Comprehensive Hematology and Hemostasis Practice Flashcards

General Characteristics and Composition of Blood

Total blood volume in the average adult ranges from $5-6\,\text{liters}$, which accounts for approximately $7-8\,\%$ of total body weight. Blood volume states are categorized as follows:

• Hypovolemia: A state of decreased blood volume, often resulting from blood loss, loss of plasma, or dehydration.

• Hypervolemia: A state of increased blood volume, which may occur during blood transfusion.

Blood is composed of two primary components:

1. Liquid Portion ($55\,\%$): Consists of plasma or serum. This portion is comprised of $90\,\%$ water and $10\,\%$ proteins, carbohydrates, salts, hormones, and other substances.

2. Formed Elements ($45\,\%$): Consists of Red Blood Cells (RBCs) and the Buffy Coat (White Blood Cells and Platelets).

Functional roles of the formed elements include:

• Red Blood Cells: Responsible for transporting oxygen to tissues and removing carbon dioxide.

• White Blood Cells: Responsible for fighting infection.

• Platelets and Clotting Factors: Responsible for plugging vessel breaks and promoting wound healing.

Specimen Collection and Anticoagulants

Specimen collection involves several tools and reagents to ensure sample integrity:

• Tourniquet: Used to provide a barrier against venous blood flow to help locate a vein. It should be applied $3-4\,\text{inches}$ above the venipuncture site and left for no longer than $1\,\text{minute}$.

• Antisepsis: The most commonly used agent is $70\,\%$ isopropyl alcohol, applied in a circular motion.

• Needles: The gauge number is inversely related to the bore size. The most common needle size is $21\text{-gauge}$, with a length of $1\,\text{inch}$.

• Clot Activators: Specimens for serum testing must clot for $30-60\,\text{minutes}$ prior to centrifugation. These accelerate clotting using glass/silica particles or thrombin.

• Separation Gel: An inert material that undergoes a temporary change in viscosity during centrifugation to serve as a separation barrier between cells and the liquid portion (serum or plasma).

• Antiglycolytic Agent: Inhibits the metabolism of glucose by blood cells (e.g., sodium fluoride).

Comparison of Plasma and Serum

Anticoagulants and Their Properties

Ethylenediamine Tetra Acetic Acid (EDTA)

• Mode of Action: Removes ionized calcium ($Ca^{2+}$) through an irreversible process called chelation, forming an insoluble calcium salt.

• Concentration: Used at $1.5\,mg/1\,mL$ of whole blood.

• Forms: $K_3\text{EDTA}$ (Tri-potassium salt, liquid, also known as tripotassium) and $Na_2\text{EDTA}$ (Disodium salt, also known as disodium). $K_2\text{EDTA}$ (Spray-dried) is recommended by the ICSH and CLSI for blood cell counting and sizing as it causes less RBC shrinkage.

• Excessive Concentration Effects: Leads to RBC shrinkage, decreased spun hematocrit, increased MCHC, and falsely low ESR. WBCs undergo degenerative changes and platelets swell, leading to false increases in automated counts.

• Platelet Satellitosis: An EDTA-induced phenomenon where platelets adhere around neutrophils, causing a false decrease in automated platelet counts. The remedy is to use sodium citrate and multiply the result by $1.1$.

Sodium Citrate

• Mode of Action: Binds calcium to form a soluble complex.

• Light Blue Top Tubes: Used for coagulation studies with a concentration of $3.2\,\%$ ($0.109\,M$) and a ratio of $1:9$ (anticoagulant to blood). Underfilling/short draws cause prolonged PT and PTT results.

• High Hematocrit Adjustment: For patients with $Hct \ge 55\,\%$, the citrate volume must be adjusted using the formula: $C = (1.85 \times 10^{-3})(100 - Hct) \times \text{volume of whole blood in mL}$.

• Black Top Tubes (Westergren): Used at a concentration of $3.8\,\%$ ($0.129\,M$) with a blood-to-anticoagulant ratio of $4:1$; not recommended for coagulation studies.

