TOPIC 3: AUTOMATION IN HEMATOLOGY Flashcards by Misaki Ayuzawa

_______started with the ingenuity of the Coulters.

Automation

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Used in order to count and size the different blood
cell

COULTER PRINCIPLE

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Why do we employ analyzers in Hematology?

● Cell counting
● Diagnosis of hemoglobinopathies
● Immunophenotyping
● Diagnosis of Leukemias and Lymphomas
● Coagulation abnormalities

CDIDC

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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

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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

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DISADVANTAGES OF AUTOMATION

Flagging of machine
RBC morphology limited
Erroneous results
Expensive

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● Measures only few parameters
● Some steps like dilution is carried out manually
○ Diluted sample will be fed to the machine

SEMI-AUTOMATED

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● 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

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● Vacuums and pressure for operating valves and also
to move the samples among the hydraulics

Pneumatics

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● 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

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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

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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

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has the aperture where the cells pass through

Aperture tube

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➔detects/does the analysis of the
blood cell that passes through here
● Internal And external electrode

Sensing zone

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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

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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

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volume of the cell

X axis

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number of the cell

Y axis

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➔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

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The machine is set to identify a ______________
on a treshold volume

particular cell based

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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

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● Incorporated with another principle
There is application of high voltage electromagnetic current
flowing between electrodes

RADIO FREQUENCY

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● Involves light scattering

OPTICAL LIGHT SCATTER

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➔Light Amplification by Stimulated Emission
of Radiation

LASER

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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

1. Forward angle scatter 2. Side Scatter Light 3. 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

1. Side scatter light 2. Fluorescence light 3. 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