**5.3. Imaging using** *ex vivo* **radiolabeled leukocytes**

Because recruitment and activation of inflammatory cells, i.e. lymphocytes, is crucial to AR, efforts have been made to image infiltration by means of labeled leukocytes. Application of *ex vivo* radiolabeled leukocytes is clinically well established particularly in the diagnostic workup of infectious disorders without a focus. Hitherto, white blood cells (WBC) are labeled using for instance 99mTc-HMPAO or 111In-oxine for SPECT and 18F-FDG or 64Cu for PET analysis, respectively [29]. These cells are considered to accumulate highly specific in inflamed tissues [30-33].

After injection of labeled leukocytes a typical distribution pattern can be observed. First, cells shortly accumulate in the lungs and then continuously migrate from the blood pool into spleen, liver, and bonemarrow, the so called reticulo-endothelial system, and certainly in inflamed sites [34-36]. After endothelial adhesion, labeled leukocytes migrate through the vessel`s wall to the focus of inflammation providing a typical radioactivity pattern indicating infiltration. For instance, Forstrom *et al.* have shown that 18F-FDG labeled leukocytes exhibit comparable distribution patterns in normal human subjects compared with 111In or 99mTc-labeled WBC [37]. Although 18F-FDG seems to exhibit the lowest labeling stability when compared to 111In and 64Cu only neglectible free 18F-FDG uptake can be observed [37]. However, labeling stability is relevant in order to assure that assessed activity refers to accumulation of labeled leukocytes and not to the unlabeled tracer only. Since half-life time of 18F-FDG is 109 min, longtime stability of 18F-FDG labeled leukocytes for clinical analysis is not of interest. However, if longtime sta‐ bility is of interest this could be addressed using other tracers like 99mTc with a half life of approximately 66h.

Successful imaging using labeled leukocytes depends on viability of labeled cells. Several studies assessed cell viability after labeling concluding satisfactory and comparable viability rates for 111In, 99mTc, 18F-FDG and 64Cu in the first 4h after labeling [38]. However, cell viability significantly decreases within one day limiting long term monitoring of AR using a single shot approach.

At present only a few preclinical and clinical studies are published dealing with labeled leu‐ kocytes and detection of AR in intestine, hearts, pancreas islets and skin. Only one study per‐ formed in a small cohort of kidney transplant recipients evaluated 99mTc-mononuclear cell scintigraphy for diagnosis of AR. In this study, the authors were able to show that AR was diagnosed correctly and successfully discriminated from ATN [39]. In a further development of their approach, we established leukocyte PET imaging using very low amounts of 18F-FDG for the diagnosis of AR in a rat kidney transplant model. *Ex vivo* 18F-FDG labeled human CTLs were able to diagnose renal AR within a time frame of 1 h after application and discriminate AR from important differential diagnoses such as acute cyclosporine toxicity or ATN (*Grabner et al. in press*) (Fig. 1).

**5.4. Imaging using** *in vivo* **radiolabeled leukocytes**

using the antibodies in a patient.

**5.5. Metabolic activity (18F-FDG)**

agnosed lately [45].

function) [46].

Instead of employing *ex vivo* labeled leukocytes, radiolabeled monoclonal antibodies (frag‐ ments) (mAbs) have been established for detection of leukocyte (related) antigens. Their ad‐ vantages include standardized production, easier storage and handling, while they are highly specific for their target leading to a good background/target ration. However, limitations might be the targeting of extravascular antigens and potential but rare allergic complications, when

Non-Invasive Diagnosis of Acute Renal Allograft Rejection − Special Focus on Gamma Scintigraphy and Positron…

http://dx.doi.org/10.5772/54737

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As discussed, CTLs are the major cell type involved in AR. Martins *et al.* used 99mTc-OKT3 targeting CTLs in recipients of renal transplants [40]. In their preliminary results they state that out of 22 patients they successfully identified 3 patients with AR using 99mTc-OKT3 scans. Apparently, their results published in 2004 have to be confirmed in further studies. A recently published attractive, being somehow better biocompatible, alternative might be CD3 targeting

<sup>18</sup>F-FDG is a daily routine tracer to assess regional glucose metabolism as a surrogate for met‐ abolic activity widely used for the PET-based routine detection of tumors, infection and in‐ flammation. The major energy source in leukocytes during the metabolic burst is glucose. Analogously, activated leukocytes highly accumulate 18F-FDG (in the same way they take up glucose but without further processing) which can be quantified by PET [42]. A clear limitation when using free 18F-FDG is that an increased uptake can be observed in any kind of cellular activation (high glycolytic activity). Hence, 18F-FDG is not a disease or target specific tracer.

Nevertheless, 18F-FDG is one of the few tracers successfully applied for the non-invasive de‐ tection of AR. Others have applied 18F-FDG in settings of lung, heart and liver transplantations. We have demonstrated very promising results for 18F-FDG-PET in diagnostics of renal AR [43;44]. Using a rat model of renal AR, 18F-FDG-PET performed well in terms of early, accurate detection and follow-up of AR [43] (Fig. 2). Using 18F-FDG, we discriminated AR non-inva‐ sively from important differential diagnoses like ATN or acute cyclosporine toxicity. More‐ over, therapy response monitoring by 18F-FDG might be useful to identify treatment unresponsive AR for earlier escalation of immunosuppressive regimen [44]. This might reduce graft damage by shortening AR episodes because at present (steroid) resistant rejection is di‐

One important issue of imaging of kidney AR with 18F-FDG is that it is eliminated with the urine in contrast to normal glucose. Thus, drainage of 18F-FDG into the renal pelvis must be taken care of when assessing 18F-FDG-uptake in the renal parenchyma. We avoided this prob‐ lem by using late acquisitions after 18F-FDG injection to reduce the instantaneous amount of tracer in the urine during the PET scan. Moreover, an impact of renal function on 18F-FDGuptake has to be excluded e.g. by renal fluoride clearance (a non-invasive measure of renal

99mTc-SHNH-visilizumab which needs to be evaluated in the future [41].

**Figure 1.** Representative PET-images of dynamic whole body acquisitions of a series of an allogeneically kidney trans‐ planted rat on postoperative day 4 60 min and 120 min after tail vein injection of 30 x 10618F-FDG labeled CTL. While the parenchyma (yellow circle) of renal allograft developing AR accumulates 18F-FDG-CTLs, the native kidney (green circle) does not show any accumulation at any time. Please note that the renal pelvis can contain eliminated 18F-FDG/ 18F-fluoride. Therefore, it has to be excluded from the measurements. ID: injected dose

Since infiltration of leukocytes, especially CTLs, in allografts appears before physiologic or mechanical manifestations of organ dysfunction is apparent, nuclear imaging employing leu‐ kocytes might be a promising tool for specific, sensitive and early detection of AR.
