Normal blood cell maturation.st.pdf

Review of normal blood cell maturation Hematology II MDLS 4226/5226 Objectives At the completion of this lecture the student is expected to use the information gained to correctly: • Analyze the function of leukopoiesis, myelopoiesis, and lymphopoisis. • Recall the types of normal white blood cel...

Review of normal blood cell maturation Hematology II MDLS 4226/5226 Objectives At the completion of this lecture the student is expected to use the information gained to correctly: • Analyze the function of leukopoiesis, myelopoiesis, and lymphopoisis. • Recall the types of normal white blood cells, their function, and predominance in normal adults. • Analyze the four functions of white blood cells. • Examine the general criteria for WBC identification, in terms of cell size, N:C ratio, cytoplasmic characteristics, and nuclear characteristics. • Evaluate each cell in the myelocytic series in order to supply the correct identification. • Evaluate the three functions of granulocytes. • Predict the movement of granulocytes from the bone marrow to the tissues. • Differentiate between mitotic, maturation, marginal, and circulating pools of leukocytes. Objectives cont. • List the granulocytic precursors in order of maturity. • Analyze the process of granulocytic maturation. • Correlate the stage of granulocytic development when given a written description of the morphology of a cell in a Wright-stained smear. • Design a study aid to learn the criteria used for the maturation process of granulocytes while examining their function, morphological features, surface markers, granular content, and location found. • Evaluate the contents of neutrophilic granules. • Assess the maturation, granular content, morphology, and function of eosinophils, basophils, and mast cells. Objectives cont. • Design a study aid to teach the criteria used for the maturation process of monocytes/ macrophages while examining their function, morphological features, surface markers, granular content, and location found. • Differentiate free and fixed macrophages. • Compare a large lymphocyte with a monocyte. • Appraise the relevant colony stimulating factors, in regards to their source and function. • Predict the identification of cells and the corresponding clinical disorders presented in this lecture, using patient history and/or clinical laboratory results. • Appraise each stage of lymphoid cell development according to its major surface markers. • Define lymphocytosis and lymphopenia. • Evaluate the three types of atypical lymphocytes in terms of their morphology, and probable disease correlation. • Compare the morphology of plasma cells with lymphocytes. • Rank white blood cells in order of their phagocytic ability. Leukopoiesis • Leukopoiesis is the production and proliferation of white blood cells, with the exception of lymphocytes, in the bone marrow, lymph nodes, & thymus. “BLT” • Myelopoiesis (granulocytopoiesis) refers to the production of neutrophils, eosinophils, and basophils • Lymphopoiesis refers to the production of lymphocytes WBC Classification • There are 5 main types of WBCs (in order of their predominance in the N. adult): • Neutrophils (Segs) 50-70% • Lymphocytes (Lymphs) 18-42% • Monocytes (Monos) 2-11% • Eosinophils (Eos) 1-3% • Basophils (Basos) 0-2% WBC Classification • Traditionally, 2 basic categories: 1. Granulocytes (develop only in bone marrow) Includes: Segs, Eos, Basos, and Monos 2. Lymphocytes or Mononuclears (develop in the bone marrow and lymphoid tissue) Includes: Lymphs, and NK cells FYI: Mononuclears can have very fine granules; it’s just that they are not granulocytes, which all have large, noticeable granules & segmented nuclei. WBC Functions • As defense against foreign (“non-self”) invaders via: • Ab production by