Week 8 Flashcards
Blood group antigens
glycolipids (carbohydrates) found on surface of ALL body cells (including RBCs)
Terminal sugars confer antigenic specificity (A,B, or O)
ABO antigen found in nature → foreign one to you, you become immunized to it
Type A blood
AA or AO genotype
Glycosyl ABO transferase allele puts additional A sugar on H antigen
→ make anti-B ab
Type B blood
BB or BO genotype
Glycosyl Transferase ABO allele adds B sugar
→ make anti-A ab
Type AB blood
Glycosyl Transferase ABO allele adds both A and B antigens on their red cells
Rarest blood group
Universal recipients
Type O blood
OO genotype
No working ABO glycosyltransferase - only have basic core H antigen
Make anti A and B ab
Most useful as blood donors - can give to any phenotype
Populations tend to drift towards O blood type - A and B babies are at higher risk for hemolytic disease of the newborn
Bombay phenotype
Lack glycosyl transferase gene that puts final sugar onto core → no H antigen expression = little h
A, B and O are all incompatible (make antibody to A, B and O)
Type as O, but they are NOT O
Isohemagglutinins
ab we make to the foreign ABO antigens to us
Appear in blood around 3-6 months of age
IgM class (ab to pure carbohydrate antigen)
Rh antigens
Rh(D) positive?
Proteins D is most important one
Rh(D) positive = D dominant over d allele (DD or Dd)
92% of US blacks, 85% of US whites are RH+
DO NOT make antibody to it unless you are Rh(D)- and become immunized with Rh(D)+ red cells
Rh not ubiquitous in nature, so we don’t normally make ab to it
Electronic Crossmatch
telling which donor units are most compatible with recipient
-Identical or compatible at ABO and Rh
Major Crossmatch
what are you testing?
how are you testing it?
what do the results mean?
Are there abs in recipient’s plasma which can react with antigens in donor’s RBCs?
Mix plasma from recipient and red cells from donor in lab
Test for hemolysis or agglutination (ab to donor red cells)
None → compatible
YES → generalized complement-mediated hemolysis, free Hg deposited in kidneys → acute renal failure
Direct antiglobulin test
is there ab ALREADY on cells?
Tests cells coated with ab IN VIVO
Patient’s RBCs → mix with ab against human IgG
If IgG present on RBC surface → agglutinate
Indirect antiglobulin tests
-is there unexpected ab to RBC antigens in recipient plasma? Can be used to crossmatch recipient/donor more thoroughly
Donor RBCs + recipient plasma → wash, add antiglobulin
Detects ab that bound cells but didn’t agglutinate them initially
Heterophile antibody
Abs induced by external antigens that cross-react with self-antigens or another antigen
EX) Monospot test: ab in serum of patient with mono reacts with horse RBCs
EX) Syphilis: ab in serum of patient with syphilis, reacts with Treponema pallidum
Hemolytic disease of the newborn
Rh(D)+ babies of Rh(D)- mothers
Pregnancy with Rh(D)+ fetus → mom exposed to RhD from baby → makes ab
Only a problem for subsequent pregnancy with Rh(D)+ fetus
→ make ab that crosses placenta and destroys second fetuses RBCs → severe hemolysis in the newborn (high bili)
Mother becomes ______ with subsequent pregnancy with Rh(D)+ fetus which ___________
sensitized
boosts response (more severe hemolytic disease of the newborn)
How can you prevent hemolytic disease of the newborn?
Mom given IgG ab to Rh(D) at 28 wks and when mother delivers her first Rh(D)+ baby
Rh(D) ab opsonizes fetal RBCs → mom not immunized to Rh(D)+
Sweeping antigen out of mom before immunization occurs
Must receive RhIg each subsequent delivery
WILL NOT work if you are already immune to Rh(D)
ABO hemolytic disease
Typically:
- Isohemagglutinins are IgM → DO NOT cross placenta
- Anti-Rh abs are IgG → DO cross placenta
ABO hemolytic disease: -Occasionally IgG isohemagglutinins made -Especially prevalent in group O people A or B fetuses of these women at risk of ABO hemolytic disease -NO RhoGAM-like ab for this
Congenital diseases of bleeding/clotting (4)
Hemophilia A and B (Factor VIII and IX Deficiency)
Factor XI Deficiency
Factor VII Deficiency
Acquired diseases of bleeding/clotting (6)
1) Liver Disease
2) Vitamin K Deficiency (Warfarin Admin)
3) Disseminated Intravascular Coagulation (DIC)
4) Thrombosis
5) Lupus Anticoagulant
Familial 6) Hypercoagulable State (Thrombophilia)
Prothrombin time (PT) test measures ________ of which factors?
normal time?
procoagulant activity
factors 7, 10, 5, 2, and fibrinogen
[extrinsic pathway and common pathway]
normal = 9-12 secs
INR
international normalized ration - used to standardize PT time
normal INR = 1
specifically sensitive for vitamin K dependent factors (7, 10, 2)
measure of Warfarin
vitamin K dependent factors include… (4)
7, 10, 9 and 2
Warfarin, Vitamin K deficiency, and inadequate liver function can be measured by…
PT or INR
Warfarin (inhibits VitK dependent rxns)
Partia Thromboplastin Time (PTT) measures __________ of…
normal = ?
