Reference Intervals for Creatinine: Enzymatic vs Modified Jaffé Method

Determination of reference intervals for serum creatinine, creatinine
excretion and creatinine clearance with an enzymatic and a
modified Jaffe
´method
Wolfgang Junge
a,b,
*, Baerbel Wilke
b
, Atef Halabi
c
, Gerhard Klein
d
a
Zentrallaboratorium des Friedrich-Ebert-Krankenhauses GmbH, Friesenstrasse 11, D-24534 Neumu
¨nster, Germany
b
Laboratorium fu
¨r Klinische Forschung GmbH, Raisdorf, Germany
c
Institut fu
¨r Klinische Pharmakologie GmbH, Kiel, Germany
d
Roche Diagnostics GmbH, Mannheim, Germany
Received 26 August 2003; received in revised form 9 February 2004; accepted 13 February 2004
Abstract
Objectives: The aim of this study was to determine the reference intervals for serum creatinine, the renal creatinine output
and the creatinine clearance (CrCl) with two new methods for accurate creatinine determination. Methods: The reference
population consisted of 252 healthy subjects (127 males and 125 females) at the age between 18 and 74 years, median 27. Urine
was collected for exactly 24 h. Creatinine in serum and urine was measured with an enzymatic assay (‘‘Creatinine Plus’’) and a
modification of the kinetic Jaffe
´reaction, named ‘‘Jaffe ´compensated’’. Results: Reference values for serum creatinine were
almost identical to previously published ones obtained with the same methods: 0.73 –1.18 and 0.55–1.02 mg/dl for males and
females, respectively, with the enzymatic, 0.72 –1.16 and 0.55 –0.96 mg/dl with the compensated Jaffe
´method (Jcomp). CrCl
values were normally distributed and showed no gender difference in contrast to some previous studies. The reference interval
for the entire group was found to be 66– 143 ml/min with the enzymatic assay and 71–151 ml/min with the chemical one.
D2004 Elsevier B.V. All rights reserved.
Keywords: Creatinine clearance; Reference intervals; Jaffe
´compensated; Enzymatic PAP method
1. Introduction
In clinical practice, creatinine clearance (CrCl) is
used as an indicator of the glomerular filtration rate
(GFR). For physiological reasons, CrCl is not iden-
tical to the ‘‘true’’ GFR because urinary creatinine
does not originate from glomerular filtration alone
but also from tubular secretion, which contributes as
much as 15–20% to the total creatinine in urine.
This condition would theoretically result in an over-
estimation of the CrCl as compared to the true GFR
by the same magnitude but the physiological inac-
curacy is—at least in healthy subjects—almost not
noticeable due to the non-specificity of the Jaffe
´
reaction. By the majority of the various Jaffe
´appli-
cations, serum creatinine, but not urinary creatinine,
0009-8981/$ - see front matter D2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.cccn.2004.02.007
Abbreviations: CrCl, creatinine clearance; Jcomp, compensated
Jaffe
´method; Jkin, (non-compensated) kinetic Jaffe ´method; Crplus,
Creatinine Plus method; MDRD, modified diet in renal disease
(study); PAP, p-amino-phenazone.
* Corresponding author. Zentrallaboratorium des Friedrich-
Ebert-Krankenhauses GmbH, Friesenstrasse 11, D-24534 Neumu
¨n-
ster, Germany. Tel.: [phone]; fax: [phone].
E-mail address: LKF_[email protected] (W. Junge).
www.elsevier.com/locate/clinchim
Clinica Chimica Acta 344 (2004) 137 – 148

