[Electrochemotherapy for multiple cutaneous tumors in immunosuppressed patients]. / Immunszupprimált betegek multiplex bordaganatainak elektrokemoterápiás kezelése.

INTRODUCTION: The risk of cutaneous malignancies is significantly higher in immunosuppressed patients compared to the general population. These high-risk skin tumors tend to be aggressive, multiplex, rapidly growing lesions. It is common to see local recurrence after surgical excision. Multiplex tumors are difficult to treat, especially in the head/neck region. OBJECTIVE: Amongst the standard treatment options, electrochemotherapy can be a suitable option. Our aim was to evaluate the efficacy of electrochemotherapy in immunocompromised patients. METHOD: In 9 immunosuppressed patients, 118 (average: 13, n = 5-27) non-melanoma skin tumors were treated with electrochemotherapy with intravenous administration of bleomycin, according to the ESOPE criteria. RESULTS: The median follow-up was 15 months. 6 months after the treatment, the objective response rate was 96%. We observed complete response in 88%, partial response in 8% and progressive disease in 2% of the treated lesions. In 2%, the response was not evaluable. CONCLUSION: In immunocompromised patients, electrochemotherapy is an effective and safe therapeutic option for non-melanoma skin tumors. In order to provide more ideal management for this special sub-group, prevention, multidisciplinary approach and optimized immunosuppressive therapy is essential. Orv Hetil. 2023; 164(37): 1462-1468.

Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology.

Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as the lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient-derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of the extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca2+ concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca2+ activated Cl- channel Anoctamin 1 (ANO1) and the epithelial Na+ channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl- measurement have improved dynamic range when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.

[History of kidney transplantation in Szeged]. / A veseátültetés története Szegeden

The first Hungarian kidney transplantation was performed in 1962, in Szeged, by András Németh (1924­1999). The first semester at the university in Szeged started in 1921, so this year we celebrate the centenary. This event inspired authors to review the history of kidney transplantation in Szeged, remembering the first one and point of the cornerstones in the transplant program. The donor of the first Hungarian kidney transplantation was the brother of the recipient. The operation itself was technically successful, but the lack of immunosuppression caused graft rejection, and the patient died after 79 days. His brother, the donor was healthy, after 50 years, and he encouraged everybody to donate organs. The organized kidney transplant program started more than 10 years later, in 1973, in Budapest. The program was supported by the Ministry of Health. Szeged joined the program in 1979 led by Erno Csajbók and Pál Szenohradszky. In the Transplant Center in Szeged, developed organizationally as well as professionally, 1701 kidney transplantation has been performed up to the end of the year 2021.

Early and late graftectomies in patients during a 5-year period - single unit data from the Department of Surgery, University of Szeged / Korai és késoi graftectomiák a Szegedi Sebészeti Klinikán - 5 év beteganyagában

Introduction. The rate of graft failure after kidney transplantation is 7­10% in the first year and 3­5% in subsequent years. The indication and exact timing of graftectomy is a matter of debate in some cases, particularly in the case of asymptomatic grafts that are no longer functioning. Methods. Data of patients who underwent kidney transplantation at the Transplantation Unit, Department of Surgery, Szeged, Hungary between January 1, 2015 and December 31, 2020 were analyzed. We reviewed the indications, timing and complications of graftectomies and compared early and late graftectomies. Results. 294 kidney transplants were performed during the study period. 37 patients (13%) of them underwent graftectomy. The most common indications were bleeding 11 (30%), arterial circulatory disorders 8 (22%), v. renal thrombosis 7 (19%), mixed active antibody and ongoing cellular rejection 7 (19%), and acute humoral rejection 4 (10%). Graftectomies were performed in 26 cases with inoperative and in 11 cases with functional graft. Comparing early and late graftectomies, 15 cases (40%) underwent early graftectomy within 30 days after transplantation and 22 cases (60%) underwent late graftectomy. Conclusions. The most common cause of graftectomies in the study period was acute bleeding, which is also due to disturbed homeostasis in chronic renal failure. In the case of the early ones, emergency surgery and in the vast majority of late graftectomies, elective surgery was performed.

