Chapter 1: Basic Principles and

Practice
School of Health and Allied Sciences
University of Saint Louis

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Units of Measure
• Unit defines physical quantity or dimension, such as
mass, length, time, or volume
• Système Internationale d’Unités (SI)
– Adopted internationally in 1960
– Preferred in scientific literature and clinical labs
– Based on metric system
– Includes basic (m, kg, s) and derived (Hz, N, C) units
– Uses standard prefixes: nano, micro, milli, kilo, mega, giga, tera

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Copyright © 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins
Copyright © 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins
Units of Measure

• Reporting of Laboratory Results

– Often expressed in terms of substance concentration
(moles) or the mass of the substance (e.g., mg/dL, g/dL,
g/L, mEq/L, and IU) rather than SI units.
– Recommended: Analytes should be reported using moles
of solute per volume of solution (substance
concentration) and that the liter be used as the reference
volume.

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Units of Measure
• Electronic Reporting of Results
– Complicating matters is that there are many
different data management systems = NO
UNIFORM STANDARD!
– Since there is no uniform standard, there is
agreement in most systems that in each test:
• Test name should be identified and should have its
unique code
• Should the value and the unit or the appropriate
abbreviation

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Reagents
• Most clinical labs use reagents prepared by
manufacturers.
• Knowledge of chemicals, standards, solutions, buffers,
and water requirements is still useful, though.
• Chemicals
– Varying grades of purity
– Analytic reagent grade (AR) and ultrapure are best for lab use.
– Specifications for AR chemicals established by American
Committee Society (ACS)
– Labels on reagents indicate grade and percentage of impurities.

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Reagents
• Chemicals used for Reagent Preparation
1. Analytical Reagent Grade (AR)
-important for qualitative and quantitative
analyses; essential for accuracy
-specifications were established by ACS
-labels on these reagents either state the
actual impurities for each chemical lot or list
the maximum allowable impurities
(percentage impurities)

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Reagents
• Chemicals used for Reagent Preparation
2. Chemically Pure (CP) or Pure Grade
-The impurity limitations of this as this type of
chemical are usually not stated– it fails to
reveal the tolerance limits of impurities.
-preparation of these chemicals in not uniform
-purity is usually delivered by measurement of
melting point or boiling point
-not recommended in the lab unless further
purification or a reagent blank is included
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Reagents
• Chemicals used for Reagent Preparation
3. Technical or Commercial Grade
- It is used primarily in manufacturing
- It should never be used in clinical laboratory
testing.
4. United States Pharmacopoeia (USP) and
Formulary (NF)
- It is approved for human consumption but
may not be applicable for laboratory analysis
- Use: for drug manufacturing
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Reagents
• Organic reagents’ Grades of Purity
1. Practical Grade with some impurities
2. Chemically Pure (which approaches the purity
level of reagent grade chemicals)
3. Spectroscopic/Spectrally pure and
Chromatographic Grade (with purity levels attained
by their respective procedure)
4. Reagent Grade – certified to contain impurities
below certain levels established by the ACS

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Reagents
• Other than purity aspects, laws related to OSHA require
manufacturers to:
1. Clearly indicate the lot number
2. Any physiologic or biologic health hazard
3. Precautions needed for the safe use and storage for any
chemical
• A manufacturer is require to provide technical aspects in
MSDS

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Reagents (cont’d)
• Reference Materials
– Clinical chemistry involves analysis of biologic fluids.
– Standards of traditional analytic chemistry don’t readily apply.
– Standard reference materials certified by NIST are used instead
of ―primary standards.‖

• Water Specifications
– Water is most-used reagent in lab.
– Labs require reagent grade water.
– Basic parameters: microbiologic count, pH, resistivity, silicate,
particulate matter, organics

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Reagents (cont’d)
• Water Specifications
– Methods of water purification:
• Prefiltration
- can remove particulate matter for municipal water
supplies before any additional treatments.
-Filtration cartridges composition: glass, cotton,
activated charcoal and submicron filters (less than or
equal to 0.2 mm)
-Use of filters depends on the quality of municipal water.
-e.g., A. Hard water may require a glass or
cotton filter
B. Submicron filter is better after treatment
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Reagents (cont’d)
• Water Specifications
– Methods of water purification (cont’d):
• Distillation
• Deionization
-has some or all ions removed, although organic
material may still be present = NEITHER PURE NOR
STERILE
-Generally, this is prepared from previously treated
water and produced using either an anion or a cation
exchange resin followed by replacement of the removed
particles with hydroxyl or hydrogen ions
-Two-bed system is excellent in removing dissolved
ionized solid and dissolved gases
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Reagents (cont’d)
• Water Specifications
– Methods of water purification (cont’d):
• Reverse osmosis
-processthat uses pressure to force water through
a semipermeable membrane
-may be used as a pretreatment of water.

