Chemistry Crash Week Notes

Monday, April 21
st


I. Matching Our Analytical Technology with the Analytes
a. Reference Ranges and Assay Sensitivities








b. Primary vs Secondary
i. Primary: Detect individual interactions between analyte (antigen) and antibody.
Primary binding/assays do not measure lattice structures.
ii. Secondary: Requires crosslinking/lattice structure formation.
c. Competitive vs Non-competitive
i. Competitive: Competition between endogenous analyte + labeled analyte. Size is
important (Small MWs, hard to make sandwiches. To make this easier, add label.
Only need 1 antibody to get a reaction).
1. Measurement of an analyte, based on competition between endogenous
analyte and labeled analyte (that is one of the reagents).
ii. Non-competitive: No labeled analytes. Example: Enzyme immunoassays, such as
sandwich assay (the most common).
1. Endogenous analyte simply binds to form an antigen:antibody complex.
Formation of the complex can then be measured.
d. Homogeneous vs Heterogeneous Assays
i. Homogeneous Assays: Generate a signal that can be detected without a separation
of the assay components. Signal (usually an enzyme) is changed by the binding of
analyte. No washing steps required.
ii. Heterogeneous Assays: Wash step required because the signal will be detected with
or without the presence of analyte. Signal isnt changed by binding of analyte.
Unbound signal is washed away so that the amount of signal is relative to the
amount of bound analyte.
e. Absorbance vs Fluorescence vs Chemiluminescence
i. Absorbance measures the amount of energy used by electrons as they are excited
to higher energy states (jump/move up). Less light measured at the end as
compared to the amount of light that was used at the beginning.
ii. Fluorescence measures amount of light emitted. Electrons jump down and produces
a flash of light (after absorbing light energy of a different wavelength).
1. As electrons jump down, energy levels:
a. Longer wavelength, Less energetic
iii. Chemiluminescence measures the light emitted by electrons as they jump down
(after being excited by a chemical reaction).
1. Same concept as fluorescence except chemical reaction involved (usually
redox reaction).
2. Both fluorescence and chemiluminescence emit light as electrons come
down.
f. Direct vs Indirect Assays
i. Direct: Detect antigen by the use of antibodies against that antigen
ii. Indirect: Detect antibody produced by individual, use antigen to detect antibody
1. Ex) Tests for autoimmune diseases
g. Choosing Assay Format Based on Molecular Size
i. Large MW molecules:
1. Non-competitive
2. Heterogeneous
3. Sandwich formats (Antibody binds at different locations)
a. Examples: Large hormones
ii. Small MW molecules:
1. Competitive
2. Homogeneous
a. Examples: Drugs, small hormones (steroid, free T3, T4)
b. Hard to attach antibodies due to small MW Do not use sandwich
h. Choosing Assay Format Based on Sensitivity
i. Hormones (of any size) tend to be present in very low quantities
1. Hormone assays often use Turbo techniques developing fluorescent or
chemiluminescent products.
i. Nephelometry and Turbidimetric Assays
i. Require lattices (Secondary).
ii. Not sensitive at all Requires high amount of analyte
1. In order to enhance the assay, put antibodies on latex particles (need to be
small to keep the turbidity low at first). Easier to produce lattices with latex
particles. Need few crosslinking events to give nephelometric signal.
j. Sandwich Formats
i. Capture System
ii. Analyte Captured
iii. Detection System (Enzyme Conjugate)
iv. Amplification System (Colored Product)
k. Turbo Formats
i. Substrate gives fluorescent/chemiluminescent product
1. This is the main difference between Turbo Assays and Sandwich Assays.
Otherwise, about the same.
l. Immune Status EIAs
i. Antigen on microwell (Main difference from other assays; normally, the well is
coated with antibody) + Patient antibody + Conjugate. Similar to Sandwich Assay.
1. Ex) Quantify autoantibodies
m. EMIT Assays
i. Enzyme Multiplied Immuno Technique Assays
1. Competitive, Direct, Homogeneous
ii. Enzyme is conjugated with the small MW analyte. Analyte is labeled with enzyme,
competes for binding to a limited amount of antibody. Enzyme is inhibited with
antibody binds to analyte.
1. If antibody binds to labeled analyte Inhibits enzyme No product
2. Endogenous analytes + Patient analytes used
3. Signal (enzyme activity is changed by binding)
4. More color change More patient analyte
5. If there is no patient analyte present No analyte No product
6. If high amount of patient analyte limited endogenous analyte binds to
patient More analyte More product
n. FPIAs
i. Fluorescence Polarization Immuno Assays
ii. Competitive, Homogeneous assay for small MW analytes
iii. Conjugate is a fluorescent-labeled analyte. Limited amount of antibody is directed
against the analyte.
1. Polarization
a. Filter with vertical slits, filters out some of the light light becomes
polarized
2. Shine polarized light on molecules, small enough molecules. Fluorescence
signal in both bound and not bound.
a. Need small MWs to cause tumbling if not bound. Only use small MWs
in this assay.
b. Will lose polarization if not bound
c. If bound to antibody Held in place, no tumbling Retains
polarization
3. Polarization decreases as patient analyte increases
a. Indirect!
4. No patient analyte Increase in polarized signal
iv. How to convert spectrometer to fluorometer
1. Spectro (Measures absorbance), Fluoro (Measures fluorescence)
2. Add another filter and bend to a right angle. Dark room. Measure only
specific wavelengths.
a. All help to minimize signal:noise ratio
o. CEDIA
i. Complementary (or Cloned) Enzyme Donor Immuno Assays
1. Identical to EMIT except 2 pieces of enzyme. If antibody binds No enzyme
activity
ii. Competitive Assays
iii. B-galactocidase enzyme requires two pieces for activity. Smaller piece is labeled
with analyte (drug, etc). This complex will compete with sample analyte for
antibodies.
1. If there is no analyte in patient sample, the small piece-complex binds to the
antibody so it cannot compete an active enzyme No signal
a. No analyte No signal
i. Direct!