NOT affected by?
prolonged by?
Measures procoagulant activity of intrinsic and common pathway
NOT affected by deficiencies of Factor VII
Normal = 25-32 sec
Prolonged by anticoagulant drugs (heparin, fibrin split products), hemophilia
Monitors Heparin therapy
Thrombin time measures ________
sensitive to?
normal = ?
prolonged by?
measures procoagulant activity of fibrinogen
Sensitive to anticoag effect of heparin or fibrin split products
Normal = 12-18 secs
Prolonged with heparin contamination or fibrinogen deficiency
Bleeding time measures ____________
______ and _______ will prolong bleeding time
normal = ?
measures platelet-vessel interaction, number / function of platelets
Decrease in platelets (
PFA-100 (Platelet Function Analyzer)
In vitro bleeding time, determines platelet response to agonists
Hemophilia A is a deficiency in _______, and Hemophilia B is a deficiency in ________.
Hemophilia A and B are inherited _______
A –> Factor 8
B –> Factor 9
XR
Labs for hemophilia
normal _______ and ________
low ________
prolonged ________
normal PT, normal bleeding time
low factor 8 or 9
Prolonged PTT
Symptoms of hemophilia
Severe, mild, moderate, and carriers
Severe → spontaneous hemorrhage in joints, muscles, soft tissues, CNS
Moderate → usually need trauma for bleeding
Mild → only bleed after trauma
-Diagnosed after bad trauma or surgery
Carrier females with bleeding = symptomatic carriers
Treatment for hemophilia A and B
recombinant factor treatment given by IV 2-3 times a week
Factor 7 deficiency:
inheritance
presentation
labs
AR
Presents as postoperative hemorrhage
Labs: Protime (PT) significantly prolonged, normal PTT
Disseminated Intravascular Coagulation (DIC) caused by…
Massive trauma, hemorrhagic/septic shock, burns, acute leukemia, and drug reactions
DIC mechanism
→ intravascular deposition of fibrin → thrombosis of small/midsize vessels with organ failure
Depletion of platelets and coagulation factors (fibrinogen, platelets, Factor VIII, V) → bleeding
Lab results in DIC (6)
1) Very low fibrinogen level
2) Low platelets
3) PT least affected, prolonged (in contrast to liver disease)
4) Greatly prolonged PTT (low factor 8)
5) Prolonged TT
6) Increased fibrin split products (D-Dimer)
Coagulopathy and Liver Disease
Liver problem → deficiency in clotting factors (Factor V, vitamin K dependent Factors II, VII, IX, and X.
Very severe liver disease, fibrinogen low
Lab results for liver disease (5)
1) Prolonged PT (INR)
2) relatively less prolonged PTT (low IX and X)
3) if fibrinogen low → TT prolonged
4) Low platelets
5) Important to CHECK VITAMIN K DEFICIENCY
Lupus Anticoagulant
Common acquired abnormality → hypercoagulable state
THROMBOTIC TENDENCY (Deep vein thrombosis, pulmonary embolism, etc.)
Antiphospholipid Antibody Syndrome (APS): IgG ab anti phospholipid platelet membrane or endothelial cell
Lab tests/results for lupus anticoagulant (3)
Prolongs PTT, BUT DO NOT have bleeding tendency
Test specifically with Russell’s Viper Venom Test (dRVVT)
Tests for lupus anticoagulant
1:1 mixing study
Mix patient and control plasma 1:1 → assay PTT
IF PTT corrects → factor deficiency (because factor in control blood)
If acquired ab to factor 8 (lupus anticoagulant)→ PTT will NOT correct
Serum protein electrophoresis
- Test function of humoral immune system
- Cheap, easily quantified
- Not very sensitive to small abnormalities
- Can be done on urine, CSF and blood
What kind of serum protein electrophoresis will you see for:
1) Plasma Cell Myeloma
2) Hypo/Agammaglobulinemia
3) Polyclonal hypergammaglobulinemia
1) M spike in IgG
2) peaks are small
3) wider peak
Single radial immunodiffusion
Measures levels of individual immunoglobulin classes or subclasses
-must be a multivalent antigen that can form precipitate with an appropriate ab
Antinuclear antibodies (ANA)
How to test for their presence?
