Week 5

Question 1

• Heparin

Answer 1

• MOA: bind to and activates AT III, which leads to thrombin inactivation
• AT III also blocks the activity of factors VIIIa, IXa, Xa, and XIa
• Administered as either HMW Heparin or fractioned in to LMW Heparin
o HMW Heparin is essentially a big sugar complex; LMW Heparin is fragments of HMW Heparin

Question 2

• Warfarin

Answer 2

• Warfarin is a slow and long-acting blood anticoagulant with a structure resembling that of Vitamin K
o Vitamin K can become carboxylated to form vitamin KH2 which carboxylates appropriate Glu residues in proenzymes to form carboxylated proenzymes (i.e. prothrombin)
• This allows Warfarin the ability to compete with Vitamin K and prevent gamma-carboxylation of Glu residues in factors II, VII, IX, X, C, and S
• Non-carboxylated blood clotting precursors increase in both the blood and plasma, but they are unable to promote blood coagulation because the clotting factors cannot bind Ca2+ which also prevents binding to a phospholipid site of activation (platelets cannot aggregate!)
o Note, Warfarin is commonly used in rat poison…
• Warfarin also blocks the activity of PS and PC which is the primary reason that one needs to “bridge with heparin” when first giving Warfarin to a patient!!!!!
o Initially patients given Warfarin are HYPERcoagulable; need to give heparin to protect them against hypercoagulable state

Question 3

o Aspirin

Answer 3

is an IRREVERSIBLE COX inhibitor
• Aspirin irreversibly binds to COX and decreases release of PLA2 and TXA2
• Since Aspirin irreversibly binds COX this will eliminate this platelet’s ability to produce PLA2 and TXA2 which blocks further potentiation in the blood coagulation pathway

Question 4

o Know how vWF works

Answer 4

• vWF = von Willebrand Factor and is involved in platelet adhesion and aggregation
• Adhesion:
o Platelets adhere to exposed collagen and release the contents of their granules; this process is mediated by vWF and GpIb-GpIX-GpV as well as GpIa and GpVI
• vWF binds to the subendothelial collagen at the site of injury and to glycoprotein Ib on the platelet surface; it also stabilizes coagulation factor VIII
• Deficiency in vWF leads to vWD:
• Three different types: I, II, and III
• Patients have reduced levels of factor VIII (hemophilia A has a reduced factor VIII)
• Aggregation
o vWF is used as a mediator for additional platelets to bind on top of the initiating platelet by binding to GpIIb-GpIIIa binding sites on the stacked platelets

Question 5

o The nature of the immune response is determined by many factors including:

Answer 5

• the form, dose, and route of administration of the antigen
• the antigen-presenting cell (APC)
• the genetic background of the individual
• any history of previous exposure (presence of memory cells?) to the cognate antigen
• any concurrent infections that the individual may have

Question 6

• Regulation by Antigen

Answer 6

o T cells and B cells are activated by antigen after effective engagement of their antigen-specific receptors coupled with appropriate co-stimulation.
• Continued antigen exposure is required to maintain T and B cell proliferation.
o At the end of an immune response, reduced antigen exposure results in a reduced expression of IL-2 and its receptor.
• This leads to apoptosis of the antigen-specific T cells.
• The majority of antigen-specific cells die at the end of an immune response.
• A small population of long-lived T and B cells survive and give rise to the memory population
o Routes of Antigen Administration:
• SQ or Intradermally given Ag → active immune response
• IV, Oral, or Aerosol given Ag → tolerance or deviation from one type of CD4+ T cell response to another

Question 7

• Regulation by APC

Answer 7

o Mature DCs can be characterized by strong expression of MHC and co-stimulatory molecules.
o Induction of mature DCs can be achieved through microbial or self-derived stimuli (danger signals).
o In the absence of stimuli, immature DCs express MHC and costimulatory molecules at low levels and antigen presentation induces T cell anergy or deletion depending upon the expression of low or high levels of self-antigen.
o DCs can either be immunogenic (able to produce an immune response by activating effector functions of immune cells) or tolerogenic (antigen binding results in clonal deletion or anergy of antigen-specific T cells)
o Molecules necessary for tolerogenic DC:T cell interactions:
• Surface molecules: E-cadherin, PD-1 L, CD103, CD152 (CTLA-4) and ICOS-L (CD275)
• Cytokines: including IL-10 and TGF-β

