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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
Q

Hydrodynamic Focusing

Purpose (2)

A

• Prevents coincident passage of cells

• Prevents recirculation of cells back to the sensing zone

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26
Q

Different Variables Measured by Electrical Impedance

A

RBCs
WBCs (3 only)
platelets

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27
Q

is incorporated with another principle.

In this example, ______ is incorporated with electrical impedance/direct current

A

Radiofrequency

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28
Q

The radio frequency signal’s purpose is to

A

analyze the Internal complexity of the cell

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29
Q

Cell internal structure density:

A

• Nucleus: cytoplasm ratio
• Nuclear density
• Cytoplasmic granulation

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30
Q

• with radio frequency, the______ of the cell will be analyzed

A

internal structures

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31
Q

Involves light scattering

There is a source of light → in the form of______ LAMP

A

TUNGSTEN HALOGEN LAMP /
HALOGEN NEON LASER

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32
Q

OPTICAL LIGHT SCATTER
can identity

A

5 parts WBCs

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33
Q

LASER →

A

Light Amplification by Stimulated
Emission of Radiation

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34
Q

• 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

A

Light scattering

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35
Q

• 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

A

Light scattering

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36
Q

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

A
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37
Q

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

A

HYDRODYNAMIC FOCUSING

Scattered light

38
Q

Different angles of light scattered detected by detectors

A
  1. Forward angle scatter
  2. Side Scatter Light
  3. Forward Low Angel Scatter
39
Q

a.0 degree scatter - straight line -
Volume of the call

A

Forward angle scatter

40
Q

Side Scatter Light
_____fight scatering - intenal stracteres
a. A.k.a…

A

90°

Orthogonal Light Scatter

41
Q

Sidescatter

b. Refraction and reflection of light
C. Internal structures of the cell

A
42
Q

Forward Low Angle Scatter/Forward High Angle Scatter

A

a. → Differential scatter
b. Cell volume

43
Q

Variables Measured by the Optical Light Scatter

A

RBC Count

Mean Cell Volume

5-part WBC differential
it is 5-part Neutrophils, eosinophils, basophils, lymphocytes, monocytes

44
Q

FLUORESCENT FLOW CYTOMETRY

2 mechanisms employed

A

Light scattering

Fluorescence

45
Q
  • 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
A

Fluorescence

46
Q

Measures multiple cellular and fluorescent properties of cells when they flow as a single cell suspension through a laser beam

A

FLUORESCENT FLOW CYTOMETRY

47
Q

FLUORESCENT FLOW CYTOMETRY

Side Scatter Light -

Side Fluorescence Light -

Forward Scatter Light -

A

internal cell structure

RNA/DNA information

cell volume

48
Q

Components of the Fluorescent Flow Cytometry

A

1 - FLUIDICS (THE FLOW SYSTEM)

#2 - OPTICS

49
Q

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

A

FLUIDICS (THE FLOW SYSTEM)

50
Q

Fluorescent flow cytometry

Two types of flow rate
High flow rate =
Low flow rate =

A

immunophenotyping analysis

DNA analysis

51
Q

Two types of flow rate
High flow rate =
Low flow rate =

A

immunophenotyping analysis

DNA analysis

52
Q

LASER light is required to excite the cells

A

Fluorescent Flow Cytometry

53
Q

A system of optical mirrors and filters then direct the specified wavelength of light to the designated photodetectors

A

Detectors for Fluorescence

54
Q

converted to an electrical signal

ELECTRONICS

Converts optical signals (photons) to corresponding electronic signals (electronics)
Electronic signal produced is _____striking a cell

A

proportional to the amount of light

55
Q

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____

A

Light electronic

Amplifier; voltage pulse

Analog-to-Digital Converter (ADC)

Computer displays

data plot

56
Q

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____

A

Light electronic

Amplifier; voltage pulse

Analog-to-Digital Converter (ADC)

Computer displays

57
Q

DATA ANALYSIS

Fluorescent Flow Cytometry

A

Data is collected and stored in the
computer

Forward Scatter, Side Scatter, Emitted Fluorescence

Data plots

58
Q

Fluorescent flow cytometry

Single parameter =
Two parameters =

A

histogram

data

59
Q

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

A

GATING

60
Q

Fluorescent flow cytometry

Consists of collecting cells of interests
• Defined through criteria of size and fluorescence

