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Blood
Fluid CT
 Blood volume is ~ 9% body volume (9ml/kg; not same in cats)
 Blood sampling (cannot take more than 1% body weight in blood)
Function
 Carries oxygen, nutrients, hormones
 Helps eliminate carbon dioxide and other wastes
 Immune function
Composition of blood
 Plasma
o 90% water, 10% other substances
o Low molecular weight molecules
 In equilibrium w/ interstitial fluids (b/c can readily penetrate membranes)
 Glucose, electrolytes
o High molecular weight molecules
 Albumin
o Maintains oncotic pressure
o Decreased albumin results in oncotic pressure and edema/ascites
 Clotting factors, immunoglobulins, enzyme, lipoproteins
o Normal appearance
 Usually colorless or straw colored
 Equine plasma is light yellow
o  bilirubin levels
 Bovine/ruminant plasma can be light yellow
o  ingestion of carotenoids in some plants
o Abnormal appearance
 Color can say a lot about the abnormality of a sample
 Hemolysis (reddish)
o Indicates intravascular RBC lysis
 Icterus (yellowish)
o Indicates  bilirubin
 Lipemia (milky opacity)
o Indicates  lipids
 Serum
o Liquid remaining after blood has clotted
 Lacks most clotting factors
  Total protein level
 Blood cells
o Most blood cells are really CT cells
o WBCs
 Use the bloodstream simply as a transport system
 Present in blood for a few hours, at most a couple of days
 Some circulating cells are immature cells who mature after taking residence in CT
o Examination of blood cells
 Cell counts
 Blood smears (cytology/hematology prep)
o Air-dried and stained with Romanovsky stain
o Look different than those in a histopath prep
o Thin smear (versus several layers)
- Cells flattened and look larger
- More cytoplasmic detail
o Wet fixation changes chromatin appearance
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Romanovsky stains
- Acidic eosin
 Stains RBCs and eosinophil granules
 Eosinophilic (yellow-orange-pink)
- Methylene blue
 Stains ribosomes, RNA, some protein
 Basophilic (blue)
- Nuclei and basophil/mast cell granules alter color of stain
 Azurophilic or metachromatic (purple)
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Hematopoeisis
 Production of blood cells (WBCs, RBCs, platelets, etc.)
o Occurs primarily in adult bone marrow
o Some extramedullary hematopoesis in spleen
 Especially in mouse, ferret, hedgehog
o Although some lymphocyte development occurs in marrow of young animals, most of the
lymphoid cells are made elsewhere
 “Yellow marrow” (lots of fat cells)
o Inactive hematopoesis
 “Red marrow”
o Active hematopoesis
 Monophyletic Theory of Hematopoesis
o Pluripotent stem cells can differentiate into other blood cell types
o If irradiate bone marrow, can re-grow all marrow provided the presence of some stem cells
 Amplification and differentiation of cells
o Erythropoesis – production of RBCs
 Howell-Jolly body (mature RBC with nucleus remnant)
o Leukopoesis (granulopoesis) – production of WBCs
 Lobulated, dark nuclei (in mature WBCs)
o Megakaryopoesis (thrombopoesis) – production of platelets
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Red blood cells
 Most common cell type in blood (7-10x106/µl)
 No mitochondria
o ATP is produced anaerobically in RBCs
 Histologic appearance
o Homogeneous orange to pink
o “Central pallor” (pale center)
o Color due to hemoglobin
 Globin (alpha and ß chains)
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 Heme (contains Fe)
Membrane proteins
o Receptors, electrolyte pumps, blood group molecules, cytoskeletal proteins
o Integral proteins create slight negative charge
 RBCs repel each other
 Loss of charge results in rouleaux (RBC stacking)
o Horses rouleaux more than other species
RBC cytoskeleton
o Reinforces membrane
o Contributes to cell flexibility
Shape
o Biconcave disk shape
  SA to volume ratio (optimal gas
exchange)
 Allows for deformability during
circulation (don’t get stuck in vessels)
o Species differences
 Dog
o Large area of central pallor
 Goat
o Very small RBCs (have more
to compensate)
 Camelid
o Oval-shaped RBCs
 Bird
o Large RBCs, probably due to presence of nucleus (inactive)
Function
o Carry oxygen to tissues (oxyhemoglobin)
 Hb takes up oxygen in lungs and releases in tissues
 Advantage of packaging Hb
o Slower turnover time
o Ability to maintain iron in ferrous state
o Ability to control Hb affinity for oxygen
o Prevents osmotic effects of free Hb