Question 8

o Molecules necessary for tolerogenic DC:T cell interactions:

Answer 8

• Surface molecules: E-cadherin, PD-1 L, CD103, CD152 (CTLA-4) and ICOS-L (CD275)
• Cytokines: including IL-10 and TGF-β

Question 9

T regs

Answer 9

o Regulatory T cells (T regs) are important for immune system regulation of T helper cells
• Functions:
Preventing and suppressing immune responses
Prevention of autoimmune diseases
Dampen responses against microbial and viral antigens, allergens, tumors, allografts, and protect fetuses (semi-allografts) during pregnancy
Suppress immune responses against both self and non-self antigens
• Facts:
T regs comprise ~10% of all CD4+ T cells
An inborn lack of T regs results in severe autoimmune inflammation in patients suffering from IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome as these individuals cannot express FOXP3 gene products
When co-cultured with CD25- effector cells (Th0) in vitro, Tregs can suppress proliferation of the effector cells
• In order to suppress, Tregs need to be stimulated via their TCR

Question 10

• T reg Effector Mechanisms:

Answer 10

Immunosuppressive cytokines → cell cycle arrest
IL-2 consumption → Bim-mediated apoptosis
• T regs express high levels of high affinity IL-2R which ‘soaks’ up IL-2
Cytolysis via direct binding to T cells (granzyme-mediated) → apoptosis
Modulation of DC maturation and function by Treg binding via CTLA-4 which causes the DC to secrete kynurenin which is toxic to Th Cells and T regs → cell cycle arrest
• Loss of T reg Effector Function results in autoimmunity

Question 11

Grave’s Disease

Answer 11

autoantibodies of thyroid hormone act as agonists for the TSH receptor

Question 12

o B Cell Tolerance

Answer 12

• B lymphocytes and plasma cells that produce antibodies that recognize self-antigens, so called auto-antibodies, pose a threat to the organism
Grave’s Disease = autoantibodies of thyroid hormone act as agonists for the TSH receptor
Pemphigus Vulgaris and Bullous Pemphigoid are blistering skin diseases that are caused by autoantibodies that recognize adhesion molecules in the epidermis
Autoantibodies are exemplified in the multi-organ autoimmune disease SLE
• Mechanisms of B Cell Tolerance:
Anergy (↓ BCR, ↑ CD5) → B cell death
Lack of T cell help or survival factors

Question 13

Pemphigus Vulgaris

Answer 13

Pemphigus Vulgaris and Bullous Pemphigoid are blistering skin diseases that are caused by autoantibodies that recognize adhesion molecules in the epidermis

Question 14

Bullous Pemphigoid

Answer 14

Pemphigus Vulgaris and Bullous Pemphigoid are blistering skin diseases that are caused by autoantibodies that recognize adhesion molecules in the epidermis

Question 15

SLE

Answer 15

Autoantibodies are exemplified in the multi-organ autoimmune disease SLE

individuals with defects in C1q, C1r, & C1s

Question 16

• Mechanisms of B Cell Tolerance

Answer 16

Anergy (↓ BCR, ↑ CD5) → B cell death

Lack of T cell help or survival factors

Question 17

• Factors that can influence Th cell differentiation

Answer 17

Site of antigen presentation, types of APCs, co-stimulatory molecules involved
Antigen density and binding affinity:
• High MHC II = Th1 and Th17; Low MHC II = Th2
Cytokine Balance, receptors on T cell, host genetic background

Question 18

• Examples of Cytokine Balance:

Answer 18

IL-12 is a potent initial stimulus for IFNγ production by T cells and NK Cells which in turn promotes differentiation of Th1 T cells. IFNα, produced early during viral infection, induced IL-12 and can switch cells from a Th2 to a Th1 profile
Early production of IL-4 favors generation of Th2 cells. Early IL-4 is produced by NKT cells, M2 macrophages, and basophils

