False-Positive Light Chain Clonal Restriction by Flow Cytometry in Patients Treated With Alemtuzumab

2018 ◽  
Vol 151 (2) ◽  
pp. 154-163 ◽  
Author(s):  
Peter P Chen ◽  
Christopher A Tormey ◽  
Stephanie C Eisenbarth ◽  
Richard Torres ◽  
Susan S Richardson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Light Chain ◽  
False Positive ◽  
Positive Light

scholarly journals Flow cytometry for fast screening and automated risk assessment in systemic light-chain amyloidosis

Leukemia ◽  
2018 ◽  
Vol 33 (5) ◽  
pp. 1256-1267 ◽  
Author(s):  
Noemi Puig ◽  
Bruno Paiva ◽  
Marta Lasa ◽  
Leire Burgos ◽  
Jose J. Perez ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Flow Cytometry ◽  
Light Chain ◽  
Light Chain Amyloidosis ◽  
Fast Screening

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.

P009 Profiling for patients with positive autologous or false positive allogenic flow cytometry cross match

2017 ◽  
Vol 78 ◽  
pp. 60
Author(s):  
Qingyong Xu ◽  
Steve Leckie ◽  
Lakshman Gunaratnam ◽  
Anthony Jevnikar ◽  
Patrick Luke
Keyword(s):  
Flow Cytometry ◽  
False Positive ◽  
Cross Match

scholarly journals High-Throughput Multiplex Flow Cytometry Screening for Botulinum Neurotoxin Type A Light Chain Protease Inhibitors

2010 ◽  
Vol 8 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Matthew J. Saunders ◽  
Steven W. Graves ◽  
Larry A. Sklar ◽  
Tudor I. Oprea ◽  
Bruce S. Edwards
Keyword(s):  
Flow Cytometry ◽  
Protease Inhibitors ◽  
High Throughput ◽  
Light Chain ◽  
Botulinum Neurotoxin ◽  
Type A ◽  
Botulinum Neurotoxin Type A

Defining Complete Response in Multiple Myeloma: Role of the Serum Free Light Chain Assay and Multiparameter Flow Cytometry.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1479-1479 ◽  
Author(s):  
Roger G. Owen ◽  
J. Anthony Child ◽  
Andy C. Rawstron ◽  
Sue Bell ◽  
Kim Cocks ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Light Chain ◽  
Plasma Cells ◽  
Complete Response ◽  
Free Light Chain ◽  
Multiparameter Flow Cytometry ◽  
High Dose ◽  
Reference Laboratory ◽  
Serum Free ◽  
Serum Free Light Chain

Abstract It is becoming increasingly clear that the use of immunofixation (IF) to define complete response (CR) in MM has its limitations. Paraprotein concentration is not a direct measure of tumour bulk and maximal responses may take many months to achieve which inevitably underestimate CR rates in therapeutic schedules that contain the sequential use of different agents. The purpose of this study was to prospectively assess the applicability and value of the serum free light chain (SFLC) assay and multiparameter flow cytometry (MFC) to assess CR in the intensive pathway of the MRC Myeloma IX Trial. In this trial patients are initially randomised to induction with CVAD or CTD and patients with stable disease or better proceed to high dose melphalan (HDM) with stem cell support. There is a second randomisation to maintenance thalidomide or no further therapy. SFLC as well as standard serum and urine paraprotein assessments were performed in a central reference laboratory at the following time points: presentation, end of induction, day 100 post HDM and 3 monthly until relapse. Similarly MFC in which neoplastic plasma cells are identified and differentiated from normal plasma cells on the basis of CD19 and CD56 expression was evaluated (again in a central laboratory) at presentation, end of induction and day 100 following HDM and annually until relapse. An initial analysis of 207/1114 randomised patients was performed and the results are detailed below - End of induction Day 100 post HDM IF negative 16.3% 49.4% SFLC normal 46.1% 78.6% MFC negative 10.2% 50.7% The SFLC assay was informative in 95% of patients and provided for a more rapid assessment of response than conventional methods. A normal SFLC assay at the end of induction appeared to predict for attainment of an IF-neg CR at day 100 (70% IF-neg CR if SFLC normal vs 30% when SFLC abnormal at the end of induction). It should however be noted that 58% of patients who failed to achieve an IF-neg CR at day 100 had a normal SFLC assay. MFC provides for a direct assessment of residual neoplastic plasma cells. The assay was informative in 96.7% of patients and has a reproducible sensitivity of 0.01%. The majority of patients (89.8%) had detectable disease at the end of induction with a median of 0.7% neoplastic plasma cells (range 0.01–14%). Further cytoreduction was provided by the HDM such that 49.3% had flow detectable disease at day 100 with a median of 0.26% neoplastic plasma cells (range 0.02–8%). 30% of patients with IF-neg CR had detectable disease while 21% of patients with a persistent paraprotein had no detectable disease in their marrow. The majority of the latter patients had IgG paraproteins and it is postulated that many of these pts will ultimately achieve an IF-neg CR. We would conclude that given the kinetics of paraprotein clearance in MM it may be more appropriate to define CR on the basis of a normal SFLC assay and the absence of minimal residual disease by MFC. In this way it should be possible to more accurately define the CR rate achieved by individual components of multi-agent sequential regimens.

