Lecture 2 and 3 leukopoiesis bone marrow wbc disorders abdulkarim aldosari
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Lecture 2 and 3 Leukopoiesis, bone marrow, WBC disorders Abdulkarim Aldosari. O bjectives. Define and describe Leukopoiesis List proper cell maturation of the myeloid and lymphoid series Characterize changes in neutrophil count and morphology that develop in response to infections

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Lecture 2 and 3 leukopoiesis bone marrow wbc disorders abdulkarim aldosari

Lecture 2 and 3

Leukopoiesis, bone marrow, WBC disorders

AbdulkarimAldosari


O bjectives

Objectives

  • Define and describe Leukopoiesis

  • List proper cell maturation of the myeloid and lymphoid series

  • Characterize changes in neutrophil count and morphology that develop in response to infections

  • Characterize the sequence of events that occur during phagocytosis

  • Define neutropenia and identify causes

  • Describe the etiology and neutrophil morphology of Chediak-Higashi syndrome, May-Hegglin, Alder-Reilly, and Pelger-Huet anomalies.

  • Describe chronic granulomatous disease

  • Compare and contrast white cell anomalies in regard to morphology

  • Define monocytosis and lymphocytosis

  • List disorders associated with monocytosis and lymphocytosis

  • Distinguish between absolute and relative, benign and malignant lymphocytosis

  • Describe infectious mononucleosis


White blood count

White blood count

Normal indices for WBC

DifferentialAbsolute counts

Lymphocytes20-44%1.2-3.4 x 103/µl

Monocytes2-9%0.11-0.59 x 103/µl

Neutrophils50-70%1.4-6.5 x 103/µl

Bands2-6%0-0.7 x 103/µl

Eosinophils0-4%0-0.5 x 103/µl

Basophils0-2%0-0.2 x 103/µl

Absolute count = %/100 x WBC


Leukopoiesis

Leukopoiesis

  • Leukopoiesis-is a form of hematopoiesis

  • White blood cells (WBC, or leukocytes) are formed in bone marrow

  • WBCs are formed from the differentiation of pluripotent hematopoietic stem cells which give rise to several cell lines with more limited differentiation potential

  • Regulated by the production of interleukins and colony-stimulating factors (CSF).

    Two main groups of WBCs:

  • Myeloid cells (granulocytes)

    • Large cells with lobed nuclei and visibly staining granules; all are phagocytic

    • Neutrophils

    • Eosinophils

    • Basophils

    • Monocytes

  • Lymphoid cells

    • Agranulocytes -lack visibly staining granules


Myelopoiesis granulocytopoiesis

Myelopoiesis (granulocytopoiesis)

  • Hematopoietic system produces enough neutrophils (~1.3 x 1011 cells per 80-kg person per day) to carry out physiologic functions

  • Also has a large reserve stored in the marrow, which can be mobilized in response to inflammation or infection

  • An increase in the number of blood neutrophils is called neutrophilia

  • Presence of immature cells is termed a shift to the left

  • A decrease in the number of blood neutrophils is called neutropenia

  • Mature neutrophils, eosinophils, basophils have similar patterns of proliferation, differentiation, division, storage in BM and delivery to PB.


Lecture 2 and 3 leukopoiesis bone marrow wbc disorders abdulkarim aldosari

Proliferation

Maturation

Storage

Functional pools


Blood cells in peripheral blood

Blood Cells in peripheral blood

  • Only formed elements that are complete cells

  • Make up less than 1% of total blood volume


Types of wbcs normally seen on smear

Types of WBCs normally seen on smear


Neutrophils in peripheral blood

Neutrophils in peripheral blood

  • Most numerous type of leukocyte = 50-70%

  • Diameter 10-12µm

  • 3000-7000 cells/mm3

  • Most mobile cell lines in human

  • Chemically attracted to sites of inflammation

  • Cytoplasmic granules – containing enzymes

    • > killing and digestion of bacteria and fungi

  • Active phagocytes of bacteria


Maturation of neutrophils

Maturation of neutrophils

  • 14 days to develop

  • Mature cells leave the marrow > moving through transiently formed pores in endothelial cells

  • Become part of functional pool > half circulating cells or half marginated cells (line blood vessel walls)

