Add-00066186 - Ruby Casebook 2019.semnat

CELL-DYN Ruby
CASEBOOK
CHOOSE TR ANSFORM ATION ™

Achieve measurably better healthcare performance.
www.corelaboratory.abbott/hematology
CONTENTS
ABOUT THE AUTHORS.......................................................................................................................5
PREFACE..................................................................................................................................................6
TECHNOLOGY AND METHODS.....................................................................................................7
SCATTERPLOTS AND HISTOGRAMS ..........................................................................................17
FLAGGING.............................................................................................................................................24
CASES......................................................................................................................................................29
REFERENCES.......................................................................................................................................53
CASEBOOK: CELL-DYN RUBY DIAGNOSTICS
PREFACE
The CELL-DYN Ruby is a technologically advanced hematology analyzer that
provides multi-dimensional blood cell analysis, based mainly on phycical technology
of flowcytometry. Automated instruments such as the CELL-DYN Ruby are used
in clinical laboratories to provide a series of analytical measurements collectively
referred to as the Complete Blood Count (CBC) or Full Blood Count (FBC). Its
software algorithms are designed to characterize, count and classify blood cells
of normal healthy individuals and of patients with a variety of diseases. In some
disease states, blood cells do occur with different physical characteristics than in
normal circumstances and these abnormal cells represent algorithmic challenges. As
the efficiency of CBC data review consequently has a significant impact on overall
performance efficiency.
Awareness of technological aspects and instrument processing mechanisms together
with associated graphical outputs is important for laboratory and subsequent clinical
interpretation. Similarly, hematology instrument reports are often accompanied
by warning indicators (flags) for certain abnormalities and it is further necessary
that reviewers have access to descriptions of flagging logic and significance so that
they can be best applied to laboratory decision processes. In those cases, visual
interpretation of the scatterplots and histograms will help to provide extended
understanding and interpretation of numerical results and warnings. Many
CELL-DYN Ruby users have expressed a need for examples in order to help them
interpreting unusual cases of their own laboratory practice. In response to this need
the authors have developed the CELL-DYN Ruby Case Interpretation book that can
be used as a guideline.
This monograph is intended for the CELL-DYN Ruby user who wants to understand
the analyzer’s technology and to interpret its analytical results. The cases shown are
examples and should not be regarded as typical for a particular disease state. The
book is not intended as a guideline for diagnosis or clinical decision making.
3
TECHNOLOGY
AND METHODS
4
INTRODUCTION
The CELL-DYN Ruby is a fully automated multiparameter hematology analyzer. It utilizes
(Multi-Angle Polarized Scatter Separation), technology, laser flow cytometry, coupled with state
of the art software for complete cellular analysis in a blood sample.
The CELL-DYN Ruby counts: red blood cells (RBC), white blood cells (WBC), platelets (PLT)
and reticulocytes optically. It also measures the hemoglobin (HGB) concentration using light
absorption. The WBC are counted using two technologies: white optical count (WOC) and
nucleated optical count (NOC). WOC is measured optically after lysing the red blood cells and
counting cells above a predetermined threshold using forward light scatter. This method of
sample analysis is used when FWBC warning flag is triggered by a sample which was run in
Normal CBC mode. Because fragile or age-deteriorated WBC may not be included in WOC count
estimate.
TECHNOLOGY - OVERVIEW
CELL-DYN Ruby makes use of two different technologies for measuring cells or c ellular
constituents. These are:
• Photometry
• Optical counting
Photometry
This technique is used for measuring hemoglobin (HGB). An aliquot of blood sample is diluted
with HGB Reagent, which lyses the RBC and converts the hemoglobin released by cyanide free
lysing process to a chromogenic substance that absorbs light of a specific wavelength. The dilution
is then transferred to the HGB Flow Cell, where the light absorption is measured and the HGB
concentration in blood is calculated applying Lambert-Beer’s law.
LED
Figure 1.1: Schematic overview of the hemoglobin flow cell
Optical counting
When cells are hit by a bundle of light, they can absorb the light or scatter it into different
directions, depending on a number of cellular properties. In this way, the principle of light
scattering can be used for cellular analysis and this is the main technology used in the CELL-DYN
Ruby.
The blood cells are diluted into a suspension that in a stream of fluid is passed through an optical
flow cell. This is a small device that, using the principle of hydrodynamic focusing, allows for
single cells interacting with the light. When light hits a blood cell in the fluid stream, it is partially
5
absorbed and partially scattered by the blood cell suspension. A set of detectors around the flow
cell measure the scattered light and powerful software enables all the scattered signals to be
combined, so that a scatter profile of single cells can be constructed. The scatter profile of each
cell depends on its characteristics, which allows distinguishing different cell types and sorting
them into groups (Figure 1.2).
CELL-DYN Ruby uses monochromatic vertically polarized light from a helium-neon laser as a
light source; its wavelength is 632.8 nm. The analyzer has four detectors for scattered light.
90 °
90 ° Dep
10 °
0°
10 °
Figure 1.2: Light scattering by a blood cell
The four scatter detectors are placed at different angles relative to the incoming laser light and
they each measure a different aspect of blood cells, as shown in Table 1.1. The measurement of
depolarized scattered light is a unique feature to CELL-DYN analyzers. It is based on the discovery
that granules of eosinophils are able to transform the polarization direction of laser light. This
property allows specific separation of eosinophilic from neutrophilic and basophilic granulocytes.1
Table 1.1. Four angles of light scatter detection in CELL-DYN Ruby
SPURIOUSLY INCREASED
0° axial light loss (ALL) cell size

10 ° intermediate angle scatter (IAS) internal structure (cell complexity)
90 ° polarized side scatter (PSS) nuclear segmentation (lobularity)
90 ° D depolarized side scatter (DSS) eosinophilic granules (granularity)
Combining the scatter signals from an individual blood cell makes accurate classification possible,
in particular of the WBC. The technology used to derive the WBC differential count is known
as Multi-Angle Polarized Scatter Separation (MAPSS), a patented process which is unique to
CELL-DYN hematology analyzers. The MAPSS technology will be discussed in detail in paragraph
1.4.
CELL-DYN Ruby not only uses optical analysis for counting and differentiating WBC, but also for
the PLT and RBC measurement. In addition, the optical analysis is also used for measuring the
Nucleated Optical Count (NOC).
6
The optical flow cell, laser and all detectors of the CELL-DYN Ruby are built together in a unit
called the optical bench, a diagram of which is shown in Figure 1.3.
Mirror
Beam expander Laser
90 ° 90 °D
Mirror
10 °detector
Optical flow cell
0 °detector
Figure 1.3: CELL-DYN Ruby optical bench
HEMOGLOBIN METHOD
For the determination of the HGB concentration, blood is diluted and mixed with a cyanide-free
reagent that lyses RBC membranes and rapidly transforms HGB into a stable chromogenic complex
that absorbs light at 555 nm. The absorbance spectrum of this complex is very similar to that of
cyanmethemoglobin.2 A photo detector measures the amount of light passing through the sample
dilution. Using the diluent as a reference, the degree of light absorbance is directly proportional to
the concentration of HGB in the sample. which lyses the RBC and converts the hemoglobin released
by cyanide free lysing process to a chromogenic substance that absorbs light of a specific wavelength.
When the WBC count supersedes 30 x 109/L (30 x 103/μL) the HGB concentration is automatically
corrected for WBC interference.
WBC METHOD
Counting and differentiating WBC is performed using MAPSS™ technology. This requires that blood
cells are in a near-native state, which is accomplished by a gentle stabilizer in the WBC reagent. The
osmotic pressure of the RBC is higher than that of the reagent. Therefore, the hemoglobin in the RBC
diffuses out of the cell and water from the reagent diffuses into the cell. The cell membrane remains
intact but the RBC now has the same refractive index as the sheath, thereby rendering it invisible
to the laser. When the WBC dilution passes the optical flow cell, up to 10,000 cells are analyzed and
from each cell 4 signals (Table 1.1) are simultaneously registered and stored as a list mode file (Table
1.2). The software analyses these 4 signals in a stepwise process for counting and classifying the
white blood cells in the blood sample and provide morphological flagging. Since cells of the same
type will scatter light similarly, each cell type will tend to form a cluster when the list mode data are
plotted in two (or more) dimensions.
250
200
150
SIZE
100
50
0
0 50 100 150 200 250

COMPLEXITY
Figure 1.4A: Before classification all events are labeled “unknown”

7
The algorithms separate and identify each of these clusters, using an appropriate combination of
parameters for each separation, in a progressive strategy. Each cell is initially labeled as “unknown”
(Figure 1.4A). At each stage of analysis, one or more groups of labels are assigned, and at the end,
every cell has been labeled as one of the possible cell types.
Table 1.2: E
xample of a list mode table after data acquisition.
0° 10 ° 90 ° 90 °D
Event Cell Type
SIZE COMPLEXITY LOBULARITY GRANULARITY
1 140 143 125 28 unknown
2 100 75 14 5 unknown
3 165 80 30 5 unknown
4 30 30 10 5 unknown
5 110 93 16 5 unknown
6 135 148 130 75 unknown
7 60 64 15 6 unknown
… … … … … …
10000 135 142 118 127 unknown
Step 1: separation of mononuclear and polymorphonuclear WBC

The scatter information is plotted with the 90° (Lobularity) scatter on the Y axis and the 10°
(Complexity) scatter on the X axis for distinguishing mononuclear from polymorphonuclear cells.
Two populations of cells are clearly seen in Figure 1.4B. The mononuclear cells fall in the cluster
in the lower left corner of the scatterplot and the polymorphonuclear cells fall in the cluster above
and to the right of them. The instrument uses a dynamic threshold to determine the best separation
between these two populations. Each cell is then identified as mononuclear or polymorphonuclear.
Once each cell is identified, it retains this classification no matter where it appears on other
scatterplots. Mononuclear cells are colored blue and polymorphonuclear cells orange, as shown in
Figure 1.4B.
250
250
200
200
150
150
90 °
90 °
100
100
50
50
0
0 50 100 150 200 250 0 50 100 150 200 250

COMPLEXITY COMPLEXITY
Figure 1.4B: Separation of mononuclear and polymorphonuclear cells
Step 2: separation of eosinophils and neutrophils

In the second step, only the polymorphonuclear cells are considered. In the 90 °D
(GRANULARITY) against 90 ° (LOBULARITY) scatterplot, eosinophils have a considerably higher
depolarized side scatter than neutrophils, allowing a dynamic threshold to separate them (Figure
1.4C). Neutrophils remain orange colored and eosinophils become green.
8
250
250
200
200
GRANULARITY
GRANULARITY
150
150
100
100
50
50
0
0
0 50 100 150 200 250 0 50 100 150 200 250
LOBULARITY LOBULARITY
Figure 1.4C: Separation of eosinophilic and neutrophilic granulocytes
Step 3: separation of monocytes, lymphocytes and basophils

In the next step, only the mononuclear cells are taken into account, but the algorithm uses
the orientation of the neutrophil cluster to aid in classifying the mononuclear cells. Dynamic
thresholds in the 0 ° against 10 ° scatterplot identify three WBC subpopulations. There are three
subpopulations of mononuclear cells because basophils are included in the mononuclear cluster.
Typically, basophils are granulated cells and therefore more complex than the mononuclear
cells. However, the basophilic granules are water soluble and dissolve in the WBC Lyse reagent.
Consequently, the degranulated basophils become less complex cells that fall into the mononuclear
cluster. Monocytes are then colored purple, lymphocytes remain blue and basophils become black
(Figure 1.4D).
250
250
200
200
150
150
SIZE
SIZE
100
100
50
50
0
0 50 100 150 200 250 0 50 100 150 200 250

Figure 1.4D: Identification of monocytes, lymphocytes and basophilic granulocytes
Step 4: elimination of cell debris and noise

Finally, the software algorithm uses a dynamic threshold based on the lymphocyte and neutrophil
populations for distinguishing intact WBC from other cells, cellular debris and noise. The
thresholds are set in 0 ° and 10 º channels as shown in Figure 1.4E. All events with a scatter signal
below these thresholds are excluded from further analysis.
The following cell types may be present in this region:*
• NRBC • Unlysed RBC
• Giant PLT • PLT clumps
*The instrument evaluates the area below below the lymphocyte cluster. All particles in this cluster are separated from lymphocytes by a dynamic
threshold. It is important to note that, these particles in this region are excluded from the WBC count and the Differential.
9
250
250
200
200
150
150
SIZE
SIZE
100
100
50
50
0
0
0 50 100 150 200 250 0 50 100 150 200 250
Figure 1.4E: Elimination of cell debris and noise
Step 5: construction of scatterplots and calculation of WBC concentrations

When all major cell types have been classified, the algorithm constructs various scatterplots.
The basic scatterplot, called the WBC differential plot, is a plot of 0 ° (SIZE) against 10 °
(COMPLEXITY) and provides an overall impression of the differential WBC count with the five
WBC subpopulations (Figure 1.4F). The scatterplots and their use will be discussed later.
250
Neutrophils
200
Monocytes
150
SIZE
100
Basophils Eosinophils
50
Lymphocytes
0
0 50 100 150 200 250

