Clinical Chemistry 1

Instructor: Mr. Michael James Z. Lat RMT, MSc. | Created by: Billy James Encallado – BSMLS 3A

Clinical Chemistry Laboratory Overview 7. Conversion Between Prefixes

1. Purpose 1. Understand the relationship between prefixes.

1. Perform analytical procedures for accurate and 2. Example: 1 liter (L) = 1,000 milliliters (mL).

precise information. 3. Example: 1,000 milliliters (mL) = 1 liter (L).

2. Aid in patient diagnosis and treatment. 8. Mass Unit

2. Requirements for Reliable Results 1. SI term for mass is kilogram.

1. Correct use of basic supplies and equipment. 2. Standard prefixes for mass use the term gram

2. Understanding of fundamental concepts critical to rather than kilogram.

analytic procedures. Category Quantity Unit Name Symbol

Units of Measure Overview Base Quantity Length Meter m
1. Components of Quantitative Results
Mass Kilogram kg
1. Number related to the test value.
Time Second s
2. Label identifying the units (e.g., mass, length, time,
Electric current Ampere A
volume).
Thermodynamic Kelvin K
2. Importance of Units:
temperature
1. Not all tests have well-defined units, but units
Amount of substance Mole mol
should be reported when possible.

3. Système International d’Unités (SI) Luminous intensity Candela cd

1. Adopted internationally in 1960. Selected Frequency Hertz Hz

Derived
2. Preferred in scientific literature and clinical
Force Newton N
laboratories.

3. Provides a uniform method for describing physical Celsius temperature Degree Celsius ℃

quantities.

4. Based on the metric system. Catalytic activity Katal kat

4. SI System Classifications Selected Minute (time) (60 s) min

Accepted Non-
1. Basic Units - Seven units, including meter (length),
SI
kilogram (mass), and mole (quantity of substance).

2. Derived Units - Mathematical functions of basic Hour (3,600 s) h

units (e.g., meters per second for velocity). Day (86,400 s) d

5. Non-SI Units Liter (volume) (1 dm³ = 10⁻³ L

1. Widely used and acceptable within the SI system m³)

(e.g., hour, minute, day, gram, liter, degrees). Angstrom (0.1 nm = 10⁻¹⁰ Å
m)
6. SI Prefixes

1. Indicate decimal fractions or multiples of a unit.

2. Example: 0.001 liter = 1 milliliter (mL) or 1 × 10⁻³ L. Factor Prefix Symbol Select Decimals

3. Example: 1,000 liters = 1 kiloliter (kL) or 1 × 10³ L. 10^-18 atto a -

 10^-15 femto f - 1. Often expressed in substance concentration (e.g.,

10^-12 pico p - moles) or mass (e.g., mg/dL, g/dL).

10^-9 nano n - 2. ISO promotes universal standards for uniform

10^-6 micro μ 0.000001 terminology and less confusion.

10^-3 milli m 0.001 3. Familiarize with both traditional and SI units for

accurate interpretation.
10^-2 centi c 0.01
Reagents
10^-1 deci d 0.1
1. Reagent Preparation
10^0 (Basic unit) - 1.0
1. Most reagents are prepackaged and ready-to-use,
10^1 deka da 10.0
requiring only water or buffer for reconstitution.
10^2 hecto h 100.0
2. Awareness of chemical hazards and regulatory
10^3 kilo k 1,000.0
requirements has led to the use of prepared
10^6 mega M -
reagents.
10^9 giga G -
2. Chemical Grades
10^12 tera T -
1. Analytic Reagent (AR): High purity, meets/exceeds
10^15 peta P -
American Chemical Society (ACS) specifications.
10^18 exa E -
2. Ultrapure: Additional purification for specific

procedures (e.g., chromatography,

SI Conversions immunoassays).
1. Conversion Principle 3. United States Pharmacopeia (USP) / National
1. Move the decimal by the difference between the Formulary (NF): Used in drug manufacturing, not
exponents of the prefixes. always suitable for lab assays.
2. Larger to smaller unit: move decimal to the right. 4. Chemically Pure (CP): Impurity limitations not
3. Smaller to larger unit: move decimal to the left. stated, not recommended for clinical labs without
2. Examples further purification.
1. Convert 1.0 L to μL: 5. Technical/Commercial Grade: Used in
1. 1.0 L (1 × 10⁰) = ? μL (micro = 10⁻⁶) manufacturing, not suitable for clinical labs.
2. Move decimal six places to the right: 3. Organic Reagents
1,000,000 μL 1. Varying grades of purity: practical, CP,
3. Reverse: 1,000,000 μL = 1.0 L spectroscopic, chromatographic, and reagent
2. Convert 5 mL to μL: grade (ACS).
1. 5 mL (milli = 10⁻³) = ? μL (micro = 10⁻⁶) 4. Safety and Regulations
2. Move decimal three places to the right: 1. Occupational Safety and Health Administration
5,000 μL (OSHA) requires manufacturers to indicate
3. Convert 5.3 mL to dL: hazards and precautions.
1. 5.3 mL (milli = 10⁻³) = ? dL (deci = 10⁻¹) 2. Safety Data Sheets (SDS) must be provided for each
2. Move decimal two places to the left: 0.053 chemical, detailing safe use, storage, and disposal.
dL

3. Reporting Laboratory Results

 Clinical Chemistry Reference Materials 3. Water Categories (CLSI Classification)

1. Clinical Chemistry 1. Clinical Laboratory Reagent Water (CLRW)

1. Analyzes biochemical by-products in biological 2. Special Reagent Water (SRW)

fluids (serum, plasma, urine). 3. Instrument Feed Water

2. Purification and exact composition are challenging. 4. Water Supplied by Method Manufacturer

2. Primary Standards 5. Autoclave and Wash Water

1. Highly purified chemicals with exact known 6. Commercially Bottled Purified Water

concentration and purity. 4. Water Monitoring Parameters

3. NIST-Certified Standard Reference Materials (SRMs) 1. Microbiological count

1. Developed by the National Institute of Standards 2. pH

and Technology (NIST). 3. Resistivity

2. Used in clinical chemistry labs due to the 4. Silicate

unavailability of ACS primary standards. 5. Particulate matter

4. Applications of NIST SRMs 6. Organics

1. Used in place of ACS primary standards in clinical 5. Prefiltration

work. 1. Removes particulate matter from municipal water.

2. Verify calibration or accuracy/bias assessments. 2. Uses glass, cotton, activated charcoal, and

5. Types of SRMs Available submicron filters.

1. Routine analytes 6. Types of Water

2. Hormones 1. Type I: Most stringent, suitable for trace metal, iron,

3. Drugs and enzyme analyses.

4. Blood gases 2. Type II: Suitable for most analytic requirements.

5. Continuously adding new SRMs. 3. Type III: Used for glassware washing, not for

Water Specifications for Laboratory Use analysis or reagent preparation.

1. Importance of Water Purification 7. Testing Procedures

1. Tap water is unsuitable for lab applications. 1. Measurements of resistance, pH, colony counts,
2. Purified water is essential for reagent and standard chlorine, ammonia, nitrate/nitrite, iron, hardness,

preparation. phosphate, sodium, silica, carbon dioxide,

2. Purification Methods chemical oxygen demand, and metal detection.

1. Distillation: Removes organic materials by boiling 2. Resistance is a key indicator of water purity.

and vaporizing water. 8. Storage and Handling

2. Ion Exchange: Uses anion or cation resins to 1. Store CLRW to minimize chemical or bacterial

remove ions. contamination.

