Cystatin C Dr. Mussap by ddh19362

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									1
ANALYTICAL METHODS
AND STANDARDIZATION




     2
CYSTATIN C BIOLOGICAL VARIABILITY

                  Keevil BG, et   Cimerman N,
                    al., 1998      et al., 2000
COMPONENTS
                    (PETIA)           (EIA)

CVA, %                8.9             8.8
CVG, %                8.1             5.7
II                    1.63            2.63
RCV (CD), %           44.3            48.2

              3
     ANALYTICAL METHODS
Immunonephelometric assay for cystatin C is
superior to other methods (Priem F, et al. Clin Chem
2001;47:2181)
A meta-analysis of correlation coefficients of 14
studies that used immunonephelometric assay
(1,698 subjects) vs 21 other techniques (1,953
subjects) showed:
     - mean r = 0.846 vs 0.784
     - 95% CI = 0.832 ÷ 0.859 vs 0.766 ÷ 0.801
Lack of superiority of 1/Cys C over 1/Cr in
previous studies may reflect differences caused
by assay methods (Dharnidharka VR, et al. Am J Kidney
Dis 2002;40:221-6
                    4
PETIA



 Hitachi                Advia 1650
 Modular P              Cobas C501
 Vitros                 Olympus
 Architect Ci8200       Monarch
           5
               …………..
                              PETIA                      PENIA

Manufacturer            DakoCytomation and          Siemens Healthcare
                            Gentian AS                  Diagnostics

Principle of method       Measurement of          Measurement of diffuse
                          transmitted light               light

Wavelength (nm)               340 - 650          840 (infrared high-
                                                 performance light-
                                                 emitting dio de)
Antibody              Rabbit antibody against    Rabbit antibody against
                      human cystatin C,          human cystatin C,
                      chemically coupled with    covalently coated with 80-
                      38-nm diameter unif orm    nm diameter uniform
                      polystirene particles      chloromethylstirene
                      (latex)                    particles (latex)
Calibration material Human cyst. C-stripped      Purified urinary cystatin
                     serum spiked with           C
                     recombinant Cy statin C
Instrument               Various automated       Siemens BN II, Vista (?)
                       biochemistry analysers

Performances          Easily adaptable on        Slightly superior in terms
                      Cobas, Modular, Hitachi,   of limit of de tection,
                      Immage, Advia,             sensitivity to
                                                 interferences, and intra-
                                                 and inter-batch precision

                                                       Séronie-Vivien S, et al. CCLM 2008;46: Ahead of print
                                    6
Séronie-Vivien S, et al. CCLM 2008;46: Ahead of print
7
Nephelometry tends to detect smaller
immunoaggregates than turbidimetry
The nephelometer monitors an increase in
light intensity against a low background
signal which accounts for non-specific
background scatter (better specificity)
The Siemens N-Latex cystatin C assay is the
only one to having FDA approval

           8
Original Report: Laboratory Investigation

Comparing Measures of Cystatin C in Human Sera by
Three Methods
Mohammad Akhtar Hossaina, b, Mahmoud Emaraa, Hamdi El moselhia,
Ahmed Shokera, c
aSaskatchewan     Transplant Program, St. Paul's Hospital
bDepartment    of Community Health and Epidemiology
cDivision   of Nephrology, Department of Medicine, University of Saskatchewan, Saskatoon, Sask.,
Canada


Am J Nephrol 2009;29:381-391 (DOI: 10.1159/000168486)

•   Sandwich human cystatin C ELISA (BioVendor)
•   PENIA on BN II
•   PETIA Dako on Hitachi 704                               80 Healthy adults
                                                            20 Patient with renal or CV diseases
                                  9
10
Original Report: Laboratory Investigation

Comparing Measures of Cystatin C in Human Sera by Three Methods
Mohammad Akhtar Hossaina, b, Mahmoud Emaraa, Hamdi El moselhia, Ahmed Shokera, c
aSaskatchewan
                  Transplant Program, St. Paul's Hospital
bDepartment    of Community Health and Epidemiology
cDivision   of Nephrology, Department of Medicine, University of Saskatchewan, Saskatoon, Sask., Canada
Am J Nephrol 2009;29:381-391 (DOI: 10.1159/000168486)




                                                            11
There is a relatively poor accuracy between PETIA and
PENIA,   suggesting   that  they  may   not  be  used
interchangeably
Regression analysis suggested that a normalization factor
of 0.6-0.7 may improve the r value between the ELISA and
the two other methods (0.66 = best performance) as well as
may decrease differences in reference values
Normalization of the ELISA method significantly improved
the precision and accuracy at low cystatin C concentrations
Normalized ELISA values may be used interchangeably with
those obtained by PETIA

