Barcode labeling minimizes human errors in sample identification by linking each specimen to patient data electronically. This improves traceability and reduces mislabeling/mix-ups.

(Other options:

• b) Faster centrifugation → Unrelated to barcodes

• c) Lower reagent costs → Not a direct benefit of barcoding

• d) Smaller sample volumes → Barcodes don’t affect volume.)

• Didymium filters contain neodymium and praseodymium, producing sharp absorption peaks at specific wavelengths (e.g., 529 nm, 586 nm).

• These peaks are used to calibrate and verify the accuracy of a spectrophotometer’s wavelength scale.

• Other options:

  • a) Fluorescence → Didymium doesn’t fluoresce.

  • c) Bacterial filtration → Not a biological filter.

  • d) Light path adjustment → Unrelated to calibration.

Key use: Quality control for instrument performance.

• Balancing the centrifuge (with symmetrically placed tubes of equal weight) is critical to prevent:

  • Excessive vibration

  • Rotor damage

  • Potential instrument failure or injury

• a) Timer setting → Important but secondary to balance.

• c) Tube material → Matters for chemical compatibility, not safety.

• d) Rotation direction → Fixed by design; not user-adjustable.

• Fluorometers detect fluorescence, where a molecule absorbs light at one wavelength (excitation) and emits it at a longer wavelength (lower energy, Stokes shift).

  • Example: Excitation at 488 nm (blue) → Emission at 520 nm (green).

• Why not others?

  • b) Conductivity → Measured by conductometers (not fluorescence).

  • c) Radioactivity → Requires scintillation counters/Geiger-Müller tubes.

  • d) Thermal energy → Calorimetry, not fluorometry.

Key principle: Filters isolate emission light from excitation light for precise quantification.

(Note: Fluorescence’s sensitivity makes it ideal for biomarkers, DNA assays, and environmental toxins.)

• Fluorescence detection is typically 10–1000x more sensitive than absorbance because:

  • It measures emitted light above background (vs. absorbance’s small Δ in light intensity).

  • Detects low analyte concentrations (e.g., nM to pM range).

• Why not others?

  • b) Linear range → Fluorescence has a narrower linear range than absorbance.

  • c) Temperature → Both methods can be temperature-sensitive (e.g., quenching in fluorescence).

  • d) Wavelength selection → Fluorescence requires two wavelengths (excitation/emission).

Key trade-off: Sensitivity vs. dynamic range. Fluorescence excels for trace analysis (e.g., DNA quantitation, biomarkers).

• Relative Centrifugal Force (RCF) is calculated using:

  RCF(G−force)=1.118×r×(RPM/1000)2RCF(G−force)=1.118×r×(RPM/1000)2

  Where $r$ = radius in cm (here, 30 cm).

• Plugging in the values:

  RCF=1.118×30×(3000/1000)2=1.118×30×9≈302 G×9≈2,718 GRCF=1.118×30×(3000/1000)2=1.118×30×9≈302G×9≈2,718G

  (Approximates to ~3,000 G for practical purposes.)

Key notes:

• Exact RCF ≈ 3,018 G, but options are rounded.

• a) 900 G → Too low (likely miscalculation).

• c) 9,000 G → Overestimates by 3x.

• d) 30,000 G → Extreme overestimation.

Remember: Always verify RCF for protocols!

• Retention time in gas chromatography (GC) is primarily influenced by:

  • Column temperature: Higher temperatures reduce retention (faster elution).

  • Stationary phase chemistry: Affects analyte interaction.

  • Carrier gas flow rate: Faster flow shortens retention.

• Why not others?

  • b) Detector wavelength → Irrelevant to retention (detection occurs post-separation).

  • c) Sample color → GC analyzes vapors; color is irrelevant.

  • d) Mobile phase pH → GC uses inert gases (no pH).

Key principle: Temperature programming optimizes separation by balancing speed/resolution.

(Note: Isothermal vs. gradient methods alter retention dynamics.)

• Köhler illumination aligns the microscope’s light path to provide even illumination, maximizing contrast and resolution while minimizing glare.

• a) Magnification → Determined by lenses, not illumination.

• c) Color reproduction → Secondary effect (depends on filters/light source).

• d) Specimen heating → Irrelevant (Köhler reduces heat with proper alignment).

• Fluorometers measure emitted fluorescence at a 90° angle to the excitation light path to:

  • Minimize interference from the intense excitation light.

