- major natural proteins in blood serum  globulins (antibodies), albumins, and proteins of
the clotting cascade such as fibrinogen
- blood often contains proteins that are not endogenous, but released upon damage to cells
- most diagnostically useful proteins are enzymes; conversion of substrates and/or generation of
products can be monitored; enzyme signals can be amplified
2. Type I and Type II protein defects
- radioimmunoassay (RIA), enzyme linked immuno-sorbent assays (ELISA) and
electrophoresis/Western blotting  other proteins have diagnostic utility; measure of total
amounts of proteins (not activity)
- physicians concerned with release of normal cellular proteins in blood; direct correlation
between activity and amount  specific activity or Kcat
- Type I protein defects  caused by altered amounts (reflected in altered activities); normal
specific activity thalassemia
- Type II protein defect  reduction in enzyme activity occurring without reduction in
amount; specific activity is reduced due to defect in enzyme
1. Importance of protein glycosylation (reference to sialic acid, in regulating levels of
exogenous proteins in blood serum)
- proteins exposed to extracellular environment (interstitium/blood) have sugar to prevent
proteolysis and aid in recognition
- glycosylation is post-translational modification; sugars are small chain of 5-8
monosaccharide units connected to proteins via asparagine (N-linked) or threonine (O-linked)
covalent bonds
- common sugars are glucose and galactose; N-acetyl glucosamine, N-acetyl galactosamine, and
sialic acid (N-acetyl neuraminic acid, NANA) is a common terminal sugar for most
oligosaccharide chains
- release of intercellular proteins into blood is continuous because of natural tissue turnover
caused by damage; proteins cleared through hepatobiliary system
- small proteins filtered by glomeruli  appear in urine
- liver cells/macrophages contain asialoglycoprotein receptors (asgR)  recognized proteins
that lack sialic acids; intracellular/foreign proteins do not contain sialic acids  recognized
by asgR, which binds them and induced receptor-mediated endocytosis  proteins internalized
by hepatocytes and macrophages  degradation by lysosome or packaged/excreted in bile
3. AST/ALT
- diagnostic potential for serum component determined by:
1.) tissue specificity
2.) concentration in tissue
3.) size of source organ
- Aspartate transaminase (AST) and alanine transaminase (ALT); catalyze interconversion of
amino acids and keto acids
- AST level higher 8000X in heart, liver, SKM, and kidney relative to serum; small damage to
these organs  large increase in serum levels of AST; AST less diagnostically useful for
damage to pancreas, spleen or lung
- ALT is highest in kidney and liver (low in all other organs); if both AST and ALT elevated 
aid in differential diagnosis of liver or kidney damage; if only AST is elevated  cardiac/SKM
damage; ALT/AST ratio diagnostic for liver disease
- some enzymes used in pathodiagnosis are tissue-specific, prostate specific antigen (PSA);
PSA is a serine protease present in seminal fluid and secreted by prostate parenchyma; levels
elevated in prostatic hyperplasia and prostate cancer
6. Sensitivity, Specificity, PPV, NPV and Receiver operator characteristic
- PPV is positive predictive value, NPV is negative predictive value
- define normal range  series of lab testes obtained from diseased (D) and healthy (H) patients
- assumption #1  H patients are healthy and D patients have disease
- diseased population has subscript i because different diseases can present with different ranges
and this can aid in differential diagnosis
- for most tests  overlap between H and D is substantial (with x-axis being distribution of
serum analyte level)
- choice can be made of any threshold to distinguish H from D
- normal ranges: R1, R2, and R3; if R1 used then there will be a lot of healthy subjects with
values out of the normal range (false positives), but a correspondingly small number of patients
with disease who have normal values (false negatives)
- range 1 used if one wants to discriminate those patients who clearly are not disease for those
who may by disease
- as range expands from R2 and R3  number of false positive decreases and number of false
negatives increases
- sensitivity  fraction of those with disease correctly identified as positive by test; sensitivity
= TP/(TP + FN)
- specificity  fraction of those without disease correctly identified as negative by test;
specificity = TN/ (TN + FP)