Question 19

Th1 Cytokines

Answer 19

IFNγ, TNFβ, IL-2) promote: macrophage activation, ADCC, and delayed-type hypersensitivity

Question 20

• Th2 Cytokines

Answer 20

(IL-4, 5, 9, 10, & 13) promote help for humoral immune responses such as IgG1 and IgE isotype switching, mucosal immunity, IgA synthesis, stimulation of mast cells, and eosinophil growth and differentiation
NKT Cells produce both Th1 & Th2 cytokines

Question 21

• Immune complex disease

Answer 21

↑ bacterial infection risk – deficiency in C3

Question 22

• Polymorphism in TLR4

Answer 22

(which normally senses gm – bacteria) has differential distributions in Africa, Asia, & Europe:
Africa – protective against cerebral malaria forms
Europe – high amounts of TNFα thought to cause septic shock (may be disadvantageous)

Question 23

• Mutation in IL-7R α-chain

Answer 23

have reduced number of T cells

Question 24

• Mutation of cytokine promoters influence levels of cytokine expression such as promoter region of TNFα

Answer 24

• Mutation of cytokine promoters influence levels of cytokine expression such as promoter region of TNFα which results in high levels of TNFα → up-regulation of ICAM-1 → ↑ adherence of infectd RBCs → blocks blood flow
This mutation is associated with Lepromatous Leprosy, Mucocutaneous Leishmaniasis, and death from Meningococcal Disease

Question 25

• HIV progression to AIDS

Answer 25

is associated with a polymorphism in CCR5. Normal CCR5 is used in macrophage-tropic strains of HIV-1 entry into cells
Individuals homozygous for this CCR5 mutation are very resistant to HIV-1 infection

Question 26

CCR5.

Answer 26

• HIV progression to AIDS is associated with a polymorphism in CCR5. Normal CCR5 is used in macrophage-tropic strains of HIV-1 entry into cells
Individuals homozygous for this CCR5 mutation are very resistant to HIV-1 infection

Question 27

• Regulation by Memory Cells and Antibody

Answer 27

o Passively administered antibody binds antigen in competition with B cells (Ab blocking)
• Only high affinity B cells will compete for antigen successfully
o Receptor Cross-linking: IgG can inhibit B cell differentiation by cross-linking the antigen receptor with the Fc receptor on the same cell
o Augmentation: IgM and IgG can act to modulate the immune response through an Fc-dependent mechanism and immune complex (IC) formation with antigen and complement which causes localized interactions with CR2 on follicular DCs (FDCs). This can facilitate the immune response by maintaining a source of antigen
o Ultimately, antibody can both help the immune system respond to antigen or it can block it

Question 28

• Immune Regulation by Selective Cell Migration

Answer 28

o Expression of different chemokine receptors on T cell subtypes allows for chemotactic signals to produce the differential localization of T cell subsets to sites of inflammation:
• Th1 cells – CXCR3 and CCR5 → monocytes
• Th2 cells – CCR3, CCR4, CCR8 → eosinophils
• Th17 cells – CCR6 → neutrophils
• CCR7 helps T cells traffic to lymph nodes
o Immune responses do not normally occur at certain sites in the body including the anterior chamber of the eye and the testes (these sites are immune privileged)
• Failure to evoke immune responses in these sites is partly due to the presence of inhibitory cytokines such as TGFβ and IL-10. Failure in the anterior chamber of the eye is due to migration inhibition factor (MIF) which inhibits NK cell activity

Question 29

o Mechanisms of Localized Immune Responses:

