1
Q
HbH
A
B4 tetramer
2
Q
HbBarts
A
y4 tetramer
3
Q
Thalassemia
A
decreased (imbalanced) production of normal globin chains (quantitative disorder0
4
Q
Thalassemias result in… (4)
A
- anemia
- Bone marrow expansion (to compensate for ineffective erythropoiesis),
- extramedullary hematopoiesis,
- increased intestinal iron absorption
5
Q
Alpha globin present on chromosome ___
A
16
6
Q
Beta globin present on chromosome ____
A
11
7
Q
Microcytic anemias (4)
A
I. Iron deficiency
ii. Thalassemia syndromes:
1. A-thal, B-thal, sickle thal, HbE syndromes
iii. Severe lead poisoning (children)
iv. Chronic disease/inflammation
8
Q
Explain the meaning of the terms thalassemia major, thalassemia intermedia, and thalassemia minor
A
Minor: mild anemia, asymptomatic trait state
Intermedia: moderate anemia, intermittent transfusions
Major: severe anemia, transfusion-dependent
9
Q
HbE
A
B-globin gene mutation (glu → lys, at position 26)) → creates unstable mRNA → less production
10
Q
A-thal trait
A
2 a gene deletion = thalassemia minor
11
Q
Hbh disease
A
3 a gene deletion = thalassemia intermedia
12
Q
Hydrops fatalis
A
4 a gene deletion = thalassemia major
13
Q
Clinical manifestations of alpha thalassemias:
___ RBCs = ___ MCV
A
Small RBCs = ↓ MCV (mean corpuscular volume)
14
Q
CM alpha thal:
____ MCHC and MCH
A
Low
15
Q
CM alpha thal:
____ RBCs = ____ RDW
A
NORMAL
16
Q
CM alpha thal:
___ in RBC production = ____ RBC
A
compensatory increase in RBC production = increased RBC
17
Q
CM alpha thal:
____ red cell survival = ____ reticulocyte count
A
decreased
increased
18
Q
CM alpha thal:
____ of intracellular RBC contents (such as: ____, _____, and ____)
A
increase
Indirect (unconjugated) bilirubin, Lactate dehydrogenase (LDH), and Aspartate aminotransferase (AST)
19
Q
spleen in alpha thalassemia
A
splenomegaly
20
Q
Peripheral smear of alpha thalassemia (3)
A
- Red cells much smaller (microcytosis, low MCV)
- Target cells
- Hypochromia
21
Q
Diagnosis of alpha thal:
2 genes deleted->
A
microcytosis
22
Q
Diagnosis of alpha thal:
3 genes deleted->
A
anemia, microcytosis
23
Q
Diagnosis of exclusion for alpha thal (3)
A
microcytosis, without iron deficiency, and with normal electrophoresis
24
Q
Clinical manifestations of Cooley’s anemia (6)
A
- dense skull (frontal bossing)
- Marrow expansion (hair on end finding)
- Splenomegaly
- Osteopenia/bone changes
- Iron overload (increased intestinal iron absorption, and dependent on blood transfusions to survive)
a. → Growth and endocrine failure (diabetes, thyroid problems, gonadal hormone issues)
b. ⅔ Cooley’s anemia pts have abnormal endocrine function
c. Hypothyroidism present in 40-60% of pts with B-thal major - Increased risk for pulmonary hypertension
25
Hemoglobin electrophoresis findings for B thal (2)
timing is important! (too soon, no B-chain being produced yet)
1.↑ HbA2, ↑ F in milder forms
2.No HbA in Cooley’s Anemia (no B-globin production)
********Must be sure person is NOT iron deficient
26
Thalassemia geographical distribution
Thalassemia most common in
- SE Asian,
- African
- Mediterranean descent.
27
SE Asians common alpha globulin genotype
(--/aa) → more likely to have child with hydrops fetalis
28
Africans common alpha globulin genotype
(a-/a-)
29
Treatment for alpha thalassemia
Most people require NO therapy
1. HbH disease: may require intermittent transfusions
2. May account for mild anemia, splenomegaly, bilirubin gallstones
3. Iron should NOT be prescribed for microcytosis (can have iron overload due to increased iron absorption)
4. Genetic counseling (avoid fetal hydrops via in utero bone marrow transplant)
30
Treatment for Cooley's anemia (3)
1. Transfusion therapy
2. lnduction of fetal hemoglobin
3. Bone marrow transplant
31
In transfusion therapy in the treatment of Cooley's anemia, you want to maintain hemoglobin at ___ g/dL (transfusions every ____ weeks)
9-10 g/dL
| 3-5 weeks
32
Problem with transfusion therapy
Results in iron overload
a. Body has NO fixed method to lose iron (fixed at 1 mg/day)
b. 1 mL: of pure RBCs = 1 mg Fe, 1 unit RBC//month → 3-4 g Fe/yr
c. Can result in:
i. Hepatic fibrosis and cirrhosis
ii. Endocrinopathies (hypothyroidism, growth failure, DM)
iii. Cardiomyopathy and conduction disturbances (sudden death)
33
How do you prevent iron overload in transfusion therapy
- chelation therapy
| - erythrocytapheresis
34
Common iron chelating agents (3)
1. deferoxamine
2. deferasirox
3. deferiprone (only one that removes Fe from heart)
35
Treatment to induce fetal hemoglobin (2)
hydroxyurea and butyrate
36
Results newborn screen:
FBart's
Genotype?
alpha thal
37
Results newborn screen:
F
Genotype?
