scholarly journals Reference Values to Assess Hemodilution and Warn of Potential False-Negative Minimal Residual Disease Results in Myeloma

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4924
Author(s):  
Noemí Puig ◽  
Juan Flores-Montero ◽  
Leire Burgos ◽  
María-Teresa Cedena ◽  
Lourdes Cordón ◽  
...  
Keyword(s):  
Reference Values ◽  
Residual Disease ◽  
False Negative ◽  
Cell Types ◽  
Pairwise Comparisons ◽  
Specific Cell ◽  
Nucleated Red Blood Cells ◽  
B Cell Precursors ◽  
Routine Assessment ◽  
Minimal Residual

Background: Whereas, in most patients with multiple myeloma (MM), achieving undetectable MRD anticipates a favorable outcome, some others relapse shortly afterwards. Although one obvious explanation for this inconsistency is the use of nonrepresentative marrow samples due to hemodilution, there is no guidance on how to evaluate this issue. Methods: Since B-cell precursors, mast cells and nucleated red blood cells are normally absent in peripheral blood, we analyzed them in 1404 bone marrow (BM) aspirates obtained in numerous disease settings and in 85 healthy adults (HA). Results: First, we confirmed the systematic detection of the three populations in HA, as well as the nonreduced numbers with aging. Pairwise comparisons between HA and MM patients grouped according to age and treatment showed significant variability, suggesting that hemodilution should be preferably evaluated with references obtained from patients treated with identical regimens. Leveraging the MRD results from 118 patients, we showed that a comparison with HA of similar age could also inform on potential hemodilution. Conclusions: Our study supports the routine assessment of BM cellularity to evaluate hemodilution, since reduced BM-specific cell types as compared to reference values (either treatment-specific or from HA if the former are unavailable) could indicate hemodilution and a false-negative MRD result.

Standardization of WT1 mRNA Quantification for Minimal Residual Disease (MRD) Monitoring in Acute Leukemia Patients: A European LeukemiaNet Concerted Action.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3295-3295
Author(s):  
Enrico Gottardi ◽  
Daniela Cilloni ◽  
Sarah Daly ◽  
S. Green ◽  
Niels Pallisgaard ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Fusion Gene ◽  
False Negative ◽  
Significant Proportion ◽  
Superior Performance ◽  
Clinical Value ◽  
Prognostic Information ◽  
Minimal Residual

Abstract Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemia. In acute myeloid leukemia (AML), the reliability of real-time quantitative PCR (RQ-PCR) and its potential clinical value for MRD studies using fusion gene (FG) transcripts as PCR targets such as PML-RARa, AML1-ETO and CBFb- MYH11 has been demonstrated, but these markers are present only in a minority of cases. In order to overcome this problem several groups looked for alternative markers and growing evidence has suggested that the high expression of WT1 in a significant proportion of acute leukemia cases provides a suitable target for therapy as well as for monitoring of MRD. However, heterogeneity of molecular approaches resulted in a lack of comparability between different MRD studies. This has been solved using RQ-PCR in a network of 9 laboratories within the European LeukemiaNet. Overall 8 primer/probe sets were evaluated including published and “in-house” sets. The assays analyzed differed significantly in terms of efficiency and sensitivity. Three assays with superior performance were identified, achieving sensitivities of at least 1 in 10,000 in serial dilutions of HL60 cells (WT1 positive). Subsequent analysis of two of the primer/probe sets, which amplify ex. 6/7 and 7/8 of WT1 respectively, revealed the potential for false negative results, following documentation of deletions of the WT1 gene in this region in primary AML samples. In two of these cases, different deletions of sequences corresponding to part of WT1 ex.8 were documented by WT1 RNA sequencing. Therefore, a primer/probe set amplifying ex. 1/2 of WT1 has been subject to further analysis in a QC round involving 9 labs. The analysis of 33 normal BM, 32 normal PB and 12 CD34 enriched PBMNCs gave the following results: BM, mean 70,32 WT1 copies/104 ABL copies (range 8,99–209,82); PB, mean 3,30 WT1 copies/104 ABL copies (range 0–13,55); CD34 enriched PBMNCs, mean 8,16 WT1 copies/104 ABL copies (range 1,55–26,16). Overall, these analyses underline the importance of standardization in the development of RQ-PCR assays for MRD detection in leukemia. There is increasing interest in identification of genes that are over-expressed in leukemia as potential MRD targets. However, it is clear that incorporation of such MRD targets into risk-directed treatment protocols is critically dependent upon establishing thresholds of expression in normal blood and marrow on regeneration following myeloablative therapy.

scholarly journals Regenerating normal B-cell precursors during and after treatment of acute lymphoblastic leukaemia: implications for monitoring of minimal residual disease

2000 ◽  
Vol 110 (1) ◽  
pp. 139-146 ◽  
Author(s):  
Elisabeth R. Van Wering ◽  
Birgit E. M. Van der Linden-Schrever ◽  
Tomasz Szczepański ◽  
Marja J. Willemse ◽  
Ed A. Baars ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
B Cell ◽  
Minimal Residual Disease ◽  
Lymphoblastic Leukaemia ◽  
Residual Disease ◽  
B Cell Precursors ◽  
Minimal Residual

scholarly journals Enhanced sensitivity of flow cytometry for routine assessment of minimal residual disease

Haematologica ◽  
2009 ◽  
Vol 95 (4) ◽  
pp. 691-692 ◽  
Author(s):  
E. Domingo ◽  
C. Moreno ◽  
A. Sanchez-Ibarrola ◽  
C. Panizo ◽  
J. A. Paramo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Enhanced Sensitivity ◽  
Routine Assessment ◽  
Minimal Residual

Impact of RNA Stabilization on Minimal Residual Disease Assessment in Chronic Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4490-4490
Author(s):  
Ingrid Thörn ◽  
Ulla Olsson-Strömberg ◽  
Cecilia Ohlsen ◽  
Anna-Maria Jonsson ◽  
Ulf Klangby ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
False Negative ◽  
Rna Degradation ◽  
Disease Assessment ◽  
Rna Stabilization ◽  
Minimal Residual

Abstract Background and objective: Accurate quantification of BCR-ABL mRNA is of critical importance for managing chronic myeloid leukemia (CML) patients receiving Imatinib therapy. RNA degradation thus constitutes a potential problem for laboratories quantifying minimal residual disease (MRD). Patient samples with long transport times between the hospital and the analyzing laboratory may be subject to RNA degradation with a corresponding loss in sensitivity and possible generation of false negative results. Recently, RNA preservation systems have been developed in order to improve RNA stability. Design and methods: The aim of the study was to investigate the performance of the PAXgene Blood RNA Kit in CML follow-up peripheral blood samples and compared the results to unstabilized parallel Trizol extracted samples. The different sample processing methods were evaluated by real-time PCR. Results: RNA isolated with the PAXgene system gave a superior yield per ml blood than with the routine Trizol extraction method. However, although of comparable quality, the RNA did not PCR-amplify as efficiently compared to equal amounts of RNA from routinely processed samples. Therefore, RNA processed with the PAXgene system showed to decreased sensitivity for MRD detection, resulting in false negatives. The sensitivity was comparable to samples processed routinely 20–30 hours after phlebotomy. Interpretation and conclusions: We conclude that routinely processed, i.e. unstabilized, peripheral blood that reaches the laboratory and is processed within 30 hours is preferable for MRD detection. Optimal results were achieved with fresh samples processed within 5 hours with the Trizol method. However, RNA stabilization may be useful if sample transit is expected to exceed 30 hours.

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