immunocytes (i.e., lymphs.) • Cytokine production by lymphocytes (aka. lymphokines.) • Inflammatory mediator production by segs & monos. • Phagocytosis (i.e., granulocytes and mononuclear cells) Normal Cell Maturation • As a normal cell matures • Overall: • • • • • cell size decreases nuclear chromatin pattern becomes denser nucleoli disappear amount and color of cytoplasm changes size of nucleus decreases greater than the size of the cell (N:C ratio) Normal Cell Maturation • Cytoplasmic Maturation: • deep blue color (high RNA content) pales • granules may appear • amount increases • Nuclear Maturation: • • • • nucleus decreases in size (exits entirely in RBCs!) chromatin becomes more condensed or clumped color changes from reddish to bluish-purple nucleoli may decrease in # or become absent Erythrokinetics • Erythron – the collection of all stages of erythrocytes throughout the body • RBC Mass – refers only to the cells in circulation Carry oxygen The role of RBCs is to ____________________________. • Hypoxia – diminished availability of oxygen to the body tissues • Occurs when the oxygen tension in the cells is decreased Erythropoietin • Erythropoietin Production and Regulation: • EPO is produced primarily in the kidney in response to ? _______________________ degree blood oxygenation • it functions to of maintain normal RBC mass • Initiates a cascade of events that lead to increased RBCs in circulation • Allows early release of reticulocytes • Increases number of mature erythrocytes • Reduces marrow transit time Six Stages of RBC Maturation: • Pronormoblast (Rubriblast or Proerythroblast) - "Blast" • Cell size: 12-20 µm • Nucleus: contains nucleoli & fine chromatin; round to slightly oval shape. High N:C ratio (8:1) • Cytoplasm: very basophilic (high RNA content, lots of organelles), with lighter-staining perinuclear area around Golgi (not always visible) Note: BFU-E for 1 week » CFU-E for 1 week » Pronormoblast for ~24 hrs. RBC Maturation Series (Peripheral Blood Tutor CD-ROM) Pronormoblast Rubriblast Proerythroblast First recognizable erythroid precursor cell Six Stages of RBC Maturation: • Basophilic Normoblast (Prorubricyte /Erythroblast) • Cell size: • Nucleus: nucleoli 10-15 µm round, coarser chromatin; no visible N:C ratio decreasing to 6:1 • Cytoplasm: temporarily even more basophilic (royal blue); Golgi may be visible as light area near nucleus RBC Maturation Series (Peripheral Blood Tutor CD-ROM) Basophilic Normoblast Prorubricyte Basophilic Erythroblast Six Stages of RBC Maturation: • Polychromatic normoblast (Rubricyte/Polychromatic erythroblast) • Cell size: • Nucleus: • Cytoplasm: 10-12 µm round, sometimes eccentric, smaller, with coarser chromatin N:C ratio of 4:1 opaque, violet-blue or grayish color (due to Hgb synthesis) polychromasia • (NOTE: Last stage capable of mitosis.) RBC Maturation Series (Peripheral Blood Tutor CD-ROM) Polychromatic Normoblast Rubricyte Polychromatic Erythroblast Six Stages of RBC Maturation: • Orthochromic normoblast (Metarubricyte/Orthochromic erythroblast) • Cell size: 8-10 µm • Nucleus: pyknotic (degenerated nuclear chromatin); eventually extruded • Cytoplasm: N:C ratio of 1:2 polychromasia • NOTE: This stage is typically called a "nucleated red", & the WBC count must be corrected if 5 or more of these are present on a 100 cell WBC differential, because automated analyzers mistake them for white blood cells! RBC Maturation Series (Peripheral Blood Tutor CD-ROM) Orthochromic Normoblast Metarubricyte Orthochromic Erythroblast ERYTHROPOIESIS Erythroblasts (NRBC) Wright-Giemsa Stain Six Stages of RBC Maturation: • Reticulocyte - "Retic“ • Cell size: 7-9 µm (nearly normal!) • Nucleus: none present • Cytoplasm: varying degrees of polychromasia (variation in cytoplasmic color, usually a bluish tinge; may still have