                     Hossain MA, et al. Am J Nephrol 2009;29:381-91
                12
The main limitation of cystatin C immunoassay
determination lies in the standardization of platforms
which will require an international reference preparation
to allow for valid comparison




1. Agreed definition of the measurand
2. Primary reference method
3. Primary and secondary reference materials
4. EQAS for cystatin C (evaluation of comparability of
   results and performance)
                13
                            STANDARDIZATION OF
                          CYSTATIN C: DEVELOPMENT
                         OF PRIMARY AND SECONDARY
                          REFERENCE PREPARATIONS

                                                  Cp (g/L) =
Primary reference                              5.197 ± 0.0078
preparation

  Purified recombinant
  human cystatin C 5.2 g/L




   High purity, homogeneity
   and identity of the intact
                                    Cystatin C is
    recombinant cystatin C          monomeric
                    14
                              STANDARDIZATION OF
                            CYSTATIN C: DEVELOPMENT
                           OF PRIMARY AND SECONDARY
                            REFERENCE PREPARATIONS

Secondary reference preparation                   Matrix of 6.220 mL
                                                  Cyst. C = 0.24 mg/L
     Human serum pool:
     - 29 healthy donors from Lund
     - 15 healthy donors from Marburg

                                        Primary reference
                                        preparation
         4468 vials sent to the
         Institute for
         Reference material
         and measurements
         (IRMM)
                      15
                      STANDARDIZATION OF
                    CYSTATIN C: DEVELOPMENT
                   OF PRIMARY AND SECONDARY
                    REFERENCE PREPARATIONS

The value assignment will be carried out using
single radial immunodiffusion, turbidimetry,
and nephelometry
The Secondary Reference Preparation is
expected to be released in the last part of 2008,
when the commercial calibrators can be
adjusted accordingly

                   Scand J Clin Lab Invest 2008;68(Suppl.241):67-70
              16
        CLINICAL ACCURACY
         EVIDENCE-BASED

Until a few years ago, few physicians outside the
field of nephrology had heard of cystatin C
Over    the   past   4   years,   several  larger
epidemiological studies have found cystatin C is
a better predictor of longitudinal outcomes than
creatinine and creatinine-based GFR estimates
                     Editorial. Am J Kidney Dis 2008;51:358-61
             17
     Clin Biochem 2008;29:47-62
18
   META-ANALYSIS STUDIES
   META-ANALYSIS
54 studies (4,492 subjects): GFR correlated better
with cystatin C than with creatinine (reciprocal
values):
    r = 0.816 vs 0.742
    95% CI = 0.804 ÷ 0.826 vs 0.726 ÷ 0.758

The mean area under the ROC curve was greater
for cystatin C (0.926) than for creatinine (0.837)
Serum cystatin C was superior                               to     serum
creatinine as a marker of GFR

                      Dharnidharka VR, et al. Am J Kidney Dis 2002;40:221-6
                 19
       DIAGNOSTIC ODDS RATIO AND SLOPE VALUES
       FOR CYSTATIN C AND SERUM CREATININE

                                     DIAGNOSTIC OR                     SLOPE*
       DIAGNOSTIC TEST
                                        (95% CI)                      (95% CI)


       Cystatin C                        54.001                      0.0199
                                   (30.175 to 96.641)           (-0.394 to 0.434)

       Serum Creatinine                 16.297                       -0.2934
                                   (8.348 to 31.785)            (-0.603 to 0.017)
TT T




*      If the slope is not significantly different from 0, the diagnostic accuracy
       does not vary with the cut-off level used

                                            Roos JM, et al. Clin Biochem 2007;40:383-91
                              20
                                   LIKELIHOOD RATIOS (LRs)           HETEROGENEITY

          TEST
                                 Positive          Negative        Positive   Negative
      (n=154)                    (95% CI)          (95% CI)          LR         LR

S-Cystatin C                    10.11                 0.23         P = 0.87   P = 0.82
                            (4.12-24.81)          (0.16-0.34)

S-Creatinine                    8.57                  0.34         P = 0.35   P = 0.35
                            (3.02-24.36)          (0.23-0.48)


Cut-off levels:   GFR = 60-79 mL/min/1.73m2 (Inulin clearance in all the groups)
                  Cystatin C = 0.9 - 1.4 mg/L

                                              Roos JM, et al. Clin Biochem 2007;40:383-91
                            21
Cystatin C’s likelihood ratio is large and
conclusive at ruling in (LR>1), but small
at ruling out (LR<1) renal impairment
Serum creatinine has a moderate ability
to rule in renal impairment compared to
cystatin C, but small ability to rule it out