B0-thal
38
Results newborn screen:
FA
Genotype?
B+-thal
39
Results newborn screen:
FE
Genotype?
HbEE or HbEB0 thal
40
Results newborn screen:
FEA
Genotype?
HbEB+ thal
41
Diagnosis?
```
decreased MCV
normal RBC
>13 MCV/RBC
increased RDW
decreased ferritin
Hb Electrophoresis normal
Response to iron? yes
```
Fe deficiency
42
Diagnosis?
decreased MCV
increased RBC
a-thal
43
Diagnosis?
decreased MCV
increased RBC
B-thal
44
Molecular basis of sickle cell
AR - qualitative hgb disorder
-both B-globin genes mutated
45
Sickle Cell Disease B-globin genes vs. Sickle Cell Anemia B-globin genes vs. sickle cell trait
Disease: HbS
1 with single AA substitution (Beta6 glu→ val), 1 that is abnormal
Anemia: HbSS
Both genes have Sickle Cell AA substitution
Trait: 1 Sickle Cell gene, 1 NORMAL gene
46
HbC mutation
glu → lys (B gene position 6) = sickle cell disease (HbSC) or Hemolytic anemia (HbCC)
47
HbE mutation
glu → lys (B gene, position 26) = Thalassemia (HbE B-thal), Sickle cell disease (HbSE)
48
Lab findings for sickle cell:
```
Anemia?
Retic count?
WBC and platelet count?
Chemistries?
RDW?
peripheral smear?
```
- Chronic anemia
- Retic count increased (compensatory)
- increased WBC and platelet count
- increased RDW
- abnormal peripheral smear
49
Peripheral smear in sickle cell (5)
1) Howell-Jolly bodies (evidence of splenic dysfunction - remnants of nuclear DNA)
- -> Purple dots in RBCs
2) Nucleated RBC (red cells that have not extruded nucleus)
3) Polychromasia (blue colored retics, larger in size)
4) Sickled RBC cells
5) Target cells: seen in Hb SBothal, Hb SB+thal, and a little in Hb SC
NOT seen in HbSS or Sickle trait
50
Target cells are seen in the peripheral smear for _______, _______, and ______ but NOT seen in _______ or _______
seen in Hb SBothal, Hb SB+thal, and a little in Hb SC
NOT seen in HbSS or Sickle trait
51
Sickle trait
genetic carrier state (Bnormal + Bsickle) - does NOT develop sickle cell disease
Normal CBC
52
Potential adverse effects or associations with sickle trait (6)
1) Microscopic hematuria
2) Renal papillary necrosis (gross hematuria)
3) Isosthenuria (mild urinary concentrating defect)
4) Increased risk of chronic kidney disease and blood clots
5) Splenic infarction (altitude of depressurized flight)
6) Exertional heat illness/rhabdomyolysis/death (sports-related)
53
Sickle cell anemia (HbSS)
```
B-globin genes?
Hb levels?
Retic count?
Size of RBC (MCV)?
Relative clinical severity?
```
B-globin genes - S + S
Hb levels - very low (6-9 g/dl)
Retic count - 5-30% (much higher)
Size of RBC (MCV)? normal
Relative clinical severity? 4+ (very severe)
54
Sickle-Bo thalassemia (HbS Bo)
```
B-globin genes?
Hb levels?
Retic count?
Size of RBC (MCV)?
Relative clinical severity?
```
B-globin genes - S + Bo (no normal B-globin)
Hb levels - very low (6-9 g/dl)
Retic count - 5-30% (much higher)
Size of RBC (MCV) - small
Relative clinical severity - 4+ (very severe)
55
Sickle-Hemoglobin C (HbSC)
```
B-globin genes?
Hb levels?
Retic count?
Size of RBC (MCV)?
Relative clinical severity?
```
B-globin genes - S + C
Hb levels - low (10-12 g/dl)
Retic count - 3-5% (higher)
Size of RBC (MCV) - normal
Relative clinical severity - 2+ (moderate severity)
56
Sickle-B+ Thalassemia (HbSB+)
```
B-globin genes?
Hb levels?
