You are on page 1of 26

General Chemistry

Immunodiagnostics

A V Differential Technology
and Case Studies.
C

Centrifugation
Disease Management
Hematology
Hemostasis
Lab Automation
Data Management
Flow Cytometry
Primary Care

Bulletin 9151

Beckman Coulter ACV Differential Technology

TABLE OF CONTENTS

DIFFERENTIAL TECHNOLOGY . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . .
Differential Methodology: Sample Preparation . . . .
Differential Methodology: Sample Flow and Technology
Flow Cell . . . . . . . . . . . . . . . . . . . .
Dual Focused Flow . . . . . . . . . . . . . . .
Focused Flow Impedance . . . . . . . . . . . .
Differential Methodology: Parameter Derivation . . .
Signal Processing . . . . . . . . . . . . . . . .
DiffPlot Characteristics . . . . . . . . . . . . . .
Lymphocytes . . . . . . . . . . . . . . . . . .
Monocytes . . . . . . . . . . . . . . . . . . .
Neutrophils . . . . . . . . . . . . . . . . . . .
Eosinphils . . . . . . . . . . . . . . . . . . .
WBC/BASO Methodology . . . . . . . . . . . . .
DIFFERENTIAL REAGENT SYSTEM OVERVIEW

. . . .
. . . .
. . . .
. . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

1
3
3
3
3
3
4
5
5
6
6
6
6
6
7

. . . . . . . . . . . .

DIFFERENTIAL ANALYTICAL/MECHANICAL SYSTEM OVERVIEW


Sequential Dilution System . . . . . . . . . . . . . . . .
DIFF Bath . . . . . . . . . . . . . . . . . . . . . . . .
Optical Bath . . . . . . . . . . . . . . . . . . . . . . .
WBC/BASO Bath . . . . . . . . . . . . . . . . . . . . .
Differential Calculation . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.

.
.
.
.
.
.

.
.
.
.
.
.

.
.
.
.
.
.

8
8
9
9
10
10

DIFFERENTIAL FLAGGING OVERVIEW


Immature Granulocytes . . . .
Band Cells . . . . . . . . . .
Blasts . . . . . . . . . . . . .
DiffPlot Flags . . . . . . . . .

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

11
11
11
11
12

CASE STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

.
.
.
.

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

TABLE

Table 1. Differential Measurement Technologies

FIGURES

Figure 1. Flow Cell . . . . . . . . . . . . . . . . . .


Figure 2. Signal Processing . . . . . . . . . . . . . .
Figure 3. Correlation of Signals to DiffPlot Populations .
Figure 4. WBC/BASO Histogram . . . . . . . . . . .
Figure 5. Sequential Dilution System . . . . . . . . . .
Figure 6. Diff Sample Flow . . . . . . . . . . . . . .
Figure 7. Optical Bench . . . . . . . . . . . . . . . .
Figure 8. WBC/BASO Bath Assembly . . . . . . . . .
Figure 9. DiffPlot Normal and Abnormal Cell Populations
Figure 10. DiffPlot Flags . . . . . . . . . . . . . . .
Figure 11. DiffPlot Flags . . . . . . . . . . . . . . .
Figure 12. DiffPlot Flags . . . . . . . . . . . . . . .

.
.
.
.
.

.
.
.
.
.

.
.
.
.
.

. . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.

3
4
5
6
7
9
9
10
10
11
12
13
14

Beckman Coulter ACV Differential Technology

INTRODUCTION

The ACT 5diff instrument is a fully automated hematology analyzer providing


a 5-part white cell differential. The 5-part white cell differential is determined
simultaneously by the differential (DIFF) and white cell/basophil (WBC/BASO)
methodologies. The differential methodology utilizes the technologies and
principles of absorbance cytochemistry and focused flow impedance.
The WBC/BASO methodology utilizes differential lysis, Coulter Principle
(impedance) and differential thresholds to provide a total white cell count
and basophil percentage. These two methodologies, listed in Table 1,
provide a robust, low cost and efficient 5-part white cell differential with
interpretive flagging for abnormal samples.
Table 1. Differential Measurement Technologies
Fluid
Dynamics
Dual Focused
Flow Aperture

Volume Aperture

Differential Methodology:
Sample Preparation

Technology

Measurement

Absorbance
cytochemistry and
Volume (ACV
Technology)

Light Absorbance
of Cytochemically
stained cells

Differential Lysis
with Coulter
Principle

Volume vs. Count

Volume vs. Count

Differential
Output
Lymphocyte
Monocyte
Neutrophil
Eosinophil
Basophil
White Cell Count

Whole blood is mixed in the DIFF bath with ACT 5diff FIX reagent incubated
and then stabilized with diluent. This reaction lyses the red cells and stains
the primary granules of monocytes, neutrophils and specific granules of
eosinophils at different intensities. This reaction also preserves the
leukocytes in their native size. The lymphocytes, monocytes, neutrophils
and eosinophils each have unique nuclear and morphologic structure,
and hence absorb light differently. The differential staining and unique
cellular structure provide unique light absorbance characteristics. The
absorbance cytochemistry method is based on the well-known reference
methods of Sheenan et al* and Kass.**

*Sheenan, H.L., Storey, G.W. (1947) An improved method of staining leukocyte


granules with Sudan Black B. Journal of Pathology and Bacteriology, 59, 336.
**Kass L. (1981) Staining of granulocyte cells by Chlorazol Black E. American
Journal of Clinical Pathology, 76, 810-812.

