Advances in Medical Sciences 60 (2015) 31–40

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Original Research Article

Which equations should and which should not be employed in calculating eGFR in children? Katarzyna Zachwieja a,*, Przemysław Korohoda b, Joanna Kwinta-Rybicka c, Monika Miklaszewska a, Anna Moczulska a, Jolanta Bugajska d, Joanna Berska d, Dorota Droz˙dz˙ a, Jacek A. Pietrzyk a a

Department of Pediatric Nephrology, Jagiellonian University Medical College, Cracow, Poland Department of Electronics, Faculty of Computer Science, Electronics and Telecommunications, AGH University of Science and Technology, Cracow, Poland c Department of Pediatric Nephrology, Children’s University Hospital of Cracow, Cracow, Poland d Clinical Biochemistry Department, Jagiellonian University Medical College, Cracow, Poland b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 March 2014 Accepted 20 August 2014 Available online 30 August 2014

Purpose: We assessed the reliability of calculating eGFR in children as compared to the iohexol disappearance test (GFR-I), which was performed 417 times in 353 children aged 2 and more. Material/methods: eGFR was estimated with equations based on serum creatinine: Schwartz (1: eGFRScr), Cockroft–Gault (2: eGFR-CG) and MDRD (3: eGFR-MDRD), and on creatinine clearance (4: eGFR-U), or relying on serum cystatin C: Hoeck (5: eGFR-H), Bokenkamp (6: eGFR-B) and Filler (7: eGFR-F), and on the three Schwartz markers (8: eGFR-S3M). Mean relative error (RE), correlation (R), Bland–Altman analysis and accuracy of GFR-I were studied in all patients and in subgroups: at GFR < 60 ml/min/ 1.73 m2; in children aged 12 and >12. Results: The results by eGFR-Scr, eGFR-S3M demonstrated no statistical difference to GFR-I at GFR < 60 ml/min/1.73 m2, but underestimated eGFR at higher filtration values by 11.6  15.1% and 19.1  16.4, respectively (p < 0.0000). The eGFR-B, eGFR-F and eGFR-MDRD equations illustrated important overestimation of reference GFR results (RE: 84  44.2%; 29.5  27.9%, 35.6  62%; p < 0.0000 for all). The MDRD and C–G formulas showed statistically better consistency in children aged >12. A good agreement was achieved by the eGFR-H equation (5.1  21.9%; p < 0.0000; R = 0.78). Conclusions: (1) Schwartz equations show a good conformity at GFR < 60 ml/min/1.73 m2, but underestimate the results at higher GFR values. (2) The Bokenkamp equation with original coefficient should not be employed in children. (3) The use of the Hoeck formula in all children and C–G and MDRD formula in children aged >12 is possible. (4) The error of eGFR calculations increases at higher GFR values. ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Keywords: eGFR calculation Children Iohexol Creatinine Cystatin C

1. Introduction Calculation of glomerular filtration rate in children aged 1 or more similarly as in adults, provides the basis for staging chronic kidney disease (CKD) and is helpful in classifying acute kidney injury [1]. As is already known, assessment of the actual GFR is

* Corresponding author at: Department of Pediatric Nephrology, Jagiellonian University Medical College, Wielicka St. 265, 30-663 Cracow, Poland. Tel.: +48 12 658 0663; fax: +48 12 658 0663. E-mail address: [email protected] (K. Zachwieja).

neither simple nor accurate [2]. Methods employed in everyday clinical practice are highly imperfect, and the golden standard – calculation of inulin clearance – is performed only in single leading centres. These problems are visible in the ever-increasing number of new formulas of eGFR estimation based on creatinine and cystatin C concentrations as well as anthropometric data both for children and adults [2–5]. At present, numerous laboratories routinely calculate eGFR using equations developed for adult patients; in children however, this yields incorrect and simply abstract values. When analysing the result arrived at in this manner, one should pay attention to the equation employed, the method of creatinine determination and standardization of the

http://dx.doi.org/10.1016/j.advms.2014.08.007 1896-1126/ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

K. Zachwieja et al. / Advances in Medical Sciences 60 (2015) 31–40

32

Table 1 The basic anthropometric data: age, body weight, height, height Z-score, BMI, BMI Z-score, BSA and glomerular filtration markers concentrations in examined cohort of children (expressed in mean value  SD, minimum–maximum). All patients

Mean  SD

Min–Max

Age (years) Body weight (kg) Height (cm) Height Z-score BMI BMI Z-score BSA (m2) Serum creatinine (mmol/l) Serum urea (mmol/l) Cystatin C (mg/l) Gender (F/M) n

12.05  4.00 44.37  19.26 147.19  21.18 0.18  1.25 19.41  4.30 0.142  1.16 1.33  0.38 77.10  70.3 5.66  3.90 0.92  0.58

