Blood Smears: Lymphocytes & Monocytes Note the differences between the nuclei
Blood within vessels of heartContains blood cells other thanerythrocytes
Blood within vessels of kidneyNotice how abundant blood cellsand vessels are in this highlyvascular organ.
General organization and components of bone marrow• Bone marrow is actually an organ comprised of different tissue components. It can be divided into a vascular compartment (40X) (arteries, veins, sinusoids) and a hemopoietic compartment (50X) (stroma of reticular tissue and free cells).
ErythropoesisDuring erythropoiesis, cell sizedecreases, the nucleus becomescondensed (and eventuallydiscarded), and the cytoplasm becomesincreasingly acidophilic.
ErythropoesisBasophilic erythroblast- Note the rim of intensely basophilic cytoplasmPolychromatophilic erythroblast- "checkerboard" nucleus and slate-colored cytoplasmNormoblast- The cytoplasm of these cells is decidedly acidophilic, due to loss of RNA and increase inhemoglobin. Look for normoblasts in the process of extruding pyknotic nucleiReticulocyte- Note that reticulocytes have the same appearance as mature erythrocytes in aconventionally-stained slide. When RNA specifically stained, you can distinguish between reticulocytesand mature erythrocytes.
Granulocytopoesis•The cells in these three lines of development can be readily identified by the presenceof pronounced cytoplasmic granules, and by the gradual lobation of the nucleus. Thestem cell is the same or similar to that for erythropoiesis.•You will only be responsible for identifying those granulocyte precursors that havespecific granules.•Note: Mature granulocytes have segmented nuclei.
Megakaryocytes & Platelet Formation •Megakaryocytes have multi-lobed nuclei •Located near sinuscytoplasmic extensions break off to form platelets
Monocytopoesis & Lymphocytopoesis• Development of the agranular leukocytes is difficult to follow at the light microscopic level. You will not be held responsible for identifying cell intermediates in these pathways (monocytes and lymphocytes).
Distinguishing Hemopoietic Stages• Erythropoiesis: – Basophilic erythroblast has basophilic/blue Basophilic and cytoplasm, while polychromatophilic erythroblast has “slate”-colored cytoplasm Poly. and a “checkerboard” pattern of granules Erythroblast – Normoblast has acidophilic/pink cytoplasm and can be seen in process of extruding nucleus• Granulocytopoiesis: Normoblast – In all stages • Eosinophilic= pink granules • Neutrophilic= neutral granules • Basophilic= blue granules – Myelocyte= round nucleus – Metamyelocyte= approaching band/stab cell curved nucleus – Band/Stab= U-shaped nucleus – Mature= segmented nucleus
Blood Clinical Correlation• Iron deficiency anemia: – Lack of adequate dietary iron results in abnormally low levels of the blood pigment hemoglobin (which binds to, and requires, iron to function). Low hemoglobin content results in smaller erythrocytes that have a thin rim of hemoglobin around their periphery• Sickle cell anemia: – Sickle cell anemia is a condition caused by a mutation in the gene that codes for hemoglobin. The pathological hemoglobin causes red cells to adopt a spiky, "sickle" shape• Chronic myelogenous leukemia: – In chronic myelogenous leukemia (CML), a mutation occurs that prevents cells of a particular line from maturing, but allows them to continue to self-renew, leading to a massive increase in their number. The immature myeloid cells (myeloblasts) accumulate in the marrow, where they replace normal elements. These abnormal cells often circulate in the peripheral blood. In the acute, "blast crisis" phase of this disease, large numbers of myeloblast-like cells appear in the peripheral blood.
Epidermis: Stratum Spinosum• this layer is formed as daughter cells from the stratum basale migrate toward the epidermal surface.• Identify the numerous artifactual intercellular "bridges" that give these cells a spiny appearance. The bridges are due to spot desmosomes
Epidermis: Stratum Granulosum• Not consistently present in thin skin, this layer (2-5 cells thick) contains darkly-staining cells.• Identify the keratohyalin granules (100X, 100X), which are responsible for the pronounced staining and granular appearance of this layer. The granules are not pigment, and are unrelated to the naturally pigmented melanin granules.• Examine keratinocyte nuclei (100X, 100X), which are beginning to degenerate in this layer.
