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PROPERTY OF MEDTECH REVIEW NOTES

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Hematology

PART I

INTRODUCTION ☻ Blood:

✓ Red color is attributed to ____________

✓ pH: __________

✓ Specific gravity: _________

☻ Composition

✓ Liquid portion (plasma – in unclotted blood; serum – in clotted blood)

✓ Solid portion (RBCs, WBCs and platelets)

✓ Gaseous portion (oxygen and carbon dioxide)

☻ Collection

✓ Most critical step in blood collection – _________________

SKIN PUNCTURE

☻ Used when there is only small amount of blood needed

☻ Recommended for

✓ infants and young children less than 1 yr old depth: ________

✓ adults with poor veins depth: _________

☻ Preferred sites

✓ Palmar surface (fleshy portion of last phalanx of 3rd or 4th finger of non-

dominant hand)

✓ Lateral plantar surface (heel portion) of newborn

☻ Best method for blood gas collection in newborn – indwelling umbilical artery

catheter

☻ For arterialized capillary blood – warming puncture site using washcloth (40-

42°C) for ________

☻ Length of lancet – ____________

☻ Distance between skin surface and bone/cartilage in middle finger –

___________

☻ Capillary tube

✓ Length – _____________

✓ Bore – __________

✓ Blood should fill – _____________

✓ Sealing clay – ___________

☻ Order of draw

1. Tube for blood glass analysis

2. Slides

3. EDTA microcollection tube

4. Other anticoagulated microcollection tubes

5. Serum microcollection tubes

VENIPUNCTURE

☻ Collection of deoxygenated blood (dark red color)

☻ Glucose level is ___________ compared to arterial blood

☻ Most common & best site: ____________________

✓ Median cubital vein

✓ Cephalic vein

✓ Basilic vein

☻ Collection

✓ Angle: __________ (bevel up)

✓ Tourniquet: ________________ above the site

✓ Needle size: __________________ (adult) or 23-25 gauge (children)

COLOR CODED NEEDLE HUBS

COLOR GAUGE

White 16

Pink 18

Cream 19

Yellow 20

Green 21

Black 22

Blue/ Light blue/ Turquoise 23

Purple 24

Orange 25

Brown 26

Gray 27

Blue-Green 28

✓ A phlebotomist can puncture a patient not more than _________


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☻ Order of Draw

1. Blood culture tubes (Yellow)

2. Coagulation tubes (Light blue)

✓ 3.2% sodium citrate (9:1 blood:anticoagulant ratio)

✓ Action: binds to calcium to form soluble complexes

✓ 3.8% sodium citrate (4:1 – used for ESR)

3. Serum tubes (without clot activator or with gel separator)

✓ Thixotropic (a change in viscosity when centrifuged)

4. Heparin tubes (Green)

✓ Action: inhibits thrombin

✓ For OFT and blood gas analysis

✓ Causes bluish coloration of background on blood smears

stained with Romanowsky attributed to pH

✓ Lithium heparin: most widely used

5. EDTA (Lavender)

✓ Most commonly used collection tube in hematology

✓ Action: chelation of calcium

✓ Optimal anticoagulant conc: 1.5 mg/mL of blood

✓ CBC and retic counts – can be performed up to ____

✓ WBC counts, hematocrit and platelet counts – up to _____

(refrigerated at 4°C)

✓ ESR – 2 hrs (room temp.) or 6 hrs (refrigerated)

✓ Blood smears – 2 hrs

6. Glycolytic inhibitor tubes (Gray)

✓ Antiglycolytic agent: ___________ – preserves glucose upto 72hrs;

AC used: ______________

✓ Antiglycolytic agent: lithium iodoacetate – preserves glucose

for 24hrs; AC used lithium heparin

HEMATOPOIESIS ☻ Continuous and regulated process of blood cell production that includes:

✓ Cell renewal

✓ Proliferation

✓ Differentiation

✓ Maturation

MESOBLASTIC PHASE

☻ Cells from mesoderm migrates to YOLK SAC

☻ Hematopoiesis begins at 19TH DAY OF EMBRYONIC DEVELOPMENT (occurs

intravascularly)

☻ PRIMITIVE ERYTHROBLAST produces:

✓ Gower -1 (______________)

✓ Gower – 2 (______________)

✓ Portland (_______________)

☻ Cells migrate to aorta-gonad-mesonephrons (AGM) give rise to

HEMATOPOIETIC STEM CELLS

HEPATIC PHASE

☻ Begins at _______________

☻

☻ Hematopoiesis occurs EXTRAVASCULARLY

✓ _______ – major site of hematopoiesis in 2ND TRIMESTER

☻ Spleen, thymus, and lymph nodes – active in blood cell production

☻ __________ IS THE FIRST FULLY DEVELOPED ORGAN IN FETUS

✓ Major site of T-cell production

☻ KIDNEY and SPLEEN – produce B cells

☻ PRODUCTION OF MEGAKARYOCYTES BEGINS IN THIS PHASE

☻ Hemoglobin___– predominant

☻ Hemoglobin ___– detectable levels

MEDULLARY (MYELOID) PHASE

☻ Hematopoiesis begins PRIOR TO 5TH MONTH OF FETAL DEVELOPMENT

(Medullary hematopoiesis)

☻ MYELOID ACTIVITY – appears in this phase

✓ Myeloid:erythroid ratio – ______

☻ ___________ – 1ST site of hematopoiesis (after 24 weeks of gestation – rodaks)

(after first 3 weeks postpartum – steininger)

☻ Hgb A1 – gradually increases its level

ADULT HEMATOPOIETIC TISSUE

BONE MARROW

☻ Located within the cavities of the cortical bone.

☻ COMPONENTS

o RED MARROW

▪ hematopoietically active marrow

▪ Consists of developing blood cells and progenitors.


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o YELLOW MARROW

▪ Hematopoietically inactive marrow

▪ Composed primarily of _____________

▪ Capable of reverting back into active marrow in cases of

increased demand.

☻ _________________ – process of replacing active marrow by adipocytes during

development. (meaning: red marrow is replaced by yellow marrow)

Other adult hematopoietic tissues

☻ Lymph node

☻ Spleen

☻ Liver

☻ Thymus

STEM CELL THEORY

☻ __________________ - all blood cells are derived from a single progenitor stem

cell called Pluripotent Hematopoietic Stem Cell.

o Most widely accepted theory among experimental hematologists

today.

☻ __________________ – each blood cell lineages is derived from its own unique

stem.

HEMATOPOIETIC STEM CELLS and CYTOKINES

☻ Common Lymphoid Progenitor – differentiates into T, B, and natural killer cells

☻ Common Myeloid Progenitor – differentiates into granulocytic, erythrocytic,

megakaryocytic lineage.

“STOCHASTIC”

• HSC randomly commits to self-renewal or differentiation

CELL CYCLE SCHEMATICS

• G0 – resting stage

• G1 – cell growth (Synthesis of components necessary for cell division)

• S – DNA replication

• G2 – premitosis phase

• M - mitosis

CD 38; HL-DR – loss of “stemness”

CD33; CD 38 – common myeloid progenitors

CD 10; CD 38 – Common lymphoid precursors

CD7 – T- progenitor cells

CD 19 – B lymphoid precursors

Colony-stimulating Factors

• Have high specificity

• Active at low concentration

• ____________ is the major source of CSF

GM-CSF + IL-3 = enhancement of megakaryocyte colony formation

Humoral agents that affects CFU-S

Positive effect (increased no., survival, proliferation)

• Phytohemagglutinin

• Cyclophosphamide

• Concanavalin A

Inhibitor

• Hydroxyurea

Colony-forming Units (CFUs)

Abbreviation Cell Line

CFU- GEMM Granulocyte, erythrocyte,

megakaryocyte, monocyte

CFU-E Erythrocyte

CFU – Meg Megakaryocyte

CFU- M Monocyte

CFU – GM Granulocyte, monocyte

CFU- BASO Myeloid to basophil

CFU- EO Myeloid to eosinophil

CFU- G Myeloid to neutrophil

CFU – pre-T T lymphocyte

CFU- pre-B B lymphocyte


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ERYTHROPOIESIS ☻ Typically occurs in erythroid island

Nomenclature System

Normoblastic

(United States)

Rubriblastic Erythroblastic

(Europe)

Pronormoblast Rubriblast Proerythroblast

Basophilic Normoblast Prorubricyte Basophilic erythroblast

Polychromatic

normoblast

Rubricyte Polychromatic

erythroblast

Orthochromic

normoblast

Metarubricyte Orthochromic

erythroblast

Polychromatic

erythrocyte

Polychromatic

erythrocyte

Polychromatic

erythrocyte

Erythrocyte

BFU-E (Burst-forming unit- erythroid)

☻ Erythroid progenitor

☻ 1 week to mature to CFU-E

☻ ____________ from BFU-E to mature RBC

CFU-E (Colony-forming unit-erythroid)

☻ Erythroid progenitor from BFU-E

☻ 1 week to mature to pronormoblast

Criteria in Identification of Erythroid Precursors

☻ Nuclear chromatin pattern

☻ Nuclear diameter

☻ N:C ratio

☻ Presence / absence of nuclei

☻ Cytoplasmic color

Trends that affects the appearance of RBCs as it matures

☻ Decreasing overall diameter of the cell

☻ Decreasing diameter of nucleus (N:C ratio also decreases)

☻ Nuclear Chromatin – coarser, clumped, and condensed.