2–18 10.2–140 83–189.5 5.2 to 3.35 12.2–45.71 3.17 to 2.53 0.49–2.68 13.2–761.8 2.3–37.5 0.45–5.2 208/209 417

laboratory, as well as remember the well-known factors affecting creatinine concentration levels (such as muscle mass, age, sex, diet, creatinine clearance and tubular creatinine secretion – which is significant at GFR dropping below 25 ml/min/1.73 m2 – but also medications that might modify the secretion). Despite the spreading of serum cystatin C determination, which is said to be a better marker of kidney function than creatinine [6–9], this method has not been universally accepted, partly because of the cost of a single test (approximately nine times higher than the cost of creatinine determination), but also of the lack of simple ‘‘bedside’’ equations allowing for prompt eGFR calculation. According to some authors, cystatin C has not met expectations as a GFR marker that would replace creatinine [8], though some still believe that it is sufficient to know serum creatinine concentration and body height alone [10]. Thus, understanding which of the available equations may be employed in children without risk of a significant error seems to be clinically important. The objective of the present study was to calculate GFR based on the reference iohexol clearance method (which has been used in our centre since 2007) and comparing eGFR values based on the selected eight formulas to the reference method. The preliminary results obtained in a smaller group of subjects were presented in 2009 [11]. 2. Material and methods The study group consisted of 353 paediatric patients of the Department of Nephrology, University Children’s Hospital of

Cracow. Detailed anthropometric data and the concentration values of glomerular filtration markers are presented in Table 1. The height and the weight of all patients were taken at the hospital by qualified nurses as a clinical routine (using the accurate hospital weight and stadiometer). In the study group, only 31 children (7.5%) did have the Z-score for height 1.5 g/day; (2) serum albumin concentration 1 mg/kg/day; (4) lack of the approval for the test. The iohexol tests were performed in all 353 patients in a hospital setting; in 64 patients the iohexol test was performed twice or thrice. The study lasted for several years. This is why each test was treated as a separate measurement and why reported mean values were calculated for 417 patients (measurements). Not all the patients had 24 h urine collection; it was performed 390 times in 350 patients. A standard dose of iohexol (5 ml) was administered to all the patients; subsequently, the venous line was rinsed with 5 ml of saline. The blood samples were collected by finger puncture (capillary sampling) at 2, 3 and 4 h following iohexol administration (in children with eGFR < 40 ml/min/1.73 m2, blood samples were collected at 2, 4 and 8 h post-administration) [13]. Plasma

Table 2 The basic anthropometric data, filtration markers and iohexol method results in subgroups of children with different CKD stages, based on GFR-I results (expressed in mean value  SD).

Age (years) Body weight (kg) Height (cm) Height Z-score BMI BMI Z-score BSA (m2) Serum creatinine (mmol/l) Serum urea (mmol/l) Cystatin C (mg/l) GFR-I (ml/min/1.73 m2) Gender (F/M) n

CKD group III

CKD group II

CKD group I

p-Value

GFR-I  60

GFR-I 60–90

GFR-I > 90

III vs. II test t/test U M–W

III vs. I test t/test U M–W

II vs. I test t/test U M–W

ANOVA Kruskal–Wallis

12.3  4.1 37.6  15.2 141.1  20.5 1.16  1.54 18.1  3.6 0.02  1.20 1.20  0.32 214.5  139.4 12.47  7.42 2.16  0.87 35.5  14.1 14/38 52 (12.5%)

11.2  4.5 39.9  18.5 141.9  23.0 0.31  1.14 18.6  4.0 0.12  1.04 1.24  0.38 79.8  26.7 5.48  1.67 0.90  0.19 77.9  8.7 45/40 85 (20.4%)

12.4  3.8 46.9  19.7 149.8  20.3 0.05  1.14 19.9  4.5 0.18  1.18 1.38  0.38 64.2  15.5 4.45  1.24 0.69  0.13 115.7  16.3 150/130 280 (67.1%)

0.1560/0.1539 0.4477/0.5361 0.8395/0.6398 0.0003/0.0064 0.4029/0.4779 0.4751/0.3920 0.5608/0.5419 0.0000/0.0000 0.0000/0.0000 0.0000/0.0000 0.0000/0.0000 0.0028*

0.8748/0.9906 0.0014/0.0008 0.0047/0.0035 0.0000/0.0000 0.0062/0.0016 0.2589/0.2006 0.0012/0.0011 0.0000/0.0000 0.0000/0.0000 0.0000/0.0000 0.0000/0.0000 0.0004*

0.0171/0.0385 0.0041/0.0041 0.0024/0.0054 0.0115/0.0040 0.0222/0.0093 0.6594/0.5072 0.0023/0.0041 0.0000/0.0000 0.0000/0.0000 0.0000/0.0000 0.0000/0.0000 0.9187*

0.1094 0.0003 0.0011 0.0000 0.0009 0.3821 0.0004 0.0000 0.0000 0.0000 0.0000 0.0017*

* According to Chi-square Pearson test. p-Value was calculated by: t test  Student’s t test; test U M–W  non-parametric U Mann–Whitney test.

K. Zachwieja et al. / Advances in Medical Sciences 60 (2015) 31–40 Table 3 The diseases category in examined group (n = 353). Diseases category Glomerulonephritis CAKUT (congenital anomalies of kidney and urinary tract) Solitary kidney Others (hypertension, kidney stone disease, hemolytic uraemic syndrome)

Number of children (%) 93 (26) 80 (23) 70 (20) 108 (31)

iohexol concentration was determined by the high-performance liquid chromatography (HPLC) method. A detailed description of this methodology was provided in a previous report [11] and it is attached in Appendix A. The iohexol test featured the method of triple blood samples collection and iohexol concentration determination developed by Bro¨chner–Mortensen, based on the slow phase of iohexol clearance [13,14]. The reassessed Bro¨chner–Mortensen formula is: Cl1 ¼

dose AUC

where dose – the iohexol dosage; AUC – the area under the curve of the plasma iohexol concentration in time based on three iohexol concentrations determination [14]. Then the calculation was done according to Jødal et al. [15]. Cl ¼

