To investigate the optimal reaction conditions of clonal rearrangements of immunoglobulin heavy chain (IgH) gene using polymerase chain reaction (PCR) with consensus primers and the feasibility of detecting this gene using SYBR green I real-time quantitative PCR with consensus primers, the systemic experiments were performed, which included annealing temperature, concentration of primer, the amount of Taq enzyme, concentration of dNTP and Mg(2+), number of PCR cycles. Detection of this gene on SYBR green I real-time quantitative PCR with consensus primers was carried out under the optimal reaction parameters obtained from previous study, and the sensitivity of IgH rearrangements gene was examined by using SYBR green I real-time quantitative PCR. The results indicated that the optimal annealing temperature was 60 degrees C, the optimal concentration of primers was 0.8 micromol/L, the satisfactory Taq enzyme amount was 0.5 U, the optimal concentration of dNTP was 100 micromol/L, the optimal concentration of Mg(2+) was 3.0 mmol/L, the suitable number of cycle was 40 cycles. Amplification of IgH rearrangement gene and detection of desired gene fluorescence signal on SYBR green I real-time PCR were performed. The sensitivity of IgH gene using this quantitative PCR was 10(4)/ml. It is concluded that the optimal reaction parameters for amplification of clonal IgH rearrangements gene by using PCR technique was determined, and stable and specific amplification of desired gene with consensus primers was performed. Basically, IgH rearrangement gene was successfully detected by SYBR green I real-time PCR.
Gene Rearrangement, B-Lymphocyte, Heavy Chain ; genetics ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Organic Chemicals ; chemistry ; Polymerase Chain Reaction ; methods ; Reproducibility of Results

<b>OBJECTIVESb>To explore a sensitive and specific method for detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma (DLBCL), and verify the credibility of the established method.

<b>METHODSb>bcl-2/IgH hemi-nested PCR primers were designed using the professional primer design software. Fifty-two samples of pathologically diagnosed DLBCL and 10 fresh tonsil tissues were amplified using hemi-nested touch down-PCR to detect bcl-2/IgH gene rearrangement. The PCR products were cloned and sequenced.

<b>RESULTSb>bcl-2/IgH gene rearrangement was detected in 6 of 52 DLBCL samples and 2 of 10 fresh tonsil tissues using one-way method. By using the hemi-nested PCR for the second round amplification, 5 of DLBCL were positive, but all of the fresh tonsil tissues were negative. The positive PCR products were sequenced and analyzed on the Internet, 3 of 8 cases obtained by one-way method were false positive, 5 positive cases amplified using hemi-nested PCR were all bcl-2/IgH gene rearrangement. PCR products of 3 false positive cases were homologous to BAC331191 and LLNLR-245D11 in human chromosome 19 and RP11-498P10 in chromosome 1.

<b>CONCLUSIONb>There are false positive results using common primers for detecting bcl-2/IgH gene rearrangement. The mechanism may be that highly homologous sequences to human genome exist in commonly used primers. The specificity of the diagnosis could be improved by hemi-nested PCR using the combination of primers we designed and the traditional ones.

Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Genes, bcl-2 ; genetics ; Humans ; Lymphoma, Large B-Cell, Diffuse ; genetics ; Polymerase Chain Reaction ; methods

<b>OBJECTIVEb>To evaluate the efficiency of the BIOMED-2 PCR assay and its implication in the diagnosis of mature B-cell non-Hodgkin's lymphomas.

<b>METHODSb>Clinical, morphological and immunohistochemical features of 72 cases of non-Hodgkin's lymphomas were studied, including 25 reactive lymphoid hyperplasia, 37 diffuse large B cell lymphomas (DLBCL) and 35 extranodal marginal zone lymphomas of mucosa associated lymphoid tissues (MALT lymphoma and in addition, 25 cases of reactive lymphoid hyperplasia were used as the controls). DNA was exacted from the paraffin embedded formalin fixed tissue blocks and the quality of DNA was assessed using the BIOMED-2 specimen control reaction. Adequate samples were then analyzed by BIOMED-2 for immunoglobulin heavy and kappa light chain rearrangements.