Bile acid- and ethanol-mediated activation of Orai1 damages pancreatic ductal secretion in acute pancreatitis.

Regardless of its aetiology, sustained intracellular Ca2+ overload is a well-known hallmark of acute pancreatitis (AP). Toxic Ca2+ elevation induces pancreatic ductal cell damage characterized by impaired ion and fluid secretion - essential to wash out the protein-rich fluid secreted by acinar cells while maintaining the alkaline intra-ductal pH under physiological conditions - and mitochondrial dysfunction. While prevention of ductal cell injury decreases the severity of AP, no specific drug target has yet been identified in the ductal cells. Although Orai1, a store-operated Ca2+ influx channel, is known to contribute to sustained Ca2+ overload in acinar cells, details concerning its expression and function in ductal cells are currently lacking. In this study, we demonstrate that functionally active Orai1 channels reside predominantly in the apical plasma membrane of pancreatic ductal cells. Selective CM5480-mediated Orai1 inhibition impairs Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. Furthermore, prevention of sustained extracellular Ca2+ influx protects ductal cell secretory function in vitro and decreases pancreatic ductal cell death. Finally, Orai1 inhibition partially restores and maintains proper exocrine pancreatic secretion in in vivo AP models. In conclusion, our results indicate that Orai1 inhibition prevents AP-related ductal cell function impairment and holds the potential of improving disease outcome. KEY POINTS: Sustained intracellular Ca2+ overload in pancreatic acinar and ductal cells is a hallmark of biliary and alcohol-induced acute pancreatitis, which leads to impaired ductal ion and fluid secretion. Orai1 is a plasma membrane Ca2+ channel that mediates extracellular Ca2+ influx upon endoplasmic reticulum Ca2+ depletion. Results showed that Orai1 is expressed on the luminal plasma membrane of the ductal cells and selective Orai1 inhibition impaired Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. The prevention of sustained extracellular Ca2+ influx protected ductal cell secretory functions in in vitro models and maintained exocrine pancreatic secretion in in vivo acute pancreatitis models. Orai1 inhibition prevents the bile acid- and alcohol-induced damage of the pancreatic ductal secretion and holds the potential of improving the outcome of acute pancreatitis.

The First 5 Years of the Newest Eurotransplant Member State: Hungarian Results of International Organ Exchange From 2014 to 2018.

INTRODUCTION: Hungary joined Eurotransplant International (ET) to improve the chance of transplantation for Hungarian patients and patient outcomes, including access and graft and patient survival. After 5 years of full membership, the evaluation of numbers and quality indicators is possible. METHOD: A comparison was made between 5 years prior to a preliminary cooperation agreement (2007-2011) and 5 years after full ET membership (2014-2018). During the 2 study periods, we analyzed numbers and circumstances of deceased organ donors, multiorgan donors, donated organs, and transplantations in Hungary and development of waiting lists along with international organ exchanges. RESULT: The number of actual organ donors increased by 22.09% (729 vs 890), an additional 823 organ removals represents an increase of 42.71% (1927 vs 2750). There were 46.51% more transplants managed in the selected periods (1561 vs 2287). The number of new patients on the waiting list increased (2305 vs 3247; 40.87%). The mean kidney mismatch number decreased from 3.21 to 2.96. CONCLUSION: Joining ET has been an effective and efficient in terms of increasing access to organs and the lives of patients on the Hungarian waiting list posttransplant. It is also a benefit for patients with special needs because the number of organ transplants is greater than the increased number of donors.

A Summary of Transplantation Activity in Hungary.

