Blood Histology PDF

 Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) It is a specialized type of connective tissue that composed of: 1. The plasma: represents the matrix. 2. The formed blood elements: represent the cells. Blood has no fibers. The formed blood elements: 1- Red b...

 Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) It is a specialized type of connective tissue that composed of: 1. The plasma: represents the matrix. 2. The formed blood elements: represent the cells. Blood has no fibers. The formed blood elements: 1- Red blood corpuscles (RBCs). 2- White blood cells (WBCs) or leukocytes including:  Granular leukocytes: neutrophils, eosinophils and basophils.  Agranular leukocytes: lymphocytes and monocytes. 3- Blood platelets. Preparation of a blood smear: 1-Giemsa's stain (methylene blue+ eosin) is used which differentiate blood cells by their nuclei and cytoplasmic granules: basophilic (violet) eosinophilic (pink) azurophilic (red purple) 2-Blood count: It is the average number of a formed blood element per cubic millimeter blood.  RBCs count.  Total leukocytic count.  Platelets count.  Differential leukocytic count: it is the percentage of each type of leukocytes relative to the total number of WBCs. Red Blood Corpuscles (RBCs) Average RBCs count:  In males: 5-5.5 millions/mm3.  In females:4.5-5 millions/mm3. Life span of RBCs:  It is 100-120 days.  Old & deformed RBCs are phagocytosed by the macrophages in the spleen & liver. Function of Red blood corpuscles: Contain hemoglobin for gas exchange. Blood & Immune system Module Page 1  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) LM: Shape: eosinophilic biconcave discs. Diameter: 7.2-7.8µm (=the size of the smallest blood capillaries). Nucleus: absent. Cytoplasm: hemoglobin occupies about 33% of the corpuscular volume & is more concentrated at the periphery. EM: Shape: membranous electron dense biconcave discs. Cell organelles: no nucleus& no organelles. RBCs Membrane: 1- From outside: It is covered by a well-developed glycocalyx which is responsible for the blood grouping. 2- From inside: It is supported by a well-developed cytoskeleton, which is responsible for the flexibility of RBCs.  It is formed of microfilaments (actin &spectrin) that are attached to a peripheral membrane protein (ankyrin). How do the RBCs adapt to their function? 1- The biconcave discoid shape increases the surface area of the RBCs by 20-30% more than if they had a spherical shape. 2- The lack of a nucleus and organelles allows more space in the cytoplasm for carrying the maximal amount of hemoglobin. 3- The concentration of hemoglobin at the periphery of the cytoplasm facilitates its binding to oxygen and carbon dioxide. 4- The structure of the cytoskeleton helps the RBCs to be squeezed through the narrowest blood capillaries without being damaged. 5- The selective permeability of the cell membrane to oxygen and carbon dioxide to enhance their diffusion rates during gas exchange. Myeloid tissue Sites: 1- The central bone marrow cavity in young long bones. 2- The marrow cavities of cancellous bones. Types of bone marrow: 1- Red bone marrow: active in hemopoiesis. 2- Yellow bone marrow: Blood & Immune system Module Page 2  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil)  inactive bone marrow in which the blood formation has stopped.  Its yellow color is due to the presence of fat cells.  It can revert to the red type in hemorrhage & anemia. Structure of myeloid tissue: I-The stroma: A. Reticular fibers: support the myeloid cells & blood sinusoids. B. Stromal cells: the fixed cell population of the bone marrow. They include: 1- Reticular cells: have 3 functions:  Produce the reticular fibers.  Secrete growth factors that stimulate the hemopoietic stem cells.  In yellow bone marrow, these cells are changed to fat cells. 2- Macrophages:  Phagocytosis of aged RBCs & the malformed blood elements.  Storage of iron for reuse. 3- Fat cells: One of the largest cells in bone marrow. II-The blood sinusoids:  Wide & irregular blood vessel.  Lined by fenestrated endothelial cells. III-The myeloid cells: The free cell population of the bone marrow. They include: Multipotent SC CFU-LY CFU-E CFU-B 1- Pluripotent hemopoietic stem cells (PHSCs): CFU-GEMM Shape: small cells with pale basophilic cytoplasm and pale nuclei. Function: they divide, give rise to daughter cells (half remains as a reserve of the HPSCs while the other half develops into multipotent hemopoietic stem cells). 