CLIN.Cl-EM. 25/6, 1005-1008 (1979)
Evaluationof the Beckman CreatinineAnalyzer
Jocelyn M. Hicks,1’2 Mariet Iosefsohn,1 and Susan A. Lewis1
We compared results obtained with the Beckman Creatinine Analyzer (Beckman Instruments, Inc.) to those with
the GEMSAEC Centrifugal Analyzer (Electro-Nucleonics,
the Jaff#{233}
reaction. The total color development appears to be
made up of three phases: firstly, an accelerated rate phase,
which includes any disturbance created by the injection of a
Inc.) and with a manual method for determination of creatinine in serum and urine. Linearity, stability, recovery,
and precision were evaluated. Interference from bilirubin,
protein. lipemia, uric acid, and hemolysis is negligible, but
acetone and acetoacetate can interfere.
sample
Additional Keyphrases: intermethod
the slow-reacting
uga! analyzer
neonatal
gency determination
#{149}
comparison
and pediatric chemistry
#{149}
centrif-
#{149}
emer-
Determination
of creatinine in serum and urine has been
a particular problem in pediatric and emergency settings.
Existing methods of analysis for creatinine have not been
entirely satisfactory:
the manual method in which Lloyd’s
reagent is used is tedious (1 the continuous-flow (Technicon
AutoAnalyzer)
method requires too much serum (2), and
centrifugal analysis methods are appropriate only for batch
),
analyses.
The Beckman
Creatinine
Analyzer
(3) (Beckman
Instruments,
Inc., Fullerton,
CA 92634), a semi-automated
instrument for determination of creatinine in serum and urine,
solves most of these difficulties.
Here we compare the determination of serum creatinine by
three techniques: the manual method with Lloyd’s reagent,
a centrifugal
analyzer (GEMSAEC;
Electro-Nucleonics,
Inc.,
Fairfield, NJ 07006) method, and the Beckman Creatinine
Analyzer. With the last, the operator has merely to pipet 25
zL of the serum or diluted urine sample into the reaction cell,
adjust the calibration controls, and read the results directly
from a digital display.
Equipment and Principles
Measurement
of creatinine with the Beckman Creatinine
Analyzer is based on a rate modification of the Jaff#{233}
reaction
(4). It utilizes an optical detection
system built around a
micro-colorimeter
(see Figure 1), which has a light source, a
520-nm interference
filter, a reaction-cell
with a 1-cm light
path, and a vacuum photodiode detector coupled to electronic
circuitry
that continuously
provides a signal for computing
the rate of change in absorbance in an alkaline picrate solution. At a fixed time, 25.6 s after the sample is introduced,
the
rate signal is locked and provides a measure of the creatinine
concentration.
Many substances are known to interfere with
‘Children’s Hospital National Medical Center, 111 Michigan Ave.,
N.W., Washington, DC 20010.
2George Washington University School of Medicine, 902 23rd St.,
N.W., Washington, DC 20037.
Presented in part at the 30th national meeting of AACC, July 27,
1978, in San Francisco.
Received June 16, 1978; accepted Mar. 13, 1979.
into
the
reagent
and
during
which
fast-reacting
chromogens, together with true creatinine, contribute to the
duration of color development
(about 5-20 s); then a pha8e
during which the observed rate is essentially due to creatinine
alone (25-60 s); and a final phase, during which the fast-reacting chromogens and creatinine have been consumed and
chromogens
contribute
production (>60 s).
The uniqueness of the Beckman
to the total
Creatinine
Analyzer
color
lies
in the measurement
step (see Figure 2). Introduction
of a
25-zL sample initiates a timing sequence,
which first shorts
the rate signal for 3.2 s to “blind” the system to any disturbances resulting from absorbance or scattering by the sample.
The electronics then continuously differentiates
the absorbance signal
with respect
to time by means
of a derivative
cir-
cuit having a time constant short compared to that of the
creatinine reaction, and provides a signal proportional to the
rate of the reaction. After the 3.2-s short, the apparent rate
signal
first rises at a rate proportional
to the difference
tween the actual rate of change of absorbance
signal
itself, passing
through
an apparent
be-
and the rate
maximum
value as
the rising rate signal overtakes the diminishing actual rate of
change of absorbance. Rather than retaining the apparent
maximum rate signal, the circuit locks and holds the rate
signal existing 25.6 s after sample introduction.
This permits
any rate contribution
attributable
to faster-reacting
interferences
to decrease
to a negligible
rate signal essentially
level, so that the retained
results only to the creatinine
present
in the sample.
