PNB2265 – PRACTICAL I – STUDY GUIDE
Tuesday February 27th 2018
Lab 1 – Blood
Components of human blood:
• Erythrocytes (red blood cells, RBC)
• Thrombocytes (platelets)
• Leukocytes (white blood cells, WBC)
Distribution (%) of Leukocytes in Blood:
If you want to learn more check out What is totalitarianism?
Lymphocytes: large, darkly stained, spherical nucleus
occupies most of the cell
Monocytes: usually have a large, dark stained “U” or
Eosinophils: large red granules in a clear cytoplasm
with a bi-lobed nucleus
Neutrophils: fine reddish granules in a pale pink
cytoplasm; nucleus has 3-5 lobes
Basophils: have a clear cytoplasm with purplish-black
granules. The nucleus is “S” or “U” shaped
Note: nuclei of neutrophils and basophils are multi
lobed, while eosinophils are bi-lobed
Sickle Cell Anemia: sickled cells
We also discuss several other topics like What happened to the anasazi?
Infectious Mononucleosis: fever, sore throat, fatigue; most commonly cause by Epstein bar virus; SKIRTING pathology; abnormal nuclei Don't forget about the age old question of Why are theories necessary?
Polycythemia: overproduction of red blood cells
Leukemia: unregulated overproduction of immature leukocytes • Identify leukemia ???? don’t need to know the different types
• RBC’s too large
• Reasons: Deficiency of certain vitamins
(folic acid, B12)
• RBC’s are too small Don't forget about the age old question of What are prophets? what roles do they play in a religion?
• Reasons: Iron deficiency or abnormal
• Pathology: low O2 carrying capacity
• Symptoms: blue lips + cold extremities Don't forget about the age old question of What is a monotonic dose-response curve?
Blood Typing/Blood Type
Blood type is determined by ???? the ANTIGENS expressed on the surface of the red blood cells If you want to learn more check out If a contract is for the sale of goods, the part of the ucc that governs the contract is what?
Question: if blood type “O” has anti-A and anti-B
antibodies, why is it a universal donor? It has no A or
B antigens, so no antibodies will attack it in other blood
Antibodies: produced by B LYMPHOCYTES in response to exposure to foreign antigens
• Released into the blood plasma
• Bind specifically to the inducing antigen
• Two antigen-binding sites per antibody – each antibody can bind to two blood cells
Hemagglutination: clumping of many RBC’s together ???? will cause hemolysis (rupture of RBC’s) and can lead to shock, kidney failure, and death
Individuals with Blood Type:
• A = B antibodies ; A antigens
• B = A antibodies ; B antigens
• AB = no antibodies ; AB antigens
• O =; no antigens
Which blood type is the universal donor and why? Type O because lack of A or B antigens
Anti-D or Rh factor ???? if Rh factor is expressed on the surface of a cell, it will
agglutinate. Agglutination means Rh factor is present = Rh positive ; if blood type
does not express Rh factor, it’s considered Rh negative
• Females: 11-16 gm% = lower
• Males: 13-18 gm% = higher
• (gm% = grams of Hb/100mL blood)
• What can effect hemoglobin value?
o Diet, geography, smoking, exercise, etc.
