Clinical Chemistry Lecture Notes

I. Introduction Overview of Clinical Chemistry

Scope of Clinical Chemistry 1. Clinical chemistry quantitative science that is

concerned with the measurement of amounts of
1. Analytical Chemistry biologically important substances (called analytes)
- Information about the composition and in body fluids.
structure of matter.  The methods to measure these substances
2. Biochemistry are carefully designed to provide accurate
- Chemical and physicochemical processes assessments of their concentrations.
- Carbohydrates, Lipids, Enzymology,
Electrolytes, Trace metals, Proteins, Tumor 2. Clinical chemistry provides biochemical
markers information for the management of patients.
3. Instrumentation
- Provide the foundation for all measurements - Such information will be of value only if it is
made in a modern clinical chemistry laboratory accurate and relevant and if its significance is
4. Toxicology
appreciated by the clinician so that it can be
- Potential toxins, intoxicating or banned used appropriately to guide clinical decision
substances, and prescription medications
making.
present in person’s body
5. Endocrinology Importance of Clinical Chemistry Analyses
- Endocrine glands and hormones
6. Computers  Confirming a clinical suspicion (which could include
- Increasing reliance of the clinician on making a diagnosis)
laboratory investigation  Excluding a diagnosis
 Assisting in selection, optimization, and monitoring
- Data processing embraces the handling of
of the treatment
information
 Providing a prognosis
Definition of Terms  Screening or disease in the absence of clinical signs
or symptoms
1. Laboratory Testing  Establishing and monitoring the severity of a
- A process conducted in a clinical laboratory to physiological disturbance
rule in or rule out a diagnosis, to select and
monitor disease treatment, to provide a
prognosis, to screen on a disease, or to
determine the severity and monitor a
physiological disturbance.
2. Matrix
- In relation to analytical methods, human
serum that contains analytes.
3. Analytes
- A substance whose chemical constituents are
being identified and measured.
4. Reference value
- A value obtained by observation or
measurement of a particular type of quantity
on a reference individual.
 Clinical Chemistry Lecture Notes
Role of Medical Technologist in Clinical Chemistry Fundamentals of the vitalism theory:

