Analytical methods

Reference ranges for total protein and albumin

Total protein

• 6.5-8.3 g/dL

Albumin

• 3.5-5.0 g/dL

Kjeldahl method

• total protein

• counts nitrogen content of protein

• nitrogen content X 6.25 = total protein plasma

Biuret method

• Cu2+ ions bind to peptide bonds

• total protein

refractometry method

• based on refractive index of light

• total protein

• different protein concentrations = different indexes

Dye-binding + two common dyes for albumin measurements

bromocresol green and bromocresol purple dye binding

Why antibody and antigen concentrations are critical in precipitin reactions

???

Principle of radial immunodiffusion (RID)

• antigen in serum diffuses from well until zone of equivalence causing immune precipitation

  • large ring = high Ag in patient

  • small ring = low Ag in patient

Define “electrophoresis”

the migration of charged solutes or particles (proteins) in liquid medium under the influence of an applied external dield

three typical support media for electrophoresis

1. Agarose gel (most used)

2. cellulose gel

3. polyacrylamide gel

migration pattern form anode to cathode for serum protein electrophoresis (SPE)

Naming from anode out

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

Gel filtration chromatography

gel acts as molecular sieve

• large particles leave first then small ones

ion exchange chromatography

• separation based on competition of different ionic compounds

• Anion: separate negative charged ions on positive surface

• Cation; separate positive charged ions on negative surface

factors affecting electrophoresis migration

• relative rate of migration based on charge/mass ration

• electrical field strength

• support medium type

• temperature of operation

Charges on protein amino acids groups and carboxylic acid groups at basic and acid pH

??

Proteins that pass through pores in agarose gel based on charge to mass ration only

true

ELP pattern characteristics: Acute “immediate” phase

• decrease albumin and beta

• increase alpha 1 and/or 2

• normal to increased gamma

ELP pattern characteristics: Chronic “delayed’ phase

• decrease albumin

• increase most all other fractions

ELP pattern characteristics: Cirrhosis

• only FOUR peaks

• beta-gamma bridge

ELP pattern characteristics: Nephrotic syndrome

• decrease in ALL fractions EXCEPT alpha 2

  • alpha 2 contains macroglobulin

ELP pattern characteristics: Hypogammaglobulinemia

all normal EXCEPT decrease gamma

ELP pattern characteristics: Hypergammaglobulinemia (monoclonal gammopathy)

all normal EXCEPT M-spike in gamma

ELP pattern characteristics: Hypergammaglobulinemia (polyclonal gammopathy)

all normal EXCEPT diffuse increase in gamma (no spike)

ELP pattern characteristics: Alpha 1 antitrypsin deficiency

all normal EXCEPT alpha 1 decrease

Why a serum sample is preferred over plasma sample for analysis in protein electrophoresis?

Serum preferred due to plasma having:

• clotting factors and fibrinogen

• gives spurious peak b/w beta and gamma regions

Why does the plasma sample contain fibrinogen and where does the fibrinogen peak appear on the electrophoesis?

it is part of the clotting factors

The spurious peak in a hemolyzed sample analyzed by ELP + where the peak occurs in ELP

avoid hemolysis because the hemoglobin will give spurious peak b/w alpha and beta regions

State the factors affecting electrophoesis

???

Explain the indications for performing immunofixation electrophoresis (IFE)

• used to further characterize M(onoclonal) proteins

  • identify bond based on position and composition

list specific protein that is detected by a urine dipstick

albumin

list three quantitative methods of urine protein

1. folin-ciocalteu method

2. turbidimetric method

3. nephelometry

list three main classifications of proteinuria that can be detected by urine protein electrophoresis patterns

1. glomerular proteinuria

2. tubular proteinuria

3. overflow/ overload

State the two general reasons for increases in cerebrospinal fluid (CSF) protein

• increase endogenous production

• damage to blood-brain barrier

• infections or hemorrhage

Explain how IgG index used to evaluate patients w/ either increases endogenous protein production (Ex: MS) vs. patient with compromised blood-brain barrier

MS:

• increase CSF [IgG] w/out increase in CSF [albumin] suggests local production of IgG

Blood-brain barrier;

• plasma cells w/in CSF and from blood getting through BBB

When can oligoclonal bands in CSF and serum protein electrophoresis can be used as screen tests for multiple sclerosis (MS)

CSF test:

• a dark band towards bottom when compared to plasma

ELP test:

• bands cannot show up on the serum protein strip or it will be invalid

four methods to quantitate CSF protein

1. Folin-ciocalteu

2. UV absorption

3. Precipitation

4. Dye-binding methods