☻ Disappearance of nucleoli

☻ Color change in cytoplasm

✓ BLUENESS (basophilia)

o Caused by ACIDIC COMPONENTS (ribosomal RNA)

o Attracts _______stains (methylene blue)

✓ PINKNESS (acidophilia)

o Caused by BASIC COMPONENTS (hemoglobin)

o Attracts _______ stains (eosin)

MATURATION SEQUENCE

N:C

RATIO

NUCLEUS;

CYTOPLASM

DIVISION

PRODUCTS

CELLULAR ACTIVITY;

INFORMATION

PRONORMOBLAST 8:1 N – purple

red

chromatin

2 prorubricyte Accumulation of

components for Hgb

synthesis

BASOPHILIC

NORMOBLAST

6:1 N –

chromatin

begins to

condense

2 rubricyte Detectable Hgb

synthesis

POLYCHROMATIC

NORMOBLAST

4:1 –

1:1

N – absence

of nucleoli

C – 1st stage

with pink

color (Hgb);

murky-gray

blue

2

metarubricyte

(____________

_____________

_____________)

Increased Hgb

synthesis

ORTHOCHROMATIC

NORMOBLAST

1:2 C – salmon-

pink; Hgb

synthesis

nearly

complete

Not capable

of division

Nucleus is ejected

from cell; loss of

______ - (responsible

for holding

organelles)

PYRENOCYTE –

extruded nucleus

PHOSPHATIDYLSERINE

– “eat me flag”


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NON-MUSCLE

MYOSIN – important

in pinching process

POLYCHROMATIC

ERYTHROCYTE

anucleated Incapable Complete Hgb

synthesis

Resides in BM for 1

day or more →

peripheral blood 1

day before maturity

ERYTHROCYTE C –

biconcave

1.5-2.5um diameter

Salmon pink

Life span: 80-120

days

__________________

☻ Dynamics of RBC production and destruction

_________________ – collection of all stages of erythrocytes throughout the body.

Stimuli of RBC Production

☻ HYPOXIA

✓ Detected by PERITUBULAR FIBROBLAST OF THE KIDNEY

▪ Produces EPO (major stimulatory cytokine for RBC)

▪ Primary O2 – sensing system of the body

Major Effects of EPO

☻ Early release of Reticulocyte

✓ Increases the width of spaces for RBC egress into the sinus (in the BM)

✓ Down regulation of expression of receptors for adhesion molecules in

the BM stroma

✓ ________________ or (stress reticulocyte)

▪ Very basophilic

▪ Shifted from BM early

☻ Inhibition of Apoptosis

✓ Increasing number of cells that will be able to mature into circulating

erythrocytes – decreasing apoptosis

☻ Reduced Marrow Transit Time

✓ Increasing the rate at which the surviving precursors can enter the

circulation

▪ Increased rate of cellular processes

▪ Decreased cell cycle times

Measurement of EPO

• Can be measured by __________________

• Reference interval – __________ (varies for each lab)

• Increased in most patients with anemia, EXCEPT FOR PATIENTS WITH ANEMIA

CAUSED BY ______________

Therapeutic use:

• Used in anemia associated with CKD and chemotherapy

• Also used prior to autologous blood donation and after bone marrow

transplantation

• Used in athletes to increase O2 -carrying capacity (illegal)

o Causes fatal arterial and venous thrombosis

Other stimuli to Erythropoiesis

•

•

•

RBC DESTRUCTION

Senescence

• Cellular aging

• Loss of glycolytic enzymes

Macrophage-mediated hemolysis (Extravascular Hemolysis)

• Spleen generates stressful environment for RBCs

Signals recognized by macrophages to differentiate senescent from young cells

o Binding of autologous IgG to band-3 membrane protein clusters

o Exposure of phosphatidylserine on the exterior of the membrane

o Inability to maintain cation balance

o Senescent changes to CD47 – binding of thrombospondin-1 – “eat

me” signal to macrophage

Mechanical hemolysis (fragmentation or intravascular hemolysis)

• Due to turbulence in the chambers of the heart or bifurcation of vessels

• Small breaks in blood vessels resulting clots can trap and rupture cells


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LEUKOPOIESIS o Overall function – _______________

Granulocytes

• WBCs whose cytoplasm is filled

• with granules different staining characteristics

• nuclei are lobulated and segmented.

Eosinophil

• Granules – basic proteins

• Stains with acid stain

Basophil

• Granules – acidic

• Stains with basic stain

Neutrophil

• React with both acidic and basic stain

o Gives them pink to lavender color.

Mononuclear cells

o Nuclei – not segmented, but round, oval, indented, or folded

o

o

GRANULOCYTES

Neutrophil

☻ Two forms in PB

✓ Segmented

✓ Band shape

☻ Life span – _________ (from myeloblast to death)

Pools of Developing Neutrophil

☻ ______________– capable of self-renewal and differentiation

✓ Consist of HSCs

☻ Proliferation (mitotic) pool – dividing cells

✓ Consists of:

▪ CFU-GEMM

▪ Granulocyte-macrophage progenitors

▪ Myeloblast

▪ Promyelocyte

▪ Myelocyte

☻ Maturation (Storage) Pool – cells undergo nuclear maturation

☻ Consists of:

✓ Metamyelocyte

✓ Band neutrophils

✓ Segmented neutrophils

MATURATION STAGES

• MYELOBLAST

o (0-3% -rodaks) (1-2% - steininger) in BM

o ___________ in diameter

o Earliest recognizable form

SUBDIVISIONS

o Type I

▪ N:C ratio – ______________

▪ Slightly basophilic cytoplasm

▪ Fine nuclear chromatin

▪ 1-4 visible nucleoli

▪ No visible granules when observed in light microscopy with

Romanowsky stain

o Type II

▪ Presence of dispersed primary (azurophilic) granules in

cytoplasm

▪ No. of granules don’t exceed 20 per cell

o Type III

▪ Darker chromatin

▪ More purple cytoplasm

▪ Contain more than 20 granules that don’t obscure the nucleus

▪ RARE in BM

▪ CAN BE SEEN IN CERTAIN TYPES OF AML

• PROMYELOCYTE (progranulocyte)

o 1-5% (rodaks) in BM

o 16-25 um in diameter

o Nucleus – round to oval; eccentric

o Synthesis of primary or (_____________) begins and ends in this stage.

▪ Rich in peroxidase


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o Paranuclear halo or _____ is present

▪ Absent in acute promyelocytic leukemia

o 1-3 nucleoli seen but obscured by the granules

• NEUTROPHIL MYELOCYTE

o (6-17% - rodaks) (10-20% -steininger) in BM

o Final stage in which mitosis occurs

o Production of _____________ granules begins

o Early Myelocyte

▪ Very similar to promyelocyte

▪ Pale pink cytoplasm begins to be evident in the area of golgi

apparatus (dawn of neutrophilia)

o Late Myelocyte

▪ Smaller than promyelocyte (15-18 um)

▪ Nucleus – more heterochromatin

▪ Nucleoli difficult to see by light microscope

• METAMYELOCYTE (JUVENILE)

o (15-30% - steininger) (3-20% - rodaks) in BM

o Synthesis of ________ granules (gelatinase granules)

o Nucleus – ___________________ shaped

o Chromatin – increasingly clumped

o Contains very little residual of RNA (little or no basophilia)

• NEUTROPHIL BAND

o 9-32% (rodaks) in BM

o Last stage before the mature cell

o Secretory granules (secretory vesicles) are formed in this stage.

o The greatest source of variation and discrepancy in leukocyte

morphology

• SEGMENTED NEUTROPHIL (seg, poly, PMN)

o 7-30% in BM

o Secretory granules continued to be formed

o 2-5 nuclear lobes connected by threadlike filaments

o NORMAL VALUES:

▪ Relative value

• 37 – 77% (steininger)

• 50 – 70% (rodaks)

▪ Absolute value

• 2.0 – 6.93 x109/ L in adults (steininger)

• 2.3 – 8.1 x 109/ L in adults (rodaks)

NOTES:

• Half of the blood neutrophils are not circulating freely, but adheres to vessel

wall (Marginal Neutrophil Pool)

• Neutrophil half-life in the blood – ____________

• _______(rolling) and _________ (stationary binding)– important in diapedesis

• __________ – stimulates release of granulocyte from BM.

• Neutrophils that don’t migrate to tissue – undergo apoptosis

NEUTROPHIL FUNCTION

• Major function –__________

o MOTILITY

▪ Wave-like or “ruffles” – moves forward

▪ ___________ – random migration

▪ Guided by ___________

• Neutrophils responds faster than monocytes

• _____ – most potent chemotactic factor

o Other chemotactic Factors

o Endotoxin – activates FXII

o Metabolic products of arachidonic acid

o Chemotactic agents released by lymphocytes and other

phagocytes

• RECOGNITION

o Opsonins

▪ Aids neutrophil on what to ingest

o Examples of opsonins

▪ Antibodies

▪ Complement substances

▪ Fibronectin

• INGESTION

• DEGRANULATION

• KILLING

o Actively metabolized O2 to produce toxic substances for

killing ingested foreign particles


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o Principal toxic metabolites

▪ Superoxide anion

▪ H2O2

o Myeloperoxidase

▪ Potentiates killing action of H2O2 presence of ascorbic

acid and halides

o Lysozymes

▪ Hydrolyzes mucopeptide cell wall of few species of

bacteria.

o Superoxide dismutase

▪ Converts superoxide into H2O2

o Catalase

▪ Destroys H2O2

o Reduced glutathione

▪ Detoxify H2O2

▪ Regenerate NADPH

• Second Function

o Generation of NETs (Neutrophil extracellular traps)

▪ Contains enzymes from the granules; shown to trap and kill

Gram (+), gram (-) bacteria, and fungi.

▪ NETosis

• Unique form of neutrophil cell death resulting a release of

NETs

o Third and Final Function

o Secretory function

o Source of Transcobalamin I or R binder protein – for proper absorption

of Vit. B12.

Tests for Neutrophil Function

• Boyden Micropore Filter Technique

o Assays neutrophil migration in response to chemotactic factor.

• Rebuck Skin Window Procedure

o Evaluates speed, type, and number of phagocytes that responds to

skin abrasion.

EOSINOPHIL

• 1-3% in BM

• 1-3% in peripheral blood

o Absolute number - 0.4 x 109/L

• First to be distinguished from neutrophil in PROMYELOCYTE STAGE

Development

• Similar to neutrophils

• Lineage is established through interactions of:

o IL-3

o IL- 5 – critical in growth & survival

o GM-CSF

o GATA-1

o PU.1

o c/EBP

STAGES

• EOSINOPHIL PROMYELOCYTE

o Identified CYTOCHEMICALLY

o Presence of __________________ proteins in primary granules

• EOSINOPHIL MYELOCYTE

o First to be identified in light microscopy and Romanowsky stain

o Cytoplasm - large, pale, reddish-orange secondary granules w/ azure

granules

o Nucleus – similar to neutrophil myelocyte

o Secondary granules – contains ___________________ (seen in TEM).

• EOSINOPHIL METAMYELOCYTE & BANDS

o Resembles neutrophil counterpart

o Increased secondary granules

o Generation of SECRETORY GRANULES/ SECRETORY VESICLES

o Has 2 organelles

▪ Lipid bodies

▪ Small granules

• MATURE EOSINOPHIL

o Bilobed nucleus

o Cytoplasm – has orange-red secondary granules

o Has extensive secretory vesicle

▪ Increased when stimulated or activated

______________ – HALLMARK OF ALLERGIC

DISORDER


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KINETICS

• From last myelocytic division to mature eosinophil – 3.5 days

• Circulating half-life – ________

• Transit time – 1-8 hours

• Survival time in tissues - 2-5 days

• Only 1% found in the blood (mostly found in tissues)

BIOCHEMISTRY

• ___________ – principal source of energy

• OUTER MATRIX OF GRANULES

o Hydrolytic enzymes

o Peroxidases (different from neutrophils)

• INNER MATRIX CORE OF GRANULES

o Major Basic Proteins

• ______________________

o Hexagonal-bipyramidal crystal

o from primary granules

o appears when large no. of eosinophils disintegrates in secretions or

exudates

o seen in

▪ allergic asthma

▪ pulmonary eosinophilic infiltrates

▪ stools in parasitic infection

FUNCTIONS

• MECHANISMS OF DEGRANULATION

o Classical Exocytosis

▪ Granules move and fuse to plasma membrane; empty contents

in EC space

o Compound Exocytosis

▪ Granules fuse first within the cell before fusing to plasma

membrane

o Piecemeal Degranulation

▪ Secretory vesicle removes specific proteins from secondary

granules; migrates and fuse to plasma membrane; specific

proteins released in EC space.

