Concise Clinical Chemistry Review PDF

Preview

Summary

I. LABORATORY SAFETYA. Chain of Infection Source  Reservoir  Portal of Entry  Mode of Transmission  Portal of Exit  Susceptible Host >>> S-R-P-M-E-HB. NFPA (Safety Diamond)C. Good Laboratory PracticesALWAYS “CHURCH” NEVER “FAME”ALWAYSC - Clean up bloody/body fluid spills (1:10 dilution...

Description

CONCISE CLINICAL CHEMISTRY REVIEW I. LABORATORY SAFETY A. Chain of Infection Source  Reservoir  Portal of Entry  Mode of Transmission  Portal of Exit  Susceptible H ost >>> S-R-P-M-E-H B. NFPA (Safety Diamond)

C. Good Laboratory Practi Practices ces

ALWAYS “CHURCH” NEVER “FAME” ALWAYS C - Clean up bloody/body fluid spills (1:10 dilution of 5% sodium hypochlorite/bleach) H – Hand wash before and after entering the lab (Water – Lather – Scrub – Rinse - Dry) U – Universal Precaution (ALL BODY FLUIDS MUST BE CONSIDERED INFECTIOUS) R – Report any accident or injury that has happened within the laboratory/workplace C - Clean up counter tops, phones and key boards (1:10 dilution of 5% sodium hypochlorite/bleach) H – Handle each specimen according to its type NEVER F – Forget to wear your PPE! (Lab gown, Mask, Gloves, Goggles, etc.) A – Apply Cosmetics or Contact lenses M – Mouth pipette E – Eat & Drink D. Common Laborat Laboratory ory Hazards 1. Blood-Borne Pathoge Pathogens ns a. OSHA “Blood-Borne Pathogen” standard requires written exposure control plan b. Categories of Expo Exposure sure Category 1 – exposed to blood and other body fluids daily Category 2 – exposed to blood and other body fluids on a regular basis Category 3 – never exposed to blood and other body fluids

>>> Hepatitis B vaccine should be offered by the employers with categories 1 & 2 2. Hazardous Chemic Chemicals als a. Material Safety Data Sheet (MSDS) – describes the properties and potential hazards of the material, how to use it safely, and what to do in an emergency. b. National Fire Protection Association (NFPA) 1) Flammable Solvents – store in flame cabinet (1 gallon or smaller containers may be stored below the fume hood) 2) Corrosives – separate from non-compatible chemicals (i.e alkali chemicals) for storage 3) Acids – separate from non-compatible chemicals

>>> NEVER store chemicals ABOVE HEAD HEIGHT

Source: University of San Francisco. NFPA Hazard Diamond. Retrieved from: https://myusf.usfca.edu/environmental-healthsafety/nfpa-hazard-diamond on 15:56; 11/9/2019.

PREPARED BY: MATT RODEL LOPEZ FERNANDO, RMT

What to do during chemical spills? Always CLEAN! C – Contain the spill L – Leave the area E – Emergency: when came in contact with eyes or skin flush with water for at least 15 minutes and call for medical attention A – Access MSDS N – Notify Supervisor 1

CONCISE CLINICAL CHEMISTRY REVIEW 3. Fire Hazard a. What to do when there is fire? RACE R – Rescue; A – Alarm; C – Contain; E – Extinguish b. How to use fire extinguishers? PASS P – Pull; A – Aim; S – Squeeze; S – Sweep

Class A B C

c. Types of Fire and Extinguishers Type of Hazard Ordinary Combustibles: Wood, Paper, Cloth Flammable Liquid: Grease, Paint Oils, Gasoline Electrical Equipment

Extinguisher Pressurized water, Dry Chemical Carbon Dioxide, Dry Chemical Halon, Carbon Dioxide, Dry Chemical Metal X Allowed to burn out Special extinguisher for Class K

Flammable Metals D Detonation/Arsenal Fire E Cooking Oils (animal and vegetable fats) K **Dry Chemical – Class A, B and C ** Carbon Dioxide – Class B and C *** Halon – substance heavier than Oxygen; displaces Oxygen on fire to extinguish

