Clin Path 3 (Plasma Proteins + Chemical Analysis)

What protein is missing in serum vs plasma?

Fibrinogen

T/F

Abnormal proteins are often secondary issues

True

What is the protein pad on a urine dipstick looking at? What can a TP miss when looking at proteins?

1. Albumin

2. Can miss abnormal proteins

T/F

Protein levels are helpful in diagnosis

True

What are the 2 major types of proteins?

1. Albumin

• Smallest + makes up 75%

• Can help w/ kidney disease, can detect earlier since small molecules will leak first

• Carrier, regulates H2O, buffer

• Made by liver

2. Globulin

• Includes 100’s of different proteins

  • IGs, complement, factors, enzymes, antibodies, etc

• Most produced in liver (not immunoglobulins - B cells)

• Typically reflect antibodies

Immune response will be seen in TP and will also be seen in A/G ratio**

What is an acute phase protein? Negative phase protein? What are the most important acute phase proteins?

Acute phase proteins

• Change significantly in inflammation

  • Fibrinogen (concern for clots)

Negative phase protein

• Decrease in inflammation

  • Albumin

Most important acute phase proteins

• Haptoglobin (carries excess iron in hemolysis, will be decreased since carrying iron)

• Fibrinogen

• CRP (most important); C-reactive protein, indicates inflammation, better than sed rate

• Serum amyloid-A and alpha

• Acid glycoprotein

What is the reference range for total protein? What are some methods for measuring?

Reference = 6-8g/dL**

Methods

1. Refractometer - many interfering substances (EX: contrast dye)

2. Spectrophotometer - measures peptide bonds

• Biuret reagent method (more purple, more protein)

What is the reference range for albumin? What are some methods for measuring?

Reference = 2.7-4.5g/dL

Methods

1. Spectrophotometer

• Bromcresol green, haba dye

• Electrophoresis - preferred in birds (used to separate molecules based on their size, charge, or shape by applying an electric current)

What is the reference range for globulin concentration?

Referenc = 1.9-3.4g/dL

What is serum protein electrophoresis? What are the different fractions?

• Can determine TP and albumin

• Blood test that separates the proteins in your serum into distinct groups so doctors can see how much of each type is present (useful for detecting abnormal or abnormal amounts of normal proteins)

• Separate proteins w/ electrical charge on agarose gel (small proteins move quickly and large move slowly, helps ID)

Fractions

• Albumin, alpha 1 and 2, beta, and gamma

What defines a turbidity test?

Reagent precipitates protein to create turbidity (SSA urinalysis)

T/F

Antibody based detection kits are used to ID a specific protein through targeting specific epitopes (specific parts of an antigen that an antibody or a T-cell receptor recognizes and binds to)

True

What can cause decreased proteins via variations? What needs to be differentiated?

• Variations in protein concentrations may be due to changes in albumin or globulin proteins

Decreased proteins

• Differentiate hypoalbuminemia or hypoglobulinemia

• Both can be decreased, can cause A/G ratio to look normal

  • Blood loss, over-hydration, protein losing enteropathy, severe skin disease (vascular permeability), effusive disease

What are 2 main causes for hypoalbuminemia?

Hypoalbuminemia

• Decreased production or

  • Hepatic failure, starvation (not getting amino acids from diet), GI parasitism, malabsorption, inflammation (negative phase protein)

• Increased loss

  • Glomerular disease, GI parasitism

Hypoglobulinemia

• Decreased immunoglobulin concentration

  • Failure of passive transfer, inherited or acquired immunodeficiency

What defines hyperproteinemia? What causes hyperalbuminemia/hyperglobulinemia? What are some causes for hyperglobulinemia specifically?

Hyperproteinemia

• Increased albumin, globulin, or both

Hyperalbuminemia/hyperglobulinemia

• Primary cause is dehydration**

  • A/G ratio not altered, hematocrit affected

Hyperglobulinemia

• Increased alpha/beta globulin

  • Acute chronic inflammation

    • Acute phase proteins in alpha and beta

What are the 2 main causes for increased gammaglobulin (gammapathies)?

1. Polyclonal - broad peak (different gamma proteins increased, many)

• Mixture of IGs - chronic inflammation, liver disease (chronic), lymphoma, CLL

2. Monoclonal - narrow peak (one specific gamma protein increased)

• Mixture of IGs - multiple myeloma (classic presentation), extramedullary plasmacytoma, lymphoma, CLL, canine ehrlichia, etc

ANTIBODIES

What can cause hyperfibrinoginemia?

• Most often inflammatory or dehydration, but also pregnancy or neoplasia

What are the 5 different types of methodologies for chemistry testing?

1. Spectrophotometry

2. Enzymatic assays

3. Optical

4. Ion-selective electrode

5. Chemoluminescence

What is the difference between a wet and dry chemistry analyzer?**

Wet chemistry analyzer

• Liquid reagents

• Cheaper

• More labor intensive (more room for error like mixing reagents wrong, run controls, mixing reagents)

• Light passing through or absorbed by final sample = how to read

Dry chemistry analyzer

• Dry slide technology (impregnated on slides, strips, or cards)

• More expensive

• Light reflected from the slide, card, or strip = how to read

How does spectrophotometry work?**

• Measures amount of light absorbed at a specific wavelength after color change reaction

• EX = hemoglobin

• Photometric = absorbance w/ dry tech

How does enzymatic assays work? What is a rate reaction? What is an endpoint reaction?

• Substrate + Enzyme = Product + Enzyme****

  • Measure substrate or product

  • Enzymes cannot be measured

  • Activity determines amount for some values or is assumed to be linear (ALT is assumed as it gets higher, quantity is higher, but test actually looks only at function)

• Rate reactions measure:

  • Rate that substrate decreases in a certain time

  • Rate that a product is produced in a certain time

• Endpoint reaction

  • To see how much product is there in the end, not timed

How do optical density tests work?

• Color change from slide tech

• Reflective index

• Similar to spec reading

How does the ion-selective electrodes technique work?

• Measures the concentration of ions

  • Based on charge

  • EX = Sodium, potassium, chloride, CO2

  • Potential is determined by the relative concentration of an analyte on each side of the membrane

What defines Beer’s Law?**

• The absorbance of a solution is directly proportional to its concentration (intensity of color of tube is proportional to amount of analyte in tube)

  • Endpoint reaction

• Standard curve

How is a standard curve established for Beer’s Law?**

1. Prepare standard solutions – make several solutions of the chemical with known concentrations (e.g., 1, 2, 3, 4 mM).

2. Measure absorbance – use a spectrophotometer to measure each solution’s absorbance at a specific wavelength.

3. Plot the points – put concentration on the x-axis and absorbance on the y-axis.

4. Draw the best-fit line – if Beer's Law is obeyed, it should be a straight line through or near the origin.

5. Use the line to find unknowns – measure the absorbance of your unknown sample, find the corresponding concentration on the curve.

What are 3 different examples of veterinary analyzers and what methodologies do they use?

1. Randox = enzymatic and immunoassay (uses antibodies to detect)

2. VetScan = enzymatic (looks at substrate or product)

3. IDEXX = dry, optical, photometric

What are calibrators/standards? How does QC differ?

1. Multiple known concentrations

2. Done infrequently

3. When major change in instrument or to validate

• Used to set up instrument and set a reference curve

Quality Control

• Used to check that assay is working correctly after calibration

• Done frequently

• 2-3 levels (normal, abnormal (low and high))

• Range of acceptable limits

• Westgard rules (allows for outliers to a degree concerning QC)

Linear limit/reportable limit

• What is my upper and lower reportable value on the instrument?