Clinical Chemistry Laboratory Notes PDF

CLINICAL CHEMISTRY LABORATORY Kim G. Sarong Laboratory Preceptor Activity 1. Phlebotomy  Patient Preparation Pre-analytical Variables – factors contributing to the variation of results 1. Exercise Different effects on analyte con...

CLINICAL CHEMISTRY LABORATORY Kim G. Sarong Laboratory Preceptor Activity 1. Phlebotomy  Patient Preparation Pre-analytical Variables – factors contributing to the variation of results 1. Exercise Different effects on analyte concentrations Transient increased: lactate, fatty acid, ammonia Long-term increased: creatinine phosphokinase (CPK), aspartate aminotransferase (AST), lactate dehydrogenase (LD) and aldolase (skeletal muscle enzymes Vigorous hand exercise (fist clenching): ↑ K+, lactate and phosphate Observed proteinuria in others 3 Activity 2. Phlebotomy  Patient Preparation 2. Fasting NPO: nil per os (Lt.) meaning “nothing by/through mouth” Fasting specimen: FBS, GTT, lipids, lipoproteins, gastrin and insulin 48 hrs fasting: ↑ serum bilirubin 72 hrs fasting: ↑ plasma triglyceride (males), ↓ glucose (healthy females, down to 45 mg/dL) Basal state collection: early morning blood collection, 12 hrs. after last food ingestion; includes glucose, lipids, lipoproteins and electrolytes Basic metabolic panel: glucose, BUN, creatinine, Na+, K+, Cl-, CO2, Ca2+ 4 Activity 2. Phlebotomy  Patient Preparation 3. Diet Increase in metabolic products of certain food in venous blood High protein diet: ↑ urea and uric acid plasma levels Atkins diet (high protein-low carb): greatly ↑ plasma urea and urine ketones Post absorptive hormonal effects: may cause variation in glucose, lipids, catecholamines Caffeine: increases glucose concentration through catecholamine release 5 Activity 2. Phlebotomy  Patient Preparation 4. Posture or position changes preferred for phlebotomy: upright position or supine (lying) Patient should be seated/supine for at least 15 minutes to 20 minutes before phlebotomy: prevent hemodilution or hemoconcentration Change from supine to standing/sitting: constriction of blood vessels, ↓ plasma volume, ↑ albumin, enzymes, and calcium 6 Activity 2. Phlebotomy  Patient Preparation 4. Posture or position changes Change from sitting to supine: hemoconcentration due to shift in water and electrolytes in tissue, ↑ proteins, lipids, BUN, iron, calcium Change from stand to supine: hemodilution due to the transfer of extravascular water to vascular system diluting non-diffusible plasma constituents, ↓ cholesterol, TAG, lipoproteins Significant ↑ of K+ after 30 minutes of standing: due to release of potassium from muscles Prolonged bed rest: ↓ plasma albumin (fluid retention) 7 Activity 2. Phlebotomy  Patient Preparation 5. Tourniquet application Recommended: 1 minute application Effects of prolonged tourniquet application: Hemoconcentration (venous stasis) Anaerobiosis Increased levels: K+, proteins (albumin), enzymes, lactate, cholesterol and ammonia Accurate measurement of lactate: Tourniquet should NOT be applied NO fist clenching during blood draw ↓venous pO2 and pH 8 Activity 2. Phlebotomy  Patient Preparation 6. Tobacco smoking (nicotine) ↑ hormone levels(ex. Catecholamines and cortisol) ↑ glucose, growth hormone, cholesterol, TAG, ammonia, urea, lactate, insulin and urinary 5-HIAA ↓ vitamin B12 plasma levels 7. Alcohol ingestion ↑ plasma levels: urate, lactate, TAG, gamma glutamyl transferase (GGT) Cause hypoglycemia chronic alcoholic patients 9 Activity 2. Phlebotomy  Patient Preparation 8. Stress ↑ levels of: catecholamines, cortisol, ACTH, prolactin, albumin, glucose and lactate Mild stress: ↑ Total cholesterol, ↓ HDL cholesterol by 15% 9. Drugs Medications affecting plasma volume: affect protein, BUN, iron, calcium