basophilic = ?) stippling Resides in marrow for 1 day, then peripheral blood for 1 day, then retained in spleen for pitting and polishing for a few days, then released as a mature cell RBC Maturation Series (Peripheral Blood Tutor CD-ROM) RETICULOCYTE (Retic) Normal ranges: Adults 0.5 - 2.0 % Children 1.0 - 3.0 % Newborns 2.0 - 6.0 % New Methylene Blue Retic Stain Six Stages of RBC Maturation: • Erythrocyte • Cell size: 7 - 8 µm • Nucleus: none present • Cytoplasm: has distinctive central pallor; no protein or Hgb made; no mitochondria present. Lifespan ? = __________ Travels how far in that 120time? days = __________ 300 miles RBC Maturation Series (Peripheral Blood Tutor CD-ROM) ERYTHROCYTE (RBC) Neutrophilic Maturation • CFU-S, or hematopoietic stem cell (HSC) • Cluster of Differentiation (CD) 34 antigen • Undergoes stimulation, mitosis, and maturation in a stem cell (CFU-GEMM) that’s specific for myeloid cells • CFU-GEMM • CD34 and CD33 antigens • Matures into CFU-GM • CFU-GM • ILs and CSFs control the stability of cell numbers and their functions • Matures into a myeloblast Colony Stimulating Factors • Multi-CSF (i.e., IL-3) production is stimulated by endotoxin released from infection • Source – secreted by marrow fibroblasts, T-lymphs, macrophages, and monocytes • Function – stimulates regeneration, maturation, and differentiation of multipotential and unipotential stem cells • GM-CSF is important for myeloid maturation in the marrow • Source – secreted by T-lymphs, marrow fibroblasts, marrow endothelial cells and monocytes • Function – stimulates neutrophils, eosinophils, and monocyte growth Colony Stimulating Factors (cont.) • G-CSF is a more specific granulocyte growth factor • Source – monocytes, marrow fibroblasts and endothelial cells • Function – stimulates neutrophils, and enhances functional response of neutrophils • M-CSF (i.e., CSF-1) is the primary monocytic growth factor • Source – secreted by mature monocytes, marrow fibroblasts, and marrow endothelial cells • Function – stimulates macrophages and the release of GCSF from monocytes. Stimulates the release of tumor necrosis factor (TNF), interferon, and IL-1 from macrophages Neutrophilic Maturation • Myeloblast: “-blast” • • • • • • • dark blue to blue cytoplasm Primary granules may begin to appear lacy, smooth, delicate, & uniformly distributed chromatin 1-2 distinct nucleoli! A large cell (15-20 µm) high N:C ratio of 4:1 (so not much? __________) cytoplasm 1-2% normal in bone marrow (none are normal in peripheral blood (p.b.)! • Express antigens CD13 and CD33 • NOTE: Using light microscopy, it’s hard for non-experts to differentiate myeloblasts / monoblasts / lymphoblasts! Neutrophilic Maturation • Myeloblast: Myeloblast Wright-Giemsa Stain Neutrophilic Maturation • Promyelocyte: “Pro-” • large, prominent, reddish-purple primary granules (described as azurophilic)* • dark blue to blue cytoplasm • uniformly distributed chromatin • N:C ratio of 3:1 • less distinct nucleoli, but still may be present • May be larger than blast form (approx. 20 µm) • 2-5% normal in bone marrow, none normal in p.b *Thus used to help distinguish blasts from pros. (Primary = pro = red-purple) Neutrophilic Maturation • Promyelocyte: Promyelocyte Wright-Giemsa Stain Neutrophilic Maturation • Myelocyte: “Myelo-” • pinkish secondary/specific granules now visible*; 1o granules less visible “Specific = Secondary” • "Dawn of neutrophilia" occurs: specific granules tend to form in Golgi area, causing pink arc* • cytoplasm losing blue color due to decreased RNA synthesis • nucleus more condensed, with chromatin clumped; nucleus starting to “round up” with one flatter side • N:C ratio of 2:1 • <10% normal in bone marrow, none normal in p.b. • Myelocytes have greatest morphological variation of all the __________.  