Exam 1 Flashcards

Question

How is thrombopoiesis different than other blood cell productions and maturation?

Answer 1

Different because they get larger as nucleated precursors mature in the marrow Mega- basophilic cytoplasm and 1-2 distinct nuclei Endocytotic division Pro- abundant RNA in cytoplasm → blue color cytoplasm Continue endocytotic division Cytoplasm increases in volume Production of cytoplasmic contents occurs Megakaryocytes (biggest) → break up into platelets

Answer 2

Constitutive productive of TPO Liver (and kidney) Platelets bind TPO → inactivates If normal platelet supports TPO binding → steady thrombopoiesis Thrombocytopenia → more free TPO Increased megakaryopoiesis Not as many platelets to bind TPO Increased TPO ⇒ increased platelet production in bone marrow Thrombocytosis Decreased megakaryopoiesis

Answer 3

Birds, reptiles, amphibians, and fish have nucleated platelets Thrombocytes- small cells with clear cytoplasm and small nuclei Have few small granules in their cytoplasm Produced in bone marrow vessel sinusoids

Answer 4

In birds, erythrocytes and thrombocytes develop in marrow blind sac sinusoids These cells do not have to move across the vessel walls to enter the blood Thrombocytes simply leave the sinusoids and enter the blood

Answer 5

Bone marrow is the primary hematopoietic organ in adults 2-3% of body weight In young animals, the marrow of all bones is red due to hemoglobin Active hematopoiesis In adult animals, the mid-shaft marrow of long bones is yellow due to fat replacement of hematopoietic cells Nutrient arteriole- surrounded by hematopoietic cells Endothelial cells- interconnected cells lining the blood vessels Granulocyte progeny Stromal or reticular cell- produce hematopoietic short range regulatory molecules Macrophage- engulf nuclei that extrude from erythroblasts when they turn into reticulocytes Megakaryocyte- lies against veins and branches discharge platelets into vein Send long pseudopodia into the blood Cytoplasm of pseudopod fragments into platelets Megakaryocytes shed platelets directly into the blood

Answer 6

Protect the host from infections Prevent invasion by pathogens removal/inactivation of pathogens after infection Protection from re-infection Supports organ system health Regulation of microbiota “Housekeeping” functions such as removal of dead and dying cells Tissue repair/remodeling

Answer 7

Primary lymphoid tissues Bone marrow- leukocyte development (hematopoiesis) Fetal liver- development of leukocyte prior to birth Thymus- generation of “T” lymphocytes Secondary lymphoid tissues- localized responses Spleen- filters blood White pulp- filtering and removal of non-self components Red pulp- filtering and removal of dysfunctional erythrocytes Lymph nodes- filters lymphatics Tonsils, peyer’s patches- filters antigens on surface of mucosal tissues of the respiratory (tonsils) and gastrointestinal (peyer’s patches) tract Appendix- species specific functions Tertiary lymphoid tissues- less developed than secondary but under right circumstances, can expand Lymphocytic aggregates- develop after strong and/or chronic exposure to microbes (bacteria/viruses/parasites) or chronic inflammation

Answer 8

Innate cell receptors- invariant in recognizing specific motifs or patterns and structures (Pathogen Associated Molecular Patterns and Danger Associated Molecular Patterns) TLR4:LPS Adaptive cells (lymphocytes)- active following binding to antigens T and B lymphocytes recognize a specific stretch of primary amino acid sequences → three dimensional structure “conformation” of an antigen These recognition motives on antigens are called epitopes T cell- linear B cell- any conformation Difference is in the types of receptors they have

Answer 9

agreed upon naming system for proteins associated with cells, especially on cell surface CD followed by a number referring to a unique structure/molecules on cells

Answer 10

antibody generation Molecules that bind to specific receptors

Answer 11

receptor on lymphocytes that binds to antigens

Answer 12

Tissue resident innate immune cells will secrete chemokines and cytokines that activate endothelial cells to express selectin ligands Leukocyte express selectins and activation changes expression of chemokine receptors. Endothelial cells express selectin ligands after injury. Leukocytes start slowing down “rolling” Slowing down of cells allows them to bind to selectin ligands and chemokines, which will lead to activation of integrins Activated integrins bind to endothelial cells, which causes firm adhesion of the cell The cell will transmigrate from blood into tissues following a chemokine gradient

Answer 13

In health, they continuously move through arterial and venous blood and in and out of secondary organs → into non-lymphoid tissues and can return into circulation If they get a signal (chemokine, cytokine), leukocytes are instructed to leave the blood and either get sequestered in secondary lymphoid tissues and/or enter a tissue/organ Migrated to tissue- activated

Answer 14

Four peptide chains together → 2 alpha and 2 beta chains Each chain has a heme → each each has iron Heme is a porphyrin ring, with iron in the middle Iron has two states Ferrous- 2+ “for us” → used for breathing Ferric- 3+ “icky” → oxidized and can’t be used for oxygenation Regulation→ affinity for oxygen is adjustable to tissue environment (Bohr environment) Lower pH and lower O2 content (eg. in actively metabolizing tissues) facilitates unloading of oxygen (lower affinity) Decreased pH Increased 2-3 DPG Increased CO2 Increased temperature Shift to the right Higher pH and higher O2 content (eg. in the lungs) facilitates loading of oxygen (higher affinity) Increased pH Decreased 2-3 DPG Decreased CO2 Decreased temperature Shift to the left

Answer 15

Methemoglobin Reductase Pathway- ensuring Fe stays in ferrous state and not ferric state Ferric iron = methemoglobin → lose all oxygen carrying capacity NADH takes the charge from ferric → becomes NAD+ and ferrous Always on and can be overwhelmed Rapoport-Luebering pathway- 2,3 DPG synthesis → regulates hemoglobin oxygen affinity

Answer 16

Highly complex, involving organic compounds Some steps happen in mitochondria and some in cytoplasm Abnormalities in heme synthesis due to Porphyrias (inherited enzymes defects in porphyrin synthesis)- very rare; lowers RBC and oxygen carrying capacity Dyserythropoiesis (acquired defects)- due to toxin or tumor Heme synthetase and Fe2+ - sticking heme onto RBC Dyserythropoiesis, specifically lead toxicity leads to this shutting down ALA dehydratase- cleaves off water from components Lead toxicity deactivates this

Answer 17

Iron absorption through diet and intestinal absorption Iron and heme absorbed through enterocyte → broken down into ferrous iron → have three final outcomes Ferritin: iron storage protein in macrophages (short term) Hemosiderin not really accessible but also in macrophages Ferroportin: iron “portal” protein, regulated by hepcidin (hormone) Master regulator of iron across body Increased hepcidin ⇒ decreased ferroportin ⇒ less iron transported in Decreased hepcidin ⇒ increased ferroportin ⇒ more iron transported in Synthesized in the liver Adjusts to changes in body iron stores Adjusts to changes in erythrocytes (hemoglobin synthesis) Released in inflammation Blocks release of iron absorption in the intestine and blocks release of iron from macrophages to developing erythroid cells Transferrin: iron transport protein in serum Can bring it to a macrophage to be stored Utilization in erythropoiesis Iron travels in plasma and binds to bone marrow macrophage → binds to transferrin receptor → internalizes Fe Can be stored in ferritin (short term) and hemosiderin (long term) storage Can be transferred through macrophage and ferroportin protein (regulated by hepcidin) to erythroid precursor cells Iron recycling from senescent red cells Circulating RBCs in peripheral blood → erythrophagocytosis of senescent (old) RBCs → in macrophage → heme is broken down → iron is taken from it and can go in the three pathways

Answer 18

HCT= hematocrit (%) Percent blood occupied by RBC → extrapolated Spun microhematocrit (PCV) Calculated HCT= RBC count x MCV (size) Most representative of mass RBC= red cell count (number of RBCs/microliter) Not much emphasis as it could be misleading → having tiny or big RBC can lead to inaccurate count HGB= hemoglobin concentration (g/dl) Used more in human medicine

Answer 19

RBCs change shape as travel capillaries → RBC membrane must be deformable ATP provides energy for membrane contractile proteins → change the shape RBC and return its normal shape when RBC reenters larger vessels Cytoskeleton (structural proteins)- in cell and anchored to the membrane Help determine and maintain RBC shape Viscoelastic properties Delimit deformability Cross linked or damage = loses elasticity Lipid bilayer- provides anchor point Permeability barrier In equilibrium with plasma lipids → plasma proteins can alter RBC membrane → alters function and flexibility Noncompressible Outer membrane- a lot of CHO → makes it tough so it can take damage Too tough or too weak → lysis Inner membrane- ion channels → move ions so that internal solutes = external solutes for osmotic pressure Membrane components (horizontal and vertical) Spectrin- spider web; really strong; can move all around; allows them to bend Actin- facilitates bending (flexing) → proactive movement Vertical- points of anchor help vertical interactions without lysing

