Between February 2008 (i.e., the date when the solid-phase Luminex assay was set up in our institution) and June 2015, a total of 298 adult patients received a liver transplant from deceased donors (DDLT) in our center. Patients excluded from the study were those that died within the first month posttransplantation (n = 34), those that needed a re-transplant during the first month (n = 2), and those that received a transplant with a preformed DSA (mean fluorescence intensity cut-off > 1000) directed against human leukocyte antigen (HLA) A, B, Cw, DR, DQ, or DP (n = 37). In order to avoid confounding factors associated with others immunosuppressive treatments, only patients that received and were maintained under Tac and mycophenolate mofetil (MMF) (with or without steroids) were included in this study (Figure 1). All patients but five received Tac given twice daily (Prograf^®). The other five received Tac once daily (Advagraf^®). We excluded patients that had Tac or MMF withdrawn. Moreover, to calculate intra-patient variability, at least three trough levels of Tac had to be available. Hence, 116 patients with a functioning liver allograft at 1 mo posttransplantation were included in this study after having given their informed consent and after we had obtained Toulouse University IRB approval.

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Figure 1 Flow chart.

The target concentration of Tac trough level was 7-10 ng/mL during the first 3 mo, and 5-10 ng/mL thereafter during the follow-up. Each participant was followed for 2 years or until re-transplantation (n = 3) or death (n = 6). The median follow-up was 24 mo (range: 6-24). All rejection episodes were biopsy proven. Biopsies were only performed for cause during the study period and were analyzed according to the Banff criteria[18-20]. Graft failure was defined as the need for re-transplantation or as death from liver failure.

Detection of cytomegalovirus was performed using real-time PCR, as previously described[21], at month 3, 6, 12, and 24, and at any other time if clinically indicated.

Tac trough levels were routinely assessed using high-performance liquid chromatography-linked tandem mass spectrometry (HPLC-MS) at discharge, then monthly between months 1-6, and thereafter at months 9, 12, 15, 18, and 24. To calculate the IPV of Tac, at least three Tac trough levels from each patient had to be available. The median number of available Tac measurements was 10 (range: 4-12).

Tac IPV was estimated using the coefficient of variability (CV). The CV-IPV was calculated as follows: CV-IPV (%) = (standard deviation/mean Tac trough-level concentration) × 100. Because all patients received the same drug dose between discharge and M24, the obtained levels were corrected for the corresponding daily dose of tacrolimus (CV C₀/D-IPV). In addition, because some patients were converted from one formulation to another during the follow-up, we calculated CV and CV C₀/D-IPV after excluding the Tac trough levels obtained during the adjustment dose period, i.e., the month following a switch.

To compare IPV with the two formulations of Tac, the Tac twice-daily CV-IPV was calculated using Tac trough levels obtained from patients that had received Tac twice daily since transplantation until last follow-up and those obtained in patients switched for Tac once daily before the switch. The Tac once-daily CV-IPV was calculated using Tac trough levels from patients that received Tac once daily since transplantation until the last follow-up, and those obtained from patients that were later switched from twice- to a once-daily formulation after the switch (this excluded Tac trough levels obtained in the month following the switch).

All patients were screened for anti-HLA DSAs at transplantation, and at month 3 and 12, and annually thereafter. Additional screening was performed in case of graft dysfunction. Luminex^® assays were used to determine the specificity of class I HLAs in A/B/Cw and class II in DR/DQ/DP IgG antibodies in the recipients’ sera (centrifuged at 10000 g for 10 min) using Labscreen single Ag HLA class-I and class-II detection tests (One Lambda, Canoga Park, CA, United States), according to the manufacturer’s instructions. The presence and specificity of antibodies were then detected using a Labscan 100^®, and the mean fluorescence (baseline) value for each sample in each bead was evaluated. The baseline value was calculated as follows: [raw sample mean fluorescence intensity (MFI)-raw negative serum control MFI-negative-bead raw MFI sample-negative-bead raw MFI negative serum control]. A baseline value of > 1000 was considered positive.

Categorical variables are expressed as percentages and comparisons between groups were made using the chi-squared test or, if appropriate, Fisher’s exact test. Continuous variables were expressed as medians and ranges, and compared using the Mann-Whitney test. Logistic regression analysis was used to determine the predictors for acute-rejection episodes and the occurrence of de novo anti-HLA DSAs. Variables with a P < 0.1 in the univariate analyses were included in the stepwise multivariable analyses. P < 0.05 was considered statistically significant.

During the follow-up, 44 (38%) patients were switched from Tac immediate-release given twice a day (Prograf^®), to Tac once a day to improve quality of life. The switch was performed at a mean of 15 (range: 1-18) mo posttransplantation.

