Improved assessment of minimal residual disease in B cell malignancies using fluorogenic consensus probes for real-time quantitative PCR

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation

“…Thus, our data demonstrate higher sensitivity of ASO IgH RQ-PCR compared to our MRD flow assay in post-transplant CLL patients. This contradicts in vitro data that had predicted sensitivities of ASO IgH RQ-PCR [21][22][23] within the same order of magnitude as for MRD flow approaches (one CLL cell in 10 4 -10 5 normal cells). 18,25,29,30 We therefore analyzed factors that influence specificity and sensitivity of MRD flow as the apparently less sensitive method.…”

“…ASO IgH RQ-PCR was performed with an ABI PRISM 7700 thermal cycler as described elsewhere. 22 Briefly, 500 ng of DNA was amplified using 300 nM of the consensus J H germ line and the individual ASO primers, 200 nM Taqman probe annealing to a downstream Table 1 Clinical details on patients from whom samples were included into the study family-specific J H region, and TaqMan Universal Mastermix (ABI). Reaction conditions were as follows: 501C 2 min, 951C 10 min followed by 45 cycles of 951C for 15 s and 59-641C (depending on ASO primer) for 60 s. Data were analyzed using the Sequence Detection Software v1.7 (ABI).…”

“…14,17,19,20 Real-time PCR using such ASO primers allows the quantitation of one CLL cell in 10 4 -10 5 cells. [21][22][23] A more rapid, but less sensitive and not quantitative method for MRD detection in CLL is PCR using consensus IgH-primers (consensus IgH-PCR). 12,16,18,24,25 This technique is reported to detect one monoclonal B-cell in 2 Â 10 1 -1 Â 10 3 benign leukocytes.…”

Stem cell transplantation for chronic lymphocytic leukemia: different outcome after autologous and allogeneic transplantation and correlation with minimal residual disease status

“…Moreover, as recently shown, real-time PCR assays may be helpful in analyzing the MRD kinetics. 47,48 In summary, the present report suggests that in CLL patients the probability of obtaining sustained MRD(−) CR after autotransplantation is low and that MRD analysis is useful to predict clinical relapse. On the other hand, the long-lasting responses observed after allogeneic transplants indicate the possibility of a true eradication of CLL in a significant proportion of patients, although delayed responses or transient relapses at MRD level may be detected in some instances.…”

Standardized MRD flow and ASO IGH RQ-PCR for MRD quantification in CLL patients after rituximab-containing immunochemotherapy: a comparative analysis

“…10,12,20 However, as the superior prognostic significance using certain MRD levels 10 or even MRD kinetics 9 has been shown, MRD quantification nowadays is clearly preferable. With attainable sensitivities of up to 10 -5 ASO IGH RQ-PCR is generally considered the more sensitive quantitative technique, 22 whereas sensitivities between 10 -5 and 10 -4 have been reported for four-color MRD flow. 10,19,23,24 Nevertheless, because of short turn-around time and broader availability, flow cytometry is considered an attractive alternative to RQ-PCR for sensitive quantification of residual CLL cells.…”