• Lab results ???? likely to be lower due to the fact that it’s diluted animal
• Hemoglobinometer: diagnostic tool to measure hemoglobin levels in blood
RBC’s are filled with hemoglobin protein (Hb)
FXN: Transports oxygen and carbon dioxide, gives RBC’s red color
Composed of: 4 polypeptide chains called globins: 2 alpha, 2 beta
• Heme: porphyrin ring with iron ion in the center ???? allows for oxygen binding and transport in blood • 4 rings = each hemoglobin molecule can carry four molecules of oxygen
Hemoglobinometer: diagnostic tool to measure hemoglobin levels in blood
Centrifuge – fraction of whole blood volume that consists of
red blood cells
PRCV = packed red cell volume
• Test used to determine what portion of a blood
sample is composed of red blood cells
• Expressed as a percentage
• In lab ???? MHCT (micro-hematocrit) ???? determined by
centrifugation of a blood vessel
• The upper layer (clear yellow) = plasma
• The lower layer (dark red) = mainly packed red blood
Normal Range of Values:
• ~47 for men – due to testosterone which produces more
• ~42 for women
Hematocrit = packed RBC/total blood x 100%
If an individual were suffering from polycythemia, how would this affect the hematocrit? • RBC’s would be more abundant, resulting in a higher hematocrit
An individual is suffering from sickle cell anemia. Describe how their test results (hematocrit, hemoglobin, blood smear) might differ, if at all, from normal blood:
• Hematocrit = smaller
• Less hemoglobin available in the blood ; apparent sickled cells in the blood smear Conceptual Question: After a car accident, a patient with type B blood was rushed to the hospital with severe hemorrhaging. Physicians decided to give Mary a blood transfusion, and an hour later she was pronounced dead from systemic circulatory agglutination. Which transfused blood types could have caused this reaction? Describe the specific antibody-antigen interaction that caused Mary’s systemic agglutination. • A or AB blood transfusions could have caused this reaction
• The specific antibody-antigen interaction that caused Mary’s systemic agglutination was ???? A antibodies in the B blood, and the A antigens in the A blood.
Hematocrit after hemorrhaging:
• Immediately after a hemorrhage usually doesn’t show extent of RBC loss because at the time of the hemorrhage, plasma and RBC’s are lost in equal proportions.
• Several hours after hemorrhage, plasma volume increases due to a shift of interstitial fluid into the vascular space ???? RBC’s cannot be replaced quickly b/c the bone marrow takes ~10 days to produce mature red blood cells ???? result: hematocrit done several hours after bleeding episode will show a more accurate picture ???? hematocrit = decreased because plasma volume has compensated for fluid loss while the red blood cells that have been lost cannot be replaced for days
LAB 2- Electrocardiogram (ECG) – Blood Supply – Sheep Heart – Human Vessels
• Measure of electrical activity of the heart
• EKG only measures electrical activity ???? meaning, the
only terms used should be depolarization and repolarization ????
no mechanical terms
• EKG does NOT tell us about contraction or relaxation
• Electrical stuff always precedes the mechanical stuff
• Not an action potential
• P wave = atrial depolarization
• QRS complex = ventricular depolarization, atrial
• T wave = ventricular repolarization
The funny current (IF) is due to HCN channels and
mediates the rhythmic electrical activity of the
heart via the influx of sodium at ~60mv (when the
membrane is hyperpolarized). HCN channels allow
the flux of sodium and potassium ions. Calcium
current (both T type and L type channels) allows
the depolarization of pacemaker cells and Sodium
and L-type Calcium current allows the
depolarization of myoctyes.
Phase 4: Resting Membrane Potential
• Resting membrane potential in contractile cardiac myocytes is typically between -80mV and -90mV. • Resting membrane potential doesn’t mean there’s no ion movement.
o Created from the continuous efflux of positively charged potassium ions through voltage gated inward rectifier potassium channels (KIR) and a small amount of sodium and chloride permeability.
o The other major contributor to resting membrane potential is the Na/K/ATPase, which serves to maintain the concentration gradients (we covered this in greater depth earlier in the text when we introduced membrane potential).
Phase 0: Depolarization:
Depolarization of contractile cardiac myocytes is similar to skeletal muscle. During this phase, depolarizing current increases sodium permeability by activating voltage gated fast sodium channels, which allow influx of positively charged sodium ions.
Phase 1: Transient Repolarization:
At the peak of the action potential, voltage gated sodium channels rapidly inactivate and sodium permeability decreases, curtailing the influx of positively charged sodium ions. As with skeletal muscle, this inactivation places cardiac myocytes in a refractory period. Since there is still transient outward current through the voltage gated potassium channels, membrane potential begins to repolarize.
