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Determination of serum creatinine by Jaffe method and how to calibrate to

eliminate matrix interference problems

Article  in  Clinical Chemistry and Laboratory Medicine · February 2008

DOI: 10.1515/CCLM.2008.224 · Source: PubMed

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 Article in press - uncorrected proof
Clin Chem Lab Med 2008;46(8):1127–1133
2008 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2008.224
Determination of serum creatinine by Jaffe method and how
to calibrate to eliminate matrix interference problems
Vratislav Chromý1,*, Kateřina Rozkošná2 and
Pavel Sedlák3
1 Jaffe method; Jaffe serum creatinine.
Institute of Chemistry, Faculty of Science, Masaryk
University, Brno, Czech Republic
2
Diagnostics Research Department, Pliva-Lachema
Diagnostika, Brno, Czech Republic
3
Department of Clinical Biochemistry, Municipal
Hospital, Čáslav, Czech Republic
Abstract
Background: The calibration of Jaffe method for
serum creatinine using one serum-based standard
complemented with artificial matrix compensating
standard’s Jaffe-interfering substances allows two-
point calibration with results well comparable with
enzymatic methods.
Method: Spectrophotometry was used.
Results: Jaffe procedures compensating serum/plas-
ma intereferents by subtracting a constant amount of
creatinine poorly overcompensate creatinine in chil-
dren. Two-point calibration with a pair of primary
serum standards certified by the reference measure-
ment procedure (isotope-dilution, mass spectrometry)
or with a pair of secondary standards linked to pri-
mary materials could provide results well agreeable
with enzymatic determination. Such a calibration
comprises an absorbance offset corresponding to oth-
er-than-creatinine Jaffe-interfering chromogens pres-
ent in standards in the calibration line, while a
two-point calibration combining one standard with
physiological saline/water always grossly distorts cal-
ibration line. We calculated/prepared artificial serum
matrices capable of compensating Jaffe-interfering
chromogens in serum standards. The combination of
even one standard with its artificial matrix also ena-
bles two-point calibration with practically the same
results as with a pair of primary standards.
Conclusions: A two-point calibration of Jaffe method
for serum creatinine combining only one serum stan-
dard with creatinine solution matching standard’s
allow matrix-present interferents present results well
comparable with enzymatic determination, providing
the standard is attested/linked to the reference meas-
urement procedure.
Clin Chem Lab Med 2008;46:1127–33.
*Corresponding author: Prof. Dr. Vratislav Chromý,
Institute of Chemistry, Faculty of Science, Masaryk
University, Komenského 2, Brno, Czech Republic
Phone: q420-549494555, E-mail: vrch@chemi.muni.cz
Received February 18, 2008; accepted April 10, 2008
2007/88
Keywords: artificial serum matrix for calibration of
Jaffe method; compensation of serum interferents in
Introduction
Most chemical methods for measuring creatinine are
still based on the reaction with alkaline picrate (Jaffe
1886). Creatinine reacts with picrate ion in an alkaline
medium to yield an orange-red adduct (1). The reac-
tion is not specific and many compounds are known
to produce Jaffe-like chromogens, including protein,
glucose, ascorbic acid, ketone bodies, pyruvate, gua-
nidine, cephalosporins (2) and bilirubin, the last one
being the negative interferent (3). The degree of inter-
ference caused by these serum-present compounds
(i.e., by serum matrix) is also dependent on the spe-
cific reaction conditions chosen. Manual methods
have traditionally been equilibrium methods with a
reaction time of approximately 15 min at room
temperature.
Kinetic assays were developed for analyses with
improved specificity, because some Jaffe-like adducts
with picrate are formed rapidly during the first 20 s
after mixing reagent and sample (such as acetoace-
tate) and some react slowly within 80–100 s after mix-
ing (such as protein) and kinetic measurement
between 20–60 or 20–80 s was attributed predomi-
nantly to the creatinine-picrate reaction (4).
