pleural effusions (reactive/carcinomatous), in patients with no history of NHL, were included. Samples were amplified by PCR, using Fr3, Fr2, LJH, and VLJH primers specific for the immunoglobulin heavy chain (IgH) gene and Vgamma-8, Vgamma9, Vgamma10, Vgamma11 and Jgamma1/Jgamma2 consensus primers specific for the T-cell receptor gamma (TCR-gamma) gene. IgH gene PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE). TCR-gamma gene PCR products were analyzed using a novel nonradioactive single-strand conformational polymorphism (SSCP) procedure. IgH gene rearrangement analysis demonstrated monoclonality in 11/12 primary low-grade B-NHLs. Identical monoclonal bands were found in both primary tumor and effusion in 9 patients. TCR-gamma gene rearrangement analysis demonstrated monoclonality in 4 of 4 primary T-NHLs. Identical monoclonal banded patterns were found in both primary tumor and effusion in 3 patients. Our results strongly support the diagnosis of concurrent/recurrent NHL in 13 of 16 (81%) cases of atypical lymphocytic effusions. IgH/PAGE and TCR-gamma/SSCP analyses are useful tools in the diagnoses of lymphocytic effusions in patients with NHL.     

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.

We describe a modified denaturing gradient gel electrophoresis (DGGE) procedure with a 40-nucleotide GC clamp in the polymerase chain reaction to improve resolution in amplifying T cell receptor-gamma (TCR-gamma) rearrangements. DNA from 46 cases of lymphoblastic leukemia/lymphoma, 5T cell lines, 2 B cell lines, 7 normal lymphocytes, and 3 cases of Hodgkin's disease was amplified by polymerase chain reaction. In addition, 20 cases of paraffin-embedded T cell lymphomas and 5 cases of reactive hyperplasia were also studied. Clonal TCR-gamma rearrangements were identified on DGGE by the presence of a predominant band. Results obtained from 5 T cell lines and 12 lymphoblastic leukemia/lymphomas containing known TCR-gamma gene rearrangements revealed 100% concordance in detecting clonal rearrangements between DGGE and traditional Southern blot analysis. Of the remaining 34 lymphoblastic leukemia/lymphoma cases studied by DGGE alone, 30 were positive. DGGE analysis of 10 lymphoblastic leukemia/lymphoma cases with known group IV gamma to J gamma 1 or J gamma 2 rearrangement sequences confirmed that the electrophoretic migration was dependent on the tumor-specific rearranged TCR-gamma sequence. In addition, 17 of 20 cases of paraffin-embedded T cell lymphomas were positive by DGGE, 6 of which had the clonal population also identified in fresh tissue DNA. DGGE analysis of GC-clamped polymerase chain reaction products can provide a way to more accurately detect TCR-gamma clonality of lymphoid tumors and can be applied to archival tissues.     

Clonality analysis of B-cell lymphoproliferative disorders using PCR and melting curve analysis.

Detection of a monoclonal immunoglobulin heavy chain gene (IgH gene) rearrangement is commonly used to support the diagnosis of B-cell non-Hodgkin lymphoma. We investigated the application of melting curve analysis as a substitute for polyacrylamide gel electrophoresis (PAGE) in the detection of monoclonal IgH gene rearrangements after PCR. A total of 140 cases were selected for this study, including 63 B-cell malignancies with a previously documented monoclonal IgH gene rearrangement. These 140 specimens were tested using PCR with melting curve analysis, and the results obtained were compared with PAGE results to calculate the relative sensitivity and specificity of melting curve analysis. Melting curve analysis detected monoclonal rearrangements in 56 of 63 specimens (relative sensitivity 88.9%). No false positives were detected (relative specificity = 100%). False-negative results were obtained only when a weak monoclonal band was present on PAGE. These results show that a positive result on melting curve analysis is specific for a monoclonal IgH gene rearrangement. However, with a sensitivity of only 88.9%, the majority of negative results would require further evaluation of the amplicons using PAGE. The application of melting curve analysis in the detection of monoclonal IgH gene rearrangements in the clinical laboratory setting is discussed.     

Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction.

