Block 2 Outline
Microanatomy
Blood
Hematocrit: measure of volume of RBCs per unit of blood
Males: 40-50% RBCs
Females: 35-45% RBCs
Newborns: 45-60% RBCs
Children (under 10): 35%
Polycythemia: increased hematocrit
Anemia: decreased hematocrit
Buffy Coat: layer above RBCs, contains white blood cells
This layer of leukocytes (WBCs) constitutes 1% of total blood volume
Leukocytosis
-increase in WBCs in peripheral blood
-often associated with leukemia
Leukocytopenia: decrease in WBCs in peripheral blood
Platelets
-fine layer on top of the white blood cells
Remember
-in a hematocrit tube, the layers from bottom to top follow this progression
Plasma RBCs WBCs platelets
Plasma
-contains 90% water
-7-9% contains the following proteins
Albumin
-maintains colloid osmotic pressure in capillaries
Globulins
Type alpha: glycoprotiens, HDL’s, ceruloplasmin and prothrombin
Type beta: transferrin and LDL’s
Type Gamma: antibodies
Fibrinogen
-major clotting protein
-1-3% contains hormones, glucose, AAs, sodium bicarbonate and calcium
Plasma: liquid portion of anticoagulant treated blood
Serum: liquid portion remaining after a clot has formed
Formed elements
-includes RBCs, WBCs and platelets
Romanovsky staining
-finds out net charge of blood based on affinity for dye
Eosinophilic: Constituents turn orange pink
Basophilic: Constituents turn blue in methylene blue
Azurophilic
-Constituents have affinity for active form of methylene blue
-stuff turns purple
Cell sizes (decreasing order)
Monocytes: 12-20 microns
Lymphocytes: 6-18 microns
Neutrophils, eosinophils and basophils: 12-15 microns
Erythrocytes: 6.5-8 microns
Platelets: 2-4 microns
Cell abundances (decreasing order)
Erythrocytes: 35-60% depending on age and sex (~4-6 x 10^6/microliter)
Platelets: 2-4 x 10^5/microliter
Leukocytes: 6000-10000/microliter
Neutrophils: 60%
Lymphocytes: 20-30%
Monocytes: 3-8%
Eosinophils: 2-4%
Basophils: <1%
Erythrocytes
-biconcave shape provides high surface area to volume ration higher gas exchange rate
In a dry smear, normal blood has…
Normocytic: normal size
Normochromic: normal color
Zone of pallor: central pale area
other terms…
Microcytic: small size
Macrocytic: large size
Hypochromic: less Hb than normal
Hyperchromic: more Hb than normal
Poikilocytosis: diversity in cell shape
Anisocytosis: abnormal variations in size
2 main causes in goofy erythrocyte shapes
1) Abnormalities in hemoglobin
HBS (sickle cell hemoglobin)
-under low O2, HBS is insoluble
-smear shows sickle cells
-HBS causes cells to get stuck in vessels, hemolysis and anemia
HBF persistence (thalassemia) and diminished globin chain synthesis
-smear shows cells looking like a target (target cells)
-result is anemia
2) defects in cytoskeletal genes
a) mutation in spectrin
-results in spherocytosis
-smear shows spherocytes (rounder RBCs)
-result is hemolytic anemia
b) mutation in band 4.1, Band III and spectrin
-results in elliptocytosis
-smear shows eliptocytes
-results in mild hemolytic anemia
Random blood facts
-RBCs last 120 days, then removed by spleen
Reticulocytes
-newly released RBCs
-contain rRNA that stains with supravital dyes (cresyl blue)
Polychromatophilic erythrocytes: stain RNA in blue (basophilic?)
