PNB2265 – PRACTICAL I – STUDY GUIDE

What is hemagglutination and when is it used?

Tuesday February 27th 2018

Lab 1 – Blood

BLOOD COMPONENTS

Components of human blood:  

• Erythrocytes (red blood cells, RBC)

• Thrombocytes (platelets)

• Leukocytes (white blood cells, WBC)  

Distribution (%) of Leukocytes in Blood:  

What causes microcytic anemia?

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  

kidney-shaped nucleus  

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  

BLOOD DISEASE

What are the components of blood?

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  

Macrocytic Anemia:  

• RBC’s too large  

• Reasons: Deficiency of certain vitamins  

(folic acid, B12)  

Microcytic Anemia:  

• 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  

hemoglobin,

• 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

HEMOGLOBIN

Hemoglobin:  

• 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  

blood

• 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  

HEMATOCRIT

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

Post-Centrifuge:  

• The upper layer (clear yellow) = plasma

• The lower layer (dark red) = mainly packed red blood  

cells (erythrocytes)  

Normal Range of Values:  

• ~47 for men – due to testosterone which produces more  

erythropoietin  

• ~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

EKG:

• 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  

Waves:  

• P wave = atrial depolarization

• QRS complex = ventricular depolarization, atrial  

repolarization

• 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.

Myocytes 

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.

SA 

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,  

intercalated discs  

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

Artery –

• Thicker tunica media, necessary for maintaining arterial pressure

• Typically maintain circular/oval shape when  

cut because of collagen

Vein –

• 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)

Superior mesenteric  

artery- blood supply to  

duodenum  

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  

iliac artery  

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

SHEEP HEART  

• External:

o Visceral pericardium  

(epicardium)

o Interventricular sulcus

o Right atrium

o Left atrium

o Left ventricle

o Right ventricle

o Aorta

o Superior vena cava

o Pulmonary trunk + arteries (r +  

l)

o Pulmonary veins

• Internal:  

o Myocardium

o Endocardium

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  

arrhythmias.

LAB 3 – FROG HEART  

FROG vs. HUMAN heart  

Frog 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  

Human Heart:  

• 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

Temperature

• 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….  

•

Ionic concentration

• 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  

will stop  

• 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  

atrial pressure  

• 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

B=atrial contraction

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  

calcium concentration.

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.

Heart Block:  

• 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

Parameter

Expected Observations

Reasoning

Decrease Temperature

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

Increased [K+]

Increase Heart Rate, then decrease  Heart Rate; no change in force

depolarizes the membrane  

potential of pacemaker cells and  decreases driving force

Isuprel

Note: why is it used to revive?

Increase heart rate and contractile  force

Activates beta-adrenergic  

receptors; causes Gs stimulation,  increases adenyl cyclase, increase  cAMP, increase HCN channel  activity (HR), increase pKA and  intracellular [Ca++] (contractile  force)

Ach

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

Mechanical stretch

Increase contractile force, no  decline phase due to pericardium

Frank-starling law

Absolute refractory

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  

SPECTROPHOTOMETRY

• Beer’s Law: linear relationship between the  

CONCENTRATION in a solution and the  

ABSORBANCE of electromagnetic radiation  

(light)  

• 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 ???

Myoglobin:  

• Oxygen binding protein in muscle tissue

• Single polypeptide globin chain

• Only one heme group ???? can only bind one oxygen molecule per  

hemoglobin

CALCULATIONS:

EXPERIMENTAL TREATMENTS:  

• 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

Quiz Questions:  

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

a. False  

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

Lymph vessels

o Lymph

Lymphoid tissues

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)

▪ Lobules  

▪ Histology:  

• Cortex = darker region of  

thymus due to lymphocyte  

presence, in comparison to  

medulla which has less  

dense concentration

• 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  

cells

▪ 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  

pulp

o Red Bone Marrow (PRIMARY)

Lymphocytes:  

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

Antibody structure:  

• Fab = antigen binding region, variable region

• Fc = constant region

ELISA

Enzyme Linked Immunosorbent Assay (ELISA)

• Utilizes basic principles of antibody-mediated  

immunity to detect antibodies or antigens in a  

sample

• Important diagnostic tool to detect infection with  

specific pathogens

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  

antigens  

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  

cells

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  

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 SRBC’s  

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