Beckman Coulter ACV Differential Technology

Differential Methodology:
Sample Flow and Technology
Flow Cell

In the flow cell, sequential analyses for cell volume by the Coulter Principle
and light absorbance are performed. The flow cell incorporates a 60 m
aperture for cellular volume analysis and an optical window of 42 m for
light absorbance.

Dual Focused Flow

Dual Focused Flow (DFF) technology is a process by which individual cells


or particles are focused in a stream of diluent (hydrodynamic focusing). Cells
are aligned and pass through the flow cell in a singular fashion.
Dual focused flow uses the sheath fluid to surround and force cells
suspended in diluent to pass in a single file through the center of the flow
cell. The first sheath flow focuses the sample through the impedance
aperture. The second sheath flow then focuses the cells as they exit the
aperture onto the optical window. The use of hydrodynamic focusing in the
ACT 5diff instrument flow cell enables accurate and rapid cell-by-cell
measurements on a large number of individual cells.
Dual Focused Flow delivers sample, cell by cell, for the analysis of cell
volume and cellular content in one flow cell. The detection of an impedance
pulse and a light absorbance pulse from a single cell as it passes through
the flow cell within 200 microseconds, prevents the counting of artifacts
such as bubbles or static.

Light
Absorbance
Impedance

Figure 1. Flow Cell

Focused Flow Impedance

Focused flow impedance, using the Coulter Principle, is the technology that
provides cell by cell volume information for each cell as it passes through
the aperture. Each time a cell passes through the aperture a change in
electrical resistance is measured. The change in resistance is proportional
to the volume of the cell.
The ACT 5diff analyzer utilizes dual focused flow in conjunction with
cytochemical staining, light absorbance and Coulter Principle to provide
cellular information for the complete WBC differential.

Differential Methodology:
Parameter Derivation

Each stained cell as it passes through the flow cell is exposed to the light
source and a measurement is taken. As the cell passes through the optical
window, light is scattered in all directions, but only the forward light scatter
is detected by the sensors. The system measures the amount of light lost
due to diffraction and absorbance as compared to full transmission when no
cell is present.
The signals collected are converted into voltage pulses and are processed.
The size and shape of the voltage pulses are equivalent to the unique
nuclear and morphologic structure of the cells being analyzed. The light
absorbance analyzes each cell for cellular content (granularity, nuclear
content) after cytochemical staining. These simultaneous measurements
provide the information for the white cell sub populations, lymphocytes,
monocytes, neutrophils, eosinophils.

Signal Processing

The signals from the impedance aperture and from the absorbance flow
cell are correlated by a window of time. The optical pulse must be detected
within a time window of the impedance pulse, otherwise the signal is
discarded.
The output signals from the focused flow impedance and the light
absorbance measurements are combined to define the white cell differential
population clusters. This combined technology provides an efficient and
robust technology for the white cell differential.
Signal processing is displayed in Figure 2. Parameter DiffPlot development
is displayed in Figure 3.

Beckman Coulter ACV Differential Technology

Incident Light
(Tungsten Lamp)

Flowcell

Detector

No Cell
No Absorbance / Scatter
Base Line Signal

Small Cell
Low Absorbance / Scatter
Low Signal

Large Granular Cell


High Absorbance / Scatter
Moderate Signal

Large Stained Cell


Very High Absorbance / Scatter
Large Signal

Volume

Volume

Figure 2. Signal Processing

Mono
Eos

Neut
Lymph

Absorbance

Debris

Absorbance
Figure 3. Correlation of Signals to DiffPlot Populations

DiffPlot Characteristics
Lymphocytes

Lymphocytes typically being small with regular shape are smaller in volume
and lower in absorbance than the other cells and are positioned in the lower
part of the DiffPlot. Normal Lymphocyte populations typically have a
homogeneous, gaussian volume distribution.

Neutrophils

Neutrophils, having cytoplasmic granules and segmented nuclei,


will absorb light depending on their morphological complexity. A
hypersegmented neutrophil will give a higher optical response when

compared to a young neutrophil population. The higher the complexity


of the cell the further to the right they appear in the DiffPlot.

