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com

J Clin Pathol 1988;41:576-581

Evaluation of an enzymatic method for determining

creatinine in plasma
H CROCKER, M D S SHEPHARD, G H WHITE From the Department of Biochemistry and
Chemical Pathology, Flinders Medical Centre, Bedford Park, South Australia

SUMMARY An enzymatic kit method for the determination of plasma creatinine was optimised for
use with a centrifugal analyser and its performance characteristics and practicability compared with
an end point and a kinetic Jaffe-based method. The enzymatic method exhibited several advantages
over Jaffe-based methods-namely, smaller sample size, rapid sample throughput (200 per hour), and
improved specificity. Glucose, acetoacetate, and cefoxitin did not interfere with the enzymatic
method, although bilirubin did cause a negative interference which depended on both creatinine and
bilirubin concentrations. The enzymatic method has particular clinical application in neonates,
diabetic ketotic patients, and those receiving cephalosporins.

Routine clinical biochemistry laboratories use several analyser (Roche Analytical Instruments, Nutley, New
methods for the estimation of plasma and urinary Jersey, USA) The analytical performance and prac-
concentrations of creatinine, most of which are based ticability of the optimised method for routine use was
on the Jaffe reaction. There are major analytical assessed and compared with both a kinetic and end
problems associated with the use of the Jaffe reaction, point Jaff-based method. The clinical utility of the
however, in particular those relating to positive and method was also examined.
negative interference by chromogens. More than 50
chromogenic interferents have been documented.' Material and methods
Many modifications of the original Jaffe method have
been developed to reduce interference by such sub- CREATININE METHODS
stances in plasma, but these vary in their degree of Enzymatic creatinine method
success. Enzymatic creatinine analyses were performed using
Enzymatic methods for creatinine determination the Creatinine PAP (CREA) enzymatic colorimetric
have been developed in an attempt to overcome some test kit (Boehringer Mannheim, Mannheim, West
of the problems inherent in the Jaffe-based methods. Germany; catalogue No 836 885).
The underlying test principle of a new commercially
available method entails a series of sequential enzyme- Preparation and stability ofreagents
mediated steps which result in the formation of Reagent 1 (buffer/enzymes/4-aminophenazone) Potas-
hydrogen peroxide. A Trinder indicator system is the sium phosphate buffer (22 ml) (100 mmol/l, pH 7.9)
final step in the reaction sequence and is responsible
for the formation of an intense red colour with a
maximum absorbance at a wavelength of 510 nm. The Table 1 Reaction sequence of enzymatic methodfor assay of
reaction sequence is summarised in table 1. creatinine
This method recently underwent a multicentre
evaluation, in which 16 laboratories, using a variety of creatininase
manual and mechanised methods and manufacturers' Creatinine + H20 - Creatine
creatinase
recommended protocols participated in the analytical Creatine + H,O - Sarcosine + urea
evaluation.2 sarcosine oxidase
Sarcosine + H20 + 02 - Glycine + HCHO +
In this report, the enzymatic method was specifically H202
optimised for use on the Cobas Bio centrifugal H202 + phenol
peroxidase
- Red benzoquinone-
derivative + 4-amino- imine dye
Accepted for publication 16 December 1987 phenazone

576
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Enzymatic methodfor determining serum creatinine 577