neutrophils * Thus used to help tell pros from myelocytes! Neutrophilic Maturation • Myelocyte: Myelocyte Wright-Giemsa Stain Mature Neutrophilic Granule Contents Primary/Nonspecific or Azurophilic Secondary/Specific or Neutrophilic Tertiary --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Red-purple Color? __________ (on Wright’s stain) (Atlas p. 45) Become light blue as cell matures Pale lavender-pink Color? ______________ (on Wright’s stain) (Atlas p. 53) Color?Invisible! _________ (on Wright’s stain) Blue-purple with special LAP*** stain Mostly visible in (blast &) promyelocyte stages Start at myelocyte stage Cause “dawn of neutrophilia” Appear very late stages Lysosomes; contains lyso- Lysosomes; contain lysozyme, Lysosomes; contain zyme, acid hydrolases, myeloperoxidase (MPO)*, proteases, & superoxide lactoferrin**, collagenases, & complement activators, but . . lysozyme, DAF, gelatinase, & LAP Stain pos. for peroxidase NO peroxidase NO peroxidase ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------* Potentiates action of hydrogen peroxide ** Combines with Fe in local environment to starve any invading microbes! *** Leukocyte Alkaline Phosphatase Neutrophilic Maturation • Metamyelocyte: “Meta-” • kidney-bean or peanut-shaped nucleus!! • nuclear chromatin more coarsely clumped • cytoplasm uniformly pink with pinky-purple 20 granules (aka. ? ________) specific (1o granules still present but don’t stain as intensely) • N:C ratio of 1.5:1 • 12-25% normal in bone marrow, none normal in p.b. • Nucleus becomes indented Neutrophilic Maturation • Metamyelocyte: Metamyelocyte Wright-Giemsa Stai Neutrophilic Maturation • Band: “band” • normal in small %'s in p.b., at about 5-10% in normal adults • Constitutes 40% normal in bone marrow • curved, band-like nucleus (C or S shape) with no segmentation • Possess full motility, active adhesion properties, and some phagocytic ability • A maturation “shift to the left” occurs when neutrophil bands are increased in p.b., in comparison to the number of segs Neutrophilic Maturation • Band: Band Neutrophil Granulocyte Wright-Giemsa Stain Neutrophilic Maturation • Segmented (polymorphonuclear) Neutrophil (PMN): “Poly-”or “Seg-” • • • • • comprise 50-70% of total WBC population in normal adult p.b. Nucleus continues indentation to form segments, a lobed nucleus Cell is completely functional should have ______ lobes 2-5 A maturation “shift to the right” occurs w/ increased number of segs, or hypersegmentation (>5 lobes) Neutrophilic Maturation • “Seg” (“Poly”) Segmented Neutrophil Granulocyte Wright-Giemsa Stain “Seg” variations Neutrophil Life Span (Peripheral Blood Tutor CD-ROM) For a neutrophil, about 10 days from myeloblast to maturation. Blast Myeloblast Pro Promyelocyte Myelo Myelocyte Meta Metamyelocyte Band Band Seg Segmented Neutrophil Granulocyte Pools • Granulocytes reside in 3 main areas of the body, moving from 1) bone marrow to 2) p.b. to 3) tissues (& movement from endothelium to tissues does not reverse itself.) • In the bone marrow, granulocytes have three functions: • Proliferation (i.e. myeloblasts, promyelocytes, and myelocytes) • Mitotic pool – 2 to 3 days • Maturation (i.e. metamyelocytes and bands) • Storage (bands and polymorphonuclear leukocytes) • Storage pool – 5 to 7 days Bone marrow holds 4-10 day supply of WBCs – about 25x amount in circulation! Granulocyte “Functional Pools” NOTE: Process of moving from bone marrow to tissues can speed up under stress (physical or emotional), causing "shift to the left": an accelerated release of immature forms into the p.b. Granulocyte Pools (cont.) • In bloodstream, granulocytes enter & then divide up equally between two other functional pools: • Circulating Pool (CP) – these are counted in a WBC count • Marginating Pool (MP) - Lies against endothelial lining