Phase 2: Plateau Phase:
Hyperpolarization is quickly interrupted as voltage gated L-type calcium channels open, increasing calcium permeability and bringing positively charged calcium ions into the cell. A role for calcium ions in the action potential is unique to cardiac myocytes. In skeletal muscle, the action potential is carried solely by sodium and potassium. This calcium influx, as we will see later in the chapter, is used to start the contractile process. The influx of positively charged calcium ions is opposed by the efflux of additional potassium ions through delayed rectifier potassium channels (KDR), which also open during this phase. These two electrical forces, working in opposition, create the plateau in membrane potential that is the defining characteristic of the cardiac action potential.
Phase 3: Rapid Repolarization
The closure of the L-type calcium channels initiates a rapid repolarization of membrane potential due to the continued efflux of potassium through voltage gated potassium channels. This repolarization brings membrane potential back to rest.
The cardiac action potential is propagated through the cardiac syncytia through gap junctions, allowing for synchronized depolarization and repolarization of the tissue. This coordination comes at the expense of speed (the cardiac action potential propagates more slowly than skeletal muscle), but this is beneficial for the mechanical functions of the heart discussed later.
Phase 1: The trace begins with a slow, incremental depolarization.
Phase 2: Incremental depolarization is followed by a rapid depolarization when threshold is reached. Phase 3: There is then a rapid repolarization and then the incremental depolarization (Phase 1) begins again and the cycle repeats.
Phase 1: Pacemaker Potential (a.k.a. Prepotential) (4 on graph)
The lack of a stable resting membrane potential is due to the efflux of potassium through delayed rectifier channels (KDR), which steadily increases to hyperpolarize membrane potential to its most negative values. However, at this negative membrane potential, specialized channels known as the hyperpolarization activated cyclic nucleotide gated channels (HCN) begin to open. These channels carry a mixed cation current which is predominantly sodium. When we have discussed action potentials in earlier chapters, we have always seen depolarizing stimuli bring channels to threshold that causes further depolarization. The HCN channels are therefore a bit strange, since they respond to hyperpolarizing stimuli and cause depolarization. This funny current reverses the direction of the change in membrane potential so that the cell begins a slow incremental depolarization, and this current is solely responsible for auto-rhythmicity.
This slow depolarization activates the voltage-gated t-type calcium channel. Though only open for a brief period of time, these t-type channels allow a temporary influx of calcium ions and a further depolarization of membrane potential.
Phase 2: Rapid Depolarization
The combined funny current (inward sodium) and transient inward calcium current continue the depolarization of membrane potential until the threshold of the voltage-gated L-type calcium channel is reached. As the t-type calcium and HCN channels close, the L-type calcium mediates a rapid depolarization of membrane potential. This makes the pacemaker cells unique from the other excitable cells discussed so far, since the rising phase of the waveform is carried entirely by calcium ions.
Phase 3: Repolarization
At the peak of the pacemaker action potential, L-type calcium channels close and inward rectifying potassium channels (KIR) open. As with the other channels, this increasing permeability to potassium efflux returns the cell to hyperpolarized membrane potentials and the cycle begins anew.
AV valves (LUB) and Semilunar Valves (DUB)
HISTOLOGY OF CARDIAC TISSUES
Normal cardiac tissue: branching,
striated fibers; centrally located nuclei,
Myocardial infarction: when blood
supply (and oxygen) to myocardium is
interrupted, the myocardial cells quickly
die. Destruction of cell membranes
results in the release of the cell contents into systemic circulation which can be
detected by elevated levels of K+ and serum enzymes
Atherosclerosis: mostly occluded coronary artery;
decreased size of arterial lumen due to the
accumulation of plaque (deposits of fat, fibrin,
cellular debris, calcium) in the interior wall.
• Impairs blood flow and hence oxygen to
the myocardium leading to coronary
artery disease and heart attack.
