Cystatin C, an Easy and Reliable Marker for Assessment

of Renal Dysfunction in Children with Liver Disease

and After Liver Transplantation
Marianne Samyn,1 Paul Cheeseman,1 Lynsey Bevis,1 Rachel Taylor,1
Beatrice Samaroo,1 Muriel Buxton-Thomas,2 Nigel Heaton,3 Mohamed Rela,3
Giorgina Mieli-Vergani,1 and Anil Dhawan1
See Editorial on Page 264
Renal dysfunction of variable severity is being increasingly recognized as a major complication of calcineurin
inhibitors (CI), in some patients even necessitating renal
transplantation. Close and effective monitoring of the
renal function is indicated. Current methods for this monitoring are calculation of the glomerular ltration rate
(GFR) based on creatinine or exogenous substances like
51
Cr-EDTA. The rst method is unreliable in children
and the second is expensive and cumbersome. Cystatin C
has been shown to be an accurate marker of glomerular
ltration but has not been evaluated in a large cohort of
pediatric patients before and after liver transplantation
(LT). We evaluated the accuracy of cystatin C in 62 children (30 male) with LT, who had their 51Cr-EDTA measured on 40 occasions prior to LT and on 47 occasions
after LT. The reciprocal of cystatin C correlated better
with 51Cr-EDTA GFR (r .78) than the reciprocal of
creatinine (r .40). Diagnostic accuracy in the identication of reduced GFR was assessed by ROC analysis.
Cystatin C yielded the highest area under the ROC curve
(AUC) in all groups assessed. From these data a cutoff
level of cystatin C predicting 51Cr-EDTA GFR < 80
ml/min/1.73m2 was calculated. A level of 1.06 mg/L was
found to have a sensitivity of 91% and a specicity of
81%. Applying this cutoff level in our patient group
would have avoided 51Cr-EDTA GFR estimation in 43 of
the 87 estimations. In conclusion, the use of this simple
test could be recommended as screening of renal dysfunction in children with liver disease and after LT. (Liver
Transpl 2005;11:344349.)

Abbreviations: CI, calcineurin inhibitors; LT, liver transplantation; GFR, glomerular ltration rate; CGFR, calculated GFR; AUC,
area under the ROC curve; 2-MG, 2-microglobin; BTP, -trace
protein; RBP, retinol-binding protein
From the 1Department of Child Health, 2Department of Nuclear
Medicine, and 3Institute of Liver Studies, Kings College Hospital, Denmark Hill, London, UK.
Address reprint requests to Anil Dhawan, FRCPCH, Paediatric
Liver Service, Department of Child Health, Kings College Hospital,
Denmark Hill, London SE5 9RS, United Kingdom. Telephone: 44-207346-3214. FAX: 44-207-346-3564. Email: anil.dhawan@kcl.ac.uk
Copyright 2005 by the American Association for the Study of
Liver Diseases
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1012/lt.20330

344

alcineurin inhibitors (CI), cyclosporin, and

tacrolimus have signicantly contributed toward
improved long-term survival of children after solid
organ transplantation.1 The current 5-year survival of
children after liver transplantation (LT) is above 85%
in most centers. Nephrotoxicity of variable severity is
one of the most serious side effects of CI treatment, at
times necessitating renal transplantation.2 The incidence of nephrotoxicity in non-renal allograft patients
is between 20% and 70%.3 Although newer immunosuppressive drugs, like mycophenolate mofetil (MMF)
and rapamicin, are not nephrotoxic, their use is still
limited to rescue therapy for renal dysfunction or resistant cellular rejection. A large cohort of children worldwide is maintained on CI as anti-rejection drugs. In
order to avoid irreversible renal damage, close and effective monitoring of their renal function is necessary. The
gold standard for measuring glomerular function is inulin clearance or 51Cr-EDTA estimation. Both methods
are cumbersome and expensive, and 51Cr-EDTA estimation requires administration of a radioactive substance. Serum creatinine is the endogenous substance
most commonly used for estimation of glomerular
ltration rate (GFR), but it is inuenced by muscle
mass, age, and gender, thus affecting its value in children.4 A simple test to evaluate renal function in pediatric patients is highly desirable. Cystatin C, a low
molecular weight protein, has recently been proposed as
a reliable marker of renal function both in adults and
children.5 15 After clearance from the plasma via glomerular ltration, cystatin C is completely reabsorbed
and catabolized in the renal tubules.7 Adult ranges are
reached by the age of 1 year and are not inuenced by
gender, height, or body composition.11 Reference
ranges for both adults and children are available.9 14,16
Cystatin C has been assessed in adult and pediatric renal
transplant recipients9,16 18 and in adults after liver
transplantation,19,20 but information is lacking in pediatric liver transplant recipients. The aim of this study
was to evaluate cystatin C as a marker of renal function
in a large cohort of pediatric patients with chronic liver
disease and after liver transplantation.

