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Editorials and Perspectives: Overview

Peripheral Blood Sampling for the Detection of Allograft Rejection: Biomarker Identification and Validation

Heidt, Sebastiaan1; San Segundo, David1; Shankar, Sushma1; Mittal, Shruti1; Muthusamy, Anand S.R.2; Friend, Peter J.2; Fuggle, Susan V.2; Wood, Kathryn J.1,3

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doi: 10.1097/TP.0b013e318218e978
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Organ transplantation is the treatment of choice for patients with end-stage organ failure. Despite excellent short-term outcomes, mainly as a result of cocktails of immunosuppressive drugs, acute and chronic rejection episodes still occur, potentially affecting long-term graft function. Currently, especially in the case of kidney transplants, rejection is suspected when graft function deteriorates and is usually confirmed by biopsy. At this stage, organ damage has long been established, and consequently, antirejection treatment starts relatively late in terms of initiation of the rejection process. In the future, noninvasive monitoring of graft-specific immune activation would allow for early diagnosis and ultimately, prediction and preemptive management of acute or chronic rejection (CR). In addition, biomarkers for assessing the patient's pretransplant immune status alongside the pretransplant crossmatch (XM) may be used to tailor immunosuppression, such that the balance between the risk of rejection and side effects of immunosuppression can be minimized.

The peripheral blood is an easily accessible source of patient material for all types of transplants on which a variety of assays can be performed to define biomarkers that accurately reflect, detect, or predict detrimental immune responses to the graft, regardless of the organ transplanted (Fig. 1). There is an ongoing debate as to whether peripheral blood can give an accurate indication of the events that are taking place within the graft. Although analysis of gene, microRNA (miRNA), or protein expression in a transplant biopsy will undoubtedly give more direct information, sequential analysis, a prerequisite for accurate diagnosis, is unlikely to be possible even for kidney transplants. For kidney transplant recipients, urine is an easily accessible source of material that is proving to be informative. However, urine biomarkers will not be applicable for patients receiving other types of transplants. Therefore, we have focused this review on peripheral blood biomarkers that are currently under investigation for determining the pretransplant immune status and for posttransplantation monitoring to predict or detect acute and chronic allograft dysfunction.

Assays to detect biomarkers in the peripheral blood of transplant recipients. Serum or plasma can be routinely assayed for anti-human leukocyte antigen (HLA) antibodies by several techniques, antiendothelial cell antibodies by the XM-ONE assay, and a variety of proteins by using ELISA. Cellular assays on peripheral blood mononuclear cells include functional assays such as the Immuknow assay for determining general immune function and interferon (IFN)-γ ELISPOT for quantifying alloimmune reactivity. The cellular component can be further characterized by using flow cytometry or mRNA/microRNA (miRNA) profiling. EC, endothelial cell; sCD30, soluble CD30; RT-PCR, reverse-transcriptase polymerase chain reaction.


Multiple assays to identify and quantitate human leukocyte antigen (HLA) antibodies are available, the majority of which are well established (1), and are therefore not covered here. In contrast, the detection and quantitation of non-HLA antibodies, particularly antiendothelial cell antibodies (AECA), a novel parameter of donor-reactive immunity, is not yet a routine practice in the majority of centers, but there is increasing evidence to suggest that pretransplant detection of AECA might aid in pretransplant risk stratification.

In transplant immunology, the notion has arisen that other antigenic systems in addition to HLA play a role in graft outcome (2). In HLA-identical sibling kidney transplantation, pretransplant panel reactive antibody (PRA) positivity resulted in lower 10-year graft survival rates (3), suggesting a role for other antibody specificities such as the angiotensin II type 1 receptor, to which antibodies were present in HLA antibody–negative kidney transplant recipients with refractory vascular rejection (4).

By using primary arterial endothelial cell (EC) cultures in flow cytometry (FCM), it was shown that AECA were present in a substantial proportion of pretransplant sera, including sera from HLA nonsensitized patients (5). Recently, an endothelial cell cross-match (ECXM) technique has been developed based on precursor ECs isolated from whole blood of the organ donor (6). In a multicenter cohort of 147 kidney transplant recipients, 24% had donor-specific AECA and experienced more rejection episodes than patients without AECA (7). However, because a significant proportion of ECXM-positive patients had no increased risk of rejection, further studies are needed to refine which AECA will be of clinical relevance.