                 Roos JM, et al. Clin Biochem 2007;40:383-91
            22
Cystatin C has greater sensitivity to detect reduced
GFR in CKD than creatinine and other low-Mr
proteins
Serum cystatin C levels increase already with mild
reduced GFR of 70 to 90 mL/min/1.43m2 in the
“creatinine-blind range”
Cystatin C serum levels rise earlier in acute kidney
injury on ICU, after cardiac surgery, liver
transplantation, cisplatin chemotherapy and
coronary angiography, following uninephrectomy
and in progressive diabetic nephropathy
              23
DIAGNOSTIC EFFICIENCIES OF SERUM CREATININE AND
DIAGNOSTIC EFFICIENCIES OF SERUM CREATININE AND
    CYSTATIN C IN RENAL TRANSPLANT PATIENTS
    CYSTATIN C IN RENAL TRANSPLANT PATIENTS
       1.00



                                                                    • GFR (mL/min/1.73 m2)
 sensitivity (TP)




                                                                      was estimated by the
       0.75                                                           inulin clearance
                                            S-Cystatin C
                                            S-Creatinine            • Serum creatinine was
                                                                      measured by isotope
                                                                      dilution         gas
       0.50
                                                                      chromatography       -
                                                                      mass     spectrometry
                                                                      (GC-MS)

       0.25




       0.00
           0.00     0.25        0.50    0.75                 1.00
                            1-specificity                  Plebani M, et al. Renal Fail 1998
                           24
   Non-parametric ROC plot for the comparison of the
diagnostic accuracy of creatinine and that of cystatin C
              1.00



                                                                                  52 type-2 diabetic
                                                                                  patients (33 males),
              0.75                              S-Cystatin C (mg/L)               aged 38-73 years
Sensitivity




                                                S-Creatinine (umol/L)
                                                                                  Renal   biopsy   was
                                                                                  evaluated to rule out
                                                                                  non-diabetic    renal
              0.50                                                                disease
                                                                                  GFR was estimated
                                                                                  by using the 51Cr-
                                                                                  EDTA          plasma
              0.25                                                                clearance



              0.00
                     0.00   0.25         0.50            0.75              1.00

                                   1-Specificity                        Mussap M, et al. Kidney Int 2002
                                    25
CYSTATIN C         LEVELS FOR DETERMINING STAGE OF              CKD

     STAGE OF                CYSTATIN C CONCENTRATION (mg/L)
       CKD            NATIVE KIDNEY DISEASE1  TRANSPLANT RECIPIENT2

          1                          Š0.80                      Š0.87
          2                   0.80 – 1.09                    0.87 – 1.23
          3                   1.10 – 1.86                    1.24 – 2.24
          4                   1.87 – 3.17                    2.25 – 4.10
          5                          >3.17                      >4.10

1   GFR = 66.8 • (Cystatin C)-1.30
2   GFR = 76.6 • (Cystatin C)-1.16
                                        Rule AD, et al. Kidney Int 2006;69:399-405
                             26
Cystatin C production is stimulated by high doses
of corticosteroids “in vitro” and “in vivo”
Untreated thyroid dysfunction is associated with
altered cystatin C levels
It has been demonstrated a positive correlation
between mildly elevated serum cystatin C and
hsCRP levels in healthy adult population: chronic
inflammatory state associated with atherosclerosis
Increased cystatin C serum levels have been
associated with obesity and smoking
             27
Early risk stratification is essential in the management
of patients with suspected or confirmed non-ST-elevation
acute coronary syndrome
Traditionally, this stratification is performed by the use
of medical history, ECG, and biochemical markers of:
       myocardial damage
       cardiac performance
       inflammation
Measurement of kidney function have been shown to
carry independent prognostic information in this
population
                28
A significant association between serum cystatin C
and a first ischemic coronary event has been found
(Luc et al.)

Two follow-up studies in patients with established
coronary heart disease did confirm the positive
correlation between cystatin C and CRP
The pathophysiological link between cystatin C
and ischemic heart disease is micro-inflammation

               29
Studies conducted in patients with established:




… in each of these studies, cystatin C had
association with future adverse outcomes
that were independent of creatinine
                          Editorial. Am J Kidney Dis 2008;51:358-61
                  30
                    CUMULATIVE PROBABILIY
QUARTILE
                     OF MI AT 12 MONTHS


  1st                         4.4%


  2nd                         5.3%


  3rd                         12.3%

  4th                         20.3%

                Jernberg T, et al. Circulation 2004;110:2342-8
           31
                                                           4663 participants
                                                           65 years of age
                                                           and older
                                                           1004 participants
                                                           (22%) had CKD




