Clinical Chemistry 3rd Year 1st Semester PDF

CLINICAL CHEMISTRY 01 - MCC1 31 EMILIO AGUINALDO COLLEGE - MEDICAL LABORATORY SCIENCE TOPIC 01 - INTRODUCTION TO CLINICAL ➔ Laboratory mathematics CHEMISTRY ➔ Separation techniques...

CLINICAL CHEMISTRY 01 - MCC1 31 EMILIO AGUINALDO COLLEGE - MEDICAL LABORATORY SCIENCE TOPIC 01 - INTRODUCTION TO CLINICAL ➔ Laboratory mathematics CHEMISTRY ➔ Separation techniques A. Units of Measurement - Systeme International Chemistry d’Unites (1960) ➔ Is the study of matter, the elements, compounds, ➔ Length (meter) and its structure ➔ Mass (kilogram) Clinical Chemistry ➔ Substance (mole) ➔ Time (second) ➔ Basic science - specialty of chemistry - studies the human body ➔ Current (Ampere) ➔ Applied science - applied science - focused in ➔ Temperature (Kelvin) diagnostic/treatment ➔ Luminous intensity (Candela) ➔ Roles: B. Reagent - “kit” or “ready-to-use” ◆ Measure chemical changes (diagnosis) Chemicals - occupational Safety and Health Administrations (OSHA); indicate lot number, hazards, Blood safety precaution (storage) ➔ Parts: ➔ Analytical Reagent Grade ◆ Plasma 35% - mostly used specimen in ◆ For laboratory use testing for Hematology (EDTA, Heparin, ➔ Ultra-pure HbA1C) ◆ Extremely pure chemicals that have ◆ Buffy coat additional purification steps for use in ◆ RBCs chromatography, immunoassays, molecular diagnostics, standardization Plasma vs Serum ➔ Chemically pure ➔ Plasma - with anticoagulant; avoids the clotting ➔ United States Pharmacopeia (USP) & National of blood Formulary (NF) ◆ Used to manufacture drugs ➔ Serum - not hemolyzed ◆ Mostly used in clinical chemistry; wait to ➔ Technical/Commercial Grade clot before centrifuge ◆ Used in manufacturing; can never be Red & yellow top used in the laboratory ◆ Classification: Normally present with a function Reference Materials in circulation ➔ Primary Standard Metabolites ◆ highly purified chemical that can be Substances released from cells measured directly as a result of cell damage ➔ Secondary Standard Drugs & toxic substances Water Specifications ➔ Distilled Water Section in Clinical Chemistry ◆ Purified to almost remove all organic ➔ Anatomical Pathology materials ◆ Histopathology ➔ Deionized ➔ Clinical Pathology ◆ Mostly used in Clinical Chemistry (ex. ◆ Immunology and Serology Testing the electrolytes) ◆ Microbiology ◆ Neither pure nor sterile ◆ Clinical Chemistry ◆ Excellent in removing dissolved ions and ◆ Hematology gasses ➔ Reverse osmosis Fundamental Concepts ◆ Uses pressure ◆ Used for pretreatment of water, does not ➔ Units of measurement remove dissolved gas ➔ Reagents ➔ Ultrafiltration & Nano-filtered ➔ Instruments ELIZA LUTH YNGENTE NOVELO CLINICAL CHEMISTRY 01 - MCC1 31 LAB ◆ Uses ultra-light oxidation to kill bacteria To contain - holds a particular and traces of organic materials volume but not the exact amount ➔ Reagent Grade Water To deliver - ◆ Type I - test methods (accuracy and ◆ Drainage characteristics precision) Blowout - ◆ Type II - analytical preparations Self-draining - ◆ Type III - autoclave/glassware washing ◆ Types of pipettes Volumetric Solution Properties Pasteur ➔ Solute Ostwald Folin ◆ Dissolved in liquid/solvent *Automatic macro/micropipette ➔ Solvent ◆ Graduated/measuring ◆ Used to dissolve the solid/solute ◆ automatic/mechanical ➔ Solution ◆ Serologic ◆ The result of solute + solvent ◆ Mohr ◆ Solute particles - thermodynamically ◆ Bacteriologic unstable ◆ Ball, Kolmer/Kahn ◆ Micropipette C. Instruments ➔ Burette ➔ Thermometer ➔ Syringe ◆ Total immersion - ex. refrigerator ➔ Desiccators ◆ Particle immersion - ex. Water bath ➔ Balances ◆ Surface immersion - ex. Incubators and ◆ Electronic Top Loading Balance oven ◆ Analytical Balance ◆ Fast reading thermometer - ex. digital thermometer Separation Techniques ➔ Glassware - pre-rinse; rinse solution depending on Terminologies the reagent to (be) use(d) ➔ Batch testing - same time, single test ◆ Glass funnel ➔ Parallel testing - more than one test ◆ Erlenmeyer flask ➔ Random access testing - any test, any sample, ◆ Florence flask any sequence ◆ Beaker ➔ Sequential testing - multiple tests, one after ◆ Pipette - should always be held at 90 another degrees ➔ Open Reagent System Types of Glassware ➔ Closed Reagent System ◆ Borosilicate (Pyrex/Kimax) - mostly used and most sturdy ➔ Centrifugation - use of centrifugal force to ◆ Aluminosilicate (Corex) separate solid matter from liquid suspension by ◆ Flint (soda lime) density of the matter ◆ High Silica ◆ Separates serum/plasma from blood ◆ Low actinic (amber colored) cells ◆ Vycor - acid and alkali resistant ◆ Separate supernatant from a precipitate ➔ Plasticware - disposable ◆ separate two immiscible liquids Types of Plasticware ◆ To expel the air from the container ◆ Tygon ➔ Filtration ◆ Teflon ➔ Dialysis ◆ Polycarbonate ◆ Polyethylene ◆ Polystyrene A. TYPES OF CLINICAL CHEMISTRY SAMPLES ◆ Polycarbonate ◆ Polyvinyl Chloride Blood ➔ Plasma - liquid part (contains salt, glucose, amino D. Laboratory Supplies acids, vitamins, urea, proteins, fats) ➔ Vessels (to contain) ➔ WBCs - part of buffy coat; engages in the immune ◆ Class A Volumetric flask system ◆ Graduated cylinder ➔ Platelets - part of the buffy coat; responsible for ◆ Erlenmeyer flask the clotting system of the blood ◆ Beaker ➔ RBCs - carries oxygen from the lungs throughout ➔ Pipettes the body ◆ Calibration marks NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB Tests performed: CBC, Blood Culture, Coagulation ➔ Also used to detect the baby’s health and genes studies… isip pa kayo :)) Tests performed: Transudates - fluid that passes through a membrane that Urine - watery; typically yellowish fluid filters out all the cells as much of protein, yielding a watery ➔ Stored in the bladder solution ➔ From the blood– filtered by the kidneys ➔ Filtrate of blood ➔ Body’s chief means of eliminating excess water ➔ Due to increased pressure in the veins and and salt capillaries that forces fluids through the vessel walls or to a low level protein of blood ➔ Contains nitrogen compounds– urea ➔ Accumulate tissues outside the blood vessels and ➔ Test: *24 hr. urine (cold urine) / midstream clean cause edema (swelling) catch (warm urine) Exudates - fluid produced by a wound as it heals ➔ Part of the healing process ➔ Composed of cells, proteins, and solid objects Tests performed: urinalysis Cerebrospinal Fluid (CSF) - flows around hollow spaces of the brain and spinal cord and two of the meninges; provides protection, cushion, nourishment and waste removal ➔ contains glucose, albumin, lactate ➔ Made by the choroid plexus in the ventricles in the Specimen Collecting and Handling Blood brain Types of Samples Tests performed: ➔ Whole blood ➔ Serum ➔ Arterial blood ➔ Sample processing - read tube, test required, requisition form of patient ➔ Sample variables - depends on