• The Hook effect (or prozone effect) occurs in two-site “sandwich” immunoassays when the analyte concentration is extremely high.

• Excess analyte saturates both the capture and signal antibodies, preventing proper “sandwich” formation and leading to a falsely low result.

Other options:

• a) Heterophilic antibodies → cause false positives or negatives by nonspecific binding, not the Hook effect.

• c) Inadequate washing → may cause high background or false positives.

• d) Substrate deterioration → causes signal loss, but not a Hook effect.

To assess random error (precision), the laboratory should perform replicate testing of a control material or patient pool over multiple runs and calculate the standard deviation and coefficient of variation.

Let’s check the options:

• a) Comparison of methods – assesses accuracy and systematic error, not purely random error.

• b) Linearity study – checks the measuring interval and proportional error, not precision.

• c) Replicate testing over multiple runs – directly estimates random error (precision).

• d) Recovery study – assesses accuracy (systematic error).

Enzymes speed up chemical reactions by lowering the activation energy required for the reaction to proceed. They do not change the reaction equilibrium or increase the product concentration at equilibrium; they only help the reaction reach equilibrium faster.

Enzyme activity is highly sensitive to:

• pH – Each enzyme has an optimal pH; too acidic or basic can denature it.

• Temperature – High temperatures can denature enzymes; low temperatures slow activity.

• Substrate concentration – Increasing substrate increases reaction rate up to a saturation point.

• Light intensity, osmotic pressure, and radiation can affect some biological systems, but they are not the main factors for enzyme activity.

ALT is a liver-specific enzyme. High levels in the blood are a strong indicator of hepatocellular damage, such as in hepatitis. While other tissues contain small amounts, its primary clinical significance is in diagnosing liver disease.

• ALT (Alanine Aminotransferase) is primarily a liver enzyme and is used as a marker for hepatocellular injury.

• Heart and muscle → AST is more common.

• Brain → ALT levels are minimal.

• Cholinesterase (acetylcholinesterase or pseudocholinesterase) breaks down acetylcholine.

• Clinical importance:

  • Organophosphate pesticide poisoning → inhibits cholinesterase, leading to toxic accumulation of acetylcholine.

  • Anesthesia sensitivity → individuals with pseudocholinesterase deficiency are at risk of prolonged paralysis with drugs like succinylcholine.

• Troponin (cTnI or cTnT) is highly specific for cardiac muscle.

• It rises 3–6 hours after myocardial infarction, peaks at 12–24 hours, and remains elevated for 7–10 days, making it excellent for both early and late diagnosis.

• CK-MB rises earlier but returns to normal within 2 days, so troponin is preferred for its specificity and longer detection window.

• A reference range (normal range) is usually defined as the central 95% of values from a healthy population.

• This corresponds to the 2.5th percentile to the 97.5th percentile, meaning 2.5% of healthy individuals may fall below and 2.5% above the range.

• Other percentiles (1–99, 5–95, 10–90) are not standard for reference intervals.

• Accuracy reflects how close a measured value is to the true or accepted reference value.

• Precision (repeatability) refers to how close repeated measurements are to each other (average value).

• While a reference method (c) can be used to assess accuracy, the definition is always in relation to the true value, not just another method.

• Normally, LD2 > LD1 in serum.

• In acute myocardial infarction, LD1 > LD2, called the “flipped pattern”, which is a classic indicator of cardiac injury.

• Liver disease → LD4 or LD5 may be elevated.

• Muscle injury → LD5 predominates.

• Hemolysis → can also raise LD1, but clinical context distinguishes it.

• Ammonia levels can rise rapidly in serum due to continued production by red blood cells and other tissues after collection.

• To ensure accurate results:

  • Collect the sample on ice,

  • Centrifuge and separate plasma/serum immediately,

  • Analyze without delay (ideally within 15–30 minutes).

Other options:

• a) Incubation at 37°C → increases ammonia falsely.

• b) Routine handling → delays cause false elevation.

• d) Room temperature storage → also causes falsely high results.

