Blood Composition and Function

• Blood is a specialized type of connective tissue with a liquid extracellular matrix
• It is a suspension of cells and cell fragments (formed elements) in fluid (plasma)
• Average human adults have a blood volume around 5 liters (4-5L in females, 5-6L in males)
• Blood circulates in a closed system of vessels
• Transports O₂, CO₂, nutrients, waste products, hormones, proteins, and ions
• Regulates body temperature
• Assists in regulating osmotic and acid-base balance

Centrifugation:

• A tube of blood after centrifugation contains ~55% plasma at the top
• A thin layer of leukocytes and platelets (<1%) forms the buffy coat in the middle
• ~45% of erythrocytes are in the bottom half of the tube
• The volume of erythrocytes is known as the hematocrit

Plasma:

• Plasma consists of 90% water, 8% protein,1% inorganic salts, 0.5% lipids, 0.1% glucose, and other minor components
• Colloid osmotic pressure is the osmotic force exerted by plasma proteins
• Regulates fluid distribution between the plasma and the extracellular space
• Keeps the fluid in the circulation

Plasma Proteins

• All proteins serve to buffer against pH changes

• Albumin makes up approximately 58% of plasma proteins

• The major protein that exerts osmotic force

• Contributes to blood's viscosity

• Made in the liver

• Binds to and transports fatty acids, electrolytes, hormones, and drugs

• Globulins make up approximately 37% of plasma proteins.

• α-Globulins transport lipids and some metal ions

• β-Globulins transport iron ions and lipids in the bloodstream

• γ-Globulins are immunoglobulins (antibodies) with immune functions

• Fibrinogen makes up approximately 4% of plasma proteins.

• The largest plasma protein at 340 kD

• Made in the liver

• Participates in blood coagulation (clotting) and is a precursor of fibrin

• Regulatory proteins make up >1% of plasma proteins.

• Consists of enzymes, proenzymes, hormones, and the complement system

• Complement proteins participate in nonspecific host defense and initiate the inflammatory process

• Fibrinogen polymerizes into insoluble, cross-linked fibers of fibrin during clotting

• Prevents blood loss from vessels

• Serum is the yellowish fluid remaining after blood has clotted

• It is similar to plasma, but lacks fibrinogen and clotting factors

Blood Smears & Stains

• Blood cells (formed elements) can be studied histologically in blood smears, which are stained for study
• Smears use special mixtures of acidic (eosin), basic (methylene blue) and azure dyes (azure A)
• Some of these special stains are Giemsa and Wright stain
• Basophilia (deep blue) has an affinity for the basic dye methylene blue in DNA in nuclei and RNA in ribosomes
• Eosinophilia/Acidophilia (pink/red) has an affinity for the acidic dye eosin
• Neutrophilia (salmon pink/lilac) has an affinity for a dye of neutral pH in specific cytoplasmic granules of neutrophils
• Azurophilia (purple) has an affinity for azure dyes in lysosomes, one of the granule types in leucocytes

Formed Elements

• Erythrocytes
• Granulocytes (basophils, eosinophils, and neutrophils)
• Agranulocytes (monocytes and lymphocytes)
• Platelets

