Comparison of immunocytochemistry, real-time quantitative RT-PCR and flow cytometry for detection of minimal residual disease in neuroblastoma

2005 ◽  
Author(s):  
Marianne Ifversen ◽  
Bertil Kågedal ◽  
Lisa Christensen ◽  
Catherine Rechnitzer ◽  
Bodil Petersen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Rt Pcr ◽  
Minimal Residual

The use and correlation with flow cytometry of fluorescent molecular beacons in real time PCR of IgH gene rearrangements for evaluation of minimal residual disease in multiple myeloma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 17552-17552
Author(s):  
O. Kara ◽  
A. Yigin ◽  
B. Sahin ◽  
S. Paydas
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Real Time ◽  
Minimal Residual Disease ◽  
Real Time Pcr ◽  
Residual Disease ◽  
Pcr Detection ◽  
Molecular Beacons ◽  
Rt Pcr ◽  
Minimal Residual

17552 Background: At present, the prognostic value of the amount of residual tumor cells in PB, BM or stem cell harvests and its changes over time is stil not clear. Also the advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by using flow cytometry. The aim of the present study was to evaluate a real time PCR test for the IgH gene using alellespecific molecular beacons as fluorescence probes to quantify residual disease and also correlate flow cytometric detection of plasma cells in MM patients during followup after treatment with high dose of chemotherapy or standart chemotherapy. Methods: After clinical diagnosis of 17 MM patients, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify its clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. We have also examined the co-expression of CD19, CD38, CD45, CD56, and CD138 molecules in cells of bone marrow aspirates in patients with multiple myeloma by flow cytometry. Results: The active disease had been accepted of whom plasma cell infiltration ratio was over 10% in bone marrow and also of whom labeled by CD38 and CD138 by FCM. The detection of the MRD was positive in 13 patients by RT-PCR, respectively. The infiltration ratio was correlated with CD138 expression (p = 0.009) and RT-PCR detection of plasma cells (p = 0.006) and also significant correlation had been found between RT-PCR detection and CD138 expression respectively. No any correlaton was found between other surface antigens (CD38, CD45, CD56). Conclusion: Our results indicated that real time PCR with specific molecular beacons provides a feasible, accurate and reproducible method for the determination of minimal residual disease in MM. By FCM only CD138 expression may have been used as disease marker in addition of the RT-PCR detection. No significant financial relationships to disclose.

Efficient Duplex Real-Time Quantitative RT-PCR of BCR-ABL and ABL Gene Transcripts for Monitoring of Minimal Residual Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4887-4887
Author(s):  
Qianli Jiang ◽  
Shan Jiang ◽  
Fanyi Meng ◽  
Ru Feng ◽  
Bing Xu ◽  
...  
Keyword(s):  
Real Time ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Myelogenous Leukemia ◽  
Rt Pcr ◽  
Exon 2 ◽  
Taqman Probes ◽  
Cancer Group ◽  
Anti Cancer ◽  
Minimal Residual

Abstract BCL-ABL fusion gene can be found in 100% chronic myelogenous leukemia (CML) and about 25% adult acute lympholid leukemia where its expression level is a crucial parameter for monitoring of minimal residual disease (MRD). Depending on the breakpoint in BCR, exon 2 of ABL (a2) joins with exons 1 (e1), 13 (b2), or 14 (b3), or rarely to exon 19 (e19) of BCR resulting in chimeric proteins of p190, p210 and p230, respectively. The c-ABL gene is one of the best controls for MRD detection by real-time quantitative RT-PCR (RQ-RT-PCR). In most studies published before, PCR probes were labeled by single fluorescence such as FAM, and BCR-ABL and ABL transcripts were detected in separate PCR reactions. Purpose: To design and evaluate a duplex, real-time quantitative RT-PCR (RQ-RT-PCR) system labeled with double fluorescence Taqman probes to simultaneously measure both BCR-ABL and ABL transcripts. Methods: Positive controls are plasmids containing full-length target sequence of BCR-ABLP210 and ABL. 70 cases of CML bone marrow samples collected in Nanfang Hospital from Jane 2005 to July 2008 were examined. Patients were untreated ones and those treated with STI571 or allo-hematological stem cell transplantation. RQ-RT-PCR value=copies of BCR-ABL/copies of ABL. The results are compared with fluorescence chromosomal in situ hybridization (FISH) for BCR-ABL. Probes and primers recommended by Europe Anti-Cancer group in 2003 were used as gold standard control; probe and primers for bcr-ablP210 gene are ENF541, ENP501 and ENR561, respectively; probe and primers for abl gene are ENPr1043, ENF1003 and ENR1063, respectively. Both Taqman probes are FAM labed. For duplex RQ-RT-PCR, HEX labeled probe is used for the ABL gene, along with corresponding primers, ENP541 labeled with FAM fluorescence and ENF501 and ENR561 were also used for BCR-ABLP210. The experiments were carried out in the same tube on a Biorad Opticon2 RQ-PCR unit. The end concentration is 0.3μmoL/L for primers and 0. 2 μmoL/L for probe. The PCR reaction was carried out in 25μL. PCR condition is at 50°C×2min+95°C×10min, then followed 95°C×15s+60°C×1min for 50cycle. Result: Testing using serial dilutions of plasmid positive control suggested that HEX-FAM duplex is readily amplified with the FAM Taqman probes of BCR-ABLP210, and the HEX-ABL results is same with those with FAM-ABL (recommeded by Europe Anti-Cancer group). Coefficiency of variation among different experiments is less than 5%. The 70 CML cases can be divide into 3 groups based on FISH value: ≥10% (n=32), 0.5% 10% (n=27) and negative (n=11). The corresponding RQ-PCR ratio of BCR-ABL/ABL are 0.590±0.264, 0.044±0.041 and (9.46±6.99)×10|4, respectively, P<0.01 between each group. Conclusion: We have established an efficient duplex RQ-RT-PCR method with FAM and HEX Taqman probes. This approach enables acquisition of more information from each test and hence reduces the amount of sample needed for each test. We believe this method will be useful to MRD monitoring in CML and BCL-ABL + B-ALL.

