A - Chapter I: INTRODUCTION TO CLINICAL CHEMISTRY Flashcards
– seeks to understand the physiologic and biochemical processes occurring in normal and abnormal states
Fundamental science
– analyses performed on body fluids or tissues to provide important information for the diagnosis and treatment of disease
Applied science
LABORATORY REAGENTS:
A. CHEMICALS
Analytical grade/Reagent grade chemicals
United States of Pharmacopoeia and National Formulary
Chemically pure/Pure grade chemicals
Technical/Commercial grade
LABORATORY REAGENTS:
B. STANDARDS
Primary Standards
Secondary Standards
Standard Reference Materials
- meet specifications set by the American Chemical Society
Analytical Grade
- of high purity and is suitable for most analytical laboratory procedures
Analytical Grade: Ultra-Pure Reagents
- Includes spectograde, nanograde and HPLC
Analytical Grade
-used to manufacture drugs
United States Pharmacopeia (USP) and National Formulary (NF)
- Less pure grade
Chemically pure (CP) / Pure Grade Chemicals
- Impurity limitations and chemical preparation are not uniform
Chemically pure (CP) / Pure Grade Chemicals
- lowest quality and should not be used for analytical work
Technical or Commercial grade
- highly purified chemicals that can be measured directly to produce a substance of exact known concentration.
Primary Standard (PS)
- used for standardization of solutions of unknown strength
Primary Standard (PS)
- stable and can be dried, preferably at 104 - 110 ̊C, without a change in composition
Primary Standard (PS)
- not hygroscopic
Primary Standard (PS)
- of lower purity with concentration determined by comparison with a primary standard
Secondary Standard
- certified by the National Bureau of Standards (NBS)
Standard Reference Materials (SRM)
- Types of Reagent Grade Water:
Distilled water
Deionized water
Type I
Resistivity (megaohm/cm (@ 25OC)
Silicate (mg/L, as SiO2)
pH
Microbiologic content (CFU/mL)
10
2.0
0.1
Type II
Resistivity (megaohm/cm (@ 25OC)
Silicate (mg/L, as SiO2)
pH
Microbiologic content (CFU/mL)
0.05
0.1
1.0
Type III
Resistivity (megaohm/cm (@ 25OC)
Silicate (mg/L, as SiO2)
pH
Microbiologic content (CFU/mL)
NS
NS
5 - 8
<10
103
NS
➢ Used in test methods requiring minimum interference and maximum precision and accuracy
Type I
➢ acceptable for most analytic procedures
Type II
trace metal , iron and enzyme analyses, electrolyte measurements
Type I
tissue or cell culture
Type I
ultramicro analysis; preparation of all standards
Type I
stored in a manner that reduces any chemical or bacterial contaminations and for short periods.
Type II
chemistry, hematology, immunology, reagent QC & standard preparation
Type II
urinalysis, parasitology, histology acceptable for washing glasswares
Type III
5 PREPARATION OF REAGENT GRADE WATER
Distillation
Filtration
Deionization
Reverse Osmosis
Unltraviolet oxidaiton/Ozone treatment
➢ Water is boiled and vaporized; Each cycle removes impurities
Distillation
➢ Some impurities sodium, potassium, manganese, carbonates and sulfates.
Distillation
➢ remove 98% of the particulate matter.
Filtration
: removes organic materials and chlorine
➢ Activated carbon
➢ : depending on the type of water
Submicron fibers (<0.2 mm) or glass or cotton fiber
: Remove particulate matters, microorganisms, pyrogens & endotoxins
➢ Ultrafiltration and Nanofiltration
➢ Uses an anion or cation exchange resin followed by replacement of the removed ions with OH- or H+.
Deionization
➢ Uses pressure to force water through a semipermeable membrane
Reverse Osmosis
➢ Does not remove dissolved gases; may be used as pre-treatment of water.
Reverse Osmosis
FACTORS DETERMINING THERMAL DURABILITY
– temperature resulting to deformation due to heat stress
- Strain Point
- °T at w/c glass is heated in order to prevent brittleness
- Annealing Point
– refers to dimension change w/ °T (ideally, it should be low)
- Coefficient of Expansion
PROPERTIES OF GLASS
- Breakabiity
- Thermal Durability
- Transparency
: dependent on silicate anion content (greater amount – more durable)
a. Breakability
: dependent on boron oxide, nickel & ferric ion content
b. Thermal Durability
: dependent on ferric ion content
c. Transparency
▪ With high degree of thermal resistance ( 510 oC)
Borosilicate w/ Low Alkaline content
▪ This should not be heated beyond its strain point
Borosilicate w/ Low Alkaline content
▪ May cloud/etch when used with strong alkalis; may be scratched
Borosilicate w/ Low Alkaline content
▪ Most common type used in volume measurements
Borosilicate w/ Low Alkaline content
▪ Corex
Alumina-silicate glasswares
▪ Strengthened chemically rather than thermally; 6X stronger than borosilicate glass but less thermally resistant
Alumina-silicate glasswares: Corex
▪ Alkali resistant; Resists some clouding and scratching
Alumina-silicate glasswares
ashing & ignition techniques; can withstand very high temperature.