Answer 20

Embden-meyerhof pathway Relies on ATP production for membrane shape, cation pump, and ionic gradient Maintains cell function and size Maintain osmotic balances Abnormalities- can lead to water going into cell → lysis or water going out of cell Membrane abnormalities Structural abnormalities (in lipids or proteins) Metabolic abnormalities (often involve ATP, Ca2+) These can lead to Decreased deformability → lysis Shape changes (poikilocytosis)- more prone to damage or not bending Premature destruction (hemolysis)

Answer 21

abnormally shaped red cell

Answer 22

has pointy edges, smaller, evenly spaced and shaped Normal or artifact: Drying artifact in smear preparation; prolonged storage in EDTA Pathologic: electrolyte abnormalities, uremia, rattlesnake envenomation

Answer 23

big, rounded projections, larger and more irregular compared to echinocytes Normal: pigs, calves, rabbits Pathologic: hepatic disease, lipid abnormalities, red cell fragmentation, neoplasia → incorporations of too little or too much CHO into the outer leaflet of the lipid bilayer causes irregular membrane protrusions

Answer 24

lose biconcase nature Normal or artifact: drying artifact in smear preparation Pathologic: anemia (nonspecific), iron deficiency, hepatobiliary disease

Answer 25

tiny fragments of RBC torn apart and membrane reanneals Normal: none Pathologic: red cell fragmentation → seen in small or large clots → fibrin is really tough → results in cleavage

Answer 26

Normal: none Pathologic: red cell fragmentation → membrane blisters and it goes outwards Common in bad trauma → results in a lot of blood clots

Answer 27

very rounded than normal; lose biconcave nature Normal: none (rabbits may have a few normally) Pathologic: immune-mediated hemolytic anemia (when spherocytes are the only poikilocyte) Agglutination- crosslinking of antibodies on RBC surface and causes them to clump together → with spherocytes, it is a hallmark of IMHA Pathologic: RBC fragmentation (when few spherocytes and schistocytes and acanthocytes, etc) → part of RBC membrane is removed by macrophage and remaining membrane reseals around the cytosol

Answer 28

Oxidation of globin chains, which clump and bind to the inner red cell membrane, protruding from the surface Cause- oxidant drugs, plants, chemicals Feline HGb is oxidant-sensitive High number of SH groups Up to 5% of RBCs may contain Heinz bodies (more with some fish diets) Staining- hard to detect with eyes but when stained with methylene blue → you can see

Answer 29

less common than Heinz Internal structure membrane oxidizes → stick together and squishes the cell → hemoglobin is pushed onto one sude Normal: none Pathologic: oxidative damage (+// Heinz bodies, eg. onion induced hemolysis)

Answer 30

Metabolic functions fails, ATP is depleted Reducing power fails, gradual oxidation of hemoglobin “Senescence” antibodies bind to altered membrane proteins Phagocytosed by splenic macrophages RBC enters macrophage → globin is broken down into AAs and heme is broken down into iron and other elements → bilirubin (chemically inert)→ bili put on mac surface → albumin takes the bili → takes to hepatocyte (liver) → liver sticks sugar on it → soluble in water and gets defecated in health In disease, hyperbili → bilirubinemia → get rid of it through GI or kidney → bili in urine (biliuria)

Answer 31

Albumin- maintains oncotic pressure; also a carrier protein Synthesized by hepatocytes (liver) Highest concentration of any single protein in plasma ~65 kDa (small) Globulins Immunoglobulins (gamma)- antibodies produced by adaptive immune cells Synthesized by plasma cells Account for most of the globulins in plasma 150-970 kDa (big) Other globulins (alpha, beta)- roles in innate inflammatory response Synthesized by hepatocytes (liver) Acute phase proteins; transport proteins; complement; clotting proteins; lipoproteins Hepcidin falls under this category Variable kDa (small)

Answer 32

Plasma- includes all proteins, including fibrinogen and clotting factors Purple top tube (CBC)- EDTA anticoagulant Proteins levels always higher compared to serum Plasma protein, plasma fibrinogen, and protein:fibrinogen ratio measured Refractometry- refractive index correlates with solute concentration Accurate to +/- 0.1 g/dl High concentrations of glucose, sodium, or urea can falsely increase TPP Hemolysis and lipemia can interfere with reading Serum- fibrinogen and clotting factors are absent, consumed in the clot Red top tube (chem panel)- no anticoagulant Total protein- biuret method Highly specific and sensitive Albumin- bromocresol green method Species differences in dye binding (doesn’t work in birds) Bilirubin and some drugs interfere with binding globulin= (total protein - albumin) a/g ratio calculated to detect disproportionate changes

Answer 33

dehydration (relative) Globulins also increase proportionately The liver never synthesizes too much albumin

Answer 34

Increased production of gamma globulins (hypergammaglobulinemia) Chronic inflammation (commonly seen) Increased immunoglobulin (antibody) production by plasma cells due to chronic antigenic stimulation Albumin WRR Polyclonal gammopathy → broad based gamma peak Lymphoid neoplasia Abnormal immunoglobulin (paraprotein) produced by neoplastic plasma cells or B lymphocytes Low albumin occasionally Monoclonal gammopathy → narrow based gamma peak

Answer 35

Occasionally, infections can cause a monoclonal spike (+/- polyclonal) Canine ehrlichiosis - most common Leishmaniasis Rarely other lymphoplasmacytic inflammatory lesions

Answer 36

can result from increased albumin, increased globulins, or both Hyperalbuminemia with normal or increased globulins → dehydration Hyperglobulinemia with normal or decreased albumin → chronic inflammatory disease* or lymphoid neoplasia

Answer 37

Acute phase response Cytokine-mediated production of “Acute phase proteins” - mostly in the liver Most of these proteins are in very low concentrations in plasma, so increases do not usually affect total protein or globulin concentrations Also increases fibrinogen and decreases albumin (minor) Hyperfibrinogenemia Fibrinogen is the most abundant acute phase protein Increases in fibrinogen can increase the total plasma protein concentration Acute (and chronic) inflammation Large animals Chronic inflammation Small animals, birds Prot:Fib ratio Proportionate increase in dehydration (more than or equal to 15) Disproportionate increase in fibrinogen is inflammation (ratio less than 15) Remember to convert mg → g (divide by 1000)

Answer 38

Decreased synthesis Hepatic insufficiency Acute phase response Compensatory response to marked hyperglobulinemia Increased loss Renal loss (glomerular) → urine Gastrointestinal loss (diarrhea, malabsorption) Blood loss, exudates, vasculitis Decreased intake and increased utilization/catabolism (malnutrition/cachexia)

Answer 39

Decreased synthesis Immunodeficiencies (hypogammaglobulinemia) Hepatic insufficiency (decreased alpha, beta globulins) Increased loss Renal loss (small globulins are lost in severe tubular disease) Gastrointestinal loss (diarrhea, malabsorption) Blood loss, exudates Decreased intake (failure of passive transfer)

Answer 40

Can result from hypoalbuminemia, hypoglobulinemia, or both Panhypoproteinemia (hypoalbuminemia + hypoglobulinemia) → liver failure or loss Hypoalbuminemia with normal or increased globulins → primarily albumin production issue Hypoglobulinemia with normal or increased albumin → primarily globulin production issue

Answer 41

primary first line defense versus bacterial infections- acute inflammation

Answer 42

Mature neutrophils have multiple nuclear lobes separated by constrictions (filaments) Contain cytoplasmic lysosomal granules that take up a little stain (with routine stains) Primary or azurophilic granules Larger and more electron dense than secondary granules Stain red/purple with Romanowsky stains but don’t stain and aren’t visible after the promyelocyte stage in many species Contents include proteases, acid hydrolases, peroxidase, lysozyme, microbicidal cationic proteins, esterases, glycosaminoglycans The microbicidal properties of neutrophils are contained primarily in the lysosomal granules Degranulation results in release of enzymes and proteins Oxygen dependent or independent Secondary or specific granules Usually not visible (in neutrophils) with Romanowsky stains Contents include lysozyme, lactoferrin, collagenase, plasminogen activator, phospholipase A

Answer 43

important in protection against helminth infections and participate in allergic reactions

Answer 44

become macrophages (in tissue); key players in phagocytosis Antigen processing and presentation Production of inflammatory mediators and cytokines

Answer 45

antibody and cytokine production; mediators in destruction of microorganisms and tumor cells distinguish between self and non self Responsible for memory Humoral and CMI

Answer 46

In some animals, heterophils instead of neutrophils Heterophils in rabbits, guinea pigs, birds, and reptiles

Answer 47

nucleated cell that travels through the blood to get to the tissues, where it functions (literally, means “white cell”, based on its unstained appearance)

Answer 48

all the leukocytes in the body including 1) marrow precursors, 2) leukocytes in the blood, 3) leukocytes and leukocyte derived cells in the tissues

Answer 49

blood test evaluating leukocytes, usually part of the CBC All nucleated cells counted, including nRBC’s