  • After few hours > leave blood for tissues and body cavities as directed by chemotactic factors in response to inflammation or infection

  • Once in tissues they do not re-enter circulation of marrow

  • As they leave blood, they are replaced by other cells from BM


Maturation of neutrophils1

Maturation of neutrophils

The myeloblast is the first recognizable precursor cell

  • Round nucleus, reddish blue, smooth nuclear membrane,

  • Fine, delicate, evenly stained chromatin,

  • Nucleoli – 0-2

  • Bluish, non-granular cytoplasm

  • N:C = 7:1 – 5:1, central

  • 15 to 20 µm

  • Not present in normal PB

  • Normal marrow – 0-2 %

  • Followed by the promyelocyte


Maturation of neutrophils2

Maturation of neutrophils

The promyelocyte evolves when the primary, or azurophil, lysosomal granules are produced

  • 12-24 µm

  • Not present in normal PB, 1-4% in BM

  • N:C = 5:1 to 3:1

  • Chromatin – finely granular

  • Nucleoli – faintly visible or not distinct

  • Blue cytoplasm, with lighter zone near nucleus

  • Cytoplasm not indented by adjacent cells

  • The promyelocyte divides to produce the myelocyte


Maturation of neutrophils3

Maturation of neutrophils

  • The myelocyte- cell responsible for the synthesis of the specific, or secondary, granules

  • Nucleus- round, oval, flattened on one side, eccentrically located

  • Chromatin strand – condensed, partly clumped, thickened, unevenly stained

  • Nucleoli – absent/rare

  • Last myeloid precursor capable of division

  • Smaller than promyelocytes

  • N:C – 2:1 to 1:1

  • 10-18 µm

  • Cytoplasm more pink

  • Not seen in normal PB, 5-20% in BM

  • End of proliferation pool of cells


What are these granules

What are these granules?

  • The primary granules contain hydrolases, elastase, myeloperoxidase, cathepsin G, cationic proteins and bactericidal/permeability-increasing protein - important for killing gram-negative bacteria

  • Azurophil granules also contain defensins, a family of cysteine-rich polypeptides with broad antimicrobial activity against bacteria, fungi, and certain enveloped viruses


What are these granules1

What are these granules?

  • Specific, or secondary, granules-contain lactoferrin, vitamin B12–binding protein, membrane components of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase required for hydrogen peroxide production, histaminase, and receptors for certain chemo-attractants and adherence-promoting factors (CR3) and other membrane receptors

  • The secondary granules do not contain acid hydrolases and therefore are not classic lysosomes

  • Secondary granule contents are released extracellularly, and their mobilization is important in modulating inflammation


Maturation of neutrophils4

Maturation of neutrophils

  • As maturation proceeds, nucleus becomes more indented (kidney bean shaped) = metamyelocyte

  • The proliferation phase from myeloblast to the metamyelocyte takes about 1 week

  • Indentation of nucleus = less than half the width of the round nucleus

  • Chromatin – clumped

  • Cells do not divide

    • Maturation pool of WBCs in BM

  • Do not have nucleoli

  • N:C = 1:1, central or eccentric

  • Small pinkish granules

  • Smaller than myelocytes = 10-18 µm

  • Absent in normal PB, 5-20% in BM


Maturation of neutrophils5

Maturation of neutrophils

  • Band neutrophil formed when the nuclear indentation is greater than the width of the nucleus

  • From metamyelocyte to mature neutrophil takes another week

  • Appearance of horseshoe, central or eccentric

  • May appear twisted or folded

  • Nuclear chromatin is pyknotic

  • Ends have a dark condensed mass

  • N:C = 1:1 to 1:2

  • Cells are smaller than metamyelocytes

  • 10-16 µm

  • Maturation pool in BM 10-35%

  • In PB 2-6%


Maturation of neutrophils6

Maturation of neutrophils

  • Segmented neutrophil – two to five nuclear lobes connected by a thin filament/strand