• Increased in parasitic helminth infection

o Destroys helminths through Major Basic Protein and Eosinophil Cationic

Protein

• Believed to prevent reinfection

• Plays important role in immune regulation

• Believed to be involved in deletion of double-positive thymocytes

(transmigrate to thymus of newborn)

• Regulates mast cell through release of MBP

o Causes mast cell to degranulate

▪ Cytokine production

▪ Nerve Growth Factor

• Promotes mast cell survival and activation

• Implicated also in airway remodeling (increased thickness of airways wall)

o Through EOSINOPHIL-DERIVED GROWTH FACTOR

BASOPHIL

• Morphologically and functionally similar to mast cells

• ______________

• Least numerous of the WBCs

o (0-2% - rodaks) (0.5-1% -steininger) of circulating WBCs

o 0.3% in the BM

DEVELOPMENT

• ______ – influences differentiation

STAGES

• IMMATURE BASOPHIL

o Nuclei

▪ Round to lobulated

▪ Slightly condensed chromatin

o Cytoplasm contains

▪ Blue-black 20 granules

• Water-soluble

• MATURE BASOPHIL

o Nuclei

▪ Bilobed or band-shaped

▪ Chromatin – clumped

o Cytoplasm


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▪ Colorless

▪ Contains large no. of large blue-black granules

o Leaves reddish-purple rim surrounding a vacuole when granules have

been dissolved

KINETICS

• Life span – 60 hours

o Due to IL-3 that initiates anti-apoptotic pathway.

FUNCTIONS

• Releases ____ and ______

o Regulates TH2 cells immune response

• Induces B cells to produce IgE

• Basophilic activation – not restricted to Ag-specific IgE. Can also be triggered

by:

o Parasitic antigen

o Lectins

o Viral superantigens binds to nonspecific IgE

• Capable of synthesizing granule protein based on activation signals

o Produces granzyme B (mediator)

o Produces retinoic acid

▪ Regulates immune and resident cells in allergic diseases

• Has a role in _____________

o Expresses Vascular Endothelial Growth Factor (VEGF)

• Involved in control of helminth infection control

o Promotes eosinophilia

MAST CELLS (TISSUE BASOPHIL)

• Not leukocyte

• Tissue effector cells of allergic responses and inflammatory reactions

• Precursors circulates on peripheral blood for brief period on their way to tissue

• Have several phenotypical and functional similarities with basophil and

eosinophil

• ______________ – major cytokine responsible for mast cell maturation and

differentiation

FUNCTION

• Releases wide variety of

o Lipid mediators

o Proteoglycans

o Proteases

o Cytokines

• can function as APC to induce differentiation on TH2 cells

• can have anti-inflammatory and immunosuppressive functions

MONONUCLEAR CELLS

MONOCYTE

• 2-11% of circulating WBCs

o Absolute No. – 1.3 x 109/ L

DEVELOPMENT

• Derived from granulocyte-monocyte progenitor

• ________ – major cytokine responsible for growth and differentiation of

monocytes

STAGES

• MONOBLAST

o Normal in BM

o Very rare

• PROMONOCYTE

o Diameter – 12 – 18 um

o Nucleus

▪ Slightly indented and folded

▪ High N:C ratio

o Cytoplasm

▪ Blue/ gray-blue

▪ Contains azure granules (heterogeneous)

o Earliest precursor cell to the monocyte that is recognizable by light

microscopy

o Undergoes 2 mitotic division in 60 hours – produces 4 monocytes

▪ If increased demand – 4 divisions in 60 hours – 16 monocytes

o In Steininger – 3 divisions taking @ 30-48 hours


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• MONOCYTE

o 15-20 um (rodaks)

o 12-15 um (steininger)

o Nucleus

▪ Round, oval, kidney-shaped

▪ U-shaped

▪ More frequently indented

o Slightly immature cell

o ULTIMATE GOAL

▪ Enter tissue and mature

o NUCLEAR CHROMATIN PATTERN

▪ Lacelike, stringy, “brain-like” convolutions, loosely woven and

linear

• MOST RELIABLE CRITERION TO DISTINGUISH A MONOCYTE

o Cytoplasm

▪ Blue-gray

▪ Has _____________ (__________appearance)

o Subsets (Flow Cytometry Immunophenotyping)

▪ Classical

▪ Intermediate

▪ Nonclassical

KINETICS

• No storage pool of mature monocyte in BM

• Monocyte in Splenic Reservoir

o Respond to tissue injury to participate in wound injury

• Marginal pool – 3.5 times the circulating pool

• Incapable of cell division

• Stays in blood for 70 hours

• Once in the tissue, it differentiates into:

o Macrophage

o Osteoclast

o Dendritic cell

o Langerhans cells

BIOCHEMISTRY

• Monocyte depends on aerobic glycolysis

• Most macrophage depends on anaerobic glycolysis except for alveolar

macrophage (aerobic glycolysis)

GRANULE CONTENTS

• Monocyte

o Acid phosphatase

o Beta- glucuronidase

o Lysozyme

▪ Bacteriolytic enzyme

▪ May enhance phagocytosis

▪ Potential antineoplastic effect

o Lipase

o Peroxidase

• Macrophage

o No peroxidase

RECEPTORS

• Monocyte and Macrophage

o Has receptors for Fc part of IgG and C3

o Receptor for IgE


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FUNCTION

• INNATE IMMUNITY

o Recognize pathogens thru _________________

o Macrophage produces ____________

▪ Cytotoxic against

• Viruses

• Bacteria

• Fungi

• Protozoa

• Helminths

• Tumor cells

o Efficient in engulfing large particles

o Influenced by Migration Inhibition Factor

▪ To remain at the site of infection

o CHEMOTACTIC FACTORS THAT ATTRACTS MONOCYTE

▪ Ag-Ab complex

▪ Complement components

▪ Kallikrein

▪ Factors released by T cells

o Killing potency of macrophage is enhanced when “activated”

▪ Refers to enhancement of

• Motility

• Metabolism

• Enzyme activity

• Killing capacity

▪ May result from

• Direct contact to microorganism

• By-products of microorganism

• Lymphokines

o Known microorganisms that parasitize macrophages

▪ Mycobacterium

▪ Listeria

▪ Salmonella

▪ Brucella

▪ Fungi

▪ Protozoa

o Shown to eliminate viruses and virus-infected cells

o Also releases _____

▪ Promotes T-cell replication

o Also releases

▪ Endogenous components (induces fever)

▪ Complement components

• ADAPTIVE IMMUNITY

o Plays role in antigen presentation (APCs)

• HOUSEKEEPING FUNCTIONS and others

o Removal of debris and dead cells at the site of infection or tissue

damage”

o Production of __________________

▪ 1o transporter of Vit. B12

o Secretion of

▪ Plasminogen activator

▪ Plasmin inhibitor

▪ Platelet activator factor

▪ Tissue-thromboplastin-like procoagulant

o Plays a role in Hgb degradation and iron transport by liberating iron

from heme.

LYMPHOCYTES

• 18-42% in the circulation

o 3- 21% in circulation – _______

o 51 – 88% in circulation – ______

o 4 – 29% in circulation – _______

▪ Absolute number – 0.8 – 4.8

x109/L

• Nucleus

o round, ovoid, kidney

• Cytoplasm – clear blue

• Chromatin – crushed velvet

• Groups

o T cells – develops in ______

o B cells – develops in _____

o NK cells – develops in _____________

o Non-T and Non-B cells – don’t develop along either T or B cells

“_____________” / “________” or “________”

Phenomenon

• Transferring iron to transferrin for

incorporation into developing RBC

• Developing RBCs surround a large

macrophage

• Happens in BM


DO NOT DISTRIBUTE

DIFFERENCES FROM OTHER WBCs

• not an end cell

o they are resting cells

o undergoes mitosis to produce

▪ ___________

▪ ___________

• recirculates from blood to tissue and back to the blood

• capable of rearranging antigen receptor genes to segments to produce

variety of antibodies and surface receptors

• T and NK cells develops and mature outside BM

DEVELOPMENT

• Colony forming unit – Lymphocyte

o Has _______ and ____

o Produces functionally diverse cells

• T and B cells subdivision

o __________________

▪ Occurs in 1o lymphoid organs

• 10 lymphoid organs develop during embryogenesis

• Occurs in fetal liver

o __________________

▪ Occurs in 2o lymphoid organs

• B CELLS

o Develops in BM

o STAGES – In these stages, immunoglobulin gene rearrangement occurs

to produce unique Ig antigen receptors

▪ Pro-B

▪ Pre-B

• has CALLA

• Earliest recognizable B cells

▪ Immature B cells

o IMMATURE B CELLS (Hematogones)

▪ Chromatin – homogeneous

▪ Cytoplasm – scanty, deep blue, devoid of granules, cleft

▪ Antigen-naïve

▪ Migrates to 20 lymphoid organs

• Resides on lymph node follicles

▪ Absence of CALLA and TdT

▪ Appearance of

• sIg (surface immunoglobulins)

• intracytoplasmic chains of IgM

• B cell – specific antigen

o B461

o S-HCL2

▪ Normally seen in newborn peripheral blood

▪ _________________________________________

• Differentiates leukemic cells from hematogones

o MATURE B CELLS

▪ Demonstrates sIg

▪ Synthesis of light chains (lambda and kappa) joined with heavy

chains inserted in the plasma membrane

▪ Has receptors for the following:

• Fc portion of the following

o

o

o

• Mouse RBC

• Complement

o C3b

o C3d

o C4b

o C1q

o C8

o C2 inhibitor

• Epstein-Barr Virus

o Resting lymphocytes

▪ Small – 9 um in diameter

▪ N:C ratio – _________

▪ Chromatin – blocks

▪ Nucleolus – rarely seen

o EFFECTOR AND MEMORY B CELLS

▪ Produced when there is contact to antigens in 20 lymphoid

organs


DO NOT DISTRIBUTE

▪ _______________ – antibody-producing

▪ Plasmacytoid lymphocyte – atypical lymphocytes

o Mitogens for B cell

▪ Pokeweed Mitogens

▪ LPS

▪ S. aureus Covan I

▪ EBV

• Independent to accessory cells

• Earliest B cell specific surface marker

o PLASMA CELLS

▪ Antibody-producing cell

▪ Nucleus

• Eccentric

• Has blocks of heterochromatin (tortoise shell)

▪ Cytoplasm

• Abundant non-granules

• Deep blue (due to ribosomes)

• Golgi apparatus

o Unstained (area of Hof)

• Periphery

o Layered look (flattened parallel sacs of RER)