Liver Transplantation, Vol 11, No 3 (March), 2005: pp 344 349

345

Cystatin C in Pediatric Liver Transplantation

Table 1. Indication for Liver Transplantation

in the 62 Children Studied
Diagnosis

Diagnosis

Biliary atresia
Alagille syndrome
Acute liver failure
Intrahepatic cholestasis
*A1AT deciency

29
7
6
6
5

Liver tumors
Cystic brosis
Criggler Najjar
GSD type 1b
Cryptogenic cirrhosis
Biliary atresia A1AT
deciency

4
1
1
1
1
1

*A1AT, alpha 1 antitrypsin deciency; GSD, glycogen storage disease.

Patients and Methods

The study group was composed of 62 children (30 male) who
underwent LT at our center between 1989 and 2000 and for
whom 51Cr-EDTA estimations and serum samples stored at
70C were available. The median age at LT was 3.1 years
(range 0.6 18.7 years). Indications for LT are listed in Table
1, biliary atresia being the most common (47%). Between
1989 and 1995, 51Cr-EDTA estimation was performed only
after transplant (22 children), while after 1995 the test was
performed also before transplant to assess baseline renal function (40 children).
Primary immunosuppression after LT consisted of cyclosporin, azathioprine, and prednisolone in 60 children. Post
LT, trough levels of cyclosporin (AxSYM Monoclonal Cyclosporin FPIA assay, Abbott UK, Kent, UK) were kept between
200 250 g/L during the rst month, 150 200 g/L from
2 to 6 months, 150 g/L from 7 to 12 months, and 100
g/L thereafter. In two patients tacrolimus and prednisolone
were used as primary immunosuppressants. Post LT, trough
levels of tacrolimus (Tacrolimus II Imx, Abbott UK, Kent,
UK) were kept between 10 12 g/L during the rst month,
8 10 g/L from 2 to 6 months, 6 8 g/L from 7 to 12
months, and 5 g/L thereafter. Twenty-four children were
switched from cyclosporin to tacrolimus as rescue for resistant
cellular rejection.
Renal function was assessed by 51Cr-EDTA estimation,
serum creatinine, calculated GFR (CGFR), and serum cystatin C measurement.
The 51Cr-EDTA estimation was carried out by using a
dose of 1.2 megabequerels of radioactive 51Cr-EDTA. Blood
samples were obtained 2 hours after injection and thereafter
every 45 minutes to a total sample number of 4. The result of
the 51Cr-EDTA clearance was then corrected to inulin and
body surface area and expressed as mL/minute/1.73m2.
Serum creatinine was measured by Jaffe reaction using the
Bayer Dax 48 and expressed in mol/L. CGFR was obtained
by applying the Schwartz formula4:
H(cm) constant (k)
creatinine ( mol/L)