of blood vessels There’s constant exchange between these 2 pools. Marginating cells can be mobilized into p.b. circulating pool during stress or exercise Granulocyte Pools (cont.) • Movement from MP to CP accounts for the elevated WBC count seen in crying children or highly stressedout adults, due to effects of hormone ?: epinephrine ____________ • Granulocytes stay in p.b. about 6-10 hrs., then move randomly into tissues via? _________, perform their diapedesis job (1-5 days), & die. - process in which they squeeze through tight junctions between endothelial cells of the blood vessel walls, & exit into the tissues. Once in the tissues, they do not return! Phagocytosis • Primary phagocytes are monocytes & neutrophils, but eos & basos ARE capable of limited phagocytosis! • Five steps: • • • • • Directed Motility (Migration) Recognition & Attachment Ingestion & Phagocytosis Degranulation/Digestion & Killing Exocytosis M R I D (K) E Phagocytosis • Step One: Directed Motility (Migration) • Chemotaxis = process of phagocytic movement along gradient of increasing [chemotaxin] (molecules generated by infection &/or inflammation.) • Phagocytes have p.m. receptors to detect chemotaxins/chemotactic factors, Abs, & fixed complement. Thus they migrate through p.b. & diapedese into tissues to reach site of infection/ inflammation • Who gets there first ? _______ Segs do! Phagocytosis • Step Two: Recognition & Attachment • Opsonization facilitates recognition & attachment by marking microorganisms for ingestion • Step Three: Ingestion & Phagocytosis • In an amoeboid motion, phagocyte uses rapid microfilament rearrangement to extend pseudopodia & surround foreign particle . . . • Endocytose it, • And then form vacuole around it (called a phagosome). https://www.bing.com/videos/search?q=phagocytosis&&view=detail&mid=E 2858A790C86D6FE2A3BE2858A790C86D6FE2A3B&&FORM=VRDGAR Phagocytosis • Step Four: Degranulation/Digestion (Killing) • WBC granules = tiny lysosomes containing lysozyme & acid hydrolases such as myeloperoxidase (MPO); these fuse with phagosome membrane garbage disposal to form a phagolysosome (functions as a ? _____________________) • Bacterial killing occurs in the phagosome by processes that are either oxygen-dependent or oxygen-independent • Oxygen dependent • The burst is triggered by superoxide (generated from MPO) in primary granules (which are still present in a mature seg, just not visible.) • Segs’ 1o granules color is? ___________ • Segs’ 1o granules specific or nonspecific? __________ • Oxygen-independent killing • Accomplished by hydrogen ions (alters the pH), lysozymes , and bactericidal proteins (cleave segments of bacterial cell wall). Phagocytosis • Step Five: Exocytosis • Essentially consists of dumping trash left over from the battle! FYI: Degranulation can also be triggered when seg (or to a lesser extent, a mono) gets “hung up” on an atherosclerotic plaque inside a blood vessel, causing release of MPO & localized inflammatory reaction. Thus MPO & other markers of inflammation are gaining interest as possible CVD markers. Eosinophils • Structurally similar to neutrophils, but differ in their unique cytoplasmic granules • Granule Contents: 2o granules = large red-orange lysosomes containing very small [peroxidase] & some acid phosphatase. These granules contain mostly crystalloid form of Major Basic Protein (MBP), which is lysine & arginine-rich (& cytotoxic to Schisto-soma.) Granules may overlie nucleus, but usually don’t • Cytoplasm is colorless • Nucleus stains less blue than neutrophils, and can be segmented (mature) or band-like (immature) Eosinophils • Move very slowly, have less intracellular killing ability than segs • Functions: • Control parasitic infections (damage larval stages of parasitic helminthic worms: flukes, tapeworms, & roundworms). • Dampen