4. Radiation a. Radiation monitoring 1. Film badge or Survey meter 2. Exposure limit (5,000 mrem/yr; whole body) 3. Wipe Test/Leak Test – surfaces are wiped with radioactive absorbent material E. Waste Disposal 1. Hazardous Waste – solid waste or mixture of solid waste which may pose a threat to human health or the environment 2. Infectious Waste – equipment, utensils or substances that may harbor pathogens 3. Medical Waste – generated from the diagnosis, treatment or immunization of humans or animals COLOR CODING DRY, NON-infectious BLACK WET, NON-infectious GEEN WET, INFECTIOUS YELLOW SHARPS RED RADIOACTIVE ORANGE MEDICAL BLUE PREPARED BY: MATT RODEL LOPEZ FERNANDO, RMT

II. LABORATORY MATHEMATICS Concept 1. % w/w 2. % w/v 3. % v/v 4. Normality 5. Molarity 6. Molality 7. Dilution 8. Dilution factor 9. Ratio 10. Absorbance 11. Equivalent Weight 12. Milliequivalen Milliequivalents ts 13. Celsius to Fahrenhe Fahrenheit it 14. Fahrenheit to Celsius 15. Celsius to Kelvin

1. Albumin/TP 2. Creatinine 3. Bilirubin 4. BUN 5. Calcium 6. Cholesterol 7. Glucose 8. Phosphorus 9. Triglycerides 10. Electrolytes 11. Thyroxine 12. Ammonia

Formula 𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑒 ⁄𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 × 100 𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑒 ⁄𝑚𝐿 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 × 100 𝑚𝐿 𝑠𝑜𝑙𝑢𝑡𝑒 ⁄𝑚𝐿 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 × 100 𝑠𝑜𝑙𝑢𝑡𝑒⁄ (𝑒𝑞. 𝑤𝑡.× 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛) 𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑒 ⁄(𝑀𝑊 𝑠𝑜𝑙𝑢𝑡𝑒 × 𝐿 𝑜𝑓 𝑠𝑙𝑢𝑡𝑖𝑜𝑛) 𝑔𝑟𝑎𝑚𝑠 𝑠𝑜𝑙𝑢𝑡𝑒 ⁄(𝑀𝑊 𝑠𝑜𝑙𝑢𝑡𝑒 × 𝑘𝑔 𝑠𝑜𝑙𝑣𝑒𝑛𝑡) 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 ⁄𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑆𝑜𝑙′𝑛 or C1V1 = C2V2 𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒⁄ 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 ⁄𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 A = 2 – log%T 𝑎𝑡𝑜𝑚𝑖𝑐 𝑤𝑒𝑖𝑔ℎ𝑡 ⁄𝑣𝑎𝑙𝑒𝑛𝑐𝑒 𝑎𝑡𝑜𝑚𝑖𝑐 𝑤𝑒𝑖𝑔ℎ𝑡⁄ 𝑣𝑎𝑙𝑒𝑛𝑐𝑒 𝑥 10000 (˚C × 1.8) + 32 (˚F - 32) × 0.556 ˚C + 273.15

CONVERSION FACT FACTORS ORS mg/dL to g/L mg/dL to umol mg/dL to umol/L mg/dL to mmol/L mg/dL to mmol/L mg/dL to mmol/L mg/dL to mmol/L mg/dL to mmol/L mg/dL to mmol/L mEq/L to mmol/L ug/dL to mmol/L ug/dL to umol/L

10 88.4 17.1 0.357 0.25 0.026 0.0555 0.323 0.0113 1 12.9 0.587

2

CONCISE CLINICAL CHEMISTRY REVIEW III. INSTRUMENTATION AND REAGENTS A. Instrumentation 1. Glassware aand nd Other Materials a. Borosilicate glass – most commonly used b. Boron free/Soft glass – less thermal resistant than Borosilicate glass c. Special aluminum silicate/Corex – more chemically resistant; 6x stronger than d. Vycor – for extreme chemical and thermal resistance (up to 900˚C) e. Flint glass – for disposable glassware; made with soda-lime glass f. Polypropylene – widely used for manufacturing pipette tips; can be autoclaved g. Polyethylene – widely used material for laboratory equipment 2. Pipettes a. Calibration Marks 1) To Contain (TC) – holds volume but does not dispense exact amount 2) To Deliver (TD (TD) – delivers exact amount