COMPLEXITY
Figure 1.4F: WBC Differential scatterplot showing five cell populations
In addition to the scatterplots, calculation of WBC count and WBC subpopulation counts are now
possible. Since the sample volume, the dilution factor and the volume of diluted blood analyzed
are exactly known, the concentrations of total WBC and all WBC subpopulations can easily be
calculated. Results are expressed in concentration units (109/L or 103/µL) as well as in percentage of
total WBC.
The WBC subpopulations are further identified by the following colors:
• Neutrophils — yellow • Monocytes — purple
• Lymphocytes — blue • Eosinophils — green
• Basophils — white
NOTE: The basophils are displayed as white dots but appear as black dots on color printouts.
A unique feature of CELL-DYN Ruby is that it can also determine a total nucleated optical count
(NOC) by analyzing the HGB dilution. After being measured in the HGB Flow Cell, the HGB
dilution is transferred to the Optical Flow Cell instead of being sent to a waste chamber as in the
10
CBC test selection. The HGB reagent lyses the cytoplasmic membrane of the white blood cells,
while it allows the nuclear membrane to remain intact. This results in a greater stability of the
white cells in the sample. The HGB dilution is lysed for approximately 15 seconds before it is sent to
the Optical Flow Cell. As the HGB dilution passes through the Optical Flow Cell, the nuclei of the
cells are counted. The result of this measurement is presented as a histogram (Figure 1.5) and the
count is stored in the datalog as NOC.
0 50 100 150 200 250

NOC
Figure 1.5: The NOC histogram
RBC METHOD
Red blood cells are measured from a separate dilution; the diluent contains reagents that make
RBC spherical while keeping their volume constant. This process, called isovolumetric sphering, is
critical for reliable optical RBC analysis.3
During the RBC analysis up to 30,000 RBC are analyzed optically and three light scatter signals
collected: 0 °, 10 ° and 90 °. These scatter signals are used for calculating, on a cell by cell basis, RBC
concentration, MCV and RDW, applying Mie theory principles. The size distribution data for
the red cells is displayed graphically as a histogram using 0° data. The size distribution data is
plotted on the X axis. The relative number of cells is normalized and plotted on the Y axis. The
RBC histogram contains two dynamic thresholds used for MCV and RDW calculation. The MCV is
derived from the RBC size distribution data on the 0°, 10°, and 90°. histograms, and is expressed in
femtoliters. The RDW is derived from the RBC histogram using the 20th and 80th percentiles.
0 50 100 150 200 250

RBC
Figure 1.6: The size distribution data for the red cells is displayed graphically as a histogram using 0 ° data.
The size distribution is plotted on the X axis. The relative n umber of cells is normalized and plotted on the Y axis.
PLT METHOD
CELL-DYN Ruby measures PLT count simultaneously with the RBC count. The PLT method uses
a two-dimensional approach for defining platelets and separating them from small non-platelet
particles. Events counted in the RBC/PLT dilution between floating thresholds are included in the
platelet
LEARNING (PLT)HEMATOLOGY
GUIDE: data, which is collected using the 0 ° and 10 ° sensors. The lower threshold floats
11
between 1 and 3 fL and the upper threshold floats between 15 and 35 fL. If there are not enough
data to determine a nice PLT population, the lower and upper thresholds are set at 2 and 35 fL
respectively. Once the thresholds have been determined, the PLT count is derived from the 10° data.
Data can be displayed as a scatterplot (0 °/RBC 10 °) including the RBC population or as a histogram
of the 10 ° data.
Events counted in the region below the lower threshold are usually either small particulate matter
or optical noise. Events counted in the region above the upper threshold are counted as RBC. If
interference with either threshold region exceeds a predetermined limit, the PLT parameters are
flagged accordingly.
250
200
150
0°
100
50
0
0 50 100 150 200 250 0 50 100 150 200 250

PLT RBC 10 °
Figure 1.7: PLT data are shown as a histogram of the 10 ° data and as a 0 °/RBC 10 ° scatterplot in the following figure. The 0 °/RBC 10 °
scatterplot colors are Orange (cells identified as PLTs); Red (cells identified as RBCs); Black (cells identified as PLT aggregates); Blue
(cells identified as generically WBCs or specifically lymphocytes) and Purple (cells identified as RBC coincidence).
RETICULOCYTE METHOD
Reticulocytes are immature RBC that are recently released from the bone marrow. In contrast
to mature RBC, reticulocytes contain ribosomal RNA. This RNA can be visualized with certain
supravital, cationic dyes that simultaneously stain and precipitate RNA to form a network or
reticulum.4
The CELL-DYN Ruby System reticulocyte method uses the dye New Methylene Blue. The
reticulocyte assay is performed in the WOC channel of the instrument. Sample preparation is
performed manually by dispensing 20 μL of blood into a tube of CELL-DYN Reticulocyte Reagent.
At room temperature, staining of reticulum is complete within approximately 15 minutes. The
stained sample is aspirated in the Open Mode and once diluted with WBC Lyse, the RBC sphere
due to the influence of the nonionic detergent incorporated into the staining solution. Sphering
is necessary to eliminate optical orientational noise that would otherwise be introduced into the
scatter measurements. The usual lytic action of the WBC Lyse is prevented by a fixative contained
in the staining solution. During data acquisition, 0 °, 10 °, and 90 ° scatter signals are collected for
up to 30,000 events. The 0 ° threshold is set high enough to exclude most platelets. Histogram
data are used to differentiate reticulocytes from mature RBC, platelet clumps, and nucleated cells.
Reticulocytes have 10 ° scatter that are similar to the scatter for mature RBC, but differ from them
by exhibiting greater 90 ° scatter. Reticulocytes are reported in percent (%R). The instrument will
automatically calculate the Reticulocyte Absolute value if an RBC concentration is entered using
the F12 – RBC Source function key.
12
250
250
200
200
150
150
90 °
90 °
100
100
50
50
0
0
0 50 100 150 200 250 0 50 100 150 200 250
RETC 0 ° RETC 10 °
Figure 1.8: RETC scatterplot. The CD-Ruby reticulocyte method is based on supravital dye staining with New Methylene Blue.
After initial off-line exposure to the dye, the blood is processed using a dedicated reticulocyte “mode” and RBC are analyzed in the
optical flow cell by three angles of laser light scatter (0 °, 10 ° and 90 °).
PROCESSING MODES
CELL-DYN Ruby offers various modes for measuring samples. For general routine operations CBC
mode is recommended as it is the most efficient. Depending on specific characteristics of the blood
sample to be measured, one of the other modes may be the mode of choice.
Routine CBC Mode
EDTA-anticoagulated blood samples are normally processed in the first instance using the CBC
operational mode. If the resulting data do not show either a FWBC (Fragile White Blood Cell) or
RRBC (Resistant Red Blood Cell) flag, then there is no need for further processing. However, if
either of these flags is triggered, then one of the following two processing options needs to be used.
NOC (Nuclear Optical Count) Mode
Samples containing fragile WBC are difficult to measure accurately because of the rapid breakdown of
cells during the measurement process. This method of sample analysis is necessary when the FWBC
warning flag is triggered by a sample run in the routine CBC mode because fragile or age-deteriorated
WBC may not be included in the WBC count. To obtain an accurate WBC count, the NOC mode uses
an alternate method using the HGB dilution (instead of the WBC dilution) to measure samples
containing fragile WBC. In NOC mode, the nuclei of WBC are counted instead of intact WBC. WBC
counts can be obtained by the NOC method with linearity by 250 x 109/L. Typically, fragile WBC are
abnormal lymphocytes that are present in chronic lymphocyte leukemia (CLL) and are the “smudge
cells” that appear when the blood smear is made.
RRBC (Resistant Red Blood Cell) Mode
The presence of lyse resistant red blood cells generates WBC and NRBC/RRBC warning indicators,
Giving an indication to the operator about the invalidated WBC counts. CD Ruby RRBC mode of
analysis overcomes the influence of lysis-resistant red blood cells and provide accurate white blood
cell counts and diffrentials. Red Blood cells containing fetal hemoglobin (premature, newborn or
hemoglobinopathy patients) or those with Target Cell morphology are relatively resistant to lysis.
In the RRBC mode, the WBC dilution is held in the mixing chamber 15 seconds longer than in the
routine CBC mode. This additional lysing time enables to lyse resistant RBC and prevent them from
interfering with the WBC count and differential.
13
SCATTERPLOTS
AND HISTOGRAMS
14
SCATTERPLOTS
As MAPSS technology uses 4 different signals for classifying every cell analyzed, WBC
differentiation is a 4-dimensional process, which cannot possibly be visualized in a single graphic.
Therefore, CELL-DYN Ruby offers a wide choice of two-dimensional scatterplots and histograms.
The most useful of them will be discussed here.
COLOR CODES
The various cell populations are color-coded throughout all screens and printed reports. The colors
used are standardized across CELL-DYN analyzers (Table 2.1):
Table 2.1: Color codes of cells used in CELL-DYN hematology analyzers.
CELL LINEAGE COLOR CELL TYPE

WBC Orange Neutrophils
Blue Lymphocytes
Purple Monocytes
Green Eosinophils
Black (white on screen) Basophils
Pink Nucleated RBC or other non-WBC
cells
RBC Red Erythrocytes
PLT Orange Platelets
Apart from basophils, cell populations in black (in white on the monitor screen) denote non-classifiable
cells; these are not included in the respective cell counts.
WBC DIFFERENTIAL PLOT

This is the standard plot that provides an overall impression of the WBC differential. It displays
0 ° scatter (cell size) against 10 ° scatter (cell complexity) and resolves the five normal WBC
subpopulations in blood (Figure 2.1). Based on information displayed in the scatterplots, the
algorithms can also trigger flags when abnormal cells are suspected (see Chapter 3). Interpreting scatter
patterns often is more informative as to the nature of the abnormal cells than a flag alone.
The WBC Differential plot is particularly useful for recognizing the presence of abnormal cells.
For example, events in region 1 are very small and non-complex, smaller and less complex than
lymphocytes. Region 1 therefore, typically is the region where lysis-resistant RBC (see case 31),
nucleated RBC or platelet aggregates (see case 29) will appear.
In region 2 events will appear that are larger in size than lymphocytes but smaller than monocytes,
and equal both cell types in complexity. In this region atypical lymphocytes (see case 15) and
sometimes blasts show up. An important region is region 3 as it represents large non-complex
cells like blasts (see case 20 – 23), plasma cells or very large platelet aggregates. Finally, in region 4
immature granulocytes (see case 8 – 10) can be found, but it is also the region where artefacts like
neutrophil aggregates (see case 26) and platelet satellites (see case 25) will show up. More in depth
interpretation of scatterplots and abnormal cells will be covered in Chapter 4.
15
250
250
200
200
150
150
SIZE
SIZE
100
100
50
50
0
0
0 50 100 150 200 250 0 50 100 150 200 250
Figure 2.1: Left: The WBC Differential scatterplot of a normal blood sample. The 0 ° scatter ( representing cell size) is plotted against the
10 ° scatter (cell complexity). Right: The WBC Differential plot with four regions where abnormal cells may be found.
MONO-POLY I PLOT
The Mono-Poly I plot shows nuclear lobularity (90 ° scatter) as a function of cell size (0 ° scatter;
Figure 2.2). This plot is particularly useful for detecting the presence of blast cells and atypical
lymphocytes. Large mononuclear cells will appear in region 1 in the lower right corner (see case
20 – 23). If more than 1 % of the WBC-population is located in this region, a blast-flag will be set.
The Mono-Poly I plot also gives a good impression of the nuclear segmentation of the neutrophils.
Cells with hypersegmentation can be found in region 2, whereas hyposegmented neutrophils show
up in region 3.
250
250
200
200
150
150
90 °
90 °
100
100
50
50
0
0 50 100 150 200 250 0 50 100 150 200 250

WBC 0 ° WBC 0 °
Figure 2.2: Left: The Mono-Poly I scatterplot of a normal blood sample, clearly showing the lobularity of
neutrophils. Right: The Mono-Poly I plot with three regions for abnormal cells. See text for explanation.
NEU-EOS PLOT
This scatterplot shows depolarized 90 ° scatter (90 °D, a marker of cytoplasmic granularity)
against nuclear lobularity (90 ° scatter). The algorithm primarily uses these two scatters for
distinguishing neutrophils from eosinophils. The NEU-EOS plot (Figure 2.3) is particularly
useful for interpretation of cases with abnormal granules, for example gross toxic granulation of
neutrophils or occasional hypogranular eosinophils (see case 12); in both cases events may be seen
in region 1. Hypersegmentation of neutrophils can be recognized when the neutrophil cluster
extends into region 2. When purple or blue colored events are seen in region 3 (see case 32) these
are mononuclear cells with abnormal depolarizing inclusions; these can occur in association with
malaria infection. Rarely hemozoin-containing malaria parasites can be found in region 4.
LEARNING GUIDE: HEMATOLOGY
16
250
250
200
200
GRANULARITY
GRANULARITY
150
150
100
100
50
50
0
0
0 50 100 150 200 250 0 50 100 150 200 250
LOBULARITY LOBULARITY
Figure 2.3: Left: The NEU-EOS scatterplot showing the separation between neutrophils and eosinophils.
Right: The NEU-EOS plot with four regions for abnormal cells. See text for explanation.
MONO-POLY II PLOT
The Mono-Poly II plot has nuclear lobularity (90 ° scatter) plotted as a function of complexity (10 °
scatter; Figure 2.4). This plot is particularly useful to observe the separation between mononuclear
and polymorphonuclear cells.
250
250
200
200
150
150
90 °
90 °
100
100
50
50
0
0
0 50 100 150 200 250 0 50 100 150 200 250

WBC 10 ° WBC 10 °
Figure 2.4: Left: The Mono-Poly II scatterplot with the threshold line separating the mononuclear and polymorphonuclear cell
populations. Right: A graphical impression of the separation between the mononuclear and polymorphonuclear population.
MONO-POLY HISTOGRAM
This histogram (Figure 2.5) shows the separation between the mononuclear and polymorphonuclear
cell populations. This threshold line is set in the valley between the two populations and is based
on the weight points of the mononuclear and polymorphonuclear population in the Mono-Poly II
scatterplot (Figure 2.5). If no clear valley is found the flag DFTL (NLMEB) will be set, indicating
that the WBC differential of CELL-DYN Ruby is not reportable.
MONO-POLY
Figure 2.5: The Mono-Poly II histogram with the threshold line (green) separating the mononuclear and polymorphonuclear populations
17
NONWBC-LYM-MONO HISTOGRAM
This histogram depicts the size of the mononuclear cells (Figure 2.6) and may occasionally be
useful for better recognizing the presence of abnormal cells that overlap with lymphocytes and
monocytes.
0 50 100 150 200 250

