urine protein measurement issues greg miller, phd professor of pathology virginia commonwealth...
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Urine Protein Measurement Issues
Greg Miller, PhD
Professor of Pathology
Virginia Commonwealth University
Richmond, VA (USA)
Outline
• Albumin molecules in urine
• Albumin measurement
• Total protein measurement
Albumin information is based on a
conference in March 2007 to address
standardization issues for urine
albumin/creatinine measurement and
reporting
Molecular forms of albumin
• Conformation is influenced by ligand binding
• Urine concentrates many ligands
• Unpaired cysteine-34 can form albumin dimers and other covalent modifications (plasma or urine)
• 1-10% is glycated in plasma (higher in diabetes)
• Glycated forms are a greater proportion in urine - attributed to differential uptake in tubules
• Large (>5 kDa) and small (500-5000 Da) fragments have been identified in plasma and urine
• C and N terminal truncation occurs
Albumin molecules in urine
• Conformation changes in plasma may influence the filtration rate at the glomerulus
• Fragmented forms in plasma should be more easily filtered by the glomerulus
• Tubular uptake is receptor mediated and may influence enrichment of modified forms in urine
• Proteases and chemical modifications occur in the urinary tract and in the urine after collection
• Influence of pH, osmolality, contact with sediment, adsorption to containers, and other sample handling factors are not well understood
Outline
• Albumin molecules in urine
• Albumin measurement
• Total protein measurement
Albumin measurement procedures
• Immunoassay
• Influenced by the albumin epitope(s) recognized by the antibody, and by reactivity with modified forms of albumin
• Evidence that polyclonal immunoassays are reactive with some modified forms
• HPLC
• Albumin may not be resolved from other urine proteins (causing overestimation)
• Hypothesis of non-immunoreactive albumin may be related to non-specificity
Measurement issues
• Influence of urine matrix variability and molecular forms of albumin are not well understood
• No urine albumin reference material
• No reference measurement procedure
• Calibration traceability is to diluted CRM 470 (ERM DA470) serum protein reference material
• Details of traceability design, dilution protocols, and measurement implementation are not standardized and appear to influence calibration uniformity between methods
• Some methods use the molar absorptivity of albumin in solution for calibration
Current status of albumin measurement
Proficiency Testing suggested:
• A range of results for the same sample
• Influenced by non-commutability of samples
• Urine dipstick results were highly variable
• A range of imprecision; with some methods having acceptable imprecision
• A variety of reporting units for albumin concentration, excretion rate, and albumin/creatinine ratio
PT example: CAP urine albumin (USA)
pooled human urine supplemented with albumin, creatinine and other substances, liquid, within method comparison
0
5
10
15
20
25
30
0 20 40 60 80 100
Peer group mean, mg/L
Am
on
g la
bo
rato
ry C
V, %
Inst. 1 (N=11)
Inst. 2 (N=79)
Inst. 3 (N=69)
Inst. 4 (N=194)
Inst. 5 (N=59)
Inst. 6 (N=59)
Inst. 7 (N=44)
Inst. 8 (N=39)
Inst. 9 (N=207)
Inst. 10 (N=112)
Inst. 11 (N=86)
Inst. 12 (N=123)
Inst. 13 (N=82)
Inst. 14 (N=18)
Inst. 15 (N=66)
mg/L CV, % ±2 SD
11 3-26 8-15
26 5-10 21-31
87 3-8 74-100
PT example: Finland, Norway, Canada
pooled or single donation human urine (may be supplemented with albumin and creatinine), liquid, among lab/method comparison
Method N Mean CV ±2SD rangemg/L % mg/L
Finland/Norway 136 19 15.4 14-25
Norway (GPs) 1012 35 12.1 27-44
Canada 28 20 16.5 14-26
Current status of albumin measurement
• Within subject biological variability (CVi) 30-40%
• Limitation: the biological variability data was not uniformly acquired nor analyzed; CVi is difficult to define for disease conditions.
• Many methods have adequate imprecision (CV); some do not (based on PT samples with a uniform molecular species)
• Reliable information on bias among methods is lacking
• Calibration uniformity among methods needs improvement
• The albumin molecular species that are being measured is not well understood
Recommendations: NKDEP/IFCC UA Conf.
• Albumin/Creatinine ratio should be reported
– “mg/mmol” or “mg/g” should be used uniformly in a country or region
• Albumin concentration (mg/L) is difficult to interpret and should not be reported alone
• First morning urine has lower biologic variability than a random collection
• Albumin should be measured on fresh (non-frozen) urine
• “urine albumin” should replace “microalbumin”
Further investigations needed: UA Conf.
• Clarify sample collection and handling requirements for minimum biologic variability
• Clarify the measurand in a urine sample; and the variability of the urine matrix
• Clarify reference intervals and decision points related to risk for kidney damage by age, gender, ethnicity, and concomitant disease
• Develop a reference system– ID/MS measurement procedure at Mayo Clinic (USA)
– Secondary urine reference material from Japan (JSCC and JCCLS)
Outline
• Albumin molecules in urine
• Albumin measurement
• Total protein measurement
Proteins in Urine
• Albumin
• Others• Immunoglobulins
• Bence-Jones
• Tamm-Horsfall
• Lysozyme
• Myoglobin
• Hemoglobin
• Bacterial origin
• Peptides
Quantitative urine protein methods
In order of clinical lab market share in USA:
• Pyrogallol red (dye binding)
• Pyrocatechol violet (dye binding)
• Benzethonium chloride (denaturation/turbidimetry)
• Biuret with precipitation (reference)
• Coomassie blue (dye binding)
Issues with urine protein methods
• Different measurement signal with different proteins
• Different chemical reaction procedures have different response ratios to different proteins
• No uniform calibration standard
Benzethonium chloride Pyrocatechol violet
Pyrogallol red #2Pyrogallol red #1
Dube et al. Clin Biochem 2005;38:479-485.
Normal urines supplemented with: albumin, gamma-globulins, polypeptides, mixed protein
Imai, Clin Chem 1995;32,1986.
Mean total protein of 12 urine samples measured by 7 methods and using 3 standard materials
Methods:SSA – sulfosalicylic acidSSA-SS - sulfosalicylic acid sodium sulfateTCA – trichloroacetic acidBC – benzethonium chlorideCBB – comassie brilliant bluePR-M – Pyrogallol red molybdenumTCA-B - Trichloroacetic acid precipitation biuret
Patients:(3) nephrotic syndrome(1) diabetic nephropathy(1) systemic lupus(1) acute glomerulonephritis(2) multiple meyoloma(4) cancer
Interfering substances with urine protein methods
A non-inclusive list:
• Creatinine
• Aminoglycosides
• Phenothiazines
• Amino acids
• Organic acids
• Peptides
• Other substances
Conclusions
• Urine albumin measurements are less variable than urine total protein measurements
• Clinical trials should use a central lab
• Data from different clinical trials may be difficult to aggregate
Questions
Comments