abs against autoantigens in the nucleus
1) Fix with agent that makes cell plasma membrane permeable so ab can penetrate
2) Wash and mix with labeled goat anti-human IgG
Immunofluorescence vs. immunohistochemistry
Immunofluorescence:
-Used to identify antibody in patient’s tissues (direct) or in their blood (indirect)
Immunohistochemistry:
-Uses final ab labeled with an enzyme that produces a brown/black product that can be observed and archived for a long time
throat swab smeared on slide, add fluorescent-labeled antibodies to known bacterial antigens
This is an example of a ________
direct immunofluorescence test
TEST FOR PRESENCE OF ANTIGEN
known bacteria placed on slide, add patient serum → label with fluorescent goat anti-human Ig
This is an example of a ________
indirect immunofluorescence
TEST FOR PRESENCE OF ANTIBODY
Passive agglutination
antigen-coated latex particles - test to detect ab
1) Couple antigens to RBCs or latex beads
2) → add dilutions of patient’s serum
3) Look for agglutination titer
EX) Used for RF which can agglutinate latex particles coated with IgG
Advantages: simple, cheap, anyone can do it
Reverse passive agglutination
Test to detect antigen
1) Latex beads coated with abs
2) Add bead to patient fluid of choice
3) If they agglutinate → antigen present
Simple Elisa
measures antibody
1) Antigen couple to a plate
2) Add test serum
3) If there is ab to antigen it will bind
4) Identified using enzyme-coupled antibody to expected serum antibody
Sandwich/Capture ELISA
6 steps
measures antigen
1) Use 2 different monoclonal abs to specific antigen of interest
2) Capture antibody: one ab on a plate so it’s stuck there
3) Add patient serum, Wash
4) Add second ab with enzyme coupled to it
5) Add colorless substrate that produces a colored product
6) Measure intensity of product color in a plate spectrophotometer
Measuring T cell number in lab - 2 ways
1) mAbs to CD3, CD4, or CD8 → count fluorescent cells under a microscope
2) Flow cytometry
Measuring T cell function in lab (6)
1) Skin test for Th1 activity - Best overall test to measure Th1 activity
2) Challenge DTH test
3) Mitogens (PHA or Con A) → stimulate T cells, observe proliferation or IL-2, IL-4, or IFNy production
4) Chest x-ray - look at thymus in infants
5) Lymphoid biopsy - check for suspected primary immunodeficiency
6) Killer cell assays
B cell function test in lab
serum protein electrophoresis
T cell function in clinic/ward
Rapid screen
EX) strep test, dipstick pregnancy test
Flow cytometry
measures number of B and T cells
- Pump cells in suspension through orifice in single file
- Cells bound to fluorescent-tagged antibody → quantify light emitted
Virchow’s Triad
1) Decreased blood flow (venous stasis)
2) Inflammation of or near the blood vessels (altered vessels)
3) Intrinsic alterations in the nature of the blood itself (altered coagulability)
Acute Iliofemoral Thrombosis of the Leg:
Symptoms? (3)
complete obstruction of venous outflow from an extremity
1) Extremely swollen
2) Blue
3) Painful
Composition of arterial thrombi vs. venous thrombi
Arterial = white thrombi
- aggregated platelets
- small amounts of fibrin
- few red cells
Venous = red thrombi
- large amounts of fibrin
- numerous red cells
Arterial thrombi are formed under conditions of _________
Venous thrombi are formed under conditions of ________
high shear stress (vWF critical for platelet adhesion)
slow blood flow
Instigating factors of arterial thrombi (3)
ATHEROSCLEROSIS
HTN
turbulent blood flow at arterial branch points
Treatment of arterial thrombi
TIME is critical!