were added to the powdered contents of phial 1, which each pool, known concentrations of the following
contained creatinase, sarcosine oxidase, peroxidase substances were added.
and 4-aminophenazone. The resulting solution had Glucose (5 mg and 10 mg) (Ajax Chemical Com-
the following composition: potassium phosphate pany, Sydney) were weighed into 2 ml aliquots of each
buffer 100 mmol/l, pH 7-9; potassium hexacyano- of the three patient pools to give final concentrations
ferrate II 5 pmol/l; 2,4,6-tribromo-3-hydroxybenzoic of 27-5 and 55 mmol/l of glucose, respectively, in each
acid 8-6 mmol/l; detergent 0-3%; 4-aminophenazone sample. The two values were consistent with those
0-8 mmol/l; creatinase >20 U/ml; sarcosine oxidase observed in uncontrolled diabetes mellitus.
> 12 U/ml; peroxidase > 1 U/ml; ascorbate oxidase Acetoacetate a stock solution (acetoacetate, lithium
> 10 U/mi. This reagent is stable for two weeks at 40 to salt, Sigma Chemical Company, St Louis, Missouri
12°C and for two days at 150 to 25°C. It must be stored 63178, USA) with a concentration of 54 g/l was
protected from light. prepared. Stock solution (5 p1 and 10 p1) were added to
Reagent 2 (starter reagent, buffer/creatininase) Three 1 ml aliquots of each of the three patient pools to give
reagent tablets were placed into the phial of buffer final concentrations of 2-5 and 5.0 mmol/l of
provided (phial 2) and allowed to dissolve. The acetoacetate, respectively. These concentrations
resulting solution contained: potassium phosphate approximated those readily attained in the plasma of
buffer 20 mmol/l pH 7 9; detergent 0-3%; creatininase patients with diabetic ketoacidosis.
> 164 U/ml. This solution is stable for two weeks at 4° Bilirubin A stock solution (bilirubin, crystalline,
to 12°C and for two days at 150 to 25°C. from bovine gallstones, Sigma Chemical Company, St
Louis, Missouri USA) of 10 mmol/l was prepared as
COMPARATIVE JAFFE-BASED METHODS follows: 117 mg of bilibrubin was dissolved in a
Endpoint Jafle (with dialysis) solution of 2 ml of 01 M sodium carbonate (Ajax
Creatinine analyses were also performed on a Tech- Chemical Co) and 1-5 ml of 0-1 M sodium hydroxide
nicon SMA 6/60 Autoanalyser (Technicon Ins- (Ajax). Once dissolved, this solution was made up to a
truments Corporation, Tarrytown, New York 10591, volume of 20 ml with pooled plasma. This stock
USA) by continuous flow analysis using an end point solution (10, 20 and 30 p1) was then added to I ml
Jaffe-based method which incorporated a dialysis step. aliquots of each of the three patient pools. Final
Colour produced was measured at a wavelength of bilirubin concentrations was measured on a Unistat
520 nm. Bilirubinometer (Reichart-Jung) and gave values of
91, 179, and 267 pmol/l, respectively.
Kinetic Jaffe Cefoxitin a cephalosporin antibiotic (0 9 mg and 1-8
Creatinine concentrations were also determined using mg) (cefoxitin sodium, Merck, Sharp, and Dohme,
the Beckman Astra 8 (Beckman Instruments Incor- Grandville, New South Wales) were added to 2 mt
porated, Clinical Instruments Division, Brea, Califor- aliquots of each of the three patient pools to give final
nia 92621, USA). Colour production was measured at cefoxitin concentrations of I and 2 mmol/l, respec-
a wavelength of 520 nm 25-6 seconds after the sample tively. These concentrations were consistent with
had been introduced into the Jaffe reagent. therapeutic concentrations for this antibiotic.
The quality control materials used in this study were
Autonorm (Nycomed AS, Oslo, Norway) and Well- Results
control Abnormal Unassayed (Wellcome Diagnostics,
Dartford, England. DA1 5AH). OPTIMISATION OF THE REACTION CONDITIONS
Technicon Diagnostics Set Point SMA Calibrator FOR THE ENZYMATIC METHOD
(Technicon Instruments Co. Tarrytown, NY. 10591) The centrifugal analyser settings recommended by the
and Beckman Astra Aqueous Calibrator (Beckman reagent kit manufacturer are listed in table 2 (column
Instruments Inc) were used as standards for the A). These variables specify that the total reaction
evaluation. The creatinine content of both solutions period should extend over eight minutes. This time
was 440 gmol/l. comprises an initial four minutes of preincubation of
reagent with sample, followed by a further four
INTERFERING SUBSTANCES minutes of reaction period after addition of the
Patient pools starting reagent containing the limiting enzyme
A series of three patient plasma pools of low, creatininase. Reaction readings are obtained at only
moderate, and high creatinine concentrations (87, 332, two points, one at the beginning and one at the end of
and 572 pmol/l, respectively) were prepared. The the four minute reaction period.
stated concentrations of analyte in each pool represen- The test variables recommended by the manufac-
ted the mean value following triplicate analysis ofeach turer were initially evaluated. The four minute prein-
pool on the Technicon SMA 6/60 Autoanalyser. To cubation period was found to be necessary, as the
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578 Crocker, Shephard, White