The selectivity of the rate Jaff#{233}
method as used in the
Beckman Creatinine Analyzer is further illustrated by Figure
2.
The GEMSAEC
Analyzer settings shown in Table 1 were
determination.
The manual method was done exactly as described by
Mattenheimer
(1 except that the volumes of all reagents and
used for creatinine
),
samples
were multiplied
by three.
Results
Linearity.
We calibrated the Beckman Creatinine Ana1yze
(y) with a 50 mg/L (440 imo1fL) creatmme standard and then
analyzed several other standards (x ) in duplicate without
recalibrating during the experiment. The resulting curve from
the direct readout of the instrument vs. the known concentration was linear to 300 mg/L (2.64 mmol/L), with a regression equation of y
1.033x - 0.156 mg/L, with a standard
error of estimate
= 0.191
mg/L and coefficient
of correlation r = 0.999. In contrast, the manual technique was linear
only to 100 mgfL (880 imoI/L).
CLINICALCHEMISTRY,Vol. 25, No. 6, 1979
1005
SIP
FILl.. -#{248}
_“I_
DRAIN
-0
Fig. 1. Diaam
Analyzer 2
of microcolorimeter
of the Beckman Creatinine
Analytical
recovery. Creatinine from an aqueous standard
of 1000 mg/L (8.80 mmol/L) was added to a serum pool to
produce concentrations
from 9.0 to 304.0 mg/L (0.08-2.68
mmol/L).
Recoveries
in the case of Beckman instrument
varied between 95.5 and 105% (Table 2).
Precision.
We assessed within-run precision by analyzing
20 samples of the same control serum without recalibrating
the Beckman instrument; this experiment was repeated with
different concentrations
of creatinine. Day-to-day precision
was evaluated by assaying five control sera with different
creatinine concentrations
on 18 consecutive days. The results
(Table 3) demonstrate a standard deviation ranging from 0.9
at the 6.0 mg/L concentration
to 1.4 at the 87 mg/L concentration.
Calibration
stability.
We calibrated the Beckman instrument with a 50 mgfL (440 imol/L) creatinine standard. Two
control sera were then assayed at 30-mm intervals. The Analyzer was not recalibrated throughout a test period of 9.5 h.
Creatinine values were very stable throughout this period. The
mean value for one control was 10 mg/L (88 zmo1/L), with a
coefficient of variation of 6.3%, and for the other control was
63 mg/L (550 tmolfL), with a coefficient of variation of
2.6%.
Correlation
studies.
Duplicate assays were performed for
each comparison method and the Analyzer calibration was
checked after each 10 injections.
Comparison
with the Jaffe manual technique
with use of
Lloyd’s reagent. Results with the Analyzer (y) were compared
to those by a manual Jaff#{233}
technique (x) in which Lloyd’s
reagent is used (1). The results for 121 serum analyses with
a range of 2 mg/L to 230 mg/L creatinine concentration
gave
the following equation for least squares regression: y = 1.028x
was 0.994, the
= 0.492
deviation for the intercept SDa = 0.142
mg/L, and the standard deviation of the slope SDb = 0.030.
For 30 urine analyses, the corresponding data were: y = 0.993x
- 1.6 mgfL, r = 0.994, S1
= 7.055 mg/L,
SDa
= 15.1 mg/L,
SDb = 0.137.
-
0.1
mg/L. The coefficient
standard
deviation
mg/L, the standard
Comparison
with
about
of correlation
the regression
centrifugal
line
analysis.
Results
obtained
Rate Signal
Rate Signal Used for
Evaluating Creatinine
Concentration
“I
25.6 Seconds
II
Time in Seconds
Fig. 2. Rate signal obtained during creatinine reaction
The two solid-line curves Illustrate typical rate curves Obtained with a 10 mgIL creatinine solution (lowersolidcurve).
and with a serum with a 10 mIL creatinine
concentration and containln9 more rapidly reacting potentially interfering substances (tper solid cuive). The shaded area between the solid curves is due to the
contribution of early-reacting substances. Thus the observed rate curve tor the serum sample may be considered to be the sum of two contributing rate curves.