Heparin

• Type: A natural acid mucopolysaccharide.

• Mode of Action: Inactivates thrombin. It is used in vitro and in vivo (e.g., during PTCA or CPB).

• Laboratory Tests: Preferred for Osmotic Fragility Tests and LAP tests.

• Limitations: Not for blood films as it destroys WBCs and platelets and creates a bluish background on Romanowsky stains.

Hematopoiesis: Evolution and Stages

Hematopoiesis is the continuous, regulated process of renewal, proliferation, differentiation, and maturation of all blood cell lines. It results in the formation and development of functional blood cells released from the bone marrow into circulation.

Stem Cell Hierarchy and Differentiation Potential

Key Terms:

• Hematopoietic Stem Cells (HSCs): Capable of self-renewal and directed differentiation.

• Progenitor: Undifferentiated hematopoietic cell committed to a cell line but not morphologically identifiable (e.g., CMP, CLP, BFU-E).

• Precursor: Differentiating cell stage that is morphologically identifiable (e.g., Pronormoblasts).

Stages of Hematopoiesis

1. Mesoblastic Stage: Begins as early as the $19^{\text{th}}\,\text{day}$ of gestation in the yolk sac blood islands. Activity is confined to erythropoiesis. RBCs produced contain embryonic hemoglobins:

   • Gower 1: $2 \zeta$ (zeta), $2 \epsilon$ (epsilon).

   • Gower 2: $2 \alpha$ (alpha), $2 \epsilon$ (epsilon).

   • Portland: $2 \zeta$ (zeta), $2 \gamma$ (gamma).

2. Hepatic Stage: Begins at the $3^{\text{rd}}\,\text{month}$. The fetal liver becomes the primary site. The spleen, kidney, thymus, and lymph nodes contribute. The thymus is the first fully developed organ and site of T cell production. Megakaryocyte production begins. $Hb F$ ($2 \alpha$; $2 \gamma$) is predominant.

3. Myeloid (Medullary) Stage: Occurs between the $5^{\text{th}}\,\text{to } 6^{\text{th}}\,\text{month}$ of gestation. The bone marrow becomes the primary site of hematopoiesis and remains so after birth. Principal adult sites are flat bones (sternum, ribs, vertebrae, skull, pelvis) and the proximal long bones.

Bone Marrow Physiology and Extramedullary Hematopoiesis

The bone marrow is one of the largest organs, located within cortical bone cavities. It has two components:

• Red Marrow: Hematopoietically active marrow consisting of developing blood cells and progenitors.

• Yellow Marrow: Hematopoietically inactive marrow composed primarily of adipocytes. It can revert to red marrow during increased demand.

M:E Ratio: This is the ratio of myeloid (granulocytic) to erythroid (normoblastic) cells. In healthy adults, it varies from $1.5:1$ to $3.3:1$ (often simplified as $2:1, 3:1, 4:1$). High ratios (e.g., $6:1$) suggest infection, and extreme ratios (e.g., $25:1$) suggest leukemia. A low ratio (e.g., $1:20$) indicates erythroid hyperplasia.

Bone Marrow Resident Cells:

• Osteoblasts: Bone-forming cells with a "water-bug" or "comet" appearance; often confused with plasma cells.

• Osteoclasts: Bone-resorbing cells; often mistaken for megakaryocytes.

• Stromal Cells: Regulate stem cell survival and differentiation.

Extramedullary Hematopoiesis (EMH): The formation and activation of blood cells outside the bone marrow (primary sites: liver and spleen). It occurs as a compensatory mechanism (secondary hematopoiesis) when the marrow is dysfunctional (e.g., aplastic anemia, leukemia) or cannot meet demands (hemolytic anemia). It results in hepatomegaly and/or splenomegaly.

Erythropoiesis and Red Blood Cell Development

Erythropoiesis is the process by which erythroid precursor cells differentiate into mature RBCs. The process from Pronormoblast to reticulocyte takes $3-5\,\text{days}$.

