28 Hematopoiesis – Formation and Roles of RBCs, WBCs and platelets

Blood Development, Blood Cells, and Blood Components

Embryonic Blood

The development of blood during embryogenesis goes through many changes.  The first blood cells are produced at day 7 of embryonic life.  The blood cell lineage expands its development in the yolk sac within the first two weeks of gestation.  As the embryo grows, blood stem cells migrate to the liver and spleen beginning at week 7.  As a fetus, blood cell formation begins to occur in the newly formed bone marrow of the developing skeleton (~week 20).  Blood stem cells remain in the bone marrow through deveopment and adulthood and continue their production from this location.  The formation of blood cells is termed hematopoiesis.  The pluripotent stem cells that continually divide to give rise to all of the blood cell types (red blood cells, RBCs and white blood cells, WBCs) are termed hematocytoblasts.

Blood Cell Lineage

Daughter cells of hematocytoblasts differentiate into various lineages.  The majority of daughter cells become erthryocytes (RBCs).  Some daughter cells become megakaryocytes and some daughter cells become leukocytes (WBCs).

Red Blood Cell Lineage:

The part of hematopoiesis that results in the production of red blood cells (erythrocytes) is termed erythropoiesis.  Daughter cells of hematocytoblasts that become erythroblasts will mature into erythrocytes after going through several stages, of maturation as they grow and become more packed full of hemoglobin.  When erythroblasts grow and differentiate into the normoblast stage, they lose their nucleus and become reticulocytes and after another 2-3 days enter the bloodstream.  Two days later, they become mature erythrocytes whose main function is the transportation of oxygen and carbon dioxide.  Lifespan of erythrocytes is approximately 120 days after which they are recycled by macrophages in the liver, bone marrow, and spleen.  The proportion of RBCs in the blood that are reticulocytes is called the “retic count” and is normally 1-2%.  If the retic count is low (e.g. 0.5%) it indicated erytrhopoiesis levels are low.

To maintain RBC homeostasis, approximately 3 million RBCs are recycle and produced per second.  Erythroipoiesis is stimulated by the hormone, erythropoietin (EPO) which is secreted by the kidneys (and some cells in the liver) in response to low blood-oxygen levels.  EPO is also secreted in reponse to growth hormone, thyroxine and testosterone.  If RBC or hemoglobin levels are too low, there will be a lower level of oxygen delivery, potentially resulting in weakness, fatigue, and confusion. RBC production requires B vitamins, folic acid (folate), amino acids, and iron.  Nutritional deficiencies in protein, vitamin B₁₂ or folate can cause reduced production in other blood cell lineages as well (i.e. WBCs and platelets).  Reduced WBC production is termed leukopenia.  Reduced platelet production is termed thrombocytopenia.

Megakaryocytes and Platelets (Thrombocytes):

Within the bone marrow, hematocytoblasts, blood stem cells, divide to produce many types of daughter cells, some of which are large cells called megakaryocytes that fragment into approximately 1000 anuclear platelets (thrombocytes).  Even though platelets lack a nucleus they contain 300 different chemicals involved in blood clotting (hemostasis) and are able to see out areas of cellular or vascular damage to provide assistance.  Platelets play a vital role in innate (non-specific defense, and are essentail in forming clots to prevent excessive bleeding from damaged blood vessels.

White Blood Cell Lineage:  

In addition to producing RBCs and platelets, hematocytoblast blood stem cells in the bone marrow also produce daughter cells that will become WBCs.  There are 2 broad categories of WBCs, the granulocytes and agranulocytes.  As the name suggests, the granulocytes, which have large granules that are easily viewable under the microscope.  The agranulocytes have smaller granules.

White Blood Cells – Granulocytes 

Granulocytes provide innate (non-specific) defense.  There are four types of granulocytes: neutrophils, eosinophils, basophils and mast cells.  All of the granulocytes are also termed polymorphonuclear leukocytes (PMNs) as they each have a nucleus that is segmented or irregularly shaped. This characteristic is useful in identifying these cells under the microscope, as is their staining characteristics.  Neutrophils stain a neutral pink colour with H&E dye.  Eosinophils stain a red colour with an acidic eosin dye. Basophils and mast cells stain blue colours with basic dye.