Erythrocytes

• Erythrocytes (red blood cells or RBCs) are round, anucleate (lacking nuclei), and biconcave when terminally differentiated
• They stain light salmon pink with either Wright or Giemsa stain
• RBCs are the only blood cells whose function does not require them to leave the vasculature
• The biconcave shape provides a large surface-to-volume ratio and facilitates gas exchange
• Erythrocytes have the classic dumbbell shape when the section plane is through the thin central zone
• Internal organelles are absent and have high electron density due to the iron molecules in the hemoglobin.
• The structure is normally quite flexible, which allows them to bend and adapt to the irregular turns and small capillary diameters
• RBCs often adhere to one another loosely in stacks called rouleaux.
• The plasmalemma of RBCs consists of approximately 40% lipid, 10% carbohydrate, and 50% protein
• Several peripheral proteins are associated with the inner surface of the membrane, including spectrin, dimers of which form a lattice bound to underlying actin filaments
• Ankyrin anchors the lattice to the glycophorins and band 3 proteins
• This submembranous meshwork stabilizes the membrane and maintains the cell shape
• The majority of the ABO antigens are expressed on the ends of long polylactosamine chains attached mainly to band 3 protein
• RBCs are filled with hemoglobin
• Mature erythrocytes possess no organelles but are filled with tetrameric hemoglobin
• Hemoglobin provides the cells' uniform acidophilia
• When combined with O₂ or CO₂, hemoglobin forms oxyhemoglobin or carbaminohemoglobin (carbamylhemoglobin)
• The combination of hemoglobin with carbon monoxide (CO) is irreversible
• A single nucleotide substitute in the hemoglobin gene produces a version of the protein that polymerizes to form rigid aggregates, which results in greatly misshapen cells with reduced flexibility from sickle cell anemia
• Homozygous individuals for the HbS gene can lead to greater blood viscosity and poor microvascular circulation, which are both features of sickle cell anemia
• Lacking mitochondria, erythrocytes use anaerobic glycolysis for minimal energy needs
• They are immotile and only serve their passively circulated function
• Lacking nuclei, they cannot replace defective proteins
• The average erythrocyte life span is 120 days (4 months)
• Defects in the aged erythrocyte's membrane's cytoskeletal lattice or ion transport systems begin to produce swelling or other shape abnormalities, as well as changes in the cells' surface oligosaccharide complexes
• They are recognized and destroyed by macrophages of the spleen, liver, and bone marrow

Leukocytes

• Leukocytes (white blood cells, WBCs) possess varying numbers of azurophilic granules, which are lysosomes
• They use the bloodstream as a means of transportation
• Once reaching their destination, they leave the blood and migrate to tissues where they become functional and perform activities related to immunity
• Lymphocytes constitute 20-25% of leukocytes
• Monocytes constitute 3-8% of leukocytes.
• Neutrophils constitute 60-70% of leukocytes
• Eosinophils constitute 2-4% of leukocytes
• Basophils constitute 0.5-1% of leukocytes
• According to the type of cytoplasmic granules and their nuclear morphology, leukocytes are separated into granulocytes and agranulocytes
• While suspended in blood plasma, both types of leukocytes are spherical
• After leaving the blood vessels and invading the tissues, they become amoeboid/motile

Granulocytes

• Granulocytes possess two major types of cytoplasmic granules including azurophilic granules (lysosomes) and specific granules that bind neutral, basic, or acidic stains and have specific functions
• Also have polymorphic nuclei with two or more distinct (almost separated) nuclear lobes

Neutrophils

• Mature neutrophils constitute 54-62% of circulating leukocytes.
• The most abundant WBC.
• The diameter in blood smears averages 12-15 µm
• Their nuclei have two to five lobes linked by thin nuclear extensions.
• Referred to as polymorphonuclear leukocytes (PMNs) or just polymorphs
• Dynamic cells undergo frequent nuclear shape changes
• Cytoplasmic granules are relatively sparse and have heterogeneous staining properties, although usually pale and do not obscure the nucleus -Azurophilic granules are lysosomes filled with lysosomal hydrolases, myeloperoxidase, bacterial permeability-increasing protein, lysozyme, and collagenase
• The azurophilic granules components are contained in large and dense vesicles
• Specific granules are less dense and stain faintly pink
• Neutrophils are active phagocytes of bacteria and other small particles
• Neutrophils usually are the first leukocytes to arrive at sites of infection
• Contain glycogen, which is processed into glucose for energy via the glycolytic pathway.
• The citric acid cycle is of less importance.
• Short-lived cells with a half-life of 6-8 hours in blood and a life span of 1-4 days in connective tissues before undergoing apoptosis

