AUTOMATED BLOOD CELL ANALYSIS
Introduction
 If there is something wrong with the machine, it
will be double checked for manual testing
 The automated blood cell analysis has virtually
replaced the manual hemoglobin, hematocrit,
and cell counting
 Because of its greater accuracy and
precision
 Release results as soon as possible
 Perform many tests in one run or one
series of testing
 Hematology blood cell analyzer are produced
by multiple manufactures including but not
limited to:
 Abbott laboratories
 HORIBA
 Siemens Healthcare Diagnostic
 Beckman Coulter
ELECTRONIC IMPEDANCE
 Electronic impedance, or low-voltage direct
current (DC) resistance, was developed by
Coulter in the 1950s
 The impedance principle of cell counting is
based on the detection and measurement of
changes in electrical resistance produced by
cells as they traverse a small aperture.
 In the counting chamber, or transducer
assembly, low-frequency electrical current is
applied between an external electrode
(suspended in the cell dilution) and an
internal electrode (housed inside the
aperture tube).
 Electrical resistance between the two
electrodes, or impedance in the current,
occurs as the cells pass through the sensing
aperture, causing voltage pulses that are
measurable
COULTER PRINCIPLE
 Detection and measurement of changes in
electrical resistance produced by cells as
they traverse a small aperture.
 Blood cell suspension it is a solution that is
composed of electrically conductive diluent
 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
(1) The cells that are suspended in an electrically
conductive diluent (saline) and are pulled through
the aperture (orifice with a glass tube)
(2) In the counting chamber or transducer
assembly, the low frequency electrical current is
applied
 Application of the electrical current between
the external electrode and electrode
 External electrode houses at the cell dilution
 Internal electrode houses at the aperture
tube
(3) Electrical resistance between two electrodes or
impedance in the current occurs as the cell passes
through the sensing aperture
(4) This causes voltage pulses that are measurable
 One pulse is equivalent to one cell
OTHER DEVICES
OSCILLOSCOPE
 Screens or displays the pulses that are
generated by the cell as they interrupt the
current
 The number of pulses is proportional to the
number of cells counted
 The height of the pulse is directly
proportional to the volume of the cell
 allows the discrimination and counting of
cells of specific volumes to the use of
threshold circuits
HISTOGRAM
 Pulses are also collected and sorted
according to their amplitude by pulse height
analyzers
 Data are plotted on a frequency distribution
graph or volume distribution histogram
o Y-axis - relative number (no. of the
cells)
o X -axis – channel number equivalent
to the specific volume or femtoliter (fL)
 It depicts the volume distribution of the cells
counted
SACDALAN, MARTY P. 3Y1 - 4
 RADIOFREQUENCY
 Low-voltage DC impedance, may be used in
conjunction with RF resistance, or resistance to
a high voltage electromagnetic current flowing
between both electrodes simultaneously.
 Alternating current resistance - A modification
used in conjunction with DC electronic
impedance
 RFDC detection method
o Shows simultaneous use of direct
current and radio frequency in one
measurement system
o Conductivity – measured by high
frequency is attenuated by nucleus or
cytoplasm ratio, nuclear density, and
cytoplasmic granulation
 Two different cell properties – to create two-
dimensional distribution cytogram or scatter
plot
o Low voltage DC impedance
o RF resistance
 Cell distribution as clusters and
concentration of cell type as number of dots
can be seen
o Clusters such as group of monocyte
or lymphocyte
 Cell internal structure density:
o Nucleus: cytoplasm ratio
o Nuclear density
o Cytoplasmic granulation
REAGENTS AND SUPPLIES
1. Electrolyte
✓ Bufferred isotonic salt solution that may
contain one or more preservatives
✓ Must be particle free
✓ Used to dilute cells and to flush the
instrument
2. Lysing Reagent
✓ A detergent solution
✓ Used to lyse erythrocytes by dissolving
their stroma when leukocyte counts are to
be performed
3. Cleaning agent
✓ Used at regular intervals to remove
protein build up from the aperture tube and
electrodes
4. Sample Container
✓ May range from small glass beakers to
plastic vials manufactured for this purpose.
✓ Must be particle free and their internal
surfaces do not react or attract cells.
FLOW CYTOMETRY
 combines fluid dynamics, optics, laser
science, highspeed computers, and
fluorochrome-conjugated monoclonal
antibodies (MAbs) that rapidly classify
groups of cells in heterogeneous mixtures\
 The principle of flow cytometry is based on
cells being stained in suspension with an
appropriate fluorochrome
 An immunologic reagent, a dye that stains a
specific component, or some other marker
with specific reactivity.
 Fluorescent dyes used in flow cytometry
must bind or react specifically with the
cellular component of interest (e.g.,
reticulocytes, peroxidase enzyme, DNA
content).
PRINCIPLES OF FLOW CYTOMETRY
 A suspension of stained cells is pressurized
using gas and transported through plastic
tubing to a flow chamber within the
instrument
 In the flow chamber, the specimen is
injected through a needle into a stream of
physiologic saline called the sheath
 The sheath and specimen both exit the flow
chamber through a 75-µm orifice.
 This laminar flow design confines the cells
to the center of the saline sheath, with the
cells moving in single file
 The stained cells then pass through the
laser beam
 The laser activates the dye and the cell
fluoresces
 Although the fluorescence is emitted
throughout a 360-degree circle, it is usually
collected by optical sensors located 90
degrees relative to the laser beam.
 The fluorescence information is then
transmitted to a computer, which controls all
decisions regarding data collection, analysis,
and cell sorting.
CELLULAR FEATURES MEASURED BY
FLOW
CYTOMETRY
Cell size or volume
DNA content
Cytoplasmic Granularity
Cell-surface antigens
Intracellular enzymes
RNA content
SACDALAN, MARTY P. 3Y1 - 4
 Emerging Clinical applications of Flow
Cytometry
Detection of small populations of cells
Determination of cell surface phenomena
Evaluation of leukocyte function
Evaluation of intracellular metabolism
Cytogenetics
Semen analysis
Detection of autoantibodies
Measurement of cytotoxicity
Analysis of platelet function