Table 2 Reaction conditions on Cobas Biofor measurement for all samples throughout the period of monitoring.
of creatinine The series of modifications described allowed the
linear range of the method to be extended (to 1600
Instrument settings A B jmol/l), the reaction monitored at shorter reading
I Units smol/l pmol/l intervals, and the total reaction time reduced by 90
2 Calculation factor 0 0 seconds from eight minutes to six and a half minutes,
3 Standard I concentration 440 440 with no difference in absolute creatinine concentration
4 Standard 2 concentration 440 440
5 Standard 3 concentration 440 440 being observed with the shorter reaction time. The
6 Limit 1600 1600 final optimised reaction conditions for the enzymatic
7 Temperature ('C) 37 37
8 Type of analysis 6 6 creatinine method are listed in table 2 (column B).
9 Wavelength (nm) 510 510
10 Sample volume (ul) 7 7 PRECISION STUDIES
11 Diluent volume (p1) 30 30
12 Reagent volume (p1) 200 200 Intrarun and interrun precision data were obtained
13 Incubation time (s) 240 240 using five different samples, comprising three patient
14 Start reagent volume (pl) 40 40
15 Timeof first reading(s) 10 60 plasma pools containing low, moderate, and high
16 Time interval (s) 240 10 concentrations of creatinine together with the two
17 Number of readings 2 9
18 Blanking mode I 1 quality control materials routinely used in the
19 Printout mode 1 I laboratory of the authors (Autonorm and Wellcom-
trol). For interrun precision studies, aliquots of the
three patient pools were frozen at 0'C. The results
reaction was non-linear during the first 240 seconds obtained for intrarun and interrun precision studies
after addition of start reagent. Using patient material are summarised in table 3.
with a high creatinine concentration (1300-1600 Coefficients of variation reported for patient sam-
pmol/l), instrument settings were further investigated. ples were smaller than those reported for the quality
Linearity with samples containing high creatinine control materials. This may be due to a combination of
concentrations was observed for only 150 seconds the different matrix of the quality control materials
after the addition of starting reagent. The time interval (lyophilised, and of bovine origin) and the contribu-
during which the reaction was monitored was tion of operator error to imprecision, as quality
therefore shortened to exclude the non-linear portion control materials were reconstituted daily from lyo-
of the reaction. Concurrently, the reaction was mon- philised powder, often by different technicians.
itored at more frequent (10 second) intervals as
opposed to the 20 second intervals used earlier. ACCURACY
Closer monitoring of the reaction kinetics using The accuracy of the optimised enzymatic method was
these modified variables showed non-linear kinetics determined by analysing 100 hospital patient samples
during the first 50 seconds following addition of in duplicate. The resulting mean value for each of the
starter, with linearity observed only during the 50 duplicate pairs was compared with the value obtained
second to 150 second period of the reaction. on the same samples using the Jaffe end point method
Variables were therefore revised again to confine the on the Technicon SMA 6/60 and the kinetic Jaffe
period of reaction monitoring to between 60 and 150 method on the Beckman Astra 8.
seconds following incubation, with a total of nine The results were assessed using linear regression and
readings being made at 10 second intervals. A series of indicated that the creatinine concentrations reported
patient samples with creatinine values ranging from 85 using the enzymatic technique were not significantly
to 1460 pmol/l were run according to these new different to those reported using Jaff&-based methods:
variables. Reaction kinetics were shown to be linear enzymatic pmol/l (y) = 0-99 (end point Jaffe) x
Table 3 Results ofprecision studiesfor optimised creatinine assay
Low Moderate High Autonorm Wellcontrol
Intrarun (n = 10):
x (pmol/l) 125 375 722 84 402
SD (pmol/l) 2-6 75 13 9 4-8 4-4
CV(%) 21 20 19 57 1*1
Interrun (n = 10):
x (pUmol/l) 123 386 741 78 410
SD (pmol/l) 8-0 18-0 21-0 8-0 10 0
CV (%) 70 45 30 105 25
Plasma with low, moderate, and high creatinine was evaluated, in addition to the two quality control materials.
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Enzymatic methodfor determining serum creatinine 579

