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Original Articles: Gastroenterology: Inflammatory Bowel Disease

Determination of Serum Infliximab Concentration by Point-of-care Devices in Children With Inflammatory Bowel Disease

Curci, Debora; Lucafò, Marianna†,‡; Cifù, Adriana§; Bramuzzo, Matteo; Martelossi, Stefano||; Favretto, Diego; De Pellegrin, Francesca; Fabris, Martina; Vascotto, Fulvia; Naviglio, Samuele; Ventura, Alessandro‡,#; Stocco, Gabriele∗∗; Decorti, Giuliana‡,#

Author Information
Journal of Pediatric Gastroenterology and Nutrition: October 2019 - Volume 69 - Issue 4 - p 474-479
doi: 10.1097/MPG.0000000000002410
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What Is Known/What Is New

What Is Known

  • Therapeutic drug monitoring of infliximab through the determination of drug trough levels allows to improve patients’ care and characterize the cause of treatment failure.
  • ELISA assays are commonly used for therapeutic drug monitoring; however, their results are not immediately available to direct clinical practice in real-time.

What Is New

  • Point-of-care devices allowed to determine serum infliximab concentrations with good accuracy, as compared with ELISA assays, especially for low drug levels.
  • Correct classification of infliximab trough levels by therapeutic interval range was achieved in most cases.

Infliximab is a chimeric monoclonal IgG1 antibody directed against tumor necrosis factor alpha (TNF) approved for the treatment of inflammatory bowel diseases (IBD), including Crohn disease (CD) and ulcerative colitis (UC), both in adults and in children. The drug has been shown to induce and maintain clinical remission and mucosal healing, and it is now considered the first choice in refractory disease (1,2). Furthermore, anti-TNF therapy seems to be promising also as a first line treatment in early-stage IBD (3). Despite its proven efficacy, a sizable proportion of patients may either not respond to treatment or lose response over time and relapse (4,5). Lack of response may be because of several factors (6–8). Among these, low blood drug levels represent one of the most causes of treatment failure, both in adult and in pediatric patients, and, in fact, adequate drug levels have been demonstrated to be strictly associated with persistent remission (9–12). The occurrence of low drug levels may be because of immunologic clearance of the drug associated with the development of antidrug antibodies (ADA) or to other causes (insufficient dosing, increased intestinal protein loss, increased drug clearance, patient compliance, etc).

Therapeutic drug monitoring (TDM) can be useful to classify the cause of treatment failure in a patient, and may be used to adjust treatment (13). Different techniques are currently used to measure infliximab levels, including enzyme-linked immunosorbent assay (ELISA), radio-immunoassay, and homogeneous mobility shift assays. Although there is not a clear gold standard available, ELISA assays represent the most commonly used assays for infliximab TDM in clinical practice, with a good agreement among different kits (14). Nevertheless, they are still not available to all clinicians, as not all hospitals have introduced the test in their test catalog. Furthermore, when available, the results are not provided rapidly, as laboratories tend to perform the test in batches, and the ELISA assay itself requires several hours to be performed by appropriately trained personnel. This may reduce the effectiveness of using TDM to guide therapy in clinical practice, as knowing infliximab blood levels immediately, at patient's bedside or in an outpatient clinic, can allow to change clinical decisions or modify (increase or decrease) the drug dose to infuse. To address these concerns, rapid point-of-care (POC) assays have been developed to measure serum infliximab concentrations. Advantages of these devices include the possibility of being used for individual sample testing, the relative ease of operation, and the quick turnaround time for results (15–20 minutes). In literature, few studies have evaluated the use of POC infliximab assays, and only in adult patients (15,16). Greater use of TDM to adjust therapy in children, however, could be very important as the consequences of sub-optimal treatment could be worse in pediatric patients, limiting their long-term quality of life (17). The importance of TDM, in fact, is increasingly being recognized in clinical practice also in pediatric IBD. Recently, Burgess et al (18) have reported the utility of a proactive dosing strategy in children on combination therapy with an immunosuppressant: IFX levels <3 μg/mL postinduction and during maintenance were associated with relapse, and levels >7 μg/mL with sustained remission. However, pediatric IBD centers could suffer from lower patient volumes in comparison with adult clinics, and this may lead to greater difficulties in obtaining prompt responses with conventional ELISA assays. We sought to compare 2 POC devices for quantification of infliximab with 2 validated ELISA assays for classification of infliximab trough levels in a pilot study on a small cohort of children with IBD treated with infliximab.



