Clin Path

Question 1

Methemoglobinemia

Answer 1

-Diminution of the oxygen-carrying capacity of circulating hemoglobin occurs due to the conversion of some or all of the four iron species from the reduced ferrous (Fe2+) state to the oxidized ferric (Fe3+) state
-Unable to bind and transport oxygen

Question 2

How do neutrophils become activated?

Answer 2

-Move from circulation into the tissues by attaching first loosely and then tightly to receptors on activated endothelial cells.
-Once adhered, they move between or through endothelial cells and pericytes into the interstitial space.
-Become activated when PRRs bind to PAMPs or DAMPs

Question 3

How do neutrophils kill pathogens?

Answer 3

1) degranulate to release destructive peptides and proteases into the extracellular matrix or into an intracytoplasmic phagosome containing ingested pathogens
2) Assemble reactive oxygen species generator (NADPH oxidase complex) on the membrane of a phagosome or on the outer cell membrane
3) Neutrophils form NETs

Question 4

NETs

Answer 4

-neutrophil extracellular traps
-deoxyribonucleic acid, histones, and other nuclear material combine with destructive peptides and proteases from intracytoplasmic granules and are expelled from the cell into the extracellular space
-forms a web of cytotoxic material that ensnares and kills pathogens while also containing the destructive molecules to prevent damage to regional tissues.

Question 5

What cytokine is most responsible for maintaining neutrophil homeostasis?

Answer 5

-granulocyte colony-stimulating factor (G-CSF)
-produced by bone marrow stromal cells, macrophages, monocytes, endothelial cells, fibroblasts

Question 6

Neutrophil production

Answer 6

-Produced by progenitor cells in the bone marrow
-in bone marrow, they mature into segmented neutrophils
-bone marrow houses a large reserve pool of mature neutrophils
-Under stimulation of growth factors and cytokines (G-CSF, granulocyte-macrophage CSF, TNF alpha, TNF beta, complement 5a -> neutrophils are released from bone marrow

Question 7

Neutrophil pools

Answer 7

1) circulating pool- neutrophils travel rapidly to through the center of larger vessels along with RBCs, sampled on CBC
2) marginated pool- neutrophils move slowly along the endothelium of smaller vessels and capillaries and tend to stagnate in postcapillary venules

Question 8

Febrile neutropenia develops by:

Answer 8

1) increased use of neutrophils
2) decreased egress of bone marrow
3) immune-mediated destruction

Question 9

Causes of bone marrow hypoplasia

Answer 9

-infectious diseases
-exposure to drugs and toxins
-radiation
-myelophthisis
-cyclic hematopoiesis (gray Collie syndrome)

Question 10

Ineffective granulopoiesis

Answer 10

-presence of adequate granulocyte precursors in the bone marrow coupled with peripheral neutropenia
-due to maturational arrest of the neutrophil cell line or retention and/or destruction of mature neutrophils in the bone marrow
-FeLV, FIV, myelodysplasia, lithium administration in cats, acute myeloid leukemia, trapped neutrophil syndrome (Border Collies)

Question 11

Drugs that cause neutropenia

Answer 11

-anti-infective agents
-antiepileptics
-cochicine
-captopril
-methimazole
-phenylbutazone
-chemotherapy agents
-estrogen analogs

Question 12

Myelopthisis

Answer 12

failure of bone marrow to continue normal hematopoiesis because of decimation by infiltrating abnormal tissue (neoplastic cells, collagen (myelofibrosis), osteoid, diffuse intramedullary inflammation (fungal osteomyelitits))

Question 13

Canine cyclic hematopoiesis

Answer 13

-Autosomal recessive genetic disorder
-AKA gray Collie syndrome
-severe neutropenia every 10-14 days

Question 15

Platelet life span

Answer 15

5-7 days

Question 16

Platelet production

Answer 16

Thrombopoesis

Mediated by hormone thrombopoietin that is synthesized in the liver

Question 17

3 stages of platelet activity

Answer 17

Primary hemostasis

1) adhesion occurs when damaged endothelial cells expose collagen that binds to platelets. Damage endothelium release vWF and binds to platelets.

2) activation occurs when there is interaction with the platelet and the collagen and BWF complexes which activate the platelet and cause granule release

3) aggregation occurs when integrin in cooperation with VWF released from the platelets leads to aggregation by bridging multiple activated platelets to form a platelet plug

Question 18

Platelet granules

Answer 18

Alpha granules are the most abundant secretary granules in platelets

Release during platelet activation

Please a roll in adhesion between platelets and other cells, inflammation, and coagulation

Question 19

Platelet plasma membrane

Answer 19

Made up of phospholipids including phosphatidylcholine (PC), phosphatodylserine (PS), phosphatidylethanolamine (PW), sphingolipid sphingomyelin (SM)

PS is negatively charged

PS and PE are inner membrane, PC and SM are outer membrane

Question 20

Platelet membrane enzymes

Answer 20

Flippase- moves phospholipids inwards

Floppase- moves phospholipids outwards

Scramblase- moves PS from the inner to the outer leaflet of the plasma membrane once platelet is activated

Activity of scramblaze is regulated by calcium release from the dense granules

Question 21

Scott Syndrome

Answer 21

Reduced pro-coagulant activity due to decreased activity of transmembrane phospholipid scramblase.

