Background: For peripheral blood stem cell transplantation (PSCT), several engraftment analysis methods have been performed including detection of restriction fragment length polymorphisms and amplification of polymorphic genetic loci. To facilitate monitoring of the engraftment, a quantitative, non-isotopic method using a short tandem repeat (STR) marker has been set up in our laboratory.
Methods: DNAs from pretransplant recipients and donors were amplified with the AmpFlSTR Profiler Plus kit that contains 9 STR markers. The fluorescent polymerase chain reaction products were then fractionated on polyacrylamide gels in an ABI PRISM 377 DNA sequencer. Results were analyzed using GeneScan 2.1 software. We selected the best markers as informative alleles which can distinguish donor from recipient. For quantitative analysis of the engraftment, we prepared a mixed chimeric sample by mixing pretransplant recipient and donor DNAs in different ratios to produce a standard curve. After amplifying the posttransplant recipient DNA, we were able to detect the extent of engraftment by interpolating the percent peak area of the informative alleles from this standard curve.
Results: We retrospectively analyzed 10 patients who had received allogeneic PSCT. Two of them showed some degree of mixed chimerism indicating leukemic relapse. In case one, 38.7% of the recipient DNA was first detected in the third month after PSCT. In case two, 6.5% of the recipient DNA was first detected in the tenth month after PSCT.
Conclusion: In summary, this method provides an accurate, quantitative, and early assessment of mixed chimerism in posttransplant patients. Such information may be useful to guide implementation of additional treatment to circumvent graft failure or relapse in the future.
(Chang Gung Med J 2002;25:734-42)

Key words: peripheral blood stem cell transplantation (PSCT), quantitative short tandem repeats (STRs), mixed chimerism.
At present, allogeneic peripheral blood stem cell transplantation (PSCT) is considered the best therapeutic option for patients with severe aplastic anemia, severe combined immunodeficiency disease, and leukemia. Detection of the extent of chimerism after transplantation is an important method for monitoring the engraftment of donor cells, thus allowing early detection of graft failure. In the past, clinical physicians used cytogenetic analysis or erythrocyte phenotyping to assess the extent of chimerism. Recently, several approaches have been published based on polymorphic DNA loci, such as restriction fragment length polymorphisms, variable number tandem repeats, and short tandem repeats (STRs).(1-3) The use of these markers not only allows an assessment of the extent of chimerism but also requires a very small amount of sample. Because STRs are interspersed throughout the genome and commercial applications are available, the use of STR loci has become a frequently used medical technology for the study of engraftment. However, the method is often limited by its ability to discriminate between donors and recipients. To facilitate the monitoring of peripheral blood stem cell engraftment, we developed a novel approach using a multiplex polymerase chain reaction (PCR) amplification of 9 STR loci and the amelogenin locus which provides a rapid, accurate, and quantitative way to determine the extent of chimerism after allogeneic PSCT.

METHODS

Patient samples
This retrospective study included 10 patients who received allogeneic PSCT at Chang Gung Memorial Hospital. Patient details are shown in Table 1.

Sample preparation
Peripheral blood samples were collected from a donor and recipient before allogeneic PSCT and from the recipient at regular intervals after the procedure. Genomic DNA was extracted from fresh blood samples using a QIAamp DNA mini kit (QIAGEN, Hidden, Germany).

Preparation of mixed chimeric DNA reconstructions
Before quantitative analysis of peripheral blood stem cell engraftment, we first had to prepare pre-PSCT recipient and donor DNAs in varying ratios of between 0% and 100% to produce a standard curve for every individual case. A reconstruction consisting of mixtures of varying percentages of pretransplant recipient and donor DNAs represented the degree of amplification of each allele in a mixed chimeric posttransplant sample. In these reconstructions, while the percentages of donor and recipient DNA changed relative to each other, the total amount of DNA in the reconstruction remained constant.