A

SORTING

61
Q

ESTIMATION OF RETICULOCYTE COUNT

Reticulocyte counting makes use of______ stains to stain the residual
RNA of the reticulocyte

A

Supravital

62
Q

+ Based upon the uptake of various dyes and fluorochromes by the RNA of reticulocytes

A

ESTIMATION OF RETICULOCYTE COUNT

63
Q

ESTIMATION OF RETICULOCYTE COUNT

FLUORESCENT DYES USED FOR SUPRAVITAL STAINING

A

Auramine O
Thiazole Orange
CD4K 530
Oxazine 750
New Methylene Blue

64
Q

Most widely used fluorochrome for [residual RNA]

A

Thiozole orange

65
Q

Methylene Blue-
Fluorescent stain -

A

manual

Automated

66
Q

Methylene Blue-
Fluorescent stain -

A

manual

Automated

67
Q

Classification of reticulocytes into 3 maturation stages

A

a. Low Fluorescence Reticulocytes
b. Middle Fluorescence Reticulocytes
C. High Fluorescence Reticulocytes

68
Q

Classification of reticulocytes into 3 maturation stages
a. Low Fluorescence Reticulocytes i.
__________
b. Middle Fluorescence Reticulocytes
C.______
High Fluorescence Reticulocytes i. Immature reticulocyte

A

More mature reticulocyte

Middle Fluorescence Reticulocytes

69
Q

= the amount of RNA found in
the reticulocyte

Classification of reticulocytes into 3 maturation stages

A

The degree of fluorescence

70
Q

OTHER METHODS

Identification and counting of granulocytes
Why granulocytes?
> Granulocytes contain the enzyme myeloperoxidase
• Lymphocytes are not stained
They will be ruled out

A

Peroxidase

71
Q

Reticulocytes and platelets

Best for detecting immature platelets

A

Fluorescence

72
Q

Accurate platelet counting using CD41/CD61 antibodies

A

Immunological

73
Q

Graphical representation of numerical data of different cell populations in a cell counter

A

Histogram

74
Q

Histogram

Gives information on:

A

• Average size of cell
• Distribution of size

75
Q

identify the type of cell based on its volume

A

Discriminators separates the distribution curve for the volume
Threshold discriminator will be able to

76
Q

Discriminators separates the distribution curve for the volume
Threshold discriminator will be

WBC Discriminator

RBC Discriminator

Platelet Discriminator

Fixed discriminator

A

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
Q

Platelets have volume between____and counted between____

A

8-12 fL

2-25 fL

78
Q

RBCs have volume____ and are counted between____

A

80-100 fL

25- 250 fL

79
Q

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

A

SHIFT TO THE LEFT

80
Q

If most of the population of this cell falls around this volume [remember the normal curve range]

NORMAL

A

MICROCYTOSIS

81
Q

More than 100
MACROCYTOSIS
There is a peak in the volume greater than 100 fL

A

CURVE SHIFTS TO THE RIGHT

82
Q

TWO PEAKS IN THE CURVE

Seen in cases of_____ and seen in______ :

TAILS: Thalassemia, anemia of chronic disease, iron deficiency, lead poisoning, sideroblastic anemia

A

DIMORPHIC POPULATION

anisocytosis

microcytic hypochromic anemias

83
Q

WBC HISTOGRAM

Cells > 35 fL = WBC in the WBC/Hb chamber

A
84
Q

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

A

WBC and Hgb chamber

85
Q

WBC HISTOGRAM

Lymphocytes =
Mononuclears =
Monocytes, blast cells, immature granulocytes, reache lymphaugtes
Neutrophils =

A

35-90 fL

90-160 fL

160-450 fL

86
Q

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

A

Neutrophilia

87
Q

WBC HISTOGRAM FOR IMPEDANCE

WBC-Histogram

Peak in the population of cells having a volume of 90-160 fL where the mononuclears are plotted

A

Monocytosis

88
Q

WBC HISTOGRAM FOR IMPEDANCE

WBC-Histogram

Eosinophils

A

Eosinophilia

89
Q
  • To ensure readings from an instrument are consistent with other measurements
  • To determine accuracy of the instrument readings
  • To establish reliability of the instrument
A

Calibration

90
Q

-Determines the accuracy and precision of the analyzers
→ “Tuning” of the instrument

A

Calibration

91
Q

Calibration is done:

A

→ Upon installation of machine
* Every 6 moths
-Periodic after major repair

HEMA 1: LEC (23 decks)

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