Chapter 1

Basic Principles and Practice of Clinical

Chemistry, part 2

C. REAGENTS; Chemical Grades

Reagent preparation in the clinical lab is decreasing most reagents are obtained from commercial
manufacturers

Objective: Identify and differentiate the different degrees of chemical

purity.

Common terms that relate to reagent purity:

Analytical Grade (purest), also called reagent grade or

ACS grades - best choice for lab work.
National Formulary (NF) or US Pharmacopeia (USP)
used for drugs, may be OK for lab work
Chemical Pure (least pure) not recommended for lab
Technical or commercial grade never for lab use

REAGENTS; Chemical Grades

Primary Standard : Highly purified solution of known

concentration. These standards are used in the clinical
lab to calibrate / standardize instruments in order to
measure other solutions of unknown concentration

Primary Standards must be 99.98% pure

Secondary Standard : Less pure substance whose

concentration was determined by comparison to a
Primary Standard
Standard Reference Material / Calibrator

The name for biological substances used as

standards
Most biological standards cannot be 99.98%
pure because the chemical processes to achieve
this level of purity would destroy the substances.

Controls

For our purposes:

A control should have the same appearance

and consistency as does the patient samples:

Defined: substance, whose physical and

chemical properties resemble the unknown
specimen

If patient sample is serum; the control should look

like and have the same consistency as serum.
If the patient sample is urine, the control is urine,
etc.

- Controls are used to verify the accuracy

and acceptability of a run.
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Control Solutions vs. Standard Solutions

A Control specimen is used to monitor Quality

Control (QC)
A Control has known acceptable ranges, established
either by the manufacturer (assayed) or the hospital
lab itself (un-assayed)
It is usually a serum/plasma based solution that is
treated just as if it were a patient specimen
Control specimens must produce results within
established ranges in order for the run to be
acceptable.

Control Solutions vs. Standard Solutions

A Standard solution is a highly purified solution that

is usually not serum / plasma based

Standard solutions have set, listed values that are

established by the manufacturer

Standard solutions are used to calibrate

instruments, that is to set instruments to measure
correctly at known concentration

Control Solutions vs. Standard Solutions

Standard solutions are also called Calibrators if

they are biological in nature

Consider for example, analytes such as bilirubin.

These substances do not come in the highly purified

state, as calcium, glucose, etc.

A bilirubin standard is biological based, and technically

a calibrator rather than the purely defined standard.

What about the hematology standards? Standard or

calibrator?

Water Specifications

Tap water is unsuitable for lab use (too many impurities)

Types of water purification techniques

Reagent Grades of water

Distillation removes most organic matter

Reverse osmosis
Filtration
Deionization ions removed

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
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Water filtration system for

Automated chemistry analyzer.

Use of Blanks

Review: Blanks used to eliminate or subtract the effects of

reagent or specimen colors that would interfere with
accurately measuring an analyte.

Water Blank DI water, used to zero the spectrophotometer.

Seen mostly in UV procedures.

Reagent Blank contains all the reagents used in the tests.

DI water sometimes used in the place of the amount of
patient specimen. Colorimetric procedures.

Patient Blank required by some procedures if patient

sample has deep color that would affect results. Name 3
situations that would warrant use of patient blank.

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Labware

Types of glass

High thermal borosilicate

Can take long periods of high temperatures
Scratches easily
Acceptable for chemistry work
Examples: Pyrex, Kimax

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Labware

Types of glass

Aluminosilicate
Can withstand heat as long as not in contact with
acids or alkalis
Resists scratching
Acceptable for chemistry work
Examples: centrifuge tubes, thermometers

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Labware

Types of glass

Soda lime not suitable for lab use

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Types of plastic resins

Polystyrene

Clear, rigid
Can withstand temperatures to 70 C
Examples: many disposables

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Types of plastic resins

Polyethylene

Translucent in appearance
Two types
One type can withstand temperatures up to 80 C,
and is flexible, i.e., reagent wash bottles
Other can withstand temperatures up to 120 C and
is rigid, i.e., droppers

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Types of plastic resins

Polyvinyl chloride

Translucent in appearance, but rigid

Withstands temperatures to 135 C
Examples: screw cap enclosures

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Types of glassware

Beakers
Flasks

Volumetric
Erlenmeyer
Graduated cylinders
Reagent bottles
Test tubes

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Pipets

Types

Volumetric

Volumetric pipets are TD, the most accurate and used to prepare
Standard solutions, Calibrators and Quality Control specimens
Ostwald-Folin

Capillary
Serologic

TD = to deliver
TC = to contain

Mohr
Transfer
Automatic and
semi-automatic
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Laboratory Vessels and Pipets

Volumetric flasks : The line indicates the level that contains an exact volume
Erlenmeyer flasks : Hold variable volumes
Graduated cylinders : Hold variable volumes
Pipet rules

TC = needs to be blown out

TD = let drain along the side of the receiving vessel
Read pipets from the bottom of the meniscus
Hold pipets straight up and down
Use suction bulbs to aspirate fluids into pipets
NEVER MOUTH PIPET !!!

