INTRO TO BB AND IMMUNOLOGY d.

Immune because of HBV vaccine

8. Valuable combination in therapy and follow-up
1. An iron-containing heme protein responsible for
in patients with germ cell tumor of the testes.
the peroxidase activity characteristic of
a. CEA and AFP
azurophilic granules: it accounts for the greenish
b. CA 125 and CA19-9
color of pus
c. AFP and B-hCG
a. Myeloperoxidase
d. HER-2 and B-hCG
b. Lactoferrin
9. Marker for gastric carcinoma:
c. Hemoglobin
a. CA 72-4
d. Myoglobin
b. CA 19-9
2. It binds iron which is an essential nutrient for
c. CA 125
microbes.
d. CA 15-3
a. Lactoferrin
10. A biomarker that exhibits greater specificity than
b. Hemoglobin
other proinflammatory markers in identifying
c. Myeloperoxidase
patients with sepsis and can be used in the
d. Myoglobin
diagnosis of bacterial infection
3. Large granular lymphocyte (LGL) is synonymous
a. Ceruloplasmin
with:
b. Cytokines
a. NK cell
c. Procalcitonin
b. B lymphocytes
d. CEA
c. T lymphocyte
11. What should be done if a noticeable clot is found
d. Cytotoxic T lymphocyte
in an RBC unit:
4. It is referred to as congenital thymic hypoplasia
a. Issue the unit; note the presence of a
which results from defective embryogenesis
clot in the release form
leading to a reduced (or absent) thymus.
b. Issue the unit: the blood will be filtered
a. Wiskott Aldrich Syndrome
c. Filter the unit in the blood bank before
b. Bruton's agammaglobulinemia
issue
c. Di George Syndrome
d. Do not issue the unit
d. X-linked agammaglobulinemia
12. The following dilutions were set up to titer an
5. Anti-dsDNA antibodies are associated with
antibody. The following results were obtained:
which of the following?
1:4 = +
a. CMV infection
1:8 = +
b. Syphilis
1:16 = +
c. SLE (Systemic Lupus Erythematosus)
1:32 = +
d. Hemolytic anemias
1:64 = (0)
6. It is a chronic progressive inflammatory
How should the titer be reported out?
autoimmune disease marked by progressive
a. 4
dryness of the eyes and the mouth
b. 16
a. SLE
c. 64
b. Rheumatoid arthritis
d. 32
c. Sjogren syndrome
13. The first line of defense in protecting the body
d. Scleroderma
from infection includes all of the following
7. Interpretation of HBV panel:
except:
HBsAg - Neg
a. Normal microbial flora
Anti-HBc - Pos
b. Unbroken skin
Anti-HBs – Pos (na dapat reactive)
c. Secretions such as mucus
a. Acute infection
d. Phagocytic leukocytes
b. Might be susceptible to a false positive
14. A primary function of the eosinophil is:
anti-HBc
a. Antigen recognition
c. Immune because of natural infection
 b. Suppression of the inflammatory
response
c. Reacting in acute, systemic
hypersensitivity reactions
d. Phagocytosis
15. Class II MHC molecules are recognized by which
of the following:
a. CD4+ T cells
b. CD8+ T cells
c. B lymphocytes
d. NK cells

NOTES:

• Wiskott Aldrich Syndrome – part of hema,

microplatelets
• Bruton's agammaglobulinemia – cannot produce
immunoglobulins
• X-linked agammaglobulinemia – cannot produce
antibodies
• Anti-dsDNA – has lot of worms
• SLE – butterfly rash
• Scleroderma – thickening of the skin
• Vaccine only contains the surface antigen, so if
you get vaccinated against hepa B, magiging
immune ka sa anti-HBs
• Terminal cell tumors, trophoblastic tumors
(germ cell) – the behavior ng testicular cancer
introduces embryo equibolin since blastic cell
ang present ang narerelease ay pregnancy
markers (AFP and B-hCG)
• Carcino Embryonic Antigen (CEA) – used for the
monitoring of colorectral cancer
• CA 125 – used to monitor ovarian cancer
• CA 19-9 – used to monitor pancreatic cancer
• HER-2 – histopathologic marker of breast cancer
• Gastric carcinoma – present in duodenum or
sometimes pericardium of the stomach
• CA 15-3 – used to monitor breast cancer
• CA 72-4 – marker for gastric carcinoma
BB Q2
This is the kind of anemia associated with a metastasis
of non myeloid cells to the bone marrow.
 Myelophthisic anemia
 Megaloblastic anemia
 Myelofibrosis
 Myelodysplastic syndrome
What is the vitamin deficiency associated with
megaloblastic anemias
o Thiamin
o Niacin
o Pyridoxine
o Cobalamin
This is an anemia characterized by a spectrin
deficiency (not sure)
 Hereditary spherocytosis
 Hereditary elliptocytosis
 None of the above
 Hereditary pyropoikilocytosis
What test differentiates anemia of chronic disease vs.
iron deficiency anemia?
 AOTA
 Transferrin saturation
 TIBC
 FEPP
Which of the ff. would most likely develop iron
deficiency anemia
o 3 months pregnant mother
o Physically active young man
o Colorectal carcinoma in situ patient
o Patient who is a chronic smoker
What is the 1st therapy for patients with megaloblastic
anemia due to Diphyllobothrium latum infection (not
sure)
o Treatment with praziquantel
o Replacement of folic acid
o PRBC transfusion
o Treatment with cyanocobalamin
This is the kind of anemia characterized by a protein
band 4.1 deficiency.
o Both
o Hereditary elliptocytosis
o Neither
o Hereditary spherocytosis
Anemia of chronic renal disease is an anemia classified
by a deficiency in ______
o Vitamin B12
o Erythropoietin
o Sodium
o Iron
Hemoglobin H disease results from:
o Absence of 1 of 4 alpha genes
o Absence of all alpha genes
o Absence of 2 of 4 alpha genes
o Absence of 3 of 4 alpha genes
The lab finding on a patient are as follows:
MCV: 55 fL MCHC: 25% MCH 17pg
A stained blood film of this patient would most likely
reveal a red cell picture that is:
o Macrocytic, hypochromic
o Normocytic, normochromic
o Microcytic, hypochromic
o Microcytic, normochromic
Excessive amount of iron that accumulate in the blood
and tissues leading to injury in the parenchymal
tissues.
o Hemosiderosis
o Sideroblastic anemia
o Porphyria
o Hemochromatosis
This is a condition in which there is deficiencies of
both CD55 and CD59 in the surface of the red cell.
o PNH
o PCH
o MDS
o CAD
What disorder is associated with erythrocytes that are
thermally instable and fragment when heated to 45 to
46 degrees celcius?

o Hereditary elliptocytosis
o Heridtary pyropoikilocytosis
o Hereditary spherocytosis
o PNH

Donath-Landsteiner antibody is present in patients

with _______.

o Paroxysmal Cold hematuria

o Diamond-Blackfan anemia
o Paroxysmal Nocturnal hemoglobinuria
o Pure red cell aplasia

This is a congenital anemia characterized by the

presence of webbing of digits. Café-au-lait spots in the
trunk and back, and congenital cardiac or renal
anomalies in patients. – Fanconi’s syndrome
BB LEC (1st week) o Blood-borne Disease Screening
o Compatibility Testing – cross match
INTRODUCTION TO BLOOD BANKING and BTRH (blood type & Rh test)
Blood Banking - Blood Transfusion
- Transfusion Reaction Investigation (If
- Collection, storage/preservation of blood and necessary) – pathologist, hematologist, RMT,
preparation of blood components patient
- Transfusion practices
o Compatibility testing RBC Biology and Preservation
o Component therapy - Normal chemical composition and structure
- AKA Immunohematology of the RBC membrane
History - Hemoglobin structure and function
- RBC metabolism
• 1492 – Pope Innocent VII; first ever recorded - RBC storage lesion
blood transfusion
• 1869 – Braxton Hicks; recommended sodium RBC Membrane
phosphate as a nontoxic anticoagulant
• 1901 – Karl Landsteiner in 1901 discovered the
ABO blood groups
• 1900's – Edward E. Lindemann; first to
successfully perform vein-to-vein transfusion
of blood
• 1900's – Unger; designed the syringe-valve
apparatus that made transfusions from
donor to patient by an unassisted physician
practical
• 1914 – Hustin; sodium citrate as an
anticoagulant solution for transfusions - RBC is composed of a double layer
• 1915 – Lewisohn; determined the minimum phospholipids w/ integrated protein and
non-toxic amount of citrate needed for carbohydrates (can be mixture or solo)
anticoagulation - Notable are:
o Protein band 4.1
• 1916 – Rous and Turner; introduced a citrate-
o Spectrin
dextrose solution for the preservation of
o Glycophorins – dito dumidikit ibang
blood
blood group
• 1930's (World War II) – Charles Drew;
▪ GP A – will carry the blood
developed techniques in blood transfusion
group M, N
and blood preservation and pioneer in the
▪ GP B – will carry the blood
establishment of a widespread system of
group Ss, U*
blood banks
- Spectrin, protein 4.1, ankyrin – RBC
• 1943 – Loutit and Mollison; formula for the
membrane proteins that are responsible for
preservative acid-citrate-dextrose (ACD).
the regulation of the shape of the cell
• 1947 – Publication of several papers on blood
- Protein band 4.1 deficiency – results in
preservation and establishment of blood
hereditary elliptocytosis
banks in many major US cities
- Spectrin deficiency – results in hereditary
• 1957 – Gibson; improved preservative
spherocytosis (hyperchromatic)
solution called citrate-phosphate-dextrose
- MCHC – average hemoglobin content of one
(CPD)
cell
Overview of Blood Bank Process o ↓ - hypochromic
▪ Micro, hypo – IDA, ACD
- Blood Donation (sometimes normo size),
- Blood Component Preparation and Storage thalassemia,
- Pre-transfusion Testing
 hemoglobinopathies, lead ▪ responsible for oxygen
poisoning* carrying capacity of
o N – normochromic hemoglobin.
▪ Aplastic anemia ▪ Once it binds to hemoglobin,
o ↑ - hyperchromic there’s no choice for
▪ Hereditary spherocytosis – hemoglobin but to release
no central pallor the oxygen
- Deformability decreases with: ▪ More likely ↓ H+
o Decreased phosphorylation of concentration
spectrin due to loss of ATP o ↓ Temp, 02/P50
o Accumulation of membrane calcium o Hyperventilating patient - ↓CO2 =
(membrane rigidity and loss of nagiging basic yung pH
pliability) o pH – 7.35-7.45
▪ In patients with o pCO2 – 45-35
hypercalcemia that o HCO3 – 22-26
demonstrates anemia - When right shifted probable
possible of cell’s burst changes/possible of:
- Permeability o ↑ DPG, Temp, O2/P50, H+
o Permeable to water and anions concentration - ↓ pH
o Relatively impermeable to cations ▪ Inverse relationship of
▪ Na+, K+, ATPase pump – +
hydrogen (H ) & pH – the
maintain cations more H+ ions present, the
▪ ↑ intracellular K+ ; ↓ lower the pH
intracellular Na+ --- ↑ - Patient with hemoglobinopathies – shift to
extracellular Na +
; ↓ the left
+
extracellular K (Potassium in - Patient with metabolic or respiratory acidosis
Sodium Outside/PISO) – shift to the right

Hemoglobin RBC Metabolism

Hemoglobin dissociation curve – sigmoidal in - Anaerobic (glycolytic) pathway

appearance o generates about 90% of the ATP
needed by the RBC
- Approximately 10% is provided by the
accessory pathway:
o Pentose phosphate pathway or
Glutathione or Hexose
Monophosphate Shunt (MOST of the
10%) – responsible for preventing the
oxidation of the hemoglobin
▪ Glucose 6 Phosphate
Pathway – responsible for
glutathione synthase (gets
the free radicals inside our
cells) and kapag kinuha nya
yung free radicals walang
mag ooxidize sa hemoglobin
natin, therefore, reducing the
- When left shifted probable changes/possible
potential to oxidize the
of:
hemoglobin.
o ↑ Abn Hb
• Oxidation = Fe2+ inside
o ↑ pH
the hemoglobin and
o ↓ DPG (2, 3-diphosphoglyceric acid)
when it gets oxidize it
 will turn into → Fe3+
(Methemoglobin)
• Deficiency – G6PD
deficiency, which results
in Heinz bodies (ferric
iron deposits in RBC)
deposition in the RBC
because of unable to
reduce the potential to
oxidize
o Methemoglobin reductase pathway or
Cytochrome b5 reductase pathway –
responsible for reverting the present
oxidation
▪ e.g., if there’s
methemoglobin present,
what will cytochrome b5 do is
to revert it from ferric to
ferrous
o Luebering-Rapoport shunt/pathway –
responsible for the production of 2,3-
DPG (responsible for oxygen carrying
capacity)
Oxygen dissociation Shift to the left (increase
curve in hemoglobin and
oxygen affinity; less
oxygen delivered to
tissues)
Plasma K+ Increased
Plasma hemoglobin Increased
• shift to the left – increases oxygen affinity;
less oxygen delivered to tissues (gahaman
yung hemoglobin)
• shift to the right – galante pero di nya kaya
mag dala ng madami
• Plasma K+ - as the cell increases
(naputol……)
• Newborns are given fresh RBC, too much or
too litle K+ is bad for the patient
• Hemoglobin is very nephrotoxic – toxic in
kidneys
RBC PRESERVATION
Anticoagulant-Preservative Solutions
NAME ABBREVIATION STORAGE TIME
(DAYS)

Acid citrate- ACD-A 21

dextrose
(formula A)*
Citrate- CPD 21
phosphate
dextrose
Citrate- CP2D 21
phosphate-
double-
dextrose
Citrate- CPDA-1 35
phosphate-
dextrose-
adenine
*ACD-A is used for apheresis components
RBC Storage Lesion
CPDA-1 – usually use in the lab
CHARACTERISTIC CHANGE OBSERVED
CHEMICAL FUNCTION PRESENT IN
% Viable cells Decreased
ACD-A CPD CP2D CPDA-1
Glucose Decreased Citrate Chelates X X X X
(sodium calcium;
ATP Decreased
citrate/citric prevents
Lactic acid Increased acid) clotting
Monobasic Maintains pH X X X X
pH Decreased sodium during
phosphate storage;
2,3-DPG Decreased necessary
for
 maintenance pH (measured at 6.6 6.5 6.5
of adequate 37°C)
levels of 2,3- 24-hour 83 85.1 80
DPG survival*(%)
Dextrose Substrate for X X X X ATP (% initial) 68 67 68.5
ATP
production 2,3-DPG (% initial) 6 6 5
(cellular
Hemolysis (%) 0.5 0.7 0.6
energy)
Adenine Production X
of ATP
(extends Long Term Preservation (Freezing)
shelf-life
from 21 to 35 1ST – maselan (personal belief, rare blood type) konti
days) lang patient na may Rh null

AMA – against medical advice

Additive Solutions How to do freezing:
- Purpose: o Need to incorporate glycerol (anti-
o Extends shelf-life (from 35-42 days) freeze, freezes only RBC not plasma)
o Allows more platelets and plasma to o Glycerol – calcium oxalate
be harvested - Autologous donation and/or RARE blood
o Makes blood less viscous; easier to types
transfuse
- Glycerol:
▪ If blood is viscous, it’s harder o High-concentration: 40% w/v
to transfuse o Low-concentration: 20% w/v
- Licensed brands: (more likely used in private
hospitals – St. Luke’s, Makati Med., The ADVANTAGES DISADVANATAGES
Medical City)
Long-term A time-consuming process
o Adsol (AS-1; Baxter Healthcare)
storage (10
o Nutricel (AS-3; Pall Corporation)
years)
o Optisol (AS-5; Terumo Corporation)
Maintenance of Higher cost of equipment and
- Saline RBC viability and materials
- Adenine function
- Glucose Low residual Storage requirements (–65°C)
- Mannitol (AS-1 & 5) leukocytes and
- Citrate & phosphate (AS-3) platelets
Removal of Higher cost of product
NAME ABBREVIATION STORAGE
significant
TIME (DAYS)
amounts of
Adsol (Baxter AS-1 42 plasma proteins
Healthcare)
Nutricel (Pall AS-3 42 High VS Low Glycerol
Corporation)
ADVANTAGE HIGH GLYCEROL LOW
Optisol AS-5 42 GLYCEROL
(Terumo
Corporation) Initial freezing –80°C –196°C
temperature
Need to control No Yes
AS-1 AS-3 AS-5 freezing rate
Storage period 42 42 42 Type of freezer Mechanical Liquid
(days) nitrogen
 Maximum –65°C –120°C
storage
temperature
Shipping Dry ice Liquid
requirements nitrogen
Effect of Can be thawed Critical
changes in and refrozen
storage
temperature

