350 page comprehensive Medical Laboratory Technologist program study guide. Includes easy to read diagrams and point form notes on Clinical Chemistry, Microbiology, Hematology, Transfusion Medicine and Histology units. EVERYTHING YOU NEED TO KNOW TO BECOME A SUCCESSFUL MLT IN ONE PLACE! I created ...
Rodak’s Hematology, 5th Edition, Elaine Keohane, Larry Smith, Jeanine Walenga,
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Medical Laboratory Science
CSMLS
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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
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