Mean tacrolimus trough level was 8 ± 3 ng/mL during the follow-up (Table 1). The mean dose of Tac was 6.8, 6.7, 6.4, 5.9, 5.4, 5.1, 4.8, and 4.6 mg/d, respectively, at discharge and at months 1, 3, 6, 9, 12, 18, and 24. Forty-five (38.8%) patients presented with a Tac trough level of < 5 ng/mL at least once during the follow-up. The overall mean Tac CV- IPV was 32 ± 12% [median CV-IPV 30.5% (7.6-80.6)]. Tac CV-IPV distribution is presented in Figure 2. The 1^th, 2^th, 3^th, and 4^th quartiles were, respectively, 25%, 30.5%, 36.5%, and 80.6%. The mean Tac CV-IPV was 30% ± 11% in patients given Tac once daily and was 32% ± 12% in patients that received Tac twice daily (P = 0.10). The mean Tac CV- IPV in the five patients that had received Tac once-daily since transplantation was 30% ± 7%. In the 44 patients that were converted from Tac twice-daily to once daily, the mean values of Tac CV-IPV were 32.3% ± 12% and 30% ± 12% before and after the switch, respectively (P = 0.21).

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Figure 2 Distribution of tacrolimus according to intra-patient variability.

Overall mean CV C₀/d- IPV was 73% ± 43%. It was 69% ± 29% with Tac twice-daily compared to 79% ± 50% for Tac given once daily (P = 0.9).

During the follow-up, 22 patients (19%) presented with at least one episode of acute rejection. The time between transplantation and a diagnosis of acute rejection (i.e., the date of the biopsy) was 3.5 mo (range: 0.5-12). Fourteen patients (12%) experienced a T-cell steroid-sensitive acute rejection, and six patients (5%) presented with a T-cell steroid-resistant acute rejection, which was treated with polyclonal antibodies. One patient presented with an acute antibody-mediated rejection at 4 mo posttransplantation. The Tac CV-IPV in this patient was high: CV-IPV of 63.2% and CV C₀/d- IPV = 68.2%. The risk factors for acute rejection after liver transplantation are presented in Table 2. The predictive factors for a biopsy-proven acute rejection were a Tac trough level of < 5 ng/mL [OR = 3.68; 95%CI (1.30-10.41), P = 0.014], the Tac CV-IPV (coded as a continuous variable) [OR = 1.1; 95%CI (1.01-1.11), P = 0.008], a CV-IPV of > 35% [OR = 3.07; 95%CI (1.14-8.24), P = 0.03], and a CV-IPV of > 40% [OR = 4.16; 95%CI (1.38-12.50), P = 0.01]. Twenty-one of the 22 patients that presented with an acute-rejection episode were receiving Tac twice daily when the rejection was diagnosed.

Thirteen patients (11.2%) presented with at least one de novo DSA during the posttransplantation follow-up (nine anti-HLA class II, three anti-HLA class I, one anti-HLA classI and II). Only one of these patients developed an antibody-mediated rejection. The median time between transplantation and detection of a de novo DSA was 3.5 mo (range: 1-12). The risk factors for a de novo DSA are presented in Table 3. The Tac CV-IPV [coded as a continuous variable: OR = 1.1, 95%CI (1.0-1.12), P = 0.006), and a CV-IPV of > 35% [OR = 4.83, 95%CI (1.39-16.72), P = 0.01] or of > 40% [OR = 9.73, 95%CI (2.65-35.76), P = 0.001] were identified as predictors for the occurrence of de novo DSAs detection.

During the follow-up, six patients died [at a mean of 13 mo (range: 6-23) posttransplantation]. The causes of death were infections (n = 3), cardiovascular (n = 2), and neoplastic (n = 1) complications. No difference in Tac CV- IPV was observed between patients that died during the follow-up (CV-IPV 33% ± 6%) and those that did not (CV-IPV 32% ± 12%; P = 0.70). Three patients required re-transplantation at month 5, 10, and 14, respectively, for ischemic cholangitis that occurred posttransplantation. During the follow-up, 24 patients presented with posttransplant replication of cytomegalovirus. No difference in Tac CV-IPV was observed between patients with replication of cytomegalovirus (CV-IPV 32% ± 9%) and those without replication (32% ± 12%, P = 0.90).

Tacrolimus (Tac) is considered a cornerstone within immunosuppression protocols to prevent T-cell and antibody-mediated rejection after liver transplantation. However, this treatment presents a narrow therapeutic index: overexposure can lead to clinically serious events, thus necessitating regular therapeutic drug monitoring, whereas underexposure can lead to acute or chronic graft rejection. The concept of intra-patient variability (IPV) refers to the fluctuations in Tac blood concentrations (and consequently episodes of over- and under-immunosuppression) that some patients experience over time.

Tac-IPV is an inexpensive assay to explore fluctuations in Tac blood concentrations. We investigated the potential usefulness of Tac-IPV to predict the incidence of donor specific antibodies and graft rejection episodes.

Our aim was to investigate the role of tacrolimus IPV in adult liver-transplant recipients.

We retrospectively assessed tacrolimus variability and analyzed its effect on the occurrence of graft rejection and de novo donor-specific antibodies.

Twenty-two patients experienced at least one acute-rejection episode (BPAR). Predictive factors for a BPAR were a tacrolimus IPV of > 35% or > 40%, and a tacrolimus trough level of < 5 ng/mL. Thirteen patients developed at least one dnDSA during the follow-up. Tacrolimus IPV and tacrolimus IPV of > 35%, and > 40% were identified as predictors to detect dnDSAs. IPV did not impact on patient- or graft-survival rates during the follow-up.

In our study higher Tac-IPV was associated with graft rejection and occurrence of DSAs.

Tacrolimus-IPV could be a useful tool to identify patients with a greater risk of graft rejection and of developing a de novo DSA after liver transplantation.