The constant factor (k) used was 30 for children 1 year of

age, 38 for children 1 year of age, and 48 for adolescent
males.4
Serum cystatin C was measured on stored samples with N
Latex Cystatin C kit on the Dade Behring Nephelometer
BN2 using the PENIA method.8 Results are expressed in
mg/L.
51
Cr-EDTA GFR was measured on 87 occasions and samples for the measurement of serum creatinine and cystatin C
were obtained within 5 days of the 51Cr-EDTA estimation.
The median age at the time of study was 5.3 years (range,
0.3 18.6 years). On 40 occasions the 51Cr-EDTA was performed at a median interval of 4 months (range, 0.1 2.75
years) prior to LT as part of LT assessment. The remaining 47
measurements were performed 1 10.3 years post LT
(median, 3.1 years). Impaired renal function was dened as a
GFR of 80 mL/min/1.73 m2.21 At our center, immunosuppression is modied by adding CI sparing agents when
51
Cr-EDTA GFR is 80 mL/min/1.73 m2.1
Statistical analysis was performed by using NCSS (Number Cruncher Statistical Systems) (Kaysbille, UT, USA).
Associations between variables (creatinine, cystatin C,
CGFR) were assessed with the correlation coefcient according to Pearson (r). The diagnostic validity of cystatin C, creatinine, and CGFR to detect reduced GFR in comparison to
51
Cr-EDTA GFR was evaluated by receiver operative curve
(ROC) analysis. NCSS was used for calculation of the area
under the ROC curve and the sensitivity/specicity data at
certain cutoffs.

Results
Median values and ranges for serum creatinine, serum
cystatin C, 51Cr-EDTA GFR, and CGFR are listed in
Table 2. Thirty-four of the 87 51Cr-EDTA GFR estimations (39%) were 80 mL/min/1.73m2 and compatible with impaired renal function.
CGFR estimations, cystatin C, creatinine, and their
reciprocals were plotted against 51Cr-EDTA GFR (Figure 1 A-F). The reciprocal of cystatin C (r .78)

Table 2. Values of Serum Creatinine, Serum Cystatin C,

51
Cr-EDTA GFR, and Calculated GFR*
in the 62 Children Studied
N 87

Units

51

Cr-EDTA GFR mL/min/1.73 m

Calculated GFR
mL/min/1.73 m2
Creatinine
mol/L
Cystatin C
mg/L
*GFR: glomerular ltration rate.

Median

Range

93
64
67
1.05

28 270
25 202
16 163
.57 2.27

346

Samyn et al.

Figure 1. Scatter plots and least-squares regression line ( ) between 51Cr-EDTA GFR and cystatin C (r .73, r2 .53),
creatinine (r .51, r2 .27), and calculated GFR (r .12, r2 .02), and the reciprocals of cystatin C (r .78, r2
.61), creatinine (r .44, r2 .16), and calculated GFR (r .017, r2 0.0003).

correlated better with 51Cr-EDTA GFR than with the

reciprocal of creatinine (r .44) or of CGFR (r .12).
To determine the value of cystatin C in predicting a
reduced GFR, ROC analysis was performed on 41 values of 51Cr-EDTA GFR 90 mL/min/1.73m2, 34
values of 51Cr-EDTA GFR 80 mL/min/1.73m2, 24
values of 51Cr-EDTA GFR 70 mL/min/1.73m2, and
17 values of 51Cr-EDTA GFR 60 mL/min/1.73m2.
The results of the area under the ROC curve (AUC) are
shown in Table 3. Cystatin C yielded the highest area
under the ROC curve in all four groups compared to
creatinine and CGFR (Figure 2 A D). The area under

the ROC curve for CGFR was the lowest compared to

cystatin C in all four groups. From these data a cutoff
level of cystatin C for predicting 51Cr-EDTA GFR
80 mL/min/1.73m2 was calculated. A level of 1.06
mg/L was found to have a sensitivity of 91% and a
specicity of 81%.

