Exam 1 lec 3- Flashcards
Hematology Instruments and Blood Cell Measurements
What parameters are included in a CBC for Erythrocytes, Leukocytes, and Platelets?
- Erythrocytes:
- Hematocrit, hemoglobin, RBC count
*Red blood cell number: Hematocrit (HCT), *Packed cell volume (PCV),
*Red blood cell count, hemoglobin (Hgb) concentration
*Red blood cell indices: Mean cell volume (MCV), mean cell hemoglobin (MCH) or optical equivalent (CH), mean cell hemoglobin concentration (MCHC) or optically measured equivalent CHCM), red blood cell distribution width (RDW).
*Nucleated RBC: How nucleated RBC are identified and how they affect the WBC count.
*Reticulocyte counts: Reticulocyte percentage, absolute reticulocyte count that are used to assess regeneration in dogs and cats.
Reticulocyte indices: Results obtained off optical- and flow-based analyzers, which can yield RBC indices for reticulocytes.
- Leukocytes
* Total leukocyte count
* Differential leukocyte count - Platelets count
* Platelet count
* some times Mean Platelet Volume (MPV).
What are some of the benefits of In-house analyzers?
- Rapid information
- Emergencies
- Client services - Analyses of fresh samples
- Rebleed if needed
- Avoids sample deterioration - Information available any time
- Cost effective
The Coulter Impedance Method
- Its inventor Wallace Coulter, is the passage of cells suspended in a known dilution through a small orifice. The electrolyte-containing diluent serves as a conductor of a constant electrical current between two electrodes.
- Change in resistance is proportional to cell volume
- Electrically conductive diluent
Coulter Impedance Instruments
- Count size and number of cells: providing cell volume distributions
- Measure Hemoglobin (Hb) spectrophotometrically
- HCT, RBC, Hb, MCV, MCH, MCHC, RDW are reported
- Can not determine intracellular characteristics of cells
- Total WBC counts (all species) and generally platelet counts (NOT CATS) are generally reported
- Only a 3-part differential based on cell size alone (limited accuracy)
Coulter Impedance Instruments
- Count size and number of cells: providing cell volume distributions
- Measure Hemoglobin (Hb) spectrophotometrically
- HCT, RBC, Hb, MCV, MCH, MCHC, RDW are reported
- Can not determine intracellular characteristics of cells
- Total WBC counts (all species) and generally platelet counts (NOT CATS) are generally reported
- Only a 3-part differential based on cell size alone (limited accuracy)
- Generally less expensive than laser flow instruments
- Can not perform reticulocyte counts
Laser Flow Cytometry
- Forward Scatter: cell size
- Side Scatter: Complexity/Ganularity
Light Scatter
- Cells passing through the laser beam will scatter
- The shadow cast by the cells is referred to as “forward angle light scatter” -forward scatter
- Cells will also scatter light in different directions
- Detectors are placed in front of the laser beam to measure forward scatter, and at angles (typically 90 degrees) to the laser beam to detect light scattered to the side
Forward Scatter, what does the detector measure?
- It measures cell’s “shadow” which is a relative indicator of the cell’s size.
- Large cell (e.g., neutrophil) casts a large shadow.
- Lymphocytes = small shadow
Laser Flow Cytometry
- What does it determine?
- Counts and classifies cells based on what?
- How many part differential counts does it provide?
Dot Plot Displays: IDEXX Procyte Dx
- Forward Scatter: cell size
- Side Scatter: Complexity/Ganularity
1. Size and complexity
2. Based on extinction of light and scattered light
3. Total WBC and 5-part differential counts (basophils not accurate in dogs). - Platelet counts, MPV, PDW (Platelet cell distribution width), PCT.
- HCT, RBC, Hb, MCV, MCH, MCHC, RDW.