Answer 29

page 5

Question 30

IPEX syndrome

Answer 30

o Signs: atopic dermatitis shortly after birth; intractable watery diarrhea, low weight, Dx of T1D, diffuse eczema, sparse hair; FTT
o Lab: normal WBC, Hgb, Platelets
• Percentage of eosinophils = high at 15% (normal < 5%)
• IgE was also elevated
These are hallmark signs of IPEX syndrome
• Autoantibodies to glutamic acid decarboxylase (GAD) & pancreatic islet cells found
• Flow revealed lack of both CD4+/CD25+ cells & CD4+ Foxp3-positive cells → no Tregs
o EGD ordered – duodenal biopsy revealed almost total villous atrophy with a dense infiltrate of plasma cells and T cells
o Tx: immunosuppressive therapy
• Cyclosporine and Tacrolimus
• BMT with correct HLA match
o IPEX Background:
• IPEX is a very rare disease caused by mutations in the gene for FOXP3 which is essential for the function of CD4+/CD25+ Treg cells
FOXP3 expression and suppressor function can be induced in human CD4+/CD25-, FOXP3- cells by cross-linking TCR and co-stimulatory receptor CD28
This suggests that de novo generation of Tregs in periphery may be natural consequence of the human immune response
Scurfy mice are used to study IPEX since these mice lack Tregs
• These patients are more prone to suffer from frequent infections; however, they have normal immunoglobulin levels (except for elevated IgE) and their ability to make antibody is intact

Question 31

• Heparin

Answer 31

• 1) binds antithrombin via its pentasaccharide sequence and induces a conformational change in the reactive center loop of antithrombin; binds to both antithrombin and thrombin simultaneously to potentiate thrombin inhibition
• 2) Inhibits Factor Xa activity by binding to Xa & antithrombin

Question 32

• LMWH

Answer 32

• Has a greater capacity to potentiate factor Xa inhibition by antithrombin than thrombin because at least ½ of the LMWH chains are too short to bridge antithrombin to thrombin

Question 33

• Fondaparinux

Answer 33

• Accelerates factor Xa inhibtion to antithrombin only!! Pentasaccharide is definitely too short to bridge antithrombin to thrombin

Question 34

• Know the importance of drug interactions and the reason to distinguish between pharmacokinetic vs pharmacodynamic interactions

Answer 34

o Drug Interactions and pharmacokinetics (PK) and pharmacodynamics (PD):
• Pharmacokinetics: ADME
• For oral anticoagulants, PK drug interactions are primarily due to:
o Enzyme induction or inhibition
o Reduced plasma protein binding (PPB)
• Pharmacodynamics: involves the biochemical and physiological effects of drugs and their MOA
• Individuals vary in the magnitude of their response to the same concentration of a drug
• For oral anticoagulants, PD drug interactions are primarily due to:
o Reduced clotting factor synthesis
o Competitive antagonism with Vitamin K
o Hereditary resistance to oral anticoagulants

Question 35

Agglutination reactions

Direct:

Answer 35

Procedure:
• Mix RBCs with antibody which will be able to bind up different RBCs resulting in agglutination (see RBC clumps); antibody binds DIRECTLY to target surface
➢ Negative reaction – no agglutination
➢ Positive reaction – agglutination has occurred
• Common Example: Hemagglutination
➢ RBCs are mixed with a decreasing concentration of antibody specific to the RBC (1 = highest concentration of Ab, 10 = negative control)
➢ Antibodies are preventing the RBCs from “sinking” to the bottom of the well and this is indicated as a positive reaction (red well)
➢ Eventually there is not enough antibody to hold the RBCs on the surface and most of the RBCs will sink to the bottom of the well (red dot in well)
• Practical Example: Influenza Antibodies
➢ To determine whether a patient has antibodies to a particular strain of influenza virus, a technician would perform a serial dilution of the patient’s antiserum into a microtiter plate
➢ Then the relevant virus (influenza) would be added along with RBCs to each well at known, constant concentrations to induce hemagglutination
• If the patient’s antiserum has anti-HA antibodies that bind the particular influenza strain being tested, the antibodies will attach to the HA molecules on the surface of the virus and prevent those molecules from inducing hemagglutination
• Therefore, the hemagglutination reaction will be inhibited at several [antibody]s