Polymerase chain reaction (PCR) technique is widely used in the diagnosis of lymphoma, and PCR amplification products are typically detected by polyacrylamide gel electrophoresis (PAGE). However, the identification of small clonal populations, or the distinction of clonal PCR products in a polyclonal milieu remains difficult, requiring technically demanding alterations to gel analysis. This study describes an alternative approach using a capillary electrophoresis (CE) system to produce an accurately sized electropherogram. A variety of patient samples were examined, including solid tissue, peripheral blood, bone marrow aspirates, and paraffin-embedded tissue. A total of 28 samples were evaluated by PCR for B-cell clonality by detection of immunoglobulin heavy chain gene rearrangement and 29 samples for T-cell clonality by detection of T-cell gamma locus gene rearrangement. Standard 10% PAGE analysis of PCR products was compared with CE. There was a 100% concordance in the assessment of both B-cell and T-cell clonality. Dilution studies with the SUP-B15 cell line showed a detection limit of 0.03% for B-cell clonality and 0.05% for T-cell clonality using CE, versus 0.2% to 1%, respectively for PAGE. Automated, fluorescent analysis of PCR products by CE seems to be at least equally as effective as gel-based analysis for the detection of clonal B-cell and T-cell populations. Moreover. CE offers superior resolution and improved sensitivity, thus representing a significant improvement over traditional gel electrophoretic techniques in these regards.     

T-cell clonality determination using polymerase chain reaction (PCR) amplification of the T-cell receptor gamma-chain gene and capillary electrophoresis of fluorescently labeled PCR products

We compared the effectiveness of polymerase chain reaction (PCR) and DNA blot analysis (DBA) for detecting clonal T-cell populations and investigated whether a nonradioactive PCR method could be used in routine clinical diagnosis. We analyzed DNA from 117 cases for T-cell clonality by PCR amplification. DBA was performed on 77 of these cases. Denaturing polyacrylamide gel electrophoresis (PCR-PAGE) of radiolabeled PCR products and capillary electrophoresis (PCR-CE) of fluorescently labeled PCR products were used for PCR product separation and quantitation. Complete agreement was obtained between PCR-PAGE and DBA in 67 of 77 cases. One case was positive by DBA and negative by PCR-PAGE, and 3 cases were positive by PAGE and negative by DBA. Five cases indeterminate by DBA were positive by PCR-PAGE, and 1 indeterminate case was negative by PCR-PAGE. In the comparison of PCR-PAGE and PCR-CE, of 63 cases with height ratios less than 2.0, all were negative by PCR-PAGE. Of 52 cases with height ratios of 2.0 or more, 50 were positive by PCR-PAGE. We conclude that PCR-CE is analytically equivalent to DBA and PCR-PAGE for detecting clonal T-cell populations. The PCR-CE method is semiquantitative and, therefore, may be more objective than gel-based methods.     

Sensitive detection of clonal immunoglobulin rearrangements in frozen and paraffin embedded tissues by polymerase chain reaction heteroduplex analysis.

Molecular detection of a clonal population of B or T cells through analysis of rearranged antigen receptor genes is an essential adjunct to the morphologic, flow cytometric, and immunohistochemical evaluation of tissue specimens for the presence of leukemia or lymphoma. Combining polymerase chain reaction (PCR) with heteroduplex annealing and polyacrylamide gel electrophoresis (PAGE) has been used to detect clonal T-cell receptor rearrangements, particularly in skin biopsy specimens. The authors have developed a similar PCR heteroduplex assay for detection of clonal VDJ immunoglobulin gene rearrangements using two sets of primers based on relatively conserved consensus regions in the J(H) and framework I and 2 regions of the immunoglobulin heavy chain V region gene. This method is able to detect a clonal rearrangement when the clone comprises as little as 1% of the population in a polyclonal B-cell background. It may be used on fresh, frozen, or paraffin-embedded tissue and detects a clonal population in a majority of lymphoma subtypes. Compared with conventional PCR analysis, this method requires only a short additional cycle of denaturation and slow renaturation before PAGE. Interpretation is simplified as the clonal PCR product migrates away from the polyclonal background products.     

