and Hematopoiesis Dr. Darren Hoffmann Recommended Reading: Junqueira Chapters 12 and 13, Ross and Pawlina Chapter 10

Lecture Objectives: After this lecture you should be able to: -Identify any formed element in a blood or smear based on its visible features. -Describe the function of any formed element in a blood or bone marrow smear, describing that cell’s life span (Where did that cell come from? Why is it here? What is it doing? How long will that cell live? What’s going to happen to it?) -Find blood cells in tissues and explain how they got there. -For an identified cell, explain the visible contents of that cell, and describe the components of that cell that are not visible with light microscopy, but are still important to understanding the cell’s function. -Use the as a “scale bar” for measuring tissues anywhere in the body. -Connect a series of blood cell progenitors to a specific mature blood cell/formed element. -Describe the sequence of cellular events that would need to take place in order to repopulate the entire blood system of a patient who received a bone marrow transplant.

Lecture Outline:

PART A: General Organization of Blood

A1. What is blood? 5L of fluid in your body It is a distribution system for: - Nutrients, Wastes, O2, CO2 - Hormones - Electrolytes - Immune Cells, Would healing machinery

Where does blood work? Sometimes blood works in the blood vessels (exchange of O2/CO2) Sometimes blood components leave blood vessels to do their jobs (Hormones, Immune/Wound healing)

What is blood made of? Blood is a Connective Tissue (Cells in a Matrix) Matrix is Plasma (no fibers!) Water (90%) Plasma Proteins (7%) - albumin (osmotic gradient) - globulins (antibodies) - clotting proteins Electrolytes (1%) Other (2%) Amino acids, vitamins, hormones, cholesterol

Cells are Erythrocytes (RBC), Leukocytes (WBC) and

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A2. Erythrocytes

- 4-6 million/µL of blood (how many in the entire circulatory system? ______) - Anucleated cells packed with hemoglobin (have a nucleus in the fetus) - 7.5 µm diameter (RBCs are a handy ruler for all cells in the body!) - Typically never leave the circulatory system

A3. Leukocytes - 6-10 thousand/µL of blood (how many in the entire circulatory system? ______) - Nucleated cells - 10-20 µm diameter - Most do their jobs outside of the circulatory system - Differentiated into two groups based on appearance (presence of protein granules in cytoplasm) Terminally differentiated cells with a short lifespan (a few days) Examples: , , (absence of protein granules in cytoplasm) Cells which may further differentiate/proliferate and with a longer lifespan Examples: ,

A4: Thrombocytes - 300 thousand/µL of blood (how many in the entire circulatory system? ______) - Anuclear cell fragments - 2-4 µm diameter - Function at sites of vascular damage to promote clotting

PART B: Where does Blood come from?

B1. Hematopoiesis: An Introduction

- 100% of hematopoiesis starts in the bone marrow (but it doesn’t all finish there)

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B2. Blood Stem Cells Two types of stem cells form from a pluripotent stem cell population early in in blood cell development:

i. Lymphoid progenitors - Form lymphocytes (B/T-cells) - Form in the bone marrow - Migrate to lymphoid organs (spleen/thymus) for further maturation

ii. Myeloid progenitors - Form multiple different specific progenitors and precursors known as “blast” cells - Give rise to ALL blood cells EXCEPT lymphocytes - Migrate into blood as mature cells - Form and mature in the bone marrow

Entire developmental process of blood cells is complex and dependent on growth factors and poietins Area of drug discovery May enable use of Bone Marrow Stem Cells to create other non-blood tissues

B3. Bone Marrow

i. Red Bone Marrow - active bone marrow (hematopoiesis IS occurring) - all fetal bone marrow is red

ii. Yellow Bone Marrow - inactive bone marrow (hematopoiesis ISNOT occurring) - can be converted to red marrow in cases of severe bleeding or oxygen deprivation

Superstructure: Stroma – Reticular fiber meshwork of bone marrow Hematopoietic Cords – Clumps of differentiating blood cells

