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MLAB 2401:Clinical Chemistry

Basic Principles and Practice of Clinical Chemistry

Part One

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 )

Basic units describe unrelated physical quantities

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Unit of Measure: Prefixes

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

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Scientific Notation

True scientific notation format: 1.22 X 104

BUT in hemo, for example a hemoglobin result would look like = 12.2 X 103

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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-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

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Water filtration system forAutomated chemistry analyzer.

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

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Concentration

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 alters the total parts, but solutes + solvents does not

Molarity: Moles / Liter

Molality: Moles / 1000 grams solvent

Normality: equivalent weight/ liter

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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 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 solutionX= 25.0 g NaOH

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Expressing Concentration:Percent Solution (parts/100) % 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

100 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

100 mL of solution

X= 15.0 %

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Expressing Concentration:Percent Solution (parts/100) % 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 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

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Expressing Concentration: Molarity 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

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Determinig Molarity: First step Molecular Weight

Sum of the atomic weights of each element in the compound What is the molecular weight of Na3PO4?

Step 1: Sodium has an atomic weight of 22.99, but there are 3 molecules so 22.99*3= 68.97

Step 2: Phosphorus has an atomic weight of 30.97, and only 1 molecule, so 30.97 *1= 30.97

Step 3: Oxygen has an atomic weight of 16, but there are 4 molecules ,so 16*4= 64.00

Step 4: Add 68.97+ 30.97+ 64.00= 163.94 gram molecular weight

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Determinig Molarity: Next Step

How many grams are contained in one mole of Na3PO4? Use the formula for mole calculations

Number grams of solute

Gram molecular weight of solute

1 mole Na3PO4 = X g Na3PO4

gram molecular weight(gmw)

X= 163.94 g Na3PO4

So, 163.94 grams of trisodium phosphate are contained in 1 mole of trisodium phosphate or 6.022 X 1023 trisodium phosphate molecules weigh 163.94 grams

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Determinig Molarity: Final Step Molarity (M) = 1 mole of solute

1L of solution

We are asked to make a 1.00 L volume of a 0.100 molar solution of trisodium phosphate. How many grams would we need?

M= grams

gmw

1.00 L of solution

0.100 molar= X grams of Na3PO4

163.94 gmw of Na3PO4

1.00 L of solution

(0.100M)(1.ooL) = X g

163.94 gmw

0.100= X

163.94

(0.100)(163.94)= X

16.39= X

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Expressing Concentration:Molality Amount of solute per one kg of solvent Expressed in terms of weight per weight or

moles per 1000 grams of solvent Used to measure the physical properties of

solutions Molality = 1 mole of solute

1 kg of solvent

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Expressing Concentration:Normality- First Step

Equivalents Weights / Liter Equivalent weight is equal to the gram molecular weight of

a substance divided by its valence Valence = the electrical charge of an ion, or the number

of moles that react with 1 Mole H+

Example The MW of calcium = 40 grams Calcium ions carry a +2 electrical charge ( valence = 2 ) Equivalent Weight of calcium = 40 / 2 = 20 gram equivalent

weight

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

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Solution Properties

Titration – Method of measuring concentration of one solution by comparing it with a measured volume of a solution whose concentration is known

General formula: 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

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Solution Properties

Density – An expression in terms (usually) of a mass per unit of volume

Many examples - including specific gravity, osmolality

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

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Temperature 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 daily. Examples

Heat blocks, water baths, and incubators shall be maintained at least +/- 1 degree C. of the desired temperature

Refrigerators shall be maintained at 2 -8 degrees C.

Each laboratory must have a NIST calibrated thermometer in order to ensure the accuracy of other thermometers in the laboratory

Out-of-range temperatures should be addressed asap

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Temperature

Scientific measurement of temperature is always expressed in the Celsius ( C) scale , not Fahrenheit ( F )

Celsius scale: 0 degrees = freezing point of water 100 degrees = boiling point of water

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Conversion: Temperature Conversion of Celsius to Fahrenheit and Fahrenheit to

Celsius

F° = ( C ° x 1.8 ) + 32

C° = ( F ° - 32 ) 1.8 For example:

Your refrigerator at home is probably around 40 ° F. What is that in Celsius? Celsius= 40-32 = 4.4

1.8

Water boils at 100 ° C. What is that expressed in Fahrenheit? (1.8)(100) +32 = 212

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Conversions

Most conversions within the metric system occur in units of TEN where changing a unit of measure to a higher or lower designation requires moving the decimal one place either to the left or to the right.

When converting measures in either the high end of the scale (example kilo to mega) or the low end of the scale (examples milli to micro, micro to nano, etc.) the decimal must be moved three places right or left as the prefix designations are assigned only to every third unit in the extreme ends.

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Example of a conversion

How many mls are there in 2.5 liters?

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The question you have to ask yourself is, what is the relationship between liters and mls? The answer : 1 liter = 1000 ml But now what?

We want to get rid of the “liters’ units and end up with “mls” … Right ?

mls 2500 Liter 1

mls 1000Liter 2.5

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1 2 51 0 0 0

11 2 5 0. L iters

m ls

L iterm ls

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 ?

1 0 01 0 0 0

11 0 0 0 0 0mg

ug

mgug

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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

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Dilutions for the Clinical Laboratory

Example:

A technician performed a laboratory analysis of patient’s serum for a serum glucose determination. The patient’s serum glucose was too high to read on the glucose instrument.

The technician diluted the patient’s serum 1:2 and reran the diluted specimen, obtaining a result of 210 g/dl. To correct for the dilution, it is necessary to multiply the result by the dilution factor (in this case x 2).

The final result is 210 g/dl x 2 = 420 g/dl.

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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

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.

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Serial Dilutions

Example: 6 tubes, each with 0.5 mL DI water Add 0.2 mL serum to first tube and serially dilute Find the dilution in tube # 6

Find the dilution factor (will be the same in each of these tubes)

1/dil factor x 1/dil factor x 1/dil factor (etc. 6 times) Result multiplying the numerator 1x1x1x1x1x1x1x = 1 Multiplying the denominators

Will give the result as 1 / 1838

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Resources

Serial dilution http://tinyurl.com/cw7e3ok

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References

Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

Doucette, L. (2011). Mathematics for the Clinical Laboratory (2nd ed.). Maryland Heights, MO: Saunders.

Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .

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