Thirty-two blood samples were obtained from 19 pediatric IBD patients (median age 15.5, interquartile range 13.6–16.9; CD = 15 and UC = 4) treated with infliximab at the Pediatric Gastroenterology and Hepatology Unit of the Institute for Maternal and Child Health IRCCS Burlo Garofolo, in Trieste, Italy, between February and November 2016. Children were being treated according to a therapeutic protocol consisting of intravenous administration of infliximab at 5 mg/kg in 2 hours infusions at weeks 0, 2, 6 (induction phase), followed by a maintenance phase in which infusions were performed every 8 weeks. Serum samples were collected immediately before infusion (trough level), both during the induction (before week 6 infusion) and maintenance phases. Samples were stored at −20°C and were analyzed retrospectively. Local Ethic Committee approval and appropriate informed consent were obtained from all patients’ parents or caregivers. As the goal was to compare analytical assays, all 32 samples were considered as independent and were analyzed with 4 different tests: 2 ELISA assays and 2 POC devices.

Enzyme-linked Immunosorbent Assays

Infliximab levels were measured using 2 commercial ELISA assays: Lisa-tracker ELISA assay (Lisa Tracker-Duo® Infliximab, Theradiag, Marne-la-Vallée, France) and Promonitor assay (Proteomika S. L., subsidiary of Progenika Biopharma S.A., Spain). Lisa-Tracker assay uses TNF-coated wells, whereas Promonitor is a capture ELISA assay, with wells precoated with anti-TNF monoclonal antibody bound to recombinant TNF. Both ELISA kits were used according to the manufacturers’ instructions. In particular, after incubation with patient sera, both assays require washings and incubation with MA-IFX6B7, a highly specific monoclonal antibody against infliximab conjugated with horseradish peroxidase. The enzymatic activity is measured by adding a chromogenic substrate and measuring the intensity of the color obtained by spectrophotometric analysis at a wavelength of 450 nm, with 620 nm as the reference wavelength.

Point-of-care Assays

Two POC assays were tested. The Quantum Blue (POC IFX/QB) (Bühlmann Laboratories, Schönenbuch, Switzerland) is a lateral flow assay, which uses a chip card to provide the reader with the test information and calibration curve. Serum samples were obtained after centrifugation, then diluted 1:20 with buffer and loaded into the port of the test cartridge according to the manufacturer's instruction. After 15 minutes, the results are shown on a display. According to the manufacturer, it has a detection limit of 0.15 μg/mL, and the lower and upper limits of quantification are 0.4 and 20 μg/mL, respectively. The RIDA Quick (POC IFX/RQ) (R-Biopharm, Darmstadt, Germany) is also a lateral flow assay based on the same principle of the POC IFX/QB assay and requires 20 minutes to display results. Serum samples were diluted 1:50 with buffer according to the manufacturer's instruction. It has lower and upper limits of quantification of 0.5 and 10 μg/mL, respectively; in case of results above 10 μg/mL, samples were further diluted to achieve a quantitative result up to 20 μg/mL.

Statistical Analysis

The results obtained with the POC assays and with Promonitor ELISA assay were compared with Lisa-Tracker ELISA assay, conventionally assumed as reference. Statistical analyses were performed using the R software (R Core Team, 2018, version 3.4.2). The threshold level of statistical significance was set to ≤0.05. Intraclass Correlation Coefficient (ICC) and Bland-Altman plots were assessed for quantitative comparison. For qualitative comparison, weighted kappa statistics were determined after stratification of results by therapeutic interval (<3 μg/mL, ≥3 to <7 μg/mL and ≥7 μg/mL). This therapeutic interval was determined according to literature data on patients treated with infliximab (19–21).


