Fibroblast Activation Protein Expression in Sarcomas.

Objectives: Fibroblast activation protein alpha (FAP) is highly expressed by cancer-associated fibroblasts in multiple epithelial cancers. The aim of this study was to characterize FAP expression in sarcomas to explore its potential utility as a diagnostic and therapeutic target and prognostic biomarker in sarcomas. Methods: Available tissue samples from patients with bone or soft tissue tumors were identified at the University of California, Los Angeles. FAP expression was evaluated via immunohistochemistry (IHC) in tumor samples (n = 63), adjacent normal tissues (n = 30), and positive controls (n = 2) using semiquantitative systems for intensity (0 = negative; 1 = weak; 2 = moderate; and 3 = strong) and density (none, <25%, 25-75%; >75%) in stromal and tumor/nonstromal cells and using a qualitative overall score (not detected, low, medium, and high). Additionally, RNA sequencing data in publicly available databases were utilized to compare FAP expression in samples (n = 10,626) from various cancer types and evaluate the association between FAP expression and overall survival (OS) in sarcoma (n = 168). Results: The majority of tumor samples had FAP IHC intensity scores ≥2 and density scores ≥25% for stromal cells (77.7%) and tumor cells (50.7%). All desmoid fibromatosis, myxofibrosarcoma, solitary fibrous tumor, and undifferentiated pleomorphic sarcoma samples had medium or high FAP overall scores. Sarcomas were among cancer types with the highest mean FAP expression by RNA sequencing. There was no significant difference in OS in patients with sarcoma with low versus high FAP expression. Conclusion: The majority of the sarcoma samples showed FAP expression by both stromal and tumor/nonstromal cells. Further investigation of FAP as a potential diagnostic and therapeutic target in sarcomas is warranted.

Thrombophilic Genetic Anomalies and Their Association With Dialysis Initiation Age in a Cohort of Lebanese Hemodialysis Patients.

OBJECTIVES: The relationship between chronic kidney disease and cardiovascular disease is complex and bidirectional. This relationship may be partially linked to thrombophilic genetic anomalies that may predispose to the progression of both diseases. MATERIALS AND METHODS: We analyzed blood samples from 102 Lebanese patients with end-stage renal disease and undergoing hemodialysis and 20 randomly selected healthy volunteers for frequencies of 12 cardiovascular disease gene mutations and traditional risk factors. The frequencies of these mutations were calculated and compared in both groups. We stratified patients by quartiles according to their mean score of genetic mutations and traditional risk factors, as well as their mean age at dialysis initiation. Correlation analyses were performed on the various patient groups. RESULTS: We observed a high frequency of mutations in patients on dialysis. Homozygous mutations (> 10% of patients) were observed in the PAI-1 (11%), MTHFR A1298C sequence variant (12.7%), and ACE genes (12%); in addition, the FXIII V34L and PAI-1 4G/5G genotypes were significantly associated with early dialysis initiation (P < .001 and P = .004, respectively). We observed a strong linear relationship between the different scores and age at dialysis initiation, with older patients exhibiting the highest genetic, traditional, and total scores versus those shown in the youngest patients (R2 = 0.72 and P < .001; R2 = 0.98 and P < .001; and R2 =0.96 and P < .001, respectively). CONCLUSIONS: Our results revealed a polygenic thrombophilic profile in our population of Lebanese patients with end-stage renal disease. This profile showed a strong association between early dialysis initiation and specific homozygous cardiovascular disease gene mutations. The cumulative load of these genetic and traditional risk factors may be partly responsible for the increased risk of cardiovascular disease and risk of progression to end-stage renal disease in patients with chronic kidney disease.

Could Salivary Cyclosporine Dosage Replace the Whole Blood Cyclosporine Measurements in Renal Transplant Patients?