Erythropoietin (EPO): The primary regulator of erythropoiesis, produced by peritubular cells of the kidney. It acts as a growth factor/hormone via signal transduction. Effects include:

1. Allowing early release of reticulocytes (shift reticulocytes).

2. Preventing apoptotic death of RBC precursors.

3. Reducing transit time in the bone marrow.

• Reference Interval: $10-30\,U/L$.

Erythrocyte Precursor Stages

Reticulocyte Details:

• Supravital Stains: New Methylene Blue (NMB) or Brilliant Cresyl Blue (BCB).

• Visual Recognition: On Wright-stained smears, reticulocytes appear as polychromatophilic macrocytes.

• Stress Reticulocytes: Prematurely released larger cells that compensate for anemia; they take $2-3\,\text{days}$ to lose their reticula.

Red Blood Cell Membrane and Metabolism

The RBC membrane maintains cell deformability, osmotic balance, and structural support for antigens. It is composed of protein ($52\,\%$), lipids ($40\,\%$), and carbohydrates ($8\,\%$). Proteins include integral (transmembrane) and peripheral (e.g., spectrin, actin).

Metabolic Pathways

1. Embden-Meyerhof Pathway (EMP): Handles $90\,\%$ of glucose utilization anaerobically. Produces $2\,\text{mol of ATP}$ for active cation transport and membrane shape preservation. The most common enzyme deficiency is Pyruvate Kinase (PK).

2. Hexose Monophosphate Shunt (HMP): Aerobically converts glucose to pentose and produces NADPH. NADPH reduces glutathione, which protects cells from oxidative damage. Functionally dependent on G6PD (the most common RBC enzyme defect affecting $\approx 400\,\text{million}$ people).

3. Methemoglobin Reductase Pathway: Maintains heme iron in the functional ferrous ($Fe^{2+}$) state using NADH and methemoglobin reductase. Deficiency leads to methemoglobinemia (accumulation of oxidized $Fe^{3+}$ iron).

4. Rapoport-Luebering Pathway: Produces $2,3-BPG$, which regulates hemoglobin affinity for oxygen.

Oxygen Dissociation Curve (ODC)

The ODC shows the relationship between oxygen saturation and partial pressure of oxygen ($PO_2$). The $P_{50}$ value is the $PO_2$ at which Hgb is $50\,\%$ saturated ($26.52\,mmHg$ standard).

• Shift to the Right (Decreased affinity/Increased release): Caused by decreased $\text{pH}$, increased $PCO_2$, increased $2,3-BPG$, and increased temperature. Seen in anemia, high altitude, and hypoxia.

• Shift to the Left (Increased affinity/Decreased release): Caused by increased $\text{pH}$, decreased $PCO_2$, decreased $2,3-BPG$, decreased temperature, CO poisoning, and fetal hemoglobin.

Red Blood Cell Destruction

As RBCs age (senescence), enzyme activity and ATP decrease, cell size decreases, and membrane flexibility is lost. Approximately $1\,\%$ of cells are destroyed daily.

1. Extravascular Hemolysis ($90\,\%$): Destruction occurs outside blood vessels via splenic macrophages. RBCs are trapped in the Cords of Billroth.

2. Intravascular Hemolysis ($10\,\%$): Destruction occurs within blood vessels, releasing free hemoglobin. Proteins like Haptoglobin (binds Hgb), Hemopexin (binds heme), and Albumin clear the debris. Laboratory findings include decreased haptoglobin/hemopexin and increased free hemoglobin, hemoglobinuria, and hemosiderinuria.

Hemoglobin Structure and Biosynthesis

Hemoglobin (MW $\approx 64,000\,\text{Daltons}$) transports oxygen ($O_2$) and carbon dioxide ($CO_2$). Concentration is highest in the morning and increases with altitude or smoking.

• Heme Structure: Protoporphyrin IX ring with a central divalent ferrous iron ($Fe^{2+}$). Each heme binds one $O_2$ molecule. Oxidation to ferric state ($Fe^{3+}$) prevents oxygen binding.

