Bone Marrow, Blood, and Objectives
Lymph Node Histology
• Become familiar with the histology of:
William Matsui, M.D.
• Blood
• Bone marrow
• Lymph node
• Spleen
• Understand
• Development and regulation of hematopoiesis
• Role of lymph nodes in immune regulation
Blood Erythrocytes
• Fluid connective tissue • Greatest cellular
• Cells (formed elements) constituent of the
blood
• Red blood cells
• White blood cells • Biconcave discs
• Platelets • Lack nucleus
• Contain Hemoglobin
• Extracellular matrix
• Transport O2, CO2
• Blood plasma serves as a • Acid-base balance
solvent for a variety of solutes
• Protein-rich matrix
Erythropoiesis Erythropoiesis
Proerythroblast Reticulocyte
Basophillic Normoblast
erythroblast
Polychromatic
erythroblast
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Red Blood Cell - Case Red Blood Cell - Case
• 6 y.o. African American male • Complete blood count
• 1 day severe leg and foot pain. Viral
White blood cells 12,000 (nl 5,000-10,000)
upper respiratory infection for 3 days. % neutrophils 38%
• No current or past medical problems % lymphocytes 60%
• Family history of anemia 20% (nl 37-42%)
Hematocrit 460 (nl 350-500)
• What’s next? Platelets
Red Blood Cell - Case Anemia
Normal Patient • Blood loss
•D ecreased RBC production - Anemia will ultimately result if the rate of RBC
production is less than that of RBC destruction.
• Lack of nutrients, such as iron, B12, or folate.
•B one marrow disorders or bone marrow suppression
• Low levels of trophic hormones which stimulate RBC production (EPO-renal
failure, thyroid hormone, androgens.
• Other diseases (anemia of chronic disease).
• Increased RBC destruction
• Inherited hemolytic anemias (eg, hereditary spherocytosis, sickle cell disease,
thalassemia major)
•A cquired hemolytic anemias (eg, Coombs'-positive autoimmune hemolytic
anemia, thrombotic thrombocytopenic purpura-hemolytic uremic syndrome,
malaria)
Sickle Cell Anemia (1) Sickle Cell Anemia (2)
• Valine for glutamic acid substitution in β-
globin chain
• Leads to α2β(s)2 hemaglobin tetramers
• Poorly soluble compared to nl α2β2 Hb
• sPiocklylemreerdizeblaotoldowcepllOs 2 and pH leading to
• Results in occlusion of venous blood vessels
• Anemia from rapid degradation of sickle
red blood cells
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White Blood Cells Neutrophils (PMNs)
• 5-10 x 106 WBC/ml in adults •4 0-75% of circulating WBC
•L ife span is 2-3 days
• Defense against foreign substances • Diameter is 10-15um
• Single multi-lobed
• Two pools of WBC in blood heterochromatic nucleus
• Circulating pool (included in WBC count)
• Non-circulating pool – marginating pool or group of cells that
rest against blood vessel walls
• Five types, classified by:
• Nuclear morphology
•Polymorphonuclear
•Mononuclear
• Presence or absence of cytoplasmic granules and their staining
properties (if present)
Neutrophils (PMNs) Basophils
• Primary granules (lysosomal granules) • Least numerous mature WBC
• Stain azurophilic • Bi-lobed nucleus
• Contain lysozymes, defensins, elastase, • Granules
acid phosphatase and myeloperoxidase
• Large and abundant
• Secondary granules (specific granules) • Stain with basic dyes
• Specific to neutrophils
• Stain pink or neutral • Contents
• Contain lactoferrin, lysozyme, phagocytins, • Heparin sulfate
collagenases and cytochrome b • Histamine
• Slow reacting substance
• Play a major role in protection against of anaphylaxis
bacterial and fungal infections
Basophils Eosinophils
• Mediate allergic responses • Bi-lobed nucleus
• Express a receptor for IgE • Granules