 Provide quality health care  Processes within living organisms were unique and
 Perform clinical chemistry test could not be duplicated in the laboratory.
 Cooperate with other health care professionals  Only living organisms can produce organic
compounds and in vitro synthesis of “organic”
History 
compounds is impossible
Only plants can synthesize complex compounds.
Animals acquire such compounds via consumption of
A. Early Beginnings
plants
- Attribution of diseases to Imbalances of Bodily
 Chemistry has no role in physiology
Humors vs. Anatomic Approach
a. Hippocrates
b. Mechanists
- Father of Medicine
- Life could be explained fully by chemical and
- Started the belief that diseases are caused by
physical principles and properties alone.
imbalances of humors in the body.
- Man is not unique as proven by the continuity
- Sparked an interest among early physicians to
between man and the animals (evolution theory)
observe bodily fluids.
in Charles Darwin’s publication ‘Origin of Species’.
b. Giovanni Morgagni
- Introduced the anatomic approach of disease
C. Animal Chemistry and How It Slowly Toppled Vitalism
process
a. Antoine Francois de Fourcroy
- Explained diseases in terms of localized pathologic
- Isolated urea from urine samples
anatomy, rather than as attributable to an
- Believed that chemical laboratories should be
imbalance of the humors diffused throughout the
located near the wards, where chemical analysis of
system.
urine and other excretions of the sick could be
c. Antoine Laurent Lavoisier
carried out.
- Father of Modern Chemistry
b. Friedrich Wohler
- Recognized and named oxygen and hydrogen
- Synthesized urea in vitro by evaporating an
- Discovered the role of oxygen in the process of
isomeric solution of ammonium cyanate
combustion and that respiration is a slow
- ‘Organic’ substance could be synthesized in vitro
combustion process.
without any ‘vital force’ in a living organism
- Started the belief that chemical analysis is a refined
- Bridge between the ‘organic’ and ‘inorganic’
type of dissection
worlds
- Sparked a renewal of interest in the examination of
c. Marcellin Berthelot
body fluids.
- Was able to synthesize organic compounds such as
B. Vitalists & Mechanists: Opposing figures
ethanol, formic acid, and benzene in vitro via
chemical treatments of inorganic compounds
a. Vitalists
d. Claude Bernard
- The very essence of life is due to a “vital force”
- Discovered that glycogen was formed by the liver
present in living organisms.
which contradicted the vitalism belief that only
- Was the popular belief among leading
plants can produce complex compounds.
physiologists and physicians including Marie
e. John Bostock
Francois Xavier Bichat, Johannes Muller, and
- Was the first to observe that urea and albumin
Justus Baron von Leibig.
concentration in plasma decreases as their
concentration increases in the urine of the patient
 Clinical Chemistry Lecture Notes
D. Chemistry in Medical Education
a. William Prout
- Credited as the first to make the true connection
between chemistry and medical practice
- Was a vitalist but advocated the benefits to be
derived from the application of chemistry to
physiology in the treatment of disease
- Favored the study of physics and chemistry by
medical students
b. Henry Bence Jones
- Stressed the practical diagnostic value of chemistry
- Urged the medical school curriculum to include a
first-rate instruction in English; “Medical men would
be much better served if they spent some time in
acquiring knowledge about chemistry and physics
instead of learning some Latin and Greek.”
Bence Jones protein
- monoclonal globulin protein or immunoglobulin light
chain found in the urine
Bence Jones protein appears in urine of multiple myeloma
patients.
1. Heat the urine between 40 – 60 , so precipitation
will occur.
2. Then when heating is continued till 100 , the
precipitation will disappear (clear).
3. If you cool the urine till 40 – 60 the precipitation
will occur
c. Thomas Hodgkin
- “Chemical studies are relevant to clinical medicine”
- Continuous exchange between the solid parts &
blood, “It is in the blood that we must look for many
important modifications in connection with disease”
Functions of Blood
1. Deliver O2 nutrients to all body cells
2. Transport waste products from cells for
elimination
3. Transport hormones
4. Maintain body temperature (distribute heat)
5. Maintain pH (carry buffers)
6. Maintain fluid volume
7. Prevent blood loss (clotting)
8. Prevent infection (WBCs, antibodies)
Blood glucose determination
Hyperglycemia- increased blood glucose
Hypoglycemia-low blood glucose levels
d. Massachusetts General Hospital
• 1847: Recognizing the powerful aid that the science of
medicine “has received from the study of organic
chemistry and the knowledge and use of the
microscope”, authorized the purchase of a
microscope at a cost
• 1851: Established the position of “Chemist-
Microscopist”
E. Clinical Chemistry Takes the Center Stage
a. Otto Knut Folin & Donald Dexter Van Slyke
- Determined reference intervals
- Correlated variations with pathologic conditions
- Elucidated metabolic pathways in health and
disease
 Clinical Chemistry Lecture Notes
b. Donald Dexter Van Slyke
- Invented a volumetric gas-measuring apparatus
for the determination of carbon dioxide
concentration.
c. Otto Knut Folin
- Together with Hsien Wu: Made a method for
production of a protein-free filtrate that can be
used for determining blood sugar
- Developed the Dubosc type colorimeter for the
measurement of creatinine in urine
d. Max Jaffe
- Developed the alkaline picrate method for
determination of creatinine concentration
Basic Laboratory Principles
I. Chemicals
 The American Chemical Society
- Established the specifications of chemicals in the
analytical reagent grade category
- They can be used for analytical laboratory
a. Analytical reagent grade
i. Very high purity
- Spectrograde
- Nanograde
- High-Performance Liquid
Chromatography (HPLC) grade
ii. States the actual impurities
