Accurate quantitation of residual B-precursor XXXX by limiting dilution and a PCR-based detection system: a description of the method and the principles involved.

@entity1 @entity1778 @entity981 @entity7513
The detection of @entity1778 cells in the bone marrow of @entity1 during morphologic remission has been greatly facilitated by use of the polymerase chain reaction (PCR) to amplify @entity1778 -specific sequences. While the current PCR strategies for estimating the @entity1778 claim a detection sensitivity of one @entity1778 cell amongst 10(5) or 10(6) normal cells, a rigorous assessment of the relative error associated with these techniques has not been presented. We have developed a method of estimating the @entity1778 in remission marrows that is analogous to the limiting dilution assays used to determine the frequency of immunocompetent cells in a responder cell population. Using this method we measured the fraction of all-or-none (i.e. positive or negative) reactions of the PCR amplification of the @entity1778 -specific @entity7513 gene rearrangement in replicate samples of serial dilutions of DNA obtained from diagnostic bone marrow specimens from 15 @entity1 with B-precursor @entity981 ( @entity981 ). A sigmoid curve representing the fraction of positive PCR reactions at a given dilution of @entity1778 DNA was found to be the best fit to the data. The narrowness of the log-linear region of this curve prevents the direct application of the analysis methodology that has previously been described for limiting dilution assays. However, the @entity1778 burden during morphological remission in these 15 @entity1 and in two additional @entity1 who experienced relapse could be estimated by the described dilution analysis method using the best-fit equation. Furthermore, the data generated for diagnostic, remission and relapse marrow samples exhibited a small interspecimen variation. The results suggest that this method can reliably estimate @entity1778 over a range of five orders of magnitude. Although the PCR reaction appears to be one of the most sensitive methods for detecting @entity1778 , all techniques based on this procedure, including our own, must exhibit limitations inherent to the amplification process. Our estimates or relative error suggest that a realistic limit for the PCR estimation of @entity1778 lies in the range of one @entity1778 cell per 10(5) normal cells. The suggested method is rapid, technically simple and relatively inexpensive. Furthermore, the principles that it is based upon can be applied to any PCR-based strategy.