NWBC-LYM-MONO
Figure 2.6: The NonWBC-LYM-MONO histogram showing the size distribution of non-WBC events (cell debris,
lyse-resistant RBC, noise), lymphocytes and monocytes. Note that this h
istogram shows mononuclear WBC only.
PLT PLOT AND HISTOGRAMS

The CELL-DYN Ruby provides a number of selectable graphical displays comprising either
histograms or two-dimensional plots. The PLT scattergram, which gives 0 º scatter (platelet size)
as a function of 10 ° scatter (mainly representing platelet granules) is perhaps the most important
as it provides an immediate impression of population separation between platelets (orange events)
and RBC (red events). Regions are marked for understanding only. In Region 1, if the overlap of two
cell populations is seen, the possible existence of RBC microcytes or schistocytes can be considered.
In Region 2, the occurance of events above the platelet population cluster suggest the possible
existence of platelet aggregates, or giant platelets, especially when accompanied by URI/NWBC
warning falgs or an invalidated MPV. In Region 3; Fragile lymphocytes of conditions such as CLL
can be found as a second blue cluster to the left of the red blood cells.
250
250
200
200
150
150
0°
0°
100
100
50
50
0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 10 ° RBC 10 °
Figure 2.7: Left: The PLT scatterplot showing separation of platelets from other small sized events.
Right: The PLT plot with three regions for abnormal cells. See text for explanation.
Of the other display options, the histograms of red cell and platelet volume distributions (Figure
2.8) are also useful for data review. PLT data are shown as a histogram of the 10° data in Figure 2.8c.
Events counted in the region below the lower threshold are usually either optical noise or small
particulate matter. Events counted in the region above the upper threshold are counted as RBC. If
interference with either threshold region exceeds a predetermined limit, the PLT parameters are
flagged accordingly. The flags are discussed in 3.9 and the cases 27, 28 and 29.
18
A B C
0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250
RBC RBC/PLT 0° PLT
Figure 2.8: showing the different RBC and PLT histograms available on CELL-DYN Ruby. All histograms can be very
indicative regarding the RBC and PLT size and helpful in determining interference, especially in the PLT-analysis.
RETC PLOTS AND HISTOGRAMS

The CELL-DYN Ruby reticulocyte measurement provides two 2-dimensional plots and two
histograms. The two scatterplots result from a 3-dimensional optical analysis (0 °, 10 ° and 90 °) and
subsequent automated algorithmic gating procedures. The various cell populations are color-coded
in the 90 °/0 ° and 90 °/10 ° scatterplots as follows:
• Red events – Red blood cells (non-reticulocytes)
• Dark blue events – Mature reticulocytes
• Light blue events – Immature reticulocytes
• Black events – White blood cells
• Orange events – Platelets (at the lower limits of the 0 ° axis and 10 ° axis)
Analytical Considerations: (1) Howell-Jolly Bodies are also stained, but they can be correctly
identified by their characteristic size and shape in mophological examinations. (2) Basophilic
stippling which can be seen in conditions like beta-thalassaemia triat, dyserythropoetic states and
some liver diseases can lead to falsely elevated Reticulocyte counts. Other erythrocyte inclusions
like Heinz Bodies, HBH inclusions and Pappenheimer Bodies can also have analytical intereference.
Details of other interferences can be found on the operation manual. Interferences affect all
automated methods to some extent and so it is important that when samples show unexpectedly
high reticulocyte counts they should be validated by alternative procedures.
Although apparently simple, the reticulocyte histograms can provide critical information regarding
the presence of analytical interferences. Even in extreme reticulocytosis, the cells with a high
90 ° signal display a gradually decreasing slope towards baseline. If there is a sharp peak or
spike in region 1 (Figure 2.9) this is strongly indicative for an interference and should be further
investigated. In contrast to the histograms, the two 2-dimensional RETC plots have little additional
value for case interpretation.
19
250
250
200
200
150
150
90 °
90 °
100
100
50
50
0
0
0 50 100 150 200 250 0 50 100 150 200 250
0 50 100 150 200 250 0 50 100 150 200 250

RETC 90 ° / 0 ° RETC 90 ° / 10 °
Figure 2.9: Showing the reticulocyte 90 °/RETC 0 ° and 90 °/RETC 10 ° scatterplots and their corresponding histograms.
Interference in the reticulocyte count is most clearly visible in the histograms as a sharp peak or spike in region 1.
20
FLAGGING
21
The scatter signals collected for the various cell populations are not only used for constructing the
differential count and the scatterplots, but also for generating flags or alerts in case the presence of
abnormal cells is suspected.
BAND AND IG FLAGS

Band neutrophils and more immature cells of the neutrophil lineage trigger the BAND and/or IG
(Immature Granulocytes) flags. Determining factors are the position and dispersion (spread) of
the neutrophil cell population along the 0°, 10° and 90° scatter axes. Immature neutrophil cells
are larger than mature neutrophils and have lower lobularity as can be seen in Figure 3.1. The
dispersion of complexity is also increased in the more immature stages.
Band and IG is one of the WBC population flag. The BAND flag is triggered if any of the following
conditions are met: 1. The CV of the neutrophil cluster on the 0 degree axis exceeds expected
criteria. 2. %BAND > 2.5% of the total WBC count. 3. The ratio of suspected bands to mature
neutrophils is >50%. The IG flag is triggered if the following condition is met: %IG is greater than
or equal to 3% of the total WBC count. Suggested action in both cases is to review the stained smear.
When bands and IGs are present, they are included in the total neutrophil count. Rules based on
appearance of flags can be created.
250
250
250
200 250
200 250
200 250
150 200
150 200
150 200
SIZE
SIZE
150
150
150
SIZE
SIZE
SIZE
SIZE
100
100
100
100
100
100
5050
5050
5050
00
00
00
0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250
COMPLEXITY COMPLEXITY COMPLEXITY

250
250
250
250
250
250
200
200
200
200
200
200
150
150
150
150
150
150
90 °
90 °
90 °
100
100
100
100
100
100
50
50
50
50
50
50
00
00
00
0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250
WBC 0° WBC 0° WBC 0°
Figure 3.1: Determinants of BAND and IG flagging, using the position and spread of the neutrophil cluster in the WBC
Differential and Mono Poly I scatterplots. Left: normal case, no flag. Center: Band flag. Right: IG flag.
BLAST FLAG
Since blasts, depending on their origin, may have highly variable scatter characteristics the flagging
algorithm for blasts is highly complex. It includes the following two major factors: percentage of
monocytes and the percentage of cells in the Blast-box. The Blast-box is defined as the area above
channel 175 of 0 ° scatter and below channel 50 of 90 ° scatter. If the Blast-box contains more than 1 %
of the total WBC count the Blast flag will be set. So the CELL-DYN Ruby algorithm defines blast cells
as large cells with low lobularity. Occasionally it can occur that blast cells, due to their size are found
in the area between lymphocytes and monocytes and that instead of the blast flag the flag for variant
lymphocytes (VAR LYM) is set.
22
250
250
200
200
150
150
90 °
90 °
100
100
50
50
0
0
0 50 100 150 200 250 0 50 100 150 200 250
WBC 10 ° WBC 10 °
Figure 3.2: Example of Blast flagging. Left: normal case, no flag. Right: Blast flag due to the presence of large mononuclear cells (> 1 %) in the blast-box
VARLYM FLAG
Abnormal lymphocytes, also called variant or atypical lymphocytes, are highly heterogeneous cells
and are comparable to blasts as to their light scatter properties. Therefore the flagging algorithm is
equally complex as blast flagging. Factors that are incorporated into the VARLYM algorithm are the
absolute lymphocyte and monocyte counts, the neutrophil-lymphocyte ratio and the presence of
fragile lymphocytes.
WBC FLAGS
The WBC warning flag is a general flag that is accompanied by another flag: these can be NWBC,
NRBC/RRBC and/or FWBC flags.
WBC FLAG IN COMBINATION WITH NRBC/RRBC FLAG

The WBC reagent contains substances that make RBC lyse, in order to prevent intact RBC
interfering in the WBC analysis. Some patients have RBC that are difficult to lyse and these resistant
RBC remain (partially) intact and can interfere in the CBC mode. The RRBC flag is triggered if there
are more than a preset number of cells with very low size and low complexity in combination with
a declining WOC kinetic rate. The NRBC/RRBC (Nucleated RBC/Resistant RBC) flag is displayed,
alerting the user to rerun the specimen in the CBC+RRBC mode. This mode applies stronger
lysis conditions and successfully removes the resistant RBC (Figure 3.3). Lyse-resistant RBC are
often found in blood from newborns and in patients with disorders affecting RBC membranes, for
example liver disease and sickle cell disease.
Resistant RBC can best be assessed using the WBC Differential scatterplot, where they are found in
region 1 (Figure 3.3). A WBC flag invalidates the WBC and WBC differential counts.
250
250
200
200
150
150
SIZE
SIZE
100
100
50
50
0
0 50 100 150 200 250 0 50 100 150 200 250

Figure 3.3: Example of resistant RBC. Left: lyse-resistant RBC are present in the lower left region of the WBC Differential scatterplot, interfering with the
lymphocytes and WBC counts. Right: In CBC+RRBC mode all erythrocytes are successfully lysed; interference is eliminated.
23
WBC FLAG IN COMBINATION WITH FWBC FLAG

When processing samples in the CBC mode, the WBC (WOC) count may be spuriously low due
to the presence of fragile WBC. These cells suffer from gradual destruction of their cytoplasmic
membrane by the lysing agents during the run cycle.
Typically fragile WBC are abnormal lymphocytes that are seen in chronic lymphocytic leukemia
(CLL) and are the “smudge cells” that appear when the blood smear is made. Also WBC in aged
blood samples may become fragile and trigger the FWBC flag.
When the FWBC flag displays, rerun the specimen using the CBC+NOC mode.
This mode uses the HGB sample dilution containing intact WBC nuclei. The Nuclear Optical Count
(NOC) provides a more accurate WBC count when fragile WBC are present.
NWBC FLAG
When a significant population of cellular debris is present (region 1) and there is no declining WOC
kinetic rate, the NWBC flag is set.
Events in this region may be indicative for platelet clumps, giant platelets or low levels of NRBC and
it is advised to set the laboratory’s review criteria accordingly. If no other suspect flags are present,
the WBC and differential are reliable and may be reported.
DFLT (NLMEB) FLAG

The DFLT flag is set to indicate that the WBC differential or a part of the WBC differential could
not be reliably reported and that the algorithm has used its default settings for the separation of the
subpopulations. This occurs in one or more of the following conditions:
• The presence of fragile cells. If the FWBC flag is triggered the DFLT (NLMEB) flag is always set.
• A too low number of cells is available for calculating the WBC differential.
• The mono-poly histogram has too much interference in the valley between both cell types.
NOTE: The letters in parentheses indicate which WBC subpopulation or group of subpopulations
is suspect. Depending on the condition, different DFLT flags ((NLMEB), (NE), (LM), (B), and (LB))
can be set indicating where the issue occurred.
A. If the DFLT (NLMEB) flag is accompanied with the FWBC flag, repeat in CBC+NOC test
selection. B. Review scatterplot for clear separation of cell cluster. C. Review a stained smear to
verify the differential values.
RBC MORPHOLOGY FLAG

The RBC Morphology flag denotes an abnormality in the RBC population and is set when there
is increase in the RDW or if the RBC indices (MCH and/or MCHC) are grossly increased or
decreased.
MCHC FLAG
The MCHC flag is set to warn the operator that the MCHC parameter shows a distinct deviation
from the normal value. This deviation may be related to a RBC abnormality but it may also be
indicative for a pre-analytical effect resulting in spurious results. It is advised to verify that the
specimen was properly mixed by following the laboratory’s protocol for flagged RBC indices.
24
PLATELET ALERTS
The Lower, Upper, or Lower and Upper Region Interference (LRI, URI and LURI) flag are added to
the PLT count and other PLT parameters for indicating the possibility of Lower Range Interference
and/or Upper Range Interference in the PLT histogram. The alerts are triggered by abnormally
sized particles in the PLT assay. Very low-volume particles may be abnormally small platelets,
fragments of other blood cells or even particles not originating from blood. At the high volume end,
interference may be caused by giant platelets, small RBC, other blood cell fragments or non-blood
cell particles.
Platelet LRI: Displayed next to MPV Cause: Interference in the lower threshold region (1-3fL) IS
GREATER THAN PREDEFINED LIMITS. Action Required: Check background counts, if that
exceeds recommended limits, trouble shoot accordingly. If the background count is acceptable,
repeat the analysis or review the smear to verify CD Ruby platelet counts to determine the cause of
interference; which can be due to Debris, Contaminated Reagent, Electronic NOise, Microbubbles,
cell fragments, protein aggregates.
Platelet URI: 1. Interference in the upper threshold region (15–35fL) > 25% of PLT peak. 2. PLT
aggregate count (PLT clumps) > 15% of PLT count. NOTE: URI may be caused by: Microcytic RBC,
Schistocytes, Giant Platelets, Sickle Cells, Platelet Clumps.
25
CASES
26
CASE 1: NORMAL
The graphical displays shown for this hematologically normal sample are those that are most
commonly used for routine laboratory review. For the SIZE/COMPLEXITY plot of WBC
distributions, the following points should be noted:
• C
lear separation between granulocytes (neutrophils plus eosinophils) and mononuclear cells
(lymphocytes plus monocytes)
• Discrete clustering of the basophil population
• Eosinophil localization below neutrophils
• Relatively few events below the size threshold line
In the second WBC plot (GRANULARITY versus LOBULARITY), the separation of eosinophils
from neutrophils can be seen. Note also that the neutrophil population barely extends to the
upper limit of the LOBULARITY axis. This is consistent with a normal degree of segmentation
or lobulation. In the middle the two mono/poly scattergrams are plotted: the left plot (90 º/10 º)
again demonstrates the clear separation between granulocytes and mononuclear cells and the right
plot (90 º/0 º) the absence of large mononuclear cells. The right plot also gives a nice indication
regarding neutrophil lobularity.
The two lower plots show the normal Gaussian distribution of RBC volumes (lower right) and the
separation between platelets and RBC (lower left) that would be expected with samples containing
normocytic RBC, normal-sized platelets and an absence of interferences. With the latter plot, the
events coded as blue (WBC) form a single cluster only.
Morphological Images
150 200 250
150 200 250