The reaction specificity is also affected by picrate/
alkali concentration. The Jaffe reaction is pseudo-first
order with respect to picrate concentration up to
30 mmol/L and hydroxide concentration more than
0.5 mol/L (4). Ferrous/ferric ions are known to catalyse
oxidation reactions. The system with ferricyanide was
used in Jaffe reaction to catalyse the oxidation of
interfering bilirubin to biliverdin (3). Similarly, cupric/
cuprous ions and Cu(II)-tartrate were also used as a
catalyser in Jaffe reaction instead of ferricyanide (5).
Several enzymatic methods have been developed
for measuring creatinine, but they are also not inter-
ference-free (4): bilirubin interference is usually
solved by adding ferricyanide into a reagent, potential
interference caused by ascorbic acid can be overcome
by inclusion of ascorbate oxidase and the influence of
intermediate creatine and urea is usually solved in the
first enzyme sequence by a preincubation with crea-
tininase. The reference measurement procedure
(RMP) employing isotope-dilution with mass spec-
trometry (ID-MS) (6) is used for creatinine assay in
Standard Reference Material (SRM) calibrators.
Nevertheless, Jaffe kinetic reaction with so-called
window measurement (selected reaction time meas-
 Article in press - uncorrected proof
1128 Chromý et al.: Calibration of serum creatinine determination by Jaffe method

urement within 20–80 s) still remains the leading sera with results well comparable with enzymatic
method for both serum and urine creatinine (4), even methods.
though it is known to overestimate ‘‘true plasma/
serum creatinine by approximately 20% at physiolog-
ical values’’, which is now undoubtedly known to be Materials and methods
caused by nonspecificity of Jaffe reaction and thus
still persisting problems which interferents are always Equipment
present in a serum matrix (4, 7, 8). The main serum
A Shimadzu UV-160 spectrophotometer (Shimadzu, Tokyo,
interferents in Jaffe reaction are proteins (9), which
Japan) with tungsten lamp, 10"0.01 mm light-path quartz
content differ significantly wcf. (10)x in adults and in cells and 6-position cell-rack changer heated to 258C or
children. Unfortunately, reaction time recommended 37"0.18C by a Peltier device (Shimadzu), with wavelength
for Jaffe procedure (4) has its lag phase and reaction 492"0.4 nm was employed. A Shimadzu spectrophotometer
time too short to be used even for small series of cre- was used only for manual Jaffe procedure with one-part re-
atinine samples made on analysers which are usually agent to obtain the data given in Figure 1 and Table 1.
preset to measure in intervals within 60–180 s. A Hitachi 911 Automatic Analyser (Hitachi Ltd., Tokyo,
Some producers of in vitro diagnostics (analytical Japan) with concave diffraction grating was employed. All
kits, standards, analysers, etc.) solved interference analyses were made at 37"0.28C at wavelengths 505"2 nm
and 570 nm (auxiliary) in 6-mm reaction cells with automatic
problems in their Jaffe-based procedures in analyser
recalculation of absorbance to 10 mm. A Hitachi analyser
software by subtracting a constant 26 mmol/L of cre-
was used for all automated Jaffe and enzymatic measure-
atinine as ‘‘an average value (11) of Jaffe-positive ments involving two-part reagents. Data are given in Figure
compounds in a serum matrix from resulting creati- 2 and Tables 2 and 3.
nine concentration estimated by their compensated
Jaffe method’’. According to Junge et al. (12), ‘‘an off-
set of 21 mmol/L of creatinine corresponds to the dif-
ferences between a modified Jaffe and enzymatic
methods’’. Wuyts et al. (9) compared Jaffe compen-
sated/uncompensated procedures for serum creati-
nine with enzymatic methods in more detail and
found a big difference in children caused mainly by
interfering proteins.
