Clinical Chemistry Lecture Notes

Clinical Chemistry – Definition & Scope

• “Clinical” derives from Greek for “bed”; highlights bedside application.
• Chemistry: science of chemical structures/interactions of matter.
• Clinical Chemistry (CC) = applied chemical science performing analyses on body fluids & tissue specimens to aid diagnosis, monitor therapy, and study disease.

Units of Measurement – SI (Système International)

• Adopted worldwide; French origin.
• Base units:
  • Mass → kilogram (kg)
  • Time → second (s)
  • Temperature → kelvin (K)
    • Philippines commonly use °C in practice, but K is SI.
  • Amount of substance → mole (mol)
  • Electric current → ampere (A)
  • Luminous intensity → candela (cd)
• Derived / commonly-used units in CC:
  • Concentration → $\text{mmol\,L}^{-1}$ (millimole per liter) for solutes.
  • Enzyme activity → $\text{U\,L}^{-1}$ (international unit per liter).
    • Key enzymes: CK, AST, LDH, ACE, etc.
  • pH → \text{[H^+]} in arterial blood gas reports.
• Volume conversions:
  • $1\,\text{L}=1000\,\text{mL}$ (essential for result interpretation).

Temperature Conversions (essential for method specifications)

• °C → K: $K = ^\circ C + 273.15$
• °C ↔ °F (recall for completeness):
  • $^\circ F = (^\circ C \times \frac{9}{5}) + 32$
  • $^\circ C = (^\circ F - 32) \times \frac{5}{9}$

Solution Terminology & Examples

• Solute: substance dissolved (e.g., NaCl).
• Solvent: medium that dissolves solute (e.g., water is universal solvent).
• Solution: homogeneous mixture of solute + solvent.

Percent Solutions

• % (w/v) → grams solute / 100 mL solution.
  • 70 % ethyl alcohol = 70 g EtOH in enough water to make 100 mL.
• % (v/v) → mL solute / 100 mL solution (both liquids).
• % (w/w) → g solute / 100 g total solution.

Quick Formulae

• Grams solute required:
  $g = \frac{\% \times V_{\text{total}}}{100}$ (for w/v)
• For v/v: replace grams w/ mL.

Safety Mnemonic – Acid to Water

• Always add Acid to Water (NOT water to acid).
  • Prevents violent exothermic reaction & splashing.
  • Large water volume absorbs heat.

Concentration Expressions

Molarity (M)

• $M = \frac{\text{moles solute}}{\text{L solution}}$
• Moles → $\frac{\text{weight (g)}}{\text{GMW}}$
  • Example: NaCl → Na (23) + Cl (35) = 58 g mol⁻¹.
• Convert % (w/v) to M:
  $M = \frac{\% \times 10}{\text{GMW}}$

Normality (N)

• $N = \frac{\text{g solute}}{\text{Eq wt} \times L}$
• Eq wt = $\frac{\text{Molecular wt}}{\text{valency}}$
• % (w/v) → N:
  $N = \frac{\% \times 10}{\text{Eq wt}}$
• Relation: $N = M \times \text{valency}$ ⇄ $M = \frac{N}{\text{valency}}$

Molality (m)

• $m = \frac{\text{moles solute}}{\text{kg solvent}}$
• Useful when temperature changes (volume-independent).

Milliequivalents / Millimoles (electrolytes)

• $1\,\text{mmol} = \frac{\text{mg}}{\text{MW}}$
• $1\,\text{mEq} = \frac{\text{mg}}{\text{Eq wt}}$

Ratios vs Dilutions

• Ratio: parts solute : parts solvent (does not include solute in denominator).
  • Bleach 1 : 9 = 1 mL bleach + 9 mL water.
• Dilution: parts solute : total parts solution.
  • Same prep above = 1 : 10 dilution.
• Reasons: economy, adjust concentration, remove interferents.

