Analytical performance of a single epitope B-type natriuretic peptide sandwich immunoassay on the Minicare platform for point-of-care diagnostics
Alexander van Reenen
a,b,*, Mario Berger
b, Emmanuel Moreau
b, Edwin Bekx
b, Tom Bruinink
b, Danielle Kemper
b, Lian van Lippen
b, Jos Weusten
b,
Anita Mrakovic
c, Etienne Michielsen
d, Joost Vissers
e, Femke de Theije
a,b, Jeroen Nieuwenhuis
a,b, Veronique Semjonow
b, Johannes Mair
caMinicare BV, Eindhoven, the Netherlands
bPhilips BG Emerging Businesses, Eindhoven, the Netherlands
cDepartment of Internal Medicine III— Cardiology and Angiology, Medical University of Innsbruck, Innsbruck, Austria
dDiagnostiek voor U, Eindhoven, the Netherlands
eFuture Diagnostics Solutions, Wijchen, the Netherlands
A R T I C L E I N F O
Keywords:
Heart failure Diagnosis
B-type natriuretic peptide Point-of-care
Capillary blood Analytical performance
A B S T R A C T
Point-of-care B-type natriuretic peptide (BNP) testing with adequate analytical performance has the potential to improve patientflow and provide primary care givers with easy-to-use advanced diagnostic tools in the management of heart failure. We present the analytical evaluation of the Minicare BNP immunoassay under development on the Minicare I-20 platform for point-of-care testing. Analytical performance was evaluated using EDTA venous whole blood, EDTA plasma and capillary whole blood. Method comparison with a lab-testing system was performed using samples from 187 patients. Normal values were determined based on 160 healthy adults, aging from 19 to 70 years. Limit of blank (LoB), limit of detection (LoD) were determined to be 3.3 ng/L, 5.8 ng/L. Limit of quantitation (LoQ) in whole blood at 20% and 10% coefficient of variation (CV) was found< 9 ng/L and <30 ng/L respectively without significant differences between EDTA whole blood and EDTA plasma. Total CV was found to be from 6.7% to 9.7% for BNP concen- trations between 92.6 and 3984 ng/L. The sample type comparison study demonstrated correla- tion coefficients between 0.97 and 0.99 with slopes between 1.03 and 1.09 between the different samples. Method comparison between Minicare BNP and Siemens ADVIA Centaur BNP demon- strated a correlation coefficient of 0.92 with a slope of 1.06. The 97.5% URL of a healthy popu- lation was calculated to be 72.6 ng/L. The Minicare BNP assay is a robust, easy-to-use and sensitive test for rapid determination of BNP concentrations that can be used in a near-patient setting.
Abbreviations: BNP, B-type Natriuretic Peptide; CI, confidence interval; CLSI, clinical laboratory standards institute; CV, coefficient of variation;
EDTA, ethylene-diamine-tetraacetic acid; fTIR, frustrated total internal reflection; HAMA, human anti-mouse antibody; HF, heart failure; K2-EDTA, dipotassium ethylene-diamine-tetraacetic acid; Li-heparin, lithium heparin; LoB, limit of blank; LoD, limit of detection; LoQ, limit of quantitation; NP, Natriuretic Peptide; NYHA, New York Heart Association; POC, point-of-care; RF, rheumatoid factor; RFID, radiofrequency identification; RT, room temperature; SD, standard deviation; URL, upper reference limit.
* Corresponding author. Philips BG Emerging Businesses, High Tech Campus 29, 5656AE Eindhoven, the Netherlands.
E-mail addresses:[email protected](A. Reenen),[email protected](F. Theije),jeroen.nieuwenhuis@minicare.
com(J. Nieuwenhuis),[email protected](V. Semjonow),[email protected](J. Mair).
Contents lists available atScienceDirect
Practical Laboratory Medicine
journal homepage:www.elsevier.com/locate/plabm
https://doi.org/10.1016/j.plabm.2019.e00119
Received 19 July 2018; Received in revised form 23 January 2019; Accepted 14 March 2019
2352-5517/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.
org/licenses/by-nc-nd/4.0/).
Practical Laboratory Medicine 15 (2019) e00119
1. Introduction
Twenty-six million adults worldwide are estimated to have heart failure and this number is expected to rise [1]. All heart failure patients, even those who are considered asymptomatic (New York Heart Association, NYHA, class I) or mildly symptomatic (NYHA class II), have a poor prognosis with a high mortality during long-term follow-up (34%–42% for class I-II and class III-IV respectively) [2].
The measurement of circulating natriuretic peptides (NP) is considered to be a useful marker of myocardial function, and inter- national guidelines recommend its use for the diagnosis of patients with heart failure (HF) [3–5], in particular ruling out the presence of HF. Point-of-Care (POC) systems for NP with a short turnaround time enable rapid clinical decision making and can thereby improve patientsflow. Furthermore, such systems have the potential to provide primary care givers such as the general practitioner access to advanced diagnostic tools, but this requires POC systems to show lab-quality test performance, be robust and easy-to use [6].
Current commercial POC B-type Natriuretic Peptide (BNP) testing systems using only samples from venous puncture are difficult to handle for non-lab-trained personnel. In addition, stability of BNP especially in plasma samples has been a concern for the reliability of NP tests [7].
Here, we demonstrate the analytical evaluation of the POC Minicare BNP assay under development, which runs on the Minicare I-20 platform with a turnaround time of less than 10 min (Fig. 1a) [8]. Aside from quantifying BNP from venous whole blood/plasma samples, Minicare BNP also accepts capillary whole blood samples fromfinger prick. This should lower the barrier for BNP testing at primary care settings, but also avoid BNP stability concerns since capillary samples are directly applied to the test system.
2. Materials and methods 2.1. Principle of the Minicare BNP test
Minicare BNP is an in vitro diagnostic POC test that is intended to be used as an aid in the diagnosis of heart failure. It uses a single test analyzer device (Minicare I-20 analyzer) and a plastic disposable cartridge containing the specific BNP assay reagents. Test results are generated within 10 min after application of the sample to the cartridge. The Minicare I-20 assays are based on proprietary Mag- notech biosensor technology [9,10]. Briefly, Minicare I-20 assays are based on the precise controlled motion of magnetic nanoparticles functionalized with analyte-specific antibodies within a small reaction volume inside each cartridge. The same magnetic particles serve as labels that are detected using frustrated total internal reflection (fTIR) imaging. The system has been designed without moving parts to create a robust and stable handheld platform.
2.2. Selection of antibodies
The Minicare BNP assay uses proprietary “Single Epitope Sandwich” (“SES”) BNP antibodies (Hytest, Turku, Finland) which recognize a single epitope within the ring structure of BNP (132) [11]. Hence, SES-BNP assays are less affected by proteolytic degradation.
