CLINICAL CHEM Lesson 2: Basic Principles and Practices in Clinical Laboratory
Lecture by Ms. Reyna May G. Cabilla, RMT
August 14, 2023
REAGENTS
● Mix according to number order and needed volume
● Follow specific instructions to create reagent
○ Ready-to-use-form
○ Kit Form
CHEMICALS
● The American Chemical Society (ACS) established the
specifications of chemicals in the analytical grade category.
● The ANALYTICAL REAGENT GRADE CHEMICALS are of very high
purity. They are labeled analytical reagent or ACS which state the
actual purity of each lot. these include chemicals such as:
○ Spectrograde
○ Nanograde
○ High Performance Liquid Chromatography (HPLC) grade
● AR GRADE IS USED IN:
○ most analytical laboratory procedures
○ making up reagents for spectrophotometry,
chromatography, trace metal analysis, atomic absorption,
atomic absorption spectrophotometry, fluorometry, and
standardization procedures.
● AR grade are of the highest
purity
● used in the prep of reagents
Analytical Reagent Grade (AR) in the clinical laboratory
● ar grade and those the
meet the standard are
designated by the ACS -
american chemical society
● Sufficiently pure to be used
in many analyses in the
clinical lab
● does not reveal limited
Chemically Pure Grade (CP) impurities that are
tolerated
● Not acceptable for research
and various clinical lab
techniques, unless
specifically analyzed for the
desired program.
REAGENTS USED IN THE LABORATORY
United States Pharmacopeia ● Generally, less pure than CP
and the National Formulary grade, chemical are not
grade chemicals (USP & NF) injurious to humans and are
used to produce drugs
● Lowest quality and should
not be used for analytical
Technical or Commercial Grade work
● Used only for industrial
purposes
STANDARD REFERENCE MATERIALS
● A primary standard (PS) is a chemical that is the highest purity and
can be measured directly to produce a substance of exact known
concentration.
● the International Union of Pure and Applied Chemistry (IUPAC)
requires that:
○ PS must be at least 99.98% pure and that the working
standards be 98.95% pure
○ Grade A - Atomic weight standards
○ Grade B - Ultimate standard
○ Grade C - Primary standards with >0.002% impurity
○ Grade D - Working standard with >0.5% impurity
○ Grade E - Secondary standards derived from using Grade
C standards
● The National Bureau of Standards, now National Institute of
Standard and Technology (NIST) purified materials known as
Standard reference materials are used as primary standard
materials in the clinical laboratory.
● Relatively expensive, must be used as reference for the
comparison of commercially obtained standards and reagents.
● Secondary standard is a substance of lower purity whose
concentration is determined by comparison to a primary
standard.
WATER SPECIFICATIONS
● REAGENT GRADE WATER is water suitable for reagent and
standard preparation.
○ Most procedures use distilled water or deionized water
● Distilled water is purified to remove almost all organic materials.
○ Water may be distilled more than once, and each
distillation cycle will remove impurities.
● Deionized 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.
● The College of American Pathologists and the National
Committee for Clinical Laboratory Standards issued minimum
specifications for RGW. The NCCLS requirements for each type
are tabulated below:
Abelgas, Labra, Machate, Rebusit notes 1
Specification Type I Type II Type III CALIBRATION

Bacterial
● The accuracy of the thermometer used to monitor the incubation
Content
temperature of an instrument should be verified every 6 to 12
(maximum <10 10^2 NA
months.
colony forming
● Reference thermometers must be certified by the NIST
unit/mL)
● For the monitoring of enzymatic reactions, thermometer should
agree within 0.1°C with the NIST reference thermometer,
pH NA NA NA
thermometers used to check refrigerator and freezer temp
should agree within 1.0°C with the reference thermometer;
Resistivity
discarded if they differ from NIST standard
(meagohms/c 1.0 (in-line) 1.0 0.1
● The temperature of the incubation area should be checked DAILY,
m at 25 degC
and the thermometer itself should be observed for splits in the
mercury column.
Silicates (mg/L 0.05 0.1 1.0
SiO2
maximum) PREVENTIVE MAINTENANCE

Particulate 0.22 um NA NA
● It is imperative that preventive maintenance procedures be
matter (filter
performed, and the results recorded for all laboratory
in system)
instrumentation.
● This includes maintenance of analytical balances, refrigerators,
Organic Activated NA NA
freezers, centrifuges, ovens, water baths, automated analyzers,
contaminants Carbon in
and all other laboratory equipment used for analyzing
system
specimens.
● Type I RGW is used for procedures that require maximum water ● PM is performed at scheduled times such as per shift, daily,
purity such as: weekly, monthly, or yearly.
○ Preparations of standard solution, ○ Celsius (°C) or centigrade - commonly used
○ Ultramicro chemical analysis, ○ Fahrenheit (°F)
○ Measurement of nanogram or subnanogram ○ Kelvin (°K) - SI Designation
concentration;
○ and tissue or cell culture (or both).