Table 4 Summary of effects on common interfering substances on enzymatic and kinetic Jaffe methods for plasma creatinine
Enzymatic (/imol/l) Kinetic Jare (umol/l)
Final concentration
Interferent ofinterferent Low Moderate High Low Moderate High
Glucose (mmol/l) 0 86 338 595 81, 311 545
27-5 85(-1)* 340(+1) 600(+1) 93(+16) 319(+3) 553(+2)
55 87(+1) 344(+2) 594(0) 96(+19) 325(+5) 565(+4)
Acetoacetate (mmol/1) 0 85 339 601 81 311 545
2-5 82 (-3) 328 (-3) 592 (-1) 142 (+ 75) 375 (+21) 619 (+ 13)
5 79(-7) 330(-3) 583(-3) 198(+144) 445(+43) 680(+25)
Bilirubin (pmol/l) 0 87 332 572 81 317 551
100 76(-13) 291 (-12) 509(-11) 64(-22) 297(-6) 535(-3)
200 60(-31) 253(-24) 456(-20) 68(-6) 290(-9) 526(-5)
300 47 (-46) 218 (-34) 396 (-31) 65 (- 20) 282 (- 11) 505 (-8)
Cefoxitin (mmol/1) 0 83 333 583 89 312 544
1 83(0) 333(0) 582(0) 269(+202) 446(+43) 711 (+31)
2 82(0) 328(-2) 576(-1) 349(+292) 598(+92) 863(+59)
*Figures in parentheses represent percentage difference in creatinine following addition of interferents.
- 0 99 umol/l; r2 = 0-99 and enzymatic Mmol/l (y) = Beckman Astra 8. Analyses were performed at least in
1-02 (kinetic Jaffe) x - 2-58 pmol/l; r2 = 0 99). duplicate. Results for all interfering agents are sum-
marised in table 4.
LINEARITY The results for glucose indicated that it does not
Linearity was confirmed up to a concentration of 1600 interfere with the enzymatic test method. A small
imol/l by serial dilution of a patient sample containing degree of positive interference (+ 19%) was observed
a high creatinine concentration. at the lowest creatinine concentration with the kinetic
Jaffe method, but higher concentrations of creatinine
REFERENCE RANGE were unaffected by the two levels ofglucose tested. The
Reference values were determined by analysing results for acetoacetate showed that while the
plasma samples from 300 presumably healthy blood enzymatic method was unaffected by the addition of
donors over a period of days in batches of 50. acetoacetate up to concentrations of 5 mmol/l, there
Reference ranges were then calculated using the was a considerable positive increase in the apparent
Hoffman plot technique.3 For adults, 55-122 ymol/l (n creatinine concentration with the kinetic Jaffe method.
= 300); for men, 70-123 pmol/l (n = 150); for women, This increase in apparent creatinine concentration was
49-108 pmol/l (n = 150). This reference range was both directly proportional to the acetoacetate concen-
comparable with that currently used in the authors' tration and inversely related to the creatinine concen-
laboratory (60-120 Mmol/l) derived using the end tration-that is, the positive effect being highest at the
point Jaffe method. The difference in the values lowest concentration of creatinine.
obtained for the male and female subpopulations can Our results indicate that the kinetic Jaffe method is
be atrributed to sex differences in muscle mass. not affected by the addition of bilirubin, but with the
enzymatic method there is a significant depression in
INTERFERING SUBSTANCES the actual creatinine concentration following the
Commonly encountered interfering substances of the addition of bilirubin. The magnitude of the decrease
Jaff-based methods include glucose, acetoacetate, depends entirely on creatinine concentration and
bilirubin, and cefoxitin.4 Glucose and bilirubin both bilirubin concentrations. Bilirubin interference in
inhibit the reaction between creatinine and alkaline peroxidase-coupled enzymatic assays entailing the
picrate. Glucose slowly reduces picric acid to spectrophotometric measurement of hydrogen perox-
picramate,5 while bilirubin, under alkaline conditions, ide has been reported previously.>" The mechanism
is oxidised to biliverdin, causing a decrease in absor- by which bilirubin interference occurs has been
bance at 520 nm.6 Acetoacetate and cefoxitin, conver- explained by a combination of both spectral and
sely, react directly with alkaline picrate. Acetoacetate, chemical effects." Spectral interference results from
in fact, reacts more rapidly with picrate than does overlap of the broad 460 nm absorption band of
creatinine.' The thiopen nucleus is the active moiety in bilirubin with the absorption band of the Trinder
the cefoxitin molecule which reacts with the Jaffe chromophore. Chemical interference is postulated to
reagent.8 The effects of these known interferents on the be due to a peroxidase reaction intermediate reacting
enzymatic method were investigated and compared with bilirubin, thereby decreasing the final amount of
with their effect on the kinetic Jaffe method used on the chomophore formed. The latter interference can be
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580 Crocker, Shephard, White

