By: Sasha Goldstein
, Light Microscopy
- Objective lenses attached to revolving nosepiece
-Objective lenses functions to provide magnification and resolution
Total magnification= objective x eyepiece (10x)
- Chromatic Abberation→ Failure of lens to focus all colours to same convergence point
- Occurs due to different refractive index to different wavelengths in lenses
- Refractive index INCREASES= wavelength DECREASES
- Condenser collect light from light source- focuses on plane of specimen
- Iris diaphragm adjusts diameter/amount of light beam passing through specimen
➢ Objectives
1) Achromatic Objectives
- Correct for 2 or 3 colours and other lens abberations
2) Parfocal Objectives
- Focal point of objective lenses are in same plane, no need to adjust focus when changing magnification
- Must adjust oculars for parafocality to be achieved
Compound Light Microscopy ( Bright-field)
- Produces image made from light when transmitted through specimen
- Contrast between specimen and background achieved by staining (ie. Gram stain)
- Kohler Illumination acts to generate extremely even illumination of sample
→Principles of Bright-field microscopy
- Resolving power→ Ability of a lense to show two adjacent objects as discrete entities d (distance)= 0.61 x / NA
- Minimum distance two objects must be separated in order to reveal as separate NA= Resolution
- Dependant on wavelength of light used and numerical aperature (characteristic of each lens) = Resolution
- Numerical aperature measures light gathering quality of lens ( NOT intensity)
- Resolving limit of light microscope= 1-10 µm
- Focal depth is thickness of specimen that can be seen when in focus
- magnification= focal depth
- NA= focal depth
- Total magnification up to 1000x with oil immersion (objective magnification x ocular)(100 x 10=1000)
→ Polarizing Microscope
- used for identification of crystals, amyloid stained with congo red
- Used for tissues exhibiting double refraction/ anisotropism/ birefringence
- Transmitting light unequally in different directions
- Uses polarizing device + second polarizing analyzer- causes light to vidrate only in one plane
→Phase- Contrast Microscope
- Examination of unstained specimens - differences in light intensities caused by different refractive indexes
- Allows visualization of transparent objects
→ Darkfeild Microscopy
- Light reflected off the specimen enters objective- Only see scattered or oblique light
- Brighlty lit specimen against dark background
→Fluorescence Microscopy
- Light of one wavelength is absorbed by a substance and emitted as a longer wavelength
- Bombard specimen with UV light and emit at visible light
- Excitation=shorter wavelength= more energy
- Emitted= loger wavelength= less energy
2
,- Uses mercury or halogen lamps
- No condenser needed
Electron Microscopy
- Better resolution (< 0.2)
- Electron gun instead of light source
- 2 types:
1) Transmission
2) Scanning
- Less magnification, greater depth of focus
3
,Quality Control
- Involves systematic monitoring of analytic processes to detect analytic errors - prevent reporting of incorrect patient results
- Quality Assurance→ Important elements of a quality management system ( Documentation, SOPs, external quality assessment)
- Preanalytical, analytical and postanalytical stages of testing
- QC vs Calibration
- QC is used to monitor the status of an analysis - maintain its performance within desired limits
- Calibration is performed using a standard ( known characteristics (concentration, activity - independent of method)) to
establish the correlation at specific points within the instrument’s operating range
Calibration
- Establishes relationship between signal and concentration
- Involves 1 or purchased calibrators (standards) of known concentration
- Signal readings are obtained and statistics for each assay are calibrated and stored - used to calculate concentrations of different
analytes in patient specimens
- Evaluation and adjustment of precision and accuracy- intended to eliminate or reduce bias over a range of continuous values
- Performed when:
- New reagents introduced
- QC is ‘out of control’
- Maintenance
- Manufacturer recommendations
- Primary Standard→ Highly purified chemical that can be measured directly to produce a substance of exact known
concentration and purity
- Stable during long-term storage
- Secondary Standard→ Concentration is determined relative to a primary standard
- Preparation of series of standards, ranges of concentrations using serial dilutions
- Most common method uses External standards→ Known concentrations prepared and analyzed
separately from samples
1) Single Point Calibration
- Measure the signal for a standard containing a known concentration of analyte A C
- Can calculate concentration of unknown samples using a single calibrator value Cu= Au x Cs / As
- Assumes analytical sensitivity is constant over a range of concentrations
- Degree of a response to a change in concentration of analyte
2) Multiple Level Calibration
- Involves at least 3 standards - each containing analye at different concentrations
- Brackets expected range for analyte ( including those above and below)
4
, - Results in production of a calibration curve- standard graph
- Functional relationship between measured and known values of the reference standards ( signal vs concentration)
- General method for determining the concentration of a substance in an unknown sample by comparing the unknown to a
set of standard samples of known concentration
- Linearity→ ability (within a given range) to obtain test results that are directly proportional to the concentration (amount) of
analyte in the sample
- Each method has linearity limit- beyond which specimen must be diluted
- use y=mx+b- to solve for Cu (A=abc; c= A/a(slope) b(1)) (?)