Place dirty pipets in soapy water with tips up

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Proper use

Use correct pipet for the job

Examine the pipet before use for cleanliness, chips,
etc.
NEVER pipet by mouth
Draw the solution slightly above the mark
Wipe the tip with a Kimwipe

Correct and incorrect pipet

positions

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Buret essentially an elaborate pipet

mounted on a stand used in titration
procedures

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Cleaning of Lab Glassware

Majority of time can simply presoak, dishwash, and

thoroughly rinse with tap and finally
distilled/deionized water

Chemically clean glassware is required for certain

chemistry procedures (enzymes, iron, heavy metals,
etc.)
dichromate acid or acid dichromate

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General Laboratory Equipment

Balances type chosen dependent on

volume/weight needed and degree of accuracy
required.

What is the best choice for clinical work?

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General Laboratory Equipment

Centrifuge

Purpose
Types
Characteristics

Fixed rotor head / swinging bucket

Closing locked closed lid now required

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General Laboratory Equipment

Other methods of separating materials

Filtration of materials
Dialysis - a method made popular by Technicon
Corporation (early manufacturer of automated lab
equipment). This method makes use of a semipermeable membrane that allows separation of
molecules using their size

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Specimen Collection and Processing

Medical ethics in specimen collection

professionalism and confidentiality at all
times

Special collection procedures

Fasting specimens: overnight for most tests, 12 hours

for lipid studies
Timed interval specimens
Examples include glucose tolerance, therapeutic
drug monitoring, and hormone stimulation testing
In some cases urine collection also required
Legal chain of evidence
Other special collection procedures

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Specimen processing

Determining specimen acceptability

Other than improper timing, identify things that can

affect chemical analysis of clinical specimens.

Specimen accessioning

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Specimen processing

Serum separators covered in summer course

Gel barrier
Beads, crystals or fibers
Plastic tube device

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Other / SPECIMEN CONSIDERATIONS

Specimen collection and processing are critical

A poor specimen = poor specimen results
Most lab errors are pre-analytical !!!
Common sources of error

Contamination with IV fluids

Hemolysis of RBCs contaminates plasma and
serum
Labeling errors
Collection with improper anticoagulants and
preservatives
Analyzers clogged by clotted specimens
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The slides that follow are from another

information source and remain here only
for general use, at this time.

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Collection tubes / Additives

Red
Red / Black
Lavender
Orange
Blue
Gray
Green

None
None Gel separator
EDTA anticoagulant
Thrombin promotes clotting
Sodium citrate anticoagulant
Sodium fluoride / Potassium oxalate
Heparin anticoagulant

Collection order ( to avoid contamination / interference )

1
2
3
4
5
6

Sterile specimens Blood Cultures (yellow)

Blue
Gold / Red / Orange
Green
Lavender
Gray

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Colligative Properties

Properties of solutions that are based only on the numbers of particles

that are dissolved in the solvent

It doesnt matter what the particles are or how big they are

Examples of colligative properties

Freezing Point
Boiling Point
Vapor Pressure
Osmotic Pressure

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Redox Potential ( Oxidation-Reduction Potential)

If a substance Gains Electrons , it is Reduced (GER)

It may also be called a Oxidizing Agent ( accepts
electrons)

Remember The lion ( LEO ) says gerr ( GER )

If a substance Loses Electrons , it is Oxidized (LEO)

It may also be called a Reducing Agent ( donates
electrons)

Conductivity: Measure of electrical current

Resistance:
Measure of resistance to current
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pH and Buffers

1
H

Buffers resist change in acidity

Buffers are usually weak acids ( or bases) and their salts
pH is the unit used to measure acidity ( Hydrogen ion
concentration )
p = negative log of the concentration of a substance in
solution.
Example: pH = - log [H+]
The Hydrogen ion concentration of deionized H2O is 1 x 10-7 M
The negative log of 10-7 = 7. The pH of H2O is 7.0

The pH scale ranges from 0 - 14

pH 7 = neutral
pH > 7 = alkaline (basic)
pH < 7 = acid

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Significant Figures Rules

All non-zeros are significant

All zeros between non-zero numbers are significant
Zeros to the right of a number with a decimal place are significant

Zeros to the right of a number without a decimal place are not significant
Zeros to the left of a number with a decimal place are not significant

Examples of significant figures

9004
101
6.2
207.0
679.01
700
24300
0.0100
0.0004

4
3
2
4
5
1
3
3
1

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Conversions

You must remember this, conversions do NOT

change the value of the concentration
Conversions only change the UNITS the value
is being expressed in.