Retic count?
Size of RBC (MCV)?
Relative clinical severity?
```
B-globin genes - S + B+ (some normal B-globin)
Hb levels - slightly lower (11-13 g/dl)
Retic count - 3-5% (higher)
Size of RBC (MCV) - small
Relative clinical severity - 2+ (moderate)
57
Sickle cell trait
```
B-globin genes?
Hb levels?
Retic count?
Size of RBC (MCV)?
Relative clinical severity?
```
B-globin genes - normal + S
Hb levels - normal (14-16 g/dl)
Retic count - 1-2% (normal)
Size of RBC (MCV) - normal
Relative clinical severity - +0 (normal)
58
Pathophysiology of cell sickling:
Deoxygenation --> ?
Re-oxygenation --> ?
glu → val (hydrophobic → charged AA)
- Deoxygenated sickle Hgb polymerizes into 14 strand helical fibers → Hgb precipitates out of solution, distorted sickle form of RBC
- Reoxygenation → polymers dissolve, RBC returns to normal shape
-After several deox-ox cycles → permanently sickled → lysed (Destroyed)
→ Increased vaso-occlusion
→ Decreased NO bioactivity
-Presence of some normal Hgbs or HbC interferes with polymerization, lessens severity
59
Normal vs. Sickle RBCs
Normal: biconcave disc-shaped, pliable, easily flow through small blood vessels, lives for 120 days
Sickle: sickle-shaped, rigid, stick (even when not sickled), lives
60
Acute complications of SCD (4)
1) Acute chest syndrome
2) Infections
3) Spleen sequestration --> infarction
4) Stroke
61
Chronic complications of SCD (6)
1) Sickle Lung Disease
2) Sickle Nephropathy
3) Retinopathy
4) Skin ulcers (legs)
5) Avascular necrosis - femoral/humeral heads
6) Splenic infarction
62
Acute Chest Syndrome in SCD occurs because...
Diagnosis? (2 criteria)
Treatment?
sickle RBCs trapped in lung circulation → damage vessel endothelium → fluid leak into lungs → compromise ability to oxygenate blood
-Most common acute cause of death in SCD
Diagnosis: new pulmonary infiltrate on CXR AND evidence of lower airway disease (cough, SOB, retractions, rales, CP)
Treatment: rapid transfusions
63
Risk for infections in SCD due to...
Treatment?
- Increased risk for encapsulated bacteria due to splenic dysfunction
- Preventable cause of death in children with penicillin prophylaxis
64
Aplastic crisis signs/symptoms (2)
1) retic count very low
| 2) severe anemia, pallor (transient)
65
Aplastic Crisis and Parvovirus B19 connection
- Aplastic crisis = sudden drop in hemoglobin
- Parvovirus B19: infects RBC precursors, arresting RBC development into mature cells
(Transient, Common in children)
Sickle cell disease patients rely on increased retics to compensate for increased RBC destruction anything that compromises bone marrow’s ability to rapidly produce RBCs → aplastic crisis
66
Splenic sequestration
prior to splenic infarction, blood can flow into sinusoids, but can’t flow out → spleen engorged and precipitous drop in Hgb → can lead to death
67
Sickle Lung Disease
- Present in 25-40% of sickle cell patients
- Severe pulmonary HTN in up to 28%
-Progressive obliteration of pulmonary vasculature
Intimal hyperplasia, micro interstitial fibrosis, plexiform lesions
-Most common cause of death in adults with sickle cell disease.
68
Sickle Nephropathy
occurs in 10-15% of sickle cell patients
Results from adhesion of sickle red cells in afferent/efferent arterioles
→ Mesangial cells phagocytose RBC fragments → get deposited on basement membrane
Findings:
- initial hyperfiltration and enlarged glomeruli (creatinine NOT a good measure)
- Microalbuminuria/proteinuria (protein loss in urine)
- Focal segmental glomerulosclerosis (FSGS)
69
Retinopathy in SCD
11-45% of sickle cell patients
retinal vessel damage→ retinal detachment, hemorrhage, and blindness
70
Skin ulcers in SCD due to...
decreased peripheral blood flow
71
Treatments of sickle cell disease (5)
1) Folic acid
2) Prohylactic penicillin
3) bone marrow transplantation
4) hydroxyurea therapy
5) transfusion therapy
72
Folic acid used in SCD to treat...
developmental delays caused by anemia
73
Bone marrow transplantation in SCD
transplantation done with HLA-matched full sibling unaffected by sickle cell disease with a greater than 90% disease free survival.
- Only 20% of eligible patients have such a donor available.
