Clinical Chemistry 1st Week PDF

CLINICAL CHEMISTRY CLINICAL CHEMISTRY SELECTED ACCEPTED NON-SI 1ST WEEK Minute (time) (60s) min...

CLINICAL CHEMISTRY CLINICAL CHEMISTRY SELECTED ACCEPTED NON-SI 1ST WEEK Minute (time) (60s) min Hour (3,600 s) h ISO – International Organization for Standards Day (86,400 s) d IUPAC – International Union of Pure and Liter (volume) (1 dm³ = 103 m³) L Applied Chemistry Angstrom (0.1 nm = 1010 A CLSI - Clinical and Laboratory Standards 1m) Institute SAMHSA – Substance Abuse and Mental Health Services Administration COMMON TEMPERATURE CONVERSIONS - Certifies laboratories to conduct forensic CELSIUS (CENTIGRADE) TO FAHRENHEIT drug testing for federal agencies °C (9/5) + 32 (Multiply Celsius temperature by 9; BASIC UNITS AND CONVERSION FACTOR divide the answer by 5, then add 32) SI UNITS Example: 37°C = 99°F Solution: recommended because compounds react on °F = 37°C (9/5) + 32 a molar basis °F = 333/5 + 32 Recommended report of analytes: mmol/L °F = 66.5 + 32 Liter is used as a reference volume °F =98.6 or 99 (always round off) Unit for enzyme: IU/L or U/L or katal unit per liter °F = 99 KU/L FAHRENHEIT TO CELSIUS (CENTIGRADE) BASE QUANTITY NAME SYMBOL (°F-32)5/9 (Subtract 32 and 4 divide the answer Length Meter m by 9, then multiply that answer by 5) Mass Kilogram kg Example: 99°F = 37°C Time Second s Solution: Electric current Ampere A °C = (99°F-32) 5/9 Thermodynamic Kelvin K °C = (67) 5/9 temperature °C = 335/9 Amount of Mole mol °C = 37.22 / 37 (always round off) substance °C = 37 Luminous intensity Candela cd SELECTED DERIVED Frequency Hertz Hz Force Newton N Celsius Degree Celsius °C temperature The predominant practice for temperature measurement uses the Celsius (°C) or centigrade scale; The Sl designation for temperature is the Kelvin (K) scale Catalytic activity Katal kat 1|L.J.P  CLINICAL CHEMISTRY Least analyte to measure: atto 5. Red color absorbs 400-500 nm light and Least analyte to measure (lumabas sa board transmits light of 600-650 nm. exam): femto (kase walang atto sa choices) 6. A green-colored solution would show highest transmittance at 525 CONVERSION FACTOR (CF) NOTE: ANALYTES Conventional SI CF Albumin g/100 mL g/L 10 Ultraviolet light = wavelength increases Bilirubin mg/dL umol/L 17.1 frequency decreases BUN mg/dL mmol/L 0.357 Wavelength and Frequency are inversely proportional Na, K, CI meq/dL mmol/L 1.0 Frequency and Energy are directly proportional meq/dl Calcium mg/dL mmol/L 0.25 Cholesterol mg/dL mmol/L 0.026 Creatinine mg/dL Umol/L 88.4 ] frequency Glucose mg/dL mmol/L 0.0555 Thyroxine ug/dL nmol/L 12.9 Total protein g/dL g/L 10 Triglyceride mg/dL mg/dL 0.0113 Uric Acid mg/dL mmol/L 0.0595 ANALYTES Conventional SI CF Hb g/dL g/L 10 HCO mEq/L mmol/L 1 Osmolality Lithium PC𝑶𝟐 mmHg kPa 0.133 P𝑶𝟐 Ammonia ug/dL umol/L 0.587 Malakas na frequency: Cosmic Cortisol ug/dL umol/L 0.276 SPECTROPHOTOMETRY Creatinine ml/min ml/s 0.0167 Clearance 1. measurement of light TRANSMITTED by a Folic Acid ng/mL nmol/L 2.27 SOLUTION Iron mg/dL umol/L 0.179 Magnesium mEq/L mmol/L 0.5 2. LIGHT/RADIANT SOURCE: source of POLYchromatic light Phosphorus mg/dL mmol/L 0.323 a. Tungsten light bulb/incandescent Vit B12 ng/mL pmol/L 0.0738 tungsten/tungsten iodide lamp most commonly used light b. Hollow Cathode lamp: AAS ANALYTICAL METHODS c. Deuterium discharge lamp and the mercury arc lamp: 1. 400-700nm = visible spectrum most commonly used source of UV 2. 