While the calibration of urine creatinine with cre-
atinine solutions seems to be without difficulties (no
proteins or traces of proteins), the improvement of
serum/plasma creatinine measurement is still the
focus of analytical and kidney function programmes
(9, 12). According to Myers et al. (13), ‘‘standardiza-
tion of calibration does not correct for analytical inter-
Figure 1 Calibration rate-curves with serum standards and
ferences (nonspecificity bias) and nonspecificity
creatinine solutions.
problems associated with some of the routine meth- One-reagent manual Jaffe procedure (MJ), time measure-
ods must be addressed’’. ment between 60 and 168 s and temperature of 378C. Full
To be explicitly defined, every calibration line must line with black squares: two-point calibration with SRM 967
have at least two calibration points. Two-point cali- LI–LII, line prolongation to x-axis intersects y/x-axes showing
bration of Jaffe reaction with two serum standards overall matrix effect as mAU/creatinine. Dashed line with
with the true creatinine value (standards attested by white circles: two-point calibration with LyoC-ASM; line pro-
longation to x-axis intersects y/x-axes showing ASM as
RMP or linked to SRM) automatically compensates
mAU/creatinine. Full line with black triangles: three-point
Jaffe interfering substances implementing them as an calibration with solutions of creatinine 50, 100 and 300
absorbance offset/nonspecificity bias into the calibra- mmol/L.
tion equation/line. Unfortunately, a lot of producers of
diagnostics supply their customers with only one
Table 1 An overall effect of SRM 967 LIqLII matrices (as
serum-based multi-purpose standard/calibrator and a creatinine, mmol/L).
user is forced to combine such a standard with water/
physiological saline to achieve two-point calibration Reaction time, s Temperature, 8C
which badly distorts Jaffe calibration line eliminating
25 37
its unavoidable absorbance offset.
We tested several approaches in serum creatinine 20–80 31 34
calibration (14) using a modified kinetic Jaffe method 30–180 33 37
60–168 32 38
with Cu(II)-tartrate as a catalyser for bilirubin oxida-
60–150a – 36a
tion (5). We believe we found a simple two-point cal- Average 32 36
ibrating procedure combining one serum standard Range 31–33 34–38
with its artificial serum matrix (ASM) compensating Manual JP (Jaffe reagent and procedure by Pliva-Lachema
standard’s matrix-present Jaffe interferents. Such cal- Diagnostika)-one-reagent procedure. aAutomated JP-two-re-
ibration could be used for both adult and children’s agent procedure.
 Article in press - uncorrected proof
Chromý et al.: Calibration of serum creatinine determination by Jaffe method 1129

Materials

Creatinine was from Sigma-Aldrich (Munich, Germany;

C4255, anhydrous G99%, Mrs113.12). For human sera,
patient sera and serum pools were used in this study. A
more detailed description of used sera is given in the legend
to Figure 2 and in the footnotes to Tables 2 and 3. Sera from
a group of 30 children used for comparing enzymatic and
Jaffe method (Table 3) was prepared from samples collected
in a children’s hospital before analysis of serum creatinine
(i.e., this group also contained Jaffe-uncompensated sam-
ples). Sera from a group of 18 children (10 samples, aged
1–5 years and 8 samples, aged 6–10 years) used for analy-
ses, presented in Figure 2, contained only samples analysed
in advance by compensated Jaffe procedure and contained
only samples with creatinine )18 mmol/L, all lower samples
being cut out by an analyser software program set to avoid
negative results.

Figure 2 Creatinine in children’s sera, comparison of Jaffe

and enzymatic methods. Reagents
Two-reagent automated Jaffe procedures, JP (uncompen-
Reagent grade chemicals were used throughout this study.
sated) and JR (enzymatic creatinine reagent and procedure
by Roche Diagnostics compensated) vs. EP. Time measure- Physiological saline (PS) was prepared by dissolving 9 g of
ment of 60–168 and 60–150 s, respectively, temperature of NaCl in distilled water in a 1000-mL volumetric flask. Creat-
378C. The same group of sera (ns18) from a children’s hos- inine stock solution of 1000 mmol/L was prepared by dis-
pital analyser, selected by their compensated JR procedure solving 113.12 mg of creatinine in a 1000-mL volumetric flask
(10 samples, aged 1–5 years plus 8 samples, aged in PS and was further diluted with PS to obtain desired
6–10 years). Enzymatic/uncompensated JPs were calibrated concentration.
