WBC

WBCs / LEUKOCYTES
precursors differentiate, proliferate and mature in the bone marrow with the exception of T lymphocytes

Normally found in the peripheral blood: Neutrophil Band neutrophil Eosinophil Basophil Monocyte Lymphocyte

WBCs / LEUKOCYTES
Wrights stain A Romanowsky-type of stain used to stain the cellular components of blood and bone marrow Major components: 1. methylene blue basic dye 2. Eosin acidic dye

WBCs / LEUKOCYTES
Wrights stain I. Polymorphonuclear cells > granulocytes (contains many granules) 1. neutrophil neutral to pinkish cytoplasm and granules 2. basophil purple black granules 3. eosinophil reddish orange granules II. Mononuclear cells > contain small amount of fine granules or no granules 1. monocyte bluish gray cytoplasm 2. lymphocyte sky blue cytoplasm

WBCs / LEUKOCYTES
Wrights stain

Leukocyte concentration in the peripheral blood

At birth, total peripheral WBC count is 9-30 x 109/L. Few immature granulocytic cells (myelocytes, metamyelocytes) can be seen Gradually declines until the age of 8 years to a level ranging from 4.5 to 11 x 109/L Affected by physiologic (e.g. pregnancy, time of day, age, sex) and pathologic events

or in the total # of WBC can be caused by an altered concentration of all WBC or more commonly by an alteration in one type of WBC

Leukocyte concentration in the peripheral blood

A.) Neutrophil 40 89 % of total WBC (1.8 7 x 109/L) Level drops to 30% by 4-6 months of age After 4 years of life, the concentration gradually increases until adult values are reached at 6 years of age

B.) Lymphocyte Concentration ranges from 25-35% (1.0 4.8 x 109/L) in adults Concentration increases to 60% at about 4-6 months of age and remains this level until 4 years of age

Leukocyte concentration in the peripheral blood

C.) Eosinophil 0 5% (up to 0.4 x 109/L) D.) Monocytes 2 10% (0.1 0.8 x 109/L)

E.) Basophil 0 1% (up to 0.2 x 109/L) An abnormal total WBC count should be followed by a leukocyte differential count which gives the realative value of each WBC Absolute value = relative value x WBC count

LEUKOCYTE FUNCTION
To protect the host from infectious agents or pathogens I. Innate immune response II. Adaptive immune response

NEUTROPHILS

NEUTROPHILS
Differentiation, Maturation & Morphology I. Myeloblast 14-20 um in diameter High N:C ratio 1 5 nucleoli visible Aganular cytoplasm, staining from a deep blue to a lighter blue CD markers: CD 34, 33,13, 38 & 45

NEUTROPHILS
Differentiation, Maturation & Morphology II. Promyelocyte 15 21 um in diameter Several nucleoli can still be visible Presence of primary/ nonspecific/ azurophilic granules a. myeloperoxidase e. bactericidal permeability b. lysozyme increasing protein (BPI) c. Cathepsin G, B & D f. Esterase N d. defensins g. elastase

NEUTROPHILS
Differentiation, Maturation & Morphology III. Myelocyte 12 -18 um in size Appearance of specific or secondary granules a. lactoferrin d. collegenase b. lysozyme e. gelatinase c. histaminase f. heparinase Secretory vesicles are scattered throughout the cytoplasm a. alkaline phosphatase b. complement receptor 1 c. cytochrome b558

NEUTROPHILS
Differentiation, Maturation & Morphology IV. Metamyelocyte 10 18 um With nuclear indentation, giving the nucleus a kidney-bean shaped Tertiary orgelatinase-containing granules are synthesized mainly in this stage a. gelatinase b. lysozyme

NEUTROPHILS
Differentiation, Maturation & Morphology V. Band Neutrophil 9 15 um a.k.a. nonsegmented neutrophil Indentation of the nucleus is more than half the diameter of the hypothetical round nucleus giving the nucleus a horseshoe shape Cytoplasm appears pinkish 1st stage normally found in the peripheral blood

NEUTROPHILS
Differentiation, Maturation & Morphology VI. Segmented neutrophil 9 15 um Segmented nucleus with 2 or more lobes connected by thin nuclear filament Cytoplasm stains a pinkish to clear color CD markers: CD15, CD16, CD11b/CD18

NEUTROPHILS
Differentiation, Maturation & Morphology

NEUTROPHILS

X chromatin body/ Barr body > an inactive X chromosome appearing as an appendage of the neutrophil nucleus