Glassware – Composition & Use

• High-thermal-resistance (borosilicate; trade names Pyrex, Kimax, Vicor)
  • Heat & sterilization.
• High-silica (> 96 % SiO₂)
  • Radiation & optical work.
• Alumina-silicate
  • Chemically strengthened.
• Standard flint (soda-lime)
  • Economical; low temp tolerance.
• Low-actinic (amber/red)
  • Light-sensitive reagents.

Measuring Vessels

• Graduated cylinder – routine volume measurement.
• Volumetric flask – single calibration mark; prepares exact volumes.
• Burette – titration, continuous delivery.
• Pipettes (see below).

Pipettes – Types & Characteristics

Calibration Terminology

• TD (To Deliver): empties exact volume when allowed to drain/blow-out.
• TC (To Contain): holds indicated volume; must be rinsed to transfer all.

Drainage Marks

• Blow-out: double/etched ring near mouth; requires last drop expelled.
• Self-draining (gravity): no rings; allow to drain on wall.

Manual Transfer Pipettes

• Volumetric (transfer)
  • Single volume; highest accuracy; non-viscous fluids (serum, plasma).
• Ostwald-Folin
  • Bulb closer to tip; viscous fluids (blood, CSF, sputum).
• Pasteur
  • No calibration; qualitative transfer.
• Serologic (graduated, blow-out)
  • Graduations to tip; general use.
• Mohr (graduated, self-drain)
  • Graduations stop before tip.

RBC/WBC & Kirk Overflow

• Special pipettes for hematology (not detailed in transcript but listed).

Mechanical / Automatic Pipettes

• Air-displacement
  • Disposable tip; piston creates air cushion; two-stop plunger.
  • Tip ejector present.
• Positive-displacement
  • Piston in direct contact with liquid (like syringe); suited for viscous/volatile samples.
• Dispenser/Diluter
  • Draws from common reservoir; repeated aliquots; combines sampling + dispensing.

General Handling Notes

• Hold vertically when aspirating.
• Bleach now preferred over old acid dichromate cleaning solutions.
• 0.1 % phenol-red solution & gravimetric methods used for performance & calibration checks.

Centrifugation

• Principle: centrifugal force separates components by density.
• Force expressed via RPM (revolutions per minute) & RCF (relative centrifugal force).
  $RCF = 1.118 \times 10^{-5} \times r \times (RPM)^2$ (where $r$ = radius in cm).

Centrifuge Types

• Benchtop / clinical.
• Fixed-angle rotor (tubes at 26°–45°, commonly 45°).
• Swing-bucket (horizontal rotor); buckets swing to 90° during spin.

Balancing Rules

• Opposite tubes equal mass/volume.
  • Even number: place aliquots 180° apart.
  • Odd number: use balance tube opposite sample; triangular or hexagonal symmetry (e.g., 1-3-5 or 2-3-5-6, etc.).

Components

• Lid (safety interlock), rotor, motor, chamber, control panel, sample, pellet, supernatant.

Quick Reference Formulae & Shortcuts

• Percent ⇄ Molarity: $M = \frac{\% \times 10}{GMW}$
• Percent ⇄ Normality: $N = \frac{\% \times 10}{Eq\,wt}$
• Dilution (single-step): $C<em>1 V</em>1 = C<em>2 V</em>2$
  • Substitute $M$ or $N$ for $C$ accordingly; rearrange for desired variable.
• Preparing acid/base solutions: always pour concentrated acid into larger water volume with constant stirring.

Connections & Practical Notes

• Accurate unit conversion underpins correct result reporting; mis-uniting (e.g., mmol/L vs mg/dL) causes clinical error.
• Solution prep & pipetting accuracy directly affect QC & patient results.
• Centrifuge imbalance → rotor failure; hence strict balancing protocols.
• Light-sensitive assays (bilirubin, vitamin A) require low-actinic containers.
• Acid cleaning replaced by safer bleach; reflects evolving lab safety standards.

Ethical & Safety Implications

• Proper handling prevents reagent waste (economic) & reduces hazard (burns from exothermic reactions).
• Accurate measurements critical for patient safety (dosing, diagnosis).
• Equipment maintenance (centrifuge lids, glassware integrity) prevents occupational injury.