700 nmB = Infrared region 4. A solution transmits light corresponding in 3. ENTRANCE SLIT: minimizes UNWANTED stray light. wavelength to its color, and usually absorbs light a. Stray light of wavelengths complementary to its color 2|L.J.P  CLINICAL CHEMISTRY the MOST COMMON cause of LOSS (or transmittance) when a substance OF LINEARITY. with a narrow natural bandpass b. Most common cause of stray light: Second (sharp absorbance or transmittance order Spectra peak) is scanned. i. Deteriorated optics ii. Didymium or holmium oxide filter ii. Light dispersed by a darkened iii. Mercury vapor lamp enveloped iii. Extraneous room light 5. EXIT SLIT: Controls the Band pass/Band width c. Absorbance error a. The narrower the bandpass, the greater the Major effect of stray light photometric resolution. Bandpass can be d. Sharp cutoff filters made smaller by reducing the width of the type of filter best for measuring stray exit slit. light Exit slit allows +/- 5 nm in a wavelength of e. Neutral Density filter and Dichromate sol'n 500 nm (495-505) verify absorbance accuracy on linearity 6. CUVET- also called absorption cell/analytical Example: A linearity study is performed cell/sample cell. It holds the solution on a visible spectrophotometer at 650 a. Square cuvette has advantage over round nm and the following absorbance cuvette readings are obtained: b. Etched c. scratched optical surfaces - Discarded Concentration of Absorbance d. alumina silica-most commonly used Standard e. glass cuvettes - for visible range, but absorbs 10.0 mg/dL 0.20 UV 20.0 mg/dL 0.41 f. Quartz-For UV radiation 30.0 mg/dL 0.62 g. Beer's law - unknown subs is directly 40.0 mg/dL 0.79 proportional to absorbed light and inversely 50.0 mg/dL 0.92 proportional to transmitted light The study was repeated using freshly prepared standards and reagents, but Concentration = absorbance results were identical to those shown. What Absorbance and Transmittance are is the most likely cause of these results? inversely proportional A. Matrix interference B. Stray light Formulas ▪ A = 2.0 – log%T 4. MONOCHROMATOR -isolates specific ▪ A = log 1/T wavelength of light. ▪ A = -log T a. Grating: most commonly used. Example: A solution that has a b. Colored glass filter: least expensive, not transmittance of 1.0%T would have an precise, they are simple, inexpensive, and absorbance of: useful ▪ A = 2.0 – log%T c. Prism: Short wavelengths are refracted more ▪ A = 2.0 – log 1.0 than long wavelengths ▪ A=2 d. Quality Assurance i. Wavelength accuracy is verified by Cu = Au/As x Cs determining the wavelength reading ▪ Where Cu = Concentration of Unknown that gives the highest absorbance ▪ Au = Absorbance of unknown 3|L.J.P  CLINICAL CHEMISTRY ▪ As = absorbance of standard a. Reagent blank (may sariling kulak ) ▪ Cs = Concentration of known Contains the same reagents used for ▪ Example: A procedure for cholesterol is the test calibrated with a serum-based reagent blank corrects for absorbance cholesterol standard that was caused by the COLOR OF THE Reagent determined by the Abell-Kendall Adjust the spectrophotometer to 0 Abs, method to be 200 mg/dL. Assuming the 100 transmittance same volume of sample and reagent No correction for interfering are used, calculate the cholesterol chromogens or lipemia (clarity concentration in the patient's sample problem) from the following results: Ultracentrifuge is used to clear the serum or plasma of chylomicrons Standard Absorbance Absorbance Absorbance Concentration of reagent of standard of patient b. Sample blank is used to subtract the intrinsic blank serum absorbance of the sample usually caused 200 mg/dL 0.00 0.860 0.740 by hemolysis, icterus, turbidity, or drug ▪ Solution: interference - it is performed by substituting the Saline for Cu = Au/As x Cs reagent Cu = 0.740/0.860 x 200 REFLECTANCE PHOTOMETRY/REFLECTOMETRY Cu = 172 mg/dL 1. Diffused light illuminates a reaction mixture in a ▪ Electricity and Patient’s concentration are carrier and the REFLECTED light is measured inversely proportional 2. The intensity of light reflected is NONLINEAR in relation to the concentration of the analyte h. Lambert law - the absorbance increases 3. 