2. If there is lots of analyte in patient sample, it binds to the antibody. So the
small piece-complex does compete the enzyme.
a. Lots of analyte Lots of signal
b. Signal = Color
p. MEIA
i. High sensitivity caused by:
1. Sandwich Heterogeneous Non-competitive Direct
2. High binding capacity on microparticles
3. Incubation in solution (everything is pulled through filter No random
movement of antigens)
4. Everything is cause in fibers of filter, form sandwiches, wash step, unbound
enzyme will wash through, add fluorescent substrate
q. Gold Particle Capture Lateral Flow Filters
i. One exception with lateral flow filters:
1. Dont need to wash Capture zones are placed early in the filters
Capillary action pulls unbound past No washing needed
a. Exception: Sandwich, but no washing required
b. Can use whole blood. Mesh can capture RBCs. Serum + plasma will
move through the mesh.
2. Form sandwich (gold/latex on top instead of enzyme)
r. Drug Screen Flow-Through Filters
i. Sandwiches form along the test strip.
II. Activating Adaptive Immune System
a. APCs (Antigen Presenting Cells) B cells, Macrophages, Dendritic Cells
i. Foreign material is recognized by APCs via toll-like receptors or opsonins
ii. B cell recognition via B cell receptors (antibodies)
1. Nave B cells have sIgM (surface IgM) and alpha + beta Igs. B cell receptor
composed of sIgM + alpha + beta Igs
2. Antibody binding in B cells No signal produced
3. T cell, CD3 signal sent down
iii. APCs phagocytize and digest material to present on MHC proteins on their
surfaces.
1. Presented on MHC II (Extracellular material)
2. MHC I (Intracellular material). Begins inside the cell.
a. Ex) Virally infected cell. No phagocytosis involved. Began as
intracellular material.
iv. T cells activated after they recognize the foreign material. Also need 2
nd

costimulator signal CD7, CD28, CD40, etc.
1. Requirements to activate T cells:
a. Recognition of foreign peptides on APCs
b. Costimulator signal
i. After both of these occur, cytokines are released and T cells
are activated
b. Macrophages vs Dendritic Cells
i. Both originate from myeloid lines, but diverge and have different roles.
ii. Macrophages: Tissue/Resident macros: Sensory cells, 1
st
to detect foreign
material. Control 1
st
line of defense. No movement, stay in the same area.
iii. Dendritic Cells: In the same area as macrophages, but in lower numbers. Primary
role: Migrate and mature to take antigen to lymphs activates adaptive response.
Must mature to change into good APCs (More MHCs and costimulators and cytokines
produced). Interact with T cells in lymphs, which activates T cells.
c. Primary/Secondary Tissues
i. Primary: Bone marrow, Thymus
ii. Secondary: Lymph, Spleen (Which is where cells become activated)
d. Inflammatory Response
i. Blood rushed to site to increase number of WBCs, allows proteins, complement,
antibodies, and coagulation factors to go to the site as well.
ii. How to relieve pressure? Lost protein gradient Have to drain through lymph
nodes. Dendritic cells move that way. B cells detect antigen on their own. Nave B
cells are found in lymph nodes in the follicular/cortex area waiting for antigen.
Antigen arrives from flow of swollen site.
iii. T cells are CD4 and CD8. After they mature and leave the thymus, they are
assigned to one of those two classes. Helper Ts or Cytotoxic Ts. Theres also
Regulator T cells: Unique type of CD4s.
1. Regulator T cells: Helpers that suppress. Normally, Helper T cells activate.
a. Thymus Testing
i. Too high affinity for self antigens or not high enough affinity
killed
ii. The pre-regulator T cells have a high affinity and almost die,
but they are saved by a little extra IL-2. They then release
suppressive chemicals. Need some self-antigen recognition.
1. Ex) Autoimmune diseases Issue with Regulator T cells
e. Proteinuria
i. Microglobulin Should get through glomerular filter should be reabsorbed.
1. If present in urine problem with reabsorption
f. Renal Tubular Acidosis II (RTA II)
i. PCT not functioning well problem with reabsorption. Leads to high amounts of
junk in urine, such as:
1. Protein, Bicarb, Amino Acids, and others
2. Everything is normally reabsorbed in the PCT
g. Helper Ts
i. MCH I Interacts with cytotoxic T cells
h. TH1 vs TH2 (Helper T 1 vs Helper T 2)
i. TH1
1. Activate cell-mediated response (phagocytosis, cytotoxic response)
a. Types of cytotoxic cells: NK cells, Cytotoxic Ts
ii. TH2
1. Promote humoral immunity
a. B cells, antibodies, increase in the amount of eosinophils, basophils,
and mast cells
III. Hypersensitivity
a. Type I
i. Immediate (Seconds), Mast-cell mediated
ii. IgE
1. Monomer (Y)
2. Mast cells are pre-coated with IgE.
a. Faster response. Just need allergen.
b. With Type III and IV, cells are not pre-coated. Takes longer.
3. IgE crosslinks to degranulate mast cells histamine released increases
inflammatory response. Bronchiole constriction.
iii. Allergies, asthma, anaphylaxis
b. Type II
i. Mediated by antibodies (Humoral)
ii. Delayed (Hours)
iii. IgG
1. Some IgM
iv. Complement activation
v. Cell surfaces/Solid surfaces/Insoluble surfaces
vi. Examples
1. Rh antibodies and HDN, Goodpastures Syndrome
c. Type III
i. Soluble antigens
ii. Delayed (Hours)
iii. IgG
iv. Complement mediated
v. Examples
1. SLE, Rheumatoid arthritis, Post-strep
d. Type IV
i. T cells (T cell receptors)
1. Cytotoxic Ts killing tissue
ii. Cell mediated
iii. Bring in other cell types
iv. Delayed response (Days)
1. Waiting for cells to become activated
v. Examples
1. Sjogrens Syndrome (Dry eyes/mouth).
a. Test for sSA and sSB antibodies. Type IV not antibody mediated.
Main mode of destruction.