3. Reverse Osmosis: Forces water through a 2. Use reagent grade water immediately to maintain

semipermeable membrane. resistivity.

4. Ultrafiltration/Nanofiltration: Removes Solution Properties

particulate matter, microorganisms, pyrogens, and 1. Definitions

endotoxins. 1. Solute: Substance dissolved in a liquid (analyte).

5. Ultraviolet Light and Ozone Treatment: Sterilizes 2. Solvent: Liquid in which the solute is dissolved

and removes trace organic materials. (biologic fluid).

 3. Solution: Combination of solute and solvent. 4. Supersaturated: More solute than saturation point;

2. Basic Properties unstable.

1. Concentration: Amount of solute in a solution. Colligative Properties

2. Saturation: Extent to which solute is dissolved. Colligative properties depend on the number of solute particles, not

3. Colligative Properties: Include vapor pressure, their type. Key properties include:

boiling point, freezing point, and osmotic pressure. 1. Osmotic Pressure

4. Redox Potential: Tendency of a chemical species to 1. Opposes osmosis through a semipermeable

acquire electrons. membrane.

5. Conductivity: Ability to conduct electricity. 2. Proportional to solute concentration.

6. Density: Mass per unit volume. 3. Measured in osmoles (osmolarity for molarity,

7. pH: Measure of acidity or alkalinity. osmolality for molality).

8. Ionic Strength: Concentration of ions in a solution. 2. Vapor Pressure

Concentration of Solutions 1. Pressure exerted by vapor in equilibrium with liquid

1. Percent Solution solvent.

1. Weight/Weight (% w/w): Grams of solute per 100 3. Freezing Point

g of solution. 1. Temperature where the first solid crystal forms in

2. Volume/Volume (% v/v): Milliliters of solute per equilibrium with the solution.

100 mL of solution. 4. Boiling Point

3. Weight/Volume (% w/v): Grams of solute per 100 1. Temperature where vapor pressure equals

mL of solution (common in clinical labs). atmospheric pressure.

2. Molarity (M) Clinical Relevance

1. Moles of solute per liter of solution. • Osmolality is preferred over osmolarity due to its stability

2. SI units: mol/L, mmol/L, μmol/L, nmol/L. against temperature and pressure changes.

3. Dependent on volume; affected by temperature • Freezing Point Depression is commonly measured to

and pressure changes. determine osmolality, as vapor pressure measurements can

3. Molality (m) be inaccurate with substances like alcohols.

1. Moles of solute per kilogram of solvent. Redox Potential

2. Expressed as mol/kg. • Definition: Redox potential (oxidation-reduction potential)

3. Not influenced by temperature or pressure. measures a solution’s ability to accept or donate electrons.

4. Normality (N) • Agents:

1. Gram equivalent weights per liter of solution. – Reducing Agents: Donate electrons.

2. Equivalent weight = gmw of substance / valence. – Oxidizing Agents: Accept electrons.

3. Used in titrations and reagent classification. • Mnemonic: LEO (Lose Electrons Oxidized) the lion says GER

4. Previously used for electrolytes, now replaced by (Gain Electrons Reduced).

mmol/L. Conductivity

5. Solution Saturation • Definition: Measures how well electricity passes through a

1. Dilute: Low solute concentration. solution.

2. Concentrated: High solute concentration. • Dependence: Depends on the number and charge of ions

3. Saturated: Maximum solute dissolved. present.

• Resistivity: Reciprocal of conductivity; measures resistance

to electrical current. Eliminating the minus sign in front of the log of the quantity

• Application: Used in clinical labs to assess water purity. results in an equation known as the Henderson-Hasselbalch

• Units: Resistivity in ohms (Ω), conductivity in siemens per equation, which mathematically describes the dissociation

meter (S/m). characteristics of weak acids (pKa ) and bases (pKb ) and the

Buffers effect on pH:

Buffers are weak acids or bases and their corresponding salts that

help stabilize pH by minimizing changes in hydrogen ion

concentration ([H+]). The pH is the negative logarithm (inverse log) Buffer Capacity

of the hydrogen ion concentration, mathematically represented as: • Greatest Buffering Capacity: When [A− ]/[HA] = 1, pH =

pKa.

• Constants: 𝐾𝑎 and pKa are constant for a given substance.

• pH Changes: Due to the ratio of conjugate base [A− ] to weak

acid [HA].

where [H+] equals the concentration of hydrogen ions in moles per

Ionic Strength
liter (M). The pH scale ranges from 0 to 14 and is a convenient way
• Definition: Concentration or activity of ions in a solution or
to express hydrogen ion concentration. The dissociation of acetic
buffer.
acid (CH3COOH), a weak acid, can be illustrated as follows:
• Effects:

– Increases ionic cloud around compounds.

– Reduces particle migration rates.

HA = weak acid, A− = conjugate base, H+ = hydrogen ions, [] =
– Enhances compound dissociation into ions.
concentration of anything in the bracket. Sometimes, the conjugate
– Increases solubility of some salts.
base (A−) will be referred to as a “salt” since, physiologically, it will
– Affects electrophoretic separation.
be associated with some type of cation such as sodium (Na + ).

Note that the dissociation constant, Ka , for a weak acid may be Clinical Laboratory Supplies
calculated using the following equation: Equipment in Clinical Chemistry Laboratories:
• Automation: Most manual techniques are now automated,

but knowledge of equipment operation is still essential.

Rearrangement of this equation reveals • Common Equipment: Includes thermometers, pipettes,

flasks, beakers, and desiccators.

Thermometers/Temperature Control:
Taking the log of each quantity and then multiplying by minus 1 (−1), • Temperature Scales: Celsius (°C) is predominant;
the equation can be rewritten as Fahrenheit (°F) and Kelvin (°K) are also used. Kelvin is the SI

unit.

• Optimal Temperature: Crucial for analytic reactions. Some

By convention, lowercase p means “negative log of”; therefore, procedures need precise control, others are flexible.
−log[H+ ] may be written as pH, and −Ka may be written as pKa . • Temperature Control Devices: Heating/cooling cells, blocks,
The equation now becomes or water/ice baths are used.

• Refrigerator Temperatures: Critical and require periodic

verification.
Types of Thermometers: • TD (To Deliver): Delivers a specified volume.

1. Liquid-in-Glass Thermometers: Types of Glassware:

1. Composition: Colored liquid (red or other) in • Borosilicate (Kimax/Pyrex): High thermal resistance.

plastic or glass. • Aluminosilicate (Corex): High strength.

2. Temperature Range: 20°C to 400°C. • High Silica: High chemical resistance.

3. Inspection: Ensure a continuous liquid line, free • Vycor: Acid and alkali resistant.

from bubbles. • Low Actinic: Amber colored, protects from light.