• Contains Russell Bodies

o Round

o Discrete globules

o Unstained, or pale-blue or red

• ___________ / _________ / __________

o Russell bodies-filled plasma cell

• T CELLS

o Develops in thymus

o ___________ – hormone that plays crucial role in maturation or pre-T to T

cells

o STAGES – in these stages, it undergoes antigen receptor rearrangement

to produce T cell receptors that are unique to each T cells

o NOTE: receptors that react to self-antigen - APOPTOSIS

▪ Pro – T

▪ Pre – T

• Has TdT

• CALLA (+)

• Ia antigen (+)

• Don’t from rosette in SRBC

• No sIg

▪ Immature T cells

• Leaves thymus; migrates to ______________

o REACTIVE / VARIANT LYMPHOCYTES

▪ Reflect on the reaction to antigen

▪ Variation of morphology of effector T cell w/ the subtype of T

cells

o Mitogens for T cells

▪ Phytohemagglutinin

▪ Concanavalin A

▪ Pokeweed Mitogen

KINETICS

▪ Average life span in blood – _________

o Some can reach up to 20 years

▪ __________ – most adherent part of the cell

• NK CELLS

o Heterogeneous group of cells w/ respect to their surface antigens

o Majorities are

▪ CD56+

▪ CD16+

▪ CD3+

▪ CD7+

▪ CD11b+

o CD – _______________

FUNCTIONS

▪ Major function – antigen recognition and appropriate immune response

▪ B CELLS

o For antibody-production (plasma cells)

o Has role in antigen presentation to T cells

o May be necessary for CD4 optimal activation

o Produces cytokines

▪ Regulates variety ot T cell and Antigen presenting cell function


DO NOT DISTRIBUTE

o End result

▪ Memory B cell

▪ Plasma cell

o Lymphokines that regulates growth and maturation of B cells

▪ B cell growth factor I & II

▪ B cell differentiation factor

▪ B cell stimulatory Factor – 2

▪ IL – 2

▪ IFN- gamma

o C3

▪ Triggers activation of early B cells

▪ Generates memory B cells

o _____________________________

▪ Regulatory role in the magnitude of B cell response in-vivo.

▪ T CELLS

o Types

▪ CD4+

• ______

o Mediates response agains intracellular pathogens

• ______

o Mediates response against EXTRACEL-LULAR

PARASITE

o Also important in induction of asthma and allergic

disease

• __________

o Against extracellular bacteria and fungi

• __________

o Maintains self-tolerance by regulating immune

response

▪ CD8+ (Cytotoxic T- Lymphocyte)

• Kills target cells by secreting perforin and granzyme or by

activating apoptotic pathway

▪ ______________

• Kills tumor cells and virus-infected cells without MHC

restriction

• Modulates function of other cells (macrophage & T cells)

• Resistance to bacterial, fungal, and parasitic agents

• Immune regulation

• Regulation of hematopoiesis

• Natural resistance to allogeneic graft

▪ KILLER CELLS

• Antigen-dependent (ADCC)

o Differentiates it from NK cells

▪ LAK CELLS (Lymphokine-activated Killer Cells)

• Activated by _____

• Don’t express some NK surface antigens

• Phenotypically different from NK cells

▪ LYMPHOKINES

o Secreted by Activated T and B cells

▪ Colony-Stimulating Factor

▪ ____________________________

• Inhibits monocyte and macrophage from migrating

▪ Lymphocyte migration inhibitory factor (LyMIF35k)

• Immobilizes lymphocyte at the site of infection

▪ Lymphocyte chemoattractant (LCF)

• Increases migration of non-essential T cells

▪ _______________________________

• Increase histamine produced by basophil and mast cell

▪ T cell activating Factor

• Antigen-independent

• Activates CTLs and NK cells

▪ Human lymphotoxin (HLT)

• Inhibits tumor cell growth

• Produced by T cells (primarily)

METHODOLOGIES

HEMATOCRIT ☻ Aka Packed Cell Volume

☻ Layers of anticoagulated whole blood (top to bottom)

a) Plasma

b) Platelets

c) White blood cells

d) Red blood cells

MICROHEMATOCRIT METHOD

☻ Capillary tube


DO NOT DISTRIBUTE

✓ Length – __________

✓ Bore – ____________

✓ Blood should fill – _________________

✓ Sealing clay – _____________

✓ Types:

o ______ tip – with 1IU of heparin

o ______ tip – no anticoagulant

☻ Centrifuge

✓ Calibration

o _________ – accuracy

o _________ – speed

✓ Speed – _________________________

☻ Trapped plasma causes 1-3% (0.01 to 0.03 L/L) increase in hematocrit

☻ Reading should be done within __________

MACROHEMATOCRIT METHOD

☻ Wintrobe tube is used

☻ Centrifuged at 2,000 to 2,300 g for 30 mins

Wintrobe tube Westergren method

Length 115 mm 300 mm

Bore 3 mm 2.55 mm

Calibration Left – ESR (0-100)

Right – Macrohematocrit (100-0)

0-200

CYANMETHEMOGLOBIN METHOD ☻ For hemoglobin determination

☻ Modified Drabkin’s reagent

✓ __________________ – permits oxidation of ferrous to ferric iron

(hemoglobin → methemoglobin)

✓ Potassium cyanide – methemoglobin → cyanmethemoglobin

✓ Dihydrogen potassium phosphate – replaces sodium bicarbonate in

original reagent (reduction of time from 15 mins to 3 mins)

✓ Non-ionic detergent – improves red cell lysis and decreases turbidity

✓ Read using spectrophotometer at ________

✓ Sample: EDTA whole blood

✓ Dilution: 1:251

✓ Sources of error:

o High WBC count (>20 x 109/L) and high platelet count (>700 x

109/L) – corrected by centrifuging sample and using the

supernatant

o Lipemic sample – prepare patient blank

o Hgb C and and Hgb S – make a 1:2 dilution with distilled water

and multiply results from standard curve by 2.

RULE OF THREE ☻ Used for normocytic, normochromic specimens

☻ ____________________________________

RED BLOOD CELL INDICES ☻ Helpful for the initial classification of anemia

☻ Mean Cell Volume(MCV) – average volume of RBC

𝑀𝐶𝑉 =hematocrit (%)

RBC count (x1012

L)

x10

☻ Mean Cell Hemoglobin (MCH) – average weight of hemoglobin in an RBC

𝑀𝐶𝐻 =hemoglobin (g/dL)

RBC count (x1012

L)

x10

☻ Mean Cell Hemoglobin Concentration – average concentration of

hemoglobin in each individual erythrocyte

𝑀𝐶𝐻𝐶 =hemoglobin (g/dL)

hematocrit (%)x100

MANUAL CELL COUNT ☻ Diluting fluids and Pipettes

RBC WBC

Diluting fluids Hayem’s

Gower’s

NSS

Formol

citrate

Toisson’s

Bethels’s

3.8% sodium

citrate

2-3% glacial acetic acid

1% HCl

Turk’s solution


DO NOT DISTRIBUTE

Characteristics Isotonic Hypotonic – lyses RBCs

except nRBCs

PIPETTE PROPERTIES

Size of bulb Larger Smaller

Color of bead Red White

Volume of bulb 100 10

Size of bore Smaller Larger

Calibration 0.5; 1; 101 0.5; 1; 11

Dilution 1:200 1:20

Dilution Factor 200 20

☻ Hemocytometer

✓ 1 ruled area – 9mm2

✓ 1 WBC square (with 16 smaller squares) - _________

o 1 square inside a WBC square – 0.0625 mm2

✓ Central square (with 25 smaller squares)

o 1 small square inside central square (with 16 smaller squares) –

0.04 mm2

o 1 smallest square – 0.0025 mm2

FORMULA : 𝑐𝑒𝑙𝑙𝑠

𝑢𝐿=

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟

𝑎𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑒𝑝𝑡ℎ (0.1)

☺ In order to memorize, list down the formula of corrected WBC count

WBC ESTIMATE

2-5 WBCs/hpf 4,000-7,000 WBCs/uL

4-6 WBCs/hpf 7,000-10,000 WBCs/uL

6-10 WBCs/hpf 10,000-13,000 WBCs/uL

10-20 WBCs/hpf 13,000-18,000 WBCs/uL

RETICULOCYTE COUNT ☻ Used to assess erythropoietic activity of bone marrow

☻ Sample: EDTA whole blood

☻ Stain: supravital stain (new methylene blue, brilliant cresyl blue)

☺ In order for you to memorize, list down all the formula for reticulocyte count, miller

disc method, absolute reticulocyte count, corrected reticulocyte count and

reticulocyte production index)

ERYTHROCYTE SEDIMENTATION RATE (ESR) ☻ Detect inflammatory process

☻ Stages:

1. Lag phase (initial rouleaux formation) – ________

2. Decantation phase (period of fast settling) – ___________

3. Final packing – ______

☻ Factors:

ESR INCREASED ESR DECREASED

Increased cholesterol, fibrinogen

and gamma globulins

Increased albumin (due to viscosity)

RBC agglutination Altered RBC shape

Leukemia Polycythemia

Increase temperature Narrow ESR column diameter

Tilted ESR tube (3 degrees tilt =

increased of 30%)

Clotted samples

OSMOTIC FRAGILITY TEST ☻ __________ OFT: Hereditary spherocytosis

☻ __________ OFT: anemias where there are target cells

BLOOD SMEARS ☻ Methods:

✓ __________ – uses 2 glass slides

✓ __________ – uses glass slide and coverslip

✓ _________ – uses 2 coverslips

✓ _____________ – if patient’s WBC count is <1x109/L

✓ Automated methods

FACTOR THIN SMEAR THICK SMEAR

Pressure

Angle Size

Speed


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Cell Abnormalities _________

☻ A decrease in either number of RBCs, hemoglobin or hematocrit

NORMOCYTIC, NORMOCHROMIC ANEMIA

☻ Examples:

✓ Aplastic anemia

✓ Hemolytic anemia

✓ Anemia of Chronic disease

o Associated with inflammatory process

o Low TIBC

✓ Sickle cell anemia

o Type of hemoglobinopathy

o β26 Glu → Val

o oxygen is released thus “sickling” of cells occur

o resistance to P. falciparum

o Primary cause of death: infectious crisis

✓ Paroxysmal Cold Hemoglobinuria

✓ Paroxysmal Nocturnal Hemoglobinuria

o Aka ___________________________

o _________ and _________ deficiency

o Tests: Ham’s acidified serum test, Sugar Water test and Flow

cytometry

MICROCYTIC, HYPOCHROMIC ANEMIA

☻ Examples:

✓ ________________

o There is a failure of iron to incorporate to protoporphyrin IX

o Excess accumulation of iron in mitochondria of normoblasts

o Dimorphic anemia

✓ ________________

o Most common cause of anemia

o Increased TIBC

o Blood picture is only evident is Stage 3 or Frank anemia

✓ Thalassemia

o Reduction or loss of synthesis of one or more of the globin chains

o Silent carrier (1/4 genes deleted), α- thalassemia (2/4 genes are

deleted), __________ (3/4 genes deleted; composed of 4 β

globin chains) and _________ (4/4 genes deleted; composed of

4 γ globin chains)

