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					   Cystatin C as a Marker of GFR

Is Cystatin C a Better Marker than Creatinine
            for Prediction of GFR?




                                                       cystatin C




eindscriptie in het kader van het vak Algemene Klinische Chemie

G.A. Harks
                                   Cystatin C as a Marker of GFR


CONTENTS

Introduction —— 1

Serum Creatinine as a Marker of GFR —————— 2

Serum Cystatine C as a Marker of GFR ——— 3

Methods to Determine Cystatine C Concentrations  5

Conclusion ——— 9

References ———— 10




                           2
                                                 Cystatin C as a Marker of GFR


Introduction
The glomerular filtration rate (GFR) is a good indicator for the renal function
and there are several methods to determine the GFR.

An ideal endogenous marker of glomerular filtration rate should be produced
at a constant rate and be eliminated exclusively by glomerular filtration. Under
such conditions its steady state concentration reflects the GFR.
Creatinine clearance is the best clinically useful test to determine GFR. This
determination is however not convenient for the patient while it requires the
collection of urine for 24 hours.
Serum creatinine is the marker most widely used to predict GFR, although
several drawbacks have been identified1.Tubular secretion plays an important
role in creatinine elimination with declining GFR and creatinine production
varies considerably intra- and interindividually. Creatinine is produced in
skeletal muscle and its release into the blood plasma is proportional to the
muscle mass. Therefore creatinine values have to be adjusted for body
composition.
Unlike creatinine, cystatin C is produced at a constant rate by all nucleated
body cells. Its elimination from the circulation is almost exclusively by
glomerular filtration. Filtered cystatin C is reabsorbed and catabolized
completely in proximal renal tubular cells2.

Measuring GFR following the injection of one or several suitable marker
substances is probably the most accurate method to determine the severity of
renal insufficiency3. The expense of radio-isotopic determinations of the GFR
(e.g. with 51Cr-EDTA, 99mTc-DTPA or 99mTc-DMSA) restricts however their
use.




                                       3
                                                 Cystatin C as a Marker of GFR


Serum Creatinine as a marker of GFR
Creatinine is most commonly used for the clinical assessment of GFR .
The use of serum creatinine as a marker for the GFR is based on a steady-
state mass balance, with the assumptions that the rate of its appearance into
the blood stream is constant and balanced solely by the rate of filtration
through the glomerulus, leading to elimination in the absence of tubular
secretion or reabsorption4.
These assumptions are however not rigorously true.

Creatinine release into the blood plasma is proportional to the muscle mass,
and may vary from 0.5-0.6 mg/dl to 1.4-1.5 mg/dl for adults with respectively
little and much muscle mass and normal renal function3.
In childhood creatinine concentrations are considerably lower and highly
variable with age and body growth in children and an impairment of GFR may
easily be overlooked in young children1,2. Serum creatinine determinations
have especially for children under 4 years an increased imprecision because
of their small muscle mass4. Assessing a normal GFR accurately even with
adjustment for body composition remains difficult.

Though creatinine is freely filtered by the glomeruli, it is to some extent both
secreted and reabsorbed by the tubulus resulting in an underestimation of the
    3,5
GFR . The tubular secretion and reabsorption increases as the GFR
decreases.

Non-renal events may influence serum creatinine levels5. Drugs compete with
creatinine concerning tubular secretion, ketoacidosis and high levels of
bilirubin interfere in some procedures for creatinine determination. Ingestion
of high amounts of cooked meat produces an increase in serum creatinine.

The above mentioned drawbacks in the use of serum creatinine as a GFR-
marker make it difficult to detect a small decrease of GFR and therefore
significantly reduce the clinical usefulness of serum creatinine and emphasize
the need for better GFR markers.




                                       4
                                                               Cystatin C as a Marker of GFR


Serum Cystatin C as a marker of GFR
Cystatin C (cysC) is a low molecular weight, 122 amino acid, nonglycosylated
basic (pI = 9.3) protein of the cystatin superfamily of cysteine proteinase
inhibitors. Cystatin C has been described as the product of a housekeeping
gene that is expressed in all nucleated cells, implying a stable production
rate. The low molecular weight (Mr = 13,359 kDa) and stable production rate,
even in inflammatory conditions 5, mean that it is freely filtered by the glomerili
and that it is neither secreted nor reabsorbed as an intact molecule.
The properties of cystatin C indicate that its serum concentration should be a
good marker for GFR and might replace serum creatinine.