Current Trends

- Improved preservation methods and

pathogen elimination (e.g., alkylating agents
that interfere with pathogen nucleic acid)
- RBC Rejuvenation (Rejuvesol)
o Reviving/rejuvenating agent
▪ e.g., as long as the blood is
not clotted, put rejuvesol and
it can still be transfuse
o The only FDA-approved rejuvenation
solution for use with CPD, CPDA-1,
and CPD/AS-1
o Contains phosphate, inosine,
pyruvate and adenine
o RBCs stored in the liquid state can be
rejuvenated at outdate or up to 3
days after outdate
- RBC Substitutes
o AKA blood substitutes, oxygen
therapeutics or artificial oxygen
carriers (AOC), plasma expanders,
hemoglobin derivatives
(DI NYA DINISCUSS YANG 3 TABLES NA YAN, NASA
o e.g., If a patient has many history of
BOOK TO BTW)
transfusion reaction or has
autoantibodies (attack it’s own RBC,
result in hemolysis in vivo) in his/her
own RBCs
o Types:
▪ Hemoglobin-based oxygen
carriers (HBOCs) -
ultrapurified stroma-free
hemoglobin (SFH) of bovine,
human or recombinant origin
▪ Perfluorocarbons (PFCs) -
synthetic hydrocarbon
structures in which all the
hydrogen atoms have been
replaced with fluorine, and
are excellent gas solvents,
effective gas carriers
BB LAB (1ST WEEK)
OVERVIEW OF THE BLOOD BANK PROCESS
• Blood Donation
• Blood Component Preparation and Storage
• Pre-transfusion Testing
• Blood Transfusion
• Transfusion Reaction Investigation (If
necessary)
• Blood Bank Inventory, Maintenance and QA
• Special Techniques
Blood Donation
Step 1: Educational Materials
- Gauge 16,17,18 is used in blood donation (like
straw of Zesto)
Step 2: The Donor Health History Questionnaire
- There are questions that a donor should
know what to answer, hindi ikaw ang mag
tatanong dapat si donor mismo ang sumagot
Step 3: The Abbreviated Physical Examination
- Should be done by a nurse or licensed
physician
Tests for Hepatitis:
- HBs Ag
o If the donor is 6-8 months into the
infection, walang madedetect na HBs
Ag pero infected sya, to detect it, it
he/she should be test to HBc Ag
- HBe Ag
- HBc Ag
- anti-HBs
- anti-HBc
- anti-HBe
Blood Component Preparation and Storage
- Whole blood
o Packed RBC (frozen, washed,
leukocyte-reduced or irradiated)
o Platelet or granulocyte concentrate
o Fresh plasma (cryoprecipitate AND
cryoprecipitate poor plasma)
- Apheresis
o Upon extraction of blood into the
donor it will go to apheresis machine
then it will be separate the platelets
into a single bag while the remaining
blood portion (plasma, rbc, wbc) will
then go back to the donor
o Deferral period for platelet apheresis
is only 3 days then after 3 days he/she
can donate whole blood because it
only takes 3 days to recuperate the
loss platelets.
▪ Resulting product – many
platelets are harvested in one
donor/unit
▪ Best to transfuse to a patient
w/ platelet
thrombocytopenia is platelet
apheresis product because
there are more contents and
is only exposed to one donor
Blood Bag Centrifuge (Cryofuge) – for the
preparation of blood products from whole blood is
called cryofuge because it is refrigerated
Blood Bag w/ Satellite Bags
• 2 bag – FFP, PRBC
• 3 bag – FFP, PC, PRBC
• 4 bag – CPP, CP, PC, PRBC
- Hindi tinatanggal ang mother bag, kadalasan
PRBC and naiiwan sa mother bag
- Every lipat ng bag dapat may label
Pre-transfusion Testing
Blood-borne Disease Screening
Current Donor Screening Tests for Infectious
Diseases
TEST DATE TEST REQUIRED
Syphilis 1950s
Hepatitis B surface 1971
antigen (HBsAg)
Hepatitis B core 1986
antibody (anti-HBc)
 Hepatitis C virus 1990 ▪ If there’s presence of
antibody (anti-HCV) hemoglobin in urine – acute
Human 19921 transfusion reaction
immunodeficiency ▪ If there’s presence of hives
virus antibodies (anti- and the result of CBC has
HIV-1/2) – HTLV III presence of hemophilia –
Human T-cell 19972 allergic transfusion reaction
lymphotropic virus
antibody (anti-HTLV- Blood Transfusion
I/II)
Human 1999 - Need 16-18 IV catheter filtered
immunodeficiency - Filter purpose
virus (HIV-1)(NAT)*,** o Is to filter blood clots in bag
Hepatitis C virus (HCV) 1999 o Clotted blood has activated platelets
(NAT) ** and when it enters the patient’s
West Nile virus (NAT) 2004 blood stream, it will result to
embolus, thrombus formation and
Trypanosoma cruzi 2007 will be deposited into pulmonary
antibody (anti-T. tract ending pulmonary embolism or
cruzi) brain stroke
• Malaria (common in Mindoro and Palawan)
• CMV (cytomegalo virus) Transfusion Reaction Investigation

Compatibility Testing - Collection of clinical samples

• ABO and Rh Typing
• Cross-matching
o Major (I) – patient serum vs. donor
RBC
▪ Done to test if it’s safe to
transfuse the RBC of the
donor to patient’s serum and
to avoid the acute hemolytic
transfusion reaction
▪ ABO incompatibility – donor blood
deadliest incompatibility
o Minor (i)
• Minor Blood Group Typing
*** Tests done in the PH (red colored fonts)
Blood Transfusion
- Clerical checks
- Blood transfusion instruments
- Rate of administration
o PRBC (packed RBC) should be
transfused within 2 hrs
- Clinical indicators of adverse transfusion
reactions
o Done by nurses
o If there’s symptom of pain automatic
stop the transfusion then report to
the lab (clerical checks, lab works –
patient’s urine, pre & post
transfusion blood, blood from donor)
o Patient post transfusion urine
o Patient pre transfusion blood
o Patient post transfusion blood
o Donor blood
- Direct and indirect Coomb's test - determine
the nature of antibody
o Direct lies inside
o Indirect – chronic (???)
- Repeat compatibility testing
o Using pre transfusion blood and
Blood Bank Inventory, Maintenance and QA
- Blood donor registry
- Blood component inventory
- Reagent preparation and QA
o Everyday monitoring
- Instrument monitoring and QA
o Everyday monitoring of ref temp.,
centrifuge, freezer, reagents, blood
component inventory, etc.
Special Techniques
- HLA typing
o Organ donation
o Tumor markers
o Types of hypersensitivities
- Paternity testing
o Should be under Human Leukocyte
Antibody (HLA) testing
BB LEC (2nd week) syndrome, patients taking
antacids.
ANEMIAS Irons is well absorbed in an
acidic environment. You need
- Anemia is a decreased in the oxygen carrying
an acidic environment for
capacity of the blood
iron to be absorbed readily.
o It can arise if there is insufficient
Antacids – lowering or
hemoglobin or the hemoglobin has
making the acidic
impaired function.
environment to become
IRON AND HEME DISORDERS basic. Therefore, iron is not
absorbed well
Iron Deficiency Anemia o Chronic blood loss
▪ Chronic hemorrhage/bleeding can
- One of the most common anemias present in a
also result to IDA
whole clinical practice
▪ Ex. Tumors, hemorrhoids, bladder
- This is the type of anemia that arises due to a
stones, kidney stones, PNH.
patient being deficient in iron.
NOTES: constant loss of RBC,
- It can occur due to many reasons:
hemoglobin = constant need
o Inadequate intake
to replace it
▪ Iron deficiency anemia can develop
when the erythron is slowly
starved of iron
▪ Seen in patients who have a poor
diet
▪ It will result in a slow developing
iron deficiency
NOTES: e.g., patients who do
not eat iron rich foods like
vegans who do not eat red
meats
o Increased need
▪ Iron deficiency can develop when
the level of iron intake is
inadequate to meet the needs of Stage 1: Iron depletion
an expanding erythron - Storage iron ay paubos lang
▪ Can be seen in rapidly growing
children Stage 2: Iron deficient erythropoiesis
NOTES: e.g., lactating
- Unti unti nang nababawasan yung iron sa
mother, why they increase
erythropoiesis or erythron iron
need? Because they need to
rump up RBC production Stage 3: Iron deficiency anemia (most chronic stage)
therefore exhausting the iron
- All of the storage and RBC iron are deficient or
stores
few
o Impaired absorption
▪ Even when diet is adequate in iron, LABORATORY DX/DIAGNOSIS
the inability to absorb that iron
through the enterocyte into the - Screening Tests for IDA
blood will result in IDA o CBC: anisocytosis, microcytosis,
▪ Medications: antacids hypochromia
(omeprazole, proton pump ▪ Declining MCV. MCH. MCHC levels
inhibitors, h2 inhibitors), etc. (first need to see for any kind of
NOTES: happens with anemia work up are these the RBC
patients with malabsorption indices, because this will give you a
lot of clues)
 ▪ Poikilocytosis codocytes and
elliptocytes
NOTE: IDA is a microhypo
kind of anemia. Small cell
with diminished hemoglobin
concentration. Anisocytosis
because of variable sizes.
- Diagnosis of IDA
o Serum iron: decreased – because it is a
direct indicators of iron levels
o TIBC: increased
o Serum ferritin: decreased – because it is a
direct indicators of iron levels
o Free erythrocyte protoporphyrin:
increased because protoporphyrin IX
requires iron to become heme. If the
patient doesn’t have iron, then
protoporphyrin IX will probably increase
and accumulate in the RBC. Is it
functional? NO.
NOTES:
Low transferrin = high TIBC because
what is measured in TIBC is what iron
it can carry. If the transferrin is
hungry for iron, then it will eat many
in the testing, therefore the result
indicates that the patient has
insufficient iron
Megakaryopoeisis – formation of
platelets
Erythropoietin – hormone
responsible for the production of
RBC, produce by the kidney and liver.
EPO is increased in iron deficiency but
in the first stages why
thrombocytosis happens? Because
EPO doesn’t play any role
In the first stages of the
megakaryoblast (MB) formation –
the first hormone to play is the EPO.
In order for the megakaryoblast to
mature, it needs EPO tapos dito na
papasok si TPO
Platelet is high in patients with IDA is
due to EPO playing a major role in the
megakaryoblast maturation.
Some patients demonstrate
thrombocytosis because of high EPO
- PBS slide of a patient with iron deficiency that
recently have a transfusion. Also known as
dimorphic kind of anemia because there’s
microhypo, hypo, normo, spherocytes
- Dimorphic anemia happens due to transfusion
and sideroblastic anemia
TREATMENT
- First therapy is to treat what is causing the loss
of iron, or inadequacy of iron
- If nutritional deficiency, oral supplements of
ferrous sulfate and if allergic to it then ferrous
gluconate, if allergic to either or then ferrous
fumarate for not more than 6 months should be
adequate because it will cause blackening of
the stool therefore a false diagnosis of
gastrointestinal bleeding
- If severe IDA needs to be treated, blood
transfusion is an option
o In chronic transfusions, an iron chelating
medication needs to be concurrently
administered (deferoxamine,
desferrioxamine – these drugs chelates
your iron in order to prevent
hemochromatosis) to avoid iron overload
- NOTES: first line – replace what is missing
RESPONSE TO RX/TREATMENT
- Reticulocyte hemoglobin content will correct
w/n 2 days but the microhypo will not correct in
2 days but rather months
- Reticulocyte count begin to increase within 5 to
10 days (there’s already symptomatic
improvement of the patient)
- PBS will show a biphasic population
o Normocytic, normochromic
o Microcytic, hypochromic
Anemia of Chronic Disease/Inflammation (ACD)
- This kind of anemia is associated with systemic
diseases (because there are acute phase
reactants possible for iron metabolism)
including inflammatory conditions (i.e. RA,
SLE), chronic infections (i.e. TB, HIV/AIDS), and
malignancies.
Pathogenesis:
- Anemia of chronic inflammation is due to the
body storing iron in response to infection.
(since bacteria need iron, the bacteria will store
the iron but since there’s no agent, your body
becomes the collateral damage)
- It is controlled by 3 acute phase reactants:
o Hepcidin
▪ This is a hormone that is produced
by the hepatocytes
▪ It regulates body iron levels,
particularly absorption of iron in
the intestines and release from
macrophages
o Lactoferrin
▪ This is an iron-binding protein in the
granules of neutrophils
▪ During inflammation, lactoferrin is
released into the plasma, leading to
low iron levels.
o Ferritin
▪ This is the main storage of iron, in
inflammation ferritin is increased
leading to more iron stored.
LABORATORY DX/DIAGNOSIS
- Mild anemia, with hemoglobin concentrations
usually 8 to 10 g/dL (tolerable) and w/o
reticulocytosis
- Iron studies show low sFe and TIBC
- Ferritin, Hepcidin are markedly increased
- ESR is markedly increased
- Platelets may be normal or markedly
increased***
NOTES: why TIBC is low in ACD? Kase naka
tago na sa kanya, ferritin and transferrin are
part of iron metabolism. If di sya gutom, naka
tago lang sa kanya. Bakit naka tago sa kanya?
Because there’s an inflammation.
The lower the RBC = the higher is the
inflammation
Platelets is increased in ACD because IL-3 is a
multiclass growth factor, interleukin increase
their concentration in inflammatory
episodes. Let say patient is chronically
inflamed, hindi tumataas and RBC kasi nga
kulang ang iron pero tumataas ang platelets
kasi inflamed.
Increase synthesis of IL-3 = increase platelet
formation
TREATMENT
- Therapeutic administration of EPO can correct
ACD, but iron must be administered
concurrently (not usually use because EPO is an
intramuscular injection, iniinject sa abdomen ng
patient)
- Anemia is typically not severe
NOTES: since in inflammation also exerts a decrease
in EPO synthesis, you also need to give EPO
For treating ACD, you need to first treat the causative
agent.
e.g., patient has rheumatoid arthritis, you should give
him/her methotrexate and anti-inflammatory drugs
in order to suppress the inflammation because if you
do not suppress the causative agent, it’s a never-
ending cycle
Sideroblastic Anemia
- These are diseases that interfere with
production of adequate amounts of
protoporphyrin
- Anemia may be microcytic and hypochromic
- In contrast to IDA, Fe is abundant in the bone
marrow
o It is present in the mitochondria,
denoting a deficiency in protoporphyrins
- Erythroblasts are positive in Perl's Prussian Blue
stain (iron stain)
NOTE: kapag maraming blue, maraming iron
What can cause SA?
- Hereditary
- Acquired
o RARS (Refractory Anemia with Ringed
Sideroblasts) – kind of myelodysplastic
syndrome
o Due to drugs:
▪ Antitubercular drugs (Isoniazid)
▪ Chloramphenicol (important, most
common antibiotic that is
associated with aplastic and
sideroblastic anemia. For patients
taking this, it is myelotoxic – toxic
in bone marrow)
▪ Alcohol
▪ Lead
 ▪ Chemotherapeutic Agents
(Doxorubicin, Vinblastine, etc.)
LEAD POISONING
- This is of special concern in children because of
the CNS effects, as well as hematologic effects
- Lead interferes with porphyrin synthesis at
several steps, but the most critical are:
o Conversion of ALA to porphobilinogen
o Incorporation of Fe to protoporphyrin IX
- Anemia, when present, is most often
normocytic and normochromic (acute in
nature)
- A classic finding is basophilic stippling (lead
poisoning)
- Treatment is administration of EDTA to chelate
Pb.
NOTES: lead poisoning is a kind of SA or
porphyria, special concern because of CNS
effect.
San nakukuha? Sa lead paints, lead
contaminated water, surfaces. Because
these are readily absorbed by skin. CNS effect
of this is mental retardation.
Lead are found sa mga toys sa Divisoria,
because their paints are cheap kase mura
lang din ang lead paint
PORPHYRIAS
- Porphyrias are inherited disorders of
deficiencies in enzymes responsible for the
production of heme
- Due to a deficiency of an enzyme, the substrate
accumulates, resulting to toxicity. (These are
the patients that should not expose to UV lights
because of these..)
o Hematologic
o Dermatologic
o Neurologic
Iron Overload
- Excess accumulation of iron results from
acquired or hereditary conditions in which the
rate of acquisition exceeds the rate of
utilization and release
- It can be acquired or hereditary
o Acquired: Chronic transfusion (IDA,
Thalassemia) leading to Hemosiderosis
(there is no parenchymal/significant
damage)
o Hereditary (Hemochromatosis – there is
tissue damage)
HEMOSIDEROSIS
- Minimal to no tissue damage
- Iron only accumulates on the mature erythron
HEMOCHROMATOSIS
- With moderate to severe tissue damage
- Iron accumulates on the erythroblasts up to the
mature erythron and organs (bronze skin)
Pathogenesis:
- Increased Iron in the serum leads to multiple
organ damage
o Cardiovascular
o Hepatic – because iron is stored in the
liver
o Pancreatic – it interferes with the ducts
function, acute pancreatitis
o Nephrotic – kidneys are the ones that
filters
TREATMENT
- Treatment for secondary tissue damage, such
as liver cirrhosis and heart failure, follows
standard protocols
- For hemochromatosis, therapeutic phlebotomy
provides a simple, inexpensive, effective means
of removing Fe
- For hemosiderosis, deferoxamine (iron
chelating) is the drug of choice.
MEGALOBLASTIC ANEMIAS
ETIOLOGY:
- The root cause of megaloblastic anemia is
impaired DNA synthesis
- The anemia is named for the very large cells of
the bone marrow due to reduction of cell
divisions
NOTES: There’s an asynchronous maturation.
The cell size in megaloblastic hindi makapag
decrease kasi yung nuclear stage nya
immature pa dahil sa DNA synthesis.
MAJOR FACTORS:
Vitamin B12
- Also known as cobalamin
 - Physiologic role: ▪ Loss of epithelium along the GI
o Vitamin B12 is a coenzyme in two tract can result in gastritis, nausea,
biochemical reactions in humans or constipation
▪ Isomerization of methylmalonyl o CVD
coenzyme COA
▪ Transfer of a methyl group from 5- NOTES: manifestation in alimentary canal is
meth (5-methyl THF) to atrophic glossitis (absence of the filiform
homocysteine, which methionine papillae, nawawala yung papillae ng dila)

VITAMIN B9 CAUSES OF VITAMIN DEFICIENCY (FOLATE)

- Also known as folate or folic acid - INADEQUATE INTAKE

- Physiology: o Folate is numerous in green leafy
vegetables
- INCREASED NEED
o Increased need of folate occurs during
pregnancy and lactation; Infants and
children also need increased folate for
growth
- IMPAIRED ABSORPTION
o Sprue, celiac disease, deficiency in the
folate transport protein
- IMPAIRED USE OF FOLATE
o Numerous drugs impair folate
metabolism (i.e. Methotrexate, 5-FU,
Vitamin B12 and B9 – both play a role in conversion of Hydroxyurea, etc.)
the uridylyl (dUMP) to thymine (dTMP) - EXCESSIVE LOSS OF FOLATE
o Physiologic loss occurs in the kidneys
NOTES: since uridylyl is present in the RNA and (dialysis who have chronic kidney
thymine is present in the DNA, if you cannot convert disease)
uridylyl to thymine hindi ito mag poproceed sa DNA
maturation CAUSES OF VITAMIN DEFICIENCY (VITAMIN B12)

OTHER CAUSES OF MEGALOBLASTOSIS OR - INADEQUATE INTAKE

MACROCYTOSIS o Best dietary sources of B12 are animal
products
- Myelodysplastic Syndromes - INCREASED NEED
- Congenital Dyserythropoietic anemia o Increased need occurs during pregnancy,
- Acute Erythroid Leukemia (AML M6) lactation, and growth
- Antiretroviral drugs – Reverse transcriptase - IMPAIRED ABSORPTION
inhibitor o Lack of IF (Pernicious anemia), lack of
o Responsible for converting RNA to DNA haptocorrin, etc.
SYSTEMIC MANIFESTATIONS OF DEFICIENCY - FAILURE TO SEPARATE B12 FROM PROTEINS
o Failure to separate B12 from proteins
- When DNA synthesis and subsequent cell occur during increased pH or decreased
division are impaired by lack of folate or B12 acid secretion, hypochlorhydria (cannot
- Systemic symptoms include: produce acid and cannot do separation,
o Symptoms related to anemia (there’s produces slightly alkaline gastric juice is
palor) produced by the patient)
▪ Fatigue, weakness, and shortness - FAILURE TO SEPARATE B12 FROM
of breath HAPTOCORRIN
o Symptoms related to alimentary canal o Achlorhydria or lack of trypsin due to
▪ Loss of epithelium on the tongue chronic pancreatitis
results in smooth surface and - LACK OF INTRINSIC FACTOR (IF)
soreness
 o Pernicious anemia (autoimmune o Howell Jolly Bodies
diseasese targeting cells that produce o Hypersegmented Neutrophils
intrinsic factors like parietal cells), Loss of - Occasional NRBCs, basophilic stipples, and
parietal cells with H. pylori infection, total cabot rings may also be observed.
or partial gastrectomy, or hereditary IF
WBC Manual Differential
deficiency.
NOTE: B12 cannot be absorb without - Hypersegmentation of neutrophils is essentially
intrinsic factors pathognomonic for megaloblastic anemia
- OTHER CAUSES: - Not all cases of megaloblastic anemia will have
o Malabsorption hypersegmented neutrophils
o Imerslund-Grasbeck syndrome (decrease
endocytosis of IF-Cobalamin complex - NOTE: hypersegmented neutrophils is
rare) suggestive of megaloblastic
o D. latum infection
o Blind loops (stenotic intestines or loop Bilirubin and LDH
syndrome as a result of surgery or - Because many RBC precursors die during
inflammation) division in the bone marrow many RBCs never
enter the circulation.
NOTES: blind loops – hindi pa alam - The usual signs of hemolysis are evident,
kung nasaan including an elevation in the levels of Total Bili,
Pernicious Anemia B1, and LDH (LD6)