  • Nuclear chromatin is purplish-red, clumped

  • N:C = 1:3

  • Cells are smaller than metamyelocytes

  • 10-16 µm

  • Maturation pool in BM 5-15%

  • In PB 50-70%


Maturation of neutrophils7

Maturation of neutrophils


Maturation of neutrophils8

Maturation of neutrophils

  • Borderline cells should be classified as the more mature cell

  • In BM – proliferation pool ( committed stem cell- blast- promyelocyte-myelocyte)

  • In BM – maturation pool (metamyelocyte and bands)

  • In BM – storage pool (mature cells for release into peripheral blood)


Neutrophil function

Neutrophil function

The main function of neutrophils – internalization of microorganisms

Phagocytosis-three phases:

  • Migration and diapedesis (outward passage of cells through intact vessel walls)

  • Opsonization and recognition

  • Ingestion, killing and digestion (phagocytosis)


Neutrophil function1

Neutrophil function

Step 1. Migration

Bacteria in tissue > sends out signal via chemoattractants > stimulate changes in neutrophil morphology and migration > neutrophil clings to the endothelial receptors > penetrates through the endothelial cells via diapedesis > neutrophil migrates to site of infection = chemotaxis

Three modes of neutrophil migration

Locomotion – random, non-directional movement of neutrophils as they roll along vessel endothelium until site of infection or injury

Chemokinesis – chemoattractants accelerate the migration speed of neutrophils

Chemotaxis – directional migration to site of infection


Neutrophil function2

Neutrophil function

Step 2. Opsonization

Neutrophils recognize and attach to infecting organism via circulating Igs and activated complement components which coat the surface of the bacteria

Neutrophil membranes carries receptors for the Fc fragment of Igs and activated complement only.

Ingestion will not take place without

the presence of membrane-bound Igs


Neutrophil function3

Neutrophil function

Step 3. Phagocytosis: ingestion, killing, digestion

Ingestion begins as soon as neutrophil receptor and bacteria bind together > pseudopods envelop microbe > form vacuole = phagosome > cytoplasmic granules fuse to phagosome, release contents > lytic action of enzymes > killing and digestion of microbe =

Killing and digestion mechanism:

  • non- oxygen dependent – lysing of bacterial cell wall by lysosomal and proteolytic enzymes

  • Oxygen dependent (respiratory burst/oxidative burst)-rapid release of NADPH oxidase > production of reactive oxygen species (superoxide and hydrogen peroxide) from neutrophils and monocytes as they come into contact with different bacteria or fungi.


Disorders of neutrophils

Disorders of neutrophils

  • Neutrophils -the first line of defense against most bacterial pathogens.

  • Requires that the host have sufficient numbers of neutrophils that respond to chemotactic stimuli and ingest and kill bacteria

  • Classified as quantitative and qualitative

  • Quantitative

    • Neutropenia = decease in number < 1.5 x 109/L

    • Neutrophilia = increase in number > 30,000/mm3or 30 x 109/L

  • Qualitative

    • Neutrophil dysfunction – impaired migration or altered bacteriocidal activity


N eutrophilia

Neutrophilia

  • Absolute count > 30,000/mm3 or 30 x 109/L

  • A classic response to infections and inflammation

  • Accelerated release of neutrophils from BM

  • Accompanied by a “left shift” = increase in the number of immature cells (metamyelocytes and bands) in the PB

  • To be differentiated from an ↑ in circulating neutrophils and immature cells due to chronic myeloid leukemia CML and other myeloproliferative disorders


Differentiate neutrophilia

Differentiate neutrophilia

  • Major distinction = immature cells limited to metamyelocytes and bands in infections but more immature cells (myelocytes, promyelocytes and blasts) present in neoplastic processes.

  • LAP – leukocyte alkaline phosphatase is ↑ in leukemoid reactions, i.e. reactions due to an infection, ↓ in CML. Done by cytochemical staining.

    A leukemoid reaction is an increase in white blood cell count similar to what occurs in people with leukemia. However, the reaction is actually due to an infection or another disease and is not a sign of cancer. Blood counts will usually return to normal when the underlying condition is treated.

  • Other cause of neutrophilia – tissue necrosis, metabolic disorders, stress, rigorous exercise, pregnancy, smoking, trauma, hemolysis, postsplenectomy

  • Various reactive changes that occur to the neutrophil during infection also occur during these others causes


Neutrophilic morphology

Neutrophilic morphology

Changes in the morphology of neutrophils that occur during a leukemoid reaction

Toxic granulation

  • Associated with sever infections

  • Granules enlarge and take on darker staining properties

  • Usually peroxidase-positive primary granules

    Dohle bodies

  • Usually accompanies toxic granulation

  • Pale blue inclusions at periphery of cytoplasm

  • Aggregated strands or rough endoplasmic reticulum

    Vacuolated cytoplasm

    Ingested microorganisms


Neutropenia

Neutropenia

Absolute decrease in the number of circulating neutrophils < 1.5 x 109/L

Mild – 1.0-1.5 x 109/L

Moderate – 0.5-1.0 x 109/L

Severe - < 0.5 x 109/L

Life threatening - <0.2 x 109/L

Not normally the only indicator of disease > should be correlated with pt history and other clinical findings and lab results.

Due to recurrent bacterial infection – Staph Aureus, Strep viridans, Gram negative enteric bacteria

Occur in the cutaneous and soft tissues > spread to the blood stream


Neutropenia1

Neutropenia

Due to increased destruction or removal of neutrophils – in PB

Infections, immune disorders

Maturation defect – impaired BM release

Megaloblastic anemia

Proliferation defect – decreased production

Aplastic anemia

BM replacement disorders

Drugs reactions

Myeloablative therapy – radiation therapy

BM fibrosis

Abnormal distribution

Hypersplenism

Can be acquired or congenital


Acquired neutropenia

Acquired neutropenia

Infections

  • mostly viral – influenza A ,B, rubella, herpes simplex, hepatitis A, B, and RSV

  • neutropenia appears during first few days of infection – within 24 to 48 hrs

    Immune mediated

  • Anti-neutrophil antibodies - alloantibodies or autoantibodies

    • Similar to RBC hemolytic disease of the newborn

      • Antigens shared by fetus and father, absent from mother, mother develops ab which cross placenta > destroys fetal neutrophils

    • Autoantibodies- rheumatoid arthritis, systemic lupus, chronic hepatitis

      Treatment – antibiotics for infections, prednisone for autoimmune response


Congenital neutropenia

Congenital neutropenia

Defects in genes encoding ribosomal proteins (SBDS, RMRP) and mitochondrial proteins (AK2, TAZ) are associated with congenital neutropenia syndromes

Some variants of congenital neutropenia may be due to mutations in genes controlling glucose metabolism (SLC37A4, G6PC3) or lysosomal function (LYST, RAB27A, ROBLD3/p14, AP3B1, VPS13B).

Patients with congenital neutropenia are prone to severe and recurrent bacterial infections such as otitis media, bronchitis, pneumonia, osteomyelitis, or cellulitis. Long-term neutropenic states also predispose to fungal infections.

http://asheducationbook.hematologylibrary.org/content/2009/1/344.full


Qualitative disorders of neutrophils

Qualitative disorders of neutrophils

Hereditary abnormalities in function > bacterial infections

Familial due to a general metabolic defect

Pathophysiology is unknown

Functional defects are classified by the general type of defect

  • Phagocytic/killing defects

  • Motility/chemotaxis defects

  • Granule function and structure defect

  • Adhesion defects


Qualitative disorders of neutrophils1

Qualitative disorders of neutrophils

Classes of qualitative neutrophil disorders

  • Cytoplasmic granules

  • Disturbances of the respiratory burst

  • Chemotaxis

    WBC counts are variable

    PB smear is not used to differentiate the disorders except for the large granules present in Chediak-Higashi Syndrome


Chediak higashi syndrome

Chediak-Higashi Syndrome

  • Rare autosomal recessive disease -one in which neutrophils, monocytes, and lymphocytes contained giant cytoplasmic granules

  • Dysfunction characterized by increased fusion of cytoplasmic granules

  • The presence of giant granules in the neutrophil interferes with their ability to traverse narrow passages between endothelial cells.


