1. Quick Compendium of CP: Chemistry
Enzymes, Serum Proteins, Acid-Base and Electrolytes
ZW 7/7/08

2. Enzymes: Basics Michaelis-Menton kinetics:
The rate of enzyme activity varies linearly with substrate concentration up to the point that the enzyme is fully saturated with substrate
At this point, enzyme working as fast as it can (Vmax)
Rate of reaction at this point varies only with enzyme concentration

3. Michaelis-Menton Kinetics

4. Enzymes Can measure enzyme concentration by: Using excess of substrate
Or, measure whether a reaction has taken place
Measure reaction products
NAD to NADH to NAD (NADH absorbs light at 340nm, NAD does not)

5. Coupled Enzyme Assay (NAD/NADH)
Aspartate (Asp) + a-ketoglutarate  oxaloacetate (OAA) + glutamate
Does not utilize NADH
Aspartate (Asp) + a-ketoglutarate  oxaloacetate (OAA) + glutamate + NADH malate + NAD
Add excess NADH and aKG, along with catalysts AST and MD
The disappearance of NADH (absorbance at 340nm) can be used as a reflection of AST (enzyme at first arrow)

6. Measurement of Enzyme Antigen
The quantity of enzyme determined by immunoassay corresponds to the enzyme ACTIVITY
Discordance between concentration and ACTIVITY usually takes the form of the immuno assay overestimating the activity
May be due to serum enzyme inhibitors
Deficiency in necessary cofactor
Defective enzyme
Proteolytically inactivated enzymes

7. Cofactors, Coenzymes Cofactors: Coenzymes:
Substances that bind to an enzyme and enhance activity
Include inorgantic cofactors like zinc, calcium, magnesium, iron
Organic: also called coenzymes
Coenzymes:
Organic cofactors, include NAD, protein S, pyridoxine (vit B6)

8. Macroenzymes Ordinary enzymes bound to antibodies Has 2 effects:
Makes it incapable of functioning
Prevents it from being cleared from blood

9. Competitive INHIBITION

10. Non-competitive Inhibition

11. Uncompetitive Inhibition

12. Enzyme Units International Unit (IU): Katal
The amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute
Katal
1 katal= the amount of enzyme that catalyzes the conversion of 1 Mole of substrate per second
1IU = 16.7 katals

13. Hepatic Enzymes Liver transaminases AST and ALT AST ALT
Cardiac muscle, liver, skeletal muscle, kidney, brain, lung, pancreas (in descending order)
Found within cytoplasm (20%)and mitochondria (80%)
ALT
MORE SPECIFIC FOR LIVER, confined to liver and kidney
Found entirely within cytoplasm

14. Hepatic Enzymes In children
AST activity is slightly higher than ALT
Pattern reverses at age 20
In adults, AST activity a little lower than ALT
May reverse with old age
Both AST/ALT activities higher in adult males over females, and in African-Americans
Hemolysis raises AST/ALT

15. Hepatic Enzymes
Intra-individual variation more significant for ALT than AST
Marked diurnal variation (highest in afternoon) and day-to-day variation up to 30%
Both AST/ALT elevated in heparin therapy to around 3X baseline
In renal failure, both significantly lower than in healthy individuals

16. Hepatic Enzymes Lactate dehydrogenase (LDH):
Present in numerous tissues, traditionally separated into 5 isoenzymes by electrophoresis
Fastest moving are LD1 and LD2
Found in heart, RBC, kidney
Slower moving are LD4 and LD5
LIVER and skeletal muscle
LD3 in lung, spleen, lymphocytes, and pancreas
LD6- “sixth” LD is sometimes seen migrating cathodal to LD5
PRESENCE THOUGHT TO BE A DIRE FINDING (hepatic insufficiency in setting of cardiovascular collapse)

17. LDH Concentrations: LD2>LD1>LD3>LD4>LD5
LD 1 elevation (with flipped LD ratio LD1>LD2):
Acute MI
Hemolysis
Renal infarction
Elevated LD4 and LD5:
LIVER DAMAGE or skeletal insult
Elevated LD1 and LD5:
Acute MI with liver congestion
Chronic alcoholism