➢ Dilutions
- if specimen has an absorbance reading higher than the absorbance of the highest calibrator (or standard)- instrument is unable
to calculate an accurate result
- by diluting the specimen & re-analyzing it, absorbance will be lower, & analyzer can perform accurate calculations
- Multiply result obtained on analyser by inverse of dilution factor used
Ex. 1:5 dilution is made then multiply result x 5
Quality Control
- Procedures used in each assay to ensure a test run is valid and results are reliable (accurate)
- Assaying stable control materials and comparing determined values with expected values ( within control limits)
- Define allowable analytic error
→ QC Materials
- Chemically and physically similar to unknown specimen- tested in exact same manner
- Solution of known concentration analyzed solely for purpose of monitoring performance of an analytical method - NOT for
calibration (standard)
- Monitors precision of test system
- Same matrix as specimens to be tested
- Concentrations span clinically important range of analyte
- Acceptable Ranges established by specific instrument by running it many times- Establish SD, %CV
- Analyte concentration is dependent on the method of assay
→ External QC
- Controlled by outside source- compares different labs
- Extends beyond laboratory
- Long term monitoring by comparison with peer groups
Ex. Regulatory agencies, QC manufacturers , proficiency testing programs
• QMP-LS
- Quality Management Program – Laboratory Services
- Government external QC program- required and mandatory to maintain licsence
- Send coded test samples to each lab to be analyzed by routine methods and results a re submitted and compared with peer
group
- Lab results which deviate are flagged and require letter of explanation, on site visit and education
→ Internal QC
- Set up and controlled by individual lab to moniter own performance
- Daily monitoring of precision and accuracy of analytical method
- Involve use of patient data
- Delta checks→ Checking current results against previous result for patient and examining extent of change
- LIS/HIS with automated systems
- Check result with peer (split sampling)
Statistics in QC
- Math calculations involved in estimating significance of deviations from test values from correct value
- Quality control programs utilized to increase accuracy and precision of lab results
→Accuracy
- Closeness of a value to the true value
- Compare obtained values with standardized test materials
5
, - Inaccuracy due to systematic error
- Evaluated using % error
% error= experimental value- expected value / expected value (1 decimal place)
- may be + or - , indicates direction of bias
*Note- Cannot have accuracy without precision!
→ Precision
- Reproducibility of test value- how close measurements are to each other
- Expressed as SD or CV
- If the mean of two methods is the same use SD
- SD is tied to a given mean
- If the means of two methods are different, use CV
- Imprecision is due to random analytical error
→Calculated Statistical Values
1) Measurements of Central Tendency
- Mean, Median, Mode
• Mean
- Average, measure of central tendency
- Same decimal places as data given
• Median
- Middle value in ordered body of data - ranked in order of increasing values
- If even number- it is mean of 2 middle numbers
• Mode
- Most common value in group of data
- Often used to describe data that seems to have 2 centers (bimodal)
2) Measure of Dispersion
- Range, variance, SD, CV
- Scatter is observed as excessive variation about the mean- evaluated using SD or CV
- Indicator of random error
- May result from poor technique, insensitive method, etc
•
( x)
Standard Deviation
x − n
2
- How much points vary around mean, measure of precision of data set 2
- One more decimal place than data provided
s=
n −1
• Coefficient of Variation
- Evaluates precision and scatter- used more often than SD
s
- ↑CV=↓ precision (↑scatter) CV = 100%
- One decimal place x
→Trend
- Gradual change over time in test results obtained from control material that suggests a
progressive problem with control material or testing system
- 4-6 consecutive values that either increase or decrease
- Continuous trend upward or downward- indicates gradual deterioration in test system
performance
- Systematic error
- Caused by: reagent deterioration, faulty instrument, loss of reagents constituents
→Shift
- Change in ONE direction
- 4-6 consecutive values lying on one side of the mean
6
, Light Microscopy
- Objective lenses attached to revolving nosepiece
-Objective lenses functions to provide magnification and resolution
Total magnification= objective x eyepiece (10x)
- Chromatic Abberation→ Failure of lens to focus all colours to same convergence point
- Occurs due to different refractive index to different wavelengths in lenses
- Refractive index INCREASES= wavelength DECREASES
- Condenser collect light from light source- focuses on plane of specimen
- Iris diaphragm adjusts diameter/amount of light beam passing through specimen
➢ Objectives
1) Achromatic Objectives
- Correct for 2 or 3 colours and other lens abberations