We use the dilution formula:

C1V1=C2V2C1​V1​=C2​V2​

Where:

• C1=500 mg/dLC1​=500 mg/dL (stock concentration)

• V1=?V1​=? (volume of stock to use)

• C2=15 mg/dLC2​=15 mg/dL (desired concentration)

• V2=100 mLV2​=100 mL (final volume)

500×V1=15×100500×V1​=15×100500×V1=1500500×V1​=1500V1=1500500=3 mLV1​=5001500​=3 mL

So, 3 mL of stock solution is needed.

• In reverse-phase chromatography:

  • The stationary phase is nonpolar (hydrophobic), e.g., C18 silica.

  • The mobile phase is polar, e.g., water with organic modifiers.

• This is called “reverse” because it’s the opposite of normal-phase chromatography, where the stationary phase is polar and the mobile phase is nonpolar.

• a) Pump direction is irrelevant.

• c) Mobile phase is not always nonpolar; it is polar in reverse phase.

• d) Mobile phase is more polar, not less polar, than the stationary phase.

A 1:100 dilution means 1 part serum + enough diluent to make 100 parts total.

So if 10 µL of serum is used:

$$\text{Total volume}=10\mu L\times 100=1000\mu L$$ $$1000\mu L=1.0mL$$

• Amperometry measures the current produced by an electrochemical reaction at a constant applied voltage.

• In the pO₂ electrode, oxygen diffuses through a membrane and is reduced at the cathode, producing a current proportional to the pO₂ in the sample.

• This current is measured to determine the partial pressure of oxygen.

Other options:

• a) Potentiometry – used for ion-selective electrodes (e.g., pH, pCO₂).

• c) Coulometry – measures total charge (used in chloride meters, for example).

• d) Conductometry – measures electrical conductivity of a solution.

• The reportable range (also called the analytical measurement range, AMR) is the range of analyte concentrations that the method can accurately and precisely measure without dilution or concentration.

• To verify this range, the lab performs a linearity study, which involves testing samples with known increasing concentrations and confirming that the instrument response remains proportional to concentration.

Lactate dehydrogenase (LDH) is a tetrameric enzyme, meaning it is composed of four polypeptide subunits. These subunits can be of two types, H (heart) and M (muscle), which combine to form five different isoenzymes (e.g., LDH1 = HHHH, LDH2 = HHHM, etc.).

The Michaelis constant (Km) is defined as the substrate concentration at which the reaction velocity is half of Vmax. It is a measure of the enzyme’s affinity for its substrate; a lower Km indicates a higher affinity.

• Km is the substrate concentration at which the enzyme works at half of its maximum velocity (Vmax).

• Vmax → the maximum velocity the enzyme can achieve.

• Ki → inhibition constant for enzyme inhibitors.

• pKa → acid dissociation constant, unrelated to enzyme kinetics.

The creatinine clearance formula is:

Creatinine Clearance=UCr×VSCr×1440Creatinine Clearance=SCr​×1440UCr​×V​

(where UCrUCr​ = urine creatinine concentration, $V$ = 24-hour urine volume in mL, SCrSCr​ = serum creatinine, and 1440 = minutes in 24 hours)

Thus, required components are:

• 24-hour urine volume

• Urine creatinine

• Serum creatinine

That matches option a.

ALT (Alanine Aminotransferase) is primarily found in the cytoplasm of liver cells. Elevated serum ALT is a more specific marker for hepatocellular damage (e.g., hepatitis) than ALP or GGT (which are more associated with bile ducts) or LDH (which is found in many tissues).

• ALT is highly concentrated in hepatocytes and is the most specific enzyme for hepatocellular injury.

• ALP (Alkaline Phosphatase) → elevated in cholestasis or bone disease, less liver-specific.

• GGT (Gamma-Glutamyl Transferase) → indicates biliary obstruction or alcohol use, not specific to hepatocyte injury.

• LDH → non-specific, found in many tissues.

An enzyme acts on a substrate, binding to it at the active site to convert it into a product.

• Product → the result of the reaction, not what the enzyme acts on.

• Inhibitor → slows down or stops enzyme activity.