Cl1 1 þ ð f  Cl1 Þ

Where Cl – total plasma clearance, Cl1 – one-pool plasma iohexol clearance,

f ¼ 0:0032  BSA1:3 BSA – body surface area And GFR-I was normalized to BSA: GFR  I ¼

CL 1:73=BSA

Serum creatinine levels were determined by the enzymatic method (VITROS FS Ortho Clinical Diagnostics). Until March 1, 2010, the method was validated relative to HPLC. From March 2010 on, the authors introduced creatinine determinations using a new calibration and the manufacturer-provided method validation was based on standard reference material SRM 967, where creatinine concentration was determined by IDMS (isotope dilution mass spectrometry). In order to compare the creatinine results obtained after March 1, 2010 (IDMS CREA) with the results obtained before standardization, creatinine concentration values were calculated using the following equation: CREA = IDMS CREA  1.0355 + 11.834, in keeping with the manufacturer’s recommendations. The comparison between these two methods of creatinine determination (before and after calibration to IDMS) was done in our laboratory and we found no difference in creatinine concentration after employing this modification. The total imprecision for low creatinine serum concentration (99 mmol/l) was 3.5% and, for high creatinine serum concentration (478 mmol/l), 2.6%. Concentrations of cystatin C were determined using the nephelometric method (a Siemens N-Latex diagnostic set was used). The total imprecision of cystatin C determination for low cystatin C concentration (1.0 mg/l) was 2.6%, and 2.9% for high cystatin C concentration (8.4 mg/l). Serum urea concentration was determined by the enzymatic method (VITROS FS Ortho Clinical Diagnostics).

33

The implementation of the study and iohexol test was approved by the Bioethical Committee, Jagiellonian University Medical College. In all the study patients, calculations of the eGFR value were performed employing the following equations based on the filtration markers: I. Serum creatinine concentration, i.e.: 1. The short (bedside) equation of Schwartz et al., developed in 2009 for children (1: eGFR-Scr) [16]; 2. The Cockroft–Gault equation (2: eGFR-CG) developed for adults [17]; 3. The shortened version of the MDRD equation for adults (3: eGFR-MDRD), following a modification for determining creatinine by the enzymatic method [18]; 4. Creatinine clearance (following 24-h urine collection – 4: eGFR-U); II. Serum cystatin C concentration, i.e.: 5. The Hoeck et al. equation (5: eGFR-H) for adults [19]; 6. Equation developed for children according to Bokenkamp et al. (6: eGFR-B) [20]; 7. Equation proposed by Filler et Lepage (7: eGFR-F) [21]; III. Concentration values of three markers (serum creatinine, urea and cystatin C): 8. The three-marker (combined) equation by Schwartz et al. for children proposed in 2012 (8: eGFR-S3M) [22]. The detailed formula description is presented in Table 4. The eGFR-CG, eGFR-MDRD and eGFR-H formulas have been developed for adults, while eGFR-Scr, eGFR-S3M as well as eGFR-B and eGFR-F have been worked out for the paediatric population. The selection of the equations was mainly a consequence of their popularity, as for example was the case of 2.eGFR-CG and 3.eGFR-MDRD, as well as of the bedside equation developed by Schwartz et al.: 1.eGFR-Scr and creatinine clearance (which is still commonly used in everyday practice). The bedside Schwartz formula is recommended by KDIGO guidelines for children [1]. The eGFR-S3M formula was supplementary because it is still needed to be verified in group of children with normal renal function [5,22]. The remaining equations were selected as comparative, and we have chosen the three cystatin-based formulas; one wellknown for adults by Hoeck (5.eGFR-H) and two established for children by Bokenkamp (6.eGFR-B) and Filler (7.eGFR-F). From among the many available eGFR equations, we have decided to compare the ones that we have been previously analyzed ourselves in a large group of children. The selected formulas using different filtration markers have been chosen on purpose to demonstrate the possibility of the underestimation and the overestimation in eGFR calculation. Surprisingly, one of the adult formulas yielded a smaller error in comparison to that in the paediatric formula. While assessing the conformity of the eGFR values calculated employing particular equations in comparison to the GFR result of the reference iohexol method, the authors used: 1) correlation coefficient, presented in Fig. 1a, for selected equations in Fig. 2a, and in Table 5 2) absolute error (bias) of eGFR-GFR-I, shown in Table 5 3) relative error (RE) according to the equation:

REðnÞ ¼ 100% 

eGFRXðnÞ  eGFRIðnÞ eGFRIðnÞ

where n is the number of all single results (patients), eGFRX(n) the nth result (for nth patient) using selected equation (X), eGFRI(n) the

K. Zachwieja et al. / Advances in Medical Sciences 60 (2015) 31–40

34 Table 4 The employed formulas description. Marker Creatinine-based formulas Schwartz eGFR-Scr (1) Creatinine

Year

Equation (ml/min/1.73 m2)

2009

41.3 x (height/Screat)

43.3

1976

[(140  age)  body weight  k/(Screat  72)], k = 1.23 for men, 1.05 for women 175  Screat1.154  age0.203  0.742 (for women)

81 77 103

Cockroft–Gaulta eGFR-CG (2)