the phase; pre- examination, examination (machine), post- examination (result, TAT) ➔ Chain of custody ➔ Electronic and paper reporting of results Keywords: Blood Amniotic Fluid - clear / slightly yellow fluid that supports ➔ Clotting time: 30 minutes an unborn baby during pregnancy ➔ Centrifugation: 10 minutes, 1000-2000 RCF ➔ Also helps baby’s lungs, digestive system, and (Relative Centrifugal Force) develops bones properly ➔ Arterial Blood Gas (ABG) -> blood pH ➔ Contains baby’s cells and other substances ◆ Heparin tube NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB ◆ Modified Allen test ➔ Hemolysis could be observed from patients with ◆ Arterial puncture syringe angle must be hemolytic anemia but could also be coming from at 90°– no tourniquet pre analytic collection variables ➔ Prolonged tourniquet application = ◆ Wrong needle gauge hemoconcentration ◆ Small veins ➔ Open and close fisting during venipuncture = ◆ Venous trauma/difficulty in specimen lactic acid collecting ➔ I.V = contamination ➔ Lipiduria/lipuria - lipid levels of patients in urine; visualized as milky appearance to serum or Keywords: Urine plasma upon centrifugation ➔ 24 urine creatinine: collected in 24hr collection in ➔ Both can affect laboratory results. The 1-2g content assessment of hemolysis, icterus, and lipaemia is ➔ Average urine output: 2L known as HIL index. ➔ Creatinine clearance: used to assess glomerular filtration rate ➔ Glomerular filtration rate (GFR): compares urine creatinine output to serum ◆ Done to test if the kidneys can still filtrate well ◆ Glomerulus - involved in the filtration of blood to urine in the kidneys ➔ Creatinine Clearance Formula UV C= P C = Creatinine clearance U = Urine Creatinine V = Volume of urine P = Plasma/Serum creatinine of urine ➔ 2 samples needed ◆ 24 hr urine ◆ Serum creatinine Keywords: CSF (3-4 bottle samples) ➔ Shared sample in hematology and microbiology ➔ Ultrafiltrate of plasma ➔ Color and glucose value should be identified ➔ Never store in refrigerator or cold temperature Keywords: Amniotic fluid ➔ Used to assess fetal lung maturity, congenital diseases, hemolytic diseases, genetic and gestational diseases. B. COLLECTION AND HANDLING OF SPECIMENS ➔ Many analytes are stable at room temperature set 24-72hrs ◆ If testing is not performed within 8hrs it is recommended that serum and for plasma be refrigerated between 2-8°C ➔ Light sensitive = bilirubin (mut be covered with foil or protective paper when transporting) ➔ Separated serum/plasma - stored in 20°C; repeated thawing should be avoided NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB TOPIC 02 – PHLEBOTOMY: OVERVIEW OF THE A. BLOOD VESSELS PROCESS ➔ Arteries - away from the heart ◆ Thick strong wall (tunica media) that can HISTORY OF PHLEBOTOMY withstand the pressure of the beating heart Phlebotomy ◆ Branches off to form arterioles and ➔ Practice of collecting blood branches more to form the capillaries ➔ Said to be therapeutic ◆ There is pulse ➔ Came from two Greek words ◆ Oxygenated blood leaves the heart, ◆ Phlebos - vein delivers oxygen to the body tissues ◆ Temnein - cut ◆ Blood absorbs carbon dioxide ◆ Also called venesection transported to the lungs ◆ Flow of blood regulates temperature History of Phlebotomy Warm body: capillaries dilate ➔ Bloodletting - first term for phlebotomy (1400 BC) Cold body: capillaries constrict– ➔ Hippocrates (460-377 BC) - believed on the conserving the heart balance of the four humors: avatar sha ➔ Veins - towards the heart ◆ Earth - blood and brain ◆ Contains valves to keep blood flowing in ◆ Air - phlegm and lungs one direction ◆ Fire - black bile and spleen ◆ No pulse ◆ Water - yellow bile and gallbladder ◆ Has a thin wall so it is easy to puncture ➔ Methods: ➔ Capillaries - connects most arteries and veins ◆ Venesection (most common) - means cutting of the vein to reduce red blood Layers of the Blood Vessels cells from the body ➔ Tunica externa/adventitia - outer layer; made of ◆ Cupping - suck blood from the skin connective tissues surface; alternative medicine to ease ➔ Tunica media - middle layer; made of smooth pain, inflammation and other health- muscle and elastic tissue related concerns ➔ Tunica interna - innermost layer; lining of ◆ Leeching “hirudotherapy” - yuck - use of epithelial cells leech to suck blood, rarely used today’s time, like surgeries; believed that Hirudo Locations of Veins Commonly Used medicinalis injects local vasodilator, ➔ Antecubital fossa (M/H) shape; point of needle anesthetic, and hirudin– anticoagulant must be 15°-45° ➔ Goals of phlebotomy practice: ◆ Median cubital vein ◆ Diagnosis treatment using blood samples Center of antecubital fossa ◆ Transfusion, remove blood from donor Forms a bridged pathway ◆ Removal of blood for polycythemia or between the cephalic and basilic therapeutic purposes– Used to cure veins disease by collecting 10mL of blood Used majority of the time Easy to palpate Phlebotomy in Healthcare Has the less tendency to roll ➔ Phlebotomist ◆ Cephalic vein ◆ Collects blood sample to get accurate Second choice results or for transfusion Not prone to rolling but difficult ◆ Representative of the laboratory - direct to palpate with patients ◆ Basilic vein “Huwag babalik sa laboratory na Third choice (approach with walang dala/kuha na dugo” de caution) wag Very difficult to feel Has the tendency to roll ➔ Centralized - collect only blood samples Underlying vein is the brachial ◆ Focused on patient care artery and median cutaneous ➔ Decentralized - rounds in sections vein ◆ Ex. 1st shift - clinical chem, next shift - ➔ Back of the hand Hematology ➔ Wrist ➔ Hybrid - all around (all sections are covered; most ➔ Ankle strenuous” ➔ Arteries ◆ Used to detect errors of decentralized NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB + Superficial vein in the hands can be an alternative Hypodermic venipuncture site; requires special techniques for Winged infusion collection ◆ Comes with color coded caps and hubs for easy identification B. BLOOD COLLECTION PROTOCOL ◆ Equipped with safety features Resheating Blunting Equipment and Supplies Retraction device ➔ Blood drawing stations ◆ Gauge; diameter of the needle, the higher ➔ Phlebotomy chair the diameter, the smaller the number ➔ Handheld carriers/phleb kit Yellow - 20 gauge ➔ Phlebotomy carts Green - 21 gauge - standard for ➔ Gloves - “good fit is essential” daw most routine adult antecubital ◆ Nonsterile venipuncture ◆ Latex Black - 22 gauge ◆ Nitrile ◆ Neoprene Evacuated Tube System ◆ Polyethylene ➔ Closed collection system ◆ Vinyl ➔ Composed of multisample needle, tube holder ◆ *Gloves with powder base are not and evacuated tubes– prevents exposure to recommended– cause of allergies contaminants ➔ Antiseptics - prevents sepsis, presence of ➔ Allows numerous tubes to be collected in a single microorganism