Whatever we are converting is just as big or

small as before we did the conversion.

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Rules for Multiplication and Division of Significant Figures

Perform the multiplication and division as written

Round off your final answer to the least number of significant figures that
occurs in the original figures

Example

0.0211 2.53 13.82

1200

0.000614794

The figure with the least number of significant figures is 1200 ( it has 2 ).
Your answer cant have more significant figures than the weakest link in the chain
The answer must also be rounded off to 2 significant figures 0.00061
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Example of a conversion

How many mls are there in 2.5 liters? ( this is an easy one )

The question you have to ask yourself is, what is the relationship between
liters and mls? The answer : 1 liter = 1000 ml This is a true statement
But now what?
We want to get rid of the liters units and end up with mls Right ?
So all you need to do is put in a truthful mathematical statement that gets rid
of the stuff you want to lose and adds the stuff you want to pick up So

1000 mls
2.5 Liter
2500 mls
1 Liter
THIS IS THE SECRET !!!
The fraction I created equals 1.0
It doesnt change the value!
I wrote it with the Liter on the bottom so it would cancel out the Liter on the top
and I also picked up the mls I need . All conversions use this strategy
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1.25 liters = _____ mls ?

Remember, write a fraction that does two things:

1. Equals 1
2. Gets rid of unwanted units and / or adds needed units

1 0 0 0 m ls
1 . 2 5 L i t e r s 1 L i t e r 1 2 5 0 m l s

100 mg =

_________ ug ?

1000 ug
1 0 0 m g 1 m g 1 0 , 0 0 0 0 u g
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Another conversion example

Physiological Saline is used in Blood Banks and Hematology to prepare

Red Blood Cell suspensions.
Physiological Saline is usually listed as being 0.9 % NaCl
0.9 grams of NaCl is added to 100 mls deionized water to make
physiological saline
What is the Normality (N) of physiological saline?

0 .9 g r a m s N a C l 1 E q W t N a C l 1 0 0 0 m l s

0 .1 5 N
1 0 0 m ls w a te r 5 8 g r a m s 1 L ite r
Unwanted units cancel out
leaving EqWt / Liter = N

Fraction = 1

Fraction = 1

Conversions are manipulations of the units not the values !!!

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Dilutions

A dilution is a numerical ratio of the original material to

the final volume ( after the addition of a diluent )

Dilutions of serum or plasma are required when the

concentration of a chemical substance being measured
exceeds the linearity of the test methodology

Example

A plasma glucose concentration exceeds the analyzers

ability to accurately measure it. The automated analyzer is
programmed to dilute the specimen 1:2.

The concentration of the diluted specimen must be

multiplied by 2 , the dilution factor ( the reciprocal of the
dilution ) to correct for the dilution of the specimen.

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Examples of dilutions and dilution factors

Parts
Specimen

Parts
Diluent

Total
Volume

Dilution

Dilution
Factor

1.0

1.0

2.0

1:2

1.0

2.0

3.0

1:3

1.0

3.0

4.0

1:4

1.0

9.0

10.0

1 : 10

10

0.5

4.5

5.0

1 : 10

10

0.2

1.8

2.0

1 : 10

10

0.2

9.8

10.0

1 : 50

50

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Making Dilutions of Concentrated Acids or Bases

Its common to make dilutions of concentrated solutions to prepare new solutions of

lower concentrations. Remember this formula:

C1V1 = C2V2

C = Concentration of solution ( M or N )
V = Volume of solution

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

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TOP 10

Know those prefixes !!!

Molarity = Moles / Liter
Molality = Moles / 1000 grams solvent

Normality = Eq Wt / Liter

Per Cent Solutions = parts / 100

( Be careful if your dealing with liquids and solid materials )

Do some simple conversions

TD pipet ( dont blow out ) TC ( blow out )

Buffers resist changes in pH ( p = - log )

A dilution is a ratio of original material to the final total volume

The lion says gerr

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