2. Type I Diabetes
e. Complement Activation in Types II and III
i. Recruiting mechanism to site. Neutrophils Move via chemotaxis Release waste
(loosen up endothelial cells digest through to get to infection site) Phagocytize
f. RAST/RIST Testing
i. RAST Determines patients IgE levels or how allergic the patient is to specific
allergens
1. Originally used radioactive labels, but this has been replaced by EIA
sandwich methods
ii. RIST Determines patients total IgE levels
g. Helper Ts
i. Helper 1: involved with Type II, III, and IV hypersensitivities
ii. Helper 2: involved with Type I hypersensitivity

Tuesday, April 22
nd


I. Antibody Structures and Functions
a. Humoral = Non-soluble (non-cellular)
i. Humoral Component of Adaptive Immunity = Not T cells. Theyre part of the
cellular components.
ii. Humoral component of innate immunity = Complement (C3b)
b. Innate vs Adaptive
i. Give mouse injection with antigen Normal adaptive immune responses
ii. Irradiate mouse, kill lymphocyte pool then give same injection May still have a
reasonable innate response, but wont have adaptive immune response due to
killing lymphs.
iii. Can give lymphocytes back from normal animal. Adaptive immune response
restored. Those are the cells involved with adaptive response Lymphocytes.
iv. Give mouse lymphocytes from mouse that was irradiated still no adaptive
immune response.
v. Give mouse other cells (ex. Granulocytes) Gives better chance, but no adaptive
response.
c. B cell Maturation
i. Maturation takes place in bone marrow
ii. Start out with lymphs that have full, intact germline (Capability of being able to
make all kinds of antibodies).
iii. Start reconstruction Recombination events. Each cell starts looping out part of
DNA to bring segments together to make unique antibody. (T cell receptors I the
case of T cells)
1. Main enzymes required for recombination: RAG1 and RAG2.
2. Disease SCID one type is deficiency in RAG enzymes
iv. Begin testing receptors, antibody that is being constructed tested against self-
antigens that are presented by Stromal cells that are cells toward the medulla of
the bone marrow that are specific for presenting as many self peptides as possible
so all antibodies are tested against as many antigens as possible.
v. Need a little bit of self-reactivity to see that antibodies react to something, but
dont want significant affinity. If so, kill them.
vi. If they pass, send out to bloodstream towards lymph node. They are now immature
B cells.
1. On cell surface, they express: sIgM (surface), IgD
vii. Head towards lymph node
1. Still considered nave B cells
a. Sitting around in follicular regions (outside region of lymph node)
b. Lymph fluid is coming from potential infection sides and flows right
past all of these B cells. Flooding B cells with antigens B cells are
most likely going to encounter antigens they react to No longer
nave.
viii. B cells come into contact with their corresponding antigen No longer naive
ix. B cells then proliferate Become activated by Helper T cells T cells are located
in the center of lymph node (Seeing antigen for first time presented by dendritic
cell) B cells and Helper T cells interact
x. B cells contain antibodies in their surfaces that are specific to specific antigens.
Will only react with particular antigens. This is why you need a lot of B cells.
Diverse population of potential receptors (B and T cell receptors), only expand the
clone that is useful at that time. Clone may not be useful again for years Need
memory B cells eventually.
1. Important to know Clonal Theory of B cells in Adaptive Immunity!
xi. One we have interaction with T cell Causes proliferation, tell some of them to
start becoming antibody factories (Plasma cells)
1. Plasma cells crank out antibodies in bone marrow
2. 2 other things that go on with antibody production in plasma cell:
a. Type switch to IgG/A/E from IgM they start with
b. Affinity Maturation
i. Germinal center is where initial proliferation takes place.
c. Somatic Hypermutation
i. Produce something thats worse. Increase number of mutations
to occur, reward cells that make better antibodies. Cells that
make bad antibodies are not rewarded. Overtime, antibodies
get better and better.
xii. Also subdivide some into memory cells.
d. What antibodies do for us
i. Bind to microbes
1. Serve as opsonins (prepare to eat), promote phagocytosis
a. NK cell response vs Neutrophil response
i. NK cells: Dont phagocytize, theyre like cytotoxic lymphs
release toxic chemicals.
1. ADCC (Antibody Dependent Cell-Mediated Cytotoxicity)
2. Block microbe binding to host cells
ii. Activate complement, promote inflammation
iii. Bind to mast cells, release histamine
iv. Increase plasma protein in liver diseases (decreased albumin production)
e. Fragments of IgGs
i. Fab fragments
1. Variable portions in this section. Also some constant regions in here.
2. Have antigen binding sites
ii. Fc fragments
1. Stock, parts that differ between Ig classes and are class specific.
2. Constant within classes.
3. Bind cell surface receptors.
iii. Digestion Enzymes
1. Pepsin Cleaves Ig heavy chain to form divalent fragment and a degraded Fc
fragment
2. Papain cuts further down, doesnt leave disulfide bonds in place. Cleaves
at hinge region yielding two Fab fragments
f. IgG
i. Monomer with 2 binding sites
ii. Found in the highest concentration in serum
iii. Primary Ig of long-term or chronic immunity
iv. Bind proteins
v. Primarily produced against protein antigens
1. T independent activation of B cells
a. Very strong interaction between carbohydrates on bacterium
(polysaccharide sort of antigen), bound strongly, crosslinking forms,
IgM on outside of B cell. Sometimes, those cells are turned on enough
by this interaction continue to make IgM, dont type switch to IgG,
referred as T-independent activation. No memory cells produced (no
long-term immunity). Primarily going to continue making antibodies
against the carbohydrate (Normally, MHC II only presents peptide
antigens).
vi. Main components of passive immunity
1. Ex) Antibodies crossing placenta, inject gamma-globulins from person who
has these antibodies in patients who are going overseas, Rhogam
g. Affinity vs Avidity
i. Avidity Total affinity. Collective binding. Most important in IgM, for example.
h. IgM
i. Pentamers with 10 binding sites
ii. Bind carbohydrates
iii. 1
st
responders
iv. Opsonins
v. Fix complement
vi. Do not cross placenta
vii. Secreted as pentamers
viii. J chain and lots of disulfide binds
i. IgA
i. Crosslinked dimers with 4 binding sites
ii. Secretory component and J chain
iii. Main antibodies secreted in fluids in intestinal tract, mucous, etc
iv. Synthesized by plasma cells and IgA travels through serum to secretion site
v. Coat mucous membranes with lots of IgA, areas where susceptible.
vi. Secreted as dimer, then gain J chain
j. IgE
i. Monomer with 2 binding sites
ii. Low IgE levels in plasma under normal conditions
1. Mainly going to be picked up and pre-bound to mast cells
iii. Mast cells have Fc receptors for IgE
iv. Mast cells respond to IgE crosslinking by releasing histamine
v. When adjacent IgEs on mast cells are bound with antigens, histamine is released,
causing increased vascular permeability and vasoconstriction
k. Penicillin as an Allergen
i. Penicillin is a hapten, small MW, that cannot stimulate an immune response by
itself because they are not presented well by the APC process.
