Module 1 – Introduction to Clinical Chemistry

Topic 1 - Scope of Clinical Chemistry

Clinical Chemistry deals with the quantitative analysis and accurate measurement of
the chemical composition of human body (called analytes) in body fluids in order to help
the physician understand the health status, obtain a diagnosis and develop a treatment
plan. It is an exciting field that combines analytics and instrumentation with information
technology, it encompasses varied chemical testing that plays a significant role in the
detection, diagnosis, and treatment of diseases as well as monitoring and maintaining
patient health.
The methods to measure these substances are carefully designed to provide accurate
and precise information of their concentration.
The results of clinical chemistry tests are compared to reference intervals or a medical
decision level (MDL) to provide diagnostic and clinical meaning for the values.
Medical Laboratory Scientists, also known as Medical Technologists or Clinical
Laboratory Scientists, perform these tests on blood and other body fluids using
analytical procedures and state-of-the-art-instrumentation.
The field is growing in parallel with the advancing technology and integrates toxicology
endocrinology and molecular diagnostics.
In addition to the traditional hospital laboratory MLS professionals work in a variety of
other laboratory settings such as research and development, environmental, and public
health laboratories.

Clinical Chemistry Laboratory Organization

➢ Core Lab Facility found at virtually all hospitals operates 24h day 7 days a week to
provide the essential most requested tests.
Highly automated environment, Instruments with Multi-analyte capabilities
• Bar-coded test tubes are loaded onto to the instrument.
• Menu Driven Test selection
• All pipetting, mixing and measurements are automatic
• Random Access (can perform specific tests on a specific sample)
• Analyzer is interfaced with Laboratory Information System (LIS).
• Once resulted are verified, they can be broadcast (sent out)
• All reagents for specific tests such as control calibrators, buffers come as kits that
a loaded directly onto the instruments.
• Instruments constantly monitors the amount of consumables on-board and volume of
liquid and solid waste generated
Type of analytical techniques found on Multi analyte analyzers
Many are immunoassays based with colorimetric detection
Ion-selective electrodes. Designed with a membrane that is specific for a particular
analyte (Na, K, Cl). Change in potential is measured when electrode is placed in sample.

➢ Special Chemistry - less frequently ordered tests, labor intensive and often manual
methods generally non-stat tests (result not required immediately). Employs
Electrophoresis, HPLC, Infrared Spectroscopy, Radioimmunoassay (RIA), GC-MS

➢ Point of Care Testing (POCT)

Instruments located outside of chemistry laboratory such as CCU, ER, ICU or satellite
center (clinic)
Tests are of urgent importance, and results will affect the immediate management of
the patient
Instruments are available that can perform certain tests at remote locations, such as at
the bedside on in a clinical care unit
Blood glucose, Urinalysis, Blood gases, Electrolytes, Cardiac markers (Troponin I & T)
Drug screens
POC are nearly always more expensive, than the same tests performed in the central
laboratory
Many are immunoassay based. Can be qualitative or quantitative

Topic 2 – Fundamental Concepts in Analytical Procedure

Water Specifications
Tap water is unsuitable for lab use (too many impurities)
Types of water purification techniques
Distillation – removes most organic matter
Reverse osmosis-removes organic, ionic, microbial, and viral contaminants
Ultrafiltration – removes particulate matter, bacteria, emulsified solids
Deionization – ions removed
Reagent Grades of water
Type I Purest – Required for sensitive tests
Type II Acceptable for most uses
Type III OK for washing glassware
CAP - QC of water: pH, electrical resistance, bacterial culture

Laboratory Glassware
1. High thermal resistant glass (borosilicate glass with low alkali content – kimax,
pyrex).
2. Alumina-Silicate glass - they are strengthened chemically rather than thermally and
six times stronger than borosilicate example is Corex, they resist clouding and
scratching and alkali resistant.
a. Corex brand – glass which has been strengthened chemically.
b. Vycor brand – recommended for use involving high temperature; drastic heat
shock. It is primarily used in ashing and ignition techniques.
3. High silica glass
4. Glass with higher resistance to alkali
5. Low Actinic Glassware
This contain materials that imparts an amber or red color to the glass and reduce the
amount of light passing to the substance within the glassware's.
It was developed to provide a highly protective laboratory glassware for handling
materials sensitive to light (bilirubin, carotene and Vitamin A).
5. Standard Flint Glass
This is a soda–lime glass composed of mixtures of the silicone, calcium and sodium.
This type of glass is lowest in cost and can readily be fabricated in a variety of shapes.
Glass of choice for weighing bottles.
Glassware can be divided into 2 groups
1. Non-volumetric glassware
Beaker
Flask
2. Volumetric Glassware
Volumetric Flask
Graduated Cylinder