Creatinine, age, body weight

Creat. enzymatic IDMS Creat. Jaffe reaction

eGFR-MDRD (3)

Creatinine, age, gender

Creat. enzymatic IDMS

2007

Cys C PENIA Cys C PETIA

2003 1998

4.32 + 80.35 x C1 30 + 162  C1

Cys C PENIA

2003

101:962þ1:123 log10 ð1=CCÞ

Creat. enzymatic IDMS. Cys C PENIA

2012

39.8  (height/Screat)0.456  (1.8/cystatin C)0.418  (30/urea)0.079  (1.076 for men)  (height/1.4)0.179

Cystatin C-based formulas Hoek eGFR-H (5) Cystatin C Cystatin C Bokenkamp eGFR-B (6) Filler eGFR-F (7) Cystatin C Combined (three markers) formula Schwartz Creatinine, urea, eGFR-S3M (8) cystatin C, height, gender

Mean eGFR (ml/min/1.73 m2)

Cys C and creat. assay

Number of patients

Examined group

Reference method

643

Children

Iohexol

72.7

236

Adults

Creatinine clearance

39.8

1628

Adults

Iothalamate

146 184

Adults Children

536

Children

Iothalamate Inulin clearance 99m Tc-DTPA

643

Children

Iohexol

43.3

C (CC), serum cystatin C concentration (mg/l); urea, serum urea, expressed in BUN (mg/dl); Screat, serum creatinine concentration (mg/dl), age (years), body weight (kg), height (m). a The results of eGFR-CG was normalized per 1.73 m2. PETIA, partly enhanced immunoturbimetry method; PENIA, partly enhanced immunonephelometry method.

nth result of GFR-I (for nth patient) and X is the equation tested for eGFR. The average value of relative error (in %) is presented in Table 5, in Fig. 1c, and for selected equations, in Fig. 2c, centiles of relative error (5, 25, 40, 50, 60, 75, 95%) are presented in Fig. 3a–c. A better agreement of a given equation to the reference method was illustrated by the proximity of the point in the graph to the line denoting zero. The closer a point corresponding to the value of a given centile was situated to the zero line (optimum value), the better conformity was achieved between a given equation and the reference method. 4) Bland–Altman plots shown in Figs. 1b and 2b. 5) The accuracy measured in 10 and 30% of the eGFR-X results in the accordance to GFR-I (iohexol method), presented in Table 5.

Table 6 presents statistical significance for the comparison of mean relative error for eGFR values calculated with all the equations in the patient subgroups listed in particular columns. Additionally, the patients were classified as to the particular CKD stages based on each of the analyzed equations (see Table 7). The statistical analysis was performed with the Matlab and Statistica v.10 software. The comparison of the eGFR results was based on the Wilcoxon nonparametric test, the Mann–Whitney’s U test and the parametric Student’s t test. The conformity of a given result to normal distribution was assessed with the Kolmogorov– Smirnov test. ANOVA was also used. The statistical significance of differences was at p < 0.05.

Results for all the comparison methods of eGFR equation to the iohexol method (GFR-I) were additionally analyzed in the following subgroups and are shown in Table 5:

In the majority of children (67.1%), the reference method demonstrated normal kidney function. Children with of GFR  90 ml/min/1.73 m2 (group I) were significantly older than group III (GFR < 60 ml/min/1.73 m2; p = 0.017). The statistical difference in the height Z-score between the three analyzed groups proved that the children from group I were the tallest comparing to patients from group II (GFR: 60–90 ml/min/1.73 m2) and group III (group I vs. II: p = 0.01, group I vs. III: p = 0.00; group II vs. III: p = 0.0003). Similarly, the group I children had larger body surface area (BSA; group I vs. II: p = 0.000; group I vs. III: p = 0.000, group II vs. III: p = 0.000). In contrast, there was no statistical difference in BMI Z-score among these three groups of the patients (see Table 2). Table 7 presents the values of eGFR calculated using all studied equations and classification of children to particular CKD stages based on the eGFR result: group I: eGFR  90; group II: eGFR: 60–90; group III: eGFR  60 ml/min/1.73 m2, respectively. There is a clear tendency for lowering the CKD classification when using both Schwartz formulas (eGFR-Scr and eGFR-3M), and a less evident one for the creatinine clearance method. The opposite

a) In children aged 12 years and younger (12 years of life) shown as group b in Table 5; b) In children above 12 years of age (>12 years of life) shown as group c in Table 5; c) In children with eGFR < 60 ml/min/1.73 m2 according to the reference method (GFR-I) shown as group d in Table 5. The selected outcomes of the comparisons which revealed an important difference (eGFR-CG, eGFR-MDRD) or lack of the difference (eGFR-3M) to the iohexol method are presented in Fig. 2a–c. For better visualization, we chose the following three formulas eGFR-3M, eGFR-CG and eGFR-MDRD. The differences between the results obtained in the group of children aged 12 to the results in the group of children aged >12 are displayed in the figures of the correlation, in the Bland–Altman plot and the relative error graphs (Fig. 2a–c, respectively).

