Medical Technology Licensure Exam Review: Clinical Chemistry PDF

MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW CLINICAL CHEMISTRY Lecturer: Roderick Balce Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King EQUIPMENTS AND SUPPLIES...

MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW CLINICAL CHEMISTRY Lecturer: Roderick Balce Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King EQUIPMENTS AND SUPPLIES 1. Gravimetric - Weight of distilled water delivered using an analytical TYPES OF GLASS MATERIAL Calibration balance; most accurate - Primarily characterized in terms of thermal and chemical resistance (similar to - Calibration temperature: 20oC 1. Borosilicate - Tolerates heating and sterilization for lengthy periods volumetric 2. Spectrophotometric (silica-containing) - May cloud with strong alkali and is subject to glasswares) - Absorbance of colored solution delivered scratching - Examples of colored solution: Potassium dichromate or - Most common brand: Pyrex; Other: Kimax para-nitrophenol 2. Aluminosilicate - Has higher silica content than Borosilicate; 6x (silica-containing) stronger than borosilicate SEMI-AUTOMATIC MICROPIPETTORS - Better able to resist scratching and alkali attack Air-Displacement Pipettes Positive-Displacement Pipettes - Most common brand: Corex - Always have air cushion (dead - Piston is in direct contact with the 3. Soft glass - Low thermal resistance (cannot be subjected to volume) between the pipette piston sample, therefore, there is no air cushion (Boron-free glass) heating) and the liquid sample and the aspiration force remains constant - Can be used with strong acids and alkali 4. Flint glass - The piston is integrated into the - The disposable piston is part of the tip - Most inexpensive (cheapest); commonly used to lower part of the pipette. When the (along with piston seal and disposable (Soda lime glass) manufacture disposables push button is pressed, air is capillary). As the push button is - Made of the oxides of silicon, calcium, and sodium displaced by the piston. The released, the piston moves up and the - Disadvantage: releases alkali causing errors in volume of air displaced is ambient pressure forces the desired certain determinations equivalent to the volume of liquid volume of liquid through the orifice into 5. Low actinic glass - Reduces light transmission aspirated. the capillary. (Amber-colored/ - Used to contain photosensitive substances - Recommended for aqueous - Recommended for very viscous or high coated glass) samples and for general laboratory density samples such as glycerol and *Silica: provides high thermal resistance or heat tolerance work. blood and other problem samples e.g. volatile, radioactive, corrosive, hot and TYPES OF PLASTIC MATERIALS cold samples Primarily characterized in terms of thermal and chemical resistance 1. Teflon - Most excellent temperature tolerance and Polytetrafluoroethylene unparalleled chemical resistance 2. Polypropylene - Resistant to most chemicals Can be autoclaved - Used for pipet tips, test tubes 3. Polycarbonate - Clear; stronger than polypropylene (resistant to shattering) and has better temperature tolerance - Chemical resistance is not as good - Used for centrifuge tubes and graduated cylinders 4. Polyethylene - Resistant to most chemicals (except Cannot be autoclaved concentrated acids) - Used for disposable transfer pipets, test tubes, bottles 5. Polystyrene - Rigid, clear; used for test tubes, graduated tubes 6. Polyvinyl chloride - Soft and flexible but porous - Frequently used as tubing CENTRIFUGES GLASS PIPETTES 1. Horizontal or Swinging bucket CALIBRATION - Tubes attain a horizontal position during spinning and a PIPET TYPE DESIGN USE MARKS vertical position when at rest 1. Volumetric Single calibration Nonviscous - Capable of speeds up to about 3000 rpm (limited by heat (most TD/SD mark samples; buildup due to air friction) accurate) Transfer standards pipets - Flat, tightly packed sediment 2. Ostwald- Viscous fluids 2. Fixed-angle or angle-head TD/BO Folin 3. Serologic Graduated Graduations down Serial dilution; - Tubes are at fixed angle when rotating TD/BO - Capable of higher speeds with much less heat build-up or to tip measuring 4. Mohr Measuring Graduations reagents - Capable of speeds up to 7,000 rpm or 9,000 g (RCF) pipets TD/SD - Sediment is slanted between two marks Types Additional: - Disadvantage: not recommended for procedures which require decantation - To contain = hold but do not deliver the volume they contain because of the 3. Ultracentrifuge tendency of liquids to cling to the inside wall of the glass - Used to separate layers of different specific gravities - Tolerance value is proportional to capacity and inversely proportional to accuracy - Usually refrigerated to counter the heat produced due to friction - Transfer pipet = only 1 calibration mark; no subdivisions - Capable of speeds up to 100,000 rpm or 165,000 g (RCF) - Graduated/measuring pipet = there are subdivisions; you can measure and 4. Cytocentrifuge deliver fractions of the capacity - Used for body fluid cell counts - Self-draining pipet (SD): contents drain by gravity; without the need of aspirator bulb - Speed = 200-2,000 rpm (low speed centrifuge) - Blow-out (BO): any amount left in the tip after drainage is blown out to the - Meant to increase the cell yield, especially for CSF analysis receiving vessel; has etched rings near the mouth - rpm: revolutions per minute - RCF (g): relative centrifugal force (gravity units) SEMI-AUTOMATIC MICROPIPETTORS Units used - S: Svedberg unit; unit used in ultracentrifugation 1. Air displacement - rpm to RCF = 1.118 x 10-5 x radius of centrifuge head in - Uses suction to draw sample into a disposable centimeter x rpm2 polypropylene tip - Parameters - Piston does not come in contact with liquid (air gap) o Speed: checked using tachometer or strobe light 2. Positive displacement Types Quality o