Quantification of donor and recipient hemopoietic cells by real-time PCR of single nucleotide polymorphisms

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  • A Corrigendum to this article was published on 24 February 2004

Abstract

Analysis of changes in recipient and donor hemopoietic cell origin is extremely useful to monitor the effect of stem cell transplantation (SCT) and sequential adoptive immunotherapy by donor lymphocyte infusions (DLI). We developed a sensitive and accurate method to quantify the percentage of recipient and donor cells by real-time PCR using single nucleotide polymorphisms (SNPs) as markers. Allele-specific PCR of seven SNPs resulted in specific markers for donor or recipient in 97% of HLA-identical sibling pairs. Both, recipient- and donor-derived hemopoietic cells can be simultaneously analyzed in 67% sibling pairs. We expect this can be increased to approximately 99% by developing three additional SNP-PCR. Serial dilution of SNP-positive DNA into either SNP-negative DNA or water revealed a detection limit of 0.1–0.01% depending on the amount of input DNA and start Ct of the used SNP-PCR. Application of our real-time SNP-PCR method for a CML patient treated by allogeneic SCT and DLI demonstrated its feasibility to follow donor T-cell chimerism and early detection of residual and recurrent autologous hemopoiesis in response to treatment. This detailed monitoring of the genetic origin of hemopoietic cells, in particular immune effector cells and target cells after SCT and DLI, may substantially contribute to understanding of the mechanisms that play a role in the success of treatment.

Introduction

Transplantation with hematopoietic stem cells from HLA-identical sibling donors has been successfully used to treat patients with hemopoietic malignancies. Allogeneic stem cell trnasplantation (SCT) results in effective replacement of eradicated recipient stem cells. Moreover, immune reactivity of donor effector cells against residual malignant cells contributes significantly to the success of treatment. This immunoreactivity of donor T-cells also limits the success rate of the therapy by transplantation-related mortality because of severe graft-versus-host disease (GVHD).1 T-cell depletion of the stem cell graft reduces the incidence and severity of GVHD but results in an increase of relapse rate, which confirms the contribution of donor-derived immunocompetent cells in eliminating residual malignant cells.2 In addition, the significantly higher relapse rates in patients who received stem cells of genetically identical twins compared to patients who received stem cells of HLA-identical siblings strongly supports the hypothesis that alloantigens significantly contribute to immune reactivity of donor lymphocytes against malignant cells.3 Attempts are made to further exploit the immune reactivity of donor cells against recipient hemopoietic cells, including leukemia and lymphoma cells using a nonmyeloablative conditioning regimen prior to transplantation, followed by delayed donor lymphocyte infusion (DLI).4,5 Interestingly, this treatment regimen has also been applied to treat solid tumors and revealed that achievement of full donor T-cell chimerism was a prerequisite for response to treatment.6,7,8,9

DLI can be very effective in preventing and curing relapse of leukemia but harbors the risk of inducing fatal GVHD.10,11,12,13 Although the response rate of CML patients to DLI is quite high, the majority of patients with acute myeloid or lymphoid leukemia (AML and ALL) do not respond to DLI.14 Success of DLI may be improved when dose and timing of the given donor T-cell infusion can be adapted to each individual patient thereby minimizing the risk of GVHD and maximizing the reactivity against malignant cells.15 Mackinnon et al 16 have shown that repetitive administration of increasing number of donor lymphocytes resulted in complete remission in CML patients who did not respond to low-dose DLI. Moreover, infusions of escalating number of donor T cells induced less GVHD compared to DLI given as bulk dose.17 In addition, in vitro activation of donor cells can improve DLI. Slavin et al18 showed that recurrent leukemia cells in patients not responding to DLI could be effectively eliminated by donor T cells activated by IL-2 ex vivo. Furthermore, early detection of relapse may enhance the success rate of DLI by treatment before the onset of overt clinical relapse.19 Increasing numbers of BCR-ABL expressing cells can indicate early relapse of CML.10,11,12,13,14,15,16,17,18,19,20,21,22 Other fusion transcripts can be indicative to determine relapse of acute leukemia patients.23,24 It has also been suggested that monitoring of chimerism in lymphoid and myeloid subsets, isolated from peripheral blood of patients after SCT, allows detection of residual or recurrent leukemia cells.25,26,27 Imminent relapse of leukemia lacking a genetic marker may be prevented by adoptive cellular immunotherapy given early after an increase of percentage of recipient cells.27

The mechanisms responsible for failure of immunotherapy after SCT are not clear. Patients with a high percentage of autologous T cells showed reduced alloreactivity of infused donor T cells, which may suggest rejection of infused T cells or induced T cell tolerance.28,29 Donor-derived regulator T cells may also suppress reactivity of infused donor lymphocytes.30 Frequent analysis of chimerism in lymphoid and myeloid cells after SCT may be of great value to identify patients with high risk for relapse or graft rejection.24,25,26,31 Several techniques have been used in these studies to monitor chimerism after SCT. PCR of DNA sequences with tandem repeats (VNTR, STR) or satellite DNA has been applied frequently.25,26,27 FISH analysis to discriminate male and female cells have been utilized in sex-mismatched sibling pairs.

We have developed a real-time PCR method based on the detection of biallelic single nucleotide polymorphism (SNP). Biallelic SNPs exist with a very high frequency in the human genome.32 We show that the method is applicable for almost all recipient/donor pairs and can accurately quantify at least 0.1% recipient cells among donor cells and vice versa. Detailed monitoring of the genetic origin of hemopoietic cells after SCT and DLI can substantially contribute to understanding of the mechanism involved in response to therapy and guide adoptive immunotherapy strategies.

Material and methods

Patient and cell samples

Chimerism of hemopoietic cells was studied in patients who received T-cell-depleted SCT with stem cells from an HLA-identical sibling donor. CML patient (UPN 480) with Philadelphia chromosome positive (Ph+) CML cells was conditioned with 120 mg cyclophosphamide per kg body weight and total body irradiation (9 Gy). Cyclosporin A was given until 2 months after SCT to prevent GVHD. PBMC from peripheral blood collected before and following SCT and DLI was isolated by Ficoll density gradient centrifugation. T cells and myeloid cells were isolated by flow cytometry (Epics Elite, Beckman Coulter, Fullerton, CA, USA) after staining with CD3-FITC-conjugated mAb (UCHT1 Beckman Coulter) or CD13/CD33-PE-conjugated mAb (WM-54, WM 47, respectively, Dako, Glostrup, Denmark). Purity of both cell populations was >99.5%.

DNA preparation and SNP-typing by RLFP analysis

Genomic DNA was isolated with QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) from sorted cell populations, PBMC of donors and recipients collected before SCT, and from EBV-transformed B cells generated from PBMC of recipients collected prior to SCT and donors (see the Appendix for detailed information). DNA fragments containing SNPs in PECAM1, ICAM1, HA1, MLH1, SUR1, and the sequence-tagged sites (STSs) G42863, G42888 were amplified as described before.33,34,35,36,37,38,39,40,41 Genotype of SNPs was determined after digestion with restriction enzyme PvuII (PECAM1 and MLH1) and PstI (SUR1) by agarose gel electrophoresis.33,40,41 Genotype of SNPs in ICAM1, HA1, G42863 and G42888 w as determined by DNA sequencing of PCR products.

Specific amplification of SNPs by real-time PCR

DNA isolated from EBV-transformed cell lines bearing homozygously the identified SNP was used to develop and optimize Taqman-based real-time PCR for each of the SNPs (Perkin Elmer Applied Biosystems, ABI Prism 7700).42,43,44 In addition to allele-specific real-time PCR, we developed an SMCY-gene (accession number: AF273841) real-time PCR to quantify male cells (see the Appendix for detailed information).

