Lab 8 – Cells and Hemopoiesis IUSM – 2016

I. Introduction Blood Cells and Hemopoiesis II. Learning Objectives III. Keywords IV. Slides A. Blood Cells 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) a. (PMNs) i. ii. iii. b. (Mononuclear) i. Lymphocytes ii. 3. Thrombocytes () B. 1. General structure 2. Cells a. b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors V. Summary SEM of a (purple) ingesting S. aureus bacteria (yellow). NIAID. Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 Blood I. Introduction II. Learning Objectives 1. Blood is a specialized type of fluid connective III. Keywords tissue that provides the body’s tissues with IV. Slides nutrition, oxygen, and waste removal and serves A. Blood Cells as a means of transportation for the activity of 1. Erythrocytes (Red Blood Cells) other body systems (e.g., carrying hormones 2. Leukocytes (White Blood Cells) from source to target for the endocrine system). a. Granulocytes (PMNs) 2. It consists of plasma (liquid ECM of blood) and i. Neutrophils formed elements (cells and platelets). ii. Eosinophils iii. Basophils 3. The “formed elements” of blood derive from b. Agranulocytes (Mononuclear) hematopoietic stem cells located in the red bone i. Lymphocytes marrow of flat bones in adults. ii. Monocytes 4. Blood cells can be classified as red blood cells 3. Thrombocytes (Platelets) (about 45% of blood) and white blood cells B. Bone Marrow (about 1% of blood) based upon their gross 1. General structure appearance upon centrifugation. 2. Cells a. Megakaryocytes 5. White blood cells (leukocytes) can be further classified as granulocytes (neutrophils, basophils, b. Hemopoietic cells eosinophils) or agranulocytes (monocytes and i. Erythroid precursors lymphocytes) based upon the presence of ii. Myeloid precursors absence of cytoplasmic secretory granules. V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 I. Introduction Classifying Blood Cells II. Learning Objectives III. Keywords Cells of the blood can be classified using several different schema depending upon what IV. Slides relationship between the sets is considered important or to be emphasized: A. Blood Cells 1. Erythrocytes (Red Blood Cells) 1. Gross appearance: upon centrifugation, blood cells tend to separate into a red-colored 2. Leukocytes (White Blood Cells) band and a white-colored band (“buffy coat”); this gross distinction allows for cells to be a. Granulocytes (PMNs) considered either red blood cells (RBCs) or white blood cells (WBCs). i. Neutrophils ii. Eosinophils 2. Location: blood cells can be categorized based upon where they are generally found in iii. Basophils the body, such as in the peripheral vasculature or only in the bone marrow, or more specific b. Agranulocytes (Mononuclear) locations, such as for lymphocytes. i. Lymphocytes ii. Monocytes 4. Microscopic appearance: microscopic examination of blood cells allows categorization based upon particular structures, such as the presence of cytoplasmic specific granules 3. Thrombocytes (Platelets) (i.e., granulocytes vs agranulocytes), organelle morphology (e.g., polymorphonuclear or B. Bone Marrow mononuclear nucleus), or based upon staining characteristics (i.e., neutrophil, , 1. General structure and ). 2. Cells a. Megakaryocytes 5. Lineage: blood cells can be categorized depending upon their lineage relationship with b. Hemopoietic cells other blood cells; all blood cells derive from hematopoietic stem cells (HSCs) but soon split i. Erythroid precursors into branching lineages based upon common progenitor cells (i.e., myeloid vs. lymphoid). ii. Myeloid precursors V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 I. Introduction Identifying “Formed Elements” II. Learning Objectives III. Keywords IV. Slides Since some “blood cells” are not actually cells when functional, the term “formed elements” is often used to describe the “cellular” content of blood. When evaluating the formed elements A. Blood Cells of blood, there are several characteristics to keep in mind when attempting to identify the 1. Erythrocytes (Red Blood Cells) type of “cell”: 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) 1. Location the sample was taken from (if known) i. Neutrophils ii. Eosinophils 2. Size of the element iii. Basophils b. Agranulocytes (Mononuclear) 3. Nucleus to cytoplasm volume ratio (N:C) i. Lymphocytes ii. Monocytes 4. Number and shape of nuclei 3. Thrombocytes (Platelets) 5. Degree of chromatin condensation B. Bone Marrow 1. General structure 6. Presence or absence of nucleoli 2. Cells a. Megakaryocytes 7. Cytoplasmic staining b. Hemopoietic cells i. Erythroid precursors 8. Presence and staining of cytoplasmic granules ii. Myeloid precursors V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Learning Objectives II. Learning Objectives III. Keywords IV. Slides 1. Understand that blood is a connective tissue with “formed elements” A. Blood Cells (cells and platelets) in a fluid matrix (plasma). 