How do you quantitate proteins?
measure nitrogen content
How do you measure the nitrogen content of proteins?
What is the Kjeldahl tech?
gold standard in quantitation of proteins by meas nitrogen content;
digest prot c acid->release NH3 ions->meas # of NH3 ions released=amt of prot
what is nephelometry?
A measurement of LIGHT SCATTER
When do you use nephelometry
when protein content is HIGH
What types of specimens can you meas protein content in?
cannot do c urine b/c prot content not high enough
What are the probs with nephelometry?
potential interferences ie hyperlipidemia, hemolysis, hyperbilirubinemia
How can you meas protein in other specimens besides serum if they have low prot [ ]?
What is turbidometry?
measurement in LIGHT TRANSMISSION
when do you use turbidometry?
in low protein media ie CSF, urine
What are the probs with turbidometry?
not very specific; anything in the speciment can block the light transmission ie debris
What else can be used to measure low prot [ ] specimens?
What is refractometry?
Measure LIGHT REFRACTION BY A SOLUTE
what are the probs with refractometry?
not specifc, same probs as with turbidometry and nephelometry
What are more specific ways to meas proteins?
What are the benefits of colorimetry?
Can be made specific to proteins and interfences are minimal
Name 3 colorimetric methods used to meas protein?
3.Coomassie brilliant blue
What is the Biuret colorimetric method?
Copper salts + prot= purple complex color
What is the Folin-Ciocalteu method?
More sensitive than Biuret method
Phenol + prot->oxid->blue color
What is the most sensitive colorimetric technique?
Coomassie brilliant blue
What 2 dyes are used in the Coomassie brilliant blue?
bromcresol green and bromcresol blue
What specific protein do you meas with Coomassie blue test?
What 2 colorimetric tech are widely used in automated analyzers in labs?
Biuret and Coomassie blue
What does the Biuret colorimetric method measure?
When do you use the ppt for proteins?
to separate prot, esp albumin from globulins to get A:G ratio
In inflammation what is the A:G ratio?
Very low because alb LOW and Ig HIGH
What is the first step to do when you want to isolate a single minor protein of interest?
What do you use to ppt prot out of urine?
TCA (tricholoracetic acid)
What is another way besides ppt to sep out protein?
How do you do EP?
charge applied across medium composed of solid support and fluid buffer
charge creates ELECTROMOTIVE FORCE
solid support has (-) charge, drawn toward (+) pole(anode)
solid support can't move
but the buffer is fluid and moves in the opp direction and goes toward (-) pole(cathode)
What is the flow of buffer called?
What are the 2 forces acting on the protein once its applied to the EP and a charge is applied?
Which way does the electromotive force pull the prot?
Towards ANODE (+)pole
Which way does the endosmotic force pull the prot?
Towards the CATHODE (-) pole
Which way do endosmotic forces pull gamma globulins?
Gamma globulins weakly (-), pulled towards CATHODE (-)pole
Gramma loves CATs
Which force is stronger in most proteins?
Electromotive force> Endosmotic force
Which way does the electromotive force pull protein?
Towards the ANODE (+)charged pole
what is the pH of the buffer in SPEP?
8.6 on agarose gel
What is the fastest moving band in an SPEP?
What is the order of bands on an SPEP?
How do you separate the gamma globulins into oligoclonal bands in CSF?
increase endosmosis (ie flow of buffer TOWARDS Cathode)
What is the most abundant protein in human plasma?
ALBUMIN (2/3 of plasma protein)
What is the congenital absence of albumin called and what is the clin sig?
Mild edema and hyperlipidemia
What is the most common allotype of albumin?
What happens if you have a variant allotype other than A?
See bisalbuminemia on EP
What is a benign condition where you see 2 peaks in the alb region?
How do you quantify albumin?
Colorimetric assay: Coomassie brilliant blue
What is the t 1/2 of albumin?
3 weeks (~20 days)
Why do you measure albumin?
liver syn fxn (only see low alb in ESLD)
diabetic control (microalbuminuria in DM q yr)
Where do you see the biggest decrease in alb?
Protein losing conditions (ie protein losing enteropathy and nephrotic synd)
What is the % of alb glycosylated in norm pts?
Where is albumin glycosylated?
Nonenzymatically in the serum
What is the % of alb glycosylated in DM pts?
up to 25% in patients with poor control of their diabetes
What type of acute phase reactant is albumin?
NEGATIVE ACUTE PHASE REACTANT (ie if inflam INC, alb DEC)
What is prealbumin?
The fastest migrating band on SPEP but there normally isn't a lot of it so you don't normally see it on SPEP
What does prealbumin do?
1. binds and carries T4 and T3 (aka thyroxine binding prealbumin TBPA)
2. binds and carries retinol-binding prot: vit A complex
What is a precursor in senile cardiac amyloidosis?
prealbumin (TTR type amyloid)
What causes a high total T4 in a euthyroid pt?
