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Instrumentation part 2
N/A
44
Medical
Undergraduate 2
03/04/2012

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Term



hemolysis: Definition 

Definition

Hemolysis (or haemolysis) from the Latin hemo (blood) and lysis (to break up) is the release of hemoglobin and other intracellular components from erythrocytes to the surrounding plasma, following damage or disruption of the cell membrane  

Term



In vivo hemolysis: Causes 

Definition

·         Typically, in vivo hemolysis does not depend on the technique of the healthcare provider and it is thus virtually unavoidable and potentially insurmountable

o   Autoimmune hemolytic anemia

o   Hemoglobinopathies

o   Drugs

o   Severe infections

o   DIC

o   Transfusion reactions

o   Heat valves 

Term




In Vitro hemolysis: Causes


Definition
·         In vitro hemolysis can occur beginning at the patient’s bedside and continue through sample handleing, processing and storage .
Term




Factors of in vitro hemolysis vary depending upon:

 

Definition

1. patients condition lifestyle

2. the skill of the person collecting the sample (training)

3. local environment (temperature, length of transport). 

Term

 

 

What are the clinical use of blanks

Definition
 Hemolysis, lipemic samples, icteric samples
Term
What is the leading cause of unsuitbale specmens for both outpatient and inpatient samples for routine and state specimens?
Definition

 

 

In vitro hemolysis

Term
 What are the clinical chem test effected by hemolysis with a consistant trend towards overesimation....
Definition

·      It generates a consistent trend towards overestimation of:

·      - ALT & AST                   lipase

·      - creatinine                    mg

·      - creatine kinase (CK)     PO4

·      - Iron                            K+

·      - LDH                            urea 

Term
What are the clin chem test effect by hemolysis that generate a consistent trend toward an underestimation of...
Definition

·      - albumin

·      - ALP

·      - chloride and sodium

·      - GGT

·      - Glucose 

Term
What are the causes of Lipemia?
Definition

·      Turbid, cloudy or milky serum (lipemic serum) may be produced by the presence of fatty substance (lipids) in the blood.

·      Bacterial contamination may also cause cloudy serum

·      A recent meal may produce transient lipemia; therefore we recommend that patients fast 12-16 hours before a blood specimen is obtained. 

Term

 

What is a icteric sample?

Definition
bright yellow/light green serum caused by liver disease (hep, cirrhosis, obstruction) or hemolytic disorders
Term
Why do we need a sample blank?
Definition

·      We have a sample with a color characteristic about it. it will absorb light. I need to get rid of this color variation so that I only get a the color of the reaction that is happening not the sample color.

Term

 

How do u creat a reagent blank?

Definition

·      Contains all substances to be measured EXCEPT SAMPLE (ALL REAGENT)

 

·      We now put the blue color tube in the spec and set to zero

·      I now measure my patient sample mixed with reagent and get an absorbance. This now is just the sample reaction with the reagent and not the color of the reagent

Term

 

How is a lamp spec check done?

Definition

·      intensity

·      select a wavelength where only minimal photons are emitted

·      ( 400 nm for tungsten – halogen lamp)

·      testing a to see if a cuvette with DI water can be set to 100%T or 0 abs. 

Term
how and why is a monochromator spec check done?
Definition

·      Checking a monochromator means making sure that thelight that emits is the same as the wavelength indicated on the control dial

·      Wavelength calibrating filters and solutions are available commercially

·      Holmium oxide and didymium filters

·      Place the filter (or solution) in the cuvette holder. The wavelength knob is adjusted through the region of wavelengths where the greatest abs is expected.

·      If the observed max. abs does not occur at the expected wavelength, then the wavelength dial must be adjusted to reflect the true wavelength passing through the cuvette. 

Term

 

 

How is band pass spec checked?

Definition

·      Using a holmium oxide or didymium filter

·      2 absorption peaks are chosen that differ in wavelength by 1 bandpass. When we scroll thru a range of wavelengths 2 distinct peaks should be apparent on the abs readout.

·      Common cause is warping of exit slit or on the surface of the monochromator

·      Dirt 

Term

What is a detector spec check checking for and what would be the causes?·      Checked for stray light

 


Definition

·      Checked for stray light

·      Stray light is any light that reaches the detector but not part of the light source

·      Misplaced cover, crack in chassis 

Term
How is the detector checked in spec checks?
Definition

·      A colored soln is put in a cuvette and a wavelengh is selected that is completely absorbed by a solution (0%T) if we see anything other that 0%T the light is coming from somewhere else

·      Commercial neutral density filters are used to check linearity response. They have a known absorbance

·      Can use a solution made of different concentrations. Should follow beers law

·      Fatique may cause loss of linearity, when the detector has been exposed to intense light that exhauseted the available electrons on the photosensitive cathode

·      Allow to reest for the period of time to allow the cathode to replenish it’s electrons may restore linearity

·      Replace. 

Term
What is flame photometry used for and how does it work?
Definition

·      Used to measure NA, K+, Mg and Li

·      Good only for elements that are easily excited and do not require very high temperatures

·      A solution containing metal ions is sprayed into a flame. The ions are energized to emit a light of a characteristic color.

