Basic Biomedical Engineering: Laboratory Apparatus Use and Maintenance

Balances in the Biomedical Laboratory

• A properly operating balance is essential for the production of high-quality reagents and standards.
• Classification of Balances: Balances are categorized based on several criteria:
  • Design and number of pans (single or double).
  • Mechanism (mechanical or electronic).
  • Operating ranges and readability:
    • Precision Balances: Readability is approximately $2\,\mu\text{g}$.
    • Analytic Balances: Readability is approximately $0.001\,\text{g}$.
    • Microbalances: Readability is approximately $0.1\,\mu\text{g}$.
• Analytic Balances:
  • These are required for preparing any primary standards.
  • Currently, analytic and electronic balances are the most popular choices in clinical laboratories.
  • Mechanical Analytic Balance: Also known as a substitution balance. It features a single pan enclosed within sliding transparent doors to minimize the impact of the environment on pan movement. The pan is attached to calibrated weights counterbalanced by a single weight at the opposite end of a knife-edge fulcrum.
  • Operation: The operator adjusts the mass setting and places the material in a tared weighing vessel on the pan. An optical scale visualizes the mass. The weight range typically spans from $0.01\,\text{mg}$ to $160\,\text{g}$.
• Electronic Balances:
  • These are single-pan balances that utilize electromagnetic force to counterbalance the mass of the sample.
  • They match mechanical balances in accuracy and precision but offer a faster response time, typically less than $10\,\text{seconds}$.
• Maintenance and Precautions:
  • Balances must be kept scrupulously clean.
  • They should be located in areas free from heavy traffic, open windows, and large pieces of electrical equipment.

Centrifugation Principles and Equipment

• Centrifugation Process: This process uses centrifugal force to separate solid matter from a liquid suspension based on relative densities.
• Applications:
  • Separating serum or plasma from blood cells during processing.
  • Separating a supernatant from a precipitate in analytic reactions.
  • Separating two immiscible liquids (e.g., lipid-laden samples).
  • Expelling air from samples.
• Components of a Centrifuge:
  • A head or rotor.
  • Carriers or shields attached to a vertical motor shaft.
  • A metal covering and a lid with an on/off switch.
  • Many models feature a brake or a built-in tachometer to indicate speed.
• Physics of Centrifugation:
  • Centrifugal force depends on three variables: mass, speed, and radius.
  • Speed is expressed in revolutions per minute ($\text{rpm}$).
  • The generated force is expressed as relative centrifugal force ($RCF$) or gravities ($g$).
  • RCF Formula: $RCF = 1.118 \times 10^{-5} \times r \times (\text{rpm})^2$.
    • $1.118 \times 10^{-5}$ is a constant determined from angular velocity.
    • $r$ is the radius in centimeters, measured from the center of the axis to the bottom of the test-tube shield.
• Centrifuge Types:
  • Refrigerated Centrifuges: Maintain samples at cool temperatures during long, high-speed runs to combat heat generated by air friction on the rotor.
  • Ultracentrifuges: Operate at speeds around $100,000\,\text{rpm}$ and provide forces up to $600,000\,g$. They often require vacuum pumps to remove air that causes friction and rotor heating.
  • Rotor Head Varieties: Fixed-head, hematocrit, swinging-bucket, or angled.

Centrifuge Maintenance and Safety

• Installation:
  • Must be installed on a rigid, flat, level surface.
  • Should not be placed next to balances or sensitive equipment due to vibration.
  • Sufficient space must be allowed around the unit for ventilation.
  • Initial calibration must be performed by a qualified service technician.
• Daily Care:
  • Clean spills (blood or glass) and debris daily.
  • Balance the load by volume and weight across the centrifuge head.
  • Wipe the inside bowl with disinfectant and rinse thoroughly.
  • If a refrigerated unit is turned off at night, leave the top open to dry; keep it closed during the day while under refrigeration to avoid condensation/ice.
• Monthly Maintenance: Clean the housing, rotor chamber, and accessories with a neutral agent. Use a $5\%$ sodium hypochlorite (bleach) solution for plastic and non-metal parts.
• Annual Maintenance: A technician should check brushes, timers, speed, and electrical leaks.
• Operational Precautions:
  • Tubes must tolerate at least $3000\,g$.
  • Use appropriate rubber/plastic cushions and manufacturer-recommended adaptors.
  • Stop operation immediately if abnormal noise occurs.
  • Never operate with the lid open to prevent aerosol contamination.