with a pair of LyoC-PS and LyoC-ASM, respectively. Circles:
JP vs. EP. Squares: compensated JR vs. EP. Creatinine Calibrators We used the National Institute of Standards
average/mean values found (mmol/L): EP 52.9/31–85; and Technology SRM 967 as primary creatinine standards
EP 51.4/27–83; JR 34.5/18–63. throughout this study (15): frozen human serum level I (LI)
was certified for creatinine 66.5"1.9 and level II (LII) for
Table 2 Preliminary experiments. The effect of calibration 346.2"7.3 mmol/L.
on serum creatinine assay by Jaffe method (mmol/L).
Lyonorm calibrator (LyoC) and its artificial serum matrix
Calibration mode/calibrator Creatinine found (ASM) We recalibrated/reset the concentration of creatinine
concentration, mmol/L in Pliva-Lachema universal human multi-purpose serum-
Low pool High pool
based standard/calibrator (Pliva-Lachema Diagnostika, Brno,
Two-point/PS-LI (66.5) 74 208 Czech Republic) against SRM using automated Jaffe proce-
Two-point/PS-LII (346.2) 123 330 dure (JP) to 331"12 mmol/L. We determined that the total
Two-point/LI-LII (66.5 and 346.2) 86* 323* amount of Jaffe interferents present in LyoC serum matrix
Three-point/creatinine solutionsa 125 362 corresponds to 38"2 mmol/L of creatinine and we prepared
Two-point/ASM-LyoC (325.0)b 85* 328* LyoC-matching ASM as a solution containing 38 mmol/L of
Automated JP-two-reagent procedure. Both pools contained creatinine. A pair of LyoC-ASM and/or LyoC-PS was used for
an equal mixture of 20 male/female adult sera. *Probably the two-point calibration for both Jaffe and enzymatic methods,
true serum creatinine free of interferents. aCreatinine solu- respectively. As an example, we also prepared and used
tions of 50, 100 and 300 mmol/L. bASM contained 38.0 ASM based on glucose and bovine serum albumin solutions,
mmol/L of creatinine. respectively (see Results).

Table 3 Comparison of Jaffe and enzymatic methods (as creatinine, mmol/L).

Procedure Adults Children

1: JP-ER 2: JP-EP 3: JR-EP 4: JP-EP 5: JP-EP

Average, Y 77 219 209 40 35
Range 45–150 103–659 100–629 8–47 6–50
Average, X 81 217 205 35 38
Range 47–144 97–649 104–659 7–44 8–49
Automated two-point calibration using two-reagent procedures combining Jaffe and enzymatic methods. For adults, proce-
dure 1: sera from a group of 100 male/female (aged 20–70 years) with normal/elevated creatinine levels. Both calibrations
with SRM LI–LII (rs0.993; ys0.996x–3.6). Procedure 2: sera from a group of 60 male/female (aged 17–64 years) with signif-
icantly elevated creatinine levels. EP calibration with LyoC-PS, JP with LyoC-ASM (rs0.991, ys1.007x–4.7). Procedure 3: the
same sera as in 2. EP calibration with SRM LI–LII, JR compensated data by subtracting 26 mmol/L of creatinine were taken
from the hospital laboratory (rs0.992; ys0.979xq0.5). For children, sera from a group of 30 children (20 samples, aged 1–5
years and 10 samples, aged 6–10 years). Procedure 4: both calibrations with SRM LI–LII (rs0.960, ys0.989x–4.5). Procedure
5: EP calibration with LyoC-PS (rs0.967; ys0.988xq2.4), JP calibration with LyoC-ASM.