The membrane glycoprotein CD30 is expressed on a variety of cells including CD4+ and CD8+ lymphocytes producing Th2 cytokines, B cells, and natural killer cells (8). On stimulation of CD30+ T cells, CD30 is enzymatically cleaved, leaving soluble CD30 (sCD30) that can be detected in the plasma or serum as a measure for immune cell activation (9).

Although healthy individuals have low serum sCD30 levels, patients with end-stage renal disease have increased levels (10, 11). The first study correlating pretransplant sCD30 levels with graft loss showed decreased 5-year graft survival rates for patients with high sCD30 levels (10). Subsequently, pretransplant sCD30 levels have been suggested to be predictive for the grade of kidney rejection (12), whereas in lung transplantation, increased pretransplant sCD30 levels were associated with decreased early graft function (13). After transplantation, sCD30 levels generally decrease, but remain relatively high, or increase in the event of rejection as shown in one cohort of kidney transplant recipients (specificity 100% and sensitivity 88%) (14) and confirmed in an independent cohort (15). Because Th2 responses are linked to antibody production by B cells, it has also been suggested that sCD30 levels may predict antibody-mediated rejection (AMR) (12). However, data presented to date fail to show an association between posttransplant sCD30 levels and HLA antibodies (15, 16) or AMR (17).

Although the association of high pretransplant sCD30 with graft failure is unlikely to be affected by the type of maintenance immunosuppression (18, 19), for reasons that are not clear, the association between pretransplant sCD30 and acute rejection (AR) was less evident in patients receiving induction therapy (20, 21). Our own preliminary data in kidney transplant recipients receiving alemtuzumab or basiliximab induction also failed to show a correlation between pretransplant sCD30 and AR (Fig. 2).

Pretransplant soluble CD30 (sCD30) levels fail to predict acute rejection (AR) in a cohort of kidney transplant recipients receiving alemtuzumab or basiliximab induction therapy. Pretransplant serum was assessed for the level of sCD30 by ELISA. Patients with an AR episode within the first year after transplantation (n=12) had similar pretransplant sCD30 levels compared with stable patients (n=52). Healthy controls (n=13) had low levels of sCD30.

A positive association between pretransplant sCD30 levels and AR or graft outcome has not been found in all studies reported (15, 18, 22, 23). Alternative explanations for these discrepant results, such as the biologic variability of sCD30 levels with time (24), may need to be taken in account. Further data are required to validate the prognostic value of pretransplant sCD30 levels for AR or graft outcome for individual patients (25, 26).


The chemokines CXC chemokine ligand 9 (CXCL9; Mig) and CXC chemokine ligand 10 (CXCL10; interferon gamma-inducible protein-10) are downstream of interferon (IFN)-γ signaling and attract CXC chemokine receptor 3 (CXCR3+) T cells into the graft. Romagnani and coworkers (27, 28) suggested that high pretransplant serum CXCL10 levels were predictive of renal graft loss and early AR of cardiac allografts (29). By analogy, high pretransplant CXCL9 levels in kidney transplant recipients were predictive for AR and 5-year graft survival (specificity 83% and sensitivity 75%) (30). However, the clinical usefulness for these assays seems to be limited because pretransplant levels of CXCL9 and CXCL10 in patients receiving alemtuzumab or basiliximab induction therapy did not predict for AR, likely due to depletion or inactivation of CXCR3+ responder cells (31).

There exist few data on the relation of posttransplant serum CXCR3-binding chemokine levels and graft outcome. In lung transplantation, it has recently been shown that posttransplant CXCL10 levels, but not CXCL9 levels, were predictive for early fibrosis recurrence (32). More research is needed to establish whether posttransplant serum CXCR3-binding chemokines are predictive for graft outcome.


The overall function of lymphocytes can be determined by their ability to proliferate (33, 34) or by their intracellular adenosine triphosphate (iATP) content after phytohemagglutinin (PHA) stimulation, the latter reflecting the “energy” levels available within the cell. Both assays have the potential to provide an indication of the level of immunosuppression, that is, lower proliferation or ATP, less ability to respond to immune stimuli. A Food and Drug Administration–approved iATP assay is available commercially, the ImmuKnow assay (Cylex, Columbia, MD).

