Biochemistry Practical (1) PDF

Biochemistry Laboratory safety PRACTICAL (1) DR. El-Sawy 0  Biochemistry Laboratory safe...

Biochemistry Laboratory safety PRACTICAL (1) DR. El-Sawy 0  Biochemistry Laboratory safety Hazards in the laboratory :  Exposure to blood, body fluids & specimens contains Biological pathogenic bacteria & viruses. Chemical  Acids, alkalis and toxic chemicals. Radiological  Ineffective radioactive waste disposal.  Fire. Accidents  Electrical. General Safety Rules : 1. Read instructions carefully before starting your experiment. 2. Wear safety goggles to protect your eyes from chemicals, heated materials, or things that might be able to shatter. 3. Wear a protecting lab coat, hand gloves, face mask and hair cap. 4. After handling chemicals, always wash your hands with soap and water. 5. During lab work, keep your hands away from your face. 6. Never mix chemicals together unless you are told to do so. 7. Notify your teacher if any spills or accidents occur. 8. Clean up your lab area at the end of your experiment. DR. El-Sawy 1  Biochemistry Laboratory safety Glassware Safety : 1. Broken glassware should be disposed of in a special glass disposal container. 2. When pouring liquids into glassware, make sure it is resting on a table at least a hands breadth from the edge. 3. If a piece of glassware gets broken, notify the teacher. Heating Safety : 1. Use protective gloves to handle hot objects. 2. Never reach across an open flame. 3. Always point top ends of test tubes that being heated away from people. 4. When heating a test tube, move it around slowly over the flame to distribute the heat evenly. 5. Only glassware that is thoroughly dry should be heated. 6. Heat glassware by placing it on a wire gauze platform on a ring stand. Do not hold it in your hand. First Aid : Burns  Immediately flush with cold water.  Do not touch an open wound without safety gloves.  Pressing directly on minor cuts will stop bleeding in Cuts & bruises a few minutes.  Apply cold compress to bruises to reduce swelling.  Flush eyes immediately with plenty of water for several minutes. eyes  If a foreign object is lodged in the eye, do not allow the eye to be rubbed. DR. El-Sawy 2  Biochemistry Sampling PRACTICAL (2) DR. El-Sawy 1  Biochemistry Sampling Specimens :  A specimen is any substance which is taken from the body for Def : testing in the laboratory. 1. Blood 2. Urine 3. Stool 4. CSF Examples : 5. Saliva 6. Semen 7. Respiratory (nasal swab, throat swab, sputum, tracheal and bronchial secretion) 8. Other body fluids (synovial, peritoneal, pleural & pericardial) Blood specimens: 1. Whole blood 2. Plasma 3. Serum includes all cellular the liquid portion of components (RBCs, WBCs, the liquid portion of blood blood (plasma) without platelets) and fluid components the clotting factors. DR. El-Sawy 2  Biochemistry Sampling Plasma separation:  A collecting tube containing anticoagulant is used to collect whole blood sample.  The cells are separated from the liquid supernatant by centrifugation Serum separation:  A collecting tube, without anticoagulant, is used to collect whole blood sample.  The blood is allowed to clot by leaving the sample at room temperature for 15-30 minutes.  The blood clot is removed by centrifuging the sample, and the resulting supernatant is the obtained serum. DR. El-Sawy 3  Biochemistry Sampling When to use whole blood, plasma or serum?  ABG  Blood culture Whole blood  Blood grouping  Hemoglobin  Coagulation tests Plasma  Clotting factors  Albumin  Creatinine Serum  Glucose  Lipid profile DR. El-Sawy 4  Biochemistry Sampling TYPES OF Collecting tubes: 1. Plain tube  NO anticoagulant, No additive Without 2. Serum separator tube (SST)  Contains clot activator & anticoagulant gel separator 1. Citrate 2. EDTA 3. Heparin With 4. Sodium citrate anticoagulant 5. Sodium fluoride  The choice of anticoagulant depends on the analysis that is to be carried out. Color Additives Purpose of use -  Serum separation for most RED biochemistry tests and  Clot activator YELLOW serology  Gel separator  Coagulation tests as PT, D.  Na citrate BLUE dime  CBC  EDTA PURPLE  HbA1C  ABG  Heparin  Ammonia GREEN  Electrolytes  Na fluoride  Estimation of Glucose level GREY DR. El-Sawy 5  Biochemistry Sampling Capillary  Required when only few drops of blood is needed  Used to measure arterial blood gases like oxygen, CO2 Arterial and pH Venous  Specimen of choice for most routine laboratory tests Sites 1. The cubital fossa (frequently selected): Superficial veins that are frequently selected for venipuncture at the cubital fossa include:  Basilic vein  Median cubital vein  Cephalic vein Because of its characteristics: a thick lumen and an easily visible confirmation. 