3. Results

K. Zachwieja et al. / Advances in Medical Sciences 60 (2015) 31–40

35

Fig. 1. (a) The correlation between results of all studied equations (eGFR-X) and iohexol method (GFR-I) in all children (typically n = 417, only for eGFR-U: n = 390). The values on the axes are presented in ml/min/1.73 m2. See group a in Tables 5 and 6. (b) Bland–Altman plots of all studied eGFR equations compared with GFR-I for all patients (typically n = 417, only for eGFR-U: n = 390). The values on the axes are presented in ml/min/1.73 m2. The bold lines represent: the average value (median line)  2SD (lower and the upper line). (c) The relative error [eGFR-X–GFR-I] referred to GFR-I values for all patients (typically n = 417, only for eGFR-U: n = 390). The relative error value (in %) is shown on vertical axis. The GFR-I value (in ml/min/1.73 m2) is presented on horizontal axis.

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K. Zachwieja et al. / Advances in Medical Sciences 60 (2015) 31–40

Fig. 2. (a) The correlation between results of three selected equations and iohexol method (GFR-I) in children with age 12 years (upper graphs, n = 203) and with age >12 years (lower graphs, n = 214). The values on the axes are presented in ml/min/1.73 m2. See groups b and c in Tables 5 and 6. (b) Bland–Altman plots of three selected equations compared with GFR-I in children with age 12 years (upper graphs, n = 203) and with age >12 years (lower graphs, n = 214). The values on the axes are presented in ml/min/1.73 m2. The bold lines represent the average value (median line)  2SD (lower and the upper line). (c) The relative error ([eGFR-X–GFR-I] referred to GFR-I) values for children with age 12 years (upper graphs, n = 203) and with age >12 years (lower graphs, n = 214). The relative error value (in %) is shown on vertical axis. The GFR-I value (in ml/min/1.73 m2) is presented on horizontal axis.

K. Zachwieja et al. / Advances in Medical Sciences 60 (2015) 31–40

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Table 5 Comparison between eGFR-X and the reference GFR-I for selected groups of patients: (a) all patients; (b) patients with age 12 years; (c) patients older than 12 years; (d) patients with eGFR-I  60 ml/min/1.73 m2. The columns present: the average error [eGFR-X–GFR-I], statistical significance (p-value) indicating differences between eGFR samples, correlation coefficient (R), average relative error (RE) along with standard deviation, percentage of RE values within 10% and 30% intervals, and size of the sample. eGFR-X

Patients selection criteria

Average error (ml/min/1.73 m2)

pt

pW

R**

Average RE  SD (%)

Accuracy  10% (%)

Accuracy  30% (%)