Monocytes

Monocytes are typically large cells with kidney shaped nucleus and a
granular cytoplasm. These cells will thus neither scatter nor absorb large
amounts of light and thus are positioned to the low end of the absorbance
axis. However due to their size are positioned high on the volume axis.
Very large monocytes will be found in the IMM (Immature cell) region.

Eosinophils

The staining of the primary and specific granules of the eosinophils


differentiate them from the monocytes and neutrophils. The lymphocytes
remain unstained.

WBC/BASO Methodology

Whole blood is mixed in the WBC/BASO bath with ACT 5diff WBC Lyse
reagent and stabilized at 35C. This reaction lyses the red cells and
specifically differentiates between basophils and other leukocytes. The
resistance of the basophils to the acidic lytic reagent is the basis for the
differentiation of the basophils from the other leukocytes. The sample is then
pulled with a constant vacuum through an aperture. As each cell passes
through the aperture, it generates a pulse proportional to its cellular volume.
This cellular data is then displayed on the WBC/BASO histogram. The total
leukocyte count and the basophil percentage are determined via the Coulter
Principle and specific thresholds. Figure 4 displays the WBC/BASO
histogram and differential thresholds.

BA1

BA2

BA3

BASO

Figure 4. WBC/BASO Histogram

Beckman Coulter ACV Differential Technology

DIFFERENTIAL REAGENT
SYSTEM OVERVIEW

The ACT 5diff system utilizes cytochemical staining and differential lysis and
the technologies of absorbance cytochemistry and the Coulter Principle to
provide the complete five-part differential. These analyses are performed
simultaneously on the ACT 5diff system.
ACT 5diff Fix reagent contains a lysing agent and the vital stain,
Chlorazole Black, which during the reaction time stains the granules of the
monocytes, neutrophils and eosinophils blue and preserves the leukocytes in
their native size. ACT 5diff Fix is an alcoholic reagent, buffered to a pH of
6.9, which gently lyses all red cells. This reaction provides the dilution used
to differentiate the leukocyte subpopulations using the absorbance
cytochemistry technology.
ACT 5diff WBC Lyse reagent is an acidic solution with a pH of 2.4 that
lyses the red cells and specifically differentiates between the basophils and
other leukocytes. This reaction provides the dilution for the total leukocyte
count and basophil percentage analyses, which are obtained by using the
Coulter Principle and using specific volume thresholds.
ACT 5diff Diluent reagent is used to: dilute the whole blood samples,
stabilize cell membranes for accurate enumeration and sizing, provide the
conductive medium, provide the sheath medium for focused flow and rinses
instrument components after analysis.

DIFFERENTIAL ANALYTICAL /
MECHANICAL SYSTEM OVERVIEW
Sequential Dilution System

The leukocyte differential reactions require mixing and delivery of sample to


the analytical hardware. The initial delivery and mixing reactions are achieved
by the Sequential Dilution System (SDS) and mixing within the DIFF and
WBC/BASO baths. This system accurately and repeatedly delivers specific
volumes of sample to the baths for the specific lytic or staining reactions.
Figure 5 displays the specific reaction baths and dilutions.

Rinse
Bath

First Dilution
Bath

DIFF
Bath

RBS/Plt
Bath

WBC/BA
Bath

Figure 5. Sequential Dilution System

Initial aspiration of whole blood occurs at the sampling probe. The SDS
system delivers specific volumes of blood to the respective baths (DIFF or
WBC/BASO) where the sampling needle aligns perfectly with the reagent
inputs. Here, the preheated reagents mix homogeneously with the whole
blood sample. The reagent cleans the sampling needle internally and
externally. Each dilution is then mixed, incubated and stabilized at 35C.
Once the reactions have taken place and the dilutions are ready for analysis,
they need to be transported to the analytical areas.

DIFF Bath

For the DIFF bath sample, the stabilized sample is transported to the
measuring bath in the optical bench. This is accomplished using the syringe
assembly for the DIFF bath. The DIFF syringe feeds the DIFF dilution to the
optical bath sensor while at the same time, two additional syringes provide
diluent sheath flow to the flow cell. Once the sensor detects the sample,
the system begins differential measurements. Figure 6 displays the DIFF
sample flow.

Beckman Coulter ACV Differential Technology

Waste Out

Sheath 1

Sample Flow
for Analysis

Sheath 2

Whole Blood Sample,


FIX Reagent and
Diluent

Diff Bath

Sample Flow
to Fill Diff
Syringe

Figure 6. DIFF Sample Flow

Optical Bench

Light absorbance measurement is accomplished within the optical bench


(see Figure 7). A tungsten halogen lamp provides the light source that is
concentrated and focused through the flow cell. As each cell passes through
the flow cell, a sensor detects light that is scattered forward. The optical
measurement is derived as a function of the amount of light lost due to
defraction and absorbance, as compared to full transmission when no cell is
present. This data is then processed and displayed as the white cell subpopulations of lymphocytes, monocytes, neutrophils and eosinophils.