BACKGROUND: Cyclosporin (CsA) has been extensively used as the immunosuppressant of choice in renal transplantation. Currently available approaches to assess CsA levels, both in serum and blood, fail to accurately reflect the concentration of the pharmacologically active drug fraction. Free CsA levels in biological fluids (blood or saliva) have been advocated to play an important role. Traditional salivary CsA monitoring tests are based on available archaic salivary techniques that are nonspecific and require large amounts of saliva. The aim of this study was to assess salivary CsA correlation using a novel and more accurate technique and to correlate with CsA levels in blood. MATERIAL AND METHODS: Patients provided blood samples of 2 ml and 2 ml of unstimulated saliva on the same day 2 h after the morning CsA dose (C2). Whole blood levels of CsA were determined using the monoclonal fluorescent polarization immunoassay (FPIA) kit. The FPIA kit was adapted to salivary testing by using a novel extraction method developed and patented under the name of Middle East Research Institute (MERI). Wilcoxon signed rank test compared the differences in blood and salivary CsA. Pearson's correlation coefficient assessed the linear association between blood and salivary CsA concentrations. All analyses were performed using IBM-SPSS version 23 (IBM Corp, Armonk, NY, USA). RESULTS: No significant correlation was observed between blood and salivary CsA levels. CONCLUSION: Salivary CsA concentrations at C2 cannot adequately replace C2 blood levels as an indicator of CsA bioavailability despite improved performance of monoclonal FPIA and application of the MERI technique. More studies may be warranted to design more reliable and less invasive procedures for therapeutic drug monitoring.

Bortezomib as a Novel Approach to Early Recurrent Membranous Glomerulonephritis After Kidney Transplant Refractory to Combined Conventional Rituximab Therapy.

We report a case of early recurrent membranous glomerulonephritis after kidney transplant from a deceased donor. The patient received induction therapy and was discharged with a serum creatinine level of 0.78 mg/dL on triple maintenance immunosuppressive therapy, which included tacrolimus, mycophenolate mofetil, and prednisone. At 7 months after transplant, a graft biopsy for new-onset isolated proteinuria (2.7 g/day) revealed stage 2 recurrent membranous glomerulonephritis. In the face of persistent proteinuria despite combined conservative rituximab therapy over several months and the total eradication of CD20-positive cells, bortezomib was introduced. This resulted in a substantial decline in proteinuria within 2 months and its subsequent disappearance several months later. This was paralleled by a considerable drop in plasma CD34-positive and CD138-positive cell counts. These preliminary observations indicate that recurrent posttransplant membranous glomerulonephritis is associated in part with a B-cell- mediated immunologic process that may involve both CD20-positive and plasma cells. Rituximab-resistant or partially responsive recurrent posttransplant membranous glomerulonephritis may benefit from a proteasome inhibitor-based therapy.

NeuCode labels for relative protein quantification.

We describe a synthesis strategy for the preparation of lysine isotopologues that differ in mass by as little as 6 mDa. We demonstrate that incorporation of these molecules into the proteomes of actively growing cells does not affect cellular proliferation, and we discuss how to use the embedded mass signatures (neutron encoding (NeuCode)) for multiplexed proteome quantification by means of high-resolution mass spectrometry. NeuCode SILAC amalgamates the quantitative accuracy of SILAC with the multiplexing of isobaric tags and, in doing so, offers up new opportunities for biological investigation. We applied NeuCode SILAC to examine the relationship between transcript and protein levels in yeast cells responding to environmental stress. Finally, we monitored the time-resolved responses of five signaling mutants in a single 18-plex experiment.

Organ trafficking and transplant tourism: the role of global professional ethical standards-the 2008 Declaration of Istanbul.

By 2005, human organ trafficking, commercialization, and transplant tourism had become a prominent and pervasive influence on transplantation therapy. The most common source of organs was impoverished people in India, Pakistan, Egypt, and the Philippines, deceased organ donors in Colombia, and executed prisoners in China. In response, in May 2008, The Transplantation Society and the International Society of Nephrology developed the Declaration of Istanbul on Organ Trafficking and Transplant Tourism consisting of a preamble, a set of principles, and a series of proposals. Promulgation of the Declaration of Istanbul and the formation of the Declaration of Istanbul Custodian Group to promote and uphold its principles have demonstrated that concerted, strategic, collaborative, and persistent actions by professionals can deliver tangible changes. Over the past 5 years, the Declaration of Istanbul Custodian Group organized and encouraged cooperation among professional bodies and relevant international, regional, and national governmental organizations, which has produced significant progress in combating organ trafficking and transplant tourism around the world. At a fifth anniversary meeting in Qatar in April 2013, the DICG took note of this progress and set forth in a Communiqué a number of specific activities and resolved to further engage groups from many sectors in working toward the Declaration's objectives.

Experimental and clinical transplantation: a commitment to integrity, accountability, and ethics in publication.