• Globin Structure: Two pairs of unlike polypeptide chains (e.g., $Hb A_1 = \alpha_2 \beta_2$). Alpha chains have $141\,\text{amino acids}$; Beta, Delta, and Gamma chains have $146\,\text{amino acids}$.

Biosynthesis:

• Heme: Occurs in the mitochondria and cytoplasm of erythroid precursors. Heme biosynthesis begins with glycine and succinyl CoA forming ALA (catalyzed by ALA synthase). The final step involves ferrochelatase inserting $Fe^{2+}$ into Protoporphyrin IX.

• Globin: Occurs on ribosomes in the cytoplasm. Chromosome $16$ dictates alpha/zeta chains; Chromosome $11$ dictates beta/epsilon/delta/gamma chains.

Hemoglobin Types and Reference Ranges

Dyshemoglobins:

• Carboxyhemoglobin (HbCO): Hgb bound to CO ($200-240 \times$ higher affinity than $O_2$). Imparts a brilliant cherry-red color. Reversible with high-flow oxygen.

• Methemoglobin (Hi): Hgb with ferric iron ($Fe^{3+}$). Imparts a chocolate-brown color. Reversible with intravenous methylene blue.

• Sulfhemoglobin (SHb): Hgb with sulfur. Imparts a lavender/green/mauve color. Irreversible; seen with sulfonamide use or $\textit{Clostridium perfringens}$ bacteremia.

Iron Metabolism and Regulation

Iron is the most abundant transition metal in the body. Total body iron is regulated via absorption in the duodenum.

• Functional Compartment: Hgb ($70\,\%$), myoglobin, cytochromes.

• Storage Compartment: Ferritin (water-soluble, major form) and Hemosiderin (water-insoluble, breakdown product of ferritin, visualized by Prussian Blue).

• Transport Compartment: Transferrin ($Fe^{3+}$).

Key Regulatory Molecules:

• Hepcidin: Master regulator peptide hormone from the liver. It binds and degrades Ferroportin (the only iron exporter) to block iron release from enterocytes and macrophages. High levels cause anemia of chronic disease; low levels cause hemochromatosis.

• DMT1: Transports $Fe^{2+}$ into enterocytes.

• Hephaestin: Oxidizes $Fe^{2+} \rightarrow Fe^{3+}$ for transferrin binding.

• Reference Data: One gram of hemoglobin carries $1.34\,mL$ of oxygen and $3.47\,mg$ of iron.

Hematology Laboratory Methods: Manual Counts

Hemacytometer (Levy chamber with Improved Neubauer ruling):

• Area/Volume: Grid is $3 \times 3\,mm$ ($9\,mm^2$). Coverslip depth is $0.1\,mm$. Total volume per side is $0.9\,mm^3$.

• WBC Areas: Four corner squares ($1\,mm^2$ each). Dilution is $1:20$ typically using $1\,\%$ ammonium oxalate or $3\,\%$ acetic acid.

• RBC Areas: Five small squares in the center ($0.2\,mm^2$ total). Dilution is $1:200$ using Isotonic Saline or Dacies fluid.

• Platelet Area: The entire center square ($1\,mm^2$). Dilution is $1:100$ using $1\,\%$ ammonium oxalate. Counted using a phase-contrast microscope.

Calculations:

• SI Unit Conversion: Multiply cells/$\mu L$ by $10^6$ to obtain cells/$L$.

• Corrected WBC Count: Necessary when $\ge 5\,\text{NRBCs/100 WBCs}$ are seen. Formula: $\frac{\text{Uncorrected WBC count} \times 100}{100 + \text{NRBCs per 100 WBCs}}$.

Hematocrit (Packed Cell Volume)

• Microhematocrit Method: Tubes are $75\,mm$ long, inner bore $1.2\,mm$. Centrifuged at $10,000\,RPM$ for $5\,\text{minutes}$. Red band = Heparinized (for skin puncture); Blue band = No anticoagulant (for anticoagulated blood).

