HEMATOLOGY 1 - WBC Anomalies PDF

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Compiled by: Vienna Cari-cariWHITE BLOOD CELLSSir Joshua Abejo’s lecture Essential to normal host defense Adults: 4-11 x10^9/L Newborn: 13.5-38 x10^9/L WBC Maturation: Chromatin Pattern Phagocytes : (MBEN) Monocyte Basophils Eosinophils Granulocyte Neutrophils Immunocyte : Lymphocytes lymphocytes - ...

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HEMA 1 | WBCs and WBC ANOMALIES Compiled by: Vienna Cari-cari

WHITE BLOOD CELLS

MYELOCYTE Sir Joshua Abejo’s lecture

- Size: 12-18 um - Secondary granules - Nucleoli not seen - Cytoplasm: “Dawn of Neutrophilia” *** --- pinkish cytoplasm, bluish periphery

- Essential to normal host defense - Adults: 4.5-11 x10^9/L - Newborn: 13.5-38.0 x10^9/L - WBC Maturation: Chromatin Pattern Phagocytes: (MBEN) - Monocyte - Basophils - Eosinophils - Neutrophils

- 10-20% in the BM - Last stage to undergo mitosis Granulocyte

METAMYELOCYTE - Aka Juvenile Immunocyte: - Lymphocytes

- Size: 10-15 um - Nucleus: indented --- Kidney / Peanut-shaped --- Less than ½

lymphocytes - predominant WBCs 4 y.o. below neutrophils - predominant WBCs 6 y.o. above

- 15-30% in BM - most common cell in bone marrow

MYELOPOIESIS MYELOBLAST

BAND/ STAB/ STAFF

- Size: 15-20 um - Nucleus: large, round - 2 or more nucleoli

- Size: 9-15 um - Nucleus: indented --- C/S shaped --- More than ½

- 1-2% in the BM - Earliest recognizable precursor in LM - Type 1: no granule - Type 2: up to 20 granules

- normally present in the PBS - 5-10% in the PBS - pigments are not present

PROMYELOCYTE - Size: 15-21 um - Azurophilic granules ---- large, prominent, reddish, ↑ MPO - 0-1 nucleoli - 2-5% in the BM Page 1 of 8

HEMA 1 | WBCs and WBC ANOMALIES

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- phagocytosis (steps: "ICED”) *** 1. Initiation 2. Chemotaxis 2.a. Recognition --- direct: Primitive Pattern Recognition Receptor to PAMPS --- indirect: Opsonin (Ig6, C3d, fibronectin)

NEUTROPHIL - Size: 9-15 um - Nucleus: segmented; 3-4 segments - Cytoplasm: Pink to tan - Violet or lilac granules

3. Engulfment (lamellopod pseudopod formation) - phagosome - neutrophil with bacteria inside - phagolysosome - phagosome + lysosomal granules

PRIMARY GRANULES

- MPO → H202 → Hypochlorite - Bactericidal Cationic Protein - Proteases - Lysozyme SECONDARY GRANULES

- Lactoferrin - Specific collagenases - Vitamin B12-binding proteins - Lysozyme TERTIARY GRANULES

- Alkaline Phosphatase - Gelatinase - Acetyltransferase

- Main function: kill bacteria - Most abundant WBC in the circulation - 50-70% of total WBC in circulation - Tissue Lifespan: 2-3 days - Circulation lifespan: 7 hours

4. Destruction/Digestion - note: neutrophil dies after (excreted via urinary system, GI tract, or respi system) - note: Superoxide dismutase (SOD) --- break down potentially harmful Oxygen molecules in cells to prevent damage to tissues

Myeloperoxidase (MPO) --- production of oxygen reactive spp (toxic to bacteria)

SOD catalase *Superoxide (oxygen reactive spp) ----------> H202 (still toxic) ------------> H + H2O

Lysozyme (aka muramidase) --- breaks down bacterial cell wall Lactoferrin --- adhesion

- Chediak-Higashi syndrome (CHS) --- inability to respond to chemotactic actors --- faulty granules = impaired killing

Alkaline phosphatase (ALP) --- unique to neutrophil --- presence means cell is mature --- in late myelocyte or in early metamyelocyte stage Leukocyte alkaline phosphatase (LAP) --- dx criteria for chronic myelogenous leukemia (CML)

EOSINOPHIL - Size: 9-15 um - Nucleus: Bilobed - Cytoplasm: filled with large, refractive granules lodges in the GI & Respiratory Tract