Utility of Multiparameter Flow Cytometry Immunophenotypic Studies In Patients with Systemic Light Chain (AL) Amyloidosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4051-4051
Author(s):  
Bruno Paiva ◽  
María-Belén Vidriales ◽  
Jose J. Perez ◽  
Maria-Consuelo López-Berges ◽  
Ramón García-Sanz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Plasma Cell ◽  
Light Chain ◽  
Cell Cycle Analysis ◽  
Disease Assessment ◽  
Multiparameter Flow Cytometry ◽  
Al Amyloidosis

Abstract Abstract 4051 Multiparameter flow cytometry (MFC) immunophenotyping has shown to be of value for differential diagnosis and minimal residual disease assessment in multiple myeloma. However, the clinical value of MFC immunophenotyping in other plasma cell disorders (PCD) remains largely unexplored. Systemic light chain (AL) amyloidosis is a rare PCD characterized by the accumulation of monoclonal light chain fragments leading to end-organ damage and short survival. Bone marrow (BM) plasma cell (PC) infiltration in AL is usually low and thus the identification of clonal PC can be often difficult by immunohistochemistry and/or immunofluorescence. In the present study we focused on 34 BM samples sent to our institution with a suspected diagnosis of AL. MFC immunophenotypic studies were performed using the following 4-color combinations of MoAbs (FITC/PE/PerCP-Cy5.5/APC): CD38/CD56/CD19/CD45 (n=34); in addition cy-Kappa/cy-Lambda/CD19/CD38 staining was add to confirm the clonal or polyclonal nature of BMPC in equivocal cases. Ploidy and cell cycle analysis were additionally performed in a subset of cases (n=12/34). From the total 34 cases included in the present study, 28 had a confirmed diagnosis of AL. The remaining 6 cases were finally diagnosed with localized - amyloidoma - (n=2) and familial (n=1) forms of amyloidosis, multiple myeloma-associated amyloid (n=2) and congestive pericarditis (n=1). Interestingly, the presence of clonal PC was detected by MFC in 27 of the 28 (96%) patients with AL; in turn, clonal PC were undetectable in the BM of all cases with localized and familial forms of amyloidosis. The median overall level of PC (M-PC plus N-PC) seen in MFC immunophenotypic analyses of BM samples of the 28 patients with AL was 1.9% (range: 0.1% - 15%), with a significant positive correlation between PC enumerated by MFC and conventional morphology (r=0.5; p=.01). Within the BMPC compartment, the median proportion of clonal PC was of 94% (mean 81% ± 29%); in 6 cases all BMPC were clonal while in the remaining 22 patients residual normal PC persisted (median of normal PC/BMPC 13% ± 31%). The most common aberrant phenotypes were down-regulation of CD19 (92%) and CD45 (83%), followed by overexpression of CD56 (56%) and infra-expression of CD38 (42%). Aneuploidy was only found in 18% of cases, all of them hyperdiploid. Cell cycle analysis showed a median % of S-phase and G2-Mitosis PC of 0.7% and 3.5%, respectively. Concerning patients' outcome, cases with undetectable normal PC (6/28, 21%) had a significantly decreased overall survival (OS) compared to patients with persistent BM normal PC at diagnosis (22/28, 79%) with 3-year OS rates of 0% vs. 59%, respectively (p=.001). In summary, these preliminary data suggests that MFC immunophenotyping investigations may be clinically relevant in patients with suspected amyloidosis for i) differential diagnosis between AL and other forms of amyloidosis and, ii) prognostication of patients with AL according to the presence or absence of baseline persistent normal PC. Disclosures: No relevant conflicts of interest to declare.

