II. MYELOID TISSUE. The blood forming tissue that consists of developing blood cells of all kinds, enmeshed in a reticular connective tissue framework surrounding blood sinusoids. Developing erythrocytes congregate around macrophages near the sinusoids. The macrophages extract iron from transferin that is brought to them by the blood. Development of granulocytes takes place more distant from the sinusoids.

A. Embryo and fetus - Blood formation begins in blood islands of yolk sac, then found in liver and spleen finally, by about 2 months gestation the bone marrow becomes the main site.

B. Adult - Myeloid tissue is mostly limited to flat adult bones where red marrow is found. When demand is high, yellow marrow as well as spleen and liver may be recruited.

C. Stem cells: It used to be debated as to whether the different kinds of blood cells required multiple stem cells or if there was a pluripotent stem cell. It is now generally accepted that there is a pluripotent stem cell that looks like a small lymphocyte and can differentiate into any blood cell. It expresses an antigen on its surface (CD34) that can be used to harvest it from peripheral blood. It gives rise to lymphoid precursors that go to lymphoid organs for differentiation while precursors of erythrocytes, megakaryocytes, monocytes, and granulocytes, remain in the marrow. These differentiate into stem cells with reduced potential. some for only erythrocytes, an erythrocyte colony-forming unit (E-CFU), others give rise to monocytes and granulocytes especially neutrophils(MG-CFU). Further differentiation gives rise to more specific precursors, those for monoctes only or for neutrophils. Eosinophil and basophil colony-forming cells arise directly from the myeloid multipotential cells.

III. ERYTHROCYTES

A. Morphology - easily deformed, enucleate, biconcave discs about 7 microns in diameter. The plasma membrane and fibrillar stroma retain the hemoglobin. Its cytoskelton consisting mostly of spectrin attached to integral proteins of the plasma membrane (glycophorins) is required to reestablish the biconcave shape after deformation.

B. Stages in formation (hematopoiesis). Requires about 5 days.

1. Proerythroblast - large cell about 25 microns in diameter; nucleus large with finely granular chromatin and nucleoli; cytoplasm contains many free polysomes, making it basophilic.

2. Erythroblast - Basophilic, actively mitotic; cytoplasm contains many free polysomes making it basophilic; the nucleus becomes progressively smaller and coarser textured. Polychromatophilic hemoglobin begins to be apparent. Orthochromatophilic hemoglobin is dominant, nucleus is pyknotic.

3. Reticulocyte - extrusion of the pyknotic nucleus of a normoblast leaves a reticulocyte which still contains some ribosomes. When supravitally stained, about 2% of the circulating erythrocytes are found to contain detectable clumps of ribosomes and mRNA, and are thus classed as reticulocytes. The reticulocyte continues to synthesize hemoglobin and loses its motility, mitochondria, and ribosomes as it matures. This requires 1 to 2 days.

4. Erythrocyte

C. Kinetics - The number of developing erythrocytes is about 1/30 the number of mature circulating cells that have a life span of about 120 days. Each stage of development takes about 1 day, so it takes 5 days from proerythroblast to erythrocyte. There is no reserve of erythrocytes in the myeloid tissue.

LEUKOCYTES

I. CLASSIFICATION AND CYTOLOGY IN PERIPHERAL BLOOD SMEARS.

A. Agranular - formed in lymphoid tissue and in marrow.

1. Lymphocytes - 20 to 45% of leukocytes; 7-8 microns up to 12 microns.

a. Morphology - nucleus is round to oval, chromatin is dense but fuzzy. The cytoplasm is blue to blue-green, gray. Azurophilic granules are present, especially in large forms.

2. Monocytes - 3-8% of leukocytes; 12-15 microns (20 microns).

a. Morphology - nucleus is ovoid to kidney shaped (folded). Chromatin is fibrous in appearance. Cytoplasm is blue-gray with azurophilic granules and sometimes vacuolated.