  • Maximize signal-to-noise ratio of the weaker emitted light.

• Why not others?

  • a) 180° → Would detect transmitted light (absorbance, not fluorescence).

  • c) 45° → Rarely used (poor signal separation).

  • d) 360° → Nonsensical for directional detection.

Key design feature: The right-angle geometry isolates fluorescence from scattered excitation light.

(Note: Some specialized systems use front-face detection for opaque samples.)

• High backpressure in HPLC usually signals a blockage (e.g., particulate buildup, column contamination, or a clogged frit).

• Other causes (less common):

  • b) Insufficient mobile phase → Air bubbles may form, but pressure drops, not rises.

  • c) Detector saturation → Causes signal issues, not pressure changes.

  • d) Sample evaporation → Unlikely to affect pressure directly.

Key troubleshooting steps:

1. Check for blocked inlet frits or column debris.

2. Flush the system with strong solvent (e.g., 100% methanol).

3. Replace/clean the guard column if used.

Prevention: Always filter samples and mobile phase!

• The ultraviolet (UV) range spans ~100–400 nm.

  • 340 nm falls within the near-UV region (300–400 nm).

• Other options are in the visible spectrum:

  • b) 450 nm → Blue light

  • c) 540 nm → Green light

  • d) 650 nm → Red light

Key point: UV spectroscopy is used for analyzing compounds with conjugated systems (e.g., nucleic acids, proteins).

• Standard microscope eyepieces (ocular lenses) typically provide 10x magnification.

• Total magnification is calculated by multiplying the eyepiece magnification by the objective lens (e.g., 10x eyepiece + 40x objective = 400x total).

(Other options:

• a) 5x → Less common (sometimes used in teaching scopes).

• c) 40x & d) 100x → Typical objective lens magnifications, not eyepiece.)

• The monochromator (with a prism or diffraction grating) selects and isolates specific wavelengths from a broad light source.

• Other components:

  • a) Photodetector → Measures light intensity, doesn’t isolate wavelengths.

  • c) Cuvette → Holds the sample; transparent to light but doesn’t filter it.

  • d) Diffraction plate → Not a standard spectrophotometer part.

Key role: Ensures only the desired wavelength reaches the sample for accurate absorbance measurements.

• The rheostat controls the voltage to the microscope’s light source, directly adjusting brightness/intensity.

• a) Condenser height affects focus and contrast, not intensity.

• c) Objective lens and d) Stage controls are unrelated to light adjustment.

• Pulsed-field gel electrophoresis (PFGE) separates large DNA fragments (up to ~10 Mb) by alternating electric fields, enabling:

  • Strain differentiation (e.g., outbreak tracking of E. coli, Salmonella).

  • Chromosomal analysis (e.g., yeast, parasites).

• Why not others?

  • b) Protein quantification → SDS-PAGE/Western blot.

  • c) Small RNA → Standard agarose/PAGE suffices.

  • d) Lipid analysis → TLC/GC-MS, not gel electrophoresis.

Key advantage: Resolves whole bacterial genomes cut with rare restriction enzymes (e.g., NotI).

(Note: “Gold standard” for epidemiological studies but labor-intensive.)

The Analytical Measurement Range (AMR) defines the range of analyte concentrations that an assay can directly measure accurately without requiring dilution, concentration, or other pretreatment of the sample.

• b) Warranty period → Irrelevant to AMR.

• c) QC limits → Related to validation, not AMR.

• d) Maintenance schedule → Instrument upkeep, not measurement range.

• Bandwidth (measured in nm) defines the range of wavelengths that pass through the spectrophotometer’s monochromator. A smaller bandwidth indicates higher spectral purity (less stray light, better resolution).

• Other options:

  • a) Photomultiplier sensitivity → Affects detection limits, not purity.

  • b) Absorbance range → Related to dynamic range, not wavelength isolation.

  • d) Digital resolution → Refers to signal digitization, not light quality.

Key point: Narrow bandwidth (e.g., 1 nm vs. 5 nm) is critical for distinguishing closely spaced absorbance peaks.

• The Rf (Retardation factor) is calculated as:

  Rf = Distance traveled by the solute ÷ Distance traveled by the solvent front​

• • It quantifies how far a compound migrates relative to the solvent front (values range 0–1).

• Why not others?

