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Abstract

Background:

High-performance liquid chromatography–tandem mass spectrometry (HPLC-TMS) is preferred to fluorescent polarisation immunoassay (FPIA) in therapeutic drug monitoring (TDM), as it only measures active drug. Our hospital replaced FPIA with HPLC-TMS for analysis of calcineurin inhibitor (CNI) levels. Preliminary studies demonstrated significantly lower CNI levels with HPLC-TMS versus FPIA in kidney transplant recipients (KTRs). Most studies evaluating CNI exposure in KTRs used FPIA, posing challenges in interpreting HPLC-TMS levels. This study aimed to compare the effectiveness and safety outcomes in the one-year period before (FPIA period) and after (HPLC-TMS period) the switch.

Methods:

A retrospective cohort study was conducted on KTRs receiving ciclosporin (CsA) or tacrolimus (FK). Primary effectiveness end points were biopsy-proven acute rejection (BPAR) rate and estimated glomerular filtration rate (eGFR); safety end points were viral infections and hospitalisation for infection. The secondary end point was change in CNI levels during the FPIA versus HPLC-TMS periods.

Results:

A total of 307 patients were analysed (CsA n=182, FK n=125). BPAR only occurred in two patients in the HPLC-TMS period among the CsA cohort (p=0.50), but it occurred in six patients in the FPIA period and one patient in the HPLC-TMS period (p=0.13) among the FK cohort. Median eGFR were similar in both periods (CsA: 52.2 mL/min/1.73 m² (interquartile range (IQR) 39.8–67.6) vs. 52.5 mL/min/1.73 m² (IQR 37.1–68.0), p=0.65; FK: 57.7 mL/min/1.73 m² (IQR 39.1–80.0) vs. 56.4 mL/min/1.73 m² (IQR 33.5–76.7), p=0.08). There were no significant differences in safety outcomes. The median change in CNI levels from the FPIA period to the HPLC-TMS period was –20.5% (IQR –32.5 to –3.9) and –6.9% (IQR –17.7 to 8.0) among CsA and FK cohorts, respectively (p<0.01).

Conclusion:

The switch from FPIA to HPLC-TMS for CNI TDM in KTRs did not yield significant changes in clinical end points.

Keywords

Renal transplant HPLC-TMS therapeutic drug monitoring

Introduction

Renal transplantation offers a life-saving treatment option for patients with end-stage renal disease (ESRD). Calcineurin inhibitors (CNIs), namely ciclosporin (CsA) and tacrolimus (FK), are the backbone of immunosuppressive therapy to prevent allograft rejection.¹ The narrow therapeutic index coupled with the presence of high intra- and interindividual variability makes therapeutic drug monitoring (TDM) of CNIs necessary in order to optimise the dose for individual kidney transplant recipients (KTRs).^2,3 TDM of CNIs can be performed using immunoassay or high-performance liquid chromatography–tandem mass spectrometry (HPLC-TMS). The latter has a major advantage over immunoassay, as HPLC-TMS is able to measure the active parent immunosuppressants specifically without interference from metabolites. Immunoassays, however, tend to overestimate drug concentrations due to non-specific cross-reactivity with the metabolites.⁴ Positive bias of up to 30% in CNI drug-level measurements determined by immunoassays compared to HPLC-TMS has been reported.⁵ HPLC-TMS also has a lower limit of quantification detection,⁶ and has been shown to be robust and efficient.⁷ In addition, it requires a small sample volume and minimal sample preparation. These advantages, together with rapid throughput and simultaneous quantification of other immunosuppressants, make HPLC-TMS a cost-effective and favoured option for TDM in clinical practice,⁸ as long as highly trained analysts are available.

Many laboratories have switched from immunoassays to HPLC-TMS to determine the concentrations of oral immunosuppressant.^7,9 Due to the absence of cross-reactivity with CNI metabolites, clinicians have to consider the implication of the lower reported drug concentrations when interpreting drug levels. This poses a challenge to clinicians to determine the therapeutic target due the paucity of studies that have evaluated the relationship between CNI exposures measured using HPLC-TMS and outcomes in kidney transplant, as the majority of studies have used immunoassays to determine CNI concentrations.

