Lecture 2 and 3

Leukopoiesis, bone marrow, WBC disorders

Abdulkarim Aldosari

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 countNormal indices for WBC

Differential Absolute counts

Lymphocytes 20-44% 1.2-3.4 x 103/µl

Monocytes 2-9% 0.11-0.59 x 103/µl

Neutrophils 50-70% 1.4-6.5 x 103/µl

Bands 2-6% 0-0.7 x 103/µl

Eosinophils 0-4% 0-0.5 x 103/µl

Basophils 0-2% 0-0.2 x 103/µl

Absolute count = %/100 x WBC

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)• 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.

1. Proliferation

2. Maturation

3. Storage

4. Functional pools

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

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• 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 neutrophilsThe 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 neutrophilsThe 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 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?

• 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 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 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 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 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 neutrophils

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 functionThe main function of neutrophils – internalization of microorganisms

Phagocytosis - three phases:

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

2. Opsonization and recognition

3. Ingestion, killing and digestion (phagocytosis)

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 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 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:

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

2. 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• 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/mm3 or 30 x 109/L

• Qualitative Neutrophil dysfunction – impaired migration or altered bacteriocidal activity

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• 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 morphologyChanges 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

NeutropeniaAbsolute 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

NeutropeniaDue to increased destruction or removal of neutrophils – in PB

Infections, immune disorders

Maturation defect – impaired BM releaseMegaloblastic anemia

Proliferation defect – decreased productionAplastic anemiaBM replacement disordersDrugs reactionsMyeloablative therapy – radiation therapyBM fibrosis

Abnormal distributionHypersplenism

Can be acquired or congenital

Acquired neutropeniaInfections

• 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 neutropeniaDefects 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 neutrophilsHereditary 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

1. Phagocytic/killing defects

2. Motility/chemotaxis defects

3. Granule function and structure defect

4. Adhesion defects

Qualitative disorders of neutrophilsClasses of qualitative neutrophil disorders

1. Cytoplasmic granules

2. Disturbances of the respiratory burst

3. 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• 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 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• 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 diseaseBest 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 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 morphologyAcquired

Hypersegmentation – five or more nuclear lobes

1. Megaloblastic anemia

2. Myeloid malignancies (AML, Myelodysplastic syndromes)

Pseudo-Pelger-Huet anomaly - Hyposegmentation

70-90% Bilobed or no lobulation

Secondary to

3. Myeloid malignancy

4. Drugs –sulfonamides etc.

Nuclear chromatin very coarse and condensed

Dumbbell shaped

Versus true Pelger-Huet – autosomal dominant, inherited disorder

Abnormal neutrophil morphologyInherited

• 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• 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

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

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• 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

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

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 BasophiliaEosinophilia

• 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

• 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 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 macrophagesMonocytes 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 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

Note the four distinct characteristics1. Dull gray-blue cytoplasm2. Convolutions of the

nucleus3. Blunt pseudopods4. Lacy chromatin

Absolute monocytosisReactive 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

LymphopoiesisLymphoid 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)

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

LymphopoiesisLymphoblast

• 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

LymphopoiesisPlasmablasts

• 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

• 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

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

Nucleus is clumpedCytoplasm clear, darker blue at periphery of cellsIndented cytoplasm

Nucleus is lacy, brain-like convolutionGround-glass appearance of cytoplasmBlunt pseudopods between cellsCompress cells - not indented by cells

Lymphocyte morphologyReactive 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. Plasmacytoid lymphocyte – prominent chromatin clumping, perinuclear

halo, eccentric nucleus similar to a plasma cell

2. Immunoblast – a reactive lymphocyte with a prominent nucleolus

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

Lymphocytosis• Relative lymphocytosis occurs 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 lymphocytosisViral

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• 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 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 examinationCBC 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 examination

LymphopeniaWhen 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 smearIf 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 field estimated total WBC count

2-4 4000-7000

4-6 7000-10,000

6-10 10,000-13,000

10-20 13,000-18,000

Correlate estimate with automated instrument count

Evaluate morphology of WBCs, record abnormalities

Peripheral blood smearOil 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