or their toxic products within the venipuncture bloodstream ◆ Prevents the growth of microorganisms, Order of Draw - BCNHES but does not kill them ◆ 70% isopropyl(isopropanol)/ethyl alcohol Yellow Blood Culture Microscopy is most used ➔ Disinfectants - can kill microorganisms Blue Citrate Coagulation ◆ corrosive, can burn the skin studies ◆ Ex. 5.25% sodium hydrochloride (bleach) 1:10 dilution Red No coagulant Clinical Chem 1:100 dilution - recommended for decontaminating non-porous Green Heparin Hematology, surfaces after Clinical ➔ Gauze pad/cotton balls Chemistry ◆ Held pressure over the site ➔ Bandages - covers the puncture site Purple EDTA Hematology ◆ Self-adhesive is used to form pressure bandage following arterial puncture or Gray Sodium Fluoride venipuncture on patients with bleeding problems Phases of Sample Testing ➔ Needles & sharps disposal container ➔ Pre-examination ◆ Should be puncture resistant, leakproof & ◆ Clinician’s request disposable ◆ Patient identification and information Should not be overfilled ◆ Correct sample collection Syringes: separate barrel to ◆ Correct primary sample identification needle ◆ Correct use of all equipment Yellow bin: used in autoclave ◆ Sample preparation or centrifugation ➔ Transillumination devices ◆ Proper preparation of sample aliquots ◆ Makes it easier to locate vein ◆ Maintaining sample integrity ➔ Tourniquet ➔ Examination ◆ Tied to restrict blood flow– makes veins ◆ Analytical phase larger ◆ Includes all processes done to sample to ◆ Rubber tourniquet is commonly used achieve result ◆ Used within one minute ➔ Needles - for withdrawing blood samples - 1-1.5 ◆Sample testing inches long ◆Maintaining testing equipment and ◆ Types: reagents Multi-sample ➔ Post-Examination NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB ◆ Postanalytical phase ◆ Process in which the results of the testing are communicated to the health care provider or physician ◆ Reporting of results ◆ Ensuring accuracy and reliability of delivery of results ◆ Follow-up to repeat testing or address physician’s concerns ◆ Storage of sample after the examination Proper Labelling ➔ Medical record number ➔ Patient’s name ➔ Initials of the person drawing the specimen ➔ Date and time of collection NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB TOPIC 03 – TYPES OF PHLEBOTOMY PROCEDURES ◆ Verify diet restrictions and latex sensitivity ◆ Sanitize hands and put on gloves SKIN PUNCTURE ◆ Position the patient ➔ Method that uses lancet to make a small incision ◆ Select the puncture site into the capillary bed of the skin to obtain a small ◆ Warm the site, if necessary volume of blood specimen ◆ Clean the air-dry site ➔ Capillary blood and peripheral/arteriolar blood ◆ Prepare the equipment ➔ Sites of choice ◆ Puncture the site and discard the lancet ◆ Ear lobe ◆ The first blood drop should be wiped ◆ Fingers (palmar surface of the 3rd and away because it may be contaminated 4th finger) with excess tissue fluid ◆ Heel and big toe ◆ Fill and mix tubes or containers in the ➔ Sites to avoid order of draw ◆ Cold and cyanotic ◆ Place gauze and apply pressure. Keep the ◆ Inflamed and pallor incision site elevated ◆ Congested and edematous ◆ Label specimen and observe special ◆ Areas with scar and calluses handling instructions ◆ Check the site and apply bandage Skin Puncture Principles ◆ Dispose used and contaminated materials ➔ Tests not compatible ◆ Thank patient, remove gloves and ◆ Erythrocyte sedimentation rate (ESR) sanitize hands ◆ Coagulation studies that require plasma ◆ Transport specimen to the laboratory specimen ◆ Blood culture ◆ Tests that need large volume of VENIPUNCTURE serum/plasma ➔ A procedure in which a needle is used to take ➔ Equipment blood from vein ◆ Lancet/Incision device - one time use ➔ Open system, closed system, and butterfly only method ◆ Laser lancet - vaporizing water in the ➔ Steps skin; eliminates risk of sharp injury ◆ Review and accession test request because cauterizing the skin is not Types: manual, computer, barcode necessary. There are two types ◆ Phlebotomist must Finger puncture lancet Check to see that all required Heel puncture lancet information is present and complete ◆ Micro collection container “microtube” - Verify tests to be collected and time hold the blood specimen and date of collection ◆ Microhematocrit tubes and sealants - Identify diet restrictions or other narrow bore tubes that are made of special circumstances either plastic or glass. Determined the test states or priority Used for hematocrit collection determinations ◆ Required information Hold 50 to 75μL Name of the physician who ordered the One end is sealed with sealants test made of clay or plastic Patient’s full name including the middle ◆ Microscope slides - used for blood films initial for hematology determinations Medical record number (if inpatient) ◆ Warming devices - used to increase the Birthday and age blood flow seven-fold by warming the Room number and bed number (in puncture site patients) ◆ Capillary blood gas (CBG) equipment - Type of test ordered used for collecting CBG specimens; Date when the test is to be performed contains CBG collection tubes, stirrers, Test status magnets, and plastic caps. Special precaution ◆ Approach, Identify, and Prepare Patient ➔ Steps Approaching the patient ◆ Review and check accession test request ○ Be organized and prepared with ◆ Approach, identify, and prepare the paperwork patient NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB ○ Look for phlebotomy-related signs Palpate patient’s dominant arm with and warning regarding the patient index finger information Roll finger side to side while pressing ○ Knock lightly on door against vein to judge size ○ Ask visitors to step out Avoid veins that feel hard and cord- ○ Identify yourself like or lack resilience ○ Obtain consent for procedure Release tourniquet and have patient ○ Put patient at ease, using open fist professional bedside manner ◆ Clean and Air-dry the site Patient identification Clean site with an antiseptic to avoid ○ Verify name and date of birth infection or contamination ○ Check ID bracelet Use 70% isopropyl alcohol ○ Notify nurse of ID discrepancies Use circular motion, moving outward ○ Search for missing IDs in widening concentric circles ○ Wake sleeping patients Clean an area about 2 to 3 inches in ○ Ask a relative or nurse to identify a diameter patient who is unconscious, young Allow area to dry 30 second to 1 mentally incompetent, or non- minute (until dry) English speaking Don’t dry alcohol with unsterile gauze Preparing the patient or fan or blow on site ○ Explain the procedure Don’t touch site after cleaning it ○ Address patient inquiries Leaving an alcohol pad pointing in the direction of the ○ Handle patient objections vein if needed for marketing the site ○ Address difficult