Basic Principles and Practice

in Clinical Chemistry

acpj
Topic Outline
• Introduction to Clinical Chemistry
• Units of Measure
• Reagents
• Clinical Laboratory Supplies
• Basic Separation Techniques
• Specimen in Clinical Chemistry
 Introduction to Clinical Chemistry

Clinical – Greek word “ Kline” meaning bed

Chemistry- the science that deals with the elements, their
compounds and the chemical structure and interaction of
matter
Clinical Chemistry- is the area of chemistry that is
generally concerned with quantitative and qualitative
analysis of bodily fluids for diagnostic and therapeutic
purposes.
Introduction to Clinical Chemistry
• Clinical Chemistry

• deals with the analysis of the chemical constituents of the blood as well as
other body fluids to facilitate diagnosis of disease.
History of Clinical Chemistry

• 300 BC: Hippocrates

• 1600: acid precipitation of urine for CHON
• 1700: study of Diabetes Mellitus
• 1850-1890s: use of Laboratory Medicine
• 1918-1920s: epinephrine, uric acid, ammonia, NPN, CHON
History of Clinical Chemistry

• 1930: enzymatic tests

• 1940: photoelectric colorimetry
• 1950s: L-J, Shewhart,
• 1960s: AAS for Calcium and Magnesium
• 1952: The International Association of Clinical Biochemists was
formed.
• 1953: IACB changed its name to The International Federation of
Clinical Chemistry (IFCC)
UNITS OF MEASURE
• Quantitative laboratory results
• 1st = Actual value
• 2nd = Label

• Système International d’Unitès (SI): preferred in scientific literature

and clinical laboratories and is the only system used in many
countries.
Image Source: Clinical Chemistry, Bishop, et.al
UNITS OF MEASURE
• Derived unit
• Derivative or a mathematical function of one of the basic units

• SI uses standard prefixes

Image Source: Clinical Chemistry, Bishop, et.al

UNITS OF MEASURE
• Reporting of laboratory results is often expressed in terms of
substance concentration, or the mass of a substance rather than in SI
units.
Image Source: Clinical Chemistry, Bishop, et.al
Electronic Reporting of Results
• Electronic transmission of laboratory data and the more routine use
of an electronic medical record, coding, billing, and other data
management systems may vary.

• Most systems agree that each test name should be clearly identified
and should have its unique code and should include the value and
the unit or the appropriate abbreviation.
REAGENTS
• A substance that is used to test for the presence of another substance
by causing reaction with it.

• Any substance employed to produce a chemical reaction

Chemicals
I. Analytical Reagent (AR)
II. Ultrapure
III. Chemically Pure (CP)
IV. United States Pharmacopeia (USP)
V. National Formulary (NF)
VI. Technical or Commercial Grade
Chemicals
vOrganic Reagents

vPractical grade with some impurities

vChemically pure
vSpectroscopic
vChromatographic
Reference Materials
• Primary standard
• highly purified chemical that can be measured directly to produce a sub-
stance of exact known concentration and purity

***NIST: Standard/Certified Reference Material (SRM/CRM)

• Secondary Standard
• substance of lower purity with concentration determined by comparison with
a primary standard
Water Specifications
• Water is the most frequently used reagent in the laboratory.
• Purification
• Distillation
• Ion exchange
• Reverse Osmosis

• Reagent Grade Water

• Clinical laboratory reagent water (CLRW), Special reagent water (SRW),
Instrument feed water, Water supplied by method manufacturer, Autoclave
and wash water, and Commercially bottled purified water.
Water Specifications
• Prefiltration: remove particulate matter for municipal water supplies
before any additional treatments
• Glass, cotton, activated charcoal, submicron filters

• A. Distillation: water has been purified to remove almost all organic

materials
• B. Ion exchange: water has some or all ions removed
• C. Reverse Osmosis: process that uses pressure to force water through a
semipermeable membrane
• D. Ultrafiltration & Nanofiltration: excellent in removing particulate
matter, microorganisms, and any pyrogens or endotoxins
Types of Water
Type I Type II Type III

Maximum colony ≤10 1000 NS

count (cfu/ml)

Silicate (mg/L) 0.05 0.1 1.0

Resistivity 10 1 0.1
(megaohm @ 25C)

pH NS NS 5-8

Organic compunds Activated carbon NS NS

CLINICAL LABORATORY SUPPLIES
• A. Thermometers

• Liquid in glass: usually measure temperatures between 20°C and 400°C

• Electronic / Thermistor probe: smaller and has millisecond response time

• Digital
CLINICAL LABORATORY SUPPLIES
• B. Glassware

• Borosilicate Glass
• Flint Glass
• Quartz Glass
• Boron-free
• Aluminosilicate
• Vycor
• Low-actinic
Image Source: Clinical Chemistry, Bishop, et.al
CLINICAL LABORATORY SUPPLIES
• C. Plasticwares

• Polyolefins

• Polycarbonate resins

• Tygon

• Teflon-fluorocarbon resin
Image Source: Clinical Chemistry, Bishop, et.al
Image Source: Clinical Chemistry, Bishop, et.al
Cleaning of Labware

• Blood clots:
• New pipets:
• Metal ion determination:
• Grease:
• Bacteriologic:
• Permanganate stains:
Image Source: Clinical Chemistry, Bishop, et.al
Laboratory Vessels
• A. Volumetric flask
• calibrated to hold one exact volume of liquid (TC)
• has a round, lower portion with a flat bottom and a long, thin neck with an
etched calibration line.

