Leukocytes D.Rezazadeh Department of Medical Laboratory Science Kermanshah,Faculty of Paramedical

Leukocytes

D.RezazadehDepartment of Medical Laboratory Science

Kermanshah,Faculty of Paramedical

RBC Membrane Proteins

peripheral alpha & beta

spectrin bands 1 & 2

actin integral (lots of

sialic acid) glycophorin A band 3

inorganic anion transport channel

cation pump regulates Na+/K+ at ratios 1:12/25:1

RBC Membrane Lipids

phospholipids lipid bilayer

unesterified cholesterol

facilitates selective cation permeability

1:1 equilibrium w/plasma cholesterol

increases in membrane cholesterol = target cells!!

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HEMATOPOIETIC FACTORS

NAME CELLULAR SOURCE CELL TYPES PRODUCED IN

INCREASED NUMBERS

Erythropoietin (EPO) Kidney cells, Kupffer cells

rbc

G-CSF Monocytes, fibroblasts, endothelial cells

n

M-CSF Monocytes, fibroblasts, endothelial cells

m

GM-GSF T cells, monocytes, fibroblasts, endothelial

cells

n, m, e, meg, rbc

IL-1 Macrophages, endothelial cells, fibroblasts

n, m, e, b, meg, rbc

IL-3 T cells n, m, e, b, meg, rbc

IL-4 T cells b

IL-5 T cells e

IL-6 Macrophages, endothelial cells, fibroblasts

n, m, e, b, meg, rbc

n, neutrophils; m, monocytes; e, eosinophils; b, basophils; meg, megakaryocytes; rbc, red blood cellsIL – interleukin , CSF – colony stimulating factor

NEUTROPHILS

Myelopoiesis - the production and development of myeloid cells in the bone marrow Growth factors (colony-stimulating

factors - CSF’s) and interleukins regulate blood cell development by:

Mediating proliferation Differentiation into cell lines Maturation of hematopoietic progenitor

cells

NEUTROPHILS

Three series of granulocytes (named for colors taken up by granules in Wright’s stain) Neutrophils

Granules stain equally with eosin and methylene blue

Normal in circulation = 42 - 75% Eosinophils

Granules stain mainly red Normal in circulation = 0-4%

Basophils Granules stain mainly with methylene blue Normal in circulation = 0-1%

Maturation of the neutrophilic series

Myeloblast Size: 10-20µm Cytoplasm

Slight amount/blue

No granules Nucleus

Round/Central or eccentric

Reddish purple

1-3 nucleoli Fine meshlike

chromatin with no clumping

N:C ratio = 4:1


Promyelocyte (progranulocyte)

Size: 10-20µm Cytoplasm

Increased amount/blue Fine, azurophilic,

nonspecific granules present

Nucleus Round/Central or

eccentric Reddish-purple Fine, meshlike

chromatin beginning to clump

1-2 nucleoli N:C ratio = 3:1


Myelocyte (last myeloid cell capable of division)


Increased, bluish-pink (“dawning of neutrophilia”)

Fine azurophilic, specific granules

Nucleus Round or

oval/eccentric Reddish-purple Chromatin loosely

clumped No nucleolus

N:C ratio = 2:1 or 1:1


Metamyelocyte (also called “meta” or “juvenile”)


Moderate amount Specific granules

(neutrophilic, eosinophilic or basophilic)

Nucleus Indented (kidney-bean

shaped) Central or eccentric Purple Clumped chromatin No nucleolus

N:C ratio = 1:1


Band Size: 10-16µm Cytoplasm

Moderate amount Specific granules

(see meta) Nucleus

Elongated, narrow band (sausage) shape with uniform thickness

Central or eccentric Clumped, coarsely

granular chromatin

WBC'S (LEUKOCYTES)

NEUTROPHILS: Most abundant 50-70%

Function: phagocytosis granules with hydrolytic

enzymes defensins - antibiotic

proteins,

EM: specific granule: 70% small, 0.3-0.4 um ovoid or irregular in shape contain: lysozyme, defensin azurophilic granule: 30% large, round or ovoid electron dense lysosome: acid phosphatase, peroxidase,

acidic hydrolase

---function: phagocytose bacterium: specific g.-kill B azurophilic g.- digest B---life span: in blood 1 day, in Tissue 2-3

days

Granule type Protein

specific granules (or "secondary granules")

alkaline phosphatase, lysozyme, NADPH oxidase, collagenase, Lactoferrin and Cathelicidin

azurophilic granules (or "primary granules")

myeloperoxidase, bactericidal/permeability-increasing protein (BPI), Defensins, and the serine proteases neutrophil elastase and cathepsin G

tertiary granules cathepsin and gelatinase

WBC'S (LEUKOCYTES) EOSINOPHILS: 2-4%

of WBC's Function: Allergies,

parasitic worms granules w/lysosomes

and digestive enzymes ( enzymes for bacteria)

attack parasitic worms - release enzymes onto the worms

lessen allergies by phagocytizing immune (Ab/antigen) complexes

inactivate certain inflammatory chemical

EM: granules: round or ovoid with cube-liked electron dense crystal contain: -ACPase-histaminase-peroxidase

---function: counteract the infection of parasite reduce allergic reaction---life span: in blood 18 hour, in Tissue 6-12

days

WBC'S (LEUKOCYTES)BASOPHILS: 0.5% of WBC'S Function: liberate heparin,

histamines histamine containing

granules = inflammatory chemical that vasodilates

attracts other WBC's to site of inflammation

mast cells - like basophils are found in Tissue = tissue basophils?