patients Note: if it is necessary to re-palpate the vein after the site ○ Address objects in patient’s mouth has been cleaned, the site must be cleaned again. Note: regardless of the difficulties involved, you must ◆ Prepare equipment and put on gloves always determine that the patient understands what is ETS equipment preparation about to take place and obtain permission before Preparation of winged infusion set proceeding. This is part of informed consent (butterfly) ◆ Verify diet restrictions and latex sensitivity Preparation of syringe equipment If the patient has eaten and you are Positioning equipment for use told to proceed with specimen ◆ Reapply tourniquet, uncap and inspect collection. It is important to write needle “non-fasting” on the requisition and According to the CLSI, when a the specimen label tourniquet has been in place for longer ◆ Sanitize hands than one minute, it should be released ◆ Position patient, apply tourniquet, and ask and reapplied after two minutes patient to make a fist ◆ Ask patient to remake a fist, anchor Positioning patient vein, and insert needle ○ Inpatients: typically, are lying down Anchoring in bed ○ Use nondominant hand to anchor ○ Outpatients: sitting up in blood- (secure firmly) the vein drawing chair ○ Place thumb at least 1 to 2 inches ○ Patients prone to fainting: reclining below and slightly to side of site chair, sofa, or bed ○ Pull skin toward wrist ○ Support hand or arm that is to be site Needle insertion of venipuncture ○ Hold collection device or butterfly Tourniquet application and fist needle in dominant hand clenching ○ With bevel facing up, position needle ○ Apply tourniquet snugly 3 to 4 inches above insertion site above intended site ○ Insert at 30-degree angle or less in ○ Never apply over open sore smooth, steady forward motion ○ Apply over a dry washcloth or gauze ◆ Establish blood flow, release if patient has sensitive skin tourniquet, and ask patient to open fist ○ Ask patient to make a fist Advance collection tube into tube ◆ Select vein, release tourniquet, and ask holder until stopper is completely patient to open fist penetrated by needle Preferred site is antecubital area of Push tube with thumb while index and arm middle fingers straddle and grasp First choices are median cubital and flanges of tube holder, pulling back median veins slightly NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB Blood will begin to flow into tube ➔ Equipment selection Release tourniquet ◆ 23-gauge butterfly needle attached to an Have patient release fist evacuated tube or syringe ◆ Fill, remove, and mix tubed ➔ Procedures immediately after removal from the ◆ Collect minimum amount of blood holder required for testing ◆ Place gauze, remove needle, activate safety feature, and apply pressure Geriatric Venipuncture ◆ Discard collection unit, syringe needle, ➔ Challenges or transfer device ◆ Skin changes ◆ Label tubes ◆ Hearing impairment Patient’s first and last names ◆ Visual impairment Patient’s identification number (if ◆ Mental impairment applicable) of date of birth ◆ Effects of disease Date and time of collection Arthritis Phlebotomist’s initials Diabetes Pertinent additional information, such Parkinson’s disease as ‘fasting’ Pulmonary function Compare information on each labeled ➔ Blood collection procedures tube with the patients with the ◆ Patient identification: don’t rely on nods patient’s wristband on the requisition of agreement; verify patient information ◆ Observe special handling instructions with a relative or attendant ◆ Check patient’s arm and apply ◆ Equipment selection: butterfly needles or bandage short-draw tubes ◆ Dispose of contaminated materials ◆ Tourniquet application: loose enough to ◆ Thank patient, remove gloves, and not damage skin sanitize hands ◆ Site selection: avoid bruised areas from ◆ Transport specimen to the lab previous venipunctures ◆ Cleaning the site: don’t rub too vigorously Pediatric Venipuncture ◆ Performing the venipuncture: anchor vein ➔ Overview firmly to avoid rolling ◆ Children 2 hours past collection process will be followed in an effort to reduce ◆ Blood for labile or time-sensitive test not clinical impact on the patient. transported properly ◆ Irretrievable Specimen Processing Notice ◆ Expired collection container must be completed by both laboratory and ◆ Blood Bank specimens lacking the collection personnel/ client. required information will be rejected and ◆ If possible, downtime testing for time- must be recollected. sensitive tests until form is completed. ◆ See Label Correction for Irretrievable Specimens – Laboratory procedure for STORAGE AND TRANSPORT instructions and access to this form. ➔ Storage and Transport: ◆ Tube Orientation: Tubes should be kept in a vertical, stopper- Reference: up position. This promotes complete clot https://riverview.org/downloads/pdfs/specimen- formation and reduces agitation of tube acceptability-and-rejection-policy.pdf contents. Clot tubes with gel should always be stored upright after mixing to prevent fibrin from attaching to the tube stopper. ◆ Exposure to light: Avoid exposing blood specimens to artificial light or sunlight for any length of time when testing for photosensitive tests like bilirubin, vitamin A, porphyrins. Protect samples by wrapping them with aluminum foil or using an amber tinted container. ◆ c. Collection sites: Unless immediately transporting samples to the laboratory after collection, serum/plasma separation must occur at the collection site. ○ Samples must be allowed to clot for 20-30 minutes prior to spinning. ○ Most samples must be spun no more than 2 hours after collection. Any secondary (aliquot) tubes used must be leak-proof. NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB TOPIC 05 – SAMPLING VARIABILITY: FACTORS ➔ Inadvertent Arterial Puncture AFFECTING SERUM CONSTITUENTS ◆ This happens when blood is filling up the tube rapidly and there is a rapid formation of hematoma on the site. PHLEBOTOMY ERRORS (TECHNIQUE PREOBLEMS) ➔ Infection ◆ Infection can be avoided by making sure that: Specimen Quality tapes or bandages are not opened ahead ➔ Hemoconcentration of time, ◆ Decrease in the fluid content or plasma volume needles are not preloaded into the tube Caused by tourniquet that stagnates the holders, normal flow of blood insertion site of the needle is not touched ○ leading to increase in concentration of after sterilization, red blood cells and other nonfilterable cap is removed just before venipuncture, large molecules and ➔ Hemolysis patients are advised to keep the bandage ◆ Also called hemolysis on the site for at least 15 minutes. ◆ Rupture of red blood cells- hemoglobin is ➔ Nerve Injury released to the surrounding fluid ◆ Nerve injuries happen when there is: ➔ Partially filed tubes or short draw improper site selection, ◆ when the phlebotomist