• Coenzyme → a helper molecule that assists the enzyme but isn’t the main target.

• A Lineweaver–Burk plot (double reciprocal plot) is a linear transformation of the Michaelis–Menten equation:

1V=KmVmax⋅1[S]+1Vmax\frac{1}{V} = \frac{K_m}{V_{max}} \cdot \frac{1}{[S]} + \frac{1}{V_{max}}V1​=Vmax​Km​​⋅[S]1​+Vmax​1​

From this plot:

• The y-intercept = 1/Vmax1/V_{max}1/Vmax​

• The x-intercept = −1/Km-1/K_m−1/Km​

Thus, it is used to determine Vmax (maximum velocity) and Km (Michaelis constant) of an enzyme-catalyzed reaction.

AST is present in high concentrations in both the liver and cardiac muscle. It is also found in skeletal muscle, kidneys, and red blood cells. Elevated levels can indicate damage to any of these tissues, most commonly the liver or heart.

• AST (Aspartate Aminotransferase) is abundant in liver, heart, skeletal muscle, and kidneys.

• It is commonly used as a marker for liver injury and myocardial infarction.

• Brain, pancreas, intestine, bone, and cartilage contain only minimal AST.

The Biuret method is a colorimetric assay for measuring total protein concentration in serum or plasma.

• In this method, peptide bonds in proteins react with copper ions (Cu²⁺) in an alkaline solution to form a violet-colored complex.

• The intensity of the violet color is directly proportional to the total protein concentration, and absorbance is usually measured at 540–560 nm.

So, Biuret = total protein, not specific fractions like albumin or immunoglobulins.

The relationship between absorbance (A) and percent transmittance (%T) is:

A=log⁡(I0I)A=log(II0​​)

where I0I0​ is the incident light intensity and $I$ is the transmitted light intensity.

Since %T = II0×100I0​I​×100,

II0=%T100I0​I​=100%T​

Thus:

A=log⁡(100%T)A=log(%T100​)$$A=\log (100)-\log (\%T)$$$$A=2-\log (\%T)$$

That matches option d.

In the Lactate Dehydrogenase (LD or LDH) assay, the reaction direction determines what is measured:

• In the commonly used “forward” reaction:
  Lactate + NAD⁺ → Pyruvate + NADH + H⁺
  → Absorbance increases at 340 nm (due to NADH formation).

• In the “reverse” reaction (used in most clinical assays):
  Pyruvate + NADH + H⁺ → Lactate + NAD⁺
  → Absorbance decreases at 340 nm (due to NADH oxidation).

• The osmolal gap = measured osmolality – calculated osmolality
  → Here: 301 – 286 = 15 mOsm/kg, which is elevated (normal ≤ 10 mOsm/kg).

• A high osmolal gap indicates the presence of unmeasured, osmotically active substances such as:

  • Ethanol

  • Methanol

  • Ethylene glycol

  • Isopropanol

  • Mannitol

• An enzyme can consist of a protein part (the apoenzyme) and a non-protein part called a cofactor (which may be a metal ion or a coenzyme).

• When the apoenzyme combines with its cofactor, it forms the holoenzyme, which is the active enzyme.

Quick breakdown:

• Apoenzyme = protein part alone (inactive).

• Holoenzyme = apoenzyme + cofactor (active).

• Coenzyme = organic molecule cofactor (like vitamins).

• Prosthetic group = tightly bound cofactor.

A sharp cutoff filter (e.g., a certain type of colored glass filter) is used in spectrophotometers to detect stray light.

• The principle: The filter transmits light well above a certain wavelength but blocks light below that wavelength.

• If the instrument detects significant light transmission when it should be blocked (at wavelengths below the cutoff), that indicates the presence of stray light.

Other options:

• a) Mercury vapor lamp – used for wavelength calibration.

• b) Holmium oxide glass – used for wavelength accuracy checks.

• c) Potassium dichromate solution – sometimes used for absorbance accuracy checks, not specifically for stray light detection.

• Holoenzyme: The complete, active form of an enzyme, consisting of the apoenzyme (protein part) + its cofactor.

