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HEMA 1: LEC > AUTOMATION IN HEMATOLOGY > Flashcards

AUTOMATION IN HEMATOLOGY Flashcards

1
Q

Automation started ..

A

Wallace H. Coulter (1913-1998)
Joseph R. Coulter, Jr (1924-1995)

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2
Q

• Used in order to count and size the different blood
One principle that hematology analyzers employ during the analysis of blood cells Automation in Hematology was developed

A

COULTER PRINCIPLE

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3
Q

Why do we employ analyzers in Hematology?

A

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

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4
Q

ADVANTAGES OF AUTOMATION

A
  1. Speed and efficient handling
  2. Greater Accuracy and Precision
  3. Multiple tests on single platform
  4. More efficient workload and management
  5. More timely diagnosis
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5
Q

DISADVANTAGES OF AUTOMATION

A
  1. Flagging
  2. RBC morphed limited
  3. Erroneous regulations
  4. Expensive
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6
Q

TYPES OF HEMATOLOGY ANALYZERS

A
  1. SEMI-AUTOMATED
  2. FULLY AUTOMATED
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7
Q

Measures only few parameters

Some steps like dilution is carried out manually

A

Semi-Automated

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8
Q

Measures multiple parameters
Requires only anticoagulated blood samples

A

Fully automated

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9
Q

BASIC COMPONENTS OF HEMATOLOGY ANALYZER

A

Hydraulics
Pnematics
Electrcals

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10
Q

Hydraulics

A

Mixing Chambers
Aperture baths/Flow cell
Hemoglobinometer
Aspirating unit
Dispensers
Diluters

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11
Q

→ where the sample is introduced
→ the sample will flow through and will be analyzed

A

Hydraulics

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12
Q

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

A

Pneumatics

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13
Q

• Analyzers and computing circuitry

A

Electricals

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14
Q

PRINCIPLES OF AUTOMATED BLOOD ANALYZERS
Electrical Impedance

A

Electrical impedance
Radio frequency
Optical light scatter
fluorescent flow cytometry

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15
Q

Detection and measurement of changes in electrical resistance produced by cells as they traverse a small aperture

A

Electrical Impedance
Makes use of the Coulter Principle

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16
Q

Where the sample IS suspended in_____

*solution is composed of electrically_____

• 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_____ is smaller than the____ aperture

A

Aperture bath

conductive diluent

RBC/platelet

WBC

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17
Q
  • has the aperture where the cells pass through
    → detects/does the analysis of the blood cell that passes through here
A

Aperture tube

Sensing zone

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18
Q

• Our cells are____ conductors of electricity; they_____
• As the cell passes through the_____ in the aperture, it resists/impedes the electrical current and the resistance / electrical current creates a_____

A

poor; impede electrical current/ resist electrical current

sensing zone; voltage pulse

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19
Q

The voltage pulse is equivalent to the property of the cell

The height of the pulse is equivalent to the =

A

volume of the cell

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20
Q

The voltage pulses will be gathered by the

A

Oscilloscope

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21
Q

The pulses will be gathered and sorted out so they will be plotted into the…

A

histogram

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22
Q

X axis - volume of the cell
Y axis - number of the cell

A

Histogram

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23
Q

• The machine is set to identify a particular cell based on a_______

Example: The threshold discrimination for the platelets is between 2-30 fL; the machine is set to recognize particles as platelets when the particles under this range

