Lab Instrumentation

What do spectrochemical methods of instrumentation in the lab measure?

• light absorbed by an analyte

• light emitted by an analyte

• light refracted by an analyte

in terms of energy, short wavelengths result in high ___ and ___

frequencies and energy

How does a spectrophotometer work?

light absorption is used to quantify the concentration of an analyte in a solution

What are the 6 parts of a spectrophotometer?

• light source

• monochromator

  • isolates individual wavelengths of light (like a prism)

• cuvette

  • contains the patient sample or QC

• photodetector

  • converts light energy into electrical energy

• amplifier

  • amplifies low electrical energy readings

• output

  • energy value (result)

Urine dipstick tests utilize what test method?

reflectometry

What does fluorimetry measure?

the intensity of fluorescent light emitted by a substance when it is excited by light

What does chemiluminescence measure?

the emission of light produced by a chemical reaction

• does not require external light

• reagent probes enter the sample —> reaction occurs —> a photomultiplier tube amplifies the signal —> output

potentiometry

• measures potential (aka voltage) between two electrodes in a solution

• used for determining electrolyte concentrations

• a reference electrode delivers a constant voltage and an indicator electrode measures

How do the separation techniques electrophoresis and densitometry work?

• charged particles migrate through a gel or liquid medium in response to an electric field

• densitometry analyzes the concentration of substances by measuring the amount of light transmitted through or reflected by the sample

How does chromatography work?

complex mixtures are separated on the basis of different physical interactions between the individual compounds and the stationary phase (part) of the system

the mobile phase carries the mixture through the stationary phase, allowing for separation based on properties like polarity and size

3 parts of a chromatography system

• mobile phase (solvent in pic)

  • holds the patient sample

• stationary phase (paper in pic)

  • held in a column, phase thru which the mobile phase flows

• eluate (colorful dots in pic)

  • the separated components of the mixture

thin layer chromatography

a filter paper is used in the stationary phase, the mobile phase moves onto the paper

high performance liquid chromatography (HPLC)

an extremely high pressure environment separates the sample in the mobile phase

How are analytes identified in mass spectrometry and what are the 5 steps of this technique?

• analytes are identified and quantified based on their structure and mass

• steps:

  • sample is introduced

  • sample is vaporized

  • sample is ionized

  • sample passes through a strong magnetic field to separate particles based on charge

  • ions reach a detector at different rates, depending on the compound

immunoassays can be used to identify:

proteins, hormones, metabolites, therapeutic drugs, and drugs of abuse in specimens

epitope

specific part of an antigen that binds to the corresponding antibody

affinity

the strength of binding of an antigen at a single site to its corresponding antibody

avidity

the cumulative strength of all binding sites between an antibody and an antigen

nephelometry

• used in immunoassays

• immune complexes form and increase turbidity, affecting absorbance and transmittance through the specimen

What is the difference between heterogenous and homogenous immunoassays?

• heterogenous

  • test requires separation of bound immune complexes from free antibody or antigen

• homogenous

  • test does not require separation, the expression of labels depends on whether or not it is bound to its target AB or Ag

competitive immunoassays

• labeled reagent antigens compete with patient antigens in a sample

• reagent AB + reagent labeled Ag + patient Ag

• more patient antigens = less labeled immune complexes

noncompetitive/sandwich immunoassays

• patient antigen binds to reagent antibody and a labeled reagent antibody binds to the immune complex

• patient Ag + reagent AB + reagent labeled AB

• more patient antigen = more labeled immune complexes

Is ELISA a heterogenous or homogenous immunoassay?

heterogenous

competitive ELISA

• utilizes:

  • bound reagent AB and a reagent labeled Ag (that competes with patient Ag)

  • OR bound reagent Ag and a labeled reagent AB (that competes with patient AB)

noncompetitive (sandwich) ELISA

• utilizes:

  • bound AB and labeled reagent ABs

  • OR bound Ag and labeled reagent AB (specific to patient AB)

fluorescence polarization immunoassay (FPIA)

• homogenous, competitive assay

• patient antigen competes with reagent antigen labeled with fluorescein

• amount of patient antigen is indirectly proportional to the light produced

  • less light produced = more patient antigen present

  • more light produced = less patient antigen present

chemiluminescent magnetic immunoassay (CMIA)

• chemiluminescent labels produce light when combined with a trigger reagent

• heterogenous, non-competitive assay (magnet is used for separation)

How does CMIA work?

• reagent AB with a magnetic component + patient Ag = bound immune complex

• magnet is applied

• excess reagent and specimen is washed away

• labeled AB is added and light is emitted when combined with the complexes

  • proportional relationship between the amount of light emitted and the concentration of patient antigen

What are 3 possible limitations to immunoassays?

• human anti-mouse AB in patient specimen

  • results in false positives in sandwich assays

• heterophile ABs in patient specimen

  • ABs formed in patients with autoimmune disorders, ABs interfere with testing

• hook effect

  • prozone effect

  • excess Ag in patient samples overwhelms the binding ability of reagent AB, resulting in low or negative results