Clinical Chemistry Lecture 1

CLIA

Clinical Laboratory Improvement Act

CAP

College of American Pathologists

Borosilicate

Pyrex (volumetric), glassware

Corex

6x stronger than borosilicate, glassware

Low actinic

Amber or red for light-sensitive materials, glassware

Polypropylene

Plastic pipette tips, some specimen & test tubes (plasticware)

Polyethylene

Test tubes, bottles, graduated tubes, disposable transfer pipettes - binds or absorbs dyes, stains & some proteins (plasticware)

Polycarbonate

Graduated cylinders & flasks (plasticware)

Polystyrene

Rigid & clear, will splinter, capped graduated tubes and test tubes (plasticware)

Teflon

Chemically inert, wide temp range, used for stirring bars, tubing, bottle cap liners (plasticware)

Centrifuge

A rapidly rotating device to separate materials within a sample

Relative centrifugal force

Force * gravity

RPM

Revolutions per minute

RCF

1.1118×10^-5 * r * rpm² (r is rotor radius in centimeters)

TD

To deliver

TC

To contain (Sahli)

TD/Blowout

To deliver/blowout

Reverse osmosis

Water forced through a semi-permeable membrane, serves as a molecular filter

Distillation

Water is vaporized and then condensed

Deionization

Ion-exchange filters remove ions using anion or cation exchange resin

Molarity

Moles of solute per liter of solution; used to calculate the concentration of a solution Molecular weight * molarity = g/l

Normality

Gram equivalents of solute per liter of solution

Percent solutions:

# parts of solute per 100 parts of solution

Dilution

Mixing parts of the solute with parts of solvent

½ dilution

(1:1) 1ml serum + 1ml water = 2ml dilution

1/3 dilution

(1:2) 1ml serum + 2ml water = 3ml dilution

1/10 dilution

(1:9) 1ml serum + 9ml water = 10ml dilution

Electromagnetic Radiation

The wavelengths of light on the visible and invisible spectrum

Spectrophotometry

Used to detect colored reactions via the intensity of the color, or the appearance or disappearance of color

Light reflection

The reason to see colors on an object

Creatinine

The measurement for absorbance is 500nm, which is in the blue-green region, reflects red.

A = abc = ebc

A = absorbance

a = absoptivity (extinction coefficient e)

b = path length of cell (cm)

c = concentration

Beer-Lambert Law

Beer-Lambert Law: Measurement Range

Spectrophotometers

A machine that measures light at a selected wavelength/spectrum

Photometers

A machine that measures light without specifying wavelength

Chromogen

An added chemical to a sample to help change the color

• light

• Wavelength selector

• Sample holder/cuvette

• Photodetector

The basic components of a spectrophotomer

Tungsten or Tungsten halogen

EMS Lamps

Deuterium

UV Radiation lamps

Monochromator

A component that selects the wavelength of light in a narrow band

Prism

A monochromator that uses refraction to create visible light

Cuvette

A component that holds the sample when reading

Photodetector

A component that converts light into electrical signals

<1%

The maximum amount of stray light allowed in a spectrophotomer

Reflectometer

A photometer that measures the amount of light reflected by a liquid sample dispensed on a non-polished surface (such as urine dipstick and dry chemistry slides)

Refractometry

Based on the refraction of light as it passes through a medium, such as glass or water.

Fluorometry

Involves exciting compounds with EMR (high energy, short wavelength) and detecting emitted EMR (lower energy, longer wavelength).

Chemiluminescence

A type of luminometry where Light that is produced from a chemical or electrochemical reaction (produces light without heat)

Electrochemiluminescence

A type of luminometry that uses an electrode, and can be detected with photomultiplier tube

Nephelometry

Measurement of light scattered by a particulate solution (Ag-Aby)

Turbidimetry

Measurement of reduction in light transmission caused by particle formation

Atomic Absorption Spectrometry (AAS)

Primarily used for metals; absorbs EMR unique to that metal, detector picks up unabsorbed monochromatic EMR

Potentiometry

The measurement of electrical potential (voltage) between two electrodes in an electrolyte solution