For the past few years, the social, economic, and political issues surrounding the field of organ transplantation have entered into many ethical discussions. Transplant tourism, and organ trade in particular, have finally received the attention they deserve and many commendable developments have ensued. The "Declaration of Istanbul on Organ Trafficking and Transplant Tourism," the result of a collective effort by hundreds of transplant professionals the world over, is one such example and is now considered the universal charter for ethical conduct in the field of transplantation. The Middle East Society for Organ Transplantation and its official journal Experimental and Clinical Transplantation were among its first endorsers, and it is our policy to ensure that all authors of articles published in our Journal adhere fully to the rules and regulations stated in The Declaration of Istanbul and by the Committee on Publication Ethics. We believe that the medical community must ensure that a foundation of ethical conduct and scientific integrity is maintained throughout the field, and we must strive toward this goal in all our clinical and scholarly efforts.

A novel approach in clinical immunosuppression monitoring: drug lymphocyte level.

Like others, we have shown a weak correlation between drug blood levels and clinical outcome and immune response. We recently established that in contrast to blood levels, drug lymphocyte levels are strongly associated with therapeutic efficacy. The discordance between the 2 methodologies regarding clinical outcome and immune response is related mainly to the weak association between drug blood level and target-cell content. This weak association explains the intra- and interindividual variabilities of the therapeutic response. These variations are related mainly to genetic and environmental factors including age, sex, body mass index, organ function, food and subsequent drug absorption, drug interactions, and the availability of extra-target-cell binding sites. These factors seem to influence the modes of action of immunosuppressive agents including drug absorption, metabolism, elimination, transport, extra-target-cell sites, as well as target-cell receptor expression and its binding affinity and specific enzyme baseline activity. Therefore, the cellular fraction of a drug at a fixed dosage is the result of the integration of out-fluxing and in-fluxing forces that are affected by genetic and environmental factors. Any redistribution of the drug between the different binding sites will ultimately affect its bioactivity with no change to its extracellular bioavailability (which is currently determined by pharmacokinetic measurements). Compared with whole-blood or plasma-level measurements, monitoring immunosuppression therapy at the site of action appears to be not only more clinically and immunologically relevant (since it measures the free fraction of the drug at its effector site), but this method also bypasses the potentially complex extracellular factors that affect bioactivity. Since the intracellular content of a drug strongly correlates with its biological effect, monitoring immunosuppression therapy at the site of action is comparable to pharmacodynamic monitoring. It is cost effective and easy to perform in clinical practice and could be used for all immunosuppressive drugs. Since it allows maximal reduction of adverse effects through dosage reduction while maintaining an optimal level of immunosuppression, it should offer a new alternative for immunosuppressive therapy monitoring and tailoring to the individual patient.

Treatment of cyclosporine-induced gingival overgrowth with azithromycin-containing toothpaste.

OBJECTIVES: Gingival overgrowth is a complication of cyclosporine therapy following organ transplantation. Oral azithromycin is frequently used to treat this complication. This study examined the efficacy of local azithromycin, in the form of toothpaste, against cyclosporine-induced gingival overgrowth. MATERIALS AND METHODS: Twenty stable renal transplanted patients (10 men and 10 women) with gingival hyperplasia were randomly assigned to a test group and a control group. Azithromycincontaining toothpaste had 85 mg azithromycin per gram of toothpaste. Both toothpastes were prescribed b.i.d., each time using 1.5 cm, for 1 month. All participants received scaling, root planing, polishing, and oral hygiene instructions, at least 4 weeks prior to initiation of the study. Gingival overgrowth index, bleeding on probing, blood urea nitrogen, creatinine, and serum cyclosporine levels were measured at baseline, and then again in the second and fourth weeks after tooth brushing. Patient satisfaction with the toothpastes was evaluated by a visual analogue scale. The stability of clinical responses was followed for 3 months after cessation of the toothpastes. RESULTS: Gingival overgrowth index decreased significantly in the azithromycin-containing toothpaste group (from 1.1+/-0.56 to 0.51+/-0.47, P<.001); however, in the control group, this decrease was not significant (P=.22). Bleeding on probing also decreased significantly in patients in the azithromycin-containing toothpaste group compared with controls (P=.001). When compared with baseline levels, trough levels of cyclosporine, blood urea nitrogen, and creatinine did not change in either of the groups. Patients in the control group were more satisfied with the toothpaste than were patients in the test group (53 vs 38). CONCLUSIONS: Azithromycin-containing toothpaste is an effective, simple, and noninvasive treatment for cyclosporine-induced gingival overgrowth.