- Reticulocyte counts
- Large instruments such as Advia 2120 offer many more measurements
Light Scatter
- Cells passing through the laser beam will scatter
- The shadow cast by the cells is referred to as “forward angle light scatter” -forward scatter
- Cells will also scatter light in different directions
- Detectors are placed in front of the laser beam to measure forward scatter, and at angles (typically 90 degrees) to the laser beam to detect light scattered to the side
Side (90 degrees = “Right angle”) Scatter
What does the detector measure?
What are fluorescent labels used for?
- It is a measure of the cell’s internal complexity or granularity.
- Neutrophils, with multilobed nucleus and granules have high amounts of side scatter.
- Lymphocytes with their high nucleus: cytoplasm ratio and scant cytoplasm have lower levels of side scatter
Fluorescent labels
-may be used to ID cell types
Side (90 degrees = “Right angle”) Scatter
What does the detector measure?
What are fluorescent labels used for?
- It is a measure of the cell’s internal complexity or granularity.
- Neutrophils, with multilobed nucleus and granules have high amounts of side scatter.
- Lymphocytes with their high nucleus: cytoplasm ratio and scant cytoplasm have lower levels of side scatter
Fluorescent labels
-may be used to ID cell types
What is RBC count used for?
- Little value by itself
- To calculate Hct electronically
- Used to calculate MCV and MCH manually
PVC and Hct Determinations
- What is one of the requirements?
- What is the formula for Hct?
- Centrifugation for PCV
- Hct (%) = MCVRBC/10
- Example: 686.6/10 = 45%
Hemoglobin Determinations
What reagent is used and what does it do?
What can cause falsely increased values (and in non-mammals)?
- Spectrophotometric assay: blood is diluted and RBC lysed to measure Protein concentration and Absorbance.
- Sodium lauryl sulfate (SLS) reagent
- Lyses erythrocytes
- Oxidizes Hb to MetHb and forms a stable SLS-MetHb product.
- Lipemia
- Heinz bodies
- Nuclei in non-mammals
Erythrocytes indices Erythrocytes Histograms (Relative number vs. Cell Volume fl).
- Mean cell Volume (femtoliters). Average volume of a single erythrocyte (fL = 10^-15 liter). Determined directly in hematology analyzers.
- Mean cell Hemoglobin (not very useful): Average amount of Hb in a single erythrocyte (picogram = 10^-12 gram). Generally correlates directly with changes in erythrocyte size (MCV). Depends to a lesser extent on internal Hb concentration. Lowest values are reported in severe iron deficiency anemia (low MCV and low internal Hb concentration).
-Mean cell Hemoglobin Concentration: determined by calculation g/dL of packed erythrocytes (NOT g/dL whole blood).
HCT = 45%
Hb = 15 g/dL
Formula: (Hb/HCT) * 100.
Example: (15/45)*100 = 33 g/dL (packed RBC).
-Red Cell Distribution Width
What are the MCV (fL) in Domestic mammals?
- Horses: 38-51
- Cattle: 38-50
- Dogs: 64-74
- Cats: 42-52
- Goats: 16-25
Red Cell Distribution Width
What is the formula?
Spuriously Increased RDW
- SD of Erythrocyte Volumes/ MCV *100
- Coefficient of variation of erythrocyte volumes and an electronic measure of anisocytosis (Anisocytosis is the medical term for having red blood cells (RBCs) that are unequal in size).
Spuriously Increased RDW
- Erythrocyte agglutination
- Platelets counted in erythrocyte histogram of severely anemic patients
What are Reticulocytes?
How is absolute Reticulocyte Counts determined?
How are they verified?
Reticulocytes are immature, anucleate erythrocytes which are released from bone marrow into the blood in increased numbers as a response to anemia caused by hemolysis (destruction) or loss (hemorrhage) of erythrocytes in most species (horses are a notable exception)
-Absolute reticulocytes counts are determined by flow cytometry and validated using manual counts
- Verify which reticulocytes are counted in cats. Only aggregate reticulocytes are counted in most instruments.
