Critically discuss the methods that
are in routine use for the
measurement of albumin in serum
and urine
Dr Frances Boa
Method questions
•
September 2012
– Write a critical discussion on the methods available for measurement of the different
forms of bilirubin.
•
March 2012
– Compare and contrast different approaches to the biochemical assessment of
glomerular filtration rate.
•
September 2011
– Write a critical description of the methods available for estimation of low-density
lipoprotein.
•
March 2011
– Write a critical description of the methods available for measurement of glycated
haemoglobin and the choice of method for specific clinical situations
•
September 2010
– Provide a critical discussion of methods available for measurement of vitamin D
concentration
•
March 2010
– Describe the analytical principles that underlie the determination of arterial blood and
venous plasma bicarbonate concentration
•
September 2009
–
Critically discuss the methods that are in routine use for the measurement of albumin in
serum and urine
(review of uALB in Clin Chem Jan 2009)
Methodology revision
• Most questions ‘critically discuss’ or ‘compare
and contrast’
• Chose an analyte
• Look at the main method groups from UKNEQAS
• Compare with methods in Tietz or similar
• Have you missed a technique / POCT?
• Imprecision, analytical range / sensitivity
• Metrological traceability
– Is there a reference method – ‘gold standard’
– How is the assay calibrated?
– Is there an international standard?
Essay structure
• Introduction
– Albumin
– What are we measuring in plasma and urine
– Different assay requirements
• Serum methods
– Table of current methods
• How they work, Advantages / disadvantages
– Reference method
– Traceability
• Urine methods
– Table of current methods (include POCT)
• How they work, Advantages / disadvantages
– Reference method
– Traceability
• Summary
Albumin
• Albumin
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66.473 kDa
585 aa’s
17 internal disulphide bonds
High content of α helical structure
4 globular domains
Binds to multiple ligands including
• Fatty acids, bilirubin, Ca2+, Mg2+
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•
•
•
•
•
•
Synthesised exclusively in the liver (~120mg/kg body wt /24h)
Both C and N terminal truncations
Significant glycation (1-10% , higher in diabetics)
T1/2 ~20 days
Has at least 5 antigenic sites
Relatively resistant to denaturation
Certified serum reference material DA470k/IFCC (2008)
UKNEQAS serum albumin
Serum albumin methods to consider
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•
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Bromocresol green
Bromocresol purple
Dry slide (bromocresol green)
Reference method – gold standard
– Immunochemical
– Mass spec – in development as candidate reference
method
• Other methods
– Electrophoresis (scanning)
– CZE
– Isoelectric focusing
• Calibrants
Useful papers - serum albumin
• Albumin standards and measurement of serum albumin with
bromocresol green
Doumas, Watson & Biggs
Clin Chim Acta 1971; 31:87-96
• New automated dye-binding method for serum albumin
determination with bromocresol purple
Pinnell & Northam
Clin Chem 1978; 24:80-6
• The measurement of albumin in serum and plasma
Hill
Ann Clin Biochem 1985; 22:565-578
Dye Binding methods
• Shift in absorption spectrum on binding to protein
• Affinity of dyes if higher for albumin than other proteins
• Specificity is increased if only the initial rate of binding is
used
• Monochromatic methods over estimate albumin in the
presence of fibrinogen and heparin – use biochromatic
methods
• Dye binding assays tend to be erroneous if the serum
protein pattern is abnormal
• Adjusted calcium calculations
Bromocresol green
Albumin + BCG
Green complex
•BCG also binds to
•
transferrin
•
lipoproteins
•
α1 & α2 globulin
•
β globulins
•Overestimates against nephelometric assays by 3 -10 g/L depending on
incubation times
•Overestimates albumin at low concentrations (<25g/L) and under
estimates at higher levels cf nephelometry
•Minimal interference from bilirubin and haemoglobin
Bromocresol purple
Albumin + BCP
Purple complex
•BCP binds to human albumin (does not react with albumin free serum,
globulin or transferrin)
•Immediate reaction
•Agrees well with immunoassays
•Considered to have interference from an endogenous uraemic toxin CMPF (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid) in
haemodialysis patients
•Bichromatic blanking – corrects for turbidity
•Increasing ionic strength of reagent reduces turbidity
•Minimal interference from bilirubin and haemoglobin
Paper most frequently quoted against
BCP method
Bromocresol purple method for serum albumin gives falsely
low values in patients with renal insufficiency
GA Maguire and CP Price
But the final sentences of the paper are usually ignored
‘the positive interference inherent in the BCG method is a more serious
drawback, applying to many more patients’ samples; its potential for
inaccuracy in any given sample is greater than in the BCP method…………Thus
we prefer to use the BCP method which for most samples gives an accurate
measurement of albumin.’
