TP and Albumin

TOTAL PROTEIN AND ALBUMIN

Clinical Chemistry I

Plasma Proteins

• Composed mainly of Albumin and globulins.

• Classification of globulin fractions:

  • α1 Globulins

  • α2 Globulins

  • β Globulins

  • γ Globulins

  • Each globulin fraction includes more than one type of protein.

• Routine Testing Includes:

  • Total protein

  • Albumin

  • Possible further testing: globulins and the albumin/globulin (A/G) ratio.

• More Specialized Tests Include:

  • Protein electrophoresis

  • Quantitation of specific proteins

Protein Electrophoresis

• Protein electrophoresis is a method used to separate proteins based on their size and charge.

  • Key fractions visualized include albumin (Alb), α1 and α2 globulins, β globulins, and γ globulins.

Total Protein Measurement

• Over 500 plasma proteins have been identified, and the Total Protein test serves as a rough measure of all plasma proteins.

• Purpose of the Test:

  • Provides general information about nutritional status, kidney disease, liver disease, and other conditions.

  • Reference Interval: 65-83 g/L.

  • Results that fall outside the reference range necessitate further testing.

Hypoproteinemia

• Defined as low protein in the blood.

• Causes:

  • Excessive Loss (Protein Loss):

  • Renal disease (e.g. nephrotic syndrome).

  • Inflammation of the digestive tract.

  • Loss of blood due to open wounds, internal bleeding, extensive burns.

  • Decreased Intake:

  • Malnutrition.

  • Intestinal malabsorption.

  • Decreased Synthesis:

  • Liver disease.

  • Inherited immunodeficiency disorders.

  • Accelerated Catabolism:

  • Seen in severe burns or trauma.

Hyperproteinemia

• A less common condition compared to hypoproteinemia.

• Causes:

  • Dehydration:

  • Relative increase in blood volume due to decreased solvent water, can occur after vomiting, diarrhea, excessive sweating, diabetic acidosis.

  • Excessive Production:

  • Mostly seen in γ-globulins linked to chronic infections, rheumatoid arthritis, lupus erythematosus, or multiple myeloma.

Protein Measurement Details

• Total Protein Testing:

  • Serum is preferred for testing but heparinized plasma can also be used.

  • Interferences:

  • Lipemia may interfere, depending on the method.

  • Hemolysis can lead to falsely elevated results due to the presence of red blood cell (RBC) proteins.

  • Reference intervals can differ:

  • Lower in infants (0-3 years), seniors, and during pregnancy.

  • Refer to specific Tables for methods (e.g., Table 11-6).

Total Protein: Biuret Method

• Definition:

  • The most widely used method for protein measurement, recommended by the International Federation of Clinical Chemistry.

  • Involves creating a violet-colored chelate between cupric ions and peptide bonds under alkaline conditions, with measurement observed spectrophotometrically at 540 nm.

  • Amount of color formed correlates to the protein concentration in the sample.

• Advantages:

  • The method is easily automated.

• Sources of Error:

  • Abnormally small proteins can lead to inaccuracies (e.g. in multiple myeloma).

  • Lipemia and severe hemolysis can also affect results.

• Reaction Requirement:

  • Must have a minimum of 2 peptide bonds to react; therefore, amino acids and dipeptides do not produce a reaction.

Specimen Storage & Stability

• Plasma/serum must be separated from cellular components within 2 hours of collection.

• Testing should occur at room temperature within 8 hours, followed by storage at 2-8°C.

• Separated samples should be frozen after 48 hours if testing is incomplete.

• Frozen samples must only be thawed once to prevent analyte deterioration. CSF should be centrifuged and analyzed immediately.

Sample Volume Requirements

• Serum/plasma: 8.0μL

• CSF: 60.0μL

Total Protein Calibration and Quality Control

• Calibrators Used:

  • Synchron Protein Calibrator Levels 1 & 2 require no special preparation.

  • Stable until the expiration date when stored unopened at -15 to -20ºC.

  • Stable for 60 days once opened and stored at 2-8ºC.

  • Perform monthly calibrator runs with each new calibration and reagent bottle.

  • Patient samples must only be reported after quality control has been evaluated and accepted.

Dynamic Range for Total Protein Measurement

• Linearity Range:

  • Serum/plasma: 10-120 g/L

  • CSF: 0.1-15 g/L.

  • Dilute samples exceeding the high end of the analytical range by half with saline and re-analyze; results require multiplication by two.

Total Protein in Urine

• This test is the most effective routine urine test for indicating renal disease.

• Protein sources include low molecular weight serum proteins and proteins produced in the genitourinary tract.

  • Renal Damage: Proteinuria Sources:

  • Glomerular Proteinuria:

    • Causes include diabetes, hypertension, atherosclerosis, and damage from abnormal substances like amyloid.