2.3. Assay format
After application of the blood sample onto the cartridge, blood cells are trapped within a semipermeablefilter membrane while plasma containing BNP enters the microfluidic system and is transported to the reaction chambers by capillary forces. Within the re- action chambers, thefirst step of the SES assay is executed as anti-BNP capture antibodies printed onto the optical imaging surface bind to the analyte molecules; seeFig. 1b. At the same time, dried magnetic particles within the reaction chamber disperse into the sample volume. The magnetic particles are attracted to the imaging surface and bind to the complex of BNP molecules and anti-BNP-antibody via the application of magneticfields to create a magnetic field gradient towards the imaging surface; seeFig. 1c. This latter step is effectively the second step of the SES assay, i.e. formation of the complete complex. After a limited amount of time to form the complex,
Fig. 1. (a) Minicare I-20 platform consisting of the Minicare I-20 analyzer and a disposable plastic cartridge. (b–d) Schematic representation of the Magnotech single-epitope sandwich assay. First (b) the analyte, i.e. BNP, binds to the functionalized optical detection surface. Subsequently (c) magnetic particles are attracted to the surface and bind the complexes. Lastly (d) unbound particles are removed from the surface to enable detection of BNP-bound particle complexes via fTIR.
unbound or weakly (non-specifically) bound particles are washed from the detection surface using a magnetic field gradient in the opposite direction. This leaves the particles which have specifically formed the complex with BNP at the detection surface to be optically detected via fTIR; seeFig. 1d.
2.4. Standardization
Due to the lack of a suitable commutable primary calibrator for BNP immunoassay standardization, new BNP standards were developed consisting of defibrinated, delipidated and charcoal-treated human plasma (Seracon II, SeraCare) and recombinant proBNP (catalogue number 8GOB2; Hytest). These standards were dose-assigned such that all sample types achieve a correlation with a target slope of 1.0 with a reference system, i.e. the Siemens ADVIA Centaur BNP. In particular for capillary whole blood, a compensation factor of 1.48 was established with respect to EDTA whole blood as determined from a paired comparison study on 117 samples reported here.
Cartridge-lot specific calibration parameters were obtained from dose-response curves generated using these calibrators and pro- grammed in the radio-frequency identification (RFID) of each cartridge.
2.5. Study samples
Samples for testing precision, detection capability, linearity, high-dose hook effect, cross-reactivity, interferences, and normal values studies were (a) left-over K2-EDTA venous blood samples from patients of the Canisius-Wilhelmina hospital (CWZ) in Nijmegen, The Netherlands, and (b) K2-EDTA venous blood samples from healthy volunteers (informed consent obtained) from Sanquin Blood bank in Nijmegen, The Netherlands. Negative and positive BNP EDTA plasma samples were selected to prepare sample pools with different levels of BNP as specified for each study.
For the sample stability and the method comparison studies, two sample types (K2-EDTA venous whole blood and K2-EDTA venous plasma) were collected for each patient at Diagnostiek voor U facilities, respectively of Sint Jans Gasthuis in Weert, The Netherlands, and central laboratory in Eindhoven, The Netherlands. Patients were selected to represent the range of BNP concentrations likely to be encountered in clinical practice, covering the measurement range of the Minicare BNP assay. Left-over samples from eligible patients were used for the study and the study was waived by local ethics committees. For the sample stability study both sample types were analyzed on Minicare BNP within 1 h after blood collection (reference time), stored at room temperature (RT, 18–26C) and tested again after 16 h. Another set of EDTA plasma samples was frozen at< -55C and tested after 2 months. EDTA plasma samples submitted to 4 freeze-thaw cycles were also tested. For the method comparison study EDTA whole blood and EDTA plasma samples were analyzed on Minicare BNP within 6 h after blood collection. EDTA plasma samples were analyzed on Siemens ADVIA Centaur BNP, Alere Triage BNP and Abbott I-STAT BNP according to manufacturer's instructions.
While the method comparison enabled a comparison between venous whole blood and venous plasma samples, a broader sample comparison study was set up to compare these sample types to capillary whole blood, as well as the anticoagulants used (K2-EDTA, Li- heparin, or none). For the broader sample type comparison study, the following sample types were collected for each patient at the Medical University Hospital in Innsbruck, Austria [1]: venous whole blood [2], venous plasma, and [3] capillary whole blood. For venous whole blood and plasma, both K2-EDTA and Li-heparin were used as anticoagulants. For capillary whole blood, samples without anticoagulants and containing K3-EDTA were tested. Samples covering the whole measurement range were measured within 2 h after sample draw. The Minicare BNP tests were executed by study nurses of the clinical trials unit of the Department of Internal Medicine III (Cardiology and Angiology), Medical University of Innsbruck. This study has been approved by all local ethical committees and written informed consent was obtained from patients prior to their participation in the study.
2.6. Precision
Assay precision was established across the dynamic range using pools of patient EDTA plasma samples in accordance with CLSI EP05- A3 [12]. Two replicates of each sample were tested twice per day in separate runs (at least 2 h apart), for 20 non-consecutive days, on two lots of Minicare BNP cartridges and using four Minicare I-20 instruments.
2.7. Detection capability
Limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ) for the Minicare BNP were established in accordance with CLSI EP17-A2 [13]. Four stripped human plasma pools (i.e. 4 different lots of SeraCon I, from SeraCare) were used to establish the LoB. Four low-concentration plasma pool samples derived from multiple donors targeting a concentration between 5 and 20 ng/L were used to establish the LoD. LoB and LoD samples were tested onfive days, with three replicates per day, on two Minicare BNP cartridge lots, usingfive analyzers. The LoB was calculated non-parametrically, i.e. the interpolated numerical result at rank 57 and rank 58 was determined to be the LoB value.
The LoQ at 10% CV and 20% CV in EDTA plasma and EDTA whole blood was established using 15 EDTA whole blood and their corresponding EDTA plasma samples targeting a range from 0 to 400 ng/L. LoQ EDTA plasma samples were tested on 5 days, in four- fold, whereas LoQ EDTA whole blood samples were each tested on a single day (different samples over multiple days), in twenty-fold.
Both EDTA plasma and EDTA whole blood samples were tested on two cartridge lots, usingfive analyzers. The LoQ was derived from a non-weighted second-order polynomialfit of the resulting precision profile.
2.8. Linearity on dilution
According to CLSI EP06-A recommendation [14], 11 EDTA plasma pools with different levels of BNP were prepared from a high and a low BNP EDTA plasma pool with steps of 10% dilution. The pool with the highest BNP level and the pool with the lowest BNP level were measured in 20-fold. All the other pools were measured in 5-fold. All tests were performed on one cartridge lot. A polynomial analysis method was applied [14].
2.9. High-dose hook effect
Eleven dilution blends prepared from pure recombinant BNP protein and a negative plasma pool were tested. All high-dose hook samples were measured in duplicate on one cartridge lot.