Acute:
1) Heparin → prevent further clot formation
2) tPA (fibrinolytic agent) → lyse existing clot
Long-Term:
1) Aspirin (inhibit COX) - ANTIPLATELET THERAPY
Instigating factors of venous thrombi
stasis, immobility, obesity, direct trauma, surgery, extrinsic compression, right-sided heart failure, presence of foreign body (IV, catheter)
Risk Factors: obesity, trauma, post-surgery, age, pregnancy
NOT HTN, hyperlipidemia → arterial problems
Pulmonary Embolus
part of thrombus breaks off and travels through major veins, past right heart, and into pulmonary artery circulation until it becomes lodged → prevents gas exchange → infarct
Signs/Symptoms: chest pain, dyspnea, anxiety, cough, syncope, cyanosis, chronic pulmonary HTN
Treatment of venous thrombosis
Anticoagulant therapy
1) Heparin - stops clot from getting bigger
2) Warfarin - prevent additional clots (slow to start working)
Clinical clues suggesting inherited hypercoaguable disorder (6)
1) First thrombosis age less than 50
2) Recurrent episodes of thrombosis
3) Family history of thrombosis
4) Thrombosis at unusual sites
5) Neonatal thrombosis
6) Thrombosis without apparent antecedent thrombogenic event (idiopathic)
D-Dimer Assay
Formed when cross-linked fibrin degraded by plasmin through fibrinolysis
Very sensitive but not specific
Positive D-dimer means further studies should be performed
high negative predictive value for DVT, but negative D-dimer rules out thrombosis
Venous ultrasound +/- US doppler is useful for diagnosing…
DVT in legs - 95% specificity and sensitivity
Spiral CT scan of chest and V/Q scan used to diagnose…
PE
Antithrombin deficiency (4)
1) Antithrombin inactivates thrombin, 10a, 9a, and 2a, so Antithrombin deficiency → hypercoagulable
2) AD - homozygotes fatal in utero
3) VENOUS thrombosis before age 50
4) Can cause heparin resistance - heparin is a cofactor of AT-III
Protein C deficiencies (3)
1) Protein C inactivates factors 5a and 8a to inhibit coagulation, VitK dependent
2) AD
3) Can be made worse by Warfarin therapy
Protein S deficiencies
1) VitK dependent, facilitates anticoagulant activity of activated protein C
2) Thrombosis in atypical areas
3) AD
Factor V Leiden (4)
1) AD mutation of factor V gene → partial resistance to inactivation by proteolytic cleavage by protein C (NOT a deficiency)
2) Factor V Leiden inactivated 10x more slowly
3) Most common inherited predisposition to hypercoagulability in Caucasian populations
4) Increased coagulation AND decreased anticoagulation
Prothrombin gene mutation
1) AD mutation in factor 2
2) Associated with VENOUS thrombosis
3) Very minor hypercoagulability
4) Causes elevated concentrations of plasma prothrombin
Clinical features of anti-phospholipid antibody syndrome (3)
1) Increased risk for thrombosis, obstetric complications, and fetal death
2) Clots in veins and arteries, any vascular bed, any age or gender
3) Prolongs APTT - due to in vitro lab artifact (see Stabler’s lecture)
Criteria for diagnosis in anti-phospholipid antibody syndrome
2 symptoms
4 blood tests
1) Vascular thrombosis
2) Complications of pregnancy
Labs:
1) Anticardiolipin antibodies (ACA)
2) Lupus anticoagulant
3) Beta2-glycoprotein-I antibodies
4) High titers for APab
If you have…
- 1 unprovoked event or provoked DVT
- 2nd unprovoked event, Cancer, APLS, high-risk thrombophilia
Then you will be on anticoagulation therapy for how long?
- 1 unprovoked event or provoked DVT –> 3 months of anticoagulation
- 2nd unprovoked event, Cancer, APLS, high-risk thrombophilia –> indefinite anticoagulation
Heparin mechanism of action (3)
- Heparin binds antithrombin III (serine protease inhibitor)→ increased rate of thrombin inactivation by 1000x
- Accelerates decay of 9a, 10a, and 12a by antithrombin III
- Heparin released by antithrombin and reused once thrombin binds complex (reversible binding to antithrombin)
Heparin is used for (6)
*Venous thrombosis, *PE, MI, during/after coronary angioplasty, during surgery requiring cardiopulmonary bypass, kidney dialysis
2 types of heparin
- unfractionated
2. Low molecular weight
Type of heparin to use durin pregnancy
unfractionated
Characteristics of unfractionated heparin (5)
- Binds antithrombin/thrombin complex
- Not absorbed from GI tract - must be given IV or IM
- Poor bioavailability, short half life → unpredictable dose response
- Requires hospital admission for careful monitoring - ONLY INPATIENT
- Does NOT cross placenta (can use during pregnancy)
Mechanism of action of low molecular weight heparin
DO NOT inhibit thrombin by antithrombin
b. Selectively binds to and inactivates factor 10a by antithrombin
i. Only has 15 saccharide units, so cannot bind thrombin, only can inactivate factor 10a
3 characteristics of low molecular weight heparin
- Given IM
- Longer half life, better bioavailability → more predictable dose response
- Requires less monitoring (OUTPATIENT treatment)