Lymphomas with follicular and monocytoid B-cell components. Evidence for a common clonal origin from follicle center cells

We investigated the clonal relationship between follicular center cell and monocytoid B-cell components of non-Hodgkin lymphoma by isolating the components and comparing the nucleotide sequences of the complementarity-determining region (CDR)3 of the rearranged immunoglobulin heavy chain (IgH) gene. Paraffin blocks from 4 cases with amplifiable DNA using the polymerase chain reaction (PCR) were identified. Multiple representative cell clusters of the 2 components were obtained by microdissection, and the IgH CDR3 was amplified using a seminested PCR. Most of the PCR products obtained from both tumor components in each case had identical lengths when analyzed with polyacrylamide gel electrophoresis (PAGE) and identical migratory patterns on denaturing gradient gel electrophoresis (DGGE). These findings indicate sequence identity of the IgH CDR3 of both tumor components. Sequence analysis showed that point mutations were responsible for bands from the same case that had nonidentical migratory patterns by DGGE. The components in each of the 4 cases studied have the same clonal origin. Intraclonal sequence variations in the IgH gene were observed in 2 cases, consistent with the presence of continued somatic hypermutation after establishment of the clone. The expression of CD10 and bcl-2, as well as the detection of bcl-2 rearrangements in 2 cases, indicate that these lymphomas are of follicular center cell origin.     

霍奇金病单个H／R—S细胞TCR—γ基因重排的研究

目的 从单个细胞水平检测Ｈ／Ｒ－Ｓ细胞是否存在Ｔ细胞受体（ＴＣＲ）－γ链基因重排,以研究其是否来源于分化早期的Ｔ淋巴细胞。方法 用显微操作仪从冷冻的新鲜活检组织切片中挑取ＣＤ３０阳性的Ｈ／Ｒ－Ｓ细胞,提取其ＤＮＡ并行聚合酶链反应（ＰＣＲ）。ＴＣＲ－γ基因重排的ＰＣＲ采用半巢式扩增法,首轮用与ＴＣＲ－γ基因Ｖγ和Ｊγ区（包括Ｎ区）互补的引物对加入β－珠蛋白引物行混合扩增,对其中β－珠蛋白扩增阳性的Ｐ     

Detection of T-cell receptor-gamma gene rearrangement in lymphoproliferative disorders by temperature gradient gel electrophoresis

OBJECTIVE: Polymerase chain reaction amplification of DNA for T-cell receptor (TCR) gene rearrangement analysis is helpful in the evaluation of T-cell lymphoproliferative disorders. Detection of polymerase chain reaction products is limited by the poor resolution of bands analyzed by agarose or polyacrylamide gel electrophoresis. To improve the detection of a clonal T-cell population, we used temperature gradient gel electrophoresis (TGGE) as an alternative method for analysis of TCR gene rearrangement. DESIGN: One hundred eighteen archival DNA samples were randomly selected based on previous Southern blot analysis results. Samples included 58 T-cell neoplasms with positivity for TCR beta gene rearrangement, 22 cases of reactive hyperplasia with germline pattern for both TCR beta and J(H), and 38 patients with B-cell lymphoma. MOLT-16, a T-cell lymphoblastic cell line, was used for the sensitivity assay. Polymerase chain reaction was performed using GC-clamped multiplex primers to amplify the TCR gamma locus and was analyzed by TGGE. The range of temperature gradients was empirically determined for optimal resolution of bands. RESULTS: The sensitivity of TGGE was 0.1% when DNA from the MOLT-16 cell line was serially diluted with DNA from reactive lymphoid tissue. Fifty-four (93%) of 58 T-cell neoplasms with TCR beta gene rearrangements showed rearrangement patterns by TCR gamma TGGE, and only 1 of 60 samples (reactive or B-cell lymphomas) showed evidence of gene rearrangement by TGGE. Patients with T-cell neoplasm and involvement of multiple sites showed an identical migration pattern by TGGE analysis. CONCLUSION: We demonstrate that TGGE is an effective method for analysis of TCR gene rearrangement in the evaluation of nodal and extranodal lymphoid lesions.     

PCR analysis of immunoglobulin heavy chain (IgH) and TcR-gamma chain gene rearrangements in the diagnosis of lymphoproliferative disorders: results of a study of 525 cases.