 Article in press - uncorrected proof
1130 Chromý et al.: Calibration of serum creatinine determination by Jaffe method
Jaffe reagent, Pliva (JP) A two-part reagent analytical kit
was used, with Creatinine Liquid 500, Cat. No. 10010227
(Pliva-Lachema Diagnostika) (Reagents; R1: NaOH 0.24
mol/L; R2: picric acid 26 mmol/L). JP was used both in man-
ual and automated procedures. In this kit, Cu(II)-tartrate is
used to catalyse bilirubin oxidation (5). Performance data
(serum): limit of detection 2.33 mmol/L, low limit of quanti-
fication 7.09 mmol/L, working range 7.09–3000 mmol/L, bias
–2.54% and –1.01% (for 80 and 334 mmol/L of creatinine,
respectively), precision (intra-/inter-assay): 1.8%/2.16%, cor-
relation with enzymatic creatinine Pliva (ns96, rs0.999,
ys1.008x–6.4 mmol/L).
Jaffe reagent, Roche (JR) A two-part reagent analytical kit
was used, with CREA, Creatinine Jaffe method, rate-blanked
and compensated, Cat. No. 11875418 (Roche Diagnostics,
Mannheim, Germany) (Reagents; R1: NaOH 0.8 mol/L; R2:
picric acid 25 mmol/L; see automated Jaffe procedure
Roche). Performance data (serum): working range
18–2210 mmol/L, low limit of quantification 18 mmol/L,
precision (inter-/intra-assay): 0.7%/2.3%, correlation with
enzymatic creatinine Roche (ns50, rs0.998,
ys1.014x–0.07 mmol/L).
Enzymatic creatinine, Pliva (EP) A two-part reagent analyt-
ical kit was used, with Creatinine Enzymatic Liquid 204, Cat.
No. 10010231 (Pliva-Lachema Diagnostika) (Reagents; R1:
Good’s buffer pH 7.5, 25 mmol/L, creatinase G12 kU/L, sar-
cosine oxidase G8 kU/L, ascorbate oxidase G2 kU/L, catalase
G200 kU/L, indicator N-ethyl-N-sulphopropyl-m-toluidine
(ESPMT) 0.47 mmol/L, detergent -1%, gentamycin -0.1%;
R2: Good’s buffer pH 7.5, 100 mmol/L, creatininase G300
kU/L, peroxidase G20 kU/L, 4-aminoantipyrine 2.95 mmol/L,
detergent -0.5%, sodium azide G0.1%). Performance data
(serum): limit of detection 1.50 mmol/L, low limit of quanti-
fication 4.60 mmol/L, working range 4.6–5700 mmol/L, bias
–3.60% and –2.20% (for 90 and 360 mmol/L of creatinine,
respectively), precision (intra-/inter-assay): 1.75%/3.14%, cor-
relation with other enzymatic creatinine (ns98, rs0.999,
ys1.051x–14.9 mmol/L).
Enzymatic creatinine, Roche (ER) A two-part reagent analyt-
ical kit was used, with Creatinine plus, Cat. No. 11775642 (Roche
Diagnostics) (Reagents; R1: N-tris(hydroxymethyl)methyl-3-
aminopropanesulphonic acid (TAPS) buffer pH 8.1,
30 mmol/L, creatinase G333 mcat/L, sarcosine oxidase
G133 mcat/L, ascorbate oxidase G33 mcat/L, indicator 3-
hydroxy-2,4,6-triiodobenzoic acid (HTIB) 5.9 mmol/L, deter-
gent and stabiliser; R2: TAPS buffer pH 8.0, 50 mmol/L,
creatininase G500 mcat/L, peroxidase G16.7 mcat/L, 4-ami-
noantipyrine 2.0 mmol/L, potassium hexacyanoferrate
163 mmol/L, detergent and stabiliser). Performance data
(serum): working range 2.7–2652 mmol/L, low limit of quan-
tification 2.7 mmol/L, precision (inter-/intra-assay): 0.8%/
2.1%, correlation with HPLC method (ns64, rs0.999,
ys0.989xq0.036 mg/dL).
Procedures
All measurements carried out manually were performed in
triplicate with one-part Jaffe reagent (MJ) at 258C and 378C,
respectively and were used only for the calculation/deter-
mination of Jaffe-interfering substances in standards and in
two types of control sera (see Figure 1 and Table 2).
