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.     

HFE genotyping using multiplex allele-specific polymerase chain reaction and capillary electrophoresis

CONTEXT: Hereditary hemochromatosis is recognized as one of the most common autosomal recessive disorders, with a prevalence of 1 in 200 to 400 in the white population. Early detection and treatment are completely effective in preventing pathology. It is anticipated that testing for hereditary hemochromatosis will increase, as will the need for a technology that can handle the demand. OBJECTIVE: To describe a high-throughput, single-tube, allele-specific multiplex polymerase chain reaction assay for identifying the 2 mutations in the HFE gene associated with hereditary hemochromatosis. DESIGN: Fluorescence-labeled polymerase chain reaction products from a multiplex polymerase chain reaction are analyzed by automated capillary electrophoresis. DATA ANALYSIS: The assay was validated by analysis of 25 blinded samples, and results were concordant with an established laboratory assay. CONCLUSION: The assay described offers a significant improvement over manual laboratory assays in throughput, reduced technologist time, and cost.     

Assessment of T-cell clonality via T-cell receptor-gamma rearrangements in cutaneous T-cell-dominant infiltrates using polymerase chain reaction and single-stranded DNA conformational polymorphism assay.

Discerning the pathologic significance of cutaneous T-cell infiltrates can pose a diagnostic challenge for dermatopathologists. Reactive conditions such as drug-associated lymphomatoid hypersensitivity and lymphomatoid lupus erythematosus can demonstrate lymphoid atypia and a phenotype resembling cutaneous T-cell lymphoma (CTCL). Further, lymphoid dyscrasias such as pityriasis lichenoides chronica, large plaque parapsoriasis, and atypical pigmentary purpura confuse the picture because they not only mimic CTCL but also represent prelymphomatous states with inherent malignant potential. Although the emergence of a dominant clone has been considered a clue indicative of a T-cell dyscrasia, there are reports concerning the identification of monoclonality in biopsies of reactive lymphoid infiltrates. We have conducted a modified single-stranded DNA conformational polymorphism (SSCP) assay using paraffin-embedded, formalin-fixed tissue on 92 T-cell-rich biopsies to determine the relative specificity and sensitivity of this methodology. In addition, laser capture microdissection (LCM) was performed on 22 of the 92 samples to isolate the area of interest and to compare its specificity and sensitivity with those SSCP assays performed without LCM. We found that monoclonality or oligoclonality is 86% specific for preneoplastic and neoplastic states, whereas the finding of polyclonality appears to be relatively specific for a reactive process. Some cases of reversible T-cell dyscrasia produced a molecular profile mimicking lymphoma or prelymphomatous states by virtue of monoclonality or oligoclonality. Although LCM appears to improve the sensitivity for detecting preneoplastic conditions, the relative specificity appears to be the same as that encountered with routine SSCP.     

The polymerase chain reaction for detection of T-cell antigen receptor expression.

Applications of the polymerase chain reaction have revolutionized the field of immunogenetics, particularly in studies of human leukocyte antigen class II polymorphism, and more recently in the analysis of T-cell receptor usage. However, the enormous diversity and variability of the T-cell receptor complex have made the amplification of the complete repertoire difficult. Several methods have been devised to address this problem. Each system is described with recent examples of its use and an assessment of its advantages and disadvantages. The use of quantitative polymerase chain reaction in T-cell receptor analysis is also discussed. The elucidation of the T-cell repertoire involved in a pathogenic process can have therapeutic implications, given the success of reversing experimental autoimmune disorders by directing specific forms of immunotherapy against V region gene products.     

A multidisciplinary approach to the diagnosis of cutaneous T-cell lymphomas.