pulls a tube before rapid needle insertion, reaching the required volume excessive redirection of the needle, and leads to the incorrect blood-to-additive blind probing. ratio. ◆ If the initial attempt is not successful, the ➔ Specimen Contamination phlebotomist should try to redirect the needle ◆ means that the specimen is compromised due by using a slightly forward or backward to incorrect handling, movement. includes allowing alcohol, powder or other ◆ The next step is to remove the needle and look materials into the sample. for an alternative site. ➔ Wrong or Expired Collection Tube ➔ Reflux of Anticoagulant ◆ should not be used because the manufacturer ◆ Blood that has already been drawn to flow could not warrant the quality of the seal and back into the vein from the collection tube may pressure after the expiration date declared in cause adverse reactions because of the the tube. presence of tube additives. ➔ Hematoma Formation ◆ To avoid this, make sure to keep the arm of the ◆ The phlebotomist should hold pressure over patient in a downward position and the tube the site immediately after discontinuing the just below the venipuncture site. draw, a cold compress or ice pack may be ➔ Vein Damage offered to help address the swelling. The ◆ Damaging the vein could be avoided by following are condition that trigger following the proper technique and avoiding hematoma: blind probing. Excessive or blind probing Inadvertent arterial puncture Troubleshooting failed venipuncture Size of the vein is too small ➔ Failure to withdraw sample The needle penetration has gone all ◆ Venipuncture attempts could fail due to through the vein improper seating of the tube and failure of the Needle is not completely inserted needle to go through the stopper. The Tourniquet is still on when the needle was phlebotomist must be aware and must take removed measures to ensure that the proper Pressure is not adequate procedures are followed: ➔ Iatrogenic Anemia ◆ The needle position is critical to the success of ◆ These results from blood loss due to blood venipuncture. The phlebotomist should ensure draw. that the following does not happen: ◆ It is important to ensure to collect only the Needle not inserted far enough required specimen volume Bevel partially out of skin because if 10% of the blood volume is Bevel partially into vein removed at once from the body, the Bevel partially through vein patients could face a threat Bevel completely through vein Bevel against vein wall Needle beside vein Undetermined position NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB ➔ Collapsed Vein ◆ occurs when conditions are less than ideal, which leads to the veins being blocked, resulting in insufficient blood flow. ◆ happens when there is a strong pressure in the vacuum of the tube or plunger, the tourniquet is too close to the site or it is too tight, or when the tourniquet was removed during the draw. ➔ Tube Vacuum ◆ To avoid failure due to loss of vacuum, the phlebotomist should make sure that the bevel is not partially out of skin and the tube itself is not damaged. PATIENT’S ERROR (PREPARATION PROBLEM) Fasting State Requirement ➔ The basal state is ideal in establishing reference range since it represents the condition of the metabolism of the body early in the morning or after approximately 12 hours of fasting. Result Variation VARIABLE EFFECT Age Red Blood Cells (RBC), White Blood Cells (WBC), Creatinine Clearance Altitude Red Blood Cells (RBC) Dehydration Hemoconcentration, Red Blood Cells (RBC), Enzymes, Iron (Fe), Calcium (Ca), Sodium (Na) Diet Glucose, Lipids, Electrolytes Diurnal Variation Thyroid Stimulating Hormone (TSH), Cortisol, Iron (Fe) Drug Therapy Enzymes. Hormones Exercise/IM pH (Potential of Hydrogen), Carbon Injection Dioxide Partial Pressure (PCO2), Creatinine Kinase (CK), Lactic Acid Dehydrogenase (LDH). Glucose Fever Hormones, Cortisol Gender Red Blood Cells (RBC), Hemoglobin (Hgb), Hematocrit (Hct), Jaundice Yellow color interfaces due to increased Bilirubin Intramuscular Creatinine Kinase (CK), and the skeletal Injection muscle fraction of LDH Position Protein, Potassium (K) Pregnancy Red Blood Cells (RBC) Smoking Cholesterol, Cortisol, Glucose, Growth Hormone, Triglycerides, White Blood Cells (WBC) Stress White Blood Cells (WBC), Iron (Fe), Adrenocorticotropic Hormone (ACTH), Catecholamine, Cortisol Temperature and Hemoconcentration Humidity NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB TOPIC 06 – CARBOHYDRATES INTRODUCTION ➔ Primary source of energy stored primarily glycogen (muscle and liver) ◆ Not produced by the body but comes from ingested foods, nutrients -> plants ◆ Plants are capable of producing their own glucose ◆ But the body (liver) can produce its own cholesterol– is needed for hormone production and cell membrane repair ➔ Disease states involve hyperglycemia and hypoglycemia ◆ Hyperglycemia More common Comes from the diet Can develop into diabetes- which depends on the health status of the patient ◆ Hypoglycemia Mimics the signs and symptoms of hyperglycemia ➔ Contains C, H, and O (Cx(H2O)y) with C=O and -OH functional groups CLASSIFICAITON DEFINITION *Amylase – enzyme produced by the pancreas and Monosaccharides ➔ Cannot be hydrolyzed to a salivary glands- helps to digest ingested foods (breaks or simple sugars simpler form down into glucose) ➔ No bonds, basic ➔ Contains 3, 4, 5, 6 carbon Terminologies atoms (triose, tetroses, ➔ Glycol aldehyde – simplest carbohydrate (CHO) pentoses, hexose, etc.) ➔ Glucose, Maltose, Fructose, Lactose, and ➔ Ex. Fructose, Glucose, Galactose – reducing substances/sugars Galactose ➔ Sucrose – most common non-reducing sugar Disaccharides ➔ Combination of 2 ➔ Non reducing sugar do not contain an active monosaccharides ketone or aldehyde group ➔ Ex. ➔ The brain is completely dependent on blood Maltose = glucose + glucose glucose for energy production- 2/3 of glucose Lactose = glucose + galactose utilization in resting adults occurs in the CNS Sucrose (table sugar) = ◆ Alams na bakit kota lagi si Reign at Jane glucose + fructose HAHAHAHAH Oligosaccharides ➔ Chain of 3 – 10 sugar units Polysaccharides ➔ Linkage of many PATHWAYS OF GLUCOSE METABOLISM monosaccharides ➔ Ex. Starch (mostly from ingested foods), and glycogen NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB ➔ As an endocrine gland, it secretes the hormones Glucose Metabolism: Terminologies insulin, glucagon, and somatostatin from ➔ Glycolysis different cells residing in the islets of Langerhans ◆ Metabolism of glucose to lactate or pyruvate in the pancreas for production of energy ◆ Two types: Insulin – Hypoglycemic Agent Aerobic/respiration glycolysis ➔ Primary hormone responsible for decreasing ○ Common pathway used by the body blood glucose ○ Having enough source of oxygen for the ➔ Synthesize by the β cells