- Pernicious anemia is due to an autoantibody NOTES: since there is an increase turnover of

against the chief cells of the stomach or cells because of bursting due to incorrect
autoantibodies against IF. The chief cells maturation, ma-sesense ito ng katawan natin
produce HCI and IF in response to gastrin kaya sisirain nya ito
LAB DIAGNOSIS Bone Marrow Exam
- Screening test - The erythroid precursors show nuclear-
o CBC cytoplasmic asynchrony
o Reticulocyte Count o The nucleus doesn't match the
o WBC manual differential cytoplasmic picture of the cell (e.g.,
o Serum bilirubin nucleus is basophilic normoblast but RBC
o Serum LDH cytoplasm is polychromatic, doesn’t
- Specific diagnostic test match)
o Bone Marrow Examination - The WBCs also appear larger than normal
o Vitamin Assay - Megakaryocytes do not show consistent
o Gastric Fluid Analysis changes in megaloblastic anemia
o Antibody Assays
o Holotranscobalamin Assay Vitamin Assay
o Deoxyuridine Suppression Test - Assays for Vitamin B9, B12, Methylmalonic acid,
o Stool Analysis for Parasites and homocysteine should be included in the
CBC and Reticulocyte Count workup for megaloblastic anemia

- Patients with uncomplicated megaloblastic NOTE: tingnan kung ano yung kulang then
anemia are expected to have decreased Hgb yun ang palitan
and Hct, pancytopenia, and reticulocytopenia
- MCV is usually 100 to 150 fL and commonly >120
fL
- RDW is increased
- OM, HJ, HN: (classic findings)
o Oval macrocytes
ALGORITHM FOR VITAMIN ASSAY
Gastric Analysis and Serum Gastrin
- Gastric analysis may be used to confirm
achlorhydria
- As a result of gastric achlorhydria, serum gastrin
levels can be markedly elevated
NOTES: work up for pernicious because if you
have autoantibodies against your chief cells,
wala kang napoproduce na HCL resulting
achlorhydria and an increase serum gastrin
Holotranscobalamin Assay
- Holotranscobalamin is the metabolically active
form of vitamin B12
- Vitamin B12 assay is a more specific than
holotranscobalamin, therefore it is not adopted
to a routine clinical algorithm.
Deoxyuridine Suppression Test
- Principle:
o Preincubation of normal BM with
deoxyuridine will suppress the
subsequent incorporation of labeled
thymidine into DNA because the normal
cells can successfully methylate uridine
→ thymidine
o In patients with either a vitamin B12 or B9
deficiency, this suppression is abnormally
low.
Stool Analysis
- A stool analysis for the presence of eggs or
proglottids of Diphyllobothrium latum can be
supportive of the diagnosis of megaloblastic
anemia.
Macrocytic Nonmegaloblastic
- Macrocytic nonmegaloblastic anemias are
macrocytic anemias in which DNA synthesis is
unimpaired
- MCV usually ranges from 100 to 110 fL
- Lacks hypersegmentation
- Seen in:
o Liver disease
o Chronic alcoholism
o Bone marrow failure
- Macrocytosis due to reticulocytosis
TREATMENT
- Treatment should be directed at the specific
vitamin deficiency and should include
addressing the cause of deficiency
- Vitamin B12 IM in patients with pernicious
anemia to bypass the IF deficiency
BONE MARROW FAILURE SYNDROMES
Aplastic Anemia
- Aplastic anemia is a pancytopenia with bone
marrow hypocellularity
- Aplastic anemia can either be:
o Acquired/Idiopathic/latrogenic: Drugs
(chloramphenicol), chemotherapy,
radiation
o Congenital: Fanconi, Diamond Blackfan,
etc.
NOTES: aplastic – there’s no tissue
Idiopathic – doesn’t know the cause
Iatrogenic – cause by a medical
procedure
ETIOLOGY:
- Radiation
o Marrow aplasia is a major acute sequela
of radiation. Radiation damages DNA.
(Who? Patients with cancer)
- Chemicals
o Benzene (clearing agent) is a notorious
cause of bone marrow failure.
- Drugs
o Chloramphenicol
- Infection
o Hepatitis is the most common preceding
infection
o Aplastic anemia can rarely follow IM
(infectious mononucleosis – caused by
CMV)
o Parvovirus causes transient aplastic crisis
in hemolytic anemias in infants and other
Pure Red Cell Aplasias
- Immunologic diseases
 o Aplasia is a major consequence of TA-
GVHD (Transfusion Associated Graft
Versus Host Disease)
o Eosinophilic fasciitis
o Thymoma and
hypoimmunoglobulinemia.
o SLE (Systemic Lupus Erythematosus)
NOTE: anti-dsDNA is usually present
in SLE
- Pregnancy
o Aplastic anemia very rarely may occur and
recur during pregnancy and resolve with
delivery or with spontaneous or induced
abortion
- Paroxysmal Nocturnal Hemoglobinuria
o An acquired mutation in the PIG-A gene
resulting in a deficiency of CD55 (Decay
accelerating factor) and CD59
(Membrane Inhibitor of Reactive Lysis) in
RBCs, causing attachment of C3b to the
RBC resulting to activation of the
complement pathway, leading lysis.
▪ NOTES: Tuwing gabi kapag
natutulog ka, nagiging acidic yung
dugo mo, ang complements nag
aactivate sa acidic environment,
naglilyse yung RBC, umiihi ka ng
hemoglobin
o Treatment: Eculizumab (monoclonal anti-
C5)
CONSTITUTIONAL DISORDERS
Fanconi anemia
- Autosomal recessive disorder, manifests as
congenital developmental anomalies,
progressive pancytopenia, and an increased risk
of malignancy.
- Patients with Fanconi anemia typically have
short stature, café au lait spots (may mga pachi-
pache yung patient), and anomalies involving
the thumb, radius and genitourinary tract.
- The most common type. Type A Fanconi
anemia, is due to a mutation in the FANCA gene.
Dyskeratosis congenita
- Characterized by the triad of mucous
membrane leukoplasia, dystrophic nails,
reticular hyperpigmentation, and with the
development of aplastic anemia in childhood.
- Due to mutations in the genes of the telomere
repair complex
Shwachman-Diamond syndrome
- Presentation is early in life with neutropenia
with pancreatic insufficiency and
malabsorption that leads to marrow hypoplasia
- Most patients have compound heterozygous
mutations in SBDS that may affect both
ribosomal biogenesis and marrow stroma
function.
PATHOPHYSIOLOGY
- Bone marrow failure results from severe
damage to the hematopoietic cell
compartment
- In aplastic anemia, replacement of the bone
marrow by fat is apparent in the morphology of
the biopsy specimen and magnetic resonance
imaging (MRI) of the spine
- Cells bearing the CD34 antigen are greatly
diminished. and in functional studies,
committed and primitive progenitor cells are
virtually absent
DRUG INJURY
- Extrinsic damage the marrow follows massive
physical or chemical insults such as high doses
of radiation and toxic chemicals
IMMUNE-MEDIATED
- Increased numbers of activated CD8+ T cells are
observed in aplastic anemia patients and
usually decline with successful
immunosuppressive therapy
LABORATORY DIAGNOSIS
- Blood
o Smear shows large RBCs and a paucity
(deficiency) of platelets and granulocytes
o MCV is commonly increased, <120 fL
o Reticulocytes are absent or few
o Presence of immature myeloid
precursors may suggest AML or MDS
- Bone Marrow
o “Dry tap” – fibrous
o Biopsy is fatty and has low cellularity
TREATMENT
- Hematopoietic Stem Cell Transplant (patient
<50 years old)
- Immunosuppression (patient >50 years old)
- Blood Transfusion (if >50, deferoxamine must
be administered to avoid hemochromatosis)
Pure Red Cell Aplasia

- This is a kind of marrow failure syndrome that is

more restricted to a single circulating cell type,
this type is the RBC
- This is characterized by anemia,
reticulocytopenia, and absent or rare erythroid
precursor cells in the bone marrow
- In adults, PRCA is acquired. In children, PRCA is
congenital (Diamond-Blackfan anemia)

Persistent Parvovirus Infection LABORATORY STUDIES

- Chronic parvovirus infection is an important, - Blood

treatable cause of PRCA o Anemia is present in most cases, either
- Parvovirus causes a transient aplastic crisis and alone or as part of bi or pancytopenia
an abrupt but temporary worsening of the o Macrocytosis is common
anemia due to failed erythropoiesis o Neutrophils are hypogranulated
- Bone marrow shoes red cell aplasia and the - Bone Marrow
presence of giant pronormoblasts. o Bone marrow is usually normal or
hypercellular
TREATMENT: PRCA o No single characteristic feature of
- Red cell aplasia is compatible with long-term marrow morphology distinguishes MDS
survival with supportive care alone
Myelophthisic Anemia
o RBC transfusions and iron chelation
- For persistent parvovirus B19 infection, almost - Myelophthisis, or secondary myelofibrosis, is
all patients respond to IVIg (intravenous reactive.
immunoglobulin) o Fibrosis can be a response to invading
tumor cells (metastasis)
Myelodysplasia
- Anemia is dominant in secondary myelofibrosis,
- The myelodysplastic syndromes are usually normocytic and normochromic.
heterogeneous group of hematologic disorders - WBCs are often elevated, sometimes mimicking
broadly characterized by both: a leukemoid reaction, with myelocytes and
o Cytopenias (1) due to bone marrow myeloblasts
o A high risk of development of AML (2)
- Five entities were defined:
HEMOLYTIC ANEMIAS: INTRINSIC AND
o Refractory anemia (RA) EXTRINSIC CAUSES
o Refractory anemia with ringed
Basics Of Hemolytic Anemias
sideroblasts (RARS)
o Refractory anemia with excess blasts - It can be due to either:
(RAEB) o Extrinsic defects
o Refractory anemia with excess blasts in o Intrinsic defects
transformation (RAEB-t) - Peripheral blood may contain the following:
o Chronic myelomonocytic leukemia o Schistocytes
(CMML) o Spherocytes
o Inclusions
NOTE: myelodysplasia – bone marrow - S/Sx are usually appropriate for hemolytic
desynchrony tissue episodes, jaundice, splenomegaly, gallstones

Dysplasia – asynchrony of the tissue NOTES: What protein decreases in patients

with hemolytic anemias? – haptoglobin
MDS ending is Acute Myeloid Leukemia (sequesters free heme in plasma)
(AML) – pre-leukemic manifestation
Heme is nephrotoxic
INTRINSIC DEFECTS
- Hereditary Spherocytosis
o Due to a deficiency of either spectrin (a or
B), ankyrin, or band 3
o Predominant cells are spherocytes
o Diagnostic strategies:
▪ Family history
▪ MCHC >36 pg
▪ Peripheral Blood Smear
▪ Osmotic Fragility Test
▪ SDS-gel electrophoresis for RBC
membrane proteins
o Treatment: No causal treatment for HS
- Hereditary Elliptocytosis
o Due to a mutation in either the Spectrin
(a or B), or Band 4.1 gene
o Some mild or even asymptomatic cases
have nearly 100% elliptocytes, whereas in
several cases, all kinds of bizarre
poikilocytes can predominate
- Pyruvate Kinase deficiency
o The most common enzyme deficiency in
the Embden-Meyerhof Pathway.
o Newborns with PK def. present with
neonatal jaundice;
▪ Jaundice persists, accompanied
with a very high reticulocytosis
o Anemia is variable in severity.
- G6PD Deficiency
o The most common enzyme deficiency in
the Hexose Monophosphate Shunt.
o Manifests with abundant schistocytes,
bite cells (pre-splenectomy), Heinz
bodies (post-splenectomy)
NOTE: What is the most common enzyme
deficiency in the world? – G6PD deficiency
What is the most common non-G6PD? –
Pyruvate Kinase
EXTRINSIC DEFECTS
- Immunologic
o AIHA
o PCH
o CAD
o PNH
- Infectious
o Malaria
o Babesia
o HUS
- Mechanical – also known as march
hemoglobinuria
Autoimmune Hemolyric Anemia (AIHA)
- This happens once a red cell is coated by an
autoantibody
o IgM - intravascular hemolysis
(complement activation)
o IgG-extravascular hemolysis (spleen
culling)
- Diagnostic test for AIHA is Coombs’s test
Paroxysmal Cold Hemoglobinuria (PCH)
- Presence of autoanti-P so called Donath-
Landsteiner antibody
- Usually triggered by a viral infection
- Diagnostic: (+) for Donath-Landsteiner
antibody
Cold Agglutinin Disease (CAD)
- Chronic AIHA that usually affects the elderly
- The antibody is usually IgM; usually it has an
anti-I specificity
- Presence of agglutination in PBS may indicate
CAD
- Blood transfusion is not very effective
- Immunosuppressive drugs can reduce the titer,
but clinical efficacy is limited
- Management of CAD has changed due to the
advent of rituximab discovery (anti-CD20)
Paroxysmal Nocturnal Hemoglobinuria (PNH)
- This is an acquired chronic HA characterized by
persistent intravascular hemolysis subject to
recurrent exacerbations
- RBCs lack CD55 (DAF) and CD59
- TRIAD: Hemolysis, Pancytopenia, Distinct
tendency to venous thrombosis.
- Screening Test: Sucrose Hemolysis test, Sugar
water test, Ham's acidified serum test
- Diagnostic Test: Flow Cytometry (CD55, CD59)
- Treatment: Eculizumab (anti-C5)
Malaria
- Caused by Plasmodium spp., most dangerous
falciparum
- Characterized by paroxysms: Chills, fever,
sweating
- Gold standard diagnostic test:
o Thick and thin blood film
- Immunoassay test:
 o Detection of parasitic LDH (Plasmodium
spp.) and HRP-II (P. falciparum)
- Treatment: Primaquine, Chloroquine
NOTES: Found in Palawan and Mindoro, part
of donor deferral
Babesiosis
- Caused by Babesia microti
- Less severe than malaria
- Transmitted by Ixodes ticks
- Diagnostic test: thick and thin blood film
- Treatment: Azithromycin/Atovaquone
Hemolytic Uremic Syndrome (HUS)
- Caused by either E. coli O157:H7 (grows in
SMAC), or Shigella spp. (nonmotile)
- Characterized by hemolysis and renal failure
(azotemia)
- Usually occurs following bloody diarrhea
HEMOGLOBINOPATHIES AND
THALASSEMIA SYNDORMES
Hemoglobinopathy
- Basic structure of hemoglobins:
o Hb A - a2B2
o Hb A2-a2D2
o Hb F-a2y2
- Major hemoglobinopathies:
o Sickle Cell Syndromes
o Hb C disease
o Methemoglobinemia
Sickle Cell Syndromes
- Caused by a mutation in the B-globin gene that
changes the sixth amino acid from glutamic acid
to valine (a2B2 6Glu-Val)
- HbS polymerizes when deoxygenated to form a
gelatinous network of fibrous polymers
- Sickle Cell Trait - HbAS
- Sickle Cell Anemia – HbSS
Sickle Cell Formation
- RBCs sickle when exposed to hypoxic
environments, forming drepanocytes or sickle
cells.
- When RBCs sickle, Ca+2 rushes in to the cell and
K+ leaks out, causing pseudohyperkalemia
- Sickle cells cause plugs that cause
microinfarctions of organ systems, leading to
multiple organ failure.
Sickle Cell Crises
- Crises are defined as the patients experience
acute onset pain, fever, tachycardia, anxiety
- All organ systems can be affected by crises:
(hypoxic and schemic)
o Cardiothoracic: Acute chest syndrome
o Nervous: Chronic subacute central
nervous system damage.
o Skeletal: Aseptic necrosis
o Male Reproductive: Priapism
- Major cause of death: Opportunistic infection
due to vaso-occlusion
SICKLE CELL DIAGNOSIS
- CBC
- PBS
- Hemoglobin electrophoresis / mass
spectroscopy
TREATMENT
- Analgesia
- Hydroxyurea
o Mainstay therapy for patients with severe
symptoms
Hb C Disease
- Caused by a mutation in the ß-globin gene that
changes the sixth amino acid from glutamic acid
to lysine (a2B2 6Glu-Lys → glutamic acid to
lysine)
- Causes inclusions characterized as hexagonal or
"Washington Monument" crystals
- Hb C may interact with Hb S to form Hb SC
crystals.
Methemoglobinemia
- Methemoglobin is generated by oxidation of
the heme iron moieties to the ferric state
- The blood has a characteristic bluish-brown
muddy color
- Congenital methemoglobinemia arises from
globin mutations that stabilize iron in the ferric
state or from mutations that impair enzymes
that reduce MetHb to Hb
- Acquired methemoglobinemia is caused by
toxins
- Treatment: IV/PO methylene blue (1 mg/kg) or
ascorbic acid
Thalassemias
- The thalassemia syndromes are inherited
disorders of a- and B-globin biosynthesis
 - The reduced supply of globin diminishes o GOLD STANDARD FOR DIAGNOSIS:
production of hemoglobin tetramers, causing HEMOGLOBIN ELECTROPHORESIS
hypochromia and microcytosis. (AGAROSE GEL PH 8.6)
- Quantitative hemoglobin disorder
PREVENTION
B-Thalassemia Syndromes
- Genetic counseling for a and 13 thalassemia
- Heterozygotes, ß-thalassemia minor, anemia is carriers, homozygotes, and heterozygotes
minimal. - Antenatal diagnosis via PCR of fetal DNA to
- Homozygotes, B-thalassemia major, causes detect mutations in the globin genes.
accumulation of highly insoluble unpaired a
TREATMENT
chains
o Forming toxic inclusions in erythroblasts - Supportive therapy
o The shortening of RBC life span produces
a response by the kidney to release EPO
o Increased EPO, Increased erythropoiesis,
Extramedullary erythropoiesis
(hepatosplenomegaly)

a-Thalassemia Syndromes

- a Thalassemia Trait
o Asymptomatic, silent carrier state (-a/aa)
- HbH Disease (Heterozygous a-thal 1 and 2)
o Causes accumulation of unpaired y chains
(Hb Barts) in children, and 34 chains in
adults (HbH)
o (--/-a)
- Hydrops fetalis (Homozygous a-thal-1)
o Causes total absence of a-globin
synthesis
o Excess y globin forms tetramers called Hb
Barts, which has very high 02 affinity
▪ It delivers almost no O2 to
peripheral tissues leading to
asphyxia, edema, CHF, and death.

