Automated Hematology Instrumentation Flashcards
Before running the sample
-CBC specimens must be checked for clots (visually, with applicator
sticks, or by automated flags), significant hemolysis, and lipemia before
reporting results
-Processing either automated or manual, should be done within 8 hours
but never longer than 24 hours after sample collection
-Samples must be thoroughly mixed before testing
Automated Blood Cell-
Counting Instruments
1.) electrical Impedance
◦ Analyzes the resistance created by each cell passing through an electrical
field
◦ Coulter principle – increased resistance occurs when poorly conductive
blood cells pass through aperture.
◦ Ex: Abbott Diagnostics, Beckman Coulter Inc., and Sysmex Corporat
Automated Blood Cell-
Counting Instruments
2.) optical light scatter
◦ Analyzes the scatter of light (forward and side) detected by each cell
passing through a beam of light (optical or laser)
◦ Ex: Siemens Healthcare, Sysmex Corporation, and Abbott Diagnosti
Electrical impendane Instruments
1st aliquot
RBC/plt dilution chamber
* External electrode
* 3 apertures (ea w/ internal electrode)
3 RBC counts are obtained, compared, and evaluated. If agreement, the
reported RBC count is an average of the 3 counts.
If RBCs are larger then normal then
Shift to the right
If RBCs are smaller then normal then
Shift to the left
Histogram
Size distribution curve
PLT size
Less then 20um
RBC size
less then 36 Um
Blood aspirated in
2-4 aliquots
1st aliquot determinations
Data determines:
◦ RBC count
◦ MCV
◦ RDW-CV and RDW-SD
◦ Plt count
◦ MPV
Calculated parameters:
◦ Hct
◦ MCH
◦ MCHC
Gaussian Curve Abnormal Curve
2nd Aliquot determinations
Delivered to what chamber
Lytic agents lyses what and converts what
Delivered to WBC/hemoglobin dilution chamber
◦ Lytic agent lyses RBCs and converts released hgb to cyanmethomoglobin,
and shrinks the leukocyte cell membrane and cytoplasm… allowing WBC
count to represent cell volume rather than cell size
2nd aliquot
WBC count
Hgb count
◦ WBC count measured by electrical impedance from 3 apertures and
reported count represents the average.
◦ Hgb determined by absorbance reading at 525 nm (Beer’s La
Lymphocytes, Monocytes basophils and eosinophils, neutrophils curve
Lymphocytes on left
Mono, eosino, baso in middle
Neutrophils on right
3rd aliquot
steps 1,2, and 3
delivered to the orbital mixing chamber
1. Blood mixed with heated lysing agent to remove RBCs
2. Stabilizing agent added to preserve WBCs
3. Cells sent through the volume-conductivity-scatter (VCS) flow cell by
hydrodynamic focusing for 5-part differenti
3rd aliquot important cell measurements
- Cell volume – by impedance
- Cell conductivity – by electromagnetic probe. Determines physical and chemical components
- Cell’s light scatter characteristics - determines internal contents and cell surface and size
Optical light scatter
-Each cell flows in a single line through a
flow cell
-A laser device is focused
-On striking cells, light is scattered in
different directions
-Sensor captures and multiplies scatter
-Forward angle light scatter (FALS) – cell
size
-Side scatter (SS) – granularity
4th aliquot
Delivered to what
Mixed with what
what solution is added
Delivered to a heated dilution chamber
◦ Mixed with new methylene blue reagent
◦ An acidic, hypotonic solution is added
4th aliquot
An acidic, hypotonic solution is added to
◦ Elutes hemoglobin
◦ Preserves precipitated RNA
◦ Spheres RBCs (eliminates interference due to variance
of shape
4th aliquot
Sent to VCS for what
important cell measurement characteristics 1,2,3
◦ Sent to VCS for analysis to classify mature vs.