of the islets of blood Langerhans (pancreas) ○ Byproduct: pyruvate / ATP ➔ Regulates blood glucose by ↑ glycogenesis, Non-aerobic glycolysis, and lipogenesis; and ↓ glycogenolysis ○ No presence of oxygen ◆ Heart problem – function of heart to Glucagon – Hyperglycemic Agent efficiently pump blood is altered; ➔ Counterpart of insulin delivery of red blood cell is ➔ Primary hormone responsible in increasing blood compromised agents ◆ Lung problem – Chronic Obstructive ➔ Synthesized by α cells of the islets of Langerhans Pulmonary Disease (COPD); may (pancreas) bara sa baga- oxygen supply is also ➔ Regulates blood glucose by ↑ glycogenolysis and compromised gluconeogenesis ○ byproduct: lactic acid / lactate ○ If prolonged, lactic acid will increase ➔ It is released during stress and fasting states and blood pH will decrease making it an ◆ Fasting unhealthy environment for the cells. No food ingested = low sugar levels ➔ Gluconeogenesis Cortisol (Glucocorticoids) ◆ Formation of glucose-6-phosphate from non-carbohydrate source ➔ No. 1 hormone related to stress “stress hormones” ➔ Glycogenolysis released by adrenal glands ◆ Breakdown of glycogen to glucose for ➔ Secreted by the cells of the zona fasciculata and use as energy zona reticularia ➔ Glycogenesis ➔ Produced by the adrenal cortex, in response to ◆ Conversion of glucose to glycogen for ACTH ↑ gluconeogenesis, glycogenolysis, and storage lipolysis ➔ Lipogenesis ➔ Pakihabaan ang pasensya sa isa’t isa lalo na sa ◆ Conversion of carbohydrates to fatty mga problema ni Jane upang di tumaas ang acids cortisol. ➔ Lipolysis ◆ Decomposition of fat Epinephrine ➔ “Adrenaline rush” REGULATION OF GLUCOSE METABOLISM ➔ Produced by the adrenal medulla, ↑ blood glucose ➔ Released during times of physical and emotional stress Hormones that Regulate Glucose ➔ Inhibits insulin secretion, ↑ glycogenolysis and ➔ Insulin* - only hormone that can decrease blood lipolysis sugar levels ➔ Glucagon Growth Hormone (Somatotrophic) ➔ Epinephrine ➔ Increases blood sugar, especially to children ➔ Cortisol ➔ Produced by the anterior pituitary gland, ↑ ➔ Growth hormone plasma glucose ➔ Thyroxine ➔ ↓ Glucose entry to cells, ↑ glycolysis, and ➔ Somatostatin glycogenolysis Pancreas Thyroxine ➔ Functions as both an endocrine and exocrine ➔ Produced by the thyroid gland, ↑ plasma glucose organ in the control CHO metabolism ➔ ↑ Glycogenolysis, gluconeogenesis, and glucose ➔ As an exocrine gland, it produced and secreted intestinal absorption amylase that is responsible for the breakdown of ingested complex CHO NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB Somatostatin Insulin antibodies ➔ Indirect rule; balance out the action of insulin and Glutamic acid decarboxylase glucagon autoantibodies ➔ Boss yan sha- taga dictate Tyrosine phosphatase IA-1 and IA-2B ➔ Produced by the Delta cells of the islet of autoabs Langerhans in the pancreas and hypothalamus There is genetic association between Type ➔ Increases plasma glucose by inhibition of insulin, 1 diabetes and HLA DR3 and DR4- the glucose, GH, etc. major focus is the MHC on chromosome no. 6 ➔ Characteristics DISEASES STATES IN GLUCOSE METABOLISM ◆ Abrupt onset ◆ Insulin dependence Hyperglycemia ◆ Ketosis tendency ➔ Is an increase in blood glucose levels ◆ Signs and Symptoms ➔ Cause: stress, severe infection, dehydration or Polydipsia (excessive thirst) pregnancy, pancreatectomy, hemochromatosis, Polyphagia (↑ food intake) insulin deficiency or abnormal insulin receptor Polyuria (excessive urine production) ➔ FBS level = > or = 126 mg/dL ◆ Idiopathic Type 1 DM ➔ All adults older than 45 years old should have a ◆ Is a for of type 1 diabetes that has no known measurement of FBS every 3 years unless the etiology; it is strongly inherited; it does not individual is diabetic have β-cell autoantibodies and have episodic requirements for insulin replacement Diabetes Mellitus Type 2 ➔ Is a group of metabolic disorders characterized by hyperglycemia resulting from defects in insulin ➔ Formerly known as secretion, insulin receptors or both ◆ Non-insulin Dependent Diabetes Mellitus ◆ Adult Type/Maturity Onset Diabetes Mellitus ➔ Fasting plasma glucose concentration > or = 162 ◆ Stable Diabetes mg/dL on more than one testing are diagnostic of ◆ Ketosis Resistant Diabetes diabetes ◆ Receptor-Deficient Diabetes Mellitus ➔ Glucosuria occurs when the plasma glucose level ➔ Epidemiology exceeds 180 mg/dL (9.99 mmol/dL) w/ normal ◆ 90% of all cases of diabetes renal function ◆ Adult onset ➔ Ketosis develops in DM from excessive synthesis ➔ Pathogenesis and Pathophysiology of acetyl-CoA, as the body attempts to obtain ◆ Insulin resistance w/ insulin secretory defect required energy from stored fat in the absence of ◆ Relative insulin deficiency an adequate supply of carbohydrate metabolites- ◆ Inc. with age, obesity, and lack of exercise the presence of ketone bodies is a frequent ◆ Insulin present (hyperinsulinemia) finding in individuals with severe, uncontrolled ◆ Glucagon secretion is attenuated diabetes ➔ Characteristics ➔ In severe DM, the ratio of β-hydroxybutyrate to ◆ Non-insulin dependent acetoacetate is 6:1 ◆ Has milder symptoms compared to type 1 ➔ The entire process of ketosis can be reversed by however, untreated type 2 DM will result to insulin administration nonketotic hyperosmolar coma- overproduction of glucose (>500 mg/dL), Classification Severe dehydration, electrolyte imbalance Type 1 and inc. BUN and creatinine ➔ Formerly known as: ◆ NOT related to an autoimmune disease ◆ Insulin Dependent Diabetes Mellitus Risk factors ◆ Juvenile Onset Diabetes Mellitus ○ Obesity, family history, advanced age, ◆ Brittle Diabetes hypertension, lack of exercise, impaired ◆ Ketosis Prone Diabetes glucose metabolism ➔ Epidemiology and Pathophysiology ◆ It has been described as geneticist’s ◆ 5-10% of all cases of diabetes nightmare ◆ Occurs in childhood and adolescence ➔ Manifestation ◆ Absence of insulin with excess in glucagon ◆ Polydipsia ➔ Pathogenesis ◆ Polyphagia ◆ β-Cell Destruction ◆ Polyuria ◆ Absolute insulin deficiency ◆ Autoantibodies Islet cell autoantibodies NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB TYPE 1 TYPE 2 ◆ Screening should be performed between 24 Pathogenesis B-cells destruction Insulin resistance and 28 weeks of gestation (1-hr Glucose Incidence rate 10-15% 90-95% Challenge Test – 50g glucose load) Onset Any; Any over 40 yrs ◆ A plasma glucose concentration of 140 mg/dL childhood/teens old or greater requires a full diagnostic glucose Risk Factors Genetic; Genetic, obesity, tolerance test (3-hr GTT with 100g glucose) autoimmune lifestyle ◆ OGTT results C-peptide levels Decreased or Detectable FBS - > or = 95 mg/dL undetectable 1-hour - > or = 180 mg/dL Pre-diabetes Autoantibodies (+) Autoantibodies (-) 2-hour - > or = 155 mg/dL Symptomatology