Timer: checked using a stopwatch - Operates like hypodermic syringe control o Temperature: if refrigerated; checked using thermometer - Piston is in direct contact with the liquid being aspirated - Frequency - Tips are reusable but must be rinsed out o Every three months or quarterly - Components: disposable piston, disposable capillary (glass or Teflon), piston seal  REAGENTS TYPES OF REAGENT WATER Characteristic Type I Type II Type III CHEMICALS Maximum 1. Analytic Grade Reagent - High degree of purity suitable for use in most colony count 1 cm frequency coupled plasma – Mass spectrometry)  PPE recommendations  Handling  Emergency and first aid procedures  Storage and transportation precautions  Chemical manufacturer’s name, address, and telephone number  Special information section CHEMICAL STORAGE REQUIREMENTS SUBSTANCE STORED SEPARATELY Flammable liquids Flammable solids Mineral acids Organic acids Caustics Oxidizers COLOR CODING SCHEME FOR HEALTH CARE WASTES Perchloric acid Water-reactive substances Type of Waste Color of Container/Bag Air-reactive substances All other chemicals 1. Non-infectious dry waste Black Heat-reactive substances requiring refrigeration 2. Noninfectious wet waste Green - TLV (Threshold Limit Value) 3. Infectious and pathological wastes Yellow o Allowable exposure value during an 8-hour shift 4. Chemical and Pharmaceutical wastes Yellow with black band o Inversely proportional to toxicity 5. Radioactive wastes Orange o ↑ Toxicity = ↓ TLV 6. Sharps and pressurized containers Red - NFPA (National Fire Protection Agency) o Standard hazards identification scheme FIRE HAZARD Combustible Class Examples Extinguishers Materials Pressurized water (A) Ordinary Wood, paper, cloth, Foam A combustibles rubber, etc. Dry chemical (multipurpose; ABC powder) Foam Flammable Grease, oil, paint, Dry chemical (ABC) B liquids and solvents, gasoline Carbon dioxide (BC) gases Halon Dry chemical (ABC) Carbon dioxide (BC) (also good for extinguishing Electrical Electrical panel, computers because it does C equipment motor, wiring, etc. not leave residue) Halon (recommended for computers) Combustible Magnesium, Special dry chemical D metals aluminum, etc. Sand Cooking oils, animal Wet chemical K Oil, grease fats, vegetable oils (saponifying agents) - Actions to take in the event of fire: o Rescue, Alarm, Confine/Contain, Extinguish/Evacuate - Correct use of fire extinguishers: DEGREES OF HAZARD (GENERAL) o Pull pin, Aim Hose/Nozzle, Squeeze Handle, Sweep from Side to Side 0 Non-hazardous 1 Slightly hazardous 2 Moderately hazardous 3 Seriously hazardous 4 Extremely hazardous  LABORATORY MATHEMATICS CALCULATIONS USED IN REAGENT PREPARATION Applications: 1. Calculation used in reagent preparation To determine the molarity of solution given 𝑚𝑜𝑙 𝑔/𝐿 M or = 2. Converting values from conventional to SI unit and vice versa the w/v (g/L): 𝐿 𝑀𝑊 To determine the normality of solution given N or 𝐸𝑞 = 𝑔/𝐿 UNITS OF MEASURE the w/v (g/L): 𝐿 𝐸𝑊 𝑀𝑊 SELECTED SI AND NON-SI UNITS EW = 𝑣𝑎𝑙𝑒𝑛𝑐𝑒 BASE QUANTITY NAME SYMBOL To determine the molarity of solution given 𝑁 M= ;N>M Length Meter m the normality: 𝑉 Mass Kilogram kg N > M: Valence is greater than Time Second s or equal to 2 Electric current Ampere A N=M: Valence is equal to one Thermodynamic Kelvin K To determine the normality of solution given temperature MxV the molarity: Amount of substance Mole mol 𝑔 To determine the w/v (g/L) given the molarity: = M x MW Luminous intensity Candela cd 𝐿 SELECTED DERIVED UNITS To determine the w/v (g/L) given the 𝑔 = N x EW Frequency Hertz Hz normality: 𝐿 Force Newton N To compute for volume or concentration of C1V1 = C2V2 Celsius temperature Degree Celsius oC diluted solutions: Catalytic activity Katal kat SAMPLE PROBLEMS: SELECTED ACCEPTED NON-SI What is the molarity of a 1-liter solution containing 73 g hydrochloric acid? Time, minute 60s min Liter (volume) 1 dm3 = 10-3 m3 L Angstrom 0.1nm = 10-10m Å PREFIXES USED WITH SI UNITS FACTOR PREFIX SYMBOL 10-18 atto a 10-15 femto f What is the molarity of a 1-liter solution containing 10 g sodium hydroxide? 10-12 pico p 10-9 nano n 10-6 micro u 10-3 milli m 10-2 centi c What is the normality of a 1-liter solution containing 24.5 g H2SO4 (sulfuric acid)? 10-1 deci d What is its molarity? 101 deka da 102 hector h 103 kilo k 106 mega M 109 giga G 1012 tera T 1015 peta P 1018 exa E What is the molarity of 0.3N H3PO4? EXPRESSIONS OF CONCENTRATION number of moles of Molarity 𝑔𝑟𝑎𝑚𝑠 (𝑔) 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 What is the normality of 3 M sulfuric acid? 𝑔𝑟𝑎𝑚 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 solute per liter (L) of solution number of moles of Molality 𝑔𝑟𝑎𝑚𝑠 (𝑔)𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 How many milliliters of 70% ethanol are needed to prepare 35 mL of 40% solute per kilogram 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (kg) of solvent ethanol? number of equivalent 𝑔𝑟𝑎𝑚𝑠 (𝑔)𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 weights of solute per Normality 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 (𝐿) liter (L) of solution amount of solute per 100 total units of % w/v % 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 How much serum is needed to prepare 2 mL of a 1:10 dilution? solution; 100 often expressed as g/100 mL or g/dL volume of sample divided by total volume Dilution 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒/𝑠𝑎𝑚𝑝𝑙𝑒 A mixture consists of 4 parts of Solution A and 6 parts of Solution B. How many mL of solution; expression 𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 of Solution A and B are needed to prepare a 3000 mL of the mixture? of relative concentration amount of something 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 relative to another; Ratio 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 usually expressed as part per part  UNITS AND CONVERSION FACTORS FOR SELECT CHEMISTRY