b. Dispensing Characteristics 1) Blowout – with etched rings on top 2) Self-draining – absence of rings; drains via gravity c. Types 1) Transfer pipettes a) Automatic macro-/micropipettes b) Ostwald-Folin – for viscous fluids c) Volumetric – for non-viscous fluids d) Pasteur pipette – for transferring fluids without specific volume 2) Measuring pipe pipettes ttes a) Serological – with graduations; blowout b) Mohr – without graduations; self-draining c) Bacteriologic d) Micropipettes (TC; >> 0.1% phenol red solution in distilled water is used to compare reproducibility of pipets 3. Basic Separation Tech Techniques niques a. Centrifugation Relative Centrifugal Force (RCF) – force acting on specimen Revolutions per Minute (RPM) – actual speed **Conversion formula: RCF = 11.18 × radius in cm × (rpm/1000)2 Divides specimen into 2 p parts: arts: 1) Supernatant – liquid 2) Sediment – solid

>>> Rule of thumb: Place tube on exact positioning or by exact opposites >>> Purpose of hole: Speed verification using Tachometer or Strobe light Types of Centrifuge 1) Refrigerated/Ultracentrifuge – used for thermal labile substances 2) Fixed angle – fixed at a certain angle around 14˚ to 40˚ 3) Horizontal/Swinging – starts vertical when off, then horizontal during rotation and returns to vertical position as it decelerates 4) Cytocentrifuge – for automated slide production >>> Maintenance: Cleaned DAILY; Timer is checked WEEKLY; Speed is checked EVERY 3

MONTHS b. Filtration – used to separate serum in from blood in place of centrifuge under special circumstances c. Dialysis – used to separate macromolecules from a solvent or smaller substance

3

CONCISE CLINICAL CHEMISTRY REVIEW B. Reagents and Reference Materials

b. Classification of Water in terms of Preparation 1)) Distilled Water – purified by distillation, purified to remove almost all organic materials

1. Chemistry Meth Methods: ods: a. Wet Method 1) Liquid 2) Lyophilized b. Dry Method 1) Dry Slide Layers: Top to Bottom Spreader

Distillation: process wherein liquid is vaporized, condensed and is used to purify or concentrate a substance or separate a volatile substance from a less volatile substance

Scavenger

Reagent

Indicator

Support

2. Analytical Chemic Chemicals als a. Analytic reagent – suitable for most analytic procedures b. Chemically pure – not recommended for clinical laboratory procedures c. Technical/Commercial grade – used primarily in manufacturing but never in the clinical lab. d. Ultrapure chemicals – used in specific procedures requiring extremely pure chemicals e. USP and NF grade chemicals – used in drug manufacturing

2) Deionized Water – water is purified by ion-exchange; purified from previously distilled water; produced using either an anion or cation resin 3) Reverse Osmosis (RO) – produced by reversed osmosis wherein water is pumped using pressure across a semi-permeable membrane, thereby producing filtered product of original water 4) Ultraviolet Oxida Oxidation tion Water – ultraviolet radiation (254nm) is used to eliminate bacteria C. Automation

3. Reference Material a. Primary Standard – highly purified chemical that can be measured directly to produce a substance of exact known concentration b. Secondary Standard – lower purity than primary substance c. Control – represent a specimen that is similar in composition to the patient’s whole blood/plasma

1. Continuous Flow Analy Analyzer zer - Liquids are pumped through a system of continuous tubing and air bubbles at regular intervals serve as separating and cleaning media - Samples flow through a common reaction vessel - Mixed by inserting a glass coil into the flow path - Disadvantage: all tests are performed in parallel

4. Water Specific Specifications ations - Water is the most frequently used reagent in the laboratory - Water quality is determined by bacteria (CFU/mL), pH, Resistivity, SiO2, Total Solids, Total Oxidisable Organic Compounds

2. Centrifugal Analyze Analyzerr - Uses force generated by centrifugation to transfer specimen and reagents - Uses acceleration and deceleration to transfer samples from one chamber to another - Advantage: capable of batch analysis

Dissolved

a. Classification of Water in terms of Purity 1) Type 1 – for test methods requiring minimum interference 2) Type 2 – acceptable for most analytic requirements, in reagent, standard and quality control preparation 3) Type 3 – autoclave wash water; only for glassware washing not for analytic procedure

PREPARED BY: MATT RODEL LOPEZ FERNANDO, RMT

3. Discrete Analyzer - Most popular and versatile analyzer – measures only requested test - Only requires 2 – 6 uL of sample - Capable of multiple-tests-one-sample-at-a-time - Advantage: random access capability for STAT samples

4

CONCISE CLINICAL CHEMISTRY REVIEW IV. ANALYTICAL TECHNIQUES

c. Frequency – number of vibrations of wave motion per second

>>> The lower the frequency, the longer the wavelength. A. Spectrophotometry Principle: measurement of light transmitted in a narrow wavelength range to determine substance concentration; wavelength selected by prisms or gratings.