Symptoms Symptoms 3-hour - > or = 140 mg/dL develop abruptly develop gradually ◆ GDM converts to DM within 10 years in 30-40% (some patients of cases asymptomatic) Ketosis Common, poorly Rare Impaired Fasting Glucose controlled ➔ It is characterized by fasting blood glucose Medication Insulin absolute Oral agents concentrations between normal and diabetic values Hyperglycemia – Laboratory Findings Impaired Glucose Tolerance ➔ ↑ Glucose in plasma and urine ➔ It is characterized by fasting blood glucose ➔ ↑ Urine specific gravity concentrations less than those required for the ➔ ↑ Serum and urine osmolality diagnosis of diabetes, but OGTT is between ➔ Ketones in serum and urine (ketonemia and normal and diabetic values ketonuria) ◆ Acetoacetate, β-hydroxybutyrate and acetoin Diabetes Insipidus is produced from FA ➔ Deficiency of ADH (vasopressin) released by ➔ ↓ Blood and urine pH (acidosis) posterior pituitary gland ➔ Electrolyte imbalance ➔ Characterized by severe polyuria w/ high SG ◆ ↓ Na – polyuria and shift of water from cells (glucosuria) ◆ ↑ K – displacement from cells in acidosis ➔ w/ signs and symptoms of GM which is polyuria and polydipsia Hyperglycemia - Disease ➔ Microvascular problems Hypoglycemia ◆ Nephropathy ➔ It results from an imbalance between glucose ◆ Retinopathy utilization and production ◆ Neuropathy ➔ Involves decreased glucose levels and can have ◆ Increased heart disease many causes ➔ The warning signs and symptoms of Other Specific Types of Diabetes hypoglycemia are related to CNS ➔ Pancreatic disorders ➔ 50 mg/dL (2.8 – 3.0 mmol/L) – observable ➔ Endocrine disorders – Cushing’s syndrome, symptoms of hypoglycemia occur pheochromocytoma, acromegaly, and ➔ < or = 50 mg/dL – diagnostic hypoglycemia value hyperthyroidism ➔ A diagnosis of hypoglycemia should not be made ➔ Drugs or chemical inducers of β-cell dysfunction unless a patient meets the criteria of Whipple’s (dilantin and pentamidine) and impair insulin triad – low blood glucose concentration with action (thiazides, glucocorticoids) typical symptoms alleviated by glucose ➔ Genetic syndrome – down syndrome, Klinefelter’s administration syndrome, Rabson-Mendengall syndrome, Symptoms Leprechaunism ➔ Neurogenic – tremors, palpitations, anxiety, diaphoresis Hyperglycemia – Diabetes Mellitus Classification ➔ Neuroglycopenic – dizziness, tingling, blurred Gestational vision, confusion, behavioral changes ➔ Pathogenesis ◆ Glucose intolerance during pregnancy Hypoglycemia - Classification ◆ Due to metabolic and hormonal changes ➔ Drug Administration – insulin, alcohol, salicylates, ◆ Infants born to diabetic mothers ate at ↑ risk sulfonamides, pentamidine for respiratory distress syndrome, ➔ Critical Illness - hepatic failure, sepsis, renal hypocalcemia and hyperbilirubinemia failure, malnutrition, cardiac failure ➔ Autoimmune hypoglycemia – insulin antibodies NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB ➔ Non-beta cell tumor – leukemia, hepatoma, ➔ Venous blood is 7mg/dL lower than capillary lymphoma blood, capillary blood glucose id dame with ➔ Hypoglycemia of infancy and childhood – arterial blood glucose galactosemia, GSD, Reye’s Syndrome ➔ Peritoneal fluid is the same with plasma glucose ➔ Alimentary (reactive) hypoglycemia – post gastric ➔ Plasma glucose increase in with age- fasting, surgery 2mg/dL/decade; postprandial, 4mg/dL/decade; ➔ Idiopathic (functional) postprandial glucose challenge, 8-13mg/dL/decade hypoglycemia Specimen Considerations Hypoglycemia – Laboratory Findings ➔ Serum or plasma must be separated from the cells ➔ ↓ Glucose in plasma within one hour to prevent losses of glucose ➔ ↑↑↑ In patients with pancreatic -cell tumors (preferably within 30 mins) (insulinoma) ➔ At RT (20-25 degree Celsius), glycolysis decreases glucose by 5-7%/hour (5-10mg/dL) in normal GENETIC DEFECTS IN CARBOHYDRATE uncentrifuged coagulated blood METABOLISM ➔ At refrigerated temp (4deg Celsius), glucose is Von Gierke Disease Glycogen buildup in the metabolized at the rate of about 1-2 mg/dL/hr (glucose-6-phosphotase liver due to inhibition of ➔ WBC and RBC metabolize glucose resulting to deficiency type 1) hepatic glycogenolysis decrease value in clotted, uncentrifuged blood Galactosemia Inhibition of (galactose-1-phosphate glycogenolysis Chemical Method uridylic transferase Oxidation Reduction Method deficiency) ➔ Alkaline Copper Reduction Methods ◆ Principle: Reduction of cuprous oxide in hot alkaline solution by glucose DIAGNOSIS OF PATIENTS WITH GLUCOSE METABOLIC ALTERATIONS Glucose Alkaline Copper Tartrate Cuprous Ions Heat Methods ➔ Fasting blood glucose Folin Wu Method ➔ POC Cuprous Ions + Phosphomolybdate ➔ 2-hr Post Prandial Sugar ➔ OGTT ➔ HbA1C ➔ Ketone Phosphomolybdic Acid or ➔ Microalbuminuria Phosphomolybdenum Blue Diabetes ➔ Mellitus Nelson Somogyi ◆ Glucose levels and insulin ○ Copper reduction method (uses BaSO4 ◆ Type 1.2, GDM to remove saccharides) ◆ Type 1 autoimmune (autoantibodies Glucose + Arseno molybdic Acid ◆ Type 2: lifestyle ◆ GDM: pregnant women ➔ Insipidus ◆ Glucose levels and ADH (vasopressin) Arseno molybdenum Blue ◆ Nephrogenic - kidney ◆ Neurogenic - brain Neocuproine Method (2,9 Dimethyl 1, 20 Diagnosis of Glucose Metabolic Phenanthroline Hydrochloride) Alterations Cuprous Ions + Neocuproine ➔ WB glucose concentration 11% lower than plasma ➔ Serum or plasma must be refrigerated and separated from the cells within 1hr Cuprous-Neocuproine Complex ➔ Sodium fluoride (gray top) can be used to inhibit (Yellow or Yellow-Orange) glycolytic enzymes ➔ FBG should be obtained in the morning after 10 Benedict’s Method (Modification of hours fasting (not longer than 16 hours) Folin-Wu NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB ○ Used for the detection and quantitation ➔ Hemolysis affects hexokinase method – false low of reducing substances in body fluids glucose value like blood and urine ➔ Hemolyzed samples containing >0.5g ○ Uses citrate or tartrate as stabilizing hemoglobin/dL are unsatisfactory for hexokinase agent because phosphate esters and enzymes released ➔ Alkaline Ferric Reduction Method (Hagedorn from RBC interfere with the assay generating Jensen) NADH. ◆ Reduction of a yellow ferricyanide to a colorless ferrocyanide by glucose Dextrostics (cellular strip) (inverse Colorimetry) ➔ Important in establishing correct insulin amount for next dose Condensation Method ➔ Effective in reducing the rate of development of ➔ Ortho toluidine (DuBowski Method) diabetic complications ➔ Impregnated w/ glucose oxidase, peroxidase, Glacial HAC and chromogen Glucose + Glycosylamine + Aromatic Amines Schiff’s base heat Enzymatic Method ➔ Acts on Glucose but not on other sugars and not on other reducing substances Glucose Oxidase Method ➔ Measures β-D glucose ➔ Also measures CSF glucose ➔ Columetric Glucose oxidase Mtd (Saifer Samples