n

1.eGFR-Scr

a. All b. Age 12 yrs c. Age >12 yrs d. eGFR-I  60

20.9 16.5 24.5 3.7

0.000* 0.000* 0.000* 0.188

0.000 0.000 0.000 0.001

0.795 0.794 0.814 0.869

19.1  16.4 14.9  16.8 22.6  15.2 10.1  20.1

18.9 21.7 16.7 36.5

74.8 83.6 67.5 82.7

417 189 228 52

2.eGFR-CG

a. All b. Age 12 yrs c. Age >12 yrs d. eGFR-I  60

16.6 26.8 8.1 13.9

0.000* 0.000* 0.013 0.001

0.000 0.000 0.000 0.000

0.736 0.726 0.798 0.834

20.4  29.1 31.9  30.3 10.9  24.3 37.7  36.5

27.6 12.2 40.4 21.2

67.4 51.9 80.3 40.4

417 189 228 52

3.eGFR-MDRD

a. All b. Age 12 yrs c. Age >12 yrs d. eGFR-I  60

31.1 68.3 0.2 20.0

0.000* 0.000* 0.955 0.001

0.000 0.000 0.268 0.000

0.454 0.493 0.745 0.689

35.6  62.0 76.0  67.7 2.2  27.5 48.9  73.1

21.3 8.5 32.0 9.6

54.7 22.8 81.1 42.3

417 189 228 52

4.eGFR-U

a. All b. Age 12 yrs c. Age >12 yrs d. eGFR-I  60

2.0 2.3 1.8 3.3

0.474* 0.559* 0.655* 0.396

0.000 0.005 0.007 0.037

0.528 0.508 0.539 0.842

0.1  38.8 0.1  39.0 0.1  38.7 8.2  28.5

24.4 24.4 24.3 27.5

70.3 69.9 70.6 72.5

390 176 214 40

5.eGFR-H

a. All b. Age 12 yrs c. Age >12 yrs d. eGFR-I  60

2.4 6.4 0.9 3.4

0.277* 0.037* 0.781 0.263

0.003 0.000 0.722 0.002

0.782 0.813 0.773 0.901

5.1  21.9 9.4  19.9 1.5  22.9 12.3  23.4

36.7 40.7 33.3 34.6

83.2 85.2 81.6 78.8

417 189 228 52

6.eGFR-B

a. All b. Age 12 yrs c. Age >12 yrs d. eGFR-I  60

83.1 89.1 78.2 21.6

0.000* 0.000* 0.000 0.000

0.000 0.000 0.000 0.000

0.782 0.813 0.773 0.901

84.3  44.2 93.5  39.0 76.7  46.8 51.1  47.7

3.6 1.1 5.7 9.6

11.8 5.3 17.1 36.5

417 189 228 52

7.eGFR-F

a. All b. Age 12 yrs c. Age >12 yrs d. eGFR-I  60

26.3 30.8 22.6 10.6

0.000* 0.000* 0.000 0.002

0.000 0.000 0.000 0.000

0.774 0.807 0.763 0.897

29.5  27.9 34.8  25.0 25.0  29.4 33.4  28.8

17.3 11.1 22.4 17.3

53.0 45.0 59.6 48.1

417 189 228 52

8.eGFR-S3M

a. All b. Age 12 yrs c. Age >12 yrs d. eGFR-I  60

14.6 12.6 16.3 1.0

0.000* 0.000* 0.000* 0.708

0.000 0.000 0.000 0.119

0.873 0.869 0.878 0.914

11.6  15.1 9.9  14.7 13.0  15.4 6.2  18.6

32.1 37.0 28.1 38.5

89.0 92.1 86.4 84.6

417 189 228 52

pt, p-value according to the Student’s t test; pW, p-value according to the unpaired Wilcoxon test. * The normal distribution of both samples not confirmed by the Kolmogorov–Smirnov test. p-Value below 0.05 indicates statistically significant error between the calculated eGFR-X and GFR-I. ** p < 0.00001 p value for all correlation coefficient (R).

Table 6 p-Value for the comparison between mean relative error ([eGFR-X–GFR-I] referred to GFR-I) in selected subcategories of patients for all studied eGFR equations. The first column (all patients vs. those with GFR-I  60 ml/min/1.73 m2) – p-value comparing relative error between group a vs. group d in Table 5. The second column (patients with GFR-I > 60 vs. those with GFR-I  60 ml/min/1.73 m2) – p-value comparing relative error between group not presented in Table 5 vs. group d. The third column (patients with age 12 yrs vs. those with age >12 yrs) – p-value comparing relative error between both groups of patients (group b vs. group c in Table 5). eGFR-X

1.eGFR-Scr 2.eGFR-CG 3.eGFR-MDRD 4.eGFR-U 5.eGFR-H 6.eGFR-B 7.eGFR-F 8.eGFR-S3M

GFR-I all vs. GFR-I  60

GFR-I > 60 vs. GFR-I  60

age 12 vs. age >12

pt

pW

pt

pW

pt

pW

0.000* 0.000* 0.154* 0.190* 0.027 0.000 0.340 0.000

0.000 0.000 0.293 0.009 0.049 0.000 0.461 0.000

0.000 0.000 0.098* 0.155* 0.011 0.000 0.278 0.000

0.000 0.000 0.234 0.004 0.026 0.000 0.403 0.000

0.000 0.000* 0.000* 0.992* 0.000 0.000 0.000 0.034

0.000 0.000 0.000 0.810 0.000 0.000 0.000 0.012

pW, p-value according to the Wilcoxon unpaired test; pt, p-value according to the Student’s t test. * Normal distribution was not found in the Kolmogorov–Smirnov test. Only nonparametric test p-value is recommended.

trend of the increasing the CKD stage is seen for the equation by Bokenkamp et al., Filler et Lepage and MDRD. 4. Discussion The main findings of the study are as follows: when comparing eGFR values to the reference method, the diversity of the particular analysis method and the difference in the obtained results should be emphasized. For example, eGFR based on creatinine clearance shows a comparatively low relative error but it is not possible to anticipate the direction of the potential error because of the significantly high standard deviation value (Table 5), the low correlation coefficient to GFR-I and disparity of the points (results) seen in Fig. 1a–c. The Bland–Altman analysis does not provide the same image as that of the relative error. The Bland–Altman plot shows the difference between the analyzed method and the reference method in relation to the average value of these two. In contrast, the relative error (RE) plot shows the same difference in relation to the reference method only and it is significantly helpful and accurate in showing the existing difference observed at low and high GFR values. These effects could be ignored on the Bland–Altman plot due to the influence of the reference value on the average result. For instance, the variation of the eGFR-Scr and eGFR-S3M figures could be indicated when ‘‘better agreement’’ to