Clin Chim Acta 1986;155:83-88
Third annual report Renal Registry 2000
Chapter 9: Serum albumin and serum bicarbonate
Accessed 12/4/13
BCG vs BCP
Identity line
Y=X
Siemens Advia 2400 BCG
albumin g/L
60
55
50
45
40
35
30
25
20
15
y = 0.9494x + 7.8103
10
10
20
30
40
50
Beckman LX20 BCP albumin g/L
60
BCG vs Nephelometric albumin
Identity line
Y=X
40
Siemens Advia 2400 BCG
Albumin g/L
35
30
25
20
y=x+5
15
10
10
20
30
PRU Beckman Immage (nephelometric)
Albumin g/L
40
BCP vs Nephelometric albumin
Identity line
Y=X
Beckman LX20 BCP
Albumin g/L
35
30
25
20
y=x-2
15
10
10
15
20
25
30
PRU Beckman Immage (nephelometric)
albumin g/L
35
Immunochemical methods
• Immunonephelometry
– Requires specialist apparatus
– Serum samples require dilution
• Immunoturbidimetry
– Can be set up on most main chemistry analysers
– Serum samples require dilution
• Historical
– Single radial immunodiffusion (RID)
– Electroimmunoassay (Laurell rocket) – original gold
standard
Other methods
• Electrophoresis
– Densitometric scan in association with total protein
assay
– calculate albumin
• CZE
– Calculate albumin
• Isoelectric focussing
• None of the above methods would be considered
as routine and have inherent inaccuracies
Calibration
• Certified Reference material ERM® DA470k/IFCC
– Material was analysed in 20 laboratories
• Metrological traceability
– Calibrated to previous material ERM-DA470
– Which was calibrated to USNRP 12-0575C (values assigned
by 24 laboratories using different in-house calibrants of
unspecified origin and purity
– Only α1 acid glycoprotein, α1 antitrypsin, transferrin and
transthyretin (pre-albumin) were calibrated against pure
proteins
BCG
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>60%
Summary
labs UK NEQAS
Measures albumin, α1 & α2
globulin, transferrin and other
proteins
Longer incubation periods lead to
increase binding of dye to other
proteins
Analytical range 15-60 g/L
Cost effective – can use animal
based sera for QC (but some
evidence for variable binding with
non – human alb.)
Easily automated
Positively biased against
nephelometric assay
Over estimates at lower albumin
levels and underestimates at
higher albumin level
BGP
• USA CAP more even distribution
between both methods
• More specific for human albumin
• Analytical range 10-70g/L
• Cost effective, but requires
human serum calibrators and
controls (low affinity of dye for
non-human albumin)
• Easily automated
• Good agreement with
nephelometric assay across the
analytical range
• Good precision
• ?Issues with renal dialysis
samples
? Can have the same reference intervals for BCG and BCP
Summary
Immunochemical assays
• Expensive reagents and
equipment
• Slow for large workload
• Nephelometric assays –
current gold standard
• Specific for albumin cf dye
binding assays
• Large functional range for
assay with good precision at
low end
Other
• Electrophoresis
– Densitometic scans
– dye binding to different
protein fractions variable
(albumin binds more dye than
per unit weight than
globulins)
• CV for albumin >10%
• Slow and not suitable for
high workload
UK NEQAS urine albumin
Urine albumin
Urine creatinine
ACR
Current Issues in the Measurement and
Reporting of Urinary Albumin Excretion
W.Greg Miller et al
National Kidney Disease Education programme –
IFCC Working Group on Standardization of Albumin
in Urine
Clinical Chemistry 55:1 24-38 (2009)
What are you measuring in urine?