2- Multipotent hemopoietic stem cells (MHSCs): They give rise to cell colonies for each of the formed blood elements. 3- Colony forming units (CFUs): 2 types: A. Colony forming unit- lymphocyte (CFU- Ly): give rise to lymphocytes. B. Colony forming unit- granulocyte, erythrocyte, monocyte, megakaryocyte (CFU- GEMM): give rise to unipotent colony: CFU-Erythrocyte (CFU-E). CFU-Megakaryocyte (CFU-Meg). CFU-monocyte (CFU-M) CFU-Granulocyte: give rise to CFU-Neutrophil. Blood & Immune system Module Page 3  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) CFU-Eosinophil (CFU-Eo). CFU-Basophil (CFU-Ba). These cells are larger with more basophilic cytoplasm (abundant ribosomes for synthesis of the specific products of each blood element). Erythropoiesis Definition: It is the process of formation of RBCs. Duration: 7 days. 1- PHSCs. 2- MHSCs. 3- Colony forming unit- erythrocytes (CFU-E). 4- Proerythroblasts:  They are the first recognizable erythrocyte precursor.  They are large cells with basophilic cytoplasm. 5- Basophilic erythroblasts:  Hemoglobin synthesis is most active.  The cytoplasm is strongly basophilic due to abundant polysomes. 6- Polychromatophilic erythroblasts:  Hemoglobin is accumulating in large amount.  Ribosomes are still abundant.  The cytoplasm shows eosinophilic areas alternating with basophilic spots.  They are the last stage in which the cells undergo cell divisions. The next phases involve morphological changes of the erythroblasts. 7- Orthochromatophilic erythroblasts (normoblasts):  The synthesis of hemoglobin is completed.  Ribosomes are much reduced in number.  The nucleus becomes small and condensed, pushed towards the periphery, extruded from the cell and phagocytosed by macrophages. 8- Reticulocytes:  Non-nucleated.  They differ from mature RBCs in that: Slightly larger. Their cytoplasm contains remnants of ribosomes and other organelles which on staining with cresyl blue stain gives a reticulate pattern.  They represent about 1% of all RBCs in a normal blood film (an increase in their percentage indicates an increase in the rate of erythropoiesis as in case of anemia or hemorrhage). 9- Mature RBCs. Blood & Immune system Module Page 4 Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) platelets Normal count: 150,000-400,000/ mm3. Structure: LM:  Shape: biconvex discs.  Diameter:2-3µm.  Nucleus: absent.  The granulomere: granular central core contains azurophilic granules.  The hyalomere: pale basophilic area at the periphery. Function of Blood Platelets: Blood platelets are concerned with the process of thrombus formation (blood clotting) in response to any vascular endothelial injury to prevent excessive blood loss. How do the blood platelets adapt to their function? EM:  Shape: irregular outline (when activated) due to presence of pseudopodia  motility.  The platelet membrane: is covered by a well-developed glycocalyx helps in platelets adhesion & aggregation.  The hyalomere contains: 1- Actin and myosin filaments  contraction of the platelet for motility and clot retraction. 2- A circumferential bundle of microtubules  maintains the discoid shape of platelets. 3- Two systems of membranes  regulation of the intraplatelet calcium concentration and thus the control of platelet degranulation. These are: The open canalicular system: small canaliculi that open on the platelet surface. The dense tubular system: remnants of the endoplasmic reticulum of the platelets mother cell (the megakaryocyte).  The granulomere contains: 1- Few ribosomes, mitochondria, and glycogen particles. 2- Three types of membrane bounded granules which are released by exocytosis during the process of thrombus formation. These are: Alpha granules: abundant, large granules which contain fibrinogen and other coagulation factors. Delta granules: less abundant, medium- sized granules which contain histamine, serotonin, ATP, ADP. Blood & Immune system Module Page 5 Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) Lambda granules (lysosomes): few small dense granules, contain lysosomal hydrolytic enzymes. Thrombopoiesis Definition: It is the formation of blood platelets. The release of mature platelets from the bone marrow into the bloodstream takes about 10 days 1- PHSCs. 2- MHSCs. 3- Colony forming unit-GEMM. 4- Colony forming unit- megakaryocyte (CFU- Meg). 5- Megakaryoblasts. 