WBC'S (LEUKOCYTES) LYMPHOCYTES: 25-

40% of WBC'S Function: immune

responses and memory, mainly found in lymph tissue

T cells - immune response to viral infected and tumor cells

B cells - give rise to plasma cells which produce Ab's

more later

EM: /free ribosome /mito. /RER---classification: TC: thymus dependent lymphocyte, 75%,

involve in cellular immune reaction and regulate immune response

BC: bone marrow dependent lymphocyte, 10-15%, become into plasma cell, involve in humoral immune response

large granular cell: KC (killer cell) and NKC( natural killer cell), 10%

---function: involve in immune response

WBC'S (LEUKOCYTES)

MONOCYTES: 3-8% of WBC's Function: phagocytosis in tissues differentiate into macrophages evident in chronic infections -

Tuberculosis defense vs. viruses and certain bacteria activate lymphocytes

EM: azurophilic granule: lysosome- contain

peroxidase, ACPase,non-spacial esterase and lysozyme

---function: actively mobile and chemotaxis---life span: in blood 1-5 days, in Tissue

which become into macrophage- mononuclear phagocytic system, MPS

3) blood platelet: cytoplasmic fragment of megakaryocyte in bone marrow

---normal number: 150-450X109/L ---structure: LM: disc-liked → irregular in

shape Stimulation

EM: /cell coat: glycosaminoglycan and glycoprotein/tubular systems: opening tubule system- increasing the exchange

area, facilitate the intaking and releasing dense tubule system- SER, collecting Ca2+ and

synthesizing prostaglandin/granules: specific granule:, with dense core, contains

clotting factor, fibrinogen, acidic hydrolase dense granule: contains serotonin(5-

hydroxytryptamine), ADP,ATP, Ca2+ and adrenalin

---function: involve in clotting and stopping the

bleed: -aggregation -release components of granule,

make protrombin become into thrombin, then later make fibrinogen become into fibrin

* stromal cells: ---including reticular cell, fibroblast,

macrophage, endothelial cell and lipid-laden cell

---function: supporting secrete cellular factors, regulate the

proliferation and differentiating of hemopoietic cells


Function of the neutrophilic series

Neutrophils - defense against bacterial infections

Eosinophils Regulate inflammation Respond to antigenic stimulation in chronic

allergies Interact with larval stages of some helminthic

parasites Basophils

Histamine in granules plays a role in acute, systemic allergic reactions (sudden release of histamine can cause anaphylactic shock)

Granules also contain heparin

MONOCYTES

Monopoiesis - the production and development of monocytes, macrophages and their precursors. Monocytes are produced in the bone marrow. Their primary function is phagocytosis.

MONOCYTES

Other names for macrophages Histiocytes in the loose connective tissue Kupffer cells in the liver Osteoclasts in the bone Microglial cells in the nervous system Also scattered throughout the body in all organ

tissues (spleen, lung, abdomen, etc.) One monocytes have grown into macrophages, they do not normally reenter the bloodstream except in cases of inflammation

Maturation of the monocytic series

Monoblast Size: 12-20µm Cytoplasm

Blue, moderate amount

No granules Nucleus

Round, oval or slightly folded

Eccentric Reddish-purple Chromatin fine and

thready Vacuoles may be

present 1-2 nucleoli

N:C ratio = 4:1


Promonocyte (difficult to discern) Size: 21-20µm Cytoplasm

Paler gray-blue and abundant “Bleb-like” pseudopods at periphery May contain fine red “dust-like” particles Vacuoles may be present

Nucleus Round with chromatin creases or brain-like folding Central Reddish-purple with light clumping of chromatin 0-2 nucleoli


Promonocyte


Mature monocyte (Normal in circulation: 2-9%) Size: 15-18µm Cytoplasm

Pale gray-blue and abundant Bleb-like pseudopods at periphery, variable shape Numerous fine pale red dustlike granule scattered

throughout Vacuoles common

Nucleus Increased folding or elongated Central Purplish with finer clumped chromatin No nucleoli


Monocyte

LYMPHOCYTES

Normal adult value: 20-45% Produced in the bone marrow

(primary lymphoid tissue)

LYMPHOCYTES

Growth factors cause lymphs to differentiate. They proliferate and mature in the primary lymphoid organs:

LYMPHOCYTES

Thymus - T cells Function

Cellular immune response (they do not produce antibodies) Regulate antibody reactions by helping or suppressing

activation of B lymphs. T cells act as “helper cells” (HIV or AIDS virus causes a destruction of these helper cells), which are instrumental in aiding B cells in antibody production, and “suppressor cells”, which act as a “thermostat” to shut off the system or keep it under control. The helper to suppressor ratio is very important and is normally 2:1.