✓ Chronic blood loss

MACROCYTIC, NORMOCHROMIC ANEMIA

☻ Examples:

✓ Megaloblastic anemia

o Vit. B12 and Folate deficiency

o _______________ – autoantibodies: anti-parietal cell antibodies

and anti-intrinsic factor antibodies

✓ Non-megaloblastic anemia

o Alcoholism, Liver disease

VARIATIONS ☻ In size: _____________

☻ In hemoglobin content: _______________

HYPOCHROMIA GRADING

Area of central pallor is ½ of cell diameter

Area of central pallor is 2/3 of cell diameter

Area of central pallor is ¾ of cell diameter

Thin rim of hemoglobin

POLYCHROMASIA GRADING

Slight

1+

2+

3+

4+

☻ in shape: _____________________


DO NOT DISTRIBUTE

RBC ANOMALIES

____________

☻ Spur cell

☻ Thorny cell

☻ Irregular spicules

☻ Found in abetalipoproteinemia and McLeod

syndrome

☻ Defect: abnormality in lecithin and

sphingomyelin ratio

Echinocyte

☻ Burr cell

☻ Sea urchin cell

☻ Evenly distributed spicules

☻ Found in uremia and PK deficiency

☻ Defect: membrane lipid content abnormality

Codocyte

☻ Target cell

☻ Mexican hat cell

☻ Leptocyte (thinner

variant)

☻ Found in thalassemia, Hemoglobinopathies

☻ Defect: deficiency in phospholipids and

cholesterol content of membrane

Elliptocyte ☻ Cigar-shaped RBC

☻ Found in hereditary elliptocytosis

☻ Defect: decreased membrane protein band

4.1

______________

☻ Mouth cell

☻ Xerocyte

(dehydrated variant)

☻ Slit-like

☻ Found in Rh null syndrome

☻ Defect: cation imbalance

Drepanocyte

☻ Sickle cell

☻ Found in sickle cell anemia

☻ Hgb S polymerization

_____________

☻ Teardrop cell

☻ Found in Myelofibrosis with myeloid

metaplasia

WBC ANOMALIES

NUCLEAR ABNORMALITIES

____________

☻ hyposegmentation

☻ Defect: mutation of lamin-beta receptor

☻ Pince-nez appearance

Hypersegmentation ☻ Neutrophils with 6 or more lobes

☻ Defect: abnormality in DNA synthesis

☻ Found in Undritz anomaly, megaloblastic

anemia

Barr bodies ☻ Appears as drumstick attached to a nucleus

by a short stalk

☻ Represents inactive X chromosome

☻ Found in Klinefelter syndrome

CYTOPLASMIC ABNORMALITIES

Auer rods ☻ Derived from fusion of primary granules

Alder-reilly ☻ Result of incomplete degradation of

mucopolysaccharides

Chediak-higashi ☻ Defect: mutation in LYST gene

☻ Cells are characterized with scanty

lysosomal vesicles

_______________ ☻ Presence of large, abnormal cytoplasmic

granules in phagocytes

_______________ ☻ Dohle-like inclusions but found in

granulocytes and monocytes

☻ Characterized with the presence of giant

platelets

Faggot Cells ☻ Mass of auer rods

Toxic granulations ☻ Found in neutrophils – altered primary

granules

LEUKOCYTE FUNCTION ABNORMALITIES

Job syndrome ☻ Mutation in STAT3 gene

☻ Neutrophils have normal random motility but

altered directional motility

Lazy leukocyte syndrome ☻ Both random and directional motility are

altered

_____________ ☻ Failure of WBCs to kill microorganisms due to

defects in respiratory burst

CELLS EXHIBITING PHAGOCYTOSIS

LE cell ☻ Neutrophil that engulfed a nuclear material

Tart cell ☻ Monocyte or macrophage that engulfed a

nuclear material

LYMPHOCYTE ABNORMALITIES

Basket cell ☻ Remnants of granulocytic cells

Smudge cell ☻ Remnants of lymphocytes

Rieder cell ☻ Cloverleaf-like

__________ ☻ TRAP (+)

☻ B lymphocyte in origin

Sezary cell ☻ Cells with cerebriform nucleus

☻ T lymphocyte in origin

PLASMA CELL ABNORMALITIES

Flame cell ☻ Aka thesaurocyte

☻ Found in IgA myeloma

Grape cell ☻ Clock face nucleaus


DO NOT DISTRIBUTE

☻ Cell with a cytoplasm completely filled with

Russell bodies

☻ Found in Multiple Myeloma

Russell bodies ☻ Aggregates of immunoglobulins

LEUKEMIA ☻ Acute leukemia - __________ in peripheral blood (based on FAB but 20%

based on WHO) and >50% blasts in bone marrow

✓ _______ – most common form of childhood leukemia

☻ Chronic leukemia - <10% blasts in PB

✓ _______ – most common form in elderly

☻ M:E ratio is _____

☻ MPO stain

✓ fresh blood is required

✓ used in differentiating AML from ALL

☻ Sudan Black B

✓ Most sensitive stain for granulocytic precursors

☻ _____ – presence of Philadelphia chromosome indicates good prognosis

ACUTE MYELOPROLIFERATIVE LEUKEMIA

M0 - Acute Undifferentiated leukemia MPO and SBB negative

M1 – AML without maturation May demonstrate auer rods

M2 – AML with maturation Most common subtype

M3 – ______________________ Assocated with DIC and faggot cells

Deadliest subtype

M4 – Acute Myelomonocytic Leukemia Aka Naegeli monocytic leukemia

2nd most common subtype

M5 – ______________________ Aka Schilling Leukemia

M6 – ______________________ Aka DiGuglielmo’s syndrome

M7 – Acute Megakaryocytic Leukemia Factor VIII stain (+)

ACUTE LYMPHOPROLIFERATIVE LEUKEMIA

Small cell, homogenous, childhood

Large cell, heterogenous, adults

Burkitt type

(-) PAS, always (+) ORO

PART II

HEMOSTASIS Greek meaning:

• “stoppage of blood flow”

• Complex interaction between blood vessels, platelets, & biochemical factors

in plasma.

• Keeps circulating blood in fluid state

• __________ – stops bleeding

• __________ – dissolves clot

HEMOSTATIC COMPONENTS

• Extravascular component

o Tissue surrounds blood vessels

• Vascular component

o Blood vessel

• Intravascular components

o Platelets

o Plasma proteins

EXTRAVASCULAR COMPONENT

• Provides back pressure on injured vessel

o Swelling

o Trapping of escaped blood

VASCULAR COMPONENTS

• Blood vessels role in hemostasis depends on:

o Size

o Amount of smooth muscle within walls

o Integrity of endothelial cell lining

INTRAVASCULAR COMPONENTS

• Key components:

o Platelets

o Biochemicals (procoagulants)


DO NOT DISTRIBUTE

• Involved in:

o Coagulation

o Fibrinolysis

CONCEPTS OF NORMAL HEMOSTASIS

• Hypocoagulation

o Abnormal bleeding

o Defective coagulation mechanism

o Inherited

▪ ____________ (FVIII deficiency)

o Acquired:

▪ DIC

▪ Liver & Kidney Disease

• Hypercoagulation

o Inappropriate formation of thrombi

o Thrombi consists of

▪ WBCs

▪ Platelets

▪ RBCs

▪ Fibrin

o Caused by defect or lack of activation of fibrinolytic system

o Most are acquired

▪ Malignancy

▪ Surgical procedure

__________________ • Respond to vascular injury or desquamation

• Rapid, short-lived response to damage

• Involves

o Blood vessels

o Platelets

ANTICOAGULANT PROPERTIES OF INTACT ENDOTHELIUM

• Rhomboid; contiguous – prevents harmful turbulence

• Secretes:

o ____________ – eicosanoid platelet inhibitor

o __________ – “relaxing” factor

▪ Promotes vasodilation

▪ Inhibits platelet activation

▪ Promotes angiogenesis and healthy arterioles

o __________ - anticoagulant glycosaminoglycan

o ____________________ – regulates extrinsic pathway

o _________ – coagulation inhibitor

o ________________ – Protein C activator

o ________________ – activates fibrinolysis

• Capillaries

o Metabolic exchange of blood and tissue takes place.

o Junctions

▪ Openings; allow passage of WBCs, O2, nutrients & wastes, in and

out of the blood

o _________

▪ Cells lie beneath endothelium

▪ May differentiate into vessel-related cells when needed.

• Arteries & Veins

o Three layers

▪ Tunica Intima (inner)

▪ Tunica media

▪ Tunica adventitia

o ________________

▪ Comes contact with blood cells

▪ Separates from subendothelium

• Basement membrane

o Elastin-rich

• Elastic connective tissue

• Collagen fibers

▪ Endothelial cells deposit von Willebrand Factor (bound to

collagen in subendothelium)

• Stored in Weibel-Palade bodies

o TUNICA MEDIA

▪ Has smooth muscles & connective tissues

• Collagen fibers

• Fibroblasts

o produces collagen


DO NOT DISTRIBUTE

o ____________________

▪ Consists of:

• Connective-tissue fibroblast

• Collagen fibers

▪ DAMAGED VESSELS

o Harmful stimulus – induces vasoconstriction

o ECs secrete vWF from its storage site _____________________

▪ Also secrete adhesion molecules (promotes platelets & WBC

binding)

• P-selectin

• ICAMs (Intercellular Adhesion Molecules) -

immunoglobulin-like

• PECAMs (Platelet Endothelial Cell Adhesion Molecules) –

promotes plt. & WBC binding

o Collagen exposure

▪ Binds and activates platelets

▪ Activates intrinsic pathway

o Tissue Thromboplastin (FIII) release

▪ Activates extrinsic pathway

o Release of Tissue Plasminogen Activators (tPAs)

▪ Initiates fibrinolysis

▪ PLATELETS’ ROLE IN HEMOSTASIS

o Adhesion to injured vessels

o Aggregation at injury site

o Promote coagulation on their phospholipid surface

o Induce clot retraction

PLATELET ULTRASTRUCTURE

o ________________________

▪ Consists of platelet’s outer membrane and related structures

▪ Bilayer composed of lipid & protein

▪ Predominant lipid – ____________

• Phosphatidylcholine

• Sphingomyelin

• Phosphatidylinositol

• Phosphatidylserine

• Phosphatidylenolamine

Phosphatidylcholine & Sphingomyelin

• Predominates in plasma layer

___________________

• Supports plt. Activation

• Supplies arachidonic acid that is converted into Prostaglandin &

Thromboxane A2

___________________

• Flips to outer surface upon activation

• charged phospholipid surface on which coenzymes especially coagulation

factors (VII and IX) & (X and V) assemble.

