CLINICAL CHEMISTRY:

INTRODUCTION

1. Definition
• Clinical Chemistry
a. A basic science that utilizes the specialty of chemistry to study human beings
b. An applied science when analysis is performed on body fluids or tissues
for diagnosis or treatment of disease
2. Malfunction
a. Trauma or by invasive agents
b. Genetic deficiency of a vital enzyme
c. Defect in the cellular recognition of signals
d. Insufficient supply of blood, nutrients & oxygen
e. Malignancy
3. Role of the CC Lab
a. Measure chemical changes in the body for diagnosis, therapy and prognosis of disease
b. Measure the concentration of a particular constituent (the analyte) in body fluids

4. Role of the Technologist

• Must understand the tools:
a. Equipment
b. Reagents
c. Principle of the testing methods
d. Knowledge of the medical uses of the determinations

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 BASIC PRINCIPLES AND PRACTICES
A. Units of Measure
▪ Quantitative measurement is expressed in defined units
▪ Components of quantitative laboratory results
1. Number; 2. Unit (a. Substance concentration (moles); b. Derived units (mg/dL, g/dL, g/L, mEq/L, IU)
B. Reagents
1. Chemicals (Grades of Purity)
a. Analytical Grade (AR)
• Suitable for most analytic procedures
• Carry designations as AR or ACS and For Laboratory Use or ACS Standard-Grade Reference Materials
b. Ultrapure Chemicals
• Suitable for techniques that require extremely pure chemicals (e.g. AAS, EIA, Mol. Dx.)
• Carry designations of HPLC or chromatographic
c. Chemically Pure (CP) or Pure Grade
• Impurity limitations are not stated. Preparation is not uniform, thus not recommended for clinical laboratories.
d. USP and NF Grade
• Used to manufacture drugs. Based on the criterion of not being injurious to man.
e. Technical or commercial grade
• Used for manufacturing
2. Reference Material
a. Primary standard - Substance of exact known concentration and purity.
b. Secondary standard - Substance of lower purity with concentration determined by comparison with a 1° standard.
3. Water Specifications
a. Distilled water - Purified by distillation
b. Deionized water - Purified by ion exchange (remove dissolved ionized solids and gases)
c. Reverse osmosis water - Uses pressure to force water through a semi permeable membrane
d. Ultrafiltration and nanofiltered water - UV oxidation, sterilization or ozone treatment
e. Reagent grade water
• Obtained by initial filter, followed by RO, deionization and a 0.2 mm filter.
i. Type I water: Trace metal analysis by FES and AAS. Gas, pH, enzyme and electrolyte analysis
ii. Type II water: For analytical preparations (E.g. reagent, QC and standard preparation)
iii. Type III/autoclave wash water (e.g. glassware washing)
C. Clinical Laboratory Supplies
1. Thermometers
• Analytical reactions occur at an optimal temperature (circulating water or heating/cooling metal blocks)
a. Liquid-in-glass: Colored liquid or mercury encased in plastic/glass material with a bulb at one end a graduated stem
i. Total Immersion - the entire thermometer (bulb and the liquid column) is exposed to the temperature being
measured (E.g. refrigerator, freezer, incubator, etc.)
ii. Partial Immersion - Has an immersion line. Should be immersed to the proper height
(E.g. heating blocks or water baths)
iii. Surface thermometer – For checking temperatures on flat surfaces (E.g. Heating oven)
b. Electronic thermometer (thermistor probe) - fast reading, millisecond respose time
c. Digital Thermometer
2. Glassware and Plasticware
• Glassware: i. Borosilicate (Kimax®/Pyrex®); ii. Aluminosilicate (Corex®); iii. Acid/alkali resistant (Vycor®)
• Plasticware: i. Polystyrene; ii. Polyethylene; iii. Polypropylene; iv. Tygon®; v. Telon®
a. Laboratory vessels
i. Volumetric flask: Calibrated to hold one exact of liquid (TC)
ii. Erlenmeyer Flasks & Beaker: Hold different volume, used in reagent preparation
iii. Graduated Cylinder: measure volumes of liquid
b. Pipets: Glass or plastic utensils used to transfer liquids

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 Pipet Classification
1. Design
A. To contain (TC) B. To Deliver (TD)
• Used for viscous samples • Used for non-viscous samples
• Uses mercury as calibrating medium • Uses distilled water as calibrating medium
• Proper use requires rinsing technique

2. Calibration Marks / Drainage Characteristics (To Deliver, TD)

A. Blowout pipet B. Self-draining
• Serologic, Ostwald Folin • Volumentric, Mohr
• With etched ring / two small • Without marking.
continuous rings • Drains completely.
• Graduated to the tip • Not graduated to the tip

3. Type / According to Use

A. Measuring or Graduated pipet B. Volumetric or Transfer pipet

• Graduated uniformly along its • Delivers an exact volume
length • Bulblike enlargement in its
• Designed to deliver any stem
amount w/in its capacity
i. Serologic pipet i. Ostwald-Folin pipet
• Has graduation marks to the • For viscous fluids
tip (blowout pipet) (blow out pipet)
ii. Mohr pipet ii. Volumetric pipet
• No graduation marks to the • For aqueous solutions
tip (self draining) (self draining)
Types of Transfer Pipet Volumetric Pipet Ostwald-Folin Pipet
Size Larger Smaller
Location of the bulb Located at the center (symmetrical) Located closer to the delivery tip
Design To deliver To contain
• Mechanical or Automatic pipets (Micropipet - deliver amount <1ml; Macropipet - >1ml; Dilutor / dispenser)
c. Burets: For dispensing liquid during titration
d. Syringes: Used to transfer small volumes in blood gas analysis, chromatography or electrophoresis
3. Dessicators and Desiccants: Uses hygroscopic substances that take up water/moisture on exposure to air.
4. Balances:
a. Top-loading balance: For knowing the mass of substances, used for preparative experiments.
b. Analytical balance: For preparation of primary standards. With sliding transparent doors. Measure exact mass but with
lower capacities (operating ranges 0.01 mg to 160 g)
D. Basic Separation Techniques
1. Centrifugation
• A process in which a centrifugal force is used to separate solid matter from a liquid suspension.
• Consist of head/rotor (Swinging Bucket or V Angled), carrier and shields
• The speed/centrifugal force is expresses by:
a. Revolution per minute (RPM)
b. Relative centrifugal force (RCF) or gravities (g)
2. Filtration: Paper, cellulose, polyester fibers and column materials