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) 2. Understand the morphology and function of red blood cells a. Granulocytes (PMNs) (erythrocytes) and the role of the spectrin membrane skeleton in i. Neutrophils maintaining their biconcave shape and flexibility. ii. Eosinophils iii. Basophils 3. Understand the relative numbers of the various types of white blood b. Agranulocytes (Mononuclear) cells in the blood of normal adults and know the major functions of i. Lymphocytes each type. ii. Monocytes 3. Thrombocytes (Platelets) 4. Understand that all leukocytes display cell motility and function in B. Bone Marrow secretion, , etc. primarily in the ECM of tissues after 1. General structure migrating across the blood vessel wall (diapedesis). 2. Cells a. Megakaryocytes 5. Be able to recognize and find neutrophils, eosinophils, basophils, b. Hemopoietic cells monocytes, and platelets in a blood smear. i. Erythroid precursors ii. Myeloid precursors V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Learning Objectives (cont.) II. Learning Objectives III. Keywords IV. Slides 6. Understand and recognize the compartments and tissues in blood A. Blood Cells marrow. 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) 7. Understand the development sequences and recognize the a. Granulocytes (PMNs) intermediate cells in the formation of red blood cells and the three i. Neutrophils types of granulocytes. ii. Eosinophils iii. Basophils 8. Be able to recognize megakaryocytes and understand how platelets b. Agranulocytes (Mononuclear) are formed and released. i. Lymphocytes ii. Monocytes 3. Thrombocytes (Platelets) B. Bone Marrow 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction II. Learning Objectives Keywords III. Keywords IV. Slides A. Blood Cells Basophil Normoblasts 1. Erythrocytes (Red Blood Cells) Bone marrow Platelets 2. Leukocytes (White Blood Cells) Eosinophil a. Granulocytes (PMNs) Erythrocytes Red blood cells i. Neutrophils Hemopoiesis Red marrow ii. Eosinophils Hemopoietic cord iii. Basophils Leukocytes Sinusoids Lymphocyte Stab cells b. Agranulocytes (Mononuclear) Stromal cells i. Lymphocytes Thrombocytes ii. Monocytes White blood cells 3. Thrombocytes (Platelets) Yellow marrow B. Bone Marrow Neutrophil 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 Slide 10: Blood Smear, Wright’s

I. Introduction II. Learning Objectives zoom in to identify leukocytes (white blood cells) amongst all the (red blood cells) III. Keywords erythrocytes IV. Slides A. Blood Cells 1. Erythrocytes (Red Blood Cells) Slide 35a (NW): Blood Smear 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) i. Neutrophils ii. Eosinophils iii. Basophils b. Agranulocytes (Mononuclear) i. Lymphocytes Slide 23 (464): Blood Smear, Wright’s ii. Monocytes 3. Thrombocytes (Platelets) B. Bone Marrow 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells notice the difference in staining of the RBCs on i. Erythroid precursors the three blood smear slides; use the coloration ii. Myeloid precursors of the acidophilic RBCs as a reference in identifying other cell types (e.g., eosinophils) V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 10: Blood Smear, Wright’s stain II. Learning Objectives III. Keywords IV. Slides A. Blood Cells Diameter 6-8 µm 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) N:C -- a. Granulocytes (PMNs) Nucleus -- i. Neutrophils ii. Eosinophils Chromatin -- iii. Basophils b. Agranulocytes (Mononuclear) Nucleoli --