- *Aggregate Reticulocytes
- *Punctuate Reticulocytes
Spurious Reticulocytosis
- Erythrocyte parasites containing RNA and DNA, e.g., hemotropic mycoplasmas, Babesia species.
- Howell-Jolly bodies (micronuclei)
- Nucleated erythrocytes
- Large immature platelets or platelet clumps
- Heinz bodies with nonspecific fluorescence
- Autofluorescence (drugs, porphyrin)
- Leukocytosis- uncommon
False Platelet Counts
What is Mean Platelet Volume the average of?
- Platelet aggregates can result in false thrombocytopenia. Platelet activation during blood collection and handling (especially in cats)
- Verify PLC by stained blood film examination
- Automated PLC are often unreliable in cats
MPV: is the volume average of a single Platelet
MPV in Domestic Mammals
- Horse: 5.9-9.9
- Cattle: 4.5-7.5
- Dogs: 6.7-11.1
- Cats: 11.0-18.1
Spuriously Increased MVP
- Platelet aggregates
- Storage of blood at 5 C
What are some errors in Blood Cell Measurement?
What is the formula for Corrected White Blood Cell Count?
- Nucleated erythrocytes (Nucleated Red Blood Cells)counted as leukocytes by manual and most automated cell counters
- *Calculate corrected total leukocyte count before calculating absolute differential leukocyte counts**
Machine WBC *(100/100+NRBC) = Corrected WBC
Example: 15x10^3 per uL *(100/100+50) = 10x10^3 per uL
Sample errors: Platelet, leukocyte, erythrocyte aggregates
Clot Formation and Cryoglobulins have what effect on cells?
- Number of cell per uL blood decreases
- Erythrocyte aggregates increase MCV, decrease electronic HCT, increase MCHC.
- Platelet aggregates may be counted as leukocytes
- MPV is increased with platelet aggregates
- Collection in citrate may reduce platelets and leukocytes aggregates
Clot formations: decrease all cell types
Cryoglobulins (precipitate below body temperature): may be counted as leukocytes or platelets
What does hemolysis, lipemia, heinz bodies do to test results?
Hemolysis: MCHC (mean cell hemoglobin concentration) increased
- In vivo = intravascular hemolysis
- In vitro = sample handling, Lipemia
- Cross-linked hemoglobin (Oxyglobin) treatment
Lipemia:
- Hb and MCHC increased
- May increase leukocyte or platelet counts
Heinz bodies
- Hb and MCHC increased
- Sometimes total leukocyte counts are increased
- Reticulocyte counts may be increased
Lecture 4
Plasma Proteins and Dysproteinemia
What is plasma comprised of?
Where are plasma proteins synthesized? Plasma proteins vs. Immunoglobulins
What is the major components of Blood?
Compare Plasma’s composition vs. Serum’s composetion
- Comprised of hundreds of proteins, many protein structures and functions
- Liver: synthesizes most plasma proteins
- Lymphoid organs: immunoglobulins
Blood
- Blood cells
- Plasma
a. Water
b. Proteins
Proteins
a. Albumin
b. Globulin
c. Fibrinogen
Blood
- Platelets VII, V, II, I = coagulation factors.
- XIII, XI, IX, VII, VIII, V, X, II, XII, I.
Serum
-XIII, XI, IX, VII, VIII, V, X, II, XII, I. I = Fibrinogen.
What are some of the functions of Plasma Proteins?
How does neonate PP compare to adults?
- Immune defense
- Hemostatic
- Acid-base
- Transport of nutrients, hormones, waste, drugs.
- Inflammation
-Neonate lacks immunoglobulin until colostrum intake and absorption. They continue to increase with age.
What are some methods of protein measurement in plasma?
What conditions lead to erroneous readings in each method?
Which protein migrates the farthest in Serum Protein Electrophoresis?
When is SPE performed?
- Physical
- Refractometry: measures total solids in plasma or urine. Routinely performed in vetmed. Quick screed of total plasma proteins. Based on the fluid’s refractive index.
