histology of blood

HISTOLOGY OF BLOOD

blood is a specialized connective tissue in which cells are suspended in fluid extracellular material called plasma . Propelled mainly by rhythmic contractions of the heart, about 5 L of blood in an average adult moves unidirectionally within the closed circulatory system.
The so-called formed elements circulating in the plasma are erythrocytes (red blood cells), leukocytes (white blood cells), and platelets .
When blood leaves the circulatory system, either in a test tube or in the extracellular matrix (ECM) surrounding blood vessels, plasma proteins react with one another to produce a clot, which includes formed elements and a pale yellow liquid called serum . Serum contains growth factors and other proteins released from platelets during clot formation, which confer biological properties very different from those of plasma.
Collected blood in which clotting is prevented by the addition of anticoagulants (eg, heparin or citrate) can be separated by centrifugation into layers that reflect its heterogeneity.
Erythrocytes make up the sedimented material and their volume, normally about 45% of the total blood volume in healthy adults, is called the hematocrit.
The straw-colored, translucent, slightly viscous supernatant comprising 55% at the top half of the centrifugation tube is the plasma. A thin gray-white layer called the buffy coat between the plasma and the hematocrit, about 1% of the volume, consists of leukocytes and platelets, both less dense than erythrocytes.

COMPOSITION OF PLASMA

Plasma is an aqueous solution, pH 7.4, containing substances of low or high molecular weight that make up 7% of its volume.
The dissolved components are mostly plasma proteins, but they also include nutrients, respiratory gases, nitrogenous waste products, hormones, and inorganic ions, collectively called electrolytes . Through the capillary walls, the low-molecular-weight components of plasma are in equilibrium with the interstitial fluid of the tissues.
The composition of plasma is usually an indicator of the mean composition of the extracellular fluids in tissues.
The major plasma proteins include the following:
■ Albumin
■ α Globulins and β Globulins.
■ γ Globulins.
■ Fibrinogen.
■ Complement proteins.


BLOOD CELLS
Blood cells can be studied histologically in smears prepared by spreading a drop of blood in a thin layer on a microscope slide. In such films the cells are clearly visible and distinct from one another, facilitating observation of their nuclei and cytoplasmic characteristics. Blood smears are routinely stained with special mixtures of acidic (eosin) and basic (methylene blue) dyes. These mixtures may also contain dyes called azures that are more useful in staining cytoplasmic granules containing charged proteins and proteoglycans.

Erythrocytes

Erythrocytes (Red Blood Cells or RBCs) are terminally differentiated structures lacking nuclei and completely filled with the O2-carrying protein hemoglobin. RBCs are the only
blood cells whose function does not require them to leave the vasculature.
Human erythrocytes suspended in an isotonic medium are flexible biconcave discs. They are approximately 7.5 μm in diameter, 2.6 μm thick at the rim, but only 0.75 μm thick in the center.
The biconcave shape provides a large surface-to-volume ratio and facilitates gas exchange. The normal concentration of erythrocytes in blood is approximately 3.9 to 5.5 million per microliter (μL, or mm3) in women and 4.1-6.0 million/μL in men.
Erythrocytes are normally quite flexible, which permits them to bend and adapt to the irregular turns and small diameters of capillaries. Observations in vivo show that at the angles of capillary bifurcations, erythrocytes with normal adult hemoglobin frequently assume a cuplike shape. In larger blood vessels RBCs often adhere to one another loosely in stacks called rouleaux.
The plasmalemma of the erythrocyte, because of its ready availability, is the best-known membrane of any cell. It consists of about 40% lipid, 10% carbohydrate, and 50% protein. Most of the latter are integral membrane proteins, including ion channels, the anion transporter called band 3 protein, and glycophorin A. The glycosylated extracellular domains of the latter proteins include antigenic sites that form the basis for
the ABO blood typing system.
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, and ankyrin, which anchors the lattice to the glycophorins and band 3 proteins. This submembranous meshwork stabilizes the membrane, maintains the cell shape, and provides the cell elasticity required for passage through capillaries.
Erythrocyte cytoplasm lacks all organelles but is densely filled with hemoglobin, the tetrameric O2-carrying protein that accounts for the cells’ uniform acidophilia. When combined with O2 or CO2, hemoglobin forms oxyhemoglobin or carbaminohemoglobin, respectively.
Erythrocyte differentiation includes loss of the nucleus and organelles, shortly before the cells are released by bone marrow into the circulation. Lacking mitochondria, erythrocytes rely on anaerobic glycolysis for their minimal energy needs. Lacking nuclei, they cannot replace defective proteins.
Human erythrocytes normally survive in the circulation for about 120 days. By this time defects in the 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. Senescent or worn-out RBCs displaying such changes are removed from the circulation, mainly by macrophages of the spleen, liver, and bone marrow.
Leukocytes
The number of leukocytes in the blood varies according to age, sex, and physiologic conditions, Healthy adults have 4500 to 11,000 leukocytes per microliter of blood.
Leukocytes (white blood cells or WBCs) leave the blood and migrate to the tissues where they become functional and perform various activities related to immunity. According to the type of cytoplasmic granules and their nuclear morphology, leukocytes are divided into two groups:
1-Granulocytes
2-Agranulocytes.
Both types are rather spherical while suspended in blood plasma, but they become amoeboid and motile after leaving the blood vessels and invading the tissues. Their estimated sizes mentioned here refer to observations in blood smears in which the cells are spread and appear slightly larger than they are in the circulation.