• Rule of Three: $3 \times RBC = Hgb$; $3 \times Hgb = Hct \pm 3\,\%$. Applies only to normocytic, normochromic specimens.

• Trapped Plasma: Falsely increases Hct results, notably in sickle cell anemia or spherocytosis.

Erythrocyte Indices and Automated Analysis

1. Mean Cell Volume (MCV): $MCV = \frac{Hct (\%)}{RBC (\times 10^{12}/L)} \times 10$. Range: $80-100\,fL$.

2. Mean Cell Hemoglobin (MCH): $MCH = \frac{Hgb (g/dL)}{RBC (\times 10^{12}/L)} \times 10$. Range: $27-31\,pg$.

3. Mean Cell Hemoglobin Concentration (MCHC): $MCHC = \frac{Hgb (g/dL)}{Hct (\%)} \times 100$. Range: $32-36\,g/dL$. Results >36\,g/dL suggest technical error or spherocytosis.

4. RBC Distribution Width (RDW): Coefficient of variation of RBC volume. Range: $11.5-14.5\,\%$. Measures anisocytosis.

Erythrocyte Sedimentation Rate (ESR):

• Principle: Measures the distance in $mm$ RBCs fall in $1\,\text{hour}$. Inflammatory proteins (fibrinogen, globulins) neutralize the zeta potential, allowing Rouleaux formation.

• Westergren Method: $200\,mm$ tube length. Modified version uses EDTA blood diluted $4:1$ with saline or sodium citrate. Reference: Male $0-10\,mm/hr$, Female $0-15\,mm/hr$.

• Error Sources: Tilting by even $1-3^{\circ}$ causes a $30\,\%$ error. Bubbles or clots invalidate the result.

The Peripheral Blood Smear (PBS)

Preparation: Wedge smear uses a $30-45^{\circ}$ angle (decreased for high Hct, increased for low Hct). The drop size is $\approx 0.05\,mL$. A good smear is $2/3$ to $3/4$ the length of the slide and finger-shaped with a "rainbow" feather edge.

Staining (Romanowsky Stain): Wright’s or Giemsa stains. Components include:

• Fixative: Methanol.

• Acid Dye: Eosin (stains basic components like Hgb pink/red).

• Basic Dye: Methylene Blue/Azure B (stains acidic components like RNA blue).

• Buffer: pH $6.4-6.8$.

Microscopy:

• $10 \times$ (LPO): Assess film quality and distribution.

• $40 \times$ (HPO): WBC estimate ($\text{Average WBC/field} \times 2000$).

• $100 \times$ (OIO): Differential count and platelet estimate ($\text{Average Platelets/field} \times 20,000$).

Red Blood Cell Abnormalities

Anisocytosis (Size Selection)

• Microcytes: <6\,\mu m (MCV < 80\,fL), often due to defective hemoglobin synthesis.

• Macrocytes: >8\,\mu m (MCV > 100\,fL), often due to impaired DNA synthesis.

Poikilocytosis (Shape Selection)

• Acanthocyte (Spur Cell): Spiked spicules; seen in Abetalipoproteinemia and liver disease.

• Echinocyte (Burr Cell): Evenly spaced blunt projections; seen in Uremia or PK deficiency.

• Target Cell (Codocyte): Bell-shaped; seen in Thalassemia and liver disease.

• Spherocyte: Sphere-shaped, no central pallor; seen in Hereditary Spherocytosis and ABO HDN. High MCHC and increased Osmotic Fragility (OFT).

• Schistocyte: RBC fragments; hallmark of Microangiopathic Hemolytic Anemias (MAHA) like DIC, TTP, and HUS.

• Dacryocyte (Teardrop): Squeezed cell; seen in Myelofibrosis.

• Sickle Cell (Drepanocyte): Crescent-shaped; hallmark of HbSS.