* *Hemolysis = Increased PPC due to Hb (plasma protein concentration)
* *Lipemia: interference with light transmission
* *Increased nonprotein solids: glucose, urea nitrogen in kidney - Biochemical: adding a reagent
- Spectrophotometric: Detector measures light absorbance of protein concentration
- Assays: a. Total globulin = subtracting albumin concentration from total protein concentration. b. Total protein concentration minus albumin concentration = total globulins concentration.
- Fraction of Serum/Plasma Proteins: various alpha, Beta, Gamma, globulin bands. Stain proteins for protein, carbohydrate, and lipids. - Fractionation
- Electrophoresis: negative charged proteins travel/migrate to positive charge (Anode +), (Cathode -).
* *Albumin migrates the farthest.
SEP (serum protein electrophoresis) is performed when
- Unexplained hyperglobulinemia is present
- Immunoglobulin deficiency is expected.
- SPE abnormalities are seldom specific for a given disease.
Albumin
How much does it weigh? What does it contain? What is the osmotic pressure relation? What functions does it have? How is low Ca++ in blood related to Albumin?
- Single homogenous protein
- 66kDa
- Contains minimal carbohydrate
- *Osmotic pressure: Hypoalbuminemia can result in edema**
- Transport functions: organic and inorganic substances. Cations (Ca++), metabolites, hormones, poorly soluble drugs, toxic substances.
- *Hypoalbuminemia = low Ca++ in blood.**
What is Acute Phase Proteins?
What inflammatory cytokines are involved?
How are they categorized?
What does increased production of positive APP indicate?
APP especially helpful in what species?
- Proteins with >25% change in serum concentrations in response to inflammatory cytokines (IL-1, TNF-alpha, IL-6).
- Categorized as positive (increase in serum) and vice-versa.
- Increased APP = indicator of inflammation, which can develop prior to leukogram.
- Species where it is helpful: cattle and manatees bc often do not exhibit prominent leukogram changes in response to inflammation.
Which plasma protein is a major acute-phase protein in all common domestic animals?
Which protein is an acute-phase protein in dogs and humans?
Which protein is a major acute-phase protein in ruminants?
Which protein is optimal in platelet aggregation and coagulation factor I?
What causes Fibrinogen to increase or decrease?
Which protein is a Plasma Copper transport and antioxidant?
Serum Amyloid A
- Family of apolipoproteins associated with high-density lipoproteins HDLs.
- Present in very low concentrations in normal animals
- Functions (supplemental information only). Transport of cholesterol to the liver for secretion into the bile. Recruitment of immune cells to inflammatory sites. Induction of enzymes that degrades extracellular matrix.
C-reactive Protein
- normally present in very low concentrations
- Functions: activates the complement system
Haptoglobin
- Binds free plasma Hb irreversibly
- Glycoprotein (20% CHO) that migrates in alpha-2 region.
- Prevents initial loss of free Hb in urine
- Protects against bacterial infections
- Antioxidant activity
Fibrinogen
- Coagulation factor I
- Precursor to fibrin in coagulation
- Scaffolding for inflammatory cells, fibroblast, and endothelial cells when deposited in tissues.
- Moderate APP that increases inflammation. Most prominent in horses, cattle, goats.
- Increase: from dehydration, active inflammation
- Decrease: liver failure, snake venoms, Disseminated Intravascular Coagulation (DIC) sometimes.
Ceruloplasmin
- Copper transport
- Ferroxidase activity
- Plasma antioxidant
- alpha-2 protein
What plasma protein is a major APP for cats?
alpha-1 Acid glycoprotein
-Drug binding, anti-inflammatory and immunomodulatory activity
What plasma protein is Iron-binding metalloprotein and NEGATIVE APP?
Iron-containing protein primarily found inside cells APP high = inflammation?
Transferrin
-correlates with total iron binding capacity (TIBC).
