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

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.

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 Validated single-tube multiparameter flow cytometry approach for the assessment of minimal residual disease in multiple myeloma

Haematologica ◽  
2020 ◽  
Vol 105 (10) ◽  
pp. e523
Author(s):  
Sandra Maria Dold ◽  
Veronika Riebl ◽  
Dagmar Wider ◽  
Marie Follo ◽  
Milena Pantic ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry ◽  
Single Tube ◽  
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.

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