100% reflectance is set with an opaque film exponentially with an increase in the light called a white reference path. 4. Examples: Bilirubinometer and Automated reagent strip reader 7. PHOTODETECTOR - detects and converts FLAME EMISSION PHOTOMETRY transmitted light to electricity a. Photomultiplier tube - Most common type, 1. measure the light emitted by a single atom excellent sensitivity BURNED in a flame (Emission) it should never be exposed to room light a. Flame color: Blue (hottest) because it will burn out 2. Uses: b. barrier layer cell/photocell/photovoltaic cell a. Sodium – Yellow → simplest detector. require no external b. Potassium – Violet Voltage source c. Lithium/Rubidium – Red c. photodiode - respond to a specific d. Magnesium/Copper – Blue wavelength UV/Visible d. phototube - Same as photocell, differs in that 3. Light intensity of atoms that are emitting an outside voltage is required for operation energy=concentration 8. METER OR READ OUT DEVICE - displays output 9. BLANKING TECHNIQUE Atomic Absorption Spectrophotometry (AAS) 4|L.J.P  CLINICAL CHEMISTRY 1. Atomizers - Convert ions to atoms c. Chemicals a. Nebulizer b. flame or a graphite furnace 2. Measures light absorbed by atoms dissociated by heat 3. Uses: Unexcited Trace metals a. Calcium b. Copper c. Magnesium d. Lead e. Aluminum f. Lithium g. Zinc 4. Flameless AAS a. Uses electricity to break the chemical bonds CHEMILUMINESCENCE instead of flame Chemical reaction = light b. Graphite cylinder holds the sample - Where 1. Emission of light is created from a chemical or electricity passes thru to evaporates electrochemical reaction. No excitation Note: Flame – Dissociation, Light – Transmittance radiation, No monochromator. 2. Not needed in a chemiluminescent immunoassay analyzer: Source lamp FLUOROMETRY/MOLECULAR LUMINESCENCE 3. Oxidation reactions of luminol, acridinium SPECTROPHOTOMETRY esters, and dioxetane (Ruthenium-included) 4. More sensitive than Fluorometry 1. measure amount of light intensity emitted by a 5. Enzyme (Bioluminescence) - Enhance of molecule after excitation by electromagnetic chemiluminescence radiation. 2. requires a Primary and Secondary TURBIDIMETRY monochromator 1. Measures LIGHT blocked by a particle in a a. primary monochromator - isolate the solution wavelength for excitation (non- 2. For measuring abundant large particles fluorescence) 3. Like spectrophotometer, the detector is behind b. secondary monochromator - isolate the the cuvette wavelength emitted by the fluorochrome 4. Dependent on: 3. Detector – detects fluorescing sample a. Concentration 4. Most spectrofluorometers use a high-pressure b. Size xenon lamp 5. 