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 LABORATORY MATHEMATICS
A. Solution Properties
▪ Introduction
i. Solute - a substance dissolved in liquid
ii. Solvent - the liquid which the solute is dissolved
iii. Biologic solutes (analytes) - substance dissolved in biologic fluids.
iv. Biologic fluids - the liquid in which analytes is dissolved.
v. Solute + Solvent = Solution
a. Concentration: Expression of analyte (solute) concentration:
i. Percent solution
• Amount of solutes per 100 total units of solution. Percent means parts per 100, represented by %
• Three expression of percent solution
1. % Weight per volume (w/v)

i. What amount of NaCl is needed to make 800 ml of 0.85% solution?

2. % Volume per volume (v/v)

i. Prepare 500 mL 40% alcohol from stock absolute alcohol solution.

3. % Weight per weight (w/w)

ii. Molarity (M)

• Number of moles per 1 liter of solution (mol/L)
• 1 mole of the substance equals its gram molecular weight (GMW)

1. How many grams are needed to make 1 L of a 2 M solution of HCL? MW of HCl = 36.5

2. A Liter of solution contains 24 g of NaOH, what is the molarity? MW of NaOH = 40

3. Make up 250 ml of a 4.8 M solution of HCL? MW of HCL = 36.5

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 iii. Normality (N)
• Number of Gram equivalent weight per 1 liter of solution
• An equivalent weight is equal to the molecular weight divided by its valence.
• Valence is the number of electrons exchanged in oxidation reduction reactions

1. What is the normality of a 500-mL solution that contains 7 g of H2SO4? MW of H2SO4 = 98 ;

Valence H (2) = 1x2 = 2

2. If a solution contains 111g CaCl2 per liter, what is the normality. MW of CaCl2 = 111 ; Valence Ca = +2

3. To make 500 mL of 3 N Na2SO4 how much substance must be weighted? MW of Na2SO4 = 142 ; Valence = 2

4. In making 2 liters of 0.3 N HCL from HCL (specific gravity of 1.185 and % purity of 36.7) what volume of HCL is
required? MW of HCL = 36.5 g ; Valence = 1

Changing Molarity to Normality

▪ N / valence = M ▪ M x valence = N
• 2 N HCL = 2/1 = 2M • 2M HCL = 2x1 = 2N
• 2N H2SO4 = 2/2 = 1M • 2M H2SO4 = 2x2 = 4N
• 2N H3PO4 = 2/3 = 0.67M • 2M H3PO4 = 2x3 = 6N
iii. Molality (m)
▪ Amount of solute per 1 kg of solvent
▪ Expressed as mol/kg

1. A solution contains 15.6 g of NaCl dissolved in 500g of water. Determine the molal concentration. NaCl = 58.5

b. Colligative Properties
i. Vapor pressure - Pressure at which the liquid solvent is in equilibrium with the water vapors.
ii. Freezing point - vapor pressures of solid or liquid is equal
iii. Boiling point - vapor pressure of the solvent reaches 1 atm
iv. Osmotic pressure - pressure that opposes osmosis when a solvent flow through a semi-permeable membrane.
c. Redox potential - Is a measure of the ability of a solution to accept or donate electrons
d. Conductivity and Resistivity
• Conductivity (ohm -1) - Measure how well electricity passes though a solution. Depends on the charges of the ions present
• Resistivity (ohms) - Resistance of substance to the passage of electrical current
e. pH and Buffers
• Buffers - Weak acids or bases and their related salts. Minimize changes in hydrogen ion concentration

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Other Laboratory Mathematics
i. Milliequevalent (mEq/L)
• For expressing electrolytes
• EW expressed in milligrams
• mg/dL → mEq/L
1. If a solution contains 350 mg/dL Na+, how many mEq/L of Na+ does it contain? MW of Na = 23; Valence of Na = 1

2. A solution containing 12 mg/dL Ca++ contains how many mEq/L calcium? MW of Ca = 40; Valence of Ca = 2

ii. Millimoles (mmol/L)

▪ Molecular weight expressed in milligrams
1. Convert 3 mg/dL magnesium to mmol/L. MW of Mg = 24.31

2. Convert 8.2 mg/dL calcium to millimoles per liter. MW of Ca = 40

iii. Ratio: Volume of solute per volume of solvent

iv. Dilution: Ratio of the volume of substance to be diluted to the final volume

1. Calculate the ratio and dilution using 50 µL of blood and 950 µL of diluting fluid.

2. Calculate the ratio and dilution using 0.5 mL of urine and 8.5 mL of isotonic saline.

3. Calculate the ratio and dilution of 0.1 mL serum in 0.9 mL water

v. Serial Dilution
▪ Multiple progressive dilutions ranging from more concentrated solutions to less concentrated solutions
• A serum sample was diluted 1:10, then 1:10, then 1:2. What is the final dilution?

1. A serum sample was diluted 1:10, then 1:10, then 1:2. What is the final dilution?

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