1. Critically discuss the methods that are in routine use for the measurement of albumin in serum and urine
Dr Frances Boa

2. Method questions September 2012 March 2012 September 2011 March 2011
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)

3. 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?

4. 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
Summary

5. Albumin
Albumin
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+
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)

6. UKNEQAS serum albumin

7. Serum albumin methods to consider
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

8. 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:

9. 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

10. Bromocresol green Albumin + BCG Green complex BCG also binds to
transferrin
lipoproteins
α1 & α2 globulin
β globulins
Overestimates against nephelometric assays by 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

11. 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

12. 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

13. Third annual report Renal Registry 2000 Chapter 9: Serum albumin and serum bicarbonate
Accessed 12/4/13

14. BCG vs BCP

15. BCG vs Nephelometric albumin

16. BCP vs Nephelometric albumin

17. Immunochemical methods
Immunonephelometry
Requires specialist apparatus
Serum samples require dilution
Immunoturbidimetry
Can be set up on most main chemistry analysers
Historical
Single radial immunodiffusion (RID)
Electroimmunoassay (Laurell rocket) – original gold standard

18. Other methods Electrophoresis CZE Isoelectric focussing
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

19. 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 C (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

20. Summary BCG BGP USA CAP more even distribution between both methods
>60% 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 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
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

21. Summary Immunochemical assays 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
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

22. UK NEQAS urine albumin

23. Urine albumin

24. Urine creatinine

25. ACR

26. 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: (2009)

27. What are you measuring in urine?

28. 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

29. 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

30. 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

31. Urine albumin methods Immunochemical POCT
Turbidimetry
Nephelometry
POCT
Quantitative / qualitative
Reference method – gold standard
?Mass spec
Calibrants

32. 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
PEG enhanced immunoturbidimetric assay
Anti-human albumin (goat) antibody
Prozone 200,000mg/L
Method traceable to internal standard using highly purified material

33. 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)

34. POCT Qualitative Semi Quantitative Quantitative
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

35. 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

36. 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

37. 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

38. 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

39. 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