Source: Taiz, Lincoln & Zeiger, Eduardo. (2015). Principle of Spectrophotometry. Retrieved from: http://6e.plantphys.net/topic07.01.html on 22:55; 11/09/2019.

1. Light Energy Energy,, Wavelength and Radiant Energy Sp Spectrum ectrum a. Energy – transmitted via electromagnetic waves that are characterized by their and wavelength b. Wavelength – is the distance between two peaks expressed in nanometers

400 – 700 nm 700 nm

Source: Scheeline, A., and Kelley, K.. Cell Phone Spectrophotometer. Department of Chemistry, University of Illinois at Urbana-Champaign. Retrieved from http://www.asdlib.org/onlineArticles/elabware/Scheeline_Kelly_Spectrophotom eter/ on 22:55; 11/09/2019.

PREPARED BY: MATT RODEL LOPEZ FERNANDO, RMT

frequency

Visible Spectrum Ultraviolet Infrared

*Beer-Lambert Beer-Lambert Law = A = 2 – log%T - states that the relationship between the absorption of light by a solution is directly proportional to its concentration and inversely proportional to transmittance. 2. Types of Spectroph Spectrophotometer otometer a. Single beam spectrophotometer – simplest type of absorption spectrometer that is designed to make one measurement at a time at one specified wavelength b. Double-beam spectr spectrophotometer ophotometer – an instrument that splits monochromatic light into two components wherein one beam passes through the sample and the other passes through a reference material – the additional beam corrects for variation in light intensity Types of Double-beam Spe Spectrophotometer ctrophotometer 1) Double beam in space – 2 photo detectors, one for the sample and reference beams 2) Double beam in time – 1 photo detector, alternately passes the monochromatic light through the sample and reference cuvettes using a chopper 3. Components of a Spectrophotomete Spectrophotometerr a. Light Source – provides polychromatic light; and energy that the sample will modify Types of Light Sources: 1) Continuous source – emits radiation that changes in intensity; widely used in the lab a) Tungsten-Halogen la lamp mp – commonly used light source for testing in the visible region b) Deuterium lamp – provides UV radiation in analytic spectrometers c) Xenon discharge lamp – covers the visible and UV range 2) Line source – emits limited radiation and wavelength a) Mercury and Sodium vap vapor or lamps (Spectrphotometers) b) Hollow Cathode La Lamp mp (AAS) c) Light Amplification by S Stimulated timulated Emission o off Radiation (LASER) b. Entrance Slit – minimizes unwanted or stray light and prevents the entrance of scattered light into the monochromator system

>>> Stray light is the most common cause of loss of linearity at high analyte concentration c. Monochromat Monochromator or – isolates specific or individual wavelength of light 1) Prisms – wedge-shaped pieces of glass, quartz or sodium chloride

5

CONCISE CLINICAL CHEMISTRY REVIEW 2) Diffraction Gratings – most commonly used; made by cutting grooves or slits into an aluminized surface of a flat piece of crown glass 3) Interference Filters – produce monochromatic light by constructive interference

D. Turbidimetry Principle: Determines the amount of light blocked by a particulate matter in a turbid solution E. Nephelometry Principle: Determines the amount of light scattered by a particulate matter suspended in a solution

>>> Used to measure antigen-antibody complexes d. Exit Slit – controls the beam (bandpass) Bandpass – is the total range of wavelengths transmitted e. Cuvet – also known as absorption cell/analytical cell/sample cell 1) Alumina Silica Gla Glass ss – most commonly used 2) Quartz/plastic – for solutions testing in the visible and UV spectra 3) Soft glass 4) Borosilicate gla glass ss f. Photodetector – detects and converts transmitted light into photoelectric energy 1) Barrier-layer cell cell/photocell /photocell – simplest and least expensive; used for measuring radiation in the visible spectrum 2) Phototube – contains cathode and anode enclosed in glass; requires external voltage 3) Photomultiplier (PM) tube – most common type; excellent sensitivity and rapid response; detects very low levels of light and amplifies radiant energy 4) Photodiode – not as sensitive as PM tube but excellent in linearity; useful as a simultaneous multichannel detector g. Meter/Read-out de device vice – displays output of the detection system B. Flame Emission Photom Photometry etry (FEP) Principle: Excitation of electrons from lower to higher energy state Light source: Flame