Epidermis: Melanin Granules• Note the widespread distribution of orange-brown granules in cells of the stratum basale, and, to some extent, in cells of the stratum spinosum (50X, 100X).• Note that melanin granules are present throughout the thickness of the epidermis, but are more difficult to see in superficial layers.
Epidermis: Melanocytes• These cells (schematic) occur with a low frequency (the melanocyte/ keratinocyte ratio is 1/36), and are difficult to see by these staining methods.• Note that melanocyte cytoplasm is lighter than the cytoplasm of surrounding keratinocytes (blue arrows, 100X). The melanocyte secretes pigment-containing melanosomes that are then taken up by the epidermal keratinocytes and degraded to add pigment to their cytoplasm.
Dermis: Papillary Layer & Reticular Layer• Papillary Layer: – The papillary and reticular layers are most easily distinguished in a trichrome-stained section. – Examine this C.T. network of fine collagenous, reticular and elastic fibers and note that dermal papillae extend upward into the epidermis . What is the function of these structures• Reticular Layer: – In this layer (and often extending into the subcutaneous region) are hair follicles, sweat and sebaceous glands, and smooth muscle (see below).
Hypodermis• Note that adipocytes predominate in this subcutaneous layer (4X, 10X, 40X), which is not a part of the skin.• Identify Pacinian corpuscles (5x, 10X, 40X) in hypodermis (and deep dermis). What is their function? – Pacinian corpuscles= encapsulated mechanoreceptors especially adapted to detect pressure and vibration – Distinctive onion-like appearance due to tight Schwann cell wrapping around the bare sensory nerve ending
Sweat (Eccrine) Glands• These are found in both thick and thin skin. The type of secretion that occurs here is merocrine (eccrine). They are the only cutaneous glands in thick skin. Identify these unbranched, coiled, tubular, serous glands located in superficial hypodermis (4X, 20X, 20X, 50X).• Identify myoepithelial cells (40X, 50X), which are associated with secretory portions (best seen in the C.P.- Trichrome slide).• Try to find an excretory duct (40X) passing straight through the dermis, and emptying at the epidermal surface.• Note that the walls of sweat gland ducts are stratified cuboidal epithelium, with two layers of cuboidal cells (40X, 50X, 100X).
Apocrine Glands (in Axilla)• Note that both eccrine and apocrine glands (4X) are present here.• Note that apocrine glands usually extend deeper into the hypodermis than do eccrine glands (4X).• Note that the secretory portion of an apocrine gland is also coiled and tubular, but has a considerably wider lumen than that of an eccrine gland (20X).
Apocrine GlandsNote the difference between apocrine glands (left) and eccrine glands (right)
Sebaceous Glands• Sebaceous glands. Hair follicles are almost always associated with sebaceous glands (20X). The secretory product, sebum, is produced by holocrine secretion. Note that the secretory portion is comprised of stratified epithelial cells that crowd into, and occlude, the lumen (50X). Identify lipid- filled vacuoles in these cells.• Note that the excretory ducts are quite short and wide, with a stratified epithelium that is continous with the epithelial hair sheath (40X).
Hair Follicles• Components of Hair Follicle: – Germinal matrix epithelial cells Hair – Vascular connective tissue papilla bulb – Hair bulbs – Hair root – Medulla, cortex and cuticle of hair. – Internal and external root sheath
Arrector Pili Muscles• Note the presence of bundles of smooth muscle cells (10X, 20X) in fortuitous sections. What is the function of these structures? – Makes hair stand erect – Pulls skin in to form dimples – Both functions “goosebumps”
Nails• Components of a Developing (Monkey) Finger: – Nail, or nail plate: body and root – Nail bed: This is comprised of an epidermis without stratum granulosum or stratum lucidum, and the underlying dermis. – Nail matrix : epithelium – Eponychium: cuticle – Hyponychium: This structure secures the free edge of the nail plate at the fingertip.
Clinical Correlation• Squamous cell carcinoma of skin: – Squamous cell carcinoma is the most common type of sun-associated tumor, although it may also be caused by chewing tobacco, carcinogenic chemicals, or ionizing radiation. Such cells may have an epithelioid (flattened) shape, or they may be rounded (1X, 5X). Squamous cell carcinomas are commonly removed surgically before they metastasize.• Basal cell carcinoma of skin: – Basal cell tumors arise when cells in the basal layer of the epithelium of skin begin growing and dividing abnormally. The cells often grow in clumps (nodular lesions) surrounded by fibroblasts and lymphocytes (10X); the cells at the periphery of these islands are often arranged radially (40X). Basal cell carcinomas grow slowly and usually do not metastasize.