Potentiometry ion selective electrodes

Membrane based electrodes that only allow specific ions to pass and be measured

pH electrodes

Glass electrodes that measure hydrogen ion activity

PCO2 electrodes

A pH electrode with sodium bicarb at the reference electrode

Direct ISE

Use undiluted samples, sensitive to free ions; doesnt detect bound ions; not affected by high protein or high lipids

Indirect ISE

Use diluted samples (chem analyzers), interfered by high protein and/or lipids; will underestimate the electrolyte concentration

Conductometry

Measures electrolytic conductivity; current is proportional to amount of ions; electrical conductance can be used to assess water purity (high reading = more ions/ low reading = pure)

Coulometry

Based on amperometry

Chromatography

Separation techniques; separates individual compounds from a mixture based on physical and chemical interactions of those compounds in the sample

Retention Time

Related to strength of interaction with stationary phase

Resolution

The ability in chromatography to separate two or more analytes in a sample

Short Retention TIme

A compound that favors the mobile phase; exits the stationary phase the quickest

Ion-exchange chromatography

Uses an ion-exchange mechanism to separate analytes based on their charge; uses a charged stationary phase, like an ion-exchange resin column

Partition/Liquid Chromatography

Based on the differential distribution of solutes between two immiscible (doesn’t mix) liquids which act as the stationary phase and the mobile phase

Adsorption Chromatography

Uses electrostatic hydrogen bonding to separate compounds by adsorption and desorption of solutes at the surface of a solid particle

Affinity Chromatography

Liquid technique that uses biological interactions like the binding of enzymes with substrates, ligands with receptors, or antigens with antibodies

Size-Exclusion Chromatography

Gel or filter pores sort molecules by size

Planar chromatography

Solid phase is a plane (flat) like paper or a layer of media, ex: thin layer chromatography

Column chromatography

Stationary phase is coated onto support particles that are packed into a tube or capillary; ex: ion-exchange chromatography

Gas chromatography

Uses a gas mobile phase (carrier gas) and a column stationary phase; solutes separate based on vapor pressure differences (volatility)

Liquid Chromatography

Uses a liquid mobile phase with very small stationary phase particles and pressure

Mass spectrometry

Used to identify unknown compounds, determine concentration of known substances, study molecular structure of organic and inorganic material

Ionization source

A device that ionizes the target molecule then separates and measures the mass of the molecule and its fragments

Electron impact ionization

Uses a beam of electrons to fragment the molecules in the sample

Matrix-assisted Laser Desrption/Ionization

Soft ionization that does not produce fragments; uses UV laser and a matrix in which the sample is embedded; used in bacterial identification

Inductively Coupled Plasma

An inert gas (argon) is inductively heated via current through a surrounding electromagnetic coil to form a plasma. The sample (typically in liquid form) is inserted, aerosolized, and digested in the plasma into ion fragments

Electrospray ionization

Ion source of choice for LC-MS; applies a high voltage to a liquid to produce an aerosol

Quadrupole mass spectrometer

Filters sample ions based on their m/z ratio; their detection is determined by the stability of their trajectory in an oscillating electrical field

Time-of-flight mass spectrometer

m/z ration is determined by a time measurement in an electrical field of known strength and a known length of detector

Tandem Mass Spectrometer (MS/MS)

Two MS end to end; the first MS selects the parent ions by m/z, the the collision cell fragments the parent ions, then the second MS acquire the mass of the fragment ions

Electrophoresis

Separation of charged compounds in a liquid medium under the influence of an electrical field

Zone electrophoresis

Uses agarose gel to separate by charge

Isoelectric focusing electrophoresis

Uses a polyacrylamide gel with a pH gradient that immobilizes proteins at their neutral pH

Capillary electrophoresis

Uses a narrow bore, fused silica capillaries to separate large and small molecules and uses high electric field strengths. Separates on charge, size, and hydrophobicity

Blotting techniques

separation of DNA and DNA fragments by agarose gel electrophoresis, then blotted on nitrocellulose paper and detected with a hybridized nucleic acid probe

Osmolality

Osmotic pressure governs movement across membranes

Osmometry

The measurement of osmolality of a solution (serum or urine)

Freezing-point osmometer

Sample is supercooled; temperature is then rised back to the freezing point, which causes crystallization)