✓ Bore – __________

Hematology ✓ Blood should fill – _____________

✓ Sealing clay – ___________
PART I ☻ Order of draw
1. Tube for blood glass analysis
2. Slides
INTRODUCTION 3. EDTA microcollection tube
4. Other anticoagulated microcollection tubes
☻ Blood: 5. Serum microcollection tubes
✓ Red color is attributed to ____________
✓ pH: __________ VENIPUNCTURE
✓ Specific gravity: _________ ☻ Collection of deoxygenated blood (dark red color)
☻ Composition ☻ Glucose level is ___________ compared to arterial blood
✓ Liquid portion (plasma – in unclotted blood; serum – in clotted blood) ☻ Most common & best site: ____________________
✓ Solid portion (RBCs, WBCs and platelets) ✓ Median cubital vein
✓ Gaseous portion (oxygen and carbon dioxide) ✓ Cephalic vein
☻ Collection
PROPERTY OF MEDTECH REVIEW NOTES
✓ Most critical step in blood collection – _________________
✓ Basilic vein
☻ Collection
SKIN PUNCTURE ✓ Angle: __________ (bevel up)
✓ Tourniquet: ________________ above the site

☻ Recommended for
DO NOT DISTRIBUTE
☻ Used when there is only small amount of blood needed ✓ Needle size: __________________ (adult) or 23-25 gauge (children)

✓ infants and young children less than 1 yr old depth: ________ COLOR CODED NEEDLE HUBS
✓ adults with poor veins depth: _________ COLOR GAUGE
☻ Preferred sites White 16
✓ Palmar surface (fleshy portion of last phalanx of 3rd or 4th finger of non- Pink 18
dominant hand) Cream 19
✓ Lateral plantar surface (heel portion) of newborn Yellow 20
☻ Best method for blood gas collection in newborn – indwelling umbilical artery Green 21
catheter Black 22
☻ For arterialized capillary blood – warming puncture site using washcloth (40- Blue/ Light blue/ Turquoise 23
42°C) for ________ Purple 24
☻ Length of lancet – ____________ Orange 25
☻ Distance between skin surface and bone/cartilage in middle finger – Brown 26
___________ Gray 27
☻ Capillary tube Blue-Green 28
✓ Length – _____________ ✓ A phlebotomist can puncture a patient not more than _________
 ☻ Order of Draw MESOBLASTIC PHASE
1. Blood culture tubes (Yellow) ☻ Cells from mesoderm migrates to YOLK SAC
2. Coagulation tubes (Light blue) ☻ Hematopoiesis begins at 19TH DAY OF EMBRYONIC DEVELOPMENT (occurs
✓ 3.2% sodium citrate (9:1 blood:anticoagulant ratio) intravascularly)
✓ Action: binds to calcium to form soluble complexes ☻ PRIMITIVE ERYTHROBLAST produces:
✓ 3.8% sodium citrate (4:1 – used for ESR) ✓ Gower -1 (______________)
✓ Gower – 2 (______________)
3. Serum tubes (without clot activator or with gel separator)
✓ Portland (_______________)
✓ Thixotropic (a change in viscosity when centrifuged)
☻ Cells migrate to aorta-gonad-mesonephrons (AGM) give rise to
4. Heparin tubes (Green) HEMATOPOIETIC STEM CELLS
✓ Action: inhibits thrombin
✓ For OFT and blood gas analysis HEPATIC PHASE
✓ Causes bluish coloration of background on blood smears ☻ Begins at _______________
stained with Romanowsky attributed to pH ☻
✓ Lithium heparin: most widely used ☻ Hematopoiesis occurs EXTRAVASCULARLY
5. EDTA (Lavender) ✓ _______ – major site of hematopoiesis in 2ND TRIMESTER
✓ Most commonly used collection tube in hematology ☻ Spleen, thymus, and lymph nodes – active in blood cell production
✓ Action: chelation of calcium ☻ __________ IS THE FIRST FULLY DEVELOPED ORGAN IN FETUS
PROPERTY OF MEDTECH REVIEW NOTES
✓ Optimal anticoagulant conc: 1.5 mg/mL of blood ✓ Major site of T-cell production
☻ KIDNEY and SPLEEN – produce B cells
✓ CBC and retic counts – can be performed up to ____
☻ PRODUCTION OF MEGAKARYOCYTES BEGINS IN THIS PHASE
✓ WBC counts, hematocrit and platelet counts – up to _____
☻ Hemoglobin___– predominant
(refrigerated at 4°C)DO NOT DISTRIBUTE
✓ ESR – 2 hrs (room temp.) or 6 hrs (refrigerated)
☻ Hemoglobin ___– detectable levels

✓ Blood smears – 2 hrs MEDULLARY (MYELOID) PHASE

6. Glycolytic inhibitor tubes (Gray) ☻ Hematopoiesis begins PRIOR TO 5TH MONTH OF FETAL DEVELOPMENT
✓ Antiglycolytic agent: ___________ – preserves glucose upto 72hrs; (Medullary hematopoiesis)
AC used: ______________ ☻ MYELOID ACTIVITY – appears in this phase
✓ Antiglycolytic agent: lithium iodoacetate – preserves glucose ✓ Myeloid:erythroid ratio – ______
for 24hrs; AC used lithium heparin ☻ ___________ – 1ST site of hematopoiesis (after 24 weeks of gestation – rodaks)
(after first 3 weeks postpartum – steininger)
☻ Hgb A1 – gradually increases its level
HEMATOPOIESIS
☻ Continuous and regulated process of blood cell production that includes: ADULT HEMATOPOIETIC TISSUE
✓ Cell renewal BONE MARROW
✓ Proliferation ☻ Located within the cavities of the cortical bone.
✓ Differentiation ☻ COMPONENTS
✓ Maturation o RED MARROW
▪ hematopoietically active marrow
▪ Consists of developing blood cells and progenitors.
 • M - mitosis
o YELLOW MARROW
▪ Hematopoietically inactive marrow Colony-forming Units (CFUs)
▪ Composed primarily of _____________ Abbreviation Cell Line
▪ Capable of reverting back into active marrow in cases of CFU- GEMM Granulocyte, erythrocyte,
increased demand. megakaryocyte, monocyte
☻ _________________ – process of replacing active marrow by adipocytes during
development. (meaning: red marrow is replaced by yellow marrow)
CFU-E Erythrocyte
Other adult hematopoietic tissues CFU – Meg Megakaryocyte
☻ Lymph node CFU- M Monocyte
☻ Spleen CFU – GM Granulocyte, monocyte
☻ Liver
☻ Thymus CFU- BASO Myeloid to basophil
CFU- EO Myeloid to eosinophil
CFU- G Myeloid to neutrophil
STEM CELL THEORY CFU – pre-T T lymphocyte
CFU- pre-B B lymphocyte
PROPERTY OF MEDTECH REVIEW NOTES
☻ __________________ - all blood cells are derived from a single progenitor stem
cell called Pluripotent Hematopoietic Stem Cell. CD 38; HL-DR – loss of “stemness”
o Most widely accepted theory among experimental hematologists CD33; CD 38 – common myeloid progenitors
today. CD 10; CD 38 – Common lymphoid precursors
DO NOT DISTRIBUTE
☻ __________________ – each blood cell lineages is derived from its own unique CD7 – T- progenitor cells
stem. CD 19 – B lymphoid precursors

HEMATOPOIETIC STEM CELLS and CYTOKINES Colony-stimulating Factors

• Have high specificity
☻ Common Lymphoid Progenitor – differentiates into T, B, and natural killer cells • Active at low concentration
☻ Common Myeloid Progenitor – differentiates into granulocytic, erythrocytic, • ____________ is the major source of CSF
megakaryocytic lineage.
GM-CSF + IL-3 = enhancement of megakaryocyte colony formation
“STOCHASTIC”
• HSC randomly commits to self-renewal or differentiation Humoral agents that affects CFU-S
CELL CYCLE SCHEMATICS Positive effect (increased no., survival, proliferation)
• Phytohemagglutinin
• G0 – resting stage • Cyclophosphamide
• G1 – cell growth (Synthesis of components necessary for cell division) • Concanavalin A
• S – DNA replication Inhibitor
• G2 – premitosis phase • Hydroxyurea
 ☻ Nuclear Chromatin – coarser, clumped, and condensed.
☻ Disappearance of nucleoli
ERYTHROPOIESIS ☻ Color change in cytoplasm
☻ Typically occurs in erythroid island ✓ BLUENESS (basophilia)
o Caused by ACIDIC COMPONENTS (ribosomal RNA)
Nomenclature System o Attracts _______stains (methylene blue)
Normoblastic Rubriblastic Erythroblastic
(United States) (Europe) ✓ PINKNESS (acidophilia)
o Caused by BASIC COMPONENTS (hemoglobin)
Pronormoblast Rubriblast Proerythroblast o Attracts _______ stains (eosin)
Basophilic Normoblast Prorubricyte Basophilic erythroblast
MATURATION SEQUENCE
Polychromatic Rubricyte Polychromatic
normoblast erythroblast N:C NUCLEUS; DIVISION CELLULAR ACTIVITY;
Orthochromic Metarubricyte Orthochromic RATIO CYTOPLASM PRODUCTS INFORMATION
normoblast erythroblast PRONORMOBLAST 8:1 N – purple 2 prorubricyte Accumulation of
Polychromatic Polychromatic Polychromatic red components for Hgb
erythrocyte erythrocyte erythrocyte chromatin synthesis
PROPERTY OF MEDTECH REVIEW NOTES
Erythrocyte BASOPHILIC 6:1 N– 2 rubricyte Detectable Hgb
NORMOBLAST chromatin synthesis
BFU-E (Burst-forming unit- erythroid) begins to
☻ Erythroid progenitor condense
☻ 1 week to mature to CFU-E DO NOT DISTRIBUTE POLYCHROMATIC 4:1 – N – absence 2 Increased Hgb
☻ ____________ from BFU-E to mature RBC NORMOBLAST 1:1 of nucleoli metarubricyte synthesis
C – 1st stage (____________
CFU-E (Colony-forming unit-erythroid) with pink _____________
☻ Erythroid progenitor from BFU-E color (Hgb); _____________)
☻ 1 week to mature to pronormoblast murky-gray
blue
Criteria in Identification of Erythroid Precursors ORTHOCHROMATIC 1:2 C – salmon- Not capable Nucleus is ejected
☻ Nuclear chromatin pattern NORMOBLAST pink; Hgb of division from cell; loss of
☻ Nuclear diameter synthesis ______ - (responsible
☻ N:C ratio nearly for holding
☻ Presence / absence of nuclei complete organelles)
☻ Cytoplasmic color PYRENOCYTE –
extruded nucleus
Trends that affects the appearance of RBCs as it matures PHOSPHATIDYLSERINE
☻ Decreasing overall diameter of the cell – “eat me flag”
☻ Decreasing diameter of nucleus (N:C ratio also decreases)
 NON-MUSCLE ▪ Increased rate of cellular processes
MYOSIN – important ▪ Decreased cell cycle times
in pinching process
POLYCHROMATIC anucleated Incapable Complete Hgb Measurement of EPO
ERYTHROCYTE synthesis • Can be measured by __________________
Resides in BM for 1 • Reference interval – __________ (varies for each lab)
day or more → • Increased in most patients with anemia, EXCEPT FOR PATIENTS WITH ANEMIA
peripheral blood 1 CAUSED BY ______________
day before maturity
ERYTHROCYTE C– 1.5-2.5um diameter Therapeutic use:
biconcave Salmon pink • Used in anemia associated with CKD and chemotherapy
Life span: 80-120 • Also used prior to autologous blood donation and after bone marrow
days transplantation
• Used in athletes to increase O2 -carrying capacity (illegal)
__________________ o Causes fatal arterial and venous thrombosis
☻ Dynamics of RBC production and destruction
Other stimuli to Erythropoiesis
_________________ – collection of all stages of erythrocytes throughout the body. •
PROPERTY OF MEDTECH REVIEW NOTES •
Stimuli of RBC Production •
☻ HYPOXIA
✓ Detected by PERITUBULAR FIBROBLAST OF THE KIDNEY RBC DESTRUCTION
DO NOT DISTRIBUTE
▪ Produces EPO (major stimulatory cytokine for RBC)
▪ Primary O2 – sensing system of the body Senescence
Major Effects of EPO • Cellular aging
• Loss of glycolytic enzymes
☻ Early release of Reticulocyte
✓ Increases the width of spaces for RBC egress into the sinus (in the BM) Macrophage-mediated hemolysis (Extravascular Hemolysis)
✓ Down regulation of expression of receptors for adhesion molecules in • Spleen generates stressful environment for RBCs
the BM stroma Signals recognized by macrophages to differentiate senescent from young cells
✓ ________________ or (stress reticulocyte) o Binding of autologous IgG to band-3 membrane protein clusters
▪ Very basophilic o Exposure of phosphatidylserine on the exterior of the membrane
▪ Shifted from BM early o Inability to maintain cation balance
☻ Inhibition of Apoptosis o Senescent changes to CD47 – binding of thrombospondin-1 – “eat
✓ Increasing number of cells that will be able to mature into circulating me” signal to macrophage
erythrocytes – decreasing apoptosis Mechanical hemolysis (fragmentation or intravascular hemolysis)
☻ Reduced Marrow Transit Time • Due to turbulence in the chambers of the heart or bifurcation of vessels
✓ Increasing the rate at which the surviving precursors can enter the • Small breaks in blood vessels resulting clots can trap and rupture cells
circulation
 ▪ Myeloblast
LEUKOPOIESIS ▪ Promyelocyte
o Overall function – _______________ ▪ Myelocyte
☻ Maturation (Storage) Pool – cells undergo nuclear maturation
Granulocytes ☻ Consists of:
• WBCs whose cytoplasm is filled ✓ Metamyelocyte
• with granules different staining characteristics ✓ Band neutrophils
• nuclei are lobulated and segmented. ✓ Segmented neutrophils