Waste
Electrodes

Aperture

Light
Source

Optical Window

Electrodes
Sample Injector

Sheath Stream 1

Figure 7. Optical Bench

10

Sample Stream

Sheath Stream 2

Detector

WBC/BASO Bath

The WBC/BASO solution is mixed, stabilized and counted in the WBC/


BASO bath. The WBC/BASO bath receives the whole blood sample and lytic
reagent. The sample is then pulled with a constant vacuum through an
80 m aperture. Each cell as it passes through the aperture generates
a pulse proportional to its cellular volume. This cellular data is then displayed
on the WBC/BASO histogram. The basophils are differentiated from the rest
of the white cells through differential lysis and histogram thresholds. The
basophil count and percentage are derived from this analysis. The
WBC/BASO bath and electrode assembly are displayed below in Figure 8.
Electrodes

Bath Wall

WBC/BASO
Bath

Figure 8. WBC/BASO Bath Assembly

Differential Calculation

The results obtained from the DIFF analysis in the flow cell and the WBC/
BASO analysis in the WBC/BASO bath provide the information for the
complete 5-part differential results. The WBC/BASO results provide the
basophil percentage and total white cell count. The DIFF results provide the
lymphocyte, monocyte, neutrophil and eosinophil results. To achieve
differential results equaling 100%, the neutrophil, lymphocyte and monocyte
results are corrected for the results obtained for the basophil analysis.

DIFFERENTIAL FLAGGING
OVERVIEW

Differential flags are generated whenever the white cell subpopulations


exceed the population statistics and interfere with separation threshold
areas of other subpopulations. The abnormal cell types and their locations
on the WBC differential DiffPlot are described in Figure 9. Figures 10, 11
and 12 describe the DiffPlot flags and the cell types associated with these
flags.

VOLUME

Beckman Coulter ACV Differential Technology

Myelocytes,
Metamyelocytes,
Imm. Neutrophils

Blasts,
Promyelocytes,

Monocytes

Imm.
Eos,
A granular
Eos,
Atypical
Neutrophils

Eosinophils

Atyp.
Lymphs,
Blasts

ds

an

Neutrophils

Lymphs
Abnormal
Lymph

Debris
NRBC's,
Lyse Resistant RBC,
Plt Aggregates

ABSORBANCE
Figure 9. DiffPlot Normal and Abnormal Cell Populations

Immature Granulocytes

Immature granulocytes are detected by their larger volume and by the


presence of granules that increase the intensity of scattered light.

Band Cells

Band cells are typically larger or of similar size to the neutrophils but due to
their low level of cellular complexity they absorb less light. This places the
band cells in the region between the Neutrophils and the Monocytes.

Blasts

Blast cells are generally larger than monocytes and may have similar
absorbance. Large blasts are included in the IMM cell area.

Atypical Lymphocytes

Small blasts will be found between the normal lymphocyte and monocyte
populations. Large lymphocytes, reactive lymphoid forms, stimulated
lymphocytes and blast cells are found in the ATL region.

NRBCs Platelet Aggregates

The presence of platelet aggregates is indicated by a distribution pattern


that moves from the DiffPlot origin into the lymphocyte region. Nucleated
red cells having very small nuclei will be situated in the debris and small
lymphocyte regions.

12

Mono
Eos
NL

Volume

DiffPlot Flags

Neut

Lymph
SL / SL1

DB

Absorbance
DB Debris
Platelet aggregates
RBCs resistant to lysis (stroma)
NRBCs
SL Small Lymphocytes
Small Lymphocytes
Plt Aggregates
NRBCs, RBCs resistant to lysis
NE, LY, MO, EO, ATL & IMM flagged
SL1 Small Lymph 1
Plt Aggregates
NRBCs, RBCs resistant to lysis
Small abnormal lymphs
NE, LY, MO, EO, ATL, IMM
% & # flagged
NL Neut/Lymph
Small Neut w/o granules
Lymps with segmented nucleus
Neuts with weak membranes
NE, LY % & # flagged
Figure 10. DiffPlot Flags

Beckman Coulter ACV Differential Technology

Volume

DiffPlot Flags
UN

UM

Mono
NE

Eos

MN

Neut
Lymph
LN

Debris
Absorbance
MN Mono/Neut
Monos with hyperbasophilic granules
Immature neuts with nonsegmented nucleus
ATL, IMM % & # flagged
MO, NE % & # inherited ( . . . . )
UM

Upper Mono
Large Monos
Myelocytes, Promyelocytes
Large Blasts
NE, MO, IMM % & # flagged

LN Lower Neut
Fragile or aged neutrophils
Plt Aggs, RBCs resistant to lysis
All WBC Diff parameters flagged
UN Upper Neut
Large neutrophils
Imm Grans (Metamyelocytes, myelocytes, promyelocytes)
NE, IMM % & # flagged
NE Neut/Eos
Immature Eos, Agranular Eos
Atypical Neutrophils
IMM % & # flagged
NE, EO % & # inhibited ( . . . . )
Figure 11. DiffPlot Flags