-mature within 24 hours after being released
-account for .8% of RBC volume
-RBCs are important for antigen profiling (A, B, O and rh)
-important for tissue transplants
Thrombocytes (platelets)
-survive 10 days in circulation
-cell fragments that lack a nucleus
-come from megakaryocytes
Contain 2 distinct regions
hyalomere
-peripheral light blue staining region
-characterized by high actin/myosin concentration
-filipodia containing microfilaments come out of this region upon activation of platelet
Granulomere: auzurophilic granule zone containing…
Alpha granules
-most abundant
-contains fibrinogen, von willebrand factor and platelet-derived growth factor and more proteins
Delta granules
-also called dense bodies
-non protein granules containing calcium, ATP, ADP
-store seratonin
Lambda granules
-lysosomes
-platelets contain a thick glycocalyx used for adhesion
-platelet ovoid shape is maintained by the marginal band of MTs
-canalicular system helps release granule contents
Clotting mechanism
1)injured endothelial cells release vonWillebrand factor and thromboplastin causing platelets to aggregate
2)platelets stick to sub endothelial (since the injury is beneath the endothelium) collogen and granule contents are released. Platelets adhere to each other (this is called platelet activation)
3)platelets release ADP and thrombospondin to attract more platelets. Platelets factors start begin to show up on plasma membrane causing further adhesion
4)fibrinogen fibrin which causes the further coagulation. Fibrin forms a net to trap formed elements
Leukocytes
Granulocytes
-contains granular components that bind to romanovsky dye
-the three types are neutrophils, eosinophils and basophils
Agranulocytes
-contain non specific azurophilic granules
-two types include lymnphocytes and monocytes
*patients between 2 weeks to 4 years have 60% lymphocytes in leukocyte population (decrease in neutrophils). Lymphocyte percentage decreases until ages 14-15 (then the population stabilizes)
Granulocytes (in depth look)
Neutrophils
-9-12 microns in diameter
-salmon pink cytoplasm and lobednucleus
-circulates for 67 hours, lives only a few days
Nucleus
-Begins as a band shape and matures to an elongated lobed structure
-contains chromatin clumps and a barr body (in females [smear indicates sex])
Cytoplasm: 2 types of granules
Azurophilic (primary)
-.5 micron diameter (larger)
-primary lysosomes containing acid phosphatases, myeloperoxidase and hydrolytic enzymes
Specific (secondary)
-.1-.2 microns (resolution of light scope)
-contain alkaline phosphatase, collagenase, lactoferrin and lysozyme
Chemotaxis: attraction to site of inflammation by molecular signals
Function
-First line of defense
-ameboid and phagocytic
-attach to endothelium of vessel and them move between cell junctions by diapedesis
-bacteria are engulfed in phagosomes and hydrolytic enzymes are dumped into vacuoles
-coating of bacteria with IgG and then a coat of C3b enhances uptake of bacterium
Opsonins: plasma components that coat bacteria (i.e. IgG and C3B)
Bacterial killing
-first specific granules fuse with phagosome and then proton pumps lower pH. Then azurophilic granules dump stuff into phagosome and bacteria dies in the following ways.
i) Reactive superoxide anions from Oxygen burst kill bacteria
ii) lysosome destroys cell wall of bacteria
iii) lactoferrin binds to iron and deprives bacteria of free iron
-can occur in low [O2] since neutrophils gain most of there energy from glycolysis. TCA cycle is not as important
-extracellular killing of bacteria is possible through neutrophil extracellular traps (NETs)
Eosinophils
-same size maybe slightly larger than neutrophils
-longevity of 8-12 days, circulates for 3-4 hours
Nucleus: segmented and bilobed
Cytoplasm
-contains 200 specific granules per cell
-elongate granules are larger than neutrophil granules
-crystalloid center of granule
-highly eosinophilic
-contains major basic protein
-contains peroxidase, eosinophilic peroxidase, acid phosphatase and ribonuclease
Function:
-increase in numbers in response to a parasite
-found near basophils and mast cells
-selectively ingest antigen/antibody complexes
-counteract the inflammatory effects of histamine
-concentrated in mucosa linings near exterior openings
Basophils
-slightly smaller than neutrophils
-non phagocytotic
-look like mast cells but come from separate bone marrow cells
Nucleus
Segmented nucleus, irregular lobes
Cytoplasm
-has largest granules of all granulocytes
-granules are water soluble
-granules contain
-heparin (accounts for metachromasia)
-no lysosomal enzymes
-histamine and peroxidase
Function
-Basophils contain IgE/IgE surface receptor complexes which increase in number during hay fever, allergies and asthma
-antigen combines with IgE to cause massive degranulation releasing tons of histamine which causes swelling/itching
Agranulocytes
Lymphocytes
-small cells with dense nucleus (size of RBC) with rim of cytoplasm
-sometimes have azurophilic granule
-migrate along lymphoid tissues and blood
3 different types of lymphocytes
B –Cells
-come from bone marrow
Plasma cells
-activated lymphocyte which constantly secretes antibodies
-don’t travel in peripheral blood
Humoral immune response: a bodily defense reaction that recognizes an invading substance (an antigen: such as a virus or fungus or bacteria or transplanted organ) and produces antibodies specific against that antigen
T-Cells
-originate from embryonic yolk sac
-can stimulate or depress activity of fellow T-cells and B-cells
Cytotoxic T cells
-recognize foreign antigens and kill cells
Cell-mediated immunity: immune response which does not involve antibodies, but rather the activation of macrophages and cytotoxic T-cells and the release of cytokines
Natural killer cells?