Quantitative comparison showed excellent ICC between the reference ELISA (Lisa-Tracker) and the 2 POC infliximab assays (ICC 0.82 and 0.87 for POC IFX/QB and POC IFX/RQ, respectively). ICC between the 2 ELISA assays was 0.87 (Fig. 1). The Bland-Altman plots showed a good dispersion between the reference ELISA assay and the POC devices, with best results achieved for lower infliximab concentrations (Fig. 2). Three and 7 samples had drug levels above the cut-off point for POC IFX/QB and POC IFX/RQ assays, respectively.

Plots representing infliximab concentrations evaluated with reference ELISA assay and point of care IFX Quantum Blue (panel A), POC IFX Rida Quick (panel B) and comparison ELISA assay (panel C). Dashed lines represent the lower and the upper detection limits of the tests. IFX = infliximab; POC = point of care; QB = Quantum Blue; RQ = Rida Quick.
Bland-Altman plots comparing the reference ELISA assay with point of care IFX/Quantum Blue (panel A), POC IFX/Rida Quick (panel B) and comparison ELISA assay (panel C). ELISA = enzyme-linked immunosorbent assay; POC = point of care; QB = Quantum Blue; RQ = Rida Quick.

Serum infliximab concentrations obtained from the 4 different assays were then compared qualitatively stratifying the results into 3 therapeutic intervals (<3 μg/mL, ≥3 to <7 μg/mL, and ≥7 μg/mL). A good agreement was shown between pairs of assays, with a kappa of 0.67 and 0.80 for POC IFX/QB and POC IFX/RQ, respectively, with reference ELISA, and 0.81 between the reference and comparison ELISA assays (Table 1).

Comparison of infliximab concentrations results obtained by enzyme-linked immunosorbent assay and point-of-care assays stratified by therapeutic interval (<3 μg/mL, ≥3 to <7 μg/mL and ≥7 μg/mL)

Overall, POC results were in agreement with the results of reference ELISA assay for 20/32 (62.5%) samples with POC IFX/QB and for 24/32 (75%) samples with POC IFX/RQ. In particular, for the 11 samples with drug levels <3 μg/mL (as measured with the reference ELISA), the agreement with the reference ELISA was achieved for 7/11 (63.6%) samples with the POC IFX/QB and for 8/11 (72.7%) samples with POC IFX/RQ; for samples with infliximab levels between 3 and 7 μg/mL with the reference ELISA agreement was obtained for 4/11 (36.4%) and 8/11 (72.7%), respectively; finally, when levels were >7 μg/mL for the reference ELISA, 9/10 (90%) and 8/10 (80%) samples were in agreement with it when tested with POC IFX/QB and POC IFX/RQ, respectively. Conversely, when the result of the POC assay was <3 μg/mL, the agreement with reference ELISA was present in all cases (7/7 and 8/8 cases for POC IFX/QB and POC IFX/RQ, respectively); for POC results between 3 and 7 μg/mL agreement was present with the reference ELISA in 4/8 (50%) cases (POC IFX/QB) and 8/13 (62%) cases (POC IFX/RQ); finally, when POC results were >7 μg/mL, agreement with reference ELISA was present in 9/17 (53%) and 8/11 (73%) cases for POC IFX/QB and POC IFX/RQ, respectively. Sensitivity, specificity, and test predictive values were then calculated for the identification of low drug levels (ie, <3 μg/mL), considering this as the most clinically relevant application of the assays (ie, identification of patients who may be failing therapy because of inadequate drug levels). For the POC IFX/QB, sensitivity was 63.6% (95% CI 31.6%–87.6%), specificity 100% (95% CI 81%–100%), positive predictive value 100% (95% CI 56%–100%), and negative predictive value 84% (95% CI 63%–95%). For POC IFX/RQ, sensitivity was 72.7% (95% CI 39.3%–92.7%), specificity 100% (95% CI 81%–100%), positive predictive value 100% (95% CI 59.7%–100%), and negative predictive value 87.5% (95% CI 66.5%–96.7%). Notably, sensitivity, specificity, and predictive values of the second ELISA assay (Promonitor), as compared to the reference ELISA, were identical to those of the POC IFX RQ.