WBC 5.90 10e3/uL
NEU 2.579 43.828 %
GRANULARITY
BLST .001 .018 %
SIZE
MONe .618 10.5 %

100
100
EOS .318 5.40 %
BASO .091 1.55 %
50
50
Normal segmented neutrophil Normal segmented eosinophil LYM 2.287 38.791 %

0
0 50 100 150 200 250 0 50 100 150 200 250

COMPLEXITY LOBULARITY
RBC 4.14 10e6/uL
HGB 12.8 g/dL
150 200 250
150 200 250
HCT 36.8 %
MCV 88.8 fL
MCH 31.0 pg
90 °
90 °
MCHC 34.9 g/dL

100
100
RDW 10.7 %
50
50
Normal lymphocyte Normal segmented basophil

PLT 340. 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250

MPV 7.01 fL WBC 10 ° WBC 0 °
150 200 250
0°
100
Normal segmented monocyte.

50
Note folded nucleus and opaque 0 50 100 150 200 250 0 50 100 150 200 250
cytoplasm with occasional small
0
RBC 10 ° RBC
cytoplasmic vacuoles
27
CASE 2A: IRON DEFICIENCY
150 200 250
150 200 250

ANEMIA WBC
NEU
6.95
3.742
10e3/uL
53.846 %
GRANULARITY
BLST 0.00 0.00 %
This sample shows RBC microcytosis
SIZE
MONe .488 7.03 %
100
100
and is further characterized by anemia EOS
BASO
.180
.043
2.60
.622
%
%
50
50
and a decreased MCHC (< 30 g/dL). LYMe 2.67 35.892 %
0
The RBC and WBC counts are relatively RBC 4.21 10e6/uL
0 50 100
COMPLEXITY
150 200 250 0 50 100
LOBULARITY
150 200 250
normal, this in combination with an
150 200 250

HGB 7.73 g/dL
150 200 250

increased platelet count. Review of HCT
MCV
29.6
70.0
%
fL RBC MORPH
the graphical displays does not reveal MCH 18.3 pg
90 °
90 °
anything of major note and even, despite MCHC 26.2 g/dL
100
100
RDW 12.8 %
the low MCV, the separation between
50
50
the platelets and RBC (lower left plot) PLT 647. 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
MPV 5.58 fL WBC 10 ° WBC 0 °
is still perfect. This profile is consistent
150 200 250

with iron deficiency anemia (IDA), but
supplementary investigations would be
required to confirm this.
0°
100
50
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
LEARNING GUIDE: HEMATOLOGY

28
CASE 2B AND C: IRON DEFICIENCY
150 200 250
150 200 250

ANEMIA AFTER THERAPY WBC 6.39 10e3/uL
NEU 3.402 53.158 %
GRANULARITY
BLST .002 .033 %
After diagnosed with a classical iron
SIZE
MONe .439 6.87 %
100
100
deficiency, the patient from case 2a EOS .301 4.71 %
BASO .110 1.72 %
50
50
received oral iron supplementation LYM 2.145 33.461 %
therapy. Due to the newly produced
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 4.72 10e6/uL
normocytic RBC’s the RBC histogram HGB 11.0 g/dL
150 200 250
150 200 250

became bimodal (Case 2b) and the RDW HCT 36.5 %
MCV 77.4 fL RBC MORPH
increased from 12.8 % to 28.8 %. After 3 MCH 23.3 pg
90 °
90 °
weeks of therapy patient still had anemia MCHC 30.1 g/dL
100
100
RDW 28.8 %
but it is clear that she responded well to
50
50
iron therapy. PLT 402. 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
MPV 7.18 fL WBC 10 ° WBC 0 °
After 5 weeks of therapy (Case 2c)
150 200 250

the bimodal RBC histogram was even
more distinct indicating that it is only a
0°
matter of time before all RBC’s will be
100
normocytic again.
50
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
Case 2b
150 200 250
150 200 250

WBC 9.13 10e3/uL
NEU 4.633 50.805 %
GRANULARITY
BLST .001 .012 %
SIZE
MONe .657 7.20 %

100
100
EOS 1.08 11.8 %
BASO .070 .772 %
50
50
LYM 2.685 29.39 %
0
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 4.82 10e6/uL
HGB 12.1 g/dL
150 200 250
150 200 250

HCT 39.3 %
MCH 25.2 pg
90 °
90 °
MCHC 30.9 g/dL
100
100
RDW 26.0 %
50
50
PLT 323. 10e3/uL

0
0 50 100 150 200 250 0 50 100 150 200 250

MPV 6.87 fL WBC 10 ° WBC 0 °
150 200 250
0°
100
50
0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 10 ° RBC
Case 2c
29
CASE 3: α-THALASSEMIA
150 200 250
150 200 250

WBC 5.67 10e3/uL
NEU 2.43 42.861 %
GRANULARITY
Case 3 differs in that the patient is BLST 0.00 0.00 %
SIZE
MONe .495 8.72 %
not particularly anemic and the RBC
100
100
EOS .179 3.16 %
count is clearly increased. This pattern BASO .045 .798 %
50
50
LYM 2.521 44.486 %
of microcytic erythrocytosis could be
0
0 50 100 150 200 250 0 50 100 150 200 250
suggestive of thalassemia, which was RBC 6.41 10e6/uL

150 200 250
150 200 250

HGB 12.0 g/dL
later confirmed by supplementary HCT 40.1 %
examination. The low MCH is MCV 62.6 fL RBC MORPH
compatible with α- or β-thalassemia,

MCH 18.7 pg
90 °
90 °
MCHC 30.0 g/dL
100
100
but does not exclude concomitant iron RDW 12.4 %
50
50
deficiency. PLT 315. 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
MPV 7.07 fL WBC 10 ° WBC 0 °
150 200 250

0°
100
50
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
CASE 4: MACROCYTIC ANEMIA
150 200 250
150 200 250

WBC 6.61 10e3/uL
This patient with a history of alcohol NEU 4.114 62.26 %
GRANULARITY
BLST 0.00 0.00 %
abuse had anemia. Her RBC were
SIZE
MONe .614 9.29 %

100
100
macrocytic and slightly hypochromic; EOS .118 1.79 %
BASO .115 1.74 %
50
50
in addition, she had elevated liver LYM 1.651 24.97 %
enzymes and a normal serum ferritin

0
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 2.89 10e6/uL
concentration. The reticulocyte count HGB 9.97 g/dL
150 200 250
150 200 250

was 84.4 x 109/L (2.9 %) and is shown as HCT
MCV
31.8
110.
%
fL RBC MORPH
case 34. MCH 34.5 pg
90 °
90 °
MCHC 31.4 g/dL

100
100
RDW 21.4 %
50
50
PLT 475. 10e3/uL

0
0 50 100 150 200 250 0 50 100 150 200 250

MPV 4.85 fL WBC 10 ° WBC 0 °
150 200 250
0°
100
50
0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 10 ° RBC
30
CASE 5: MEGALOBLASTIC ANEMIA
150 200 250
150 200 250

WBC 7.72 10e3/uL
This 68 year old male patient presented NEU 4.653 60.226 % BAND
GRANULARITY
BLST .001 .014 %
with a mild anemia in combination
SIZE
MONe .656 8.49 %
100
100
with a distinct macrocytosis, a
EOS .181 2.34 %
BASO .054 .698 %
50
50
decreased RBC concentration and an LYM 2.175 28.255 %
0
elevated MCH. Remarkably also is
0 50 100 150 200 250 0 50 100 150 200 250
RBC 2.67 10e6/uL
the neutrophil position in the SIZE/ HGB 12.1 g/dL
150 200 250
150 200 250

SUSPECT
HCT 34.0 %
COMPLEXITY scatterplot indicating MCV 127. fL RBC MORPH
that the neutrophils are larger MCH 45.4 pg
90 °
90 °
MCHC 35.6 g/dL
than normal and based on this the
100
100
RDW 13.4 %
CELL-DYN Ruby generated the BAND
50
50
PLT 332. 10e3/uL
flag. Another notable observation is the
0
0 50 100 150 200 250 0 50 100 150 200 250
MPV 6.54 fL WBC 10 ° WBC 0 °
high lobularity signal of the neutrophils,
150 200 250

very well visible in the 90 º/WBC 0 º
and the GRANULARITY/LOBULARITY
scatterplots. The blood smear confirmed
0°
100
the presence of large (giant) neutrophils
50
in combination with hypersegmentation.
0
0 50 100 150 200 250 0 50 100 150 200 250
After additional investigations the patient RBC 10 ° RBC
was diagnosed with megaloblastic anemia

due to vitamin B12 deficiency.
CASE 6: AUTO-IMMUNE SUSPECT

150 200 250
150 200 250

HEMOLYTIC ANEMIA WBC
NEU
11.3
3.436
10e3/uL
30.522 %
GRANULARITY
BLST .001 .010 %
This patient was a 76 year old man
SIZE
MONe .448 3.98 %

100
100
with a history of chronic lymphocytic EOS
BASO
.062
.109
.553
.965
%
%
50
50
leukemia, initially diagnosed 2 years LYM 7.20 63.985 % VAR LYM
0
0
ago. Over the past three weeks RBC 1.78 10e6/uL
0 50 100
COMPLEXITY
150 200 250 0 50 100
LOBULARITY
150 200 250
the patient had noted progressive

150 200 250
150 200 250

HGB 8.09 g/dL
fatigue and shortness of breath with HCT

MCV
22.3
125.
%
fL
exertion. Laboratory examination MCH 45.4 pg
90 °
90 °
showed a severe anemia and a MCHC 36.3 g/dL

100
100
RDW 22.2 %
disproportionately low RBC count.
50
50
MCV was strongly increased, due to PLT 244. 10e3/uL

0
0 50 100 150 200 250 0 50 100 150 200 250

MPV 6.84 fL WBC 0 °
the massive reticulocytosis (see case
36). Morphology showed numerous
spherocytes as well as polychromatic
macrocytic erythrocytes. Additional
studies revealed 35 % reticulocytosis, an
elevated LDH (960 IU/L), and a positive 0 50 100 150 200 250 0 50 100 150 200 250
direct Coomb’s test. PLT RBC
31
CASE 7A AND B: GRANULOCYTOSIS
150 200 250
150 200 250

WBC 36.5 10e3/uL
NEU 34.6 94.9 % BAND
GRANULARITY
Both cases (a and b) show a significant MONe 1.39 3.82 %
granulocytosis (neutrophilia). For
SIZE
EOS .011 .030 %
100
100
BASO .047 .130 %
Case 7a, the neutrophil cluster retains LYM 0.409 1.12 %
50
50
a relatively normal shape but its
0
RBC 2.70 10e6/uL 0 50 100 150 200 250 0 50 100 150 200 250
position is slightly higher on the SIZE HGB 7.79 g/dL
axis compared to normal. The sample HCT 23.9 %
150 200 250
150 200 250

MCV 88.5 fL
also shows monocytosis, moderate MCH 28.8 pg
normocytic anaemia and a high normal MCHC 32.6 g/dL
90 °
RDW 13.4 %
100
100
platelet count. The BAND flag suggests
50
50
the possibility of a left shift. PLT 461. 10e3/uL
MPV 6.44 fL
0
0 50 100 150 200 250 0 50 100 150 200 250
For comparison, Case 7b (51 year old RBC 10 ° RBC
male following nephrectomy for renal Case 7a

cell carcinoma) shows a less marked
neutrophilia, with relatively normal
150 200 250
150 200 250

RBC and platelet results. With regards WBC 16.3 10e3/uL
NEU 13.4 82.0 %
GRANULARITY
to the graphical displays, the neutrophil MONO .835 5.12 %
SIZE
population cluster (upper left plot) is EOS .025 .151 %
100
100
BASO .057 .351 %
also relatively normal but the extended LYM 2.02 12.4 %
50
50
location of the cluster along the
0
RBC 4.22 10e6/uL 0 50 100 150 200 250 0 50 100 150 200 250
LOBULARITY axis in the other WBC HGB 13.1 g/dL
plot (upper right) indicates that the HCT 39.6 %
150 200 250
150 200 250

MCV 93.8 fL
neutrophils are hypersegmented. MCH 31.1 pg
MCHC 33.1 g/dL
90 °
RDW 11.0 %
100
100
50
50
PLT 229. 10e3/uL
MPV 9.78 fL
0
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
Case 7b
Non-segmented neutrophil (Band Cell) Neutrophil hypersegmentation
32
CASE 8A AND B: IMMATURE
150 200 250
150 200 250