14

Volume

IMM

Mono
Eos
ATL

Neut
Lymph
Debris
Absorbance
ATL
Large Lymphs
Reactive lymphs
Plasma cells
Blasts
IMM Immature cells
Large Monocytes
Promyelocytes, Myelocytes,
Metamyelocytes
Blasts
Figure 12. DiffPlot Flags

Beckman Coulter ACT 5diff Analyzer Case Study


NORMAL PATIENT

WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

5.5 L
5.03
15.2
44.5
89.0
30.3
34.2
17.7

103/L
106/L
g/dL
%
fL
pg
g/dL
%

Range (1)
6.0 / 11.0
4.00 / 6.20
11.0 / 18.8
35.0 / 55.0
80.0 / 100.0
26.0 / 34.0
31.0 / 35.0
10.0 / 20.0

PLT
MPV

169
9.1

103/L
fL

150
6.0

/ 400
/ 10.0

NE
LY
MO
EO
BA

53.8
37.4
6.5
1.6
0.7

%
%
%
%
%

Range (1)
50.0 / 80.0
25.0 / 50.0
2.0 / 10.0
0.0 / 5.0
0.0 / 5.0

NE#
LY#
MO#
EO#
BA#

2.77
1.92
0.33
0.08
0.04

103/L
103/L
103/L
103/L
103/L

2.0
1.0
0.1
0.0
0.0

RBC

VOL

/
/
/
/
/

FLAGS

8.0
5.0
1.0
0.4
0.2

PLT

WBC/
BASO

ABS

History
A normal patient.

Beckman Coulter ACT 5diff Analyzer and Manual Differential Results

Commentary
The ACT 5diff differential correlates

Manual Differential

well with the manual differential. The

Neutrophils

differential DiffPlot displays the white

Bands
Lymphocytes

cell populations with each cell type well


defined with no abnormalities. The red
cell and platelet histograms are
unremarkable with normal CBC
parameter results.

16

Morphology
54%
1%
39%

Monocytes

5%

Eosinophils

1%

LYMPHOCYTOSIS

WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

3.6 L
5.14
13.8
41.4
81.0
26.8
33.3
15.1

103/L
106/L
g/dL
%
fL
pg
g/dL
%

Range (1)
6.0 / 11.0
4.00 / 6.20
11.0 / 18.8
35.0 / 55.0
80.0 / 100.0
26.0 / 34.0
31.0 / 35.0
10.0 / 20.0

PLT
MPV

188
8.7

103/L
fL

150
6.0

/ 400
/ 10.0

NE
LY
MO
EO
BA

31.3 LL
58.1 HH
6.2
4.0
0.4

%
%
%
%
%

Range(1)
50.0 / 80.0
25.0 / 50.0
2.0 / 10.0
0.0 / 5.0
0.0 / 5.0

NE#
LY#
MO#
EO#
BA#

1.13
2.10
0.22
0.14
0.01

103/L
103/L
103/L
103/L
103/L

2.0
1.0
0.1
0.0
0.0

RBC

VOL

/
/
/
/
/

8.0
5.0
1.0
0.4
0.2

FLAGS

Lymphocytosis
Neutropenia

PLT

WBC/
BASO

ABS

History
A 4-year-old female patient admitted
to the emergency room with fever and

Beckman Coulter ACT 5diff Analyzer and Manual Differential Results

vomiting. Enlarged lymph nodes noted


on examination.

Manual Differential

Morphology

Neutrophils

32%

Commentary

Lymphocytes

60%

The ACT 5diff differential correlates

Monocytes

5%

well with the manual differential. The

Eosinophils

1%

lymph region of the DiffPlot

Basophils

1%

demonstrates an increased density


of lymphocytes, even though the white
cell count is low. The MCHC is
somewhat elevated and is believed
to be related to the recent onset of
vomiting and fever. Otherwise the
differential results and the histograms
appear unremarkable.

Beckman Coulter ACT 5diff Analyzer Case Study


EOSINOPHILIA

WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

4.5
2.42
8.5
24.8
102
35.0
34.1
12.8

PLT
MPV

312
7.8

L
L
L
L
H
H

103/L
106/L
g/dL
%
fL
pg
g/dL
%

Range (1)
6.0 / 11.0
4.00 / 6.20
11.0 / 18.8
35.0 / 55.0
80.0 / 100.0
26.0 / 34.0
31.0 / 35.0
10.0 / 20.0

103/L
fL

150
6.0

/ 400
/ 10.0

NE
LY
MO
EO
BA

41.5
22.6
12.7
21.9
1.3

LL%
L
HH
HH

NE#
LY#
MO#
EO#
BA#

1.87 L
1.02
0.57
0.99HH
0.06

Range (1)
50.0 / 80.0
%
25.0
%
2.0
%
0.0
%
0.0

/ 50.0
/ 10.0
/ 5.0
/ 5.0

103/L
103/L
103/L
103/L
103/L

/
/
/
/
/

2.0
1.0
0.1
0.0
0.0

8.0
5.0
1.0
0.4
0.2

FLAGS
WBC: IMM
RBC:

Neutropenia
Eosinophilia
Monocytosis
Macrocytes

PLT

RBC
VOL

WBC/
BASO

ABS

History
A 38-year-old female patient with
admitting diagnosis: Renal failure.

