EXERCISE 7: Automated Differential
MLAB 1315 Hematology
AUTOMATED DIFFERENTIAL
LAB OBJECTIVE
Dry lab
1.
The student will interpret 5 differentials from the Coulter STKS, the Bayer Advia and the
Abbott Cell-Dyne by examining the instrument printout of the CBC as well as the
histograms and scattergrams.
2.
The student will state the theory of the technology used to perform the automated
differential from 4 different instruments.
Wet lab
The student will examine the scatterplots of CBCs performed at CPL and interpret the
results stating whether the automated differentials are normal or abnormal.
PRINCIPLE
Coulter STKS (currently in use as a primary instrument at CPL and as a backup instrument at
Seton Medical Center.)
The Coulter STKS performs a five-part differential consisting of monocyte, lymphocyte,
granulocyte, eosinophil and basophil. The Coulter VCS established WBC differential
technology uses three measurements: individual cell volume, high-frequency conductivity and
laser-light scatter as explained below:
Volume Analysis -
Conductivity -
Scatter -
LAB EXERCISE #7
Electronic leukocyte volume analysis uses a low-frequency current
measure volume of the WBCs as they impede the current when they pass
through the aperture.
Cell walls act as conductors to high-frequency current. As the current
passes through he walls and through each cell interior, it detects
differences in the insulating properties of cell components. It
characterizes the nuclear and granular constituents and the chemical
composition of the cell interior.
Leukocytes are hydrodynamically focused and passed in a steady stream
through a sensing zone on which a laser light is focused. As each cell
passes through the sensing zone of the flow cell, it scatters and reflects the
focused light which is detected by a photodetector. The patterns of scatter
are measure at various angles (forward scatter at 180 and right angle
scatter at 90). Scattered light provides information about cell structure,
shape and reflectivity. The characteristics are used to differentiate the
various types of WBCs and to produce scatterplots with a five-part
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EXERCISE 7: Automated Differential
MLAB 1315 Hematology
differential.
Bayer Advia (currently at Seton Medical Center and Brackenridge)
The Bayer Advia utilizes 3 technologies to enumerate and identify cells: optical flow cytometry,
cytochemistry and light scattering. WBCs are fixed with formaldehyde and stained with
peroxidase in the peroxidase reaction chamber. The high heat in the chamber lyses platelets and
RBCs and causes the WBCs to be fixed and dehydrated. Forward-angle light scatter and
tungsten light optics are used to measure WBC size and peroxidase activity. Myeloperoxidase is
a granulocyte enzyme marker that is present in varying degrees in neutrophils, eosinophils and
monocytes but is absent from basophils, lymphocytes and blasts. A specific basophil count is
determined separately in the basophil/lobularity chamber. Whole blood is exposed to an acid
buffer that selectively lyses all cells except basophils. The resulting particles are sorted and
quantified by measuring the forward angle and light scattering properties. An additional
category of cells is reported by the Advia as LUC (large unstained cells). This category reflects
atypical lymphocytes or blasts.
Sysmex TOA
The Sysmex TOA uses radio frequency (RF) and direct current (DC) to enumerate and identify
WBC populations. The RF method detects and sizes lyse-treated cells based on density and
nuclear size, whereas the DC method sizes the entire cell, nucleus, and cytoplasm. Cells pulses
detected by RF and DC methods are then displayed as a 3-dimensional WBC scattergram which
contains information about the distribution of lymphs, monos and granulocytes. Separate
histograms are generated for basophil and eosinophil populations.
LAB EXERCISE #7
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MLAB 1315
EXERCISE 7: Automated Differential
MLAB 1315 Hematology
Abbott Cell-Dyne (currently at Seton Northwest)
This system uses multiangle polarized scatter separation (MAPSS) flow cytometry with
hydrodynamic focusing of the cell stream. The leukocyte differential is accomplished by light
scatter. It features three independent measurements and focused flow impedance.
Multidimensional light scatter and fluorescent detection are used as well.
SPECIMEN
EDTA-anticoagulated blood
QUALITY CONTROL
Commercial low, normal and high controls
Monitor the CBC and differential parameters. Latron controls monitor the performance of the
volume, conductivity and light scatter for the automated differential. Control values are stored in
the instrument computer and can be monitored with the generation of a Levey-Jennings graph for
each parameter.