B. Granular - formed in bone marrow (myeloid tissues).

1. Neutrophilic polymorphonuclear leukocytes, PMNs, neutrophils, "Polys."

a. Proportion - 50-70% of leukocytes; Size - 9-12 microns

b. Morphology - nucleus is dense and lobulated; horseshoe shaped when the cell is young. Cytoplasm is pale pink; many poorly stained neutrophil granules and a few azurophilic granules (lysosomes).

2. Eosinophils.

a. Proportion - 2-4% of leukocytes; Size - 8-10 microns

b. Morphology - nucleus is usually bilobed the cytoplasm is filled with large, uniform eosinophil granules.

3. Basophils.

a. Proportion 0.5% of leukocytes

b. Morphology - large, irregularly-shaped basophil granules obscure the nucleus

C. Platelets (Thrombocytes); 150,000-300,000 per cubic millimeter. They are disks 2-4 micrometers in diameter

1. Come from megakaryocytes in myeloid tissue. These very large cells have a polyploid nucleus resulting from several cycles of amitosis. The plasma membrane forms clefts (demarcation channels) that release fragments of cytoplasm (platelets) through gaps in the sinusoid endothelium..

2. Platelets have a central dark stained chromomere (granulomere) surrounded by a light blue hyalomere where a bundle of microtubules maintains the discoid shape. The granules in the granulomere differ in their content. Some are lysosomes, some contain growth factor and some store bioactive substances such as serotonin and ATP.

3. Participate in initiation of clotting, clot retraction and clot resolution.

II. STUDY OF LEUKOCYTES

A. Counting - total WBC and differential 6,000-10,000 per cubic millimeter

1. Total concentration of leukocytes is obtained by manual counting using a hemocytometer or by using an automatic particle counter.

2. Differential count - count all cells encountered (100-500). Determine proportion in each class of leukocytes.

3. Suffixes - -penia = too few; -cytosis too many

4. Exercise - increases PMNs due to mobilization of marginated cells. It also increases lymph flow and this returns lymphocytes to the blood.

B. Separation of leukocytes

1. Buffy coat - leukocytes settle slower than erythrocytes.

2. RBC sedimentation; rouleaux formation speeds up settling; dextran causes clumping of RBC.

C. In vivo

Labeled leukocytes used to study turnover and fate of leukocytes. PMN's 6-8 days in marrow, 10 hours in blood; some lymphocytes are long-lived; others live for only a few weeks or months.

III. FORMATION - LEUKOPOIESIS IN MYELOID TISSUE (MARROW).

A. Agranular leukocyte development

1. Lymphopoiesis - Large, medium and small lymphocytes are described. The large are typical blast cells with finely textured chromatin in the nucleus and basophilic cytoplasm (free polysomes). These cells divide, get smaller and the nucleus becomes more condensed until only a thin rim of cytoplasm remains around a dense nucleus. They migrate out of the marrow to populate the thymus where they differentiate into T cells or they remain in the bone marrow and differentiate into B cells. All lymphocytes have a few azurophil granules, lysosomes. When a small lymphocyte is stimulated to enlarge and proliferate, it is said to undergo blastogenic transformation.

2. Monocytopoiesis - Monocyte formation from the monoblast (M-CFU) goes through a stage recognized as a promonocyte. Mature monocytes circulate in the blood for about 8 hours before they enter the tissues to serve as part of the mononuclear phagocytic system.

B. Stages in granulocyte formation.

1. Myeloblast - 15 microns; oval nucleus with nucleoli, no cytoplasmic granules. They are similar to the proerythroblast.

2. Promyelocyte - larger; azurophilic granules (lysosomes) increase in number as cell gets larger and divides

3. Myelocyte - specific granules appear and the cells from this point on are classified as neutrophil, eosinophil or basophil. The nucleus is oval and more dense

4. Metamyelocyte - also called a juvenile cell, has an indented nucleus. In the later stages, just before the nucleus becomes lobulated, it is deeply indented and is called a band cell.

