Chapter 1

Basic Principles and Practice of Clinical

Chemistry, part 2

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 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

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 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.

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 Controls
 For our purposes:
 Defined: substance, whose physical and
chemical properties resemble the unknown
specimen …
 A control should have the same appearance
and consistency as does the patient samples:
 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.

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 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

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 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?

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 Water Specifications

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

 Types of water purification techniques

 Distillation – removes most organic matter

 Reverse osmosis
 Filtration
 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

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Water filtration system for
Automated chemistry analyzer.

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 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 semi-
permeable 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 None
 Red / Black None – Gel separator
 Lavender EDTA anticoagulant
 Orange Thrombin promotes clotting
 Blue Sodium citrate anticoagulant
 Gray Sodium fluoride / Potassium oxalate
 Green Heparin anticoagulant

 Collection order ( to avoid contamination / interference )

 1 Sterile specimens – Blood Cultures (yellow)
 2 Blue
 3 Gold / Red / Orange
 4 Green
 5 Lavender
 6 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 doesn’t 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 Loses Electrons , it is Oxidized (LEO)

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

 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 )

 Conductivity: Measure of electrical current

 Resistance: Measure of resistance to current

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 1
 pH and Buffers 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-zero’s are significant
 All zeros between non-zero numbers are significant
 Zero’s 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 4
 101 3
 6.2 2
 207.0 4
 679.01 5
 700 1
 24300 3
 0.0100 3
 0.0004 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  0.000614794
1200

The figure with the least number of significant figures is 1200 ( it has 2 ).
Your answer can’t 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 doesn’t 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

 1000 mls 
125
. Liters 
 1 Liter 
  1250 mls

100 mg = _________ ug ?

 1000 ug 
100 mg  1 mg   10,0000 ug
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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 grams NaCl   1 EqWt NaCl   1000 mls 

     015
. N
 100 mls water   58 grams   1 Liter 

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 analyzer’s

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 Parts Total Dilution Dilution

Specimen Diluent Volume Factor

1.0 1.0 2.0 1:2 2

1.0 2.0 3.0 1:3 3

1.0 3.0 4.0 1:4 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

It’s 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 ( don’t 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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