REAGENTS, SUPPLIES AND EQUIPMENT
As indicated for specific instrument
INTERPRETATION OF RESULTS
LEUKOCYTE HISTOGRAM ANALYSIS
Size-referenced leukocyte histograms display the classification of leukocytes according to size
following lysis. It does not display the native cell size. The lytic agent lyses the cell and the
cytoplasm collapses around the nucleus, producing differential shrinkage. The histogram
subpopulations reflect the sorting of the cells by their relative size, which is primarily their
nuclear size. As each leukocyte passes through the aperture, it causes a change in electrical
resistance which is proportional to its volume. The histogram is a representation of the sizing of
the leukocytes. The differentiation is as follows:
Cell type
Size range
Cells that fall within this size range
Lymphocytes
35-90 fL
lymphs and atypical lymphs
Mononuclear
90-160 fL
Monos, promyelocytes, myelocytes, plasma cells and blasts
Granulocyte
160-450 fL
segs, bands, metas, eos and basos
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EXERCISE 7: Automated Differential
MLAB 1315 Hematology
Leukocyte Region Codes
A valley or depression should be seen between each population. Abnormal sized cells or
abnormal particles can cause abnormal patterns and the instrument will print an alert specific for
the region where the abnormal pattern exists. The following table lists the region (R) flags and
the abnormalities they may represent:
R Flag
Region
Abnormality
R1
Far left
Erythrocyte precursors (NRBCs)
Cryoglobulins
Nonlysed erythrocytes
Giant and/or clumped platelets
R2
Between lymphs and
monos
Blasts
Basophilia
Eosinophilia
Plasma cells
Abnormal/variant lymphs
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MLAB 1315
EXERCISE 7: Automated Differential
R3
Between mons and
granulocytes
Abnormal cell populations
Eosinophilia
Immature granulocytes
R4
Far right
Increased absolute granulocytes
RM
LAB EXERCISE #7
MLAB 1315 Hematology
Multiple flags
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EXERCISE 7: Automated Differential
MLAB 1315 Hematology
SCATTERPLOT ANALYSIS
Coulter Model STKS and MAXM
V
O
L
U
M
E
N
EU
TR
O
MONOCYTE
EO
SI
N
PH
IL
O
PH
I
L
BASOPHIL
LYMPHOCYTE
NRBC, PLT CLUMPS, MALARIA,
DEBRIS
DF 1
On a scatterplot, individual cells are depicted as points reflecting cell volume and light-scattering
characteristics. Clusters of cells are identified as monocytes, eosinophils, lymphocytes and
neutrophils based on their relative position on the scatterplot. Separate scatterplots comparing
cell volume and conductivity properties are generated to quantify basophils.
Floating discriminators examine areas between different cell populations. Abnormalities are
identified by specific region flags. The STKS flagging system is enhanced by providing specific
alphanumeric codes and three types of messages including suspect, definitive and condition
messages. Suspect messages flag abnormal cell populations or distributions such as platelet
clumping, variant lymphocytes and immature granulocytes. These messages appear in the cell
classification window on the sample analysis display and abnormalities should be confirmed by a
microscopic review. On the DMS screen, you can see the results two dimensions at a time and
rotate the screen to view the cells in three different ways. The density of the cells is represented
by colors with yellow being the highest density followed by red, green and blue.
Light Scatter DF 1
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MLAB 1315
EXERCISE 7: Automated Differential
MLAB 1315 Hematology
Bayer
ADVIA
Leukocyte Differential cell identification by peroxidase content and cell
size. (Technicon)
The Advia uses cytochemical as well as light scatter to differentiate WBCs. It provides two
scattergrams - one for peroxidase and one for basophil/lobularity. The WBCs are lysed, fixed
and stained with a peroxidase reagent. A dark precipitate forms in the primary granules of
leukocytes containing peroxidase when a chromogen is added with hydrogen peroxide as the
substrate. Eosinophils and neutrophils are strongly positive and monocytes are weakly positive.
Peroxidase is not present in basophils, lymphocytes, blasts or large unstained cells (LUC) The
clusters of dots on the scattergram are defined and analyzed, the cells are counted and classified
LAB EXERCISE #7
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EXERCISE 7: Automated Differential
MLAB 1315 Hematology
based on information stored in the computer. This information is used to generate the total WBC
and differential except for basophils.
The nuclear channel is used to measure the conformation of the nucleus of WBCs. When
exposed to a surfactant at a low pH, the membranes and cytoplasm disintegrate and only the
nucleus is left. The nuclear channel cytometer distinguishes leukocytes by differences in nuclear
shape and counts basophils. A fixed horizontal threshold separates basophils from the nuclei of
other leukocytes.
An immunoperoxidase reaction is employed for lymphocyte subtyping. Two monoclonal
antibodies are added to the whole blood followed by a peroxidase reagent. Lymphocytes which
have been labeled by the immunoperoxidase reaction, appear between the unlabeled lymphocyte
population and the monocytes. Cells with endogenous peroxidase such as neutrophils stain
intensely and appear far to the right.
Abbot Cell-Dyne
The five-part differential is performed using multi-angle polarized scatter separation technology
(MAPPS). Sheath fluid surrounds the sample stream for laminar flow and hydrodynamic
focusing of the cells into single file for passage through the optically clear quartz flow cell where
the vertically polarized helium neo laser beam is interrupted by passage of each individual cell.
The light scattered by cell passage through the laser beam is collected by sensors at 4 different
angles. The four measurements from each cell are stored for step by step analysis and cell
identification for the five-part differential.
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MLAB 1315
EXERCISE 7: Automated Differential
MLAB 1315 Hematology
AUTOMATED DIFFERENTIAL STUDY QUESTIONS
Name___________________________________
Date____________________________________
1.
Draw and label R regions on a WBC histogram. (10 pt)
2.
List the abnormalities that cause a flag in each region (10 pt)
3.
List the cells that are located in each region of the following WBC histogram. (10 pts).
LAB EXERCISE #7
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EXERCISE 7: Automated Differential
4.
MLAB 1315 Hematology
Locate each WBC region on the Coulter STKS and Bayer Scatterplots
Light Scatter DF 1
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LAB EXERCISE
#7 Differential cell identification
Leukocyte
by peroxidase
content and cell size. (Technicon)
MLAB 1315
EXERCISE 7: Automated Differential
5.
MLAB 1315 Hematology
Read the article attached and answer the questions at the end.
LAB EXERCISE #7
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