6- Megakaryocytes. Megakaryocyte LM: Shape: Giant cells (50-150µm). Nucleus: large, lobulated containing the polyploid number of the ordinary DNA sets. Cytoplasm: basophilic contains many organelles together with the alpha, delta, and lambda granules. EM: the cytoplasm shows membranous demarcation lines surrounding groups of granules (lines of cleavage of the megakaryocytes into about 1200 platelets per megakaryocyte). Megakaryocytes are located near the bone marrow sinusoids; they extend their processes to release the platelets in bloodstream. Megakaryoblast Blood & Immune system Module Page 6  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) White Blood Cells (Leukocytes) 3 Normal count: 4000-10,000 / mm blood. Types:  Granular leukocytes (granulocytes) which have specific granules in their cytoplasm: 1- Neutrophils. 2- Eosinophils. 3- Basophils.  Agranular leukocytes (agranulocytes) which lack specific granules in their cytoplasm: 1- Monocytes. 2- Lymphocytes. Function of white blood cells: They do not function within the bloodstream but, they circulate in the blood stream and when they receive a stimulus, they leave the bloodstream to enter the connective tissue and perform an immune function. Granular Leukocytes I-Neutrophils (polymorphonuclear leukocytes): Why are they named neutrophils? because their granules have a neutral staining affinity to the stains in blood film. Why are they named PNLs? Because the cells may contain from 2-5 lobes of nuclei according to its age. Differential count: 60-70% of the total leukocytic count. LM:  Cytoplasm: lightly stained.  Nucleus: multilobed formed of 2-5 segments that are connected with a thin chromatin thread.  In females with a normal karyotyping (44+XX chromosomes), one of the X- chromosomes is condensed and attached to the nucleus forming a drumstick appearance (Barr body). EM: (How do the neutrophils adapt to its function?)  Shape: have an irregular outline due to extension of pseudopodia  motility& phagocytosis  Cytoplasm: contains two types of granules  phagocytosis: 1- Small specific granules: contain protease enzymes e.g., collagenase. 2- Larger azurophilic granules (non-specific): containing lysosomal hydrolytic enzymes and the antibacterial defensins. Blood & Immune system Module Page 7  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) Function of the neutrophils: (The first line of defense against any invading micro-organism) Killing strategy 1- Go to battlefield: they leave the circulation and migrate to the site of action in connective tissue. 2- Identify the enemy. 3- Phagocytosis (so they are called microphage). During the inflammatory reaction, neutrophils themselves are damaged by the bacterial toxins causing their death and they are called pus cells. II-Eosinophils: Why are they called eosinophils? because their granules have a strong affinity to the eosin component of the stains. Differential count:1-4% of the total leukocytic count. LM:  Nucleus: dense, bilobed, C-shaped.  Cytoplasm: contains large, shiny reddish granules. EM: (How do the eosinophils adapt to its function?)  The cytoplasm contains two types of granules: 1- Large specific granules: with electron dense crystalloid core), containing major basic protein and histaminase enzyme. 2- Small nonspecific azurophilic granules: containing lysosomal hydrolytic enzymes. Functions of the eosinophils: 1- They are responsible for regulation of allergic reactions (secrete histaminase). 2- In parasitic infections: killing of parasites. Killing Strategy 1- Go to battlefield: They circulate in the blood and then migrate to the connective tissue (especially mucosa of digestive, respiratory, genital, urinary & skin). 2- Identify the enemy. 3- The proteins in their specific granules act on the surface of the parasitic worm  forming pores  facilitating entry of lethal substances in the granules to the parasite  death of parasite. III-Basophils = mast cell of the blood: Why are they called basophils? Because their granules have strong affinity to the basic component of the stains. Why are they called mast cell of the blood? Because they secrete heparin and histamine like the connective tissue mast cells. Blood & Immune system Module Page 8 Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) Differential count: 0.5- 1 % of the total leukocytic count. LM:  Nucleus: bilobed, S-shaped, obscured by the abundant basophilic granules in the cytoplasm.  Cytoplasm: contains abundant basophilic granules  Basophils show a metachromasia when stained with toluidine blue (the granules appear red instead of blue because of their content of heparin). EM: (How do the basophils adapt to its function?)  The cytoplasm contains two types of granules: 1- Large specific granules: containing heparin and histamine. 