Hematology II notes - Anna Rose Magtalas PDF

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By: Anna Rose D. MagtalasCompiled notes on Megakaryopoiesis, Primaryand Secondary Hemostasis, Laboratoryevaluation, and Mixing studiesMagtalas ‖ Hematology 2Clinical Hematology: Principles, Procedures, Correlations by Steininger####### HEMATOPOIESIS / HEMOPOIESISHemopoiesis is a complex process of b...

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By: Anna Rose D. Magtalas Compiled notes on Megakaryopoiesis, Primary and Secondary Hemostasis, Laboratory evaluation, and Mixing studies

Rodak’s Hematology: Clinical Principles and Applications Clinical Hematology: Principles, Procedures, Correlations by Steininger

Magtalas ‖ Hematology 2 2.

HEMATOPOIESIS / HEMOPOIESIS Hemopoiesis is a complex process of blood formation of bone-forming organs like the long bones of sternum and femur(most important bone-forming organ). The lungs may also form blood cells such as megakaryocytes.

Cellular Derived Stimulators: Cytokines Cytokines are cellular secretions that gives signal in order for the cell to proliferate and differentiate. Among which are:  IL3 - acts synergistically with TPO to induce early differentiation of stem cells. Results in the progenitor lineage.  IL6 with IL11 - acts in the presence of TPO to enhance the later phenomenon of the following processes: Endomitosis, Megakaryocyte maturation, and Platelet release/shedding Different cells produce different cytokines depending on what cell it is: Monocyte produce monokines and lymphocyte produce lymphokines.

Multipotential stem cell - the most stem cell line of blood located and produced in the bone marrow. It is capable of becoming several types of blood cells. Cytokines - in the presence of these, the maturation process of the blood cells are greatly affected.

MEGAKARYOPOIESIS Megakaryopoiesis is a process wherein there will be megakaryocyte maturation and eventually the production of platelets. It is a part of hemopoiesis.

Megakaryocyte  Primary cell involved in megakaryopoiesis and the bone marrow cell responsible for the production of platelets.  Largest cell in the body the accounts for 1% of total nucleated cells in the bone marrow.  Considered as polyploid cell -possess multiple chromosomes  Characteristics: a. The only cell line where cell become smaller in size as they mature, because it has to prepare itself for platelet shedding phase (a process wherein meg. fragments to produce platelets). b. Do not go mitosis. It undergo ENDOMITOSIS c. Do not increase in number as they mature d. Can be harvested in the lungs and BM e. On blood films (Wright stained smear), 2-4 megakaryocyte per LPF f. A single megakaryocyte may shed 2000-4000 platelets where 1/3 of it is being sequestered by the spleen.

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Growth Factors Growth factors are necessary for the growth, maturation and proliferation of megakaryocytes. Among which are:  Stem cell factors/Kit Ligand/Mast cell growth factor  GM-CSF  G-CSF  EPO - atleast 20% of circulating epo is needed for meg and platelet maturation

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Inhibitors of Megakaryopoiesis Inhibitors control and regulate production of platelet in the circulation because accumulation of platelet in the blood may lead to formation of thrombus that cuts the oxygen supply in the body causing tissue necrosis and eventually death.  Platelet factor 4  β-thromboglobulin  NAP2 (Neutrophil activating peptide 2)  IL8 - the only cytokine that inhibits megakaryopoiesis

HO HORM RM RMON ON ONES ES AN AND DC CYT YT YTOKIN OKIN OKINES ES TTHA HA HATT C CONT ONT ONTROL ROL ROLSS ME MEGAK GAK GAKARY ARY ARYOP OP OPOIESI OIESI OIESISS

FACT ACTOR OR ORSS TTHA HA HATT AF AFFFE CT M MEGA EGA EGAK KARY RYOP OP OPOIESI OIESI OIESIS: S: 1.

Hormone: Thrombopoietin (TPO)  It is a glycoprotein hormone produced mainly by liver, smooth muscle cells, and kidney that regulates the production of platelets by the bone marrow.  Stimulates production and differentiation of meg. It means that from a primitive cell line, TPO affects the growth of megakaryocyte until it becomes a mature one.