VEIN, ARTERY, NERVE
• Thicker tunica media, necessary for maintaining arterial pressure
• Typically maintain circular/oval shape when
cut because of collagen
• Usually wider than arteries
• Tend to collapse when cut in cross section
(not circular), or in absence of flow
• Thicker tunica externa
Nerve – distinguishable by lack of lumen (center)
HUMAN BODY BLOOD SUPPLY
Common Carotid artery- Arteries that branch off from the aortic arch and feed the brain with blood (left and Right)
Internal jugular veins- Brings blood back from the brain to the brachiocephalic vein then to superior vena cava
Cephalic vein- Brings blood from the arm to the subclavian vein then to the brachiocephalic vein then to the superior vena cava
Subclavian Artery- (Right below the clavicle) (Left and Right) takes blood from the aortic arch (Right come off of the brachiocephalic artery) to the arms (Left and Right)
Subclavian Vein- Takes blood back from the arm (Axillary vein) and brings it to the brachiocephalic vein then to the superior vena cava
Brachiocephalic Trunk- First branch of the aortic arch and braches into the right common carotid artery and the right subclavian artery (One artery and two veins)
Axillary Vein- Brings blood from the armpit area (blood from brachial artery) to the subclavian vein then to the brachiocephalic vein then to the superior vena cava
Superior Vena Cava- Larger of the Vena Cava/ brings blood from the brachiocephalic veins (left and right) to the right atrium
Aorta is the main supply
of blood in upper body
and all other artery
structures listed below
originate from it
Celiac artery – blood
supply to stomach (NOT
SHOWN ON DIAGRAM)
artery- blood supply to
Renal arteries- blood supply to kidneys – 1/3 of blood flow from heart
Inferior mesenteric artery- blood supply to the large intestine
(The mesentery is a fold of membranous tissue that arises from the posterior wall of the peritoneal cavity and attaches to the intestinal tract)
Common Iliac artery- Brings
blood from the aortic bifurcation
to both the internal and external
Internal Iliac artery- Brings blood
from the common iliac artery to
the but and reproductive organs
External Iliac artery- Brings
blood from the common Iliac
artery to the femoral artery and
supplies blood to the legs
o Visceral pericardium
o Interventricular sulcus
o Right atrium
o Left atrium
o Left ventricle
o Right ventricle
o Superior vena cava
o Pulmonary trunk + arteries (r +
o Pulmonary veins
o Right atrium
o Tricuspid valve
o Right ventricle
o Left atrium
o Bicuspid (mitral) valve
o Left ventricle
o Chordae tendinae
o Papillary muscles
o Pectinate muscles
o Pulmonary semilunar valve
o Aortic semilunar valve
o Interventricular septum
Sotatol attenuates the efflux of
potassium ions from ventricular
myocytes and prolongs the
repolarization of the ventricles leading
to a longer cardiac action potential and
leading to a longer QT segment. This
drugs helps to reset the ectopic foci of
the heart and is used to treat ventricular
LAB 3 – FROG HEART
FROG vs. HUMAN heart
• Has three chambers, instead of 4 ???? 2 atrium, 1 ventricle
• Pacemaker cells equivalent are located in the sinus vinosus (SV node); pacemaker cells are also found in frog ventricle
• One ventricle ???? some mixing of o2 and deoxy-O2 blood in ventricle
• Trabeculae ???? columns of muscular tissues
o Provide site of attachment for papillary muscles, give ventricle spongy texture; may reduce suction against heart wall, and to limit mixing of O2 and Deoxy-O2 blood flows
• Spiral folds ???? in vessels leading out of heart ; guide blood flow from atria to systemic and pulmucutaneous arteries; maintaining separation of O2 and deoxyO2 blood
• Pacemaker in Frog heart = SV node
• Pacemaker cells located in SA and AV nodes
• 2 separate ventricles divided by a septum
• Pacemaker in mammalian heart = SA node
Mechanical AND electrical signals will be recorded to study the frog’s cardiac functions A frogs ECG is recorded using: an electrical stimulator
How would you differentiate atrial contractions from ventricular contractions?
