scholarly journals Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry

2017 ◽  
Vol 31 (3) ◽  
pp. 385-394 ◽  
Author(s):  
Ling Guo ◽  
Zhen Wang ◽  
Courtney M Anderson ◽  
Emerald Doolittle ◽  
Siobhan Kernag ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
B Cell ◽  
Light Chain ◽  
Lambda Light Chain ◽  
Rna In Situ Hybridization ◽  
Cell Clonality

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.

Detection of t(14;18)(q32;q21) in B-Cell Chronic Lymphocytic Leukemia

2005 ◽  
Vol 129 (3) ◽  
pp. 410-411
Author(s):  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Susanne Schnittger ◽  
Claudia Schoch
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Chronic Lymphocytic Leukemia ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Lymphoma Therapy ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Cytomorphologic testing and multiparameter flow cytometry are the mainstays in diagnosing B-cell chronic lymphocytic leukemia, whereas fluorescence in situ hybridization that targets the translocation t(14;18)(q32;q21) often is used to identify follicular lymphoma. Therapy is highly diverse between both diseases. We describe a case with cytomorphologically and immunologically proven B-cell chronic lymphocytic leukemia in which t(14;18)(q32;q21) was found.

scholarly journals Branched-chain in situ hybridization for κ and λ light chains: A powerful ancillary technique for determining B-cell clonality in cytology samples

2015 ◽  
Vol 124 (3) ◽  
pp. 203-212 ◽  
Author(s):  
Kshitij Arora ◽  
Ivan Chebib ◽  
Lawrence Zukerberg ◽  
Manoj Gandhi ◽  
Miguel Rivera ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
B Cell ◽  
Light Chains ◽  
Branched Chain ◽  
Cell Clonality

Gene Expression Profiling, Frozen and Paraffin Section Immunohistochemistry, and In Situ Hybridization for Determination of Monoclonality in Diffuse Large B-Cell Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4553-4553
Author(s):  
Matthew W. Andres ◽  
Robin A. Roberts ◽  
Debbie J. Mustacich ◽  
Randy D. Gascoyne ◽  
Deborah A. Fuchs ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Light Chain ◽  
Expression Profiling ◽  
Paraffin Section ◽  
B Cell Lymphoma ◽  
Light Chain Restriction

Abstract Background: Determining monotypia of surface immunoglobulin (SIg) is often an important step in the diagnosis of B-cell lymphomas. Many competing methods have been developed and are commonly used in clinical practice. The role of gene expression profiling (GEP) in determining light chain restriction is yet to be clarified. The goal of this study was to compare 4 methods by which light chain restriction can be determined: frozen section immunohistochemistry (FS-IHC), paraffin section IHC (PS-IHC), in-situ hybridization (ISH), and GEP. Design: 40 cases of DLBCL, part of a previous GEP study of DLBCL (Rosenwald et al, NEJM 2002), were made into a tissue microarray (TMA). FS-IHC slides, previously stained using by-hand streptavidin and diaminobenzidine, were re-examined for κ:λ restriction. PS-IHC was done on the TMA using the Benchmark system according to manufacturer’s protocols for κ/λ staining (Ventana Medical Systems VMSI, Tucson, AZ). ISH was performed on the TMA with a newly developed sensitive ISH procedure from VMSI which uses a purified streptavidin reagent to reduce background and increase sensitivity. The FS-IHC, PS-IHC, and ISH arrays were reviewed and scored as κ/λ monotypic, SIg-negative or indeterminate by 3 pathologists. The averaged gene expression ratios for microarray elements probing for κ and λ on each case were plotted on a log2 scale. Monoclonality was determined using 2 different GEP criteria: (1) κ-monoclonal if κ:λ expression was >2log2 or λ-monoclonal if expression was λ>κ or (2) κ and λ light chain relative expression on either side of the median. These data from all 4 techniques were used to determine a consensus clonality in which the majority of the results agreed. The results for each technique were then compared to the consensus for that case. Results: 7 Cases had a 4/4 consensus, 9 cases a 3/4, 11 cases a 3/3, 7 cases a 2/3, and 1 case a 2/2. 5 cases were excluded from the study because there was no majority consensus. 19 cases (47.5%) were κ monoclonal, 10 (25%) λ monoclonal, 6 (15%) SIg-negative, and 5 cases (12.5%) indeterminate. Compared to the consensus clonality FS-IHC was accurate in 21/26 (81%), PS-IHC in 28/32 (87%), sensitive ISH in 29/29 (100%) cases. GEP was accurate in 31/35 (89%) cases using either criteria (1) or (2); in 27/35 (77%) using criterion (1) alone, in 28/35 (80%) using criterion (2) alone, and 24/35 (69%) of cases when both criteria (1) and (2) were met. 6 SIg-negative cases failed all GEP criterion, but were considered correctly classified as neitherκ nor λ monoclonal. Discussion: The technique with the highest accuracy compared to consensus was the sensitive ISH assay, in part because samples were eliminated if the mRNA control (polyT probe) staining was suboptimal. GEP was also accurate using either of our criteria. The SIg-negative cases were not classified as either κ or λ monoclonal using any of our criteria. Given the increasing role GEP is likely to play in hematopathology, the application of GEP to B cell clonality may have diganostic utility.

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