Answer 50

increase in leukocyte numbers above the reference limit (neutrophilia or heterophilia is the usual case) Because neutrophils are the most common leukocyte Heterophils in rabbits, guinea pigs, birds, and reptiles

Answer 51

decrease in leukocyte numbers below the reference limit (neutropenia is the usual case)

Answer 52

decrease in all leukocyte types in the blood below their respective reference limits

Answer 53

decrease in all blood cell types (leukocytes, rbc’s, platelets) in the blood below their respective reference limits

Answer 54

neutrophils (heterophils), eosinophils, basophils

Answer 55

lymphocytes, monocytes

Answer 56

Infections specific to leukocytes Neoplasia involving leukocytes Inherited conditions specifically affecting leukocytes Multisystemic inherited diseases (storage diseases), including leukocytes (leukocytes are useful marker for some of these diseases

Answer 57

The major players are IL-3, GM-CSF, and G-CSF (primary specific regulator) IL-3 and GM-CSF- potent stimulator of all leukocytes Act directly on the bone marrow precursors ro Increase stem cell commitment and cellular proliferation Promote neutrophil differentiation and maturation Result in differentiated cellular function Inflammation results in release of cytokines by activated T cells, macrophages, endothelium, and other cells In concert with other inflammatory mediators (IL-1, TNF), also stimulate marrow release into blood and emigration of neutrophils into tissues The (essential) net result is to increase the numbers of functioning neutrophils at the site of injury

Answer 58

Anemia- decreased red cell mass Decreased RBC mass → hypoxia (lower blood oxygen tension in kidney) → erythropoietin production → stimulation of erythropoiesis (increased red cell production) → release of reticulocytes and replaces RBC mass (regenerative response) Regeneration depends on Severity of anemia (hypoxic) A lower HCT (more hypoxia) elicits increased response Duration of the anemia Ability of the bone marrow to upregulate erythropoiesis

Answer 59

Anemia (general) Low HCT, low Hgb, low RBCC Regenerative- high MCV and low or WRI MCHC Regeneration evidence- high anisocytosis (and RDW), high polychromasia (and high reticulocyte count), high or normal MCV, low MCHC, high nucleated RBCs, and +/- basophilic stippling Most non-regenerative- WRI MCV and WRI MCHC Mechanisms- Decreased erythropoiesis and ineffective erythropoiesis Causes- extramedullary cause (system disease process→ could be anything and everything since the erythropoiesis is so complicated) and medullary cause (bone marrow disease, pure red cell aplasia, immune mediated anemia → slowing down production or ineffective) Most common cause is chronic inflammation Decreases EPO, decreases RBC lifespan, hepcidin release, iron sequestration, and decreased erythropoiesis Medullary causes are less common Bone marrow destruction/altered hematopoietic precursors and niche- often affects myeloid, erythroid, and megakaryocytic lineages Concurrent cytopenias → often severe CBC- schistocyte, keratocyte, dacrocytes, elliptocytes; atypical nRBCs, myeloid cells, and lymphoid cells Bone marrow- highly variable findings Features- absence of regeneration (minimal polychromasia) and RBC shapes (usually none) Iron deficiency: hypochromasia, target cells, schistocytes, keratocytes Bone marrow disorder: Elliptocytes, dacrocytes, schistocytes, keratocytes Hemolysis- high MCHC Seen with high bilirubin, bilirubinuria, hemoglobinemia, and hemoglobinuria Three different causes Trauma- external wounds and bruising Alimentary- GI blood loss Acute will be regenerative Chronic will cause iron deficiency → degenerative anemia Hemostatic abnormality- issues with platelets and coagulation factors Types of hemolysis Immune-mediated Antibodies bind and coat RBC → splenic macrophage phagocytosis the RBC (extravascular) or complement cascade punches hole into it to lyse (intravascular) or crosslink (agglutination) Seen in spherocytes and agglutination Could be microorganisms in RBC → triggers immune response → target RBCs and they get damaged Oxidative Heavy oxidative materials cause RBCs to become Heinz bodies → harder to bend and they lyse when they bend Become ghost cells → after lyse, just see faint remnants Eccentrocytes also seen Exogenous Heavy metals* Acetaminophen (tylenol) Skunk musk (sulfur intake) Allium family plants (onions) Endogenous- build up of internal things Ketoacids Uremic acids Hyperthyroidism Mechanical lysis Keratocytes and schistocytes Disseminated intravascular coagulation, hemangiosarcoma, vasculitis, and thrombi Iron deficiency- low MCV and WRI or low MCHC Fe needed for Fe synthesis Cellular Hgb guides RBC division Causes- Chronic hemorrhage*, dietary deficiency (less Fe and Cu) Morphology- hypochromasia, schistocytes, keratocytes, thrombocytosis Over time, cells become more microcytic and hypochromic Extra cell divisions and less Fe

Answer 60

high MCV and low or WRI MCHC Regeneration evidence- high anisocytosis (and RDW), high polychromasia (and high reticulocyte count), high or normal MCV, low MCHC, high nucleated RBCs, and +/- basophilic stippling

Answer 61

WRI MCV and WRI MCHC Mechanisms- Decreased erythropoiesis and ineffective erythropoiesis Causes- extramedullary cause (system disease process→ could be anything and everything since the erythropoiesis is so complicated) and medullary cause (bone marrow disease, pure red cell aplasia, immune mediated anemia → slowing down production or ineffective) Most common cause is chronic inflammation Decreases EPO, decreases RBC lifespan, hepcidin release, iron sequestration, and decreased erythropoiesis Medullary causes are less common Bone marrow destruction/altered hematopoietic precursors and niche- often affects myeloid, erythroid, and megakaryocytic lineages Concurrent cytopenias → often severe CBC- schistocyte, keratocyte, dacrocytes, elliptocytes; atypical nRBCs, myeloid cells, and lymphoid cells Bone marrow- highly variable findings Features- absence of regeneration (minimal polychromasia) and RBC shapes (usually none) Iron deficiency: hypochromasia, target cells, schistocytes, keratocytes Bone marrow disorder: Elliptocytes, dacrocytes, schistocytes, keratocytes

Answer 62

high MCHC Seen with high bilirubin, bilirubinuria, hemoglobinemia, and hemoglobinuria Three different causes Trauma- external wounds and bruising Alimentary- GI blood loss Acute will be regenerative Chronic will cause iron deficiency → degenerative anemia Hemostatic abnormality- issues with platelets and coagulation factors

Answer 63

Immune-mediated Antibodies bind and coat RBC → splenic macrophage phagocytosis the RBC (extravascular) or complement cascade punches hole into it to lyse (intravascular) or crosslink (agglutination) Seen in spherocytes and agglutination Could be microorganisms in RBC → triggers immune response → target RBCs and they get damaged Oxidative Heavy oxidative materials cause RBCs to become Heinz bodies → harder to bend and they lyse when they bend Become ghost cells → after lyse, just see faint remnants Eccentrocytes also seen Exogenous Heavy metals* Acetaminophen (tylenol) Skunk musk (sulfur intake) Allium family plants (onions) Endogenous- build up of internal things Ketoacids Uremic acids Hyperthyroidism Mechanical lysis Keratocytes and schistocytes Disseminated intravascular coagulation, hemangiosarcoma, vasculitis, and thrombi

Answer 64

low MCV and WRI or low MCHC Fe needed for Fe synthesis Cellular Hgb guides RBC division Causes- Chronic hemorrhage*, dietary deficiency (less Fe and Cu) Morphology- hypochromasia, schistocytes, keratocytes, thrombocytosis Over time, cells become more microcytic and hypochromic Extra cell divisions and less Fe

Answer 65

Relative (common) of erythrocytosis Dehydration: HCT + plasma proteins increase Splenic contraction EPI induced, mainly horses and cats HCT and platelets increase Absolute (rare) Primary (RBC neoplasia = polycythemia vera) Secondary (EPO secretion due to chronic hypoxia or by rare tumors)

Answer 66

Innate Macrophage Neutrophil Dendritic cell- link innate and adaptive Eosinophil Basophil Natural killer Adaptive Lymphocytes Chemokines, cytokines- pro + anti inflammatory; chemoattractant or chemorepulsion

Answer 67

Breach of barrier Recognition by sentinels Recruitment of reinforcement Pathogen elimination and resolution

Answer 68

Mechanical barrier- epithelia form specialized barriers; form tight junctions; differ based on the physiological requirements of tissue Chemical barrier- antimicrobial substances (lysozyme, deferins, cathelicidins,...) kills/inhibits microbes Microbial barrier- resident flora prevents colonization of pathogenic bacteria