The first kidney transplantation was performed in Hungary by András Németh in 1962. It was a living donor procedure. After many years of silence, organized cadaveric programs were established in Budapest (1973), Szeged (1979), Debrecen (1991), and Pécs (1993). The heart program was initiated by Professor Zoltán Szabó in 1992 and the liver transplant program by Professor Ferenc Perner in 1993. The pancreas transplantation program was started in Pécs in 1998 by Károly Kalmár-Nagy, followed another in Budapest by Robert Langer in 2004. The lung transplant program was started in cooperation with Vienna in 1996. This fruitful collaboration continues today, even though that the national Hungarian program was established by Ferenc Rényi-Vámos and Professor György Lang in 2015, as it is detailed in this special issue. As a framework, the Hungarian Society of Organ Transplantation was founded in 1997 to give a scientific background for the transplant professionals. The coordination and organ allocation from deceased donors is carried out in collaboration with Eurotransplant. Usually more than 200 potential cadaveric donors are reported yearly, and 168 actual donation after brain death (DBD) donors (17.17 pmp) were utilized in 2018. The multiorgan donor rate was 65.5% among all DBDs in 2018; 505 organs were donated for transplant purposes. To date, more than 10,000 organ transplantations have been performed. The living related kidney transplant program was established in all transplant centers, led by Budapest. In this paper the authors summarize the activity of the Hungarian transplant community and of the Society over the last few decades.

New-onset Diabetes Mellitus Following Successful Kidney Transplantation Facilitates Aortic Stiffening.

BACKGROUND: Following kidney transplantation (KT), new-onset diabetes mellitus (NODM) is one of the most common complications. NODM usually occurs early after KT, and is diagnosed according to the general guidelines relevant for general diabetes mellitus patients. Arterial stiffness is a surrogate marker of cardiovascular risk. According to the literature, a successful KT has only limited and late beneficial effects on aortic elastic properties. The present study aimed to assess whether NODM has any additive value on the worsening of echocardiography-derived aortic elastic properties in transplanted patients. METHODS: We have included 28 nondiabetic post-KT patients in the study, older than 18 years (mean age: 48.2 ± 6.9 years; 13 men, 15 women). After an oral glucose tolerance test, 8 patients were diagnosed with NODM, and their results were compared to 23 age-, sex-, and risk factor-matched controls (mean age: 54.9 ± 11.0 years; 9 men, 14 women). All post-KT patients and matched controls underwent a complete transthoracic 2-dimensional Doppler echocardiography, together with an assessment of echocardiographic aortic elastic properties. The assessments included aortic strain, aortic distensibility, and aortic stiffness index. RESULTS: Aortic elastic properties showed alterations in post-KT patients compared to matched controls (aortic strain: .084 ± .039 vs .057 ± .032, P < .05; aortic distensibility: 2.36 ± 1.09 cm2/dynes 10-6 vs 1.83 ± 1.18 cm2/dynes 10-6, P = .07; aortic stiffness index: 7.15 ± 3.58 vs 11.2 ± 6.1, P < .05). Further deterioration in the aortic stiffness index (14.8 ± 7.6 vs 9.68 ± 4.88, P < .05) was detected in the presence of NODM. CONCLUSIONS: NODM following successful KT facilitates aortic stiffening.

[Changes in carbohydrate metabolism after kidney transplantation and their effects on cardiovascular risk]. / Vesetranszplantáció utáni szénhidrátanyagcsere-változások és annak hatásai a cardiovascularis rizikóra.

INTRODUCTION: Cardiovascular disease is the major cause of deaths after transplantation, with diabetes mellitus being the main risk factor in development. AIM: The aim of our study was to assess the prevalence of new onset diabetes mellitus in connection with the cardiovascular risk predicted by the HEART Score. METHOD: 44 patients were involved in our study; after overview of baseline data, OGTT was performed, followed by patient classification into the following groups: normal, impaired fasting glucose/impaired glucose tolerance, and new onset diabetes mellitus. Insulin resistance and kidney function were also assessed. RESULTS: Concerning baseline data, cold ischemic time (p = 0.016), body weight (p = 0.035), BMI (p = 0.025), and HbA1C (p = 0.0024) proved to be significantly different between normal and diabetic patients. Significant difference was found based on HOMA IR between the two groups 1.69±0.51 vs 6.46±1.42; p = 0.0017). Based on the HEART Score, patients with new onset diabetes mellitus were put into Group 3, which also reflects the risk which diabetes carries for the development of cardiovascular diseases. CONCLUSION: Cardiovascular risk can be decreased with increased allograft survival by early diagnosis and management of diabetes. Orv Hetil. 2017; 158(38): 1512-1516.