2. Wrist and dorsum of the hands  also commonly used to perform venipuncture. DR. El-Sawy 6  Biochemistry Sampling METHODS Needle and Syringe Butterfly needle Evacuation tube system EQUIPMENT 1. Tourniquet 5. Sharps container (Safety box) 2. Gloves 6. Permanent ink pen 3. Syringe needle & syringe 7. Appropriate collection tubes 4. Alcohol & Gauze 8. Adhesive bandage or tape Precautions  Ensure that patient is in relaxed position: 1. Patient is sitting or lying down 2. Place arm at downward angle to prevent reflux 3. Remind patient to hold still  Verify patient conditions: fasting or non/medications DR. El-Sawy 7  Biochemistry Sampling Lab tests that require fasting: 1. Fasting blood glucose 5. Iron tests 2. HOMA-IR 6. Vit B12 3. C-peptide 7. Gamma glutamyl transferase 4. Lipid profile (triglycerides) Fasting means no food or drinks (except water) for 8-12 hours before taking the sample. Procedure steps : 1. Sensitization. 2. Tourniquet Application. 3. Site cleanse. 4. Equipment preparation. 5. Tourniquet reapplication and needle insertion. 6. Needle removal. 7. Blood transfer to collecting tube. 8. Collecting tube labeling. 9. Sample transport and processing. DR. El-Sawy 8  Biochemistry Sampling Sensitization  Wash your hands  Use alcohol-based hand sanitizers  Put on your gloves Tourniquet Application  Locate tourniquet 3 to 4 inches above venipuncture site (for vein visualization – increase amount of available blood)  tourniquet should be tight to impede venous return but not restrict arterial flow.  Maximum time is one minute  Ask the patient to make a fist (the veins become more prominent, making them easier to locate)  Palpate the area with the tip of the index finger  Vein enhancement: message arm upward from wrist  Then release the tourniquet and ask the patient to open fist Site cleanse  Clean the site with a gauze pad soaked with 70% isopropyl alcohol or a commercially prepared alcohol prep pad (by a circular motion cleanse from inside to outside)  Allow to air dry, 30–60 seconds (assemble syringe and needle in this period)  Failure to let alcohol dry causes a stinging sensation for patient DR. El-Sawy 9  Biochemistry Sampling Equipment preparation  Select a syringe and needle size compatible with age of the patient, the size and condition of the patient’s veins and the amount of blood to be collected.  Attach the needle to the syringe but do not remove the needle sheath (cap) at this time.  To ensure that it moves freely, move the plunger back and forth slightly a few times Tourniquet reapplication and needle insertion  Reapply the tourniquet (help needle entry), being careful not to touch the cleaned area.  Remove the needle cap and visually inspect the needle.  At this time the patient is asked to again make a fist.  The nondominant hand is used to anchor the vein, with the thumb 1-2 inches below the site and other fingers on the back of the patient arm  The collection equipment is held and the needle inserted using dominant hand.  Insert the needle (bevel up), 15-30 degree angle  Establishment of blood flow is normally indicated by blood in hub of syringe.  In some cases blood will not flow until the syringe plunger is pulled back.  Release the tourniquet and ask patient to open the fist, so that blood flow returns to normal  A syringe is filled by slowly and steadily pulling back on the plunger until the barrel is filled to the appropriate level DR. El-Sawy 10  Biochemistry Sampling Needle removal  Withdraw the needle from the vein while simultaneously applying pressure to the site with your free hand, for 3 to 5 minutes or until the bleeding stops  Do not ask the patient to bend the arm up. The arm should be kept extended or even raised.  Apply an adhesive gauze or tape on the site  A needle must be promptly discarded in a safety box Never bend or recap needle  When transfer is complete, the syringe also must be discarded in safety box  If recapping needle is absolutely necessary: 1. Never recap needle with both hands 2. Use a one-handed technique Blood transfer to collecting tube  Select tubes according to the tests that have been ordered.  