i. Lymphocytes Cytoplasm Eosinophilic ii. Monocytes 3. Thrombocytes (Platelets) B. Bone Marrow 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors erythrocytes (red blood cells) are the most numerous type of cell found in blood; before leaving the bone ii. Myeloid precursors marrow where they are formed, they lose their nucleus and most organelles and become filled with V. Summary hemoglobin, giving them acidophilic cytoplasm; they circulate for about 120 days and are essential for transport of carbon dioxide and oxygen around the body; they serve as a useful size reference for other cells Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 10: Blood Smear, Wright’s stain II. Learning Objectives III. Keywords IV. Slides A. Blood Cells % of WBCs 40-75% 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) Diameter 10-15 µm a. Granulocytes (PMNs) N:C 1:3 i. Neutrophils ii. Eosinophils Nucleus Multi-lobed in a iii. Basophils variety of shapes b. Agranulocytes (Mononuclear) Chromatin Condensed i. Lymphocytes ii. Monocytes Nucleoli No 3. Thrombocytes (Platelets) Cytoplasm Non-distinct staining B. Bone Marrow of granules 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells neutrophils (polymorphonuclear neutrophils, polymorphs, or segmented neutrophils) are the most i. Erythroid precursors abundant type of leukocyte with approximately 1011 leaving the bone marrow each day; they circulate for 6- ii. Myeloid precursors 10 hours before exiting into the tissues where they live for 1-2 days; like most mature leukocytes, they have V. Summary condensed chromatin and few cytoplasmic organelles; they are phagocytic and the first-responders at sites of inflammation to destroy damaged tissue and bacteria Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 35a (NW): Blood Smear, Wright’s stain II. Learning Objectives III. Keywords IV. Slides A. Blood Cells % of WBCs < 5 % 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) Diameter 10-15 µm a. Granulocytes (PMNs) N:C 1:3 i. Neutrophils ii. Eosinophils Nucleus “Horseshoe” iii. Basophils b. Agranulocytes (Mononuclear) Chromatin Condensed i. Lymphocytes Nucleoli No ii. Monocytes 3. Thrombocytes (Platelets) Cytoplasm Non-distinct staining B. Bone Marrow 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells band cells are the final stage of all development before becoming mature cells but are usually i. Erythroid precursors only seen for neutrophils; they generally have a characteristic “horseshoe”-shaped nucleus, instead of the ii. Myeloid precursors segmented (“string of sausages”) nucleus of mature neutrophils; they are normally only seen in low V. Summary numbers in the peripheral circulation but may become more numerous during inflammation or infection; this increase is referred to as a “left-shift” (etymology of the term is uncertain) or bandemia Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 10: Blood Smear, Wright’s stain II. Learning Objectives III. Keywords IV. Slides A. Blood Cells % of WBCs 1-6% 1. Erythrocytes (Red Blood Cells) Diameter 12-17 µm 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) N:C 1:3 i. Neutrophils Nucleus Bi-lobed ii. Eosinophils iii. Basophils Chromatin Condensed b. Agranulocytes (Mononuclear) i. Lymphocytes Nucleoli No ii. Monocytes Cytoplasm Large, acidophilic 3. Thrombocytes (Platelets) granules that stain B. Bone Marrow the same color as 1. General structure surrounding RBCs 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors eosinophils are granulocytes with intensely-staining eosinophilic (acidophilic) cytoplasmic granules; they circulate for a few hours before exiting into tissues; eosinophils are important in killing parasitic worms V. Summary (helminths) and modulating inflammation Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 23 (464): Blood Smear, Wright’s stain II. Learning Objectives III. Keywords IV. Slides % of WBCs < 1% A. Blood Cells 1. Erythrocytes (Red Blood Cells) Diameter 14-16 µm 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) N:C 1:3 i. Neutrophils Nucleus Bi-lobed (if seen) ii. Eosinophils iii. Basophils Chromatin Condensed b. Agranulocytes (Mononuclear) Nucleoli No i. Lymphocytes ii. Monocytes Cytoplasm Large, basophilic 3. Thrombocytes (Platelets) granules that often B. Bone Marrow obscure everything else in the cell 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells basophils are granulocytes and the rarest type of circulating leukocyte; they appear similar to mast cells i. Erythroid precursors (only in tissues) with a bi-lobed nucleus that is usually obscured by the numerous, large, strongly-basophilic ii. Myeloid precursors granules that fill the cytoplasm; basophils play a role in parasitic infections and are part of the inflammatory V. Summary response (contain histamine and heparin); a neutrophil can be seen in the lower left of the slide Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 10: Blood Smear, Wright’s stain II. Learning Objectives III. Keywords IV. Slides A. Blood Cells % of WBCs 20-50% 1. Erythrocytes (Red Blood Cells) Diameter 6-12 µm (90%) 2. Leukocytes (White Blood Cells) 15-20 µm (10%) a. Granulocytes (PMNs) N:C 4:1 (small cells) i. Neutrophils 2:1 (large cells) ii. Eosinophils