3 complications of heparin therapy
- excessive bleeding
- heparin induce thrombocytopenia
- allergic reactions
Treatment of hemorrhage due to heparin therapy
Stop use → anticoagulation effect disappears within hours
Can reverse life-threatening bleeding with protamine sulfate
Heparin-Induced Thrombocytopenia
(Thrombosis)
- Platelet count decreases
- Abs to platelet factor 4/heparin complexes → platelets activated into prothrombotic state → venous thromboembolism, stroke, MI, arterial thrombosis
- Less likely with LMWH
Allergic reactions to heparin are due to _____
chondroitin sulfate
contaminant in heparin supply
Alternative anticoagulant therapies for thrombocytopenia:
Argatroban (Novastan) - small molecule inhibitor of thrombin
Lepirudin (Refludan) - recombinant form of hirudin anticoagulant from leeches → inhibits thrombin
Advantages of new oral anticoagulants (3)
rapid onset of action, absence of food interactions, do not require monitoring
Disadvantages of new oral anticoagulants (5)
contraindicated with kidney disease, greater GI bleeding, shorter half life (problem with compliance), cost, NO ANTIDOTE
Pradaxa
for afib, approved in 2015 - thrombin inhibitor
Eliquis
for afib approved in 2012 - factor Xa inhibitor
Warfarin
Vitamin K analogue - inhibits enzymes that use vitamin K as a cofactor
1.Factor 2, 7, 9, 10 undergo vitamin K dependent gamma carboxylation of N-terminal glutamates → cannot bind calcium → non functional
Warfarin is used for (4)
Venous thromboembolism, systemic embolism (pts with prosthetic heart valves or afib), stroke, recurrent infarction
5 complications of warfarin
i. Hemorrhage
- Takes 24-48 hrs for reversal - coagulation factors must be resynthesized
- Can transfuse plasma to replace coagulation factors or Vit. K
ii. Cannot be used during pregnancy - crosses placenta
iii. Drug and food interactions
iv. Requires Monitoring - genetic variability in ability to metabolize Warfarin
v. Delayed onset of action
Heparin onset of action
Rapid onset - Heparin acts quickly, but transitioned into an oral anticoagulant
Warfarin onset of action
SLOW onset of action - must have turnover of factors before effective
3 Fibrinolytic agents
I. t-PA (Tissue Plasminogen Activator): serine protease that binds fibrin, cleaves plasminogen → plasmin (degrades fibrin, degrades clot)
- Used to dissolve clots in emergency setting
ii. u-PA (Urokinase): converts plasminogen to plasmin (does NOT bind fibrin)
iii. Streptokinase: non-enzymatic protein from B-hemolytic strep - Forms complex with plasminogen → activates it → converts to plasmin
4 uses of fibrinolytic agents
i. Acute MI (in combo with ASA)
ii. Ischemic stroke (within 3 hrs after stroke)
iii. DVT (in combo with heparin/warfarin)
iv. PE
high risk agents (hemorrhage, systemic lytic state)
3 types of antiplatelet agents
- Aspirin
- ADP receptor antagonists
- Gp IIb/IIIa inhibitors
Aspirin
inhibit formation of platelet products
- Irreversibly inactivates COX1 → inhibit formation of thromboxane A2
- Effects last life of platelet (7-10 days)
- Used for prevention of AMI and stroke pts with atherosclerosis
ADP receptor antagonists
prevent platelet aggregation (Plavix)
1.Block platelet activation by ADP
→ inhibits secretion of alpha granules, blocks expression of adhesion proteins GPIIb/IIIa
- Slow onset (5-7 days)
- Effects last for life span of platelet (7-10 days)
Gp IIb/IIIa inhibitors
block adhesion proteins
- Block adhesion protein (integrin) on surface of platelets that is a receptor for fibrinogen → block platelet aggregation
- Given IV
- Before, during and after angioplasty
Difference between HIV-seropositive and AIDS
HIV-Seropositive: people with antibody to HIV-1 virus
AIDS: once symptoms of opportunistic infections, Kaposi’s sarcoma, or Th (CD4+) cells fall below 200/uL
Virus that causes AIDS (hard one, I know)
HIV-1
i. Lentivirus: Non-transforming retrovirus
1. RNA virus with no oncogene - copies its RNA into DNA (reverse transcriptase)
ii. DNA not being transcribed in host cell → latent until T cell is activated
Origin of AIDS virus
i. Evolved from SIV in the 1940s in Zaire (Congo)
ii. First in US in 1978, in Africa in 1959
Origin of current AIDS epidemic
Congo → rest of Africa → Caribbean → Europe → USA
Approximate number of AIDS cases in USA and the world
i. USA: 1.1 million people living with AIDS in US (16% don’t know it)
1. 50,000 new cases a year
ii. World: 35 million people living with HIV in 2013
1. 36 million people have died from AIDS so far
2. 5th leading cause of death
Two protective alleles to HIV
- People with two CCR DELTA32 alleles do NOT express any surface CCR5 → can become chronically infected with HIV but DO NOT become ill
- “Long Term Survivors” - HLA-B57:
- “Elite Controllers” → become infected with HIV but did not progress to AIDS
- Make effective CTL cells
Pathogenesis of AIDS
