Laboratory Protocols PDF

Laboratory Protocols Blood Sampling Anticoagulants: ○ EDTA: Used for hematology; binds calcium to prevent coagulation. Excess storage can cause swelling of blood cells. ○ Heparin: Preferred for plasma biochemistry; used in synthetic or lyophilized forms to...

Laboratory Protocols Blood Sampling Anticoagulants: ○ EDTA: Used for hematology; binds calcium to prevent coagulation. Excess storage can cause swelling of blood cells. ○ Heparin: Preferred for plasma biochemistry; used in synthetic or lyophilized forms to measure electrolytes. ○ Na2-citrate: Common for coagulation studies due to reversible calcium binding. Key Notes: Correct filling and gentle mixing of tubes are crucial to ensure sample integrity. Packed Cell Volume (PCV) Definition: Ratio of blood volume to red blood cell volume, expressed as l/l or %. Methods: ○ Microhematocrit Method: Centrifuge blood-filled capillaries and read results. ○ Automated Counter: Calculates PCV using mean corpuscular volume (MCV) and RBC count. ○ Handheld Meters: Quick PCV and hemoglobin measurements using species-specific chips. Clinical Interpretation: ○ Decreased PCV: Indicates anemia, overhydration, or bone marrow suppression. ○ Increased PCV: Can signify dehydration, chronic stress, or erythropoietin-related polycythemia. Physiological Principles Isovolaemia (Fluid Volume) Water Compartments: ○ Total water content ≈ 600-650 ml/kg body weight, distributed across extracellular, intracellular, and transcellular spaces. Volume Disturbances: ○ Hypovolaemia: Caused by blood loss or inadequate circulation (e.g., heart failure).  ○ Hypervolaemia: Results from excessive fluid intake or retention (e.g., renal issues). Clinical Signs: Skin turgor, mucous membrane condition, sunken eyes, capillary refill time (CRT), and blood pressure are assessed. Laboratory Indicators: Changes in PCV, hemoglobin (Hb), plasma proteins, and osmolality. Isoionia (Electrolyte Balance) Serum Osmolality: ○ Reflects osmotic pressure of fluids. Typical range: 270-310 mOsm/kg. ○ Hyperosmolality: Linked to dehydration, hyperglycemia, or renal failure. ○ Hypoosmolality: Indicates fluid overload or electrolyte loss. Electrolytes: ○ Sodium (Na+): Main extracellular ion; affects osmolality and water movement. Hypernatremia: Caused by dehydration, salt poisoning, or hormonal disorders. Hyponatremia: Results from fluid overload, sodium loss, or adrenal insufficiency. ○ Potassium (K+): Critical for nerve and muscle function. Hypokalemia: Occurs with anorexia, diuretics, or alkalosis. Hyperkalemia: Associated with kidney failure, acidosis, or excessive intake. ○ Chloride (Cl-): Balances sodium and bicarbonate levels. Hyperchloremia: Often linked to salt poisoning or infusion overdose. Hypochloremia: Caused by vomiting, diarrhea, or sweating. ○ Calcium (Ca2+): Regulates muscle contractions and blood clotting. Hypocalcemia: Common in lactation, vitamin D deficiency, or alkalosis. Hypercalcemia: Seen in neoplastic diseases, hyperparathyroidism, or excess vitamin D. ○ Magnesium (Mg2+): Influences ATP metabolism and neuromuscular activity. Hypomagnesemia: Caused by poor intake or absorption. Hypermagnesemia: Linked to kidney dysfunction or infusion overdose. Clinical and Diagnostic Tools Dehydration Evaluation Clinical signs (e.g., skin elasticity, mucous membrane dryness) and laboratory parameters (e.g., urine output, osmolality) guide diagnosis. Severity Levels: ○ Mild: 10%, shock symptoms, critical care required. Volume and Osmotic Changes Isotonic Dehydration: Loss of both water and solutes (e.g., vomiting, diarrhea). Hypertonic Dehydration: Water loss exceeds solute loss (e.g., panting, fever). Hypotonic Dehydration: Solute loss exceeds water loss (e.g., prolonged diarrhea). Acid-Base Disorders and Blood Gas Analysis Fundamentals of Acid-Base Balance Isohydria: Maintenance of a tightly controlled hydrogen ion concentration ([H+]), with a pH range of 7.35–7.45 being critical for cellular functions. Buffering Systems: Resist pH changes using chemical buffers: ○ Carbonic acid-bicarbonate system (dominant buffer system). ○ Phosphate buffers. ○ Protein-proteinate buffers (albumin, hemoglobin, etc.). Vital Role of Lungs and Kidneys: ○ Lungs: Rapid pH adjustments by regulating CO2 levels. ○ Kidneys: Slower adjustments via H+ excretion and HCO3- reabsorption. Acid-Base Analysis Parameters pH: Indicates acidemia (7.45). pCO2 (Respiratory parameter): Measures CO2 levels, normal range 35–45 mmHg. HCO3- (Metabolic parameter): Normal range 21–24 mmol/L. Actual Base Excess (ABE): Indicates metabolic disturbances, normal ±3.5 mmol/L. Anion Gap: Differentiates metabolic acidosis causes, normal 8–16 mmol/L. Types of Acid-Base Disturbances  1. Metabolic Acidosis: ○ Causes: HCO3- loss (e.g., diarrhea), increased acid production (lactic acidosis, ketoacidosis), or reduced acid excretion (renal failure). ○ Effects: Hyperkalemia, Kussmaul breathing, and cardiac arrhythmias. ○ Treatment: Ventilation support and bicarbonate infusion if pH < 7.2. 2. Metabolic Alkalosis: ○ Causes: Excess bicarbonates, vomiting, or hypokalemia. ○ Effects: Hypoventilation, muscle weakness, and arrhythmias. ○ Treatment: Correcting electrolyte imbalances. 3. Respiratory Acidosis: ○ Causes: Hypoventilation from airway obstruction, pleural diseases, or neuromuscular issues. ○ Effects: Dyspnea, cyanosis, and hypoxemia. ○ Treatment: Assisted ventilation, oxygen therapy, and treating underlying conditions. 4. Respiratory Alkalosis: ○ Causes: Hyperventilation from anxiety, fever, or compensation for metabolic acidosis. ○ Effects: Hyperoxia and increased renal bicarbonate excretion. ○ Treatment: Breathing techniques to retain CO2 or mild sedatives. Blood Gas Analysis Purpose: Assess gas exchange effectiveness and ventilation. Key Parameters: ○ pO2: Reflects oxygenation, arterial range 88–118 mmHg. ○ SaO2: Oxygen saturation, arterial 90–100%. ○ FiO2: Fraction of inspired oxygen, varies with O2 supplementation. Interpretation: Evaluates normoventilation, hypoventilation (pCO2 > 45 mmHg), or hyperventilation (pCO2 < 35 mmHg). Diagnostic Steps 1. Determine acidosis or alkalosis based on pH. 2. Identify the primary cause (metabolic or respiratory) using pCO2, HCO3-, and ABE. 3. Evaluate compensation efforts (e.g., Kussmaul breathing in metabolic acidosis). 4. Consider specific causes (e.g., lactic acidosis, kidney failure). Introduction to Haemostasis Haemostasis is the physiological process that stops bleeding upon tissue or blood vessel injury. It involves: 1. Vasoconstriction: Immediate narrowing of blood vessels to reduce blood flow. 2. Platelet Plug Formation: Platelets aggregate and adhere to the injury site, forming a temporary plug. 3. Coagulation Cascade: Sequential activation of clotting factors leading to fibrin mesh formation, stabilizing the platelet plug. Major Haemostasis Disorders 1. Vasculopathy: Impaired vasoconstriction due to blood vessel dysfunction. 2. Thrombocytopathy: Defective platelet function despite adequate platelet counts. 3. Thrombocytopenia: Reduced platelet count, affecting clot formation. 4. Coagulopathy: Abnormalities in the coagulation cascade involving intrinsic, extrinsic, or common pathways. Diagnostic Tests for Haemostasis General Tests: Bleeding Time (BT/BMBT): Assesses platelet and capillary function. Normal: 3-5 minutes. Capillary Resistance Test: Indicates capillary fragility, useful in vasculitis. Coagulation Time (CT): Detects coagulopathies using fresh whole blood. ○ Normal clotting times vary by method: Glass Tube: 4-5 minutes. Plastic Syringe: 10-12 minutes. Watch Glass: 7-15 minutes. Specific Coagulation Tests: 1. Prothrombin Time (PT): ○ Measures extrinsic pathway functionality. ○ Normal: 10-15 seconds. 2. Activated Partial Thromboplastin Time (APTT): ○ Evaluates the intrinsic pathway. ○ Normal: 20-30 seconds. 3. Thrombin Time (TT): ○ Assesses fibrinogen levels and conversion to fibrin. Advanced Tests: Fibrin Degradation Products (FDPs) and D-Dimer: Indicate fibrinolysis activity. Elevated levels are markers of disseminated intravascular coagulation (DIC). Platelet Aggregation Test: Measures aggregation ability using stimulants like ADP or epinephrine. Thrombocyte (Platelet) Disorders Thrombocytopenia: Low platelet counts (