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Sinusoidal capillaries – Leaky capillaries - feed bone marrow O2 and nutrients - allow blood cells to enter bloodstream to leave bone

B4. Process of erythrocyte development (about 7 days) - cell volume decreases - nucleus decreases in size - chromatin condenses - nucleus is extruded from cell - number of ribosomes decreases (basophilic purple stain decreases) - hemoglobin levels increase (acidophilic pink stain increase) - organelle number drops

Process occurs in “Erythroblastic Islands” in the Bone marrow stroma - islands can be identified by the clumps of heavily condensed nuclei with RBCs nearby - other blastic islands ( development e.g granulopoiesis) are less nuclear-dense

To leave the bone marrow, the mature erythrocyte enters the capillary through sinusoidal gaps Exits bone through blood vessel – nutrient foramen

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Stages of Erythrocyte Development

Entire process is 3-5 Cell Divisions Stages are identified based on staining characteristics and nuclear characteristics

1. Large cells with visible nucleolus, loose chromatin, basophilic cytoplasm (ribosomes)

2. Erythroblast (Basophilic Erythroblast) Chromatin condensing, basophilic cytoplasm (ribosomes)

3. Polychromatophilic Erythroblast Checkerboard nucleus, mixed color cytoplasm (some ribosomes [baso]/some hemoglobin [acido])

4. Orthochromatophilic Erythroblast Condensed nucleus, acidophilic cytoplasm (hemoglobin, few ribosomes)

5. Anuclear, acidophilic cytoplasm (very few ribosomes, only detectable through Cresyl blue stain)

6. Mature Erythrocyte Anuclear, acidophilic cytoplasm, trademark biconcave disk shape

FOR A MORE DETAILED LOOK AT HOW TO IDENTIFY THESE CELLS – SEE SWAILES’ GUIDE TO IDENTIFYING BONE MARROW CELLS

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B5. Thrombopoiesis Process of development (less than three days)

i. (15-50 µm) - polyploid nucleus (30x normal DNA) - multiple nucleoli - highly basophilic cytoplasm

ii. (35-150 µm) - irregular lobulation of nucleus - no visible nucleoli - plasma membrane invaginates into hundreds of ‘demarcation membranes’ - membrane extensions project into sinusoidal capillaries - “membrane shedding” - membrane pieces are shed (4-8000/megakaryocyte)

B6. Granulopoiesis Process of development (7 days) - cell must develop granules - first to develop is azurophilic granules (pale blue lysosomal proteins) - second to develop are specific granules (granules unique to each granulocyte type) - as specific granules develop, azurophilic granules are less prevalent - golgi apparatus wastes away ( production comes to an end) - nucleus becomes lobulated (cell is done dividing)

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Stages of Granulocyte Development

1. - no granules present, large nucleus with a visible nucleolus

2. - nucleolus - azurophilic granules (pale blue) - developed Golgi apparatus

3. - mixed azurophilic (blue) and specific (pink) granules - round/oval nucleus - developed Golgi

4. - specific granules only: pink for eosinophils, purple for basophils, neutral for neutrophils - bean-shaped nucleus - atrophic Golgi

5. Band/Stab Cell - nucleus looks like a horse-shoe

6. Mature Segmented Granulocyte (Seg) - Segmented nucleus (two segments for baso/; multiple for )

Note that we will look at how lymphocytes and monocytes develop when we discuss the immune system

FOR A MORE DETAILED LOOK AT HOW TO IDENTIFY THESE CELLS – SEE SWAILES’ GUIDE TO IDENTIFYING BONE MARROW CELLS

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PART C: Cells of Circulating Peripheral Blood

C1. Erythrocytes - well-characterized plasma membrane proteins that maintain the biconcave disk shape - highly flexible – important for capillary flow - bags of hemoglobin, with no organelles - because they have no organelles: - they depend on anaerobic metabolism of glucose - they cannot make more hemoglobin - they will eventually wear out (about 120 days)