ii. Penicillin has the ability to couple with other proteins, which serve as a carrier
molecule. Presented to stimulate immune response.
l. Haptens and Antigenicity
i. Small hapten molecules cannot stimulate an immune response alone
ii. Haptens are first conjugated to a carrier molecule (BSA) so that is it processed and
peptide with hapten is presented properly to stimulate an immune response.
iii. These conjugates produce a good antibody response
II. ANAs and ENAs
a. Defined by what lights up when you do a fluorescence assay
b. ANA Antinuclear Antibodies
i. Ex) SLE
c. ENA Extractable Nuclear Antigens
i. Dont show up unless you do a gentle washing process during ANA procedures
d. ANA antibodies Speckled pattern
i. Normally corresponds with Sm and ENA antibodies
ii. Also SS-A and SS-B antibodies common in Sjogrens Syndrome
iii. Diagnostic for SLE, but not specific
e. ANA antibodies Nucleolar pattern
i. Only a few larger discrete areas in nucleus (red counter stain)
ii. Scleroderma patients
iii. Nucleolus (RNA material)
f. ANA antibodies Rim pattern
i. Rim/peripheral pattern, bright on edges
ii. Associated with histones staining
g. Testing for SLE
i. Anti-ds-DNA test
1. Specific. Monitor flare-ups.
h. ANCA antibodies
i. Anti-neutrophilic cytoplasmic antibodies
ii. Staining everything except the nucleus (Opposite of ANA)
iii. Wegeners Granulomatosis
iv. Churg-Strauss Syndrome
III. Autoimmune Diseases
a. Occurrence of Autoimmune Diseases
i. Occurs more in females
ii. Vitiligo is a condition that causes depigmentation of parts of the skin
1. Michael Jackson
b. Systemic Diseases
i. SLE, Wegeners
c. Hashimotos Disease
i. Hypothyroid
1. Thyroid attacked Goiter.
2. Thyroid peroxidase (anti-microsomal antibodies)
a. Enzyme that puts iodine on, specific for thyroid. Other tissues dont
use iodine but when we are making the thyroglobulin, add iodine.
b. Iodine added to outside, shipped out as completed thyroglobulin.
3. Thyroglobulin colloid
ii. T4 replacement as treatment. May do immune suppression also.
iii. Primary thyroid problems
1. Primary: Low T3/T4, Normal TSH
2. Secondary: Low T3/T4, Low TSH
d. Graves Disease
i. Hyperthyroid Most common cause
1. Thyroidtoxicosis (enlargement)
2. Heat intolerance, weight loss
ii. Follow-up Testing involves antibodies
1. TSI stimulates release of T4
2. TGSI stimulates proliferation of thyroid cells
iii. Binding of antibody causes receptor to think it has bound TSH Constantly being
stimulated. High level of TSH all the time. Due to antibodies binding to receptors.
iv. Primary thyroid problems
v. High T3 and T4, TSH would be low (Primary)
1. If this were secondary, TSH and T3/T4 would be high. (Pituitary)
2. If this were tertiary, TSH, T3, T4 would be high and also overactive
hypothalamus.
e. Addisons Disease
i. Autoantibodies to adrenal cells lead to destruction of adrenal cortex
1. Hypoglycemia, dehydration, shock
ii. Low production of cortisol (glucocorticoid), usually aldosterone is also low
(mineralocorticoid), high ACTH (corticotropin) as system tries to stimulate more
cortisol production
1. Cortisol: increases glucose by gluconeogenesis. Increases metabolism.
Breakdown of glycogen if cortisol is high and also epinephrine.
2. ACTH: made in pituitary
f. Pernicious Anemia
i. Macrocytic anemia, caused by deficiency of B12
1. Caused by absorption problem
2. B12 requires intrinsic factor (binding protein made in parietal cells in
stomach)
ii. Caused by autoimmune destruction of parietal cells in stomach (antibodies seen)
iii. Autoantibodies to intrinsic factor itself (blocking antibodies)
iv. Autoantibodies to complex of IF and B12 (binding antibodies)
g. Type I Diabetes (IDDM)
i. Insufficient insulin production caused by destruction of B-cells
ii. Autoantibodies lead to selective destruction and inflammation
1. Anti-islet cells, a-GAD, a-IA-2, a-insulin
iii. T cells also damage pancreas by infiltration
iv. Early onset (10-14 years)
1. Used to be considered Juvenile Diabetes
v. HLA antigens DR3 and DR4 associated
vi. Frequently preceded by viral infections
vii. Type II diabetes is not autoimmune
1. Dont respond to insulin (insulin resistance)
h. Multiple Sclerosis
i. Faulty T-cell regulation appears to be involved
ii. Decreased T-cell suppression observed during acute episodes
iii. Demyelination occurs during active episodes by combination of suppressor and
helper T cells and macrophages releasing myeline into CSF
iv. Active inflammatory episodes are associated with increased CRP and ESR values and
a polyclonal increase in IgG in the serum.
1. Look for increased IgG in CSF
2. Oligo bands in protein electrophoresis
i. Myasthenia Gravis
i. Muscle weakness
ii. Antibodies to acetylcholine receptors in neuromuscular junctions
1. Antibody-mediated damage causes progressive muscle weakness
2. Frequently associated with SLE, RA, etc
3. Treated with anticholinesterase drugs
j. Autoimmune Hemolytic Anemias
i. Several types of anemias induced by:
1. Warm antibodies
2. Cold antibodies
3. 4 types of drug mechanisms
a. Drug dependent (immune complex)
b. Drug adsorption (hapten)
c. Membrane modification
d. Autoantibody formation
ii. Idiopathic thrombocytic purpura caused by similar destruction of platelets
iii. Intravascular**
k. Sjogrens Syndrome
i. Lymphocyte infiltration and destruction
l. Autoimmune Hepatitis
i. Autoimmune hepatitis and primary biliary cirrhosis are two forms of autoimmune
liver disease
ii. Antibodies against F-actin are particularly important in the diagnosis
iii. Antibodies against F-actin represent highly specific marker for AIH type I
iv. Antibodies cannot be determined using EIA or Western Blot
m. Primary Biliary Cirrhosis
i. Initial inflammation begins with T lymphocytes
ii. T cells invade and destroy epithelial cells lining small bile ducts
iii. T cells also produce cytokines and other chemicals that stimulate epithelial cells to
secrete chemotactic agents so more T cell infiltrate, thereby creating an ongoing
cycle of damage
iv. Anti-mitochondrial Primary biliary cirrhosis
v. Anti-microsomal Thyroid
n. Systemic Autoimmune Diseases
i. SLE
1. Skin, joints, kidney, brain, heart, lungs
2. Causes
a. Decreased levels of CR1 on RBCs (poor complement clearance)
b. Circulating immune complexes
i. Binds to C3b receptor on RBCs
ii. Taken to spleen
iii. Phagocyte can release binding of RBC RBC is freed and may
live
c. Body isnt good at clearing these soluble immune complexes. Not
enough C2 or mutation in receptors, person is likely to have increased
amounts and have immune reaction, producing autoantibodies
towards the complexes.