Pipets (Pipettes)
Laboratory instrument used to transport a measured volume of liquid.
Three types of glass pipets used in the laboratory
1. Volumetric - Designed to deliver a single volume precisely, the volume will be
indicated near the top of the pipet, At the top of the pipet is an etched ring.
has a large bulb, and is calibrated for a single volume. Typical volumes are 10, 25, and
50 mL
Fluid must be drawn up the pipet to above the ring indicating the volume and then
released slowly until the bottom of the meniscus is exactly at the ring.
To transfer this volume to a second container, touch the pipette tip to the inside of the
new container and allow the liquid to drain out.
Types of Volumetric pipettes:
a. Ostwald–Folin pipette
A modified volumetric pipette with their bulbs closer to the delivery tip. They are used
for measuring from 0.5 to 10 ml of viscous fluids such as whole blood, serum or plasma.
The pipette has an etched ring near the mouthpiece indicating that the last drop be
blown out (blow–out pipette).
b. Van Slyke pipette
Constructed of thick-walled capillary tubing with a bulb in the center and one mark
above and one below. The specified volume is delivered between these two marks.

2. Graduated Pipet
a. Mohr - TC - sometimes called “drain out pipettes”, a pipet with this marking has been
calibrated to contain a specified volume of liquid. These pipets have a single painted or
frosted ring at the top and are allowed to simply drain with the tip placed against the
side of the receiving vessel.
To accurately transfer fluid with this type of pipet, the meniscus must be precisely on a
calibration mark both at the beginning and at the end of a transfer.
b. Serological - A pipet marked TC, also known as a “blow out” pipet, has been
calibrated "to contain" a specified volume of liquid.
These pipets will have no base mark and graduations continue onto the tip. This means
that ALL the measured liquid in the pipet must be delivered.
A 5 mL serological pipette with one-tenth milliliter graduations can be used to obtain
any desired volume of liquid in one-tenth milliliter increments between 0.1 mL to 5.0mL,
although volumes smaller than 0.2 mL are best measured with automatic pipets.
3. Pasteur Pipettes
Pasteur pipettes are made from glass. With its bulb-shaped top, a Pasteur pipette
resembles an archetypal liquid dropper. Pasteur pipettes are considered fairly
inaccurate today. They are neither calibrated nor graduated, and are more often used in
biology – rather than chemistry – laboratories as a way of transferring aqueous
solutions from one container to another. Named after French physician Louis Pasteur,
Pasteur pipettes are often disposed of after use.
4. Micropipettes – available for measuring very small volumes (0.5 ml or less).
Volumes are often expressed in lambdas. One lambda equals 0.1 ml or 0.001 ul. It is
calibrated either TC or TD. The TC or the wash out pipette are more accurate of the two.
When using micropipettes, the sample is drawn just above the mark, the outer surface
of the pipette is wiped clean with tissue paper and the sample is adjusted to the mark
by touching the tip repeatedly to the paper. The contents are then delivered rinsing
several times into a diluent. The diluent should not be drawn above the calibration mark
because this will add a film of sample in excess of normal value.
a. Sahli hemoglobin pipette – calibrated to contain .02 ml (20ul).
5. Automatic pipettes – dispenses a pre–rated sample when plunger is moved through
its complete stroke. Used to accurately deliver very small volumes, microliters, of liquid.
A volume of 0.1 mL is equal to 100 microliters
a. Peristaltic type b. Piston type c. Seligson type
Beaker
Used for transferring liquid to another container or to transfer a small amount of
reagent for use in procedures. Volume is not accurate, just an estimate.
NEVER PLACE A REAGENT IN ANOTHER CONTAINER WITHOUT LABELING THE
CONTAINER FIRST.
Erlenmeyer Flask
Features a conical base, a cylindrical neck and a flat bottom.
They are marked on the side (graduated) to indicate the approximate volume of their
contents. This is NOT used for ACCURATE measurement
Graduated Cylinder
For rapid measurement of liquid volume. They are generally more accurate and precise
for this purpose than flasks. This is a semi accurate liquid measuring vessel.
Volumetric Flask
A volumetric flask is used to measure very precisely one specific volume of liquid (100
ml, 250 ml, etc., depending on which flask you use).
This flask is used to prepare a solution of known concentration.
To make up a solution, first dissolve the solid material completely, in less fluid than
required to fill the flask to the mark.
After the solid is completely dissolved, very carefully fill the flask to the mL mark.
The top is then sealed and the flask is inverted several times to mix.