2) Parfocal Objectives
- Focal point of objective lenses are in same plane, no need to adjust focus when changing magnification
- Must adjust oculars for parafocality to be achieved
Compound Light Microscopy ( Bright-field)
- Produces image made from light when transmitted through specimen
- Contrast between specimen and background achieved by staining (ie. Gram stain)
- Kohler Illumination acts to generate extremely even illumination of sample
→Principles of Bright-field microscopy
- Resolving power→ Ability of a lense to show two adjacent objects as discrete entities d (distance)= 0.61 x / NA
- Minimum distance two objects must be separated in order to reveal as separate NA= Resolution
- Dependant on wavelength of light used and numerical aperature (characteristic of each lens) = Resolution
- Numerical aperature measures light gathering quality of lens ( NOT intensity)
- Resolving limit of light microscope= 1-10 µm
- Focal depth is thickness of specimen that can be seen when in focus
- magnification= focal depth
- NA= focal depth
- Total magnification up to 1000x with oil immersion (objective magnification x ocular)(100 x 10=1000)
→ Polarizing Microscope
- used for identification of crystals, amyloid stained with congo red
- Used for tissues exhibiting double refraction/ anisotropism/ birefringence
- Transmitting light unequally in different directions
- Uses polarizing device + second polarizing analyzer- causes light to vidrate only in one plane
→Phase- Contrast Microscope
- Examination of unstained specimens - differences in light intensities caused by different refractive indexes
- Allows visualization of transparent objects
→ Darkfeild Microscopy
- Light reflected off the specimen enters objective- Only see scattered or oblique light
- Brighlty lit specimen against dark background
→Fluorescence Microscopy
- Light of one wavelength is absorbed by a substance and emitted as a longer wavelength
- Bombard specimen with UV light and emit at visible light
- Excitation=shorter wavelength= more energy
- Emitted= loger wavelength= less energy
2
,- Uses mercury or halogen lamps
- No condenser needed
Electron Microscopy
- Better resolution (< 0.2)
- Electron gun instead of light source
- 2 types:
1) Transmission
2) Scanning
- Less magnification, greater depth of focus
3
,Quality Control
- Involves systematic monitoring of analytic processes to detect analytic errors - prevent reporting of incorrect patient results
- Quality Assurance→ Important elements of a quality management system ( Documentation, SOPs, external quality assessment)
- Preanalytical, analytical and postanalytical stages of testing
- QC vs Calibration
- QC is used to monitor the status of an analysis - maintain its performance within desired limits
- Calibration is performed using a standard ( known characteristics (concentration, activity - independent of method)) to
establish the correlation at specific points within the instrument’s operating range
Calibration
- Establishes relationship between signal and concentration
- Involves 1 or purchased calibrators (standards) of known concentration
- Signal readings are obtained and statistics for each assay are calibrated and stored - used to calculate concentrations of different
analytes in patient specimens
- Evaluation and adjustment of precision and accuracy- intended to eliminate or reduce bias over a range of continuous values
- Performed when:
- New reagents introduced
- QC is ‘out of control’
- Maintenance
- Manufacturer recommendations
- Primary Standard→ Highly purified chemical that can be measured directly to produce a substance of exact known
concentration and purity
- Stable during long-term storage
- Secondary Standard→ Concentration is determined relative to a primary standard
- Preparation of series of standards, ranges of concentrations using serial dilutions
- Most common method uses External standards→ Known concentrations prepared and analyzed
separately from samples
1) Single Point Calibration
- Measure the signal for a standard containing a known concentration of analyte A C
- Can calculate concentration of unknown samples using a single calibrator value Cu= Au x Cs / As
- Assumes analytical sensitivity is constant over a range of concentrations
- Degree of a response to a change in concentration of analyte
2) Multiple Level Calibration
- Involves at least 3 standards - each containing analye at different concentrations
- Brackets expected range for analyte ( including those above and below)
4
, - Results in production of a calibration curve- standard graph
- Functional relationship between measured and known values of the reference standards ( signal vs concentration)
- General method for determining the concentration of a substance in an unknown sample by comparing the unknown to a
set of standard samples of known concentration
- Linearity→ ability (within a given range) to obtain test results that are directly proportional to the concentration (amount) of
analyte in the sample
- Each method has linearity limit- beyond which specimen must be diluted
- use y=mx+b- to solve for Cu (A=abc; c= A/a(slope) b(1)) (?)