Correlation and sensitivity
Several studies have shown that the reciprocal of cystatin C correlated
significantly better with GFR than the reciprocal of creatinine. This better
correlation was not only measured in adult populations 6,7,8, but also in
children2,5 (Table 1).
It should however be noticed that these correlations only indicate a relative
agreement, because the serum concentrations are compared with a “gold
standard” for GFR. The correlation       also depends on the range of
concentration values used. The correlation coefficient is better if a wider
concentration range is used.

Table 1. Correlation of Serum CysC and Serum Creatinine with GFR.
    gold standard      correlation (r)        correlation (r)      meth.   No. of    age    ref.
                      1/cysC vs GFR         1/creat. vs GFR                subj.     (yr)
Cr-EDTA clearance           0.83                   0.67            PETIA     69      1-16      5
 inulin clearance           0.88                   0.72            PETIA    184      0-18      2
                                      a                       a
 inulin clearance     0.765 (4-12yr)        0.841 (4-12 yr)        PETIA     26      4-12      4
                                        a                      a
                     0.869 (12-19 yr)       0.892 (12-19 yr)       PETIA     34     12-19
Tc-DTPA clearance           0.88                   0.53             EIA     206                8
 iohexol clearance          0.87                   0.71            PETIA     31     19-61      7
Cr-EDTA clearance           0.81                   0.50            PETIA     51      8-81      6
a
 in plasma
EIA    = enzyme immunoassay
PETIA = particle-enhanced turbidimetric immunoassay

Grubb (1992), Pergande and Jung (1993), Kyhse-Andersen (1994), Newman
(1995) and Tian (1997) have indicated that cystatin C is a more sensitive
GFR-marker in adult populations than creatinine.
A superior diagnostic accuracy in predicting reduced GFR of serum cystatin C
compared to that of serum creatinine was also observed in children by Helin
(1998) and Bökenkamp (1998).

age
An essential advantage of serum cystatin C compared with the conventionally
used serum creatinine is the age independence of this parameter. No
significant correlation between Cys-C concentrations and age was observed
unlike the strong correlation between creatinine and age 1,2,5. An increase in
the cystatin C levels with age was noted by Norlund, corresponding to the age
related decrease of the GFR9.



                                                 5
                                                Cystatin C as a Marker of GFR



gender
Serum creatinine has showed to display a large concentration difference
between sexes9.
In most studies no sex difference between cystatin C levels was
observed5,6,7,9,10. Pergande and Jung however found cystatin C
concentrations in sera that were significantly higher in men than in women 8.

stability
Storage time proved to have no systematic effect on cystatin C
measurements.
Cystatin C has proved to be very stable against physical and chemical
degradation2,8.




                                      6
                                                 Cystatin C as a Marker of GFR


Methods to Determine Cystatin C Concentrations
To be able to exploit the value of serum cystatin C as a GFR marker reliable
reference intervals are required. The quality of practical usefulness depends
on the precision of the method used for quantitation, selection of reference
population and intra-individual variation of CysC9.

Löfberg and Grubb developed the first enzyme immunoassay for quantifying
cystatin C in human biological fluids in 1979 and later recommended this as a
kidney function test, despite of its time consuming procedure and limited
analytical sensitivity.
Subsequently more-sensitive radio-fluorescence and various enzyme
immunoassays were developed.
In 1993 a sandwich immunoassay was developed to determine cystatin C
using commercially available antibodies.
 In 1994-1995 two rapid fully automated latex particle-enhanced turbidimetry
assays for cystatin C were introduced6,7 and in 1997 a rapid automated
method based on particle-enhanced nephelometry was developed10.

Sandwich immunoassay
The quality of an enzyme immunoassay is decisively influenced by the purity
of antigen used. Different procedures cause heterogeneity of
immunoreactants.
Heterogeneity of calibration materials used causes a wide range of normal
values8.
Pergande and Jung (1993) developed a direct sandwich immunoassay for
determining cystatin C in serum and this was the first time that commercially
available antibodies were used8. Before antibodies and antigens were
prepared by the laboratory wanting to use them.
Rabbit anti-cystatin C antibodies (DAKO Diagnostika) were used in the assay
that takes 2 hours and absorbances were measured at 492 nm.
The within-assay variation (CV) was <5% and the between assay variation
was 8.8%. The detection limit, defined as the mean + 3SD of the blank value
expressed as cystatin C concentration and calculated from the measured
absorbances of 20 determinations of the buffer solution without antigen in the
assay, was 0.9g/ml.
The cystatin C concentrations in sera of 31 outpatients with suspected kidney
damages were measured. The values were correlated with GFR determined
       99m
by         Tc-diethylenetriaminopentaacetic    acid   (DTPA)     (Table     1).
Concentrations in sera from men were significantly higher than in women
(mean and SD: 2.14  0.31 vs 1.78  0.26 mg/L). The diagnostic sensitivity of
cystatin C was 88.2% of that of the standard isotope clearance method.