• Apoenzyme: The inactive protein part without the cofactor.

• Isoenzyme: Different forms of an enzyme that catalyze the same reaction but are encoded by different genes.

• Substrate: The molecule upon which an enzyme acts.

• Sodium fluoride is an antiglycolytic agent that inhibits the enzyme enolase in the glycolytic pathway.

• By inhibiting glycolysis, it prevents blood cells (especially erythrocytes) from consuming glucose after the blood sample is drawn.

• This helps maintain the glucose concentration at the in vivo level until analysis.

Other options:

• a) Incorrect — sodium fluoride is not a coenzyme; it’s an enzyme inhibitor.

• b) Incorrect — fluoride does not primarily precipitate proteins (though it may have some effects, that’s not its purpose here).

• d) Incorrect — preventing reactivity of non-glucose reducing substances is not the role of fluoride.

Acid phosphatase, particularly the prostatic isoenzyme (PAP), was a classic tumor marker for prostate cancer. It is released into the blood when prostate tissue is damaged, such as by a malignant tumor. It has now been largely replaced by the more specific PSA (Prostate-Specific Antigen).

LDH catalyzes the reversible conversion of pyruvate to lactate using NADH as a coenzyme, a critical step in anaerobic glycolysis. This reaction regenerates NAD⁺, allowing glycolysis to continue in the absence of oxygen.

• a) Glucose ↔ pyruvate is catalyzed by glycolytic enzymes, not LDH.

• c) Creatine ↔ creatinine is catalyzed non-enzymatically.

• d) Alanine ↔ pyruvate is catalyzed by alanine aminotransferase (ALT).

• One International Unit (IU) of enzyme activity is defined as the amount of enzyme that catalyzes the conversion of 1 micromole (μmol) of substrate per minute under specified standard conditions (usually temperature, pH, and substrate concentration).

• 1 mol, 1 mmol, 1 nmol are incorrect scales for IU.

Enzymes are proteins (mostly) that accelerate chemical reactions in living organisms without being consumed in the process. They lower the activation energy required for reactions, making biological processes much faster and efficient.

• a) Structural proteins – These provide support and shape (e.g., collagen), but don’t speed up reactions.

• c) Hormones – These regulate metabolism but are not catalysts.

• d) Vitamins – Some act as coenzymes, but they themselves are not enzymes.

Myoglobin is released into the bloodstream most rapidly after muscle damage, including a heart attack. Its levels can begin to rise within 1–3 hours, making it the earliest marker. However, it is not specific to heart muscle. Troponin and CK-MB are more specific cardiac markers but rise later.

• CK-MB → rises after about 3–6 hours.

• Troponin → rises after 3–6 hours but is more specific and remains elevated longer.

• LDH → rises much later (12–24 hours), not useful for early detection.

• Pre-analytical errors in potassium measurement usually result from sample handling or collection issues, not diet.

Here’s why:

• a) Prolonged tourniquet application → causes hemoconcentration and potassium leakage.

• b) Hemolysis → releases intracellular potassium, falsely elevating results.

• d) Delayed separation → allows leakage of potassium from cells into serum/plasma.

• c) Dietary intake → normally has minimal short-term effect on serum potassium due to tight renal regulation.

• Indirect ISE (ion-selective electrode) methods can be falsely affected by lipemia, because the sample’s aqueous phase is effectively reduced by the lipid fraction.

• Direct ISE measures ions in the plasma water without dilution, avoiding lipemic interference.

• Ultracentrifugation can remove lipids, also correcting interference.

• Diluting or filtering may not reliably correct the error.

• Simply reporting the result with a comment does not address the analytical inaccuracy.

GGT is a sensitive marker for hepatobiliary disease, particularly useful for detecting alcohol-induced liver injury. It is often elevated in chronic alcoholism and biliary obstruction. While not entirely specific, its combination with other liver enzymes helps in diagnosis.

• GGT (Gamma-Glutamyl Transferase) is a liver enzyme that rises in hepatobiliary disease and is particularly sensitive to alcohol-induced liver injury.