The cutaneous T-cell lymphomas (CTCLs) comprise a spectrum of non-Hodgkin lymphomas with a predilection for the skin. This heterogeneous group of CTCLs include the prototypic CTCL mycosis fungoides (MF) and the recently described Ki-1+ lymphomas. MF is notoriously difficult to diagnose in its early stages. The histologic appearance of early MF is indistinguishable from that of chronic dermatitis. The limitations of light microscopy in the diagnosis of the CTCLs have led to the development of other diagnostic laboratory techniques. The best approach to the diagnosis of the CTCLs is a multidisciplinary one and should include ultrastructural morphometry, immunophenotyping, immunogenotyping, and histologic evaluation whenever possible. It is the purpose of this overview to point out the strengths and weaknesses of each of these techniques and, together with clinical input, to provide a comprehensive and rational approach to patient care.     

中线T细胞淋巴瘤的TCR—γ基因重排研究

目的对中线Ｔ细胞淋巴瘤的克隆性进行研究。方法用一组针对Ｔ细胞受体γ链基因Ｖ区片段的家族特异性引物和ＰＣＲ方法,对１１例（２２个标本）中线Ｔ细胞淋巴瘤病例进行了Ｔ细胞受体γ基因重排的检测。结果２２个标本中２１个有Ｔ细胞受体γ链基因的克隆性重排（９４４５％）。在９例原发灶的连续活检和１例原发灶及其转移灶的标本中均未发现增生的克隆家族的改变。结论中线Ｔ细胞淋巴瘤的病变组织中存在Ｔ细胞的单克隆性增生,为该肿瘤的Ｔ细胞起源提供了分子生物学的证据。在中线Ｔ细胞淋巴瘤的疾病过程中（包括转移灶）增生Ｔ细胞的家族未发生变化,支持肿瘤的单克隆起源学说     

毛细管电泳多重PCR诊断杜氏/贝氏进行性肌营养不良

目的用毛细管电泳技术结合多重PCR方法分析DMD基因缺失位点,以建立一种快速、准确、适用于临床的DMD的诊断技术.方法将对缺失热区18对引物分成两套,用毛细管电泳分析7个DMD/BMD家系中的7名患者的二步多重PCR产物.结果毛细管电泳能快速、准确分离18对引物多重PCR产物,判断出DMD基因缺失位点.结论毛细管电泳定量PCR方法能高效、准确、快速诊断缺失型DMD患者,具有很好临床应用价值.     

Adoptive immunotherapy for cutaneous T-cell lymphomas.

The pathology of cutaneous T-cell lymphomas is predominantly in the skin. Allogeneic bone-marrow transplantation results in graft-versus-host disease involving the skin in many cases. It has been suggested that it would be interesting to see the effect of allotransplantation, even though no series has been reported so far. Adoptive immunotherapy with donor lymphocyte infusions has been increasingly used to treat post-transplant relapse and even prevention. The present hypothesis suggests that infusion of human leucocyte antigen (HLA)-matched donor lymphocyte infusions would result in a graft-versus-leukemia/lymphoma (GVL) effect in these disorders. Therefore, clinical trials are suggested.     

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.     

Immunopathology of cutaneous T-cell lymphomas.

In this study the authors attempted to establish immunopathologic criteria for the distinction of various T-cell lymphomas affecting the skin. We studied skin specimens from 27 patients with mycosis fungoides (MF) (n = 12), the Sézary syndrome (SS) (n = 6), adult T-cell leukemia (ATL) (n = 4), and nonepidermotropic T-cell lymphoma of large cell (n = 4) and lymphoblastic (n = 1) types. Identification of tumor cells in mixed cell populations and detection of weak expression of surface antigens by tumor cells was facilitated by immunoelectron microscopy. The mature helper T-cell phenotype (T11+ T3+ T4+) was found in 14 of 18 cases of MF/SS. One case of MF had a cytotoxic/suppressor (T4- T8+ 3A1+) phenotype; one with frequent blastic cells showed only weak expression of T4 antigen; 2 cases of SS were T11-. Tumor cells infiltrating the skin expressed 3Al antigen in 44% and cellular activation antigens Ia and/or Tac in 78% of patients with MF/SS. No consistent phenotypic differences were found between ATL cells from ATLV (HTLV) antibody-positive patients and tumor cells of patients with MF/SS who lacked this antibody. In contrast, a group of nonepidermotropic T-cell lymphomas showed phenotypic differences from MF/SS and ATL in all but 1 case. These cases were distinguished by the frequent absence of T3, T4, and Leu 1 antigens in 3 large-cell lymphomas; frequent expression of Ki-1 antigen, a Hodgkin's disease-associated antigen, in 2 cases with RS-like cells; and an immature thymocyte phenotype in lymphoblastic lymphoma. These findings demonstrate that tumor-cell phenotypes can be useful in distinguishing different histologic types of cutaneous T-cell lymphoma.     