Clinical Correlation• Melanoma: – Malignant melanoma is a cancer that results from uncontrolled growth of melanocytes. It occurs most commonly in skin that has been heavily exposed to sunlight. Melanoma cells are large, have large nuclei, and can be found in any layer of the skin (5X, 40X). These cells have a high incidence of metastasis.• Bullous pemphigoid: – In this autoimmune disorder, antibodies attack one of the protein components ("bullous pemphigoid antigen") of the hemidesmosome (schematic; EM). Since the hemidesmosome anchors the epithelium to the underlying connective tissue, its destruction loosens that connection and results in the formation of blisters.
ThymusThe thymus, a central lymphoid organ, is the organ in which T-cell precursors differentiate to become immunocompetent T-lymphocytes capable of responding to antigen in the context of "self". The thymus is not a site where immune responses to foreign antigen occur, and thus it differs from the peripheral lymphoid tissues in structure and organization; specifically, it contains neither nodules nor germinal centers. It is a bi-lobed organ whose size and appearance varies with age.
Thymus: Overall Structure• Identify the connective tissue capsule and trabeculae that divide the thymus into lobules (4X).• Note the variation in lobule size.• Identify cortex and medulla of lobules (10X).• Note that lobules are not completely separated by trabeculae. Each lobe of the thymus contains a medullary core that extends into each lobule (4X).• Note the absence of lymphoid nodules and sinuses; you should be careful not to confuse circular regions of medulla with germinal centers.• Note that trabeculae are cortical structures; they do not extend into the medulla.
Thymic Cortex• Identify large, mitotic thymocytes (100X, thymic lymphocytes), and note that they decrease in size as the medulla is approached.• Identify reticular epithelial support cells (schematic, 100X); these have a stellate shape, and pale-staining nuclei with distinct nucleoli.• Note that, unlike other lymphoid organs, the thymus has an entirely cellular stroma; no reticular fibers are present. Green arrows= reticular epithelial support cells NOTE: These cells appear to be “outlined”
Thymic Medulla• Note that lymphocytes are less dense here (medulla is less stained than the cortex on previous slide…), so that reticular epithelial cells are more readily identified.• Identify reticular epithelial cells (100X), which have large, oblong nuclei.• Locate a Hassalls corpuscle (50X), and identify the concentric reticular cells that are hyalinized (100X, EM) and degenerated toward the center of the corpuscle.
Thymus: Blood Vessels• Note that only capillaries (50X) are present in the cortex. Epithelial reticular cells ensheath thymic cortical capillaries, thereby contributing to a blood-thymic barrier (schematic) that keeps the cortex free of circulating antigens. Epithelial reticular cells cover thymic cortical capillaries blood-thymic barrier
Diffuse Lymphoid Tissue• This tissue is often present as an infiltration of the lamina propria of mucous membranes, especially in gastrointestinal and respiratory tracts. Lymphocytes, plasma cells, macrophages and some other blood cells are scattered within the reticular connective tissue among the components of the lamina propria. Scan these slides of various regions of the digestive tract and locate diffuse lymphoid tissue (10X, 40X). Diffuse lymphoid tissue is particularly abundant in the lamina propria of colon. In all slides, identify:• Lymphocytes (100X). These are the most abundant cell type.• Macrophages (100X, lamina propria of small intestine). Both fixed and free macrophages are generally present.• Plasma cells (20X, 100X). You may also see these antibody-producing cells.
Lymphoid Nodules• Lymphoid tissue arranged in a dense, spherical aggregation is called a lymphoid nodule. These can occur as solitary nodules, as loosely-organized aggregations, or as part of an encapsulated lymphoid organ.
Solitary Lymphoid Nodules• Solitary One nodule on its own, at a distance from other nodules• Note that nodules can vary considerably in size.• Identify lymphocytes in the nodules.• Identify lymphoid nodules (4X, 10X, 10X, 10X) and secondary nodules (4X, 10X). Note that secondary nodules have lighter-stained germinal centers.• Note that nodules may be surrounded by diffuse lymphoid tissue.