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Reagents (cont’d)
• Water Specifications
– Methods of water purification (cont’d):
• Ultrafiltration and nanofiltration
-are excellent in removing particulate matter,
microorganisms, and any pyrogens or endotoxins.
-UV oxidation (removes some trace organic material) or
sterilization processes (use specific wavelengths),
together with ozone treatment, can destroy bacteria but
may leave behind residual products)

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Reagents (cont’d)
• Water Specifications
– Combination of purification methods is needed to produce
reagent grade water.
– Three Grades of Reagent Water
• Type I
• Is used for test methods requiring minimum interference
• Is used for procedures that require maximum water
purity for accuracy and precision
• Should be used immediately!
• Use: FEP, AAS, Blood gases and pH, enzyme studies,
electrolyte testing, HPLC, trace elements and iron studies

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Reagents (cont’d)
• Water Specifications
– Three Grades of Reagent Water (cont’d)
• Type II
• For hematology, microbiology, immunology and chemistry
• Is a acceptable for preparation or reagents and quality
control materials.
• Type III
• For urinalysis, parasitology, and histology.
• For washing glasswares.

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Reagents (cont’d)
• Solution Properties
– Terms and definitions
• Solute: a substance dissolved in a liquid
• Analytes: biologic solutes
• Solvent: liquid in which solute is dissolved
• Solution: solute plus solvent
– Concentration
• Expressed in percent solution, molarity, molality, normality,
moles

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Reagents (cont’d)
•

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Reagents (cont’d)
•

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Reagents (cont’d)
•

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Reagents (cont’d)
•

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Reagents (cont’d)
•

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Reagents (cont’d)
•

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Reagents (cont’d)
•

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Reagents (cont’d)
•

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Reagents (cont’d)
•

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Reagents (cont’d)
• Solution Properties
– Colligative properties: osmotic pressure, vapor pressure,
freezing point, boiling point
– Redox potential: measure of ability of solution to accept or
donate electrons
– Conductivity: measure of how well electricity passes through a
solution
– pH: inverse log of hydrogen ion concentration
– Buffers: weak acids or bases and their related salts that
minimize changes in hydrogen ion concentration

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Clinical Laboratory Supplies
• Thermometers/Temperature
– Scales: Celsius (C) (most common), Fahrenheit (F), Kelvin (K)
– Types: liquid-in-glass, electronic (thermistor probe), digital
– All thermometers must be calibrated for accuracy.
– Heating/cooling cell or block, or water/ice bath control
temperature for certain reactions

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Clinical Laboratory Supplies (cont’d)
• Glassware and Plasticware
– Borosilicate glass
• Used for heating and sterilization purposes
– Boron-free glassware/Soft glass
• It has high resistance to alkali
• Its thermal resistance is less as compared to borosilicate glass
– Corex (Corning)
• A special alumina-silicate glass that has been strengthened
chemically than thermally, six times stronger than borosilicate

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Clinical Laboratory Supplies (cont’d)
• Glassware and Plasticware
– Vycor (Corning)
• It is utilized for thermal, drastic heat shock and
extreme chemical treatment with acids (except
hydrofluoric) and dilute alkali; it can be heated to
900C
– Flint glass
• Made up of soda-lime glass and a mixture of
calcium, silicon and sodium oxides
• It has poor resistance to high temperature
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Measuring vessels
• Graduated cylinder
– Used where less accurate measurements are
required
• Burets
– Used for titration purposes only
– Stopcock: glass for acid, rubber for alkali
• Volumetric flask
– Frequently used for the preparation of
standard solutions (known concentration)
– Measuring liquid accurately
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Measuring vessels
• Pipets
– Classification According to Calibration Mark Design
• To deliver (TD)
• Delivers the exact amount it holds into a container
• Calibration is usually performed by measuring the amount
of water delivered by the pipet
• Water is commonly used as the calibration medium

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Measuring vessels
• Pipets
– Classification According to Calibration Mark Design
• To contain (TC)
• Holds the particular volume but does not dispense the exact
volume
• Calibrated by introducing exact volume or weight by mercury

– Classification According to Drainage Characteristics

• Blowout
• Has a continuous etched rings on top of the pipet

• Self-draining
• Absence of etched rings
• Liquid is allowed to drain by gravity
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Measuring vessels
• Pipets
– Types
• Transfer pipet
• Volumetric pipet
• For nonviscous fliud—self draining
• Has a cylindrical bulb located midway the
mouthpiece and the tip
• Used to deliver an exact amount of fluid
• Oswald Folin
• Has bulb like enlargement and with etched ring
• For viscous fluid
• Blowout pipet
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Measuring vessels
• Pipets
– Types
• Transfer pipet
• Pasteur pipet
• Transfers fluid without consideration of a specific
volume
• Automatic macro or micropipette
• Graduated or Measuring pipet
• Serological
• With graduation marks up to the tip
• Generally a blowout pipet
• Mohr pipet
• Without graduations to the tip; Self-draining
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Measuring vessels
• Pipets
– Types
• Graduated or Measuring pipet
• Bacteriologic pipet
• Ball, Kolmer and Kalin
• Micropipet- TC pipets