Peritubular capillary basement membrane multilayering in early and advanced transplant glomerulopathy: quantitative parameters and diagnostic aspects.

The ultrastructural quantitative aspects of peritubular capillary basement membrane multilayering (PTCBML) were examined in 57 kidney transplant biopsies with transplant glomerulopathy (TG). The measurements included three cutoffs [permissive: 1 PTC with 5 basement membrane (BM) layers, intermediate: 3 PTCs with 5 layers or 1 PTC with 7 layers, strict: 1 PTC with 7 layers and 2 PTCs with 5 layers] and the mean number of BM layers (PTCcirc). Two groups were assigned, namely patients with mild TG (Banff cg1a and cg1b) and those with moderate-to-severe TG (cg2 and cg3). Their respective clinical, serological, and morphological characteristics were then compared. The clinical data revealed that mild TG corresponded to early chronic antibody-mediated rejection (cABMR), while moderate-to-severe TG corresponded to the advanced stage of the disease. The permissive threshold displayed the lowest specificity (73 %) and the highest sensitivity (83 %) for moderate-to-severe TG, and its corresponding PTCcirc value was 3 layers. In contrast, the strict threshold-adopted by the Banff 2013 classification-displayed a specificity and sensitivity of 93 and 52 %, respectively, and the corresponding PTCcirc was 4 layers. In mild TG, 26 % of the cases met the permissive cutoff and 6 % the strict cutoff. Mild TG was associated with a lower PTCcirc (2.6 layers vs 4.5 layers in moderate-to-severe TG; p < 0.0001). Amongst the various criteria, the permissive criterion was associated most frequently with mild TG, and had prognostic relevance. Because of this, we propose its usage as a marker of early cABMR-induced PTCBML if non-alloimmune causes of PTCBML can be ruled out.

[Banff score changes in kidneys from marginal donors]. / Banff-score-változások a marginális donorokból származó veséknél.

INTRODUCTION: Despite an increase in the number of cadaver donors and the number of overall organ transplantations, the dramatic increase in the waiting list makes it necessary to reconsider donor criteria. AIM: The authors examined whether differences could exist in the function and/or morphology of transplanted kidneys originated from marginal and ideal donors one and five years after transplantation. METHOD: Kidney function and histopathologic findings were analysed and compared one and 5 years after transplantation in 97 patients having marginal donor kidneys and 178 patients who received ideal donor kidneys. RESULTS: Serum creatinine level was significantly higher (p = 0.0001) and estimated glomerular filtration rate was significantly lower (p = 0.003) in patients having marginal donor kidneys as compared to those with ideal donor kidneys 5 years after transplantation. Morphological changes in the transplanted kidneys such as tubulitis (p = 0.014) and interstitial inflammation (p = 0.025) were significantly more frequently present in patients with marginal donor kidneys than in those with ideal donor kidneys one year after transplantation. CONCLUSION: Despite an absence of differences in kidney function one year after kidney transplantation between patients having marginal and ideal donor kidneys, morphologic differences in the transplanted kidneys can be detected between the two groups of patients.

Morphologic Features and Clinical Impact of Arteritis Concurrent with Transplant Glomerulopathy.

