Hydrasys 2 Training Manual - Protein Assays

What is Pre-Albumin and its significance?

• This is a small protein fraction anodic to albumin

• The measurement of this protein is reflective on the state of your nutrition

• If, Pre-albumin is low, other proteins and substances in the blood may also be low

• Typically not seen in agarose gel tech

What conditions can cause Decreased Pre-albumin?

1) Severe or chronic illnesses such as cancer

2) Hyperthyroidism

3) Liver Disease

4) Serious infections

5) Malnutrition

What conditions can cause Increased Pre-Albumin?

1) High dose of corticosteroid therapy

2) Hyperactive adrenal glands

3) Hodgkin’s Disease

4) High dose of nonsteroidal anti-inflammatory meds

5) Malnutrition

What is the Albumin fraction and its significance?

• This fraction represents the largest fraction seen on a normal protein pattern

• This protein is responsible for:

  • Maintaining oncotic pressure within the body

  • General transport protein

  • Source of amino acids

What Conditions can cause Hypoalbuminemia?

1) Liver disease

2) Malnutrition

3) Protein Loss (ex. urine, GI tract, burns, neoplasia)

What Conditions can cause Hyperalbuminemia?

1) Normal healthy variation

2) Dehydration is the only pathological association

What Conditions can cause Bisalbuminemia?

1) Congenital

2) Induced by drugs

• These are temporary and go away once meds are discontinued

3) In acute pancreatitis

How does Bisalbuminemia presented as a fraction?

• This fraction will present as two peaks that can vary in sizes from Peak 1 to Peak 2

What is the Alpha-1 Fraction and its significance?

• A small fraction that acts as an inflammatory protein

  • Can be divided into two smaller proteins:

    • Alpha-1 acid glycoprotein (AGP or orosomucoid protein)

    • Alpha-1 antitrypsin (alpha-1 protease inhibitor)

What is the Alpha-1 AGP and its significance?

• One of the major acute phase proteins

• Concentration increases in response to systemic tissue injury, inflammation, infection.

• This fraction is underestimated with staining

What is the Alpha-1 antitrypsin and its significance?

• Produced by the liver

• Considered an Acute Phase Reactant Protein

• This protein is responsible for the ability to inhibit the activity of elastase, cathepsin G, trypsin, and other proteolytic enzymes

• Deficiency of this protein is associated with development of emphysema

How does the Alpha-1 heterozygous phenotype pattern appear?

• This appears as a double band of equal intensity

How does the Alpha-2 fraction appear?

• Normally appears as single peak cathodic to alpha-1 fraction

What proteins migrate in the Alpha-2 region?

• Haptoglobin

  • Double band may be indicator of phenotype

• Alpha-2 macroglobulin

How does the Alpha-2 fraction on protein electrophoresis affect the alpha-2 values?

• The values are decreased due to intravascular hemolysis

What Conditions cause increase in the alpha-2 values?

1) Acute inflammation

2) Nephrotic syndrome

3) Hemolyzed samples (in vitro)

What is the Significance of the Alpha-2 macroglobulin?

• This protein acts as a Protease inhibitor

• This is not eliminated through the kidneys because of its large size

What causes an increase is Alpha-2 macroglobulin?

• Nephrosis (secondary to synthesis); 10x increase

• Diabetes

• Patients with alpha-1 antitrypsin deficiency and emphysema

What causes a decrease in Alpha-2 macroglobulin?

• No specific diseases associated

How is the Beta-1 and Beta-2 fractions normally presented in the Protein B1B2 gel?

• Beta-1 typically presents as a slightly higher peak than Beta-2

What is the significance of the B-1 fraction?

• This represents Transferrin (TRF)

  • The principle protein for iron transport from the GI tract and from the breakdown of Hb to the BM

• Synthesized in the liver

How is the B-1 Lipoproteins migration zone presented in the gel?

• This can present with a very sharp or “flying bird” or “mustached” squiggly band

What conditions can cause Increased Beta-1 fraction?

• Iron deficiency anemia

What conditions can cause Decreased Beta-1 Fraction?

1) Alcoholic cirrhosis

2) Renal disease and thermal injuries

3) Iron overload

4) Acute or chronic inflammation

What is the Primary Protein found in the Beta-2 fraction?

• Complement

  • Involved in inflammatory response

What is the Beta-2 protein fraction sensitive to?

• Very sensitive to storage conditions such as freezing

  • Degradation starts just after 3 days when stored at 2-8 C

  • So fresh samples are recommended

What immunoglobulin can migrate in the Beta-2 fraction?

IgA

What is indicated when the Beta-2 fraction is equal or larger than the Beta-1?

• This can indicate that a monoclonal protein is present

How does Beta-2 react with the gamma zone?

• This fraction can form a “bridge” with the gamma zone

  • In this case, it is necessary to rule out a co-migrating monoclonal that hide within the “bridging” pattern

What causes Increased Beta-2 fraction?

1) Acute Phase Response

2) Co-migrating monoclonal band

What causes decreased Beta-2 fraction?

1) Older sample in which the complement has degraded

How does a Cirrhosis pattern present?

• Typically the IgA will be increased in a patient

• There will be bridging between the beta-gamma regions

How does Fibrinogen present?

• This protein should not be present in properly processed normal serum

• It is only present when a sample experiences insufficient clotting

• The band may be mistaken for a monoclonal protein

What does the Gamma fraction consist of?

• This fraction consists of the immunoglobulin proteins:

  • IgG, IgA, IgM. IgD. IgE

  • It has been demonstrated that immunoglobulins can migrate from the Alpha region to the Gamma region

    • IgA → close to B2 fraction

    • IgM → between beta and gamma

    • Light chains kappa/lambda → a-2 to gamma

How is the gamma region normally presented?

• Smooth, rounded symmetry without any bumps, sharp restrictions, or peaks

• Any change in symmetry is a good indicator that additional testing should be performed

What causes decreased gamma fraction?

1) In Elderly

2) Congenital immunodeficiency

3) Immunosuppressive treatment (HIV, transplants)

What causes increased gamma fraction?

1) In lymphocytic syndromes such as Multiple Myeloma

2) Hyperimmunization

3) Acute infections

4) Chronic liver disease

5) Chronic inflammatory disease

When should you suspect the presence of light chains based on the pattern?

• In Hypogammagobulinemia

  • The gamma fraction demonstrates a decreased pattern with a flat area

How is the Oligoclonal pattern presented and what is its significance?

• This pattern typically demonstrates multiple immunoglobulin bands

• This effect is due to:

  • Transplants

  • Immunosuppresion

  • Infections

• Important to rule out any underlying monoclonal gammopathy in these patients

The B1B2 gel separates the Beta fraction into two distinct fractions named ______ and ______?

Beta-1 and Beta-2

Typically which immunoglobulin migrates in the B region?

• Immunoglobulin IgA

Which Protein can be seen in serum samples when the sample has not completely clotted prior to centrifugation?

• Fibrinogen

This protein typically appears as a “squiggly” pattern in the beta region

• Beta lipoprotein

What is the term used when the Albumin fraction appears as two peaks?

Bis Albuminemia

The complement fraction is very sensitive to storage conditions:

True of False

True

These proteins precipitate at temperatures less than 37 C

• Cryoglobulin

Which fraction contains the majority of immunoglobulins?

• Gamma fraction

Which immunoglobulin is typically associated with beta-gamma bridging pattern?

IgA

Which protein fraction is decreased in patients that developed emphysema?

• Alpha-1 antitrypsin