WBC 17.1 10e3/uL
GRANULOCYTES (IG) NEU 16.1 94.2 % IG/BAND
GRANULARITY
MONe .221 1.29 %
In contrast to the previous two cases (7a
SIZE
EOS .00 .00 %
100
100
BASO .041 .243 %
and 7b), the granulocytosis in these two LYM .736 4.31 %
50
50
patients is characterised by distinctively
0
RBC 2.61 10e6/uL 0 50 100 150 200 250 0 50 100 150 200 250
abnormal neutrophil population cluster HGB 8.47 g/dL
shapes. This cluster pattern is suggestive HCT 24.1 %
150 200 250
150 200 250

MCV 92.2 fL
of immature granulocytes (IG), and this MCH 32.4 pg
possibility is further indicated by the MCHC 35.1 g/dL
90 °
RDW 13.6 %
100
100
presence of IG/BAND flagging alerts.
50
50
There are no other major abnormalities PLT 68.2 10e3/uL
MPV 10.2 fL
with regards to the WBC differentials
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
although the increased monocyte and
Case 8a
basophil counts in Case 8b may have
relevance in the context of possible
150 200 250
150 200 250

WBC 56.9 10e3/uL
myeloproliferative disease. Both patients NEU 50.3 88.4 % IG/BAND
GRANULARITY
also show a moderate normocytic MONe 4.28 7.53 %
SIZE
EOS .029 .050 %
anaemia and mild to moderate
100
100
BASO .520 .913 %
reductions in the absolute platelet count. LYM 1.75 3.08 %
50
50
Reference microscopic review of these
0
RBC 3.12 10e6/uL 0 50 100 150 200 250 0 50 100 150 200 250
two samples revealed the presence of HGB 8.80 g/dL

HCT 27.7 %
150 200 250
150 200 250

19% IG in Case 8a and 16% IG in Case 8b. MCV 88.8 fL
MCH 28.2 pg
MCHC 31.8 g/dL
90 °
RDW 17.4 % 100
100
50
50
PLT 135. 10e3/uL
MPV 8.85 fL
0
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
Case 8b
Immature Granulocyte Immature Granulocyte

(neutrophilic) showing a (neutrophilic) with a single, slightly
single nucleus, nucleolation, indented, nucleus (Myelocyte/
and cytoplasmic granulation Metamyelocyte)
(Promyelocyte/Myelocyte)
33
CASE 9: LEFT SHIFT OF SUSPECT
150 200 250

150 200 250
GRANULOPOIESIS WITH IG FLAG WBC 14.0 10e3/uL
GRANULARITY
NEU 8.569 61.04 % IG
BLST 0.00 0.00 %
In this patient a clearly increased
SIZE
MONe 2.53 18.0 %
100
100
WBC count was found, accompanied EOS .003 .020 %
50
50
BASO .155 1.10 %
by an IG flag. In all scatterplots the LYM 2.776 19.84 %
neutrophil cluster was more dispersed
0
0
0 50 100 150 200 250 0 50 100 150 200 250
than normal, indicating increased RBC 3.20 10e6/uL
150 200 250
150 200 250

HGB 10.1 g/dL
heterogeneity of the neutrophil series. HCT 29.2 %
The size of most neutrophils was MCV

MCH
91.4
31.7
fL
pg
90 °
90 °
more or less normal but at the same
100
100
MCHC 34.7 g/dL
time part of the neutrophils show RDW 17.2 %
50
50
larger size in combination with lower PLT 74.4 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
complexity. Looking at the lobularity it MPV 7.80 fL WBC 10 ° WBC 0 °
is also evident that the position of the
150 200 250

neutrophil population on the 90 ° signal
was lower than in a normal sample,
0°
100
indicating that most neutrophils tend to
have a decreased lobularity.
50
0
0 50 100 150 200 250 0 50 100 150 200 250
This combination of results is typical of RBC 10 ° RBC
neutrophilia with more severe left shift.
CASE 10: EOSINOPHILIA

150 200 250
150 200 250

WBC 9.17 10e3/uL
This patient had an increased WBC
GRANULARITY
NEU 3.529 38.505 %
BLST 0.00 0.00 %
count as a consequence of eosinophilia.
SIZE
MONe .500 5.45 %

100
100
There were no other hematological EOS 2.86 31.2 %
50
50
abnormalities. The GRANULARITY/ BASO
LYM
.119
2.161
1.30 %
23.606 %
LOBULARITY plot demonstrated the
0
0
0 50 100 150 200 250 0 50 100 150 200 250
clear separation of eosinophils and RBC 4.34 10e6/uL
150 200 250
150 200 250

HGB 13.1 g/dL
neutrophils using the polarized and HCT 38.7 %
depolarized 90 ° scatter. MCV 89.1 fL
MCH 30.2 pg
90 °
90 °
Eosinophilia of this magnitude is

100
100
MCHC 33.9 g/dL
RDW 11.4 %
well compatible with severe allergy,
50
50
parasitic infections or with eosinophilic PLT 192. 10e3/uL

0
0 50 100 150 200 250 0 50 100 150 200 250
malignancies. MPV 7.68 fL WBC 10 ° WBC 0 °

150 200 250
Eosinophilia, greater than 450 to

500 eosinophils/μL in peripheral
blood, is a hallmark of or a related
0°
100
finding in many allergic, infectious,

50
autoimmune, idiopathic, malignant,

0
0 50 100 150 200 250 0 50 100 150 200 250

and miscellaneous clinical scenarios. RBC 10 ° RBC
The clinical history is often the most

important clue in discovering a pathway
by which the patient is possibly affected
by eosinophilia. Tailored evaluation
based on scenario, including allergy
testing, laboratory testing, imaging, and
pathologic biopsy of affected areas can
be useful in confirming a diagnosis.
34
CASE 11: EOSINOPHILIA*
150 200 250
150 200 250

WBC 6.90 10e3/uL
In this patient eosinophilia was seen on
GRANULARITY
NEU 3.51 50.839 %
BLST 0.00 0.00 %
a background of an otherwise normal
SIZE
MONe .443 6.42 %
100
100
WBC count, although the eosinophilia EOS 1.20 17.3 %
50
50
was still markedly elevated. Note that an BASO
LYM
.054
1.699
.784 %
24.624 %
eosinophil count > 0.40 x 109/L already
0
0 50 100 150 200 250 0 50 100 150 200 250
is defined as eosinophilia and that RBC 4.20 10e6/uL
150 200 250
150 200 250

HGB 13.5 g/dL
increased eosinophil counts generally HCT 37.6 %
are less than 1.0 x 109/L. Noticeable MCV

MCH
89.5
32.1
fL
pg
90 °
90 °
in this case is also the location of the
100
100
MCHC 35.9 g/dL
eosinophils in the GRANULARITY/ RDW 11.9 %
50
50
LOBULARITY scatterplot, indicating
0
PLT 153. 10e3/uL 0 50 100 150 200 250 0 50 100 150 200 250
that both the eosinophilic granularity MPV 9.68 fL WBC 10 ° WBC 0 °
and lobularity are less than normal.
150 200 250

When performing a manual differential
special attention should be paid if the
0°
100
eosinophils show signs of degranulation
50
as the combination of an elevated
eosinophil concentration with
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
degranulation can be indicative for more
severe pathology.
CASE 12: BASOPHILIA

150 200 250
150 200 250

WBC 5.47 10e3/uL
Basophilia is often seen in relatively
GRANULARITY
NEU 2.965 54.275 %
BLST 0.00 0.00 %
complex hematological pictures in
SIZE
MONe .617 11.3 %

100
100
which also basophils mostly are involved EOS .194 3.56 %
50
50
BASO .175 3.20 %
and may present themselves in an LYM 1.517 27.679 %
unpredictable way. In this case only
0
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 4.71 10e6/uL
a mild basophilia of 0.175 x 109/L was
150 200 250
150 200 250

HGB 12.4 g/dL
present, but clearly showed where to HCT 37.9 %
expect basophils in CELL-DYN Ruby MCV

MCH
80.4
26.4
fL
pg
90 °
90 °
scatterplots.
100
100
MCHC 32.8 g/dL
RDW 14.3 %
50
50
Non-malignant conditions associated

PLT 296. 10e3/uL
with mild to moderate basophilia
0
0 50 100 150 200 250 0 50 100 150 200 250

MPV 7.46 fL WBC 10 ° WBC 0 °
include hypothyroidism, IgE-mediated
150 200 250
hypersensitivity reactions, inflammatory

disorders such as rheumatoid arthritis
and ulcerative colitis, and some viral
0°
100
infections. Basophilia is a common

50
finding in chronic myeloid leukaemia

0
0 50 100 150 200 250 0 50 100 150 200 250

(CML) and (to a lesser extent) the RBC 10 ° RBC
other myeloproliferative disorders.

Basophils in these conditions may have
atypical morphological features, and an
increasing basophil count is an indicator
of disease progression/acceleration.
*Eosinophilia, greater than 450 to 500 eosinophils/µL in peripheral blood, is a hallmark of or a related finding in many allergic, infectious,
autoimmune, idiopathic, malignant, and miscellaneous clinical scenarios. The clinical history is often the most important clue in discovering a
pathway by which the patient is possibly affected by eosinophilia. Tailored evaluation based on scenario, including allergy testing, laboratory
LEARNING GUIDE:
testing, imaging, and HEMATOLOGY
pathologic biopsy of affected areas can be useful in confirming a diagnosis.
35
CASE 13: MONOCYTOSIS
150 200 250
150 200 250

WBC 8.16 10e3/uL
This patient had monocytosis of 1.90 x
GRANULARITY
NEU 4.54 55.715 %
BLST .002 .026 %
109/L. The SIZE/COMPLEXITY and
SIZE
MONe 1.90 23.3 %
100
100
the 90 º/WBC 0 º scatterplots show EOS .047 .575 %
a clear separation of the lymphocyte
50
50
BASO .058 .709 %
LYM 1.616 19.77 %
and the monocyte populations. The
0
0 50 100 150 200 250 0 50 100 150 200 250
monocytosis did not trigger a flag because RBC 3.38 10e6/uL
150 200 250
150 200 250

HGB 9.43 g/dL
their size distribution was within pre- HCT 29.1 %
defined limits. The monocytes were MCV 86.3 fL
pg
morphologically normal. This picture MCH 27.9
90 °
90 °
100
100
MCHC 32.4 g/dL
might be associated with certain RDW 14.0 %
50
50
infections, notably tuberculosis. PLT 211. 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
WBC 10 ° WBC 0 °
Monocytosis can be manifested in chronic MPV 6.76 fL
150 200 250

inflammatory conditions, Infections:
tuberculosis, brucellosis, listeriosis,
subacute bacterial endocarditis, syphilis,
0°
100
and other viral infections and many
50
protozoal and rickettsial infections .
Blood and immune causes can be: chronic
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
neutropenia and myeloproliferative
disorders.
Autoimmune diseases and vasculitis:
systemic lupus erythematosus,
rheumatoid arthritis and inflammatory
bowel disease. Malignancies: Hodgkin’s
disease and certain leukaemias, such
as chronic myelomonocytic leukaemia
(CMML) and monocytic leukemia.
CASE 14: LYMPHOCYTOSIS SUSPECT

150 200 250
150 200 250

WBC 22.0 10e3/uL
SIZE
This patient had lymphocytosis,

GRANULARITY
NEU 2.633 12.006 %
BLST .056 .255 %
accompanied by a VAR LYM flag. In the MONe .203 .923 %
100
100
WBC Differential plot the lymphocytes EOS .084 .382 %

50
50
BASO .091 .414 %

and monocytes seemed to form a LYM 18.97 86.09 % VAR LYM
single coherent population. The SIZE/
0
0 50 100 150 200 250 0 50 100 150 200 250

COMPLEXITY and the 90 º/WBC 0 º RBC 4.70 10e6/uL
150 200 250
150 200 250
HGB 12.4 g/dL

scatterplot demonstrated that part HCT 37.4 %
of the lymphocytes were classified as MCV 79.5 fL

MCH 26.3 pg
90 °
90 °
monocytes. It were actually the larger

100
100
MCHC 33.1 g/dL

lymphocytes that were considered as RDW 12.9 %
50
50
monocytes, fully in agreement with the PLT 374. 10e3/uL

0
morphological observation of atypical, MPV 4.22 fL 0 50 100 150 200 250 0 50 100 150 200 250
monocytoid lymphocytes. WBC 10 ° WBC 0 °

150 200 250
This combination of findings is

highly characteristic of Infectious
Mononucleosis, a diagnosis that was
0°
100
confirmed in this patient with serological

50
testing.
0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 10 ° RBC
36
CASE 15A: CHRONIC LYMPHOCYTIC SUSPECT
150 200 250
150 200 250

LEUKEMIA (CLL) WBC 67.8* 10e3/uL WBC
GRANULARITY
NEU 5.268* 77.717* %
BLST .003* .052* % FWBC
This sample analysis shows a severe
SIZE
MONe .006* .086* %
100
100
lymphocytosis of 67.8 x 109/L in EOS .004* .063* %
50
50
BASO .217* .320* % DFLT (NLMEB)
combination with a mild hypochromic LYM 62.32* 91.59* % VARLYM
anemia. Several warning flags are
0
0 50 100 150 200 250 0 50 100 150 200 250
triggered. RBC 4.13 10e6/uL
150 200 250
150 200 250

HGB 10.0 g/dL
HCT 31.5 %
The FWBC flag suggests the presence of MCV 76.3 fL
fragile white blood cells and consequently MCH 24.2 pg
90 °
90 °
100
100
MCHC 31.7 g/dL
the WBC and DFLT(NLMEB) flags RDW 16.1 %
50
50
are triggered, thus the WBC count and
associated differential may be incorrect. PLT 244. 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
MPV 7.74 fL WBC 10 ° WBC 0 °
Strongly indicative for the presence of
150 200 250

fragile white cells is also the additional
blue (WBC) population, located at the left
from the RBC population in the 0º/RBC
0°
100
10º scatterplot.
50
Combined triggering of WBC and FWBC
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
flag means that it is necessary to perform
a nuclear optical count (NOC) to provide
an accurate white blood cell count. This
is because fragile white cells may not
be “seen” by the optical analysis and
may thus be underestimated. The NOC
count however counts all white cells
irrespectively of whether or not they are
fragile.
CASE 15B: CHRONIC LYMPHOCYTIC

150 200 250
SUSPECT
150 200 250

LEUKEMIA (CLL) WBC 85.6 10e3/uL NOC GRANULARITY
NEU 5.786 6.761 %
BLST .017* .020* % FWBC
This analysis corresponds to the
SIZE
MONe .861* 1.01* %

100
100
previous sample (case 15a) when EOS .052* .060* %

50
50
BASO 2.32* 2.72* % DFLT (NLMEB)

processed with the CELL-DYN Ruby LYM 76.52* 89.41* % VAR LYM
0
CBC+NOC mode. The instrument lyses

0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 4.17 10e6/uL
the cytoplasm of all white blood cells
150 200 250
150 200 250
HGB 10.1 g/dL

and counts the nuclei thus providing a HCT 31.7 %
correct WBC count MCH 24.1 pg
90 °
90 °
100
100
MCHC 31.7 g/dL

(85.6 x 109/L) compared to that obtained RDW 16.3 %
50
50
in the normal CBC mode (67.8 x 109/L).