<b>RESULTSb>Adequate DNA was obtained in 83 of 97 samples, including 60 mature B cell lymphomas and 23 reactive lymphoid hyperplasia. Clonal B-cell gene rearrangements were detected in 57 of 60 (95%) lymphomas. In contrast, clonal Ig gene rearrangements were not detected in any of the 23 cases of reactive lymphoid hyperplasia.

<b>CONCLUSIONb>BIOMED-2 assay is highly sensitive and specific for the detection of clonal B cell gene rearrangement using routine paraffin embedded formalin fixed specimens.

Antigens, CD20 ; metabolism ; CD79 Antigens ; metabolism ; DNA, Neoplasm ; genetics ; Gene Rearrangement, B-Lymphocyte ; genetics ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; genetics ; Gene Rearrangement, B-Lymphocyte, Light Chain ; genetics ; Genes, Immunoglobulin ; Humans ; Immunophenotyping ; Lymphoma, B-Cell ; genetics ; immunology ; pathology ; Lymphoma, B-Cell, Marginal Zone ; genetics ; immunology ; pathology ; Lymphoma, Large B-Cell, Diffuse ; genetics ; immunology ; pathology ; Paraffin Embedding ; Pseudolymphoma ; genetics ; immunology ; pathology ; Sensitivity and Specificity

<b>OBJECTIVEb>To evaluate the significance of immunoglobulin heavy chain (IgH) gene rearrangement for B-cell lymphoma and T-cell receptor (TCR) gene rearrangement for T-cell lymphoma and NK/T-cell lymphoma in diagnosing and typing of primary non-Hodgkin's lymphoma (NHL) in nasal cavity.

<b>METHODSb>Semi-nested polymerase chain reaction ( PCR) with two pairs of primers was used to detect monoclonal IgH gene rearrangement in paraffin-embedded tissues from 11 patients with B-cell lymphoma, and one-stepped PCR with two pairs of primers was used to detect T-cell receptor gene rearrangement from 23 patients with NK/T-cell lymphoma and 20 patients with T-cell lymphoma. Ten patients with nasal polyp were detected with all the primers by PCR respectively.

<b>RESULTSb>Among the 54 patients with an evaluable PCR results, 10 of 11 (90. 9% ) B-cell lymphomas were positive for monoclonal IgH gene rearrangement, 17 of 20 (85. 0% ) T-cell lymphomas and 10 of 23 (43. 5% ) NK/T-cell lymphomas were positive for monoclonal TCR gene rearrangement. Ten patients with nasal polyp were negative for all detection.

<b>CONCLUSIONSb>Detecting gene rearrangement was an efficient method in auxiliary diagnosing and typing of primary NHL in nasal cavity; Using semi-nested PCR or one-stepped PCR with two pairs of primers can enhance the positive rate of gene rearrangement detection.

Adolescent ; Adult ; Aged ; Child ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Gene Rearrangement, T-Lymphocyte ; Humans ; Lymphoma, Non-Hodgkin ; diagnosis ; pathology ; Male ; Middle Aged ; Nasal Cavity ; Nose Neoplasms ; diagnosis ; pathology ; Young Adult