Eosinophil MATURATION STAGES

• Granules – basic proteins
• Stains with acid stain • MYELOBLAST
Basophil o (0-3% -rodaks) (1-2% - steininger) in BM
• Granules – acidic o ___________ in diameter
• Stains with basic stain o Earliest recognizable form
Neutrophil
• React with both acidic and basic stain SUBDIVISIONS
o Gives them pink to lavender color. o Type I
PROPERTY OF MEDTECH REVIEW NOTES ▪ N:C ratio – ______________
Mononuclear cells ▪ Slightly basophilic cytoplasm
o Nuclei – not segmented, but round, oval, indented, or folded ▪ Fine nuclear chromatin
o ▪ 1-4 visible nucleoli
o DO NOT DISTRIBUTE ▪ No visible granules when observed in light microscopy with
Romanowsky stain
GRANULOCYTES o Type II
▪ Presence of dispersed primary (azurophilic) granules in
Neutrophil cytoplasm
☻ Two forms in PB ▪ No. of granules don’t exceed 20 per cell
✓ Segmented o Type III
✓ Band shape ▪ Darker chromatin
☻ Life span – _________ (from myeloblast to death) ▪ More purple cytoplasm
▪ Contain more than 20 granules that don’t obscure the nucleus
Pools of Developing Neutrophil ▪ RARE in BM
☻ ______________– capable of self-renewal and differentiation ▪ CAN BE SEEN IN CERTAIN TYPES OF AML
✓ Consist of HSCs • PROMYELOCYTE (progranulocyte)
☻ Proliferation (mitotic) pool – dividing cells o 1-5% (rodaks) in BM
✓ Consists of: o 16-25 um in diameter
▪ CFU-GEMM o Nucleus – round to oval; eccentric
▪ Granulocyte-macrophage progenitors o Synthesis of primary or (_____________) begins and ends in this stage.
▪ Rich in peroxidase
 o Paranuclear halo or _____ is present • 50 – 70% (rodaks)
▪ Absent in acute promyelocytic leukemia ▪ Absolute value
o 1-3 nucleoli seen but obscured by the granules • 2.0 – 6.93 x109/ L in adults (steininger)
• 2.3 – 8.1 x 109/ L in adults (rodaks)
• NEUTROPHIL MYELOCYTE
o (6-17% - rodaks) (10-20% -steininger) in BM NOTES:
o Final stage in which mitosis occurs • Half of the blood neutrophils are not circulating freely, but adheres to vessel
o Production of _____________ granules begins wall (Marginal Neutrophil Pool)
• Neutrophil half-life in the blood – ____________
o Early Myelocyte • _______(rolling) and _________ (stationary binding)– important in diapedesis
▪ Very similar to promyelocyte • __________ – stimulates release of granulocyte from BM.
▪ Pale pink cytoplasm begins to be evident in the area of golgi • Neutrophils that don’t migrate to tissue – undergo apoptosis
apparatus (dawn of neutrophilia)
o Late Myelocyte NEUTROPHIL FUNCTION
▪ Smaller than promyelocyte (15-18 um) • Major function –__________
▪ Nucleus – more heterochromatin o MOTILITY
▪ Nucleoli difficult to see by light microscope ▪ Wave-like or “ruffles” – moves forward
▪ ___________ – random migration
• METAMYELOCYTE (JUVENILE) PROPERTY OF MEDTECH REVIEW NOTES ▪ Guided by ___________
o (15-30% - steininger) (3-20% - rodaks) in BM • Neutrophils responds faster than monocytes
o Synthesis of ________ granules (gelatinase granules) • _____ – most potent chemotactic factor
o Nucleus – ___________________ shaped o Other chemotactic Factors
DO NOT DISTRIBUTE
o Chromatin – increasingly clumped o Endotoxin – activates FXII
o Contains very little residual of RNA (little or no basophilia) o Metabolic products of arachidonic acid
o Chemotactic agents released by lymphocytes and other
• NEUTROPHIL BAND phagocytes
o 9-32% (rodaks) in BM
o Last stage before the mature cell • RECOGNITION
o Secretory granules (secretory vesicles) are formed in this stage. o Opsonins
o The greatest source of variation and discrepancy in leukocyte ▪ Aids neutrophil on what to ingest
morphology o Examples of opsonins
▪ Antibodies
• SEGMENTED NEUTROPHIL (seg, poly, PMN) ▪ Complement substances
o 7-30% in BM ▪ Fibronectin
o Secretory granules continued to be formed • INGESTION
o 2-5 nuclear lobes connected by threadlike filaments • DEGRANULATION
• KILLING
o NORMAL VALUES: o Actively metabolized O2 to produce toxic substances for
▪ Relative value killing ingested foreign particles
• 37 – 77% (steininger)
 o Principal toxic metabolites • First to be distinguished from neutrophil in PROMYELOCYTE STAGE
▪ Superoxide anion
▪ H2O2 ______________ – HALLMARK OF ALLERGIC
o Myeloperoxidase DISORDER
▪ Potentiates killing action of H2O2 presence of ascorbic Development
acid and halides • Similar to neutrophils
o Lysozymes • Lineage is established through interactions of:
▪ Hydrolyzes mucopeptide cell wall of few species of o IL-3
bacteria. o IL- 5 – critical in growth & survival
o Superoxide dismutase o GM-CSF
▪ Converts superoxide into H2O2 o GATA-1
o Catalase o PU.1
▪ Destroys H2O2 o c/EBP
o Reduced glutathione
▪ Detoxify H2O2 STAGES
▪ Regenerate NADPH
• Second Function • EOSINOPHIL PROMYELOCYTE
o Generation of NETs (Neutrophil extracellular traps) o Identified CYTOCHEMICALLY
PROPERTY OF MEDTECH REVIEW NOTES
▪ Contains enzymes from the granules; shown to trap and kill o Presence of __________________ proteins in primary granules
Gram (+), gram (-) bacteria, and fungi.
▪ NETosis • EOSINOPHIL MYELOCYTE
• Unique form of neutrophil cell death resulting a release of o First to be identified in light microscopy and Romanowsky stain
NETs DO NOT DISTRIBUTE o Cytoplasm - large, pale, reddish-orange secondary granules w/ azure
granules
o Third and Final Function o Nucleus – similar to neutrophil myelocyte
o Secretory function o Secondary granules – contains ___________________ (seen in TEM).
o Source of Transcobalamin I or R binder protein – for proper absorption
of Vit. B12. • EOSINOPHIL METAMYELOCYTE & BANDS
o Resembles neutrophil counterpart
Tests for Neutrophil Function o Increased secondary granules
• Boyden Micropore Filter Technique o Generation of SECRETORY GRANULES/ SECRETORY VESICLES
o Assays neutrophil migration in response to chemotactic factor. o Has 2 organelles
• Rebuck Skin Window Procedure ▪ Lipid bodies
o Evaluates speed, type, and number of phagocytes that responds to ▪ Small granules
skin abrasion.
• MATURE EOSINOPHIL
EOSINOPHIL o Bilobed nucleus
• 1-3% in BM o Cytoplasm – has orange-red secondary granules
• 1-3% in peripheral blood o Has extensive secretory vesicle
o Absolute number - 0.4 x 109/L ▪ Increased when stimulated or activated
KINETICS • Increased in parasitic helminth infection
• From last myelocytic division to mature eosinophil – 3.5 days o Destroys helminths through Major Basic Protein and Eosinophil Cationic
• Circulating half-life – ________ Protein
• Transit time – 1-8 hours • Believed to prevent reinfection
• Survival time in tissues - 2-5 days • Plays important role in immune regulation
• Only 1% found in the blood (mostly found in tissues) • Believed to be involved in deletion of double-positive thymocytes
(transmigrate to thymus of newborn)
BIOCHEMISTRY • Regulates mast cell through release of MBP
• ___________ – principal source of energy o Causes mast cell to degranulate
▪ Cytokine production
• OUTER MATRIX OF GRANULES ▪ Nerve Growth Factor
o Hydrolytic enzymes • Promotes mast cell survival and activation
o Peroxidases (different from neutrophils)
• Implicated also in airway remodeling (increased thickness of airways wall)
• INNER MATRIX CORE OF GRANULES o Through EOSINOPHIL-DERIVED GROWTH FACTOR
o Major Basic Proteins
BASOPHIL
• ______________________ PROPERTY OF MEDTECH REVIEW
• Morphologically and NOTES
functionally similar to mast cells
o Hexagonal-bipyramidal crystal • ______________
o from primary granules • Least numerous of the WBCs
o appears when large no. of eosinophils disintegrates in secretions or o (0-2% - rodaks) (0.5-1% -steininger) of circulating WBCs
exudates DO NOT DISTRIBUTE o 0.3% in the BM
o seen in
▪ allergic asthma DEVELOPMENT
▪ pulmonary eosinophilic infiltrates • ______ – influences differentiation
▪ stools in parasitic infection
FUNCTIONS STAGES
• IMMATURE BASOPHIL
• MECHANISMS OF DEGRANULATION o Nuclei
o Classical Exocytosis ▪ Round to lobulated
▪ Granules move and fuse to plasma membrane; empty contents ▪ Slightly condensed chromatin
in EC space o Cytoplasm contains
o Compound Exocytosis ▪ Blue-black 20 granules
▪ Granules fuse first within the cell before fusing to plasma • Water-soluble
membrane • MATURE BASOPHIL
o Piecemeal Degranulation o Nuclei
▪ Secretory vesicle removes specific proteins from secondary ▪ Bilobed or band-shaped
granules; migrates and fuse to plasma membrane; specific ▪ Chromatin – clumped
proteins released in EC space. o Cytoplasm
 ▪ Colorless FUNCTION
▪ Contains large no. of large blue-black granules • Releases wide variety of
o Leaves reddish-purple rim surrounding a vacuole when granules have o Lipid mediators
been dissolved o Proteoglycans
o Proteases
KINETICS o Cytokines
• Life span – 60 hours
o Due to IL-3 that initiates anti-apoptotic pathway. • can function as APC to induce differentiation on TH2 cells
• can have anti-inflammatory and immunosuppressive functions
FUNCTIONS
• Releases ____ and ______ MONONUCLEAR CELLS
o Regulates TH2 cells immune response
• Induces B cells to produce IgE MONOCYTE
• Basophilic activation – not restricted to Ag-specific IgE. Can also be triggered • 2-11% of circulating WBCs
by: o Absolute No. – 1.3 x 109/ L
o Parasitic antigen
o Lectins DEVELOPMENT
o Viral superantigens binds to nonspecific IgE • Derived from granulocyte-monocyte progenitor
PROPERTY OF MEDTECH REVIEW NOTES
• Capable of synthesizing granule protein based on activation signals • ________ – major cytokine responsible for growth and differentiation of
o Produces granzyme B (mediator) monocytes
o Produces retinoic acid STAGES
▪ Regulates immune and resident cells in allergic diseases • MONOBLAST
• Has a role in _____________ DO NOT DISTRIBUTE o Normal in BM
o Expresses Vascular Endothelial Growth Factor (VEGF) o Very rare
• Involved in control of helminth infection control • PROMONOCYTE
o Promotes eosinophilia o Diameter – 12 – 18 um
o Nucleus
MAST CELLS (TISSUE BASOPHIL) ▪ Slightly indented and folded
• Not leukocyte ▪ High N:C ratio
• Tissue effector cells of allergic responses and inflammatory reactions o Cytoplasm
• Precursors circulates on peripheral blood for brief period on their way to tissue ▪ Blue/ gray-blue
• Have several phenotypical and functional similarities with basophil and ▪ Contains azure granules (heterogeneous)
eosinophil o Earliest precursor cell to the monocyte that is recognizable by light
microscopy
• ______________ – major cytokine responsible for mast cell maturation and o Undergoes 2 mitotic division in 60 hours – produces 4 monocytes
differentiation ▪ If increased demand – 4 divisions in 60 hours – 16 monocytes
o In Steininger – 3 divisions taking @ 30-48 hours