  • b) Time → TLC doesn’t measure time; it’s distance-based.

  • c) Temperature → Irrelevant to Rf (unless specified for controlled conditions).

  • d) Particle size → Affects resolution but not Rf directly.

• Side scatter (SSC) measures light refracted orthogonally (90°) to the laser, correlating with:

  • Granularity/organelle content (e.g., granules in neutrophils, vacuoles).

  • Nuclear shape (e.g., lobed vs. round nuclei).

• Why not others?

  • b) Membrane potential → Requires fluorescent dyes (e.g., DiOC₆).

  • c) Total protein → Measured by assays (e.g., Bradford), not SSC.

  • d) Mitochondrial activity → Requires probes (e.g., JC-1).

Key use: Differentiates cell types by internal structure (e.g., granulocytes vs. lymphocytes).

(Note: Combined with forward scatter (FSC), SSC enables basic cell population gating.)

• Flow cytometry identifies and sorts cells by detecting fluorescence from labeled antibodies bound to:

  • Surface markers (e.g., CD4, CD19).

  • Intracellular proteins (e.g., cytokines, phospho-proteins).

• Why not others?

  • b) X-ray diffraction → Analyzes crystal structures, not cells.

  • c) Ultrasonic waves → Used in imaging (e.g., ultrasound), not cell analysis.

  • d) Infrared spectroscopy → Measures molecular vibrations, not cell markers.

Key feature: Multiplexing with multiple fluorophores enables simultaneous analysis of dozens of parameters per cell.

(Note: Combined with scatter signals (FSC/SSC), it provides comprehensive cell profiling.)

• Beer’s Law (A = εlc) states absorbance (A) is proportional to concentration (c).

• To find an unknown concentration:

  1. Measure absorbance of a standard solution (known concentration).

  2. Measure absorbance of the unknown sample.

  3. Calculate concentration using the ratio:
     ^Aunknown / ^Astandard = ^cunknown / ^cstandard​​

Why not others?

• a) Multiply standard absorbance by unknown → Illogical (unknown concentration is what you’re solving for).

• c) Averaging standards → Useful for calibration but doesn’t directly calculate unknowns.

• d) Subtract blank → Corrects for background but doesn’t solve for concentration alone.

Key point: Requires a calibration curve or direct ratio comparison for accuracy.

• The retention factor (Rf) is calculated as:

  Rf = Distance traveled by the solute ÷ Distance traveled by the solvent front​

  • It quantifies how far a compound migrates relative to the solvent front (values range 0–1).

• Why not others?

  • b) Time → TLC is distance-based, not time-dependent.

  • c) Temperature → May affect Rf but doesn’t define it.

  • d) Porosity → Influences separation but isn’t measured by Rf.

Key use: Identifies compounds by comparing Rf values to standards under identical conditions (e.g., same solvent, adsorbent).

(Note: Rf is unitless and reproducible only with controlled variables.)

• Atomic absorption spectrophotometry (AAS) quantifies elements by measuring the absorption of light by ground-state atoms in a flame or graphite furnace.

• a) Light emission → Describes atomic emission spectroscopy (AES), not AAS.

• c) Molecular vibrations → Infrared spectroscopy, not AAS.

• d) Electrical conductivity → Related to conductometry, not absorption.

Key principle: AAS relies on the Beer-Lambert Law for concentration determination, using element-specific wavelengths.

• Dual-beam photometry splits light into two paths: one through the sample and one through a reference blank, measuring both simultaneously.

• This compensates for fluctuations in light source intensity (e.g., lamp drift) by continuously comparing the sample to the reference.

Why not others?

• a) Sample viscosity → Affects flow cells but not compensated by dual-beam.

• c) Cuvette diameter → Errors are minimized by using matched cuvettes, not beam design.

• d) Ambient temperature → Controlled externally (dual-beam doesn’t address this directly).

Key advantage: Improves accuracy and stability in real-time measurements.

• Nephelometry measures light scattered by particles (e.g., immune complexes, proteins) at a 90° angle to the incident beam.

  • Ideal for detecting small aggregates in solution (e.g., antigen-antibody reactions).

• Why not others?

  • b) Complete absorption → Spectrophotometry, not nephelometry.

  • c) Electrical impedance → Flow cytometry/Coulter counters.

  • d) Magnetic susceptibility → MRI/magnetic separation techniques.

