A Single-Tube Seven-Colour Flow Cytometry Assay for Detection of Minimal Residual Disease In Myeloma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1688-1688
Author(s):  
Soraya Wuilleme ◽  
Nelly Robillard ◽  
Steven Richebourg ◽  
Marion Eveillard ◽  
Laurence Lodé ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Flow Cytometry Assay ◽  
Single Tube ◽  
Cell Identification ◽  
Colour Flow ◽  
Multiparameter Analysis ◽  
Minimal Residual

Abstract Abstract 1688 The eradication of minimal residual disease (MRD) in myeloma predicts for improved outcome. A number of different approaches to myeloma MRD detection are available; these vary widely in sensitivity and cost. Flow cytometric assessment of MRD may be preferable in practice because of lower cost and easier feasibility. Myeloma MRD flow cytometry requires at least three markers for plasma cell identification (CD38, CD138 and CD45) and combination of several additional markers to detect phenotypic abnormality including CD19, CD20, CD27, CD28, CD45, CD56 and CD117. Also, assessment of immunoglobulin light-chain restriction (cytoplasmic K and L) combined with myeloma-associated phenotypic plasma cell abnormalities, is very important. Four-tube four-colour flow cytometry combine markers CD38/CD138/CD45 with markers for plasma cell phenotypic abnormalities and clonality. Six –colour flow cytometry combines the same markers (markers for plasma cell identification) plus clonality markers; it potentially increases the sensitivity of the method through coincident multiparameter analysis. However, the single-tube six-colour flow cytometry, proposed by others studies, excludes the myeloma-associated phenotypic plasma cell abnormalities and consequently decreases specificity of the assay. We propose a new single-tube seven-colour flow cytometry, including plasma cell identification antigens, clonality markers and myeloma-associated phenotypic plasma cell abnormalities markers. In this new method, PCs are stained with antibodies: (i) CD38, CD138, CD45 used for identified plasma cells and percentage plasma cells to total leucocytes. (ii) CD19 and CD56+CD28 used to identify normal and abnormal plasma cells; and (iii) cy-IgK and cy-IgL, for confirm the plasma cells clonality. We analysed normal bone marrow provided from healthy individuals. Our results showed a presence myeloma-associated phenotypic plasma cell abnormalities at low levels in healthy individual. The monotypy studies confirm polyclonality of this normal plasma cells. Then we compared MRD assessement with single-six colour flow cytometry assay (plasma cells markers, clonality markers and exluding myeloma-associated phenotypic markers) and seven-colour flow cytometry assay (including myeloma-associated phenotypic markers). Six –colour flow cytometry has a better sensitivity and showed efficacy for quantification MRD in myeloma patients. However, the single-tube six-colour flow cytometry excluded the myeloma-associated phenotypic plasma cell abnormalities and in some cases the seven-colour flow cytometry will be more informative because it detected myeloma-asociated phenotypic marquers combined with clonality marquers. Finally, the single-tube seven colour flow cytometry assay provides reduction in antibody cost and increases sensitivity and specificity of the method through coincident multiparameter analysis. Disclosures: No relevant conflicts of interest to declare.

Flow Cytometric Evaluation Of Minimal Residual Disease In Adult Acute Lymphoblastic Leukemia Using a Simplified, Single-Tube Approach

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1378-1378
Author(s):  
Roger Belizaire ◽  
Olga Pozdnyakova ◽  
Daniel J. DeAngelo ◽  
Betty Li ◽  
Karry Charest ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Turnaround Time ◽  
Tube Flow ◽  
Flow Cytometry Assay ◽  
Single Tube ◽  
Negative Results ◽  
Minimal Residual