18. Alkaline phosphatase Two types of phosphatases:
Alkaline (optimum pH is 9):
Bone, bile ducts, intestine, placenta
Separate reference ranges for women and children
Acid (optimum pH is 5):
Found in prostate, RBC, and bone
RBC acid phosphatase is susceptible to inhibition by 2% formaldehyde and resistance to inhibition by tartrate (this is also seen in hairy cell leukemia)

19. Alkaline phosphatase 4 isoenzymes by electrophoresis:
Each displays characteristic degrees of inactivation by heating, urea incubation, and l-phenylalanine
Heating produces significant inactivation of bone alk phos (bone burns), 50% inactivation of biliary alk phos, and NO inactivation of placental alk phos.

20. Alkaline phosphatase Biliary alk phos: Bone alk phos:
Most sensitive marker of hepatic metastases
Bone alk phos:
Produced by osteoBLASTS and reflects bone reforming activity
Highest levels seen in Paget’s disease of bone
A specific immunoassay for bone alk phos available

21. Regan Isoenzyme Observed in about 5% of individuals with carcinoma
Appears identical to placental alkaline phosphatase

22. Intestinal Alk Phos Elevation
Can be factitious in non-fasting individuals, particularly in Lewis positive type B or O pts
Ingesting a meal can elevate alk phos by 30% in 2-12 hours
Repeat fasting alk phos

23. Alk Phos Minor elevations are a common clinical problem
Usually higher in men than women
Higher in African-Americans
Threshold of 1.5 times normal limit for further investigation (repeat in 6 months if borderline)
Causes:
Pregnancy, CHF, hyperthyroidism, drugs

24. Alkaline phosphatase Sensitive indicator for hepatic metastases
In women, investigation should include assay for anti-mitochondrial antibodies

25. Gamma-glutamyl transferase (GGT)
best test to confirm if elevate alk phos if of biliary tree origin
Found in biliary epithelial cell, particularly those of the small interlobular bile ducts and ductules
Exquisitely sensitive to biliary injury
Also elevated in:
Steatosis, diabetes, hyperthyroidism, RA, acute MI, COPD

26. GGT Present within the smooth endoplasmic reticulum of hepatocytes
Whenever there is induction due to excess toxin, GGT levels increase
This includes warfarin, barbiturates, dilantin, valproic acid, methotrexate, EtOH
2-3X normal limit in heavy drinkers
Returns to normal after 3 weeks abstinence and can be followed as marker for alcohol consumption

27. 5’ Nucleotidase Main source is biliary epithelium
Levels highest in cholestatic conditions
Another test to confirm if elevated alk phos is due to hepatobiliary disease
Low sensitivity, best as confirmatory test, utility less than GGT

28. Ammonia Hyperammonemia nearly always due to liver failure
In children, it should raise the suspicion for an INBORN ERROR IN METABOLISM
Sources of ammonia:
Skeletal muscle and gut
Bacteria in GI tract produce ammonia
Normally functioning liver removes this ammonia and discards it in the form of urea which is excreted in urine

29. Ammonia Blood ammonia can become disastrously high when:
Too much collateral circulation
Excess protein in gut (excess hemoglobin from variceal bleed)
SIGNIFICANT HEPATOCYTE DISFUNCTION
In cirrhotic patients, these conditions are often met, and neurotoxicity can result

30. AMMONIA
Measurement requires a FRESH specimen which has been CHILLED during transport and has undergone NO hemolysis
Smoking patients must abstain for several hours before draw
Anyone care to comment on the current state of the ammonia level as it can or cannot be ordered?

