Proteins

Question 1

Albumin

• amount in serum
• analbuminemia results in
• albumin alleles
• clinical utility

Answer 1

• Most abundant serum protein
• Analbuminemia results in mild edema and hyperlipidemia
• Several albumin alleles (most common = albumin A); variant alleles may result in bisalbuminemia (benign)
• Clinical utility:
  
  1. Assessing nutritional status: halflife of albumin is 17 days
  2. Assessing hepatic synthetic function
  3. Assessment of renal glomerular function
  4. Negative acute phase reactant
  5. Diabetic control: glycated albumin is indicator of short term glycemic control
  6. Maintains serum oncotic pressure

Question 2

Prealbumin

• SPEP
• function
• clinical utility

Answer 2

• AKA transthyretin
• Not normally seen on SPEP
• Functions in serum to bind
  
  • thyroxine
  • retinol binding protein: vitamin A complex
• Clinical utility
  
  • Assessment of nutritional status
  • Negative acute phase reactant
  • Prealbumin band is a hallmark of CSF protein electrophoresis

Question 3

Alpha1-antitrypsin

• SPEP appearance
• genetics (number of alleles, most common alleles)
• clinical utility of SPEP

Answer 3

• Major component of the alpha1 band
• AAT gene (SERPINA1) highly polymorphic with > 100 alleles
• Most common allele is PiM
• Most common genotype is PiMM
• SPEP can detect AAT deficiency (homozygotes for PiZZ) showing a diminished alpha1 band

Question 4

Electrophoretic band

alpha1-alpha2 interface

Answer 4

• Gc globulin: binds vitamin D
• alpha1-antichymotrypsin: positive acute phase reactant
• alpha1-acid glycoprotein: minor component of alpha1 band normally but major component of the increased alpha1 band in acute inflammatory states

Question 5

alpha2 electrophoretic band

Answer 5

• alpha2-macroglobulin:
  
  • relative concentration elevated in liver and renal disease
  • large size prevents its loss in nephrotic syndrome, leading to a relative 10 fold rise in concentration
• Haptoglobin:
  
  • binds free hemoglobin
  • rapidly depleted in intravascular hemolysis
  • does not bind myoglobin
  • positive acute phase reactant
• Ceruloplasmin
  
  • binds copper
  • low ceruloplasmin not detectable with SPEP
  • positive acute phase reactant
  • decreased in Wilson disease

Question 6

alpha2-beta interface electrophoretic band

Answer 6

• Usually empty
• Hemoglobin, usually absent from serum, may be present here when there is hemolysis (a possible pseudo M spike)

Question 7

Beta1 electrophoretic band

Answer 7

Transferrin

• Transports ferric (Fe³⁺) iron
• Normally 30% saturated
• Markedly increased in iron deficiency
• Asialated transferrin (tau protein) and a double transferrin peak are hallmarkes of CSF electrophoresis
• Carbohydrate deficient transferrin a marker for alcohol use

Question 8

beta1-beta2 interface

Answer 8

Beta-lipoprotein

• LDL

Question 9

Beta2 electrophoretic band

Answer 9

• IgA
• Fibrinogen, usually absent from serum
  
  • May be present in the beta-gamma interface when there is incomplete clotting (a possible pseudo M spike)
• C3
  
  • positive acute phase reactant
  • C3 breakdown products may produce a pseudo M spike

Question 10

gamma1 electrophoretic band

Answer 10

gamma globulins

• positive acute phase reactants

Question 11

gamma2 electrophoretic band

Answer 11

CRP

• Marker of inflammation
• produces a small band
• high sensitivity assays can detect CRP as little as <0.5 mg/L resulting in 3 tiers of CRP
  
  • Normal CRP: <2-3 mg/L
  • High level CRP elevation: >10 mg/L usually indicates active inflammation such as collagen vascular disease, infection, etc.
  • Low level CRP elevation: 3-10 mg/L; indicates cellular stress and correlated with higher all cause mortality, poor outcome following cardiovascular events

Question 12

Answer 12

Question 13

Answer 13

Question 14

Answer 14

Question 15

Answer 15

Question 16

What does a normal serum electrophoresis look like

Answer 16

• Nearly invisible prealbumin band
• Large albumin band
• Small peaked alpha1
• Broad alpha2
• Biomodal beta
• Broad gamma

Question 17

Bisalbuminemia

Answer 17

• Heterozygotes for albumin alleles
• SPEP shows double albumin spike
• No clinical consequence

Question 18

alpha1-antitrypsin deficiency electrophoresis

Answer 18

Can be detected with SPEP (but not the most S and S assay)

Question 19

Nephrotic syndrome electrophoresis

Answer 19

• Loss of small serum proteins, particularly albumin
• Large protein molecules retained
• Decrease of all bands except alpha2 band that contains alpha2 macroglobulin