Tian (1997) measured serum cystatin C levels with an enzyme-linked
immunosorbent assay (ELISA) in 33 healthy people and 35 with various renal
diseases11. The reference interval with 95% ranges was 0.47-1.03 mg/L in the
serum of healthy people.




                                       7
                                                     Cystatin C as a Marker of GFR


Paritcle-enhanced immunoturbidimetry assay (PETIA)
Turbidity causes the attenuation of the intensity of the incident beam of light
as it passes through a solution of particles. The measurement of the
decrease in intensity of the incident light beam that is caused by scattering,
reflectance and absorption of the light is called turbidimetry12.

In most recent studies cystatin C was determined using a commercially
available kit     (DAKO Inc.1,2,5,9) for an automated particle enhanced
turbidimetric method. Analyses were performed in a Hitachi 717 analyzer 1,2,4,
on a Monarch 2000 centrifugal analyzer6 on a COBAS MIRA PLUS
instrument (Roche Inc. Basel, Switzerland)5,9 or on a COBAS FARA
                                           7
instrument ( Roche Inc, Basel, Switzerland) .
With the development of an automated immunoturbidimetric method, cystatin
C serum concentrations can now be measured with little technical effort on
standard laboratory equipment2. PETIA technology can be applied to all
clinical analyzers used for the measurement of creatinine.

Kyhse-Anderson (1994) described a fully automated PETIA for cystatin C in
undiluted serum and EDTA -plasma7. The change in absorbance at 340 nm
was measured and the assay range was 0.4-14.1 mg/L cystatin C. Ninety
samples could be analyzed per hour and urgent samples in 7 minutes. The
detection limit, defined as the concentration corresponding to the mean of 10
determinations of the zero calibrator signal + 3 SD was 0.15 mg/L.
The within and between run imprecision were respectively 0.9% and 2.2%
No interference with rheumatoid factors, bilirubin, hemoglobin and
triglycerides was observed.

Newman (1995) developed an assay for serum cystatin C using latex particle-
enhanced immunoturbidimetry6. Latex particles were covalently coupled to
rabbit anti-human cystatin C and the samples were monitored in a disposable
cuvette rotor at 340 nm. Intra- and interassay imprecision were <3% and <5%
respectively. The detection limit of the assay was 0.027 mg/L cystatin C.
The PETIA method was not influenced by icterus or hemolysis, both of which
cause interference in creatinine assays.

Norlund (1997) measured serum and plasma cystatin C concentrations of 259
healthy individuals (both sexes) and recommended reference intervals for
practical clinical use (Table 2 and 3)9.

Table 2. Statistical Data for the Serum and Plasma levels of Cystatin C in the
Reference Population of Healthy Individuals.
                                                n         mean (mg/L)        SD
    S-cystatin C           men                 121           1.08           0.19
                          women                121           1.03           0.18
    P-cystatin C           men                 118           1.02           0.21
                          women                118           0.99           0.18

Table 3. Recommended Serum Reference Intervals for GFR.
                                   age range                95% reference interval
                                                                   (mg/L)
        Cystatin C                  20-50 yr                      0.70-1.21



                                       8
                                                 Cystatin C as a Marker of GFR


                                     50+ yr                    0.84-1.55

Filler (1997) measured cystatin C concentrations in 216 patients (0.8-18
years) with PETIA and calculated a 95% reference interval with an upper limit
of 1.38 mg/L cystatin C1. A cystatin C concentration > 1.38 mg/L suggests an
impaired GFR irrespective of age.

Bökenkamp (1998) examined serum cystatin concentrations in 184 children 2.
A cutoff cystatin C concentration of 1.39 mg/L had 90% sensitivity and 86%
specificity for detecting abnormal GFR.
The reference interval in a different group of 195 healthy children and
adolescents was 0.70-1.38 mg/ml13.
One cystatin C measurement cost DM 8.27 compared with DM 0.60 per
creatinine determination.