One of the early studies using this approach reported that high CD4+ T-cell iATP levels were associated with a risk of AR in recipients of kidney, liver, heart, and small bowel transplants, whereas low levels were a risk factor for infections (35). These data have been confirmed for kidney, liver, and heart transplantation in separate studies (36–38) and were interpreted as indicating that the quantification of iATP levels may be a tool to determine the immune status of a patient. Not all studies have found correlation with AR but have found a correlation between iATP levels and infection with varying levels of specificity and sensitivity (39–42). In addition, it has been hypothesized that high pretransplant iATP levels correlating with both AR and poor early function in kidney transplant recipients might be predictive for transplant outcome (43), but thus far there are no published data confirming this hypothesis. This type of assay has also been shown to reflect the level of immunosuppression in kidney transplant recipients with long-term surviving allografts; recipients with squamous cell carcinoma showed impaired responses toward phytohemagglutinin compared with patients without malignancy (44).

Overall, the main utility of assays based on mitogenic stimulation of lymphocytes is for detection of overimmunosuppression. A potential drawback of these techniques is that they determine a patient's general immune status but not the T-cell reactivity directed toward the allograft. The published data are inconsistent and suggest that results should be interpreted with caution when trying to relate them to rejection, infection, or long-term graft outcome.


Determining donor-reactive memory T-cell reactivity is of great interest for risk stratification in transplantation, because memory T cells respond more rapidly to stimulation by alloantigen (45) and are more difficult to suppress compared with their naïve counterparts (46, 47). Thus far, the IFN-γ ELISPOT is one of the few functional assays available for determining donor-specific memory T-cell reactivity.

The IFN-γ ELISPOT was originally described for the quantification of pretransplant alloreactive memory T cells in kidney transplant recipients (48). In subsequent studies, increased levels of donor-reactive IFN-γ–producing T cells prekidney and postkidney transplantation were shown to be a risk factor for AR and a predictor of graft function, independent of PRA level (49–51). Moreover, the IFN-γ ELISPOT detected alloreactive T cells in lymphopenic patients (specificity 80% and sensitivity 83%), making this a broadly applicable assay (52).

Analogous to PRA detection, panels of HLA-typed target cells have been used to determine pretransplant T-cell alloreactivity (53, 54). Similarly, but independent of PRA, a high level of panel reactive T cells has been associated with immune-mediated graft injury in a study of 30 kidney transplant recipients (specificity 78% and sensitivity 86%) (55). On balance, current data suggest that the IFN-γ ELISPOT is a potentially powerful technique to determine the level of donor-specific and general alloimmunization. However, not all studies have found a correlation between a positive IFN-γ ELISPOT and transplant outcome (43), emphasizing the critically important steps of optimization, standardization, and validation in developing any biomarker assay. The European RISET consortium ( has implemented a rigorous approach to validate the IFN-γ ELISPOT, enabling it to be used by multiple laboratories (Volk and coworkers, unpublished results, 2010) (56).


A huge advantage of FCM is the capacity to quantify many immune cell subsets with great flexibility. Alterations in a variety of subsets have been described in patients experiencing AR or in patients with long-term surviving grafts. Although standard multiparameter FCM does not provide information about cell function, there are now strategies for determining the antigen specificity of T and B cells, signaling pathway activity, transcription factor expression, and cytokine production that are being investigated in the setting of transplantation.

As mentioned earlier, memory T cells are relatively resistant to immunosuppression (57), a finding confirmed by our own clinical data (58). Furthermore, we and others have shown that effector memory T cells were associated with AR in kidney transplant recipients (57–59) and in heart transplant recipients (60). Memory T cells can be detected by FCM because of an altered cell surface phenotype compared with naïve T cells, including CD45RA, CCR7, and CD62L. FCM analysis showed that the percentage of activated CD8+CD69+ T cells was increased during both acute renal (specificity 85% and sensitivity 86%) (61) and cardiac allograft rejection (62). Likewise, frequencies of CD28- and CD38-expressing T cells were higher in liver transplant recipients with AR compared with patients with stable function or infection (63). Lun et al. (64) found that the percentage of CD25+ T cells was increased in liver transplant recipients during AR (CD4+CD25+: specificity 91% and sensitivity 63%; CD8+CD25+: specificity 90% and sensitivity 56%).