2- Small nonspecific azurophilic granules: containing lysosomal enzymes. Function of basophils: Basophils are responsible for the release of histamine and heparin in systemic hypersensetivity allergic reaction. Cell type Neutrophil Eosinophil Basophil Differential 60-70% 1-4% 0.5-1% leukocytic count LM Nucleus Multilobed, Barr body in females. Bilobed C shaped. Bilobed S shaped. Cytoplasm Lightly stained. Shiny reddish Basophilic granules granules. obscuring the nucleus. Metachromatic staining. EM Small=collagenase Large=major basic Large= histamine, Specific granules Large protein, histaminase. heparin. Azurophilic Small Small granules Functions First line of defense against any 1-Activated in parasitic Involved in invading microorganism; microphage. infection. hypersensitivity reaction. 2- down regulate allergic reaction. Blood & Immune system Module Page 9  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) Granulopoiesis Promyelocytes:  Cytoplasm is more basophilic: as active protein synthesis occurs and the production of azurophilic granules begins. Myelocytes:  The specific granules for each type of the granulocytes start to appear in addition to the azurophilic granules.  The cytoplasmic affinity changes: it shows decreased basophilia and increased eosinophilia in neutrophils and eosinophils, whereas basophilia dominates in basophils.  This stage represents the last stage in which the cell can undergoes cell division, the next stages represent only morphological changes. Metamyelocytes:  The specific granules are more abundant.  The cytoplasm shows the staining affinity characteristic for each of the granular leukocytes.  Specifically, the neutrophils present by the end of these stage with an additional phase known as the band cells: 1- immature neutrophils 2- with a band-shaped nucleus. 3- They are 0-3% in the blood. This percentage increases if there is an acute infection. Mature granulocytes:  The nucleus becomes segmented in the pattern characteristic for each type of the granulocytes. Blood & Immune system Module Page 10  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) Agranular Leukocytes I-Monocyte: Why are they called monocyte? Because they have a single, non-segmented nucleus. Differential count: 3-8 % of the total leukocytic count. LM:  Size: the largest leukocytes.  Nucleus: large, eccentric and kidney shaped or horseshoe.  Cytoplasm: abundant, pale basophilic and finely granular. EM: (How does the monocyte adapt to its function?)  Shape: has an irregular outline due to extension of pseudopodia  motility& phagocytosis  Cytoplasm: contains small dense azurophilic granules containing lysosomal hydrolytic enzymes. Function of monocytes: Monocytes circulate in the blood and then migrate to the different body tissues where they differentiate to specific tissue phagocytic cells that constitute the mononuclear phagocytic system:  CT: Macrophages.  Bone: Osteoclasts.  CNS: Microglia.  Liver: Von Kupffer cells.  In chronic infections: macrophages fuse with one another forming multinucleated giant cells.  Phagocytosis of foreign antigens. Killing Strategy: 1- Go to battlefield. 2- Differentiate into specific phagocytic cell. 3- Identify the enemy. 4- Phagocytosis.  Antigen presenting cells: they process antigens and present them to T lymphocytes. Monopoiesis 1- PHSCs. 2- MHSCs. 3- CFU-GEMM. 4- CFU-M. 5- Monoblast. Blood & Immune system Module Page 11  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) 6- Promonocyte: it has large indented nucleus & basophilic cytoplasm. It is the last cell capable of division. 7- Monocyte. II-Lymphocytes: Why are they called lymphocyte? Because they are the main cell population of the lymphoid organs. Differential count: 20-30 %of the total leukocytic count. Life span: for several months. The memory cells can live for several years. LM: Shape& size: rounded cells with variable diameter. Nucleus: large, dense and slightly indented. Cytoplasm: narrow rim of basophilic cytoplasm. EM:  Cytoplasm: small dense azurophilic granules containing lysosomal enzymes.  The cell coat: surface molecules acting as: A- Receptors for the antigen. B- The cluster of differentiation (CD markers)  classifying the lymphocytes into subtypes according to their functions. Types of lymphocytes: According to their size:  Large lymphocytes.  Medium sized lymphocytes.  Small lymphocytes: represents most of the circulating lymphocytes in blood. They represent the mature form of lymphocytes. According to the function: 1- T lymphocytes: small lymphocytes,60-80% of circulating lymphocytes. 