Neutrophils and eosinophils are phagocytes and are able to protect against pathogens.

Mature neutrophils arise from immature band cells and live approximately 2-5 days.  Neutrophils are distinguishable under the microscope, as they contain a 2-5 lobed nucleus.  Neutrophils are the most plentiful WBC, making up 50-70% of circulating WBCs, and contain extensive lysosomes.  Neutrophils use amoeboid movement (as do all WBCs) and are amongst the first cells to arrive at sites of cellular damage.  Neutrophils play important roles in engulfing bacteria and cellular debris. The granules of neutrophils contain lactoferrin and proteases (proteolytic enzymes) for killing pathogens and the secretory vesicles contain cytokines and cytotoxic compounds such as ROSs which are used to destroy pathogens.  Once activated, neutrophils live 24-48 hours.  When neutrophils are active, their rate of production increases and there is an increase in number of neutrophils in the blood, which is revealed in a blood cell count.  An increased number of neutrophils per mL of blood is called neutrophilia.  Bacterial infections often cause neutrophilia and are also associated with a shift to the left which refers to an increased number of band cells (immature neutrophils) in the blood.  At times, very severe bacterial or viral infections can lead to lower numbers of neutrophils, as the rate of mitosis of neutrophils is outpaced by their ability to defend against the infection.  Low numbers of neutrophils per mL of blood is termed neutropenia.  Nutritional deficiencies, some congenital defects, autoimmune diseases (Systemic Lupus Erythematosus) and cancers can also cause neutropenia.

Eosinophils stain pink with eosin dye, and appear at the site of infection or cellular damage 2-3 hours after neutrophils.  Not only are eosinophils able to phagocytose debris and pathogens, they are also able to exocytose toxins destroying pathogens that are too large to phagocytose (e.g. helminths or parasitic worms).  Eosinophils circulate the bloodstream, patrol tissues, and account for 2-3% of WBCs.  The granules of eosinophils are filled with toxins (e.g. major basic protein).  A large number of eosinophils will be produced in response to helminth (parasitic worm) infections.  Eosinophilia (high number of eosinophils per mL blood) will also occur in cases of allergies and autoimmune diseases

Mast Cells are located in tissues throughout the body and most prevalent in sites that are most at risk for infection: the dermis of the skin, the mucosa of the lungs and the mucosa of the gastrointestinal (GI) tract.  Basophils are very similar to mast cells as both contain granules filled with heparin and histamine.  However, while mast cells are fixed patrolling certain tissue beds, basophils circulate the bloodstream.  Both mast cells and basophils fulfill similar roles.  In response to cellular injury, basophils and mast cells are activated, migrate to the affected area, and degranulate releasing pro-inflammatory cytokines (e.g. histamine, bradykinins, prostaglandins, and leukotrienes).  Heparin is an anti-coagulant that is thought to decrease the number of microthrombi formed at sites of injury.

White Blood Cells – Agranulocytes (White Blood Cells with smaller granules)

There are 2 major types of agranulocytes: monocytes and lymphocytes. The monocytes mature to become macrophages and dendritic cells.  There are 3 main types of lymphocytes: Natural Killer cells, T cells (T lymphocytes) and B cells (B lymphocytes).

Blood Composition – Plasma and Formed Elements

Blood is considered a fluid connective tissue and contains a liquid extracellular matrix called plasma. The formed elements within blood is the cellular portion and contain RBCs, WBCs, and platelets.

If one were to sit a test tube of blood in a tray, it would settle out by weight, with the ligher plasma on the top, the buffy coat containing leukocytes and platelets forming a small white layer, and then a dark red bottom layer consisting of the heavier iron-rich erythrocytes.  By volume, blood is approximately 55% plasma, <1% leukocytes and platelets, and depending on biological sex, 41%-45% erythrocytes.