Chromatographic Separations

• General Principle: Separations depend on the different rates at which substances in a mobile phase (a moving stream) are retarded by a stationary phase (stationary material) as they pass through it.
• Mobile Phase: Can be a volatilized sample transported by an inert gas (e.g., helium) or a liquid transported by an organic solvent (e.g., acetone).
• Stationary Phase: Usually contained in a thin tube (column). Substances with a greater affinity for the stationary phase move slower, allowing them to be detected as individual peaks at the end of the column.
• Gas Chromatography (GC):
  • Most common clinical laboratory instrumental method is the gas-liquid chromatograph.
  • Columns: Packed columns (liquid coated onto inert support) or capillary columns (liquid coated onto inner walls). Capillary columns have the highest resolving power but lower sample capacity.
  • Carrier Gas: Typically helium or nitrogen. The column is housed in an oven.
  • Detectors: Thermal conductivity, flame ionization, nitrogen/phosphorous, and mass spectrometry ($MS$).
  • Mass Spectrometry: Provides excellent sensitivity by breaking volatilized molecules into ionized fragments, then separating them by mass-to-charge ratios ($m/z$).
• High-Performance Liquid Chromatography (HPLC):
  • Uses high pressure to force the mobile phase through a long, thin column for rapid, excellent resolution.
  • Detectors: UV/visible spectrophotometers are common. Mercury arc lamps ($254\,\text{nm}$) detect aromatic compounds. Diode array detectors ($190\,\text{nm}$ to $600\,\text{nm}$) capture a spectrum in $10\,\text{msec}$.

Spectral Methods and Spectrophotometry

• Principles: These methods rely on the absorption or emission of electromagnetic radiation.
• Wavelength Calculation: $\lambda = \frac{c}{\nu}$.
  • $\lambda$ = wavelength in meters.
  • $c$ = speed of light ($3 \times 10^8\,\text{m/s}$).
  • $\nu$ = frequency in $Hz$.
• Spectral Ranges: Visible ($390\text{–}780\,\text{nm}$) and Ultraviolet/UV ($180\text{–}390\,\text{nm}$).
• Beer’s Law: Absorbance of a solution is proportional to the concentration of the absorbing compound and the path length of light through it.
• Spectrophotometer Components:
  • Light Sources: Tungsten-halogen ($360\text{–}950\,\text{nm}$) for visible light; Deuterium lamp ($220\text{–}360\,\text{nm}$) for UV light. Dual-lamp systems switch at approximately $360\,\text{nm}$.
  • Wavelength Selection: Filters, prisms (requires quartz for UV), and diffraction gratings (reflective versions are popular to avoid light attenuation). Gratings have $1000\text{–}3000\,\text{grooves/mm}$ and achieve pass bands of $0.1\,\text{nm}$.
  • Detectors: Photomultiplier tube ($PMT$). It contains a photocathode that emits electrons and a series of $10\text{–}15$ dynodes (each $50\text{–}100\,\text{V}$ higher than the last). This creates an electron amplification of $4\text{–}6$ per stage, with overall gains reaching a million or more.
• Optical Configurations:
  • Dual-beam spectrophotometer: Uses a beam splitter (half-silvered mirror) and a chopper (rotating mirror) to alternately direct light to sample and reference.

Advanced Photometry and Spectroscopy

• Fluorometry:
  • Molecules absorb energy and emit photons with longer wavelengths (less energy). When this happens in $<10^{-8}\,\text{s}$, it is called fluorescence.
  • Assays are 10–100 times more sensitive than absorption because light is measured against a black background.
  • Equipment: Uses two monochromators (one for excitation, one for emission). The detector is placed off-axis (usually $90^{\circ}$) to minimize scatter.
• Flame Photometry:
  • Measures Sodium ($589\,\text{nm}$, yellow), Potassium ($767\,\text{nm}$, violet), and Lithium ($671\,\text{nm}$, red).
  • Uses a propane and compressed air flame to heat electrons until they emit characteristic wavelengths.
• Atomic Absorption Spectroscopy (AAS):
  • Measures absorption of optical radiation by free atoms in the gas phase. It is about 100 times more sensitive than flame photometry.
  • Sample is aspirated into a flame where atoms remain in the ground state to absorb light.
  • Requires a specific radiation source for the element, an atomizer, a monochromator, and a detector.
• Atomic Emission Spectroscopy (AES): Detects radiation emitted as excited species relax to the ground state after being atomized in a hot source.
• Turbidimetry and Nephelometry:
  • Turbidimetry: Measures the attenuation of a beam of light passing through a solution ($\alpha = 0$).
  • Nephelometry: Measures scattered light at an angle to the central beam for maximum sensitivity.
  • Both are used to detect antigen-antibody complexes.

Particle Counting and Identification

• Coulter Principle: Automates blood cell counts by monitoring electrical impedance between two fluid compartments. As cells pass through a small aperture, impedance increases proportionally to the cell volume.
• Red and White Cell Counting: Red cells are counted using diluted blood; white cells require destroying red cells and using a more concentrated sample.
• Hydrodynamic Focusing: A sheath fluid creates a thin sample column where cells pass in single file, preventing aperture sticking or multiple cells passing at once.
• Flow Cytometry:
  • Characterizes, counts, and separates suspended cells based on scattering and fluorescent properties.
  • Utilizes a quartz flow chamber and complex optics. Light is measured at the excitation wavelength (scattering) and longer wavelengths (fluorescence).
  • Sources: Argon laser ($488\,\text{nm}$) is preferred for high performance.
  • Sorting: Piezoelectric vibration breaks the stream into droplets, which are charged and diverted by deflection plates into collection vessels.