Transfer sample from syringe to the tube.  If the tube contains an additive, mix it by gently inverting it several times.  (Lack of, delayed, or inadequate mixing can lead to clot formation and necessitate recollection of the specimen)  Do not shake or vigorously mix blood specimens, as this can cause haemolysis  Nonadditive tubes do not require mixing. DR. El-Sawy 11  Biochemistry Sampling Collecting tube labeling  Tubes must be labeled in the presence of the patient immediately after blood collection  Label information: 1. Patient’s first and last name 2. Patient’s ID number 3. Date and time of collection 4. Collector’s initials 5. Additional information (e.g. fasting) Sample transport and processing  Rapid delivery to the laboratory protects specimen integrity.  Follow handling requirements (Place specimens that must be cooled (e.g., ammonia) in crushed ice slurry  Wrap specimens that require protection from light (e.g., bilirubin) in aluminum foil or other light-blocking material).  Plasma and serum separation within 2 hours of collection DR. El-Sawy 12  Biochemistry Sampling NOTE Don’t Forget to : 1- Dispose  all contaminated materials in the proper biohazard containers or according to facility protocol. 2- Discard  other used disposable items in the regular trash. 3- Remove gloves  aseptically and discard them 4- Sanitize your hands Complications of blood collection Fainting Hematoma Petechiae Bleeding Seizures DR. El-Sawy 13  Biochemistry Types of Solutions & Osmolarity PRACTICAL (3) DR. El-Sawy 1  Biochemistry Types of Solutions & Osmolarity Homogenous Heterogeneous True solution (Crystalloid) Suspensions Colloids Solute Solvent Substance dissolved Dissolving medium in in a solution a solution DR. El-Sawy 2  Biochemistry Types of Solutions & Osmolarity Crystalloid (True solution) Colloid Suspension  Homogenous mixture  Heterogeneous mixture that  Heterogeneous mixture in composed of two or more has insoluble dispersed which the solid particles are Definition substances. particles. spread throughout the liquid without dissolving in it.  Invisible under microscope.  Visible under microscope.  Visible to naked eyes. solute particles  ˂1 nm (nm = 10-9 of meter)  1 - 1000 nm  ˃1000 nm  Flour suspended in water.  Oral antibiotics: for children  Sodium Chloride (NaCL) in  Fat globules dispersed in milk. are manufactured in powder Example water.  Plasma proteins. form, so require reconstitution into oral suspensions (e.g amoxicillin). DR. El-Sawy 3  Biochemistry Types of Solutions & Osmolarity Definition : The distribution of electrical charge around a molecule. Types : Polar molecule Nonpolar molecule  Charge is unevenly distributed → has a dipole,  Charge is evenly distributed in → no positive or Charge where part of the molecule has a partial positive negative poles. charge and part has a partial negative charge. Example  Water: universal solvent.  Benzene.  Polar solvents dissolve polar solutes (as salt).  Nonpolar solvents dissolve nonpolar solutes (as naphthalene). DR. El-Sawy 4  Biochemistry Types of Solutions & Osmolarity Definition :  It is the amount of solute dissolved in a solvent. Dilute: if it has a low concentration of solute dissolved Concentrated: if it has a high concentration of solute dissolved solute To quantitatively express concentration, we use: Molarity (M) Molality Normality (N)  The number of moles of solute  The number of moles of solute  Equivalent weight of solute (grams) in present in one liter of solution. present in one kilogram of solution. one liter of solution. M = Moles of solute / Volume of m = Moles of solute / Mass of solution Equivalent weight = molecular weight / solution (in Liter) (in Kg) valance  Example:  Example:  Valence is measure of the atom if 2 moles of NaCl are if 3 moles of KOH are dissolved combining power with other atoms when dissolved in 1 liter of water → in 3 Liters of water (density of it forms chemical compounds or molecules molarity of resulting solution = water 1 kg/L) → molality of (the number of electrons needed to fill 2M. solution = 1 m, as there is 1 mole the outermost shell of an atom). of KOH present in each Kg of  Ex: in H2O, oxygen has a valence 2; and water. in HCL, CL has a valence of 1 DR. El-Sawy 5  Biochemistry Types of Solutions & Osmolarity Classification of Solutions : according to the concentration of solute Molar solution  MW of solute (g) in one liter of solvent. (M) Molal solution  MW of solute (g) in one Kg of solvent. Normal solution  Equivalent weight of solute (g) in one liter of solvent. (N)  Equivalent weight = MW / valance To prepare a solution that contains a specified concentration of a substance, it is necessary to dissolve the desired number of moles of solute in enough solvent to give the desired final volume of solution. 