immature RBCs
1. Cell volume – by impedance
2. Cell conductivity – by electromagnetic probe.
Determines physical and chemical components
3. Cell’s light scatter characteristics - determines internal
contents and cell surface and size
Indicators that may appear
after the data
@
data is outside the linearity limit
*
Data is doubtful
+ or -
Data is outside the reference limits
—– minus minus minus minus
Data doesn’t appear due to analysis error or abnormal sample
++++
Data exceeds display limit
Hematocrit
MCV/ RBC count
MCH
Hb/ RBC count
MCHC
Hb/ Mct
Automated pt count problems
- Plt clumping - recollect in Heparin
- Satellitosis – warm sample
- RBC microcytes
- Giant plts
What to do when controls are out of limit
out of control
stop testing
identify and correct problems
Repeat testing on patient samples and controls
Do not report results until problem is solved and controls indicate proper performance
Coagulation testing
Today, numerous semi-automated & fully automated instruments to perform coagulation testing
Instruments today are likely to have sophisticated front end capabilities
Coagulation testing
Modern congulation analyzers have
reduced what
Random what
Modern coagulation analyzers have
greatly ↑ performance via improved
accuracy & precision
Reduced rgt & sample volumes required
Random, discrete sampling capabilities
Flagging (sample & instrument)
Electromechanical instruments
Instruments
- Fibromter (BBL Microbiology Systems, Becton Dickinson)
Semi-automated: reagents & samples usually added manually by operator
Principle & Operation - Electromechanical clot detection
Stationary & moving probes
Electromagnetic monitoring
- Electromagnetic monitoring of movement
of a steel ball in plasma
Ball moves because of applied magnetic
force
Clot = ↓ movement sensed by electronic
sensor set to predefined limit
Photo optical systems
*Detection of sample optical
density due to formation of fibrin
*Records decreased light at the
forward 180 ̊ angle
Nephelometry End point Detection
-Modification of photo-optical end point detection
-90 degree or forward angle scatter contributes to measurement
-Ag-Ab form precipitates that scatters incident light
-Nephelometric instruments read loss of intensity of exiting beam as increasing amounts of
impinging light scattered agglutinates form
Thromboelastography
Whole blood clotting assay
Allows real-time comprehensive evaluation of hemostasis
Can analyze fibrinogen, factor activity, platelet function, and fibrinolysis
Quality Control for Coag Testing
specimen
Proper collection (anticoagulant, order of
draw, meds)
Phlebotomy technique
Delays in processing
Improper storage
Centrifugation
Quality Control for Coag Testing
Reagent
Human error
Reagent:
Shipping conditions
Storage conditions
Reconstitution
Contamination
Deterioration
Lot changes
Human error
Choosing wrong test
Analyzing wrong sample
Using improper sample
Using incorrect reagents
The possibilities are staggering
Platelet testing
Automated testing replaced
Measures pt what
detects what
the Bleeding Time
Measures plt function in whole blood using:
- collagen/epinephrine
- collagen/ADP
Detects platelet plug formation
Platelet aggregation studies
Patient’s PRP is warmed to 37°C, stirred, and an
agonist/stimuli is added
◦ ADP
◦ Epinephrine
◦ Collagen
◦ Ristocetin – depends upon vWF and GPIb/IX/X and targets
agglutination (RIPA)
◦ Arachidonic acid
Plt agregation is recorded on a what
Plt aggregation is recorded on a graph
◦ Biphasic curve – analyzing both waves of aggregation ADP and epinephrine
◦ Monophasic curve – only one wave of aggregation Collagen and Ristocetin
Flow cytometry
cytometry=
Flow cytometry=
Multi parameter flow cytometry
-Cytometry = measurement of physical/ chemical characteristics of cells
-Flow cytometry = measurements are made upon cells while suspended
in a fluid stream
◦ Cells must be able to be separated and not cohesive
-Multi-parameter flow cytometry = technology that simultaneously
measures multiple parameters of single cells at a rapid rate
◦ Permits detailed analysis of markers of cellular differentiatio
Specimen processing
Suspension of what
Suspension of individual cells
◦ Often after lysing RBCs
aspirated and injected into flow chamber containing 2 columns of fluid