hypersensitivity reactions (allergies). • Lifespan: Eos remain < 1 week in p.b. MYELOPOIESIS Classic Eosinophil (Peripheral Blood Tutor CD-ROM) Eosinophil Granulocyte Wright-Giemsa Stain Basophils • Phagocytic ability much < segs & eos. Classified only into immature or mature forms (based upon degree of nuclear segmentation.) • Functions: • Mediate inflammatory responses via IgE receptors on their plasma membrane (including allergies) • Granule Contents: Large bluish-black lysosomes containing histamine and heparin(released in allergic reactions). These are water-soluble, so may disintegrate during staining (& so may appear as empty areas) • Granules usually DO overlie nucleus Classic Basophil Basophil Granulocyte Wright-Giemsa Stain Monocytes First major function is phagocytosis - even though a monocyte’s speed of mobility is slow compared to that of segs • Even though speed of mobility is slower, phagocytosis is much quicker • Require less opsonization, and phagocytosis can be initiated by contact • Play a minor role in processing specific antigens for lymphocyte recognition • A mono in the tissues is no longer a mono, but called a macrophage, either free or fixed • Monos stay in p.b. ~ 3 d., then move into tissues & stay several months or more. Monocyte/Macrophage Maturation Monocyte Maturation • Monoblast “blast” • Same stimulating factors active for maturation, are involved in osteoclast activity, suggesting link in certain malignant marrow diseases (i.e. Multiple Myeloma) • Usually seen with a single large nucleolus • Strongly positive for CD33, weakly pos. for CD34 • Also pos. for CD4 (seen in T-lymphs) • Promonocyte “pro” • Capable of some phagocytosis, but lack range of activity MONOPOIESIS Monocyte Maturation Classic Monocyte (Peripheral Blood Tutor CD-ROM) Monocytes Wright-Giemsa Stain Monocytes (cont.) • Granule Contents: • Monos contain MANY lysosomal enzymes: includes lysozyme (released continuously), acid phosphatase (where was alk. phosphatase? _________________), in 3o granules of segs & a small amount of peroxidase (< << < seg contains!) • Monos also stain + for nonspecific esterases (NSEs.) • Thus so far, where have we seen peroxidase? ____________________________________ 4+ in early segs , 2+ in later segs, 1+ in monos Monocytes (cont.) • • • • • Nucleus is indented or curved Chromatin is lacy w/ small clumps Largest-sized cell in p.b. Phagocytic vacuoles are common Regarded as a transitional cell • Easily confused with variant / atypical lymphs or large bands. (Always look at nuclear chromatin pattern: is it frilly or chunky?) Macrophage • As a mono leaves the circulation and enters the tissue, it evolves into a lysozyme-filled macrophage • Undergo a sudden increase in metabolic energy, phagocytic activity, lysosomes, IgG surface receptors, and mobility • Cytoplasm is highly vacuolated and has foamy appearance • Two categories – free and fixed • Free macrophages are found in various sites of inflammation and repair, and body fluids • Fixed macrophages are fund in specific sites of concentration (i.e., Kupffer cells, bone marrow, and lymph nodes) • Develop different minor characteristics depending upon the organ they occupy Bone Marrow Macrophages Macrophages in body fluid The roles of the players • Monocytes and Macrophages function as a secondary line of defense to clean-up and remove microbial organisms or cellular debris, and to remove the bodies own aged and damaged cells • Some macrophages are motile and respond to chemotactic factors such as complement and factors from activated lymphs • Monocytes become immobilized by migration-inhibition factor (MIF) released from activated lymphs • Become activated to phagocytize by complement, prostaglandins, or in response to previously phagocytized material The roles of the