Ferritin
- Low concentration in plasma
- Correlates with total body iron stores
Which Proteins are Hormone Binding Proteins that prevents them from being rapidly filtered by the kidney?
- Corticosteroid-binding globulin
- Thyroxine-binding globulin
- Sex hormone-binding globulin
What protease inhibitor inhibit thrombin and other coagulation factors?
Antithrombin: requires GAGs (Heparin) for optimal activity
alpha1-antitrypsin: inhibits protease release during inflammation, and certain coagulation factors.
alpha-2-Macroglobulin: inhibits protease release during inflammation, pancreatic proteolytic enzymes, and certain coagulation factors.
Negative Acute-Phase Proteins noticeable during chronic inflammation
Albumin
- Concentration falls gradually
- Reduction of Albumin allows increase in AAs available for positive APP production.
Transferrin
- Decrease is not marked.
- May help limit iron availability for microbes
What effect does dehydration and hyperglobulinemia have on changes in plasma protein?
Hyperproteinemia
What effect does dehydration, dilution, decreased production, increased loss, and sequestration in body cavities have on changes in plasma protein?
Hypoproteinemia
What effect does inflammation on plasma protein increase, decrease, and synthesis?
- Increased loss of Albumin
- Increased vascular permeability
- Increased synthesis of some proteins due to cytokines: +APP- Positive acute-phase proteins, Immunoglobulins
- Decreased synthesis of other proteins due to altered cytokines: -APP- Negative acute-phase proteins (albumin, transferrin).
What effect does inflammation on plasma protein increase, decrease, and synthesis?
- Increased loss of Albumin
- Increased vascular permeability
- Increased synthesis of some proteins due to cytokines: +APP- Positive acute-phase proteins, Immunoglobulins
- Decreased synthesis of other proteins due to altered cytokines: -APP- Negative acute-phase proteins (albumin, transferrin).
Hypoproteinemia
What is it called when both hypoalbuminemia and hypoglobulinemia are decreased present?
Panhypoproteinemia
- Albumin to globulin (AG) ratios are often calculated and provided in clinical chemistry panels.
- The AG ratio is expected to be normal in panhypoproteinemia
Fluid balance
What is lost during dehydration and what is not lost?
What does external hemorrhage result in?
- Only fluid component of blood is lost
- Proteins and erythrocytes are NOT lost
- Causes relative hyperproteinemia and erythrocytosis
External hemorrhage
- All components of blood lost equally
- Fluid is replaced more rapidly than proteins
- Results in Hypoproteinemia and anemia
What factors/conditions can cause Hypoalbuminemia?
- Excessive fluid therapy
- globulins also decrease - Decreased synthesis
- Chronic liver failure (synthesis albumin). Example: Liver failure - Pyruvate Kinase Deficiency, Ascites- Hypoalbuminemia
- Inflammation (negative APP)
- Marked hyperblogulinemia (osmotic balanced?) - Loss from the body
- Glomerulopathy protein loss (globulins may be normal bc albumin is much smaller protein and more easily lost).
- Hemorrhage
- Protein loss enteropathy
- Severe exudative dermatopathies - Sequestration
- Peritonitis
Hyperproteinemia
- Dehydration: AG ratio increased
- Artifact- problem with the assay
- Dogs with hepatocellular carcinoma that produces albumin (very rare).
Hypoglobulinemia
- Overdehydration (albumin also low)
- Hemorrhage
- Protein-losing enteropathy
- Failure of passive transfer
- Humoral immunodeficiency (rare)
Hyperglobulinemia polyclonal and monoclonal
- Hemoconcentration: dehydration
- Increased immune-mediated response
- Neoplastic lymphoid cells (usually plasma cells)
- Increased APP concentrations when inflammation is present: Haptoglobin, Fibrinogen
Polyclonal
- Blunt broad peak
- IgG from many lymphoid clones
- Antigenic stimulation: infections, immune-mediated, immune response to neoplasia.
