CHEM - Osmometry, Immunochemical Techniques, Laboratory Automation, Carbohydrates

What is osmometry?

A technique for measuring the concentration of solute particles that contribute to the osmotic pressure of a solution.

What does osmotic pressure govern?

The movement of a solvent across a membrane that separates two solutions.

In physiologic conditions, what is the solute?

Water.

What are the clinical applications of osmometry?

Analysis of osmolality in urine, plasma, and serum.

What does osmolality express?

Concentrations relative to the mass of the solvent.

What is a 1 molal solution?

Contains 1 mole of solute per 1 kg of water.

What units is measured osmolality usually expressed in?

mOsm/kg of water.

What are colligative properties?

Physical properties that are a function of the number of solute particles suspended in the solution and not the chemical nature of the particles.

What are the four colligative properties?

Elevation in the boiling point, elevation in the osmotic pressure of the solution, depression of the freezing point, and depression of the vapor pressure.

What are the components of a freezing point osmometer?

1) Cooling bath or block; 2) Rapid stir mechanism to initiate freezing of the sample; 3) A thermistor to measure temperature of the sample; 4) LED display that indicates the final result.

What happens to the sample in a freezing point osmometer?

The sample is lowered into the bath and super-cooled to a temperature several degrees below its freezing point.

What happens after sufficient super-cooling in a freezing point osmometer?

The sample is raised above the liquid in the cooling bath; the stirrer is changed from a gentle rate to a more vigorous one, which initiates freezing of the super-cooled solution.

How much of the solvent solidifies during freezing point osmometry?

About 2% to 3%.

What happens to temperature after freezing begins in freezing point osmometry?

Released heat of fusion initially warms the solution and then the temperature plateaus and remains stationary.

What is the equilibrium point in freezing point osmometry?

The point at which both freezing and thawing are occurring.

What does vapor pressure osmometry measure?

The decrease in the dew point temperature of the solvent (water) caused by the decrease in vapor pressure of the solvent by the solutes.

What substances increase vapor pressure instead of lowering it?

Volatile substances such as ethanol, methanol, and isopropanol.

Is vapor pressure osmometry practical for most clinical uses?

No.

What are the clinical uses of serum, plasma, and urine osmolality?

To assess fluid and electrolyte balance related to hydration, acid-base balance, and toxic screening, and to assist in diagnosing disease such as diabetes.

What sample types are used for osmometry?

SST/PST and random urine.

What is the reference range for serum osmolality?

275-295 mOsm/kg.

What is the reference range for urine osmolality?

250-900 mOsm/kg.

What are the major osmotic substances in serum?

Na+, Cl-, glucose, and urea (Na+ is the biggest contributor).

What is the formula for calculated serum osmolality in SI units?

Calculated serum osmolality (mOsm/kg) = 2(Na mmol/L) + Glucose (mmol/L) + Urea (mmol/L).

What are immunochemical techniques also known as?

Immunoassays.

What do immunochemical techniques involve?

Principles of antibody-antigen interactions.

What allows immunochemical techniques to identify and quantify specific substances?

The specificity and high affinity of antibody-antigen interactions.

What is affinity?

The strength of the bond between the antigen and the antibody.

What is avidity?

The overall stability of an antibody-antigen complex, based on affinity of the antibody for the antigen, the number of binding sites available, and the way the two molecules combine.

What is specificity?

A property of T cell and B cell receptors that results in their ability to recognize one, and only one, epitope.

What is lattice formation?

One antigen molecule crosslinks more than one antibody, resulting in the formation of a lattice structure.

What happens when the lattice reaches a certain size?

It loses its solubility and precipitates.

When does maximum precipitation occur?

When the relative concentration of the antigen and antibody allows each antibody and each antigen molecule to participate in the lattice.

What is a precipitation curve?

A graphic representation of precipitation reactions that occur when the concentration of one reactant is constant while the concentration of the second reagent is increased serially.

What is the zone of antibody excess (prozone)?

At all stages, free antibody is present; antigen/antibody interactions occur and immune complexes form, but there is insufficient antigen to form large immune complexes.

What happens as more antigen is added in the prozone?

Precipitation is observed.

What is the zone of equivalence?

More precipitation is observed until all antigen molecules and all antibody molecules interact and form a lattice.

What is present in the zone of equivalence?

No free antigen and no free antibody molecules in solution.

What is the zone of antigen excess (postzone)?

When an increasing amount of antigen is added beyond the zone of equivalence, there is a gradual decrease in precipitation until no precipitation is observed.

What is present in the zone of antigen excess?

Free antigen in the solution.

What is the difference between heterogeneous and homogeneous immunoassays?

Heterogeneous assays require a step to physically separate free from bound analyte; homogeneous assays do not need a separation step.

What happens in a homogeneous immunoassay when binding occurs?

The activity of the label attached to the antigen is diminished.

What is a competitive immunoassay?

Unlabeled analyte in the test sample is measured by its ability to compete with labelled antigen for a limited number of antibody binding sites.

What is the relationship between label measured and unlabeled antigen in a competitive immunoassay?

Less label measured means more unlabeled antigen is present.

What is the relationship between antigen in the test sample and label measured in a competitive immunoassay?

The amount of antigen in the test sample is inversely related to the amount of label measured.

What is a noncompetitive immunoassay also called?

A sandwich immunoassay.

Why is a noncompetitive immunoassay called a sandwich assay?

The analyte is bound (sandwiched) between two highly specific antibody reactions.

What does the reaction mixture typically include in a noncompetitive immunoassay?

