(FINAL) CC1 LAB L6

Proteios

Total Protein and Albumin Determination

• “__________” – first rank of importance

Nitrogen

Total Protein and Albumin Determination

• Composed of carbon, hydrogen, oxygen, __________ & sulfur

  • __________ – element that distinguished protein from other macromolecules

Amphoteric

Total Protein and Albumin Determination

• __________; can yield a positive or negative charge

  • Good buffer

Liver

Total Protein and Albumin Determination

• Mostly synthesized by the __________

Amino acids

Total Protein and Albumin Determination

• __________ are the basic structural units of protein

Albumin + Globulins

Total Protein and Albumin Determination

• Total Protein = __________

Albumin

Total Protein and Albumin Determination

• Major plasma proteins:

  • __________ – maintains oncotic pressure; transports hormones, drugs, fatty acids.

Globulins

Total Protein and Albumin Determination

• Major plasma proteins:

  • __________ – immune response (immunoglobulins), transport (transferrin), enzymatic activity.

Fibrinogen

Total Protein and Albumin Determination

• Major plasma proteins:

  • __________ – clotting protein (absent in serum).

Immunoglobulins

Total Protein and Albumin Determination

• Synthesized primarily in the liver (except __________—produced by plasma cells).

Synthetic function

Total Protein and Albumin Determination

• Assess __________ of the liver

Albumin

Total Protein and Albumin Determination

• Usually tested with __________

Increased Total Protein (Hyperproteinemia)

Clinical Significance: __________

• Dehydration (hemoconcentration)

• Multiple myeloma (↑ immunoglobulins)

• Chronic inflammation

Decreased Total Protein (Hypoproteinemia)

Clinical Significance: __________

• Overhydration

• Malnutrition, malabsorption

• Liver disease (↓ synthesis)

• Nephrotic syndrome (protein loss)

• Severe burns (loss through damaged skin)

Biuret Reaction

TP Laboratory Determination: __________ (Routine Method)

• Principle: In an alkaline medium, peptide bonds of proteins react with copper (II) ions to form a violet-colored complex. The intensity of the color is directly proportional to protein concentration

• Reaction: Protein + Cu²⁺ (in alkaline medium) → Violet Cu-protein complex

• Measured spectrophotometrically at 540–560 nm.

540–560

TP Laboratory Determination: Biuret Reaction (Routine Method)

• Principle: In an alkaline medium, peptide bonds of proteins react with copper (II) ions to form a violet-colored complex. The intensity of the color is directly proportional to protein concentration

• Reaction: Protein + Cu²⁺ (in alkaline medium) → Violet Cu-protein complex

• Measured spectrophotometrically at __________ nm.

Ellitrol I, Ellitrol II

Biuret Reaction Reagents/Materials/Instrumentation

• __________ (Normal Control)

• __________ (Pathologic Control)

• Total Protein Reagent Kit:

  • Sodium hydroxide, potassium sodium tartrate, copper sulfate, potassium iodide

• Standard:

  • Protein: 8 g/dL or 80 g/L, Sodium azide

8,80

Biuret Reaction (Manual Method) Reagents/Materials/Instrumentation

• Ellitrol I (Normal Control)

• Ellitrol II (Pathologic Control)

• Total Protein Reagent Kit:

  • Sodium hydroxide, potassium sodium tartrate, copper sulfate, potassium iodide

• Standard:

  • Protein: ___ g/dL or ___ g/L, Sodium azide

Sodium hydroxide, potassium sodium tartrate, copper sulfate, potassium iodide

Biuret Reaction (Manual Method) Reagents/Materials/Instrumentation

• Ellitrol I (Normal Control)

• Ellitrol II (Pathologic Control)

• Total Protein Reagent Kit:

  • __________

• Standard:

  • Protein: 8 g/dL or 80 g/L, Sodium azide

520-580

Biuret Reaction (Manual Method)

• Wavelength: ________ nm

37

Biuret Reaction (Manual Method)

• Temperature: ___°C

1

Biuret Reaction (Manual Method)

• Optical Path: ___cm

Reagent blank

Biuret Reaction (Manual Method)

• Read against __________

Total Protein (g/dL) = Asample / Astandard x Cstandard (g/dL)

Biuret Procedure (Manual Method) Formula

6.0, 6.4-8.3

Biuret Procedure (Manual Method)

• Standard value: typically ___ g/dL

• Normal Serum Range: ______ g/dL

• Plasma values slightly higher due to fibrinogen

Fibrinogen

Biuret Procedure (Manual Method)

• Standard value: typically 6.0 g/dL

• Normal Serum Range: 6.4-8.3 g/dL

• Plasma values slightly higher due to __________

Kjeldahl

Other Methods for Total Protein: __________

• Digestion of protein and measurement of nitrogen content; reference method

Refractometry

Other Methods for Total Protein: __________

• Measurement of refractive index due to solutes in serum (rapid but not commonly used)