GAUSSIAN/NORMAL DISTRIBUTION ANALYTES - Data points are distributed symmetrically around the mean (bell curve) with ANALYTE CONVENTIONAL RECOMMENDED CONVERSION most values close to the center; mean, median, and mode are identical UNIT SI UNIT FACTOR - 95% confidence limit: desired; control observations should fall within this limit Glucose 0.0555 BUN 0.357 Uric acid 0.0595 mg/dL Cholesterol 0.026 mmol/L Triglyceride 0.0113 Calcium 0.25 Phosphorus 0.323 Magnesium (Mg2+) 0.5 mEq/L Na+ /K+ /Cl-/HCO3- 1 Creatinine 88.5 mg/dL Bilirubin 17.1 µmol/L - 68-95-99 rule (if the data set is normally distributed) Ammonia 0.588 o 68% of values fall within 1SD Iron µg/dL 0.179 o 95% of values fall within 2SD Cortisol 0.0276 o 99% of values fall within 3SD T4 12.9 T3 ng/dL nmol/L 0.0154 REFERENCE INTERVAL STUDIES Folic acid ng/mL 2.27 - Verifying a reference interval Vitamin B12 pg/mL pmol/L 0.738 o Done to confirm the validity of an existing or published RI for an analyte Proteins (TPAG) g/dL g/L 10 o Requires at least 20 study individuals mg/dL (for fibrinogen 0.01 o RI is adopted if QA > QC - Quality system: refers to all of the laboratory’s policies, processes, procedures, and resources needed to achieve quality testing - Quality assurance/assessment: process by which the laboratory ensures quality results by closely monitoring pre-analytical, analytical, and post – analytical stages of testing; under QA is QC QUALITY ASSURANCE - 3 Phases of QA WESTGARD CONTROL RULES o Pre-analytical: include test requisition, patient preparation, patient Rule Description Use identification, specimen collection, labeling, specimen transport, One control value exceeds Warning rule; trigger the handling, and processing the 𝑥̅ + 2SD inspection of the control o Analytical: include reagents, preventive maintenance of equipment, data calibration, analysis of samples, and quality control o Post-analytical: include verification of calculations and reference Accept the run if there is no Detect random error ranges, flagging and notification of panic/critical values, delta checks, 12s other violation reporting of results - Delta Check: an algorithm in which a current laboratory result is compared with results obtained on a previous specimen from the Not recommended for use same patient; important for detecting pre-analytical and analytical as a rejection rule because errors of high false alert rate One control value exceeds Detects imprecision or 13s QUALITY CONTROL the + 3SD systematic bias - Goal of QC: ensure reliability; to maintain both accuracy and precision Range between two levels Detects imprecision or - Accuracy R4S of control within a run random error o closeness of the result to the true or actual value exceeds 4SD - Precision Two consecutive values Detects bias o ability to produce a series of results that agree closely with each other 22s exceed the + 2SD in the o commonly expressed in terms of coefficient of variation; inversely same direction Detect systematic error proportional with CV Four consecutive control Detects bias trend o also called reproducibility 41s values exceed 𝑥̅ + 1SD - Internal/lntralaboratory QC Eight consecutive o Involves the analysis of at least 2 levels of control every 24 hours for at least 20 days observations for the same 81s o Important for the daily monitoring of accuracy and precision of analytical level of control exceed 1SD methods in the same direction - External/Inter-laboratory QC Ten consecutive control Detects bias; but not o Proficiency testing (EQAS or NEQAS in the Philippines) 10x values are on the same side recommended o Involves testing samples of unknown concentration of analytes sent of the mean periodically by regulatory agencies (national reference laboratories) to CUSUM Cumulative sum of SDI Detects bias trend participating laboratories Exponentially weighted  Lung Center of the Philippines – NRL for Clinical Chemistry EWMA moving average o SDI >2.0 indicates poor performance; outliers; not in agreement with other participating laboratories 𝐿𝑎𝑏 𝑚𝑒𝑎𝑛 − 𝑃𝑒𝑒𝑟 𝑔𝑟𝑜𝑢𝑝 𝑚𝑒𝑎𝑛 𝑺𝑫𝑰 = 𝑃𝑒𝑒𝑟 𝑔𝑟𝑜𝑢𝑝 𝑆𝐷 ANALYTICAL VARIATION TYPES OF ERROR IN LABORATORY TESTING RANDOM ERROR SYSTEMATIC ERROR - due to chance or an - influences observations consistently unpredictable cause in one direction - affects precision - affects accuracy - affects few control - persists until troubleshooting is observations or samples performed - affects all observations in a run - shift and trend - Examples: - Examples: o instrument instability o deterioration of reagents or o dirty optics change in reagent lot, o improper mixing of o unstable reagent blanks sample and reagent o calibration error o temperature and o changes in the standard voltage fluctuations concentration o operator variability o contaminated control solutions e.g. pipetting error o diminishing lamp power or deteriorating light source o pipette or maintenance error - Patterns in QC charts that indicate errors o Shift  formed by control values that distribute themselves on one side or either side of the mean for 6 consecutive days; abrupt or sudden change  most common cause: calibration error o Trend  formed by control values that continue to increase or decrease (gradually) over a period of 6 consecutive days passing through the mean  most common cause: deterioration of reagents o Outliers  highly deviating control values caused by random or systematic errors; loss of precision QUALITY MANAGEMENT APPROACHES BLOOD COLLECTION AND SPECIMEN CONSIDERATIONS VENIPUNCTURE - GENERAL PRECAUTIONS AND TECHNIQUES o Sites of puncture:  Veins in the antecubital fossa  Median cubital  Cephalic  Basilic (close to the median nerve and brachial artery) - Internal benchmarking  Alternatives: dorsal hand, wrist, ankle, and foot veins o setting standards and try to meet or exceed such standards for your o Tourniquet application: 5,000 ft) = ↓ draw volume  Depriving the palm/hand with oxygenated blood, after which, the ulnar