Analytical grade chemicals are used in:
- Most analytical laboratory procedures
- Making up reagents for:
o Spectrophotometry
o Chromatography
o Trace-metal analysis
o Atomic absorption
o Fluorometry
o Standardization
b. United States Pharmacopeia and National
Formulary grade
- Not injurious to humans
- Production of drugs
- Food
- Not pure enough for use in most
procedures
c. Laboratory grade
- Most popular grade for use in educational
applications
- Exact levels of impurities are unknown
- Excellent for teaching and training
d. Less pure grade chemicals
- Referred to as purified and technical
- Meets no official standard
- Do not state the impurity limitations
- Preparation of chemicals is not uniform
- Not recommended for reagent preparations
e. Technical or commercial grade
- Primarily used for manufacturing
- Should not be used in clinical laboratory
- Lowest quality
- Not for analytical work
II. Standards
- Reference materials
- Basis
- Essential for monitoring the accuracy and
precision of analytical measurement
- Instrumental qualification and calibration
- Validation of method
- Estimation of measurement uncertainty
- The National Institute of Standard and
Technology (NIST) developed certfified
reference materials/ SRMs for use in clinical
chemistry laboratories
Types of Standard
a. Primary Standard
- Highest Purity
- Can be measured directly
- Exact known concentration
- Should be at least 99.98 % pure
b. Secondary Standard
- Substance of lower purity
- Concentration is determined by
comparison to a primary standard
Examples of Standards
- Pure substances characterized for chemical
purity and/or trace impurities
- Standard solutions and gas mixtures
 Clinical Chemistry Lecture Notes
- For determination of the most true value of
the analyte
III. Water
A. Reagent grade water
- Suitable for reagent and standard preparation
- Most procedures use distilled or deionized
water
a. Distilled water
- Purified to remove almost all organic
materials
- Water maybe distilled more than once and
each distillation cycle remove impurities
b. Deionized water
- Produced from distilled water anion or
cation exchange resin
- Replacement of hydroxyl or hydrogen ion
respectively
c. Prefiltration
- Can remove particulate water from
municipal water supplies before any
additional treatments.
d. Reverse osmosis
- Pumps water across a semi permeable
membrane, produces RO water
e. Ultrafiltration
- Excellent in removing particulate matter,
microorganisms and any pyrogens or
endotoxins
f. Ultraviolet light sterilization or ozone
treatment
- Sterilization process (uses specific
wavelengths)
- Together with ozone treatment
- Destroy bacteria but may leave behind
residual products
Clinical and Laboratory Standards Institute (CLSI)
- Classified into one of six categories
- Based on the specifications needed for its use
 These categories include:
- Clinical Laboratory reagent water (CLRW)
- Special reagent water (SRW)
- Instrument feed water
- Water supplied by method manufacturer
- Autoclave and wash water
- Commercially bottled purified water
Specifications
o Most water monitoring parameters include at
least microbiological count
i. pH
ii. Resistivity (measure of resistance in ohms
and influence by the number of ions present)
iii. Silicate
iv. Particulate matter
v. Organics
Types of Water
 Type I
- Used for test methods requiring minimum
interference
 Type II
- Acceptable for most analytic requirements
including reagent, quality control and
standard precaution
 Type III
- Autoclave wash water is acceptable for
glassware washing but not for analysis
reagent preparation
IV. Laboratory vessels
A. Flasks, beakers and graduated cylinders are
used to hold solutions
- Volumetric and Erlenmeyer flask are two types
of containers in general use in the clinical
laboratory
 Glassware and Plastic ware
 Kimax/ Pyrex (borosilicate)
- A type of glass and glassware that is resistant
to heat, chemicals, and electricity.
 Corex (aluminosilicate)
- is a aluminosilicate composition with
mechanical strength and scratch resistance
superior to Pyrex.
- It is used in the manufacture of breakage
resistant pipets and centrifuge tubes.
- It is a bit expensive than borosilicate
 High silica
- High purity
- High heat resistance (1, 000 - 1,700ºC)
- Extremely chemically stable and it has
excellent chemical resistance.
 Vycor (acid and alkali resistant)
- high-silica, high-temperature glass
- It provides very high thermal
shock resistance
-
 Clinical Chemistry Lecture Notes
 Low actinic (amber colored)
- Provides protection for materials sensitive
to light.
 Flint (soda lime) glass
- Used for disposable material
- Should consist of high thermal borosilicate or
aluminosilicate glass
- Meet the class A tolerance recommended by the NIST/
ASTM/ ISO 9000
B. Plasticware
- Replace glassware in the laboratory setting
- The unique high resistance to corrosion and
breakage
- Varying flexibility
- Completely disposable

Major types of resin

 Polystyrene, polyethylene, polypropylene,

tygon, teflon, polycarbonate and polyvinyl
chloride
C. Cleaning and washing
- Require special techniques
- Immediate rinsing
- Followed with a powder or liquid detergent
- Several distilled water rinses
- Pre soaking
- Compatibility
- Multiple rinses

V. Centrifuge
- Centrifugation is a process in which centrifugal
force is used to separate solid matter from a liquid
suspension.
- It consists of a head or rotor, carriers, or shields that
are attached to the vertical shaft of a motor and
enclosed in a metal covering.
- The centrifuge always has a lid and an on/off
switch; however, many models include a brake or a
built-in tachometer, which indicates speed, and
some centrifuges are refrigerated.

 Centrifugal force depends on three variables: mass,

speed, and radius. The speed is expressed in
revolutions per minute (rpm), and the centrifugal
force generated is expressed in terms of relative
centrifugal force (RCF) or gravities (g).

The speed of the centrifuge is related to the RCF by the

following equation: