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Technical Briefs |
Departments of
1
Chemical Pathology,
2 Medicine & Therapeutics, and
3
Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR
4 Department of Cardiology, Grantham Hospital, Hong Kong SAR
5 Department of Cardiology, Green Lane Hospital, Auckland 1003, New Zealand
aaddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR; fax 852-2194-6171, e-mail loym{at}cuhk.edu.hk
Despite current interest in the biology and diagnostic application of plasma cell-free DNA (1), there is little knowledge regarding the cellular origin of this DNA. Recently, we have used a sex-mismatched bone marrow transplantation model to study the relative contributions of hematopoietic and nonhematopoietic cells to circulating DNA (2). We have demonstrated that the predominant proportion of plasma DNA originates from the hematopoietic system (2). However, the proportions of cell-free plasma DNA originating from other organs (e.g., heart, liver, and kidneys) remain unknown. We therefore investigated the contribution of the heart, liver, and kidneys to circulating DNA with use of sex-mismatched heart, liver, and renal transplantation models, respectively.
Sex-mismatched heart, liver, and renal transplantation patients were recruited for the study. Twenty-one patients who had received heart transplants were recruited from the Grantham Hospital, Hong Kong. Fourteen of these heart transplantation patients were females with male donors, whereas the remaining 7 were males with female donors. Four sex-mismatched liver transplantation patients at the Pediatric Surgical Unit of the Department of Surgery, Prince of Wales Hospital were recruited; two of these patients were females with male donors and the other two were males with female donors. Six sexmismatched renal transplantation patients were recruited from the Department of Medicine and Therapeutics of the Prince of Wales Hospital. Three of these patients were females with male donors, whereas the remaining three were males with female donors. Informed consent was obtained from all individuals. None of these transplantation recipients had evidence of graft rejection or graft-vs-host disease. Ten healthy individuals were also recruited with informed consent.
Peripheral blood samples were collected into EDTA tubes from all participants. The blood samples were subjected to centrifugation at 1600g for 10 min, followed by microcentrifugation at 16 000g for 10 min (Eppendorf Centrifuge 5415D) to obtain cell-free plasma (3). DNA was extracted with use of the QIAamp Blood Kit (Qiagen) according to the "blood and body fluid protocol" as recommended by the manufacturer (4). We used 400 µL of plasma per column for DNA extraction. We then subjected 5 µL (of an elution volume of 50 µL) of the extracted plasma DNA to real-time quantitative PCR for the ß-globin and SRY genes as described previously (5), using a PE Applied Biosystems 7700 Sequence Detector. The percentage of male DNA in each plasma sample, denoted as Y%, was calculated as described previously (2). Statistical tests were carried out with SigmaStat 2.0 software (SPSS).
The results are illustrated in Fig. 1
. The assay imprecision for the Y% values was described previously (2). Analysis of the Y% of the plasma indicated that it was different between the group of female patients receiving organ transplants from male donors and the group of male patients receiving organ transplants from female donors (Fig. 1
). The median Y% of plasma samples in the former and the latter groups was 0.0% and 83.8%, respectively. The difference between these two groups was highly significant (Mann–Whitney rank-sum test, P <0.001). Three male recipients with female donors had a calculated Y% value >100%, possibly as a result of the imprecision of the assay. In the group of female patients receiving organ transplants from male donors, the SRY sequences detected originated predominantly from the graft of the male donors. Hence, the Y% of plasma in this group of patients indicated the percentage of plasma cell-free DNA originating from the respective organ. On the other hand, in the male patients receiving organ transplants from female donors, the SRY sequences detected mainly originated from hematopoietic cells of the male recipients, with a minority originating from nonhematopoietic cells of the male recipients. Thus, the Y% of plasma in these cases reflected the percentage of free plasma DNA originating from all cells of the body other than those in the graft. We therefore concluded that the heart, liver, and kidneys account for only a minority of the plasma cell-free DNA.
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To investigate whether organ transplantation was associated with a quantitative aberration in the concentrations of circulating DNA, we compared total plasma DNA concentrations in the transplant patients and the 10 healthy controls. The median DNA concentrations of the controls and the patients studied were 916.4 and 1336.3 genome-equivalents/mL, respectively. No significant difference was observed between these values (Mann–Whitney rank-sum test, P = 0.076).
In this study, we have provided a quantitative estimation of the proportions of plasma cell-free DNA originating from different nonhematopoietic tissues, namely, the heart, the liver, and the kidneys. These nonhematopoietic cells accounted for only a minority of the free circulating DNA, agreeing with our previous findings that the hematopoietic system is the predominant origin of plasma cell-free DNA (2).
Although our previous study revealed the presence of donor-specific DNA in the plasma of kidney and liver transplant recipients (6), the present work provides the first quantitative study of donor-specific DNA sequences in the plasma of patients receiving solid organ transplants. The quantitative information in this report provides normative values for clinically stable posttransplantation patients with no evidence of graft rejection or other pathologies. Because plasma DNA has been associated with cell death (7), plasma donor DNA may be released as a result of graft rejection or other sources of tissue damage in the transplanted organ, such as graft infection and neoplastic involvement of the graft. This suggests the theoretical possibility that the concentration of donor DNA in the recipient’s plasma may be a marker for these processes. Of relevance is the recent demonstration that urinary cell-free DNA is increased in patients with renal graft rejection (8)(9). In this regard, it would be interesting to obtain quantitative data for serial samples from patients receiving solid organ transplants, especially those who are suffering from graft rejection episodes.
Acknowledgments
Y.M.D.L. is supported by the Innovation and Technology Fund (AF/90/99 and ITS/195/01), the Hong Kong Research Grants Council, and the Direct Grants Scheme of the Chinese University of Hong Kong.
References
The following articles in journals at HighWire Press have cited this article:
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  K. Saukkonen, P. Lakkisto, V. Pettila, M. Varpula, S. Karlsson, E. Ruokonen, K. Pulkki, and for the Finnsepsis Study Group Cell-Free Plasma DNA as a Predictor of Outcome in Severe Sepsis and Septic Shock Clin. Chem., June 1, 2008; 54(6): 1000 - 1007. [Abstract] [Full Text] [PDF]
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T. R. Gingeras, R. Higuchi, L. J. Kricka, Y.M. D. Lo, and C. T. Wittwer Fifty Years of Molecular (DNA/RNA) Diagnostics Clin. Chem., March 1, 2005; 51(3): 661 - 671. [Full Text] [PDF]
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 Y. Li, X. Y. Zhong, A. Kang, C. Troeger, W. Holzgreve, and S. Hahn Inability to Detect Cell Free Fetal DNA in the Urine of Normal Pregnant Women nor in Those Affected by Preeclampsia Associated HELLP Syndrome Reproductive Sciences, December 1, 2003; 10(8): 503 - 508. [Abstract] [PDF]
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, 
, and 
, respectively. The percentage of Y-chromosome DNA in the plasma (Y% of plasma) is plotted on the y axis.