Key advantage: More sensitive than turbidimetry for low-concentration particles (e.g., CRP, immunoglobulins).

(Note: Turbidimetry measures reduced transmitted light, while nephelometry detects scattered light.)

• Potentiometry measures the electrical potential difference between a pH-sensitive glass electrode (ion-selective electrode) and a reference electrode, directly correlating to pH.

• b) Spectrophotometry → Used for other analytes (e.g., hemoglobin), not pH.

• c) Conductometry → Measures electrolyte conductivity, not pH.

• d) Fluorometry → Detects fluorescence (e.g., for glucose or certain metabolites).

Key point: Blood gas analyzers rely on ion-selective electrodes (ISEs) for pH, pCO₂, and pO₂ measurements. The pH electrode specifically uses potentiometry.

• Ion-exchange chromatography separates molecules based on their net charge at a given pH.

  • Cation exchangers bind positively charged molecules (e.g., proteins with pH < pI).

  • Anion exchangers bind negatively charged molecules (e.g., proteins with pH > pI).

• Why not others?

  • b) Hydrophobicity → Reverse-phase chromatography.

  • c) Molecular weight → Size-exclusion chromatography.

  • d) Light absorption → Spectrophotometry, not separation.

Key application: Purifying proteins, nucleotides, or charged metabolites by adjusting pH/salt gradients.

• Linearity verification ensures the analyzer’s results are proportional across concentrations. If out-of-range:

  1. Confirm with manual methods (e.g., hemocytometer for counts, spun hematocrit).

  2. Identify if the issue is instrument-specific (e.g., sensor drift) or sample-specific (e.g., interference).

• Why not others?

  • a) Dilute all samples → Maskes the problem; doesn’t address root cause.

  • c) Adjust calibration → Only after confirming true linearity failure.

  • d) New reagents → Unnecessary unless reagent degradation is proven.

Key principle: Always validate aberrant results before corrective actions.

(Note: Linearity testing uses serial dilutions of high-concentration samples.)

• Anhydrous cobalt chloride (CoCl₂) is blue when dry and turns pink when hydrated (moisture absorbed).

• This color change is reversible if dried.

Other options:

• b) Clear to white → Silica gel (not cobalt chloride).

• c) Green to yellow → No common desiccant shows this change.

• d) Red to black → Incorrect for cobalt chloride.

• Reverse-phase HPLC (RP-HPLC) employs a nonpolar stationary phase (e.g., C18 or C8 silica) and a polar mobile phase (e.g., water/acetonitrile or water/methanol).

  • Nonpolar analytes interact more strongly with the column, eluting later.

  • Polar analytes elute first.

• Why not others?

  • b) Cationic resins → Ion-exchange chromatography.

  • c) Aqueous mobile phases only → RP-HPLC often uses organic modifiers (e.g., acetonitrile).

  • d) High temperatures → Used in some methods but not defining for RP-HPLC.

Key advantage: Dominates analytical HPLC due to versatility (e.g., drug testing, peptides).

• A sudden pressure increase in HPLC typically signals a physical obstruction, such as:

  • Blocked frit (e.g., from particulate matter or precipitated sample).

  • Column clogging (e.g., due to matrix buildup or bacterial growth).

• Why not others?

  • b) Mobile phase depletion → Causes pressure drop (air bubbles enter system).

  • c) Detector malfunction → Affects signal, not pressure.

  • d) Sample evaporation → Unlikely to impact pressure directly.

Key troubleshooting steps:

1. Check inlet frit/guard column for debris.

2. Flush column with strong solvent (e.g., 100% methanol).

3. Filter samples/mobile phase to prevent future clogs.

Prevention: Always use 0.22 µm filters on samples!

• Gel filtration chromatography (also called size-exclusion chromatography) separates molecules by their size and shape as they pass through porous beads.

  • Larger molecules elute first (cannot enter pores, shorter path).

  • Smaller molecules elute later (enter pores, longer path).

• Why not others?

  • a) Polarity → Reverse-phase or normal-phase chromatography.

  • c) Ionic charge → Ion-exchange chromatography.

  • d) Fluorescence → Detection method, not separation mechanism.

Key use: Protein purification, polymer analysis, and desalting.

• Buffer solutions in electrophoresis:

  • Stabilize pH: Critical for maintaining molecule charge (e.g., proteins in SDS-PAGE).