An excess of labelled antibody so that all analyte is bound.

What is the relationship between labeled analyte and antigen in a noncompetitive immunoassay?

The measurement of labeled analyte is directly proportional to the amount of antigen present in the sample.

What is radial immunodiffusion (RID)?

A passive diffusion method in which a concentration gradient is established for a single reactant, usually the antigen.

Where is the antibody in RID?

Dispersed uniformly in the gel matrix.

What happens in RID?

Antigen is allowed to diffuse from a well into the gel until antibody excess exists and immune precipitation occurs.

What indicates the presence of antigen in RID?

A well-defined ring around the well.

What isotopes are used as labels in radioimmunoassay (RIA)?

Radioactive isotopes of iodine and tritium.

What is the basis of RIA?

Competition between radiolabeled and unlabeled antigen or antibody in a reaction to determine the concentration of the unlabeled antigen or antibody.

What does RIA require?

The antigen must be available in a pure form and be labeled with a radioactive isotope.

Why has the use of RIAs declined?

Concerns of safe handling and disposal of radioactive reagents and waste.

What is immunoradiometric assay (IRMA)?

An alternative assay design to RIAs that uses labelled antibody and does not require purified antigen.

Why are antibodies used in IRMA?

Antibodies are more stable proteins and are easier to label without damage to the protein's function.

What is ELISA?

Enzyme-linked immunosorbent assay.

What happens in ELISA?

Antibodies are absorbed to the surface of a solid phase (microtiter plate well); samples containing antigen are added and incubated; wells are washed; enzymes are added; wells are washed again; a substrate is added; absorbance is measured.

What is the relationship between sample absorbance and analyte concentration in ELISA?

Sample absorbance is inversely proportional to the analyte concentration.

What is EMIT?

Enzyme-Multiplied Immunoassay Technique.

What happens in EMIT?

Antibody against the analyte is added together with substrate to the patient's sample; binding of antibody and analyte occurs; an enzyme with the attached analyte is added and binds with any excess antibody, altering the enzyme activity.

What is the relationship between enzymatic activity change and analyte concentration in EMIT?

Change in enzymatic activity is proportional to the analyte concentration in the patient's sample.

What is CMIA?

Chemiluminescent microparticle immunoassay.

What does CMIA use to detect analytes?

A chemiluminescent compound.

What do chemiluminescent labels produce when combined with a trigger reagent?

Light.

What type of assay is CMIA?

A non-competitive sandwich assay.

What is the relationship between analyte and signal intensity in CMIA?

The amount of analyte present in the sample is directly proportional to the intensity of the measured signal.

What is FPIA?

Fluorescence Polarization Immunoassay.

What type of assay is FPIA?

A type of fluoroimmunoassay (FIA).

What happens in FPIA?

Labeled, antibody-bound antigen in the sample competes with labeled, free antigen for antibody binding sites.

What does FPIA measure?

Therapeutic drugs, DOA, toxic substances, and hormones.

What is the label of choice in FPIA?

Fluorescein.

At what wavelength does fluorescein produce fluorescent light when activated?

520 nm.

How do Ab-Ag-fluorescein molecules behave when excited by polarized light?

They are large and rotate slowly in solution.

How do smaller Ag-fluorescein molecules behave when excited by polarized light?

They rotate rapidly.

What wavelength do smaller Ag-fluorescein molecules emit at?

520 nm.

What wavelength is absorbed by the smaller Ag-fluorescein molecules?

490 nm.

What is the relationship between signal and analyte concentration in FPIA?

There is an inverse relationship.

What is the high-dose hook effect?

Seen when sandwich immunoassays are performed; very high antigen concentrations in the patient sample bind to all available antibody binding sites, in both the antibody solid phase and the antibody-labeled conjugate.

What does the high-dose hook effect prevent?

Formation of the sandwich complex.

What does the high-dose hook effect cause?

Falsely decreased test results.

What is HAMA?

Human anti-mouse antibody.

What is HAMA a problem for?

Sandwich immunoassays.

What causes HAMA?

Circulating human antibodies that react with animal immunoglobulin, particularly HAMAs.

What can HAMA cause in two-site antibody-based sandwich assays?

Positive or negative interference.

How does HAMA cause a false positive?

By bridging between a mouse immunoglobulin antibody and mouse conjugate, thus mimicking the specific analyte.

How does HAMA cause a false negative?

HAMA reacting with one of the assay reagents and preventing formation of the sandwich.

What is the goal of laboratory automation?

To produce more results more quickly and with better precision and accuracy.

What are other benefits of laboratory automation?

Decreases the lab worker's exposure to hazardous chemicals and uses less sample and reagents than manual testing.

What is throughput?

Number of tests performed per hour.

What is carry-over?

The transport of a quantity of analyte or reagent from one specimen reaction into and contaminating a subsequent one.

What is dwell time?

The minimum time from initial sampling to the production of a result.

What is point of care testing (POCT)?

A mode of testing in which the analysis is performed at the site where healthcare is provided.

What are other names for POCT?

Bedside, near-patient, decentralized, and off-site testing.

What is continuous flow analysis?

Pumps reagent non-stop through large amounts of plastic tubing; samples are injected at regular intervals; bubbles are used to separate samples and scrub the tubing clean.

What was the problem with continuous flow analysis?

The bubbles were found to be ineffective.

What is a centrifugal analyzer?

Specimen and reagent are pipetted into rotor compartments containing cuvettes; as the rotor spins, centrifugal force pushes specimen and reagent into the cuvette where they mix and react.