Singapore General Hospital’s (SGH) biochemistry department replaced fluorescent polarisation immunoassay (FPIA) with HPLC-TMS as the standard analytical method of quantification of CsA and FK levels on 15 February 2016. Two preliminary studies were performed to compare the performance of FPIA against HPLC-TMS for measurement of CsA and FK concentrations in KTRs in SGH. The median CsA two-hour peak concentration measured by HPLC-TMS was 62.0 μg/L (range –406.3 to 334.7 μg/L) lower compared to FPIA, corresponding to a percentage difference of 12.6% (range –50.5% to 62.9%; p<0.05). Similarly, the median FK level measured by HPLC-TMS was 0.7 μg/L (range –4.0 to 2.0 μg/L) lower compared to FPIA, corresponding to a percentage difference of 11.7% (range –41.0% to 32.8%; p<0.01).

We conducted this study to evaluate the impact on clinical outcomes and the change in immunosuppressant levels with the switch in analytical method from FPIA to HPLC-TMS for CNI drug-level determination among KTRs. The hypothesis of the study was that there were no differences in the effectiveness and safety outcomes among KTRs on CNIs between the FPIA period and the HPLC-TMS period. The primary aim of the study was to compare the effectiveness outcomes as determined by the incidence of biopsy-proven acute rejection (BPAR) and estimated glomerular filtration rate (eGFR), as well as the safety outcomes as determined by infective outcomes among KTRs on CNIs in the one-year period before (15 February 2015 to 14 February 2016; FPIA period) and one-year period after (15 February 2016 to 15 February 2017; HPLC-TMS period) the change in analytical method of quantification for CNI drug level from FPIA to HPLC-TMS. A secondary objective was to compare the change in CsA and FK levels measured using FPIA versus HPLC-TMS while patients were maintained at the same dose within the same study period.

Methods

Study design

This was a retrospective cohort study involving a review of patient charts available both electronically and on file from 15 February 2015 to 15 February 2017. The study was approved by SGH’s Institutional Review Board. Charts of KTRs aged ⩾21 years who were treated with FK or CsA and followed up at SGH were screened. CNI drug levels and clinical outcomes of recruited patients were collected as part of their routine clinical care. KTRs receiving CsA or FK with at least one drug level drawn during both the FPIA period and the HPLC-TMS period at SGH were included. Recipients of multi-organ transplants; KTRs on prolonged duration of major cytochrome P450 interacting drugs such as diltiazem, ketoconazole, clarithromycin and Viekira Pak^® (ombitasvir, paritaprevir, ritonavir, dasabuvir); KTRs on concurrent MTOR inhibitors as an immunosuppressive regimen; pregnant KTRs; KTRs on tailing doses of CNI due to a failing kidney graft; and KTRs whose CNI dose was changed prior to the first available HPLC-TMS level were excluded from the study.

Drug concentration measurement

Drug levels were collected if they were drawn within 15 minutes of FK trough or CsA two-hour peak timing. Whole-blood trough concentrations of FK and CsA were determined using the validated FPIA before 15 February 2016 and using HPLC-TMS from 15 February 2016 performed by the Department of Pathology at SGH.

Data collection

KTRs’ baseline demographics such as age, sex, ethnicity, cause of ESRD and transplant immunological risk status were retrieved from their medical charts. These records were maintained as part of the KTRs’ routine post-transplant care at SGH. In addition, baseline, 12-month and 24-month serum creatinine were collected. Information on delayed graft function (DGF) status, renal biopsy results, cytomegalovirus (CMV) and BK virus infections, hospitalisation for infections, as well as induction and maintenance immunosuppression regimens were also collected. Diagnosis of BPAR was based on histological findings using the 1997 Banff classification criteria,¹⁰ including the modifications and updates at the actual period of biopsy.^11
–15 DGF was defined by the requirement of haemodialysis within the first week post renal transplant. Expanded criteria donor in deceased donor transplants was defined as the presence of both diabetes and hypertension regardless of age, donation after cardiac death, cold ischaemia time exceeding 24 hours, aged ⩾60 years or aged >50 years with at least two of the following: hypertension, serum creatinine >133 µmol/L or death from a cerebrovascular accident.^16,17