  • Conduct current: Enables ionic movement (e.g., Tris-glycine for SDS-PAGE).

• Why not others?

  • b) Viscosity → Adjusted by gel matrix (e.g., polyacrylamide), not buffer.

  • c) Visualization → Requires stains/dyes post-run, not buffer.

  • d) Denaturation prevention → Achieved by additives (e.g., urea), not buffer alone.

Key point: Buffers ensure consistent migration and resolution. Example: TAE (Tris-acetate-EDTA) for DNA

• Optical platelet counting (flow cytometry-based) outperforms impedance by:

  • Accurately sizing giant platelets (often undercounted by impedance as RBCs/WBCs).

  • Distinguishing platelets from microcytic RBCs or debris.

• Why not others?

  • b) Faster analysis → Both methods are similarly rapid.

  • c) Lower cost → Optical typically requires fluorescent dyes (higher cost).

  • d) Sample volume → Comparable for both methods.

Key use-case: Essential for patients with thrombocytopenia or MPN (e.g., essential thrombocythemia).

(Note: Combines fluorescence (CD61) and light scatter for precision.)

• Affinity chromatography isolates molecules based on highly specific biological interactions, such as:

  • Antibody-antigen binding.

  • Enzyme-substrate or receptor-ligand pairs.

  • Tagged proteins (e.g., His-tag binding to nickel resins).

• Why not others?

  • b) Molecular weight → Size-exclusion chromatography.

  • c) Electrical charge → Ion-exchange chromatography.

  • d) Solubility → Reverse-phase chromatography.

Key advantage: Unmatched purity in isolating target biomolecules (e.g., antibodies, enzymes).

• Ion-selective electrodes (ISEs) rely on a specialized membrane that selectively binds the target ion (e.g., glass for H⁺ [pH], crystalline for F⁻, liquid polymer for K⁺).

  • The membrane’s ionophore (ion-binding molecule) dictates specificity.

• Why not others?

  • b) Reference electrode → Maintains stable potential but doesn’t select ions.

  • c) Sample volume → Affects detection limits, not specificity.

  • d) Voltage amplitude → Output depends on ion activity, not selectivity.

Key principle: The membrane’s chemical design ensures response only to the target ion (e.g., Na⁺ ISE ignores K⁺).

(Note: Interferences can occur from ions with similar properties, e.g., K⁺ on Na⁺ ISE.)

• Automated basophil counts are prone to false elevations due to:

  • Interferences (e.g., nucleated RBCs, platelet clumps, cryoglobulins).

  • Artifactual staining (rare but possible).

• Microscopic review of a Wright-stained smear is critical to:

  • Identify true basophils (distinct granules).

  • Rule out pseudobasophilia.

• Why not others?

  • a) Report immediately → Risk of false results.

  • c) Warmer reagents → Unlikely to resolve (not temperature-related).

  • d) IgE levels → Clinical correlation, not diagnostic for basophil count.

Key step: Correlate with clinical context (e.g., allergies, myeloproliferative disorders).

(Note: True basophilia is rare—always verify!)

• Gas chromatography (GC) separates compounds based on their volatility and interaction with the stationary phase.

  • Analytes must vaporize at the GC oven temperature (typically 50–300°C).

  • Non-volatile compounds (e.g., sugars, salts) require derivatization (e.g., silylation) to become volatile.

• Why not others?

  • b) Fluorescent tags → Used in HPLC/fluorescence detection, not GC.

  • c) Water solubility → Irrelevant (water is rarely used as a solvent in GC).

  • d) Electrical charge → GC separates neutrals; ions require MS detectors or derivatization.

Key limitation: GC cannot analyze thermally labile or non-volatile compounds without modification.

• The first step in developing a spectrophotometric method is identifying the wavelength (λmax) where the analyte exhibits peak absorbance (via a scanning spectrum). This ensures maximum sensitivity and specificity.

• Later steps address:

  • b) Sample volume → Optimized after wavelength selection.

  • c) Cuvette material → Chosen based on wavelength (e.g., quartz for UV, glass for visible).

  • d) Reagent stability → Evaluated during method validation.

Key principle: λmax is foundational—all other parameters depend on it. Use a spectral scan (e.g., 200–800 nm) to identify it.

• Guard columns are short, disposable columns placed before the analytical column to:

  • Trap particulate matter, chemical contaminants, or strongly retained compounds.