1000 X more sensitive than spectrophotometer NEPHELOMETRY 6. Disadvantage - QUENCHING EFFECT (nawawala 1. determines the amount of scattered LIGHT. yung fluorescence/diminishing) 2. Rayleigh's Law - Light scatters when the a. pH wavelength is greater than 10 times the b. Temp – Hotter = low/decrease fluorescence particle diameter 3. More sensitive than Nephelometry 4. The detector response is directly proportional to concentration 5|L.J.P  CLINICAL CHEMISTRY 5. For measuring Ag-Ab complexes ii. Ideal for separating proteins of 6. Depends on: IDENTICAL size but w/DIFFERENT NET a. Wavelength CHARGES b. Particle size f. High-resolution protein electrophoresis - separate proteins into as many as 12 zones LASER LIGHTS Flow cytometry 5. Detection and Quantitation 1. Narrow spectral width and small cross-sectional a. Densitometry - measures absorbance of - I area with LOW divergence (do not scatter) stain. Scan and quantitative electrophoretic 2. Application: Coulter counter pattern 3. More sensitive than Spectrophotometer b. UV 4. Determination of structure and identification of samples ELECTROPHORESIS 1. Ampholyte – a molecule, such as protein, whose net charge can be either positive or negative 2. Isoelectric point (pl) – pH where protein has no net charge 3. Particle with No net charge won't migrate 4. MIGRATION of Charged particles a. Particles migrate towards anode (positive) ➔ At pH 8.6 proteins migrate and divide to: Albumin, a1 globulin, a2 globulin, beta globulin, and gamma globulin b. Iontophoresis – migration of small ions CHROMATOGRAPHY c. Zone electrophoresis – migration of charged Based on solubility / soluble components macro molecules (protein) 1. Separation of soluble COMPONENTS in a d. Capillary Electrophoresis solution. 2. Rf (retention factor) value = distance leading edge of component moves/total distance of solvent 3. Paper Chromatography (Whatman paper) – sugar & amino acid 4. Thin Layer Chromatography (silica gel or alumina) – Drugs/ Drug screening 5. Gas Chromatography i. separation is performed in narrow-bore a. the elution order of volatiles is usually based fused silica capillaries upon the Boiling Point ii. molecules are separated by electro- b. Sample: blood / serum / plasma osmotic flow (EOF) c. Mobile phase – Water/Gas iii. Positive (+) charge → moves faster d. Stationary phase – Paper Negative (-) charge → moves slower e. GC-MS (Gas Chrom-Mass Spectrometry) - e. Isoelectric Focusing (isolate the charge) Gold standard for DRUG TESTING/drug of i. molecules migrate thru a pH abuse gradient 6|L.J.P  CLINICAL CHEMISTRY i. test shall be challenge within 3. Freezing point depression osmometry – most SPECTROPHOTOMETRY 15 days after commonly used method for measuring the receipt of the result through GAS changes in colligative properties in a solution. CHROMATOGRAPHY/MASS 4. Osmometer Reference solution – NaCl ii. If the sample is not challenged for 15 5. Each osmole Vapor pressure is lowered by 0.3 days it should be discarded mmHg/g iii. East Avenue: reference lab for DT 6. Each osmole Boiling point is raised by 0.52°C 7. Freezing point osmometer is sensitive to ethanol 6. MS/MS (Tandem Mass spectrometry) – detect while vapor pressure osmometer is not 20 inborn errors of Metabolism. 8. Osmolal gap, which is the difference between a. Vacuum - needed to prevent collision the measured osmolality and the calculated between fragments osmolality. b. Principle of New born screening a. Osmolal gap indirectly indicates the presence of osmotically active substances 7. Liquid Chromatography b. Osmolal gap formula: a. HPLC (High Performance Liquid Measured osmolality - calculated osmolality Chrom)/normal phase – most widely used, uses _____ for fast separations. 9. Not a contributor to OSMOLALITY: Lipid and i. Polar stationary protein Polar – water soluble , to separate polar and non-polar NOTES: substances. H2O with salt: Boiling point - >100, Freezing point ii. Uses: Rapid HbA1c -