Question 36

Passive agglutination

Answer 36

agglutination reaction occurs but it is not easily detected by the naked eye
Typically this process involves using latex beads (with antibodies bound to it already, the Fc portion) to bind antigen
• Negative reaction = antibody is NOT bound to its antigen and it doesn’t bind to latex beads → no agglutination
• Positive reaction = antibody IS bound to latex and IS bound with antigen

Question 37

Direct ELISA

Answer 37

Starts with antibody on the surface of the well
Protein antigen is added and is then washed with buffer
Add fluorescent labelled, enzyme-conjugated antibodies that bind the antigen added in the previous step; wash with buffer
Add substrate for the enzyme and measure amount of colored product
Results:
• Positive = well changes color (intensity of color ↑ with increased Ag)
• Negative = no color change occurs

Question 38

Indirect ELISA

Answer 38

Starts with antigen on the surface of the well
Add patient’s serum sample which includes that individual’s antibodies; wash with buffer
Add anti-IgG antibodies conjugated to an enzyme; wash with buffer
Add substrate for enzyme and measure the amount of colored product
Results:
• Positive = well changes color (intensity of color ↑ with increased Ag)
• Negative = well does NOT change color

Question 39

ELISA Competitive

Answer 39

most sensitive!
Mix patient’s specimen containing antigen with known amount of antibody which is commercially purchased (antigen-antibody complexes form which cause the commercial antibody to not be able to bind to the surface antigen, as it is bound to the patient antigen)
Add specimen-antibody complex to antigen-coated microtiter well; wash with buffer
Add anti-IgG conjugated to an enzyme; wash with buffer
Add substrate for enzyme and measure the amount of colored product
Results:
• Positive = no color change in well
• Negative = color change (decreased color change with ↑ amounts of Ag)

Question 40

What are the different interpretations of being able to detect IgM or IgG antibodies in a patient’s sera?

Answer 40

PAGE 9

Question 41

What are the functions of the three essential chemical components found in HAT media used for creating monoclonal antibodies?

Answer 41

HAT medium selects for myeloma cells that did not fuse with mouse splenic cells to die; the splenic B cells from the mouse will naturally die in culture since these are not immortal like the myeloma cells
HAT = Hypoxanthine Aminopterin Thymidine
• Hypoxanthine = precursor for GTP; this precursor needs to have HGPRT enzyme to convert hypoxanthine to inosine monophosphate (IMP) and it can also convert guanine to GMP
➢ HGPRT catalyzes the formation of the bond between the base and the first position of the ribose sugar
➢ Thus, the fusion cells will be able to have “GTP rescue”
• Aminopterin = the selecting agent as it blocks synthesis of TTP and GTP (specifically blocks DHFR)
• Thymidine = a precursor to a thymidine base however it has a deoxy sugar and is missing the necessary phosphates; this allows the fusion cells to have “TTP rescue”

Question 42

• Definition of multiple myeloma

Answer 42

o	Multiple myeloma is a malignant proliferation of plasma cell dysplasia which often involves osteolytic lesions within the bone marrow
o	Things Need To Know:
•	monoclonal plasma cell proliferation
•	monoclonal gammopathy
•	decreased normal immunoglobulins
•	osteolytic lesions

Question 43

• Presentation of multiple myeloma

Answer 43

o 80% of patients present with bone pain (low back, pelvis, or ribs). Pain is associated with multiple lytic bone lesions.
o Bruising or bleeding from decreased platelets
o Infections from decreased levels of normal immunoglobulins
o Hypercalcemia from bone destruction (problem for kidneys and heart)
o 50% of patients present with renal failure
o Hyperviscosity syndrome – caused by large amounts of circulating immunoglobulins causing purpura, confusion, decreased vision
o Major causes of death – infection, renal failure
o Classic triad – anemia, bone pain, renal failure
o Average age of diagnosis – 69 years

Question 44

• Recognize the Criteria for a Multiple Myeloma Diagnosis.