Monoclonal
- Narrow sharp peak
- Ig (Bense Jones protein) from a single lymphoid clone.
- Neoplasia: multiple Myeloma, Lymphoma/leukemia.
- Rarely biclonal, non-neoplastic.
Lipoproteins
- Synthesis in GI and Liver
- Transport water insoluble lipids in blood
- Water-miscible (triglycerides, cholesterol, phospholipids).
- Pseudomicellar particules
- Apolipoproteins: High MW
Chylomicrons
What is Postprandial lipemia?
- Low density lipoproteins
- Formed in the mucosal cells of the duodenum and jejunum following digestion of fat in the diet.
- Their core triglycerides are rapidly hydrolyzed by lipoprotein lipase in capillary beds.
Postprandial lipemia: white cloudy plasma after eating meal in carnivores and omnivores.
- Density less than water, Chylomicros form a cream layer in refrigerated blood.
- Chylomicrons: very short half-life
Very Low Density Lipoproteins
- Synthesized by liver and some in the intestine
- Transport endogenous triglycerides
- Plasma remains cloudy/milky, but no creamy layer after overnight refrigeration.
Low Density Lipoproteins
- LDLs arise as metabolic products from VLDLs
- Major mechanism cholesterol transport to peripheral tissues
- Cholesterol component needed for steroid hormones formation
Lecture 5 Bone Marrow Development
When does it develop?
What is cartilage replaced by?
What happens in the liver and bone marrow to hematopoietic cells?
What are the types of hematopoietic cells?
What does the Extravascular space create?
- Second trimester
- By bone
- Vascular sinuses and extravascular mesenchymal stromal cells from connective tissue meshwork
- Hematopietic cells from liver seed marrow once hematopoietic microenvironment forms.
Extravascular Space creates hematopoietic microenvironment
Cells
- Stem cells
- Progenitor cells
- Recognizable blood cell precursors
- Mature leukocytes
Stromal elements
- Endothelial cells: regulate transendothelial movement of cells between blood and the marrow extravascular space. Synthesis of growth factors and extracellular matrix.
- Reticular fibroblast-like cells: provide structural support for the bone marrow. Synthesis of growth factors and extracellular matrix.
- Adipocytes: some inhibitory effects on hematopoiesis. Accumulate increased lipid when the numbers of hematopoietic cells in the marrow decrease (reversible).
- Extracellular matrix: Collagen fibers. Scaffolding for other components of the microenvironment.
- What supports the vasculature? the Basal laminae of the sinuses in the matrix
- VCAM-1: adhesion molecule glycoproteins that binds the hematopoietic cells to appropriate environment
- Proteoglycans (proteins with repeating GAGs attached such as Heparan Sulfate) bind growth factors and hematopoietic cells
Accessory cells
- Macrophages: *most important for erythropoiesis
- T and B cells lymphocytes help regulate hematopoiesis. Subsets produce positive growth factors. Can inhibit proliferation of hematopoietic cells
- Osteoblasts: Stem cell maintenance
Erythrocytes and thrombocytes produced in vascular spaces in birds.
Hematopoietic Niches
- Quiescent HSCs: near endosteal and trabecular bone (osteoblasts)
- More active HSCs and HPCs: near vascular sinuses.
- Erythroid cells: around central macrophages, often near sinuses
- Megakaryocytic cells: near sinuses
- Granulocytic cells: around arterioles, immature near endosteum, mature near vascular sinuses.
What do Hematopoietic Stem cells and Hematopoietic Progenitor cells look like?
How often do they replicate?
Do they have the capacity to differentiate into all blood cell types and some tissue cells?
What do HSCs produce?
- Lymphocyte-like appearance
- HSCs: generally positive for CD34 surface antigen
- Early HPCs are also positive for CD34 (Cluster of Differentiation - CD)
- Every 8-10 weeks
- Yes, e.g., macrophages, dendritic cells, osteoclasts, mast cells.
- < 0.0001% of nucleated cells in cat marrow.