 • 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 PROPERTY OF MEDTECH REVIEW NOTES
BIOCHEMISTRY
▪ Intermediate • Monocyte depends on aerobic glycolysis
▪ Nonclassical • Most macrophage depends on anaerobic glycolysis except for alveolar
macrophage (aerobic glycolysis)
KINETICS DO NOT DISTRIBUTE
• No storage pool of mature monocyte in BM GRANULE CONTENTS
• Monocyte in Splenic Reservoir • Monocyte
o Respond to tissue injury to participate in wound injury o Acid phosphatase
• Marginal pool – 3.5 times the circulating pool o Beta- glucuronidase
• Incapable of cell division o Lysozyme
• Stays in blood for 70 hours ▪ Bacteriolytic enzyme
• Once in the tissue, it differentiates into: ▪ May enhance phagocytosis
o Macrophage ▪ Potential antineoplastic effect
o Osteoclast o Lipase
o Dendritic cell o Peroxidase
o Langerhans cells • Macrophage
o No peroxidase
RECEPTORS
• Monocyte and Macrophage
o Has receptors for Fc part of IgG and C3
o Receptor for IgE
FUNCTION o Shown to eliminate viruses and virus-infected cells
• INNATE IMMUNITY o Also releases _____
IL-1
o Recognize pathogens thru _________________
TOLL-LIKE RECEPTORS ▪ Promotes T-cell replication
o Macrophage produces ____________
Nitric oxide o Also releases
▪ Cytotoxic against ▪ Endogenous components (induces fever)
• Viruses ▪ Complement components
• Bacteria
• Fungi • ADAPTIVE IMMUNITY
• Protozoa o Plays role in antigen presentation (APCs)
• Helminths
• Tumor cells • HOUSEKEEPING FUNCTIONS and others
o Efficient in engulfing large particles o Removal of debris and dead cells at the site of infection or tissue
o Influenced by Migration Inhibition Factor damage”
▪ To remain at the site of infection o Production of __________________
Transcobalamin II
▪ 1o transporter of Vit. B12
o CHEMOTACTIC FACTORS THAT ATTRACTS MONOCYTE o Secretion of
▪ Ag-Ab complex ▪ Plasminogen activator
▪ Complement components ▪ Plasmin inhibitor
▪ Kallikrein PROPERTY OF MEDTECH REVIEW NOTES ▪ Platelet activator factor
▪ Factors released by T cells ▪ Tissue-thromboplastin-like procoagulant
o Killing potency of macrophage is enhanced when “activated” o Plays a role in Hgb degradation and iron transport by liberating iron
▪ Refers to enhancement of from heme.
• Motility DO NOT DISTRIBUTE
• Metabolism LYMPHOCYTES
• Enzyme activity “_____________” Suckling or “________”
Suckling Pig / “________” Nursing • 18-42% in the circulation
• Killing capacity Phenomenon o 3- 21% in circulation – _______
B cells
▪ May result from • Transferring iron to transferrin for o 51 – 88% in circulation – T______
cells
• Direct contact to microorganism incorporation into developing RBC o 4 – 29% in circulation – NK
_______
cells
• By-products of microorganism • Developing RBCs surround a large ▪ Absolute number – 0.8 – 4.8
• Lymphokines macrophage x109/L
• Happens in BM • Nucleus
o Known microorganisms that parasitize macrophages o round, ovoid, kidney
▪ Mycobacterium • Cytoplasm – clear blue
▪ Listeria • Chromatin – crushed velvet
▪ Salmonella • Groups
▪ Brucella o T cells – develops in Thymus
______
▪ Fungi o B cells – develops in _____
BM
▪ Protozoa o NK cells – develops in BM
_____________
and Thymus
o Non-T and Non-B cells – don’t develop along either T or B cells
 • Resides on lymph node follicles
DIFFERENCES FROM OTHER WBCs ▪ Absence of CALLA and TdT
• not an end cell ▪ Appearance of
o they are resting cells • sIg (surface immunoglobulins)
o undergoes mitosis to produce • intracytoplasmic chains of IgM
___________
▪ Memory cells • B cell – specific antigen
___________
▪ Effector cells o B461
• recirculates from blood to tissue and back to the blood o S-HCL2
• capable of rearranging antigen receptor genes to segments to produce ▪ Normally seen in newborn peripheral blood
variety of antibodies and surface receptors
• T and NK cells develops and mature outside BM ▪ _________________________________________
FLOW CYTOMETRY IMMUNOPHENOTYPING
• Differentiates leukemic cells from hematogones
DEVELOPMENT o MATURE B CELLS
• Colony forming unit – Lymphocyte ▪ Demonstrates sIg
o Has _______
HLA-DR and ____
TdT ▪ Synthesis of light chains (lambda and kappa) joined with heavy
o Produces functionally diverse cells chains inserted in the plasma membrane
• T and B cells subdivision ▪ Has receptors for the following:
o Antigen-independent
__________________ • Fc portion of the following
PROPERTY OF MEDTECH REVIEW NOTES
▪ Occurs in 1o lymphoid organs o IgG
• 10 lymphoid organs develop during embryogenesis o IgE
• Occurs in fetal liver o IgA
o Antigen-dependent
__________________ • Mouse RBC
DO NOT DISTRIBUTE
▪ Occurs in 2o lymphoid organs • Complement
o C3b
• B CELLS o C3d
o Develops in BM o C4b
o C1q
o STAGES – In these stages, immunoglobulin gene rearrangement occurs o C8
to produce unique Ig antigen receptors o C2 inhibitor
▪ Pro-B • Epstein-Barr Virus
▪ Pre-B
• has CALLA o Resting lymphocytes
• Earliest recognizable B cells ▪ Small – 9 um in diameter
▪ Immature B cells 5:1 – 2:1
▪ N:C ratio – _________
▪ Chromatin – blocks
o IMMATURE B CELLS (Hematogones) ▪ Nucleolus – rarely seen
▪ Chromatin – homogeneous
▪ Cytoplasm – scanty, deep blue, devoid of granules, cleft o EFFECTOR AND MEMORY B CELLS
▪ Antigen-naïve ▪ Produced when there is contact to antigens in 20 lymphoid
▪ Migrates to 20 lymphoid organs organs
 ▪ _______________
Plasma cells – antibody-producing • CALLA (+)
▪ Plasmacytoid lymphocyte – atypical lymphocytes • Ia antigen (+)
• Don’t from rosette in SRBC
o Mitogens for B cell • No sIg
▪ Pokeweed Mitogens ▪ Immature T cells
▪ LPS paracortical area
• Leaves thymus; migrates to ______________
▪ S. aureus Covan I
▪ EBV o REACTIVE / VARIANT LYMPHOCYTES
• Independent to accessory cells ▪ Reflect on the reaction to antigen
• Earliest B cell specific surface marker ▪ Variation of morphology of effector T cell w/ the subtype of T
cells
o PLASMA CELLS o Mitogens for T cells
▪ Antibody-producing cell ▪ Phytohemagglutinin
▪ Nucleus ▪ Concanavalin A
• Eccentric ▪ Pokeweed Mitogen
• Has blocks of heterochromatin (tortoise shell)
▪ Cytoplasm KINETICS
• Abundant non-granules ▪ Average life span in blood – _________
4 years
PROPERTY OF MEDTECH REVIEW NOTES
• Deep blue (due to ribosomes) o Some can reach up to 20 years
• Golgi apparatus ▪ __________
UROPOD – most adherent part of the cell
o Unstained (area of Hof)
• Periphery
DO NOT DISTRIBUTE
o Layered look (flattened parallel sacs of RER) • NK CELLS
• Contains Russell Bodies o Heterogeneous group of cells w/ respect to their surface antigens
o Round o Majorities are
o Discrete globules ▪ CD56+
o Unstained, or pale-blue or red ▪ CD16+
• ___________
MORULA / _________
GRAPE / __________
MOTT CELLS ▪ CD3+
o Russell bodies-filled plasma cell ▪ CD7+
▪ CD11b+
• T CELLS Cluster Designation
o CD – _______________
o Develops in thymus FUNCTIONS
___________ – hormone that plays crucial role in maturation or pre-T to T
o THYMOSIN ▪ Major function – antigen recognition and appropriate immune response
cells
o STAGES – in these stages, it undergoes antigen receptor rearrangement ▪ B CELLS
to produce T cell receptors that are unique to each T cells o For antibody-production (plasma cells)
o NOTE: receptors that react to self-antigen - APOPTOSIS o Has role in antigen presentation to T cells
▪ Pro – T o May be necessary for CD4 optimal activation
▪ Pre – T o Produces cytokines
• Has TdT ▪ Regulates variety ot T cell and Antigen presenting cell function
 o End result • Regulation of hematopoiesis
▪ Memory B cell • Natural resistance to allogeneic graft
▪ Plasma cell ▪ KILLER CELLS
o Lymphokines that regulates growth and maturation of B cells • Antigen-dependent (ADCC)
▪ B cell growth factor I & II o Differentiates it from NK cells
▪ B cell differentiation factor ▪ LAK CELLS (Lymphokine-activated Killer Cells)
▪ B cell stimulatory Factor – 2 • Activated by _____
IL-2
▪ IL – 2 • Don’t express some NK surface antigens
▪ IFN- gamma • Phenotypically different from NK cells
o C3
▪ Triggers activation of early B cells ▪ LYMPHOKINES
▪ Generates memory B cells o Secreted by Activated T and B cells
o _____________________________
Prostaglandin E2 (PGE2) ▪ Colony-Stimulating Factor
▪ Regulatory role in the magnitude of B cell response in-vivo. ____________________________
▪ Macrophage-inhibiting factor (MIF)
• Inhibits monocyte and macrophage from migrating
▪ T CELLS ▪ Lymphocyte migration inhibitory factor (LyMIF35k)
o Types • Immobilizes lymphocyte at the site of infection
▪ CD4+ ▪ Lymphocyte chemoattractant (LCF)
• ______
Th1 PROPERTY OF MEDTECH REVIEW NOTES • Increases migration of non-essential T cells
o Mediates response agains intracellular pathogens ▪ Histamine-producing cell-stimulating factor (HCSF)
_______________________________
• ______
Th2 • Increase histamine produced by basophil and mast cell
o Mediates response against EXTRACEL-LULAR ▪ T cell activating Factor
PARASITE DO NOT DISTRIBUTE • Antigen-independent
o Also important in induction of asthma and allergic • Activates CTLs and NK cells
disease ▪ Human lymphotoxin (HLT)
• __________
Th17 (DHTL) • Inhibits tumor cell growth
o Against extracellular bacteria and fungi • Produced by T cells (primarily)
Treg
• __________ METHODOLOGIES
o Maintains self-tolerance by regulating immune
response HEMATOCRIT
☻ Aka Packed Cell Volume
▪ CD8+ (Cytotoxic T- Lymphocyte) ☻ Layers of anticoagulated whole blood (top to bottom)
• Kills target cells by secreting perforin and granzyme or by a) Plasma
activating apoptotic pathway b) Platelets
▪ ______________
NK CELLS c) White blood cells
• Kills tumor cells and virus-infected cells without MHC d) Red blood cells
restriction
• Modulates function of other cells (macrophage & T cells) MICROHEMATOCRIT METHOD
• Resistance to bacterial, fungal, and parasitic agents ☻ Capillary tube
• Immune regulation
 ✓ Length – __________ ✓ Sources of error:
✓ Bore – ____________ o High WBC count (>20 x 109/L) and high platelet count (>700 x
✓ Blood should fill – _________________ 109/L) – corrected by centrifuging sample and using the
✓ Sealing clay – _____________ supernatant
✓ Types: o Lipemic sample – prepare patient blank
o ______ tip – with 1IU of heparin o Hgb C and and Hgb S – make a 1:2 dilution with distilled water
o ______ tip – no anticoagulant and multiply results from standard curve by 2.
☻ Centrifuge
✓ Calibration
o _________ – accuracy RULE OF THREE
o _________ – speed ☻ Used for normocytic, normochromic specimens
✓ Speed – _________________________ ☻ ____________________________________
☻ Trapped plasma causes 1-3% (0.01 to 0.03 L/L) increase in hematocrit
☻ Reading should be done within __________