d. **Clearance problem
3. Apoptosis vs Necrosis
a. Necrosis: Cell begins to swell, membrane disintegrates, intracellular
junk that wasnt handled well is now free more likely to cause SLE.
Intracellular contents out in general circulation. Immune system can
react.
b. Apoptosis: Nuclear condensation, small bite-sized pieces released. Not
released into circulation, released as closed pieces that can be
phagocytized very easily my nearby macrophages. Not as dirty of a
process. Programmed cell death.
4. ANA
a. ANA screening test 98% sensitive. Homogeneous, rim patterns specific
for SLE. Sensitive, but not very specific.
b. Confirm with testing for auto-dsDNA
c. Confirm inflammation and complement activation by testing for ESR,
CRP, C3 and C4 (complement proteins, tell us complement has been
activated. Levels should decrease if activated. If C3 is decreased and
C4 doesnt, alternative pathway), IgG levels
i. Classic pathway C3 and C4 used
ii. Alternative pathway C3 only used
ii. Rheumatoid arthritis
1. Complement activation by immune complexes caused by inflammatory
process
a. Rheumatoid factor Antibodies directed against the Fc portion of IgG
i. Bind to normal IgGs in plasma and form small immune
complexes, including IgG dimers
ii. Tissue complexes activate complement and initiate
inflammatory processes
2. Joint pain is symmetrical because its systemic
iii. Scleroderma
1. Antibodies to connective tissues of skin, lungs, kidneys, and GI tract
2. Primary antibodies are ANA (atypical speckled or nucleolar patterns)
iv. Goodpastures Syndrome
1. Caused by autoantibodies to basement membranes
2. Renal basement membrane becomes inflamed following complement
activation and cytotoxic response
a. Causes glomerular nephritis, hematuria, proteinuria, RBC casts
b. Pulmonary basement membranes also attacked in similar manner
c. Cause dyspnea alveolar damage (bleeding and fibrosis)
3. Kidney disease and lung disease (similar tissues in both and both are
attacked)
4. How does pathogenesis differ from that of glomerular nephritis following
strep infection?
a. Post-Strep infection Self-limiting
i. Caused by antibodies, do ASO titer to confirm (streptolysin).
Antibodies against organism. Filtered, too big, stick to
glomerular filter, activate complement, Type III
hypersensitivity (soluble antigens)
b. Goodpastures Autoimmune, life-threatening
i. Type II hypersensitivity
c. SLE Type III (soluble antigens)
IV. Cell Characteristics
a. Macrophage
i. Markers
1. TLR (Not specific most cells have these)
2. MHC I and II (APC)
3. Fc-y receptors (APC)
4. B7 (costimulator)
ii. Location
1. Residence in tissues
2. Infection sites
iii. Roles
1. Sentries for tissues
2. Phagocytosis
3. APC
iv. Exports
1. Cytokines
b. Monocyte
i. Markers
1. TLR
2. MHC I
3. Fc-y receptors (Not granulocyte, eosinophil, or mast)
ii. Location
1. Blood stream (Could be mono)
2. Infection site
iii. Roles
1. 2
nd
responders (Mono)
a. 1
st
responders (Neutro)
2. Develop into macrophages
3. Respiratory burst
iv. Exports
1. Cytokines
c. Neutrophil
i. Markers
1. TLR
2. MHC I
ii. Location
1. Bloodstream
2. Infection site
iii. Roles
1. 1
st
responders
2. Phagocytosis
3. Respiratory burst
iv. Exports
1. Digestive enzymes
d. Mast cell
i. Markers
1. TLR
2. MHC I
3. Fc-E receptors
ii. Location
1. Tissues
iii. Roles
1. Inflammation
iv. Exports
1. Histamine
e. Eosinophil
i. Markers
1. TLR
2. MHC I
3. Fc-y
4. Fc-E
ii. Location
1. Blood
2. Tissues
iii. Roles
1. Inflammation
iv. Exports
1. Digestive enzymes
f. Dendritic Cell
i. Markers
1. TLR
2. MHC I, II
3. B7
ii. Location
1. Residence in tissues
2. Migrate to lymph nodes
iii. Roles
1. Migrate to lymph nodes
2. APC
iv. Exports
1. Cytokines
g. B cell
i. Markers
1. TLR
2. MHC I, II
3. CD 19, 20, 21
4. BCR (B cell receptor), sIg
ii. Location
1. Bone marrow
2. Spleen, lymph
iii. Roles
1. Secrete antibodies
iv. Exports
1. IgG
h. T cell
i. Markers
1. TLR
2. MHC I, II
3. TCR (T cell receptor)
4. CD 3, 4, 5, 8
ii. Location
1. Thymus
iii. Roles
1. Develop into T cell
2. Choose CD4, CD8
iv. Exports
1. Helper Ts: CD4 exports cytokines
2. Cytotoxic Ts: CD8 exports cytokines and toxins
i. Pre-B cell
i. Markers
1. TLR
2. MHC I, II
3. HC-Ig with surrogate LC**
ii. Location
1. Bone marrow
iii. Roles
1. Recombination
2. Maturation to mature B cell
iv. Exports
1. Nothing
j. Helper T Type I (TH1)
i. Markers
1. TLR
2. MHC I
3. TCR
4. CD4
ii. Location
1. Lymph nodes
2. Blood
3. Infection site
iii. Roles
1. Proinflammatory
2. Activates macrophages
iv. Exports
1. INF-y, TNF, IL-2
k. Helper T Type 2 (TH2)
i. Markers
1. TLR
2. MHC I
3. TCR
4. CD4
ii. Location
1. Lymph nodes
2. Blood
3. Infection sites
iii. Roles
1. Promotes defense against parasites
2. Promotes eosinophil and mast cell production
3. Promotes type switch to IgEs
iv. Exports
1. IL-4, IL-5, IL-13
l. Follicular Dendritic cell
i. Markers
1. TLR
2. MHC I, II
3. B7
ii. Location
1. Germinal center of lymph nodes
iii. Roles
1. Antigen presentation
2. Promotes antibody type switching
iv. Exports
1. Cytokines
m. Cytotoxic T cell
i. Markers
1. TLR
2. MHC I
3. TCR
4. CD-8
ii. Location
1. Lymph nodes
2. Blood
3. Infection site
4. Site of tumor
iii. Roles
1. Cytotoxicity
iv. Exports
1. INF-y
2. Granzymes
3. Perforin
n. Natural Killer cell
i. Markers
1. TLR
2. MHC I
3. Fc-y Receptor
4. No TCR Definitely NK cell
ii. Location
1.