- positive predictive value (PPV)  fraction of people with positive tests who actually have
the condition; PPV = TP / (TP + FP)
- negative predictive value (NPV)  fraction of people with negative tests who actually do not
have the condition; NPV = TN / (FN + TN)
- specificity increased from R1  R3; ratio of healthy to diseased populations to right of
threshold
- sensitivity decreased from R1  R3; determined by diseased population to right of threshold,
divided by total disease population
- optimal choice for Normal range determined by a receiver-operator characteristic (ROC) 
plot of the likelihood ratio  asks, If you have a positive test, how many times more likely are
you to have the disease?; likelihood ratio of 6  someone with positive tests is six times more
likely to have the disease than someone with a negative test
- likelihood ratio = sensitivity/ (1.0-specificity)
- ROC analysis  sensitivity plotted against (1.0 – specificity) ; best diagnostic tests are those
with greatest area under curve; optimum choice is takes as point at which ROC curve crosses
diagonal
- sometimes many false positive OK, if there is high sensitivity  small Normal range (R1 used);
false positives then separated from true positives by CT/MRI
4. ISOZYMES
- isozymes are two or more forms of enzymes that catalyze the same reaction yet have distinct
primary sequences; products of different genes; pancreatic (P) and salivary (S) amylases 
both catalyze hydrolysis of starch or glycogen in the chain (endo-saccharidases)
- amylases are small enzymes and is one of few serum proteins that can be cleared by kidney
(urine activity is measurable); kidney disease  increase in urine amylase without change in
serum amylase
- both P and S amylase undergo post-translational modification (glycosylation); S-form
glycosylated, P-form is not
Electrophoresis
- electric current makes protein samples move according to size, shape, and charge; small,
negatively charged proteins move faster; separation can be determined just by molecular mass
by adding denaturants to impart a constant shape and charge/mass ratio
- transfer of proteins from electrophoresis gel to a membrane  higher specificity; then stained
with antibodies (Western blot)
Alkaline phosphatase (ALP)
- ALP is a plasma membrane protein both liver/bone (same MW) forms are sialidated
(glycosylated with sialic acid residues at termini)
- sialic acid  negative charge; since both are same MW  both liver and bone forms migrate
as a single band; treatment of serum with neuraminidase (sialidase) removes sialic acid termini
and native differences in charge are revealed; bone isozymes travel more slowly than liver
isozymes towards anode
- ALP is diagnostically important in hepatobiliary disease and bone diseases associated with
increased osteoblastic activity
- hepatobiliary disease  serum liver ALP rises 10X upon extrahepatic obstruction, such as
bile stone are cancer; serum levels of bone ALP increase during bone remodeling; very ALP
high levels in bone cancer
5.
Creatine kinase
- CK for creatine kinase; CPK for creatine phosphokinase
- used to diagnose damage to heart muscle (MI)
- CK present in all muscle tissues and in brain
- specific isoforms for brain, skeletal muscle and cardiac muscle
- CK is a dimer  comprised of two polypeptide chains derived from either brain (B) or
skeletal muscle (M) isoforms
- three possible combinations: BB (CK-1) in brain, MB (CK-2) in cardiac muscle, MM (CK3) in skeletal muscle
- B subunit from chromosome 14; M subunit from chromosome 19
- polypeptides  monomeric units  dimers organize according to expression pattern in given
cell
- brain  only B gene active; in skeletal muscle  only M gene is predominant
- in heart  both M and B genes active; isozyme pattern in heart is 1:2:1 MM:MB:BB
- CK-2 isozyme increases in serum 3 hour following a myocardial infarction and persists for 3
days; CK-3 may increase, as well; CK-1 not detectable in serum
- cardiac involvement is indicated y area under curve
Troponins
- CK-2 increases upon onset of MI, but takes 3-4 hours before serum levels exceed normal
range
- cardiac-specific troponins; troponins are proteins involved in functioning of myofibril (not
enzymes)
- cardiac specific troponins  c-TnT and c-TnI elevate in serum at same rate as CK-2;
however, levels of cTnT and cTnI are undetectable in normal patients  takes little time for
cardiac troponins to exceed normal values
- release of cardiac specific troponins takes occurs over days and have a longer half life  good
markers for late detection
- CK-2, cTnT, and cTnI reach maximum diagnostic potential at 6 hours