In this study, we show that infliximab trough levels for TDM can be determined with good accuracy with 2 POC assays in children with IBD treated with infliximab. The results of the POC assays showed good agreement with both ELISA assays in the quantitative analysis. Notably, the Bland-Altman plots showed a good dispersion between the reference ELISA assay and the POC devices, in particular, at lower concentrations, which represent also the most interesting testing range from a clinical point of view. Also, when patients were stratified according to a proposed therapeutic window, a good agreement between tests was found. These observations allow to identify POC devices as a reasonable option for infliximab quantification in clinical practice in pediatric patients, especially when ELISA tests are not available, or an immediate result is needed to direct clinical decisions. Several factors, however, need to be taken in consideration. In particular, the agreement was not perfect, and both tests performed better at lower drug concentrations, with a slightly better overall performance of the POC IFX/RQ. Notably, in our cohort, the POC IFX/QB seemed to overestimate the concentration of infliximab compared with the other assays. This observation needs, however, to be confirmed in other studies.

With both assays, from a practical point of view, it should be noted that a result by the POC device of infliximab <3 μg/mL predicted with a high grade of certainty that the drug levels were actually low, whereas a result >3 μg/mL was less predictive, as in some cases, drug levels were overestimated. In this regard, both tests had a better performance in terms of specificity rather than in sensitivity, with very good positive predictive values (100%) and acceptable negative predictive values (84%–87.5%). From a clinical point of view, therefore, although it is certainly important to be able to know with a high degree of certainty that a patient has low drug levels, the possibility of a false negative result (ie, the possibility of an overestimation) must be taken in consideration. It should be noted, nevertheless, that the agreement of the 2 ELISA assays was also not perfect (kappa = 0.81 between the reference and comparison ELISA assays, vs kappa of 0.80 between POC IFX/RQ and reference ELISA, and kappa of 0.67 between POC IFX/QB and the reference ELISA), implying that quantification of infliximab is probably not perfectly accurate with all assays. Considering that the POC assays are less reliable for samples with high drug concentrations, they may be more useful to personalize therapy in the postinduction phase, when the infliximab levels tend to be lower as compared with the induction phase, and detection of very low concentrations of infliximab is more relevant.

So far, few studies have compared POC infliximab assays and ELISA assays, only in adult patients. Afonso et al showed the POC IFX/QB could represent a reliable alternative to ELISA assays in adult IBD patients (15). Similar results were obtained by Nasser et al, who compared ELISA assays and 2 rapid tests (POC IFX/QB and POC IFX/RQ), finding a good accuracy (16). POC assays have several advantages. First of all, they allow to quantify a single serum sample efficiently, whereas ELISA assays, given the standard 8-well or 96-well assay format and the need to use several wells for the calibration, need to run on several samples for cost-effectiveness, and this may represent a cause of delay in obtaining the results of the analysis for individual IBD patients. Furthermore, ELISA assays have a turnaround time of around 4 to 8 hours, whereas POC assays are completed in 15 to 20 minutes. POC testing has also several disadvantages, including the lack of external and internal quality control results, and greater difficulty in error tracking and monitoring.