31. Bilirubin Unconjugated (indirect) bilirubin:
Water-insoluble form produced by breakdown of heme
Taken to liver tightly bound to albumin where it under goes glucuronidation to produce water-soluble (as in bile) conjugated (direct) bilirubin
Conjugated bili excreted in bile where intestinal bacteria convert to urobilinogen

32. Bilirubin
Urobilinogen ends up in feces, some of which is reabsorbed and excreted in urine
Some urobilinogen is converted by colonic bacteria into brown pigments (complete biliary obstruction leads to yellow-white stool- the Silver Stool of Thompson)

33. Bilirubin
Unconjugated bilirubin, even when it is quite high, does NOT appear in urine
Thus, bilirubinuria indicates CONJUGATED hyperbilirubinemia
2 test methods
Diazo-colorimetric methods:
Rely on formation of colored dye through reaction of bili with diazo compound
Without the addition of an accelerator (alcohol), only conjugated bilirubin is measured
Addition of accelerators measures combined unconj and conjugated (total) bilirubin
Direct spectrophotometry
Bilirubin concentration measured by absorbance (455nm)

34. Causes of Hyperbilirubinemia
Unconjugated:
Hemolysis (extravascular)
Blood shunting (cirrhosis)
Right heart failure
Gilbert syndrome
Drugs: rifampin
Crigler-Najjar syndrome
Hypothyroidism

35. Causes of Hyperbilirubinemia
Conjugated:
Dubin-Johnson syndrome
Hepatitis
Endotoxin (sepsis)
Pregnancy (estrogen)
Drugs: estrogen, cyclosporine
Mechanical obstruction:
PBC, PSC, tumor, stricture, stone

36. Additional Hepatic Function Tests
PT:
Factor VII has half life of 12 hours (ON RISE EXAM)
Sensitive marker for impaired hepatic synthetic function
Impaired bile secretion can lead to Vit K deficiency (bile salts required for absorption)
How do you distinguish between a prolonged PT because of cholestasis/impaired Vit K absorption and hepatocyte injury?
Add parental vit K
Gammaglobulins
Serum gammaglobulins elevated in liver injury, especially autoimmune

37. Neonatal Jaundice
Most cases of neonatal jaundice are entirely benign (“physiologic jaundice”)
Hepatic enzymes not yet at full capacity leading to build-up of unconjugated bilirubin
Usually noted between days 2-3 of neonatal life
Usually peaks at 4-5 days; rarely exceeds 5-6 mg/dL

38. Severe hyperbilirubinemia in neonates
Most common causes:
Hemolytic disease of the newborn (HDN)
Sepsis
Poorly developed blood-brain barrier causes unconjugated bili to pass to CNS and cause damage (kernicterus)

39. When to worry about neonatal jaundice?
Appearance in first 24 hours of life
Rising bili beyond 1 week
Persistance of jaundice past 10 days
Total bili that exceeds 12 mg/dL
Single-day increase of >5 mg/dL
Conjugated bili (direct) that exceeds 2 mg/dL

40. Therapy for neonatal jaundice
Phototherapy:
Consider when bili exceeds 10 mg/dL before 12 hours of age; 12 mg/dL before 18 hours of age; 14 mg/dL before 24 hours of age
Phototherapy converts unconj bili to a molecule that can be excreted WITHOUT conj
Not useful for conj hyperbili
Exchange transfusion:
When bili exceeds 20 mg/dL

41. DDX of neonate hyperbili
Jaundice in 1st 24 hours:
Erythroblastosis fetalis
Concealed hemorrhage
Sepsis
TORCH infection
Jaundice between 3rd and 7th day:
Bacterial sepsis (usually UTI origin)
Arising after 1st week:
Breast milk jaundice, sepsis, extrahepatic biliary atresia, cystic fibrosis, congenital paucity of bile ducts (Alagille syndrome), neonatal hepatitis, glactosemia, inherited hemolytic anemia (PK def, hered. Spherocytosis, G6PD def)

42. Lab Eval of Acute Liver Injury
May be symptomatic, Jaundice, Elevated transaminases
May be due to viral hepatitis (HAV, HBV, HCV), autoimmune hep, toxin, drug, ischemia, or Wilson disease
Labs:
Hepatitis serologies, ANA, ceruloplasmin, clinical history (new drugs usually cause damage within 4 months of starting)