Nucleus Large, spherical iii. Basophils b. Agranulocytes (Mononuclear) Chromatin Condensed i. Lymphocytes ii. Monocytes Nucleoli No (until activated) 3. Thrombocytes (Platelets) Cytoplasm Minimal and B. Bone Marrow basophilic; Golgi is 1. General structure usually visible 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors lymphocytes are agranulocytes generally similar in size to RBCs but have a very large, round nucleus which ii. Myeloid precursors fills most of the cell, leaving little cytoplasm with few organelles; the cells function as part of the adaptive V. Summary immune system but specific subtypes (B and T cells) are not distinguishable in light microscopy (majority are T cells, however); activated lymphocytes may enlarge and appear similar to monocytes but are still distinguishable by their nuclei Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 10: Blood Smear, Wright’s stain II. Learning Objectives III. Keywords IV. Slides A. Blood Cells % of WBCs 2-10% 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) Diameter 15-20 µm a. Granulocytes (PMNs) N:C 3:1 i. Neutrophils ii. Eosinophils Nucleus Large and indented or iii. Basophils kidney-shaped b. Agranulocytes (Mononuclear) Chromatin Condensed i. Lymphocytes Nucleoli Maybe ii. Monocytes 3. Thrombocytes (Platelets) Cytoplasm “Dishwater” B. Bone Marrow appearance with fine 1. General structure purple/pink granules 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors monocytes are agranulocytes and the largest leukocytes; they are -precursor cells that ii. Myeloid precursors circulate in blood for about a day before migrating into tissues and becoming ; they may be V. Summary confused with large lymphocytes (two small lymphocytes are seen below), but the distinctive shape of the respective nuclei should help to differentiate them Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 10: Blood Smear, Wright’s stain II. Learning Objectives III. Keywords IV. Slides A. Blood Cells Diameter 1-3 µm 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) N:C -- a. Granulocytes (PMNs) Nucleus -- i. Neutrophils ii. Eosinophils Chromatin -- iii. Basophils Nucleoli -- b. Agranulocytes (Mononuclear) i. Lymphocytes Cytoplasm Basophilic ii. Monocytes 3. Thrombocytes (Platelets) Granulocyte? -- B. Bone Marrow 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors platelets are small cellular fragments derived from megakaryocytes in the bone marrow; when in contact ii. Myeloid precursors with collagen (exposed CT), platelets adhere and degranulate, triggering the formation of a blood clot; V. Summary around 1011 platelets are produced each day in a healthy adult, and they survive in the circulation for 7-10 days Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 104: Bone, H&E Slide 34: Bone, H&E II. Learning Objectives III. Keywords IV. Slides A. Blood Cells 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) i. Neutrophils ii. Eosinophils iii. Basophils b. Agranulocytes (Mononuclear) i. Lymphocytes ii. Monocytes 3. Thrombocytes (Platelets) B. Bone Marrow 1. General structure 2. Cells a. Megakaryocytes yellow(er) bone marrow red bone marrow b. Hemopoietic cells (less hematopoietically active) (more hematopoietically active) i. Erythroid precursors by adulthood, by adulthood, ii. Myeloid precursors 85% adipose tissue 40% adipose tissue found in medullary cavity (trabecular bone) of long found in flat bones and trabecular bone near V. Summary bones epiphysis of long bones Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 Slide 12 (464): Decalcified Bone

I. Introduction look at additional bone marrow slides to II. Learning Objectives compare the amount of adipose and III. Keywords hemopoietic cells seen IV. Slides A. Blood Cells 1. Erythrocytes (Red Blood Cells) Slide 20a (464): Bone Marrow 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) i. Neutrophils ii. Eosinophils iii. Basophils b. Agranulocytes (Mononuclear) i. Lymphocytes Slide 77 (464): Bone Marrow, Giemsa ii. Monocytes 3. Thrombocytes (Platelets) B. Bone Marrow 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 153: Bone Marrow, Needle Biopsy, H&E II. Learning Objectives III. Keywords IV. Slides A. Blood Cells 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) i. Neutrophils trabecula of bone ii. Eosinophils iii. Basophils b. Agranulocytes (Mononuclear) i. Lymphocytes “spaces” are adipocytes ii. Monocytes 3. Thrombocytes (Platelets) B. Bone Marrow 1. General structure hemopoietic cord 2. Cells with clusters of blood cell a. Megakaryocytes precursors and megakaryocytes b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 20a (464): Bone Marrow, H&E II. Learning Objectives III. Keywords IV. Slides A. Blood Cells 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) i. Neutrophils ii. Eosinophils iii. Basophils adipocyte b. Agranulocytes (Mononuclear) i. Lymphocytes ii. Monocytes 3. Thrombocytes (Platelets) hemopoietic B. Bone Marrow cord 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors V. Summary sinusoids Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 34: Bone, H&E II. Learning Objectives III. Keywords endosteum lining inner IV. Slides surface of trabecular bone A. Blood Cells 1. Erythrocytes (Red Blood Cells) sinusoid 2. Leukocytes (White Blood Cells) full of RBCs a. Granulocytes (PMNs) i. Neutrophils hemopoietic cord ii. Eosinophils iii. Basophils endothelial cell b. Agranulocytes (Mononuclear) lining sinusoid i. Lymphocytes ii. Monocytes macrophage 3. Thrombocytes (Platelets) B. Bone Marrow stromal cell 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors in red bone marrow, the stroma consists of reticular connective tissue cradling collections of hemopoietic ii. Myeloid precursors cells called hemopoietic cords; within the stroma, there are macrophages and specialized fibroblast-like cells V. Summary called stromal cells (reticular or adventitial cells); the cords are situated between specialized blood vessels called sinusoids, lined by simple squamous epithelium (endothelium), which blood cells must enter into in order to leave the bone marrow and enter the peripheral circulation Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction Slide 153: Bone Marrow, Needle Biopsy, H&E II. Learning Objectives III. Keywords IV. Slides A. Blood Cells 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) megakaryocyte i. Neutrophils ii. Eosinophils iii. Basophils b. Agranulocytes (Mononuclear) i. Lymphocytes ii. Monocytes 3. Thrombocytes (Platelets) B. Bone Marrow 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors megakaryocytes are very large cells (50-70µm) with a single, multi-lobed nucleus from endomitosis V. Summary (up to 48N); they are generally found located adjacent to sinusoids in the bone marrow where they bud off platelets, releasing them into the sinusoids to enter the peripheral blood; each megakaryocyte produces between 1000-3000 platelets Lab 8 – Blood Cells and Hemopoiesis Developing Blood Cells: IUSM – 2016 and Granulopoieisis I. Introduction Identifying ( ) in bone marrow samples can be very challenging: there are lots of cell II. Learning Objectives hemopoietic hematopoietic cells types, lots of terms, and lots of ambiguity. It is certainly not expected to be able to identify all the cells observable on a slide III. Keywords (at least not for this course), so focus should be on understanding the overall processes of erythropoiesis ( IV. Slides differentiation) and (granulocyte differentiation) and appreciating the respective readily identifiable cell A. Blood Cells stages and the defining characteristics for each. 1. Erythrocytes (Red Blood Cells) Be sure to look at lots of cells on each slide to gain a comparative perspective amongst the cells. Every bone marrow smear 2. Leukocytes (White Blood Cells) looks different. Find easily identifiable cells first (e.g., band cells) and use them as a reference when identifying other cells. a. Granulocytes (PMNs) Early cell types (e.g., proerythroblasts vs ) are very difficult to differentiate, especially without better slides and i. Neutrophils higher magnifications, so focus on identifying the later cell stages as highlighted in the lab guide. ii. Eosinophils Terminology iii. Basophils Understanding, instead of just memorizing, the cell names is key to identifying the cells and retaining the material b. Agranulocytes (Mononuclear) (hopefully a goal). A few ideas to keep in mind: i. Lymphocytes ii. Monocytes 1. As seen in other cell lineages (e.g., osteoblasts and osteocytes), “blasts” precede “cytes”. For example, myeloblasts occur before myelocytes (i.e., myelocytes are more differentiated). 3. Thrombocytes (Platelets) B. Bone Marrow 2. The term myeloid (Gr. “bone marrow”) is used generally to refer to granulocyte precursor cells. This usage reflects an 1. General structure historic misunderstanding of hemopoiesis, but the terminology remains and is seen in the naming of granulocyte precursor cells, e.g., myelocytes. The normal ratio of myeloid cell to erythroid cells (M:E) is about 3:1. 