General Laboratory Equipment

Centrifuge
Purpose
Separating solids from a liquid suspension by means of centrifugal force
Types
Benchtop
Swing-bucket
Fixed-head or fixed-angle
Ultracentrifuge
Maintenance
Interior and exterior cleaning
Verification of accuracy of timers and speeds
Plastic wares
1. Polyolefins (polyethylene, polypropylene)
2. Polycarbonate resins
3. Tygon
4. Teflon Fluorocarbon resins

Cleansing solutions for glassware:

1. Diluted detergent
2. 10% KOH
3. potassium dichromate
UNITS OF MEASURE
Measurement requires a numerical value and a unit
Laboratory results almost always have units of measurement associated with them
SI units:
length ( meter )
mass ( gram )
quantity ( mole )
Volume ( liter )
Time ( second )
Common prefixes and abbreviations that are added to units of measure:
deci (d) 10-1
centi (c) 10-2
milli (m) 10-3
micro ( μ) 10-6
nano (n) 10-9
pico (p) 10-12
femto (f) 10-15
Example:
A common unit of liquid measurement is a deciliter (dl), or one – tenth of a liter
Combine a prefix with a basic unit results in a statement of a specific length, weight or
Volume Reporting clinical chemistry results may be in units such as :
mg / dL
g / dL
mEq / L
Solutions
The clinical lab almost always uses solutions. A solution means that something has
been dissolved in a liquid. In the clinical laboratory the solvent we measure most of the
time is human plasma. The solute is whatever the substance is we want to measure.
Mixtures of substances – the substances in a solution are not in chemical combination
with one another.
Dispersed phase - the substance is dissolved (the solute)
The substance in which the solute is dissolved is the solvent.
Solute + Solvent = Solution
Concentration of Solution
Amount of one substance relative to the amounts of the other substances in the
solution.
Concentration can be measured in many different units:
% Solutions: w/w, v/v , w/v (parts of solute / 100 totals parts )
Note:
liquids + liquids and solids + solids alter the total parts, but solutes + solvents does not
Molarity: Moles / Liter
Molality: Moles / 1000 grams solvent
Normality: equivalent weight/ liter

Expressing Concentration: Percent Solution (parts/100)

% w/w – percentage weight per weight
Most accurate method of expressing concentration, but can be cumbersome (especially
with liquids), not often used in clinical labs.
% w/w = gram of solute OR gram of solute per 100.0 g of solution
How many grams of NaOH are needed to make a 25.0% w/w solution using deionized
water as the solvent?
25.0% w/w = X g of solute in 100 g of solution
X= 25.0 g NaOH
% w/v – percentage weight per volume
Easiest & most commonly used, very accurate if temperature controlled.
%w/v= g of solute OR g of solute per 100.0 mL of solution
What is the %w/v of a solution that has 15.0 g of NaCl dissolved into a total volume of
100 mL deionized water? X% w/v = 15.0 g NaCl or 15%

% v/v – percentage volume per volume

Least accurate, but used when both substances are liquids
Note: volumes of liquids are not necessarily additive
%v/v= mL of solute OR milliliter of solute per 100 mL of solution
How many milliliters of ethanol are needed to make a 75.0% v/v solution using
deionized water as the solvent?
75.0% v/v EtOH = X mL EtOH in 100 mL of solution
= 75.0 mL EtOH
Three components of Molarity
Gram weight of solute
Solute’s gram molecular weight
Solvent quantity
Number of moles per one liter of solution
Mole = 6.022 X 1023 number of atoms or molecules OR Mole= Molecular weight in
grams
Normality:
N= number of grams of solute
Gram equivalent weight of solute__
1.00 L of solution
Normality (N)
N = Molarity (M) x valence
Molarity = N / valence
M is always < N
When you have a volume and concentration of one, and either the volume or the
concentration of the other: V1 C1 = V2 C2
For Example:
How many mls of 1.0 N HCl is required to prepare 25 mls of 0.5 N HCl
( 1.0 N ) ( ? mls ) = ( 0.5 N ) ( 25 mls)
? mls = 12.5 mls
You would need to add 12.5 mls of 1.0 N HCl to 12.5 mls of deionized water
( a total volume of 25 mls) to prepare 25 mls of 0.5 N HCl

Conversion:

How many mls are there in 2.5 liters?

 1000 mls 
 2.5 L iter   2500 mls
 1 L iter 

Dilutions
A ratio of the concentrate to the total (final) volume.
A 1:4 dilution has a 1 volume of sample and 3 volumes of diluent mixed together.
Any volume can be used to create this dilution, but it must be the same unit of volume
Keep in mind the sample size when making your dilution
For example: a 2:3 dilution could contain:
2 mL serum: 1 mL pure water
20 µL of serum: 10 µL of pure water
0.2 mL of serum: 0.1 mL of pure water

Serial Dilutions
In these types of questions, you are given a series of tubes. Each tube having a
measured amount of a diluent. You are instructed to add a specified amount of
specimen into the first tube, mix well and transfer a specified amount of the mixture to
the next tube, etc.