Quantification of the percentage of cells containing allele-specific sequences

Calibration functions from Ct obtained by real-time PCR of serially diluted DNA isolated from cell samples homozygous, heterozygous or hemizygous harboring one of the marker sequences were generated. These calibration curves were used to calculate the percentages of recipient and donor cells in the blood samples collected after SCT (see the Appendix for detailed information). The amount of input DNA was accurately defined by real-time PCR using a DNA fragment encoding albumin.

Calculation of the discriminating capacity of biallelic SNPs

Genotype frequencies obtained by SNP-PCR from 80 SCT recipients and their HLA-identical sibling donors were compared with SNPs frequencies described earlier.33,34,35,36,37,38,39,40,41 The genotype frequencies we obtained by real-time PCR of SNPs were used to analyze the discriminative capacity of the seven SNPs. Calculation of probability that siblings have different genotypes with alleles A and B that are located on the autosomes revealed the following formula: [(Freq AA+5/8 Freq AB+Freq BB) × Freq AB]. The probability that siblings have different genotypes of SNPs located on the X chromosome was calculated by the formula: [5/8 Freq AB+1/2 Freq A × Freq BB+1/2 Freq B Freq AA].

Results

Development of SNP-specific real-time PCR for identification of recipient and donor cells

Seven SNPs of which high frequency in human populations have been described were selected for the identification of recipient- and donor-derived cells after SCT (Table 1). SNPs located on chromosome 6, that contain the MHC complex encoding the HLA molecules, were excluded. RFLP analysis and DNA sequencing of amplified fragments were used to define cells bearing the selected SNPs, homozygously, heterozygously or hemizygously. Using DNA isolated from these cells, we developed real-time PCR with SNP-allelespecific primers and gene-specific probes. To hinder amplification of the noncomplementary alleles, we synthesized SNP-allele specific primers containing one extra mismatch in one of the two adjacent nucleotides of the polymorphic nucleotides. We replaced these nucleotides for one of the three alternative nucleotides and determined which of these mismatched primers resulted in highest specific amplification and lowest background amplification. We observed significant differences in amplification efficiency and target specificity of allele-specific primers as shown for the A allele of MLH1 (Figure 1). High fluorescence signal early after amplification of the positive allele (MLH1-A, Ct 24.5) and low fluorescence during amplification of the negative allele (MLH1-G, Ct 35.9) was observed using the allele-specific primer ending with ATT (primer set 1 in Figure 1). In contrast, allele-specific primer ending with TAT (primer set 2 in Figure 1) showed lower sensitivity for MLH1-A (Ct 28.2) and allele-specific primer ending with CTT (primer set 3 in Figure 1) showed high background amplification of the negative allele MLH1-G (Ct 29.1). Using this approach we developed primer sets that reached the detection threshold prior to 25 cycles of amplification, and showed background amplification of the non-complementary allele after 35 cycles using 500 ng of genomic DNA. Table 2 shows the developed primer sets, the optimum annealing temperature and the amplification efficiency (Ct, and ΔRn after 45 cycles of amplification), obtained by real-time PCR for seven SNPs. Cycle threshold of amplification of repeated experiments ranged within 0.5 cycle, starting with the same amount of target DNA. Log dilutions of target DNA resulted in Ct differences of approximately three cycles. Theoretically, differences of more than 10 cycles in reaching the threshold between the positive and negative allele allowed quantification of at least 0.1% target DNA. Fixed annealing/elongation temperature of 60°C to screen 100 ng DNA for all seven SNP in one real-time PCR run resulted in Ct<30 using allele-specific primers for ICAM1, HA1, MLH1 and SUR1. Allele-specific primers for PECAM1 and G42863; G42888 were shortened to reach Ct<30 after amplification of 100 ng DNA.

