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Histology of Blood tissue

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1 Histology of Blood tissue
Dr. Nabil Khouri 1

2 Learning Objectives Be able to recognize all of the formed elements found in peripheral blood by light. Know the approximate abundance and life span of the formed elements. Understand the general functions of of the formed elements and major plasma proteins. Be familiar with the general process of hematopoeisis. Describe the organization of the bone marrow.

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5 Major Plasma Proteins Protein Function
Albumin Maintain colloid osmotic pressure; transport insoluble metabolites Globulins  and  Transport metal ions, protein bound lipids, lipid-soluble vitamins  Antibodies for host defense Complement proteins Destruction of microorganisms Clotting factors Formation of blood clots Plasma lipoproteins Transport of triglycerides and cholesterol to/from liver

6 Cells of the blood Erythrocytes (red blood cells, RBC)
Platelets (thrombocytes) Leukocytes (white blood cells, WBC) Granulocytes (with specific granules) Neutrophil (~60% of WBC) Eosinophil (~4% of WBC) Basophil (<1% of WBC) Agranulocytes (without specific granules) Lymphocyte (B-cell, T-cell) (~27% of WBC) Monocyte (~8% of WBC)

7 Blood smear procedure Remember that the cells you see in a blood smear have not been sectioned. Instead you are seeing whole cells dried down on the glass After the smear is made, it is air-dried and then stained. Common stains are Wright's stain and Giemsa stain. The stains generally include two or more dyes, one of them a basic dye (often methylene blue) and another an acidic dye (usually eosin). Reddish-blue azures are formed when methylene blue is oxidized. Cells usually stain pink/red with acidic dye and nuclei stain purple/black with basic dye, while specific granules stain characteristically. . Procedure for preparing a blood smear. From Junqueira's histology text, 6th ed., page BloodSmear-23J91(2).tif. Original Source: Junqueira's histology text, 6th ed., page BloodSmear-23J91(2).tif.

8 Human blood smear, with RBCs, WBCs and platelets
Human blood smear at low magnification. The field is covered with red blood cells (RBCs = erythrocytes --note their light centers, since the biconcave shape makes the centers thinner). There are two white blood cells (WBC) in the field, a neutrophil at left, and a lymphocyte at right (the lymphocyte's cytoplasm appears reddish but should be light blue). Platelets occur as very small bluish clumps seen occasionally between the RBCs. AKC. From "Misc" slide collection, 1x3" glass slide labeled "Blood, Man, May-Grunwald Giemsa" (of the two slide labeled that way, this label has two black spots).

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10 Erythrocyte (red blood cell, RBC)
Life span in blood: About 120 days. Size and shape: biconcave disk, 8 µm diameter, 2m at thickest point, 1 m at thinnest The shape is maintained by a cytoskeletal complex inside the plasma membrane (involving spectrin, actin and other components) flexible: RBC’s normally bend to pass through small capillaries LM appearance in smear: Pink circle with light center (center is thinner because of the biconcave shape). NO NUCLEUS. TEM appearance: Solid dark gray cytoplasm, because of highly concentrated hemoglobin. Function: Transport of oxygen and carbon dioxide bound to hemoglobin (oxyhemoglobin and carboxyhemoglobin)

11 Red blood cells in a blood smear
RBC Red blood cells (erythrocytes) in a human blood smear. A small group of platelets is in the center of the field. Giemsa stain. Platelet

12 RBC, transmission electron microscopy
Platelet Electron micrograph of red blood cells and a platelet. Inside the red blood cells is a solution of hemoglobin so concentrated that it appears essentially solid and homogeneous in electron micrographs. The platelet contains dense granules, dense tubular system, and at the two ends (in this section) you can see cross sections of microtubules that form a ring around the periphery of the platelet disk (in 3 dimensions, rather than the 2 dimensions of this EM section).

13 RBCs, scanning electron microscopy
Junqueira's Basic Histology, 10th edition, page 235

14 RBC Cytoskeleton and Membrane-Associated Proteins
Hereditary spherocytosis: defective spectrin; RBCs are fragile and destroyed in spleen leading to anemia. A,B,O blood antigens: antigenic carbohydrate chains on extracellular domain of glycophorins Rh antigen: multipass integral membrane protein (similar to band 3), also comprises a blood group Red blood cells (erythrocytes) maintain a consistent biconcave shape, and yet must be very flexible, capable of bending to pass through small capillaries. These characteristics are produced by cytoskeletal complexes on the inner face of the plasma membrane. For details, see the legend of this diagram. From Ross' Histology, 4th edition, page RBCmemb-Ross4-219.tif.