Despite data suggesting that general activation markers may be useful for detecting AR, the discriminatory potential of such assays is likely to be poor as the same activation markers will be increased on any T cell as it responds to antigen. Our own preliminary data showed that the frequencies of CD25+, CD38+, and HLA-DR+ CD8+ T cells were increased not only during AR but also during infection in kidney transplant recipients (Heidt et al., unpublished data, 2010). These data are consistent with the demonstration that cytomegalovirus (CMV) infection in renal transplant patients coincided with increased levels of CD8+CD38+ T cells and the up-regulation of CD69 on CD8+ T cells correlating with CMV viremia but not with acute renal allograft rejection (65–67).

Regulatory T cells (Tregs) have been shown to prevent allograft rejection in many animal models of transplantation (68). Therefore, monitoring of Treg in the peripheral blood may detect unresponsiveness to the graft. We and others have shown lower percentages of Treg in the peripheral blood of renal transplant recipients with chronic graft dysfunction compared with stable or operationally tolerant patients (69–71). A proportion of liver transplant recipients who are electively weaned off immunosuppressive drugs exhibit operational tolerance to donor alloantigens, making these patients valuable for defining biomarkers of tolerance (72). In adult and pediatric liver transplant patients who were successfully weaned off immunosuppressive drugs, increased numbers of CD4+CD25+ cells with regulatory activity were present in the peripheral blood compared with the levels present in patients remaining on immunosuppression or healthy controls (73).

Although increased numbers of Treg have been found in the peripheral blood of immunosuppression-free liver transplant recipients, this has not been the case for immunosuppression-free kidney transplant recipients (74, 75). Thus, findings for a defined clinical outcome for one organ type cannot be extrapolated to another.

Detection of T cells with the phenotype characteristic of Treg by FCM using CD25 as a marker in humans is well known to be difficult and prone to investigator bias. The addition of other markers to the panel, including CD127 (a chain of the interleukin [IL] 7 receptor) or CD45 (leukocyte common antigen) can reduce this. The activation marker CD127 facilitates clear-cut gating, with CD127low T cells being regulatory and CD127high T cells being activated (76, 77). Human CD4+CD25+CD127lowFOXP3+ cells have been shown to have higher regulatory activity compared with CD4+CD25highFOXP3+ cells in a humanized mouse model of transplant vasculopathy, supporting the hypothesis that addition of CD127 as a marker facilitates in defining Treg (78). Including one of the isoforms of CD45, the leukocyte common antigen has also been reported to be beneficial. CD4+CD25+CD45RO+CD127low Treg levels were low in liver and kidney transplant recipients compared with healthy subjects, whereas concomitantly, levels of CD4+CD25+CD45RO+CD127high activated T cells were increased with the highest levels found in patients with chronic AMR (79). Alonso-Arias et al. have confirmed the increased percentages of CD4+CD25+CD45RO+CD127high activated T cells in kidney transplant recipients. In addition, relatively high levels of CD127low cells without regulatory properties correlated with renal failure (80).

It is well recognized that immunosuppressive drugs have differential impact on the frequency of Treg in transplant patients (81, 82). Surprisingly, immunosuppressive drugs can also obscure FCM detection of Treg. Our laboratory found highly variable FOXP3 expression with time after venesection only in samples from patients on calcineurin inhibitor-based immunosuppression (Carroll and Wood, unpublished observations, 2010), emphasizing the importance of standardizing and validating workflow, such that time between sample collection and processing is kept to an absolute minimum for biomarker detection.

B cells may also have a role in regulating immune responses. Increased numbers of B cells with a regulatory phenotype, defined as CD1d+CD5+, have been found in the peripheral blood of immunosuppression-free kidney transplant recipients compared with the number present in patients with stable graft function remaining on immunosuppression. However, no differences between stable patients and those undergoing CR were found (83). These findings have been confirmed in the larger cohorts of the Reprogramming the Immune System for Establishment of Tolerance (RISET) and Immune Tolerance Network (ITN) networks (74, 75). Similar observations have been made in pediatric liver transplant patients, where the percentage of B cells was increased in stable drug-free patients compared with patients under immunosuppression and healthy controls (73). Data on the contribution of B cells in clinical tolerance and rejection are exciting and warrant more in-depth analysis of B-cell subsets in transplant patients in the near future.