0
PLT 238. 10e3/uL

0
0 50 100 150 200 250 0 50 100 150 200 250

MPV 7.64 fL WBC 10 ° WBC 0 °
Note that when analyzed in the CD
150 200 250
Ruby CBC+NOC mode, the VAR LYM

and FWBC flags remain. Additional, the
(NOC) flag indicates that the WBC count
0°
100
and differential are now referred to the

50
NOC analysis.
0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 10 ° NOC
37
CASE 16: MANTLE CELL LYMPHOMA
100 150 200 250

100 150 200 250
SUSPECT
WBC 16.7 10e3/uL
This 60 year old male patient was referred NEU 4.08 24.4* %
LYM 10.3* 61.6* % VAR LYM
by his general practitioner to a specialist
SIZE
90 °
MONO 2.03* 12.2* %
because of persisting lymphadenopathy. EOS .201 1.20 %
50
50
BASO .102 .611 % DFLT (L M)
The peripheral blood count on
0
0
0 50 100 150 200 250 0 50 100 150 200 250
CELL-DYN Ruby showed RBC 4.23 10e6/uL COMPLEXITY WBC 0°
HGB 12.5 g/dL
thrombocytopenia and leukocytosis HCT 39.2 %
100 150 200 250

which was accompanied by the DFLT MCV 92.6 fL
MCH 29.5 pg
(LM) flag, indicating that the algorithm MCHC 31.9 g/dL
0°
was unable to make a clear separation RDW 13.9 %
between the lymphocyte and monocyte
50
PLT 98.2 10e3/uL
population. Visual interpretation of the MPV 12.7 fL
0
0 50 100 150 200 250 0 50 100 150 200 250
WBC scatterplots and the NWBC-LYM- NWBC-LYM-MONO WBC 10 °
MONO histogram suggested that the

default threshold lines spuriously classified
part of the “lymphocyte” population as
monocytes. This finding was confirmed by
microscopic examination, which showed
mainly medium-sized pleomorphic
lymphocytes with irregular nuclei.
Immunological typing of the leukemic cells
and lymph nodes confirmed the diagnosis
of mantle cell lymphoma.
CASE 17: ACUTE LYMPHOCYTIC

LEUKEMIA (ALL) SUSPECT
150 200 250
150 200 250

WBC 82.0 10e3/uL
GRANULARITY
NEU 2.61 3.18 %
This young girl presented with an evident BLST .056 .068 %
SIZE
MONe .203 .248 %

leukocytosis (82.0 x 109/L), primarily
100
100
EOS .084 .103 %
due to lymphocytosis (78.9 x 109/L). The
50
50
BASO .091 .111 %
leukocytosis was combined with anemia LYM 78.95 96.3 % VAR LYM
0
0
0 50 100 150 200 250 0 50 100 150 200 250
and thrombocytopenia. The graphical WBC RBC 3.27 10e6/uL
150 200 250
essentially showed a single population 150 200 250

HGB 9.12 g/dL
HCT 28.5 %
cluster with moderate size variability, MCV 87.2 fL
triggering the VAR LYM flag. MCH 27.9 pg

90 °
90 °
100
100
MCHC 32.0 g/dL

RDW 12.9 %
Although the predominant population was
50
50
nominally classified as lymphoid, the total PLT 36.5* 10e3/uL

0
0 50 100 150 200 250 0 50 100 150 200 250

WBC 10 ° WBC 0 °
picture was highly indicative for high- MPV 3.95* fL
grade malignancy such as lymphoblastic

150 200 250
lymphoma or acute leukemia. Microscopic LRI
assessment of the smear revealed that

0°
100
practical all leukocytes were blasts.

50
Immunological typing of the leukemic cells

revealed them to be of the T-cell lineage.
0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 10 ° RBC
Finally, the LRI flag indicated the presence

of lower range interference in the platelet
count. The LRI flag is relatively commonly
seen in the condition where a low platelet
concentration is accompanied by high-grade
proliferations that have circulating cell
fragments or abnormal plasma components.
38
CASE 18: ACUTE LEUKEMIA
150 200 250
150 200 250

WBC 38.4 10e3/uL WBC
NEU .460 1.20 %
The sample shows all the features
GRANULARITY
LYM 37.2 96.9 % VAR LYM
of a high-grade malignancy such as
SIZE
MONO .317 .825 % FWBC
100
100
EOS .004 .010 %
lymphoblastic lymphoma or acute BASO .398 1.04 % DFLT (NLMEB)
50
50
leukaemia. There is an increased WBC
0
count, a marked neutropenia and severe RBC 2.11 10e6/uL 0 50 100 150 200 250 0 50 100 150 200 250
HGB 7.27 g/dL
thrombocytopenia, and an anaemia that HCT 22.0 %
150 200 250
150 200 250

is slightly macrocytic. The graphical MCV
MCH
104.
34.4
fL
pg
RBC MORPH
WBC displays essentially show a single MCHC 33.0 g/dL
90 °
population cluster with moderate size RDW 17.7 %
100
100
variability and evidence of WBC fragility
50
50
PLT 15.2 10e3/uL
(FWBC flag). MPV 11.5 fL LURI
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
Although the predominant population

is nominally classified as lymphoid, it
is possible for blast cells in the more
immature (agranular) acute myeloid Morphological Images
leukaemias to show similar optical
characteristics. It is also not uncommon
for the abnormal cells in acute leukaemias
to show abnormally increased osmotic
fragility (FWBC).
Finally, the LURI flag indicates the
presence of both lower and upper range
interferences in the platelet count. This Acute lymphoblastic leukaemia Acute myeloid leukaemia (AML)
(ALL) with immature blasts showing showing predominance of immature
is a relatively common feature in some nuclear clefts blast cells. Note the occasional
high-grade proliferations that have cytoplasmic Auer rods.
circulating cell fragments or abnormal
plasma components.
CASE 19: ACUTE MYELOID

150 200 250
150 200 250

SUSPECT
LEUKEMIA (AML) WBC 28.8 10e3/uL
GRANULARITY
NEU 16.856 58.393 % IG/BAND

BLST 7.89 27.3 %
The patient was a 49 year old man with a 2
SIZE
MONe 2.15 7.46 %

100
100
week history of fever, weight loss and easy EOS .039 .135 %
50
50
bruising. The graphical data of this patient BASO

LYM
.039
1.827
.135 %
6.338 % BLAST
0
show hardly any comparison with case 19

0
0 50 100 150 200 250 0 50 100 150 200 250

but also in this case it concerned a patient RBC 2.76 10e6/uL
150 200 250
150 200 250
HGB 8.13 g/dL

diagnosed with an acute myeloid leukemia, HCT 25.1 %
M2 type, indicating again how different the MCV

MCH
91.0
29.4
fL
pg
RBC MORPH
90 °
90 °
manifestation of acute leukemia can be.

100
100
MCHC 32.3 g/dL

RDW 18.8 %
50
50
The WBC was elevated and the WBC

differential revealed predominantly
0
PLT 38.1 10e3/uL 0 50 100 150 200 250 0 50 100 150 200 250
MPV 9.56 fL WBC 10 ° WBC 0 °
neutrophils, but there was also an increase
150 200 250
in monocytes (with an absolute blast of

7.89 x 109/L). The BLAST flag was set
in combination with the IG/BAND flag,
0°
100
indicating the presence of immature

50
cells of myeloid lineage. Leukocytosis

0
0 50 100 150 200 250 0 50 100 150 200 250

was accompanied by anemia and RBC 10 ° RBC
thrombocytopenia.
39
CASE 20A: ACUTE MONOCYTIC SUSPECT
100 150 200 250
100 150 200 250

LEUKEMIA (AML)
WBC 30.1 10e3/uL
NEU 6.512* 21.62* %
GRANULARITY
BLST 10.0* 33.4* %
This samples was from a 49 year old male
SIZE
MONe 9.48* 31.5* %
patient previously diagnosed with acute EOS

BASO
.018*
1.81*
.060* %
6.01* % DFLT (NL MEB)
50
50
monocytic leukemia. The patient was initially LYM 2.261* 7.507* % BLAST
0
treated with combination chemotherapy with
0 50 100 150 200 250 0 50 100 150 200 250
RBC 3.87 10e6/uL
good response, but now presented in a first
100 150 200 250
100 150 200 250

HGB 11.2 g/dL
relapse. HCT 34.6 %
MCV 89.5 fL
The WBC was moderately increased and
90 °
90 °
MCH 28.9 pg
MCHC 32.3 g/dL
was accompanied by a mild anemia and RDW 18.3 %
50
50
thrombocytopenia. The WBC differential
0
0 50 100 150 200 250 0 50 100 150 200 250
showed monocytosis (19.5 x 109/L), half PLT 36.6* 10e3/uL
WBC 10 ° WBC 0 °
MPV >>>> fL URI
of which were classified as blast cells.
100 150 200 250

The complete WBC differential was
nevertheless marked with asterisks
0°
because the algorithm was unable to make
50
a clear separation between mononuclear
0
and polymorphonuclear cells. This issue 0 50 100 150 200 250
RBC 10 °
0 50 100 150 200 250
RBC
was clearly visible in the 90 º/WBC 10 º
scatterplot and resulted in the flag DFLT Case 20a
(NLMEB). Visual interpretation of the
WBC scatterplots suggested that the default
150 200 250

SUSPECT
150 200 250

threshold lines spuriously classified a part of WBC 19.2 10e3/uL
GRANULARITY
NEU .862 4.502 %
the “monocyte” population as basophil and BLST 4.93* 25.8* %
neutrophil. This finding was confirmed by MONe 11.4* 59.4* % SIZE
100
manual microscopy where the majority of 100
EOS .015 .080 %
50
50
BASO .726 3.79 % DFLT (LM)
the cells were classified as immature blast LYM 1.246* 6.5* % BLAST
0
0
0 50 100 150 200 250

cells, displaying monocytoid features. The
0 50 100 150 200 250
RBC 3.53 10e6/uL
URI warning flag signifies that there was a
150 200 250
150 200 250

HGB 10.5 g/dL
possible interference in the platelet count. HCT 32.7 %
Most likely this interference was due to cell MCV

MCH
92.5
29.7
fL
pg
90 °
fragments or plasma components and were 90 °

100
100
MCHC 32.1 g/dL
visible in the 0 º/RBC 10 º scatterplot as black RDW 16.6 %
50
50
dots. PLT 22.3* 10e3/uL

0
0 50 100 150 200 250 0 50 100 150 200 250

MPV 12.7* fL LRI WBC 10 ° WBC 0 °
150 200 250
CASE 20B: ACUTE MONOCYTIC

LEUKEMIA (AML)
0°
Three days after the collection of sample 21a

100
this sample was taken and noticeable is that

50
the WBC count decreased to 19.2 x 109/L and

0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 10 ° RBC
that the DFLT (NLMEB) flag has disappeared.
Interpretation of the 90 º/WBC 10 º scatterplot Case 20b
clearly demonstrated a much better separation
than the same graph of case 20a. Despite
the less problematic separation between
mononuclear and polymorphonuclear
cells, the algorithm still had difficulties
differentiating mononuclear cells into
monocytes, lymphocytes and basophils. The
problematic separation of mononuclear cells
resulted in a DFLT (LM) flag.
40
CASE 21: CHRONIC MYELOID
150 200 250
150 200 250

SUSPECT
LEUKEMIA (CML) WBC 44.6 10e3/ul
GRANULARITY
NEU 40.47 90.59 % IG/BAND
BLST .120 .269 %
This patient had evident leukocytosis with a
SIZE
MONe 2.09 4.69 %
100
100
mild anemia and thrombocytopenia. In the EOS .215 .482 %
50
50
BASO .233 .522 %
SIZE/COMPLEXITY graph a notably disperse LYM 1.526 3.419 % BLAST
0
0 50 100 150 200 250 0 50 100 150 200 250
neutrophil cluster was seen, extending RBC 3.63 10e6/uL
towards the upper central region of the plot.
150 200 250
150 200 250