Aggregate Lymphoid Nodules• Aggregate multiple nodules adjacent to one another• Examine this section for loose aggregates of lymphoid nodules. In the ileum, these characteristic aggregates may include 10-100 lymphoid nodules and are called Peyers patches (4X, 20X). They aid in the identification of this region of small intestine (to be studied later).• Identify lymphoid nodules and note secondary nodules
Peripheral Lymphoid Organ• A lymphoid organ is an aggregation of lymphoid tissue that has a capsule of dense connective tissue. Lymph nodes, spleen and thymus are lymphoid organs that have complete capsules; tonsils are partially encapsulated lymphoid organs.
Lymph Nodes: General Structure• Note the overall bean-like shape, with a hilus at the concavity.• Identify the dense irregular connective tissue capsule with septa (20X, 40X) or trabeculae extending into the organ.• Identify both afferent (4X, 10X, 40X) and efferent lymphatic vessels. This feature is unique to lymph nodes.• Identify both cortical and medullary regions.
Lymph Nodes: Cortex• Identify lymphoid nodules surrounded by diffuse lymphoid tissue.• Lymphoid nodules in their inactive state are primary nodules; secondary nodules are easily identified by their germinal centers.• Note that cells within the germinal centers (40X) are larger, and have paler-staining, more euchromatic nuclei (100X), than do the cells surrounding them.
Lymph Nodes: Medulla• Note that the lymphoid tissue is arranged here as cords (20X).• Identify medullary sinuses.
Lymph Nodes: Framework• Note that the capsule (50X) and trabeculae (50X) are composed of dense C.T.• Identify fibroblasts in the dense C.T. (50X, 50X)• Study the delicate reticular tissue (50X) of the stroma. Note the reticular fibers stained specifically on especially slide 0-45.• Examine the reticular cells (50X), which are responsible for producing reticular fibers. The reticular cells form a network of cell processes associated with the supporting reticular fiber network.• Note that the reticular framework extends into sinuses, as well as into nodules and cords (50X, 50X).• Identify fixed macrophages (100X).
Lymph Nodes: Sinuses• Lymph enters the node via afferent vessels, passes through the node via sinuses, and exits at the hilus via efferent vessels. The sinuses have discontinuous walls lined by reticular cells and fixed macrophages, allowing lymphocytes to move into sinuses, and, eventually, into efferent lymphatics.• Identify afferent lymphatics (4X, 10X, 40X) piercing the capsule at a number of sites.• Identify subcapsular sinuses (20X).• Identify cortical (trabecular) sinuses (20X).• Identify medullary sinuses (50X).• Identify efferent lymphatics at the hilus. See Medulla slide for medullary sinus
Lymph Nodes: Blood Vessels and Nerves• Identify arteries at the lymph node hilus that provide branches to medulla, cortex, and trabeculae.• Identify the dense network of capillaries (40X) in the medullary cords.• Note that the cuboidal endothelium of post-capillary (high-endothelial) venules (100X, 100X, 100X, animation) is the site where B- and T-lymphocytes leave the blood to enter the lymph stream. They eventually leave the lymph node via efferent lymphatics.• Locate nerves entering the lymph node at the hilus.
Tonsils• Although considered lymphoid organs by virtue of their partial connective tissue capsule, tonsils are not much more organized than Peyers patches. Three groups of tonsils form a ring of lymphoid tissue around the pharynx. All are aggregates of nodules embedded in diffuse lymphoid tissue. They have an invaginated surface epithelium and an underlying connective tissue capsule.
Palatine Tonsils• Identify lymphoid nodules (4X).• Note that the surface epithelium extends into deep invaginations, the tonsillar crypts (10X).• Identify lymphocytes invading the epithelium (40X).• What type of C.T. is present in the hemi-capsule (40X) that separates the tonsil from underlying muscle? – Fibrous connective tissue
Lingual Tonsils• These tonsils are actually aggregations of epithelial pits surrounded by lymphoid tissue.• Identify crypts (4X).• Identify surface epithelium (40X, 40X).• Identify nodules and internodular spaces (4X, 40X).• Examine the hemi-capsule (4X, 40X).