All Clones Are Not Equal: Somatic Hypermutation Significantly Enhances The Utility Of Tumor Tagging Rearrangements At IG Light Chain Loci In B Cell Malignancy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1376-1376
Author(s):  
Christopher S Carlson ◽  
Bryan Howie ◽  
Alfred L. Garfall ◽  
Eline Luning Prak
Keyword(s):  
B Cells ◽  
B Cell ◽  
Minimal Residual Disease ◽  
Light Chain ◽  
False Positive ◽  
Residual Disease ◽  
B Cell Malignancies ◽  
Tumor Tracking ◽  
Equity Ownership ◽  
Minimal Residual

Abstract Select tumor tag sequences, informed by the probability of generating the same sequence in an independent recombination event, are likely to be useful in tracking mature B cell malignancies where the IGH locus has been deleted. We considered the use of IGK and IGL for this purpose, but found that 2-4% of these rearrangements were shared between different individuals. The relatively high frequencies of such public rearrangements at IGK and IGL prompted us to search for other forms of sequence diversity that could be used as private clonotypic tags. Somatic hypermutations (SHM) may serve such a function. Our analysis of IGK and IGL suggests that tumor tracking sequences for detecting minimal residual disease should be selected with care, and these loci may be best suited for lymphoid malignancies that are characterized by high levels of SHM. Tracking minimal residual disease for B cell malignancies is an established technology, traditionally using either flow cytometry or a custom quantitative PCR assay for each patient. Recent technical developments in the massively parallel sequencing of somatically rearranged IG loci allow for a standard assay to be applied to screen for residual tumor burden in all patients, by first identifying the clonal IG rearrangements tagging the tumor in an index sample taken from the patient during active disease, and then screening for these tumor tagging sequences in follow up samples. A crucial assumption in these tagging strategies is that the tumor tagging sequences are idiosyncratic to the tumor, and unlikely to be generated independently in a recurrent rearrangement. In order to screen for recurrent sequences between two healthy individuals, we generated IG heavy and light chain libraries from 100,000 antigen experienced B cells (CD19+CD27+) isolated from whole blood by FACS. 130 bp reads were collected, starting within the J segment and extending across the CDR3 into the V segment. Unique sequences were compared between individuals to assess the frequency of nucleotide identical, “public” rearrangements shared between individuals. Less than 0.01% of unique IGH sequences overlapped between individuals, so the risk of a false positive MRD result from recurrent recombination at IGH is minimal. However, 4.3% and 1.9% of unique sequences at IGK and IGL, respectively, were shared between individuals. The shared sequences had significantly higher average copy numbers than unshared sequences, accounting for 20% of total sequences at IGK and 12% of total sequences at IGL. These data suggest that B cells carrying public sequences undergo higher levels of clonal expansion, and/or they are recurrently produced. Public sequences carried by B cell malignancies are likely to be of limited utility as tumor-tagging sequences, as it may be impossible to distinguish between low-level residual disease and benign, recurrent rearrangements in the patient. Therefore we assessed if we could predict whether a given sequence in the memory repertoire would be public using solely information derived from that sequence. We used logistic regression to screen for variables to predict the likelihood of a given sequence to be public, and identified a number of expected variables as significant predictors, including the identity of the V and J segments, the length of the non-templated insertion at the junction, and the number of somatic hypermutations within the V or J segment. By far the most important of these factors was the number of SHM events in the clone; consequently, the most useful light chains for tumor tracking will be those with significant SHM. We continue to explore factors contributing to the public IG repertoire. Particularly at IGK, there is an unexpectedly narrow range of CDR3 lengths, and we are determining if this might be attributable to low diversity in the primary repertoire, or due to positive selection in favor of this length in the mature naïve or mature repertoires. In conclusion, a high frequency of public IG light chain sequences in the antigen- experienced peripheral B cell repertoire suggests that naïve application of light chain clones for tracking MRD can generate false positive results, but that careful selection of tumor tracking sequences with SHMs can minimize this risk. Disclosures: Carlson: Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Howie:Adaptive Biotechnologies: Employment, Equity Ownership.