Granulocytes
possess two major types of cytoplasmic granules:
1) lysosomes (often called azurophilic granules in blood cells).
2) specific granules that bind neutral, basic, or acidic stains and have specific functions.

Granulocytes have polymorphic nuclei with two or more distinct (almost separated) nuclear lobes, granulocytes include the following:
1-Neutrophils.
2-Eosinophils.
3-Basophils.
All granulocytes are terminally differentiated cells with a life span of only a few days. Their Golgi complexes and rough ER are poorly developed. They have few mitochondria
and depend largely on glycolysis for their low energy needs.
Granulocytes normally die by apoptosis in the connective tissue and billions of neutrophils alone die by apoptosis each day in the adult human. The resulting cellular debris is removed by macrophages and, like all apoptotic cell death, does not itself elicit an inflammatory response.
Agranulocytes
Do not have specific granules, but they do contain azurophilic granules (lysosomes), with affinity for the basic stain azure A. The nucleus is spherical or indented but not lobulated. This group includes:
1- Lymphocytes.
2- Monocytes.
The differential count( is the percentage of all leukocytes for each type of leukocyte).
All leukocytes are key players in the defense against invading microorganisms, and in the repair of injured tissues, specifically leaving the microvasculature in injured or infected tissues.

Neutrophils (Polymorphonuclear Leukocytes)

Mature neutrophils constitute 54% to 62% of circulating leukocytes; circulating immature forms raise this value by 3% to 5%. Neutrophils are 12-15 μm in diameter in blood smears, with nuclei having two to five lobes linked by thin nuclear extensions.
In females, the inactive X chromosome may appear as a drumstick-like appendage on
one of the lobes of the nucleus although this characteristic is not obvious in every neutrophil. Neutrophils are inactive and spherical while circulating but become actively
amoeboid during diapedesis and upon adhering to solid substrates such as collagen in the ECM.
Neutrophils are active phagocytes of bacteria and other small particles and are usually the first leukocytes to arrive at sites of infection, where they actively pursue bacterial cells using chemotaxis.
The cytoplasmic granules of neutrophils provide the cells’ functional activities and are of two main types.
1) Azurophilic primary granules.
2) Specific secondary granules.


In a process called diapedesis (Gr. dia, through + pedesis, to leap), the leukocytes send extensions through the openings between the endothelial cells, migrate out of the venules into the surrounding tissue space, and head directly for the site of injury or invasion. The attraction of neutrophils to bacteria involves chemical mediators in a process of chemotaxis, which causes leukocytes to rapidly accumulate where their defensive actions are specifically needed.
Neutrophils are short-lived cells with a half-life of 6 to 8 hours in blood and a life span of 1 to 4 days in connective tissues before dying by apoptosis.