Study end points

The primary end points for effectiveness were the incidence of BPAR and eGFR using CKD-EPI.¹⁸ The primary safety end points were the incidence of CMV infections, BK virus infections and hospitalisations for infection. The secondary end point of the study was the change in mean CNI levels drawn during the FPIA period versus the HPLC-TMS period computed for each KTR while being maintained at the same dose.

Statistical analysis

All statistical tests were two-sided and were conducted using the 0.05 level of significance. Frequency distributions are provided for categorical variables; mean and standard deviation (SD) or median and interquartile range (IQR) are presented for continuous variables, as appropriate. Categorical variables, including the incidence of BPAR and infections, were compared using McNemar’s test. eGFR comparisons and the difference in drug levels between the two analytical assays were determined using Wilcoxon’s signed ranked test. All data analyses were performed using IBM SPSS Statistics for Windows v22 (IBM Corp., Armonk, NY). Study outcomes were analysed separately for KTRs on CsA and FK.

Results

A total of 246 KTRs and 178 KTRs prescribed with CsA or FK, respectively, were screened. Fifty-seven patients on CsA were excluded due to a change in CsA dose prior to the first HPLC-TMS (n=18), TDM performed using trough levels (n=15), missing CsA level (n=10), inconsistent drug level timing (n=6), transplant after study start date (n=3), concurrent major interacting drugs (n=3), pregnancy (n=1) and discontinuation of CsA (n=1). Fifty KTRs prescribed with FK were excluded due to transplant or transfer of care to SGH after the study start date (n=17), inconsistent drug level timings (n=12), change of FK dose prior to the first HPLC-TMS level (n=10), concurrent major interacting drugs (n=5), missing FK level (n=3) and FK initiation after study start date (n=3). Seven and three KTRs on CsA and FK, respectively, discontinued the respective CNI before the end of the study. Reasons for premature discontinuation were death (n=3), switch in immunosuppressive agent (n=2) and failed graft (n=2) among KTRs on CsA; death (n=2) and failed graft (n=1) among KTRs on FK. Hence, 182 KTRs on CsA and 125 KTRs on FK were analysed at two years (Figure 1). All results in this report are presented in the sequence of FPIA period versus HPLC-TMS period.

Figure 1.

Disposition of patients in the study.

Baseline characteristics

The baseline characteristics as well as the transplant and immunosuppression details of KTRs are presented in Tables 1 and 2, respectively. The mean age of the KTRs was 53.9±10.1 years. The majority of KTRs were Chinese (80%) and on haemodialysis prior to transplant (80%). Fifty-six per cent of KTRs received deceased donor renal transplants, and 40% of KTRs had DGF.

Table 1.

Baseline characteristics of KTRs receiving CsA or FK.

Characteristics (units)	CsA (N=182)	FK (N=125)
Age (years)	55.5±9.7	51.6±10.2
Sex
Male	88 (48.4)	64 (51.2)
Ethnicity
Chinese	152 (83.5)	93 (74.4)
Malay	19 (10.4)	24 (19.2)
Indian	7 (3.9)	4 (3.2)
Other	4 (2.2)	4 (3.2)
Cause of ESRD
Glomerulonephritis	83 (45.6)	58 (46.4)
Immunoglobulin A nephropathy	21 (11.54)	20 (16.0)
Diabetes	12 (6.6)	10 (8.0)
Hypertension	13 (7.1)	12 (9.6)
Focal segmental glomerulosclerosis	4 (2.2)	5 (4.0)
Polycystic kidney disease	7 (3.9)	2 (1.6)
Systemic lupus erythematosus	3 (1.7)	8 (6.4)
Other	18 (9.9)	6 (4.8)
Unknown	21 (11.5)	4 (3.2)
Pre-transplant dialysis modality
Haemodialysis	149 (81.9)	100 (80.0)
Peritoneal dialysis	10 (5.5)	12 (9.6)
Not started on dialysis	10 (5.5)	10 (8.0)
Unknown	13 (7.1)	3 (2.4)
Laboratory results
eGFR (mL/min/1.73 m²)	56.4±20.5	60.0±25.1

All data are presented as the mean±standard deviation or number (%.)