2.10. Cross-reactivity and interferences
Various blood components (hemoglobin, human albumin, bilirubin, triglycerides, human anti-mouse antibodies (HAMA), rheu- matoid factor (RF), and total protein) and drugs (allopurinol, acetaminophen, ampicillin, ascorbic acid, acetylsalicylic acid, atenolol, caffeine, captopril, diclofenac, digoxin, dopamine, enalapril maleate, erythromycin, furosemide, heparin, ibuprofen, isosorbide dini- trate, methyldopa, niphedipine, phenytoin, propranolol, theophylline, verapamil, warfarin) were tested for interference andα-atrial natriuretic polypeptide 1–28, arg vasopressin, C-type natriuretic peptide and urodilatin (CCD/ANP) for cross-reactivity according to CLSI EP07-A2 recommendations [15] in an EDTA plasma pool with a BNP concentration between 80 and 120 ng/L. As a reference, only the dilution matrix of the substances was tested in the EDTA plasma pool. All samples were tested in 5-fold on one cartridge lot, divided over 18 analyzers.
2.11. Sample stability
A total of 36 EDTA whole blood and EDTA plasma samples covering the dynamic range were used to establish the room temperature (18–26C) stability of samples on the Minicare BNP assay. A total of 44 and 45 EDTA plasma samples covering the dynamic range were used to establish respectively the long-term stability (at< 55C) and freeze-thaw stability (4 cycles). Every sample was measured at least twice using four analyzers. The reference concentration (t¼ 0) was measured within 1 h after blood draw. Stability was assessed by comparing results to the reference (t¼ 0) using Passing-Bablok regression.
2.12. Method comparison
The Minicare BNP was compared with the Siemens ADVIA Centaur BNP, Alere Triage BNP and Abbott I-STAT BNP by testing and comparing EDTA whole blood samples (on Minicare, Triage and I-STAT) and corresponding EDTA plasma samples (on Minicare and Centaur) from respectively 187 to 216 individuals with a BNP concentration range as measured using Minicare from 10 to 2890 ng/L in accordance with CLSI EP09-A3 [16]. EDTA whole blood samples were applied to the Minicare BNP test both from blood tubes using SmearSafe (Typenex Medical) blood dispensers and using a pipette. Analysis was done using Passing-Bablok regression.
2.13. Sample type comparison
Sample type comparison was done in accordance with CLSI EP09-A3 recommendations [5]. The sample type comparison performed at the Medical University Hospital in Innsbruck, Austria, was done in two parts, I and II. Part I aimed to compare (a) different sample types (venous whole blood/venous plasma/capillary whole blood), (b) different anticoagulants (K2-EDTA/Li-Heparin/none), and (c) different blood tube sampling devices (blood-tube dispenser/pipette). Part I data was analyzed with an assay standardization (see section2.4) that did not include a compensation for capillary whole blood samples. Part II aimed to confirm the compensation for capillary whole blood samples in the assay standardization and the use of different sampling devices (capillary tubes/blood-tube dispenser/pipette). Li-Heparin samples were excluded in part II. In each part at least 100 patient samples were collected for each comparison. Additional comparison data, i.e. 232 paired patient samples, between EDTA venous whole blood and EDTA venous plasma was also generated at the Eindhoven site during the method comparison studies. Results were analyzed using Passing-Bablok regression.
EDTA whole blood samples were applied to the Minicare BNP test from blood tubes (BD Vacutainer tubes and S-Monovette tubes from Sarstedt) using blood dispensers (SmearSafe, Typenex Medical/Haemo-Diff, Sarstedt) and/or via a pipette. Capillary samples were transferred using capillary tubes (Microsafe, Safe-Tec, uncoated; and POCT Minivette, Sarstedt, uncoated and EDTA-coated).
Excluded from the data sets were samples with observed concentrations below the LoQ with 20%CV (i.e. 10 ng/L).
2.14. Normal values
Samples from blood donors, presenting voluntarily at the Eindhoven MMC Sanquin post in the Netherlands, were used to determine normal values. Healthy adults were selected based on Sanquin standard questionnaire and were invited to take part in this study. All subjects enrolled signed an informed consent form.
In total 160 blood donors (94 males and 66 females; with 84 aged more than 55 years and 76 aged 55 years or less) qualified as final
study population for the study. Normal values were determined by testing EDTA whole blood and EDTA plasma samples. Reference intervals were estimated using a non-parametric method [17].
3. Results 3.1. Precision
Total imprecision and within-run imprecision expressed as CV were calculated at various BNP levels for the Minicare BNP assay; see Table 1. Total imprecision of 7% and within-run imprecision of 6% was obtained for BNP concentrations around 100 and 300 ng/L. For high BNP levels (i.e. around 3000 ng/L) a total imprecision of less than 10% and within-run imprecision of 7% was obtained.
3.2. Detection capability
The LoB for lot 1 and lot 2 was found to be respectively 3.3 ng/L and 2.5 ng/L.
The highest value of LoB was used to calculate the LoD. The LoD was determined to be 5.6 ng/L and 5.8 ng/L for respectively lot 1 and lot 2. For EDTA whole blood the CV profile is shown inFig. 2. For EDTA whole blood, the LoQ at 10%CV was below 30 ng/L and the LoQ at 20%CV was below 9 ng/L for both cartridge lots; seeTable 2.
3.3. Linearity
Weighted linear regression between observed and expected concentrations, resulted in sustained linearity over the 9.3–5766 ng/L tested range, with recovery within 15% of expected values.
3.4. High-dose hook effect
No High-dose hook effect was found for samples up to a BNP concentration of 20,000 ng/L.
3.5. Cross-reactivity and interference
Results were considered acceptable if samples enriched with the possible interferents were within 10% of the results for the control samples. None of the tested blood components and/or drugs showed interference beyond 90% and 110%. The observed percentage of cross-reactivity of Minicare BNP was<0.2% for all tested cross-reactants.
3.6. Sample stability
Samples were considered stable if a bias smaller than 10% was observed. Results are shown inTable 3. It was found that Minicare BNP can measure BNP concentrations in EDTA whole blood and EDTA plasma samples stored at room temperature for 16 h (95% CI of Passing-Bablok slope for all cases within 0.92 and 1.02). Frozen EDTA plasma samples showed stable results up to 2 months (95% CI of Passing-Bablok slope within 0.96 and 1.02). EDTA plasma samples showed no instability after 4 freeze-thaw cycles (95% CI of Passing- Bablok slope within 0.97–1.02).
3.7. Method comparison
Fig. 3shows the method comparison results for the different methods tested. InTable 4the estimated bias based on Blant-Altman analysis is shown for the different method comparisons. Comparing Minicare BNP to the Siemens Centaur BNP, a slope was found of 1.06 (95% CI of 0.98–1.12) and a Pearson correlation coefficient was found of 0.92. Compared to the Alere Triage BNP, the slope was 1.06 (95% CI of 1.01–1.10) and the Pearson correlation coefficient was 0.97. Compared to the Abbott I-STAT BNP, the slope was 0.72 (95% CI of 0.68–0.74) and the Pearson correlation coefficient was 0.95. No significant differences were observed between Minicare BNP whole blood measurements using a pipette or SmearSafe dispenser: a Passing-Bablok slope of 1.00 (95% CI of 0.98–1.02) with a Pearson correlation coefficient of 1.00 based on 245 measurement pairs.