The study was purpose to evaluate the value of real time quantitative-PCR for monitoring IgH level in patients with B-cell malignancy after hematopoietic stem cell transplantation (HSCT). Quantification of IgH levels was performed on bone marrow mononuclear cells from 9 patients with B-cell malignancy before and after HSCT by PCR using the consensus JH TaqMan probe in combination with an allele-specific oligonucleotide (ASO) upstream primer. The IgH levels was normalized by control gene GAPDH. The results indicated that the reproducible sensitivity of RQ-PCR was 1 copy, the significant reduction of IgH copies was observed in bone marrow samples of 9 patients at one month post HSCT (6.67x10(3)/10(6) GAPDH vs 29/10(6) GAPDH, p<0.01). 3 out of 9 patients who achieved complete clinical and molecular cytogenetic remission (CCyR) contained persistently measurable low IgH level of 10(2)/10(6) GAPDH within 15 months and no detectable IgH at 18 months post HSCT. Whereas 5 out of 9 patients whose IgH copies were less than 10(2)/10(6) GAPDH within 3 months and less than 10(3)/10(6) GAPDH 3 months post HSCT achieved a sustained complete remission (CR). IgH copies in one patient were 4.5x10(3)/10(6) GAPDH at 3 months post HSCT, who relapsed at 4 months post HSCT. The median levels of tumor contamination in the stem cell harvests from 8 patients measured by RQ PCR were 3.68x10(2) (0-1720)/10(6) GAPDH. RQ PCR showed that PBPC harvests were less contaminated than BM harvests [75 (0-890)/10(6) GAPDH vs 1.1x10(3) (527-1720)/10(6) GAPDH, p<0.05]. 8 patients whose stem cell harvest were avaiable for RQ PCR were still in CR despite of the tumor contamination. The level of tumor contamination in stem cell harvest well correlated with IgH levels at diagnosis and one month after HSCT (r=0.810, r=0.708, p<0.05). It is concluded that RQ PCR can effectively monitor the IgH levels in patients with B-cell malignancy after auto-HSCT. 10(3)/10(6) GAPDH within 3 months post HSCT may be a cut-off level of IgH copies, which may be used to evaluate different prognoses of patients.
Adolescent ; Adult ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Hematopoietic Stem Cell Transplantation ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Male ; Neoplasm, Residual ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; therapy ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

<b>OBJECTIVEb>To analyze and optimize the gene rearrangement primers of different frame regions (FR) of immunoglobulin heavy chain (IgH) genes by bioinformatic methods and explore the application of these primers in the detection of paraffin-embedded lymphoma tissues.

<b>METHODSb>Three pairs of primers from IgH FR1, FR2 and FR3 regions (P1c, P2A and P31, respectively) were selected as the B cell gene rearrangement primers after comparison of the gene fragments in 44 IgH variable and 6 joining regions. Using one pair of T cell receptor (TCR) gamma primer as the T cell gene rearrangement primer, 101 histopathologically confirmed lymphoproliferative samples including 80 B cell lymphomas, 14 T cell lymphomas, and 7 reactive proliferative lymph nodes were examined by PCR for gene arrangement. The DNAs from DG75 and Jurkat cell lines were used as the positive controls for B and T cell lymphoma, respectively, with those from reactive proliferative lymph nodes as the negative control.

<b>RESULTSb>The positivity rates of IgH primers (P1c, P2A and P31) in the 80 B cell lymphomas were 37.5% (30/80), 52.5% (42/80) and 70.0% (56/80), respectively, and only one of the 14 T cell lymphoma cases was positive for the primers, suggesting significant differences in the detection rates of B cell lymphomas by the 3 primers. The detection rate was increased to 83.9% by combining the results by P31 and P2A primers. No positivity was found in the proliferative reaction tissues.

<b>CONCLUSIONb>Primers from IgH FR3 region genes are more sensitive than that from the FR1 and FR2 regions in the detection of gene rearrangement in paraffin-embedded lymphoma tissues. The detection rates can be increased by combining the results with the primers for IgH FR3 with that of FR2.

DNA Primers ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; genetics ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Lymphoma, B-Cell ; diagnosis ; genetics ; pathology ; Lymphoma, Non-Hodgkin ; diagnosis ; genetics ; pathology ; Lymphoma, T-Cell ; diagnosis ; genetics ; pathology ; Male ; Paraffin Embedding

<b>OBJECTIVEb>To investigate the overrepresentation of specific gene segments of immunoglobulin heavy chain variable region (IgVH) among unmutated and mutated chronic lymphocytic leukemia (CLL) patients and its prognostic implication.

<b>METHODSb>Multiplex PCR was used to identify the expression of IgVH segment and its mutation status in CLL.

<b>RESULTSb>Analyses were successfully performed in 80 of 85 samples. Marked skewed IgVH families were disclosed. The most commonly used VH was VH3 (40.0%), followed by VH4 (30.0%), VHI (13.8%), VH2 (10.0%) and VH5, VH7 (2.5%). Fifty-six patients (70.0%) had mutated VH, 24 (30.0%) unmutated VH. Nine cases (11.3%) were with 100% germline sequence. Fifteen cases (15/24, 62.5%) in VH4, 29 (29/32, 90.7%) in VH3, and 4 (4/11, 36.3%) in VH1 had mutated VH. The most frequently used IgVH gene was VH4-39 (13.8%), and VH4-34 (8.8%). J4 (36/66, 54.5%) and D3 (25/66, 37.8%) were the most frequently used in J and D genes. The progression-free survival (PFS) was 82 and 17 months (P = 0.000), and the overall survival (OS) was 90 and 41 months (P = 0.009), respectively, for mutated and unmutated cases. Recurrent CDR3 sequences were found in our patients and 2 patients with VH1-69 had CDR3 sequences highly similar to those reported in literature.

