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

RBC Membrane Proteins peripheral integral (lots of sialic acid) 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 unesterified cholesterol lipid bilayer unesterified cholesterol facilitates selective cation permeability 1:1 equilibrium w/plasma cholesterol increases in membrane cholesterol = target cells!!

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 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 IL-4 b IL-5 e IL-6 n, neutrophils; m, monocytes; e, eosinophils; b, basophils; meg, megakaryocytes; rbc, red blood cells IL – 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

Maturation of the neutrophilic series 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

Maturation of the neutrophilic series Myelocyte (last myeloid cell capable of division) Size: 10-18µm Cytoplasm 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

Maturation of the neutrophilic series Metamyelocyte (also called “meta” or “juvenile”) Size: 10-18µm Cytoplasm 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

Maturation of the neutrophilic series 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

---normal number: 150-450X109/L ---structure: LM: 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

Maturation of the neutrophilic series

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

Maturation of the monocytic series 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 N:C ratio = 3:1 or 2:1

Promonocyte

Maturation of the monocytic series 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 N:C ratio = 2:1 or 1:1

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

Maturation sequence of lymphocytes Prolymphocyte (difficult to distinguish) Size: 9-18µm Cytoplasm 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

Maturation sequence of lymphocytes 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

Maturation sequence of lymphocytes 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 Embryonic haemopoietic stem cells-mesenchymal cells in yolk sac After 12 week fetal liver and spleen becomes the main site From week 20, bone marrow starts to become important and by the time of birth it is the main haemopoietic organ

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

Intravascular Erythrocyte 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

نوتروفیل G.CSF C/EBPα,ε IL5 C/EBPα,ε,β بازوفیل IL3,Kit ligand مونوسیت ائوزینوفیل IL5 C/EBPα,ε,β بازوفیل IL3,Kit ligand مونوسیت M.CSF PU.1 , C/EBPβ لنفوسیتB IL4 PAX5,IKAROS لنفوسیتT IL7,IL2 GATA_3,notch1 NKسل SCF,IL15 C/EBPγ اریتروئید EPO,GMCSF,IL3 GATA_1,NFE2 مگاکاریوسیتیک TPO,IL3,IL11,IL6 GATA_1,GATA_2

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

Thymic processing of T-cells See notes