Manual Jaffe procedure, Pliva (MJ) The manual Jaffe pro-
cedure, Pliva (MJ), was used only in one-reagent mode. MJ-
reagent was prepared from JP by mixing 4 parts of R1 with
1 part of R2, see Jaffe reagent JP. In a cell heated to the
selected temperature, mix 1.00 mL of MJ reagent with
0.050 mL of a sample and register immediately the rate curve
against picrate reagent with PS at 492 nm. Calculate the dif-
ference DAs(A2 –A1) for reaction time between 20–80 and
60–168 s, respectively.
Automated Jaffe procedures Automated Jaffe procedures
were used only in two-reagent mode with both Jaffe/enzy-
matic methods. Analyses, which were carried out in dupli-
cate, were used for all data presented in Tables 2 and 3 and
in Figure 2. The analyser was programmed accordingly for
two-reagent procedure JP and JR, respectively, with a wave-
length of 505 and 570 nm, respectively.
Automated Jaffe procedure, Pliva (JP) Mix 0.015 mL of a
sample with 0.240 mL of R1 and incubate for 5 min, add
0.060 mL of R2 and after 60 s read the absorbance A1 and
after 168 s read the absorbance A2, reaction time measure-
ment between 60 and 168 s.
Automated Jaffe procedure, Roche (JR) Mix 0.006 mL of
sample with 0.250 mL of R1 and incubate for 5 min, add
0.125 mL of R2 and after 60 s read the absorbance A1 and
after 150 s read the absorbance A2, reaction time measure-
ment between 60 and 150 s. Rate-blanked and compensated
JR and Roche calibrator was used only in a hospital labo-
ratory which supplied us with patient sera together with their
creatinine values.
Automated enzymatic procedure, Pliva (EP) Mix 0.018 mL
of sample with 0.81 mL of R1, incubate for 5 min and read
the first absorbance (A1), add 0.270 mL of R2, incubate for
10 min and read the second absorbance (A2) and calculate
the difference. Wavelength: 546 nm.
Automated enzymatic procedure, Roche (ER) Mix 0.006 mL
of a sample with 0.65 mL of R1, incubate for 5 min and read
the first absorbance (A1), add 0.20 mL of R2, incubate for
5 min and read the second absorbance (A2) and calculate the
difference. Wavelength: 546 nm.
Results
Preliminary experiments
Calibration lines and equations in Figure 1 were
obtained by JP-one-reagent manual procedure and
were used to calculate the amount of serum/matrix
interfering substances (Table 1) present in used stan-
dards. Figure 1 shows the overall effect of interfering
substances in a pair of SRM both as absorbance/
creatinine nonspecificity bias. Compared with a curve
constructed with creatinine solutions, SRM curve is
shifted to a higher absorbance which is caused by
other-than-creatinine positive chromogens present in
SRM matrices. All calibration lines combining a
serum standard and/or standards with water or PS as
zero will distort calibration curves, a distortion being
dependent on the standard and the calibration mode
chosen. Figure 1 also shows that a pair of LyoC-ASM
enables determination of serum creatinine with accu-
racy well comparable with SRM, bringing the calibra-
tion line tightly to that constructed with a pair of
primary standards.
 Article in press - uncorrected proof
Chromý et al.: Calibration of serum creatinine determination by Jaffe method 1131
In our preliminary experiments (Table 2), we tested
various standards/calibration modes on two serum
pools with low/high level of creatinine using JP-two-
reagent automated procedure. Jaffe method was cal-
ibrated either with a pair of SRM (15) or with a
secondary standard LyoC paired with its ASM or with
solutions of pure creatinine. Calibration with pure cre-
atinine and also with a serum standard combined
with PS/water used to establish zero will always dis-
tort calibration line and overestimate true serum cre-
atinine, the overestimation being major in serum
samples with low creatinine.