2. Cells a. Megakaryocytes 3. Erythroid terminology: the cells are named primarily based upon the staining of their cytoplasm. As the cells b. Hemopoietic cells differentiate, rER is lost and hemoglobin is gained, shifting the cytoplasm from basophilic to acidophilic. Keeping this i. Erythroid precursors is mind should help in remembering the names and the order of the cell types. ii. Myeloid precursors 4. Myeloid terminology: based upon the appearance of secondary granules, the stage is the first stage which V. Summary is recognizable to a specific granulocyte lineage (e.g., an eosinophilic myelocyte looks different than a neutrophilic myelocyte). Myelocytes therefore serves as the “center” of the terminology. Cells that precede the myelocyte stage are (Gr. “before”), and cells that follow the myelocyte stage are metamyelocytes (Gr. “after”). Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 Slide 39a (NW): Bone Marrow Smear

I. Introduction hemopoietic cells can be seen in the bone II. Learning Objectives marrow slides previously studied or on the III. Keywords bone marrow blood smear slides seen here IV. Slides A. Blood Cells 1. Erythrocytes (Red Blood Cells) Slide 127: Bone Marrow Smear 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) i. Neutrophils ii. Eosinophils iii. Basophils b. Agranulocytes (Mononuclear) i. Lymphocytes Slide 39b (NW): Bone Marrow Smear ii. Monocytes 3. Thrombocytes (Platelets) B. Bone Marrow 1. General structure 2. Cells a. Megakaryocytes b. Hemopoietic cells how do these blood smear slides appear different i. Erythroid precursors from the peripheral blood smear slides seen ii. Myeloid precursors earlier? V. Summary Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 As the RBC differentiation pathway advances, there is: (1) a decrease in overall size of the cell and the nucleus (chromatin I. Introduction condenses), (2) a decrease in N:C ratio, (3) a loss of organelles (and basophilia), and (4) an increase in hemoglobin (and eosinophilia). II. Learning Objectives ProEb III. Keywords (ProEb) Proerythroblasts (earliest recognizable precursor) IV. Slides • large cell with sparse, basophilic cytoplasm (lack hemoglobin) large, intensely-staining granular nucleus with one or more A. Blood Cells • nucleoli 1. Erythrocytes (Red Blood Cells) • thin Golgi adjacent to nucleus 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) (BaEb) Basophilic erythroblast • smaller than with decreasing N:C ratio i. Neutrophils • more abundant basophilic cytoplasm due to abundance of rER ii. Eosinophils for synthesizing hemoglobin iii. Basophils • condensing nucleus and chromatin with no clear nucleoli b. Agranulocytes (Mononuclear) (PolEb) Polychromatophilic erythroblast i. Lymphocytes • reduced cell volume ii. Monocytes OrEb • polychromatic = basophilic (rER) and acidophilic (hemoglobin) PolEb 3. Thrombocytes (Platelets) cytoplasm • nucleus continuing to condense with clumped chromatin B. Bone Marrow 1. General structure (OrEb) Orthochromatophilic erythroblast (normoblast) 2. Cells • small cell with mainly acidophilic cytoplasm (hemoglobin) a. Megakaryocytes • orthochromatic = “right color” = typical cytoplasm color of RBC • highly-condensed nucleus, soon to be ejected from cell b. Hemopoietic cells BaEb i. Erythroid precursors (not seen) ii. Myeloid precursors • nucleus has been extruded V. Summary • cytoplasm still contains small amount of rER but difficult to see • reticulocyte = specialized stains cause ribosomes to clump and Image adapted from: Wheater’s Functional Histology, 5 ed. form reticular network Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 As the granulocyte differentiation pathway advances, there is: (1) I. Introduction a decrease in N:C ratio, (2) condensation of the nucleus, II. Learning Objectives Pro eventually becoming indented and finally segmented, (3) the III. Keywords appearance of non-specific and then specific cytoplasmic granules, (4) a loss of organelles (and cytoplasmic basophilia). IV. Slides A. Blood Cells (not seen) 1. Erythrocytes (Red Blood Cells) • 14-20µm cell with high N:C ratio (i.e., not much cytoplasm) • euchromatic nucleus with generally 3-5 prominent nucleoli 2. Leukocytes (White Blood Cells) • deeply-basophilic cytoplasm lacking granules; Golgi is visible a. Granulocytes (PMNs) Mye (Pro) i. Neutrophils • large, azurophilic (purple) primary (non-specific) granules ii. Eosinophils • largest cell size of the series (15-24µm) iii. Basophils • decreasing N:C ratio with basophilic cytoplasm b. Agranulocytes (Mononuclear) • nucleus becoming heterochromatic with less prominent nucleoli