- “The cure”
74
Hydroxyurea therapy
1) oral chemotherapy agent that induces production of HbF
2) Interferes with sickle hemoglobin polymerization
3) Improves anemia, increases MCV, decreases WBC count (decreased adhesion molecules)
4) Reduces frequency of acute pain crises and reduces mortality
5) No evidence of reduction in chronic organ injury
6) Only FDA approved drug for sickle cell disease
75
Transfusion therapy
| two types
“Dilutes out” sickle RBCs (keep sickle
76
Risks of transfusion
Increased hyperviscosity if transfused to a hemoglobin >10 g/dL
Associated with transmission of infection, allo-immunization (antibody formation to donor blood), iron overload
77
Exchange vs. Simple transfusion therapy
- Exchange transfusion: remove pts sickle RBCs, replace with normal RBCs
- Simple transfusion: dilution by transfusion of normal RBCs
78
Iron Chelation Therapy
chelating agent binds excess iron
Indications: people who receive multiple transfusions
Drawbacks: compliance with therapy is challenging
Infused subcutaneously over 8-12 hours, usually in abd area, 5-7 times a week
79
Sickle Solubility Testing (aka sickledex)
limitations?
Patient blood sample → hemolyze it → release Hgb into fluid → sickle Hgb precipitates out, normal Hgb is translucent
Detects sickle Hgb in concentrations as low as 8-20%
→ CANNOT distinguish sickle trait from disease or type of disease
80
Hemoglobin separation
limitations?
Lysate RBCs - gel electrophoresis → different charges of hemoglobins → travel at different rates
Measure relative % of types of Hgb in sample as a whole
Does NOT necessarily reflect relative % of types of hemoglobin within each RBC
81
Isoelectric Focusing
Similar to Hemoglobin separation
Benefits: higher resolution (able to better separate bands), able to run a lot of samples at same time
Different hemoglobins migrate to isoelectric point
82
High performance liquid chromatography (HPLC)
Abnormal Hgb electrophoresis → HPLC → peaks and spikes that allow for accurate depiction of relative quantities of different hemoglobins
83
Newborn screening result:
FS
Genotype?
SS or SBo-thal
84
Newborn screening result:
FSA
Genotype?
SB+-thal
85
Newborn screening result:
FSC
Genotype?
SC
86
Newborn screening result:
FAS
Genotype?
AS
A = normal
87
Newborn screening result:
FA
Genotype?
normal
88
Serum vs. plasma
serum = plasma after it is clotted
89
Heavy (H) chain
- MW=50,000
- each antibody has 2 H chains
- Each H chain has 1 variable domain (VH) and 3-4 constant domains (CH1, CH2, CH3, (CH4))
- 5 kinds of H chains (gamma, alpha, mu, epsilon, delta—each corresponds to the appropriately named antibody: (EX-IgA has alpha chains)
90
Light (L) Chain
MW= 25,000, each antibody has 2 L chains
-Each L chain has 1 variable domain (VL) and 1 constant domain (CL)
91
Kappa and Lambda chains
2 types of L chains
Each cell that makes antibody has a choice, but it uses only one kind.
92
Hinge region of antibody
allows for flexibility so when bound to antigen, constant part of antibody can change conformation
93
Fab vs. F(ab')2
Fab = S--S bonds between the H and L chains - can be fully reduced (key part of key)
F(ab')2: 2 Fabs joined by S—S bond
94
Fc
non-antigen binding region of the antibody, makes antibody participate in complement (handle of key) - can bind to cells
95
Complementarity-Determining Regions (CDRs) - aka?
aka Hypervariable region
- comprise the actual antigen-binding site.
- region in V domain with most of the variability for antigen specificity
- 3 hypervariable regions on V domain of L and H chains
- not all of hypervariable region needs to interact with each antigen
96
Epitope
- specific part of antigen that interacts (non-covalently) with specific part of antibody (dependent on protein folding)
- Can be continuous or discontinuous
- Can be carbohydrates, nucleic acids, or most commonly, proteins
97
Variable (V) domain
at N-terminal of antibody
- determines specificity for one antigen or another
- VL and VH interact with antigen in their hypervariable regions (3 per chain)
98
Constant (C) domain
region that is essentially identical from antibody to antibody
99
IgG structure
2 light and 2 gamma (heavy) chains
100
IgE structure
light and 2 epsilon (heavy) chains
| extra constant domain
101
IgD structure
light and 2 delta (heavy) chains
| extra long hinge region
102
IgA structure
- 4 light, 4 alpha (heavy) chains (2+2 dimer) + 1 Joining chain + 1 secretory component
- Designed to be secreted in mucosa, protected from digestion via secretory component
- 10 total polypeptide chains (4L, 4H, 1J, 1SC)
- Valence is 4
103
IgM structure
10 light, 10 mu (pentameter with 2L + 2mu chains) and 1 joining chain (J chain)
Valence is 10
104
IgG size and serum concentration
150,000
1000 mg/dL
105
IgA size and serum concentration
Secreted 400,000 (monomer is 160,000, J chain is 15,000 and SC is 70,000)
200 mg/dL
106
IgM size and serum concentration
900,000 (5x 180,000, an extra CH4 domain + J chain)
100 mg/dL
107
Antibody combining site
where antibody binds to antigen
Made up of V domains of both the H and L chain (VH and VL)
108
Ab Subclass
immunoglobulins are divided into subclasses because of slight differences in the amino acid sequences of their H chain C regions.