- Produce CLP (common lymphoid progenitor), CMP (common myeloid progenitor: non lymphoid blood cells.
Hematopoietic Progenitor Cells
Do they have unlimited replication?
Do they replicate faster or slower than HSCs?
- No, it is limited
- Slower
- They have differentiation but more restricted lineage than potential HSCs
- Colony forming units (GFUs) and burst-forming units (BFUs) in culture.
- About 1% of bone marrow cells in adults, but higher in neonates.
Mesenchymal Stem Cells
-Frequency in marrow = 0.001% to 0.0002% of all marrow cells Types: -Reticular cells -Adipocytes -Endothelial cells -Osteoblasts and chondrocytes
What cells are glycoproteins that promote the proliferation, maturation, and survival of hematopoietic cells?
**Must have an appropriate surface receptor to respond to a specific HGF.
Hematopoietic Growth Factors
- They are produced locally in the bone marrow (paracrine and autocrine) and/or by cells in peripheral tissues (endocrine) Autocrine means “relating to a cell-produced substance that has an effect on the cell by which it is secreted” while paracrine means “relating to a hormone which has effect only in the vicinity of the gland secreting it”.
- Nomenclature: Cytokines including colony stimulating factors (CSFs), interleukins, and poietins.
Which are the EARLY-acting Hematopoietic Growth Factors?
-Stem cell factor and flt3 ligand. (SCF, FL). They require the presence of other cytokines
Which are the Intermeidate-acting Hematopoietic Growth Factors?
-IL3, GM-CSF
Which are the Late-acting Hematopoietic Growth Factors?
-G-CSF, M-CSF, EPO, TPO, and IL5.
Which are the Overlap in action, and Indirect stimulation Hematopoietic Growth Factors?
- There is variable overlap with cell types stimulated by growth factors.
- Indirect stimulation during INFLAMMATION, TNF-alpha, IL1 stimulate stromal and accessory cells to release varios HGFs.
Erythropoiesis, Erythroid Islands functions
Where do erythroid cells develop?
What is the early, intermediate, and late acting growth factors?
- They develop around macrophages, which help create the environment.
- Phagocytize expelled nuclei and damaged or aged erythrocytes
- Early/intermediate growth factors created by macrophages
- Erythropoietin: late acting factors, and Inflammatory cytokines (IL1, TNF) inhibit erythropoiesis.
- Islands store iron released from phagocytized erythrocytes
What are the stages or cell names during erythrocyte maturational division?
- Rubriblast
- Prorubricytes
- Rubricytes
- Metarubricytes
- Reticulocytes (In dogs: bone marrow release). Formed when metarubricytes expel their nuclei, still bound to macrophages. Produced by Rubriblast from 1=16 reticulocytes in 4 days. Maturation occurs in marrow, spleen, blood, depending on species. Synthesize 20% of Hb. If RNA is degraded; therefore, loss of basophilia. Mitochondria removed by autophagy. Membrane remodeling is extensive, with loss of some proteins.
- Erythrocytes (In horses: bone marrow release).
Erythropoieting (EPO). What type of protein is it and what is its function? What is its main mechanism of action? Where is it synthesized? How does hypoxia affect Renal EPO? What does lack of EPO can result in?
- Glycoprotein
- Growth factor: promotes proliferation, differentiation, and survival erythroid progenitor cells and early erythroid precursors (Rubriblasts).
- Inhibition of apoptosis is the main mechanism of action
- Synthesized in Renal interstitial cells and liver, bone marrow.
- Hypoxia increases number of cells producing EPO, Proximal tubules, Epithelial cells.
- Lack of EPO = apoptosis. Inhibitors (inflammatory cytokines) may also induce apoptosis, component of pathogenesis of the anemia of inflammatory disease
Granulopoiesis
Neutrophil Production
- Myeloblast
- Promyelocytes
- Myelocytes
- Metamyelocytes
- Bands
- Neutrophils
Neutrophilic Growth Factors
How is it inhibited and by what cells?