Question 20

Beta-gamma bridging

Answer 20

• Indicative of cirrhosis and caused by increased serum IgA
• Hypoalbuminemia
• Blunted alpha1 and alpha2 bands

Question 21

Monoclonal gammopathy

Answer 21

• Immunochemically homogeneous immunoglobulin M protein in the serum
• SPEP shows a prominent, discrete, dark band within gamma, beta, or rarely alpha2
• M protein usually intact immunoglobulin, composed of 2 heavy and 2 light chains
  
  • Sometimes light chain only
  • Rarely a heavy chain only
  • Biclonal gammopathy in 3-4% of cases (if IgA, more likely due to the appearance of both monomers and dimers, especially if IgA light chain)
• 10% of patients show only hypogammaglobulinemia
• Immunofixation or immunosubtraction is indicated to characterize the M protein
  
  • urine should be screened for monoclonal free light chains
  • serum free light chain levels should be evaluated
• M spike is most commly the result of PCN, Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma) or CLL

Question 22

Pseudo M spikes

Answer 22

1. Fibrinogen (incompletely clotted sample)
2. Hemoglobin (hemolyzed sample)
3. Elevated CRP
4. Elevated transferrin
5. Antibiotics
6. Radiocontrast agents
7. Serum tumor markers (e.g., CA19-9)
8. C3

Question 23

Answer 23

Question 24

Answer 24

Question 25

Answer 25

Question 26

Answer 26

Question 27

Answer 27

Question 28

Answer 28

Question 29

CSF protein electrophoresis

Answer 29

* CSF normally contains proteins in serum, but in smaller quantities * Prominent prealbumin band and a double beta (transferrin) band * Electrophoresis used to support a diagnosis of multiple sclerosis by finding oligoclonal bands, which should be absent in the serum

Question 30

Urine Protein Electrophoresis

Answer 30

* Glomerular proteinuria pattern: * strong albumin alpha1 and beta bands * large proteins (persistence of some filtering function) absent * small proteins (tubular reabsorption) absent * Intermediate proteins are left in urine * Albumin * AAT * Transferrin * Tubular proteinuria * Weak albumin band * strong alpha1 and beta bands * Overflow proteinuria pattern * Monoclonal light chain (Bence Jones proteinuria) usually * Could also be myoglobin or hemoglobin

Question 31

Cryoglobulins - define - detection

Answer 31

* Immunoglobulins that precipitate reversibly at low temperature * Detection * Draw blood at 37 degrees until clotted * Centrifuge at 37 degrees * Place serum at 4 degrees for 3 days * Centrifuge at 4 degrees * Any precipitate can be washed and electrophoresed and immunofixed

Question 32

Type I, II, and III cryoglobulins

Answer 32

1. monoclonal immunoglobulins associated with MM or Waldenstrom 2. Mixture of monoclonal IgM and polyclonal IgG * IgM has RF activity (anti IgG) * most common type of cryoglobulin 3. Mixture of 2 polyclonal immunoglobulins, typically IgG and IgM; IgM has RF activity

Question 33

Mixed cryoglobulinemia

Answer 33

* Associated with * Lymphoproliferative disorders * Chronic infection * Chronic liver disease * Autoimmune disease * HCV is most common cause * Systemic immune complex disease * palpable purpura (leukocytoclastic vasculitis) * arthralgias * HSM * LAD * anemia * sensorineural deficits * glomerulonephritis * Renal involvement: * Nephrotic syndrome or nephritic syndrome * Associated with severe hypocomplementemia * MPGN type II most common * +/- thrombotic microangiopathy * electron microscopy: subendothelial immune complex deposits with a fibrillary or tubular structure in a fingerprint like pattern

Question 34

Lab method of protein electrophoresis

Answer 34

* Performed at pH 8.6 for serum, resulting in 5 bands: alpha1, alpha2, beta, and gamma * bands are measured by densitometry

Question 35

Immunoelectrophoresis (IEP)

Answer 35

* No longer commonly used * Patient serum placed in every other of a series of wells * Remaining wells contain normal serum * Gel undergoes EP * Antiserum added to each trough * Precipitation arcs form between antisera in the troughs and the electrophoresed proteins in the gel * Interpretation depends on visual comparison of the arcs formed with patient serum

Question 36

Immunofixation electrophoresis

Answer 36

* Patient serum placed into 6 wellsin agarose gel * Gel undergoes EP * 5 different antisera applied to gel: anti IgG, IgA, IgM, kappa, and lambda

Question 37

Immunotyping

Answer 37

IT, immunosubtraction * Often used in conjunction with capillary EP * Serum sample incubated with different solid phase sepharose beads attached to antibodies against gamma, alpha, mu, kappa, or lambda * supernatants undergo EP to determine which reagent resulted in removal of the abnormal spike

Question 38

Answer 38

Question 39

Answer 39

Question 40

Answer 40