scholarly journals Minimal residual disease monitoring in neuroblastoma patients based on the expression of a set of real-time RT-PCR markers in tumor-initiating cells

2013 ◽  
Vol 29 (4) ◽  
pp. 1629-1636 ◽  
Author(s):  
TRI BUDI HARTOMO ◽  
AIKO KOZAKI ◽  
DAIICHIRO HASEGAWA ◽  
THI VAN HUYEN PHAM ◽  
NOBUYUKI YAMAMOTO ◽  
...  
Keyword(s):  
Real Time ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Disease Monitoring ◽  
Rt Pcr ◽  
Pcr Markers ◽  
Tumor Initiating Cells ◽  
Minimal Residual Disease Monitoring ◽  
Minimal Residual

scholarly journals Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR

Leukemia ◽  
2000 ◽  
Vol 14 (2) ◽  
pp. 324-328 ◽  
Author(s):  
B Cassinat ◽  
F Zassadowski ◽  
N Balitrand ◽  
C Barbey ◽  
JD Rain ◽  
...  
Keyword(s):  
Real Time ◽  
Minimal Residual Disease ◽  
Acute Promyelocytic Leukemia ◽  
Residual Disease ◽  
Promyelocytic Leukemia ◽  
Rt Pcr ◽  
Minimal Residual

scholarly journals DETECTION OF MINIMAL TUMOR CELLS FOR THE DIAGNOSIS AND TREATMENT MONITORING OF CHILDREN WITH NEUROBLASTOMA

2021 ◽  
Vol 20 (3) ◽  
pp. 10-16
Author(s):  
I. Zh. Shubina ◽  
N. A. Burlaka ◽  
A. P. Kazantsev ◽  
Yu. I. Dolzhikova ◽  
A. A. Petkevich ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Multicolor Flow Cytometry ◽  
Qrt Pcr ◽  
Minimal Residual

Diagnosis, treatment and designing an adequate strategy of neuroblastoma (NB) therapy in children is still a complicated tasks for pediatric oncology and hematology. One of the key aspects of NB control is detection and monitoring of minimal residual disease.The authors make a concise review of the up-to-date methods, such as immunocytochemistry, fluorescent in situ hybridization (FISH), flow cytometry, the methods of qualitative and quantitative polymerase chain reaction (PCR) to estimate mRNA (RT-PCR and QRT-PCR), which are currently used for minimal residual disease detection in patients with NB. Disialoganglioside GD2, a specific NB marker, is traditionally determined by immunocytochemistry with fluorochromes that enhance its specificity, and by flow cytometry, as well. At present, FISH test is a gold standard for evaluation of the MYCN gen status in NB. A widely used multicolor flow cytometry method allows achieving high specificity of the analysis for NB diagnosis. RT-PCR may search for various targets to reveal NB cells, however, at the moment the only accepted immune target is tyrosine hydroxylase mRNA. Moreover, the studies established that quantitative QRT-PCR has more advantages than traditional qualitative RT-PCR, since this method allows a more accurate and quantitative detection of one or several mRNAs in clinical samples. The review discusses advantages and disadvantages of the main methods currently used for minimal residual disease evaluation of NB cells, such as RT-PCR, flow cytometry, FISH, etc. Comparative studies included multicolor flow cytometry with various combinations of CD9/CD81/CD56/CD45/GD2 monoclonal antibodies, conventional RT-PCR and quantitative QRT-PCR to reveal circulating/disseminated NB cells in the clinical samples of cancer patients and healthy volunteers.The authors analyze the results of various studies that compared accuracy and sensitivity of diagnostic methods such as RT-PCR, flow cytometry, FISH and some others. Despite the advantages of each method, the authors emphasize that multicolor flow cytometry is the optimal approach for the rapid and reliable detection of minimal residual disease and micrometastases of NB. 

Quantitative nested real-time RT-PCR specific for tyrosinase transcripts to quantitate minimal residual disease

2002 ◽  
Vol 317 (1-2) ◽  
pp. 39-46 ◽  
Author(s):  
Nicole Max ◽  
Karin Wolf ◽  
Eckhard Thiel ◽  
Ulrich Keilholz
Keyword(s):  
Real Time ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Rt Pcr ◽  
Minimal Residual
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