Jaffe-like interferents in standards
A calibration line in Figure 1, constructed with a pair
of SRM (LI and LII) enables estimation of an overall
effect of matrices-present interferents as its intersec-
tion with y/x-axes or calculation of either the overall
(Table 1) or even individual matrix effects combining
appropriate equations for SRM with those for creati-
nine solutions. We calculated the following individual
matrix effects (as creatinine, mmol/L): SRM-LI ;13,
SRM-LII ;23, LyoC ;38, and two control sera, low
;9 and high ;40. As expected, the higher matrix
effect was always found in multi-purpose standards/
controls spiked generally to higher levels. An overall
SRM matrix could also be found by an analyser using
two-reagent procedures: a Hitachi analyser was
two-point calibrated with a pair of SRM, then several
creatinine solutions with known concentration
(10–60 mmol/L) were analysed as ‘‘unknown sam-
ples’’. The zero intersection between two straight
lines which the analyser printed as found/known cre-
atinine in ‘‘unknown samples’’ corresponded to the
interferents present in SRM matrices. A matrix cor-
responding to an absorbance matching ;36 mmol/L
of creatinine could also be simulated by solutions of
glucose (;280 mmol/L) or by bovine serum albumin
(;109 g/L). However, ASM with creatinine is the best
because of the same reaction rate. Proteins (9) are the
major interferents in Jaffe method. It is easy to cal-
culate that an average offset of 21 mmol/L of Jaffe-
serum-creatinine found by Junge et al. (12)
corresponds to the interference caused by ;64 g/L of
protein (as bovine albumin).
Reaction time measurement
We were not able to confirm any positive effect of a
reaction time on the compensation of serum interfe-
rents in Jaffe kinetic methods (Table 1). All matrix-
present interferents, even when measured at different
times, were situated in a very narrow range. The
greater matrix effect found at 378C is caused by tem-
perature-increased reaction velocity of some interfe-
rents. Moreover, DA measured within the generally
recommended 20–80 s (4) is low. In this respect, it is
more convenient to read the absorbance within
;60–180 s, where DA is approximately 50% higher
and which timing is also convenient for most of the
automatic analysers.
Creatinine determined by Jaffe and enzymatic
methods
The two-point calibration of Jaffe method with a pair
of SRM and/or with LyoC-ASM we used in preliminary
experiments compensates for serum matrix effects
(Table 2). We also verified it in more detail by two
enzymatic procedures, EP/ER, on two groups of adult
sera, the first one with normal/elevated creatinine, the
second one with greatly elevated creatinine level
(Table 3, procedures 1 and 2, see footnotes). It is evi-
dent that the enzymatic procedures can be two-point
calibrated both with a pair of SRM and with one sec-
ondary standard (such as LyoC) complemented with
PS. A Jaffe method can be calibrated either with a pair
of serum standards or with one standard comple-
mented with its ASM. Averages/ranges of creatinine
concentration and also regression lines/relationships
found did not show notable differences. We used the
same group of samples as in procedure 2 also for
comparing enzymatic method with compensated JR
procedure using compensation by subtracting the
constant amount of 26 mmol/L of creatinine from all
samples (Table 3, procedure 3). We were surprised by
the very good results we found with compensated
Jaffe method (11) in adults.
Special attention was given to the analysis of sera
from children (Table 3, procedures 4 and 5). Results
achieved by uncompensated Jaffe and enzymatic cre-
atinine correlate closely and regression data are quite
acceptable. It is evident a two-point calibration, either
with a pair of SRM and/or with a pair of LyoC-ASM,
respectively, used for uncompensated Jaffe assay in
adults that functions well even in children. In our sec-
ond experiment with samples from children, we tried
to compare compensated (JR) and uncompensated
(JP) procedures with enzymatic creatinine (Figure 2).
For such comparison, we used sera in which creati-
nine was analysed in advance by compensated Jaffe
procedure in a hospital laboratory, which means this
group contained only sera with creatinine )18
mmol/L. A correlation graph comparing enzymatic
and compensated Jaffe assays indicates overcompen-
sation in creatinine of approximately ;16 mmol/L in
children. The correlation between EP/JP procedures is
quite good and a two-point calibration used allows
much better results in children, compared with com-
pensated Jaffe method.