  • Tubular Proteinuria:

    • Characterized by decreased reabsorption of albumin and other low molecular weight proteins; often coincides with glomerular issues. Causes may include toxins and viral infections.

Urine Protein Testing Procedures

• Quantitative Testing:

  • Timed samples (e.g., 24-hour collections), account for circadian rhythms.

  • Proper collection technique must be followed, and volume must be recorded.

  • Test an aliquot and measure in g/L, ultimately reporting in g/day.

  • Reference value for normal protein: < 0.10 g/L.

• Methods Summary:

  • Common urine protein methods include turbidimetric (sulfosalicylic acid, TCA, benzethonium chloride), Biuret, Folin-Lowry, and dye-binding methods.

Total Protein in CSF

• Total protein is one of the most common chemical tests performed on CSF samples.

• An increase in protein can be due to:

  • Increased permeability of the capillary endothelium, often associated with conditions such as meningitis or hemorrhage.

  • Increased production of immunoglobulins in CSF, examples include those associated with multiple sclerosis (MS).

  • Decreased clearance of normal proteins.

  • Neurologic disorders.

• Conversely, decreased CSF protein can indicate:

  • Decreased dialysis from plasma base.

  • Rapid production of CSF.

  • Fluid leaks from recent punctures.

  • Conditions like water intoxication or hyperthyroidism.

CSF Protein Measurement Methods

• Common methods for total protein include turbidimetric (nephelometric if automated), TCA, SSA, and benzethonium chloride.

• Other methods include dye-binding and Biuret.

• Reference Interval: 0.15-0.45 g/L.

• Potential Interferences:

  • Hemolysis and the presence of RBCs can affect results.

Total Protein in Other Body Fluids

• Can include analyses of:

  • Peritoneal fluid

  • Pleural fluid

  • Seminal fluid

  • Vaginal fluid

  • Tears

Albumin

• Description:

  • Small, globular protein synthesized daily in the liver without any reserve or storage.

  • Half-life is approximately 15-20 days.

  • Most abundant protein in plasma and also exists in the interstitial space.

  • Responsible for about 80% of the intravascular colloid osmotic pressure, helping to maintain fluid balance in tissues.

  • Acts as a negative acute phase reactant.

Functions of Albumin

• Acts as a buffer to maintain pH.

• Binds and transports various substances within the blood, including:

  • Thyroid hormones, fat-soluble hormones, iron, fatty acids, unconjugated bilirubin, salicylic acid, calcium, magnesium, glucose (including in the form of glycated albumin), many drugs, and dyes (though dyes should not be present in blood).

Causes of Hypoalbuminemia

• Causes Include:

  • Malnutrition and malabsorption

  • Liver diseases that lead to diminished synthesis

  • Protein-losing enteropathy or gastrointestinal loss, e.g., from diarrhea

  • Renal diseases, such as nephrotic syndrome

  • Skin losses due to burns

  • Hypothyroidism

  • Dilution from polydipsia or excessive IV fluid administration

  • Acute diseases or inflammatory conditions

  • Fluid redistribution due to hemodilution, increased capillary permeability, and decreased lymph clearance

  • Rare genetically inherited disorders like analbuminemia or bisalbuminemia.

Causes of Hyperalbuminemia

• Typically caused by:

  • Dehydration and excessive albumin infusion.

Albumin Measurement Techniques

• Most Widely Used Methods:

  • Dye-binding methods, where the pH is adjusted to impart a positive charge on albumin, facilitating its binding to anionic dye through electrostatic attraction.

  • The resulting shift in absorption maximum is measured; the absorbance is directly proportional to the albumin concentration in the sample.

  • Common dyestuffs used include bromcresol green (BCG) and bromcresol purple (BCP), with BCP being preferred for accuracy.

• Serum is the Recommended Sample Type:

  • Note that albumin concentrations can seemingly rise due to the influence of fibrinogen and heparin.

• Interferences with BCG Method:

  • Hemolysis, which alters results as hemoglobin binds to dye, can lead to overestimation of albumin in patients exhibiting low levels of albumin and high levels of α-globulin (e.g. in nephrotic syndrome).

• Interferences with BCP Method:

  • Bilirubin can lead to underestimation of albumin in renal insufficiency.

• Reference Range for Albumin: 35-50 g/L.

Albumin/Globulin Ratio (A/G)

• The A/G ratio indicates possible disruptions in kidney and liver diseases.

• The formula to calculate is:

  • $A/G = \frac{Albumin}{Total~~ Protein - Albumin}$

• Normal A/G ratio is slightly greater than 1.