-Early and intermediate acting factors
-G-CSF: granulocyte progenitors to myeloblasts. Increased cell division, decrease marrow transit time.
-Cell production of growth factors: Fibroblast and endothelial cells
T-lymphocytes and mononuclear phagocytes.
Inhibition
- Mature neutrophils in marrow can release substances that inhibit granulopoiesis. They can also remove bacterial stimulus in peripheral tissue for granulopoiesis.
- Neutrophil clearance of circulating G-CSF by binding to receptors on neutrophils
Eosinophil, Basophil, Mast Cell Production
Eosinophil Production
- 1 week marrow transit time
- Storage pool like neutrophils
- Early and intermediate acting factors
- IL-5 from Th2 lymphocytes for terminal maturation
Basophil Production
- GMP produces bipotential basophil/mast cell progenitor
- Basophil progenitor for basophils
- Granules at myelocyte stage
- Specific factor not recognized- growth factors similar to those for eosinophils
Mast Cells Production
- Mast Cells develop in tissues
- Mast cell progenitor (MaP) released from marrow to blood
- SCF is especially important for mast cell formation, but multiple other cytokines involved.
Monocytopoiesis
What is the activating factor?
- M-CSF is a late activating factor stimulating monocyte production
- Monocytes may become macrophages or dendritic cells (inflammatory) in tissues or Osteoclasts in bone marrow depending on the amounts of various cytokines present.
Osteoclasts
Where do they develop from?
- Develop from monocyte fusion
- Osteoclasts are multinucleated cells which degrade bone
Lymphopoiesis
Which are the early lymphoid progenitor cells in marrow?
Where are B and T cells produce in most mammals?
- Sten cell factor (SCF) and FL (flt3 ligand).
- B cell production: marrow in most mammals. Peyer’s patches in dogs, pigs, ruminants. Bursa of Fabricius in birds.
- T cell production in Thymus: progenitors in marrow. Final development in Thymus
What growth factor is especially important for B-lymphocyte Production?
- IL-7 needed
- Aprox 2-3 days for pre-B lymphocytes to become naive in marrow.
- Inhibited by certain cytokines including interferons.
- They migrate co cortex of lymph nodes and follicles in Peyer’s patches and spleen
- May develop into mature plasma cells
What growth factor is especially important for T-lymphocyte Production?
- Flt3L and IL-7 (also B-lymph) promote development
- Homing of progenitor cells to thymus
- Accumulate in paracortical areas of lymph nodes, periarteriolar lymphoid sheaths of spleen, and interfollicular areas of Preyer’s patches.
NK cell production
- Primarily produced and developed in bone marrow for a week or more
- Various growth factors are required
- subsets develop in thymus, and possibly lymph nodes, liver, spleen.
Thrombopoiesis
Where are Megakaryocytes located (Niche)?
What is the major site of production?
- Generally nuclear reduplication without division
- Mitosis - Endomitosis
- Megakaryocytes are located just outside the vascular sinuses.
- Proplatelet processes extend into the vascular sinuses and platelets form from the proplatelets processes within the vasculature.
Stimulation of Thrombopoiesis
- TPO: Thrombopoietin: promotes differentiation of progenitor cells into megakaryocytes. Stimulates increased endomitosis, resulting in increased ploidy and size
- Proliferation and expansion of megakaryocyte progenitor cells: early and intermediate-acting growth factors.
Major site of production is endothelial cells of liver.
- Relatively constant production of TPO. TPO receptors on platelets, providing negative feedback.
- IL-6 stimulates TPO synthesis by liver during inflammation
What have Recombinant Growth Factors have been used for?
- Human EPO and G-CSF have been used to treat animals.
- They work for a short time, but antibodies made against these glycoproteins limits their effectiveness. The antibodies can cross-react and make things worse.
- Dog and cat recombinant growth factors have been made, but not commercially available.