• Antigen binds to and crosslinks the IgE molecules
• Large refractile red or orange
• Substances released from granules play a major role • Major basic protein
in the hypersensitivity reactions
• May lead to cardiovascular and respiratory collapse • Toxic to parasites
(anaphylaxis) • Vasoactive agents
• Agents that limit inflammatory
response
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Eosinophils Monocytes
• Immunity • Life span of months to years
• Undergo chemotaxis in response to bacterial products • 3-8/100 WBCs
and complement components
• Ingest and destroy antigen-antibody complexes • Nucleus
• Important in defense against parasites • Large, eccentrically place
• Stains deep blue to purple
• Mediate allergic response • Indention becomes more
• Express receptors for IgE pronounced with maturity
• Attenuate inflammatory responses • Cytoplasm
• Preferentially attracted by substances released from • Ground glass appearance
basophils and mast cells • Blue/gray
• Release of histaminase moderates the potentially • Azurophilic granules
deleterious effects of the vasoactive agents
Monocytes Myeloid development
• Little function in circulating blood Myelocyte
• Migrate to peripheral tissues where they assume the
role of macrophages Metamyelocyte
• Respond to presence of necrotic material and invading
microorganisms Promyelocyte
• Large content of lysosomal enzymes Band Myeloblast
• Engulf and destroy tissue debris and foreign Neutrophil
material
• Present antigens to adaptive immune system Segmented Neutrophil
Lymphocytes Lymphocytes
• Variable size (6-18um) • Main functional cell of adaptive immune system
• smallest are • Most found in blood or lymph are “recirculating
quiescent immunocompetent cells”
• Nucleus • T Lymphocytes (thymus-dependent)
• Intensely stained • Have a long life span
• Slightly indented • Involved in cell mediated immunity
• Spherical in shape
• B Lymphocytes (B cells)
• Cytoplasm appears as • Variable life span
a pale blue rim around • Involved in the production of circulating antibodies
nucleus
• Indistinguishable in blood smears or tissue section
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Platelets Megakaryocytes
• Life span of 10 days
• 2-4um in diameter
• Exhibit an intensely stained core
• α-lysosomal granules : red-violet -
azurophilic
• Clotting factors - fibrinogen, von
Willebrand factor, thrombospondin, and
fibronectin, adenine nucleotides
• Growth factors - platelet derived
growth factor = PDGF
• Chemotactic factors
• Vasoactive substances - serotonin
Hematopoiesis Bone marrow histology
Regulate peripheral blood cell types and numbers •Y ellow Marrow – Mostly fat cells
•R ed Marrow – Formation of blood cells
• Maintain adequate numbers of blood cell (homeostasis) • Bone marrow cellularity = 100 - age
• Proper number of specialized cell types
• Increase cell production with changing needs
• Granulocytes - bacterial infections
• Eosinophils - parasitic infections
• Erythrocytes - high altitude
• Prevent excessive cell production (leukemia)
Bone marrow histology Hematopoietic development
• Hematopoietic cords Mesoblastic phase (1-2 mos) Kids and young adults (birth - 20 years)
• Hematopoietic cells • Blood islands of the yolk • Hematopoiesis in both long bones and axial
• Stroma sac skeleton
• Microenvironment • Cells produce hemaglobin
Older adults (20 years - )
• Vasculature and become nucleated • Most hematopoiesis in axial skeleton
• Sinusoidal capillaries RBC's
• Endothelial cells
• Reticular (adventitial cells) Hepatic phase (2-5 mos)
• Normal RBC's produced in
reticuloendothelial system
(liver, spleen, thymus, etc.)