ERRORS IN AUTOMATION

INSTRUMENTAL ERRORS:
 Negative Errors - Excessive Lysis of RBC’s
 Positive Errors
▪ Bubbles caused by shaking
▪ Extraneous electrical pulses from improperly grounded equipment

▪ Aperture plugs
▪ improper setting of aperture current (positive or negative)
CAUSED BY NATURE OF SPECIMEN:
 Giant platelets: counted as RBC or WBC
 Leukocyte cytoplasmic fragment: counted as platelets or RBC’s
 Increased number of fragmented RBC’s: inaccurate RBC and platelet counts
 Agglutination of cells: false decrease
 Agglutinated RBC and platelets: false increase WBC count
 Platelet satellitism: decreased platelet count
 RBC’s that resists lysis: increased WBC count

SACDALAN, MARTY P. 3Y1 - 4

TABLE: Methods for Hemogram, Reticulocyte, Nucleated RBC, and WBC Differential Counts on
Four Major Instrument

Parameter Beckman Coulter UniCel Sysmex XN Series Abbot CELL-DYN Sapphire Siemens ADVIA 2120i
DxH 800

Impedance volume and Fluorescent staining;

WBC conductivity and five-angle forward light scatter Light scatter (primary count), Light scatter and absorption
light scatter measurement and side fluorescent impedance (secondary count)
light detection

RBC Impedance Impedance Impedance Low-angle and high-angle laser light

scatter

Modified
HGB cyanmethemoglobin (525 Sodium lauryl sulfate- Modified cyanmethemoglobin Modified cyanmethemoglobin (546
nm) HGB (555 nm) (540 nm) nm)

HCT (RBC X MCV)/10 Cumulative RBC pulse (RBC X MCV)/10 (RBC X MCV)/10
height detection

MCV Mean of RBC volume (HCT/RBC) x 10 Mean of RBC volume distribution Mean of RBC volume distribution
distribution histogram histogram histogram

MCH (HGB/RBC) x 10

MCHC (HGB/HCT) x 100

Impedance volume and
Platelet conductivity and five-angle
Count
light scatter measurement
Impedance; light
scatter; fluorescent Dual-angle light scatter
analysis; impedance count for Low-angle and high-angle light
staining, forward light scatter; refractive index
scatter, and side verification; optional CD61
fluorescent light monoclonal antibody count
detection

RDW RDW as CV (%) of RBC RDW-SD (fL) or RDW- Relative value; equivalent to CV CV (%) of RBC histogram
histogram or RDW-SD (fL) CV (%)

Supravital staining; Fluorescent staining; low-angle Supravital staining (oxazine 750);

Reticulocyte
impedance volume and
count
conductivity and light scatter
measurement
Impedance volume and
NRBC* conductivity and five-angle
light scatter measurement
Impedance volume and
WBC
conductivity and five-angle
differential
light scatter measurement
Fluorescent staining;
scatter, and fluorescent light low- angle and high-angle light
forward light scatter
detection scatter and absorbance
and side fluorescent
light detection
Red fluorescent dye staining;
Multiangle light scatter measurements
forward light scatter and
in the two WBC differential channels
fluorescent light detection
Fluorescent staining; Multiangle polarized scatter Peroxidase staining, light scatter and
forward and side light separation (MAPSS) and absorption; for basophils, differential
scatter, and side three- color fluorescence lysis, low-angle and high-angle laser
detection
fluorescent light light scatter
detection

SACDALAN, MARTY P. 3Y1 - 4

HISTOGRAM  The importance of this different threshold is
 Graphical representation of numerical data to distinguish the platelets from the small
of different cell populations in a cell counter red blood cells that is located in the upper
 Gives information on: end of the platelet population, and the
o Average size of cell debris at the lower end testing.
o Distribution of size  To distinguish their particular boundaries.
 X axis = volume of cell
 Y axis = number of cells

 Microcytosis
 Shift to the Left
 Presence of microcytosis - smaller RBCs
 Discriminators - separates the distribution  Macrocytosis
curve for the volume
 Shift to the right
 WBC Discriminator
 More than the normal distribution
Lower Discriminator = 30-60 fL  Macrocytosis
Upper Discriminator = fixed at 300 fL  Dimorphic Population
 RBC Discriminator  There is a bimodal peak of the curve.
 Two population of sizes of red blood cells.
Lower Discriminator = 25-75 fL
Upper Discriminator = 200-250 fL
WBC HISTOGRAM
 Platelet Discriminator
 Cells >35 fL - WBC in the WBC/Hb
Lower Discriminator = 2-6 fL chamber
Upper Discriminator 12-30 fL  Lymphocytes = 35-90 Fl
 MID cells = 90-160 fL
Fixed discriminator 12 fL  Refers to monocytes, basophil, and
eosinophils
 Neutrophils = 160-450 fL
RBC HISTOGRAM

 Platelets have volume between 8-12 fL and

counted between 2-25 fL
 The value here in the figure is within the
lower and upper discriminator
 RBCs have volume 80-100 fL and are
counted between 25- 250 fL
 The value here in the figure is within the
lower and upper discriminator
 A platelet size distribution plot is produced
using a threshold.
 One fixed at 12 fL and the other two are
allowed to hunt the upper and lower ends of
the platelet population between certain limits. Abnormal WBC Distribution Curve
 The lower platelet size threshold may move
between 2-6 fL, the higher between 12 - 30  When a particular parameter peaks
fL.
SACDALAN, MARTY P. 3Y1 - 4