43. Acute liver injury labs
Acute viral hepatitis due to HAV, HBV most often leads to complete recovery
Acute HCV goes to chronic HCV in >80% of cases
Serologic testing for HAV, HBV are very dependable for diagnosing acute infx (IgM anti-HAV, IgM anti-HBc, HBsAg)
Anti-HCV test only about 60% sensitive for acute infx
HCV RNA testing 90-95% sensitive

44. Transaminases
Acute hepatic injury due to ischemic or toxic injury produce PROFOUND elevations in transaminases- often >100X upper limit of normal (RARE in acute hepatitis)
AST > 3,000 U/L = toxin in 90% of cases
AST 10X upper limit of normal in acute viral hepatitis, but reaches this level RARELY in alcoholic hepatitis
AST:ALT ratio is over 2 in 80% of pts with toxic, ischemic, and EtOH hepatitis (<1 in viral hep)
Amount of transam elevation poorly correlates with LEVEL of injury

45. PT/BILIRUBIN PT (protime)- BILI
Probably the best indicator of prognosis in acute hepatic injury
>4.0 secs indicates severe injury/unfav prog
BILI
Jaundice in 70% of pts with EtOH, HAV
Jaundice in <20% of pts with HBV, HCV
Jaundice rare in kids with acute viral hep, rare in toxic or ischemic injury
>15 mg/dL indicates severe liver injury, bad prognosis

46. Pancreatic Enzymes AMYLASE:
Serum amylase = salivary and pancreatic isoenzymes
On electrophoresis 6 bands result
1st three are salivary
Slower 3 are pancreatic
Can be separated by inhibition as well
Salivary amylase sensitive to inhibition by wheat germ lectin (treticum vulgaris)
Assays based on monoclonal Abs directed against specific isoenzymes are very accurate

47. Serum Amylase Rises within 2-24 hours of onset of acute pancreatitis
Returns to normal in 2-3 days
Higher levels don’t correlate with severity
Higher levels are more specific for acute pancreatitis
Persistance in elevation suggests complication like pseudocyst

48. Urine amylase
Nearly all pts have concomitant increase in urine amylase
Amylase primarily cleared by glomeruli
Renal insufficiency = spurious amylase elevation
Fractional excretion of compound (x) = FEx

49. Amylase Sensitivity of serum amylase for acute pancreatitis is 90-98%
Specificity is only around 70-75%
Specificity of urine amylase and FEamylase is higher

50. Additional causes of increased amylase
Diabetic ketoacidosis, peptic ulcer dz, acute cholecystitis, ectopic pregnancy, salpingitis, bowel ischemia, intestinal obstruction, macroamylasemia, and renal insufficiency, opioid analgesics (contraction of sphincter of oddi)

51. Pancreatic Enzymes LIPASE:
Unlike amylase, essentially specific for pancreas
Rise parallels rise in amylase, but remains elevated for 14 days
Less reliant on renal clearance than amylase
Often considered superior to amylase in dx of acute pancreatitis

52. Lab Eval for Acute Pancreatitis
Amylase limited in sensitivity and specificity
Hypertriglyceridemia (common cause of acute pancreatitis) interferes with amylase assay (false negative)
Lipase remains elevated longer giving greater sensitivity
Others: trypsinogen-2 and elastase-1 (not used often) but have excellent negative predictive value

53. Acute Pancreatitis Prognosis
Ranson Criteria:
Aggressive management
ICU admission
Parenteral feeding
Systemic antibiotics
Provides specificity of 90%
Cannot be assigned until 48 hours after admission
Serum amylase/lipase are poor predictors of outcome

54. Etiology of Acute Pancreatitis
Stones
EtOH
Viruses
Inherited diseases:
Mutations in
Cationic trypsinogen (PRSS-1)
Pancreatic secretory trypsin inhibitor (PSTI)
Cystic fibrosis transmembrane conductance regulator (CFTR)

55. Pancreatic EXOCRINE function
Tests include:
Secretin-cholecystokinin (secretin CCK, secretin pancreozymin), fecal elastase-1, fecal fat
FECAL FAT:
Oil-red O stain and 72 hour fecal fat quant
Stain has sensitivity of 70%
Fecal fat quant quite sensitive for panc. Insuff.