players • Monocytes and Macrophages perform many vital functions • A defense against microorganisms • Monocytes present and deliver IL-1 (a T-lymphocyte activating factor) to specific membrane surface receptor sites • Have a role in the daily destruction of aged blood cells, denatured plasma proteins, and lipids • Macrophages possess heme oxidase activity, which enables them to break down hemoglobin, and store iron • Serve as important effector cells by secreting IL-1, and regulators and complement, coagulation, and Kinin cascades Lymphoid Maturation Lymphoid Maturation • The 2 Primary Lymphoid Organs: • Bone marrow & thymus (“BMT”) • Lymphocyte production from these sites is continuous & Ag-independent. These send partially differentiated lymphs to . . . • The Secondary Lymphoid Tissues: • Lymph nodes, spleen, tonsils, & MALT (Mucosal Associated Lymphoid Tissues in respiratory & GI tracts, such as Peyer's patches (in ? ___________________). the small intestine • These act as main repositories for already differentiated lymphs Lymphocyte Maturation • Lymphoblast “blast” • Small to medium sized cell • Round-to-oval nucleus containing loose chromatin and one or more active nucleoli • Cytoplasm is scanty and has basophilia in proportion to amount of RNA • Prolymphoblast “Pro” • Clumped chromatin • Cytoplasm is more abundant than blast, but deeply basophilic Lymphoblast Lymphoblast Wright-Giemsa Stain Prolymphocyte Lymphocyte Characteristics • Can vary in size from 9 µm to 15 µm • Small (resting) lymphs - 7-10 μm; scanty cytoplasm, large N:C ratio. • Large lymphs - 11-25 μm; have abundant cytoplasm. • • • • Both have round or oval nuclei Chromatin appears blocked or “smudgy” Cytoplasm stains blue; may include some azurophilic granules Both types are NORMAL LYMPHOPOIESIS Small Lymphocyte Wright-Giemsa Stain • 8-12µm in diameter with high N:C ratio • nucleus is round with coarse, dense chromatin • cytoplasm is weakly basophilic, homogenous and clear without inclusions • plays a big role in the body’s immune response, particularly in viral infections Small Lymphocyte Wright-Giemsa Stain LYMPHOPOIESIS Large Lymphocyte Wright-Giemsa Stain • 12-16µm in diameter with an irregular outline • nucleus can appear irregular and the chromatin is not as coarse as in small lymphocytes • cytoplasm is abundant and tends to be light blue • plays a big role in the body’s immune response, particularly in viral infections Large Lymphocyte Wright-Giemsa Stain Small Resting Lymph with Darker Cytoplasm (Peripheral Blood Tutor CD-ROM) Lymphocytes Functions of Lymphocytes A. B-cells • Differentiation occurs in the adult bone marrow • Seen primarily in p.b. as small, resting lymphs. • Upon Ag exposure in the 2o lymphoid tissues, these enlarge, differentiate into 1. Plasma cells - secrete Igs. & responsible for antibody production 2. Memory cells Plasma Cells Functions of Lymphocytes B. T cells act in 2 major roles: 1. Effector cells - responsible for cellmediated immunity: defense vs. which 5 major types of “not-self pathogens"? viruses, fungi, bacteria, parasites, & tumor cells • Ex., Cytotoxic T cells (Tcyto) – usually CD8+. Destroy Ag-specific target cells on contact. Functions of Lymphocytes B. T cells act in 2 major roles (cont.): 2. Regulatory cells - induce or suppress proliferation / differentiation of effector cells; for example: • Helper/Inducer T cells (TH) - CD4+. Induce other lymphs to carry out certain functions (Ex., inducing B cells to produce Abs.) • Delayed hypersensitivity T cells (TD) – pro-duce chemotactic lymphokines in response to Ags. • Suppressor T cells (TS) - CD8 +; regulate humoral & cell-mediated responses. (Not solely suppressive, despite name.) AND . . . Functions of Lymphocytes C. Natural Killer (NK) cells - type of large granular lymphocyte, or LGL (LGL is