Chediak higashi syndrome1

Chediak-Higashi Syndrome

  • The decrease in phagocytosis results in recurrent pyogenic infections, partial albinism and peripheral neuropathy

  • Patients have light skin and silvery hair, solar sensitivity and photophobia – albinism

  • Other features of the disease include neutropenia, thrombocytopenia, natural killer cell abnormalities

  • Patients with this syndrome exhibit an increased susceptibility to infection due to defects in neutrophil chemotaxis, degranulation, and bactericidal activity.


Clinical features of chs

Clinical Features of CHS

  • The infections involve the mucous membranes, skin, and respiratory tract.

  • Susceptibility to both gram-positive and gram-negative bacteria as well as fungi, with Staphylococcus aureus being the most common infecting organism.

  • The neuropathy may be sensory or motor in type, and ataxia may be a prominent feature.

  • Covers two of the four disorders – granule structure/function and chemotaxis defect

  • Treatment – antimicrobial therapy, ascorbic acid


Chronic granulomatous disease

Chronic granulomatous disease

Best understood disease of neutrophil function

  • Mutation in NADPH oxidase > ineffective bacterial killing

  • Mostly X-linked recessive – affects mostly boys by first year

  • Failure in the activation of the respiratory burst > little or no superoxide production

  • Mutation in the gene encoding the NADPH oxidase


Chronic granulomatous disease1

Chronic granulomatous disease

  • Clinical picture – recurrent pulmonary infections, infected eczematous rash, neutrophilia instead of neutropenia

  • Diagnosis – nitroblue tetrazolium test NBT

    • It is negative in CGD= does not turn blue. The higher the blue score, the better the cell is at producing reactive oxygen species

    • Depends upon the direct reduction of NBT to the insoluble blue compound formazan by NADPH oxidase; NADPH is oxidized in the same reaction

  • Treatment – prophylactic antibiotic therapy, gamma interferon, BM and stem cell transfusions


Abnormal neutrophil morphology

Abnormal neutrophil morphology

Acquired

Hypersegmentation – five or more nuclear lobes

  • Megaloblastic anemia

  • Myeloid malignancies

    (AML, Myelodysplastic syndromes)

    Pseudo-Pelger-Huet anomaly - Hyposegmentation

    70-90% Bilobed or no lobulation

    Secondary to

  • Myeloid malignancy

  • Drugs –sulfonamides etc.

    Nuclear chromatin very coarse and condensed

    Dumbbell shaped

    Versus true Pelger-Huet – autosomal dominant, inherited disorder


Abnormal neutrophil morphology1

Abnormal neutrophil morphology

Inherited

  • Alder-Reilly anomaly

    • Prominent, dark staining coarse cytoplasmic granules,

    • Similar to toxic granulation but larger

  • May-Hegglin anomaly

    • Dark blue staining cytoplasmic inclusions in neutrophils

    • Larger than Dohle bodies; thrombocytopenia, giant platelets

  • Chediak-Higashi syndrome

    • Giant granules in granulocytes

  • Pelger-Huet anomaly –

    • Bilobed or non-segmented nuclei


Eosinophils

Eosinophils

  • Large, round, secondary, refractile granules staining orange to reddish-brown > takes on the acid eosin stain

  • Relatively uncommon – 0-3% in BM, 0-4% in PB

  • Granules contain hydrolytic enzymes – peroxidase, acid phosphatase, aryl sulfatase, beta-glucuronidase, phospholipase, cathepsin, ribonuclease


Eosinophils1

Eosinophils

  • Diurnal variation in circulating eosinophils- % ↑ at night and ↓ in the morning

  • Bilobed nucleus

  • 10-14µm

  • Lasts 5 days in circulation

  • Stem cell kinetics not as well known as for neutrophils


Eosinophils2

Eosinophils

  • Substances in the granules of Eosinophils become cytotoxic when they are released on the surface of parasites.

  • Eosinophils are not phagocytic, but they intoxicate nematodes and other parasites and bacteria.

  • The cytotoxic substances are major basic protein, which kill helminthes, eosinophil cationic protein (an extremely efficient killer of parasites and potent neurotoxins) and eosinophil peroxidase (kills bacteria, helminthes and tumor cells).

  • Eosinophils are involved in hypersensitivity reactions.