Predisposed in German shepherds

Bleeding occurs because platelets are unable to adhere to damaged endothelium

Question 22

Platelet function testing

Answer 22

BMBT

Platelet function analyzers

Platelet aggregometry

Question 23

Viscoelastic coagulation assay

Answer 23

Global assessment of clot formation kinetics and strength in which platelet function plays a pivotal role.

TEG, ROTEM, VCM

Question 24

DOGiBAT Score

Answer 24

Grades ITP

Comprises site-specific grades of nine different anatomical sites

0- none
1- mild
2- severe

Question 25

What is the role of lipid rafts in platelets?

Answer 25

Lipid wraps are a subdomain of the phospholipid bilayer, role is to carry signaling molecules such as integrands and glycoproteins along the plasma membrane to initiate signal transduction Disturbance of these lipid rafts can lead to unnecessary cell signaling and platelet activation

Question 26

Platelet integrin receptors

Answer 26

-must undergo confirmational change for cell signaling to occur -seven transmembrane receptor family -includes thrombin receptors, ADP receptors (P2Y1, P2Y12), prostaglandin/prostacycline receptors

Question 27

What are the two types of platelet granules?

Answer 27

Alpha granules Platelet dense granules

Question 28

Primary hemostasis

Answer 28

Comprises the endothelium, platelets, extracellular matrix components Goal of primary hemostasis is the formation of platelet aggregates that facilitate thrombus growth and stabilization Occurs in three stages: initiation, extension, stabilization

Question 29

Initiation stage of primary hemostasis

Answer 29

Vascular injury leads to the release of fibronectin, laminin, and von willebrand factor vwf plays a role in platelet adhesion by binding to GP1b-alpha -> results in the tethering and rolling of platelets along the endothelium. Once bound, the platelets are activated

Question 30

Extension of primary hemostasis

Answer 30

Undergoes further activation resulting in secretion, shape change, formation of the pro-coagulant membrane, and integrin activation Needs to local accumulation of thrombin and secretion of agonist such as thromboxane a2 and ADP which act as autocrine and paracrine factors to recruit and activate nearby platelets -> inside out signaling leading to confirmational changes of the platelets

Question 31

Stabilization of primary hemostasis

Answer 31

Firm adhesion to fibrinogen via platelet integrins results in outside in signaling -> leads to the clustering of platelet integrin within the lipid rafts and protein complexes that are linked to actin/myosin filaments. Contraction of these filaments narrows the gap between platelets. Also causes clot retraction when fibrin is deposited which stabilizes the platelet plug without premature disaggregation.

Question 32

Secondary hemostasis

Answer 32

Goal is the formation of a stable fibrin clot A series of enzymatic reactions activates thrombin (factor II). Intrinsic, extrinsic, common

Question 33

Cell based model of coagulation

Answer 33

Secondary hemostasis takes place on phospholipids on the external leaflets of intact cells These cells include fibroblast, platelets, leukocytes Phospholipids provide the docking sites for the assembly of coagulation complexes to form Scramblase causes the external movement of phosphatidylserine. PS is a docking site for factors 9A and 8a, along with factors 10A and 5A Cell-based model of coagulation comprises four phases: initiation, amplification, propagation, termination

Question 34

Initiation phase of cell-based model of coagulation

Answer 34

Factors VII and TF are considered the main initiators of secondary hemostasis. VII is produced by the liver -> activated to VIIa in the blood. In the presence of vascular injury, TF is exposed and binds to VIIa, activating IX and X. Xa causes activation of V, and Xa binds to Va, forming prothrombinase. Prothrombinase converts some prothrombin to thrombin.

Question 35

Amplification phase of cell based coagulation

Answer 35

The thrombin formed in the first phase works to maximize platelet aggregation and formation of pro-coagulant membrane. Thrombin activates factors XI, V, VIII which dissociates from vwf and attaches to platelets

Question 36

Propagation phase

Answer 36

Production of a substantial amount of thrombin via formations of tenaae (factor VIIIa and IXa) and prothrombinase (Xa and Va) on activated platelets. Thrombin cleaves fibrinogen into fibrin. Factor VIII becomes activated by thrombin and is responsible for crosslinking fibrin

Question 37

Termination phase

Answer 37

Endothelium produces TFPI which regulates coagulation through inhibition of a quarernary complex of TFPI, VIIa, FXa, TF AT is produced by the liver- inhibits thrombin generation by forming complexes with FIxa, Xa, Xia, XIIa Protein C and S also play a role in anticoagulation -> inhibits coagulation through proteolysis of factors Va and VIIIa

Question 38

What factor plays a key role in fibrinolysis?

Answer 38

Plasminogen

Question 39

Plasminogen is cleaved into plasmin by:

Answer 39

tPA and uPA