• Correlate it with the electrical trace, if the electrical trace is typical
When applying Ach to the heart, if the frog heart stops beating, you should do each of the following: • Wash the heart with warm ringers
• Apply a few drops of Isuprel
• Massage the heart
Factors Impacting Cardiac Function
• Q10: Pressure Coefficient: measure of the rate of change of a biological or electrical system as a consequence of increasing the temperature by 10 degrees C.
• How much the rate of a process increases with a temp increase of 10 degrees.
o For most biological systems, the Q10 value is ~2-3
o Consider what a Q10 of ½ means….
• Extracellular POTASSIUM concentrations are increased?
o Note: K concentrations inside cell = higher than outside cell…. = high inward to outward concentration gradient ???? allows spontaneous K efflux from the cell, contributing to a maintenance of negative resting membrane potential, as positive charge is leaving the cell
o Even a slight – moderate increase in extracellular potassium ions should DECREASE the concentration gradient because it will bring the concentration of outside and inside the cell closer in value
o This decrease in gradient ???? reduces Potassium efflux which depolarizes the resting membrane potential ???? increase rate of
action potential firing
HIGH dose of potassium can make
extracellular potassium higher than
that of inside ???? reverses
concentration gradient; cardiac
myocyte will not be able to repolarize
because potassium will not flow out of
the cell spontaneously ???? contraction
• Extracellular CALCIUM concentrations are increased?
o Notes: skeletal muscle ???? calcium enters cytosol through RYR on SR
o In cardiac muscle ???? calcium enters cytoplasm through L-type calcium in the plasma membrane, which also binds to the RYR on the SR = calcium-induced calcium release; while the majority of cylocotic calcium responsible for contraction comes from the myocardial SR, the influx of extracellular calcium through surface membrane channels is really important for maintaining the sustained plateau phase depolarization of the cardiac action potential.
o Because extracellular calcium entry is important for initiating cardiac muscle contraction, the strength and speed of cardiac myocyte contraction proportional to the amount of calcium o Hypothesize what will happen if extracellular calcium is increased…..
ex: a moderate increase in extracellular potassium will _______ heart rate due to a more _______ Ek. Increase; negative
Parasympathetic and sympathetic innervation
• Parasympathetic (acetylcholine release) has less effect on contractile force due to the lack of parasympathetic innervation to the ventricles
• Sympathetic (norepinephrine release)
• Atropine =
o Blocks parasympathetic : increases HR
o M-AChR antagonist ????
o cholinergic antagonist ???? prevent activation of muscarinic acetylcholine receptors • Isoproterenol=
o Synthetic amine ???? structurally related to epinephrine.
o Agonist almost exclusively at beta receptors
o Stimulate Sympathetic ???? increase HR
Refractory Period of Cardiac Muscle:
• Action potentials in myocardial cells have a long plateau phase before repolarization. • Why? The calcium channels in the myocardial membrane allow significant calcium influx, preventing membrane potential from repolarizing quickly.
o This sustained depolarization prolongs the inactivation of sodium channels ???? with this delay in the removal of sodium channel inactivation gates, the cardiac action potential experiences a longer refractory period, which prevents firing of adittional action potential, preventing tetanuss (which we see in skeletal muscle, not cardiac).
• How is refractory period different in cardiac muscle compared to skeletal muscle? o Long repolarization is caused by L-type Ca2+ channels. Higher [Ca2+] out vs. in, so during AP, Ca2+ enters cells, contributing to depolarization and persistence of plateau phase.
o It is important because it causes longer inactivation of Na+ channels which prevents heart from over-excitation and summation.
o Long absolute RP of cardiac cells prevents the heart from reaching tetanus.
Mechanical Stretch ????
Frank-Starling Law of the Heart:
• Describes the length-tension relationship
• Degree of stretch of ventricular muscle (length) and the
strength of cardiac contraction (measured as tension
produced by contraction).