Alumina-silicate glasswares: Vycor
- with good thermal endurance (900 - 1200OC), chemical stability and electrical characteristics
High Silica Glasswares (96% silica)
- good optical and temperature characteristics
High Silica Glasswares (96% silica)
- With poor heat resistance but has high resistance to alkali
Boron-free glass (“Soft Glasswares”)
- Thermally resistant and with a red or amber color
Low-Actinic glass
- soda lime glass composed of a mixture of oxides of Silicon, Calcium and Sodium
Flint glass
- cheapest and with poor resistance to high temperatures
Flint glass
Class A tolerances according to NIST :
high thermal borosilicate or aluminosilicate glass
SPECIAL GLASSWARES
- Colored and Opal Glasses
- Coated Glasses
- Optical Glass
- Glass Ceramics
- Radiation
- has metallic oxides; used for filters and light bulbs
Colored and Opal Glasses
- has a thin, metallic oxide permanently fire-bonded to its surface; can conduct electricity
Coated Glass
- made of soda lime, lead and borosilicate
Optical Glass
- has a high optical activity; prisms, lenses and optical mirrors
Optical Glass
- with high thermal resistance, chemical stability and corrosion resistance
Glass Ceramics
- for hot plates, table tops and heat exchangers
Glass Ceramics
- made of soda lime and lead
Radiation-Absorbing Glass
LABORATORY PLASTIC WARES
- Polystyrene (PS)
- Polyethylene
- Polypropylene (PP)
- Teflon
- Tygon
- Polycarbonate (PC)
- Polyvinyl chloride (PVC)
- Clear and rigid; not autoclavable
Polystyrene (PS)
- Used for disposable wares
Polystyrene (PS)
- Not recommended for use with acids, aldehydes, ketones, ethers, hydrocarbons or essential oils
Polystyrene (PS)
- Chemically resistant to most substances except for aldehydes, amines, ethers, hydrocarbons and essential oils
Polyethylene
: translucent and flexible; not autoclavable
- Conventional Polyethylene (CPE)
- Has the same chemical resistant as polyethylene
Polypropylene (PP)
- Translucent and rigid; autoclavable
Polypropylene (PP)
- Resin that has excellent chemical resistance to almost all chemicals in the lab.
Teflon
- Clear, translucent and flexible; autoclavable
Teflon
- Used for stopcocks, wash bottles and tubings
Teflon
- Translucent and flexible; autoclavable
Tygon
- Very susceptible to damage by most chemicals.
Polycarbonate (PC)
Resistant to water, aqueous salts and inorganic acids for a long period.
Polycarbonate (PC)
- Very clear and rigid; autoclavable
Polycarbonate (PC)
- Used for carboys, test tube racks
Polycarbonate (PC)
- Used for most bottles and tubings
Polyvinyl Chloride (PVC)
STERILIZATION OF HIGH QUALITY PLASTICWARE:
- Autoclaving:
- Chemical sterilization:
- Gas sterilization:
121°C at 15 psi for 15-20 mins
Benzalkonium chloride
Ethylene oxide
: 121°C at 15 psi for 15-20 mins
Autoclaving
: Benzalkonium chloride
Chemical sterilization
: Ethylene oxide
Gas sterilization
- CLASSIFICATION ACCORDING TO GRADUATIONS:
A. TRANSFER PIPETS
B. GRADUATED OR MEASURING PIPETS
C. “Blow Out”
D. “Between Two Marks”
A. TRANSFER PIPETS
- Volumetric or Transfer pipet
- Ostwald-Folin pipet
- Pasteur pipets
- Automatic (macro or micropipets)
B. GRADUATED OR MEASURING PIPETS
Mohr
Serological
- used to transfer aqueous solutions & non-viscous samples
Volumetric or Transfer pipet
- self-draining
Volumetric or Transfer pipet
Mohr
- has the greatest degree of accuracy and precision
Volumetric or Transfer pipet
- read at the lower meniscus
- with bulb at the center
Volumetric or Transfer pipet
Should be used when diluting standards, calibrators, or QC material.