A

treshold volume

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24
Q

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

A

Hydrodynamic Focusing

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25
Hydrodynamic Focusing Purpose (2)
• Prevents coincident passage of cells • Prevents recirculation of cells back to the sensing zone
26
Different Variables Measured by Electrical Impedance
RBCs WBCs (3 only) platelets
27
is incorporated with another principle. In this example, ______ is incorporated with electrical impedance/direct current
Radiofrequency
28
The radio frequency signal's purpose is to
analyze the Internal complexity of the cell
29
Cell internal structure density:
• Nucleus: cytoplasm ratio • Nuclear density • Cytoplasmic granulation
30
• with radio frequency, the______ of the cell will be analyzed
internal structures
31
Involves light scattering There is a source of light → in the form of______ LAMP
TUNGSTEN HALOGEN LAMP / HALOGEN NEON LASER
32
OPTICAL LIGHT SCATTER can identity
5 parts WBCs
33
LASER →
Light Amplification by Stimulated Emission of Radiation
34
• Light source will emit a light and the light will heat the blood cell that is being analyzed The blood cell will scatter the light in different directions
Light scattering
35
• Light source will emit a light and the light will heat the blood cell that is being analyzed The blood cell will scatter the light in different directions
Light scattering
36
The light scattered by the cell will be captured Absorption, diffraction, refraction, and reflection The scattered light will convert the light signals into an electrical signal The electrical signal will be analyzed by the electrical systems, and will be converted to results
37
There is a single file of cell; because this principle also employs_______ The cells will be analyzed one cell at a time A light will be emitted by the source As the light hits the cell, there will be a corresponding scattering of light _______will be gathered by the detectors and converted to an electrical signal
HYDRODYNAMIC FOCUSING Scattered light
38
Different angles of light scattered detected by detectors
1. Forward angle scatter 2. Side Scatter Light 3. Forward Low Angel Scatter
39
a.0 degree scatter - straight line - Volume of the call
Forward angle scatter
40
Side Scatter Light _____fight scatering - intenal stracteres a. A.k.a…
90° Orthogonal Light Scatter
41
Sidescatter b. Refraction and reflection of light C. Internal structures of the cell
42
Forward Low Angle Scatter/Forward High Angle Scatter
a. → Differential scatter b. Cell volume
43
Variables Measured by the Optical Light Scatter
RBC Count Mean Cell Volume 5-part WBC differential it is 5-part Neutrophils, eosinophils, basophils, lymphocytes, monocytes
44
FLUORESCENT FLOW CYTOMETRY 2 mechanisms employed
Light scattering Fluorescence
45
- 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 • There is a loss of energy, and that loss of energy is in the form of light
Fluorescence
46
Measures multiple cellular and fluorescent properties of cells when they flow as a single cell suspension through a laser beam
FLUORESCENT FLOW CYTOMETRY
47
FLUORESCENT FLOW CYTOMETRY Side Scatter Light - Side Fluorescence Light - Forward Scatter Light -
internal cell structure RNA/DNA information cell volume
48
Components of the Fluorescent Flow Cytometry
#1 - FLUIDICS (THE FLOW SYSTEM) #2 - OPTICS
49
Fluorescent flow cytometry The sample is injected into a stream of sheath fluid within the flow chamber 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
FLUIDICS (THE FLOW SYSTEM)
50
Fluorescent flow cytometry Two types of flow rate High flow rate = Low flow rate =
immunophenotyping analysis DNA analysis
51
Two types of flow rate High flow rate = Low flow rate =
immunophenotyping analysis DNA analysis
52
LASER light is required to excite the cells
Fluorescent Flow Cytometry
53
A system of optical mirrors and filters then direct the specified wavelength of light to the designated photodetectors
Detectors for Fluorescence
54
converted to an electrical signal ELECTRONICS Converts optical signals (photons) to corresponding electronic signals (electronics) Electronic signal produced is _____striking a cell
proportional to the amount of light
55
Fluorescent Flow Cytometry ___is converted to____ signal _____receives the electric current and converts to____ _____assigns the voltage pulse is assigned a digital value representing a channel The channel number is transferred to the computer ______the channel number to the appropriate position in the____
Light electronic Amplifier; voltage pulse Analog-to-Digital Converter (ADC) Computer displays data plot
56
Fluorescent Flow Cytometry ___is converted to____ signal _____receives the electric current and converts to____ _____assigns the voltage pulse is assigned a digital value representing a channel The channel number is transferred to the computer ______the channel number to the appropriate position in the____