α - / ααα trait TRAIT HbA

α-/α- Mild/heterozygous HbA
α-/-- HbH HbH
--/-- Hb Hydrops fetalis Hb Barts

DIAGNOSIS OF THALASSEMIA SYNDROMES

- The diagnosis of B-thalassemia major is readily

made during childhood
o Hb electrophoresis shows inc. Hb A2,
HbF, or both. Little to no HbA is present
o B-thalassemia minor
▪ Hb electrophoresis shows inc/nor.
HbA2, HbF
o a-thalassemia trait
▪ HbA2 and HbF are normal, mild
hypochromia and microcytosis
BLOOD BANK o Elderly – expect ↓ Ab = ↓ reaction
potential
ABO BLOOD GROUP
ABO Antibodies
Blood Groups
IgM
• Major blood groups – usually use for blood
- “Naturally occurring form”
transfusion and organ transplantation
- COLD-reacting
o ABO (most important)
- BIND complement – activated by IgM & IgG
o Rh
- DO NOT CROSS placenta
• Minor blood groups
IgG and IgA
ABO Blood Group System
- “Immune forms” or secretory forms
• Discovered by Landsteiner, von Descatello and
- Results from foreign red cell stimulation by
Sturle
transfusion or pregnancy
o Drew blood from himself and 5
o Fetal Maternal Hemorrhage (FMH) –
colleagues
bleeding caused by labor
o Separated serum from the red cells
and mixed them up ABO Antigens
o Unknowingly performed the first ever
ward and reverse blood typing General characteristics

Landsteiner’s Experiment - Glycoproteins or glycolipids

Red Cells Sera from A Sera from B - Present in all organs of the body – blood
person (Anti- person (Anti- transfusion & organ transplantation
B) A) (histoblood group antigens)
O person (No NEG NEG - Phenotypic or physical expression affected by
Ag) several factors:
A (A Ag) NEG + o Age - Develop as early as 37th day of
B (B Ag) + NEG fetal life; Newborn red cells have only
AB (A and B + + 25 to 50% of the number of antigenic
Ag's) sites found on adult red cells
o Race – but some races do not
demonstrate ABO usually Bombay
Blood Typing:
blood group
• Forward – unknown Ag + known Ab o Genetic interaction – A1, A2
• Reverse – unknown Ab + known Ag o Disease states – acquired A, acquired B
- Inheritance
Landsteiner's Experiment o Alleles involved: A, B, O, H and Se
- Individuals have "naturally occurring" Blood Group Genes
antibodies in their serum directed against the A AA, AO (homozygous A,
missing antigens on the surface of their RBCs heterozygous A)
- Naturally occurring antibodies against the ABO B BB, BO (homozygous B,
blood group heterozygous B)
- Significance: once you’re exposed to wrong AB AB (heterozygous AB)
blood type there will be an immune reaction O OO (homozygous OO)
Dominant – AB blood groups
What We Know Today
Recessive – O blood groups
- ABO antibodies are not really "naturally
occurring". Their production is stimulated by - Formation
ubiquitous substances such as: o Precursor substance: paragloboside
o Bacteria ▪ Red cell surface
o Pollen • Type 2 (terminal
o Other substances galactose and N-
- Production of ABO antibodies starts at birth acetylglucosamine in
but titers are too low for detection until 3 to 6 beta 1 → 4 linkage) =
months of age. (postpartum) Glycolipid is formed.
- Antibody production peaks at 5 to 10 years but ▪ Body fluids and secretions.
decreases with age. • Type 1 (terminal
- Persons above 65 have low titers for detection. galactose and N-
acetylglucosamine in
 beta 1 → 3 linkage) = Glycosyl Transferases
Glycoprotein is Ge Enzyme Nucle Sugar Anti
formed. ne otide gen
(Sugar
NOTES: Donor
)
Red cell surface – glycolipid
H α-2-L- GDP- L-fucose H
Body fluids and secretions – glycoprotein fucosyltransferase Fuc
A α-3-N- UDP- N- A
o Addition of Sugars acetylgalactosami GaINA acetyl-
▪ Glycosyltransferases nyltransferase C D-
▪ Type of glycosyltransferase galactos
depends on inherited alleles amine
B α-3-D- UDP- D- B
GEN GLYCOSYLTRANSFERASE IMMUNODOMIN galactosyltransfera Gal galactos
E ANT SUGAR se e
H α-2-L-fucosyltransferase L-fucose O None None None O
A α-3-N- N-acetyl-D- NOTES: O is none because it only has H
acetylgalactosaminyltrans galactosamine
ferase Glycosyl Transferases
B α-3-D- D-galactose
• GDP-Fuc: guanosine-diphosphate L-fucose
galactosyltransferase
• UDP-GalNAC: uridine diphosphate-N-acetyl-D-
galactose
NOTES: • UDP-Gal: uridine diphosphate galactose

Sugar A – Soluble A, B and H Antigens (present in secretions)

Sugar H – Secretion

- You need L-fucose for your A or B blood group
sugars to bind.
o e.g., Patient (A+)
▪ in the RBC there should be a
present of L-fucose before it
binds to N-acetyl-D-
galactosamine
▪ if the case is you do not have
L-fucose, whether A or B yung
na-iinherit mo, you will
present as O blood group.
Why? Because there’s no
binding antigen and therefore
there’s no reaction to anti-
sera.
▪ You need L-fucose to bind A or
B
- Secretor status (Sese or SeSe): α-2-L-fucosyl
transferase expression in tissues related to
exocrine secretions
o Secretes A, B, H antigens in saliva,
urine, mucus secretions. How? It’s a
histoblood group. In all of our
secretory organs, there’s a present α-
2-L-fucosyl transferase. If you have a
presence of L-fucose in your
secretions/secretory organs, may mag
bibind dito na A or B Ag resulting in
secretions of either A, B, AB, O Ag in
saliva, milk, mucus secretions, etc.

- Non-seceretors (sese) – heterozygous

Fluids in which A, B and H substances can be

detected in secretors

- Saliva, urine, bile, amniotic fluid, tears,

digestive juices, milk and pathologic fluids
(pericardial, peritoneal, pleural)
 Differences Between Red Cell and Soluble A, B Aa, Ab, Ac and Ad Antigens
and Antigens
Red cell A, B Soluble A, B - Based on the four different forms of the H
and H antigens and H antigens antigen
Biomolecule Glycolipids Glycoproteins o All boils down to anti-H antisera. Is it
First sugar Glucose N-acetyl problematic in blood transfusion? NO.
galactosamine because A1 and A2 are usually the
Chain Type 2 Type 1 most often associated with weak
Gene FUT1 FUT2 reactions, delayed HTRS
- H antigen forms: H1, H2, H3 and H4
o H1 and H2 are unbranched straight
**bakit nagkakaroon ng subgroups? Dahil may chains
mahihinang reaction. o H3 and H4 are complex branched chains
ABO Subgroups o H1 and H2 can be converted to Aa and
Ab by both A1 and A2 enzymes
A Subgroups o H3 and H4 can only be converted to Ac
and Ad by A1 enzyme and very poorly
- Main subgroup (2 schemes)
by A2 enzyme
o A1 and A2
o So: A1: has Aa, Ab, Ac and Ad
o Aa, Ab, Ac and Ad
A2: has Aa, Ab, low Ac and no Ad
- Weak subgroups
o Occur in 1% of the population Reagents Used to Detect A Subgroups
o A3, Am, Ax, Ay, Aend and Ael
Anti-A (ASUL → BLUE)
A1 and A2
- Extracted from sera of type B persons
- First described by von Dungern in 1911 - Contains both Anti-A and Anti-A1
- Quantitative and qualitative differences:
o Concentration of α-3-N-acetyl- Adsorbed Anti-A
galactosaminyltransferase - Sera from type B persons are adsorbed with A 2
o Number of antigen sites on red cell cells
o Antibodies produced - Contains only Anti-A1
A1 o Tinatanggal mismo sa solution, ang
natitira Anti-A1
- A1 gene elicits production of high
concentrations of α-3-N-acetyl- Dolichos biflorus lectin/Anti-A1 lectin
galactosaminyltransferase - Seed extract that agglutinates A1 cells
o Resulting in ↑ concentration of - Similar to Anti-A1
GALNAC - Used to detect Anti-A1 patients
▪ e.g., Sa RBC mo marami kang
GALNAC A Subgroup Typing
- Production of 810,000 to 1,170,000 antigen
sites Anti-A (from B Adsorbed Anti-
sera) = BLUE A/Anti-A1
- Presence of A and A1 antigens
lectin =
- Production of Anti-H cold agglutinin
Dolichos
o Sa sobrang daming A Ag, hindi na biflorus
naeexpose sa sariling H Ag A1 + +
A2 A2 + NEG

- Less activity of α-3-N-acetyl-

galactosaminyltransferase than A1 Weak A Subgroups (page 131 – table 6-15)
- Production of 240,000 to 290,000 antigen sites
- Presence of only the A antigen
- Production of Anti-A1
o Merong presence ng H and A na
nakikita ng immune cells mo. Problem
is few A, kaya nagpoproduce ng Anti-
A1

A subgroup Ag Ab
A1 A, A1, *H Anti-H, anti-B
A2 A, H Anti-A1, anti-B
B Subgroups Hypogammaglobulinemia Weakened Anti-A and B
Agammaglobulinemia
Main subgroup Very young and old
populations
- α-3-D-galactosyl transferase:
Polysaccharides from E. Acquired B antigen
o Requires Mn2+ as cofactor
coli (Due to any
o Optimum pH 6.5 condition that increases
o Two types: pl’s 4.8 to 5.2 and 8.2 to permeability of
8.8 intestinal wall)
- Also based on the four different forms of the H Excessive amounts of Absence of antigens
antigen: BI, BII, BIII, and BIV (not usually used in blood group-specific during forward typing;
the lab) soluble substances (Due Patients appear to be
to gastric and blood type O
Weak subgroups (page 134 – table 6-16) pancreatic cancers –
because of increase
- B3, BM, BX, Bel
secretion)

NOTES:

For babies – only forward typing

Some E. coli produce enzyme responsible for

converting GALNAC to galactosamine. The
galactosamine cross-reacts with Anti-B

FORWARD REVERSE
AB Subgroups A – 4+ RA - O
B – 2+ RB – 4+
- Related to A subgroups D – 4+
- A1B and A2B ** THIS TABLE IS FOR ACQUIRED B ANTIGEN**

Bombay Phenotypes If there is an increase consumption in the secretion

therefore wala kang nacoconsume sa RBC antigens
- Lack A, B and H antigens (doesn’t have
anchoring body) Pancreatic cancers
- Production of anti-H that react at 37°C (IgG)
- ↑ secretory phases (pancreatic juice or gastric
- Two types:
o Classical Bombay (doesn’t produce H enzymes) = ↑ secretions of A, B, H Ag = ↓ RBC
Ag) carbohydrates = +/- absence of Ag
▪ hh FORWARD REVERSE
o Para-Bombay A–O RA – 2+
▪ Ah, Bh and ABh B–O RB – O
▪ Weak expression of A and B D – 4+
▪ Mutant H (FUT) gene results in **THIS TABLE IS FOR GASTRIC/PANCREATIC CANCERS**
low levels of H antigen
▪ Usually 1+, 2+

Anti-H QUESTIONS AND ANDSWERS PORTION:

- A1 and A1B Anti-H are cold agglutinins BTRH (for forward and reverse typing)
- Bombay Anti-H react strongly at 37°C
Specimen: EDTA/red top
- Reactivity: O > A2 > B > A2B > A1 > A1B
1. Centrifuge the blood to separate plasma and
NOTES:
RBCs.
O – has greatest H antigen and so on… 2. Prepare 6 tubes. Each labeled with A, B, D, RA,
RB, RBC.
Disease States and Conditions that Alter ABH 3. Get the RBC in the bottom to make 5% RBC
Antigens suspension, get 0.5mL RBC and dilute it with
Condition Alteration 4.5 mL NSS to have 5mL (estimate).
Leukemia Weakened A and B 4. Get 1 drop of RBC suspension and put it on
Conditions related to antigen expression forward typing (A, B, D) to know the Ag in the
stress hematopoiesis
patient’s blood.
(e.g., thalassemia)
5. Get patient’s plasma if EDTA and serum if red
Hodgkin's disease
top. Then put it on reverse typing (RA, RB).
 6. Get anti-sera and put it on their respective
tubes.

Anti-A for tube A

Anti-B for tube B
Anti-D for tube D
7. Get a prepared known cell. These known cells
are blood type to know what antigen are they.
Get known A and put it on Reverse A (RA) and Example given (problem to solve):
known B on Reverse B (RB). What you need to
know what’s on RA and RB? To know what Ab 6 months old, female:
patient has.
FORWARD REVERSE
8. The known A and B that you’ve get will be A–O RA – O
centrifuge to 3,500 rpm for 30 seconds. B – 4+ RB – O
Centrifuge the tube A, B, D, RA, RB. D – 2+
9. The IgG that are present in the anti-sera are Blood Type (BT): B pos
going to be potentiate (pagsasama-samahin).
If they are in only one suspension, hindi sila Reverse is negative because the patient is only 6
gaanong magkakalapit, hindi magiging malakas months old, new born baby and it still doesn’t produce
yung reaction. antibodies.

37 years old, male:

FORWARD REVERSE
A–O RA – 4+
B–O RB – 4+
D – 4+ RO – 4+
Blood Type (BT): Bombay

Because it has Reverse O (RO) which has L-fucose (H

After centrifugation dislodged the RBC button. If the
RBC button has reaction or none, naka dikit ito sa
basement ng tube. After that, grade their
agglutination reactions then dito na papasok yung 4+,
3+, etc.
Ag). Nagkaroon ng reaction sa Reverse H, sa Reverse H
na demonstrate na mayroong anti-H production si
patient which only happens in A2 and Bombay. Walang
forward na A2 so it’s not A2. So kahit ano yung ma-
inherit na A or B Ag hindi ito mag dedemonstrate sa
forward because walang anchoring (L-fucose).
NOTES:
- RO or anti-H is the same
- Why we do reverse typing?
o For confirmation if forward is right and
if possible of it having other Ab
present. For the safety of the patient.
- BTRH = A positive
o Rh Blood Group are seen in anti-D anti-
sera, Rh is composed of DCEce, D is the
major antigen of rhesus.

Blood Type: A pos

 Blood Antigen Antibody (Ab)
Group (Ag)
(BG)
A A, H anti-B, A1 = anti-H, A2 = anti-A1
B B, H anti-A
AB A, B, H none
O H anti-A, anti-B
Bombay none anti-A, anti-B, anti-H
**anti-H → weak

FORWARD REVERSE
A–O RA – 4+
B–O RB – 4+
D–O
Blood Type (BT): O neg

For blood transfusion:

- Patient’s identification
- BTRH (both forward reverse is done) MT
- Cross-matching (I, II) → IS, LISS, AHG (37°C)
- Pre-transfusion
- Transfusion RN, MD
- Post-transfusion
BLOOD BANK subscripts written as superscripts (Rh0,
Rh1, Rh2, RhZ, rh, rh', rh”, rhy).
Rh Blood Group - Rules for longhand:
o Rh0 refers to “D”
Rh Antigens o Single prime (') refers to “C or c”
o Double prime (“) refers to “E or e”
- Nonglycosylated transmembrane proteins o If “r” precedes “h” (rh' or rh”), we are
- Started out with just 5 antigens (D, C, c, E and referring to the uppercase “C and E”
e) o If “h” precedes “r” (hr' or hr”), we are
- 51 antigen specificities (refer to Table 6-5 of referring to the lowercase “c and e”
Harmening) - Example:
- Immunogenicity: D > c > E > C > e o Dce converts to Rh0hr'hr”
- Genetics still unclear - Rules for shorthand:
o “R” denotes presence of “D”, “r”
Rh Antibodies
denotes absence.
- Production due to sensitization o “C” denoted by subscript “1 (one)” or
- IgG (subclasses 1 to 4) by a single prime (')
o “E” denoted by subscript “2 (two)” or
- Do not usually bind complement
by a double prime (“)
- Extravascular hemolysis
o When both “C and E” are uppercase,
- IgG1 and IgG3 subclasses are of greatest clinical
the subscript “z” or superscript “y” is
significance
used.
- React optimally at 37°C or after antiglobulin o When both “c and e” are lowercase,
testing the subscript “0” or no superscript is
- Can cross placenta used.
- IgA and transient IgM forms o Subscripts are used with uppercase
“R”, while superscripts and primes
Nomenclature
with lowercase “r”.
- 4 schemes: Fisher-Race, Wiener, Rosenfield - Example:
and ISBT o R1 converts to DCe

