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1 Bone Marrow, Blood, and Lymph Node Histology William Matsui, M.D. Objectives • Become familiar with the histology of: •Blood • Bone marrow

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Published by , 2017-02-03 05:15:03

Objectives Bone Marrow, Blood, and Lymph Node Histology

1 Bone Marrow, Blood, and Lymph Node Histology William Matsui, M.D. Objectives • Become familiar with the histology of: •Blood • Bone marrow

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

2

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

3

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

5

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

6

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)

7

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

8

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

9

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

10

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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