This report summarizes a cumulative 4-year experience in polymerase chain reaction (PCR) analysis of immunoglobin heavy chain (IgH) and TcR-gamma chain gene rearrangements in 525 cases of lymphoproliferative disorders. Because the sensitivity of the PCR methodology was found to be tissue dependent, in the study of the presence of clonal cell population in tissues containing a small number of polyclonal lymphocytes, such as skin and gastrointestinal biopsy specimens, we used the multiple-PCR run approach. In this latter methodology, we repeat the PCR reaction from the same sample at least three times to confirm the reproducibility of the results. In the study of 273 cases of B- or T-cell lymphomas with characteristic immunomorphological and clinical features, a clonal IgH or TcR-gamma chain gene rearrangement was detected in approximately 80% of cases. A clonal rearrangement involving both IgH and TcR-gamma chain genes was found in 10% of cases of both B-cell and T-cell lymphomas. The study of 167 cases of nonneoplastic lymphoid tissue samples showed the presence of clonally rearranged cell populations for IgH or TcR-gamma genes in 3 and 9% of cases, respectively. We also applied PCR for the study of 85 cases of lymphoproliferations with no definite diagnosis (i.e., benign versus malignant) after immunomorphological analysis. In 65 cases (76%), the correlation of immunomorphological features with the presence (48 cases) or the absence (17 cases) of clonal lymphoid cell populations led to a definite diagnosis. In almost all these cases, the final diagnosis was found to be in agreement with the clinical course. In the 20 remaining cases (24%), no definite diagnosis could be made. We also assessed the value of PCR in detecting bcl-2/J(H) gene rearrangement as an additional clonal marker in the diagnosis of follicular lymphoma. Bcl-2/J(H) rearrangement and/or IgH gene rearrangement was found in approximately 85% (71/85) of follicular lymphoma cases studied.     

A novel four-color PCR assay to assess T-cell receptor gamma gene rearrangements in lymphoproliferative lesions.

We describe a novel 4-color polymerase chain reaction (PCR) assay combined with GeneScan analysis to assess for T-cell receptor gamma chain gene (TCRgamma) rearrangements and evaluate its usefulness in 86 lymphoproliferative lesions. In this assay, each variable region (Vgamma) family primer is 5' end-labeled with a different fluorescent dye, allowing determination of the Vgamma family involved in each TCRgamma rearrangement. PCR products were analyzed by capillary electrophoresis. We detected clonal TCRgamma rearrangements in 60 (98%) of 61 T-cell lymphomas, 2 (15%) of 13 B-cell lymphomas, and 3 (25%) of 12 reactive lesions. These results compared favorably with conventional PCR methods using denaturing gradient gel electrophoresis, which revealed clonal TCRgamma rearrangements in 37 (90%) of 41 T-cell lymphomas, 1 (25%) of 4 B-cell lymphomas, and 2 (25%) of 8 reactive lesions. This 4-color PCR assay is at least equivalent to conventional PCR methods and is convenient, allows accurate size determination of TCRgamma rearrangements, and identifies the specific Vgamma family involved, providing more specific information about TCRgamma rearrangement.     

Automated high-resolution polymerase chain reaction fragment analysis: a method for detecting T-cell receptor gamma-chain gene rearrangements in lymphoproliferative diseases.

Southern blot analysis and the polymerase chain reaction (PCR) are powerful tools for detecting clonal antigen receptor gene rearrangements. However, a number of limitations restrict the predictive value of the results obtained by these techniques as they are commonly used. We describe a new method, automated high-resolution PCR fragment analysis, that can partially overcome many of the limitations of analyzing the T-cell receptor (TCR) gamma-chain gene. Analysis of TCR-gamma is performed using PCR with four sets of primers, previously described by others, specific for all variable (V) and joining (J) regions of the TCR gamma-chain gene. In addition, the four V region primers are 5' end-labeled with a fluorescent compound, 5-carboxyfluorescein. After amplification, the labeled PCR products are separated with an automated sequencing system, ABI 373 (Applied Biosystems, Weiterstadt, Germany). With the help of the Gene-Scan software ABI 672 (Applied Biosystems) and fluorescent-labeled DNA length markers, the exact size of each peak can be displayed and analyzed. The resolution of this method allows separation of PCR products differing in length by as little as 1 bp. Semiquantitative estimation of specific clones also can be performed. Infiltrate-specific gene rearrangement patterns can be identified and recognized in different tissue specimens at the time of diagnosis or in subsequent biopsy specimens. We conclude that automated high-resolution PCR fragment analysis allows more accurate and convenient analysis of the TCR gamma-chain gene.     

Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions.

Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.     

High-resolution analysis of gene rearrangements in lymphoid malignancies.

As T-cell receptor and immunoglobulin gene rearrangements provide specific clonal markers for lymphoid cell proliferations, analysis of these genes is useful for distinguishing between reactive and malignant disease. We have developed an automated, high-resolution analysis of PCR fragments to identify clonally rearranged TCR-gamma (TCR gamma) genes and IgH genes. Consensus primers are used to detect the majority of possible rearrangements in multiplex PCR assays, and the PCR products are fluorescently labelled for visualisation with ABI Genescan software. Polyclonal populations of lymphoid cells are represented by a spectrum of fragments, whereas a monoclonal population of cells is represented by one or two discrete bands, indicating rearrangement of one or both alleles. For TCR-gamma PCR, the rearranged DNA fragment from a monoclonal population of T-cells diluted to 0.1% in DNA from a polyclonal population of cells is still readily distinguishable from the polyclonal background. Similarly, for IgH PCR, the gene rearrangement from a monoclonal population of B-cells is still distinguishable to 0.5% in a polyclonal background. As this technique allows semi-quantitative resolution of fragments one base different in size, it is ideal for detecting monoclonal and oligoclonal populations of B- and T-cells. The accurate size determination of PCR fragments also minimises the risk of false positives resulting from contamination, as individual monoclonal rearrangements are frequently patient-specific on the basis of size alone.     

Evaluation of IS6110 as amplification target for direct tuberculosis diagnosis

We describe in the present study an evaluation of the IS6110 repetitive element in the rapid diagnosis of pulmonary and extrapulmonary tuberculosis by polymerase chain reaction (PCR). A pair of oligonucleotide primers was designed to amplify a 201-bp DNA fragment of IS6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by Sal I digestion and Southern blot hybridization with a 32P-labeled probe. To detect the presence of amplification inhibitors, an internal control DNA that used the same primers as for the target sequence was added to each PCR reaction. PCR results were compared with the results of acid fast stained smears, cultures, and clinical data in 102 sputum and 41 extrapulmonary specimens. With the exception of four samples, M. tuberculosis was detected by PCR in all smear- and culture-positive cases and in all smear-negative, culture positive cases. Additionally, PCR was able to detect 6 cases that were smear and culture negative but clinically strongly suspected of tuberculosis. The final PCR sensitivity and specificity were 93.1% and 95.18%, respectively. One M. tuberculosis strain isolated from a sputum was found to lack IS6110. This study shows that (1) PCR diagnosis based on IS6110 reached the best sensitivity and specificity but must be considered carefully since some M. tuberculosis strains lack IS6110; and (2) PCR must be interpreted in conjunction with clinical and radiological data when it is discordant with conventional methods results.     

Profiling of bacterial flora in gastric biopsies from patients with Helicobacter pylori-associated gastritis and histologically normal control individuals by temperature gradient gel electrophoresis and 16S rDNA sequence analysis

The aim of this study was to establish bacterial profiles in gastric biopsy specimens from patients with Helicobacter pylori-associated gastritis by means of temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Specimens from eight patients with asymptomatic gastritis and five histologically normal controls revealed a Helicobacter-specific band in the TTGE profile with increased amounts of Helicobacter-specific DNA in the biopsies from most of the gastritis patients. DNA from other genera including Enterococcus, Pseudomonas, Streptococcus, Staphylococcus and Stomatococcus was also found in the stomach. In the absence of gastric inflammation, Helicobacter spp. appeared to be part of a complex, presumably indigenous microbial flora found in the biopsy specimens from the stomach.     