Abstract Flow cytometry for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) has been widely used in pediatric patients to quantify therapeutic response and to assess the risk of relapse. Flow cytometry for MRD provides roughly the same level of sensitivity (0.01%) as molecular methods but at lower cost and with faster turnaround time. MRD assessment in ALL currently requires an evaluation of 20 or more parameters divided among multiple tubes. In part due to the assessment complexity, the use of flow cytometry for MRD detection in adult ALL patients has been relatively limited. We developed a 6-color, single-tube, flow cytometry assay to detect MRD in bone marrow (BM) aspirate specimens from adult ALL patients. The 73 patients included 52 patients with B-ALL (71%), 19 patients with T-ALL (26%) and 2 patients with T/myeloid leukemia (3%) and were treated with one of several standard chemotherapeutic regimens or targeted therapies. Patients were tested for MRD by flow cytometry after induction or re-induction therapy and serially thereafter. The 6-marker MRD panel was customized for each patient based on the 18-20-marker diagnostic immunophenotype. Sixty-three percent of B-ALL patients (n=33) had lymphoblasts with an aberrant immunophenotype; expression of a myeloid marker (e.g., CD13, CD15 or CD33) was the most common aberrancy. The remaining 37% of B-ALL patients (n=19) had disease with a hematogone immunophenotype, which comprised surface expression of CD10, CD19, CD20, CD34, CD38 and CD45; in the majority of these cases, leukemic cells were distinguishable from normal hematogones based on the intensity of surface marker expression. Forty-seven percent of T-ALL patients (n=9) had an aberrant immunophenotype, most often characterized by CD33 expression. One-hundred forty-six consecutive specimens analyzed for MRD by flow cytometry were classified as positive (23%), negative (72%) or uncertain (5%). Of the 34 samples classified as positive, 14 (41%) showed morphologic (i.e., BM aspirate or biopsy) evidence of disease; nineteen (65%) samples did not show morphologic evidence of disease and 1 sample did not have a concurrent morphologic assessment. Of the 105 samples classified as negative by flow cytometry, 103 (98%) were also negative by morphology and 1 sample did not have a concurrent morphologic assessment. One sample that was negative by flow cytometry had morphologic evidence of disease in the biopsy (10-20% blasts) but not the aspirate, suggesting that aspirate sampling artifact was responsible for the discrepancy. None of the 7 samples classified as uncertain by flow cytometry had morphologic evidence of disease; five out of 7 uncertain classifications were in B-ALL patients with hematogone immunophenotypes. Overall, MRD flow cytometry showed 86% concordance with the results of morphologic assessment. We evaluated outcomes in all patients with negative morphologic results and any positive MRD flow cytometry result(s). Of the 73 patients in this study, 61 had morphology-negative results that were either MRD-negative (n=45) or MRD-positive (n=16). Patients in this group were at various points of treatment post-induction or re-induction. Four out of 45 patients (9%) with MRD-negative results relapsed during a median follow-up period of 22 months, and 8 out of 16 patients (50%) with an MRD-positive result relapsed during a median follow-up period of 15 months (odds ratio for relapse 10.3, 95% confidence interval 2.5-42.4, P=0.001). In addition, relapse-related and overall mortality (Figure 1) were higher in patients with MRD-positive results (P=0.0023 and P=0.0016, respectively, by the log-rank test). In summary, we present a simplified, single-tube, flow cytometry assay that can be used to detect MRD in adult ALL at relatively low cost with rapid turnaround time; our approach was applicable to cases with either hematogone or aberrant immunophenotype, yielding a definitive result in 95% of cases. Notably, the presence of MRD was associated with relapse and mortality, suggesting that our method of MRD assessment could be used to guide treatment of adult ALL. Further analysis of the correlations between MRD results, clinical management and patient outcomes is ongoing. Disclosures: No relevant conflicts of interest to declare.

Evaluation of a 5-Color Flow Cytometric Technique for Immunophenotyping and Quantitation of Plasma Cell Myeloma in Post-Therapy Bone Marrows.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5036-5036
Author(s):  
Tove Isaacson ◽  
Andrzej Jakubowiak ◽  
Lloyd Stoolman ◽  
Usha Kota ◽  
William Finn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Plasma Cell Myeloma ◽  
Color Flow ◽  
Flow Cytometric ◽  
Minimal Residual

Abstract Multiparameter flow cytometry is a useful tool for comprehensive immunophenotyping of plasma cell myeloma, and has been proposed as a sensitive method for the evaluation of minimal residual disease in patients following treatment. This study aimed to assess the value of flow cytometry in quantitation of residual disease, in comparison to routine morphologic examination of first-pull bone marrow aspirate smears, in myeloma patients post-therapy. Heparinized bone marrow aspirates were obtained from 27 treated patients with plasma cell myeloma. Cells were prepared for 5-color flow cytometric analysis within 24-hours of specimen draw. Surface membrane staining with anti-CD19, CD20, CD38, CD45, CD56, and CD138 was followed by ammonium chloride lysis of red cells. Fixed and permeabilized cells were analyzed for cytoplasmic light chains to confirm clonality. Data were acquired using an FC500 flow cytometer (Beckman-Coulter), analyzed with CXP software with plasma cells isolated based on bright CD38+ or CD138+ expression. A median of 97,639 cellular events (range 14,279 to 262,508) were collected per analysis. Flow cytometric enumeration of plasma cells was compared to 500-cell differential counts of Wright-Giemsa-stained first-pull aspirate smears from the same cases. The median plasma cell count as determined by flow cytometry was 0.5% (range 0–7.9%). The median plasma cell count estimated by morphologic review was 8.0% (range 0–84.4%). Flow cytometry underestimated the plasma cell content in all but one case. Clonal plasma cells expressed CD38 and CD138 in all cases; 87.5% (21/24) coexpressed CD56, 25% (6/24) coexpressed CD45, and 4.2% (1/24) coexpressed CD19. None was positive for CD20. Although detection of minimal residual disease after therapy for acute leukemia is routinely achieved by flow cytometric analysis, successful quantitation of minimal residual disease in treated myeloma patients using flow cytometry remains limited as it usually underestimates the plasma cell content of bone marrow samples compared to routine morphology of first-pull aspirates. We have observed that this holds true for both pre-treatment and post-treatment specimens. Causes for the discrepancy may include hemodilution of second-pull aspirates used for flow cytometry, fragility and loss of plasma cells during preparation for flow cytometry, and incomplete disaggregation of plasma cells from bone marrow spicules. With improved outcome of treatments, better and more reliable methods of detection of minimal residual disease are needed for optimal prognostic stratification. We are currently validating alternative methods, which may offer more sensitivity while at the same time allow more objectivity, for assessing the amount of minimal residual disease in myeloma patients.