is overestimated by as much as 15–25% caused by
the presence of interfering constituents, mainly pro-
teins, in blood. As a consequence, one factor largely
offsets the other one resulting in a close agreement
of the CrCl and inulin clearance values as has been
found in many studies.
A variety of methodological modifications of the
Jaffe
´reaction were suggested aiming at minimizing
the unspecificity but none of the effective ones were
suitable for routine analyses. A solution of that
problem would be the use of accurate enzymatic
creatinine assays, but these are expensive and there-
fore not popular. However, in two recent studies [1,2],
it was demonstrated that a new methodological ap-
proach to the Jaffe
´procedure results in an excellent
agreement with an enzymatic assay. The modified test
is named compensated Jaffe
´method (Jcomp).
There are two facts which cause some practical
problems if specific creatinine assays are applied in
the routine laboratory. First, only scarce data are
available for a CrCl reference interval based on such
assays and, second, the various equations proposed
for an estimation of the CrCl from the serum
creatinine [3–6] are useless because they were
derived from results obtained by the non-specific
Jaffe
´methods.
Due to the growing requirement for accurate and
specific diagnostic tests and their traceability to ref-
erence methods, we assume that both the enzymatic
and/or compensated Jaffe
´method will find increasing
acceptance. This view prompted us to determine the
reference intervals for the CrCl applying both, the
Jcomp and a new version of the enzymatic p-amino-
phenazone (PAP) method.
2. Materials and methods
2.1. Reference population
The reference population comprised 252 healthy
individuals (127 males and 125 females) from the area
of Kiel, Germany. The age ranged from 18 to 74 years
(median 27). The majority of subjects participates
regularly in phase I clinical trials.
The condition of good health was checked by a
brief physical examination, by laboratory tests (stan-
dard clinical chemistry profile and urinalysis) and by a
brief medical interview based on a questionnaire.
Subjects taking any drugs were not included, but
smokers were. Participants were instructed about
appropriate consumption and nature of meals and
drinks, but no special dietary recommendations were
given.
Twelve subjects were excluded from the CrCl
evaluation due to obvious collection errors. The
decision for elimination was based on an implausible
low creatinine excretion in relation to body weight
( < 8 mg/kg body weight) and/or a 24-h collection
volume of less than 500 ml.
2.2. Specimen collection and handling
For practical reasons, blood was taken only once
on the day before start of the 24-h urine collection.
Blood was collected using Monovettes (Sarstedt,
Germany). For preparation of plasma, lithium heparin-
ate containing Monovettes were used. Plasma and
serum were prepared simultaneously 30 min after
blood collection by centrifugation at 1800 gand
analyzed within 8 h. Urine was collected according to
a detailed written instruction on the participants’
responsibility. Collection volumes ranged from 500
to 4830 ml (median: 1560 ml). If not analyzed
immediately, the specimens were stored in a refriger-
ator at 2–8 jC until analysis, which was performed
within 48 h.
CrCl values were calculated according to the
commonly used clearance formula Cl
Crea
=(C
U
Vo l
U
)/(C
Se
t) where C
U
and C
Se
are the creatinine
concentrations in urine and serum in mg/dl, Vol
U
= ur-
ine collection volume in ml, t= urine collection time
in minutes, and corrected for standard body surface
area of 1.73 m
2
, which was calculated from height and
Fig. 1. Standardization of the Jkin and the Jcomp methods. (a) Comparison of the Jaffe
´rate-blanked method (Jkin = y) using a preliminary
calibrator set-point as determined with the reference method and the ID-MS reference method (x) in six human pool sera. Final c.f.a.s. set points
are calculated as follows: non-compensated: set point = (preliminary set pointintercept)/slope; compensated: set point = preliminary set point/
slope. (b) Comparison of the Jaffe
´rate-blanked compensated method (Jcomp = y) using the final calibrator set-point and the ID-MS reference
method. Compensation ( = subtraction of 0.3 mg/dl) is performed after analysis of the patient samples only.
W. Junge et al. / Clinica Chimica Acta 344 (2004) 137–148138