▪ Glycocalyx

• Surface coat

o (20 – 30 nm – rodaks)

o (10 – 50 nm – steinininger)

• Absorbs albumin, fibrinogen, and other plasma proteins

using ENDOMITOSIS

• Consist of glycoprotein

o Ia

o Ib

o Ic

o IIa

o IIb

o III

o IV

o V

o ______________________

▪ Links the membrane and the inner cell body

▪ Underlies plasma membrane

▪ Specific area

• Organelles in inner matrix of unaltered platelet never

come in contact w/ internal side of platelet cell wall


DO NOT DISTRIBUTE

▪ Contains organized system of filaments

▪ contributes to regulate the normal discoid shape

▪ act as base of pseudopod

▪ modulates platelet adhesion and clot retraction

o ___________________

▪ Contributes the matrix or muscle and skeletal portion of the

platelet

▪ For contractile process

▪ Underlies submembrane filaments

▪ Consists of:

• Circumferential microtubule system

• Microfilaments

o Provides force after activation that directs the

organelles towards center of cell w/ control and

direction from microtubules

▪ Stable gel

• regulates arrangement of internal organelles

▪ influences communication of organelles w/ plt. External

surroundings

▪ MICROTUBULAR SYSTEM

• Cytoskeletal support system

• Contractile response of plts. to stimulation.

o ____________________

▪ Major portion of plt’s cytoplasm

▪ DENSE-GRANULES

• 250 – 350 nm

• 2-7 granules

• release contents directly into plasma upon platelet

activation

• supports:

o P-selectin

o Alpha IIb Beta3

o Gp Ib/X/V

• Components

o Calcium

o Adenosine

o Guanosine

o Diphosphates

o Triphosphates

o Serotonin

o Magnesium

o ATP

o ADP

Calcium & Magnesium

• Activation of phospholipase

• Plt. Activation and coagulation

________

• Most important in dense granules

• Binds specific receptors and initiates plt. Aggregation

• Rapidly degraded to adenosine

o Inhibits plt. function by enhancing cAMP levels

▪ ALPHA GRANULES

• 300 – 500 nm

• Contents:

o Coagulation Factors

o Permeability factor

o Cationic proteins

o PDGF

o Plt. Factor 4

o B-thromboglobulin

o Fibrinogen

o Factor V

o Fibronectin

o Thrombospondin

o Protein C inhibitor

o PAI-1

_________

• Most important cationic protein released from platelet (mitogenic agent)

• Stimulates smooth cell growth and proliferation

• Not specific for plts. (also in endothelial and other blood cells)

Platelet factor 4

• Binds to heparin (neutralize)


DO NOT DISTRIBUTE

Permeability Factor

• Influences vessel wall permeability and tone

Factor V

• Likely not synthesized by platelet

o PLASMA MEMBRANE SYSTEM

▪ SURFACE-CONNECTING CANNALICULAR SYSTEM

• Delivery route for substances ingested by platelet

• Route of extrusion of substance released from stimulated

plts.

• Readily visible in

o Platelet shape change

o Contraction

o Adhesion

o Aggregation

• Allows enhanced interaction of the platelet w/ its

environment

o Increase access to platelets’ interior as well as

increase egress of plt. release production.

▪ ________________________

• CONTROL CENTER FOR PLATELET ACTIVATION

• Condensed remnants of rough ER

• Randomly dispersed in the platelet cytosol

• Close to circumferential band of microtubules

• Has important rol in influencing microtubules supporting

discoid platelet shape

• Contains peroxidase activity

o For prostaglandin synthetase activity

• Sequesters Ca2+

• Bears series of enzymes that support platelet activation

ENZYMES IN DENSE TUBULAR SYSTEM

• Phospholipase A2

• Cyclooxygenase

• Thromboxane A2 (TxA2)

• Phospholipase C - Supports production of inositol triphosphate (I3)

PLATELET FUNCTIONS

• ADHESION

o Adhere to:

▪ Detached endothelial cells

▪ Collagen fibril

▪ Fibronectin

▪ Basement membrane

o NOTE: Platelets don’t release constituents nor promote aggregation

____________________________

• Endothelial cell-derived chemorepellent

• Inhibits plt. adhesion

• Underlies the endothelium

_____________________

• Promotes plt. adhesion

• Facilitates aggregation and release

______________________

• Promotes adhesion

• Don’t causes plt. aggregation under specific conditions

___________________

• Link between specific platelet glycoprotein Ib and subendothelial cell

connective tissue (reversible binding process – decelerates platelets)

• Released by damaged endothelial cells

NOTE: COLLAGEN TYPE I binds to GP VI

• Triggers plt. activation pathways: releases

o TxA2

o ADP

• AGGREGATION

o Key part of 10 hemostasis

o Induced by:

▪ ADP

▪ TxA2

▪ Thrombin


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▪ Collagen

▪ Epinephrine

▪ Endotoxin

o ADP, TxA2, Thrombin

▪ Independent mediators

ADP-induced Aggregation

• Dependent to GpIIB/IIIa

• Causes conformational change to IIb-IIa complex (inactive IIb/IIIa to active

IIb/IIIa)

o Allows binding of fibrinogen complex

Thrombin (produced because of Tissue Factor)

• Reduced plt. aggregation in three mechanism

o Stimulates plt. to release ADP

▪ Mediates ADP-induced aggregation

o Activates plt. membrane phospholipase

▪ Initiates formation of TxA2

o Can induce platelet aggregation independently; poorly understood

________________________

• Arachidonic acid metabolite

• Induces plt. aggregation DIRECTLY

• May also act synergistically w/ ADP

• POTENT VASOCONSTRICTOR

• ASSISTS HEMOSTASIS AND COAGULATION INDIRECTLY

__________________ • Delayed, long-term response

COAGULATION FACTORS

• Enzyme precursors (zymogens)

• Nonenzymatic cofactors

• Calcium

• Phospholipid (from platelet)

•

Serine Protease – active enzymes

• Have exposed serine-rich, active enzyme sites.

• Hydrolyze arginine- or lysine-containing peptide of zymogens

_____________ – no biological activity until converted to active enzymes (serine

proteases).

Cofactors:

• II

• VII

• IX

• X

• XI

• XII

• Prekallikrein

_______________ – assist in activation of zymogens by:

• Altering zymogen conformation

o To permit more efficient cleavage by serine protease

• Binding the zymogen and appropriate serine protease on plt. phospholipid

o To enhance & accelerate zymogen activation process.

Cofactors:

• V

• VIII

• III

• HMWK

Zymogen activation (conversion into serine protease) is either

• Conformational change (twist, turn, bend) in zymogen molecule

• Hydrolytic cleavage of specific peptide bond of zymogen.

Zymogens of X and II requires Va & VIIIa

• Va & VIIIa is activated by small amounts of thrombin.

COAGULATION FACTORS • Referred in roman numerals

o Except for kallikrein & HMWK

o Assigned by International Committee of Nomenclature of Blood

Coagulation Factors


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• In the order of its discovery.

• “a” denotes for “activated serine protease”

• “f” denotes for “fragmented factor XII” (XIIf)

• Factor VI – discovered to be activated Factor V

o Nonexistent

COAGULATION FACTORS

Numeral Preferred name Synonyms

I Fibrinogen

II Prothrombin Prethrombin

III Tissue Factor Tissue thromboplastin

IV Calcium

V Proaccelerin Labile factor

Accelerator globulin

VII Proconvertin Stable factor

Serum Prothrombin

Conversion Accelerator

(SPCA)

Autoprothrombin

VIII:C Antihemophilic Factor (AHF) Antihemophilic globulin

Antihemophilic factor A

Platelet Cofactor 1

IX Plasma Thromboplastin Component Christmas Factor

Antihemophilic Factor B

Platelet Cofactor 2

X Stuart-Prower Factor Stuart Factor

Prower Factor

Autoprothrombin III

XI Plasma thromboplastin antecedent Antihemophilic factor C

XII Hageman factor Glass factor

Contact Factor

XIII Fibrin-Stabilizing factor Laki-Lorand factor

Fibrinase

Plasma transglutaminase

Fibrinoligase

- Prekallikrein Fletcher factor

- HMWK Fitzgerald factor

Contact activation factor

Williams factor

Flaujeac factor

____________________

• Present in adsorbed plasma (slightly

reduced level)

o Partially adsorbed by BaSO4

COAGULATION GROUPS

• Contact group

• Prothrombin group (Vit. K-dependent group)

• Fibrinogen group

_______________ (Prekallikrein, HMWK, 11, 12)

• Adsorbed by contact with negatively charged surface

o Collagen

o Subendothelium (in-vivo)

• Causes slow conversion of XII → XIIa

o Initiates

▪ Intrinsic pathway

▪ Fibrinolysis

Prekallikrein & HMWK play role in

• Intrinsic pathway activation

• Activation of fibrinolysis

• Kinin formation

• Complement system activation

_____________________ (9, 10, 7, 2, Protein C & S)

• Vitamin K – dependent Group

• Adsorbed by BaSO4 and other salts

• Factors are synthesized in the liver in the presence of Vitamin K

o Vit. K – quinone; fat-soluble

▪ Ingested in diet (green leafy vegetables)

▪ Manufactured by gut flora (Bacteroides fragilis & Escherichia

coli)

▪ No substantial storage in the body

• ___________ is necessary to gamma-carboxylate the preformed enzyme

precursors of IX,X,VII,II.

Phosphatidylserine of platelet was once

called ________________


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o Gamma-carboxylation of glutamic acid residue at N-terminal or amino-

end of factor polypeptide chain.

o Calcium binds to factor forming calcium bridge with acidic

phospholipid surface of activated platelets.

o Ca2+ and PL (phospholipid) – essential for enzyme and substrate function

in coagulation pathways

• FACTORS THAT INHIBITS GAMMA-CARBOXYLATION REACTION

o Dietary Vitamin K deficiency

o Administration of antibiotics that sterilize intestinal tract

o Oral anticoagulant therapy (coumarin/warfarin)

▪ Interferes gamma-carboxylation

• Des-gamma-carboxyl Proteins or proteins in Vit. K antagonism (PIVKAs)

o procoagulants released from liver without second carboxyl group

added to gamma carbon.