Discussion
Our results indicate that cystatin C is a safe, childfriendly, and accurate measurement of renal function in

Cystatin C in Pediatric Liver Transplantation

Table 3. Area Under the Receiver Operative Curve

Obtained by ROC Analysis when Patients were Divided in
Four Groups According to Their 51Cr-EDTA GFR Results

51

Cr-EDTA GFR

90 mL/min/1.73
m2
80 mL/min/1.73
m2
70 mL/min/1.73
m2
60 mL/min/1.73
m2

Cystatin C CGFR Creatinine

AUC*
AUC
AUC

41

.89

.53

.76

34

.93

.52

.76

24

.90

.59

.80

17

.89

.60

.78

*AUC: area under the ROC curve; GFR: glomerular ltration rate; CGFR: calculated GFR.

children with chronic liver disease and in those exposed

to nephrotoxic drugs after grafting.
With increased long-term survival following pediatric liver transplantation, the monitoring of renal function has become essential to avoid serious late compli-

347

cations. The gold standard for GFR estimation is 51CrEDTA, but this procedure is costly, invasive, timeconsuming, and stressful for children and parents.
Attempts have been made to use non-invasive techniques, such as simple creatinine measurement or calculation of GFR with the Schwartz formula.1,3,22
Though considered satisfactory by some,1,3,22 our experience is that these techniques are not sufciently accurate. Prior to LT most patients with chronic liver disease
are malnourished, especially those with cholestatic conditions like biliary atresia,23 making creatinine measurement, which is inuenced by body muscle mass,
particularly unreliable. Though after transplantation a
marked improvement of the nutritional state and
growth is usually observed,23 growth failure may persist.24
Several low molecular weight proteins, like cystatin
C, 2-microglobin (2-MG), -trace protein (BTP),
and retinol-binding protein (RBP), have been evaluated
as possible replacements for creatinine in the assessment
of GFR.10,15,26 Cystatin C is a cysteine-prokinase inhibitor with widespread distribution in human biological

Figure 2. ROC analysis with calculation of area under the ROC curve (AUC) for cystatin C (), creatinine (), and
calculated GFR (F) for different cutoffs of 51Cr-EDTA GFR. (A) 51Cr-EDTA GFR < 60 mL/min/1.73m2, (B) 51CrEDTA GFR < 70 mL/min/1.73m2, (C) 51Cr-EDTA GFR < 80 mL/min/1.73m2, (D) 51Cr-EDTA GFR < 90 mL/min/
1.73m2

348

Samyn et al.

uids.27 The gene encoding for cystatin C was cloned

by a Swedish group in 1990 and Northern blot experiments have revealed that it is expressed in all human
tissues examined, including kidney, liver, pancreas,
intestine, stomach antrum, lung, and placenta.27 Cystatin C is produced at a constant rate and its levels are
not inuenced by inammation or malignancy,28 being
almost exclusively determined by glomerular ltration7
and being identical to those detected in adult subjects
from the rst year of life.11,13,14 Height, gender, and
body composition do not inuence serum cystatin C
levels.11,15 In contrast, other markers, like 2-MG,
BTP, and RBP, are affected by the presence of infections or malignancies and their value has not been
assessed in pediatric patients.15,26 Moreover, cystatin C
levels are easy to measure by commercially available
latex immunoassays, based on turbidimetry (PETIA) or
on nephelometry (PENIA).8
Cystatin C levels as measure of renal function have
been evaluated in adults with cirrhosis29 31 and after
LT.19 In 44 adult patients with advanced cirrhosis,29
the reciprocal of cystatin C correlated well with GFR
measured by inulin clearance. Orlando et al. reported
that plasma cystatin C was a more accurate GFR marker
in cirrhotic adult patients than were plasma creatinine
concentrations and CGFR, the values of which varied
according to the severity of liver disease.30 Similar
results were obtained by Hermida et al.,19 and a further
study showed serum cystatin C to be a valuable tool for
the early diagnosis of moderately impaired renal function in adult patients with cirrhosis.31
A possible drawback in the use of cystatin C is the
reported effect of steroid and cyclosporin in increasing
its levels in asthmatic patients.32 In a study from Bokenkamp et al., however, in 44 children after renal transplantation no signicant effect of these medications on
cystatin C levels was reported.16 In another study in
children with corticosteroid-sensitive nephrotic syndrome, cystatin C levels remained unaffected by a standard high dose corticosteroid therapy.33
This study is the rst to address the value of cystatin
C levels for assessing renal function in a large cohort of
pediatric patients with chronic liver disease before and
after LT. Our results show that cystatin C levels are
closely correlated with 51Cr-EDTA GFR estimation
and are much more reliable than creatinine and especially CGFR (Schwartz formula) in assessing renal function.
Using the data obtained from the ROC curve analysis, we have calculated a cystatin C cutoff level of 1.06
mg/L for predicting a GFR 80 mL/min/1.73m2,
which has a sensitivity of 91% and a specicity of 81%.