Table 1
Total imprecision and within-run imprecision for measurements (with n repeats) of BNP on pooled EDTA plasma samples. Total imprecision contains within-run, run-to-run and day-to-day variability.
EDTA plasma sample n Mean conc Within-run imprecision Total imprecision
ng/L CV CV
Pool I 80 92.6 6.1% 6.7%
Pool 2 80 327 5.8% 7.0%
Pool 3 80 3984 7.3% 9.7%
3.8. Sample type comparison
Sample type comparison studies were done to establish and confirm the assay standardization for all sample types and transfer devices. Studies were done in 2 parts, afirst part to establish possible systematic biases and a second study to verify the results. Data in Table 5shows the sample types where systematic biases (slopes of the Passing-Bablok regression outside the range of 0.9–1.1) were observed.Table 6shows sample type comparisons without systematic biases when including a compensation factor of 1.48 for capillary samples in the assay standardization.
3.9. Normal values
The primary objective of this study was to establish the reference ranges for Minicare BNP in healthy volunteers EDTA plasma and EDTA whole blood samples.
Results inTable 7show that the distribution of the BNP values measured in EDTA whole blood and EDTA plasma are similar for the two sample types. One sample yielded invalid results for both EDTA whole blood and EDTA plasma, and another one for EDTA plasma only.
The 97.5th percentile for EDTA whole blood and EDTA plasma were 72.6 ng/L and 71.6 ng/L respectively. The reference ranges for the two sample types were comparable (overlapping confidence intervals).
Fig. 2. CV profile for EDTA whole blood. On the horizontal axis the average Minicare BNP concentration in ng/L is depicted. The line is a guide to the eye.
Table 2
Limit of Quantitation (LoQ) for whole blood and plasma.
Lot Whole blood LoQ (ng/L) Plasma LoQ (ng/L)
10% CV 20%CV 10% CV 20% CV
Lot 1 22 8.4 39 4.3
Lot 2 30 8.8 22 7.6
Table 3
Sample stability data from comparison of measured concentrations from stored samples to reference measurements done within 1 h after blood drawn.
Passing-Bablok slopes including 95% confidence intervals were computed.
Sample type Storage time Storage temperature N freeze-thaw cycles Passing-Bablok slope 95% CI
EDTA Whole blood 2 h Room temperature NA 0.98 [0.95–1.00]
EDTA Whole blood >16 h Room temperature NA 0.96 [0.92–0.99]
EDTA Plasma 4 h Room temperature NA 0.98 [0.97–1.00]
EDTA Plasma >16 h Room temperature NA 1.00 [0.97–1.02]
EDTA Plasma NA < 55C 4 1.00 [0.97–1.02]
EDTA Plasma 1 month < 55C 1 0.99 [0.97–1.02]
EDTA Plasma 2 months < 55C 1 0.97 [0.96–0.99]
Fig. 3. Method comparison between Minicare BNP whole blood (WB) and (A) Siemens ADVIA Centaur BNP, (B) Alere Triage BNP, and (C) Abbott I- STAT BNP. Data is shown on a logarithmic scale. Passing-Bablok regression results are shown in the inset. The solid black line corresponds to the regression line.
Table 4
Blant-Altman analysis of mean differences of method comparison results.
Method A Method B mean difference 95% C.I.
Minicare BNP Centaur BNP 6.5% 1.0% to 12.0%
Minicare BNP Biosite BNP 9.2% 5.0% to 13.3%
Minicare BNP I-stat BNP 30% 34% to 25%
Table 5
Summary of the sample type comparison results for assay standardization without compensation for capillary samples. C.I.: 95% confidence interval.
Different comparison types are labeled: A: anticoagulants, B: sample types, and C: transfer devices.
Result“X” Result“Y” Type comparison N Passing-Bablok regression Pearson correlation (r)
Slope Intercept
estimate C.I. estimate C.I.
EDTAwhole blood pipette
Capillary whole blood Sarstedt (uncoated)
ABC 117 1.48 1.38
1.55
0.4 6.9
6.1 0.98
EDTA whole blood pipette
EDTA capillary whole blood Sarstedt
BC 117 1.32 1.25
1.43
2.6 12
3.9 0.93
EDTAwhole blood pipette
LiHepwhole blood pipette
A 117 1.44 1.37
1.51
1.9 7.2
2.0 0.97
EDTAplasma pipette
LiHepplasma pipette
A 119 1.40 1.33
1.47
1.1 5.7
5.3 0.98
EDTAwhole blood pipette
EDTAwhole blood Haemodiff
C 116 0.929 0.895
0.982
1.3 2.1
3.7 0.98
Table 6
Summary of the sample type comparison results for assay standardization with compensation of capillary samples with a factor 1.48 as found in thefirst part of the Innsbruck study (seeTable 5). C.I.: 95% confidence interval. Different comparison types are labeled: A: anticoagulants, B: sample types, and C: transfer devices.
Result“X” Result“Y” Type
comparison
N Passing-Bablok regression Pearson
correlation (r)
Slope Intercept
estimate C.I. estimate C.I.
EDTA whole blood pipette Capillary whole blood Safe-Tec (uncoated)
ABC 150 1.05a 0.991
1.09
2.1 2.6
6.2 0.98
EDTA whole blood pipette Capillary whole blood Sarstedt (uncoated)
ABC 148 1.03a 0.990
1.07
0.3 3.7
4.6 0.98
Capillary whole blood Sarstedt (uncoated)
Capillary whole blood Safe-Tec (uncoated)
C 150 1.01 0.992
1.04
1.7 1.0
4.2 0.99
EDTA whole blood pipette EDTA whole blood Haemodiff C 116 0.93 0.895
0.982
1.3 2.1
3.7 0.98
EDTA whole blood Pipetteb EDTA whole blood SmearSafe C 245 1.00 0.98
1.02
0.2 0.9
1.5 1.00
EDTA whole blood pipette EDTA plasma pipette B 151 1.09 1.06
1.12
2.0 5.6
0.9 0.98
EDTA whole blood pipetteb EDTA plasma pipetteb B 232 1.04 1.02
1.06
0.2 1.8
1.3 0.99
aCorrected for the systematic bias of 1.48 as found in a preceding sample type comparison study.
b Measured at the Eindhoven site.
Table 7
Descriptive statistics of the Minicare BNP normal values.