  • Extend the life of the expensive main column.

• Why not others?

  • b) Increase flow rate → Guard columns slightly reduce flow (minimal effect).

  • c) Reduce backpressure → They may increase pressure slightly.

  • d) Enhance sensitivity → No direct role in detection.

Key tip: Replace guard columns regularly to maintain performance!

• Overloaded samples cause distorted bands due to:

  • Overlapping proteins (poor resolution).

  • Smearing (exceeds gel’s separation capacity).

• Other causes (less common):

  • b) Low ionic strength → Can cause overheating/band bending (not primary distortion cause).

  • c) Short run time → Incomplete separation (bands too close, but not distorted).

  • d) High voltage → Heat artifacts (e.g., “smiling” bands), but distortion typically stems from overloading.

Key fix: Dilute samples or reduce loading volume for crisp bands.

(Note: Always include a protein ladder for reference!

• Low automated platelet counts require microscopic verification to rule out:

  • Pseudothrombocytopenia (e.g., EDTA-induced clumping).

  • Platelet satellitism (platelets adhering to WBCs).

  • True thrombocytopenia (e.g., DIC, ITP).

• Why not others?

  • a) Report immediately → Risk of false results without confirmation.

  • b) New sample → Only if collection error is suspected (e.g., clot).

  • d) Higher dilution → Used for high counts, not low.

Key step: Perform a peripheral smear with Giemsa stain to assess platelet clumping/estimate true count.

(Note: If clumping is seen, recollect in sodium citrate or heparin tubes.)

Modern automated analyzers are predominantly random access analyzers, which allow flexible, on-demand testing of multiple samples without predefined batches. They improve efficiency by processing different tests per sample simultaneously and reducing turnaround times, unlike batch processing or sequential systems. Manual loading platforms are outdated for high-throughput labs.

• Electrical impedance counting (Coulter principle) requires:

  • A conductive diluent (e.g., isotonic saline) to carry current through the aperture.

  • Cells suspended in the diluent disrupt current flow, generating measurable pulses.

• Why not others?

  • a) Fluorescent labels → Used in flow cytometry, not impedance.

  • c) Centrifugation → Unnecessary (analyzes raw suspensions).

  • d) Staining → Only for manual microscopy, not impedance counts.

Key requirement: The diluent must maintain cell integrity and conductivity (e.g., avoid lytic agents for WBC counts).

(Note: Aperture size determines the cell size range measured!)

• Hollow cathode lamps (HCLs) emit element-specific wavelengths when the target element’s atoms are excited in a low-pressure gas discharge.

  • Example: A copper HCL emits light at 324.7 nm for copper analysis.

• Why not others?

  • a) Tungsten-halogen → Broad spectrum (visible/near-IR), used in colorimetry.

  • c) Deuterium lamp → UV continuum source, used in molecular spectrophotometry.

  • d) LED → Narrowband but lacks element specificity for AAS.

Key feature: HCLs provide the sharp, monochromatic lines required for atomic absorption’s high selectivity.

• The Coulter principle counts and sizes cells by measuring changes in electrical resistance as they pass through a small aperture:

  • Each cell displaces electrolyte solution, causing a momentary voltage drop proportional to its volume.

• Why not others?

  • a) Light scatter → Flow cytometry, not Coulter counters.

  • c) Fluorescence → Requires labels (e.g., antibodies/dyes).

  • d) Magnetic resonance → MRI/NMR techniques, unrelated to impedance.

Key application: Foundational to automated hematology analyzers (e.g., RBC/WBC/platelet counts).

(Note: Aperture clogging is a common issue—requires regular maintenance!)

• NRBC flags indicate nucleated red blood cells in peripheral blood, which:

  • Must be quantified manually (microscopic review of Wright-stained smear).

  • Require correction of WBC count (analyzers falsely include NRBCs in WBC totals).

• Why not others?

  • a) Report directly → NRBCs are clinically significant (e.g., hypoxia, hemolysis).

  • c) Warming → Unnecessary (NRBCs are real cells, not artifacts).

  • d) Test plasma → Irrelevant (NRBCs are cellular, not plasma components).

Key steps:

1. Manual differential to count NRBCs/100 WBCs.

2. Adjust WBC count:

   Corrected WBC = Automated WBC × 100 ÷ 100 + NRBCs per 100 WBC

(Note: NRBCs in adults suggest pathology—always investigate!)