STR amplification
We used the AmpFlSTR Profiler Plus PCR amplification kit (Applied Biosystems, CA, USA) to perform STR-PCR. The tetranucleotide STR loci amplified in this reaction included: D3S1358, vWA, and FGA(4-6) (all labeled with 5-FAM); CSF1PO, TPOX, and TH01(7-9) (all labeled with JOE); and D5S818, D13S317, and D7S820(10,11) (all labeled with NED). In addition, the amelogenin locus was analyzed, which discriminates between X and Y chromosomes (labeled with JOE). For each PCR reaction, the final volume was 25 ml and included 5 ng of DNA as recommended by the manufacturer. The cycle conditions were: 95 oC for 11 min, followed by 28 cycles of 94 oC for 1 min, 59 oC for 1 min, and 72 oC for 1 min. The final elongation step was 45 min at 60 oC.

Electrophoresis and GeneScan analysis
The amplified PCR products were separated and detected using an ABI377 automated DNA sequencer (Applied Biosystems). A denaturing polyacrylamide gel containing 10X TBE, 6 M urea, and 5% Long Ranger gel solution (FMC Biosystems, Maine, USA) was used. For each sample, an internal size standard (GeneScan Rox 500, ABI, Warrington, UK) and a formamide loading dye solution were added. After denaturation, 1.5 μl of this mixture was loaded onto the gel, which was run for 2 h at 2.2 kV and 56 oC. Then the results were analyzed using the GeneScan 2.1 software.

Calculation of mixed chimerism
The recipient percentage peak area of the reconstruction samples was calculated and plotted versus the percentage of recipient DNA amplified to determine the engraftment of the posttransplant samples. The recipient percentage peak area was calculated as follows: %Peak area = [(AR1+AR2)×100%] / (AD1+AD2+AR1+AR2),(12) where A is the peak area; D is the donor alleles; and R is the recipient alleles (Fig. 1). In cases of shared alleles, only the informative alleles (i.e., those that distinguish donor from recipient) were used in the calculation. We detected the extent of engraftment or relapse by interpolating the peak area of the informative alleles using this standard curve, and the engraftment or relapse was expressed as either percent donor or percent recipient DNA.

RESULTS

Precision, accuracy, and the standard curve
Because shorter alleles may amplify more efficiently than longer alleles, it is important to simulate a range of mixed chimerism that might be encountered in actual clinical posttransplant samples. We performed dilution experiments in order to test the linearity, reproducibility, precision, and sensitivity of the STR-PCR for quantitative assessment of mixed chimerism. Figure 2 shows the GeneScan data for the reconstruction samples in this experiment. The donor peak showed increasing donor allele peak area with increasing percentage of donor DNA. Conversely, the upper recipient allele peak area decreased as the percentage of recipient DNA decreased. The percent peak area data for the informative alleles of the reconstruction samples were calculated and used to plot a standard curve (Fig. 3). Although the donor peak area of the standard curve for each marker was directly proportional to the percent of donor DNA added, the curve for each locus varied slightly. Because amplification of the alleles differed from each other, this variation emphasizes the importance of preparing standard curves for the informative markers for each set of samples.

Sensitivity
In dilution experiments for plotting the standard curve, a minor cell population representing 5% was reproducibly detected. Even below 5%, signals could be found depending on the constellation of peaks present. In other words, alleles for which 1 individual was homozygous could be seen down to 1%. In addition to this, the use of multiple alleles increased the reliability of results, because of the higher chance of encountering an informative peak constellation.

Informative STR alleles
There were many combinations of donor and recipient alleles, and some of these combinations were informative for the detection of recipient alleles, whereas others were not. Although some loci were technically informative, they were not optimal for use in determining mixed chimerism because of the existence of stutter bands that appeared predominantly as 2 minor peaks alongside the major allele peaks. In general, stutter peaks are 1 repeat smaller or larger than the major allele peaks and typically 10% or less than the area of the major allele. They can influence the choice of informative loci for engraftment analysis.