优化PCR体系结合毛细管电泳技术检测FMR1基因前突变与全突变

目的 通过优化PCR并结合毛细管电泳,建立高扩增效率、高分辨率的FMR1基因CGG重复序列异常扩增检测体系.方法 选择标准样本和经Southern印迹技术确定(CGG)n的正常、前突变、全突变男性和女性样本15例,进行PCR检测体系的优化.优化的PCR扩增产物经琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳和毛细管电泳等多种方法进行结果比较.结果 经优化的PCR体系可以检测出(CGG)n大于260个拷贝的全突变男性和(CGG)n达到183个拷贝的前突变女性.毛细管电泳能够清晰分辨出相差1个CGG的两个等位基因,结果具有良好的可重复性.结论 该PCR检测体系大幅度提高了普通PCR方法的扩增效率和分辨率,明显降低了对于Southern印迹技术的依赖,可以作为临床筛查FMR1基因突变的首选方法.     

Development of the polymerase chain reaction for diagnosis of chancroid.

The published nucleotide sequences of the 16S rRNA gene of Haemophilus ducreyi were used to develop primer sets and probes for the diagnosis of chancroid by polymerase chain reaction (PCR) DNA amplification. One set of broad specificity primers yielded a 303-bp PCR product from all bacteria tested. Two 16-base probes internal to this sequence were species specific for H. ducreyi when tested with 12 species of the families Pasteurellaceae and Enterobacteriaceae. The two probes in combination with the broad specificity primers were 100% sensitive with 51 strains of H. ducreyi isolated from six continents over a 15-year period. The direct detection of H. ducreyi from 100 clinical specimens by PCR showed a sensitivity of 83 to 98% and a specificity of 51 to 67%, depending on the number of amplification cycles.     

Multiplex polymerase chain reaction-based analysis of T-cell receptor gamma gene rearrangements for the determination of T-lymphocyte clonality

Determination of the frequency of mutations at hprt or other loci in human lymphocytes provides a useful biomarker for human exposure to mutagens. One problem, however, is distinguishing between unique mutants and sibling mutants arising as progeny of an earlier mutant cell. We have developed a multiplex polymerase chain reaction (PCR)-based method to analyze T-cell receptor (TCR) gamma gene rearrangements for determination of T-cell clonality in mutational spectrum analysis. PCR primers for different subgroups of the V gene segment of the TCR gamma gene were selected at different sites in the TCR gamma gene so that the size of PCR products could define which V subgroup was involved in rearranged TCR gamma genes; gamma genes involving different V and J subgroups could be determined directly by PCR. Mutant T-lymphocytes with rearranged TCR gamma genes containing the same V and J subgroups were analyzed using PCR-based denaturing polyacrylamide gel electrophoresis. All of the 161 hprt mutant clones analyzed contained rearranged TCR gamma genes. Rearrangements among all subgroups of the V and J gene segments of the TCR gamma gene could be detected. VgammaI and Jgamma1/2 subgroups were involved in 69 and 71% of rearranged TCR gamma genes, respectively. This PCR-based analysis of TCR gamma gene rearrangements provides a simple and comprehensive method for identifying the clonality of mutant T-lymphocytes in human hprt mutant lymphocyte assay and mutational spectrum analysis.     