After adjustment for traditional risk factors, prevalent stroke,
heart failure, coronary heart disease, and CRP, the persons with
no CKD and high cystatin C levels had statistically significantly
increased risk for each outcome (50% more likely to die)

                         Shlipak MG, et al. Ann Int Med 2006;145:237-46
                    32
  10
                                                                          I quartile
   9
                                                                          II quartile
   8                                                                      III quartile
                                                                          IV quartile
   7

   6

   5

   4

   3

   2

   1

   0
       All-Cause Mortality        Cardiovascular Events       Incident Heart Failure
                             Outcome by Quartiles of Cystatin C
Probability value for trend <0.001 for each outcome
                                                                         CV EVENTS
                   I: ≤0.91 mg/L                                          1.   CHD death
                  II: 0.92 - 1.05 mg/L                                    2.   Non fatal MI

                 III: 1.06 - 1.29 mg/L                                    3.   Stroke

                 IV: ≥1.30 mg/L
                                                    Ix JH, et al. Circulation 2007;115:173-9
                             33
990 ambulatory persons with Coronary
Heart Disease (CHD):


Higher serum cystatin C levels predict all-cause
mortality, CV events, and incident heart failure
independent of traditional CV risk factors
25% of persons with cystatin C levels in the highest
quartile did not have an eGFR<60 mL/min/1.73m2
Higher serum cystatin C level has similar predictive
value for adverse clinical outcomes among persons
with and without low eGFR or microalbuminuria

                           Ix JH, et al. Circulation 2007;115:173-9
              34
                         Severity of CAD at angiography according to cystatin C
In patients with non-    tertile in patients randomized to the EIS (P = 0.12).
ST elevation acute       1 VD = 1-vessel disease; EIS = early invasive strategy
coronary syndrome
who have an
increased cTn T
concentration, mild
to moderate renal
dysfunction is
associated with a
higher risk of death
and spontaneous MI
during long-term
follow-up (5.5% 1st
tertile, 7.5% 2nd, and
9.8% 3rd)
                             Windhausen F, et al. Clin Chem 2009;55:1118-25
                   35
CYSTATIN C FOR ENHANCEMENT OF RISK STRATIFICATION IN
NON-ST ELEVATION ACUTE CORONARY SYNDROME PATIENTS
            WITH AN INCREASED TROPONIN T




                       Windhausen F, et al. Clin Chem 2009;55:1118-25

               36
            QuickTime™ and a
              decompressor
     are needed to see this picture.




37     Naruse H, et al. Am J Med 2009;122:566-73
            QuickTime™ and a
              decompressor
     are needed to see this picture.




       Naruse H, et al. Am J Med 2009;122:566-73
38
                                                   Cystatin C may be
                                                   considered a strong
                                                   independent predictor
                                                   of cardiac mortality in
                                                   hospitalized patients
                       QuickTime™ and a
                                                   for worsening CHF
                         decompressor
                are needed to see this picture.    with normal to
                                                   moderately impaired
                                                   kidney function;
                                                   Cystatin C may
                                                   substantially improve
                                                   the admission risk
                                                   stratification


ROC curves of cystatin C and modified MDRD-       Naruse H, et al.
derived GFR between patients who died from
                                                  Am J Med 2009;122:566-73
cardiac causes and those39who did not.
     Shishehbor MH. Am J Cardiol 2008;101:1741-6
40
             PATIENTS WITH CKD                   MARKERS WITH MECHANISTIC LINKS

1. Significant lipoprotein derangements         1.   Apolipoproteins, total cholesterol, LDLc,
                                                     HDLc, tryglicerides

2. Heightened levels of oxidative stress        2. NO, MDA

3. Endothelial damage                           3. Homocysteine

4. Thrombosis                                   4. Lp(a), Fibrinogen

5. Inflammation                                 5. hsCRP

6. Insulin resistance and glucose intolerance   6. Fasting glucose



Reduced GFR is strongly associated with the risk of new-onset coronary
artery disease (CAD), as well as with the risk of death following an
initial miocardial infarction (MI)
                              41
Of a total of 4,680 patients seen as a tertiary preventive
cardiology program, 524 met the criterion for CKD
Homocysteine and Fibrinogen levels explained 38% of
the association between CKD and mortality despite
multiple adjustments for traditional as well as novel CV
risk factors
Homocysteine and Fibrinogen can be considered as 2
potential therapeutic targets that may affect CKD-
associated mortality beyond addressing traditional CV
risk factors
                         Shishehbor MH, et al. Am J Cardiol 2008;101:1741-6
                42
   CYSTATIN C IN ALZHEIMER’S
           DISEASE
Cystatin C has a protective role in neurodegeneration
Cystatin C binds amyloid-β and inhibits its oligomerization
and amyloidogenesis
As cystatin C is implicated in proliferation of stem cells,
reduced cystatin C in Alzheimer’s disease brain may led to
failure in neural stem cell replacement, which in turn may
contribute to progression of the disease
In various brain injuries, such as ischemia, surgery, and
epilepsy, cystatin C expression increase; however, it is not
clear whether low or high levels of cystatin C are beneficial