Fisher-Race (DCE) SUMMARY OF WIENER TERMINOLOGY

Antigen Longhand Shorthand Fisher-
- Theory: Race
o Antigens produced by three closely Rh0 Rh0hr'hr” R0 Dce
linked alleles (D, C/c and E/e) Rh1 Rh0rh'hr” R1 DCe
o One antigen per allele Rh2 Rh0hr'rh” R2 DcE
o Codominant expression RhZ Rh0 rh'rh” RZ DCE
- Antigen designations: D, d, C, c, E and e rh hr'hr” r dce
- Rules: rh' rh'hr” r' dCe
o Antigens have same designations as rh” hr'rh” r” dcE
genes. Genes are italicized. rhy rh'rh” ry dCE
o “d” refers to the absence of D antigen.
o “-” represents other missing antigens Rosenfield (Alpha/Numeric)
o Haplotypes are inherited from each
parent - Theory: No genetic basis. Simply states
- Example: presence or absence of Rh antigens.
o Genotype: DCe/dce - Rules:
o Phenotype (antigens produced): D, C, o Minus sign (-) before the number
c and e designates absence.
o If an antigen hasn't been tested for, its
Wiener (Rh-Hr) number will not appear.
o Designations:
- Theory: three antigens produced by single
 D: Rh1
gene (Rh0)
 C: Rh2
- Antigens: Rh0, Rh1, Rh2, Rhz, rh, rh', rh”, rhy
 E: Rh3
- Longhand and shorthand designations
 c: Rh4
- Rules for writing genes:  e: Rh5
o Antigens have same designations as o Examples:
genes. Genes are italicized, with  D +, C + E +, c -, e - converts
to Rh: 1, 2, 3, - 4, - 5
 - Drawback: Other blood groups such as: Kell, - Limited amount of precursor 1 converted to
Duffy, Kidd, Lutheran, Sciana, etc. have precursor 2
similar nomenclature scheme. - Weak expression of CDE or LW antigens
- Individuals may produce antibodies to Rh
D. ISBT
antigens they lack
- Six digit numbers
Clinical Considerations
- 004 was assigned to Rh blood group system
(e.g., Rh1 → “D” → 004001) - Transfusion reactions
- Similar to Rosenfield, only difference: o Circulating Rh antibodies appear
o RH instead of Rh within 120 days of primary exposure
o Takes genetics into account. and 2 to 7 days after secondary
Characters are italicized, followed by exposure.
a space or an asterisk, and then o Unexplained fever, mild bilirubin
numbers of the antigens are separated elevation and decreased hemoglobin
by commas. and haptoglobin
- Example: Rh1 converts to DCe and RH 1,2,5 or o Positive DAT
RH*1,2,5 - HDN
o RhoGAM (IM injection)
Proposed Genetic Pathways
Hemolytic Disease of the Newborn (HDN)
- Fisher and Race theory
- Wiener theory - Destruction of fetal red blood cells by
- Currently accepted model maternal antibodies
o Two linked genes on chromosome 1 - Mother is stimulated to produce antibodies by
control Rh expression previous pregnancy or during 2nd or 3rd
 Codes for presence or absence trimester of pregnancy
of “D” - Two common types:
 Codes for Ce, cE, ce and CE o Rh HDN
o Codominant expression o ABO HDN
- Cause: Usually antibodies (IgG) to the Rh(D)
Mechanisms of Antigen Expression
antigen
- Couples usually affected:
o Rh negative female
o Rh positive male
- Second child is usually affected
- Prevention: Rh immune globulin (RhIg or
Rhogam)
Rh Subgroups Factors Affecting Immunization and Severity
- Rhnull - Antigenic exposure
- Rhmod - Host factors
Rhnull - Immunoglobulin class
- Antibody specificity
- Regulator/ Inhibitor - ABO group influence
o Genes: homozygous X0r/X0r
o Precursor 1 not converted to precursor Antigenic exposure
2
o No CDE or LW antigens - Fetomaternal hemmorhage
o Individuals may produce antibodies to o Pregnancy (Transplacental)
any of the Rh antigens o Delivery
- Amorphic - As little as 1ml of fetal RBC can already
o Genes: rr immunize mother
o Precursor 1 converted to precursor 2 Conditions that Promotes Fetomaternal
o Unaltered precursor 2 Hemmorhage
o Individuals may produce antibodies to
any of the Rh antigens - Amniocentesis
- Chorionic villus sampling
Rhmod
- Abortion
- Genes: homozygous XQr/XQr - Ectopic pregnancy
- Abdominal trauma
 - Accidental or inadvertent transfusion Pathology: Bilirubin Elevation
- Greater than 40 weeks gestation
- Newborn liver unable to conjugate bilirubin
Host factors - This results in elevated indirect
(unconjugated) bilirubin
- Depends on complex genetic factors - Kernicterus – permanent brain damage
Immunoglobulin Class Diagnosis: Serologic and Clinical Tests
- IgG starts crossing the placenta during the - Serologic tests
second trimester o ABO and Rh typing
- IgG1 and IgG3 are more efficient in RBC o Antibody screen: 37ºC and potentiator
hemolysis (PEG or LISS)
- IgG half-life: 25 days o Treatment with dithiotreitol or 2-
mercaptoethanol destroys J-chain of
Antibodies that causes HDN
IgM; and differentiates IgG from IgM
Common: - Clinical tests
- Amniocentesis and cordocentesis
- Anti-D o Amniotic fluid is read
- Anti-D + C spectrophotometrically at 450nm
- Anti-D + E o Readings are plotted in a Liley graph
- Anti-C o Bilirubin causes a change in optical
- Anti-E density
- Anti-c
New Diagnostic Techniques:
- Anti-e
- Anti-K - Ultrasonography
- Doppler assessment of middle cerebral artery
Rare:
peak velocity
- Anti-Fya - Percutaneous umbilical blood sampling
- Anti-s - Allele-specific gene amplification studies on
- Anti-M fetal cells
- Anti-N - Intrauterine transfusion
- Anti-S Treatment
- Anti-JKa
- Intrauterine transfusion:
Antibody Specificity o Amniotic fluid change in OD450 results
- Anti-D is most potent are in high zone II or III
o Cordocentesis blood sample has
- Other Rh antibodies: Anti-E and anti-c causes
hemoglobin less than 10 g/dL
severe HDN
o Fetal hydrops is noted on ultrasound
- Other blood group antibodies: Anti-Kell is most
examination
potent
- Early delivery
ABO Group Influence - Phototherapy
- Newborn transfusions
- ABO incompatibility between mother and fetus
prevents mother from being sensitized. Newborn Transfusion:

Pathology - Purpose:
o Removal of bilirubin
- Erythroblastosis fetalis and hydrops fetalis o Removal of sensitized RBCs and
- Bilirubin elevation incompatible antibody
Pathology: Erythroblastosis Fetalis o Suppression of erythropoiesis
- Blood must be:
- Red cell destruction →Overworked bone o CMV-negative
marrow → Spleen and liver produces red cells o Hematocrit 70%
→ Hepatosplenomegaly → Hypoproteinemia
→ Hydrops fetalis (Cardiac failure with
edema)
Prevention: Rh Immune Globin

- Dose: Volume of fetomaternal hemorrhage

divided by 30
- Fetomaternal hemorrhage volume:

Number of fetal cells X Maternal blood volume

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑚𝑎𝑡𝑒𝑟𝑛𝑎𝑙 𝑐𝑒𝑙𝑙𝑠

ABO vs. Rh HDN

Characteristics ABO Rh
First pregnancy YES RARE
Disease NO YES
predicted by
titers
IgG YES YES
Bilirubin at NORMAL ELEVATED
birth
Anemia at birth NO YES
Phototherapy YES YES
Exchange RARE SOMETIMES
transfusion
Intrauterine NONE SOMETIMES
transfusion
Spherocytosis YES RARE
MT-IMMUNO 1 (Immunology and Serology) LEC Self- Very good; no Very good;
non/self microbe-specific occasional failures
Innate Immunity discriminati self/non-self of discrimination
on patterns in host result in
Immunity autoimmune
 Vertebrates are protected by two systems of disease
immunity. Soluble Many antimicrobial Antibodies and
- Innate immunity components properties, cytokines
 First and second line of defense of blood proteins, and other
- Adaptive immunity mediators,
 Takes time but has memory including cytokines
 Third line of defense Major cell Phagocytes T cells, B cells,
 Innate immunity can be found in all multicellular types (monocytes, antigen-presenting
plants and animals macrophages, cells
- Response remain the same even after neutrophils,
encountering the same microorganism/foreign dendritic cells), NK
bodies cells, other
 Adaptive immunity evolved in jawed vertebrates leukocytes,
- Improves over time and acquires this response epithelial and
and throughout its lifetime endothelial cells
 Humans have both innate and adaptive immunity
Innate Immunity
Innate and Adaptive Immunity  Innate immune system
Attribute Innate immunity Adaptive - Physical/Anatomical barriers
(Non- immunity  Skin and mucous membranes – impermeable
specific/Natural) (Specific/Acquired as long as intact, contains appendages such as
) the hair which trap and prevent pathogens
Response Minutes/hours Days (because it from entering the system
time involves activation - Mechanical barriers
of B cells and T  Peristalsis, tearing of eyes, urinating and
helper cells); sweating, coughing and sneezing
antigen has to - Chemical barriers
encounter the B/T  Acidity of stomach, antimicrobial molecules
cell in secreted by sweat and oil glands
corresponding - Cellular barriers (Second line of defense)
receptors  Cells with sensitive receptors that can detect
Specificity For molecules and Highly-specific; microbial invaders
molecular patterns discriminates  Include normal indigenous microflora that
associated with between even minor compete with pathogens for nutrients and
pathogens; foreign differences in space
bodies are molecular structure  Antimicrobial proteins – inhibitory peptides
discovered through of microbial or provided by the body’s microflora (chemical barriers)
pattern recognition nonmicrobial - Psoriasin – produced by the skin (only works for
receptors molecules; gram-negative becteria)
discovered through  Antimicrobial activity to E. coli – help when
antigen-antibody skin is scratched or cut to prevent infection
interaction since we are exposed to E. coli in fecal
Genetic A limited number Highly diverse; a material
diversity of conserved, germ- very large number - Saliva, tears, and mucous membranes (contain IgA
line encoded of receptors arising and lysozyme) help to wash invaders away as well
receptors from genetic as contain antimicrobial peptides
recombination of  Normal flora – help to out-compete pathogens for
receptor genes in space and nutrients
each individual  Innate responses can be found in:
Memory Some (observed in Persistent memory, - Skin
responses invertebrates with faster response - Mouth
innate responses of greater - Airway
and mouse/human magnitude on - Lungs
NK cells) subsequent - Epithelial linings of the airway and lungs
exposure - Epithelial lining of alimentary canal