• Higher right atrial pressure = cardiac output increases to
a max value
• Cardiac output is highly sensitive to changes in right
• When additional blood enters the heart, the additional
blood volume stretches muscle tissue, which makes
heart contract more forcefully, and thus ejects more blood
• Intrinsic mechanism in which stroke volume can match venous return to heart
A= atrial depolarization, P wave
C= atrial repolarizaton and ventricular depolarization, QRS
D= ventricular contraction
1. This will cause increase in HR, not contractile
force, by depolarizing resting membrane potential.
2. This will cause an increase in pressure in the right
atrium, thereby stretching cardiac myocytes and
increasing contractile force (a la Frank-Sterling law)
to increase cardiac output.
3. Like all muscle, contractile force is dependent on
4. Atropine acts to block parasympathetic
innervation from vagus nerve through its antagonistic action on muscarinic ACh receptors. Normally ACh will lower heart rate, so blocking those sites would cause an increase of heart rate but not affect ventricular contractile force.
5. Norepinephrine activates beta-1 adrenergic receptors, and is mimicked in this experiment using isuprel. It acts on both pacemaker nodes and directly on ventricular myocytes.
• Partial heart block: beat of atria and ventricle comprise a ratio of 3:1 (three beats atria to one beat ventricle)
• Complete heart block: beating of atria and ventricle are completely independent of each other • Ventricular standstill: cease to beat completely; happens more frequently than a complete heart block
Decrease in Heart Rate
Protein and enzymes require higher temperatures to work efficiently
Incrase in [Ca++]
Obvious increase in force with little or no increase in Heart Rate
Facilitates the cross-bridge
formation between thick and thin filaments, increase contractility
Increase Heart Rate, then decrease Heart Rate; no change in force
depolarizes the membrane
potential of pacemaker cells and decreases driving force
Note: why is it used to revive?
Increase heart rate and contractile force
receptors; causes Gs stimulation, increases adenyl cyclase, increase cAMP, increase HCN channel activity (HR), increase pKA and intracellular [Ca++] (contractile force)
Decrease heart rate
Activate muscarinic receptors; cause Gi stimulation, decrease adenyl cyclase, decrease cAMP. Decrease cAMP causes decrease HCN channel activity (HR),
decrease PKA and decrease K channel
Atropine then Ach
Normal heart rate and force
Plant alkaloid, block muscarinic receptors
Increase contractile force, no decline phase due to pericardium
Prevents occurrence of temporal summation
Na channel inactivation
How cold ringers affected heart rate: DECREASED heart rate by decreasing rate of Ca2+ diffusion, leading to a slower depolarization and repolarization of the heart; t contractile force decreased
Affects of Isuprel (isoproterenol) and acetylcholine (Ach) on heart rate. Identify their receptors: • Isuprel = increases HR
o Receptor = Ach muscarinic receptors
• Acetylcholine = decreases HR
o Receptor: beta adrenergic receptors
What receptor does atropine block to increase HR? blocks the acetylcholine muscarinic receptor • Useful in bradycardia
Peak 1 = atria contracting/at peak of contraction
Peak 2 = ventricle contracting/at peak of contraction
LAB 4- PHYSIOLOGY OF HEMOGLOBIN-OXYGEN BINDING
• Beer’s Law: linear relationship between the
CONCENTRATION in a solution and the
ABSORBANCE of electromagnetic radiation
• Absorbance = ELC
Absorbance maximum ???? some substances may have more than
one peak of absorbance
Peak of greatest absorbance is the max wavelength
Absorbance = 660nm
Ambient air pressure = 760 mm Hg
Deoxygenated hemoglobin has greater absorbance at 660nm than oxygenated hemoglobin
OXYGEN HEMOGLOBIN DISSOCIATION CURVE
Cooperative Binding: when an oxygen molecule binds to one
subunit of hemoglobin, the affinity of the remaining subunits for
oxygen is increased
P50 = Partial pressure of oxygen at which 50% of the hemoglobin is
bound to oxygen
“CADET face right”
“CADET face right”
“CADET face right”
CADET = CO2, Acidity, DPG, Exercise, Temp
Right Shift = RELEASE of Oxygen
• Higher P50
• Decreased O2 affinity
• Increased temp
• Low pH/High acidity
• High 2-3 DPG
• Increased partial pressure of carbon dioxide
Left Shift = LOADING oxygen (onto hemoglobin)
• No DPG
• Increased O2 affinity
• Decreased Temp
• High pH/Low acidity
• Lower P50
Human Fetal Hemoglobin:
• Stronger affinity for O2 than adult hemoglobin
• Advantage: fetal hemoglobin needs to extract oxygen from ???