Answer 69

Dendritic cells and macrophages act as sentinels at epithelial barriers Mac- phagocytic cell that consumes foreign pathogens and cancer cells. Stimulates response of other immune cells Migrates from blood vessels into tissues Origin- in embryonic development, made in yolk sac and fetal liver → migrate to tissue and regenerate; in adults, made in bone marrow, generate monocyte → in bloodstream and becomes macrophage in tissue Dendritic cell- presents antigens on its surface, thereby triggering adaptive immunity Present in epithelial tissue, including skin, lung, and tissues of the digestive tract. Migrates to lymph nodes upon activation Macrophage surveillance (steady state vs. injury) In healthy tissue, macrophages take up and clear apoptotic cells etc. without recruiting other cells by using scavenger receptors Skin and GI: macrophages are located behind epithelial barrier Lung (lower airways): No great physical barrier present, alveolar macrophages is in front of epithelium/in lumen of alveoli Liver- kupffer cells and CNS- microglia Survey for damage and are first responders following an insult Sensory organ of the cell is the receptor → antigen recognition Innate- one cell has many receptor types and many specificities (not specific) Recognition of conserved epitope (ubiquitous) → present on different pathogen Using pattern recognition receptors (PRRs) Can mass produce cells since all of them have similar antigen recognition → identical genomes Exogenous ligands → PAMPS- repetitive structures that are found across classes of pathogens (bacteria, viruses, fungi, etc) Signal “stranger” and are recognized by pattern recognition receptors (PRRs) Toll like receptor- phylogenetically conserved PRR family Surveil compartments- extracellular, cytosolic, endosome Triggered when encountering molecules that either don’t exist in the host (eg. dsRNA) or are not normally present in a given compartment (eg. ssRNA) Endogenous ligands → DAMPS- released by cellular trauma (physical damage- cuts, tears, etc or damage by heat/cold) Release of cellular materials into the extracellular environment Endogenous molecule but in the wrong compartment Altered self→ natural killer cells Major function- cytotoxicity = destruction of target cells Bind Major Histocompatibility Complexes (MHC) on the surface of the cells NK receptors are either activating or inhibitory Lack of killing requires an inhibitory signal from the target cell Killing occurs by pore formation (perforin) and release of cytotoxic granules (perforins) Steady state Balance between inhibitory and activating signal from target cell NO target cell destruction Cancer or viral infection Downregulation of inhibitory signal on target cell (MHC I) Activating signal dominates Cell lysis

Answer 70

TLR engagement leads to activation of transcriptional regulator NF-kB Signal transduction is produced → causes cascade of signaling molecules and changes in gene expression One of the responses is release of cytokines and chemokines Pro-inflmmatory- released by macrophages upon activation via PRR; stimulate inflammation Tumor necrosis factor (TNF alpha) Interleukin 1 (IL-1) Interleukin 6 (IL-6) anti-inflammatory cytokines: released by engagement of scavenger receptors during healing phase of response Transforming growth factor beta (TGF beta) Interleukin (IL-10) Acute phase response in liver APPs are a class of proteins whose plasma concentrations Increase positive acute phase proteins or Decrease negative acute phase proteins In response to inflammation Roles- opsonization, complement activation, chemotaxis, and trapping of microbes Extravasation of leukocytes- vasodilation and induction of adhesion molecules → rolling → adhesion → extravasation/diapedesis → migration Neutrophils: within 6 hour of inflammatory response; chemokine CXCL8 Macrophage: more slowly; chemokine CCL2

Answer 71

Sentinel cells in different tissues (liver and CNS) Antigen recognition Signal transduction and chemotaxis to tissue Kill all pathogens By phagocytosis Microbe is bound by receptor on surface of phagocytosis Internalization Fusion with lysosome → phagolysosome Formation of oxide radicals → killing By NETosis- neutrophil Extracellular Traps Regulated form of neutrophil cell death Nuclear chromatin is released into extracellular space creating neutrophil extracellular traps (NETs) NETs capture microbes and facilitate phagocytosis by macrophages and neutrophils By complement system Enhances the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen’s cell membrane Origin- proteins synthesized in liver, present in blood as inactive precursors, and is 10% of the globulin fraction of blood serum C3a- inflammation C3b- opsonization and phagocytosis Increases vascular permeability Causes gaps in the endothelium for components of blood to go inside C5a- inflammation

Answer 72

Phagocytosis and microbial action Done through adherence and emigration through vessel wall- involves rolling, sticking (adhesion), and extravasation via selectins, integrins, chemokines, and ICAMS Chemotaxis- C5a, C567, FActor XIIa, LTB4, IL-8 (specific for neutrophils (CXCL8), PAF, bacterial products Ingestion, degranulation, and bacterial killing Bacterial killing Oxygen independent (microbicidal cationic proteins - defensins, cathepsins, BPI, etc) Oxygen dependent Respiratory “burst” and activation of NADPH oxidase Hydrogen peroxide (H2O2) Superoxide anion (O2-) Hydroxyl radical (OH) Singlet oxygen (O2) These ROS denature proteins, oxidize lipids ie. bactericidal H2O2 complexes with MPO (myeloperoxidase) to form a VERY potent bactericidal system MPO-H2O2-Halide complex that results in production of HOCL, a powerful toxic radical There are protective mechanisms to “detoxify” reactive species (SOD, catalase, GPx/GR system, NADPH)

Answer 73

Compartment or “pools” Progenitor pool (stem cells, committed stem cells) Proliferative pool (~20% of precursors, undergo mitosis, 4-5 mitoses, some attrition at myelocyte stage, transit time (tt), ~2.5 days) Maturation-storage pool (80% of precursors, don’t undergo mitosis, tt~2.5 days, size of storage pool varies with spp. → dogs have biggest storage pool, oldest cells enter the blood first) Sequential unidirectional movement through pools Size of pools and rate of traffic between them determines both number and degree of maturity of neutrophils in blood Marginal pool (MP)- neutrophils accumulate near the walls of small vessels (capillaries, post capillary venules)- attach, roll, detach intermittently before migrate into tissues Random exchange between MP and CP Marginal pool cells not measures in the hemogram Circulating pool (CP)- neutrophils flow freely in the center of (larger) vessels CP is the only pool sampled by phlebotomy CP = MP in dogs, horses, and ruminants MP ~ 3X > CP in cats Total blood = CP + MP Average blood transit time is ~ 8-10 hours (all blood neutrophils replaced ~2.5x daily) Tissue emigration random (NOT longest circulating leaves first), adhesion mediated, unidirectional, and “upregulatable” (inflammation) Important- need to be in marginal pool to be in tissues (circulating → marginal → tissue)

Answer 74

Health Tissue survival ~1-4 days Effete neutrophils (apoptotic) in tissues removed by macrophages Disease Tissue has purulent inflammation (pus) → most common cause of neutrophilia Extravascular accumulation of neutrophils Increased extravasation (endothelial activation and adhesion) and migration of neutrophils (chemotaxis) via inflammatory mediators towards a noxious stimulus Inflammation → increased peripheral use, which results in Decreased blood T1/2 Increased marrow release Increased marrow production Cytokine mediated

Answer 75

Bone marrow release is first response Increased marrow release mediated by G-CSF, IL-1, and TNF (inflammation) and corticosteroids Manifests in blood rapidly, <1-2 days Release is orderly - more mature released first As mature neutrophils depleted (storage pool), younger cells are released → LEFT SHIFT Indicative of depletion of mature storage pool Typically in response to inflammation (increased demand) Therefore, left shift = inflammation but nor necessarily the converse (can get inflammation without left shift) Neutrophilia +/- left shift is the usual response to inflammation Neutropenia with left shift (degenerative) indicates “exhaustion” of marrow due (most often) to rapidly developing overwhelming infection (poor prognosis) Increased effective granulopoiesis Increased cell divisions at the myelocyte stage Decreased attrition at myelocyte stage Manifests in blood for 2-3 days (slower than increased release) Stem cell input increase IL-3 and GM-CSF increase the number of stem cells becoming myeloblasts Total neutrophil production can be markedly increased, takes 3-5 days (longer first 2 mechanisms) to manifest in the blood - biggest effect Rate of egress from blood Inflammation results in decreased circulating T1/2 transit time Corticosteroids increase circulating T1/2 (decreased adhesion)/transit time Hypersegmented neutrophils (6 or more lobes) may be seen Most common cause of hypersegmented neutrophils in blood, but there are other causes Pool shifts Shifts to circulating pool Epinephrine Corticosteroids Shift to marginal pool endotoxin

Answer 76

Species with larger maturation/storage pools can mount larger responses Dogs, cats, pigs = large storage pool species Also rodents, birds, marine mammals Horses, ruminants = small storage pool species Neutropenia with left shift is common during early phase of (usually septic, purrelent) marked inflammation Indicates serious disease but NOT marrow exhaustion Hence, not as bad prognostically as same findings in large storage pool species (dog, cat, pig) If an animal survives the first 2-3 days of disease, neutrophil count “recovers” to normal or neutrophilia

Answer 77

Epinephrine mediated Increased heart rate and blood pressure → displaces neutrophils → mild neutrophilia (2X) without left shift Typically seen in excited, healthy young animals, especially cats and pigs Excitement or exercise “fight or flight” Marginal pool “shifts” to circulating pool Total pool hasn’t changed but moved to marginal Lymphocytosis may be present, especially in cats (may be up to 20,000/ microliter) Neutrophilia most pronounced in cats because they have large marginal pools (3:1 ratio M:C) Short term changes