• Steric exclusion chromatography (also called size-exclusion chromatography or gel filtration) separates molecules based on size differences.

  • Large molecules cannot enter the pores of the stationary phase beads and elute first.

  • Small molecules enter the pores, take a longer path, and elute later.

• Why not others?

  • b) Charged vs. uncharged → Ion-exchange chromatography.

  • c) Hydrophobic compounds → Reverse-phase chromatography.

  • d) Metal ions → Chelation or ion-exchange chromatography.

Key use: Purifying proteins, polymers, or separating complexes by molecular size.

• Nephelometry measures light scattered by immune complexes (antigen-antibody interactions) in solution, making it ideal for:

  • Quantifying proteins (e.g., immunoglobulins, CRP).

  • Clinical serology (e.g., autoimmune diseases).

• Why not others?

  • b) DNA → Typically measured by UV absorbance or fluorescence.

  • c) Electrolytes → Analyzed via ion-selective electrodes or atomic spectroscopy.

  • d) Enzyme activity → Usually assessed by spectrophotometry (absorbance changes).

Key advantage: Detects small aggregates in real-time without precipitation steps.

(Note: Turbidimetry, its cousin, measures reduced light transmission due to scattering.)

• Forward scatter (FSC) in flow cytometry measures the amount of light diffracted along the laser path, which correlates with:

  • Cell size/diameter (larger cells scatter more light forward).

• Why not others?

  • b) Cytoplasmic granularity → Detected by side scatter (SSC).

  • c) Surface markers → Measured via fluorescence (antibody tags).

  • d) DNA content → Requires DNA-binding dyes (e.g., propidium iodide).

Key application: Distinguishing cell populations by size (e.g., lymphocytes vs. monocytes in blood).

(Note: FSC/SSC plots provide basic cell classification before fluorescence analysis.)

• Bichromatic analysis measures absorbance at two specific wavelengths at the same time:

  • Primary wavelength: Analyte’s peak absorbance.

  • Secondary wavelength: Corrects for background interference (e.g., turbidity, reagent color).

• Why not others?

  • b) Alternating wavelengths → Describes dual-wavelength (not bichromatic) analysis.

  • c) Multiple time points → Kinetics studies, unrelated.

  • d) Different temperatures → Rarely used in standard spectrophotometry.

Key advantage: Compensates for matrix effects, improving accuracy. Example: Bilirubin assays often use 460 nm (primary) and 540 nm (secondary).

• Hematocrit (Hct) is calculated by automated analyzers as:
  Hct (%) = MCV (fL) × RBC count (1012/L) ÷ 10

  • MCV (Mean Corpuscular Volume) = Average RBC size.

  • RBC count = Red blood cells per volume.

• Why not others?

  • a) Direct centrifugation → Manual method (“gold standard” but not used by analyzers).

  • c) Hemoglobin → Used for MCHC (Mean Corpuscular Hemoglobin Concentration), not Hct.

  • d) Plasma proteins → Irrelevant to Hct calculation.

Key point: This formula leverages electrical impedance/optical measurements of RBCs for speed and precision.

(Note: Always validate with manual centrifugation if results are questionable.)

• Gas chromatography (GC) separates compounds based on their ability to vaporize without decomposing at the oven temperature (typically 50–300°C).

  • Volatility: Analytes must transition to the gas phase.

  • Thermal stability: Must resist degradation (e.g., sugars often require derivatization).

• Why not others?

  • b) Fluorescent labels → Used in HPLC/fluorescence, irrelevant to GC.

  • c) Aqueous solutions → GC avoids water (damages columns; solids/liquids are injected directly).

  • d) Magnetic properties → No role in GC separation.

Key limitation: GC cannot analyze non-volatile or thermally labile compounds without chemical modification (e.g., silylation).

• A tachometer measures rotational speed (RPM) in centrifuges, ensuring accurate and safe operation.

• b) Thermometer → Monitors temperature (useful but unrelated to speed).

• c) Voltmeter → Measures electrical voltage (irrelevant to centrifugation).

• d) Barometer → Measures air pressure (not used in centrifuges).

Key point: Regular tachometer checks prevent overspeed/underspeed errors. Modern centrifuges often include built-in digital tachometers.