i. Lymphocytes Band (Mye) Myelocyte (first cell unique for each granulocyte type) ii. Monocytes • secondary (specific) granules (pink/lilac for neutrophils) • more abundant cytoplasm; N:C ~2:1 3. Thrombocytes (Platelets) • nucleus becomes ovoid and eccentrically-placed B. Bone Marrow • coarse chromatin and nucleoli become indistinct 1. General structure (Meta) (different granulocyte types are easily seen) 2. Cells • nucleus is indented (“kidney-bean” shaped) a. Megakaryocytes • cytoplasm becomes less basophilic (continued loss of rER) b. Hemopoietic cells • secondary granules are prominent; very few primary granules i. Erythroid precursors Band cell (stab cell) ii. Myeloid precursors Meta • nucleus typically has “horseshoe” appearance V. Summary • indentation of nucleus is > ½ the diameter of the nucleus • nucleus has uniform width and is not yet segmented Myeloid precursors of neutrophil granulocytes. Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 Common Confusion:

I. Introduction Band vs. Monocyte II. Learning Objectives III. Keywords Band cells: final precursor cells of mature granulocytes but generally only readily seen for neutrophils; seen mostly in IV. Slides bone marrow; once nucleus becomes segmented, no longer A. Blood Cells considered a band 1. Erythrocytes (Red Blood Cells) 2. Leukocytes (White Blood Cells) Look for: (1) 10-12µm diameter; (2) small, elongated nucleus with N:C ratio of 1:2; (3) nucleus has a “horseshoe” a. Granulocytes (PMNs) shape; (4) granular cytoplasm i. Neutrophils ii. Eosinophils iii. Basophils b. Agranulocytes (Mononuclear) Band cell i. Lymphocytes ii. Monocytes Monocytes: largest type of leukocyte; they are circulating 3. Thrombocytes (Platelets) precursors of macrophages B. Bone Marrow 1. General structure Look for: (1) 20µm diameter; (2) larger nucleus with N:C ratio of ~3:1; (3) agranular, blue-gray cytoplasm 2. Cells (“dishwater” cytoplasm); (4) small “holes” (vacuoles) of a. Megakaryocytes unstained areas may be seen in the cytoplasm b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors V. Summary Monocyte Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 Common Confusion:

I. Introduction Large Lymphocyte vs. Monocyte II. Learning Objectives III. Keywords Large lymphocytes: usually immunoblasts that have enlarged after activation from antigen stimulation; some IV. Slides increase in the size of the nucleus occurs, but most of the A. Blood Cells size increase is in the cytoplasm, resulting in a decreased N:C 1. Erythrocytes (Red Blood Cells) ratio; plasma cells are not normally seen in the blood 2. Leukocytes (White Blood Cells) Look for: (1) nucleus is round or ovoid, but not generally a. Granulocytes (PMNs) indented; (2) more abundant cytoplasm with a variable N:C i. Neutrophils ratio, but ~2:1 is common; (3) chromatin is usually fine with ii. Eosinophils visible nucleoli; (4) cytoplasm is light blue and may contain azurophilic granules; (5) cell borders may be distorted by iii. Basophils surrounding RBCs b. Agranulocytes (Mononuclear) Large Lymphocyte i. Lymphocytes ii. Monocytes Monocytes: largest type of leukocyte; they are circulating 3. Thrombocytes (Platelets) precursors of macrophages and are generally more common B. Bone Marrow than large lymphocytes 1. General structure Look for: (1) nucleus is indented; (2) N:C ratio of ~3:1; (3) 2. Cells coarser chromatin without nucleoli; (4) agranular, blue-gray a. Megakaryocytes cytoplasm (“dishwater” cytoplasm); (5) small “holes” b. Hemopoietic cells (vacuoles) of unstained areas may be seen in the cytoplasm i. Erythroid precursors ii. Myeloid precursors V. Summary Monocyte Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 Common Confusion:

I. Introduction Megakaryocyte vs. II. Learning Objectives III. Keywords Megakaryocytes: large cells in bone marrow that give rise to thrombocytes (platelets) in peripheral blood IV. Slides A. Blood Cells Look for: (1) 20-60µm diameter; (2) large, single nucleus 1. Erythrocytes (Red Blood Cells) may be irregularly lobulated, but lobes are connected and 2. Leukocytes (White Blood Cells) not separated by cytoplasm; (3) generally located in bone marrow adjacent to sinusoids, not immediately adjacent to a. Granulocytes (PMNs) bone; (4) finely-granular cytoplasm i. Neutrophils ii. Eosinophils iii. Basophils b. Agranulocytes (Mononuclear) Megakaryocyte i. Lymphocytes ii. Monocytes : very large cells responsible for bone 3. Thrombocytes (Platelets) resorption; they are derive from the fusion of mononuclear B. Bone Marrow hemopoietic precursor cells 