• Initial production of myeloid
and lymphoid cells
Myeloid phase (5-9 mos)
• Bone marrow is primary site
of blood cell production
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Hematopoiesis Regulation of HSC
Stem cells Moore and Lemischka. Science. 2006 Cells
Committed • Bone marrow stromal cells
progenitors • Osteoblasts
• Endothelial cells
Differentiated
cells Extracellular matrix
• Fibronectin, collagen, vitronectin,
tenascin
Molecules involved
• Cytokines - IL-3, SCF, Tie2
• Chemokines - CXCR4/SDF-1
• Morphogens - Notch, Wnt, BMP
• Adhesion molecules - VLA4, LFA-2,
E-selectin, ICAM-1, VCAM-1
Regulation of HSC Regulation of Progenitors
Function of stem cell niche
• Cellular homing
• Protection and survival of HSC
• Maintain pluripotency (prevent differentiation)
• Regulate self-renewal (asymetric vs symetric cell division)
Progenitors Stem Cells
Symmetrical Division Asymmetrical Division
• Cells mature • One daughter cell matures
• Lose some ability to divide as the other remains as an exact
copy of parent (self-renewal)
• Maintains stem cell pool
Bone Marrow Case #1 Bone Marrow Case #1
• 28 y.o. female • Complete blood count
• 3 months of heavy menstrual bleeding, 2
White blood cells 800 (nl 5,000-10,000)
weeks of progressive fatigue % neutrophils 5%
• No current or past medical problems % lymphocytes 95%
• No family history of bleeding 21% (nl 37-42%)
Hematocrit 35 (nl 350-500)
Platelets
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Bone Marrow Case #1 Bone Marrow Failure (1)
Normal Patient • Aplastic anemia
• Acquired - immune destruction of hematopoiesis
• Induced by drugs
• Following infections (hepatitis)
• Inherited -
• Fanconi Anemia - DNA repair mechanisms
• Schwachman-Diamond - RNA processing
• Dyskeratosis congenita - Telomerase
Bone Marrow Case #2 Bone Marrow Case #2
• 75 y.o. male • Complete blood count
• 2 months of malaise, fevers for 3 days, no
White blood cells 62,000 (nl 5-10,000)
abnormal bleeding % neutrophils 1%
• History of hypertension, smoking % lymphocytes 1%
• No family history of cancer or bleeding % other 98%
18% (nl 37-42%)
Hematocrit 15 (nl 350-500)
Platelets
Bone Marrow Case #2 Bone Marrow Failure (2)
• Infiltrative - marrow replacement
• Leukemias
• Myelodysplastic syndrome
• Lymphoma
• Other cancers
• Fibrosis
• Nutritional deficiencies - Vit B12
• Bad infections (viral)
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Acute Myeloid Leukemia Lymphoid Anatomy
• Expansion of myeloid cells in bone marrow
and/or blood with blocked differentiation
• Arise from normal hematopoietic stem
cells
• Many different genetic lesions
• Chromosomal translocations, genetic
instability
• Clinical decisions based on chromsomal
abnormalities
Lymphoid Anatomy Peripheral (Secondary) Lymphoid Tissue
• Two major functional regions: • Lymph nodes
• Primary immune organs
• Sites of initial maturation and development of • Encapsulated
immune competent cells • Interposed between lymphatic vessels (only)
• B cells - bone marrow • Spleen lymphatic tissue
• T cells - thymus
• Encapsulated
• Secondary immune organs • Interposed between lymph and blood circulation
• Sites of interaction between antigens and
immune cells • Diffuse lymphatic tissue
• Antigen driven replication and differentiation into • Unencapsulated
effector cells • GI (tonsils, Peyer’s patches, appendix); Respiratory
tract; Genitourinary
• Found in the lamina propria where pathogens are likely
to invade
Lymph Node Anatomy Lymph Node Histology
• Capsule: Dense, Irregular CT
(collagen type I stains red
brown)
• Trabeculae are extensions of
the capsule
• Reticular tissue - framework
(collagen III stains black with
the Silver stain)
• Hilum - efferent lymph; entry
and exit of blood vessels
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Lymph Node Parenchyma Lymph Node Cortex
• Cortex • Primary follicle
• Primary, secondary nodules
• B cells • Naïve B cells
• No germinal center
• Deep cortex
• Paracortex, juxtamedullary cortex, • Secondary follicle
thymus dependent cortex
• No nodules • Germinal Center
• T cells • Antigen has been
• Medullary cords encountered
• Plasma cells synthesize and • Looks pale because of
release anitbodies into lymph
flowing through the sinuses plasmablasts: large
euchromatic nuclei
• Surrounded by T cells
Reactive Germinal Center Medullary Cord and Sinus
MZ • Dark zone • Medullary cord
LZ • Densely packed B • Lymphocytes
lymphocytes separated by • Plasma cells
DZ reticular cells • Blood vessels
• Macrophages
• Light zone
• Immunoblasts/plasmablasts • Medullary sinus
• Large cells, central nucleus, • Macrophages
prominent nucleolus
• T cells at periphery
• Mantle zone
• Naive B and T cells
Lymph Flow Lymphocyte Trafficking
Afferent Lymphatics à subcapsular sinus à trabecular sinuses à • Naive B and T lymphocytes
medullary sinus à efferent lymphatics home to specific sites
within the lymph node via
peripheral blood
• B cell homing include:
• The primary and
secondary cortical
follicles
• Medullary cords:
plasma cells release
immunoglobulins into
the efferent lymph
• T cell homing
• primarily paracortex
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Lymph Node Case #1 Lymph Node Case #1
• 8 y.o. female
• “Lump” in neck growing over 3 weeks
• No past medical problems
• No family history of cancer
• Physical exam - enlarged, rubbery,
movable 6 cm lymph node in lateral neck
• What next?