COMMON TEMPERATURE CONVERSIONS
○ Type I RGW is discouraged for storage, as contamination
chances are increased (higher resistivity)
Celsius to Fahrenheit °C(9/5) + 32
■ Proper procedure and handling of RGW
(especially Type I) should be followed.
Fahrenheit to Celsius (°F - 32)5/9
● Type II RGW is used in most laboratory determination in
chemistry, hematology, microbiology, immunology, and other
chemical laboratory areas.
● Type III RGW is used in most qualitative GLASSWARE AND PLASTICWARE
measurement/examinations, most procedures in urinalysis,
parasitology and histology, washing glassware and procedures
not requiring Type I or Type II water GLASSWARE PLASTICWARE
● Carbon-dioxide-free water is obtained by boiling Type II water.
○ This is used when carbon dioxide, ammonia and oxygen ● Breakable ● Highly
may affect analysis. ● Reuseable shockproof/shatterproof
● Ideal for acidic solutions ● Disposable
● High thermal and corrosion ● Ideal for alkaline solutions
CLINICAL LABORATORY SUPPLIES resistance ● Relatively inert
● Good Optical properties ● Flexible
THERMOMETERS ● Suitable for Cryogenic
(Freezing) experiments
● Are either an integral part of an instrument or need to be placed
in the device for temperature maintenance. ● Most laboratory supplies must satisfy certain tolerances of
● Three major types of thermometers: accuracy and fall into two classes or precision tolerance
○ Liquid in-glass, (American Society for Testing and Materials)
○ Electronic thermometer or thermistor probe, and ○ Class A: preferred for laboratory application
○ Digital thermometer ○ Class B: have twice the tolerance limits of class A
○ All temp reading devices must be CALIBRATED to ascertain
accuracy.

Abelgas, Labra, Machate, Rebusit notes 2

 GLASS: 5 GENERAL TYPES
1 HIGH THERMAL RESISTANT GLASS
● Borosilicate glass with low alkali content
● Resists heat, corrosion and thermal shock
● Most common are Pyrex, Kimax and Exax
● Corex
○ 6x stronger than borosilicate glass
○ Alumina-silicate glass
○ Better able to resist clouding due to alkali or
scratching
○ Ideal for higher temperature thermometers,
graduated cylinders and centrifuge tubes.
● Vycor
○ Recommended for use in application involving high
temperature and drastic heat shock and extreme
chemical treatment with acids and dilute alkalis
○ Heatable to 900 degC and withstand down shock
from 900 degC to ice water
○ Ideal for ashing and ignition techniques
● Should be Class A
2 HIGH SILICA GLASS
● 96% silica content comparable to fused quartz in its
thermal endurance, chemical stability and electrical
characteristics
● Radiation resistant, good optical qualities and temperature
capabilities
● Ideal for high precision analytical work and can also be
used for optical reflectors and mirrors
3 GLASS WITH HIGH RESISTANCE TO ALKALIES
● aka soft glass
● Boron-free glassware for strong alkali solutions and
digestion with strong alkalies
● Less thermal than borosilicate glass
4 LOW ACTINIC GLASS
● Amber, red colored to reduce the amount of light passing
through the substance within the glassware
● Highly protective laboratory glassware for handling
heat-labile substances in the 300-500 nm range (bilirubin,
carotene and vitamin A)
5 STANDARD FLINT GLASS
● Soda lime glass which is composed of a mixture of oxides of
Si, Ca and Na
● Lowest in cost and readily fabricated
● Poorly resistant to high temperatures and sudden changes
in temperature
● Easy to melt and shape thus used as reagent bottle and
disposable laboratory glassware
● Release alkali into the pipetted liquid
PLASTICWARE TYPES
1 POLYOLEFINS (POLYETHYLENES, POLYPROPYLENES)
● Unique group of resins with relatively inert chemical
properties
● Usually unaffected by acids (however, concentrated sulfuric
acid slowly attacks polyethylene at RT), alkalies, salt
solutions and most aqueous solutions
● Aromatic, aliphatic, and chlorinated hydrocarbons cause
moderate swelling at RT
● Organic acids, essential oils and halogens slowly penetrate
these plastics
● Strong oxidizing agents attack this group at elevated
temperatures only.
○ Polypropylene is more expensive than
polyethylene therefore the latter is used in most
disposable plasticwares
○ Polypropylene is sterilizable however it absorbs
pigment and tends to become discolored
2 POLYCARBONATE RESIN
● Twice as strong as polypropylene (from -100 °C to 160 °C)
● Chemical resistance is NOT as wide as the polyolefins
● UNSUITABLE for use with bases as amines, ammonia and
alkalies as well as oxidizing agents
● Glass-clear and shatterproof
● Ideal for centrifuge tubes and graduated cylinders
3 TYGON
● Nontoxic, clear plastic of modified PVC (polyvinylchloride)
● Used extensively for the manufacture of ACA tubings
● Flexible and used to handle most chemicals
● Can be steam-autoclaved or chemically sterilized
● Tubing is soft and flexible and quickly slips over tubulatures
but gripping tightly on glass or metals.
