September 2012

September 2012

• 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)

Most questions ‘critically discuss’ or ‘compare and contrast’

• 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?

Introduction

• 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

• 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+

• 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)

Bromocresol green

• 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

Albumin standards and measurement of serum albumin with bromocresol green Doumas, Watson & Biggs Clin Chim Acta 1971; 31:87-96

• 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

Shift in absorption spectrum on binding to protein

• 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

Albumin + BCG Green complex

• Albumin + BCG Green complex

Albumin + BCP Purple complex

• Albumin + BCP Purple complex

Bromocresol purple method for serum albumin gives falsely low values in patients with renal insufficiency GA Maguire and CP Price

• 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

Immunonephelometry

• 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

Electrophoresis

• 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

Certified Reference material ERM® DA470k/IFCC

• 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

• BCG

Immunochemical assays

• Immunochemical assays

Large amounts are filtered at the glomerulus, despite acidic charge at physiological pH

• 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 exposed to a wider range of pH and ionic strength

• Albumin in urine exposed to a wider range of pH and ionic strength

• 
  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

Immunochemical

• 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

• 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