Eosinophils

• Eosinophils constitute 1-3% of leukocytes, and increase during helminth and parasitic infections as well as during allergic reactions
• Eosinophils in blood smears are about the same size as a neutrophil or slightly larger They have a characteristic bilobed nucleus
• The main identifying characteristic is the abundance of large, acidophilic (eosinophilic) specific granules, which stain pink or red
• Eosinophils are particularly abundant in the connective tissue of the intestinal lining and sites of chronic inflammation, exemplified by lung tissues of asthma patients
• The life span is a few hours in the blood, 2 weeks in connective tissue
• The number of circulating eosinophils increases during helminthic infections and allergic reactions
• Eosinophils modulate inflammatory responses by releasing chemokines, cytokines, and lipid mediators, playing an important role in the inflammatory response triggered by allergies
• Eosinophilic specific granules are oval-shaped with flattened crystalloid cores
• They contain major basic proteins (MBP) which constitutes up to 50% of the total granule protein by weight
• MBP is a potent toxin for helminths and mammalian cells and may have important roles in allergic and inflammatory reactions.
• It can release histamine from mast cells and damage epithelial cells of bronchial tubes
• MBP is also involved in antiparasitic defense mechanisms and immune hypersensitivity reactions

Basophils

• Basophils averaging 12-15 µm in diameter, and making up less than 1% of blood leukocytes are difficult to find in normal blood smears
• The nucleus is divided into two irregular lobes
• Specific granules overlying the nucleus usually obscure its shape
• Stain purple with the basic dye of blood smear stains
• Are fewer, larger, and more irregularly shaped than other granulocytes’ granules
• Basophilia, when stained with eosin, of the granules is due to heparin and sulfated GAGs, which also contain histamine and inflammatory mediators
• Basophilic specific granules also contain much histamine, platelet activating factor, and eosinophil chemotactic factor
• By migrating into connective tissues, basophils appear to supplement the functions of mast cells (a type of granulocyte in connective tissues throughout the body)
• Basophils and mast cells have metachromatic granules that take up a color different from that of the stain due to their heparin and histamine contents and secrete their granular components in response to certain antigens and allergens

Agranulocytes

• Agranulocytes do not have specific granules
• Contain lysosomes (azurophilic granules) with an affinity for the basic stain azure A
• The nucleus is spherical or indented, not lobulated
• There are two types of agranulocytes: Lymphocytes and Monocytes
• Lymphocytes constitute 20-25% of leukocytes
• Monocytes constitute 3-8% of leukocytes

Lymphocytes

• Lymphocytes are the smallest leukocytes with spherical nuclei, and are abundant, since they accounting for one-third of leukocytes
• Their presence is variable, according to their specific functions, and some only live a few days. however other can survive in the circulating blood or other tissues a number of years
• Lymphocytes often show an unusual trait with small cytoplasm and dark-staining nuclei.
• Small lymphocytes are slightly larger than neighboring erythrocytes and have only a thin rim of cytoplasm around their spherical nucleus
• Medium lymphocytes & large lymphocytes are distinctly larger than erythrocytes
• Large lymphocytes may represent activated cells that have returned to the circulation
• Medium and large lymphocytes have larger, slightly indented nuclei and more cytoplasm that’s mildly basophilic with few azurophilic granules (lysosomes) and other organelles
• Lymphocytes are morphologically similar cells, but they do have subdivisions based on functional groups with distinctive surface areas (called “cluster of differentiation” or CD)
• The three types are B lymphocytes (B cells), T lymphocytes (T cells), and null cells
• T cells are for cellular immune activity B cells function in the humoral response (produce antibody)
• Null cells are few in number, do not possess immune determinates; and are of two types pluripotential hemopoietic stem cells (PHSCs) and natural killer (NK) cells

Monocytes

• With a diameter of 12-15 μm, circulating monocytes are the largest circulating blood cells
• The nucleus is large and often kidney or C-shaped.
• The chromatin is less compacted than in lymphocytes; consequently, it stains lighter
• Monocyte cytoplasm is basophilic and holds many small azurophilic granules and stains with a faint bluish-gray Monocytes are precursor cells of macrophages (alveolar), osteoclasts (bone), and other cells of the mononuclear phagocyte system in connective tissue
• These highly phagocytic cells ingest microorganisms, cellular remnants, and particulate matter

Platelets

• Platelets (thrombocytes) are very small nonnucleated, membrane-bound cell fragments with a diameter of 2-4 µm.
• Their fragments are displayed more often in the blood smears around the cell periphery
• Each individual platelet showcases a generally discoid region with a lighter peripheral region known as the hyalomere and darker staining section holding the granules, called granulomere