hemolysis: 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  

In vivo hemolysis: Causes 

·         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 

In Vitro hemolysis: Causes

Factors of in vitro hemolysis vary depending upon:

1. patients condition lifestyle

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

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

What are the clinical use of blanks

In vitro hemolysis

·      It generates a consistent trend towards overestimation of:

·      - ALT & AST                   lipase

·      - creatinine                    mg

·      - creatine kinase (CK)     PO4

·      - Iron                            K+

·      - LDH                            urea 

·      - albumin

·      - ALP

·      - chloride and sodium

·      - GGT

·      - Glucose 

·      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. 

What is a icteric sample?

·      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.

How do u creat a reagent blank?

·      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

How is a lamp spec check done?

·      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. 

·      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. 

How is band pass spec checked?

·      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 

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

·      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 

·      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. 

·      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 

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

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

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

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. 

describe monochromators in flame photometry

·      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.

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

·      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) 

AA vs FPAA

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 

·      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 

·      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

·      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.

·      Gas discharge (mercury and Xenon)

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

Fluorometry cuvette

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

·      Placed between lamp and the sample

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

The fluorescing sample emits light in all directions

·      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 

Fluorometry filters and monochromators·     

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

photomultiplier tubes bc their higher sensitivity to low light intensities.

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

Fluroresence concentration measurements are related to

·  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 

fluoremetry used to measure...

·      Used to measure bilirubin, quinine 

·      Quinine is isolated from chinona bark, used against malaria

·      Suppresses abnormalities of heart rhythm

·      Tonic water

·      Can be toxic

Variables that effect results in fluorometry·  

·      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 

what are the advantages of fluorometry

·      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 

sensitive to environmental changes (pH, temperature