Overall, FCM is a useful monitoring technique providing a wealth of data. However, because flow cytometer configuration and setup, and data acquisition and analysis, will be different among centers, standardization to enable multiparameter phenotyping to be used in biomarker studies is crucial (84).


The use of molecular biology in the search for biomarkers allows the screening of vast numbers of genes by microarray techniques, whereas the more sensitive polymerase chain reaction–based techniques are better suited for hypothesis-driven research (85). This section will mainly discuss the hypothesis-driven studies.

Perforin, granzyme B, and FasL are molecules involved in cytotoxicity that have been studied extensively in relation to rejection (Table 1). Up-regulation of two or more of these genes in the peripheral blood correlated with AR in kidney transplant recipients (minimum specificity 60% and sensitivity 100%) (86), a finding that has been confirmed and extended in other studies. For example, granzyme B and HLA-DR expression combined yielded high specificity (100%) and poor sensitivity (50%) to diagnose AR (87), whereas in other studies, altered expression of perforin, granzyme B, or FasL correlated with AR (88, 89) or CR (90, 91)with varying specificity and sensitivity. In addition, some studies have shown by serial measurements that AR can be detected by increased granzyme B and perforin expression approximately a week before diagnosis with specificity up to 97% and sensitivity up to 73%, depending on timing and patient cohort (92, 93), but this has not been replicated in all studies (94).

Studies describing perforin, granzyme B, and FasL mRNA levels in peripheral blood of kidney transplant recipients

As with other potential biomarkers already discussed, not all studies have produced consistent findings. For example, Shin and coworkers (94) found an association between acute kidney rejection and perforin expression levels, but they observed no association with granzyme B or FasL. In addition, Graziotto et al. (95) failed to find an increase in granzyme B, perforin, or FasL expression in the peripheral blood during kidney rejection, whereas perforin and FasL expression was increased in biopsies, raising the question whether peripheral blood mRNA levels for these molecules are universally applicable to different patient cohorts.

The studies referred earlier compare patients experiencing rejection with those who have stable graft function or patients with nonimmunologic pathology (Table 1). We assayed serial samples from kidney transplant recipients with AR and patients with confirmed BK virus or CMV infection and found that increased mRNA expression levels of both granzyme B and perforin in the peripheral blood often coincide with AR and with infection (Fig. 3). These preliminary results are consistent with data from islet allograft recipients, in whom expression of cytotoxic molecules often correlated with infection (96), and from kidney transplant recipients, in whom mRNA levels of urinary cytotoxic molecules were up-regulated during viral infections (97). This may not be surprising because any form of immune stimulation, not least viral infection, may lead to detectable changes of cytotoxic cell function.

Peripheral blood perforin and granzyme B mRNA levels are regularly increased on infection and acute rejection. Example of a kidney transplant recipient who was treated with basiliximab induction, tacrolimus, azathioprine, and steroids and experienced acute rejection (AR) at 39 days posttransplantation, followed by a cytomegalovirus (CMV) infection at day 225. Both perforin and granzyme B mRNA levels, as determined by reverse-transcriptase polymerase chain reaction, showed a peak at time of rejection and at time of infection. HPRT, hypoxanthine phosphoribosyltransferase.

The changes in gene expression of general markers of immune activation, such as costimulatory molecules and cytokines, have also been correlated with rejection, such as CD154 (98, 99). In addition, expression levels of cytokines such as IL-4, IL-5, IL-6, and IFN-γ in mitogen-stimulated peripheral blood mononuclear cells have been found to correlate with AR of renal allografts (100, 101). Using daily sampling of peripheral blood, IL-4 and tumor necrosis factor-α (TNF-α) mRNA expression in peripheral blood mononuclear cell were shown to be predictive for AR in kidney transplantation 2 days before clinical diagnosis (101).