Beckman Coulter ACT 5diff Analyzer and Manual Differential Results


Commentary
The ACT 5diff differential DiffPlot

Manual Differential

demonstrates an increased number of

Neutrophils

Morphology
47%

cells in the eosinophil region. The WBC

Bands

IMM flag signifies the presence of an

Lymphocytes

abnormal number of cells in the upper

Monocytes

6%

monocyte, upper neutrophil and Channel

Eosinophils

26%

127 regions of the DiffPlot. The IMM

Basophils

flag indicates that a white cell count


greater than 0.4 103/L or 2.5% can be
found in this area. The MCV is elevated
and is represented very well on the red
cell histogram. The platelet histogram
is unremarkable.

18

1%
19%

1%

Macrocytosis +2

MONOCYTOSIS

WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

15.8 HH 103/L
3.41 L 106/L
11.0
g/dL
31.3 L %
92
fL
32.1
pg
35.0
g/dL
12.2
%

PLT
MPV

190
8.2

103/L
fL

Range
6.0 /
4.00 /
11.0 /
35.0 /
80.0 /
26.0 /
31.0 /
10.0 /
150
6.0

11.0
6.20
18.8
55.0
100.0
34.0
35.0
20.0

/ 400
/ 10.0

(1)
NE
LY
MO
EO
BA

64.1
11.1 LL
22.2 HH
1.1
1.5

Range
%
%
%
%
%

(1)
50.0
25.0
2.0
0.0
0.0

/
/
/
/
/

80.0
50.0
10.0
5.0
5.0

FLAGS
WBC:*WBC, IMM
RBC:
PLT:

NE#
LY#
MO#
EO#
BA#

10.1 HH
1.75
3.51 HH
0.17
0.24 H

103/L
103/L
103/L
103/L
103/L

2.0
1.0
0.1
0.0
0.0

/
/
/
/
/

8.0
5.0
1.0
0.4
0.2

Leukocytosis
Lymphopenia
Neutrophilia
NRBCs
Monocytosis
Platelet
Aggregates

PLT

RBC

VOL

WBC/
BASO

ABS

History
A 64-year-old female admitted with
advanced degenerative bone disease.

Beckman Coulter ACT 5diff Analyzer and Manual Differential Results

Commentary
The ACT 5diff DiffPlot displays a

Morphology

Manual Differential
Neutrophils
Bands

65%
5%

Lymphocytes

13%

Monocytes

17%

Monocytes with
increased
granulation
and numerous
vacuoles.

population of monocytes extending into


the upper monocyte and neutrophil
regions, generating the IMM flag. The
cells associated with the IMM flag
include large monocytes and hyperbasophilic monocytes. The manual
differential results agree with the
automated differential results and the
white cell morphology correlate with the
IMM flag. The *WBC flag indicates white
cell count interference with possibly
platelet aggregates or nucleated red
cells. This flag is generated from the
WBC/BASO histogram. Red cell and
platelet morphology are unremarkable.
The red cell count, hemoglobin and
hematocrit results appear decreased.

Beckman Coulter ACT 5diff Analyzer Case Study


LEFT SHIFT

WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

16.3
3.11
10.4
30.1
97
33.3
34.5
13.3

HH
L
L
L

PLT
MPV

82
8.7

103/L
106/L
g/dL
%
fL
pg
g/dL
%

Range (1)
6.0 / 11.0
4.00 / 6.20
11.0 / 18.8
35.0 / 55.0
80.0 / 100.0
26.0 / 34.0
31.0 / 35.0
10.0 / 20.0

103/L
fL

150
6.0

/ 400
/ 10.0

NE
LY
MO
EO
BA

91.5 HH
3.0 LL
2.5
2.0
1.0

%
%
%
%
%

Range (1)
50.0 / 80.0
25.0 / 50.0
2.0 / 10.0
0.0 / 5.0
0.0 / 5.0

NE#
LY#
MO#
EO#
BA#

14.9 HH
0.49 LL
0.41
0.33
0.16

103/L
103/L
103/L
103/L
103/L

2.0
1.0
0.1
0.0
0.0

RBC

VOL

/
/
/
/
/

8.0
5.0
1.0
0.4
0.2

FLAGS
WBC:*WBC, IMM
RBC:
PLT:

