Basic Principles and Practice of Clinical Chemistry

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Chapter 1

• Basic Principles and Practice of Clinical
  Chemistry

2

• Introduction
• So, you want to be a clinical chemist? Thats
  terrific !!!
• I am assuming that you have taken ( and passed )
  the first semester of Inorganic Chemistry
• Chapter 1 is intended as a review ( hopefully )
  of some basic chemistry terminology and equipment
  It is not intended to replace first semester
  inorganic chemistry
• A review is in order because Clinical Chemistry
  often uses these basic chemistry terms

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Key Terms

• Buffer
• Centrifugation
• Colligative property
• Conductivity
• Deionized water
• Density
• Dilution
• Distilled water
• Equivalent weight ( EqW )
• Molality
• Molarity ( M )
• Normality ( N )
• Osmotic Pressure
• Oxidation / Reduction

• Oxidizing / Reducing Agent
• Percent Solution
• pH
• pK
• Pipet
• Primary / Secondary Standard
• Serial Dilution
• Solute / Solvent
• Specific Gravity
• Standard Reference Materials
• Whole Blood / Serum / Plasma
• Valence
• Celsius / Fahrenheit
• To Deliver / To Contain

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• Objectives
• Define the prefixes commonly used in units of
  measurements
• Perform simple conversions ( temperature and
  common measurements )
• Identify different grades of purity for chemical
  reagents
• Define different types of standards that are used
  in clinical chemistry
• Define all the terms in the Key Terms list
• Describe different pipets and their proper uses
• Define 3 different types of specimens that are
  commonly used in a clinical laboratory
• Identify the anticoagulants and colored tops
  associated with blood collection tubes

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• UNITS OF MEASURE
• Measurement requires a numerical value and a unit
• SI units
• length ( meter )
• mass ( gram )
• quantity ( mole )
• Basic units describe unrelated physical
  quantities
• Laboratory results almost always have units of
  measurement associated with them ( It is
  important to know these units )

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• Common prefixes 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
• Reporting clinical chemistry results may be in
  units such as
• mg / dL
• g / dL
• mEq / L

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• REAGENTS
• Reagent preparation in the clinical lab is
  decreasing as most reagents are obtained from
  commercial manufacturers
• However , here are some common terms that relate
  to reagent purity
• Analytical Grade(purest)USPNFChemical Pure
  (least pure)
• Primary Standards must be 99.98 pure
• Most biological standards cannot be 99.98 pure
  because the chemical processes to achieve this
  level of purity would destroy the substances.
• Biological substances are referred to as SRM (
  Standard Reference Material )
• Primary Standard Highly purified solution of
  known concentration. These standards are used in
  the clinical lab to calibrate instruments in
  order to measure other solutions of unknown
  concentration
• Secondary Standard Less pure substance whose
  concentration was determined by comparison to a
  Primary Standard

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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 or the
  hospital lab itself
  
• Every lab is responsible for the establishment of
  normal ranges
• It is usually a plasma based solution that is
  treated just as if it were a patient specimen
• Control specimens must produce results within
  established ranges
• A Standard solution is a highly purified solution
  that is usually not 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 concentrations
• Standard solutions are also called Calibrators

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• Water Specifications
• Tap water is unsuitable for lab use ( too many
  impurities )
• Types of water purification techniques
• Distillation
• Reverse osmosis
• Filtration
• Deionization
• Reagent Grades of water
• Type I Purest Required for sensitive tests
• Type II Acceptable for most uses
• Type III OK for washing glassware
• QC of water pH, electrical resistance,
  bacterial culture

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Water filtration system for Automated chemistry
analyzer.
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• Concentrations
• The clinical lab almost always uses solutions. A
  solution means that something has been dissolved
  in a liquid
• Solute Substance being dissolved into a
  liquid
• Solvent The liquid the substance is being
  dissolved into
• Solute Solvent Solution
• A solutions concentration is a measurement of
  how much solute is dissolved in a given volume of
  the solvent Thats pretty much what
  clinical chemistry is all about !!!
• 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

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• Solution Properties
• 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 alters the total parts, but solutes
  solvents does not
• Molarity Moles / Liter
• Molality Moles / 1000 grams solvent
• Normality Equivalents Weights / Liter

Equivalent Weight MW / Valence Valence the
electrical charge of an ion, or the number of
moles that react with 1 Mole H
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• But what is a mole? ( No, its not a cute little
  furry animal with bad eyes )
• Mole 6.022 X 1023 number of atoms or
  molecules
• Molecular Weight
• The molecular weight( MW ) of hydrogen 1.0
  
• That means that 6.022 X 1023 hydrogen atoms
  weighs 1.0 gram
• The MW of H2O (1)(2) (16) 18
• 1 mole of water weighs 18 grams
• That means that 6.022 X 1023 H2O molecules weigh
  18.0 grams

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• Equivalent Weight
• Equivalent Weight Molecular Weight / Valence
• The valence is the electrical charge of the
  substance
• 1 Equivalent weight of any substance reacts with
  1 Equivalent Weight of hydrogen ions
• Example
• The MW of calcium 40 grams
• Calcium ions carry a 2 electrical charge (
  valence 2 )
• Equivalent Weight of calcium 40 / 2 20 grams

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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 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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• pH and Buffers
• 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 gt 7 alkaline (basic)
• pH lt 7 acid

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• CLINICAL LABORATORY SUPPLIES
• Temperature
• Scientific measurement of temperature is always
  expressed in the Celsius ( C) scale , not
  Fahrenheit ( F )
• Measurement of temperature is an important
  component of the clinical lab. Instruments,
  refrigerators and incubators are required to
  operate within specific temperatures that must be
  maintained and monitored.
• Each laboratory must have a NIST calibrated
  thermometer in order to ensure the accuracy of
  other thermometers in the laboratory
• Celsius scale 0 degrees freezing point of
  water
• 100 degrees boiling point of
  water
• Conversion of Celsius to Fahrenheit and
  Fahrenheit to Celsius
• F ( C x 1.8 ) 32
• C ( F - 32 )
• 1.8

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Examples of Temperature Conversions
Your refrigerator at home is probably around 40
F. What is that in Celsius?
Water boils at 100 C. What is that expressed
in Fahrenheit?
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• Glassware and Plasticware
• Pipets
• To Contain ( TC ) Blow out
• TO Deliver ( TD ) Let drain
• Serological pipets are usually TC
• Volumetric pipets are TD, the most accurate and
  used to prepare Standard solutions, Calibrators
  and Quality Control specimens
• Place dirty pipets in soapy water with tips up
• A micropipet delivers less than 1.0 ml ( MLA
  micropipets are common )

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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 !!!

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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 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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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

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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• Conversions
• You must remember this , a kiss is just a .
  Opps! Lets try again!
• 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.
• Many students go into convulsions when it comes
  to conversions. They throw their arms up and get
  spastic. Really!
• Take a deep breath and chill out because Im
  going to tell you a secret about conversions.
  Ready?

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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
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
100 mg _________ 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?

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 12.
• 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
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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• 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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• 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
• 2 Red / Orange
• 3 Green
• 4 Lavender
• 5 Gray

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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