➢ Dilutions
- if specimen has an absorbance reading higher than the absorbance of the highest calibrator (or standard)- instrument is unable
to calculate an accurate result
- by diluting the specimen & re-analyzing it, absorbance will be lower, & analyzer can perform accurate calculations
- Multiply result obtained on analyser by inverse of dilution factor used
Ex. 1:5 dilution is made then multiply result x 5
Quality Control
- Procedures used in each assay to ensure a test run is valid and results are reliable (accurate)
- Assaying stable control materials and comparing determined values with expected values ( within control limits)
- Define allowable analytic error
→ QC Materials
- Chemically and physically similar to unknown specimen- tested in exact same manner
- Solution of known concentration analyzed solely for purpose of monitoring performance of an analytical method - NOT for
calibration (standard)
- Monitors precision of test system
- Same matrix as specimens to be tested
- Concentrations span clinically important range of analyte
- Acceptable Ranges established by specific instrument by running it many times- Establish SD, %CV
- Analyte concentration is dependent on the method of assay
→ External QC
- Controlled by outside source- compares different labs
- Extends beyond laboratory
- Long term monitoring by comparison with peer groups
Ex. Regulatory agencies, QC manufacturers , proficiency testing programs
• QMP-LS
- Quality Management Program – Laboratory Services
- Government external QC program- required and mandatory to maintain licsence
- Send coded test samples to each lab to be analyzed by routine methods and results a re submitted and compared with peer
group
- Lab results which deviate are flagged and require letter of explanation, on site visit and education
→ Internal QC
- Set up and controlled by individual lab to moniter own performance
- Daily monitoring of precision and accuracy of analytical method
- Involve use of patient data
- Delta checks→ Checking current results against previous result for patient and examining extent of change
- LIS/HIS with automated systems
- Check result with peer (split sampling)
Statistics in QC
- Math calculations involved in estimating significance of deviations from test values from correct value
- Quality control programs utilized to increase accuracy and precision of lab results
→Accuracy
- Closeness of a value to the true value
- Compare obtained values with standardized test materials
5
, - Inaccuracy due to systematic error
- Evaluated using % error
% error= experimental value- expected value / expected value (1 decimal place)
- may be + or - , indicates direction of bias
*Note- Cannot have accuracy without precision!
→ Precision
- Reproducibility of test value- how close measurements are to each other
- Expressed as SD or CV
- If the mean of two methods is the same use SD
- SD is tied to a given mean
- If the means of two methods are different, use CV
- Imprecision is due to random analytical error
→Calculated Statistical Values
1) Measurements of Central Tendency
- Mean, Median, Mode
• Mean
- Average, measure of central tendency
- Same decimal places as data given
• Median
- Middle value in ordered body of data - ranked in order of increasing values
- If even number- it is mean of 2 middle numbers
• Mode
- Most common value in group of data
- Often used to describe data that seems to have 2 centers (bimodal)
2) Measure of Dispersion
- Range, variance, SD, CV
- Scatter is observed as excessive variation about the mean- evaluated using SD or CV
- Indicator of random error
- May result from poor technique, insensitive method, etc
•
( x)
Standard Deviation
x − n
2
- How much points vary around mean, measure of precision of data set 2
- One more decimal place than data provided
s=
n −1
• Coefficient of Variation
- Evaluates precision and scatter- used more often than SD
s
- ↑CV=↓ precision (↑scatter) CV = 100%
- One decimal place x
→Trend
- Gradual change over time in test results obtained from control material that suggests a
progressive problem with control material or testing system
- 4-6 consecutive values that either increase or decrease
- Continuous trend upward or downward- indicates gradual deterioration in test system
performance
- Systematic error
- Caused by: reagent deterioration, faulty instrument, loss of reagents constituents
→Shift
- Change in ONE direction
- 4-6 consecutive values lying on one side of the mean
6