>>>Internal standard: Lithium/Cesium – to correct for variations in flame and atomizer characteristics C. Atomic Ab Absorption sorption Spec Spectrophotometry trophotometry (AAS) Principle: Element is not excited by merely dissociated from its chemical bond and place in an unionized, unexcited, ground state. Light source: Hollow-cathode lamp

>>>Lanthanum/Strontium chloride is added to samples to form complexes with phosphate to avoid calcium interferences. PREPARED BY: MATT RODEL LOPEZ FERNANDO, RMT

F. Electrophoresis Principle: Physical separation of proteins based on their ionic charge and molecular size 1. Iontophoresis – migration of small ions 2. Zone electrophoresis – migration of charged macromolecules in a porous support medium 3. Capi Capillary llary electrophoresis – separation is performed in narrow-bore fused with silica capillaries Error Cause No Migration Instrument Not Connected, Wrong pH, Electrodes Connected Backwards Bands Too Large/Small Over/Underloading Very Slow Migration High MW, Low Charge, Ionic Strength Too High, Voltage Too Low Sample Precipitates in Support pH Too High or Low, Volatge Too High Split or Broken Bands Excessive Pressure Applied to Wire Applicators Crescent-Shaped Bands Bent Applicators, Overloarding G. Chromatography Principle: Mixture can be separated into individual components on the basis of specific differences in physical characteristics 1. Liquid Chromatography – for better separation of thermolabile substances; uses low temp. 2. Gas Chromatograp Chromatography hy – useful for compounds that are naturally volatile

>>> GC/MS – gold standard for drug testing 3. Thin-layer Chromat Chromatography ography (TL (TLC) C) – used to identify drugs, lipids, carbs and amino acids 4. Ion exchange Chromatography – separation based on MW, molecular size and charge 5. High Pe Performance rformance Liquid Chromatography (HPLC) – uses pressure for separation, thereby achieving better separation of thermolabile compounds H. Flourometry Principle: It determines the amount of light emitted by a molecule after excitation by electromagnetic radiation. I. Chemiluminescence Principle: The chemical yields an electronically excited compounds that emits light as it returns to its ground state, or that transfers its energy to another compound, which then produces emission J. Electrochemistry – the measurement of current or voltage generated by the activity of a specific ion. 1. Amperometry - measurement of the current flow produced by an oxidation-reduction rxn. 2. Coulometry – quantity of electricity needed to convert an analyte to a different oxidation state 3. Potentiometry – measurement of potential/voltage between two electrodes in a solution 6

CONCISE CLINICAL CHEMISTRY REVIEW 4. Voltametry – a potential is applied to an electrochemical cell and the resulting current is measured K. Mass Spectrometry – based on fragmentation and ionization of molecules using suitable source of energy. Compounds for MS must be first isolated by GC or HPLC

9. Chemiluminescence 10. Amperometry

>>> Tandem Mass Spectroscopy (MS/MS) – can detect 20 inborn errors of metabolism from a single blood spot

11. Coulometry

L. Osmometry Principle: Based on the measuring changes in the colligative properties of solutions that occur owing to variations in particle concentrations. Colligative propertie properties: s: 1. Boiling Point – temperature at which vapor pressure of the solvent reaches 1 atm. 2. Freezing Point – temperature at which the vapor pressure of the solid and liquid phases are the same 3. Osmotic Pressu Pressure re – pressure that allows solvent to flow through a semipermeable membrane to establish equilibrium between compartments of different concentration 4. Vapor Pressure – pressure at which the liquid solvent is in equilibrium with the water vapor

12. Potentiometry 13. Voltametry

Any substance dissolved in a solvent will do the following: DECREASE/DEPRESS Freezing Point Vapor Pressure

INCREASE/ELEVATE Osmotic Pressure Boiling Point

Summary of Analy Analytical tical Techniques Analytical Method Measures 1. Spectrophotometry Light transmitted by a solution 2. Flame...