Scharf S J, Smith A G, Hansen J A, McFarland C, Erlich H A
Department of Molecular Genetics, Roche Molecular Systems, Inc, Alameda, CA 94501, USA.
Blood. 1995 Apr 1;85(7):1954-63.
We have developed a quantitative, nonisotopic method using variable number tandem repeat (VNTR) and short tandem repeat (STR) markers for monitoring donor cell engraftment in marrow transplant recipients. Posttransplant DNA from the recipient is amplified with fluorescent polymerase chain reaction (PCR) primers for polymorphic markers that distinguish donor alleles from recipient alleles. The fluorescent PCR products are then separated on agarose or acrylamide gels on the Applied Biosystems 373A Sequencer (Foster City, CA). Using GeneScan 672 software (Applied Biosystems) to analyze the separated alleles, we can correlate allele peak areas to the percentage of donor or recipient DNA. We quantitate engraftment in a mixed chimeric sample by mixing pretransplant recipient and donor DNAs in a range of percentages and amplifying the mixtures to produce a standard curve. By amplifying and analyzing the posttransplant sample DNA(s), we can determine the extent of engraftment by interpolating the percent peak area of the informative allele(s) from this standard curve. This approach provides a precision of measurement ranging, depending on the marker, from 3.5% to 8.0% (percent coefficient of variation) and an accuracy of engraftment determination ranging from 97% to 99%, with a sensitivity of detection of 1% donor or recipient DNA. We retrospectively analyzed a panel of 32 patients and found seven to be informative for some degree of mixed chimerism, indicative of either residual normal host cells or leukemic relapse. An analysis of different cell lineages obtained posttransplant showed different degrees of engraftment in myeloid and T-cell populations. In summary, this method can provide an accurate, quantitative assessment of mixed chimerism in patients posttransplant. Such information may be useful in the future in guiding early implementation of additional treatment designed to circumvent graft failure or suppress relapse.
我们开发了一种定量、非同位素方法，利用可变数目串联重复序列（VNTR）和短串联重复序列（STR）标记来监测骨髓移植受者体内供体细胞的植入情况。用荧光聚合酶链反应（PCR）引物扩增受者移植后的DNA，这些引物针对的是能区分供体等位基因和受者等位基因的多态性标记。然后，将荧光PCR产物在Applied Biosystems 373A测序仪（加利福尼亚州福斯特城）上的琼脂糖或丙烯酰胺凝胶中进行分离。使用GeneScan 672软件（Applied Biosystems）分析分离后的等位基因，我们可以将等位基因峰面积与供体或受者DNA的百分比相关联。我们通过将移植前受者和供体DNA按一系列百分比混合，并扩增这些混合物以生成标准曲线，来定量混合嵌合样本中的植入情况。通过扩增和分析移植后样本DNA，我们可以通过从该标准曲线中插值信息性等位基因的峰面积百分比来确定植入程度。这种方法的测量精度根据标记不同，变异系数百分比范围为3.5%至8.0%，植入情况测定的准确度范围为97%至99%，检测灵敏度为1%的供体或受者DNA。我们回顾性分析了一组32例患者，发现其中7例在某种程度的混合嵌合方面具有信息价值，这表明存在残留的正常宿主细胞或白血病复发。对移植后获得的不同细胞谱系进行分析显示，髓系和T细胞群体中的植入程度不同。总之，该方法可以对移植后患者的混合嵌合情况进行准确、定量的评估。这些信息未来可能有助于指导早期实施旨在避免移植失败或抑制复发的额外治疗。