KTR: Kidney transplant recipients; CsA: ciclosporin; FK: tacrolimus; ESRD: end-stage renal disease; eGFR: estimated glomerular filtration rate.

Table 2.

Transplant and immunosuppression details of KTRs receiving CsA or FK.

Characteristics (units)	CsA (N=182)	FK (N=125)
Immunological risk and transplant details
Panel reactive antibodies^a	9.4±19.3	19.2±32.3
Cold ischaemia time (hours)^b	8.6±6.9	10.1±7.5
DGF^c	36 (41.4)	32(37.2)
Donor type
Deceased – standard criteria donor	31 (17.0)	32 (25.6)
Deceased – extended criteria donor	17 (9.3)	16 (12.8)
Deceased – unknown	66 (36.3)	11 (8.8)
Living	51 (28.0)	61 (48.8)
Unknown	17 (9.3)	5 (4.0)
Previous rejection, including borderline changes	45 (24.7)	31 (24.8)
Immunosuppressive therapy
Antibody induction^d
No induction	12 (22.2)	5 (6.4)
Non-depleting antibodies	28 (51.9)	45 (57.7)
Depleting antibodies	14 (25.9)	28 (35.9)
Primary immunosuppressive regimen^e
CNI+mycophenolate+steroid	71 (64.6)	91 (90.1)
CNI+azathioprine+steroid	24 (21.8)	4 (3.9)
CNI+MTOR-I+steroid	4 (3.6)	2 (2.0)
CNI+steroid	7 (6.4)	2 (2.0)
MTOR-I+mycophenolate+steroid	3 (2.7)	0
Other	1 (0.9)	2 (2.0)

All data are presented as the mean±standard deviation or number (%).

Values available in 82 patients on CsA and 62 patients on FK.

Values available in 92 patients on CsA and 55 patients on FK.

Values available in 87 patients on CsA and 86 patients on FK.

Values available in 54 patients on CsA and 78 patients on FK.

Values available in 110 patients on CsA and 101 patients on FK.

KTR: Kidney transplant recipients; CsA: ciclosporin; FK: tacrolimus; DGF: delayed graft function; CNI: calcineurin inhibitor; MTOR-I: mammalian target of rapamycin inhibitors.

Primary outcomes

BPAR

A similar proportion of KTRs developed BPAR during the FPIA and HPLC-TMS periods in both the CsA and FK cohorts (CsA: 0% vs. 1.1%, p=0.50; FK: 4.8% vs. 0.8%, p=0.13; Table 3). The BPAR episodes were classified as early chronic antibody-mediated rejection (AMR; n=1) and a borderline change (n=1) in the HPLC-TMS period of the CsA cohort, as borderline changes (n=4), type 2A (n=1) and suspected AMR (n=1) in the FPIA period and as a borderline change (n=1) in the HPLC-TMS period among KTRs on FK.

Table 3.

Primary Outcomes.

		Effectiveness				Safety
Cohort	Period	BPAR	p-Value	eGFR (mL/min/1.73 m²)	p-Value	BK virus infections	p-Value	CMV infections	p-Value	Hospitalisation for infections	p-Value
CsA (N=182)	FPIA	0	0.5	52.2 (39.8–67.6)	0.65	2 (1.1)	1.00	11 (6.0)	0.24	57 (31.3)	0.49
CsA (N=182)	HPLC-TMS	2 (1.1)	0.5	52.5 (37.1–68.0)	0.65	2 (1.1)	1.00	17 (9.3)	0.24	51 (28.0)	0.49
FK (N=125)	FPIA	6 (4.8)	0.13	57.7 (39.1–80.0)	0.08	6 (4.8)	0.22	13 (10.4)	0.18	31 (24.8)	1.00
FK (N=125)	HPLC-TMS	1 (0.8)	0.13	56.4 (33.5–76.7)	0.08	2 (1.6)	0.22	7 (5.6)	0.18	31 (24.8)	1.00

All data are presented as median (IQR) or number (%).

