Proteins 2 Flashcards
cryoglobulins specimen
**immunoglobulins that precipitate at temperatures below 37 degrees
specimen :
serum form red ( non-additive tube ) *
tube must be pre-warmed
sample must be kept at 37 degrees
Cryoglobulins
qualitative testing
- patient specimen if placed in an incubator after being received in the lab
- sample is allowed to clot
- serum removed & aliquoted into 2 labelled tubes( 1
at 4 degrees & 1 at 37 degrees )
- normal patient control of pooled serum undergoes
the same treatment
- samples are checked for 7 days
results
negative : clear serum
positive: turbid serum that clears at 37 degrees
Cryoglobulinemia
cryoglobulins can cause organ & vascular damage
they are often associated with:
autoimmune disorders
infections
malignancies
cryoglobulins can form globular or cylindrical precipitates at room temp that will be counted as cells by automated hematology analyzers
to avoid reporting false elevations of WBCs & Plts in patients suspected to have cryoglobulins, samples should be kept at 37 degrees and analyzed immediately
Total protein - Biuret method
***most widely used method for total protein analysis
alkaline medium protein+cupric ions (Cu2+) ---------> purple /blue color
a minimum of 2 peptide bonds is necessary ( 3 amino acids )
absorbance is measured at 540 nm **
color produced is directly proportion al to the number if peptide bonds which reflects the total protein concentration in the sample
points about biuret method
**biuret reagent contains :
CuSO4 - source of Cu2+ ( provides copper ions )
Sodium potassium tartrate - keeps Cu2+ in solution
NaOH - provides an alkaline pH
a fasting serum or plasma should be used
- bilirubin & lipemia can interfere
**avoid:
hemolysis - falsely elevated protein due to the release of hemoglobin
prolonged tourniquet use- hemoconcentration will cause false elevation of protein & lipids
- not sensitive enough for urine or CSF
- concentration of each is too low
biuret assay can be calibrated using solutions of albumin**
Total Protein ( refractometry )
*Measures refractive index
used to quickly assess high concentrations of protein
serum is composed mostly of protein*
- the refractive index is largely dependent on the protein concentration
when concentration < 35 g/L accuracy decreases**
- salt, glucose, other low-molecular weight compounds contribute more to refractive index
used more often to assess total solutes in urine than to measure total serum protein( RR: 65-85g/L)
Total protein - Kjeldahl method
reference method for protein ***
measures the amount of nitrogen in a specimen
acid is used to digest the sample & convert nitrogen in the protein to ammonium ions
the ammonium nitrogen value is multiplied by 6.25 to account for the average nitrogen content in protein
this method is time consuming, inconvenient, & impracticial for routine use
Dye- binding method for albumin
***most widely used method for determining albumin
pH of a solution is adjusted to make albumin positively charged & able to bind an anionic dye
albumin + dye —> albumin - dye complex + free dye
the bound dye has a different absorbance maximum than the free dye
the absorbance of the albumin -dye complex is proportional to the albumin concentration of the sample
Points about dye- binding
the dye used must be specific for albumin
the dye must have a high binding affinity ( not easily split apart from albumin )
there must be a substantial absorbance change between the free dye & the bound dye
the wavelength of peal absorbance of the bound dye should be away from the peak absorbance of bilirubin & hemoglobin ( so that they wont interfere )
Salt fractionation ( precipitation )
Globulins are separated from albumin by a precipitation process using sodium salt
Total protein = Albumin + Globulins
the precipitate will contain globulins, while the supernatant will contain albumin
Albumin can be measured on the supernatant by a Biuret reaction
Globulins can be calculated using the following formula: Globulins = Total Protein - Albumin ***
this method is not commonly used today
Serum Protein electrophoresis (SPE)
Electrophoresis is the migration of charged particles within a liquid medium under the influence of an electric field
in SPE proteins are separated based on their electric charge & density
Proteins in an electric current will move according to their charge which is determined by the pH of surrounding buffer
Protein Charges
Proteins have the ability to be negatively or positively charged (AKA amphoteric )
the pH of the liquid medium ( buffer) determines their charge
isoelectric point = net charge is zero ( pH at which protein has equal # of pos & neg charges )