• A black ring on the barrel of a microscope objective typically marks it as an oil immersion lens (usually 100x magnification).

• Oil immersion is required to reduce light refraction and improve resolution at high magnification.

Other options:

• a) Low power → Usually black ring is not used for low power (e.g., 4x, 10x).

• c) Damaged lens → Damage isn’t indicated by color coding.

• d) Staining capability → Objectives are optical, not chemical, tools.

• Abnormal MCV in controls warrants immediate repeat testing to:

  • Rule out transient errors (e.g., bubble, clot, or control mishandling).

  • Confirm persistent deviation before troubleshooting.

• Why not others?

  • a) Calibration adjustment → Premature without verifying control integrity.

  • c) Shut down → Overreaction unless repeated failures occur.

  • d) Reagent change → Only if other parameters (e.g., Hb/RBC) are also off.

Next steps if repeat fails:

1. Check reagent expiration/mixing.

2. Inspect aspiration system for clots.

3. Run fresh controls or calibrate if needed.

(Note: MCV shifts may indicate RBC swelling/shrinking due to old samples or improper storage.)

• Absorbance (A) and percent transmittance (%T) are related by the equation:

  $$A=2-\log (\%T)$$

  *(Derived from the Beer-Lambert Law: $A=-\log (T)$, where $T$ is fractional transmittance (0–1). Since %T = $T\times 100$, $A=-\log (\%T/100)=2-\log (\%T)$.)*

Why not others?

• a) $1-\log (T)$ → Incorrect constants/log placement.

• b) $2+\log (T)$ → Sign error.

• d) $1+\log (T)$ → Incorrect constants.

• Phase contrast microscopy enhances contrast in transparent, unstained samples (e.g., live cells, microorganisms) by converting phase shifts in light waves into brightness differences.

• a) Stained slides → Brightfield microscopy is typically used.

• c) Cell counts → Possible but not the primary purpose.

• d) Chemical analysis → Requires other techniques (e.g., spectroscopy).

• Real-time PCR (qPCR) quantifies DNA by measuring the cycle threshold (Ct), the cycle number at which fluorescence exceeds background.

  • Lower Ct = Higher starting template concentration.

  • Standard curves relate Ct to known concentrations.

• Why not others?

  • b) Electrophoretic mobility → Used in endpoint PCR (gel electrophoresis).

  • c) Temperature gradients → Critical for melting curve analysis (post-amplification).

  • d) pH changes → Irrelevant to qPCR quantification.

Key advantage: Enables absolute/relative quantification (e.g., viral load, gene expression).

(Note: Fluorescent probes/dyes (e.g., SYBR Green, TaqMan) generate the signal.)

• Immersion oil must be wiped off the objective lens and specimen after use to prevent:

  • Dust accumulation

  • Lens damage (oil can degrade coatings)

  • Cross-contamination between samples

• a) Left on lenses → Incorrect; causes residue buildup.

• c) Only 40x objectives → False; used with 100x (high-power) objectives.

• d) Refrigeration → Unnecessary; store at room temperature.

• Chromatography separates components based on their differential affinity between two phases:

  • Stationary phase (e.g., silica gel, C18 column).

  • Mobile phase (e.g., solvent/gas).

• Compounds partition (dissolve/distribute) differently between these phases, leading to separation.

Why not others?

• b) Charge separation → Electrophoresis, not chromatography.

• c) Magnetic properties → Irrelevant in standard chromatography.

• d) Thermal conductivity → Detector property (e.g., in GC), not separation mechanism.

Key principle: “Like dissolves like”—polar/nonpolar interactions drive partitioning (e.g., HPLC, TLC).

• OD260 (Optical Density at 260 nm) is used to quantify nucleic acids:

  • 1.0 OD260 ≈ 50 μg/mL for double-stranded DNA (dsDNA).

  • 1.0 OD260 ≈ 40 μg/mL for single-stranded DNA/RNA.

• Why not others?

  • a) 10 μg/mL → Too low (typical for diluted samples).

  • b) 20 μg/mL → Incorrect for DNA (used for RNA under some conventions).

  • d) 100 μg/mL → Overestimates by 2x.

Key formula:

DNA concentration (μg/mL)=OD260×50×dilution factor

Note: Contaminants (e.g., protein/phenol) can skew readings—check OD260/280 ratio for purity (~1.8 for DNA, ~2.0 for RNA).