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