EX) IgG1, IgG2, IgG3, IgG4. IgA1, IgA2. IgM1, IgM2. IgD and IgE
109
Ab Allotype
minor allelic differences in the sequence of immunoglobulins between individuals
Determined by allotypes of your parents, useful in determining relatedness
110
Ab Idiotype
unique combining region, made up of CDR aa of its L and H chain - can create an antibody that can recognize another antibody (becomes the antigen)
Anti-idiotype: antibodies made that recognize the unique sequence of that combining site.
111
Define valence in regards to antibodies
number of antigenic determinants that an individual antibody molecule can bind
i. Valency of all antibodies is at least two (divalent) and in some instances more (multivalent).
112
Define affinity in regards to antibodies
strength with which an antibody molecule binds an epitope (= antigenic determinant)
113
Define precipitation in regards to antibodies
large immune complexes that are formed at or near equivalence tend to become insoluble and fall out of solution, when the antigen is a molecule, it is called precipitation.
114
Define agglutination
large immune complexes that are formed at or near equivalence tend to become insoluble and fall out of solution, when the antigen is a cell or cell-sized particle, it is called agglutination
115
Epitope
part of antibody that actually interacts with antigen (aka antigenic determinant)
i. Usually 10-20 amino acids long.
ii. Proteins have several epitopes which bind to different antibodies.
116
Ab that can cross placenta
IgG
117
Ab with greatest ability to activate complement
IgM
118
Characteristics of IgG (4)
i. Comes up later than IgM after primary immunization, but levels go higher and last longer
ii. Plasma half life = 3 weeks
iii. Phagocytic cells have receptors for the Fc of bound IgG→ opsonizing (vital for clearance of most extracellular bacteria)
iv. Takes 2 IgG’s close together to activate complement (need high density of epitopes on antigen)
119
First immunoglobin seen in blood after immunization and only antibody made in the fetus
IgM
120
Characteristics of IgM (4)
i. Decavalent, but shape rarely allows more than 2 of its 10 binding sites to interact with antigenic determinants (epitope)
- Best at complement because it always has 2 adjacent Fc’s to begin complement cascade.
ii. large → trouble getting from blood into tissues
iii. viscous in solution (because of its size), so if we only had IgM we couldn’t pump our blood
iv. No useful IgM receptors on phagocytes.
121
IgA
made by plasma cells in lymphoid tissues near mucous membranes, assembled into dimer by addition of J chain while in plasma cell and then secreted into interstitial space.
122
Process of IgA generation and distribution to interstitial space
Adjacent epithelial cells have receptors for IgA → binds to them, taken up and moved to luminal side → IgA exocytosed, still bound to receptor (now called Secretory Component (SC))
NOTE: SC protects IgA from digestion in gut → first line of immunological defense against invading organisms.
123
IgD role
only important role is as a B cell receptor - trigger for activation of antibody forming cells
124
IgE
its Fc adheres to mast cells and basophils→ trigger histamine loaded cells, causes immediate hypersensitivity or allergy.
125
IgE and parasites
Important for resistance to parasites when it triggers mast cells to release eosinophil chemotactic factor→ eosinophils come and kill parasites
126
Quantitative precipitin test
mix antigen + antibody in different ratios, see how much precipitate forms
i. Antigen or antibody excess → less precipitate because complexes are smaller, not every molecule gets bound
ii. Antigen + antibody bound = immune complex → optimal equivalence at max precipitate level
127
Immunodiffusion
i. Antibody in one well, antigen in another → diffuse radially out of wells
ii. Precipitate forms in agar where antigen and antibody meet in optimal proportions (more immune complex forms and precipitates out of solution)
128
Define complement
main inflammatory mediator of humoral immune system
i.Large number of proteins - exists in inactive form → first is activated, then rest follow in cascade
129
Now complement Maddie, cause she is awesome
So many to choose from
130
3 ways to activate complement cascade
1. Classical pathway
2. Alternative pathway
3. Lectin pathway
131
Classical pathway
- Used by IgG/IgM for bacterial invaders
- Antibody interaction with antigen → change in Fc end of antibody → allows binding and activation of C1q
- C1 → activate C4 → activate C2 → C2+C4 activate C3 → activate C5
132
C1q must interact with..
