Annals of Clinical & Laboratory Science 38:264-272 (2008)
© 2008 Association of Clinical Scientists
Systematic Analysis of Stutters to Enhance the Accuracy of Chimerism Testing
Ding-Ping Chen1,
Ching-Ping Tseng2,
Shu-Hui Tsai1,
Tsu-Lan Wu1,
Pi-Yueh Chang1 and
Chien-Feng Sun1,3
1 Department of Clinical Pathology, Chang Gung Memorial Hospital, Taoyuan County, Taiwan2 Graduate Institute of Medical Biotechnology and Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan County, Taiwan3 Department of Pathology, School of Medicine, Chang Gung University, Taoyuan, Taiwan
Address correspondence to Dr. Chien-Feng Sun, Department of Clinical Pathology, Linkou Medical Center, Chang Gung Memorial Hospital, 5 Fushin Street, Kueishan, Taoyuan, 333, Taiwan; tel 886 3 328 1200 ext. 2554; fax 886 3 397 1827; e-mail: suncgj{at}adm.cgmh.org.tw.
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Abstract
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Post-transplantation chimerism testing is important to monitor the engraftment of donor stem cells and for the diagnosis of relapse. Detecting the presence of donor/recipient-specific short tandem repeats (STRs) is a frequently used method for engraftment study. Unfortunately, the interpretation of the STR-based chimerism tests is often subject to interference by the presence of a stutter peak, which is one 4-base repeat unit smaller than an authentic allele. The aim of this study was to systematically analyze and resolve the effect of stutter peaks on the interpretation of STR-based chimerism tests. The AmpFlSTR Identifiler Amplification kit (Applied Biosystems)was used to amplify 15 STR loci using genomic DNA from 30 randomly selected, healthy donors. We found that the stutter peaks had locus-specific characteristics. The stutter percentage was defined as the percentage of the stutter peak area/main STR peak area. Based on mean values for the 30 DNA samples, the stutter percentage varied from locus to locus and ranged from 3.12% to 10.71% for 15 STR loci. The locus-specific stutter effect can be eliminated through appropriately adjusted equations. The usefulness of these equations in the prediction of relapse was confirmed by the 5% sensitivity test. Hence, this report offers a valuable scheme to enhance the accuracy of chimerism testing.
Keywords: allogeneic hematopoietic stem cell transplantation, chimerism testing, stutter peak
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Introduction
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Allogeneic hematopoietic stem cell transplantation (HSCT) is usually performed in patients with high-risk or advanced hematological malignancies and aplastic anemia. Complete donor-derived hematopoiesis in the recipient patient is essential for sustained engraftment and for preventing relapse of the underlying disease. In this regard, detecting the existence of chimerism, the progressive presence of recipient-derived cells in the recipient patient, is important for monitoring the engraftment of donor stem cells and for the diagnosis of relapse [1–8]. Appropriate medical action can then be implemented to improve the management of patient care. For example, adoptive immunotherapy by donor lymphocyte infusion can be performed for patients with relapse of chronic myelogenous leukemia to produce clinical remission [9–12]. Alternatively, a second HSCT can be performed for patients with relapse of acute leukemia. Hence, chimerism testing is crucial to confirm complete engraftment, mixed chimerism, or recipient relapse.
Several chimerism testing methods have been developed to analyze the polymorphic DNA loci of the donor and recipient cells. These methods include restriction fragment length polymorphisms, variable number tandem repeats, and short tandem repeats (STRs). Because STRs are interspersed throughout the genome and commercial analytical systems are available, STR loci are commonly used for the study of engraftment. Although STR-based analysis is both qualitative and quantitative, the accuracy of this method is impaired by the presence of stutter peaks that are often derived from polymerase slippage during PCR amplification and appear as one 4-base repeat unit smaller than the authentic allele [13–16].
The effects of stutters on chimerism tests are usually apparent at the early stage of relapse when the STR locus of the recipient is 4-bp smaller than that of the donor. Consequently, high rates of misinterpretation may occur. Systematic analysis of stutter effects on monitoring donor engraftment and diagnosis of relapse is thus warranted. This study is designed to develop a method for improving the accuracy of a commercially available analytical system for chimerism testing.