4. Calibration: Against NIST-certified or traceable • Flint (Soda Lime): Used for disposable items.

thermometers. NIST SRM thermometers and Plasticware Advantages:

gallium can be used for verification. • Corrosion and Breakage Resistance: High resistance,

2. Electronic Thermometers (Thermistors): flexible, and often disposable.

1. Advantages: Smaller size, faster response time. • Common Resins: Polystyrene, polyethylene, polypropylene,

2. Calibration: Similar to liquid-in-glass Tygon, Teflon, polycarbonate, polyvinyl chloride.

thermometers, calibrated against SRM Cleaning and Maintenance:

thermometers. • Biohazardous Material: Disposable or decontaminated.

Conversion Formula Explanation • Cleaning Techniques: Immediate rinsing, detergent

Type
washing, distilled water rinses.
Celsius to ∘C×9/5+32 Multiply the Celsius temperature by • Automatic Dishwashers: Compatible detergents and
Fahrenheit 9, divide the result by 5, then add 32.
temperatures.
Fahrenheit to (∘F−32) × Subtract 32 from the Fahrenheit
• pH Check: Ensure detergent removal by comparing rinse
Celsius 5/9 temperature, multiply the result by
5, then divide by 9. water pH.

Key Points: • Special Applications: Use disposable glassware for heavy

• Calibration: Essential for accuracy in all temperature- metals or molecular testing.

reading devices. Reusable Pipettes:

• Advancements: Automation and miniaturization have • Soaking: In soapy water with tips up.

increased the use of electronic thermometers. • Cleaning: Use appropriate brushes and fresh reagent grade
water for rinsing.
Glassware and Plasticware
Plasticware Cleaning:
Transition from Glass to Plastic:
• Nonwettable Surface: Easier to clean but not suitable for all
• Traditional Glassware: Pipettes, flasks, beakers, and
applications.
burettes were primarily made of glass.
• Avoid Harsh Cleaners: No brushes or abrasive cleaners.
• Modern Plasticware: Increasingly used due to refined plastic
• Ultrasonic Cleaners: Effective for debris removal.
materials.

Precision Tolerance: Laboratory Vessels

• Class A: High precision, marked with “A,” preferred for Types of Vessels:

laboratory use. • Flasks: Volumetric and Erlenmeyer.

• Class B: Lower precision, often used in student labs for • Beakers: Griffin beakers.

durability. • Graduated Cylinders: Various sizes.

Designations: Volumetric Flasks:

• TC (To Contain): Holds a specified volume. • Purpose: Calibrated to hold one exact volume of liquid (TC).
• Design: Round bottom, flat base, long thin neck with Types of Pipettes:

calibration line. • Measuring/Graduated Pipettes: Dispense various volumes;

• Usage: Used to bring reagents to final volume with precise used for reagents and dilutions.

diluent addition. – Mohr Pipette: No graduations to the tip, self-

Erlenmeyer Flasks and Griffin Beakers: draining.

• Erlenmeyer Flasks: – Serologic Pipette: Graduations to the tip, blowout

– Design: Wide bottom, smaller neck. type.

– Usage: Holds various volumes, used in reagent – Micropipette: Holds < 1 mL can be Mohr or

preparation. serologic.

• Griffin Beakers: • Transfer Pipettes: Dispense one volume without subdivisions.

– Design: Flat bottom, straight sides, wide opening – Ostwald-Folin Pipette: For viscous fluids, blowout

with spout. type.

– Usage: Holds various volumes, used in reagent – Volumetric Pipette: For aqueous solutions, self-
preparation. draining, high accuracy.

Graduated Cylinders: • Automatic Pipettes: Most common in clinical labs.

• Design: Long cylindrical tubes with calibration marks, – Types: Fixed volume, variable volume,

upright base. multichannel.

• Usage: Measures volumes of liquids, less accurate than – Mechanisms: Air-displacement, positive-

volumetric labware. displacement, dispenser pipettes.

• Sizes: Commonly 10, 25, 50, 100, 500, 1,000, and 2,000 mL. Calibration:

Precision and Accuracy: • Class A Pipettes: Do not need recalibration.

• Class A Utensils: Preferred for critical measurements to • Others: Require periodic calibration to ensure accuracy and

ensure accuracy and precision. precision.

– Gravimetric Method: Weighing a known volume of

Pipettes
water.
Types and Usage:
– Photometric Methods: Using absorbance changes
• Materials: Glass or plastic, reusable or disposable.
to verify volume.
• Volume: Typically for volumes ≤20 mL; larger volumes use
Daily Checks:
automated devices.
• Use volumetric flasks for quick checks.
Classification:
• Ensure pipette tips are snug and free from deformities.
• To Contain (TC): Holds a specific volume but does not
Key Points:
dispense it exactly.
• Proper technique ensures accuracy.
• To Deliver (TD): Dispenses the exact volume indicated.
• Different pipettes for specific tasks.
Usage Technique:
• Regular calibration maintains reliability.
1. Immerse tip in liquid without touching vessel walls.

2. Use a pipette bulb to draw liquid above the graduation line. Syringes

3. Wipe the tip, allow liquid to drain to the calibration mark. Usage:
4. Hold pipette vertically, drain contents into the receiving • Small Volume Transfer: < 500 μL.

vessel. • Applications: Blood gas analysis, chromatography,

electrophoresis.
Design: Calibration:

• Material: Glass with fine barrels. • Use test weights from ANSI/ASTM Classes 1-4.

• Plunger: Made of fine wire. • Frequency dictated by accreditation/licensing guidelines.

Tips: Maintenance:

• Chromatography: No tips used for gas or high-pressure • Keep balances clean.

liquid systems. • Place away from heavy traffic, large electrical equipment,

• Electrophoresis: Disposable Teflon tips may be used. and open windows.

• Correct level checkpoint before weighing.

Desiccators and Desiccants

Key Concepts: Centrifugation

• Hydrate: Compound with water molecules forming loose Definition: Centrifugation uses centrifugal force to separate solids

crystals. from liquids in suspension. It’s crucial in clinical chemistry for

• Anhydrous: Compound without water of crystallization. preparing samples and concentrating urine sediment for

• Hygroscopic: Substances that absorb water from the air. microscopic analysis.
• Deliquescent: Hygroscopic substances that dissolve in Key Points:

absorbed water. • Purpose: Separates mixtures based on mass and density.

Uses: • Components: Includes a rotor, carriers, shields, and a motor.

• Desiccants: Drying agents to prevent hydration of other Modern centrifuges have safety features like locking lids and

chemicals. brakes.

• Desiccators: Sealed containers with desiccants to store • Variables: Centrifugal force depends on mass, speed (rpm),

hygroscopic substances. and radius. The force is expressed as relative centrifugal

Applications: force (RCF) or gravities (g).

• Storage: Used in sealed packets or shipping containers,

Equation
especially those requiring refrigeration, to prolong storage by
• RCF: Relative Centrifugal Force
keeping contents dry.
• Formula: RCF = 1.118 × 10−5 × 𝑟 × (rpm)2

Balances – 𝑟: Radius in centimeters

Importance: – rpm: Revolutions per minute

• Essential for producing high-quality reagents and standards. This formula calculates the force exerted on particles in a

Types: centrifuge, essential for separating components based on density.

• Analytic Balances: Types of Centrifuges:

– Single pan with sliding transparent doors. • Benchtop or Floor Models: Vary by size and application.

– Tared weighing vessel on the sample pan. • Rotor Heads: Fixed, hematocrit, cytocentrifuge, swinging

– Optical scale for visualizing mass. bucket, angled.

