What are the 2 major components of blood? What is contained within each component?
– the fluid phase of blood 90% water, 9% proteins, and 1% inorganic salts, ions, nitrogenous compounds, nutrients and gases
– Erythrocytes, Leukocytes and Platelets
What are the 3 parts of sedimented blood? What is contained within each?
What is hematocrit?
When blood cells sediment in a centrifuge, three layers are formed with the densest cells at the bottom, the least dense at the top.
Middle: Buffy Coat
- White blood cells: 6-10,000/Pl in normal blood
- Platelets: 200-400,000/ Pl in normal blood
Bottom: Packed RBCs (erythrocytes)
HEMATOCRIT – volume of packed red blood cells:
- Male 40-50% (4.1-6 million/μl)
- Female 35-45 % (3.9-5.5 million/μl)
What are the 3 compartments where blood cells can be found? What type is found in each compartment?
• Compartment 1 – Bone Marrow (and Thymus for T-lymphocytes)
- All Blood Cells (where blood cells originate)
• Compartment 2 – Blood Circulation
- All Blood Cells
• Compartment 3 – Connective Tissue
- Leukocytes only
- RBCs should not (normally) leave circulation. leukocytes get into CT to perform their functions. could be an organ, CT space such as epidermis, etc
What are the 3 classifications of blood cell types? State what types of cells fall under each category.
• Erythrocytes (RBCs)
• Thrombocytes (Megakaryocytes)
State where the normal sites of hematopoiesis are during fetal life and in the adult. State when each of the sites begin and cease their functions in hematopoiesis.
Yolk Sac: Beginning 2 weeks post-conception in mesoderm which forms both vessels and erythrocytes
Liver: Begins 6th week of gestation and continues until birth. Can occur abnormally in postnatal life
Bone Marrow: Beginning fifth month of gestation and continues throughout life
Effective hematopoiesis requires what 2 components?
Effective hematopoiesis requires a functional hematopoietic microenvironment and hematopoietic cells.
What are the 2 types of bone marrow? In which marrow does hematopoiesis occur?
State what is contained in each type of marrow.
• Found in all bones
- Contains hematopoietic stroma (stromal cells and their connective tissue), adipocytes, mesenchymal stem cells (MSCs which are capable of differentiation into a wide variety of tissues), osteoblasts and hematopoietic cells
- Around 20 years of age most red marrow converts to to yellow marrow which contains hematopoietic stroma, MSCs, osteoblasts and adipocytes
In what part of the bone do nutrient arteries enter? What do the nutrient arteries supply?
What kind of capillaries are found in bone marrow?
How do newly formed blood cells enter the circulation?
• Nutrient arteries enter through diaphysis (long part of bone) which supply blood to both bone and to marrow
• In the marrow, arteries supply a sinusoid network (of capillaries)
• Sinusoids feed venous system which receives newly formed blood cells (new blood cells leave through venous system)
When in development do hematopoietic stem cells (HSCs) differentiate?
What are the 2 functions of HSCs?
Hematopoietic Stem Cells
Hematopoietic stem cells (HSCs) differentiate in the early embryo when hematopoiesis
begins. In postnatal life pluripotent hematopoietic stem cells fulfill two requirements.
In addition to providing the precursor cells for
hematopoiesis, these stem cells may be
multipotential cells with regenerative capacity
for other organ systems.
The multipotent cells differentiate to give rise to all the circulating blood and lymphoid
Stem cells (HSCs) and cells that give rise to the
blood cells require a unique environment for
hematopoiesis to proceed. Both cellular and acellular components form this environment.
State the roles of the following cellular components of the hematopoietic environment.
• A subset of these act as a binding site for HSCs.
• Secrete hematopoietic growth factors--Direct contact with stem cells is important for their growth and development.
• Bind HSCs through N-cadherin.
Osteoclasts - Protease secretion breaks down extracellular matrix components, which assists in mobilization (release to the circulation) of stem cells.
Stromal Cells – express growth factors which aid in homing and regulation of stem cells.
Endothelial Cells – possible growth factor contributor.
Adipocytes – possible growth factor contributor.
Sympathetic Neurons – signaling down regulates osteoblast chemokine expression.
How is calcium sensed by hemotopoietic cells?
What is the role of heparin sulfate proteoglycans in the hematopoietic environment? How are they linked to bone and cartilage matrix?
What is the role of annexin2 in the hematopoietic microenviroment? What cells secrete annexin2?
• Calcium – The rich reservoir of calcium may be
detected by Calcium Sensing Receptor on hematopoietic stem cells.
• Heparin Sulfate Proteoglycans - linked to bone and cartilage matrix via collagen X. Binds hematopoietic cell growth factors (to keep them in place).
• Annexin2 – secreted by osteoblasts, serves as a binding protein for stem cells.
Explain the mobilization of HSCs and the cells involved.