Basophils

Basophils

  • Maturation, division and proliferation similar to neutrophils

  • Nucleus is masked by large basophilic granules

  • Granules = histamine and heparin, are water soluble

  • Respond to acute and delayed allergic reactions

  • Granules formed in the myelocytic stage and continue to be produced throughout later stages of maturation


Basophils1

Basophils

  • Mature basophils rarely have more than two segments

  • Circulate for few hours then migrate into skin, mucosa and other serous membranes

  • All stages are smaller than neutrophils

  • In BM 0-1%, In PB 0-2%

  • Diurnal variation in circulating basophils- % ↑ at night and ↓ in the morning

  • Mast cells = tissue basophils


Basophils2

Basophils

  • Circulating basophils and mast cells residing in the tissues are morphologically similar

  • Granules contain histamine and other vasoactive amines.

  • Cells are involved in hypersensitivity reactions.

  • The binding of IgE to the cells stimulate the release of histamine, but also of prostaglandins, leucotrienes and cytokines.

  • Some mast cell contains trypsin and cytoplasmic IgE and others contains both trypsin and chymotrypsin.


Eosinophilia and basophilia

Eosinophilia and Basophilia

Eosinophilia

  • Mostly caused by – allergic reactions in developed countries

  • Parasitic infection – helminthic

  • Also in malignant hematopoietic disorders, skin disorders, pulmonary disorders, inflammatory disorders

  • Disappears with the resolution of the disease

    Basophilia

  • Secondary to allergic reactions

  • Malignant hematological disorders - CML


Monocytes and macrophages

Monocytes and macrophages

  • In PB monocytes 12-18 µm

  • 2-9% of WBC in PB

  • 0-2% in BM

  • Larger than a mature neutrophil

  • N:C = 1:1 or 2:1

  • With wrights stain = cytoplasm- dull gray blue

  • Numerous fine small reddish/purplish stained granules > ground-glass, cloudy appearance to the cytoplasm


Monocytes and macrophages1

Monocytes and macrophages

  • Digestive vacuoles may be present in cytoplasm

  • In cytoplasm > see phagocytized RBC, bacteria, fungi, cell fragments, nuclei

  • Nuclei – kidney shaped,, folded or indented, lobular

  • Distinct convolutions in the nucleus (brain-like)

  • Lacy chromatin with small chromatin clumps

  • Cells may be round or have blunt pseudopods


Monocytes and macrophages2

Monocytes and macrophages

Monocytes circulate from 8hrs to 3 days before entering tissues > macrophages

  • Pulmonary alveolar macrophages, peritoneal macrophages, splenic macrophages, Kupffer cells in liver, connective tissue macrophages

  • Macrophages are large - 20-80 µm

  • Irregular shaped tissue cells

  • One to two nucleoli, clumped chromatin, abundant cytoplasm with vacuoles

  • Numerous azurophilic granules

  • Macrophages = histiocytes

  • Mononuclear phagocytic system MPS = monocytes, macrophages and their precursors (promonocytes and monoblasts)


  • Monocytes and macrophages3

    Monocytes and macrophages

    • Inactivated and circulating macrophages are called monocytes

    • When they migrate to extravascular tissues they are known as macrophages

    • Macrophages contain lysosomes filled with various catabolic enzymes

    • The macrophage membrane contains receptors for binding complement components and immunoglobulins

    • Macrophages destroy other phagocytized organisms or molecules by production of free radicals and digestive enzymes

    • Tumor necrosis factor (TNF) is produced by macrophages stimulated by bacterial cell wall components. TNF turns a tumor into hemorrhagic necrosis


    Monocytes

    Monocytes

    Note the four distinct characteristics

    Dull gray-blue cytoplasm

    Convolutions of the nucleus

    Blunt pseudopods

    Lacy chromatin


    Absolute monocytosis

    Absolute monocytosis

    Reactive monocytosis

    • Secondary to chronic infection, inflammatory conditions

    • Accompanied by neutrophilia, mild to moderate anemia

    • Mature monocytes with clumped nuclear chromatin

    • Nucleus is folded, indented

    • Cytoplasm spread out and vacuolated, fine granules

      Monocytosis due to malignancy

    • More immature forms

    • Fine nuclear chromatin

    • Nucleoli present


    Lymphopoiesis

    Lymphopoiesis

    Lymphoid progenitor > T or B cell

    T cell in thymus

    B cell in adult bone marrow

    Null cells (natural killer cell NK) in bone marrow – unknown maturation sequence

    T, B, Null cells morphologically identical

    Distinguished functionally and by immunological markers

    In thymus and bone marrow lymphocytes differentiate, proliferate and mature into fully functional immune cells