Answer 44

o 1) Bone marrow with >20% plasma cells OR
o 2) Plasmacytoma plus one of the following:
• monoclonal protein in serum > 3 g/dl
• monoclonal protein in urine
• lytic lesions
o 3) Usual clinical features of myeloma
o 4) Exclude connective tissue diseases, chronic infections, carcinoma, lymphoma, leukemia

Question 45

• Recognize pathophysiology of multiple myeloma

Answer 45

o Biology of Normal Plasma Cells
• Plasmablasts in lymph node (IgM) → activated B cells in bone marrow (IgG, IgA) →
1) Differentiate into plasma cells (small in number, well-differentiated, characteristic phenotype, die by apoptosis); or
2) Plasmablasts do not differentiate into plasma cells, continue to proliferate and accumulate in marrow, produce large amounts of immunoglobulins, normal death of cells doesn’t occur, crowds out other cells – i.e. RBC precursors. Suppress antibody synthesis by normal plasma cells.

Question 46

• Understand what an M spike is and how it is used for diagnosis and follow-up.

Answer 46

o Initial Lab Tests to Order:
• CBC → low Hgb, platelet count
• Creatinine – elevated
• UA → look for trace amounts of protein (abnormal finding!)
o Specific Myeloma Tests:
• Serum protein electrophoresis: large M spike
M-spike (monoclonal or myeloma spike)
Type of Ig:
• IgG in 60% of cases
• IgA in 20% of cases
• IgD or IgE in rare cases
• Virtually never IgM
• Serum immunoelectrophoresis: look for decreased normal IgX
• Urine immunoelectrophoresis: IgA kappa fragment
Bence-Jones protein in urine (light chain fragments that are filtered out in the urine)
• These do not stay in the blood so it is important to obtain a urine specimen as well

Question 47

• Describe the Treatment and Prognosis of Multiple Myeloma

Answer 47

o Myeloma is staged based on amount of β2-microglobulin and serum albumin
o Treatment:
• Symptomatic disease (active) should be treated immediately
• Treatment strategy related to age and coexisting conditions (heart, lungs, kidneys)
• Classic Agents: will reduce M spike, but people do not live longer… Benefit??
Dexamethasone - steroid
Melphalan – alkylating agent
Cyclophosphamide – alkylating agent
• Autologous peripheral blood stem cell transplant (PBSC)
• Newer Agents:
Thalidomide – antiangiogenic agent
• First used with advanced and refractory myeloma (2001)
Lenalidomide – newer antiangiogenic agent
Bortezomib – proteasome inhibitor
• Supportive Therapies:
Biphosphonates – inhibit bone resorption, treats bone lesions and hypercalcemia.
Erythropoietin – helps anemia and decreases need for transfusions
o Prognosis:
• Conventional chemotherapy – 3-4 years
• Intensive therapy – variable depending on age. Under age 45 → 50% ten year survival
• Causes of death- marrow replacement with pancytopenia (16%), renal failure (10%), sepsis (14%), acute leukemia (5%), other chronic illnesses unrelated to myeloma (23%)

Question 48

o Solitary plasmacytoma

Answer 48

collection of neoplastic plasma cells that is localized to one area of the body

Question 49

o Plasma cell leukemia

Answer 49

this can occur rarely at the end of a case of myeloma; can be de novo

Question 50

o Waldenström macroglobulinemia

Answer 50

• Lymphoplasmacytoid lymphoma (lymphocytes that appear like plasma cells); these cells can make IgM which causes the blood to be hyperviscous (commonly occurs in the eye which results in visual changes; however, this phenomenon can occur anywhere)
• IgM
• Hyperviscosity syndrome

Question 51

o MGUS

Answer 51

(Monoclonal gammopathy of undetermined significance)
• Small M spike with no myeloma symptoms
• Occasionally transforms into myeloma

Question 52

• Be able to describe in detail the three steps involved in making a clot (i.e., be able to describe the steps in making a platelet plug).