concentrations. Hepatotoxic drugs: ↑ liver function enzymes Diuretics: ↓plasma Na+ and K+ Opiates: ↑ liver and pancreatic enzymes 10 Activity 2. Phlebotomy  Patient Preparation Physiologic variation Changes that occur w/in the body (ex. Cyclic changes, gender, age, underlying medical conditions, etc.) Age: ↑ albumin, ALP, cholesterol and phosphorus Recent Food ingestion: ↑ glucose, TAG, gastrin, free Ca2+ ↓ electrolytes, ALP and AMS Gender: Male = ↑ albumin, ALP, creatinine, uric acid, cholesterol, BUN Female = ↑ HDL, iron, cholesterol 11 Activity 2. Phlebotomy  Patient Preparation Laboratory tests affected by Diurnal Variation Cortisol Peaks 4am-6am; lowest 8pm-12am; 50% lower at 8pm than 8 am; Increased with stress ACTH Lower at night; increased with stress Plasma renin activity Lower at night; higher levels when standing than supine Aldosterone Lower at night Insulin Lower at night GH Higher in afternoon and evening ACP Higher in afternoon and evening Thyroxine Increases with exercise Prolactin Higher with stress; higher levels at 4am and 8am, and at 8pm and 10pm Iron Peaks early to late morning; decreases up to 30% during the day Calcium 4% decrease when supine 12 Activity 2. Phlebotomy  Order of Draw (Evacuated Tube and Syringe) Potassium Oxalate/ 2mg Sodium 0.2 mg/mL of blood Fluoride/mL of Lithium Heparin blood Sodium 1-2 mg/mL of blood Polyanethol- K2EDTA (plastic) sulfonate (SPS) K3EDTA (glass) 13 Activity 2. Phlebotomy  Anticoagulants Sodium Polyanethol-sulfonate (SPS) prevents the killing of bacteria by innate cellular and humoral factors Tube inversions: 8 times Trisodium Citrate combines with calcium in a non-ionized form Concentration: 1 part citrate to 9 parts blood (3.2% or 3.8%) Short draw: falsely increase clotting time Tube Inversions: 3-4 times 14 Activity 2. Phlebotomy  Anticoagulants Serum Tubes Red Top (silicone coated, glass) Blood clotting time: 60 minutes Tube Inversions: 0 Red Top (clot activator + silicone coated, plastic) Blood clotting time: 60 minutes Tube Inversions: 5 times (to mix with clot activator) 15 Activity 2. Phlebotomy  Anticoagulants Serum Tubes Clot activator and Gel Separator (SSTs with gold top or red/gray top) Blood clotting time: 30 minutes Tube Inversions: 5 times (to mix with clot activator) Gel separator or polymer barrier (thixotropic gel) has SG between blood clot and serum: between 1.03-1.09 (preferably 1.04) Must NOT be used in collecting blood samples for therapeutic drug monitoring (TDM) as some gels absorb drugs causing falsely low results Re-spinning of gel tubes can falsely increase potassium 16 Activity 2. Phlebotomy  Anticoagulants Heparin Also known as mucoitin polysulfuric acid Ideal universal anticoagulant Acts as anti-thrombin and anti-thromboplastin preventing fibrin formation Tests: K+, NH3, carboxy/methemoglobin, pH and blood gas, cytogenetic studies Lithium heparin: glucose, BUN, ionized calcium, electrolytes and creatinine (most chem tests) Sodium heparin: injectable form used in anticoagulant therapy Lithium and ammonium heparin: preferred for microcollection tubes Not used for nucleic acid testing: coextracted with DNA, inhibits polymerase in PCR 17 Activity 2. Phlebotomy Tube Inversions: 8 times  Anticoagulants EDTA Ethylenediamine tetraacetic acid Combines with calcium (chelation) Preparations: K2EDTA – spray dried (plastic) K3EDTA – liquid form (glass) Tests: CBC, Carcinoembryonic antigen (CEA), TDM and lead Excess EDTA (due to underfilled tubes): cause RBCs to shrink thus changing blood count results Used for nucleic acid testing to inhibit enzymes that may lyse nucleic acid EDTA contamination: ↑ Potassium, ↓calcium and magnesium Tube Inversions: 8 times 18 Activity 2. Phlebotomy  Anticoagulants Potassium Oxalate Combines