15 Platelets (thrombocytes)
Life Span: about 10 days Shape, size, and origin: Small, biconvex disks, 2-3 µm in diameter. Non-nucleated cell fragments derived from cytoplasm of a very large cell, the megakaryocyte, in bone marrow. Platelets have a life span of about 10 days. LM appearance in smears: Small basophilic fragments, often appearing in clusters. Function: Platelets initiate blood clots (stop bleeding). Not uncommonly, trauma victims, whose platelets are lost due to bleeding and related processes, require platelet transfusions. Also patients battling leukemia and other cancers – especially if they undergo transplantation with bone marrow (or related stem cell sources) – will require transfusions to make up for their platelets that are destroyed by chemotherapy and/or radiation.

16 Platelets in aggregates

17 Platelets Electron Microscopy
Platelets Light Microscopy Platelets in a human blood smear. Giemsa stain. From the Mizobuti histology slide set (J). J LymSm-J-186.tif. Platelets Electron Microscopy

18 Transmission electron micrographs of a platelet seen in cross section (above) and in a section in the plane of the disk (below) A platelet is shaped like a biconvex disk. This figure shows two electron micrographs of the disk, one cut in cross section (above), and the other cut in the plane of the disk (below). granule membrane tubule Fawcett's Histology, 11th edition, page 118.

19 Structure of a platelet
Peripheral microtubule bundle (maintains shape) Actin and myosin (clot contraction) Organelles facilitate clotting: Mitochondria for ATP production Granules contain clotting factors Dense tubular system sequesters Ca++ for signaling (similar to SR in skeletal muscle) Open canalicular system facilitates signaling and secretion

20 Platelets and blood clot formation
When a blood vessel wall is damaged, factors from the damaged endothelial cells and the ECM induce the clotting cascade. Platelets aggregate and release proteins for clot formation and resolution: 1. Vasoconstriction –via release of serotonin 2. Further platelet aggregation –mediated via thromboxane A2 and ADP 3. Fibrin polymerization –initiated by thromboplastin and free Ca++ 4. Clot contraction –via actin, myosin, and ATP released into the matrix of the clot 5. Clot resolution –platelet plasminogen activator (pPA, converts plasminogen into active fibrinolytic plasmin) 6. Tissue repair –platelet derived growth factor (PDGF, stimulates smooth muscle and fibroblast proliferation) Prothrombin thrombin Thrombin Fibrinogen Fibrin Fibrin polymerization thromboplastin Ca++

21 Leukocytes (white blood cells, WBC)
Granulocytes (with specific granules) Neutrophil (~60% of WBC) Eosinophil (~4% of WBC) Basophil (<1% of WBC) Agranulocytes (without specific granules) Lymphocyte (B-cell, T-cell) (~27% of WBC) Monocyte (~8% of WBC)

22 Neutrophil (polymorphonuclear leukocyte)
Life Span: < 1 week Granulocyte with specific and non-specific granules LM appearance in smear: About 9-12 µm in diameter (thus larger than RBC). Nucleus long and multi-lobed (usually 2-4 lobes). The Cytoplasm has small, neutrally stained specific granules. Non-specific granules are azurophilic. Function: Primarily antibacterial Neutrophils leave the blood and follow chemotaxic signals to sites of wounding or other inflammation, and phagocytose foreign agents such as bacteria. Pus is composed largely of dead neutrophils. Specific granules Type IV collagenase (aids migration) Lactoferrin (sequesters iron) Phospholipase A2 (leukotriene synthesis) Lysozyme (digests bacterial cell wall) Non-specific granules (lysosomes) Lysozyme Acid hydrolase Myeloperoxidase Elastase

23 Two neutrophils in a blood smear
Two neutrophils (polymorphonuclear leucocytes, PMN) in a human blood smear. Note that their diameter is larger that of red blood cells. The nucleus is long, and thins out ins some places, producing lobes (each of these cells has 2 lobes, but there are often 3 or 4). In the cytoplasm are indistict small granules that take the neutral stain (neither strongly basic nor acidophilic). Mizoguti slide set (J). J-196. LM appearance in smear: About 9-12 µm in diameter (thus larger than RBC). Nucleus long and multi-lobed (usually 2-4 lobes). Cytoplasm has small, neutrally stained specific granules. Non-specific granules are azurophilic.

24 Neutrophil, transmission electron micrograph
Specific granule Lysosome (=azurophilic granule) TEM appearance: Multi-lobed nucleus and numerous specific granules and lysosomes (=azurophilic granules in LM). Electron micrograph of a neutrophil (polymorphonuclear leucocyte, PMN). The strand connecting two lobes of the nucleus is included in the section. Note the small specific PMN granules in the cytoplasm. The larger granules are lysosomes (="azurophilic granules"), while smaller ones are specific granules (contents including lysozyme and alkaline phosphatase).