W. Junge et al. / Clinica Chimica Acta 344 (2004) 137–148 139

weight according to the equation of Dubois and
Dubois [7].
2.3. Methods, reagents and instruments
Serum, plasma and urinary creatinine was mea-
sured by the conventional kinetic Jaffe
´(Jkin) meth-
od, the new Jcomp method and by a modified
version of the established enzymatic creatinine PAP
method called Creatinine Plus (Crplus). All reagents,
including calibrators and controls were provided by
Roche Diagnostics (Mannheim, Germany). Measure-
ments were carried out on a Hitachi 717 analyzer
throughout.
The Crplus method is based on the same reaction
sequence as the creatinine PAP method consisting of
four consecutive enzymatic steps via creatininase,
creatinase, sarcosine oxidase and peroxidase [8], but
using a different chromophore (2,4,6-triiodo-3-
hydroxybenzoic acid), and a modified detergent com-
position. Endogenous creatine is completely removed
during a preincubation step. Lipemic samples are
rapidly cleared by the detergent system, which is
effective at 37 jC only. Bilirubin interference is
eliminated by the combined effect of detergents and
the use of a new chromophore. The concentration of
reagents was optimized for completion of the reaction
after 10 min. The assay was standardized with a
isotope dilution mass spectrometry method for creat-
inine. Measuring range from lower detection to dilu-
tion limit is 0.03–30 mg/dl creatinine.
In both, the Jkin and the Jcomp method a reduction
of bilirubin interference is achieved by means of a
rate-blanked mode during the preincubation period.
To further increase the specificity, an additional cor-
rection is made in the Jcomp method for non-creati-
nine Jaffe
´-reactive components, e.g. proteins, glucose,
ascorbic acid, ketone bodies, by subtraction of 0.3 mg/
dl from the result obtained for all Jaffe
´-reactive serum
components. This numerical term reflects the average
contribution of the serum matrix to the Jaffe
´reaction
signal as estimated in a comprehensive multicenter
study [9] and later confirmed by Mazzachi et al. [1].
This means that creatinine-free serum samples will be
measured by the Jkin method as having on the average
0.3 mg/dl creatinine; the existence of such a ‘‘blank
value’’ has been reported earlier [10,11], but never
been quantified.
The calibrator (c.f.a.s.) set points for both Jaffe
´
procedures are determined by measuring several hu-
man serum pools with the reference and the Jkin
method. As preliminary set point the creatinine con-
centration of the calibrator as determined with the
reference method is used. Due to the presence of non-
creatinine Jaffe
´reactive constituents in the calibrator
matrix, a distinct difference is found between the
matched data pairs as illustrated in Fig. 1a.The
correct set point for the Jkin method is then calculated
from the regression equation of that curve.
If the Jkin values of the serum pools are divided by
the slope of the regression line shown in Fig. 1a and
plotted versus the reference method values, the result-
ing regression curve parallels the identity line, but in a
distance which is determined by the non-creatinine
Jaffe
´reactants in the serum pools (Fig. 1b). Division
of the preliminary calibrator value by the slope of the
regression line in Fig. 1a then defines the Jcomp set
point. However, the results of serum samples obtained
with this set point have finally to be corrected for the
non-specificity of the Jaffe
´method by subtraction of
0.3 mg/dl.
The measuring range for the Jcomp method is
0.2– 25 mg/dl.
2.4. Statistics
Reference intervals were calculated using an Excel
compatible non-parametric program for descriptive
statistics. Significance was tested with a ‘‘Wilcoxon
two-sample test’’ for the differentiation between the
male and the female group and with a ‘‘Wilcoxon
two-sample test in case of dependent samples’’ for
the differentiation between the results obtained with
the two creatinine assays for the same reference
groups. Regression analysis in method comparisons
was done with non-parametric Passing/Bablok calcu-
lation and a linear regression model to establish
correlation coefficients.
3. Results and discussion
3.1. Analytical variability
Standard deviations of both Jaffe
´methods are the
same, CVs necessarily higher for the Jcomp due to the
W. Junge et al. / Clinica Chimica Acta 344 (2004) 137–148140

Fig. 2. Comparison between the enzymatic ‘‘Plus’’ (x) and the Jaffe
´method for the determination of serum creatinine in the reference group
(n= 240) on Hitachi 717. (a) y= Jaffe
´rate-blanked compensated (Jcomp). (b) y= Jaffe ´rate-blanked without compensation (Jkin).
W. Junge et al. / Clinica Chimica Acta 344 (2004) 137–148 141