Chapter Eight

Cell Counting Automations

Learning Objective
At the end of this chapter students should be able to:
 Define the instrumentation principle of cell counting
automations
 Explain the optical principles of light scatter in cell counting
 Discuss the measurement principle of hematological
parameters
 Describe methods of quality control in automation
 Discuss the detection principle of fluorescence signal of flow
cytometry
 Explain the interpretation Histograms and Scattergrams of
flow cytometry
Chapter Outline:

1. Automated Hematology analyzers

1.1. Introduction
1.2. Sources of parameters
1.3. principle of automation
 Electrical Resistance
 Optical Detection
 Radio Frequency
 Hydrodynamic focusing
1.4. Interpretation of histogram and scattergram
1.5. Common errors
1.6. Quality Control
Chapter Outline

2. Flowcytometry
2.1. Basic concept
2.2. Essential components
 Illumination
 Fluidics
 Optics
 Detectors
 Interpretation of flow data
Automated Hematology Analyzers
Introduction
Different models of automated hematology analyzers have somewhat
similar principle of cell counting.
Introduction to Automated Hematology,
Cont’d…
 Automated analyzers can perform;
 Counting of WBCs, RBCs and Platelets
 Measurement of Hemglobin
 Calculation of Hematological Indices
 Some can perform Differential counts
 Some can also indicate abnormalities of RBCs,
Platelets and WBCs (Flags)
Analyzers provide an electronic measurement
of several parameters
Sources of parameters

Parameters may be:

 Directly measured using optical scatter (optical counts),
electrical impedance (impedance counts), or Radio
frequency
 Derived from computerized analysis of histograms or
scatterplots/scattergrams.
 Photometric measurements - HGB using a modified
cyanmethemoglobin method.
 Calculated – MCH, MCV and MCHC are computed from
other parameters Hgb, RBC, ...
Principles of Automation
Electrical Resistance / Impedance/Coulter
 The Coulter method of sizing and counting particles is
based on measurement of an electric current resistance
(impulse) produced as a result of nonconductive
particles (cells) suspended in electrolytes (isotonic
saline) passes between two electrodes.
 A small opening (aperture) between electrodes is the
sensing zone that the suspended particles pass. In the
sensing zone each particle displaces volume of
electrolyte. Volume displaced is measured as a voltage
pulse; the size of the pulse being proportional to the
volume of the particle.
Electrical Resistance / Coulter,
Cont’d…
 DC current is applied between the two
electrodes.
 Electrical resistance or impedance
occurs as the cells pass through the
aperture causing a change in voltage.
 Each cell momentarily increases the
electrical resistance between two
electrodes
 The amplitude and size of the pulse
depends on the cell volume
Electrical Resistance / Coulter,
Cont’d…
 The number of pulses is
proportional to the number of
cells counted.
 The size of the voltage pulse
is also directly proportional to
the volume or size of the cell.

 Threshold limits are

established for the
enumeration of each cell
population based on the cell
volume
 Pulses are channelized by
their height or amplitude
Electrical Resistance, Cont’d…
Histogram
 Histograms are
created for the red
blood cell, white blood
cell and platelet
populations based on
cell volume measured
in femtoliters (fl) and
relative number
Histogram, cont’d…
 A display of the distribution of cell volume and frequency
 Each channel on the X axis represents size in fl
 The Y axis represents the relative number
Optical Detection Principle of
Cell Counting
 In the optical or hydrodynamic focusing method of cell
counting and cell sizing, laser light is used
 A diluted blood specimen passes in a steady stream through which
a beam of laser is focused
 As each cell passes through sensing zone of flow cell, it scatters
focused lights
 Scattered light is detected by a photodetector and converted to an
electrical pulse
 Number of pulses generated is directly proportional to the number
of cells passing through the sensing zone in a specific time period
Optical Detection Principle,
Cont’d…
 Measures laser beam of Side scatter
forward & side scatter.
 The intensity of forward Incident Forward
scattered beam measures
size; Laser Beam scatter
 lager cell size will have more
intense forward scatter.
 The intensity of side scattered
beam measures granularity;
 more granular cell will have more Sample
intense side scatter right.
(whole blood)