Clinical samples
Clinical DNA samples from the panel of 10 transplant patients' peripheral blood cells were amplified for STR markers. Information on the patients is given in Table 1. Figure 4 uses 1 of these patients as an example. Donor and patient pretransplant samples were found to be informative with D18S51. The D18S51 markers were sized and genotyped with the D18S51 allelic ladder from the Applied Biosystems AmpFlSTR Profiler Plus kit. Of the 10 patients examined, all patients showed complete engraftment after PBST, but the recipient DNA in 2 patients appeared to relapse after a period of time (Fig. 5). One recipient's DNA appeared at 3 months post-transplantation, while the other appeared after 10 months. In the first relapsed recipient, interpolation of the reconstruction plot shows that 27.82% of the recipient peak area of the posttransplant sample corresponded to 38.7% of the recipient DNA (Fig. 6). The other showed that 1.67% of the recipient peak area of the posttransplant sample corresponded to 6.5% of the recipient DNA.

DISCUSSION

Peripheral blood stem cell transplantation has been established as a lifesaving procedure in selected hematologic malignancies and bone marrow failure syndromes, and it may be valuable in other types of neoplastic disease. Usually, relapse is a significant cause of transplantation failure. Because most relapses occur by expansion of a surviving leukemic clone of recipient origin, early detection of the progressive reappearance of recipient cells by bone marrow engraftment analysis may help to predict clinical relapse. For patients with relapse of chronic myelogenous leukemia, the effectiveness of adoptive immunotherapy by donor lymphocyte infusion to produce clinical remission has been well documented.(13-16) With acute leukemia, a second PSCT is a reasonable therapeutic approach for the treatment of a relapse. So it is necessary to carry out peripheral blood stem cell engraftment analysis of allogeneic PSCT patients to confirm the engraftment and to detect mixed chimerism or recipient relapse after transplantation.
DNA polymorphisms are used to monitor engraftment after transplantation from a related or unrelated donor. DNA polymorphisms are not useful after autologous BMT. If the donor is an identical twin, DNA polymorphisms are not useful, either. The most valuable polymorphism for this purpose is caused by variations in certain repeated sequences that are known as short tandem repeats (STRs). An informative STR locus is one for which at least 1 recipient allele has a different number of repeats than the donor allele(s). Because the recipient and donor are often related, they share many alleles. Therefore, multiple STR loci need to be tested to identify 1 or more informative loci.(17) The simultaneous amplification of multiple loci in a single reaction tube facilitates the identification of informative loci.
Although some loci are technically informative, they might not be optimal for the determination of mixed chimerism primarily because of the existence of stutter bands that appear predominantly as 2 minor peaks alongside the major allele peaks. The minor peaks are predominantly 1 repeat smaller or larger than the major allele peak. Stutter peaks have been proposed to result from slipped strand mispairing during PCR amplification,(18) which results in a peak that is 1 repeat smaller or larger than the major allele peak. The existence of stutter peaks influences the choice of informative loci for engraftment analysis. In a state of recipient DNA relapse, the percentage of recipient DNA is less than 5% to 10%, which is the same size as a typical stutter peak. Hence it is necessary to use the following criterion to choose the best locus: the recipient-specific allele must be at least 2 repeats larger or smaller than the nearest donor allele.