1. General structure Look for: (1) 50-150µm diameter; (2) multinucleated – 2. Cells nuclei are usually round and distinctly separate, average of a. Megakaryocytes 4-8 per cell; (3) generally located adjacent to bone, b. Hemopoietic cells especially in Howship’s lacunae; (4) granular cytoplasm i. Erythroid precursors ii. Myeloid precursors V. Summary Osteoclast Lab 8 – Blood Cells and Hemopoiesis IUSM – 2016 Summary I. Introduction II. Learning Objectives 1. Blood is a specialized type of connective tissue with a liquid extracellular matrix (ECM) called III. Keywords plasma; the cellular elements of blood are referred to as formed elements and consist of red blood IV. Slides cells (erythrocytes), which are non-nucleated cells that contain hemoglobin, and white blood cells A. Blood Cells (leukocytes), which are nucleated cells that lack hemoglobin. 1. Erythrocytes (Red Blood Cells) 2. Leukocytes are traditionally divided into granulocytes (have cytoplasmic granules that affect the 2. Leukocytes (White Blood Cells) staining of the cell), which include neutrophils, eosinophils, and basophils, and agranulocytes a. Granulocytes (PMNs) (generally lack readily discernable cytoplasmic granules), which include lymphocytes and i. Neutrophils monocytes. ii. Eosinophils iii. Basophils 3. The prevalence of leukocytes in the blood is often remembered by the perhaps misguided b. Agranulocytes (Mononuclear) mnemonic: never let monkeys eat bananas (for neutrophils, lymphocytes, monocytes, eosinophils, i. Lymphocytes and basophils); the relative percentages can be remembered approximately by the “6/3 rule”: 60% ii. Monocytes for neutrophils, 30% for lymphocytes, 6% for monocytes, 3% for eosinophils, and 1% (remainder) 3. Thrombocytes (Platelets) for basophils. B. Bone Marrow 4. The principal functional site of leukocytes is outside the blood in tissues; in loose CT, they are either 1. General structure permanent or wandering cells and generally appear different than as seen on blood smears; see Lab 2. Cells 5 – Connective Tissue for examples. a. Megakaryocytes b. Hemopoietic cells 5. Hemopoietically-active bone marrow is referred to as red bone marrow; in adults it is primarily i. Erythroid precursors located within the cancellous bone of flat bones (such as the bones of the skull, sternum, vertebrae, ii. Myeloid precursors and pelvis). Erythroid precursors and granulocyte precursors are generally recognizable based upon V. Summary the characteristic appearance and changes of their nuclei and cytoplasm during differentiation. Lab 8 – Blood Cells and Hemopoiesis Appearance of Blood Cells in Electron and Light Microscopy IUSM – 2016 Characteristic Appearance I. Introduction II. Learning Objectives Structure Electron Microscopy Light Microscopy (H&E stain) III. Keywords Neutrophil IV. Slides A. Blood Cells 1. Erythrocytes (Red Blood Cells) Lymphocyte 2. Leukocytes (White Blood Cells) a. Granulocytes (PMNs) Monocyte i. Neutrophils ii. Eosinophils Eosinophil iii. Basophils b. Agranulocytes (Mononuclear) i. Lymphocytes Basophil ii. Monocytes Orthochromatophilic 3. Thrombocytes (Platelets) erythroblast B. Bone Marrow

1. General structure Reticulocyte 2. Cells a. Megakaryocytes Promyelocyte b. Hemopoietic cells i. Erythroid precursors ii. Myeloid precursors Myelocyte V. Summary Band cell Lab 8 – Blood Cells and Hemopoiesis Questions about Blood Cells and Hemopoiesis IUSM – 2016

I. Introduction How does the shape and appearance of II. Learning Objectives the nuclei of erythroid precursors change in comparison to the nuclei of III. Keywords myeloid precursors? IV. Slides A. Blood Cells Why are mature granulocytes also referred to as polymorphonuclear 1. Erythrocytes (Red Blood Cells) leukocytes? What types of leukocytes 2. Leukocytes (White Blood Cells) are mononuclear? a. Granulocytes (PMNs) How does the cytoplasm of erythroid i. Neutrophils precursors change in comparison to ii. Eosinophils the cytoplasm of myeloid precursors? iii. Basophils b. Agranulocytes (Mononuclear) If viewing a blood smear, what clues can be used to determine if it is from i. Lymphocytes peripheral blood or bone marrow? ii. Monocytes 3. Thrombocytes (Platelets) Why do hemopoietic cells experience a B. Bone Marrow decreasing N:C and loss of nucleoli as 1. General structure they differentiate? 2. Cells a. Megakaryocytes How are eosinophils and basophils b. Hemopoietic cells differentiated in electron micrographs? i. Erythroid precursors ii. Myeloid precursors The principal functional location of V. Summary which formed elements is in the blood?