K2-EDTA whole blood K2-EDTA plasma
N 159 158
Median Age, years (Min-Max) 56 (19–70) 56 (19–70)
Male/Female 94/65 93/65
Median BNP level, ng/L (Min-Max) 15.8 (0–101) 16.7 (0.6–109)
2.5th percentile, ng/L (95% CI) 4.4 (0.0–5.5) 2.8 (0.6–5.2)
95th percentile, ng/L (95% CI) 54.5 (34.8–90.7) 59.8 (35.7–81.5)
97.5th percentile, ng/L (95% CI) 72.6 (43.1–101) 71.6 (52.1–109)
4. Discussion
The Minicare BNP detection capability (LoQ 20%CV< 9 ng/L and LoQ 10% CV < 30 ng/L for whole blood) was tested both on whole blood samples and plasma samples, and showed no significant difference. In relation to the normal values study, at least 80% of the healthy population quantified above the LoQ 20%CV ¼ 30 ng/L. At least 99% of healthy donors quantified below the 100 ng/L cut-off classically used for the rule out of acute HF [3].
Sample stability data show that whole blood can be reliably used up to at least 16 h at room temperature conditions, in agreement with earlier reports [18].
The sample type comparison study between capillary whole blood, K2-EDTA venous whole blood and K2-EDTA venous plasma samples demonstrated good correlation. The data confirm that the standardization approach leads to a comparable test performance for the three sample types. This enables interchangeable use of the sample types, under the condition that the user pre-selects the sample type (venous/capillary) to be used before testing due to a correction factor of 1.48 between capillary and venous blood samples (based on Part I study data inTable 5). The origin of this difference is unknown.
In the part I study, the slope between Li-Heparin whole blood and EDTA whole blood was found to be 1.44 (95% CI of 1.37–1.51, N¼ 117). A bias between Li-Heparin and EDTA has been observed before. Santos et al. [19] found a mean bias ofþ65% for Li-Heparin BNP samples over EDTA samples on the Beckman Coulter Access 2 system. Others however [18] found that heparinized and non-anticoagulated samples quantify with a negative bias of39% compared to EDTA samples on the Centaur system. The differences are generally attributed to the sample handling, and most importantly in relation to the sample device used [20]. In our studies, wefind that for specific sample types, the use of different sampling devices does not lead to significant quantification differences when the same sample type is used. For EDTA venous whole blood, this concerned the use of different tubes (vacutainer and S-Monovette) in com- bination with different dispensers (SmearSafe and Haemodiff) and a pipette. For non-anticoagulated capillary whole blood, this con- cerned the use of two different capillary transfer devices (Safe-Tec and POCT Minivette).
Method comparison between Minicare BNP and the reference BNP assay (Siemens ADVIA Centaur BNP) demonstrated comparable BNP results. The Minicare assay uses a single epitope sandwich (SES) format, in contrast to the conventional assays used by the other systems in the method comparison, which bind to two epitopes of the BNP structure. With the SES assay targeting a single epitope within the ring structure of BNP(1–32), the SES assay is less affected by proteolytic degradation [11] and therefore differences should be observable compared to conventional assays. Systematic measurement differences as observed before [21], were not observed comparing Minicare to the Centaur BNP assay. However, any systematic difference would be masked in this study since the Minicare BNP was standardized to quantify with a slope of approximately one with respect to the Centaur BNP test. Method comparison results show that the I-STAT reports significantly higher BNP levels compared to the other three BNP systems. This possibly leads to higher incidence of false positive BNP outcomes when using the I-STAT device with the same cut-off (i.e. 100 ng/L). These differences are likely due to the absence of BNP standards or standard references for assay standardization. This also supports not using a single cut-off value for diagnosis without considering the method used.
Considering BNP as a marker for heart failure, the FDA approved heart failure drug LCZ696 (Entresto™, Novartis) has led to speculation about the reliability of BNP over NT-proBNP. LCZ696 combines a neprilysin inhibitor and an angiotensin II receptor, of which the former is known to inhibit BNP degradation and thereby expected to raise circulating BNP levels. It has however been argued that before concluding on the effects on circulating BNP levels, more understanding is needed on the effect of the drug on the complex biochemistry of proBNP-derived peptides [22]. For example, a recent study showed that the precursor proBNP is not degraded by neprilysin, while it is the major circulating BNP immunoreactive form [23]. The study showed that single epitope BNP assays are much less sensitive to BNP degradation by neprilysin than sandwich assays targeting the region 14–21 of BNP. Based on these data one may speculate that the Minicare BNP may be less susceptible to the use of LCZ696 than other BNP assays. This should be confirmed in further studies.
Minicare BNP is an easy-to-use POC test that can accurately measure BNP near the patient with a turnaround time of less than 10 min, using a single droplet of blood (30μL). With a CV of 7% near the 100 ng/L cut-off recommended for rule out of acute HF [3], the test is competitive with state-of-the-art POC BNP tests. Next to EDTA whole blood and EDTA plasma, the test supports capillary samples which allows for simple and easy blood collection viafinger prick that can be done by minimally trained care-givers, in contrast to whole blood/plasma sample collection procedures. Support of capillary samples also provides robustness against BNP instability since capillary samples are applied directly afterfinger prick, and hence avoid any instability effects. The absence of any required sample preparation steps reduces turnaround time and the possibility of user errors. Robustness is also improved via the selected single-epitope sandwich format which makes the assay less susceptible to degradation of BNP molecules. This makes the test suitable for use by healthcare professionals and/or healthcare facilities with limited/no access to advanced diagnostic tools such as BNP lab tests or echocardiography.
In conclusion, the Minicare BNP assay under development is a fast, robust and sensitive test that can be used in a near-patient setting on capillary or EDTA venous whole blood sample. This offers for the Minicare BNP test the potential for good clinical performance in the diagnosis of HF in various patient settings, which is to be evaluated in a large clinical study.
Acknowledgements
We thank Canisius Wilhelmina Ziekenhuis (CWZ), Nijmegen, The Netherlands and Sanquin Blood Bank, Nijmegen, The Netherlands for their support in the analytical studies. We thank the clinical trials unit (M. Spechtenhauser, G. Laschober, C. Mayerl) of the Department of Internal Medicine III (Medical University of Innsbruck, Innsbruck, Austria) for assistance in performing this study at this study site.
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Baseline urinary KIM-1 concentration in detecting acute kidney injury should be interpreted with patient pre-existing nephropathy
Yun Huang
a,*, Yuan Tian
d, Sergei Likhodii
e, Edward Randell
b,caKingston General Hospital and Department of Pathology and Molecular Medicine, Queen's University, 76 Stuart Street, Kingston, ON, Canada
bDiscipline of Laboratory Medicine, Eastern Health Authority, 300 Prince Philip Dr, St. John's, NL, Canada
cMemorial University of Newfoundland, 300 Prince Philip Dr, St. John's, NL, Canada
dDepartment of Endocrinology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, PR China
eB.C. Provincial Toxicology Centre, Provincial Health Services Authority, Vancouver, BC, Canada
A R T I C L E I N F O
Keywords:
Urinary KIM-1 Kidney injury Nephropathy Reference interval
A B S T R A C T
Objectives: To determine whether pre-existing nephropathy impacts urinary KIM-1 levels, urinary KIM-1 were measured in patients with normal kidneyfiltration function but either with or without proteinuria. The reference intervals of urinary KIM-1 in adults with normal kidneyfiltration function but without urine proteinuria were established.