scholarly journals Comparison of minimal residual disease detection in multiple myeloma between the DuraClone and EuroFlow methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takeshi Yoroidaka ◽  
Kentaro Narita ◽  
Hiroyuki Takamatsu ◽  
Momoko Fujisawa ◽  
Shinji Nakao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cells ◽  
Residual Disease ◽  
Total Cell ◽  
Multiparameter Flow Cytometry ◽  
Single Tube ◽  
The Cost ◽  
Minimal Residual

AbstractIn this study, the minimal residual disease (MRD) levels in patients with multiple myeloma (MM) were assessed by comparing the new 8-color single-tube multiparameter flow cytometry method (DuraClone), which reduces the cost of antibodies and labor burden of laboratories, with the EuroFlow next-generation flow (NGF) method. A total of 96 samples derived from 69 patients with MM were assessed to determine the total cell acquisition number (tCAN), percentages of total and normal plasma cells (PCs), and MRD levels using two methods. We found that the tCAN was significantly higher with EuroFlow-NGF than with DuraClone (median 8.6 × 106 vs. 5.7 × 106; p < 0.0001). In addition, a significant correlation in the MRD levels between the two methods was noted (r = 0.92, p < 0.0001). However, in the qualitative analysis, 5.2% (5/96) of the samples showed discrepancies in the MRD levels. In conclusion, the DuraClone is a good option to evaluate MRD in multiple myeloma but it should be used with caution.

scholarly journals A single-tube six-colour flow cytometry screening assay for the detection of minimal residual disease in myeloma

Leukemia ◽  
2007 ◽  
Vol 21 (9) ◽  
pp. 2046-2049 ◽  
Author(s):  
R M de Tute ◽  
A S Jack ◽  
J A Child ◽  
G J Morgan ◽  
R G Owen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Screening Assay ◽  
Single Tube ◽  
Colour Flow ◽  
Minimal Residual

Improved Concordance of Minimal Residual Disease Measurements By Quantitative PCR and 10-Color Flow Cytometry in Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2614-2614
Author(s):  
Mary Sartor ◽  
Draga Barbaric ◽  
Tamara Law ◽  
DR Anuruddhika Dissanayake ◽  
Nicola C Venn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Residual Disease ◽  
Rank Correlation ◽  
Color Flow ◽  
Single Tube ◽  
Time Points ◽  
Colour Flow ◽  
Risk Patients ◽  
Minimal Residual

