TOPIC 3: AUTOMATION IN HEMATOLOGY Flashcards
_______started with the ingenuity of the Coulters.
Automation
Used in order to count and size the different blood
cell
COULTER PRINCIPLE
Why do we employ analyzers in Hematology?
● Cell counting
● Diagnosis of hemoglobinopathies
● Immunophenotyping
● Diagnosis of Leukemias and Lymphomas
● Coagulation abnormalities
CDIDC
Complement system
- Anemia caused by a deficiency in _______ and ________
- With the use of hematology analyzing machines,
physicians may be able to directly diagnose based on
the results from a particular laboratory analyzer
Paroxysmal nocturnal hemoglobinuria
CD55 and CD59
ADVANTAGES OF AUTOMATION
- Speed and efficient handling of samples
- Greater Accuracy and Precision
- Multiple tests on single platform
- More efficient workload and management
- More timely diagnosis
DISADVANTAGES OF AUTOMATION
- Flagging of machine
- RBC morphology limited
- Erroneous results
- Expensive
● Measures only few parameters
● Some steps like dilution is carried out manually
○ Diluted sample will be fed to the machine
- SEMI-AUTOMATED
● All of the steps during analysis are performed by the
machine itself
● what MTs do is load the sample on the machine and
press button - analysis will start
● Measures multiple parameters
● Requires only anticoagulated blood samples
- FULLY AUTOMATED
● Vacuums and pressure for operating valves and also
to move the samples among the hydraulics
Pneumatics
● Analyzers and computing circuitry
○ In order to process the data during analysis
● Responsible to do the analysis and then the
calculation, and also the reflection of the sample of
the result generated
● Will be able to control the operational sequences of
the total system
● has an algorithm on how to analyze and process the
data during analysis of the blood samples/ blood cells
Electricals
Makes use of the Coulter Principle
○ Detection and measurement of changes in
electrical resistance produced by cells as
they traverse a small aperture
Electrical Impedance
Where the sample is suspended
*solution is composed of electrically conductive diluent
● There is a flow of electric current within the solution
● 2 chambers filled with a conductive buffered
electrolyte solutions separated by glass tube having
a small aperture
● direct current is generated between the internal and
external electrode
● Aperture for RBC/platelet is smaller than the WBC
aperture
APERTURE BATH
has the aperture where the cells pass through
Aperture tube
➔detects/does the analysis of the
blood cell that passes through here
● Internal And external electrode
Sensing zone
BASIC COMPONENTS OF HEMATOLOGY ANALYZER
Hydraulics
○ Mixing Chambers
○ Aperture baths/Flow cell
■ Where the samples are suspended
for anlaysis
○ Hemoglobinometer
■ Where Hgb is measured
○ Aspirating unit
○ Dispensers
○ Diluters
The height of the pulse is equivalent to the volume of
the cell
● Higher pulse, higher volume
● Number of pulses is equivalent to the number of cells
analyzed in the machine
● The pulses will be gathered and sorted out so they
will be plotted into the histogram
Oscilloscope
volume of the cell
X axis
number of the cell
Y axis
➔Through this analysis, the cells will be able to be counted
and will be discriminated/differentiated
➔Basis of differentiation will be through their CELL VOLUME
THRESHOLD DISCRIMINATOR
The machine is set to identify a ______________
on a treshold volume
particular cell based
Provides a sample stream surrounded by a sheath
fluid as cells pass through the aperture
● Allows the alignment of cells into a single-file
passage through the sensing zone
Hydrodynamic Focusing
● Incorporated with another principle
There is application of high voltage electromagnetic current
flowing between electrodes
RADIO FREQUENCY
● Involves light scattering
OPTICAL LIGHT SCATTER
➔Light Amplification by Stimulated Emission
of Radiation
LASER
There is a source of light → in the form of _______________
TUNGSTEN HALOGEN LAMP / HALOGEN NEON
LASER
The blood cell will scatter the light in
different directions ; hence the name _______________
Light scattering
The light scattered by the cell will be captured
___________, ________, ___________, _____________
● Absorption, diffraction, refraction, and reflection
There is a single file of cell; because this principle also
employs
HYDRODYNAMIC FOCUSING
Different angles of light scattered detected by
detectors
1.a. 0 degree scatter – straight line
b. Diffraction of light
c. Cell volume
2.a. A.k.a Orthogonal Light Scatter
b. Refraction and reflection of light
c. Internal structures of the cell
3.a. → Differential scatter
b. Cell volume
- Forward angle scatter
- Side Scatter Light
- Forward Low Angel Scatter/Forward High Angle
Scatter
_______________ - directly count RBC
● ________________ – direct measurement
o It is able to directly measure the cell
through forward angle scatter
● __________________
o It may be 3-part or 5-part differential; but
with optical light scatter; it is 5-part
RBC Count
Mean Cell Volume
5-part WBC differential
– light emission when electrons are
raised to an excited state; as they go back to their
ground state; they emit a light of a particular
wavelength
Fluorescence
FLUORESCENT FLOW CYTOMETRY
1.
