Clin-Chem Lab AA Part 4

Principle:

• Measures total protein based on the refractive index of serum/plasma

REFRACTOMETRY

What’s the concept of Refractometry?

The higher the protein concentration, the greater the ________________?

light refraction

Disadvantages of Refractometry:

• Affected by other molecules.

• Less accurate than spectrophotometric

Albumin remains soluble, while globulins precipitate in the presence of salts (sodium sulfate, ammonium sulfate)

Most widely used methods for determining Albumin.

DYE-BINDING

Principle:

• Albumin binds to dyes, causing color change proportional to concentration

DYE-BINDING

What are the Dyes under DYE-BINDING for Albumin Testing?

• Methyl orange

• 2,4’-hydroxyazobenzene-benzoic acid (HABA)

• Bromocresol Green (BCG)

• Bromocresol Purple (BCP)

• Nonspecific for albumin

• Beta-lipoproteins, alpha-1 and alpha-2 globulins also bind to dye

Methyl Orange

• Has low sensitivity but more specific for albumin

• Salicylates, penicillin, conjugated bilirubin, and sulfonamides -interfere with the binding of albumin to this dye

2,4’-hydroxyazobenzene-benzoic acid (HABA)

Formula for Bromocresol Green (BCG):

Albumin + BCG → Green-blue complex (628 nm)

is an anionic dye that binds to albumin in an acidic environment, forming a green-blue complex (628 nm)

Bromocresol Green (BCG)

Limitations of Bromocresol Green (BCG):

• Hemoglobin binds to BCG (for every 100 mg/dL of Hgb, albumin increased by 0.1 g/dL)

• Has been reported to overestimate low albumin values (nephrotic syndrome or end-stage renal disease)

Formula for Bromocresol Purple (BCP):

Albumin + BCP → Purple complex (603 nm)

is a more specific anionic dye that binds only to albumin, forming a purple complex.

Bromocresol Purple (BCP)

Limitations of Bromocresol Purple (BCP):

• Underestimates albumin in renal insufficiency.

• Bilirubin interferes with BCP binding, leading to falsely low albumin results in jaundiced patients.

Principle:

• Glyoxylic acid, in the presence of copper ions (Cu²⁺) and an acidic medium (acetic acid & sulfuric acid, H₂SO₄), reacts with tryptophan residues in globulins

TOTAL GLOBULIN: DIRECT COLORIMETRIC

What is the formula of TOTAL GLOBULIN: DIRECT COLORIMETRIC?

Globulin (Tryptophan) + Glyoxylic Acid + Cu²⁺ (Acid Medium) → Purple Complex (Measured at 540 nm)

What is the Indirect calculation of Albumin using Total Globulin?

Albumin (g/dL)=Total Protein (g/dL)−Total Globulin (g/dL

What are the 5 bands in ELECTROPHORESIS?

5 bands:

Albumin

Alpha-1 globulins

Alpha-2 globulins

Beta-globulins

Gamma-globulins

in ELECTROPHORESIS the Width of the band = ?

number of proteins present in that fraction.

Computation for protein fractions:

• Specialized scanner: Densitometer

• Uses dye to measure a specific wavelength (Coomassie Blue at 595 nm)

Absorbance Measurement and Strip-Chart Recorder