i. HIV-1 is VERY antigenically variable
- Reverse transcriptase highly error prone → many variants of HIV
ii. Single exposure infection → Blood virus level peak at 6 weeks, Antibody to HIV peaks at 9 weeks → virus levels fall (but not to zero) = “Set Point”
- Set point determined by patient’s immune system
- Systemic infection between 6-9 weeks with loss of CD4 cells in gut mucosa, increased gut permeability
HIV target cells (2)
- Dendritic cells
2. Th CD4+ cells
HIV interaction with dendritic cells
a. Upon initial infection adheres to lectin on DC = DC-SIGN
b. Trojan horse → lymph nodes for Th cells
HIV interactions with Th cells
Mode of entry of virus into cell:
i. HIV binds by its envelope glycoprotein gp120 to CD4 molecule on surface of Th cells
ii. → conformational change in gp120 and CD4 on cell surface
iii. → conformational change allows CD4 to bind co-receptor CCR5
iv. → gp41 binds cell membrane and allows fusion of virus with cell membrane
v. Binding to gp120/gp41 and conformational change creates hydrophobic region on T cell that allows virus to fuse with T cell membrane
Injects core into cell, activates reverse transcriptase
i.RNA → DNA → insert into nucleus of T cell with viral integrase
HIV exit from cells
Viruses bud en masse from infected cell, tearing holes in membrane → cell dies
Latency of HIV
- Seropositive without symptoms
2. Therapy can prevent next stages of disease progression
Th1, Th2 and Tfh cells in HIV infections
- Th1: site of HIV infection
- Orchestrates CTL killing - has molecules that bind GP120 → generates IL-2 → stimulates HIV transcription - Tfh: site of HIV persistence in lymph node - harbors virus reservoir, has molecules that bind GP120, resides in lymph node follicles
i. Suppress viral replication but can’t eliminate virus DNA from nuclei
ii. When Tfh activated by its correct antigen → clone of virus producing cells - Th2: has molecules that bind GP120, required for production of antibodies for antibody class switching required to stimulate antibody production
3 Opportunistic infections common in AIDS patients
- Candida Albicans (yeast) - mouth, esophagus, rectum
a. Fevers, night sweats, weight loss, fatigue - TB: leading cause of death in people infected with HIV
- Malignancy (Kaposi sarcoma, Burkitt lymphoma)
Late AIDS dementia complex:
due to brain cells infected with HIV (macrophages, microglia)
Infections common in AIDS patients are primarily
Infections that require T cell mediated immunity
- Cytomegalovirus, hepatitis, HSV, VZV
- TB, Fungi (Candida albicans, pneumocystis jirovecii)
Why do CD4 cells decline in AIDS patients
i. Virus able to replicate faster and longer than you can re-make CD4 cells
- Estimated that entire population of virus replaced daily, and CD4 cells every 3 days
- Eventually CD4 cells lost, unable to make them as fast
ii. Eventually can’t keep up, and CD4/CD8 ratio falls in blood
iii. → Immune system can no longer cope, opportunistic infection takes hold
Ineffectiveness of antibody in HIV infection (2)
- When virus is replicating gp120/gp41 made early and inserted into cell plasma membrane → allows fusion of infected cell to nearby uninfected CD4 cells → syncytia
- Antibodies patient make are not protective but they do bind the virus and block attachment to and infection of Th cells
- BUT neutralizing epitope on virus shielded by carbohydrate
Lab diagnosis of AIDS
- Antibody measured by an ELISA, confirmed by Western Blot analysis
- Virus then sequenced to see what drugs it is susceptible to ($$)
- PCR identification of virus RNA
6 treatments of HIV/AIDS
i. Reverse Transcriptase Inhibitors:
Nucleosides (NRTI): competitive inhibitors, chain terminators
Non-Nucleoside (NRTI): binds enzyme, changes conformation at catalytic site (common resistance mutation)
ii. Protease Inhibitors
iii. Fusion inhibitors
iv. CCR5 antagonist: blocks viral entry into CD4+ cells by changing conformation of CCR5 preventing engagement of gp120
v. Integrase inhibitor: blocks viral DNA insertion into cell DNA
vi. Antiretroviral Therapy (ART): two NRTIs and a third drug from a different class
Problem with producing HIV vaccine
Need a vaccine that can preferentially stimulate Th1 cells and CTL
Currently vaccines best at inducing ab response - ab not protective
Prospects for HIV vaccine
Broadly neutralizing antibodies (bnAb) can block infection by almost all HIV strains and mutant forms
Directed against gp120 that binds CD4
6 challenges for HIV vaccine
- V(D)J of bnAb mutates very quickly and thus becomes ineffective at binding site on HIV
- Tremendous global genetic diversity of HIV
- Immense mutational capacity (evasion of T and B cell immunity)
- Latency in host genome - can’t be eliminated by antiretroviral drugs
- No known example of spontaneous immune clearance
- bnAbs have been found but are rare and take years to develop
Basic process of blood collection
i. Volunteer donation