HSCs interact with osteoblastic cells at the bone
interface. These cells home to this site from the circulation during embryonic
development, and may also home to this site in the adult. HSC’s when stimulated by
appropriate growth factors, and with alteration of niche components by protease (MMP-
9) digestion can be mobilized. Mobilized HSCs may enter the circulation, or may go
through hematopoietic differentiation in the marrow prior to release into the circulation.
Stem cell mobilization has important clinical applications because it allows
collection of stem cells from peripheral bloods in treatment of malignancies.
Attached figure: This figure illustrates a mechanism for regulating movement of HSCs into and out of the
hematopoietic niche. Stimulation of osteoblasts by parathyroid hormone can increase
HSC adhesion and expansion. Activation of osteoclast protease secretion by other
factors, both hormonal such as RANKL, or due to stress to the individual, promotes
release of HSC from their niche.
What is a lineage?
What are the 2 hematopoietic lineages? Name all of the cell types that are derived from these lineages.
A lineage is a family of cells derived from an immature progenitor that lead to a mature end stage cell.
Hematopoietic lineages are:
Common Myeloid Progenitor – cells leading to all non lymphoid cells
- Erythroid – cells leading to erythrocytes
- Granulocytic – a subset of myeloid that leads to all three granulocyte
- Megakaryocyteic – cells leading to platelets
Common Lymphoid Progenitor – cells leading to lymphocytes (T, B and NK) and dendritic cells
see page 10 of course notes
What are colonies? What are colony forming units (CFUs)?
Colonies were discovered in irradiated mice that were injected with bone marrow hematopoietic cells after irradiation. After several days the spleen has nodules of cells. These are colonies of cells derived from stem cells that settled into the spleen (a friendly environment) and grew. Different colonies contain different combinations of cells, reflecting the different lineages of hematopoietic cells. By definition a single cell that has the capacity to divide and give rise to a colony of cells is called a Colony Forming Unit, of CFU. It has also been possible to grow these colonies in cell culture, which has helped to identify the hematopoietic growth factors that stimulate the growth of different hematopoietic lineages. There are many different CFU’s as seen in the lineage diagram (pg 10 of course notes). Note that there are different CFUs for each lineage (see attached pic)
What is the site of secretion for hematopoietic growth factors?
What is their specificity for stimulation of the various hematopoietic lineages?
Hematopoietic Growth Factors
• Proteins produced by several different organs.
• Varying degree of specificity for the stimulation of cells
of different hematopoietic lineages.
• May also alter normal mature blood cell and leukemic
• Characterized first in tissue culture experiments, some
are now multibillion dollar drugs.
What growth factors are implicated in the formation of RBCs, monocytes, granulocytes, and platelets? State where each growth factor acts.
- IL-3 plays an important role in earlier development of multiple lineages.
- Erythropoietin (made in the kidneys) is a major regulator of erythrocyte development.
- Thrombopoietin (made in the liver) is a major regulator of megakaryocyte development.
- G-CSFand GM-CSF are major regulators of granulocyte and monocytes development
What does erythropoietin stimulate? (formation of what cell types in erythrocyte development)
In the early embryo, what is the role of erythropoietin in erythrocyte formation?
What stimulates the formation of proerythroblasts and subsequent progeny?
During erythrocyte development, what changes occur to the cytoplasm, nucleus, and organelles? Why?
• Primary regulator of erythropoiesis is erythropoietin, which is made in the kidney. Production increases if RBC number decreases (Early Embryonic: Erythropoietin independent, formation of nucleated RBC’s)
• Erythropoietin, with other factors, stimulates formation of BFU-E and CFU-E and proerythroblasts
• Proerythroblast and subsequent progeny assisted in development by macrophages which produce growth factors and consume nuclei by phagocytosis
• The development of red blood cells centers on the elaboration of hemoglobin. In general, progressively the cytoplasmic affinity for stain changes, the chromatin becomes more heterochromatic, and the nuclear volume decreases until the nucleus is totally extruded.
• During maturation there is change in cytoplasmic staining. Affinity for basic dyes (blue) decreases as RNA is decreased whereas affinity for acidic dyes increases (as globin chain is synthesized).
List the stages of erythrocyte development beginning with proerythroblast and ending with an erythrocyte.
Describe the appearance of cells in each stage of development.
1) Proerythroblast- For now, don’t worry about what it looks like. It is a progenitor cell committed to further maturation in the erythroblast lineage through the following stages
2) Basophilic Erythroblast - small cell with circular nucleus and blue, basophilic cytoplasm. Similar in appearance to a lymphocyte but the cytoplasm is usually more intense blue.
3) Polychromatophilic Erythroblast (no further cell division after this stage) – More condensed nucleus than the basophilic erythroblast, sometimes with a checkerboard appearance, and a mottled, pink and blue cytoplasm.
4) Orthochromatophilic Erythroblast (extrusion of the nucleus from these cells results in the next stage). Very condensed, circular nucleus and a cytoplasm almost the same color as an erythrocyte.