CML = decreased ALP activity

- 2 stages in bone marrow: - Mitotic stages: Myeloblast, Promyelocyte, Myelocyte - Maturation stages: metamyelocyte, band, segmenter (aka neutrophil)

Inner granule: Peroxidase - arginine-rich - contains lysine, phospholipase, melanin

- 2 types in circulation: (ratio 1:1) - Circulating - Marginating - has adhesion molecules (L-selectin, Beta-2-integrin, P-selectin glycoprotein ligand) - (Loose) L-selectin + P-selectin - (Strong) B2 Integrin + P-selectin - (Circulation to tissue) P-selectin 6P Ligand + P-selectin - note: mixes with circulatory during stress, ex: crying

Outer granules: Major basic protein (MBP) - Charcot-Leyden crystals - composed of phospholipase - H&E staining: reddish-black - Trichrome: reddish-purple

- random movement: zigzag - directed movement: towards chemotactic agent --- note: C5a - most potent chemotaxin Page 2 of 8

Q: in bronchial asthma, what are the 3 crystals seen? *** A: (3C’s) - Charcot-Leyden crystals - Creola bodies - Curschmann's spirals

HEMA 1 | WBCs and WBC ANOMALIES

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- IL-5 specific interleukin - Prefer chemotactic factors produced by Mast cells & Basophil (ECF-A)

MONOCYTE

- Main function: kill parasites - Secondary function: immediate hypersensitivity reaction

- Size: 14-20 um ; same with pronormoblast - Nucleus: horse-shoe shaped - Chromatin pattern: loosely-woven or lace-like - Cytoplasm: “ground glass” appearance - Fine distinct granules

- How do they kill parasites? antibody-mediated. • Fc epsilon receptor (FcεR) – binds to FC portion of antibodies bound to helminths → degranulation / exocytosis • chemotactic factors

- largest cell in the PBS - rich in lysozyme - lysozyme in urine = monocytosis due to monocytic leukemia - strong positive: NSE

BASOPHIL Stages: Monoblas → Promonocyte → Monocyte

- Size: 10-16 um - Nucleus: Bilobed or unsegmented - Granules: Dark violet to purple-black

Monoblast: - Chromatin stains lighter than myeloblast - 1-2 nucleoli

Granules: 1. Histamine – normal activity = localized vasodilation – hyper = systemic vasodilation

Promonocyte: - Nucleus: moderately indented - Cytoplasm: grayish-blue - Fine lilac granules aka “azure dust”

2. Chondroitin sulfate – ex. of chondroitin sulfate: heparin

- Earliest recognizable precursor in monocytic series -- Monoblast vs Myeloblast: cannot be differentiated morphologically :

Primary function: immediate type hypersensitivity

Chemotactic factors: - Ag-Ab complexes - Complement components - Kallikrein - Lymphokines Costimulatory molecules in APC: in T cell: - B7 -------- CD28 - CD40 ---- CD154 - CD58 ---- CD2

Most common bacteria related to monocytes: - w/ increased monocytosis = M. tuberculosis - w/o increased monocytosis: = Listeria monocytogenes

Note: BASOPHIL and EOSINOPHIL are closely related When basophil becomes overactive → Basophil produces Eosinophil chemotactic factor → Eosinophil pakalmahon si Basophil via MBP (amine oxidase) which contrasts histamine

- Both a phagocyte and an antigen-presenting cell (APC) - does not die after phagocytosis - modifies antigenic determinant of the pathogen - presents antigen to dendritic cells

Dendritic cells - most potent and most efficient Ag-presenting cell

* Explains why in CBC, (during inflammation) Eosinophil is increased instead of Basophil

- phagocytosis: I, C, E, D → modify Ag structure of pathogen → modified structure incorporated in plasmalemma → → presents it to T-cells → T cells produce lymphokines → calls other macrophages * Negative feedback: (when no more invaders are detected) - monocytes release prostaglandin → T-cells are suppressed

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HEMA 1 | WBCs and WBC ANOMALIES

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Small / Medium / Large

MACROPHAGE

Small lymphocyte --- most numerous - Size: 7-10 um - Nucleus: spherical - Cytoplasm: thin rim

- Size: 30 um - Most abundant cell in the body --- because seen anywhere in body - IL-1 – resets hypothalamic thermostat; – causes fever - Transcobalamin II – transport protein for vitamin B12 - Liver: Kuppfer cells - Brain: Microglial cells - Lungs: Alveolar macrophage- aka dust cell - Skin: Langerhan’s cells - Bone: Osteoclast - Kidneys: mesangial - Spleen: littoral cells - Connective tissue: histiocytes - Placenta: hoff baeur cells