eliminated by the addition of ferrocyanide to the gmol/l was assayed by the routine Beckman Astra. To
reaction mixture, which stabilises the reaction inter- assist the clinical decision on appropriate fluid man-
mediate.'2 Ferrocyanide is present in the reaction agement the specimen was reanalysed using the
mixture of the reagent kit under evaluation, but only at enzymatic method; a value of 81 pmol/l was found,
a final concentration of 5 pmol/l. This concentration suggesting normal renal function. The high concentra-
may be too low to effect adequate stabilisation. tion of acetoacetate in the patient's plasma had
The addition of the cephalosporin antibiotic cefox- artefactually raised the apparent creatinine concentra-
itin resulted in a significant positive increase in creatin- tion to 235 pmol/1 when assayed by the kinetic Jaffe
ine concentrations with the kinetic Jaffe method. The method.
positive interference observed was greatest at -the
lowest concentration of creatinine. No such inter- Case 2
ference was recorded when the enzymatic method was An 18 month old infant presented with a three day
assessed. history of persistent rhinorrhoea and modest
anorexia, together with a 12 hour history of high fever,
CLINICAL UTILITY OF THE ENZYMATIC METHOD irritability, episodic drowsiness and vomiting.
The advantages in specificity of the enzymatic method Examination showed prominent neck stiffness and
were also shown in the following two clinical cases. possibly slight dehydration. A presumptive diagnosis
of meningitis was subsequently confirmed by examina-
Case I tion of cerebrospinal fluid. The patient was given
A 16 year old female, an insulin dependent diabetic, cephalosporin cefotaxime (500 mg intravenously) and
presented to casualty in a confused state. She was intravenous fluids at half normal maintenance
volume depleted and pre-renal failure was suspected. volume. Plasma electrolytes over the next 24 hours are
The results of initial biochemical investigations are shown in table 6.
shown in table 5. The creatinine concentration of 235 The admission hyponatraemia was thought to be

Table 5 Biochemistry results on 16 year old insulin dependent diabetic (A) on admission; (B) and (C) three and 24 hours after
starting treatment

A B C
(0855 h) (1200 h) (0900 h) Units Reference range
Sodium 135 137 135 mmol/l 132-144
Potassium 53 4.7 3.7 mmol/I 30-4 7
Chloride 100 109 107 mmol/l 93-108
Bicarbonate 9 6 14 mmol/l 21-32
Urea 8-3 7-3 2-9 mmol/l 30-8-0
Creatinine
Kinetic Jaffe 235 225 76 pmol/1 60-120
Enzymatic 81 pmol/l 55-122
Glucose 39-8 190 11-7 mmol/l 30-5-5
Lactate 3-1 mmol/l < 2-0
,B Hydroxybutyrate 7-6 mmol/l <0 3
Acetoacetate 31 mmol/l <0 1
Anion gap 31 27 18 mmol/l 7-17

Table 6 Plasma biochemistry results in 18 month old baby receiving cephalosporin for meningitis