Albumin in urine
• Large amounts are filtered at the glomerulus, despite acidic
charge at physiological pH
• Filtration is increased if acidic groups blocked by glycation (cf
diabetes)
• Most filtered albumin is reabsorbed by proximal tubular cells
• Reabsorption - receptor mediated
• Normal urine will contain ~ 20mg albumin / L
• Dimers and fragments have been found in urine
Albumin in urine
• Albumin in urine exposed to a wider range of pH and ionic
strength
– <pH4 and >pH8 albumin undergoes major conformational changes
– Other potential modifying factors - urea, glucose ascorbate and
cleavage by peptidases
• Urine albumin stable for one week (4 – 20oC)
• Freezing at -20oC causes fragmentation
• Single freeze thaw cycle may cause an apparent albumin loss
of 40%
• Polyvalent vs monovalent antibodies
Albumin in Urine
• Measuring mg’s rather than g’s
• Serum dye binding methods lack analytical
sensitivity
• Current routine methods include
– Dipsticks (only if urine albumin >150mg/L)
– Immunoassays
– 2-site immunometric assays (LOD 2-10mg/L)
– Size-exclusion liquid chromatography
Urine albumin methods
• Immunochemical
– Turbidimetry
– Nephelometry
• POCT
– Quantitative / qualitative
• Reference method – gold standard
– Immunochemical
– ?Mass spec
• Calibrants
Immunochemical methods
• Turbidimetry
– Majority in UK
– Can run on routine chemistry analysers
– Need to be aware of potential carry over from serum
albumin samples
– Hook effect
• Siemens Advia 2400
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PEG enhanced immunoturbidimetric assay
Anti-human albumin (goat) antibody
Prozone 200,000mg/L
Method traceable to internal standard using highly
purified material
Immunochemical methods
• Nephelometry
– Need specific instrumentation
– Tend to have better precision and a lower LOD
than turbidimetric methods
– High dose hook
• Depending on choice of antibodies,
immunoassays can underestimate albumin
(particularly in diabetic patients)
POCT
• Qualitative
– Only for an initial screen. Need to confirm
presence of albumin with a quantitative method
• Semi Quantitative
– Roche Micral dipstick: neg, 20, 50 &100mg/L
– Diagnostic Chemicals ImmunoDip <18mg/L
• Quantitative
– DCA 2000T (will do ACR)
– Haemocue
Reference method procedure (RMP)
• RMP for urine albumin should specifically measure albumin molecule(s) in
native urine
• Define measurand
• Immunological procedures not suitable
• Mayo Clinic developing multiplexed LC-MS/MS measurement procedure
that measure N-terminal 24-amino acid fragment of albumin
• NIST working on 15N-labelled HSA CRM as an internal standard for RMP
for albumin in urine
• Need to think about RMP for urine creatinine
Calibration
• Most routine methods are traceable to diluted ERM-DA470,
but considerable variation
• 5-10% of calibrators used with routine immunoassay contain
polymerised albumin
• JSCC has developed secondary reference material for urine
albumin
– Will submit to JCTLM Q3 2013
Assessing performance
• Little data on uniformity of results between
methods and laboratories using freshly
collected samples
• EQA samples frequently prepared using
purified albumin and creatinine
– Not such a complex matrix as native urine
• Unable to assess bias of methods as reference
method / material are not fully developed
Issues requiring further investigation for the
standardisation of measurement and reporting
of urine albumin
• pre-analytical requirements
• molecular forms of albumin in urine
• degree of urine albumin degradation under different
storage conditions
• Variation in urinary matrix composition
• RMP and secondary reference material
• Appropriate EQAS
• Etc etc
Summary
• Traceability of urine albumin methods to an international
standard
• Lack of a defined measurand
• Polyclonal vs monoclonal antibodies
• Commutability of EQA materials and ability to compare
methods
• Cost
• High dose hook
• Standardisation of collection procedures
– AER
– ACR (need to have standardisation of urine creatinine measurement)
– ACR ranges ?same in males/females/ethnicity