TWO Fcs simultaneously (2IgGs close together or one IgM)
133
C3 and C5 role in the complement (3)
they are responsible for opsonizing, chemotaxis and anaphylastoxic
134
Classical pathway sequence (simplified)
1,4,2,3,5,6,7,8,9
135
Alternative pathway
structures of microorganisms (dextrans, levans, zymosan, endotoxin) activates cascade
a. Bacterium can activate C3b this way in ABSENCE of antibody→ Part of innate immune system
b. Cell wall structures + IgA provide surface for binding of properdin (P) → anchor for assembly of C3b, factor B, and factor D → stable C2bDbC3b complex → activates C5 → 6-7-8-9 activated
136
Lectin pathway is mediated by....
Mannose-binding protein (MBP), or MBL (a lectin)
a. Mannose = found in carbs of bacteria, but NOT humans
- Functionally similar to C1q
b. Lectins = proteins that bind foreign carbohydrates
137
Lectin pathway steps
MBP + proteases (MASPs) → activate C2 and C4 → 3-5-6-7-8-9
| *** Innate immunity
138
Complement components that are opsonizing (2)
1. C3b adheres to antigen membrane → phagocytic cells have C3b receptors → firm grip on antigen opsonized with C3b.
2. IgG is also opsonizing because phagocytes have receptors for its Fc end called FcR.
139
Complement components that are lytic
membrane attack complex (MAC) activated when C5 activates C6-C7-C8-C9 (attack complex)
140
C8 and C9 form...
a lesion on target cell membrane (looks like a hole on electron microscopy) → cell loses ability to regulate osmotic pressure and lyses or pops.
iEX) Neisseria (gonorrhea, meningitis) most susceptible to C lysis
141
Complement components that are anaphylatoxic (3)
C3a, C4a and C5a can all release histamine from mast cells by binding → increase blood flow to area of antigen deposition, better chance for inflammatory cells to get out of blood and into tissues.
142
Complement components that are chemotactic (1)
the C5 activation product, C5a, is chemotactic for phagocytes, especially neutrophils.
143
What happens if a bacterium is resistant to lysis by C9?
- Not all bacteria need to complete complement pathway all the way to C9
- Many can be killed via opsonizing with just C5 activation
144
The most susceptible family of bacteria to lysis is...
Neisseria (gonorrhea and meningitis)
145
Hemolysis (define)
decrease in red cell survival or increase in turnover beyond standard norms
146
Normal RBC turnover:
most turnover in _______, small amount (10%) ___________
spleen (extravascular)
intravascularly
147
Intravascular Hemolysis (4 steps)
1) RBC releases hemoglobin into circulation
2) Hgb dissociates into dimer
3) binds haptoglobin
4) removed by liver
148
If haptoglobin is overwhelmed, what happens to intravascular hemolysis? (4 steps)
1) Hgb iron oxidized to methemglobin
2) dissociation of globin releases metheme
3) metheme binds albumin
4) converted to bilirubin
149
Extravascular hemolysis (8 steps)
1) RBC ingested by macrophage in spleen
2) heme separated from globin
3) iron removed and stored in ferritin
4) prophyrin ring converted to bilirubin
5) bilirubin released from cell and taken up by liver
6) bilirubin made water soluble with addition of glucuronic acid and secreted into SI
7) glucuronic acid removed and bilirubin converted to urobilinogen
8) urobilinogen cycles between gut and liver or is excreted by kidney into urine
150
Classification of Hemolytic anemia:
2 questions
1) Is anemia associated with other hematologic abnormalities?
NO → Is there an appropriate reticulocyte response to anemia?
2) YES → Is there evidence of hemolysis? (↑ bilirubin, ↑LAD, ↓ haptoglobin, hemosiderin in urine)
YES → evaluate for cause of hemolysis
NO → evaluate for hemorrhagic cause of anemia
151
CBC findings for hemolytic anemia:
```
retic count
bilirubin
haptoglobin
metheme/methemalbumin
housekeeping enzymes
Hemoglobin in urine?
```
retic count ↑
bilirubin ↑ (unconjugated fraction bilirubin ↑)
serum haptoglobin ↓
metheme/methemalbumin ↑
housekeeping enzymes (LDH, SGOT) ↑
Hemoglobin in urine? YES
152
Hereditary spherocytosis characteristics (4) and hallmark
- anemia
- jaundice (intermittent)
- splenomegaly
- responsiveness to splenectomy
153
Hereditary spherocytosis hallmark and pathophysiology
Hallmark: loss of plasma membrane and formation of the microspherocyte
Patho:
spectrin, ankyrin or band 3 defects weaken the cytoskeleton and destabilize the lipid bilayer → microspherocyte → decreased deformability, entrapment in spleen (red cell survival
154
Treatment for hereditary spherocytosis (2)
supportive care for anemia
splenectomy
155
Lab findings of hereditary spherocytosis:
Variable HCT and HGB
↑ retic count/index
↓ MCV, ↑ MCHC
Spherocytes on smear (NOT diagnostic)
Unconjugated hyperbilirubinemia
Increased osmotic fragility
156
Clinical complications for hereditary spherocytosis (2)
aplastic crisis (rapid, severe, life-threatening anemia)
bilirubin stones (increased bilirubin in biliary tree leads to stones in gall bladder).