NEUTROPHILS
Distribution, Concentration and Kinetics I. Bone Marrow a. Mitotic pool/ proliferating pool Cells spend about 3-6 days in this pool undergoing 4-5 cell divisions Includes cells capable of DNA synthesis: myeloblasts, promyelocytes, myelocytes

NEUTROPHILS
Distribution, Concentration and Kinetics I. Bone Marrow b. Postmitotic pool/ maturation and storage pool Cells spend about 5-7 days in this compartment before they are released into the peripheral blood (shortened during infection) 3x the size of the mitotic pool Includes metamyelocytes, bands and segmented neutrophils Once precursor cells have matured in the bone marrow, they are released into the bone marrow

NEUTROPHILS
Distribution, Concentration and Kinetics II. Peripheral blood > average neutrophil circulates at an average 7.5 hours in the blood before diapedesing to the tissues (Ferrata cells) a. Marginating pool b. Circulating pool III. Tissues > in response to chemotactic stimulation

NEUTROPHILS
Function Primarily in the tissues where microbial invasion typically occurs 4 steps: 1. adherence 2. migration 3. phagocytosis 4. bacterial killing

NEUTROPHILS
Function

NEUTROPHILS
Function

NEUTROPHILS
Terminologies: Leukocytosis Leukopenia Agranulocytosis Granulocytosis Neutrophilia Neutropenia

EOSINOPHILS

EOSINOPHILS
Differentiation, Maturation & Morphology Eosinophil originates from the IL-5 responsive CD34+ myeloid progenitor cells IL-5 released by TH2 lymphocytes is the major cytokine required for eosinophil production and terminal differentiation Morphologic maturation is similar to neutrophil with same 6 stages of maturation identified CD markers: CD9, CD11a & CD13

EOSINOPHILS
Differentiation, Maturation & Morphology Morphologic maturation is similar to neutrophil with same 6 stages of maturation identified 3 types of granules: 1. Primary granules 2. Small granules a. acid phosphatase b. aryl-sulphatase 3. Eosinophil-specific granules a. major basic protein (MBP) b. eosinophil cationic protein (ECP) c. eosinophil peroxidase (EPO) d. eosinophil-derived neurotoxin (EDN)

EOSINOPHILS
Functions: 1. Host defense against helminth parasites 2. Down modulate the allergic response by neutralizing mast cell and basophil products Eosinophilia Eosinophil concentration > 0.4 x 109/L Associated with: 1. allergic diseases 4. GI diseases 2. parasitic infections 5. respiratory tract disorders 3. toxic reactions 6. neoplastic disorders T-cell dependent

EOSINOPHILS
Charcot-Leyden crystals Are bipyramidal crystals commonly found in fluids or tissues in associateion with eosinophilic inflammatory reactions Consists primarily of phospholipase

BASOPHILS

BASOPHILS
Originate from the CD34+ myeloid progenitor IL-3 is the main cytokine involved in growth and differentiation Requires 2.5 - 7 days maturation in the bone marrow before they are released into the circulation CD markers: CD9, CD11a & CD13

BASOPHILS
Basophil granules: purple black in color Water soluble (+) with Periodic Acid Schiff (PAS) reaction ( + for peroxidase)

Contain: 1. histamine 2. heparin 3. cathepsin G

4. MBP 5. lysophospholipase

BASOPHILS
Mast Cell (Tissue Basophil)

BASOPHILS
Mast Cell (Tissue Basophil)

BASOPHILS
Functions: 1. Involved in allergic reactions by releasing histamine and heparin when activated via the binding og IgE to membrane Fc receptors 2. CD 40L interacts with B lymphocte CD40 to induce IgE synthesis

MONOCYTES

MONOCYTES
Produced in the bone marrow from a bipotential progenitor cell (CFU-GM)

Maturation: I. Monoblast 12 20 um Pale blue-purpe nuclear chromatin which is finely dispersed (lacy), and with several nucleoli Has nonspecific esterase

MONOCYTES
Maturation: II. Promonocyte Nucleus most often irregular and indented with a fine chromatin network (coarser than the monoblast) Azurophilic granules can be present

III. Monocyte 12 20 um in diameter Largest mature cell in the peripheral blood Ground glass cytoplasm with fine azurophilic granules Nucleus is frequently horseshoe- or bean-shaped and possesses numerous folds, giving it the apperance of brainlike convolutions MURAMIDASE positive

MONOCYTES

MONOCYTES
Maturation: IV. Macrophages Together with monocytes, makeup the mononuclear phagocyte system (MNP) 1. 2. 3. 4. 5. Liver: Kupffer cells Lung: alveolar macrophages Skin: Langerhans cells Brain: Microglial cells Kidney: Mesangial cells