artery is released and complete blood return is - Humidity evaluated/observed o Affects plastic tubes only (permeable to water vapors) o Amount of anticoagulant (heparin): 0.05mL/mL of blood (excessive o ↑ Humidity = migration of water vapor inside a tube amount leads to falsely decreased pH) o ↓ Humidity = escape of water vapor from a tube containing a wet additive SPECIAL SITUATIONS IN PHLEBOTOMY - Light - IV o Affects glass CTAD (Citric Acid/Citrate, Theophylline, Adenosine, o Alternative: use the opposite arm, perform fingerstick Dipyridamole) tube, a photosensitive additive for coagulation testing o Turn off the IV line for 2 minutes to avoid contamination and dilution which minimizes platelet activation after blood collection - CVA (Central Venous Access Collection) o Dipyridamole prevents platelet activation but is photo sensitive o Discard the first 3-5mL of blood which are likely to contain the infused medications - Cannula/Fistula, Mastectomy o Draw blood from the opposite arm - Burns, edema, scars, sclerosed veins, tattoos o Select another site - Hematoma o Draw below PHLEBOTOMY COMPLICATIONS - Vascular: bleeding from venipuncture site and hematoma formation - Infections: second most common complication - Anemia (iatrogenic): results from blood loss for testing; particular problem with pediatric patients - Neurologic: post-phlebotomy seizure or pain - Cardiovascular: orthostatic hypotension, syncope, shock, and cardiac arrest - Dermatologic: allergic reaction to iodine, adhesives, or latex  ANALYTICAL TECHNIQUES AND INSTRUMENTATION - Photodetector/Photomultiplier o Light transmitted is the light which was not absorbed SPECTROPHOTOMETRY o Transforms light into an equivalent amount of electrical energy which will be quantified by the meter (part of the read-out device) - Measurement of the amount of light from a specific wavelength of the o Most commonly used and most sensitive: photomultiplier electromagnetic wavelength - Regions of Interest in Electromagnetic Spectrum: QUALITY ASSURANCE o Ultraviolet light (700nm) - Absorbance check: done using glass filters and solutions that have known absorbance values - Linearity: a change in concentration results in a straight-line calibration curve (Beer’s Law); conformity with Beer’s law = as the concentration increases, absorbance should also increase (using neutral density filters and dichromate solution) - Stray light: any wavelength outside the band of interest; causes absorbance error and loss of linearity; detected using sharp cutoff filters (ex. NiSO4, NaNO2, - Relationship between wavelength and energy (inverse) and acetone) o Wavelength: distance between 2 successive peaks o In the image above: DOUBLE-BEAM SPECTROPHOTOMETRY  Left to Right: Increasing wavelength but decreasing energy and - Designed to compensate for variations in intensity of the light source by frequency splitting the light beam from the lamp and directing one portion to a reference  Gamma rays and X-rays are called ionizing radiation which damage DNA because they are highly energetic cuvet and the other to the sample cuvet  Right to left: Shorter wavelength, but higher energy and frequency - Only 1 light source but split into two (strikes the reference cuvet and sample  Microwave and radio waves cuvet); meant to compensate for variations in the intensity of the light source - Beer's Law (Beer-Lambert’s Law): The concentration of a substance is - 2 types: directly proportional to the amount of light absorbed and is inversely o Double-beam-in-space: 2 cuvets are struck by light at the same time; 2 proportional to the logarithm of transmitted light photodetectors are used, one for each cuvet o Absorbance (A) = abc  a = molar absorptivity  b = light path (diameter of cuvette) in centimeters; 1cm  c = concentration o A = 2 — log %T (percent transmittance)  %T = 10(2-A) o Given that A = abc, the concentration of an unknown analyte can be obtained by running a standard whose concentration is known, cu = Aucs/As 𝐴𝑢 (𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 𝑠𝑎𝑚𝑝𝑙𝑒) 𝑥 𝐶𝑠 (𝑘𝑛𝑜𝑤𝑛 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑) 𝐶𝑢 = 𝐴𝑠 (𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑) SOLUTIONS USED IN SPECTROPHOTOMETRY - Blank o Double-beam-in-time: 1 photodetector; light is alternately passed o Distilled water, reagent, or sample used to subtract absorbances not due through the sample and the reference cuvet by using a chopper to the analyte of interest o Sets the spectrophotometer to 0 absorbance - Standard/Calibrator o Substance of known purity and concentration used to calibrate an assay method or determine the concentration of the unknown analyte (for a single analyte only) - Control o Solution containing various analytes with known target values  Assayed = published reference range of target values  Unassayed = laboratory should set or determine the target values o Monitor the performance of an assay to ensure accuracy and precision o Bovine origin; preferred because of biohazard issues associated with human sera o Must not be hemolyzed, Non-lipemic/non-lactescent, Non-icteric FLUOROMETRY COMPONENTS OF A SINGLE-BEAM SPECTROPHOTOMETER: - Also known as Molecular Emission Spectroscopy - Principle: Measurement of the amount of light emitted by excited molecules - Important components: gas discharge lamps (source); 2 monochromators or filters (primary and secondary monochromator); Photomultiplier tube (detector) o Primary monochromator: selects wavelength of light from the source (excitation energy) - Advantages: High specificity and sensitivity (a thousand-fold more sensitive - Light source than spectrophotometry) o provides the light which the analyte in the sample will absorb (polychromatic; consists of various wavelengths) - Disadvantage: Quenching o Ex. Deuterium, xenon, tungsten (for visible and near infrared regions) (any decrease in - Entrance slit fluorescence due to o ensures that the light