Answer 52

o Steps in clot formation = vessel constriction + platelet plug + fibrin formation
• Vasoconstriction allows for:
• Decreased blood loss
• Platelets and coagulation factors to meet
• Platelet Plug (Primary Hemostasis)
• Proteins/collagen is exposed
• Platelets adhese and release their granular contents (i.e. ADP, vWF, fibrinogen)
• Platelets aggregate
• Phospholipids are exposed (necessary for cascade to work)
• Fibrin Formation (Secondary Hemostasis)
• Tissue Factor (TF) is exposed → cascade begins
• Cascade makes fibrin which solidifies the plug (“soft clot”)
o In order to make a “hard clot,” factor XIII needs to be activated by thrombin (IIa) so that XIIIa can cross link the fibrin monomers

Question 53

• Draw out a simplified version of the coagulation cascade as it happens in vivo, with the intrinsic pathway on the left and the extrinsic pathway on the right. Make sure to include all the factors and cofactors that are important in vivo. Name the factors that are affected by TFPI, ATIII and proteins C and S.

Answer 53

page 13

Question 54

o (Template) Bleeding Time:

Answer 54

• Used to:
• Evaluate platelet response to vascular injury
• Some platelet disorders have a long bleeding time
• How is this test performed?
• A blood pressure cuff is inflated and two incisions are made
• The patient is observed for how long it takes for the incisions to stop bleeding
• Be aware that there are a lot of different factors that affect this test and that for the most part this test is considered to be unreliable!

Question 55

o Platelet Aggregation

Answer 55

• Used to find platelet function abnormalities
• How is this test performed?
• A sample of the patient’s blood is mixed with different aggregating agents and aggregation is observed and measured based on decreasing turbidity
• Beware that an abnormal result is not always abnormal; these tests must always be repeated!!
• Note, this test is not ordered frequently

Question 56

PT

Reagents:

Features:

Increased due to:

When Ordered?

Answer 56

Reagents:
Patient plasma + Ca2+ & phospholipid surface)
Thromboplastin (acts like TF)

Features:

• Measures Extrinsic Pathway
• Most sensitive to coag issues due to VII’s short t ½

Increased due to:

• ↓ VII, X, V, II, I
• Pt on Warfarin or heparin
• DIC

When Ordered?
NEVER!!!
Order an INR

Question 57

INR

When Ordered?

Answer 57

Reagents:

Features:

Increased due to:

When Ordered?

• Access liver fxn
• Monitor Warfarin therapy
• Diagnose DIC
• Assess pre-op status

Question 58

PTT

Reagents:

Features:

Increased due to:

When Ordered?

Answer 58

Reagents:
(Patient plasma + Ca2+ & phospholipid surface)

Features:
- Measures Intrinsic Pathway

Increased due to:

• Hemophilia A or B
• DIC
• Pt on Heparin
• Presence of inhibitors (aka anti-phospholipid Ab)

When Ordered?

• To investigate Hx of abnormal bleeding
• Monitor Heparin tx
• Diagnose DIC or an anti-phospholipid Ab
• Assess pre-op status

Question 59

TT

Reagents:

Features:

Increased due to:

When Ordered?

Answer 59

Reagents:
(Patient plasma + Ca2+) Thrombin

Features:

• Measures conversion of fibrinogen to fibrin
• Bypasses Intrinsic & Extrinsic Pathways

Increased due to:

• ↓ fibrinogen
• ↑ FDPs

When Ordered?
- Pt has a prolonged PTT and want to rule out a fibrinogen problem (rare!)

Question 60

PTT Mixed Study

Reagents:

Features:

Increased due to:

When Ordered?

Answer 60

Reagents:
(Patient plasma + phospholipid surface)
Pooled Plasma

Features:

Increased due to:
If PTT corrects → something was missing from either pathway → factor deficiency
If PTT doesn’t correct → inhibitor present in patient’s plasma

When Ordered?
- PTT is prolonged, but TT is normal

Question 61

FDP

Reagents:

Features:

Increased due to:

When Ordered?

Answer 61

Reagents:

• Measures FDPs (including D-dimers which cross-link fibrin)
• VERY sensitive!

Features:

Increased due to:

• Thrombi
• Minor clotting

When Ordered?
Used to rule out a clot rather than to rule in a clot

Question 62

• Describe Virchow’s triad, and give some clinical findings that would make you consider a hereditary thrombotic disorder.