We suggest that only children with this cystatin C cutoff level should be further investigated by 51Cr-EDTA
GFR estimation to obtain a more accurate assessment
of renal dysfunction. Had this cutoff level been used in
the present cohort, 43 of the 87 51Cr-EDTA GFR
estimations (49%) would have been avoided.
In conclusion, cystatin C is a simple test that could
be used as screening for severe renal dysfunction in
children with chronic liver disease and after LT. If its
accuracy is conrmed in a larger cohort of patients with
severe renal dysfunction, it may become a substitute for
51
Cr-EDTA GFR estimation.

References
1. Aw M, Samaroo B, Baker A, Verma A, Rela M, Heaton N, et al.
Calcineurin-inhibitor related nephrotoxicity-reversibility in paediatric
liver
transplant
recipients.
Transplantation
2001;72:746 749.
2. Neau-Cransac M, Morel D, Bernard P-H, Revel P, Potaux L,
Saric J. Renal failure after liver transplantation. Outcome after
calcineurin inhibitor withdrawal. Clin Transpl 2002;16:368
372.
3. McDiarmid S. Renal function in pediatric liver transplant
patients. Kidney Int Suppl. 1996;53:S77 S84.
4. Schwartz G, Brion L, Spitzer A. The use of plasma creatinine
concentration for estimating glomerular ltration rate in infants,
children and adolescents. Pediatr Clin North Am 1987;34:571
590.
5. Simonsen O, Grubb A, Thysell H. The blood serum concentration of cystatin C (gamma-trace) as a measure of the glomerular
ltration rate. Scand J Clin Lab Invest 1985;45:97 101.
6. Grubb A, Simonsen O, Sturfelt G, Truedsson L, Thysell H.
Serum concentration of cystatin C, factor D and 2-microglobulin as a measure of glomerular ltration rate. Acta Med
Scand 1985;218:499 503.
7. Grubb A. Diagnostic value of analysis of cystatin C and protein
HC in biological uids. Clin Nephrol 1992;38(suppl 1):S20
S27.
8. Finney H, Newman D, Gruber W, Merle P, Price C. Initial
evaluation of cystatin C measurement by particle-enhanced
immunonephelometry on the Behring nephelometer systems
(BNA, BN II). Clin Chem 1997;43:1016 1022.
9. Laterza O, Price C, Scott M. Cystatin C: an improved estimator
of glomerular ltration rate? Clin Chem 2002;48:699 707.
10. Filler G, Witt I, Priem F, Ehrich J, Jung K. Are cystatin C and
2-microglobulin better markers than serum creatinine for prediction of a normal glomerular ltration rate in pediatric subjects? Clin Chem 1997;43:1077 1078.
11. Bokenkamp A, Domanetzki M, Zinck R, Schumann G, Byrd D,
Brodehl J. Cystatin Ca new marker of glomerular ltration
rate in children independent of age and height. Pediatrics 1998;
101:875 881.
12. Randers E, Krue S, Erlandsen EJ, Danielsen H, Hansen L. Reference interval for cystatin C in children. Clin Chem 1999;45:
1856 1858.
13. Finney H, Newman D, Thakkar H, Fell J, Price C. Reference
ranges for plasma cystatin C and creatinine measurements in

Cystatin C in Pediatric Liver Transplantation

14.

15.

16.

17.

18.

19.

20.

21.