Multiparameter Flow Cytometry (MFC) for Identification of the Waldenström's Clone in IgM MGUS and Waldenstrom's Macroglobulinemia (WM): New Criteria for Differential Diagnosis and Risk Stratification

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 936-936
Author(s):  
Bruno Paiva ◽  
Maria-Carmen Montes ◽  
Ramón García-Sanz ◽  
Jennifer Alonso ◽  
Natalia de las Heras ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Conflicts Of Interest ◽  
Phenotypic Characterization ◽  
International Prognostic Scoring System ◽  
Multiparameter Flow Cytometry ◽  
Risk Of Progression

Abstract Abstract 936 Demonstration of bone marrow (BM) infiltration by lymphoplasmacytic lymphoma is essential to the diagnosis of WM, and a trephine biopsy is considered mandatory for this assessment. Multiparameter flow cytometry (MFC) has demonstrated its clinical relevance in MGUS and myeloma; however, immunophenotypic studies on IgM monoclonal gammopathies are scanty, and focus only in patients with WM. Herein, MFC immunophenotyping was performed on BM samples from 244 patients, including 67 IgM MGUS, 77 smoldering, and 100 symptomatic WM newly diagnosed patients according to the Second International Workshop. A four color panel that systematically allowed the identification of B cells and plasma cells (PC), and their phenotypic characterization for a total of 24 antigens was used. We first analyzed the percentage of B cells and PC in BM and the percentage of light chain restricted cells in both compartments. Our results show a progressive increment of B cells from IgM MGUS to smoldering and symptomatic WM (medians of 2%, 9% and 12%; P<.001), as well of light chain restricted B cells (75%, 96% and 99%; P<.001). In contrast, no differences were found for the percentage of PC (median of 0.3%), but light chain restricted PC progressively increased from IgM MGUS to smoldering and symptomatic WM (70%, 85% and 97%; P<.001). Accordingly, only 1% of IgM MGUS patients showed >10% B cells, in contrast to 34% and 55% of smoldering and symptomatic WM (P<.001). Likewise, only 1% of IgM MGUS patients showed 100% light chain restricted B cells, in contrast to 19% and 40% of smoldering and symptomatic WM (P<.001); similar results being also found using a cutoff of 100% light chain restricted PC. Subsequently, we explored whether the percentages of BM and light chain restricted B cells and PC could predict time to progression (TTP) from smoldering into symptomatic WM, as well as overall survival (OS) in symptomatic WM. In smoldering WM, B cells (>10% vs ≤10%: median TTP of 47m vs 145m; P=.016) and light chain restricted B cells (100% vs <100%: 26m vs 145m; P<.001) but not PC, predicted risk of progression. On the multivariate analysis that included serum M-spike (±3g/dL), BM infiltration (±50% lymphoplasmacytic cells), BM B-cells and light chain restricted B cells (by MFC), only the later retained independent prognostic value (HR: 19.8, P=.001). Upon analyzing factors influencing survival in symptomatic WM patients, cases with >10% B cells showed a trend for inferior OS (P=.080), and significant differences emerged when comparing patients with 100% vs <100% light chain restricted B cells (median OS 44m vs 78m; P=.001). The later marker was independent (HR: 2.6; P=.004) of the International Prognostic Scoring System (HR: 2.2; P=.006). Focusing on the antigenic profiles of B cells and PC, we noted that within the B-cell compartment there was a progressive increment of CD22dim (69%, 92% and 88%; P<.001), CD25+ (61%, 88% and 90%; P<.001) and sIgM+ (88%, 95% and 97%; P=.002) B cells from IgM MGUS to smoldering and symptomatic WM. This underlies that the accumulating light chain restricted clonal B cells show a characteristic Waldenstrom's phenotype (CD22dim/CD25+/IgM+). Of note, a bimodal (from - to +) expression for the B cell memory marker CD27 was found in >50% of WM patients, which raises the possibility that the WM clone may arise, at least in some cases, before antigenic stimulation; subsequent maturation of the clone into PC would explain the typical presence of somatic hypermutations. On the other hand, B-cells from IgM MGUS and WM patients were negative in ≥90% of cases for CD5, CD10, CD11c and CD103, which can be useful to differentiate between WM and other B-NHL. Finally, the antigenic profile of PC in IgM MGUS and WM was similar to that of normal PC, and different from myeloma PC by consistently showing a CD27+ and CD56- phenotype, in addition to sIgM+ expression in ≥87% of all cases. Similarly to B-cells, we also noted that within the PC compartment there was a progressive increment of CD19+, CD45+ and sIgM+ CD20+ PC from IgM MGUS to smoldering and symptomatic WM. This underlies that this transition is asssociated with an accumulation of light chain restricted clonal PC displaying an immature/plasmablastic phenotype. In summary, our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document