Clinical Chemistry 52: 379-382, 2006.
First published January 5, 2006; 10.1373/clinchem.2005.058974
(Clinical Chemistry. 2006;52:379-382.)
© 2006 American Association for Clinical Chemistry, Inc.
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Molecular Diagnostics and Genetics |
Soluble Donor DNA Concentrations in Recipient Serum Correlate with Pancreas-Kidney Rejection
Vijayakrishna K. Gadi1,2,a,
J. Lee Nelson1,2,
Nicholas D. Boespflug1,
Katherine A. Guthrie1,3 and
Christian S. Kuhr1,4
1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA.
Departments of2
Medicine, 3
Biostatistics, and4
Surgery, University of Washington, Seattle, WA.
aAddress correspondence to this author at: 1100 Fairview Ave N, D2-00, Seattle, WA 98109. Fax 206-667-5255; e-mail vkgadi{at}u.washington.edu.
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Abstract
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Background: There is no reliable serum marker available to monitor incipient pancreas or islet-cell rejection. We tested the hypothesis that quantification of donor-specific genomic DNA in serum (from tissue damage) can serve as a marker of rejection.
Methods: Using a recently developed panel of HLA-specific quantitative PCR assays (Q-PCR), we tested 158 sera from 42 pancreas-kidney transplant recipients. Temporally related biopsies for 65 sera permitted analysis for correlation of donor DNA concentrations with rejection.
Results: Donor DNA concentrations were higher in sera from recipients who had experienced allograft rejection (n = 31) than from those who had not (n = 34). Median concentrations, expressed as the genome-equivalent (gEq) number of donor cells per 106 host cells, were 2613 and 59 gEq/106, respectively (P = 0.03).
Conclusion: Q-PCR for donor-specific genetic polymorphisms merits further investigation as a noninvasive approach to monitor pancreas-kidney as well as other types of allograft rejection.
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Introduction
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For patients with type I diabetes, the only reliable method to restore euglycemia is allotransplantation of functional islets containing insulin-producing ß cells. Although 1-year pancreas graft survival exceeds 80%, nearly one half of patients experience at least 1 episode of acute graft rejection (1). Acute cellular rejection (ACR)
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represents a form of tissue injury mediated by the host adaptive immune system that, if untreated, is ultimately fatal to the grafted tissue. Percutaneous biopsy with ultrasound guidance to diagnose pancreas rejection has an overall diagnostic sensitivity of only 79%–88% (2)(3) and carries a major complication risk of 
2%–7% (4).
Unfortunately, reliable serum markers for ACR are lacking in pancreas transplantation. Serum enzymes such as amylase and lipase lack sufficient specificity or sensitivity to serve as sole markers for ACR (5), and recurrent hypoglycemia is a late consequence of graft rejection (6). In diabetic patients receiving pancreas grafts, the reemergence of anti-islet antibodies has been proposed as a serum marker for ACR (7). However, the reappearance of autoantibodies was more specific for chronic loss of ß cells and less specific for ACR or for loss of exocrine function of the pancreas. Thus, the need for noninvasive, sensitive, and inexpensive tests as markers for pancreas allograft rejection remains unmet.
Our laboratory has developed a panel of 16 real-time quantitative-PCR (Q-PCR) assays targeting specific HLA sequences that can be used to quantify DNA from a specific allogeneic source; the assay has a detection limit of 1 chimeric genome in 105 host genomes (8). Multiple HLA mismatches are present in nearly all pancreas-kidney transplants. We investigated whether higher quantities of cell-free donor-specific HLA DNA in recipient serum correlated with allograft destruction as corroborated by biopsy.