56. Myocardial Enzymes CK (creatine kinase):
Three isoenzymes distinguishable by electrophoresis
CK-MM, CK-MB, CK-BB
Fastest migrating is BB (CK1), then MB (CK2), then MM (CK3)
CK-BB: found primarily in brain, with lesser amounts in bladder, stomach, and prostate.
CK-MM (CK3) found in skeletal muscle and cardiac muscle (Skel.muscle: 99% MM, cardiac: 70%MM). In normal subjects, serum CK is 100% MM
CK-MB (CK2) found in cardiac and skeletal muscle (card: 30%, skel: 1%), skeletal muscle is source of nearly all MB in circulation in serum

57. CK Now use immunoassays to measure CK
Much faster and more accurate than electrophoresis (particularly in low/clinical range)
Total CK enzymatic assay
Ratio of CK-MB to total CK (RI: relative index) adds to ability to distinguish MI
RI of 2% is usually cut-off

58. Troponin I Group of enzymes consisting of
Troponin T (TnT)
Troponin I (TnI)
Troponin C (TnC)
Involved in mediating the actin-myosin interactions that result in muscle contraction
Immunoassays distinguish cardiac troponins (cTnI and cTnT) from skeletal muscle troponins

59. Troponin
Vast majority of cardiac muscle troponin is bound to actin/mysosin
Very small amount free in cytoplasm
So:
Immediate release of cytoplasmic troponin in MI (4-8 hours)
And sustained release of bound troponin over next days

60. Cardiac Troponin I
Only cTnI is currently widely available for use in clinical diagnosis
cTnT marginally less cardiospecific
cTnI NOT elevated in skeletal muscle injury
cTnI may be elevated in other forms of cardiac muscle injury(contusion, myocarditis)

61. Myoglobin Mgb is THE MOST SENSITIVE of the cardiac markers
Earliest marker of acute MI
Myoglobin should be elevated as soon as an infarcting patient present to ED
LEAST CARDIOSPECIFIC of cardiac markers!

62. Ischemia-modified albumin (IMA)
When albumin circulates through harsh environments (acidosis, hypoxemia, free radicals, altered calcium- all seen in ischemia), its ability to rapidly and tightly bind cobalt is altered
Altered Cobalt Binding (ACB) assay:
Add cobalt, measure unbound amount
Measurement reflects ischemia modified albumin

63. B-type Natriuretic Peptide (BNP)
Natriuretic peptides:
Cause vasodilation and sodium excretion
A-type: stored in granules in atrial myocytes, affected by atrial filling pressure, ventrilar wall tension
B-type: synthesized in ventricular myocytes
Correlates directly with ventricular wall tension
N-terminal peptide fragment (N-terminal pro-BNP) is cleaved from pro-BNP to make active hormone BNP
N-terminal pro-BNP is more stable, provides more longitudinal info
Elevated in heart failure
Distinguishes between cardiac/non-cardiac dyspnea
Provides prognostic info for pts with CHF and acute coronary syndrome (ACS)

64. ACS Acute coronary syndrome:
Encompasses many clinical situations with myocardial ischemic damage
Stable angina, unstable angina, acute MI, sudden cardiac death
Lab assays good at diagnosing MI, bad at diagnosing the remainder
Usual biomarkers for necrosis (MB, myoglobin, troponin) are overall poor markers for non-AMI ACS
BNP is predictive for both recurrence and higher likelihood of sudden cardiac death in non-AMI ACS
C-reactive protein good predictor of development of ACS in healthy individuals