ADayl BDay2 CDay2 DDay2

(2100h) (1115 h) (1500 h) (2315 h) Units Reference range
Plasma
Sodium 125 139 143 136 mmol/l 132-144
Potassium 4-6 4.4 53 3-8 mmol/l 3-0-4-7
Chloride 100 110 113 110 mmol/l 93-108
Bicarbonate 14 9 12 15 mmol/l 21-32
Urea 44 6-1 5-9 4-1 mmol/l 30-70
Creatinine
Kinetic Jaffe 63 137 132 99 pmol/l 20-50
Enzymatic NA NA 28 23 imol/l
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Enzymatic methodfor determining serum creatinine 581

secondary to the syndrome of inappropriate References
antidiuretic hormone secretion, but the raised creatin- 1 Cook JGH. Factors influencing the assay of creatinine. Ann Clin
ine concentrations of 63, 137, 132 and 99 umol/l Biochem 1975;12:219-32.
suggested pre-renal failure. The biochemistry 2 Gruder WG, Hoffmann GE, Hubbuch A, Poppe WA, Siedel J,
laboratory was consulted and enzymatic creatinine Price CP. Multicentre evaluation of an enzymatic method for
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Chem Biochem 1986;24:889-902.
plasma samples. The results were found to be normal 3 Hoffman RG. Establishing quality control and normal ranges in the
for an infant of this age (table 6), which allowed clinical laboratory. New York: Exposition Press, 1971.
intravenous fluid administration to be continued. 4 Spencer K. Analytical reviews in clinical biochemistry: the estima-
tion of creatinine. Ann Clin Biochem 1986;23:1-25.
5 Bowers LD, Wong ET. Kinetic creatinine assays. II. A critical
PRACTICABILITY evaluation and review. Clin Chem 1980;26:555-61.
The practicability of the enzyme-based method was 6 Knapp ML, Hadid 0. Investigations into negative interference by
also assessed. The assay is simple to perform, readily jaundiced plasma in kinetic Jaffe methods for plasma creatinine
adaptable to the emergency or on-call situation, determinations. Ann Clin Biochem 1987;24:85-97.
7 Gerard SK, Khayam-Bashi H. Characterization of creatinine
utilises a very small sample size (7 p1) and is rapid, error in ketotic patients. Am J Clin Pathol 1985;84:659-64.
being capable of analysing 200 samples perthour. A 8 Letellier G, Desjarlais F. Analytical interference of drugs in
minor disadvantage of the method is its relatively high clinical chemistry. III. The interference of three cephalosporins
cost. with the determination of serum creatinine concentration by the
Jaffe reaction. Clin Biochem 1985;18:352-6.
9 Gochman N, Schmitz JM. Application of a new peroxidase
Discussion indicator reaction to the specific, automated determination of
glucose with glucose oxidase. Clin Chem 1972;18:943-50.
The enzymatic method for creatinine evaluated in this 10 Perlstein MT, Thiberc RJ, Watkins R, Zak B. Spectrophotometric
study of bilirubin and haemoglobin interactions in several
study showed considerable improvement on the hydrogen peroxide generating procedures. Clin Chem
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ough a problem still exists with interference from 1 1 Witte DL, Brown LF, Feld RD. Effects of bilirubin on detection of
bilirubin. The modified enzymatic method was both hydrogen peroxide by use of peroxidase. Clin Chem
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precise and accurate, used a small sample size, and was 12 Spain MA, Wu AHB. Bilirubin interference with determination of
capable of a rapid throughput of patient samples. We uric acid, cholesterol, and triglycerides in commercial perox-
conclude that the enzymatic method is suitable as a idase-coupled assays, and the effect of ferrocyanide. Clin Chem
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measurement of plasma creatinine, particularly for
diabetic ketotic patients, neonates, and patients
receiving cephalosporins.
We thank Miss Maria Vallis for secretarial assistance Requests for reprints to: Dr GH White, Department of
and Dr Chris Munt for providing clinical information Biochemistry and Chemical Pathology, Flinders Medical
for the two case studies presented. Centre, Bedford Park, South Australia 5042, Australia.
 Downloaded from http://jcp.bmj.com/ on April 30, 2015 - Published by group.bmj.com

Evaluation of an enzymatic
method for determining creatinine
in plasma.
H Crocker, M D Shephard and G H White

J Clin Pathol 1988 41: 576-581

doi: 10.1136/jcp.41.5.576

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