– Weight range: 0.01 mg to 160 g. • Speed: Includes ultracentrifuges for high-speed applications.

• Electronic Balances: Applications:

– Single pan using electromagnetic force. • Separating serum/plasma from blood cells.
– Fast response time (< 10 seconds). • Isolating supernatants from precipitates.

– Accuracy and precision equal to mechanical • Separating immiscible liquids.

balances. • Expelling air.

Maintenance: pH (Negative Logarithms)

• Cleaning: Daily removal of spills and debris. Definition

• Balancing: Ensuring even load distribution to prevent • pH: Negative logarithm of the hydrogen ion concentration.

vibrations. • Formula: pH = −log[𝐻+ ]

• Safety: Keep the cover closed until the centrifuge stops to Example Calculation

avoid aerosol contamination. • Given: Hydrogen ion concentration = 5.4 × 10−6

• Checks: Regularly verify timer, brushes, and speed using a • Steps:

tachometer or strobe light. – Identify 𝑥 (negative exponent) and 𝑁 (decimal

Balancing Tips: portion): 𝑥 = 6, 𝑁 = 5.4

• Use “balance” tubes to match sample volumes. – Calculate log(𝑁): log(5.4) = 0.73

• Follow the rule of even placement and opposition for tube – Determine pH: pH = 𝑥 − log(𝑁) = 6 − 0.73 = 5.27

positioning. Reverse Calculation (Finding [𝐇+ ] from pH)

Accreditation: • Given: pH = 5.27

• Periodic verification of centrifuge speeds is required by • Steps:

accreditation agencies. – Identify 𝑥 (next largest whole number): 𝑥 = 6

– Calculate 𝑥 − pH: 6 − 5.27 = 0.73

Laboratory Mathematics and Calculations: Significant Figures
– Find antilog: [𝐻+ ] = 10−0.73 × 10−6 = 5.4 × 10−6
Definition: Significant figures are the minimum digits needed to
Shortcut
express a value in scientific notation without losing accuracy.
• Subtract pH from 𝑥 and take the antilog: 6 − 5.27 = 0.73
Rules:
• Antilog of 0.73: 5.4 × 10−6
1. All nonzero numbers are significant (1-9).
Note: Rounding can alter the answer slightly. Use precise
2. Zeros between nonzero numbers are significant.
calculations for accuracy.
3. Zeros to the right of the decimal are significant if followed by
Concentration Review
a nonzero number.
Percent Solution
4. Zeros to the left of the decimal are not significant.
• Percent (%): Parts per 100, independent of molecular
Examples:
weight.
• 814.2: Four significant figures (8.142 × 10²).
Weight/Weight (w/w)
• 0.000641: Three significant figures (6.41 × 10⁻⁴).
• Example: 5% aqueous HCl solution.
• 10.00: Four significant figures (zeros after the decimal are
– To make 250 g of 5% HCl using 12 M HCl:
significant).
• Calculation: 250 g × 0.05 = 12.5 g HCl

Logarithms Weight/Volume (w/v)

Definition: Logarithms are the inverse of exponential functions. • Example: 10% NaOH solution.

– To make 1,000 mL of 10% NaOH:

• Calculation: 0.10 × 1,000 mL =

Calculations: 100 g NaOH

• Logarithms: Use the log function on calculators without • Add 100 g NaOH to a 1,000-mL flask,

converting to scientific notation. dilute to mark with water.

• Antilogarithms: To find the original number from a log value, Volume/Volume (v/v)

use the inverse or shift function on calculators. • Example: 2% HCl solution.

– To make 50 mL of 2% HCl:
 4.8 mol HCl 250 mL
• Calculation: 0.02 × 50 mL = 1 mL HCl – Equation: 36.5 g HCl × × =
1 mol 1000 mL
• Add 1 mL HCl to a 50-mL flask, dilute to 43.8 g HCl
mark with water. – Procedure:

• Note: Always add acid to water! • Add 200 mL of reagent grade water to a

Molarity Review 250-mL volumetric flask.

Definition • Add 43.8 g of HCl and mix.

• Molarity (M): Moles of solute per liter of solution (mol/L) or • Dilute to the calibration mark with reagent

millimoles per milliliter (mmol/mL). grade water.

• 1 mol: Equal to the gram molecular weight (gmw) of the Key Technique

substance. • Unit Cancellation: Useful for solving clinical chemistry

Steps to Determine Amount Needed problems involving molarity, normality, or concentration

1. Final Units: Decide the final concentration units (mol/L or conversions.

mmol/mL).
Normality
2. Convert Units: Ensure all units are consistent and cancel out
Definition
unnecessary units.
• Normality (N): Number of equivalent weights per liter (Eq/L)
Key Relationships
or milliequivalents per milliliter (mmol/mL).
• Molarity: Moles/Liter
• Equivalent Weight: Gram molecular weight (gmw) divided
• Molality: Moles/Kilogram (equivalent to molarity for water)
by valence (V).
Example Calculation
• Usage: Common in acid-base calculations due to its
• Problem: How many grams are needed to make 1 L of a 2 M
equivalence to combining weight.
solution of HCl?
Examples
– Step 1: Final units needed: Grams per liter (g/L).
Example 1.7: Equivalent Weight Calculation
– Step 2: Determine gmw of HCl (36.5 g/mol).
1. NaCl: gmw = 58 g/mol, valence = 1
– Calculation:
2 mol 1. Equivalent weight = 58 g
36.5 g HCl × = 73 g HCl
1L
2. H₂SO₄: gmw = 98 g/mol, valence = 2
– Result: 73 g HCl per liter for a 2 M solution.
1. Equivalent weight = 49 g
Example 1.5: Determining Molarity of NaOH Solution
Example 1.8: Normality of H₂SO₄ Solution
• Given: 24 g of NaOH in a 1-L volumetric flask.
• Given: 7 g of H₂SO₄ in 500 mL.
• Goal: Find molarity (mol/L).
• Steps:
• Steps:
– Units needed: Eq/L.
– Units needed: Moles per liter (mol/L).
– Conversion: 49 g per equivalent (from H₂SO₄
– Convert grams to moles:
equivalent weight).
• Molar mass of NaOH = 40 g/mol. 7g 1 Eq
24 g NaOH 1 mol – Equation: × = 0.286 N
0.5 L 49 g
• Equation: × = 0.6 mol/L
1L 40 g NaOH
Example 1.9: Normality of 0.5 M H₂SO₄ Solution
– Result: 0.6 M or 0.6 mol/L.
• Conversion Formula: 𝑁 = 𝑀 × 𝑉
Example 1.6: Preparing 250 mL of 4.8 M HCl Solution
– For H₂SO₄ (V = 2): 𝑁 = 0.5 M × 2 = 1 N
• Goal: Find grams of HCl needed.
Example 1.10: Molarity of 2.5 N HCl Solution
• Steps:
• Given: 2.5 N HCl.
– Units needed: Grams (g).
• Steps:
– Molar mass of HCl: 36.5 g/mol.
 – Valence of HCl: 1. Problem:
𝑁 2.5 N
– Conversion: 𝑀 = = = 2.5 N What is the molarity of this stock solution? The final units desired
𝑉 1
are moles per liter (mol/L). The molarity of the solution is?
Key Technique
Solution:
• Unit Cancellation: Essential for solving problems involving
1. Molecular Weight of HCl:
normality, molarity, and concentration conversions.
Molecular Weight of HCl = 36.46 g/mol

Specific Gravity 2. Moles of HCl per mL:

Definition 0.4403 g/mL

Moles of HCl per mL = = 0.01208 mol/mL
36.46 g/mol
• Density: Mass per unit volume (g/mL).