Cyclosporine lymphocyte maximum level: a new alternative for cyclosporine monitoring in kidney transplantation.

OBJECTIVES: To determine the relationship between clinical outcome, lymphocyte count (LC), and cyclosporine (CsA) lymphocyte maximum level (LT(m)L) in kidney transplant recipients. MATERIALS AND METHODS: CsA LT(m)L was determined in patients with biopsy-proven graft dysfunction and in patients with normal graft function. Clinical outcome was compared according to CsA LT(m)L, dosage, blood trough (C(0)) and maximum (C(max)) levels, hematocrit level, and LC. RESULTS: Rejecting patients had significantly lower LT(m)L than did those with normal graft function (27 -/+ 11 pg/Lc vs 71 -/+ 79 pg/Lc; P < 0.01) and similar LTmL to those with nephrotoxicity (27 -/+ 8 pg/Lc). Patients with normal graft function exhibited significantly lower LC (0.001292 -/+ 696 x 10(9)/L) and serum creatinine levels (88.4 -/+ 35 micromol/L) when compared with rejecting patients (0.001717 -/+ 364 x 10(9)/L, 132.6 -/+ 8.8 micromol/L) and those with nephrotoxicity (0.001884 -/+ 582 x 10(9)/L, 123.7 -/+ 8.8 micromol/L) (P < 0.03, P < 0.001). No significant difference was observed among the 3 groups with regard to CsA dosage, C(0), C(max), mycophenolate mofetil (MMF) dosage, and mycophenolic acid (MPA) plasma levels. CsA LT(m)L closely correlated in an exponential (R(2) = 0.98) and linear (R(2) = 0.35) fashion with LC and hematocrit level, respectively. Conversely, CsA C(max) failed to correlate with C(0) and these 2 latter parameters. Weak correlations were observed between CsA C(max) and its corresponding LT(m)L. CONCLUSIONS: CsA LT(m)L appears to correlate better than CsA C(max) with rejection-free outcome and LC. An increase in hematocrit appears to have an adverse effect on CsA lymphocyte binding. CsA LT(m)L may offer a new alternative for CsA monitoring in kidney transplantation.

Pharmacokinetic conversion study of a new cyclosporine formulation in stable adult renal transplant recipients.

Cyclosporine A (CyA) is a standard component of immunosuppressive regimens. It is a critical-dose drug for which a minor change in absorption can have important clinical consequences. The aim of the study was to compare the pharmacokinetics and safety of the new generic CyA formulation, Equoral capsules, after a switch from original formulation, Neoral capsules, in seventy stable adult renal transplant recipients. The extent and rate of pharmacokinetic parameters for bioequivalence were compared in a non-randomized, steady-state clinical study with fixed non-replicate study design. Pharmacokinetic analysis of CyA have shown that both the rate and extent of absorption of Equoral does not differ significantly from that of Neoral. At identical dosing, the new formulation was found to have geometric means of C(max) 717 ng/ml and AUCtau 3108 ng/ml.h, while corresponding results of comparator were 725 ng/ml and AUCtau 3039 ng/ml.h, respectively. The 90 % confidence intervals of C(max) and AUCtau were within 80- 125 % interval of the mean values. The results suggest that Equoral capsules can be used as an alternative treatment to Neoral capsules in CyA regimen.

Middle East Society for Organ Transplantation (MESOT) Transplant Registry.

During the seventies, sporadic renal transplants were performed in few MESOT-region countries, mainly Turkey, Iran, Egypt, and Lebanon. Since the introduction of cyclosporine in the early eighties, transplantation has become the preferred therapeutic modality for end-stage renal failure. In 1986, the Islamic theologians (Al Aloma) issued what became known as the Amman declaration, in which they accepted brain death and retrieval and transplantation of organs from living and cadaveric donors. Based on this and similar declarations, all Middle Eastern countries except Egypt passed laws that allow cadaveric transplantation and regulate live donations. Iran, Turkey, Saudi Arabia, Kuwait, Tunisia, Jordan, and Lebanon all have current active cadaveric programs and perform liver, heart, pancreas, and lung transplants. More than 5088 renal transplants/year are performed in the region with Iran leading with 1600. The cumulative number of renal transplant patients is now nearly 60,000. With a 2003 population of 600,682,175, the rate/million for renal transplantation in the MESOT region is a mere 9/million. Rates of renal transplantation range from 31/million in some countries to 0 in others. The major obstacle in establishing an accurate number of transplants is "tourist transplantation," in which the same transplanted patients are registered in different countries. Although cadaveric programs have been active for more than 10 years, live-related and nonrelated transplants account for nearly 85% of the total transplants. The data presented were collected from MESOT representatives in the region and from publications. For proper compilation of the registry, a format is being proposed that will be presented at the Congress for review and adaptation. Even with the limited resources in the region, immunosuppressive drugs for induction and maintenance therapy are available and are used. Costs for transplantation and immunosuppressive therapy are either totally or heavily supported by governmental agencies.