MACROHEMATOCRIT METHOD
RED BLOOD CELL INDICES
☻ Helpful for the initial classification of anemia
☻ Wintrobe tube is used PROPERTY OF MEDTECH REVIEW NOTES
☻ Centrifuged at 2,000 to 2,300 g for 30 mins
☻ Mean Cell Volume(MCV) – average volume of RBC
hematocrit (%)
𝑀𝐶𝑉 = x10
Wintrobe tube Westergren method x1012
RBC count ( )
L
Length 115 mm 300 mm ☻ Mean Cell Hemoglobin (MCH) – average weight of hemoglobin in an RBC
Bore
Calibration
DO NOT DISTRIBUTE
3 mm
Left – ESR (0-100)
2.55 mm
0-200 𝑀𝐶𝐻 =
hemoglobin (g/dL)
x1012
x10
RBC count ( )
Right – Macrohematocrit (100-0) L
☻ Mean Cell Hemoglobin Concentration – average concentration of
hemoglobin in each individual erythrocyte
CYANMETHEMOGLOBIN METHOD 𝑀𝐶𝐻𝐶 =
hemoglobin (g/dL)
x100
hematocrit (%)
☻ For hemoglobin determination
☻ Modified Drabkin’s reagent
✓ __________________ – permits oxidation of ferrous to ferric iron
MANUAL CELL COUNT
(hemoglobin → methemoglobin) ☻ Diluting fluids and Pipettes
✓ Potassium cyanide – methemoglobin → cyanmethemoglobin RBC WBC
✓ Dihydrogen potassium phosphate – replaces sodium bicarbonate in Diluting fluids Hayem’s Toisson’s 2-3% glacial acetic acid
Gower’s Bethels’s 1% HCl
original reagent (reduction of time from 15 mins to 3 mins)
NSS 3.8% sodium Turk’s solution
✓ Non-ionic detergent – improves red cell lysis and decreases turbidity
Formol citrate
✓ Read using spectrophotometer at ________
citrate
✓ Sample: EDTA whole blood
✓ Dilution: 1:251
 Characteristics Isotonic Hypotonic – lyses RBCs
except nRBCs ERYTHROCYTE SEDIMENTATION RATE (ESR)
PIPETTE PROPERTIES ☻ Detect inflammatory process
Size of bulb Larger Smaller ☻ Stages:
Color of bead Red White 1. Lag phase (initial rouleaux formation) – ________
Volume of bulb 100 10 2. Decantation phase (period of fast settling) – ___________
Size of bore Smaller Larger
3. Final packing – ______
Calibration 0.5; 1; 101 0.5; 1; 11
☻ Factors:
Dilution 1:200 1:20
ESR INCREASED ESR DECREASED
Dilution Factor 200 20
Increased cholesterol, fibrinogen Increased albumin (due to viscosity)
☻ Hemocytometer
and gamma globulins
✓ 1 ruled area – 9mm2
RBC agglutination Altered RBC shape
✓ 1 WBC square (with 16 smaller squares) - _________
Leukemia Polycythemia
o 1 square inside a WBC square – 0.0625 mm2 Increase temperature Narrow ESR column diameter
✓ Central square (with 25 smaller squares) Tilted ESR tube (3 degrees tilt = Clotted samples
o 1 small square inside central square (with 16 smaller squares) – increased of 30%)
0.04 mm2
o 1 smallest square – 0.0025 mm2PROPERTY OF MEDTECH REVIEW NOTES
𝑐𝑒𝑙𝑙𝑠
OSMOTIC FRAGILITY TEST
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
FORMULA : = ☻ __________ OFT: Hereditary spherocytosis
𝑢𝐿 𝑎𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑒𝑝𝑡ℎ (0.1)