When an informative locus is selected, thee STR-PCR method can be used to qualitatively evaluate the engraftment.(3,12,19,20) A quantitative method for BME analysis using fluorescently labeled primers for PCR amplification of individual VNTR and STR loci was first described in 1995 by Scharf et al.(12) An electrical peak area for each end-labeled fragment detected is directly proportional to the number of DNA molecules present. To simulate a posttransplant MC or a relapsed state, we mixed 2 different DNA samples that displayed recipient and donor in various ratios of between 0% and 100% using the same total amount of 5 ng DNA to prepare the amplification reconstruction standard curves.
Because shorter alleles may amplify more efficiently than longer alleles, it is often not possible to use a simple visual estimation of the peak intensity to accurately determine the extent of mixed chimerism. In addition, it is critical to determine engraftment because of the potential for preferential amplification in mixtures of alleles. These preferential amplification effects depend on a variety of factors: the amount of DNA or enzyme used, the inherent efficiency of the amplification system, and the number and the relative size difference of the alleles present in the sample. We have found that the reconstruction standard curves obtained for a specimen can vary among different markers. In addition, different donor or recipient samples can produce different standard curves for the same marker. So it is very important to establish individual reconstruction standard curves for each marker in each donor-recipient couple.
From our experience in preparing standard curves, we found that the sensitivity of detection of small percentages of recipient DNA is related to the amount of genomic DNA in the PCR reaction. The use of 10 to 15 ng of DNA may improve the sensitivity of detecting small amounts of recipient cells by sampling more DNA, but this amount often results in nonspecific peaks that ultimately decrease the sensitivity by increasing the background, especially in multiplex reactions. The use of 1 to 3 ng of DNA generally gives a clean baseline above which recipient-specific peaks can easily be seen, but the sensitivity is lower. As a result of these findings, we routinely use 5 ng of DNA for PCR amplification in engraftment analysis to obtain the best results. In addition to this, we strongly suggest that before interpreting a result as negative for a recipient, it is important to carefully search at a low scale for recipient-specific peaks.
Because relapsing cells are originally from bone marrow rather than from peripheral blood cells, we also suggest that it is better to use DNA of bone marrow cells to survey the engraftment condition rather than using that of peripheral blood cells. In fact in 1998, Bader presented bone marrow transplant data in which the recipient DNA of only 2 of 7 patients could be detected in bone marrow samples but not in peripheral blood.(19) In addition to this, a longer time interval in the follow-up of these patients after allogeneic PSCT is a major limitation of the sensitivity of this assay. Consequently, it is better to investigate patients at short time intervals. With frequent monitoring, the detection of mixed chimerism by chimerism analysis may alert clinicians to a high risk of relapse and allow early intervention with rapid treatment using immunosuppression or donor lymphocyte infusion therapy, or even a second PSCT. Although many methods have been used for engraftment analysis, PCR amplification of STR loci is the best choice for many clinical laboratories because it is informative, quantitative, relatively rapid, and sensitive.
In conclusion, the greatest advantage of the STR-PCR procedure over other published procedures seems to be its high rate of discrimination. It represents an easy and accurate means for quantitatively assessing relapsed DNA. Furthermore, the use of fluorescent primers enables reliable quantification without the need for additional densitometry. The method can be especially helpful in monitoring patients undergoing transplantation.