Volumetric or Transfer pipet
- used for biologic fluids having viscosity greater than water
Ostwald-Folin pipet
- blowout pipets
Ostwald-Folin pipet
- read on the upper meniscus
Indicated by two etched continuous rings at the top.
Ostwald-Folin pipet
- bulb is closer to the delivery tip
Ostwald-Folin pipet
- no calibration mark
Pasteur pipets
used to transfer solutions or biologic fluids without consideration of a specific volume
Pasteur pipets
capable of dispensing; used to deliver a pre- determined volume of liquid
GRADUATED OR MEASURING PIPETS
➢ calibrated between two marks; deliver between their calibration marks
Mohr pipet
➢ Tip should NOT touch the receiving vessel
Mohr pipet
➢ self-draining; with smaller orifice
Mohr pipet
➢ graduated down to the tip
Serological pipet
➢ blow-out pipet; with larger orifice
Serological pipet
– exact volume is calibrated to fill the volume between two calibration points on the pipet
“Between Two Marks”
CLASSIFICATION ACCORDING TO USE:
“To-Contain” (TC)
“To-Deliver” (TD)
➢ Holds the particular volume but does not dispense the exact volume
“To-Contain” (TC)
➢ Requires rinsing
“To-Contain” (TC)
➢ calibrated with mercury
“To-Contain” (TC)
➢ usually a micropipette; Volumes are expressed in microliter
“To-Contain” (TC)
Example: Sahli pipet
Example: Mohr, Serologic, Volumetric Transfer pipets
“To-Contain” (TC)
“To-Deliver” (TD)
➢ Delivers the exact volume indicated; Calibrated for the volume delivered
“To-Deliver” (TD)
➢ fluid is allowed to flow freely with the pipet tip touching the inner wall of receiving vessel
Designed to be drained by gravity
“To-Deliver” (TD)
Safer, less time consuming, precise & convenient
Automatic macropipets or micropipets
➢ mechanism draws up and dispenses the liquid
Automatic macropipets or micropipets
Automatic macropipets or micropipets types
: relies on a piston for suction creation to draw the sample into a disposable tip
Air-displacement
: operates by moving the piston in the tip or barrel
Positive displacement
- Sample enters directly upon contact without air interference
Positive displacement
- No need to replace delivery tip
Positive displacement
: obtain the liquid from a common reservoir and dispense it repeatedly
Dilutor/Dispenser pipets
combines sampling & dispensing functions
Dilutor/Dispenser pipets
process in which centrifugal force is used to separate solid matter from a liquid suspension; also separate two liquid phases of different densities
Centrifugation
RCF in grams=
1.118 x 10-5 x r x (rpm)2 ; or use nomogram
RPM =
tachometer or strobe light
CENTRIFUGE TYPES
- Horizontal-head or swinging-bucket centrifuge
- Angle-head or fixed angle centrifuge
- Ultracentrifuge
➢ tubes placed in the cups of the rotor assume a horizontal plane when the rotor is in motion and vertical position when at rest
Horizontal-head or swinging-bucket centrifuge
➢ Tubes are held at a fixed angle from 25-40 degrees to the vertical axis of rotation
Angle-head or fixed angle centrifuge
➢ Particles are driven outside and bottom of the tube and the surface of the sediment packs against the side and bottom of the tube and the surface of the sediment is parallel to the shaft of the centrifuge
Angle-head or fixed angle centrifuge
➢ High-speed centrifuge ( its rotor can spin as high as 1000000 x g)
mainly fixed angle rotors
For the separation of lipoproteins
Ultracentrifuge
➢ requires a refrigerated chamber
Ultracentrifuge
5% HCl or 5% HNO3
New pipets
10% NaOH (12 - 24 hours)
Blood clots
20% HNO3
Metal ion determinations
50% KOH
Grease
50% HCl; Mixture of 1% FeSO4 in 25% H2SO4
Permanganate stains
2 - 4% cresol autoclaving
Bacteriologic glasswares
HCL solution (1:2); HNO3 solution (1:3)
Iron determination
General washing procedures:
- each individual lot is analyzed and the actual amount of impurity is reported.
A. Lot-Analyzed Reagents
- the maximum impurities are listed.
B. Maximum Impurities Reagents
not pure enough for use in most chemical procedures
United States Pharmacopoeia (USP) and National Formulary (NF)
Primarily used in manufacturing
Technical or Commercial grade
IUPAC requires PS to be at least 99.98% pure; working standard be 99.95%
Primary Standard (PS)
concentrations cannot be exactly known by direct measurement
Secondary Standard
Is often used to verify calibration or accuracy/bias assessments.