1. Weigh out a sample of solute that contains the necessary moles of solute. 2. Transfer to a volumetric flask. 3. Add a portion of solvent to the flask to dissolve the solute. 4. Additional solvent is added up to the mark on the flask (to complete the required volume of solvent). DR. El-Sawy 6  Biochemistry Types of Solutions & Osmolarity Exercise Prepare 1 molar solution of sodium chloride (NaCl). MW of Na = 22.99 g MW of Cl = 35.45 g Molarity (M) = Moles of solute / Volume of solution (in Liter) MW of NaCL = 22.99 + 35.45 = 58.44 gram Weigh 58.44 gram of NaCL and complete to 1 liter of water. Prepare 1 N of NaOH. MW of Na = 22.99 MW of O = 16 MW of H = 1 Valence of NaOH = 1 MW of NaOH = 22.99+16+1 = 39.99 gram N= equivalent weight of solute (grams) in one liter of solution. Equivalent weight = molecular weight (MW) / valance Equivalent weight of NaOH = 39.99 / 1 = 39.99 Weigh 39.99 gram of NaOH and complete to 1 liter of water. DR. El-Sawy 7  Biochemistry Types of Solutions & Osmolarity Definitions :  Diffusion of solvent across semipermeable membrane from low solute concentration to high solute concentration, until the solution are of equal concentrations. Osmosis  The number of solute particles per 1 L of solvent.  It is a unit of solute concentration. Osmolarity  It is the minimum pressure applied to a solution to stop the flow of solvent molecules through a semipermeable membrane.  The osmotic pressure of a solution is proportional to the molar concentration of the solute particles in the solution and temperature. Osmotic pressure DR. El-Sawy 8  Biochemistry Types of Solutions & Osmolarity Clinical relevance of osmotic effects : RBCs have an osmotic pressure equivalent to that of the plasma (isotonic) surrounding them → neither gain nor lose water. RBCs In order to avoid discomfort on administration of solutions to the delicate membranes of body (as eyes) these solutions are made isotonic with the relevant tissues → prevent irritation and cell damage, and to maximize drug efficacy. Isotonic eye drops When fingers are placed in water for a longer period of time, they become wrinkled due to the flow of water inside the cells. Finger in water DR. El-Sawy 9  Biochemistry Types of Solutions & Osmolarity The causative agent of the pandemic disease cholera. Overpopulate the intestines and reverse the flow of absorption of water, which results in diarrhea & dehydration. V. cholera DR. El-Sawy 10  Biochemistry Types of Solutions & Osmolarity Significance of Osmotic pressure : 1. Maintenance of the shape, volume, internal environment of cells. 2. Absorption of water. 3. The osmotic pressure of plasma proteins maintain blood volume. 4. Osmotic laxatives: for treatment or prevention of constipation (Mg citrate). They draw extra water into the stool, making it softer and easier to pass. DR. El-Sawy 11  Biochemistry Measurement of Glucose in Body Fluids PRACTICAL (4) DR. El-Sawy 0  Biochemistry Measurement of Glucose in Body Fluids Glucose Measurement : Glucose is a simple form of sugar. The terms glucose and sugar are usually used interchangeably when referred to blood, urine and body fluids contents of glucose. This method is highly specific for glucose only. It is a double sequential enzymatic reaction in the presence of an indicator that changes in color in the presence of glucose. It measures true glucose content in body fluid. Glucometers & autoanalyzers based on this principle. The intensity of the colored product is proportional to glucose concentration. Glucose Oxidase Glucose Gluconic acid + H2O2 Peroxidase 2 H2O2 2 H2O + O2 o-toluidine + O2 colored products Different lab techniques based on Glucose Oxidase Method 1. Urine test strips or dipstick test. 2. Glucometer = Glucose meter. 3. Colorimeter. DR. El-Sawy 1  Biochemistry Measurement of Glucose in Body Fluids Components :  Ribbon made of plastic or paper of 5 millimeter wide.  Plastic strips have pads impregnated with chemicals that react with the compounds present in urine producing a characteristic color. Active reagent:  Glucose oxidase (100U/ml).  