that enters the monochromator only comes from the prolonged exposure to UV light source; prevents stray light light, increased - Monochromator temperature, pH changes, o selects a specific wavelength of the light emitted by the source (makes presence of contaminants, polychromatic light monochromatic) very high analyte o Prisms and diffraction gratings (holographic and diffraction gratings; concentration [loss of diffraction gratings are commonly used) linearity]) - Exit slit o further prevents stray lights and controls the band pass o ensures that only a narrow fraction of the spectrum will strike the sample CHEMILUMINESCENCE - Cuvet - Principle: Measurement of light emitted by a chemical reaction; involves o Should be transparent to the wavelength of interest oxidation of an organic compound catalyzed by an enzyme, a metal, or hemin o Light should pass through and not absorb any part of it - Substrates used: luciferin, luminol, acridinium ester o Can be quartz, plastic, or glass. - Advantages: subpicomolar detection limits (sensitive), speed, ease of use,  Quartz is for UV and IR simple instrumentation  Plastic is for UV and visible light  Glass is for visible light  If the wavelength is UV, don’t use glass, because glass absorbs UV SCINTILLATION COUNTING CARBOHYDRATES - Detection of scintillations (flashes of light) using a PM tube and counting of the electrical impulses HORMONAL REGULATION - Label is a radioisotope Insulin - The only hypoglycemic agent; lower plasma glucose level o Crystal scintillation: gamma counter; ligands labelled with iodine 125 or - Pancreatic hormone (produced by the beta cells of the islets iodine 131 of Langerhans) o Liquid scintillation: beta counter; ligands labelled with hydrogen 3 of - Promotes cellular uptake of glucose, glycolysis, glycogenesis, carbon 14 and lipogenesis - Inhibits glycogenolysis OTHER IMMUNOASSAY TECHNIQUES Glucagon - Major hyperglycemic hormone; increases glucose level - Microparticle capture enzyme immunoassay (MEIA) - Pancreatic hormone (produced by the alpha cells of the islets o uses microparticles as solid phase, an enzyme label, and a fluorogenic of Langerhans) substrate - Promotes gluconeogenesis (synthesis of glucose from non- o heterogenous immunoassay technique carbohydrate sources) and glycogenolysis (reverse of o separation technique is used glycogenesis) o application: for high molecular weight substances - Inhibits glycolysis o Microparticle enzyme immunoassay: technique that is similar to ELISA ACTH - Promotes gluconeogenesis and glycogenolysis in that there is a double antibody system that forms a sandwich with the - Anterior pituitary hormone analyte of interest e.g. hormones Growth - Inhibits glycolysis hormone - Anterior pituitary hormone - Fluorescence polarization immunoassay (FPIA) o uses polarized light to excite a fluorescent label Thyroxine - Promotes intestinal absorption of glucose o rate of rotation is inversely proportional to the degree of polarization and - Produced by the thyroid analyte concentration Cortisol - Promotes gluconeogenesis and lipolysis o homogenous immunoassay is used - Produced by the adrenal cortex o separation is not needed Epinephrine - Promotes glycogenolysis and lipolysis o application: low molecular weight substances - Produced by the adrenal medulla - Both used in therapeutic drug monitoring (specially for immunosuppressants) HYPOGLYCEMIC DISORDERS TURBIDIMETRY AND NEPHELOMETRY - Hypoglycemia: plasma glucose level < 50 mg/dL - Common application: measurement of immune - Panic value for hypoglycemia: 30 2. FRUCTOSAMINE kg/m2) - Used to assess glycemic control over the past 2-3 weeks in cases when HbA1c - Habitually physically inactive is unreliable - High-risk minority population - Represents glycosylated albumin - Hypertension (>140/90 mmHg) - Has the advantage of using serum samples - Risk factors Dyslipidemia (HDL: 200mg/dL) - Reference values: 205-285 umol/L - History of CVD - HbA1C >5.7% 3. WHOLE BLOOD GLUCOSE - IGT or IFG - Difference is significant between plasma glucose and whole blood glucose - History of GDM or delivering a baby o Whole blood glucose is 10-15% lower than plasma glucose weighing more than 9 lb o When interpreting blood glucose levels, you should not use reference - PCOS interval for plasma glucose - Other clinical conditions o Plasma glucose is less than 5% than serum glucose associated with insulin resistance o Capillary blood glucose is higher than venous blood glucose by 2-5mg/dL (e.g. severe obesity and acanthosis - Most commonly analyzed using POCT devices at home or at the patient’s nigricans) bedside - Opposite of insulinoma - Nonketotic hyperosmolar state - Recommendation: 3-4 times per day including pre-prandial and post-prandial; - C-peptide levels low then (coma) possible (plasma levels - ADA glycemic goals: Clinical findings undetectable become uncontrolled) o Pre-prandial: 70-130 mg/dL - Autoantibodies - Panic value for hyperglycemia: >500 o Post-prandial: < 180 mg/dL - Diabetic Ketoacidosis mg/dL METHODS OF GLUCOSE MEASUREMENT (increased beta- hydroxybutyric acid and SPECIMEN CONSIDERATIONS other ketone bodies) - Insulin injection - Lifestyle changes - Use of NaF (Sodium Fluoride) or SST to prevent glycolysis (only used when prompt analysis is not possible; if specimen will stand for a considerable Therapy - Oral hypoglycemic agents amount of time) - May require insulin o Gray top: Sodium fluoride o Otherwise, glucose will be lost to glycolysis at a rate of:  7 mg/dL/hour at room temperature TESTS AND DIAGNOSTIC CRITERIA FOR DM  2 mg/dL/hour at 4oC (refrigerator temperature) RPG FPG o Additional! High WBC count will also lead to a substantial decrease in (Random Blood (Fasting Plasma 2-hr PG plasma glucose HbA1C Glucose/ Casual Glucose/Fasting (OGTT) Plasma Glucose) Blood Sugar) - Effect of dextrose contamination o 10% contamination with 5% dextrose will increase plasma glucose by Significant