Design and methods: 188 urine samples were obtained from adults with normal kidneyfiltration. 83 of the 188 showed negative urine protein, erythrocytes and leucocytes were used as normal controls. The remaining 105 samples showed at least one abnormal result suggesting possible pre- existing nephropathy. Urinary KIM-1 concentrations were measured using an enzyme-linked immunosorbent assay. Urinary KIM-1 was normalized with urine creatinine concentration. The reference interval for urinary KIM-1 was determined by non-parametric methodology on 147 individuals.
Results: The results showed significantly increased urinary KIM-1 concentration in protein positive (proteinþ, erythrocyte þ/, leucocyteþ/-) samples compared to controls (protein-, erythrocyte -, leucocyte -). Urinary KIM-1 concentrations were significantly higher when proteinuria was at trace concentration (0.25 g/L) and correlated with the severity of proteinuria. The creatinine normal- ized urinary KIM-1 was significantly higher when urine protein was 1 þ to 3þ (0.75–5 g/L). The reference interval for urinary KIM-1 was 0.00 (90%CI: 0-0) to 4.19 (90%CI: 3.11–5.62)μg/L, and for creatinine normalized urinary KIM-1 0.00 (90%CI: 0-0) to 0.58 (90%CI: 0.44–0.74)μg/mmol.
Conclusions: Baseline urinary KIM-1 concentrations were increased when there was detectable urine protein and correlated with its severity. The urinary KIM-1 concentrations should be interpreted with consideration of urine protein levels in individual patients.
1. Introduction
Kidney injury molecule-1 (KIM-1) wasfirst identified in a rat by Ichimura et al. in 1998 as a type 1 transmembrane protein with an immunoglobulin and mucin domain, upregulated in the renal proximal tubule of the kidney after acute kidney injury (AKI) [1]. It was subsequently shown that extensive KIM-1 expression occurs in proximal tubule cells in patients with confirmed acute tubular necrosis
* Corresponding author. Kingston General Hospital, 76 Stuart Street, Kingston, ON, K7L 2V7, Canada.
E-mail address:[email protected](Y. Huang).
Contents lists available atScienceDirect
Practical Laboratory Medicine
journal homepage:www.elsevier.com/locate/plabm
https://doi.org/10.1016/j.plabm.2019.e00118
Received 20 November 2018; Received in revised form 15 February 2019; Accepted 4 March 2019
2352-5517/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.
org/licenses/by-nc-nd/4.0/).
Practical Laboratory Medicine 15 (2019) e00118
(ATN) [2]. Normalized urinary KIM-1 concentrations were significantly higher in patients with ischemic ATN compared to patients with other forms of AKI [2]. A number of other studies have supported that KIM-1 could be a useful biomarker for early detection and diagnosis of AKI caused by exposure to nephrotoxin, complications of post-cardiac surgery and ischemia. In contrast to non AKI patients, urinary KIM-1 is significantly increased in patients with AKI 2 h after cardiac surgery and may predict AKI with good performance in patients within 24 h of cardiac surgery [3]. Overall, the body of evidence on KIM-1 as a biomarker was sufficient for the Federal Drug Administration and the European Medicines Agency to qualify KIM-1 for preclinical assessment of nephrotoxicity and for clinical evaluation on a case-by-case basis [4].
Recent studies also identified urinary KIM-1 as a biomarker for the assessment of nephropathy in various chronic kidney diseases (CKD). In patients with clinically mild IgA nephropathy, high urinary KIM-1 was linked to relatively severe pathologic changes in renal biopsy [5]. In patients with systemic lupus erythematosus, the number of tissue KIM-1 positive cells strongly correlated with histological findings of glomerular and interstitial inflammation. Urinary KIM-1 concentrations were also significantly correlated with the expression of tissue KIM-1 in systemic lupus erythematosus patients. Further, patients with active lupus nephritis exhibited elevated urinary KIM-1 concentrations compared to inactive lupus nephritis patients. In addition, urinary KIM-1 concentrations were also correlated with severity of proteinuria and tubular damage in active lupus nephritis group [6].
Thesefindings increase the potential use of urinary KIM-1 in the diagnosis or prognosis of CKD, but also results in difficulties in the interpretation of urinary KIM-1 when it is used in early detection of AKI. For best laboratory practice it is necessary to determine reference intervals specific to the local patient population, with partitioning by gender and age, where required, according to the recommendations of Clinical and Laboratory Standards Institute (CLSI) guidelines [7]. In most previous clinical studies, the baseline urinary KIM-1 concentration was measured in nominally healthy individuals or individuals who were being treated but did not develop AKI, which does not represent appropriate reference intervals for clinical decision making. In this study we measured and compared urinary KIM-1 concentrations in adults with normal kidneyfiltration function but with or without pre-existing kidney injury to clarify if they should be considered as different patient populations in the establishment of reference intervals. Moreover, the effect of age and gender on urinary KIM-1 and creatinine normalized urinary KIM-1 concentration was investigated. Finally, we determined urinary KIM-1 reference intervals from subjects with normal kidneyfiltration function and without proteinuria.
2. Subjects and methods
2.1. Subject selection and urine sample collection
Urine samples were obtained from adults (17 year-old) submitting samples for routine urine analysis, between July 2015 and January 2016. Samples were aliquoted into clean tubes and frozen at20C within 4 h of collection for later use. A total of 188 (71 male and 117 female, 17–95 year old) samples were obtained from adults with normal kidney filtration as evidenced on basis of two recent serum creatinine measurements using age- and sex-specific reference limits (male: 54–113μmol/L, female:37–91μmol/L). The study was approved by the local Health Research Ethics Authority and the Research Proposals Approval Committee.
2.2. Urine analysis and urine creatinine
Routine urine analysis was done on an Urysis 2400 analyzer (Roche diagnostics) in the hospital core laboratory, using multi- parameter test cassette that measures pH, protein (albumin), glucose, ketones, bilirubin, urobilinogen, nitrite, erythrocyte, leucocyte esterase, and specific gravity. Only urine protein, erythrocyte, leucocyte esterase results that reflecte possible pre-existing nephropathy were recorded for data analysis in this study. The following cutoffs were used to classify results into test-positives. Urine protein positive:
trace (0.25 g/L), 1þ (0.75 g/L), 2þ (1.5 g/L) and 3þ (5 g/L). Erythrocyte positive: trace (10Ery/μL), 1þ (25Ery/μL), 2þ (50Ery/μL), 3þ (150Ery/μL) and 4þ (250Ery/μL). Leucocyte esterase positive: trace (25Leu/μL), 1þ (100Leu/μL), 2þ (500Leu/μL). Urinary KIM-1 results were normalized to urine creatinine concentrations determined by analyzing all urine samples within 4 h of collection using an enzymatic assay on the Architect Ci8200 chemistry analyzer (Abbott Laboratories).