Red Cell Inclusions

• Howell-Jolly Bodies: Dark blue DNA remnants; seen post-splenectomy.

• Basophilic Stippling: Precipitated RNA granules; seen in Lead Poisoning.

• Pappenheimer Bodies: Siderotic iron granules; seen in Sideroblastic Anemia.

• Heinz Bodies: Denatured Hgb; visible ONLY by supravital stain; seen in G6PD deficiency.

• Cabot Rings: Remnants of mitotic spindles.

• $Hb C$ Crystals: Hexagonal "bars of gold."

• $Hb SC$ Crystals: Finger-like "Washington Monument" projections.

Anemias

Anemia is a manifestation of underlying disease characterized by decreased RBC, Hgb, and Hct resulting in low oxygen delivery.

Classifications

• Morphological: Determined by Wintrobe indices (MCV/MCHC).

  • Microcytic/Hypochromic: IDA, Thalassemia, Chronic Inflammation (ACD).

  • Macrocytic/Normochromic: Megaloblastic (B12/Folate deficiency) and Non-megaloblastic.

  • Normocytic/Normochromic: Aplastic anemia, acute blood loss, most hemolytic anemias.

• Pathophysiological: Decreased production (Bone marrow failure, DNA/Hgb synthesis defects) vs. Increased destruction (Intrinsic vs Extrinsic hemolytic anemia) vs. Blood loss.

Key Anemic Entities

• Aplastic Anemia: BM failure caused by idiopathic, secondary (benzene, drugs), or inherited (Fanconi Anemia - most common inherited form) factors.

• Paroxysmal Nocturnal Hemoglobinuria (PNH): An acquired stem cell disorder where cells lack complement-protective proteins ($CD55/CD59$). Screening: Sugar water test. Confirmation: Flow cytometry.

• Iron Deficiency Anemia (IDA): Most common worldwide. Stages include storage depletion (low ferritin), transport depletion (low iron/high TIBC), and functional depletion (low Hgb).

• Sickle Cell Anemia (HbSS): Valine replaces glutamic acid at position $6$ of the beta chain. Symptoms include vaso-occlusive crises and autosplenectomy. Screening: Solubility test (Dithionite). Confirmation: Alkaline electrophoresis.

• Thalassemia: Quantitative globin defect. Alpha-Thal (gene deletions) ranges from silent carrier to Hydrops Fetalis ($Hb Bart's = \gamma_4$). Beta-Thal (mutations) includes Minor and Major (Cooley's Anemia), featuring "hair-on-end" skull appearance.

Leukocytes: Physiology and Development

The adult reference interval for WBCs is $4.5-11.5 \times 10^9/L$. Mature neutrophils are the predominant type in adults, whereas lymphocytes predominate in children under $4\,\text{years}$ of age.

Granulopoiesis (Neutrophil Series)

1. Myeloblast: First recognizable stage; reticulated chromatin; $2-5$ nucleoli.

2. Promyelocyte: Features large non-specific primary azurophilic granules (contain MPO). Has a "hof" (paranuclear halo).

3. Myelocyte: Last stage capable of mitosis. First appearance of secondary specific granules (neutrophil-identifying).

4. Metamyelocyte: First stage NOT capable of mitosis. Kidney bean-shaped nucleus.

5. Band (Stab): Sausage/C-shaped nucleus. Youngest cell normally in peripheral blood.

6. Segmented Neutrophil: Fully mature with $2-5$ nuclear lobes connected by thread-like filaments.

Other Leukocyte Series

• Monopoiesis: Monoblasts evolve into Promonocytes, then Monocytes (largest in peripheral blood, "lace-like" chromatin, "ground-glass" cytoplasm). Tissue monocytes are Macrophages (e.g., Kupffer cells in liver, Osteoclasts in bone).

• Lymphopoiesis: Lymphoblasts evolve into Prolymphocytes, then Lymphocytes (resting cells, non-end cells). T cells ($80\,\%$) and B cells ($20\,\%$) are the primary components.