 Has 23% homology with erythropoietin  Concentration of TPO is inversely proportional with megakaryocyte and platelet mass. 2

Rodak’s Hematology: Clinical Principles and Applications Clinical Hematology: Principles, Procedures, Correlations by Steininger

Platelets  Come from mature megakaryocytes  Anucleated  Triggers primary hemostasis especially in exposed in endothelial lining, subendothelium, and presence of plasma procoagulants in the blood vessels.  Characteristics: DIAMETER: 2-5 um and commonly mistaken as debris MEAN PLATELET VOLUME (MPV): 8-10 fL SHAPE: disc-shaped or circular that appears lavander and granular under wright’s stain.  Normal values: (Under OIO): 7-21 platelets/10fields 9 (Metric): 150-400 x10 /L 3 (SI): 150-400 x10 /ul STR STRUCT UCT UCTUR UR URE E OF PPLLATEL ELET ET ET:: I. PERIPHERAL ZONE: Outermost layer Consist of 2 major portions: A. Pla Plasma sma Mem embr br brane ane - made up of a phospholipid bilayer where each and every phospholipid especially in the inner layer has their own functions especially during primary hemostasis. 1. Outer layer is composed of: a. Pho Phosphat sphat sphatidylc idylc idylcholin holin holinee b. Sphi Sphingo ngo ngom myelin 2. Inner layer consists of: a. Pho Phosphat sphat sphatidyl idyl idylsseri erin ne b. Pho Phosphat sphat sphatidyl idyl idyletha etha ethanola nola nolamin min minee - capable of flipping out during platelet activation. It is a phospholipid layer where coagulation enzymes can assemble. These enzymes that will adhere to phosphatidylethanolamine are called PROTHROMBINASE COMPLEX consisting of Factors Va, Xa, IV-Calcium. Prothrombinase complex is essential for the formation of a stable fibrin clot. c. Phosph Phosphatid atid atidylino ylino ylinossit itol ol - supports platelet activation by supplying arachidonic acid which is needed to produce thromboxane A2, an enzyme that is strictly needed for platelet activation.

Magtalas ‖ Hematology 2 Classification of Plasma membrane receptors that provide for Cell Adhesion Molecule (CAM):  CAM Integrin Family - clinically significant CAM. This is made up of Glycoprotein IIb-IIIa, a calcium-dependent membrane protein complex for fibrinogen receptor, necessary for platelet aggregation.  CAM Leucine Rich Family - clinically significant CAM because this is the CAM that makes up the Glycoprotein Ib which serves as binding site for vWF, necessary for platelet adhesion process. Another members of CAM Leucine rich family: GPV and GPIX  CAM Ig gene Rich Family  CAM Selectin Family  CAM Seven Transmembrane Receptor(STR) Family II. SOL-GEL ZONE It lies beneath the platelet membrane. Sol-gel zone provides support to entire platelet especially during changing of shape and during activation process. Composed primarily of 2 different proteins: A. Microtubule contains protein tubulin that maintains the disc shape of the platelet B. Microfilaments this contains the actin and myosin which upon the stimulation of platelet will interact to form actomyosin (thrombosthenin) for clot retraction. It enables the platelet to change in shape. *Clot retraction Formed because of the aggregated platelets in the red blood cells due to presence of microfilaments.

III. ORGANELLE ZONE: Centromere The part where the secretory products of platelet come from. These secretory products are components of the platelet itself, needed for platelet activation: a. alpha granules 1. Platelet Factor 4 2. β-thromboglobulin 3. Platelet-derived growth factor 4. Fibrinogen 5. Factor V 6. Von Willebrand Factor (vWF)- platelet can also produce vWF aside from liver. vWF acts as a ligand with GPIb especially in platelet adhesion. 7. Thrombospondin Fibronectin b. Dense granules - provides energy for the platelets during platelet activation. 1. ADP 2. ATP 3. Calcium and Magnesium 4. Serotonin c. Mitochondria - for ATP synthesis used in platelet metabolism d. Lysosomal granules 1. Acid phosphatase 2. Hydrolytic enzyme - enables the platelet to fight bacterial infection 8.