Fasting of at CHO intake of Part of the 500mg/dL results must least 8 hours is >150g/day for diagnostic always be in required 3 days criteria A. CHEMICAL METHODS conjunction with (Non-specific; Based on Redox and Condensation reactions) Important considerations symptoms of Levels that Fasting for 8- Specimen: DM: exceed the 14 hours EDTA whole 1. COPPER REDUCTION - Polyuria cutoff at 2 blood - Polyphagia occasions = DM Standard load - Redox; based on Benedict’s principle, which involves the reduction of cupric - Polydipsia is confirmed of 75 grams Sample is a ions to cuprous ions by glucose in the presence of heat and alkaline or 1.75g/kg lysate from environment; cuprous ions are oxidized by PMA, AMA Indicative but Preferred as body weight the whole - Any reducing agent may replace glucose; falsely increased when there are not diagnostic; confirmatory blood other reducing substances must be test 2 samples are o FoIin-Wu (Colorimetric; blue) confirmed by a collected 2hr post load o Nelson Somogyi (Colorimetric; blue) glucose Normal 200 mg/dL >6.5% symptoms - Gestational DM (100 g; 3-hour OGTT) o Ferricyanide is yellow orange, but becomes colorless when reduced into o Screening test: 50 g; 1-hour OGTT ferricyanide o Diagnosis is made if any 2 or more of the following glucose levels are met or exceeded: 3. CONDENSATION - FPG > 95 mg/dL - Dubowski reaction, intensity of color reaction is proportional to glucose - 1-h PG > 180 mg/dL concentration; colorimetric - green) - 2-h PG > 155 mg/dL - 3-h PG > 140 mg/dL o Reaction of glucose with aromatic amines (ortho-toluidine) MEASURES OF GLYCEMIC CONTROL 1. GLYCOSYLATED HEMOGLOBIN/ HbA1c - Hb A with glucose irreversibly attached to one or both N-terminal valines of the β-chains - Provides an index of average blood glucose levels over the past 2-3 months o Long term monitoring: 2-3 months (lifespan of RBCs) - Unreliable in patients with hemolytic disorders and hemoglobinopathies (shortened RBC lifespan) - Sample: hemolysate (from EDTA whole blood) - Methods: Electrophoresis, chromatography, immunoassays B. ENZYMATIC METHODS (more popular; has high sensitivity and specificity) LIPIDS AND LIPOPROTEINS 1. GLUCOSE OXIDASE COMPONENTS OF LIPOPROTEINS - SURFACE o Apolipoproteins - 2 major isomers in plasma: 65% is β-D-glucose while 35% is α-D-glucose  Protein moieties of lipoproteins (allow their transport in plasma) - Glucose oxidase is only specific to β-D-glucose, so α-D-glucose is converted  Lipids are water-insoluble so the particles must contain protein to beta by mutarotase moieties to facilitate their transport in plasma - Detection Methods:  Basic role: o Peroxidase-coupled/Trinder reaction — subject to many interferences  Structural support; primary/basic role of apolipoproteins, to maintain the integrity of the particle  Aka. Trinder assay  Lipoprotein metabolism (enzyme cofactor or inhibitor)  Colorimetric, spectrophotometric  Ligands for cell receptors  Nonspecific may be affected by interfering agents o Phospholipids  Oxidizing agents: false increase  Primarily makes up the surface layer along with cholesterol;  Reducing agents: false decrease apolipoproteins are just embedded  Chromogen is added which reacts with H2O2, producing a  Amphipathic (has polar/hydrophilic and non-polar groups) colored compound (oxidized chromogen) o Non-esterified cholesterol o Polarographic (Amperometric) - measurement of the degree of O2  30% of cholesterol; also amphipathic consumption using a pO2 (Clark) electrode; requires addition of molybdate and iodide or catalase and ethanol to prevent reformation of - CORE hydrogen from hydrogen peroxide; the amount of oxygen detected is o Triglycerides inversely proportional to glucose  Hydrophobic; insoluble in water o Cholesterol esters/Cholesteryl esters 2. HEXOKINASE  A result of the attachment of fatty acid to non-esterified cholesterol  The formation is catalyzed by LCAT  Esterified or Non-esterified  70% of cholesterol: esterified - Reference method; not subject to interferences and the coupling is highly  When a fatty acid is attached to the cholesterol, it attaches to specific the polar group, therefore the polarity is lost (which explains - G6PD is highly specific why esterified cholesterol is found in the core) - Increase in absorbance at 340nm is proportional to the level of NAPDH and o Free fatty acids level of glucose  Non-esterified fatty acids - Sources of errors:  Fatty acids that are not forming parts of phospholipids and o Hemolyzed sample: falsely decreased result; due to the interference of triglycerides; they form part of the core hemoglobin with the activity of the enzymes in the assay o Icteric samples: false decrease INBORN ERRORS OF CARBOHYDRATE METABOLISM - GALACTOSEMIA o due to deficiency of the enzyme GALT (Galactose-1-phosphate uridyltransferase) o one of the most common enzyme deficiencies o affected children present with mental retardation, failure to thrive, and galactosuria (screened using Clinitest; positive since galactose is a reducing sugar) o part of newborn screening - GLYCOGEN STORAGE DISEASES/GLYCOGENOSES SIGNIFICANT APOLIPOPROTEINS o Hepatic glycogenosis Plasma  usually manifest with hepatomegaly, hypoglycemia, and growth Main Apo Conc. Function retardation distribution (mg/dL)  von Gierke disease (GSD Type Ia): most common; due to glucose- A-I 100-200 HDL LCAT activator 6-phosphatase deficiency  presents with hypoglycemia, ketonuria, lactic acidosis, A-II 20-50 dyslipidemia, and hyperuricemia A-IV 10-20 CM, VLDL, HDL o Muscle glycogenosis B-100 70-125 VLDL, IDL, LDL LDL-R ligand Structural  Manifest with exercise intolerance, muscle cramps, fatigue and Remnant B-48 500 mg/dL - Abell-Kendall method: consists of saponification, extraction, and color development using Liebermann-Burchard - Appearance of plasma (reflects the level of TGs in the sample): - Modified Abell-Kendall; used to be the CDC reference method which o Clear: 300 mg/dL o