Helin (1998) used the serum cystatin C concentrations of 56 children with
normal GFR to define a preliminary reference interval (mean ± 1.96 SD) 5.
This was found to be 0.63-1.33 mg/l and the imprecision of the method was
3.2% using a control sample with 1.28 mg/l.. The reference interval for an
adult population was found to be 0.61-1.21 mg/l. The diagnostic sensitivity of
this interval is 100% and the specificity 98% for the identification of patients
with reduced GFR.

Paritcle-enhanced immunonephelometry assay (PENIA)

Nephelometry is defined as the detection of light energy scattered or reflected
towards a detector that is not in the direct path of the transmitted light 12.

Nephelometry assays have been proposed as being potentially more
sensitive than turbidimetric assays. Nonspecific background scatter of
biological samples however requires high sample predilutions and reduces
achievable detection limits.
Finney (1997) described a fully automated and rapid particle-enhanced
nephelometric immunoassay (PENIA) for measuring serum cystatin C on
                                              5
Behring nephelometer systems (BNA, BN II) . The sample volume was 80l
and the sample had to be diluted 100 times before analysis. The light source
was an infrared high-performance light-emitting diode (840nm). The signal
change between 10 seconds and 6 min was monitored in a cuvette within a
fixed cuvette rotor segment. Each sample was analyzed in 6 minutes with 75
samples per batch.
The assay covered the range 0.23-7.25 mg/L up to 7 times the normal value.
The intra- and interassay imprecision were <3.3% and 4.5% respectively.
Hemoglobin, bilirubin, triglycerides, rheumatoid factor and myeloma
paraprotein did not interfere with the assay.
For clinical use of PENIA for detecting cystatin C are reference ranges
determined with PENIA necessary.




                                       9
                                                       Cystatin C as a Marker of GFR

                                                                                 10
Table 4. Comparison of Immunoassays for Determining Cystatin C in Serum .
                                       a
    method      LOD (g/ml)   CV (%)       ref. inter., mean ± SD    No. of       proc.
                                                    (mg/ml)         subjects   duration (h)
     RID           300          11          1.3 ± 0.26 (0.72-1.7)      46          ~38
     EIA            30        10-12         1.1 ± 0.42 (0.63-2.5)      30          ~16
     EIA           1.3         n.d.         0.96 ± 0.20 (0.6-1.7)     100         16-21
     FIA           ~1          n.d.                   n.d.                        1or 3
     EIA           n.d.        n.d.               1.10 ± 1.15          20          ~4.5
     EIA           1.9         4-5           0.75 ± 0.65 < 20 yr       85           ~5
                                             1.34 ± 0.95 > 20 yr      189
     EIA           0.195       4-8         1.25 ± 0.22 (0.86-1.7)      50         ~1.5
     EIA            0.9        3-9           1.78 ± 0.26 female        33          2
                                              2.14 ± 0.31 male
       11
     EIA           n.d.         n.d.              (0.47-1.03)          68         ~2.5
         9
    PETIA          n.d.       2.5-3.1       (0.70-1.21) 20-50 yr      259
                                             (0.84-1.55) 50+ yr
            1
    PETIA                                            < 1.38           216
         2
    PETIA                                            < 1.39           184             1
         2
    PETIA                                         (0.70-1.38)         195             1
         5
    PETIA                       3.2               (0.63-1.33)          56
                                                             b
    PETIA          150        2.0-3.2            (0.61-1.21)           27        7 mins
    PETIA           27          3-5                  <1.25                       5 mins
                                                             b
    PENIA          170          3-5              (0.64-1.04)          30         6 mins
a
  Range of intra- and interassay variations
b
  Range, mg/L
RID, radial immunodiffusion; EIA, enzyme immunoassay; PETIA, particle-enhanced
turbidimetric immunoassay; PENIA, particle-enhanced nephelometric immunoassay;
n.d., not done.




                                           10
                                                 Cystatin C as a Marker of GFR


Conclusion
The use of serum creatinine as a marker for GFR has serious drawbacks. Its
muscle mass and therefore age dependency and partial reabsorption by the
tubulus restricts the use of serum creatinine.

Cystatin C has a stable production rate and is solely filtered by the glomerili.
Its reciprocal serum concentration correlates significantly better with GFR
than the reciprocal serum creatinine concentration. In most studies a superior
diagnostic accuracy in predicting reduced GFR of serum cystatin C compared
to serum creatinine was observed for adults as well as for children.
The apparant age and gender independency of serum cystatin C make it
together with the above mentioned advantages a better marker for GFR than
serum creatinine.