dane.
 - Stomach o C4b will bind/form a complex with C2a,
- Large and small intestine producing C4b2a (C3 convertase of the
- Rectum classical pathway) which will split C3,
- Urogenital tract which will form C3a and C3b.
- Salivary, lacrimal, and mammary glands o C3b will bind to C4b2a to form C4b2a3b
(C5 convertase), which will activate C5,
Organ or Tissue Innate mechanisms which will split into C5a and C5b.
protecting o C5b binds to C6, C7, C8, and C9.
skin/epithelium Altogether, they form the membrane
Skin Antimicrobial peptides, attack complex (C5b789).
fatty acids in sebum  Alternative
Mouth and upper Enzymes, antimicrobial o C3 is a relatively fragile protein. It
alimentary canal peptides, sweeping of activates on its own, splitting into C3a and
surface by directional flow C3b.
of fluid towards the o Upon stabilization by Factor D, Factor B,
stomach and properdin, C3b becomes C3
Stomach Low pH, digestive enzymes, convertase. C3b activates a protein,
antimicrobial peptides, fluid Factor B, into Bb and Ba. The Bb
flow toward intestine component binds C3b so it can activate
Small intestine Digestive enzymes, more C3.
antimicrobial peptides, fluid o If C3b binds with C3 convertase (C3bBb),
flow toward intestine it becomes C5 convertase (C3bBbBb)
Large intestine Normal intestinal flora which activated C5.
compete with invading o C5 splits into C5a and C5b, and C5b binds
microbes, fluid/feces to C6, C7, C8, and C9. Altogether, they
expelled from rectum form the membrane attack complex
Airway and lungs Cilia sweep mucus outward, (C5b789).
coughing, sneezing expel  MB-lectin pathway
mucus, macrophages in o Same with classical pathway with one
alveoli of lungs exception which is the initiating factor.
 Pattern Recognition Receptors (PRRs) Instead of the antigen-antibody, this has
- In contrast, antibodies and T cell receptors the mannose-binding protein and the
recognize finer detail of molecular structure mannose on the surface of the bacteria.
- PRRs recognize Pathogen-Associated Molecular o Lectin is a protein that can bind
Patterns (PAMPs) that are found on a variety of carbohydrates. In this case, it is a
microbes, instead of antigens (which is for mannose-binding protein.
Adaptive response) o Mannose acts as a substitute for the
antigen, while mannose-binding protein
 Complement System
acts as substitute for the antibody.
- One part is a collection of proteins that form
 Complement proteins are labeled C1 to C9.
aggregates that punch holes in pathogen’s cell
Activation is achieved when the protein is
membrane causing lysis
cleaved into two fragments, a larger
- Include serum glycoproteins that promote uptake
fragment (B) and smaller fragment (A),
of pathogens by phagocytes (opsonization –
applicable for all proteins except C2.
enhancement of phagocytosis)
 The larger fragments (B) are responsible for
 Complement system components coat cells to
punching holes in the in the bacterial cell
be recognized by phagocytes
walls/membranes, except for C2, where the
 Chemotaxis – signal for directed migration of
larger fragment is designated A. On the other
WBCs
hand, the smaller fragments (A) are
 Anaphylatoxin – mediate or trigger
responsible for mediating the immune effects,
inflammation
(chemotaxis, anaphylatoxin etc.), except for
- Complement system ties innate and adaptive
C3b which acts as opsonin.
immunity
 C3a, C4a, and C5a are anaphylatoxins.
- Complement activation
 C3b is an opsonin.
 Classical
 C3a and C5a are chemoattractants.
o Upon binding of antigen-antibody
complex (immune complex), activation of  Innate initiation of adaptive response
C1 will follow. C1 will split into C1a and - When pathogens infect cells, intracellular bacteria,
C1b. Simultaneously; C1a will then parasites, and virus-infected cells produce
activate C4 into C4b and C4a, and C2 to cytokines and other microbials that kill
C2a and C2b. intracellular pathogens. They can already die at
this point. However, if this is still not enough,
dane.
 virus-infected cells can be killed by NK cells, if it
(virus-infected cell) is coated with antibodies.
- When an infected by intracellular pathogens, a cell
can secrete cytokines and chemokines which can
initiate inflammatory response.
- For extracellular pathogens, it can result in
activation of complement proteins, which will
cause it to lyse (antimicrobial proteins and
peptides, MBL, CRP, complement proteins activate
complement pathways which induce lysis) or
produce opsonins. Once opsonized, this can be
phagocytosed by macrophages and neutrophils.
- If phagocytosed, phagocytes can produce
cytokines and chemokines can activate T cells, or
at the same time, present the phagocytosed
antigens to T-helper cells, which will then trigger
an adaptive immune response
Inflammation
 Its goal is to arrest the pathogen or to limit its spread
to allow for more focused innate immune response.
 Upon arresting a pathogen, all WBCs will go to that
location to facilitate tissue repair.
 Hallmarks
- Swelling (tumor)
- Redness (rubor)
- Heat (calor)
- Pain (dolor)
- Loss of function (functio laessa)
 Within minutes of tissue injury:
- Vasodilation – blood vessels dilate, resulting to
rise of blood volume to area, which explains why
there is redness and heat.
 Tissue damage causes a release of vasoactive
and chemotactic factors that trigger a local
increase in blood flow and capillary
permeability
- Vascular permeability increases – accumulation
of fluid (plasma in the blood leaks from the blood
vessel to the extravascular space/tissues, causing
the swelling)
 Edema
 Permeable capillaries allow an influx of fluid
(exudates) and cells
- Leukocytes adhere to endothelial cells and
pass through walls of capillaries and tissues –
extravasation
 Diapedesis/Extravasation – squeezing of
WBCs through the gaps of endothelial cells
 Phagocytes migrate to the site of
inflammation (chemotaxis)
 Phagocytes and antibacterial exudates
destroy bacteria
 Othereffects
- Chronic inflammation – persisting inflammation
 Cancer
 Necrosis
- Fibrosis – replacing of healthy tissue with scar
tissue
 Extravasation
- Inflammatory response develops – various
cytokines and inflammatory mediators act on
endothelium of blood vessels
 Increased expression of Cell Adhesion
Molecules (CAMs)
 Cells, such as neutrophils, adhere to
endothelium using these CAMs strongly
enough not to be swept away by flowing
blood
 Then they must penetrate the wall of the
vessel to move into the tissue
 Neutrophil Extravasation
- Rolling
 Selectin-mucin interactions mediate rolling
- Activation of chemoattractant stimulus
 Chemokines/chemoattractants induce change
in integrins
- Arrest and adhesion
 Integrins adhere firmly to ICAMs
- Transendothelial migration to the tissue
(diapedesis)
Antimicrobial Peptides
 Isolated from humans, frogs, flies, nematodes, plants
 Range from 6-59 amino acids long
 Good source in humans is the neutrophil
 Work by disrupting microbial membrane
- How do they discriminate between microbial and
host membrane?
- Big area of research
C Reactive Protein
 Inflammation-associated protein
 Recognizes ligands on surface of microbes
- Produced by liver in response to inflammation
 Elevated levels are indicative of disease
- Opsonin
 Helps in phagocytosis
- Activates complement-mediated attack
Type I Interferons
 When interferons (kind of cytokine that has particular
effect on viruses) bind to the receptors in immune
cells, it induces the cell to produce anti-viral proteins
that
- Blocks viral protein synthesis
- Degrades viral DNA
- Blocks assembly of viral peptides.
Pattern Recognition Receptors – Toll like Receptors
 1980s
- Toll in flies
 Important in fly development
 1996
- Toll in fruit flies
 Mutation caused susceptibility to infection of
fungus
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 1997 (Janeway) - Opsonization
- Found out that Toll-like receptor activated  Fc receptors – antibody binds antigen with
expression of immune human genes Fab portion, Fc portion is free to bind with Fc
 Made of leucine-rich repeat sequences receptors on immune cells for ADCC,
phagocytosis, etc
Cell Types of Innate Immunity  Enhancement of phagocytosis
 Neutrophils  Phagocytes have receptors for Fc portion of
- Phagocytosis anitbody
- Reactive oxygen and nitrogen species - Complement fixation
- Antimicrobial peptides  Activation of complement by antigen-
 Macrophages antibody complex
- Phagocytosis - Antibody-dependent cell-mediated
- Inflammatory mediator cytotoxicity (ADCC)
- Antigen presentations  Cells that have cytotoxic potential (NK cells,
- Reactive oxygen and nitrogen species macrophages, etc) express receptors for Fc
- Cytokines portion of antibody
- Complement proteins  Antibodies bind antigens on the surface of
 Dendritic cells target cells
- Antigen presentation  NK cell CD16 Fc receptors recognize cell-
- Costimulatory signals bound antibodies
- Reactive oxygen species  Cross-linking of CD16 triggers degranulation
- Interferon into a lytic synapse
- Cytokines  Tumor cells die by apoptosis
 Natural killer cells (T-cytotoxic cells) - Degranulation
- Lysis of viral-infected cells  As seen in basophils during type 1
- Interferon hypersensitivity, occurs when antibodies
- Macrophage activation bound to WBCs via their Fc receptors, bind to
the same antigen
Signal Transduction Pathways  This cross-linkage will result in the same WBC
 Signal – microbial product releasing it granular contents (mostly
 Receptor – extracellular portion of TLR anaphylatoxins)
 Signal transductor – interactions of intracellular  Antibody isotypes mediate different effector
molecules—phosphorylation; signal transduction functions
pathway—promotes phosporylation of transcription - IgM
factors in nucleus  Fixing complement
 Effector mechanism – production of proteins, cell  Very good at grabbing and agglutinating
differentiation, inflammation, antigen-presentation, pathogens
etc. - IgG
 Fix complement, although not as efficiently as
Adaptive Immunity IgM
 Mediate ADCC
Components  Opsonin
- IgA
 Humoral – mediated by antibodies
 Neutralize toxins and pathogens (first line of
 Cell-mediated – mediated by T-cytotoxic cells
defense)
 In mucous membranes and secretions
Antibody-Mediated Effector Functions
- IgE
 Antibodies don’t directly kill pathogens by themselves.
 Inflammation, parasite
They are able to do so because of the following
 Interactions with basophils and anaphylatoxin
- Neutralization
histamine
 Occurs when antibodies coat a virus or other
antigen, preventing it from binding other cells
Cell Mediated Immunity
or tissue
 Important in detecting and eliminating cells that
- Agglutination
harbor intracellular pathogens
 Involves antibody binding antigen
 Antigen-specific
 Although in such cases, the antibody-antigen
- CD8+ and CD4+ T cells
complex is still bound to the cells, cross-link
with one another, causing cells or pathogens  Effector responses
to aggregate or agglutinate - Importance is evident when system is defective
 DiGeorge syndrome
o Born without thymus, lack T cell
requirement
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 o Can fight extracellular pathogens but have
issues with intracellular pathogens (virus,
intracellular bacteria)
- Effector T cells express variety of effector
molecules that are crucial in effectiveness of
effector response
 Cytotoxic T cells
o CTLs or Tc cells, CD8+
o Class I MHC restricted
- Effector phase of CTLs
 CTL-mediated death
o Initiated by conjugate formation of CTL
with infected cell
o Membrane attack, CTL dissociation
o Target cell destruction either through
perforin (cell membrane disruption and
subsequent lysis) or Fas-FasL (binding of
Fas receptor to Fas ligand) pathway
(induces apoptosis in target cell)
 NK cells
- Defend against viruses, other intracellular
pathogens, and tumors
- Produce important cytokines
- Lymphoid cells
 Share early lineage to T cells
 NK cells don’t develop exclusively in thymus
- Killing of target cell is similar to that of CTL
- Follow the opposing signals model
 Many different receptors for activation and
different inhibitory ones
- NK receptors
 Lectin-like – actually bind proteins instead of
carbohydrates
 Immunoglobulin-like
- Balance between activation and inhibitory signals
allows NK to distinguish between self and non-
self, very complicated
- Activation and inhibition of NK cell is achieved
depending which ligand
 NKT cells
- Cells that have characteristics common to CTLs
and NK cells
- Considered as part of the innate immune system
- Role remains to be completely defined
Adaptive Immunity
 Adaptive immunity tends to be found in vertebrates
 However, do find innate immunity in non-vertebrates,
even plants
- Sea squirt (chordate) – complement, TLRs
- Fruit fly – TLRs, antimicrobial proteins
- Tomato – oxidative bursts, enzymes that digest
fungi, plant can isolate isolation by strengthening
cell wall
Vaccination
Immunization/Vaccination
 Immunity can be achieved by active or passive
immunization
- Active – long term production, immunologic
memory, actual exposure
 Coming into contact with the foreign
substances
 Vaccines
- Passive – transfer of preformed antibodies
 Maternal antibodies to fetus (IgG to the fetus;
IgA colostrum)
 Antibody therapy for bites, immunodeficiency
 Temporary; antibodies will only last so long,
but it can help someone recover from a
current infection
 To achieve lifetime immunity, it must be
through active immunization
 There is a chance of side effects in a small number of
population
- In the case with any treatment/drug
- However, if the benefits to the population
outweigh the risk of side effects, vaccines must be
used to protect the majority of the population
- Herd immunity is important
 There might be some people who can’t be
vaccinated sue to cancer, allergies, etc
 Just because you got the vaccine, it doesn’t
mean it worked on you
 Designing effective vaccine
- Protective immunity must be achieved
 Must pay attention to how the antigen
activates the humoral and cell-mediated
branches
- Must produce immunologic memory
 Vaccine that produces primary response but
fails to produce secondary response is not
effective
Classification of Common Vaccines for Humans
 Live, Attenuated Vaccines
- Microorganisms can be attenuated so that they
lose ability to cause significant disease
- Retain capacity for growth in host
- Bacteria are grown for prolonged period in
adverse conditions; those that survive will not be
suited to grow in better conditions in host
- A virus might be grown in cell type that is not
normal host; accumulates mutations that might
weaken it
- Measles, mumps, rubella vaccines
- Advantages
 Can grow in host therefore producing
immunologic memory with only a single
vaccination
 Produces memory T cells
 Good for distribution in third world countries
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 - Disadvantages
 Possibility that it will revert to virulent form
o Polio – 1 in 2.4 million chance that this
will happen
 Complications
o Measles vaccine – encephalitis
o Out of 75 million patients between 1970
and 1993, only 48 cases
 Danger from remaining unvaccinated and
getting disease is much greater than
complications to these proven vaccines
 Inactivated or “Killed” Vaccines
- Inactivation of pathogen by heat or chemical
means
 Not capable of replication in host
 Epitopes have to be maintained after killing
process
- Often require boosters
- Risks
 Pathogen has to be grown in large numbers
prior to inactivation – individuals involved in
manufacturing are at risk
 Some of the pathogen may not be killed
- Pertussis, typhoid, flu vaccine
 Subunit Vaccines
- Purified macromolecules derived from pathogens
- Toxoids
 Some bacteria are pathogenic because of
exotoxins they produce
 Purify exotoxin, inactivate it with
formaldehyde to form toxoid that can be used
to immunize
 The immune system is then able to neutralize
the toxin when an infection occurs
- Conjugate
 When subunit vaccines are poor immune
stimulators, they can be coupled with strong
immune activators
 Polysaccharide on H. influenzae (not
immunogenic) with tetanus toxoid which is
highly immunogenic
o Results in immunity to Haemophilus
- Recombinant vector vaccines
 Still in clinical testing
 Viruses and other microbes that replicate in
cells but don’t cause disease can be used to
deliver fragments of a pathogenic microbe for
vaccination
- DNA vaccines
 Tend to insert microbes’ DNA into host cells,
allowing host cells to manufacture microbial
proteins and antigens that will serve as the
vaccine
 Adjuvants – enhances immune system
response and time with the immunogen
o Virosome – reconstituted virus envelope
without any genetic information (used in
influenza and hep A vaccine)
o AS04 – alum salt containing LPS (highly
recognized by TLR4)
o Freund’s incomplete adjuvant – used in
research
Preservatives in Vaccine Vials
 Thimerosal – preservative in vaccine vials (ethyl
mercury not methyl mercury which is the bad kind
that builds up in fishes and is bad for humans)
Hypersensitivity Reactions and Allergies
Hypersensitivity Reactions
 Hypersensitivity – responding inappropriately to an
antigen
 Inflammatory response can have deleterious effects
- Tissue injury
- Disease
- Death
 May develop in course of humoral or cell-mediated
response
- Immediate hypersensitivity
 Anaphylactic
 Antigen-antibody complexes
 Manifests in minutes
- Delayed type hypersensitivity
 May occur in days
 4 types of hypersensitive reaction
- Types I-III – humoral
- Type IV – cell-mediated
Type I (IgE–Mediated) Hypersensitivity
 Antigen induces cross-linking of IgE bound to mast
cells and basophils with release of vasoactive
mediators
 Typical manifestations
- System anaphylaxis
- Localized anaphylaxis such as hay fever, asthma,
hives, big bites food allergies, and eczema
 Induced by antigens referred to as allergens
 Induces humoral responses but induces high secretion
of IgE
- Fc portion of IgE binds with Fc receptors on mast
cells and basophils
- Degranulation occurs
 Common components
- Allergens
 Atopy – hereditary predeposition to
development of immediate hypersensitivity
reactions to common antigens
o Allows non-parasitic antigens to easily
induce IgE response
- IgE
 Normally lowest of all antibody classes in
serum
 Half-life is 2-3 days but once bound to mast
cells or basophils, can last for weeks
- Mast cells and basophils
- IgE binding receptors
 High affinity
 Low affinity
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 o Atopic individuals have higher amount of  Categories of Type I Hypersensitivity
soluble IgE receptor that has been shown - Systemic anaphylaxis
to increase IgE production by B cells  Quick, can be fatal
 Common allergens associated with type I  Respiration labored, blood pressure drops,
hypersensitivity bronchiole constriction, edema, shock
Plant pollens Drugs  Epinephrine treats, relaxes smooth muscle
Rye grass Penicillin and increases cardiac output (prevents
Ragweed Sulfonamides vascular collapse)
Timothy grass Local anaesthetics - Localized hypersensitivity reactions
Birch tree Salicylates  Limited to tissue or organ
Foods Insect products  Types
Nuts Bee venom o Allergic rhinitis – “hay fever”
Seafood Wasp venom o Asthma
Eggs Ant venom o Atopic dermatitis – eczema
Peas, beans Cockroach calyx o Atopic urticaria - hives
- Food allergies – can cause local reactions or
Milk Dust mites
anaphylaxis
Other allergens
 Asthma
Animal hair and dander
- Inflammatory disease
Latex
- Induce expression of adhesion molecules on
Mold spores endothelial cells for eosinophils and neutrophils
 IgE cross-linkage initiates degranulation  Cause significant injury because of toxic
- Once cross-linkage of antigen has occurred, enzymes, cytokines
intracellular signaling result in mast cell  Notice sloughing of the pseudostratified
degranulation ciliates columnar epithelial cells lining the
- Cooperation among protein and lipid kinases, bronchiole
phosphatases, rearrangement of cytoskeleton  Clinical Methods to detect type 1
1. Allergen cross-linkage of cell-bounf IgE - Skin testing
2. Antibody cross-linkage of IgE  Histamine – negative control
3. Chemical cross-linkage of IgE  Feather – plane tree pollen
4. Cross-linkage of IgE receptors by anti-receptor  Cat – birch tree pollen
antibody  Dog – grass pollen
5. Enhanced Ca2+ influx by ionosphere that increases  Horse – daisy pollen
membrane permeability to Ca2+  Sheep wool – alternaria (mold)
 Granulocytes produce molecules responsible for - Checking serum level of IgE
Type 1 Hypersensitivity  Susceptibility to Type 1
- Primary mediators - Air pollution can increase allergies
 Preformed and stored in granules – ready to - Farm animals and bacteria exposure early in life
go can decrease allergies
 Examples: histamine, proteases, eosinophil  Hygiene hypothesis
chemotactic factor, heparin - Diet
- Secondary mediators - Genetics
 Synthesized after reaction is initiated  Control of Type I
 Examples: platelet-activating factor,
- Avoiding contact
leukotrienes, prostaglandins, bradykinins,
- Immunotherapy
some cytokines and chemokines  Subcutaneous injections of allergens –
- Histamine
desensitization
 Formed by decarboxylation of amino acid
o Causes shift to IgG production instead of
histidine
IgE
 Major component of granules  Monoclonal anti-human IgE
 Effects observed in minutes
- Drug therapies
 Contraction of smooth muscle (intestinal and
 Antihistamines
bronchial), increase permeability of venules,
 Antileukotienes
increased mucus secretion by goblet cells  Steroids
- Leukotrienes and Prostaglandins
 Drugs that enhance production of second
 Increased vascular permeability
messenger cAMP
 Smooth muscle contraction o Prevents degranulation of mast cells
 Mucus production
 Epinephrine and epinephrine agonists
- Cytokines and chemokines
(albuterol)

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Type II (Antibody-Mediated Cytotoxic) Hypersensitivity  Large amounts of these complexes can lead to tissue
 Antibody directed against cell surface antigens damage
mediates cell destruction via complement activation
or ADCC Type IV (Cell-Mediated) Hypersensitivity
 Typical manifestations  Sensitized TH1 cells release cytokines that activate
- Blood transfusion reactions macrophages or TC cells that mediate direct cellular
- Erythroblastosis fetalis (hemolytic disease of the damage
newborn)  TH2 cells and CTLs mediate similar responses
- Autoimmune hemolytic anemia  Typical manifestations
 Transfusion reactions - Contact dermatitis
- Due to exposure to microorganisms in gut, - Tubercular lesions
individuals have antibodies to blood types not - Graft rejection
their own  Some subpopulations of TH cells encounter antigen,
- Antibody attaches to RBC and initiates secrete cytokines and induce localized inflammatory
complement system to lyse RBC response
- After lysis  Most cases are not detrimental
 Hemoglobin detected in plasma, starts to filter  Intracellular pathogens and contact antigens that
through kidneys and found in urine induce delayed-type (type IV) hypersensitivity
(hemoglobinuria) Intracellular bacteria Intracellular viruses
 Hemoglobin converted to bilirubin – toxic at Mycobacterium tuberculosis Herpes simplex virus
high levels Mycobacterium leprae Variola (smallpox)
 Fever, chills, blood clotting Brucella abortus Measles virus
 Hemolytic disease of newborn Listeria monocytogenes
- Rh+ fetus, Rh- mother Intracellular fungi Contact antigens
- IgG antibodies cross placenta
Pneumocystus carinii Picric chloride
 Some of these antibodies may be anti-Rh
Candida albicans Hair dyes
antibodies
Histoplasma capsulatum Nickel salts
o Can have severe consequences
Cryptococcus neoformans Poison ivy
 Antibodies against ABO blood groups produce
less consequences, can be easily treated Poison oak
- Rhogam shot Intracellular parasites
 Given to mother Leishmania sp.
 Anti-Rh antibodies bind to fetal cells that  Sensitization phase and effector phase of DTH
might have entered mother’s system during - Sensitization phase
birthing process, facilitates clearing before  Antigen-presenting cells: Macrophages
there is a B cell response o Langerhans cells
 DTH-mediating cells
Type III (Immune Complex-Mediated) Hypersensitivity o TH1 cells generally
 Antigen-antibody complexes deposited in various o CD8 cells occasionally
tissues induce complement activation and an ensuing - Effector phase
inflammatory response mediated by massive  TH1 secretions
infiltration of neutrophils o Cytokines – IFN-γ, TNF-β, IL-2, IL-3, GM-
 Typical manifestations CSF, MIF
- Localized Arthus reaction o Chemokines – IL-8/CXCL8, MCP-1/CCL2
- Generalized reactions such as serum sickness,  Effects of macrophage activation
necrotizing vasculitis, glomerulonephritis, o Class II MHC molecules
rheumatoid arthritis, and systemic lupus o TNF receptors
erythematosus o Oxygen radicals
- Pathogens o Nitric oxide
 Meningitis  Prolonged DTH can lead to formation of granuloma
 Hepatitis  Contact dermatitis
 Malaria
- Drugs Chronic Inflammation
 Sulfonamides  Caused by infections, continuing physical damage to
 Penicillin tissue, obesity, and autoimmunity
- Autoimmune
 Rheumatoid arthritis
 Lupus
 Complexing of antigen plus antibody facilitates
phagocytosis and clearing of antigen

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Tolerance, Autoimmunity, and Transplants  Peripheral tolerance – kills lymphocytes in
secondary lymphoid tissue
Horror Autotoxicus - Also, life span of lymphocytes regulated by
 Failure of host’s humoral and cellular immune systems apoptosis
to distinguish self from non-self  CTLA-4 on Treg cells play a role in inhibiting activated
- Autoimmunity effector T cells
- Can result in tissue and organ damage, can be fatal  This keeps the effector T cell population at a
 Some organ-specific autoimmune diseases reasonable level
Disease Self-antigen Immune - If there is more antigen to deal with, then more
response effector cells will be activated
Addison’s disease Adrenal cells Auto-antibodies - If not, it brings the activated population down,
Autoimmune RBC membrane Auto-antibodies helping to prevent autoimmunity and unnecessary
hemolytic anemia proteins
inflammation
Goodpasture’s Renal and lung Auto-antibodies
syndrome basement  Some antigens can produce tolerance, termed
membranes tolerogens than immunogens
Graves’ disease Thyroid- Auto-antibodies - High dosages of antigen in host
stimulating (stimulating) - IV or oral introduction
hormone receptor - Absence of adjuvants
Hashimoto’s Thyroid proteins TH1 cells, auto- - Low levels of costimulators
thyroiditis and cells antibodies  CD28 will bind to B7 and provide activating
Idiopathic Platelet membrane Auto-antibodies signals; however, it was discovered that
thrombocytopenia proteins another receptor, CTLA-4 will bind to B7 and
purpura
inhibit
Insulin-dependent Pancreatic beta TH1 cells, auto-
diabetes mellitus cells antibodies
Myasthemia gravis Acetylcholine Auto-antibodies
Organ-Specific Autoimmune Diseases
receptors (blocking)  Target antigen specific to organ or gland
Myocardial infarction Heart Auto-antibodies  Cellular lysis and chronic inflammation that can
Pernicious anemia Gastric parietal Auto-antibody damage organ
cells; intrinsic  Hashimoto’s thyroiditis
factor - Mainly middle-aged women
Poststreptococcal Kidney Antigen-antibody - Target is thyroid antigens
glomerulonephritis complexes - Goiter can form
Spontaneous Sperm Auto-antibodies
- Hypothyroidism – decrease
infertility
 Autoimmune anemias
 Systemic Autoimmune Diseases
- Pernicious anemia
Disease Self-antigen Immune  Antibody against membrane bound intestinal
response protein that uptakes B12 needed for
Ankylosing Vertebrae TH1 cells and TC
hematopoiesis
spondylitis cells, auto-
antibodies - Hemolytic anemia
Multiple sclerosis Brain or white Auto-antibodies,  Antibodies to red-blood cell antigens
matter immune complexes  Type II hypersensitivity
Rheumatoid Connective tissue, Auto-antibodies - Drug-induced anemia
arthritis IgG  Goodpasture’s Syndrome
Sclerodoma Nuclei, heart, lungs, Auto-antibodies - Antibodies against basement membranes in
gastrointestinal glomeruli and alveoli
tract, kidney - Leads to kidney damage and pulmonary
Sjogren’s syndrome Salivary gland, Auto-antibodies hemorrhage
liver, kidney,
- Type II hypersensitivity
thyroid
Systemic lupus DNA, nuclear Auto-antibodies,  Insulin-Dependent Diabetes Mellitus (Type 1)
erythematosus protein, RBC and immune complexes - Antibodies against beta cells that produce insulin
(SLE) platelet - Insulin is needed by cells to uptake glucose
membranes needed for cellular respiration
- Type IV hypersensitivity – cell-mediated
Tolerance destruction
 Number of mechanisms are in place to protect  In some autoimmune diseases, antibodies act as
individual from self-reactive lymphocytes antagonists
 Central tolerance – deleting T or B clones before - Bind inappropriately to receptor, resulting in
maturity if they have receptors that recognize self- overproduction
antigens with great affinity