2.
Light scattering
Fluorescence
_______________ - internal cell structure Side
● ____________ - RNA/DNA information
Forward
●_____________- cell volume
- Side scatter light
- Fluorescence light
- Scatter light
o The sample is injected into a stream of
sheath fluid within the flow chamber
o The cells are hydrodynamically focused so
that they will line in a single file; so these
cells will be analyzed one cell at a time
1 - FLUIDICS (THE FLOW SYSTEM)
Two types of flow rate
_____________________= immunophenotyping
analysis
______________________ = DNA analysis
High flow rate
Low flow rate
__________light is required to excite the cells
o Argum/neon helium lamp
Laser
____________ is proportional to the
amount of light striking a cell
ELECTRONICS
___________ is collected and stored in the computer
Data
Data plots
o ____________ = histogram
o ____________ = data plot
Single parameter
Two parameters
Boundary that can be set to restrict the analysis to a
specific population within the sample
Data selected by the gate is then displayed in
subsequent plots
GATING
● Consists of collecting cells of interests
o Defined through criteria of size and
fluorescence
SORTING
Reticulocyte counting makes use of
___________________ to stain the residual RNA
of the reticulocyte
supravital stains
FLUORESCENT DYES USED FOR SUPRAVITAL STAINING
1.
2.
3.
4.
5.
● Auramine O
● Thiazole Orange
o Most widely used fluorochrome for
[residual RNA]
● CD4K 530
● Oxazine 750
● New Methylene Blue
Classification of reticulocytes into 3 maturation stages
1.
2.
3.
a. Low Fluorescence Reticulocytes - more mature
b. Middle Fluorescence Reticulocytes
c. High Fluorescence Reticulocytes - immature
● Identification and counting of granulocytes
Peroxidase
contain the enzyme myeloperoxidase
Granulocytes
● Reticulocytes and platelets
● Best for detecting immature
platelets
Fluorescence
● Accurate platelet counting using
CD41/CD61 antibodies
Immunological
Makes use of Von Behren’s plate
Abbott CELL-DYN
With MAPSS technology, the light that is scattered at
90 degrees will be directed to a __________
polarizer
● Makes use of a unifluidics instead of hydraulics
Siemens ADVIA 2120i
It employs peroxidase staining for identification of
granulocytes, particularly neutrophils
Siemens ADVIA 2120i
RBCs, platelets, neutrophils, lymphocytes,
monocytes, eosinophils will be
disintegrated upon exposure to _____________, and basophils will remain intact
non-ionic surfactant
________, ________, ________make use of Modified
Cyanmethemoglobin which makes use of Drabkin’s
reagent
Beckman, Abott, and Siemen
For _________, it makes use of Sodium lauryl sulfateHGB technique
Sysmex
Graphical representation of numerical data of
different cell populations in a cell counter
● Gives information on:
o Average size of cell
o Distribution of size
HISTOGRAM
● The normal curve peaks between _______
80-100
________________ will be able to identify the
type of cell based on its volume
Threshold discriminator
● Platelets have volume between 8-12 fL and counted
between ________
o Some references say 2-30
● RBCs have volume 80-100 fL and are counted
between __________
● Peak of RBC MCV – ________
2-25
25- 250 fL
80-100 fL
Small cytosis/cells
o Most of the cells analyzed by the machine
have a very small volume- so if we take a
look at this under the microscope, we will
be able to observe cells that are very small
o Cells having a volume of around 50 fL – very
small (normal volume is around 80-100)
● MICROCYTOSIS
There is a peak in the volume greater than
100 fL
MACROCYTOSIS
Significant number of cells having smaller
volume
● DIMORPHIC POPULATION
To ensure readings from an instrument are
consistent with other measurements
- To determine accuracy of the instrument readings
- To establish reliability of the instrument
When the machine is able to give measurements that
are accurate over a long period of time, that indicates
that the machine is reliable – we can trust the results
generated to be___________ and ______________
Accurate and precise
● The values are known
● Done
o Upon installation of machine
o Every _________
o Periodic after major repair
Commercial Calibrators
6 months
Lipemic samples will cause __________________
FALSELY INCREASED MCHC
________________ the sample is milky due to the
high lipid content in the sample of the
patient
o Causes turbidity in the sample
o False increase in the hemoglobin reading
o MCHC will be falsely increased – this is
directly affected by hemoglobin
Lipemia
_________________the cells are rupturing
o Causes false low increase
o Increase in the hematocrit during
hematologic analysis – Hematocrit is a
computed value(not directly measured)
▪ Equal to MCV x RBC count /10
Hemolysis
___________________There will be false increase because
multiple cells are analyzed at the same time
o The cells seemingly become one big cell
● Cold agglutinins, RBCs appear big