Rare variant of prealb has very high affinity for T4
What mutant version causes familial amyloid polyneuropathy?
Mutant version of prealb (transthyretin)
What is prealb used for clinically?
assessment of nutritional status
what is the t 1/2 of alb?
Can prealb x the BBB?
Yes, its actively secreted into CSF by choroid plexus
what are the 2 hallmarks of CSF protein EP?
1. Sharp prealb band
2. double transferrin peak
Why do you see a "prealbumin" band in heparinized patients on SPEP?
this is really a beta lipoprot (LDL) band that migrates in the same spot where prealb would be
what type of acute phase reactant is prealb?
NEGATIVE ACUTE PHASE REACTANT
What is alpha 1 antitrypsin?
major component of the alpha 1 SPEP band
What does A1AT do?
inactivates proteases (ie trypsin and elastase)
What genotype of A1AT are MOST people?
PiMM-homozygous M allele on ch14
Which genotype of A1AT gives clin dz?
PiZZ--you are pizzed off you have lung and liver probs
What are the clin fx of PiZZ A1AT?
EARLY ADULT EMPHYSEMA
How do you screen for PiZZ?
What does PiZZ look like on SPEP?
VERY DECREASED ALPHA 1 band
what type of acute phase reactant is A1AT?
MARKEDLY POSITIVE APR
What is alpha 1 acid glycoprotein (Orosomucoid)?
very fast + acute phase reactant
minor component of Alpha 1 band
What is alpha 2 macroglobulin?
Where do you see alpha 2 macroglobulin?
very increased in liver and renal dz
how do you interpret alpha 2 macroglobulin?
10x increased in [ ] in NEPHROTIC SYND
large molecule, can't get out of kidney
What type of acute phase reactant is ceruloplasmin?
Where do you see ceruloplasmin on SPEP?
alpha 2 band
What does ceruloplasmin do?
What is the ceruloplasmin level in Wilson's dz?
Important marker in Wilson's dz
What is the ddx for decreased ceruloplasmin?
Menke's kinky hair synd
When do you get falsely normal or increased levels of ceruloplasmin?
What are the characteristic findings in Wilson dz?
INCREASED hepatic copper
INCREASE urine copper
do you use serum copper levels to dx Wilson dz?
NO! Serum Cu can be inc, dec or normal in Wilson's dz
What is haptoglobin?
Binds free Hb
What are the 3 components of the alpha 2 band on SPEP?
1. alpha 2 macroglobulin
What is a VERY SENSITIVE MARKER of HEMOLYSIS?
Why is haptoglobin such a sensitive marker of hemolysis?
because only a very tiny amt of free Hb is needed to deplete serum of haptoglobin
Why else, besides marker of hemolysis, is haptoglobin useful?
It doesn't bind to myoglobin helps ddx + Hb urine dipstick-->rhabdomyolysis
What type of acute phase reactant is haptoglobin?
What is the major Beta globulin in SPEP?
What does transferrin do?
transports FERRIC (FE 3+) IRON
norm about 30% saturated
What are transferrin levels in pts with Fe def?
If have inc levels of transferrin what does that look like on SPEP?
Abnml band that looks like pt has M-protein
what else besides Fe def causes inc transferrin?
What do the transferrin levels look like in increased inflammatory states?
like Ig: first decreases in acute phase BUT
if inflam persists it INCREASES
How does transferrin x BBB and get into CSF?
% gets modified to make asialated transferrin (AKA Tau protein)
When do you see fibrinogen on SPEP?
heparinized pt, spec doesn't completely clot
Where do you see fibrinogen on the SPEP?
straddles the Beta gamma interface
What does fibrinogen look like on SPEP?
What makes up most of the gamma region on SPEP?
Which serum proteins are in the alpha 1 band on SPEP?
1. alpha 1 AT
2. alpha 1 acid glycoprotein
3. alpha lipoprotein (HDL)
4. alpha fetoprotein
Which serum proteins are in the alpha 2 band on SPEP?
1. alpha 2 macroglobulin
Which serum proteins are in the beta band on SPEP?
1. beta 2 microglobulin
3. complement (C3)
4. beta lipoprotein (LDL)
which serum proteins are in the gamma band on SPEP?
3. C-reactive protein
what does the SPEP/densitometry look like in nephrotic synd?
decreases in all globulins except alpha 2 macroglobulin--looks like pseudo M protein on densitometry but no restriction band on IFE
Where do IgM paraproteins (ie IgM kappa) migrate near on densitometry?
IgM paraproteins migrate NEARER Beta region
Where do IgA M proteins (ie IgA lambda) migrate on densitometry?
INTO the beta region
What does the SPEP and densitometry look like in acute inflammation?
