scholarly journals Comparison of Reverse Transcription Quantitative Real-Time PCR, Flow Cytometry, and Immunohistochemistry for Detection of Monoclonality in Lymphomas

ISRN Oncology ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-6
Author(s):  
Anders Ståhlberg ◽  
Pierre Åman ◽  
Linda Strömbom ◽  
Neven Zoric ◽  
Alfredo Diez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Real Time ◽  
B Lymphocytes ◽  
Light Chain ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Light Chains ◽  
Quantitative Real Time Pcr ◽  
Healthy Humans

In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription quantitative real-time PCR analysis has complementary values. In a considerable number of cases tumor cells produced both kappa and lambda light chain transcripts, but only one type of light chain peptide was produced.

scholarly journals Monoclonal anti-light chain idiotype as a tumor-specific probe for human neoplastic B lymphocytes

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 919-923 ◽  
Author(s):  
M Wrightham ◽  
AL Tutt ◽  
MJ Glennie ◽  
TJ Hamblin ◽  
GT Stevenson ◽  
...  
Keyword(s):  
B Cell ◽  
Tumor Cells ◽  
B Lymphocytes ◽  
Light Chain ◽  
Normal Serum ◽  
Lymphocytic Leukemia ◽  
Light Chains ◽  
Specific Probe ◽  
Tonsillar Cells ◽  
Binding Curves

Abstract Tumor cells from patients with B cell neoplasms often secrete small amounts of free monoclonal light chains that can be found in the urine. Such tumor-derived light chains of the lambda type from a patient with typical chronic lymphocytic leukemia have been used to raise mouse monoclonal antibodies (MoAbs). A hybridoma-secreting antibody that recognized the idiotypic lambda chain but not normal lambda chains by a preliminary screen but which also reacted with idiotypic IgM from the patient's tumor cells was selected. This MoAb in fact recognized 1 in 20 X 10(3) molecules of pooled normal lambda chains, thus establishing its specificity for a private idiotypic determinant. It failed to give a detectable reaction with normal IgM, normal serum, or a panel of IgM paraproteins. The antibody bound to the patient's neoplastic B cells but not to normal tonsillar cells. The site of binding of the antibody to idiotypic IgM is clearly separate from that of another MoAb specific for idiotypic determinants on heavy plus light chains, since the two showed additive binding curves. The determinant also appeared to be less available in dimeric lambda chains than in monomeric lambda chains or in idiotypic IgM. Antibodies to idiotypic determinants on light chains show some technical advantages and should be useful for monitoring and possibly treating B cell tumors, either alone or together with the more conventional anti-idiotypic antibodies that usually recognize the heavy and light chain combination.

scholarly journals Monoclonal anti-light chain idiotype as a tumor-specific probe for human neoplastic B lymphocytes

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 919-923
Author(s):  
M Wrightham ◽  
AL Tutt ◽  
MJ Glennie ◽  
TJ Hamblin ◽  
GT Stevenson ◽  
...  
Keyword(s):  
B Cell ◽  
Tumor Cells ◽  
B Lymphocytes ◽  
Light Chain ◽  
Normal Serum ◽  
Lymphocytic Leukemia ◽  
Light Chains ◽  
Specific Probe ◽  
Tonsillar Cells ◽  
Binding Curves

Tumor cells from patients with B cell neoplasms often secrete small amounts of free monoclonal light chains that can be found in the urine. Such tumor-derived light chains of the lambda type from a patient with typical chronic lymphocytic leukemia have been used to raise mouse monoclonal antibodies (MoAbs). A hybridoma-secreting antibody that recognized the idiotypic lambda chain but not normal lambda chains by a preliminary screen but which also reacted with idiotypic IgM from the patient's tumor cells was selected. This MoAb in fact recognized 1 in 20 X 10(3) molecules of pooled normal lambda chains, thus establishing its specificity for a private idiotypic determinant. It failed to give a detectable reaction with normal IgM, normal serum, or a panel of IgM paraproteins. The antibody bound to the patient's neoplastic B cells but not to normal tonsillar cells. The site of binding of the antibody to idiotypic IgM is clearly separate from that of another MoAb specific for idiotypic determinants on heavy plus light chains, since the two showed additive binding curves. The determinant also appeared to be less available in dimeric lambda chains than in monomeric lambda chains or in idiotypic IgM. Antibodies to idiotypic determinants on light chains show some technical advantages and should be useful for monitoring and possibly treating B cell tumors, either alone or together with the more conventional anti-idiotypic antibodies that usually recognize the heavy and light chain combination.

scholarly journals Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using Reverse Transcription Quantitative real-time PCR

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Ana Érika Inácio Gomes ◽  
Leonardo Prado Stuchi ◽  
Nathália Maria Gonçalves Siqueira ◽  
João Batista Henrique ◽  
Renato Vicentini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Quantitative Real Time Pcr ◽  
Expression Studies ◽  
Gene Expression Studies

scholarly journals Ultrasensitive RNA In Situ Hybridization for Kappa and Lambda Light Chain mRNA in Marginal Zone Lymphoma Demonstrates Comparable Performance to Flow Cytometry

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S109-S109
Author(s):  
Michael Franklin ◽  
Chelsey Deel ◽  
Mohammad Vasef
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
B Cell ◽  
Light Chain ◽  
Marginal Zone ◽  
Marginal Zone Lymphoma ◽  
Ffpe Tissue ◽  
Light Chains ◽  
Light Chain Restriction