The dashed-line cw-ve,
Which
peakS
earlier
and decreases sooner, is dueto fast-reacting,potentialtyinterferingsubstances.whereasthecreatinine contribution
(lowersoildcurve)peaks later and decreases lessrapidly.Byusingtheratevalueat a fixed
a rate proportioaI
1006
time. chosen so that the non-creatinine contribution has disappeared.
to the initial creatinine concentration is obtained. (Figure cotifesy Beckman
CLINICAL CHEMISTRY, Vol. 25, No. 6, 1979
Instrument
Co.)
Table 1. GEMSAEC Settings for Serum Creatinine Assay
Analyzer/control
module Instructions
Rotoloador Instructions
Pump
Volume
Sample
Flush
Reagent
Computer
Insiructions
well
20
20
100
B
Reaction temp., 30 #{176}C
80
200
40
B
500
1000
50
C
Wavelength, 510 nm
Filter position, 430-560
IA
RI
30
30
Reaction mode, Auto-Rate
NR
5
SC = value of st
KT = 1
TF = 1
TC = 5
AD = 3
CD =
First readings, 30 s
Use metal sample tip
Blank switch reagent
Reading intervals, 30 $
Standard position 2: a protein-based std.
No. readings, 5
xx =
DI =
Unknown positions 3-16
DA
ER
with the creatinine
analyzer were
GEMSAEC
analyzer with use of
agent (Harleco,
Gibbstown,
NJ
creatinine
concentrations
ranging
the results were: r
0.992, y
compared
to those with the
a commercial
creatinine
re08027). For 163 sera with
from 2.0 mg/L to 224 mg/L,
1.056x - 0.4 mg/L,
0.492 mg/L, SDb
0.026, and SDa
0.111 mg/L. For 46
urines, r = 0.995, y = O.952x - 1.0 mgfL,
5.515 mg/L,
SDb
0.09, and SDa
9.14 mg/L.
Carryover.
Two serum pools with creatinine values of 8
mg/L
(70 zmo1/L)
and 14 mg/L
(120 zmo1/L)
were assayed
before and after a specimen having a creatinine concentration
of 80 mg/L (700 zmol/L) and one with 200 mg/L (1.76
mmolfL).
The values were unaltered,
indicating
no carryover.
Interference.
We evaluated
possible interference
by some
substances
that have caused interference
in other methods
for the determination
of creatinine (5-7), to see whether they
affected creatinine
readings obtained
with the Beckman
instrument. Pooled sera, analyzed for creatinine after increasing
anounts
of bilirubin were added to give values up to 250 mg/L
(425 mmol/L),
the results.
showed no measurable
effect of bilirubin
on
We similarly assessed the effects of grossly lipemic serum,
hemoglobin
(up to 2 g/L), glucose (up to 10 g/L), ascorbic acid
Table 2. Analytical Recoveries with the Beckman
Creatinine Analyzer
Caic.
Creatinine, mg/L
D.td.
1
I-Il = 1.4
LO = 0.2
SA = 0.2
RM = 2
1
1
= 0.0
= 1
(up to 1 gIL), protein (up to 150 mg/L), acetoacetate (up to 1.5
g/L), and acetone (up to 1 gIL). Only the last two caused interference.
Acetoacetate,
1 gIL, increased a creatinine
reading
of 9 mg/L (79 zmo1/L) to one of 36 mg/L (317 tmo1/L);
for
acetoacetate at 250 mgfL, the corresponding
value was 16
mg/L (141 zmo1/L). Acetone at 1 g/L increased the apparent
creatinine
mo1/L).
from
9 mgIL
(79 tmo1/L)
to 13 mg/L
Discussion
The Analyzer offers several advantages
creatinine:
precision and accuracy,
speed,
in the analysis for
and small sample
volume (25 zL). Results compare well with those by the
manual procedure for serum, less well for urine. We cannot
explain the difference between results for urine by the manual
procedure and with the Beckman instrument. Our finding that
bilirubin does not interfere with results obtained with the
Analyzer
(8).
is supported
Bilirubin
creatinine
particularly
by the work of Osberg
probably
readings
suitable
is oxidized
and Hammond
to biliverdin
are taken. This
for measurement
before the
makes the instrument
of creatinine
in new-
Table 3. Precision of Creatinine Determination
with the Beckman Creatinine Analyzer
Croatlnlne, mg/L
SD
Mean
Within-run (n
0.8
Recovery,
4
CV,%
=
20)
18.5
6.4
0.5
0.8
1.0
1.8
18
23
54
95.5
99.0
105.0
96.6
98.2
8
50
88
209
104
105
100.0
6
0.9
15.0
152
152
100.0
8
0.5
6.3
170
180
104.0
18
1.0
5.6
250
247
98.6
296
97.0
1.2
1.4
1.9
304
62
87
9
13
18
24
55
9
13
(114
Oay-to-day(n=
INCAL
1.6
1.2
0.9
18)
1.6
CHEMISTRY.Vol. 25, No. 6, 1979 1007
horns, who frequently have bilirubin concentrations
that exceed those for older individuals.