4 TEFLON FLUOROCARBON RESINS
● Almost chemically inert with high corrosion resistance at
extreme temperatures
● For cryogenic experiments and work at high temperatures
over extended periods (-270 to 255 degC)
● Pure translucent white and inert
● Easy to clean and fast drying but it can be scratched and
misshaped
PIPETS
1 VOLUMETRIC OR TRANSFER PIPETTE
● Designed to deliver a fixed volume of liquid
● Consists of a cylindrical bulb joined at both ends to
narrower glass tubing
● Calibration mark is etched around the upper suction tube
● Lower delivery tube is drawn out to a fine tip
● Used for accurate measurements of aliquots of
nonviscous samples, filtrates, controls, and standard
solutions.
Abelgas, Labra, Machate, Rebusit notes 3
 ● Ostwald-Folin Pipet
○ Similar to volumetric pipet, but has their bulb closer
to the delivery tip
○ Used for measuring viscous fluids as blood or serum
○ Has an etched ring near the mouth piece
○ Blown out only when the fluid has drained to the
last drop in the delivery tip
2 GRADUATED OR MEASURING PIPETTE
● Plain, narrow tube drawn out to a tip
● Graduated uniformly along its length
● Two types which are calibrated for delivery are available
○ Mohr pipet
■ Calibrated between two marks on the stem
○ Serological Pipet
■ Intended for the delivery of predetermined
volumes
■ Principally used for the measurements of
reagents and are not generally considered
accurate enough for measuring viscous
samples and standards
■ Serological pipets have larger orifice than the
Mohr pipet, thus drains faster.
● Micropipets
○ Sahli (TC)
○ Kirk (TC)
○ Lang-levy (TD/TC)
○ Overflow (TC)
○ Capillary (TC)
○ Sanz pipet (TC)
○ Unopette (TC)
○ Semiautomatic Eppendorf (TD/TC)
Cleaning Glasswares and Plastic Wares
● Routine washing may be done by soaking in dilute bleach
followed by drying in an oven, soaking in 20% nitric acid solution
for 12-24 hours and soaking in acid-dichromate solution
○ BLOOD CLOTS - soak in 10% NaOH
○ NEW PIPETS - soak in 5% HCl or 5% HNO3
○ METAL ION Determination - soak in 20% nitric acid
○ GREASE - soak in any organic solvent or 50% KOH
○ PERMANGANATE STAINS - soak in 50% HCl or mixture of
1% ferrous sulfate in 25% sulfuric acid
○ BACTERIOLOGIC GLASSWARE - soak in 2% to 4% cresol
solution followed by autoclaving and thorough washing
○ IRON Determination - soak in 1:2 dilution of conc. HCl
solution or 1:3 dilution of conc. HNO3.
BALANCES
● Analytic Balances
○ Used for the preparation of any primary standard. Weight
range of 0.01mg to 160g
■ Mechanical analytic balance aka substitution
balance
● Electronic Balances
○ Single-pan, uses an electromagnetic force to
counterbalance the weighed sample’s mass.
CALIBRATIONS
● The NIST recognizes the use of five specific types of calibrating
weights.
○ Class S Weights - for monthly calibration of analytic
balances
○ Class M Weights- to calibrate the wei themselves since
their quality is that of primary standard.
○ Class S-1 - used for routine analytical work
○ Class P Weights - are allowed greater tolerance levels
than class S-1
○ Class J Weights - used for microanalytical work.
● Test weights used for calibrating balances should be selected
from the appropriate ANSI/ASTM Classes 1 through 4.
○ Class 1- used for calibrating high-precision analytical
balances in the weight range 0.01 to 0.1mg
○ Class 2- weight range of 0.001 to 0.01 g
○ Class 3 - Weight range 0.01 to 0.1g
Balances should be kept scrupulously clean and be located in an area
away from heavy traffic, large pieces of electrical equipment, and
open windows.
BASIC SEPARATION TECHNIQUES
● Centrifugation
● Filtration
● Dialysis
CENTRIFUGATION
● Process in which the centrifugal force is used to separate solid
matter from a liquid suspension.
● Consists of a head/rotor, carriers, or shields that are attached to
the vertical shaft of a motor or air compressor and enclosed in a
metal covering.
● The relative centrifugal force is obtained by using a nomogram
ort by using this formula:
○ RCF = 1.118x10^-5 x r x rpm^2
○ where:r = radius in cm between axis of rotation and the
center of the centrifuge tube
○ rpm = revolutions per minute
● The CAP recommends cleaning of any spills or debris such as
blood, glass and others daily, checking the timer, brushes, and
speed every three months. The speed of the centrifuge is
checked using the tachometer or strobe light.
FILTRATION
● Can be used instead of centrifugation for the separation of solids
from liquids. Uses filter paper
DIALYSIS
● Is another method for separating macromolecules from a solvent
or smaller substances.
Abelgas, Labra, Machate, Rebusit notes 4