65. ACUTE MI
Typical rise and fall of CK-MB or troponin with ischemic symptoms
ECG changes
Or interventionally demonstrated coronary artery abnormality
TROPONINS: single positive troponin is highly specific for AMI
Single low troponin has low sensitivity for negative predictor
CK-MB: increase detectable within 3-6 hours of AMI, peaks at hours, returns to normal in 72 hours; sensitivity using serial measurements approaches 100%
Myoglobin: rapidly released from damaged muscle, early indicator, negative predictive value 2 hours post symptoms approaches 100%

66. SERUM PROTEINS Protein quantitation:
Nitrogen count (Kjeldahl technique) is gold standard; involves acid digestion of protein to release ammonium ions which are quantified
Assumption is serum proteins are 16% nitrogen by mass
Colorimetry (Biuret technique) is recommended routine method for measuring total protein
Absorbance from chelate with copper at 540 nm is proportional to total protein

67. Protein Separation Use PRECIPITATION Electrophoresis:
Movement of proteins due to electrical potential
Charge applied across a medium composed of solid support (gel) and fluid buffer. Charge creates electromotive force
Solid support has slight neg charge and is drawn towards the anode (+ pole), but being solid, cannot move
Compensatory flow of fluid buffer towards negative pole (cathode)
This flow is called endosmosis- has capacity to carry substances suspended in medium

68. Protein Separation If proteins added, two forces are on the proteins:
Electromotive force
Endosmotic force
Most proteins have negative charge
Electromotive force pulls towards anode
Endosmosis pulls towards cathode
Gammaglobulins= weak net negative charge
Electromotive force exceeds endosmotic force
Move to variable extent towards anode

69. Serum Protein Electrophoresis
SPEP:
When electrophoresis is carried out on serum at pH 8.6 on agarose gel, then fixed and stained:
Five distinct bands can be seen
Fastest moving band is albumin
Next fastest- two a bands (a1 and a2)
Then the b band
Then the g band (move very slowly)

70. Serum Protein Electrophoresis

71. Protein Electrophoresis
Increasing endosmosis is used to separate gamma globulins into oligoclonal bands in CSF electrophorsis
Capillary electrophoresis can also be used if there is
Small sample size
Needed automation
Need for speed

72. Immunofixation Electrophoresis
IFE
Method for characterizing a suspected monoclonal band observed in SPEP or UPEP
Much simpler to interpret than IEP
Place pt serum into six wells in agarose gel
Five different monospecific antisera are applied
Anti-IgG, IgA, IgM, Kappa, and lambda
Entire gel is stained
IEP (immunoelectrophoresis)- not commonly used anymore

73. IFE

74. Serum Proteins Albumin Most abundant protein in human plasma
2/3 of total plasma protein
Many functions:
Maintains serum osmotic pressure, carries multiple substances
Congenital absence NOT a serious problem (mild anemia and hyperlipidemia)
Several allotypes:
Most common is Albumin A
When variant is present, might get 2 peaks

75. Albumin Clinical utility: Nutritional status (half-life is 17 days)
Hepatic synthetic function (ESLD)
Diabetic control
In normal pts, up to 8% of albumin is glycosylated
In diabetics with poor control, up to 25% may be glycosylated
Negative acute phase reactant (decreases in inflammatory conditions)

76. Prealbumin Fastest migrating protein on SPEP
Sparse, not normally seen on traditional SPEP
Binds T4 and T3
Binds and carries retinal-binding protein:vitamin A complex
Also, prealbumin is the precursor protein in senile cardiac amyloidosis
Short ½ life of 48 hours
True elevations seen in chronic EtOH, steroids
Negative acute phase reactant

77. a1 antitrypsin (AAT) Major component of the a1 band
Main function is to inactivate various proteases like tyrpsin and elastase
SPEP can be used to screen for AAT deficiency
Markedly positive acute phase reactant

78. a1-acid glycoprotein (orosomucoid)
Briskly positive acute phase reactant
Minor component of a1 band
Major component of increased a1 band seen in inflammation
May be used to monitor chronic inflammatory conditions like ulcerative colitis