• Oxygen binding protein in muscle tissue
• Single polypeptide globin chain
• Only one heme group ???? can only bind one oxygen molecule per
• Control hemoglobin sample
o Room temperature, pH = 7.4
• Experimental Treatments:
o Cold temperature (flask on ice)
o Low pH
o Hemoglobin stripped of 2,3-BPG
1. Which calculation is used to determine the concentration of a sample, if the Absorbance of the sample is measured using spectrophotometry?
a. A = (extinction coefficient) x (optical length) x (concentration of sample)
2. In lab this week, we will use the spectrophotometer to measure the _____ of a sample
a. Absorbance of light
3. Which of the following has a greater absorbance of light at 660 nm?
a. Deoxygenated hemoglobin
4. What is lambda max?
a. Wavelength at which a sample has the greatest absorbance of light
5. When you walk into the lab this week, what is the first thing you should do?
a. Make sure the spectrophotometer is turned on
6. In the systemic capillaries in muscle tissue, hemoglobin releases virtually all the oxygen it is carrying and becomes completely desaturated. T/F
7. When one oxygen molecule binds to hemoglobin, the affinity of the remaining hemoglobin subunits for oxygen is increased. This is due to:
a. Cooperative binding
8. One molecule of hemoglobin can bind to 4 molecules of oxygen
PRACTICE QUESTION: KNOW HOW TO CALCULATE!!!!
% Saturation = (A-B)/(A-C) x 100%
A = Absorbance of completely deoxygenated blood
B = Absorbance of partially deoxygenated blood
C= Absorbance of completely oxygenated blood
LAB 5 – LYMPHATIC AND IMMUNE SYSTEM
STRUCTURES OF LYMPHATIC SYSTEM
o Tonsils (mucosal lining in oral cavity)
Lymphoid organs: (lymphatic organs)
Primary Lymphoid organs: sites where cells of immune system are generated and mature Secondary Lymphoid organs: sites where the cells of the immune system aggregate and initiate a specific immune response
o Thymus (PRIMARY)
▪ Development of T lymphocytes (site)
▪ site of final maturation of T
lymphocytes (which begin
development in bone marrow)
• Cortex = darker region of
thymus due to lymphocyte
presence, in comparison to
medulla which has less
• Hassal’s corpuscles formed by epithelial cells arranged in a predictable pattern o Lymph Node (Secondary lymphoid organ)
▪ Lymphocytes in nodes assist in activating immune response
▪ If they detect a pathogen in the lymph, cells will initiate an immune response agsint pathogen
▪ Lymph flows into nodes through afferent vessels
▪ Lymph exits nodes through 1-3 efferent lymphatic vessels
• Fewer efferent than afferent vessles ???? allows lymph to slow down as it passes though
o Spleen (SECONDARY)
▪ Main function: cleanse/filter blood of defective blood
▪ Largest lymphoid organ in the body
▪ Primary site of lymphocyte development
▪ Initiation of immune responses
▪ New white blood cells can be picked up
▪ Red pulp = site of storage of RBC’s and filters them
▪ White pulp = clusters of T cells, B cells, and macrophages
(darker spots on histology slide)
▪ Left side of body
▪ Central arteries surrounded by T cells, run through white
o Red Bone Marrow (PRIMARY)
o B cells (humoral immunity/antibody mediated immunity)/ B lymphocytes
▪ Production of antibodies
o T cells (cellular immunity/cell-mediated immunity)/ T lymphocytes
▪ Helper (CD4) T cells secrete cytokines that regulate functions of cells of the immune system
FUNCTIONS OF LYMPHATIC SYSTEM
• Maintain fluid balance
o Lymphatic capillaries collect interstitial fluid (lymph) from tissue spaces, and merge larger lymph vessels
▪ An excess of ~3L interstitial fluid per day
o Lymph vessels ultimately merge into right lymphatic duct and thoracic ducts
o Ducts return fluid to the blood circulation at the subclavian veins
▪ Right Side = right lymphatic duct drains lymph from right arm and right side of head neck and thorax and returns it to the right subclavian veins
▪ Left Side = thoracic duct ???? receives lymph from left upper body and from all the lymph vessels of the lower body below diaphragm. Returns lymph fluid via the left subclavian vein
▪ **continuous recycling of fluid between blood and interstitial spaces ** ???? maintain fluid balance
o Lymph circulates through lymph nodes scattered through the body
• Participate in immune responses to protect against foreign pathogens
• Absorb lipids (in small intestine through vessels called lacteals)
PATHOGENS AND ANTIGENS
Pathogens: infectious microbe that causes disease
Antigen: molecule that elicits the production of antibodies as part of the immune response • “ANTIbody GENerator
Antibodies are produced by B cells in response to infection with a pathogen ????