 

·      Li = red

·      NA = yellow

·      K+ = violet

·      Mg = blue 

Term
in flame photomery how is concentration measure?
Definition

·      The intensity of the color is proportional to the amount of element in the sample 

Term
In flame photometery what is an atomizer (nebulizer)?
Definition
sprays the sample into fine droplets into the flame. Flame is made by combining sample with air and fuel. 
Term
What are the types of fuel in flame photometry?
Definition

·      Various combos of acetylene, propane, oxygen, natural gas and compressed air

·      The combo and types of fuel used determine the temp of the flame. 

Term
what is the theory behind flame photometry? 
Definition

The sample matrix evaporates followed by atomization of the sample. Atoms present in the high temperature zone of the flame are excited to higher energy levels by absorbing energy form the flame. As excited atoms return to the ground state that emit radiation in definite wavelength depending on the energy level from which each atom drops. 

Term

 

describe monochromators in flame photometry

Definition

·      Flame photometry a pre-selected filter (depending on the atom in question) is used and it is the intensity of the emission line that is practically measured and is related to the original concentration of the sample in solution.

 

Term
What type of photodetector is used in flame photometry
Definition

·      The detector is usually a phototube or a photomultiplier tube depending on the quality of the instrument. 

Term
In summary what is flame photometry?
Definition

·      In short, flame photometry (FP) is an atomic technique which measures the wavelength and intensity of light emitted by atom in a flame resulting from the drop from the excited state (formed due to absorption of energy from the flame) to lower states. No light source is required since the energy imparted to the atoms comes from the flame. Thus. FP is different from atomic absorption spectroscopy (AA) 

Term

 

 

AA vs FPAA

 


Definition

AA

·      Absorption – light absorbed by unexcited atoms in flame

·      Atomization

·      Light source

 

FP

·      Emission- light emitted by ecited atoms in flame

·      Atomization and excitation

·      No light source 

Term
How is fluorometry the same and different compared to a basic spec?
Definition

·      Similar to  a basic spec in its light source, cuvette and detector

·      Differs in two ways:

o   1. Has 2 monochromator

o   2. The detector is at a right angle to avoid measuring transmitted light 

Term
What is fluorometry?
Definition

·      using uv radiation some substances absorb the radiation and become excited

·      after excitation they return to ground state and emit a photon of light

·      fluorescent light is the result of the absorbance of a photon of radiant energy by a molecule.

·      Once the photon is absorbed by the molecule, the mol. Has an increased level of energy. It will eject this energy because the energy of the mol. Is greater than the energy of its environment

·      When this excess energy is ejected as a  photon the result is fluorescence emission

 

Term
what is stokes shift?
Definition

·      Is the difference (in wavelength or frequency units) between positions of the band maxima of the absorption and emission spectra. It is named after Irish physicist George g stokes 

 

*Energy from candy bar - energy you end with*


·     

When a system (be it a molecule or atom) absorbs a photon, it gains energy and enters an excited state. One way for the system to relax is to emit a photon, thus losing its energy. When the emitted photon has less energy than the absorbed photon, this energy difference is the stokes shift.

Term
fluorometer Lamp
Definition

·      Gas discharge (mercury and Xenon)

·      Incandescent lamps can not be used as they release little UV light 

Term

 

 

Fluorometry cuvette

 

Definition

Must be made of quartz because they are transparent to UV wavelengths of light 

Term
Fluorometry primary filter
Definition

·      Placed between lamp and the sample

·      Selects the wavelength that is best absorbed by the solution to be measured. 

Term
fluorometry sample holder (cuvette)
Definition

The fluorescing sample emits light in all directions

Term
fluorometry secondary filter and detector
Definition

·      Placed at right angles to the sample cell

·      The detector and secondary filter are placed at right angles from the sample cell so that the secondary filter can pass longer wavelengths of fluorescent light and prevent incident light (from lamp) from hitting the photodetector 

Term

 

 

Fluorometry filters and monochromators·     

 

Definition

·      Use grating, prisms, or filters for isolation of radiation

Term
Fluorometery photodetectors
Definition

photomultiplier tubes bc their higher sensitivity to low light intensities.

·      The electrical output of the photodetector is proportional to the intensity of fluorescent energy. 

Term


Fluroresence concentration measurements are related to

Definition

·  1.    Molar absorptivity of the compound

·    2.  Intensity of the incident radiation

·      3.quantum efficiency of the energy emitted per quantum absorbed

·    4.  Length of cuvette path 

Term

fluoremetry used to measure...

 

 

 

 

Definition

·      Used to measure bilirubin, quinine 

Term
what is quinine
Definition

·      Quinine is isolated from chinona bark, used against malaria

·      Suppresses abnormalities of heart rhythm

·      Tonic water

·      Can be toxic

 

Term

 

 

Variables that effect results in fluorometry·  

Definition

·      pH has a marked effect on a substance to fluoresce

·      temp- higher temp molecules move more rapidly and collide, this will dissipate energy that would otherwise produce fluorescence

·      increased reduces results, decreased increases results 

Term

 

 

what are the advantages of fluorometry

Definition

·      more sensitive and specific than conventional spectrophotometry

·      greater specificity because secondary monochromator selects only the emitted wavelength of light

·      interferene could only occur if sample has 2 compounds that fluoresce at same wavelength (very, very, rare)

·      1000x more sensitive 

Term
disdvantages of fluorometry 
Definition

sensitive to environmental changes (pH, temperature

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