                 43
CYSTATIN C IN PREECLAMPSIA




     44
Cystatin C has higher pooled sensitivity (81% vs 69%)
Cystatin C has similar pooled specificity (88% vs 88%)
Cystatin C has a higher chance of detecting a true
positive renal impairment
However, the 95% CIs for the pooled diagnostic odds
ratio for the biomarkers overlap
The slope (β) for cystatin C was not significantly
different from 0

                       Roos JM, et al. Clin Biochem 2007;40:383-91
               45
 CYSTATIN C-BASED
EQUATIONS FOR eGFR




    46
Cystatin C-based equations have been
generated and validated in small data set
of patients, in single center settings, and
by   using     different   gold    standard
measurements for GFR (MDRD equation was
calculated from a data set of 1,628 Patients in a multicenter
study)

This partially explains the                     variation
between individual equations
                   47
Chew JSC, et al. Clin Biochem Rev 2008;29:47-61
48
Chew JSC, et al. Clin Biochem Rev 2008;29:47-61
49
Chew JSC, et al. Clin Biochem Rev 2008;29:47-61
50
The marked heterogeneity of:
  Cohorts, ranging from:
  - infants to adults
  - normal to markedly reduced GFR in healthy subjects
  - CKD and renal transplant patients
  Gold standard GFR measurement
  Cystatin C-based equations


    Do not permit a definitive conclusion
    whether these equations are superior
                  51
       Can be cystatin C-based equations used
       with all cystatin C measurement methods?




•   PENIA Siemens on BN II
•   PETIA Gentian on Hitachi 917
•   PETIA Dako on Hitachi 917
                   52
     Delanaye P, et al. Clin Chim Acta 2008;398:118-24
53
No internationally recommended calibrator or procedure for
determination of cystatin C levels are available
These tools are urgently needed to be able to reach more
uniform results with different cystatin C assay systems
The Dako PETIA is significantly less precise than the two
other assays, reflecting potential consequences on the
precision of cystatin C-based equations
No difference between the Dako and the Gentian PETIA for
cystatin C below 2.5 mg/L
Results from Siemens are not interchangeable with the two
other PETIA assays
                     Delanaye P, et al. Clin Chim Acta 2008;398:118-24
                54
GFR = 66.8 x (Cystatin C)-1.30

GFR = 273 x (Creatinine)-1.22 x age-0.299
if female: x 0.738




                     GFR = 76.6 x (Cystatin C)-1.16



                             Rule AD, et al. Kidney Int 2006;69:399-405

                55
GFR = (43.820.003 x height)/Cyst C0.635 x S-Creat                       0.547

*   if renal transplant: x 1.165
*   if spina bifida: x 1.57 x S-Creat   0.92



                              Zappitelli M, et al. Am J Kidney Dis 2006;48:221-30




GFR = 63.2 x (Cr/96)-0.35 x (CysC/1.2)-0.56 x (Kg/45)0.30 x (y/14)0.40

                              Bouvet Y, et al. Pediatr Nephrol 2006;21:1299-1306
                         56
… Perhaps, the most promising use of cystatin C will
be as a marker of preclinical or early kidney disease
among persons with creatinine-based eGFR in the
“normal” range (eGFR≥60 mL/min/1.73m2) but elevated
cystatin C
                Michael G. Shlipak. Am J Kidney Dis 2008; 51:358-61
                57
RECOMMENDATIONS FOR THE DIFFERENTIATED USE OF MARKERS
TO DETECT REDUCE GFR

      GFR                MARKER                     SPECIFIC INDICATION

60-90        Serum Cystatin C
20-60        MDRD equation                   In adults
             Cockcroft-G. equation           In adults
             Schwartz equation               In childrfen
             Serum Cystatin C                BMI>30 Kg/m2, nephrotic
                                             sindrome, acute kidney injury
                                             superimposed on CKD
<20          Creatinine clear.+Urea cl.
Acute kidney Serum Cystatin C
injury


                         Herget-Rosenthal S, et al. Clin Biochem 2007;40:153-61
                    58

								
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