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Materials and Methods
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Sample preparation.
This study was approved by our hospital’s Review Board. Peripheral blood samples of 30 randomly selected healthy donors were the remnants of 5–6 ml specimens collected by venipuncture into EDTA tubes for routine paternity testing at Chang Gung Memorial Hospital. The iage and gender of these donors is shown in Table 1
. Blind testing was assured by omitting the donors’ names, so the specimens were not individually identifiable. In a mixing experiment, DNA specimens were obtained from a 10-yr-old child with acute lymphoblastic leukemia and the child’s bone marrow donor. Genomic DNA was extracted from 200 µl of whole blood using a QIAamp DNA mini kit according to the instruction of the manufacturer (Qiagen, Hidden, Germany). DNA concentration and purity were determined by measuring the absorbance at 260 nm and A260/A280 ratio, respectively. A maximum amount of 50 µg of genomic DNA could be obtained by the DNA purification procedure.
STR amplification.
We used the AmpFlSTR Identifiler amplification kit (Applied Biosystems) to perform STR-PCR. The tetranucleotide STR loci amplified in this reaction included: D8S1179, D21S11, D7S820, and CSF1PO (all labeled with 6-FAM blue dye); D3S1358, TH01, D13S317, D16S539, and D2S1338 (all labeled with VIC green dye); D19S3433, vWA, TPOX, and D18S51 (all labeled with NED yellow dye); and D5S818 and FGA (all labeled with PET red dye). In addition, the amelogenin locus was analyzed to differentiate X and Y chromosomes (labeled with PET red dye). PCR was performed using 1 ng of genomic DNA in a final reaction volume of 25 µl as suggested by the manufacturer. The PCR cycle conditions were: 95° for 11 min, followed by 28 cycles with 94° for 1 min, 59° for 1 min and 72° for 1 min. The final elongation step was 45 min at 60°. The PCR products were analyzed with an ABI PRISM 310 DNA sequencer (Applied Biosystems) as described by the manufacturer.
Statistical analyses of stutter.
The STRs were analyzed with the GeneScan Analysis software. Allele size and designation as well as peak height and peak area values for each allele in the profiles were exported from Genotyper 2.0 into a spreadsheet for statistical calculations. The stutter percentage was defined as the percentage of the stutter peak area/main STR peak area. The mean stutter percentage was the mean value for the stutter percentage of the 30 samples. According to the stutter distribution patterns, adjusted equations were used to calculate the results of 5% sensitivity test to determine whether the accuracy of the test was improved.
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Results
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The AmpFlSTR Identifiler amplification kit was used in this study to analyze 15 STR loci of 30 randomly selected blood samples. The age and sex of the 30 sample donors are listed in Table 1
. The locus designation is described in Table 2. After resolving the amplified PCR product on a DNA sequence analyzer, we found that a stutter peak was often present in addition to the main STR peak (Fig. 1). Stutter peak pattern analysis indicates that the stutters have locus-specific characteristics. Each locus has a specific and usually narrow range of stutter percentage as revealed by the means of stutter percentage ± SD (Table 2). The means of stutter percentage varied from locus to locus and ranged from 3.12% to 10.71% for the 15 STR loci (Table 2). Besides, the longer alleles appear to have a higher stutter percentage than the shorter alleles.
The unique but not the shared STR loci of the donor and recipient are usually used to evaluate chimerism and to determine relapse percentage. Hence, when the relapse peak and the stutter peak are present at the same position for the respective STR locus, the area of the stutter peak has to be deducted to obtain an accurate relapse percentage. Analysis of the peak patterns revealed that at least 6 different types of recipient-donor relationship, designated as type A to type F, were affected by the presence of stutters (Fig. 2). Dependent on the type of recipient-donor relationship, different adjusted equations were proposed for the calculation and adjustment of the relapse percentage (Fig. 2). Consequently, the accuracy of chimerism testing can be greatly enhanced as validated by the 5% sensitivity test.