Abstract Objectives Evaluation of light chain restriction is critical to establish clonality in B-cell lymphoproliferative disorders (LPDs). Immunohistochemistry (IHC) and in situ hybridization (ISH) are commonly used to assess light chain restriction in formalin-fixed, paraffin-embedded (FFPE) tissues. However, except for cases with plasma cell differentiation, these techniques often fail to identify immunoglobulin light chains. An ultrasensitive technique, RNAscope, has been recently introduced that can identify light chains in cases of B-cell LPDs. We analyzed the utility of this ultrasensitive method in detection of clonality and correlated with flow cytometry results when available. Methods A tissue microarray was constructed using 1.6-mm diameter tissue punches of 31 FFPE tissue blocks from 27 cases that were previously characterized as marginal zone lymphoma (MZL) by a combination of morphology, IHC, and/or flow cytometry. Cases included 8 nodal and 19 extranodal MZLs. In two cases, additional blocks were included to assess reproducibility. For ultrasensitive ISH RNAscope assay, 4-µm thickness tissue sections were hybridized using kappa and lambda probes, incubated overnight, counterstained with hematoxylin, cover-slipped, and reviewed blindly without knowledge of prior flow cytometry results. Results Of 18 cases with evaluable staining, 15 were clonal and 3 were polytypic. Flow cytometry was available in 14 of these 18 cases with concordance in 13 of 14 (93%). The discordant case was polytypic by flow cytometry but kappa restricted by RNAscope. The false-negative flow results could be due to sampling issues. In six cases, staining failed and could not be evaluated. Conclusion Ultrasensitive RNAscope is a reliable assay in the detection of clonality in FFPE tissue, particularly where fresh tissue is not available for flow cytometry. In addition, our results confirm and further expand prior observations that RNAscope is a highly sensitive and specific assay with high concordance with flow cytometry.

scholarly journals A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)

3 Biotech ◽  
2020 ◽  
Vol 10 (12) ◽  
Author(s):  
Siti Suriawati Badai ◽  
Omar Abd Rasid ◽  
Ghulam Kadir Ahmad Parveez ◽  
Mat Yunus Abdul Masani
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Oil Palm ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr

Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests

2006 ◽  
Vol 53 (8) ◽  
pp. 195-202 ◽  
Author(s):  
G. Garcés ◽  
M. Effenberger ◽  
M. Najdrowski ◽  
C. Wackwitz ◽  
A. Gronauer ◽  
...  
Keyword(s):  
Anaerobic Digestion ◽  
Real Time ◽  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Combined Treatment ◽  
Quantitative Real Time Pcr ◽  
Anaerobic Digesters ◽  
Hsp70 Mrna ◽  
Cryptosporidium Parvum Oocysts

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic–thermophilic–mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of ≥2 and ≥5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

Phenotyping Studies of Clonotypic B Lymphocytes From Patients With Multiple Myeloma by Flow Cytometry

2009 ◽  
Vol 133 (10) ◽  
pp. 1594-1599 ◽  
Author(s):  
E. Joseph Conway ◽  
Jianguo Wen ◽  
Yongdong Feng ◽  
Albert Mo ◽  
Wan-Ting Huang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
B Lymphocytes ◽  
Light Chain ◽  
Peripheral Blood ◽  
Remission Rate ◽  
Light Chains ◽  
Immunoglobulin Light Chain ◽  
Peripheral Blood Stem Cells ◽  
Myeloma Cells

Abstract Context.—Clonotypic B lymphocytes, monoclonal B lymphocytes sharing identical, rearranged IGH-CDR3 sequences with the patient's myeloma cells, have been detected in the peripheral blood of patients with multiple myeloma. These cells have been postulated to act as a therapy-resistant tumor reservoir that drives recurrence. Objective.—To characterize clonotypic B lymphocytes for future investigation of their role in myeloma pathogenesis. Design.—Harvests of cryopreserved peripheral blood stem-cells from 20 myeloma patients were enriched for clonotypic B lymphocytes. Cytoplasmic immunoglobulin light chain and surface immunophenotype were analyzed by flow cytometry. IGH-CDR3 gene-rearrangement pattern was performed to determine clonality. Posttransplant remission rate was compared with the percentage of clonotypic B lymphocytes. Results.—Clonotypic B lymphocytes expressing CD34±, CD38+, CD184+, CD31±, CD50±, CD138−, CD19−, CD20−, and the same immunoglobulin light chain as the patients' known myeloma cells were identified in 12 of 20 patients (60%). Progenitor B lymphocytes expressing similar surface immunophenotype but opposite light chains were identified in the same patients. Polymerase chain reaction for IGH rearrangement showed clonal rearrangement pattern in clonotypic lymphocytes but not in B lymphocytes expressing light chains opposite to myeloma cells. There was no statistically significant correlation between the percentage of clonotypic B lymphocytes and response to autologous transplant. Conclusions.—Clonotypic B lymphocytes expressing CD34, but not CD19, were identified in stem cell harvests from patients with myeloma and could represent progenitor cells of neoplastic plasma cells. However, the same or similar immunophenotyping can be detected in both clonotypic B lymphocytes and benign progenitor B cells, suggesting clonality analysis might be needed to determine clonotypic B lymphocytes in patients with myeloma. Further studies are warranted to study the role of clonotypic B lymphocytes in the pathogenesis of myeloma.

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