o Transports carbon dioxide to lung (carbaminohemoglobin)
Polychromasia
o Large, immature RBCs called different names depending on the stain
o Wright Stain
 RBCs have cytoplasm with blue-grey tinge (ribosomes present)
 Cells called polychromatophils
o New Methylene Blue
 RBCs appear to have clumped ribosomes
 Cells called reticulocytes
Sensecent RBCs
o RBCs circulate for several months
o Worn out or damage RBCs are eliminated by macrophages in spleen and bone marrow
RBC Abnormalities
o Polycythemia
  [] of RBCs per volume of blood
o Anemia
  [] of RBCs or hemoglobin per volume of blood
 Regenerative anemia
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Characterized by immature cells in circulation
Indicates marrow response to blood loss or hemolysis (RBC destruction)
White Blood Cells
 ~ 6-16x103/ul of blood
 Classification
o Granulocytes
 Neutrophils, eosinophils, basophils
o Agranulocytes
 Lymphocytes, monocytes
 Motile cells
o Function in CT
o Body’s defense against infection
o Huge number produced, but quickly move out of blood stream into tissue
o Move into tissue in a sequence of events
 Margination – adhesion of WBCs to endothelium of blood vessels
 Diapedesis – degradation of blood vessel basement membranes in order for WBCs
to pass through into interstitial fluid
 Migration – WBCs move along chemotactic gradient to the site of injury/infection
 WBC count
o Only count free WBCs (circulating pool)
o Don’t count WBCs stuck to wall (marginal pool)
o Marginal pool = circulating pool
 Dogs, calves, horses, humans
o Marginal pool is 2.5 times
circulating pool (cat)
 Leukocytosis (increased WBC)
 Leukopenia (decreased WBC)
Granulocytes
 Lobulated, ribbon-shaped nucleus
 Azurophilic granules (primary granules)
o Lysosomes (present in both
granulocytes and agranulocytes)
 Specific granules (secondary granules)
o Differentiate from primary granules
o Specific for type of granulocyte
 Neutrophils, eosinophils,
basophils
 Tertiary granules (only in neutrophils)
o Facilitate migration through CT
Neutrophils
 Most numerous WBC
 Duration in blood (6-10 hours)
 Lifespan in tissue (1-2 days)
 Typical Appearance
o “Neutral” staining
o Indistinct and almost colorless
(sometimes pale pink to pale lilac)
o Tightly clumped chromatin
o 20 granules usually indistinct
o Polymorphonuclear leukocytes
(PMNs), segmented neutrophils,
immature neutrophils (bands)
o Defined by shape of nucleus
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Function
o Released from marrow in response to inflammation
o First responders to any inflammation reaction
o First line of defense against pathogenic bacteria
o Phagocytose bacteria
o Oxygen-dependent systems
 Myeloperoxidase in 10 granules (H2O2  HOCl)
 Respiratory burst (free radical formation)
o Oxygen-independent systems
 Lysozyme (degrades cell walls)
 Lactoferrin (binds iron)
 Low pH in phagosomes
o Synthesize and release immunoregulatory molecules (20 granules)
 Pro-inflammatory factors
 Anti-inflammatory factors
o Anaerobic metabolism allows survival in inflamed and necrotic tissues
o Release of degradative enzymes leads to tissue damage
 Neutrophilia
o Increased circulating neutrophils
o Often a sign of inflammation
 Neutropenia
o Decreased circulating neutrophils
o Patient is at risk of bacterial infection
Eosinophils
 Only remain in blood for ~20 minutes
 Survive several weeks in tissues
 Typical appearance
o Ribbon-shaped nucleus
o “Eosinophilic” granules
o Orange to pink (similar to RBCs)
o Orange 20 granules
 Function
o Defense against parasites
 Not very phagocytic, but kill parasites by emptying toxic substances onto them
o Major basic protein, eosinophil peroxidase, eosinophil cationic protein
 Regulation of allergic reactions
 Eosinophilia
o Increased circulation of eosinophils
o Often a sign of parasite infection
o May be a sign of allergic disease
 Eosinopenia
o Decreased circulation of eosinophils
Basophils
 Least common leukocyte (<1%)
 Function
o Promote inflammation
 Heparin and histamine affect
smooth muscle, make
capillaries leaky
 Hypersensitivity reactions
o Allergic reactions
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Wasp stings
Typical appearance
o Ribbon or horse-shoe shaped nucelus
o Granules are usually dark purple (metachromatic)
o Granules more difficult to identify in dogs and cats
 Cytoplasm and granules may be lavender or gray color
 Similar to color of the nucleus
How do mast cells compare to basophils?