________________ (1, 5, 8, 13)

• Have highest molecular weights of all factors

• Most labile

• Consumed in coagulation

• Acts as subtrates for plasmin

• Only factors that are found in platelets’ alpha granules, except:

o XIII – found in cytoplasm

o VIII:C - NOT FOUND IN PLATELETS

Factor VIII (VIII/vWF) – large multimeric molecule

• Has two principal parts:

o Coagulant portion (VIII:C)

▪ Cofactor in intrinsic pathway

▪ Antihemophilic Factor

• Defective in Hemophilia A

o ____________________________

▪ important to normal platelet function

• High Molecular Weight Portion of Factor VIII

o Synthesized in

▪ endothelial cells

▪ megakaryocyte

o has two parts:

▪ antigenic portion (VIII:Ag)

▪ von Willebrand Factor (VIII:vWF)

• results to von Willebrand’s Disease if either both are

decreased

NOMENCLATURE FOR FACTOR VIII (International Committee on Thrombosis and

Hemostasis)

• _________ – entire molecule as it circulates in plasma.

o Composed of:

▪ VIII:C

▪ VIII:vWF

• _________ – for binding to endothelium

o Supports normal platelet adhesion & function

o Tested by bleeding time

• _________ – cofactor to IXa (with Ca2+) in conversion of X → Xa.

o Tested with partial thromboplastin time

• _________ – antigenic property of procoagulant portion

o Measured by immunologic monoclonal antibody

• _________ – F VIII-related antigen, which is a property of large vWF portion of

molecule

o Measured by:

▪ Immunologic techniques of Laurell rocket or

▪ Immunoradiometric assay

• _________ – ristocetin cofactor activity

o Required for aggregation of human platelets with ristocetin.

_________ (a disintegrin and metalloproteinase with a thrombospondin type 1 motif,

member 13)

▪ vWF-cleaving protease

▪ Defective ADAMTS-13 is assoc. w/

o Presence of ultralarge vWF

o Platelet aggregation

o Microvascular thrombosis


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Properties of Coagulation Group

Contact Prothrombin Fibrinogen

Factors XI, XII, Prekallikrein,

HMWK

II, VII, IX, X, Protein

C and Protein S

I, V, VIII, XIII

Function Cofactor: HMWK Cofactor: Protein S Cofactor: V, VIII

Transamidase:

XIII

Stability Fairly stable Heat labile: _______

Well preserved in

___________

Heat labile:

___________

Storage labile:

________

Vitamin K-

dependent for

synthesis?

No Yes No

Adsorbed by

BaSO4 and

other salts

Partially Yes No

Consumed in

coagulation?

Partially No (except_____) Yes

Site Plasma/serum Plasma/serum

(except II – not

present in serum)

Plasma

Destroyed by

plasmin or high

concentrations

of thrombin?

No No Yes

Found in

platelets?

No No Alpha granules:

____________

General

cytoplasm: _____

Not present: ____

Acute-phase

reactant?

No No Yes

Production

reduced by

anticoagulants?

No Yes No

PHOSPHOLIPID CONTRIBUTING TO COAGULATION

Tissue Thromboplastin

• Lipoprotein

o Complex of two parts:

▪ Phospholipid (PL)

▪ Protein

• Initiates extrinsic pathway

o Binding its PL to VII

▪ Converting VII → VII

• In Prothrombin Time:

o Reagent used

▪ Rabbit brain or

▪ Lung tissue Thromboplastin

▪ Ca2+

Partial Thromboplastin & Platelet Phospholipid

• Reagent used as a platelet substitute in evaluating intrinsic pathway

• “partial thromboplastin”

o Partial – reagent consist only of PL portion of tissue thromboplastin

o Platelet substitute

• Intrinsic pathway requires PL for FX activation in-vitro.

o Test requires PPP

PHYSIOLOGIC VARIATIONS OF COAGULATION FACTORS

_____________

o Normally deficient in Vitamin K

o Moderate deficiency exists in Vitamin K-dependent factors

ADULTS

NOTE: THROMBOPLASTIC

Thrombo – pertains to clotting Plastic – Greek word: “plassein” – to mold


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Conditions associated w/ physical variations in Coagulation and Fibrinolytic

System

Condition Related Factors

increases

Related Factor

decreases

Stress I

Tissue necrosis I

Inflammation I

Pregnancy I, VIII, IX, X XIII, XI, AT-III

Oral contraceptives I, VIII, VII, IX, X

Hypermetabolism

(Hyperthyroidism)

I, VIII, Plasminogen

Vigorous exercise VIII, XI, XII

Chronic Thrombocytopenia VIII

Hypothyroidism IX, XI, Plasminogen

Childbirth I, VIII

Surgical Procedure I, VIII

Trauma I, VIII

Myocardial Infarction I, VIII

Acute Illness I, VIII

o Don’t cause coagulation abnormalities

INTRINSIC PATHWAY

• Initiation occurs by exposure to negatively charge surface (such as glass;

damaged endothelium)

• Initiation begins with contact phase of coagulation. Involved are contact

group:

o XII

o XI

o HMWK

o Prekallikrein

• Coagulation factors in intrinsic:

o XII

o XI

o IX

o VIII

FACTOR XII ACTIVATION

o Begins with XII absorption to the negatively charged surface of vascular

collagen

o Prekallikrein and HMWK and XI are absorbed also in the collagen

XII -----------------------------------------------> XIIa

(Kallikrein accelerates the conversion rate)

o Enzymes of basophils and endothelial cells can activate FXII.

o _________ enhances proteolytic effect of kallikrein on XIIa

o _________ can activate prekallikrein to kallikrein.

ROLES OF FACTOR XIIa (CONTACT ACTIVATION)

o Initiates intrinsic pathway of coagulation

o Initiates fibrinolysis

o Forms complex with kallikrein to convert plasminogen → plasmin

o Initiates kinin and complement system

o Formation of kallikrein by XIIf and HMWK, causes HMWK to be

converted into kinins (such as bradykinin).

o Plasmin resulted from kallikrein can initiate complement system

• IMPORTANT ROLES OF KALLIKREIN

o Perpetuates FXII activation and its own production

o Initiates kinin system

o Initiates fibrinolytic and complement system with XIIa

o Activates IX directly

o Can activate IX → IXa on its own.

• IMPORTANT ROLE OF PLASMIN

o Promotes clot dissolution

o Activates complement system

o cleaves XIIa to XIIf (also kallikrein)

FACTOR XI ACTIVATION

XI -----------------------------------------------> XIa

• ____ can also activate plasminogen.

contact group

Kallikrein \

HMWK

XIIa


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FACTOR IX ACTIVATION

IX ---------------------------------------------------> IXa

• Kallikrein can also activate Factor IX

EXTRINSIC PATHWAY

• Factors other than those in plasma are required for the initiation.

• Coagulation factors in extrinsic:

o Tissue Factor (FIII)

o VII

o Ca2+

• Gamma-carboxyglutamic acid residue of FVII binds to PL portion of FIII in the

presence of Ca2+ (act as a bridge between FVII and FIII)

o VII → VIIa

COMMON PATHWAY

• ___________ – beginning of common pathway

X -------------------------------------------> Xa

II -------------------------------------------> IIa

V -------------------------------------> Va

Fibrinogen -----------------------> Fibrin

Coagulation Complexes

Complex Components Activates

Extrinsic Tenase VIIa, Tissue Factor,

Phospholipid, Ca2+

IX and X

Intrinsic Tenase IXa, VIIIa, Phospholipid,

Ca2+

X

Prothrombinase Xa, Va, Phospholipid,

Ca2+

Prothrombin

THROMBIN FEEDBACK MECHANISM

• _________– generated through prothrombinase complex

o Autocatalytic – enhances rate of prothrombinase production

▪ Self-perpetuating

o Enhances FV and FVII in small amounts

o Stimulates platelet aggregation

XIII ------------------------------------> XIIIa

THROMBIN AS COAGULATION INHIBITOR

• Destroys FV and FVIII in high concentration

• Activates _______ – potent anticoagulant

o Enhanced by

▪ Ca2+

▪ Thrombomodulin (cofactor) in endothelial surface

▪ Protein S (cofactor)

o Protein C-S complex

▪ Inhibits Va and VIIIa

▪ Stimulates fibrinolysis

• Increases plasminogen activator activity

o Protein C

▪ Inhibits Tissue Plasminogen Activator Inhibitor (TPAI)

FINAL CLOT FORMATION & STABILIZATION

• action of thrombin to fibrinogen – final steps of coagulation

• _____________

o Ultimate substrate of coagulation pathway

o glycoprotein composed of three nonidentical but intricately

interwoven paired chains:

▪ A alpha

▪ B beta

▪ Gamma

Cofactor: Ca2+

XIa

VIIa-Tissue Factor-Ca2+ complex

Prothrombinase complex

IIa

IIa

IIa


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• Chains are linked by _____________

• Alpha and beta chains have fibrinopeptides

o Fibrinopeptide A

o Fibrinopeptide B

▪ Total of four fibrinopeptides (two A and B)

• CONVERSION OF FIBRINOGEN TO FIBRIN

1. There is cleavage of four fibrinopeptides from alpha & beta ends by

thrombin.

a. Once fibrinopeptides A and B are removed – it becomes soluble

fibrin monomer (unstable clot)

2. _________ of fibrin monomers interact within the ___________of other fibrin

monomers forming fibrin polymers (unstable)

a. Note: FIBRIN POLYMERS ARE SOLUBLE TO ______ OR

_________________________

3. Factor XIIIa (transglutaminase) crosslinks adjacent fibrin monomers

through formation of covalent bond in alpha and gamma chains forming

stable clot

a. Stabilized fibrin clot is insoluble to 5M urea & weak acids.

___________________________

• Final event of hemostasis

• Body’s defense against occlusion of blood vessels.

ACTIVATION OF PLASMINOGEN TO PLASMIN

• _______________ – synthesized in the liver

o Possesses five glycosylated loops (kringles)

o Stored and transported in eosinophil

o Increased in inflammation

o Part of any clot (fibrin tends to absorb plasminogen)

o Plasmin is also formed within the clot

PLASMINOGEN ------------------------------------> PLASMIN

PLASMINOGEN ACTIVATORS

• Intrinsic Plasminogen Activators

o XIIa

o Kallikrein

o HMWK

• Extrinsic Plasminogen Activators

o Tissue Plasminogen Activators

▪ Secreted by ECs

• Plasminogen Activators in Secretory Ducts

o Present in body fluids

▪ Urine (Urokinase)

▪ Tears

▪ Saliva

▪ Semen

▪ Milk

• Exogenous Plasminogen Activators

o For therapeutic destruction of thrombi

▪ Urokinase Plasminogen Activator

• Secreted by

o UT epithelial cells

o Monocytes

o Macrophages

▪ Streptokinase

▪ Tissue plasminogen activators (tPA)

MULTIPLE ROLES OF PLASMIN

Plasmin’s role Comments

Activates fibrinolysis Cleaves fibrin & fibrinogen into

fibrinogen degradation products

Activates intrinsic coagulation XII → XIIa is amplified indirectly by

plasmin

Interferes intrinsic & common pathway Destroys _____ and _____

Blocks thrombin conversion of

fibrinogen to fibrin

Fibrinogen degradation products

interfere with thrombin influence on

fibrinogen

Activates kinin system Enhances conversion of prekallikrein →

kallikrein and kininogen → kinin

Activates complement system Cleaves C3 → C3a and C3b; activates

C1 (first component of complement

system

Plasminogen activator


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• Free plasmins are destroyed by the ___________ found in the circulation.