• infection w/ pathogen
• antigens on pathogen are detected by B cells in the lymphoid organs
• B cells differentiate into plasma cells and secrete antibodies that bind only to that specific antigen on the invading pathogen
• Antibodies can only bind to one specific antigen
• Fab = antigen binding region, variable region
• Fc = constant region
Enzyme Linked Immunosorbent Assay (ELISA)
• Utilizes basic principles of antibody-mediated
immunity to detect antibodies or antigens in a
• Important diagnostic tool to detect infection with
o Extremely sensitive
o Extremely specific
In lab ???? indirect ELISA
• Step 1: coat with ANTIGEN (BSA)
• Step 2: Wash
• Step 3: Block with Gelatin
• Step 4: Add primary antibody
• Step 5: wash
• Step 6: add secondary antibody (goat anti-rabbit antibody linked to enzyme = HRP) • Step 7: wash
• Step 8: add substrate (ABTS)
• Analysis: color change = Does contain anti-BSA antibodies ; no color chain = doesn’t contain
Why is a direct ELISA used rather than an indirect ELISA
for at home pregnancy tests?
• Tests the urine, not blood; no antibodies in
urine, so must do a direct test to screen directly for
Why is it called an indirect ELISA? It tests indirectly for
exposure to antigen by testing for antibodies
Why isn’t a Direct ELISA used to test for HIV?
• Because the HIV virus can go latent inside of
A patient was recently potentially exposed to HIV. His ELISA test for HIV came back negative. The doctor told him to be tested again in 6 months. Why?
• Patient may not have seroconverted yet ???? takes time to develop enough antibodies to detect
Hemolytic Plaque Assay:
• Used to detect antibody-producing plasma cells
• Mice are immunized with sheep red blood cells (SRBC)
• Stimulates B cells in the mouse spleen to differentiate into plasma
cells and secrete antibodies against SRBC
Sources: Images and certain questions in this study guide: via
KuraCloud, UConn 2265 Lab PPTS, and Pre-Lab Quizzes – I take no
credit for them in any way ☺
Activation of complement results in: lysis of cells, bacteria, viruses, Opsonization which promotes phagocytosis of pathogens, Triggers inflammation, Immune clearance
Control well – no plaques
Hemolytic plaque assay:
• Used to detect antibody-producing plasma cells
• Mice are immunized with sheep red blood cells (SRBC)
• Stimulates B cells in the mouse spleen to differentiate into plasma cells and secrete antibodies against SRBC
• Mix together:
o Mouse spleen ceclls
o Complement (added to this mixture)
▪ Classical complement pathway ** ???? dependent on presence of antibodies on invading pathogen
• Incubate in slide chambers
Classical Complement Pathway:
• C3b initiates formation of
membrane attack complex (MAC)
• Requires this antibody binding to
surface of pathogen to get started