Answer 62

o Triad: Endothelial damage, Stasis, Hypercoagulability
• Endothelial damage:
Atherosclerosis, HTN, hyperlipidemia, obesity, smoking
• Stasis:
Immobilization, varicose veins, cardiac dysfunction
• Hypercoagulability:
Trauma/surgery, carcinoma, estrogen/postpartum, thrombotic disorders

Question 63

• Know the underlying mechanism that causes factor V Leiden. How would the patient present? How would you test for it?

Answer 63

o Factor V Leiden Disorder:
• Things need to know:
Most common cause of unexplained thrombosis
Point mutation in factor V gene
Factor V can’t be turned off
Need genetic testing for diagnosis
• Mechanism:
A single point mutation occurs in factor V gene (position 506) which causes it to be constitutively turned on and CANNOT be turned off → constantly activates common pathway
This point mutation produces an abnormal factor V which participates in the coagulation cascade and is unable to be cleaved by protein C
• Clinical Presentation:
Half of patients with unexplained thromboses!
5% of Caucasians have it
VERY rare in non-Caucasians
Risk factors:
• Heterozygotes: 7 times normal
• Homozygotes: 80 times normal
• Normal risk = 1-2 patients per 1000 (per year)
• Lab Tests:
Need genetic testing! (this is a cheap test since same primers can be used on all patients)
PTT and INR are not helpful since this would give normal values since factor V is still able to work (overactive) instead of having reduced ability to function
• Treatment:
Only if patient presents with thrombosis – give anticoagulant
If have multiple episodes – give long-term anti-coagulation

Question 64

• What are warfarin-induced skin necrosis and purpura fulminans? In what type of patients are they most likely to occur?

Answer 64

o Warfarin-induced skin necrosis and Purpura Fulminans are conditions that serve as warning signs that patients might have or are forming clots/thromboses.
• Warfarin-induced skin necrosis is caused by taking Warfarin without having bridging doses of heparin which results in a HYPERcoaguable state in which patients can develop petechiae that progresses to ecchymoses and hemorrhagic bullae
Dermatopathological findings are diffuse dermal microthrombi progressing to full-thickness skin necrosis
• Purpura fulminans progresses from purpura to necrotic/gangrenous tissue and is associated with coagulopathies and sepsis (HUGE mortality rate!)

Question 65

• Describe what antiphospholipid antibodies do and list a few conditions in which you might see them. What is the paradox surrounding the action of these antibodies?

Answer 65

o Things Need to Know:
• Autoantibodies against phospholipids
• Falsely prolong INR
• May cause thrombosis
• Antiphospholipid syndrome is serious
o Antiphospholipid antibodies are IgG antibodies against phospholipids and come in three different variants:
• Anticardiolipin and lupus anticoagulant antibodies and antibodies against other molecules
o These antiphospholipid antibodies bind to phospholipids and screw up coagulation tests by binding up PTT/PT reagent which causes the specimen to not clot, thus the test result appears to be prolonged
• Other tests it interferes with include: direct antiglobulin test (DAT) and syphilis test
o These antibodies are called inhibitors:
• Promote coagulation in vivo
• Inhibit coagulation in vitro
Thus, test results appear contradictory!
o Who has a risk for developing these antibodies?
• No risk = elderly patients on certain drugs
• Mild risk = children following infection
• Moderate risk = adults with autoimmune diseases
o Antiphospholipid antibodies can become Antiphospholipid Antibody Syndrome when patients develop:
• Recurrent thrombosis
• Recurrent spontaneous abortions
• Increased risk of stroke
• Pulmonary HTN
• Renal failure
o How are these antibodies detected?
• 1. Order a PTT
• 2. If PTT is prolonged, order a PTT mixed study
If PTT corrects, then this is usually due to a factor deficiency…
If PTT doesn’t correct, then this means there are antibodies present in the patient’s blood
• 3. If normal, antibody may still be present!!
Order fancy tests (from a coag lab)