• Plasmin can’t distinguish between fibrinogen and fibrin.

Plasmin can destroy:

• Fibrin – covalent bonds slows the degradation.

• Fibrinogen – quickly destroyed because of instability

• FVIII

• V

• Other factors

FIBRIN(OGEN) DEGRADATION

• Products:

o Fibrin(ogen) Degradation Products (FDP) or Fibrin(ogen) Split Products

(FSP)

▪ Products are removed by reticuloendothelial system and other

organs.

• Principal Products:

o Early Degradation Products

▪ X (D-E-D

▪ Y (D-E)

o Late Degradation Products

▪ D-D dimer

▪ E

Fragment ___________

• First and last fragment formed

• Result of plasmin cleavage of terminal portion of alpha chains from fibrin

polymer

o Cleaved to form

▪ YY fragment (Y is D-E)

▪ DXD fragments – intermediate complex

DXD complex is cleaved into

• DED

• DY/YD Fragments

NOTE: Presence of D-D dimer – specific indicator of in-vivo fibrinolysis

• Indicative only for Fibrin Degradation products

• Most FDPs inhibits coagulation

• All four fragments have affinity to coating platelet membrane

o Cause a clinically significant platelet dysfunction (aggregation

inhibition)

NATURAL COAGULATION INHIBITORS

______________________

• Alpha2 globulin

• Synthesized in the liver

• Half-life: 2.7 days

• Action:

o Inhibits

▪ XIIa

▪ XIa

▪ Xa

▪ IXa

o Inhibiting by forming enzyme inhibitor complexes with activated

factors

▪ Neutralizing them to prevent their action on other zymogens.

• Also has an inhibitory effect in plasmin and kallikrein

• Action is enhanced by cofactor __________

o Once bound to the lysine site, it causes conformational change to the

arginine residue of AT-III reactive site

▪ The site will be more accessible to the active site of serine

proteases.

• Decreased its therapeutic use by heparin

o Because heparin accelerates binding of AT-III to serine protease

__________________

• Naturally occurring relative to heparin

• Glycosaminoglycan (mucopolysaccharide) anticoagulant

• Identified in

o Surface of platelets

o Vascular endothelium

• Together with AT-III, it protects uninjured vessels from abnormal clotting by

neutralizing proteases.

o Without heparin – slow

o With heparan sulfate – neutralization is accelerated from 2000 –

10,000x faster


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______________________________

• Large

• Binds to thrombin (doesn’t completely inhibit)

• Rate of inhibition is slower than AT-III

• Protects its bound enzyme from other circulating inhibitors

o Thrombin bound to it may still activate small amounts of FV and FVII.

• Also inhibits fibrinolysis

o Inhibits (but not totally eliminates) plasmin’s function to fibrinolysis.

• Inhibits kinin system

o Inhibiting kallikrein

_____________________________

• Potent inhibitor of FXIa

• Also inactivates thrombin (slow rate)

• Inactivates fibrinolytic system by

o Inactivation of plasmin

▪ Least significant of the three naturally occurring fibrinolytic

system inhibitors

• Important to pulmonary functions

• Deficiency is associated to

o Liver cirrhosis

o Emphysema

_________________________

• Inhibitor of C1 esterase

• Inhibits

o XIIa

o XIIf

o XI

o Plasmin

o Kallikrein

_________________________

• Vitamin k – dependent

• Coagulation inhibitors

• Protein C is activated slowly by THROMBIN

o Enhanced when

▪ Thrombin binds to thrombomodulin (in surface of endothelial

cells)

▪ Forms 1:1 stoichiometric complex with Protein S (cofactor)

• EC Protein C Receptor (EPCR)

o Transmembrane protein in EC membrane that binds Protein C and

activated Protein C (APC)

• ______________ – transmembrane protein

• Thrombin cofactor

• Expressed in vascular ECs

• Activated Protein C-S complex – enhances fibrinolysis

o Destroys Factor Va and VIIIa

o Inactivates plasminogen activator inhibitors.

▪ Enhances formation of plasmin

• __________ – cofactor that binds and stabilizes Activated Protein C (APC)

o 40% - free

o 60% - bound to C4bBP (complement control protein)

• __________ – cofactor to Z-dependent protease inhibitor (ZPI) (coagulation

inhibitor)

• ____ - potent inhibitor of Factor Xa

o Also inhibits FXI

NOTE: When thrombin is bound to thrombomodulin:

• Its specificity is altered

• Once altered: o It will not activate Factor V and VIII or platelets o It will not convert fibrinogen to fibrin.


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• PROTEIN C INHIBITOR

o Inhibits

▪ APC

▪ Thrombin

▪ Xa

▪ XIa

▪ Urokinase

NATURAL FIBRINOLYSIS INHIBITORS

_____________________

• Most important fibrinolysis inhibitor

o First to bind with plasmin in plasma

o When it’s depleted

▪ Alpha-2-Macroglobulin binds to plasmin

▪ ALPHA-1-ANTITRYPSIN is the last to bind plasmin

• Principal inhibitor of fibrinolysis

o Neutralizing free plasmin (1:1 stoichiometric complex with any plasmin)

▪ Prevents binding of plasmin to fibrin

▪ Prevents premature digestion and uncontrolled digestion of

• Fibrin

• Fibrinogen

• V

• VIII

• Inhibits Tissue Plasminogen Activator

o Prevents plasminogen → plasmin

• Inhibits:

o XIIf

o XIa

o IIa

o Xa

o Clot-promoting activity of kallikrein

• Permits a slow and orderly dissolution of clot and adequate time for repair of

damaged tissues

Plasmin-Alpha-2-antiplasmin complex

• Binds to serine active sites

• Inactivates serine protease & prevents enzymatic action on its usual substrate

_________________________

• Synthetic fibrinolytic inhibitor

• Used to treat bleeding disorders (urinary tract bleeding)

o Inhibits plasminogen activation

________________________

• Inhibits coagulation and fibrinolytic system

• effectively inhibits plasmin after Alpha2-antiplasmin depletion.

________________________

• third most important natural fibrinolytic inhibitor

• does not inhibit plasmin until both alpha-2-antiplasmin and alpha-2-antitrypsin

are saturated.

• more important in coagulation inhibition

OTHER FIBRINOLYTIC INHIBITORS

• ____________

o Inhibits plasmin and kallikrein

• ____________

o Also inhibits plasmin

FIBRINOLYTIC INHIBITORS SECRETED FROM ENDOTHELIAL CELLS

• ____________________________________

o TPA control protein; inhibits plasmin generation and fibrinolysis

• ____________________________________

o Synthesized in the liver.

o Activated by thrombin bound to thrombomodulin.

PHYSIOLOGIC COAGULATION CONTROL MECHANISM

• Inhibition process at site of clot formation

o Fibrin itself restricts active coagulants to the interior of fibrin clot

o Platelet and endothelium also restrict coagulation at site of injury

• Hepatic clearance prevents fibrinolytic and coagulation components from

circulating in the venous system

o Liver impairment may cause

▪

▪

A-2-Antiplasmin → A-2-Macroglobulin → A-1-Antitrypsin


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TESTS FOR PRIMARY HEMOSTASIS ☻ Platelet count

✓ Indirect method

o Platelets counted in relation to 1000 RBC in the smear

o Dameshek mtd, Fonio’s mtd, Olef’s mtd and Cramer and

Bannerman

o Significant bleeding occurs at a platelet count of <50 x 103/uL

✓ Direct method

o Most accurate way of platelet count

o Whole blood is diluted using an RBC pipet and counted in the

WBC square of hemocytometer

o Light microscopy methods

▪ Rees-Ecker

▪ Guy and Leake method

o Phase contrast microscopy methods

▪ Brecker-Cronkite – uses 1% ammonium oxalate; GOLD

STANDARD

o Automated

☻ ________________

✓ Reflects both platelet number and platelet functional integrity

✓ Affected by

o Platelet count and function

o Thickness and vascularity of the skin

o Quality of blood vessels

o Medications: aspirin (avoid 7 days before test) and NSAIDs

(avoid 24hrs before test)

o Methods:

▪ Duke Method

▪ Ivy Method

▪ Copley-Lallitch Immersion Method

▪ Aspirin tolerance test

☻ Clot retraction

✓ Influenced by

o number and activity of platelets

o concentration of fibrinogen

o PCV

o Calcium + ATP


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✓ Clot retraction begins within 30 seconds and almost complete by the

end of 4hrs.

☻ Platelet Aggregation

PLATELET AGGREGATION STUDIES

Aggregating agents Normal response Abnormal response

ADP, Collagen and

Epinephrine

VWD and Bernard-Soulier

syndrome

Glanzmann’s

thrombasthenia

Ristocetin Glanzmann’s

thrombasthenia

VWD and Bernard-Soulier

syndrome

Tests for secondary hemostasis ☻ ________________

✓ Screening method for problems associated with clotting and

coagulation mechanism

✓ Prolonged in all coagulation disorders

☻ _________________

✓ For monitoring of extrinsic and common pathways

✓ Used to monitor Coumadin/Warfarin/Coumarin therapy – oral

anticoagulants

☻ _____________________

✓ For monitoring of intrinsic and common pathways

✓ Used to monitor heparin therapy – intravenous anticoagulant

✓ Activators used: Micronized silica, Elagic acid, Cellite and Kaolin

☻ ________________

✓ Reagent: East Indian Viper (Vipera russelli)

✓ Detects deficiencies in the common pathway only

☻ ________________

✓ Detects fibrinogen deficiency

✓ Affected by heparin therapy

☻ _______________

✓ Detects fibrinogen deficiency

✓ Unaffected by heparin therapy

✓ From Bothrops atrox

☻ ______________

✓ Detects Factor XIII deficiency

✓ Presence of Factor XIII – clot should be insoluble for 24hrs at 37°C

☻ Mixing Studies

TESTS FOR FIBRINOLYSIS ☻ _______________________

✓ Clot lysis after 48hrs – NORMAL

✓ Clot lysis before 48hrs – INCREASED FIBRINOLYSIS

☻ _______________________

✓ Euglobulin – protein that precipitates when plasma is diluted with

water and acidified

☻ ___________________

✓ Detects presence of fibrin monomers

✓ Paracoagulation

☻ ____________________

✓ More specific than protamine sulfate but less sensitive

☻ ____________________

✓ Positive if there is secondary fibrinolysis (eg. DIC)

REFERENCES

Henry’s Clinical Diagnosis and Management by Laboratory Methods

Hematology: Principles, Procedures, and Correlations by Cheryl Lotspeich-Steininger

Hematology: Principles and Procedures by Barbara Brown

Hematology: Clinical Principles and Applications by Rodak et.al

Hematology notes by Mr. Rene Jesus Alfredo Dinglasan, RMT

Intensive Review Notes of University of the Immaculate Conception – Medical

Laboratory Science Program

Hematopoiesis and Hemostasis notes of Mr. Elton John O. Bayalas, RMT


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