A novel polymerase chain reaction assay to detect Mycoplasma genitalium.

AIMS: To design and validate a polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Mycoplasma genitalium. METHODS: Primers were designed that were complementary to the 16S rRNA gene sequence of M genitalium. After optimisation of the reaction conditions, the PCR was tested against nine M genitalium strains, a dilution series of M genitalium DNA, and a panel of common microorganisms. The PCR was also challenged in parallel with a published assay against 54 urine specimens from men with urethritis. RESULTS: The expected 341 bp product was produced on amplification of material from all M genitalium strains and from none of the other microorganisms tested. The lower limit of detection was 50 genome copies. The new assay detected M genitalium DNA in nine of 54 men with urethritis, in comparison with eight positive specimens detected with the alternative PCR. CONCLUSIONS: This novel PCR targeting the M genitalium 16S rRNA gene has been optimised and now provides a sensitive and specific alternative or addition to the available MgPa gene targeting assays.     

Molecular diagnosis of Beckwith-Wiedemann syndrome using quantitative methylation-sensitive polymerase chain reaction.

PURPOSE: Beckwith-Wiedemann Syndrome is caused by defects in imprinted gene expression at 11p15. Currently, quantitative Southern analysis using DNA methylation-sensitive restriction enzymes is used in molecular diagnosis of this syndrome. METHODS: We describe a rapid and highly quantitative test for assessing DNA methylation at 11p15 using sodium bisulfite treatment of genomic DNA coupled with quantitative TaqMan methylation-sensitive polymerase chain reaction. RESULTS: TaqMan MSP can assess DNA methylation at both differentially methylated region (DMR)1 and DMR2 at 11p15. In addition, by using TaqMan MSP we were able to determine the parent of origin of a duplication of 11p15 by quantification of both DMR1 and DMR2 DNA methylation. CONCLUSION: TaqMan MSP method is a robust and rapid method for detecting changes in DNA methylation that compares favorably to the current standard of Southern blot for DNA methylation analysis. Assessment of DMR1 and DMR2 provides the most comprehensive assay for methylation defects in Beckwith Wiedemann Syndrome, accounting for more than 70% of the cases. The advantages of TaqMan MSP are that it requires less DNA and that it is rapid, less labor-intensive, and amenable to high-throughput analysis. Moreover, this approach can be modified to assess DNA methylation changes anywhere in the genome.     

Determination of lineage and clonality in diffuse lymphomas using the polymerase chain reaction technique

Malignant lymphomas of the diffuse mixed or diffuse large cell subtype are immunologically heterogeneous and morphologic features do not allow prediction of lineage. Applications of immunophenotypic and gene rearrangement techniques has greatly improved our ability to determine clonality and lineage. Nevertheless, some diffuse lymphomas are composed of numerous reactive cells accompanying a small clonal population, thereby making determination of clonality difficult, even with gene rearrangement techniques. In this study, we report two malignant lymphomas in which immunophenotypic and genotypic studies failed to elucidate evidence of clonality. In both cases, the polymerase chain reaction amplified segments of DNA containing the bcl-2/JH sequence, providing evidence of clonality and suggesting B-cell lineage. We conclude that the polymerase chain reaction technique may be useful in the diagnosis of some diffuse mixed and large cell lymphomas.     

CSF pretreatment and the diagnosis of herpes encephalitis using the polymerase chain reaction.

A number of techniques for extraction of DNA prior to polymerase chain reaction (PCR) amplification of herpes simplex virus (HSV) DNA in cerebrospinal fluid (CSF) were compared to the use of "native" CSF in the PCR reaction. The results indicate that extraction of DNA (which allows efficient removal of inhibitors of Taq polymerase) is an essential pre-requisite of the PCR detection of CSF HSV DNA.