Pharyngeal Tonsils• Located in the nasopharynx, rather than the oropharynx, these tonsils differ somewhat from palatine and lingual tonsils.• Examine the surface invaginations (4X). These are actually longitudinal folds, rather than crypts.• Identify the surface epithelium (20X, 40X).• Identify nodules (4X).• These tonsils have a hemi-capsule, although the capsule is not visible in these particular slides because it was not dissected along with the specimen.
Spleen: Overall Structure• Identify the capsule and branching trabeculae (4X).• Distinguish white pulp and red pulp. Identify splenic nodules (10X) within the white pulp.
Spleen: Capsule and Trabeculae• Note the presence of blood vessels (10X) in the trabeculae.• Within the connective tissue, identify elastic fibers (100X) and smooth muscle cells (20X, 100X).
Spleen: Reticular Framework• Slide E-47, with reticular stain, demonstrates the presence of a delicate reticular tissue stroma, upon which free cells are supported.
Spleen: White Pulp• Identify lymphocytes (100x).• Locate a secondary lymphoid nodule in the white pulp. Identify the central artery/arteriole (20X, 20X, 100X). The nodule is actually a local expansion of the lymphoid ensheathment of the artery.• Note the eccentric placement of the central artery (20X, 40X) in those nodules that have a germinal center. All secondary lymphoid nodules, which are activated for a humoral response, have germinal centers.• Identify the Periarteriolar Lymphoid Sheath (PALS) of lymphocytes surrounding the central artery (20X, 20X, 20X, 40X, 100X). T-lymphocytes principally surround the central artery, while B-lymphocytes are primarily located in the secondary nodules with germinal centers (100X). A germinal center is a site of B- lymphocyte proliferation.• Why is white pulp called white pulp? – White pulp contains lymphocytes (WHITE blood cells) and can be distinguished from red pulp, which contains sinusoids filled with RED blood cells. B-lymphocytes T-lymphocytes
Spleen: Red Pulp• Two components are present in red pulp: venous sinuses and pulp cords.
Red Pulp: Pulp Cords (aka Cords of Bilroth)• These structures consist of a spongy network of cells arranged on the reticular stroma. Identify the principal cell types (40X, 100X, 100X) present in the cords (20X): lymphocytes, macrophages (40X, 100X), plasma cells (100X), and erythrocytes.• Note that red pulp cords gradually merge into white pulp.• Note that lymphocytes (of all sizes) are less numerous in red than white pulp (20X, 40X).• Note that the pulp cords contain a large population of highly phagocytic cells (macrophages; 100X). What do these cells do here? – Macrophages destroy aged/damaged RBCs that move freely through the cords and venous sinuses.• Why is red pulp called red pulp? – Much less lymphocytes than white pulp; greater number of RBCs
Splenic Phagocytosis• Slide O-18 demonstrates the presence of phagocytic cells in spleen (100X) by uptake of India ink (only phagocytic cells can sequester the ink, which is made up of tiny carbon particles).
Red Pulp: Venous Sinuses• These lie between pulp cords (20X, 100X). Note that they are lined by endothelial cells that form a discontinous sinus wall (100X, 100X, EM).• Study the contents of the venous sinuses.
Spleen: Blood Circulation• You should make sure that you can identify the following splenic structures in your slides:• Splenic artery.• Trabecular arteries.• Central arteries of white pulp.• Penicillar arterioles of red pulp (with pulp arteriole, sheathed arteriole, and terminal arterial capillary).• Venous sinuses.• Pulp veins.• Trabecular veins.• Splenic veins.
Clinical Correlation1. Follicular lymphoma (HD047). A follicular lymphoma is a tumor of lymphoid cells (most often B-cells), in which the cells cluster into nodules that resemble normal lymphoid follicles. All lymphoid tumors derive from a single transformed cell, so they are monoclonal, which means that all of the tumor cells are identical. As it fills with abnormal lymphocytes, the lymph nodes normal structure is destroyed (1X).2. Hodgkin lymphoma (HD045). Hodgkin lymphoma is a lymphoid cancer that begins as an enlargement of a single node, then spreads to neighboring nodes. Its most characteristic feature is the giant, neoplastic Reed-Sternberg (RS) cell. This form of the disease (Nodular Sclerosis) shows a modified form of the RS cell, the lacunar cell (5X, 40X). 40x 5x
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