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Materials and Methods
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hla-specific q-pcr
Methods for DNA analysis were identical to those reported previously (8) with the exception of modifications to permit measurement from sera. Briefly, a calibration curve for the HLA-specific assay of interest was generated with known quantities of genomic DNA [0, 0.5, 1, 5, 10, 50, 100, and 500 genome-equivalents (gEq)] derived from Epstein–Barr virus–transformed cell lines that were previously HLA typed and known to be homozygous for the allele of interest. A separate ß-globin calibration curve was created to quantify the total amount of genomic DNA derived from both host and donor within each specimen. Total genomic DNA was isolated from patient sera (200 to 500 µL) by use of a DNA Mini Kit (Qiagen) with a final elution volume of 50 µL. Specimen HLA Q-PCR reactions contained 10 µL of template DNA (or 5 µL for the ß-globin assay to maximize the amount of DNA eluate available for HLA assays), 25 µL of TaqMan Universal Master Mix (Applied Biosystems), 300 nM each of the forward/reverse primers (MWG Biotech), 100 nM dual-labeled probe (MWG Biotech), and DNase/RNase-free water to a final volume of 50 µL. Of note, to prevent PCR competition for the reagents between the more prevalent ß-globin PCR product and the less prevalent HLA-specific product, assays were performed in a nonmultiplexed format with HLA-specific and ß-globin assays contained in separate wells on the same plate. Four wells were measured, on average, for each serum sample for HLA and 1 well for ß-globin. PCR reactions were incubated in an ABI Prism 7000 thermocycler for 2 min at 50 °C, followed by 45 cycles of 95 °C for 15 s and 60 to 64 °C, depending on the HLA assay, for 1 min. HLA (or ß-globin) quantities were determined for sample wells by plotting on the calibration curve the point at which a fluorescence threshold for a given assay was exceeded. Results were rejected and assays repeated if either calibration curve correlation coefficient (r2) was <0.99.
HLA quantities were expressed as the total number of cell-free gEq/mL of serum as calculated by the equation:
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The equation accounts for the proportion of the DNA extract that was amplified for each target allele (elution volume/sum of sample volume). In addition, we determined the ratio of donor DNA to host cell-free DNA (as determined by simultaneous ß-globin Q-PCR) in the serum to control for nonspecific increases in total soluble DNA. The ratio was subsequently corrected to reflect whether the assayed HLA allele was present in 1 or 2 copies in the donor genome.
samples
Serum samples from 42 simultaneous kidney-pancreas transplant recipients (age at transplantation, 22–54 years) were obtained from a transplantation serum repository established at the University of Washington. A total of 158 sera were available for analysis with 2 to 7 time points per patient, ranging from before transplantation to nearly 5 years after transplantation. Chart reviews were performed by investigators blinded to the results of donor DNA measurement to ascertain each patient’s posttransplantation course to identify episodes of kidney or pancreas allograft rejection as determined by standardized histologic grading of biopsies. Pancreas-kidney transplant recipients were selected without previous knowledge of rejection status or outcome, based on donor HLA-mismatch with the recipient that could be targeted by an assay in our panel of HLA-specific Q-PCR assays. The local Institutional Review Board for human studies approved the study.
statistical analysis
Analyses were performed with SAS statistical software. Donor-specific DNA in recipient serum at a given time point was analyzed in a logistic regression model, and measurements based on multiple blood draws per patient were entered into the analysis as repeated measures to adjust for possible correlation between values within a patient (9).
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Results
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pretransplantation recipient dna and early posttransplantation donor dna concentrations
HLA-specific Q-PCR was preformed on pretransplantation specimens to ensure that the HLA sequence chosen to detect donor DNA was specific to the donor. Most patients had no detectable donor HLA genomic DNA in pretransplantation specimens (Table 1
). Four patients had low concentrations of non-self HLA DNA (<5 gEq/mL) in pretransplantation sera, a finding attributable to preexisting DNA from other sources (e.g., maternal/fetal microchimerism or previous blood transfusion) (10)(11)(12)(13). Additional serum (or DNA) was not available in the repository; we therefore were unable to retest time points with more than one HLA-specific assay.
Serum samples from the first week after transplantation were available for 36 patients. Donor-specific DNA was detectable in 94% of patients, with a median concentration of 11.3 gEq/mL (range, 0–461 gEq/mL) or 1350 gEq/106 recipient genomes (range, 0–115 000 gEq/106 recipient genomes). This result was concordant with our expectation that large numbers of cells damaged by ischemia-reperfusion injury were likely being cleared in the period immediately after transplantation. We do not know whether early procedural events are reflected in increased donor-specific DNA as information regarding donor organ ischemic times and cadaveric donor medical conditions was unavailable for the current studies.
donor dna and acute rejection
Day 8 and beyond was defined as the at-risk period for acute rejection. Sixty-five serum specimens from 39 patients (3 patients were excluded because all records preceded day 7) with temporally matched biopsies were available for analysis. Donor DNA concentrations were higher in sera associated with rejection (n = 31) than in sera not associated with rejection (n = 34). The median concentrations of donor DNA were 10.4 and 0.9 donor gEq/mL of host serum in rejection and nonrejection samples, respectively (Table 1
). When corrected for total cell-free serum DNA, the median concentrations for rejection and nonrejection specimens were 2613 and 59 donor gEq/106 host cell free genomes, respectively.