<b>CONCLUSIONb>There is difference in IgVH gene segment usage and mutational status in different area CLL patients. Recurrent CDR3 sequences were found in specific IgVH gene segments, which highlights the importance of immunoglobulin mediated stimulation in the development of CLL.

Adult ; Aged ; Aged, 80 and over ; DNA Mutational Analysis ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; immunology ; pathology ; Male ; Middle Aged ; Mutation

<b>OBJECTIVEb>To investigate the cell features of 6 Castleman's disease patients and evaluate their prognosis.

<b>METHODSb>The resected tumors were investigated by routine histopathology and immunohistochemistry. Reverse-transcript PCR (RT-PCR) and sequencing of RT-PCR products were used to assess the clonal characters of the main tumor cells.

<b>RESULTSb>Histologically, all of the 6 tumors were classified as the hyaline vascular type. B-cells dominated the follicular germinal centers, with T-cells dispersing inter-follicularly. The results of RT-PCR each obtained a single band of either 128 bp or 122 bp and sequencing showed that there was highly homogeneity within the same length sequences, accompanied by fewer different nucleotide acids.

<b>CONCLUSIONb>Monoclonal and/or oligoclonal B cells were identified in Castleman's disease. These B cells were originated from germinal center cells.

Adolescent ; Adult ; Antigens, CD20 ; analysis ; B-Lymphocytes ; metabolism ; pathology ; Castleman Disease ; genetics ; metabolism ; pathology ; Clone Cells ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; genetics ; Humans ; Immunohistochemistry ; Leukocyte Common Antigens ; analysis ; Male ; Prognosis ; Reverse Transcriptase Polymerase Chain Reaction

<b>OBJECTIVEb>To assess the practical value of BIOMED-2 primers in the diagnosis of ocular adnexal lymphoma by PCR.

<b>METHODSb>DNA was extracted from 63 formalin-fixed paraffin-embedded (FFPE) ocular adnexal lymphoma specimens. The DNA quality was evaluated by PCR-based amplification of housekeeping gene beta-actin. IgH(B) and IgK(B) primers of BIOMED-2 standardized clonality analysis system were used to evaluate the immunoglobin gene rearrangements. PCR products were analyzed using capillary electrophoresis and GeneScan software.

<b>RESULTSb>76.2% (48/63) of FFPE samples produced amplifiable DNA for detection of Ig gene rearrangements.Positive detection rates by BIOMED-2 IgH(B) and IgK(B) primers were 79.2% (38/48) and 68.8% (33/48), respectively, with a combined positive detection rate of 91.7% (44/48).

<b>CONCLUSIONSb>IgH(B) and IgK(B) primers of BIOMED-2 are suitable for the detection of clonal rearrangements of Ig gene using FFPE specimens of ocular adnexal lymphomas.

Actins ; genetics ; Adult ; Aged ; DNA Primers ; Eye Neoplasms ; diagnosis ; genetics ; pathology ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Gene Rearrangement, B-Lymphocyte, Light Chain ; Humans ; Lymphoma, B-Cell ; diagnosis ; genetics ; pathology ; Lymphoma, Follicular ; diagnosis ; genetics ; pathology ; Male ; Middle Aged ; Paraffin Embedding ; Pilot Projects ; Young Adult

Base Sequence ; DNA, Neoplasm ; genetics ; DNA-Binding Proteins ; metabolism ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Genes, Immunoglobulin Heavy Chain ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Interferon Regulatory Factors ; metabolism ; Lymphoma, Large B-Cell, Diffuse ; genetics ; metabolism ; Molecular Sequence Data ; Neprilysin ; metabolism ; Proto-Oncogene Proteins c-bcl-6