Plasma is a yellowish fluid and contains water, electrolytes (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^–, HCO₃^–, HPO₄^–, SO₄^2-), dissolved gases, amino acids, fatty acids, glycerides, cholesterol, glucose, hormones, organic wastes (urea, uric acid, creatinine, bilirubin) and proteins which include antibodies, complement proteins, enzymes, and transport proteins such as albumin and lipoproteins. It is likely not surprising that interstitial fluid (the fluid between tissue cells) is very similar in composition to plasma as an equilibrium does exist particularly for small water soluble ions and molecules.  In comparison with plasma, intersitial fluid contains similar concentrations of water, electrolytes, dissolved gases, amino acids and hormones.  However plasma contains a lot of dissolved proteins, whereas interstitial fluid does not.

Plasma Proteins:

1. Albumin: 60% of plasma protein: The most abundant protein in the plasma, albumin is involved in transporting lipid soluble substances (i.e. fatty acids and steroid hormones) in the blood, and is produced in the liver. Albumin is responsible for the majority of the blood colloid osmotic force.
2. Globulins: 35% of plasma protein: Globulins includes antibodies (Immunoglobulins) produced by B cells (B lymphocytes) and transport globulins that are produced in the liver that transport vitamins, lipids, metal ions, and hormones (e.g. lipoproteins).
3. Antibodies: Antibodies are specialized proteins involved in immunity that are produced in lymph tissue by B cells.
4. C-reactive proteins (CRP): are small proteins produced by liver cells (hepatocytes) and circulate the blood binding to the surface of dead cells and bacteria in order to activate the complement system.  The amount of damage often correlates with the number of CRP per mL of blood as CRP is produced in response to cytokines released by macrophages.
5. Complement proteins:  are produced by hepatocytes and circulate the blood as part of the immune system, capable of: a) acting as opsonins, b) creating membrane attack complexes on bacterial surfaces, and c) stimulating mast cell activity during infection.
6. Fibrinogen and Prothrombin: 4% of plasma protein: Fibrinogen and prothrombin are produced in the liver and are involved in blood clotting. Require vitamin K during synthesis.
7. Lipoproteins: Produced in the liver and involved in transporting triglycerides and cholesterol in the blood.
8. Hormones and enzymes: which perform specialized functions

*Note: Hormones are important signalling molecules that regulate complex biological processes, growth, development, maturation, and behavioiur.  Hormones are classified into 5 main categories: lipids (e.g. prostaglandins, thromboxanes), steroids (estrogen and testosterone), amino acids (e.g. epinephrine), peptides (e.g. insulin), and gases (e.g. nitric oxide, NO)

Serum is the fluid plasma component of blood, with all of the clotting factors, platelets, and cells removed.    Serum is used in blood typing as well as several diagnostic tests.  Serum is obtained by allowing blood to clot and then centrifuing to separate the blood by weight.  The top liquid portion (that has not coagulated) is serum.  Serum contains water, electrolytes and soluble proteins (e.g. antibodies),  Diagnostic tests have been developed for some cancers, autoimmune reactions, and other diseases that analyze the presence or level of various serum biomarkers (e.g. C-reactive protein, CRP; specific antibodies; prostate specific antigen, PSA).

Hematocrit:

Hematocrit is defined as the percentage of blood that is formed elements.  Biological males typically have a higher hematocrit than biological females due to testosterone stimulating EPO and red blood cell production.  Hematocrit for biological males is approximately 46% and is 42% for biological females.  To some extent a greater concentration of erythrocytes in biological males might be expected, as testosterone also stimulates a larger amount of skeletal muscle to form during puberty, which as a metabolically active tissue type requires sufficient levels of oxygen and nutrients to maintain and support. 

Blood Disorders – Examples:

Anemia is defined as condition in which there is a reduced oxygen-carrying capacity,.  Anemia can result from various causes, such as a low number of red blood cells or sickle-shaped cells (e.g. Sickle Cell Anemia).  The word anemia originates from an- meaning without, and -emia referring to blood. Risk factors include: blood loss, systemic lupus erythematosus, autoimmune diseases that affect intestinal absorption of food (e.g. Crohn diseae, ulcerative colitis), blood cancers (e.g. lymphoma), chemotherapy and long term infections (e.g. HIV, osteomyelitis, hepatitis).