Electrochemical and Ion-Specific Methods

• Potentiometry: Measures voltage across electrochemical cells with little or no current flow.
• Nernst Equation: $V = \frac{RT}{zF} \ln\left(\frac{a_2}{a_1}\right)$.
  • $R$ = gas constant ($8.314\,\text{J/K}\cdot\text{mol}$).
  • $T$ = temperature in Kelvin.
  • $z$ = ionization number.
  • $F$ = Faraday constant ($9.649 \times 10^4\, \text{C/mol}$).
  • $a_n$ = activity of ion in solution $n$.
• Ion-Selective Electrodes (ISE): Use membranes permeable only to specific ions like $H^+$, $Na^+$, $K^+$, $Li^+$, $Ca^{++}$, and $Cl^-$.
  • $CO_2$ and $NH_4^+$ electrodes: Modified pH electrodes using a thin layer of solution (sodium bicarbonate for $CO_2$, ammonium chloride for $NH_4^+$) separated by a membrane.
• Amperometry (Clark Electrode): Measures partial pressure of oxygen ($pO_2$) by measuring the current produced by the reaction $O_2 + 2H^+ + 2e^- \rightarrow H_2O_2$ at a platinum electrode with $\approx -0.65\,\text{V}$ applied.

Radioactive Detection Methods

• Isotopes: Atoms with the same atomic number but different mass. Used to label molecules for detection at very low concentrations (e.g., radioimmunoassays).
• Radiation Types:
  • Alpha: Helium nucleus ($2$ protons, $2$ neutrons). Rarely used.
  • Beta: Electrons or positrons. Low penetration ability.
  • Gamma: High-energy electromagnetic radiation. High penetration.
• Units and Activity:
  • Activity is measured in disintegrations per second ($\text{dps}$).
  • Curie (Ci): $3.7 \times 10^{10}\,\text{dps}$.
  • Becquerel (Bq): $1\,\text{dps}$.
• Half-Life ($t_{1/2}$): Time for activity to reduce by half. $t_{1/2} = \frac{0.693}{\lambda}$.
  • Example: Carbon-14 is $5760\,\text{years}$; Iodine-131 is $8.1\,\text{days}$.
• Counters:
  • Gamma Counters: Use sodium iodide crystals doped with thallium ($Tl$) which act as scintillators.
  • Liquid Scintillation Counters (Beta Counters): Sample is dissolved in a liquid fluor. Uses a coincidence circuit with two phototubes to reduce noise and chemiluminescence false counts.
  • Quenching: Any process reducing scintillation efficiency (counts per minute / decays per minute).

Osmometry and Coagulation

• Osmometers: Measure colligative properties (proportional to total solute particles).
  • Freezing Point Depression: Most accurate method. A solute concentration of $1\,\text{osmol/kg}$ water lowers the freezing point by $1.858^{\circ}\text{C}$. Sample is supercooled, then frozen by vigorous stirring to reach enthalpy equilibrium.
  • Vapor Pressure Depression: Requires smaller samples but is less precise and does not measure volatiles like ethanol.
  • Colloid Osmotic (Oncotic) Pressure: Measures pressure of molecules $>30,000\,\text{amu}$ across a semipermeable membrane using a pressure transducer.
• Coagulation Timers: Automate clotting time tests by monitoring changes in viscosity or light scattering due to fibrin formation.

pH Meter Use and Maintenance

• Construction: Consists of a silver wire coated with $AgCl$, immersed in $0.1\,\text{mmol/L HCl}$. The glass membrane tip is sensitive only to $H^+$ and contains oxides of lithium, cesium, lanthanum, barium, or aluminum.
• Reference Electrodes: Commonly a Calomel ($Hg/Hg_2Cl_2$) or silver/silver chloride ($Ag/AgCl$) electrode immersed in saturated $KCl$.
• Calibration: Must be done daily using buffers at $pH\,4.0, 7.0, \text{and } 10.0$. Values must be within $0.1\,pH$ units to be acceptable.
• Maintenance:
  • Keep glass electrodes immersed in salt solution for long-term storage.
  • Keep Calomel electrodes in $KCl$ buffer when not in use.
  • Rinse after use and do not touch the sensitive membrane.

Laboratory Automation and Trends

• Automation Classes:
  • Continuous Flow: Reagents and samples pass through a single tube separated by air bubbles.
  • Centrifugal: Uses plastic rotors as cuvettes; mixing/transport happens via spinning.
  • Pack-based: Uses specialized packs containing all reagents for specific tests.
  • Dry Chemistry: Reagents are layered on a slide; liquid sample is added, and color is read by reflectance photometry.
• Future Trends: Increased use of whole blood analysis, shift from radioisotopes to enzyme-linked fluorescent assays, and replacement of flame photometry with $ISE$ methods.
• Fume Hoods: Essential safety devices that pull air away from the user to exhaust building-out. Used for toxic gases, volatile radioactive materials, and exothermic reactions. Users should work $6\,\text{inches}$ inside the hood and keep the sash closed.