5) Reticulocyte – Newly formed RBC with some vesitigial RNA that forms a “reticulum”. Appears the same as an RBC expect with special stains. (will not be expected to ID)
Too much erythropoietin leads to what condition? Not enough erythropoietin leads to what condition?
What disease processes may these occur in conjunction with?
The major regulator of erythropoiesis is erythropoietin which is produced in the kidney. Not enough leads to anemia (which occurs during renal failure). Too much can cause secondary polycythemia (too many RBCs) which can occurr from tumors of erythropoietin forming cells.
Explain the cell division process of megakaryocytes and the number of copies of DNA they contain.
What are demarcation membranes?
What is the purpose of the cytoplasmic granules in megakaryocytes?
What is the major regulator of megakaryopoiesis? Where is it synthesized? What does this regulator bind to?
Megakaryopoiesis refers to the development of megakaryocytes (huge polyploid cells containing large nuclei). Megakaryocytes are the precursor cells which give rise to platelets.
Cell division in megakaryopoiesis is incomplete. At each cycle the megakaryocyte chromosomes duplicate, cytoplasmic volume increases but the cell does not divide into two daughter cells (karyokinesis but no cytokinesis). The outcome is a gigantic polyploid [x (2N)] cell. 2N refers to 22 pairs of autosomes and a pair of sex chromosomes contained in all of the somatic cells of the body; these therefore are diploid. In the megakaryocyte, however, this number is multiplied by 2 at each cell cycle. Therefore a megakaryocyte can contain anywhere from a 4N to a 64 N nucleus; thus the megakaryocyte is polyploid.
Megakaryocyte maturation is characterized by:
Development of "Demarcation Membranes"- These are extensive membrane systems that are continuous with the plasma membrane and extend inward close to the nucleus. Thus they are extensions of the plasme membrane. In this manner the megakaryocyte provides abundant surface membrane required for perhaps thousands of platelets that arise from a single megakaryocyte.
Production of cytoplasmic granules- These granules will be packaged into future platelets and are essential to platelet functions.
The major regulator is thrombopoietin, which is made in the liver. Thrombopoietin is bound to platelets. If platelet count decreases, unbound thrombopoietin increases.
see slides 38-40
Name and describe the developmental stages in megakaryopoiesis.
Developmental stages in megakaryopoiesis:
Megakaryoblast- The least mature. The “blast” suffix indicates an immature stage
Basophilic megakaryocyte- basophilic refers to the staining properties of the cytoplasm (and not the nucleus). This, in general, is the convention in describing cells as the nucleus is always basophilic anyway. The basophilia of cytoplasm is due to abundance of RNA. We know that the cytoplasm of this cell is rich in RNA which is required for the synthesis of the protein content of the granules.
Granular megakaryocyte- Here granularity is due to abundance of granules; that is many granules have already been produced in this cell.
Platelet producing megakaryocyte- This is the latest stage of megakaryocyte development. The cell is now mature and ready to produce platelets. This occurs by the development of long cytoplasmic processes which protrude across the endothelium of bone marrow sinusoids, which break into small fragments, each becoming a platelet.
What cell type to monocytes share a common progenitor with? What stimulates this common progenitor to differentiate into monocytes?
What are the developmental stages of monocytes?
Monocytopoiesis- Monocytopoiesis refers to the development of monocytes. Share a common progenitor cell with granulocytes. This common progenitor is stimulated by Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). This takes place in the bone marrow from the pluripotent hemopoietic stem cell through the following stages:
3. Monocyte (mature form)
Describe the developmental stages of granulocytopoiesis. Explain the appearance of cells at each stage.
How many lobes of nuclei are present in mature neutrophils, eosinophils, and basophils?
• Important regulators are GM-CSF and G-CSF
• Each granulocyte type has a unique progenitor
• Maturation characterized by loss of primary granules, accumulation of specific granules, lobulation of nucleus
• Newly formed neutrophils marginate in blood circulation and enter connective tissue as needed.
1. Myeloblast (least mature)-lacks granules (do not have to ID)
2. Promyelocyte- This cell contains primary granules (aka azurophilic granules) ; these granules are larger than the neutrophilic granules in neutrophils but smaller than the specific granules in eosinophils or basophils and are irregular in size and deep blue in color.
3. Myelocyte- Primary granules are still present, but the specific granules (secondary granules) begin to develop at this stage. Thus with at this stage and thereafter the three major types of granulocytes can be distinguished. Nucleus is typically eccentric and oval to round.
4. Metamyelocyte - The nucleus is indented and only secondary granules are present.
5. Band cell - The nucleus is elongated and forms a ‘U’ or ‘S’ shape—called a band shape (neutrophilic).
6. Granulocytes: neutrophil, eosinophil, basophil - mature forms have a segmented nucleus; neutrophils have 2-5 lobes, eosinophils and basophils have 2 lobes.
see pg 15 of course notes