Other potential biomarkers include the expression of genes linked to the presence of Treg as discussed earlier. Aquino-Dias et al. (89) showed that an increased FOXP3 mRNA expression was associated with AR of kidney allografts (specificity 95% and sensitivity 94%), whereas in CR, decreased FOXP3 expression levels have been described (90). However, in long-term surviving renal transplant recipients, FOXP3 expression did not correlate with rejection (91). In addition, Dijke et al. (102) have shown that peripheral blood FOXP3 expression did not correlate with cardiac allograft rejection, whereas FOXP3 levels in biopsies did. The markers tolerance associated gene-1 (TOAG-1) and α-1,2-mannosidase were initially discovered in experimental studies (103) and have been translated to clinical transplantation. The ratio of FOXP3:α-1,2-mannosidase mRNA expression was shown to be substantially lower in kidney transplant patients with CR compared with stable patients and tolerant drug-free patients (74). These potential biomarkers require further validation to determine, again, whether there are specific clinical situations where they are uninformative as the lower ratio of FOXP3:α-1,2-mannosidase expression has not been confirmed in a separate study, in which expression levels of CTLA4 and transforming growth factor-β were also unaltered (90).

In the large multicenter study by the RISET and ITN consortia, 30 genes discovered by microarray analysis and confirmed by polymerase chain reaction were associated with operational tolerance in immunosuppression-free kidney transplant recipients, many of which were B cell related (75). It will be interesting to see whether these genes are predictive for specific unresponsiveness to donor alloantigens. Interestingly, the microarray-derived gene signatures for tolerant kidney and liver transplant recipients are distinct (74, 75, 104, 105).


A novel area of biomarker research aims at detecting miRNAs, small noncoding single-stranded RNA molecules involved in posttranslational regulation of gene expression (106). Two studies on miRNA expression in renal allograft biopsies during AR so far have identified specific, but distinct patterns of miRNA expression correlating with AR in small patient groups (107, 108). It will be interesting to see whether quantifying miRNA levels in the peripheral blood will provide relevant biomarkers.


The past 2 decades have seen much progress in defining the immunologic mechanisms involved in allograft rejection. By taking this knowledge to generate hypotheses as to which genes and molecules are predictive of rejection and by using a vast array of techniques to identify new molecules and pathways that could play a role, many laboratories have identified potential biomarkers for rejection and tolerance in different sources of patient material. Peripheral blood biomarkers are of particular interest, because, at least theoretically, they can be applied irrespective of the type of transplant and can be used both in the short and long term after transplantation to profile the immune and metabolic status of transplant recipients.

Studies on biomarkers for rejection are promising, but, as the field stands today, not without caveats. Most studies describe patients with defined conditions, such as (acute) rejection or stable function, sometimes ignoring other causes for immune activation such as infection. For any biomarker to be clinically applicable for predicting rejection, its capacity to differentiate between immune activation toward the allograft and infectious agents must be established. Because many parameters of immune activation will not be specific to alloimmunity, we hypothesize that a combination of markers will be needed to provide sufficient specificity and sensitivity. For immune tolerance, it has already been shown that a combination of markers provided the highest specificity for defining operationally tolerant patients (74). Obviously, disadvantages of using multiple parameters in a clinical setting are increased analysis time and data complexity, which has to be taken into account when translating biomarker as a diagnostic entity into the clinical setting.

Validation of findings from single-center analyses in multicenter studies is a critical step in defining biomarkers that can be used to predict rejection in the future (109). Currently, striking discrepancies in the biomarker data exist, as reported by various laboratories investigating the same parameter. This is likely due to differences in recipient and donor populations, clinical management, and the actual laboratory techniques used to perform the assays. Therefore, it is of great importance that the assays used to detect biomarkers are optimized, validated, and standardized, and subsequently, candidate biomarkers are investigated in large patient cohorts from different transplant centers using the same, validated, assays (110). International research networks such as the RISET, ITN, and Genome Canada networks have already begun this process and are invaluable for discovering and validating biomarkers to enable them to be introduced into clinical practice (111).

The establishment of noninvasive monitoring techniques will greatly improve patient care, facilitating the development of personalized medicine for transplant recipients. Biomarkers will likely enable the detection of rejection episodes at an earlier stage than is currently possible, a identification of drug toxicity and signs of specific unresponsiveness to donor alloantigens to modify the immunosuppressive regimen. Recent advances in the identification of peripheral blood biomarkers are encouraging even if further work is required to validate and implement these new approaches. Nevertheless, we anticipate that it is only a matter of time before noninvasive monitoring is implemented as a part of routine clinical practice in transplantation.


The authors thank Sally Ruse for patient enrollment and Ian Toogood-Johnson for technical assistance with the data reported from the authors own laboratory.


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Immune monitoring; Transplantation; Biomarkers; Allograft rejection; Chronic allograft dysfunction

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