When exposed to a specific antigen
1)Lymphocyte now only reacts to that specific antigen
2)Lymphocyte divides
3)Progeny act as helper T-cells, differentiate into plasma cells or become “memory” cells
*T-cells live longer than B-cells and can last for years
*in circulating blood T cells make up most of lymphocytes while B-cells only account for 15-30%
Monocytes (other form of agranulocyte)
-largest WBC in blood smear
-circulate in blood for 1.5 days before invading tissues
-phagocytosis of foreign stuff
-act as antigen presenting cells for lymphocytes
-can divide and differentiate into macrophages (survive long in tissues [months])
Nucleus
-round to horshoe shaped
-chromatin is lightly condensed
Cytoplasm
-stains grey-blue
-contain azurophilic granules
Blood cell formation
-remember that blood cells survive 120 days, platelets 8-11 days and neutrophils a few days
Hemopoesis: formation of peripheral blood (except lymphocytes) from bone marrow
-Lymphocytes come from yolk sac and marrow but mature in the lymphoid organs
Fetal hemopoiesis
2-6 weeks
-occurs in blood islands in body stalk of yolk sac
-primitive erythrocytes have nuclei
6 weeks+
-liver and spleen make definitive erythrocytes
-definitive erythrocytes have no nuclei
bone marrow forms after 5 months
Postnatal hemopoesis
-after birth up to 4-5 years of age, hemopoesis takes place in “red marrow”
-after 5 years, adipocytes replace red marrow making “yellow marrow”
-adult red marrow is found in vertebrae, ribs, skull, sternum and pelvis
-in certain diseases, yellow marrow and liver execute hemopoiesis
Structure of bone marrow
-capillaries of endosteum and cortical bone turn into marrow vessels (sinuses)
-some radial arteries connect with the sinuses
-sinuses lined with continuous endothelium (regulate entry and exit into marrow)
-marrow cells exit and enter through endothelial cells not between them
parynchyma- blood cells and maturing stem cells
stroma
-extracellular matrix and reticular fibars
-important for coordinating blood cell development
adventitial cells : cover outside of endothelial cells of sinus
adipocytes: come from adventitial cells
macrophages: found on erythroblastic islands
fibroblastoid cells: construct the basement membrane
marrow organization
-megakaryocytes (make platelets) lie next to sinuses
-erythrocytes are made next to sinuses
-granulocytes produced away from sinuses (formation regulated by stroma!!!)
cell hemopoises stages (look at table on page 240 and know it!)
Pleuripotent hematopoetic stem cells (PHSCs)
-constitute less than .1 % of total cells in marrow
-can divide or differentiate into multipotent stem cells
-multipotent stem cells give rise to progenitor cells
-pleuripotent, multipotent and progenitor cells all have surface antigen CD 34
marrow composition
-20-30% erythroid precursors
-60-70% myloid precursors
-10% lymphocytes, plasma cells, macrophages and stem cells
*stem cells DO circulate in blood
Erythropoesis
-important to review page 241 for pictures
Mitotic cells
Proerythroblast
-14-19 microns
-fine chromatin with nucleoli
-appear light with basophilic cytoplasm
Basophilic erythroblast
-12-17 microns
-clumped chromatin (spoked wheel appearance)
-no nucleoli and cytoplasm is very basophilic
Polychromatic erythroblast
-10-14 microns
-more condensed chromatin
-HB synthesis begins here
Non mitotic precursors
Orthochromatophilic erythroblast
-8-12 microns
-extremely dense chromatin with no light spots
-cytoplasm is eosinophilic
Reticulocyte (polychromatophilic erythroblast)
-larger than mature erythrocyte
-reticulum present from RNA
*macrophages in spleen eat up old RBCs and send iron back to marrow via transferring
platelet generation
Megakaryoblast
-Basophilic cytoplasm devoid of granules
-endomitosis occurs (replication of DNA but no nuclear division that results in lobulated nucleus)
Non platelet forming megakaryocyte
-cytoplasmic basophilia reduced and azurophilic granules appear
-granules clump and are separated by plasma membranes (however, plasmonella is still continuous with these new membranes)
Platelet forming megakaryocyte
-sheds platelets into sinue lumen through cytoplasmic extensions through endothelial lining
-4k-8k platelets formed from one megakaryocyte
Granulocytopoiesis
Mitotic cells
Myloblast
-basophilic cytoplasm, no granules, light nucleus, nucleoli present
Promyelocyte
-cytoplasm with primary azurophilic granules and nucleoli
Mylocyte
-no nucleoli, but secondary granules are apparent
Non Mitotic cells
Metamyelocyte
-kidney bean nucleus
-little basophilia
Band form (aka stab cell)
-horshoe or U shaped nucleus
segmented neutrophil (eosinophil or basophile)
-lobulated nucleus
Monopoesis
-monocytes come from CFU-M cells and leave immediately into connective tissue after differentiation
-most macrophages come from bone marrow, few from connective tissue
Blood cell compartments
Granulocytes
-stored and produced in bone marrow
-stored and functional in peripheral blood