5. Segmented granulocyte - this is the mature form. The nuclei of neutrophils may have 3-5 lobes while eosinophils have only 2. Because the nucleus of the basophil is obscured by the granules its segmentation is difficult to discern. In females, the nucleus of netrophils have a small projection that contains one of the X chromosomes (Barr body). It is heterochromatic (inactive) but not always seen.

C. Kinetics - Explain larger numbers of developing granulocytes in marrow than erythrocytes. Granulocytes, especially neutrophils, have a short life-span and there is a need for a reserve that can be rapidly mobilized.

1. PMN - 6-10 hours in blood and 3-4 days in the tissues. There are 15X more mature and band cells in the marrow than in the circulation.

2. Eosinophils 200-300 in marrow for each one in blood.

IV. FUNCTIONS OF LEUKOCYTES

A. Lymphocytes

1. Thymic or T-lymphocytes differentiate in thymus from precursor cells derived from the bone marrow. They are long-lived cells that have to do with immunological memory and cellular immunity and comprise 75% of circulating lymphocytes. They recirculate.

2. "Bursal" or B-lymphocytes - in mammals they differentiate directly in fetal and adult bone marrow. In birds they differentiate in the Bursa of Fabricius near the cloaca and thus their name. They populate the secondary lymphoid organs and when activated transform into plasma cells and produce antibodies.

3. A very small population of cells that look like small lymphocytes can actually be the source of all blood cells. They are the pluripotent stem cells.

B. Monocytes

1. Large, slowly moving phagocytic cells (macrophages); azurophilic granules are lysosomes which contain peroxidase.

2. Phagocytic activity makes these cells particularly suited to wall off and digest TB bacilli and to remove injured and dead tissues (apoptotic bodies)..

3. Become macrophages (histiocytes) in tissues - also foreign body giant cells; Mononuclear phagocytic (reticuloendothelial) system includes tissue macrophages, liver and spleen macrophages, macrophages in lymphoid tissues and the microglia of the CNS. Antigen presenting cells are derived from this population. They present to T cells antigenic fragments of foreign molecules on their surface complexed with major histocompatability complex antigens (MHC II). Osteoclasts are thought to be derived from these cells or, more likely, from a precursor which is common to these cells.

C. Neutrophils

1. Microphages - They migrate out of the blood through post capillary venules and phagocytose and kill invading bacteria. As a result they die and accumulate at the site forming pus.

2. Adherence to endothelial cells (first stage in migration out of blood stream) is reduced by aspirin, coffee, and ETOH

3. Chemotaxis is directed motion, not change in speed; AMP gradient; factors from lymphocytes cause chemotaxis. There is a hereditary defect in chemotaxis in some severe forms of periodontal disease.

4. Phagocytosis is actin dependent

5. Killing - result of the action of two types of granules supported by reactive oxygen arising from an oxidative burst. These give rise to superoxide, hydrogen peroxide and hypochlorous acid.

a. Neutrophil granules - contain alkaline phosphatase, lysozyme, phagostatin, collagenase, and lactoferrin and are the first to fuse with a phagosome.

b. Azurophil granules - add myeloperoxidase and lysosomal enzymes to the phagosome.

D. Eosinophils

1. Mobile

2. Numbers increase in allergy and parasitic infections; decrease with corticosteroid, which inhibits release from marrow and enhances diapedesis.

3. Eosinophil granules are special lysosomes that contain histaminase that breaks down histamine and in their crystaline inclusion major basic protein that attacks parasitic worms. These cells digest antigen-antibody complexes and slow reacting substance (SRS-A) released by mast cells.

4. Azurophil granules are typical lysosomes.

E. Basophils

1. Similar to mast cells but may contain chondroitin sulfate instead of heparin. May have a common precursor.

2. Function - similar to mast cells, but have capacity to migrate to sites of allergic inflammation.

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