• Specific Gravity (SG): Ratio of the density of a material to 3. Molarity (M):

the density of pure water at a given temperature. Molarity is moles per liter (mol/L).

• Usage: Commonly used for concentrated materials like Molarity = 0.01205 mol/mL × 1000 mL/L = 12.05 M

commercial acids (e.g., sulfuric and hydrochloric acids). The molarity of the solution is 12.08 M.

Calculation
Conversions and Dilutions
• Formula: Actual Concentration = Specific Gravity ×
Conversions: To convert one unit into another, the approach of
Assay as decimal
canceling out like units can be applied. For instance, a chemistry
Example 1.11
laboratory might report a given analyte, like calcium, using two
Problem:
different concentration units—millimoles per liter (mmol/L) and
What is the actual weight of a supply of concentrated HCl whose
milligrams per deciliter (mg/dL).
label reads specific gravity 1.19 with an assay value of 37%?

Solution:
Example 1.13: Convert 8.2 mg/dL calcium to millimoles per liter
To find the actual weight:
(mmol/L). The gram molecular weight (gmw) of calcium is 40
1. Specific Gravity (SG):
g/mol. Therefore, 40 g per mol translates to 40 mg per mmol. The
Specific gravity is the ratio of the density of the substance to
desired final units are mmol/L.
the density of water. 1 dL 1000 𝑚𝐿 1 mmol
Equation: 8.2 mg/dL × ( )×( )𝑥 ( ) =
100 mL 𝐿 40 mg
Density of HCl solution
SG = = 1.19 2.05 mmol/L
Density of water
Solution: The systematic, stepwise approach of canceling similar
2. Density of Water:
units is used for this conversion.
Assume the density of water is 1 g/mL.
Dilution: A common problem in the laboratory involves dilution,
3. Density of HCl Solution:
where a weaker concentration or different volume is needed from
Density of HCl solution = 1.19 × 1 g/mL = 1.19 g/mL
a stock solution, provided the concentration terms remain the
4. Weight of HCl per mL:
same. The following formula is useful for such dilution problems
Assay value indicates that 37% of the solution by weight is
when three of the four variables are known:
HCl.
𝐶1 × 𝑉1 = 𝐶2 × 𝑉2
Weight of HCl per mL = 1.19 × 0.37 = 0.4403 g/mL
Where:
So, the actual weight of HCl per mL of solution is 0.4403 g.
• 𝐶1 = Concentration of the initial substance
• 𝑉1 = Volume of the initial substance
Example 1.12
• 𝐶2 = Concentration of the final substance

• 𝑉2 = Volume of the final substance

Example 1.14: What volume is needed to make 500 mL of a 0.1 M Important Clarifications:

solution of Tris buffer from a 2 M Tris buffer solution? • Ratio vs. Dilution: A ratio is always expressed with a colon

Identify Known Values: (e.g., 1:4), whereas a dilution can be expressed as either a

• 𝐶1 = 2 M fraction (e.g., 1/4) or a ratio.

• 𝑉2 = 500 mL • Dilution Factor vs. Dilution Expression: The dilution factor is

• 𝐶2 = 0.1 M the reciprocal of the dilution. For example, a "1-in-4" dilution

𝐶2 ×𝑉2 0.1 M×500 mL
Equation: 𝑉1 = = = 25 mL means adding one part stock to a total of four parts (i.e., one
𝐶1 2M

Solution: To make 500 mL of a 0.1 M solution, 25 mL of the 2 M part stock and three parts diluent), which is a dilution factor

stock solution is required. Subtracting this volume from the final of 1/4. However, a "1-to-4" dilution means one part stock in

volume gives the amount of diluent needed, which is 475 mL. four parts total, a dilution factor of 1/5.

Note: This problem differs from basic conversions as it involves Example 1.16: Scaling Up the Dilution

diluting a stock solution. The approach helps determine how much Problem:

stock is needed to make the final solution, and the remaining If 150 mL of the 100 mmol/L sodium solution is required, how do we
volume must be filled with the diluent. maintain the dilution ratio?

Solution:
Dilutions 1
The original dilution factor is . To find the amount of stock needed:
30
Definition and Basic Concept: 150 mL
Stock Volume = = 5 mL
30
A dilution represents the ratio of a concentrated or stock solution
To make the solution, add 5 mL of stock to 145 mL of diluent. This
to the total final volume of a solution. It consists of the volume or
keeps the dilution factor consistent, where the ratio of stock to
weight of the concentrated substance plus the volume of the 5
diluent is .
145
diluent, with the concentration units remaining unchanged. The

ratio of the concentrated solution to the total solution volume is Simple Dilutions

known as the dilution factor. Overview:

Key Points: In a laboratory setting, a simple dilution involves mixing a specific

• Dilution Factor: The dilution factor is the inverse of volume of a concentrated solution (stock) with a diluent to achieve

concentration—when the dilution factor increases, the a desired concentration in a larger total volume. The dilution factor

concentration decreases. is the ratio of the final volume to the volume of the stock solution.
• Calculation of Dilution Factor: To determine the dilution Key Points:

factor, divide the desired concentration by the stock • Dilution Factor: The dilution factor is the ratio of the stock

concentration, reducing the result to its simplest form. material to the total final volume of the solution. It can be

Example 1.15: Determining the Dilution Factor expressed as a fraction (e.g., 1/10) or a ratio (e.g., 1:9).

Problem: • Calculation: To determine how much diluent is needed,

What is the dilution factor needed to make a 100 mmol/L sodium subtract the volume of the stock solution from the total

solution from a 3,000 mmol/L stock solution? volume. Multiply the diluent volume by the number of parts

Solution: specified in the dilution ratio.

100 mmol/L 1 Example 1.18: Achieving a 1:10 Dilution

Dilution Factor = =
3000 mmol/L 30 Problem:
This dilution factor indicates that the stock solution is 1 part in a Create a 1:10 dilution of serum using different volumes.

total volume of 30 parts. To make this dilution, add 1 mL of stock to Approaches:

29 mL of diluent to achieve a total final volume of 30 mL. • 100 μL of serum + 900 μL of saline
• 20 μL of serum + 180 μL of saline Serial Dilutions

• 1 mL of serum + 9 mL of saline Serial dilution refers to the process of making multiple, progressive

• 2 mL of serum + 18 mL of saline dilutions starting from a more concentrated solution to achieve less

Explanation: concentrated solutions. This technique is particularly useful in

Each of these approaches maintains the ratio of 1 part serum to 9 situations where the quantity of the concentrate or diluent is

parts diluent, achieving the desired 1:10 dilution. limited, or when multiple dilutions are required, such as in

determining the titer of a solution.