• Decreases in A/G ratio indicate decreased albumin production (e.g. in cirrhosis) or overproduction of globulins (e.g. in multiple myeloma or autoimmune diseases).

• Increases in A/G ratio can indicate underproduction of globulins (e.g. in leukemia).

Specimen Storage & Stability for Albumin Tests

• Again, serum/plasma requires separation from cells within 2 hours of collection.

• Testing must transpire at room temperature within 8 hours, followed by refrigeration at 2-8°C.

• Separated samples should be frozen after 48 hours if tests are incomplete, with the stipulation that frozen samples should only undergo a single thawing process to avoid loss or degradation of analytes.

Sample Volume for Albumin

• Serum/plasma required: 5.0μL.

Albumin Calibration and Quality Control

• Utilize Synchron Protein Calibrator Levels 1 & 2 without preparation.

• Storing properly allows stability until the printed expiration date; once opened, they maintain stability for 60 days in cold storage (2-8ºC).

• Monthly performance checks are required, and calibrators 1, 2 and 3 should be run once per shift.

• It’s critical to validate QC results before any patient samples are reported, ensuring checks against lot numbers and expiration dates.

Albumin Measurement: Dynamic Range

• Dynamic Range:

  • Serum/plasma: 10-70 g/L.

  • High concentration samples over the analytical range must be diluted by half with saline and re-analyzed, ensuring to multiply the result by two.

Calibration Failure Insights

• Potential Causes:

  • Range failure (calibration accuracy), switch of calibrators, instrument malfunction, expired or poorly prepared reagents, or sample probe blockages.

  • Precision issues may relate to bubbles in reagent lines or sample.

  • Sensitivity could be affected by incorrect or contaminated calibrators.

QC Failure Considerations

• Leading cause for QC failures is improper temperature management of controls.

• A systematic troubleshooting process should include questions such as:

  • What occurred?

  • What has changed recently?

  • What part failed - calibration or QC?

References

• Clinical Chemistry: Principles, Techniques, and Correlations. 8th Edition. Bishop, M.L.; Fody, E.P.; Schoeff, L.E.. Lippincott Williams & Wilkins. 2013.

• Tietz: Fundamentals of Clinical Chemistry. 6th Edition. Burtis, C.A.; Ashwood, E.R.; Bruns, D.E.. Saunders Elsevier. 2008.

• Clinical Chemistry 3 Course Manual. Saskatchewan Institute of Applied Science and Technology. 2002.

• Proteins. CSMLS Mode Module. Chapman, John C. 2009.

Practice Questions

1. The plasma protein mainly responsible for maintaining colloidal osmotic pressure in vivo is:

   • A. Hemoglobin

   • B. Fibrinogen

   • C. Myoglobin

   • D. Albumin

2. Which of the following is not a cause of hypoproteinemia?

   • A. Dehydration

   • B. Glomerulonephritis

   • C. Malnutrition

   • D. Liver disease

3. The Biuret method measures the presence of which of the following in the protein molecule?

   • A. Amino acids

   • B. Nitrogen

   • C. Peptide bonds

   • D. Tyrosine

4. Blood is collected from a patient who has been fasting since midnight; the collection time is 7 am. Which of the following tests would not give a valid test result?

   • A. Glucose

   • B. Triglyceride

   • C. Creatinine

   • D. Bilirubin

5. Which statement below describes a non-kinetic enzyme assay?

   • A. Initial absorbance is measured, followed by a second reading after 5 minutes

   • B. Absorbance is monitored continuously for 1 minute

   • C. Reflectance is measured from a xenon source lamp

   • D. Absorbance is measured at 10-second intervals for 100 seconds

6. What is being measured in nephelometry?

   • A. Light produced by excitation of ground state atoms

   • B. Light absorbed by particles in suspension

   • C. Light produced by fluorescence

   • D. Light scattered by particles in suspension

7. In dry slide technology, unabsorbed light is reflected back to the photodetector from the bottom of which layer in the slide?

   • A. Spreading layer

   • B. Reagent layer

   • C. Support layer

   • D. Indicator layer

8. What effect does blood in a CSF specimen have on a total protein result?

   • A. Falsely decreased

   • B. Falsely increased

   • C. None

9. What is the most common cause of hyperproteinemia?

   • A. Nephrotic syndrome

   • B. Malnutrition

   • C. Dehydration

   • D. Liver disease

10. Albumin methodologies include all of the following EXCEPT:

    • A. Bromcresol purple

    • B. Methyl orange

    • C. Bromcresol green

    • D. Phosphotungstic acid

11. The red-colored complex developed in the creatinine Jaffe reaction is caused by the complexing of creatinine with:

    • A. Diacetyl monoxide

    • B. Alkaline picrate

    • C. Sulfuric acid

    • D. Sodium hydroxide