HGB 10.6 g/dL
This corresponded to the presence of large- HCT 33.3 %

MCV 91.7 fL
sized granular cells with characteristics
90 °
90 °
MCH 29.1 pg
(nuclear shape, nuclear/cytoplasmic ratio and
100
100
MCHC 31.7 g/dL
RDW 12.5 %
granularity) that were different from normal
50
50
mature neutrophils. These were immature
0
PLT 71.3 10e3/uL 0 50 100 150 200 250 0 50 100 150 200 250
WBC 10 ° WBC 0 °
granulocytes (myelocytes, metamyelocytes
MPV 9.69 fL
150 200 250

and non-segmented neutrophils). The
suspected presence of immature precursors
was indicated by the IG/Band flag. All
0°
100
four WBC scatterplots demonstrated
50
large neutrophils, compatible with (meta)
0
0 50 100 150 200 250 0 50 100 150 200 250
myelocytes that were seen in the smear. In RBC 10 ° RBC
the 90º/WBC 0º scatterplot there was a

population of large mononuclear cells visible
in the lower right corner (purple events). This
population, classified as monocytes/blasts,
corresponded well with the 4 % blast counted
by manual microscopy. Using molecular
analysis, in this patient a diagnosis of chronic
myeloid leukemia was made.
CASE 22: MYELODYSPLASTIC

150 200 250
SUSPECT
150 200 250

SYNDROME (MDS RAEB) WBC 2.12 10e3/uL
GRANULARITY
NEU .969 45.79 % BAND
BLST .031 1.45 %
This sample was from a patient who was
SIZE
MONe .333 15.7 %

100
100
regularly monitored since myelodysplastic EOS .022 1.04 %
50
BASO .072 3.39 % 50

syndrome (MDS) had been diagnosed LYM .692 32.68 % BLAST
0
previously. The hemogram showed

0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 3.69 10e6/uL
pancytopenia: neutropenia, normocytic
150 200 250
150 200 250
HGB 10.0 g/dL
normochromic anemia and thrombocytopenia. HCT 30.2 %

MCV 81.8 fL
Typically this disease is characterized by two- MCH 27.2 pg
90 °
90 °
or three-lineage cytopenia with dysplastic

100
MCHC 33.2 g/dL

100
RDW 15.4 %
features in one or more cell lines. The SIZE/
50
50
COMPLEXITY scatterplot displayed a cluster PLT 65.2 10e3/uL

0
0 50 100 150 200 250 0 50 100 150 200 250

MPV 10.3 fL WBC 10 ° WBC 0 °
of large granulocytes, triggering the BAND
150 200 250
flag. Notable was also the quite low lobularity

of the neutrophils in the GRANULARITY/
LOBULARITY and the 90º/WBC 0º plots.
0°
100
The scatterplots showed no signs of abnormal

50
granulation, but manual microscopy

0
demonstrated hypogranulation and dysplastic

0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
features in almost all granulocytes. Also
immature granulocytes were observed as well
as 5 % blast cells. All these findings are well
compatible with the MDS diagnosis.
41
CASE 23: MONOCLONAL
150 200 250
150 200 250

SUSPECT
GAMMOPATHY WBC 6.39* 10e3/uL WBC
GRANULARITY
NEU 3.03* 47.546 %
BLST .001* .010 % NRBC/RRBC
This blood sample was from a patient
SIZE
MONe .502* 7.87 %
100
100
who presented with Waldenströms
EOS .062* .963 %
50
50
BASO .112* 1.75 %
macroglobulinemia with a serum IgM LYM 2.671* 41.93 %
0
0 50 100 150 200 250 0 50 100 150 200 250
concentration of 23.4 g/L (reference range RBC 4.59 10e6/uL

150 200 250
150 200 250

0.4 to 2.4 g/L). HGB
HCT
13.2
38.8
g/dL
%
MCV 84.7 fL
On the SIZE/COMPLEXITY scatterplot MCH 28.7 pg
90 °
90 °
the presence of interfering events (pink)
100
100
MCHC 33.9 g/dL
RDW 13.6 %
can be seen beneath the threshold. Also the
50
50
lymphocyte population had an abnormal PLT 260. 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
MPV 5.10 fL WBC 10 ° WBC 0 °
shape, indicating that most likely this
150 200 250

population was affected by the presence of
the interference. Based on the position of the
0°
interference, the algorithm generated the
100
NRBC/RRBC flag and triggered the WBC
50
flag to indicate that the total white cell count
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
and differential count were not reliable.
Running the sample in CBC+RRBC mode
did not improve the results. Most likely the
interfering population is due to precipitation
of M-proteins in the WBC reagent.
Also the black (noise) events in the 0º/RBC
10º scatterplot are strongly indicative for
the presence of M-proteins. The presence
of M-proteins did not interfere with the
platelet count in this sample.
42
CASE 24: PLATELET SATELLITES
150 200 250
150 200 250

SUSPECT
WBC 3.46 10e3/uL
GRANULARITY
This patient had a moderately low platelet
NEU 3.029 87.48 % IG/BAND
BLST .013 .367 %
SIZE
count. Of special note were abnormal MONe .088 2.55 %
100
100
EOS .036 1.05 %
populations in the SIZE/COMPLEXITY
50
50
BASO .008 .237 %
and the 90 º/WBC 0 º scatterplots: LYM .288 8.331 %
0
0 50 100 150 200 250 0 50 100 150 200 250
there were many neutrophils dispersed RBC 3.86 10e6/uL

150 200 250
150 200 250

in the upper right corner of the SIZE/ HGB
HCT
11.6
34.5
g/dL
%
COMPLEXITY plot and these large and MCV 89.2 fL
MCH 30.1 pg
highly complex neutrophils also seemed to
90 °
90 °
100
100
MCHC 33.7 g/dL
have a high lobularity. RDW 15.2 %
50
50
0
0
PLT 41.4* 10e3/uL
Inspection of the blood smear revealed the MPV 10.6* fL
0 50 100 150 200 250
WBC 10 °
0 50 100 150 200 250
WBC 0 °
presence of so-called platelet satellites.
150 200 250

These are platelets that aggregate together
with neutrophils in the form of rosettes.
0°
100
Similarly to “pure” platelet aggregates,
50
EDTA-dependent antibodies are involved
in the mechanism of these mixed platelet
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
and neutrophil aggregates. Spurious

thrombocytopenia is a problem in clinical
practice that may sometimes lead to
unnecessary evaluations and treatment.
Platelet satellitism, which is an in vitro
phenomenon, is associated with EDTA-
treated blood at room temperature. It is
seen in patients with vasculitis, lupus,
mantle cell lymphoma, and marginal zone
B-cell lymphoma, and most commonly in
healthy individuals. One of the proposed
mechanisms is IgG autoantibodies directed
against platelet glycoprotein IIb/IIIA
complex and the neutrophil Fc γ receptor
III.
Clinicians should be familiar with this
spurious cause of thrombocytopenia. These
antibodies, which are present in some
normal individuals, might occur naturally.
Due to the exposure of certain antigenic
structures present on EDTA-modified
platelets and neutrophils, they may manifest
themselves by triggering the Platelet
Satellitism phenomenon.
43
CASE 25: NEUTROPHIL
150 200 250
150 200 250

SUSPECT
AGGREGATES WBC 15.9 10e3/uL
GRANULARITY
NEU 11.84 74.63 % IG/BAND
BLST .002 .010 %
This 78 year old patient was hospitalized
SIZE
MONe 1.10 6.95 %
100
100
at the Intensive Care Unit with sepsis. EOS .118 .743 %
50
50
BASO .038 .241 %
As this sample gave an IG/BAND flag, a LYM 2.767 17.469 %
0
blood smear was made and examined.
0 50 100 150 200 250 0 50 100 150 200 250
RBC 5.70 10e6/uL
The microscopist noted that the
150 200 250
150 200 250

HGB 17.1 g/dL
neutrophils tended to form small groups HCT 49.7 %

MCV 87.2 fL
and when she investigated the feather MCH 30.1 pg
90 °
90 °
end of the smear she observed large
100
100
MCHC 34.5 g/dL
RDW 11.4 %
clusters of neutrophils. It was concluded
50
50
that this was a case of neutrophil PLT 241. 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
MPV 9.18 fL WBC 10 ° WBC 0 °
aggregation. This is an in vitro artefact,
150 200 250

often caused by EDTA-dependent
antibodies to neutrophils.
0°
100
In the SIZE/COMPLEXITY, 90 º /
50
WBC 10 º and 90 º /WBC 0 º plots,
neutrophils are not only located in
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
the normal position, but there is an
additional cluster, presumably consisting
of two or more aggregated neutrophils.
As the aggregated neutrophils are
counted as single neutrophils, the total
WBC and neutrophil count might be
underestimated.
CASE 26: URI FLAG

150 200 250
150 200 250

WBC 7.20 10e3/uL
The URI flag in this specimen indicated

GRANULARITY
NEU 4.179 58.081 %
BLST 0.00 0.00 %
that there was an abnormality detected
SIZE
MONe .520 7.22 %

100
in the upper region of the platelet 100

EOS .179 2.48 %
50
50
BASO .090 1.26 %
cluster. The 0 º/RBC 10 º scatterplot LYM 2.223 30.93 %
0
0 50 100 150 200 250 0 50 100 150 200 250

demonstrated the presence of large RBC 5.84 10e6/uL
platelets and this was in agreement

150 200 250
150 200 250
HGB 10.5 g/dL
with the MPV of 10.0 fL. The RBC HCT

MCV
35.6
61.0
%
fL RBC MORPH
are microcytic and hypochromic, but MCH 18.0 pg
90 °
90 °
100
100
despite the small volume of the RBC, the MCHC 29.6

RDW 12.8
g/dL
%
50
50
separation between the RBC and PLT

cluster was still perfect. PLT 221.* 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250

MPV 10.0* fL URI WBC 10 ° WBC 0 °
This picture may be compatible with

150 200 250
reactive platelets that are generally

larger than normal, in combination with
0°
100
a disorder of microcytic RBC, probably

50
thalassemia trait.
0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 10 ° RBC
44
CASE 27: NWBC FLAG
150 200 250
150 200 250

SUSPECT
WBC 6.45 10e3/uL
This sample had a reasonably normal
GRANULARITY
NEU 2.811 43.56 %
BLST 0.00 0.00 % NBWC
WBC count and differential, but the results
SIZE
MONe .603 9.35 %
100
100
were accompanied by an NWBC (non- EOS .169 2.61 %
50
50
BASO .075 1.16 %
white blood cell) flag. The Size versus LYM 2.794 43.32 %
Complexity plot was abnormal and showed
0
0 50 100 150 200 250 0 50 100 150 200 250
a clear population of events adjacent to RBC 4.15 10e6/uL
150 200 250
150 200 250

HGB 12.6 g/dL
the lymphocyte population and below the HCT 36.1 %
size threshold. These events triggered the MCV

MCH
86.9
30.3
fL
pg
90 °
90 °
NWBC flag.
100
100
MCHC 34.8 g/dL
RDW 10.7 %
50
50
The NWBC flag can be triggered by a
number of different factors including PLT 79.7 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
MPV 7.69 fL WBC 10 ° WBC 0 °
low numbers of nucleated red blood cells
150 200 250

(NRBC), lyse-resistant RBC (RRBC),
platelet clumps, or giant platelets.
Examination of scatterplots, together with
0°
100
consideration of the other hematological
50
parameters, can often provide clues as to the
0
0 50 100 150 200 250 0 50 100 150 200 250
reason for an NWBC flag. In this example, RBC 10 ° RBC
the white blood cell count and the red cell

parameters were normal, but there was an
apparent moderate thrombocytopenia of
79.7 x 109/L. In addition, the 0 º/ RBC 10 º
scatterplot demonstrated a cluster of black
events, just above the platelet cluster. All of
these are suggestive of platelet clumps and
this was confirmed by reference microscopy.
The low platelet count was consequently
interpreted as pseudo-thrombocytopenia.
CASE 28: PLATELET AGGREGATION SUSPECT

150 200 250
150 200 250

WBC 2.29 10e3/uL
This sample was a more distinct example

GRANULARITY
NEU 1.279 55.816 %

BLST .001 .039 % NBWC
of platelet aggregation. The NWBC flag
SIZE
MONe .445 19.5 %

100
100
was set and was accompanied by an URI EOS .082 3.57 %

50
50
BASO .034 1.48 %

flag, indicating interference in the region LYM .450 19.634 %
0
0 50 100 150 200 250 0 50 100 150 200 250

between the PLT and RBC populations. RBC 2.63 10e6/uL
This interference was clearly visible in

150 200 250
150 200 250
HGB 7.88 g/dL
the 0 º/RBC 10 º scatterplot as a cluster of HCT

MCV
23.2
88.2
%
fL RBC MORPH
black events above the platelets and was MCH 30.0 pg
90 °
90 °
100
100
strongly suggestive for the presence of MCHC 34.0

RDW 19.8
g/dL
%
50
50
platelet aggregation.
PLT 65.2* 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
When a low platelet count is detected in MPV 10.3* fL URI WBC 10 ° WBC 0 °
combination with the NWBC and URI

150 200 250
flag, it is advised to check the sample for

the presence of platelet aggregation.
0°
100
50
0
0 50 100 150 200 250 0 50 100 150 200 250

RBC 10 ° RBC
45
CASE 29: COLD AGGLUTINATION
150 200 250
150 200 250

WBC 6.54 10e3/uL
In this blood sample all RBC and PLT
GRANULARITY
NEU 4.211 64.446 %
BLST .002 .033 %
results were invalidated by the MCHC
SIZE
MONe .570 8.72 %
100
100
alarm, triggered by the extremely EOS .088 1.35 %
50
50
high MCHC. This was due to a large
BASO .036 .544 %
LYM 1.629 24.954 %
discrepancy between HGB concentration
0
0 50 100 150 200 250 0 50 100 150 200 250
and RBC count. The sample contained RBC 3.53* 10e6/uL
150 200 250
150 200 250