Leukocytosis
Neutrophilia
Lymphopenia
Myelemia
Immature Cells
Platelet
Aggregates

PLT

WBC/
BASO

ABS

History
A 48-year-old female admitted with
respiratory failure.
Commentary
The ACT 5diff differential results agree
with the findings from the manual
differential. The automated differential
results (91.5% neutrophils) correlate
well with the thirty-three percent (33%)
bands and sixty percent (60%)
neutrophils reported in the manual
differential. The DiffPlot demonstrates
an increased intensity of cells in the
neutrophil and upper neutrophil regions.
This population of cells generates the
IMM flag. The cells associated with
this flag include myleocytes,
metamyleocytes, promyleocytes, large
neutrophils and blasts. The *WBC flag
indicates white cell count interference
with possibly platelet aggregates or
nucleated red cells. Red cell count
hemoglobin and platelet values are
decreased.

Beckman Coulter ACT 5diff Analyzer and Manual Differential Results

Manual Differential

20

Morphology

Neutrophils

60%

Bands

33%

Lymphocytes

7%

NRBC

1%

GRANULOCYTOSIS, MICROCYTOSIS

WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

17.8 HH 103/L
5.13
106/L
12.9
g/dL
37.1
%
72
LL fL
25.1 L pg
34.8
g/dL
16.9
%

PLT
MPV

440 H
7.9

103/L
fL

Range (1)
6.0 / 11.0
4.00 / 6.20
11.0 / 18.8
35.0 / 55.0
80.0 / 100.0
26.0 / 34.0
31.0 / 35.0
10.0 / 20.0
150
6.0

/ 400
10.0

NE
LY
MO
EO
BA

86.2 HH
4.8 LL
7.3
1.0
0.7

%
%
%
%
%

Range (1)
50.0 / 80.0
25.0 / 50.0
2.0 / 10.0
0.0 / 5.0
0.0 / 5.0

NE#
LY#
MO#
EO#
BA#

15.3 HH
0.85 LL
1.30 HH
0.18
0.12

103/L
103/L
103/L
103/L
103/L

2.0
1.0
0.1
0.0
0.0

RBC

VOL

/
/
/
/
/

8.0
5.0
1.0
0.4
0.2

FLAGS
WBC:*WBC
RBC:
PLT:

Leukocytosis
Lymphopenia
Neutrophilia
Monocytosis
NRBC
Microcytes
Platelet
aggregates

PLT

WBC/
BASO

ABS

History
A 70-year-old female admitted in

Beckman Coulter ACT 5diff Analyzer and Manual Differential Results


Manual Differential
Neutrophils

83%

Bands

9%

Lymphocytes

4%

Monocytes

4%

respiratory failure.

Morphology

Commentary

Platelets
increased,
marked
microcytosis.

The ACT 5diff differential results agree


with the findings from the manual
differential. The automated differential
results (86.2% neutrophils) correlate
well with the ninety-two percent (92%)
neutrophils (bands and segs) reported in
the manual differential. The DiffPlot
displays a small population of cells in
the lower lymph region, possibly platelet
aggregates. The instrument generates
the microcytosis interpretive flag based
on the customer definable limits and the
MCV of 72 fL.

Beckman Coulter ACT 5diff Analyzer Case Study


ACUTE MYELOID LEUKEMIA

WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

8.6
3.99 L
11.1
33.9 L
85
27.9
32.9
13.9

103/L
106/L
g/dL
%
fL
pg
g/dL
%

Range (1)
6.0 / 11.0
4.00 / 6.20
11.0 / 18.8
35.0 / 55.0
80.0 / 100.0
26.0 / 34.0
31.0 / 35.0
10.0 / 20.0

PLT
MPV

18 RLL
8.4 R

103/L
fL

150
6.0

/ 400
/ 10.0

NE
LY
MO
EO
BA

6.1 RLL
58.0 HH
33.6 RHH
0.5
1.8

%
%
%
%
%

50.0
25.0
2.0
0.0
0.0

/
/
/
/
/

80.0
50.0
10.0
5.0
5.0

NE#
LY#
MO#
EO#
BA#

0.53 RLL
5.01 H
2.90 RHH
0.04
0.16

103/L
103/L
103/L
103/L
103/L

2.0
1.0
0.1
0.0
0.0

/
/
/
/
/

8.0
5.0
1.0
0.4
0.2

Lymphocytosis
Neutropenia
Immature Cells
Atypical Lymp,
Monocytosis
Plt interpretation
impossible

PLT

RBC

VOL

Range (1)
FLAGS
WBC: UM,ATL,IMM
RBC:
PLT:
SCH

WBC/
BASO

ABS

History
A 78-year-old female admitted with
Acute Myelogenous Leukemia.