saponification with alcoholic KOH o Opaque or milky: >600 mg/dL o extraction with petroleum ether (N-hexane) o colorimetry using the Liebermann-Burchard reaction which comprises CLINICAL SIGNIFICANCE OF ABNORMAL RESULTS sulfuric and acetic acids and acetic anhydride - HYPERTRIGLYCERIDEMIA - GCMS (Gas Chromatography-Mass Spectrometry) o Hyperlipoproteinemia types 1, 2b, 3, 4, 5 (all except 2a), nephrotic o Current reference method; specifically measures cholesterol and does syndrome, alcoholism, pancreatitis, hypothyroidism not detect related sterols o Shows good agreement with the gold standard Definitive method using - HYPOTRIGLYCERIDEMIA IDMS o Malnutrition, malabsorption syndrome, hyperthyroidism B. ENZYMATIC METHOD LIPOPROTEIN CHOLESTEROL - peroxidase-catalyzed dye oxidation HDL-C - Cholesterol content of HDL which is routinely measured o Ultracentrifugation – reference method o Direct Method – mostly based on immunoassay; homogenous assays o Two-step method (routinely done): - The enzyme cholesterol esterase (cholesterase) catalyze the hydrolysis of  Precipitation of apoB-containing lipoproteins by polyanion- cholesterol esters liberating free cholesterol divalent cations (e.g. heparin sulfate-Mn2+, dextran sulfate and - Once, the cholesterol is liberated, it is oxidized in the presence of cholesterol sodium phosphotungstate-Mg2+, heparin-Ca2+) oxidase producing cholestenone and H2O2 (hydrogen peroxide).  apoB is precipitated and removed from the sample  Lipid profile requires fasting sample → we’re getting VLDL, IDL, LDL, - Once we have H2O2, the reaction can be coupled with a peroxidase catalyzed reaction. HDL  Purpose of fasting is to clear chylomicrons from the plasma - Oxidation of a chromogen (4-aminophenazone) by H2O2 (one of the products  After centrifugation, HDL is suspended in the supernatant while other of preceding reaction) producing an oxidized chromogen (quinoneimine; red- lipoproteins are in the sediment → supernatant is taken for purple; increased absorbance at 500nm) that is colored and whose color cholesterol determination. intensity is proportional to the level of cholesterol.  POD (Peroxidase)-coupled reaction - C/S (Colorimetric/Spectrophotometric) method [TRINDER REACTION] - Reference values: 1. COLORIMETRIC o High risk for CHD: 400 mg/dL o Errors are noticeable at TG of > 200 mg/dL - NAD is measured as decrease in absorbance at 340nm o Unreliable and unacceptable when the TG level exceeds > 400 mg/dL - Not colorimetric since it uses UV - Total Cholesterol = HDL + LDL + VLDL - NAD does not absorb light at 340nm - Friedewald equation: LDL = TC – (HDL + TG/5) o Use 5 for mg/dL - The reduced form NADH is the one that absorbs light maximally at 340nm o Use 2.175 for mmol/L 2. CONDUCTIMETRIC o This equation is more commonly used - measurement of the increase in conductivity due to NH4+ and CO32- ions o VLDL = TG/5 or 2.175 - De Long equation: LDL = TC – (HDL + TG/6.5) - Non-ionized is hydrolyzed and ions is liberated that will increase the o Use 6.5 for mg/dL conductivity that is proportional to the urea in the sample o Use 2.825 for mmol/L CLINICAL SIGNIFICANCE OF ABNORMAL VALUES - Reference values: - Reference intervals: 6 - 20 mg/dL (BUN) o Optimal: < 100 mg/dL o Near to above optimal: 100 - 129 mg/dL - Panic value: >80 mg/dL o Borderline high: 130 - 159 mg/dL - AZOTEMIA o High: 160 - 189 mg/dL o Prerenal: ↓ renal blood flow/volume (dehydration, hemorrhage, shock), o Very high: >190 mg/dL ↑ protein diet, ↑ protein catabolism, dehydration (relative ↑ in hematocrit) o Renal: ↓ excretion (glomerulonephritis, renal failure) o Postrenal: urinary tract obstruction (nephrolithiasis, tumors, severe infection) - UREMIA/UREMIC SYNDROME o Very high plasma urea concentration accompanied by renal failure o Associated with hematologic abnormalities o Production of urine smelling sweat NONPROTEIN NITROGEN COMPOUNDS CREATININE (5%) UREA (45-50%) PHYSIOLOGY - Most abundant NPN - Constitutes 5% of total plasma NPNs PHYSIOLOGY - Formed from creatine in the muscles and excreted into plasma at a constant - NPN present in highest concentration in the blood rate (body surface area is considered in testing for creatinine) - Formed in the liver from CO2 and ammonia (deamination of amino acids) – - Factors that affect concentration: product of the detoxification of ammonia o Muscle mass (males have larger values than females) - Major excretory product of the metabolism of proteins and other nitrogen- o Renal function (glomerular filtration function) containing chemicals - Removed from the circulation by glomerular filtration; not reabsorbed and - Major factors that affects plasma urea and BUN concentration minimally secreted by the tubules o Protein content in the diet - A better marker of GFR than Urea o Renal function; freely filtered but 40-60% is reabsorbed - Concentration is inversely related to GFR (renal function) o State of hydration o Plasma creatinine levels is always inversely proportional to creatinine o Rate of protein catabolism (proteins are broken down into amino acids, clearance/GFR amino acids are deaminated and ammonia is formed, ammonia is o Creatinine clearance (CC) is a routine procedure for estimating GFR converted to urea through the urea cycle in the liver) o Thus, ↑Plasma Creatinine (PC), ↓ CC - Concentration is expressed in terms of nitrogen content: o If the rate of removal is low, creatinine remains in the circulation and it BUN (Blood Urea Nitrogen) x 2.14 = Urea accumulates (inverse relationship). o If BUN to Urea: Multiply by 2.14 - Blood volume is directly proportional to cardiac output which is proportional to o If Urea to BUN: divide by 2.14 renal blood flow which is also proportional to GFR UREA: CO(NH2)2 with a molecular weight of 60 g/mol - Increased creatinine clearance: high cardiac output o Taking only Nitrogen: - Decreased creatinine clearance: impaired kidney function such as in cases of  N2 = 28 g/mol