The broad range of linearity
and the good precision allow virtually all serum samples to be
analyzed without dilution. Another advantage of this analyzer
over the centrifugal analyzer is that sera and diluted urines
can be run interspersed with each other without interference
or carryover or without the need for standards with different
matrices-i.e.,
serum-based
for sera and aqueous-based
for
urines. The Beckman Creatinine Analyzer is very suitable and
appears to be ideal for emergency work and for pediatric
samples.
Laboratory,
MI, p 69.
2nd ed., Ann Arbor
Science Publishers, Inc., Ann Arbor,
A. L., Grady, H. J., and Stanley, M. A., Determination
creatinine by means of automatic chemical analysis. Am. J. Clin.
Pat ho!. 35,83 (1961).
:L Hearn, D. J., McLean, M. H., and Flores, 0. R., The evaluation of
a new rate system for the determination
of creatinine. Clin. Chem.
23, 1172 (1977).
4. Fabini, D. L., and Ertingshausen,
G., Automated reaction-rate
method for determination of serum creatinine with the CentrifiChem.
(‘tin. Chem. 17,696 (1971).
5. Koenest, M., and Frier, E., An evaluation of two methods for the
determination
of true creatinine.
Am. J. Med. Technol. 37, 473
2. Chasson,
of
(1971).
We appreciate the help supplied by Mr. Monte
Orlando Flores (Beckman Instrument Co.).
McLean
and Mr.
References
1. Mattenheimer,
H., Micromethods
6. Narayanan, S., and Appleton, H. D., Specificity of accepted profor urine creatinine. Clin. Chem. 18, 270 (1972).
7. Mitchell, R. J., Improved method for specific determination
of
creatinine
in serum and urine. Clin. Chem. 19, 408 (1973).
8. Osberg, I. M., and Hammond, K. B., A sotution to the problem of
hilirubin
interference with the kinetic Jaff#{233}
method for serum creatinine. Clin. Chem. 24, 1196 (1978).
cedures
for the Clinical and Biochemical
Corrections
Gremlins
take particular
delight in seeing that errors
creep into Corrections,
thus doubly embarrassing
us.
Thus on p 2045 of vol. 22, p 52 should read p 528.
And
on p 2202 of vol. 24, the first figure in the equation
should read “O.194”
Other corrections:
Volume 24
p 1848, col.
p 158: “Mahmoodian”
is the correct spelling of the
second authors’ name.
p 200, column
2: Publication
date for the CRC
Handbook
should read “1978” instead of “1959.”
p 321: Under “Effect ofiodide . . . the phrase should
read
between 0.1 and 1.0 mmol/L . . . and the year
of reference 8 should read “1978.”
Contents
page (March): Titles and page numbers for
the last two Letters are interchanged.
p 359: An explanation
of the symbols at the bottom
of column one was received too late for inclusion:
,“
“.
1: “200
mL/liter”
should
read
“200
gIL.”
p 1954: A postal delay resulted in two authors’ corrections to this paper not being made. The correct name
of the hospital in footnote 2 is “The Birmingham
and
Midland Hospital for Women.” On p 1995, under Procedure, part of the second sentence should read
dilute 2 zL of standard
or sample
with 200 zL Of buffer
into . .
Although 2 tL seems a small volume to
measure accurately, these authors could do so (Micromedic diluter used) successfully as the data in Table 1,
which were in fact obtained with 2-zL samples, show.
The procedure as printed presumably would work satisfactorily also.
p 1969: In the information
under Fig. 1, the number
“197” should read “1970.”
“.
.“
1008
Volume 25
CLINICALCHEMISTRY,Vol. 25. No. 6, 1979
.
.
,“
.
so, ground
Si, excited
state
singlet
level of ligand
T(T1), triplet levels of ligand
REX(T), central ion energy level above ligand triplet
level
RE, central ion resonance level, emissive
p 360: The third sentence
in the first full paragraph
should read: “A mixed chelate
of metal, ethylenediaminetetraacetate,
and second
ligand can be made
. . The
authors of ref. 19 are “Wieder, J., and Hodgson,
K. 0.”