    In secondary lymphoid organs – lymphocytes interact with antigen-presenting cells (APC), phagocytes and macrophages in an active immune response

    Secondary organs = lymph nodes, spleen, mucosal tissues (tonsils, Peyer’s patch)


    Lymphopoiesis1

    Lymphopoiesis

    • T-lymphocytes, which undergo maturation in the thymus = 75% of lymphocytes

    • T-lymphocytes possess distinct cell surface antigens (CD3)

      • Play a central role in cell-mediated immunity

      • T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells

    • B-lymphocytes produce the antibodies after antigen exposure

      • Become plasma cells.

      • B-lymphocytes comprise 25% of all lymphocytes.

    • NK cells – large granular lymphocytes - ~5% of blood and splenic lymphocytes

      • Play important role in innate immune response to infections and some tumors


    Lymphopoiesis2

    Lymphopoiesis

    Lymphoblast

    • Large round nucleus, small basophilic cytoplasm N:C = 7:1 to 4:1

    • Nuclear chromatin – thin, loose evenly stained strands, not clumped

    • Nucleoli – 1-2

    • 10-20 µm

      Prolymphocyte

    • Intermediate chromatin pattern with clumping

    • 9-18 µm

    • N:C = 5:1 to 3:1

    • Nucleoli – 0-1

    • Slightly different from lymphoblast


    Lymphopoiesis3

    Lymphopoiesis

    Plasmablasts

    • Similar to other blast cells

    • N:C – 5:1 to 4:1

    • Cytoplasm – blue

    • 16-25 µm

      Plasmacytes

    • End stage of B-lymphocyte lineage

    • Not seen in normal PB, 1% in BM

    • 10-20 µm

    • Round, oval, slightly irregular margins

    • Cytoplasm deep blue with perinuclear clear zone

    • Secretory vesicles at cell periphery

    • Nucleus – eccentrically placed


    Lymphocytes

    Lymphocytes

    • Second most numerous white cell in the blood 20-44%

    • 5-15% in BM

    • Mostly small - 8-12 µm

    • Small resting lymphocytes = size of a RBC

    • Some intermediate, and large

    • Cytoplasm – scant, colorless or light blue

    • Nucleus – coarse, absent nucleoli

    • Size varies based on thickness of smear – in thin area of smear – appear large


    Lymphocytes1

    Lymphocytes

    • Large lymphocytes are indented by RBCs > holly-leaf shaped

      • Cytoplasm – blue, clear, darker blue at periphery of the cell

    • Most do not have granules – some large cells may have very distinct reddish granules

    • N:C – 4:1 to 2:1

    • May have nucleoli – capable of growth and replication


    Large lymphocyte versus monocyte

    Large lymphocyte versus monocyte

    Nucleus is clumped

    Cytoplasm clear, darker blue at periphery of cells

    Indented cytoplasm

    Nucleus is lacy, brain-like convolution

    Ground-glass appearance of cytoplasm

    Blunt pseudopods between cells

    Compress cells - not indented by cells


    Lymphocyte morphology

    Lymphocyte morphology

    Reactive lymphocytes = transformed or benign lymphocytes

    Not the same as atypical = malignant-appearing cells

    Reactive lymphocytes occur in normal patients

    < 10% of total lymphocytes

    Large – 9-30 µm N:C – low to moderate

    Nucleus round to irregular

    Cytoplasm – colorless to dark blue, uneven staining

    Chromatin – coarse to moderately fine

    Nucleoli absent to distinct


    Lymphocyte morphology1

    Lymphocyte morphology

    • Plasmacytoid lymphocyte – prominent chromatin clumping, perinuclear halo, eccentric nucleus similar to a plasma cell

    • Immunoblast – a reactive lymphocyte with a prominent nucleolus


    Lymphocytosis

    Lymphocytosis

    • Increase in lymphocytes in the peripheral blood

    • Lymphocytosis - detected when a complete blood count is routinely obtained.