Sheath fluid
Radiofrequency RF
 Conductivity or radio frequency
measurements provide
information about the internal
characteristics of the cell
 RF resistance is a high voltage
electromagnetic current flowing
between the electrodes to detect
the size of cells based on the
cellular density
 The nuclear to cytoplasmic ratio,
nuclear density, and cytoplasmic
granulation are determined.
Hydrodynamic Focusing
 A process to generate a narrow channel through which
cells separate and align in a single file in front of a
detection system one at a time; eliminates blockage due to
flowing of multiple cells.
 It involves injecting the cell suspension into the center of a
wide, rapidly flowing stream (the sheath stream, or
isotonic fluid)
Interpretation of Hematoanalyzers
Histograms and Scattergrams
Review of histograms and scatterplots can alert the operator to
abnormal cell distribution/scatter patterns.
Conditions That May Interfere or Produce Errors in
Automated Parameters:
WBC: NuclRBC's; presence of unlysed red cells; clumped
platelets; giant platelets
RBC: RBC agglutination; hemolysis
HGB: Gross lipemia or bilirubinemia; high WBC count
MCV: RBC agglutination; hemolysis
PLT: Schistocytes; microcytic red cells; anucleate
cytoplasmic fragments; giant platelets or clumped
platelets (may cause high MPV); hemolysis
Interpretation of Histograms
RBC histogram
 It can be used to determine MCV or dispersion of cells
around the average size (RDW). Microscopic review of
blood smear should be done if abnormal MCV or RDW
values are observed.
Interpretation, Cont’d…
PLT Histogram
 Mean platelet volume (MPV) is usually obtained from the
PLT histogram.
Interpretation, Cont’d…
WBC histogram and Scatterplot
 WBC scatterplot is a graphic display of single cell property
(e.g., cell size) on the Y axis plotted against another
single cell property (e.g., internal cell structures) on the X
axis.
 The density of dots in each cluster represents cell
population and the existence of relatively similar cellular
subpopulation
Interpretation, Cont’d…
WBC histogram and Scatterplot
Common Errors in Automated analyzers :

WBC: NuclRBC's; presence of unlysed red cells;

clumped
platelets; giant platelets
RBC: RBC agglutination; hemolysis
HGB: Gross lipemia or bilirubinemia; high WBC count
MCV: RBC agglutination; hemolysis
PLT: Schistocytes; microcytic red cells; anucleate
cytoplasmic fragments; giant platelets or clumped
platelets (may cause high MPV); hemolysis
Example of Automated Report
Flagging

 Condition Flags
 Normal - Abnormal
 WBC Suspect Flags
 Immature Granulocytes
 Variant Lymphs
 Blasts
 RBC Suspect Flags
 Dimorphic RBC
 Micro
 Fragments
 Anis
Flagging

 Platelet Suspect Flags

 Giant Platelets
 Platelet Clumping
 Small platelets
 Definitive Flags
 Predetermined lab limits
 Neutrophillia
 Monocytosis
 Anemia
Slide Review

 Scan  Manual Diff

 WBC  WBC <4,00>15,00
 Neut >80% W/suspect  Baso >3%
flag/Band
 Mono> 15%
 Lymphs >60% (>6 yrs
age)
 Eos>15%
 RBC
 Variant Lymphs
 MCV <70 > 110
 Suspect Blast
 RDW >20
 All R Flags
 Plt <50,000>600,000
Automation Considerations

 RBC agglutinin:
 dec RBC, HCT, MCH , inc MCV
 Lipemia:
 inc HGB, MCV, MCH, MCHC
 Platelet Clumps:
 inc WBC, dec plt count
 High WBC:
 inc RBC, HGB, HCT, MCH
 NRBC:
 inc WBC
Automation Considerations

 Cryoproteins:
 inc WBC, RBC, HGB, HCT, MCH, MCHC
 dec MCV
 Platelet Satellitosis:
 inc WBC, dec plt count
 Extremely small microcytes:
 inc plt count

 Dec RBC
 Old Specimen
 Inc MCV, MPV,
 Dec WBC, plt
Quality Control

 The process of monitoring the testing system for

accuracy and precision
 Instrumentation function adequately
 Accuracy of unknowns
 Methods:
 Calibration of the instrument
 Controls or assayed material
 Previous patients
 Delta checks and XB analysis
Quality Control