if the pH of buffer > pI, the protein has a net neg charge (anion )
of pH of buffer
protein charges - migration
in an electrical field, proteins with a neg charge move to the anode( positive electrode )
the further the protein if from the pI , the greater the net charge on the protein
greater the charge on the protein the faster it moves
with buffer pH of 8.6, albumin is the most negatively charged protein & moves the fastest towards the anode
Protein charges at pH 8.6
SPE if performed at 8.6 pH
gamma os at its isolectric point ( zero charge )
all other fractions have a neg charge
-albumin is the furthest from pI ( fastest ) followed by
- alpha 1
- alpha 2
- beta
Types of electrophoresis
***Serum Protein Electrophoresis ( zone electrophoresis)
High- Resolution protein electrophoresis
Capillary electrophoresis
Isoelectric focusing
Immunochemical methods
-radial immunodiffusion
-immunoelectrophoresis
- immunofixation electrophoresis ***( suspected
multiple myeloma)
-immunoturbidimetry
- immunonephelometry
Serum protein electrophoresis (set up )
most common electrophoresis is zone electrophoresis on agarose gel* or cellulose acetate*
serum samples are applied close to the cathode end of the support medium ( gel) using a serum applicator
gel is placed in an alkaline buffer in an electrophoresis chamber & connected to 2 electrodes
of gel does not maintain contact with the buffer, the proteins will not be able to migrate
Serum protein electrophoresis ( migration )
the negatively charged proteins migrate to the anode(seperation occurs)
after a period of time th egel is removed & immersed in fixative ( acids/alcohol) to denature & immobilized proteins )
the gel is stained with paragon blue or amido black
to visualize bands
then it is dried at rm temp or in an oven
Serum Protein electrophoresis ( inspection )
after drying gel can be inspected virtually or ith a densitometer
the most significant finding is monoclonal immunoglobulin
- AKA monoclonal protein or M- peak
hypergammaglobulinemia ( spread out band with feathered edge ) Monoclonal peak ( one, clean edge band )
Quantitation of Protein Bands
Qualitative visual inspection made by comparison to normal control that has been analyzed on same gel
- gel scanned on denistometer for quantitation
- determines the relative % of each of the protein fractions ( bands)
Quantitative result obtained for each fraction by multiplication:
%(band) x total protein = results in g/L for each band
can be achieved via:
colorimetric
densitometer
Densitometer Quantitation
gel is scanned by densitometer
light shines up through each band to a photometer
amount of light transmitted is determined by the intensity of the staining ( concentration of each fraction)
darker the band= inc. concentration = dec. light transmitted
the photometer measures light transmitted
absorbance changes are graphed ( gives tracings of peaks )
Factors affecting SPE
electroendosmosis buffers temperature amperage voltage membrane pore size
electroendosmosis
- first layer of gel has fixed neg charges that attract pos charges ( immobile layer)
- next layer has more positive than neg charges & flows toward cathode ( mobile layer)
- material without any charge is carried with mobile layer
- gamma globulin is carried to cathode in this manner ( electroendosmosis)
buffers
function :
carry applied current
establish pH
determine the electric charge on the solute
effect on ionic strength of buffer :
- ->increase in ionic strength - protein bands denser & closer together (condensed)
- -> decrease in ionic strength - protein bands move faster & further apart ( diffuse)
Temperature/Amperage/Voltage
An increase in any of these will increase the migration of the proteins
except if the temp is too high( protein will be denatured)
Net charge:
the larger the net charge of a protein, the faster it will move
Size & Shape:
larger the molecule, the slower it will move
Summary of factors affecting migration
migration increased :
increased temp, amperage, voltage, pores size
decreased ionic strength
migration decreased:
increased temp( too high),ionic strength, viscosity
decreased temp, amperage, voltage
gel loses contact with buffer
Urine electrophoresis
protein concentration is usually low in urine
specimen must be concentrated first
- this can be done with a minicon concentrator
electrophoresis of urine can reveal causes of proteinuria
- Bence Jones Proteins ( multiple myeloma)
- tubular Proteinuria
- Glomerular Proteinuria
it is useful to compare serum & urine electrophoresis
- low MW proteins can be lost in the urine & may not show up in the serum
CSF electrophoresis
protein concentration is low in CSF & needs to be concentrated prior to electrophoresis as well