Table 1 Characteristics of SNPs used as specific markers for recipient and donor cells
Figure 1
figure1

Amplification curves of the MLH1 SNP-alleles by real-time PCR. Amplification plots of PCR using three reverse primers specific for the A allele and a common forward primer are shown. Primer set 1, with reverse primer 5′-TCGTGCTCACGTTCTTCCATT-3′ reached threshold after 24.5 cycles for the A allele (▪) and after 35.9 cycles for the G allele (□) (ΔCt=11.4). Primer set 2, with reverse primer 5′-TCGTGCTCACGTTCTTCCTA T-3′ reached threshold after 28.2 cycles for the A allele () and after 41.8 cycles for the G allele () (ΔCt=13.6). Primer set 3, with reverse primer 5′-TCGTGCTCACGTTCTTCCCTT-3′ reached threshold after 24.4 cycles for the A allele (•) and after 29.1 cycles for the G allele () (ΔCt=4.7). Primer set 1 gave the best results for the MLH1-A allele.

Table 2 Amplification characteristics of developed SNP allele-specific primers in real-time PCR

The results shown here demonstrate that real-time PCR of target sequences with SNPs can be used to identify the genetic origin of cells. Moreover, this method is highly reproducible and applicable for detection of very small percentage of cells with specific SNP-markers.

Typing of allelic differences in recipient and donor cells by SNPs

To determine the capacity of the SNPs to discriminate between siblings, 80 SCT recipients and their HLA-identical donors were screened for the presence of the seven SNPs. First of all the genotype frequency of SNPs in 160 paired siblings was defined (Table 3). This analysis showed that genotype frequency of SNPs in either donors or recipients was similar (data not shown). Moreover, all SNP genotype frequencies that we determined correlated with those published, except one (Table 3). The frequency of the SNP in the MLH1 gene, we found, differs significantly from that determined in the Japanese population by Ito et al.39 As expected, frequencies of heterozygous genotypes for both SNPs located on the X chromosome were low because of single alleles in males (Table 3).

Table 3 Genotype frequencies of SNPs

Next, we analyzed the contribution of each SNP regarding their capacity to reveal specific molecular markers in sibling pairs. Exclusive appearance of one of the two polymorphic alleles in either recipient or donor was determined. The seven SNPs revealed a specific marker for either recipient or donor in 24–50% of the pairs (Table 4). The SNP in MLH1 showed the lowest capacity (24%) to mark genetically recipient and donor cells, because of high frequency of homozygous MLH1-G in the analyzed population. SNPs located on the X chromosome contributed as effective in specific recipient or donor marking as SNPs located on autosomes (Table 4). A significant number of sibling pairs (28%) allowed recipient- and donor-specific discrimination by both biallelic variants of one SNP. Hemizygous appearance of allelic SNP-variants on the X chromosome contributed dominantly to this phenomenon (Table 4). Furthermore, the probability of the seven biallelic SNPs to reveal specific markers for recipient and donor was calculated using the genotype frequencies that we had determined in 80 sibling pairs. The analyzed frequencies of SNPs as genomic marker correlate highly with that calculated suggesting that all SNP-markers segregate in a Mendelian fashion that is not influenced by the close relationship of siblings (Table 4).

Table 4 Recipients and donors (%) that can be discriminated based on SNP markers

The high percentages that each SNP could discriminate between siblings resulted in specific markers for either recipient or donor in 97% of these pairs (Table 4). The probability calculations based on genotype frequencies revealed the same discrimination capacity of the seven SNP-markers (Table 4). Each dimorphic SNP increased the percentage of pairs with a specific marker for both recipient and donor significantly (8–14%). Location of two SNPs on the same chromosome (19 and X) did not affect the accumulation of the percentage of sibling pairs with specific SNP markers (Table 4). The seven SNPs we utilized revealed a specific genomic marker for both recipient and donor in 67% of the 80 analyzed sibling pairs (Table 4). The PCR specific for SMCY located on the Y chromosome discriminated recipient and donor cells in all sex-mismatched sibling pairs and increased the percentage of pairs that have a specific marker for both recipients and donors with 12% (data not shown).

These results demonstrate that a restricted number of SNPs in the human genome can be used to genotype the vast majority of sibling pairs. Each SNP with a high heterozygous frequency has the ability to discriminate 30–50% of sibling pairs with either a recipient- or donor-specific marker. More importantly, all SNPs used for this analysis act as additional makers specific for either donor or recipient, which results in specific markers for 67% of both members of sibling pairs. Addition of three biallelic SNP- markers that appear to be highly discriminative will lead to specific markers for both recipient and donor in 99% of sibling pairs.

Quantification of recipient and donor cell ratios

We developed a quantitative assay to measure the percentage of recipient or donor cells by allele-specific real-time PCR in different cell populations. Calibration curves for each SNP were performed. Homozygous and heterozygous DNA for all targeted alleles were diluted either in DNA homozygous for the alternative (negative) alleles or in water. Amplification signals (Ct) were plotted against amount of input DNA. Real-time PCR of DNA encoding the albumin gene was simultaneously performed to normalize the amount of input DNA of test samples to the calibration samples. Figure 2 shows calibration curves of amplified SNP-positive DNA (PECAM1-GG and PECAM1-GC) diluted in negative DNA (PECAM1-CC) (Figure 2a) and amplified SNP-positive DNA diluted in water (Figure 2b). All calibration curves of DNA amplified by SNP-specific primer sets reached slopes of −3.3 (±0.2). Calibration curves for samples diluted in both negative DNA and water gave similar results for all seven SNP-PCR (data not shown). Deviation in Ct was stable and average of two-fold standard error of repeated samples did not exceed 0.4 cycle. The deviation of Ct obtained by real-time PCR is independent of the concentration target DNA in the samples that reached the threshold between 20 and 35 cycles of amplification. Therefore within this range, accuracy was directly related to the concentration and calculated confidential intervals were approximately +30% and −25% for all measured values. As shown in Table 2, all SNP-PCR showed low amplification of DNA containing the allele that was not complementary with the specific SNP-primers (Ct>35). Therefore, specific amplification of 500 ng DNA results in detection up to 0.1% SNP-positive cells in SNP-negative cells. Specific amplification of DNA with SNPs that reached the threshold prior to 22 cycles of amplification (ΔCt>13) can be quantified up to 0.01%.

Figure 2
figure2

Calibration curves of DNA. Serial dilutions of DNA containing homozygous (▪) or heterozygous (□) the G allele of PECAM1 (PECAM1-GG, and PECAM1-GC, respectively). Standard errors of Ct are ±0.25 cycle and presented by sizes of the boxes. (a) SNP-target sequence-positive DNA diluted in target sequence negative DNA (PECAM1-CC). (b) SNP-target sequence-positive DNA diluted in water.

The results show that real-time PCR of SNPs can be used to quantify chimerism in cell samples taken after SCT. Quantification of low percentage target-SNP-positive cells in mixed samples is very accurate, but standard errors are high for samples with high percentage target-SNP-positive cells. Analysis of hemopoietic cell populations of patients after SCT by both recipient- and donor-specific SNP-markers may result in significant information about chimerism present in these patients.

Analysis of recipient-derived hemopoietic cells following SCT and adoptive immunotherapy

To apply our SNP method for the determination of the origin of lymphocytes and myeloid cells after allogeneic transplantation, we studied in detail chimerism of one CML patient who relapsed and was subsequently treated with DLI (Figure 3). The patient relapsed at 9 months after SCT and received 0.7 × 108 T cells/kg body weight 2 weeks later. Acute GVHD was not observed, but the patient developed extensive chronic GVHD 3 months after DLI. The percentages of recipient-derived T cells and myeloid cells in blood were determined by real-time PCR of the ICAM1-G allele. Remarkably, a high percentage of T cells of recipient origin was detected in this patient at 1, 3 and 6 months after SCT. Within 1 month after DLI the percentage of recipient-derived T cells decreased very rapidly and fell below the detection limit prior development of chronic GVHD (Figure 3). A small percentage of autologous cells could be detected in the purified myeloid cells at 1 and 3 months after SCT (0.3 and 0.6%, respectively). However, myeloid cell samples were 99.5% pure, thus contaminating T cells may contribute to this signal. The percentage of myeloid cells of recipient origin was significantly increased at 7 months after SCT. Shortly after DLI, the percentage of recipient-derived myeloid cells still increased (Figure 3). At 2 months after DLI the myeloid cells of recipient origin dropped very fast to below 0.05% (Figure 3). A high percentage (50%) Ph+ cells was observed in bone marrow by FISH analysis at 6 months after SCT. The percentage of Ph+ cells in bone marrow increased up to 90% prior DLI and did not decrease until 1.5 months after DLI. All Ph+ cells disappeared within 5 months after DLI (data not shown).

Figure 3
figure3

Analysis of cells of recipient origin after SCT. T cells and myeloid cells are purified by flow cytometry after staining PBMC with cell lineage-specific mAb. The percentage recipient derived T cells () and myeloid cells (▪) are detected by real-time PCR with allele-specific primers for polymorph DNA of the ICAM1 gene. Gray bars represent the confidential interval of real-time PCR of SNP.

The data show persistence of high percentage of recipient-derived T-cells in this patient after SCT. The percentage of these T-cells decreased immediately after DLI prior to the onset of clinical GVHD symptoms, which suggests that alloreactivity of infused donor cells can be detected early by this method. Moreover, kinetics of myeloid cells of recipient origin in blood after SCT and DLI parallels that of Ph+ cells in BM as analyzed by FISH. This indicates that monitoring of chimerism in myeloid cells may be informative regarding relapse and response to treatment for those myeloid leukemia patients whose leukemia cells do not bear a specific tumor marker.

Discussion

The aim of the present study was to develop an accurate, sensitive and fast method to quantify the percentage of recipient and donor cells present after transplantation of HLA-identical stem cells. Following SCT, the majority of transplanted patients have hemopoietic cells of both recipient and donor origin (mixed chimerism) at least for a short period of time. Hemopoietic cells of some of these patients remain of mixed origin for a long period of time or revert to autologous hemopoiesis.46 The influence of mixed chimerism on leukemic relapse, grafts rejection, and treatment failure after SCT have been studied extensively.47,48,8 It has become clear that the hemopoietic cell lineage in which mixed chimerism occurs contribute to the choice for adoptive immunotherapy after SCT.49,50 T-cell mixed chimerism contributes clearly to graft tolerance and resistance against GVHD.28,30,51 Mixed myeloid cell chimerism in acute and chronic myeloid leukemia patients may reflect residual disease and may early indicate imminent relapse of disease.24,25,26 Particularly, the kinetics of recipient and donor ratios in these cell lineages would be very informative about immune reactivity and course of disease and will be useful in determining strategies of additional treatment.26,27

We developed a real-time PCR method using seven genomic SNPs markers that are capable of discriminating either recipient or donor cells in 95% of HLA-identical sibling pairs. Logarithmic diluted concentrations of input target DNA are proportionally linear with Ct. Deviation in Ct is independent of the concentration DNA ranging between 500 and 0.05 ng. Therefore, the sensitivity is very high for low concentrations of target DNA but low for high concentrations of target DNA. However, to reach high sensitivity in both directions, two discriminative SNPs can be used with two calibration curves. One calibration curve to quantify a range from 0.1 to 50% recipient cells in donor cells using one SNP-PCR and one calibration curve to quantify a range from 0.1 to 50% donor cells in recipient cells by another SNP-PCR. One recipient-specific and one donor-specific SNP-PCR can be utilized for sensitive monitoring after SCT and DLI. By this approach seven biallelic SNP markers resulted in very sensitive monitoring of chimerism in 67% of our patients. Additional real-time PCR analysis with three SNPs allows a screening that can routinely be applied to determine specific markers for 99% of both recipients and donors. In addition to SNP-PCR, we developed an SMCY-gene-specific real-time PCR to quantify male cells in sex-mismatched recipient donor pairs. A similar approach has been described for the DFRY gene by Fehse et al.52 Male-specific PCR are markers in 50% of randomly selected recipient and donor pairs and can be utilized as biallelic marker for either recipient or donor. Dimorphic SNPs revealed to be specific markers for either recipient or donor of sibling pairs comparable to male-specific markers.