2.3. Urinary KIM-1 measurement
The urinary KIM-1 concentrations were measured in duplicate for each sample using the Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). Manufacturer's instructions were followed in sample preparation, reagent reconstitution and testing procedures. A calibration curve was generated for each testing plate to report urinary KIM-1 in concentration units ofμg/L, which were subsequently normalized usingμg/mmol creatinine units. The analytical performance of the assay such as linearity, within plate and between plate precision, and recovery were determined to be consistent with manufacturer's claims. The limit of detection was 0.009μg/L.
2.4. Statistical analysis
The normality of data distribution was assessed by Kolmogorov-Smirnov test. Urinary KIM-1 concentration values were expressed as median with interquartile range. The reference interval was determined following CLSI guideline (C28-A3) [7] by non-parametric method. The outliers were checked by Reed/Dixon method. The KIM-1 concentration between the groups was compared by Mann-Whitney U test of SPSS software version 19.0 for Windows (SPSS Inc. Chicago, USA). P< 0.05 was considered as statistically
significant.
3. Results
3.1. Subject general information
A total of 188 urine samples were collected from 117 females and 71 males between 17 and 95 years old. 83 samples showed no evidence of pre-existing nephropathy by urine analysis were negative for urine protein, erythrocytes and leucocytes. The remaining 105 samples showed at least one abnormal result for these three parameters suggesting possible pre-existing nephropathy.
3.2. Increased concentration of urinary KIM-1 in proteinuria
The urinary KIM-1 concentrations ranged from undetectable to 11.93μg/L, from undetectable to 4.26μg/mmol creatinine and were not normally distributed. The urinary KIM-1 concentrations were compared between the following groups clustered using results of routine urine analysis: (1) Protein positive: the group of samples screened positive for urinary protein and/or erythrocytes and/or leucocytes (proteinþ, erythrocyte þ/, leucocyteþ/-); (2) Protein negative: the group with undetectable urinary protein but screened positive for erythrocytes and/or leucocytes (protein-, erythrocyteþ/-, leucocyte þ/); and (3) Control: the group which screened negative for urine protein, erythrocyte and leucocyte (protein-, erythrocyte-, leucocyte -) (Table 1). The results showed significantly increased urinary KIM-1 concentration in protein positive samples (group 1) compared to control samples (group 3). A similar trend was observed when samples were normalized for urine creatinine, but without achieving statistical significance. Compared to control samples there was no significant difference in urinary KIM-1 in the urine samples only positive for erythrocytes and/or leucocytes (group 2).
In addition, the increase of urinary KIM-1 was related to the concentration of urinary protein (Table 2). The urine samples with more protein detected showed higher concentration of KIM-1. For urine samples with positive protein, KIM-1 concentration increased significantly even when the protein in urine was at trace concentration (0.25 g/L). The creatinine normalized urinary KIM-1 was significantly higher when urine protein was 1 þ to 3þ (0.75–5 g/L), but not when urine protein was present as trace. Urinary KIM-1 concentrations were not significantly related to erythrocyte and leucocyte positivity if urinary protein was negative.
3.3. The effect of age and gender on KIM-1 concentration in urine samples
The concentrations of absolute and urine creatinine normalized urinary KIM-1 were analyzed by age and sex in 147 individuals with normal kidneyfiltration function and with negative urinary protein, negative or positive erythrocytes or leucocytes (Table 3). No statistically significant age difference of urinary KIM-1 concentration and creatinine normalized KIM-1 concentration in male and fe- male groups were observed inFig. 1; and there was no significant difference of urinary KIM-1 and creatinine normalized KIM-1 con- centration between the genders (KIM-1: p¼ 0.084, KIM-1/Cre: p ¼ 0.234) (Table 3).
The median and interquartile range of urinary KIM-1 and creatinine normalized KIM-1 concentrations in 147 adults without pro- teinuria were 0.54 (0.18–1.18μg/L) and 0.08 (0.03–0.17μg/mmol). The laboratory specific reference interval for urinary KIM-1 was 0.00 (90%CI: 0-0) to 4.19 (90%CI: 3.11–5.62)μg/L, and for creatinine normalized urinary KIM-1 0.00 (90%CI: 0-0) to 0.58 (90%CI:
0.44–0.74)μg/mmol.
4. Discussion
This study examined baseline urinary KIM-1 concentrations in adults with normal kidneyfiltration but with or without pre-existing nephropathy. Urinary KIM-1 concentrations were significantly higher when urine protein was detected and increased with the severity of urine proteinuria but not hematuria or pyuria. No significant effect of age and sex on urinary KIM-1 concentration was observed. A laboratory specific reference interval for urinary KIM-1 in adults with normal kidney function and without proteinuria was established.
KIM-1 presents at very low concentration in healthy normal kidneys. It is expressed in proliferating bromodeoxyuridine-positive and dedifferentiated vimentin-positive epithelial cells in regenerating proximal tubules and is recognized as a biomarker for early detection of kidney tubular damage [1,2,12]. The increase of urinary KIM-1 has been observed to correlate with proteinuria or albuminuria in diabetic patients [8–10]. Urine KIM-1 is increased in type 2 diabetic patients even with normal (urinary albumin/creatinine ratio
Table 1
Urinary KIM-1 concentration in urine samples with normal and abnormal urine analysis.
Number KIM-1(μg/L) KIM-1/Cre (μg/mmol)
Protein positive (Proteinþ, erythrocyte þ/-,leucocyte þ/-) 40 1.51(0.78–2.55)** 0.12(0.08–0.25) Protein negative (Protein-, erythrocyteþ/-, leucocyte þ/-) 65 0.48(0.14–1.13)## 0.08(0.03–0.13)
Control (Protein-, erythrocyte-, leucocyte -) 83 0.60(0.19–1.26) 0.09(0.02–0.19)
Results are expressed as median (interquartile range). (**): P< 0.01 significant difference when compared to control group (Protein-, erythrocyte -, leucocyte-). (##): P< 0.01 significant difference when compared to protein positive group (protein þ, erythrocyteþ/-, leucocyte þ/). KIM-1: kidney injury molecule-1, KIM-1/Cre: creatinine normalized kidney injury molecule1.
<10 mg/g creatinine) or mildly (urinary albumin/creatinine ratio 10–30 mg/g creatinine) increased albuminuria [8]. Positive corre- lation was found between KIM-1 and urine albumin and urine albumin/creatinine ratio in type 2 diabetic patients (r¼ 0.479 P < 0.001, r¼ 0.400, P < 0.001 respectively) [9]. Urinary KIM-1 concentration was significantly associated with the regression of
Table 2
Association of urinary KIM-1 concentration with the severity of proteinuria, hematuria and pyuria.