renal failure, hemorrhage, shock, dehydration  60 g/mol divided by 28 = 2.14 CLINICAL APPLICATION: ANALYTICAL METHODS CLINICAL APPLICATION: ANALYTICAL METHODS - Reference method: Isotope Dilution Mass Spectrometry (IDMS) - CHEMICAL (JAFFE) - non-specific, colorimetric, endpoint: A. CHEMICAL - Direct, Diacetyl Monoxime method, or Fearon Method; Non-specific - Alkaline picrate: saturated picric acid and sodium hydroxide - False increase: plasma chromogens - Inexpensive/cheap - Diacetyl directly reacts with urea → diazine making a yellow color end-product - Techniques to improve specificity: which can be measured spectrophotometrically. o Kinetic: timed-rate; taking absorbance measurement intervals or by - No hydrolysis using a continuously recording spectrophotometer - Unaffected by smoking o Use of adsorbents: by removing interfering substances using adsorbents like: B. ENZYMATIC (INDIRECT)  Lloyd’s (sodium aluminum silicate)  Fuller’s Earth (magnesium aluminum silicate) - Indirect because enzymatic methods detect ammonia/ammonium and not really UREA; more specific - Urea is hydrolyzed, in the presence of urease, to ammonium and carbonate ions → the product ammonium will be detected and made to react with some reagents - False increase: smoking - False decrease: fluoride tubes  GFR ESTIMATES - ENZYMATIC/CREATININASE METHOD - Cockcroft-Gault formula (140 − 𝑎𝑔𝑒)(𝑤𝑒𝑖𝑔ℎ𝑡 𝑖𝑛 𝑘𝑔) = 𝑥0.85 (𝑖𝑓 𝑓𝑒𝑚𝑎𝑙𝑒) 72 𝑥 𝑆𝑒𝑟𝑢𝑚 𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 (𝑚𝑔/𝑑𝐿) - Superior in terms of sensitivity and specificity o Women have 15% lower CC than men o Urine collection is not needed - Modification of Diet in Renal Disease formula o Original formula in 1999 and modified in 2000 o Original MDRD formula: Creatinine, BUN, Albumin, Age, Race, and Gender o Newer formula: only Creatinine, Race, Age, and Gender - NAD → decreased absorbance of NAD at 340nm (UV) - Reference intervals o Jaffe:  M 0.9—1.3 mg/dL  F 0.6—1.1 mg/dL o Enzymatic:  M 0.6—1.1 mg/dL - CKD-EPI (2009) formula is better; has less bias;  F 0.5—0.8 mg/dL o k = 0.7 in females and 0.9 in males o Panic value: >5mg/dL o alpha = -0.329 in females and -0.411 for males o Males have higher values due to bigger muscle mass and body surface area CLINICAL SIGNIFICANCE OF ABNORMAL VALUES METHODS FOR ESTIMATING GFR - BUN/creatinine ratio: 10-20: 1 Increased (↑) creatinine muscular diseases, impaired renal function 𝑈𝑉 1.73 (low GFR), chronic nephritis - Creatinine clearance: 𝑥 𝑃 𝐴 BUN/Creatinine ratio > 20:1 pre-renal (dehydration) o Most widely used clearance test with normal creatinine o Creatinine is endogenous; not significantly reabsorbed nor secreted; that BUN/Creatinine ratio > 20:1 renal or post-renal azotemia is why CC is the recommended method for estimating GFR with elevated (↑) creatinine o In case where the patients’ body surface area deviates greatly from the average (obese, big, child) then use a correction factor by multiplying BUN/Creatinine ratio 13 mg/dL Β2- o A component of MHC-Class I and present in all o Conversion factor: 0.0595 microglobulin nucleated cells o Dissociates from the MHC at a constant rate CLINICAL SIGNIFICANCE OF ABNORMAL VALUES o Falsely elevated: multiple myeloma, lymphoma, - HYPERURICEMIA and malignancies o gout, leukemia, chemotherapy, chronic nephritis, Lesch-Nyhan syndrome o A low molecular weight protein (11,800 daltons) (inborn error of metabolism resulting from the deficiency of HGPT – o Freely filtered, reabsorbed, and metabolized hypoxanthine guanine phosphoribosyltransferase presenting as orange completely by the PCT sand in diaper), alcoholism, lactic acidosis, and von Gierke’s disease o Has a tendency to fold into a beta sheet - HYPOURICEMIA configuration which results to amyloid formation o Fanconi syndrome (failure/decrease of tubular reabsorption), severe liver (dialysis associated amyloidosis) disease, purine inhibitors, allopurinol (drug use to treat hyperuricemia) AMMONIA (0.2%) LEVELS OF STRUCTURE PHYSIOLOGY - Smallest in concentration; only comprises 0.2% of total plasma NPNs - Ammonia is neurotoxic and the body does not allow its accumulation in the blood - Produced from the catabolism of amino acids - Toxic compound metabolized exclusively in the liver (via the Krebs Henseleit or - Primary – number and types of amino acids in a specific amino acid sequence urea cycle) - Secondary – regularly repeating structures stabilized by hydrogen bonds - Liver function test (hepatic detoxification function), not a renal function test between the amino acids (beta pleated sheets and alpha-helix) - Tertiary – overall shape or conformation of the protein molecule; 3- CLINICAL APPLICATION: ANALYTICAL METHODS dimensional - The preferred specimen: arterial blood - Quaternary – results from the interaction of more than one protein molecule - Although venous blood is not recommended, if used, tourniquets should be or subunit (held together by noncovalent forces) used minimally, and fist clenching and relaxing avoided during collection - Specimens should be kept in ice water until separation of cells from plasma PROTEIN ELECTROPHORESIS occurs - Serum samples are applied close to the cathode end (sample wells) of a support medium that is saturated with an alkaline buffer (pH 8.6) at which pH A. CHEMICAL proteins carry a net negative charge and migrate toward the anode (+) - Potentiometric: measurement of pH change as NH3 diffuses through a - pH > pI = negative charge; pH < pI = positive charge; pH = pI = zero charge selective membrane - Support media: - Colorimetric: using an indicator dye or nesslerization and berthelot o Agarose o Cellulose acetate o PAGE (polyacrylamide gel) B. ENZYMATIC (GLDH METHOD) - Stains used: - NAD is measured as the decrease in absorbance at 340nm o Coomassie Brilliant Blue - GLDH: Glutamate dehydrogenase o Amido black o Ponceau S o Lissamine green - Reference intervals: 19—60 ug/dL - Densitometer – specialized colorimeter designed