Answer 78

Neutrophilia due to increased entry from storage pool, shift from marginal to circulating pool, and prolonged circulation time Neutrophilia Usually no left shift (unless depleted storage pool) Lymphopenia (less marked in cats) Difference in EPI induced → lymphophilia Eosinopenia Monocytosis (only in dogs) Can be caused by exogenous or endogenous steroids When sick, may induce this response Can be seen in dogs with cushings disease → hyper adrenal corticosteroids Occurs in hours-days while EPI takes minutes to seconds May be seen concurrently with neutrophilia of other causes

Answer 79

Decreased blood neutrophil concentration Patients with <500 neutrophils/microliter are considered at risk for sepsis Causes Decreased production of neutrophils Could be due to viral infection or radiation and chemotherapy No left shift Increased demand Increased demand in tissue than bone marrow can supply Left shift Peripheral destruction of neutrophils (immune mediated neutropenia) Ineffective granulopoiesis Common cause of myelodysplasia Sequestration in the marginal pool → initial response to endotoxin

Answer 80

Leukergy- sticky leukocytes Seen in horse with endotoxemia Morphologic abnormalities of neutrophils and their precursors Due to the presence of increased inflammatory mediators/cytokines +/- presence of bacterial toxins Most often an indicator of “sepsis”/infection (esp. bacterial) when marked EDTA can produce artifacts (time dependent) that complicate evaluation - “fresh” smear essential

Answer 81

Things seen Döhle bodies (blue inclusions in the cytoplasm) Diffuse cytoplasmic basophilia (blueness) Cytoplasmic vacuolation (foaminess) Abnormally large neutrophils (especially cats) Persistent azurophilic or purple granules (“toxic granulation”) in the cytoplasm (rare finding most often seen in horses)

Answer 82

1+ - Döhle bodies only (spp. differences) 2+ - Döhle bodies + Diffuse cytoplasmic basophilia OR Cytoplasmic vacuolation 3+ - Döhle bodies + Diffuse cytoplasmic basophilia AND Cytoplasmic vacuolation +/- Toxic granulation 4+- Too toxic to differentiate from monocytes Generally, 2+/3+ or 4+ toxic change indicates sepsis/endotoxemia 1+ to occasionally 2+ toxic change can occur secondary to ny strong marrow stimulation of granulopoiesis (immune mediated hemolysis, necrosis, etc)

Answer 83

Morphological changes indicate increased cytoplasmic contents (ribosomes, endoplasmic reticulum) and lysosomal enzymes and/or bacterial substances denaturing/damaging cytoplasmic proteins and organelles Compromised function?

Answer 84

Bacteria in circulating neutrophils of septicemic animals Uncommon Poor prognosis: overwhelming infection Intracytoplasmic organisms- specific infections, specific diseases Can recognize on routine hematology

Answer 85

Bright orange granules in cytoplasm Staining is due to their highly basic (cationic) proteins, predominantly MBP and ECP Not recognizable before the promyelocyte/myelocyte stage

Answer 86

Eosinophil granules vary in size and shape between species Horse- most distinctive with giant, bright granules Feline- rice shaped and rod shaped granules → looks like a heterophil

Answer 87

Develop from distinct progenitor in marrow, CFU-Eo (colony forming unit- eosinophil) Variable time for development (2-6 days) IL-3, GM-CSF, and IL-5 are the major players IL-3 and GM-CSF are for all leukocytes IL-5- specifically stimulates eosinophils A maturation/storage pool exists Marrow reserve variable (spp. Specific - eg. guinea pig has marrow:blood ratio of 300:1 versus 3.4:1 humans) Blood T1/2 very short (species variable) Marginal pool exists Tissue life span longer than neutrophils (6 days +) and can be increased under the influence of cytokines Don’t recirculate (like neutrophils) Eosinophils preferentially reside in skin, GI tract, and lung (blood:tissue ~1:200-300) Adherence and emigration through endothelium occurs via similar mechanisms to neutrophils, but different set of chemotactins Chemokines, esp eotaxin antigen/antibody complexes- chemoattraction ECF-A (from mast cells)- downregulating mast effect Histamine- mast cells are full of it LTB4- arachidonic acid metabolites C5a, C567- complement proteins PAF- platelet attractant factor Parasite and damaged tissue products

Answer 88

Helminth killing (+Ab + complement = opsonization) Type I hypersensitivity / allergic reactions (regulatory role)- phagocytosis immune complexes and “neutralize” mast cell products (histamines) Phagocytosis and killing of bacteria (Very minor)

Answer 89

Corticosteroids (main cause) Part of the “stress” leukogram Corticosteroids may attenuate or dampen eosinophilia of other causes Ex. there are always low concentrations of histamine in blood all the time and steroids stop Eo from coming out

Answer 90

Parasitism (helminth parasites) Allergies or hypersensitivities- esp. Skin, gut, lung locations (IgE, IgG leads to mast cell granule release: chemotactic to Eos) +/- fungal disease (esp. horses) “Sensitized” T cells produce a more marked eosinophilia upon second exposure Paraneoplastic Mast cell tumors Spontaneously degranulate → eosinophilia attracted to it Lymphoma (especially T cell) Many other Eosinophilic leukemia is rare

Answer 91

Easily recognizable with blue purple granules Staining due to highly acidic (negatively charged) proteins in secondary granules High affinity for basic dyes (basophilic color) Histamine, heparin, sulfated mucopolysaccharides Granules typically fill cytoplasm but less numerous in the dog, “paler” in the cat Maturation parallels that of neutrophils and they have a similar nuclear morphology Not recognizable before the promyelocyte/myelocyte stage

Answer 92

Dog- not many granules Horse- very granulated (dark purple) Cat- pale, lavender colored granules

Answer 93

Distinct marrow progenitor- CFU-Baso Similar cytokines as involved with eosinophil production (IL-3, GM-CSF, IL-5). Marrow storage minimal Kinetics poorly understood - blood half life ~ 6 hours, tissue lifespan 2 weeks? Inverse relationship between blood basophil counts and tissue mast cell numbers (ie. cats versus rabbits) Cats- lots of blood basophils and not many mast cells Rabbits are opposite

Answer 94

Similar contents to mast cells and likely similar functions Degranulation secondary to antigen binding and crosslinking surface Ig (release of inflammatory mediators) Only known source of heparin and activators of lipoprotein lipase Basophils go up when there are changes to lipid metabolism

Answer 95

Not clinically recognized as so few present normally

Answer 96

Uncommon, usually mild Often accompanies eosinophilia Lipid disorders, feline heartworm disease, myeloid neoplasms (mechanism unknown)

Answer 97

Largest cells in the blood (on a blood smear) Artifact of smearing → likes to stick to glass Variably shaped, “ameboid nuclei” with abundant, blue-gray cytoplasm, often vacuolated Precursors difficult to ID- monoblasts present in very low frequency in marrow and difficult to distinguish from myeloblasts with routine stains Increased vacuolation may indicate activation (ddx artifact due to excessively long EDTA storage) Becomes activated → macrophage in tissue

Answer 98

Monocytes are immature macrophages, capable of mitosis, undergo further differentiation Develop from bi-potenital progenitor cell (CFU-GM) Major factors are IL-3, GM-CSF, and M-CSF (specific) Marrow production is rapid (2-3 days) No marrow storage pool (blood is the storage pool) Blood circulation time longer than neutrophils ~24 hours Marginal pool exists in some spp. (human, mouse, dog, rabbit) Leave blood randomly, don’t recirculate and become macrophages in the tissues- “fixed” and “wandering” (mesothelial, joints, lung) Long tissue T1/2 and can divide in the tissues

Answer 99

Immature macrophages Macrophage functions: Phagocytosis Antigen processing and presentation Cytokine production (inflammation, hematopoiesis) Destruction of effete cells and debris Pinocytosis and catabolism of plasma proteins Specialized “fixed” function ie. Kupffer cells

Answer 100

Present with inflammation Monocytosis has correlation, albeit relatively poor, to tissue macrophage accumulations (chronic inflammation) Monocytosis may occur during acute purulent inflammation (esp. If there is associated tissue destruction) as well as chronic inflammation Corticosteroids cause monocytosis, esp. Dogs, less so other spp. Response to disease, secondary to Persistent inflammatory stimuli- viral, fungal, atypical bacterial infections Component of steroid-induced leukogram (primarily dogs) Diseases with increased tissue demand for macrophages Immune mediated diseases, necrosis, malignancy, hemolysis, pyogranulomatous disease