iii. Roles
1. Stimulate cytotoxicity
iv. Exports
1. INF-y
2. Granzymes



Wednesday, April 23
rd


I. ABG Techniques
a. Procedure
i. Taken from wrist. Filled by blood pressure. Syringe has heparin, dont want
clotting to occur. Put on ice. Eject any possible clot/air bubbles.
b. pH Meters
i. Analytes measured/reported out (5):
1. pO2, pCO2, pH
ii. Other items reported out:
1. HCO3 (Bicarb) instrument calculates based on Henderson-Haselback (pH =
pKa (pKa = 6.1 for Hgb) + log[HCO3]/[pCO2]
a. Concentrations in molarity
i. Except pCO2 units: mmHg (pressure)
ii. Change 40 x 0.03 Converted to molarity
2. O2 Saturation
a. Normal range: 94-100
c. Potentiometric
i. Measures voltage between H sensitive electrode and reference electrode
ii. Voltage difference between reference and indicator electrodes is the potential
and is related to [H]
iii. In blood gas instruments and electrolyte analyzers, several indicator electrodes
may use common reference electrode
iv. Other potentiometric electrodes
1. All other electrolytes Na, K, Ch, Free Ca
2. Except total calcium in BNP dibinding, color change. No electrode used
d. PCO2 Electrodes
i. Measured with a modified pH electrode
1. Gas permeable membrane allows only gaseous CO2 to get through
membrane
2. Only the form that is already as CO2 gets through
3. Once inside membrane, CO2 redissolves in H2O and releases H, which is
then measured by the pH electrode
e. O2 Electrodes
i. Oxygen electrodes (AKA Clark electrodes) based on current
1. O2 + 2H2O + 4 es 4 OH
f. pO2 and O2 Saturation
i. Measured pO2 is the amount of O2 dissolved in solution and not the O2 bound to
Hgb or the calculated % O2 saturation
ii. Under conditions with less O2 binding (ex CO-poisoning), pO2 may be high, but real
% O2 saturation may be much lower than the calculated
1. Examples that cause discrepancies between pO2 and O2 Saturation
a. CO Poisoning
i. High free O2, but O2 saturation on Hgb is low. Because ABG
calculated the O2 sat, its going to say its high, but its really
not. Theyre in respiratory distress.
b. Methemoglobin
g. Capillary ABGs
i. Neonates
ii. Not a very good form of specimen collection
iii. Try to draw from artery, difficult to draw from
iv. Magnet which mixes to promote mixing and avoid clotting
h. Bubbles and ABGs
i. How bubbles affect ABG
1. CO2 will be drasticallly lower affects Bicarb in equation. pH affected
(increases).
2. O2 not affected as much
II. Co-Ox Measurements
a. Procedures
i. Spectrophotometric, emphasis on Spectro-.
1. Spectrum range of wavelengths. Key to how this is read. Read wavelengths
spectrophotometrically.
ii. Sample used Whole blood from ABG
iii. Met-Hgb blood will appear darker
1. Iron oxidized to Fe3+ Body tries to reduce back down to Fe2+. Met-Hgb
doesnt bind oxygen very well or at all.
iv. CO bright red blood
b. Reducing Power
i. Glutathione gives oxidizing power. Reduces by giving up sulfhydryl group forms
dimer able to regenerate it back to SH group. Intracellular reducing agent, used
in various processes.
1. Enzyme used to make glutathione GGT
2. GGT increased in alcoholics and other kinds of drug detoxifications.
III. Serum Proteins Electrophoresis
a. Running the Gels
i. Most gels are cellulose acetate or agarose, not polyacrylamide
ii. Why pre-cast gels?
1. Dont want expense to prove that the gels we made work. No QC involved
b. Protein Electrophoresis
i. Serum and urine proteins are routinely separated based on their differing mobility
in an electronic field
ii. Travels from cathode (-) to anode (+)
1. Immunoglobulins are not a homogeneous group; every clone of B cell is
going to make own antibodies. Some antibodies will recognize negative
epitopes on the antigen they recognize theyre probably positively
charged at binding site. If they have (+) charges, they will move backwards
towards the cathode.
2. Albumin must have a large number of negative charges PI (isoelectric
point, where the overall molecule is neutral) will be around 5.1 have to
lower pH from 8.5 during electrophoresis all the way down to 5.1
c. Normal Order of Peaks
i. (Left to right): Albumin, alpha1-globulin, alpha2-globulin, beta-globulin, gamma-
globulin.