22.

premature infants, neonates and older children. Arch Dis Child

2000;82:71 75.
Harmoinen A, Ylinen E, Ala-Houhala M, Janas M, Kaila M,
Kouri T. Reference intervals for cystatin C in pre- and full-term
infants and children. Pediatr Nephrol 2000;15:463 465.
Filler G, Priem F, Lepage N, Sinha P, Vollmer I, Clark H, et al.
-trace protein, cystatin C, 2-microglobulin and creatinine
compared for detecting impaired glomerular ltration rates in
children. Clin Chem 2002;48:729 736.
Bokenkamp A, Domanetzki M, Zinck R, Schumann G, Byrd D,
Brodehl J. Cystatin C serum concentrations underestimate glomerular ltration rate in renal transplant recipients. Clin Chem
1999;45:1866 1868.
Krieser D, Rosenberg A, Kainer G, Naidoo D. The relationship
between serum cystatin C, serum creatinine and glomerular ltration rate in pediatric renal transplant recipients: a pilot study.
Pediatr Transplant 2002;6:392 395.
Plebani M, DallAmico R, Mussap M, Montini G, Ruzzante N,
Marsi R, et al. Is serum cystatin C a sensitive marker of glomerular ltration rate? A preliminary study on renal transplant
patients. Ren Fail 1998;20:303 309.
Hermida J, Romero R, Tutor J. Relationship between serum
cystatin C and creatinine in kidney and liver transplant patients.
Clin Chim Acta 2002;316:165 170.
Schuck O, Gottfriedova H, Maly J, Jabor M, Stollova M, Bruzkova I, et al. Glomerular ltration rate assessment in individuals
after liver transplantation based on serum cystatin C levels. Liver
Transpl 2002;8:594 599.
Rigden SPA. Chronic renal failure. In: Postlethwaite RJ, ed.
Clinical Pediatric Nephrology. Oxford, UK: Butterworth-Heinemann, 1994:266.
Berg U, Ericzon G, Nemeth A. Renal function before and long
after liver transplantation in children. Transplantation 2001;72:
631 637.

349

23. Holt RI, Baker AJ, Miell JP. The pathogenesis of growth failure
in paediatric liver disease. J Hepatol 1997;27:413 423.
24. Holt RI, Baker AJ, Jones JS, Miell JP. The insulin-like growth
factor and binding protein axis in children with end-stage liver
disease before and after orthotopic liver transplantation. Pediatr
Transplant 1998;2:76 84.
25. Holt RI, Broide E, Buchanan CR, Miell JP, Baker AJ, Mowat
AP, Mieli-Vergani G. Orthotopic liver transplantation reverses
the adverse nutritional changes of end-stage liver disease in children. Am J Clin Nutr 1997;65:534 542.
26. Donadio C, Lucchesi A, Ardini M, Giordani R. Cystatin C,
2-microglobulin and retinal-binding protein as indicators of
glomerular ltration rate: comparison with plasma creatinine.
J Pharm Biomed Anal 2001;24:35 42.
27. Abrahamson M, Barrett AJ, Salvesen G, Grubb A. Isolation of six
cysteine proteinase inhibitors from human urine. J Biol Chem
1986;261:11282 11289.
28. Leung N. A tale of two markers. Liver Transpl 2002;8:600 602.
29. Woitas P, Stoffel-Wagner B, Flommersfeld S, Poege U, Schiedermaier P, Klehr H, et al. Correlation of serum concentrations of
cystatin C and creatinine to inulin clearance in liver cirrhosis.
Clin Chem 2000;46:712 714.
30. Orlando R, Mussap M, Plebani M, Piccoli P, De Martin S,
Floreani M, et al. Diagnostic value of plasma cystatin C as a
glomerular ltration marker in decompensated liver cirrhosis.
Clin Chem 2002;48:850 858.
31. Gerbes A, Gulberg V, Bilzer M, Vogeser M. Evaluation of cystatin C concentration as a marker of renal function in patients
with cirrhosis of the liver. Gut 2002;50:106 110.
32. Cimerman N, Mesko Brguljan P, Krasovec M, Suskovic S, Kos J.
Serum cystatin C, a potent inhibitor of cysteine proteinases, is elevated in asthmatic patients. Clin Chim Acta 2000;300:8395.
33. Bokenkamp A, van Wijk J, Lentze M, Stoffel-Wagner B. Effect
of corticosteroid therapy on serum cystatin C and 2-microglobulin concentrations. Clin Chem 2002;48:1123 1126.