The operational characteristics of POC assays could improve the possibility of implementing real proactive dose adjustment in clinical practice. Proactive dose adjustment strategy refers to the use of infliximab levels to guide therapy as compared on clinical parameters alone. The recent TAXIT and TAILORIX trials tested the clinical utility of proactive strategy to dose infliximab on the basis of TDM (13,22). Although TDM-based dosing had some advantages, these studies did not identify significant differences in terms of clinical remission in patients treated with standard of care or proactive monitoring strategies. One of the reasons for the lack of improvement in clinical outcomes with the proactive strategy in the TAILORIX study could possibly be the delay in dose adjustment because of the use of ELISA assays that allowed dose adjustment only at subsequent infusions. Notably, a recent study on children and young adults demonstrated the utility of proactive adjustment of infliximab therapy during induction therapy (week 10) (23). Patients receiving TDM-guided infliximab monotherapy had improved results compared with standard of care monotherapy. At this timepoint, the infliximab concentration threshold used was 20 and 25 μg/mL, as during induction therapy, infusions are closer in time compared with maintenance therapy. Therefore, if this proactive approach is confirmed, POC assays should be adapted to quantify higher concentrations of infliximab (ie, >20 μg/mL) whereas in the present study, we did find a better accuracy for low drug levels. Notably, POC IFX/QB has an upper quantification limit of 20 μg/mL whereas the POC IFX/RQ up to 10 μg/mL, and, whenever necessary, serum had to be diluted to quantify more precisely IFX concentrations above this limit.

We have chosen a cut-off level of 3 μg/mL in accordance to several works identifying this as the minimum level associated with remission, therefore, this level could be the most relevant in clinical practice. Most data on infliximab trough levels come from adult studies, and in fact, different cut-off levels have been identified. Several studies identified trough levels above 3 μg/mL postinduction as associated with clinical remission (19–21). Ungar et al (24) showed that levels of infliximab above 5 μg/mL are associated with mucosal healing. More recently, Burgess et al (18) showed that levels <3 μg/mL postinduction were associated with relapse in children. These data confirm the results of our group on pediatric IBD patients, in which infliximab levels at week 14 above 3.11 μg/mL emerged as the strongest predictor of sustained clinical remission (11).

In terms of costs, for POC assays is around 500 € for 25 tests (20 €/test; tests can be performed singularly), whereas the cost of ELISA is around 1400 € for 96 wells. Considering the standard curve (16 wells) and that for each patient, the sample is analyzed at 2 dilutions, the maximum number of patients that can be analyzed with 1 plate is 40, with a cost of 35 € for each test. However, it should be considered that this cost applies only if a full 96-well plate is analyzed, whereas if less samples are tested, the cost is obviously higher. This further limits the cost-effectiveness of ELISA assay, particularly in contexts were number of samples to be analyzed is low.

This study has several limitations. The study sample was small, and we did not correlate results obtained with POC assays to clinical outcomes. Another limitation was that we did not test for ADA. Several studies demonstrated that the simultaneous measurement of infliximab trough levels and ADA titers significantly increase the diagnostic accuracy in the therapeutic decision in IBD patients (20,25,26). The dosage of ADA is usually carried out in case of low infliximab trough levels. However, their appearance, even when infliximab levels are in range, may be an early sign for clinical deterioration, especially if they are persistently present (20). Therefore, it may be more cost-effective to evaluate both ADA and drug levels simultaneously. POC testing for ADAs has recently become available as well and seem to be promising in terms of accuracy (16).


In conclusion, our data suggest that POC assays can represent a feasible alternative for TDM of infliximab in children with IBD, especially when ELISA assays are not yet available among the routine laboratory tests or when an immediate result is desirable in order to change a clinical decision, eg, in a patient who is losing response to treatment. The use of POC is also cost effective for the analysis in particular in the context of a limited number of samples. Limitations of the tests should nevertheless be considered, especially considering that the assays had very good positive predictive values for the detection of low drug levels but suboptimal sensitivity; ELISA assay should be, therefore, performed in doubtful cases, especially for results >3 μg/mL, as POC assays showed worse performances in this concentration range. Our results should, however, be validated in larger populations, and correlated to clinical outcomes.


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antitumor necrosis factor; enzyme-linked immunosorbent assay; pharmacokinetics; therapeutic drug monitoring

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