– **Mechanical or Automatic Pipet

• Air displacement
• Relies on piston for suction creation to draw the
sample into a disposable tip
• The piston does not come in contact with the liquid
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Measuring vessels
• Pipets
– **Mechanical or Automatic Pipet
• Positive displacement pipet
• Operates by moving the piston in the pipet tip or
barrel much like a hypodermic syringe
• Does not require a different pipet for each use

• Dispenser/Dilutor pipet
• Obtains the liquid from a common reservoir and
dispense it repeatedly
• Combine sampling and dispensing functions

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Measuring vessels

• Notes to remember
– Acid dichromate or concentrates sulfuric acid or
water: cleaning solution for glasswares
– 0.1% phenol red solution in distilled water: used
to compare the reproducibility of brands of pipet
tips
– Class A pipets do not require recalibration;
gravimetric method is utilized for calibration of
non Class A pipets
– Pipets should be held vertically when delivering
liquids

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Clinical Laboratory Supplies (cont’d)
• Desiccators and Desiccants
– Desiccator: a closed, airtight chamber
– Desiccant: a drying agent that removes moisture from air and
other substances

• Balances
– Classified by:
• Design
• Number of pans (single or double)
• Mechanical vs. electronic
• Operating range: precision, analytic, microbalance

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Basic Separation Techniques
• Centrifugation
– A process in which centrifugal force is used to separate solid
matter from a liquid suspension
– Carried out by a centrifuge, which consists of following:
• Head or rotor
• Carriers or shields attached to vertical shaft of motor
• Metal enclosure
– Force depends on mass, speed, and radius.

– Speed is measured by RPM

– RCF is computed as: 1.118 x 10^-5 x r x RPM^2
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Basic Separation Techniques (cont’d)
• Centrifugation
– Centrifuges classified according to the following:
• Benchtop vs. floor
• Refrigeration
• Rotor head type (fixed, hematocrit, swinging-bucket, angled)
• Maximum speed attainable
– Common applications
• Separating serum or plasma from blood cells
• Separating supernatant from a precipitate in analytic reaction
• Separating two immiscible liquids or expelling air
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Horizontal vs angle head centrifuge

Horizontal head Angle head

• Vertical when not in use • Fixed angle whether in
use or not
• Horizontal when in use
• Lower air friction; lower
• Higher air friction; air resistance
higher air resistance
• Speed: 9000g
• Speed: 1650g
• Remixing is possible

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Centrifugation
• AHC is used in rapid separation of plasma or serum from
cells
• Tachometer: calibrator for speed
• Stopwatch: calibrator for time
• Centrifuge should be cleaned weekly and calibrated every
after 3 months

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Basic Separation Techniques (cont’d)
• Properly balanced centrifuge

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Basic Separation Techniques (cont’d)
• Filtration
– Used only occasionally in labs today
– Filler materials: paper, cellulose, polyester fibers, glass, resin
column materials
– Paper is folded and fitted into funnel; solution (filtrate) drains
through filter into receiving vessel.

• Dialysis
– Solution is put in bag or on one side of semipermeable
membrane.
– Larger molecules remain in bag or on one side of membrane;
smaller molecules and solvents are diffused out.

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Laboratory Mathematics and Calculations
(cont’d)
• Concentration Calculations
– Percent solution
– Molarity
– Normality
– Specific gravity
– Conversions

• Dilutions Calculations
– Simple dilutions
– Serial dilutions

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Simple Dilution

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Serial Dilutions

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Specimen Considerations
• Types of Samples
– Whole blood: both liquid portion (plasma) and cellular
components (red and white blood cells, platelets)
– Plasma: clear yellow supernate
– Serum: remaining liquid after clotting
– Arterial blood: to measure blood gases and pH
– Urine
– Cerebrospinal fluid
– Paracentesis fluids: pleural, pericardial, peritoneal
– Amniotic fluids

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Specimen Considerations (cont’d)
• Blood sample

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Specimen Considerations (cont’d)
• Sample Processing
– Correct matching of blood collection tube with appropriate
analyte request and patient identification labels
– Centrifugation
– Noting presence of any serum or plasma characteristics
(hemolysis or icterus) or turbidity (associated with lipemia)
– Analysis within 4 hours
– Proper capping and protection from areas of rapid airflow, light,
and heat to minimize evaporation
– Refrigeration at 4C for 8 hours, or freezing at -20C for longer
periods, if later testing is required

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Specimen Considerations (cont’d)
• Sample Variables
– Physiologic considerations
– Proper patient preparation
– Problems in collection, transportation, processing, and storage

• Chain of Custody
– Lab tests related to crime or accident are forensic.
– Documented specimen identification is required at each phase of
process.
– Tamper-proof seal

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