Little is known about the morphology and clinical relevance of arteritis in renal allograft biopsies with transplant glomerulopathy. We retrospectively reviewed the morphologic findings and clinical course of 59 patients with cg, 16 of which featured concurrent arteritis (fibrosing intimal arteritis with luminal narrowing in 15, and acute intimal arteritis in 1 case). Fifteen out of the 16 cases with arteritis fulfilled the morphological diagnostic criteria for chronic active antibody-mediated rejection, and 11 cases with arteritis showed morphological evidence of concurrent, ongoing T-cell-mediated alloimmune response (acute T-cell-mediated rejection in 5, borderline changes in 6 cases). Further, the Banff grades of interstitial inflammation in scarred and nonscarred cortex, total cortical inflammation, and arterial luminal narrowing were significantly higher in biopsies with arteritis. By immunohistochemistry, T-lymphocyte predominance over macrophages was found in the intimal infiltrates in 14 out of 16 cases, and cytotoxic T-lymphocytes were identified among intimal mononuclears in 10 cases. Patients with arteritis demonstrated a significantly shorter renal survival (7.5 vs. 29 months). In conclusion, T-cell-mediated mechanisms could play a role in the development of arteritis concurrent with cg. However, this finding does not exclude the possibility that antibody-mediated rejection can also contribute to the evolution of the lesion. Importantly, the lesion carries negative prognostic value likely via severe arterial luminal narrowing.

[Frequency of T-cell mediated rejection in new-onset diabetes after kidney transplantation]. / T-sejt-mediált rejectio gyakorisága vesetranszplantáció után, újonnan kialakult diabetes mellitusban.

INTRODUCTION: New-onset diabetes is one of the most common complications after kidney transplantation. AIM: The aims of the authors were to examine the frequency of new-onset diabetes mellitus in kidney transplanted patients receiving cyclosporine A (n = 95) and tacrolimus (n = 102) and to analyze the occurrence of T-cell mediated rejection in these two groups of patients. METHOD: Age, laboratory results, renal function and histological findings were evaluated one year after kidney transplantation. Histological evaluation was performed according to the 2007 modification of the Banff 1997 classification. RESULTS: New-onset diabetes developed in 12% of patients receiving cyclosporine A-based immunosuppression and in 24% of patients taking tacrolimus (p = 0.002). Uric acid level (p = 0.002) and the age of the recipient (p = 0.003) were significantly different in the new-onset diabetic and non-diabetic groups, while renal function showed no significant difference. Evaluation of tissue samples revealed a significant difference in T-cell mediated rejection between the new-onset diabetic and non-diabetic groups (13 vs. 8 patients; 37% vs. 6%; p = 0.001). CONCLUSIONS: The results indicate an early development of the pathological effect of new-onset diabetes after kidney transplantation on the morphology of the renal allograft.

The role of body image integrity and posttraumatic growth in kidney transplantation: A 3-year longitudinal study.

The aim of this study was to explore the role of body image, posttraumatic growth, and emotional state in recovery after transplantation. A total of 53 kidney transplant patients were assessed using our Self-Test and Organ Drawing Test, the Spielberger Anxiety Inventory, the Beck Depression Inventory, and the Posttraumatic Growth Inventory in a 3-year follow-up. Logistic regression analysis showed that lower levels of integrity of the body image and posttraumatic growth, and higher pre-discharge serum creatinine levels were significant predictors of graft rejection. Our results suggest that the integrity of the body image and posttraumatic growth might contribute to better health outcomes in organ transplantation.

[50-year history of kidney transplantation in Hungary]. / A veseátültetés elso 50 éve Magyarországon.

The first Hungarian kidney transplantation was performed by András Németh in Szeged in 1962, approximately 50 years ago. A preliminary agreement with Eurotransplant was signed in 2011, and special patient groups gained benefit from this cooperation in 2012, wnich lead to a full membership to Eurotransplant. This event inspired the authors to review the history of Hungarian kidney transplantation of the past 50 years, from the first operation to recent via the specific cornerstones of the transplant program. The donor of the first Hungarian kidney transplantation was the brother of the recipient. The operation itself was technically successful, but the lack of immunosuppression caused graft rejection, and the patient died after 79 days. His brother, the donor, is still healthy, after 50 years, and he encourages everybody to donate organs. Organized kidney transplant program started more than 10 years later, such as 1973, in Budapest. The program was supported by the Ministry of Health. New centers joined the program later, Szeged in 1979, Debrecen in 1991 and Pécs in 1993. These four transplant centers work currently in Hungary, and 6611 kidney transplantation has been performed up to the end of year 2012.