25 Neutrophil antibacterial activity
Chemotaxis and migration (chemokine synthesis and matrix proteolysis) Phagocytosis and bacterial destruction Digestion via lysozymes Production of reactive oxygen compounds (respiratory burst) Iron sequestration via lactoferrin Release factors to increase inflammatory response (and increase neutrophil production) superoxide hydrogen peroxide hypochlorous acid NADPH oxidase* superoxide dismutase myeloperoxidase O2 O2- H2O2 HOCl Cl- *deficiency increases risk of persistent bacterial infections Neutrophils migrate to a site of tissue injury and infection, in order to phagocytize and digest bacteria and other noxious agents. This diagram shows the process of phagocytosis. See the legend for details. Pus in a healing wound is mostly dead neutrophils.

26 Eosinophil Life Span: < 2 weeks
Granulocyte with specific and non-specific granules LM appearance in smear: About µm in diameter. Bi-lobed nucleus. The cytoplasm has prominent pink/red specific granules (stained with eosin dye). If the smear is not stained properly, the granules may be brownish. Function: Anti-parasitic activity Mediators of inflammatory/allergic responses in tissues Inactivate leukotrienes and histamine secreted by basophils Engulf and sequester antigen-antibody complexes Inflammatory stimulus increases production/release of eosinophils from bone marrow, whereas inflammatory suppression decreases eosinophil numbers in peripheral blood. Specific granules Major basic protein Eosinophilic cationic protein Neurotoxin Histaminase Non-specific granules (lysosomes) Lysozyme Acid hydrolase Myeloperoxidase Elastase

27 Eosinophil in a human blood smear
Eosinophil in a human blood smear. The nucleus is bilobed. The cytoplasm is filled with reddish granules of relatively uniform size. If the smear is improperly stained, the granules may be brown. Image from the 86X normal blood smear slide from University of Michigan Virtual Slide Collection University of Michigan Virtual Slide Collection LM appearance in smear: About µm in diameter. Bilobed nucleus. The cytoplasm has prominent pink/red specific granules (stained with eosin dye). If the smear is not stained properly, the granules may be brownish.

28 Eosinophil, transmission electron microscopy
internum externum TEM appearance: The specific granules are ovoid in shape, and contain a dark crystalloid body composed of major basic protein (MBP), effective against parasites. The rest of the granule contains other anti-parasitic substances and histaminase. The cytoplasm also contains lysosomes (=azurophilic granules). Electron micrograph of a human eosinophilic leucocyte. Note the large specific granules,each containing a dense crystalloid body. The crystals contain major basic protein (MBP), important in combatting parasites.

29 Basophil Life Span: 1-2 years (?)
Granulocyte with specific and non-specific granules 2. LM appearance in smear: About 8-10 µm in diameter. The cytoplasm contains large, purple/black specific granules (stained with the basic dye) that are larger but not as numerous as those of eosinophils. The nucleus is usually bilobed, but usually is partially obscured by granules, which can lie over it. Function: Allergies and anaphylaxis (hypersensitivity reaction) Binding of antigens to membrane-bound IgE antibodies induces degranulation of specific granules, which leads to allergic reaction. In hypersensitivity reaction, widespread vasodilation (arteriolar) and vessel leakiness induce circulatory shock. Bronchial spasms cause respiratory insufficiency; combined effect is anaphylactic shock. Similarity to tissue mast cells: Tissue mast cells also have IgE receptors and similar (though not identical) granule content. Mast cells and basophils have a common precursor in bone marrow. Specific granules Histamine Heparin Eosinophil chemotactic factor Phospholipids for synthesis of leukotrienes, e.g. slow-reacting substance of anaphylaxis ( SRS-A ) Non-specific granules (lysosomes) Lysozyme Acid hydrolase Myeloperoxidase Elastase

30 Comparison of basophil and eosinophil in a blood smear
Basophil and eosinophil, with surrounding red blood cells, in a human blood smear. Wright's blood stain. Note that the basophil's granules are black or dark purple, large, scattered (some lying over the nucleus), and of somewhat variable shape. The eosinophil granules, on the other hand, are reddish, rather uniform in size, and are abundant, filling the cytoplasm. The nucleus of an eosinophil is usually bilobed, although the lower lobe in this one seems to have some subdivisions. The nucleus of a basophil appears large and of irregular shape, which can't be seen very well because of the granules. Blood smear from a U-M Histology course set (I ran on to this one day teaching blood in the histology course, and subsequently photographed it). Eosinophil Basophil J.M. Velkey.

31 Basophil, transmission electron microscopy
Human basophil seen by electron microscopy. Granules are large, and contain histamine and other strong vasoactive mediators. In some of the granules you can see myelin figures, which usually result from phospholipids in the granule). Myelin figure Granule Erlandsen's slide set (MH). MH-2G2. TEM appearance: The specific granules vary in size and shape, and have occasional myelin figures (usually formed from phospholipids). The cytoplasm also has some lysosomes (=azurophilic granules).