Medium lymphocyte: - Size: 10-12 um --- distinctly larger than erythrocytes - Cytoplasm: scanty

Q: how does a cell recognize what to phagocytose? *** A: - if bacterium has no capsule: chemotactic factors - if bacterium has capsule: opsonin (ex: IgG, C3b, fibronectin)

Large lymphocyte: - Size: 12-25 um --- twice the size of erythrocytes - Activated (lymphocytes that had encountered antigen)

Q: What is the CD marker whenever a lymphocyte encounters an antigen? A: CD25 (true to both T and B cell) ***

IgG - most potent opsonin

T cell vs B cell --- differentiated via flow cytometry based on CD markers T cell - 60-80% - Found in: paracortex of lymph node - End products: cytokines

LYMPHOCYTES

- Has CD 2,3,4,8 (and 25 if activated) ***

---- CD4:CD8 ratio = 2:1 Stages: lymphoblast → prolymphocyte → lymphocyte

Pan-T cell stage (before it becomes a T cell)

- CD2 - rosette formation using sheep RBCs

Lymphoblast: - Nucleus: large, round - Chromatin: thin and loosely stained - 1-2 nucleoli

double negative stage

- NEITHER CD4 nor CD8

double positive stage

- BOTH CD4 and CD8

mature stage

- EITHER CD4 or CD8 - CD3 – marker of mature T cell

Prolymphocyte: - Nucleus: eccentric - Chromatin: blue-purple chromatin Lymphocyte: - Nucleus: deep purple, round or indented - Cytoplasm: Robins Egg blue Variants: - Type 1 – Turks irritation cell / plasmacytoid cell - Type 2 – Downey cells/ ballerina skirt cell/ fried egg cell - Type 3 – finely reticulated nuclear pattern

- CD4 – helper - CD8 – cytotoxic

- T-helper 1 – intracellular - T-helper 2 – extracellular - Not an end cell

B cell

- May become Effector/memory/blast - Cannot be determined which - End stage maturation: plasma cell

- Nucleus: tortoise shell appearance*** - Secretes Ab - Type 1 is seen in German measles (Rubella infection) *** Page 4 of 8

- 20-30% - Has surface immunoglobulins - Found in: cortex of lymph node - End products: Antibodies - Progenitor B cell: CD 19, 24, 45R - Precursor B cell: CD 19, 24, 45r, and MU chain - Immature B cell: CD 19, 24, 45r, 21, 40; IgM, MHC class II - Mature B cell: CD 19, 24, 45r, 21,40; IgM, MHC class II; and IgD

IgM: - both a pentamer (in circulation) and a monomer (in B cell)

HEMA 1 | WBCs and WBC ANOMALIES

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NK cell

FLAME CELL

- aka Lymphokine-activated killer cell (LAK cell) - produces: perforins (function: kill pathogens) *** ---- presence of calcium = crystalize → pore formation

- Aka saurocyte - Abnormal plasma cell with flaming cytoplasm

CD 16 and 56 ***

- Seen in IgA myeloma Recap from Monocyte: Costimulatory moleules

Monocyte – has B7, CD40, CD58 - B7 attaches to CD28 in T cell - CD40 attaches to CD154 in T cell - CD58 attaches to CD2 in T cell

HAND MIRROR CELL

*Attachment is suppressed in immunosuppression

- Lymphocyte with hand mirror appearance - Seen in infectious mononucleosis IM (Epstein-Barr virus) - Note: to remember in EBV infection: - CD21*** - hand mirror cell - Burkitt’s lymphoma - type 2 lymphocyte

WHITE BLOOD CELL ANOMALIES SMUDGE CELL - Thumbprint appearance - Seen in CLL

- Note: hand mirror appearance when seen in…. - Neutrophil = normal when padulong phagocytosis - Lymphocyte = IM

- Nuclear remnants of lymphocytes - Structureless Chromatin - Spinner-type smear preparation – reduces formation of smudge cell (vs. manual wedge technique)

FOAM CELL - Deficiency in sphingomyelinase - Macrophage with fat droplets - Greatly affects liver and spleen

BASKET CELL - Nuclear remnants of granulocytes - Netlike pattern

- Seen in - Niemann-Pick disease ---- organs affected: spleen and liver - Hemophilia C ---- because of Ashkenazi Jews ** *

- Seen in Leukemia of myeloid lineage (mnemonics: GE 4M) - Granulocytes (BEN) - Erythrocyte - Monocytes - Macrophages - Megakaryocytes - Mast cells Page 5 of 8