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Fig. 2. Six different types of recipient-donor relationship were affected by the presence of stutters. Each type of the stutter effect on the calculation of relapse percentage can be eliminated using the indicated equation. The small open peak R represents the recipient STR peak area; the large open peak D represents the donor STR peak area; the small closed peak that is 4-bp smaller than the donor STR peak represents the stutter peak derived from the donor. R, recipient peak area; D, donor peak area; S%, stutter percentage.
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We present a case to illustrate the application of the proposed equations for the adjustment of relapse percentage (Fig. 3). A total of 15 STR loci were characterized for a 10-yr-old child with acute lymphoblastic leukemia and for the child’s bone marrow donor. In addition, a mixing experiment was performed by spiking 0.05 ng recipient DNA into 0.95 ng donor DNA followed by STR loci analysis using the AmpFlSTR Identifiler amplification kit. Except the loci of D21S11, TH01, and TPOX, which had overlapping recipient and donor peaks that cannot be used to distinguish recipient and donor, the other 12 markers were informative. Analysis of the donor and recipient peak patterns revealed that D8S1179, CSF1PO, and D16S539 had the peak pattern B; vWA had the peak pattern C; D7S820 had the peak pattern D; and D3S1358 had the peak pattern F. D13S317, D2S1338, D19S433, D18S51, D5S818 and FGA were not affected by stutters and no adjustment was required for these STR markers. In the mixing experiment, the relapse percentages before and after equation adjustment were 7.83% and 5.62% for D8S1179, 5.48% and 2.63% for D7S820, 7.54% and 4.40% for CSF1PO, 11.16% and 4.1% for D3S1358, 6.48% and 4.17% for D16S539, 10.93% and 2.84% for vWA, respectively (Fig. 3). These data suggest that the indicated equations can be used to calculate and adjust the relapse percentage by eliminating the stutter effect. Consequently, the accuracy of chimerism testing was enhanced as demonstrated by the 5% sensitivity test.
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Discussion
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Chimerism testing after allogeneic HSCT is an important diagnostic method for engraftment monitoring [17]. An optimal evaluation method should ensure high sensitivity and precision in chimerism quantification. At present, PCR amplification of microsatellite markers is the standard method to evaluate post-transplant chimerism. However, the presence of stutters may interfere with the informative peaks, leading to misdiagnosis (false-positive result). The stutter effect is more prominent in low concentration of chimerism testing. In some circumstances, the STR and stutter peak positions are overlapping, so that we may have to eliminate the marker in the final interpretation [7]. Consequently, the number of informative markers could be significantly reduced for sibling donor/recipient pairs because they share the same genetic background as their parents. In this study, we systematically analyzed the stutter effect with an aim to enhance the accuracy of chimerism testing. According to our findings, the stutter peak usually occurs with a peak area that is at the range from 3.12% and 10.71% of the corresponding STR alleles and the stutter effects are STR locus-specific (Table 2).
We recommend the following two strategies to discount the stutter effect and enhance the accuracy of chimerism testing. First, we suggest considering the unique but not the shared STR markers of the donor and recipient in the determination of relapse percentage. Second, depending on the type of recipient-donor relationship, different adjusted equations can then be used to calculate the relapse percentage. Through these adjustments, the accuracy of the chimerism test can be greatly enhanced as demonstrated by the mixing experiment we describe in this report.
In addition to stutter peaks, pull-up peaks and template-independent nucleotide additions can also affect the interpretation of chimerism tests [18]. Fortunately, we can eliminate pull-up peaks by decreasing the amount of PCR product to prevent specific peaks from being off-scale. To eliminate double peaks resulting from the presence or absence of terminal adenosine residues, it is recommended to use primers with optimal tailing and to include a final extension step of 45 min at 60° at the end of the PCR reaction [18].
In conclusion, the main difficulty posed by stutter peaks is in the interpretation of mixed samples; faint peaks at a position 4 bases shorter than intense peaks could be either stutter peaks or real alleles from a minor component in a mixture. This report represents a systematic study to resolve the stutter effect and it offers a method to enhance the accuracy of chimerism testing.
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Acknowledgement
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Grant NSC95-2320-B-182-003 from the National Science Council of Taiwan supported this work.
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References
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Abstract
Introduction