MONOCYTES
Functions: 1. Ingest and kill microorganisms 2. Secrete a variety of cytokines that affect the function of other cells, especially that of lymphocytes 3. Act as APCs 4. Scavengers for cellular debris, effete cells and other particulate matter 5. Direct cytotoxic effect to tumor cells

LYMPOCYTES

LYMPOCYTES

HSC CLP
B lymphocyte T lymphocyte

NK cell

LYMPOCYTES
Phases of T & B lymphopoeisis: 1. Antigen-independent lymphopoiesis Takes place within the primary lymphoid tissue Results in the formation of immunocompetent T & B lymphocytes (referred to as virgin or nave lymphocytes)

2. Antigen dependent lymphopoiesis Begins with recognition of and interaction with antigens by specific antigen receptors on the surface of immunocompetent T & B lymphocytes Results in the formation of efector T (Tc, Ts, Tdhs, Treg, Th) and B lymphocytes

LYMPOCYTES
Differentiation: Morphology of immature lymphocytes 1. Lymphoblast About 10 18 um in diameter with a high N:C ratio lacy and fine nuclear chromatin With 1 or 2 well-defined pale blue nuccleoli

Stain negative for peroxidase, lipid and esterase (differentiating it from myeloblast) but contain ACP and sometimes deposits of glycogen (PAS +)
Contain Tdt (terminal deoxynucleotidyl transferase)

2. Prolymphocyte

LYMPOCYTES
Differentiation: Morphology of immature lymphocytes 3. Lymphocyte Has extensive size variability, 7 16 um a. Small lymphocytes Range in size from 7-10um Nucleus is about the size of an RBC and occupies about 90% of the cell area Few azurophilic granules and vacuoles can be present b. Large lymphocytes Range in size from 11 16 um in diameter

LYMPOCYTES
Differentiation: Morphology of immature lymphocytes 3. Lymphocyte Large granular lymphocytes (LGL) 3% of blood lymphocytes So named because of the presence of large granules in their cytoplasm Consist of a mixed population of both NK cells and CD8+ Tc

LYMPOCYTES

LYMPOCYTES

LYMPOCYTES
Morphology of Activated lymphocytes Contact and binding of specific antigens to receptors on nave T and B lymphocytes begin a complex sequence of cellular events known as blast transformation (blastogenesis) Transformed cells, called immunoblasts, Have the option to differentiate into effector cells or memory cells

Reactive lymphocytes and immunoblasts were referred to in the past as Downey cells and were classified as Type I, II or III

LYMPOCYTES
Morphologically identifiable forms of antigen-stimulated lymphocytes: 1. Reactive lymphocytes Also referred to as a stimulated, transformed, atypical, activated, or variant lymphocyte Found in increase number in viral infections, hence also called a virocyte Increase in size (16-30 um) and have a decreased N:C ratio Nucleus is more frequently elongated or irregular

LYMPOCYTES
Morphologically identifiable forms of antigen-stimulated lymphocytes: 2. Immunoblast 12 25 um Prominent nucleoli and a fine nuclear chromatin pattern B immunoblast plasmacytoid lymphocyte & plasma cell T immunoblast Tc, Ts, Th 3. Plasmacytoid lymphocyte Central or slightly eccentric nucleus with deeply basophilic cytopasm Has some cytoplasmic Ig (cIg), as well as surface membrane Ig (sIg)

LYMPOCYTES
Morphologically identifiable forms of antigen-stimulated lymphocytes: 4. Plasma cells Round or slightly oval with 9 20 um diameter Eccentrically placed nucleus Chromatin has cartweel or spoke wheel arrangement No nucleoli Hof area cIg but sIg is usually absent Committed to secreting large amounts of specific Ig of the same isotype (Ig class) and idiotype (antigen specificity)

LYMPOCYTES
Morphologically identifiable forms of antigen-stimulated lymphocytes:

LYMPOCYTES
Morphologic variations of plasma cells 1. Flame cells Named fo their reddish-purple cytoplasm Red tinge is caused by a glycoprotein, and the purple tinge is caused by the presence of ribosomes

LYMPOCYTES
Morphologic variations of plasma cells 2. Mott cells A.k.a. grape cells Plasma cells filled with globules containing immunoglobulin (Russell bodies)

LYMPOCYTES
Morphologic variations of plasma cells Dutcher bodies Intranuclear membrane-bound inclusion bodies most often found in neoplastic plasma cells