• Plasma Cells: Derived from B cells; show eccentric nucleus with "cartwheel" chromatin and a large well-defined "hof".

Leukocyte Anomalies and Storage Diseases

• Pelger-Hu\u00ebt Anomaly: Failure of nuclear segmentation (pince-nez nuclei); autosomal dominant.

• Pseudo-Pelger-Hu\u00ebt: Acquired condition mimicking Pelger-Hu\u00ebt; associated with AML and MDS.

• May-Hegglin Anomaly: Large gray-blue spindle-shaped inclusions (D\u00f6hle-like) with giant platelets and thrombocytopenia.

• Chediak-Higashi Syndrome: Large lysosomal granules; leads to albinism and recurrent infections.

• Alder-Reilly Anomaly: Coarse azurophilic granules in all leukocytes due to mucopolysaccharidosis.

• Storage Diseases: Gaucher's (glucocerebrosidase deficiency; "wrinkled tissue paper" macrophages) and Niemann-Pick (sphingomyelinase deficiency; "foam cells").

Leukemias and Myeloproliferative Neoplasms

Leukemia is the malignant overproduction of leukocytes classified as Acute (blasts; fast) or Chronic (mature cells; slow).

• AML: Most common in adults. FAB categories $M0-M7$. $M3$ (APL) is associated with DIC and Faggot cells (bundles of Auer rods).

• ALL: Most common in children. Categories $L1, L2, L3$ (Burkitt Lymphoma, "starry-sky" pattern).

• CLL: Most common in elderly; features smudge cells.

• CML: Features the Philadelphia Chromosome [t(9;22), BCR-ABL1 fusion]. Characterized by extremely high WBC counts and a low LAP score ($N = 20-100$).

Myeloproliferative Neoplasms (MPNs):

• Polycythemia Vera (PV): Clonal disorder featuring JAK2 V617F mutation, pancytosis, low EPO, and "ruddy" skin.

• Essential Thrombocythemia (ET): Platelets >1000 \times 10^9/L.

• Primary Myelofibrosis: BM fibrosis, splenomegaly, and Dacryocytes (Teardrops).

Hemostasis: Primary and Secondary

Hemostasis keeps blood fluid and stops bleeding through the interaction of blood vessels, platelets, and coagulation factors.

Primary Hemostasis

Refers to vasoconstriction and platelet plug formation. Platelets ($150-400 \times 10^9/L$) undergo:

1. Adhesion: Platelets bind collagen via VWF and $GP Ib/IX/v$ receptors.

2. Aggregation: Platelets bind each other via Fibrinogen and $GP IIb/IIIa$ receptors.

3. Secretion: Release of granules ($\alpha$ granules = factors I, V, VIII; dense granules = CAPASM [Calcium, ADP, Phosphate, ATP, Serotonin, Magnesium]).

• Aspirin: Impairs function by permanently inactivating cyclooxygenase (THX A2 synthesis).

Secondary Hemostasis (Coagulation Cascade)

1. Intrinsic Pathway: Contact activation (factors XII, XI, IX, VIII). Monitored by APTT ($26-38\,\text{seconds}$).

2. Extrinsic Pathway: Tissue Factor (factor III) and VII. Monitored by PT ($10-12\,\text{seconds}$).

3. Common Pathway: Convergence at factor X; activates Prothrombin ($II$) to Thrombin ($IIa$), which converts Fibrinogen ($I$) to Fibrin. Monitored by PT and APTT.

• Inhibitors: Protein C and S (Vitamin K-dependent) inhibit factors V and VIII. Antithrombin inhibits thrombin and Xa.

Laboratory Tests and Findings

• Mixing Studies: Uses Fresh Normal Plasma (FNP) to distinguish factor deficiencies (corrects with FNP) from inhibitors (does not correct).

• Fibrinolysis: Breakdown of fibrin clots into FDPs and D-dimers (specific for cross-linked fibrin) via Plasmin. DIC is a state of secondary fibrinolysis with excessive clotting and subsequent bleeding.