38

K. Zachwieja et al. / Advances in Medical Sciences 60 (2015) 31–40

iohexol method is seen in the RE plot. It may be also noted for the eGFR-MDRD and eGFR-B figures, when a worse agreement for higher GFR values is presented in Bland–Altman plot (bigger dispersion of the results), which is not so clear on RE plots (see Fig. 1b and c). The eGFR calculation error depends on the GFR value and it is increasing with higher GFR results; the lowest error was noted for GFR < 60 ml/min/1.73 m2, which was not proven for MDRD and Filler et Lepage equations (Tables 5 and 6; Fig. 1b and c). The significant error difference was observed in eGFR calculation by employing some of the formulas in group of children 12 years in relation to the older group (>12 years), the lowest precision was found for the MDRD equation and for the Cockroft– Gault (C–G) formula in younger children (Tables 5 and 6; Figs. 2a–c and 3b and c). In contrast, the agreement with the iohexol method in children >12 years was satisfying. There was no age influence on the creatinine clearance application (Table 6). The eGFR results by both Schwartz et al. equations revealed the best concordance with the reference method for GFR-I  60 ml/ min/1.73 m2, but eGFR results were significantly underestimated at higher GFR values, especially by the eGFR-Scr formula (Figs. 1–3; Tables 5–7). The eGFR calculation by eGFR-B, eGFR-F and eGFRMDRD formulas (when the originally published coefficient was used) clearly overestimated the GFR-I value, and the largest error was noted for the eGFR-B application. In turn, the eGFR-H formula showed pretty good agreement with the reference method (Fig. 1a–c; Tables 5 and 7). There is a tendency towards slight local modifications of the original equations in publications presently available [23–25]; this makes it very difficult to compare different authors’ results. As not every nephrology centre could perform direct measurements of GFR, it is widespread practice to use ‘‘a classical coefficient of the published formula’’. This was one of the reasons why the present authors decided not to modify equations coefficient – in full awareness of the possibility of deterioration of their conformity to the reference method. Our observation of the increasing eGFR calculation error with the GFR value is in concordance with Sharma et al. [26] and may be also explained by the increased disparity of the GFR values observed at higher GFR results. The Schwartz combined (eGFR-S3M) equation were previously assessed by Chechade et al. [23] and Andersen et al. [24], who demonstrated, in contrast to our findings, a good conformity to the inulin clearance method. But others (including Sharma et al.) also noted the tendency for underestimation of eGFR using this formula at high GFR values. Recently, a similar opinion was published by Westland et al. [27] in a group of children with solitary kidney and also by Chechade et al. [28]. Some authors [29–32], and Siddique et al. in a group of children after renal transplant [33], noted a similar tendency for underestimation by using eGFR-Scr at higher GFR values, but contrary opinions of good agreement or even overestimation of eGFR results can also be found [34,35]. Moreover, others, like De Souza et al. [25] and Pottel et al. [30], proposed an additional age-dependent coefficient that would improve the accuracy of the GFR-Scr equation. This age dependency of eGFR-3M equation was not so evident but it is significant for other formulas except the creatinine clearance. These differences could be partly explained by the diversity of the studied group of children and the alternative reference method which was used as a GFR gold Fig. 3. (a) Relative error centiles (of bias GFR-X–GFR-I) of all studied eGFR equations for all patients (n = 417, see Figs. 1a and 2a). The centiles (5, 25, 40, 50, 60, 75, 95%) values are shown on vertical axis. On the horizontal axis the eGFR equations are presented as follows: 1 – eGFR-Scr; 2 – eGFR-CG; 3 – eGFR-MDRD; 4 – eGFR-U; 5 – eGFR-H; 6 – eGFR-B, 7 – eGFR-F, 8 – eGFR-S3M. (b) Relative error centiles (of bias GFR-X–GFR-I) of all studied eGFR equations for patients =12 years (n = 203, see Figs. 1b and 2b). The relative error centiles (5, 25, 40, 50, 60, 75, 95%) values are shown on vertical axis. On the horizontal axis eGFR equations are presented as

follows: 1 – eGFR-Scr; 2 – eGFR-CG; 3 – eGFR-MDRD; 4 – eGFR-U; 5 – eGFR-H; 6 – eGFR-B, 7 – eGFR-F, 8 – eGFR-S3M. (c) Relative error centiles (of bias GFR-X–GFR-I) of all studied eGFR equations for patients >12 years (n = 214, see Figs. 1c and 2c). The relative error centiles (5, 25, 40, 50, 60, 75, 95%) values are shown on vertical axis. On the horizontal axis eGFR equations are presented as follows: 1 – eGFR-Scr; 2 – eGFR-CG; 3 – eGFR-MDRD; 4 – eGFR-U; 5 – eGFR-H; 6 – eGFR-B, 7 – eGFR-F, 8 – eGFR-S3M.

K. Zachwieja et al. / Advances in Medical Sciences 60 (2015) 31–40

39

Table 7 The mean value, SD, median, minimum and maximum value of eGFR calculated according to all equations and number of children classified to CKD stages based on eGFR results: group I: I CKD: eGFR > 90 ml/min/1.73 m2; group II: II CKD: eGFR 60–90 ml/min/1.73 m2; group III (3, 4 and 5 CKD): eGFR  60 ml/min/1.73 m2 (there are low number of children with 4 and 5 CKD stages).

Mean value  SD Median Min–max Group I: eGFR > 90 (n) Group II: eGFR 60–90 (n) Group III: eGFR  60 (n) a

GFR-I

eGFR-Scr

eGFR-CG

eGFR-MDRD

eGFR-Ua

eGFR-H

eGFR-B

eGFR-F

eGFR-S3M

98.0  31.6 102.8 6.8–157.7 280

77.1  24.0 80.3 5.9–154.7 112

114.5  38.3 117.5 6.7–276.2 325

129.0  65.1 117.5 5.6–628.9 316

97.7  46.3 91.9 3.7–320.0 208

100.4  32.5 105.7 11.1–174.2 290

181.1  65.5 191.9 1.2–330.0 372

124.3  42.2 130.5 14.4–224.6 335

83.3  22.8 88.7 9.4–129.4 198

85

227

57

64

116

77

18

42

161

52

78

35

37

66

50

27

40

58

For GFR-U: n = 390.

standard. The equations of Schwartz were developed in a group of children with stage 3–5 of CKD and thus our findings are not surprising. The majority of the authors [32,36–38] negated the use of the C–G formula in children; only Pierrat et al. [39] share the present authors’ opinion that the C–G equation allows for a precise calculation of eGFR in a group of children >12 years of life. Even better agreement for MDRD equation was noted in this age category. It may be explicated by the anthropometric features of the analyzed group of children. The average age of the whole studied group was close to 12 years and the children with higher eGFR (>90 ml/min/1.73 m2) had higher body weight in comparison to children with lower GFR values (90 ml/min/1.73 m2. From the clinical point of view, overestimation of GFR value may be dangerous and may result in administration of an excessive medication dose, not adjusted to the kidney function or in missing deteriorating kidney function. It might be worthwhile to provide the users with detailed information on the potential error and its direction they have to deal with using a given equation. It should be also known that each of eGFR calculation method has its own limitation which has been indicated above in main conclusions based on the results of the present study. Future research should contain an attempt for the local laboratories validation of the creatinine and cystatin C assays to the international standard. It should help in standardization of the eGFR calculation. What is more, it may be essential to seek and determine the proper coefficient for eGFR formula in children at different GFR values and maybe for dedicated groups of children instead of establishing new formulas. Conflict of interests The authors report no conflict of interest. Financial disclosure The study was in part financially supported from the Jagiellonian University Medical College grant (No K/ZDS/000438).