Cyclosporine blood level monitoring. Cross-reactivity of anti-cyclosporine A monoclonal with its sulphate metabolite: an in vitro study.

Cyclosporine is metabolized in the liver by the Cytochrom P450 system. Many of the metabolites have been identified and thoroughly studied. However the sulphate metabolite is the least known. The metabolite is hydrophilic and thus it is not easily detectable by HPLC and it is not available in pure form for analysis. We have isolated the metabolite from transplant patients receiving CYA as part of their immunosuppressive therapy. The amino acid sequence was determined and the molecule was synthesised in our laboratories. We have tested the molecule in vitro using cell culture to determine its activity. 1/2 ml of blood+1/2 ml of RPMI was incubated with the following concentration of either CSA or CSS: 0, 25, 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 ng/ml for 2 and 1/2h at 37 degrees C with 5% CO2. The blood was then stimulated with Ionomycine and PMA (phorpol 12 myristate 13-acetate) for additional 2h. Supernatant was collected and assayed for the concentration of the following cytokines: IL-1a, IL-2, Interferon (IFNa), IL-6, IL-12, TNFa. The cells were used to evaluate the expression of cell activation marker CD69. Blood was assayed for CYA concentration using Abbott TDx assay. Results the level of the metabolite was actually higher than the parent compound indicating that the monoclonal antibodies detected the S form preferentially. The results with other CYA monoclonal was also similar. These results suggest that blood level monitoring of CYA many not be accurate as all the monoclonal antibodies currently used cross react with the metabolites.

The mosaic of immunosuppressive drugs.

Graft rejections as well as tolerance are true representation of the specificity, sophistication and redundancy of an elegantly and meticulously designed immune system. Tolerance is in a way similar to the process of self-recognition where lymphoid clones, during development, baring self-reactive receptor are eliminated or rendered in active by "clonal deletion" leading to a state of accommodation and acceptance (anergic). On the other hand, both acute and chronic rejections are manifestation of the purpose of existence of the immune system, which is to defend the host against foreign invaders. Thus, in order to treat (control) graft rejection it is necessary to determine and understand the steps leading to recognition, stimulation, activation, and amplification of the immune system. The first step leading to the initiation of the immune system cascade is recognition. Which can either be direct where donor antigens of the major histocompatibility complex (MHC) expressed on the donor cells (passenger leukocytes) or tissues are recognised by the host immune system. The direct recognition pathway initiates acute graft rejection. Alternatively processed donor MHC peptides presented by the recipient antigen presenting cells (APC) initiate the indirect pathway of immune response, which is as important as the direct recognition especially in chronic rejection. Recognition is followed by the ligation of a series of adhesion molecules starting with an antigen to its specific T-cell receptor (TCR)/cluster of differentiation (CD) complex, expressed on the surface of the T cell. In order for the activation to precede additional costimulatory signals, such as ligation of the CD28/B7, CD4/HLA class II and CD/HLA class I antigens are required. The activation process is accompanied by an increase of cytokines production such as interleukin (IL)-2, IL-12, interferon (INF) and tumour necrosis factor (TNF) by the primed T cell. The complexity and the polymorphic nature of the immune system have necessitated designing agents that inhibit the immune system at different levels. Cyclosporine and Tacrolimus, collectively known as calcineurin inhibitors, seems to act on the IL-2 by inhibiting its production thus leading to a decrease in the proliferation of the activated lymphocyte. Rapamycin, which is similar to Tacrolimus, inhibits graft rejection by blocking IL-2 activation and phosphorylation of 70 S6 kinase thus inhibiting the progression of T-cell from G to S phase. While Cellcept (MMF) reduce the proliferation of T cell by inhibiting purine synthesis and by its action on ionosine monophosphate dehydrogenase. Anti-lymphocyte antibodies (ATG) deplete circulating lymphocytes while selective monoclonal antibodies are directed against IL-2 receptor thus reducing the rate of proliferation of activated T cells. Recently, antibodies to the CD40/CD40 ligand have been shown to induce long-term graft survival with the inhibition of the Th1 cytokines (INF), IL-2 and IL-12 and upregulating the Th2 cytokines IL-4 and IL-10. Lastly graft rejection can be reduced by blockade of the B7/CD28 costimulation pathway with the fusion protein CTLA-4Ig. With the availability of such potent and diverse agents it is now possible to develop multi drug regiments that can depress the immune system at the different steps of the activation cascade, with minimal side effects, thus improving graft and patient survival rates.