DO NOT DISTRIBUTE
☺ In order to memorize, list down the formula of corrected WBC count ☻ __________ OFT: anemias where there are target cells
WBC ESTIMATE
2-5 WBCs/hpf 4,000-7,000 WBCs/uL
4-6 WBCs/hpf 7,000-10,000 WBCs/uL BLOOD SMEARS
6-10 WBCs/hpf 10,000-13,000 WBCs/uL ☻ Methods:
10-20 WBCs/hpf 13,000-18,000 WBCs/uL ✓ __________ – uses 2 glass slides
✓ __________ – uses glass slide and coverslip
✓ _________ – uses 2 coverslips
✓ _____________ – if patient’s WBC count is <1x109/L
RETICULOCYTE COUNT ✓ Automated methods
☻ Used to assess erythropoietic activity of bone marrow FACTOR THIN SMEAR THICK SMEAR
☻ Sample: EDTA whole blood Pressure
☻ Stain: supravital stain (new methylene blue, brilliant cresyl blue) Angle
Size
☺ In order for you to memorize, list down all the formula for reticulocyte count, miller Speed
disc method, absolute reticulocyte count, corrected reticulocyte count and
reticulocyte production index)
 ✓ Thalassemia
Cell Abnormalities 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
☻ A decrease in either number of RBCs, hemoglobin or hematocrit
MACROCYTIC, NORMOCHROMIC ANEMIA
NORMOCYTIC, NORMOCHROMIC ANEMIA
☻ Examples:
☻ Examples:
✓ Megaloblastic anemia
✓ Aplastic anemia
o Vit. B12 and Folate deficiency
✓ Hemolytic anemia
o _______________ – autoantibodies: anti-parietal cell antibodies
✓ Anemia of Chronic disease
and anti-intrinsic factor antibodies
o Associated with inflammatory process
✓ Non-megaloblastic anemia
o Low TIBC
o Alcoholism, Liver disease
✓ Sickle cell anemia
o Type of hemoglobinopathy
o β26 Glu → Val PROPERTY OF MEDTECH REVIEW NOTES
VARIATIONS
o oxygen is released thus “sickling” of cells occur
o resistance to P. falciparum ☻ In size: _____________
o Primary cause of death: infectious crisis ☻ In hemoglobin content: _______________
✓ Paroxysmal Cold Hemoglobinuria DO NOT DISTRIBUTE
✓ Paroxysmal Nocturnal Hemoglobinuria HYPOCHROMIA GRADING
o Aka ___________________________ Area of central pallor is ½ of cell diameter
o _________ and _________ deficiency Area of central pallor is 2/3 of cell diameter
o Tests: Ham’s acidified serum test, Sugar Water test and Flow Area of central pallor is ¾ of cell diameter
cytometry Thin rim of hemoglobin
POLYCHROMASIA GRADING
Slight
MICROCYTIC, HYPOCHROMIC ANEMIA 1+
☻ Examples: 2+
✓ ________________ 3+
o There is a failure of iron to incorporate to protoporphyrin IX 4+
o Excess accumulation of iron in mitochondria of normoblasts
o Dimorphic anemia ☻ in shape: _____________________
✓ ________________
o Most common cause of anemia
o Increased TIBC
o Blood picture is only evident is Stage 3 or Frank anemia
 RBC ANOMALIES Auer rods ☻ Derived from fusion of primary granules
____________ ☻ Irregular spicules Alder-reilly ☻ Result of incomplete degradation of
☻ Spur cell ☻ Found in abetalipoproteinemia and McLeod mucopolysaccharides
☻ Thorny cell syndrome Chediak-higashi ☻ Defect: mutation in LYST gene
☻ Defect: abnormality in lecithin and ☻ Cells are characterized with scanty
sphingomyelin ratio lysosomal vesicles
Echinocyte ☻ Evenly distributed spicules _______________ ☻ Presence of large, abnormal cytoplasmic
☻ Burr cell ☻ Found in uremia and PK deficiency granules in phagocytes
☻ Sea urchin cell ☻ Defect: membrane lipid content abnormality _______________ ☻ Dohle-like inclusions but found in
Codocyte ☻ Found in thalassemia, Hemoglobinopathies granulocytes and monocytes
☻ Target cell ☻ Defect: deficiency in phospholipids and ☻ Characterized with the presence of giant
☻ Mexican hat cell cholesterol content of membrane platelets
☻ Leptocyte (thinner Faggot Cells ☻ Mass of auer rods
variant) Toxic granulations ☻ Found in neutrophils – altered primary
Elliptocyte ☻ Cigar-shaped RBC granules
☻ Found in hereditary elliptocytosis LEUKOCYTE FUNCTION ABNORMALITIES
☻ Defect: decreased membrane protein band Job syndrome ☻ Mutation in STAT3 gene
4.1
______________
PROPERTY OF MEDTECH REVIEW NOTES
☻ Slit-like
☻ Neutrophils have normal random motility but
altered directional motility
☻ Mouth cell ☻ Found in Rh null syndrome Lazy leukocyte syndrome ☻ Both random and directional motility are
☻ Xerocyte ☻ Defect: cation imbalance altered
(dehydrated variant)
Drepanocyte DO NOT DISTRIBUTE
☻ Found in sickle cell anemia
_____________ ☻ Failure of WBCs to kill microorganisms due to
defects in respiratory burst
☻ Sickle cell ☻ Hgb S polymerization CELLS EXHIBITING PHAGOCYTOSIS
_____________ ☻ Found in Myelofibrosis with myeloid LE cell ☻ Neutrophil that engulfed a nuclear material
☻ Teardrop cell metaplasia Tart cell ☻ Monocyte or macrophage that engulfed a
WBC ANOMALIES nuclear material
NUCLEAR ABNORMALITIES LYMPHOCYTE ABNORMALITIES
____________ ☻ Defect: mutation of lamin-beta receptor Basket cell ☻ Remnants of granulocytic cells
☻ hyposegmentation ☻ Pince-nez appearance Smudge cell ☻ Remnants of lymphocytes
Hypersegmentation ☻ Neutrophils with 6 or more lobes Rieder cell ☻ Cloverleaf-like
☻ Defect: abnormality in DNA synthesis __________ ☻ TRAP (+)
☻ Found in Undritz anomaly, megaloblastic ☻ B lymphocyte in origin
anemia Sezary cell ☻ Cells with cerebriform nucleus
Barr bodies ☻ Appears as drumstick attached to a nucleus ☻ T lymphocyte in origin
by a short stalk PLASMA CELL ABNORMALITIES
☻ Represents inactive X chromosome Flame cell ☻ Aka thesaurocyte
☻ Found in Klinefelter syndrome ☻ Found in IgA myeloma
CYTOPLASMIC ABNORMALITIES Grape cell ☻ Clock face nucleaus
 ☻ Cell with a cytoplasm completely filled with
Russell bodies PART II
☻ Found in Multiple Myeloma
Russell bodies ☻ Aggregates of immunoglobulins
HEMOSTASIS
LEUKEMIA Greek meaning:
☻ Acute leukemia - __________ in peripheral blood (based on FAB but 20% • “stoppage of blood flow”
based on WHO) and >50% blasts in bone marrow • Complex interaction between blood vessels, platelets, & biochemical factors
✓ _______ – most common form of childhood leukemia in plasma.
☻ Chronic leukemia - <10% blasts in PB • Keeps circulating blood in fluid state
✓ _______ – most common form in elderly __________ – stops bleeding
• Coagulation
☻ M:E ratio is _____ __________ – dissolves clot
• Fibrinolysis
☻ MPO stain
✓ fresh blood is required HEMOSTATIC COMPONENTS
✓ used in differentiating AML from ALL • Extravascular component
☻ Sudan Black B o Tissue surrounds blood vessels
✓ Most sensitive stain for granulocytic precursors
PROPERTY OF MEDTECH REVIEW NOTES
☻ _____ – presence of Philadelphia chromosome indicates good prognosis
• Vascular component
o Blood vessel
ACUTE MYELOPROLIFERATIVE LEUKEMIA • Intravascular components
M0 - Acute Undifferentiated leukemia MPO and SBB negative o Platelets
M1 – AML without maturation
M2 – AML with maturation
DO NOT DISTRIBUTE
May demonstrate auer rods
Most common subtype
o Plasma proteins

M3 – ______________________ Assocated with DIC and faggot cells EXTRAVASCULAR COMPONENT