Monoclonal analysis in B-cell neoplasms by the semi-nested polymerase chain reaction using consensus primers

Monoclonality on B-cells is well known to be determined on the basis of presence of a rearranged-IgH gene, which is detected by Southern blot hybridization (SBH) remaining to be elucidated in respects of not only time-consumed, labour and cost benefit and also the use of much DNA samples. Alternative to this SBH, we examined the clinical usefulness of monoclonal analysis by the polymerase chain reaction technique which amplifies rearranged-CDR III region of IgH gene (IgH-PCR). The detective sensitivity of the IgH-PCR was different dependently upon each analysis for amplified products, namely 10(-2) per mononuclear cells in agarose gel analysis and 10(-3) in polyacrylamide gel and single strand conformation polymorphism analysis (PAGE and SSCP). Then, using the IgH-PCR and PAGE/SSCP analysis, 75 Japanese patients with B-neoplasm and 23 with T-cell neoplasms were examined for clonal IgH rearrangements. The diagnostic sensitivity in each group of B-ALL, B-CLL, B-lymphoma, HCL, AML with B-cell antigens, and non-T cell neoplasms was 88%, 92.3%, 71.4%, 100%, 57.1%, and 0%, respectively, with an overall sensitivity and specificity of 88% and 100%. This indicates that PCR analysis is very useful in detecting the clonal rearrangement of IgH genes on B-cell neoplasms, especially on ALL and CLL corresponding to neoplasms counterparting to naive B-cells.     

Molecular typing of the bacterial flora in sputum of cystic fibrosis patients

Despite recent advances in therapy, lower airway infections remain the major cause of morbidity and mortality in cystic fibrosis (CF) patients. Bacterial colonisation of the lower airways in CF is limited to a few bacterial species, commonly Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. Burkholderia cepacia colonisation is much rarer, but it has been thought to be associated with more advanced lung disease and increased mortality. A rapid characterisation of the bacterial flora in sputum of CF patients is of great importance for proper treatment. The aim of this study was to establish bacterial profiles and to identify pathogenic bacteria in respiratory specimens by means of molecular methods including temporal temperature gradient gel electrophoresis (TTGE) and DNA sequencing of PCR amplicons derived from 16S rDNA variable V3 and V6 regions. Sputa of 13 CF patients (7 males/6 females, age 19-59 years) collected at the Stockholm CF centre were analysed. TTGE revealed the presence of complex bacterial profiles in all samples. The V3 and V6 PCR amplicons were cloned and sequenced by real-time DNA Pyrosequencing. DNA from Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, respectively, was identified together with sequences from normal oral cavity flora. The results were in reasonable agreement with those obtained by conventional bacterial culture, considering that only known CF pathogens are included in routine reports. However, the methodology seems too elaborate to be introduced into daily routine     

A T-cell receptor gamma polymerase chain reaction assay using capillary electrophoresis for the diagnosis of cutaneous T-cell lymphomas.

Detection of clonal T-cell receptor gamma rearrangements by polymerase chain reaction (TCRgamma PCR) followed by high-resolution electrophoresis has now become a valuable tool in the diagnosis of cutaneous T-cell lymphoma (CTCL). The identification of clonal TCRgamma PCR products by fluorescent fragment analysis (FFA) on a capillary DNA sequencer is described here and compared with an established hetero-duplex temperature gradient gel electrophoresis (HD-TGGE). Of 55 CTCL derived lesional skin samples, clonality was obtained in 46 samples by FFA (83.6%) and in 45 samples by HD-TGGE (81.8%). Of 35 control skin specimens from various nonmalignant dermatoses, two samples (pityriasis lichenoides chronica) showed clonality by both methods, one sample (chronic dermatitis) only by FFA. The sensitivity of FFA was established using three clonal T-cell lines and peripheral blood mononuclear cells. The detection limit for clonal material was approximately 1% to 2.5% in mixtures of DNA and 1% to 3% in cell dilutions. For cell dilution series, we confirmed a linear correlation between the clonal/polyclonal peak-size ratios and the portion of clonal cells up to about 10%. Thus, the initial ratio between mono-and polyclonal template is correctly displayed by FFA within that concentration range. In conclusion, FFA on capillary DNA sequencer is a well-suited separation technique in TCRgamma PCR-based clonality analysis also exhibiting quantitative properties.