Answer 101

No clinical significance

Answer 102

Usually the smallest and most featureless of the blood leukocytes Typically have a high N/C ratio with sparse cytoplasm that is variably blue Lymphocytes are often the predominant leukocyte in the peripheral blood of ruminants (in health) Activated lymphocytes are larger Can be granular - seen in dogs and cats Differentiation of lymphocytes is manifest by acquisition and loss of lineage specific cell surface antigens (CD antigens) T cells B cells Morphologically, it is usually not possible to determine to what subpopulation and stage of differentiation a lymphocyte belongs Plasma cells are recognizable as terminally differentiated, Ig secreting B cells

Answer 103

Using CD markers and testing

Answer 104

Blood lymphocytes originate and recirculate from numerous sites (distribution and relocation) Thymus Marrow Lymph nodes Spleen MALT Recirculation mechanisms complex and involve molecules we’ve already discussed Selectins, integrins, ICAMS, chemokines, lymphatics, and HEVs Recirculation is crucial and facilitates Generalized distribution Re-location (immune surveillance) Many lymphocytes are long lived cells Recirculation is common via lymphatics and specialized vasculature (HEVs) Appearance may change under influence of antigenic or cytokine stimulation (become “reactive” when stimulated) The majority of circulaitng lymphocytes in most species are T cells ~70% T, 20% B, 10% NK Blood lymphocyte numbers have relatively poor correlation to tissue lymphocyte numbers Most lymphocytes are in tissue Blood lymphocyte counts are higher in young, growing animals and decrease with age Younger animals encounter many novel stimuli

Answer 105

Ig production- B cells Cytokine production (predominantly CD4+)- T cells Cytotoxicity (CD8+, NK cells)

Answer 106

More common finding on CBCs of sick animals than lymphocytosis Corticosteroid mediated (usually) Lymphocytes apoptosis in response to corticosteroids Get hung up in tissue and not as much recirculation Depletion Lymph loss (chylothorax, lymphangiectasia, inflammatory or neoplastic intestinal disease resulting in lymphatic obstruction) Lymphoid hypoplasia/aplasia Hereditary immunodeficiency (CID or arabian foals), acquired immunodeficiency, thymic aplasia/atrophy Immunosuppressive therapy, radiation Acute systemic infections- esp. Viral infections K9 distemper, K9 parvovirus, FIP Disruption of normal lymph node architecture Prevents recirculation eg. lymphoma, granulomatous disease (lymphadenitis)

Answer 107

Young animals have higher counts - may be out of adult reference interval Epinephrine - mediated (physiologic), esp. Cats Due to increased BP → increased lymphatic pressure Thoracic duct empties → lymphocytosis Immune stimulation (fairly rare situation - typically increases numbers, but stays within reference interval) Persistent inflammation Increased lymphocytes as a result of increased lymphopoiesis, lymph node hyperplasia can be noted Also called “reactive” lymphocytosis (antigenic stimulation, chronic infection) Considered viral, atypical bacterial, fungal, rickettsial, protozoal infections among others (“persistent” infections) Concurrent hematologic abnormalities may include mature neutrophilia and monocytosis Neoplasia Neoplastic proliferation of lymphocytes in marrow, lymph nodes or other lymphoid tissue Not all lymphomas are associated with lymphocytosis In fact, most are not!! Persistent lymphocytosis in cattle subclinical , non neoplastic manifestation of BLV infection in cattle

Answer 108

Reactive lymphocytes Immune stimulation, including infections Atypical lymphocytes (neoplasia) Plasma cells

Answer 109

Recognizes specific epitopes on antigens that they did not encounter during development (“non-self”) B cell- BCR can bind to an epitope on many different types of antigens: proteins, lipids, carbohydrates, nucleic acid (DNA), and it can be either It may bind to a stretch of amino acids (continuous or LINEAR epitope) OR it may bind to a particular tertiary or quaternary structure (conformation) on an antigen = CONFORMATIONAL epitope T cell- the majority of T cells recognize peptide epitopes Small subsets of T cells Lipids (NKT) Metabolites (MAIT) T cells bind a particular amino acid sequence. These are linear epitopes in the context of an antigen-presenting cell Activating a naive T cell- DC Activated T cells- lots of cells

Answer 110

B cell- 2 light chains and 2 heavy chains and associated with signaling chains Heterodimer associated with two signaling chains Variable region- light and heavy chains Constant region- heavy chains

Answer 111

T cell- TCR resembles a membrane bound Fab-fragment 2 variable 2 constant regions Encoded by T-cell alpha and beta or gamma and delta chains Heterodimeric protein linked by a disulfide bridge Each chains has a variable (V) and constant (C) region Highly homologous to immunoglobulin TCR has one antigen binding site TCR is never secreted

Answer 112

The BCR defines specificity of a B cell for its “cognate antigen” Every B cell develops one distinct BCR that when secreted by the cell is called an antibody

Answer 113

CD4- MHC II (class II restricted) CD8- MHC I (class I restricted)

Answer 114

MHC are highly variable and present varying peptides to T cells I- can fit 8-10 amino acids long II- can fit 13-25 amino acids long

Answer 115

B cells- Class switch recombination Process of generating the Ig is recombination Requires recombination activating gene (RAG1 and RAG2), and DNA repair enzymes Defects in these enzymes → severe combined immunodeficiency (SCID) as B- and T-cells develop A diverse repertoire of due to multiple genes encoding the variable region Complementarity determining regions (CDR) CDR3: region that is hypervariable Recombination + Non-genome encoded nucleotides

Answer 116

Selection of T cells T cell progenitors from the bone marrow home to thymus → enters the corticomedullary junction → cortex → medulla → exit to periphery (as a single positive- only CD4+ or CD8+) In the cortex, Cell is DP (double positive) and through initial TCR:MCH interaction (in thymus), it can be either CD4+ or CD8+ Can also be double negative → apoptosis In the medulla, antigen presented by epithelial cells (medullary), or dendritic cells (DC) Interaction leads to removal from body Aire is a transcription factor that allows the transcription of tissue antigens by medullary thymic epithelial cells (mTEC) If no interaction, mature cells are released from the thymus into blood

Answer 117

Selection of B cells- three checkpoints for rearranged Ig chains for functionality and self-reactivity Pre-B cell: Is heavy chain functional? Expression a “pre-BCR” on the cell surface consists of VDJH-IgM constant region, surrogate light chains (VpreB, Lamda5), CD79a/b BCR signaling chains Outcomes Weak signal- cell proliferate and undergo light chain rearrangement Signaling too strong- cell is eliminated (reacts with antigens in bone marrow) No signaling- cell undergoes apoptosis (cannot signal) Immature B cells: is the BCR functional? Surface expression of the BCR VDJh-IgM constant region and VJL kappa light chain fully rearranged CD79a/b BCR signaling chains Outcomes Weak signal- cells proliferate and leave bone marrow Signaling strong- cell undergo rearrangement on lamda light chain locus Signaling too strong- cell undergoes apoptosis (reacts too strongly with antigens in bone marrow) Spleen- transitional B-cell: is the BCR reactive to antigens in the spleen? VDJh-IgM constant region and VJL kappa light chain fully rearranged Start to have IgD constant region BCR replace the IgM CD79a/b BCR signaling chains Outcomes Weak signal- cells fully mature resting B cells Signaling too strong- cell undergoes apoptosis (reacts too strongly with antigens in the spleen)

Answer 118

Selection process eliminates B cells that recognize self-antigens “self-reactive” Central tolerance- in the bone marrow Peripheral tolerance- in the spleen

Answer 119

Allows for safe administration of compatible transfusions Also allows for avoidance of neonatal isoerythrolysis

Answer 120

Blood groups defined by genetically determined markers (known as antigens) on the surface of erythrocytes Varies from species to species If it has the marker, it is positive If it doesn’t have the marker, it is negative The markers are considered “antigenic” (recognized as non self) in individuals lacking the same marker Antigenicity- likelihood that the immune system will react and make antibodies against the foreign substance If we give wrong donor blood to a transfusion recipient → can result in hemolytic transfusion reactions Surface antigens + opposing antibodies ⇒ agglutination (clumping) and hemolysis Important to know which blood types the donor and recipient are and which blood types and circumstances are likely to cause immune reaction Pre-transfusion testing minimizes the risk of incompatible transfusions and minimizes exposure of transfusion recipients to foreign red cell antigens they lack to prevent new antibody formation Blood typing- identifies the primary rec cell surface antigens of the donor and recipient Crossmatching- identifies any blood groups incompatibilities Major- tests for alloantibodies in recipient’s plasma against donor cells Minor- tests for alloantibodies in donor plasma against recipient’s RBCs

Answer 121

estimated 13 blood types DEA (dog erythrocyte antigen) DEA 1 most important → strong alloantibody response after sensitization → most clinically significant to test for DEA 3, 4, 5, 6, 7, 8 Others Dal Kai-1, Kai-2 Dogs lack clinically important natural alloantibodies pre-sensitization DEA-1 has no naturally occurring alloantibodies Variable naturally occurring antibodies in other DEA negative dogs (3, 5, 7) No known natural antibodies to Dal or Kai-1/Kai-2 First transfusion → cross matching is not required but recommended, especially if IMHA If repeat transfusion (or unknown history), required cross matching for transfusion DEA 1- donor → recipient can be DEA 1- or DEA 1+ DEA 1+ donor → only to DEA 1+ dogs