2- B lymphocytes: small lymphocytes, 20-30% of circulating lymphocytes. Blood & Immune system Module Page 12  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) 3- Natural Killer cells: 5-10% of circulating lymphocytes. Lymphopoiesis 8- PHSCs. 9- MHSCs. CFU-LY 10- Colony forming unit- lymphocyte (CFU- Ly):  Some daughters of these cells: migrate during fetal and CFU-B CFU-T early postnatal life to the cortex of the thymus where they differentiate into colony forming unit -T lymphocyte.  The rest: remains in the bone marrow and differentiate to colony forming unit- B lymphocyte. 11- Colony forming unit- T lymphocyte: They divide in the thymus giving rise to T- lymphoblasts. T lymphoblasts divide to give immunocompetent T- lymphocytes (expressing specific surface receptors). Colony forming unit- B lymphocyte: They divide in the bone marrow giving rise to B- lymphoblasts. They divide to give immunocompetent B- lymphocytes (expressing specific surface receptors). The Immune System There are three main lines of defense mechanisms: I-Surface protective mechanisms: 1- Skin: constitutes a barrier to the entry of microorganisms. 2- The mucous surfaces: as the conjunctiva and oral cavity are protected by antibacterial lysozyme secreted in tears and saliva. 3- The acidic environment in the stomach and vagina: inhibits the growth of pathogens. 4- The ciliated respiratory epithelium: is protected by a layer of mucus which is continuously removed with the trapped particles. The first line of defense. When these defenses fail, the two other types of defense mechanisms are activated. II-Non-specific immune response (innate immune system): 1- The complement system: mediates a wide range of inflammatory processes. 2- The phagocytic cells. 3- Natural killer cells. Blood & Immune system Module Page 13  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) III-Specific immune response: This depends on the recognition of specific antigens by specific surface receptors on the immune cells. There are two types of specific immune responses:  Humoral immune response.  Cell mediated immune response. Cells of the immune system: All lymphocytes are similar in either light or electron microscopic pictures. They can be distinguished by immunohistochemical methods into 3 different types according to their surface receptors or surface markers:  T-Lymphocyte: Origin: HPSCs in bone marrow and then their daughter cells migrate to thymus. Development: in the thymus where they proliferate, mature, and acquire T cell receptors (TCR) on their surface specific for certain antigen. Site of action: T cells migrate to the peripheral lymphoid tissues (lymph nodes, spleen, and Peyer’s patches in the ileum) where they come into contact with the antigens When T cells are activated by the specific antigen, they proliferate and differentiate into four functional subtypes:  Cytotoxic T cells (killer cells): They recognize foreign cells and destroy them by release of perforins (create holes in the membrane of the target cells) and granzymes (induce apoptosis of the target cells).  T helper cells: they produce lymphokines that activate B and cytotoxic T lymphocytes.  T regulatory (suppressor cells): they decrease the activities of other T and B lymphocytes in order to suppress immune response.  T memory cells: long lived cells responding more rapidly and strongly upon subsequent exposure to the same antigen.  B-lymphocytes: Origin & development: the bone marrow where they proliferate, mature and acquire surface immunoglobulins receptors specific for certain antigen recognition. Site of action: B cells migrate to the peripheral lymphoid tissues. Blood & Immune system Module Page 14  Histology of the Blood, Immune & Lymphoid system(By Prof. Dr. Iman Nabil) where they come into contact with antigens. When they are activated by the specific antigen, they proliferate and differentiate into: Plasma cells: secrete antibodies (humoral immune response). B memory cells: long lived cells responding more rapidly and strongly upon subsequent exposure to the same antigen.  Natural killer cells: Origin: in bone marrow, from the same precursors of T and B cells. They lack the surface markers characteristic of B and T cells. They act nonspecifically to kill virally infected cells and malignant cells. Origin: the bone marrow. They recognize, phagocytose and present the foreign antigens to T lymphocytes. They include:  Macrophages.  Langerhans cells of the skin.  B lymphocytes.  Epithelial reticular cells of the thymus. Blood & Immune system Module Page 15