Light electronic Amplifier; voltage pulse Analog-to-Digital Converter (ADC) Computer displays
57
DATA ANALYSIS Fluorescent Flow Cytometry
Data is collected and stored in the computer Forward Scatter, Side Scatter, Emitted Fluorescence Data plots
58
Fluorescent flow cytometry Single parameter = Two parameters =
histogram data
59
Fluorescent flow cytometry Boundary that can be set to restrict the analysis to a specific population within the sample Data selected by the ___ is then displayed in subsequent plots
GATING
60
Fluorescent flow cytometry Consists of collecting cells of interests • Defined through criteria of size and fluorescence
SORTING
61
ESTIMATION OF RETICULOCYTE COUNT Reticulocyte counting makes use of______ stains to stain the residual RNA of the reticulocyte
Supravital
62
+ Based upon the uptake of various dyes and fluorochromes by the RNA of reticulocytes
ESTIMATION OF RETICULOCYTE COUNT
63
ESTIMATION OF RETICULOCYTE COUNT FLUORESCENT DYES USED FOR SUPRAVITAL STAINING
Auramine O Thiazole Orange CD4K 530 Oxazine 750 New Methylene Blue
64
Most widely used fluorochrome for [residual RNA]
Thiozole orange
65
Methylene Blue- Fluorescent stain -
manual Automated
66
Methylene Blue- Fluorescent stain -
manual Automated
67
Classification of reticulocytes into 3 maturation stages
a. Low Fluorescence Reticulocytes b. Middle Fluorescence Reticulocytes C. High Fluorescence Reticulocytes
68
Classification of reticulocytes into 3 maturation stages a. Low Fluorescence Reticulocytes i. __________ b. Middle Fluorescence Reticulocytes C.______ High Fluorescence Reticulocytes i. Immature reticulocyte
More mature reticulocyte Middle Fluorescence Reticulocytes
69
= the amount of RNA found in the reticulocyte Classification of reticulocytes into 3 maturation stages
The degree of fluorescence
70
OTHER METHODS Identification and counting of granulocytes Why granulocytes? > Granulocytes contain the enzyme myeloperoxidase • Lymphocytes are not stained They will be ruled out
Peroxidase
71
Reticulocytes and platelets Best for detecting immature platelets
Fluorescence
72
Accurate platelet counting using CD41/CD61 antibodies
Immunological
73
Graphical representation of numerical data of different cell populations in a cell counter
Histogram
74
Histogram Gives information on:
• Average size of cell • Distribution of size
75
identify the type of cell based on its volume
Discriminators separates the distribution curve for the volume Threshold discriminator will be able to
76
Discriminators separates the distribution curve for the volume Threshold discriminator will be WBC Discriminator RBC Discriminator Platelet Discriminator Fixed discriminator
LD = 30-60 fL UD = fixed at 300 fL LD = 25-75 fL UD = 200-250 fL LD = 2-6 fL UD 12-30 fL 12 fL
77
Platelets have volume between____and counted between____
8-12 fL 2-25 fL
78
RBCs have volume____ and are counted between____
80-100 fL 25- 250 fL
79
The normal curve peaks between 80-100 This curve peaks at around peaks around 30-60 There is a shift going to the left, skewed more to the left That means most of the cells in the sample are within this volume - between around 30-100
SHIFT TO THE LEFT
80
If most of the population of this cell falls around this volume [remember the normal curve range] NORMAL
MICROCYTOSIS
81
More than 100 MACROCYTOSIS There is a peak in the volume greater than 100 fL
CURVE SHIFTS TO THE RIGHT
82
TWO PEAKS IN THE CURVE Seen in cases of_____ and seen in______ : TAILS: Thalassemia, anemia of chronic disease, iron deficiency, lead poisoning, sideroblastic anemia
DIMORPHIC POPULATION anisocytosis microcytic hypochromic anemias
83
WBC HISTOGRAM Cells > 35 fL = WBC in the WBC/Hb chamber
84
In the WBC chamber, it involves hemolysis of RBCs RBCs will be hemolyzed so that only WBCs will remain in the solution When RBCs are hemolyzed, hemoglobin will be released
WBC and Hgb chamber
85
WBC HISTOGRAM Lymphocytes = Mononuclears = Monocytes, blast cells, immature granulocytes, reache lymphaugtes Neutrophils =
35-90 fL 90-160 fL 160-450 fL
86
WBC HISTOGRAM FOR IMPEDANCE The plot has more number of cells that have a volume > 100 Around 160 - 200 fL Normal: 160-450 More neutrophils vs. other cells Lymphocytosis
Neutrophilia
87
WBC HISTOGRAM FOR IMPEDANCE WBC-Histogram Peak in the population of cells having a volume of 90-160 fL where the mononuclears are plotted
Monocytosis
88
WBC HISTOGRAM FOR IMPEDANCE WBC-Histogram Eosinophils
Eosinophilia
89
- To ensure readings from an instrument are consistent with other measurements - To determine accuracy of the instrument readings - To establish reliability of the instrument
Calibration
90
-Determines the accuracy and precision of the analyzers → "Tuning" of the instrument
Calibration
91
Calibration is done:
→ Upon installation of machine * Every 6 moths -Periodic after major repair

HEMA 1: LEC (23 decks)

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