Example 1.19: Creating a Specific Protein Standard

Importance of Serial Dilution
Problem:
Serial dilutions are essential when only a small volume of the
You have a 10 g/dL stock of protein standard and need a 2 g/dL
patient sample is available (e.g., pediatric samples). This method
standard. You have 0.200 mL of the 10 g/dL stock, but the
allows sufficient sample volume to be spread across multiple tests.
procedure requires only 0.100 mL.
2 g/dL 1 The procedure begins similarly to a simple dilution but requires that
Solution: Dilution Factor = =
10 g/dL 5
each subsequent dilution is made from the previous one.
You need 1 part of stock to a total of 5 parts, meaning 4 parts of
Key factors that must be considered when performing serial
diluent are required. With 0.200 mL of stock available, the dilution
dilutions include:
can be made in several ways:
• Total volume needed
• 0.050 mL stock + 0.200 mL diluent (4 parts × 0.050 mL)
• Available amount of diluent or concentrate
• 0.100 mL stock + 0.400 mL diluent (4 parts × 0.100 mL)
• Dilution factor
• 0.200 mL stock + 0.800 mL diluent (4 parts × 0.200 mL)
• Final concentration required
Example 1.21: Determining Final Concentration
• Necessary support materials
Problem:

Calculate the concentration of a 200 μmol/mL hCG standard after Example 1: A Three-Tube, Twofold Serial Dilution
a 1/50 dilution. In this example, a three-tube, twofold serial dilution is performed,
200 μmol/mL
Solution: Final Concentration = = 4 μmol/mL meaning each tube will have half the concentration of the previous
50

Explanation: tube.
The final concentration is obtained by multiplying the original • Step 1: Label the tubes. The total volume per tube will be 1
concentration by the inverse of the dilution factor. mL.
Example 1.22: Calculating Actual Serum Concentration • Step 2: Add 0.5 mL of diluent and 0.5 mL of the patient
Problem: sample to Tube 1. This results in a 1:2 dilution.
A 1:2 dilution of serum with saline results in a creatinine • Step 3: Transfer 0.5 mL of the well-mixed content from Tube
measurement of 8.6 mg/dL. Determine the actual serum creatinine 1 into Tube 2, which already contains 0.5 mL of diluent. This
concentration. creates a 1:4 dilution.
Solution: Actual Concentration = 8.6 mg/dL × 2 = 17.2 mg/dL • Step 4: Transfer 0.5 mL from Tube 2 into Tube 3, which
Explanation: already contains 0.5 mL of diluent. This results in a 1:8
Since the result represents half the concentration (due to the 1:2 dilution.
dilution), multiply by the inverse of the dilution factor to get the Calculation: The dilution for each tube is calculated by multiplying
actual concentration. the dilution factor (1:2) by the previous tube’s dilution. Hence, Tube

1 is 1:2, Tube 2 is 1:4, and Tube 3 is 1:8.

Example 2: Mixed Dilution Factors 2. Moles Calculation:

This example explores a scenario where dilution factors are not We need 0.5 moles of CuSO₄ per liter to make a 0.5 M

multiples of one another, such as 1:10, 1:20, 1:100, and 1:200. There solution:

are different approaches to solving this: Moles of CuSO₄ = 0.5 mol/L × 1 L = 0.5 mol
• Approach 1: Treat each consecutive pair of dilutions as a 3. Mass Calculation:

separate serial dilution problem (e.g., 1:10 → 1:20, 1:20 → To obtain 0.5 moles of CuSO₄·5H₂O, we must weigh:

1:100, 1:100 → 1:200). Mass = 0.5 mol × 250 g/mol = 125 g

• Approach 2: Calculate the dilution factor needed to reach Thus, 125 g of CuSO₄·5H₂O is needed to prepare 1 L of 0.5 M CuSO₄.

the final dilution directly from the concentrate.

Example 1.26: Conversion from Anhydrous to Hydrated Form
For the example:
In this case, a procedure requires 0.9 g of anhydrous CuSO₄, but
1. Initial Dilution (1:10): Add 1 mL of concentrate to 9 mL of
only the pentahydrate form (CuSO₄·5H₂O) is available.
diluent, yielding a 10 mL solution. This satisfies the initial 1:10
1. Percentage of CuSO₄ in CuSO₄·5H₂O:
dilution.
The percentage of CuSO₄ in the hydrated form is given by the
2. Second Dilution (1:20): To make the 1:20 dilution from the
ratio of the molar masses:
1:10 solution, add 2 mL of the 1:10 solution to 2 mL of diluent gmw of CuSO₄ 160 g
%CuSO₄ = × 100 = × 100 = 64%
gmw of CuSO₄·5H₂O 250 g
(a 1:2 dilution of the 1:10).
2. Weight of Hydrated Form Needed:
3. Third Dilution (1:100): Make a 1:5 dilution of the 1:20 solution
Since 1 g of CuSO₄·5H₂O contains 0.64 g of CuSO₄, the
by adding 0.5 mL of the 1:20 solution to 2 mL of diluent.
required mass of CuSO₄·5H₂O to provide 0.9 g of CuSO₄ is:
4. Final Dilution (1:200): Perform a 1:2 dilution of the 1:100
0.9 g
Required mass = = 1.406 g
0.64
solution by adding 2 mL of the 1:100 solution to 2 mL of
Thus, 1.406 g of CuSO₄·5H₂O is required to obtain 0.9 g of CuSO₄.
diluent.

Graphing and Beer's Law

Water of Hydration
The Beer-Lambert Law, commonly referred to as Beer's law,
Many compounds exist in a hydrated form, meaning water
describes the relationship between concentration and absorbance
molecules are attached to the compound. When calculating the
in photometric assays. The law is expressed as:
gram molecular weight (gmw) of such compounds, the water of
𝐴 = 𝑎·𝑏·𝑐
hydration must be accounted for. The presence of water changes
Where:
the effective mass of the substance, which must be reflected in
• 𝐴 is the absorbance,
calculations for accurate measurements.
• 𝑎 is the absorptivity constant (specific to the compound,

Example 1.25: Preparing a Solution of Hydrated Copper Sulfate wavelength, temperature, pH, etc.),

(CuSO₄·5H₂O) • 𝑏 is the length of the light path, and

In this example, we aim to prepare 1 L of a 0.5 M CuSO₄ solution • 𝑐 is the concentration of the solution.

using the hydrated form, CuSO₄·5H₂O.

Key Points of Beer's Law:
1. Gram Molecular Weight Calculation:
1. Proportionality: Absorbance (𝐴) is directly proportional to
The gmw of anhydrous CuSO₄ alone is 160 g/mol. However,
the concentration (𝑐) of the compound, provided that the
since we are using the pentahydrate form (CuSO₄·5H₂O), we
absorptivity constant (𝑎) and light path length (𝑏) remain
must include the mass of the five water molecules. The gmw
constant.
of CuSO₄·5H₂O is therefore:
2. Linearity: In practice, the linearity of the absorbance-
gmw of CuSO₄·5H₂O = 160 g + 5 × 18 g = 250 g/mol
concentration relationship is tested over a wide
 concentration range. The range within which this relationship unchanged. If any element in the system changes, a new

holds true defines the "reportable range" of the assay. standard graph must be created.