ii. Answer questions about current/past illnesses, surgery, travel, vaccination, high-risk behavior
iii. Call back with symptoms
iv. Abbreviated physical exam
v. Test hematocrit and platelet count
vi. Skin prep for phlebotomy (reduce bacterial contamination)
6 components derived from blood donation
- whole blood
- packed red blood cells (PRBCs)
- Fres frozen plasma (FFP)
- Cryopreciptate
- Platelet concentrates
- Granulocyte (WBC) concentrates
Whole blood
- Keep at 4-6 C for 35 days
- Hct is 36-40%
- Platelet / neutrophil function degenerate by 24-48 hrs of storage
- Red cells well maintained
- Loss of clotting factors more slowly
Packed red blood cells (PRBCs):
- Keep at 4-6 C for 35 days or longer (42 days with special solutions)
- Hct is 70% without plasma
- RBCs only cell you want in there
a. Can get some WBCs in there that you don’t want → use leukodepleted units - Frozen/glycerolized RBC can last 10 years in freezer
Fresh frozen plasma (FFP):
- Acellular product
- Keep at -18 C for one year
- > 80% all clotting and anti-coagulant proteins
a. Contains all plasma proteins (procoagulant, anti-coagulant, complement proteins)
Cryoprecipitate
- Made from fresh plasma frozen quickly at -80 C → sit for 18hrs at 4 C
- Cryoprecipitable proteins isolated and frozen at -18 C for one year
Cryopreciptate contains (6)
a. Cryopreciptiable proteins
b. 80-100 U factor 8 per bag
c. vW Ag
d. Fibrinogen
e. Factor 13
f. Fibronectin
Platelet concentrates
tx for pts with low count or poor function
- Random donor unit (RDUs) or Apheresis platelet concentrates
a. Leukoreduced
b. Stored at 22-24 C for 5-7 days in gas permeable bags
Granulocyte concentrates
- Collected by apheresis
- No storage allowed
- Keep at room temp - transfuse within 8-12 hours
- Use for severe infections in neutropenic patients
Blood groups
serologic determination of ABO and Rh(D) antigens of patients/donors
i. A: H antigen + fucose + N-acetylgalactosamine
ii. B: H antigen + fucose + D-galactose
iii. AB: H antigen + fucose + D-galactose + N-acetylgalactosamine
iv. O: H antigen + fucose
v. Rh alleles
Indications for whole blood use
Massive transfusions to replace oxygen carrying capacity AND blood volume
ii. ABO, Rh(D) type specific
iii. Must be crossmatched
Indications for packed red cells use
Transfused for oxygen carrying capacity: chronic anemia, acute blood loss
ii. ABO, Rh(D) type specific
iii. Must be crossmatched
Indications for fresh frozen plasma use
Coagulopathy related procoagulant deficiency (DIC, liver failure, VitK deficiency)
ii. Use specific anticoags for inherited deficiencies
iii. ABO type specific
iv. No crossmatch
Indications for platelet use
Bleeding associated with thrombocytopenia and/or platelet dysfunction
ii.ABO type specific or compatible
Basic rules of blood administration (3)
1) Typed for ABO and Rh(D)
- Weak expression of D can be mistake for Rh-
2) Screened for antibodies other than ABO
3) Major Crossmatch
Major crossmatch
Mix donor cells and recipients serum in vitro → evaluate for agglutination
- Add Coombs reagent to look for IgG or complement
- Remarkably reduces possibility of immediate hemolytic transfusion reactions
What do you do in an urgent situation when a patient needs a transfusion
- Give O Rh(D) negative
- For males and non-childbearing females can use O Rh(D)+ - Abbreviated testing of ABO and Rh(D) types and ab screen without crossmatch
Infectious risks (7)
Syphilis, Hep A, Hep B, Hep C, HIV, WNV, CMV
What is tested for when blood is collected? (3)
HIV, West Nile Virus, Hep C
7 non infectious adverse events of transfusion reactions
- Febrile nonhemolytic transfusion reactions and mild allergic reactions
- Acute hemolytic transfusion reactions
- Delayed hemolytic reactions
- Anaphylactic reactions
- Dilutional coagulopathy
- GvH disease
- Iron overload
Febrile nonhemolytic transfusion reactions and mild allergic reactions
- Caused by leukoagglutinins in recipient cytokines
2. TX: antipyretics, antihistamine
Acute hemolytic transfusion reactions:
preformed alloantibodies and autoantibodies after infusing incompatible blood products (ABO) → intravascular hemolysis of transfused cells with activation of clotting (DIC)
1.Life threatening, severe → renal failure
Delayed hemolytic reactions:
alloantibody production and slow destruction of transfused RBCs by extravascular hemolysis
Anaphylactic reactions:
bronchospasm, airway response
1.TX: epinephrine, benadryl, steroids
Dilutional Coagulopathy:
massive blood loss and transfusion with replacement of fluids or blood components - deficient clotting factors → Bleeding
TX: replace clotting factors or platelets
Graft vs. Host Disease
lymphocytes from donor transfused in an immunoincompetent host
Iron overload:
no physiologic mechanism to excrete excess iron
Immune surveillance Theory