[Psychological factors of successful kidney transplantations. The effects of anxiety and intrapsychic integration of the organ on recovery]. / A vesetranszplantáció sikerességének pszichológiai tényezoi. A szorongásszint és a szerv intrapszichés integrációjának hatása a gyógyulási folyamatra.

UNLABELLED: Transplant patients' attitudes and representations related to their illness, their body, and the healing process have a significant impact on their recovery. AIMS: The study involved 51 patients from the Department of Surgery, University of Szeged, Hungary. The primary aim was to examine the possible connections between emotional and mood factors, illness and body representations, and the successful onset of renal functions after surgery. METHODS: Patients were tested with a combination of 4 instruments: Spielberger's anxiety scale and Beck depression scale, self and organ drawings, and a questionnaire designed by the authors. RESULTS: Our data suggest that high distress correlates with kidney disfunction after transplantation. Patients with higher anxiety drew the kidney larger in their projective drawing test. It was a remarkable result that post-transplant blood test on Day 10 showed significantly lower creatinine and urea levels in those patients who had drawn the kidney smaller in their projective drawing test. This might indicate that the organ's normal intrapsychic integration and the related kidney functions are disturbed. CONCLUSIONS: The results of this study provide useful information about the psychological background, which has received relatively little attention so far. It can also give important clues for further research on clinical health psychology in supporting the healing process.

Peritubular capillary basement membrane changes in chronic renal allograft rejection: Comparison of light microscopic and ultrastructural observations.

Marked peritubular capillary basement membrane (PTCBM) multilayering, the ultrastructural feature of chronic antibody-mediated rejection (ABMR) of kidney allografts, was found to correspond histologically to PTCs with thickened BMs; such PTCs have been suggested as a novel histological marker of chronic rejection. We investigated whether scoring of PTCBM thickening can substitute the ultrastructural search for PTCBM multilayering. The thickening was graded in PAS- and Jones-stained sections in 110 biopsies from recipients with a late dysfunction, all examined ultrastructurally for transplant capillaropathy (≥3 PTCs with ≥5 BM layers). Grade 0 indicated no thickening. Grade 1 and grade 2 were assigned when the PTCBMs were as thick as or thicker than those of the non-atrophic tubules, and duplication/chain-like lamination of the PTCBM was noted in ≤3 or ≥4 high-power fields, respectively. The series was enrolled in subgroups of those with and those without histopathological lesions of chronic rejection. Fifty-six biopsies displayed lesions of chronic ABMR. Transplant capillaropathy was demonstrated in 40 biopsies. Grade 2 thickening furnished a substantial interobserver concordance rate (κ = 0.803) and correlated with the transplant capillaropathy. Jones staining performed somewhat better in scoring than PAS staining. Grade 2 thickening was verified in 35 biopsies involving chronic ABMR, and in one control biopsy (sensitivity 61.4%, specificity 0.98). Grade 1 thickening was not suggestive of chronic ABMR at all. In conclusion, grade 2 thickening can be regarded as the histopathological lesion of chronic ABMR; however, electron microscopy remains the gold standard in the assessment of PTCBM changes.

Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry.

BACKGROUND: Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required. METHODS: Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1% v/v formic acid and 0.1 mmol/L Cs+. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [2H12]-Cyclosporin A [13C, 2H3]-Rapamycin, [13C, 2H2]-Tacrolimus, and [13C2, 2H4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards. RESULTS: The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8 - 102.5% for cyclosporin A, 101.6 - 103.0% for sirolimus, 100.0 - 101.2% for tacrolimus, and 99.5 - 102.4% for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17 - 2.60% for cyclosporin A in the concentration range between 8.1 and 979 microg/L, 0.92 - 1.72% for sirolimus in the concentration range between 2.1 and 33.2 microg/L, 0.44 - 1.06% for tacrolimus in the concentration range between 2.0 and 30.8 microg/L and 0.82 - 4.34% for everolimus in the concentration range between 2.1 and 31.4 microg/L. CONCLUSIONS: An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.