157
G-6-PD enzyme deficiency and hemolytic anemia
inheritance?
clinical features?
- X-linked recessive
- protective for malaria
intermittent episodes of acute hemolytic anemia, hyperbilirubinemia associated with oxidant stress, hemolysis, reticulocytosis
Peripheral Smear: occasionally shows microspherocytes, blister or bite cells.
158
G-6-PD deficiency pathophysiology (4 steps)
1) G-6-PD deficiency → can’t restore reduced glutathione
2) Oxidant stress → denatured Hgb attaches to membrane (Heinz bodies) and spectrin damaged (oxidized)
3) → Decreased deformability of RBC
4) → splenic trapping and extravascular hemolysis (sometimes intravascular)
159
G-6-PD deficiency treatment
avoid oxidant food/drugs, supportive care, folate, transfusion for severe anemia
160
Pyruvate Kinase deficiency presentation (5)
variable chronic anemia, hemolysis, splenomegaly, gallstones, aplastic crises.
161
Pyruvate Kinase deficiency labs and treatment
Labs: mild-severe anemia, ↑ retic, no specific morphology.
TX: supportive care, folate, transfusions. Splenectomy may help with disorder.
162
Autoimmune hemolytic anemia
Cold vs. warm antibodies
-auto-antibodies to universal red cell antigens causes hemolysis
COLD:
IgG or IgM (“cold antibodies”) transiently bind RBC membrane in cool areas of body
→ Move back to central circulation → avidly activate complement through C5-9 and create holes in plasma membrane → INTRAvascular hemolysis
IgG (“warm antibodies”) bind RBC membrane with high affinity → very little and incomplete complement activation → EXTRAvascular hemolysis
163
Test for autoimmune hemolytic anemia with the _______ aka _______ test
Antiglobulin or Coombs test
164
Clinical findings of autoimmune hemolytic anemia
acute or chronic anemia
pallor
jaundice
dark urine
165
Lab findings for autoimmune hemolytic anemia
Smear shows spherocytes, teardrop or “bite” cells.
Presence of DAT
Mild to severe decrease in Hgb
increase in retic
increase in bilirubin, hemoglobinemia/uria (depending on presence of intravascular component)
166
Splenectomy complications and amelioration of complications
Complication:
1) overwhelming bacterial sepsis
2) spleen = origin of IgM agglutins --> problems with immunity
AVOID splenectomy at all costs in kids under 5 years
Amelioration:
1) pre-surgery vaccination
2) prophylactic abx
3) close monitoring of fevers
167
Direct antiglobulin test (DAT)
direct Coombs test
measure cell directly for presence of IgG, C3d, C4d on surface of RBC
-Autoimmune hemolytic anemia = positive DAT
168
Indirect antiglobulin test
indirect Coombs test
measure what is in the plasma
detect ability of patient's serum to bind IgG and/or complement to test normal RBCs
169
Cross-reactivity
give good and bad example
endency of one antibody to react with more than one antigen at CDRs - good and bad
EX) immunize with cowpox → antigenic determinants of smallpox also recognized and person will be immunized
EX) heart valves contain lamin antigen that cross-reacts with streptococci - infection can lead to inflammatory process in heart = Rheumatic heart disease
170
Instructive Theory
Lamarckian
Antigen tells immune system to make antibody of appropriate conformation by some change in genetic info
WRONG
171
Clonal Selection Theory
Darwinian
Entire population of potential antibody making cells preexists in a normal individual, prior to contact with antigens
Each cell of immune system programmed to make only ONE antibody - choice is random, not dependent on outside information
Antigen gets into body → eventually will come into contact with receptor it binds with sufficient affinity to activate it → expansion of that clone and production of that antibody
The best fitting clones are SELECTED by antigen
172
Allotypic exclusion
Only 1 H chain (maternal or paternal in origin) and one L chain (either kappa or lambda, either maternal or paternal) are synthesized in and ONE B cell
Choose: 1 H chain (from 2 choices) and 1 L chain (from 4 choices)
173
Heavy chain V domain contains _____ gene regions
Light chain V domain contain ______ gene regions
VDJ + C (mu, delta, gamma, alpha, etc.)
VJ (no D) + C (kappa, lambda)
174
How are heave chains made from genes?