INCREASED alpha 1 and alpha 2 globulins
What does the SPEP and densitometry look like in chronic inflammation?
MILDLY DECREASED albumin
INCREASE alpha 1, alpha 2 AND GAMMA globulins
What does the SPEP, IFE, and densitometry look like in PBC?
Beta-gamma bridging (no restriction band on IFE) and changes of chronic inflam
What is bisalbuminemia?
heterozygote with albumin allotype; see 2 spikes in alb
no clin sig
What is lost in minimal change dz neprotic synd?
SELECTIVE LOSS OF ALBUMIN (AKA Selective proteinemia)
What do you see on SPEP in nephrotic syndrome?
decreased amt of all bands esp albumin EXCEPT alpha 2 macroglobulin retained
What is nephrotic syndrome?
loss of protein due to glomerular damage
massive loss of SMALL proteins
What is the hallmark of cirrhosis on SPEP?
Bridging beta gamma band (due to increased serum IgA)
Why do you get a bridging beta gamma band on SPEP in cirrhosis?
Increased serum IgA (IgA migrates into Beta region)
What do you see on SPEP of cirrhosis?
Bridging Beta-gamma band
blunted alpha1 and 2 bands
What are the monoclonal proteins called?
M-proteins or paraproteins
Where do you see M-proteins on SPEP or densitometry?
Discrete dark band in gamma region
What does an M spike represent?
Can you see 2 M-proteins on one patient sample?
Yes, biclonal gammopathy in 3-4% of cases
After you identify the presence of an M-prot what do you do?
quantify and determine composition
how do you quantifiy an M-protein?
Densitometry or nephelometry
How do you characterize an M-protein?
Do you need to see an M spike to do IFE?
No, do it if:
strong clin suspicion of myeloma or
If pt has neg serum scrn and you still have a high suspicion of myeloma, what can you do?
Urine EP and IFE
What should you look for in pts with M-prot?
How does normal CSF EP compare to SPEP?
same as serum just lower quanities of prot
What are the characteristic fx in CSF EP?
1. prominent PREalbumin band
2. DIM Alb, alpha 2 and gamma bands
3. Broad Beta band
What is the main reason to do CSF EP?
look for oligoclonal banding in MS
What does oligoclonal banding look like in CSF EP?
mult bands in gamma region
these bands represent several clones of Ig
Does urine have a lot of protein normally?
No, if anything all you normally see is trace albumin
What are the 3 main prob in a pt with proteinuria?
1. glomerular prob
2. tubular prob
3. overflow prob
What cause glomerular proteinuria?
What causes tubular proteinuria?
Tubulointerstitial nephritis or
What causes overflow proteinuria?
excess serum prot overwhelms kidneys and flows into urine (ie Bence Jones proteinuria-monoclonal light chain)
What besides excess prot causes overflow proteinuria?
Hemoglobin and myoglobin--toxic to tubules
What is the pattern of glomerular proteinuria on UPEP?
VERY inc ALBUMIN
increased alpha 1 and 2
What is the pattern of tubular proteinuria on UPEP?
INC alpha 1 and B bands
what are immunoglobulins that ppt at reversibly low temps?
How do you detect presence of cryoglobulins?
draw blood and keep at 37C til clots->
centrifuge at 37C->
store at 4C x 3 days->
any ppt that forms is cryoppt and can do EP to charact it
How many types of cryoglobulins are there?
3: type I, II and III (II and III are mixed types)
What type of cryoglobulin is assoc with mult myeloma and Waldestrom's macroglobulinemia?
What is the most common type of cryoglobulin?
What type of cryoglobulin is a mix of monoclonal IgM and polyclonal IgG?
What Ig has rheumatoid fx activity (anti-IgG)?
Type II IgM
What type of cryoglobulin is a mix of 2 polyclonal Ig?
What types of cryoglobulins are seen in pts with lymphoprolif d/o, chronic infxns, chronic liver dz, and autoimmune dz (esp SLE)?
What patient demographic are mixed cryoglobulins commonly found in?
Women in 4th or 5th decade
what is the most common cause of mixed cryoglobulins?
What is the distinct clinical synd made up of in pts with cryoglobulinemia?
Where do you consistently see purpura on the body?
How long does it take for renal syndromes to develop in pts with cryoglobulinemia?
What type of renal probs do pts develop who have cryoglobulinemia?
Nephrotic synd or Nephritic synd
assoc with SEVERE HYPOCOMPLEMENTEMIA
What type of renal dz most common on bx in pts with cryoglobulinemia?
MPGN type II
acutely looks like thrombotic microangiopathy
What is the main pathologic lesion in all tissues in a patient with cryoglobulinemia?
What is the tx for cryoglobulinemia?
Alpha interferon for HCV
What do you see on EM in MPGN type II?
Large SUBENDOTHELIAL immune complex deposits