• Muscle damage, thyroid disease, and kidney function → GGT is not a reliable marker for these conditions.

A constant systematic error means that the new method consistently gives results that are shifted by a fixed amount compared to the reference method, regardless of the concentration.

In regression analysis $y=mx+b$:

• Slope (m) relates to proportional error.

• Intercept (b) relates to constant error.

So a constant systematic error will appear as a difference in the intercept between the new method and the ideal (y = x) line.

That matches option b.

• Coenzymes are organic cofactors (often derived from vitamins) that bind loosely to enzymes and help in the catalytic process, often by transferring chemical groups.

• Metal ions can also be cofactors but are usually inorganic and sometimes tightly bound.

• Apoenzyme is the protein part of the enzyme without any cofactor.

• Inhibitor is a molecule that decreases enzyme activity.

Transferases catalyze the transfer of a specific functional group (e.g., a phosphate, methyl, or amino group) from one molecule (the donor) to another (the acceptor). Isomerases rearrange atoms, hydrolases cleave bonds with water, and lyases remove groups to form double bonds or add groups to double bonds.

• Transferases → catalyze the transfer of functional groups (like methyl, phosphate, or amino groups) from one molecule to another.

• Isomerases → rearrange atoms within a molecule to form isomers.

• Hydrolases → break bonds by adding water.

• Lyases → break bonds without water or oxidation, often forming double bonds or rings.

• Amylase is produced by both the pancreas and salivary glands.

• In salivary gland disorders (e.g., mumps), serum amylase rises, but lipase remains normal because lipase is pancreas-specific.

• Acute pancreatitis → both amylase and lipase are elevated.

• Hepatitis → affects liver enzymes (ALT, AST), not amylase/lipase.

• Renal failure → may mildly elevate amylase due to reduced clearance but is non-specific.

The three main CK isoenzymes are:

• CK-MM (found in skeletal and heart muscle)

• CK-MB (primarily in heart muscle)

• CK-BB (found in the brain and smooth muscle)

CK-MB is the cardiac-specific form and is a key biomarker for myocardial infarction.

• ALP (Alkaline Phosphatase) is elevated in conditions involving bile duct obstruction (cholestasis) or increased bone turnover (e.g., Paget’s disease, bone growth, fractures).

• Myocardial infarction → does not significantly raise ALP.

• Liver failure → may raise ALT/AST more than ALP.

• Diabetes mellitus → ALP is usually unaffected.

Hydrolases catalyze hydrolysis reactions, which break chemical bonds by adding water. Lyases remove groups without hydrolysis, isomerases rearrange molecules, and oxidoreductases handle electron transfer reactions.

• Hydrolases → catalyze hydrolysis reactions, breaking chemical bonds by adding water. Examples include lipases, proteases, and nucleases.

• Lyases → break bonds without water, often forming double bonds.

• Isomerases → rearrange atoms within a molecule.

• Oxidoreductases → catalyze redox (electron transfer) reactions.

• This is Beer–Lambert law:

$$A=\epsilon \cdot l\cdot c$$

Where:

• A → absorbance (unitless)

• ε → molar absorptivity (L·mol⁻¹·cm⁻¹)

• l → path length of the cuvette (cm)

• c → concentration of the solution (mol/L)

Oxidoreductases are the class of enzymes that catalyze oxidation-reduction reactions, which involve the transfer of electrons between molecules. Transferases move functional groups, hydrolases use water to break bonds, and ligases join molecules together.

• Oxidoreductases → catalyze oxidation-reduction (redox) reactions, where electrons are transferred between molecules.

• Transferases → transfer functional groups (like phosphate or methyl groups) between molecules.

• Hydrolases → catalyze hydrolysis reactions, breaking bonds with water.

• Ligases → join two molecules together, usually using ATP.

The active site is the specific region on an enzyme where the substrate binds, allowing the enzyme to catalyze the reaction.

• Regulatory site → site where molecules can increase or decrease enzyme activity.

• Allosteric site → a type of regulatory site away from the active site that can change enzyme shape.

• Cofactor site → location where cofactors (like metal ions) may bind, not the substrate.