• B. Erlenmeyer flasks and Griffin beakers

• designed to hold different volumes rather than one exact amount
• Erlenmeyer flask has a wide bottom that gradually evolves into a smaller,
short neck
• Griffin beaker has a flat bottom, straight sides, and an opening as wide as the
flat base, with a small spout in the lip.
Laboratory Vessels
• C. Graduated cylinders
• has calibration marks along its length and is used to measure volumes of
liquids
• long, cylindrical tubes usually held upright by an octagonal or circular base
Image Source: Clinical Chemistry, Bishop, et.al
Pipets
• Glass or plastic utensils used to transfer liquids; they may be reusable
or disposable

• Designed to contain (TC) or to deliver (TD) a particular volume of

liquid
Pipets

Image Source: Clinical Chemistry, Bishop, et.al

Pipets
• A. To Contain (TC)
• holds or contains a particular volume but does not dispense that exact
volume

• B. To Deliver (TD)
• will dispense the volume indicated
Pipets
• A. Blowout
• has a continuous etched ring or two small, close, continuous rings located
near the top of the pipet

• B. Self-draining
• the user allows the contents of the pipet to drain by gravity
Pipets
• Measuring or Graduated Pipets

A. Mohr: does not have graduations to the tip

• self-draining pipet, but the tip should not be allowed to touch the vessel while
the pipet is draining
B. Serologic: has graduation marks to the tip
• used for the measurements of reagents and are not generally considered
accurate enough for measuring viscous samples and standards
• Blowout pipet
Pipets
• Measuring or Graduated Pipets

C. Micropipet: with a total holding volume of less than 1 mL

• may be designed as either a Mohr or serologic pipet
Pipets
• Transfer Pipets

A. Ostwald-Folin: used with biologic fluids having a viscosity greater

than that of water
• blowout pipets, indicated by two etched continuous rings at the top
B. Volumetric: designed to dispense or transfer aqueous solutions and
is always self-draining
• has the greatest degree of accuracy and precision and should be used when
diluting standards, calibrators, or quality-control material
Pipets
• Transfer Pipets

C. Pasteur: do not have calibration marks and are used to transfer

solutions or biologic fluids without consideration of a specific volume
• not used in any quantitative analytic techniques
D. Automatic: most routinely used pipet in today’s clinical chemistry
laboratory
• Safe, stable, high precision, saves time, less cleaning required
• Fixed or variable
Pipets
• Transfer Pipets

D. Automatic:
• Air Displacement: relies on a piston for suction creation to draw the sample into a
disposable tip that must be changed after each use

• Positive Displacement: operates by moving the piston in the pipet tip or barrel, much
like a hypodermic syringe

• Dispenser: obtain the liquid from a common reservoir and dispense it repeatedly

ØCalibration: Gravimetric or Photometric

Image Source: Clinical Chemistry, Bishop, et.al
Burets
• looks like a wide, long, graduated pipet with a stopcock at one end
• used to dispense a particular volume of liquid during a titration

Image Source: Clinical Chemistry, Bishop, et.al

Syringes
• Sometimes used for transfer of small volumes in blood gas analysis or
in separation techniques such as chromatography or electrophoresis

• Glass and have fine barrels

Dessicators and Dessicants
• Dessicants
• keep other chemicals from becoming hydrated
• most effective when placed in a closed, airtight chamber called a desiccator
• In the laboratory, desiccants are primarily used to prevent moisture
absorption by chemicals, gases, and instrument components.

Image Source: Clinical Chemistry, Bishop, et.al

Balances
A. Analytical Balance: required for the preparation of any primary
standards
• mechanical analytic balance is also known as a substitution balance
B. Electronic Balance: uses an electromagnetic force to
counterbalance the weighed sample’s mass
• measurements equal the accuracy and precision of any available mechanical
balance, with the advantage of a fast response time

ØCalibration
ØClass I: 0.01 mg to 0.1 mg
ØClass II: 0.001–0.01 g
ØClass III: 0.01–0.1 g
Image Source: Clinical Chemistry, Bishop, et.al
Basic Separation Techniques
• A. Centrifugation
• A process in which centrifugal force is used to separate solid matter from a
liquid suspension
• Centrifugal force: mass, speed, and radius
• Speed: rpm
• Centrifugal force generated: RCF or g

• used to separate serum or plasma from the blood cells

• separate a supernatant from a precipitate during an analytic reaction
• separate two immiscible liquids
• to expel air
Image Source: Clinical Chemistry, Bishop, et.al
• Centrifuge Care
• daily cleaning of any spills or debris
• balancing the centrifuge load
• centrifuge cover should remain closed until the centrifuge has come to a
complete stop
vTachometer or Strobe Light: used to check the speed of a centrifuge

Image Source: Clinical Chemistry, , Bishop, et.al

Basic Separation Techniques
• B. Filtration
• Used for the separation of solids from liquids
• Filter material is made of paper, cellulose and its derivatives, polyester fibers,
glass, and a variety of resin column materials
• Filter papers differ in pore size
• Filter paper should not be used when using strong acids or bases

vFILTRATE

Image Source: Clinical Chemistry, Bishop, et.al

Basic Separation Techniques
• C. Dialysis
• Used for separating macromolecules from a solvent or smaller substances
• Larger molecules are retained within the sack or on one side of the
membrane, while smaller molecules and solvents diffuse out
Basic Separation Techniques
• D. Lyophilization
• Freeze drying

• E. Extraction
• separation of compounds, based on their relative solubilities in two different
immiscible liquids or solid matter compound
Specimens in Clinical Chemistry
• Blood
• Urine
• CSF
• Pleural fluid
• Pericardial fluid
• Peritoneal fluid
• Amniotic fluid
• References

• Clinical Chemistry: Techniques, Principles, Correlations, Bishop, M.L., et.al

• Clinical Chemistry Handbook for Medical Technologists, Rodriguez, MT.