Appendix A A.1. Methodology of determining plasma iohexol by HPLC. HPLC determinations were performed using a WATERS kit and Millenium 32 software. The distribution was performed using an Xterra column (C18 3.5 mm, 3.0 mm  150 mm) at 30 8C employing isocratic elution with the mobile phase composed of acetonitrile and water at the ratio of 4:96, at a flow of 0.36 ml/min. The analysis time was 15 min; iohexol was detected at the wavelength of 254 nm. The standard mixture was Omnipaque 300 mgJ/ml (647 mg iohexol/ml). The internal standard was iohexol compound B manufactured by LGC Promoche.

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After iohexol application to healthy patients, serum iohexol recovery was calculated in the blood samples. The following values were obtained: For 10.0 mg – 109.2%; 65.0 mg – 96.6%; 300.0 mg – 93.7%; 647.0 mg – 96.8%. For each series of determinations, residual control serum was employed with mean iohexol value of 159.89, SD = 6.48. The extraseries coefficient of variation CV was 4.05%. The intra-series coefficient of variation equaled CV = 0.69. References [1] Kidney Disease. Improving global outcomes (KDIGO) CKD work group. KDIGO 2012 clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int Suppl 2013;3:1–150. [2] Filler G, Yasin A, Medeiros M. Methods of assessing renal function. Pediatr Nephrol 2014;29:183–92. [3] Gretz N, Schock D, Sadick M, Pill J. Bias and precision of estimated glomerular filtration rate in children. Pediatr Nephrol 2007;22(2):167–9. [4] Andersen TB, Eskild-Jensen A, Frokiaer J, Brochner-Mortensen J. Measuring glomerular filtration rate in children; can cystatin C replace established methods? A review. Pediatr Nephrol 2009;24(5):929–41. [5] Harmon WE. Glomerular filtration rate in children with chronic kidney disease. Clin Chem 2009;55(3):400–1. [6] Filler G, Bokenkamp A, Hofmann W, Le Bricon T, Martinez-Bru C, Grubb A. Cystatin C as a marker of GFR – history, indications, and future research. Clin Biochem 2005;38(1):1–8. [7] Dharnidharka VR, Kwon C, Stevens G. Serum cystatin C is superior to serum creatinine as a marker of kidney function: a meta-analysis. Am J Kidney Dis 2002;40(2):221–6. [8] Filler G, Huang SH, Yasin A. The usefulness of cystatin C and related formulae in pediatrics. Clin Chem Lab Med 2012;50(12):2081–91. [9] Barnfield MC, Burniston MT, Reid U, Graham AM, Henderson M, Picton SV. Cystatin C in assessment of glomerular filtration rate in children and young adults suffering from cancer. Nucl Med Commun 2013;34(6):609–14. [10] Martini S, Prevot A, Mosig D, Werner D, van Melle G, Guignard JP. Glomerular filtration rate: measure creatinine and height rather than cystatin C! Acta Paediatr 2003;92(9):1052–7. [11] Zachwieja K, Korohoda P, Kwinta-Rybicka M, Miklaszewska M, Berska J, Bugajska J, et al. A comparison of various methods of GFR estimation in children; the experience of a single center. Nefrologia i Dializoterapia Polska 2009;13(4):234–40. [12] Haycock GB, Schwartz GJ, Wisotsky DH. Geometric method for measuring body surface area: a height-weight formula validated in infants, children, and adults. J Pediatr 1978;93(1):62–6. [13] Nilsson-Ehle P. Iohexol clearance for the determination of glomerular filtration rate – 15 years experience in clinical practice. J Int Fed Clin Chem 2002; 13(2):1–5. [14] Brochner-Mortensen J. A simple method for the determination of glomerular filtration rate. Scand J Clin Lab Invest 1972;30(3):271–4. [15] Jødal L, Brøchner-Mortensen J. Reassessment of a classical single injection 51Cr-EDTA clearance method for determination of renal function in children and adults. Part I: Analytically correct relationship between total and one-pool clearance. Scand J Clin Lab Invest 2009;69(3):305–13. [16] Schwartz GJ, Munoz A, Schneider MF, Mak RH, Kaskel F, Warady BA, et al. New equations to estimate GFR in children with CKD. J Am Soc Nephrol 2009;20(3):629–37. [17] Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976;16(1):31–41. [18] Levey AS, Coresh J, Greene T, Marsh J, Stevens LA, Kusek J, et al. Expressing the modification of diet in renal disease study equation for estimating glomerular filtration rate with standardized serum creatinine values. Clin Chem 2007; 53(4):766–72.

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