Answer 122

AB blood group system Type A (majority of DSH/DLH and non pedigree cats) Type B (uncommon but varies with breed/location) Type AB (rare) MiK antigen Patients are positive or negative Most DSH have MiK Pre-existing alloantibodies Type A: weak anti-B antibodies Type B: strong anti-A antibodies Type AB: no A or B antibodies Mik negative cats: anti-Mik antibodies Cats should be typed and cross matched before transfusion- confirm type B or AB due to rarity Potential for hemolytic transfusion reactions on first transfusion Transfuse Type A → type A cats Type B → type B cats Type A → type AB cats because there are not many AB donors Neonatal isoerythrolysis Litters from type B queens (+type A toms) Type A or AB kittens are at risk Kittens receive anti-A alloantibodies through colostrum → hemolysis Contributes to “fading kitten syndrome” Prevention Blood type prior to mating Remove kittens from type B queens for first ~24 hours

Answer 123

8 major blood group systems (7 internationally recognized) A, C, D, K, P, Q, U, (T) >30 different RBC antigens (factors) recognized within these groups Type-specific blood transfusions nearly impossible Aa, Ca, and Qa most important clinically for their role in hemolytic transfusion reactions and neonatal isoerythrolysis naturally occurring alloantibodies → ~90% do not have them, but ~10% do to Aa and Ca Antibodies can develop after transfusion or pregnancy (most common for Aa and Qa) There is no universal donor so attempt to match donor and recipient types as closely as possible Especially A, C, Q Other testing options Donors should have negative antibody screen Crossmatch Testing limitations Access to donors and rapid typing? Mostly Aa and Ca positive donors (majority of horses have this blood type) Emergencies First transfusion might be performed without compatibility testing If typed donors not available → use healthy gelding of same breed Usually well tolerated, unknown RBC lifespan Neonatal isoerythrolysis Higher incidence in Foals born to Aa or Qa negative mares Some breeds (eg. Friesans) Mule foals due to “donkey factor” Prevention typing/antibody screening Muzzle foal for 24-48 hours + alternative colostrum

Answer 124

Matching of blood types is not practical First transfusion risk- typically minimal Choose blood donor of same species transfusion Crossmatch if high risk

Answer 125

Dc are professional antigen presenting cells Uptake antigen from peripheral sites and migrate to the afferent lymphatics Guided by chemokines DC can be activated by many types of PAMPs/DAMP Presentation in the lymph node activates adaptive immune responses Therefore, DC are a bridge between adaptive and innate immunity

Answer 126

If activated by PAMPs/DAMPs or pro-inflammatory cytokines, endocytosis /environmental sampling is reduced Increases expression of chemokine receptors Migrate towards afferent lymphatics Increase expression of cell surface proteins Migrate to T cell zones Present antigens by MHC I and MHC II (MHC I: antigen is turned into smaller fragments → kept in ER; MHC II: fuse of phagolysosome and goes out into MHC II) Activate antigen-specific CD8 and CD4 T cells

Answer 127

Signal 1- antigen specific stimulation Ag peptides/MHC complex TCR interacting with MHC In naive T cell, this isn’t enough to activate Signal 2- Co-stimulation Membrane bound proteins CD28 on T cells CD80/CD86 (aka B7.1/B7.2) on DC Signal 3- polarization Determine the “character” of effector mechanism Induce correct gene transcription to deal with problem at hand Membrane bound molecules Cytokines - “instructions” with how to deal with this antigen and tells it where to go

Answer 128

CD4+- T helper cell Makes cytokines Can be either T follicular helper cells or T regulatory (induced) Different signal 3’s can give rise to different effects and cytokines produced CD8+- cytotoxic Punch holes (using perforin) in target cells and enzyme goes in there and kill the target cell

Answer 129

NK cells- kill host cells that lack MHC I Acts as an inhibitory signal in the NK cell Not antigen specific Often consequence of viral infection Some viruses decreases MHC I presentation → no more stop signal → cell death CD8 cells- recognition of antigen:MHC I complex Antigen specific killing → stimulation to kill

Answer 130

Naive cells- function as pool for activation Activation requires all three signals Memory T cells- after initial response, waiting to see antigen again to mount response Effector T-cells die off after insult is cleared Three main types of memory Tcm- central memory (reside in LN, spleen, circulate) Tem- effector memory (circulate in/out of tissues) Trm- Tissue resident memory (reside in tissue, site of insult) Antigen-experienced CD4+ or CD8+ T cells that can be reactivated by “signal 1” alone Increase the number of antigen-specific T cells for faster and stronger recall responses

Answer 131

Following release of pre-platelets → mature platelets 30% of platelets storage in spleen Life span: ~4-6 days Sensensence leads to desialylation Loss of sialic acid exposes galactose which triggers Phagocytosis by Kuffer cells TPO production

Answer 132

Regulation → mediated by hormone, thrombopoietin (TPO), which is made in the liver primarily (kidneys, smooth muscle) Levels of TPO are regulated by platelet numbers (“sponge theory”) Platelets express receptors that bind to TPO and internalize TPO TPO does not cause fragmentation of proplatelets from MK Other stimulants of TPO Senescent “aged” platelets Inflammatory cytokines IL-6 IL-11 GM-CSF CLL5

Answer 133

Sites of platelet formation Bone marrow Bloodstream Lung Megakaryoblasts undergo endometriosis (endoreplication) to become polypoid Endomitosis- all the steps of mitosis except they don’t split Each MK releases ~10^4 platelets Production of all necessary membrane proteins, granules, organelles, and genetic material (RNA) for a mature platelet to survive circulation MK extends long cytoplasmic processes with platelet-sized beads, known as proplatelets Proplatelets further undergo fragmentation to form mature platelets in circulation

Answer 134

Platelet membrane- fluidity of phospholipid bilayer maintained by cholesterol, phospholipids, and influenced by temperature Receptors “float on” the bilayer and act in concert when given the signal When temperature <15 to 18 degrees celsius, fluidity of membrane declines (phase transition) Gel consistency → R can’t move on cell membrane → causes isolation or crash together (too little or too much signaling) This shift activates platelets inappropriately or decreases their ability to respond Phospholipids on platelet membrane are heterogenous Inactive membrane- neutral phospholipids are located in the exterior while electronegative phospholipids face the cytoplasm Electronegative phospholipids are “flipped” when platelets are activated Integrins are heterodimeric (alpha and beta subunits) transmembrane receptors linking to the cytoskeleton Crucial role in platelet plug formation and clot retraction Integrin alpha IIb beta 3 (pka GPIIb/IIIa) In unstimulated platelets, they remain in “low affinity” state Platelet agonist activated the integrin in the cytoplasmic tail (beta3) → opening of ectodomain → “high affinity” state

Answer 135

initiation extension stabilization

Answer 136

platelet adhesion to damaged endothelium Exposure of collagen and release of vWF (von Willebrand factor) from endothelium vWF cleaved and immobilized on collagen fibers Platelets bind to vWFs (tethering and rolling) Facilitates formation of initial platelet monolayer

Answer 137

activation of platelets due to adhesion to the monolayer. Release of agonist for self-activation and recruitment of additional circulating platelets to the initial monolayer Secretion of... ADP Binds to P2Y12 and P2Y1 → causes Ca mobilization, TxA2 generation, and platelet aggregation Inhibits Prostaglandins Sources- denes granules, damaged cells at vascular site of injury, breakdown of ATP, works synergistically with other platelet agonists P2Y12 can be inhibited by a class of drug called thienopyridines (clopidogrel, prasugrel) Thromboxane A2 Arachidonic acid is cleaved by the enzyme, phospholipase A2 (PLA2), activated by many other agonists TxA2 is generated from the fatty acid, arachidonic acid (AA), in the cell membrane via COX-1 TxA2 generated can self-activate platelets or activate nearby platelets via TR Aspirin inhibits platelets by inhibiting COX-1 decreasing generation of TxA2 Non-steroidal anti-inflammatory drugs (NSAIDs) Thrombin Thrombin is generated via secondary hemostasis Most potent activator of platelets Thrombin activates protease activated receptors (PAR) on platelets Strong couple of phospholipase C resulting in profound elevation of intracellular Ca++ Granules are also secreted during activation Platelet shape change Occurs secondary to activation or changes in blood flow (shear stress) Transformation from biconcave disk to fully spread cells Increases surface area to facilitate further adhesion to site of vascular injury and other platelets Must occur prior to platelet aggregation Formation of procoagulant membrane Membrane is heterogeneous Platelet activation externalizes electronegative phospholipids They provide “docking” surface for coagulation factors to form complexes for thrombin generation (Factor II) Integrin activation