-peripheral blood storage of granulocytes is found in the lining of blood vessels
Platelets (thrombocytes)
-produced in bone marrow but not stored
-stored and functional in peripheral blood
-storage of platelets in peripheral blood occurs in the spleen
Erythrocytes
-produced in bone marrow but not stored
-only functional RBCs circulate the periperal blood
Regulation of hemopoiesis
-occurs via cell-cell signaling through Bone marrow stroma
-occurs humorally through hormones and stuff outside marrow
Steel factor
-stromal cells produce and secreted onto plasmonella and binds to tyrosine kinase receptor (c-kit ligand) on pleuripotent cells, multipotent cells and progenitor cells
-stimulates blood cell production
-progenitor cells are sensitive to several growth factors but as differentiation occurs, fewer growth factors produce a response
look at figure 9-14 for illustration on page 245
*IL-1, IL-3, IL-6 are interleukins that maintain populations of Pleuripotent and multipotent stem cells
Erythropoiten (Epo)
-humoral factor that effects later stage cells of erythropoesis
-Higher O2 tension means less erythropoietin
-secreted by kidney
When going to higher altitudes
Polycythemia: increase in RBC count occurs (higher hematocrit)
-reticulocyte count initially increases only a little bit, but goes back to normal after acclimatization occurs
Circulation
General organization
Tunica intima
Endothelium
-simple squamous layer of endothelial cells
-Nuclei of this layer bulge into the lumen
-smooth layer helps blood flow
Subendothelial layer
-made of loose connective tissue
-has smooth muscle bundles (sometimes)
Tunica media
-contains elastic fibers
-contains circular smooth muscle bundles
Tunica adventitia
-contains mostly collagen and a little elastin
Internal elastic lamina
-separates tunica intima from tunica media
-found in arteries and large arterioles and sometimes veins
External elastic lamina
-separates tunica media from tunica adventitia in muscular arteries
Vasa vasoram
-blood vessels supplying blood vessels
Sympathetic stimulation
-primary autonomic nervous input (causes contraction)
Parasympathetic stimulation
-causes dilation of arteries indirectly by causing endothelial cells to secrete nitrous oxide
-some arteries contain baro and chemoreceptors
Types of vessels
Arteries
Large conducting or elastic arteries
-Example: pulmonary arteries or aorta
-have thick tunica intima (endothelium + muscular subendothelium)
-Tunica media has a good amount of smooth muscle with fenestrated elastin sheets
*elastic recoil of these vessels retains blood pressure
-tunica adventitia is pretty thin
-hard to see the internal and external elastic lamina
Medium, muscular or distributing arteries
-example: found in intercostals or thoracoacromial
-tunica intima: endothelium + muscular subendothelium
-tunica media: very thick with 40 layers of circular muscles
-tunica adventitia: blends in with surrounding connective tissue
-these arteries distribute blood flow to body
-easy to see internal elastic lamina due to overall lack of elastin
-can see external elastic lamina in largest muscular arteries
Small arteries + arterioles
-tunica intima: endothelium + a non muscular (but connective) subendothelium
-Tunica media: small arteries have 2 layers of muscle while arterioles can have 6
-these guys respond most to autonomic input
-Tunica adventitia is thin and blends with surrounding connective tissue
-can see internal elastic lamina in largest arterioles
-endothelial cells contain weibel-palade granules which help in blood clotting
-these granules are important for getting platelets to stick to endothelium
Capillaries
-consist of endothelial cells and basal lamina
-endothelial cells joined together by tight junctions and gap junctions
Pericytes
-surround endothelial cells
-their basal lamina is fused with the basal lamina of the endothelial cells
-contain contractile filaments and can differential to repair damaged endothelium
-generally are about 3-7 microns thick (about the size of an RBC)
Sinusoids
-have larger diameter (30-40 microns)
-provide sluggish movement for blood
-found in liver, anterior pituitary, spleen, bone marrow and lymph
-3 types of sinusoids
Continuous (least “leaky”) capillaries and continuous sinusoids
-endothelial cells joined by tight junctions and have continuous basal lamina
-lipid soluble stuff passes through cells while water soluble stuff passes through pores
-endothelial cells have pinocytotic vesicles used for transcytosis
Continuous capillaries
-found in blood brain barrier, muscle, nervous and CT
Continuous sinusoids
-found in bone marrow
Fenestrated (intermediate “leakiness”) capillaries and sinusoids
-endothelial cells have pores (Fenestrations) with diaphragms in pores
-continouous basal lamina
-found in anterior pituitary
-also found in glomerulus (no diaphragms though)
Discontinuous (most leaky) capillaries and discontinuous sinusoids