CsA: ciclosporin; FK: tacrolimus; BPAR: biopsy-proven acute rejection; CMV: cytomegalovirus; FPIA: fluorescent polarisation immunoassay; HPLC-TMS: high-performance liquid chromatography–tandem mass spectrometry; IQR: interquartile range.

eGFR

Median eGFR values at 12 months (FPIA period) and 24 months (HPLC-TMS period) were not significantly different in both the CsA and FK cohorts (CsA: 52.2 mL/min/1.73 m² (IQR 39.8–67.6) vs. 52.5 mL/min/1.73 m² (IQR 37.1–68.0), p=0.65; FK: 57.7 mL/min/1.73 m² (IQR 39.1–80.0) vs. 56.4 mL/min/1.73 m² (IQR 33.5–76.7), p=0.08; Table 3).

Infections

Two (1.1%) KTRs in both the FPIA and HPLC-TMS periods had BK virus infections in the CsA cohort (p=1.00). There was also similar proportion of patients with a BK virus infection among KTRs on FK (4.8% vs. 1.6%, p=0.22). A similar occurrence of CMV infections was reported during the FPIA and HPLC-TMS periods for both the CsA and FK cohorts (CsA: 6.0% vs. 9.3%, p=0.24; FK: 10.4% vs. 5.6%, p=0.18). There were no significant differences between the two periods in the incidence of hospitalisations for any infections in both the CsA and FK cohorts (CsA: 31.3% vs. 28.0%, p=0.49; FK: 24.8% vs. 24.8%, p=1.00; Table 3).

Secondary outcome

Change in drug level

The median difference in CsA peak levels performed during the HPLC-TMS period compared to the FPIA period was –119.3 μg/L (IQR –213.1 to –19.8), while maintained at the same CsA dose, corresponding to a percentage difference of –20.5% (IQR –32.5 to –3.9; p<0.01). The median difference in FK trough levels drawn during the HPLC-TMS period compared to the FPIA period was –0.4 μg/L (IQR –1.2 to 0.4), corresponding to a percentage difference of –6.9% (IQR –17.7 to 8.0; p<0.01; Table 4).

Table 4.

Comparison of CNI concentrations measured using FPIA versus HPLC-TMS.

	Median CNI concentrations (μg/L), median (IQR)
	FPIA	HPLC-TMS	Inter-method difference	Percentage difference (%)	p-Value
CsA peak	628.1 (524.5–709.0)	488.0 (422.0–576.9)	–119.3 (–213.1 to –19.8)	–20.5 (–32.5 to –3.9)	<0.01
FK trough	6.2 (5.3–7.4)	5.8 (4.9–6.8)	–0.4 (–1.2 to 0.4)	–6.9 (–17.7 to 8.0)	<0.01

All data are presented as median (IQR).

CNI: calcineurin inhibitor; FPIA: fluorescent polarisation immunoassay; HPLC-TMS: high-performance liquid chromatography–tandem mass spectrometry; CsA: ciclosporin; FK: tacrolimus; IQR: interquartile range.

Discussion

HPLC-TMS is an ideal method for the measurement of CNIs, as it is specific for the parent drug and meets the criteria for a reference method.^19,20 With the change in our hospital’s method of CNI level quantification, this study was undertaken to compare the clinical effectiveness and safety end points with the use of HPLC-TMS versus FPIA in KTRs.