Deadliest subtype • Provides back pressure on injured vessel
M4 – Acute Myelomonocytic Leukemia Aka Naegeli monocytic leukemia o Swelling
2nd most common subtype o Trapping of escaped blood
M5 – ______________________ Aka Schilling Leukemia
M6 – ______________________ Aka DiGuglielmo’s syndrome VASCULAR COMPONENTS
M7 – Acute Megakaryocytic Leukemia Factor VIII stain (+) • Blood vessels role in hemostasis depends on:
ACUTE LYMPHOPROLIFERATIVE LEUKEMIA o Size
Small cell, homogenous, childhood o Amount of smooth muscle within walls
Large cell, heterogenous, adults o Integrity of endothelial cell lining
Burkitt type
(-) PAS, always (+) ORO INTRAVASCULAR COMPONENTS
• Key components:
o Platelets
o Biochemicals (procoagulants)
 • Involved in: o __________
Nitric oxide – “relaxing” factor
o Coagulation ▪ Promotes vasodilation
o Fibrinolysis ▪ Inhibits platelet activation
▪ Promotes angiogenesis and healthy arterioles
CONCEPTS OF NORMAL HEMOSTASIS __________
o Heparin sulfate - anticoagulant glycosaminoglycan
• Hypocoagulation ____________________
oTissue Factor Pathway Inhibitor– regulates extrinsic pathway
o Abnormal bleeding _________
o Protein C – coagulation inhibitor
o Defective coagulation mechanism o ________________
Thrombomodulin – Protein C activator
o Inherited ________________
o plasminogen
Tissue activator– activates fibrinolysis
____________
▪ Hemophilia A (FVIII deficiency)
o Acquired: • Capillaries
proteins that control blood clotting
▪ DIC become overactive o Metabolic exchange of blood and tissue takes place.
▪ Liver & Kidney Disease o Junctions
▪ Openings; allow passage of WBCs, O2, nutrients & wastes, in and
• Hypercoagulation out of the blood
o Inappropriate formation of thrombi o _________
Pericyte
o Thrombi consists of ▪ Cells lie beneath endothelium
▪ WBCs ▪ May differentiate into vessel-related cells when needed.
▪ Platelets PROPERTY OF MEDTECH
• REVIEW NOTES Arteries & Veins
▪ RBCs o Three layers
▪ Fibrin ▪ Tunica Intima (inner)
o Caused by defect or lack of activation of fibrinolytic system ▪ Tunica media
o Most are acquired DO NOT DISTRIBUTE ▪ Tunica adventitia
▪ Malignancy
▪ Surgical procedure o ________________
Tunica Intima
▪ Comes contact with blood cells
▪ Separates from subendothelium
__________________
Primary hemostasis • Basement membrane
• Respond to vascular injury or desquamation o Elastin-rich
• Rapid, short-lived response to damage • Elastic connective tissue
• Involves • Collagen fibers
o Blood vessels ▪ Endothelial cells deposit von Willebrand Factor (bound to
o Platelets collagen in subendothelium)
• Stored in Weibel-Palade bodies
ANTICOAGULANT PROPERTIES OF INTACT ENDOTHELIUM
• Rhomboid; contiguous – prevents harmful turbulence o TUNICA MEDIA
▪ Has smooth muscles & connective tissues
• Secretes: • Collagen fibers
____________ – eicosanoid platelet inhibitor
o Prostacyclin • Fibroblasts
o produces collagen
 • Phosphatidylinositol
o ____________________
Tunica Adventitia • Phosphatidylserine
▪ Consists of: • Phosphatidylenolamine
• Connective-tissue fibroblast
• Collagen fibers Phosphatidylcholine & Sphingomyelin
• Predominates in plasma layer
▪ DAMAGED VESSELS
o Harmful stimulus – induces vasoconstriction ___________________
Phosphatidylinositol
o ECs secrete vWF from its storage site Weibel
_____________________
– Palade Bodies • Supports plt. Activation
▪ Also secrete adhesion molecules (promotes platelets & WBC • Supplies arachidonic acid that is converted into Prostaglandin &
binding) Thromboxane A2
• P-selectin
___________________
Phosphatidylserine
• ICAMs (Intercellular Adhesion Molecules) - • Flips to outer surface upon activation
immunoglobulin-like • charged phospholipid surface on which coenzymes especially coagulation
factors (VII and IX) & (X and V) assemble.
• PECAMs (Platelet Endothelial Cell Adhesion Molecules) –
promotes plt. & WBC binding ▪ Glycocalyx
o Collagen exposure PROPERTY OF MEDTECH REVIEW NOTES
• Surface coat
▪ Binds and activates platelets o (20 – 30 nm – rodaks)
▪ Activates intrinsic pathway o (10 – 50 nm – steinininger)
o Tissue Thromboplastin (FIII) release • Absorbs albumin, fibrinogen, and other plasma proteins
DO NOT DISTRIBUTE
▪ Activates extrinsic pathway using ENDOMITOSIS
o Release of Tissue Plasminogen Activators (tPAs) • Consist of glycoprotein
▪ Initiates fibrinolysis o Ia
o Ib
▪ PLATELETS’ ROLE IN HEMOSTASIS o Ic
o Adhesion to injured vessels o IIa
o Aggregation at injury site o IIb
o Promote coagulation on their phospholipid surface o III
o Induce clot retraction o IV
o V
PLATELET ULTRASTRUCTURE
o ______________________
Submembrane zone
o ________________________
Peripheral zone ▪ Links the membrane and the inner cell body
▪ Consists of platelet’s outer membrane and related structures ▪ Underlies plasma membrane
▪ Bilayer composed of lipid & protein ▪ Specific area
▪ Predominant lipid – Phospholipid
____________ • Organelles in inner matrix of unaltered platelet never
• Phosphatidylcholine come in contact w/ internal side of platelet cell wall
• Sphingomyelin
 ▪ Contains organized system of filaments o Diphosphates
▪ contributes to regulate the normal discoid shape o Triphosphates
▪ act as base of pseudopod o Serotonin
▪ modulates platelet adhesion and clot retraction o Magnesium
o ATP
o ___________________
Sol-gel zone o ADP
▪ Contributes the matrix or muscle and skeletal portion of the
platelet Calcium & Magnesium
▪ For contractile process • Activation of phospholipase
▪ Underlies submembrane filaments • Plt. Activation and coagulation
▪ Consists of:
• Circumferential microtubule system ________
ADP
• Microfilaments • Most important in dense granules
o Provides force after activation that directs the • Binds specific receptors and initiates plt. Aggregation
organelles towards center of cell w/ control and • Rapidly degraded to adenosine
direction from microtubules o Inhibits plt. function by enhancing cAMP levels
▪ Stable gel
• regulates arrangement of internal organelles
▪ ALPHA GRANULES
PROPERTY OF MEDTECH REVIEW NOTES
▪ influences communication of organelles w/ plt. External
• 300 – 500 nm
surroundings • Contents:
▪ MICROTUBULAR SYSTEM o Coagulation Factors
• Cytoskeletal support system o Permeability factor
DO NOT DISTRIBUTE
• Contractile response of plts. to stimulation. o Cationic proteins
o PDGF
o ____________________
Organelle zone o Plt. Factor 4
▪ Major portion of plt’s cytoplasm o B-thromboglobulin
o Fibrinogen
▪ DENSE-GRANULES o Factor V
• 250 – 350 nm o Fibronectin
• 2-7 granules o Thrombospondin
• release contents directly into plasma upon platelet o Protein C inhibitor
activation o PAI-1
• supports: _________
PDGF
o P-selectin • Most important cationic protein released from platelet (mitogenic agent)
o Alpha IIb Beta3 • Stimulates smooth cell growth and proliferation
o Gp Ib/X/V • Not specific for plts. (also in endothelial and other blood cells)
• Components
o Calcium Platelet factor 4
o Adenosine • Binds to heparin (neutralize)
o Guanosine
Permeability Factor PLATELET FUNCTIONS
• Influences vessel wall permeability and tone
• ADHESION
Factor V o Adhere to:
• Likely not synthesized by platelet ▪ Detached endothelial cells
▪ Collagen fibril
▪ Fibronectin
o PLASMA MEMBRANE SYSTEM ▪ Basement membrane
o NOTE: Platelets don’t release constituents nor promote aggregation
▪ SURFACE-CONNECTING CANNALICULAR SYSTEM
• Delivery route for substances ingested by platelet 13- Hydroxyoctadecadienoic acid (13-HODE)
____________________________
• Route of extrusion of substance released from stimulated • Endothelial cell-derived chemorepellent
plts. • Inhibits plt. adhesion
• Readily visible in • Underlies the endothelium
o Platelet shape change
o Contraction _____________________
Collagen Type I & III
o Adhesion • Promotes plt. adhesion
o Aggregation PROPERTY OF MEDTECH REVIEW NOTES • Facilitates aggregation and release
• Allows enhanced interaction of the platelet w/ its
environment ______________________
Collagen IV & V
o Increase access to platelets’ interior as well as • Promotes adhesion
DO NOT DISTRIBUTE
increase egress of plt. release production. • Don’t causes plt. aggregation under specific conditions
▪ ________________________
DENSE TUBULAR SYSTEM
• CONTROL CENTER FOR PLATELET ACTIVATION ___________________
von Willebrand Factor
• Condensed remnants of rough ER • Link between specific platelet glycoprotein Ib and subendothelial cell
• Randomly dispersed in the platelet cytosol connective tissue (reversible binding process – decelerates platelets)
• Close to circumferential band of microtubules • Released by damaged endothelial cells
• Has important rol in influencing microtubules supporting
discoid platelet shape NOTE: COLLAGEN TYPE I binds to GP VI
• Contains peroxidase activity • Triggers plt. activation pathways: releases
o For prostaglandin synthetase activity o TxA2
• Sequesters Ca2+ o ADP
• Bears series of enzymes that support platelet activation
ENZYMES IN DENSE TUBULAR SYSTEM • AGGREGATION
• Phospholipase A2 o Key part of 10 hemostasis
• Cyclooxygenase o Induced by:
• Thromboxane A2 (TxA2) ▪ ADP
• Phospholipase C - Supports production of inositol triphosphate (I3) ▪ TxA2
▪ Thrombin
 ▪ Collagen • Hydrolyze arginine- or lysine-containing peptide of zymogens
▪ Epinephrine
▪ Endotoxin _____________ – no biological activity until converted to active enzymes (serine
o ADP, TxA2, Thrombin proteases).
▪ Independent mediators Cofactors:
• II
ADP-induced Aggregation • VII
• Dependent to GpIIB/IIIa • IX
• Causes conformational change to IIb-IIa complex (inactive IIb/IIIa to active • X
IIb/IIIa) • XI
o Allows binding of fibrinogen complex • XII
• Prekallikrein
Thrombin (produced because of Tissue Factor)
• Reduced plt. aggregation in three mechanism _______________ – assist in activation of zymogens by:
o Stimulates plt. to release ADP • Altering zymogen conformation
▪ Mediates ADP-induced aggregation o To permit more efficient cleavage by serine protease
o Activates plt. membrane phospholipase • Binding the zymogen and appropriate serine protease on plt. phospholipid
▪ Initiates formation of TxA2 o To enhance & accelerate zymogen activation process.
PROPERTY OF MEDTECH REVIEW NOTES
o Can induce platelet aggregation independently; poorly understood Cofactors:
• V
________________________
Thromboxane A2 (TxA2) • VIII
• Arachidonic acid metabolite • III
• Induces plt. aggregation DIRECTLY DO NOT DISTRIBUTE • HMWK
• May also act synergistically w/ ADP
• POTENT VASOCONSTRICTOR Zymogen activation (conversion into serine protease) is either
• ASSISTS HEMOSTASIS AND COAGULATION INDIRECTLY • Conformational change (twist, turn, bend) in zymogen molecule
• Hydrolytic cleavage of specific peptide bond of zymogen.

__________________ Zymogens of X and II requires Va & VIIIa

• Delayed, long-term response • Va & VIIIa is activated by small amounts of thrombin.

COAGULATION FACTORS
• Enzyme precursors (zymogens) COAGULATION FACTORS
• Nonenzymatic cofactors • Referred in roman numerals
• Calcium o Except for kallikrein & HMWK
• Phospholipid (from platelet) o Assigned by International Committee of Nomenclature of Blood
• Coagulation Factors
Serine Protease – active enzymes
• Have exposed serine-rich, active enzyme sites.
 • 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 Phosphatidylserine of platelet was once • Present in adsorbed plasma (slightly
called ________________
o Nonexistent reduced level)
COAGULATION FACTORS o Partially adsorbed by BaSO4
Numeral Preferred name Synonyms
I Fibrinogen COAGULATION GROUPS
II Prothrombin Prethrombin • Contact group
III Tissue Factor Tissue thromboplastin • Prothrombin group (Vit. K-dependent group)
IV Calcium • Fibrinogen group
V Proaccelerin Labile factor
Accelerator globulin _______________ (Prekallikrein, HMWK, 11, 12)
VII Proconvertin Stable factor • Adsorbed by contact with negatively charged surface
Serum Prothrombin o Collagen
Conversion Accelerator o Subendothelium (in-vivo)
(SPCA) • Causes slow conversion of XII → XIIa
o Initiates
VIII:C
PROPERTY OF MEDTECH REVIEW NOTES
Antihemophilic Factor (AHF)
Autoprothrombin
Antihemophilic globulin ▪ Intrinsic pathway
Antihemophilic factor A ▪ Fibrinolysis
Platelet Cofactor 1
IX Plasma Thromboplastin Component Christmas Factor Prekallikrein & HMWK play role in
DO NOT DISTRIBUTE
Antihemophilic Factor B • Intrinsic pathway activation
Platelet Cofactor 2 • Activation of fibrinolysis
X Stuart-Prower Factor Stuart Factor • Kinin formation
• Complement system activation
Prower Factor
Autoprothrombin III
_____________________ (9, 10, 7, 2, Protein C & S)
XI Plasma thromboplastin antecedent Antihemophilic factor C
• Vitamin K – dependent Group
XII Hageman factor Glass factor
• Adsorbed by BaSO4 and other salts
Contact Factor
• Factors are synthesized in the liver in the presence of Vitamin K
XIII Fibrin-Stabilizing factor Laki-Lorand factor
Fibrinase
o Vit. K – quinone; fat-soluble
Plasma transglutaminase
▪ Ingested in diet (green leafy vegetables)
Fibrinoligase
▪ Manufactured by gut flora (Bacteroides fragilis & Escherichia
- Prekallikrein Fletcher factor
coli)
- HMWK Fitzgerald factor ▪ No substantial storage in the body
Contact activation factor • ___________ is necessary to gamma-carboxylate the preformed enzyme
Williams factor precursors of IX,X,VII,II.
Flaujeac factor
 o Gamma-carboxylation of glutamic acid residue at N-terminal or amino- ▪ antigenic portion (VIII:Ag)
end of factor polypeptide chain. ▪ von Willebrand Factor (VIII:vWF)
o Calcium binds to factor forming calcium bridge with acidic • results to von Willebrand’s Disease if either both are
phospholipid surface of activated platelets. decreased
o Ca2+ and PL (phospholipid) – essential for enzyme and substrate function
in coagulation pathways NOMENCLATURE FOR FACTOR VIII (International Committee on Thrombosis and
Hemostasis)
• FACTORS THAT INHIBITS GAMMA-CARBOXYLATION REACTION
o Dietary Vitamin K deficiency • _________ – entire molecule as it circulates in plasma.
o Administration of antibiotics that sterilize intestinal tract o Composed of:
o Oral anticoagulant therapy (coumarin/warfarin) ▪ VIII:C
▪ Interferes gamma-carboxylation ▪ VIII:vWF