REFERENCES

1. Lawler M, Humphries P, McCann SR. Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction. Blood 1991;77:2504-14.
2. Leclair B, Fregeau CJ, Aye MT, Fourney RM. DNA typing for bone marrow engraftment follow-up after allogeneic transplant: a comparative study of current technologies. Bone Marrow Transplant 1995;16:43-55.
3. Bader P, Holle W, Klingebiel T, Handgretinger R, Niethammer D, Beck J. Quantitative assessment of mixed hematopoietic chimerism by polymerase chain reaction after allogeneic BMT. Anticancer Res 1996;16:1759-63.
4. Li H, Schmidt L, Wei MH, Hustad T, Lerman MI, Zbar B, Tory K. Three tetranucleotide polymorphisms for loci: D3S1352, D3S1358, D3S1359. Hum Mol Genet 1993;2:1327.
5. Kimpton C, Walton A, Gill P. A further tetranucleotide repeat polymorphism in the vWF gene. Hum Mol Genet 1992;1:287.
6. Mills KA, Even D, Murray JC. Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus (FGA). Hum Mol Genet 1992;1:779.
7. Edwards A, Hammond HA, Jin L, Laskey CT, Chakraborty R. Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 1992;12:241-53.
8. Anker R, Steinbrueck T, Donis-Keller H. Tetranucleotide repeat polymorphism at the human thyroid peroxidase (hTPO) locus. Hum Mol Genet 1992;1:137.
9. Hammond HA, Jin L, Zhong Y, Laskey CT, Chakraborty R. Evaluation of 13 short tandem repeat loci for use in personal identification application. Am J Hum Genet 1994;55:175-89.
10. Hudson TJ, Stein LD, Gerety SS, Ma J, Castle AB, Silva J, Slonim DK, Baptista R, Kruglyak L, Xu SH. An STS-based map of the human genome. Science 1995; 270: 1945-54.
11. Green ED, Mohr RM, Idol JR, Jones M, Buckingham JM, Deaven LL, Moyzis RK, Olson MV. Systematic generation of sequence-tagged sites for physical mapping of human chromosomes: application to the mapping of human chromosome 7 using yeast artificial chromosomes. Genomics 1991;11:548-64.
12. Scharf SJ, Smith AG, Hansen JA, McFarland C, Erlich HA. Quantitative determination of bone marrow transplant engraftment using fluorescent polymerase chain reaction primers for human identity markers. Blood 1995;85: 1954-63.
13. Collins RH, Jr., Shpilberg O, Drobyski WR, Porter DL, Giralt S, Champlin R, Goodman SA, Wolff SN, Hu W, Verfaillie C, List A, Dalton W, Ognoskie N, Chetrit A, Antin JH, Nemunaitis J. Donor leukocyte infusion in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997;15:433-44.
14. Kolb HJ, Mittermuller J, Clemm C, Holler E, Ledderose G, Brehm G, Heim M, Wilmanns W. Donor leukocyte transfusion for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990;76:2462-5.
15. Kolb HJ, Schattenberg A, Goldman JM, Hertenstein B, Jacobsen N, Arcese W, Ljungman P, Ferrant A, Verdonck L, Niederwieser D. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 1995;86:2041-50.
16. Porter DL, Roth MS, McGarigle C, Ferrara JL, Antin JH. Induction of graft-versus-host disease as immunotherapy for relapsed chronic myeloid leukemia. N Engl J Med 1994;330:100-6.
17. Kimpton CP, Gill P, Walton A, Urquhart A, Millican ES, Adams M. Automated DNA profiling employing multiplex amplification of short tandem repeat loci. PCR Methods Appl 1993;3:13-22.
18. Walsh PS, Fildes NJ, Reynolds R. Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Res 1996;24:2807-12.
19. Bader P, Beck J, Frey A, Schlegel PG, Hebarth H, Handgretinger R, Einsele H, Niemeyer C, Benda N, Faul C, Kanz L, Niethammer D, Klingebiel T. Serial and quantitative analysis of mixed hematopoietic chimerism by PCR in patients with acute leukemias allows the prediction of relapse after allogeneic BMT. Bone Marrow Transplant 1998;21:487-95.
20. Bader P, Holle W, Klingebiel T, Handgretinger R, Benda N, Schlegel PG, Niethammer D, Beck J. Mixed hematopoietic chimerism after allogeneic bone marrow transplantation: the impact of quantitative PCR analysis for prediction of relapse and graft rejection in children. Bone Marrow Transplant 1997;19:697-702.

From the Clinical Pathology; 1Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Taipei; 2School of Medical Technology, Chang Gung University, Taoyuan.
Received: May 30, 2002; Accepted: Jul. 26, 2002
Address for reprints: Dr. Chien-Feng Sun, Department of Clinical Pathology, Chang Gung Memorial Hospital. 5, Fu-Shing Street, Kweishan 333, Taoyuan, Taiwan, R.O.C. Tel.: 886-3-3281200 ext. 2554; Fax: 886-3-3284376; E-mail: suncgj@cgmh.org.tw