Abstract Introduction: Detection of minimal residual disease (MRD) after induction and consolidation therapy is highly predictive of outcome for childhood acute lymphoblastic leukaemia (ALL) and is used to identify high risk patients in most current ALL clinical trials. Two methods broadly applicable for MRD analysis in ALL cases are real-time quantitative PCR based detection of unique immunoglobulin and T-cell receptor gene rearrangements (Ig/TCR PCR-MRD) and the multi-parameter flow cytometry based quantitation of Leukemia Associated Immunophenotypes (LAIP Flow-MRD). We compared the two techniques using samples from patients referred for PCR-MRD analysis initially using 4-tube 4-colour flow and more recently 1-tube 10-color flow. Methods: Newly diagnosed consented ALL patients enrolled on ANZCHOG ALL8 (2002-2011) or AIEOP-BFM ALL 2009 (2012-2014) had duplicate bone marrow aspirates, collected at diagnosis, day 15, day 33 and day 79, and analysed by PCR-MRD and Flow-MRD techniques. PCR-MRD analysis utilized clone specific primers and generic probes for Ig/TCR rearrangements according to EuroMRD guidelines. Flow-MRD which detects levels of aberrant combinations of cell-surface proteins using fluorescently labelled antibodies was performed until 2009 with 4-tube 4-colour flow before we adopted a 1-tube approach (9-colour for BCP-ALL and 10-colour T-ALL) based on the AIEOP-BFM harmonised protocol for 2012-2014. Results: Our early comparison showed a relatively poor correlation of 4-colour Flow-MRD results with PCR-MRD (Spearman rank correlation coefficient rho = 0.516, n=267) for patients enrolled at a single centre on ANZCHOG ALL8 in 2002-2009. Only the PCR-MRD results were used for the MRD risk-adapted stratification for patients on this trial. Flow-MRD for subsequent patients on this trial (2010-11) was improved by using more antibodies and adopting a single tube approach. In our current trial, day 15 Flow-MRD results are used for the early identification of low risk patients for a randomized treatment reduction. In bone marrow samples from patients enrolled on this trial, the correlation of the PCR-MRD and Flow-MRD methods is high when considered for all time points (rho = 0.803 n=418; Figure 1). In the same set of patient samples, the concordance between 2 different PCR markers based on different rearrangements was even better (rho = 0.929, n=390). A comparison of time points found that the best correlation between the two methods was observed at day 15 when MRD is often higher and the bone marrow is not regenerating (Table 1). Both PCR and 10-colour flow enabled MRD to be performed for 94% of ALL patients, and only one patient did not have a sensitive MRD assay. Conclusion: The adoption of new approaches to measurement of Flow-MRD, using a single tube and 10-colors, for ALL patients has greatly improved the concordance of Flow-MRD and PCR-MRD results. It is not surprising given the different nature of the techniques that the correlation of results produced by two different markers for PCR-MRD is higher than that with Flow. However we conclude that these two methods can now be used interchangeably at day 15 in BFM-style protocols for ALL patients. The concordance at later time points is weaker and warrants investigation in the whole trial cohort to enable effects of ALL subtype and patient outcomes to be evaluated. Table 1. Concordance of MRD levels at different time points in the same set of patients (Spearman's Rank correlation coefficient rho). MRD by PCR first Ig/TCR marker versus MRD by 10-colour flow MRD by first Ig/TCR PCR marker versus second Ig/TCR marker All timepoints 0.803 (n=418)** 0.921 (n=390)** Day 15 0.795 (n=155)** 0.950 (n=129)** Day 33 0.417 (n=137) 0.826 (n=132)** Day 79 0.383 (n=126) 0.842 (n=129)** ** Correlation is significant at the 0.01 level (2 tailed) Support: NHMRC Australia APP1057746 and Tour De Cure Foundation Figure 1. Comparison of MRD levels measured by 1-tube 10-color Flow MRD versus PCR MRD (left) or by two different PCR Ig/TCR MRD markers (right) in the 418 and 390 paired measurements in the same set of patients. Figure 1. Comparison of MRD levels measured by 1-tube 10-color Flow MRD versus PCR MRD (left) or by two different PCR Ig/TCR MRD markers (right) in the 418 and 390 paired measurements in the same set of patients. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Marked Variability in Reported Minimal Residual Disease Lower Level of Detection of 4 Hematolymphoid Neoplasms: A Survey of Participants in the College of American Pathologists Flow Cytometry Proficiency Testing Program

2015 ◽  
Vol 139 (10) ◽  
pp. 1276-1280 ◽  
Author(s):  
Michael Keeney ◽  
Jaimie G. Halley ◽  
Daniel D. Rhoads ◽  
M. Qasim Ansari ◽  
Steven J. Kussick ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Plasma Cell Myeloma ◽  
Minimal Residual