Discussion
SRMs used in this study were based on two frozen
SRMs which are intended (15) ‘‘primarily for use in
evaluating the accuracy of procedures for the deter-
mination of creatinine in human serum and for use in
validating secondary reference materials’’. We used
SRMs as primary standards for all procedures com-
pared, for creatinine validation in the secondary stan-
dard LyoC and also for the calculation/determination
of the Jaffe-matrix-effect in some other serum mate-
rials. Creatinine in SRM, being certified by RMP (6), is
supposed to be free of interferences caused by Jaffe-
 Article in press - uncorrected proof
1132 Chromý et al.: Calibration of serum creatinine determination by Jaffe method
like serum constituents, while the absorbance found
by Jaffe procedure with SRM and/or with serum
samples should also include the effect of other-than-
creatinine Jaffe-like chromogens as an absorbance
offset.
Our study proved that two-point calibration of a
Jaffe method using a pair of serum standards with
true creatinine value and/or a secondary standard
paired with its artificial matrix yield results well com-
parable with enzymatic methods. Such two-point cal-
ibration respects natural and unavoidable absorbance
offset representing nonspecificity Jaffe bias and
incorporates it into the calibration equation/line. Any
calibration combining a serum standard with PS/
water closes calibration line incorrectly to zero result-
ing in falsely increased serum creatinine.
In agreement with Wuyts et al. (9), we confirmed
that the greatest errors/nonspecificity bias in Jaffe
methods is caused by proteins. Reference interval (10)
for total serum protein varies greatly in adults and its
variation in newborns is even greater ("15% and
"35% from the mean value, respectively). Moreover,
children’s sera and particularly sera of toddlers can
contain only a fraction of creatinine compared with
adults wcf. (10)x. In this respect, to find a universal
compensation constant for compensated Jaffe meth-
od valid for both adults/children is impossible. While
compensated Jaffe method (11) applied to adults
yielded serum creatinine well comparable with enzy-
matic procedure, its application to children poorly
overcompensates creatinine in children and a cut-out
filter implemented to compensated Jaffe procedures
even disallows the determination of a real low creat-
inine level in children within 1–5 years of age. In fact,
the overcompensation we found in creatinine levels
in children must be even greater, because our tested
group contained only selected sera from children with
creatinine levels )18 mmol/L. Without such an ana-
lyser made correction, a lot of creatinine levels in chil-
dren found by compensated Jaffe procedure should
have been negative. Both types of two-point calibra-
tion we used in this study for uncompensated Jaffe
method function well for both adults and children.
Unfortunately, the scatter of serum creatinine levels
in children will always be large because of a generally
very low DA measured with children’s samples.
The daily calibration with a pair of SRMs is expen-
sive and is not common. We used SRMs in our study
only to compare various creatinine procedures, to
determine/calculate an absorbance offset/nonspecifi-
city bias caused by Jaffe-interfering substances in
serum standards and to prepare matched artificial
serum matrices.
Thus far, many companies producing diagnostics
offer only one type of multi-purpose serum-based
standard/calibrator insufficient for two-point calibra-
tion of Jaffe method, and we hope our study would
help to solve calibration problems still connected with
Jaffe procedure. Unfortunately, the concentration of
creatinine in all multi-purpose calibrators is usually
set too high. For reliable serum creatinine glomerular
filtration rate and for the treatment of chronic kidney
diseases, it would be more suitable to calibrate
serum/plasma creatinine with standards in which cre-
atinine levels range between 50 and 150 mmol/L. It
would also be more convenient for the standardised
calibration of sera for both children and adults.
Acknowledgements
We would like to express our gratitude to Drs. Ludmila
Malášková and Miroslava Beňovská, Department of Clinical
Biochemistry, Faculty Hospital, Brno-Bohunice, Czech
Republic, for patient sera analysed by their Jaffe-based, rate-
blanked and compensated Roche analytical method. We
would also like to thank Dr. Jan Havliš, Department of Func-
tional Genomics and Proteomics, Institute of Experimental
Biology, Faculty of Science, Masaryk University, Brno, for
advice and help during manuscript preparation. V.C. would
like to express thanks to PLIVA-Lachema Diagnostika, Barr
Group, Brno, Czech Republic, for financial support of this
work.
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