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Technical Briefs |
1 Department of Laboratory Medicine, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul, Korea;2 Department of Diagnostic Laboratory, Center for Clinical Services, National Cancer Center, Goyang-si, Gyeonggi-do, Republic of Korea;
aaddress correspondence to this author at: Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, Korea; fax 82-2-3410-2719, e-mail jwonk{at}smc.samsung.co.kr
Monitoring of chimerism after allogeneic stem cell transplantation is important for the early diagnosis of graft failure or disease relapse. Two main approaches used for monitoring chimerism are fluorescence in situ hybridization and PCR of short tandem repeats (STRs) expressing high degrees of polymorphism (1). Although both methods are useful, the STR assay has been increasingly used because fluorescence in situ hybridization can be used only in cases with specific genetic aberrations or a sex-mismatched donor (2)(3)(4). The STR assay, which produces quantitative results within 1 day, uses fluorescence-labeled primers and a capillary electrophoresis system (5)(6). However, the precision of the assay’s performance with respect to the chimeric stages of hematopoietic cells has not been fully investigated. We therefore aimed to evaluate the assay imprecision (CV) for seven STRs, D7S820, D8S1179, D16S539, D18S51, D21S11, TH01, and TPOX, to determine whether the precision changes according to the degree of chimerism. We used cell mixtures at various concentrations to simulate hematopoietic chimerism, and we also determined the detection limit.
In an initial screening to find adequate samples for evaluation, we obtained peripheral blood specimens from 96 volunteer donors. Genomic DNA was isolated by use of Wizard Genomic DNA purification reagents (Promega) and was assayed for allele determination for seven different STRs. PCRs were set up in a final volume of 25 µL containing 10x buffer, 200 µM deoxynucleotide triphosphates, 5 pmol of each primer labeled with a fluorescence dye, 1 U of Taq polymerase (Roche), and template DNA. PCR was carried out in a GeneAmp PCR System 9600 (Applied Biosystems), and PCR products were analyzed by capillary electrophoresis on an ABI 3100 (Applied Biosystems) with performance-optimized polymer 4 (POP-4), a 47-cm capillary, and GA buffer plus EDTA. We mixed 1 µL of PCR product with 12 µL of deionized formamide containing 0.3 µL of GeneScan-500 ROX Size Standard (Applied Biosystems). Each sample was heated at 93 °C for 3 min to denature the DNA, chilled for 3 min at 4 °C, and then separated on ABI 3100. The sizes of the PCR products were determined by use of GeneScan software (Applied Biosystems).
After we determined the alleles for the 96 donors based on PCR product size, we calculated the number of alleles, the heterozygosity, and useful statistical values for application to STR analysis, by use of the PowerStat program (Promega).
On the basis of the allele data, we chose two unrelated individuals among volunteers who shared only one allele for each STR. We excluded individuals who had stutter bands because interpretation could be difficult when stutter bands were present. We drew whole blood from the selected volunteers and determined leukocyte counts on the XE-2100 automated hematology analyzer (Sysmex). To simulate mixed chimerism, we calculated the volumes required to achieve a constant 107 leukocytes with a targeted proportion of each sample. For example, when A and B had 5000 and 8000 leukocytes/µL, respectively and we had planned to make 1:1 mixtures of A and B, we took 1000 µL of the well-mixed whole blood from A and 625 µL from B. The mixture of the two would then contain 5 x 106 leukocytes from A and 5 x 106 leukocytes from B. After mixtures had been prepared, DNA extraction was performed as described above.
Samples targeting five different concentrations (1%, 5%, 50%, 95%, and 99% of one selected donor) were used to calculate the precision of each STR assay. Each sample was processed separately, and the measurement protocol consisted of two runs per day for 7 days. The results are presented as the ratio of the donor peaks area, which was calculated as follows: ratio = area of donor peaks/area of donor and recipient peaks (7). To determine the detection limit, we prepared 17 samples with concentrations ranging from 0% to 100% and assayed them twice. The detection limit was defined as the lowest dilution concentration at which the peak-area ratio of the minor cell population was >0.01 in each of two estimations.
The information for the observed alleles and statistical values for the seven STRs are represented in Table 1
. The detection limits of the seven STR assays were between 0.5% and 5%, and the imprecision results are shown in Fig. 1
. The imprecision ranged from 5.4% for TPOX to 12% for D7S820, on average, for all concentrations, and was inversely related to the proportion to the concentration of cell mixtures.
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This study shows that the precision differed among STR assays and, for each marker, was related to the concentration of cell mixtures simulating chimeric stages of hematopoietic cells. This was especially evident at low-range concentrations <5%, where the imprecision of the seven STR assays was high and ranged from 6.3% to 34% for TPOX and D18S51, respectively.
There was no common guideline for interpretation of STR assays with respect to clinical response. Many studies have emphasized that a change or trend of mixed chimerism can have clinical consequences. Although no consensus has been reached for the clinically relevant decision cutoff in the STR assay, a previous study showed that in five patients who suffered early graft rejection, the concentration of recipient cells was 5–10% at days 30–40 after bone marrow transplantation (7). The authors of that study also described a patient with acute myeloid leukemia who had recipient cells concentrations of 2.5–5% at days 30–40 without any clinical consequence. Although we could not decide on a clinically relevant decision cutoff based on a single study result, it is probable that the STR marker with high imprecision at cell concentrations of 5–10% may be inappropriate for monitoring chimerism. In our study, D7S820 showed imprecision as high as 21% for a 5% cell mixture. Considering that this marker has been included in many STR systems and is used in many laboratories, we suggest that investigations of the imprecision of STR assays need to be performed in each laboratory.
The detection limits of the seven STR assays were <5% in this study and were comparable to those from previous studies using fluorescently labeled primers (5)(6)(8)(9)(10). Each STR assay, however, had a different detection sensitivity. For example, whereas both D8S1179 and D18S51 were represented in our study as good informative markers having a high power of exclusion and low matching probability, they showed considerable differences. Aside from the type of STR, detection limits can be influenced by the allele types of the donor and recipient. In a case in which at least one recipient allele was shorter than the donor alleles, the STR assay tended to display a better detection sensitivity (8), and a distinct recipient STR allele with an 8- to 10-bp difference from the donor showed the best results in a recent study (11).
Because the selection of STRs for follow up of hematopoietic chimerism may be critical in clinical applications, effective strategies for choosing the appropriate STR are essential for each laboratory. Moreover, it is necessary to determine how many alleles will be tested. Although we think the best way to decide on the kind and number of alleles to be used for STR assays is to observe large actual donor-recipient data on each population, the minimum number for a given STR assay may be suggested by use of combined match probability. In the present study, the combined match probability ranged from 1.5 x 10–3 for two STRs to 1.7 x 10–8 for seven STRs, magnified in order of heterozygosity. Considering the fact that the size of the donor registry necessary to find at least one HLA-matched donor for 90% of random patients was 400 000 in Korea (Y.-S. Yang, unpublished data), the six STR markers (D7S820 excluded because of high imprecision) with a combined matching probability of 2.0 x 10–7 would cover sufficient donor-recipient pairs among patients receiving transplants of stem cells from unrelated individuals in our population. However, these data could not be used for patients receiving stem cells transplanted from relatives or in other populations.
Because data from one laboratory cannot be used in other laboratories or populations, a performance evaluation of the STR assay adopted by each laboratory is important. Additionally, we suggest that cell mixtures containing low-range concentration be included in the imprecision evaluation protocol.
In conclusion, the STR assay for hematopoietic cell chimerism assessment has advantages such as sex independence and a small sample size requirement. However, careful consideration is required because imprecision may differ among STR assays and may potentially be high, especially in early graft failure or disease relapse.
Acknowledgments
This work was supported by National Research Laboratory Grants from the Korea Institute of Science & Technology Evaluation and Planning, Korea.
References
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