Eryptosis: removal of dead/dying erythrocytes - occurs by in spleen, bone marrow and liver - signaled through defective oligosaccharides on the RBC surface - occurs at the same rate as erythropoiesis - erythropoiesis is stimulated by erythropoietin (EPO) from the kidney - diseases can effect the balance between eryptosis and erythopoietin - sepsis, haemolytic uremic syndrome, malaria, sickle cell anemia, beta-thalassemia

C2. Leukocytes: an overview

Classified visually: Leukocytes are divided into two groups: - Granulocytes and Agranulocytes Presence/color of granules or absence of granules - Granulocytes: neutrophils (PMNs), eosinophils, basophils - Agranulocytes: lymphocytes, monocytes

Classified developmentally: All Leukocytes except lymphocytes have a common progenitor - Lymphoid progenitor: Lymphocytes (T and B Cells) - Myeloid progenitor: all other Leukocytes (granulocytes and monocytes) and other blood cells

Important functional principles of granulocytes: They are terminally differentiated (the last cell in the lineage) - They have low protein synthesis rates and few mitochondria (so they use glycolysis for energy) - High glycolysis means they can function in inflamed areas with low oxygen (anaerobic)

They often have to migrate out of the blood vessel to do their work Chemotaxis: the movement of a cell due to a chemical signal - Directs leukocytes to sites of infection/ Diapedesis: The process of squeezing out of an intact capillary to enter the tissues - Provides a way for leukocytes to access inflamed tissues - Typically occurs in post-capillary venules (lower pressure and lower shear forces) - Mediated by inflammatory signals and receptors on and leukocytes - Most leukocytes never return to the blood once they’ve entered an inflamed area

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C3. Neutrophils - aka Polymorphonucleocytes – PMNs) - 60-70% of leukocytes

ID characteristics: - multi-lobed nucleus - presence of both azurophilic and specific granules, but specific - granules are more prevalent but since both are present, stain appears Neutral = Neutrophil - presence of ‘chicken drumstick’ indicates cell is from a female (genetics: female = XX therefore one X chromosome is inactivated, the inactivated X chromosome appears as a chicken drumstick appendage)

Lifespan: - 6-7 hour half-life in the blood - 1-4 days in connective tissues - Death occurs by apoptosis

Functions: - and killing of bacteria - Granules are used to kill phagocytosed bacteria

C4. Eosinophils - 2-4% of leukocytes

ID characteristics: - Bi-lobed nucleus - Large eosinophilic specific granules

Lifespan: - 8-12 hours in blood or 8-12 days in connective tissues - Longest lasting of the granulocytes

Functions: - Killing of parasitic worms – large specific granules - Many other less characterized functions in inflammation

C5. Basophils <1% of leukocytes

ID characteristics: - Irregular nucleus that is hard to see because of- - Large, abundant basophilic granules (contain heparin and histamine)

Lifespan: Unclear due to scarcity in blood

Functions: Histamine and heparin release at inflammation sites

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C6. Monocytes 5% of leukocytes

ID characteristics: - Kidney/Horseshoe nucleus

Lifespan: - Months to years

Functions: - When they leave the capillary and enter tissues, they become phagocytic macrophages

C7. Lymphocytes 25% of leukocytes

ID characteristics - Round nucleus that occupies much of the cell - Condensed chromatin - Very little cytoplasm - Not much bigger than an RBC

Functions: - Immune cells with many functions - Life span is variable (days to years) - This is the ONLY leukocyte that can re-enter the circulation

C8. Thrombocytes (platelets) Features: - Core is dense (granulomere) and contains granules - Periphery is less dense (hyalomere) - Open canalicular system inside platelets connect to the outside which facilitates release of factors stored in the platelet - Marginal bundle of microtubules maintains shape of platelet - Dense tubules (actin/myosin) allows for movement of platelet during clotting

Functions: Facilitates all steps in clotting event 1. Forms a plug at site of injury 2. Releases adhesive glycoprotein and ADP 3. Releases clotting factors 4. Contraction of dense tubules – retracts clot from the vessel lumen 5. Contributes to degradation of the clot during healing

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