In a logistic regression model, higher concentrations of donor-specific DNA in recipient serum at a given time point were associated with greater chance of ACR in biopsy specimens. Because distributions were heavily skewed, data were log2-transformed to approximate a gaussian distribution. Rejection status was treated as a binary variable to estimate the odds of rejection for 2 HLA quantities, one twice as large as the other. For the absolute measure of gEq/mL of patient serum, as DNA quantity doubled, the odds for rejection increased 1.24-fold (95% confidence interval, 1.05- to 1.45-fold; P = 0.01). For the relative measure of gEq/106 host cell-free genomes, the odds for rejection increased 1.09-fold (95% confidence interval, 1.01- to 1.18-fold; P = 0.03).
One potentially powerful application of HLA-based donor DNA measurement in serum would be the ability to longitudinally follow ACR and the outcome of treatment. The relative concentrations of donor DNA in host serum (gEq/106 host genomes) in an illustrative case (patient 28) over time, obtained with the DQB1*06 assay, are shown in Fig. 1
. Donor DNA concentrations were increased during rejection but were undetectable after its resolution. A similar plot was obtained for the measurement of donor gEq/mL of host serum (not shown). Because few serum specimens from the same individual could be matched with several concomitant biopsies over time, we could meaningfully correlate ACR and donor DNA concentrations longitudinally in only a subset of individuals in this initial study.
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Figure 1. Longitudinal assessment of donor DNA concentrations and acute rejection.
High concentrations of donor DNA (given in gEq/106 host genomes), as measured by the DQB1*06 assay, were detected in serum from patient 28 at points correlating with ACR in pancreas and kidney specimens (*) or pancreas alone (**). Day 196 was negative for ACR in the pancreas.
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Discussion
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We demonstrate for the first time that detection and quantification of donor DNA by HLA-specific Q-PCR in recipient serum may serve as a noninvasive surrogate biomarker for identifying pancreas-kidney allograft damage. By either of 2 measures, the absolute quantity of donor gEq/mL or the fraction of donor gEq compared with host gEq, we found a significant concordance of increased values and biopsies positive for ACR. Whether donor-derived serum DNA is specific to graft rejection (vs other causes of graft damage) is not known. In the current cohort, only one instance of nonimmune-mediated graft damage without concomitant rejection was identified (patient 13, day 15 biopsy with thrombotic microangiopathy) and had modest concentrations of donor DNA (4.1 gEq or 1934 gEq/mL of serum). Although the presence of allogeneic donor DNA is not exclusive to rejection, this approach offers potential as a marker from which to base decisions to biopsy or to treat when biopsy is not possible. On the basis of the current findings, we do not know whether donor DNA might serve as surrogate biomarker by itself or alternatively as a component in a clinical prediction algorithm for rejection. Because this new approach is potentially applicable for noninvasive monitoring for rejection in transplant recipients, a careful prospective evaluation is warranted.
The present study focuses on pancreas-kidney transplantation; however, noninvasive HLA-specific Q-PCR assay use could be generalized to other types of transplantation (liver, heart, tissue, and cellular). Quantification of donor DNA by the approach described here may be particularly useful in the management of immune suppression in the emergent field of islet cell transplantation, a situation in which no organ is available for biopsy.
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Acknowledgments
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Drs. Gadi, Nelson, and Kuhr conceived and designed the experiments and were primarily responsible for the writing of this manuscript. Dr. Gadi and Nicholas Boespflug performed sample processing and Q-PCR assays. Dr. Guthrie performed the statistical analysis. The funding sources had no involvement with any aspect of design of experiments or with the writing of this manuscript. This work was supported by Grants AI-45659, AI-41721, and NCRR-MD1-RR-00037 from the National Institutes of Health.
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Footnotes
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1 Nonstandard abbreviations: ACR, acute cellular rejection; Q-PCR, quantitative PCR; and gEq, genome equivalent(s). 
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References
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Abstract
Introduction