• Des-gamma-carboxyl Proteins or proteins in Vit. K antagonism (PIVKAs) • _________ – for binding to endothelium
o procoagulants released from liver without second carboxyl group o Supports normal platelet adhesion & function
added to gamma carbon. o Tested by bleeding time

________________ (1, 5, 8, 13) • _________ – cofactor to IXa (with Ca ) in conversion of X → Xa. 2+

PROPERTY OF MEDTECH
• Have highest molecular weights of all factors REVIEW NOTES
o Tested with partial thromboplastin time
• Most labile
• Consumed in coagulation • _________ – antigenic property of procoagulant portion
• Acts as subtrates for plasmin o Measured by immunologic monoclonal antibody
DO NOT DISTRIBUTE
• Only factors that are found in platelets’ alpha granules, except:
o XIII – found in cytoplasm • _________ – F VIII-related antigen, which is a property of large vWF portion of
o VIII:C - NOT FOUND IN PLATELETS molecule
o Measured by:
Factor VIII (VIII/vWF) – large multimeric molecule ▪ Immunologic techniques of Laurell rocket or
• Has two principal parts: ▪ Immunoradiometric assay
o Coagulant portion (VIII:C)
▪ Cofactor in intrinsic pathway • _________ – ristocetin cofactor activity
▪ Antihemophilic Factor o Required for aggregation of human platelets with ristocetin.
• Defective in Hemophilia A
o ____________________________ _________ (a disintegrin and metalloproteinase with a thrombospondin type 1 motif,
▪ important to normal platelet function member 13)
▪ vWF-cleaving protease
• High Molecular Weight Portion of Factor VIII ▪ Defective ADAMTS-13 is assoc. w/
o Synthesized in o Presence of ultralarge vWF
▪ endothelial cells o Platelet aggregation
▪ megakaryocyte o Microvascular thrombosis
o has two parts:
 Properties of Coagulation Group Production No Yes No
Contact Prothrombin Fibrinogen reduced by
Factors XI, XII, Prekallikrein, II, VII, IX, X, Protein I, V, VIII, XIII anticoagulants?
HMWK C and Protein S
Function Cofactor: HMWK Cofactor: Protein S Cofactor: V, VIII PHOSPHOLIPID CONTRIBUTING TO COAGULATION
Tissue Thromboplastin
Transamidase: • Lipoprotein
o Complex of two parts:
XIII
▪ Phospholipid (PL)
Stability Fairly stable Heat labile: _______ Heat labile:
▪ Protein
___________
• Initiates extrinsic pathway
Well preserved in
o Binding its PL to VII
___________ Storage labile:
▪ Converting VII → VII
________
• In Prothrombin Time:
o Reagent used
Vitamin K- No Yes No
▪ Rabbit brain or
dependent for
▪ Lung tissue Thromboplastin
synthesis?
▪ Ca2+
Adsorbed by
BaSO4 and
Partially PROPERTY
Yes No OF MEDTECH REVIEW NOTES
NOTE:
THROMBOPLASTIC
other salts Thrombo – pertains to clotting
Consumed in Partially No (except_____) Yes Plastic – Greek word: “plassein” – to mold
coagulation?
Site Plasma/serum
DO NOT DISTRIBUTE
Plasma/serum Plasma Partial Thromboplastin & Platelet Phospholipid
(except II – not
• Reagent used as a platelet substitute in evaluating intrinsic pathway
present in serum) • “partial thromboplastin”
Destroyed by No No Yes o Partial – reagent consist only of PL portion of tissue thromboplastin
plasmin or high o Platelet substitute
concentrations • Intrinsic pathway requires PL for FX activation in-vitro.
of thrombin? o Test requires PPP
Found in No No Alpha granules: PHYSIOLOGIC VARIATIONS OF COAGULATION FACTORS
platelets? ____________
_____________
General o Normally deficient in Vitamin K
cytoplasm: _____ o Moderate deficiency exists in Vitamin K-dependent factors
ADULTS
Not present: ____

Acute-phase No No Yes
reactant?
 Conditions associated w/ physical variations in Coagulation and Fibrinolytic o Prekallikrein and HMWK and XI are absorbed also in the collagen
System
Condition Related Factors Related Factor contact group Kallikrein
\
increases decreases XII -----------------------------------------------> XIIa
Stress I
Tissue necrosis I (Kallikrein accelerates the conversion rate)
Inflammation I
Pregnancy I, VIII, IX, X XIII, XI, AT-III o Enzymes of basophils and endothelial cells can activate FXII.
Oral contraceptives I, VIII, VII, IX, X o _________ enhances proteolytic effect of kallikrein on XIIa
Hypermetabolism I, VIII, Plasminogen o _________ can activate prekallikrein to kallikrein.
(Hyperthyroidism)
Vigorous exercise VIII, XI, XII ROLES OF FACTOR XIIa (CONTACT ACTIVATION)
Chronic Thrombocytopenia VIII o Initiates intrinsic pathway of coagulation
Hypothyroidism IX, XI, Plasminogen o Initiates fibrinolysis
o Forms complex with kallikrein to convert plasminogen → plasmin
Childbirth I, VIII
o Initiates kinin and complement system
Surgical Procedure I, VIII
o Formation of kallikrein by XIIf and HMWK, causes HMWK to be
Trauma I, VIII
converted into kinins (such as bradykinin).
Myocardial Infarction
Acute Illness
PROPERTY OF MEDTECH REVIEW NOTES
I, VIII
I, VIII
o Plasmin resulted from kallikrein can initiate complement system
o Don’t cause coagulation abnormalities • IMPORTANT ROLES OF KALLIKREIN
o Perpetuates FXII activation and its own production
INTRINSIC PATHWAY DO NOT DISTRIBUTE
• Initiation occurs by exposure to negatively charge surface (such as glass;
o Initiates kinin system
o Initiates fibrinolytic and complement system with XIIa
damaged endothelium) o Activates IX directly
• Initiation begins with contact phase of coagulation. Involved are contact o Can activate IX → IXa on its own.
group:
o XII • IMPORTANT ROLE OF PLASMIN
o XI o Promotes clot dissolution
o HMWK o Activates complement system
o Prekallikrein o cleaves XIIa to XIIf (also kallikrein)
• Coagulation factors in intrinsic:
o XII FACTOR XI ACTIVATION
o XI
o IX XIIa HMWK
o VIII XI -----------------------------------------------> XIa
FACTOR XII ACTIVATION • ____ can also activate plasminogen.
o Begins with XII absorption to the negatively charged surface of vascular
collagen
 Intrinsic Tenase IXa, VIIIa, Phospholipid, X
FACTOR IX ACTIVATION Ca2+
Prothrombinase Xa, Va, Phospholipid, Prothrombin
XIa Cofactor: Ca2+ Ca2+
IX ---------------------------------------------------> IXa
• Kallikrein can also activate Factor IX THROMBIN FEEDBACK MECHANISM