for Glucose Measurement Gernstenfield) RBS – random - Requested during insulin ◆ β-D glucose + O2+H2O – glucose oxidase - blood sugar shock, hyperglycemic > gluconic acid + H2O2 ketonic coma ◆ H2O2 + chromogenic subs. – peroxidase -> FBS – Fasting - NPO (non-Per Orem) at least oxidized chromogen + H2O Blood Sugar 8 hrs before the test ➔ Polarographic Glucose Oxidase 2-Hr PPBS – 2 ◆ Measures rate of oxygen consumption hour post w/c is proportional to glucose prandial blood concentration sugar ◆ Glucose oxidase in the reagent catalyzes GTT – Glucose - Multiple blood sugar test the oxidation of glucose by oxygen under Tolerance Test - To determine how well the first order conditions, forming hydrogen body metabolizes glucose peroxide over a given time required ◆ The hydrogen peroxide is prevented from re-forming oxygen by adding molybdate, Kinds of Glucose Tolerance Test iodide, catalase, and ethanol Oral Glucose Tolerance Test ➔ Janney Isaacson Method (single-dose method) Hexokinase Method ◆ Most common ➔ Most specific glucose method; reference method ➔ Exton Rose Method (Divided oral dose or Double- ➔ Plasma collected using heparin, EDTA, fluoride, dose method) oxalate or citrate may be used for this test ➔ Other samples: urine, CSF, and serous fluids Added Plasma Glucose after OGTT: ➔ Glucose + ATP – hexokinase -> glucose 6-PO4 + ➔ 30 mins = 30-60 mg/dL above fasting ADP ➔ 1 hour = 20-50 mg/dL above fasting ➔ Glucose 6-PO4 + NADP+ - G-6-PD -> NADPH + H+ + ➔ 2 hours = 5-15 mg/dL above fasting 6-phosphogluconate ➔ 3 hours = fasting level or below NOTES Intravenous Glucose ➔ Elevated amounts of bilirubin, uric acid, and ➔ Used for diabetes patients with gastrointestinal ascorbate – false decrease values of glucose disorders (glucose oxidase method) NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 01 - MCC1 31 LAB ➔ 0.5g of glucose/kg body weight given within 3 ➔ 3-6% of Hgb id glycosylated; 18-20% is prolonged mins) administers IV hyperglycemic ➔ Fasting sample is also required ➔ 7% HbA1C = cutoff value ➔ The first blood collection is after 5 mins of IV glucose Methods of HbA1C Measurement ➔ Indications: ➔ Based on charged differences between ◆ Those who are unable to tolerate a large glycosylated and non-glycosylated hemoglobin CHO load ➔ Structural characteristics of gyrogroups on ◆ Those with altered gastric physiology hemoglobin ◆ Those who had undergone previous operation or surgery in the intestine Diagnosis of Glucose Metabolic Alterations ◆ Those with chronic malabsorption syndrome ➔ Glucose Load ◆ 75g (WHO standard glucose load) ◆ 100 gms ◆ 1.75 g of glucose/kg body weight (children) Procedure for OGTT ➔ The patient should avoid exercise, eating, drinking (except water), and smoking during the test ➔ For non-pregnant women and adults, only the fasting and 2-hr sample may be measured (or according to the physician’s request) 1. Collect the fasting blood sample (urine may also be collected) 2. Instruct the patient to drink the glucose load within 5 mins) Fructosamine 3. Collect blood sample after 30 mins, 1 hour,2 hours, ➔ Also called glycosylated or glycated 3 hours, respectively albumin/plasma protein ketoamine ➔ It is a reflection of short-term glucose control (2- Criteria for Fasting Plasma Glucose (FPG) 3 weeks) ➔ Non-diabetic (Prediabetes) = or = 126 mg/dL variants (Hb S or Hb C_ - decreased RBC lifespan ➔ It should not be measured in cases of low plasma HbA1C albumin ( 4.5 to 8.0% ➔ For every 1% change in the HbA1C value, 35mg/dL is added to plasma glucose ➔ Older RBC and iron deficiency Anemia – High HbA1C levels ➔ Not suitable for patients with shortened RBC lifespan disorders – low HbA1C value ➔ Dietary status on the day of the tst has no effect on HbA1C NOVELO, ELIZA LUTH YNGENTE CLINICAL CHEMISTRY 1 Adapted from: PPT’s LECTURE 4: LIPIDS & LIPOPROTEINS OUTLINE Note: I. Lipoprotein physiology and metabolism Measuring blood cholesterol level not need fasting? II. Lipid Disorders Cholesterol level is not affected by single meal but affected by long term pattern of eating (change from high fat diet to low fat diet for several weeks) Cholesterol level is elevated during pregnancy (till 6 weeks after delivery) FATTY ACIDS Some drugs are known to increase cholesterol levels as anabolic steroids, Linear chains of C—H bonds that terminate with a carboxyl group (—COOH) beta blockers, epinephrine, oral contraceptives and vitamin D. Majority Of plasma fatty acids are instead found as a constituent of triglycerides or phospholipids. Most fatty acids are synthesized in the body from carbohydrate precursors, TOTAL CHOLESTEROL (TC) except linoleic and linolenic acids, which are referred to as essential fatty acids Directly linked to risk of heart and blood vessel disease. Goal values: TRIGLYCERIDES o 75-169 mg/dL for those age 20 and younger Contain three fatty acid molecules o 100-199 mg/dL for those over age 21 attached to one molecule of glycerol PREPARATION: by ester bonds in one of three This test may be measured anytime of the day without fasting. However, if the stereochemically distinct bonding test is drawn as part of a total lipid profile, it requires a 12-hourfast (no food or positions drink, except water). For the most accurate results, wait at least two months after From plant sources, such as corn, a heart attack, surgery, infection, injury or pregnancy to check cholesterol levels. sunflower seeds, and safflower seeds, are rich in polyunsaturated CHYLOMICRONS fatty acids and are oils at room The largest and the least dense of the lipoprotein particles temperature Turbidity or milky appearance of postprandial plasma specimens Triglycerides from animal sources contain mostly saturated fatty acids and are Principal role of chylomicrons is the delivery of dietary lipids to hepatic and usually solid at room temperature. peripheral cells Triglyceride is body storage form of fat and energy Most TG found in adipose tissue GENERAL LIPOPROTEIN STRUCTURE Give energy in ease of absence of carbohydrates Some triglycerides circulate in the blood to provide fuel for muscles to work. VERY-LOW DENSITY LIPOPROTEIN INTERMEDIATE-DENSITY Extra triglycerides are found in the blood after meal TG "gut" >>>> blood>>>> LIPOPROTEINS adipose Elevated in obese or diabetic patients. Level increases from eating simple sugars Produced primarily by the liver Also referred to as VLDL remnants, or drinking alcohol. Associated with heart and blood vessel disease. and contains Apo BIOO, the main normally only exist transiently TG test needs 12 hrs fasting because its level is affected by meal (fatty meal, high apolipoprotein, Apo E, and Apo during the conversion of VLDL to carbohydrates meal) Cs; like chylomicrons, they are LDL Level should be: Less than 150 mg/dl also rich in triglycerides IDLS are not typically present in High TG leads to fatty liver