32 Leukocytes (white blood cells, WBC)
Granulocytes (with specific granules) Neutrophil (~60% of WBC) Eosinophil (~4% of WBC) Basophil (<1% of WBC) Agranulocytes (without specific granules) Lymphocyte (B-cell, T-cell) (~27% of WBC) Monocyte (~8% of WBC)

33 Lymphocyte Life Span: variable (few days to several years)
LM appearance in smear: Small lymphocyte (about 90% of lymphocytes you will see) are ~8 µm in diameter, while Large lymphocytes may be up to about 15 µm. Round, dense nucleus (abundant heterochromatin). The cytoplasm of a small lymphocyte is a narrow rim around the nucleus, and when well stained is pale blue. T-lymphocytes and B-lymphocytes cannot be distinguished in a smear. Function: Cellular and humoral immunity (more detail in the lecture and lab on lymphatic system histology). In general: B-lymphocytes (B-cells): may differentiate into tissue plasma cells which make antibodies. Some B-cells become memory cells. T-lymphocytes (T-cells): cytotoxic T cells and helper T cells.

34 Small lymphocyte in a blood smear
Small lymphocyte in a human blood smear. Note the dense nucleus and the pale blue cytoplasm. The cell is not that much larger than the surrounding red blood cells. There are also some platelets at right. Giemsa stain. LM appearance in smear: Small lymphocyte (about 90% of lymphocytes you will see) are ~8 µm in diameter, while large lymphocytes may be up to about 15 µm. Round, dense nucleus (abundant heterochromatin). The cytoplasm of a small lymphocyte is a narrow rim around the nucleus, and when well-stained is pale blue.

35 Large lymphocyte in a blood smear
Large lymphocyte in a human blood smear. The cytoplasm is more extensive than in a small lymphocyte. This cell somewhat resembles a monocyte, but the nucleus here is darker than that usually seen in monocytes. LM appearance in smear: Small lymphocytes (about 90% of lymphocytes you will see) are ~8 µm in diameter, while large lymphocytes may be up to about 15 µm with ovoid, dense nuclei (abundant heterochromatin).

36 Electron micrograph of a lymphocyte
Mitochondrion Centriole Lymphocyte seen in an electron micrograph. The cytoplasm contains a few mitochondria, a centrioles, ribosomes (actually polysomes = ribosomes on mRNA) but few other organelles, and usually no granules. TEM appearance: The cytoplasm doesn't appear to be very active, containing mainly mitochondria and free ribosomes.

37 Tissue plasma cells (derived from B-lymphocytes)
Electron micrograph of a plasma cell. Plasma cells arise by the differentiation of B-lymphocytes. In the cytoplasm is an extensive rough endoplasmic reticulum (RER), which is involved in the synthesis and transport of antibody.

38 Monocyte Life Span: few days in blood, several months in connective tissue LM appearance in smears: About 16 µm in smears, thus the largest leukocyte. Large, eccentric nucleus either oval, kidney-shaped or horseshoe-shaped, with delicate chromatin that is less dense than that of lymphocytes. Pale cytoplasm, often grayish, may contain occasional stained granules (lysosomes = azurophilic granules). Large lymphocytes may resemble monocytes, but the lymphocyte nucleus is usually more dense. Function Migrate into tissues and constitute mononuclear phagocyte system that help destroy foreign bodies and maintain or remodel tissues and Mediate inflammatory response Tissue macrophages Kupfer cells (liver) Osteoclasts (bone), Dust cells (lungs),Microglia (brain), Antigen presenting cells: Dendritic Cells, Langerhans cells

39 Monocyte in a blood smear
Monocyte in a human blood smear. The nucleus is large, relatively pale and is bean-shaped (it is often oval). The cytoplasm is also pale, and some of the indistinct spots may be small lysosomes. Monocytes migrate from the blood into tissues and differentiate into various types of macrophages. Large lymphocytes may resemble monocytes, but the lymphocyte nucleus is usually darker. Giemsa stain. LM appearance in smears: About 16 µm in smears, thus the largest leukocyte. Large, eccentric nucleus either oval, kidney-shaped or horseshoe-shaped, with delicate chromatin that is less dense than that of lymphocytes. Pale cytoplasm, often grayish, may contain occasional stained granules (lysosomes = azurophilic granules). Large lymphocytes may resemble monocytes, but the lymphocyte nucleus is usually more dense.

40 Monocyte, transmission electron microscopy
Lysosome (=azurophilic granule) Mitochondrion Centriole Golgi Electron micrograph of a human monocyte. The cytoplasm contains mitochondria, a Golgi element, a section through a centriole, and small lysosomal granules (= azurophilic granules). Monocytes migrate from the blood to tissues, where they differentiate into macrophages of various kinds. TEM appearance: Cytoplasm contains mitochondria and some small lysosomes.


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