Answer 138

Binds to extracellular matrix proteins Activation of alpha IIb beta3 → fibrinogen, fibronectin, and vWF Platelet aggregation- platelet to platelet interaction through fibrin mesh Once alpha IIb beta3 binds to its ligand, platelets undergo “final activation process” Clot retraction and stabilization of the platelet plug

Answer 139

Thrombin is generated by secondary hemostasis Alpha granules released from platelets are important for secondary hemostasis Facilitates fibrin formation

Answer 140

Primary- like letting the tap drip slowly, so symptoms are Petechiae Ecchymosis Epistaxis melena/GI Hemorrhage (dog) Scleral hemorrhage Secondary- like not turning off the tap at all Hematomas Hemoabdomen Hemothorax Hemarthrosis Both could be due to trauma or surgically induced Primary- platelets, endothelium, vWF Secondary- coagulation factors, fibrinolysis (removal of clots after formation)

Answer 141

Mean platelet volume (MPV)- the mean volume of all platelets Large platelets in circulation due to increased fragmentation and increased demand (higher TPO) Indication of active bone marrow response Manual count- count # of platelets in 10 100x oil immersion field monolayer Estimated platelet count X 10^3/ul = average # of platelets in 10 fields x 15 If there are clumps, estimated count should be considered a minimum value

Answer 142

Low platelet count- can be either artifact/pseudothrombocytopenia, macrothrombocytopenia, or thrombocytopenia Artifact Dilution Clumping due to in vitro platelet activation and aggregation Increased duration from sampling to anticoagulant → clot formation Inadequate mixing of blood and anticoagulant Poor venipuncture Pseudothrombocytopenia Anticoagulant (EDTA)-mediated alteration in integrin causing agglutination by antibodies, IgG or IgM Use other anticoagulants like heparin or citrate (account for ~10% drop in platelet count for citrated samples) Macrothrombocytopenia - breed dependent mutations in beta 1 tubulin leading to “Giant Platelet Disorder” Defective fragmentation of proplatelets from megakaryocytes Characterized by Low platelet count (as low as 20,000/ul) High MPV No bleeding diathesis Normal function, no clinical signs in dogs Thrombocytopenia Increased consumption Acute onset of hemorrhage → loss +/- consumption of platelets from trauma or surgery Disseminated intravascular coagulation (DIC)- ongoing consumption of coagulation and platelets due to underlying diseases Snake envenomation Moderate to marked thrombocytopenia Increased destruction- immune mediated thrombocytopenia (ITP/IMTP) Platelets are phagocytized by splenic/hepatic macrophages mediated by anti-platelets antibodies and complement Primary is due to spontaneous autoimmune disease, while secondary is due to underlying disease Marked thrombocytopenia → spontaneous hemorrhage Decreased production Thrombopoiesis affected at the level of the bone marrow Myelophthisis caused by fibrosis, neoplasia, or granulomas chemotherapy, radiation, viral infection (canine parvovirus, feline panleukopenia virus) Other types of blood cells are likely affected Moderate to marked thrombocytopenia Increased sequestration Spleen harbors up to 30% of circulating platelets (seen in splenomegaly) endotoxemia/sepsis → platelet activation → sequestration in pulmonary vasculature or other microvasculature Thrombocytopenia is mild

Answer 143

increase in number of platelets in circulation (>500 x 10^3 /L) Primary- when idiopathic, while secondary- from other diseases Inflammation, neoplasia, endocrine (hyperadrenocorticism)

Answer 144

Initiation- platelet adhesion to damaged vessels vWF and exposure of collagen is important Platelets express receptors that bind to collagen and vWFs

Answer 145

Tier 1 Tests Template bleeding time (TBT)- making cuts in the animal (standardized) and recording the time it takes to stop bleeding Buccal mucosal bleeding time (BMBT)- standardized cut into lip and recording time it takes for bleeding to stop Crude assessment of initiation phase of coagulation Platelet function assay- 100/200 Cartridge based assay that mimics in vivo high-shear environment. Whole blood flown through an aperture and membrane coated with collagen +/- ADP or epinephrine Time takes for aperture to close measured as “platelet closure time” Assess vWFs and platelet function Highly automated with high precision and repeatability Not widely available and non-specific (screening test) Quantity- immunodetection using anti-vWF antibodies (vWF:Ag) Patient plasma compared to species-specific pooled plasma from healthy animals Diagnostic = vWF:Ag < 50% Equivocal or borderline = vWf:Ag 50 to 70% Normal = vWF:Ag > 70% Multimeric assay Analysis of multimers by separation of vWFs based on molecular weight by electrophoresis and immunoblot Rarely performed in clinical patients Functional assay Rarely performed in clinical patients vWF to collagen binding (vWF:col) For known mutations Genetic testing- test for potential carriers, patients with equivocal vWF:Ag Tier 2 Tests Aggregometry Collects platelet rich plasma and measures light that can pass through Flow cytometry Characteristics: cell size/granularity Quantity specific population of cells Expression of cell membrane receptors Intracellular protein expression Intracellular protein phosphorylation

Answer 146

T independent- non protein antigens can activate B cells Typically structures with repeating epitopes Polymers, carbohydrates, lipids T dependent- most effective B cell responses, supported by CD4+ T cells After activation and clonal expansion, Migration to B-T cell border Receive signals (CD40 ligand on T cell) Differentiate into long lived plasma cells or memory B cells Step 1- antigen binds to BCR Step 2- antigen is internalized and processed for presentation on MHC II Step 3- B cell with epitope on MHC II interacts with T cell that recognizes that specific antigen B cell co-stimulation signaling: interaction of T cell CD40L and CD40 on B cell Cytokines from T cell skew or induce isotype in the B cell

Answer 147

Antigen goes into afferent lymphatics → in the subcapsular sinus, complement receptor binds to it from subcapsular macrophage → B cells pass it along to each other until it gets to the B cell follicle → passes antigen to follicular dc → mature B cells see if it can bind to antigen epitope → proliferates if it does → goes out efferent lymphatics Similar process in the spleen → passing of antigen in marginal zone until it gets to follicular dc in the primary follicle Naive B cell → activation (binding to antigen) → clonal expansion → activated B cells migrate to T-B border zone Development of B cell (VDJ) → somatic hypermutation (random point mutations in V region) → class switch (constant regions)

Answer 148

Prior immunization increases the number of specific B and T cells Some of these will become memory B and T cells Respond faster to BCR and TCR activation No need for costimulation to activate memory T cells Memory B cells rapidly differentiate in an extrafollicular response = fast production of antigen specific class-switched antibodies Challenge (re infection or booster immunization) gives faster and stronger responses This is the basis for vaccine induced immune protection

Answer 149

Activation induces “Activation-induced deaminase” (AID) expression Switch the Ig heavy chain constant region → produce different Ig isotypes Loops different parts → parts that aren’t in loop → constant that influences Ig type This is based on cytokine signaling Constant region dictates structure → structure lends to function (# of binding sites) Receptors for these isotypes are differentially expressed and may have unique affinities for each isotype Gives rise to what it can bind In early response, Ig is mostly IGM, but as time as goes on, more class switching → more IgG response

Answer 150

T independent- after activation, B cells differentiate into antibody-producing plasma cells → short lived cells At border of B-T cell zone Cells reside in medullary cords of LN or red pulp of spleen Rapid generation (days-weeks) Generation of short-lived plasma cells No/little memory

Answer 151

T dependent Lymph tissue follicles Develop slowly (1-2 weeks) Introduction of point mutations in the variable region that encodes BCR “somatic hypermutation” Changes BCR - random point mutations that are most frequent in the VDJ region Since random, some BCR will have low affinity to helper T → apoptosis Some will have high affinity → T cell helps B cell mature and proliferate → memory B cell and plasma cell Generates long-lived high-affinity plasma cells that migrate to bone marrow Leads to memory B cells

Answer 152

Memory Do not secrete antibodies Circulate between blood and secondary lymphoid tissues Can reside in other tissues Often Ig class switched (not IgM+) Plasma cells Migrate to bone marrow Secrete class switched and high affinity antibodies into the blood Antibodies persist for months to years in serum and plasma

Answer 153

clinical test for exposure to antigen by determining frequency of antigen specific IFNg secreting T cells Using the whole blood from patient, which contains APC and CD4+ T cells Add antigen to the cells Antigen phagocytosed by APC Presented to T cells by MHC II On plates that are coated capture with antibodies that bind IFNg After detection, antibody and substrate addition, count stops

Answer 154

Antibody capture (indirect ELISA) Antigen coated well → addition of patient serum → specific antibody binds to antigen → wash and enzyme linked antibody binds to specific antibody → wash and substrate is added and converted into colored product and measured with absorption Antigen capture (sandwich ELISA) Monoclonal antibody coated well → wash and addition of patient serum → antigen binds to antibody → wash and second monoclonal antibody, linked to enzyme, binds to immobilized antigen → wash and substrate is added and converted by enzyme into colored product and measured as absorption

Exam 1 Flashcards

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