1. Beta peak is sometimes divided
d. Albumin vs Globulins
i. TP Albumin = Globulins
1. A/G ratio = 1
e. Protein Electrophoresis
i. Differences in mobility are due to:
1. Molecular size, shape, overall charge, and charge density
ii. Most proteins are negative between pH 6-8
1. Proteins move towards positive electrode (anode)
2. Faster proteins are said to be anodal to others
3. Numbering of proteins is from fastest to slowest, or (+) to (-)
iii. SDS Gels different from these lab gels because proteins are denatured
1. Detergent, fatty acid with sulfate
2. SDS also surrounds protein with a negative charge. Everything moves as a
very negative complex inherent charge doesnt matter.
f. Normal Protein Pattern
i. More than 500 different plasma proteins
1. IgG slowest
g. Total Serum Protein Assays
i. Most common assays are dye-binding assays
1. Coomassie Blue 250
IV. Assessing serum Protein Patterns
a. Active Phase Reactants
i. Indicative of inflammatory response
1. CRP (C-reactive protein) is most volatile, increasing up to 3000x
2. AAT (Alpha-1-antitrypsin), haptoglobin, fibrinogen (plasma), ceruloplasmin,
and C3 complement are all positive APRs, usually incrasing 2-5x
3. Albumin and transferrin are negative APRs
a. Sequester iron so bacteria cant get enough. Intentionally decrease
transferrin.
4. CRP and fibrinogen have been suggested as much better indicators of
inflammation than ESRs
a. Old, non-specific, and still very popular
b. Acute vs Chronic Inflammation Patterns
i. Acute: alpha 1 and alpha 2 increased, albumin decreased a little
ii. Chronic: alpha 1 and alpha 2 increased as well as beta peak and usually gamma
peak
1. Large increase in gamma peak from immunoglobulins
c. Cirrhosis Pattern
i. Beta-gamma-bridge: large decrease in albumin (livers not functioning well)
plasma proteins also decreased except for immunoglobulins, which does not
involve the liver.
ii. Cirrhosis marked by major reduction in liver protein synthesis (and other metabolic
processes)
1. Reductions include coag factors and many other proteins
2. Reducations partially compensated for by the IgGs, IgMs
iii. Albumin and other peaks reduced, only beta-lipoproteins and gamma-region
proteins increased
d. Polyclonal Gammopathies
i. Overproduction by large number of plasma cell clones. Similar pattern to cirrhosis
or chronic inflammation.
ii. Plasma cells normally reside in bone marrow when producing antibodies
e. Hypogammaglobulinemias
i. Serious immune deficiency, relating to the total or partial lack of antibody
production
ii. Virtually eliminates gamma-peak and obviously causes susceptibility to infections
iii. Brutons
f. AAT Deficiency
i. Alpha-1-antitrypsin deficiency, relating to the total or partial lack of AAT
ii. Virtually eliminates alpha-peak and results in emphysema in adults and liver
disease in children
1. Alligator mouth???
2. Criteria for emphysema vs bronchitis: Dead space in emphysema. Alveoli are
useless
3. Liver disease in children: Happens after infection.
g. Monoclonal Gammopathies
i. Single clone of plasma cells overproduces a single Ig
1. Ig can be IgG, IgA, or IgM
2. Can increase the TP amount
ii. Ex) Multiple Myeloma
1. Bone pain, cells in bone marrow overproduced running out of room
2. Clones of B cells out of control
3. No room for other cells thrombocytopenia, lots of penias.
h. Immunofixations (IFE)
i. Electrophoresis of serum or urine proteins is followed by staining with anti-
human Ig
ii. Separate anti-sera can identify IgG, IgA, IgM, gammopathies
iii. Light chains (kappa or lambda) are also stained separately for Bence-Jones
proteins
i. Nephritic vs Nephrotic
i. Nephrotic: Red color, strong positive for blood Wont see many/any RBCs,
broken up. A lot of hemoglobin. Lysed RBCs
ii. Nephritic: Blood in urine, will see RBCs. RBC casts also seen. Maybe even granular
casts.
j. Nephrotic
i. Massive loss of protein through basement membrane
ii. BM has lost ability to restrict all but very large proteins (ex. Alpha-2-
macroglobulin)
iii. Decreased albumin and total protein, results in edema
k. Tubular Proteinuria
i. Caused by decreased tubular function, including decreased protein reabsorption
1. PCT is where protein reabsorption occurs
l. Beta-2-microglobulin
i. Beta-2-microglobulin is very small
ii. Comprises light chain of Class I MHC antigen on surface of all cells
V. Important Plasma Proteins
a. Pre-albumin
i. Migrates faster than albumin, not a precursor to albumin, also a negative APR,
carries around thyroid hormones and binds to RBG to carry vitamin A
ii. Assesses malnutrition
b. Transferrin
i. Transports iron, binds 2 Fe3+ ions, does not bind Fe2+
ii. Measured by nephelometry or indirectly by TIBC
c. Complement Proteins
i. C3 and C4 are primary factors measured unless coagulation problem suspected
1. Both are positive APRs
ii. Decrease in C3 in post strep glomerular nephritis, decreases with severe liver
disease and SLE with renal disease
iii. Decrease in C4 with ANA and anti-dsDNA diagnostic for SLE
d. Fibrinogen
i. Factor 1, required for fibrin clot formation
ii. No fibrinogen found in serum, or very little
iii. Large protein, indicator of inflammation
iv. Positive APR
e. Haptoglobin
i. Positive APR
ii. Binds alpha + beta dimers of Hgb
iii. Prevents nephrotoxicity by clearing Hgb from plasma
iv. Decreased as it binds dimers (Haptoglobin digested in process)
1. Used up during intravascular
2. Extravascular: dont use haptoglobin because RES system used. Cleaner
process.
f. Ferritin
i. Serum ferritin reflects amount of iron stored. Earliest and best indicator of iron
deficiency
ii. Positive APR, concentrations increase in presence of infection or cancer
g. Ceruloplasmin
i. Scavenger for copper
ii. Deficiency of ceruloplasmin causes Wilsons disease
1. Leads to childhood liver disease
2. Copper accumulates in brain and liver, unable to excrete into bile
3. Treatment involves coper chelator, penicillamine
iii. Donation of copper to cells, requires uptake and destruction of whole protein
h. CRP Major APR
i. CRP: Indicator of inflammation. Its an opsonin (Molecular tag to tell phagocytes
know what to eat).