B. Gl Glyc yc ycocal ocal ocalyx yx - an adhesive layer that responds readily to hemostatic requirement. It provides a distinct connection between the inside part of the platelet to the outside environment. * It is made up of different glycoproteins (V, VIII, and fibrinogen) that are necessary for the 2 major process of primary hemostasis: 1. Platelet Adhesion- attachment of the platelet to the site of injury. 2. Platelet Aggregation- adhesion of platelets with one another to form a temporary platelet clot.

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Rodak’s Hematology: Clinical Principles and Applications Clinical Hematology: Principles, Procedures, Correlations by Steininger

IV. PLATELET MEMBRANE SYSTEM Consist of 2 parts: 1. Den Dense se TTub ub ubu u lar Sy Syst st stem em (D (DTS) TS)  control center for platelet activation  derived from smooth endoplasmic reticulum and sequesters calcium for platelet activation process.  it is also the site of platelet cyclooxygenase and of prostaglandin synthesis  Contains series of enzymes necessary for platelet activation: A. PHO PHOSP SP SPHO HO HOLIP LIP LIPAS AS ASE E A- enzyme that activates phosphatidylinositol to release arachidonic acid. B. CYC CYCLLOOXY OXYGENA GENA GENASE SE SE- acts on arachidonic acid to synthesize Prostaglandin 2. C. PER PEROXID OXID OXIDAS AS ASE E - cleaves PG2 to synthesize PG3 D. Thro Thrombo mbo mboxa xa xane ne sy synth nth nthet et eta ase- takes action on PG3 to release TXA2 which is needed for platelet activation. It is the last enzyme secreted by DTS to make vasoconstriction happen. E. PHO HOSP SP SPHOLI HOLI HOLIP PASE C- cleaves phosphatidylinositol to release Inositoltriphosphate and diacylglycerol (DAG) which is another pathway of platelet activation. 2. Op Open en Ca Canalic nalic nalicular ular Sys ysttem ((SCCS) SCCS)  surface connecting canalicular system  an invagination of the plasma membrane  acts as a canal for the release of the granule constituents and cytoplasm to the exterior of the platelet PLA PLATEL TEL TELET ET FU FUNCTI NCTI NCTION: ON: Participates in a sequence of events that lead to the formation of a platelet plug and ultimately to the formation of a stable fibrin clot at the site of vessel interruption. a.P a.Primar rimar rimaryy he hemo mo mosta sta stassis by adhesion, secretion and aggregation with the end view of hemostatic plug. (Platelet plug formation) b.Bl b.Bloo oo ood d ccoag oag oagulati ulati ulation on by releasing platelet factor 3 that plays a big role in forming fibrin clot. c. c.Clot Clot retr retracti acti action on by its actomysin d. d.Hel Hel Helps ps in loc localiz aliz alizatio atio ation n of bact bacteri eri eriaa aand nd ot other her small ob object ject jectss and producing aggregates too large to pass through capillaries.

Magtalas ‖ Hematology 2 Co Comp mp mpos os ositi iti ition on of a TTe emp mpora ora orary ry Pl Platel atel atelet et Pl Plug: ug:  RBCs  WBCs  Platelet  Trapped bacteria/microbes ●●●