Alterations in immunoglobulin production: 1. Hypogammaglobulinemia 2. Monoclonal gammopathy 3. Polyclonal gammopathy

LYMPOCYTES
Memory cells Morphologically similar to resting small lymphocytes; retain the ability to react with the stimulating antigen, and are capable of eliciting a rapid 2ndary immune response when challenged again by the same antigen

LYMPOCYTES
Lymphocyte Function 1. T-cell (Th, Ts & Tc) > cell-mediated immunity 2. B-cell > humoral immunity 3. NK cell > non-MHC restricted cytotoxicity > kill tumor cells and those cells infected with a virus > ADCC

LYMPOCYTES
Distribution, Concentration and Kinetics Peripheral concentration in adults ranges from 1.0 4.8 x 109/L Peripheral blood lymphocytes comprise only 5% of the total body lymphocytes. 95% are in the lymph nodes and spleen Life span range from few months to many years

LYMPOCYTES
Membrane markers: 1. T lymphocytes TCR, CD2/LFA-3, CD3, CD5, CD7, CD4 OR CD8 2. B lymphocyte a. pro-B-cell > CD34, CD19 (pan B antigen) & Tdt b. pre-B-cell > CD19, CD20, CD24, CD10 (CALLA) c. Mature B cell > CD19, -20, -21, -22, -24, sIgM, sIgD 3. NK cell: CD16 & CD56

Peripheral Blood Smear Preparation

Manual Methods: 1. Coverglass smear Provides a smear with even distribution of the leukocytes Disadvantages: 1. Difficulty in mastering the technique 2. Fragility of the coverglass 3. Difficulty in staining the coverglass

Peripheral Blood Smear Preparation

Manual Methods: 2. Wedge smear Method most commonly used in routine laboratory practice Advantages: 1. The technique is easily mastered. 2. The smears are not fragile 3. It can be stored for extended periods of time

Disadvantage: 1. subject to poor WBC distribution

Peripheral Blood Smear Preparation

Peripheral Blood Smear Preparation

Sample: EDTA anticoagulated blood or capillary blood Stain: Romanowsky stain 1. Wrights 3. Jenners 2. Giemsa 4. Leisman

5. Wrights-Giemsa 6. May-Grunwald

pH:

pH 7.2 for blood/bone marrow smear pH 6.8 for malarial smear

Peripheral Blood Smear Preparation

Peripheral Blood Smear Preparation

Peripheral Blood Smear Preparation

WBC Differential Count Reported in % Relative WBC count 50 - 70%: segmented neutrophils 2 6%: band neutrophils 1 4%: eosinophils 0 1%: basophil 2 6%: monocytes 22 40 %: lymphocytes

Absolute WBC count = relative WBC count x WBC count

Shift to the left (Regenarative or Degenerative): immature cells Shift to the right: hypersegmented neutrophils

Peripheral Blood Smear Preparation

Estimation of WBC count

Peripheral Blood Smear Preparation

EOSINOPHIL COUNT
Eosinophil in allergy and parasitic infections in hyperadrenalism/ Cushings syndrome Reagents for Direct Eosinophil Count: 1. Phloxine/Eosin/ Neutral red stains eosinophil 2. Propylene glycol lyses RBCs 3. Na2CO3 lyses WBCs except eosinophil 4. Heparin inhibits clumping of WBC

EOSINOPHIL COUNT
Methods 1. Pilot 2. Randolph 3. Manners 4. Hinkleman 5. Discombe & Dunger n 6. Tannen Stain Phloxine Phloxine Phloxine Eosin Eosin Neutral Red

WBC pipet : 1:10 dilution

EOSINOPHIL COUNT
Thorns Test Used to assess adrenocortical function 1st specimen: Fasting = do eosinophil count (1) 2nd specimen: After 4 hours = administer ACTH & do eosinophil count (2) Normal: Eo (2) is 50% less than Eo (1) Hypoadrenalism: Eo (2) = Eo (1)

RAST (Radioallergosorbent Test)

Makes use of a radioactive isotope containing goat antihuman IgE which binds to the IgE/Antigen complex

Total IgE can be used to diagnose allergies 1. Allergic patients often have low IgE 2. Elevated IgE can be a result of other things such as parasitic infection

BASOPHIL COUNT
Direct: Copper-Cruickshank Reagents: 1. Copper Toluidine Blue stain basophil 2. Cetylpyridium chloride lysis 3. Aluminum sulfate mordant 4. EDTA prevents platelet aggregration No dilution needed Basophils are seen as purple-red metachromatically stained cells