Thursday, April 24
th


I. Methods of Clinical Toxicology


Friday, April 25
th


I. Drug Metabolism
a. Dose Response
i. Effects of drugs are based on concentrations at specific tissues.
ii. Concentrations at tissues depends on absorption, metabolism, excretion,
distribution, and protein binding
b. Multi-Doses
i. Steady state levels reached after 3-5 doses, even with the loading dose.
c. Pharmacokinetics
i. Therapeutic drug monitoring is complicated by several factors
1. Plasma concentration is not always directly related to pharmacologic action
2. Protein concentration is important to certain drugs
3. Tissue distribution to site of action
d. Drug Binding to Plasma Proteins
i. Many drugs bind very well to plasma proteins
1. Acidic drugs primarily bind to albumin
2. Basic drugs primarily bind to globulins such as alpha-1-acid glycoprotein
(AAG)
ii. Changes in concentration of these proteins can affect drug activity and toxicity
iii. Drugs free in solution are the active form, but excreted quickly
iv. Protein-bound drugs are usually inactive (longer half-lives)
e. Drugs Competing for Protein Binding
i. Protein-binding by drugs can be in competition for other things
1. Other drugs
2. Free fatty acids
3. Electrolytes and pH changes that affect charges
ii. Some serious drug interactions involve drugs that compete for protein binding sites
1. If many of a drugs normal binding sites are full, the free drug will be
increased, maybe to a toxic range
f. Renal or Liver Diseases can Affect Drugs
i. Renal failure can change binding to proteins
1. Example in PowerPoint
ii. Liver disease can change protein similarly
iii. Metabolic conditions can change binding site availability
g. TDMs Quantify [Total Drug] in Plasma
i. Protein-bound drugs are usually inactive, but the TDM assays usually quantify total
amount of drug in plasma
ii. Assays often involve releasing steps similar to that used for serum Fe assays
iii. Patients with hypoalbuminemia might have a normal therapeutic level of drug, but
toxic level of active drug
1. Lidocaine, used to control arrhythmias, binds to AAG
h. pH affects absorption in GI tract
i. Many drugs are not recognized by transport proteins
ii. Drugs that do not hijack a transporter must diffuse across the membrane
iii. Since ions cant diffuse, the drug must be at a proper pH to become neutral
i. pKa and Charges
i. Acidic groups negative whenever pH > pKa, neutral when pH < pKa
ii. Basic groups positive whenever pH < pKa, neutral when pH > pKa
iii. Drug absorption and cellular intake are affected by pH and pKa of drug
1. Proton mostly on at pKas below the pH
2. Protein is mostly off at pKas above the pH
iv. Definition of pKa = Half protonated, Half unprotonated (50% mark)
1. Below that, mostly protons on
2. Above that, mostly protons off
j. Metabolism Kinetics
i. First Order Metabolism
1. Dependent on drug concentration
ii. Zero Order Metabolism
1. Independent of drug concentration
a. Only example of something that is zero order metabolism: Alcohol
metabolism
i. Use equation to see how long to hold someone in ER
ii. Rate = 0.015%/hour
2. Zero Order Alcohol Metabolism
a. Alcohol detox is prime example of zero order metabolism
b. Has nothing to do with starting concentration of alcohol
k. GFR and Renal Elimination
i. Rate of renal elimination is proportional to GFR (creatinine clearance)
1. Usually, only the free or unbound drug is filtered
2. Drugs bound to albumin will not be cleared through kidney
3. Drugs must not be secreted or reabsorbed
ii. Change in GFR greatly impacts half-life of drug
1. Low GFR = Longer Half-Life
iii. Drugs with high tissue uptake will not be as affected by changes in GFR (ex.
Digatoxin)
l. Metabolism of Acetominaphen
i. Different ways to begin metabolizing
1. If we use oxidase, convert to potentially toxic molecule intermediate.
Under normal circumstances (glutathione), it will help us reduce this
intermediate to a non-toxic alcohol from the ketone No problem.
2. If we overwhelm all of the systems and go different way in flow chart, form
covalent bonds with macromolecules, proteins, etc and kills the cells.
3. Acetaminophen overdoses hot topic for causing liver problems
a. Main problem: Acetaminophen is added to various drugs
b. So many OTC drugs with this added
m. Drug Metabolism
i. Phase I: Redox System
1. Cytochrome P-450
2. GGT is elevated in alcoholics and druggies because its part of this system
a. If system is being used a lot More enzymes Higher GGT, for
example
b. GGT = Good marker for alcoholics.
ii. Phase II: Conjugation System
1. Adds glucuronides, sulfides, etc
2. Makes drug metabolites more soluble and easier to excrete through kidneys
n. Drug Conjugation
i. Glycosylation
1. System similar to bilirubin conjugation
2. Utilizes liver microsomal enzymes
II. Therapeutics and TDMs
a. Beneficial drugs will become toxic outside of their therapeutic ranges
i. Some have smaller ranges, so its easier to become toxic
b. Analgesics
i. Opiates
1. Morphine, Codiene, Hydrocodone, Oxycodon, Demerol, Darvon
(Propoxiphene)
a. First synthetic opiate drug was demerol
ii. NSAIDS
1. Aspirin (Salicilate)
2. Naproxin
3. Ibuprofin
4. Indomethicin (Indocin)
iii. Tylenol (Acetaminophen)
c. Pain Management
i. NSAIDs and Tylenol act at site, to block generation of prostaglandins
ii. Local anasthetics block neuronal transmission
iii. Opiates act at various receptors in brain and spinal cord
d. Anti-Seizure Drugs
i. Phenobarbitol
1. Febrile and neonatal seizures
ii. Phenytoin (Dilantin)
1. Epileptic drug of choice
iii. Valproic Acid (Deprakene)
1. Generalized tonic-clonic seizure
iv. Carbamazepine
1. Psychomotor seizure
e. Aminoglycosides
i. Gentamicin, Tobramicin, Streptomicin
ii. Given IV in hospital, gram negatives, for GPCs with B-lactams
iii. Nephrotoxic effects can result monitored in hospital
f. Vancomycin
i. Glycopeptide, given IV, last resort
ii. Nephrotoxic
g. Psychics
i. Lithium
1. Manic depressives
2. Measured by ISEs
ii. Tri-Cyclic Antidepressants
1. Imipramine
2. Amitriptyline
3. Block reuptake or serotonin and norepinephrine


Review Day

I. Immunoassays
a. Hetero vs Homo
i. Hetero = Wash because of UTIs
ii. Homo = No Ho, No Wash
b. Immunoassays in General
i. Signal vs Analyte concentration
1. Inversely proportional to
2. Directly proportional to
ii. Sandwiches are always heterogeneous!
c. MEIA
i. We want to absorb the sandwich.
ii. Sandwich goes directly into my mouth, which will be absorbed in my stomach.