Group Number KIM-1(μg/L) KIM-1/Cre (μg/mmol)
Proteinþ (erythrocyteþ/-, leucocyteþ/-)
Control 83 0.60(0.19–1.26) 0.09(0.02–0.19)
Trace 26 1.29(0.58–2.24)**## 0.11(0.05–0.19)
1–3þ 14 1.70(1.27–3.63)** 0.15(0.09–0.32)*
Leucocyteþ (protein-, erythrocyte þ/)
Control 83 0.60(0.19–1.26) 0.09(0.02–0.19)
Trace 15 0.87(0.07–1.91) 0.08(0.02–0.27)
1þ 12 0.27(0.06–1.04) 0.07(0.00–0.11)
2þ 10 0.29(0.18–0.85) 0.08(0.04–0.17)
Erythrocyteþ (protein-, leucocyteþ/-)
Control 83 0.60(0.19–1.26) 0.09(0.02–0.19)
Trace 14 0.30(0.00–0.89) 0.06(0.00–0.11)
1þ 14 0.76(0.00–1.22) 0.07(0.00–0.15)
2–4þ 18 0.40(0.15–1.37) 0.09(0.04–0.19)
Results are expressed as median (interquartile range). (*): P< 0.05, (**): P < 0.01 significant difference when compared to control group (protein-, erythrocyte-, leucocyte-). (##): P< 0.01 significant difference when compared to protein positive (1–3 þ group), KIM-1: kidney injury molecule-1, KIM-1/Cre: creatinine normalized kidney injury molecule-1.
Table 3
Effect of gender on urinary KIM-1 concentration.
Age Female Male
Number KIM-1(μg/L) KIM-1/Cre (μg/mmol) Number KIM-1(μg/L) KIM-1/Cre (μg/mmol)
17-95y 100 0.42(0.15–1.07) 0.08(0.02–0.16) 47 0.68(0.23–1.36) 0.09(0.03–0.22)
Results are expressed as median (interquartile range). KIM-1: kidney injury molecule-1, KIM-1/Cre: creatinine normalized kidney injury molecule-1.
Fig. 1. The distribution of urinary KIM-1 and urine creatinine normalized KIM-1 by age
microalbuminuria over the subsequent 2 years [10]. Moreover, in patients with type 1 diabetes and proteinuria, baseline blood KIM-1 concentration strongly predicted the rate of eGFR loss and risk of ESRD during 5–15 years of follow-up, after adjustment for baseline urinary albumin/creatinine ratio, eGFR, and HbA1C [11]. These studies indicate that urinary KIM-1 concentration is elevated when urine microalbumin is normal or mildly increased and associated with progress of chronic kidney injury. In this study we measured urinary KIM-1 in adults with normal kidneyfiltration but with pre-existing nephropathy indicated by positive urine protein and/or erythrocyte and/or leucocyte in routine urine analysis. The results showed that urinary KIM-1 significantly increased when urine protein was at even trace concentrations, and the KIM-1 concentrations increased with the degree of urine proteinuria. Although, the creatinine normalized urinary KIM-1 concentrations also increased, it was to a lesser degree as absolute KIM-1 concentrations. No increase in KIM-1 concentrations was observed if hematuria or pyuria was present without significant proteinuria. The urinary KIM-1 could be 2–3 times higher in patients with urine protein positive than patients with negative urine protein. Therefore, when using urinary KIM-1 to early detect AKI, patient baseline urinary KIM-1 should be interpreted carefully with appropriate reference intervals or cutoff values if patient is possible with chronic kidney diseases. Routine urine analysis is not a sensitive method for detecting urine albumin, however it has a fast turnaround time, can be used for POCT, and provide preliminary information to facilitate decisions on whether KIM-1 measurement would be useful.
The effect of age and sex on urinary KIM-1 was investigated in at leastfive studies since 2013 and reference intervals were estab- lished for urinary KIM-1 in different population. For healthy full term newborns atfirst or second day of life, urinary KIM-1 correlated with gestational age but did not correlate with birth weight and gender. Median urinary KIM-1 was reported as 1.326 ng/mL for female and 1.248 ng/mL for male[13]. In healthy infants aged 1 day to 1 year old urinary KIM-1 concentrations were substantially lower in almost all 106 subjects (median 0.08 ng/mL) and not related to age, sex or ethnicity [14]. However, a study by McWilliam including 120 and 171 healthy children recruited in USA and UK respectively, showed creatinine normalized urinary KIM-1 concentrations to be significantly associated with age and ethnicity but not with sex. Urinary KIM-1 also showed diurnal variation, with higher concen- trations found in morning samples. The mean urinary KIM-1 concentrations were 0.43 ng/mg creatinine in the UK group and 0.18 ng/mg creatinine in the USA group, which might be due to the difference of participant ethnicity mix and the time at sample collection [15]. Similarly, Bennett MR et al. [16] established the reference intervals for urinary KIM-1 for 368 healthy children ages 3–18 years old and showed an overall median of urinary KIM-1 concentration of 0.410 ng/mL. The urinary KIM-1 concentration did not show significant difference between male and female groups. However, urinary KIM-1 was significantly higher in 15–18 year old children (0.5156 ng/mL) when compared to 3–5 year (0.3368 ng/mL) and 5–10 year olds (0.3864 ng/mL). The reference intervals established by Pennemans V et al. included 338 healthy, non-smoking subjects between 0 and 95 year old. In their study, the subjects with elevated alpha 1- microglobulin values that indicating tubular damage were excluded. Both age and gender differences were found in urinary absolute KIM-1 results [17]. Including adults from 17 to 95 year old with normal kidney function and without proteinuria that excluded glomerular and tubular kidney damage, our reference intervals did not show statistical significance on the increase of urinary KIM-1 concentrations with age increasing. The differences in male versus female urinary KIM-1 concentrations also failed to achieve statistical significance. The impact of differences in the number of male (47) versus female (100) subjects on this finding is unclear.
Furthermore, there was no significant difference on creatinine normalized urinary KIM-1 based on age or sex. The currently reference intervals of urinary KIM-1 cannot be standardized due to subject population, methodology and the statistical analysis used in the calculation of reference intervals. Laboratory specific reference intervals for urinary KIM-1 need to be established with subjects excluded chronic kidney diseases before the test is used in the laboratory service.
In conclusion, this study highlighted that the increase of urinary KIM-1 in the patient with pre-existing nephropathy is related to the severity of proteinuria. Patient baseline urinary KIM-1 should be interpreted with caution if patients have chronic kidney diseases when urinary KIM-1 is used for early detection of AKI. Different reference intervals or cutoff values may need to be determined for patients with versus without pre-existing nephropathy. Urine protein should be assessed to distinguish subjects with versus without proteinuria when establishing reference intervals for urinary KIM-1, and might be considered as an adjunct test to determine whether the increase of KIM-1 is of clinical significance.
Conflict of interest
The authors declare no conflict of interest.
Funding
The study was funded by Health Care Foundation Research Funding, Eastern Health Authority, 2013. The founding sponsors had no role in the design and completion of the study and the publication.
Acknowledgements
The authors thank the technologists in the Core Lab of St. Clare's Mercy Hospital and the Renal Lab of Health Sciences Center, Eastern Health Authority for their great assistance in sample collection and measurement.