The generics in transplantation and the rules on their use.

By definition a product identified by its official chemical name rather than an advertised brand name is called a generic. If a drug exert its pharmacological effects at the same site, have the same potency, same dosage form and same bioavailability as a brand name, reference listed drug (RLD), is considered as a generic. However inactive ingredients can differ between brand name and generic. It is through the regulations of the FDA that the generics gained many ground in the drug market, they currently account to more than 42% of the total prescription in the USA. These regulations include the abbreviated new drug application (ANDA) for the registration process and drug substitution at the pharmacy level without patient or physician consent. This coupled with a keen interest of third party payers and the health authorities to reduce the high transplant health budget (over 2 Billion US $) made it a necessity to introduce the generics into the field of transplantation. Using the above mentioned definition we can theoretically say that all anti-lymphocytes, produced in the same animal species, are generic of each. Moreover, monoclonal antibodies that are directed against the same target and have the same bioavailability are also consider generics to each other. Of all the immunosuppressive drugs that have been introduced into the field of transplantation none has been as dominant as Cyclosporine. Cyclosporine became and still is the backbone for any immunosuppressive protocol. In the year 1992, Consupren, the first, non-FDA approved, generic to Sandimmun was introduced. Although Consupren was not bioequivalent to Neoral, however, long-term results in kidney transplantation have been similar for both drugs. The introduction of Consupren resulted in a near 40% reduction in the total cost of immunosuppressive therapy. Interestingly the cost of the brand name drug Neoral was also reduced by 20%. The cost reduction allowed the introduction of the new immunosuppressive agents MMF and Rapamycin. Currently there are 5 FDA approved Cyclosporine generics with a 20% market share in the USA and a mere 0% in Europe. Alternatives formulations to both Rapa and for MMF would be available soon. These forms are not by definition generics and are considered by the FDA to be new brand names act on the same site as Cell Cept and Rapaimmune. Their introduction would be a great welcome and would definitely results in cost saving in transplantation cost. In conclusion, generics efficacy and safety is similar to that of the brand name and their use is cost effective.

Bioavailability versus bioequivalence: the cyclosporine model.

The quest for a fixed-dose immunosuppressive drug continues. Experience with cyclosporine, tacrolimus and mycophenolate mofetil has taught us that there is no correlation between dose and clinical events. These data indicate that the concentration of the drug at the site of action (bioavailability) of each of these agents differs from one patient to the next. In addition, the bioequivalence (concentration of intact drug at the site of action resulting in a measurable response [effect]) may differ among individuals. Technically, it is very difficult to measure the drug concentration at a particular site, especially in organs or tissues that are not directly accessible. Therapeutic drug-blood-level monitoring is a simple indirect method that is used to estimate both bioavailability and bioequivalence. However, the immunosuppressive effect of all these drugs is initiated by binding to receptors on the surface of lymphocytes, which leads to inhibition of cytokine production and proliferation of activated lymphocytes. Thus, it would be more advantageous to monitor the level and effect of these drugs at the site of action (bioequivalence), the lymphocyte. This report describes an assay of this type that was developed for monitoring transplant patients at one center. The assay is based on measuring drug levels in the cytoplasm of lymphocytes. It is quick and easy to perform (20 samples per hour), inexpensive, and reproducible. The between-run Coefficient of Variance (CV) is 5.4 and a within-run CV is 3.1. For this study, blood and lymphocyte drug levels in transplant patients were determined and correlated with graft function and clinical events (biopsy-proven rejection and/ or toxicity).