 Similar metachromatic granules and function
o Basophil (bean to ribbon-shaped nucleus)
o Mast cell (round nucleus)
 Arise from difference precursors in marrow
o Basophil leaves the bone marrow already mature
o Mast cell circulates in an immature form, only maturing once at a tissue site
 Usually in CT near blood vessels (especially skin, respiratory and GI mucosa)
 Important cause of allergic reactions
Agranulocytes
 Have lysosomes, but lack specific and secondary granules found in granulocytes
 Lymphocytes, monocytes
Lymphocytes
 Predominant agranulocytes
 Predominant WBC in some species
 Precursors produced in marrow but then “seed” lymphoid tissue where new cells are produced
o Thymus, lymph node, spleen
 Most time spent in the lymphoid tissues, but can re-circulate
o Move from lymph node to lymph node via lymphatics and blood stream
 Typical appearance
o Usually round nucleus (occasionally cleaved)
o High nuclear–to–cytoplasm ratio with small amount of smooth, light blue cytoplasm
o Smooth, slightly clumped, pinkish purple chromatin
o Vary in size
 Small (smaller than neutrophil)
 Medium
 Large (larger than neutrophil)
 Reactive lymphocytes
o Ability for mitosis
 Stimulated lymphocytes can activate their nucleus and proliferate
 Cytoplasm can become a deep royal blue
 Function
o Important for protection against microbes, viruses, and neoplasia (cancer)
 Production of antibodies (B lymphocytes)
 Destruction of infected or neoplastic cells (T lymphocytes)
o Variable life span (some live for years)
 Lymphocytosis
o Increased circulating lymphocytes
o May indicate a response to a foreign agents (virus, neoplasm)
o Response to vaccination
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Monocytes
 Precursors for long-lived macrophages
(live for months in tissue)
 Typical Appearance
o Vary in size (usually the
largest WBC)
o Nucleus is round to lobated
o Cytoplasm
 Grainy, blue to bluegrey, may have
vacuoles
 Color is distinctly
different from nucleus
 Functions
o Phagocytosis of large, particulate debris
o Antigen processing
o Cytotoxicity
o Production of monokines
 Interleukins
 Tumor necrosis factor
 Factors that promote tissue repair and angiogenesis
o Production of Tissue Factor Procoagulant
 Link between inflammation of coagulation
 Monocytosis
o Increased circulating monocytes
o Often indicated inflammation or tissue necrosis
Platelets
 200,000 – 500,000/µl
 Small cytoplasmic fragments
 Survive 8-10 days
 Megakaryocytopoesis
o Platelets derived from the cytoplasm of bone marrow megakaryocytes
 Typical appearance
o Light-stained periphery (hyalomere)
o Central zone with granules (granulomere)
o NO NUCLEUS
 Structure
o Microtubules, microfilaments, glycogen particles for energy
o Alpha granules
 Contain coagulation factors and growth factors (i.e. fibrinogen)
o Dense bodies (ADP, calcium, Mg, serotonin)
o Lambda granules (lysosomes)
 Function
o Clot formation
 Form temporary aggregate to patch over damaged endothelium
 Common for platelets to clump during blood collection
 Degranulation releases clotting factors and a vasoconstrictor
o Triggers clotting cascade (formation of fibrin plug)
 Activation of contractile cytoskeleton results in clot retraction
o Allows tissue to be re-vascularized