a descriptive term derived from flow cytometry studies; it may NOT be obviously granular on Wright’s stain!) Lyses some tumor cells & virus-infected cells - Majority are CD56+ CD16+ NOTE: Not all LGLs seen in p.b. are NK cells, but some are. You couldn’t tell unless you did flow analysis . . . LGL (also “atypical” due to number of granules) (Courtesy Keila Poulsen) Cells That Confuse Lymphocyte ID • Blasts • Large lymphs may be similar in size to blasts, & occasionally may even contain nucleoli. • Monocytes • Have lacy chromatin (with brain-like convolutions); the nucleus is not stained as darkly as it does in lymphs. Monocyte cytoplasm tends towards blue-gray, & has an opaque, “ground-glass appearance.” • Rubricytes/polychromatic normoblasts • Similar in size to lymphs, but rubricyte cytoplasm has a grayish-blue appearance, whereas lymph cytoplasm is a clearer blue. Also, rubricyte chromatin is much denser than lymphocytes’ chromatin. Definitions A. Lymphocytosis - absolute ↑ in # of circulating lymphs above normal range: • ↑ 4.0 x 103/uL in adults • ↑ 9.0 x 103/uL in children (Note difference!) B. Lymphopenia - absolute ↓ in # of circulating lymphs below normal range (due to ↓ production, alterations in lymph traffic, and/or ↑ lymph destruction.) Morphology of Reactive/Atypical Lymphs Type I – some call this “Plasmacytoid lymph” • Smallest of the atypicals (9-20 um). • Variable basophilic cytoplasm; vacuolated, may contain granules. • “Foamy” or “frilly” nuclear appearance, but chromatin still dense. Most likely a triggered (i.e., immunocompetent) B cell. • Classically, this is type most commonly confused with monocytes, but it can also appear in an easier to ID form . .. More Easily Identified Plasmacytoid Lymph (less monocytic looking) (Courtesy Keila Poulsen) Morphology of Reactive/Atypical Lymphs • Type II – aka. “Downey cells”. • These are the classical reactive or atypical lymphs • Classically seen in Infectious Mononucleosis! • Abundant cytoplasm, irregularly- shaped & edges indented by surrounding structures. • Overall “fried egg” or “flared skirt” type of appearance. • No nucleoli “Downey Cells” – atypical lymphs Reactive Lymphocyte Morphology of Reactive/Atypical Lymphs • Type III • Largest of the atypicals (12-35 um). • Vacuolated, very basophilic cytoplasm. • Immature chromatin with nucleoli! • So how do you tell these highly reactive lymphs (reactive lymphocytosis) from blasts (in a malignant lymphocytosis)? The extreme pleomorphism of the reactive lymphs a malignant lymphocytosis looks more clonal, with a more homogeneous cell population. Large Atypical Lymph Morphology of Reactive/Atypical Lymphs NOTE: Plasma cells – generally smaller than Type III atypicals (10-28 um). • Abundant, deep blue, & agranular cytoplasm. • Eccentrically located nucleus. • General “cometary” type appearance. Plasma Cells Plasma Cells Megakaryocytopoiesis Megakaryocytopoiesis Megakaryoblast Promegakaryocyte Basophilic/Granular Megakaryocyte Megakaryocyte Late Stage Megakaryocyte in Bone Marrow, Showing Budding (Courtesy Rodak,2nd ed.) Megakaryocyte Thrombocyte Wright-Giemsa Stain • A normal platelet measures 1,5–3 µm in diameter. • round or oval in EDTA-anticoagulated blood. In blood films made directly from capillary blood they appear aggregated. • Thrombocytes are fragments of the of the megakaryocyte cytoplasm. • Platelet size is of diagnostic significance particularly when considered in relation to the platelet count. • Thrombocytes play a big role in the body coagulation process Thrombocyte Wright-Giemsa Stain Band Neutrophil or Segmented Neutrophil? Wright-Giemsa Stain Small Lymphocyte or Nucleated RBC? Wright-Giemsa Stain RECAP: • Place the following WBCs in order of their phagocytic activity (not speed!) Eos, Basos, Monos, & Segs _____ > ____ > ____ > _____