HEM HEMOS OS OSTTASI ASISS - process wherein upon tissue injury the body responds to promote stoppage of blood - a complex process wherein there will be an interaction of blood vessels, cellular components (red cells, platelet, wbc), coagulation factors and fibrinolytic factors that are being secreted by the liver, fibrinolysis and tissue repair. Why the body has to perform hemostasis: 1. To produce clot formation to stop the bleeding 2. To confine the clot that has been formed by the platelets & different coag factors 3. If the wound is already stable within 48 hours the body should respond in order to lyse the clot that has been formed as the wound heals. Components of hemostasis: 1.. Extr Extrav av ava ascul scular ar tis tisssue fact factor or or// TF facto ctorr - It is the tissue surrounding the vessel. The subendothelium and the endothelial lining that is being exposed upon vessel injury. 2.. Vas ascula cula cularr inti intima ma - The blood vessel through which the different cell components (platelets) would flow 3. Intr Intrav av avas as ascula cula cularr co com mpon onent ent - made up of different plasma proteins (coagulation factors I-XII including pr pre e kallikr allikrein ein & high molecular weight kininogen, inhibitors of fibrinolysis, factors for fibrinolysis, VWF, platelets) Co Conce nce ncept pt of normal blo blood od ccoa oa oagul gul gulation ation The body has its vessels through which the blood flow and it should be maintained in balance because upon excessive production of coagulation factors, the patient is susceptible in having thrombotic episode and upon excessive production of fibrinolytic factor, there will be increased lysis of plasma protein on the clot formed which will lead to bleeding tendencies Hypocoagulation Increased prod of fibrinolytic factor – these are the plasma proteins that will lyse the clot that has been formed inside the body which will make the body susceptible to bleeding tendencies. There is poor clot formation and excessive fibrinolysis. Hypercoagulation There will be uncontrolled thrombi formation – if your body’s platelet is capable of adhesion and aggregation, this will further be reinforced by the different coagulation factors (prothrombinase complex), if there is continuous activity of coagulation factors, the body will be susceptible in developing thrombotic episodes and hypercoagulable disease.

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Rodak’s Hematology: Clinical Principles and Applications Clinical Hematology: Principles, Procedures, Correlations by Steininger

PRIMARY HEMOSTASIS Primary hemostasis is based on: 1. Subendothelium/ endothelial lining 2. Platelets 2 most important phases in primary hemostasis: 1. platelet adhesion phase 2. platelet aggregation phase Sequence of events in primary hemostasis: EV E NT EVE 1

COMM ENT OMMENT

Controlled by vessel muscle Happens in exposed smooth muscles and enhanced by chemicals secreted by platelets as well as signals secreted by the brain Adheres to exposed subendothelial 2 Platelet Adhesion connective tissue 3 Platelet Aggregation Interaction of platelets to one another to create a temporary platelet plug at the site of injury. 4 Fibrin Platelet Plug Coagulation factors interact on Formation platelet surface to produce fibrin. Fibrin clot must be stabilized by 5 Fibri Fibrin n St Stabili abili abilizati zati zation on coagulation factor XIII *Steps 4-5 are included in secondary hemostasis

Magtalas ‖ Hematology 2 Process of cell adhesion: 1. Upon injury, there is an exposed subendothelial tissue, causing bleeding. 2. The brain secretes serotonin in order to give signal to the subendothelial tissue to expose collagen 4&5 and 1&3 at the same time. 3. Once it’s already exposed, circulating platelet should be able to bind in the site of injury only with the help of vWF which is produced by the liver (together with factor VIII) and alpha granules of active platelets. 4. VWF will bound to GP1B and this will promote adhesion of platelet to the damaged subendothelial tissue. 5. The platelet that adheres to the site of injury will undergo a change in shape – the Pseudopod extrusion – which is needed for the next phase of hemostasis which is the platelet secretion phase.

Vasoconstriction

ST STEP EP 11:: V VASO ASO ASOCO CO CONSTRIC NSTRIC NSTRICTION TION Upon vessel injury your brain is capable of secreting a stimulus which is known as the serotonin (other source: dense granules of platelet). It is capable of causing the very first stage of hemostasis which is vasoconstriction. Vasoconstriction occurs as a neurogenic response and causes the site of injury to swell so platelets could easily adhere to the actual site. It is capable of exposing the different types of collagen that would serve as a ligand for platelet adhesion and aggregation. Collagen promotes thrombus formation by causing the adherence of platelets to the area of injury. 2 kinds of collagen that are exposed upon vasoconstriction: 1. for platelet adhesion phase – collagen types 4 & 5 2. for aggregation – collagen 1 & 3 * upon adhesion and aggregation, there will now be a temporary platelet plug. The body’s response would be reinforcement to produce a stable fibrin clot and then there will be secondary hemostasis activity. * vWF – the most important ligand