• Regular verification of linearity and calibration is required,

Standard Graph or Curve:
especially if there are changes in the system's stability.
Regulatory agencies often specify how often and under what
To determine the concentration of unknown samples using Beer's
conditions this verification should occur.
law, a standard graph (or curve) is constructed by plotting
Enzyme Assay Calculations Using Beer's Law
absorbance against the concentration of known standards. In
Overview
clinical labs:
Beer's Law can be applied to enzyme assays by monitoring the rate
• The x-axis usually represents concentration.
of change in absorbance (∆A) to determine enzyme concentration.
• The y-axis represents absorbance.
The key components include:
Photometric assays often start with an initial absorbance set to
• Delta Absorbance (∆A): The change in absorbance during
zero using a reagent blank, leading to a data point at (0, 0).
the reaction.
Once a standard graph is established, future measurements can
• Molar Absorptivity (ε): A constant specific to the product
use a one-point calibration method, as long as the system remains
being measured.
stable (e.g., same instrument, reagent lots, etc.). A one-point
Formula
calibration compares the absorbance of a known
1. Basic Beer's Law for Enzymes:
standard/calibrator to the absorbance of the unknown sample.
1. When absorptivity constant (a) is given, replace it

One-Point Calibration: with molar absorptivity (ε) and use ∆A for

The one-point calibration method is expressed mathematically as: absorbance in Beer's law:
𝛥𝐴
Concentration =
Concentration of Standard Cs Concentration of Unknown Cu 𝜀·𝑏
=
Absorbance of Standard As Absorbance of Unknown Au 2. Where:

Solving for the concentration of the unknown: 1. 𝜀 is molar absorptivity (L·mol⁻¹·cm⁻¹)

Concentration of Unknown 2. 𝑏 is path length (usually 1 cm)

2. Enzyme Activity (IU):

Concentration of Standard x Absorbance of Unknown
=( )
Absorbance of Standard 1. Defined as the amount of enzyme that catalyzes 1
μmol of substrate per minute per liter.
Example 1.27:
2. Reported as units per liter (U/L) or international
In this example, the biuret protein assay is used, which follows
units (IU).
Beer's law. A standard with a known concentration of 6 g/dL had
3. Conversion to Different Units:
an absorbance of 0.400, and an unknown sample had an
1. For IU:
absorbance of 0.350. (𝛥𝐴)10−6 ·TV
Activity IU = (𝜀)(𝑏)(SV
Using the one-point calibration equation:
1. TV = Total volume (mL)
6 g/dL 2. SV = Sample volume (mL)
Concentration of Unknown = ( ) × 0.350
0.400
2. For katal:
Concentration of Unknown = 5.25 g/dL
1. Convert micromoles to moles and
Thus, the concentration of the unknown sample is 5.25 g/dL.
minutes to seconds.
Important Considerations:
Example Calculation
• The one-point calibration method is valid as long as the
1. Given Data:
assay conditions (instrument, reagents, etc.) remain
1. ∆A = 0.250 per minute
 0.250·1.050·10−6
2. Molar absorptivity (ε) = 12.2 × 10^3 L·mol⁻¹·cm⁻¹ at Enzyme Activity IU =
12.2×103 ·0.050
0.2625
425 nm Enzyme Activity IU =
610

3. Reagent volume = 1 mL Enzyme Activity IU ≈ 430 U/L

4. Sample volume = 0.050 mL Summary

2. Calculate Enzyme Activity in IU: The enzyme activity is approximately 430 IU/L under the given
0.250·Total Volume
Activity IU = conditions.
12.2×103 ·0.050·10−6

1. Note: Path length (b) is usually 1 cm and is constant,

Specimen Considerations
so it is often omitted in calculations.
Preanalytical Error Prevention
Important Points
• Importance: Proper specimen collection, handling, and
• Enzyme activity is temperature-dependent; different
processing are crucial to avoid preanalytic errors.
temperatures affect the reaction rate.
• Accreditation Requirements: Laboratories must define
• SI unit for enzyme activity is the katal (mol·L⁻¹·s⁻¹). Ensure
procedures for collection, transport, and processing, and
proper conversions for accurate reporting.
document error resolution.
Enzyme Activity Calculation • CLIA 88: Specifies documentation for specimen submission,

Given Data handling, and disposition of unacceptable specimens.

• Change in absorbance (ΔA) per minute: 0.250 Types of Samples

• Molar absorptivity (ε): 12.2 × 10³ L/mol·cm at 425 nm, 30°C 1. Phlebotomy (Venipuncture)

• Path length (b): 1 cm (constant) 1. Definition: Obtaining blood from a vein using a

• Volume of reagent: 1 mL needle and collection device.

• Volume of sample: 0.050 mL 2. Sites: Medial antecubital vein of the arm.

Objective 3. Equipment: Tourniquet, 23- or 21-gauge needle, IV

Calculate the enzyme activity in international units (IU), where 1 IU infusion set (butterfly).

is the amount of enzyme that catalyzes 1 μmol of substrate per 4. Considerations: Avoid sites adjacent to IV therapy;

minute. discard initial 5 mL if drawn below IV site.

Calculation Steps 2. Skin Puncture

1. Beer's Law: 1. Sites: Heel stick for infants, finger stick for adults.

𝐴= 𝜀·𝑏·𝑐 2. Equipment: Sharp lancet, capillary or microtainer

Rearrange to solve for concentration (c): tubes.

𝐴
𝑐= Blood Sample Analysis
𝜀·𝑏

2. Substitute Given Values: • Whole Blood: Contains plasma and cellular components;
0.250
𝑐= requires anticoagulant.
12.2×103 ·1
0.250
𝑐= • Plasma: Liquid portion after centrifugation; contains clotting
12.2×103

𝑐 = 2.05 × 10−5 mol/L factors.

3. Convert to Enzyme Activity (IU): • Serum: Liquid portion after clotting; lacks fibrinogen, slightly
𝛥𝐴·𝑇𝑉
Enzyme Activity IU = higher potassium.
𝜀·𝑆𝑉·10−6

Where: Centrifugation
1. 𝑇𝑉 = Total volume (reagent + sample) = 1 mL + 0.050 • Purpose: Separates plasma and cells.

mL = 1.050 mL • Procedure: Centrifuge for 10 minutes at 1,000 to 2,000 g;

2. 𝑆𝑉 = Sample volume = 0.050 mL avoid hemolysis.

Arterial Blood Samples Post-Processing Observations

• Use: Measure blood gases and pH. • Characteristics: Note any hemolysis, icterus (increased

• Sites: Radial, brachial, femoral arteries. bilirubin), or turbidity (lipemia).

• Challenges: Arterial pressure, bleeding, hematoma risk. • Analysis Timing: Analyze samples within 4 hours to

Patient Identification and Collection Protocols minimize evaporation effects. Properly cap samples and

• Identification: Proper patient ID is crucial. keep them away from rapid airflow, light, and heat.

• Collection Protocols: Use correct tubes, avoid prolonged Storage

tourniquet application, proper labeling. • Short-Term: Refrigerate at 4°C for up to 8 hours.

• Antiseptic Use: Isopropyl alcohol for general use; soap and • Long-Term: Freeze at -20°C for longer storage. Avoid

water for blood alcohol levels. repeated freezing and thawing cycles.