79. a2 macroglobulin Protease inhibitor
Serum concentration elevated in liver and renal disease
Large size prevents its loss in nephrotic syndrome, leading to a 10-fold increase in concentration

80. Ceruloplasmin a2 protein Functions in copper transport
Decreased serum ceruloplasmin important marker for Wilson disease, ddx includes:
Hepatic failure
malnutrition
Menke syndrome
Acute phase reactant:
Elevated in inflammation and pregnancy

81. Haptoglobin Third major component of a2 Binds free hemoglobin
Decreased or absent in acute intravascular hemolysis
Very sensitive marker for hemolysis
Haplotypes I and II:
Phenotypes 1-1, 1-2, 2-2
2-2 phenotype is independent risk factor for CAD in diabetes
Acute phase reactant

82. Transferrin Major b globulin
Functions to transport ferric iron (Fe3); normally 30% saturated
Marked increase in Fe def.; abnormally masquerades as an M-protein
Increased in pregnancy and estrogen tx
Decreases in acute phase; but rises is inflammation persists
Blood brain barrier transports transferrin to CSF in a modified form (Tau-protein)
Carbohydrate-deficient transferrin: superior to GGT as marker for EtOH abuse

83. Fibrinogen Also called b globulin
In normal course of events, no fibrinogen in serum as it is consumed by formation of clot
If specimen clots incompletely (heparinized pt) fibrinogen may be seen
Can straddle the b-g interface
When present in serum, may be misinterpreted as an M-protein
May be seen in serum in:
Dysfibrinogenemia, APL syndrome, liver dz, vitamin K def, or heparin

84. C-reactive protein (CRP)
Produced in liver
Predictive value of low level elevations (>2-3 mg/L) for cardiac events
High sensitivity CRP (hsCRP) now available with sensitivity of <0.5 mg/L
Distribution NOT Gaussian curve-
Curve skewed significantly with dense cluster in very lowest CRP levels and long tail extending into >10 range
Half of population has CRP >2

85. CRP Three categories based on CRP: Low level elevation may indicate:
Normal CRP <3 mg/L
High CRP >10 mg/L (active inflammation)
Low-level elevations 3-10 mg/L (cellular stress)
Low level elevation may indicate:
Minor disease states
Genetic factors
Demographic variables
Behavioral patters
Individuals normal set point is inherited

86. CRP
Low-level CRP elevation predicts poor outcome following cardiovascular events
Also correlates with mortality in non-cardiac dzs

87. SPEP Patterns Normal serum: Bisalbuminemia: Invisible prealbumin band
Very large albumin band
Then, small peaked a1, a2, a bimodal B, and a broad gamma band
Bisalbuminemia:
Seen in heterozygotes for albumin allotypes
Double albumin spike
No clinical consequence

88. SPEP Patterns a1-antitrypsin (AAT) deficiency: Nephrotic syndrome:
AAT is the major component of a1 band
Genotype PiZZ individuals have a visibly and quantitatively decreased band
Nephrotic syndrome:
Massive loss of small proteins, particularly albumin
Minimal change disease: especially high loss of albumin
With other forms of nephrotic syndrome, gamma globulins also lost
LARGE PROTEIN MOLECULES RETAINED
Result: dimming of all electrophoretic bands, with exception of a2 which contains a2-MACROglobulin

89. SPEP Patterns Acute inflammation:
Acute phase reactants account for increases in a1, a2 bands
Albumin slightly increased
Prolonged inflammation shows polyclonal gamma globulin increase
Beta-gamma bridging (BODOR noted this has been on several Board Exams):
Hallmark of CIRRHOSIS
Also, hypoalbuminemia, blunted a1 and a2
Beta-gamma bridging mainly due to increase serum IgA

90. Monoclonal Gammopathy
SPEP shows a prominent discrete dark band (M-spike, m-protein) usually within the gamma region (sometimes in B or a2)
A monoclonal gammopathy (paraprotein) shows immunochemically homogeneous immunoglobulin (M-protein) in serum
May be result of:
Multiple myeloma
Neoplastic proliferations like solitary plasmactyoma, MGUS, Waldenstroms macroglobulinemia (lymphoplasmacytic lymphoma), and CLL/SLL