Theory that adaptive immune response evolved less for dealing with foreign substances than as a way of detecting changes in the body’s own cell surfaces
Evidence for immune surveillance theory (4)
1) Immunodeficient/ Immunosuppressed people (particularly of T cells) have higher incidence of tumors (organ transplant, AIDS, chemo drugs)
2) Activated T cells in a tumor is a good prognostic sign
3) Spontaneous regression of some tumors
4) Paraneoplastic syndromes - symptoms at distant organ that doesn’t contain tumor cells
3 steps of immunoediting
1) Elimination
2) Equilibrium
3) Escape
Elimination step of immunoediting
- elimination of most cells that get initiated by a mutagenic event via immune surveillance
- Malignant clone recognized as abnormal by both innate and adaptive immune systems, and thus eliminated
Equilibrium step of immunoediting
- lymphocytes infiltrate tumor but do not fully destroy it
- Remain in equilibrium until drop in host immune response - can occur for any number of reasons → further mutations accumulate and lead to reactivation
Escape step of immunoediting
-tumor cells fight back
Examples:
1) Checkpoint inhibitory surface receptor ligands upregulated (CTLA-4 and PD1 receptors)
2) Immunosuppressive factors expressed by tumor cells (TGF-B)
3) Downregulate expression of MHC class I on tumor → less CTL recognition
Tumor-Associated Antigens (TAA)
Antigens overexpressed or abnormally expressed by the tumor
-Driver or passenger mutations
Tumor Rejection Antigens (TRA)
TAA recognized by T cells → destruction of tumor
small subset of TAAs
Carcinoembryonic antigen (CEA)
Example of a TAA
- Oncofetal antigen
- Found in blood of patients with colon carcinoma and other cancers
- Can test for CEA with high suspicion of colon cancer, to confirm complete excision, or to warn of recurrence
Role of CTL cells in killing tumor cells - 3 steps
1) recognize TRA presented by MHC class I
2) CTL activated in nodes (not at tumor site) via interactions with APCs
3) Activated TRA-specific T cells leave lymph node and migrate to tumor → induce apoptosis via perforin (secretion-based) or Fas-mediated (transmembrane signaling) pathways
PROBLEM: tumors down regulate MHC class I receptors on their cells
Role of Th1 cells in killing tumor cells
CD4+ cells recognize tumor antigens, make lymphokines and attract M1 macrophages
-Problem is tumor make themselves resistant to M1 and only susceptible to M2 macrophages which protect tumors
NK cells have receptors for….
1) Stress markers –> NK killing
2) MHC class I –> suppress NK cells
3) Receptors for Fc for IgG –> ADCC killing
Implications of tumor down regulation of MHC class I
Tumor cells downregulate MHC to avoid CTL → makes them NK targets
PD-1
- inhibitory receptor on T cell surface, engaged by PD-L1 on APC
- Upregulated late in T cell activation
- Keeps immune response in check
CTLA-4
-Inhibitory receptor that appears on T cell surface that effectively turns off CTL
_____ and _______ are over-expressed by tumor cells AND tumor infiltrating cells → turn off T cells that are present at tumor site!
PDL1 and CTLA-4 ligands
T cells there, just have their off buttons pushed
BCG treatment
- innocent bystander killing
- Inject directly into tumor
- Causes delayed-type hypersensitivity reaction to BCG
- Tumor cells killed as innocent bystanders by M1 macrophages
- Treatment of choice for superficial bladder carcinoma
Nivolumap and Pembrolizumab are…
monoclonal antibodies against PD-1
monoclonal antibodies against PD-1 act by…
- bind PD-1 and prevent it from receiving inhibitory signal from PD-L1
- Cover up off switch so tumor can’t push it
- Not very toxic, and highly effective
Ipilimumab
Monoclonal Abs against CTLA-4
- effective, but toxic (big immune responses, including autoimmunity)
- Prevent immune system from being turned off → autoimmunity
Response to mAbs related to…
extent of PD-L1 expression on tumor associated cells (more PD-L1 = better response) AND directly correlated to number of tumor mutations
Tumor-Infiltrating Lymphocytes
cells directly from tumor
Can be used in Autologous cell therapy
T cells expanded in culture → irradiate patient’s immune system → reintroduce T cells into the immune-depleted patient to kill remaining tumor cells
Driver Mutation
- activate oncogenes, inactivate tumor-suppressor genes
- The reason the tumor grows
- “Undruggable”
Passenger Mutations
Greatly outnumber drivers
- Not causative, but incidental to disorder that exists in transformed cells
- “Neoantigens”
- The more passenger mutations a tumor has the more likely it is to be well-controlled by therapies that relieve the CTL from “Checkpoint Blockade”
Chimeric antigen receptor
Re-arms CTLs making them have higher affinity for MHC-peptide-TCR system
- Light chain/heavy chain variable domain + transmembrane domain + T cell stimulatory domain
- NO MHC RESTRICTION
- Triggered via normal TCR-associated pathway to become fully-cytotoxic