Other Samples
Sample Variables
1. Urine
Key Considerations
1. Common Fluid: Used for quantitative analyses.
• Physiologic Factors: Includes cyclic changes (diurnal or
2. Timed Samples: Usually 24 hours; creatinine
circadian), exercise, diet, stress, gender, age, medical
analysis for completeness.
conditions (e.g., fever, asthma, obesity), drugs, and posture.
2. Other Body Fluids
• Patient Preparation: Ensure patients follow specific
1. Types: CSF, paracentesis fluids, amniotic fluid.
instructions for timed samples or diets. Elderly patients may
2. Handling: Note color and characteristics before
need extra guidance.
centrifugation; verify sample designation for clinical
• Collection Issues: Problems can arise during collection,
chemistry.
transportation, processing, and storage.
CSF Analysis
Minimizing Preanalytic Errors
• Comparison: Analyze blood sample concurrently for glucose
• Assessment and Action Plans: Identify weak areas, predict
and protein.
potential problems, and implement policies, procedures, or
Amniotic Fluid
checkpoints.
• Uses: Assess fetal lung maturity, congenital diseases, genetic
• Communication: Maintain good communication with all
defects, gestational age.
personnel involved to ensure plans meet laboratory needs
Sample Processing and patient care standards.

Initial Processing • Quality Assurance: Accreditation agencies require

• Matching: Ensure blood collection tubes match the test consideration of all preanalytic variations as part of quality

requisition and patient ID labels. assurance plans, including effective problem-solving and

• Error Prevention: Use bar code labels on primary sample documentation.

tubes to detect and minimize clerical errors. Physiologic Variation

• Sample Acceptability: Check for sufficient volume, proper • Fasting: Patients may become dehydrated, leading to

anticoagulants or preservatives, appropriate timing, and higher-than-expected results.

intact transport conditions (e.g., cooled, on ice, protected • Medications: Determine if any medications may interfere

from light). with tests. Common influences include:

Centrifugation – Smoking: Increases glucose, growth hormone,

• Procedure: Centrifuge the sample to separate serum or cortisol, cholesterol, triglycerides, and urea.

plasma from cells if not analyzed immediately.

 – Alcohol: Causes hypoglycemia, increased Chain of Custody

triglycerides, and elevated liver enzymes. Forensic Laboratory Tests

– Intramuscular Injections: Increase creatine kinase • Specimen Identification: Required at each phase when

and lactate dehydrogenase. linked to a crime or accident.

– Opiates: Increase liver and pancreatic enzymes. • Forms and Protocols: Each facility has its own; patient and

– Oral Contraceptives: Affect various analytic usually a witness must identify the sample.

results. • Tamper-Proof Seal: Sample should be collected and sealed

– Diuretics: Decrease potassium and cause with this.

hyponatremia. Documentation Process

– Thiazide Medications: Cause hyperglycemia and • Receipt Documentation: Any individual in contact with the

prerenal azotemia. sample must document:

Postcollection Variations – Receipt of the sample

• Common Issues: Clerical errors, inadequate separation of – Condition of the sample at the time of receipt
cells from serum, improper storage, and collection. – Date and time it was received

Variables Affecting Select Chemistry Analytes • Witness Verification: In some cases, one witness verifies the

• Age: Affects creatinine clearance and hormone levels. entire process and cosigns as the sample moves along.

• Gender: Influences hemoglobin, ALP, cholesterol, uric acid, Analytic Tests and Legal Testimony

and hormone levels. • Attention to Samples: Laboratory scientists should give

• Diurnal Variation: Alters ACTH, cortisol, Fe, aldosterone, each sample the same attention as a forensic sample, even

ACP, growth hormone, PTH, and TSH. without documentation, as any analytic test could be used in

• Day-to-Day Variation: Affects ALT, bilirubin, Fe, TSH, and legal testimony.

triglycerides.
Electronic and Paper Reporting of Results
• Recent Food Ingestion: Impacts glucose, insulin,
Electronic Transmission and Data Management
triglycerides, gastrin, ionized Ca2+, chloride, phosphorus,
• Debate on Standards: The use of electronic medical records,
potassium, amylase, and ALP.
coding, billing, and data management systems has sparked
• Posture: Changes albumin, cholesterol, aldosterone, and
debates on appropriate standards for reporting guidelines
Ca2+ levels.
and data privacy safeguards.
• Activity: Increases CK, lactic acid, creatine, protein, AST, LD,
• Variety of Systems: Different health care agencies use
K+, glucose, and decreases cholesterol, triglycerides, and pH.
various data management systems, complicating
• Stress: Elevates ACTH, cortisol, catecholamines, and TSH.
standardization. Examples include:
• Race: Affects total protein, albumin, IgG, IgA, CK, LD,
– Logical Observation Identifiers Names and Codes
cholesterol, triglycerides, and glucose incidence in different
(LOINC)
ethnic groups.
– International Federation of Clinical
• Fasting: Required for accurate fasting blood sugar, glucose
Chemistry/International Union of Pure and Applied
tolerance test, triglycerides, lipid panel, gastrin, insulin,
Chemistry (IFCC/IUPAC)
aldosterone, and renin levels.
– ASTM
• Anaerobic Conditions: Necessary for lactic acid, ammonia,
– Health Level 7 (HL7)
blood gas, and ionized Ca2+ samples.
– Systematized Nomenclature of Medicine,
• Hemolysis: Increases K+, ammonia, PO4, Fe, Mg2+, ALT, AST,
Reference Technology (SNOMED RT)
LD, ALP, ACP, catecholamines, and CK levels.
 – Proprietary systems protein, sodium, alanine, and aspartate

Standardization Efforts transaminases.

• HIPAA Compliance: To protect patient information Reporting Requirements

confidentiality, the Healthcare Common Procedure Coding • Minimum Information: Any system must include a unique

System (HCPCS) was developed for recognition by all patient identifier, test name, and code related to the HCPCS

insurers. and ICD databases.

• ICD Codes: The International Classification of Diseases (ICD) • Report Contents: Whether paper or electronic, reports

by the World Health Organization (WHO) identifies patient should include:

diseases and conditions. In the U.S., ICD-10 is currently used, – Unique patient identifier

with clinical modifications maintained by the National – Test name and appropriate abbreviations

Center for Health Statistics. – Test value with the unit of measure

• CPT Codes: The Current Procedural Terminology (CPT) – Date and time of collection

codes, developed by the American Medical Association, – Sample information

identify almost all laboratory tests and procedures. These – Reference ranges

codes are divided into subcategories with five-digit numbers. – Any other pertinent information for proper test

interpretation

Clinical Laboratory Procedures – Indication of results subject to autoverification

• CPT Category I: Coding numbers between 80,000 and Accreditation Requirements

89,000. Multiple codes may exist for a given test based on • Name and address of the laboratory performing the analysis

the reason and type of testing. • Patient name and identification number or unique identifier

• Example Codes: • Name of the physician or person ordering the test

– Blood glucose testing: 82947 (quantitative except • Date and time of specimen collection and release of results

for strip reading), 82948 (strip reading), 82962 • Specimen source or type

(self-monitoring by FDA-cleared device) • Test results and units of measure

– Comprehensive metabolic panel (80053): Includes • Reference ranges

albumin, alkaline phosphatase, total bilirubin, • Comments on any sample or testing interferences that may
blood urea nitrogen, total calcium, carbon dioxide, alter interpretation

chloride, creatinine, glucose, potassium, total