• Specificity = the ability of a test to correctly identify those who do not have the disease.

  Specificity=True NegativesTrue Negatives+False Positives\text{Specificity} = \frac{\text{True Negatives}}{\text{True Negatives} + \text{False Positives}}Specificity=True Negatives+False PositivesTrue Negatives​

• A highly specific test → few false positives.

In contrast:

• Sensitivity measures the ability to correctly identify those with the disease (true positives).

The Jaffe reaction is a colorimetric method in which creatinine reacts with picric acid in an alkaline solution to form an orange-red complex (creatinine–picrate complex).

This reaction is the basis of many routine creatinine assays, though it can be affected by interfering substances such as glucose, ketones, and proteins.

• Indirect ISE dilutes the sample before measurement.

• If the sample is lipemic, lipids displace plasma water but are not electrolytes.

• The sodium concentration in the aqueous phase is normal, but the reported concentration (based on total volume including lipids) is falsely low.

• Hyperglycemia (a) can cause pseudohyponatremia by water shift, but that’s a real physiological effect, not an analytical error with indirect ISE.

• Hemolysis (c) increases potassium but not a common cause of falsely low sodium.

• Alkalosis (d) may affect sodium slightly but not a major cause of falsely low sodium.

• CK-MB (Creatine Kinase–MB isoenzyme) is highly specific for cardiac muscle and is used to diagnose myocardial infarction (heart attack).

• AST (Aspartate aminotransferase) → found in liver, heart, and muscle, less specific.

• LDH (Lactate dehydrogenase) → found in many tissues, not specific.

• ALT (Alanine aminotransferase) → primarily a liver enzyme, not cardiac-specific.

• A Levey-Jennings chart plots quality control values over time against the mean and standard deviations.

• It helps laboratories detect shifts, trends, or random errors in an assay, ensuring ongoing precision and accuracy.

• a) Method comparison → scatter plots or Bland-Altman plots

• c) Sensitivity/specificity → diagnostic performance studies

• d) Reference intervals → established from population studies, not QC charts

• Amylase → breaks down starch; Lipase → breaks down fats.

• Both are elevated in acute pancreatitis and are used as diagnostic markers.

• Myocardial infarction → CK-MB, troponin

• Hepatitis → ALT, AST

• Muscle injury → CK, LDH

• A reagent blank contains all reagents except the analyte (sample).

• Its purpose is to measure any absorbance caused by the reagents or cuvette, not by the analyte.

• This value is then subtracted from the total absorbance of the test sample to ensure the result reflects only the analyte’s contribution.

The Michaelis–Menten equation describes how the rate (velocity) of an enzyme-catalyzed reaction depends on substrate concentration. It helps determine:

• Vmax → maximum reaction velocity

• Km → substrate concentration at half-maximal velocity

• Henderson–Hasselbalch equation → relates pH to acid/base ratio

• Beer–Lambert law → relates absorbance to concentration

• Lineweaver–Burk equation → is a linearized form of Michaelis–Menten for plotting purposes

• Isoenzymes (isozymes) → are different molecular forms of the same enzyme found in different tissues or developmental stages. They catalyze the same reaction but may differ in kinetic properties or regulatory features.

• a) is incorrect because isoenzymes are not chemically identical.

• c) refers to inhibitors, not isoenzymes.

• d) refers to cofactors, which are unrelated.

• 1-3s rule (Westgard):

  • If a single control measurement falls outside ±3 standard deviations (SD) from the mean, it signals a possible random error.

• Other Westgard rules detect systematic errors, trends, or shifts:

  • 2-2s → two consecutive controls >2SD → systematic error

  • R-4s → difference between two controls >4SD → random error

  • 4-1s → four consecutive controls >1SD → systematic error

• In competitive inhibition, the inhibitor competes with the substrate for the active site of the enzyme.

• This means:

  • Vmax → unchanged, because adding more substrate can outcompete the inhibitor.

  • Km → increased, because a higher substrate concentration is needed to reach half of Vmax.

So the enzyme’s affinity for the substrate appears lower, but the maximum rate can still be achieved.