Iron-deficiency Anemia: most commone in biological femals, as females have lower iron reserves than biological males.  Other contributing factors can include vegetarian diet, poor diet, menstruation, and frequent blood donors.  A low retic count, indicating low levels of erythropoiesis may occur.  Prevention involves healthy diet that includes iron-rich foods such as: eggs, meat, leafy green vegetables,

Sickle Cell Anemia: is due to an autosomal recessive disorder in which both inherited genes for hemoglobin’s  Hb beta-chain on chromosome 11 are mutated.  This results in misfolding of hemoglobin and the rigid sickle-shape of RBCs.  The RBCs have a shortened life expectancy and reduced flexibility.  Sickled RBCs cause blockages and downstream hypoxia throughout the body.  Blood transfusions and the donation of bone marrow stem cells is used to treat this disease.

Alpha and Beta Thalassemia: are autosomal recessive diseases in which hemoglobin gene mutations have occured in either the alpha or beta chain respectively.  Signs and symptoms range from mild to severe based on the types of mutations present.

Pernicious Anemia: is a rare auto-immune disease in which auto-antibodies binding to and inhibiting the action of Intrinsic Factor. Intrinsic Factor is produced by intestinal cells and is required for the absorbtion of dietary vitamin B₁₂.  A low retic count, indicating low levels of erythropoiesis may occur.  Without treatment vitamin B₁₂ deficiency leads to anemia.

Hemorrhagic Anemia: as the name suggests is due to excessive blood loss.

Polycythemia is defined as a condition in which there is excessive red blood cell production.  The word polycythemia originates from poly- meaning many, cyte referring to cell, and -emia referring to blood.  This condition can be dangerous as it thickens blood, increases the risk of blood clots, and also puts a strain on the heart to pump harder.  Unfortunately many elite athletes  have been found to have blood dope by injecting themselves with erythropoietin (EPO) to artificially increase red blood cell count for improved oxygen delivery and athletic performance.

On the other, moving to a higher altitude, with lower atmospheric oxygen levels, can naturally increase levels of EPO.  In this case a higher hematocrit level may also be present.

Erythrocyte Sedimentation Rate (ESR): is a diagnostic blood test, that involves calculating the rate at which erythrocytes settle to the bottom of a tube of blood.  RBCs are the heaviest component of blood and will settle to the bottom of the tube, however their rate of settling is affected by: the number of RBCs per mL of blood as well as the number of plasma proteins present.  For example, an adult male (XY) typically has a higher hematocrit compared to an age-matched female (XX).  Having more RBCs per mL results in slower settling as the erythrocytes are negatively charged and repel each other which slows down sedimentation.  On average the ESR of young adult males is 12mm/hr and the average ESR of age-matched females is 18mm/hr.  During injury or infection the number of plasma proteins (e.g. CRP, prothrombin, plasminogen, fibrinogen, complement proteins) per mL increases.  These proteins allow the RBCs to stick closer together and fall faster.  Therefore a ESR is often proportional to the amount of damage or inflammation that is present in the body.  Serious infections, autoimmune disease, and chronic ailments can result in ESRs that are 100mm/hr or more.

Summary

• Describe plasma components & define vocabulary words:
  • Plasma – liquid matrix containing water, electrolytes, and plasma proteins
  • Plasma proteins – antibodies, complement proteins, clotting factors, albumen and transporter proteins
  • Platelets/Thrombocytes – a nuclear cell fragments formed from large megakaryocytes;  involved in clotting (hemostasis)
  • Leukocytes – WBCs
  • Lymphocytes: type of WBC involved in antibody production (B lymphocytes), targeted immune response (T lymphocytes), and surveillance (NK lymphocytes)
  • Neutrophils: The most abundant phagocyte in the blood; contain extensive lysosomes
  • Eosinophils: Destroy parasitic worms & immune complexes
  • Basophils & Mast cells: Release histamine, heparin, prostaglandins, and leukotrienes in process known as degranulation
  • Erythrocytes – RBCs; transport oxygen & carbon dioxide
  • Hematocrit –  % by volume of blood that is   formed elements
  • Anemia: reduced oxygen-carrying capacity of blood due to low levels of functional RBCs or hemoglobin.
  • Polycythemia; greater than normal # of RBCs
  • EPO, erythropoietin: hormone that stimulates production of RBCs