Immunoassays were developed for cystatin C and the introduction of the use
of commercially available antibodies improved their quality. The time
consuming procedures however restrict their clinical use.
Serum cystatin C concentrations can now be measured with little technical
effort on standard laboratory equipment by automated rapid particle-
enhanced immunotubidimetry or nephelometry. Interferences with serum
components that deteriorated serum creatinine determinations were not
observed with this method.

To exploit the value of serum cystatin C as a GFR marker reliable reference
intervals for the different methods are necessary. More research has to be
done to obtain enough data to be able use serum cystatin C for standard
clinical assesment of GFR.




                                       11
                                                    Cystatin C as a Marker of GFR


References

1     Filler, G., Witt, I., Priem, F., Ehrich, J.H., Jung, K., Are Cystatin C and 2-
      Microglubulin Better Markers than Serum Creatinine for Prediction of a
      Normal Glomerular Filtration Rate in Pediatric Subjects?, Clinical Chemistry,
      43 (1998) 1077-1078.
2     Bokenkamp, A., Domanetzki, M., Zinck, R., Schumann, G., Byrd, D., Brodehl,
      Cystatin C- A New Marker of Glomerular Filtration Rate in Children
      Independent of Age and Height, J., Pediatrics, 101 (1998) 875-881.
3     Kortleven, J., http://www.imicos.be/hosted/vjz/teksten/nierfunctie.htm.
4     Stickle, D., Cole, B., Hock, K., Hruska, K.A., Scott, M.G., Correlation of
      Plasma Concentrations of Cystatin C and Creatinin to Inulin Clearance in a
      Pediatric Population, Clinical Chemistry, 44 (1998) 1334-1338.
5     Helin, I., Axenram, M., Grubb, A., Serum Cystatin C as a Determinant of
      Glomerular Filtration Rate in Children, Clinical Nephrology, 49 (1998) 221-
      225.
6     Newman, D.J., Thakkar, H., Edwards, R.G., Wilkie, M., White, T., Grubb,
      A.O., Price, C.P., Serum Cystatin C Measured by Automated Immunoassay:
      A More Sensitive Marker of Changes in GFR than Serum Creatinine, Kidney
      International, 47 (1995) 312-318.
7     Kyhse-Anderson, J., Schmidt, C., Nordin, G., Andersson, B., Nilsson-Ehle,
P.,   Lindstrom, V., Grubb, A., Serum Cystatin C, Determined by a Rapid,
      Automated Particle-Enhanced Turbidimetric Method, Is a Better Marker than
      Serum Creatinine for Glomerular Filtration Rate, Clinical Chemistry, 40 (1994)
      1921-1926.
8     Pergande, M., Jung, K., Sandwich Enzyme Immunoassayt of Cystatin C in
      Serum with Commercially Available Antibodies, Clinical Chemistry, 39 (1993)
      1885-1890.
9     Norlund, L., Fex, G., Lanke, J., Von Schenck, H., Nilson, J.E., Leksell, H.,
      Grubb, A., Reference intervals for the glomerular filtration rate and cell-
      proliferation markers: serum cystatin C and 2-microglobulin/cystatin C ratio,
      Scan J Clin Lab Invest, 57 (1997) 463-470.
10    Finney, H., Newman, D.J., Gruber, W., Merle, P., Price, C.P., Initial
      evaluation     of     cystatin    C    measurement       by   particle-enhanced
      immunonephelometry on the Behring nephelometer systems (BNA, BN II),
      Clinical Chemistry, 43 (1997)           1016-1022.
11    Tian, S., Kusano, E., Ohara, T., Tabei, K., Itoh, Y., Kawai, T., Asano, Y.,
      Cystatin C measurement and its practical use in patients ith various renal
      diseases, Clinical Nephrology, 48 (1997) 104-108.
12    Burtis, Ashwood, Tietz Fundamentals of Clinical Chemistry, 4th ed., W.B.
      Saunders Company, Philadelphia, 1996 78-79.
13    Bokenkamp, A., Domanetzki, M., Zinck, R., Schumann, G., Brodehl, J.,
      Reference Values for Cystatin C Serum Concentrations in Children, Pediatr.
      Nephrol., 12 (1998) 125-129.
14    Grubb, A., Clinical Nephrology, 38 Suppl. 1 (1992) S20-S27.




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