As mentioned earlier, very small percentages of recipient cells in donor cells and vice versa can be detected by real-time PCR. A detection limit of 0.01% can be reached using SNP-specific primers that efficiently amplify the positive allele (Ct22) and show low amplification (Ct>35) of the negative alleles. Standard errors in repeated PCR are small (±0.2 cycle) which results in confidence interval of roughly +30 and −25% of the measured values. However, quantification of SNP-specific DNA by real-time PCR is more accurate than quantification of products by conventional PCR that amplify DNA samples to a fixed number of cycles. PCR products that differ in length by the number of tandem repeats sequences (STR/VNTR) utilize electrophoresis to quantify both recipient- and donor-specific products amplified in the same PCR. Analysis of recipient cells and donor cells by separate real-time PCR of SNPs exclude interference with nonspecific signals. Moreover, real-time PCR excludes spectral overlap of recipient- and donor-specific fluorescence signals or stutter peaks of amplified recipient DNA that comigrate with donor specific peaks or vice versa, as sometimes observed by the amplification of STR.53,54 In addition, real-time PCR of SNPs is less hampered by the competition for reagents that may occur by the amplification of PCR products that significantly differ in length. Moreover, the use of reference gene amplified by real-time PCR may better define the amount of input DNA compared to conventional PCR. However, sensitive quantification of recipient- and donor-derived cells in leukocyte subsets may be limited by efficiency of purification.

The percentage recipient and donor pairs that have an SNP- marker is significantly higher than the number of patients and donors that have markers applicable for FISH analysis and can compete with STR markers.55,56,57 Use of specific PCR primers for about 10 SNPs with high heterozygous frequencies will result in a discriminative marker for both recipient and donors in approximately 99% of all sibling pairs. The reproducibility of our SNP-marker analysis is very high and the method is significantly less laborious compared to marker analysis by FISH. High percentage of recipient and donor cells can be quantified more exactly using FISH analysis.58 Real-time PCR can sensitively detect low percentage of cells with recipient -or donor-specific sequences because of the restricted number of cells that is analyzed after FISH. Specific target sequences in recipient and donor DNA can be separately utilized for quantification by real-time PCR and thus result in sensitive quantification of both recipient and donor DNA present in one-cell samples.

In summary, monitoring of hemopoietic chimerism after SCT may early indicate graft rejection and relapse. Moreover, it gives significant information about the immunological response after SCT and DLI. We show that real-time PCR of SNPs is a sensitive and very reliable method to analyze chimerism. The method is less laborious compared to FISH analysis and quantification is more accurate than that performed after gel electrophoresis and conventional PCR.

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Acknowledgements

We are indebted to Marieke Overdijk, Paulien Polderman, Adrian van der Heijden, and Rob Woestenenk for screening recipient and donor DNA for SNPs. We thank Dr Joop Jansen for critically reading the manuscript. This work was supported by the Dutch Cancer Foundation Grant KUN 96-1363.

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Correspondence to E van de Wiel-van Kemenade.

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Maas, F., Schaap, N., Kolen, S. et al. Quantification of donor and recipient hemopoietic cells by real-time PCR of single nucleotide polymorphisms. Leukemia 17, 621–629 (2003) doi:10.1038/sj.leu.2402856

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Keywords

  • transplantation
  • adoptive immunotherapy
  • chimerism
  • SNP-markers
  • real-time PCR

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