Peroxidase (100U/ml). Steps : 1. Immerse the test strip in a well mixed sample of urine for 1- 2 min. 2. Extract the strip from the container and support its edge over the container to remove excess urine. 3. Left to stand for 1-2 minutes (the time needed for the reactions to occur). 4. Compare the color that appear against the chromatic scale (color chart) provided by the manufacturer. 5. The color chart value are mg/dl for glucose. Note Normally, Glucose is absent in urine samples. Glucose appears in urine when blood glucose increases beyond 180 mg/dl (the renal threshold for glucose). DR. El-Sawy 2  Biochemistry Measurement of Glucose in Body Fluids DR. El-Sawy 3  Biochemistry Measurement of Glucose in Body Fluids To determines the amount of glucose in blood, the sample usually coming from a finger. The device analyzes a drop of capillary blood, typically obtained from one finger of patient. It provides a numerical reading that indicates the amount of glucose in the blood. Hypoglycemia Normal Hyperglycemia 140 mg/dl. DR. El-Sawy 4  Biochemistry Measurement of Glucose in Body Fluids Principle of Glucometers : Use test strips containing glucose oxidase that reacts to glucose in the blood droplet and an interface to an electrode inside the meter. Requirements : Glucose meter. Glucose meter test strips. Lancet or lancet device. Alcohol swabs. Test method : 1. After washing your hands, put gloves, withdraw a strip, turn monitor on confirm code if needed. 2. Choose finger, wipe with alcohol swab. 3. Lancet a finger. 4. Squeeze blood onto the strip fill area fully. 5. Wait for result. 6. Dispose of all materials in the proper container DR. El-Sawy 5  Biochemistry Measurement of Glucose in Body Fluids Comment on the provided readings. DR. El-Sawy 6  Biochemistry Measurement of Glucose in Body Fluids A device used in clinical laboratories to determine the concentration of compounds in solution. Certain reagents are added on the biological samples to produce colored products whenever certain compounds are present in the sample. Then by measuring the absorbance of the colored products at a specific wavelength of light, the concentration can be calculated.  Colored solutions can absorb lights at certain wavelengths. Principle & method Glucose can be measured by colorimeter depending on Glucose oxidase method. Glucose Oxidase Glucose Gluconic acid + H2O2 Peroxidase 2 H2O2 2 H2O + O2 o-toluidine + O2 colored products DR. El-Sawy 7  Biochemistry Measurement of Proteins in Body Fluids PRACTICAL (5) DR. El-Sawy 1  Biochemistry Measurement of Proteins in Body Fluids  Biological fluids (Serum, Plasma, Urine , Synovial fluid , Saliva, CSF , Amniotic fluid, Bile & Tears) are in close contact with the tissues.  Protein analysis of human body fluids is important as disease biomarker because the protein components of the tissues may be liberated and affected by diseases. Principle : Based on binding of Cu2+ to N atoms involved in peptide bonds. The formation of copper-protein complex requires two peptide bonds in an alkaline media and produces a violet-colored product. The intensity of the violet color is proportional to the number of peptide bonds which reflects protein concentration. Cannot detect urine or cerebrospinal fluid protein except in high concentrations only. DR. El-Sawy 2  Biochemistry Measurement of Proteins in Body Fluids Normal range : 6.0-8.0 g/dl. Types : 1. Albumin (50%-60%): Normal plasma albumin is 3.5 to 5.5 g/dl. 2. Globulin. 3. Fibrinogen.  One principle of colorimetric assays of total plasma proteins is Biuret test.  Biuret test is a traditional method used to determine the Colorimetric plasma protein concentration (currently used as a assay reference method).  The measured absorption of the wavelength 540 nm is proportional to concentration of total plasma protein.  Depends on the different mobility of proteins in the electric field.  In an alkaline media, proteins migrate from cathode (-) to anode (+).  Six fractions of plasma proteins can be distinguished by protein mobility: albumin, α-1, α-2, β-1, β -2 and γ globulins. Electrophoresis DR. El-Sawy 3  Biochemistry Measurement of Proteins in Body Fluids  These methods are the most accurate.  Depending on the reaction of protein with specific antibody for detection of proteins present in very low concentrations. ELISA  Normally very little protein is found in urine.  A normal total protein amount in urine is