Within the overall two-year follow-up duration, there were no significant differences in the proportion of KTRs who developed BPAR and in the mean eGFR between the FPIA and HPLC-TMS periods. A revision in the SGH post-renal transplant immunosuppression protocol occurred in early 2016, which coincided with the switch in analytical technique from FPIA to HPLC-TMS. In the updated protocol, FK is the first-line CNI for maintenance immunosuppression regimens for all KTRs. Within the FK cohort for our study, there was a trend towards lower BPAR occurrence and lower eGFR values during the HPLC-TMS period compared to the FPIA period. The lower BPAR rate in the HPLC-TMS period may be attributed in part to the higher FK trough target range in the 2016 protocol, that is, 5–8µg/L for standard and extended criteria donor KTRs beyond one year post transplant compared to the target range of 5–6 µg/L in the previous protocol. This may also explain the lower eGFR trend in the HPLC-TMS period within the FK cohort, as higher FK levels predispose patients to CNI nephrotoxicity and potentially lower eGFR. However, analysis of primary safety outcomes of CMV and BK infections, as well as hospitalisations for any infections, yielded no significant differences between the two analytical methods of quantification.

Our previous studies comparing the two analytical techniques performed using the same blood sample yielded a median inter-method reduction of 62 μg/L for CsA peak and 0.7 μg/L for FK trough with the use of HPLC-TMS versus FPIA. Interestingly, in the current study, the median drug levels were 119 and 0.4 μg/L lower in the HPLC-TMS period compared to the FPIA period for CsA peak and FK trough, respectively. The disparity in observed inter-assay drug levels may be a result of greater intra-patient variability when drug levels were drawn using separate samples due to the cohort nature of the current study. The observed reduction in median CsA peak levels from 628 to 488 μg/L may not prompt most physicians to increase the CsA dose for stable chronic KTRs, as the latter is still within the recommended CsA peak target of 400–600 μg/L stipulated in the protocol. On the contrary, the observed reduction of median FK trough levels from 6.2 to 5.8 μg/L may prompt physicians to increase the FK dose for higher-risk transplant patients, for example ABO-incompatible KTRs, to attain the higher target trough of 6–8 μg/L among KTRs beyond a year post transplant.

A limitation of the study was its retrospective nature, which restricted data collection to information that was available in the database. We were also unable to control for potential confounders that influenced the drug levels attained, such as revisions to the immunosuppression protocol, the occurrence of malignancies and medication compliance. Although the unforeseen protocol revision is a potential source of bias, our study findings provide affirmation that targeting slightly higher FK drug levels during the HPLC-TMS period did not increase the incidence of infective adverse events while maintaining comparable efficacy end points. The follow-up duration of one year for each analytical method also limits the applicability of the study findings beyond a year.

Conclusion

The effectiveness and safety clinical end points were comparable when using FPIA versus HPLC-TMS in the quantification of CNI levels among KTRs. The study findings imply that the current CNI target range appears to be appropriate with the switch to HPLC-TMS for CNI level quantification at our institution.

Footnotes

Acknowledgements

None.

Authors’ contributions

L.F.G. and P.H.L. researched the literature and conceived the study, and were involved in protocol development, gaining ethical approval and data analysis. L.F.G. wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript.

Availability of data and materials

Data sharing is not applicable to this article, as no data sets were generated or analysed during the current study.

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Ethical approval

Singhealth Centralised Institutional Review Board (CIRB). CIRB Ref: 2016/2945.

Funding

The authors received no financial support for the research, authorship and/or publication of this article.

Informed consent

Informed consent was not sought for the present study because the data collected were part of routine drug monitoring and follow-up care through the hospital computerised system (Citrix, Sunrise Clinical Manager). After the completion of data collection, the KTRs were de-identified.

ORCID iDs

Li Fang Goh

Terence Kee

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Impact of transiting from fluorescent polarisation immunoassay to high-performance liquid chromatography–tandem mass spectrometry for immunosuppressant levels analysis in kidney transplant patients

Abstract

Background:

Methods:

Results:

Conclusion:

Keywords

Introduction

Methods

Study design

Drug concentration measurement

Data collection

Study end points

Statistical analysis

Results

Baseline characteristics

Primary outcomes

BPAR

eGFR

Infections

Secondary outcome

Change in drug level

Discussion

Conclusion

Footnotes

Acknowledgements

Authors’ contributions

Availability of data and materials

Conflict of interest

Ethical approval

Funding

Informed consent

ORCID iDs

References