91. Biclonal Gammopathy 2 M-proteins Occurs in 3-4% of cases
If biclonal gammopathy has IgA spikes having a single light chain (by IFE):
Most likely due to appearance of both monomers and dimers in SPEP
Should be considered monoclonal

92. SPEP Hypogammaglobulinemia
10% of SPEPs
When not due to myeloma:
May be due to congenital deficiency, lymphoma, nephrotic syndrome, or corticosteroids
Pts with myeloma and hypogammaglobulinemia are likely to have free-light chains in urine (Bence-Jones proteins)

93. IFE Indicated to characterize M-protein on SPEP
Even if no m-spike is seen, do IFE if:
Strong suspicion of myeloma (lytic bone lesions in 80 year old male)
Systemic AL amyloidosis
Hypogammaglobulinemia
In pts with negative serum screens with high suspicion, do UPEP (for light chain disease)
May be losing all of light chain in urine

94. M-protein
Usually an intact immunoglobulin composed of 2 heavy and 2 light chains
Sometimes, it is light chain only or very rarely, heavy chain only
Systemic amyloidosis may result from monoclonal gammopathy, when M protein produced has unusual (amyloidogenic) properties

95. After diagnosis:
Protein electrophoresis is used to follow disease progression and efficacy of treatment
3 quantitive results are important:
M-protein concentration
Degree of suppression of other immunoglobulins (IgG, IgA, IgM, whichever is not involved)
Quantity of free serum light chain, especially in light chain only myelomas (extremely sensitive to myeloma recurrence after tx)

96. Hyperviscosity syndrome
Normally, viscosity of serum is centipoise (cp)
Hyperviscosity syndrome: exceeds 3.0 cp
Symptoms:
Nasal bleeding, blurred vision, retinal vein dilation, neurologic symptoms

97. Cryoglobulins May be looked for in pts with M-proteins
Precipitate reversibly at low temps
Blood is drawn and kept at 37 degrees C until clotted
Centrifuged at 37 degrees
Remaining serum stored at 4 deg C for at least 3 days, then centrifuged at 4 deg C
Any precipitate that is formed is CRYOPRECIPITATE and can be subject to electrophoresis

98. Cryoglobulins
3 types:
Type I: monoclonal immunoglobulins associated with MM or Waldenstroms
Type II: mix of monoclonal IgM and polyclonal IgG. IgM has Rheumatoid factor activity (anti-IgG). This is the most common type of cryo
Type III: mix of two polyclonal cryoglobulins

99. Mixed Cryoglobulinemia
Types II & III
Found in variety of conditions:
Lymphoproliferative d/o, chronic infx, chronic liver dz, autoimmune dz (SLE)
Most common in women in 4th-5th decades of life
30-50% of cases have underlying HepC virus (most common cause)
Clinical manifestation: palpable purpura, arthralgias, hepatoslenomegaly, lymphadenopathy, anemia, sensoineural defects, and glomerulonephritis
Tx: corticosteroids, plasmapheresis, a-interferon

100. UPEP In proteinuria, UPEP can determine source of protein
Glomerular proteinuria pattern:
Strong albumin, a1, B bands
Very large and very small proteins don’t make it into urine, leaving albumin, AAT, and transferrin
Tubular proteinuria pattern:
Weak albumin band, strong a1 and B bands
Impaired tubular reabsorption of LMW proteins normally filtered freely by glomerulus (a2-macroglobulin, b2 microglobulin, light chains)
Overflow proteinuria pattern:
Monoclonal light chain (Bence Jones)
Remember that these back up into SPEP in MYELOMA KIDNEY

101. CSF Protein electrophoresis
Different than serum
All proteins in serum, but smaller quantities
Prominent prealbumin band and double beta (transferrin) band (transferrin conjugated to Tau-protein on way into CSF)