◦ Cells forced into single file and sent into path of laser beam (hydrodynamic
focusing)
◦ Gradient between sample and sheath fluid keeps fluids separate
◦ Light scatter is measured by photodetectors (both forward and side scatter)
◦ PMT (photomultiplier tube) detects fluorescent molecul
Flourochromes
Fluorochromes – molecules that are excited by light at one wavelength and
emit light at another wavelength
◦ Can be attached to cells via antibodies or DNA
Cytometer components
Fluidics
laser
Electronics
optics- that gather the light
Light detectors- to sense the light
Computer- to output the data into a form that can be used
Optics and electronics
laser light
blue argon laser
3 fluorochromes
additional fluorochromes
Thershold
minimal fluorescence
neccessary
Photodetectors
adjustable for sensitivity
Amplification
logarithmic
Flourescence compensation
statistically predictable & mathematically correctable
Data collection
Side scatter
Granularity or internal complexity
Forward scatter
Cell size
what graph is used for Flow cytometry
two dimensional histogram
White cells and other cells fall into distinct populations
according to their size and granularity
Classic flow cytometry uses
- Classic Flow Cytometry uses very specific antibodies to label cellular antigens
(extra and intracellular). The antibodies are labeled in most cases directly with
fluorescent dye(s) to generate a fluorescent signal. - Can take antibodies against various surface antigens and conjugate each antibody
to a different fluorophore. Can determine what antigens are expressed on the cell
surface and, thereby, their phenotype and function.
Immune panel
Immune panel: Peripheral blood
Evaluate immune competency
Innumerate (% and absolute #) CD3+ T cells, including CD4+ and CD8+
subsets, CD19+ B cells, CD56+ NK cells, CD45RA/RO Naïve and Memory
cells
Oxidative burst
Oxidative Burst
Chronic granulomatous disease due to diminished or absent NADPH
oxidase activity
Leukocyte adhension markers
and what workup
Leukocyte Adhesion Markers
Leukocyte adhesion deficiencies- poor wound healing, delayed umbilical
cord separation
Leukemia/lymphoma workup
Satellitosis.
Platelets surrounding WBCs
Microcytes will cause
A shift to the left
Giant platelets
Some platelets will be counted as RBCs
Platelets clumping will
Will cause instrument to think they are RBC
Red extension on length
Giant platelets
A extension of curve width and smaller in height in plT curve
check samples in heme before
Running test on them
Processing in heme should be done within
8 hours
In hematology we have what to keep the specimen mixed
rockers
Optical light scatter
Analyze scatter of light at a forward and side angle direction
in heme we have what number of aliquots
4 and 2 with four being the most common
difference between the external and internal electrode
Apenture measures the cells that pass through the external and internal electrode. ( current difference).
Histogram
Look at cell size compared to cell number
Width of the curve will give the
RDW
if cell is less then 20 then it is a
plt
if cell is greater then 36
RBC
Broad (large) range of forward scatter is dependent on
size of cell
Side scatter has a
Small range
PCW
Platelets RDW
Platelets aggregate around WBC will cause
An increased size measurement of the WBC or increased vol of WBC
12s rule
West guard rule
The fibrometer was the standard in
Heme for many years
- the two probes worked together to detect the fibrin clot
- there will be tension resistance between the two probes and that resistance will stop the test
Thromboelastography
ROTEM
accesses the tensile strength
rate of formation
maximum firmness and stability
clot lysing/ breakdown
ROTEM formula
measures claw amplitude/ time
ROTEM
R time
reflects fibrin generation at the onset of clot formation
prolonged then coagulation factor deficient
short R time= hypercoagulatable
K time
time it takes for a clot to reach 20 min in amplitude
A angle
Tanget of curve at the K time
decreased A angle then fibrinogen decreased
Represents the speed of clot buildup
MA=
Represents the greastest firmness and stability of clot ( function of plts)
Iy30=
Fibrinolysis
PFA 100
primary hemostatis test
Plaletet plug formation