Pre-albumin band will be seen with CSF but not normally in serum
- good indication of nutritional status
CSF electrophoresis is done to detect oligoclonal banding found in multiple sclerosis patients
- 2-5 bands are normally seen
- 70-90% of MS patients have these bands (in gamma region )
inflammatory electrophoresis pattern
decrease in albumin
increase in ⍺1, ⍺2, & ß
Chronic infection electrophoretic pattern
decrease in albumin
increase in y-globulins ( bc antibodies are located here)
Hypogammaglobulinemia Electrophoretic pattern
decrease in y- globulins
Nephrotic syndrome electrophoresis pattern
decrease in albumin & IgG ( lost in urine
increase in ⍺2, ß
(⍺2 - macroglobulin, ß- lipoprotein, complement, haptoglobin)
Liver Cirrhosis Electrophoretic pattern
ß-y bridge
fast moving y-globulins prevent resolution of ß- & y-globulins
Multiple Myeloma electrophoretic pattern
a spike in the y or ß regions
Multiple Myeloma
occurs due to malignancy of a clone of plasma cells
increase in immunoglobulin
electrophoresis should be performed on serum & urine to diagnose
- if Bence Jones protein is being produced it may show up in the urine but not the serum
confirmatory test :
immunoelectrophoresis
immunofixation (IFE)
Interferences in SPE
Fibrinogen
- plasma collected instead of seru,
- migrates to the ß/y region
Free Hemoglobin
- small band in late ⍺2 or ß region
Hemoglobin -Haptoglobin complexes
- small band in ⍺2 region
High resolution electrophoresis
standard electrophoresis separates into 5 distinct bands
HRE separates proteins into as many as 12 bands
in HRE:
- higher voltage is used
- more concentrated buffer
- agarose gel
- patterns can be scanned with densitometer to find semiquantitative estimates of proteins in each band
useful for :
- detecting small monoclonal bands
- differentiating unusual bands from monoclonal gammopathies
Capillary electrophoresis
protein separation takes place in silica capillaries
-30-50 cm long, 25-100µm diameter
Capillaries filled with aqueous buffer
sample injection end connected to high- voltage power supply
Positively charged buffer flows to the negatively charged anode ( detection end)
- this net flow is called electro-osmotic force ( EOF)**
- **Negatively charged particles have a tendency to migrate back towards positively charged injector end by electrophoretic mobility- but EOF is stronger
- therefore all molecules move toward cathode at different rates , based on sized & charge
- molecules detected by their absorbance as they pass through small window near detection end of capillary
Capillary Electrophoresis
Advantages:
capillaries allow heat dissipation
- higher voltages can be used
- faster analysis
sample volume needed is small
isoelectric focusing
proteins are separated based on their isoelectric point (pI)
a pH gradient (3.5-10) is used
when an electric field is applied , charged particles will move to their pI on the gel & remain there
clinical applications:
- phenotyping of ⍺1-antitrypsin deficiencies
- determination of genetic variants of enzymes & hemoglobulins
- detection of paraproteins in serum or oligoclonal bands in CSF
- isoenzyme determinations
immunofixation electrophoresis (IFE)
can be performed on serum, urine or CSF
process:
- electrophoresis separates protein in the serum
sample
-Antisera against the protein of interest is applied to
the gel
- The protein precipitates in the gel matrix
- a wash step removes other proteins
- a stain is used to visualize the precipitated protein
a comparison can be made of the location of the precipitate bands & the location on the reference lane
- this is used to identify the specific immunoglobulin
present
immunofixation
a normal distribution of immunoglobulins will have some diffused stain or “blush”
to evaluate an immunofixation pattern, look for well
- defined bands for both heavy & light chains
IgM can sometimes produce a thin, narrow band that is hard to see
if a light chain is seen with no apparetn heavy chain ( IgG, IgM, or IgA) the process should be repeated using an anti-IgD & IgE
90% of IgD have ℷ light chains
- IgE is very rare
Clinical significance
RR protein : 63-85g/L
Hypoproteinemia: excessive loss - in urine ( renal disease ) - in GI tract ( inflammation of digestive system ) - in open wounds, bleeding, burns
decreased intake
- malnutrition
- malabsorption
decreased synthesis
- liver diesease
- in adequate dietary proteins
increased protein catabolism
- burns, traumas, injuries
Hyperproteinemia :
dehydration
Monoclonal gammopathies
- multiple myeloma ( IgG,IgA, light chains )
- Waldenstroms Macroglobulinemia (IgM)
Polyclonal gammopathies
- Many chronic diseases