Context Flow cytometry is often applied to minimal residual disease (MRD) testing in hematolymphoid neoplasia. Because flow-based MRD tests are developed in the laboratory, testing methodologies and lower levels of detection (LODs) are laboratory dependent. Objectives To broadly survey flow cytometry laboratories about MRD testing in laboratories, if performed, including indications and reported LODs. Design Voluntary supplemental questions were sent to the 549 laboratories participating in the College of American Pathologists (CAP) FL3-A Survey (Flow Cytometry—Immunophenotypic Characterization of Leukemia/Lymphoma) in the spring of 2014. Results A total of 500 laboratories (91%) responded to the supplemental questions as part of the FL3-A Survey by April 2014; of those 500 laboratories, 167 (33%) currently perform MRD for lymphoblastic leukemia, 118 (24%) for myeloid leukemia, 99 (20%) for chronic lymphocytic leukemia, and 91 (18%) for plasma cell myeloma. Other indications include non-Hodgkin lymphoma, hairy cell leukemia, neuroblastoma, and myelodysplastic syndrome. Most responding laboratories that perform MRD for lymphoblastic leukemia reported an LOD of 0.01%. For myeloid leukemia, chronic lymphocytic leukemia, and plasma cell myeloma, most laboratories indicated an LOD of 0.1%. Less than 3% (15 of 500) of laboratories reported LODs of 0.001% for one or more MRD assays performed. Conclusions There is major heterogeneity in the reported LODs of MRD testing performed by laboratories subscribing to the CAP FL3-A Survey. To address that heterogeneity, changes to the Flow Cytometry Checklist for the CAP Laboratory Accreditation Program are suggested that will include new requirements that each laboratory (1) document how an MRD assay's LOD is measured, and (2) include the LOD or lower limit of enumeration for flow-based MRD assays in the final diagnostic report.

scholarly journals Novel Single Panel Method for Minimal Residual Disease Detection for Plasma Cells By Flow Cytometry

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
David Kimmel ◽  
Mohammed A Aljama ◽  
Stephen Ronan Foley ◽  
Hira S Mian ◽  
Catherine A Ross
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Large Data ◽  
Final Analysis ◽  
Distinct Advantage ◽  
Limit Of Quantitation ◽  
Minimal Residual

Introduction: Advancement in myeloma therapy has significantly improved outcomes including minimal residual disease (MRD) negativity that may be a surrogate for overall survival. We describe an assay using a single 10-colour panel to detect minimal residual disease (MRD) in plasma cell neoplasms to a level &lt; 10-5. Methodology: Bone marrow aspirate specimens for this MRD assay must be from the first pull of 1.0 to 1.5 mL of bone marrow. A 1 in 10 dilution of the bone marrow is prepared and run on the instrument using a WBC count from the following calculation to determine the volume of specimen to process: For the MRD method, a corrective factor of 4 has been empirically determined to provide a sufficient number of cells to achieve the goal of 10-13 million viable cells during analysis. The required volume of bone marrow is added directly to Versalyse (Beckman Coulter) with 10% bovine serum albumin (BSA) in a bulk RBC lysis step. The cells are incubated for 15 minutes at room temperature while rocking. The cells are centrifuged and the following antibodies are added (Table 1): The cells and surface antibodies are incubated for a total of 20 minutes, specimen is gently vortexed at 10 minutes. Intracellular staining is achieved using the IntraPrep Kit (Beckman Coulter) using our standardized laboratory process. Cells are suspended in approximately 2mL of RPMI 1640 with 10% FCS. The specimen is loaded on to the Navios EX flow cytometer (Beckman Coulter)and data is acquired at approximately 5000 to 10000 events per second. The Navios EX is not capable of collecting more 1 700 000 events at acquisition when all 10 fluorescent detectors are in use plus light scatter detectors, so the specimen is repeatedly reloaded a total of 7 or 8 times. All data files are opened in Kaluza (Beckman Coulter) and merged in to a single file. This large data file is then imported in to the analysis template and analyzed for plasma cells. Analysis: A pilot of 20 specimens from patients with varying plasma cell disorders have been analyzed. Half of these specimens contained populations of monoclonal plasma cells. Ranked in order of smallest to largest (Figure 1): The smallest clone detected at 10x10E-4 is comprised of 1311 events. If a theoretical lower limit of quantitation of 50 events or 5x10E-6 in 10 000 000 total cells analyzed is required, this method will meet this criteria .Notably, all bone marrow specimens of adequate quality (not clotted, non-hemodilute) required less than 1.5mL of bone marrow to achieve &gt; 10 000 000 nucleated cells in the final analysis. Analysis is complex using several dozen plots. Plasma cells are identified using CD38 and CD138. Gated plasma cells are analyzed for the immunophenotype of CD56, CD117, CD27, CD45, CD81 and cytoplasmic light chains simultaneously using n-dimensional radar plots (Figures 2, 3a, 3b): Qualitative results can be calculated from adjusted gates (Table 2): Conclusion: This rapid, high-sensitivity assay for immunophenotypically abnormal and clonal plasma cells requires low volumes of bone marrow. Results are ready in approximately 4 hours which is a distinct advantage and sensitivity can be shown to reach 5 X10-6. Disclosures Foley: Amgen,CelgeneJanssen: Honoraria. Mian:Takeda: Consultancy, Honoraria; Sanofi: Consultancy; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy.

Sign in / Sign up

Export Citation Format

Share Document