Lymph Node Case #1 Lymph Node Case #1
• Lymph node biopsy • Cat Scratch Disease
• Caused by Bartonella henselae
• Most cases resolve spontaneously
• May disseminate to organs (liver, spleen)
• Unclear role for antibiotics in localized
disease
Lymphadenopathy (1) Lymph Node Case #2
• Bacterial - Localized Streptococcal pharyngitis; skin infections; • 58 y.o. male
tularemia; plague; cat scratch disease; diphtheria; chancroid; rat bite • “Lump” in neck growing over 2 weeks. Fever for
fever; brucellosis; leptospirosis; lymphogranuloma venereum;
typhoid fever 1 month, night sweats for 2 weeks, 20 lbs weight
loss
• Viral - Human immunodeficiency virus; Epstein-Barr virus; herpes • No past medical problems
simplex virus; cytomegalovirus; mumps; measles; rubella; hepatitis • No family history of cancer
B; dengue fever • Physical exam - enlarged, rubbery, movable 3
cm lymph node in lateral neck, 2 cm
• Mycobacterial - Mycobacterium tuberculosis; atypical mycobacteria supraclavicular lymph node on right
• Fungal - Histoplasmosis; coccidioidomycosis; cryptococcosis
• Protozoal - Toxoplasmosis; Leishmaniasis • What next?
• Spirochetal - Secondary syphilis; Lyme disease
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Lymph Node Case #2 Lymph Node Case #2
• Lymph node biopsy • Hodgkin’s disease
• Characterized by Reed-Sternberg cells
• Derived from B cells
• Affects kids and elderly
• May be caused by EBV
• May be associated with “B” symptoms - fever,
night sweats and weight loss
• Highly curable with chemotherapy
Lymphadenopathy (2) The Spleen
• Cancer - Squamous cell cancer head and neck; Functions
metastatic; lymphoma; leukemia • Site of fetal hematopoiesis
• Reservoir for red blood cells
• Lymphoproliferative angioimmunoblastic • Filters blood
lymphadenopathy with dysproteinemia • Iron retrieval
• Immune regulation
• Autoimmune lymphoproliferative disease -
Rosai-Dorfman's disease, hemophagocytic Spleen
lymphohistiocytosis
• White pulp
• Immunologic - Serum sickness; drug reactions
(phenytoin) • lymphoid tissue
• Endocrine - Hypothyroidism; addison's disease • Red pulp
Spleen • sinusoids
• red blood cells
• Capsule
• Periarterial
• Dense connective lymphoid sheath
tissue
• lymphocytes
• White pulp
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• lymphoid tissue
• Red pulp
• sinusoids
Spleen Splenic Vasculature
• Central artery
• Periarterial
lymphoid sheath
• Mantle zone
• Trabecular vein
MALT
Mucosal associated lymphoid tissue
• GALT: gut-associated lymphoid tissue
• BALT: bronchial/tracheal-associated lymphoid tissue
• SALT: skin associated lymphoid tissue
• etc...
• Antigen presentation of luminal antigens to MALT by
specialized epithelial cells (M cells)
• Primary immunoglobulin isotype produced is IgA
(secretory)
• Intraepithelial lymphocytes also play a role in immune
surveillance at mucosal surfaces.
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