Beckman Coulter ACT 5diff Analyzer and Manual Differential Results

Commentary
The ACT 5diff DiffPlot demonstrates
an intense population of cells beginning
in the lymphocyte region extending
through the lymphocytes, atypical
lymphocytes, monocyte and upper
monocyte regions. The lack of
separation within this cellular population
generates the WBC flags including UM,
ATL, IMM and diff parameter R flags.
The cells associated with these flags
include large monocytes, myelocytes,
promyleocytes and blasts, which is in
agreement with the manual differential
results. The platelet histogram displays
marked interference from schistocytes,
generating the platelet interpretation
impossible flag. Reassessment of the
results listed by a reference method
is required. Reference method platelet
results were five.

Manual Differential
Neutrophils

14%

Bands

11%

Metamyelocytes

2%

Blasts

7%

Lymphocytes
Atypical Lymphs
Monocytes

22

Morphology

47%
5%
14%

Numerous
schistocytes, +2
anisocytosis,
platelets
decreased and
numerous
smudge cells.

HB S, SICKEL-CELL CRISIS

WBC
RBC
HGB
HCT
MCV
MCH
MCHC
RDW

5.0
2.39
8.3
23.8
100
34.8
34.9
17.8

PLT
MPV

257
9.7

L
LL
LL
LL
H
H

103/L
106/L
g/dL
%
fL
pg
g/dL
%

Range (1)
6.0 / 11.0
4.00 / 6.20
11.0 / 18.8
35.0 / 55.0
80.0 / 100.0
26.0 / 34.0
31.0 / 35.0
10.0 / 20.0

103/L
fL

150
6.0

/ 400
/ 10.0

NE
LY
MO
EO
BA

33.9
59.4
2.2
3.8
0.7

RLL
RHH
R
R

%
%
%
%
%

Range (1)
50.0 / 80.0
25.0 / 50.0
2.0 / 10.0
0.0 / 5.0
0.0 / 5.0

NE#
LY#
MO#
EO#
BA#

1.69
2.96
0.11
0.19
0.03

RL
R
R
R

103/L
103/L
103/L
103/L
103/L

2.0
1.0
0.1
0.0
0.0

8.0
5.0
1.0
0.4
0.2

Lymphocytosis
Neutropenia
NRBCs
Anemia

PLT

RBC

VOL

/
/
/
/
/

FLAGS
WBC:*WBC,SL,SL1
RBC:
PLT:

WBC/
BASO

ABS

Beckman Coulter ACT 5diff Analyzer and Manual Differential Results

Manual Differential

Morphology

Neutrophils

26%

Lymphocytes

72%

Numerous sickle
cells, +3 target
cells, +1 anisocytosis, rare
large platelet.

Eosinophils

1%

Basophils

1%

NRBC

18/100
WBC

History
A 27-year-old female presented to the
emergency room with shortness of
breath and severe joint and abdominal
pain. Patient has a known history of
Hb S, Sickle-Cell disease. Current
diagnosis: Hb S, Sickle-Cell disease,
crisis phase.
Commentary
The ACT 5diff differential DiffPlot
demonstrates a continuous population
of cells beginning in the lower
lymphocyte region. The lack of
separation within this cellular population
generates the SL and SL1 flags. The
cells associated with these flags include
small lymphocytes, nucleated red cells
and red cells resistant to cell lysis.
Additionally, on the WBC/BASO
histogram a population of cells appears
to the left of the lower threshold. This
population of cells generates the *WBC
flag and differential R flags. These flags
are in agreement with the manual
differential results. The corrected white
cell count is 4.2x103/L. The red cell
histogram displays a markedly wide
distribution in agreement with the mean
cell volume and presence of target
cells. The platelet histogram is
unremarkable.

S I M P L I F Y A U T O M AT E I N N O VAT E
Eastern Europe, Middle East, North Africa: Switzerland, Nyon (41) 22 994 0707. Australia, Gladesville (61) 2 9844 6000. Canada, Mississauga (1) 905 819 1234.
China, Beijing (86) 10 6515 6028. Hong Kong (852) 2814 7431, 2814 0481. France, Villepinte (33) 1 49 90 90 00. Germany, Krefeld (49) 2151 33 35.
Italy, Milan (39) 02 953921. Japan, Tokyo (81) 3 5404 8424. Latin America (1) (305) 380 4709. Mexico, Mexico City (525) 575 6805. Netherlands, Mijdrecht (31) 297 230630.
Singapore (65) 6339 3633. South Africa/Sub-Saharan Africa, Johannesburg (27) 11 805 2014. Sweden, Bromma (46) 8 564 85 900. Switzerland, Nyon 0800 850 810.
Taiwan, Taipei (886) 2 2378 3456. Turkey, Istanbul (90) 216 309 1900. UK, High Wycombe (44) 01494 441181. USA, Brea, CA (1) 800 352 3433, (1) 714 993 5321.

www.beckmancoulter.com

Copyright 2002 Beckman Coulter, Inc.

PRINTED IN U.S.A.