 This diagram shows the different types of
leucocytosis.
 Increased neutrophil = neutrophilia
 Increased lymphocyte = lymphocytosis
CALIBRATION
 MID cells is difficult to detect or distinguish
when there is a presence of leukocytosis
 WBC distribution of that particular curve
are all distributed in the same region.
 It cannot be distinguished whether there
is an increase in monocyte, basophil or
eosinophil.
 all three cells could be the reason of the
increase of that particular parameter.
 Lymphocytes (red) – smaller and less
granular
 smaller in have few internal components or
granules than the monocyte (yellow) and
granulocyte (purple).
 Granulocyte (purple)
 most granular population and have the most
side scatter, and appear farthest to the right
of the scatter plot.
 Calibrate every morning before running
tests
 To ensure readings from an instrument are
consistent with other measurements
 To determine accuracy of the Instrument
readings
 To establish reliability of the instrument
 Determines the accuracy and precision of
the analyzers
 “Tuning” of the instrument

WBC Scatterplot

 X axis = side scatter and will detect the

granularity of the cell
 The more it is going to the right, the more
granular
 Y axis = forward scatter that will reflect the
size of the cell.
 Going upward, the larger the size
Interpretation:
TABLE: Conditions that cause interference on most hematology analyzer

SACDALAN, MARTY P. 3Y1 - 4

 Parameters
Condition Rationale Instrument Indicators Corrective Action
Affected

Dual RBC population on RBC

RBC , MCV ,
map (dimorphic population), Warm specimen to 37 degrees
Cold Agglutinins MCHC , Grainy Agglutination of RBCs
or right shift on RBC Celsius and rerun
appearance
histogram (macrocytosis)

 Turbidity affects
spectrophotometric
reading for HGB
HGB x 3 ≠ HCT ± 3, abnormal
Lipemia, icterus HGB , MHC  Plasma replacement
histogram/cytogram
(Increase turbidity
more absorbed light,
increase absorbance
and result)

HGB x 3 ≠ HCT ± 3, may

RBC lysed and not
Hemolysis RBC , HCT  show lipemia pattern on Request new specimen
counted
histogram/cytogram

Lysis-resistant RBCs RBCs with haemoglobin Interference at noise-WBC Perform manual dilutions, allow
with abnormal WBC á, HGB á S, C or F may fail to lyse; interface on histogram/cytogram incubation time for lysis
hemoglobins will be counted as WBCs

Volume of RBCs or RBC

Microcytes or fragments less than Left shift on RBC histogram,
schistocytes RBC â, PLT á lower RBC threshold, MCV flagged if below LIMITS; Review blood film
an/or within PLT abnormal PLT histogram may
threshold be flagged

Nucleated RBCs, Newer instruments eliminate this

megakaryocyte WBC á (older Nucleated RBCs or Nucleated RBC flag resulting error count nucleated RBCs and
fragments or instruments) megakaryocytes counted from interference at noise correct the WBC count
micromegakryoblasts as WBCs lymphocyte interface on count in micromegakaryoblasts per
histogram/cytogram 100 WBCs and correct

Large clumps counted as Platelet clumps/ N flag, Redraw specimen in sodium citrate,
Platelet clumps PLT â, WBC á WBCs and not as interference at noise- multiply result by 1.1
platelets lymphocyte interface on
histogram/ cytogram

á Turbidity affects Manual HCT; perform manual HGB

WBC>100,000/μL HGB á, RBC á, spectrophotometric HGB x 3 ≠ HCT ± 3, WBC count (spin/read supernatant) correct RBC
HCT incorrect reading for HGB, WBCs may be above linearity count, recalculate indices; if above
counted with RBC count linearity, dilute for correct WBC
count
Leukemia, especially WBC â, PLT á Fragile WBC, fragments Platelet count inconsistent with Review film, perform phase platelet
with chemotherapy counted as platelets previous results count or CD61 count

MCV á, MPV, á RBC swell as specimen

PLT â, automated ages, platelets swell and Abnormal clustering on WBC Establish stability and specimen
Old specimen differential may be degenerate, WBCs histogram/cytogram rejection criteria
incorrect affected by prolonged
exposure to EDTA

SACDALAN, MARTY P. 3Y1 - 4