EXTRINSIC PATHWAY • _________– generated through prothrombinase complex

• Factors other than those in plasma are required for the initiation. o Autocatalytic – enhances rate of prothrombinase production
• Coagulation factors in extrinsic: ▪ Self-perpetuating
o Tissue Factor (FIII) o Enhances FV and FVII in small amounts
o VII o Stimulates platelet aggregation
o Ca2+
• Gamma-carboxyglutamic acid residue of FVII binds to PL portion of FIII in the IIa
presence of Ca2+ (act as a bridge between FVII and FIII) XIII ------------------------------------> XIIIa
o VII → VIIa THROMBIN AS COAGULATION INHIBITOR
• Destroys FV and FVIII in high concentration
COMMON PATHWAY • Activates _______ – potent anticoagulant
• ___________ – beginning of common pathway PROPERTY OF MEDTECH REVIEW NOTES o Enhanced by
▪ Ca2+
VIIa-Tissue Factor-Ca2+ ▪ Thrombomodulin (cofactor) in endothelial surface
complex
▪ Protein S (cofactor)
X -------------------------------------------> Xa DO NOT DISTRIBUTE o Protein C-S complex
▪ Inhibits Va and VIIIa
▪ Stimulates fibrinolysis
Prothrombinase complex • Increases plasminogen activator activity
II -------------------------------------------> IIa o Protein C
▪ Inhibits Tissue Plasminogen Activator Inhibitor (TPAI)
IIa
V -------------------------------------> Va FINAL CLOT FORMATION & STABILIZATION
• action of thrombin to fibrinogen – final steps of coagulation
IIa
Fibrinogen -----------------------> Fibrin • _____________
o Ultimate substrate of coagulation pathway
Coagulation Complexes o glycoprotein composed of three nonidentical but intricately
Complex Components Activates interwoven paired chains:
Extrinsic Tenase VIIa, Tissue Factor, IX and X ▪ A alpha
Phospholipid, Ca2+ ▪ B beta
▪ Gamma
 • Chains are linked by _____________
• Alpha and beta chains have fibrinopeptides • Extrinsic Plasminogen Activators
o Fibrinopeptide A o Tissue Plasminogen Activators
o Fibrinopeptide B ▪ Secreted by ECs
▪ Total of four fibrinopeptides (two A and B)
• Plasminogen Activators in Secretory Ducts
• CONVERSION OF FIBRINOGEN TO FIBRIN o Present in body fluids
1. There is cleavage of four fibrinopeptides from alpha & beta ends by ▪ Urine (Urokinase)
thrombin. ▪ Tears
a. Once fibrinopeptides A and B are removed – it becomes soluble ▪ Saliva
fibrin monomer (unstable clot) ▪ Semen
2. _________ of fibrin monomers interact within the ___________of other fibrin ▪ Milk
monomers forming fibrin polymers (unstable)
a. Note: FIBRIN POLYMERS ARE SOLUBLE TO ______ OR • Exogenous Plasminogen Activators
_________________________ o For therapeutic destruction of thrombi
3. Factor XIIIa (transglutaminase) crosslinks adjacent fibrin monomers ▪ Urokinase Plasminogen Activator
through formation of covalent bond in alpha and gamma chains forming • Secreted by
stable clot o UT epithelial cells
PROPERTY OF MEDTECH REVIEW NOTES
a. Stabilized fibrin clot is insoluble to 5M urea & weak acids. o Monocytes
o Macrophages
___________________________ ▪ Streptokinase
• Final event of hemostasis ▪ Tissue plasminogen activators (tPA)
DO NOT DISTRIBUTE
• Body’s defense against occlusion of blood vessels.
MULTIPLE ROLES OF PLASMIN
ACTIVATION OF PLASMINOGEN TO PLASMIN
Plasmin’s role Comments
• _______________ – synthesized in the liver Activates fibrinolysis Cleaves fibrin & fibrinogen into
o Possesses five glycosylated loops (kringles) fibrinogen degradation products
o Stored and transported in eosinophil Activates intrinsic coagulation XII → XIIa is amplified indirectly by
o Increased in inflammation plasmin
o Part of any clot (fibrin tends to absorb plasminogen) Interferes intrinsic & common pathway Destroys _____ and _____
o Plasmin is also formed within the clot Blocks thrombin conversion of Fibrinogen degradation products
fibrinogen to fibrin interfere with thrombin influence on
Plasminogen activator fibrinogen
PLASMINOGEN ------------------------------------> PLASMIN Activates kinin system Enhances conversion of prekallikrein →
PLASMINOGEN ACTIVATORS kallikrein and kininogen → kinin
• Intrinsic Plasminogen Activators Activates complement system Cleaves C3 → C3a and C3b; activates
o XIIa C1 (first component of complement
o Kallikrein system
o HMWK
 o Cause a clinically significant platelet dysfunction (aggregation
• Free plasmins are destroyed by the ___________ found in the circulation. inhibition)
• Plasmin can’t distinguish between fibrinogen and fibrin.
NATURAL COAGULATION INHIBITORS
Plasmin can destroy:
• Fibrin – covalent bonds slows the degradation. ______________________
• Fibrinogen – quickly destroyed because of instability • Alpha2 globulin
• FVIII • Synthesized in the liver
• V • Half-life: 2.7 days
• Other factors • Action:
o Inhibits
FIBRIN(OGEN) DEGRADATION ▪ XIIa
• Products: ▪ XIa
o Fibrin(ogen) Degradation Products (FDP) or Fibrin(ogen) Split Products ▪ Xa
(FSP) ▪ IXa
▪ Products are removed by reticuloendothelial system and other o Inhibiting by forming enzyme inhibitor complexes with activated
organs. factors
• Principal Products: ▪ Neutralizing them to prevent their action on other zymogens.
o Early Degradation Products PROPERTY OF MEDTECH REVIEW NOTES • Also has an inhibitory effect in plasmin and kallikrein
▪ X (D-E-D
▪ Y (D-E) • Action is enhanced by cofactor __________
o Late Degradation Products o Once bound to the lysine site, it causes conformational change to the
▪ D-D dimer DO NOT DISTRIBUTE arginine residue of AT-III reactive site
▪ E ▪ The site will be more accessible to the active site of serine
Fragment ___________ proteases.
• First and last fragment formed
• Result of plasmin cleavage of terminal portion of alpha chains from fibrin • Decreased its therapeutic use by heparin
polymer o Because heparin accelerates binding of AT-III to serine protease
o Cleaved to form __________________
▪ YY fragment (Y is D-E) • Naturally occurring relative to heparin
▪ DXD fragments – intermediate complex • Glycosaminoglycan (mucopolysaccharide) anticoagulant
• Identified in
DXD complex is cleaved into o Surface of platelets
• DED o Vascular endothelium
• DY/YD Fragments • Together with AT-III, it protects uninjured vessels from abnormal clotting by
neutralizing proteases.
NOTE: Presence of D-D dimer – specific indicator of in-vivo fibrinolysis o Without heparin – slow
• Indicative only for Fibrin Degradation products o With heparan sulfate – neutralization is accelerated from 2000 –
• Most FDPs inhibits coagulation 10,000x faster
• All four fragments have affinity to coating platelet membrane
______________________________ • EC Protein C Receptor (EPCR)
• Large o Transmembrane protein in EC membrane that binds Protein C and
• Binds to thrombin (doesn’t completely inhibit) activated Protein C (APC)
• Rate of inhibition is slower than AT-III NOTE: When thrombin is bound to thrombomodulin:
• Protects its bound enzyme from other circulating inhibitors • Its specificity is altered
o Thrombin bound to it may still activate small amounts of FV and FVII. • Once altered:
o It will not activate Factor V and VIII or platelets
• Also inhibits fibrinolysis o It will not convert fibrinogen to fibrin.
o Inhibits (but not totally eliminates) plasmin’s function to fibrinolysis.
• Inhibits kinin system • ______________ – transmembrane protein
o Inhibiting kallikrein • Thrombin cofactor
• Expressed in vascular ECs
_____________________________
• Potent inhibitor of FXIa • Activated Protein C-S complex – enhances fibrinolysis
• Also inactivates thrombin (slow rate) o Destroys Factor Va and VIIIa
• Inactivates fibrinolytic system by o Inactivates plasminogen activator inhibitors.
o Inactivation of plasmin ▪ Enhances formation of plasmin
▪ Least significant of the three naturally occurring fibrinolytic
system inhibitors
• Important to pulmonary functions PROPERTY OF MEDTECH REVIEW NOTES
• Deficiency is associated to
o Liver cirrhosis
o Emphysema
DO NOT DISTRIBUTE
_________________________
• Inhibitor of C1 esterase
• Inhibits
o XIIa
o XIIf
o XI
o Plasmin
o Kallikrein
_________________________
• Vitamin k – dependent • __________ – cofactor that binds and stabilizes Activated Protein C (APC)
• Coagulation inhibitors o 40% - free
• Protein C is activated slowly by THROMBIN o 60% - bound to C4bBP (complement control protein)
o Enhanced when • __________ – cofactor to Z-dependent protease inhibitor (ZPI) (coagulation
▪ Thrombin binds to thrombomodulin (in surface of endothelial inhibitor)
cells) • ____ - potent inhibitor of Factor Xa
▪ Forms 1:1 stoichiometric complex with Protein S (cofactor) o Also inhibits FXI
 • PROTEIN C INHIBITOR
o Inhibits _________________________
▪ APC • Synthetic fibrinolytic inhibitor
▪ Thrombin • Used to treat bleeding disorders (urinary tract bleeding)
▪ Xa o Inhibits plasminogen activation
▪ XIa
▪ Urokinase ________________________
NATURAL FIBRINOLYSIS INHIBITORS • Inhibits coagulation and fibrinolytic system
• effectively inhibits plasmin after Alpha2-antiplasmin depletion.
_____________________
• Most important fibrinolysis inhibitor ________________________
o First to bind with plasmin in plasma • third most important natural fibrinolytic inhibitor
o When it’s depleted • does not inhibit plasmin until both alpha-2-antiplasmin and alpha-2-antitrypsin
▪ Alpha-2-Macroglobulin binds to plasmin are saturated.
▪ ALPHA-1-ANTITRYPSIN is the last to bind plasmin • more important in coagulation inhibition

A-2-Antiplasmin → A-2-Macroglobulin → A-1-Antitrypsin OTHER FIBRINOLYTIC INHIBITORS

• ____________
• Principal inhibitor of fibrinolysis PROPERTY OF MEDTECH REVIEW NOTES o Inhibits plasmin and kallikrein
o Neutralizing free plasmin (1:1 stoichiometric complex with any plasmin) • ____________
▪ Prevents binding of plasmin to fibrin o Also inhibits plasmin
▪ Prevents premature digestion and uncontrolled digestion of
• Fibrin DO NOT DISTRIBUTE FIBRINOLYTIC INHIBITORS SECRETED FROM ENDOTHELIAL CELLS
• Fibrinogen • ____________________________________
• V o TPA control protein; inhibits plasmin generation and fibrinolysis
• VIII • ____________________________________
• Inhibits Tissue Plasminogen Activator o Synthesized in the liver.
o Prevents plasminogen → plasmin o Activated by thrombin bound to thrombomodulin.
• Inhibits:
o XIIf PHYSIOLOGIC COAGULATION CONTROL MECHANISM
o XIa • Inhibition process at site of clot formation
o IIa o Fibrin itself restricts active coagulants to the interior of fibrin clot
o Xa o Platelet and endothelium also restrict coagulation at site of injury
o Clot-promoting activity of kallikrein • Hepatic clearance prevents fibrinolytic and coagulation components from
• Permits a slow and orderly dissolution of clot and adequate time for repair of circulating in the venous system
damaged tissues o Liver impairment may cause
▪
Plasmin-Alpha-2-antiplasmin complex ▪
• Binds to serine active sites
• Inactivates serine protease & prevents enzymatic action on its usual substrate
 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
PROPERTY OF MEDTECH REVIEW NOTES o Automated
☻ ________________
✓ Reflects both platelet number and platelet functional integrity
✓ Affected by
DO NOT DISTRIBUTE 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
 ✓ Clot retraction begins within 30 seconds and almost complete by the ☻ Mixing Studies
end of 4hrs.
☻ Platelet Aggregation
PLATELET AGGREGATION STUDIES
Aggregating agents Normal response Abnormal response
ADP, Collagen and VWD and Bernard-Soulier Glanzmann’s
Epinephrine syndrome thrombasthenia
Ristocetin Glanzmann’s VWD and Bernard-Soulier
thrombasthenia syndrome
TESTS FOR FIBRINOLYSIS
☻ _______________________
Tests for secondary hemostasis ✓ Clot lysis after 48hrs – NORMAL
☻ ________________ ✓ Clot lysis before 48hrs – INCREASED FIBRINOLYSIS
✓ Screening method for problems associated with clotting and ☻ _______________________
coagulation mechanism ✓ Euglobulin – protein that precipitates when plasma is diluted with
✓ Prolonged in all coagulation disorders water and acidified
☻ _________________ ☻ ___________________
✓ Detects presence of fibrin monomers
PROPERTY OF MEDTECH REVIEW NOTES
✓ For monitoring of extrinsic and common pathways
✓ Used to monitor Coumadin/Warfarin/Coumarin therapy – oral ✓ Paracoagulation
anticoagulants ☻ ____________________
☻ _____________________ ✓ More specific than protamine sulfate but less sensitive
✓ For monitoring of intrinsic and common pathways ☻ ____________________
DO NOT DISTRIBUTE
✓ Used to monitor heparin therapy – intravenous anticoagulant ✓ Positive if there is secondary fibrinolysis (eg. DIC)
✓ Activators used: Micronized silica, Elagic acid, Cellite and Kaolin
☻ ________________ REFERENCES
✓ Reagent: East Indian Viper (Vipera russelli)
✓ Detects deficiencies in the common pathway only Henry’s Clinical Diagnosis and Management by Laboratory Methods
☻ ________________ Hematology: Principles, Procedures, and Correlations by Cheryl Lotspeich-Steininger
✓ Detects fibrinogen deficiency Hematology: Principles and Procedures by Barbara Brown
✓ Affected by heparin therapy Hematology: Clinical Principles and Applications by Rodak et.al
☻ _______________ Hematology notes by Mr. Rene Jesus Alfredo Dinglasan, RMT
✓ Detects fibrinogen deficiency Intensive Review Notes of University of the Immaculate Conception – Medical
✓ Unaffected by heparin therapy Laboratory Science Program
✓ From Bothrops atrox Hematopoiesis and Hemostasis notes of Mr. Elton John O. Bayalas, RMT
☻ ______________
✓ Detects Factor XIII deficiency
✓ Presence of Factor XIII – clot should be insoluble for 24hrs at 37°C
PROPERTY OF MEDTECH REVIEW NOTES

DO NOT DISTRIBUTE