dane.
  For example, upregulating a hormonal
response without the presence of that
hormone
 Grave’s Disease – auto-antibodies bind to
receptor for thyroid stimulating hormone
resulting in over-stimulation of thyroid
o Type II hypersensitivity
 Myasthenia gravis – antibodies bind
acetylcholine receptors on motor end plate of
muscles – progressively weakened skeletal
muscles
o Type II hypersensitivity
Systemic Autoimmune Diseases
 Response is directed toward wide range of target
antigens
 Systemic Lupus Erythematosus
- Typically middle-aged women
- Fever, weakness, arthritis, skin rash, kidney
problems
- Produce autoantibodies to DNA, histones,
platelets, leukocytes, clotting factors
- Excessive complement activation
- Type III hypersensitivity – immune complexes
 Multiple sclerosis
- Numbness, paralysis, vision loss
- Inflammatory lesions in myelin sheath caused by
T cells
- Epidemiology
 Frequent in African-American and Hispanic
women
 Environmental components as well as genetic
components
 Rheumatoid arthritis
- Chronic inflammation of joints
- Produce auto-Abs that bind Fc portion of IgG
circulating in blood that creates immune
complexes
- Type III hypersensitivity
 Proposed mechanisms for induction of
autoimmunity
- Release of sequestered antigens
 Blood-brain barrier, sperm released into
tissues during vasectomy
- Molecular mimicry
- Inappropriate expression of Class II MHC
 Non-antigen presenting cells will for some
reason express Class II MHC
o Can be caused by viral infection
 This allows them to present self antigen to T
helper cells – leads to inappropriate reaction
Treatment
 Immunosuppressive drugs
 Removal of thymus (for example, with myasthenia
gravis)
 Plasmapheresis – removing plasma and then returning
RBCs (removes extra immune complexes)
 Treating the inflammation
 Antigen given orally can induce tolerance
AIDS and other Immunodeficiencies
Autoimmunity VS Immunodeficiency
 Autoimmunity – system attacks host cells and tissues
 Immunodefiency – system fails to protect
- Primary immunodeficiency
 Genetic or developmental defect
- Secondary immunodeficiency – acquired
Primary Immunodeficiencies (Lymphoid
Immunodeficiencies)
 Combined – effects both B and T cells
 B-cell immunodeficiency
- Range from absence of B cells, plasma cells,
immunoglobulins to absence of only certain
classes of antibodies
- Subject to bacterial infections but do well against
viral since T-cell branch is ok
 T-cell immunodeficiency
- Can affect both humoral and cell-mediated
 Primary immunodeficiencies are often detected early
in life
Primary Immunodeficiencies – Combined
Immunodeficiencies (CD)
 Severe combined immunodeficiency (SCID)
- Deficiency in RAG1, RAG2, Artemis, or DNA-PK
 No TCR or Ig gene rearrangement
 Inheritance mode – AR
- Reticular dysgenesis – defect in mitochondrial
enzyme AK2 required for hematopoiesis
 Block in differentiation of hematopoietic stem
cells into myeloid and lymphoid lineages
 Inheritance mode – AR
- ADA and PNP deficiency
 Toxic purine metabolites in lymphoid
progenitor cells. Leading to loss of B, T, and
NK cells
 Inheritance mode – AR (ADA), XL (PNP)
- Common γ-chain deficiency, JAK3 deficiency,
IL-7Rα deficiency
 Defective signals from IL-2, -4, -7, -9, -15, -21
lead to blocks in development of T and NK
cells
 Inheritance mode – AR
- CD3δ, ε, or ζ deficiency
 Defective signals from TCR block T-cell
development
 Inheritance mode – AR
 Leaky SCID
- Hypomorphic mutations in proteins required for
V(D)J gene rearrangements, AK2, common γ
chains
- T cells present but with reduced repertoire
- B cells normal or decreased
- Igs reduced but may have increased IgE
- May include oligoclonal T cells and autoimmunity
(Omenn syndrome)
- Inheritance mode – AR, AD, or XL
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 Bare-lymphocyte syndrome (BLS):
deficiencies
- Class I – β2-microglobulin, TAP peptide
transporter, tapasin
 No/reduced MHC class I expression, reducing
number of CD8+ T cells
- Class II – defect in transcription factors activating
MHC II gene expression
 No MHC class II molecules, reducing numbers
of CD4+ T cells
- Inheritance mode – AR
 Wiskott-Aldrich syndrome (WAS)
- Cytoskeletal protein (WASP)
- Defective T cells and platelets
- Inheritance mode – AR
 Mendelian susceptibility to microbacterial
diseases (MSMD)
- IFN-γR, IL-12, IL-12R, STAT1
- Impaired immunity to mycobacteria
- Inheritance mode – AR or AD
 DiGeorge syndrome
- Thymic aplasia
- Decreased number of T cells
- Inheritance mode – AC
 Hyper-IgM syndrome
- Defective CD40 ligand or CD40
- Elevated IgM due to loss of isotypes other than
IgM; defective APC activation leading to reduced
T-cell responses to intracellular pathogens
- Inheritance mode – XL, AR
Primary Immunodeficiencies – Agammaglobulinemias
 X-linked (Bruton’s) agammaglobulinemia
- Defective Bruton’s tyrosine kinase (Btk)
- Block in B cell development; no mature B cells
- Inheritance mode – XL
 Reduction in one or more immunoglobulin
isotypes
- Defective activation-induced cytidine deaminase
(AID) or B cell surface proteins
- No/low IgG, IgA, IgE – various effects on IgM, IgG,
or IgA
- Elevated or normal IgM
- No APC or T-cell deficiency
- Inheritance mode – AR
 Common variable immunodeficiency
- Complex/unknown defect
- Low IgG, IgA; variable IgM
- Inheritance mode – complex
 Selective IgA deficiency
- Complex/unknown defect
- Low or no IgA
- Inheritance mode – complex
Primary Immunodeficiencies – Innate Immune
Deficiencies
 Chronic granulomatous disease
- Mutations in phagosome NADPH oxidase subunits
- No ROS or RNS for killing of phagocytosed
pathogens
MHC - Inheritance mode – XL, AR
 Chediak-Higashi syndrome
- Defective lysosomal trafficking regulator protein
(LYST)
- Decreased CTL- and NK-mediated cytolytic
activity
- Inheritance mode – XL, AR
 Leukocyte adhesion deficiencies
- Defective integrins
- Leukocyte extravasation and chemotaxis
- Inheritance mode – AR
 Selective NK-cell deficiencies
- Mutations in STAT5b, GATA2, MCM4, and RTEL1
genes
- Absent or decreased total NK cells or NK subsets
- Increased susceptibility to viral infections
- Inheritance mode – AR
Primary Immunodeficiencies – Immune Regulation
Deficiencies
 Autoimmune polyendocrinopathy with candidiasis
and ectodermal dystrophy (APECED)
- AIRE defect
- Reduced thymic negative selection and T reg
formation
- Autoimmunity
- Inheritance mode – AR
 Immune dysregulation, polyendocrinopathy,
enteropathy, X-linked (IPEX) syndrome
- FoxP3 defect
- Absence of Tcreg cells
- Autoimmunity
- Inheritance mode – XL
Warning Signs of Primary Immunodeficiency
 4 or more new ear infections within 1 year
 2 or more serious sinus infections within 1 year
 2 or more months on antibiotics with little effect
 2 or more pneumonias within 1 year
 Failure of an infant to gain weight or grow normally
 Recurrent, deep skin or organ abscesses
 Persistent thrust in mouth or fungal infection on skin
 Need for intravenous antibiotics including septicemia
 A family history of PI
Combined Immunodeficiencies
 Severe combined immunodeficiency (SCID)
- Low number of circulating lymphocytes
- Non-proliferating T cells
- Thymus doesn’t develop
- Usually fatal early years of life
 Infant will have viral and fungal infections
 Bacteria don’t show up until later because of
placental transfer of antibodies from mother
 Chronic diarrhea, pneumonia, lesions
- Many genetic defects can contribute to SCID
 Usually defects that target the early steps of
hematopoiesis
 MHC defects
- Sympyoms can resemble SCID
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 - Bare-lymphocyte syndrome
- Failure to train T cells correctly, it affects both B
and T cells
 Thymus
- DiGeorge syndrome – decreased or absent
thymus
 Results from deletion of region on
chromosome 22 in developing embryo,
developmental anomaly
 Lowered T cell numbers, results in B cells not
producing sufficient antibodies
 Wiskott-Aldrich syndrome (WAS)
- X-linked disorder
 Results in issues with cytoskeleton
components in hematopoietic cells
- Initially B and T cell numbers are normal but will
decrease with age
- Treated with passive antibodies or stem cell
transfer
- Can result in fatal infection or lymphoid
malignancy
 X-linked Hyper-IgM syndrome
- Deficiency of IgG, IgE, IgA but elevated levels of
IgM
- Defect in T cell surface marker CD40L
 This is needed for interaction between TH and
B cell for class switching for T-dependent
antigens
 T independent antigens are not effected
therefore there is production of IgM
 Hyper-IgE syndrome (Job syndrome)
- Autosomal dominant
- Skin abscesses, pneumonia, eczema, facial
abnormalities
- High number of eosinophils and IgE but don’t
show increased allergies
B cell Immunodeficiencies
 X-linked agammaglobulinemia
- B cell defect
 Defect in kinase that keeps B cells in pre-B
stage with H chains rearranged but L chains
not
- Low levels of IgG and absence of other classes
- Recurrent bacterial infections
 Common variable immunodeficiency (CVI)
- Low levels of immunoglobulin – different from genetic condition
hypogammaglobulinemia
- Manifests later in life
- Undefined genetic component
 Selective deficiencies of immunoglobulin classes
- IgA deficiency is most common
 Recurrent respiratory and urinary tract
infections, intestinal problems
Innate Immunodefiencies
 Leukocyte adhesion deficiency (LAD)
- Integrin proteins needed for adhesion and cellular
interaction
 Defect limits recruitment of cells into areas of
inflammation
 Chediak-Higashi syndrome
- Autosomal recessive disease
- Phagocytes don’t have ability to kill bacteria
 Issue with lysosome activity
 Interferon-Gamma-Receptor defect
- Autosomal recessive trait – results from
inbreeding
- Defect in receptor for IFN-γ and subsequent
pathways
 Patients suffer from infection with
mycobacterium, showing importance of this
receptor in fighting mycobacterium
Myeloid Immunodeficiencies
 Reduction in neutrophil count
- Low concentration – granulocytopenia or
neutropenia
- Congenital neutropenia – frequent bacterial
infections
- Acquired neutropenia
 Certain drug or chemotherapy can cause this
 Autoimmune disorder – destruction of
neutrophils
 Complement defiencies
- Fairly common
- Mostly associated with bacterial infections or
immune-complex diseases
Treatments for Immunodeficiencies
 Replacement of missing protein
- Administering immunoglobulin
- Express genes in vitro (in bacteria) for cytokines
 Replacement of missing cell type
- Bone marrow transplantation
 Replacement of missing or defective gene
- Gene therapy
 Still far out from clinical use
 Can it be corrected in vitro and then introduce
cells back into patient
Acquired Immunodeficiencies
 No genetic component
 Examples
- Hypogammaglobulinemia – unknown cause,
 AIDS caused by HIV
 HIV
- Retrovirus (Lentivirus genus)
- Viral envelope derives from host
 Recognizes CD4 antigen on T cell
- Recognizes CD4 antigen on T cell
- HIV-1 – main causative agent for AIDS
 Many different subtypes
 Probably evolved from SIV and transferred to
humans during the “bushmeat trade” in early
1900s
- HIV-2 – cousin to HIV-1 but disease progresses
slowly, if at all
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 HIV Treatment
- Chemokine receptor antagonists
- Inhibit fusion
- Inhibit reverse transcription
- Inhibit integrase
- Inhibit protease
 AIDS does not fit the paradigm for classic vaccine
development
- Classic vaccines mimic natural immunity against
reinfection generally seen in individuals
recovered from infection; there are no recovered
AIDS patients
- Most vaccines protect against disease, not against
infection; HIV infection may remain latent for long
periods before causing AIDS
- Most vaccines protect for years against viruses
that change very little over time; HIV-1 mutates at
a rapid rate and efficiently selects mutant forms
that evade immunity
- Most effective vaccines are whole killed or live
attenuated organisms; killed HIV-1 does not
remain antigenicity, and the use of a live
retrovirus vaccine raises safety issues
- Most vaccines protect against infections that are
infrequently encountered; HIV may be
encountered daily by individuals at high risk
- Most vaccines protect against infections through
mucosal surfaces of the respiratory or
gastrointestinal tract; the great majority of HIV
infection is through the genital tract
- Most vaccines are tested for safety and efficacy in
an animal model before trials with human
volunteers; there is no suitable animal model for
HIV/AIDS at present

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MT-IMMUNO 1 (Immunology and Serology) LEC - Immunogen – a substance capable of eliciting an
immune response
Introduction and Overview - All immunogens are antigens but not all antigens
are immunogens (i.e., haptens, incomplete
Immunology antigen incapable of eliciting immune response)
 Study of the immune system or immunity - Epitope – portion of the antigen that is recognized
 Immunity by an antibody or T cell receptor
- “Immunis” = exempt (Latin)
Systems of Immunity
History  Innate immunity (Non-specific)
 Discipline of immunology grew out of observation that - 1st and 2nd lines of defense
individuals who recovered from infectious diseases  Barriers that protect host (e.g., skin, acidity
were protected from disease of stomach, lysozymes in fluids)
 15th Century  Phagocytic cells
- Chinese and Turks tried to prevent smallpox  Antimicrobial peptides (e.g., interferons,
 Dried crust from pastules were inhaled or complements)
inserted into small cuts  Temperature
 1718 - Molecular and cellular mechanisms deployed
- Lady Montagu had that technique done on her before an infection
children - Distinguishes between self and pathogens but not
 1798 specialized to distinguish small differences in the
- Edward Jenner foreign particles
 Noticed that milkmaids that contracted - Less specific
cowpox were immune to smallpox  Adaptive immunity (Acquired and Specific)
 Inoculated a small boy with fluid from cowpox - Develops in response to infection
pustule - Adapts to recognize, eliminate, and remember
 Intentionally infected the boy with small pox – pathogen
the child did not develop smallpox - Highly specific
 1881 - 3rd line of defense
- Louis Pasteur  Antigenic specificity
 Vaccinated sheep with heat-attenuated  Diversity
anthrax  Immunologic memory
 Infected sheep with virulent strain of anthrax  Self-, non-self recognition
– the sheep did not develop anthrax - Effective adaptive immune response involves two
 1883 groups of cells
- Metchnikoff demonstrated that certain WBCs  Lymphocytes
were able to phagocytize microorganisms o B cells
 1901  Mature in bone marrow
- Von Behring and Kitasato demonstrated that  Antigen binding receptor: antibody
serum (noncellular component of blood) from made of glycoproteins
animals immunized to diphtheria could transfer  Glycoprotein structure
that immunity to non-immunized animals  Heavy chains – 2 identical
 1977 polypeptides
- Last naturally acquired case of smallpox  Light chains – 2 shorter identical
polypeptides
 In industrialized nations, measles, mumps, whooping
cough, tetanus, polio, and diphtheria are extremely o T cells
 Arise in bone marrow but mature in
rare or nonexistent
- This is due to vaccine thymus
- Prevents death, paralysis, deafness, blindness,  Two well defined subpopulations of
T cells
mental retardation
 T helper cells
Immune System  T cytotoxic cells
 T cells recognize antigen presented in
 Evolved to protect multicellular organisms from
MHC molecule
pathogens
 MHC – major histocompatibility
 Does this by two related activities – recognition and
complex
response
 MHC Class I – found on all of our
 Definition of Terms
nucleated cells (cytotoxic T cells
- Pathogen – something that causes disease
recognize this)
- Antigen – any foreign substance that binds
 MHC Class II – found on antigen
specifically to an antibody or T cell receptor
presenting cells (B cells, dendritic
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 cell and macrophages). Helper T - Myeloid progenitor cell
cells recognize this  Progenitor cells have lost ability for self renewal and
o Cytokines secreted by TH cells can are committed to particular cell lineage
activate phagocytic cells  Most complex stem cell system in the mammalian
o TC cells can kill altered self-cells body
 Cells infected by viruses  Regulated at gene level
 Tumor cells - Transcription factors play important roles in
 Antigen presenting cells hematopoiesis
o Phagocytosis of enemy cell (antigen) - Studies using knockout mice:
o Fusion of lysosome and phagosome  Gene inaction – if RBC or a particular WBC
o Enzymes start to degrade enemy cell fails to develop, it is concluded that protein
o Enemy cell broken into small fragments was involved in development of that cells
o Fragments of antigen presented on APC  Ikaros – transcription factor needed for
surface choice between B and T cell development
o Leftover fragments released by exocytosis  Notch – transcription factor needed for choice
between B and T cell development
Antibodies  Notch signaling is evolutionary conserved
 Antigen coated by antibody is eliminated in several pathway in multicellular organisms that
ways regulates cell fate determination during
- Can cross-link several antigens, making it easier to development and maintains adult cell
be ingested by phagocytic cells homeostasis
- Activate complement system resulting in lysis of
microorganism Hematopoietic Homeostasis
 Erythrocyte
Primary VS Secondary Immune Response - Average life span – 120 days
 Initial encounter with antigen causes primary  Leukocytes
response - Life spans from 1 day to 20-30 years
 Later contact with antigen will result in more rapid  Apoptosis – programmed cell death
response – secondary response
Cells of the Immune System
Immune Dysfunction  Myeloid lineage
 Allergies and Asthma - Monocytes that become macrophages (innate)
 Graft rejection - Dendritic cells (innate)
 AIDS - Granulocytes (innate) – neutrophils, eosinophils,
 Immunodeficiency basophils, mast cells
- Megakaryocytes (platelets) and erythrocytes
Cells and Organs of the Immune System  Lymphoid lineage – lymphocytes
- 20-40% of WBC
Stem Cells - 3 populations
 Ability to “self-renew” and to differentiate into  B cells (adaptive)
different cell types  T cells (adaptive)
 Embryonic stem cells  NK cells (innate)
- Capacity to generate almost all cell types
- Pluripotent Myeloid Lineage Cells
 Adult stem cells  First responders
- Capacity to give rise to the cell types that specify a  Mononuclear phagocytes
particular tissue - Monocytes circulate in blood and then migrate
- Multipotent into tissue and differentiate into specific
macropahage
Hematopoiesis - Activated macrophages are more effective than
 All blood cells (RBC and WBC) arise from resting ones
Hematopoietic Stem Cells (HSC)  Macrophages
 Study of these cells s difficult - Most tissue-resident macrophages arise early in
- Scarce embryonic development and have the ability to
- Difficult to grow in vitro continue to self-renew (multipotent)
- Isolation requires monoclonal antibodies and flow Tissue Name Tissue-specific
cytometry function
 Early in hematopoiesis, stem cell differentiates to Brain Microglia Neural circuit
either development
- Lymphoid progenitor cell (synaptic
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 pruning) - Antigen Presentation by Macrophage
Lung Alveolar Remove
macrophage pollutants and
microbes, clear
surfactants
Liver Kupffer cell Scavengr RBCs,
clear part
Kidney Resident kidney Regulate
macrophage inflammatory
responses to
antigen filtered  Dendritic Cells
from blood - Long membranous extensions, look like dendrites
Skin Langerhans cell Skin immunity on nerve cells
and tolerance - Antigen presentation – take a “snapshot” of what
Spleen Red pulp Scavenge RBCs, is happening in the tissues and carry this image to
macrophage recycle iron the lymph node
Peritoneal Peritoneal Maintain IgA - Four major groups
cavity cavity production by B-  Langerhans
macrophage 1 B cells  Interstitial
Intestine Lamina propria Gut immunity  Monocyte-derived
macrophage Regulate  Plasmatocytoid-derived
Intestinal peristalsis - Follicular dendritic cells
muscularis  Involved with B cell maturation
macrophage  Granulocytes
Bone marrow Bone marrow Maintain niche - Neutrophils
macrophage for blood cell  Multi-lobes nucleus, light colored granules
development,  First to arrive at site of inflammation
clear  High numbers is first indication of infection
neutrophils  Phagocytic
Lymph node Subcapsular Trap antigen  Generate antimicrobial agents
sinus particles  Very short-lived when compared to
macrophge macrophages
Heart Cardiac Clear dying  Come out of blood vessels ready to kill, would
macrophage heart cells cause too much damage to tissues if long-lived
- Eosinophils
- Includes:
 Phagocytic
 Intestinal macrophages in gut
 Play a role against parasitic organisms
 Alveolar macrophages in lung
- Basophils
 Histiocytes in connective tissue
 Non-phagocytic
 Kupffer cells in the liver
 Play a role in allergic reactions
 Mesangial cells in the kidney
- Mast cells
 Microglial cells in the brain
 Play important role in development of
 Osteoclasts in bone
allergies
- Monocyte VS Macrophage
 Similar to basophil cells