HEMA 1 | WBCs and WBC ANOMALIES

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GAUCHER CELL

TOXIC GRANULATIONS - Altered primary granules - Seen in blood poisoning, lead poisoning note: in WBC = toxic granulation in RBC = basophilic stippling / cabot rings

- Crumpled tissue paper appearance - Deficiency in B-glucocerebrosidase - Macrophage with small, eccentric nucleus - Only seen in BM

AUER RODS

LE CELL - Neutrophil/Macrophage that has ingested a nuclear mass of a destroyed cell (aka LE body) - Seen in Systemic Lupus Erythematosus (SLE)

- Crystals of coalesced nonspecific granules - Seen in AML

DOHLE BODIES - Composed of rRNA - Round or oval blue staining cytoplasmic inclusions

REIDER CELL - Nucleus: notched, lobulated or cloverleaf-like - Seen in CLL

MAY-HEGGLIN CELL - Composed of mRNA - Gray-blue spindle shaped inclusions in phagocytes

GRAPE CELL - Aka morula cell or Mott cell - Russel bodies – granulations which contains Ab or Ig

PELGER-HUET CELL - Pince-nez nucleus - Failure to segment; spectacle form

TART CELL - Monocyte that has ingested lymphocyte - Seen in drug sensitivity

- most common genetic disorder

Tart cell vs LE cell: - Tart: Monocyte; LE: Neutrophil - Ingested nuclei in Tart cell have not reacted with LE cell factor, thus retaining their chromatin pattern and stain blue Page 6 of 8

HEMA 1 | WBCs and WBC ANOMALIES

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WHITE BLOOD CELL COUNT

ALDER-REILLY CELL

- Hemocytometer have 2 identical sides and both sides are counted - WBC: 10-12 counts (allowable correction) - RBC: 15-16 counts

- Dense, azurophilic granulations in all types of leukocytes - Abnormal deposition of mucopolysaccharides

- Neubauer Chamber is 3 mm x 3 mm - 4 corner large squares are subdivided into 16 squares - used for Manual WBC count - WBC Pipet Markings: 0.5, 1, 11

CHEDIAK-HIGASHI CELL

- Fuch’s Rosenthal: specific for Eosinophil

- Large, abnormal cytoplasmic granules in phagocytes - Defective chemotaxis

WBC Count Formula:

- Partial Albinism – impaired packing of melanosome in Golgi apparatus

= WBC counted x 10 x 20 4 Where 10 is the depth correction factor 20 is the dilution factor 4 is the area correction factor

HAIRY CELL - Lymphocytes with cytoplasmic projections

WBC count > 30,000 = 1:100 >/= 100,000 = 1:200

- TRAP + - isoenzyme 0, 1, 2, 3a, 3b, 4, 5

WBC CORRECTION COUNT >10 NRBCs Formula: uncorrected x 100/ 100 + NRBC

REED-STERNBERG CELL - Aka owl eye - Lymphoid cell that may demonstrate two nuclei - Seen in Hodgkin’s lymphoma

WBC DILUTING FLUIDS - must be hypotonic (capable of lysing erythrocytes without destroying leukocytes) - 2 v/v Glacial HAc – automation - 1 v/v Hydrochloric acid - Turk’s solution – composed of 3 mL Glacial HAc, 1 mL CV, 100 mL DW

HYPERSEGMENTED NEUTROPHIL - 5-10 lobes - Seen in Pernicious Anemia and Folic Acid Deficiency

Gentian Violet dye Methyl Violet dye

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HEMA 1 | WBCs and WBC ANOMALIES

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RELATIVE COUNT

ABSOLUTE COUNT

- # of specific WBC type per 100 WBCs

# of specific WBC type per mm3 of blood

Formula:

Formula:

= No. of specific WBC x 100 100 - Neutrophil: - Lymphocyte: - Monocyte: - Eosinophil: - Basophil:

51-67 % 25-33 % 2-6 % 1-4 % 0-1 %

= Relative Count x WBC count Neutrophil: Lymphocyte: Monocyte: Eosinophil: Basophil:

1, 600-7,260 mm3 760-4,400 mm3 180-880 mm3 45-440 mm3 45-110 mm3

- best in determining whether the patient has too few or too many cells

WBC DIFFERENTIAL COUNTING 100-cell differential: Routine 200-cell differential: results are divided by two --- indicate in the report that 200 WBCs were counted >10% Eosinophil >2% Basophil >11 monocyte Lymphocyte > Neutrophil...