Dye-binding

Other Methods for Total Protein: __________

• Protein binds to dye and cause a spectral shift in the absorbance maximum of the dye

Fibrinogen

Pre-analytical Considerations for Total Protein

• Non-fasting test

• Free from hemolysis, lipemia

• Concentration is higher in plasma than in serum

  • Presence of __________

  • Increased from 0.2-0.4 g/dL (2-4 g/L)

Albumin

• Major protein found in plasma

• Primary Function: maintenance of oncotic pressure and transport of large number of compounds

• Aid in the diagnosis of protein loss

• GI loss, nephrotic syndrome or decreased synthesis during hepatic insufficiency

Bromocresol Green (BCG)

Common Dye-Binding Methods

• __________ – widely used; forms a green complex at pH 4.2.

  • Less specific than BCP

Bromocresol Purple (BCP)

Common Dye-Binding Methods

• __________ – more specific for albumin, less interference from α- and β-globulins.

  • Reference Method for Albumin

628, 600

Common Dye-Binding Methods

• Principle: At acidic pH, albumin binds to an anionic dye to form a colored complex. Intensity of color (measured at ___ nm for BCG or ___ nm for BCP) is proportional to albumin concentration.

Methyl Orange

OTHER DYES FOR DYE BINDING METHOD

• __________: Nonspecific for albumin

HABA [2,4’-hydroxyazobenzenebenzoic acid]

OTHER DYES FOR DYE BINDING METHOD

• __________: Many interference (salicylates, bilirubin)

Bromocresol purple

OTHER DYES FOR DYE BINDING METHOD

• __________: Specific, sensitive, precise

Salt precipitation

Other Methods for Albumin Analysis

• __________: Globulins are precipitated in high salt concentration and the albumin in the supernatant is quantitated by biuret reaction

Electrophoresis

Other Methods for Albumin Analysis

• __________: Proteins are separated based on their electric charge

Ellitrol I, Ellitrol II

BCG Reagents/Materials/Instrumentation

• __________ (Normal Control)

• __________ (Pathologic Control)

• Albumin Reagent Kit

  • Succinate buffer (pH 4.2), Bromocresol green, Surfactant

  • Standard: Albumin: 3.5g/dL or 35 g/L (may vary)

  • Sodium azide

Succinate buffer, albumin, sodium azide

BCG Reagents/Materials/Instrumentation

• Ellitrol I (Normal Control)

• Ellitrol II (Pathologic Control)

• Albumin Reagent Kit:

  • __________ (pH 4.2), Bromocresol green, Surfactant

  • Standard: __________: 3.5g/dL or 35 g/L (may vary)

  • __________

620

Dye-Binding Methods: Bromocresol Green (BCG)

• Wavelength: ___nm

37

Dye-Binding Methods: Bromocresol Green (BCG)

• Temperature: ___°C

1

Dye-Binding Methods: Bromocresol Green (BCG)

• Optical path: ___cm

Reagent blank

Dye-Binding Methods: Bromocresol Green (BCG)

• Read against __________

Albumin (g/dL) = Asample / Astandard x Cstandard (g/dL)

BCG Method Procedure Formula

3.5-5.0

BCG Method Procedure Normal Range

• Adults: ______ g/dL

• Children: 3.8-5.4 g/dL

3.8-5.4

BCG Method Procedure Normal Range

• Adults: 3.5-5.0 g/dL

• Children: __________ g/dL

Globulin = Total Protein - Albumin

BCG Method Procedure Calculation of Globulin

Albumin / Globulin Ratio = Albumin / Globulin

BCG Method Procedure Calculation of A/G Ratio

1.2-2.0

BCG Method Procedure Calculation of A/G Ratio

• Normal A/G Ratio: ≈ ______ g/dL

Increased Albumin (Hyperalbuminemia)

__________:

• Dehydration

• Prolonged tourniquet application

• After excessive albumin infusion

Decreased Albumin (Hypoalbuminemia)

__________:

• Malnutrition

• Liver disease

• Nephrotic syndrome

• Chronic inflammation

Hemolysis

Biuret Method Sources of Error: __________

• Effect: False ↑ protein

• Preventive Measure: Avoid traumatic draw

Lipemia

Biuret Method Sources of Error: __________

• Effect: Turbidity interferes with readings

• Preventive Measure: Ultracentrifuge sample

High bilirubin

Biuret Method Sources of Error: __________

• Effect: Color interference

• Preventive Measure: Use bichromatic analysis

Incorrect wavelength

Biuret Method Sources of Error: __________

• Effect: Inaccurate absorbance

• Preventive Measure: Calibrate spectrophotometer

Improper blanking

Biuret Method Sources of Error: __________

• Effect: Baseline drift

• Preventive Measure: Run reagent blank first