Standard Reference Materials (SRM)
: Is a water suitable for reagent and standard preparation.
REAGENT GRADE WATER
: is purified to remove almost all organic materials.
- Distilled Water
: is produced from distilled water using either an anion or cation exchange resin followed by replacement of the removed particles with hydroxyl or hydrogen ions respectively.
- Deionized Water
For trace metal analyses , iron and enzyme analyses, electrolyte measurements, tissue or
cell culture , Ultra-micro chemical analysis, and preparation of all standards
Type I
not requiring Type I or Type I water.
Type III
Used for most qualitative measurements/examinations.
Type III
Oldest method of water purification.
Distillation
are composed of glass, cotton, activated charcoal which removes organic materials and chlorine
Filtration cartridges
➢ The use of UV radiation at the biocidal wavelength of 254nm eliminates many bacteria and cleaves many ionizing organics that are then removed by deionization.
Ultraviolet oxidation; Ozone treatment
Highly protective for handling heat-labile substances in the 300-500 nm range
LOW-ACTINIC GLASS
Alcohols and bases can be used but not to be stored longer than 24 hours.
Polystyrene (PS)
: unique group of resins with relatively inert chemical properties.
Polyolefins (polyethylene & polypropylene)
Unaffected by acids
Polyethylene
Used for screw-cap closures
Polypropylene (PP)
Twice as strong as polypropylene ( from -100⁰C to 160⁰C)
Polycarbonate (PC)
DISADVANTAGES OF PLASTICWARES:
1.[?] – increase in concentration
2.[?] – decreased reaction accuracy
3.[?] is difficult to describe
Evaporation of solutions
Absorb dyes/pigments
Color
- Routine washing:
dilute bleach followed by drying in an oven, soaking in 20% Nitric Acid solution for 1224 hours and soak in Acid dichromate solution.
- For blood clots:
soak in 10% NaOH
- For new pipets:
soak in 5% HCl or 5% HNO3
- For metal ion determination,
soak in 20% HNO3
- For grease,
soak in any organic solvent or 50% KOH
- For permanganate stains,
soak in 50% HCl or a mixture of 1% Fe2SO4 in 25% H2SO4
- For bacteriologic glassware:
soak in 2-4% cresol solution followed by autoclaving and thorough washing.
- For iron determination,
soak in 1:2 dilution of Conc. HCl solution or 1:3 dilution of Conc. HNO3.
GENERAL WASHING PROCEDURES:
1. Soak glassware in soapy water or dilute bleach detergent Rinse with tap H2O 3X Rinse with dist. H2O oven dry @ > 140OC
2. Soak glassware in acid dichromate overnight rinse with dilute ammnonia rewash according to the procedure. Acid Dichromate preparation: Dissolve 50g sodium dichromate in 50 mL dist. H2O; Add to 500 mL conc. H2SO4
3. Soak glassware in 20% HNO3 for 12 – 24 hrs rewash according to the first procedure.
: ✓ they are usually used to transfer volumes of 20 mL or less
PIPETS
: is designed to transfer a KNOWN volume of liquid.
TRANSFER PIPETS
✓ Is the most routinely used pipet in today’s Clinical Chemistry Laboratory.
Automatic (macro or micropipets)
✓ Automated/self-automated
Automatic (macro or micropipets)
✓ Advantages: safe to use, stable, ease of use, increased precision,
Automatic (macro or micropipets)
✓ time-saveing, less cleaning required.
Automatic (macro or micropipets)
✓ time-saveing, less cleaning required.
Automatic (macro or micropipets)
= constant( determined from the angular velocity)
1.118 x 10-5
= in cm ( measured from the center of the centrifuge axis to the bottom of the test tube shield or bucket)
r
✓ The rotor looks like CROSS with bucket
Horizontal-head or swinging-bucket centrifuge
✓ The surface of the sediment is flat
Horizontal-head or swinging-bucket centrifuge
PIPET CLASSIFICATION ACCORDING TO DRAINAGE CHARACTERISTICS:
- BLOW-OUT
- SELF-DRAINING
✓ Has a continuous etched ring or two small, close, continuous rings located near the top of the pipet.
- BLOW-OUT
✓ No markings, the pipet is drained by gravity.
- SELF-DRAINING
used as primary standard materials in the clinical laboratory.
Standard Reference Materials
Should not be used in any quantitative analytic technique.
Pasteur pipet
Has an etched-ring ( or pair of rings) near the bulb end of the pipet
Serological pipet
used for tubings
Tygon
Does not have graduations to the tip.
Mohr
Does not have graduations to the tip.
Mohr
Used for screw-cap closures
Polypropylene