    • Lymphocytes normally represent 20 to 44% of circulating white blood cells.

    • In absolute lymphocytosis, the total lymphocyte count is elevated.

      • In adults= lymphocyte count >4000 per microliter, in older children >7000 per microliter and in infants >9000 per microliter.

      • Decreases with age

    • The absolute lymphocyte count (ALC) can be directly measured by flow cytometry or calculated by multiplying the total WBC count by the percentage of lymphocytes found in the differential count


    Lymphocytosis1

    Lymphocytosis

    • Relative lymphocytosisoccurs when the percentage of lymphocytes is > 40%, while the absolute lymphocyte count is within normal range.

    • During neutropenia when lymphocytes are not affected

    • When patient is dehydration

    • Relative lymphocytosis is normal in children under age 2


    Causes of reactive lymphocytosis

    Causes of reactive lymphocytosis

    Viral

    Bacterial

    Drugs reactions

    Miscellaneous (allergic reactions, autoimmune diseases)

    Most notable cause – Infectious mononucleosis (IM)

    • Large mononuclear cells first thought to be monocytes

    • Now known to be lymphocytes

    • Found that serum from patients with IM contained antibodies against sheep RBCs – basis for the Monospot test

    • Test used to measure IgM heterophile abs (abs that react with cells of other species)


    Infectious mononucleosis

    Infectious mononucleosis

    • Virus responsible for IM = Epstein-Barr virus (EBV) – DNA virus

      • Associated with Burkitt’s lymphoma

    • More common in young people – 17-25yrs

    • Transmitted through saliva > kissing disease

    • Onset is abrupt > sore throat, lymphadenopathy, fever, malaise, excessive fatigue

    • Nausea, headache, sweats etc., last 2-3wks

    • Multiple organ involvement


    Infectious mononucleosis1

    Infectious mononucleosis

    • Multiple organ involvement

    • Uncommon in older adults

    • Diagnosis of lymphocytosis is influenced by age of patient

    • CLL more likely cause of lymphocytosis in older adults than IM

    • 80-90 % of adults have had exposure and are immune to IM

    • Treatment - Self limiting disease, supportive therapy -bed rest, acetaminophen or ibuprofen


    Laboratory examination

    Laboratory examination

    CBC with differential

    • WCB count is ↑ 12-25 x 109/L = absolute lymphocytosis

      Peripheral blood smear

    • Presence of reactive lymphocytes >20%

    • Differentiate between lymphoblast, monoblasts and reactive lymphs

      Serologic studies

  • Monospot – heterophile antibody test (+) for Infectious mono

  • Repeat in 1 week if initially negative and IM suspected

  • Negative Monospot > ELISA for EBV viral capsid antigen

  • If negative do further cultures, serologic tests, lymph node or BM biopsy


  • Laboratory examination1

    Laboratory examination


    Lymphopenia

    Lymphopenia

    When absolute count < 1000 lymphocytes/mm3 (< 1.0 x 109/L)

    Normocytic normochromic anemia

    Granulocytopenia may also be present

    Causes – infections, auto immune diseases, systemic diseases, malignant disorders, other disorders ( immunodeficiency disorders, nutritional deficiencies), radiation therapy

    Most have a decrease in T lymphocytes, CD4+ helper T cells.


    Peripheral blood smear

    Peripheral blood smear

    If abnormalities in white blood cell count > perform a blood smear

    Under low power: Scan the edges and center for abnormal cells

    High power: perform WBC estimate. Count 10 fields and average

    no./high power fieldestimated total WBC count

    2-44000-7000

    4-67000-10,000

    6-1010,000-13,000

    10-2013,000-18,000

    Correlate estimate with automated instrument count

    Evaluate morphology of WBCs, record abnormalities


    Peripheral blood smear1

    Peripheral blood smear

    Oil immersion 100x

    Perform 100 WBC differential count

    Moving in a zig-zag

    Count all WBCs until 100 cells are counted

    Correct total WBC based on the number of nucleated red cells

    Corrected WBC = WBC x100

    100 + No. of NRBCs/100 WBCs


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