with calcium to form insoluble salt Interferes with Na+, K+, and most BUN (urease) measurements Concentration: 1-2 mg/mL of blood Used as an anticoagulant in Grey Top tubes Tube inversions: 8 times 19 Activity 2. Phlebotomy  Anticoagulants Sodium Fluoride Forms weakly dissociated calcium components Antiglycolytic agent: prevents the glycolytic enzyme enolase Blocks glycolysis for up to 48-72 hrs Interferes with Na+, K+, and most BUN (urease) measurements Used to collect ethanol specimens to prevent ↓ (glycolysis) or ↑ (bacterial fermentation) in alcohol concentration Used for lactate test sample collection Tube inversions: 8 times 20 Activity 2. Phlebotomy  Order of Filling Microcollection Tubes: 21 Activity 2. Phlebotomy  Risks of arterial puncture Arterial puncture an invasive procedure with the potential for significant complications must be performed with priority given to the safety of the patient Uses: blood gas analysis and pH measurement Sites: radial artery, brachial artery, femoral artery, scalp artery and umbilical artery Femoral artery: relatively large and easy to puncture BUT can bleed more in older patients Arterial bleeding is the hardest to control and requires special attention. If a phlebotomist accidentally punctures an artery, he/she should immediately apply pressure and report the case to the supervisor. 22 Activity 2. Phlebotomy  Risks of arterial puncture Major complications: Thrombosis (common in radial) may lead to distal arterial embolism leading to loss of function of hand may lead to ischemia and gangrene distal to the puncture site Hemorrhage (common in femoral and brachial) Nerve damage Possible Infection Never perform arterial puncture on diabetics. 23 Activity 2. Phlebotomy  Notes to remember: IV Fluid contamination: ↑ infused substances: glucose, chloride, Na+, K+, ↓ urea and creatinine as little as 10% contamination: ↑ glucose in blood sample by >500 mg/dL Traumatic draw (vessel wall injury): ↑ CK, myoglobin and potassium 24 Activity 2. Phlebotomy  Notes to remember: Rapid separation of blood after centrifugation Separate serum or plasma from cells w/in 1 hour Adequate time for clotting must be observed before centrifugation (>30 minutes) to prevent latent fibrin formation, which may clog automated analyzers. Plasma should not remain in contact with cells overnight (protein aggregation can occur in plasma even if refrigerated or frozen) Centrifugation requirement: 3000 relative centrifugal force (RCF) for 10 minutes 25 Activity 2. Phlebotomy  Notes to remember: Rapid separation of blood after centrifugation 1. To prevent glycolysis 2. Certain substances are very unstable Failure to separate after 24 hrs (due to hemoconcentration): ↑ Total bilirubin, electrolytes, urea, albumin and total protein Least stable in serum if not removed from the clot w/in 30 minutes: Potassium Phosphorus Glucose 26 Activity 2. Phlebotomy  Notes to remember: Rapid separation of blood after centrifugation 1. To prevent shift in electrolytes False ↑ Potassium and false ↓ Sodium in serum and plasma 2. To prevent hemolysis ↑ levels of analytes: enzymes (LD, ACP, ALT, AST); electrolytes (magnesium, phosphorus, potassium); total protein; albumin; cholesterol and iron Interferes with color reactions Increases bilirubin levels (>25 mg/L bilirubin interferes with albumin, cholesterol and total protein tests) Inhibits lipase enzyme 27 Activity 2. Phlebotomy Name of Student: _________________ Block: __________ Date and Time: ___________________ Activity No. 1: Phlebotomy Name of Patient: _________________ Gender: _________ Date of Birth: ____________________ Age: ____________ Date and Time of Collection: _______________________________ Medical Technologist: ____________________________________