ST STEP EP 22:: PLA PLATE TE TELET LET AD ADHE HE HESIO SIO SION N Factors that should be present in for platelet adhesion: 1. VWF 2. Glycoprotein1B –always exposed in the plasma membrane

PLA PLATTELET SE SECRE CRE CRETIO TIO TION N/R RELEA ELEA ELEASE SE M ME ECH CHANIS ANIS ANISM M Platelet will extend its pseudopod so that the open canalicular system will be open resulting the granules to localize at the center for it to readily be secreted by the platelets. Contents of the granules that are released outside: 1. Inhibitors of megakaryopoiesis (factor 4 and β-thromboglobulin) 2. ADP – for aggregation and energy 3. Serotonin – for vasoconstriction 4. VWF – ligand for adhesion 5. Platelet derived growth factor – will support megakaryopoiesis Causes of Release mechanism: 1. Exposed collagen 1 & 3 2. Secretion of thrombin – an activated factor II 3. Thromboxane A2 – for platelet activation and vasoconstriction 4. Secretion of epinephrine – for aggregation

ST STEP EP 33:: PLA PLATE TE TELET LET A AGGR GGR GGRE EGA GATIO TIO TION N It is the recruitment of additional platelets, which interact with each other to form clumps. Requires fibrinogen and calcium; Involves binding of fibrinogen to specific platelet receptor complex GpIIb-IIIa Platelet stimulating agents (collagen, epinephrine , thrombin, ADP) binds to platelets, causing them to adhere to one another. Platelet to platelet interaction is necessary to produce a temporary platelet plug. 3 IImp mp mporta orta ortant nt Me Mediat diat diators ors of Pl Platel atel atelet et Ag Aggr gr greg eg egati ati ation: on: ADP –causes secondary, irreversible platelet aggregation by inducing a change in the conformation of the IIb-IIIacomplex allowing the binding of fibrinogen Thr Throm om ombin bin –stimulates the platelets to release ADP and activates the platelet membrane phospholipases TXA2 TXA2–promotes platelet aggregation directly or in synergy with ADP; acts as a potent vasoconstrictor

Summary of Primary Hemostasis  Initiated by the exposure of platelets to the subendothelium connective tissue components of blood vessels (Collagen, microfilaments, basement membrane)  If acute injury occurs, the small vessels constrict and platelets immediately adhere to the exposed surfaces and release ADP and ATP.  Thromboxane A2 is also released to promote further vasoconstriction. 5

Rodak’s Hematology: Clinical Principles and Applications Clinical Hematology: Principles, Procedures, Correlations by Steininger

 A reversible primary platelet aggregation takes place during which platelet adhere to one another.  Platelets also change in shape and then organelles become centralized. At this point, platelets may disaggregate in the absence of further stimulation.  Important substances released during platelet aggregation includes:  ADP – promotes secondary platelet aggregation and recruits additional platelets to the site of injury.  ATP  Serotonin – promotes further vasoconstriction.

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Lab Labor or orat at atory ory EEvval alua ua uattio ion n of Pri Primary mary Hem Hemost ost ostasi asi asiss

Magtalas ‖ Hematology 2 C. C Copl opl oplyy LLalit alit alitch ch Met Method hod Procedure involves: (1)Puncture of finger to a depth of 6mm (2)Immersion of punctured finger in sterile physiologic saline solution warmed at 37degC Normal Values: >3minutes D. Adels delson-Cr on-Cr on-Crosb osb osbyy M Me ethod – same with Copley-lalitch E. Macf metho Macfarla arla arlane’ ne’ ne’ss metho hod d - same principle with Adelson-Crosby method but it only uses ear lobe as the site of puncture. F. Aspiri spirin n tole olera ra rance nce Test est- assesses the effect of a standard dose of aspirin on the Duke’s Bleeding Time. BLEEDING TIME IS PROLONGED IN THE FF. CONDITIONS: 1.When the blood platelets are greatly reduced. a. Thrombocytopenic purpura- dec. In platelets b. Acute Leukemia c. Aplastic anemia 2. Injury of capillary wall a. Scurvy b. Toxins (infection, chemical, snake venom) c. Allergy 3. PT deficiency a. Destructive dise...