HGB 13.8* g/dL
cold agglutinins, causing aggregates HCT 33.4* %
of RBC that fell outside the window MCV

MCH
94.8*
39.1*
fL
pg
RBC MORPH
90 °
90 °
of analysis. This could easily be seen
100
100
MCHC 41.3* g/dL MCHC
in the RBC histogram, where there RDW 15.7* %
50
50
was an abnormal irregular area and a PLT 150.* 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
distinct peak on the right side of the MPV 5.38* fL WBC 10 ° WBC 0 °
RBC population indicating the presence
150 200 250

of large RBC (as a result of the RBC
agglutination).
0°
100
50
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
CASE 30: MALARIA (PL.
150 200 250
150 200 250

FALCIPARUM)
SUSPECT
WBC 11.3 10e3/uL
GRANULARITY
NEU 2.253 19.888 %
This patient was hospitalized in BLST .030 .262 %

SIZE
MONe 1.21 10.7 %
a critically ill condition on return 100
100
EOS .214 1.88 % ATY DEP
from a visit to Ghana with a fever of
50
50
BASO .065 .577 %
LYM 7.574 66.7 % VAR LYM
40 ºC, violent headaches, myalgia and
0
0
0 50 100 150 200 250 0 50 100 150 200 250
severe diarrhea. His admission blood RBC 4.32 10e6/uL
150 200 250
150 200 250

HGB 14.2 g/dL
sample showed mild lymphocytosis in HCT 39.1 %
combination with thrombocytopenia. The MCV 90.5 fL
lymphocytosis was accompanied with MCH 32.8 pg

90 °
90 °
100
100
MCHC 36.3 g/dL
an ATYP DEP (atypical depolarization) RDW 13.2 %
50
50
and VAR LYM flag alert. In the blood PLT 73.4 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250

smear about 23 % of his erythrocytes were MPV 5.42 fL WBC 10 ° WBC 0 °
infected by malaria parasites. A diagnosis

150 200 250
of Plasmodium falciparum malaria was

made.
0°
100
In the GRANULARITY/LOBULARITY
50
plot, numerous purple-colored cells with

0
atypical depolarization were visible, above

0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
the normal monocyte position. Events
coded by MAPSS as monocytes (purple)
normally never depolarize light. This
pattern is compatible with monocytes that
have ingested malaria-derived hemozoin
pigment.
Patient was treated with an experimental
anti-malaria drug and released after 6 days
from the hospital without complications.
46
CASE 31A: RESISTANT RBC FLAG
150 200 250
150 200 250

SUSPECT
WBC 14.5* 10e3/uL WBC
This case shows a sample that was
GRANULARITY
NEU 1.50* 10.3* %
BLST 0.00* 0.00* % NRBC/RRBC
processed in routine CBC mode. The
SIZE
MONe .159* 1.10* %
100
100
WBC count (14.5 x 109/L) and the EOS .053* .366* %
50
50
BASO .040* .276* % DFLT (NL ME B)
absolute lymphocyte count (12.7 x 109/L) LYM 12.7* 87.6* %
0
0 50 100 150 200 250 0 50 100 150 200 250
both appeared to be increased, but these RBC 3.78 10e6/uL
results were accompanied by WBC and
150 200 250
150 200 250

HGB 8.25 g/dL
NRBC/RRBC flags and data-invalidating HCT

MCV
26.5
70.0
%
fL RBC MORPH
* asterisks. Examination of the Size MCH 21.8 pg
90 °
90 °
100
100
versus Complexity plot showed a distinct MCHC 31.2
RDW 23.0
g/dL
%
50
50
population of pink events below the size
threshold. The RRBC flag indicates the PLT 79.1 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
MPV 6.07 fL WBC 10 ° WBC 0 °
need for sample rerun in the CBC+RRBC
150 200 250

mode.
0°
100
50
0
0 50 100 150 200 250 0 50 100 150 200 250
RBC 10 ° RBC
CASE 31B: RESISTANT RBC FLAG SUSPECT
150 200 250
150 200 250

WBC 3.22 10e3/uL
When the sample of case 31a was rerun
GRANULARITY
NEU 1.444 45.03 %
BLST 0.00 0.00 % NWBC
in the CBC+RRBC mode, a much lower
SIZE
MONe .312 9.70 % 100
100
WBC count of 3.22 x 109/L was obtained EOS .057 1.76 %
50
50
BASO .094 2.93 %
and the absolute lymphocyte count LYM 1.30 40.6 %
0
0
0 50 100 150 200 250 0 50 100 150 200 250
dropped from 12.7 x 109/L to 1.30 x RBC 3.93 10e6/uL
109/L. But there was still a persistence

150 200 250
150 200 250

HGB 8.26 g/dL
of residual pink events suggesting that HCT

MCV
27.6
70.0
%
fL RBC MORPH
not all resistant RBC were lysed or that MCH 21.0 pg
90 °
90 °
100
100
NRBC could be present. Consequently, MCHC 30.0
RDW 23.7
g/dL
%
50
50
the NWBC flag was set.

PLT 88.5 10e3/uL
0
0 50 100 150 200 250 0 50 100 150 200 250
However, even though these flags MPV 6.89 fL WBC 10 ° WBC 0 °
indicate the need for independent

150 200 250
validation, visual review of the Size

versus Complexity plot suggests that
0°
100
the results are unlikely to be affected to

50
any great extent by the residual RRBC.

0
Eventually, reference microscopy 0 50 100 150 200 250

RBC 10 °
0 50 100 150 200 250
RBC
confirmed the presence of 4 NRBC/100

WBC.
47
CASE 32: RETICULOCYTES NORMAL
200 250
200 250
HEALTHY SUBJECT %R 1.48 %
RBCr 4.61 10e6/uL
RETC 68.3 10e3/uL
The numerical and graphical displays
150
150
90 °
90 °
100
100
shown were from a healthy subject
without anemia and with all RBC
50
50
parameters within the reference ranges.
0
0 50 100 150 200 250 0 50 100 150 200 250
The normal reticulocyte reference range

for adults is between 0.5 % and 1.6 %,
with absolute counts in the order of 25
to 100 x 109/L. In a 3-month fetus, 90 %
of the red cells are reticulocytes and this
drops to 15 % – 30 % at the 6th month 0 50 100 150 200 250 0 50 100 150 200 250
of fetal life. Compared to adults, cord RETC 90 °/0 ° RETC 90 °/10 °
blood samples from full-term infants

still show increased reticulocyte counts,
with a reported mean of 3.11 ± 0.75 %
(SD) and an absolute count of 137.3 ± 33.3
x 109/L. These fall to adult levels within
5 days. Premature infants tend to have
higher reticulocyte counts than full-term
infants.
CASE 33: RETICULOCYTES

200 250
200 250
MACROCYTIC ANEMIA %R 2.92 %
RBCr 2.89 10e6/uL
RETC 84.4 10e3/uL
This patient, earlier discussed in
150
150
90 °
90 °
100
100
case 4, had a macrocytic and slightly
hypochromic anemia. In addition,
50
50
she had a normal serum ferritin and
0
0
0 50 100 150 200 250 0 50 100 150 200 250
elevated liver enzyme concentrations,
most likely due to her history of alcohol
abuse. The reticulocyte count was
84.4 x 109/L, around the upper limit
of the reference range. Nevertheless
looking to the RBC concentration of
2.89 x 1012/L in combination with a 0 50 100 150 200
RETC 90 °/0 °
250 0 50 100 150 200
RETC 90 °/10 °
250
HGB concentration of 9.97 g/dL, a more

distinct reticulocytosis would be likely.
Therefore it was concluded that this
patient had decreased erythropoiesis.
48
CASE 34: RETICULOCYTES
200 250
200 250
NEONATE %R 7.28 %
RBCr 5.62 10e6/uL
RETC 409. 10e3/uL
A newborn boy was 1 day old when a
150
150
90 °
90 °
100
100
blood sample was collected. Essentially
all results were normal for a neonate
50
50
of that age. He had no anemia (HGB
0
0 50 100 150 200 250 0 50 100 150 200 250
was 16.3 g/dL) and his erythropoiesis
was active as shown by the reticulocyte
count, which was even increased for a
healthy neonate of his age.
0 50 100 150 200 250 0 50 100 150 200 250

RETC 90 °/0 ° RETC 90 °/10 °
CASE 35: RETICULOCYTES AUTO-
200 250
200 250
IMMUNE HEMOLYTIC ANEMIA %R >>>> %
RBCr 1.78 10e6/uL
RETC >>>> 10e3/uL
A 76 year old man had severe anemia
150
150
90 °
90 °
100
100
and a disproportionately low RBC count.
MCV count was strongly increased,
50
50
due to the massive reticulocytosis.
0
0
0 50 100 150 200 250 0 50 100 150 200 250
The percentage of reticulocytes was
suppressed by the CELL-DYN Ruby
because it exceeded the upper limit of
23 %. Manual examination revolved a
reticulocyte percentage of 35.
0 50 100 150 200 250 0 50 100 150 200 250

RETC 90 °/0 ° RETC 90 °/10 °
49
REFERENCES
1. Terstappen L.W.M.M., de Grooth B.G., Visscher K., van Kouterik F.A., Grew. J. Four-Parameter
White Blood Cell Differential Counting Based on Light Scattering Measurements. Cytometry
1987;9:39 – 43.
2. Kim YR, Stroupe SD. Cyanide-free reagent and method for the determination of hemoglobin. US
Patent 5,612,223 1997.
3. K
im YR, Ornstein L. Isovolumetric sphering of erythrocytes for more accurate and precise cell
volume measurement by flow cytometry. Cytometry 1983;3:419 – 427.
4. C
LSI. Methods for reticulocyte counting (Automated blood cell counters, flow cytometry, and
supravital dyes); approved guideline – second edition. NCCLS document H44-A2. Wayne, PA:
Clinical and Laboratory Standards Institute, 2004.
50
CHOOSE TRANSFORMATION™
Achieve measurably better healthcare performance.
www.corelaboratory.abbott/hematology
For in vitro diagnostic use only. CELL-DYN Ruby is a Class 1 Laser Product.
CELL-DYN, CELL-DYN Ruby and Choose Transformation are trademarks of Abbott
Laboratories in various jurisdictions.
© 2019 Abbott Laboratories. ADD-00066186.

Add-00066186 - Ruby Casebook 2019.semnat

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Add-00066186 - Ruby Casebook 2019.semnat

Uploaded by

Copyright:

Available Formats

CELL-DYN Ruby

CHOOSE TR ANSFORM ATION ™

TECHNOLOGY AND METHODS.....................................................................................................7

SCATTERPLOTS AND HISTOGRAMS ..........................................................................................17

Figure 1.1: Schematic overview of the hemoglobin flow cell

Figure 1.2: Light scattering by a blood cell

Table 1.1. Four angles of light scatter detection in CELL-DYN Ruby

0° axial light loss (ALL) cell size

Beam expander Laser

Figure 1.3: CELL-DYN Ruby optical bench

0 50 100 150 200 250

Figure 1.4A: Before classification all events are labeled “unknown”

Step 1: separation of mononuclear and polymorphonuclear WBC

0 50 100 150 200 250 0 50 100 150 200 250

Figure 1.4B: Separation of mononuclear and polymorphonuclear cells

Step 2: separation of eosinophils and neutrophils

Figure 1.4C: Separation of eosinophilic and neutrophilic granulocytes

Step 3: separation of monocytes, lymphocytes and basophils

0 50 100 150 200 250 0 50 100 150 200 250

Figure 1.4D: Identification of monocytes, lymphocytes and basophilic granulocytes

Step 4: elimination of cell debris and noise

Figure 1.4E: Elimination of cell debris and noise

Step 5: construction of scatterplots and calculation of WBC concentrations

0 50 100 150 200 250

Figure 1.4F: WBC Differential scatterplot showing five cell populations

0 50 100 150 200 250

Figure 1.5: The NOC histogram

0 50 100 150 200 250

0 50 100 150 200 250 0 50 100 150 200 250

Table 2.1: Color codes of cells used in CELL-DYN hematology analyzers.

CELL LINEAGE COLOR CELL TYPE

WBC DIFFERENTIAL PLOT

0 50 100 150 200 250 0 50 100 150 200 250

0 50 100 150 200 250 0 50 100 150 200 250

0 50 100 150 200 250

PLT PLOT AND HISTOGRAMS

0 50 100 150 200 250 0 50 100 150 200 250

RBC RBC/PLT 0° PLT

RETC PLOTS AND HISTOGRAMS

0 50 100 150 200 250 0 50 100 150 200 250

BAND AND IG FLAGS

COMPLEXITY COMPLEXITY COMPLEXITY

WBC 0° WBC 0° WBC 0°

WBC FLAG IN COMBINATION WITH NRBC/RRBC FLAG

0 50 100 150 200 250 0 50 100 150 200 250

WBC FLAG IN COMBINATION WITH FWBC FLAG

DFLT (NLMEB) FLAG

RBC MORPHOLOGY FLAG

150 200 250

MONe .618 10.5 %

Normal segmented neutrophil Normal segmented eosinophil LYM 2.287 38.791 %

0 50 100 150 200 250 0 50 100 150 200 250

150 200 250

MCHC 34.9 g/dL

Normal lymphocyte Normal segmented basophil

0 50 100 150 200 250 0 50 100 150 200 250

Normal segmented monocyte.

CASE 2A: IRON DEFICIENCY

150 200 250

150 200 250

normal, this in combination with an

150 200 250

150 200 250

Table 2.1: Color codes of cells used in CELL-DYN hematology analyzers.