Each cell will chose one of its V’s, one D, and one J to make a VH domain gene region
1) Developing B cell brings one random D segment close to one J → DNA cut → intervening DNA discarded → ends joined, D2 now next to J2
2) Then brings V segment up to recombined DJ → cut and join → V7 next to D2 and J2
3) Entire VDJ unit (including constant domain of both mu and delta) transcribed into nuclear RNA = primary RNA transcript
4) Primary RNA transcripts processed by RNA splicing → makes one VDJ-mu (VDJC) and one VDJ-delta (VDJC)
175
How are light chains made from genes?
Same concept as heavy chains
except only V and J segments + C domain gene (kappa or lambda)
176
RAG recombinase
-enzyme that does recombination of antibody and T cell receptor DNA
-Bind splice signals (to right of D segment and left of J segment)
→ pull them together, cut, and splice
- Bind splice sequence (to right of V segment) → pull, cut, splice
- RAG knocked out → make no B or T cells = Omenn Syndrome
177
Class switching
ALL B cells start by making IgM and IgD → may switch later to IgG, IgE, or IgA
V domain stays the same, but the C region on H chain changes
A cell which is making IgM can go on to make IgG, BUT a cell making IgG CANNOT go back to making IgM because the mu gene is physically GONE.
order of C genes: mu, delta, gamma1, gamma2, epsilon, alpha
178
Hypermutation
each time B cell divides after antigenic stimulation, there is a good chance 1 of the daughter cells will make a slightly different antibody → selection by antigen for best-fitting mutants allows for gradual increase of affinity during immune response = affinity maturation
179
Hypermutation mechanism
1) Antigen binds Ab
2) Ab begins dividing
3) → Activation-Induced (cytidine) Deaminase (AID) converts a random cytosine in the CDR region → uracil (CG pair → UG mismatch)
4) Uracil removed by repair enzyme and error-prone DNA polymerases fill in the gap creating mostly single-base substitution mutations so at the end of cell division the daughter may be making a different antibody.
180
N region diversity
somatic mutation mechanism that produces antibody diversity - "sloppiness of V-D, D-J end joining"
1: exonucleases chew away a few nucleotides after DNA is cut but before gene segments (D to J and V to DJ) are joined.
2: enzyme Terminal Deoxynucleotidyl Transferase (TdT) adds nucleotides without a template (random!) → can’t predict sequence at joining area “N region”
181
Bursa of Fabricius
where precursors from bone marrow go to finish their development
182
B cells start and continue to mature in __________
T cells start in ________ and move to ________ where they are selected for _________
bone marrow
bone marrow --> thymus
selected for self+antigen reactivity
183
B cell development (4 types of cells)
1) Pro B cell progenitor
2) PreB cell
3) Immature B cell
4) Mature B cell
184
Pro B cell progenitor - identifiable when they begin to make _______________
detectable cytoplasmic my chains
185
Pre B Cell - cell with ________, but no _______
cytoplasmic IgM
NO surface IgM
186
Immature B cell - cell with _______
Also undergoes _________
surface IgM only - can interact with outside world
clonal abortion/selection for self+antigen
187
Clonal Abortion
explains why we don't make antibody to self
In bone marrow pre-B cells differentiating into immature B cells and any cell whose receptors happen to be anti-self will almost surely encounter “self” in the bone marrow and will either make a new receptor or will be aborted.
If it does not encounter antigen (not anti-self) then it will mature further so it expresses IgM and IgD.
Then when it meets antigen, it will be stimulated.
188
Mature B cell has ______ and _______
IgM and IgD on cell surface
189
Antibody response to antigen - primary vs. secondary response
1) Primary response:
IgM secreted first, then T cells help B cells switch to IgG (or IgA, or IgE) - delayed IgG production
2) secondary response (after already being exposed to antigen)
IgM response same, but IgG response is sooner, faster, higher, and more prolonged due to immunological memory
190
Antibody levels before birth and after
Before birth:
- IgM made by the fetus before birth (IgM cannot cross placenta)
- Mom’s IgG actively transported across placenta so at birth baby has 100% of adult levels
After birth:
-mother’s IgG levels drop (half life=3 weeks).
-3-6 months after birth: baby begins to make its own IgG.
Most vulnerable time for babies at 6 mos, when mom’s IgG is low and baby’s IgG is low
-IgA starts at 2-3 months.
191
If a newborn has a positive antibody titer it's important to ask if it is positive to IgM or IgG. Why?
If the antibody is IgM, we know that it was produced by the baby (the baby was exposed to something) because mom’s IgM cannot cross the placenta
If the antibody is IgG, it is from the mother (IgG can cross the placenta).
192
IgG has a half life of...
3 weeks
193
What happens to your immune response as you get old?
Old people have fewer naive cells, but lots of memory cells
-Can't completelty reconstitute their T cell numbers and diversity by age 40
Young people: have more naive cells, but fewer memory cells
Blood and Lymph
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