Units 10&11 - Instrumentation/Automation Flashcards
The Coulter Principle
Counting particles/cells by electrical impedence
Principle of Coulter electrical impedence counters
- Particles in a conductive fluid with current running through
- Particles are bad conductors
- Particles are sent in single file, they interrupt the current and that increases resistance creating a voltage pulse
- Amount of voltage pulses = cell count
- Amplitude (height) of each pulse = cell size
Early Coulter counters
One type of cell counted at a time
(ex. RBC at 60-120 fL)
Dilutions done manually, either for RBC WBC or PLT
Later Coulter Multiparameter Instruments
Counted different cells in separate counting chambers
Machine made dilutions and directed cells to appropriate channels
Coulter Model S in 1960s
Later multi parameter Coulter instrument
Modern Analyzers using Coulter principles
Lasers - fwd & side scatter
High freq. probe for conductivity
Retic count added
Slide makers added
Stainers added
Types of specimen for heme analyzers
Whole blood - WBC, RBC, PLT
Body fluids
How do cell counts differ in body fluids from whole blood
Body fluids have LOW cell counts
Proportion of WBC and RBC often inverted
RBC cell counts performed in what kind of solution
Isotonic solution (saline diluent)
Dilution very high so only one RBC read at a time
What must be done if high amount of WBC due to leukemia when automated RBC count?
Mathematical correction
WBC cell counts performed how
Diluted in fluid that lyses RBC
Similar to hemacytometer method
Same channel used to measure hgb; WBC diluent contains cyanide reagent for cyanmethemoglobin method
PLT cell counts performed how
In the same channel as RBCs
Instrument separates populations as to size
1fL = xliter
10^-15 L
1fL = xmililiter
10^-12 mL
1fL = xmicroliter
10^-9 mcL
x axis and y axis of histogram for cell counts
X axis: size
Y axis: frequency/count
Usual units for cell volume
femtoliter
Uses of histograms
RBC:
MCV, RDW, Visible interferences like clumped PLTs
WBC:
3 part diff on low end models
PLT:
MPV
Coincidence
Multiple cells passing through aperture at same time
How do coincidences skew values
False increase pulse height
False decrease cell count
How to fix coincidences
Decrease aperture size
Hydrodynamic focusing
Instrument software that edits out bad pulses
Correction tables
Dilute sample
Hydrodynamic focusing
Forcing cells into single file
Coincidences more common in specimen with..
High cell counts
Thresholds
Setting upper and lower size limit of each cell type
How thresholds are set for RBCs
Separate RBCs and PLT
How thresholds are set for WBCs
Granulocytes - Largest
Monos
Lymphs - Smallest
Action levels
Ranges for which counts are acceptable
Below action level
SLide review
Manual count
Above action level
Dilute
Repeat
Multiply by DF
Critical Values
Instrument result acceptable but has serious consequences for patient
Phone doctor immediately
Instrument flags/Prompts
Abnormalities detected
MLS must investigate
If two or more of the triplicate counts are not in agreement range –>
No result reported
3 Part Differential
Histogram of:
Lymphs
Monos
Granulos
Lymphocyte size after diluent applied
35-90fL
Monos size after diluent applied
90-160 fL
Granulos size after diluent applied
160-450 fL
5 Part Differential
Scatter plot - Coulter VCS
Volume
Conductivity
Scatter
Diluent keeps them in near native state
Which differential keeps the cell sizes in near native state
5 part diff
Volume (VCS)
Direct current
Essentially impedence
Cell size in fL
Conductivity (VCS)
High frequency current
Measures opacity based on N/C ratio, nuclear density, granularity
Scatter (VCS)
Laser light scatter
Internal structure
Granularity
Surface Characteristics
Components of multi parameter Coulter like instrument
Specimen loading bay and rocker
Dilutor, tubing, channels to route cells
Counting chambers for WBC RBC Hgb
Power supply
Coulter Reticulocyte Method
In separate channels, RBCs mixed with new methylene blue and diluent
Retics counted and reported as absolute amount
Comparison of retic maturity on scattergram and PBS
Coulter Reticulocyte Diluent
Contains acid to remove hgb, leaving mRNA to stain
Hypotonic to convert all RBC to spherocytes - removes error based on shape
How are measured CBC values run
Triplicates
What are measured CBC values
MCV
WBC
RBC
Hgb
PLT count
What are calculated CBC values
MCHC
MCH
Hct
RDW
MPV
3 or 5 part diff
Percent/abs counts for each WBC type
Which hct is higher? calculated or spun?
Spun because of trapped plasma between RBCs
Errors with Coulter-like instruments
Carryover
False increase in hgb
increased MCV
decreased RBC
How is hgb measured in CBC
Cyanmethemoglobin
Same as manual
When is carryover a problem?
If next specimen has low counts
Why would false increase in hgb occur
Elevated WBC
Elevated lipids
(false inc in absorbance)
Why would increase in MCV and decrease RBC happen
Cold agglutinins
Rouleaux
(clumping)
What would you do to a specimen that has an issue with its plasma?
Replace plasma with saline
What occurs in specimen with too much EDTA
RBC shrinkage
Hct false decrees
MCV and MCHC decreased
When would a specimen have too much EDTA?
Underselling a tube
MCHC calculated formula
(Hgb) * 100 / Hct
MCH calculated formula
Hgb*10 / RBC
Hct Calculated formula
MCV * RBC / 10
RDW formula
CV of RBC size distribution
Sysmex hematology analyzers scatterplot axes
X -Side Scatter
Y -Side Fluorescence
Techniques used by Sysmex
Absorption spectrometry - SLS hgb method
Hydrodynamic focusing = single file (no physical aperture)
Counts cells by direct current (impedance)
Fluorescent flow cytometry (stains DNA)
Radio frequency
Absorption spectrometry - SLS hgb method
Separate channel on instrument for hgb
Diluent lysis RBC
SLS (sodium laurel sulfate) converts Fe++ to Fe+++ for methemoglobin
Methgb combines with SLS –> chromatin absorbs at 555 nm
Run is fast with NO CYANIDE
Hydrodynamic focusing and impedence
Same as Coulter only no physical aperture