 Three levels of control are run on each shift

 Control material have known values
 Normal
 Low
 High
Delta Check
 Compare patient parameters with previous run
parameters
 If a difference considered an analytical error
 precision and accuracy
 Example
 Hemoglobin and MCV
 Failed Delta within 3 days
FLOW CYTOMETRY
Basic Concept
 A flow cytometer is the process of passing cells (or other
particles) singly in a fluid stream infront of a beam of
light.
 As the cells pass in front of laser beam, photons of light
are emitted and scattered. These beam of light will be
detected and stored in a data file for subsequent
analysis.
 Particles without intrinsically autofluorescence are
stained with fluorescent dyes to make the non-
fluorescent particles “visible” to the cytometer
Flow Cytometery, Cont’d…
Essential Component
Illumination
 The illumination should be bright enough to produce
scattered light or fluorescence emission of detectable
intensity.
 Current research flow cytometers may include two or three
lasers because of the increased requirement of
fluorochromes with different emission spectra
Essential component, Cont’d…
Fluidics
 The fluidics in a cytometer is designed to decrease the
probability that multiple cells will coincide in the analysis
point by the principle of hydrodynamic focusing.
 Hydrodynamic focusing is the strategy of confining cells
in a focused, narrow flow stream (the sheath stream, or
isotonic fluid), where, the cells will remain confined to a
narrow core at the center of the wider stream.
(Refer to hydrodynamic focusing diagram in the hematology section)
Optics
Signals from Cells, SSC and FSC
 Side scatter (SSc) and Forward Scatter (FSc) provide
information about the general physical characteristics
of the cell. , i.e. about the granularity and relative
volume.
Signals from Cells, cont’d…
Fluorescent signals
 Maximum absorbance of both Fluorescein and
Phycoerythrin dye is at 488-nm, and the maximum
emission is at approx 530 nm and 580nm, respectively.
 If a cell has been stained with antibodies of a particular
specificity conjugated to fluorescein and with antibodies
of different specificity conjugated to phycoerythrin, when
the cell passes through a 488-nm laser beam, it will emit
light of 530 nm and 580 nm.
 Detecting fluorescent light is similar to detecting side-
scatter light, but with the addition of wavelength-specific
mirrors and filters – dichroic mirrors.
Signals from Cells, cont’d…
Detectors
 Flow cytometers today can have from 3 to 15 photodetectors
and thus are capable of detecting and recording the intensity
of forward scatter light, side scatter light, and fluorescent light
of 1–13 different colors.
Interpretation of Data
 After the data about a group of cells are stored into a
data file, all the remaining processes of flow cytometry
are computing. Different softwares analyze the data
acquired on instruments; they all allow a display of the
distribution of any one parameter value for the cells in
the data file (in a histogram or dot plot).
 A histogram is used to display the distribution of one
parameter over the cells in the data file.
Interpretation of Data, Cont’d…Histogram

 With the data from a five-parameter flow cytometer, there will be five
numbers describing each cell (e.g., the intensities of forward scatter,
side scatter, intensities of the three fluorescence dyes). It display the
distribution of each of those five parameters (in five separate
histograms), so that it is possible to see the distribution for each
parameter.
Interpretation of Data, Cont’d…
Gating
 One of the unique aspects of flow cytometry is the possibility of
“gating.” Gating is the term used for the designation of cells of
interest within a data file for further analysis. It permits the analysis
of subsets of cells from within a mixed population.
 A mixed population of white blood cells that have been stained with
fluorescent labeled antibodies can be further sub populated. B/c
these mixed population of WBC can be distinguished from each
other by the separate clusters they form in a plot of forward scatter
vs side scatter light, the fluorescence of one cluster can be further
analyzed independently without interferences from other cell
population.
Interpretation of Data, Cont’d..
Gating
Review Questions

1. Defined the principle of electrical impedance in cell

counting
2. Described the use of radio frequency in cell counting
3. Stated the principles of light scatter in cell counting
4. Describe the essential components of flowcytometry
5. Identified sources of error in automated cell counting
6. Discussed methods of quality control in automation
 Next Chapter (nine) is dealing with
general laboratory automation

Most analytical techniques can be fully

automated.
What does fully automated mean?