Eosinophils

Eosinophils are far less numerous than neutrophils, constituting only 1% to 3% of leukocytes. In blood smears, this cell is about the same size as a neutrophil or slightly larger, but with a characteristic bilobed nucleus. The main identifying characteristic is the abundance of large, acidophilic specific granules typically staining pink or red.
Eosinophils act to kill parasitic worms or helminthes, modulate inflammatory responses,
have important role in the inflammatory response triggered by allergies and particularly abundant in connective tissue of the intestinal lining and at sites of chronic inflammation, such as lung tissues of asthma patients.
These leukocytes also remove antigen-antibody complexes from interstitial fluid by
phagocytosis.

Basophils

Basophils are also 12 to 15 μm in diameter but make up less than 1% of blood leukocytes and are therefore difficult to find in normal blood smears. The nucleus is divided into two irregular lobes, but the large specific granules overlying the nucleus
usually obscure its shape.
The specific granules (0.5 μm in diameter) typically stain purple with the basic dye of blood smear stains and are fewer, larger, and more irregularly shaped than the granules of other granulocytes.
The strong basophilia of the granules is due to the presence of heparin and other sulfated GAGs. Basophilic specific granules also contain much histamine and various other mediators of inflammation.

Lymphocytes

By far the most numerous type of agranulocyte in normal blood smears or CBCs, lymphocytes constitute a family of leukocytes with spherical nuclei.
Lymphocytes are typically the smallest leukocytes and are abundant, constituting up to a third of these cells.
Although they are morphologically similar, lymphocytes can be subdivided into functional groups by distinctive surface molecules (called “cluster of differentiation” or CD markers) that can be distinguished using antibodies with immunocytochemistry or flow cytometry. Major classes Include:
1- B lymphocytes.
2- T lymphocytes {Helper and Cytotoxic (CD4+ and CD8+, respectively)}.
3- Natural Killer (NK) cells.
These and other types of lymphocytes have diverse roles in immune defenses against invading microorganisms and certain parasites or abnormal cells.
Though generally small circulating lymphocytes have a wider range of sizes than most leukocytes. Many have diameters similar to those of RBCs; medium and large lymphocytes are 9 to 18 μm in diameter, with the latter representing activated lymphocytes or NK cells. The small lymphocytes are characterized by spherical nuclei with highly condensed chromatin and only a thin surrounding rim of scant cytoplasm,
making them easily distinguishable from granulocytes.
Larger lymphocytes have larger, slightly indented nuclei and more cytoplasm that is slightly basophilic, with a few azurophilic granules (lysosomes), mitochondria, free polysomes, and other organelles.
Lymphocytes vary in life span according to their specific functions; some live only a few days and others survive in the circulating blood or other tissues for many years.


Monocytes
Monocytes are agranulocytes that are precursor cells of macrophages, osteoclasts, microglia, and other cells of the mononuclear phagocyte system in connective tissue. All monocyte-derived cells are antigen-presenting cells and have important roles in immune defense of tissues. Circulating monocytes have diameters of 12 to 15 μm, but macrophages are somewhat larger.
The monocyte nucleus is large and usually distinctly indented or C-shaped. The chromatin is less condensed than in lymphocytes and typically stains lighter than that of large lymphocytes.
The cytoplasm of the monocyte is basophilic and contains many small lysosomes or azurophilic granules, some of which are at the limit of the light microscope’s resolution. These granules are distributed through the cytoplasm, giving it a bluish-gray color in stained smears.
Platelets
Blood platelets (or thrombocytes) are very small non-nucleated, membrane- bound cell fragments only 2 to 4 μm in diameter.
Platelets originate by separation from the ends of cytoplasmic processes extending from giant polyploid bone marrow cells called megakaryocytes. Platelets promote blood clotting and help repair minor tears or leaks in the walls of small blood vessels, preventing loss of blood from the microvasculature. Normal platelet counts range from 150,000 to 400,000/μL (mm3) of blood. Circulating platelets have a life span of about 10 days.
In stained blood smears, platelets often appear in clumps. Each individual platelet is generally discoid, with a very lightly stained peripheral zone, the hyalomere, and a darker-staining central zone containing granules, called the granulomere.
A sparse glycocalyx surrounding the platelet plasmalemma is involved in adhesion and activation during blood coagulation.