Protect against viral, bacterial, fungal or protozoan infection Responsible for chronic organ graft rejection. (T cells have

many unique antigens on their cell surfaces, some of which are the HLA - human lymphocyte antigen - markers. Cytotoxic and helper T cells act against these HLA markers if they are foreign to the host organism. HLA markers are instrumental in chronic graft rejection and that is why tissue typing is done to establish a good donor match.)

LYMPHOCYTES

Bone marrow - B cells Function

Humoral (antibody) immune response by transforming into antibody-producing plasma cells

Defense against encapsulated bacteria such as streptococci

Mediate hyperacute organ rejection

Secondary lymphoid tissue Lymph node, spleen, Peyer’s patches in

intestine and mucosal tissues such as tonsils

Active immune response occurs in above tissues where lymphs communicate and interact with antigen-presenting cells, phagocytes and macrophages

Maturation sequence of lymphocytes

Lymphoblast (which is preceded by a lymphoid stem cell)

Cell size: 10-20µm Cytoplasm

Blue/scanty No granules, Auer

rods are never present

Nucleus Purple, smooth

chromatin Round, central or

eccentric 1-2 nucleoli

N:C ratio = 4:1


Prolymphocyte (difficult to distinguish)


Blue, scanty Usually granules are

absent, but a few azurophilic granules may be present

Nucleus Purple, condensed

chromatin Round or indented,

eccentric 0-1 nucleoli

N:C ratio = 4:1


Mature lymphocyte Cell size: 7-10µm (a typical

normal lymph has a nucleus that is the size of a normal RBC)

Cytoplasm Light blue, scanty to

moderate Few azurophilic granules

may be present Nucleus

Purple, dense, clumped chromatin

Round or indented, eccentric

No nucleoli

Large lymphocytes versus monocytes

Size Large lymph: 12-15µm Mono: 15-18µm

Nucleus Large lymph: clumped, condensed Mono: lacy, brainlike folds

Granules Large lymph: large azurophilic, easy to count Mono: red, fine


Variant lymphs Other terms used are reactive, atypical, Downey

cell, transformed, virocyte, plasmacytoid Caused by antigenic stimulants such as viruses,

post-transfusion reactions and organ transplants

Reactive lymphocyte

Plasma cells

Plasma cells Function is the synthesis and excretion of antibodies

(immunoglobulins) Normally not present in the peripheral blood; comprise

2% of bone marrow cells. (May be seen in the peripheral blood in the disease called multiple myeloma, a disease of uncontrolled production of immunoglobulins.)

End stage of the B lymphocyte Appearance

Size: 10-18µm Cytoplasm is dark blue with perinuclear halo and may contain vacuoles

indicating antibody synthesis Nucleus is round, eccentric, dark purple with dense clumped chromatin

Plasma cells

Thank you

Sites of Haemopoiesis Yolk sac

Liver and spleen

Bone marrow Gradual replacement

of active (red) marrow by tissue inactive (fatty)

Expansion can occur during increased need for cell production

Extravascular Erythrocyte Destruction Normal end-of-life

event Inherited RBC Defects

Membrane abnormalities

Enzyme deficiencies Hemoglobinopathies Thalassemias

Acquired RBC Defects B12 Deficiency

Immune-mediated destruction

Drug-induced Autoimmune

IntravascularErythrocyte Destruction Intravascular hemolysis

Normal end-of-life event

Complement Activation PNH PCH

Physical/mechanical Trauma

DIC Prosthetic heart

valves Toxins

Arsenic poisoning Bacterial Infections

Lab Evidence of Hemolysis

Extravascular Urine urobilinogen Fecal urobilin Yellow-colored

plasma

Intravascular Increased Reticulocyte

Index Decreased haptoglobin Decreased hemopexin Presence of

Methemoglobin/methalbumin

Increased indirect bilirubin Increased urobilinogen Decreased RBC Increased iron stores

Hemosiderinuria Ferritin

C/EBPα,ε G.CSF نوتروفیلC/EBPα,ε,β IL5 ائوزینوفیل

IL3,Kit ligand بازوفیلPU.1 , C/EBPβ M.CSF مونوسیت

PAX5,IKAROS IL4 لنفوسیتBGATA_3,notch1 IL7,IL2 لنفوسیتT

C/EBPγ SCF,IL15 NKسلGATA_1,NFE2 EPO,GMCSF,IL3 اریتروئید

GATA_1,GATA_2 TPO,IL3,IL11,IL6 مگاکاریوسیتیک

Lymphocytes and Lymphoid Tissues

Primary lymphoid tissues

(thymus and bone marrow), Secondary lymphoid tissues

(lymph nodes, Peyer’s patches, and spleen) Tertiary lymphoid tissues

(skin and mucosal)

Thymus

Cortex

Medulla

Thymicprocessingof T-cells

See notes

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Leukocytes D.Rezazadeh Department of Medical Laboratory Science Kermanshah,Faculty of Paramedical