Lymphoid Lineage Cells

 B cells and T cells
- Adaptive immunity
- Small cells
- Those that have not interacted with antigen are
called naïve
 Interactions with antigen – proliferation into
effector cells (i.e. plasma cells) and memory
cells
 Natural killer cells – innate immunity
 Cluster of differentiation (CD) Markers – identifies
certain cell types

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  Subpopulations
o T helper (TH)
o T cytotoxic (TC)
o T regulatory (Treg)
o Recent research shows there are even
more subpopulations
 T helper cells
 Lymphocytes o CD4 glycoprotein
- Once they have encountered antigen to which they o Recognizes antigen presented in MHC
are specific for, they will proliferate, making an Class II
army of B and T cells that are specific for an o “Help” activation of B cells, TC cells,
antigen macrophages in immune response
- Plasma or effector cells  T cytotoxic cells
 Plasma B cells – actively secrete antibody o CD8 glycoprotein
against the antigen o Recognizes antigen presented in MHC
 Effector T cells – actively search cells Class 1
presenting the antigen they are specific for o Eliminates infected cells or cancerous
- Memory B and T cells – last for decades, cells
watching for that same antigen to come back  T regulatory cells
 What causes a cell to become a plasma or o CD4 and CD25 glycoproteins
memory cell after activation is not yet known o Help suppress the immune system after
- Lymphocyte activation cycle (B and T cells) its been upregulated, after the infection is
cleared
 NK cells
o Innate immune response
o Large, granular
o Recognize tumor or virus-infected cells
o Antibody-dependent cellular cytotoxicity
(ADCC) or “kiss of death.”

Lymphoid Tissues and Organs

 Tissues and organs of the Immune System (AKA
 Primary
- B Lymphocytes (B cells)
 Site of maturation
o Bursa of Fabricius in birds
o Bone marrow in mammals
 Display membrane-bound immunoglobulin
(antibody)
 Once antigen is encountered
o Differentiation
o Plasma cells – antibody can be secreted,
die within 1-2 weeks
o Memory B cells – same-membrane
bound antibody as parent B cell, longer
life span
- T Lymphocytes (T cells)
 Site of maturation - thymus
 T cell receptor
o Only recognize antigen that is bound to
cell membrane proteins called major
histocompatibility complex (MHC)
 Once antigen bound to MHC is encountered, it
differentiates into
o Effector T cells
o Memory T cells
Lymphoid)
- Thymus
 T cell development and maturation
 Bilobed organ above heart
 Surrounded by capsule and divided into
lobules (outer cortex; inner medulla)
 Network of epithelial cells, dendritic cells, and
macrophages
 Thymus will induce death of those cells that:
o Can’t recognize self-MHC molecules
o Those that interact with MHC molecules
too strongly
 Function decreases with age
- Bone marrow
 Lymphocytes arise there, but T cells go to
thymus to mature
 B cells mature here
 90% of plasma IgA comes from B cells in the
bone marrow
- Place of maturation of lymphocytes
- Lymphatic system
 Interstitial fluid (the portion that doesn’t
enter venous system) is returned to
circulatory system by lymphatic vessels
 Largest lymphatic vessel – Thoracic duct
o Enters left subclavian vein

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 o Lymph from right arm and right side of  Innate immune cells – use pattern
head enters through right lymphatic duct, recognition receptors (PRRs)
drains into right subclavian  Lots of immune cells will express cytokine
 Antigen is carried by lymph to lymph nodes receptors
 Secondary o Cytokines are molecules that will elicit a
- Lymph nodes, spleen, mucosa-associated response in the immune cell
lymphoid tissues such as gut-associated lymphoid  Receptor-Ligand Interaction
tissues - Multiple noncovalent bonds between receptor and
- Provide venue for mature lymphocytes to interact ligand
with antigen  Hydrogen bonds
- Lymph nodes  Ionic bonds
 Encapsulated  Van der Waals
 Divided into 3 regions  Hydrophobic interactions
o Cortex – primary follicles (B cells, - Univalent interaction
macrophages, dendritic cells) - Bivalent interaction
o Paracortex – T cells, dendritic cells  Affinity
o Medulla - plasma cells secreting - As an immune response progresses, cells that
antibody have been activated will produce and express
- Lymph nodes more receptors
 Primary follicle – unactivated lymphoid - The receptors have higher affinity as the immune
follicle response progresses
 Secondary follicle  Receptor locations
o Activated by antigen - Immune antigen receptors can be transmembrane,
o Germinal center – area of activated B cytosolic or secreted
cells  Antibody can be secreted or transmembrane
- Spleen (B-cell receptor)
 Filters blood, traps blood-borne pathogens  T-cell receptor is transmembrane
 Important in systemic infections  Some innate immune system receptors are
 Blood enters through splenic artery cytosolic (we have receptors in our cells that
 Encapsulated might recognize viral particles)
 Structure
o Projections from capsule from trabeculae Adaptive Immune System Receptors
o Compartments  B-cell receptors
 Red pulp – macrophages, RBCs - B cells produce antibodies (Abs) that are secreted
 White pulp - Some antibodies are membrane bound and are
 Surrounds branches of splenic associated with other proteins
artery - Collectively known as the B-cell receptor (TCR)
 Forms PALS (periarteriolar which is membrane bound
lymphoid sheath)  Antibodies and T-cell receptors
 Primary follicles rich in B cells - Recognizes epitopes
- MALT (Mucosa-associates Lymphoid Tissue) - Immunologically active regions of immunogen
 Can be specified further that bind to antigen-specific antibodies or T-cell
o Gut-associated lymphoid tissues (GALT) receptors
o Bronchus-associated lymphoid tissue  Epitopes
(BALT) - Antigenic determinants recognized by B cells and
 Organized areas along digestive, respiratory, T cells
and urogenital tracts - B cell epitopes tend to be on the outside of the
 Very well organized areas in intestine are antigen
referred to as Peyer’s patches  For example, the hydrophilic amin acids on a
 Includes tonsils and appendix protein’s surface
- T cell epitopes from proteins derived from
Immune Recognition and Response enzymatic digestion of peptide and then
association with MHC
Introduction - Most antigens offer multiple epitopes
 Cells of the immune system must constantly recognize - However, a single B cell will only produce
molecular signals from the external environment antibody specific to single epitope
- The immune cells use several different receptors
to do so:
 Adaptive immune cells (B and T cells) – use
specific B-cell receptors (BCRs) and T-cell
receptors (TCRs)
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Antibodies (Abs)
 Epitope-binding proteins
- Membrane bound on B cells (as part of the B-cell
receptor) OR  Overall structure of Immunoglobulin
- Secreted in blood (Humoral immunity) - Primary – sequence of amino acids
- Secondary – folding into series of β-pleated
 Share structural features, bind to antigen, and
sheets
participate in number of effector functions
- Tertiary – compact globular domains
 Known collectively as Immunoglobulins (Igs)
- Quarternary – adjacent light and heavy chains
 Antibodies don’t kill anything, their job is to serve as
interact
target markers for other immune system components
 Hypervariable Regions
 Basic structure
- Complimentarily-determining regions (CDRs)
- Known since late 19th century that antibodies are
- Complimentary to epitopes that they will bind to
in serum
 Hinge region
 Serum is fluid phase that remains after
- ɣ (gamma), δ (delta), and α (alpha) heavy chains
plasma is allowed to clot
have extended peptide sequence
 Antibodies are also found in other secretions
- Rich in proline and cysteine
- Heterodimers
- Gives flexibility
 2 light chains – 22,000 daltons each
 B Cell Receptor (BCR)
 2 heavy chains – 55,000 daltons each
- Immunoglobulins can be secreted or membrane-
 First 110 aa of amino terminal end of heavy
bound
and light chain vary depending on antibody
- Membrane-bound differs from secreted in the
specificity
carbonyl-terminal end:
- Light chains
 Extracellular “spacer” of 26 aa
 When aa sequences of light chains from
 Hydrophobic transmembrane sequence
several individuals were sequenced, pattern
 Cytoplasmic tail
emerged:
- Membrane-bound antibody + accessory
o Amino terminal end (110 aa) varies
membrane-bound molecules = BCR
o Other part remained constant
- Heavy chain portion of membrane-bound
 Were found to be either kappa (κ) or
antibody does not extend far enough through the
lambda (λ)
cell membrane for signaling
 In mice and humans, different
- Also associated with CD21, CD19, and CD81
lambda subtypes have been
found  Antibody-mediates effector functions
- Heavy chains - Remember, they “mark” an invader
 Amino-terminal end also shows variability - In addition to binding antigen, Abs can:
 Each antibody has 2 identical heavy chains, 2  Promote phagocytosis (opsonization)
identical light chains  Activate complement
 5 different heavy chain constant regions  Antibody dependent cell mediated toxicity
(isotypes) (ADCC)
o IgM – µ  NK cells have receptor for Fc portion of
o IgG – ɣ antibody
o IgA – α  Some can cross epithelial layers to be
o IgD – δ excreted through mucous or across placenta
o IgE – ε  Antibody class switching
 Some subisotypes have been discovered in - Changing the constant region of an antibody is
some species class switching
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 - Makes an IgG from an IgM
Antibody Isotypes
 IgM
- Monomeric IgM expressed on B cells
- Secreted is pentameric
- 1st class produced in primary response
- Activates complement
- Very good at agglutination
 IgA
- Predominant class in secretions
- J chain and secretory component helps with
transport across intestinal wall
- J chain makes IgA more resistant to acids and
enzymes found in digestive tract
- IgA and macrophages restrict commensal bacteria
that occasionally enter the tissues from the
intestines
- Better for IgA than IgG to interact with
macrophage—because the Fc portio of IgG has
high affinity for receptors of immune cells and
would trigger inflammatory responses
- Exists as a dimer
- Can cross-link large antigens

 IgD
- Membrane bound on B cells

 Iso-, Allo-, and Idiotype

 IgG
- Most abundant
- 4 human subclasses
- Crosses placenta
- Involved in complement activation
- Opsonin
- IgG subclasses

 IgE
- Involved in allergic reactions
- Involvement in parasitic infections
T-Cell Receptor
 Expressed on surface of T-cells
 Recognize processed antigen complexed with MHC
molecules
 T cell VS B Cell Receptor
- T cell receptor is only membrane bound – doesn’t
appear in soluble form like antibodies so more
difficult to assess its structure
- Antigen binding of T cell receptor is weaker than
that of antibodies
- Antigen recognized by T cells is not antigen alone
but antigen associated with MHC molecules
 TCR-MHC interaction
- (a) T cell receptor is specific for peptide A
- (b) Right MHC haplotype but wrong antigen
(peptide B)
- (c) Right antigen (peptide A) but wrong haplotype

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 domain wall
(NOD)- peptidoglycans
like
receptor
(NLR)
Absent- Cytosol and Viral and Production of
in- nucleus bacterial DNAs interferons
melanom and cytokines
a (AIM)-
like
 TCR heterodimers are similar to immunoglobulins receptor
 Therefore they are classified in immunoglobulin (ALR)
superfamilt  Cytokines
 Resembles Fab portion of antibody - Low-molecular weight regulatory proteins or
 TCR Coreceptors glycoproteins
- Accessory molecules help in signal transduction - Secreted by and through which WBC and various
after interaction of T cell with antigen (e.g., CD4, other cells communicate
CD8, CD28) - Assist in development of immune effector cells
- Assist with signal transduction - Some possess direct effector functions of their
- Affinity of TCR for peptide MHC complexes is own
enhanced by coreceptors - Some referred to as interleukins (IL)
- IL-1 through IL-29 have been described
Innate Immune System Receptors - Chemokines
 Remember that Abs and TCRs recognize specific  Subpopulation of cytokines
epitopes  Mobilize immune cells from one area to
 Innate immune system receptor are Pattern another (help move WBCs into tissues)
Recognition Receptors (PRRs) that recognize  Class of chemoattractants
Pathogen Associated Molecular Patterns (PAMPS) - Cytokine action
 Pattern Recognition Receptors (PRRs)  Mainly functions by autocrine and paracrine
Full Cellular Ligands Cellular action
name locations functions
Toll-like Plasma Microbial Production of
receptor membrane, carbohydrates, antimicrobials,
(TLR) endosomes, lipoproteins, antivirals, and
lysosomes fungal cytokines;
mannans, inflammation
bacterial
flagelline, viral
RNA, self-
components of
damaged
tissues, etc  Cytokines bind to specific receptors
C-type Plasma Carbohydrate Phagocytosis,  Trigger signal transduction pathways that
lectin membrane components of production of alter gene expression in target cells
receptor fungi, antimicrobials
(CLR) mycobacteria, and cytokines;
viruses, inflammation
parasites, and
some allergens
Retinoic Cytosol Viral RNA Production of
acid- interferons
inducible and cytokines
gene-I
(RIG-I)-
like
receptor
Nucleoti Cytosol Fragments of Production of
de intracellular or antimicrobials  Exhibit pleiotropy, redundancy, synergy,
oligomer extracellular and cytokines; antagonism, cascade induction
ization bacteria cell inflammation

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  Non-specificity of cytokines contradicts with
established specificity of immune system
 How does this work?
o Careful expression of the receptors for
those cytokines on specific cells
o Receptors are often only expressed after
exposure to antigen
- Cytokine Families
 Cytokines belong to 6 families
o Interleukin 1 (IL-1)
o Hematopoietin family (Class i)
o Interferon family (Class II)
o Chemokine family
o Tumor necrosis family
o Interleukin 17 (IL-17)
 Based on structural studies
 All have molecular mass less than 30kDa
 All have similarities and few rarely act alone
- Cytokine Receptors
 Cytokine receptors fall into families
o Immunoglobulin superfamily receptor
o Class I cytokine receptor family
(hematopoietin)
o Class II cytokine receptor family
(interferon)
o TNF receptor family
o Chemokine receptor family
 IL-2R is the most thoroughly studied cytokine
receptor
o Expressed by T cells for proliferation
(also called CD25, surface marker in
mature T cells)
o Complete receptor has 3 subunits –
subunits together have higher affinity for
IL-2
- Cytokine-Cytokine Receptor Interaction
 Initiates a signal cascade in the cell resulting
in a nuclear response (activation of
transcription factors resulting in production
of proteins, resulting in mitosis/proliferation,
etc.)
Immunogen VS Antigen
 Immunogenicity
- Ability to induce humoral (B cell antibody) and/or
cell-mediates (T cell) immune response
- Immunogen is the substance that induces
response
 Antigenicity
- Ability to combine specifically with Abs or T-cell
receptor/MHC
- Not all antigens are immunogenic (e.g., haptens)
 Properties of Immunogen contribute to
immunogenicity
- Foreignness
 Lymphocytes that do not bind to self-antigens
are allowed to further develop
 Therefore, they will later only recognize non-
self antigens
 For example:
o Bovine serum albumin (BSA) is not
immunogenic when injected into cow but
it is when injected into chicken
 Some macromolecules are highly conserved
throughout evolution and display little
immunogenicity
o Cytochrome C and collagen
- Molecular size
 Active (good) immunogens - >100,000
daltons
 Poor immunogens - <5,000-10,000 daltons
- Chemical composition and complexity
 Polymers composed of multiple copies of
same amino acid or sugar tend to be poor
immunogens
 Lipids are haptens and need to be conjugated
with carrier to produce antibodies –
important for assays for detection of some
steroids and vitamins
- Ability to be processes and presented on MHC
 Susceptibility to antigen processing
 Genotype of recipient animal
 Material presentation
 Susceptibility to Antigen Processing
- Large, insoluble macrophages are more likely to
be phagocytized for processing
 Genotype of recipient animal
- Genes of MHC
- Genes encoding for specific antibodies
 Material presentation
- Immunogen dosage
 Too low or high of dosage can induce
tolerance
 Single dose is often not enough, booster is
needed
- Route of administration
 Intravenous (IV) – travel to spleen
 Intradermal (ID)
 Subcutaneous (SC) – travel to lymph nodes
 Intramuscular (IM)
 Intraperitoneal (IP)
 Adjuvants
- Enhance immunogenicity
- Not exactly sure how they work but are
recognized by Toll-like receptors
- Water-in-oil adjuvants
 Freund’s incomplete adjuvant
- Antigen in aqueous solution, mineral oil, and
emulsifying agent
- Antigen is then released very slowly from
injection site
- Based on Freund’s complete adjuvant which
contains heat-killed Mycobacteria
 Haptens
- Too small, lack immunogenicity
- If hapten is coupled to carrier protein, immune
response can be induced
 Hapten-Carrier conjugate
- Produces 3 types of antigenic determinants
 Antibodies to hapten
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 Antibodies to carrier
 Antibodies to hapten-carrier conjugate

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